All right are reserved to the Iraqi Journal of Pharmaceutical Sciences 

CONTENTS 

ARTICLES Page 

Effect of Some Storage Conditions upon the Survival of Some Fungal Spores. 

Raghad A.Al-Shikli , Alaa A.Abdulrasool and Mustafa M. Al-Hiti 

Improving an Ovulation Rate in Women with Polycystic Ovary Syndrome by 

Using Silymarin . 

Mohammed A.Taher , Yaser A.Atia and Manal K.Amin 

A Comparative Biochemical Study of Proteins Profile in Iraqi Children and 

Adolescent with β–Thalassemia. 

Ali M. Malik ,Emad M. Malik , Nawal MJ Al-Shammaa and Zeinab M. Al-Rubaei 

Comparative Effects of Fentanyl, Medazolam, Lignocaine and Propranolol on 

Controlling the Hemodynamic Pressor Response during Laryngoscopy and 

Intubation. 

May S. Al-Sabbagh 

Validity of Generalized Standard Addition Method for a Mixture of Amino 

Acid Analysis . 

Azhar M. Jasim 

Lithotripsy of Different Urinary Tract Stones by Using Seeds of Carum 

copticum. 

Ahmed G. Sabar 

Evaluation of Stability of Cefamandol and Ceftazidime with Clavulanic Acid 

Against Extended Spectrum β- Lactamase. 

Siham S. Shaokat and Hamoudi A. Hameed 

Gravimetric Estimation of Caffeine in Different Commercial Kinds of Tea 

Found in the Iraqi Market. 

Maha N. Hamad and Dhuha A. Abdul-Hussain 

Goserelin versus Norethisterone in the Management of Menorrhagia with 

Uterine Fibroid. 

Faris A. Rasheed , Jwan N. Sulaiman and Yousif Abdul-Raheem 

Anti-bacterial Properties of Melatonin against Mycobacterium Tuberculosis in 

vitro. 

Thamer M. Jasim , Mustafa G.Alabbassi , Suhad F. Hatem Almuqdadi and 

Jinan K. Kamel 

In vitro Evaluation of Tinidazole Bioadhesive Vaginal Gels. 

Zainab T.Salih 

Urine Protein SDS-PAGE Reveals Different Profiles in Iraqi Children with 

Kala-azar. 

Yassir MK Al-Mulla Hummadi 

Development of Modified Release Nicotine Tablet Formulation for Treatment 

of Ulcerative Colitis. 

Marwan Y. Al-hurr 

1 

11 

19 

24 

31 

38 

42 

48 

54 

59 

64 

70 

75

Iraqi J Pharm Sci, Vol.19(2) 2010 Fungal contamination and storage conditions 

Effect of Some Storage Conditions upon the Survival of 

Some Fungal Spores 

Raghad A. Al-Shikli *,1 , Alaa A.Abdulrasool ** and Mustafa M. Al-Hiti * 

* Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad, Baghdad, Iraq. 

** Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. 

Abstract 

Folic acid and multivitamin tablets containing Aspergillus flavus Penicillia spp. and Cladosporia 

spores were prepared at a compression pressure of 148 MN/m 2 and stored at 35°C under different 

relative humidifies (75,85, and 95)% within air tight containers, to study the effect of storage condition 

on the m, as well as ,the estimation of the microbial level of the raw materials intended to be used in the 

two kinds of tablets . Result showed that some raw materials derived from natural origin were heavily 

contaminated with microorganism compared to that of synthetic origin ,the results also indicated the 

effect of relative humidity , types of fungal spore , and the hygroscopic nature of exicpient upon 

survival. Multivitamin tablets showed more survival than folic acid tablets and this is due to the 

presence of more nutrients. No aflatoxin was obtained from both multivitamin and folic tablets at 35°C 

temperature; this is due to the temperature which is not an optimum temperature for aflatoxin B1 

production. 

Key words: Storage conditions of tablet, fungal spores. 

ﺔﺻﻼﺨﻟﺍ 

ﺓﺭﺍﺮﺣ ﺔﺟﺭﺩ ﻲﻓ ﺎﻬﻧﺰﺧ ﻢﺗﻭ 2ﻡ 

/ ﻦﺗﻮﻴﻧ ﺎﻜﻴﻣ 148 ﻂﻐﺿ ﺖﺤﺗ ﺕﺎﻨﻴﻣﺎﺘﻴﻔﻟﺍ ﻦﻣ ﺔﻋﻮﻤﺠﻣ ﺏﻮﺒﺤﻣﻭ ﻚﻴﻟﻮﻔﻟﺍ ﺾﻣﺎﺣ ﺏﻮﺒﺣ ﺮﻴﻀﺤﺗ ﻢﺗ 

ﺞﺋﺎﺘﻨﻟﺍ ﺖﻨﻴﺑ ﺪﻘﻟﻭ ﺏﻮﺒﺤﻟﺍ ﻦﻣ ﻦﻴﻋﻮﻧ ﺮﻴﻀﺤﺗ ﻲﻓ ﺔﻣﺪﺨﺘﺴﻤﻟﺍ ﻡﺎﺨﻟﺍ ﺩﺍﻮﻤﻠﻟ ﻲﺑﻭﺮﻜﻴﻤﻟﺍ ﺙﻮﻠﺘﻟﺍ ﺏﺎﺴﺣ ﻢﺗ ﺎﻤﻛ ﺔﻔﻠﺘﺨﻣ ﺔﻴﺒﺴﻧ ﺔﺑﻮﻃﺭﻭ ﻡ° 

35 

ﺩﺍﻮﻤﻟﺍ ﻦﻣ ﺔﺛﻮﻠﻣ ﺮﺜﻛﺃ ﺔﻴﻧﻻﺪﻴﺼﻟﺍ ﺕﺍﺮﻀﺤﺘﺴﻤﻟﺍ ﺔﻋﺎﻨﺼﻟﺍ ﻲﻓ ﻞﺧﺪﺗ ﻲﺘﻟﺍ ﺔﻴﺗﺎﺒﻨﻟﺍ ﻭﺍ ﻲﻧﺍﻮﻴﺤﻟﺍ ﻞﺻﻷﺍ ﺕﺍﺫ ( ﻡﺎﺨﻟﺍ ) ﺔﻴﻟﻭﻷﺍ ﺩﺍﻮﻤﻟﺍ ﻥﺃ 

ﺪﻗ ﻉﺍﻮﻧﻷﺍ ﺾﻌﺑ ﻥﺇ ﺚﺤﺒﻟﺍ ﻝﻼﺧ ﻦﻣ ﻦﻴﺒﺗ ﺪﻗﻭ .. ﺔﻴﺿﺮﻣ 

ﺎﻳﺮﺘﻜﺑ ﻰﻠﻋ ﺎﻬﺋﺍﻮﺘﺣﺍ ﺚﺤﺒﻟﺍ ﻦﻣ ﻦﻴﺒﺗ ﻲﺘﻟﺍ ﺍﺪﻋﺎﻣ ﻲﻋﺎﻨﺼﻟﺍ ﻞﺻﻷﺍ ﺕﺍﺫ ﺔﻴﻟﻭﻷﺍ 

ﻲﺘﻟﺍ ﺏﻮﺒﺤﻟﺍ ﻥﺍ ﺞﺋﺎﺘﻨﻟﺍ ﺕﺮﻬﻇﺃ ﺪﻗﻭ ﺕﺎﻳﺮﻄﻔﻟﺍ ﻩﺬﻫ ﻰﻠﻋ ﺎﻫﺮﻴﺛﺄﺗﻭ ﺔﻔﻠﺘﺨﻣ ﺕﺪﺑ ﻑﻭﺮﻇ 

ﺔﺳﺍﺭﺩ ﻰﻟﺍ ﺖﻋﺩ ﻚﻟﺬﻟﻭ ﺲﺒﻜﻟﺍ ﺔﻴﻠﻤﻋ ﺖﻣﻭﺎﻗ 

ﺔﺒﺴﻧ ﻰﻠﻋ ﺮﻬﻇﺃ ﺱﻼﺟﺭﺎﺒﺳﺍ ﺮﻄﻔﻟﺍ ﻥﺍﻭ ﺔﻴﺋﺍﺬﻏ ﺩﺍﻮﻣ ﻦﻣ ﻪﻳﻮﺤﺗ ﺎﻣ ﺐﺒﺴﺑ ﺙﻮﻠﺘﻟﺍ ﻦﻣ ﻰﻠﻋﺍ ﺔﺒﺴﻨﺑ ﺮﻬﻈﺗ ﺕﺎﻨﻴﻣﺎﺘﻴﻔﻟﺍ ﻦﻣ ﻉﻮﻤﺠﻣ ﻱﻮﺤﺗ 

. 1 ﺏ ﻦﻴﺴﻛﻮﺗﻼﻓﺍ ﺝﺎﺘﻧﻹ ﺔﻤﺋﻼﻣ ﺮﻴﻏ ﻡ° 

35 ﺔﺟﺭﺩ ﻥﺇ ﻰﻟﺇ ﺩﻮﻌﻳ ﺍﺬﻫﻭ ﻦﻴﺴﻛﻮﺗﻼﻓﻼﻟ 

ﺔﺒﺴﻧ ﻱﺍ ﻞﺠﺴﻳ ﻢﻟﻭ ﺕﺎﻳﺮﻄﻔﻟﺍ ﺔﻴﻘﺒﺑ ﺔﻧﺭﺎﻘﻣ ﺙﻮﻠﺘﻠﻟ 

Introduction 

The microbiological quality of nonsterile 

pharmaceuticals (tablets) is largely 

determining by the microbial contamination of 

a raw materials. The effect of the 

manufacturing process and the fate of 

contaminating microorganisms during storage. 

Several infection outbreaks which would be 

traced back to the use of heavily contaminated 

raw materials of natural origin have been 

reported (1) (2) (3) and (4) .During manufacturing 

the viability of microbial cells can be 

significantly affected by the drying process of 

granules (5) (6) (7) 

and by the actual compaction 

.The availability of water probably plays an 

important role. As long as tablets are stored 

under dry conditions spoilage due to growth of 

micro organisms is unlikely to occur (8) . 

1 Corresponding author E- mail : raghad razook@yahoo.com 

razook@yahoo.com 

Received : 7 /2 / 2009 

Accepted : 24 /5 / 2009 

1 

However, in regions with a hot and humid 

conditioned, growth of contaminating microorganisms 

cannot be excluded. More ever ,in 

such countries pharmaceutical preparations are 

frequently stored under uncontrolled 

conditions and may be dispended in non 

protective packaging or even without any 

packaging at all. Few studies that investigate 

the effect of storage on the microbiological 

quality of tablets 

(9) (10) (11) 

,but little 

informations are available upon the fungi as 

contaminants in pharmaceutical industries and 

possible toxicogenic power (13) (15) (16) .The aim 

of this study was to investigate the effect of 

storage under different conditions on the 

growth of contaminating fungal spores..


Materials and Methods 

Chemicals 

Acetonitrile, Acetone , Ammonium 

hydroxide, Anhydrous Sodium Sulfate, 

Benzene, chloroform , glacial Acetic acid , 

hydrous disodium hydrogen phosphate 

(Na2HPO4 . 12H2O) . hydrochloric acid, 

methanol, potassium hydroxide, potassium 

hydroxide, potassium chloride, sulfuric acid, 

Sodium 1-hexana sulfonate sodium 

perchlorate, sodium chloride, and Tween 80 

(supplied by BDH England monobasic 

potassium phosphate from fluka-swizerland 

Hexana supplied by Merch-w Germany 

Microcrystalline cellulse (Avicel PH 101, 

Avicel PH 301), Folic Acid, Maize Starch, 

Vitamin B1 (Thiamin mononitrite), Vitamin 

B2 (Riboflavin). Vitamin B6 (pyridoxine 

HCl). Methionine, talc and Magnesium Stearte 

supplied by FMC corporation and Kindly 

supplied from (Samara Drug Industries SDl. 

Iraq). 

Microorganisms 

Aspergillus flavus, Penicillia SPP. And 

Cladsporoia Cladosporoids were obtained from 

College of Agriculture, University of Baghdad. 

Cultures were stored on Sabouraud Agar slants 

following incubation at 25°C for five days 

Fresh cultures were prepared every four weeks. 

Culture Media 

Sabouraud Dextose Agar. 

Rose Bengal Agar 

macConkey Broth 

solution used for dilutions and preparation of 

spore suspension. 

Relative humidity of the prepared solution. 

Extraction solvent of Aflatoxin. 

Relative humidity Containers 

2 

Relative humidity of the prepared solution. 

Table (1) represents the percentage of the 

relative humidity RH% of the prepared 

solutions as prescribed by AL Taher, 1990 

Extraction solvent of Aflatoxin 

According to howell and Taylor(1980) the 

extraction solvent of aflatoxin consists of 

acetonitrile : KCl 4% w/v: HCl 5N a ratio of 

450 ml: 10ml. 

Relative humidity Containers 

Relative humidity containers consist of two 

glass containers connected one above the other 

and joined together by the cover of them and 

the cover is punched to allow the exchange of 

humidity between the two containers . 

Table 1 : Relative Humidity of Various 

Solution 

Substance 

H2SO4 

KCl 

Na2HPO4.12H2O 

% of Substance 

in Solution 

23% 

Saturated Solution 

Saturated Solution 

Assessment of Microbial Levels of the Raw 

Materials and active Ingredients 

Sample of the following raw materials 

were collected from various sources and were 

subjected to microbiological assay according 

to the BP 1998 , as shown in table (2) in order 

to determine their microbial contents. Three 

types of raw materials were examined (Gelatin, 

Talc, and Starch) representing animal , 

mineral, and botanical origin, respectively As 

well as those of synthetic origin such as avicel, 

methionine, thiamin, pyridoxine, riboflavin 

and folic acid . 

Samples were taken aseptically using the pour 

plate and memberane filtration techniques to 

determine the microbial level in the raw 

materials . 

Table 2 : Isolation and Ide tification Tests for Spe cified Microorganisms (BP , 1998) 

RH% 

Organism Enrichment Primary test Secondary test Confirmation 

Enterobacteriaceae Lactose broth EEB-Mossel 

VRBGLA 

Growth of Gram- 

35-37 °C for 2-5 hr 35-37 °C for 24-48 hr 35-37 °C for 16-24 hr negatives 

E.coli As above MacConkey broth MacConkey agar 

Indole 43.5-44.5°C 

43-45°C for 18-24 hr 43-45°C for 18-24 hr Biochemical 

Salmonella As above 

TBBG broth 

TSI agar 

Biochemical 

For 5-24 hr. 

42-43°C For 18-24 hr. 

Then subculture on: 

DCA,XLDA or BGA 

35-37 °C for 24-48 hr. 

35-37 °C for 18-24 hr. serological 

P s.aeruginosa Saline peptone Casein digest broth Cetrimide agar 

Oxidase test 

35-37 °C for 2-5 hr 35-37 °C for 24-48 hr. 35-37 °C for 24-48 hr. 

Staph.aureus As for P s.aeruginosa As for Ps.aeruginosa Baird-Parker 

Coagulase. 

above 

above 

35-37 °C for 24-48 hr. Catalase,DNase Test 

EEB-m-Mossel Enterobacteriaceae enrichment broth - Mossel ;VRVGLA,violet red bile agar 

75% 

85% 

95%


Preparation of dried spore powder 

A 0.1 ml aliquots of 7 days cultures of A. 

flavus, Cladospoa and pencillia were 

inoculated onto the surfaces of predried 

sabouraud dextrose ager plates, these were 

incubated at 25°C for 5 days. After this spores 

were clearly visible on all the plates. Three 

millimeters of stetrile water containing 0.1% 

Tween 80 (as a dispersing agent) were added 

to each plates. The spores were then dislodged 

by using glass spreader. The spore suspensions 

were obtained then stirred by using a vortex 

mixer for one minute. The spore suspensions 

were then filtered through a sterile cotton wool 

in order to get rid of the hypha. The filtrates 

were then harvested by centrifugation at 

(10,000xy) for 10 min ) the superntanat liquids 

were then decanted and the residues were 

resuspended in 20 ml of sterile distilled water, 

washing were repeated three times . The 

number of spores of the resultant spore 

suspensisons was determined by aviable count 

technique, spore suspensions were adjusted so 

that the following suspensions were obtained, 

as 2.16x106 spore/ml for A flavus, 1.68x106 

spore/ml forpenicillia spp. And 1.98x106 

spore/ml for c.cladosporoids. 

Tablet formulation 

Multivitamin tablets and folic acid tablets 

were prepared using the formulas listed in 

tables (3) and (4), respectively. 

The following excipients were used. Avicel PH 

301 as a direct compression excipient, starch 

(5% w/w) as a disintegrant, magnesium 

stearate and stearic acid as lubricants. They 

were mixed with the active ingredient and 

compressed directly using a single punch 

tabletting machine with 7-mm flat-faced 

punches 

Table 3 : The Formula of the Pre pared 

Folic Acid Tablet. 

Ingre dient Amount/tab. 

Folic Acid 1 mg 

Avicel PH 301 

118.6 mg 

Maize Starch 6.5 mg 

Mg. Stearte 2.6 mg 

Talc 1.3 mg 

Total 130 mg 

3 

Table 4: The Formula of the Prepare d 

Multivitamin Tablet 

Ingredie nt Amount / tab 

Tianmin Mononitrite 1.5 mg 

Riboflavin U.S.P. 2.0 mg 

Pyridoxine HCl U.S.P 2.0 mg 

Methionine 2.0 mg 

Avicel PH 301 105 mg 

Maize Starch 6.5 mg 

Taic U.S.P 6 mg 

Stearic Acid ( Powdered) 3.0 mg 

Mg.Stearte ( Powdered ) 2.0 mg 

Total 130 mg 

Preparation of contaminated tabltes: 

For preparation of 500 contaminated 

tablets (0.3 ml. 30 ml) of A. flavus (0.4 ml 

40ml) of penicillia spp and (0.3ml, 30ml) of C. 

cladosporoids were transferred to a sterile 

mortars and placed in the incubator until 

completely evaporation of water. Dried spores 

were scraped off and were included in direct 

compression formulations by dry mixing to get 

10 2 spore /gram and 10 4 spore/gram for each of 

A. flavus pencicilia spp and C. cladosporoids 

respectively. Ingredients including dried 

microorganisms spores were weighed and 

lightly mixed in a glass morter by the method 

of geometric dilution technique for 20 minutes 

preliminary experiments had established that 

this method gives a uniform distribution of the 

microorganisms within the formuation screen 

in the lubricant (magnesium stearte or stearic 

acid) and mixed for an additional 5 minutes . 

Quantities each of 130 mg were accurately 

weighed and poured into 7-mm diameter 

compresed between flat –Faced punces using a 

single punch tableting machine which was 

disinfected with 70% alcohol and the feed shoe 

was heat sterilized before use. 

Determination of viable number of spores in 

the prepared tablets: 

Viable number of spores in prepareted 

tablets was determined immediately after their 

production at different compression forces and 

after storage up to 8 weeks eight tablets (total 

wt.=1gm) were disintegrated in tryptic soy 

broth (9ml) according toBP 1998 using a flask 

shaker and suitable serial dilution in tryptic 

broth were prepred. One –ml sample of each 

dilution was poured in sterile petridish and 

then 15 ml of molten dextrose agar was added 

to the plate.


The sample and molten sabouraud dextrose 

agar were mixed together in forward and 

backward movement and swirled movement. 

The plate were allowed to solidify on surface . 

the plates were incubated at 35°C for 2-5 days. 

Survivals as colony forming units were 

estimated as the mean of triplicate 

determination and expressed as a percentage 

relative to an uncompressed control samples of 

the contaminated formulation. 

Physical properties of tablets: 

Thirty tablets were prepared using 

different compression pressure (137.9, 144.8 

and 148.3MN/m 2 ) the physical properties of 

the tablets were determine (plumpton, 1982), 

these are tablet weight, thickness, friability, 

hardness (breaking strength), and 

disintegration time 

Assay of Tablets: 

An HPLC method was used for the assay 

of multivitamin tablets and folic acid tablets. 

Assay for pyridoxine hydrochloride and 

thiamin multivitamin Tablets 

The assay for pyridoxine hydrochloride 

and thiamin in multivitamin tablets was done 

according to the U.S.P XXIV method. 

Effect of storage under different Relative 

Humidities upon the Survival of A. flavus, 

Penicilline, and Cladospori in folic acid and 

Multivitamin Tablets 

Folic acid and multivitamin tablets 

containing Aspergillus.flavus Cladosporia ,and 

penicillia spores prepared at a compression of 

148 MN/m2 and stored at 35°C under different 

relative humidity's (RH). The relative humidity 

were 75%, 85% and 95% within airtight 

containers. Survival of the spores within the 

tablets was assessed as the mean viability for 

each group at time 0 and after 1,2, 4,6 and 8 

weeks. The total viable counts of the 

uninoculated (control) folic acid and 

multivitamin tablets were measured directly 

after preparation and after 4,8 weeks of storage 

at 35°C and 75% RH, 85% RHor 95% RH. 

Aflatoxin assay 

The amount of aflatoxin produced after 

storage the tablets (multivitamin and folic 

acid) at different relative humidities (75,85 and 

95)% were assed at different time intervals 

using a modified method of Howel and Taylor 

(1981), the modification includes the use of 

twenty five grams of multivitamin and flic acid 

tablets stored at 4,6 and 8 weeks intervals. 

4 

Results and Discussion 

Microbiological quality of some raw 

materials used in the production of tablet and 

tablet ingredients 

Microbiological evaluation of the raw 

materials used in the production of tablets is 

presented in table (5), the result show that 

synthetic materials such as folic acid, 

magnesium stearate ,thiamin, riboflavin, 

pyridoxine, methionine and microcrystalline 

cellulose (avicel PH 301) had no microbial 

contaminants thus they meet the requirement 

of the B.P 98 which specify that atotal viable 

aerobic count bacteria should be equivalent to 

or less than 103 c.f u/gm and a total viable 

count for fungi equivalent to or to less than 

102 c.f.u/gm is accepted. Microcrystalline 

cellulose PH 101 although it is a syntheyic raw 

material , it showed the presence of 

pseudoumonas aeruginosa (2x 10 2 cfu/gm) . 

samples taken from different parts of the 

microcrystalline PH 101 container showed 

apresence of pathogens in upper part of the 

container this is because microcrystalline 

(MCC) is a highly hygroscopic material (17) 

through its capillary action when it is exposed 

to air .Table (5) also shows that raw materials 

of natural origin(maize starch ,gelatin and talc) 

had relatively higher microbial levels which 

are (7*10 2 ,9*10 2 and 102 C.F.U/gm)for starch, 

gelatin and talc, respectively than that of the 

synthetic origin. This finding is similar to that 

of lbrahim Y.K.E 1991 which is due to the 

fact that materials of natural origin is rich in all 

the necessary requirement of growth needed by 

the microorganism. in addition, the results 

indicate that raw materials derived from 

animal and botanical origin had a higher 

microbial level than that o f mineral origin. 

This is in agreement with the reported 

hypothesis of Bonomi and Negrriti,Baggerman 

and Kannegiter 

(22),(23),(3) 

.However, the 

microbial level obtained still below the B.P 

1998 requirements.


Table 5 : Microbial Contamination Levels in Some Pharmaceutical Raw Mate rials for the 

Production of Folic Acid and Multivitamin Table ts 

* C.F U/gm Starch Ge latin Talc 

Total Viable Aerobic Count 710 2 

910 2 

bacillus 

5 

Avicel 

PH PH 

301 101 

Mag 

Steara. 

Folic acid 

B1,B2,B3 

Me thionine 

10 2 ** ** **** **** 

Total Viable Count for Fungi **** **** **** ** ** **** **** 

Enterobacteriaces **** **** **** ** ** **** **** 

Escherchia coli **** **** **** ** ** **** **** 

Staphylucocus aureus **** **** **** ** ** **** **** 

Pseudomonas aerugenosa **** **** **** ** ** **** **** 

Salomonella **** **** **** ** ** **** **** 

Tablet Evaluation: 

Folic acid and multivitamin tablets were 

prepared as previously mentioned (tables 3 and 

4 respectively). The prepared folic acid and 

multivitamin tablets were evaluated physically, 

chemically and microbiologially. The results 

are shown in table 6. 

Table 6 : Physical Che mical and Microbiological ( Control ) Evaluation of Folic acid and 

Multivita min Tablet 

Tablet Evaluation Multivitamin Folic acid 

Weight 

(7 mm ) 

130 mg 130 mg 

C. Pressure 148.3MN/m 2 148.33MN/m 2 

Wt. Uniformity 0.9% 0.9% 

Hardness (Kp) 6.6 0.4 6.5 0.6 

Thickness(mm) 2.85 2.85 

Friability % 0.2 0/2 

DisintegrationTime Assay 2.3 min 2 min 

B Pyridoxine % 133.78% 

B1 Thiamin % 148.2% 

Folic acid % 90% 

Microbiological Quality 

One day after preparati less than 10 * CFU/gm 

After storage for 8 weeks at 35 C , 75 % RH less than 10 CFU/gm 



Effect of relative humidity upon the survival 

of .A flavus in multivitamin and folic Acid 

tablets 

Figure1,2 show the effect of storage 

under different relative humidities (95,85 and 

75)% upon survival of Aflavus. spores in 

multivitamin and folic acid tablets. The results 

indicate that at a contamination level of 10 4 

spore/gm as shown in figure(1) and storage at 

75% R.H a decrease in survival over the eight 

weeks storage period to 13% whilest storage at 

95% RH, caused an initial reduction in 

viability followed by a substantial increase to 

86% at the end of 8 weeks in multivitamin 

tablets. Agermination, mycelia growth and 

sporulation occurred.The same behavior with 

folic tablets but with lower percentages. 

Visible fungal growth and sporulation were


apparent on the tablets after 6 weeks of 

storage. Further storage for 8 weeks caused a 

tablets to afragment. Storage at 85% R.H also 

showed visible fungal growth after 6 week. 

Viability of the organisms decreased during 

the first week of storage. This was 

accompanied by visible signs of mycelia 

growth. The decreased viability was probably 

due to the transition from dormant to mycelia 

state. Tablets containing hygroscopic materials 

are much more to physically, adsorb 

substantial amounts of water. Avicel has the 

ability to pick up moisture by capillary action 

and loosening of inter particulate hydrogen 

bonds on exposure to high humidities (8) .The 

water requirements for microorganisms varies 

depending on the organism (Blair,1988) as 

mentioned before. For different mould spores 

the minimum R.H required for germination 

varies from 70 to 98% and the optimum 

temperature for growth of moulds vary from 

(23-40) °C.For Aspergillus, 80% humidity 

aw=0.81 (23) is essential for spore germination 

and the optimum temperature is (30-40) 

°C.Multivitamin tablets show higher growth 

than folic acid tablets within the storage time 

especially 95%, R.H this may be due to 

presence of more nutrients like vitamins 

(B1.B2 and B3)carbon source (starch) and 

amino acids (metithionine) or what is called 

nutrient availability. When nutrients are 

abundant, growth will be sustained but when 

only atrace nutrients are present, growth will 

be minimal. Fungi need various nutrients in 

order to meet their energy needs and to form 

macromolecules such as proteins and DNA. 

Since fungi cannot synthesize carbohydrate, so 

the substrate showed contain these compounds 

; however, they can growth in a substrate rich 

in proteins without carbohydrates , e.g. cheese 

, by using amino acids as carbon source. 

Another important nutrient is nitrogen . All 

fungi can assimilate organic nitrogen 

compounds, depending on the species , certain 

vitamins must also be present in substrate, 

while the fungus itself synthesized others. The 

most important factors for growth are 

temperature , water activity and oxygen 

besides the presence of nutrients. Figure 2 

shows the effect of storage upon the percent of 

survival using 10 2 spore/gm of A. flavus in 

multivitamin and folic acid tablets.The results 

indicate that storge at 75% and 85%R.H 

showed a decrease in number of spores to zero 

percent in eight weeks duration whilst storage 

at 95% showed increase in number of spores to 

420 percent for the same time. The result also 

indicate that there is a significant different 

(p


The effect of R.H upon survival of Penicillia 

spp. Spores in multivitamin and folic acid 

Tablets: 

Figures 3,4 show the effect of relative 

humidities upon survival of penicillia spp. 

Spores in multivitamin and folic acid tablets. 

Figure 3 : Effect of relative humidity upon 

survival of Pe nicillia SPP. (10 4 spore / gm ) 

compacted in multivita min (mv) tablet and 

folic acid (fa ) tablet at (148.3 MN/m 2 ) and 

store d at 35C . L.S.D0.05 = 27.87. 

100 


survival of Pe nicillia SPP. (10 2 spore / gm ) 

compacted in multivita min (mv) tablet and 

folic acid (fa ) tablet at (148.3 MN/m 2 ) and 

store d at 35C . L.S.D0.05 = N.S. 

The results indicate that for both 

contamination levels of 10 4 and 10 2 spore/gm, 

storage at 75%RH, more decrease in survival 

of penicillia over the eight weeks storage 

period than A. flavus. was obtained i.e the 

decrease was to 1.8% and zero for 10 4 and 10 2 

spore/gram, respectively. Whilst storage at 

95% R.H however caused an initial reduction 

in viability followed by a substantial increase 

as germination mycelial growth and 

sporulation occurred for 10 4 spore/gram. 

Visible fungal growth and sporulation were 

apparent on the tablets after six weeks storage 

but the survival level was less than A. flavus. 

Further storage for eight weeks caused the 

tablets to fragment. On the other hand, storage 

at 85% R.H both contamination level 10 2 and 

10 4 spore/gm, no visible fungal growth was 

apparent on the tablets after storage for eight 

weeks. Penicillia spp.. 80% humidity is 

essential for spore germination aw=0.84 (23) 

and the optimal temperature is (25-30) °C for 

most penicillia spp. The maximal temperature 

is (28-35) °C .the result also show that 

multivitamin tablets have more survival than 

folic acid for the three relative humidites used. 

So, the over all data indicate that there is a 

significant difference (p


100 


survival of Penicillia SPP C. Clado (10 4 

spore / gm ) compacte d in multivita min 

(mv) tablet and folic acid (fa ) table t at 

(148.3 MN/m 2 ) and stored at 35C . L.S.D0.05 

= N.S. 

100 


survival of Penicillia SPP C. Clado (10 2 

spore / gm ) compacte d in multivita min 

(mv) tablet and folic acid (fa ) table t at 

(148.3 MN/m 2 ) and stored at 35C . L.S.D0.05 

= N.S. 

The obtained results are in contrast to the 

results obtained by Fassihi and Parker (24) ,who 

found that tablets stored at a lower temperature 

(25°C) and at 96% R.H did not spoil due to 

mould growth (Aspergillus niger and penicillia 

spp) when stored under these condition and the 

viability of mould spores decreased slightly . 

On the other hand the results are in agreement 

with that of the study of Bos (19) in which a 

visible growth of A. Niger during storage at 

100 and 95% R.H of sta-RX tablets and 

lactose/starch tablets stored at 25°C and 31°C 

respectively was obtained. 

If contaminants are introduced into tablets 

prior to processing (i.e. from the raw materials) 

then they might sitll eventually be responsible 

for the spoilage of the finished products . The 

nature of contaminating organism , the relative 

humidity at which tablets are stored and the 

tablet nutrient all contribute to survival of the 

organisms . 

Aflatoxin Assay 

Aflatoxin production is highly affected 

by type of substrate, the presence of minerals, 

the humidity and temperature (26 - 29) .Results 

were obtained from thin layer chromatography 

(TLC) and in camparison with standard 

showed that both multivitamin tablets and folic 

acid tablets contain no aflatoxin B1.If the 

environmental conditions (temperature, and 

relative humidity) are not suitable for fungal 

growth this will lead to decrease in aflatoxin 

production to a level that cannot be detected. 

Storage of tablets at 75% RH showed no 

aflatoxin B1 production, this result is in 

agreement with WHO (30) which determins that 

83-85% RH is an optimum RH for AFB1 

production by A. Flavus also this result is 

consistent with that obtained by Austwish (31) 

that determined 85% RH and more at 

temperature of 25°C is an optimum RH for A. 

flavus growth in addition, lakshinarasimham, 

and (32) determined that 20°C and RH 73.5% 

are considered as an optimum conditions for 

storage without fungal contamination. 

Furthermore storage at 85% RH showed no 

aflatoxin production, although RH is considerd 

as optimum for A. flavus growth but storage at 

35°C is not optimum temperature for aflatoxin 

production since the optimum temperature for 

aflatoxin is (25-28)°C (33, 34) .Also, no aflatoxin 

production was noticed when storage at 95% 

RH although RH is considered as an optimum 

for A. flavus growth but 35°C is not an 

optimum temperature for aflatoxin production . 

Multivitamin tablet which contanins amino 

acid also show no aflatoxin production this 

because the storage temperature is not an 

optimum temperature for aflatoxin production 

so both an optimum temperature and relative 

humidity required for aflatoxin production . 

Conclusions 

The results showed the existence of 

relationship between type of the raw materials 

used in pharmaceutical production and its 

microbial level . The results showed the effect 

of various storage conditions upon survival, 

which depends upon the type of fungal spore, 

the hygroscopic nature of the excipient and the 

relative humidity of storage. Multivitamin 

tablet showed more survival than folic acid 

tablet and this is due to the presence of more 

nutrient. And finally no aflatoxin was obtained 

for both multivitamin and folic acid tablets at 

35°C temperature, this is due temperature


which is not an optimum temperature for 

aflatoxin B1 production. 

Reference 

1. Kalliugetal. Kallings, L.O: ringeretz o 

silverstorage : and emr feldt F. 

Microbiologgical contamination of 

medical preparations, Actapharma 

Suec1966: 3:219-228. 

2. Sinugh p1 arirastaia B, kuma. A and 

dubey, N.K., Microb E.coli, , 2008 (56) : 

555-560. 

3. Kimiko farmati cheskii ehumnali, 

surivalal of microscopic fungi mnonsierile 

medical prrpation and auxillary subsiances 

, 2006: 40: 54-56 

4. Obuexaeco, obekwe I.F. , ogbimi AO 

actapol. Pharm- drag res. , 2001: 53 

5. Parker MT. Jowrnal of the Society of 

cosmetic chemists, 1978 : 23 :415 

6. Fassihi, A.R., and parker, M.S inimicable 

effects of compaction speedon micro 

organisms in powder systems with 

dis_simiilar compaction mechanisms 

J.phase SCI , 1987 :76: 466-470 

7. Plumpton EJ Gilbat,p, and fell JT the 

survival of micro organisms during 

tableting INT.J pharm, 1986a, 30 :241- 

146. 

8. Blair, T.C, buckton, g, and bloomfiled, 

SF baird RJ hR. heak R.f and leachr (edj) 

microbial quality assurance pharmacent 

calts cosmeticals cosmetics and tioletric 

eis horwood chichest ppi 1988,104-116. 

9. Blair , T.C graham bucton, sally F. 

bloomfield on the mechanism of kill of 

microbial of contanints during tablet 

compression int –jpharm ,1991:72: 111- 

115. 

10. Fassihi A.R ,and palkar M.S the influence 

water activity and oxygentension upon the 

survival of aspergilly and penisillia 

species and tablet INT bio detior bull B, 

1977:75-80 

11. Waterman R.f sumner EFbldwn JN and 

warren F.W survival of stupply lococcus 

aureson pharmaceutical solid dosage form 

J. pharmsei , 1973 ,621 1317-1320. 

12. Goda, n. k.s v. malath and R.uu suganthi 

effect of some chemical and herbal como 

under ongrowth of aspergillus parsiticesv 

and of latoxin production Animal feed 

science of technology , 2004,116:281-291. 

13. abdullaMH1988 the isolation of aflatoxin 

from acacia and the incidence of 

Aspergillus flavus in the Sudan 

Mycopathologia, ; 1988, 104: 143-144. 

14. Hitokoto, H 1978 H: muruzomi, s: Wauke, 

Tsakai, S: and Kuratah, Fungal 

9 

contaminand mycotoxindetection of 

powdered herbal drugs Appl. 

15. Wall heaver K,H micrological as aspects 

on the subject of oral solid dosage from 

pharm . Ind , : 1977 , 39 491-497. 

16. AL- HiTi 1998 M.M.A Al janabi S. the 

effect of some peservits on preservative on 

Aspergilliaus flavus growth and its 

aflatoxin production Irqi J.pharm Scivol 

(10): 1998. 

17. Aulton 2000 et., Me,: Tebby. H,G white 

p.J.p journal pharm, pharmacol, 26 suppl, 

59p-60p 2000 Blair, T.C Graham Bucton; 

Sally F,: bloomfield on the mechanism of 

kill of microbial contaminants during 

tablet compression – Int –J- pharm , 1991. 

72: 111- 115 . 

18. Alfonoso 1985 

19. Bos 89, C,E van doorne ,H lerk C.F. on 

microbiological stability of tablets stored 

under tropical conditions, : 1989 , 55: 175- 

183 . 

20. Ibrahim Y.K.E, 1991Y.K.E; orinolou- pdf. 

Comparative microboial levels in levels in 

wet granulation and direct compression 

method of tablet production, ACTA 

HELV. : 1991 66: 11. 

21. Bonomi, E. and nergetti, F. 1977 Bonomi 

E. and Nergetti, F. studies on the 

microbial content of raw materials used in 

pharmaceutical preparation Ann. 1 st . 

super Sonita: 1977, 13:802-832. 

22. Panlo. J Brasillianj of microbiology , 2006 

,vol. 37.no 1. 

23. Northolt MD and bull ermine prevention 

of moldgrothed and Bullerman prevention 

of moldgroth and tex production through 

control of environmental condition .J. too 

production , 1982 ,45: 519- 526. 

24. Fassihi and parker 1977 fassihi A.R and 

parker M.S the of water activity and 

oxygen tension upon the survival of 

aspergillus and penicllia tablet INT 

Biodeterior Bull.1977 , 13-80 

25. Plumpton 1982 E.J studies upon the 

survival of various microorganism in solid 

dosage froms Ph.D. thesis university of 

Manchester. 1982 

26. Reddy S.V M.D Kiram R.M. uma, K. thir 

umaladai and D.V.R ready aflatoxin B 

different grades of chillies, 2001, 18: 553- 

558, 

27. Elad. D, .risk uma, assessment grades of 

malicious bio contamination of food 

tournal food protection, 2005, 68:1302- 

1305. 

28. Shar pirar.o. control of mycotoxin storage 

and technigagus for their decontaminal my 

cotoxin in food detection and control.


Wood head publishing Cambridge u.K- 2004, 190-223. 

29. Northolt M.D. the effect water activity and 

temperature the production of some my 

cotoxines Diss wageninger 1979 

30. WHO, 2003. Laboratory manual 2 nd 

ediction interim guidelines WHO. 

31. Austwish , p.k.c; and ayerst, G.groundaut 

micrflora and toxicily –chem. Ind 1963 

,55 -61. 

32. Mukher 95 :and lakshminarasimiham A.B 

aflatoxin contamination of storage under 

10 

controlled conditions int J med Microbiol 

Virol parassitol infect dis ., 1995 , 282(3): 

387-43. 

33. Bennet Jw, klichm my cotoxins- chinical 

microbiology review ; 2003,16:497-516. 

34. Paterson rrm; venancio A; liman solution 

to penicillium taxonomy crucy to my 

eotoxin rerearch and heath research in 

microbiology ; 2004 ,155: 507-513.

Iraqi J Pharm Sci, Vol.19(2) 2010 Polycystic ovary syndrome and silymarin 

Improving an Ovulation Rate in Women with Polycystic Ovary 

Syndrome by Using Silymarin 

Mohamme d A.Taher* ,1 , Yaser A.Atia** and Manal K.Amin*** 

*Department o f Clinical Laboratory Sciences, College of Pharmacy, University of Baghdad,Baghdad,Iraq 

** College of Alkindney Medicine ,University of Baghdad, Baghdad, Iraq . 

*** Ministry of Health, Baghdad, Iraq. 

Abstract 

Polycystic ovary syndrome(PCOS) is a heterogeneous disorder of uncertain etiology , it is the most 

common endocrinopathy in women and most common cause of anovulatery infertility ,characterized by 

chronic anovulation and hyperandrogenemia .The present study was designed to investigate the effect of 

silymarin which is known to have antioxidant and insulin sensitivity effects on the levels of glucose, 

insulin ,testosterone ,leutinizing hormone(LH) and progesterone .Ovulation rate and Homeostasis Model 

Assessment of insulin Resistance (HOMA) ratio were determined .A 3-months of treatment were conducted 

in 60 PCOS patients in three well-matched groups .The first one (n=20),received silymarin(750mg/day) 

.The second group received metformin(1500mg/day) while the third group treated by 

combination of metformin (1500mg/day )and silymarin (750mg/day). All these groups had taken the 

drugs in divided doses. The results showed significant improvement in all parameters at the end of treatment 

.The percentage of increment in progesterone levels after completion of treatment were 12.12, 15.9, 

and 17.51 in groups 1, 2, and 3 respectively and the number of patients ovulated after 3 months of treatment 

were 4,5, and 10 in groups 1,2, and 3 respectively. However they are more better in group of patients 

who were treated with combination of silymarin with metformin.In conclusion the addition of silymarin 

to metformin in treatment of PCOS patients has improving effect on disturbed hormones and 

ovulation rate. 

Key words: Polycystic ovary syndrome, silymarin, ovulation rate,metformin 


ﺐﺒﺳ ﺮﺜﻛﺃﻭ ءﺎﺴﻨﻟﺍ ﺪﻨﻋ ﻊﺋﺎﺷ ﻲﻧﻮﻣﺭﻮﻫ ﻝﻼﺘﻋﺍ ﻪﻧﺍ. 

ﺓﺪﻛﺆﻣ ﺮﻴﻏ ﺏﺎﺒﺳﻷ ﺮﻳﺎﻐﺘﻣ ﺏﺍﺮﻄﺿﺍ ﻮﻫ ﺱﺎﻴﻛﻷﺍ ﺩﺪﻌﺘﻣ ﺾﻴﺒﻤﻟﺍ ﺔﻣﺯﻼﺘﻣ ﻥﺇ 

ﺮﻴﺛﺄﺗ ﺺﺤﻔﻟ ﺖﻤﻤﺻ ﺔﻴﻟﺎﺤﻟﺍ ﺔﺳﺍﺭﺪﻟﺍ ﻥﺃ. 

ﻱﺮﻛﺬﻟﺍ ﻥﻮﻣﺭﻮﻬﻟﺍ ﻉﺎﻔﺗﺭﺍﻭ ﻦﻣﺰﻣ ﺔﺿﺎﺑﺍ ﻡﺪﻌﺑ ﺰﻴﻤﺘﻳﻭ ﺔﺿﺎﺑﻻ ﺍ ﻡﺪﻌﺑ ﺏﻮﺤﺼﻤﻟﺍ ﻢﻘﻌﻠﻟ ﻊﺋﺎﺷ 

, ﻦﻴﻟﻮﺴﻧﻷﺍ , ﺮﻜﺴﻟﺍ ﺕﺎﻳﻮﺘﺴﻣ ﻰﻠﻋ ﻦﻴﻟﻮﺴﻧﻸﻟ ﺔﻣﻭﺎﻘﻤﻟﺍ ﻞﻴﻠﻘﺗﻭ ﻦﻴﻟﻮﺴﻧﻷﺍ ﺲﺴﺤﺘﻟ 

ﺓﺩﺎﻳﺯ ﻭ ﺪﺴﻛﺄﺘﻟﺍ ﺪﺿ ﺔﻴﺻﺎﺧ ﻚﻠﻤﻳ ﻱﺬﻟﺍ ﻮﻫﻭ ﻦﻳﺭﺎﻤﻠﻴﺴﻟﺍ 

(HOMA ) ﻦﻴﻟﻮﺴﻧﻸﻟ ﺔﻣﻭﺎﻘﻣ ﻞﻴﻟﺩﻭ ﺔﻇﺎﺑﻻﺍ ﺔﺒﺴﻧ ﺏﺎﺴﺘﺣﺍ ﻢﺗ . ﻥﻭﺮﻴﺴﺘﺴﻴﺟﻭﺮﺒﻟﺍﻭ ﻲﻨﻴﺗﻮﻠﻟﺍ ﻥﻮﻣﺭﻮﻬﻟﺍ , ﻱﺮﻛﺬﻟﺍ ﻥﻮﻣﺭﻮﻬﻟﺍ 

20 ﻦﻣ ﺔﻧﻮﻜﻣ ﻰﻟﻭﻷﺍ ﺔﻋﻮﻤﺠﻤﻟﺍ . ﻊﻴﻣﺎﺠﻣ ﺔﺛﻼﺛ ﻲﻓ ﺱﺎﻴﻛﻷﺍ ﺩﺪﻌﺘﻣ ﺾﻴﺒﻤﻟﺍ ﺔﻣﺯﻼﺘﻤﺑ ﺔﻀﻳﺮﻣ 60 ﻝ ﻲﻄﻋﺃ ﺝﻼﻌﻟﺍ ﻦﻣ ﺮﻬﺷﺃ ﺔﺛﻼﺛ. 

ﺎﻀﻳﺃ 

ﺔﻋﺮﺠﺑﻭ ﻦﻴﻣﺭﻮﻔﺘﻤﻟﺍ ﺭﺎﻘﻌﺑ ﻦﺠﻟﻮﻋ ﺔﻀﻳﺮﻣ 20 ﻦﻣ ﺔﻧﻮﻜﻣ ﺔﻴﻧﺎﺜﻟﺍ ﺔﻋﻮﻤﺠﻤﻟﺍ ﻭ ﺎﻴﻣﻮﻳ ﻢﻐﻠﻣ 750 ﺔﻋﺮﺠﺑ ﻦﻳﺭﺎﻤﻴﻠﻴﺴ ﻟﺍ ﺭﺎﻘﻌﺑ ﻦﺠﻟﻮﻋ ﺔﻀﻳﺮﻣ 

ﻦﻳﺭﺎﻤﻠﻴﺴﻟﺍﻭ ﺎﻴﻣﻮﻳ ﻢﻐﻠﻣ 1500 ﻦﻴﻣﺭﻮﻔﺘﻴﻤﻟﺍ ﺞﻳﺰﻤﺑ ﻦﺠﻟﻮﻋ ﺎﻀﻳﺃ ﺔﻀﻳﺮﻣ 20 ﻦﻣ ﺔﻧﻮﻜﻤﻟﺍﻭ ﺔﺜﻟﺎﺜﻟﺍ ﺔﻋﻮﻤﺠﻤﻟﺍ ﺎﻤﻨﻴﺑ ﺎﻴﻣﻮﻳ ﻢﻐﻠﻣ 1500 

ﻥﺍ. 

ﺝﻼﻌﻟﺍ ﺔﻳﺎﻬﻧ ﺪﻌﺑ ﻞﻴﻟﺎﺤﺘﻟﺍ ﻞﻜﻟ ﻯﻮﺘﺴﻤﻟﺍ ﻦﺴﺤﺗ ﺕﺮﻬﻇﺃ ﺞﺋﺎﺘﻨﻟﺍ. 

ﺔﺋﺰﺠﻣ ﻉﺮﺠﺑ ﺝﻼﻌﻟﺍ ﺕﺬﺧﺍ ﻊﻴﻣﺎﺠﻤﻟﺍ ﻩﺬﻫ ﻞﻛ. 

ﺎﻴﻣﻮﻳ ﻢﻐﻠﻣ 750 ﺔﻋﺮﺠﺑ 

ﻦﻳﺭﺎﻤﻴﻠﻴﺴﻟﺍ ﺞﻳﺰﻤﺑ ﻦﺠﻟﻮﻋ ﻲﺗﺍﻮﻠﻟﺍ ﺕﺎﻀﻳﺮﻤﻟﺍ ﺔﻋﻮﻤﺠﻣ ﻲﻓ ﻞﻀﻓﺃ ﺖﻧﺎﻛ ﺔﺿﺎﺑﻻﺍ ﺔﺒﺴﻧﻭ ﻥﻭﺮﻴﺘﺴﻴﺟﻭﺮﺒﻟﺍ ﺕﺎﻳﻮﺘﺴﻣ ﺔﺒﺴﻧ ﺓﺩﺎﻳﺯ 

ﺮﻴﺛﺄﺗ ﻪﻟ ﺱﺎﻴﻛﻷﺍ ﺩﺪﻌﺘﻣ ﺾﻴﺒﻤﻟﺍ ﺔﻣﺯﻼﺘﻤﺑ ﺕﺎﺑﺎﺼﻤﻟﺍ ﺕﺎﻀﻳﺮﻤﻟﺍ ﺝﻼﻌﻟ ﻦﻴﻣﺭﻮﻔﺘﻤﻠﻟ ﻦﻳﺭﺎﻤﻴﻠﻴﺴﻟﺍ ﺔﻓﺎﺿﺇ ﻥﺇ ﺝﺎﺘﻨﺘﺳﻻﺍ ﻲﻓﻭ . ﻦﻴﻣﺭﻮﻔﺘﻴﻤﻟﺍ ﻭ 

. ﺔﺿﺎﺑﻻﺍ ﺔﺒﺴﻧﻭ ﺔﺑﺮﻄﻀﻤﻟﺍ ﺕﺎﻧﻮﻣﻮﻬﻟﺍ ﻰﻠﻋ ﻦﺴﺣ 

Introduction 

Polycystic ovary syndrome (PCOS) is a 

heterogeneous disorder of uncertain aetiology; 

it is the most common endocrinopathy in women 

and most common cause of anovulatory infertility,affecting 

5-10% of population of reproductive 

age. (1) It is characterized by chronic 

anovulation and hyperandrogenism. (2). Insulin 

resistance and associated hyperinsulinemia also 

have been recognized as important pathogenic 

factors in determining the majority of PCOS 

women particularly when obesity is present . (3) 

Most but no t all women with PCOS have hyperinsulinemia 

with insulin resistance (4) .The association 

between hyperinsulinemic insulin resis- 

1Corresponding author E- mail : Mohammed_taher34@yahoo.com 

Received : 16/1/2010 

Accepted 26/5/2010 

11 

tance and PCOS well recognized and play an 

import role in the development of 

PCOS (5) .Hyperinsulinemia has been shown to 

reduce sex hormone binding globuline (SHBG) 

synthesis in liver (6) and insulin has a direct effect 

on ovarian steroidogenesis in theca cell. (7) 

Metformin is the oldest and still most insulin 

sensitizer world wide in the treatment o f type2 

diabetes mellitus and PCOS-associated with 

insulin resistance. It is a biguanide derivative 

and considered as an insulin sensitizer since it 

lowers glucose levels without increasing insulin 

secretion . (8)


Silymarin is an active polyphenolic flavenoid 

extracted from fruits(seeds) of medicinal plant 

silybum marianum (milk thistle), extracts were 

standardized to contain 70-80% silymarin complex 

which comprised mainly of three major 

flavolignans , silybinin silychristin and silydianin 

of which silybinin is the most biological 

active. Silymarin is considered to be very safe 

and there are only few reports on its adverse 

effects,mainly a mild laxative effect has been 

observed in occasional instances and there are 

no known contraindications or side effects reported 

during its regular use. (9) According to the 

multiple pharmacological actions of silymarin, 

silybinin have been clinically evaluated in diabetics 

for their therapeutics value reduces the 

lipoperoxidation of cell membrane and insulin 

resistance significantly, decreasing endogenous 

insulin overproduction and the need for exogenous 

insulin administration. (10) So this study 

was designed to evaluate the efficacy of silymarin 

as insulin sensitizer improving an ovulation 

rate by treatment of PCOS and consequently 

its effect on hormonal and biochemical profile 

of the patients and comparing it with a classical 

one, metformin. 


Patients 

This study was conducted into Baghdad 

city ,in al-Elwia maternity teaching hospital 

from 12/2006-6/2007.The study groups included 

80 women selected randomly, 60 patients 

with PCOS aged (19-39) years with a 

mean age ( 27.5) years and 20 healthy control 

women aged (21-32) years with mean age (24) 

years.The diagnosis of PCOS was made by the 

gynaecologists depending on ultrasound examination 

,clinical features and laboratory tests 

according to diagnosis criteria of ( Rotterdam 

2003) (11) . Table-1 shows that the clinical presentations 

of patients in present study like those 

reported in other studies of polycystic ovary 

syndrome in that it is a heterogeneous disorder 

Investigations included : serum fasting glucose 

levels, fasting insulin levels, serum testosterone, 

serum progesterone and serum leutinizing hormone 

(LH).All patients participitated in this 

study were diagnosed having PCOS and were 

non-diabetic, not hypertensive, not pregnant , 

and not taking any medications that affect the 

reproductive or metabolic functions with 90 

days of study. The patients were followed 

weekly regularly under gynecologist supervision 

during the period of treatment.The women 

were grouped into 4 groups as follow: 

Group 1: included 20 PCOS patients ,with BMI 

(31.22±1.138 Kg/m2),and age (19-31) years. 

They received Sylimarin tablets (750mg/day) in 

3 divided doses after meals for 3 months. 

12 

Group 2: included 20 patients with BMI 

30.84±1.23kg/m2) and age (20-35) years. The 

treatment was including metformin tablets 

1500mg/day in 3 divided doses (500mg after 

meals for 3 moths. 

Group 3: included 20 patients with BMI 

32.83±1.37 kg/m2), age (22-39) years. The 

treatment was consisting of combination of 2 

drugs (sylimarin 750 mg/day ) and metformin ( 

1500 mg /day) in 3 divided doses for 3 months. 

Group 4: included 20 healthy women with 

BMI 28.4±1.01kg/m2) ,age (21-32) years and 

these women were with regular cycle (21-32 

days) who were taken fro m outside of the hospital 

and selected as controls. 

Sample collection 

Venous blood sample withdrew after 

overnight fasting ( at least 12 hours of fasting ) 

fro m PCOS women and the control group . For 

the subjects with regular cycles ,the samples 

were taken at 3-5 days after the cycle for determination 

of serum LH and the sample for 

progesterone were taken at 21 days of the cycle 

.The other patients with irregular cycle ,the 

samples were taken randomly. The samples 

were taken from the patients before starting 

and after one month of treatment . 

Biochemical analysis 

Determination of serum glucose and insulin 

levels 

Fasting serum glucose and insulin levels 

were measured by commercial kit obtained 

fro m Randox using enzymatic method (12,13) . 

Determination of Homeostasis Model Assessment 

of insulin Resistance (HOMA-IR) , 

HOMA - IR was calculated using the 

following formula (14) : 

HOMA-IR=Fasting glucose (mmol/L)× Fasting 

insulin (pmol/ml)/22.5. 

Insulin resistance patients were defined as having 

HOMA>2.7. 

Determination of serum testosterone (15) and 

LH levels (16) : 

Serum testosterone and LH levels were 

determined by radioimmunoassay(RIA) method 

using a kit provided by Sigma-Aldrich. 

Determination of serum progesterone & Ovulation 

Rate 

Serum progesterone levels were determined 

using kit obtained from Sigma-Aldrich , 

using (RIA) method, and the ovulation rate was 

determined according to mid-luteal phase 

progesterone level that was equal to or more 

than 16nmol/L (5ng/ml) . (17) 

Determination of body mass index(BMI) 

BMI was calculated using standard 

formula : BMI= weight (kg)/high (m2).


Obese patients were defined as having MBI > 

27kg/m2 (18). 

Ultrasound study 

Transvaginal ultrasound study scan is 

performed for each patient at about day 12 of 

the cycle in order to to confirm follicular 

changes that appear through biochemical and 

hormonal changes , also it was repeated for 

each patient who had serum progesterone levels 

higher than or equal to 16nmol/L in order to 

confirm improvement of fertility and response 

of patients to treatment and follow up follicular 

development . (19) 

Diagnosis 

Hyperandrogenism 

Based on criteria of Androgen Excess 

Society (AES 2006), which recommended the 

following diagnostic criteria for PCOS hyperandrogenemia. 

(20) 

1. Hyperandrogenism ( hirsutism and /or 

hyperandrogenemia ) 

2. Ovarian dysfunction (oligo-anovulation 

and /or PCOS). 

3. Exclusion of related disorders such as 

hyperprolactenemia and congenital adrenal 

hyperplasia. 

Hirsutism 

Based on Ferriman-Gallwey score , evaluates 

nine body sites including the face, chest , 

areolae , linea alba , upper back, lower back, 

buttocks, inner thighs and external genetalia . (21) 

Infertility 

Inability of any couple to conceive a child 

within a 12 months period of unprotected coitus 

( sexual intercourse) . (22) 

Statical analysis 

Student t-test was used to examine the 

quantitative differences in the mean parameters. 

The results are expressed as mean±SD and the 

P-values

Groups 

1 

2 

3 


Table 2: Effect of me tformin and /or silymarin on Insulin ,glucose ,HOMA-IR ratio, total testosterone 

and proge sterone in wo men with PCOS. 

Analyte s Control Base line Afte r 1 M Afte r 2M After 3M 

Insulin(pmol/L) 57.5±0.359 92.18±4.73 89.35±0.35* 85.65±4.28* 81.44±3.66* 

Glucose(mg/dl) 5.1±0.17 5.29± 0.29a 5.01± 0.192NS 4.88±0.128* 4.73±0.128* 

HOMA 2.13±0.015 3.11± 0.244a 2.865±0.233* 2.673±0.178* 2.02±0.178* 

Testosterone(nmol/L) 1.45±0.03 4.59± 0,223a 4.427±0.242* 4.242±0.303* 3.396±0,318 

Progesterone(nmol/L) 17.15±0,02 12.84±0,612a 13.39±0.682NS 13.96±0.804* 14.41±0.942* 

LH(u/L) 5.2±0.365 9.38± 0.317a 9.18± 0.284NS 9±0.245* 8.71±0,376* 

Insulin(pmol/L) 57.5±0.359 83.7±4.49a 82.1±3.468 80.8±3.01* 74.5±4.73* 

Glucose(mg/dl) 5.1±0.17 5.35± 0.362a 4.49± 0.209* 4.63±0.35* 4.25±0.229* 

HOMA 2.13±0.015 2.68± 0.226a 2.59± 0.212* 2.39±0.199* 2.02±0.178* 

Testosterone(nmol/L) 1.45±0.03 4.07± 0.199a 3.938±0.213* 3.765±0.185 3.9±0.167* 

Progesterone(nmol/L) 17.15±0,02 12.95±0.967a 13.517±0.941* 14.04±1.01* 15.01±1.33* 

LH(u/L) 5.2±0.365 111.13±0.87a 10.56±0.839NS 10.10±0.644* 9.52±0.741* 

Insulin(pmol/L) 57.5±0.359 106±6.6 94.05±4.26* 84.16±4.50* 77.22±3.09 

Glucose(mg/dl) 5.1±0.17 5.12±0.301a 4.58±0.330* 4.27±0.369* 3.87±0.22* 

HOMA 2.13±0.015 3.55±0.172a 2.75±0.144* 2.298±0.245* 1.91±0.135* 

Testosterone(nmol/L) 1.45±0.03 4.59±0.942a 4.18±0.176* 4.06±0.159* 3.9±0.167* 

Progesterone(nmol/L) 17.15±0,02 13.88±0.875a 14.46±0.792* 15.10±0.673* 16.31±0.916* 

LH(u/L) 5.2±0.365 10.08±0.510a 9.33±0.480* 8.89±4.22* 8.54±0.515* 

Values are Mean±SD , a P< 0.05 for comparison with control group ,*P0.05 

Table 3: Comparison among me an % of increame 

nt in progesterone levels (nmol/L) and 

number of women who had ovulate d during 

the study in a ll groups of PCOS patients. 

Group1 

(sylimarin) 

Group2 

( Metformin) 

Group 3 

(combination) 

% 

of 1 st 

Month 

% 

of 2 nd 

Month 

% 

of 3 rd 

Month 

No.of 

wome n 

ovulated 

4.28 8.72 12.22 4 

4.324 8.42 15.9 5 

4.179 8.79 17.51 10 

Discussion 

The percentage of patients with hirsutism 

and acne was 43.3% and 36% respectively (table-1) 

and this finding was consistence with 

other study performed in diagnosis of 

PCOS.Cutaneous manifestations of hyperandrogenism 

in PCOS include hirsutism,acne or 

14 

combination , and male –pattern hair loss ( androgenic 

alopecia); whereas acanthosis nigrigans 

is a cutaneous marker of hyperinsulinemia 

. (23) The study demonstrated that percentage of 

obese patients was 68.6% while it was 31.6% 

for lean , this is common in PCOS and it is in 

line with other studies which demonstrated that 

40-60% of women with PCOS are obese 

(BMI>27 kg/m2). (24,25) The present study 

showed that (51.6%) of the patients were infertile 

, 31.6% with amenorrhea, 56% with oligomenorrhea 

,11.6% with regular cycle,78.3% 

with insulin resistance and 85% with hyperandrogenemia 

,these results are in agreement partly 

with other results which demonstrate the 

presence of infertility by (55-75%) , amenorrhea 

(26-15%) ,oligomenorrhea (50-90%) regular 

cycle (22%) and hirsutism (60-90%) in 

women with PCOS . (24,26) The high levels of 

androgens lead to chronic anovulation , menstrual 

disturbances and hirsutism. PCOS patients 

typically have elevated LH levels and 

LH:FSH ratios. (27) because hyperandrogenism 

leads to abnormal folliculogenesis and endome-


trial development . (28,29) Hyperandrogenemia is 

a key feature of the syndrome; but it is not always 

linked to hyperandrogenic symptoms such 

as acne or hirsutism; indeed ,ethnic groups such 

as Asian shown insulin resistance and associated 

hyperinsulinemia are also now recognized 

as important pathogenic factors in determining 

hyperandrogenism in the majority of 

PCOS women ,particularly when obesity is 

present . (30) The present study illustrated a significant 

(P


rone and LH concentration. (41) Therefore it is 

probaple that effect of silymarin on progesterone 

levels were consequences of its effect on 

insulin resistance and hyprinsulinemia. There 

was remarkable response to combination treatment 

because each drug act by its own mechanism 

and higher increment in progesterone and 

ovulation rate exerted by each drug alone may 

be enhanced by their combination . The base 

line LH levels in this work increases significantly 

(P


reisstance &beta cell fuction from fasting 

plasma glucose &insulin concentration in 

man.Diabetolgia.1985;28:412-419. 

15. Ratccliffe WA, Corrie JE Dalziel et 

al.Clinical chem..J.1982;28:1341-1318.As 

cited by sigma-Aldrich Diagnostic. 

16. ThomasCM,Segers MF.Clin 

Chem.Apr.1988;34:768.As cited by Sigma- 

Aldrich Diagnostic. 

17. Yilmas S,Unaly Y, et al.The effect of metformin 

on insulin resistance ,clomipheninduced 

ovulation &pregnancy rate in 

women with polycystic ovary syndrome 

.European 

J.Obst.&Gynecol,&Reprod.Biology.2004;1 

13:214-220. 

18. Peter n.Herbert.Eating disorders.In: Andeoli,Carpenter 

(editor).Cecil essentials of 

medicine.Saunders Company,5 th end. 2001, 

515-521. 

19. Yeh HC,Futterweit W,Thornton JC. Polycystic 

ovarian disease:US features in 140 

patients.Radiology.19987;163:111-116. 

20. TaskForce on phenotype of PCOS of the 

Androgen Excess Society.Psition statement:The 

androgen Excess Society evidence-based 

criteria for defining PCOS as 

apredominantly hyperandrogenic syndrome.J.clinical 

Endeocr Meta 2006;91: 

9237-9245. 

21. Ferriman D &Gallway J.D. Clincal assessment 

of body hair growth in women.J Clincal 

Endocr Meta.1961;21:1440-1447. 

22. Text book of gynecology,L. Copel and, 

W.B.Saunders Co.,1993. 

23. Tsilchorozidou T,Overton C, Conway 

GS.The pathophysiology of polycystic 

ovary syndrome.Clin Endocrinol (0xf). 

2004;60:1-17. 

24. Futterweit W.Polycystic ovary syndrome 

clinical perspective&management.Ostet 

Gynecol Surv.1999;54:402-413. 

25. Campbell PJ, Geriich JE.Impact of obesity 

on isulin action in volunteers with normal 

glucose tolerance demonstration of a threshold 

for the adverse effect of obesity.J 

Clin Endocrnol Metabb.1990;70:1114- 

1118. 

26. Aziz R,Dunaif A,Giudice LC, et 

al.Diagnosis & management of polycystic 

ovary syndrome.Contemp Obstet Gynecol.1998;43(supp11):1-29. 

27. Legro RS,Kunselman AR,Dodson WC,et 

al.Prevenlance and predictors of risk for 

type 2 diabetes mellitus and impaired glucose 

tolerance in polycystic ovary syndrome 

:a prospective, controlled study in 

254 affected women.J clin Endocrinol Metab.1999;84:165-169. 

17 

28. Robert D.Utiger.Insulin &the polycystic 

ovary syndrome.N Engl J Med.1996; 

9(335):657-658. 

29. Franks S.Polylstic ovary syndrome.N.Eng J 

Med. 1989;333:853-861. 

30. Clifford K,Rai R,Watson H, et al. An informative 

protocol for the investigation of 

recurrent miscarriage:preliminary experience 

of 500 consecutive cases.Hum Reprod.1994;9:1328-1332. 

31. Laure C.Morin-Papunen,1lka Vauhkonen, 

et al.Insulin sensitivity ,insulin secretion & 

metabolic & hormonal parameters in 

healthy women & women with PCOS. 

Human Reproduction.2000; 15(6): 1266- 

1274. 

32. Landian K,Tengborn L,Smith U.Metformin 

and metoprolol CR treatment in non-obese 

men.J Intern Med.1994;235:335-341. 

33. Hundal RS&Inzucchi SE.Metformin : new 

understanding, new uses. Drugs. 2003; 63: 

1879-1894. 

34. 4)Soto C, Recoba R,Barron H et al. Silymarin 

increases antioxidant enzymes in alloxan-induced 

diabetes in rat pancrease.Comp 

Biochem Physiol Toxicol 

Pharmacol.2003;136:205-212. 

35. Maddux BA,See W, et al. Protection 

against oxidative stress-induced insulin resistance 

in rat L6 muscle cells by micro 

molar concentrations of alpha-lipoic acid.Diabetes 

. 2001;50:404-410. 

36. Gonzalez F, Thusu K, abdel-Rahman E, et 

al. Elevated serum levels of tumor necrosis 

factor in normal –eight women with polycystic 

ovary syndrome.Metabolism. 1999; 

48:437-441. 

37. Conway GS , Honour JW,Jacobs HS. Heterogenety 

of polycystic ovary syndrome: 

clinical , endocrine and ultrasound feaures 

in 556 patients.Clin Endocrinol (Oxf) 

1989;30:459-470. 

38. Valezquezz EM, Mendoza S, Hamer T, et 

al. Metformin therapy in PCOS reduces 

hyperinsulinemia , insulin resistancd, 

hyperandrogenemia , and systolic blood 

pressure , while facilitating normal menses 

and pregnancy.Metabolism. 1996;43:647- 

654. 

39. Acbay O, Gundogdu S. Can metformin 

resuce insulin reistance in polycystic ovary 

syndrome? Fertil Steril.1996;65:946-949. 

40. Velazqquez E,Acosta A, Mendoza SG. 

Menstrual cylicity after metformin therapy 

in PCOS.Obsest Gynecol.1997;90:392-395. 

41. Meenakumari KJ,Agarwal S, Krishna A, et 

al. Effect of metformin in treatment of luteal 

phase progesterone concentration in 

PCOS.Brazilin J Medical & Biological Research 

.2004;37:1637-1644.


42. Derelli D, Derelli T, Bayraktar F, et al. 

Endocrine & metabolic effects of rosiglitazone 

in non- obese women with 

PCOS,Endocrinol J.2005;52(3):299-308. 

43. Karlo BN, Loucks TL, Begra 

SL.Neuromodulation in polycystic ovary 

syndrome.Obset Gynecol Clin North 

Am.2001; 28:35-62. 

44. Frank S: polycystic ovary syndrome . N 

Engl J Med. 1989; 333: 853-861. 

18 

45. Waldstreicher J, Santoro NF, Hall 

JE,Filicari M, Crowley WF 

Jr.Hyperfunction of the hypothalamicpituitary 

axis in women with polycystic 

ovarian disease.Idirect evidence for partal 

gonadotrophin desensitization . J Clin Endocrinol 

Metab . 1988; 66:165-172. 

46. Vincenzo De Leo, Antonio La Marca and 

Felice petraglia. Insulin lowering agents in 

the management of polycystic ovary syndrome 

.2003;24(5):633-667.

Iraqi J Pharm Sci, Vol.19(2) 2010 Study proteins profile with β–thalassemia 

A Comparative Biochemical Study of Proteins Profile in Iraqi 

Children and Adolescent with β–Thalassemia 

Ali M. Malik* , Emad M. Malik**, Nawal MJ Al-Shammaa*** and 

Zeinab M. Al-Rubaei*** 

*Central Children Hospital,Ministry of Health,Baghdad,Iraq . 

**Departement of Pharmaceutical Chemistry,Risafa Directurate,Ministry of Health,Baghdad,Iraq . 

*** Department of Che mistry,College of Education , University of Baghdad ,Baghdad,Iraq . 

Abstract 

The aim of the present research is to study different protein fractions in sera of children and 

adolescent with β –thalassemia major and minor and to compare the results with that of healthy 

control.One hundred fifty children and adolescents were enrolled in this study,including 50 patients 

with β- thalassemia major , 50 patients with β- thalassemia minor as pathological control group and 

another apparently 50 healthy individuals as a control group. The age of all studied groups ranged 

from (4-18)years.Total protein, albumin and immunoglobulins were estimated in sera of all subjects. A 

Significant decrease was found in the total protein and albumin levels in sera of both major and 

minor thalassemic patients compared to normal groups. A Significant increase in immunoglobulin 

levels (IgG, IgM and IgA) was found in the sera of major and minor β-thalassemia patients compared 

to control.Different protein parts in sera of all subjects were detected using cellulose acetate 

electrophoresis.The results revealed significant reduction in β- globulin fractions in β- thalassemia 

major patients compared to the normal and pathological control groups. Significant elevations in γ- 

globulins fractions were observed in both major β- thalassemia and minor β- thalassemia as compared 

to normal control group. As a Conclusion the alteration in some protein parts occurred which is more 

obvious in major thalassemia patients compared to the normal and pathological control groups. 

Key words: Protein Parts , Elecrtrophoresis , β-Thalassemia. 


ﻂـﺳﻮﺘﻤﻟﺍ ﺾﻴـﺑﻻﺍ ﺮـﺤﺒﻟﺍ ﻡﺩ ﺮـﻘﻓ ﺽﺮﻤﺑ ﺎﺑﺎﺼﻣ ﻢﻬﻨﻣ ﻥﻮﺴﻤﺧ ٬ ﻕﺍﺮﻌﻟﺍ ﻲﻓ ﺭﺎﻐﺼﻟﺍ ﻦﻣ" 

ﺎﺼﺨﺷ 150 ﻦﻣ ﻡﺪﻟﺍ ﺝﺩﺎﻤﻧ ﺖﻌﻤﺟ 

50ﻭ 

٬ ﺔﻴـﺿﺮﻣ ﺓﺮﻄﻴـﺳ ﺔـﻋﻮﻤﺠﻤﻛ ﺍﻭﺮﺒﺘﻋﺍ ﻦﻳﺬﻟﺍ ﻭ ﻒﻴﻔﺨﻟﺍ ﻂﺳﻮﺘﻤﻟﺍ ﺾﻴﺑﻻﺍ ﺮﺤﺒﻟﺍ ﻡﺩ ﺮﻘﻓ ﺽﺮﻤﺑ ﺎﺑﺎﺼﻣ ﻢﻬﻨﻣ ﻥﻮﺴﻤﺧﻭ ٬ﺪﻳﺪﺸﻟﺍ 

ﻒﻠﺘﺨﻣ ﺔﺳﺍﺭﺩ ﺚﺤﺒﻟﺍ ﻦﻣ ﻑﺪﻬﻟﺍ . ﺔﻨﺳ( 

184 

) ﻦﻴﺑ ﺔﺳﻭﺭﺪﻤﻟﺍ ﻊﻴﻣﺎﺠﻤﻟﺍ ﺭﺎﻤﻋﺍ ﺡﻭﺍﺮﺘﺗ. 

ﺔﻴﻌﻴﺒﻃ ﺓﺮﻄﻴﺳ ﺔﻋﻮﻤﺠﻤﻛ ءﺎﺤﺻﻻﺍ ﻦﻣ ﺎﺼﺨﺷ 

ﺔـﻋﻮﻤﺠﻤﻟﺍ ﻊـﻣ ﺎـﻬﺘﻧﺭﺎﻘﻣﻭ ﻕﺍﺮـﻌﻟﺍ ﻲـﻓ ﺭﺎﻐـﺼ ﻟﺍ ﺹﺎﺨـﺷﻻﺍ ﻦﻣ ﺎﺘﻴﺑ ﻉﻮﻧ ﻂﺳﻮﺘﻤﻟﺍ ﺾﻴﺑﻻﺍ ﺮﺤﺒﻟﺍ ﻡﺩ ﺮﻘﻓ ﻰﺿﺮﻣ ﻲﻓ ﻦﻴﺗﻭﺮﺒﻟﺍ ءﺍﺰﺟﺍ 

ﺕﺭﺎـﺷﺍ . ﺔﻄﺑﺎﻀﻟﺍﻭ ﺔﻴﺿﺮﻤﻟﺍ ﻊﻴﻣﺎﺠﻤﻟﺍ ﻦﻣ ﻞﻛ ﻞﺼﻣ ﻲﻓ ﺕﺎﻨﻴﻟﻮﻴﺑﻮﻠﻛﻮﻨﻴﻣﻻﺍﻭ ﻦﻴﻣﻮﺒﻟﻻﺍﻭ ﻲﻠﻜﻟﺍ ﻦﻴﺗﻭﺮﺒﻟﺍ ﻦﻣ ﻞﻛ ﺱﺎﻴﻗ ﻢﺗ . ﺔﻄﺑﺎﻀﻟﺍ 

) ﺕﺎﻨﻴﻟﻮﻴﺑﻮﻠﻛﻮ ﻨﻴﻣﻻﺍ ﻦﻣ ﻞﻛ ﻯﻮﺘﺴﻣ ﻲﻓ ﺔﺤﺿﺍﻭ ﺓﺩﺎﻳﺯ ﻊﻣ ﻦﻴﻣﻮﺒﻟﻻﺍﻭ ﻲﻠﻜﻟﺍ ﻦﻴﺗﻭﺮﺒﻟﺍ ﻯﻮﺘﺴﻣ ﻲﻓ ﻱﻮﻨﻌﻣ ﻥﺎﺼﻘﻧ ﺩﻮﺟﻭ ﻰﻟﺍ ﺞﺋﺎﺘﻨﻟﺍ 

ﻲﻓ ﻦﻴﺗﻭﺮﺒﻟﺍ ءﺍﺰﺟﺍ ﺔﺳﺍﺭﺩ ﻢﺗ . ﺔﻴﻌﻴﺒﻄﻟﺍ ﺔﻄﺑﺎﻀﻟﺍ ﺔﻋﻮﻤﺠﻤﻟﺍ ﻊﻣ ﺔﻧﺭﺎﻘﻣ ﺔﺳﻭﺭﺪﻤﻟﺍ ﻰﺿﺮﻤﻟﺍ ﻊﻴﻣﺎﺠﻣ ﻦﻣ ﻞﻛ ﻲﻓ( 

IgAﻭ 

IgGﻭ 

IgM 

ﻡﺪـﻋ ﻰﻟﺍ ﺞﺋﺎﺘﻨﻟﺍ ﺕﺭﺎﺷﺍ ﻭ ﻲﺋﺎﺑﺮﻬﻜﻟﺍ ﻞﻴﺣﺮﺘﻟﺍ ﺔﻴﻨﻘﺘﺑ ﺔﻴﻌﻴﺒﻄﻟﺍ ﺔﻄﺑﺎﻀﻟﺍ ﺔﻋﻮﻤﺠﻤﻟﺍ ﻭ ﻂﺳﻮﺘﻤﻟﺍ ﺾﻴﺑﻻﺍ ﺮﺤﺒﻟﺍ ﻡﺪﻟﺍ ﺮﻘﻓ ﻰﺿﺮﻣ ﻝﻮﺼﻣ 

ﻰﺿﺮﻤﻟﺍ ﻊﻴﻣﺎﺠﻣ ﻦﻴﺑ ﺎﻣﺎﻛ ﻦﻴﻟﻮﻴﺑﻮﻠﻛ 

ﻲﻓ ﺔﺤﺿﺍﻭ ﺓﺩﺎﻳﺯ ﻊﻣ ﺎﺘﻴﺑ ﻦﻴﻟﻮﻴﺑﻮﻠﻛ 

ﻲﻓ ﺢﺿﺍﻭ ﺺﻘﻧ ﻙﺎﻨﻫ ﺎﻤﻨﻴﺑ ﺎﻔﻟﺍ– 

ﻦﻴﻟﻮﻴﺑﻮﻠﻛ ﻲﻓ ﺺﻘﻧ ﺩﻮﺟﻭ 

ﺔﻧﺭﺎﻘﻣ ﺓﺪﻳﺪﺸﻟﺍ ﻂﺳﻮﺘﻤﻟﺍ ﺾﻴﺑﻻﺍﺮﺤﺒﻟﺍ ﻡﺩ ﺮﻘﻓ ﻰﺿﺮﻣ ﻲﻓ ﺔﺻﺎﺧﻭ ﺔﻳﻮﻨﻌﻣ ﻦﻴﺗﻭﺮﺒﻟﺍ ءﺍﺰﺟﺍ ﻲﻓ ﺮﻴﻐﺘﻟﺍ ﻥﺍ ﺝﺎﺘﻨﺘﺳﻻﺍ ﻢﺗ . ﺔﻔﻴﻔﺨﻟﺍﻭ ﺓﺪﻳﺪﺸﻟﺍ 

. ﺔﻴﻌﻴﺒﻄﻟﺍ ﺔﻄﺑﺎﻀﻟﺍ ﺔﻋﻮﻤﺠﻤﻟﺍﻭ ﻒﻴﻔﺨﻟﺍ ﻂﺳﻮﺘﻤﻟﺍ ﺾﻴﺑﻻﺍﺮﺤﺒﻟﺍ 

ﻡﺩ ﺮﻘﻓ ﻰﺿﺮﻣ ﻊﻣ 

Introduction 

Thalassemia is the name of a group of 

geneticlly (inherited), blood disorders , all of 

which involve under production of 

haemoglobin , and partial or complete failure 

of synthesis a specific type of globin chain.The 

defect may affect the α, γ and δ chain or may 

affect some combination of the β, γ and δ 

chains in the same patient, but never α and β 

chain together, unmatched globins could 

precipitate and damage RBC membranes 

causing their destruction while still in the 

marrow [1,2] .Beta (β)- thalassemia manifest 

clinically has three major groups: 1-βthalassemia 

major, 2- β- thalassemia 

1Corresponding author E- mail : Elaf95@yahoo.com 

Received : 11/1/2010 

Accepted : 2/6/2010 

19 

intermedia and 3-β- thalassemia minor 

(trait) [2] .β -thalassemia major occurs at a high 

gene frequency throughout the Mediterranean 

populations, the Middle East, India and 

Southern China through Thailand 

populations [3] .The prevalence of β– 

thalassemia in Iraq have not taken much 

intention in previous studies in spite of the 

large population affected by this 

haematological disease.Proteins are substances 

that made up of smaller building blocks called 

amino acids [3] , which are an important 

constituents of all cells and tissues. Human 

serum contains more than 125 well identified


proteins. So there are many different kind of 

proteins in the body with many different 

functions,for the example:- enzymes, some 

hormones, hemoglobin, immunoglobulin 

(antibodies) [4] . The major sites of synthesis of 

plasma proteins are the liver and the immune 

system [5] . Total protein level depend on the 

balance between their synthesis and their 

catabolism or loss from body . A test for total 

serum protein measures total amount of protein 

in blood serum as the amounts of albumin and 

globulins [6] . Albumin has a single polypeptide 

chain of (580) amino acids. It is a very stable 

protein with a high net negative charge at the 

physiological pH. It has a molecular weight of 

(66)KDa . Albumin molecule could serve as 

hormones and various metabolites as well as 

drugs and antibiotics carrier. Albumin also 

functions in the maintenance of proper osmotic 

pressure [7] .The immunoglobulins which are 

antibodies, are a heterogeneous group of 

plasma proteins produced by B- 

lymphocytes . .These proteins are important in 

preventing and fighting infections. Elevation in 

the serum levels of immunoglobulin are seen 

in infectious diseases and thalassemia [8] .Many 

studies have been carried out to evaluate 

changes of the immune system in thalassemia 

patients, considering the humoral and cellular 

immune system; but no consistent defect in 

white cells or immune functions had been 

documented [8] .The aim of the present research 

is to study different protein fractions in sera of 

children and adolescent with β -thalassemia 

major and minor and to compare the results 

with that of healthy control. 


Selection of subjects and blood sampling 

Six ml of venous blood sample was 

obtained from 150 children and adolescent 

attending Ibn Al-Baladi Hospital . 50 patients 

were with β -thalassemia major, 50 patients 

were with β- thalassemia minor (as 

pathological control group) and 50 apparently 

healthy individuals as control group.The age of 

all studied groups were ranging from (4-18) 

years.The blood samples were transferred into 

plain tubes, allowed to stand for 15 minutes at 

room temperature then centrifuged at 3500 rpm 

for (10) minutes. The resulting serum was 

separated and frozen at (-20 0 C) till used for 

the estimation of total ptotein (TP), albumin, 

IgM, IgA, IgG and performing electrophoresis 

for sera. 

Determination of Total Protein(TP) 

The concentration of total proteins was 

determined according to the colorimetric 

method described by Gornall A. [9-10] The 

20 

peptide bonds of proteins react with Cu 2+ in 

alkaline solution to form a colored complex in 

which the absorbance at 550 nm was 

proportional to the concentration of total 

protein in the specimen. The biuret reagent 

contains sodium potassium tartrate to complex 

cupric ions and maintains their solubility in 

alkaline solution . 

Determination of Albumin 

Albumin concentration in serum was 

measured using manual procedure, TECO 

diagnostics kit.Serum albumin binds 

selectively to the dye bromcresol green at the 

pH 4.2. The absorbances of the resulting 

albumin-dye complex, was read at 630 nm, 

was proportional to the albumin 

concentration [9] . 

Determination of IgM, IgA and IgG 

Immunoglobulins (IgM, IgA and IgG) 

were determined by using 

immunoturbidimetric 

method [11] .Immunoglobulins (IgM, IgA and 

IgG) form a complex with antibodies in 

solution. The absorbance of the complex could 

be measured spectrophotometrically at 340 nm. 

Immunoglobulin concentrations for each 

sample was estimated by the equation obtained 

from a comparable standard curve for each 

type . 

Serum Protein Electrophoresis 

Electrophoresis is referring to the 

transport of electrically charged particles in an 

electric field,that can be utilized for the 

characterization of their components after a 

comparison to references.To perform an 

electrophoretic separation requires the support 

material (cellulose acetate) which was made 

with buffer previously placed in the electrode 

chamber then sample (serum) was applied to 

the support, and electrophoresis was performed 

by conducting to electric power for 40 min, 

using a constant voltage (150 v). The support 

was then removed from the electrophoresis 

unit stained with 1% penceau S. After washing 

out excess dye, the support media was dried 

then the electrograms were scanned [9] . 

Statistical Analysis 

Data presented as means and standard 

deviation. Study of T-Test (p) was used to 

compare the significance of the difference in 

the mean values of any groups( p ≤ 0.05) were 

considered statistically significant. The overall 

predictive values for the results in all studied 

groups were performed according to 

[12] . 

biostatistics by Daniel in 1987



Table (1) summarizes the results of 

estimated levels of serum total protein (TP) 

,albumin ,globulin and (A/G) ratio expressed 

21 

as (mean ± SD) in sera of normal 

control,thalassemia minor and β- thalassemia 

major . 

Table 1 : Total protein (TP), albumin,globulin and (A/G) ratio le ve ls in se ra of β- thalasse mia 

major, minor thalassemia patients and normal control . 

TP Albumin Globulin (A/G)ratio t-Test 

Groups 

gm/dl gm/dl gm/dl 

(No.=50) (No.=50) (No.=50) 

thalassemia major 5.46± 0.54* 3.83± 0.54* 1.626± 0.011* 2.35 * P≤ 0.05 

(thalassemia minor) 6.88 ± 0.66* 4.55± 0.49* 2.335± 0.150* 1.94 * P≤ 0.05 

Control 7.99 ± 0.59 5.21 ± 0.48 2.775 ± 0.402 1.88 P≤ 0.05 

P significant difference from control values. 

*: significant difference between minor and major thalassemia patients. 

The (Mean ± SD) level of TP in sera of control 

group, and β- thalassemia patients (minor 

&major) in gm /dl were (7.99 ±0.59), (6.88 ± 

0.66) and (5.46 ± 0.54), respectively. The 

results showed a significant decrease of TP in 

β- thalassemia major and minor comparing 

with control. Also a significant decrease in TP 

of β- thalassemia major compared to minor 

was found . These results are in agreement 

with one study who claimed that the decrease 

in serum total protein is due to secondarily 

decreased synthesis of protein by the 

liver [13] .The results of serum albumin 

measurements revealed that the mean values of 

albumin in the three studied groups in gm/dl 

were (5.21 ± 0.48) , (4.55 ± 0.49) and (3.83 ± 

0.54) , respectively.The low levels of albumin 

may roughly be balanced by arise in 

immunoglobulin levels . This is quite common 

combination;where most of individual 

proteins , other than albumin , make relatively 

small contribution to total protein because of 

quite large percentage change in the 

concentration of one of them may not be 

detected as change in total protein so only 

low albumin levels are of clinical 

importance [14] .The (A/G)ratio of β- thalassemia 

patients (major &minor) were 2.35 and 1.94 

respectively decreased significantly to 1.88 in 

the control group. A ratio much less than one 

can give clues about problems in the 

body [15,16]. The serum levels of IgG, IgM and 

IgA in sera of control,minor and β- thalassemia 

major patients are shown in table (2). 

Furthermore,the results showed high levels of 

IgG, IgM and IgA in sera of thalassemia 

patients compared to control. Also a significant 

higher increase in immunoglobulins in sera of 

major thalassemic patients was noticed 

compared to thalassemia minor patients. 

Table 2 : Serum immunoglobulin le vels (IgA, IgM and IgG) of control, pathological control and 

β- thalassemia major patients. 

IgG 

IgM 

IgA 

Groups 

mg/dl mg/dl 

mg/dl t-Test 

(No.=50) (No.=50) (No.=50) 

thalassemia major patients 1519 ± 37.60* 220 ± 6.55* 241.6 ± 7.48* P≤ 0.05 

thalassemia minor Patients 1182 ± 18.41* 

Control 1004 ± 19.00 

148.68 ± 4.13* 202 ± 11.12* 

P significant difference from control values. 

* significant difference between minor and major thalassemia patients 

Such observations can be attributed to many 

factors. For instance repeated blood transfusion 

in β- thalassemia patients would result in a 

continuous exposure to various antigens and 

might lead to increased levels of serum 

immunoglobulins.Repeated infections also 

P≤ 0.05 

120.6 ± 3.04 168.6 ± 5.05 P≤ 0.05 

stimulate the immune system and may result 

in increased immunoglobulin levels [17,18] . 

Iron overload was suggested by some 

investigators as an important contributing 

factor in altering the immune parameters in 

thalassemia patients that results in increased


migration of T helper cells to the gut and 

lymph nodes and this causes an increase in 

serum immunoglobulin levels in thalassemia 

patients [19,20] .Figure (1) showed sera 

electrophoresis pattern of thalassemia major 

,minor and control groups. Abnormal pattern 

of serum protein in sera of patients groups is 

obvious by the decrease shown in the albumin 

band and change in the pattern of β and γ 

globulins which include main proteins 

specially those of immunoglobulins.The 

decrease in albumin levels of patient groups 

could be due to decrease rate of synthesis ,or to 

an increase in catabolism rates which occurred 

Albumin 

Alfa-1 fraction 

Alfa-2 fraction 

Beta fraction 

Gamma fraction 

A 

+ 

- 

A c c B 

Figure 1 : Serum prote in Electrophoresis Patte rn (c control) 

A- (le ft):Normal control and major β-tha lassemia patients 

B-(right):Normal control and patho logicalcontrol (minor) 

References 

1. Noguchi,C.,Butterwoth,J.,Karawajew,L.,K 

uppers,R.,Favaloro, F. ,and Jacobsohn , D. : 

Hematologica , 2004,89,1281-1283. 

2. Hope R., Longmore J., Mc Maunus S., and 

Wood A.: "xford Hand Book of Clinical 

Medicine"15 th ed.Oxford University 

Press,2001,22-25. 

3. Cappolini N., CohenA., and Proter 

J.:"Guidelines for the Clinical Monagement 

of Thalassemia". Thalassemia International 

Federaton,2000,1-29, 79-92. 

4. Lissaure T., and Colayden G.: "Illustration 

Text Book of Paeidiat rics",2 nd ed. , Mosby 

International Lmt.,2001,305. 

5. Kanieco J., Harvey J., and Bruss. M. : 

"Clinical Biochemistry of Domestic 

Animals " 5 th ed. , Academic Press. ,1990 , 

120-226. 

22 

in many diseases [14] .Meanwhile results 

revealed a mild reduction in β globulin that 

may be due to the presence of transferrin 

(the iron bound protein)in this band,which is 

considered as to be the major component of 

the β-globulin fraction and appears as a distinct 

band on high resolution serum protein 

electrophoresis [22] . Also figure (1A) of 

electrophoresis showed a highly significant 

increase in γ- globulins in major β- thalassemia 

major patients compared to normal control, 

while figure (1B) showed a mild increase in γ- 

globulins in pathological control compared 

to normal control.The increase in γ-globulins 

related to hemoglobinopathy diseases [9] . 

6. Michael L., Janet L., and Edward P.: 

"Clinical Chemistry", 4 th ed. Lippincott 

Williams & Wilkins,2002,172. 

7. Nelson D. , Cox M. : "Lehninger Principles 

of Biochemistry" 3 rd ed. ,Worth Publishers 

, 2000 , 2, 877. 

8. Ahmad A., Susan J., Reza A., Soheila A., 

Nima J.,MehranK.:Evalusion of serum 

levels of immunoglobulins in thalassemia 

patients,British Journal of Haematology , 

2005,214: 220-223. 

9. Bishop M.,Pody E.,Schoff L.:"Clinical 

Chemistryprocedures , correlations ",5 th ed., 

Lippincott Williams&Wilkins,2005,105- 

106, 194- 212. 

10. Gornall A.:Biolabo Regents ,biolabo. 

Manufacturer : Biolabo SA, Ref. 

8216,Maizy,France.


11. Shivananda Nayak B.: "Manipal Manual of 

Clinical Biochemistry ", 3 rd ed., Medical 

publishers LTD., New Delhi,2007,96-100. 

12. Dainel W.: "Biostatestics: A Foundation 

for Analysis in the Health Science", 4 th 

ed.,1987, 127-139. 

13. Ismaail H.: Sialic acid and other 

biochemical parameters level in βthalassemia 

major child patients. M. 

Sc.Thesis College of Science.Al- 

Mustansiriyah University,2001.. 

14. Zilva P.M., and Philip D. M.: "Clinical 

chemistry in Diagnosis and treatment'' 6 th 

ed.,2006,159-160. 

15. International myloma foundation : The 

Scientific and Medical Communties, 2008, 

818, 4487-7455. 

23 

16. 16-Dambro, Goldman:Cecil Textbook 0f 

Medicine,1 st ed,2000,300-309. 

17. Henk, S.,Leo,M. and Anneke ,B.: 

allontibodies after blood transfusions in a 

nonhematologic allommunized patients, 

Transfusion,2006,46(4),630-635. 

18. Weatherland D.,Clegg j.:"The Thalassemia 

Syndromes", 4 th ed.,London: Black well 

Scientific Publications,2000,120- 124. 

19. Weatherland D., Clegg J.,Higgs D., Wood 

W.:The hemoglobinopathies. In: The 

metabolic basis of inherited disease. 8 th ed. 

Mc Graw-Hill,2000,4000– 4656. 

20. Ranjna C.: "Practical Clinical 

Biochemistry ", 3 rd ed.,New Delhi,Medical 

publishers,2003,600-605.

Iraqi J Pharm Sci, Vol.19(2) 2010 Preanesthetic medications and hemodynamic changes 

Comparative Effects of Fentanyl, Medazolam, Lignocaine and 

Propranolol on Controlling the Hemodynamic Pressor Response 

during Laryngoscopy and Intubation 

May S. Al-Sabbagh* ,1 

*Department of Clinical Pharmacy, College of Pharmacy, University of Baghdad, Baghdad, Iraq. 

Abstract 

Laryngoscopy and tracheal intubation are considered the most invasive stimuli in anesthesia. 

They provoked cardiovascular responses that include hypertension, tachycardia and dysrhythmias. 

Various pharmacological approaches have been used to blunt or attenuate such pressor responses. The 

present study was designed to evaluate the effect of medazolom, lignocaine and propranolol as a 

valuable adjuvant to fentanyl in attenuating hemodynamic responses to endotracheal intubation in 

normotensive patients. Thirty two patient with physical status I or II according to the score of 

American Society of Anesthesiologist (ASA), scheduled for elective surgery under standard general 

anesthesia, were randomly allocated into four groups (8 patients in each group), assigned as F, M, L 

and P groups. Each patient in the four groups received 1 µg/kg i.v fentanyl. Patients in groups M, L 

and P are treated with 0.2 mg/kg i.v medazolam, 1.5mg/kg i.v lignocaine and 0.01mg/kg i.v 

propranolol respectively. Induction of anesthesia was then accomplished with 2mg/kg thiopental 

sodium followed by1.5mg/kg succinylcholine. Tracheal intubation was performed 2 minutes after 

induction of anesthesia. Heart rate, systolic blood pressure, diastolic blood pressure, mean arterial 

pressure and rate pressure product were measured before induction, after induction and at 2, 4, 6 and 8 

minutes after intubation. The results indicated no significant variation in the hemodynamic pressor 

response in all four groups with tracheal intubation. In conclusion, a minimum effective dose of i.v 

pre-medications (fentanyl, medazolom, lignocaine and propranolol) were found to be individually 

successful in attenua ting and providing a reliable control of all hemodynamic response changes 

accompanied the process of laryngoscopy and intubation. Therefore, all are proved effective 

premedication and no one being superior. 

Key words: fentanyl, medazolom, lignocaine, propranolol, endotracheal intubation, 

hemodynamic response. 


ﺔﻴﻋﻭﻷﺍﻭ ﺐﻠﻘﻟﺍ ﻝﺎﻌﻓﺃ ﺩﻭﺩﺭ ﺮﻴﺜﻳ ﺎﻤﻫﻼﻛ , ﺮﻳﺪﺨﺘﻟﺍ ءﺎﻨﺛﺃ ﻱﻭﺰﻏ ﺰﻔﺤﻣ ﺖﻴﺒﻨﺘﻟﺍ ﻚﻟﺬﻛﻭ ﺔﻴﺋﺍﻮﻬﻟﺍ ﺔﺒﺼﻘﻟﺍﻭ ﺓﺮﺠﻨﺤﻟﺍ ﺮﻴﻈﻨﺗ ﺮﺒﺘﻌﻳ 

ﻭﺃ ﺪﺤﺗ ﻥﺃ ﺎﻬﻧﺎﺷ ﻦﻣ ﺔﻔﻠﺘﺨﻣ ﺐﻴﻟﺎﺳﺃ ﺖﻣﺪﺨﺘﺳﺍ ﺪﻗﻭ , ﻢﻈﻨﻟﺍ ﻞﻠﺧﻭ ﺐﻠﻘﻟﺍ ﺕﺎﺑﺮﺿ ﻡﺎﻈﺘﻧﺍ ﻡﺪﻋﻭ ﻡﺪﻟﺍ ﻂﻐﺿ ﻉﺎﻔﺗﺭﺍ ﻞﻤﺸﺗ ﻲﺘﻟﺍ ﺔﻳﻮﻣﺪﻟﺍ 

ﺩﺍﻮﻣ ﺎﻫﺭﺎﺒﺘﻋﺎﺑ ﻝﻮﻟﻮﻧﺍﺮﺑﻭﺮﺒﻟﺍﻭ ﻦﻴﻛﻮﻨﻜﻴﻟﺍ , ﻡﻻﻭﺯﺍﺪﻴﻤﻟﺍ ﻦﻣ ﻞﻛ ﺮﻴﺛﺄﺗ ﻢﻴﻴﻘﺘﻟ ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﻢﻴﻤﺼﺗ ﻢﺗ ﺪﻘﻟ . ﻞﻴﺒﻘﻟﺍ ﺍﺬﻫ ﻦﻣ 

ﻞﻌﻔﻟﺍ ﺩﻭﺩﺭ ﻒﻔﺨﺗ 

ﻭﺫ ﻰﺿﺮﻤﻠﻟ ﻲﻣﺎﻏﺮﻟﺍ ﺖﻴﺒﻨﺘﻟﺍ ﺔﻴﻠﻤﻋ ﻝﻼﺧ ﺎﻬﻴﻓ ﺏﻮﻏﺮﻤﻟﺍ ﺮﻴﻏ ﻞﻌﻔﻟﺍ ﺩﻭﺩﺭ ﻒﻴﻔﺨﺘﻟ ﺔﻳﻮﻣﺪﻟﺍ ﺓﺭﻭﺪﻟﺍ ﻲﻓ ﻞﻴﻧﺎﺘﻨﻔﻠﻟ ﺔﻤﻴﻗ ﺕﺍﺫ ﺓﺪﻋﺎﺴﻣ 

ﻭﺃ ﻝﻭﻷﺍ ﻝﺎﺣ , ﺮﻳﺪﺨﺘﻟﺍ ءﺎﺒﻃﻷ ﺔﻴﻜﻳﺮﻣﻻﺍ ﺔﻴﻌﻤﺠﻟﺍ ﻢﻈﻧ ﻰﻠﻋ ًﺍﺩﺎﻤﺘﻋﺍ ﺖﻔﻨﺻ ﺔﻟﺎﺣ ﻦﻴﺛﻼﺛﻭ ﻦﻴﻨﺛﻻ ﻲﺋﺍﻮﺸﻌﻟﺍ ﻊﻳﺯﻮﺘﻟﺍ ﻢﺗ . ﻲﻌﻴﺒﻄﻟﺍ ﻂﻐﻀﻟﺍ 

8 ) ﻊﻴﻣﺎﺠﻣ ﻊﺑﺭﺃ ﻰﻟﺇ ﻢﻬﻤﻴﺴﻘﺗ ﻢﺗ . ﻲﺳﺎﻴﻘﻟﺍ ﺏﺎﺨﺘﻧﻻﺎﺑ ﻡﺎﻌﻟﺍ ﺮﻳﺪﺨﺘﻟﺍ ﺖﺤﺗ ﻢﻬﻟ ﺔﻴﺣﺍﺮﺟ ﺔﻴﻠﻤﻋ ءﺍﺮﺟﺇ ﺭﺮﻘﺗ ﻦﻳﺬﻟﺍ ﻦﻣ , ﻰﺿﺮﻤﻟﺍ ﻦﻣ ﻲﻧﺎﺜﻟﺍ 

ﻦﻣ ﻢﻐﻛ 

/ ﻡﺍﺮﻏﻭﺮﻜﻴﻣ 1 ﺕﺎﻋﻮﻤﺠﻣ ﻊﺑﺭﻷﺍ ﻲﻓ ﺾﻳﺮﻣ ﻞﻛ ﻰﻘﻠﺗ . ءﺎﻳﻭ ﻡﻻ ٬ ﻢﻴﻣ ٬ ءﺎﻓ ﻑﺮﺣﻷﺎﺑ 

ﻢﻬﻟ ﺰﻣﺭﻭ ٬ ( ﺔﻋﻮﻤﺠﻣ ﻞﻛ ﻲﻓ ﻰﺿﺮﻣ 

ﻦﻴﻛﻮﻨﻜﻟ ﻢﻐﻛ/ 

ﻢﻐﻠﻣ 1.5 ﺎﻳﺪﻳﺭﻭ ﻡﻻﻭﺯﺍﺪﻴﻣ ﻢﻐﻛ / ﻢﻐﻠﻣ 0.2 ﺭﺍﺪﻘﻤﺑ ءﺎﻳ ٬ ﻡﻻ ٬ ﻢﻴﻣ ﺕﺎﻋﻮﻤﺠﻣ ﻲﻓ ﻰﺿﺮﻤﻟﺍ ﺞﻟﺎﻌﺗﻭ . ﺎﻳﺪﻳﺭﻭ ﻞﻴﻧﺎﺘﻨﻔﻟﺍ 

ﻪﻌﺒﺘﻳ ﻥﻮﺘﻨﺑﻮﻳﺎﺛ ﻡﻮﻳﺩﻮﺼﻟﺍ ﻦﻣ ﻢﻐﻛ/ 

ﻢﻐﻠﻣ 2 ﻊﻣ ﺮﻳﺪﺨﺘﻟﺍ ءﺍﺮﻘﺘﺳﺍ ﺎﻫﺪﻌﺑ ﻢﺗ . ﻲﻟﺍﻮﺘﻟﺍ 

ﻰﻠﻋ ﺎﻳﺪﻳﺭﻭ ﻝﻮﻟﻮﻧﺍﺮﺑﻭﺮﺑ ﻢﻐﻛ / ﻢﻐﻠﻣ 0.01 ﺎﻳﺪﻳﺭﻭ 

ﻡﺪﻟﺍ ﻂﻐﺿﻭ ﺐﻠﻘﻟﺍ ﺕﺎﻀﺒﻧ ﻝﺪﻌﻣ ﺱﺎﻴﻗ ﻢﺗﻭ ٬ ﺮﻳﺪﺨﺘﻟﺍ ﺾﻳﺮﺤﺗ ﻦﻣ ﺔﻘﻴﻗﺩ 2 ﺪﻌﺑ ﻲﻣﺎﻏﺮﻟﺍ ﺖﻴﺒﻨﺘﻟﺍ ﺬﻔﻧ. 

ﻦﻴﻟﻮﻛ ﻞﻴﻨﺴﻜﺴﻟﺍ ﻦﻣ ﻢﻐﻛ/ 

ﻢﻐﻠﻣ1.5 

ﻖﺋﺎﻗﺩ 4٬6٬8 ٬ 2 ﺪﻌﺑ ﻢﺛ ﺚﺤﻟﺍ ﺪﻨﻋ ﻚﻟﺫﻭ ٬ ءﺍﺮﻘﺘﺳﻻﺍ ﻞﺒﻗ ﺞﺘﻨﻤﻟﺍ 

ﻂﻐﺿ ﻝﺪﻌﻣﻭ ﻲﻧﺎﻳﺮﺸﻟﺍ ﻂﻐﻀﻟﺍ ٬ ﻲﻃﺎﺴﺒﻧﻻﺍ ﻡﺪﻟﺍ ﻂﻐﺿ ٬ ﻲﺿﺎﺒﻘﻧﻻﺍ 

ءﺎﻨﺛﺃ ﺔﻌﺑﺭﻷﺍ ﺕﺎﺌﻔﻟﺍ ﻊﻴﻤﺟ ﻲﻓ ﺔﻳﻮﻣﺪﻟﺍ ﺓﺭﻭﺪﻟﺍ ﻭﺃ ﺐﻠﻘﻟﺍ ﺩﻭﺩﺭ ﺓﺭﺎﺛﺇ ﺔﺑﺎﺠﺘﺳﺍ ﻲﻓ ﺮﻴﺒﻛ ﻑﻼﺘﺧﺍ ﺩﻮﺟﻭ ﻡﺪﻋ ﺞﺋﺎﺘﻨﻟﺍ ﺖﺤﺿﻭﺃ ٬ ﺖﻴﺒﻨﺘﻟﺍ ﺪﻌﺑ 

ﻦﻴﻛﻮﻨﻜﻟﺍ ٬ ﻡﻻﻭﺯﺍﺪﻴﻤﻟﺍ ٬ ﻞﻴﻧﺎﺘﻨﻔﻟﺍ ) ﺔﻌﺑﺭﻷﺍ ﺔﻳﻭﺩﻷﺍ ﻞﺒﻗ ﻦﻣ ﺔﻟﺎﻌﻔﻟﺍ ﻰﻧﺩﻷﺍ ﺪﺤﻟﺍ ﺔﻋﺮﺟ ﻥﺇ ﻦﻴﺒﺗ ﻡﺎﺘﺨﻟﺍ ﻲﻓ . ﺔﻴﺋﺍﻮﻬﻟﺍ ﺔﺒﺼﻘﻟﺍ ﺖﻴﺒﻨﺗ 

ﻞﻜﻓ ﺍﺬﻟ ﺎﻬﺑ ﻕﻮﺛﻮﻣ ﺔﺒﻗﺍﺮﻣ ﺮﻴﻓﻮﺗﻭ ﺖﻴﺒﻨﺘﻟﺍﻭ ﺓﺮﺠﻨﺤﻟﺍ ﺮﻴﻈﻨﺗ ﺔﻴﻠﻤﻋ ﺐﺣﺎﺼﺗ ﻲﺘﻟﺍ ﺕﺍﺮﻴﻐﺘﻟﺍ ﻊﻴﻤﺟ ﺓﺪﺣ ﻒﻴﻔﺨﺗ ﻲﻓ ﺔﺤﺟﺎﻧ ( ﻝﻮﻟﻮﻧﺍﺮﺑﻭﺮﺒﻟﺍﻭ 

. ﺎﻤﻬﻨﻴﺑ ﺮﺧﻵﺍ ﻦﻣ ﻞﻀﻓﺃ ﻮﻫ ﻦﻣ ﻚﻟﺎﻨﻫ ﺲﻴﻟﻭ ﺔﻠﻀﻔﻣ ﺮﺒﺘﻌﺗ ﺪﻋﺎﺴﻤﻟﺍ ﺩﺍﻮﻤﻟﺍ ﻩﺬﻫ ﻦﻣ 

Introduction 

Laryngoscopy and intubation are 

mandatory for most patients undergoing 

surgery under general anesthesia, often 

accompanied by a hemodynamic pressor 

response (1,2,3) . The rise in pulse rate and blood 

pressure is usually transient, variable and 

unpredictable; these changes are usually 

1Corresponding author E- mail : may_sabbagh @ yahoo.com 

Received : 13/4/2010 


24 

tolerated by healthy individuals, however, they 

may be deleterious in patients with 

hypertension, coronary artery diseases or 

intracranial hypertension, culminating 

perioperative myocardial ischemia, cardiac 

arrhythmias, acute heart failure and 

cerberovascular accident (4) .


Various drugs including calcium channel 

(5) (6) 

blockers , vasodilators , β-adrenergic 

blockers (7,8) , topical and intravenous 

(9,10) (11,12) 

lignocaine , opioids and deep 

inhalational anesthesia (13,14) have been used in 

an attempt to attenuate or prevent pressor 

responses that accompanied endotracheal 

intubation, but non have been satisfactory. 

Fentanyl, a synthetic opioid, is one of the 

potent analgesics, when used before induction 

helps to attenuate hemodynamic response to 

(1) 

intubation . Medazolam, a short acting 

benzodiazepine, most commonly used for its 

anxiolytic, muscle relaxant and sedative 

properties (15,16) , has slow onset of action with 

more gradual effects on circulation and greater 

degree of antegrade amnesia than thiopental; 

so it may offer an advantage in situation where 

hemodynamic stability is crucial (17,18) . Recent 

studies suggested that propranolol and osmolol 

can also provide consistent and reliable 

protection against the increase in both heart 

rate and systolic blood pressure that 

accompany intubation, and may reduce the 

risk of adverse cardiac events in patient 

(8,19) 

undergoing major surgical operation . 

Lignocaine hydrochloride, an amine 

ethylamide local anesthetic and class I B- 

antidysrrhythmic drug is also acceptable for 

attenuation of cardiovascular response to 

intubation, and can also diminish cough 

reflexes, dysrhythmias and increase in 

intracranial pressure (4) . The present study was 

designed to evaluate the effects of medazolam, 

lignocaine and propranolol, as adjuvants to 

fentanyl, on the hemodynamic pressor 

response during endotracheal intubation in 

normotensive patients. 

Patients and Methods 

The present study was conducted at the 

Neurosurgery Hospital in Baghdad in 2007 

and involved thirty two ASA physical status I 

or II patients, with age range of 18-45 years, 

scheduled for elective surgery, requiring 

general anesthesia with endotracheal 

intubation. Patients with abnormal 

electrocardiogram, significant bronchospastic, 

neurologic or cardiovascular diseases, 

including those receiving medication known to 

affect blood pressure and heart rate were 

excluded. On arrival to the operating room, 

electrocardiograph monitoring, pulseoximetry 

and noninvasive arterial blood pressure 

monitoring were applied, and baseline values 

of heart rate (HR), systolic blood pressure 

(SBP), diastolic blood pressure (DBP), mean 

arterial pressure (MAP), and the rate pressure 

product (RPP) were obtained. Patients were 

randomly allocated into four groups (each 

25 

include 8 patients) treated as follow: group F 

considered as control received only 1µg/kg i.v 

fentanyl, group M; patients in this group 

received 1µg/kg i.v fentanyl plus 0.2mg/kg i.v 

medazolam, group L; received both 1µg/kg i.v 

fentanyl and 1.5mg/kg i.v lignocaine, while 

group P received 1µg/kg i.v fentanyl together 

with 0.01mg/kg i.v propranolol. After 2 

minutes of administrating pre-medications, 

induction of anesthesia was achieved with 

2mg/kg thiopental and 1.5 mg/kg 

succinylcholin (all steps and doses were 

utilized according to the guidelines adopted in 

the neurosurgery hospital, Baghdad). After 

loss of eyelash reflex, the lungs were manually 

ventilated with oxygen. Direct laryngoscopy 

was performed at 2 minutes and trachea was 

intubated with proper sized disposable cuffed 

tube and fixed after confirmation of proper 

position. Following intubation, anesthesia was 

maintained with O2, 1% halothane and 

pancuronium according to the requirements of 

surgery. The follow up of targeted parameters 

was started after administration of preanesthetic 

medications up to 8 minutes later 

on. HR, SBP, DBP and MAP were recorded 

before and after induction, then after 

intubation every 2 minutes for 8 minutes 

interval. The rate pressure product (RPP) was 

calculated by multiplying SBP by HR (20) . The 

data were statistically evaluated utilizing 

paired Student's t-test to compare pre- and 

post-treatment values. Intergroup comparison 

was performed using unpaired t-test and 

ANOVA. Results were considered 

significantly different at P


intubation. Compared to base line values, both 

HR and RPP showed gradual but non 

significant increase at 2, 4, 6 and 8 minutes 

Table 1: Demographic data of patie nts included in the prese nt study 

26 

after intubation and reach optimal value at 6-8 

minutes of intubation in all groups. 

Groups Sex Age (year) Weight (kg) Hb (%) HCT (%) 

Gr F 6 M 25.8 ± 8.2 64.0 ± 11.6 13.0 ± 1.7 41.1 ± 5.1 

(n=8) 2 F NS 

NS 

NS 

NS 

Gr M 5 M 25.6 ± 7.4 64.6 ± 13.9 12.1 ± 1.5 37.0 ± 4.7 

(n=8) 3 F NS 

NS 

NS 

NS 

Gr L 6 M 27.6 ± 9.0 64.1 ± 18.2 13.1 ± 1.1 38.0 ± 4.2 

(n=8) 2 F NS 

NS 

NS 

NS 

Gr P 6 M 28.0 ± 9.4 61.9 ± 13.6 13.1 ± 1.5 39.4 ± 5.2 

(n=8) 2 F NS 

NS 

NS 

NS 

F: Fentanyl group; M: Medazolam group; L: Lignocaine group; P: Propranolol group; Data are 

presented as mean ± SD; n=number of patients. NS: non significant 

Table 2: Effects of Fentanyl or its combination with Medazolam, Lignocaine or Propranolol on 

systolic blood pressure during intubation in surgery 

Stages 

Systolic Blood Pressure (mmHg) 

Groups 

F M L P 

Pre-induction 

(base line) 

129.8 ± 22.4 a 135.5 ± 15.1 a,b 125.5 ± 21.5 a 137.4 ± 10.5 b 

Induction 121.3 ± 21.2 a,b 129.6 ± 15.7 a,b 126.3 ± 20.8 a 134.6 ± 10.2 b 

2 min 126.1 ± 20.7 

Postintubation 

a 140.9 ± 20.1 a 132.0 ± 20.0 a 139.4 ± 10.7 a 

4 min 117.3 ± 11.1 a 127.8 ± 16.3 a,b 127.5 ± 21.2 a,b 134.3 ± 13.1 b 

6 min 116.4 ± 8.4 a 124.4 ± 23.1 a 130.9 ± 26.0 a 132.0 ± 33.8 a 

8 min 110.8 ± 26.3 a 113.8 ± 24.6 a 117.9 ± 21.9 a 128.6 ± 23.2 b 

Data are presented as mean ± SD; number of patients was 8 in each group; no significant difference 

existing with respect to induction value; non-identical superscripts (a,b) within the same time represent 

significant difference (P


Table 4: Effects of Fentanyl or its combination with Medazolam, Lignocaine or Propranolol on 

mean arterial pressure during intubation in surgery 

Stages 

Mean Arterial Pressure (mmHg) 

Groups 

F M L P 

Pre -induction 

(base line) 

93.6 ± 12.7 a 97.9 ± 12.9 a 91.8 ± 12.2 a 98.5 ± 8.5 b 

Induction 90.0 ± 13.3 a 96.0 ± 17.2 a 93.1 ± 12.9 b 96.4 ± 18.3 c 

2 min 94.5 ± 22.4 

Postintubation 

a 101.3 ± 30.3 a 95.4 ± 10.6 b 100.1 ± 8.6 b 

4 min 89.6 ± 15.7 a 96.6 ± 13.7 a 91.9 ± 13.4 b 98.3 ± 8.9 b 

6 min 88.9 ± 8.0 a 93.8 ± 14.2 a 99.4 ± 19.2 a 95.8 ± 21.9 a 

8 min 83.8 ± 13.9 a 82.5 ± 14.3* a 82.6 ± 16.5 a 94.4 ± 25.4 a 

Data are presented as mean ± SD; number of patients was 8 in each group; *P


Discussion 

Laryngoscopy and tracheal intubation 

produced stressful hemodynamic changes in 

the form of hypertension and tachycardia, 

attributed to increase in the circulating levels 

of catecholamines 

(21,22) . Control of such 

hemodynamic changes are very important to 

prevent detrimental effects, and the need for 

safe and effective therapeutic agents that may 

attenuate, blunt, suppress or abolish such 

changes became an important intervention 

during surgical procedures under general 

anesthesia. The results obtained from the 

present study revealed that all studied patients 

groups showed quantitatively and qualitatively 

similar hemodynamic pressor response at 

induction, intubation and post-intubation; the 

differences, if present, failed to reach 

statistically significant values. In the present 

study, failure to predict superiority for each 

pattern of drug intervention may be attributed 

to the limited number of patients in each 

group, and increase the number of patients 

may lead to more predictable values. 

However, pre-operative use of minimum 

effective doses of pre-anesthetic medications 

(1μg/kg fentanyl, 0.2mg/kg medazolam, 

1.5mg/kg lignocaine and 0.01mg/kg 

propranolol) in the present study was found to 

be effective in restricting the non-significant 

increase in SBP, DBP and MAP values during 

short period of time (up to 2 minutes postintubation), 

then each parameter start to 

decrease gradually (but non-significantly) until 

8 minutes postintubation; this means that all 

studied medications produce consistent and 

reliable protection against the abnormal 

increase in hemodynamic pressor response 

during laryngoscopy and intubation, similar to 

observations reported by other investigators 

(4,8,19,23) . Additionally in the present study, a 

non-significant increase in mean pulse rate and 

rate pressure product (good indicator for 

oxygen consumption) was reported in all 

groups of operated patients, starting from 

intubation and reach optimal values after 6-8 

minutes post-intubation; this could be 

explained by the fact that surgical intervention 

usually starts after 6-8 minutes postintubation, 

which is by itself a stressful 

procedure, predominantly suppresses the 

pressor response more effectively than 

tachycardia as a response (24) . Light anesthesia 

(fewer drugs by the intravenous route or via 

inhalational means) is claimed to be the major 

factor responsible for pre-operative awareness 

and hemodynamic instability (17) ; to overcome 

this problem, fentanyl and/or medazolam are 

administered for the purpose of analgesia, 

sedation and anxiolysis (16) . Many evidence 

28 

indicated that each of them, when used alone 

or in combination, enables reduction of the 

thiopental dose required to produce induction, 

and consequently limit potential side effects 

and help in attenuating the hemodynamic 

response to laryngoscopy and intubation 

(16,25,26) 

. Despite the potential advantages of the 

drug combination, reluctance to incorporate 

medazolam during light anesthesia persists 

due to concern regarding the potential for 

prolonged recovery (27,28) . However, a small 

pre-induction bolus dose of medazolam 

utilized in the present study did not prolong 

both recovery and discharge time from the day 

care unit following general anesthesia; this can 

be explained by the fact that the effects of 

medazolam on CNS is dose dependent (27) . In 

the present study, although fentanyl was 

administrated in relatively small doses, it 

produces sufficient analgesia for short surgical 

procedures, and no one of the operated 

patients experienced pain of relatively long 

duration or great severity. Although there is a 

possibility that administration of narcotic 

analgesic like fentanyl may affect 

pharmacokinetics of the anesthetic agents 

during induction, which is mostly due to 

changes in hemodynamic response (29,30,31) , the 

patients in F and M groups showed nonsignificant 

increase in HR and RPP during 

laryngoscopy up to 6 minutes post-intubation; 

this increase seems to be suppressed in 

medazolam-treated group compared to 

fentanyl-treated group. This indicates that 

administration of medazolam before induction 

lead to hemodynamic stability most probably 

by mutual potentiation (32) .Many studies have 

reviewed the effect of lignocaine to blunt the 

hemodynamic response after endotracheal 

intubation (4) . It has been reported that the 

strength and timing o f lignocaine 

administration are equally important to 

prevent hemodynamic changes (33) , however, 

irrespective of the dose and time of 

administration of lignocaine, there are still 

significant increase in hemodynamic 

parameters after intubation (34) . Kindler et al 

and Durrani et al reported that i.v. 

administration of 1.5mg/kg lignocaine did not 

prevent the increase in hemodynamic response 

associated with laryngoscopy and intubation 

(35,36) 

. Meanwhile, other investigators reported 

that 1.5mg/kg lignocaine effectively blocked 

the increase in SBP, DBP and HR after 

intubation 

(4,9) . In the present study, i.v 

administration of 1.5mg/kg lignocaine, 2 

minutes before intubation provide reliable 

protection against the rise in hemodynamic 

response that associated with intubation 

process; this result was in accordance with


many previously reported data, but not 

consistent with others (4,9) . Such effect may be 

attributed to rapid equilibration of lignocaine 

between blood and brain with production of 

sedative effect when administrated in 

appropriate dose (37) . Blocking and blunting 

adrenergic responses of tracheal intubation is 

the key pathophysiological step connecting βblockers. 

Most of the studies concerned with 

evaluating the benefit of β-blockade on 

mortality and myocardial ischemia after 

tracheal intubation are based on using ultrashort 

acting selective β-blockers (38,39) , while 

very limited reports were available about using 

propranolol in this respect (19) . In the present 

study, 0.01mg/kg i.v propranolol was used, 

and no significant differences were reported in 

HR and RPP between patients groups. Even a 

slight rise in HR and RPP that occurs at 6 

minutes postintubation (a time of surgical 

intervention) was non significantly slowered 

in the β-blockade group in compare to other 

groups. These results are in accordance with 

those reported by Hussain et al and Yutaka et 

al (7,39) . The results of the present study shed a 

light on the possibility of using minimum 

doses of thiopental sodium for induction and 

maintenance of light anesthesia, for the aim of 

decreasing the time to discharge the patient 

from the recovery room and the day care unit; 

this situation seems to be compatible with the 

condition of shortage in medications required 

for anesthesia. In conclusion, minimum 

effective doses of pre-anesthetic medications 

(fentanyl, medazolam, lignocaine and 

propranolol) can maintain hemodynamic 

stability during laryngoscopy and intubation. 

References 

1. Channaiah VB, Chary K, Vik JL, Wang 

Y. Low-dose fentanyl: hemodynamic 

response to endotracheal intubation in 

normotensive patients. Arch Med Sci 

2008; 4:293- 299. 

2. Boralessa H, Senior DF. Cardiovascular 

response to intubation. Anaesthesia 1983; 

38: 623-627. 

3. King BD, Harris LC, Grieleuestein FE, 

Elder JD. Reflex circulating response to 

direct laryngoscopy and tracheal 

intubation performed during general 

anesthesia. Anesthesiology 1951; 12:556. 

4. Malde AD, Sarode V. Attenuation of the 

hemodynamic response to endotracheal 

intubation: fentanyl versus lignocaine. 

The Internet J Anesthesiol 2007; 12:1. 

5. Govindaiah MH, Suryanaayana VG. Can 

calcium and sodium channel blockers 

attenuate hemodynamic response to 

29 

endotracheal intubation? Eur J Gen Med 

2008; 5(4):198-207. 

6. Kamra S, Wig J, Sapru RP. Topical 

nitroglycerin. A safeguard against pressor 

responses to endotracheal intubation. 

Anesthesia 1986; 41:1087-1091. 

7. Hussain AM, Sultan ST. Efficacy of 

fentanyl and esmolol in the prevention of 

hemodynamic response to laryngoscopy 

and endotracheal intubation. J Coll 

Physicians Surg Pak 2005 

; 15(8):454-457. 

8. Ogurlu UB, Erdal MC, Aydin ON. Effects 

of Esmolol, Lidocaine and Fentanyl on 

haemodynamic responses to endotracheal 

intubation: A comparative study. Clin 

Drug Invest 2007; 27:269-277. 

9. Kim WY, Lee YL, Ok SJ, Chang MS. 

Lidocaine does not prevent bi-spectral 

index increases in response to 

endotracheal intubation. Anesthesia Analg 

2006; 102: 156-159. 

10. Hamill JF, Bedford RF, Weaver DC, 

Colohn AR. Lidocaine before 

endotracheal intubation: intravenous or 

laryngotracheal? Anesthesiology 1981; 

55:578-581. 

11. Dahlgren N, Messeter K. Treatment of 

stress response to laryngoscopy and 

intubation with fentanyl. Anesthesia 2007; 

36:1022-1026. 

12. Adachi YU, Satomoto M, Higuchi H. 

Fentanyl attenuates the hemodynamic 

response to endotracheal intubation more 

than the response to laryngoscopy. 

Anesthesia Analg 2002; 95:233-237. 

13. Sklar BZ, Lurie S, Ezri T, Krichelli DL. 

Lidocaine inhalation attenuates the 

circulatory response to laryngoscopy and 

endotracheal intubation. J Clin Anesthesia 

1992; 4(5):382-385. 

14. Kautto UN, Saarnivaaral A. Attenuation 

of the cardiovascular intubation with NO2, 

Halothane or enflurane. Acta Anaesthesiol 

Scand 1983; 27:289-293. 

15. Kim HK, Chung YJ, Lee MS. 

Comparison of medazolam and thiopental 

as an induction agent. Korean J 

Anaesthesiol 1991; 24(4):826-832. 

16. Lohakare R, Sangawar AV, Ghosh AA. 

Influence of intravenous fentanyl and/or 

Medazolam on induction of anesthesia 

with thiopentone. J Anaesth Clin 

Pharmacol 2004; 20(3): 273-278. 

17. Khan AM, Akan F. Midazolam and 

thiopentone co-induction: looking for 

important in quality of anaesthesia. JPMA 

2003; 53:542-547.


18. Reves JG, Fragen R J, Vinik HR, et al. 

Midazolam: pharmacology and uses. 

Anesthesiology1985 ; 62:310-324. 

19. Chae DH, Park KJ. Effect of verapamil 

and propranolol on heaemodynamic 

response to laryngoscopy and tracheal 

intubation in hypertension patients. 

Korean J Anesthesiol 1990; 23(3) :366- 

372. 

20. Rathore A, Gupta HK, Tanwar GL. 

Attenuation of the pressure response to 

laryngoscopy and endotracheal intubation 

with different doses of osmolol. Indian J 

Anaesth 2002; 46(6):449-452. 

21. Asad N, Ai K, Qayyum A. Effect of 

Nalbuphin and midazalam on 

hemodynamic response to intubation. 

Canadian J Anesthesia 2006; 53: 26192. 

22. Kyung Y, Un LJ, Hak SK. Hemodynamic 

and Catecholamine response to 

Laryngoscopy and Tracheal intubation in 

patient with complete spinal cord injuries. 

Anesthesiology 2001; 95(3):647-65. 

23. Im ES, Jeon DG, Shine HC. Effect of 

fentanyl, medazolam and fentanyl- 

medazolam on the cardiovascular system 

and blood glucose during general 

anesthetic. J Korean Soc Anesthesiol 

1994; 27(9):1083-1091. 

24. Constant I., naghe M.C, Boudt L,B. 

Reflex papillary dilatation in response to 

skin incision and alfantanil in children 

anaesthetized with sevoflurane : a more 

sensitive measure of noxious stimulation 

than the commonly used variables. British 

Journal of anesthesia 2006;96(5);614- 

619. 

25. Dundee JW, Halliday NJ. Pretreatment 

with opiods. The effect of thiopentone 

induction requirements and overset of 

action of medazolam. Anesthesia 1986; 

41:159-161. 

26. Vinik HR. Anesthetic interactions. Eur J 

Anesthesiol 1995; 12:3-4. 

27. Miller DR, Blew PG, Martineau RJ. 

Midazolam and awareness with recall 

during total intravenous anesthesia. Can J 

Anaesth 1996; 439(9):946-953. 

28. Delucia JA, White PF. Effect of 

medazolam on induction and recovery 

characteristics of propranolol. Anesth 

Analg 1992; 74:563. 

29. Adachi YU, Watanabe K, Higuchi H, 

Satoh T. The determinants of propofol 

induction of anesthesia dose. Anesth 

Analg 2001; 92:656-661. 

30 

30. Kazama T, Ikeda K, Morita K. et al. 

Relation between initial bloods 

distribution volume and propofol 

induction dose requirement. 

Anesthesiology 2001; 94:205-210. 

31. Cocksholl ID, Briggs LP, Douglas EJ, 

White M. Pharmacokinetics of propofol in 

female patients: studies using single bolus 

injections. Br J Anaesth 1987; 59:1103- 

1110. 

32. Shlomo B, Abdelkhalim H. Midazolam 

act synergistically with fentanyl for 

induction of anaesthesia. Br J Anaesth 

1990; 64:45-47. 

33. Wang YM, Chung KC, Huang YM. 

Lignocaine the optimal timing of 

intravenous administration in attenuation 

of increase of intraocular pressure during 

tracheal intubation. Acta Anaesthesiol Sin 

2003; 41(2):71-75. 

34. Miller CD, Warren SJ. I.V Lignocaine 

fails to attenuate the cardiovascular 

response to laryngoscopy and tracheal 

intubation. Br J Anaesth 1990; 65(2):216- 

219. 

35. Kindler CH, Schumacher PG, Schneider 

MC. Effects of intravenous lidocaine 

and/or esmolol on hemodynamic response 

to laryngoscopy and intubation; a doubleblind, 

controlled clinical trial. J Clin 

Anesth 1996; 8:491-496. 

36. Durani M, Barwise JA, Johnson RC, et al. 

Intravenous chloroprocaine attenuates 

hemodynamic changes associated with 

direct laryngoscopy & tracheal intubation. 

Anesth Analg 2000; 90:1208-1212. 

37. Nishino T, Hiraga k, Sugimori K. Effects 

of i.v. Lignocaine on airway reflexes 

elicited by irritation of the tracheal 

mucosa in humans anaesthetized with 

enflurane. Br J Anaesth 1990; 64:682- 

687. 

38. Sugiura S, Seki S, Hidaka K. The 

hemodynamic effects of landiolol, an 

ultra-short-acting beta1-selective blocker, 

on endotracheal intubation in patients 

with and without hypertension. Med Ac 

Jp 2007; 22:123-126. 

39. Yutaka O, Nishikawa K, Hase I. The 

short-acting β1-adrenoceotor antagonists 

esmolol and landiolol suppress the 

bispectral index response to tracheal 

intubation during sevoflurane anesthesia. 

Anesth Analg 2005; 100:733-737.

Iraqi J Pharm Sci, Vol.19(2) 2010 Modification of GSAM 

Validity of Generalized Standard Addition Method for a Mixture of 

Amino Acid Analysis 

Azhar M. Jasim* ,1 

*Departement of Pharmaceutical Chemistry,College of Pharmacy,University of Baghdad,Baghdad, Iraq. 

Abstract 

A Modified version of the Generlized standard addition method ( GSAM) was developed. This 

modified version was used for the quantitative determination of arginine (Arg) and glycine ( Gly) in 

arginine acetyl salicylate – glycine complex . According to this method two linear equations were 

solved to obtain the amounts of (Arg) and (Gly). The first equation was obtained by spectrophotometic 

measurement of the total absorbance of (Arg) and (Gly) colored complex with ninhydrin . The second 

equation was obtained by measuring the total acid consumed by total amino groups of (Arg) and ( Gly). 

The titration was carried out in non- aqueous media using perchloric acid in glacial acetic acid as a 

titrant. The developed method is accurate, precise, and free from interferences and may provide a 

useful approach to calibrate in the direct analysis of solid sample and it is suitable method to be used as 

a quality control procedure. 

Key words: GSAM , amino acid analysis . 

31 

ةصلاخلا 

ٌخٗمم ىو ضٍموموخ ُ م خوخ تطمضو طٌٕخماخم أ هٕمله هممشب GSAM تمٌط وخ ت م ط ةوخ تاطمساخ تمة طض اممىي ط ٗم ح نطملاخ ًمح 

تةوم ط ووخ ومٌا ًمح ُ مخ ل ُ خواطم ٌ امىي نٗمل ىوٖ ُ مل لال – ج ى ل وموط هوم طوخ أ ُوم ّويسأ مةوم ٍووخ ـم لطح ضما تىلخموموخ تم ّ ٌاخ 

ٌطممض يخماخمم خب أ ض طممٌ مم ٌ ضمما مملخوخ تممة طضٖ امموٖاخ تممواط ٍوخ اممىي نٗممل ىو ُ سمممّّٕوخ نٗممى ٌ يخماخمم خب أ ت ِٗوممىوخ 

ٌطمض تاطمسخب ممة ٍوخ ُمٌ م م أ كى مل وط ه طخ م اخ نيمي مم ب كموذٖ٬ 

تم ِط وخ تمواط ٍوخ امىي نٗمل ىو ملٌ نٗمى ٍل كم سٗىلط بوخ 

تممة ط ب ٓمخ ٍ ل ط مممة حٖ ط م اخ ٗ مو وخ ـ ممٍ وخ ت م خٗ ب س ط م خٍ وخ م مم أ كى مل وط ه طخ م اخ ( الا سطم ياخمم 

٥٫٠أك 

أٖ سٗىلٖسمم ٕوخ 

ُمٌ تم وطلٖ تة قا طخّوخٖ ت ط تة ط وخ ْ ٔ َٗل اىي جوا طٕ وخ هصٗخوخ ًح ضخوخ طخّوخ . ضيطيساخٖأ طشطبٍوخ لخوخ أ لخوخ 

. ةمة ٌ ةيٕيا جطخ ح ٖ ت ٖط ٍ موخ ثلالخمخوخ 

Introduction 

Numerous methods for the analysis of 

acetylsalicylic acid could be found in the 

literature (1-10) . However, these methods are not 

suitable for the analysis of acetylsalicylic acid 

mixture with arginine and glycine. Argnine 

and glycine interfere with direct titrimetric 

determination of acetylsalicylic acid therefore, 

it was intended to separate acetylsalicylic acid 

quantitatively from its salts before it is 

determined by titrimetric method. It was 

intended to use spectrophotometry or titrimetry 

for determination of arginine and glycine . 

These two methods are simple and therefore 

the most widely used in routine drug and many 

compounds analysis as could be seen from the 

latest version of British , Swiss and United 

State Pharmacopeias. However, direct 

application of these methods for determination 

of arginine and glycine in a mixture containing 

both of them is not possible because they 

interfere with each other. As a remedy for this 

situation it was decided to use the GSAM 

which has wide applications (11-17) . It is based 

on the principle of varying both of the sample 

molar concentration and pH of solution. 

Aimed at the validation and standardization of 

analytical procedures with direct solid sample 

contain two types of amino acids without 

1Corresponding author E- mail : azharmjk@yahoo.com 

Received : 11/5/2010 

Accepted : 27/6/2010 

previous isolation. Many methods were used 

for the determination of arginine and other 

amino acid involved previous isolation like the 

classical analytical technique uses automated 

amino acid analyzers, However these methods 

require expensive dedicated equipment duo to 

the post- column derivatization of the amino 

acids , long assay times and large samples 

volumes ( 18 ) .Also the studies of PITC as the 

derivatizing agent, following the Pico-Tag 

method used for the determination of arginine 

& other amino acid , those studies require the 

establishment of standard–added curve (by 

application of the standard addition method )to 

avoid proportional errors (19) . The non-aqueous 

titration was used for detection of free amino 

group in amino acid (20) .This method used for 

the quantitative determination of many 

chemical compounds and drugs in 

pharmaceutical forms, providing precise and 

accurate results, which could be verified by 

statistical methods (21-23) . A spectrophotometric 

method are widely used for determination of 

amino acid based on the reaction with 

coloring agent at different pH forming colored 

complex that measured at specific 

wavelength (24-27) .


Experimental 

Material and Methods 

Glacial acetic acid (99.8 %) was obtained 

from sigma. Standard perchloric acid (0.1 ml) 

in glacial acetic acid was prepared by diluting 

(4.25 ml) of perchloric acid (72%) to (500 ml) 

with glacial acetic acid containing (10 ml) 

acetic anhydride. The prepared solution was 

standardized by titration with standard 

anhydrous sodium carbonate (0.106 gm) in 

glacial acetic acid (50 ml) the titration end 

point was determined potentiometrically with 

potentiometric titration equipped with glass – 

calomel combined electrode. The prepared 

standard was used in the titrimetric procedure 

for the determination of arginine and glycine 

in arginine acetylsalicylate–glycine complex. 

Anhydrous sodium carbonate (99.5 %) , 

sodium hydroxide (0.5 M) and hydrochloric 

acid (0.5 M) were Aldrich standards . Arginine 

(98%, sigma) was standardized to determine 

the exact percentage of arginine by titration 

with standard hydrochloric acid (0.5 M). 

Glycine (99%, fluka) was used after drying in 

an oven for 1hr at (105ºC). Acetylsalicylic acid 

was purified by recrystilization from ethanol/ 

water mixture and standardized with standard 

(0.5 M) sodium hydroxide (10) . The determined 

percentage of acetylsalicylic acid was (99.4 

%).The standards arginine , glycine and 

acetylsalicylic acid were used to prepare 

arginine acetylsalicylate – glycine complex . 

The later was used to test the validity of the 

proposed method. Sodium acetate buffer (4M, 

pH 5.5) was prepared by dissolving sodium 

acetate trihydrate NaOAc.3H2O (BDH 

“Analar “99.9) in 200 ml of deionized water. 

The solution was heated with stirring at (60ºC 

) until a clear solution was obtained. Glacial 

acetic acid (50 ml) was added and the volume 

was completed to (500 ml) with distilled water. 

The pH was adjusted by a drop wise addition 

of sodium hydroxide (4M) to pH (5.5). 

Ninhydrin reagent was prepared as 

following : ninhydrine ( I gm) in 2 - methoxy 

ethanol (25 ml) and stannous chloride 

SnCl2.2H2O (0.07 g) with continuous stirring 

until completely dissolved (28) . Sodium acetate 

buffer (8.3 ml) was added and the resulting 

solution was immediately transferred to dark 

glass reservoir bottle and a steam of nitrogen 

gas was babbled into the reagent solution for 

approximately (20 min). Titration was made 

with potentiometric titrator (Metrohm , 

Switzerland ) titro process or equipped 

with(Metrohm).Dosimate and glass calomel 

combined electrodes. Spectrophotometric 

measurement was performed on carry (100 

conc) uv- visible spectrophotometer using (1 

cm) cells. 

32 

Determination of acetyl salicylic acid in this 

complex 

(1gm) of arginine acetylsalicylate– 

glycine complex was weighed and dissolved in 

(25 ml) of distilled water. The salt was 

converted to free acetyl salicylic acid by 

acidification with hydrochloric acid (3 M) until 

pH (2.7) was reached. The acetyl Salicylic 

acid was extracted by ether (3 × 20 ml). The 

ether portions were pooled together and ether 

was evaporated by a rotary evaporator under 

vacuum. The acetyl salicylic acid was 

collected to be determined quantitatively by 

back titration method or by direct titration 

method (5, 10) . The result obtained for acetyl 

salicylic acid was corrected for the presence of 

salicylic acid as an impurity during liberating 

and separating acetyl salicylic acid form the 

complex. Salicylic acid was then determined in 

the liberated materials (29) . 

Determination of arginine and glycine in this 

complex 

This was achieved by constructing and 

solving equations (2) and (4) using ninhydrin 

spectrophotometric and titrimetric method in 

non – aqueous media respectively. 

Spectrophotometric Method 

(36 mg) of the complex was precisely 

weighed and dissolved in (50 ml) distilled 

water to prepare a stock solution. An aliquot (1 

ml) of this stock solution was diluted to (25 

ml) with distilled water. An aliquot ( 1 ml ) of 

this solution was mixed with ( 1ml ) of 

ninhydrin reagent in a stoppered test tube , 

shacked and placed in a boiling water bath for 

( 15 min ). Ethanol (2.5 ml) and sodium 

acetate buffer (2.5 ml) were added to the 

mixture which was then cooled below (30 ºC ). 

The sample was shacked thoroughly for (30 

second) and absorbance was measured at (570 

nm) against a reagent blank using (1 

cm).Standard curves for arginine and glycine 

were constracted using (0.05 = 0.2 mM) 

standard solution for each. The values of the 

molar absorpitivity coefficient aA and aG in 

equation (2) were determined from slops of 

these calibration curves. 

Titrimetric Method in non – aqueous media 

(0.3 gm) of the complex was dissolved in 

glacial acetic acid (50 ml) in a dry flask. The 

glass electrode of the titroprocessor was 

immersed in the solution and the mixture was 

titrated against standard perchloric acid 

(reagent 1). A plot of pH against the titrant 

volume was constructed to obtain the titration 

end point. 

Stability study 

Identical aqueous solution (0.1 g/ 100 

ml) of the complex was made. The stability 

was determined at different temperatures (25,


40, 50, 60 & 70 ºC ) incubated in ovens for 

certain intervals. The decomposition of 

arginine acetylsalicylate – glycine complex 

was indicated by the release of acetylsalicylic 

acid , arginine and glycine as appears on TLC 

using a solvent system of ( n-propanol : 34% 

ammonia 7 :3 ) . The appearance of three 

spots on TLC plates for acetyl salicylic acid, 

arginine and glycine when the samples were 

incubated at (50, 60 & 70 ) ºC . While 

incubation at (25 and 40) ºC showed only one 

spot for arginine acetylsalicylate – glycine 

complex. 

Effect of Buffer 

Glycine was used as a buffer to stabilize 

arginine acetylsalicylat complex. The 

concentration of glycine is (0.5 M) in respect 

to arginine (1M) to maintain the pH of the 

preparation at (4.7). 

Result and Discussion 

Method of calculation 

The GSAM is a traditional vector – 

matrix notation to construct and to solve a 

system of (n) linear equation in order to 

determine the concentration of a mixture of (n) 

substances. The mixture of compounds 

interferes with each other when measured by 

(n) different sensors that belong to any suitable 

analytical technique. According to GSAM, the 

following system of two linear equations is 

required to determine arginine and glycine: 

A1 = a11%A+ a12 % G 

A2 = a21%A+ a22 % G 

……… (1) 

…….زز 

Where ; A1 and A2 are the total responses of 

two analytical sensors ( 1 and 2 ) to the 

percentage of arginine ( % A ) and glycine ( % 

G ) in the sample the factors a11 – a22 are 

absorptivity constant multiplied by the optical 

path length ( i.e of the sample ) .Theory of 

GSAM equations (1) gives precise result for 

%A and %G when the following two 

conditions are fulfilled: Firstly there should be 

a large difference in magnitude between the 

ratio a11/a12 and the ratio a21/a22. Secondly 

the precision is measuring A1 and A2 should 

be high. These requirements arise from the fact 

that the mathematical manipulation magnify 

the random error in measurement , so that 

large random error is produced in the 

calculated concentration of the measured 

substances ( i.e %A and %G ) (11,12) .According 

to the requirements stated in these two 

conditions, it was decided to construct one of 

the equations of the system by using 

spechtrophotometric technique, while the 

second equation is to be constructed using 

titrimetic method. Ninhydrin was chosen as a 

33 

color developing reagent in the 

spechtrophtometric procedure, as it is the most 

selective among other coloring agent for 

spechtrophotometric determination for amino 

acids (30-31) .The titrimetric method was used to 

obtain the second equation which could not be 

performed in aqueous medium because of the 

neutral behavior of salts in aqueous medium 

that prevent the occurrence of a clear titration 

end point . Accordingly, a titrimetric method 

in non- aqueous media was adopted using 

glacial acetic acid as a solvent and a solution 

of perchloric acid in glacial acetic acid as a 

titrant. This method was used for quantitative 

determination of salt of amines with carboxylic 

acids (32-33) .The presence of an acidic salts in 

non-aqueous solvent behave as bases and 

therefore produce a clear titration end point 

when titrated with strong acid (34) .This 

experiment of procedure was intended to 

produce two linear equations in which the ratio 

a11/a12 and a21/a22 are very different in 

magnitudes. This was expected from the fact 

that arginine and glycine have nearly the same 

efficiency to produce colored complexes with 

ninhydrin reagent in spectrophotometric 

procedure 

(31) .While in the titrimetric 

procedure arginine molecules have two 

titratable amino groups in contrast to the 

glycine molecules which have one titratable 

group. The first equation which is obtained 

from the spectrophotometric procedure was 

derived starting from Lambert – beer law as 

follows : 

… (2) 

Equation (2) can be abbreviated as follows: 

A = a11 %A + a12 %G ……… (3) 

In equation (2) Wasg gm of arginine acetyl 

salicylate – glycine complex sample was 

dissolved to prepare (vo ml) of stock solution. 

This stock solution was then subjected to serial 

dilutions (n) with f1,f2…fn dilution factors to 

reach the required final concentration. 

Mw A and Mw G are the molecular weight of 

arginine and glycine respectively. aA and aG 

are the molar absorpitivity constants of 

arginine and glycine respectively.The use of a 

combination of titrimetry and 

spechtrophotometry satisfies the requirements 

stated in (2). The precision of the 

spectrophotometric method is moderate in


general.While; the precision of the titrometric 

method is very high. Therefore, the use of non- 

aqueous titration method improves the overall 

precision of the results obtained from such 

combination of methods. The second equation, 

was obtained from titrimetric procedure as 

follows: 

Equation (4) can be abbreviated as below: 

…(4) 

MpVp = a21%A + a22%G …….. (5) 

Equation (4) V ml of glacial acetic acid 

containing V asg of the analyzed arginine 

acetyl Salicylic-glycine complex was titrated 

with Mp molar standard of perchloric acid, so 

that Vp ml of the standard was required to 

reach the end point . Equations (3) and (5) are 

mathematically compatible and can be solved 

linearly. The ratio a11/a12 and a21/a22 have 

large differences in their magnitudes since 

a11/a12 ≈ 2 while a 21/a 22 ≈ 1. 

Development of Titrimetric Procedure for 

Acetyl salicylic acid determination 

The complex sample solution must be 

acidified to liberate acetyl salicylic acid form 

its salt with arginine before extraction by 

ether. The optimum pH of the acidified 

aqueous solution was determined 

experimentally by applying the sample 

complex at different pH values of the extracted 

sample solution. The result of this study 

indicates that pH (2.7) is the optimum pH for 

acetyl salicylic acid extraction. At pH higher 

than (2.7) low result for acetyl salicylic acid 

were obtained due to incomplete liberation 

acetyl salicylic acid from its salt with 

arginine.The extraction step is vital in the 

development procedure because an attempt to 

perform direct titration with hydrochloric acid 

without performing apparent titration end 

point as could be seen from (Figure 1). 

Figure 1: Titration curve of arginine 

acetylsalicylate -glycine solution with 

standard hydrochloric acid (0.5). 

34 

Analysis of Arginine Acetyl salicylate – 

Glycine complex 

Quantitative Determination of Acetyl salicylic 

acid. 

Acetyl salicylic acid was determined 

quantitatively by hydrolysis and back titration 

method (29) . The result of the two methods was 

summarized in table (1). 

Table 1: result of acetylsalicylic acid, 

arginine and glycine in arginine acetyl 

salicylate – glycine complex 

Item 

Acetylsalicylic 

Acid 

Arginin 

Glycine 

Expected 

Percentage 

W/W 

50.0 

40.0 

10.0 

Calculated 

Percentage 

( w/w ) ± SD 

49.6 ± 0.2 * 

49.6 ± 0.3 * 

40.3 ± 0.6 

9.7 ± 0.2 

* Hydrolysis and back titration method. 

* *Direct titration method. 

Quantitative determination of arginine and 

glycine in the complex 

The quantitative determination of 

arginine and glycine in the complex using a 

modified version of the GSAM was achieved 

by a combination of colorimetric method ( to 

obtain the first equation ) and non-aqueous 

titration method ( to obtain the second 

equation).The colorimetric method using the 

colored complex of arginine and glycine with 

ninhydrin was applied once at different pH 

media and once by using different 

wavelengths; the results of these experiments 

were summarized in table (2) and (3) and in 

figure (2) and (3) respectively. 

Table 2: Molar absorptivity constant a* of 

arginine and glycine colored complex with 

ninhydrin at different pH values measured 

at 570nm 

pH of aA 

ninhydrin 

solution 

5.5 0.83 

7.0 1.07 

9.0 1.73 

aG 

0.62 

0.78 

0.97 

*Represent the Molar absorptivity 

multiplied by the cell width 

aA / aG 

1.34 

1.37 

1.41 

C.V 

0.4 % 

0.6 % 

1.5 % 

2.0 %


Table 3: Molar absorptivity constant a* 

of arginine and glycine colored complex 

with ninhydrin at different wavelengths. 

pH of 

ninhydrin 

solution 

Wavelength 

hs nm 

244 

aA 

1.19 

aG 

0.92 

5.5 

409 0.74 0.54 

570 0.83 0.63 

244 1.28 0.97 

7.0 

409 0.87 0.62 

570 1.02 0.75 

244 1.40 1.08 

9.0 

409 0.92 0.67 

570 1.32 1.00 

*Represent the Molar absorptivity 

multiplied by the cell width 

aA/aG 

1.29 

1.37 

1.32 

1.32 

1.40 

1.36 

1.30 

1.37 

1.32 

Figure 2: standard curve of arginine and 

glycine colored complexes with ninhydrin 

at different pH values measured at 570 nm 

35 

Figure 3: uv-visible spectra of arginine and 

glycine colored complexes with ninhydrin at 

different pH values 

According to the non-aqueous titration 

method; the titration end point of the titration 

curve between standard perchloric acid with 

the complex was determined 

potentiometrically. A typical plot for the 

titration curve was exhibited in figure ( 4 )


Figure 4: titration curve between standard 

perchloric acid (0.1N) and arginine 

acetylsalicylate –glycine complex. 

The result of stability study indicates the 

appearance of three spots on TLC at (50, 60, 

79)ºC and only one spot at (25, 40) ºC as 

summarized in table (4). 

Table 4: *Rf values of the arginine 

acetylsalicylate – glycine complex at different 

temperatures for certain time intervals 

C Intial 1 st 2 nd 4 th 

25 0.42 0.42 0.42 0.41 

40 0.42 0.42 0.42 0.41 

50 0.42 0.43 0.46 0.55 

60 0.42 0.44 0.45 0.57 

70 0.42 0.45 0.55 0.56 

*The solvent system is (n-propanol : 34% 

ammonia 7 :3 ) 

Interference 

As mentioned earlier the degradation 

product of arginine acetylsalicylate – glycine 

complex might interfere with the 

determination of arginine acetyl Salicylic acid 

and glycine by the proposed method. The 

evacuation step in the acetylsalicylic acid 

analysis procedure ensures complete removal 

of acetic acid thus prevents its influence on the 

titration of acetyl salicylic acid with sodium 

hydroxide. Beside, the determination of the 

amount of salicylic acid (20) and the subsequent 

correction eliminates its interference on the 

acetyl Salicylic acid determination. The non – 

aqueous titration procedure is not affected by 

all the degradation procedure as this procedure 

is selective for basic groups such as the amino 

group. 

Conclusion 

The proposed procedure is suitable to be 

used as a quality control procedure for the 

36 

determination of arginine acetylsalicylate – 

glycine complex, as well as many formulas 

that contain essential and non essential amino 

acids (free or in combination)in different 

preparation like food supplement or other 

pharmaceutical preparation.This proposed 

procedure is simple, fast and accurate in 

compares with other procedures. 

Refrences 

1. Xie Y., Wang T. and Goodeng R., 

Xuexiao Huaxue Xuebao, 1980; 14(2) : 

175 -179. 

2. Kiungel OH.,Ensing K., Hegge H., Franke 

JP. And Zeeuw D., J Planer Chromatogrmod 

TLC, 1933;6(2):112-116. 

3. Kmeted VJ., Pharm Biomed Anal., 1992; 

10(10-12):1073-1076. 

4. Edgar E. Themer and Emil W. Cinrczak, J 

Pharm Sci, 2006; 66(1):139-140. 

5. Britsh Pharmacopoeia, The Stationery 

Offices, London, 2008:192-193. 

6. Juskowiak B., Spectrochim Octa, 1993; 

49(2): 173-182. 

7. Erdal Dinc, Abdil Ozdemir and Dumitru 

Baleanu, Talanta, 2005; 65(1):36-47. 

8. Erdal Dinc, , Talanta,1999;48(5):1145- 

1157. 

9. Abbas Afghani and Morteza Bahram, 

,Talanta, 2005;66(3):712-720. 

10. D.C.Harris, Quantitative Chemical 

Analysis, 6th Ed., 2003:130, 15. 

11. Sexberg H. and Kowalski R., Anal Chem, 

1979; 51:1031. 

12. Kalivas H. and Kowalski R., Anal Chem, 

1981; 53:2212. 

13. Margaret Raymond, Clemens Jachum and 

Bruce R. Kowalski, J Chem Fdu, 1988; 

60(12):1072-1073. 

14. Michael Berglund and Douglas C. Baxter, 

J Microchimica Acta, 1955; 119(3-4):311- 

312. 

15. Marjanovic L,McCrindle RI,Botha 

BM,Potgieter HJ,Anal.bioanal chem, 

2004; 379(1):104-107. 

16. Matschat R,Hassler J,Traub H,Dette 

A,Anal Bioanal chem,2005;383(7- 

8):1060-1070. 

17. Ulberth F, Anal Bioanal chem, 2006; 

386(4):1121-1136. 

18. Ignacio Ferrandez-Figares,Luis Cuadros 

Rodriguez,Antonio Gonzalez-Casado,j of 

chromatography B,2004;799:73-74. 

19. L.Cuadrose Rodriguez,L.Gawiz Gracia, 

E.M.Amansa Lopez,J.M.Bosque Sandra, 

Trends Anal chem.,2001(20):620. 

20. N.R.Meyckik, Y.U.Nikolaeva, I.P. 

Ermakov , Biochemistry (Moscow), 2009; 

74 (8): 933-937.


21. Herida R.N. Marowa,Cristian 

C.C.Lopes,Simone G.Cardoso ,Lat.Am. J. 

Pharm., 2003;22(4):339-342. 

22. Yamamoto M,Taguchi K,Journal of The 

Japanese Association for Petroleum 

Technology,2000;65(5)469-476. 

23. Leigh M.Clemow,W.Roy Jackson, Fuel, 

2002; 81(7):959-961. 

24. Ieggli, Carine Viana Silva; Cardoso, 

Simone Goncalves; Belle, Luziane Potrich 

, Journal of AOAC International , 2005; 

September 1. 

25. Sheng Yun LI, Yin Zhe REN, Feng Lin 

ZHAO, Chinese Chemical Letters, 2006; 

17( 8): 1065-1068. 

26. J. Demeester, M. Bracke, R. Vochten, A. 

Lauwers,j pharm sci,2006;67(5):729-730 . 

27. D. Kowalczuk, R. Pietraś, J. Baran, 

Annales Universitatisaari Aecurie- 

37 

sklodowska Lublin-Polonia, 2007,VOL. 

XX, N 2, 11:83-87. 

28. British Pharmacopoeia, the Stationery 

Offices, London, 2008:vol.4.A87. 

29. Jin X.,Yaovw Fenxi Zazhi,1992; 12(3): 

169-170 

30. Pin HS. Organic Chemistry,5th Ed., MC 

Graw-Hill,Inc,USA,1988:817,823. 

31. Holme J. and Peck H., Analytical 

Biochemistry,2nd ed., Longman Group, 

UK, 1933;49:384-396. 

32. Mihajlovic R., Anal Chimicia Acta, 1990; 

229:287-290. 

33. Skoog AD.,West MD. And Holler JF. 

Fundamentals of Analytical Chemistry, 

5th ed., WB-Saunders Company, USA, 

1988:249. 

34. Kontoyannis G., J Pharm Biomedical 

analysis, 1993; 13(1):73.

Iraqi J Pharm Sci, Vol.19(2) 2010 Lithotripsy of urinary stone by Carum copticum seeds 

Lithotripsy of Different Urinary Tract Stones by Using Seeds of 

Carum copticum 

Ahmed G. Sabar* ,1 

* Department o f Community Health ,College of Health and Medical Technology, Baghdad,Iraq . 

Abstract 

It has been a well-known practice to use seeds and the essential oil of Carum copticum as a 

strongly antiseptic , antispasmodic , aromatic , bitter , diaphoretic , digestive , diuretic , 

expectorant and tonic. Also used for cure influenza, asthma, and rheumatoid arthritis. To our 

knowledge it will be the first time to use the seeds of this herb as a urinary tract stone lithotripsy.This 

research aimed to the use of these seeds as a lithotripsian against different types of urinary stones and 

determine the efficiency of these preparation against which types of stone.A liquid solution was 

prepared from dissolving the seeds powder in cow milk and then concentration this preparation was 

done by boiling at 100 ° C to reduce the volume of solution to the half.The treatment was given via oral 

administration for successive 9 days before breakfast. 350 patients with urinary stone of different type 

took part in this research. All patients were subjected to ultrasonography and intravenous pyelography 

examinations to localized the position and detect diameter of stone. The above examination and also 

biochemical tests for diagnosis of stones ingredients were repeated after the administration of treatment 

and excretion of stone fragments in urine. The results were so promising especially against pure caoxalate 

stone. 

Key words: Carum copticum; Lithotripsy; Ca-Oxalate stone; mixed stone 


, ﺓﺭﺪﻣ , ﺺﻐﻤﻠﻟ ﺓﺩﺎﻀﻣ ﺩﺍﻮﻤﻛ ﺭﻭﺬﺒﻟﺍ ﻦﻣ ﺔﺼﻠﺨﺘﺴﻤﻟﺍ ﺓﺭﺎﻴﻄﻟﺍ ﺕﻮﻳﺰﻟﺍ ﻚﻟﺬﻛﻭ ( ﻲﻛﻮﻠﻤﻟﺍ ﻥﻮﻤﻛ) 

ﺓﻮﺨﻨﻟﺍ ﺕﺎﺒﻧ ﺭﻭﺬﺑ ﺖﻣﺪﺨﺘﺳﺍ 

ﺍﺬﻫ ﺭﻭﺬﺑ ﺎﻨﻣﺪﺨﺘﺳﺍﻭ . ﻲﻣﺰﺗﺎﻣﻭﺮﻟﺍ ﻞﺻﺎﻔﻤﻟﺍ ﺏﺎﻬﺘﻟﺍﻭ ﻝﺎﻬﺳﻻﺍﻭ ﻮﺑﺮﻟﺍﻭ ﻝﺎﻌﺴﻟﺍﻭ ﺩﺮﺒﻟﺍ ﺝﻼﻌﻟ ﺎﻀﻳﺍ ﺖﻠﻤﻌﺘﺳﺍﻭ , ﺔﻌﺸﻘﻣ , ﻢﻀﻬﻠﻟ ﺓﺪﻋﺎﺴﻣ 

ﺕﺎﺒﻧ ﺭﻭﺬﺑ ﻡﺍﺪﺨﺘﺳﺍ ﻰﻟﺍ ﺚﺤﺒﻟﺍ ﺍﺬﻫ ﻑﺪﻬﻳ . ﻝﺎﺠﻤﻟﺍ ﺍﺬﻫ ﻲﻓ ﺓﺮﻣ ﻝﻭﻻ ﺎﻨﺗﺎﻣﻮﻠﻌﻣ ﺐﺴﺣ ﺔﻴﻟﻮﺒﻟﺍ ﻱﺭﺎﺠﻤﻟﺍ ﻰﺼﺣ 

ﺖﻴﺘﻔﺘﻟ ﺝﻼﻌﻛ ﺕﺎﺒﻨﻟﺍ 

ﻢﺗ . ﻰﺼﺤﻟﺍ ﻉﺍﻮﻧﺍ ﻦﻣ ﻉﻮﻧ ﻱﺍ ﻩﺎﺠﺗ ﺮﺒﻛﻻﺍ ﺔﻴﻟﺎﻌﻔﻟﺍ ﺪﻳﺪﺤﺗﻭ , ﺔﻔﻠﺘﺨﻤﻟﺍ ﺎﻬﻋﺍﻮﻧﺎﺑ ﺔﻴﻟﻮﺒﻟﺍ ﻱﺭﺎﺠﻤﻟﺍ ﻰﺼﺣ ﺖﻴﺘﻔﺘﻟ ﺝﻼﻌﻛ ﺔﻳﺪﻨﻬﻟﺍ ﺓﻮﺨﻨﻟﺍ 

ﻰﻟﺍ ﺔﻴﻤﻜﻟﺍ ﻝﺍﺰﺘﺧﻻ ﺔﻳﻮﺌﻣ ﺔﺟﺭﺩ 100 ﻰﻟﺍ ﻪﻧﺎﻴﻠﻐﺑ ﻊﻴﻘﻨﻟﺍ ﺰﻴﻛﺮﺗ ﻢﺗﻭ ﺭﺎﻘﺑﻻﺍ ﺐﻴﻠﺣ ﻡﺍﺪﺨﺘﺳﺎﺑ 

ﺕﺎﺒﻨﻟﺍ ﺭﻭﺬﺑ ﻕﻮﺤﺴﻣ ﻦﻣ ﻊﻴﻘﻧ ﺮﻴﻀﺤﺗ 

ﺩﻮﺟﻭ ﻦﻣ ﻥﻮﻧﺎﻌﻳ ﻦﻤﻣ ﺎﻀﻳﺮﻣ 350 ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﻲﻓ ﻙﺭﺎﺷ. 

ﺭﺎﻄﻓﻻﺍ ﻞﺒﻗ ﺔﻴﻟﺎﺘﺘﻣ ﻡﺎﻳﺍ 9 ﺓﺪﻤﻟ ﻢﻔﻟﺍ ﻖﻳﺮﻃ ﻦﻋ ﻂﻴﻠﺨﻟﺍ ﻲﻄﻋﺍ . ﻒﺼﻨﻟﺍ 

ﻢﺛ , ﻰﺼﺤﻟﺍ ﻢﺠﺣﻭ ﻊﻗﻮﻣ ﺪﻳﺪﺤﺘﻟ ﺔﻧﻮﻠﻤﻟﺍ ﺔﻌﺷﻻﺍﻭ , ( ﺔﻴﺗﻮﺼﻟﺍ ﻕﻮﻓ ﺕﺎﺟﻮﻤﻟﺍ) 

ﺭﺎﻧﻮﺴﻟﺍ ﺕﺎﺻﻮﺤﻓ ﻰﻟﺍ ﺍﻮﻌﻀﺧﺍ ﻲﻟﻮﺒﻟﺍ ﻯﺮﺠﻤﻟﺍ ﻲﻓ ﻰﺼﺤﻟﺍ 

ﺔﻳﻭﺎﻴﻤﻴﻛﻮﻳﺎﺒﻟﺍ ﺕﺎﺻﻮﺤﻔﻟﺍ ﺖﻳﺮﺟﺍ ﻚﻟﺬﻛ ﺝﻼﻌﻟﺍ ﻡﺍﺪﺨﺘﺳﺍ ﺪﻌﺑ ﺕﺎﺻﻮﺤﻔﻟﺍ ﻩﺬﻫ ﺕﺪﻴﻋﺍ. 

ﻡﺎﻌﻟﺍ ﺭﺍﺭﺩﻻﺍ ﺺﺤﻓ ﺚﺤﺒﻟﺍ ﺕﺎﻨﻴﻋ ﺔﻓﺎﻜﻟ ﻱﺮﺟﺍ 

ﺕﻻﺰﻛﻭﺍ ﺓﺎﺼﺣ ﻩﺎﺠﺗ ﺔﻴﻟﺎﻋ ﺓءﺎﻔﻜﺑ ﻝﺎﻌﻓ ﺝﻼﻌﻟﺍ 

ﺍﺬﻫ ﻥﺍ ﻦﻴﺒﺗ ﺚﻴﺣ ﺎﻬﺗﺎﻧﻮﻜﻣ ﺺﻴﺨﺸﺘﻟ ﺭﺍﺭﺩﻻﺍ ﻲﻓ ﺔﻟﺯﺎﻨﻟﺍﻭ ﺔﺘﺘﻔﺘﻤﻟﺍ ﺕﺎﻴﺼﺤﻟﺍ ﻰﻠﻋ 

. ﻯﺮﺧﻻﺍ ﻉﺍﻮﻧﻻﺍ ﻦﻣ ﺕﺍﻮﺼﺤﻠﻟ ﻞﻗﺍ ﺔﺟﺭﺪﺑﻭ ﺔﻴﻘﻨﻟﺍ ﻡﻮﻴﺳﺎﺗﻮﺒﻟﺍ 

Introduction 

Renal stones (nephrolithiasis) are 

concretion composed of crystalline 

components and organic Matrix [1]. Although the 

symptomatic presentations may be similar ;the 

disorder is heterogeneous as to composition 

and etiology.Today, most urinary stones in 

patients in most countries are renal stones. 

About 1-4% of the population is believed to 

have kidney stones every year in USA and 

Europe. About 2-5% of population in Asia, 8- 

15% in Europe and North America and 20% in 

saudia Arabia develop kidney stone in their 

lifetime [2, 3, and 6] . Renal stones tend to recur, 

and the rate of recurrence is about 75% during 

20 years environmental and genetic factors [4-5] . 

A specific diagnosis for every patient with 

kidney stones, may give very important 

information about the stone-formation 

mechanism and the pharmaceutical manner to 

38 

[5-6] 

prevent recurrent stone formation 

.Carum copticum with herbarium number 293- 

0303-1 is a plant in Umbelliferae family with a 

white flower and small, brownish seeds. This 

plant is commonly grows in India,Iran,Egypt 

and Europe [7] .The seeds and especially the 

essential oil are strongly antiseptic, 

antispasmodic, diuretic, and used in the 

treatment of so many diseases.The seeds 

contains about 4-6% essential oil, of which 45- 

55% is thymol [8] , while the essential oil in the 

dried fruits (2.5-5% ) is dominated by thymol 

(35-60%) [9] .From South India Carum copticum 

fruits, almost pure thymol has been isolated 

(98%) , but the leaf oil was found to be 

composed of monoterpenoids and 

sesquiterpenoids: 43%cadinene, 11% 

longifolene, 5% thymol, 3% camphor and 

others [9] . The effective components of this 

1Corresponding author E- mail : ahmedalqaicy@yahoo.com , ahmed@hotmail.com 

Received : 16/11/2009 

Accepted : 1/8/2010


Plant , responsible for the observed 

bronchodialatory effect [6] ,despite the availability 

of modern medication the propensity towards the 

traditional medications is growing through out 

the word [7,8] which needs scientific investigation 

for evaluating the therapeutic effects and their 

mechanism of action. Indeed no acute toxicity 

data were available for Carum copticum 

,although animal studies of putative beneficial 

effect of this plants seeds involved the use of a 

dose of 500mg/kg body weight in mice and rat 

without morbidity or mortality ascribable to the 

herb, suggesting that the oral LD50 of Carum 

copticum (fruit/seeds) is likely to be higher than 

that figure [9] . The present study was carried out to 

determine the role of Carum copticum seeds as a 

herbal medication for treatment urinary tract 

stones. 


The study of effects of Carum copticum 

seeds as a lithotripsian agent was carried out in 

2008 on (350) patients diagnosed by specialist 

physician in private clinic from different sites of 

Iraq (Baghdad,Diala,Mousol) and the 

experimental part was undertaken during the 

period (2001-2008). 

Materials 

Seeds of Carum copticum (other Latin 

name : Trachyspermum ammi ), were collected 

fro m local market in Baghdad, identified under 

expert guidance ,taxonomy was performed by 

national herbarium of Iraq at 1997,depended on 

Ayurvedic pharmacopoeia of India (API) [10] , 

and kew herbarium [11] , liquid milk, sugar. The 

dose normally recommended in traditional 

Ayurvedic use is 3-6 gm / day , presumably 

being 3 gm once or twice a day [12] . Also this 

dose was documented in Arabic antique 

manuscript (Tathkarat Daood AL-Antaky) [13] . 

Methods 

Before started giving the preparation we 

obtained the written consent of the patients who 

included in the study; (15gm) of seeds were 

ground to a very fine powder.(Total dose for 

each patients taken with in 9 days) ;(5gm) o f 

grounded seeds were boiled with 150 ml of liquid 

cow milk and 25 gm sugar, until half of the 

volume was obtained; the preparation was kept 

cool [13] ;This preparation divided to ( 3 ) equal 

doses, each patients was given single dose a day 

before breakfast for a period of 3 days ; This 

procedure was repeated for remainder (10gm) of 

grounded seeds ; Ultrasonography (U /S) and 

Intravenous pyelography (IVP), were performed 

pre-, and post treatment to be sure of curing 

urinary tract from any stone; Urinary tract stones 

were of different sizes ranging from 5mm and 

1.2 cm and they were seen in the kidney (renal 

stone) at upper pole calyx, mid renal part and 

39 

lower pole calyx, also they were seen in the 

ureter (uretral stones) , and finally they were seen 

inside urinary bladder (vesical stones) . In 

addition general urine examination (GUE) was 

done for all patients ; Qualitative analysis of 

stone / fragments passed after herbal treatment, a 

procedure described by Hodgkinson [14] , was 

employed to figure out the chemical constituent 

of urinary stone 

Results 

The present study included (350) patients 

with different urinary tract stones were treated 

with liquid extract of Carum copticum seeds. 

Table 1 : Chemical constituent of stones 

Type of stone 

Pure Ca-oxalate 

(Ca-oxalate/uric 

acid) mixed 

stone 

( Ca-oxalate/ 

Hydroxyapatite) 

mixed stone 

Total 

No. of 

patients 

170 

100 

80 

350 

Table 2 : Lithotripsy events 

Type of stone 

Pure ca-oxalate 

(Ca-oxalate./ 

Uric acid) 

mixed stone 

( Ca-oxalate./ 

hydroxyapatite) 

mixed stone 

Total 

No. of 

patie nts 

170 

100 

% 

48,57 

28.57 

22,86 

100% 

No. of 

lithotripsed 

stone 

170 

53 

Table 3 : Duration of lithotripsy 

Pure ca-oxalate 

80 

350 

Type of stone 

Ca-oxa./uric acid)(Mixed stone. 

25 

Ca-oxalate./hydroxyapatite) Mixed stone. 

Patients age 

range (years) 

(20-45) 

( 20-50) 

(22-50) 

% 

100 

53 

31.25 

Time 

(days) 

2-7 

7-12 

7-15


Table 4 : Response the types of stones to the 

treatme nt with Carum copticum 

Type of stone 

Pure ca-oxalate. 

Ca-oxalate./uric 

acid mixed stone. 

(1) 

Caoxa./hydroxyapatite 

mixed stone. (2) 

Response 

(%) 

170 

(100%) 

53 

(53%) 

25 

(31.25%) 

* H.S: Highly Significant 

C.S (p-value) 

Be tween each pairs of 

types 

Ca-oxa.×mixe d(1) 

Ca-oxa.×mixe d(2) 

mixe d(1)×mixed(2) 

0.000 

*H.S 

0.000 

H.S 

0.003 

H.S 

Discussion 

The results of this study including the 

investigation of the efficiency of Carum 

copticum seeds (liquid solution), locally prepared 

on urinary stone among (350) patients.The 

results present in table (1) showed that pure caoxalate 

consist the larger percentage among types 

of stones. Since a (170) patients out of (350) 

patients (48.57%) have a pure ca-oxalate urinary 

stone.Results of table (1) revealed that (28.57), 

(22.86) of patients got mixed stone (caoxalate\uric 

acid) and (ca-oxalate 

\hydroxyapatite) respectively.The results cleared 

out in table (2) indicated that the local 

preparation of Carum copticum seeds have a 

good affectivity on ca-oxalate stones , since a 

hundred percent of this type of urinary stone had 

been lithotripsed , comparing to (53%) for mixed 

(ca-oxalate\uric acid) and (31.25%) for mixed 

(ca-oxalate\hydroxyapatite) . Recently in India 

had successfully purified an anticalcifying 

protein from the seeds of Carum copticum using 

oxalate depletion assay and deciphered its 

inhibitory activity against ca-oxalate crystals 

growth .The antilithiatic potential of Carum 

copticum was confirmed by its ability to maintain 

renal functioning, reduce renal injury and 

decrease crystal excretion in urine and retention 

in renal tissue [15] .It was obvious from the results 

presented in table (3) that pure ca-oxalate 

required the minimum duration to complete 

lithotripsy. While mixed stone of both types 

required a longer time to complete lithotripsy. 

The response of types of stone was indicated in 

table (4) which showed that pure ca-oxalate was 

the most affected types of stones by the treatment 

with Carum copticum seeds compared with other 

two types of urinary stones mainly (caoxalate\uric 

acid) mixed stone and (caoxalate\hydroxyapatite) 

mixed stone. On the 

other hand type two of stone (ca-oxalate\uric 

40 

acid) mixed stone was affected by treatment with 

Carum copticum seeds more than the mixed 

stone (ca-oxalate\hydroxyapatite). Our results 

confirmed the antilithiatic properties of Carum 

copticum seeds that authorized by many 

researchers [16-19] . Biostatistical analysis 

(Binomial-test and Z-test) confirmed these 

results. It could be argued for that the effect of 

herb extract (crude extraction) is depending 

totally on chemical structure of urinary tract 

stones, also it is clear from the results presented 

in table (4) that ca-oxalate whenever existed as 

one of the constituent of urinary tract stones it 

will provoke or stimulate the action of herbs 

seeds extract, in other word ca-oxalate is decisive 

component of urinary tract stone that encourage 

lithotripsy whenever Carum copticum seeds 

extract are available.In this view, mixed stones 

which are either (ca-oxalate\uric acid) or (caoxalate\hydroxyapatite) 

showed different 

response to the treatment with Carum copticum 

seeds extraction and that in our view is 

corresponding to the amount of ca-oxalate 

present in the mixture .In general, the lithotripsic 

effects of Carum copticum seeds against urinary 

tract stones were mentioned by Arabic scientist 

sheikh Dawood Antaki (about 1008 A. H) [13] . 

Conclusion 

Thus, the present study suggests the 

potential of Carum copticum seeds in lithotripsy 

of urinary stone, especially calcium oxalate and 

forms the basis for the development of 

antilithiatic drug interventions against 

urolithiasis. 

Acknowledgment 

The author is grateful to Dr. Nabeel Fadel , 

Dr. Nasseer Abed Agha, and Dr. Akram Mohd 

Al-mahdawy, for help in the diagnosis of the 

caseas. Thank also to Dr. Zuhair Nouman Al-Ani 

a Professor of pharmacogenetic for his scientific 

advices. 

References 

1. Pak CYC. Kidney stones. Williams Textbook 

of Endocrinology 1992; 1519-36. 

2. Pak CYC.Kidney stones. Lancet 1998; 351: 

1797-801. 

3. Kamoun A, Daudon M, Abdelmoula J, 

Hamzaoui M,Chouachi B, Houissa T, Zghal 

A, Ben Ammar S, Belkahia C, Lakhoua R. 

Urolithiasis in Tunisian Children. Pediater 

Nephrol 1999; 13(9):920-5. 

4. Gault MH, Chafe L. Relationship of 

frequency, age, sex, stone weight and 

composition in 15 624 stones. J Urol 2000; 64 

(2): 302-7. 

5. Kourambas J, Aslan P, The CL, Mathias BJ, 

Preminger M. Role of stone analysis in 

metabolic evaluation and medical treatment of


nephrolithiasis. J Endocrinol 2001; 15 (2): 

181-6. 

6. Thomas SE, Stapleton FB. Leave no (stone) 

untreated; understanding the genetic bases of 

calcium-containing urinary stones in 

childhood. Adv Pediatr 2000; 47: 199- 221. 

7. Seyed Hassan Hejazian; Mohd Hossein ,and 

Mohd Dashti,.(Antinociceptive effect of 

Carum copticum extract in mice using 

formalin test).,world Appl.Sci.J. 

2008,3(2):215-219 

8. Chopra. R. N., Nayar. S. L. and Chopra. I. C. 

Glossary of Indian Medicinal Plants 

(Including the Supplement). Council of 

Scientific and Industrial Research, New Delhi. 

1986. 

9. Versita,Warsaw. Fumigant toxicity of 

essential oil from Carum copticum against 

Indian meal moth,journal of plant protection 

Research,Dec. 2008, 48(4);1427- 4345. 

10. Ayurvedic Pharmacopoeia of India,A 

jamoda.Government of india.Part 1980; 

Vol.I,First Edition:2-3. 

11. Bull.of Miscellaneous information(Royal 

Garden,Kew), 1931 ; 1931(6); 299- 344. 

12. The Ayurvedic Pharmacopoeia of 

India,Appendix.,Government of india.Part 

I,Volume II,First Edition: 1999; 189-93. 

13. Shaikh Dawood Antaki. Tadhkirat Uli Al 

Albab Wal jamia lil Ajab Alujab. Medieval 

medical book. 10 th century, product code : 

1568. 

41 

14. Hodgkinson A. A combined qualitative and 

quantitative procedure for the chemical 

analysis of urinary tract calculi. J.clin.path. 

1971, 24,147-151. 

15. Tan Zeer Kaur , Rakesh, et al., In vivo 

efficacy of Trachyspermum ammi 

anticalcifying protein in urolithiatic rat model. 

Jour. of Ethnopharmacology .2009, 126(3) 

459-462. 

16. A k Pathak, N Nainwal et al., Pharmacological 

activity of Trachyspermum ammi : A review. 

Jour.of Pharmacy Research, 2010; 3(4); 895- 

899. 

17. Gurinder J and Daljit S.A., Bioactive potential 

of Anethum graveolens,Foeniculum vulgare 

and Trachyspermum ammi belonging to the 

family Umbelliferae-current status. Jor. Of 

Medicinal plant research 2010; 4(2), 87-94. 

18. Shazia S. ,Mir A.K, Mushtaq A. and 

Muhammed Z., Indigenous Knowledge of folk 

medicines by the women of District chakwal, 

Pakistan . Jor.Ethnobotanical Leaflets 2006; 

10: 243-253. 

19. Muhammed H.,Sumera A.,Mir Ajab K, 

Ethnopharmacology, indigenous collection 

and preservative technique of some frequently 

used medicinal plants of utror and 

gabral,district swat,Pakistan. Afric.Jor.of 

Tradition. ,Complem.,and Altenet. Med. 2006 

; 3(2) , 57-73.

Iraqi J Pharm Sci, Vol.19(2) 2010 Stability of Cfm and Cfz with CA against β- lactamase 

Evaluation of Stability of Cefamandol and Ceftazidime with 

Clavulanic Acid Against Extended Spectrum β- Lactamase 

Siham S. Shaokat * and Hamoudi A. Hameed* ,1 

* Ministry of Industry and Minerals, Food and Drugs Sector, Baghdad, Iraq. 

Abstract 

The aim of this study is to evaluate in-vitro activity of Cefamandol (Cfm) and Ceftazidime (Cfz), 

in combination with Clavulanic acid (CA) against ten complicated multiresistant uropathogenic E.coli 

.One hundred clinical strains were isolated from patients with chronic urinary tract infections (UTIs), 

these isolates were identified by the Api identification systems. The antimicrobial susceptibility tests 

were determined by Kirby-Bauer method, all of them were sensitive to Imipenem (Imp). Ten strains 

were chosen for the present study, they were resistant to Ampicillin (Amp), Amoxicillin (Amo), 

Carbenicillin (Cb), Ticarcillin (Tic), Azlocillin (Azl), Amoxicillin\ Potassium Clavulanate 

{Augmentin(Amc)}, (Amo\CA), Ticarcillin\ Potassium Clavulanate {Timentin} (Tic\CA) ,Cefazolin 

(Cfo) ,Cefaloridin (Cfr), Cefamandol, (Cfm),Ce foxitin, Ceftazidime (Cfz), Cefixime (Cxm), 

Cefoperazone( Cfp) and Aztreonam (Atm), also resistant to other antibiotics, 

Tetracycline(Tc),Cloramphenicol(Cm),Gentamycin(G),Amikacin (Amk), Ciprofloxacin (Cip) and 

Trimethoprim. 50% of the isolates were resistant to Nalidixic acid and Rifampicin. The minimum 

inhibitory concentrations of Cefamandol and Ceftazidime were determined, by tube method. Transfer 

of plasmids were done by direct conjugation test to sensitive standard E.coli ,cell free β- lactamases 

were prepared and detected by macro-iodometric method. The activity of each cell free ß – lactamases 

extract against Cfm and Cfz were determined by disks diffusion method (microbiological Masuda 

method) .Excellent activities were obtained against these strains when Cfm and Cfz, combined with 

CA, therefore complete zones of inhibition were obtained indicated the prevalence of extended 

spectrum β- lactamases in E.coli. The stability of Cfm and Cfz in the presence of CA were useful in the 

treatment of chronic urinary tract infections caused by multiresistant β- lactamase (ESBL) producer 

E.coli. 

Key words: Exte nde d spectrum β- lactamases, Imipe nem, Aztreonam, Ce ftaz idime . 


E- ) ﻩﺎﺠﺗ ﻚﻧﻻﻮﺠﻓﻼﻜﻟﺍ ﺾﻣﺎﺣ ﺔﻓﺎﺿﺎﺑ ( Cfz) 

ﻢﻳﺩﺯﺎﺘﻔﻴﺴﻟﺍﻭ ( Cfm) 

ﻝﻭﺪﻨﻣﺎﻔﻴﺴﻟﺍ ﺔﻴﻟﺎﻌﻓ ﺔﺳﺍﺭﺩ ﻰﻟﺍ ﺚﺤﺒﻟﺍ ﻑﺪﻬﻳ 

ﻥﺍ ﺔﺳﺍﺭﺪﻟﺍ ﺖﻨﻴﺑ ﺪﻗﻭ ﻦﻣﺰﻤﻟﺍ ﺔﻴﻟﻮﺒﻟﺍ ﻱﺭﺎﺠﻤﻟﺍ ﺏﺎﻬﺘﻟﺍ ﻦﻣ ﻥﻮﻧﺎﻌﻳ ﻰﺿﺮﻣ ﻦﻣ ﺔﻟﻭﺰﻌﻣ ﻡﺎﺘﻛﻻﺎﺘﻴﺒﻟﺍ ﺕﺍﺩﺎﻀﻣ ﺔﻋﻮﻤﺠﻤﻟ ﺔﻣﻭﺎﻘﻤﻟﺍ ( Coli 

ﻢﻳﺩﺯﺎﺘﻔﻴﺴﻟﺍﻭ ﻝﻭﺪﻧﺎﻣﺎﻔﻴﺴﻟﺍ ﻦﻣ ﻞﻛ ﺔﻴﺗﺎﺒﺛ ﻥﺍﻭ ﻚﻧﻻﻮﺠﻓﻼﻜﻟﺍ ﺾﻣﺎﺣ ﺩﻮﺟﻮﺑ ﻥﻮﻜﺗ ﻢﻳﺩﺯﺎﺘﻔﻴﺴﻟﺍﻭ ﻝﻭﺪﻧﺎﻣﺎﻔﻴﺴﻟﺍ ﻦﻣ ﻞﻜﻟ ﻰﻠﺜﻤﻟﺍ ﺔﻴﻟﺎﻌﻔﻟﺍ 

ﺕﺍﺩﺎﻀﻤﻟ ﺔﻣﻭﺎﻘﻤﻟﺍ ( E-Coli ) ﻦﻋ ﺔﺠﺗﺎﻨﻟﺍ ﺔﻴﻟﻮﺒﻟﺍ ﻱﺭﺎﺠﻤﻟﺍ ﺏﺎﻬﺘﻟﺍ ﺔﺠﻟﺎﻌﻣ ﻲﻓ ﺎﻤﻬﻣﺍﺪﺨﺘﺳﺍ ﺔﻴﻧﺎﻜﻣﺍ ﻰﻟﺍ ﺮﻴﺸﺗ ﺾﻣﺎﺤﻟﺍ ﺩﻮﺟﻮﺑ 

. ﺚﺤﺒﻟﺍ ﻲﻓ ﺖﻣﺪﺨﺘﺳﺍ ﻲﺘﻟﺍ ﺺﻴﺨﺸﺘﻟﺍﻭ ﻢﻴﻴﻘﺘﻟﺍ ﻖﺋﺍﺮﻃ ﻝﻼﺧ 

ﻦﻣ ﻡﺎﺘﻛﻻﺎﺘﻴﺒﻟﺍ 

Introduction 

Clavulanic acid is a β- lactam; 

structurally it differs from Pnicillins in two 

respects, the replacement o f sulfur in the 

Penicillin thiazolidine ring with oxygen in the 

clavam oxazolidine ring and the absence of the 

side chain at position 6. Clavulanic acid a 

naturally occurring clavam isolated from 

Streptomyces clavuligerus has poor 

antibacterial activity but exerts a potent and 

irreversible inhibitory effect on β- lactamases 

especially penicillinase by blocking the active 

sites of these enzymes and is strongly 

synergistic with most o f the β- lactamines in 

vitro (1) lactam antibiotics 

. Due to this combination, Amoxicillin 

is protected from degradation and its spectrum 

is therefore extended to include bacteria 

normally resistant to amoxicillin and other β- 

(2) . In the case of β- lactam 

resistant bacteria a bacterial enzyme, β- 

lactamase, cleaves the β- lactam ring and 

renders the antibiotic inactive. β-lactamases 

are a large and diverse group of enzymes in 

which four clinically relevant classes are 

known (3) . β-lactamase continues to be the 

leading cause of resistance to β- lactam 

antibiotics among Gram-negative bacteria. In 

recent years there has been an increased 

incidence and prevalence of extendedspectrum 

β-lactamases (ESBLs), enzymes that 

hydrolyze and cause resistance to Oxyimino- 

Cephalosporins and Aztreonam.The majority 

of ESBLs are derived from the widespread 

broad - spectrum β- lactamases TEM-1 and 

SHV-1. 

1Corresponding author E- mail : hamodiabas@yahoo.com 

Received : 11/6/2009 


42


ESBLs have become widespread throughout 

the world and are now found in a significant 

percentage of E.coli and Klebsiella 

pneumoniae strains in certain countries (4, 5, 6, 7) . 

There are also new families of ESBLs, 

including the Cefotaximase (CTX-M) and 

OXA- type enzymes, Ceftazidimase, as well as 

novel unrelated β- lactamases (8, 9, 10) . The 

stability of different Cephalosporins to the 

most important β- lactamases was assessed 

and many clinical studies have shown that up 

to 75% of the β- lactamases responsible for β- 

lactam resistance in G-negative bacteria were 

R-plasmid mediated (11) . Recently, new fourth 

generation cephalo-sporins, such as Cefepime, 

Cefpirome, Cefoselis, Cefditoren, Cefozopran 

(12) , were introduced into antibacterial 

chemotherapy and their activities were 

compared with other β-lactams such as 

Ceftazidime,Imipenem and Carbapenem, 

against..P.aeruginosa, Enterobacteriaceae 

(E.coli, Klebsiella pneumoniae) and Gpositive 

bacteria. In addition several drug 

combinations have been produced which 

contain both a β- lactam antibiotic and a β- 

lactamase inhibitor; the inhibitor has high 

affinity for β- lactamase, irreversibly binds to 

it, and thereby preserves the activity of the β- 

lactam. Currently, four penicillin inhibitor 

combinations are in clinical use: Ampicillin- 

Salbactam (Unasyn), Amoxicillin- Clavulanate 

(Augmentin), Ticarcillin –Clavulanate 

(Timentin) and Pipracillin- Tazobactam 

(Zosyn) (12) . Urinary tract infections (UTIs) are 

cause a significant health problem and E.coli 

has been reported to be the primary pathogen 

in approximately 80% of cases. E.coli, express 

structures called adhesins fimbriae or pili that 

help them bind to specific tissue (13) . The aims 

of the study are: 

1. To know the prevalence of extended 

spectrum β- lactamase (ESBL) in multi 

drug resistant (MDR) strains of E.coli 

isolated from complicated urinary tract 

infections. 

2. To evaluate the following combinations: 

Cefamandol/Clavulanateand Ceftazidime 

/ Clavulanate for their in vitro 

antimicrobial activity against complicated 

urinary tract infections caused by ESBLs 

β- lactamases. 


Standard strains with plasmid – mediated 

beta – laectamases were used: 

1-E.coli K12 (TEM-1 type β- lactamase with 

isoelectric point 5.4) confer plasmid(R 111) 

and E.cloacae P99 ). 2-E.coli K12 (SHV-1 

type β- lactamase Pitton (type II) I.p 7.7 

(10).3-E.coli K12 600 Rif and E.coli K12 600 

43 

Nal Sensitive to antibiotics (10) . 4-Clinical 

isolates of E.coli. 5-Pure enzyme of Med Labs. 

6- E.coli ATCC 25922 provided by Medical 

city Identification of E.coli. A total of 100 

strains of E.coli were selected and identified 

by Api 20 E . System (Biomerieux vitek, 

Inc) (14) . 

Antibiotic susceptibility test (Disk diffusion 

method (10) 

The resistance pattern for antibiotics 

were determined by Kirby/Bauer diffusion 

assay on Mueller – Hinton agar (20ml / plate) 

the inoculum was 10 4 – 10 5 CFU / ml, of 6 

hours cultures at 37ºC for 24 hours.The 

antibiotics used were as follow: Amoxicillin 

(Amo) 30 µg, Augmentin(Amc) (Amo 20µg + 

CA10µg),(Tic),Azlocillin 100µg, Timentin 

(Tim) (75µgTic+CA 10 µg), Cefaloridin(Cfr) 

30µg ,Cefamandol (Cfm) 30µg and 

Ceftazidime (Cfz) 30µg, Cefixime(Cxm) 

30μg, Ceftriaxone (Ctr) 30µg, Cefoperazon 

(Cfp) 30µg ,Aztreonam (Atm) 30µg 

Rifampicin (Rif) 30µg, Nalidixic acid 

(Nal)30µg, Ciprofloxacin(Cip) 10mcg, 

Amikacin (Amik)10µg (Tc)30µg, 

Chloramphenicol (Cm)30µg, Gentamicin 

(Gm)30µg, and Cotrimoxazole 

(Trimethoprime 2.5 µg + Sulfamethaxazole 

22.5 µg) (Tm). 

Minimum inhibitory concentrations (MICs) 

MICs were determined by dilutions of 

different concentrations of Cfm, Cfz, alone 

and in the presence of Clavulanic acid (CA). 

According to the method recommended by the 

National committee for microbiology 

Laboratory standards (FRANCE) Powders of 

β -lactam antibiotics were obtained from 

(Russell and Beecham). (15) 

Transfer of genetic information by direct 

conjugation method 

Conjugal transfer of 3GC resistant ESBL 

producing strains was done at 35°C -37°C in 

liquid medium {Brain heart infusion (B.H)} or 

in solid media {Trypticase Soya agar (T.S.A) 

or Mueller – Hinton (M.H)} using E. coli 

K12 600 Rif and E.coli K12 600 Nal as 

recipient. Equal volumes (1 mL) of culture of 

the donor and the recipient strain (108-109 

CFU/mL) grown with agitation in tryptic soya 

broth were mixed and incubated statically for 

18 hours at 35°C. Transconjugants were 

selected on M.H agar containing 64-µg/mL 

Nalidixic acid to inhibit the growth of donor 

and 2.5 µg/mlCfz to inhibit the growth of 

recipient strain (11) .


Phenotypic confirmatory disc diffusion test 

(PCDDT) for ESBL (18) 

Ten µl of CA solution was added to discs 

of Cfz and Cxm one hour before culture, these 

were applied to the surface of a Muller Hinton 

agar, seeded with a suspension of 10 4 - 

10 5 /CFU of bacteria under test.. An increase in 

zone diameter for either antimicrobial agent 

tested in combination with CA versus its zone 

when tested alone was observed. For Cfz an 

increase in zone diameter of> 5mm and for 

Cxm > 3 mm was considered as an ESBL 

producer. 

Extraction of ß – lactamase 

Cell free beta –lactamases were prepared 

from strains known to be good producers of 

the desired enzymes, (ß –lactamases, type 

TEM-1 and SHV-1, R-plasmid mediated 

enzymes) and ß –-lactamase from E.cloacae 

P99 ( cephalospornase) as references. Crud 

enzymes also prepared from test isolates of 

E.coli (10) . 

Detection of ß – lactamase by Macro - 

iodometric method. 

This test is based on the reaction of the 

(oic) acid of penicillin with iodine. ß – 

lactamasehydrolyze penicillin to penicilloic 

acid, which in turn react with iodine, the 

presence of ß – lactamase in a test system was 

shown by decolorization of starch-iodine 

complex, observed in 1-18hours at 4°C (16) . 

Assessment of stability of ß – lactams to cellfree 

ß – lactamases (17) 

The surface of Muller Hinton agar was seeded 

with a suspension of sensitive indicator E.coli 

ATCC. Four discs containing ß – lactams 

under test were placed near filter papers discs; 

each of them was impregnated with 30μl of 

the extract enzymatic. The plates were 

incubated at 37°C for 18hours, the ß – 

lactamase activity was observed like half 

moon zone of inhibition. 

Masuda microbiological method (17) 

Ten clinical isolates were screened for ß – 

lactamase inhibitors using 10μl CA in 

combination with 30 μl of Cfz or Cfm. 

Sensitivity discs containing Cfz or Cfm and a 

filter disc incorporated with10μl enzyme and 

10μl (CA) as potassium clavulanate were 

placed on agar plate on which a bacterial 

suspension of sensitive E.coli ATCC 

(standard) was spread the inoculum was 10 4 – 

10 5 CFU / ml, of 6 hours cultures at 35 C 0 - 

37C 0 for 24 hours. Unchangeable inhibition 

zones demonstrate stability of the antibiotic to 

the enzyme. 

44 


Extended –spectrum ß – lactamases ( ESBLs) 

are derivatives of enzymes such as SHV-1 and 

TEM-1 that have undergo site specific 

mutation that enable them to hydrolyze , and 

thus inactivate , oxyimino – cephalosprins 

such as, cefotaxime and ceftazidim. (19) . All 

clinically important reactions of ß – lactamase 

inhibitors , such as tazobactam , sulbactam, 

and calvulanic acid , involve ß – lactam ring 

cleavage during acylation of an active site . 

Although other clavams produced in nature 

may possess antibacterial and antifungal 

properties, clavulanic acid is the only one 

known clavam with potent ß – lactamase 

inhibitory activity owing in part to its 3R ,5R 

stereochemistry , it is a potent inhibitor of ß – 

lactamase enzymes produced by many strains 

of Staphylococcus aureus , E.coli , Klebsiella , 

Proteus , Sheglla , Pseudomonas , and 

Haemophilus influenzae (20,21,) . 

100% of the isolates were found to be resistant 

to Amp, Amo,Cb,Tic, Azl, Cfr,Cfo, Tc,Cm 

and Tm , 10% were resistant to Cfm Cxm, 

Cfz,Cfp,Ctr , Atm Tim, and Amc.Also 

resistant to G,Amk,Cip and Tm , 50% of the 

isolates were resistant to Nal and Rif as shown 

in Table 1. ESBL was detected in 10 isolates 

by PCDDT the zone of inhibition increased 

in presence of CA. For Cfz >10mm, and for 

Cfm and Cxm>5mm, potentiation of the 

inhibiton zone of 3GC in the presence of CA 

was observed.. indicated ESBL production in 

ten strains; the diameters zone of inhibition for 

Amc and Tim were range from 0 - 5mm while 

the normal diameters zones of inhibition were 

for Amc 14-21mm and for Tim is 13mm. The 

critical normal MICs for Tim and Amc were 

(4-16) and (128) respectively. The MICs were 

studied for ten clinical isolates of E.coli in 

comparison with standard resistant strains, the 

range of MICs for Cfm was 512 - 2048 μg/ml 

and for Cfz 32-64 μg/ml, while for non ESBL 

producer it ranged from 0.02-8 µg/mL. After 

the addition of CA eight-fold reduction or 

more in MICs (Table 2 ) . These results was in


agreement with the investigation of Chaudhary 

,U, Aggarwal-R (17) indicated ESBL producers. 

All the isolates were sensitive to Imp, but 

among the non ß – lactam antibiotics Cip and 

Amk were most effective drugs 90 strains 

were sensitive. Resistance to Cfz was 

transferred to recipient E. coli K12 C600 Rif or 

E. coli K12 C600 Nal strains, along with 

resistance to other ß – lactam antibiotics, ESBL 

production is coded by genes on conjugation 

plasmids which are easily transmitted among 

different members of Enterobacteriaceae, all 

ESBLs have serine at their active sites. The 

results of detection of ß – lactamases by 

iodometric method were positive for 10 strains 

comparing with standard negative and positive 

ß – lactamases R111 (TEM-1) and E.coli 

ESBL producer. The inhibition of betalactamase 

production by CA has been 

NO of 

isolates 

45 

demonstrated with many strains of bacteria, 

this effect potentiates the action of many beta 

lactams, such as Amp, Amo, Cb and Azl. 

Many clinical reports of combination of Amo 

with CA have been encouraging, in urinary 

tract infections due to ß – lactamase-producing 

organisms type TEM and SHV, whilst Amo 

alone had no effect, the addition of CA (as 

salt) dramatically change the half moon 

inhibition zone to complete inhibition zone 

(3,4,11) . Figure 1 indicate the activity of ß – 

lactamase extracts against ß -lactams 

antibiotics, figure 2 indicate the Antibiotic – 

enzyme Interaction by the highly sensitive 

double disks technique, demonstrated their 

hydrolysis, however ß -lactamase of E.cloacae 

not effected by Amc and inhibited by Azl and 

hydrolyzed all cephalosporins. ( 5,6,20) . 

Table 1: Se nsitivity Tests of Ten Strains De termine d by Disk Diffusion Te st . 

Diameters of zone of inhibition / MM 

Amo Amc Tic Tim Cxt Cfm Cfz Cxm Ctr Amk Cip Rif Nal 

1 0 4 0 5.5 16 2 3 10 11 10 12 19 0 

2 0 4.5 0 7 16 3 3 12 10 11 10 0 18 

3 0 3.5 0 8 18 2 5 8 5 14 12 19 0 

4 0 4 0 8.5 17 0 4 4 13 12 11 10 0 

5 0 5 0 9 17 6 14 15 12 12 11 19 0 

6 0 5.5 0 9 19 10 14 15 13 12 18 0 0 

7 0 6 0 7 20 11 11 15 12 18 20 0 18 

8 0 6 0 6.5 20 5 10 13 10 18 24 0 19 

9 0 6 0 9.5 20 5 11 12 10 15 22 19 19 

10 0 7.5 0 9 17 7.5 13 10 10 12 10 19 18 

E.coli 

(ESBL) 

E.coli 

0 0 0 5 17 7 13 12 10 12 18 11 17 

ATCC 

25922 

21 21 13 13 22 22 21 21 21 32 40 32 33 

Abbreviation's : Amo: Amoxicillin Amc:Amoxiclave; Cb: Carbenicillin;Azl: Azlocillin; Tim: Timentin; Cxt Cefoxitin ; Cfm: 

Cefamandol; Cfz : Ceftazidime; Cxm:Cefixime,Ctr:Ceftriaxone, Cfp :Cefoperazon; Amk: Amikacin, Cip: Ciprofloxacin , Rif: 

Rifampicin; Nal: Nalidixic acid; the diameters of zone of inhibition for Amp icillin, Amoxic illin, Carbenicillin,Ticarcillin, 

Azlocillin,Cefazolin(Cfo), Cefaloridin(Cfr),Cefoperazone(Cfp) Aztreonam(Atm) , Tetracycline ; Chloramphenicol; 

Trimethoprim and Gentamicin were zero. All of them sensitive to Imipenem (Imp) 17-23mm and Cefoxitin 15-22mm .Normal 

zone for Amc: 14-21mm; Tim:13mm; Cfz,Ctr,Ctx: 15-21mm; Cfm:15-22mm. Amk:25-32mm; Rif :19-32mm;Cip:30-40mm. 

Table 2: Minimum Inhibitory Concentrations of Ten Uropathoge nic E.Coli Co mparing with 

Standard Strains . 

MICs mcg/ml 

No . o f Iso late E.coli 

Imp Cfm Cfz Cfm+CA Cfz+CA 

1,2,3 2 512 64 2 1 

4,5,6 1 1024 64 0.5 2 

7,8,9,10 4 2048 32 0.25 0.5 

E.coli (453)* SHV-1 (7.7) (19) 16 16 0.05 0.25 0.25 

E.coli (R111) TEM-1 (5.4)* ) 16 32 0.02 0.25 0.25 

E.cloacae(P99** (8.3)* 64 512 64 32 64 

E.coli (ESBL) 4 128 64 0.25 2 

* Isoelectric points. ** Cephalosporinase. 

Normal values of MICs : Cfm S32mcg\ml 

Cfz S16mcg\ml


Cefoxitin Cefazolin Cefamandol Amoxicillin 

Ceftriaxone Azthreonam Ceftazidime Cefixime 

Imipenem Cefalo ridin Azlocillin Carbenicillin 

Figure 1: Activity of ß – lactamase against ß - 

lactams antibiotics 

Figure 2: Antibiotic –enzyme Interaction ,by 

the highly se nsitive double disks technique (24) 

demonstrate d 

A : hydrolysis of ceftazidime and Cefalmandol 

by β-Iactamases producing E.coli 

B: Inhibition by Clavulanic acid (CA) . 

Conclusions 

The Ten clinical isolates in this study 

were very resistant to Amc, Tim,Cfm 

,Cfz,Cxm,Cfp, Ctr and Atm, but sensitive to 

Imipenem comparing with standard TEM-1 

and SHV-1 (plasmidic penicillinases) and 

E.clocae P99 ) (Chromosomic 

Cephalosporinase) indicating the prevalence of 

extended-spectrum β-lactamases (ESBLs) 

enzymes that hydrolyze and cause resistance 

to oxyimino-cephalosporins and aztreonam. 

Our study shows presence of ESBL producer 

E.coli in ten clinical isolates. The routine 

antimicrobial sensitivity test may fail to detect 

ESBL, mediated resistance against 3GC and 

detection of ESBL production should be 

carried out as a routine in diagnostic 

laboratories by PCDDT as it is a simple and 

cost effective test, the combination with 

Clavulanic acid bringing the susceptibility 

back, confirms the ESBLs. ESBLs have 

become widespread throughout the world and 

are now found in a significant percentage of 

E.coli and Klebsiella pneumoniae strains in 

certain countries, (6, 7, 9, 10) . The increasing 

emergence of cephalosporins resistant E.coli 

has leaded to concern about the use of various 

combination therapies. 

References 

1. Jensen, -S-E; Paradkar, -A-S; Mosher, -R- 

H; Anders, -C; Beatty, -P-H; Brumlik, - 

M-J; Griffin, -A; Barton, -B.Five 

additional genes are involved in 

clavulanic acid biosynthesis in 

Streptomyces clavuligerus.Antimicrob- 

Agents-Chemother. 2004; 48(1): 192-202 

2. Dumon L., Adriaens P., Anne J., Eyssen 

H. Effect of Clavulanic acid on the 

minimum inhibitory concentrations of 

benzylpenicillin, ampicillin, carbenicillin, 

or cefalothin against clinical isolates 

resistant to beta-lactam antibiotics. 

Antimicrob. Agents and Chemother., 

1979,N2,315-317 

3. Bush, K., Jacoby,G.,A and Medeiros.A.,A 

. A functional classification scheme for 

beta – lactamases and its correlation with 

molecular structure.Antimicrobial agents 

chemotherapy 1995 p1211-1233. 

4. Poeschl, -P-W; Eckel,-D; Poeschl,-E. Post 

operative prophylactic antibiotic 

treatment in third molar surgery--a 

necessity? J-Oral-Maxillofac-Surg. 2004; 

62(1): 3-8; discussion 9IS 

5. Bradford, -P-A. Extended-spectrum βlactamases 

in the 21 st century: 

characterization, epidemiology, and 

detection of this important resistance


threat.Clin-Microbiol-Rev. 2001; 4(4) 

933-51. 

6. Sturenburg, -E; Mack, -D.Extendedspectrum 

beta-lactamases: implications 

for the clinical microbiology laboratory, 

therapy, and infection control. J-Infect. 

2003; 47(4): 273-95 

7. Bell,-J-M;Turnidge,J-D;Jones,R-N. 

Prevalence of extended-spectrum betalactamase-producing 

Enterobacter cloacae 

in the Asia-Pacific region: results from 

the sentry Antimicrobial Surveillance 

Program, 1998 to 2001. Antimicrob- 

Agents-Chemother.2003; 47(12): 3989-93 

8. Edelstein, -M; Pimkin, -M; Palagin, -I; 

Edelstein,-I; Stratchounski,-L .Prevalence 

and molecular epidemiology of CTX-M 

extended-spectrum beta – lactamase - 

producing E.coli and Klebsiella 

pneumoniae in Russian hospitals. 

Antimicrob- Agents- Chemother. 2003; 

47(12): 3724-32 

9. Bauernfeind, A., H. Grimm, and 

S.Schweighart. A new – plasmidic 

cefotaximase in clinical isolate of E.coli. 

Infection. 1990 18: 294-298 . 

10. Siham., S.S, Joly,B, . Phillipon ,A. 

,Sirot,D.,Cluzel.R. Resistance al’a 

carbenicilline des bacilles a Gram- 

negatife , frequance, determinisme 

biochemique et genetique, 1985 Pathol 

,Biol., 33, 825-829. 

11. El-Sukhon,-S-N; Faiza-Boukhatem,-Z. 

Activity of combinations of ceftazidime, 

imipenem and pefloxacin against 

Staphylococcus aureus, E. coli and P. 

aeruginosa. Int-J-Antimicrob-Agents. 

2003 Dec; 22(6): 613-7. 

12. Kamidono, S et al. A comparative study 

on the clinical utility of cefozopran and 

cefpirome against complicated urinary 

tract infections. Jap.J. Antibiot.2000; 

53(6): 430-450. 

13. Dromigny, -J-A; Ndoye, -B; Macondo, - 

E-A; Nabeth, -P; Siby, -T; Perrier-Gros- 

Claude, -J-D.Increasing prevalence of 

antimicrobial resistance among 

47 

Enterobacteriaceae uropathogens in 

Dakar, Senegal: a multicenter study. 

Diagn-Microbiol-Infect-Dis. 2003; 47(4): 

595-600. 

14. Cowan, S.T, Manual for identification of 

medical bacteria 1977. P106-Cambridge 

university press. Cambridge New York. 

15. Baur A. W., Kirby, W. M.M., Sherris 

K.C. and Turck, M.Antibiotic 

susceptibility testing by a standardized 

single disc method.,Amer. J. clin.path. 

1966, 45,493-496 

16. Labia, R., Barthelemy.M. L enzymogram 

des ß -lactamases .A daptation en gel .La 

method iodometrique, Ann. Macrobiol., 

1979, 130B, 236-240. 

17. Masuda G., Tomioka s and Haregawa M., 

Detection of ß -lactamases production by 

Gram-negative bacteria.The journal of 

Antibiotics, 1976, 29(6),662-664 

18. Chaudhary,U,Aggarwal-R. Extendedspectrum 

beta-lactamases.An emerging 

threat to clinical therapeutics.Indian J- 

IMed. Microbiol2004; 22(2): 75-80 

19. Matthew Kalp et al. Why extended – 

spectrum ß – lactamases SHV-2 and SHV- 

5 are ' Hypersuceptible' to Mechanism 

based Inhibitors, Biochemistry , 2009, 48 

(41) , 9912-9920 

20. Mattew Kalp et al . Efficient Inhibition of 

Class A and Class D ß – lactamases by 

Michaelis Complexes , Journal of 

Biological Chemistry , 2007.28(30) . 

21. Mary L. Raber , Micheal F. Freeman and 

Criag A.Twonsend , Journal of Biological 

Chemistry , 2009, 284 (1). 

22. Siham-SS. Marc O. Sirot-D- Joly-B. and 

Cluzel –R Spread of SHV-1 betalactamases 

in E.coli isolated from fecal 

samples in Africa.1987. Antimicro. Agent 

and Chemother.vol 31 (6)943-945. 

23. -Ma, -Y; Li, -J-Y; Yao, -L; Zhang, -L; 

Hu,-C-Q; Jin,-S-H Zhonghua-Yi-Xue- 

Za-Zhi. Antimicrobial resistance of 

Escherichia coli isolates collected from 

inpatients and outpatients] 2003 25; 

83(12): 1046-8.

Iraqi J Pharm Sci, Vol.19(2) 2010 Gravimetric estimation of caffeine 

Gravimetric Estimation of Caffeine in Different Commercial Kinds 

of Tea Found in the Iraqi Market 

Maha N. Hamad* ,1 and Dhuha A. Abdul-Hussain* 

*Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad,Iraq . 

Abstract 

Caffeine (1,3,7-trimethylxanthine), which is the most widely consumed stimulant in the world, 

had been isolated and estimated gravimetrically in fifteen different commercial kinds of tea found in 

the Iraqi market.The kinds of tea were chosen according to their differences in the degree of 

fermentation and the method of processing i.e. black , gray and green . The isolated caffeine was 

identified by melting point, sublimation, TLC, chemical tests, UV , IR , HPLC and CHNO analysis. 

Key words: Caffeine , Purine, te a. 


ﺔﻘﻳﺮﻄﺑ ﻪﺘﻴﻤﻛ ﻦﻴﻴﻌﺗﻭ ﻪﻠﺼﻓ ﻢﺗ , ﻢﻟﺎﻌﻟﺍ 

ﻲﻓ ًﻻﺎﻤﻌﺘﺳﺇ ﺔﻬﺒﻨﻤﻟﺍ ﺩﺍﻮﻤﻟﺍ ﺮﺜﻛﺃ ﻦﻣ ﺮﺒﺘﻌﻳ ﻱﺬﻟﺍ ( ﻦﻴﺜﻧﺍﺯ ﻞﻴﺜﻣ ﻲﺛﻼﺛ – 7 , 3 , 1) 

ﻦﻴﻳﺎﻓﺎﻛ 

ﺮﻴﻤﺨﺘﻟﺍ ﻦﻣ ﺔﻔﻠﺘﺨﻣ ﺕﺎﺟﺭﺩ ﻰﻠﻋ ﻱﺎﺸﻟﺍ ﻦﻣ ﺝﺫﺎﻤﻧ ﺭﺎﻴﺘﺧﺃ ﻢﺗ . ﺔﻴﻠﺤﻤﻟﺍ ﻕﺍﻮﺳﻷﺍ ﻲﻓ ﺩﻮﺟﻮﻤﻟﺍ ﻱﺎﺸﻟﺍ ﻦﻣ ًﺎﻔﻠﺘﺨﻣ ًﺎﻋﻮﻧ ﺮﺸﻋ ﺔﺴﻤﺧ ﻲﻓ ﺔﻴﻧﺯﻭ 

, ﻥﺎﺑﻭﺬﻟﺍ ﺔﺟﺭﺩ ﺱﺎﻴﻗ ﺎﻬﻨﻣ ﺔﻔﻠﺘﺨﻣ ﻕﺮﻄﺑ ﻝﻭﺰﻌﻤﻟﺍ ﻦﻴﻳﺎﻓﺎﻜﻟﺍ ﺺﻴﺨﺸﺗ ﻢﺗ . ﺮﻀﺧﻻﺍﻭ ﻲﺻﺎﺻﺮﻟﺍﻭ ﺩﻮﺳﻻﺍ ﻱﺍ ﺮﻴﻀﺤﺘﻠﻟ ﺔﻔﻠﺘﺨﻣ ﻕﺮﻃ ﻭ 

ءﺍﺮﻤﺤﻟﺍ ﺖﺤﺗ ﺔﻌﺷﻷﺍﻭ ﺔﻴﺠﺴﻔﻨﺒﻟﺍ ﻕﻮﻓ ﺔﻌﺷﻷﺍ ﻒﻴﻃﻭ , ﺔﻳﻭﺎﻴﻤﻴﻛ ﺕﺎﺻﻮﺤﻓﻭ , ﺔﻘﻴﻗﺮﻟﺍ ﺔﻘﺒﻄﻟﺍ ﺎﻴﻓﺍﺮﻏﻮﺗﺎﻣﻭﺮﻛﻭ , ﻲﻣﺎﺴﺘﻟﺍ 

. ﻦﻴﺠﺴﻛﻭﻻﺍﻭ ﻦﻴﺟﻭﺮﺘﻴﻨﻟﺍ , ﻦﻴﺟﻭﺭﺪﻴﻬﻟﺍ , ﻥﻮﺑﺮﻜﻟﺍ ﺮﺻﺎﻨﻋ ﺐﺴﻧ ﺏﺎﺴﺣﻭ ﺔﻴﻟﺎﻌﻟﺍ ﺓءﺎﻔﻜﻟﺍ ﺕﺍﺫ ﺔﻠﺋﺎﺴﻟﺍ ﺎﻴﻓﺍﺮﻏﻮﺗﺎﻣﻭﺮﻜﻟﺍﻭ 

Introduction 

Thea or tea consists of the prepared 

leaves and leaf buds of camellia sinensis 

(formerly known as Thea sinensis) of the 

Theaceae family. There are three main 

commercial types of tea: green , oolong (gray) 

and black, depending on the method of 

processing. The leaves may be fermented or 

left unfermented. Fermented teas are referred 

to black tea, unfermented teas as green tea and 

partially fermented teas as oolong tea.Black tea 

is prepared by heaping the fresh leaves until 

fermentation has begun, then they are rapidly 

dried artificially with heat, while green tea is 

prepared by rapidly drying the freshly picked 

leaves in copper pans over a mild artificial 

heat, or the leaves are often rolled in the palm 

of the hand as they dry. Gray tea is partially 

fermented by heaping then they are dried on 

N 

N 

N 

H 

N 

O 

NH 

O 

H 

N 

48 

artificial heat. Tea contains caffeine (theine) 

and small amounts of adenine, theobromine, 

theophylline, gallotannic acid and volatile oil. 

1,2 Caffeine (1,3,7-trimethylxanthine), the 

molecular formula of which is C8H10N4O2 , is 

the most widely consumed stimulant in the 

world can be considered to be constructed 

from the purine ring system, which is 

important biologically, being found in nucleic 

acids and nucleotide and in few organisms as 

alkaloids. 3 Caffeine was first discovered in tea 

in 1827, and was named theine , and later it 

was found in mate , coffee and various other 

plants and the term theine was then 

dropped. 4 Purines are considered pseudo 

alkaloids since they are not derived from an 

amino acid precursor. 5 

N 

H 

N 

H 3C-N 

Purine X anthine C affeine 

1Corresponding author E- mail : manoha_1957@yahoo.com 

Received : 28/4/2010 


O 

O 

N 

CH 3 

N 

N 

CH 3


Caffeine acts as a CNS stimulant , mild 

diuretic, it increases the heart rate and blood 

pressure and stimulate gastric secretions. It 

also acts as a natural pesticide that paralyze 

and kills certain insects feeding on the plants. 

6,7 Caffeine with UV can kill some kinds of 

algae and there are evidences that it enhances 

mutations in bacteria and viruses and also 

induce chromosome damage. 8,9,10,11 Caffeine is 

an ingredient of several dozen proprietary 

products, for the most part, these combination 

with acetyl salicylic acid, ascorbic acid, 

codeine, paracetamol, and other analgesics 

and antipyretics.Caffeine is found in a number 

of beverages ingested by people in addition to 

tea as coffee, and to some extent cocoa. Other , 

less commonly used sources of caffeine 

include the plants guarana, and yerba mate' 

which are sometimes used in the preparation of 

teas, and recently ,energy drinks. Tea leaves 

contain 1-4% caffeine, while coffee 1-2% yet a 

cup of brewed coffee contains about 100-150 

mg caffeine while a cup of tea contains 60-75 

mg. caffeine is also a common ingredient of 

soft drinks such as cola. Soft drinks typically 

contain about 10-50 mg of caffeine per serving 

. The range of caffeine contents in various 

beverages is shown table 1 

Table 1 : range of caffeine in various 

beve rages 

Approximate caffe ine 

content of various 

beverages 

Coffee (5oz. cup) 

Range of mgs 

of caffeine 

40-170 

Soft drinks (12 oz. can) 10-50 

Black tea (one tea bag) 25-110 

Oolong tea (one tea bag) 12-55 

Green tea (one tea bag) 8-30 

Decaffeinized tea(one tea 

bag) 

1-4 

Energy drinks ( 12 0z. can) 75-90 

Diagram 1 : Isolation of caffe ine from te a 

49 

In this paper we have estimated caffeine 

gravimetrically in fifteen different kind of tea 

found in the market black, gray and green tea . 

Materials and method 

Samples of tea were chosen randomly to 

represent black, gray and green tea in the 

form of tea bags or unpacked form. 

All reagents are anhydrous solvents were of 

analar type and generally used as received 

from the commercial suppliers. 

Silica gel used in the form or ready made 

aluminum plates of silica gel GF254 , Merck 

Co. 

UV was run in methanol , IR was run in KBr 

disk. 

HPLC was done using Knauer/ Germany 

HPLC. 

Standard caffeine is from Evans Medical Ltd 

, Liverpool. 

Isolation of caffeine 

25 gm of tea were boiled with 200ml of 

water for fifteen minutes in a covered beaker . 

The extract was filtered through muslin and the 

mark was re boiled with 120ml of water for 

five minutes, filtered and the mark over the 

muslin was washed with 70ml of boiling water, 

the muslin was then squeezed till exhaustion. 

The combined extracts were cooled and mixed 

with 4gm of sodium carbonate with stirring, 

then transferred to a separatery funnel and 

partitioned with three successive portions of 

methylene chloride each of 50ml (i.e. 

3X50ml), each time the funnel was inverted 

back and forth ten times. The methylene 

chloride layers were combined together and 

dried over anhydrous sodium sulfate , filtered 

and evaporated to dryness under vacuum. The 

obtained caffeine was re crystallized from 

boiling ethanol, filtered and weighed. The 

percentages of caffeine was calculated as w/w. 

The extraction procedure is shown in 

diagram(1).


Two samples of each kind were used and the 

average weights of the isolated caffeine were 

taken for calculation of the percentages and 

comparison. 

Identification of Caffeine 

The isolated caffeine was identified by 

several methods : 

It was identified by measuring its melting 

point and compare it with standard caffeine 

and also using a mixed sample from isolated 

caffeine and the standard . 12 The results are 

shown in table (2). 

Table 2 : Melting points of isolated and 

standard caffe ine 

Sample 

Isolated caffeine 

Standard caffeine 

Mixed isolated and 

Standard caffeine 

Melting point 

236.7°C 

237.3°C 

237°C 

Then caffeine was identified by two chemical 

tests : 

1- Murexide test : Isolated caffeine gave a 

purple color. 

2- Isolated caffeine was treated with hydrogen 

peroxide and 2% HCL. After evaporation 

to dryness , a bright red color was obtained. 

The color turn purple upon addition of 

drops of 5% ammonia. 13 

Also caffeine was identified by sublimation 

and this process was achieved by introducing a 

small quantity of caffeine in a porsalen dishand 

covered with a watch glass , the porsalen dish 

was subjected to heat while the watch glass 

was covered with a plastic sack containing ice 

.Upon heating caffeine started to sublime and 

condense on the lower surface of the watch 

glass. Caffeine was also identified by TLC 

using silica gel GF254 plates developed in three 

different mobile phases and comparing the Rf 

Figure 2 : IR spectrum of the isolate d caffeine 

50 

values of isolated caffeine with standard using 

single and mixed spots , and detection was 

done under UV254nm. 14,15,16,17 

Mobile phases used are: 

Mobile phase I : Ethyl acetate : acetic acid 

95:5 . 

Mobile phase II : Chloroform : ethyl acetate : 

formic acid 5: 4 : 1 

Mobile phase III : petroleum ether : methylene 

chloride : ethyl acetate 1: 1 : 2. 

The result of TLC are shown in table (3) 

Table 3 : Rf value s of isolate d and standard 

caffeine 

Mobile 

phase 

Isolated 

caffe ine 

Rf value 

Standard 

Caffeine Rf 

values 

I 

0.226 0.257 

II 0.490 0.516 

III 0.110 0.110 

The UV absorption spectrum exhibits a pair 

of absorption bands peaking at (209) and 

(272)nm with a shoulder between them 18 . 

The UV spectrum of the isolated caffeine is 

shown in fig.(1) 

Figure 1 UV spe ctrum of the isolated 

caffeine 

Also caffeine was identified by IR 19 and the 

spectrum is shown in fig. (2)


Also caffeine was identified by CHNO 

analysis ,whereby the percentage of each 

element measured and compared with the 

calculated one.The results are shown in table 4. 

Table 4 : CHNO analysis of caffeine 

Ele ment Calculate d Found 

perce ntage s pe rcentages 

Carbon 49.484 49.877 

Hydrogen 5.155 5.199 

Nitrogen 28.866 29.109 

Oxygen 16.495 16.709 

Figure 3 : HPLC of isolated caffeine 

Figure 4 : HPLC of standard 


The average weights and percentages of the 

caffeine isolated from each kind of tea are 

shown in table (5) and fig. (5). The idea of this 

method of isolation of caffeine is to extract the 

water soluble materials in the tea leaves in a 

hot water . (the solubility of caffeine in water 

is 22 mg/ml at 25 o c , 180 mg/ml at 80 o c and 

760 mg/ml at 100 o c ) . The caffeine is 

extracted from the water after cooling with 

dichloromethane (140 mg/ml) than in water 

(22 mg/ml), it readily dissolves in the 

51 

Caffeine was finally identified by HPLC 18 

using C18 5x150mm column and a mobile 

phase composed of methanol/water 90:10 with 

flow rate of 0.8ml/minute and detection with 

UV detector at 275nm. The retention time of 

the isolated caffeine was compared with that of 

the standard. The results are shown in figures 3 

and 4. 

dichloromethane.However, the tannins are 

slightly soluble in dichloromethane but upon 

addition of sodium carbonate to the extract the 

tannins will be converted to phenolic anions 

(since phenols are acidic enough to be 

converted to phenolic salts i.e. deprotenation of 

OH group ) upon addition of sodium 

carbonate). The phenolic salts thus formed are 

not soluble in dichloromethane , soluble in 

water, as shown below: 

ArOH + Na + 2CO3 -2 → ArO - Na + + Na + HCO3 - 

tannins soluble tannins salts 

in water, dichloromethane soluble in water 

insoluble in dichloromethane


Table 5 :The perce ntage of caffe ine in differe nt kinds of te a 

no 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

% Percentage of cafe in 

2.5 

Tea brand 

Al-Otoor (black) 

Al-Rabeea (black) 

Mahmood (black) 

Ahmad (black ) 

Al-Wazza (black ) 

Al-Tuffaha (black ) 

Ahmad (black , Tea bags ) 

Ration tea ( black ) 

Lipton (black , tea bags ) 

Al-Okozay ( gray , tea bags ) 

Al-attar ( green , tea bags ) 

Lipton (green , tea bags ) 

Ahmad (green , tea bags ) 

Green tea (un packed) 

Alokozay ( green , tea bags ) 

2 

1.5 

1 

0.5 

0 

Figure 5 : percentages of caffeine in diffe rent kinds of tea 

Caffeine being a xanthine derivative was first 

identified by the murexide test. The core of the 

reaction is apparently that caffeine through a 

number of steps , is oxidized into the 

intermediate that ultimately condenses to 

murexoin. The ammonium salt of murexoin is 

the principal contributor to the purple color of 

the final solution. 

20 The quantitative 

differences obtained in different kind of tea is 

probably due to the method of processing of 

each kind since caffeine sublimes with out 

decomposition upon exposure to heat, 

therefore it should be expected that caffeine 

could be lost during fermentation and 

processing. Also the differences of caffeine 

quantity and consequently the percentage may 

be due to different time of harvesting of leaves 

of the plant. 

52 

Weight of 

caffeine 

In 25 gm of tea 

0.481 

0.351 

0.322 

0.310 

0.294 

0.238 

0.2 

0.180 

0.090 

0.110 

0.341 

0.327 

0.221 

0.130 

0.120 

% of caffe ine 

1.924 

1.40 

1.288 

1.240 

1.176 

0.952 

0.800 

0.720 

0.360 

0.440 

1. 364 

1.308 

0.884 

0.520 

0.480 

Acknowledgement 

We are deeply greatfull to Mr. Dhulfiqar 

A. Abid ( MSc.) and Mrs. Sahar M. Shakir ( 

MSc. ) and Mr. Zaid M. Abdul-Khalik for 

running the UV , IR , and HPLC spectrum. 

References 

1. Tyler V.E. , Brady L.E., "Pharmacognosy" 

9 th edition, Lea and Febiger Philadelphia 

1988. 

2. Trease G.E. and Evans W.C., 

"Pharmacognosy" 15 th edition , WB 

Saunders Co. Ltd London 2002. 

3. Dey P.M. , Harborne J.B. , "Plant 

Biochemistry" Academic press 1997. 

4. Weinberg BA., Bealer BK., "The world of 

caffeine" Routledge, Newyork and 

London 2001.


5. Cordell G.A., "Introduction to alkaloids" 

John Wiley and Sons , Inc Canada 1981, 6. 

6. Jinno, D. , "Comperhensive Medical 

chemistry" Pergamon Press 1996. 

7. Eaton K., "Caffeine could be healpful" Las 

Vegas Review Journal , 2010, 23. 

8. Srivastava B.S., Kumar H.D., Singh H.N. , 

"The effect of caffeine and light on killing 

of the blue-green algae Anabaena doliolum 

by UV radiation" Archives of 

Microbiology 1971, 78(2), 139-144. 

9. Siderpoulos A.S., Shankel D.M., 

"Mechanism of caffeine enhancement of 

mutations induced by sub lethal UV 

dosages" J. Bacteriology 1968, 96(1), 198- 

204. 

10. Lytle C.D., "The effect of caffeine on the 

survival of UV-irradiated Herpes simplex 

Type 1 virus" J. General virology 1974, 24, 

381-383. 

11. Cremer C., Cremer T., and Gray J. W., 

"Introduction of chromosome damage by 

UV light and caffeine" Cytometry 1982, 

2(5), 287-290. 

12. The Merck index , 8 th edition Merck and 

Co. Inc. Rahway, NJ, USA 1996. 

13. Cave' A., "Pharmacognosy , 

Phytochemistry, medicinal plants" 3 rd 

53 

edition , Intercept Ltd, England 1995, 882- 

883. 

14. Fenske M., "caffeine determination in 

human saliva and urine by TLC andUV 

absorption densitometry" 

Chromatographia 2007, 65(3-4) , 233-238. 

15. Pavlik J.W., "The detection of caffeine in 

commercial products" J. of Chemical 

education 1973, 50(2), 134. 

16. Franciszek B., Sabina A., Urszula H., 

"Densitometric determination of caffeine in 

tea beverages after TLC e-separation" 

Chemia analityczna, 2006, 51(4), 603-611. 

17. Stahl, E., "Thin layer chromatography" 

Springer-Verlag Berlin. Heidlberg N.Y. 

1969. 

18. Pavlova V., Petrovsk S., "Simultaneous 

determination of amphetamine, 

methamphetamine, and caffeine in seized 

tablets and HPLC" Acta 

Chromatographica, 2007, 18, 157-164. 

19. Cook D., Regnier Z.R., "The infrared 

spectra of caffeine salts" Canadian Journal 

of Chemistry, 1967, 45, 2895-2897. 

20. Pedersen O., "Pharmaceutical chemical 

analysis : Methods for identification and 

limit tests" 2006, 86-88.

Iraqi J Pharm Sci, Vol.19(2) 2010 Goserlin in the management of fibroid 

Goserelin versus Norethisterone in the Management of 

Menorrhagia with Uterine Fibroid 

Faris A. Rasheed * ,1 , Jwan N. Sulaiman* and Yousif Abdul-Raheem 

* Departement of Obs/Gyn, Al-Kindy Medical College, University of Baghdad, Baghdad, Iraq. 

Abstract 

Menorrhagia is common in patients with uterine fibroids, if operation needs to be delayed for a 

particular reason, goserelin can be used safely to reduce bleeding and the size of the tumor.The 

objective is to compare between goserelin acetate and norethisterone on patients with menorrhagia and 

uterine fibroid. A randomized controlled study conducted in Elwiya maternity teaching hospital, 

Baghdad from the first of November 2007 to the end of April 2009. 90 patients from the consultant 

outpatient clinic with menorrhagia and fibroid, and their operations were delayed for medical reason 

were allocated in two groups, the first group, was given 3.2 mg goserelin acetate subcutaneously 

monthly for 3 months and the second group was given 5 mg norethisterone orally three times daily 

during the attack of bleeding and 5 mg once daily, cyclically if no bleeding for 3 months. The fibroid 

was measured in two dimensions, using convex real-time ultrasound before treatment and three months 

after treatment. Haemoglobin and the number of pads used were also reported before and after 

treatment, also the side effects in both groups and the need for operations.The size of fibroid in two 

dimensions measurement was reduced from 28.24 cm 2 ± 6.14 to 12.3 cm 2 ± 3.45 in the goserelin group 

(P=0.0001) versus 26.56 cm 2 ± 5.96 to 25.22 cm 2 ± 5.01 in the norethisterone group (P= 0.2589). The 

haemoglobin level was 9.28 gm/100ml ± 2.44 pre-treatment in the goserelin group and 11.2 gm/100ml 

± 1.88 post-treatment (P= 0.0001) versus 10.08 gm/100ml ± 2.86, and 10.24 gm/100ml ± 2.46 

respectively in the norethisterone group (P= 0.7798). The need for operation was decreased significantly 

in the goserelin group. Goserelin showed better patient response and reduction in the tumor size than 

norethisterone in treatment of patients with menorrhagia and uterine fibroids if operation is delayed for 

medical or other reasons. 

Key words: Goserelin, Nore thiste rone , Menorrhagia, Uterine Fibroid 


ﺺﻴﻠﻘﺗﻭ ﺔﺠﻟﺎﻌﻣ ﻲﻓ Norethisterone ( ﻥﻭﺮﻴﺘﺳ ﻲﺛﺍﺭﻮﻧ ) ءﺍﻭﺩ ﻦﻣ ﻞﻀﻓﺃ ﺞﺋﺎﺘﻧ ﺮﻬﻈﻳ ( Goserlin ) ﻦﻴﻟﺭﺯﻮﻛ ءﺍﻭﺩ ﻥﺇ 

ﻰﻟﺇ ﻱﺩﺆﺗ ﺔﻴﺒﻃ ﺏﺎﺒﺳﺃ ﻙﺎﻨﻫ ﻭﺃ ﺔﻳﺮﻬﺸﻟﺍ ﺓﺭﻭﺪﻟﺍ ءﺎﻨﺛﺃ ﺪﻳﺪﺷ ﻑﺰﻧ ﻦﻣ ﻦﻴﻧﺎﻌﻳ ﻲﺗﺍﻮﻠﻟﺍ ﺕﺎﻀﻳﺮﻤﻟﺍ ﺪﻨﻋ ﺔﺻﺎﺧ ﻭ ﻢﺣﺮﻟﺍ ﻲﻓ ﺔﻴﻔﻴﻠﻟﺍ ﺪﻘﻌﻟﺍ ﻢﺠﺣ 

ﺔـﺠﻟﺎﻌﻣ ﻲـﻓ ﻝﺎـﻌﻓ ﺮﻬـﺷﺃ 3 ﺓﺪـﻤﻟ ( Goserlin ) ( ﻦﻴﻟﺭﺯﻮـﻜﻟﺍ ) ءﺍﻭﺩ ءﺎـﻄﻋﺇ 

ﻥﺃ ﺪـﺟﻭﻭ . ًﺎـﻴﺣﺍﺮﺟ ﺔـﻴﻔﻴﻠﻟﺍ ﺪـﻘﻌﻟﺍ ﻊـﻓﺭ ﺔـﻴﻠﻤﻋ ﻞـﻴﺟﺄﺗ 

ﻊﻓﺮﻳﻭ ﻑﺰﻨﻟﺍ ﺓﺪﺷ ﻦﻣ ﻞﻠﻘﻳ ﻪﻧﺍ ﺪﺟﻭ ﺫﺇ ﻢﺣﺮﻟﺍ ﻰﻠﻋ ﺔﻴﻔﻴﻟ ﺪﻘﻋ ﺩﻮﺟﻭ ﺐﺒﺴﺑ ﺔﻳﺮﻬﺸﻟﺍ ﺓﺭﻭﺪﻟﺍ ءﺎﻨﺛﺃ ﺪﻳﺪﺷ ﻑﺰﻧ ﻦﻣ ﻦﻴﻧﺎﻌﻳ ﻲﺗﺍﻮﻠﻟﺍ ﺕﺎﻀﻳﺮﻤﻟﺍ 

. ﺔﻔﻴﻔﻃ ﺔﻴﺒﻧﺎﺟ ﺽﺍﺮﻋﺇ ﺩﻮﺟﻭ ﻊﻣ ﻲﺣﺍﺮﺠﻟﺍ ﻞﺧﺍﺪﺘﻟﺍ ﻰﻟﺇ ءﻮﺠﻠﻟﺍ ﻭﺃ ﻡﺩ ءﺎﻄﻋﺇ ﻰﻟﺇ ﺔﺟﺎﺤﻟﺍ ﻞﻠﻘﻳ ﺎﻤﻣ ﻡﺪﻟﺎﺑ ﻦﻴﺑﻮﻠﻛﻮﻤﻴﻬﻟﺍ ﺔﺒﺴﻧ ﻦﻣ 

Introduction 

Uterine fibroids are the most common 

tumor in the female reproductive system. They 

are estimated to occur in 25% of women of 

reproductive age. (1) In the USA, 30% of women 

will have had a hysterectomy by the age of 60 

years and 60% will be performed to treat 

(2) 

fibroids. Hysterectomy or myomectomy 

remain the most common types of treatment 

and it is associated with high level of 

satisfaction. Myomectomy is carried out when 

fertility is to be preserved, it can relief 

symptoms associated with myoma. Goserelin 

acetate, a Gonadotrophin releasing hormone 

(GnRH) agonist is a synthetic form of 

gonaderelin. It acts on the luteinizing hormone 

releasing hormone (LHRH) receptors in the 

pituitary gland, in the same way as natural 

gonadorelin. The available data seems to 

1Corresponding author E- mail : faris_54@yahoo.com 

54@yahoo.com 

Received : 19/4/2010 

Accepted : 18/8/2010 

54 

support the use of GnRH agonist treatment 

before surgery for uterine fibroids in selected 

circumstances. (3) Administration of GnRH 

agonist for only two or three months 

preoperatively in cases of uterine fibroid 

decreases the bleeding, mucus debris and 

diameter, limiting side effects and cost, (3) and 

increase the haematocrit value with no 

metabolic side effects or detectable bone 

demineralization . 

(4) Norethisterne is a 

synthetic progestin. It has several indications in 

gynaecology and primary care. At low dose ≤ 

1mg it can be used in contraception and 

hormone replacement therapy. At higher dose ≥ 

5mg it can be used in menorrhagia. (5) In our 

study we compare the effect of goserelin 

acetate versus norethisterone on patients with 

menorrhagia and uterine fibroid.



This study is a randomized controlled 

study conducted in Elwiya maternity teaching 

hospital, Baghdad from the 1 st of November 

2007 to the 30 th of April 2009. The patients 

enrolled in the study were 102 women, with 

menorrhagia and the presence of uterine 

fibroid(s). Patients with pervious myomectomy 

and those with known or suspected to have 

breast carcinoma were excluded from the 

study.All patients were not suitable for near 

surgery because of medical problem, long 

waiting lists or refusal of surgery by the patient. 

Uterine bleeding was considered abnormal 

according to the patients subjective evaluation 

in comparison with previous menstrual status. 

The degree of the bleeding was assessed by the 

number of the pads used and hemoglobin 

estimation before and after treatment. 52 

patients received monthly SC injection of 

Goserelin acetate 3.6 mg (Zoladex, 

AstraZeneca, UK ) for three months, the second 

group was 50 patients received 5 mg of 

norethisterone tablets (Primolut N, Schering, 

Germany) orally three times daily during the 

attack of bleeding and 5 mg daily if there is no 

bleeding to complete 21 days per cycle for 

three cycles. Twelve patients failed to complete 

the study, two from the goserelin group and ten 

from the norethisterone group, so the final 

number was 50 in the goserelin group and 40 in 

the norethisterone group.The measurement of 

the fibroid was done by taking two dimensions 

of the largest fibroid, including the biggest 

diameter using ultrasound with a convex 3.5 

MHz probe (Hunda, Japan) pretreatment and 

after three months. Pretreatment hemoglobin 

was checked, and then after three months. 

General investigations were carried out for both 

groups including complete blood picture, 

fasting blood sugar, blood urea, and liver 

function tests. Patient's acceptance and 

response were subjectively registered on a 

special questionnaire with any side effects 

occurred in that period.The results were 

statistically analyzed, using the Statistical 

Package for Social Sciences (SPSS) version 11. 

Descriptive statistical analyses (frequency 

distributions and percentages) were used, while 

inferential statistics limited to t test, for 

comparison of means, and chi-square test of 

proportions. P


difference reduction in number in the goserelin 

group, but not in the norethisterone group. 

Table 2 : The effect of goserelin (group 1) 

and norethisterone (group 2) 

Treatment P value 

Groups 

Before After 

Size o f 

fibroid (cm 2 ) 

Mean 

SD 

Hb level 

(mg/dl) 

Mean 

SD 

Number o f 

Pads changed 

per day 

Group I 

Group II 

Group I 

Group II 

Group I 

≤5 

6-7 

8-9 

≥10 

Group II 

≤5 

6-7 

8-9 

≥10 

5 

8 

23 

14 

4 

6 

21 

9 

28.24 

6.14 

26.56 

5.96 

9.28 

2.44 

10.08 

2.86 

12.3 

3.45 

25.22 

5.01 

11.2 

1.88 

10.24 

2.46 

29 

15 

6 

0 

6 

13 

18 

3 

0.0001 

0.2589 

0.0001 

0.7798 

0.000 

0.102 

Table-3 showed the side effects of both groups, 

there was no statistical significant difference 

between the two groups regarding all the side 

effects ( P= 0.119 ), but there was more 

menopausal symptoms in the goserelin group, 

15 versus 7 in the norethisterone group.Table-4 

showed the operations that were done after 

finishing the treatment up to about one year. 

There were more operations in the 

norethisterone group.Twenty five (50%) of 

patients in the goserelin group had no 

operations versus four (10%) in the 

norethisterone group. Seven had hysterectomy 

in the goserelin group and 12 in the 

norethisterone group and myomectomy in 

nineteen and twenty four respectively. All 

operations showed statistical significant 

reduction in the goserelin group. 

Table 3 : the side effects of Gose relin and the 

Norethisterone treated groups 

Side e ffect Goserelin Nore thisterone P 

group group value 

No. ( % ) No. ( % ) 

Menopausa 

l symptoms 

15 (30 ) 7 ( 17.5 ) 

Joint pain 13 (26) 5 ( 12.5 ) 

Skin 

allergy 

7 (14) 4 ( 10 ) 

Increase 

weight 

5 (10 ) 6 ( 15 ) 

acne 4 ( 8 ) 6 ( 15 ) 

No 

complaint 

6 (12 ) 12 ( 30 ) 

56 

total 50 40 

Table 4 : the ope rations in both groups 

Type of surgery Group 1 Group 2 P value 

Hysterectomy 7 12 

Myomectomy 18 24 0.000 

No operation 25 4 

Discussion 

The (GnRH) agonists are an effective 

medical approach for the management of both 

dysfunctional uterine bleeding (DUB) and 

uterine fibroids. However, their use is restricted 

to short courses due to its long effect on bone 

mass. (6) Norethisterone is a common treatment 

of menorrhagia in our clinical practice; it is 

cheap, always available and well tolerated by 

the patients. After the introduction of Goserelin 

in our clinical practice, we designed this study 

to compare both drugs in cases of menorrhagia 

with uterine fibroids. Regarding blood loss, the 

increase in haemoglobin is significant after 

Goserelin use, (7) (8) with about 1 and 1.5 

gm/100ml increase in both studies respectively. 

In our study the increase was about 2 gm/100 

ml in the Goserelin group, it was not significant 

in the norethisterone group. In assessing the 

blood loss we use the subjective method by 

patient observation in comparison with her 

previous menses and the objective methods by 

counting pads and hemoglobin estimation, all 

showed significant improvement in the 

Goserelin group. Ranke HR, found no 

significant difference in adding 

estradiol/norethisterone to goserelin in 

reduction of blood loss. (9) Pre operative 

goserelin has been shown to decrease blood 

transfusion during operation and increase the 

post operative hemoglobin. (10) Goserelin was 

found to decrease the size of uterine fibroids. 

(11-15) In our study we used two dimensions 

measurement of the fibroid by ultrasound, as it 

can be done by the usual ultrasound equipment 

available in gynecology clinics; the reduction in 

the area of the fibroid was from 28.24± 6.14 

cm 2 to 12.3±3.45 which represent more than 

50%. Bozzini, 2004 found in a randomized 

controlled trial that Goserelin use in monthly 

injection for 3 months reduce the size of fibroid 

by 43%. (16) Adding goserelin after uterine 

artery embolization was found not to increase 

in the reduction of the size of leiomyoma, (17) in 

the same study the reduction of size of fibroid 

in Goserelin group was 45%. In a study done 

by Lumsden, 1994, on 71 ladies scheduled for 

hysterectomy for fibroid, and were divided in 2 

groups, one was given Goserelin and the other 

placebo before operation and they found that 

the size of the fibroid is smaller in the


Goserelin group, also the haemoglobin level 

and the duration of the operation. (18) Goserelin 

was also found to increase pregnancy rates if 

given before hysteroscopic resection of fibroid 

in cases of sub fertility. (19) Many studies found 

that blood loss during operation for fibroid after 

Goserelin use was less than without it. (17)(20) 

Recent study showed that the use of triple 

tourniquets is associated with less blood loss 

than the use of pre-operative Goserelin in open 

myomectomy. (21) Goserelin was found also to 

shorten the operative time. (13) In our study 

these parameters were not measured.No 

significant medical problems were found after 

the short time use of Goserelin. (4) In our study 

there was no significant difference in the side 

effects of both groups (P=0.119), but more 

vaso-motor symptoms noted in the Goserelin 

group, 39% versus 17.3% in the n 

group.Regarding the need for operations, 25 of 

the Goserelin group ( 50% ) had operations, 7 

hysterectomy and 18 myomectomy, versus 36 

out of 40 in the norethsterone group, 12 

hysterectomy and 24 myomectomy, so the need 

for operation is decreased with Goserelin , 

mostly due to improvement of symptoms, and 

the desire of most of the women to preserve the 

uterus. Hysterectomy rates for leiomyoma 

decreased significantly from 2.13 per 1000 to 

1.91 (P < .0001), due to increase in uterine 

artery embolization and uterine ablation. (22) 

GnRH agonists shrink the uterus and fibroids, 

and this effect make it possible to change some 

of abdominal hysterectomies to vaginal 

hysterectomy, in one study, 76% of GnRH 

agonist-treated patients had vaginal 

hysterectomy versus 16% of non treated 

patients. (23) The use of GnRH agonists can 

make possible a conversion from abdominal 

hysterectomy to either vaginal hysterectomy or 

laparoscopic-assisted vaginal hysterectomy or 

laparoscopic supracervical hysterectomy. (23) In 

conclusion, Goserelin for three months is a 

reasonable choice of treatment for patients 

having menorrhagia with uterine fibroid, as it 

increases the hemoglobin concentration, 

decrease the need for operation, decrease blood 

transfusion during operation, and with limited 

side effects. 

References 

1. Buttram, V. and Reiter, R. Uterine 

leiomyomata: etiology, symptomatology 

and management. Fertil. Steril: 1981; 36, 

433–445.) 

2. AHRQ (2000) Management of uterine 

fibroids. Evidence report. Technology 

assessment: Number 34. Agency for 

Healthcare Research and Quality of Life. 

http://www.ahcpr.gov/ 

http://www.ahcpr.gov/.. 

57 

3. Crosignani PG, Vercellini P, Meschìa M, 

Oldani S, Bramante T GnRH agonists 

before surgery for uterine leiomyomas. 

Report Med 1996 ; 41(6):415-21.) 

4. Cagnacci A, Paoletti AM, Soldani R, 

Angiolucci M, Arangino S, Falqui A, Melis 

GB Role of goserelin-depot in the clinical 

management of uterine fibroids. Obstet 

Gynecol 1994; 21(4):263-5.) 

5. Irvine GA; Campbell-Brown MB; Lumsden 

MA; Heikkila A; Walker JJ; Cameron IT, 

Randomised comparative trial of the 

levonorgestrel intrauterine system and 

norethisterone for treatment of idiopathic 

menorrhagia. Br J Obstet Gynaecol 

1998;105(6):592-8. 

6. Thomas EJ, Add-back therapy for long-term 

use in dysfunctional uterine bleeding and 

uterine fibroids. Br J Obstet Gynaecol. 

1996; 103 Suppl 14:18-21 

7. Gerris J, Degueldre M, Peters AA, Romao 

F, Stjernquist M, al-Taher H 

The place of Zoladex in deferred surgery for 

uterine fibroids. Zoladex Myoma Study 

Group.] Horm Res 1996; 45(6):279-84. 

8. Stovall TG, Summit RL, Washburn SA, 

Ling FW Gonadotropin-releasing hormone 

agonist use before hysterectomy. Am J 

Obstet Gynecol 1994 ; 170(6):1744-8; 

discussion 1748-51. 

9. Franke HR, Snaaijer FF, Houben PW, van 

der Mooren MJ Treatment of dysfunctional 

uterine bleeding in the perimenopause: The 

effects of adding combined estradiol / 

norethisterone acetate therapy to goserelin 

acetate treatment: Gynecol Endocrinol 

2006 ; 22(12):692-7 

10. Lim SS, Sockalingam JK, Tan PC: 

Goserelin versus leuprolide before 

hysterectomy for uterine fibroids. Int J 

Gynaecol Obstet 2007 27. 

11. Moris,EP, RymerJ, RobinsonJ, FogelmanI 

Efficacy of tibolone as "add-back therapy" 

in conjunction with a gonadotropinreleasing 

hormone analogue in the treatment 

of uterine fibroids. Fertil Steril 2007 15. 

12. Russo P, Ciolli P, Atlante M, Carico E, 

Mancini R, Russo R, Vecchione A [Clinical 

use of leuprolide acetate depot in a group of 

gynecologic patients in the preoperative 

period; Minerva Ginecol 1998 ; 50(11):499- 

502. 

13. Tiufekchieva E, Nikolov A: 

Hysteroresection of submucous myomas 

after treatment with zoladex; Akush 

Ginekol (Sofiia) 2006; 45(1):19-24. 

14. Donnez J, Hervais Vivancos B, Kudela M, 

Audebert A, Jadoul P A randomized, 

placebo-controlled, dose-ranging trial 

comparing fulvestrant with goserelin in


premenopausal patients with uterine fibroids 

awaiting hysterectomy. Fertil Steril 2003 ; 

79(6):1380-9. 

15. Baytur YB, Ozbilgin K, Cilaker S, Lacin S, 

Kurtul O, Oruc S, Koyuncu FM A 

comparative study of the effect of raloxifene 

and gosereline on uterine leiomyoma 

volume changes and estrogen receptor, 

progesterone receptor, bcl-2 and p53 

expression immunohistochemically in 

premenopausal women. Eur J Obstet 

Gynecol Reprod Biol 2006 Sep 12 

16. BozziniN, MessinaML, BorsariR, 

HilarioSG, PinottiJA Comparative study of 

different dosages of goserelin in size 

reduction of myomatous uteri.J Am Assoc 

Gynecol Laparosc 2004 ;11(4): 462-3.I 

17. VilosGA,VilosAG, , Abu-RafeaB, PronG, 

KozakR, GarvinG, Administration of 

goserelin acetate after uterine artery 

embolization does not change the reduction 

rate and volume of uterine myomas. Fertil 

Steril 2006 ; 85(5):1478-83. 

18. Lumsden MA, West CP, Thomas E, Coutts 

J, Hillier H, Thomas N, Baird DT 

Treatment with the gonadotrophin releasing 

58 

hormone-agonist goserelin before 

hysterectomy for uterine fibroids. Br J 

Obstet Gynaecol 1994 ; 101(5):438-42. 

19. Narayan R, Rajat, Goswamy K Treatment 

of submucous fibroids, and outcome of 

assisted conception. J Am Assoc Gynecol 

Laparosc 1994 ;1(4 Pt 1):307-11. 

20. Crosignani PG, Vercellini P, Meschìa M, 

Oldani S, Bramante T GnRH agonists 

before surgery for uterine leiomyomas. ]J 

Reprod Med 1996 ; 41(6):415-21 

21. Al-Shabibi N, Chapman L, Madari S, 

Papadimitriou A, Papalampros P, Magos A 

Prospective randomised trial comparing 

gonadotrophin-releasing hormone analogues 

with triple tourniquets at open 

myomectomy. BJOG 2009 Feb 4. 

22. Jacobson GF, Shaber RE, Armstrong MA, 

Hung YY. Links Changes in rates of 

hysterectomy and uterine conserving 

procedures for treatment of uterine 

leiomyoma. . Am J Obstet Gynecol. 2007; 

196(6):601.e1-5; discussion 601.e5-6. 

23. Carter JE. Alternatives to total abdominal 

hysterectomy. JSLS. 1997; 1:259-262.

Iraqi J Pharm Sci, Vol.19(2) 2010 

Anti-bacterial Properties of Melatonin against Mycobacterium 

Tuberculosis in Vitro 

Thamer M. Jasim*, Mustafa G.Alabbassi** , Suhad F. Hatem Almuqdadi* 

and Jinan K. Kamel*** 

* Department of Microbiology and Biotechnology,Al-Mustansyria,University,College of Pharmacy, 

Baghdad, Iraq. 

** Department of Pharmacology and Therapeutics, Al-Mustansyria University,College of Pharmacy, 

Baghdad, Iraq. 

*** National Reference Laboratory 

Abstract 

57 isolates of Mycobacterium tuberculosis and Mycobacterium bovis were identified; they were 

isolated from different clinical sources which included sputum, bronchial wash, abscess, pleural fluid, 

gastric fluid, eye fluid, and CSF, also urine and ear swab. This investigation was carried out on 198 

patient attended National Reference Laboratory for T.B during September 2009. Also the study 

declared that the ratio of separation of this bacterium from male was (67.6%) and it’s higher than the 

ratio of separation this bacterium from females which was (32.3%). The susceptibility of 

Mycobacterium tuberculosis to melatonin was evaluated. Many concentrations of melatonin were 

prepared to investigate it as antibacterial drug against multidrug resistant Mycobacterium tuberculosis 

and Mycobacterium bovis. Suspension bacteria (10 -1 , 10 -3 and 10 -5 ) were cultured on Lowenstein- 

Jensen media (LJ) contains melatonin, while control media without this drug. Six isolates were chosen 

according to their susceptibility patterns; they were resisting to Rifampicin, Streptomycin, Isonicotinic 

–hydrazide and sensitive to Ethambutol. In conclusion, these in vitro studies clearly demonstrate antibacterial 

effects of melatonin. Among possible mechanisms, it is concluded that melatonin showed 

antibacterial effects against multidrug resistant T.B by reducing intracellular substrates. Identifying the 

mode of action could be of great help in developing and researching new anti-bacterial drugs. 

Key words: Antibacterial, Melatonin, Mycobacterium tuberculosis. 


ﻲﺘﻟﺍﻭ ﺔﻔﻠﺘﺨﻤﻟﺍ ﺔﻳﺮﻳﺮﺴﻟﺍ ﺭﺩﺎﺼﻤﻟﺍ ﻦﻣ ﺔﻳﺮﻘﺒﻟﺍ ﺔﻴﻧﺭﺪﻟﺍ ﺕﺎﻴﺼﻌﻟﺍﻭ ﺔﻳﺮﻄﻔﻟﺍ ﺔﻴﻧﺭﺪﻟﺍ ﺕﺎﻴﺼﻌﻟﺍ ﻦﻣ ﺔﺒﺟﻮﻣ ﺔﻟﺰﻋ 57 ﺺﻴﺨﺸ ﺗ ﻢﺗ 

ﻰﻠﻋ ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﺕﺬﻔﻧ . ﻥﺫﻷﺍ ﺔﺤﺴﻣﻭ ﺭﺍﺭﺩﻻﺍ ﻚﻠﻟﺬﻛ ﻲﻋﺎﺨﻨﻟﺍﻭ ﻦﻴﻌﻟﺍﻭ ﺓﺪﻌﻤﻟﺍﻭ ﺐﻨﺠﻟﺍ ﻞﺋﺎﺳ ٬ﺝﺍﺮﺨﻟﺍ ٬ﻲﺒﺼﻘﻟﺍ ﻞﺴﻐﻟﺍ ٬ ﻊﺸﻘﻟﺍ ﺖﻨﻤﻀﺗ 

ﻲﻫ (% 67.6) 

ﺭﻮﻛﺬﻟﺍ 

ﺔﺑﺎﺻﺍ ﺔﺒﺴﻧ ﻥﺍ ﺔﺳﺍﺭﺪﻟﺍ ﺖﺤﺿﻭﺃ 

. 2009 ﻝﻮﻠﻳﺍ ﺮﻬﺷ ﻝﻼﺧ ﻥﺭﺪﺘﻠﻟ 

ﻲﻌﺟﺮﻤﻟﺍ ﻲﻨﻃﻮﻟﺍ ﺮﺒﺘﺨﻤﻟﺍ 

ﻊﺟﺍﺭ ﺾﻳﺮﻣ 198 

ﻦﻴﻧﻮﺗﻼﻴﻤﻟﺍ ﻦﻣ ﺰﻴﻛﺍﺮﺗ ﺓﺪﻋ ﻡﺍﺪﺨﺘﺳﺍ ﻢﺗ . ﻦﻴﻧﻮﺗﻼﻴﻤﻟﺍ ﺭﺎﻘﻌﻟ ﺔﻴﻧﺭﺪﺘﻟﺍ ﺕﺎﻴﺼﻌﻟﺍ ﺔﻴﺳﺎﺴﺣ ﻢﻴﻴﻘﺗ ﻢﺗ (% 32.3) 

ﺙﺎﻧﻷﺍ ﺔﺑﺎﺻﺍ ﺔﺒﺴﻧ ﻦﻣ ﻰﻠﻋﺍ 

ﻒﻴﻓﺎﺨﺗ ﺓﺪﻋ ﺖﻋﺭﺯ 

. ﺕﺍﺩﺎﻀﻤﻠﻟ ﺓﺩﺪﻌﺘﻤﻟﺍ ﺔﻣﻭﺎﻘﻤﻟﺍ ﺕﺍﺫ M. bovis ﻭ M. Tuberculosis ﻥﺭﺪﺘﻟﺍ ﺕﺎﻴﺼﻋ ﺪﺿ ﺎﻳﺮﺘﻜﺒﻠ ﻟ ﺩﺎﻀﻤﻛ 

( 10 ﻱﺮﻴﺘﻜﺒﻟﺍ ﻖﻟﺎﻌﻠﻟ 

-5 ,10 -3 ,10 -1 ﺍﺬﻫ ﻱﻮﺤﻳﻻ ﺓﺮﻄﻴﺴﻟﺍ ﻂﺳﻭ ﺎﻤﻨﻴﺑ ﻦﻴﻧﻮﺗﻼﻴﻤ ﻟﺍ ﺭﺎﻘﻋ ﻰﻠﻋ ﻱﻮﺘﺤﻳ ﻱﺬﻟﺍﻭ (LJ) ﻂﺳﻭ ﻰﻠﻋ 

) 

٬ﻦﻴﺴﻳﺎﻣﻮﺘﺑﺮﺘﺴﻟﺍ ٬ﻦﻴﺴﺒﻣﺎﻔﻳﺮﻠﻟ ﺔﻣﻭﺎﻘﻣ ﺕﻻﺰﻌﻟﺍ ﻩﺬﻫ ﺖﻧﺎﻛ ﺚﻴﺣ ﺔﻴﺴﺴﺤﺘﻟﺍ ﻁﺎﻤﻧﻷﺍ ﻰﻠﻋ ﺩﺎﻤﺘﻋﻷﺎﺑ ﺕﻻﺰﻋ ﺔﺘﺳ ﺕﺮﻴﺘﺧﺍ . ﺭﺎﻘﻌﻟﺍ 

ﺎﻳﺮﺘﻜﺒﻟﺍ ﻰﻠﻋ ﻦﻴﻧﻮﺗﻼﻴﻤﻠﻟ ﻲﻄﻴﺒﺜﺘﻟﺍﺮﻴﺛﺎﺘﻟﺍ 

ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﻦﻣ ﺞﺘﻨﺘﺴﻧ . ﻝﻮﺘﻴﺒﻣﺎﺜﻳﻼﻟ ﺔﺳﺎﺴﺣﻭ ٬INHﺪﻳﺍﺯﺍﺭﺪﻳﺎﻫ 

ﻚﻨﺗﻮﻜﻴﻧﻭﺰﻳﺍ 

. ﺪﻳﺪﺟ ﻱﺮﻴﺘﻜﺑ ﺩﺎﻀﻤﻟ ﻕﺎﻓﻵﺍ 

ﺢﺘﻔﻳ ﻚﻠﻟﺬﺑﻭ ﺎﻳﺮﺘﻜﺒﻠﻟ ﺔﻴﻠﺨﻟﺍ ﻞﺧﺍﺩ ﻰﻠﻋ ﻩﺮﻴﺛﺄﺗ 

ﺔﻄﺳﺍﻮﺑ ﺔﻠﻤﺘﺤﻤﻟﺍ ﺔﻴﻜﻴﻧﺎﻜﻴﻤﻟﺍﻭ ﺎﻳﺮﺒﺘﺨﻣ 

Introduction 

Although tuberculosis (TB) is a 

preventable and treatable disease, it causes 3 

million deaths annually. The current situation 

of TB is unique, mainly due to two aspects: the 

association between TB and human 

immunodeficiency virus (HIV) infection, and 

the global spread of virulent strains resistant to 

key antituberculous drugs. There is a direct 

association between HIV infection and the 

reactivation of latent TB or the progression of 

TB following newly acquired infection (1) . 

Melatonin originally identified as an effector 

for circadian rhythms, is now known to be a 

hormone involved in a vast range of 

homeostasis maintenance activities, for 

example seasonal timing, sexual development, 

the antioxidant defense system and immune 

1Corresponding author E- mail : 

Received : 10/4/2010 

Accepted : 18/9/2010 

59 

response (2) . Melatonin is synthesized from 

tryptophan within the serotonin pathway 

mainly in the pineal gland, and in a number of 

extrapineal organs such as retina, lens, bone 

marrow, intestine, skin and so on. To date, 

three mammalian melatonin receptors, Gprotein 

coupled receptors MT1 and MT2, and a 

quinone reductase family receptor, MT3, have 

been identified (3) . Melatonin is a highly 

studied endogenous molecule. This indolamine 

has a variety of beneficial effects within cells 

and organisms, including cell cycle regulation 

(4) . Although there are a plethora of studies on 

melatonin, only a few of them relate to it in 

vitro antimicrobial activities (5–7) on to its 

effects in infectious diseases (8-10) .


New in vitro antimicrobial studies using 

melatonin suggested that it has limited 

antimicrobial properties (11, 12) while one study 

found that melatonin inhibited Candida 

albicans at a concentration of 300 µg/ml (12) .In 

contrast to the in vitro studies, virtually all in 

vivo studies performed with melatonin in 

infectious disease models documented it as a 

successful therapy (9, 13) . Melatonin is a highly 

versatile molecule. One example is its ability 

to limit the growth of a variety of tumor types. 

One of several proposed mechanisms to 

explain melatonin’s inhibitory actions on 

cancer growth is its ability to curtail the uptake 

of growth factors which promote cell 

proliferation 

(14) . Linoleic acid (LA), an 

essential omega-6 polyunsaturated fatty acid is 

a growth factor for a number of tumor types. 

Via an action on the cell membrane, melatonin 

prevents the uptake of this fatty acid by cancer 

cells which reduces the activation of genes that 

promote cell proliferation (15) . Similar actions 

of melatonin on the bacterial wall thus may 

restrict the survivability of bacteria. 

Additionally, melatonin has a high metal 

binding capacity. Melatonin binds iron (III), 

copper and zinc thereby reducing their 

cytoplasmic availability. Bacteria are strongly 

dependent on free metals, in particular, free 

iron for growth (16) . Clearly, there are several 

potential mechanisms that may explain the 

possible antibacterial efficacy of melatonin. In 

the current study we tested the antimicrobial 

effects of melatonin against resistant strains of 

Mycobacterium tuberculosis. 


Samples 

A total of 198 clinical samples from 

National Reference Laboratory for T.B, were 

included in this study. They were collected 

during November 2009. Identification of these 

isolates as Mycobacterium tuberculosis and 

Mycobacterium bovis were based on 

biochemical properties (17) . The most samples 

were sputum; they are transfer to the 

laboratory in sterile container. Three samples 

from each patient were taken to diagnose 

tuberculosis. Susceptibility test was done to 13 

isolates from 57 positive samples to 

tuberculosis according to routine work of 

national reference laboratory for tuberculosis. 

All samples treat with sodium hydroxide and 

hydrochloric acid to remove all 

microorganisms and epithelial cells (Petroffs 

method). Zeihl-Neelsen stain was done for all 

specimens. 

Culture the Specimens 

The specimens was cultured on 

Lowenstein-Jensen media (LJ) pouring in 

60 

screw –capped tubes in final volume 6ml, these 

tubes put in oven at 80-85 C for 45 min. The 

media left for 24 hr to be insure that there is no 

contamination. The treated specimens will 

culturing by using Pasture pipette, (0.4-0.2) ml 

from inoculme of specimen must be transferred 

to LJ media and let the culture for 72 hr at 37 

C in incubators( in slope position), then for 50 

days at 37 C ( in vertical position). 

Susceptibility Test 

Susceptibility test was done by using the 

proportional method (18) , four antibiotic 

solutions were used; Rifampicin 40 µg/ml, 

Streptomycin 4 µg/ml, Ethambutol 2 µg/ml, 

Isonicotinic hydrazide 0.2 µg/ml. 

Mycobacterium tuberculosis suspension was 

prepared in many dilutions 10 -1 -10 -5, the 

primary dilution adjusted with MacFerland 

solution(3×10 8 ) CFU/ml. 100 µl from 10 -1 , 10 - 

3 and 10 -5 dilution was cultured on media (LJ) 

contain antibiotics and without antibiotics as 

control. After 6-8 weeks the result must be 

read, if the growth on media contains 

antibiotics more than control media this means 

resistant bacteria. While the growth consider 

sensitive when the growth less than control 

media, for this test we chose 4 isolates from 

Mycobacterium tuberculosis and 2 isolates 

from Mycobacterium bovis, according to their 

typing (catalase, nitrase test, niacin test and 

colony form). 

Melatonin drug 

In this study, many concentration of 

melatonin dissolved in 96% ethanol (0.4, 0.2, 

0.1, 0.05) mg/ml were prepared to investigate 

it as antibacterial drug against multi-drug 

resistant Mycobacterium tuberculosis and 

Mycobacterium bovis.Suspension bacteria (10 - 

1 , 10 -3 and 10 -5 ) were cultured on LJ media 

contains melatonin, while control media 

without this drug. Six isolates were chosen 

according to their susceptibility patterns, they 

were resist to Rifampicin, Streptomycin, 

Isonicotinic –hydrazide and sensitive to 

Ethambutol (19) . 

Results 

Table -1 showed us the number of 

Mycobacterium tuberculosis isolated from 

many sources. It was found that 57 samples 

were positive samples. Table-2 shows number 

and percentage of infection with M. 

tuberculosis and M. bovis according to the sex, 

it was found that 134(67.6%) of patients were 

males and 64(32.3%) were females with 

present of statistical significant. Table-3 

shows the percentage of infected with M. 

tuberculosis and M.bovis according to the age. 

The higher percentage rate (46.2%) was found 

in the group of (30-39 year) while the lowest


percentage rate (6.7%) was found in the group 

(70≥ year) with present of statistical 

differences. Table-3 shows the number and 

percentage of infection with Mycobacterium 

SPP. According to the sex, it was found that 

134 (67.6%) of patients were males and 

64(32.3%) were females with present of 

statistical significant differences. Table-4 

shows the effect of many concentration of 

melatonin on M. tuberculosis and M.bovis 

isolates. It was found that M. tuberculosis and 

M.bovis sensitized to the concentrations 0.05, 

0.1, 0.2 and 0.4mg/ml. Table-5 shows the 

susceptibility patterns of many dilution of M. 

tuberculosis and M. bovis to different 

concentration of melatonin. It was found that 

dilution of bacteria 10 -1 , 10 -3 and 10 -5 were 

sensitized to all concentrations of melatonin 

that were used. Figure -1 shows the growth of 

M. tuberculosis and M bovis. Resistant to 

Rifampicin, streptomycin and INH and were 

sensitize to Ethambutol.and Melatonin. 

Table 1 : Number of M.tuberculosis and 

M.bovis isolated from many sources 

Isolates source 

Numbe r of 

Examine d 

Samples 

Numbe r 

Of Positive 

Sample 

No. (%) 

Sputum 145 55 96.5 

Bronchial wash 19 1 1.8 

Abscess 5 1 1.8 

Pleural Fluid 13 - - 

Gastric Fluid 3 - - 

Eye Fluid 2 - - 

Knee Fluid 1 - - 

C.S.F 1 - - 

Ear Swabs 1 - - 

Urine 8 - - 

Total 198 57 

Table 2 : Number and perce ntage of 

infe ction with M. tuberculosis and M. bovis 

according to the sex 

Sex 

of 

Patients 

Examined 

samples 

Number 

of positive 

M. 

tuberculosis 

iso lates 

Number 

of positive 

M. bovis 

isolates 

No. % No. % No. % 

Male 134 67.7 23 65.7 14 63.6 

female 64 32.3 12 34.3 8 36.4 

Total 198 35 22 

61 

Table 3 : Percentage of infected with M. 

tuberculosis and M.bovis according to age . 

Age 

Groups 

Numbe r of 

Examined 

Samples 

Number of Positive 

Sample 

No. (%) 

10< year 6 0 0 

10-19 year 16 6 *37.5 % 

20-29 year 36 14 38.9 % 

30-39 year 39 18 46.2 % 

40-49 year 29 6 37.5 % 

50-59 year 41 8 19.5 % 

60-69 year 16 4 25 % 

70≥ year 15 1 **6.7 % 

Total 198 57 

** Statistically significant difference 

Table 4 : Effect of many conce ntration of 

melatonin on Mycobacterium SPP. Isolates 

Code Me latonin concentration (mg/ml) 

of 

isolates 

(0.4) (0.2) (0.1) (0.05) Control 

S S S S R 

L2 S S S S R 

L3 S S S S R 

L4 S S S S R 

L5 * 

S S S S R 

L6 * 

S S S S R 

L =isolate, S =Sensitive, R=Resist, *= M. bovis 

Table 5 : Susceptibility patterns of many 

dilutions M.tuberculosis and M.bovis to 

many concentration of Melatonin 

Dilution 

of 

Bacteria 

3×10 7 

CFU/ml 

(10 -1 ) 

3×10 5 

CFU/ml 

(10 -3 ) 

3×10 3 

CFU/ml 

(10 -5 ) 

Melatonin concentration 

mg/ml 

(0.4) (0.2) (0.1) (0.05) Contro l 

S S S S R 

S S S S R 

S S S S R


Figure 1 : Growth of M.tube rculosis resists 

to Rifampicin,Stre ptomycin, and INH and 

was sensitized to Ethambutol and Me latonin 

on a Lowesten agar slant (from the left to 

the right). 

Discussion 

The results of the present study indicate 

that melatonin has in vitro antimicrobial 

activity against strains of antibiotic-resistant 

mycobacterium tuberculosis. As melatonin is 

weakly soluble in water, investigators 

generally use ethanol to dissolve the 

indolamine. The antibacterial effects of 

melatonin could be a result of the metal 

binding capacity of the indolamine. Normally, 

tissue fluids contain unsaturated iron-binding 

proteins including transferrin in plasma and 

lymph and lactoferrin in other secretions such 

as milk or mucous (20, 21) . These proteins ensure 

that the concentration of free iron in these 

fluids is virtually zero. This is essential for the 

normal bactericidal and bacteriostatic effects 

of plasma and extracellular fluids. If iron 

becomes freely available, the antibacterial 

effects of these fluids are lost. This can lead to 

rapid extracellular bacterial growth and a 

major increase in bacterial virulance. 

Pathogenic bacteria have also ways of 

extracting essential iron from the low iron 

environment in vivo via siderophores such as 

enterochelin, which can remove iron from 

unsaturated transferrin or lactoferrin (22) . An 

intriguing aspect of this issue is that, because 

iron is absolutely essential for bacteria, it could 

make the development of bacterial resistance 

very difficult for any organism deprived of 

iron. Of the concern being expressed over the 

increasing resistance to antibiotics now being 

encountered, particularly within hospitals, 

studies on the development of novel drugs 

against both iron transport and/or intrabacterial 

free iron availability should be undertaken. 

Melatonin reportedly has a high metal binding 

capacity including iron. Limson et al. (23) 

62 

observed that melatonin and its precursors 

exhibited the ability to bind metals in situ. 

Gulcin et al. (24) also noted that melatonin is an 

effective metal chelating agent. This feature of 

melatonin has been typically thought to be 

related to the antioxitant properties of the 

indole by making transition metals unavailable 

for the Fenton reaction. However, in case of 

bacterial growth, an agent which binds free 

iron in the cytoplasm has great importance. As 

melatonin easily passes all biological barriers 

including bacterial cell wall, it may bind free 

iron in the cytoplasm and restrict bacterial 

growth via this mechanism. In the present 

study, melatonin was tested against resistant 

mycobacterium tuberculosis. In gram-negative 

bacteria, the cell envelope is composed largely 

of protein glycopeptide, lipopolysaccharide, 

and also, substantial amounts of lipid (25) . 

Melatonin has been shown to limit the uptake 

of LA and total fatty acids by human breast 

cancer cells. This feature may also work 

against an extremely fast dividing prokaryote. 

Konar et al. (7) reported that melatonin, at the 

concentration of 1000 µg/ml, significantly 

reduced the lipid level of Sacchoramyces 

cerevisae. Furthermore in the same study, 

melatonin, at concentration of 300 µg /ml, was 

shown to be one of the most effective agents in 

reducing lipid levels of Candida albicans. In 

organisms, melatonin can be administered via 

any of several routes, e.g. orally, sublingually, 

etc., and it is available either as an over-thecounter 

supplement or as a prescription drug 

(depending on the country). The molecule has 

a long shelf-life and is inexpensive relative to 

conventional drugs used to treat the variety of 

bacterial infections.Melatonin is also very safe 

with few side effects and a very wide safety 

margin. The current in vitro studies should be 

expanded to investigate the efficacy of 

melatonin as an in vivo antibiotic. It has been 

previously shown that melatonin is beneficial 

in newborn humans suffering fro m septicemia 

(26) . Those findings coupled with the data 

reported here suggest further investigations of 

the role of melatonin in reducing bacterial 

growth. 

References 

1. Abate G, Hoffner S, Stegard V, Mqrner H. 

Characterization of Isoniazide-Resistant 

Starins of Mycobacterium tuberculosis on 

the basis of Phenotypic Properties and 

Mutations in KatG. Eur J Clin Microbiol 

Infect Dis , 2001; 20: 239-333. 

2. Mohd. A, Yoshinao A, Hajime F, Tomoyuki 

M et al. Serotonin and melatonin, 

neurohormones for homeostasis, as novel


inhibitors of infections by the intracellular 

parasite Chlamydia. Journal of 

Antimicrobial Chemotherapy,2005;10:1-8. 

3. Chan AS, Lai FP, Lo RK et al. Melatonin 

MT1 and MT2 receptors stimulate c-Jun 

N-terminal kinase via pertussis toxinsensitive 

and insensitive G proteins. Cell 

Signal, 2002; 14: 249–57. 

4. Reiter RJ, Gultekin F, Flores LJ et al. 

Melatonin: potential utility for improving 

public health. Kor Hek, 2006; 5:131–158. 

5. Wang HX, Liu F, NG TB. Examination of 

pineal indoles and 6-methoxy-2- 

benzoxazolinone for antioxidant and 

antimicrobial effects. Comp Biochem 

Physiol C Toxicol Pharmacol, 2001; 

130:379–388. 

6. Ozturk AI, Yilmaz O, Kirbag S, Arslan M. 

Antimicrobial and biological effects of 

ipemphos and amphos on bacterial and 

yeast strains. Cell Biochem Funct, 2000 ; 

18 : 117–126. 

7. Robertson GT, Doyle TB, Du Q, Duncan 

L, Mdluli KE, Lynch AS: A novel indole 

compound that inhibits Pseudomonas 

aeruginosa growth by targeting MreB is a 

substrate for MexAB-OprM. J Bacteriol, 

2007; 189: 6870–6881. 

8. Grandgirard D, Leib SL. Strategies to 

prevent neuronal damage in paediatric 

bacterial meningitis. Curr Opin Pediatr, 

2006; 18:112–118. 

9. Valero N, Espina LM, Mosquera J. 

Melatonin decreases nitric oxide 

production , inducible nitric oxide 

synthase expression and lipid peroxidation 

induced by Venezuelan encephalitis 

equine virus in neuroblastoma cell 

cultures.Neurochem Res , 2006 ; 31: 925– 

932. 

10. Valero N, Marinaespina L, Bonilla E, 

Mosquera J. Melatonin decreases nitric 

oxide production and lipid peroxidation 

and increases interleukin-1 beta in the 

brain of mice infected by the Venezuelan 

equine encephalomyelitis virus. J Pineal 

Res, 2007; 42:107–112. 

11. Wang HX, Liu F, NG TB. Examination of 

pineal indoles and 6-methoxy-2- 

benzoxazolinone for antioxidant and 

antimicrobial effects. Comp Biochem 

Physiol C Toxicol Pharmacol, 2001; 

130:379–388. 

12. Konar VV, Yilmaz O, Ozturk AI et al. 

Antimicrobial and biological effects of 

bomphos and phomphos on bacterial and 

yeast cells. Bio-Organic Chemistry, 2000; 

28:214–225. 

13. Reynolds FD, Dauchy R, Blask D et al. 

The pineal gland hormone melatonin 

63 

improves survival in a rat model of sepsis/ 

shock induced by zymosan A. Surgery 

2003; 134:474–479. 

14. Reiter RJ. Mechanisms of cancer 

inhibition by melatonin. J Pineal Res, 

2004; 37:213–214. 

15. Blask DE, Dauchy RT, Sauer LA. Putting 

cancer to sleep at night: the 

neuroendocrine / circadian melatonin 

signal. Endocrine, 2005; 27:179–188. 

16. Ward CG, Bullen JJ, Rogers HJ. Iron and 

infection: new developments and their 

implications. J Trauma, 1996; 41:356– 

364. 

17. Vestal AL. In: Procedure for the isolation 

and identification of mycobacteria. US 

Department of Health, Education and 

Welfare Pub no. (CDC) 77 - 8230. 

Atlanta, Georgia: Centers for Disease 

Control and Prevention; 1977. p. 15-90. 

18. Guidelines for surveillance of drug 

resistance in TB 1997, WHO, IUATLD. 

19. Canetti G, Fox W, Khomenko A, Mahler 

HT, Menon NK,Mitchison DA, et al. 

Advances in techniques of testing 

mycobacterial drug sensitivity and the use 

of sensitivity tests in tuberculosis control 

programmes. Bull World Health Organ, 

1969; 41 : 21-43. 

20. Omer Faruk, RecaiOgur1, Ahmet 

Korkmaz, Abdullah Kilic,Russel J.Reiter . 

Melatonin as an antibiotic: new insights 

into the actions of this ubiquitous 

molecule. J.Pineal Res, 2008; 44:222–226. 

21. Bullen JJ, Rogers HJ, Spalding PB, Ward 

CG. Iron and infection: the heart of the 

matter. FEMS Immunol Med Microbiol, 

2005; 43:325–330. 

22. Bullen JJ, Rogers HJ, Spalding PB, Ward 

CG. Natural resistance, iron and infection: 

a challenge for clinical medicine. J Med 

Microbiol 2006; 55:251–258. 

23. Limson J, Nyokong T, Daya S. The 

interaction of melatonin and its precursors 

with aluminium, cadmium, copper, iron, 

lead, and zinc: an adsorptive voltammetric 

study. J Pineal Res, 1998; 24:15–21. 

24. Gulcin I,Buyukokuroglu ME, Kufrevioglu 

OI.Metal chelating and hydrogen peroxide 

scavenging effects of melatonin. J Pineal 

Res 2003; 34:278–281. 

25. Hebeler BH, Chatterjee AN, Young FE. 

Regulation of the bacterial cell wall: effect 

of antibiotics on lipid biosynthesis. 

Antimicrob Agents Chemother 1973; 

4:346–353. 

26. Gitto E, Karbownik M, Reiter RJ et al. 

Effects of melatonin treatment in septic 

newborns. Pediatr Res, 2001; 50:756–760.

Iraqi J Pharm Sci, Vol.19(2) 2010 Tinidazole bioadhesive vaginal gels 

In vitro Evaluation of Tinidazole Bioadhesive Vaginal Gels 

, 1 

Zainab T.Salih* 

*Department of Pharmaceutics, College of Pharmacy, University of Baghdad,Baghdad, Iraq. 

Abstract 

Many trials were made to prepare Tinidazole 2% as bioadhesive vaginal gels using different gel 

bases including hydroxypropyl methyl cellulose (3 and 4% w/w), methylcellulose (3 and 4%w/w) and 

carboxymethylcellulose (2 and 3% w/w) .Swelling index of the polymers,pH , viscosity , bioadhesive 

force , and in-vitro drug release to the simulating vaginal fluid (S.V.F.) were investigated for all the 

prepared bioadhesive gels . The mechanism of drug release from the gel bases was also investigated. 

The results revealed that C MC 3% gave the highest viscosity and bioadhesive strength with the lowest 

release rate while lowest viscosity and bioadhesive force was obtained with HPMC gel base with the 

highest release rate. The mechanism of drug release was affected by the type of gel base. Fickian 

diffusion was obtained with all gel bases. 

Key words: Tinidaz ole, bioadhesive polymers, gels 


ﻞﻴﺜﻣ ﻞﻴﺑﻭﺮﺑ ﻲﺴﻛﻭﺭﺪﻴﻫ ﻞﻤﺸﺗ ﺔﻔﻠﺘﺨﻣ ﺔﻴﻣﻼﻫ ﺪﻋﺍﻮﻗ ﻡﺍﺪﺨﺘﺳﺎﺑ ﻕﺎﺼﺘﻟﻻﺍ ﻲﻃﺎﺨﻣ ﻲﻠﺒﻬﻣ ﻡﻼﻫ ﻞﻜﺷ ﻰﻠﻋ ﺮﻀﺣ % 2 ﻝﻭﺯﺍﺪﻨﻴﺗ 

ﺓﻮﻗﻭ ﺔﺟﻭﺰﻠﻟﺍﻭ ﻲﻨﻴﺟﻭﺭﺪﻴﻬﻟﺍ ﺱﻻﺍ ﺱﺎﻴﻗ ﻢﺗ ﺎﻤﻛ 

. ﺎﻬﻟ ﺥﺎﻔﺘﻧﻻﺍ ﺔﺒﺴﻧ ﺱﺎﻴﻗ ﻢﺗ ﻲﺘﻟﺍﻭﺯﻮﻠﻴﻠﻴﺳ ﻞﻴﺜﻣ ﻲﺴﻛﻮﺑﺭﺎﻜﻟﺍﻭ ﺯﻮﻠﻴﻠﻴﺳ ﻞﻴﺜﻤﻟﺍ, 

ﺯﻮﻠﻴﻠﻴﺳ 

ﺕﺮﻬﻇﺍ ﺪﻗﻭ. 

4.2 ﻲﻨﻴﺟﻭﺭﺪﻴﻫ ﺱﺎﺑ ﻲﻠﺒﻬﻤﻟﺍ ﻂﻴﺤﻤﻠﻟ ﻪﺑﺎﺸﻤﻟﺍ ﻂﺳﻮﻟﺍ ﻲﻓ ﺓﺮﻀﺤﻤﻟﺍ ﺔﻴﻣﻼﻬﻟ ﺍ ﺐﻴﻛﺍﺮﺘﻟﺍ ﻦﻣ ءﺍﻭﺪﻟﺍ ﺭﺮﺤﺗﻭ ﻕﺎﺼﺘﻟﻻﺍ 

ﻕﺎﺼﺘﻟﺍ ﻰﻠﻋﺍﻭ ﺔﺟﻭﺰﻟ ﻰﻠﻋﺍ ﻰﻄﻋﺍ ﺯﻮﻠﻴﻠﻴﺳ 

ﻞﻴﺜﻣ ﻲﺴﻛﻮﺑﺭﺎﻛ % 3 ﻥﺃﻭ, 

ﻡﺪﺨﺘﺴﻤﻟﺍ ﺮﻤﻴﻟﻮﺒﻟﺍ ﺰﻴﻛﺮﺗﻭ ﻉﻮﻨﺑ ﺮﺛﺎﺘﻳ ءﺍﻭﺪﻟﺍ ﺭﺮﺤﺗ ﻥﺍ ﺞﺋﺎﺘﻨﻟﺍ 

ﻲﻓ ﺥﺎﻔﺘﻧﺍ ﺔﺒﺴﻧ ﻯﺍ ﻲﻄﻌﻳ ﻢﻟﻭ ءﺍﻭﺪﻠﻟ ﺭﺮﺤﺗ ﻰﻠﻋﺍ ﻰﻄﻋﺍ ﺪﻘﻓ ﺯﻮﻠﻴﻠﻴﺳ ﻞﻴﺜﻣ ﻞﻴﺑﻭﺮﺑ ﻲﺴﻛﻭﺭﺪﻳﺎﻬﻟﺍ ﺎﻣﺍ . ﺭﺮﺤﺗ ﺎﻄﺑﺍﻭ ﺥﺎﻔﺘﻧﺍ ﺔﺒﺴﻧ ﻰﻠﻋﺍﻭ 

Introduction 

The vaginal route of administration has 

many advantages compared to other routes (1). 

The vaginal epithelium is permeable to a wide 

range of drugs like hormones, peptides, 

proteins and antimycotics agents that need to 

reside at the site of infection for a prolonged 

period to be more effective. The vagina 

provides a promising site of systemic drug 

delivery because of its large surface area 

and its rich blood supply. In addition , a 

prolonged contact of a delivery system with 

the vaginal mucosa may be achieved 

more easily than at other route of drug 

absorption site like rectum or intestinal 

mucosa (2). . Tinidazole ( TND ) is {1- [ 2- 

(ethylsulphonyl) ethyl ] – 2 – methyl – 5 - 

nitroimidazole} has been proved to be very 

effective for the therapy of amoebiasis , 

trichomonas , lambliasis and anaerobic 

infections (3) .The drug is well tolerated and 

widely used in clinical practice in the form of 

I.V infusion , tablets and vaginal pessaries (4). 

The efficacy of TND in the treatment of 

trichomonas vaginalis has been well 

documented (5) . TND is similar to 

metronidazole intravaginal gel can be used 

to treat bacterial vaginosis (6,7), it is effective 

, safe , easy to apply and has fewer side 

effect compared to oral dosage forms of 

1Corresponding author E- mail : Zainabthabit@yahoo.com 

Received : 3/5/2010 

Accepted : 18/9/2010 

64 

. ﻡﺪﺨﺘﺴﻤﻟﺍ ﺰﻴﻛﺮﺘﻟﺍ 

this medication (8,9) patients tolerate gels 

better than vaginal inserts or ointments. (10) 

This study was conducted to formulate TND in 

a suitable mucoadhesive vaginal gel through 

studying different variables affecting the 

physicochemical properties and the in vitro 

release of the drug from these preparations. 


Materials 

Tinidazole ( TND ) was supplied by 

Sigma chemical co., methylcellulose( MC) 

and carboxymethylcellulose(CMC)from(BDH 

limite ( HPMC) from ( Shin-Etsu chemical 

co.– Germany) . All other materials used were 

of analytical grade. 

Methods 

Preparation of simulating vaginal fluid 

(S.V.F) 

Simulating vaginal fluid (S.V.F ) was 

prepared by dissolving 3.51gm of sodium 

chloride ,1.4gm of potassium hydroxide 

,0.22gm of calcium hydroxide , 0.018gm of 

bovine serum albumin, 2 gm of lactic acid , 

1gm of acetic acid ,0.16gm of glycerol 

,0.4gm of urea and 5gm of glucose in 1 liter 

distilled water then the pH was adjusted at 

4.2 (11).


Preparation of TND vaginal aqueous gel 

bases 

The tested aqueous gel bases were 

prepared by dispersing the polymers 

powder of 3 and 4%(w/w) MC ,2 and 

3%(w/w) CMC and 3 and 4%(w/w) HPMC in 

cold freshly distilled water with the aid of a 

high speed stirrer , until a solutions were 

complete (12) .The products were refrigerated 

for 1hour before use to obtain a suitable 

consistency .TND gels were prepared by 

dispersing 2% drug in different gel bases as 

shown in table(1). 

Table 1:Composition of TND bioadhe sive 

vaginal ge l 

In vitro dissolution of TND from gel bases 

The release study was carried out in a 

USP apparatus type І at 100 rpm ,a basket 

of 2.5 cm in diameter was enclosed with a 

multifold filter paper (dialysis cell) in order to 

be filled with 1gm of each base containing 

2%w/w TND. After connecting to a stirrer 

motor , the dialysis cell was immersed to 

about 1 cm of its surface in 500 ml of 

simulating vaginal fluid pH4.2 at 37 ◦ C for 5 

hours .Samples were withdrawn after 

0.5,1,2,3,4 and 5 hours and replaced with 

an equal volume of the same fluid solution . 

The samples were analyzed for their drug 

content at its λmax 320nm (13, 14). . 

The effect of polymer type on the release of 

TND from the gel bases 

The effect of MC, CMC and HPMC 

bases on the release of TND from gel bases 

was studied. 

The effect of polymer concentration on 

the release of T ND from the gel bases 

The effect of 3 and 4% w/w MC , 2 

and 3% w/w CMC and 3 and 4% w/w 

HPMC on the release of TND from gel 

bases were studied. 

Measurement of the swelling index of dry 

polymer in S.V.F 

The swelling index is the volume in 

ml occupied by 1 gm of polymer after it 

has swollen in aqueous liquid (S.V.F used 

in the study) for four hours. The process is 

carried out by placing 1gm of dry polymer 

in 25ml graduated cylinder , moisten it with 

1ml (ethanol 96%) and with an addition of 25 

ml S.V.F shaking vigorously every 10 

minute for one hour ,then allowing to stand 

for three hours . After1.5houre from 

beginning the test any volume of liquid 

retained in the layer of the polymer and any 

particles of polymer floated at the surface 

of the liquid were released (15) .The swelling 

was measured according to this equation: 

Swelling = 

(final volume- initial volume) 

initial volume 

Mechanism of TND release from gel bases 

To understand the mechanism of the 

drug release , the release profiles from 

different gel bases, the % fraction drug 

released verses time profiles were plotted 

using linear fitted equations proposed by 

Koresmeyer - Peppas 

( 16 ) 

F = Mt/Mo = ktⁿ at time t (1) 

ln F = ln k + n ln t (2) 

The equations express the fraction (F) release 

of drug from gel Where Mt is the amount 

released at time t ,Mo is the initial amount of 

drug ,K is the rate constant and n is the 

releasing exponent value indicative the 

mechanism of drug release( if n is 0.5 or 

less (fickian ) holds the release, while 

when n greater than 0.5 this indicate nonfickian 

(anomalous) releasing mechanism), 

n value could be result from the slope 

obtained by the drawing of ln. Fraction 

released verses ln. Time. 

Viscosity measurement 

A Brook Field digital viscometer with a 

suitable sample adaptor was used to measure 

the viscosity in centipoises of the bioadhesive 

prepared gel. (17) 

Determination of pH 

The pH of the bioadhesive gels were 

determined by digital pH meter .One gram of 

gel was dissolved in 25 ml of dislilled water 

and the electrode dipped into gel formulation 

for 30 minute..The measurements of pH of 

each system were replicated three times (18)


Determination of mucoadhesive force 

The mucoadhesive potential of each 

system was determined by measuring the force 

required to detach the gel from sheep vaginal 

mucosal tissue by using a modified chemical 

balance. Asection of vaginal mucosa was cut 

from the sheeps vaginal cavity and instantly 

fixed with mucosal side out onto each glass 

vial using a rubber band. The vials with 

vaginal mucosa were stored at 37 ◦ C for 5 

minutes .Then next vial with a section of 

mucosa was connected to the balance in 

inverted position, while first vial was placed 

on a height adjustable pan. Fixed amount of 

sample of each gel system were placed onto 

the vaginal mucosa of first vial. Then the 

height of second vial was adjusted so that 

mucosal surfaces of both vials come in 

intimate contact .Five minutes contact time 

was given to ensure intimate contact between 

tissues and the sample. Then weight was kept 

rising in the pan until vials get detached. The 

bioadhesive forces as the detachment stress in 

dyne/cm 2 , was determined from the minimal 

weights that detached the tissues from the 

surface of each gel using the following 

equation (19): 

Detachment stress(dyne/cm 2 )= m g/A 

Where, m= weight required for detachment of 

two vials in grams. 

g= Acceleration due to gravity [980cm/s 2 ]. 

A=area of tissue exposed. 

The vaginal mucosa was changed for each 

measurement .Measurements were repeated 

three times for each of the gel system. 


In order to fortify the adhesion of 

administered drug onto the mucosal 

surface, TND was formulated in a suitable 

mucoadhesive polymers , MC, CMC and 

HPMC bases to take a full advantageous of 

66 

the contact time , the drug can be 

dispersed in the gel giving a concentration 

that is higher than corresponding to the 

solubility of the drug (20) ,as well as ,it can be 

considered as a way for sustaining the release 

of the drugs from gel dosage form . MC, CMC 

and HPMC were used as a gel base 

because of there safety use for vaginal 

application as well as there compatibility with 

TND, with other vaginal secretion. 

The effect of polymer type and concentration 

on the releasing of TND from the gel bases 

Figures 1, 2 and 3 showed the amount 

of TND released from gel bases in 

S.V.F medium .It was found that the amount 

of TND released from gel bases were 

significantly influenced (p

F fraction released 



Figure 1 : The effect of MC Concentration 

on the release profile of TND at pH 4.2 and 

37 time(minute) 

C 

Figure 2: The effect of CMC on the release 

profile of TND at pH 4.2 and 37 C 


0.4 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0.4 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

0 

0 100 200 300 400 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

2% w/w CMC 

3% w/w CMC 

0 100 200 300 400 

time(minute) 

0 

3% w/w HPMC 

4% w/w HPMC 

3% w/w MC 

4% w/w MC 

0 100 200 300 400 

time(minute) 

Figure 3 : The effect of HPMC on the 

release profile of TND at pH 4.2 and 37 C 

67 

Figure 4 : The swe lling mechanism of 3 & 

4% w/w MC in simulating vaginal fluid pH 

4.2 and 37 C 

t/v (hr/ml fluid solution /ml CMC ) 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

3% CMC 

2% CMC 

1 2 3 4 5 

time(hr) 

Figure 5 : The swelling mechanism of 2 & 

3% w/w C MC in simulating vaginal fluid 

pH 4.2 and 37 C 

Releasing mechanism of TND from gel bases 

It was found that formulation with higher 

swelling index retard the release of drugs more 

than those with lower swelling indices , this 

swelling also depends on the pH of the 

medium and the presence of electrolytes , 

where the S.V.F pH 4.2 has a lot of 

electrolytes content such as : sodium chloride , 

potassium hydroxide , calcium hydroxide (25). 

This swelling is important since its 

significantly decreases the releasing rate of the 

drug and increases in the amount of the drug 

release after five hours ,as shown in table-3- 

.The reason behind this increasing in the 

amount released due to erosion of the gel as a 

result of severe hydration or relaxation of the 

polymer , so interchain intermolecular force 

will no longer be able to resist any external 

forces ,once gel erodes ,it breaks up into 

smaller and smaller particles ,more surfaces 

will be exposed to the fresh swelling medium


and hence more drug will be released leads to 

an increasing in the amount of the drug 

released from CMC gel base more than other 

swollen MC gel base after five hours (18) .As a 

result , CMC gel bases hold a Fikian release 

mechanism. 

Table 3 :The release rate, amount released and the releasing mechanism of the TND from gel bases 

Polymer type 

and 

concentration 

MC 3% 

MC 4% 

CMC 2% 

CMC 3% 

HPMC 3% 

HPMC 4% 

n expone nt 

release value 

0.41 

0.49 

0.509 

0.52 

0.29 

0.5 

Viscosity measurment 

Viscosity is an important parameter for 

characterizing the gel as it affects the 

spreadibility ,extrudability and release of 

drug (26) .CMC gel base show the highest 

viscosity, and as the polymer concentration 

increase ,the viscosity will be increased as 

shown in table 4. On the other hand , minimum 

level of viscosity was seen with F5 and F6 

explain the rapid release of drug fro m this gel 

system and this due to non swelling property 

of HPMC polymer at these concentration . 

Table 4:The viscosity and pH of the 

prepared TND bioadhe sive vaginal gel 

Formula Viscosity pH 

No. (cps) 

F1 1450 6.02 

F2 3240 5.2 

F3 1700 5.03 

F4 6800 5.17 

F5 14.5 5.02 

F6 21.6 5.2 

Determination of pH 

The pH of gels were found to be within 

the range of 5-6.2 which is within the limit of 

semisolid specification and at this pH the gel 

will be non irritant to vagina .(12). The physical 

appearance of the gel was evaluated with 

naked eye . The gel was smooth without any 

lumps and of uniform color ,no color change 

/liquification /separation were observed . 

Determination of mucoadhesive force 

Scheme 1 indicates the vaginal bioadhesive 

properties of the prepared gels in sheep 

vagina,and the resultes showed that all vaginal 

bioadhesive strengths were found in the 

K % minⁿ 

re leasing 

rate 

5.3974 

3.5744 

4.1594 

3.582 

6.1472 

3.1864 

amount re le ase 

(mg/5hr) 

(mean±SD,n=3) 

7.05±0.14 

4.466±0.13 

7 ±0.15 

4.35±0.12 

5.2±0.03 

4.58±0.11 

Mechanism of 

TND release 

Fickian 

Fickian 

Fickian 

Fickian 

Fickian 

Fickian 

following order F2,F4>F3>F1>F6>F5 

Indicating that (F2)and (F4) showed the 

highest bioadhesive properties. 

Sche me 1: Bioadhesive stre ngth of TND 

Bioadhesive vaginal gels 

Conclusion 

Results of this study confirm that, the 

physical properties of the prepared gel were 

affected by the type and concentration of the 

polymer used in the preparation. The more 

sustained preparation with highest bioadhesive 

force and highest viscosity was obtained by the 

use of 3% CMC as a gel base.In addition to 

that the mechanism of drug release from 

tinidazole 3%CMCgel base was diffusion 

mixed with erosion. 

References 

1. Richardson JI Illum L The vaginal route 

of peptide & protein drug delivery .Adv. 

drug delivery rev 1992 ; 8: 341-366 . 

2. Claudia, KV, Constantiae, Irene H. , 

Andreas, SB . Development and in vitro 

evaluation of a mucoadhesive vaginal 

delivery system for progesterone. J. 

controlled release 2001; 77: 323-332.


3. Trac JW. Webster LT. Drugs used in 

the chemotherapy of protozoal 

infection .In:Hardman, J.,G., Limbird,L. 

,E.,the pharmacological basis of 

therapeutics 9 th ed. Mc Graw –Hill,New 

York 1996 ; 995-998 

4. Martindale. The complete drug 

reference, 32 ed., pharmaceutical press 

1999; 594: 1099, 1541. 

5. BNF, British national formulary, British 

medical association and Royal 

Pharmaceutical society of Great Britain 

2004; 47 ed.: 287, U.K. 

6. Milani,M., Barcellona,E., Agnello,A., 

Efficacy of the combination of 2g oral 

tinidazole and acidic buffering vaginal 

gel in comparison with vaginal 

clindamycin alone in bacterial vaginosis 

:a randomized ,investigator -blinded, 

controlled trial .Euro .J. of obst. and gyne 

2003 ;109: 67-71. 

7. Livengood, . G. H. Mc Gregor , ., J.,A., 

Soper, D., E, Newton, E., Thomason, J.,L., 

Bacterial vaginosis : efficacy and 

safety of intravaginal metronidazole 

treatment .Am.J.Obstet.Gynecol 1994 

;170: 759-764. 

8. Hanson, JM., McGregor, JA, Hiller, 

SL., Eschenbach, DA., Kreutner A.K., 

Galask, RP., Martens, M.. 

Metronidazole for bacterial vaginosis. 

Acomparision of vaginal gel vs. oral 

therapy .J.Reprod.Med 2000; 45: 889-896. 

9. Dubonchet, L. , McGregor, J.A. , Is mail 

, M. , McCormack, W.M. , A pilot study 

of metronidazole vaginal versus oral 

metronidazole for tretmeant of 

trichomonas vaginalisvaginitis ,sex. 

Transm. di 1998 ;25:176-179. 

10. Edsman, K. Carlfors J. Peterson R. 

Reological evaluation of poloxamer as 

an in situ gel for ophthalmic use. 

Eur.J.Pharm.Sci. 1998; 6: 105-112. 

11. Owen,DH., Peters JJ., Lavine L.L., Katz 

D.F., Effect of temperature and pH on 

the contraceptive gel viscosity 

.Contraceptive 2003;67: 57-64 . 

12. Farhan, J.A , Mohd ,A .A., Zeenat .I K , 

Roop, K. K. and Mushir, A . Development 

and in vitro evaluation of an acid 

buffering bioadhesive vaginal gel for 

mixed vaginal infection . Acta pharm. 

2008 ; 58: 407-419 . 

13. Mariee, N.K. Factors affecting the in 

vitro diffusion of diclofenac sodium 

from ointment. Iraqi .J. of pharmacy 

2001; 1: 62-71 

14. Hui, H.W., Robinson, J.R., Effect of 

particle dissolution rate on ocular 

69 

drugbioavailability .J. pharma. sci. 1986; 

75: 280-287. 

15. BP ,British Pharmacopoeia 2008 ;vol. IV, 

Appendix XI E A273. 

16. Martin A.N. “Physical Pharmacy”, 4 th ed , 

Waverly, New Delhi. 1993; 497. 

17. Ritger, PL, Peppas, NA. Modelling of 

water transport solute release in 

physiologically sensitive gels. J.control 

release 1987; 5:37-40 . 

18. Benoy, B.B.,Bhalani, S.N. and Arkenduc . 

Formulation development and 

characterization of metronidazole 

microencapsulated bioadhesive vaginal 

gel. Int. J. of pharmacy and 

pharmaceutical sciences 2009; 1, Issue1. 

19. Yong, C S, Choi, T S, Quan, Q Z . Rhee, 

J D, Kim, CK , Lim, S J, Effect of Na 

chloride on the gelation temperature,gel 

strength and bioadhsive force of 

poloxamer gels containing diclofenac Na. 

Int. J .pharm. 2001; 226:195-205. 

20. Veyries ,M.,L., Couarraze,G., Geiger,S., 

Agnely,F., Massias,L., Kunzli,B., 

Faurisson,R., Rouveix,B. , 

Controlled release of vancomycin 

from poloxamer 407 gels 

.Int.J.Pharm. 1999; 192: 183-193. 

21. Mirchandan HL ,Chien YW ,Bruce PD, 

Senshang L ,Effect of HPMC and 

carbopol on the release and floating 

properties of gastric floating drug delivery 

system using factorial design Int.J.Pharm. 

2003; 253: 3-22. 

22. Jaleh, V, Nasser T., Sharona ,S 

.,Development and physical 

characterization of a periodontal 

bioadhesive gel of metronidazole, Drug 

deliv.2002; 9, issue 2: 127-133. 

23. Martinez, N Vazquez, Antoniocruz, R .del 

C ,Alvarez Castillo A ,Mendoza, A M , 

Martinez, A B, Morales Cepeda , 

Swelling kinetic of hydrogels from methyl 

cellulose and poly acrylamide .I S S N 

2007 ; 6 : 337-345. 

24. Naim, S., Samuel, B., Chauhan, B., 

Paradkar, A. Effect of potassium chloride 

and cationic drug on swelling, erosion and 

release from κ-carrageenan matrices. 

AAPS PharmSciTech. 2004; 5(2): article 

25. 

25. Omidian, H, Park, k., Swelling agents and 

devices in oral drug delivery ,J.drug 

.del.sci.tech. 2008; 18(2): 83-93. 

26. Sanap, G.S., Smita, T., Tarannum, S. and 

Sangita, G. Formulation and evaluation of 

mucoadhesive beads of glipizide by 2 3 

factorial design .jou. of phar. Res. 2009; 

2(5): 934- 938.

Iraqi J Pharm Sci, Vol.19(2) 2010 Protein profiles in children with Kala-azar 

Urine Protein SDS-PAGE Reveals Different Profiles in Iraqi 

Children with Kala-azar 

Yassir MK Al-Mulla Hummadi* ,1 

* Department of Pharmaco-therapeutics, College of Pharmacy, Al-Mustansiriyah University, Baghdad, Iraq. 

Abstract 

Urine proteomics have been an area of interest and recently in Kala-azar as an alternative sample 

type for serum or plasma. Because of simplicity, noninvasiveness of collection and simpler matrix. 

Many studies had detected an increased protein excretion in the urine of patients with active Kala-azar 

due to renal involvement particularly by an immunological related mechanism(s). This study have 

demonstrated the presence of three different protein profiles in Iraqi children (Patients: including 60 

children aged 4-60 months) with defined Kala-azar using the conventional SDS-PAGE on urine 

samples. Urine protein profile in Kala-azar patients revealed three groups of banding patterns: group- 

1(33.4)% of the patients show the pattern of 5 bands with a MW. Ranged (512.861-158.489), groups-2, 

3 were (55, 11.6) % of the patients showing 2 banding proteins with a MW. ranged (512.861-199.526) , 

(199.526-181.97) respectively. These findings may be correlated with other epidemiological studies 

that revealed the presence of different clinical presentations like fever, spleenomegally, hepatomegally, 

thro mbocytopaenia, and different response to leishmania therapy. Furthermore, the presence of 

different protein patterns may also be related to the chronicity of infection and the degree of renal 

involvement. The presence of a similar protein band between group-1 and 2 may be of diagnostic 

purpose and further studies on expanded number of patients are required to identify that kind of protein 

or other urine protein profiles. 

Key words: Protein band, prote in pattern, sodium dode cyl sulphate -poly acrylamide gel 

electrophoresis (SDS-PAGE), urine proteome, visceral leishmaniasis. 


ﻭَﺃ ِﻞﺼﻤﻟﺍ ﺕﺎﻨﻴﻌﻟ ﻞﻳﺪﺑ ﺝﺫﻮﻤﻨﻛ ﻪﻴﺋﺎﺸﺣﻻﺍ ﺕﺎﻴﻧﺎﻤﺸﻠﻟﺍ ءﺍﺩ ﻲﻓ ًﺍﺮﺧﺆﻣﻭ ﻡﺎﻤﺘﻫﺍ ﺓﺮﺋﺍﺩ 

ﻝﻮﺒﻟﺍ ﺕﺎﻨﻴﻋ ﻲﻓ ﺕﺎﻨﻴﺗﻭﺮﺒﻟﺍ ﻞﻜﺸﺗ 

ﻪﺗﺩﺎﻣ ﺔﻃﺎﺴﺑ ﻰﻟﺍ ﻪﻓﺎﺿﺍ ﻪﺣﺭﺎﺠﻟﺍ ﺕﺍﻭﺩﻻﺎﻛ ﻪﻌﺿﺎﺒﻟﺍ ﻞﺋﺎﺳﻮﻟﺍ ﺐﻨﺠﺘﺑ ﻪﻌﻤﺟ ﺔﻟﻮﻬﺳﻭ ﻪﻴﻠﻋ ﻝﻮﺼﺤﻟﺍ ﻲﻓ ِﺔﻃﺎﺴﺒﻟﺍ ﺐﺒﺴﺑ . ﺎﻣﺯﻼﺒﻟﺍ 

ﺽﺮﻤﻠﻟ ﺔﻄﻴﺸﻨﻟﺍ ﻩﺮﺘﻔﻟﺍ ﻲﻓ ﻪﻴﺋﺎﺸﺣﻻﺍ ﺎﻴﻧﺎﻤﺸﻠﻟﺎﺑ ﻦﻴﺑﺎﺼﻤﻟﺍ 

ﻰﺿﺮﻤﻟﺍ ِﻝﻮﺑ ﻲﻓ ﺪﻳﺍﺰﺘﻣ ِﻦﻴﺗﻭﺮﺑ َﺡﺮﻃ ِﺕﺎﺳﺍﺭِﺪﻟﺍ ْﻦِﻣ ﺪﻳﺪﻌﻟﺍ ْﺖﻔﺸﺘﻛﺇ . ﻪﻴﺳﺎﺳﻻﺍ 

ﻝﻮﺑ ﺝﺫﺎﻤﻧ ﻲﻓ ِﺔﻔﻠﺘﺨﻣ ِﻦﻴﺗﻭﺮﺑ ﻁﺎﻤﻧﺍ ﻊﻴﻣﺎﺠﻣ ﺙﻼﺛ َﺩﻮﺟﻭ ِﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﺖﻔﺸﻛ . ﻪﻴﻋﺎﻨﻤﻟﺍ ِﺔﻴﻟﻵﺎﺑ ﻖﻠﻌﺘﻳ ﺎﻤﺑ ًﺎﺻﻮﺼﺧ ِﻱﻮﻠﻜﻟﺍﺮﺛﺎﺘﻟﺍ ﺐﺒﺴﺑ 

ﻲﺋﺎﺑﺮﻬﻜﻟﺍ ﻞﻴﺣﺮﺘﻟﺍ ﺔﻴﻨﻘﺗ ﻡﺍﺪﺨﺘﺳﺎﺑ ﻚﻟﺫﻭ ( ﺮﻬﺷ 604 

ِﺮﻤﻌﺑ ﻞﻔﻃ 60 : ﻪﻴﺋﺎﺸﺣﻻﺍ ﺎﻴﻧﺎﻤﺸﻠﻟﺎﺑ ﻦﻴﺼﺨﺸﻤﻟﺍ ﻰﺿﺮﻤﻟﺍ) 

ِﻦﻴﻴﻗﺍﺮﻌﻟﺍ ِﻝﺎﻔﻃﻷﺍ 

ﻡﺰﺣ ﺲﻤﺧ ﺕﺮﻬﻇﺍ ﻰﺿﺮﻤﻟﺍ ﻦﻣ % ( 33.4) 

1 ﺔﻋﻮﻤﺠﻣ : ﻲﻠﻳ ﺎﻤﻛ ﺖﻧﺎﻛ ﺪﻗﻭ . ِﻝﻮﺒﻟﺍ ِﺕﺎﻨﻴﻋ ﻰﻠﻋ ( ﺪﻳﺎﻣﺍ ﻞﻳﺮﻛﻻﺍ ﺩﺪﻌﺘﻣ ﻡﻼﻫ) 

ﻱﺪﻴﻠﻘﺘﻟﺍ 

ﺕﺮﻬﻇﺍ ﻰﺿﺮﻤﻟﺍ ﻦﻣ % ( 11.6 ٬ 55) 

ْﺖﻧﺎَﻛ 3 ﻭ 2 ﻪﻋﻮﻤﺠﻣ ٬ﻥﻮﺘﻟﺍﺩ ( 158.489 

512.861 ) ﻦﻴﺑ َﺡﻭﺍﺮَﺗ ﻲﺌﻳﺰﺟ ﻥﺯﻭ ﻊَﻣ ﻪﻴﻨﻴﺗﻭﺮﺑ 

. ﻲﻟﺍﻮﺘﻟﺍ ﻰﻠﻋ ﻭ ﻥﻮﺘﻟﺍﺩ ( 181.97 

199.526) 

٬ ( 199.526 512.861 

) ﻦﻴﺑ َﺡﻭﺍﺮَﺗ ﻲﺌﻳﺰﺟ ﻥﺯﻭ ﻊَﻣ ﺎﻤﻬﻨﻣ ﻞﻜﻟ ِﻦﻴﺗﻭﺮﺒﻟﺍ ﻦﻣ ﻦﻴﺘﻣﺰﺣ 

ﺪﺒﻜﻟﺍ ﻢﺨﻀﺗ 

‘ ﻰﱠﻤُﺤﻟﺍ ﻞﺜﻣ ِﺔﻔﻠﺘﺨﻤﻟﺍ ِﺔﻳﺮﻳﺮﺴﻟﺍ ِﺽﺭﺍﻮﻌﻟﺍ ﺩﻮﺟﻭ ْﺖﻔﺸَﻛ ﻲﺘﻟﺍﻭ ﻪﻘﺑﺎﺴﻟﺍ ﻯﺮﺧﻷﺍ ﻪﻴﺋﺎﺑﻮﻟﺍ ِﺕﺎﺳﺍﺭِﺪﻟﺎﺑ ُﻂَﺒْﺗﺮُﺗ ْﺪَﻗ ِﺞﺋﺎﺘﻨﻟﺍ ﻩﺬﻫ 

ًﺎﻀﻳﺃ ﺎَﻤﱠﺑُﺮَﻟ ِﺔﻔﻠﺘﺨﻤﻟﺍ ِﻦﻴﺗﻭﺮﺒﻟﺍ ِﻁﺎﻤﻧﺃ ﺭﻮﻀﺣ ٬ﻚﻟﺫ ﻰﻠﻋ ﺓﻭﻼﻋ . ﻪﻴﺋﺎﺸﺣﻻﺍ ﺎﻴﻧﺎﻤﺸﻠﻟﺍ ِﺝﻼﻌﻟ ﻪﻔﻠﺘﺨﻣ ّﺕﺎﺑﺎﺠﺘﺳﺍﻭ ٬ﺕﺎﺤﻴﻔﺼﻟﺍ ﺔﻠﻗ ٬ﻝﺎﺤﻄﻟﺍﻭ 

ﻥﺎﻜﻤﺑ ﺓﺪﺋﺎﻔﻟﺍ ْﻦِﻣ ﻥﺎﻧﻮُﻜَﺗ ْﺪَﻗ 2ﻭ 

1 ِﺔﻋﻮﻤﺠﻤﻟﺍ ﻦﻴﺑ ِﺔﻠﺛﺎﻤﻣ ِﻦﻴﺗﻭﺮﺑ ﺔﻣﺰﺣ ﺩﻮﺟﻭ ﻥﺇ ﺎﻤﻛ . ِﻱﻮﻠﻜﻟﺍ ِﻞّﺧﺪﺘﻟﺍ ِﺔﺟﺭﺩﻭ ﺽﺮﻤﻟﺍ ﻥﺎﻣﺯﺎﺑ ﺔﻘّﻠَﻌَﺘُﻣ 

ﻁﺎﻤﻧﺃ ﻒﺸﻛ ﻭَﺃ ِﻦﻴﺗﻭﺮﺒﻟﺍ ْﻦِﻣ ِﻉﻮﻨﻟﺍ ﻚﻟﺫ ﺰﻴﻴﻤَﺘﻟ ﻰﺿﺮﻤﻟﺍ ْﻦِﻣ ِﻊّﺳﻮﻣ ِﺩﺪﻋ ﻰﻠﻋﻭ ﻯﺮﺧﺃ ِﺕﺎﺳﺍﺭِﺩ ءﺍﺮﺟﺍ ﻲﻋﺪﺘﺴﻳ ﺎﻤﻣ ِﻲﺼﻴﺨﺸﺘﻟﺍ ِﺽﺮﻐﻠﻟ 

Introduction 

Visceral leishmaniasis and cutaneous 

leishmaniasis caused by Leishmania donovani 

and Leishmania tropica or Leishmania major 

respectively are known endemic diseases in 

Iraq.Visceral leishmaniasis had been prevalent 

in Iraq for the last 2-3 decades causing serious 

problems (1) . It represents an important factor 

in infant's mortality in this country 

.Diagnosis of visceral leishmaniasis generally 

based on clinical presentation and 

microscopical demonstration of the parasite in 

smears, aspirates of bone marrow or culture of 

these aspirates on certain culturing media. 

However, the techniques are painful, 

1Corresponding author E- mail : yassir.almullahummadi@ymail.com 

Received : 25/5/2010 

Accepted : 29/9/2010 

(2) 

70 

. ِﻝﻮﺒﻟﺍ ﺕﺎﻨﻴﻋ ﻲﻓ ﺕﺎِﻨﻴﺗﻭﺮﺒﻟﺍ ﻦﻣ ﻯﺮﺧﺃ 

hazardous and need skilled personnel and 

equipped hospitals some times difficult to be 

obtained (3) . Since visceral leishmaniasis is 

occurring in places of poor socioeconomic 

conditions where health services are poorly 

developed (4) , the diagnosis in this case is 

difficult to be obtained using these 

techniques (3) . However, presence of 

Leishmania donovani soluble antigen, 

corresponding antibody and component of the 

complement in the serum of patient with active 

kala-azar has been demonstrated in a number 

of studies (1) , Al-Bashir (5) .


On the other hand, the detection of Leishmania 

donovani parasite antigen in the urine samples 

of patient with active Kala-azar has been 

demonstrated in a number of studies by 

(6) 

immunoblotting ; enzyme linked 

(7) 

immunosorbent assay (ELISA) ; latex 

agglutination test (LAT) 

(8) and direct 

agglutination test (DAT) (8, 9) .Furthermore, 

urine samples are more easily to be collected 

especially from young children, as well as 

facilitating field studies (9) .Therefore, this 

study aims to demonstrate the presence of 

variations in protein profiles in a number of 

Iraqi patients with defined visceral 

leishmaniasis using the conventional sodium 

dodecyl sulphate-poly acrylamide gel 

electrophoresis (SDS-PAGE) in urine samples. 


This study was divided into two groups: 

1. Patients: including 60 children aged 4-60 

months, admitted to paediatric ward of Alkadhimiyah 

teaching hospital-Al-Nahrain 

University from December 2007- 

Febriwary 2009. All were proven cases of 

Kala-azar by: clinical manifestation of 

fever, hepato-spleenomegally, the 

demonstration of the parasite - the 

amastigotes from indirect smear of bone 

marrow and serological diagnosis by 

indirect immunofluorescence antibody test 

IFAT. 

2. Control: consist from 10 healthy children 

all were seronegative. 

Urine samples 

24 hours urine specimens were collected 

from each child in the control or the diseased 

group. Urine samples from all children were 

concentrated 100 times using Amicon 

Apparatus (Amicon Corporation Davers, MA) 

and kept at -20 o C till use to avoid bacterial 

contamination as described by Baqir et al. 

(2002) (6) ; Al-Bashir and Ali (2003) (8) .Total 

protein for each sample was measured 

according to the method of Lowery et al. 

(1951) (10) . The protein electrophoresis was 

dependent on the conventional polyacrylamid 

gel electrophoresis PAGE as described by 

Fehrnstrom and Morberg (1977) (11) . 

Urine protein profile 

Urine protein electrophoresis was 

performed as described by Fehrnstrom and 

Morberg (1977) (11) , using Coomaise brilliant 

blue staining.Migration distance of the 

calibration proteins were measured after 

staining the protein bands. The relative 

mobility (RM) was measured too according to 

the following equation: 

71 

The calibration curve was constructed by 

plotting RMs versus log. Molecular weights 

for calibration proteins Fig.(1). The molecular 

weight of proteins of interest was determined 

from the position of its RM value on the 

calibration curve. 

Figure 1: Standard curve for approximate 

estimation of molecular weights of various 

prote ins inurine samples. 

Results 

Conventional polyacrylamide gel 

electrophoresis has been used to differentiate 

between protein patterns in serum of normal 

and patients with Kala-azar. Coomassie 

brilliant blue stain has been used to visualize 

the band separated on the gel (12) . Urine of the 

control group has a specific protein pattern 

with one protein bands fig.(2). 

Control group (1) group (2) group (3) 

Figure 2 : Urine prote in profiles in normal 

and kala-azar children. 

The relative mobility value (RM) was 0.48 

with a molecular weight 199.525.Urine protein 

profile in Kala-azar patients revealed three 

banding patterns ranged from (2-5) protein


bands. The (RM) values were ranged from 

(0.22-0.52). See fig.(2). The first protein 

pattern demonstrates five bands. The RM 

values were ranged from (0.22-0.52), with 

molecular weights ranged from (512.861 - 

158.489) ×10 3 Dalton respectively. Four out of 

these bands were shown to be abnormal from 

the control these are bands (1, 2, 3 and 5) as 

shown in table (1). This band picture was 

found in 20 patients (33.4 %) Table (2). The 

second protein pattern shows two bands the 

RM values were ranged from (0.22-0.48), with 

72 

a molecular weights (512.861 and 199.526) 

×10 3 Dalton respectively. One of these two 

bands (band-1) was shown to be different from 

the control as shown in table (1). This profile 

was found in 33 patients (55 %) Table (2).The 

third protein shows two bands the RM values 

were ranged from (0.48-0.50), with a 

molecular weights (199.526 and 181.970) ×10 3 

Dalton respectively. One of these two bands 

(band-2) was shown to be different from the 

control as shown in table (1). This profile was 

found in 7 patients (11.66 %) Table (2). 

Table 1 : RM values and mole cular weight for each band in different urine prote in profile groups. 

Band 

No. 

Control 

RM 

Control 

MW×1 3 

Dalton 

G1- 

RM 

G1- 

MW×10 3 

Dalton 

G2- 

RM 

G2- 

MW×10 3 

Dalton 

G3- 

RM 

G3- 

MW×1 3 

Dalton 

1 0.48 199.526 0.22 512.861 0.22 512.861 0.48 199.526 

2 0.32 316.227 0.48 199.526 0.50 181.970 

3 0.37 236.026 

4 0.48 199.526 

5 0.52 158.489 

RM: relative mobility, MW: molecular weight in Daltons. 

Table 2 : Diffe re nt urine protein profiles with the pe rcentage of patie nts in each group. 

Group Total No. bands No. abnormal bands No. Patie nts % patient 

1 5 4 20 33.4 

2 2 1 33 55 

3 2 1 7 11.6 

Discussion 

The detection and identification of trace 

amounts of proteins in complex samples is a 

major challenge in biomarkers discovery and 

validation (13, 14) . Samples of interest for 

detection of protein biomarkers are typically 

serum or plasma. However, with the rapidly 

growing interest in human urine proteome (15, 

16) , because of simplicity and 

noninvasiveness of collection, making urine an 

alternative sample type for many diagnostic 

tests (17) .Furthermore, urine contains a 

relatively small number of proteins typically 

present at low concentrations and thus simpler 

matrix for detecting proteins as compared to 

serum (17) . Some human diseases, excess 

proteins are found in the urine as can occur in 

patients with compromised kidney function. 

As a result, many of the proteins normally 

present in blood will also be excreted in to the 

urine. This condition is known as proteinuria, 

is often observed in acute inflammation, acute 

urinary tract infection, amyloidosis, diabetic 

nephropathy, kidney failure, multiple 

myeloma, nephrotic syndrome and severe 

yeast infection (18, 19 ) . Kidney involvement 

has been demonstrated in patients with Kalaazar 

with significant proteinuria (20) .The 

detection of Leishmania donovani soluble 

antigen and antibody (IgM and IgG) has also 

been shown in urine samples of kala-azar 

patients (21, 22).Abnormal renal functions 

particularly, glomerular filtration rate, urinary 

concentration, acidification that may be 

correlated to an immunological mechanisms 

and the evidence of renal proximal tubular 

damage have been demonstrated recently (23, 

24) . Lima et al. (2009) (24) have also detected 

the presence of different kinds of proteins in 

the urine of patients with Kala-azar 

particularly, albumin, IgG, β2-microglobulin, 

α1 , α2 , β and γ-globulins. All these studies 

had confirmed renal involvement during active 

Kala-azar disease. However, to the best of our 

knowledge, no study till now tried to 

demonstrate the types of protein patterns or 

profiles that may be observed in patients with 

the active disease. Therefore, in this study we 

tried to investigate-using urine samples- the 

presence of different protein profiles in a 

number of defined children with Kala-azar in


Iraq using the conventional SDS-PAGE.The 

results of this study revealed the presence of 

one protein band in the control healthy 

children and the presence of three different 

protein patterns groups in the defined children 

with Kala-azar. As shown in the results, 

patients involved in this study were divided 

into three groups according to their urine 

protein profile (see the above results).Groups 2 

and 3 showed only 2 banding proteins in 55 

and 11.6 % of the studied patients, while group 

1 showed 5 different banding proteins in 33% 

of the studied patients. These differences in the 

number of bands for each banding pattern 

group may be attributable to the chronicity of 

infection (24) and to the difference in the 

degree of renal tubular dysfunction with 

differences in the types and amounts of 

proteins excreted in the urine (23, 24) 

.Furthermore, clinical and epidemiological 

studies in Iraq showed a large differences in 

the clinical picture of the disease in terms of 

fever, hepatomegally, spleenomegally (2, 25, 

26), anaemia, thrombocytopaenia and response 

to therapy (sodium stibogluconate) (26) . 

Similar findings have been reported in other 

endemic areas with Kala-azar like Sudan (27), 

India (28) and Iran (29). This may explain the 

different protein patterns among different 

groups in this study.An interesting finding was 

the presence of common band between group 1 

and 2 with a molecular weight (512.861×103) 

Dalton.Further investigations with expanded 

number of patients as well as identifying the 

types of protein for each banding pattern group 

especially for the common band which could 

be of diagnostic value. 

References 

1. Kharazmi, A., H. R. Rezai, M. Fani, and 

N. C. Behforouz. Evidence for the 

presence of circulating immune complexes 

in serum and C3b and c3d on red cells of 

Kala-azar patients. Trans Roy. Soc. Trop. 

Med. Hyg. 1982;76:793-6. 

2. Atia, L. A.. Hepatic involvement in 

visceral leishmaniasis and its relation to 

the severity of infection. Msc. Theses 

College of science Baghdaduniversity.1995. 

3. Jensen, A. T. R., A. Gaafar, A. Ismail, C. 

B. V. Christensen, M. Ke mp, A. M. El- 

Hassan, and T. Theauder. Serodiagnosis of 

leishmania donovani infections. Trans 

Roy. Soc. Trop. Med. Hyg. 1999;93:157- 

60. 

4. Cerf, B. J., T. C. Jones, R. Badaro, D. 

Sampaio, R. Teixeira, and, W. D. Jr 

Johnson. Malnutrition as a risk factor for 

73 

severe visceral leishmaniasis. J. Inf. Dis. 

1987;156:1030-33. 

5. Al-Bashir, N. M. T. Plasma protein 

profiles in Iraqi children with Kala-azar. 

Indian Journal of Clinical 

Biochemistry.(in press). 

6. Baqir, H. I., T. I. Al-Jeboori, and N. T. Al- 

Bashir. Detection of parasite antigen in the 

urine of patients with active visceral 

leishmaniasis. Diagnostic potential. 

2002;2(1):22-5. 

7. Islam, M.Z., M. Itoh, , S. M 

Shmsuzzaman, R. Mirza, F. Matin, I. 

Ahmed, A. K. M. S. Choudry, M. A. 

Hossain, X. G. Qiu, N. Begam, M. 

Furuya, J. L. Leafasia, Y. Hashinguchi, 

and, E. Kimura. Diagnosis of visceral 

leishmaniasis by ELISA using urine 

samples. Clinical diagnostic laboratory 

immunology. 2002;9:789-94. 

8. Al-Bashir, N. M. T., and S. H. Ali. The 

use of urine as a sample for the diagnosis 

of visceral leishmaniasis. The Iraqi journal 

of medical sciences. 2003;2(3):332-39. 

9. Islam, M. Z., M. Itoh, R. Mirza, I. Ahmed, 

A. R. M. S. Ekram, , A. H. Sarder, S. M. 

Shamsuzzaman, Y. Hashiguchi, and E. 

Kimura. Direct agglutination test with 

urine samples for the diagnosis of visceral 

leishmaniasis. Am J Trop Med Hyg. 

2004;70: 78–82. 

10. Lowery, Q. L., N. S. Rosenbrough,, A. L. 

Farr, and R. J. Randall. Protien 

mesurement with Folin-Phenol reagent. 

Journal of Biochemistry.1951;193:265-75. 

11. Fehrnstrom, H., and U. Morberg. SDS 

and conventional polyacrylamide gel 

electrophoresis with LKB2117 multiphor. 

LKB application note 306. Bromam, 

Sweden1977. 

12. Johnson, A., E. Rohfs, and M. Siverman. 

Protein. In. Tietz textbook of clinical 

chemistry. Eds. Burtis, C. A. & Ashwood. 

3rd ed. R. E. Saunders W. B. CO. London. 

1999;pp.477-530. 

13. Echan, L. A., H. Y. Tang, N. Ali-Khan, K. 

Lee, and D.W. Speicher. Depletion of 

multiple high-dependence protein 

improves protein profiling capacities of 

human serum and plasma. Proteomics. 

2005;5:3292-303. 

14. Sigdel, T. K., K. Lau, J. Schilling, and M. 

Sarwal. Optimizing protein recovery for 

urinary proteomics, a tool to monitor renal 

trasplantation. Clinical transplant. 

2008;22(5):617-23. 

15. Soldi, M., C. Sarto, and C. Valsecchi. 

Proteome profile of human urine with 

two-dimentional liquid phase 

fractionation. Proteomics.2005;5:2641-47.


16. Thongboonkerd, V., T. Semangoen, and S. 

Chutipongtanate. Enrichment of 

basic/cationic urinary proteome using ion 

exchange chromatography and bath 

adsorption. Journal of Proteome 

Research.2007;6:1209-14. 

17. Kushnir, M. M., P. Morzinski, A. L. 

Rockwood, and D. K. Crockett. Depletion 

strategy for improved detection of human 

proteins from urine. Journal of 

Biochemical Techniques.2009;20: 101-08. 

18. Biesenbach G., K. Hommer, G. Stadler, 

R. Kramar, G. Syré, and J. Zazgornik. 

["Diabetic" proliferative retinopathy and 

nodular glomerulosclerosis without 

diabetes mellitus]. Dtsch Med 

Wochenschr. 1988;16,113(50):1968-71. 

19. van Ypersele de Strihou, C., and Y. 

Pirson. Indications for dialysis and 

trasplantation in end-stage renal diseae. 

Contrib Nephrology. 1989;71:1104-10. 

20. Weisinger, José R., A. Pinto, G. A. 

Velazquez, I. Bronstein, J. J. Dessene, J. 

F. Duque, J. Montenegro, F. Tapanes, and 

A. R. de Rousse. Clinical and Histological 

Kidney Involvement in Human Kala-Azar. 

Am. J.Trop. Med. Hyg.1978 ;27(2) : 357- 

359. 

21. Kohanteb, J., S. M. Ardehali, and H. R. 

Rezai. Detection of Leishmania donovani 

soluble antigen and antibody in the urine 

of visceral leishmaniasis patients. Trans R 

Soc Trop Med Hyg. 1987; 81(4):578-80. 

22. Mebrahtu, Y. B., L. D. Hendricks, C. N. 

Oster, P. G. Lawyer, P. V. Perkins, H. 

Pamba, D. Koech, and C. R. Roberts. 

Leishmania donovani parasites in the 

nasal secretions, tonsillopharyngeal 

mucosa, and urine centrifugates of visceral 

leishmaniasis patients in Kenya. Am J 

Trop Med Hyg. 1993;48(4):530-5. 

74 

23. Lima Verde, F. A., F. A. Lima Verde, I. 

A. Lima Verde, G. B. Silva Junior, E. F. 

Daher, and E. M. Lima Verde. Evaluation 

of renal function in human visceral 

leishmaniasis (kala-azar): a prospective 

study on 50 patients from Brazil. J 

Nephrol. 2007;20(4):430-6. 

24. Lima Verde, F. A., F. A. Lima Verde, E. 

F. Daher, G. M. Dos Santo, and E. M. 

Lima Verde. Renal tubular dysfunction in 

human visceral leihmaniasis (Kala-azar). 

Clinical Nephrology, 2009;71(5):492-500. 

25. Abas, M. Z., and A. M. Jabri. Clinicoepidemiological 

study of kala-azar in Iraq. 

A thesis submitted to the scientific council 

of Paediatrics. Baghdad.1998. 

26. Ali, S. H., N. T. Al-Bashir, and D. M. 

Thamiri. Clinical and epidemiological 

study of visceral leishmaniasis in a group 

of hospitaized children in Baghdad. Iraqi 

paediatric Medicine Journal, 2003;2(4): 

371-77. 

27. Zijlstra, E. E., M .S. Ali, A. M. el- 

Hassan, I. A. el-Toum,, M. Satti, H.W. 

Ghalib, E. Sondorp, and A. Winkler. 

Kala-azar in displaced people from 

southern Sudan: epidemiological, clinical 

and therapeutic findings. Trans R Soc 

Trop Med Hyg. 1991;85(3):365-9. 

28. Mittal, V., R. Bahatia, and S. Sehgal. 

Clinocoepidemiological profile of Kalaazar 

patients in Delhi. Journal of 

Communicable Diseases, 1989;21(4) : 

255-61. 

29. Soleimanzadech, G., G. H. Edrissian, A. 

M. Movahhed, Danesh, and A. Nadim. 

Epidemiological aspect of Kala-azar in 

Meshkin-Shahr, Iran (human infection). 

Bull WHO. 1993;71(6): 759-62.

Iraqi J Pharm Sci, Vol.19(2) 2010 Modified release nicotine tablet 

Development of Modified Release Nicotine Tablet Formulation for 

Treatment of Ulcerative Colitis 

Marwan Y. Al-hurr *,1 

* Department of Pharmaceutics, College of Pharmacy, University of Baghdad,Baghdad,Iraq. 

Abstract 

One of the therapeutic effects of nicotine is used as a protective against developing ulcerative 

colitis . ulcerative colitis is an inflammatory disease of the bowel affecting the superficial lining 

mucosa in the rectum and large intestine. In this study nicotine tablets were formulated as modified 

release tablets targeted to the colon. All formulas were studied for drug release , effect of diluent, 

retardant concentration, avicel grade,and compression force, the selected formula was then further 

studied for drug release in 3 different pH ( coated tablets) .The kinetic study revealed acceptable shelf 

life . Finally the selected formula was given to 6 patients in a pre-liminary clinical study which showed 

that nicotine can stabilize mild to moderate ulcerative colitis attacks. 

Key words: Ulcerative colitis, Nicotine, Modifie d release , Colon delivery. 


ﻪﻈﻴﻠﻐﻟﺍ ءﺎﻌﻣﻻﺍ ﻭ ﻢﻴﻘﺘﺴﻤﻠﻟ ﺔﻴﺤﻄﺴﻟﺍ ﺔﻴﻃﺎﺨﻤﻟﺍ ﺔﻘﺒﻄﻟﺍ ﺐﻴﺼﺗ ﻲﺘﻟﺍ ﻲﻤﻀﻬﻟﺍ ﺯﺎﻬﺠﻟﺍ ﺕﺎﺑﺎﻬﺘﻟﺍ ﺪﺣﺍ ﻲﺣﺮﻘﺘﻟﺍ ﻥﻮﻟﻮﻘﻟﺍ ﺏﺎﻬﺘﻟﺍ 

ﻰﻟﺍ ﻪﺟﻮﻣ ﺭﺮﺤﺘﻟﺍ ﺓﺭﻮﺤﻣ ﻦﻴﺗﻮﻜﻴﻨﻟﺍ ﺕﺎﻃﻮﻐﻀﻣ ﺮﻴﻀﺤﺗ ﻢﺗ ﺚﺤﺒﻟﺍ ﺍﺬﻫ ﻲﻓ . ﺽﺮﻤﻟﺍ ﺍﺬﻫ ﺭﻮﻄﺗ ﺪﺿ ﺔﻳﺎﻤﺣ ﻞﻣﺎﻋ ﻦﻴﺗﻮﻜﻴﻨﻟﺍ ﺮﺒﺘﻌﻳﻭ 

ﺓﻮﻗ ﺮﻴﺛﺄﺗﻭ , ﺓﺮﻀﺤﻤﻟﺍ ﻎﻴﺼﻠﻟ ﺭﺮﺤﺘﻠﻟ ﻪﻄﺒﺜﻤﻟﺍ ﺩﺍﻮﻤﻟﺍ ﺰﻴﻛﺮﺗ ﻭ ﺔﻟﺎﻌﻔﻟﺍ ﺮﻴﻏ ﺔﻓﺎﻀﻤﻟﺍ ﺩﺍﻮﻤﻟﺍ ﻉﻮﻧ ﺮﻴﺛﺄﺗ ﻭ ءﺍﻭﺪﻟﺍﺭﺮﺤﺗ ﺔﺳﺍﺭﺩ ﻢﺗﻭ . ﻥﻮﻟﻮﻘﻟﺍ 

ﻭ ﻲﻨﻴﺟﻭﺭﺪﻴﻬﻟﺍ ﺱﻻﺍ ﺔﻔﻠﺘﺨﻣ ﻁﺎﺳﻭﺍ ﻲﻓ ﻦﻴﺗﻮﻜﻴﻨﻟﺍ ﺭﺮﺤﺗ 

ﺚﻴﺣ ﻦﻣ ﺓﺭﺎﺘﺨﻤﻟﺍ ﻪﻐﻴﺼﻠﻟ ﻊﺳﻭﺍ ﺔﺳﺍﺭﺩ ءﺍﺮﺟﺃ ﻢﺗﻭ . ﻦﻴﺗﻮﻜﻨﻟﺍ ﺭﺮﺤﺗ ﻰﻠﻋ ﺲﺒﻜﻟﺍ 

ﺕﺮﻬﻇﺃ ﺚﻴﺣ ﺓﺭﺎﺘﺨﻤﻟﺍ ﺔﻐﻴﺼﻠﻟ ﻰﺿﺮﻣ 6 ﻰﻠﻋ ﺔﻴﻟﻭﺍ ﻪﻳﺮﻳﺮﺳ ﺔﺳﺍﺭﺩ ءﺍﺮﺟﺍ ﺎﻀﻳﺍ ﻢﺗﻭ . ﺔﻔﻠﺘﺨﻣ ﺓﺭﺍﺮﺣ ﺕﺎﺟﺭﺩ ﻲﻓ ﺎﻬﺗﺎﺒﺛ ﺚﻴﺣ ﻦﻣ 

. ﺓﺪﺸﻟﺍ ﺔﻟﺪﺘﻌﻤﻟﺍ ﻭ ﺔﻄﻴﺴﺒﻟﺍ ﻞﺣﺍﺮﻟﺍ ﻲﻓ ﺽﺮﻤﻟﺍ ﺍﺬﻫ ﺔﻳﺭﺍﺮﻘﺘﺳﺍ ﻦﻣ ﺪﻳﺰﻳ ﻭ ﺽﺮﻤﻟﺍ ﺭﻮﻄﺗ ﻊﻨﻤﻳ ﻦﻴﺗﻮﻜﻴﻨﻟﺍ ﻥﺍ ﺔﺳﺍﺭﺪﻟﺍ 

Introduction 

Colonic drug delivery has gained 

increased importance not just for the delivery 

of the drugs for the treatment of local disease 

associated with the colon like Crohns disease , 

ulcerative colitis and irritable bowel 

syndrome..etc., but also for the potential 

systemic delivery of proteins and therapeutic 

peptides. The large intestine, though difficult 

to reach by preoral delivery is still deemed to 

the delivery of agents to cure the local disease 

of the colon (1,2) . Colonic delivery formulations 

are in general may be designed to provide 

either the burst release or for sustained/ 

prolonged release once reaching the colon (3) . 

The proper selection of a formulation approach 

depends upon several important factors (4) 

which are : the pathology and pattern of the 

disease , the physicochemical and 

biopharmatical properties of the drug and 

finally the desired release profile of the active 

ingredient. The most common physiological 

factors considered in the design of delayed 

release colonic formulations is pH gradient of 

the gastrointestinal tract (5,6) .delayed release 

formulations such as single unit or 

multiparticulate system for colon targeting , 

nanoparticulate system, microspheres, 

pelletsand beads, coating with pH sensitive 

polymers, embedding in matrices and 

bioadhesive systems (7) can be considered for 

75 

the design of colon deliveryformulations. A 

wide array of polymers has been employed as 

drug release retarding agents each of which 

presents a different approach to matrix 

concept. Plastic matrix system , due to their 

chemical inertness and drug embedding ability 

, have been widely used for sustaining the 

release of drugs. Plastic polymers e.g. 

ethylcellulose and acrylates, which are capable 

of forming insoluble or skeleton matrices, have 

been widely used for controlled release of 

drugs due to their inertness and drug 

embedding ability . Acrylate polymers are 

widely used as tabletcoating and as retardents 

for drug release in sustained release 

formulations (8) . Ulcerative colitis (U.C) is an 

inflamma tory disease of the bowel affecting 

the superficial lining mucosa in rectum and 

large intestine. The disease typically starts 

from the rectum and continues through the 

large bowel sparing the deeper layers of the 

intestinal wall 

(9) . A variety of anti- 

inflamma tory, immunosuppressive, and 

biological agents have been used to induce and 

/ or maintain remission in UC . Sulfasalazin, 

olsalazine, balsalazide, oral and rectal 

mesalamine and topical corticosteroids are the 

standard first line therapies for UC.Patients 

who fail to respond to these agents are usually 

treated with oral corticosteroids. 

1Corresponding author E- mail : m@marwanpharm.com , mngateway2000@yahoo.com 

Received : 1/ 9/ 2010 

Accepted : 11/12/2010


There is a need for additional first line 

treatments in patients with UC and for 

alternatives to corticosteroid therapy in 

refractory patients. Nicotine may be such an 

agent for which epidemiologic studies have 

shown that smoking protects against the 

development of UC and controlled clinical 

trials have demonstrated that transdermal 

nicotine is efficacious for active UC (10-15) . 

Nicotine is a drug obtained from the plant 

Nicotina tabacum. It’s a weak base. Its 

available as a colorless to pale yellow oily 

liquid with an unpleasant tobacco-like odour 

and burning taste (16) . Its most preferred that 

absorption of nicotine occurs predominantly in 

the colon. So post-gastric delayed release 

composition, the composition will pass through 

the small intestine in about 4-8hr and will 

resides in the colon for about 48hr. further 

more nicotine is absorbed more slowly in the 

colon than in the small intestine, therefore, 

nicotine delivered for absorption 

predominantly in the colon will be absorbed 

more slowly over a sustained period and will 

give rise to more uniform plasma 

concentration. By predominant absorption 

from the colon we mean to include preferably 

80-90% of the total dose of nicotine (17) .The 

present study is to develop modified release of 

20mg nicotine tablets as a targeted delivery 

system in the colon with pre-liminary clinical 

study. Eudragit RS PM as a retardant polymer 

which is responsible for the sustained release 

behavior of the drug was used for preparation 

of the core matrix and the selected formula was 

76 

targeted to release in the colon by using enteric 

coating ( a mixture of Eudragit L and S ). 

Materials and Method 

Nicotine (Sigma) gifted from 

Pharmacognosy Department. College of 

Pharmacy/ University of Baghdad, Sodium 

hydroxide (Fluka chemie AG. Buchs/Scheiz), 

Dibutylphthalate ( USB, B. Brussels,Belgium), 

Hydrochloric acid , Iospropanol, and 

Orthophosphoric acid (Riedal De Haen Ag 

Seelze Hanover), Polyvinylpyrrolidone ( PVP 

K30 ) , Acetone, Potassium dihydrogen 

phosphate, Ethanol 99% ( BDH chemicals, 

Ltd, Liverpool, England )Microcrystalline 

cellulose- Avicel ® - PH101, PH302, PH200 

(FMC Corperation, Pennsylvania, USA), 

Eudragit ® L100, S100, RS PM – Rhö m 

Pharma GmbH Weiterstadt, 

Germany),Trisodium phosphate, Talc 

(Hopkins and Williams Ltd. England), coloring 

agent ( deep orange lakes) Zinc stearate 

(Barlocher, GmbH, Germany), Disodium 

hydrogen phosphate, Mannitol, Starch ( Merk, 

Germany). Table (1) summarizes 8 formulas to 

prepare modified release nicotine tablets by 

wet granulation method with alcohol. A known 

weight of the granules were mixed with a 

speicified amount of Zn stearate ( 1%) in a 

well closed container and compressed into 

tablets using 9mm punches ( tablet machine 

single punch – Korch, type EKO, Erweka 

GmbH, Kr Offenbanch/ Germany ). 

Table 1 : Different formulas of nicotine pre pared as modifie d re le ase tablets. 

Constituents 

1 2 3 

Formulas ( mg) 

4 5 6 7 8 

Nicotine 20 20 20 20 20 20 20 20 

PVP (10%) 20 20 20 20 20 20 20 20 

Avicel ® PH302 40 40 40 40 40 40 

Eudragit ® RS PM 20 20 40 60 20 20 20 20 

Starch 100 100 100 100 100 100 100 

Zinc stearate 1% 1% 1% 1% 1% 1% 1% 1% 

Mannitol 100 

Avicel ® PH101 40 

Avicel ® PH200 40 

Compression force 4Kg 4Kg 4Kg 4Kg 4Kg 4Kg 6Kg 8Kg 

Total weight of final tablet 200 200 200 200 200 200 200 200 

Evaluation of the prepared tablets 

The following parameters were used to 

compare the prepared formulas to obtain the 

final selected formula. 

1. Effect of dilue nts type on the pe rcent 

re leased of nicotine . 

Formula 1 and 2 were used to study the 

effect of two different diluents ( starch 

andmannitol ) on drug release. 

2. Effect of Eudragit RS PM conce ntration. 

Formula 1,3 and 4 were utilized to study 

the effect of different concentrations (10% 

, 20% and 30% respectively) of Eudragit 

RS PM on the drug release.


3. Effect of Avicel grade. 

Formulas 1,5 and6 which contain Avecil 

PH 302, PH101,and PH200 respectively 

were used to study the effect of different 

grades of Avecil as channeling agent on the 

drug release. 

4. Effect of compre ssion force on nicotine 

re lease. 

Formulas 1, 7and 8 were used to study the 

effect of changing the compression force 

4Kg, 6Kg and 8Kg respectively on the drug 

release. 

Drug release : ( USP dissolutuion apparatus 

type II, Coply scientific Ltd, England) 

The Medium used : pH 6.8 phosphate 

buffer 750ml, Apparatus II , rotation 75 rpm, 

with a Sampling time: 1,2,3,4 and 6hr. The 

amount of nicotine dissolved was determined 

spectrophotometecally at λmax. 260nm of 

filtered samples.( UV visible 

spectrophotometer, Carrywin UV. Varian, 

Australia). The samples were diluted with 

dissolution medium if necessary and compare 

with a standard solution having a known 

concentration of nicotine in the same medium. 

Assay: HPLC analytical method 

The chromatographic separation was 

achieved on a C-18 colum with UV detection 

at 260nm the HPLC system comprised a 

(Waters 1500 series HPLC pump( USA) , 

waters 2487 dual λ absorbance detecter, water 

breeze soft ware.) was operated at ambient 

temperature and used citrate buffer: methanol 

(85: 15 % v/v ) with an appearent pH 2.4 as the 

mobile phase. The flow rate was maintained at 

0.7 ml / min. and the retention time 6.94 

min. (18) . 

Preparation of coating formula 

The coating solution was prepared 

according to the Rhom pharma 

recommendations (the manufacturer) as 

follows: 

Formula : 

Eudragit * 6gm 

isoprpanol 115.7gm 

acetone 77.1gm 

Dibutylphthalate 1.2gm 

talc 3.25gm 

Magnesium stearate 0.25gm 

Color 0.25gm 

Titanium oxide 1.55gm 

Semithicone Q.S. 

*mixture of Eudragit L 100 and S 100 in a 

ratio 1:2 

77 

The final coating solution formula prepared 

was 205.3gm. 

Procedure 

The formula was prepared by mixing the 

solvents together with the plasticizer ( 

dibutylphthalate) in a high shear mixer MLW 

type LR10 ( VEB ML W Prufgerate-werk, 

Medingen/ Stizfrtal/ Germany). Eudragit 

mixture was added slowly at room temperature 

, the powder was thoroughly wetted and care 

was taken to ensure that nothing settled at the 

bottom or formed lumps . mixing lasted for at 

least 30mins, until the solution was clear, the 

fillers were added step by step . 

Calculation 

( 19) 

required 

of the amount of lacquer 

A specific thickness of coating is 

required based on the purpose of the coating 

and the amount needed depending on the 

surface area of the cores which may be 

calculated from the following equation 

assuming tha t the tablets are cylindrical in 

shape: 

S.A= Л ( d.h + ½ d 2 ) 

Where d is the diameter (mm) 

h is the height (mm) 

S.A is the surface area (mm 2 ) 

The nicotine tablets had a diameter of 9mm 

and a surface area approximately equals to 

240mm 2 . 

Since 3-5mg lacquer / cm 2 of tablet cores 

required to produce a core resistant to acidic 

environment ( enteric coated tablets). So 

multiplying the surface area of the tablet core 

by the amount required and dividing it by the 

weight of tablet, the quantity o f the lacquer to 

be applied as a percentage will be obtained. 

The amount to be applied 

( % dry lacquer substace)= S.A( mm 2 ) / w ( 

mg) x ( mg/ mm 2 ) 

= 240 / 200 x 5 

= 6% 

Tablet coating 

The selected Formula was coated by 

dipping method . each tablet was held by 

forcipes and dipped in the coating lacquer in 

and out 15-20 times , the coat was dried by a 

.( 20) 

stream of warm air between each dip 

( 21) 

Dissolution study of the coated tablets 

The dissolution rate of the selected 

formula for nicotine ( coated tablets) was 

determined using USP apparatus at 37+ 0.5 o C 

with paddle and the rotation speed was set at 

75rpm in order to simulate the pH change of


the GIT , pH change dissolution procedure was 

applied as follows: 

2hr. testing in 0.1N HCl solution followed by 

testing for one hour in phosphate buffer pH 4 

obtained by adding 195ml 0.2M tribasic 

sodium phosphate solution during which 

samples were withdrawn at specified times and 

replaced immediately by fresh medium. Then 

the medium was changed to pH 6.8 by adding 

55ml 0.2 M tribasic sodium phosphate adjusted 

by 2N NaOH or 2N HCl if required . samples 

were withdrawn at different time intervals and 

analyzed spectrophotometrically at 260nm. 

Kinetic study 

Effect of temperature: 

The effect of temperature on the 

degradation of the selected formula of nicotine 

modified release tablets was studied . The 

study was done by storing 90 tablets in ovens 

(Mermert UL 80 ( Rostfrei, Schwach, 

Germany) )at different temperatures 50 o C, 

60 o C and 70 o C. 

Samples were taken at specified time intervals 

and analyzed for nicotine. Since the 

degradation of the drug follows 1 st order 

kinetics, the expiration date t10% at 25 o C could 

be calculated using the following equation : 

T10% = 0.105/ k 25 o C 

Pre-liminary clinical study 

Before giving the preparation we 

obtained a written consent of the patients who 

were included in this study. The modified 

release nicotine tablets of the selected formula 

was given to 6 patients suffering from mild to 

moderate ulcerative colitis (high mucus 

secretions, irritable bowl syndrome, mild to 

moderate bleeding and gases). All patients 

were put on 20mg single dose of nicotine for 2 

weeks. The patients were evaluated 

clinically(physical examination and endoscopy 

) before and during treatment ( physical 

examination ) under the supervion of Dr. 

Mumtaz k. Hanna at his private clinic. 

Results and discussion 

Effect of diluent’s type on nicotine release 

Although diluents are normally thought 

to be inert ingredients they can significantly 

affect the biopharmaceutical, chemical and 

physical properties of the final tablets .(22) 

Formula 1and 2 which contain maize starch 

and mannitol as diluents, it was seen that 

starch gave the best drug release compared 

with mannitolas shown in fig. 1 .This behavior 

may be attributed to the swellability property 

of starch when compared with mannitol which 

the release is due to water solubility. (23) 

78 

% Drug Released 

Figure 1: Effect of dilue nt type on the 

release of nicotine at pH 6.8 and 37 o C 

Effect of Eudragit concentration: 

Eudragit RS PM can be in corperated in 

a percent of 10-30% (w/w) by weight to 

provide suitable granules and matrix tablets. 

The amout of Eudragit RS PM to be added , 

depends upon the solubility characteristics of 

the drugs and the rate required (20). Formulas 1 , 

3 and 4 which contain 10% , 20% and 30 % 

w/w of Eudragit ( as a retardant) respectively 

were evaluated . formula 1 gave the best 

modified release of nicotine when compared 

with the requirements of drug release to the 

colon (17). The results from dissolution profiles 

of formulas 1 ,3 and 4 indicates that the 

retardant content affects the release of nicotine 

from the tablet, this result is in a consistent 

with the results obtained when Eudragit RSPM 

polymer was used as a retardant material for 

diclofenac sodium and indomethacin tablets. (24) 

It appears that the amount of retardant needed 

is 10 % as shown in fig. 2 .This is in 

agreement with the reported data which 

indicated that the retardation effect on the 

release of drug is dependent on the amount of 

Eudragit included (25) 

%Drug Released 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 1 2 3 4 6 

Time ( hr.) 

0 1 2 3 4 6 

Time ( hr.) 

10% eudragit 

RSPM 

20% eudragit 

SRPM 

30% eudragit 

RSPM 

starch 

Figure 2 : Effect of Eudragit RS PM 

concentration on the percent re leased of 

nicotine at pH 6.8 and 37 o C. 

mannitol


Effect of Avicel grade 

Avicel is microcrystalline cellulose , it is 

partially depolymerized cellulose prepared by 

treating alpha- cellulose obtained as a pulp 

fibrous plant material with acids. The grade of 

Avicel depends on its normal loss on drying , 

bulk density and degree of polymerization 

values. (26 ) The results showed that formula 1 

in which Avicel PH 302 present, gave the best 

dissolution profiles while the difference in the 

release that occurred in the other formulas 5 

and 6 which contain Avicel PH 101 and PH 

200, respectively. is due to the difference in the 

porosity , surface area, particle size and density 

of Avicel as stated (27 ) as shown in fig. 3 

%Drug Released 

100 

80 

60 

40 

20 

0 

0 1 2 3 4 6 

Time ( hr.) 

Figure 3 : Effect of Avicel grade on the perce nt 

release d of nicotine at pH6.8 and 37 o C. 

Effect of compression force 

Compression forces affect the hardness 

of a tablet and its thickness at a constant die fill 

as additional compression force is applied , the 

hardness values increase and the thickness 

decrease and the decrease as shown in fig.4 

( 19) 

, thereby the dissolution of the drug decrease in 

surface area and increase in hardness. This 

may be related to the low porosity resulted 

from the increase in compression force. (8) 

Therefore, formula 1 was selected because it 

gave the best drug release profile which 

complies with absorption properties of nicotine 

from the colon and it was further investigated 

for enteric coating, kinetic study and preliminary 

clinical study (17). 

Figure 4 : Effect of compression force on 

perce nt release d of nicotine at pH 6.8 and 

37 0 C. 

Avicel 

PH302 

Avicel PH 

101 

Avicel PH 

200 

79 

Coating formula 

The tablets showed good appearance with 

no signs of craking or splitting or peeling. 

Induction of hydrophobic materials and inert 

fillers ( Mg stearate , talc , titanium oxide , 

aluma lakes with an orange color ) these fillers 

facilitate processing of the lacquer by reducing 

its stickness, help to smooth the permeability 

to water and decrease the tackiness of the 

drying lacquer. In addition they reduce the 

permeability of the film as long as the 

mechanical strength is maintained thereby 

enhancing the enteric properties of the film ( 

28,29 ). Formula 1 was coated to target the drug 

to the colon. The coated tablets showed no 

drug release in 0.1 N HCl for 2hr. period of 

the test and the release of the drug increased 

rapidly when the pH changed to 6.8 as shown 

in fig 5. 

% Drug Released 

100 

pH1.2 

80 

pH 4 pH 6.8 

60 

40 

20 

0 

1 2 3 4 5 6 7 8 9 10 

Time (hr) 

Figure 5 : The cumulative percent of enteric 

coate d nicotine table ts release at diffe rent 

pH-media at 37 o C. 

Kinetic study 

Effect of temperature 

The stability of the coated modified 

release tablets were studied at different 

exaggerated temperatures ( 50 o C , 60 o C and 

70 o C ) for 3 months. Fig 6 shows the change in 

the log percentage remaining of nicotine versus 

time at different temperatures. The obtained 

profiles were linear , indicating that nicotine 

degradation follows 1 st order kinetics. The 

slopes of these lines were determined and the 

calculated rate constants (k) are summarized in 

fig (6). Arrhenius plot was then constructed as 

shown in fig 7. the linearty of the curve 

indicates its utility in predicting the rate of 

degradation at lower temperatures. since the 

degradation of the drug followed 1 st order 

kinetics the expiration date can be calculated at 

25 o C for nicotine coated matrix tablets and it 

was 52 month.


ln percent remaining 

Figure 6 : Perce nt remaining of drug versus 

time. 

log K 

4.65 

4.6 

4.55 

4.5 

4.45 

4.4 

4.35 

0 

-0.5 

-1 

-1.5 

-2 

-2.5 

-3 

0 1 2 3 

Time( month) 

Fig7: Arrhinous plot for expiration date 

estimation of nicotine tablet (Formula 1). 

50 C 

60 C 

70 C 

0.0028 0.0029 0.003 0.0031 0.0032 0.0033 0.0034 

Clinical study 

Nicotine was given to 6 non smoking 

patients ( 5 males and 1 female ) with an age 

range of 27- 65 yr. The patients took 20mg 

once a day for 10 days suffering from mild to 

moderate ulcerative colitis. The out come of 

this preliminary study indicates that 67% ( 4 

out of 6 patient ) were responsive to nicotine 

therapy ( relief of bleeding and most sign and 

sympto ms) although all patients suffered from 

adverse effect reaction towards nicotine 

therapy because the patient were non smokers 

(lightheadedness or dizziness, nausea, 

headaches, central nervous system stimulation 

and tachycardia). (30) further studies in the 

future should be done including in vivo 

nicotine blood concentration to optimize the 

dose. 

References 

1. Davis SS. Overcoming barriers to the oral 

administrationof peptide drugs. Trends 

Pharm Sci 1990;11:353-5. 

2. Vandenmooter G, Kinget R. Oral colonspecific 

drug delivery: A review. Drug 

Delivery 1995;2:81-93. 

3. Pinhasi A, Gomberg M, Avramoff A. 

US200467030442004. 

1/T 

4. Brahma NS. Modified – release solid 

formulations for colonic delivery. Recent 

pat drug deliv formul 2007;1:53-63. 

5. Evans DF, Pye G, Bramley R,Clark AG, 

Dyson TJ, Hardcastle JD. Measurement 

of gastrointestinal pH profiles in normal 

ambulant human subjects. Gut 1988; 

29:1035-41. 

6. Nugent SG, Kumar D, Rampton DS, 

Evans DF. Intestinal luminal pH in 

inflamma tory bowel disease: Possible 

determinants and implications for therapy 

with aminosalicylatesand other drugs. 

Gut 2001;48:571-7. 

7. Das S, Deshmukh R, Jha AK. Role of 

natural polymers in the development of 

multipartculate systems for colon drug 

targeting. Systematic Reviews in 

pharmacy 2010;1:1:70-85. 

8. Uddin MB, Chowdhury JA, Azam KR, 

Islam MK. Investigation of the effects of 

different physiochemical parameters on 

in vitro release kinetics of theophylline 

from Eudragit NE 30 and Eudraget RS 

30D matrix tablets. J. pharm. Sci. and Res 

2010; 2(4):240-46. 

9. Friedmans S, Blumberg R, Inflamma tory 

bowel disease In ; Fauci AS , Braunwald 

E, Kasper DL, Hauser SL, Longo DL, 

Jameson JL, et al , editors Harrison’s 

principles of internal medicine 17 th ed. 

Mc Graw Hill; 2008 p. 1777-89. 

10. Wolf JM, Lashner BA, Inflamma tory 

bowel disease: sorting out treatment 

options. Cleveland Clin. J. Med. 

2002;69:621-31. 

11. Kozuch PL, Hanauer SB. Treatment of 

inflamma tory bowel disease: a review in 

medical therapy. World J. Gastroenterol. 

2008;14:354-77. 

12. Lashner BA, Hanauer SB, Silverstien 

MD. Testing nicotine gum for ulcerative 

colitis patients: experience with single 

patient trials. Dig. Dis. Sci. 1990;35:827- 

32. 

13. Pullan RD, Rhodes J, Ganesh S, Manni 

V, Morris JS, Williams GI, et al. 

Transdermal nicotine for active ulcerative 

colitis . N Eng. J. Med. 1994; 330: 811-5. 

14. Guslandi M, Nicotine treatment of 

ulcerative colitis . Br. J. Pharmacol. 

1999;48:481-4. 

15. Boyko EJ, Perera DR, Koepsell TD, et al. 

Effect of cigarette smoking on the clinical 

course of ulcerative colitis. Scand. J. 

Gastroenterol. 1988;23:1147-52. 

16. Remington: The science and practice of 

pharmacy, 21 st ed. Lippincot,Williams 

and Wilkins, USA (2006), 1391


17. Rhodes J., Evans B., Rhodes P., Sandorn 

W.; Intestinal absorption of nicotine 

responsive conditions; J. Mayo education 

and research: 1999; 6:238. 

18. Dash A.K., Wong S.T., Liquid 

chromatographic method for the 

determination of nicotine in 

pharmaceutical formulation. J. 

Chromatogr. A. 1996: 749: 81-85. 

19. Rohm pharma information sheets , Rohm 

Pharma GMbH, Weiterstadt; info. 

Eudragit L100-55 a/e , Eudragit S100 4/e 

, 1/s-7e 2006. 

20. Lachman L., Lieberman H.A., Kanig 

J.A; the theory and practice of industrial 

pharmacy 3 rd ed. Leo and febiger . chap. : 

1986;12:372. 

21. USP 2007 compact disk. 

22. Banker G., Baley P., baley G.; tablet 

formulation and design, liberman and 

Lachman (Eds) pharmaceutical dosage 

form tablets, vol.1 , Marcel Deckker, 

New York 1982; 61-108. 

23. Laako R., Eerkainen S.;Effect of core 

components on indomethacin release 

fro m film-coated granules; int.J. Pharm. 

:1991; 67(1):79-88. 

24. Al-hurr M.Y. ; formulation andclinical 

study of diclofenac sodium and 

81 

indomethacin as an oral modified release 

tablets, thesis for M.Sc. degree, College 

of Pharmacy, University of Baghdad, 

2003. 

25. Khanfar M.S., Salem M.S. Najib N.M., 

Pillai G.K, Dissolution Behavior of 

sustained release formulation of 

indomethacin with Eudragit RS Acta. 

Pharm. Hung 1997; 67(6):235-239. 

26. Handbook of pharmaceutical excipients 

6 th ed. Washington DC., American 

pharmaceutical association 2009. 

27. Al-Ebady M.N. ; Design and in vitro 

evaluation of Prednisolone tablets as a 

potential colon delivery system, thesis for 

M.Sc. degree, college of pharmacy, 

university of Baghdad, 2006. 

28. Chatfield H.W,; Science of surface 

coating, Van Nostrand, NewYork 1962. 

29. Kassab H.J., Formulation and clinical 

study of sulphasalazine tablets, M.Sc. 

thesis College of Pharmacy, University of 

Baghdad, 1999. 

30. Sandborn WJ, Tremaine WJ, Offerd KP, 

Transdermal nicotine for mildly to 

moderately active ulcerative colitis . A 

randomized double – blind , placebo- 

controlled trial . Ann Intern Med :1997; 

126: 364 -71.

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