Journal of Theoretical and Experimental Biology-Volume 6 (3 and 4.pdf

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Theoretical and Experimental Biology 

(An International Journal of Basic and Applied Biology) 

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ISSN: 0972-9720 

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Dr.M.VIVEKANANDAN, Department of Biotechnology, Bharathidasan University, 

Tirchirappalli-620024, INDIA. (bard_vivek@yahoo.com). 

Dr.P.BALASUBRAMANIAN, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, 

Coimbatore-641003, INDIA. (balasubrap@hotmail.com). 

Dr.V. B. HOSAGOUDAR, Tropical Botanic Garden and Research Institute, Palode-695562, 

Thiruvananthapuram, Kerala, INDIA (vbhosagoudar@rediffmail.com). 

Dr.M.JAYAKUMAR, Department of Botany, VHNSN College, 

Virudunagar-626001, INDIA. (jayakuma_99@yahoo.com). 

Dr. JOSEPH A. J. RAJA, Department of Plant Pathology (Unit of Molecular Virology), College of 

Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan (R.O.C). 

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Dr.C.VIJAYALAKSHMI, Department of Crop Physiology, Tamil Nadu Agricultural University, 

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Dr.A.K.M. NAZRUL ISLAM, Department of Botany, Dhaka University, 

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Dr. APN LIPTON, Central Marine Fisheries Research Institute, 

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Dr.M.EYINI, Department of Botany, Thiyagarajar College, Madurai-625009, INDIA. (eyini@eth.net). 

Dr.P.K.JHA, Department of Botany, Tribuvan University, Kirtipur, Kathmandu, NEPAL. 

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Dr.A.SELVI, Division of Crop Improvement, Sugar Cane Breeding Institute, 

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Dr.G.ANNIE JULIET, Department of Molecular Genetics and Microbiology, 

University of Texas at Austin, Texas 78712, USA. (ganniejuliet@yahoo.com). 

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Theoretical 

and 

Experimental Biology 


ISSN: 0972-9720 

www.ejteb.org 

Volume 6 No. 3 and 4 February and May 2010 

G. Kulandaivelu 

Editor-in-Chief 

E. John Jothi Prakash 

Executive Editor 

Elias Academic Publishers 

India

Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 6 (3 and 4): 167-176, 2010 

© 2010 Elias Academic Publishers 

www.ejteb.org 

Hypoglycaemic Activity and Modulatory Effect on Glucose 

Metabolism by Artificially Cultivated Ganoderma lucidum in 

Streptozotocin Induced Diabetic Rats 

A. Usha Raja Nanthini 1 ٭ , M. Rajasekara Pandian 2 and G.Kavitha 3 

1 Department of Biotechnology, Noorul Islam College of Arts and Science, Kumaracoil- 629 180, Tamil Nadu, India. 

2 Department of Zoology, Arignar Anna Government Arts College, Namakkal-637001, Tamil Nadu, India. 

3 Vinayaka Mission’s University, Salem-637408, Tamil Nadu, India. 

Received: 24 November, 2009; revised received: 10 January, 2010 

Abstract 

In Chinese medicine the fruit bodies of Ganoderma lucidum (Fr.) Karst is used to treat and 

prevent various diseases, including diabetes mellitus. Present investigation studied whether 

artificially cultivated Ganoderma lucidum collected from Kollimalai, India possesses 

modulatory effect on glucose metabolism and hypoglycaemic activity. Treatment with 

aqueous extract of artificially cultivated Ganoderma lucidum fruit bodies (10-30mg/kg 

body weight) on streptozotocin (STZ)-induced type 1 diabetic rats for 45 days reduced 

blood glucose and urine sugar levels and increased the insulin level in diabetic rats. A 

reduction in glucose-6-phosphatase, fructose-1, 6-bisphosphatase and elevation in 

hexokinase was observed. Glycogen content in liver and muscle was reduced and in kidney 

it was increased. The effect was dose-dependent and maximum effect was obtained in the 

dose 30mg/kg. It can be understood that aqueous extract of artificially cultivated 

Ganoderma lucidum exhibited a significant antihyperglycaemic activity and improved the 

metabolic alterations in STZ-diabetic rats. These results provide a rationale for the use of 

artificially cultivated Ganoderma lucidum collected from India to treat diabetes mellitus. 

Key words: Ganoderma lucidum, streptozotocin, diabetes mellitus, hypoglycaemic 

activity. 

Introduction 

Mushrooms have a notable place in the folklore throughout the world and in the traditions of 

many cultures. Ganoderma lucidum (Fr.) Karst is a rare mushroom which was considered 

precious during ancient times. It was once the provenance of the emperors of China, since the 

Ganoderma lucidum is extremely rare and difficult to find in the wild. Because the husks of the 

spore are very hard, the spores can’t germinate as readily as the spores of other mushrooms 

(George,2007).The mycelia and fruiting bodies of Ganoderma lucidum are used as Chinese 

traditional medicine to treat diseases such as tumours (Peng et al., 2005), hypertension, 

hyperglycaemia, hepatitis, chronic bronchitis, bronchial asthma (T.K, 1999; Kyo et al., 2002), 

Liver fibrosis (Wu et al., 2004), Lupus erythematosis, nephritis, dysmenorrhoea, anorexia, 

migraine, arthritis, haemorrhoids, hypercholesterolemia, constipation (Shiao et al., 1994), 

neurasthenia, insomnia (Lin, 2002) gastric ulcer (Kim and Kim,1999), cough (Yan et al.,1999) 

inflammation, cardio vascular disorders and acts as antiviral (eg., anti-HIV), antibacterial, 

antiparasitic, immunomodulator, kidney toxic, nerve toxic, sexual potentiator (Wasser and 

Weis, 1999), antiaging (Gan et al., 1998), antiangiogenic, anti-metastasis and anti angiogenesis 

(Kimura et al.,2002; Shiao, 2003) Wound healing agent (Lia et al., 2001). So this mushroom is 

*Corresponding author: Email address: biotechurn@gmail.com 

167

Usha et al / Ganoderma lucidum on Glucose Metabolism 

considered as a symbol of success and well being meaning “marvellous herbs” or “mushroom of 

immortality”. Traditionally, it is taken as powder in hot water or in whisky, or by boiling the 

fruiting body and drinking the Ganoderma “tea” (Quimio, 1986). However there is no 

previously published report on the use of aqueous extract of artificially cultivated Ganoderma 

lucidum collected from India for the anti-diabetic properties. The present investigation was 

designed to evaluate the antihyperglycaemic activity and modulatory effect on glucose 

metabolism of aqueous extract of artificially cultivated Ganoderma lucidum collected from 

Kollimalai, India in streptozotocin induced diabetic rats. 

Materials and Methods 

The Mushroom, Ganoderma lucidum 

The identity of Ganoderma lucidum (Fr.) Karst, collected from the Kollimalai, Tamilnadu, India 

was confirmed using the Simon and Schuster’s Guide to Mushrooms. The mycelium observed 

under microscope was compared with MTCC strains. The mushroom had stipe and cap with 

pores beneath. They had shiny surface and the flesh was brown in colour. The cap covered with 

a shiny crust was circular or kidney shaped. Cap showed zones from yellow to dark red, and the 

margin was white or yellow. They had rust-brown colour spores. 

Artificial cultivation of Ganoderma lucidum and Preparation of Aqueous Extract 

Spawn preparation was done in Shorgum vulgare grains and wooden chips of various plants 

were used to cultivate G. lucidum. The wood chips were cut into pieces (1-2 cm), soaked in 

water trough for about 12 hours, boiled for 30 min, shade dried and used with optimum 

moisture. The wooden chips and spawn were filled in polythene bags as 4-5 alternate layers. 

The plugged polythene bags were kept in dark room at 28±2°C and 70-90% humidity. After 15- 

20 days the fruit bodies of G. lucidum emerged from the mouth of the polythene bag. The 

harvested fruit bodies of G.lucidum (250 gm) was made into small pieces, shade dried, 

powdered and homogenized in a wareing blender with 500 ml of distilled water. The extraction 

was carried out with constant stirring overnight. The homogenate was then centrifuged at 2000 

rpm for 10 min at 0-4°C. The supernatant was concentrated and used for the treatment of STZ 

induced diabetic rats. 

Test Animals 

Wistar albino rats (200-250gm) of either sex were used. These animals were housed in an airconditioned 

animal room at 23±2°C with 12 h light/dark photoperiod and maintained with free 

access to water and ad libitum feeding. All animal experiments were in accordance with the 

guidelines of the National Institute of Health Guide (1985). 

Chemicals 

Streptozotocin was procured from Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India. All 

other chemicals used were of analytical grade. 

Induction of Diabetes 

Diabetes was induced in overnight fasted adult Wistar strain albino male rats weighing 200–250 

g by a single intraperitoneal injection of 55 mg/kg Streptozotocin. Streptozotocin (55 mg/kg) 

was dissolved in 0.1 M citrate buffer (pH 4.5). Hyperglycaemia was confirmed by the elevated 

glucose levels (Above 250 mg/dl) in plasma, determined at 72 h after injection. Those animals 

with hyperglycaemia were used in the experiment. 

After successful induction of experimental diabetes, the rats were randomly divided into 

six groups each comprising a minimum of six rats. These were: Group 1, Normal control with 

healthy rats without diabetes; Group 2, Normal rats administered with G. lucidum aqueous 


168


extract (30 mg/kg/b.wt.) in aqueous solution orally for 45 days; Group 3, Diabetic control (STZ 

induced); Group 4, Diabetic rats administered with G. lucidum aqueous extract (10 mg/kg/b.wt.) 

in aqueous solution orally for 45 days; Group 5, Diabetic rats administered with G.lucidum 

aqueous extract (20 mg/kg/b.wt. ) in aqueous solution orally for 45 days ; Group 6, Diabetic rats 

administered with G. lucidum aqueous extract (30 mg/kg/b.wt. ) in aqueous solution orally for 

45 days. At the end of the experimental period, rats were fasted overnight, anaesthetized and 

sacrificed by cervical decapitation. The blood was collected with or without EDTA 

(ethylenediaminetetraacetic acid) for plasma or serum separation, respectively. 

Biochemical Analysis 

The level of blood glucose was estimated following glucose oxidase method (Triender, 1969). 

Insulin was estimated using Boerhringer Mannheim kit. Liver, kidney and skeletal muscles were 

immediately dissected, washed in ice-cold saline to remove the blood and homogenised in 0.1 

M Tris–HCl buffer, pH 7.4. The supernatant was used for enzyme activity assays. Hexokinase, 

glucose-6-phosphatase and fructose-1, 6-bisphosphatase were assayed by the method of 

Brandstrup et al., (1957), Baginsky et al., (1974) and Gancedo and Gancedo (1971) 

respectively. Glycogen was assayed by the method of Ong and Khoo (2000). 

Statistical Analysis 

All the grouped data were statistically evaluated with SPSS/ 10.0 software. Hypothesis testing 

methods included one way analysis of variance (ANOVA) followed by least significant 

difference (LSD) test; p value of less than 0.05 were considered to indicate statistical 

significance. All the results were expressed as the mean ± S.D. for six animals in each group. 

Results 

Effect of Daily Administration of Ganoderma Lucidum Aqueous Extract on Blood Glucose, 

Insulin and Urine Sugar in Normal and Diabetic Rats 

Table 1 shows blood glucose, plasma insulin and urine sugar levels of control and experimental 

group of rats. The blood glucose level in the diabetic control rats was significantly (p < 0.05) 

increased. Inversely, the insulin level was decreased significantly (p < 0.05). In diabetic rats, 

elevated urine sugar level was observed. Treatment of diabetic rats with aqueous extracts of 

G. lucidum elicited significant decrease in blood glucose and urine sugar levels and increase in 

the insulin level in dose dependent manner, when compared with diabetic control rats. No 

change in blood glucose, insulin and urine sugar level was observed in rats grown under control 

conditions. 

Effect of Daily Administration of Ganoderma Lucidum Aqueous Extract on 

Gluconeogenic Enzymes (Hexokinase, Glucose-6-Phosphate Dehydrogenase and 

Fructose-1,6-Bisphosphatase) in Normal and Diabetic Rats 

Table 2 summarizes the level of glucose-6- phosphatase in the liver and muscle tissues of 

control and experimental groups of rats. A significant (p < 0.05) increase in glucose-6- 

phosphatase level was observed in STZ-diabetic rats and it was normalized after treatment with 

G. lucidum aqueous extract. Table 3 depicts the level of the enzyme fructose-1, 

6-bisphosphatase level in liver, kidney and muscle of control and experimental rats. The level of 

fructose-1,6-bisphosphatase was significantly (p


the activities of the enzyme. In all the three enzymes the maximum modulatory effect was 

observed in 30mg/kg extract. No significant change in the levels of Glucose-6-phosphatase, 

Fructose-1,6-bisphosphatase and Hexokinase was observed in normal control rats administered 

with G. lucidum aqueous extract. 

Table 1: Effect of Ganoderma lucidum aqueous extract on blood glucose, insulin and urine sugar in 

control and diabetic rats 

Groups 

Blood glucose 

(mg/dL) 

Insulin 

(μU/ml) 

Urine sugar 

Normal 82.44 ± 2.68 b 15.52 ± 1.51 a Nil 

Normal + given extract 74.39 ± 4.17 a 15.83 ± 1.47 a Nil 

STZ-control 289.28 ± 3.18 f 6.38 ± 0.89 b ++++ 

STZ-induced+extract(10mg/kg) 194.63 ± 3.74 e 7.81 ± 0.77 b ++ 

STZ-induced+extract(20mg/kg) 164.87 ± 2.68 d 10.21 ± 1.10 c + 

STZ-induced+extract 30mg/kg) 107.23 ± 7.23 c 12.34 ± 1.21 d Nil 

Values are mean ± SD for 6 rats in each group 

Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT) 

Table 2: Effect of Ganoderma lucidum aqueous extract on glucose 6-phosphatase in normal and STZinduced 

experimental rats. 

Groups 

Liver 


Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT) 


Muscle 

(mmol of glucose phosphorylated/h/mg protein) 

Normal 20.00 ± 1.52 a 17.30 ± 1.30 a 

Normal + given extract 19.50 ± 1.20 a 17.00 ± 1.22 a 

STZ-control 39.51 ± 2.51 b 35.01 ± 2.65 b 

STZ-induced + extract (10mg/kg) 37.21 ± 2.39 c 32.41 ± 1.96 c 

STZ-induced + extract (20mg/kg) 33.32 ± 3.31 c 28.23 ± 1.82 c 

STZ-induced + extract (30mg/kg) 27.30 ± 2.08 d 22.10 ± 1.90 d 

Effect of Daily Administration of Ganoderma Lucidum Aqueous Extract on Tissue 

Glycogen Content in Normal and Diabetic Rats 

Table 5 showed the changes in the level of glycogen in the liver, kidney and muscle of control 

and experimental rats. The level of glycogen was significantly (p < 0.05) decreased in the liver 

and muscle tissues of diabetic rats when compared with normal control rats, whereas kidney 

tissue had elevated glycogen level. Oral administration of G. lucidum aqueous extract to 

diabetic rats significantly (p < 0.05) increased the liver and muscle glycogen content and 

decreased the kidney glycogen content. The effect was the maximum in 30mg/kg extract. The 

administration of G. lucidum aqueous extract to normal rats resulted in no significant changes in 

the level of tissue glycogen. 

170


Table 3: Effect of Ganoderma lucidum aqueous extract on fructose 1,6-bisphosphatase in normal and 

STZ-induced experimental rats. 

Groups 

Liver Kidney Muscle 

(μmole of Pi liberated/min/mg protein) 

Normal 11.52 ± 0.60 a 16.62 ± 1.10 a 2.90 ± 0.12 a 

Normal + given extract 11.06 ± 0.51 a 16.55 ± 1.02 a 2.86 ± 0.10 a 

STZ-control 23.50 ± 1.20 b 29.04 ± 1.66 b 5.50 ± 0.43 b 

STZ-induced+extract(10mg/kg) 22.10 ± 1.09 b 27.56± 1.39 c 5.01± 0.51 c 

STZ-induced+extract(20mg/kg) 19.11 ± 1.51 c 24.44 ± 1.91 c 4.21 ± 0.42 c 

STZ-induced+extract(30mg/kg) 16.04 ± 0.80 d 21.80 ± 1.33 c 3.80 ± 0.30 c 


Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT). 

Table 4: Effect of Ganoderma lucidum aqueous extract on hexokinase in normal and STZ-induced 


Groups 


(mmol of glucose phosphorylated/h/mg protein) 

Normal 98.02 ± 4.46 a 79.11 ± 6.02 a 114.02 ± 8.68 a 



STZ-induced + extract(10mg/kg) 60.13 ± 4.39 c 64.19± 4.39 c 86.89 ± 6.56 c 

STZ-induced + extract (20mg/kg) 75.56 ± 5.31 c 67.56 ± 5.91 c 91.23 ± 7.42 c 

STZ-induced + extract (30mg/kg) 90.02 ± 6.85 d 71.01 ± 4.65 d 102.00 ± 7.70 d 



Table 5: Effect of Ganoderma lucidum aqueous extract on glycogen content in normal and STZinduced 


Groups 


(mg/g tissue) 

Normal 2.88 ± 0.26 a 1.62 ± 0.12 a 2.32 ± 0.18 a 



STZ-induced + extract (10mg/kg) 1.92 ± 0.11 c 2.59± 0.18 c 1.77 ± 0.16 c 

STZ-induced + extract (20mg/kg) 2.15 ± 0.21 d 2.26 ± 0.19 d 1.89 ± 0.12 cd 

STZ-induced + extract (30mg/kg) 2.32 ± 0.20 d 1.91 ± 0.11 a 2.00 ± 0.10 d 




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Discussion 

Diabetic rats induced by STZ shows an increased sensitivity to oxygen free radicals and 

hydrogen peroxide, the breakdown products of liver, which impose oxidative stress in diabetes 

and would damage inner endothelial tissue; this would eventually be directly responsible for 

high blood glucose (Reddi and Bollineni, 2001). The present investigation showed that 

treatment with Ganoderma lucidum aqueous extract reduces the blood sugar level and it may be 

due to stimulating effect on insulin release from regenerated beta cells of the panaceas or may 

be due to increased cellularity of the islet tissues and regeneration of the beta cells. The aqueous 

extract might be exerting its hypoglycaemic effect by an extra-pancreatic action (Dabis et al., 

1984), e.g. possibly by stimulating glucose utilization in peripheral tissues (Naik et al., 1991; 

Obatomi et al., 1994). Also, it could be the result of an increase in glycolytic (Steiner and 

Williams, 1959) and / or glycogenic enzymes activity in peripheral tissues (Naik et al., 1991). It 

might be also possible that the aqueous extract may decrease the secretion of the counter 

regulatory hormones (glucagons, corisols and growth hormones) (Roman-Ramos et al., 1995). 

The STZ-induced diabetic control rats showed decreased level of insulin in the plasma 

than the normal control rats. The treatment with G. lucidum aqueous extract had increased the 

insulin level to near normal level. The elevation of plasma insulin in the G. lucidum treated STZ 

diabetic rats could be due to the insulinotropic substances present in the extract, which induce 

the intact functional β-cells of the langerhans islet to produce insulin (Jeong-Sook, 2006). 

Insulin deficiency is clearly associated with change in hepatic metabolism (Consoli et al., 

1989). 

Insulin decreases gluconeogenesis by decreasing the activities of key enzymes such as 

glucose-6-phosphatase, fructose 1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase and 

pyruvate carboxykinase (Murray et al., 2000). The liver and skeletal muscle is the major organ 

for glucose disposal. Glucose-6-phosphatase, a key enzyme in the homeostatic regulation of 

blood glucose concentration, is expressed mainly in the liver and kidney and is critical in 

providing glucose to other organs during diabetes, prolonged fasting or starvation (Bouché et 

al., 2004). It catalyzes the dephosphorylation of glucose-6-phosphate to free glucose as the 

terminal step in gluconeogenesis and glycogenolysis. This reaction occurs in the lumen of the 

endoplasmic reticulum and the enzyme complex is composed of glucose-6- phosphate 

transporter that transports glucose-6-phosphate from the cytoplasm into the lumen of the 

endoplasmic reticulum and a glucose-6-phosphatase catalytic subunit that hydrolyzes the 

glucose-6-phosphate to glucose and phosphate (Chou et al., 2002). Glucose is transported out of 

the liver to increase blood glucose concentration. STZ increases the expression of glucose-6- 

phosphate (Massillon et al., 1996; Liu et al., 1994). In contrast, insulin inhibits the hepatic 

glucose production by suppressing glucose-6-phosphate activity (Chen et al., 2000; 

Wiernsperger and Bailey, 1999). Our results demonstrated that hepatic and muscle glucose-6- 

phosphatase activity in diabetic rats was significantly higher than that of normal rats and the 

oral feeding of G. lucidum aqueous extract markedly lowered its activity. The reduction in 

enzyme activity can lead to a decrease in gluconeogenesis and blood glucose concentration. 

Fructose-1, 6-bisphosphatase is a highly regulated, rate-limiting enzyme that catalyzes 

the dephosphorylation of fructose-1, 6- bisphosphate to fructose-6-phosphate, the second to last 

step in the gluconeogenic pathway (Pilkis and Claus, 1991). Under normal conditions, insulin 

functions as a suppressor of gluconeogenic enzymes (Baquer et al., 1998). This results in a 

decrease in the glycolytic flux. An increase in the activity of fructose-1,6-bisphosphatase has 

been suggested as a possible mechanism for the production of increased endogenous glucose 

after it was shown that diabetics have an increase in gluconeogenesis (Nurjhan et al., 1992). The 

increased activity of fructose-1, 6-bisphosphatase has been observed in animal models of 

diabetes, insulin resistance and obesity and suggests a principal role for fructose-1,6- 

bisphosphatase in the flux of gluconeogenesis and endogenous glucose production 


172


(Andrikopoulos et al., 1993). The increased activities of these gluconeogenic enzymes in 

diabetic rats were decreased to near-normal levels after the administration of Ganoderma 

lucidum aqueous extract. The possible mechanism by which Ganoderma lucidum aqueous 

extract bring about the normalization of enzyme activity may be by potentiation of insulin 

release from β-cells of the islets of Langerhan’s which might enhance glucose utilization. 

Hexokinase (HK) is an isoenzyme that catalyzes phosphorylation of glucose to glucose- 

6-phosphate, thus playing a crucial function in tissue intermediary metabolism. Hexokinase is 

an insulin-dependent and insulin-sensitive enzyme and is almost completely inhibited or 

inactivated in diabetic tissues in the absence of insulin (Gupta et al., 1997). There are four 

isoforms of mammalian hexokinases involved in the oxidation of glucose (Wilson, 1995). 

Hexokinases I–III have a high affinity for glucose and are feedback-inhibited by physiologic 

concentrations of glucose-6-phosphate. Whereas, glucokinase (HK-IV or GK), the major 

glucose- phosphorylating enzyme, has a lower affinity for glucose and its abundance is 

regulated transcriptionally by insulin and glucagon and post-translationally by the GK 

regulatory protein (GKRP) (Collier and Scott, 2004). Among four isoforms of hexokinases, HK- 

I and GK are expressed in the liver. Reports on animal models and isolated hepatocytes 

established that hexokinase exerts a strong impact on glucose utilization and glycogen synthesis 

in liver (Postic et al., 2001) and muscle (Murray et al., 2000) and their levels are very low in 

both human and rodent diabetes; insulin administration rapidly reinstates hexokinase activity to 

the hepatocytes (Ferre et al., 1996). Because of these observations, restoration of hepatic 

hexokinase activity provides a possible therapeutic strategy for diabetes treatment. The 

markedly decreased level of insulin in the streptozotocin-induced diabetic animals ultimately 

leads to the impairment in the activity of hexokinase, since insulin deficiency is a hall mark of 

diabetes (Postic et al., 2001). However, the modest increase in the activity of hexokinase as 

observed in the diabetic animals administered with Ganoderma lucidum aqueous extract 

protects the hepatic and extrahepatic tissues against streptozotocin-induced diabetes by 

stimulating insulin from the remnant β -cells, since streptozotocin selectively destroys 

pancreatic β -cells. This study also demonstrated that a modest augmentation of hexokinase 

activity in the liver, kidney and muscle enhances glucose metabolism and promotes overall 

glucose homeostasis similar to the studies of Palsamy and Subramanian (2009). 

Glycogen, a branched polymer of glucose residues synthesized by the enzyme glycogen 

synthase, is the primary intracellular storable form of glucose and its quantity in various tissues 

is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition 

by stimulating glycogen synthase and inhibiting glycogen phosphorylase (Pederson et al., 

2005). The activity of glycogen synthase is regulated by decreased cellular glycogen content, 

hormone signaling, subcellular localization, targeting of phosphatase and allosteric activation by 

glucose-6-phosphate (Parker et al., 2004). Glycogen phosphorylase, a rate-limiting enzyme of 

glycogenolysis, cleaves β (1→4) linkages to remove glucose molecules from the glycogen. This 

enzyme exists as a dimer with each subunit linked to the essential cofactor pyridoxal phosphate, 

which donates the phosphate as an electron donor for release of glucose-1-phosphate 

(Greenberg et al., 2006). Its activity is regulated by phosphorylation and by allosteric binding of 

AMP, ATP, glucose-6-phosphate and glucose (Bollen, 1998). Since streptozotocin causes 

selective destruction of pancreatic β -cells resulting in apparent decline in insulin levels, it is 

responsible for the decreased glycogen levels in major storage tissues such as liver and skeletal 

muscle as they depend on insulin for entry of glucose (Whitton and Hems, 1975; Golden et al., 

1979; Bishop, 1970). During diabetic conditions, the glycogen levels, glycogen synthase 

activity and responsiveness to insulin signalling are diminished and glycogen phosphorylase 

activity is significantly increased (Parker et al., 2004). Glycogen levels in various tissues 

especially skeletal muscle are direct reflection of insulin activity. Insulin promotes intracellular 

glycogen deposition by stimulating glycogen synthase and inhibiting glycogen phosphorylase. 

Since STZ selectively damages β-cells of islets of Langerhans resulting in marked decrease in 


173


insulin levels, it is rational that glycogen levels in tissues (skeletal muscle and liver) decrease as 

they depend on insulin influx of glucose (Whitton and Hems, 1975). Moreover, this alteration in 

muscle and hepatic glycogen was normalized by insulin treatment (Weber et al., 1966). The 

entry of renal glucose is not dependent on action of insulin and, therefore, in the event of 

hyperglycemia there is an increase in the entry of glucose (Belfiore et al., 1986). This has been 

postulated to cause increased intra-renal glycogen deposition, which leads to glycosylation of 

basement membrane collagen in the kidney (Anderson and Stowring, 1973). From the present 

study it is clear that STZ induced diabetic rats had increased glycogen level in kidney and 

decreased level in Liver and skeletal muscle. Treatment with G. lucidum aqueous extract had 

reversed it by decreasing the renal glycogen and increasing the hepatic and skeletal muscle 

glycogen. It may be assumed by the above explained reasons. 

In conclusion results of the present investigation indicate that the aqueous extract of 

artificially cultivated G. lucidum collected from Kollimalai, India has antidiabetic effect, 

possibly due to insulin like effect of G. lucidum aqueous extract on peripheral tissues. The 

present study draws out a sequential metabolic correlation between increased glycolysis and 

decreased gluconeogenesis and normal glycemia stimulated by Ganoderma lucidum aqueous 

extract which may have the biochemical mechanism through which glucose homeostasis is 

regulated. 

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www.ejteb.org 

Influence of Hormone Induced Spawning in 

Etroplus suratensis 

S. Albin Dhas 1 *, M. Michael Babu 1 , T. Selvaraj 1 , T. Citarasu 1 , V. A. J. Huxley 2 and 

S. Mary Josephine Punitha 1 

1 Centre for Marine Science and Technology, Manonmaniam Sundaranar University, 

Rajakkamangalam - 629 502, Tamil Nadu, India. 

2 Department of Zoology, Thiru Vi Ka Government Arts College, Thiruvarur- 610003, Tamil Nadu, India. 

Received: 27 February, 2010; revised recieved: 29 May, 2010. 

Abstract 

A study was carried out to understand the influence of synthetic hormones such as ovaprim, 

HCG+LHRH on induced spawning in Etroplus suratensis grown in aquarium tanks of 5 ton 

capacity. Biochemical parameters such as triglyceride, total protein and cholesterol level in 

the blood, liver and gonads were estimated in hormone treated ones and it was compared 

with the control. The length and width of the egg development stages such as oocyte, previtellogenic 

and matured eggs were also analyzed in different hormone treated fishes and 

were compared with the control. The percentage of eggs in the ovary of control and 

hormone treated fishes were also compared. In all these parameters, the combined hormone 

HCG+LHRH administered experimental fishes showed the highest increased level. It was 

suggested that administration of the synthetic hormone HCG+LHRH induced spawning in 

E. suratensis. 

Key words: Hormones, Etroplus suratensis, ovaprim, HCG+LHRH, spawning 

Introduction 

The fish Etroplus suratensis belonging to the family chichilidae is commonly found in the 

estuaries and inland waters of India and Sri Lanka (Talwar and Jingran, 1992; Rao, 1995; 

Blaber, 1997). It grows in brackish as well as fresh waters and has been observed to breed in 

these habitats (Rishi and Singh, 1982). It involves in commercial fisheries (Gopakumar, 1997), 

yet this fish is preferred as the candidate species for aquarium. This fish is dioecious and breeds 

freely both in fresh and brackish waters (Pethiyagoda, 1991; Arkipehuk, 1999). Among the fish 

species, it has low fecundity rate with about 500 eggs laid in single spawning (Jayaprakas et al., 

1990). The eggs are attached to submerged logs, rocks or sometimes roots of aquatic weeds. 

These guardian parents take care of the eggs until hatching and within four days, the eggs will 

hatch. The fry shoal around their parents during the first week of growth in natural conditions. 

Although all the fish species are spawned in the natural environment, only a limited 

species are successfully spawned through induced breeding in laboratory conditions. The 

success of induced spawning depends upon several factors, which were not clearly understood 

in most of the fishes (Stuart et al., 1988). During the past three to four decades, induced 

breeding technique has been attempted in many of the fresh water and marine fishes. For this 

technique, many of the alternative hormones such as human chronic Gonadotropin (HCG) 

(Adebayo., 2004); Inyang and Hettiarachchi, 1994), luteinizing hormone – releasing hormonoeo 

(De Leeuw et al., 1985; Fermin, 1992) and ovaprim (Alok et al., 1993; Haniffa et al., 1996) 

were used. Treatments using the above hormones are effective in many of the fish species. But 

*Corresponding author; Email address: salbindhas@yahoo.com 

177

Albin Dhas et al / Hormone Induced Spawning in Etroplus suratensis 

so far, there were only limited attempts in induced breeding of E. suratensis. Only very few 

works were carried out in the direction of larval propagation in E. suratensis (Eschmeyer, 

1990). Few works were focused on the induced breeding by applying hormones since the 

attempts were not encouraging (Karnfield, 1984). The present study is an attempt to induce 

breeding in E. suratensis using synthetic hormones such as HGG and LHRH and to document 

the earlier larval stages of the fish. 


Brood Stock Management 

E. suratensis brooders with the size of 15-20 cm length and weight group of approximately 

110 ± 10 g were collected from the backyard estuarine waters at Centre for Marine Science and 

Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District, 

India. The collected fishes were stocked in aquarium tanks (5 T capacities) for 7 days to ensure 

the disease free status of the experimental fish. The healthy fishes were transferred to the 

circular brood stock tanks (1 T capacity). The water quality parameters such as temperature, 

salinity and oxygen level were maintained at 27-30ºC, 5 pit and 5 mg/l, respectively. A 100% 

water exchange was made daily. During this period, the fishes were fed with lap-lap at the rate 

of 3% of fish body weight daily. After 5 days, the gonad maturity of fish was determined in both 

male and female fishes by performing catheter biopsy in the gonads through the genital opening. 

The collected biopsy samples were observed for the gonad maturity with the parameters of eggs 

diameter and stage of the eggs in female, as well as sperm motility in the male. 

Administration of Synthetic Hormones 

There were three groups with five female replicates in each group that were stocked in 

individual spawning tanks (1.5 ton capacity) for hormone administration. In the first group, 

ovaprim (Syndel Co., Canada) was administered with the optimum concentration of 1 ml/kg 

fish. The second group was administered with HCG (Profess and SIGMA, USA) 1000 U/kg fish 

with the combination of LHRH (SIGMA, USA) 60 µg/kg fish (Mai, 1998). The third group was 

treated as control which received sterile saline injection (0.81% NaCl). 

After 48 h of hormonal administration the blood samples from the three groups of 

experimental fish were collected using sterile syringe. Thereafter, the fishes were sacrificed- the 

liver and gonad samples were dissected carefully from each group and individually stored at – 

20ºC until further use. The biochemical parameters such as triglyceride and cholesterol were 

estimated in all the blood, liver and gonad samples using ELISA–micro plate method (Palacious 

et al., 1998). Total protein content in the same samples were estimated by the method described 

by Bradford (1976). 

To characteristic the stages of maturity in females, oocyte count as well as gonad 

somatic index (GSI) were recorded in all the groups. Three stages of oocytes were found in the 

gonad (Previtellogenic, vitellogenic and matured egg) and the number of oocytes belong to each 

stage was counted for 100 mg of gonad sample in each group of fishes. The size of the egg 

(length and width) was also determined using Ocular Micrometry method. 


The data obtained in the present study were subjected for statistical 2-way ANOVA and 

regression analysis followed by Zar (1974). 

Results 

Biochemical indices like triglyceride, total protein and cholesterol were estimated in the blood, 

liver and gonad samples of E. suratensis brooders treated with synthetic hormones like ovaprim 

and HCG+LHRH. The level of triglyceride was more in all the tested samples (63.23 µg/ml in 

blood, 487.23 µg/g in liver and 51.2 µg/g in gonad) of fish administered with HCG+LHRH. But 

theseparameters exhibited lower values in fishes administered with ovaprim (30 to 41.0 mg/g) 

and control (22.0 to 435 µg/g) groups (Table 1). 

Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 6 (3): 187-183, 2010 

178


Table 1: Biochemical parameters of different tissues in E. suratensis in both experimental and control 

groups. 

Parameters Treatments Blood (mg/ml) Liver (mg/g) Gonad (mg/g) 

Control 1.31 ± 0.05 7.02 ± 0.06 2.0 ± 0.08 

Total Proteins Ovaprim 1.14 ± 0.04 8.98 ± 0.28 3.75 ± 0.05 

HCG+LHRH 1.66 ± 0.11 9.12 ± 0.19 4.02 ± 0.09 

Control 50.0 ± 4.0 435.0 ± 5.0 22.0 ± 1.0 

Triglycerides Ovaprim 37.0 ± 3.0 410.0 ± 8.0 30.0 ± 2.0 

HCG+LHRH 63.23 ± 2.0 487.23 ±2.7 51.20 ± 1.50 

Control 12.2 ± 1.3 61.21± 3.0 3.0 ± 0.20 

Cholesterol Ovaprim 9.0 ± 1.0 144.0 ± 4.0 1.50 ± 0.05 

HCG+LHRH 14.4 ± 1.5 212.20 ±7.0 5.0 ± 0.50 

Table 2: Length and width of E. suratensis eggs. 

Egg Stage Length (µm) Width (µm) 

Oocyte 186.62 ± 12.25 176.62 ± 14.20 

Pre-vitrllogenic egg 599.85 ± 11.21 333.25 ± 25.33 

Mature eggs 1932.85 ± 82.81 1039.72 ± 92.22 

Table 3: Percentage of eggs in 100 mg of ovary. 

Stage of egg 

Control Egg % 

(in 100 mg 

ovary) 

Hormone treated 

Egg % (in 100 mg 

ovary) 

Oocyte 64.45 + 6.5 19.24 + 1.8 

Pre-vitellogenic 

egg 

21.96 + 2.3 38.96 + 3.7 

Vitellogenic egg 

(matured egg) 

13.58 + 1.2 41.78 + 4.3 

Like the triglyceride, a similar trend of result was observed in total protein level of 

blood (1.66 mg/ml), liver (9.12 mg/g) and gonad (4.02 mg/g) samples of E. suratensis 

administered with HCG+LHRH than the ovaprim (1.14 mg/g) as well as control (1.31 mg/ml to 

7.02 mg/g) treatment (P


Length-Width Relationship of E. suratensis Oocytes 

200 

195 

190 

y = 1.199x - 42.521 

R 2 = 0.9548 

185 

Width (μm) 

180 

175 

170 

165 

160 

170 175 180 185 190 195 200 

Length (μm) 

Figure 1a: Length-width relationship of E. suratensis pre-vitellogenic egg. 

Length-Width Relationship of E.suratensis Pre-vitrllogenic egg 

370 

360 

350 

Width (μm) 

340 

330 

y = 1.9578x - 845.04 

R 2 = 0.6779 

320 

310 

300 

585.00 590.00 595.00 600.00 605.00 610.00 615.00 

Length (μm) 

Figure 1b: Length-width relationship of E. suratensis oocytes. 

Percentage Distribution of Different Egg Stages 

From matured gravid females of E. suratensis, based on the maturity, three different stages were 

identified (Figure 1a, b and c). They are oocytes which are spherical in shape with the length 

and width of 186.62 µm and 176.62 µm respectively. The second stage was pre-vitellogenic 


180


oocytes, which had the vitellogenic package of transparent yellow spheres which were 599.85 

µm long and 333.25 µm wide. Likewise, the third stage was vitellogenic (matured) eggs, 

prominent shape with 1932.85 µm length and 1039.72 µm wide (Table 2). Freshly ovulated 

mature eggs were slightly spherical in shape, visible to the naked eye and strong yellow in 

colour and opaque. The oocyte was obviously enhanced by hormone treatment as indicated by 

the increase of oocyte diameter. The percentage of matured egg was maximum (47.03%) in E. 

suratensis that received HCG+LHRH hormone. At the same time, the control group of fishes 

had 13.58% matured eggs, followed by fishes administered with ovaprim hormone, 41.78%. 

Vitellogenic eggs (38.97%) and previtellogenic eggs (64.45%) were more in ovaprim 

administered fish and control fish, respectively (Table 2, Fig 1a, b, c). 

The data recorded for percentage of eggs in 100 mg ovary showed that the hormonetreated 

experimental fishes showed the highest percentage (41.78%), followed by previtellogenic 

(38.96%) and the oocyte (19.24%) (Table 3). 

Length-Width relationship of E.suratensis mature eggs 

360 

350 

340 

y = 0.2567x - 163.48 

R 2 = 0.6823 

Width (μm) 

330 

320 

310 

300 

1840 1860 1880 1900 1920 1940 1960 1980 2000 2020 2040 

Length (μm) 

Figure 1c: Length-width relationship of E. suratensis mature eggs. 

Discussion 

For the assessment of internal milieu of fishes during reproduction several biochemical indices 

should be clearly resolved (Svoboda et al., 2001). The present study also had a part to analyze 

the possible biochemical variables such as triglycerides, total protein and cholesterol from 

blood, liver and ovary tissues, while administration with commercial synthetic hormones was 

carried out. Significant differences were observed between the control land hormone 

administered spawners. Results from the examination of triglyceride in the spawners tissues 

(liver and ovary) and blood indicate the significant positive regulation and high titer value in 

HCG+ LHRH treated group. The group administered with ovaprim failed to reserve the 

significant quality of triglycerides in blood and tissue, which is clearly reflected in the number 

of matured eggs in the ovary. Similar trend was observed in the blood plasma of fish tench 

(Tinca tinca L.) during pre and post-spawning period under the condition of hormonally induced 

artificial reproduction (Svoboda, 2001). Possible role of triglyceride in fish reproduction is to 

serve as the higher energy source as well as the precursor for yolk protein synthesis (Luskova, 

1997; Kovaqcheva and Tchekov, 1993). 

Moreover, cholesterol which is the precursor in the synthesis of steroid hormones 

involved in fish maturation was significantly regulated in the hormone treated animals than the 

control groups. The cholesterol that is incorporated in the membranes and the endogenous 

structures of the egg and its concentration in blood plasma of females were found to increase 


181


during the administration of hormones (Diwan and Krishnan (1986). Diwan and Krishnan 

(1986) observed a fluctuation of serum cholesterol in male and females of E. suratensis as 

related to maturity. In the present study, cholesterol concentration in liver tissues of females 

that are found to be lowest in the control females was reflected in the egg maturity. 

The total protein level in the tissue samples from the hormone injected groups exhibited 

significant higher values than the control groups in the present study. This result is in 

accordance with the data reported for some other fish species like trout – Salmo trutta 

(Mulcahy, 1971); carp-Cyprinus carpio (Svobodova and Parova, 1977); trout – Salmo 

gairdeneri (Hille, 1982); rainbow trout (Jirasek et al., 1993) and common carp (Rehulka, 1996). 

Total protein level in the blood determines the health status and reproductive ability in cichilids. 

In the present study also, higher levels of total protein were registered in HCG+LHRH group as 

it caused improved health status or reproductive ability of the fish E. suratensis. 

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Decolorization of Textile Dye Reactive Black HFGR Using a 

Novel Isolate Paenibacillus lautus SK21 

S. Senthil Kumar 1* , M. S. Mohamed Jaabir 1 , A. Veeramani 2 and R. Ravikumar 3 

1 Department of Biotechnology, Jamal Mohamed College (Autonomous), 

Tiruchirappalli-620 020, Tamil Nadu, India. 

2 Department of Botany, Aringar Anna Govt. Arts College, Namakkal-637 002, Tamil Nadu, India. 

3 Research Department of Botany, Jamal Mohamed College (Autonomous), 

Tiruchirappalli-620 020, Tamil Nadu, India. 

Received: 27 February, 2010; revised recieved: 6 June, 2010. 

Abstract 

Paenibacillus lautus strain SK21 was isolated from the textile effluent polluted soil in 

Tirupur, Tamil Nadu and identified based on Biochemical and 16S RNA Sequence. The 

present study was carried out in an attempt to decolorize a commonly used yet tougher dye 

to decolorize, Reactive Black HFGR. The decolorization percentage was calculated from 

UV-Vis spectrophotometric analysis. Dye decolorization was probably due to the 

biotransformation and depended upon the biomass. Replacement of nutrient broth with 

minimal media did not show any decolorization property. Decolorization was optimized 

and found to be up to 95% at 7pH, 40°C under static and non-aerated condition. 

Key words: Reactive dyes, Azo dyes, Bacterial isolates, dye decolorization, 

Paenibacillus lautus 

Introduction 

Synthetic dyes find use in a wide range of industries such as textile dyeing, paper printing, 

cosmetics and pharmaceuticals (Erdal and Taskin, 2010). Approximately 10,000 different dyes 

and pigments are used in industries and over 7 × 10 5 tons of these dyes are annualy produced 

world-wide. Due to inefficiencies of the industrial dyeing process, 10 - 15% of the dyes are lost 

in the effluents of textile units, rendering them highly coloured. Among the various classes of 

dyes, reactive dyes are more difficult to remove. They contain chromophoric groups such as 

azo, anthraquinone, triarylmethane, etc. and reactive groups e.g. vinylsulphone, chlorotriazine, 

trichloropyrimidine etc. that form covalent bonds with the fiber. Azo reactive dyes are the 

largest class of water soluble synthetic dyes with the greatest variety of colors and structure and 

are generally resistant to biodegradation processes (Lin and Peng, 1994; Sanghi et al., 2006; 

Daneshvar et al., 2007). 

Water pollution control is at present one of the major areas of scientific activity. While 

colored organic compounds generally impart only a minor fraction of the organic load to 

wastewater, their colour renders them aesthetically unacceptable. Colour is one of the most 

obvious indicators of water pollution and discharge of highly coloured synthetic dye effluents 

can be damaging to the receiving water bodies (Nigam et. al., 1966). Two percent of dyes that 

are produced are discharged directly in aqueous effluent and 10% are subsequently lost during 

*Corresponding author; Email address: envsenthil@gmail.com 

185

Senthil Kumar et al / Decolorization of Textile Dye Reactive Black HFGR 

the textile colouration process (Pearce et al., 2003). Some of the azo dyes, xanthene dyes and 

anthroquinone dyes are known to be very toxic and mutagenic to living organisms. 

Microbial decolorization has been proposed as a less expensive and less 

environmentally intrusive alternative. In the present study, we focused our attention on the 

isolation of dye decolorizing microorganisms from contaminated soil of an industrial estate and 

analyzed the ability of these isolates to degrade Reactive Black HFGR. 


Chemicals 

All chemicals and reagents used in this investigation were of Analytical grade. The common 

name of the dye (Reactive Black HFGR) has been used for convenience and was procured from 

A.K. Chemi Dyes Enterprises, Mumbai, India. The stock solutions of the dyes were filter 

sterilized and added to the growth medium in the concentration of 100ppm (mg/litre). 

Spectrum Study of the Dye 

The dye procured from the industry was initially studied for absorption maxima in a Double 

Beam UV-Vis Spectrophotometer from 350nm to 800nm (Schimadzu, UV-Vis 1800). 

Isolation of Bacterial Cultures 

Soil sample taken from the dumping grounds of the sludge was used for the isolation of dye – 

decolorizing microorganisms owing to long – term usage of the location for over 5 decades 

since the establishment. Bacteria from the soil sample were isolated by pour plate method and 

serial dilution technique using nutrient agar medium. All the plates were incubated at 37°C for 

24 hours. 

Study of Decolorization Activity 

All decolorization experiments were performed in triplicates. A loopful of each isolated 

bacterial culture was inoculated into a separate 250 ml Erlenmeyer flask containing the Reactive 

Black HFGR (100 mgl -1 ) in Nutrient broth and incubated for 24 h at 37°C for initial screening 

of the isolates for the ability to decolorize the dye. Aliquots of the culture (3 ml) was withdrawn 

at different time intervals, centrifuged at 5000rpm for 15 min to separate the bacterial cell mass. 

Decolorization was determined by measuring the absorbance of the supernatant at 520 nm (λ 

max) and percentage of decolorization was calculated (Saratale et al., 2006) as follows: 

(%) Decolorization = (Initial absorbance – Observed absorbance) / Initial absorbance X 100 

A loopful of culture was inoculated in 250 ml Erlenmeyer flask containing 100 ml 

nutrient broth. Separate study was carried out for different temperatures (20, 30, 40 and 50) and 

pH (3-10). Decolorization was also studied under shaking (150 rpm/min) and static (nonaerated) 

conditions. Decolorization was also studied in minimal media (mgl -1 ): Glucose 1800; 

MgSO 4 .7H 2 O 250; KH 2 PO 4 2,310; K 2 HPO 4 5,550; (NH 4 ) 2 SO 4 1,980. Effect of the source of 

nitrogen in the medium for decolorization of the dye was studied using Yeast extract and 

Peptone. 

Identification of the Isolate SK20 by 16SrRNA Gene Amplification and Sequencing 

DNA was extracted from pure culture of the isolate SK20 that showed prospective application. 

A partial DNA sequence for 16SrRNA gene was amplified by using ATG GAT CCG GGG 

GTT TGA TCC TGG CTC AGG(forward primer) and TAT CTG CAG TGG TGT GAC GGG 

GGG TGG (reverse primer) (Jing et al., 2004). Amplifications performed in 50µl reactions 

mixtures containing the template DNA, 40ng, 0.2µM, for each of the primers, dNTPs 200µM, 

Taq DNA polymerase 2.5U and 10X buffers 5µl. The mixture was subjected to the following 

amplification conditions; 2min at 94°C for 1min, and ended by a final extension step at 72°C for 

7min. The PCR products mixture was purified and sequenced at Chromous Biotech, Bangalore, 


186


India. The identity of the bacterium determined by sequencing method was verified and 

confirmed through biochemical tests. 

Results and Discussion 

From the isolated soil sample, twenty four bacterial isolates were obtained and named as SK 1 

to SK24 based on the Author’s name. The absorption maxima for Black HFGR were found to 

be at 600 nm (Table 1). Decolorization occurred only when nutrient broth was available in the 

medium. This was confirmed when the minimal media was replaced by the Nutrient Broth. 

Among the 24 isolates, SK03, SK20 and SK 21 demonstrated decolorization beyond 50% while 

the other isolates did not show any decolorization activity beyond 23.25% (Table 2). 

Table 1: Absorption Maxima (λ max) of Reactive Black HFGR dye. 

Dye 

λ max (nm) 

Reactive Blue 604 

Table 2: Showing % of decolorization of Reactive Black HFGR dye by 24 bacterial isolates. 

S. No Isolate Black HFGR 

1 SK1 4.65 % 

2 SK2 10.46 % 

3 SK3 53.48 % 

4 SK4 6.97 % 

5 SK5 4.65 % 

6 SK6 6.97 % 

7 SK7 10.46 % 

8 SK8 6.97 % 

9 SK9 23.25 % 

10 SK10 6.97 % 

11 SK11 9.30 % 

12 SK12 930 % 

13 SK13 3.48 % 

14 SK14 15.11 % 

15 SK15 4.65 % 

16 SK16 0.00 % 

17 SK17 0.00 % 

18 SK18 0.00 % 

19 SK19 0.00 % 

20 SK20 54.65 % 

21 SK21 51.16 % 

22 SK22 0.00 % 

23 SK23 1.16 % 

24 SK24 0.00 % 

The optimum temperature for the selective SK03, SK20 and SK21 demonstrated better 

decolorization at a temperature of 40°C but the magnitude of decolorization differed with 

57.8%, 63% and 97.9% respectively (Figure 1). The optimum pH among the three isolates 

SK03, SK20 and SK21 were 6, 7 and 8 respectively. However, highest decolorization was 

obtained in SK21 (Figure 2). For all the three isolates, static condition demonstrated the highest 


187


percentage of decolorization and SK21 was found to be the best isolate for decolorizing reactive 

black HFGR upto 95.5% (Figure 3). When choice of nitrogen source was tested, yeast extract 

was suitable for SK03 and SK20 showing 45.8% and 50.8% of decolorization respectively. 

Peptone was seen to be suitable for SK21 producing 25% of decolorization. 

Figure 1: Effect of Temperature on the Decolorization of reactive black HFGR by SK21. 

Figure 2: Effect of different pH on the Decolorization of reactive black HFGR by SK21. 


188


Figure 3: Effect of shaking and static (non-aerated) condition on the Decolorization of reactive black 

HFGR by SK21. 

UV-Vis Spectral Analysis of Biodecolorization 

The UV-Vis spectra of the media containing the dye before decolorization showed a maximum 

absorption at 600 nm (0 hour). In the final stage after decolorization, the absorption maxima 

totally disappeared from 600 nm (12 hours). Disappearance of the peak from 600 nm is a clear 

evidence of molecular rearrangements in the dye structure and degradation thereof (Figure 4). 

As reported by Asad et al. (2007) decolorization of dyes by bacteria is due to adsorption 

by microbial cell as a surface phenomenon or to biodegradation. In case of adsorption, the 

UV-Vis absorption peak would tend to decrease approximately in proportion to each other, 

whereas, in biodegradation either the major visible light absorbance peak disappears completely 

or a new peak appears. The observation of Paenibacillus lautus strain SK21cells mass retained 

their natural colour after decolorization of reactive black HFGR. Based on this, it is confirmed 

that the reactive black HFGR has undergone biotransformation and not due to simple adsorption 

over the surface. 

Physiological differences among the bacterial isolates may account for differences in 

the decolorization abilities (Reddy, 1995; Asgher et al., 2006). The complex enzymatic system 

responsible for the dye degradation and pattern of its expression may also vary among the 

isolates (Nagai et al., 2002; Boer et al., 2004); however, the relative rates of decolorization for 

the reactive blue dye cannot be easily explained. Degradation of dye involves aromatic ring 

cleavage which is dependent on the identity of the ring substituents with the presence of 

phenolic, amino, acetamido, 2-methoxy phenol or other easily biodegradable functional groups 

resulting in a greater extent of degradation (Spadaro et al., 1992; Mazmanci and Unyayar, 

2005). In the present study, reactive black HFGR was found to be decolorized to different 

extents by the individual bacterial isolates. Different isolates have degraded the dye to different 

levels following a different pattern during the incubation period as is commonly observed in 

studies elsewhere (Knapp et al., 1997; Toh et al., 2003). However, overall complexity of 

structure alone is not an indicator of the difficulty of decolorization of a particular dye (Maas 

and Choudary 2005). 


189


Table 3: Biochemical profile of Paenibacillus genus. 

S.No. Biochemical Test Result 

1. Glycerol + 

2. Esculin + 

3. Maltose + 

4. D-Arabinose + 

5. L-Arabinose + 

6. Ribose + 

7. D-Xylose + 

8. a-Methylxyloside + 

9. Rhamnose - 

10. Dulcitol - 

11. Inositol - 

12. Sorbitol - 

13. 1-Methyl-Dmannoside - 

14. Arbutin + 

15. Salicin + 

16. Lactose + 

17. Starch + 

18. Glycogen + 

19. D-Tagatose - 

20. D-Arabitol - 

21. 5-Ketogluconate - 

22. 2-Ketogluconate - 

23. Gentiobiose + 

24. Raffinose + 

25. Trehalose + 

26. Sucrose + 

27. Melibiose + 

28. Cellobiose + 

29. Amygdalin + 

30. N-Acetylglucosamine + 

31. Mannose + 

32. Glucose + 

33. Galactose + 

34. Adonitol + 

35. D-Fucose + 

36. Glycerol + 

37. Esculin + 

38. Maltose - 

39. D-Arabinose - 

40. L-Arabinose + 

+ Positive: - Negative 


190


Figure 4: UV-Vis spectra of Reactive Black HFGR biodegradation at 0 hr (a) and 12 hrs (b). 

Nucleotide Sequence Accession Number 

The bacterial isolate SK21 was observed to be gram-positive, rod-shaped bacteria and identified 

to belong to Paenibacillus genus based on the biochemical tests (Albert and Anciet, 1999) 

(Table 3). This was verified and confirmed with the sequence analysis of the amplified 16S 

ribosomal DNA and therefore the isolate SK21 was determined (using the BLAST tool on 

http://www.ncbi.nlm.hih.gov) to be Paenibacillus lautus SK21. The sequence data has been 

deposited in the GenBank nucleotide sequence databases under accession number FJ974057. 

Reactive black HFGR was completely biotransformed by the novel Paenibacillus lautus 

SK21 bacterial strain isolated from textile industry waste land. UV-Vis spectroscopic studies has 

revealed the molecular rearrangement of the dye and therefore confirmed to have undergone 

biodecolorization and biodegradation. Our study indicates the use of our novel isolate for 

environmentally safe disposal of the textile dyes as compared to the existing physico chemical 

methods. 

References 

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Applied and Environmental Microbiology, 65-10: 4425-4430. 

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Technol., 98: 2082–2088. 

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solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main 

lignolytic enzyme. Bioresource Technology, 94: 107–112. 

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solution containing Malachite Green by microalgae Cosmarium sp. Bioresour. Technol., 98: 

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semi continuous anaerobic reactors. Process Biochemistry, 40: 699–705. 

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immobilized on Luffa cylindrica sponge. Process Biochemistry, 40: 337-342. 

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characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and 

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textile effluent containing azo, diazo and reactive dyes. Process Biochemistry, 31: 435–442. 

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www.ejteb.org 

Field Study for the Management of Rice Blast with 

Minimum Fungicides 

P. Krishnan* 

Post Graduate and Research Department of Botany, The Madura college, Madurai- 625 011, Tamil Nadu, India 

Received: 17 January, 2010; revised received: 30 March, 2010. 

Abstract 

Rice blast caused by Pyricularia oryzae Cav. strikes whenever and wherever the climatic 

factors are favourable. It attacks rice plants at various stages of growth in rice fields and 

causes great production loss. Fungicide application is still the preferred control measure 

against blast, but to minimize the fungicide use different treatment practices were 

investigated to control the leaf and neck blast effectively under field conditions. Among the 

various treatments given, the seed treatment with Pyroquilon (4 g ai/kg of seed) with 

additional foliar sprays (1%) gave maximum leaf blast protection (77%). Foliar spray of 1% 

Pyroquilon, one at the time of panicle emergence and another at 15 days after the formation 

of panicle, gave maximum neck blast protection (>90%) over the control. 

Keywords: Rice blast, Pyricularia oryzae , pyroquilon, foliar spray, seed treatment. 

Introduction 

The blast disease of rice (Oryza sativa L.) caused by Pyricularia oryzae Cav. is considered the 

most important disease of rice. The disease outbreaks whenever and wherever the climatic 

factors are favourable. The blast fungus attacks rice plants at various stages of growth in rice 

fields 

and produces lesions on leaves, nodes, panicles and grains (Suzuki, 1975). The 

aggregated annual toll from even light infections may cause greater production losses (Bhatt, 

1988). Till date, the primary control measures largely followed in the rice fields are cultural 

practices, fungicides application, biological control and raising the resistant cultivars (Ou, 1980; 

Muralidharan et al., 2004). It is obvious that fungicide is still the preferred control measure 

against blast (Teng, 1994). 

Nagarajan (1988) has reported that the blast disease can be effectively controlled by the 

application of Bavistin, Topsin, Edifenphos, Fongorine and Tricyclazole as foliar spray and seed 

treatment. Seed treatment with the fungicide Pyroquilon at the concentration of 4 g ai/kg of seed 

and foliar spray with 1% solution gave maximum protection against this disease (Narasimhan et 

al., 1991; Nagarajan, 1988; Surin et al., 1988; Sharma and Sood, 1990; Bhatt and Singh, 1990). 

Drawbacks to the use of chemicals to control blast include increased cost of rice 

production due to the spraying of fungicides frequently throughout the growing season, addition 

of pollutants to the environment by recurrent application of agrochemicals, and development of 

resistance by the pathogen to the fungicides. To counter these problems, an attempt is made to 

investigate the possible methods to minimize the fungicide application for managing the blast 

disease effectively. 


Present investigation was carried out in Kulamangalam, situated 09 o 59.3 N 078 o 07.1 E and 

494 ft. above the sea level, 20 km away from Madurai city, TamilNadu. The experiment was 

conducted in the field trial plots during second crop seasons of 2006-2007 and 2007-2008 using 

the rice cv. IR 50. The optimum concentration, for seed treatment (4g ai / Kg seed) and for 

*Corresponding author; Email address:funkittu@rediffmail.com 

193

Krishnan / Management of Rice Blast with Minimum Fungicides 

foliar spray (1% aqueous solution), of the fungicide Pyroquilon was alone used in this study 

with the rice cv. IR 50. 

Time of Fungicide Application to Control Leaf Blast 

To find out the appropriate time of fungicide application to achieve maximum control of leaf 

blast, Pyroquilon treatments were given as follows: 

T 0 = Control (crops without fungicide treatment), 

T 1 = Five foliar sprays on the seedlings with >0.20 disease proportion at 7 day 

intervals and the spray initiated at 27 days after sowing (DAS), 

T 2 = Four foliar sprays on the seedlings with the proportion of disease incidence 

ranging between 0.10 - 0.20 at 10 day intervals and the spray initiated at 

20 DAS, 

T 3 = Seed treatment, 

T 4 = Seed treatment with one foliar spray at 35 DAS and 

T 5 = Seed treatment with two foliar sprays at 35 and 50 DAS . 

The IR 50 seeds pretreated with fungicides as well as untreated seeds were sown 

directly in the field plot (1 m 2 ) prepared in a randomized complete block design with triplicates 

and exposed for natural blast infection. 

Time of Fungicide Application to Neck Bast Control 

Paddy crops (IR 50) were raised on the microplots (1 m 2 ) prepared in a randomized complete 

block design with triplicates; however, to procure the crops of panicle initiation stage the 

planting was done 50 days before the onset of the favourable conditions for the blast disease. To 

control neck blast timely, Pyroquilon sprays were given as follows 

NB 0 = Control (crops without fungicide treatment), 

NB 1 = One spray at the time of panicle emergence and another spray at 15 days 

after the panicle formation and 

NB 2 = Two sprays at 10 day intervals and the spraying was begun after the 

observation of >0.10 neck blast disease proportion on the plants. 

The crops in the field plots were exposed for natural blast infection. 

Assessment of Blast Disease Incidence 

Ten hills per plot were randomly fixed and the blast incidence was assessed at 7 day intervals on 

the leaves and neck regions (Loganathan and Ramaswamy, 1984). The AUDPC was calculated 

(Shaner and Finny, 1977) for each treatment and percent disease protection was also computed 

as: 

where, 

DC – DT 

DP = X 100 

DC 

DC = proportion of disease incidence in control plants and 

DT = proportion of disease incidence in fungicides treated plants. 

Results 

The rice blast symptom appeared on the rice cv. IR 50 leaves of 20 d old seedlings. The results 

of disease incidence (Table 1) showed that the fungicide treatments (T 1 , T 2 , T 3 , T 4 and T 5 ) 

suppressed the blast disease progress over the check (T 0 ). The seed treatment (T 3 ) of 

Pyroquilon gave the maximum protection till 30 DAS and it also reduced subsequent disease 

progress. Moreover, the seed treatment with additional foliar sprays (T 4 and T 5 ) resulted in 

further reduction in blast disease progress over T 3 . The same trend was seen in each season 

studied (Table 1) from 2006 to 2008. 


194


Figure 1: Area under disease progress curve of leaf blast (A) and neck blast (B) against various 

treatments of Pyroquilon on the rice CV IR 50 during 2006 to 2008 seasons. 

T 0 , T 1 , T 2 , T 3 , T 4 , and T 5 indicate control, five foliar sprays at 7 day intervals, four sprays at 10 day 

intervals, seed treatment, seed treatment plus one spray and seed treatment plus two sprays respectively. 

NB 0 , NB 1 and NB 2 indicate control, two foliar sprays, (one spray at the time of panicle emergence and 

another at 15 days after the panicle formation) and two sprays at 10 day intervals (after the panicle 

emergence) respectively. Values above the box denote the % disease protection over control. 


195


The AUDPC calculated for leaf blast on the rice cv.IR50 against each treatment showed 

that the seed treatment reduced the AUDPC with a protection of above 65% during 2006-2007 

and 73% during 2007–2008 seasons when compared to the foliar spray given alone. The 

differences in disease control were significant (P < 0.01) according to Duncan’s Multiple Range 

Test. Further, the seed treatment with additional foliar spray(s) rendered greater protection (up 

to 77%), but the difference was not significant among T 4 and T 5 (Fig. 1). Of the foliar sprays 

given at definite time intervals (T 1 and T 2 ) from the onset of blast, T 2 (foliar spray of 1% 

Pyroquilon at 10 day intervals) yielded considerable restriction of blast (over 40% disease 

protection) and T 1 (foliar spray of 1% Pyroquilon at 7 day intervals) appeared to be less efficient 

(maximum of 16% protection). Further, the studies revealed that the treatment effect was same 

in the crop seasons of both years in spite of disease pressure being higher during 2007-2008 

season than in 2006-2007 season. 

Table 1: Leaf blast disease incidence against various fungicide treatments in the rice cultivar IR 50 

during 2006-2008 seasons. 

Season Treatment # Days after sowing 

Proportion of disease incidence @ 

2006-2007 

2007-2008 

20 27 34 41 48 55 

T 0 0.09a 0.18a 0.29a 0.37a 0.42a 0.43a 

T 1 0.09a 0.18a 0.28a 0.33a 0.40a 0.42b 

T 2 0.09a 0.17a 0.26a 0.31a 0.38a 0.38b 

T 3 0.00a 0.00b 0.10b 0.16b 0.19b 0.23c 

T 4 0.00a 0.00b 0.09b 0.13b 0.17b 0.19c 

T 5 0.00a 0.00b 0.12b 0.14b 0.16b 0.16c 

T 0 0.15a 0.24a 0.44a 0.53a 0.60a 0.66a 

T 1 0.16a 0.25a 0.36a 0.42b 0.50b 0.52b 

T 2 0.13a 0.19a 0.25bc 0.30c 0.33c 0.38c 

T 3 0.00b 0.00b 0.14c 0.17d 0.23cd 0.28d 

T 4 0.00b 0.00b 0.17c 0.18d 0.22d 0.25ed 

T 5 0.00b 0.00b 0.14c 0.16d 0.19d 0.19e 

# T 0 , T 1 , T 3 , T 4 and T 5 indicate control, five foliar sprays at 7 day intervals (spray 

initiated at 27 days after sowing), four foliar sprays at 10 day intervals (spray initiated at 

20 Days after sowing), seed treatment, seed treatment plus one spray and seed treatment 

plus two sprays respectively. 

@ Each value is the mean of three replicates. 

Disease incidence followed by a common letter are not significantly different at 1% level 

by Duncan’s Multiple Range test. 

The study of the blast disease incidence on the neck region revealed that NB 1 and NB 2 

treatments reduced the neck blast progress over the check. In control, the blast disease 

progressed to the maximum proportion of 0.28 and 0.35 during 2006-2007 and 2007-2008 

seasons respectively. The proportion of disease was less (0.10 in the neck regions) treatment, the disease 

progressed to the maximum proportion of 0.14 after first spray and the subsequent spray also 

reduced the disease progress (Table 2). Further, the AUDPC of neck blast for each treatment of 

Pyroquilon reveals that NB 1 treatment yielded maximum disease protection (>90%) over the 

control. The NB 2 treatment reduced AUDPC to 31% and 41% during 2003-2004 and 2004-2005 

seasons respectively (Fig. 1). In general, both the NB 1 and NB 2 treatments reduced the blast 

disease incidence significantly (P


Discussion 

The results on fungicide application as a measure to protect rice crops against blast revealed 

differential effect of selected fungicides with respect to the mode of application. 

Fungicide sprays are unnecessary when the conditions are unfavourable for disease 

onset. But, application of fungicides during favorable conditions for the disease in the field is 

undoubtedly important for efficient control of crop disease (Madden et al., 1978). Hence, 

fungicide applications were timed to optimize fungicide use (Fry, 1977; Vincelli and Lorbeer, 

1987) according to the conditions conducive for disease development and / or disease intensity. 

Castor et al. (1975) have suggested weather-based or disease intensity-based fungicide 

scheduling system to control potato late blight with minimum utility of fungicides. The timing 

of fungicide application to control blast epidemics in Japan and Colombia were reported by 

Kobayashi (1984) and Ahn and Rubiano (1984). 

Table 2: Neck blast disease incidence against various fungicide treatments in the rice cultivar IR 50 

during 2006-2008 seasons. 

Season Treatment # Days after sowing 

Proportion of disease incidence @ 

2006-2007 

2007-2008 

60 67 74 81 55 

NB 0 0.06a 0.09a 0.17a 0.26a 0.28a 

NB 1 0.00b 0.00b 0.00b 0.02b 0.02b 

NB 2 0.06ac 0.10ac 0.13c 0.14c 0.15c 

NB 0 0.10a 0.18a 0.27a 0.32a 0.35a 

NB 1 0.00b 0.00b 0.02b 0.03b 0.03b 

NB 2 0.11ac 0.14c 0.15c 0.16c 0.16c 

# NB 0 – control; NB1 - two foliar sprays, one spray at the time of the panicle emergence and 

another spray at 15 days after the panicle formation; NB 2 - two sprays at 10 day intervals and 

the spray initiated when the observation of >0.10 disease proportion 

@ Each value is the mean of three replicates 

Disease incidence followed by a common letter are not significantly different at 1% level by 

Duncan’s Multiple Range test 

In the present study, the IR 50 rice crops raised from seeds pre-treated (T 3 ) with 

Pyroquilon were protected against the blast to the extent of 65% during 2006-2007 and 73% 

during 2007-2008. Further, enhanced protection to the extent of 77% was achieved by 

subsequent foliar application (T 3 and T 4 ). But, the application of fungicide in terms of foliar 

spray alone (T 1 and T 2 ) after the disease onset at 10 day interval gave the maximum of 40% 

disease protection. Shoemaker and Lorbeer (1977) have also suggested fungicide-scheduling 

system based on critical disease level to control leaf blight of onions. 

Of the Pyroquilon spray schedules followed to control neck blast, the spray given at the 

time of emergence of inflorescence (NB 1 ) protected the rice cv. IR 50 to the extent of 95%. In 

NB 2 treatment, where fungicide application started only after the appearance of visual blast 

symptom, the maximum protection achieved was only 41%. The results demonstrate that both 

the treatments reduced the disease incidence significantly (P


In general, the studies on the time dependent application of fungicides suggest that the 

seed treatment with weather-based additional sprays could be recommended to the farmers for 

controlling leaf blast of susceptible rice cvs., if the conducive climatic conditions for blast 

development are observed before sowing. If this could not be done in time, foliar sprays at 

definite time intervals according to the disease intensity can be adopted. Further, to control neck 

blast, the farmers may follow fungicide spray before the panicle emergence according to 

weather conditions and if not, fungicide application at 10 day intervals should be necessary 

according to disease severity. 

References 

Ahn, S.W., and Rubiano, M. 1984. Methods and timing of fungicide application to control rice blast 

under favourable upland conditions in colombia. Int. Rice Res. Newsl. 9: 5. 

Bhatt, J.C. 1988. Yield loss in five rice varieties due to blast disease. J. Hill Res., 1: 115-118. 

Bhatt, J.C., and Singh, R.A. 1990. Fungicidal control of blast in hills. Pp228. Proc.Int.Symp. Rice Res. 

New Frontiers. Nov. 15-18. Directorate of Rice Research. Hyderabad. pp.465. 

Castor, L.L., Ayers, J.E., MacNab, A.A., and Kranze, R.A. 1975. Computer forecasting system for 

Stewart’s bacterial disease on corn. Plant. Dis. Rep., 59: 533-536. 

Fry, W.E.1977. Integrated control of potato late blight: Effects of polygenic resistance and techniques of 

timing of fungicide applications. Phytopathology, 67: 415-420. 

Kobayashi, J. 1984. Studies on epidemics of rice leaf blast Pyricularia oryzae Cav. in its early stage [In 

Japanese, English summary]. Bull. Akita Agric. Exp. Stn., 26: 1-84. 

Loganathan, M., and Ramaswamy, V. 1984. Effect of blast (BI) on IR 50 in late Samba. Int. Rice Res. 

Newsl., 9: 6. 

Madden, L., Pennypacker, S.P., and Macnab, A.A. 1978. Fast, a forecast system for Alternaria solani on 

tomato. Phytopatholgy, 68: 1354-1358. 

Muralidharan, K., Reddy, C.S., Krishnaveni, D., and Laha G.S. 2004. Field application of fluorescent 

Pseudomonas products to control blast and sheath blight diseases in rice. J. Mycol. Pl. Pathol., 34: 

411-414. 

Nagarajan, S. 1988. Epidemiology and crop loss of rice, wheat and pearl millet diseases in India. 5 th Int. 

Cong. Pl. Pathol., Kyoto, Japan.. Aug. 20-27. 88pp. 

Narasimhan, V., Ramdoss, N., Chandrasekaran, A., and Abdul Kareem, A. 1991. Chemical management 

of blast disease of rice. Symp. New. Front. Chemi. Cont. October 9-10. Centre for advanced study in 

Botany, University of Madras, India. 25 pp. 

Ou, S.H. 1980. Look at worldwide rice blast disease control. Plant Dis., 64: 439-445. 

Shanner, G.E. and Finney, R.E. 1977. The effect of nitrogen fertilizers on the expression of slowmildewing 

resistance in knox wheat. Phytopathology, 67: 1051-1056. 

Sharma, O.P., and Sood, G.K. 1990. Evaluation of fungicides against glume blight control. Proc. Int. 

Symp. Rice. Res: New Frontiers. November, 15-18. Directorate of Rice Res. Hyderabad. 465 pp. 

Shoemaker, P.B., and Lorbeer, J.W. 1977. Timing of initial fungicide application to control botrytis leaf 

blight epidemics on onions. Phytopathology, 67: 409-414. 

Surin, A, Arunyanart, P., Dhitkiattipong, R., Rojanahusdin, W., Disthapron, S., and Soontrajarn, K. 1988. 

Rice yield loss to sheath rot. Int. Rice Res. Newsl., 13: 6. 

Suzuki, H. 1975. Meterological factors in the epidemiology of rice blast. Annu. Rev. Phytopath., 13: 239- 

255. 

Teng, P.S. 1994. The epidemiological basis for blast management. In: The Rice Blast Disease. Edited by: 

R.S. Zeighler. CAB, International. pp 409-433. 

Vincelli, P.C and Lorbeer, J.W. 1987. Sequential sampling plan for timing initial fungicide application to 

control Botrytis leaf blight of onion. Phytopathology, 77: 1301-1303. 


198



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Anamorphs of Asterinales 

V. B. Hosagoudar* 

Tropical Botanic Garden and Research Institute, Palode 695 562, Thiruvananthapuram, Kerala, India 

Received: 30 October, 2009; revised received: 30 December, 2009. 

Abstract 

The order Asterinales includes two families, Asterinaceae and Lembosiaceae with 29 

genera. Of these, 11 teleomorphic genera have 12 anamorphic genera. The key has been 

provided here to facilitate the identification of anamorphic genera with their teleomorphic 

connection. Each anamorphic genus supplemented with generic characters and line 

drawings. 

Key words: Black mildews, Asterinales, anamorphs, taxonomy. 

Introduction 

Asterinaceous fungi are ectophytic, obligate biotrophs infecting wide range of flowering plants 

ranging from herbs to trees, weeds to economically important cultivated plants, etc. These fungi 

produce thin to dense black colonies on the surface of the leaves. Structurally brown superficial 

mycelium produces appressoria which in turn produce haustoria or nutritive hyphae into the 

epidermal cells of the host plants for the nourishment. The fruiting body is flattened with 

radiating cells known as thyriothecium, which splits radially like a star (aster), hence they are 

known as Asterinaceous fungi. The family Asterinaceae was raised to an order Asterinales 

(Barr, 1976). The order Asterinales includes four families: Asterinaceae, Englerulaceae, 

Parmulariaceae and Parodiopsidaceae (Eriksson and Hawksworth, 1986). Muller & Arx (1962) 

and Arx and Muller (1975) have clearly distinguished Asterinaceae from Microthyriaceae. 

Apparently, these two unrelated families show similarity. The former is with non-ostiolate 

thyriothecia, dehisce stellately at the center and have oval to globose asci. While, the latter has 

ostiolate thyriothecia with cylindrical asci. The family Asterinaceae includes 27 genera (Arx 

and Muller, 1975). Subsequently, Asterinaceae segregated and a new family Lembosiaceae was 

proposed to include the genera having ellipsoidal to elongated or X or Y shaped thyriothecia 

split or dehisce longitudinally (Hosagoudar et al., 2001). Ishwaramyces and Maheshwaramyces 

have been added to this group (Hosagoudar et al., 2004, 2009). 

Pleomorphy is a common phenomenon with almost all higher fungi in which 

teleomorph belongs to either Basidiomycetes or Ascomycetes and the anamorph belongs to 

Deuteromycetes or Fungi Imperfecti. All teleomorphs are not represented with their anamorphs. 

It may be: the fungus would have lost this stage or we are unaware of this. However, it is well 

studied in case of Schiffnerula (Hughes, 1987). Anamorphs will give vital clue in the process of 

identification. Hence, an attempt has been made here to ease the role of identification of 

teleomorphs of Asterinales with the help of their anamorphs. Key for the identification of 

anamorphs of the genera: Asterina, Asterodothis, Batistinula, Eupelte, Prillieuxina, 

Schiffnerula, Symphaster, Trichomelia and Uleothyrium is provided. 

*Corresponding author; Email address:vbhosagoudar@rediffmail.com 

199

Hosagoudar / Anamorphs of Asterinales 

Key to the Anamorph Genera 

1. Reproduction by conidia produced on conidiophores … 1 

1. Reproduction by pycnidiospores / pycnothyriospores … 8 

2. Conidia 3-4-armed … 

Triposporium 

(cf. Batistinula) 

2. Conidia not so … 3 

3. Conidia brown to black, sarciniform … 

Sarcinella 

(cf. Schiffnerula) 


4. Conidia oval, mostly 0-4-6-septate, on Ziziphus … 

Mitteriella 



5. Conidia fusiform, sickle shaped, always 3-septate, pale brown … 

Questieriella 



6. Conidia cheiroid, with 4-5 closely appressed arms … 

Digitosarcinella 


6.Conidia not so … 7 

7. Appressoria present … 

7. Stomopodia present … 

8. Mycelium appressoriate … 11 

8. Mycelium non-appressoriate … 9 

9. Appressoria formed only around the stomata of the host plant … 

9. Appressoria totally absent … 10 

10. Pycnothyriospores unicellular … 

10. Pycnothyriospores one to few septate and of different shapes … 

11. Pycnothyriospores ovate, clavate, margin entire … 

11. Pycnothyriospores conoid, angular to slightly depressed margin … 


200 

Clasterosporium 

(cf. Trichomelia, 

Eupelte, 

Asterodothis, 

Maheswaramyces 

Septoidium 

(cf. Eupelte) 

Bramhamyces 

(cf. Symphaster) 

Asterostomula 

(cf. Prillieuxina) 

Septothyrella 

(cf. Uleothyrium) 

Asterostomella 

(cf. Asterina) 

Mahanteshamyces 

(cf. Asterina)


Figure 1: The genus Asterostomella. a -Appressoriate mycelium, b- Pycnothyrium, c-Pycnothyriospores. 

The genus Asterostomella 

Asterostomella Speg., Ann. Soc. Cien. Arg. 22: 198, 1886. 

Leaf parasites. Mycelium ectophytic, appressoria lateral, setae absent. Pycnothyria 

orbicular with radiating cells, astomatous, dehisce stellately at the center; pycnothyriospores 

unicellular, ovate, pyriform, brown. 

Type: A. paraguayensis Speg. 

The genus Asterostomula 

Asterostomula Theiss., Ann. Mycol. 14: 270, 1910. 

Leaf parasites. Mycelium ectophytic, appressoria and setae absent. Pycnothyria 

orbicular with radiating cells, astomatous, dehisce stellately at the center; pycnothyriospores 

unicellular, ovate, pyriform, brown. 

Type: A. loranthi Theiss. 


201


Figure 2: The genus Asterostomula. a-Non-appressoriate mycelium, b-Pycnothyrium, 

c- Pycnothyriospores. 

The genus Bramhamyces 

Bramhamyces Hosag., Indian J. Sci. Techn. 2(6): 17, 2009. 

Leaf parasites. Hyphae brown, branched, septate, ramify in the grooves only around 

stomata to form ‘areole’ to produce 1-3-appressoria. Remaining hyphae devoid of appressoria. 

Appressoria produced on the guard cells of the stoma produce corolloid haustoria in the 

neighboring cells. Stomata often plugged with mycelium. Pycnothyria grown below the 

mycelium, orbicular, connate; pycnothyriospores unicellular, brown, oval, pyriform. 

Type: B. ilecis Hosag. & Chandra. 

Mycelium devoid of appressoria but are produced around the stomata of the host plant 

by forming ‘areole’ is the character of this anamorphic genus. 

The genus Clasterosporium 

Clasterosporium Schweinitz, Trans. Am. Phil. Soc., N.S. 4: 300, 1832. 

Colonies usually effuse dark brown to black, often velvety. Mycelium superficial, 

stroma absent. Setae absent or sometimes present only in old colonies, simple, dark, smooth, 

straight to uncinate. Appressoria present. Conidiophores macronematous, mononematous, 

straight to flexuous, simple. Conidiogenous cells monoblastic, integrated, terminal, determinate, 

percurrent, cylindrical. Conidia solitary, acrogenous, simple, straight to curved, cylindrical to 

obclavate. 

Type: C. caricinum Sch. 


202


Figure 3: The genus Bramhamyces. a and b-Non-appressoriate mycelium produce ‘areole’ around 

stomata and with appressoria produced at the apical portion of the mycelium around the guard cells, c- 

Pycnothyrium, d- Pycnothyriospores. 

Figure 4: The genus Clasterosporium: a- Appressoriate mycelium, b- Conidiophore, c- Conidia 


203


Figure 5: The genus Digitosarcinella (After Hughes, 1984). a - Hyphae, b- Developing conidia on 

conidiophores, c- Cheiroid conidium 

The genus Digitosarcinella 

Digitosarcinella Hughes, Can. J. Bot. 62: 2208, 1984. 

Colonies foliicolous. Hyphae superficial, brown to dark brown, branched, appressoriate, 

appressoria sessile, lateral, unicellular. Conidiogenous cells lateral, sessile, monoblastic. 

Conidia cheiroid, with 4-5 closely appressed arms, up to 7-septate, constricted at the septa. 

Type: D. caseariae Hughes 

The genus Mahanteshamyces 

Mahanteshamyces Hosag., J. Econ. Taxon. Bot. 28: 189, 2004. 

Foliicolous, ectophytic parasites. Mycelium brown, superficial, appressoriate. 

Pycnothyria scutate, dimidiate, radiate, orbicular, stellately dehisce at the center; 

pycnothyriospores unicellular, brown, angular, wall straight to sinuate. 

Type sp.: M. agrostistachydis Hosag. & C.K. Biju 

The genus Mahanteshamyces differs from Asterostomella in having roundedly 

projected and shallowly lobate, angular and thick walled pycnothyriospores (Batista and 

Cifferri, 1959; Sivanesan, 1983; Sutton, 1980). 


204


Figure 6: The genus Mahanteshamyces Hosag. a-Appressoriate mycelium, b-Thyriothecium, 

c- Pycnothyriospores. 

Figure 7: The genus Mitteriella. a - Appressoriate mycelium, b- Conidia. 


205


The genus Mitteriella 

Mitteriella Sydow, Ann. Mycol. 31: 95, 1933. 

Colonies black. Hyphae superficial, brown, branched, septate, appressoriate. 

Appressoria lateral, unicellular. Conidiophores macronematous, mononematous, short, simple. 

Conidiogenous cells polyblastic, integrated, terminal, sympodial, denticulate. Conidia solitary, 

simple, ellipsoidal to limoniform, black, 0-4-septate. 

Type: M. ziziphina Sydow 

Figure 8: The genus Questieriella. Appressoriate mycelium, b- Germinating conidia. 

The genus Questieriella 

Questieriella Arn. ex Hughes, Can. J. Bot. 61: 1729, 1983. 

Colonies black, hyphae superficial, brown, branched, septate, appressoriate. 

Appressoria lateral, unicellular. Conidiophores micronematous, mononematous to 

macronematous, lateral, 0-2-septate. Conidiogenous cells monoblastic to polyblastic, integrated, 

terminal, lateral or incorporated in the hyphae. Conidia blastic, terminal, solitary, narrowly 

ellipsoidal to obovoidal, curved, falcate, sigmoid, truncate at the base, 3-septate. 

Type: Q. pulchra Hughes 


206


Figure 9: The genus Sarcinella. a-Appressoriate mycelium, b-Conidia on conidiophores, c- Conidia 

Sarcinella Sacc., Michelia 2: 31, 1880. 

The genus Sarcinella 

Colonies black. Hyphae superficial, branched, septate, appressoriate. Appressoria 

lateral, unicellular. Conidiophores macronematous, semi-macronematous, simple to branched. 

Conidiogenous cells monoblastic, integrated, terminal, intercalary, determinate. Conidia 

solitary, acrogenous or acropleurogenous, subspherical, sarciniform, dark brown to reddish 

brown, smooth, constricted at the septa. 

Type: S. heterospora Sacc. 


207

Septoidium Arn., Ann. Epiphyt. 7: 106, 1921. 


The Genus Septoidium 

Colonies effuse, reddish brown, olivaceous brown or black. Mycelium superficial. 

Hyphae thick, often golden brown to reddish brown, smooth, branched, intertwined and 

anastomosing to form a close network. Stroma none, Setae absent. Stomatopodia present, simple 

to lobed. Conidiophores macronematous to semi-macronematous, mononematous, simple, 

straight to flexuous, pale to mid golden brown to reddish brown, smooth. Conidiogenous cells 

monoblastic, integrated, terminal, percurrent, cylindrical.Conidia solitary, dry, acrogenous, 

simple, clavate, cylindrical, rounded at the apex to almost ellipsoidal, always truncate at the 

base, pale to golden brown to reddish brown, smooth, with one or several transverse septa. 

Type: S. clusiaceae Arn. 

Figure 10: The genus Septoidium (After Ellis, 1971). a-Conidia on conidiophores, b- Conidia. 


208


The Genus Septothyrella 

Septothyrella Höhn., Sitz. der Kais. Akad. der Wiss., Math.-naturw. Kl., Abt. 120: 393, 1911. 

Mycelium brown, septate, nonappressoriate, branched. Pycnothyria orbicular, brown, 

glabrus, ostiolate, upper surface with radiating cells. Pycnothyriospores clavate, ellipsoidal, 

entire to horizontally septate. 

Type: S. microthyrioides (Henn.) B. Sutton 

This genus appears to be synonymous to Asterothyrium Henn. 

Figure 11: The genus Septothyrella (Batista & Ciferri, 1959). a-Reticulate mycelium, b-Hyphae, 

c-Pycnothyrium, d-T.S. through the pycnothyrium, e-Pycnothyriospores on the hymenium, 

f- Pycnothyriospores. 

The genus Triposporium 

Triposporium Corda, Icon. Fung. 1: 16, 1837. 

Colonies effuse, black, hairy or velvety. Mycelium mostly immersed. Stroma none. 

Setae and appressoria absent. Conidiophores macronematous, mononematous, scattered, simple, 

straight to flexuous, almost cylindrical, broadened at the base to form a flat plate, brown, 

smooth. Conidiogenous cells monoblastic, integrated, terminal, percurrent, cylindrical, 

doliiform to lageniform. Conidia solitary, dry, acrogenous, branched, usually made up to a small 

calvate, doliiform or cylindrical stalk cells and 3 or occasionally 4 conical smooth, septate arms 

joined by their wide, rounded branch; the arms are dark brown near the centre of the conidium, 

hyaline or sub hyaline at the tips. 

Type: T. elegans Corda 


209


Figure 12: The genus Triposporium (Ellis, 1971). a-Conidiophores, b-Conidia. 

Table 1: Anamorphs and their teleomorphs. 

S.no. Anamorphs Teleomorphs 

1. Asterostomella Asterina 

2. Asterostomula Prillieuxina 

3. Bramhamyces Symphaster 

4. Clasterosporium Asterodothis 

Eupelte 

Maheshwaramyces 

Trichomelia 

5. Digitosarcinella Schiffnerula 

6. Mahanteshamyces Asterina 

7. Mitteriella Schiffnerula 

8. Questieriella Schiffnerula 

9. Sarcinella Schiffnerula 

10. Septoidium Eupelte 

11. Septothyrella Uleothyrium 

12. Triposporium Batistinula 


210


Acknowledgement 

Thanks are due to the Director, Tropical Botanic Garden and Research Institute, Palode, Kerala 

State, India for the facilities. 

References 

Arx, J.A.V., and Muller, E. 1975. A re-evaluation of the bitunicate Ascomycetes with keys to families 

and genera. Stud. Mycol., 9: 1-159. 

Barr, M.E. 1976. Perspectives in the Ascomycotina. Mem. New York Bot. Gard., 28: 1-128. 

Batista, A.C., and Cifferri, R. 1959. Sistematica dos fungos imperfectos de picnostromas con himenio 

invertido (Peltasterales). Mycopath. Mycol. Appl., 11: 1-102. 

Ellis, M.B. 1971. Dematiaceous Hyphomycetes. CMI Kew Surrey, England. 

Eriksson, O., and Hawksworth, D.L. 1986. An alphabetical list of the generic names of Ascomycetes. Systema 

Ascomycetum, 5: 4-184. 

Hosagoudar, V.B., Abraham, T.K., and Biju, C.K. 2001. Re-evaluation of the family Asterinaceae. J. 

Mycopathol. Res., 39: 61-63. 

Hosagoudar, V.B., Archana, G.R., and Mathew Dan 2009. Maheshwaramyces, a new genus of the family 

Lembosiaceae. Indian J. Sci. Technol., 2 (6): 12-13. 

Hosagoudar, V.B., Biju, C.K., and Abraham, T.K. 2004. Studies on foliicolous fungi- II. J. Econ. Taxon. 

Bot., 28: 183-186. 

Hughes, S.J. 1984. Digitosarcinella caseariae sp. nov. and Questieriella synanamorphs of the so-called Amazonia 

caseariae. Canadian J. Bot., 62: 2208-2212. 

Hughes, S.J. 1987. Pleomorphy in some hyphopodiate fungi. In: Pleomorphic fungi - The diversity and its 

taxonomic implications. Edited by: Sugiyama. Kodansa and Elsevier,Tokyo. pp. 103-139. 

Muller, E., and Arx, J.A.von 1962. Die Gattungen der didymosporen Pyrenomyceten. Beitr. 

Kryptogamenfl. Schweiz, 11:1-922. 

Sivanesan, A. 1983. The Bitunicate Ascomycetes. International Books and Periodical Supply Service, 

New Delhi. pp. 701. 

Sutton, B.C. 1980. The Coelomycetes: Fungi imperfecti with pycnidia, acervuli and stromata. CMI, Kew. 

pp. 696. 


211



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Plant Antioxidants Mediated Protein Alterations in 

Clarias batrachus Linn. 

C. Suseela Bai 

Department of Plant Biology and Plant Biotechnology, Women’s Christian College, 

Nagercoil-629001, Tamil Nadu, India. 

Received: 17 August 2009; revised received: 12 January, 2010 

Abstract 

An investigation on the effect of plant antioxidants supplemented feed on blood protein of 

the fresh water cat fish Clarias batrachus was carried out in the laboratory. Fifty fingerlings 

of the fish were reared using supplementary diet for a period of 120 days in outdoor cement 

cisterns. Along with the basic feed fish meal, the plant antioxidants Punica granatum and 

Phyllanthus emblica were given as supplementary feeds to the fishes. Effect of plant 

antioxidants at a dosage of 10 mg/ 100 mg feed on blood protein characteristics of the 

experimental animal was found significant. 

Key words: Plant antioxidants, Clarias batrachus, blood protein 

Introduction 

Plants and plant products significantly build up in the food chain of aquatic organisms 

(Direkbusarakom, 2004) and move up in the food chain of human. It has been observed that 

there are many quantitative relationships between plant products and biological activities of 

fishes established in fresh water aquatic systems. Wild satavari, Asparagus racemosus widely 

used to promote human health produced a similar effect in shrimps (Mony, 2002) and Labeo 

rohita 

( Major Carp) fry (Sharma, 1996). Herbal extracts of Stellaria aquatica, Impatiens 

biflora, Oenothera biennis, Artemisia vulgaris and Lonicera japonica were reported to exhibit 

antimicrobial activity against bacterial and viral fish pathogens (Shagnliang et al ., 1990). 

Many herbal preparations were able to control diseases due to their antioxidant and 

antimicrobial activities (Prasad et al., 1993., Citarasu et al., 1998,2001, 2002; Pundarikakshudu 

et al ., 2001; Sivaram et al., 2004). Moreover, Babu and Marian (2001) and Citarasu et al. 

(2002) demonstrated disease resistant larval production in Penaeus monodon reared on herbal 

supplemented diets. 

The fish, C. batrachus is a popular delicacy because of its faster growth rate, higher 

protein and iron content. In view of it, this paper is aimed at determining the responses of plant 

antioxidants on concentration and profile of blood protein of C. batrachus reared under outdoor 

culture conditions. 


Fifty fingerlings of C. batrachus (average body weight, 4.3 to 5.1g ; average body length 6.1 to 

7.2 cm ) were obtained from a commercial fish farm and transported to the culture site in plastic 

bags filled with aerated water. The fishes were fed daily on a formulated fish feed containing 

65% crude protein, acclimatized for a period of 30 days before the commencement of the 

experiment and they were reared in cement cisterns under outdoor culture conditions during 

which feeding was done with plant antioxidants supplementary feed prepared in dry pelleted 

*Email address: suseela.bai@yahoo.com 

213

Suseela Bai / Plant Antioxidants Mediated Protein Alterations in Clarias batrachus 

form using fruit rind of Punica granatum and dried leaves of Phyllanthus emblica (10 mg/100 

mg feed) separately. The culture medium was changed once in three days before adding the 

feed. Simultaneously a control tank receiving diet without plant products was kept. All the 

experiments were subjected to 12 hr day/ night cycle. 

Five individuals were selected at random from each tank at the end of the experimental 

period of 120 days, subjected to fasting for 12 hrs and blotted dry with soft absorbent paper. 

Blood samples were collected without any anticoagulant and estimations of total protein content 

were carried out using plasma following the procedures of Young (1997). The protein 

separation was done using SDS - PAGE and comparisons were made. 


The total protein content of blood plasma of fishes reared on control and plant antioxidants 

supplementary feed is given in Fig.1 The fishes fed with P. granatum supplemented feed had 

the maximum protein content of 13.70 ± 0.23 g/dl whereas those reared using P.emblica 

supplemented feed registered 6.87 ± 0.56g/dl. The animals reared on control diet had 4.43 ± 

0.13g/dl protein in their blood samples. The statistical treatment of the data (Student ‘t’ test) 

revealed significant differences (P < 0.005). 

(g/dl) 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Control 

P.granatum 

P.emblica 

Figure 1: Effect of plant antioxidants supplementary feed on protein (g/dl) content of C. batrachus. 

General protein profile of fresh blood samples of C.batrachus exhibited no homology as 

in Fig.2. Striking differences could be seen in the protein profile of fishes fed with plant 

antioxidants supplemented diets. Further study is needed to reveal the exact mechanism of plant 

antioxidant mediated free radical scavenging physiological activities. 

Figure 2: SDS-PAGE of blood samples of C.batrachus grown using plant antioxidants supplemented 

feed. 


214

Suseela Bai / Plant Antioxidants Mediated Protein Alterations in Clarias batrachus 

Supplementation of plant products promise a cheaper and viable solution to many 

problems aquaculture industries face today. Methanolic extracts of many herbs successfully 

controlled Vibrio pathogen and improved the immune system of the grouper larviculture 

(Sivaram et al., 2004). Various plant species such as Hygrophila spinosa , Withania somnifera, 

Zingiber officinalis, Solanum trilobatum, Andrographis paniculata, Phyllanthus niruri and 

Tinospora cordifolia promoted growth and served effectively as antistress, antibacterial agents 

and immunostimulants in shrimp / fish larviculture (Citarasu et al., 1998, 2003a, 2003b). The 

herbal extracts of Daemia extensa and Leucas aspera were effective antibacterial agents against 

Vibrio parahaemolyticus and Vibrio harveyi (Latha, 2008). Moreover, species such as Ocimum 

sanctum, W. somnifera and Myristica fragrans improved immune parameters such as 

phagocytic activity, albumin – globulin ratio and leucocrit values in fishes. 

The increase in total protein content of blood serum and the differential protein profile 

of C.batrachus obtained in the present study may possibly be due to plant antioxidants 

supplemented feed. 

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www.ejteb.org 

Quercitrin, a Bioflavonoid Increases Glucose Utilization and 

Altering The Carbohydrate Metobolic Enzymes in 

Streptozotocin-Induced Diabetic Rat Tissues 

Ranganathan Babujanarthanam 1* , Purushothaman Kavitha 1 , Sarika Sasi 2 and 

Moses Rajasekara Pandian 3 

1 Department of Biochemistry, K.M.G.College of Arts and Science, 

Gudiyattam – 632 602, Tamil Nadu, India 

2 Vinayaga Missions University, Salem -600010, Tamil Nadu, India 

3 Department of Zoology, Arignar Anna Government Arts College, 

Namakkal – 637 001, Namakkal District, Tamil Nadu, India. 

Received: 24 November, 2009; revised received: 20 February, 2010. 

Abstract 

The present study is an investigation into the role of quercitrin on carbohydrate metabolism 

in normal and streptozotocin (STZ)-induced diabetic rats. Administration of STZ leads to a 

significant increase (P < 0.05) in fasting plasma glucose and a decrease in insulin levels. 

The content of glycogen is significantly decreased (P < 0.05) in liver and muscle, but 

increased in the kidney. The activity of hexokinase decreased whereas the activities of 

glucose 6-phosphatase and fructose 1,6-bisphosphatase significantly increased (P < 0.05) in 

the tissues. Oral administration of quercitrin (30 mg/kg) to diabetic rats for a period of 30 

days resulted in significant (P < 0.05) alterations in the parameters studied but not in 

normal rats. A decrease of plasma glucose and increase in insulin levels were observed 

along with the restoration of glycogen content and the activities of carbohydrate metabolic 

enzymes in quercitrin-treated diabetic rats. The histopathological study of the pancreas 

revealed the protective role of quercitrin. There was an expansion of the islets and 

decreased fatty infiltrate of the islets in quercitrin treated diabetic rats. In normal rats 

treated with quercitrin, we could not observe any significant change in all the parameters 

studied. Combined, these results show that quercitrin plays a positive role in carbohydrate 

metabolism in diabetic rats. 

Key words: Quercitrin, streptozotocin, diabetes mellitus, gluconeogenesis, pancreas. 

Introduction 

Diabetes mellitus is the world’s largest endocrine disorder resulting in multiple etiologies, 

involving metabolic disorders of carbohydrate, fat and protein. All forms of diabetes are due to a 

decrease in the circulating concentration of insulin (insulin deficiency) and a decrease in the 

response of peripheral tissues to insulin, that is, insulin resistance. According to World Health 

Organization’s projections, the prevalence of diabetes is likely to increase by 35% by the year 

2025 (Boyle et al., 2001). 

Alterations in glucose metabolism in diabetes are frequently accompanied by changes in 

the activities of the enzymes that control glycolysis and gluconeogenesis in liver and muscle, 

such that the latter process becomes favored (Gerich et al., 1993) Increased rates of hepatic 

* Corresponding author; Email address: kmrbabugym@yahoo.com 

217

Babujanarthanam et al / Quercitrin, a Bioflavonoid Increases Glucose Utilization in Rats 

glucose production result in the development of overt hyperglycemia, especially fasting 

hyperglycemia, in patients with type 2 diabetes (DeFronzo et al., 1988). There are several 

important enzymatic checkpoints that act to control hepatic glycolysis and glycogen synthesis 

(glucokinase, glycogen synthase kinase-3), glycogenolysis (phosphorylase), gluconeogenesis 

(phosphoenolpyruvate carboxykinase, fructose 1,6bisphosphatase), or steps that are common to 

the pathways (glucose-6-phosphatase). Some of them are directly controlled by insulin via 

phosphorylation and dephosphorylation (Zhang et al., 2004). 

Plants have always been usable sources of drugs, and many currently available drugs 

are directly or indirectly derived from plants. Many of the oral agents that are presently in use 

for the treatment of diabetes mellitus suffer from implication in a number of serious and adverse 

effects (Zhang et al., 2000). Therefore, it is important to investigate the biologically active 

components of plants with hypoglycemic actions which include flavonoids, alkaloids, 

glycosides, polysaccharides, and peptidoglycans (Grover et al., 2002; Mao et al ., 2002) 

Flavonoids comprise a large group of compounds occurring widely throughout the plant 

kingdom. Daily flavonoid intake (typically present in onion, apple, grape, wine, herbs and 

spices) in the human diet is highly variable, with estimations ranging from 23 mg/day (Hertog et 

al., 1993) to more than 500 mg/day (Manach et al., 1996). Flavonoids exert several biological 

activities, which are mainly related to their ability to inhibit enzymes and their antioxidant 

properties, and are able to regulate the immune response (Hollman et al., 1995). Among 

flavonoids, quercetin is the most common flavonoid in nature, and it is mainly present as its 

glycosylated forms such as quercitrin (5,7,3c,4c-OH, 3-rhamnosylquercetin). 

A wide variety of pharmacological activities of quercitrin was reported, that is, antiinflammatory 

(Sanchez et al., 2002; Taguchi et al 1993) antidiarrhoeals (Galvez), antiinociceptive 

property (Gadotti et al., 2005), antileishmanial activity (Muzitano et al., 2006), and 

neuroprotective (Hollman et al., 1999). However, the majority of the studies have been carried 

out with the aglycone (Quercetin) form and little is known about the biological properties of 

glycoside forms, due to the lack of commercial standards. Therefore, we undertook the present 

study to evaluate the role of quercitrin on the glycogen content and the activities of some 

carbohydrate metabolic enzymes, lipid peroxidation and antioxidant status in normal and STZinduced 

diabetic rats. 


Chemicals 

Adenosine triphosphate, magnesium chloride, ammonium molybdate, fructose 1,6- 

bisphosphate, carboxymethyl cellulose sodium salt, phosphotungstic acid, thiobarbituric acid, 

1,1c,3,3c tetramethoxy propane, butylated hydroxy toluene, xylenol orange, dithionitro bis 

benzoic acid, ascorbic acid, 2,2c dipyridyl, p-phenylene diamine hydrochloride and sodium 

azide were obtained from SD Fine Chemicals, Mumbai, India. Quercitrin and streptozotocin 

were purchased from Sigma Chemical Co., St Louis, MO, USA. All the other chemicals used in 

the present study were of high analytical grade. 

Experimental Animals 

Male albino Wistar rats (150–180 g) were used in this study. The animals were fed on a standard 

pellet diet (Pranav Agro Industries, Pune, India) and water ad libitum. The pellet diet consisted 

of 22.02% crude protein, 4.25% crude oil, 3.02% crude fiber, 7.5% ash, 1.38% sand silica, 0.8% 

calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins and 56.17% carbohydrates. It 

provided a metabolisable energy of 3600 kcal/kg. They were maintained in a controlled 

environment (12 : 12 h light/dark cycle) and temperature (30 ± 2 C). The experiment was 

carried out according to the guidelines of the Committee for the Purpose of Control and 

Supervision of Experiment on Animals (CPCSEA), New Delhi, India. 

Induction of Experimental Diabetes 

Diabetes was induced in 12 h fasted rats with streptozotocin (50 mg/kg) dissolved in citrate 

buffer (0.01 M, pH 4.5) intraperitoneally and the injection volume was 1 mL/rat. Control 


218


animals were injected with citrate buffer alone. After 72 h of STZ injection, blood was 

withdrawn from animals (sinocular puncture) fasted overnight in tubes containing potassium 

oxalate and sodium fluoride as anticoagulant and plasma glucose was estimated using a 

commercial glucose kit (Product No. 72101) provided by Qualigens Diagnostics, Mumbai, 

India. Rats that had a fasting plasma glucose value of above 13.89 mmol/L (250 mg/dL) were 

included in the study as diabetic rats. 

Experimental Design 

A pilot study was conducted previously with three doses of quercitrin (10, 20 and 30 mg/kg 

body weight) to determine the dose dependent effects in STZ-induced diabetic rats. We found 

that 10, 20 and 30 mg/kg of quercitrin significantly (P < 0.05) decreased plasma glucose levels 

and quercitrin at doses 30 mg/kg was more effective in reducing plasma glucose levels 

significantly (P < 0.05) after 30 days of experimental study. Hence, we chose the dose 30 mg/kg 

of quercitrin for further studies. 

For the present study, the animals were grouped as follows: Group I, normal control; 

Group II, normal + quercitrin (30 mg/kg); Group III, diabetic control; Group IV, diabetic + 

quercitrin (30 mg/kg). Quercitrin was suspended in carboxymethyl cellulose (CMC) (0.01 

g/mL) and was orally administered to rats (1 mL/rat) using an intragastric tube. Normal control 

and diabetic control rats received CMC alone (1 mL/rat). 

The treatment period was 30 days, and after the last treatment, rats were fasted 

overnight and sacrificed by cervical decapitation. Blood was collected and plasma was obtained 

after centrifugation and used for various biochemical estimations. Tissues such as liver, kidney, 

muscle and pancreas were excised immediately from the animals and stored in ice-cold 

containers. They were then homogenized with appropriate buffer, centrifuged at low speed (705 

g), and the supernatant was collected. Biochemical estimations were carried out using these 

homogenates. 

Biochemical Assays 

Plasma insulin was assayed by ELISA method using a commercial kit (Catalog No. SP-401) 

from United Biotech Inc., Mountain View, CA, USA. Liver, kidney and muscle glycogen were 

estimated by the method of Morales et al. (Morales et al., 1973). The activity of hexokinase in 

the tissues was assayed by the method of Brandstrup et al.(1957). Glucose 6-phosphatase in the 

tissues was assayed by the method of Koida and Oda (Koida et al., 1959), Fructose 1,6- 

bisphosphatase in the tissues was assayed by the method of Gancedo and Gancedo (Gancedo et 

al., 1971), Phosphorus content of the supernatant was estimated by the method of Fiske and 

Subbarow (Fiske et al., 1925). 

In pancreas, the protein-bound hexoses concentration were estimated by the method of 

Dubois and Gillesl. (Dubois et al., 1956), Protein-bound hexosamine was estimated by the 

method of Wagner (Wagner et al., 1979] Sialic acid in plasma and tissues was estimated by the 

method of Warren et al. (Warren et al., 1959), Fucose in plasma and tissue was estimated by the 

method of Dische and Shettle (Dische et al., 1948). 

Histopathological Studies 

For histopathological studies, animals of different groups were perfused with 10% neutral 

formalin solution. Pancreas was removed immediately from the animals; paraffin sections were 

made and stained using hematoxylin– eosin (H&E) stain. After staining, the sections were 

observed under light microscope and photographs were taken (20x). 


Statistical analysis was done by one-way ANOVA followed by Duncan’s multiple range test 

(DMRT) (Duncan et al., 1957), using SPSS software package, version 9.05. P values < 0.05 

were considered as significant and included in the study. 


219


Results 

The body weight of the experimental rats was recorded throughout the study (data not shown). 

At the end of the experimental period, a significant (P < 0.05) increase in the body weight of 

normal control rats and normal + quercitrin (30 mg/kg) treated rats were observed. The diabetic 

control rats showed a significant (P < 0.05) decrease in body weight when compared with 

normal control rats. Diabetic rats treated with quercitrin (30 mg/kg) showed a significant (P < 

0.05) increase in body weight when compared with diabetic control rats. The levels of fasting 

plasma glucose and insulin are shown in Table 1. In diabetic control rats, the fasting plasma 

glucose levels were significantly (P < 0.05) high (23.17 ± 2.02 mmol/L). Diabetic rats when 

treated with quercitrin (30 mg/kg) had significantly (P < 0.05) decreased plasma glucose levels 

(8.06 ± 0.60 mmol/L). Normal rats treated with quercitrin (30 mg/kg) did not show any 

significant effect on plasma glucose levels (4.07 ± 0.30 mmol/L). The levels of plasma glucose 

in normal control were found to be 4.21 ± 0.29 mmol/L. A significant (P < 0.05) decrease in 

plasma insulin levels was observed in diabetic control rats (7.57 ± 0.22 lU/mL) and on treatment 

with quercitrin (30 mg/kg), the levels significantly (P


Table 1: Effect of quercitrin on the content of glycogen in the tissues of normal and diabetic 

rats. 


Groups 

(mg/g tissue) 

Normal control 3.22 ± 0.20 a 2.19 ± 0.12 a 3.45 ± 0.11 a 

Normal + quercitrin (30 mg/kg) 3.11 ± 0.29 a 2.22 ± 0.10 a 3.49 ± 0.12 a 

Diabetic control 2.01 ± 0.12 b 3.74 ± 0.18 b 2.92 ± 0.10 b 

Diabetic + quercitrin (30 mg/kg) 3.17 ± 0.18 c 2.08 ± 0.16 c 3.21 ±0.14 c 

Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter (a,b,c) 

differ significantly with each other (P


Table 4: Effect of quercitrin on the activity of glucose 6-phosphate in the tissues of normal and diabetic 

rats. 

Groups 

Liver 

Kidney 

(μ mole of Pi liberated/min/mg protein) 

Normal control 23.10 ± 1.62 a 19.80 ± 1.21 a 

Normal + quercitrin (30 mg/kg) 22.20 ± 1.30 a 18.30 ± 1.32 a 

Diabetic control 42.31 ± 2.91 b 37.21 ± 2.47 b 

Diabetic + quercitrin (30 mg/kg) 31.10 ± 2.19 c 24.11 ± 1.52 c 

Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter 

(a,b,c) differ significantly with each other (P


Discussion 

In this study, we found that quercitrin has the ability to increase glucose utilization and 

normalize the carbohydrate metabolic enzymes in STZ-induced diabetic rats. Blood glucose 

level is strictly controlled by insulin secretion from pancreatic b-cells and insulin action on liver, 

muscle and other target tissues(Hii et al., 1984), Quercitrin by its ability to scavenge free 

radicals and to inhibit lipid peroxidation, prevents STZ-induced oxidative stress and protects b- 

cells resulting in increased insulin secretion and decreased blood glucose levels. In this context, 

research by Vessal et al. (Vessal et al., 2003), has shown that quercetin, the aglycone of 

quercitrin decreased blood glucose concentration and increased insulin release in STZ-induced 

diabetic rats. Coskun et al. (Coskun et al., 2005) have also reported that, in STZ-induced 

diabetic rats, quercetin protected pancreatic b-cells by decreasing oxidative stress and 

preserving pancreatic b-cell integrity. Increased insulin levels could also be due to the 

stimulatory effect of quercitrin, thereby potentiating the existing b-cells of the islets of 

Langerhans in diabetic rats. Hii and Howell (Hii et al., 1985), showed increased number of 

pancreatic islets in quercetin treated animals. Hyperglycemia and decreased insulin levels are 

characteristics of diabetic rats in this study. Quercitrin treatment to diabetic rats significantly 

reduced plasma glucose levels and increased insulin levels. Quercitrin, being a flavonoid, could 

induce the intact functional b-cells to produce insulin and/or protect the functional b-cells from 

further deterioration so that they remain active and produce insulin. 

Table 7: Effect of quercitrin on glycoproteins in the kidney of normal and diabetic rats. 

Groups 

Hexose Hexosamine Fucose Sialic acid 

(mg/g defatted tissue) 

Normal control 32.07 ± 3.02 a 24.22 ± 1.49 a 11.37 ± 1.04 a 6.21 ± 0.42 a 

Normal + quercitrin 

(30 mg/kg) 

32.74 ± 3.10 a 24.56 ± 1.67 a 11.29 ± 1.02 a 6.02 ± 0.44 a 

Diabetic control 54.08 ± 4.26 b 34.11 ± 3.08 b 23.27 ± 2.04 b 12.31 ± 1.08 b 

Diabetic + quercitrin 

(30 mg/kg) 

39.21 ± 3.67 c 28.27 ± 2.46 c 16.63 ± 1.44 c 8.03 ± 0.65 c 

Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter (a,b,c) 

differ significantly with each other (P


deficiency as they depend on insulin for influx of glucose. In contrast, kidney glycogen content 

is increased and this is due to the entry of glucose in a hyperglycemic state as renal tissue is 

independent of insulin action (Belfiore et al., 1986), Increased insulin and consequently 

decreased blood glucose levels due to treatment with quercitrin could positively alter the 

glycogen content in the diabetic tissues. 

Alterations in glucose metabolism in diabetic are frequently accompanied by changes in 

the activities of the enzymes that control glycolysis and gluconeogenesis in liver and muscle, 

such that the latter process becomes favored (Gerich 1986), Laakso (Laakso et al., 1995), has 

reported that hexokinase is the first regulatory enzyme of glycolytic pathway that converts 

glucose into glucose 6-phosphate. Glucose 6-phosphatase plays a key role in the regulation of 

blood glucose levels by catalyzing the hydrolysis of glucose 6-phosphate in the common 

terminal step of the gluconeogenic and glycogenolytic pathways (Wallert et al., 2001), Fructose 

1,6-bisphosphatase catalyses the conversion of fructose 1,6-bisphosphate to fructose 6- 

phosphate, a step necessary to achieve a reversal of glycolysis (Maye, 1996). 

Hexokinase insufficiency in diabetic rats can cause decreased glycolysis and decreased 

utilization of glucose for energy production. Oral administration of quercitrin to diabetic rats 

resulted in a significant reversal in the activity of hexokinase. The increased plasma insulin and 

decreased glucose in diabetic rats given quercitrin may also be as a result of increased hepatic 

hexokinase activity, resulting in increased glycolysis. The gluconeogenic enzyme glucose-6- 

phosphatase is a crucial enzyme of glucose homeostasis because it catalyses the ultimate 

biochemical reaction of both glycogenolysis and gluconeogenesis (Mithievre et al., 1996). 

Increased glucose 6-phosphatase activity in diabetic rats provides hydrogen, which binds with 

NADP+ in the form of NADPH and enhances the synthesis of fats from carbohydrates (i.e. 

lipogenesis) Bopanna et al., 1997) and, finally, contributes to increased levels of glucose in the 

blood. Increased hepatic glucose production in diabetes mellitus is associated with impaired 

suppression of the gluconeogenic enzyme fructose 1,6-bisphosphatase. Activation of 

gluconeogenic enzymes is due to the state of insulin deficiency, because under normal 

conditions, insulin functions as a suppressor of gluconeogenic enzymes. 

In the present study, the concentration of glycoproteins were found to be significantly 

(P


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Ethnopharmacol, 90: 39–43. 


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Assessment of Antibacterial Activity and Detection of 

Small Molecules in Different Parts of 

Andrographis paniculata 

R.Arunadevi 1 *, S. Sudhakar 1 and A.P. Lipton 2 

1 Department of Biotechnology, Manonmaniam Sundararnar University, Tirunelveli-627012, Tamil Nadu, India 

2 Marine Biotechnology Laboratory, Vizhinjam Research Centre of CMFRI, Vizhinjam-695521 Kerala, India 

Received: 15 October 2009; revised received: 20 January, 2010. 

Abstract 

Andrographis paniculata (Acanthaceae) is a medicinal plant used in India, China and other 

tropical countries for ailments such as upper respiratory tract infections, inflammations and 

diabetics. The major constituents of plants are reported to contain diterpenoids, flavonoids 

and polyphenols. The present study was to investigate the antibacterial activity of the 

different parts of the plant using different solvents by well diffusion method and also to 

screen the small molecules which are present in different parts such as: leaves, stem, 

branches, seed, root and buds both in fresh and dried form. The results suggest that the 

crude extracts of the leaves, stem and branches of Andrographis paniculata could be 

potential lead sources of broad spectrum antibiotic - resistance modifying compounds. A 

total of twenty nine small molecular compounds were screened and details presented. 

Keywords: Antibacterial activity, Andrographis paniculata, small molecules, Thin Layer 

Chromatography. 

Introduction 

Microbial infections represent the world’s leading cause of premature death and the general well 

being of humans depends on the production of new clinically useful antibiotics to curtail or 

manage the pathogens (Hugo and Russell, 2003). For over a decade, the pace of development of 

new antimicrobial agents has slowed down while the prevalence of resistance has grown at an 

astronomical rate. The rate of emergence of antibiotic resistant bacteria is not matched by the 

rate of development of new antibiotics to combat them (Prescott and Kelin, 2002). There are 

indications that some herbal materials can act as antibiotic resistant inhibitors. Combinations of 

some herbal materials and different antibiotics might affect the inhibitory effect of these 

antibiotics (Aiyegoro et al., 2009). 

Medicinal plants have a long history of use both in developing and developed countries. 

Among the few advantages of using antimicrobial compounds of medicinal plants include fewer 

side effects, better patient tolerance and relatively less expensive. All these data highlights the 

need for developing alternative new regimens. The plant chosen, Andrographis paniculata 

(Acanthaceae) is commonly known as “King of Bitters”. The plant is an annual herb. It is 

branched, erect, growing up to 1 meter in height. The leaves and the stems of the plant are used 

to extract the active phytochemicals. 

*Corresponding author; Email address: arunaanurag@gmail.com 

227

Arunadevi et al / Antibacterial Activity of Andrographis paniculata 

Earlier reports indicated that the primary medicinal component of Andrographis is 

andrographolide. It has a very bitter taste, is a colourless, crystalline in appearance, and is called 

a diterpene lactone. As per the reports the leaves contain the highest amount of andrographolide, 

the most medicinally active phytochemical in the plant, while the seeds contain the lowest. 

Andrographis paniculata demonstrated significant activity in fighting common cold, flu and 

upper respiratory infections (Coon et al., 2004). Considering these, the present study was 

initiated with the objective of screening for antibacterial properties and to detect the compounds 

recovery in different parts of the plant. 


Collection and Preparation of Plant Material 

Andrographis paniculata plants (Fig.1) were collected from Pottalpudhur village in Tirunelveli 

district (8°43’ N 77°29’ E Latitude and Longitude), India and were confirmed by local medical 

practitioners and available literature. The plant parts were thoroughly washed with water and the 

different parts like, leaf, stem, root, side branches, seed and buds were separated. 

Figure 1: Andrographis paniculata. 

Preparation of Fresh Extract 

250g of each plant part (root, stem, side branches, leaves, seed and buds) were ground by mortar 

and pestle and extracted with 250ml methanol and water respectively. The solvents were 

evaporated to dryness to obtain crude extracts. The various crude extracts were stored at 4°C 

and subjected to further analysis. 

Preparation of Dry Extract 

250g of each plant part (root, stem, side branches, leaves, seed and buds) were air dried, 

powdered and extracted with 250ml of water and methanol. The extracts were stored at 4°C 

until further use. 


228


Test Organisms 

The test organisms such as Bacillus subtilis, Salmonella typhi, Staphylococcus aureus, 

Escherichia coli, Klebsiella pneumoniae, Enterobacter faecalis and Pseudomonas aeruginosa 

were used for the bioassay. These strains were isolated from clinical samples collected from 

M/S. Vivek Scientific Laboratory Nagercoil. The organisms were characterized by biochemical 

tests. 

Evaluation of Antibacterial Activity 

Inoculum Preparation 

Overnight broth culture (in nutrient broth- HiMedia) of the test bacteria was made and the 

turbidity was compared with Mc Farland Nephalometer. 

Antibacterial Activity 

The sensitivity testing of the crude extracts of the plant was performed using agar well diffusion 

method. The bacterial isolates were first grown in nutrient broth and inoculam was prepared as 

described above. The Muller Hinton Agar medium was prepared, sterilized and the molten 

medium at 50° C was poured into sterile petridishes and the medium was allowed to solidify. 

The organisms were uniformly swabbed on the plates and wells were made in the agar medium 

using a sterile 6mm cork borer. The wells were later filled with the extract at a concentration of 

20µl. The plates were allowed to stand on for 1 hour to allow proper diffusion of the extract and 

to prevent spillage onto the surface of the agar medium and then incubated at 37° C for 24 hours 

after which they were observed for zone of inhibition. Kanamycin and ampicillin at the 

concentration of 0.1mg/ml each were used as controls. 

Screening of Small Molecules 

Preparation of Plates 

The slurry was prepared by mixing silica gel with water in the ratio 3:2 and a few drops of 

Ammonia were added into the slurry to separate the nitrate compounds in the sample. The slurry 

was coated on the glass plate at a thickness of about 0.25mm and then plates were allowed to 

dry at room temperature for 15-20 minutes. Then the plates were kept in hot air oven at 100- 

120 ο C for 1-2 hrs to remove the moisture and to activate the absorbent on the plate. The samples 

were loaded on the plate about 1.5-2 cm from the bottom, the spots were allowed to dry and 

spotting were done repeatedly to obtain a more concentrated spot. 

Chromatogram Development 

The solvent chloroform and acetone in the ratio 4:1 was used as mobile phase. The solvent was 

poured into the tank and allowed to stand for an hour to ensure that the atmosphere within the 

tank become saturated with solvent vapours. After equilibration, the plate was placed vertically 

in the tank; the solvent moves upwards due to capillary action and thus compound get separated. 

Identification of Compounds 

The chromatogram was allowed to dry and the plate was exposed to UV light source. Some of 

the compounds were fluoresced in different colours. Then the plates were exposed to iodine 

vapour. The iodine vapour reacted with the spots and formed reddish brown colour. 


Antibacterial Activity 

Fresh Extract 

The results of the agar well diffusion assay for the fresh extract of different parts of 

Andrographis paniculata are presented in Table 1. 

The fresh methanol extract of both leaf and stem exhibited the maximum inhibitory 

activity against the G +ve organisms. The organisms were resistant to the buds extract. The 

G –ve bacterial sensitivity studies revealed the following result: the leaves extract of 


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Andrographis paniculata showed maximum inhibitory activity against Salmonella typhi and 

Enterobacter faecalis which are causative organisms for typhoid (enteric fever) and other 

diseases, The stem and branch extract showed activity against Klebsiella pneumoniae. The root 

extract showed activity only to Salmonella typhi. The fresh water extracts of leaves showed 

maximum inhibitory activity against G +ve and G –ve organisms such as Klebsiella 

pneumoniae, Escherichia coli, Pseudomonas aeruginosa and lesser activity to Salmonella typhi 

and Enterobacter faecalis. The extracts of branch, stem, seed and bud extract did not have any 

bactericidal activity to the G +ve organisms. Salmonella typhi showed maximum inhibitory 

activity to leaf, stem, branch, and seed whereas Klebsiella pneumoniae showed activity only to 

the leaves extract. 

Table 1: Effect of different parts of Andrographis paniculata fresh methanol and aqueous extracts on 

Gram +ve and Gram -ve bacteria. 

Sl. 

No 

Name of 

Pathogen 

Plant parts 

Leaf Stem Branch Root Seed Buds 

M W M W M W M W M W M W 

1 Bacillus subtilis 20 18 13 NS NS NS NS NS NS NS R R 

2 

3 

4 

Staphylococcus 

aureus 

Klebsiella 

Pneumoniae 

Salmonella 

typhi 

15 12 10 NS NS NS NS NS NS 10 R NS 

10 12 12 NS 10 NS NS NS NS NS R NS 

12 10 10 10 NS 10 10 NS NS 10 R R 

5 Escherichia coli 10 12 NS NS NS 10 NS 12 NS 10 NS R 

6 

7 

Enterobacter 

faecalis 

Pseudomonas 

aeruginosa 

12 10 10 NS NS NS NS 10 NS NS NS NS 

10 12 NS NS NS NS NS 10 NS NS NS NS 

R – Resistant NS – Not Significant M - Methanol W - Water (values are mean of replicates) 

Dry Extract 

The zone of inhibition for the dry aqueous and methanol extracts on G +ve and G –ve organisms 

are given in Table 2. The leaf extract showed maximum activity to Staphylococcus aureus and 

Pseudomonas aeruginosa and moderate activity towards Bacillus subtilis, Klebsiella 

pneumoniae, Salmonella typhi, Escherichia coli and Enterobacter faecalis. All the other 

extracts showed insignificant activity towards G + ve and G negative organisms. 

While comparing the fresh water and methanol extracts, fresh methanol extract showed 

maximum inhibitory activity towards G +ve and G – ve organisms. Also while comparing to the 

different parts of fresh methanol and dry methanol extracts, leaves extracts only showed 

significant effect in dried form (Table 3). 

The water extracts from dried parts of Andrographis paniculata exhibited no positive 

result to all the organisms. This showed that the active compounds present in the plant materials 

are not soluble in water when the plant parts are dried, whereas, methanol extract of dried parts 

showed positive results. 


230


Table2: Effect of different parts of Andrographis paniculata dry methanol and aqueous extracts on 

Gram +ve and Gram -ve bacteria. 

Sl. 

No 

1 

2 

4 

5 

6 

7 

3 

Name of 

Pathogen 

Bacillus 

subtilis 

Staphylococc 

us aureus 

Klebsiella 

Pneumoniae 

Salmonella 

typhi 

Escherichia 

coli 

Enterobacter 

faecalis 

Pseudomonas 

aeruginosa 

Plant parts 

Leaf Stem Branch Root Seed Buds 

M W M W M W M W M W M W 

10 5 NS R NS R NS NS NS R R NS 

15 8 NS R NS NS NS R NS NS R R 

10 5 R R R NS NS NS NS R NS R 

10 9 NS R NS NS NS R 10 NS R R 

10 R NS R R R R NS R R R R 

10 R NS NS NS R R R NS NS NS R 

12 8 NS R R NS NS NS R R R R 

R – Resistant NS – Not Significant M - Methanol W – Water (values are mean of replicates). 

Table 3: Antibacterial effect of the leaves extract of Andrographis paniculata compared with common 

antibiotics. 

Sl No 

Name of Pathogen 

(Gram +ve and 

Gram –ve) 

Fresh Leaf 

methanol extract 

Inhibition zone (mm) 

Dry Leaf 

methanol 

extract 

Kanamycin 

Ampicilin 

1 Bacillus subtilis 15 20 18 13 

2 Staphylococcus aureus 5 13 20 18 

3 Klebsiella pneumoniae 10 15 18 15 

4 Salmonella typhi 9 12 15 13 

5 Escherichia coli 13 16 15 16 

6 Enterobacter faecalis 10 17 17 15 

7 Pseudomonas aeruginosa 8 R NS NS 

R – Resistant NS – Not Significant M - Methanol W - Water (values are mean of replicates). 

Screening of Small Molecules 

The small molecular compounds which were screened by thin layer chromatography are 

presented in Figure 2. Figure 2A showed the compounds from the fresh methanol 

extract of different parts. Figure 2B represented the compounds from the fresh water 

extract of different parts. The small molecular compounds which are screened from the 

dried different parts of Andrographis paniculata are shown in figure 2C. 


231


A B C 

Figure 2: Photographs of thin layer chromatograms showing the small molecular compounds screened 

by thin layer chromatography. A-Fresh methanol; B-fresh water; C-dry methanol. The extracts from 

different parts of the plant are loaded at: 1. Main stem, 2.Branch, 3.Root, 4.Leaves, 5.Seed, 6. Buds. a, 

b, c, d, e, f, g, h, I, j, k, l, m, n – the compounds screened . 

The fresh methanol extract of different parts of Andrographis paniculata showed eight 

compounds. The buds and root extract showed one single compound. Two same compounds 

were identified in the stem and leaves extract. The branches and the seed extract showed the 

same compound (Table 4). 

Table 4: Screening of small molecules. 

Sl. No 

Extracts 

Plant parts 

Stem Branch Root Leaves Seed Buds 

1 Fresh methanol 2 1 1 2 1 1 

2 Fresh water 1 3 1 2 - - 

3 Dry methanol 3 2 3 4 1 1 

4 Dry water - - - - - - 

With reference to the fresh water extract, the two spots which were seen in fresh 

methanol leaves extract were also seen in fresh water leaves extract. Only one spot was 

visualized in the stem extract. Three spots were identified in the extract of branches. No 

significant spots were identified in the flower and seed extract. 

In the dry methanol extract, the leaves showed four compounds. Three compounds were 

visualized in the root extract. The water extract of the dried different parts did not show 

significant antibacterial activity and also no significant spots. 

The antimicrobial effect of plant extract could be due to the presence of some of these 

phyto constituents (Ebana et al., 2005). The secondary metabolites exert antimicrobial activity 

through different mechanisms. The chloroform, methanol and aqueous extracts of Andrographis 

paniculata showed antibacterial sensitivity against Staphylococcus aureus in impetigo (Rajani 

et al., 2000). It could be inferred from the results that the extracts of Andrographis paniculata 

could be used for treating skin infections. The bacteria which are used in the study are in general 


232


considered as common pathogens, causing various infective ailments. Klebsiella pneuemonia is 

a commonest pathogen for respiratory infections, urinary tract infections, wound infections 

(Martin and Ernst, 2003). 

Thin layer chromatographic techniques were also described for the estimation of 

andrographolide in Andrographis paniculata extracts (Schneiders et al., 2003). Three main 

diterpenoid lactones identified in the Andrographis paniculata leaves were andrographolide, 

neoandrographolide and deoxyandrographolide as compared with the results of Srivasthava 

et al., (2004). 

Conclusion 

Plant extracts have great potential as antimicrobial compounds. The synergistic effect from the 

association of antibiotic with plant extracts against resistant bacteria leads to new choices for the 

treatment of infectious diseases. Our study has shown that crude extract of the leaves, stem, 

branches and root extracts of Andrographis paniculata exhibited potentials of synergy in 

combinations with some antibiotics against pathogenic bacteria often presenting problems of 

drug resistance. The results of the preliminary screening of small molecules of these extracts 

showed the presence of 29 compounds. 

Acknowledgements 

The authors are thankful to Dr. R.T. Sabapathy Mohan, Vice Chancellor, Manonmaniam 

Sundaranar University, Tirunelveli for providing necessary facilities and encouragement. 

Reference 

Aiyegoro, O. A., Afolayan, A. J. and Okoh, A. I. 2009. Invitro antibacterial activities of crude extracts of 

the leaves of Helichrysum longifolium in combination with selected antibiotics. African Journal of 

Pharmacy and Pharmacology, 3(6): 293-300. 

Coon, J.T., and Ernest, E. 2004. Andrographis paniculata in the treatment of upper respiratory tract 

infections: A systematic review of safety and efficacy. Planta. Med., 70: 293-298. 

Ebana, R.U.B., Madunagu, B.E., and Ekpe, E.D. 2005. Microbiological exploitation of cardiac glycosides 

and alkaloids from Garcinia kola,Borreli ocymoides,Kola nitida, Citrus aurantifolia. J. Appl. 

Bacterial., 71: 398-401. 

Hugo, W.B., and Russell, A.D. 2003. Pharmaceutical Microbiology; 6 th Edn. Blackwell Science 

Publishers, Oxford, United Kingdom. pp.91-129. 

Martin.K.,W., and Ernst, E. 2003. Herbal medicine for the treatment of bacterial infections-A review of 

controlled clinical traits. J. Antimicrobial chemotherapy, 51: 241-246. 

Prescott, H., and Klein, J.O. 2002. Microbiology 6th ed. Macgraw Hill Publishers, USA. pp.808-823. 

Rajani.M., Shrivastava, N., and Ravishankara, M. N. 2000. A rapid method for isolation of 

Andrographolide from Andrographis paniculata Nees (Kalmegh). Pharmaceut. Biol., 38: 204-209. 

Schneiders, T., Amyes, G. B., and Levy. 2003. Role of Acr. R and Rams in fluproguinolone resistance 

in clinical Klebsiella pneumonia isolates from Singapore. Antimicrobial agents and chemotherapy, 

47(a): 2831- 2837. 

Srivasthava, A., Misra, H., and Verma, R.K., and Gupta, M.M. 2004. Chemical finger printing of 

Andrographis paniculata using HPLC, HPTLC and densitometry. Phytochem. Anal., 15: 280-285. 


233



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Studies on Morphometrical Relationships and Growth in 

Uca annulipes Milne Edwards 

I. Jayakumari 

Department of Zoology, Sree Ayyappa College for Women, Chunkankadai-629807, Tamil Nadu, India. 

Received: 10 July, 2009; revised received: 15 January, 2010. 

Abstract 

The morphometrical relationship between total length and weight and total length and 

carapace length were investigated in detail to estimate the growth pattern in Uca 

annulipes.The‘t’ test values for length- weight in males and females were 1.62916 (P > 

0.05) and 3.80732( P < 0.05) respectively. This indicates that males exhibit isometric 

growth while the females depart significantly from the isometric growth pattern. The 

relationship between total length and carapace length was linear in both sexes with a high 

degree of correlation (males r = 0.97 and females r = 0.93) 

Key words: Length-weight, regression coefficient, isometric growth. 

Introduction 

The data on length - weight is a prime requisite for estimating growth rates, age structure and 

for assessing other aspects of fish population such as the relative well being of the population, 

estimating the standing stock biomass etc (Petrakis and Stergiou,1995).The relationship between 

length and weight differs from species to species based on their body shape and within a species 

according to the condition or robustness of the species based on the availability of food and 

environmental factors essential for growth .Length - weight studies also provides a 

mathematical relationship between the two parameters since, length is a linear measure and 

weight a measure of volume. The general belief is that the weight of an individual species vary 

with the cube of its length (Brown, 1957 and Lagler, 1968).Hence theoretically it is expressed 

by the cube law W=CL 3 where W=weight of fish, L = length of fish and C= a constant. This 

formula can be applied to fin and shell fishes which exhibit isometric growth throughout the life 

span. In nature, the body proportion continuously change with ageing, so the cube law cannot be 

applied as the value of C is not constant but subject to great variation. Therefore a modified 

equation was suggested by Le Cren (1951) where W=aL n or Log W =n Log L + Log a where W 

=weight of fish, L = length of fish and ‘a’ and ‘n’ are constants. The constants ‘a’ and ‘n’ can be 

estimated empirically from the data on length-weight. 

The present paper provides a mathematical relationship between length - weight and 

length - length parameters in Uca annulipes inhabiting the intertidal sandy shore of the coastal 

waters of Neendakara in Kerala. Such a study is useful for comparing the population of the 

species in space and time for the proper conservation and management of the species. 


341 crabs were collected from the coast of Neendakara for a period of one year of which 270 

were males and 71 were females. Morphometric measurements such as total length, carapace 

length and weight of each specimen were recorded separately for the sexes. Analysis of 

covariance was used to study whether there is any significant variation between the sexes as the 

‘b’ value may vary among the sexes (Snedecor and Cochran, 1975). 

*Corresponding author; Email address: jayakumari_i@yahoo.co.in 

235

Jayakumari / Morphometrical Relationships and Growth in Uca annulipes 

For an ideal fish which maintains constant shape ‘n’ will be equal to 3.0 (Allen, 1938). 

According to Hile, 1936 and Martin (1949) the value of ‘n’ lies between 2.5 and 4.0.So the 

regression coefficient was analysed using‘t’ test (Bailey, 1959) to find out if there is any 

departure from the isometric growth value of 3.0 proposed by Allen, 1938.The relationship 

between total length and carapace length were also determined according to the linear 

regression model. 

Results 

Size Composition and Sex Ratio 

The total length of Uca annulipes obtained for the present study ranged from 9 to 25 cm with a 

mean of 19.21 ± 2.72 and the weight varied from 0.120 to 4.220 gm with the mean value of 

1.872 ± 0.889.The minimum length observed in males was 12cm and maximum 25cm with a 

mean of 19.8 ± 2. The size range of females varied from 9cm to 24cm with a mean value of 

16.9 ± 4.The average weight of a male crab was 2.118 ± 0.802 while the female weighed 0.948 

± 0.575. 

The sex ratio of males to females was 1:0.26.The males out numbered the females 

throughout the year. 

Relationship between Total Length and Weight 

Analysis of variance for length - weight relations in the population and sexes of uca annulipes 

are present in Tables 1, 2 and 3. 

Table 1: Analysis of variance for length-weight relationship in 

male Uca annulipes. 

Anova df SS MS F value 

Regression 

1 

4.976279 

4.976279 

256.8931** 

Residual 

268 

5.191429 

0.019371 

Total 

269 

10.167708 

**Significant (P < 0.01) 

Table 2: Analysis of variance for length-weight relationship in female Uca annulipes. 

Anova df SS MS F value 

Regression 

Residual 

Total 

1 

69 

70 

5.321642 

0.431712 

5.753354 

5.321642 

0.006257 

850.551** 

* * Significant (P < 0.01) 

Table 3: Estimated values of regression (b) and correlation coefficient (r) for length-weight relationship 

in Uca annulipes 

Sex b value r value r 2 value 

Total population 3.1374 0.84909 0.7210 

Males 2.72431 0.69959 0.4894 

Females 2.65358 0.96175 0.9249 


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The relation between total length and weight in the population is expressed as Log W = 

3.1374 Log TL - 0.860 and r = 0.849 .The F value is significant at 1% level. The percentage of 

fitness to the trend line is 72.10% (Fig1). Since the value of ‘b’ was greater than 3.0 in the total 

population the length - weight relationship was calculated separately for each sex. The ‘b’ value 

varied between the sexes and is expressed as follows: 

Log weight 

3.8 

LogW= 3.1374LogTL - 0.860 

3.6 

3.4 

3.2 

3 

2.8 

2.6 

2.4 

2.2 

2 

0.9 1 1.1 1.2 1.3 1.4 1.5 

Log total length 

Figure 1: Length weight relationship in the total population of uca annulipes. 

Males: Log W = 2.7243 Log TL - 0.2413 and r = 0.6996 .The F value is significant at 1% level. 

The percentage of fit is only 48.94 % (Fig. 2). 

3.7 

3.5 

Log W = 2.7243 Log TL - 0.2413 

3.3 

Log weight 

3.1 

2.9 

2.7 

2.5 

1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 


Figure2: Length weight relationship in male Uca annulipes. 

Females: Log W =2.6536 Log TL - 0.3621 and r =0.9618 .The F value is significant at 1% level. 

The percentage of fit is 92.49 % (Fig: 3). It is closer to the trend line. 


237


3.6 

3.4 

Log W = 2.6536 LogTL - 0.3621 

Log weight 

3.2 

3 

2.8 

2.6 

2.4 

2.2 

2 

0.9 1 1.1 1.2 1.3 1.4 1.5 


Figure 3: Length weight relationship in female Uca annulipes. 

Since the value of ‘b’varied in the population and in the sexes (3.1374, 2.7243(males) and 

2.6536(females) respectively) the‘t’ test was conducted and the results are as follows: 

Population: t = 1.29579 (n = 3.1374 and S b = 0.106014) P >0.05. 

Males : t = 1.62916 (n = 2.7243, S b = 0.169973) P > 0.05. 

Females : t = 3.80732 (n =2.6536, S b = 0.909877) P < 0.05. 

Relationship between Total Length and Carapace Length 

The regression analysis for total length of the body and carapace length varied between the 

sexes. 

Males: Log CL = 1.0552 LogTL - 0.3638.The coefficient of correlation r is 0.9691 and the F 

value is significant at 1 % Level .The percentage of fitness to the trend line is 93.92 % (Fig 4). 

Log carapace length 

1.2 

1.15 

1.1 

1.05 

1 

0.95 

0.9 

0.85 

0.8 

0.75 

0.7 

Log CL=LogTL 1.0552 - 0.3638 

1 1.1 1.2 1.3 1.4 1.5 


Figure 4: Relationship between total length and carapace length in male Uca annulipes. 


238


1.2 

1.1 

LogCL = Log TL1.0343 - 0.3403 

Log carapace length 

1 

0.9 

0.8 

0.7 

0.6 

0.9 1 1.1 1.2 1.3 1.4 1.5 


Figure 5: Relationship between total length and carapace length in female Uca annulipes. 

Females: Log CL =1.0343 TL - 0.3403and the r value is 0.9289 .The F value is significant at 1% level 

.The percentage of fitness to the trend line is only 86.29% (Fig 5). 

Discussion 

Growth is a progressive increase in mass which is restricted to the short period of intermoult in 

the shelled fishes like the prawns and crabs. In Uca annulipes, it is evident from the data on 

length-weight that the body weight of both males and females increases with the total length 

exhibiting a linear relationship. 

The regression coefficient ‘b’ is an indicator in length-weight relationship to estimate 

the growth and find out if there is any deviation from isometric growth in the population and 

sexes. In the total population of Uca annulipes the ‘b’ value exhibited a slight increase of 

3.1374 against the isometric value of 3.0.The regression coefficient ‘b’ was lesser than the 

isometric growth value in both the sexes.. It was greater in males (2.7243) than in the females 

(2.6536). Similar results were observed for Scylla serrata by Lalitha, 1985 (males 2.71832 and 

females 2.6589) and Jayakumari, 2006 for Ocypode platytarsis (males 2.9365 and females 

2.6641). 

The‘t’ test values for the total population and for the male sex of Uca annulipes was 

insignificant while in the females it was significant at 5%level .This indicates that growth 

departs significantly from isometry in the females of Uca annulipes while males exhibit 

isometric growth (Jayachandran, 1984). This difference in the growth pattern of males and 

females may be due to various factors such as temperature, food, size, sex, time of year and 

stage of maturity (Hasan and Selcuk, 2003). 

The length-length relationship is also of prime importance for comparative growth 

studies. The relationship between total length and carapace length was linear in both sexes with 

a high degree of correlation (males 0.97 and females 0.93) .The regression coefficient ‘b’ was 

less than 3.0 in both the sexes (males 1.0552 and females 1.0343) indicating that carapace 

length increases proportionately with gradual increase in total length (Vijayakumar et al, 

2000).The percentage of fitness to the best fit line was higher in the males (93.92%) compared 

to the females (86.29%). 


239


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www.ejteb.org 

ISSN: 0972-9720 

Volume 6 Numbers 3 and 4 February and May 2010 

Contents 

Hypoglycaemic Activity and Modulatory Effect on Glucose Metabolism by 

Artificially Cultivated Ganoderma lucidum in Streptozotocin Induced 

Diabetic Rats 

A. Usha Raja Nanthini, M. Rajasekara Pandian and G.Kavitha 

Influence of Hormone Induced Spawning in Etroplus suratensis 

S. Albin Dhas, M. Michael Babu, T. Selvaraj, T. Citarasu, V. A. J. Huxley and 

S. Mary Josephine Punitha 

Decolorization of Textile Dye Reactive Black HFGR Using a Novel Isolate 

Paenibacillus lautus SK21 

S. Senthil Kumar, M. S. Mohamed Jaabir, A. Veeramani and R. Ravikumar 

Field Study for the Management of Rice Blast with Minimum Fungicides 

P. Krishnan 

Anamorphs of Asterinales 

V. B. Hosagoudar 

167-176 

177-183 

185-192 

193-198 

199-211 

Plant Antioxidants Mediated Protein Alterations in Clarias batrachus Linn. 

C. Suseela Bai 

213-215 

Quercitrin, a Bioflavonoid Increases Glucose Utilization and Altering 

The Carbohydrate Metobolic Enzymes in Streptozotocin-Induced Diabetic 

Rat Tissues 

Ranganathan Babujanarthanam, Purushothaman Kavitha, Sarika Sasi and 

Moses Rajasekara Pandian 

Assessment of Antibacterial Activity and Detection of 

Small Molecules in Different Parts of Andrographis paniculata 

R.Arunadevi, S. Sudhakar and A.P. Lipton 

Studies on Morphometrical Relationships and Growth in 

Uca annulipes Milne Edwards 

I. Jayakumari 

217-226 

227-233 

235-340 

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