preliminary phytochemical and antibacterial screening of the ...

Katsayal and Lamai Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 121 – 125 

Nigerian Journal of Pharmaceutical Sciences 

Vol. 8, No. 2, October, 2009, ISSN: 0189-823X 

All Rights Reserved 

PRELIMINARY PHYTOCHEMICAL AND ANTIBACTERIAL 

SCREENING OF THE ETHANOLIC STEM BARK EXTRACT OF 

PHYLLANTHUS MUELLERIANUS 

*U. A. Katsayal and R. S. Lamai 

Department of Pharmacognosy and Drug development, Ahmadu Bello University, Zaria, Nigeria 

*Author for Correspondence: uakatsayal@abu.edu.ng, 0803 674 5451 

ABSTRACT 

Throughout the history of drug development, natural products have provided a fundamental source of drugs for 

fighting infections. In this paper therefore, the phytochemical constituents present in the stem bark extract of 

Phyllanthus muellerianus were screened and its antibacterial properties in vitro were determined using agar cup 

plate method on selected organisms, namely Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and 

Staphylococcus aureus. The phytochemical screening of the ethanolic extract of the stem bark revealed the 

presence of carbohydrates, saponins, flavonoids, tannins, alkaloids, steroids and triterpenes, cardiac glycosides 

and anthraquinones. The extract revealed a good concentration dependent antibacterial activity against the test 

organisms similar to that of the standard drug, gentamicin with the exception of Ps. aeruginosa. The observed 

effects could not be unrelated to the presence of the chemical constituents in the extract. The outcome of this 

study could therefore justify the ethnomedicinal uses of P. muellerianus. 

Keywords: 

Phyllanthus muellerianus, Euphobiaceae, phytochemical screening, antibacterial screening 

INTRODUCTION 

The Phyllanthus genus contains over 600 

species of shrubs, trees and annual or 

biennial herbs distributed throughout the 

tropical and subtropical regions of both 

hemispheres (Taylor, 2003). The plant, 

Phyllanthus muellerianus (Kuntze) Exell, 

is a glabrous shrub or woody climber, 

occasionally arborescent, found in 

savannah and drier secondary forests 

common in coastal thickets and scrub and 

widespread in parts of tropical Africa 

(Hutchinson, 1954) (Plate 1). It has been 

used traditionally in the treatment of 

various ailments. In Tanganyika, a root 

decoction is taken by draught for hard 

abscesses, and the powdered dried root and 

bark is sprinkled on wounds as a dressing 

(Burkill, 1985). After removal of surface 

spines, twigs are used as chewing sticks in 

Nigeria and in Ivory Coast to prevent 

toothache (Burkill, 1985). In Congo, the 

dried bark powder is taken for colds and 

sinusitis (Burkill, 1985). Together with 

powdered roots, a bark decoction in 

draught and enema is used for throat 

troubles and glandular fevers (Burkill, 

1985). Other species include Phyllanthus 

amarus, Phyllanthus sellowianus (now 

varieties of Phyllanthus niruri), 

Phyllanthus niruri and Phyllanthus 

121


simplex of which Phyllanthus niruri has 

been studied extensively and shown to 

possess antibacterial property (Taylor, 

2003). The past few decades have 

experienced an overwhelming increase in 

global interest on the practice of traditional 

medicine and its use of medicinal plants to 

treat illness (Akarele, 1994). Plant-derived 

preparations and isolated phytochemicals 

or their model derivatives may be 

potentially useful to treat infectious 

diseases, especially in the light of the 

emergence of drug-resistant 

microorganisms and the need to produce 

more efficacious and cost-effective 

antimicrobial agents (Ncube et al., 2008). 

The use of antibiotics has revolutionized 

the treatment of various bacterial 

infections. However, their indiscriminate 

use has led to an alarming increase in 

antibiotic resistance among 

microorganisms (Hart and Karriuri, 1998). 

This necessitates the need for development 

of novel antimicrobials (Chopra et al., 

1997). One way of preventing antibiotic 

resistance of pathogenic species is 

development of new compounds that are 

not based on existing synthetic 

antimicrobial agents (Rojas et al., 2006). 

The present study was therefore carried out 

to evaluate the preliminary phytochemical 

and antibacterial properties of the 

ethanolic extract of P. muellerianus stem 

bark, with the aimed of providing a lead 

compound(s) for the development of new, 

novel antibacterial agent(s). 

Plate 1: Phyllanthus muellerianus in its Natural Habitat 

MATERIALS AND METHODS 

Collection of Plant Material 

The plant, Phyllanthus muellerianus was 

collected from behind Ramat Hal, Ahmadu 

Bello University main campus in the 

month of February, 2009 and authenticated 

at the Herbarium Unit of the Department 

of Biological Sciences, Ahmadu Bello 

University, Zaria where it was assigned a 

voucher specimen number (925) by 

Mallam Umar Gallah. 

Solvent and Drugs and Test Organisms 

The solvent used for the extraction of the 

plant material is of Analar grade, and was 

purchased from Sigma-Aldrich 

representative in Nigeria. The standard 

drug used in this experiment, gentamicin, 

122


was obtained from Lek Pharmaceutical 

Company Limited, Slovenia. The test 

organisms were obtained from the 

Department of Pharmaceutics and 

Pharmaceutical Microbiology, Ahmadu 

Bello University, Zaria. 

Processing of the Plant Material 

The stem bark was removed manually; air 

dried under shade and powdered using a 

wooden pestle and mortar. The preferable 

solvent used by the local people is mostly 

water and/or local gin; therefore, the 

extraction of the plant material was 

performed by weighing 100 g of the air 

dried, milled root-bark of Phyllanthus 

muellerianus and exhaustively extracted 

by repeated maceration with 70 % v/v 

aqueous-ethanol at room temperature. The 

alcoholic solutions were combined and 

evaporated to dryness at 45 °C under 

reduced pressure to obtain a dry extract 

which was weighed to yield 8.7 % and 

stored in a desiccator before used. 

Phytochemical Screening 

Phytochemical screening of the ethanolic 

extract was carried using standard methods 

(Evans, 2002; Sofowora, 2008) to screen 

for the presence of various chemical 

constituents. 

Test Organisms 

Standard type cultures of Bacillus subtilis 

(NCTC 10342), Escherichia coli (ATCC 

11775), Pseudomonas aeruginosa (ATCC 

10145) and Staphylococcus aureus (ATCC 

021001) were kindly obtained from the 

Department of Pharmaceutics and 

Pharmaceutical Microbiology, Ahmadu 

Bello University, Zaria were used in this 

study. 

Standardization of Inoculum 

The bacterial cultures were prepared by 

transferring 2 to 3 colonies into bacteria 

growth medium (Luria broth agar) and 

incubated at 37° C for 14 hrs before use as 

described by Eloff (1998). The cells were 

maintained in Luria agar (10 g/l Bacto 

tryptone, 5 g/l Bacto yeast extract, 10 g/l 

NaCl at pH 7.0) in glass slant bottles. 

Antimicrobial Activity Determination 

Antibacterial screening was performed 

using an Agar diffusion method as 

described by Hugo and Russell, 1998. The 

plates were prepared in duplicate using 

nutrient agar. Three concentrations (10 

mg/ml, 50 mg/ml and 100 mg/ml) of the 

extract in sterile water were prepared. 

Standard gentamicin, 2.5 mg/ml was 

employed as positive control. The plates 

were inoculated with standardized cultures 

of B. subtilis, S. aureus, E. coli and Ps. 

aeruginosa by flooding. Using the cup 

plate method described by Hugo and 

Russell (1998), a sterile cork borer was 

used to make 4 holes of about 8 mm in 

diameter into the solidified agar in each 

plate. 0.1ml of each concentration of the 

extract and the reference drug were applied 

in separate holes in each prepared plate. 

The plates were covered and kept for an 

hour to allow the extract and the standard 

drug to diffuse, before transferring to an 

incubator at 37° C for 18 hours after which 

zones of inhibition were measured to the 

nearest millimetre. 

RESULTS AND DISCUSSION 

The preliminary phytochemical screening 

revealed the presence of alkaloids, tannins, 

flavonoids, anthraquinones, saponins, 

steroids and reducing sugars (Table 1). 

Plants used in the treatment of disease are 

said to contain active principles which are 

phytochemicals with biological activity, 

some of which are responsible for the 

characteristic odours, pungencies and 

colours of plants while others give a 

particular plant its culinary, medicinal or 

poisonous virtues (Evans, 2002). From the 

result of the phytochemical screening, the 

extract was found to contain saponins, 

flavonoids and tannins which are well- 

123


known to possess antimicrobial activities 

(Akiyama et al, 2001). Flavonoids have 

been reported to be synthesized by plants 

in response to microbial infection and have 

been shown to have antibacterial activities 

(Kujumgiev et al., 1999). Tannins were 

also reported have demonstrated activity 

against bacteria (Akiyama et al, 2001). 

These kinds of compounds were found to 

be present in the extract of this plant 

(Table 1). The extract was found to 

demonstrate a good antibacterial activity 

against Bacillus subtilis, Staphylococcus 

aureus and Escherichia coli with 

exception of Pseudomonas aeruginosa 

(Table 2). This was measured using zone 

of inhibition with a measurement of 

greater than or equal to 10 mm indicating 

good antibacterial activity (Kujumgiev et 

al., 1999). A concentration dependent 

activity was exhibited by the extract 

against these organisms, an increase in 

concentration giving rise to an increase in 

the zone of inhibition. Evaluation of the 

leaf extract was also found to show good 

antibacterial activity at high 

concentrations, with lowest activity against 

S. aureus. Both leaf and bark extracts have 

been shown to have high antibacterial 

activity (Doughhari and Sunday, 2008). 

Chloroform leaf extract of this plant was 

also shown to have good activity against 

C. albicans, S. aureus and E. coli (Onocha 

et al, 2003). Other species of this genus 

were also found to demonstrate 

antibacterial and even antiviral activity 

(Taylor, 2003). The findings of this study, 

could therefore justify the use of this plant 

in traditional medicine in the treatment of 

bacterial infections. 

Table 1: 

Preliminary Phytochemical screening of the ethanolic stem barks extract 

of P. muellerianus 

Constituents 

Inference 

Alkaloids + 

Anthraquinones + 

Cardiac glycosides + 

Flavonoids + 

Reducing sugars + 

Saponins + 

Tannins + 

Unsaturated steroids 

And triterpenes + 

Key: + = Present 

Table 2: 

Antibacterial activity of the ethanolic stem bark extract of P. muellerianus 

Diameter of zone of inhibition (mm) 

Extract/Drug B. subtilis S. aureus E. coli Ps. aeruginosa 

(Mg/ml) 

10 16.5 14.0 0.0 0.0 

50 19.0 17.5 14.5 0.0 

100 19.0 20.0 16.0 0.0 

Gentamicin 

(2.5) 

40.0 42.0 40.0 42.5 

Cork Borer Diameter: 8 mm 

124


ACKNOWLEDGEMENT 

We thank the staff of the Herbarium Unit 

of the Department of Biological Sciences, 

Ahmadu Bello University, Zaria for 

authenticating the identity of the plant and 

Mr. Ezekiel of the Department of 

Pharmaceutics and Pharmaceutical 

Microbiology, Ahmadu Bello University, 

Zaria for his technical assistance in 

carrying out the antimicrobial screening. 

REFERENCES 

Akerele, O. (1994). Nature's Medicinal Bounty: Do 

not throw it away. World Health Forum 14: 390- 

395. 

Akiyama, H.; Fujii, K.; Yamasaki, O.; Oono, T. 

and Iwatsuki, K. (2001). Antibacterial Action of 

Several Tannins Against Staphylococcus aureus, 

Journal of Antimicrobial Chemotherapy, 48 (4): 

487- 491. 

Burkill, H. M. (1985). The Useful Plants of West 

Tropical Africa, Vol. 2, Royal Botanic Gardens, 

Kew. 

Chopra, I., Hodgson, J., Metcalf, B., Poste, G. 

(1997). The Search for Anti-bacterial Agents 

Effective Against Bacterial Resistance to Multiple 

Antibiotics. Antimicrob. Agents Chemother. 41: 

497-503. 

Doughari, J. H. and Sunday, D. (2008). 

Antibacterial Activity of Phyllanthus muellerianus, 

Pharmaceutical Biology, 46 (6): 400-405. 

Evans, W. C. (2002). Trease and Evans 

Pharmacognosy, (15 th edition), W.B. Saunders 

Company Ltd., London, Pp. 191-393. 

Hart, C. A., Karriuri, S. (1998). Antimicrobial 

Resistance in Developing Countries. Br. Med. J., 

317: 421-452. 

Hugo, W. B. and Russell, A. D. (1998). 

Pharmaceutical Microbiology, 6 th edition, 

Blackwell Sciences Ltd, Oxford, P. 24. 

Eloff, J. N. (1998). A Sensitive and Quick Method 

to Determine the Minimum Inhibitory 

Concentration of Plant Extracts for Bacteria. Planta 

Medica, 60: 1-8. 

Hutchinson, J., and Dalziel, J.M. (1954). Flora of 

West Tropical Africa, 2 nd edition, Vol. 1 Part 1, 

Crown agents for Oversea Governments and 

administrations, Mill bank, London, p 384. 

Kujumgiev A., Tsvetkoval, Serkedjievay, Bankova 

V., Christo, R. and Popov, S. (1999). Antibacterial, 

antifungal and antiviral activity of propolis 

geographic origin, Journal of Ethnopharmacology 

44: 35- 40. 

Ncube, N. S., Afolayan, A. J., Okoh, A. I. (2008). 

Assessment Techniques of Antimicrobial 

Properties of Natural Compounds of Plant Origin: 

Current methods and future trends. Afr. J. Biotech. 

7: 1997-1806. 

Onocha, P. A., Opegbemi, A. O., Kadri A. O., 

Ajayi, K. M. and Okorie, D. A. (2003). 

Antimicrobial Evaluation of Nigerian 

Euphobiaceae Plants 1: Phyllanthus amarus and 

Phyllanthus muellerianus leaf extracts, Nigerian 

Journal of Natural Products and Medicine 7: 9-12. 

Rojas, J. J., Ochoa, V. J., Ocampo, S. A., Munoz, J. 

F. (2006). Screening for Antimicrobial Activity of 

Ten Medicinal Plants Used in Colombian Folkloric 

Medicine: A possible alternative in treatment of 

nonnosocomial infections. BMC Complement. 

Altern. Med., 6: 2 doi: 10.1186/1472-6882-6-2. 

Sofowora, A. (2008). Medicinal Plants and 

Traditional Medicine in Africa, (3 rd edition), 

Spectrum Books Ltd. Ibadan, Nigeria, Pp. 199- 

205. 

Taylor, L. (2003). Herbal Secrets of the Rainforest, 

(2 nd edition), Sage Press, Inc. Texas, USA, P. 1. 

125

preliminary phytochemical and antibacterial screening of the ...

Create successful ePaper yourself

Delete template?

Save as template?