Journal of Theoretical and Experimental Biology-Volume 6 (3 and 4.pdf
Journal of Theoretical and Experimental Biology Volume 6 (3 and 4)
Journal of Theoretical and Experimental Biology Volume 6 (3 and 4)
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<strong>Journal</strong> <strong>of</strong><br />
<strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong><br />
(An International <strong>Journal</strong> <strong>of</strong> Basic <strong>and</strong> Applied <strong>Biology</strong>)<br />
www.ejteb.org<br />
ISSN: 0972-9720<br />
Editor-in-Chief:<br />
Dr.G.KULANDAIVELU, Emeritus Pr<strong>of</strong>essor, Center for Advanced Studies in Botany, Guindy<br />
Campus, University <strong>of</strong> Madras, Chennai-600025, INDIA. (gkplant1@sify.com).<br />
Executive Editor:<br />
Dr.E.JOHN JOTHI PRAKASH, Department <strong>of</strong> Plant <strong>Biology</strong> <strong>and</strong> Plant Biotechnology,<br />
TDMNS College, T.Kallikulam-627113, INDIA. (john.jothiprakash@rediffmail.com).<br />
Editors:<br />
Dr.M.VIVEKANANDAN, Department <strong>of</strong> Biotechnology, Bharathidasan University,<br />
Tirchirappalli-620024, INDIA. (bard_vivek@yahoo.com).<br />
Dr.P.BALASUBRAMANIAN, Centre for Plant Molecular <strong>Biology</strong>, Tamil Nadu Agricultural University,<br />
Coimbatore-641003, INDIA. (balasubrap@hotmail.com).<br />
Dr.V. B. HOSAGOUDAR, Tropical Botanic Garden <strong>and</strong> Research Institute, Palode-695562,<br />
Thiruvananthapuram, Kerala, INDIA (vbhosagoudar@rediffmail.com).<br />
Dr.M.JAYAKUMAR, Department <strong>of</strong> Botany, VHNSN College,<br />
Virudunagar-626001, INDIA. (jayakuma_99@yahoo.com).<br />
Dr. JOSEPH A. J. RAJA, Department <strong>of</strong> Plant Pathology (Unit <strong>of</strong> Molecular Virology), College <strong>of</strong><br />
Agriculture <strong>and</strong> Natural Resources, National Chung Hsing University, Taichung, Taiwan (R.O.C).<br />
(jajraja@yahoo.com)<br />
Dr.C.VIJAYALAKSHMI, Department <strong>of</strong> Crop Physiology, Tamil Nadu Agricultural University,<br />
Coimbatore-641003, INDIA. (vijicv@yahoo.co.uk).<br />
Dr.A.K.M. NAZRUL ISLAM, Department <strong>of</strong> Botany, Dhaka University,<br />
BANGLADESH. (asnazrul@bangla.net).<br />
Dr. APN LIPTON, Central Marine Fisheries Research Institute,<br />
Vizhingam, Triv<strong>and</strong>rum-695521, INDIA. (liptova@yahoo.com).<br />
Dr.M.EYINI, Department <strong>of</strong> Botany, Thiyagarajar College, Madurai-625009, INDIA. (eyini@eth.net).<br />
Dr.P.K.JHA, Department <strong>of</strong> Botany, Tribuvan University, Kirtipur, Kathm<strong>and</strong>u, NEPAL.<br />
(ecos@wlink.com.np).<br />
Dr.RUP KUMAR KAR, Department <strong>of</strong> Botany, Visva-Bharati,<br />
Santiniketan-731235, INDIA. (r_kkar@rediffmail.com).<br />
Dr.A.SELVI, Division <strong>of</strong> Crop Improvement, Sugar Cane Breeding Institute,<br />
Coimbatore-641007, INDIA. (selviathiappan@yahoo.co.in).<br />
Dr.G.ANNIE JULIET, Department <strong>of</strong> Molecular Genetics <strong>and</strong> Microbiology,<br />
University <strong>of</strong> Texas at Austin, Texas 78712, USA. (ganniejuliet@yahoo.com).<br />
Dr. SANTANU RAY, Department <strong>of</strong> Zoology, Visva-Bharathi,<br />
Santiniketan-731235. INDIA. (santanu_5@yahoo.com)<br />
Dr. A. THANGA RAJ, Global engineering Systems, FZC, P6-073, SAIF Zone,<br />
P.O. Box No. 7913, Sharjah, United Arab Emirates (drthangaraj@environment.ae)<br />
Dr. JULIET VANITHARANI, Department <strong>of</strong> Animal Sciences, Sarah Tucker College,<br />
Palayamkottai-627002, INDIA. (juliet@sancharnet.in).<br />
Dr. N.GEETHA, Department <strong>of</strong> Entomology, Sugar Cane Breeding Institute,<br />
Coimbatore – 641007, INDIA. (mvsbi@yahoo.com).<br />
Dr. S.S. YADAV, Division <strong>of</strong> Genetics, Indian Agricultural Research Institute<br />
New Delhi-110012, INDIA. (shyamsinghyadav@yahoo.com).<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> is an international journal for current research in<br />
Basic <strong>and</strong> Applied <strong>Biology</strong> <strong>and</strong> is issued quarterly. It is published by Elias Academic Publishers, ELMA-ZION,<br />
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<strong>Journal</strong> <strong>of</strong><br />
<strong>Theoretical</strong><br />
<strong>and</strong><br />
<strong>Experimental</strong> <strong>Biology</strong><br />
(An International <strong>Journal</strong> <strong>of</strong> Basic <strong>and</strong> Applied <strong>Biology</strong>)<br />
ISSN: 0972-9720<br />
www.ejteb.org<br />
<strong>Volume</strong> 6 No. 3 <strong>and</strong> 4 February <strong>and</strong> May 2010<br />
G. Kul<strong>and</strong>aivelu<br />
Editor-in-Chief<br />
E. John Jothi Prakash<br />
Executive Editor<br />
Elias Academic Publishers<br />
India
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Hypoglycaemic Activity <strong>and</strong> Modulatory Effect on Glucose<br />
Metabolism by Artificially Cultivated Ganoderma lucidum in<br />
Streptozotocin Induced Diabetic Rats<br />
A. Usha Raja Nanthini 1 ٭ , M. Rajasekara P<strong>and</strong>ian 2 <strong>and</strong> G.Kavitha 3<br />
1 Department <strong>of</strong> Biotechnology, Noorul Islam College <strong>of</strong> Arts <strong>and</strong> Science, Kumaracoil- 629 180, Tamil Nadu, India.<br />
2 Department <strong>of</strong> Zoology, Arignar Anna Government Arts College, Namakkal-637001, Tamil Nadu, India.<br />
3 Vinayaka Mission’s University, Salem-637408, Tamil Nadu, India.<br />
Received: 24 November, 2009; revised received: 10 January, 2010<br />
Abstract<br />
In Chinese medicine the fruit bodies <strong>of</strong> Ganoderma lucidum (Fr.) Karst is used to treat <strong>and</strong><br />
prevent various diseases, including diabetes mellitus. Present investigation studied whether<br />
artificially cultivated Ganoderma lucidum collected from Kollimalai, India possesses<br />
modulatory effect on glucose metabolism <strong>and</strong> hypoglycaemic activity. Treatment with<br />
aqueous extract <strong>of</strong> artificially cultivated Ganoderma lucidum fruit bodies (10-30mg/kg<br />
body weight) on streptozotocin (STZ)-induced type 1 diabetic rats for 45 days reduced<br />
blood glucose <strong>and</strong> urine sugar levels <strong>and</strong> increased the insulin level in diabetic rats. A<br />
reduction in glucose-6-phosphatase, fructose-1, 6-bisphosphatase <strong>and</strong> elevation in<br />
hexokinase was observed. Glycogen content in liver <strong>and</strong> muscle was reduced <strong>and</strong> in kidney<br />
it was increased. The effect was dose-dependent <strong>and</strong> maximum effect was obtained in the<br />
dose 30mg/kg. It can be understood that aqueous extract <strong>of</strong> artificially cultivated<br />
Ganoderma lucidum exhibited a significant antihyperglycaemic activity <strong>and</strong> improved the<br />
metabolic alterations in STZ-diabetic rats. These results provide a rationale for the use <strong>of</strong><br />
artificially cultivated Ganoderma lucidum collected from India to treat diabetes mellitus.<br />
Key words: Ganoderma lucidum, streptozotocin, diabetes mellitus, hypoglycaemic<br />
activity.<br />
Introduction<br />
Mushrooms have a notable place in the folklore throughout the world <strong>and</strong> in the traditions <strong>of</strong><br />
many cultures. Ganoderma lucidum (Fr.) Karst is a rare mushroom which was considered<br />
precious during ancient times. It was once the provenance <strong>of</strong> the emperors <strong>of</strong> China, since the<br />
Ganoderma lucidum is extremely rare <strong>and</strong> difficult to find in the wild. Because the husks <strong>of</strong> the<br />
spore are very hard, the spores can’t germinate as readily as the spores <strong>of</strong> other mushrooms<br />
(George,2007).The mycelia <strong>and</strong> fruiting bodies <strong>of</strong> Ganoderma lucidum are used as Chinese<br />
traditional medicine to treat diseases such as tumours (Peng et al., 2005), hypertension,<br />
hyperglycaemia, hepatitis, chronic bronchitis, bronchial asthma (T.K, 1999; Kyo et al., 2002),<br />
Liver fibrosis (Wu et al., 2004), Lupus erythematosis, nephritis, dysmenorrhoea, anorexia,<br />
migraine, arthritis, haemorrhoids, hypercholesterolemia, constipation (Shiao et al., 1994),<br />
neurasthenia, insomnia (Lin, 2002) gastric ulcer (Kim <strong>and</strong> Kim,1999), cough (Yan et al.,1999)<br />
inflammation, cardio vascular disorders <strong>and</strong> acts as antiviral (eg., anti-HIV), antibacterial,<br />
antiparasitic, immunomodulator, kidney toxic, nerve toxic, sexual potentiator (Wasser <strong>and</strong><br />
Weis, 1999), antiaging (Gan et al., 1998), antiangiogenic, anti-metastasis <strong>and</strong> anti angiogenesis<br />
(Kimura et al.,2002; Shiao, 2003) Wound healing agent (Lia et al., 2001). So this mushroom is<br />
*Corresponding author: Email address: biotechurn@gmail.com<br />
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Usha et al / Ganoderma lucidum on Glucose Metabolism<br />
considered as a symbol <strong>of</strong> success <strong>and</strong> well being meaning “marvellous herbs” or “mushroom <strong>of</strong><br />
immortality”. Traditionally, it is taken as powder in hot water or in whisky, or by boiling the<br />
fruiting body <strong>and</strong> drinking the Ganoderma “tea” (Quimio, 1986). However there is no<br />
previously published report on the use <strong>of</strong> aqueous extract <strong>of</strong> artificially cultivated Ganoderma<br />
lucidum collected from India for the anti-diabetic properties. The present investigation was<br />
designed to evaluate the antihyperglycaemic activity <strong>and</strong> modulatory effect on glucose<br />
metabolism <strong>of</strong> aqueous extract <strong>of</strong> artificially cultivated Ganoderma lucidum collected from<br />
Kollimalai, India in streptozotocin induced diabetic rats.<br />
Materials <strong>and</strong> Methods<br />
The Mushroom, Ganoderma lucidum<br />
The identity <strong>of</strong> Ganoderma lucidum (Fr.) Karst, collected from the Kollimalai, Tamilnadu, India<br />
was confirmed using the Simon <strong>and</strong> Schuster’s Guide to Mushrooms. The mycelium observed<br />
under microscope was compared with MTCC strains. The mushroom had stipe <strong>and</strong> cap with<br />
pores beneath. They had shiny surface <strong>and</strong> the flesh was brown in colour. The cap covered with<br />
a shiny crust was circular or kidney shaped. Cap showed zones from yellow to dark red, <strong>and</strong> the<br />
margin was white or yellow. They had rust-brown colour spores.<br />
Artificial cultivation <strong>of</strong> Ganoderma lucidum <strong>and</strong> Preparation <strong>of</strong> Aqueous Extract<br />
Spawn preparation was done in Shorgum vulgare grains <strong>and</strong> wooden chips <strong>of</strong> various plants<br />
were used to cultivate G. lucidum. The wood chips were cut into pieces (1-2 cm), soaked in<br />
water trough for about 12 hours, boiled for 30 min, shade dried <strong>and</strong> used with optimum<br />
moisture. The wooden chips <strong>and</strong> spawn were filled in polythene bags as 4-5 alternate layers.<br />
The plugged polythene bags were kept in dark room at 28±2°C <strong>and</strong> 70-90% humidity. After 15-<br />
20 days the fruit bodies <strong>of</strong> G. lucidum emerged from the mouth <strong>of</strong> the polythene bag. The<br />
harvested fruit bodies <strong>of</strong> G.lucidum (250 gm) was made into small pieces, shade dried,<br />
powdered <strong>and</strong> homogenized in a wareing blender with 500 ml <strong>of</strong> distilled water. The extraction<br />
was carried out with constant stirring overnight. The homogenate was then centrifuged at 2000<br />
rpm for 10 min at 0-4°C. The supernatant was concentrated <strong>and</strong> used for the treatment <strong>of</strong> STZ<br />
induced diabetic rats.<br />
Test Animals<br />
Wistar albino rats (200-250gm) <strong>of</strong> either sex were used. These animals were housed in an airconditioned<br />
animal room at 23±2°C with 12 h light/dark photoperiod <strong>and</strong> maintained with free<br />
access to water <strong>and</strong> ad libitum feeding. All animal experiments were in accordance with the<br />
guidelines <strong>of</strong> the National Institute <strong>of</strong> Health Guide (1985).<br />
Chemicals<br />
Streptozotocin was procured from Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India. All<br />
other chemicals used were <strong>of</strong> analytical grade.<br />
Induction <strong>of</strong> Diabetes<br />
Diabetes was induced in overnight fasted adult Wistar strain albino male rats weighing 200–250<br />
g by a single intraperitoneal injection <strong>of</strong> 55 mg/kg Streptozotocin. Streptozotocin (55 mg/kg)<br />
was dissolved in 0.1 M citrate buffer (pH 4.5). Hyperglycaemia was confirmed by the elevated<br />
glucose levels (Above 250 mg/dl) in plasma, determined at 72 h after injection. Those animals<br />
with hyperglycaemia were used in the experiment.<br />
After successful induction <strong>of</strong> experimental diabetes, the rats were r<strong>and</strong>omly divided into<br />
six groups each comprising a minimum <strong>of</strong> six rats. These were: Group 1, Normal control with<br />
healthy rats without diabetes; Group 2, Normal rats administered with G. lucidum aqueous<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
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Usha et al / Ganoderma lucidum on Glucose Metabolism<br />
extract (30 mg/kg/b.wt.) in aqueous solution orally for 45 days; Group 3, Diabetic control (STZ<br />
induced); Group 4, Diabetic rats administered with G. lucidum aqueous extract (10 mg/kg/b.wt.)<br />
in aqueous solution orally for 45 days; Group 5, Diabetic rats administered with G.lucidum<br />
aqueous extract (20 mg/kg/b.wt. ) in aqueous solution orally for 45 days ; Group 6, Diabetic rats<br />
administered with G. lucidum aqueous extract (30 mg/kg/b.wt. ) in aqueous solution orally for<br />
45 days. At the end <strong>of</strong> the experimental period, rats were fasted overnight, anaesthetized <strong>and</strong><br />
sacrificed by cervical decapitation. The blood was collected with or without EDTA<br />
(ethylenediaminetetraacetic acid) for plasma or serum separation, respectively.<br />
Biochemical Analysis<br />
The level <strong>of</strong> blood glucose was estimated following glucose oxidase method (Triender, 1969).<br />
Insulin was estimated using Boerhringer Mannheim kit. Liver, kidney <strong>and</strong> skeletal muscles were<br />
immediately dissected, washed in ice-cold saline to remove the blood <strong>and</strong> homogenised in 0.1<br />
M Tris–HCl buffer, pH 7.4. The supernatant was used for enzyme activity assays. Hexokinase,<br />
glucose-6-phosphatase <strong>and</strong> fructose-1, 6-bisphosphatase were assayed by the method <strong>of</strong><br />
Br<strong>and</strong>strup et al., (1957), Baginsky et al., (1974) <strong>and</strong> Gancedo <strong>and</strong> Gancedo (1971)<br />
respectively. Glycogen was assayed by the method <strong>of</strong> Ong <strong>and</strong> Khoo (2000).<br />
Statistical Analysis<br />
All the grouped data were statistically evaluated with SPSS/ 10.0 s<strong>of</strong>tware. Hypothesis testing<br />
methods included one way analysis <strong>of</strong> variance (ANOVA) followed by least significant<br />
difference (LSD) test; p value <strong>of</strong> less than 0.05 were considered to indicate statistical<br />
significance. All the results were expressed as the mean ± S.D. for six animals in each group.<br />
Results<br />
Effect <strong>of</strong> Daily Administration <strong>of</strong> Ganoderma Lucidum Aqueous Extract on Blood Glucose,<br />
Insulin <strong>and</strong> Urine Sugar in Normal <strong>and</strong> Diabetic Rats<br />
Table 1 shows blood glucose, plasma insulin <strong>and</strong> urine sugar levels <strong>of</strong> control <strong>and</strong> experimental<br />
group <strong>of</strong> rats. The blood glucose level in the diabetic control rats was significantly (p < 0.05)<br />
increased. Inversely, the insulin level was decreased significantly (p < 0.05). In diabetic rats,<br />
elevated urine sugar level was observed. Treatment <strong>of</strong> diabetic rats with aqueous extracts <strong>of</strong><br />
G. lucidum elicited significant decrease in blood glucose <strong>and</strong> urine sugar levels <strong>and</strong> increase in<br />
the insulin level in dose dependent manner, when compared with diabetic control rats. No<br />
change in blood glucose, insulin <strong>and</strong> urine sugar level was observed in rats grown under control<br />
conditions.<br />
Effect <strong>of</strong> Daily Administration <strong>of</strong> Ganoderma Lucidum Aqueous Extract on<br />
Gluconeogenic Enzymes (Hexokinase, Glucose-6-Phosphate Dehydrogenase <strong>and</strong><br />
Fructose-1,6-Bisphosphatase) in Normal <strong>and</strong> Diabetic Rats<br />
Table 2 summarizes the level <strong>of</strong> glucose-6- phosphatase in the liver <strong>and</strong> muscle tissues <strong>of</strong><br />
control <strong>and</strong> experimental groups <strong>of</strong> rats. A significant (p < 0.05) increase in glucose-6-<br />
phosphatase level was observed in STZ-diabetic rats <strong>and</strong> it was normalized after treatment with<br />
G. lucidum aqueous extract. Table 3 depicts the level <strong>of</strong> the enzyme fructose-1,<br />
6-bisphosphatase level in liver, kidney <strong>and</strong> muscle <strong>of</strong> control <strong>and</strong> experimental rats. The level <strong>of</strong><br />
fructose-1,6-bisphosphatase was significantly (p
Usha et al / Ganoderma lucidum on Glucose Metabolism<br />
the activities <strong>of</strong> the enzyme. In all the three enzymes the maximum modulatory effect was<br />
observed in 30mg/kg extract. No significant change in the levels <strong>of</strong> Glucose-6-phosphatase,<br />
Fructose-1,6-bisphosphatase <strong>and</strong> Hexokinase was observed in normal control rats administered<br />
with G. lucidum aqueous extract.<br />
Table 1: Effect <strong>of</strong> Ganoderma lucidum aqueous extract on blood glucose, insulin <strong>and</strong> urine sugar in<br />
control <strong>and</strong> diabetic rats<br />
Groups<br />
Blood glucose<br />
(mg/dL)<br />
Insulin<br />
(μU/ml)<br />
Urine sugar<br />
Normal 82.44 ± 2.68 b 15.52 ± 1.51 a Nil<br />
Normal + given extract 74.39 ± 4.17 a 15.83 ± 1.47 a Nil<br />
STZ-control 289.28 ± 3.18 f 6.38 ± 0.89 b ++++<br />
STZ-induced+extract(10mg/kg) 194.63 ± 3.74 e 7.81 ± 0.77 b ++<br />
STZ-induced+extract(20mg/kg) 164.87 ± 2.68 d 10.21 ± 1.10 c +<br />
STZ-induced+extract 30mg/kg) 107.23 ± 7.23 c 12.34 ± 1.21 d Nil<br />
Values are mean ± SD for 6 rats in each group<br />
Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT)<br />
Table 2: Effect <strong>of</strong> Ganoderma lucidum aqueous extract on glucose 6-phosphatase in normal <strong>and</strong> STZinduced<br />
experimental rats.<br />
Groups<br />
Liver<br />
Values are mean ± SD for 6 rats in each group<br />
Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT)<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
Muscle<br />
(mmol <strong>of</strong> glucose phosphorylated/h/mg protein)<br />
Normal 20.00 ± 1.52 a 17.30 ± 1.30 a<br />
Normal + given extract 19.50 ± 1.20 a 17.00 ± 1.22 a<br />
STZ-control 39.51 ± 2.51 b 35.01 ± 2.65 b<br />
STZ-induced + extract (10mg/kg) 37.21 ± 2.39 c 32.41 ± 1.96 c<br />
STZ-induced + extract (20mg/kg) 33.32 ± 3.31 c 28.23 ± 1.82 c<br />
STZ-induced + extract (30mg/kg) 27.30 ± 2.08 d 22.10 ± 1.90 d<br />
Effect <strong>of</strong> Daily Administration <strong>of</strong> Ganoderma Lucidum Aqueous Extract on Tissue<br />
Glycogen Content in Normal <strong>and</strong> Diabetic Rats<br />
Table 5 showed the changes in the level <strong>of</strong> glycogen in the liver, kidney <strong>and</strong> muscle <strong>of</strong> control<br />
<strong>and</strong> experimental rats. The level <strong>of</strong> glycogen was significantly (p < 0.05) decreased in the liver<br />
<strong>and</strong> muscle tissues <strong>of</strong> diabetic rats when compared with normal control rats, whereas kidney<br />
tissue had elevated glycogen level. Oral administration <strong>of</strong> G. lucidum aqueous extract to<br />
diabetic rats significantly (p < 0.05) increased the liver <strong>and</strong> muscle glycogen content <strong>and</strong><br />
decreased the kidney glycogen content. The effect was the maximum in 30mg/kg extract. The<br />
administration <strong>of</strong> G. lucidum aqueous extract to normal rats resulted in no significant changes in<br />
the level <strong>of</strong> tissue glycogen.<br />
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Table 3: Effect <strong>of</strong> Ganoderma lucidum aqueous extract on fructose 1,6-bisphosphatase in normal <strong>and</strong><br />
STZ-induced experimental rats.<br />
Groups<br />
Liver Kidney Muscle<br />
(μmole <strong>of</strong> Pi liberated/min/mg protein)<br />
Normal 11.52 ± 0.60 a 16.62 ± 1.10 a 2.90 ± 0.12 a<br />
Normal + given extract 11.06 ± 0.51 a 16.55 ± 1.02 a 2.86 ± 0.10 a<br />
STZ-control 23.50 ± 1.20 b 29.04 ± 1.66 b 5.50 ± 0.43 b<br />
STZ-induced+extract(10mg/kg) 22.10 ± 1.09 b 27.56± 1.39 c 5.01± 0.51 c<br />
STZ-induced+extract(20mg/kg) 19.11 ± 1.51 c 24.44 ± 1.91 c 4.21 ± 0.42 c<br />
STZ-induced+extract(30mg/kg) 16.04 ± 0.80 d 21.80 ± 1.33 c 3.80 ± 0.30 c<br />
Values are mean ± SD for 6 rats in each group<br />
Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT).<br />
Table 4: Effect <strong>of</strong> Ganoderma lucidum aqueous extract on hexokinase in normal <strong>and</strong> STZ-induced<br />
experimental rats.<br />
Groups<br />
Liver Kidney Muscle<br />
(mmol <strong>of</strong> glucose phosphorylated/h/mg protein)<br />
Normal 98.02 ± 4.46 a 79.11 ± 6.02 a 114.02 ± 8.68 a<br />
Normal + given extract 98.62 ± 4.51 a 80.01 ± 4.06 a 109.00 ± 6.02 a<br />
STZ-control 50.76 ± 4.40 b 58.46 ± 4.42 b 80.28 ± 6.09 b<br />
STZ-induced + extract(10mg/kg) 60.13 ± 4.39 c 64.19± 4.39 c 86.89 ± 6.56 c<br />
STZ-induced + extract (20mg/kg) 75.56 ± 5.31 c 67.56 ± 5.91 c 91.23 ± 7.42 c<br />
STZ-induced + extract (30mg/kg) 90.02 ± 6.85 d 71.01 ± 4.65 d 102.00 ± 7.70 d<br />
Values are mean ± SD for 6 rats in each group<br />
Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT).<br />
Table 5: Effect <strong>of</strong> Ganoderma lucidum aqueous extract on glycogen content in normal <strong>and</strong> STZinduced<br />
experimental rats.<br />
Groups<br />
Liver Kidney Muscle<br />
(mg/g tissue)<br />
Normal 2.88 ± 0.26 a 1.62 ± 0.12 a 2.32 ± 0.18 a<br />
Normal + given extract 2.92 ± 0.20 a 1.60 ± 0.16 a 2.20 ± 0.20 a<br />
STZ-control 1.76 ± 0.10 b 2.84 ± 0.21 b 1.64 ± 0.13 b<br />
STZ-induced + extract (10mg/kg) 1.92 ± 0.11 c 2.59± 0.18 c 1.77 ± 0.16 c<br />
STZ-induced + extract (20mg/kg) 2.15 ± 0.21 d 2.26 ± 0.19 d 1.89 ± 0.12 cd<br />
STZ-induced + extract (30mg/kg) 2.32 ± 0.20 d 1.91 ± 0.11 a 2.00 ± 0.10 d<br />
Values are mean ± SD for 6 rats in each group<br />
Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT).<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
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Discussion<br />
Diabetic rats induced by STZ shows an increased sensitivity to oxygen free radicals <strong>and</strong><br />
hydrogen peroxide, the breakdown products <strong>of</strong> liver, which impose oxidative stress in diabetes<br />
<strong>and</strong> would damage inner endothelial tissue; this would eventually be directly responsible for<br />
high blood glucose (Reddi <strong>and</strong> Bollineni, 2001). The present investigation showed that<br />
treatment with Ganoderma lucidum aqueous extract reduces the blood sugar level <strong>and</strong> it may be<br />
due to stimulating effect on insulin release from regenerated beta cells <strong>of</strong> the panaceas or may<br />
be due to increased cellularity <strong>of</strong> the islet tissues <strong>and</strong> regeneration <strong>of</strong> the beta cells. The aqueous<br />
extract might be exerting its hypoglycaemic effect by an extra-pancreatic action (Dabis et al.,<br />
1984), e.g. possibly by stimulating glucose utilization in peripheral tissues (Naik et al., 1991;<br />
Obatomi et al., 1994). Also, it could be the result <strong>of</strong> an increase in glycolytic (Steiner <strong>and</strong><br />
Williams, 1959) <strong>and</strong> / or glycogenic enzymes activity in peripheral tissues (Naik et al., 1991). It<br />
might be also possible that the aqueous extract may decrease the secretion <strong>of</strong> the counter<br />
regulatory hormones (glucagons, corisols <strong>and</strong> growth hormones) (Roman-Ramos et al., 1995).<br />
The STZ-induced diabetic control rats showed decreased level <strong>of</strong> insulin in the plasma<br />
than the normal control rats. The treatment with G. lucidum aqueous extract had increased the<br />
insulin level to near normal level. The elevation <strong>of</strong> plasma insulin in the G. lucidum treated STZ<br />
diabetic rats could be due to the insulinotropic substances present in the extract, which induce<br />
the intact functional β-cells <strong>of</strong> the langerhans islet to produce insulin (Jeong-Sook, 2006).<br />
Insulin deficiency is clearly associated with change in hepatic metabolism (Consoli et al.,<br />
1989).<br />
Insulin decreases gluconeogenesis by decreasing the activities <strong>of</strong> key enzymes such as<br />
glucose-6-phosphatase, fructose 1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase <strong>and</strong><br />
pyruvate carboxykinase (Murray et al., 2000). The liver <strong>and</strong> skeletal muscle is the major organ<br />
for glucose disposal. Glucose-6-phosphatase, a key enzyme in the homeostatic regulation <strong>of</strong><br />
blood glucose concentration, is expressed mainly in the liver <strong>and</strong> kidney <strong>and</strong> is critical in<br />
providing glucose to other organs during diabetes, prolonged fasting or starvation (Bouché et<br />
al., 2004). It catalyzes the dephosphorylation <strong>of</strong> glucose-6-phosphate to free glucose as the<br />
terminal step in gluconeogenesis <strong>and</strong> glycogenolysis. This reaction occurs in the lumen <strong>of</strong> the<br />
endoplasmic reticulum <strong>and</strong> the enzyme complex is composed <strong>of</strong> glucose-6- phosphate<br />
transporter that transports glucose-6-phosphate from the cytoplasm into the lumen <strong>of</strong> the<br />
endoplasmic reticulum <strong>and</strong> a glucose-6-phosphatase catalytic subunit that hydrolyzes the<br />
glucose-6-phosphate to glucose <strong>and</strong> phosphate (Chou et al., 2002). Glucose is transported out <strong>of</strong><br />
the liver to increase blood glucose concentration. STZ increases the expression <strong>of</strong> glucose-6-<br />
phosphate (Massillon et al., 1996; Liu et al., 1994). In contrast, insulin inhibits the hepatic<br />
glucose production by suppressing glucose-6-phosphate activity (Chen et al., 2000;<br />
Wiernsperger <strong>and</strong> Bailey, 1999). Our results demonstrated that hepatic <strong>and</strong> muscle glucose-6-<br />
phosphatase activity in diabetic rats was significantly higher than that <strong>of</strong> normal rats <strong>and</strong> the<br />
oral feeding <strong>of</strong> G. lucidum aqueous extract markedly lowered its activity. The reduction in<br />
enzyme activity can lead to a decrease in gluconeogenesis <strong>and</strong> blood glucose concentration.<br />
Fructose-1, 6-bisphosphatase is a highly regulated, rate-limiting enzyme that catalyzes<br />
the dephosphorylation <strong>of</strong> fructose-1, 6- bisphosphate to fructose-6-phosphate, the second to last<br />
step in the gluconeogenic pathway (Pilkis <strong>and</strong> Claus, 1991). Under normal conditions, insulin<br />
functions as a suppressor <strong>of</strong> gluconeogenic enzymes (Baquer et al., 1998). This results in a<br />
decrease in the glycolytic flux. An increase in the activity <strong>of</strong> fructose-1,6-bisphosphatase has<br />
been suggested as a possible mechanism for the production <strong>of</strong> increased endogenous glucose<br />
after it was shown that diabetics have an increase in gluconeogenesis (Nurjhan et al., 1992). The<br />
increased activity <strong>of</strong> fructose-1, 6-bisphosphatase has been observed in animal models <strong>of</strong><br />
diabetes, insulin resistance <strong>and</strong> obesity <strong>and</strong> suggests a principal role for fructose-1,6-<br />
bisphosphatase in the flux <strong>of</strong> gluconeogenesis <strong>and</strong> endogenous glucose production<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
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(Andrikopoulos et al., 1993). The increased activities <strong>of</strong> these gluconeogenic enzymes in<br />
diabetic rats were decreased to near-normal levels after the administration <strong>of</strong> Ganoderma<br />
lucidum aqueous extract. The possible mechanism by which Ganoderma lucidum aqueous<br />
extract bring about the normalization <strong>of</strong> enzyme activity may be by potentiation <strong>of</strong> insulin<br />
release from β-cells <strong>of</strong> the islets <strong>of</strong> Langerhan’s which might enhance glucose utilization.<br />
Hexokinase (HK) is an isoenzyme that catalyzes phosphorylation <strong>of</strong> glucose to glucose-<br />
6-phosphate, thus playing a crucial function in tissue intermediary metabolism. Hexokinase is<br />
an insulin-dependent <strong>and</strong> insulin-sensitive enzyme <strong>and</strong> is almost completely inhibited or<br />
inactivated in diabetic tissues in the absence <strong>of</strong> insulin (Gupta et al., 1997). There are four<br />
is<strong>of</strong>orms <strong>of</strong> mammalian hexokinases involved in the oxidation <strong>of</strong> glucose (Wilson, 1995).<br />
Hexokinases I–III have a high affinity for glucose <strong>and</strong> are feedback-inhibited by physiologic<br />
concentrations <strong>of</strong> glucose-6-phosphate. Whereas, glucokinase (HK-IV or GK), the major<br />
glucose- phosphorylating enzyme, has a lower affinity for glucose <strong>and</strong> its abundance is<br />
regulated transcriptionally by insulin <strong>and</strong> glucagon <strong>and</strong> post-translationally by the GK<br />
regulatory protein (GKRP) (Collier <strong>and</strong> Scott, 2004). Among four is<strong>of</strong>orms <strong>of</strong> hexokinases, HK-<br />
I <strong>and</strong> GK are expressed in the liver. Reports on animal models <strong>and</strong> isolated hepatocytes<br />
established that hexokinase exerts a strong impact on glucose utilization <strong>and</strong> glycogen synthesis<br />
in liver (Postic et al., 2001) <strong>and</strong> muscle (Murray et al., 2000) <strong>and</strong> their levels are very low in<br />
both human <strong>and</strong> rodent diabetes; insulin administration rapidly reinstates hexokinase activity to<br />
the hepatocytes (Ferre et al., 1996). Because <strong>of</strong> these observations, restoration <strong>of</strong> hepatic<br />
hexokinase activity provides a possible therapeutic strategy for diabetes treatment. The<br />
markedly decreased level <strong>of</strong> insulin in the streptozotocin-induced diabetic animals ultimately<br />
leads to the impairment in the activity <strong>of</strong> hexokinase, since insulin deficiency is a hall mark <strong>of</strong><br />
diabetes (Postic et al., 2001). However, the modest increase in the activity <strong>of</strong> hexokinase as<br />
observed in the diabetic animals administered with Ganoderma lucidum aqueous extract<br />
protects the hepatic <strong>and</strong> extrahepatic tissues against streptozotocin-induced diabetes by<br />
stimulating insulin from the remnant β -cells, since streptozotocin selectively destroys<br />
pancreatic β -cells. This study also demonstrated that a modest augmentation <strong>of</strong> hexokinase<br />
activity in the liver, kidney <strong>and</strong> muscle enhances glucose metabolism <strong>and</strong> promotes overall<br />
glucose homeostasis similar to the studies <strong>of</strong> Palsamy <strong>and</strong> Subramanian (2009).<br />
Glycogen, a branched polymer <strong>of</strong> glucose residues synthesized by the enzyme glycogen<br />
synthase, is the primary intracellular storable form <strong>of</strong> glucose <strong>and</strong> its quantity in various tissues<br />
is a direct manifestation <strong>of</strong> insulin activity as insulin supports intracellular glycogen deposition<br />
by stimulating glycogen synthase <strong>and</strong> inhibiting glycogen phosphorylase (Pederson et al.,<br />
2005). The activity <strong>of</strong> glycogen synthase is regulated by decreased cellular glycogen content,<br />
hormone signaling, subcellular localization, targeting <strong>of</strong> phosphatase <strong>and</strong> allosteric activation by<br />
glucose-6-phosphate (Parker et al., 2004). Glycogen phosphorylase, a rate-limiting enzyme <strong>of</strong><br />
glycogenolysis, cleaves β (1→4) linkages to remove glucose molecules from the glycogen. This<br />
enzyme exists as a dimer with each subunit linked to the essential c<strong>of</strong>actor pyridoxal phosphate,<br />
which donates the phosphate as an electron donor for release <strong>of</strong> glucose-1-phosphate<br />
(Greenberg et al., 2006). Its activity is regulated by phosphorylation <strong>and</strong> by allosteric binding <strong>of</strong><br />
AMP, ATP, glucose-6-phosphate <strong>and</strong> glucose (Bollen, 1998). Since streptozotocin causes<br />
selective destruction <strong>of</strong> pancreatic β -cells resulting in apparent decline in insulin levels, it is<br />
responsible for the decreased glycogen levels in major storage tissues such as liver <strong>and</strong> skeletal<br />
muscle as they depend on insulin for entry <strong>of</strong> glucose (Whitton <strong>and</strong> Hems, 1975; Golden et al.,<br />
1979; Bishop, 1970). During diabetic conditions, the glycogen levels, glycogen synthase<br />
activity <strong>and</strong> responsiveness to insulin signalling are diminished <strong>and</strong> glycogen phosphorylase<br />
activity is significantly increased (Parker et al., 2004). Glycogen levels in various tissues<br />
especially skeletal muscle are direct reflection <strong>of</strong> insulin activity. Insulin promotes intracellular<br />
glycogen deposition by stimulating glycogen synthase <strong>and</strong> inhibiting glycogen phosphorylase.<br />
Since STZ selectively damages β-cells <strong>of</strong> islets <strong>of</strong> Langerhans resulting in marked decrease in<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 167-176, 2010<br />
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insulin levels, it is rational that glycogen levels in tissues (skeletal muscle <strong>and</strong> liver) decrease as<br />
they depend on insulin influx <strong>of</strong> glucose (Whitton <strong>and</strong> Hems, 1975). Moreover, this alteration in<br />
muscle <strong>and</strong> hepatic glycogen was normalized by insulin treatment (Weber et al., 1966). The<br />
entry <strong>of</strong> renal glucose is not dependent on action <strong>of</strong> insulin <strong>and</strong>, therefore, in the event <strong>of</strong><br />
hyperglycemia there is an increase in the entry <strong>of</strong> glucose (Belfiore et al., 1986). This has been<br />
postulated to cause increased intra-renal glycogen deposition, which leads to glycosylation <strong>of</strong><br />
basement membrane collagen in the kidney (Anderson <strong>and</strong> Stowring, 1973). From the present<br />
study it is clear that STZ induced diabetic rats had increased glycogen level in kidney <strong>and</strong><br />
decreased level in Liver <strong>and</strong> skeletal muscle. Treatment with G. lucidum aqueous extract had<br />
reversed it by decreasing the renal glycogen <strong>and</strong> increasing the hepatic <strong>and</strong> skeletal muscle<br />
glycogen. It may be assumed by the above explained reasons.<br />
In conclusion results <strong>of</strong> the present investigation indicate that the aqueous extract <strong>of</strong><br />
artificially cultivated G. lucidum collected from Kollimalai, India has antidiabetic effect,<br />
possibly due to insulin like effect <strong>of</strong> G. lucidum aqueous extract on peripheral tissues. The<br />
present study draws out a sequential metabolic correlation between increased glycolysis <strong>and</strong><br />
decreased gluconeogenesis <strong>and</strong> normal glycemia stimulated by Ganoderma lucidum aqueous<br />
extract which may have the biochemical mechanism through which glucose homeostasis is<br />
regulated.<br />
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Wu, Y.W., Chen, K.D., <strong>and</strong> Lin, W.C. 2004. Effect <strong>of</strong> Ganoderma tsugae on chronically carbon<br />
tetrachloride-intoxicated rats. Am. J. Chinese Med., 32: 841-50.<br />
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© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Influence <strong>of</strong> Hormone Induced Spawning in<br />
Etroplus suratensis<br />
S. Albin Dhas 1 *, M. Michael Babu 1 , T. Selvaraj 1 , T. Citarasu 1 , V. A. J. Huxley 2 <strong>and</strong><br />
S. Mary Josephine Punitha 1<br />
1 Centre for Marine Science <strong>and</strong> Technology, Manonmaniam Sundaranar University,<br />
Rajakkamangalam - 629 502, Tamil Nadu, India.<br />
2 Department <strong>of</strong> Zoology, Thiru Vi Ka Government Arts College, Thiruvarur- 610003, Tamil Nadu, India.<br />
Received: 27 February, 2010; revised recieved: 29 May, 2010.<br />
Abstract<br />
A study was carried out to underst<strong>and</strong> the influence <strong>of</strong> synthetic hormones such as ovaprim,<br />
HCG+LHRH on induced spawning in Etroplus suratensis grown in aquarium tanks <strong>of</strong> 5 ton<br />
capacity. Biochemical parameters such as triglyceride, total protein <strong>and</strong> cholesterol level in<br />
the blood, liver <strong>and</strong> gonads were estimated in hormone treated ones <strong>and</strong> it was compared<br />
with the control. The length <strong>and</strong> width <strong>of</strong> the egg development stages such as oocyte, previtellogenic<br />
<strong>and</strong> matured eggs were also analyzed in different hormone treated fishes <strong>and</strong><br />
were compared with the control. The percentage <strong>of</strong> eggs in the ovary <strong>of</strong> control <strong>and</strong><br />
hormone treated fishes were also compared. In all these parameters, the combined hormone<br />
HCG+LHRH administered experimental fishes showed the highest increased level. It was<br />
suggested that administration <strong>of</strong> the synthetic hormone HCG+LHRH induced spawning in<br />
E. suratensis.<br />
Key words: Hormones, Etroplus suratensis, ovaprim, HCG+LHRH, spawning<br />
Introduction<br />
The fish Etroplus suratensis belonging to the family chichilidae is commonly found in the<br />
estuaries <strong>and</strong> inl<strong>and</strong> waters <strong>of</strong> India <strong>and</strong> Sri Lanka (Talwar <strong>and</strong> Jingran, 1992; Rao, 1995;<br />
Blaber, 1997). It grows in brackish as well as fresh waters <strong>and</strong> has been observed to breed in<br />
these habitats (Rishi <strong>and</strong> Singh, 1982). It involves in commercial fisheries (Gopakumar, 1997),<br />
yet this fish is preferred as the c<strong>and</strong>idate species for aquarium. This fish is dioecious <strong>and</strong> breeds<br />
freely both in fresh <strong>and</strong> brackish waters (Pethiyagoda, 1991; Arkipehuk, 1999). Among the fish<br />
species, it has low fecundity rate with about 500 eggs laid in single spawning (Jayaprakas et al.,<br />
1990). The eggs are attached to submerged logs, rocks or sometimes roots <strong>of</strong> aquatic weeds.<br />
These guardian parents take care <strong>of</strong> the eggs until hatching <strong>and</strong> within four days, the eggs will<br />
hatch. The fry shoal around their parents during the first week <strong>of</strong> growth in natural conditions.<br />
Although all the fish species are spawned in the natural environment, only a limited<br />
species are successfully spawned through induced breeding in laboratory conditions. The<br />
success <strong>of</strong> induced spawning depends upon several factors, which were not clearly understood<br />
in most <strong>of</strong> the fishes (Stuart et al., 1988). During the past three to four decades, induced<br />
breeding technique has been attempted in many <strong>of</strong> the fresh water <strong>and</strong> marine fishes. For this<br />
technique, many <strong>of</strong> the alternative hormones such as human chronic Gonadotropin (HCG)<br />
(Adebayo., 2004); Inyang <strong>and</strong> Hettiarachchi, 1994), luteinizing hormone – releasing hormonoeo<br />
(De Leeuw et al., 1985; Fermin, 1992) <strong>and</strong> ovaprim (Alok et al., 1993; Haniffa et al., 1996)<br />
were used. Treatments using the above hormones are effective in many <strong>of</strong> the fish species. But<br />
*Corresponding author; Email address: salbindhas@yahoo.com<br />
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Albin Dhas et al / Hormone Induced Spawning in Etroplus suratensis<br />
so far, there were only limited attempts in induced breeding <strong>of</strong> E. suratensis. Only very few<br />
works were carried out in the direction <strong>of</strong> larval propagation in E. suratensis (Eschmeyer,<br />
1990). Few works were focused on the induced breeding by applying hormones since the<br />
attempts were not encouraging (Karnfield, 1984). The present study is an attempt to induce<br />
breeding in E. suratensis using synthetic hormones such as HGG <strong>and</strong> LHRH <strong>and</strong> to document<br />
the earlier larval stages <strong>of</strong> the fish.<br />
Materials <strong>and</strong> Methods<br />
Brood Stock Management<br />
E. suratensis brooders with the size <strong>of</strong> 15-20 cm length <strong>and</strong> weight group <strong>of</strong> approximately<br />
110 ± 10 g were collected from the backyard estuarine waters at Centre for Marine Science <strong>and</strong><br />
Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District,<br />
India. The collected fishes were stocked in aquarium tanks (5 T capacities) for 7 days to ensure<br />
the disease free status <strong>of</strong> the experimental fish. The healthy fishes were transferred to the<br />
circular brood stock tanks (1 T capacity). The water quality parameters such as temperature,<br />
salinity <strong>and</strong> oxygen level were maintained at 27-30ºC, 5 pit <strong>and</strong> 5 mg/l, respectively. A 100%<br />
water exchange was made daily. During this period, the fishes were fed with lap-lap at the rate<br />
<strong>of</strong> 3% <strong>of</strong> fish body weight daily. After 5 days, the gonad maturity <strong>of</strong> fish was determined in both<br />
male <strong>and</strong> female fishes by performing catheter biopsy in the gonads through the genital opening.<br />
The collected biopsy samples were observed for the gonad maturity with the parameters <strong>of</strong> eggs<br />
diameter <strong>and</strong> stage <strong>of</strong> the eggs in female, as well as sperm motility in the male.<br />
Administration <strong>of</strong> Synthetic Hormones<br />
There were three groups with five female replicates in each group that were stocked in<br />
individual spawning tanks (1.5 ton capacity) for hormone administration. In the first group,<br />
ovaprim (Syndel Co., Canada) was administered with the optimum concentration <strong>of</strong> 1 ml/kg<br />
fish. The second group was administered with HCG (Pr<strong>of</strong>ess <strong>and</strong> SIGMA, USA) 1000 U/kg fish<br />
with the combination <strong>of</strong> LHRH (SIGMA, USA) 60 µg/kg fish (Mai, 1998). The third group was<br />
treated as control which received sterile saline injection (0.81% NaCl).<br />
After 48 h <strong>of</strong> hormonal administration the blood samples from the three groups <strong>of</strong><br />
experimental fish were collected using sterile syringe. Thereafter, the fishes were sacrificed- the<br />
liver <strong>and</strong> gonad samples were dissected carefully from each group <strong>and</strong> individually stored at –<br />
20ºC until further use. The biochemical parameters such as triglyceride <strong>and</strong> cholesterol were<br />
estimated in all the blood, liver <strong>and</strong> gonad samples using ELISA–micro plate method (Palacious<br />
et al., 1998). Total protein content in the same samples were estimated by the method described<br />
by Bradford (1976).<br />
To characteristic the stages <strong>of</strong> maturity in females, oocyte count as well as gonad<br />
somatic index (GSI) were recorded in all the groups. Three stages <strong>of</strong> oocytes were found in the<br />
gonad (Previtellogenic, vitellogenic <strong>and</strong> matured egg) <strong>and</strong> the number <strong>of</strong> oocytes belong to each<br />
stage was counted for 100 mg <strong>of</strong> gonad sample in each group <strong>of</strong> fishes. The size <strong>of</strong> the egg<br />
(length <strong>and</strong> width) was also determined using Ocular Micrometry method.<br />
Statistical Analysis<br />
The data obtained in the present study were subjected for statistical 2-way ANOVA <strong>and</strong><br />
regression analysis followed by Zar (1974).<br />
Results<br />
Biochemical indices like triglyceride, total protein <strong>and</strong> cholesterol were estimated in the blood,<br />
liver <strong>and</strong> gonad samples <strong>of</strong> E. suratensis brooders treated with synthetic hormones like ovaprim<br />
<strong>and</strong> HCG+LHRH. The level <strong>of</strong> triglyceride was more in all the tested samples (63.23 µg/ml in<br />
blood, 487.23 µg/g in liver <strong>and</strong> 51.2 µg/g in gonad) <strong>of</strong> fish administered with HCG+LHRH. But<br />
theseparameters exhibited lower values in fishes administered with ovaprim (30 to 41.0 mg/g)<br />
<strong>and</strong> control (22.0 to 435 µg/g) groups (Table 1).<br />
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Table 1: Biochemical parameters <strong>of</strong> different tissues in E. suratensis in both experimental <strong>and</strong> control<br />
groups.<br />
Parameters Treatments Blood (mg/ml) Liver (mg/g) Gonad (mg/g)<br />
Control 1.31 ± 0.05 7.02 ± 0.06 2.0 ± 0.08<br />
Total Proteins Ovaprim 1.14 ± 0.04 8.98 ± 0.28 3.75 ± 0.05<br />
HCG+LHRH 1.66 ± 0.11 9.12 ± 0.19 4.02 ± 0.09<br />
Control 50.0 ± 4.0 435.0 ± 5.0 22.0 ± 1.0<br />
Triglycerides Ovaprim 37.0 ± 3.0 410.0 ± 8.0 30.0 ± 2.0<br />
HCG+LHRH 63.23 ± 2.0 487.23 ±2.7 51.20 ± 1.50<br />
Control 12.2 ± 1.3 61.21± 3.0 3.0 ± 0.20<br />
Cholesterol Ovaprim 9.0 ± 1.0 144.0 ± 4.0 1.50 ± 0.05<br />
HCG+LHRH 14.4 ± 1.5 212.20 ±7.0 5.0 ± 0.50<br />
Table 2: Length <strong>and</strong> width <strong>of</strong> E. suratensis eggs.<br />
Egg Stage Length (µm) Width (µm)<br />
Oocyte 186.62 ± 12.25 176.62 ± 14.20<br />
Pre-vitrllogenic egg 599.85 ± 11.21 333.25 ± 25.33<br />
Mature eggs 1932.85 ± 82.81 1039.72 ± 92.22<br />
Table 3: Percentage <strong>of</strong> eggs in 100 mg <strong>of</strong> ovary.<br />
Stage <strong>of</strong> egg<br />
Control Egg %<br />
(in 100 mg<br />
ovary)<br />
Hormone treated<br />
Egg % (in 100 mg<br />
ovary)<br />
Oocyte 64.45 + 6.5 19.24 + 1.8<br />
Pre-vitellogenic<br />
egg<br />
21.96 + 2.3 38.96 + 3.7<br />
Vitellogenic egg<br />
(matured egg)<br />
13.58 + 1.2 41.78 + 4.3<br />
Like the triglyceride, a similar trend <strong>of</strong> result was observed in total protein level <strong>of</strong><br />
blood (1.66 mg/ml), liver (9.12 mg/g) <strong>and</strong> gonad (4.02 mg/g) samples <strong>of</strong> E. suratensis<br />
administered with HCG+LHRH than the ovaprim (1.14 mg/g) as well as control (1.31 mg/ml to<br />
7.02 mg/g) treatment (P
Albin Dhas et al / Hormone Induced Spawning in Etroplus suratensis<br />
Length-Width Relationship <strong>of</strong> E. suratensis Oocytes<br />
200<br />
195<br />
190<br />
y = 1.199x - 42.521<br />
R 2 = 0.9548<br />
185<br />
Width (μm)<br />
180<br />
175<br />
170<br />
165<br />
160<br />
170 175 180 185 190 195 200<br />
Length (μm)<br />
Figure 1a: Length-width relationship <strong>of</strong> E. suratensis pre-vitellogenic egg.<br />
Length-Width Relationship <strong>of</strong> E.suratensis Pre-vitrllogenic egg<br />
370<br />
360<br />
350<br />
Width (μm)<br />
340<br />
330<br />
y = 1.9578x - 845.04<br />
R 2 = 0.6779<br />
320<br />
310<br />
300<br />
585.00 590.00 595.00 600.00 605.00 610.00 615.00<br />
Length (μm)<br />
Figure 1b: Length-width relationship <strong>of</strong> E. suratensis oocytes.<br />
Percentage Distribution <strong>of</strong> Different Egg Stages<br />
From matured gravid females <strong>of</strong> E. suratensis, based on the maturity, three different stages were<br />
identified (Figure 1a, b <strong>and</strong> c). They are oocytes which are spherical in shape with the length<br />
<strong>and</strong> width <strong>of</strong> 186.62 µm <strong>and</strong> 176.62 µm respectively. The second stage was pre-vitellogenic<br />
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oocytes, which had the vitellogenic package <strong>of</strong> transparent yellow spheres which were 599.85<br />
µm long <strong>and</strong> 333.25 µm wide. Likewise, the third stage was vitellogenic (matured) eggs,<br />
prominent shape with 1932.85 µm length <strong>and</strong> 1039.72 µm wide (Table 2). Freshly ovulated<br />
mature eggs were slightly spherical in shape, visible to the naked eye <strong>and</strong> strong yellow in<br />
colour <strong>and</strong> opaque. The oocyte was obviously enhanced by hormone treatment as indicated by<br />
the increase <strong>of</strong> oocyte diameter. The percentage <strong>of</strong> matured egg was maximum (47.03%) in E.<br />
suratensis that received HCG+LHRH hormone. At the same time, the control group <strong>of</strong> fishes<br />
had 13.58% matured eggs, followed by fishes administered with ovaprim hormone, 41.78%.<br />
Vitellogenic eggs (38.97%) <strong>and</strong> previtellogenic eggs (64.45%) were more in ovaprim<br />
administered fish <strong>and</strong> control fish, respectively (Table 2, Fig 1a, b, c).<br />
The data recorded for percentage <strong>of</strong> eggs in 100 mg ovary showed that the hormonetreated<br />
experimental fishes showed the highest percentage (41.78%), followed by previtellogenic<br />
(38.96%) <strong>and</strong> the oocyte (19.24%) (Table 3).<br />
Length-Width relationship <strong>of</strong> E.suratensis mature eggs<br />
360<br />
350<br />
340<br />
y = 0.2567x - 163.48<br />
R 2 = 0.6823<br />
Width (μm)<br />
330<br />
320<br />
310<br />
300<br />
1840 1860 1880 1900 1920 1940 1960 1980 2000 2020 2040<br />
Length (μm)<br />
Figure 1c: Length-width relationship <strong>of</strong> E. suratensis mature eggs.<br />
Discussion<br />
For the assessment <strong>of</strong> internal milieu <strong>of</strong> fishes during reproduction several biochemical indices<br />
should be clearly resolved (Svoboda et al., 2001). The present study also had a part to analyze<br />
the possible biochemical variables such as triglycerides, total protein <strong>and</strong> cholesterol from<br />
blood, liver <strong>and</strong> ovary tissues, while administration with commercial synthetic hormones was<br />
carried out. Significant differences were observed between the control l<strong>and</strong> hormone<br />
administered spawners. Results from the examination <strong>of</strong> triglyceride in the spawners tissues<br />
(liver <strong>and</strong> ovary) <strong>and</strong> blood indicate the significant positive regulation <strong>and</strong> high titer value in<br />
HCG+ LHRH treated group. The group administered with ovaprim failed to reserve the<br />
significant quality <strong>of</strong> triglycerides in blood <strong>and</strong> tissue, which is clearly reflected in the number<br />
<strong>of</strong> matured eggs in the ovary. Similar trend was observed in the blood plasma <strong>of</strong> fish tench<br />
(Tinca tinca L.) during pre <strong>and</strong> post-spawning period under the condition <strong>of</strong> hormonally induced<br />
artificial reproduction (Svoboda, 2001). Possible role <strong>of</strong> triglyceride in fish reproduction is to<br />
serve as the higher energy source as well as the precursor for yolk protein synthesis (Luskova,<br />
1997; Kovaqcheva <strong>and</strong> Tchekov, 1993).<br />
Moreover, cholesterol which is the precursor in the synthesis <strong>of</strong> steroid hormones<br />
involved in fish maturation was significantly regulated in the hormone treated animals than the<br />
control groups. The cholesterol that is incorporated in the membranes <strong>and</strong> the endogenous<br />
structures <strong>of</strong> the egg <strong>and</strong> its concentration in blood plasma <strong>of</strong> females were found to increase<br />
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during the administration <strong>of</strong> hormones (Diwan <strong>and</strong> Krishnan (1986). Diwan <strong>and</strong> Krishnan<br />
(1986) observed a fluctuation <strong>of</strong> serum cholesterol in male <strong>and</strong> females <strong>of</strong> E. suratensis as<br />
related to maturity. In the present study, cholesterol concentration in liver tissues <strong>of</strong> females<br />
that are found to be lowest in the control females was reflected in the egg maturity.<br />
The total protein level in the tissue samples from the hormone injected groups exhibited<br />
significant higher values than the control groups in the present study. This result is in<br />
accordance with the data reported for some other fish species like trout – Salmo trutta<br />
(Mulcahy, 1971); carp-Cyprinus carpio (Svobodova <strong>and</strong> Parova, 1977); trout – Salmo<br />
gairdeneri (Hille, 1982); rainbow trout (Jirasek et al., 1993) <strong>and</strong> common carp (Rehulka, 1996).<br />
Total protein level in the blood determines the health status <strong>and</strong> reproductive ability in cichilids.<br />
In the present study also, higher levels <strong>of</strong> total protein were registered in HCG+LHRH group as<br />
it caused improved health status or reproductive ability <strong>of</strong> the fish E. suratensis.<br />
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Maccullochella peeli (Mitchell) (Percichthyidae) Aquaculture Issue, 4: 371-389.<br />
Svoboda, M., Kouril, J., Hamackova, J., Kalab, P., Savina, L., Svobodova, Z., <strong>and</strong> Vykusova, B. 2001.<br />
Biochemical pr<strong>of</strong>ile <strong>of</strong> blood plasma <strong>of</strong> tench (Tinca tinca L.) during pre- <strong>and</strong> prost spawning period.<br />
Acta Vet Brno., 70: 259-268.<br />
Svobodova, Z., Parova, J., 1977. The use <strong>of</strong> somephysiological parameters <strong>of</strong> fish for the evaluation <strong>of</strong><br />
feeding tests. Bulletin VURH Vodnany, 13: 12.<br />
Talwar, P. K. <strong>and</strong> Jhingran, A. G. 1992. Inl<strong>and</strong> fishes <strong>of</strong> India <strong>and</strong> adjacent countries. <strong>Volume</strong> 2.<br />
Balkema, A. A., Rotterdam.<br />
Zar, J. H. 1974. Biostatistical Analysis. Englewood Cliffs, New Jersey: Prentice-Hall. pp. 620.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3): 187-183, 2010<br />
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© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Decolorization <strong>of</strong> Textile Dye Reactive Black HFGR Using a<br />
Novel Isolate Paenibacillus lautus SK21<br />
S. Senthil Kumar 1* , M. S. Mohamed Jaabir 1 , A. Veeramani 2 <strong>and</strong> R. Ravikumar 3<br />
1 Department <strong>of</strong> Biotechnology, Jamal Mohamed College (Autonomous),<br />
Tiruchirappalli-620 020, Tamil Nadu, India.<br />
2 Department <strong>of</strong> Botany, Aringar Anna Govt. Arts College, Namakkal-637 002, Tamil Nadu, India.<br />
3 Research Department <strong>of</strong> Botany, Jamal Mohamed College (Autonomous),<br />
Tiruchirappalli-620 020, Tamil Nadu, India.<br />
Received: 27 February, 2010; revised recieved: 6 June, 2010.<br />
Abstract<br />
Paenibacillus lautus strain SK21 was isolated from the textile effluent polluted soil in<br />
Tirupur, Tamil Nadu <strong>and</strong> identified based on Biochemical <strong>and</strong> 16S RNA Sequence. The<br />
present study was carried out in an attempt to decolorize a commonly used yet tougher dye<br />
to decolorize, Reactive Black HFGR. The decolorization percentage was calculated from<br />
UV-Vis spectrophotometric analysis. Dye decolorization was probably due to the<br />
biotransformation <strong>and</strong> depended upon the biomass. Replacement <strong>of</strong> nutrient broth with<br />
minimal media did not show any decolorization property. Decolorization was optimized<br />
<strong>and</strong> found to be up to 95% at 7pH, 40°C under static <strong>and</strong> non-aerated condition.<br />
Key words: Reactive dyes, Azo dyes, Bacterial isolates, dye decolorization,<br />
Paenibacillus lautus<br />
Introduction<br />
Synthetic dyes find use in a wide range <strong>of</strong> industries such as textile dyeing, paper printing,<br />
cosmetics <strong>and</strong> pharmaceuticals (Erdal <strong>and</strong> Taskin, 2010). Approximately 10,000 different dyes<br />
<strong>and</strong> pigments are used in industries <strong>and</strong> over 7 × 10 5 tons <strong>of</strong> these dyes are annualy produced<br />
world-wide. Due to inefficiencies <strong>of</strong> the industrial dyeing process, 10 - 15% <strong>of</strong> the dyes are lost<br />
in the effluents <strong>of</strong> textile units, rendering them highly coloured. Among the various classes <strong>of</strong><br />
dyes, reactive dyes are more difficult to remove. They contain chromophoric groups such as<br />
azo, anthraquinone, triarylmethane, etc. <strong>and</strong> reactive groups e.g. vinylsulphone, chlorotriazine,<br />
trichloropyrimidine etc. that form covalent bonds with the fiber. Azo reactive dyes are the<br />
largest class <strong>of</strong> water soluble synthetic dyes with the greatest variety <strong>of</strong> colors <strong>and</strong> structure <strong>and</strong><br />
are generally resistant to biodegradation processes (Lin <strong>and</strong> Peng, 1994; Sanghi et al., 2006;<br />
Daneshvar et al., 2007).<br />
Water pollution control is at present one <strong>of</strong> the major areas <strong>of</strong> scientific activity. While<br />
colored organic compounds generally impart only a minor fraction <strong>of</strong> the organic load to<br />
wastewater, their colour renders them aesthetically unacceptable. Colour is one <strong>of</strong> the most<br />
obvious indicators <strong>of</strong> water pollution <strong>and</strong> discharge <strong>of</strong> highly coloured synthetic dye effluents<br />
can be damaging to the receiving water bodies (Nigam et. al., 1966). Two percent <strong>of</strong> dyes that<br />
are produced are discharged directly in aqueous effluent <strong>and</strong> 10% are subsequently lost during<br />
*Corresponding author; Email address: envsenthil@gmail.com<br />
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the textile colouration process (Pearce et al., 2003). Some <strong>of</strong> the azo dyes, xanthene dyes <strong>and</strong><br />
anthroquinone dyes are known to be very toxic <strong>and</strong> mutagenic to living organisms.<br />
Microbial decolorization has been proposed as a less expensive <strong>and</strong> less<br />
environmentally intrusive alternative. In the present study, we focused our attention on the<br />
isolation <strong>of</strong> dye decolorizing microorganisms from contaminated soil <strong>of</strong> an industrial estate <strong>and</strong><br />
analyzed the ability <strong>of</strong> these isolates to degrade Reactive Black HFGR.<br />
Materials <strong>and</strong> Methods<br />
Chemicals<br />
All chemicals <strong>and</strong> reagents used in this investigation were <strong>of</strong> Analytical grade. The common<br />
name <strong>of</strong> the dye (Reactive Black HFGR) has been used for convenience <strong>and</strong> was procured from<br />
A.K. Chemi Dyes Enterprises, Mumbai, India. The stock solutions <strong>of</strong> the dyes were filter<br />
sterilized <strong>and</strong> added to the growth medium in the concentration <strong>of</strong> 100ppm (mg/litre).<br />
Spectrum Study <strong>of</strong> the Dye<br />
The dye procured from the industry was initially studied for absorption maxima in a Double<br />
Beam UV-Vis Spectrophotometer from 350nm to 800nm (Schimadzu, UV-Vis 1800).<br />
Isolation <strong>of</strong> Bacterial Cultures<br />
Soil sample taken from the dumping grounds <strong>of</strong> the sludge was used for the isolation <strong>of</strong> dye –<br />
decolorizing microorganisms owing to long – term usage <strong>of</strong> the location for over 5 decades<br />
since the establishment. Bacteria from the soil sample were isolated by pour plate method <strong>and</strong><br />
serial dilution technique using nutrient agar medium. All the plates were incubated at 37°C for<br />
24 hours.<br />
Study <strong>of</strong> Decolorization Activity<br />
All decolorization experiments were performed in triplicates. A loopful <strong>of</strong> each isolated<br />
bacterial culture was inoculated into a separate 250 ml Erlenmeyer flask containing the Reactive<br />
Black HFGR (100 mgl -1 ) in Nutrient broth <strong>and</strong> incubated for 24 h at 37°C for initial screening<br />
<strong>of</strong> the isolates for the ability to decolorize the dye. Aliquots <strong>of</strong> the culture (3 ml) was withdrawn<br />
at different time intervals, centrifuged at 5000rpm for 15 min to separate the bacterial cell mass.<br />
Decolorization was determined by measuring the absorbance <strong>of</strong> the supernatant at 520 nm (λ<br />
max) <strong>and</strong> percentage <strong>of</strong> decolorization was calculated (Saratale et al., 2006) as follows:<br />
(%) Decolorization = (Initial absorbance – Observed absorbance) / Initial absorbance X 100<br />
A loopful <strong>of</strong> culture was inoculated in 250 ml Erlenmeyer flask containing 100 ml<br />
nutrient broth. Separate study was carried out for different temperatures (20, 30, 40 <strong>and</strong> 50) <strong>and</strong><br />
pH (3-10). Decolorization was also studied under shaking (150 rpm/min) <strong>and</strong> static (nonaerated)<br />
conditions. Decolorization was also studied in minimal media (mgl -1 ): Glucose 1800;<br />
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 <strong>of</strong> the source <strong>of</strong><br />
nitrogen in the medium for decolorization <strong>of</strong> the dye was studied using Yeast extract <strong>and</strong><br />
Peptone.<br />
Identification <strong>of</strong> the Isolate SK20 by 16SrRNA Gene Amplification <strong>and</strong> Sequencing<br />
DNA was extracted from pure culture <strong>of</strong> the isolate SK20 that showed prospective application.<br />
A partial DNA sequence for 16SrRNA gene was amplified by using ATG GAT CCG GGG<br />
GTT TGA TCC TGG CTC AGG(forward primer) <strong>and</strong> TAT CTG CAG TGG TGT GAC GGG<br />
GGG TGG (reverse primer) (Jing et al., 2004). Amplifications performed in 50µl reactions<br />
mixtures containing the template DNA, 40ng, 0.2µM, for each <strong>of</strong> the primers, dNTPs 200µM,<br />
Taq DNA polymerase 2.5U <strong>and</strong> 10X buffers 5µl. The mixture was subjected to the following<br />
amplification conditions; 2min at 94°C for 1min, <strong>and</strong> ended by a final extension step at 72°C for<br />
7min. The PCR products mixture was purified <strong>and</strong> sequenced at Chromous Biotech, Bangalore,<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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India. The identity <strong>of</strong> the bacterium determined by sequencing method was verified <strong>and</strong><br />
confirmed through biochemical tests.<br />
Results <strong>and</strong> Discussion<br />
From the isolated soil sample, twenty four bacterial isolates were obtained <strong>and</strong> named as SK 1<br />
to SK24 based on the Author’s name. The absorption maxima for Black HFGR were found to<br />
be at 600 nm (Table 1). Decolorization occurred only when nutrient broth was available in the<br />
medium. This was confirmed when the minimal media was replaced by the Nutrient Broth.<br />
Among the 24 isolates, SK03, SK20 <strong>and</strong> SK 21 demonstrated decolorization beyond 50% while<br />
the other isolates did not show any decolorization activity beyond 23.25% (Table 2).<br />
Table 1: Absorption Maxima (λ max) <strong>of</strong> Reactive Black HFGR dye.<br />
Dye<br />
λ max (nm)<br />
Reactive Blue 604<br />
Table 2: Showing % <strong>of</strong> decolorization <strong>of</strong> Reactive Black HFGR dye by 24 bacterial isolates.<br />
S. No Isolate Black HFGR<br />
1 SK1 4.65 %<br />
2 SK2 10.46 %<br />
3 SK3 53.48 %<br />
4 SK4 6.97 %<br />
5 SK5 4.65 %<br />
6 SK6 6.97 %<br />
7 SK7 10.46 %<br />
8 SK8 6.97 %<br />
9 SK9 23.25 %<br />
10 SK10 6.97 %<br />
11 SK11 9.30 %<br />
12 SK12 930 %<br />
13 SK13 3.48 %<br />
14 SK14 15.11 %<br />
15 SK15 4.65 %<br />
16 SK16 0.00 %<br />
17 SK17 0.00 %<br />
18 SK18 0.00 %<br />
19 SK19 0.00 %<br />
20 SK20 54.65 %<br />
21 SK21 51.16 %<br />
22 SK22 0.00 %<br />
23 SK23 1.16 %<br />
24 SK24 0.00 %<br />
The optimum temperature for the selective SK03, SK20 <strong>and</strong> SK21 demonstrated better<br />
decolorization at a temperature <strong>of</strong> 40°C but the magnitude <strong>of</strong> decolorization differed with<br />
57.8%, 63% <strong>and</strong> 97.9% respectively (Figure 1). The optimum pH among the three isolates<br />
SK03, SK20 <strong>and</strong> SK21 were 6, 7 <strong>and</strong> 8 respectively. However, highest decolorization was<br />
obtained in SK21 (Figure 2). For all the three isolates, static condition demonstrated the highest<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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percentage <strong>of</strong> decolorization <strong>and</strong> SK21 was found to be the best isolate for decolorizing reactive<br />
black HFGR upto 95.5% (Figure 3). When choice <strong>of</strong> nitrogen source was tested, yeast extract<br />
was suitable for SK03 <strong>and</strong> SK20 showing 45.8% <strong>and</strong> 50.8% <strong>of</strong> decolorization respectively.<br />
Peptone was seen to be suitable for SK21 producing 25% <strong>of</strong> decolorization.<br />
Figure 1: Effect <strong>of</strong> Temperature on the Decolorization <strong>of</strong> reactive black HFGR by SK21.<br />
Figure 2: Effect <strong>of</strong> different pH on the Decolorization <strong>of</strong> reactive black HFGR by SK21.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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Figure 3: Effect <strong>of</strong> shaking <strong>and</strong> static (non-aerated) condition on the Decolorization <strong>of</strong> reactive black<br />
HFGR by SK21.<br />
UV-Vis Spectral Analysis <strong>of</strong> Biodecolorization<br />
The UV-Vis spectra <strong>of</strong> the media containing the dye before decolorization showed a maximum<br />
absorption at 600 nm (0 hour). In the final stage after decolorization, the absorption maxima<br />
totally disappeared from 600 nm (12 hours). Disappearance <strong>of</strong> the peak from 600 nm is a clear<br />
evidence <strong>of</strong> molecular rearrangements in the dye structure <strong>and</strong> degradation there<strong>of</strong> (Figure 4).<br />
As reported by Asad et al. (2007) decolorization <strong>of</strong> dyes by bacteria is due to adsorption<br />
by microbial cell as a surface phenomenon or to biodegradation. In case <strong>of</strong> adsorption, the<br />
UV-Vis absorption peak would tend to decrease approximately in proportion to each other,<br />
whereas, in biodegradation either the major visible light absorbance peak disappears completely<br />
or a new peak appears. The observation <strong>of</strong> Paenibacillus lautus strain SK21cells mass retained<br />
their natural colour after decolorization <strong>of</strong> reactive black HFGR. Based on this, it is confirmed<br />
that the reactive black HFGR has undergone biotransformation <strong>and</strong> not due to simple adsorption<br />
over the surface.<br />
Physiological differences among the bacterial isolates may account for differences in<br />
the decolorization abilities (Reddy, 1995; Asgher et al., 2006). The complex enzymatic system<br />
responsible for the dye degradation <strong>and</strong> pattern <strong>of</strong> its expression may also vary among the<br />
isolates (Nagai et al., 2002; Boer et al., 2004); however, the relative rates <strong>of</strong> decolorization for<br />
the reactive blue dye cannot be easily explained. Degradation <strong>of</strong> dye involves aromatic ring<br />
cleavage which is dependent on the identity <strong>of</strong> the ring substituents with the presence <strong>of</strong><br />
phenolic, amino, acetamido, 2-methoxy phenol or other easily biodegradable functional groups<br />
resulting in a greater extent <strong>of</strong> degradation (Spadaro et al., 1992; Mazmanci <strong>and</strong> Unyayar,<br />
2005). In the present study, reactive black HFGR was found to be decolorized to different<br />
extents by the individual bacterial isolates. Different isolates have degraded the dye to different<br />
levels following a different pattern during the incubation period as is commonly observed in<br />
studies elsewhere (Knapp et al., 1997; Toh et al., 2003). However, overall complexity <strong>of</strong><br />
structure alone is not an indicator <strong>of</strong> the difficulty <strong>of</strong> decolorization <strong>of</strong> a particular dye (Maas<br />
<strong>and</strong> Choudary 2005).<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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Table 3: Biochemical pr<strong>of</strong>ile <strong>of</strong> Paenibacillus genus.<br />
S.No. Biochemical Test Result<br />
1. Glycerol +<br />
2. Esculin +<br />
3. Maltose +<br />
4. D-Arabinose +<br />
5. L-Arabinose +<br />
6. Ribose +<br />
7. D-Xylose +<br />
8. a-Methylxyloside +<br />
9. Rhamnose -<br />
10. Dulcitol -<br />
11. Inositol -<br />
12. Sorbitol -<br />
13. 1-Methyl-Dmannoside -<br />
14. Arbutin +<br />
15. Salicin +<br />
16. Lactose +<br />
17. Starch +<br />
18. Glycogen +<br />
19. D-Tagatose -<br />
20. D-Arabitol -<br />
21. 5-Ketogluconate -<br />
22. 2-Ketogluconate -<br />
23. Gentiobiose +<br />
24. Raffinose +<br />
25. Trehalose +<br />
26. Sucrose +<br />
27. Melibiose +<br />
28. Cellobiose +<br />
29. Amygdalin +<br />
30. N-Acetylglucosamine +<br />
31. Mannose +<br />
32. Glucose +<br />
33. Galactose +<br />
34. Adonitol +<br />
35. D-Fucose +<br />
36. Glycerol +<br />
37. Esculin +<br />
38. Maltose -<br />
39. D-Arabinose -<br />
40. L-Arabinose +<br />
+ Positive: - Negative<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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Senthil Kumar et al / Decolorization <strong>of</strong> Textile Dye Reactive Black HFGR<br />
Figure 4: UV-Vis spectra <strong>of</strong> Reactive Black HFGR biodegradation at 0 hr (a) <strong>and</strong> 12 hrs (b).<br />
Nucleotide Sequence Accession Number<br />
The bacterial isolate SK21 was observed to be gram-positive, rod-shaped bacteria <strong>and</strong> identified<br />
to belong to Paenibacillus genus based on the biochemical tests (Albert <strong>and</strong> Anciet, 1999)<br />
(Table 3). This was verified <strong>and</strong> confirmed with the sequence analysis <strong>of</strong> the amplified 16S<br />
ribosomal DNA <strong>and</strong> therefore the isolate SK21 was determined (using the BLAST tool on<br />
http://www.ncbi.nlm.hih.gov) to be Paenibacillus lautus SK21. The sequence data has been<br />
deposited in the GenBank nucleotide sequence databases under accession number FJ974057.<br />
Reactive black HFGR was completely biotransformed by the novel Paenibacillus lautus<br />
SK21 bacterial strain isolated from textile industry waste l<strong>and</strong>. UV-Vis spectroscopic studies has<br />
revealed the molecular rearrangement <strong>of</strong> the dye <strong>and</strong> therefore confirmed to have undergone<br />
biodecolorization <strong>and</strong> biodegradation. Our study indicates the use <strong>of</strong> our novel isolate for<br />
environmentally safe disposal <strong>of</strong> the textile dyes as compared to the existing physico chemical<br />
methods.<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 185-192, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 193-198, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Field Study for the Management <strong>of</strong> Rice Blast with<br />
Minimum Fungicides<br />
P. Krishnan*<br />
Post Graduate <strong>and</strong> Research Department <strong>of</strong> Botany, The Madura college, Madurai- 625 011, Tamil Nadu, India<br />
Received: 17 January, 2010; revised received: 30 March, 2010.<br />
Abstract<br />
Rice blast caused by Pyricularia oryzae Cav. strikes whenever <strong>and</strong> wherever the climatic<br />
factors are favourable. It attacks rice plants at various stages <strong>of</strong> growth in rice fields <strong>and</strong><br />
causes great production loss. Fungicide application is still the preferred control measure<br />
against blast, but to minimize the fungicide use different treatment practices were<br />
investigated to control the leaf <strong>and</strong> neck blast effectively under field conditions. Among the<br />
various treatments given, the seed treatment with Pyroquilon (4 g ai/kg <strong>of</strong> seed) with<br />
additional foliar sprays (1%) gave maximum leaf blast protection (77%). Foliar spray <strong>of</strong> 1%<br />
Pyroquilon, one at the time <strong>of</strong> panicle emergence <strong>and</strong> another at 15 days after the formation<br />
<strong>of</strong> panicle, gave maximum neck blast protection (>90%) over the control.<br />
Keywords: Rice blast, Pyricularia oryzae , pyroquilon, foliar spray, seed treatment.<br />
Introduction<br />
The blast disease <strong>of</strong> rice (Oryza sativa L.) caused by Pyricularia oryzae Cav. is considered the<br />
most important disease <strong>of</strong> rice. The disease outbreaks whenever <strong>and</strong> wherever the climatic<br />
factors are favourable. The blast fungus attacks rice plants at various stages <strong>of</strong> growth in rice<br />
fields<br />
<strong>and</strong> produces lesions on leaves, nodes, panicles <strong>and</strong> grains (Suzuki, 1975). The<br />
aggregated annual toll from even light infections may cause greater production losses (Bhatt,<br />
1988). Till date, the primary control measures largely followed in the rice fields are cultural<br />
practices, fungicides application, biological control <strong>and</strong> raising the resistant cultivars (Ou, 1980;<br />
Muralidharan et al., 2004). It is obvious that fungicide is still the preferred control measure<br />
against blast (Teng, 1994).<br />
Nagarajan (1988) has reported that the blast disease can be effectively controlled by the<br />
application <strong>of</strong> Bavistin, Topsin, Edifenphos, Fongorine <strong>and</strong> Tricyclazole as foliar spray <strong>and</strong> seed<br />
treatment. Seed treatment with the fungicide Pyroquilon at the concentration <strong>of</strong> 4 g ai/kg <strong>of</strong> seed<br />
<strong>and</strong> foliar spray with 1% solution gave maximum protection against this disease (Narasimhan et<br />
al., 1991; Nagarajan, 1988; Surin et al., 1988; Sharma <strong>and</strong> Sood, 1990; Bhatt <strong>and</strong> Singh, 1990).<br />
Drawbacks to the use <strong>of</strong> chemicals to control blast include increased cost <strong>of</strong> rice<br />
production due to the spraying <strong>of</strong> fungicides frequently throughout the growing season, addition<br />
<strong>of</strong> pollutants to the environment by recurrent application <strong>of</strong> agrochemicals, <strong>and</strong> development <strong>of</strong><br />
resistance by the pathogen to the fungicides. To counter these problems, an attempt is made to<br />
investigate the possible methods to minimize the fungicide application for managing the blast<br />
disease effectively.<br />
Materials <strong>and</strong> Methods<br />
Present investigation was carried out in Kulamangalam, situated 09 o 59.3 N 078 o 07.1 E <strong>and</strong><br />
494 ft. above the sea level, 20 km away from Madurai city, TamilNadu. The experiment was<br />
conducted in the field trial plots during second crop seasons <strong>of</strong> 2006-2007 <strong>and</strong> 2007-2008 using<br />
the rice cv. IR 50. The optimum concentration, for seed treatment (4g ai / Kg seed) <strong>and</strong> for<br />
*Corresponding author; Email address:funkittu@rediffmail.com<br />
193
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foliar spray (1% aqueous solution), <strong>of</strong> the fungicide Pyroquilon was alone used in this study<br />
with the rice cv. IR 50.<br />
Time <strong>of</strong> Fungicide Application to Control Leaf Blast<br />
To find out the appropriate time <strong>of</strong> fungicide application to achieve maximum control <strong>of</strong> leaf<br />
blast, Pyroquilon treatments were given as follows:<br />
T 0 = Control (crops without fungicide treatment),<br />
T 1 = Five foliar sprays on the seedlings with >0.20 disease proportion at 7 day<br />
intervals <strong>and</strong> the spray initiated at 27 days after sowing (DAS),<br />
T 2 = Four foliar sprays on the seedlings with the proportion <strong>of</strong> disease incidence<br />
ranging between 0.10 - 0.20 at 10 day intervals <strong>and</strong> the spray initiated at<br />
20 DAS,<br />
T 3 = Seed treatment,<br />
T 4 = Seed treatment with one foliar spray at 35 DAS <strong>and</strong><br />
T 5 = Seed treatment with two foliar sprays at 35 <strong>and</strong> 50 DAS .<br />
The IR 50 seeds pretreated with fungicides as well as untreated seeds were sown<br />
directly in the field plot (1 m 2 ) prepared in a r<strong>and</strong>omized complete block design with triplicates<br />
<strong>and</strong> exposed for natural blast infection.<br />
Time <strong>of</strong> Fungicide Application to Neck Bast Control<br />
Paddy crops (IR 50) were raised on the microplots (1 m 2 ) prepared in a r<strong>and</strong>omized complete<br />
block design with triplicates; however, to procure the crops <strong>of</strong> panicle initiation stage the<br />
planting was done 50 days before the onset <strong>of</strong> the favourable conditions for the blast disease. To<br />
control neck blast timely, Pyroquilon sprays were given as follows<br />
NB 0 = Control (crops without fungicide treatment),<br />
NB 1 = One spray at the time <strong>of</strong> panicle emergence <strong>and</strong> another spray at 15 days<br />
after the panicle formation <strong>and</strong><br />
NB 2 = Two sprays at 10 day intervals <strong>and</strong> the spraying was begun after the<br />
observation <strong>of</strong> >0.10 neck blast disease proportion on the plants.<br />
The crops in the field plots were exposed for natural blast infection.<br />
Assessment <strong>of</strong> Blast Disease Incidence<br />
Ten hills per plot were r<strong>and</strong>omly fixed <strong>and</strong> the blast incidence was assessed at 7 day intervals on<br />
the leaves <strong>and</strong> neck regions (Loganathan <strong>and</strong> Ramaswamy, 1984). The AUDPC was calculated<br />
(Shaner <strong>and</strong> Finny, 1977) for each treatment <strong>and</strong> percent disease protection was also computed<br />
as:<br />
where,<br />
DC – DT<br />
DP = X 100<br />
DC<br />
DC = proportion <strong>of</strong> disease incidence in control plants <strong>and</strong><br />
DT = proportion <strong>of</strong> disease incidence in fungicides treated plants.<br />
Results<br />
The rice blast symptom appeared on the rice cv. IR 50 leaves <strong>of</strong> 20 d old seedlings. The results<br />
<strong>of</strong> disease incidence (Table 1) showed that the fungicide treatments (T 1 , T 2 , T 3 , T 4 <strong>and</strong> T 5 )<br />
suppressed the blast disease progress over the check (T 0 ). The seed treatment (T 3 ) <strong>of</strong><br />
Pyroquilon gave the maximum protection till 30 DAS <strong>and</strong> it also reduced subsequent disease<br />
progress. Moreover, the seed treatment with additional foliar sprays (T 4 <strong>and</strong> T 5 ) resulted in<br />
further reduction in blast disease progress over T 3 . The same trend was seen in each season<br />
studied (Table 1) from 2006 to 2008.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 193-198, 2010<br />
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Figure 1: Area under disease progress curve <strong>of</strong> leaf blast (A) <strong>and</strong> neck blast (B) against various<br />
treatments <strong>of</strong> Pyroquilon on the rice CV IR 50 during 2006 to 2008 seasons.<br />
T 0 , T 1 , T 2 , T 3 , T 4 , <strong>and</strong> T 5 indicate control, five foliar sprays at 7 day intervals, four sprays at 10 day<br />
intervals, seed treatment, seed treatment plus one spray <strong>and</strong> seed treatment plus two sprays respectively.<br />
NB 0 , NB 1 <strong>and</strong> NB 2 indicate control, two foliar sprays, (one spray at the time <strong>of</strong> panicle emergence <strong>and</strong><br />
another at 15 days after the panicle formation) <strong>and</strong> two sprays at 10 day intervals (after the panicle<br />
emergence) respectively. Values above the box denote the % disease protection over control.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 193-198, 2010<br />
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The AUDPC calculated for leaf blast on the rice cv.IR50 against each treatment showed<br />
that the seed treatment reduced the AUDPC with a protection <strong>of</strong> above 65% during 2006-2007<br />
<strong>and</strong> 73% during 2007–2008 seasons when compared to the foliar spray given alone. The<br />
differences in disease control were significant (P < 0.01) according to Duncan’s Multiple Range<br />
Test. Further, the seed treatment with additional foliar spray(s) rendered greater protection (up<br />
to 77%), but the difference was not significant among T 4 <strong>and</strong> T 5 (Fig. 1). Of the foliar sprays<br />
given at definite time intervals (T 1 <strong>and</strong> T 2 ) from the onset <strong>of</strong> blast, T 2 (foliar spray <strong>of</strong> 1%<br />
Pyroquilon at 10 day intervals) yielded considerable restriction <strong>of</strong> blast (over 40% disease<br />
protection) <strong>and</strong> T 1 (foliar spray <strong>of</strong> 1% Pyroquilon at 7 day intervals) appeared to be less efficient<br />
(maximum <strong>of</strong> 16% protection). Further, the studies revealed that the treatment effect was same<br />
in the crop seasons <strong>of</strong> both years in spite <strong>of</strong> disease pressure being higher during 2007-2008<br />
season than in 2006-2007 season.<br />
Table 1: Leaf blast disease incidence against various fungicide treatments in the rice cultivar IR 50<br />
during 2006-2008 seasons.<br />
Season Treatment # Days after sowing<br />
Proportion <strong>of</strong> disease incidence @<br />
2006-2007<br />
2007-2008<br />
20 27 34 41 48 55<br />
T 0 0.09a 0.18a 0.29a 0.37a 0.42a 0.43a<br />
T 1 0.09a 0.18a 0.28a 0.33a 0.40a 0.42b<br />
T 2 0.09a 0.17a 0.26a 0.31a 0.38a 0.38b<br />
T 3 0.00a 0.00b 0.10b 0.16b 0.19b 0.23c<br />
T 4 0.00a 0.00b 0.09b 0.13b 0.17b 0.19c<br />
T 5 0.00a 0.00b 0.12b 0.14b 0.16b 0.16c<br />
T 0 0.15a 0.24a 0.44a 0.53a 0.60a 0.66a<br />
T 1 0.16a 0.25a 0.36a 0.42b 0.50b 0.52b<br />
T 2 0.13a 0.19a 0.25bc 0.30c 0.33c 0.38c<br />
T 3 0.00b 0.00b 0.14c 0.17d 0.23cd 0.28d<br />
T 4 0.00b 0.00b 0.17c 0.18d 0.22d 0.25ed<br />
T 5 0.00b 0.00b 0.14c 0.16d 0.19d 0.19e<br />
# T 0 , T 1 , T 3 , T 4 <strong>and</strong> T 5 indicate control, five foliar sprays at 7 day intervals (spray<br />
initiated at 27 days after sowing), four foliar sprays at 10 day intervals (spray initiated at<br />
20 Days after sowing), seed treatment, seed treatment plus one spray <strong>and</strong> seed treatment<br />
plus two sprays respectively.<br />
@ Each value is the mean <strong>of</strong> three replicates.<br />
Disease incidence followed by a common letter are not significantly different at 1% level<br />
by Duncan’s Multiple Range test.<br />
The study <strong>of</strong> the blast disease incidence on the neck region revealed that NB 1 <strong>and</strong> NB 2<br />
treatments reduced the neck blast progress over the check. In control, the blast disease<br />
progressed to the maximum proportion <strong>of</strong> 0.28 <strong>and</strong> 0.35 during 2006-2007 <strong>and</strong> 2007-2008<br />
seasons respectively. The proportion <strong>of</strong> disease was less (0.10 in the neck regions) treatment, the disease<br />
progressed to the maximum proportion <strong>of</strong> 0.14 after first spray <strong>and</strong> the subsequent spray also<br />
reduced the disease progress (Table 2). Further, the AUDPC <strong>of</strong> neck blast for each treatment <strong>of</strong><br />
Pyroquilon reveals that NB 1 treatment yielded maximum disease protection (>90%) over the<br />
control. The NB 2 treatment reduced AUDPC to 31% <strong>and</strong> 41% during 2003-2004 <strong>and</strong> 2004-2005<br />
seasons respectively (Fig. 1). In general, both the NB 1 <strong>and</strong> NB 2 treatments reduced the blast<br />
disease incidence significantly (P
Krishnan / Management <strong>of</strong> Rice Blast with Minimum Fungicides<br />
Discussion<br />
The results on fungicide application as a measure to protect rice crops against blast revealed<br />
differential effect <strong>of</strong> selected fungicides with respect to the mode <strong>of</strong> application.<br />
Fungicide sprays are unnecessary when the conditions are unfavourable for disease<br />
onset. But, application <strong>of</strong> fungicides during favorable conditions for the disease in the field is<br />
undoubtedly important for efficient control <strong>of</strong> crop disease (Madden et al., 1978). Hence,<br />
fungicide applications were timed to optimize fungicide use (Fry, 1977; Vincelli <strong>and</strong> Lorbeer,<br />
1987) according to the conditions conducive for disease development <strong>and</strong> / or disease intensity.<br />
Castor et al. (1975) have suggested weather-based or disease intensity-based fungicide<br />
scheduling system to control potato late blight with minimum utility <strong>of</strong> fungicides. The timing<br />
<strong>of</strong> fungicide application to control blast epidemics in Japan <strong>and</strong> Colombia were reported by<br />
Kobayashi (1984) <strong>and</strong> Ahn <strong>and</strong> Rubiano (1984).<br />
Table 2: Neck blast disease incidence against various fungicide treatments in the rice cultivar IR 50<br />
during 2006-2008 seasons.<br />
Season Treatment # Days after sowing<br />
Proportion <strong>of</strong> disease incidence @<br />
2006-2007<br />
2007-2008<br />
60 67 74 81 55<br />
NB 0 0.06a 0.09a 0.17a 0.26a 0.28a<br />
NB 1 0.00b 0.00b 0.00b 0.02b 0.02b<br />
NB 2 0.06ac 0.10ac 0.13c 0.14c 0.15c<br />
NB 0 0.10a 0.18a 0.27a 0.32a 0.35a<br />
NB 1 0.00b 0.00b 0.02b 0.03b 0.03b<br />
NB 2 0.11ac 0.14c 0.15c 0.16c 0.16c<br />
# NB 0 – control; NB1 - two foliar sprays, one spray at the time <strong>of</strong> the panicle emergence <strong>and</strong><br />
another spray at 15 days after the panicle formation; NB 2 - two sprays at 10 day intervals <strong>and</strong><br />
the spray initiated when the observation <strong>of</strong> >0.10 disease proportion<br />
@ Each value is the mean <strong>of</strong> three replicates<br />
Disease incidence followed by a common letter are not significantly different at 1% level by<br />
Duncan’s Multiple Range test<br />
In the present study, the IR 50 rice crops raised from seeds pre-treated (T 3 ) with<br />
Pyroquilon were protected against the blast to the extent <strong>of</strong> 65% during 2006-2007 <strong>and</strong> 73%<br />
during 2007-2008. Further, enhanced protection to the extent <strong>of</strong> 77% was achieved by<br />
subsequent foliar application (T 3 <strong>and</strong> T 4 ). But, the application <strong>of</strong> fungicide in terms <strong>of</strong> foliar<br />
spray alone (T 1 <strong>and</strong> T 2 ) after the disease onset at 10 day interval gave the maximum <strong>of</strong> 40%<br />
disease protection. Shoemaker <strong>and</strong> Lorbeer (1977) have also suggested fungicide-scheduling<br />
system based on critical disease level to control leaf blight <strong>of</strong> onions.<br />
Of the Pyroquilon spray schedules followed to control neck blast, the spray given at the<br />
time <strong>of</strong> emergence <strong>of</strong> inflorescence (NB 1 ) protected the rice cv. IR 50 to the extent <strong>of</strong> 95%. In<br />
NB 2 treatment, where fungicide application started only after the appearance <strong>of</strong> visual blast<br />
symptom, the maximum protection achieved was only 41%. The results demonstrate that both<br />
the treatments reduced the disease incidence significantly (P
Krishnan / Management <strong>of</strong> Rice Blast with Minimum Fungicides<br />
In general, the studies on the time dependent application <strong>of</strong> fungicides suggest that the<br />
seed treatment with weather-based additional sprays could be recommended to the farmers for<br />
controlling leaf blast <strong>of</strong> susceptible rice cvs., if the conducive climatic conditions for blast<br />
development are observed before sowing. If this could not be done in time, foliar sprays at<br />
definite time intervals according to the disease intensity can be adopted. Further, to control neck<br />
blast, the farmers may follow fungicide spray before the panicle emergence according to<br />
weather conditions <strong>and</strong> if not, fungicide application at 10 day intervals should be necessary<br />
according to disease severity.<br />
References<br />
Ahn, S.W., <strong>and</strong> Rubiano, M. 1984. Methods <strong>and</strong> timing <strong>of</strong> fungicide application to control rice blast<br />
under favourable upl<strong>and</strong> conditions in colombia. Int. Rice Res. Newsl. 9: 5.<br />
Bhatt, J.C. 1988. Yield loss in five rice varieties due to blast disease. J. Hill Res., 1: 115-118.<br />
Bhatt, J.C., <strong>and</strong> Singh, R.A. 1990. Fungicidal control <strong>of</strong> blast in hills. Pp228. Proc.Int.Symp. Rice Res.<br />
New Frontiers. Nov. 15-18. Directorate <strong>of</strong> Rice Research. Hyderabad. pp.465.<br />
Castor, L.L., Ayers, J.E., MacNab, A.A., <strong>and</strong> Kranze, R.A. 1975. Computer forecasting system for<br />
Stewart’s bacterial disease on corn. Plant. Dis. Rep., 59: 533-536.<br />
Fry, W.E.1977. Integrated control <strong>of</strong> potato late blight: Effects <strong>of</strong> polygenic resistance <strong>and</strong> techniques <strong>of</strong><br />
timing <strong>of</strong> fungicide applications. Phytopathology, 67: 415-420.<br />
Kobayashi, J. 1984. Studies on epidemics <strong>of</strong> rice leaf blast Pyricularia oryzae Cav. in its early stage [In<br />
Japanese, English summary]. Bull. Akita Agric. Exp. Stn., 26: 1-84.<br />
Loganathan, M., <strong>and</strong> Ramaswamy, V. 1984. Effect <strong>of</strong> blast (BI) on IR 50 in late Samba. Int. Rice Res.<br />
Newsl., 9: 6.<br />
Madden, L., Pennypacker, S.P., <strong>and</strong> Macnab, A.A. 1978. Fast, a forecast system for Alternaria solani on<br />
tomato. Phytopatholgy, 68: 1354-1358.<br />
Muralidharan, K., Reddy, C.S., Krishnaveni, D., <strong>and</strong> Laha G.S. 2004. Field application <strong>of</strong> fluorescent<br />
Pseudomonas products to control blast <strong>and</strong> sheath blight diseases in rice. J. Mycol. Pl. Pathol., 34:<br />
411-414.<br />
Nagarajan, S. 1988. Epidemiology <strong>and</strong> crop loss <strong>of</strong> rice, wheat <strong>and</strong> pearl millet diseases in India. 5 th Int.<br />
Cong. Pl. Pathol., Kyoto, Japan.. Aug. 20-27. 88pp.<br />
Narasimhan, V., Ramdoss, N., Ch<strong>and</strong>rasekaran, A., <strong>and</strong> Abdul Kareem, A. 1991. Chemical management<br />
<strong>of</strong> blast disease <strong>of</strong> rice. Symp. New. Front. Chemi. Cont. October 9-10. Centre for advanced study in<br />
Botany, University <strong>of</strong> Madras, India. 25 pp.<br />
Ou, S.H. 1980. Look at worldwide rice blast disease control. Plant Dis., 64: 439-445.<br />
Shanner, G.E. <strong>and</strong> Finney, R.E. 1977. The effect <strong>of</strong> nitrogen fertilizers on the expression <strong>of</strong> slowmildewing<br />
resistance in knox wheat. Phytopathology, 67: 1051-1056.<br />
Sharma, O.P., <strong>and</strong> Sood, G.K. 1990. Evaluation <strong>of</strong> fungicides against glume blight control. Proc. Int.<br />
Symp. Rice. Res: New Frontiers. November, 15-18. Directorate <strong>of</strong> Rice Res. Hyderabad. 465 pp.<br />
Shoemaker, P.B., <strong>and</strong> Lorbeer, J.W. 1977. Timing <strong>of</strong> initial fungicide application to control botrytis leaf<br />
blight epidemics on onions. Phytopathology, 67: 409-414.<br />
Surin, A, Arunyanart, P., Dhitkiattipong, R., Rojanahusdin, W., Disthapron, S., <strong>and</strong> Soontrajarn, K. 1988.<br />
Rice yield loss to sheath rot. Int. Rice Res. Newsl., 13: 6.<br />
Suzuki, H. 1975. Meterological factors in the epidemiology <strong>of</strong> rice blast. Annu. Rev. Phytopath., 13: 239-<br />
255.<br />
Teng, P.S. 1994. The epidemiological basis for blast management. In: The Rice Blast Disease. Edited by:<br />
R.S. Zeighler. CAB, International. pp 409-433.<br />
Vincelli, P.C <strong>and</strong> Lorbeer, J.W. 1987. Sequential sampling plan for timing initial fungicide application to<br />
control Botrytis leaf blight <strong>of</strong> onion. Phytopathology, 77: 1301-1303.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 193-198, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Anamorphs <strong>of</strong> Asterinales<br />
V. B. Hosagoudar*<br />
Tropical Botanic Garden <strong>and</strong> Research Institute, Palode 695 562, Thiruvananthapuram, Kerala, India<br />
Received: 30 October, 2009; revised received: 30 December, 2009.<br />
Abstract<br />
The order Asterinales includes two families, Asterinaceae <strong>and</strong> Lembosiaceae with 29<br />
genera. Of these, 11 teleomorphic genera have 12 anamorphic genera. The key has been<br />
provided here to facilitate the identification <strong>of</strong> anamorphic genera with their teleomorphic<br />
connection. Each anamorphic genus supplemented with generic characters <strong>and</strong> line<br />
drawings.<br />
Key words: Black mildews, Asterinales, anamorphs, taxonomy.<br />
Introduction<br />
Asterinaceous fungi are ectophytic, obligate biotrophs infecting wide range <strong>of</strong> flowering plants<br />
ranging from herbs to trees, weeds to economically important cultivated plants, etc. These fungi<br />
produce thin to dense black colonies on the surface <strong>of</strong> the leaves. Structurally brown superficial<br />
mycelium produces appressoria which in turn produce haustoria or nutritive hyphae into the<br />
epidermal cells <strong>of</strong> the host plants for the nourishment. The fruiting body is flattened with<br />
radiating cells known as thyriothecium, which splits radially like a star (aster), hence they are<br />
known as Asterinaceous fungi. The family Asterinaceae was raised to an order Asterinales<br />
(Barr, 1976). The order Asterinales includes four families: Asterinaceae, Englerulaceae,<br />
Parmulariaceae <strong>and</strong> Parodiopsidaceae (Eriksson <strong>and</strong> Hawksworth, 1986). Muller & Arx (1962)<br />
<strong>and</strong> Arx <strong>and</strong> Muller (1975) have clearly distinguished Asterinaceae from Microthyriaceae.<br />
Apparently, these two unrelated families show similarity. The former is with non-ostiolate<br />
thyriothecia, dehisce stellately at the center <strong>and</strong> have oval to globose asci. While, the latter has<br />
ostiolate thyriothecia with cylindrical asci. The family Asterinaceae includes 27 genera (Arx<br />
<strong>and</strong> Muller, 1975). Subsequently, Asterinaceae segregated <strong>and</strong> a new family Lembosiaceae was<br />
proposed to include the genera having ellipsoidal to elongated or X or Y shaped thyriothecia<br />
split or dehisce longitudinally (Hosagoudar et al., 2001). Ishwaramyces <strong>and</strong> Maheshwaramyces<br />
have been added to this group (Hosagoudar et al., 2004, 2009).<br />
Pleomorphy is a common phenomenon with almost all higher fungi in which<br />
teleomorph belongs to either Basidiomycetes or Ascomycetes <strong>and</strong> the anamorph belongs to<br />
Deuteromycetes or Fungi Imperfecti. All teleomorphs are not represented with their anamorphs.<br />
It may be: the fungus would have lost this stage or we are unaware <strong>of</strong> this. However, it is well<br />
studied in case <strong>of</strong> Schiffnerula (Hughes, 1987). Anamorphs will give vital clue in the process <strong>of</strong><br />
identification. Hence, an attempt has been made here to ease the role <strong>of</strong> identification <strong>of</strong><br />
teleomorphs <strong>of</strong> Asterinales with the help <strong>of</strong> their anamorphs. Key for the identification <strong>of</strong><br />
anamorphs <strong>of</strong> the genera: Asterina, Asterodothis, Batistinula, Eupelte, Prillieuxina,<br />
Schiffnerula, Symphaster, Trichomelia <strong>and</strong> Uleothyrium is provided.<br />
*Corresponding author; Email address:vbhosagoudar@rediffmail.com<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Key to the Anamorph Genera<br />
1. Reproduction by conidia produced on conidiophores … 1<br />
1. Reproduction by pycnidiospores / pycnothyriospores … 8<br />
2. Conidia 3-4-armed …<br />
Triposporium<br />
(cf. Batistinula)<br />
2. Conidia not so … 3<br />
3. Conidia brown to black, sarciniform …<br />
Sarcinella<br />
(cf. Schiffnerula)<br />
3. Conidia not so … 4<br />
4. Conidia oval, mostly 0-4-6-septate, on Ziziphus …<br />
Mitteriella<br />
(cf. Schiffnerula)<br />
4. Conidia not so … 5<br />
5. Conidia fusiform, sickle shaped, always 3-septate, pale brown …<br />
Questieriella<br />
(cf. Schiffnerula)<br />
5. Conidia not so … 6<br />
6. Conidia cheiroid, with 4-5 closely appressed arms …<br />
Digitosarcinella<br />
(cf. Schiffnerula)<br />
6.Conidia not so … 7<br />
7. Appressoria present …<br />
7. Stomopodia present …<br />
8. Mycelium appressoriate … 11<br />
8. Mycelium non-appressoriate … 9<br />
9. Appressoria formed only around the stomata <strong>of</strong> the host plant …<br />
9. Appressoria totally absent … 10<br />
10. Pycnothyriospores unicellular …<br />
10. Pycnothyriospores one to few septate <strong>and</strong> <strong>of</strong> different shapes …<br />
11. Pycnothyriospores ovate, clavate, margin entire …<br />
11. Pycnothyriospores conoid, angular to slightly depressed margin …<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
200<br />
Clasterosporium<br />
(cf. Trichomelia,<br />
Eupelte,<br />
Asterodothis,<br />
Maheswaramyces<br />
Septoidium<br />
(cf. Eupelte)<br />
Bramhamyces<br />
(cf. Symphaster)<br />
Asterostomula<br />
(cf. Prillieuxina)<br />
Septothyrella<br />
(cf. Uleothyrium)<br />
Asterostomella<br />
(cf. Asterina)<br />
Mahanteshamyces<br />
(cf. Asterina)
Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 1: The genus Asterostomella. a -Appressoriate mycelium, b- Pycnothyrium, c-Pycnothyriospores.<br />
The genus Asterostomella<br />
Asterostomella Speg., Ann. Soc. Cien. Arg. 22: 198, 1886.<br />
Leaf parasites. Mycelium ectophytic, appressoria lateral, setae absent. Pycnothyria<br />
orbicular with radiating cells, astomatous, dehisce stellately at the center; pycnothyriospores<br />
unicellular, ovate, pyriform, brown.<br />
Type: A. paraguayensis Speg.<br />
The genus Asterostomula<br />
Asterostomula Theiss., Ann. Mycol. 14: 270, 1910.<br />
Leaf parasites. Mycelium ectophytic, appressoria <strong>and</strong> setae absent. Pycnothyria<br />
orbicular with radiating cells, astomatous, dehisce stellately at the center; pycnothyriospores<br />
unicellular, ovate, pyriform, brown.<br />
Type: A. loranthi Theiss.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 2: The genus Asterostomula. a-Non-appressoriate mycelium, b-Pycnothyrium,<br />
c- Pycnothyriospores.<br />
The genus Bramhamyces<br />
Bramhamyces Hosag., Indian J. Sci. Techn. 2(6): 17, 2009.<br />
Leaf parasites. Hyphae brown, branched, septate, ramify in the grooves only around<br />
stomata to form ‘areole’ to produce 1-3-appressoria. Remaining hyphae devoid <strong>of</strong> appressoria.<br />
Appressoria produced on the guard cells <strong>of</strong> the stoma produce corolloid haustoria in the<br />
neighboring cells. Stomata <strong>of</strong>ten plugged with mycelium. Pycnothyria grown below the<br />
mycelium, orbicular, connate; pycnothyriospores unicellular, brown, oval, pyriform.<br />
Type: B. ilecis Hosag. & Ch<strong>and</strong>ra.<br />
Mycelium devoid <strong>of</strong> appressoria but are produced around the stomata <strong>of</strong> the host plant<br />
by forming ‘areole’ is the character <strong>of</strong> this anamorphic genus.<br />
The genus Clasterosporium<br />
Clasterosporium Schweinitz, Trans. Am. Phil. Soc., N.S. 4: 300, 1832.<br />
Colonies usually effuse dark brown to black, <strong>of</strong>ten velvety. Mycelium superficial,<br />
stroma absent. Setae absent or sometimes present only in old colonies, simple, dark, smooth,<br />
straight to uncinate. Appressoria present. Conidiophores macronematous, mononematous,<br />
straight to flexuous, simple. Conidiogenous cells monoblastic, integrated, terminal, determinate,<br />
percurrent, cylindrical. Conidia solitary, acrogenous, simple, straight to curved, cylindrical to<br />
obclavate.<br />
Type: C. caricinum Sch.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 3: The genus Bramhamyces. a <strong>and</strong> b-Non-appressoriate mycelium produce ‘areole’ around<br />
stomata <strong>and</strong> with appressoria produced at the apical portion <strong>of</strong> the mycelium around the guard cells, c-<br />
Pycnothyrium, d- Pycnothyriospores.<br />
Figure 4: The genus Clasterosporium: a- Appressoriate mycelium, b- Conidiophore, c- Conidia<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
203
Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 5: The genus Digitosarcinella (After Hughes, 1984). a - Hyphae, b- Developing conidia on<br />
conidiophores, c- Cheiroid conidium<br />
The genus Digitosarcinella<br />
Digitosarcinella Hughes, Can. J. Bot. 62: 2208, 1984.<br />
Colonies foliicolous. Hyphae superficial, brown to dark brown, branched, appressoriate,<br />
appressoria sessile, lateral, unicellular. Conidiogenous cells lateral, sessile, monoblastic.<br />
Conidia cheiroid, with 4-5 closely appressed arms, up to 7-septate, constricted at the septa.<br />
Type: D. caseariae Hughes<br />
The genus Mahanteshamyces<br />
Mahanteshamyces Hosag., J. Econ. Taxon. Bot. 28: 189, 2004.<br />
Foliicolous, ectophytic parasites. Mycelium brown, superficial, appressoriate.<br />
Pycnothyria scutate, dimidiate, radiate, orbicular, stellately dehisce at the center;<br />
pycnothyriospores unicellular, brown, angular, wall straight to sinuate.<br />
Type sp.: M. agrostistachydis Hosag. & C.K. Biju<br />
The genus Mahanteshamyces differs from Asterostomella in having roundedly<br />
projected <strong>and</strong> shallowly lobate, angular <strong>and</strong> thick walled pycnothyriospores (Batista <strong>and</strong><br />
Cifferri, 1959; Sivanesan, 1983; Sutton, 1980).<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 6: The genus Mahanteshamyces Hosag. a-Appressoriate mycelium, b-Thyriothecium,<br />
c- Pycnothyriospores.<br />
Figure 7: The genus Mitteriella. a - Appressoriate mycelium, b- Conidia.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
205
Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
The genus Mitteriella<br />
Mitteriella Sydow, Ann. Mycol. 31: 95, 1933.<br />
Colonies black. Hyphae superficial, brown, branched, septate, appressoriate.<br />
Appressoria lateral, unicellular. Conidiophores macronematous, mononematous, short, simple.<br />
Conidiogenous cells polyblastic, integrated, terminal, sympodial, denticulate. Conidia solitary,<br />
simple, ellipsoidal to limoniform, black, 0-4-septate.<br />
Type: M. ziziphina Sydow<br />
Figure 8: The genus Questieriella. Appressoriate mycelium, b- Germinating conidia.<br />
The genus Questieriella<br />
Questieriella Arn. ex Hughes, Can. J. Bot. 61: 1729, 1983.<br />
Colonies black, hyphae superficial, brown, branched, septate, appressoriate.<br />
Appressoria lateral, unicellular. Conidiophores micronematous, mononematous to<br />
macronematous, lateral, 0-2-septate. Conidiogenous cells monoblastic to polyblastic, integrated,<br />
terminal, lateral or incorporated in the hyphae. Conidia blastic, terminal, solitary, narrowly<br />
ellipsoidal to obovoidal, curved, falcate, sigmoid, truncate at the base, 3-septate.<br />
Type: Q. pulchra Hughes<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 9: The genus Sarcinella. a-Appressoriate mycelium, b-Conidia on conidiophores, c- Conidia<br />
Sarcinella Sacc., Michelia 2: 31, 1880.<br />
The genus Sarcinella<br />
Colonies black. Hyphae superficial, branched, septate, appressoriate. Appressoria<br />
lateral, unicellular. Conidiophores macronematous, semi-macronematous, simple to branched.<br />
Conidiogenous cells monoblastic, integrated, terminal, intercalary, determinate. Conidia<br />
solitary, acrogenous or acropleurogenous, subspherical, sarciniform, dark brown to reddish<br />
brown, smooth, constricted at the septa.<br />
Type: S. heterospora Sacc.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
207
Septoidium Arn., Ann. Epiphyt. 7: 106, 1921.<br />
Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
The Genus Septoidium<br />
Colonies effuse, reddish brown, olivaceous brown or black. Mycelium superficial.<br />
Hyphae thick, <strong>of</strong>ten golden brown to reddish brown, smooth, branched, intertwined <strong>and</strong><br />
anastomosing to form a close network. Stroma none, Setae absent. Stomatopodia present, simple<br />
to lobed. Conidiophores macronematous to semi-macronematous, mononematous, simple,<br />
straight to flexuous, pale to mid golden brown to reddish brown, smooth. Conidiogenous cells<br />
monoblastic, integrated, terminal, percurrent, cylindrical.Conidia solitary, dry, acrogenous,<br />
simple, clavate, cylindrical, rounded at the apex to almost ellipsoidal, always truncate at the<br />
base, pale to golden brown to reddish brown, smooth, with one or several transverse septa.<br />
Type: S. clusiaceae Arn.<br />
Figure 10: The genus Septoidium (After Ellis, 1971). a-Conidia on conidiophores, b- Conidia.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
The Genus Septothyrella<br />
Septothyrella Höhn., Sitz. der Kais. Akad. der Wiss., Math.-naturw. Kl., Abt. 120: 393, 1911.<br />
Mycelium brown, septate, nonappressoriate, branched. Pycnothyria orbicular, brown,<br />
glabrus, ostiolate, upper surface with radiating cells. Pycnothyriospores clavate, ellipsoidal,<br />
entire to horizontally septate.<br />
Type: S. microthyrioides (Henn.) B. Sutton<br />
This genus appears to be synonymous to Asterothyrium Henn.<br />
Figure 11: The genus Septothyrella (Batista & Ciferri, 1959). a-Reticulate mycelium, b-Hyphae,<br />
c-Pycnothyrium, d-T.S. through the pycnothyrium, e-Pycnothyriospores on the hymenium,<br />
f- Pycnothyriospores.<br />
The genus Triposporium<br />
Triposporium Corda, Icon. Fung. 1: 16, 1837.<br />
Colonies effuse, black, hairy or velvety. Mycelium mostly immersed. Stroma none.<br />
Setae <strong>and</strong> appressoria absent. Conidiophores macronematous, mononematous, scattered, simple,<br />
straight to flexuous, almost cylindrical, broadened at the base to form a flat plate, brown,<br />
smooth. Conidiogenous cells monoblastic, integrated, terminal, percurrent, cylindrical,<br />
doliiform to lageniform. Conidia solitary, dry, acrogenous, branched, usually made up to a small<br />
calvate, doliiform or cylindrical stalk cells <strong>and</strong> 3 or occasionally 4 conical smooth, septate arms<br />
joined by their wide, rounded branch; the arms are dark brown near the centre <strong>of</strong> the conidium,<br />
hyaline or sub hyaline at the tips.<br />
Type: T. elegans Corda<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Figure 12: The genus Triposporium (Ellis, 1971). a-Conidiophores, b-Conidia.<br />
Table 1: Anamorphs <strong>and</strong> their teleomorphs.<br />
S.no. Anamorphs Teleomorphs<br />
1. Asterostomella Asterina<br />
2. Asterostomula Prillieuxina<br />
3. Bramhamyces Symphaster<br />
4. Clasterosporium Asterodothis<br />
Eupelte<br />
Maheshwaramyces<br />
Trichomelia<br />
5. Digitosarcinella Schiffnerula<br />
6. Mahanteshamyces Asterina<br />
7. Mitteriella Schiffnerula<br />
8. Questieriella Schiffnerula<br />
9. Sarcinella Schiffnerula<br />
10. Septoidium Eupelte<br />
11. Septothyrella Uleothyrium<br />
12. Triposporium Batistinula<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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Hosagoudar / Anamorphs <strong>of</strong> Asterinales<br />
Acknowledgement<br />
Thanks are due to the Director, Tropical Botanic Garden <strong>and</strong> Research Institute, Palode, Kerala<br />
State, India for the facilities.<br />
References<br />
Arx, J.A.V., <strong>and</strong> Muller, E. 1975. A re-evaluation <strong>of</strong> the bitunicate Ascomycetes with keys to families<br />
<strong>and</strong> genera. Stud. Mycol., 9: 1-159.<br />
Barr, M.E. 1976. Perspectives in the Ascomycotina. Mem. New York Bot. Gard., 28: 1-128.<br />
Batista, A.C., <strong>and</strong> Cifferri, R. 1959. Sistematica dos fungos imperfectos de picnostromas con himenio<br />
invertido (Peltasterales). Mycopath. Mycol. Appl., 11: 1-102.<br />
Ellis, M.B. 1971. Dematiaceous Hyphomycetes. CMI Kew Surrey, Engl<strong>and</strong>.<br />
Eriksson, O., <strong>and</strong> Hawksworth, D.L. 1986. An alphabetical list <strong>of</strong> the generic names <strong>of</strong> Ascomycetes. Systema<br />
Ascomycetum, 5: 4-184.<br />
Hosagoudar, V.B., Abraham, T.K., <strong>and</strong> Biju, C.K. 2001. Re-evaluation <strong>of</strong> the family Asterinaceae. J.<br />
Mycopathol. Res., 39: 61-63.<br />
Hosagoudar, V.B., Archana, G.R., <strong>and</strong> Mathew Dan 2009. Maheshwaramyces, a new genus <strong>of</strong> the family<br />
Lembosiaceae. Indian J. Sci. Technol., 2 (6): 12-13.<br />
Hosagoudar, V.B., Biju, C.K., <strong>and</strong> Abraham, T.K. 2004. Studies on foliicolous fungi- II. J. Econ. Taxon.<br />
Bot., 28: 183-186.<br />
Hughes, S.J. 1984. Digitosarcinella caseariae sp. nov. <strong>and</strong> Questieriella synanamorphs <strong>of</strong> the so-called Amazonia<br />
caseariae. Canadian J. Bot., 62: 2208-2212.<br />
Hughes, S.J. 1987. Pleomorphy in some hyphopodiate fungi. In: Pleomorphic fungi - The diversity <strong>and</strong> its<br />
taxonomic implications. Edited by: Sugiyama. Kodansa <strong>and</strong> Elsevier,Tokyo. pp. 103-139.<br />
Muller, E., <strong>and</strong> Arx, J.A.von 1962. Die Gattungen der didymosporen Pyrenomyceten. Beitr.<br />
Kryptogamenfl. Schweiz, 11:1-922.<br />
Sivanesan, A. 1983. The Bitunicate Ascomycetes. International Books <strong>and</strong> Periodical Supply Service,<br />
New Delhi. pp. 701.<br />
Sutton, B.C. 1980. The Coelomycetes: Fungi imperfecti with pycnidia, acervuli <strong>and</strong> stromata. CMI, Kew.<br />
pp. 696.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 199-211, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 213-215, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Plant Antioxidants Mediated Protein Alterations in<br />
Clarias batrachus Linn.<br />
C. Suseela Bai<br />
Department <strong>of</strong> Plant <strong>Biology</strong> <strong>and</strong> Plant Biotechnology, Women’s Christian College,<br />
Nagercoil-629001, Tamil Nadu, India.<br />
Received: 17 August 2009; revised received: 12 January, 2010<br />
Abstract<br />
An investigation on the effect <strong>of</strong> plant antioxidants supplemented feed on blood protein <strong>of</strong><br />
the fresh water cat fish Clarias batrachus was carried out in the laboratory. Fifty fingerlings<br />
<strong>of</strong> the fish were reared using supplementary diet for a period <strong>of</strong> 120 days in outdoor cement<br />
cisterns. Along with the basic feed fish meal, the plant antioxidants Punica granatum <strong>and</strong><br />
Phyllanthus emblica were given as supplementary feeds to the fishes. Effect <strong>of</strong> plant<br />
antioxidants at a dosage <strong>of</strong> 10 mg/ 100 mg feed on blood protein characteristics <strong>of</strong> the<br />
experimental animal was found significant.<br />
Key words: Plant antioxidants, Clarias batrachus, blood protein<br />
Introduction<br />
Plants <strong>and</strong> plant products significantly build up in the food chain <strong>of</strong> aquatic organisms<br />
(Direkbusarakom, 2004) <strong>and</strong> move up in the food chain <strong>of</strong> human. It has been observed that<br />
there are many quantitative relationships between plant products <strong>and</strong> biological activities <strong>of</strong><br />
fishes established in fresh water aquatic systems. Wild satavari, Asparagus racemosus widely<br />
used to promote human health produced a similar effect in shrimps (Mony, 2002) <strong>and</strong> Labeo<br />
rohita<br />
( Major Carp) fry (Sharma, 1996). Herbal extracts <strong>of</strong> Stellaria aquatica, Impatiens<br />
biflora, Oenothera biennis, Artemisia vulgaris <strong>and</strong> Lonicera japonica were reported to exhibit<br />
antimicrobial activity against bacterial <strong>and</strong> viral fish pathogens (Shagnliang et al ., 1990).<br />
Many herbal preparations were able to control diseases due to their antioxidant <strong>and</strong><br />
antimicrobial activities (Prasad et al., 1993., Citarasu et al., 1998,2001, 2002; Pundarikakshudu<br />
et al ., 2001; Sivaram et al., 2004). Moreover, Babu <strong>and</strong> Marian (2001) <strong>and</strong> Citarasu et al.<br />
(2002) demonstrated disease resistant larval production in Penaeus monodon reared on herbal<br />
supplemented diets.<br />
The fish, C. batrachus is a popular delicacy because <strong>of</strong> its faster growth rate, higher<br />
protein <strong>and</strong> iron content. In view <strong>of</strong> it, this paper is aimed at determining the responses <strong>of</strong> plant<br />
antioxidants on concentration <strong>and</strong> pr<strong>of</strong>ile <strong>of</strong> blood protein <strong>of</strong> C. batrachus reared under outdoor<br />
culture conditions.<br />
Materials <strong>and</strong> Methods<br />
Fifty fingerlings <strong>of</strong> C. batrachus (average body weight, 4.3 to 5.1g ; average body length 6.1 to<br />
7.2 cm ) were obtained from a commercial fish farm <strong>and</strong> transported to the culture site in plastic<br />
bags filled with aerated water. The fishes were fed daily on a formulated fish feed containing<br />
65% crude protein, acclimatized for a period <strong>of</strong> 30 days before the commencement <strong>of</strong> the<br />
experiment <strong>and</strong> they were reared in cement cisterns under outdoor culture conditions during<br />
which feeding was done with plant antioxidants supplementary feed prepared in dry pelleted<br />
*Email address: suseela.bai@yahoo.com<br />
213
Suseela Bai / Plant Antioxidants Mediated Protein Alterations in Clarias batrachus<br />
form using fruit rind <strong>of</strong> Punica granatum <strong>and</strong> dried leaves <strong>of</strong> Phyllanthus emblica (10 mg/100<br />
mg feed) separately. The culture medium was changed once in three days before adding the<br />
feed. Simultaneously a control tank receiving diet without plant products was kept. All the<br />
experiments were subjected to 12 hr day/ night cycle.<br />
Five individuals were selected at r<strong>and</strong>om from each tank at the end <strong>of</strong> the experimental<br />
period <strong>of</strong> 120 days, subjected to fasting for 12 hrs <strong>and</strong> blotted dry with s<strong>of</strong>t absorbent paper.<br />
Blood samples were collected without any anticoagulant <strong>and</strong> estimations <strong>of</strong> total protein content<br />
were carried out using plasma following the procedures <strong>of</strong> Young (1997). The protein<br />
separation was done using SDS - PAGE <strong>and</strong> comparisons were made.<br />
Results <strong>and</strong> Discussion<br />
The total protein content <strong>of</strong> blood plasma <strong>of</strong> fishes reared on control <strong>and</strong> plant antioxidants<br />
supplementary feed is given in Fig.1 The fishes fed with P. granatum supplemented feed had<br />
the maximum protein content <strong>of</strong> 13.70 ± 0.23 g/dl whereas those reared using P.emblica<br />
supplemented feed registered 6.87 ± 0.56g/dl. The animals reared on control diet had 4.43 ±<br />
0.13g/dl protein in their blood samples. The statistical treatment <strong>of</strong> the data (Student ‘t’ test)<br />
revealed significant differences (P < 0.005).<br />
(g/dl)<br />
16<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
Control<br />
P.granatum<br />
P.emblica<br />
Figure 1: Effect <strong>of</strong> plant antioxidants supplementary feed on protein (g/dl) content <strong>of</strong> C. batrachus.<br />
General protein pr<strong>of</strong>ile <strong>of</strong> fresh blood samples <strong>of</strong> C.batrachus exhibited no homology as<br />
in Fig.2. Striking differences could be seen in the protein pr<strong>of</strong>ile <strong>of</strong> fishes fed with plant<br />
antioxidants supplemented diets. Further study is needed to reveal the exact mechanism <strong>of</strong> plant<br />
antioxidant mediated free radical scavenging physiological activities.<br />
Figure 2: SDS-PAGE <strong>of</strong> blood samples <strong>of</strong> C.batrachus grown using plant antioxidants supplemented<br />
feed.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 213-215, 2010<br />
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Suseela Bai / Plant Antioxidants Mediated Protein Alterations in Clarias batrachus<br />
Supplementation <strong>of</strong> plant products promise a cheaper <strong>and</strong> viable solution to many<br />
problems aquaculture industries face today. Methanolic extracts <strong>of</strong> many herbs successfully<br />
controlled Vibrio pathogen <strong>and</strong> improved the immune system <strong>of</strong> the grouper larviculture<br />
(Sivaram et al., 2004). Various plant species such as Hygrophila spinosa , Withania somnifera,<br />
Zingiber <strong>of</strong>ficinalis, Solanum trilobatum, Andrographis paniculata, Phyllanthus niruri <strong>and</strong><br />
Tinospora cordifolia promoted growth <strong>and</strong> served effectively as antistress, antibacterial agents<br />
<strong>and</strong> immunostimulants in shrimp / fish larviculture (Citarasu et al., 1998, 2003a, 2003b). The<br />
herbal extracts <strong>of</strong> Daemia extensa <strong>and</strong> Leucas aspera were effective antibacterial agents against<br />
Vibrio parahaemolyticus <strong>and</strong> Vibrio harveyi (Latha, 2008). Moreover, species such as Ocimum<br />
sanctum, W. somnifera <strong>and</strong> Myristica fragrans improved immune parameters such as<br />
phagocytic activity, albumin – globulin ratio <strong>and</strong> leucocrit values in fishes.<br />
The increase in total protein content <strong>of</strong> blood serum <strong>and</strong> the differential protein pr<strong>of</strong>ile<br />
<strong>of</strong> C.batrachus obtained in the present study may possibly be due to plant antioxidants<br />
supplemented feed.<br />
References<br />
Babu, M.M., <strong>and</strong> Marian, M.P. 2001. Developing bioencapsulated herbal products for maturation <strong>and</strong><br />
quality larval production in Penaeus monodon. Special Publication, European Aquaclture Society,<br />
Oostende, Belgium, 36: 40-43.<br />
Citarasu, T., Immanuel, G., <strong>and</strong> Marian, M.P. 1998. Effects <strong>of</strong> feeding Artemia enriched with Stresstol<br />
<strong>and</strong> cod liver oil on growth <strong>and</strong> stress resistance in the Indian white shrimp Penaeus indicus<br />
postlarvae. Asian Fisheries Science 12:65-75.<br />
Citarasu, T., Babu, M.M. Punitha, S.M.J. Venket Ramalingam K., <strong>and</strong> Marian, M.P. 2001. Control <strong>of</strong><br />
pathogenic bacteria using herbal biomedicinal products in the larviculture system <strong>of</strong> Penaeus<br />
monodon. Proceedings <strong>of</strong> International Conference on Advanced Technologies in Fisheries <strong>and</strong><br />
Marine Sciences. M.S. University, India.<br />
Cirtarasu, T., Venket Ramalingam., K. Raja Jeya Sekar, R., Micheal Babu, M., <strong>and</strong> Marian, M. P.<br />
2003a. Influence <strong>of</strong> the antibacterial herbs, Solanum trilobatum, Andrographis paniculata <strong>and</strong><br />
Psoralea corylifolia on the survival, growth <strong>and</strong> bacterial load <strong>of</strong> Penaeus monodon postlarvae.<br />
Aquaculture International, 11: 583-595.<br />
Citarasu, T., Raja Jeya Sekar, R., Venket Ramalingam, K., Dh<strong>and</strong>apani, P. S., <strong>and</strong> Marian., M. P. 2003b.<br />
Effect <strong>of</strong> wood apple Aegle marmelos, Correa (Dicotyledons, Sapindales, Rutaceae) extract as an<br />
antibacterial agent on pathogens infecting prawn (Penaeus indicus) larviculture. Indian <strong>Journal</strong> <strong>of</strong><br />
Marine Sciences, 32 (2): 156-161.<br />
Direkbusarakom, S., 2004. Application <strong>of</strong> Medicinal herbs to Aquaculture in Asia. J. Sci. <strong>and</strong> Tech., 1(1):<br />
7-14.<br />
Latha, S.M.F. 2008. Screening <strong>and</strong> partial characterization <strong>of</strong> the herbal antibacterial active principle<br />
against shrimp pathogenic bacteria Vibrio harveyi <strong>and</strong> Vibrio parahaemolyticus. M.Phil. Dissertation,<br />
Vinayaka Mission’s University, Salem, Tamil Nadu, India.<br />
Mony, C. S. 1998. Studies on the use <strong>of</strong> some ayurvedic products for improving the reproductive<br />
performance in parthenogenetic Artemia from Thamaraikulam, South India. Ph. D. Thesis, M. S.<br />
University, Tirunelveli, India.<br />
Prasad, S., Variyur, K., <strong>and</strong> Padhyoy, J. 1993. Chemical investigation <strong>of</strong> some commonly used spices.<br />
Aryavaidyan, 6(4): 262-267.<br />
Pundarikakshudu, K., Jayvadan, K. Munira, P., Bodar S., <strong>and</strong> Deans, S. G. 2001. Short Communication –<br />
Antibacterial activity <strong>of</strong> Galega <strong>of</strong>ficinalis L. (Goat’s rue). J. Ethnopharmacol., 77:211 – 112.<br />
Sharma, K. K. 1996. Use <strong>of</strong> herb (Asparagus racemosus, Wild) supplemented diet for promoting growth<br />
in the fry <strong>of</strong> Labeo rohita. The fourth Indian Fisheries Forum. pp. 136.<br />
Sivaram., V., Babu, M. M. Citarasu, T. Immanuel, G. Murugadass, S., <strong>and</strong> Marian, M. P. 2004. Growth<br />
<strong>and</strong> Immune response <strong>of</strong> juvenile greasy groupers (Epinephelus tauvia) fed with herbal antibacterial<br />
active principle supplemented diets against Vibrio harveyi infections. Aquaculture, 237: 9-20.<br />
Young, D. 1997. Effect <strong>of</strong> Preanalytical Variables on Clinical Laboratory Tests. 2 nd Ed., AACC Press,<br />
Washington.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 213-215, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 217-226, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization <strong>and</strong><br />
Altering The Carbohydrate Metobolic Enzymes in<br />
Streptozotocin-Induced Diabetic Rat Tissues<br />
Ranganathan Babujanarthanam 1* , Purushothaman Kavitha 1 , Sarika Sasi 2 <strong>and</strong><br />
Moses Rajasekara P<strong>and</strong>ian 3<br />
1 Department <strong>of</strong> Biochemistry, K.M.G.College <strong>of</strong> Arts <strong>and</strong> Science,<br />
Gudiyattam – 632 602, Tamil Nadu, India<br />
2 Vinayaga Missions University, Salem -600010, Tamil Nadu, India<br />
3 Department <strong>of</strong> Zoology, Arignar Anna Government Arts College,<br />
Namakkal – 637 001, Namakkal District, Tamil Nadu, India.<br />
Received: 24 November, 2009; revised received: 20 February, 2010.<br />
Abstract<br />
The present study is an investigation into the role <strong>of</strong> quercitrin on carbohydrate metabolism<br />
in normal <strong>and</strong> streptozotocin (STZ)-induced diabetic rats. Administration <strong>of</strong> STZ leads to a<br />
significant increase (P < 0.05) in fasting plasma glucose <strong>and</strong> a decrease in insulin levels.<br />
The content <strong>of</strong> glycogen is significantly decreased (P < 0.05) in liver <strong>and</strong> muscle, but<br />
increased in the kidney. The activity <strong>of</strong> hexokinase decreased whereas the activities <strong>of</strong><br />
glucose 6-phosphatase <strong>and</strong> fructose 1,6-bisphosphatase significantly increased (P < 0.05) in<br />
the tissues. Oral administration <strong>of</strong> quercitrin (30 mg/kg) to diabetic rats for a period <strong>of</strong> 30<br />
days resulted in significant (P < 0.05) alterations in the parameters studied but not in<br />
normal rats. A decrease <strong>of</strong> plasma glucose <strong>and</strong> increase in insulin levels were observed<br />
along with the restoration <strong>of</strong> glycogen content <strong>and</strong> the activities <strong>of</strong> carbohydrate metabolic<br />
enzymes in quercitrin-treated diabetic rats. The histopathological study <strong>of</strong> the pancreas<br />
revealed the protective role <strong>of</strong> quercitrin. There was an expansion <strong>of</strong> the islets <strong>and</strong><br />
decreased fatty infiltrate <strong>of</strong> the islets in quercitrin treated diabetic rats. In normal rats<br />
treated with quercitrin, we could not observe any significant change in all the parameters<br />
studied. Combined, these results show that quercitrin plays a positive role in carbohydrate<br />
metabolism in diabetic rats.<br />
Key words: Quercitrin, streptozotocin, diabetes mellitus, gluconeogenesis, pancreas.<br />
Introduction<br />
Diabetes mellitus is the world’s largest endocrine disorder resulting in multiple etiologies,<br />
involving metabolic disorders <strong>of</strong> carbohydrate, fat <strong>and</strong> protein. All forms <strong>of</strong> diabetes are due to a<br />
decrease in the circulating concentration <strong>of</strong> insulin (insulin deficiency) <strong>and</strong> a decrease in the<br />
response <strong>of</strong> peripheral tissues to insulin, that is, insulin resistance. According to World Health<br />
Organization’s projections, the prevalence <strong>of</strong> diabetes is likely to increase by 35% by the year<br />
2025 (Boyle et al., 2001).<br />
Alterations in glucose metabolism in diabetes are frequently accompanied by changes in<br />
the activities <strong>of</strong> the enzymes that control glycolysis <strong>and</strong> gluconeogenesis in liver <strong>and</strong> muscle,<br />
such that the latter process becomes favored (Gerich et al., 1993) Increased rates <strong>of</strong> hepatic<br />
* Corresponding author; Email address: kmrbabugym@yahoo.com<br />
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Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
glucose production result in the development <strong>of</strong> overt hyperglycemia, especially fasting<br />
hyperglycemia, in patients with type 2 diabetes (DeFronzo et al., 1988). There are several<br />
important enzymatic checkpoints that act to control hepatic glycolysis <strong>and</strong> glycogen synthesis<br />
(glucokinase, glycogen synthase kinase-3), glycogenolysis (phosphorylase), gluconeogenesis<br />
(phosphoenolpyruvate carboxykinase, fructose 1,6bisphosphatase), or steps that are common to<br />
the pathways (glucose-6-phosphatase). Some <strong>of</strong> them are directly controlled by insulin via<br />
phosphorylation <strong>and</strong> dephosphorylation (Zhang et al., 2004).<br />
Plants have always been usable sources <strong>of</strong> drugs, <strong>and</strong> many currently available drugs<br />
are directly or indirectly derived from plants. Many <strong>of</strong> the oral agents that are presently in use<br />
for the treatment <strong>of</strong> diabetes mellitus suffer from implication in a number <strong>of</strong> serious <strong>and</strong> adverse<br />
effects (Zhang et al., 2000). Therefore, it is important to investigate the biologically active<br />
components <strong>of</strong> plants with hypoglycemic actions which include flavonoids, alkaloids,<br />
glycosides, polysaccharides, <strong>and</strong> peptidoglycans (Grover et al., 2002; Mao et al ., 2002)<br />
Flavonoids comprise a large group <strong>of</strong> compounds occurring widely throughout the plant<br />
kingdom. Daily flavonoid intake (typically present in onion, apple, grape, wine, herbs <strong>and</strong><br />
spices) in the human diet is highly variable, with estimations ranging from 23 mg/day (Hertog et<br />
al., 1993) to more than 500 mg/day (Manach et al., 1996). Flavonoids exert several biological<br />
activities, which are mainly related to their ability to inhibit enzymes <strong>and</strong> their antioxidant<br />
properties, <strong>and</strong> are able to regulate the immune response (Hollman et al., 1995). Among<br />
flavonoids, quercetin is the most common flavonoid in nature, <strong>and</strong> it is mainly present as its<br />
glycosylated forms such as quercitrin (5,7,3c,4c-OH, 3-rhamnosylquercetin).<br />
A wide variety <strong>of</strong> pharmacological activities <strong>of</strong> quercitrin was reported, that is, antiinflammatory<br />
(Sanchez et al., 2002; Taguchi et al 1993) antidiarrhoeals (Galvez), antiinociceptive<br />
property (Gadotti et al., 2005), antileishmanial activity (Muzitano et al., 2006), <strong>and</strong><br />
neuroprotective (Hollman et al., 1999). However, the majority <strong>of</strong> the studies have been carried<br />
out with the aglycone (Quercetin) form <strong>and</strong> little is known about the biological properties <strong>of</strong><br />
glycoside forms, due to the lack <strong>of</strong> commercial st<strong>and</strong>ards. Therefore, we undertook the present<br />
study to evaluate the role <strong>of</strong> quercitrin on the glycogen content <strong>and</strong> the activities <strong>of</strong> some<br />
carbohydrate metabolic enzymes, lipid peroxidation <strong>and</strong> antioxidant status in normal <strong>and</strong> STZinduced<br />
diabetic rats.<br />
Materials <strong>and</strong> Methods<br />
Chemicals<br />
Adenosine triphosphate, magnesium chloride, ammonium molybdate, fructose 1,6-<br />
bisphosphate, carboxymethyl cellulose sodium salt, phosphotungstic acid, thiobarbituric acid,<br />
1,1c,3,3c tetramethoxy propane, butylated hydroxy toluene, xylenol orange, dithionitro bis<br />
benzoic acid, ascorbic acid, 2,2c dipyridyl, p-phenylene diamine hydrochloride <strong>and</strong> sodium<br />
azide were obtained from SD Fine Chemicals, Mumbai, India. Quercitrin <strong>and</strong> streptozotocin<br />
were purchased from Sigma Chemical Co., St Louis, MO, USA. All the other chemicals used in<br />
the present study were <strong>of</strong> high analytical grade.<br />
<strong>Experimental</strong> Animals<br />
Male albino Wistar rats (150–180 g) were used in this study. The animals were fed on a st<strong>and</strong>ard<br />
pellet diet (Pranav Agro Industries, Pune, India) <strong>and</strong> water ad libitum. The pellet diet consisted<br />
<strong>of</strong> 22.02% crude protein, 4.25% crude oil, 3.02% crude fiber, 7.5% ash, 1.38% s<strong>and</strong> silica, 0.8%<br />
calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins <strong>and</strong> 56.17% carbohydrates. It<br />
provided a metabolisable energy <strong>of</strong> 3600 kcal/kg. They were maintained in a controlled<br />
environment (12 : 12 h light/dark cycle) <strong>and</strong> temperature (30 ± 2 C). The experiment was<br />
carried out according to the guidelines <strong>of</strong> the Committee for the Purpose <strong>of</strong> Control <strong>and</strong><br />
Supervision <strong>of</strong> Experiment on Animals (CPCSEA), New Delhi, India.<br />
Induction <strong>of</strong> <strong>Experimental</strong> Diabetes<br />
Diabetes was induced in 12 h fasted rats with streptozotocin (50 mg/kg) dissolved in citrate<br />
buffer (0.01 M, pH 4.5) intraperitoneally <strong>and</strong> the injection volume was 1 mL/rat. Control<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 217-226, 2010<br />
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animals were injected with citrate buffer alone. After 72 h <strong>of</strong> STZ injection, blood was<br />
withdrawn from animals (sinocular puncture) fasted overnight in tubes containing potassium<br />
oxalate <strong>and</strong> sodium fluoride as anticoagulant <strong>and</strong> plasma glucose was estimated using a<br />
commercial glucose kit (Product No. 72101) provided by Qualigens Diagnostics, Mumbai,<br />
India. Rats that had a fasting plasma glucose value <strong>of</strong> above 13.89 mmol/L (250 mg/dL) were<br />
included in the study as diabetic rats.<br />
<strong>Experimental</strong> Design<br />
A pilot study was conducted previously with three doses <strong>of</strong> quercitrin (10, 20 <strong>and</strong> 30 mg/kg<br />
body weight) to determine the dose dependent effects in STZ-induced diabetic rats. We found<br />
that 10, 20 <strong>and</strong> 30 mg/kg <strong>of</strong> quercitrin significantly (P < 0.05) decreased plasma glucose levels<br />
<strong>and</strong> quercitrin at doses 30 mg/kg was more effective in reducing plasma glucose levels<br />
significantly (P < 0.05) after 30 days <strong>of</strong> experimental study. Hence, we chose the dose 30 mg/kg<br />
<strong>of</strong> quercitrin for further studies.<br />
For the present study, the animals were grouped as follows: Group I, normal control;<br />
Group II, normal + quercitrin (30 mg/kg); Group III, diabetic control; Group IV, diabetic +<br />
quercitrin (30 mg/kg). Quercitrin was suspended in carboxymethyl cellulose (CMC) (0.01<br />
g/mL) <strong>and</strong> was orally administered to rats (1 mL/rat) using an intragastric tube. Normal control<br />
<strong>and</strong> diabetic control rats received CMC alone (1 mL/rat).<br />
The treatment period was 30 days, <strong>and</strong> after the last treatment, rats were fasted<br />
overnight <strong>and</strong> sacrificed by cervical decapitation. Blood was collected <strong>and</strong> plasma was obtained<br />
after centrifugation <strong>and</strong> used for various biochemical estimations. Tissues such as liver, kidney,<br />
muscle <strong>and</strong> pancreas were excised immediately from the animals <strong>and</strong> stored in ice-cold<br />
containers. They were then homogenized with appropriate buffer, centrifuged at low speed (705<br />
g), <strong>and</strong> the supernatant was collected. Biochemical estimations were carried out using these<br />
homogenates.<br />
Biochemical Assays<br />
Plasma insulin was assayed by ELISA method using a commercial kit (Catalog No. SP-401)<br />
from United Biotech Inc., Mountain View, CA, USA. Liver, kidney <strong>and</strong> muscle glycogen were<br />
estimated by the method <strong>of</strong> Morales et al. (Morales et al., 1973). The activity <strong>of</strong> hexokinase in<br />
the tissues was assayed by the method <strong>of</strong> Br<strong>and</strong>strup et al.(1957). Glucose 6-phosphatase in the<br />
tissues was assayed by the method <strong>of</strong> Koida <strong>and</strong> Oda (Koida et al., 1959), Fructose 1,6-<br />
bisphosphatase in the tissues was assayed by the method <strong>of</strong> Gancedo <strong>and</strong> Gancedo (Gancedo et<br />
al., 1971), Phosphorus content <strong>of</strong> the supernatant was estimated by the method <strong>of</strong> Fiske <strong>and</strong><br />
Subbarow (Fiske et al., 1925).<br />
In pancreas, the protein-bound hexoses concentration were estimated by the method <strong>of</strong><br />
Dubois <strong>and</strong> Gillesl. (Dubois et al., 1956), Protein-bound hexosamine was estimated by the<br />
method <strong>of</strong> Wagner (Wagner et al., 1979] Sialic acid in plasma <strong>and</strong> tissues was estimated by the<br />
method <strong>of</strong> Warren et al. (Warren et al., 1959), Fucose in plasma <strong>and</strong> tissue was estimated by the<br />
method <strong>of</strong> Dische <strong>and</strong> Shettle (Dische et al., 1948).<br />
Histopathological Studies<br />
For histopathological studies, animals <strong>of</strong> different groups were perfused with 10% neutral<br />
formalin solution. Pancreas was removed immediately from the animals; paraffin sections were<br />
made <strong>and</strong> stained using hematoxylin– eosin (H&E) stain. After staining, the sections were<br />
observed under light microscope <strong>and</strong> photographs were taken (20x).<br />
Statistical Analysis<br />
Statistical analysis was done by one-way ANOVA followed by Duncan’s multiple range test<br />
(DMRT) (Duncan et al., 1957), using SPSS s<strong>of</strong>tware package, version 9.05. P values < 0.05<br />
were considered as significant <strong>and</strong> included in the study.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 217-226, 2010<br />
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Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
Results<br />
The body weight <strong>of</strong> the experimental rats was recorded throughout the study (data not shown).<br />
At the end <strong>of</strong> the experimental period, a significant (P < 0.05) increase in the body weight <strong>of</strong><br />
normal control rats <strong>and</strong> normal + quercitrin (30 mg/kg) treated rats were observed. The diabetic<br />
control rats showed a significant (P < 0.05) decrease in body weight when compared with<br />
normal control rats. Diabetic rats treated with quercitrin (30 mg/kg) showed a significant (P <<br />
0.05) increase in body weight when compared with diabetic control rats. The levels <strong>of</strong> fasting<br />
plasma glucose <strong>and</strong> insulin are shown in Table 1. In diabetic control rats, the fasting plasma<br />
glucose levels were significantly (P < 0.05) high (23.17 ± 2.02 mmol/L). Diabetic rats when<br />
treated with quercitrin (30 mg/kg) had significantly (P < 0.05) decreased plasma glucose levels<br />
(8.06 ± 0.60 mmol/L). Normal rats treated with quercitrin (30 mg/kg) did not show any<br />
significant effect on plasma glucose levels (4.07 ± 0.30 mmol/L). The levels <strong>of</strong> plasma glucose<br />
in normal control were found to be 4.21 ± 0.29 mmol/L. A significant (P < 0.05) decrease in<br />
plasma insulin levels was observed in diabetic control rats (7.57 ± 0.22 lU/mL) <strong>and</strong> on treatment<br />
with quercitrin (30 mg/kg), the levels significantly (P
Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
Table 1: Effect <strong>of</strong> quercitrin on the content <strong>of</strong> glycogen in the tissues <strong>of</strong> normal <strong>and</strong> diabetic<br />
rats.<br />
Liver Kidney Muscle<br />
Groups<br />
(mg/g tissue)<br />
Normal control 3.22 ± 0.20 a 2.19 ± 0.12 a 3.45 ± 0.11 a<br />
Normal + quercitrin (30 mg/kg) 3.11 ± 0.29 a 2.22 ± 0.10 a 3.49 ± 0.12 a<br />
Diabetic control 2.01 ± 0.12 b 3.74 ± 0.18 b 2.92 ± 0.10 b<br />
Diabetic + quercitrin (30 mg/kg) 3.17 ± 0.18 c 2.08 ± 0.16 c 3.21 ±0.14 c<br />
Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter (a,b,c)<br />
differ significantly with each other (P
Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
Table 4: Effect <strong>of</strong> quercitrin on the activity <strong>of</strong> glucose 6-phosphate in the tissues <strong>of</strong> normal <strong>and</strong> diabetic<br />
rats.<br />
Groups<br />
Liver<br />
Kidney<br />
(μ mole <strong>of</strong> Pi liberated/min/mg protein)<br />
Normal control 23.10 ± 1.62 a 19.80 ± 1.21 a<br />
Normal + quercitrin (30 mg/kg) 22.20 ± 1.30 a 18.30 ± 1.32 a<br />
Diabetic control 42.31 ± 2.91 b 37.21 ± 2.47 b<br />
Diabetic + quercitrin (30 mg/kg) 31.10 ± 2.19 c 24.11 ± 1.52 c<br />
Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter<br />
(a,b,c) differ significantly with each other (P
Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
Discussion<br />
In this study, we found that quercitrin has the ability to increase glucose utilization <strong>and</strong><br />
normalize the carbohydrate metabolic enzymes in STZ-induced diabetic rats. Blood glucose<br />
level is strictly controlled by insulin secretion from pancreatic b-cells <strong>and</strong> insulin action on liver,<br />
muscle <strong>and</strong> other target tissues(Hii et al., 1984), Quercitrin by its ability to scavenge free<br />
radicals <strong>and</strong> to inhibit lipid peroxidation, prevents STZ-induced oxidative stress <strong>and</strong> protects b-<br />
cells resulting in increased insulin secretion <strong>and</strong> decreased blood glucose levels. In this context,<br />
research by Vessal et al. (Vessal et al., 2003), has shown that quercetin, the aglycone <strong>of</strong><br />
quercitrin decreased blood glucose concentration <strong>and</strong> increased insulin release in STZ-induced<br />
diabetic rats. Coskun et al. (Coskun et al., 2005) have also reported that, in STZ-induced<br />
diabetic rats, quercetin protected pancreatic b-cells by decreasing oxidative stress <strong>and</strong><br />
preserving pancreatic b-cell integrity. Increased insulin levels could also be due to the<br />
stimulatory effect <strong>of</strong> quercitrin, thereby potentiating the existing b-cells <strong>of</strong> the islets <strong>of</strong><br />
Langerhans in diabetic rats. Hii <strong>and</strong> Howell (Hii et al., 1985), showed increased number <strong>of</strong><br />
pancreatic islets in quercetin treated animals. Hyperglycemia <strong>and</strong> decreased insulin levels are<br />
characteristics <strong>of</strong> diabetic rats in this study. Quercitrin treatment to diabetic rats significantly<br />
reduced plasma glucose levels <strong>and</strong> increased insulin levels. Quercitrin, being a flavonoid, could<br />
induce the intact functional b-cells to produce insulin <strong>and</strong>/or protect the functional b-cells from<br />
further deterioration so that they remain active <strong>and</strong> produce insulin.<br />
Table 7: Effect <strong>of</strong> quercitrin on glycoproteins in the kidney <strong>of</strong> normal <strong>and</strong> diabetic rats.<br />
Groups<br />
Hexose Hexosamine Fucose Sialic acid<br />
(mg/g defatted tissue)<br />
Normal control 32.07 ± 3.02 a 24.22 ± 1.49 a 11.37 ± 1.04 a 6.21 ± 0.42 a<br />
Normal + quercitrin<br />
(30 mg/kg)<br />
32.74 ± 3.10 a 24.56 ± 1.67 a 11.29 ± 1.02 a 6.02 ± 0.44 a<br />
Diabetic control 54.08 ± 4.26 b 34.11 ± 3.08 b 23.27 ± 2.04 b 12.31 ± 1.08 b<br />
Diabetic + quercitrin<br />
(30 mg/kg)<br />
39.21 ± 3.67 c 28.27 ± 2.46 c 16.63 ± 1.44 c 8.03 ± 0.65 c<br />
Each value is mean ± S.D. for 8 rats in each group. Values that have a different superscript letter (a,b,c)<br />
differ significantly with each other (P
Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
deficiency as they depend on insulin for influx <strong>of</strong> glucose. In contrast, kidney glycogen content<br />
is increased <strong>and</strong> this is due to the entry <strong>of</strong> glucose in a hyperglycemic state as renal tissue is<br />
independent <strong>of</strong> insulin action (Belfiore et al., 1986), Increased insulin <strong>and</strong> consequently<br />
decreased blood glucose levels due to treatment with quercitrin could positively alter the<br />
glycogen content in the diabetic tissues.<br />
Alterations in glucose metabolism in diabetic are frequently accompanied by changes in<br />
the activities <strong>of</strong> the enzymes that control glycolysis <strong>and</strong> gluconeogenesis in liver <strong>and</strong> muscle,<br />
such that the latter process becomes favored (Gerich 1986), Laakso (Laakso et al., 1995), has<br />
reported that hexokinase is the first regulatory enzyme <strong>of</strong> glycolytic pathway that converts<br />
glucose into glucose 6-phosphate. Glucose 6-phosphatase plays a key role in the regulation <strong>of</strong><br />
blood glucose levels by catalyzing the hydrolysis <strong>of</strong> glucose 6-phosphate in the common<br />
terminal step <strong>of</strong> the gluconeogenic <strong>and</strong> glycogenolytic pathways (Wallert et al., 2001), Fructose<br />
1,6-bisphosphatase catalyses the conversion <strong>of</strong> fructose 1,6-bisphosphate to fructose 6-<br />
phosphate, a step necessary to achieve a reversal <strong>of</strong> glycolysis (Maye, 1996).<br />
Hexokinase insufficiency in diabetic rats can cause decreased glycolysis <strong>and</strong> decreased<br />
utilization <strong>of</strong> glucose for energy production. Oral administration <strong>of</strong> quercitrin to diabetic rats<br />
resulted in a significant reversal in the activity <strong>of</strong> hexokinase. The increased plasma insulin <strong>and</strong><br />
decreased glucose in diabetic rats given quercitrin may also be as a result <strong>of</strong> increased hepatic<br />
hexokinase activity, resulting in increased glycolysis. The gluconeogenic enzyme glucose-6-<br />
phosphatase is a crucial enzyme <strong>of</strong> glucose homeostasis because it catalyses the ultimate<br />
biochemical reaction <strong>of</strong> both glycogenolysis <strong>and</strong> gluconeogenesis (Mithievre et al., 1996).<br />
Increased glucose 6-phosphatase activity in diabetic rats provides hydrogen, which binds with<br />
NADP+ in the form <strong>of</strong> NADPH <strong>and</strong> enhances the synthesis <strong>of</strong> fats from carbohydrates (i.e.<br />
lipogenesis) Bopanna et al., 1997) <strong>and</strong>, finally, contributes to increased levels <strong>of</strong> glucose in the<br />
blood. Increased hepatic glucose production in diabetes mellitus is associated with impaired<br />
suppression <strong>of</strong> the gluconeogenic enzyme fructose 1,6-bisphosphatase. Activation <strong>of</strong><br />
gluconeogenic enzymes is due to the state <strong>of</strong> insulin deficiency, because under normal<br />
conditions, insulin functions as a suppressor <strong>of</strong> gluconeogenic enzymes.<br />
In the present study, the concentration <strong>of</strong> glycoproteins were found to be significantly<br />
(P
Babujanarthanam et al / Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization in Rats<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 217-226, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Assessment <strong>of</strong> Antibacterial Activity <strong>and</strong> Detection <strong>of</strong><br />
Small Molecules in Different Parts <strong>of</strong><br />
Andrographis paniculata<br />
R.Arunadevi 1 *, S. Sudhakar 1 <strong>and</strong> A.P. Lipton 2<br />
1 Department <strong>of</strong> Biotechnology, Manonmaniam Sundararnar University, Tirunelveli-627012, Tamil Nadu, India<br />
2 Marine Biotechnology Laboratory, Vizhinjam Research Centre <strong>of</strong> CMFRI, Vizhinjam-695521 Kerala, India<br />
Received: 15 October 2009; revised received: 20 January, 2010.<br />
Abstract<br />
Andrographis paniculata (Acanthaceae) is a medicinal plant used in India, China <strong>and</strong> other<br />
tropical countries for ailments such as upper respiratory tract infections, inflammations <strong>and</strong><br />
diabetics. The major constituents <strong>of</strong> plants are reported to contain diterpenoids, flavonoids<br />
<strong>and</strong> polyphenols. The present study was to investigate the antibacterial activity <strong>of</strong> the<br />
different parts <strong>of</strong> the plant using different solvents by well diffusion method <strong>and</strong> also to<br />
screen the small molecules which are present in different parts such as: leaves, stem,<br />
branches, seed, root <strong>and</strong> buds both in fresh <strong>and</strong> dried form. The results suggest that the<br />
crude extracts <strong>of</strong> the leaves, stem <strong>and</strong> branches <strong>of</strong> Andrographis paniculata could be<br />
potential lead sources <strong>of</strong> broad spectrum antibiotic - resistance modifying compounds. A<br />
total <strong>of</strong> twenty nine small molecular compounds were screened <strong>and</strong> details presented.<br />
Keywords: Antibacterial activity, Andrographis paniculata, small molecules, Thin Layer<br />
Chromatography.<br />
Introduction<br />
Microbial infections represent the world’s leading cause <strong>of</strong> premature death <strong>and</strong> the general well<br />
being <strong>of</strong> humans depends on the production <strong>of</strong> new clinically useful antibiotics to curtail or<br />
manage the pathogens (Hugo <strong>and</strong> Russell, 2003). For over a decade, the pace <strong>of</strong> development <strong>of</strong><br />
new antimicrobial agents has slowed down while the prevalence <strong>of</strong> resistance has grown at an<br />
astronomical rate. The rate <strong>of</strong> emergence <strong>of</strong> antibiotic resistant bacteria is not matched by the<br />
rate <strong>of</strong> development <strong>of</strong> new antibiotics to combat them (Prescott <strong>and</strong> Kelin, 2002). There are<br />
indications that some herbal materials can act as antibiotic resistant inhibitors. Combinations <strong>of</strong><br />
some herbal materials <strong>and</strong> different antibiotics might affect the inhibitory effect <strong>of</strong> these<br />
antibiotics (Aiyegoro et al., 2009).<br />
Medicinal plants have a long history <strong>of</strong> use both in developing <strong>and</strong> developed countries.<br />
Among the few advantages <strong>of</strong> using antimicrobial compounds <strong>of</strong> medicinal plants include fewer<br />
side effects, better patient tolerance <strong>and</strong> relatively less expensive. All these data highlights the<br />
need for developing alternative new regimens. The plant chosen, Andrographis paniculata<br />
(Acanthaceae) is commonly known as “King <strong>of</strong> Bitters”. The plant is an annual herb. It is<br />
branched, erect, growing up to 1 meter in height. The leaves <strong>and</strong> the stems <strong>of</strong> the plant are used<br />
to extract the active phytochemicals.<br />
*Corresponding author; Email address: arunaanurag@gmail.com<br />
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Earlier reports indicated that the primary medicinal component <strong>of</strong> Andrographis is<br />
<strong>and</strong>rographolide. It has a very bitter taste, is a colourless, crystalline in appearance, <strong>and</strong> is called<br />
a diterpene lactone. As per the reports the leaves contain the highest amount <strong>of</strong> <strong>and</strong>rographolide,<br />
the most medicinally active phytochemical in the plant, while the seeds contain the lowest.<br />
Andrographis paniculata demonstrated significant activity in fighting common cold, flu <strong>and</strong><br />
upper respiratory infections (Coon et al., 2004). Considering these, the present study was<br />
initiated with the objective <strong>of</strong> screening for antibacterial properties <strong>and</strong> to detect the compounds<br />
recovery in different parts <strong>of</strong> the plant.<br />
Materials <strong>and</strong> Methods<br />
Collection <strong>and</strong> Preparation <strong>of</strong> Plant Material<br />
Andrographis paniculata plants (Fig.1) were collected from Pottalpudhur village in Tirunelveli<br />
district (8°43’ N 77°29’ E Latitude <strong>and</strong> Longitude), India <strong>and</strong> were confirmed by local medical<br />
practitioners <strong>and</strong> available literature. The plant parts were thoroughly washed with water <strong>and</strong> the<br />
different parts like, leaf, stem, root, side branches, seed <strong>and</strong> buds were separated.<br />
Figure 1: Andrographis paniculata.<br />
Preparation <strong>of</strong> Fresh Extract<br />
250g <strong>of</strong> each plant part (root, stem, side branches, leaves, seed <strong>and</strong> buds) were ground by mortar<br />
<strong>and</strong> pestle <strong>and</strong> extracted with 250ml methanol <strong>and</strong> water respectively. The solvents were<br />
evaporated to dryness to obtain crude extracts. The various crude extracts were stored at 4°C<br />
<strong>and</strong> subjected to further analysis.<br />
Preparation <strong>of</strong> Dry Extract<br />
250g <strong>of</strong> each plant part (root, stem, side branches, leaves, seed <strong>and</strong> buds) were air dried,<br />
powdered <strong>and</strong> extracted with 250ml <strong>of</strong> water <strong>and</strong> methanol. The extracts were stored at 4°C<br />
until further use.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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Test Organisms<br />
The test organisms such as Bacillus subtilis, Salmonella typhi, Staphylococcus aureus,<br />
Escherichia coli, Klebsiella pneumoniae, Enterobacter faecalis <strong>and</strong> Pseudomonas aeruginosa<br />
were used for the bioassay. These strains were isolated from clinical samples collected from<br />
M/S. Vivek Scientific Laboratory Nagercoil. The organisms were characterized by biochemical<br />
tests.<br />
Evaluation <strong>of</strong> Antibacterial Activity<br />
Inoculum Preparation<br />
Overnight broth culture (in nutrient broth- HiMedia) <strong>of</strong> the test bacteria was made <strong>and</strong> the<br />
turbidity was compared with Mc Farl<strong>and</strong> Nephalometer.<br />
Antibacterial Activity<br />
The sensitivity testing <strong>of</strong> the crude extracts <strong>of</strong> the plant was performed using agar well diffusion<br />
method. The bacterial isolates were first grown in nutrient broth <strong>and</strong> inoculam was prepared as<br />
described above. The Muller Hinton Agar medium was prepared, sterilized <strong>and</strong> the molten<br />
medium at 50° C was poured into sterile petridishes <strong>and</strong> the medium was allowed to solidify.<br />
The organisms were uniformly swabbed on the plates <strong>and</strong> wells were made in the agar medium<br />
using a sterile 6mm cork borer. The wells were later filled with the extract at a concentration <strong>of</strong><br />
20µl. The plates were allowed to st<strong>and</strong> on for 1 hour to allow proper diffusion <strong>of</strong> the extract <strong>and</strong><br />
to prevent spillage onto the surface <strong>of</strong> the agar medium <strong>and</strong> then incubated at 37° C for 24 hours<br />
after which they were observed for zone <strong>of</strong> inhibition. Kanamycin <strong>and</strong> ampicillin at the<br />
concentration <strong>of</strong> 0.1mg/ml each were used as controls.<br />
Screening <strong>of</strong> Small Molecules<br />
Preparation <strong>of</strong> Plates<br />
The slurry was prepared by mixing silica gel with water in the ratio 3:2 <strong>and</strong> a few drops <strong>of</strong><br />
Ammonia were added into the slurry to separate the nitrate compounds in the sample. The slurry<br />
was coated on the glass plate at a thickness <strong>of</strong> about 0.25mm <strong>and</strong> then plates were allowed to<br />
dry at room temperature for 15-20 minutes. Then the plates were kept in hot air oven at 100-<br />
120 ο C for 1-2 hrs to remove the moisture <strong>and</strong> to activate the absorbent on the plate. The samples<br />
were loaded on the plate about 1.5-2 cm from the bottom, the spots were allowed to dry <strong>and</strong><br />
spotting were done repeatedly to obtain a more concentrated spot.<br />
Chromatogram Development<br />
The solvent chlor<strong>of</strong>orm <strong>and</strong> acetone in the ratio 4:1 was used as mobile phase. The solvent was<br />
poured into the tank <strong>and</strong> allowed to st<strong>and</strong> for an hour to ensure that the atmosphere within the<br />
tank become saturated with solvent vapours. After equilibration, the plate was placed vertically<br />
in the tank; the solvent moves upwards due to capillary action <strong>and</strong> thus compound get separated.<br />
Identification <strong>of</strong> Compounds<br />
The chromatogram was allowed to dry <strong>and</strong> the plate was exposed to UV light source. Some <strong>of</strong><br />
the compounds were fluoresced in different colours. Then the plates were exposed to iodine<br />
vapour. The iodine vapour reacted with the spots <strong>and</strong> formed reddish brown colour.<br />
Results <strong>and</strong> Discussion<br />
Antibacterial Activity<br />
Fresh Extract<br />
The results <strong>of</strong> the agar well diffusion assay for the fresh extract <strong>of</strong> different parts <strong>of</strong><br />
Andrographis paniculata are presented in Table 1.<br />
The fresh methanol extract <strong>of</strong> both leaf <strong>and</strong> stem exhibited the maximum inhibitory<br />
activity against the G +ve organisms. The organisms were resistant to the buds extract. The<br />
G –ve bacterial sensitivity studies revealed the following result: the leaves extract <strong>of</strong><br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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Arunadevi et al / Antibacterial Activity <strong>of</strong> Andrographis paniculata<br />
Andrographis paniculata showed maximum inhibitory activity against Salmonella typhi <strong>and</strong><br />
Enterobacter faecalis which are causative organisms for typhoid (enteric fever) <strong>and</strong> other<br />
diseases, The stem <strong>and</strong> branch extract showed activity against Klebsiella pneumoniae. The root<br />
extract showed activity only to Salmonella typhi. The fresh water extracts <strong>of</strong> leaves showed<br />
maximum inhibitory activity against G +ve <strong>and</strong> G –ve organisms such as Klebsiella<br />
pneumoniae, Escherichia coli, Pseudomonas aeruginosa <strong>and</strong> lesser activity to Salmonella typhi<br />
<strong>and</strong> Enterobacter faecalis. The extracts <strong>of</strong> branch, stem, seed <strong>and</strong> bud extract did not have any<br />
bactericidal activity to the G +ve organisms. Salmonella typhi showed maximum inhibitory<br />
activity to leaf, stem, branch, <strong>and</strong> seed whereas Klebsiella pneumoniae showed activity only to<br />
the leaves extract.<br />
Table 1: Effect <strong>of</strong> different parts <strong>of</strong> Andrographis paniculata fresh methanol <strong>and</strong> aqueous extracts on<br />
Gram +ve <strong>and</strong> Gram -ve bacteria.<br />
Sl.<br />
No<br />
Name <strong>of</strong><br />
Pathogen<br />
Plant parts<br />
Leaf Stem Branch Root Seed Buds<br />
M W M W M W M W M W M W<br />
1 Bacillus subtilis 20 18 13 NS NS NS NS NS NS NS R R<br />
2<br />
3<br />
4<br />
Staphylococcus<br />
aureus<br />
Klebsiella<br />
Pneumoniae<br />
Salmonella<br />
typhi<br />
15 12 10 NS NS NS NS NS NS 10 R NS<br />
10 12 12 NS 10 NS NS NS NS NS R NS<br />
12 10 10 10 NS 10 10 NS NS 10 R R<br />
5 Escherichia coli 10 12 NS NS NS 10 NS 12 NS 10 NS R<br />
6<br />
7<br />
Enterobacter<br />
faecalis<br />
Pseudomonas<br />
aeruginosa<br />
12 10 10 NS NS NS NS 10 NS NS NS NS<br />
10 12 NS NS NS NS NS 10 NS NS NS NS<br />
R – Resistant NS – Not Significant M - Methanol W - Water (values are mean <strong>of</strong> replicates)<br />
Dry Extract<br />
The zone <strong>of</strong> inhibition for the dry aqueous <strong>and</strong> methanol extracts on G +ve <strong>and</strong> G –ve organisms<br />
are given in Table 2. The leaf extract showed maximum activity to Staphylococcus aureus <strong>and</strong><br />
Pseudomonas aeruginosa <strong>and</strong> moderate activity towards Bacillus subtilis, Klebsiella<br />
pneumoniae, Salmonella typhi, Escherichia coli <strong>and</strong> Enterobacter faecalis. All the other<br />
extracts showed insignificant activity towards G + ve <strong>and</strong> G negative organisms.<br />
While comparing the fresh water <strong>and</strong> methanol extracts, fresh methanol extract showed<br />
maximum inhibitory activity towards G +ve <strong>and</strong> G – ve organisms. Also while comparing to the<br />
different parts <strong>of</strong> fresh methanol <strong>and</strong> dry methanol extracts, leaves extracts only showed<br />
significant effect in dried form (Table 3).<br />
The water extracts from dried parts <strong>of</strong> Andrographis paniculata exhibited no positive<br />
result to all the organisms. This showed that the active compounds present in the plant materials<br />
are not soluble in water when the plant parts are dried, whereas, methanol extract <strong>of</strong> dried parts<br />
showed positive results.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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Arunadevi et al / Antibacterial Activity <strong>of</strong> Andrographis paniculata<br />
Table2: Effect <strong>of</strong> different parts <strong>of</strong> Andrographis paniculata dry methanol <strong>and</strong> aqueous extracts on<br />
Gram +ve <strong>and</strong> Gram -ve bacteria.<br />
Sl.<br />
No<br />
1<br />
2<br />
4<br />
5<br />
6<br />
7<br />
3<br />
Name <strong>of</strong><br />
Pathogen<br />
Bacillus<br />
subtilis<br />
Staphylococc<br />
us aureus<br />
Klebsiella<br />
Pneumoniae<br />
Salmonella<br />
typhi<br />
Escherichia<br />
coli<br />
Enterobacter<br />
faecalis<br />
Pseudomonas<br />
aeruginosa<br />
Plant parts<br />
Leaf Stem Branch Root Seed Buds<br />
M W M W M W M W M W M W<br />
10 5 NS R NS R NS NS NS R R NS<br />
15 8 NS R NS NS NS R NS NS R R<br />
10 5 R R R NS NS NS NS R NS R<br />
10 9 NS R NS NS NS R 10 NS R R<br />
10 R NS R R R R NS R R R R<br />
10 R NS NS NS R R R NS NS NS R<br />
12 8 NS R R NS NS NS R R R R<br />
R – Resistant NS – Not Significant M - Methanol W – Water (values are mean <strong>of</strong> replicates).<br />
Table 3: Antibacterial effect <strong>of</strong> the leaves extract <strong>of</strong> Andrographis paniculata compared with common<br />
antibiotics.<br />
Sl No<br />
Name <strong>of</strong> Pathogen<br />
(Gram +ve <strong>and</strong><br />
Gram –ve)<br />
Fresh Leaf<br />
methanol extract<br />
Inhibition zone (mm)<br />
Dry Leaf<br />
methanol<br />
extract<br />
Kanamycin<br />
Ampicilin<br />
1 Bacillus subtilis 15 20 18 13<br />
2 Staphylococcus aureus 5 13 20 18<br />
3 Klebsiella pneumoniae 10 15 18 15<br />
4 Salmonella typhi 9 12 15 13<br />
5 Escherichia coli 13 16 15 16<br />
6 Enterobacter faecalis 10 17 17 15<br />
7 Pseudomonas aeruginosa 8 R NS NS<br />
R – Resistant NS – Not Significant M - Methanol W - Water (values are mean <strong>of</strong> replicates).<br />
Screening <strong>of</strong> Small Molecules<br />
The small molecular compounds which were screened by thin layer chromatography are<br />
presented in Figure 2. Figure 2A showed the compounds from the fresh methanol<br />
extract <strong>of</strong> different parts. Figure 2B represented the compounds from the fresh water<br />
extract <strong>of</strong> different parts. The small molecular compounds which are screened from the<br />
dried different parts <strong>of</strong> Andrographis paniculata are shown in figure 2C.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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A B C<br />
Figure 2: Photographs <strong>of</strong> thin layer chromatograms showing the small molecular compounds screened<br />
by thin layer chromatography. A-Fresh methanol; B-fresh water; C-dry methanol. The extracts from<br />
different parts <strong>of</strong> the plant are loaded at: 1. Main stem, 2.Branch, 3.Root, 4.Leaves, 5.Seed, 6. Buds. a,<br />
b, c, d, e, f, g, h, I, j, k, l, m, n – the compounds screened .<br />
The fresh methanol extract <strong>of</strong> different parts <strong>of</strong> Andrographis paniculata showed eight<br />
compounds. The buds <strong>and</strong> root extract showed one single compound. Two same compounds<br />
were identified in the stem <strong>and</strong> leaves extract. The branches <strong>and</strong> the seed extract showed the<br />
same compound (Table 4).<br />
Table 4: Screening <strong>of</strong> small molecules.<br />
Sl. No<br />
Extracts<br />
Plant parts<br />
Stem Branch Root Leaves Seed Buds<br />
1 Fresh methanol 2 1 1 2 1 1<br />
2 Fresh water 1 3 1 2 - -<br />
3 Dry methanol 3 2 3 4 1 1<br />
4 Dry water - - - - - -<br />
With reference to the fresh water extract, the two spots which were seen in fresh<br />
methanol leaves extract were also seen in fresh water leaves extract. Only one spot was<br />
visualized in the stem extract. Three spots were identified in the extract <strong>of</strong> branches. No<br />
significant spots were identified in the flower <strong>and</strong> seed extract.<br />
In the dry methanol extract, the leaves showed four compounds. Three compounds were<br />
visualized in the root extract. The water extract <strong>of</strong> the dried different parts did not show<br />
significant antibacterial activity <strong>and</strong> also no significant spots.<br />
The antimicrobial effect <strong>of</strong> plant extract could be due to the presence <strong>of</strong> some <strong>of</strong> these<br />
phyto constituents (Ebana et al., 2005). The secondary metabolites exert antimicrobial activity<br />
through different mechanisms. The chlor<strong>of</strong>orm, methanol <strong>and</strong> aqueous extracts <strong>of</strong> Andrographis<br />
paniculata showed antibacterial sensitivity against Staphylococcus aureus in impetigo (Rajani<br />
et al., 2000). It could be inferred from the results that the extracts <strong>of</strong> Andrographis paniculata<br />
could be used for treating skin infections. The bacteria which are used in the study are in general<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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Arunadevi et al / Antibacterial Activity <strong>of</strong> Andrographis paniculata<br />
considered as common pathogens, causing various infective ailments. Klebsiella pneuemonia is<br />
a commonest pathogen for respiratory infections, urinary tract infections, wound infections<br />
(Martin <strong>and</strong> Ernst, 2003).<br />
Thin layer chromatographic techniques were also described for the estimation <strong>of</strong><br />
<strong>and</strong>rographolide in Andrographis paniculata extracts (Schneiders et al., 2003). Three main<br />
diterpenoid lactones identified in the Andrographis paniculata leaves were <strong>and</strong>rographolide,<br />
neo<strong>and</strong>rographolide <strong>and</strong> deoxy<strong>and</strong>rographolide as compared with the results <strong>of</strong> Srivasthava<br />
et al., (2004).<br />
Conclusion<br />
Plant extracts have great potential as antimicrobial compounds. The synergistic effect from the<br />
association <strong>of</strong> antibiotic with plant extracts against resistant bacteria leads to new choices for the<br />
treatment <strong>of</strong> infectious diseases. Our study has shown that crude extract <strong>of</strong> the leaves, stem,<br />
branches <strong>and</strong> root extracts <strong>of</strong> Andrographis paniculata exhibited potentials <strong>of</strong> synergy in<br />
combinations with some antibiotics against pathogenic bacteria <strong>of</strong>ten presenting problems <strong>of</strong><br />
drug resistance. The results <strong>of</strong> the preliminary screening <strong>of</strong> small molecules <strong>of</strong> these extracts<br />
showed the presence <strong>of</strong> 29 compounds.<br />
Acknowledgements<br />
The authors are thankful to Dr. R.T. Sabapathy Mohan, Vice Chancellor, Manonmaniam<br />
Sundaranar University, Tirunelveli for providing necessary facilities <strong>and</strong> encouragement.<br />
Reference<br />
Aiyegoro, O. A., Afolayan, A. J. <strong>and</strong> Okoh, A. I. 2009. Invitro antibacterial activities <strong>of</strong> crude extracts <strong>of</strong><br />
the leaves <strong>of</strong> Helichrysum longifolium in combination with selected antibiotics. African <strong>Journal</strong> <strong>of</strong><br />
Pharmacy <strong>and</strong> Pharmacology, 3(6): 293-300.<br />
Coon, J.T., <strong>and</strong> Ernest, E. 2004. Andrographis paniculata in the treatment <strong>of</strong> upper respiratory tract<br />
infections: A systematic review <strong>of</strong> safety <strong>and</strong> efficacy. Planta. Med., 70: 293-298.<br />
Ebana, R.U.B., Madunagu, B.E., <strong>and</strong> Ekpe, E.D. 2005. Microbiological exploitation <strong>of</strong> cardiac glycosides<br />
<strong>and</strong> alkaloids from Garcinia kola,Borreli ocymoides,Kola nitida, Citrus aurantifolia. J. Appl.<br />
Bacterial., 71: 398-401.<br />
Hugo, W.B., <strong>and</strong> Russell, A.D. 2003. Pharmaceutical Microbiology; 6 th Edn. Blackwell Science<br />
Publishers, Oxford, United Kingdom. pp.91-129.<br />
Martin.K.,W., <strong>and</strong> Ernst, E. 2003. Herbal medicine for the treatment <strong>of</strong> bacterial infections-A review <strong>of</strong><br />
controlled clinical traits. J. Antimicrobial chemotherapy, 51: 241-246.<br />
Prescott, H., <strong>and</strong> Klein, J.O. 2002. Microbiology 6th ed. Macgraw Hill Publishers, USA. pp.808-823.<br />
Rajani.M., Shrivastava, N., <strong>and</strong> Ravishankara, M. N. 2000. A rapid method for isolation <strong>of</strong><br />
Andrographolide from Andrographis paniculata Nees (Kalmegh). Pharmaceut. Biol., 38: 204-209.<br />
Schneiders, T., Amyes, G. B., <strong>and</strong> Levy. 2003. Role <strong>of</strong> Acr. R <strong>and</strong> Rams in fluproguinolone resistance<br />
in clinical Klebsiella pneumonia isolates from Singapore. Antimicrobial agents <strong>and</strong> chemotherapy,<br />
47(a): 2831- 2837.<br />
Srivasthava, A., Misra, H., <strong>and</strong> Verma, R.K., <strong>and</strong> Gupta, M.M. 2004. Chemical finger printing <strong>of</strong><br />
Andrographis paniculata using HPLC, HPTLC <strong>and</strong> densitometry. Phytochem. Anal., 15: 280-285.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 227-233, 2010<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
© 2010 Elias Academic Publishers<br />
www.ejteb.org<br />
Studies on Morphometrical Relationships <strong>and</strong> Growth in<br />
Uca annulipes Milne Edwards<br />
I. Jayakumari<br />
Department <strong>of</strong> Zoology, Sree Ayyappa College for Women, Chunkankadai-629807, Tamil Nadu, India.<br />
Received: 10 July, 2009; revised received: 15 January, 2010.<br />
Abstract<br />
The morphometrical relationship between total length <strong>and</strong> weight <strong>and</strong> total length <strong>and</strong><br />
carapace length were investigated in detail to estimate the growth pattern in Uca<br />
annulipes.The‘t’ test values for length- weight in males <strong>and</strong> females were 1.62916 (P ><br />
0.05) <strong>and</strong> 3.80732( P < 0.05) respectively. This indicates that males exhibit isometric<br />
growth while the females depart significantly from the isometric growth pattern. The<br />
relationship between total length <strong>and</strong> carapace length was linear in both sexes with a high<br />
degree <strong>of</strong> correlation (males r = 0.97 <strong>and</strong> females r = 0.93)<br />
Key words: Length-weight, regression coefficient, isometric growth.<br />
Introduction<br />
The data on length - weight is a prime requisite for estimating growth rates, age structure <strong>and</strong><br />
for assessing other aspects <strong>of</strong> fish population such as the relative well being <strong>of</strong> the population,<br />
estimating the st<strong>and</strong>ing stock biomass etc (Petrakis <strong>and</strong> Stergiou,1995).The relationship between<br />
length <strong>and</strong> weight differs from species to species based on their body shape <strong>and</strong> within a species<br />
according to the condition or robustness <strong>of</strong> the species based on the availability <strong>of</strong> food <strong>and</strong><br />
environmental factors essential for growth .Length - weight studies also provides a<br />
mathematical relationship between the two parameters since, length is a linear measure <strong>and</strong><br />
weight a measure <strong>of</strong> volume. The general belief is that the weight <strong>of</strong> an individual species vary<br />
with the cube <strong>of</strong> its length (Brown, 1957 <strong>and</strong> Lagler, 1968).Hence theoretically it is expressed<br />
by the cube law W=CL 3 where W=weight <strong>of</strong> fish, L = length <strong>of</strong> fish <strong>and</strong> C= a constant. This<br />
formula can be applied to fin <strong>and</strong> shell fishes which exhibit isometric growth throughout the life<br />
span. In nature, the body proportion continuously change with ageing, so the cube law cannot be<br />
applied as the value <strong>of</strong> C is not constant but subject to great variation. Therefore a modified<br />
equation was suggested by Le Cren (1951) where W=aL n or Log W =n Log L + Log a where W<br />
=weight <strong>of</strong> fish, L = length <strong>of</strong> fish <strong>and</strong> ‘a’ <strong>and</strong> ‘n’ are constants. The constants ‘a’ <strong>and</strong> ‘n’ can be<br />
estimated empirically from the data on length-weight.<br />
The present paper provides a mathematical relationship between length - weight <strong>and</strong><br />
length - length parameters in Uca annulipes inhabiting the intertidal s<strong>and</strong>y shore <strong>of</strong> the coastal<br />
waters <strong>of</strong> Neendakara in Kerala. Such a study is useful for comparing the population <strong>of</strong> the<br />
species in space <strong>and</strong> time for the proper conservation <strong>and</strong> management <strong>of</strong> the species.<br />
Materials <strong>and</strong> Methods<br />
341 crabs were collected from the coast <strong>of</strong> Neendakara for a period <strong>of</strong> one year <strong>of</strong> which 270<br />
were males <strong>and</strong> 71 were females. Morphometric measurements such as total length, carapace<br />
length <strong>and</strong> weight <strong>of</strong> each specimen were recorded separately for the sexes. Analysis <strong>of</strong><br />
covariance was used to study whether there is any significant variation between the sexes as the<br />
‘b’ value may vary among the sexes (Snedecor <strong>and</strong> Cochran, 1975).<br />
*Corresponding author; Email address: jayakumari_i@yahoo.co.in<br />
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Jayakumari / Morphometrical Relationships <strong>and</strong> Growth in Uca annulipes<br />
For an ideal fish which maintains constant shape ‘n’ will be equal to 3.0 (Allen, 1938).<br />
According to Hile, 1936 <strong>and</strong> Martin (1949) the value <strong>of</strong> ‘n’ lies between 2.5 <strong>and</strong> 4.0.So the<br />
regression coefficient was analysed using‘t’ test (Bailey, 1959) to find out if there is any<br />
departure from the isometric growth value <strong>of</strong> 3.0 proposed by Allen, 1938.The relationship<br />
between total length <strong>and</strong> carapace length were also determined according to the linear<br />
regression model.<br />
Results<br />
Size Composition <strong>and</strong> Sex Ratio<br />
The total length <strong>of</strong> Uca annulipes obtained for the present study ranged from 9 to 25 cm with a<br />
mean <strong>of</strong> 19.21 ± 2.72 <strong>and</strong> the weight varied from 0.120 to 4.220 gm with the mean value <strong>of</strong><br />
1.872 ± 0.889.The minimum length observed in males was 12cm <strong>and</strong> maximum 25cm with a<br />
mean <strong>of</strong> 19.8 ± 2. The size range <strong>of</strong> females varied from 9cm to 24cm with a mean value <strong>of</strong><br />
16.9 ± 4.The average weight <strong>of</strong> a male crab was 2.118 ± 0.802 while the female weighed 0.948<br />
± 0.575.<br />
The sex ratio <strong>of</strong> males to females was 1:0.26.The males out numbered the females<br />
throughout the year.<br />
Relationship between Total Length <strong>and</strong> Weight<br />
Analysis <strong>of</strong> variance for length - weight relations in the population <strong>and</strong> sexes <strong>of</strong> uca annulipes<br />
are present in Tables 1, 2 <strong>and</strong> 3.<br />
Table 1: Analysis <strong>of</strong> variance for length-weight relationship in<br />
male Uca annulipes.<br />
Anova df SS MS F value<br />
Regression<br />
1<br />
4.976279<br />
4.976279<br />
256.8931**<br />
Residual<br />
268<br />
5.191429<br />
0.019371<br />
Total<br />
269<br />
10.167708<br />
**Significant (P < 0.01)<br />
Table 2: Analysis <strong>of</strong> variance for length-weight relationship in female Uca annulipes.<br />
Anova df SS MS F value<br />
Regression<br />
Residual<br />
Total<br />
1<br />
69<br />
70<br />
5.321642<br />
0.431712<br />
5.753354<br />
5.321642<br />
0.006257<br />
850.551**<br />
* * Significant (P < 0.01)<br />
Table 3: Estimated values <strong>of</strong> regression (b) <strong>and</strong> correlation coefficient (r) for length-weight relationship<br />
in Uca annulipes<br />
Sex b value r value r 2 value<br />
Total population 3.1374 0.84909 0.7210<br />
Males 2.72431 0.69959 0.4894<br />
Females 2.65358 0.96175 0.9249<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
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Jayakumari / Morphometrical Relationships <strong>and</strong> Growth in Uca annulipes<br />
The relation between total length <strong>and</strong> weight in the population is expressed as Log W =<br />
3.1374 Log TL - 0.860 <strong>and</strong> r = 0.849 .The F value is significant at 1% level. The percentage <strong>of</strong><br />
fitness to the trend line is 72.10% (Fig1). Since the value <strong>of</strong> ‘b’ was greater than 3.0 in the total<br />
population the length - weight relationship was calculated separately for each sex. The ‘b’ value<br />
varied between the sexes <strong>and</strong> is expressed as follows:<br />
Log weight<br />
3.8<br />
LogW= 3.1374LogTL - 0.860<br />
3.6<br />
3.4<br />
3.2<br />
3<br />
2.8<br />
2.6<br />
2.4<br />
2.2<br />
2<br />
0.9 1 1.1 1.2 1.3 1.4 1.5<br />
Log total length<br />
Figure 1: Length weight relationship in the total population <strong>of</strong> uca annulipes.<br />
Males: Log W = 2.7243 Log TL - 0.2413 <strong>and</strong> r = 0.6996 .The F value is significant at 1% level.<br />
The percentage <strong>of</strong> fit is only 48.94 % (Fig. 2).<br />
3.7<br />
3.5<br />
Log W = 2.7243 Log TL - 0.2413<br />
3.3<br />
Log weight<br />
3.1<br />
2.9<br />
2.7<br />
2.5<br />
1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45<br />
Log total length<br />
Figure2: Length weight relationship in male Uca annulipes.<br />
Females: Log W =2.6536 Log TL - 0.3621 <strong>and</strong> r =0.9618 .The F value is significant at 1% level.<br />
The percentage <strong>of</strong> fit is 92.49 % (Fig: 3). It is closer to the trend line.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
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Jayakumari / Morphometrical Relationships <strong>and</strong> Growth in Uca annulipes<br />
3.6<br />
3.4<br />
Log W = 2.6536 LogTL - 0.3621<br />
Log weight<br />
3.2<br />
3<br />
2.8<br />
2.6<br />
2.4<br />
2.2<br />
2<br />
0.9 1 1.1 1.2 1.3 1.4 1.5<br />
Log total length<br />
Figure 3: Length weight relationship in female Uca annulipes.<br />
Since the value <strong>of</strong> ‘b’varied in the population <strong>and</strong> in the sexes (3.1374, 2.7243(males) <strong>and</strong><br />
2.6536(females) respectively) the‘t’ test was conducted <strong>and</strong> the results are as follows:<br />
Population: t = 1.29579 (n = 3.1374 <strong>and</strong> S b = 0.106014) P >0.05.<br />
Males : t = 1.62916 (n = 2.7243, S b = 0.169973) P > 0.05.<br />
Females : t = 3.80732 (n =2.6536, S b = 0.909877) P < 0.05.<br />
Relationship between Total Length <strong>and</strong> Carapace Length<br />
The regression analysis for total length <strong>of</strong> the body <strong>and</strong> carapace length varied between the<br />
sexes.<br />
Males: Log CL = 1.0552 LogTL - 0.3638.The coefficient <strong>of</strong> correlation r is 0.9691 <strong>and</strong> the F<br />
value is significant at 1 % Level .The percentage <strong>of</strong> fitness to the trend line is 93.92 % (Fig 4).<br />
Log carapace length<br />
1.2<br />
1.15<br />
1.1<br />
1.05<br />
1<br />
0.95<br />
0.9<br />
0.85<br />
0.8<br />
0.75<br />
0.7<br />
Log CL=LogTL 1.0552 - 0.3638<br />
1 1.1 1.2 1.3 1.4 1.5<br />
Log total length<br />
Figure 4: Relationship between total length <strong>and</strong> carapace length in male Uca annulipes.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
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Jayakumari / Morphometrical Relationships <strong>and</strong> Growth in Uca annulipes<br />
1.2<br />
1.1<br />
LogCL = Log TL1.0343 - 0.3403<br />
Log carapace length<br />
1<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.9 1 1.1 1.2 1.3 1.4 1.5<br />
Log total length<br />
Figure 5: Relationship between total length <strong>and</strong> carapace length in female Uca annulipes.<br />
Females: Log CL =1.0343 TL - 0.3403<strong>and</strong> the r value is 0.9289 .The F value is significant at 1% level<br />
.The percentage <strong>of</strong> fitness to the trend line is only 86.29% (Fig 5).<br />
Discussion<br />
Growth is a progressive increase in mass which is restricted to the short period <strong>of</strong> intermoult in<br />
the shelled fishes like the prawns <strong>and</strong> crabs. In Uca annulipes, it is evident from the data on<br />
length-weight that the body weight <strong>of</strong> both males <strong>and</strong> females increases with the total length<br />
exhibiting a linear relationship.<br />
The regression coefficient ‘b’ is an indicator in length-weight relationship to estimate<br />
the growth <strong>and</strong> find out if there is any deviation from isometric growth in the population <strong>and</strong><br />
sexes. In the total population <strong>of</strong> Uca annulipes the ‘b’ value exhibited a slight increase <strong>of</strong><br />
3.1374 against the isometric value <strong>of</strong> 3.0.The regression coefficient ‘b’ was lesser than the<br />
isometric growth value in both the sexes.. It was greater in males (2.7243) than in the females<br />
(2.6536). Similar results were observed for Scylla serrata by Lalitha, 1985 (males 2.71832 <strong>and</strong><br />
females 2.6589) <strong>and</strong> Jayakumari, 2006 for Ocypode platytarsis (males 2.9365 <strong>and</strong> females<br />
2.6641).<br />
The‘t’ test values for the total population <strong>and</strong> for the male sex <strong>of</strong> Uca annulipes was<br />
insignificant while in the females it was significant at 5%level .This indicates that growth<br />
departs significantly from isometry in the females <strong>of</strong> Uca annulipes while males exhibit<br />
isometric growth (Jayach<strong>and</strong>ran, 1984). This difference in the growth pattern <strong>of</strong> males <strong>and</strong><br />
females may be due to various factors such as temperature, food, size, sex, time <strong>of</strong> year <strong>and</strong><br />
stage <strong>of</strong> maturity (Hasan <strong>and</strong> Selcuk, 2003).<br />
The length-length relationship is also <strong>of</strong> prime importance for comparative growth<br />
studies. The relationship between total length <strong>and</strong> carapace length was linear in both sexes with<br />
a high degree <strong>of</strong> correlation (males 0.97 <strong>and</strong> females 0.93) .The regression coefficient ‘b’ was<br />
less than 3.0 in both the sexes (males 1.0552 <strong>and</strong> females 1.0343) indicating that carapace<br />
length increases proportionately with gradual increase in total length (Vijayakumar et al,<br />
2000).The percentage <strong>of</strong> fitness to the best fit line was higher in the males (93.92%) compared<br />
to the females (86.29%).<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
239
Jayakumari / Morphometrical Relationships <strong>and</strong> Growth in Uca annulipes<br />
References<br />
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Ecol., 7: 33-49.<br />
Bailey, H. T. J.1959 . Statistical methods in <strong>Biology</strong> (The English University Press). pp.200.<br />
Brown, M.E.1957. <strong>Experimental</strong> studies on growth. In: The Physiology <strong>of</strong> Fishes, Vol-1. Edited by:<br />
H.E.Brown. Academic Press,Newyork. pp. 361-400.<br />
Hasan Huseyin Atar <strong>and</strong> Selcuk Secer.2003.Width / Length–weight Relationships <strong>of</strong> the blue crab<br />
(Callinectes sapidus Rathbun, 1896). Population living in Beymelek Lagoon Lake.Turk<br />
J.Vet.Anim.Sci., 27:443-447.<br />
Hile, R.1936.Age <strong>and</strong> growth <strong>of</strong> Cisco Leucichthys astedi (Lesuer) in the lakes <strong>of</strong> the north-eastern<br />
highl<strong>and</strong>s,Wisconscin.Bull.U.S.Bur. Fish, 48:311-317.<br />
Jayach<strong>and</strong>ran, K.V.1984.Studies on the breeding biology <strong>of</strong> the Palaemonid prawns <strong>of</strong> the South west<br />
coast <strong>of</strong> India. Ph.D Thesis.University <strong>of</strong> Kerala, India.<br />
Jayakumari, I.2006. Length –weight relationship <strong>of</strong> Palaemon (Palaemon) concinnus, Macrobrachium<br />
latimanus <strong>and</strong> Ocypode platytarsis .Proc .Zoo. Soc.India, 5(2):1-10.<br />
Lagler, K.F. 1968.Capture, Sampling <strong>and</strong> Examination <strong>of</strong> Fishes W.Ricker (Ed).Methods for assessment<br />
<strong>of</strong> fish production in Fresh waters.IBP H<strong>and</strong>book 111:7-45.<br />
Lalitha Devi, S.1985.The fishery <strong>and</strong> biology <strong>of</strong> the crabs <strong>of</strong> the Kakinada region.Indian J. Fish., 18-32.<br />
Le Cren, E.D.1951.The length-weight relationship <strong>and</strong> seasonal cycle in gonad weight <strong>and</strong> condition in<br />
the Perch, Perca fluviatilis.J.Anim.Ecol., 20(2):201-219.<br />
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Petrakis,G., <strong>and</strong> StergiouK.I.1995.Weight–length relationship for 33 fish species in Greek<br />
waters.Fish.Res.21:465-469.<br />
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Vijayakumar,R., Das,S., Chatterji, A., <strong>and</strong> Parukkar, A.H.2000. Morphometric characteristics in the<br />
Horse shoe crab Tachypleus gigas .Indian .J. mar. Sci., 29:333-335.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Theoretical</strong> <strong>and</strong> <strong>Experimental</strong> <strong>Biology</strong> (ISSN: 0972-9720), 6 (3 <strong>and</strong> 4): 235-240, 2010<br />
240
<strong>Journal</strong> <strong>of</strong><br />
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<strong>Volume</strong> 6 Numbers 3 <strong>and</strong> 4 February <strong>and</strong> May 2010<br />
Contents<br />
Hypoglycaemic Activity <strong>and</strong> Modulatory Effect on Glucose Metabolism by<br />
Artificially Cultivated Ganoderma lucidum in Streptozotocin Induced<br />
Diabetic Rats<br />
A. Usha Raja Nanthini, M. Rajasekara P<strong>and</strong>ian <strong>and</strong> G.Kavitha<br />
Influence <strong>of</strong> Hormone Induced Spawning in Etroplus suratensis<br />
S. Albin Dhas, M. Michael Babu, T. Selvaraj, T. Citarasu, V. A. J. Huxley <strong>and</strong><br />
S. Mary Josephine Punitha<br />
Decolorization <strong>of</strong> Textile Dye Reactive Black HFGR Using a Novel Isolate<br />
Paenibacillus lautus SK21<br />
S. Senthil Kumar, M. S. Mohamed Jaabir, A. Veeramani <strong>and</strong> R. Ravikumar<br />
Field Study for the Management <strong>of</strong> Rice Blast with Minimum Fungicides<br />
P. Krishnan<br />
Anamorphs <strong>of</strong> Asterinales<br />
V. B. Hosagoudar<br />
167-176<br />
177-183<br />
185-192<br />
193-198<br />
199-211<br />
Plant Antioxidants Mediated Protein Alterations in Clarias batrachus Linn.<br />
C. Suseela Bai<br />
213-215<br />
Quercitrin, a Bi<strong>of</strong>lavonoid Increases Glucose Utilization <strong>and</strong> Altering<br />
The Carbohydrate Metobolic Enzymes in Streptozotocin-Induced Diabetic<br />
Rat Tissues<br />
Ranganathan Babujanarthanam, Purushothaman Kavitha, Sarika Sasi <strong>and</strong><br />
Moses Rajasekara P<strong>and</strong>ian<br />
Assessment <strong>of</strong> Antibacterial Activity <strong>and</strong> Detection <strong>of</strong><br />
Small Molecules in Different Parts <strong>of</strong> Andrographis paniculata<br />
R.Arunadevi, S. Sudhakar <strong>and</strong> A.P. Lipton<br />
Studies on Morphometrical Relationships <strong>and</strong> Growth in<br />
Uca annulipes Milne Edwards<br />
I. Jayakumari<br />
217-226<br />
227-233<br />
235-340<br />
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