Studies on the Degradation of Prosopis Juliflora (Sw.) D.C

Studies on the degradation of Prosopis juliflora (Sw.) D.C. by marine cyanobacterium (Oscillatoria laetevirens) and its assessment of bioactive property on animal model

Thesis submitted for the award of the degree of

DOCTOR OF PHILOSOPHY IN MICROBIOLOGY

By D. S. PRABHA (10891/Ph.D-1/Micro/FT/July.2006/dt.27/09/06)

DEPARTMENT OF MARINE BIOTECHNOLOGY NATIONAL FACILITY FOR MARINE CYANOBACTERIA (Sponsored by DBT, Govt. of India) BHARATHIDASAN UNIVERSITY TIRUCHIRAPALLI, TAMILNADU, INDIA.

JANUARY 2011 BHARATHIDASAN UNIVERSITY, TIRUCHIRAPPALLI – 620 024. TAMIL NADU, INDIA.

Dr. P. MALLIGA Course Director & Convener Associate Professor Model Organic Farm National Facility for Marine Cyanobacteria (Sponsored by Ministry of Agriculture, Department of Marine Biotechnology Government of India)

CERTIFICATE

I have pleasure in forwarding this doctoral thesis entitled “Studies on the

degradation of Prosopis juliflora (Sw.) D.C. by marine cyanobacterium (Oscillatoria

laetevirens) and its assessment of bioactive property on animal model” submitted by

Ms. D. S. PRABHA for the award of the degree of Doctor of Philosophy in

Microbiology, Bharathidasan University, Tiruchirapalli, India.

Ms. D. S. PRABHA (10891/Ph.D-1/Micro/FT/July.2006/dt.27/09/06) has

completed the research work under my supervision for the full period prescribed in the

ordinance of this University for the said degree. The work was entirely carried out by

the candidate and any part of this thesis work has not been submitted elsewhere for the

award of any other degree, diploma or associateship of any University.

(P. MALLIGA) Research Advisor

Phone / Fax : 0431 – 2407084 (O) 0431 – 2418591 (R) 94432-08345, 9487549118 Email : [email protected]

DECLARATION

I hereby declare that this thesis entitled “Studies on the degradation of

Prosopis juliflora (Sw.) D.C. by marine cyanobacterium (Oscillatoria laetevirens) and its assessment of bioactive property on animal model” has been originally carried out by me under the guidance and supervision of Dr. P. MALLIGA, Associate

Professor, Dept. of Marine Biotechnology, National Facility for Marine Cyanobacteria,

Bharathidasan University, Tiruchirapalli, Tamilnadu, India and any part of this thesis work has not been submitted elsewhere for the award of any other degree, diploma or associateship of any Indian or foreign University.

Tiruchirappalli

January 2011 (D. S. PRABHA)

ACKNOWLEDGEMENT

It is my immense pleasure to thank Dr. P. Malliga, Associate professor, Department of Marine Biotechnology, National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirapalli, for her valuable guidance, constant supervision of this thesis and scientific encouragement. The integrity and sincerity to work which she inculcated me will take me a long way in my life.

I am profoundly indebted to Dr. G. Subramanian, Founder Director of NFMC for establishing the excellent facility of our department.

I would like to extend my special thanks to Dr. L. Uma, Director, Department of Marine Biotechnology, National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirapalli for her constant encouragement and support.

I thank Dr. N. Jayabalan, Professor and Head, Dept. of Plant Sciences, Bharathidasan University, Tiruchirapalli. As a doctoral committee member, he helped me with his valuable advice for my queries. His precious suggestions helped me to approach research problems in different ways.

I am very grateful to all the faculty members of this department Dr. D. Prabakaran, Dr. M. Sundararaman and Dr. K. Anbarasu for the dissemination of knowledge and their best wishes.

My cordial thanks to Dr. G. Archunan, Associate Professor, Dept. of Animal Science, Dr. G. Muralitharan, Assistant Professor, Department of Microbiology, Dr. K. Kala, Director, Student Counselling and Grievance Redress Cell, Bharathidasan University for their kind support and suggestions for compiling my thesis.

I am thankful to all research scholars of my department and my juniors Mr. S. Durairaju, Mr. V. Subramaniyan, Mr. S. Krishnamoorthy, Mr. G. Manoharan and Ms. K. Chitra for their support during my course work. I also extend my thanks to Mrs. P. Muthulakshmi and Mrs. P. Susila, Field workers, Model organic farm for the timely help provided to me during my project work.

I must express my sincere thanks to my best friends Mr. T. Boopathi, Ms. S. Sathia, Mr. S. Gopinath, Ms. M. Razia and Ms. R. Menaka for their constant encouragement, support, gracious and tireless enthusiasm at every stage of my work which cannot be expressed in words.

I owe special thanks to Mr. T. Rajavel, Lecturer, Nandha College of Pharmacy, Erode and Research Scholars Mr. N. Mahesh kumar, Mr. P. Ponmanickam, Mr. K. Karthikeyan, Mr. A. Amarnath, S. Muniasamy, Mr. G. Bhupesh, Mr. E. Prabakaran and Mr. G. Venkateshwaran for their timely help.

Words are insufficient to express my gratefulness to my uncles Mr. V. Rajamanickam, Mr. T. Paridevan, Mr. K. Ammasi, Mr. T. Ulagadevan, Mr. T. Saravanan and Mr. T. Ramar for their constant support and encouragement.

I express my heartfelt love, thanks and sense of gratitude to my beloved grandparents Mr. P. Chithamparam, Mrs. C. Anjalai, Mr. M. Thangaraju, Mrs. T. Savithiri and my parents Mr. C. Sethuraman, Mrs. S. Devaki and other family members for their support, love and affection.

I praise almighty who has bestowed me the courage and strength to achieve this target. Last but not least I want to thank all those who have helped me directly or indirectly to make this work a success but missed a mention.

D. S. PRABHA Page CONTENTS No. A. LIST OF TABLES ……………………………………………………. I B. LIST OF FIGURES ………………………………………………...... I C. LIST OF PLATES ……………………………………………………. V D. ABBREVIATIONS …………………………………………………… VI

1. INTRODUCTION 1.1 Lignocellulosic waste …………………………………………………….. 1 1.2 Prosopis juliflora …………………….…………………………………….. 2 1.3 Cyanobacteria ………………………..…………………………………… 3 1.4 Biodegradation ……………………….…………………………………. 3 1.5 Phenolic compounds ……………………..………………………………… 4 Objectives …………………………………………………………………… 5 Work Flow ……………………………………………………………………. 6

2. REVIEW OF LITERATURE 2.1 Lignocellulose-a valuable resources……………………………… 7 2.1.1 Structural feature of lignocellulose …………………...... 8 2.1.1a Cellulose………………………………………………... 8

2.1.1b Hemicellulose ……………………………………...... 9

2.1.1c Lignin …………………………………………………... 10

2.2 Lignocellulosic waste-Prosopis juliflora …………………………….. 12

2.2.1 Botanical description ………………………………………….. 14 2.2.2 Chemical composition ………………………………………. 15 2.2.3 Natural durability ……..……………………………………..... 16 2.2.4 Prosopis in traditional medicine …………………………….... 17 2.2.5 Toxicity of Prosopis sp. ………………………………………. 18 2.3 Lignin Degradation …………………………………………………… 20

2.3.1 Microbiology of lignin degradation ………………………… 21

2.3.1a Bacteria ………………………………………………… 21

2.3.1b Cyanobacteria ...... 23

2.3.1c Fungi ...... 25 2.3.2 Lignin degrading enzymes ……………………………………. 27 2.3.2a Laccase …………………………………………………... 27 2.3.2b Polyphenol oxidase ……………………………………… 28 2.3.2c Manganese independent peroxidase …………………… 28 2.3.2d Other enzymes …………………………………………… 29 2.4 Cyanobacteria – potential for applied purposes …………………………… 29 2.4.1 Biotechnological potential …………………………………….. 31 2.4.2 Bioactive compounds from cyanobacteria…………………….. 32 2.4.3 Pharmacological potential …………………………………… 33 2.5 Bioactive compounds tested with animal models…………………….... 35 2.5.1 Phenolic compounds ………………………………………… 36 2.5.2 Metabolites from plant sources ...... 38 2.5.3 Antioxidant properties of bioactive compounds ...... 38

3. MATERIALS AND METHODS 3.1 Growth of Cyanobacteria ………………………………………………………..... 42 3.1.1 Cultures used ………………………………………………… 42

3.1.2 Media and growth conditions …………………………………. 42

3.1.3 Lignocellulosic material ………………………………………. 44

3.1.4 Screening of cyanobacteria …………………………………… 44 3.2 Degradation of Prosopis juliflora by O. laetevirens ………………………….. 44 3.2.1 Optimization of P. juliflora wood particle size and dry weight ratio………...... 44 3.2.2 Biochemical estimations of degraded P. juliflora …………… 45 3.2.2a Chlorophyll a …………………………………………… 45

3.2.2b Reducing sugar ………………………………………… 46

3.2.2c Phenol …………………………………………………. 46

3.2.2d Lignin …………………………………………………. 47 3.2.3 Biodegradation with using optimized conditions…………… 48 3.2.4 Microscopic observation of P. juliflora degradation………. 48 3.2.5 Estimation of growth parameters ………………………….. 48

3.2.5a Chlorophyll a ………………………………………… 48

3.2.6 Colorimetric enzyme assay ………………………………….. 48 3.2.6a Laccase ………………………………………………… 49 3.2.6b Polyphenol oxidase …………………………………… 49 3.2.6c Manganese independent peroxidase …………………… 49

3.2.6d Hydrogen peroxide …………………………………… 50

3.2.7 Analysis of biochemical parameters ...... 50

3.2.7a Reducing sugar ...... 50 3.2.7b Phenol ...... 50 3.2.7c Spectrum analysis ...... 51

3.2.7d Nitrate ...... 51

3.2.7e Ammonia ...... 52 3.2.7f Protein ...... 53 3.2.7g Lignin ...... 54 3.2.7h Holocellulose ...... 55

3.2.8 Phytochemical analysis ...... 56

3.2.8a Alkaloids ...... 56

3.2.8b Flavonoids ...... 56 3.2.8c Terpenoids ...... 56 3.2.8d Saponin ...... 56 3.2.8e Steroids ...... 56

3.3. Compound Identification ...... 56

56 3.3.1 Preparation of extract ...... 3.3.1a Spectrum analysis ...... 57 3.3.1b Thin Layer Chromatography (TLC)...... 57 3.3.1c High Performance Thin Layer Chromatography (HPTLC) ...... 59

3.3.1d High Performance Liquid Chromatography (HPLC) ..... 60

3.3.1e Gas Chromatography Mass Spectroscopy (GC-MS) ...... 61

3.3.1f Fourier Transform Infrared Spectroscopy (FTIR) ……. 62 3.3.1g Nuclear Magnetic Resonance Spectroscopy (NMR) …. 63 3.4. Pharmacological Studies...... 63 3.4.1 Acute toxicity study...... 63

3.4.2 Subacute toxicity study...... 64

65 3.4.3 Morphological observation ………………………………. 3.4.3a Body weight ………………………………………… 65 3.4.4 Haematological and biochemical analysis by auto 65 analyzer…………………………...... 3 4.5 Sperm count …………………………………………… 66 3.4.6 Histopathology ………………………………………….. 67 3.4.6a Kidney …………………………………………….. 67 3.4.6b Liver ………………………………………………. 69 3.4.7 Antioxidant properties ……………………………………. 69

4. RESULTS AND DISCUSSION 4.1. Growth and Degradation ………………………………………………………….... 71 4.1.1 Screening of cyanobacteria ……………………………… 71 4.1.2 Optimization of growth conditions ……………………… 71 4.1.2a Biochemical analysis ……………………………… 72

4.1.3 Biodegradation of optimized P. juliflora …………………. 75

4.1.3a Microscopic view of degraded P. juliflora …………. 75

4.1.3b Growth parameters ………………………………….. 76

4.1.3c Enzyme assay ……………………………………… 77 4.1.3d Biochemical estimations ……………………………. 79 4.1.3e Phytochemical analysis …………………………… 83 4.2. Compound Identification ………………………………………………………...... 84 4.2.1 Spectrum analysis …………………………………………. 84

4.2.2 Thin Layer Chromatography (TLC)……………………… 84

4.2.3 High Performance Thin Layer Chromatography 85

(HPTLC)…………………………………………………...

4.2.4 High Performance Liquid Chromatography (HPLC)……… 85

4.2.5 Gas Chromatography Mass Spectroscopy (GC-MS)……… 86 4.2.6 Fourier Transform Infrared Spectroscopy (FTIR)………… 86 4.2.7 Nuclear Magnetic Resonance Spectroscopy (NMR)……… 87 4.3. Pharmacological Studies…………………………………………………………………. 88 4.3.1 Experimental animal-Rattus norvegicus ………………… 88

4.3.1a Mode of drug administration...... 88

4.3.2 Acute toxicity study ………………………………………. 89

4.3.3 Subacute toxicity study…………………………………… 89

4.3.3a Morphological observation -Body weight …………. 89 4.3.4 Haematology ……………………………………………… 90 4.3.4a Haemoglobin ……………………………………….. 90 4.3.4b Erythrocyte Sedimentation Rate (ESR) ……………. 92 4.3.4c Packed Cell Volume (PCV)………………………… 93 4.3.4d Red Blood Cells (RBC)…………………………… 94

4.3.4e White Blood Cells (WBC)………………………… 94

4.3.4f Neutrophil ………………………………………… 96

4.3.4g Lymphocyte ……………………………………… 96

4.3.4h Eosinophil ………………………………………… 97 4.3.5 Biochemical studies ……………………………………….. 98 4.3.5a Protein …………………………………………… 98

4.3.5b Glucose ………………………………………… 99 4.3.5c Albumin and Globulin ………………………… 100 4.3.6 Lipid analysis……………………………………………… 101 4.3.6a Cholesterol ……………………………………... 101 4.3.6b Triglycerides (TGL)…………………………….. 103 4.3.7 Renal function test ……………………………………… 104 4.3.7a Urea …………………………………………… 104 4.3.7b Uric acid ……………………………………… 105 4.3.7c Creatinine ……………………………………..... 106 4.3.8 Liver function test ………………………………………… 107 4.3.8a Serum Glutamic Pyruvic Transaminase (SGPT) …… 107 4.3.8b Serum Glutamic Oxaloacetic Transaminase (SGOT).. 108 4.3.8c Total and Indirect bilirubin……………………... 109 4.3.8d Alkaline Phosphatase (ALP)…………………… 110 4.3.9 Sperm count ……………………………………………….. 111 4.3.10 Histopathology ………………………………………….. 112 4.3.10a Kidney ………………………………...... 112 4.3.10b Liver ……………………………………………… 113 4.3.11 Antioxidant properties …………………………………… 114

5. STATISTICAL ANALYSIS [ANOVA] ………………………………. 116

6. SUMMARY AND CONCLUSION …………………………………… 119

7. BIBLIOGRAPHY ……………………………………………………… i

LIST OF TABLES

Table 1. Growth of marine cyanobacterial species with Prosopis juliflora. Table 2. Qualitative analysis of the phytochemicals present in the ethanolic extract of degraded P. juliflora by O. laetevirens (30th day sample). Table 3. Standard chemicals matching with compounds separated from O. laetevirens treated P. juliflora sample. Table 4. High Performance Thin Layer Chromatography (HPTLC) data of P. juliflora and O. laetevirens treated P. juliflora. Table 5. Acute toxicity study of ethanolic extract of P. juliflora.

LIST OF FIGURES

Fig. 1. Structure of lignocellulose. Fig. 2. Structure of cellulose. Fig. 3. Structure of hemicellulose. Fig. 4. Structure of lignin precursors. Fig. 5. Structure of lignin. Fig. 6. Approximate dispersal and present distribution of the genus Prosopis originated from America. Fig. 7. Plant secondary metabolites. Fig. 8. Estimation of chlorophyll a in Oscillatoria laetevirens treated with different particle size and ratio of Prosopis juliflora on 15th day. Fig. 9. Estimation of sugar during degradation of P. juliflora by O. laetevirens on 15th day. Fig. 10. Estimation of phenol during degradation of P. juliflora by O. laetevirens on 15th day. Fig. 11. Estimation of lignin in degraded P. juliflora by O. laetevirens on 15th day. Fig. 12. Estimation of chlorophyll a in O. laetevirens treated P. juliflora in different days. Fig. 13. Effect of temperature on laccase activity of O. laetevirens treated P. juliflora on 30th day.

Fig. 14. Effect of pH on laccase activity of O. laetevirens treated P. juliflora on 30th day. Fig. 15. Effect of temperature on polyphenol oxidase activity of O. laetevirens treated P. juliflora on 30th day. Fig. 16. Effect of pH on polyphenol oxidase activity of O. laetevirens treated P. juliflora on 30th day. Fig. 17. Effect of temperature on manganese independent peroxidase activity of O. laetevirens treated P. juliflora on 30th day. Fig. 18. Effect of pH on manganese independent peroxidase activity of O. laetevirens treated P. juliflora on 30th day.

Fig. 19. Quantitative analysis of hydrogen peroxide (H2O2) production by O. laetevirens when treated with P. juliflora (30th day). Fig. 20. Estimation of sugar released during degradation of P. juliflora by O. laetevirens in different days. Fig. 21. Estimation of phenol released during degradation of P. juliflora by O. laetevirens in different days. Fig. 22. UV-Vis spectrum analysis during degradation of P. juliflora by O. laetevirens in different days. Fig. 23. Estimation of nitrate released during degradation of P. juliflora by O. laetevirens in different days. Fig. 24. Estimation of ammonia released during degradation of P. juliflora by O. laetevirens in different days. Fig. 25. Estimation of protein released during degradation of P. juliflora by O. laetevirens in different days. Fig. 26. Estimation of lignin in degraded P. juliflora by O. laetevirens in different days. Fig. 27. Estimation of holocellulose in degraded P. juliflora by O. laetevirens in different days. Fig. 28. UV-Vis spectra of ethanolic extract of P. juliflora and degraded P. juliflora in different days. Fig. 29. Separation of compounds from O. laetevirens treated P. juliflora sample by Thin Layer Chromatography (TLC) (30th day sample)

Fig. 30. High Performance Thin Layer Chromatography (HPTLC) of P. juliflora and O. laetevirens treated P. juliflora (30th day sample). Fig. 31. Identification of compound by High Performance Liquid Chromatography (HPLC) (30th day sample). Fig. 32. Gas Chromatography Mass Spectrometry (GC-MS) of ethanolic extract of degraded P. juliflora (30th day sample). Fig. 33. Fourier Transform Infrared (FTIR) spectrometry spectrum of ethanolic extract of degraded Prosopis juliflora (30th day sample). Fig. 34. Nuclear Magnetic Resonance (NMR) spectroscopy spectrum of ethanolic extract of degraded P. juliflora (30th day sample). Fig. 35. Effect of degraded P. juliflora by O. laetevirens on body weight of Rattus norvegicus. Fig. 36. Effect of degraded P. juliflora by O. laetevirens on haemoglobin content of Rattus norvegicus. Fig. 37. Effect of degraded P. juliflora by O. laetevirens on Erythrocyte Sedimentation Rate (ESR) of Rattus norvegicus. Fig. 38. Effect of degraded P. juliflora by O. laetevirens on Packed Cell Volume (PCV) of Rattus norvegicus. Fig. 39. Effect of degraded P. juliflora by O. laetevirens on Red Blood Cells (RBC) count of Rattus norvegicus. Fig. 40. Effect of degraded P. juliflora by O. laetevirens on White Blood Cells (WBC) count of Rattus norvegicus. Fig. 41. Effect of degraded P. juliflora by O. laetevirens on neutrophil level of Rattus norvegicus. Fig. 42. Effect of degraded P. juliflora by O. laetevirens on lymphocyte level of Rattus norvegicus. Fig. 43. Effect of degraded P. juliflora by O. laetevirens on eosinophil level of Rattus norvegicus. Fig. 44. Effect of degraded P. juliflora by O. laetevirens on protein level of Rattus norvegicus. Fig. 45. Effect of degraded P. juliflora by O. laetevirens on glucose level of Rattus norvegicus.

III

Fig. 46. Effect of degraded P. juliflora by O. laetevirens on albumin and globulin level of Rattus norvegicus. Fig. 47. Effect of degraded P. juliflora by O. laetevirens on cholesterol level of Rattus norvegicus. Fig. 48. Effect of degraded P. juliflora by O. laetevirens on triglycerides level of Rattus norvegicus. Fig. 49. Effect of degraded P. juliflora by O. laetevirens on urea level of Rattus norvegicus. Fig. 50. Effect of degraded P. juliflora by O. laetevirens on uric acid level of Rattus norvegicus. Fig. 51. Effect of degraded P. juliflora by O. laetevirens on creatinine level of Rattus norvegicus. Fig. 52. Effect of degraded P. juliflora by O. laetevirens on Serum Glutamic Pyruvic Transaminase (SGPT) level of Rattus norvegicus. Fig. 53. Effect of degraded P. juliflora by O. laetevirens on Serum Glutamic Oxaloacetic Transaminase (SGOT) level of Rattus norvegicus. Fig. 54. Effect of degraded P. juliflora by O. laetevirens on total and indirect bilirubin level of Rattus norvegicus. Fig. 55. Effect of degraded P. juliflora by O. laetevirens on Alkali Phosphatase (ALP) level of Rattus norvegicus. Fig. 56. Effect of degraded P. juliflora by O. laetevirens on sperm count of Rattus norvegicus. Fig. 57. Antioxidant activity of degraded P. juliflora by O. laetevirens.

LIST OF PLATES

Plate-1 a. Oscillatoria laetevirens b. Prosopis juliflora

Plate-2 a. Different particle size of P. juliflora b. O. laetevirens treated P. juliflora c. Ethanolic extract of degraded P. juliflora by O. laetevirens

Plate-3 Microscopic observation of P. juliflora degradation by O. laetevirens (10x). a. Adherence of O. laetevirens on wood particles b. Colonization of O. laetevirens on wood particles c. Mineralization of wood particles by O. laetevirens d. Degraded wood particles by O. laetevirens

Plate-4 Effect of ethanolic extract of degraded P. juliflora by O. laetevirens on kidney cells of R. norvegicus.

Plate-5 Effect of ethanolic extract of degraded P. juliflora by O. laetevirens on liver cells of R. norvegicus.

ABBREVIATIONS

% - Percentage °C - Degree Celsius μg - Microgram μL - Microliter μL/mm3 - Microliter per cubic millimeter μm - Micrometer µE m-2 s-1 - Microeinstein per second and square meter µg/mL - Microgram(s) per milliliter µM/g - Micromolar per gram μg/kg - Microgram per kilogram μM - Micromolar 4-HBA - 4-hydroxybenzoic acid 4-NQO - 4-nitroquinoline-1-oxide a. m. u - Atomic Metric Units ABTS - 2, 2´-azinobis (3-ethylbenzthiazoline-5-sulphonate) ACT - Apple Condensed Tannins AD - Atopic Dermatitis AIBN - 2, 2’-azobis 2-methylpropionitrile ALP - Alkaline Phosphatase ALT - Alanine Transaminase ANOVA - Analysis of variance AOM - Azoxymethane APS - Ammonium persulphate ASN III - Artificial Sea Nutrients III AST - Aspartate Transaminase BDU - Bharathidasan University BG 11 - Blue Green 11 BHT - Butylated hydroxytoluene CAPE - Caffeic Acid Phenethyl Ester

CCl4 - Calcium tetrachloride Cd - Cadmium cm - Centimeter Cr - Chromium Cont… Cu - Copper

mm3 - Cubic millimeter DC - Differential Count DEAE - Diethylaminoethyl DNA - Deoxyribonucleic acid DNSA - Dinitro salicylic acid DPPH - 1, 1-diphenyl-2-picrylhydrazyl DPX - Dibutyl Phthalate Xylene EAC - Ehrlich Ascites Carcinoma EDTA - Ethylenediamine tetraacetate ESR - Erythrocyte Sedimentation Rate eV - Electron volt F - Experimental value Fcrit - Tabulated value FTIR - Fourier Transform Infrared Spectroscopy g - Gram g/dL - Gram/deciliter GC-MS - Gas Chromatography Mass Spectroscopy GPT - Glutamic pyruvic transaminase GOT - Glutamate oxaloacetate transaminase GSH - Glutathione

H2O2 - Hydrogen peroxide HDL - High Density Lipoprotein HL-60 - Human monocytic leukaemia cells HPLC - High Performance Liquid Chromatography HPTLC - High Performance Thin Layer Chromatography hr - Hour IgE - Immunoglobulin E KAU - King-Armstrong Unit kBr - Potassium bromide

LD50 - Lethal Dose 50

LDH - Lactate Dehydrogenase LDL - Low Density Lipoprotein LiP - Lignin peroxidase m - Meter M - Molar Cont...

VII m/z - Mass to charge mAU - Milli Absorbance Units mcg - Micrograms mg - Milligram mg/dL - Milligram/ decilitre mg/g - Milligram/gram mg/kg - Milligram/kilogram mg/L - Milligram/Litre mg/ml - Milligram/millilitre MHz - Meter heterz min - Minute mL - Millilitre mL/min - Milliliter/minute mM - Millimolar mm - Millimeter mm/hr - Millimeter per hour MnP - Manganese peroxidase MTT - Methyl Thiazol Tetrazolium N - Normal

N2 - Nitrogen Ni - Nickel nm - Nanometer NMR - Nuclear Magnetic Resonance Spectroscopy O. l - Oscillatoria laetevirens O. l+P. j - Oscillatoria laetevirens treated Prosopis juliflora OD - Optical density OECD - Organisation for Economic Cooperation and Development OH - Hydroxyl group P. j - Prosopis juliflora

P2O5 - Phosphorus oxide

PPE - Polyphenolic extract PCV - Packed Cell Volume pH - Hydrogen potential ppm - Parts per million RBC - Red Blood Cell Cont… VIII

Rf - Retention factor rpm - Revolution per minute

Rt - Retention time sec - Second SGOT - Serum Glutamic Oxaloacetic Transaminase SGPT - Serum Glutamic Pyruvic Transaminase TBARS - Thiobarbituric Acid Reactive Substances TC - Total Count TCA - Trichloroacetic Acid TFA - Trifluoroacetic Acid TGL - Triglycerides TLC - Thin Layer Chromatography TPA - 12-O-tetradecanoylphorbol-13-acetate UV - Ultraviolet U/L - Units/Litre v/v - Volume by volume VLDL - Very Low Density Lipoprotein w/v - Weight by volume WBC - White Blood Cell

INTRODUCTION

"So mesquite is something more than a tree, it is almost an elemental force, comparable to fire - too valuable to extinguish completely and too dangerous to trust unwatched" (Peattie, 1953).

Introduction

Lignin is an important constituent of plant materials and the second most abundant renewable polymeric component of biomass. Disposal of the recalcitrant is very difficult because of its lignocellulosic nature and thereby its slow degradation in the natural environment. Cyanobacteria are free living photoautotrophic microorganisms that are widely distributed in the natural environment. Some cyanobacteria are able to fix atmospheric nitrogen and are therefore inexpensive to maintain in the natural conditions. With such potentials they have been exploited in the field of lignocellulosic waste degradation, pesticide degradation, liquid waste treatment and degradation of aromatic compounds. Thus, lignin depolymerization is an efficient for conversion of cellulose and hemicelluloses into single-cell protein, into soluble sugar being used for fermentation, into fuel or direct use as an energy source (feed) for ruminants. Biological treatment using cyanobacterial strains enhances the digestibility and increases the availability of energy resources to fulfill some demanding needs, which is a cheap and economically feasible process.

1.1 Lignocellulosic waste Lignocellulose, the major component of plant biomass, derives from wood, grass, agricultural residues, forestry waste and municipal solid waste that constitute half of the matter produced by photosynthesis. It consists of three types of polymers which are cellulose, hemicellulose and lignin. These polymers are strongly intermeshed and chemically bonded by non-covalent forces and by covalent cross linkages where lignin is an amorphous heteropolymer consisting of phenylpropane units joined together by different types of linkages. The major problem in studying the chemistry of lignin has been the difficulty in isolating intact lignins from plant materials. It is the most abundant natural organic compound on earth besides cellulose. Lignin is deposited as an encrusting and protecting material on the cellulose or hemicellulose matrix and it

Introduction sets up a complex and acts as a kind of glue that cements the fibrous cell walls together. The complex structure of lignocellulose in plants gives strength and rigidity to the plants and forms a protective barrier to cells from destruction by microorganisms. Thus, only organisms possessing lignolytic enzymes with different activities acting in concert can completely degrade the plant cell wall. The decomposition of lignin constitutes an important process in the total biological deterioration of wood. Recently, there has been a growing interest in studying optimal lignin degrading systems for their use in various biotechnological applications.

1.2 Prosopis juliflora Prosopis juliflora is native to the West Indies, Central America and Northern South America. In India, it is an invader species that compete with native species. It is a fast-growing deciduous tree or shrub that is thorny, has deep roots, grows in very hot, dry climates with temperature up to 48°C and annual precipitations of 15.0 to 167.0 cm. It can grow in a variety of soils including saline, alkaline, sandy and rocky soils. Its low nutritional requirements and its resistance to hydric deficits provide P. juliflora with a great plasticity of response, which allows its wide distribution in arid and semiarid zones of tropical areas. The wood of P. juliflora is hard, heavy and resistant to rotting. The constituents of woody biomass can be divided into cellulose, hemi-cellulose, lignin, extractives, ash and water. The levels of chemical constituents in P. juliflora have been estimated as 25-30% hemicellulose, 40-45% cellulose, 11-28% lignin and 3-15% extractives. Extractive chemicals from woody biomass include sugar, resins, volatile oils, fatty acids, tannins, alcohols and phenols with tannin content up to 9% of woody material. Although P. juliflora is a potential source of fuel wood, timber, honey and pods as animal forage, it become a rather competitive weed and it has been declared as a noxious weed in many countries due to its invasiveness, with subsequent negative ecological, economic and social impacts. Environmental effects of this species include

Introduction

 Increased land degradation and loss of soil moisture due to their extensive and deep root system.  P. juliflora competes with and takes over native vegetation causing a change in habitat and led to a loss of biodiversity.  Impairs the growth of forage thus provision of refuges for feral animal population was prohibited.  Damage to environmentally sensitive areas such as water courses.

1.3 Cyanobacteria Cyanobacteria, also known as blue-green algae, blue-green bacteria or cyanophyta are the most widely distributed organisms in aquatic or moist habitats. They belong to oxygen producing Gram negative photosynthetic prokaryotes. They are found in conceivable habitats, from oceans to fresh waters, from bare rock to soil as well as in association with other organisms. Cyanobacteria occur as unicellular, filamentous and colonial forms. They constitute a vast potential resource in varied applications such as in food, feed, fuel, fertilizers, medicine and in combating pollution because of their simple nutrient requirements and flexibility to survive in various environmental conditions. Cyanobacteria are potential degraders of various organic pollutants and it is being used in many polluted areas to alleviate the effects of pollutants. For example, cyanobacteria and their enzymes thought to be useful in the delignification of lignin and will be a better candidate for treating lignocellulosic wastes with a higher potential than any other organism. This is because cyanobacteria do not deoxygenate the environment, instead providing oxygen as a photosynthetic by- product.

1.4 Biodegradation The degradation of organic materials is always due to the characteristic succession of the microbial activity. The biological degradation of cellulose, hemicellulose and lignin has attracted the interest of microbiologists and biotechnologists for many years for their utilization. The diversity of cellulosic and lignocellulosic substrates has contributed to the difficulties found in enzymatic studies. Microorganisms have two types of extracellular enzyme systems, the hydrolytic system

Introduction which produces hydrolases and is responsible for cellulose and hemicellulose degradation and a unique oxidative and extracellular lignolytic system, which depolymerizes lignin. Fungi are the best-known microorganisms capable of degrading these three polymers and lignin peroxidases, manganese peroxidases and laccases are the three families of enzymes that are implicated in the biological degradation of lignin. The complexicity of the lignin structure provided most of the challenges for its degradation. Hence, the present investigation was carried out to study the degradation of the lignocellulosic waste Prosopis juliflora by the marine cyanobacterium Oscillatoria laetevirens and to estimate the renewable products released during the process of degradation.

1.5 Phenolic Compounds Phenolic compounds are widely distributed in the plant kingdom. Plant tissues may contain up to several grams per kilogram. External stimuli such as microbial infections, ultraviolet radiation and chemical stress induce their synthesis. Phenolic compounds are phytochemicals that are ubiquitous in plants which are a dominant part of human diets worldwide. Phenolic compounds are also of considerable interest due to their antioxidant properties. These compounds posses an aromatic ring bearing one or more hydroxyl groups and their structures may range from that of a simple phenolic molecule to that of a complex high-molecular weight polymer. Flavonoids, which bear the C6–C3–C6 structure, account for more than half of the over eight thousand different phenolic compounds. The antioxidant activity of phenolic compounds depends on their structure, number and position of the hydroxyl groups and on the nature of substitutions on the aromatic rings. Fruits, vegetables and beverages are the major sources of phenolic compounds in human diets. The food and agricultural products processing industries generate substantial quantities of phenolic rich by-products, which could be valuable natural sources of antioxidants. Some of these by-products have been the subject of investigations and have proven to be effective sources of phenolic antioxidants. Furthermore, phenolic rich extracts have shown better antioxidant activities comparable to that of synthetic antioxidants. This study was carried out to identify and characterize the antioxidant potentials of P. juliflora extracts and could

Introduction

therefore be of valuable interest as a potential source of antioxidants from a renewable origin. Hence, the study was carried out to degrade lignocellulosic waste of P. juliflora by marine cyanobacterium for its considerable utilization.

Objectives The main objective of this study was to make use of the invasive species P. juliflora to develop potential new resources for local dependents through the development its efficient utilization by the following aspects

 Selection of efficient cyanobacteria to degrade the lignocellulosic material.

 Degradation of lignocellulosics by marine cyanobacterium to obtain value added compounds.

 Identification of the compound by TLC, HPTLC, HPLC, GC-MS, FTIR and NMR.

 Bioactive / Bioinhibitory activity of the identified compound in animal model by

. Haematological study.

. Biochemical study

. Immunological study

. Analysis of male reproductive system

. Histopathological study.

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LITERATURE

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2.1. Lignocellulose - a valuable resources The lignocellulosic material mainly composed of cellulose along with lignin and hemicellulose. Cellulose and hemicellulose are macromolecules from different sugars, whereas lignin is an aromatic polymer synthesized from phenylpropanoid precursors. The composition and percentages of these polymers vary from one plant species to another. Moreover, the composition within a single plant varies with age, stage of growth and other conditions (Jeffries, 1994).

Lignin

Hemicellulose

Cellulose

Fig. 1. Structure of lignocellulose.

The chemical properties of the components of lignocellulosics make them a substrate of enormous biotechnological value (Malherbe and Cloete, 2003). Large amounts of lignocellulosic waste are generated through forestry and agricultural practices, paper-pulp industries, timber industries and many agro industries and they pose an environmental pollution problem (Howard et al., 2003). However, the huge amounts of residual plant biomass considered as waste can potentially be converted into various different value added products including biofuels, chemicals, cheap energy sources for fermentation, improved animal feeds and human nutrients (Howard et al., 2003). Lignocellulolytic enzymes also have significant potential applications in various

Review of Literature industries including chemicals, fuel, food, brewery, wine, animal feed, textile, laundry, pulp, paper and agriculture (Joseph and Ronald, 2009).

2.1.1. Structural feature of lignocellulose 2.1.1a. Cellulose Basically, the cellulose is the most widely distributed skeletal polysaccharide and represents about 50% of the cell wall material of plants. Beside hemicellulose and lignin, cellulose is the major component of agricultural wastes and municipal residues (Wood, 1992). The cellulose molecules are composed of longer chains of β-D- glucopyranose residues linked by 1-4 glucosidic bonds called ‘elementary fibrils’ which are linked together by hydrogen bonds and van der waals forces to form long chains (Beguin and Aubert, 1994). Within each elementary fibril the cellulose molecules are laterally bound and the adjacent molecules run in opposite directions but in parallel with various degree of orientation (Zarnea, 1994).

Fig. 2. Structure of cellulose.

The average degree of polymerization of plant cellulose varies between 7000 to 15,000 glucose units depending on the source (Fengel and Wegener, 1983). Cellulose can appear in crystalline form, called crystalline cellulose. In addition, there is a small percentage of non-organized cellulose chains, which form amorphous cellulose comprises less-oriented molecules. Many of these elementary fibrils form together a microfibril and furthermore several microfibrils joined together to form a macrofibril (Sandhu and Bawa, 1992; Wood, 1992). In this conformation, cellulose is more susceptible to enzymatic degradation (Kadla and Gilbert, 2000).

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For the complete hydrolysis of cellulose to glucose, the cellulase system must contain the following enzymes: endoglucanase, exoglucanase and β-glucosidase. Only the synergistic effect of these enzymes makes the hydrolysis of cellulose to glucose (Sandhu and Bawa, 1992; Jeffries, 1994; Sanchez, 2009).

2.1.1b. Hemicellulose Hemicellulose is a complex carbohydrate polymer and makes up 25-30% of total wood dry weight. It is a polysaccharide with a lower molecular weight than cellulose. It consists of D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-glucuronic, D-galacturonic and D-glucuronic acids (Jeffries, 1994). Sugars are linked together by β-1,4 and occasionally β-1,3 glycosidic bonds. The principal component of hardwood hemicellulose is glucuronoxylan, whereas glucomannan is predominant in softwood (Perez et al., 2002).

Fig. 3. Structure of hemicellulose

Hemicelluloses are more soluble than cellulose and they can be isolated from wood by extraction. However, alkali extractions deacetylate the hemicelluloses completely (Sjostrom, 1981). The average degree of polymerization of hemicelluloses varies between 70 and 200 depends on the plant species (Fengel and Wegener, 1983).

Hemicelluloses are of particular industrial interest because they are a readily available bulk source of xylose from which xylitol and furfural can be derived. Xylitol is used instead of sucrose in food as a sweetener, has odontological applications such as teeth hardening, remineralisation and as an antimicrobial agent. It is used in chewing

Review of Literature gum and toothpaste formulations (Parajo et al., 1998; Roberto et al., 2003). Furfural is used in the manufacture of furfural-phenol plastics, varnishes and pesticides (Montane et al., 2002). Over 200,000 tones of furfural with a market price of about $1700 per ton is annually produced (Zeitch, 2000).

2.1.1c. Lignin Lignin is an important constituent of plant materials and the second most abundant renewable polymeric component of biomass. Over 95% of lignin is used presently as an energy source or disposed of as waste. On the other hand, it seems to be an attractive material for modification and used for the purpose of increasing environmental sustainability. It can provide a number of aromatic chemicals for use in paints, food industry and agriculture (Rohella et al., 1997). It is an extremely complex three-dimensional polymer formed by dehydrogenase polymerization of p-hydroxycinnamyl, coniferyl and sinapyl alcohols (Camaerero et al., 1999).

The process of lignification is initiated when a phenolic hydrogen atom is removed by peroxidase to form a phenoxy free radical. The radical centre can be decolorized to aromatic and side chain carbons. Such radicals when coupled together lead to polymerization and form a beta-0-4-bond which is the most common inter unit linkage in lignin (Rohella et al., 1997).

p-coumaryl alcohol coniferyl alcohol sinapyl alcohol

Fig. 4. Structure of lignin precursors.

Lignin has several important functions in the cell wall matrix of plants (Eriksson et al., 1990). It strengthens the cell wall structure, thus lending mechanical support to the plant. It also serves as a barrier to protect the plant against microbial attack. The water permeation-reducing property of lignin plays an important role in the internal transport of water, nutrients and metabolites in the plant (Kuhad et al., 1997).

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Lignin is responsible for the decay resistance that enables woody plants to live for centuries in an environment teeming with parasitic and saprophytic organisms capable of degrading non lignified plant cells (Humphrey et al., 1999; Perez et al., 2002). Unfortunately, the same strength, stability and hydrophobic nature make plants and trees to have an adverse impact upon mankind and the environment in many ways (Eriksson, 2000).

Fig. 5. Structure of lignin.

The chemical structure of native lignin is essentially changed under high temperature and acidic conditions, such as the conditions during steam pretreatment. At higher temperature greater than 200ºC, lignin has shown to be agglomerated into smaller particles and separated from cellulose (Tanahashi et al., 1983). Early studies on hardwood lignin have shown that the β-O-4 aryl ether linkages are cleaved in steam- explosion causing a decrease in molecular weight and an increase in phenolic content (Marchessault et al., 1981). A study on steam exploded softwood has shown that lignin becomes more condensed and the reactive groups at the α-position, such as hydroxyl groups and ethers, are oxidized to carbonyl groups or generate benzylic cations, which form C-C bonds, leading to loss of reactivity at the α-positions (Shevchenko et al., 1999).

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Recent observations have given indications that all lignin is not homogeneous in structure. Lignin seems to consist of amorphous regions and structured forms such as oblong particles and globules (Novikova et al., 2002). Lignin in higher plant cell wall is not amorphous. Phenyl rings of softwood lignin have been shown to be aligned preferentially in the plane of the cell wall (Agarwal and Atalla, 1986). There are also indications that both the chemical and three dimensional structure of lignin are strongly influenced by the polysaccharide matrix (Houtman and Atalla, 1995). Molecular dynamic stimulations have suggested that the hydroxyl and methoxyl groups in lignin precursors and oligomers may interact with cellulose microfibrils despite the fact that lignin is hydrophobic in character (Sanchez, 2009).

2. 2. Lignocellulosic waste - Prosopis juliflora Prosopis juliflora is a shrub or small tree native to Mexico, South America and the Caribbean. It has become established as a weed in Asia, Australia and elsewhere. Its uses include forage, wood and environmental management. The tree grows to a height of up to 12 metres and has a trunk with a diameter of up to 1.2 metres. It is known to hold the record for depth of penetration by roots. Thus Prosopis juliflora roots were found growing at a depth of 53.3 meters at an open-pit mine near Tucson, Arizona (Duke, 1983)

In the last 200 years, species of Prosopis have been introduced or reintroduced to certain areas of Argentina, Chile, Peru, Mexico and the USA, as well as in some regions of Asia, Africa, India and Australia (Pasiecnik et al., 2001). The majority of introduced species were P. juliflora, P. pallida, P. glandulosa and P. velutina. The former two species are prevalent in tropical zones, while the later two are found in more sub-tropical zones. Species such as P. alba and P. chilensis have proved to be well adapted and are locally common in some regions (Pasiecnik et al., 2001). .

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America. originated from from originated Prosopis nd present distribution of the genus genus the of distribution nd present Fig. 6. Approximate dispersal a dispersal Approximate 6. Fig.

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Prosopis juliflora grows in all kinds of soil conditions, including wastelands at altitudes ranging from 0 to 1,500 m above sea level, under mean annual temperature of 14 to 34°C and annual rainfall of 50 to 1,200 mm (Pasiecnik et al., 2001). Mature trees grow up to 17 m in height. This species is characterized by twisted stem with axial invasion of grasslands, protected forests and nature reserves has alarmed the ecologists thorns situated on both sides of the nodes and branches. It is reported to dry out the soil and compete with grasses, particularly in dry areas and is therefore considered as a weed in some areas (Choge et al., 2007).

P. juliflora wood has been described as a source of lumber, firewood, activated carbon and charcoal. There is a considerable potential for P. juliflora as a source of fiber for the paper, paperboard and hardboard industries (Kailappan et al., 2000; Goel and Behl, 2001). Currently, it has escaped plantations and dominates many plant communities thus considered as weed. The tree can grow in a variety of areas, including those with saline, alkaline, sandy and rocky soils and the roots penetrate to great depths in the soil (Tiwari, 1999; Al-Rawai, 2004). Drought tolerant genes were identified from P. juliflora through analysis of expressed sequence tags. It has been presently used as source of drought tolerant genes for transformation in crop plants (George et al., 2007).

2.2.1 Botanical description The genus Prosopis comprises 44 species of which 40 are native to the America. Of the remaining species P. africana is indigenous to Africa, whereas P. kodziana, P. farcta and P. cineraria are natives to the Middle East and Pakistan (Burkart, 1976). The placing of Prosopis in the wider taxonomic classification system was based on Lewis and Elias (1981).

Prosopis species grow in a wide array of environments and are not restricted by soil type, pH, salinity and fertility. It grows in semi arid and arid tracts of tropical and sub-tropical regions of the world and is spreading fast because the leaves are unpalatable and animals do not digest its seeds (Sawal et al., 2004).

In India this species has been found to be of great value for rehabilitation of saline and alkaline soils especially in the northern parts of the country (Goel and Behl,

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1996 and 2001). Apart from poor soils, Prosopis trees withstand exceptionally high temperatures (45°C) in summer and cool temperatures in winter (Shirke and Pathre, 2004).

Senthilkumar et al. (2005) examined the soil and plant samples (root and shoot) of P. juliflora in the vicinity of metal based foundry units in Coimbatore, India and assessed their metal content (Cu and Cd) to ascertain the use of P. juliflora as a green solution to decontaminate soils contaminated with copper and cadmium. Results showed that Cu and Cd content was much higher in plant components compared to their extractable level in the soil and there was a strong correlation between the distance of the sources of industrial units and the accumulation of heavy metals in plants. P. juliflora was recommended for the decontamination of heavy metal contaminated soils (Cu, Cd, Ni and Cr) in view of its ability to accumulate heavy metals and its ability to establish and proliferate in such soils with long roots covering larger areas (Nivethitha et al., 2002).

2.2.2 Chemical composition The nutrient concentrations in component of P. juliflora were quite high compared with the values reported for many temperate trees, but are comparable to multispecies averages obtained in the analysis of tropical forest ecosystems and tropical forage legumes (Jummane et al., 2004). The heartwood of different Prosopis species contains significant amounts of wood extracts and polyphenol compounds (Goldstein et al., 1972). Many phenolic compounds are accumulated in heartwood, whereas they are found only in trace amounts in the corresponding sapwood (Toshiaki, 2001). Extracts of tree wood, bark and leaves range from simple compounds such as vanillin to polymeric condensed tannins (Taylor et al., 2006). Such features provide the basis of chemotaxonomy of woody plants (Toshiaki, 2001).

Juliflorine, the main alkaloid of Prosopis juliflora was isolated and its partial structure was reported by Ahmad et al. (1979). Later Longoni et al. (1980) reported the complete structure of juliflorine (juliprosopine) from P. juliflora. Juliflorine has been reported to possess significant antidermatophytic (Khan et al., 1986) and antibacterial activity (Ahmad et al., 1986).

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Leaves of P. juliflora contain alkaloids such as tryptamine, piperidine, phenethylamine and juliprosopine has antifungal and plant growth inhibiting properties as well as it is capable of inducing neuronal damages in animals (Tapia et al., 2000). Leaf extractives analyzed using GC-MS showed the presence of an important quantity of fatty acids such as hexadecanoic, octadecanoic acids, glucopyranose, hydroquinone, glucopyranosides and galactose sugar (Sirmah, 2009).

A large amount of flavonoid has been extracted and isolated from the heartwood of Prosopis juliflora. Structural and physicochemical elucidation based on FTIR, 1H and 13C NMR, GC-MS and HPLC analysis clearly demonstrated the presence of (-)-mesquitol as the sole compound without any noticeable impurities. The product was able to slow down the oxidation of methyl linoleate induced by 2, 2’-azobis 2-methylpropionitrile (AIBN) and this important amount and high purity of (-)- mesquitol could therefore be of valuable interest as a potential source of antioxidants from a renewable origin (Sirmah et al., 2009).

High amount and purity of the rare flavonoid (-)-mesquitol was identified as a major metabolite in heartwood extractives of P. juliflora, while (+)-epicatechin, (+) - catechin, gallocatechins, methylgallocatechins, fatty acids and free sugar are present in the bark. P. juliflora pods contain important quantity of galactomanans, mannose, saturated and unsaturated fatty acids and free sugar which are used as a food supplement and medicine for animals and humans (Sirmah, 2009).

2.2.3 Natural durability Wood is a natural composite constituted mainly of cellulose, lignin and hemicellulose. In addition to these polymeric materials, it may contain low molecular weight compounds present in different quantities in extracts. Wood extractives have been described to have a crucial effect on the natural durability of wood, explaining the resistance of some wood species to biodegraders (Windeisen et al., 2002; Haupt et al., 2003).

Biological deterioration of wood is of concern to the timber industry due to economic losses caused to wood in service or in storage. Fungi, insects, termites, marine borers and bacteria are the principal wood biodegraders. They attack different

Review of Literature components of wood at different rates giving rise to a particular pattern of damage (Silva et al., 2007). Degradation is influenced by environmental conditions of the wood, whether in storage or in use. The degraded wood material is returned into the soil to enhance its fertility (Silva et al., 2007).

2.2.4 Prosopis in traditional medicine Many plants of the genus Prosopis are known to have medicinal properties and are used in folk medicine as astringent, in rheumatism and as remedies against scorpion stings and snake bites (Wassel et al., 1972). The powdered flowers mixed with sugar are eaten by women during pregnancy as a safeguard against miscarriage. Its ash when rubbed over the skin removes the hair (Chopra et al., 1956).

According to Mitchell and Rook (1979) the thorn from mesquite on penetrating the eye, causes more inflammation than expected from physical injury. The irritation may be due to waxes and using the wood in a fireplace caused dermatitis. Lewis and Elvin (1977) reported that ingestion of pods over long periods of time will result in death of cattle. Further, they report that the pollen may cause allergic rhinitis, bronchial asthma and hypersensitivity pneumonitis.

Isolation and structural determination of two new alkaloids from this plant, which have been named as juliprosinene and juliflorinine showed better antibacterial activity against strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Shigella sonnei (Uddin et al., 1989). The antimicrobial activity of benzene insoluble alkaloid mixture of the leaves of P. juliflora was measured in vitro against 22 bacterial strains including 9 Gram positive and 13 Gram negative. It was found more effective than bacitracin, Gentamycin, Chloromycetin and Trimethoprim against S. aureus, S. lactis, S. faecalis, S. pyrogenes and C. diptheria. Alkaloids of P. juliflora produced significant haemolysis against rats and humans which are dose dependent and showed 90% lethality when concentration of alkaloids is about 150 mcg that damage the erythrocyte membrane (Kandasamy et al., 1989).

The extract of Prosopis juliflora leaves showed better antibacterial activity against three phytopathogenic Xanthomonas pathovars viz., Xanthomonas axonopodis

Review of Literature pv. malvacearum, X. a. pv. phaseoli and X. campestris pv. vesicatoria associated with angular leaf spot of cotton, common blight of bean and bacterial spot of tomato, respectively, and 14 human pathogenic bacteria (Raghavendra et al., 2009).

The heartwood extractives of Prosopis juliflora had higher antioxidant properties in comparison with bark and sapwood extractives. Hydrophilic extractives from the more polar solvents such as acetone and toluene/ethanol mixture gave higher antioxidant properties than lipophilic extractives present in dichloromethane extractives, suggesting that flavanols such as mesquitol present in these extracts are responsible for the antioxidant property (Sirmah, 2009). Indeed, correlation of phenolic contents in plants to their antioxidant activities has reported earlier (Haupt et al., 2003 and Wang et al., 2004).

2.2.5 Toxicity of Prosopis sp. Mesquite (Prosopis juliflora) is a major cause of allergic disease in the southwestern United States (Novey et al., 1977; Bieberdorf and Swinny, 1952), Mexico (Bessega et al., 2000), Saudi Arabia, South Africa (Al-Frayh et al., 1999; Ezeamuzie et al., 2000), Kuwait (Davis , 1969), United Arab Emirates (UAE) (Bener et al., 2002), and India (Thakur, 1991). Kingsbury (1964) reported some details on mesquite poisoning in cattle, where autopsies showed pods and seeds in the rumen.

Aqeel et al. (1991), investigated the antidermatophytic activity of juliflorine and a benzene insoluble alkaloidal fraction obtained from P. juliflora against Trichophyton mentagrophytes infection in rabbits. Tropical application of 2.5% juliflorine was found to heal 75% of dermatophytic lesions in three weeks. Benzene insoluble alkaloidal fraction was found comparatively more effective than juliflorine.

Furthermore, several studies showed that the leaf extract of P. juliflora inhibited mycelial growth and spore germination of several pathogenic fungi including Pyricularia oryzae, which cause blast disease in rice (Kamalakannan et al., 2001), as well as Colletotrichum capsici and Gloeosporium piperatum infecting Capsicum annum (Gomathi and Kannabiran, 2000).

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Nakano et al. (2002), isolated and identified two plant growth inhibitors (syringin and lariciresinol) from aqueous leachates of mesquite as candidates for allelopathic substances. The allelopathic potential of L-tryptophan in the leachate from the foliage of mesquite was investigated by Nakano et al. (2003) and reported that 17.9 μM of L-tryptophan caused 69.8% inhibition of the root growth of barnyard grass (Echinochloa crus-galli L.).

P. juliflora leaf alkaloids and their different isomers have the ability to inhibit growth of surrounding plants (Kishore and Pande, 2005). Fractionated alkaloids isolated from leaves have been shown to induce cytotoxicity leading to neuronal damages, neuromuscular alterations and gliosis in animals (Silva et al., 2007). The leaves are reported to contain crude protein levels of 14-22%, crude fibre 21-23%, nitrogen free extract 43-50%, calcium 1.5% and phosphorus 0.2% while mineral content is directly related to the levels of minerals in the soil (Pasiecnik et al., 2001).

Assessment of growth inhibition by alkaloids from P. juliflora against the shoot and root growth of monocotyledonous plants - barnyard grass, rice and timothy and dicotyledonous plant - amaranth, lettuce and cress exhibited growth inhibition. Among the alkaloids, the highest active compound appeared to be julisporine followed by a (1:1) mixture of 3-oxo and 3’-oxo-juliprosine and juliprosopine (Nakano et al., 2004a). Plant growth inhibitory alkaloids were isolated from the leaf extract of P. juliflora and identified by 13C-NMR spectral analysis which exhibited root growth inhibition on Lepidium sativum L. (Nakano et al., 2004b).

Quintas-Junior et al. (2004) in their preliminary study on the total alkaloid fraction of the pods of P. juliflora reported that they induced behavioral changes in rodents, suggesting some effect on the central nervous system. They also showed an acute toxicity (LD50) of 10.3 mg/kg intraperetonially and 637 mg/kg orally. Cytotoxic effects of the extract containing alkaloids on GL-15 (Glial cells of lineage) cell line as confirmed by the MTT (Methyl Thiazol Tetrazolium) test, LDH (Lactate Dehydrogenase) activity and Trypan blue staining. The cytotoxic effects of alkaloid extract from P. juliflora pods upon the viability were confirmed by MTT test, LDH activity and Trypan blue staining (Hughes et al., 2005).

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Different flavonoids such as naringenin and tamoxifen are reported to enhance the antibacterial, antiviral, or anti-cancer activities (Arima et al., 2002). Combination with some drugs lead to synergistic effects. Some flavonoids from Larix leptolepis such as taxifolin and quercetin show antifeedant activity against subterranean termites (Ohmura et al., 2000).

Thakur (1991) isolated two antigenically active glycoprotein fractions from crude allergen extract of the pollen of P. juliflora using DEAE-cellulose ion exchange chromatography. Killian and McMichael (2004) performed a computerized statistical analysis of skin allergy test results correlating patient reactivities in the cross reactive allergens of mesquite tree pollen. Dhyani et al. (2006) reported that among pollen allergens P. juliflora was an important sensitizer in allergic patients with 37% skin positivity for total IgE and detected thirteen human allergens of mesquite pollen by Western blotting.

2.3. Lignin Degradation Lignin, although quite resistant to microbial attack is ultimately degraded to humus, carbon dioxide and water (Baldrian and Gabriel, 2002). Lignin degrading 14 14 ability of a microorganism is commonly evaluated by measuring CO2 evolution C labelled lignin preparation (Temp et al., 1999). The enzymatic machinery for degrading cellulose, hemicellulose and lignin is possessed only by microorganisms (Rathore et al., 2001).

A wide range of microorganisms are involved in degradation of lignin. These include bacteria, actinomycetes, cyanobacteria, algae and fungi. Each lignin degrading microbial strain attacks lignin in its own specific way, although it is known that the patterns of attack on the structure of lignin are often similar within a specific group of organisms (Kirk and Farell, 1987). Gutierrez et al. (1996) analyzed lignin polysaccharide complexes and found that the aromatic fractions of lignin polysaccharide complexes were derived from lignin and break down of p-hydroxyphenyl propane, guiacylpropane and syringyl propane.

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The expression of manganese peroxidase in nitrogen limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by hydrogen peroxidase and various chemicals, including ethanol, sodium arsenate and 2,4-dichlorophenol as well as by manganese and heat shock. During hydrolysis of lignocellulosic material appreciable amount of sugar, degradation products, organic acids and phenolic compounds are produced (Buswell and Odier, 1987). Aromatic and side chain radicals when coupled together leading to polymerization and form a ß-o-4-bond which is the most common under unit linkage in lignin (Rohella et al., 1997). The process of lignification is initiated when phenolic hydrogen atom is removed by peroxidase to form a phenoxy free radical and the radical centre can be decolorized (Eriksson, 2000). Two families of lignolytic enzymes are widely considered to play a key role in the enzymatic degradation, phenol oxidase (laccase), peroxidases (lignin peroxidase (LiP) and manganese peroxidase (MnP) (Krause et al., 2003; Malherbe and Cloete, 2003). Other enzymes with not fully elucidated functions include H2O2 producing enzymes like glyoxal oxidase (Kersten and Kirk, 1987), glucose oxidase (Kelley and Reddy, 1986), veratryl alcohol oxidases (Bourbonnais and Paice, 1988), methanol oxidase (Nishida and Eriksson, 1987) and oxido-reductase (Bao and Renganathan, 1991). Enzymes involved in lignin breakdown suggest that lignases employ low-molecular, diffusible reactive compounds to affect initial changes to the lignin substrate (Call and Mucke, 1997).

In a biodegradation study, the selected lignocellulosics showed highest lignin content in coir pith (37%) followed by P. juliflora (23%) and L. camara (22%). However, Oscillatoria annae treated lignocellulosics showed maximum reduction of lignin content in L. camara (18.2%) followed by P. juliflora (17.4%) and coir pith (16.9%) after 30 days of incubation (Viswajith, 2008).

2.3.1. Microbiology of lignin degradation 2.3.1a. Bacteria Bacterial degradation of the cell wall occurs by tunnelling the pit membranes in the sapwood and deterioration of ray parenchyma cells (Clausen, 1996; Fengel and Wegener, 1984). Though bacteria are more tolerant to high lignin and extractive contents in wood with low levels of oxygen requirement than fungi, they may be the sole agents of decay in situations where other decay organisms are excluded. The three

Review of Literature different forms of bacterial decay are recognized as erosion, tunnelling and cavitations (Singh and Kim, 1997; Daniel and Nilsson, 1998; Jing et al., 2009).

Nonfilamentous bacteria usually mineralize less than 10% of lignin preparations and can degrade only low-molecular weight part of lignin as well as degradation products of lignin, play some role in final mineralization of lignin (Ruttimann et al., 1991). Among nonfilamentous and eubacteria, Pseudomonas sp. are the most efficient degraders (Vicuna, 1988; Zimmermann, 1990). However, since these bacteria do not produce extracellular oxidoreductases and large molecules apparently cannot be taken up into the cell, they are obviously unable to attack polymeric lignin.

Odier et al. (1981) reported that several non cellulolytic gram negative aerobic bacteria are capable of degrading dioxane and milled wood poplar lignins at the rates ranging between 4% and 20% within a 7 day period. These strains were identified as Pseudomonas, Xanthomonas and Acinetobacter. Deschamps et al. (1981) have reported that mixed cultures of Bacillus and Cellulomonas strains delignified pine bark chips whereas no delignification was observed in pure cultures treated with bark chips.

Ramachandra et al. (1987) reported that extracellular lignin peroxidases are involved in lignin degradation in actinomycetes. Pasti et al. (1991) reported the lignin solubilizing ability of eleven novel Streptomyces strains isolated from the gut of worker termite. Adhi et al. (1989) noticed that Streptomyces viridosporus 77A produces extracellular enzymes that extensively degrade both lignin and carbohydrate components when grown on corn lignocellulosic material. The enzymatic ability to cleave alkyl-aryl ether bonds enable bacteria to degrade oligomeric and monomeric aromatic compounds released during lignin degradation by fungi (Vicuna, 2000).

Bacteria of the class Actinomycetes are quite active in lignin degradation and were the primary lignin degraders. They create conditions necessary for fungal attack by altering the nutrient status of the wood and production of synergistic secondary metabolites (Holt et al., 1979). Since Actinomycetes do not produce extracellular oxido reductase and large molecules apparently cannot be taken up into the cell, they are obviously unable to attack polymeric lignin (Crawfold and Sutherland, 1980). Two actinomycetes, Thermomonospora mesophila and Streptomyces sp. were particularly

Review of Literature effective in degrading ball milled straw or utilize lignin related aromatic compounds (McCarthy et al., 1984).

However, it has been shown that certain actinomycetes strains are able to readily solubilise the lignin component (Haider and Martin, 1981; Jing et al., 2009). McCarthy and Broda (1984) have identified a range of lignin degrading actinomycetes using (14C) lignin labelled lignocelluloses. Crawford (1980) reported that two strains of Bacillus polymyxa caused a loss of 42% in lignin content of Scots pine sapwood.

2.3.1b. Cyanobacteria A marine cyanobacterium Phormidium valderianum BDU 30501 was found to degrade phenol completely at 100 mg/L by its intracellular oxidase and laccase enzymes. Such strain could be effectively used for treatment of phenol containing waste and effluents (Subramanian and Uma, 1996). The degradation of phenol by the marine cyanobacterium Phormidium valderianum was also studied (Shashirekha et al., 1997). Investigations by Wurster et al. (2003) of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability to metabolize phenol under non-photosynthetic conditions up to 100 mg/L.

Malliga et al. (1996) have reported that Anabena azollae while being used as a biofertilizer exhibited lignolysis and released phenolic compounds which include profuse sporulation of the organism. This report gives the usefulness of coir waste as carrier for cyanobacterial biofertilizer with supporting enzyme studies on lignin degrading ability of cyanobacteria and use of lignocellulosic coir waste as an excellent and inexpensive carrier for cyanobacterial biofertilizers.

Phenol degradation by the freshwater cyanobacteria Anabaena cylindrica and Phormidum foveolarum was first reported by Ellis (1977), but no metabolites or cleavage of the aromatic ring were found. The metabolism of naphthalenes, anilines, biphenyl and phenanthrene and the formation of different hydroxylated metabolites by one unicellular, two filamentous and a hypersaline cyanobacterial strains have been reported (Cerniglia et al., 1980; Naro et al., 1992). Cyanobacteria also contribute to the detoxification of pesticides, insecticides and fungicides in soil and in the aquatic

Review of Literature environment (Khalil and Mostafa, 1987; Mostafa et al., 1991; Megharaj et al., 1994).

Although fewer studies were carried out with microalgae, it has been reported that some eukaryotic algae and cyanobacteria are capable of biodegrading aromatic pollutants commonly found in wastewaters (Cerniglia et al., 1980; Craigier et al., 1965; Semple and Cain, 1995; Semple et al., 1999). Chlorella sp. was found not only to decolourize certain azo-dyes, but to use them as carbon and nitrogen sources (Jingi and Houtian, 1992). Other authors have reported that three species of Chlorella were found to degrade pentachlorophenol in a light-dark photo-regime (Tikoo et al., 1997). However, the metabolic degradation pathways occurring in these organisms have not been widely addressed (Lovell et al., 2002). Lima et al. (2003) reported that when Coenochloris pyrenoidosa was associated with Chlorella vulgaris in a 3:1 ratio, complete removal of the nitro-aromatic compound (p-nitrophenol, 50 mg/L) occurred within three days.

Barton et al. (2004) reported that under aerobic, photosynthetic conditions the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p- nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Some strains of Nostoc, Oscillatoria and Phormidium have also been reported to grow in media supplemented with methyl parathion or other organophosphorus pesticides, but presumptive degradation of these compounds has not been supported by the evidence of their transformation (Mishra and Pandey, 1989; Orus and Marco, 1991; Meharaj et al., 1994; Subramanian et al., 1994). The potential transformation of trinitrotoluene by auxenic Anabaena sp. was also reported (Pavlosthatis and Jackson, 1999, 2001).

Parikh and Madamwar (2005) isolated cyanobacterial cultures from sites polluted by industrial textile effluents and screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B by 55%.

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Lignolytic enzyme profile of Oscillatoria annae was studied by Viswajith (2008) for justifying the lignin degrading ability of O. annae. The results revealed the presence of manganese independent peroxidase, laccase, polyphenol oxidase and other cellulolytic enzymes like endogluconase and xylanase. Also H2O2 production was markedly enhanced in O. annae exposed to lignocellulosic waste, which indicates the increase in ligninolytic activity characteristic of cultures grown under stress conditions.

Anbuselvi and Rebecca (2009) reported that the selected three different cyanobacterial spp such as Phormidium sp., Oscillatoria sp. and Anabaena azollae sp. degrade high lignin containing coir waste efficiently and estimated that 89% of lignin and 92% of hemicelluloses were found to be reduced.

2.3.1c. Fungi Fungi play a pivotal role to degrade lignin extensively (Kirk and Farrell, 1987). Because lignin is an insoluble polymer, the initial steps in its biodegradation must be extracellular. Lignin degrading fungi are classified into three major categories based on the type of wood decay caused by these organisms: white-rot fungi, brown- rot fungi and soft-rot fungi (Giardina et al., 1999; Saparrat et al., 2002). Of these three groups, white-rot fungi are the most effective lignin degraders and have been studied most extensively (Hatakka, 1994).

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by hydrogen peroxidase and various chemicals including ethanol, sodium arsenate and 2,4-dichlorophenol as well as by manganese and heat shock (Li et al., 1995).

White-rot and brown-rot fungi can be found in the same genera and are taxonomically similar. Most wood rotters belong to the order Agaricales and Aphyllophorales. Brown-rot fungi mainly decompose the cellulose and hemicellulose components in wood, but they can also modify lignin to a limited extent (Eriksson et al., 1990). In this group the most commonly used model organism in lignin

Review of Literature biodegradation studies is Phanerochaete chrysosporium (Boominathan and Reddy, 1992).

White-rot fungi comprise a heterogeneous collection of several hundreds of species of basidiomycetes. They are able to completely mineralize both the lignin and carbohydrate components of wood. They metabolize lignin only in the presence of an alternate energy source, such as cellulose, hemicellulose or simple carbohydrates, which is called co-metabolism (Ainsworth et al., 1973). Most white-rot fungi degrade lignin only during secondary metabolism, which is triggered by a limitation of nutrients. However, some fungal species have been reported to degrade lignin even in the presence of sufficient primary nutrients (Leatham and Kirk, 1983).

Lignin can be degraded by white rot fungi; some bacterial species can also degrade lignin (Akin et al., 1995). Lignin can be degraded by wood-rot fungi but is completely degraded only by white-rot fungi. Kuan and Tien (1993) reported that addition of small quantities of manganese peroxidase to lignocellulosic material could increase biodegradability.

Basidiomycetes are the largest group of fungi that degrade wood. In North America there are 1600-1700 species of wood-degrading basidiomycetes have been described. Reproduction of basidiomycetes usually occur by haploid basidiospores and thus the primary mycelium developed after spore germination is also haploid (Gilbertson, 1980).

Nagarajan et al. (1985) reported that the incubation of coir pith with Pleurotus sp. mother spawn for 26 days at room temperature resulted in drastic reduction in the lignin content from 30% in the raw coir pith to 4.6% in the inoculated and incubated coir pith. The cellulose content also decreased from 26.52 to 10.10%, indicating degradation of lignocelluloses by Pleurotus sp. During hydrolysis of lignocellulosic material, a significant amount of sugar, degradation products, organic acids and phenolic compounds are produced (Buswell and Odier, 1987).

Microfungi or molds, i.e., deuteromycetes and certain ascomycetes that are usually thought to degrade mainly carbohydrates in soil, forest litter and compost can

Review of Literature also degrade lignin in these environments (Rodriguez et al., 1996). Thus, some microfungi are able to mineralize grass lignins up to 27% (Haider and Trojanowski, 1975). Although actinomycetes were predominant among 82 strains selected in a screening for ligninolytic microorganisms in a forest soil, also some microfungi were identified, e.g., Penicillium chrysogenum, Fusarium oxysporum and Fusarium solani (Rodriquez et al., 1996). Kuhad et al. (1997) reported that lignin degradation by white rot fungi was an oxidative process and phenol oxidases were the key enzymes.

Pelaez et al. (1995) studied 90 cultures representing 68 species of Basidiomycetes and found laccase activity in 50% of fungi tested. Cullen (1997) implicated four classes of extracellular enzymes in lignin degradation viz, lignin peroxidases (LiP), manganese peroxidases (MnP), laccases and H2O2 generating enzyme glyoxal oxidase (GLOX). Fillingham et al. (1999) noticed that these enzymes act synergistically with xylanases to disturb the hemicellulose-lignin association, without mineralisation of lignin. Lignin Peroxidases (LiP), manganese peroxidases (MnP) and laccases were studied extensively in white rot fungi Phanerochaete chrysosporium, Pleurotus ostreatus and Trametes versicolor (Malherbe and Cloete, 2003).

2.3.2. Lignin degrading enzymes Lignin degradation is in a central position in the earth's carbon cycle, because most renewable carbon is either in lignin or in compounds such as cellulose and hemicellulose protected by lignin from enzymatic degradation (Kirk, 1983). Lignin peroxidases, manganese peroxidases and laccases are three families of enzymes that were implicated in the biological degradation of lignin (Jeffries, 1994; Perez et al., 2002)

2.3.2a. Laccase This enzyme is a copper containing oxidase and it does not require peroxide (Thurston, 1994). Like manganese peroxidase, it normally oxidizes only those lignin model compounds with a free phenolic group, forming phenoxy radicals. However, in the presence of the artificial substrate ABTS (2, 2´-azinobis (3-ethylbenzthiazoline-5- sulphonate) or some other synthetic mediators, laccase can also oxidize certain non- phenolic compounds, veratryl alcohol and manganese (II) (Collins and Dobson, 1997).

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Laccase is produced by most of the white-rot fungi, but normally not present in Phanerochaete chrysosporium (Kirk and Farrell, 1987). It has the capability to both polymerize and depolymerize lignin model compounds. Despite of the fact that laccase was the first enzyme found to have a function in the degradation of lignin, its complete role was still not known (Eriksson et al., 1990).

Cullen (1997) reported two major families of enzymes, peroxidases and laccases, involved in ligninolysis by white-rot fungi. Apparently, these enzymes use low-molecular weight mediators to carry out lignin degradation. In addition, reductive enzymes including cellobiose oxidizing enzymes, aryl alcohol oxidases and aryl alcohol dehydrogenases also seem to play major roles in ligninolysis.

2.3.2b. Polyphenol oxidase Phenol oxidases are excreted mainly by microorganisms and extensively exist in bacteria, fungi, plants and animals (Alfred, 2006). Based on substrate specificity, phenol oxidases can be divided into laccases and polyphenol oxidases (Jing et al., 2009). They use oxygen as the final electron acceptor to catalyze the oxidation of recalcitrant aromatic compounds such as lignin into more readily available substrates (Cullen and Kersten, 1996). In vitro studies have proven that polyphenol oxidases were involved in the degradation of natural phenols with more complex structures such as anthocyanins and flavanols (Finger, 1994). Many studies reported that purified phenol oxidases are involved in the biodegradation and detoxification processes of some aromatic pollutants (Carine et al., 2007; Farnet et al., 2004). Moreover, these enzymatic activities can be inhibited or induced by various xenobiotic compounds, such as heavy metals (Baldrian and Gabriel, 2002; Tuomela et al., 2005).

2.3.2c. Manganese independent peroxidase Glenn and Gold (1985) reported that manganese peroxidase (MnP) is another type of heme peroxidase that produce extracellularly in the culture media by fungi. This enzyme shows a strong preference for Mn(II) as its reducing substrate. The product Mn(III) forms a complex with organic acids and diffuses away from the enzyme to oxidize other materials, such as lignin. MnP oxidizes Mn(II) to Mn(III) which then

Review of Literature oxidizes phenolic rings to phenoxyl radicals which lead to a decomposition of compounds (Gold et al., 1989; Dominic and Wong, 2009).

2.3.2d. Other enzymes Lignin peroxidase (LiP) was first discovered in the Phanerochaete chrysosporium (Glenn et al., 1983; Tien and Kirk, 1983) and is produced by many white-rot fungi. This enzyme is an extracellular hemeprotein, dependent on H2O2 with an unusually high redox potential and low optimum pH (Gold and Alic, 1993). It shows little substrate specificity, reacting with a wide variety of lignin model compounds and even unrelated molecules (Barr and Aust, 1994). It can oxidize methoxylated aromatic rings without a free phenolic group, generating cation radicals that can react further with a variety of pathways, including Cα-Cβ cleavage, ring opening, demethylation and phenol dimerisation. The first report on purification and characterization of lignin peroxidase from Penicillium sp. P6 was reported by Yang et al. (2005).

The peroxidases are heme-containing enzymes with catalytic cycles that involve the activation by H2O2 and substrate reduction of compound I and compound II intermediates (Ralph et al., 2004). Lignin peroxidases have the unique character to catalyze oxidative cleavage of C-C bonds and ether (C-O-C) bonds in non-phenolic aromatic substrates of high redox potential. Versatile peroxidases are hybrids of lignin peroxidase and manganese peroxidase with a bifunctional characteristic (Dominic and Wong, 2009).

2.4. Cyanobacteria - potential for applied purposes Cyanobacteria are free living photoautotrophic micro organisms widely distributed in soil, fresh water and marine habitats. They are gram negative, filamentous or unicellular, and grow as mats on the surface of bare soil as primary colonizers (Roger and Reynand, 1979). The cyanobacteria are a well recognized source with a vast range of pharmacologically important bioactive compounds like antimicrobial (Patterson, 1996) and anti-neoplastic compounds (Suda et al., 1986).

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Cyanobacteria from marine origin show a wide range of bioactive compounds. Microcystins and nodularins are the most common among them. They produce two types of toxins, namely hepatotoxins and neurotoxins. Microcystins toxicosis may exhibit jaundice, shock, abdominal pain/distention, weakness, nausea/vomiting, severe thirst, rapid/weak pulse and eventually death. Among the neurotoxins, especially those from Anabaena affinis, show toxicity against tumuor cells and can be applied in tumour treatment. Most microcystins display a LD50 of 50-100 µg/kg of mice (Namikoshi et al., 1994).

Cyanobacteria can produce a wide variety of linear (e.g. aerugenosins and microginins) and cyclic peptides (e.g. anabaenopeptins, anabaenopeptilides, microviridins, nostopeptilides). These may not be acutely toxic but have other bioactivities such as serine protease inhibition (Rantala et al., 2004). They are responsible for oral and gastrointestinal inflammation, allergic reactions and skin irritation, resulting from the ingestion of cyanobacterial cells or after contact with water blooms (Voloshko et al., 2008).

Cyanobacteria represent a vast resource of biologically active compounds that may find tremendous application in agriculture and the pharmaceutical industry. Toxic water blooms comprising genera such as Microcystis, Anabaena and Nostoc, produce a diverse array of bioactive compounds exhibiting antibiotic, antifungal, algicidal, cytotoxic, immunosuppressive and enzyme inhibiting activities (Pranitha et al., 2008).

Saxitoxin and neosaxitoxin are neurotoxins which block sodium channels of nerve cells making them incapable of generating a nerve impulse. Saxitoxin/ neosaxitoxin toxicosis may exhibit weakness, staggering, loss of muscle coordination, difficulty in swallowing, labored respiration, complete muscle paralysis and death. Humans may also exhibit tingling around the mouth and finger tips, as well as slurred speech (Crayton, 1997).

Other bioactive compounds from plants such as jervinone from the rhizomes of Veratrum album exhibited antihypertensive effects in a dose-dependent manner. On the other hand, a new triterpenoidal saponin of hederagenin named Symphytoxide-A has been isolated from the roots of Symphytum officinale. This compound also exhibited

Review of Literature hypotensive activity in anaesthetized rats. Ebeinone has been isolated from the bulbs of Fritillaria imperialis which shows anticholinergic activity in isolated tissue experiments. Ebeinone also completely block inhibitory responses of acetylcholine (Sener, 1994).

For cytotoxic assays using HL-60 cells (Human monocytic leukaemia cells) strong apoptotic effects were observed when cells were exposed to methanol or dichloromethane extracts of cyanobacteria. It is interesting to note that cyanobacterial strains and extracts that caused a higher percentage of apoptotic HL-60 cells were the ones that exhibited antibacterial activities. It is also possible that the same cyanobacteria strain produces different bioactive substances targeted against different organisms or biochemical processes (Rosario et al., 2008).

2.4.1 Biotechnological potential The search for new photosynthetic organisms from different environment with high growth rates, high biomass yields and a high utilization potential is essential in order to produce bioactive compounds cost effectively. The new source organism could be mass cultured in waste water and may play a dual role as feed or fertilizer.

Cyanobacteria are ideally suited to perform these functions by virtue of their high flexibility to adapt to a varied array of environments and their known nutritional and fertilizer value (Walsh and Merill, 1984; Subramanian and Sundaram, 1986). Blue green algal extracts are known to stimulate the growth of plants, and this may be due to the presence of growth hormones (Borowitzka, 1988).

Cyanobacteria are capable of abating various kinds of pollutants and have been used in the production of energy, fertilizers, human food, animal feed, polysaccharides, biochemicals and pharmaceuticals (Hall et al., 1995). Immobilized as well as free living cyanobacterial applications were found to be distinctly advantageous over control as it enhanced various parameters of growth significantly such as shoot length, root length, fresh and dry weight of the plants, chlorophyll and protein content over a period of 30 days (Sophiarajini, 1995).

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Ardelean and Zamea (1996) reported that cyanobacteria have a very versatile metabolism (photosynthesis, either oxygenic or anoxygenic, respiration fermentation, nitrogen fixation, etc.) thereby, generating a response against many environmental factors. The cyanobacteria contain organic carbon and nitrogen that are considered as efficient biofertilizers in increasing soil fertility.

Namikoshi and Rinehart (1996) found a number of compounds in cyanobacteria which are inhibitors of proteases like micropeptins, cyanopeptolins, oscillapeptin, microviridin, aeruginosins and other enzymes, while still other compounds have no recognized biological activities. In general are cyclic peptides and depsipeptides the most common structural types, but a wide variety of other types are also found: linear peptides, guanidines, phosphonates, purines and macrolides. The close similarity or identity in structure between cyanobacterial products and compounds isolated from sponges, tunicates and other marine invertebrates suggests that the latter compounds may be derived from dietary or symbiotic blue-green algae.

2.4.2 Bioactive compounds from cyanobacteria The importance of cyanobacteria has been realized since long. In recent years people throughout the world are focussing on cyanobacteria in the production of biofuels, ammonia, various metabolites, vitamins, toxins, therapeutic substances and animal feeds (Mohanty, 2004). The discovery of new compounds decreased steadily over the past few decades from these families (Actinomycetes and Lypomycetes). Hence, it became important to discover new sources of bioactive natural products to treat newly emerging infections, diseases and also those that have become resistant to currently available drugs. In the last decade, microalgae have drawn much attention for their potential as excellent sources of biologically active constituents (Moore et al., 1988).

Algae are photosynthetic prokaryotes used as food by humans; they have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores throughout the world. They are also reported to be a source of fine chemicals, renewable fuel and bioactive compounds with antiviral, anti-tumor, antibacterial, anti-HIV and food additive effects (Singh et al., 2005).

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Many cyanobacteria produce compounds with potent biological activities. These compounds are generally considered to be secondary metabolites that are not essential for general metabolism or growth of the organism and are present in restricted taxonomic groups (Sivonen and Jones, 1999). Many potent toxins are secondary metabolites, causing health problems for animals and humans when the producer organisms occur in masses in water bodies. The toxins produced by cyanobacteria (cyanotoxins) are grouped into two categories on the basis of the bioassay methods used to screen them: cytotoxins and biotoxins (Carmichael, 1997).

Many species of cyanobacteria (blue-green algae) have potent biotoxic or cytotoxic properties. These metabolites differ from the intermediates and cofactor compounds that are essential for cell structural synthesis and energy transduction. Thus, cyanobacteria produce a large number of compounds with varying bioactivities (Rao et al., 2002). Based on the reports of potential toxicity associated with cyanobacteria, new ventures have been initiated to screen materials from natural populations of cyanobacteria (Rao et al., 2002).

Highly encouraging preliminary results on antiviral (Boyd, 1988) antibacterial (Sundararaman et al., 1992) and antifungal (Decaire et al., 1993) activities of different marine cyanobacterial strains have resulted in expanding the scope of the work towards the isolation, purification and identification of active compounds and also the development of novel drugs. Oscillatoria formosa BDU 40261 and Phormidium argustissium BDU 40061 were possessing antibacterial compounds against gram positive and gram negative pathogens (Sundararaman et al., 1992).

2.4.3 Pharmacological potentials The chemical compounds isolated from cyanobacteria are of biotechnological interest especially for clinical applications since they show antibiotic, algicidal and cytotoxic properties and hence it could be used as biocontrol agents of bacterial and fungal pathogens (Borowitzka, 1995). In addition to neurotoxins and hepatotoxins, cyanobacteria are known to produce several antibacterial compounds (Carmeli et al., 1990), antifungal compounds (Frankmolle et al., 1992), antiviral agents, anticancerous and (or) anti- neoplastic agents (Smith et al., 1994; Nianjun et al.,

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2004; Voloshko et al., 2008) and compounds useful in the treatment of HIV (Gerwick et al., 1994; Harrigan et al., 1998).

Burja et al. (2001) reported that many of the cyanobacterial secondary metabolites are of pharmaceutical importance that includes hepatotoxic, neurotoxic, cytotoxic compounds and toxins responsible for allergic reactions (Carmichael, 1994; Volk and Mundt, 2006). In recent years, there has been a tremendous enhancement in studies of their biological significance, especially those produced by cyanobacteria, as they have proven to be exciting molecules with immune modulatory, bioregulatory, and therapeutic potential (Singh et al., 2005).

Mundt et al. (2001) reported that cyanobacteria produce a variety of secondary metabolites with antibiotic, algicidal, cytotoxic, immunosuppressive and enzyme inhibiting activities. Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria isolated from German lakes and the Baltic sea showed antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems. Cyanobacteria including microcystin-producing strains produce a large number of peptide compounds, e.g. micropeptins, cyanopeptolins, microviridin, circinamide, aeruginosin, with varying bioactivities and potential pharmacological applications (Rao et al., 2002).

It was reported that Curacin A and the dolastatins are examples of important marine cyanobacterial metabolites possessing exquisite anticancer properties. Genetic studies on the biosynthetic capacity of these marine microalgae revealed many novel biochemical properties pertaining to the enzymes of secondary metabolism (Tong, 2007).

Recent report by Rajesh et al. (2009) revealed that cyanotoxins have ecological roles as allelochemicals and could be employed for the commercial development of compounds with applications such as algaecides, herbicides and insecticides. Also he added that cyanobacteria have become an attractive source of innovative classes of pharmacologically active compounds showing interesting biological activities that range from antibiotic, immunosuppressant and anticancer, antiviral, antiinflammatory and proteinase-inhibiting agents (Rajesh et al., 2009).

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2.5. Bioactive Compounds tested with animal models Plant-derived phenolic compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis in vivo. In a study, the efficacy of several plant derived phenolics, including caffeic acid phenethyl ester (CAPE), curcumin, quercetin and rutin were investigated for the prevention of tumors in C57BL/6J-Min/+ (Min/+) mice. These animals bear a germline mutation in the Apc gene and spontaneously develop numerous intestinal adenomas by 15 weeks of age (Mahmoud et al., 2000). At a dietary level of 0.15%, CAPE decreased tumor formation in Min/+ mice by 63%. Curcumin induced a similar tumor inhibition. Quercetin and rutin, however, both failed to alter tumor formation at dietary levels of 2%. Examination of intestinal tissue from the treated animals showed that tumor prevention by CAPE and curcumin was associated with increased enterocyte apoptosis and proliferation. CAPE and curcumin also decreased the expression of oncoprotein ß-catenin in the enterocytes of the Min/+ mouse, an observation previously associated with an antitumor effect. These data place the plant phenolics CAPE and curcumin among a growing list of anti-inflammatory agents that suppress Apc-associated intestinal carcinogenesis (Mahmoud et al., 2000).

The effects of dietary phenolic compounds on inhibition of intestinal sucrase were investigated in brush border membrane vesicles purified from rat small intestine. Screening experiments with different classes of phenolic compounds in both oxidized and native forms were tested. The most potent inhibitor was native tannic acid at 0.1 mg/ml, resulting in an 80% loss of activity. Oxidized tannic acid had no effect. Other phenolic compounds such as ferulic, p-coumaric and caffeic acids tend to be slightly inhibitory, while no inhibition was observed with vanillin or chlorogenic acid at the concentrations tested and the results confirmed the enzymatic inhibitory action of tannic acid and also demonstrated that some individual dietary phenolic monomers have the potential to modulate enzyme activity in a brush border membrane vesicle of a model system (Welsch et al., 1989).

Juliflorine, an antimicrobial alkaloid isolated from Prosopis juliflora possesses some immuno-modulating activity. This activity was tested in rabbits and compared with Freund's complete adjuvant in which Listeria hemolysin (antigen) was injected intramuscularly along with varying concentrations of juliflorine and a dose related

Review of Literature immune response was noted. After four weeks, following weekly doses of 30 mg/kg of juliflorine, the antihemolysin titre was found high (1:1280) and more than that with Freund's complete adjuvant. On the other hand repeated injections of 30 mg/kg of juliflorine were found to be toxic and produced tissue degeneration, increased lymphocyte count as high as 95% and high level of lactate dehydrogenase, cholesterol and triglyceride (Ahmad et al., 1992).

Mazzuca et al. (2003) reported that extracts of different polarities from Prosopis alpataco, Prosopis denudans var. denudans, Prosopis denudans var. patagonica and Prosopis denudans var. stenocarpa were screened in order to evaluate their antibacterial, antifungal, antifeedant, antihelminthic, molluscicidal and toxic activities. The extractions of the plant materials were carried out successively with petroleum ether, dichloromethane, ethyl acetate, methanol and water. All petroleum ether extracts showed antibacterial activity. The dichloromethane extract of P. alpataco showed antibacterial and antifungal activities. Methanol and aqueous extracts of P. denudans var. denudans and P. denudans var. patagonica showed antifungal activities and a slight response to the toxicity test. Fatty acids and a group of pentacyclic triterpenes were identified as being responsible for antibacterial activities in some of the active extracts.

The methanolic extract of Prosopis juliflora bark (MEPJ) 100, 200 and 400 mg/kg exhibited significant anti-inflammatory activity in acute and chronic inflammatory models. At the dose level 400 mg/kg showed maximum inhibition of 55.32% in carrageenan induced rat paw oedema while the standard diclofenac inhibited it by 61.33% after 3hr of carrageenan injection. All the doses of MEPJ showed dose dependent inhibition against histamine and serotonin induced rat paw oedema as compared with control animals. Furthermore, the same dose levels successfully reduced the formation of granulation tissues by cotton pellets in rats (Sivakumar et al., 2009).

2.5.1 Phenolic compounds Phenolic compounds comprise one of the largest and most ubiquitous groups of plant metabolites. They are formed to protect the plant from photosynthetic stress, reactive oxygen species, wounds, infections and herbivores. Phenolic compounds are an important part of the human diet. The most commonly occurring ones in foods are

Review of Literature flavonoids and phenolic acids. Early interest in polyphenols was related to their antinutritional effects but current interest stems from the observations that dietary polyphenolic compounds have antioxidative, antiinflammatory and anticarcinogenic activities (Chung et al., 2001).

All plant phenolic compounds arise from the common intermediate, phenylalanine or its close precursor, shikimic acid. They can be divided into at least ten different classes based on their general chemical structures (Strack, 1997). 4-HBA is a phenylpropanoid compound which act as a key intermediate in the biosynthesis of several secondary metabolites in plants including red napthoquinone pigment shikonin (Yazaki et al., 1991).

Common feature of phenolic compound is the presence of at least one hydroxyl substituted aromatic ring system. Phenolic compounds seem to be universally distributed in plants and they have been the subject of a great number of chemicals, biological, agricultural and medical studies. Also phenolic acids form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids. Hydroxycinnamic acid compounds occur most frequently as simple esters with hydroxy carboxylic acids or glucose. Hydroxybenzoic acid compounds are present mainly in the form of glucosides (Chung et al., 2001).

Ferulic acid is a major cinnamic acid derivative in the cell walls of a number of monocotyledons particularly in Poaceae (Parr et al., 1996). This phenolic acid is thought to play an important role in altering the mechanical properties of cell walls and digestibility of grasses by acting as a cross-link between polysaccharides and lignin (MacAdam and Grabber, 2002). Moreover, derivatives of ferulic acid are well known for their antioxidant properties (Garcia et al., 1997).

Hye et al. (2009) isolated a biologically important polyphenol (+)-catechin from catechu, the extract of the red heartwood of Acacia Catechu tree and reported the chief constituents of the red heartwood are catechin and catechu tannic acid. It also contains tannin, flavotannin, gallotannin, phloratannin, etc. (+)-Catechin is a substance, which diminish or arrest the action of some hormones. The tanning property of catechin in human skin may be supposed to be the active ingredient for the treatment of

Review of Literature leucoderma (shiti). Catechin has antihormone activity and its further activity has also been correlated with those of vitamin.

2.5.2 Metabolites from Plant sources Plant secondary metabolites are usually classified according to their biosynthetic pathways. Phenolics, steroids and alkaloids are the widespread metabolite family that provides secondary compounds which constitutes the basis for chemotaxonomy and chemical ecology (Harborne, 1999). These compounds are known to play a major role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals (Bourgaud et al., 2001).

Polyphenols like caffeic acid, ellagic acid, chlorogenic acid and ferulic acid (0.02–0.05% in the diet) are shown to inhibit 4-nitroquinoline-1-oxide (4-NQO)- induced tongue carcinogenesis in rats (Tanaka et al., 1993). Also studies reported that curcumin (0.2% and 0.6% in the diet), administered to azoxymethane (AOM)-treated rats during the promotion or progression stage, inhibited colon tumorigenesis (Kawamori et al., 1999). The multiplicity of both the invasive and noninvasive adenocarcinomas was lower and the apoptosis of colonic tumor cells was higher in the curcumin-treated group. Curcumin (0.1% in the diet) also inhibited intestinal tumorigenesis in the APCmin mice (Mahmoud et al., 2000).

Phenol Steroids Alkaloid

Fig. 7. Plant Secondary metabolites.

2.5.3 Antioxidant properties of bioactive compounds Antioxidant compounds recently have attracted attention due to their broad spectrum of activities in disorders of multiple origin viz. coronary heart disease, cancer,

Review of Literature diabetes, rheumatic disorders and inflammatory conditions where free radicals play an important role (Rao et al., 2004).

Research in the recent past has accumulated enormous evidences revealing that enrichment of body systems with natural antioxidants may correct the vitiated homeostasis and can prevent the onset as well as treat diseases caused and/or fostered due to free-radical mediated oxidative stress. These developments accelerated the search for antioxidant principles that lead to the identification of natural resources, the isolation of active principles and further modification and refinement of active antioxidant molecules (Halliwell, 1994; Tiwari, 1999; Pietta, 2000).

In foods, antioxidants have been defined as a substance that in small quantities are able to prevent or greatly retard the oxidation of easily oxidizable materials such as fats (Chipault, 1962). However, in biological systems the definition for antioxidants has been extended to any substance that when present at low concentrations compared to those of an oxidizable substrate significantly delays or prevents oxidation of that substrate like lipids, proteins, DNA, and carbohydrates (Halliwell, 1990). Currently however, biological antioxidants have further assumed a broad definition to include repair systems such as iron transport proteins (e.g. transferrin, albumin, ferritin and caeruloplasmin), antioxidant enzymes and factors affecting vascular homeostasis, signal transduction and gene expression (Frankel and Meyer, 2000).

Antioxidants may exert their effects by different mechanisms such as suppressing the formation of active species by reducing hydroperoxides and H2O2 and also by sequestering metal ions, scavenging active free radicals and repairing damage. Similarly, some antioxidants also induce the biosynthesis of other antioxidants or defense enzymes. The bioactivity of an antioxidant is dependent on several factors like their structural criteria, physico-chemical characteristics and in vivo radical generating conditions (Tiwari, 2001).

The biological activity of the leaves and pods of Prosopis chilensis and Prosopis tamarugo has been determined for free radical scavenging activity. The results suggest that the activity is related to polyphenols. The alkaloids β-phenethylamine and tryptamine were isolated from P. chilensis, and phenethylamine

Review of Literature was detected in P. tamarugo. At the concentration of 0.50 mg/ml both compounds showed DNA binding activity with values of 18.5 and 11% respectively. The exudates of Prosopis alba showed a strong free radical scavenging effect. The activity was related with the total phenolic content which consists mainly of catechin (Astudillo et al., 2000).

Rao et al. (2004) reported an antioxidant compound (-)-mesquitol from an new source Dichrostachys cinerea which has been compared with existing pharmacologically acceptable additives and proved to be useful antioxidant molecule than the presently used medicinally important lipophilic antioxidants probucol and α- tocopherol (Madhusudana et al., 2004). Thus studies have reported the possibility of developing new biocides, pharmaceutical products and analogous to natural products from the extracts (Inamori et al., 2000; Baya et al., 2001). Such products if developed synthetically would be more environmentally acceptable due to their composition based on analogies with natural extracts present in plants. Indeed the search for potent natural antioxidants from plant sources as nutritional supplements for health foods is gaining a lot of interest worldwide. Antioxidants are also used as a key component for inhibiting or reversing carcinogenesis (Wang et al., 2004).

Many publications have described the inhibition of tumorigenesis by plant polyphenols. The inhibitory effect of topically applied caffeic acid, ferulic acid, chlorogenic acid and curcumin on tumor promotion by 12-O-tetradecanoylphorbol-13- acetate (TPA) has been demonstrated by Huang et al. (1997).

Tapia et al. (2000) reported the biological activity of the alkaloids tryptamine, piperidine and phenethylamine derivatives isolated from the extracts of aerial parts of Prosopis alpataco, Prosopis argentina, Prosopis chilensis, Prosopis flexuosa and Prosopis pugionata. The isolated compounds were assessed for DNA binding, β-glucosidase inhibition and free radical scavenging effect using the DPPH decolouration assay. At the concentration of 0.50 mg/ml, DNA binding activities ranged from 28% for tryptamine to 0-27% for the phenethylamine and 47-54% for the piperidine derivatives. Tryptamine and 2-β-methyl-3-β-hydroxy-6-β-piperidinedo- decanol showed a moderate inhibition (27-32%) of the enzyme β-glucosidase at 100 μg/ml. The exudate of P. flexuosa which has the active constituent catechin displayed a

Review of Literature strong free radical scavenging activity in the DPPH decolouration assay (Tapia et al., 2000).

Almaraz et al. (2007) suggest that pollen of P. juliflora is an important source of flavonoids, which can be considered as natural antioxidants. Mesquite pollen extracts showed antioxidant activity related to the flavonoid concentration in both the in vitro biological system and in vivo system with a lower activity in the latter of these systems. Under in vivo conditions and in those in which a state of oxidation is not induced, a high concentration of flavonoid in the extract of mesquite pollen can have a pro-oxidant effect.

MATERIALS AND

METHODS

Materials and Methods

3.1 GROWTH OF CYANOBACTERIA 3.1.1 Cultures used Unicellular and filamentous non-heterocystous marine cyanobacteria were obtained from the germplasm of National Facility for Marine Cyanobacteria (NFMC), Bharathidasan University, Tiruchirappalli, Tamilnadu, India.

1. Gleocapsa sp. BDU 110711 2. Chroococcus turgides BDU 142111 3. Synechocystis pevalekii BDU 130051 4. Spirulina subsalsa BDU 141021 5. Oscillatoria salina BDU 92071 6. Oscillatoria laetevirens BDU 20801 7. Phormidium tenue BDU 141753 8. Phormidium valderianum BDU20041 9. Phormidium corium BDU 60121 10. Phormidium fragile BDU 42911 11. Lyngbya sp. BDU 90181 12. Lyngbya sp. BDU 141961

3.1.2 Media and growth conditions Marine cyanobacteria were grown and maintained in Artificial Sea Nutrients- III (ASN III) (Rippka et al., 1979) medium under white fluorescent light of 13.8 µE m-2 s-1 at 25 ±2ºC with 14/10 hr Light/Dark cycle.

Materials and Methods

3.1.2a Composition of ASN III Medium

Name of the Chemicals Amount (g L -1)

NaCl 25.0

MgSO4 .7H2O 3.5

MgCl2.6H2O 2.0

KCl 0.5

CaCl2.2H2O 0.5

NaNO3 0.75

K2HPO4.3H2O 0.02

EDTA disodium salt 0.0005

Citric acid 0.003

Ferric ammonium citrate 0.003

*A5 micronutrients 1.0 mL

Distilled water 1000 mL

pH 7.2 – 7.8

*A5 Micronutrients

Name of the Chemicals Amount (g L-1)

H3BO3 2.86

MgCl2.4H2O 1.81

ZnSO4.7H2O 0.22

NaMoO4.2H2O 0.39

CuSO4.5H2O 0.079

Co(NO3)2.6H2O 0.494

Distilled water 1000 mL

Materials and Methods

3.1.3 Lignocellulosic material Woody stems of Prosopis juliflora were collected from Bharathidasan University campus, Trichirapalli, Tamilnadu. The finely chopped wood was dried under sunlight and ground into powder in a ball mill and passed through a 100 µm- 1mm, 1 mm-2 mm, 2 mm-3 mm mesh sieves.

Scientific classification Kingdom Plantae Division Magnoliophyta Class Magnoliopsida Subclass Rosidae Order Fabales Family Fabaceae Subfamily Mimosoideae Tribe Mimoseae Genus Prosopis Species juliflora

3.1.4 Screening of cyanobacteria The listed marine cyanobacteria were grown with the lignocellulosic waste P. juliflora separately at a fresh:dry weight ratio of 0.1:0.1 (cyanobacteria: lignocellulosic) in ASN III media under above mentioned conditions (Ref. 3.1.2) for 15 days. Effective strain was selected based on the growth of cyanobacteria along with the lignocellulose.

3.2 DEGRADATION OF PROSOPIS JULIFLORA BY O. LAETEVIRENS 3.2.1 Optimization of P. juliflora wood particle size and dry weight ratio The ability of the selected cyanobacterium O. laetevirens to grow in the presence of the different wood particle size (100 µm-1 mm, 1-2 mm, 2-3 mm) and at varying dry weight ratios ( 0.05, 0.1, 0.2, 0.3 and 0.4) were tested in ASN III media as above mentioned earlier (Ref. 3.1.2) for 15 days. The pellet and supernatants were separated and subjected to biochemical analysis to check the degradability of P. juliflora wood particle by O. laetevirens.

Materials and Methods

3.2.2 Biochemical estimations of degraded P. juliflora

3.2.2a Estimation of Chlorophyll a (Mackinney, 1941)

Principle Chlorophyll content provides the physiological status of the cyanobacteria and the estimation of chlorophyll usually involves the extraction of this fat-soluble pigment in methanol.

Reagents 80% methanol

Procedure  One gram of O. laetevirens culture was taken and centrifuged at 5000 rpm for 10 min.  Pellet was washed twice in distilled water.  Pellet was resuspended in 4 mL of methanol and vortexed thoroughly.  Tubes were incubated in a water bath at 60°C for 1 hr, in dark with occasional shaking.  The suspension was centrifuged at 5000 rpm for 10 min and the supernatant was stored.  The process was repeated to ensure complete extraction.  Absorbance of the supernatant was read at 663 nm in JASCO UV-Vis spectrophotometer against 80% methanol as blank.  The amount of chlorophyll a in the sample was calculated using the formula

Calculation

A663 X 12.63 X weight of sample Chl a = ------mg/g Volume of methanol

A663 - absorbance at 663nm 12.63 - correction factor and the amount was expressed as mg/g

Materials and Methods

3.2.2b Estimation of Reducing Sugar (Miller, 1959)

Principle 3,5-dinitrosalicylic acid (DNSA) reagent appears yellow due to its nitro group. An alkaline solution of DNSA is reduced to 3-amino, 5-nitrosalicylic acid with the reducing sugar to form orange brown coloration which was read at 540 nm.

Reagents a) Dinitrosalicylic acid (DNSA): 1g DNSA was dissolved in 20 mL 2 N NaOH ( Sodium hydroxide) which was made upto 100 mL with distilled water b) 30 g of Sodium potassium tartrate

Procedure  To 1 mL of sample, 1 mL of reagent was added.  The tubes were kept in boiling water bath for 10 min.  After cooling it was made up to 10 mL by adding distilled water.  Absorbance was measured spectrophotometrically at 540 nm.  Concentration of sugar was determined by plotting optical density against the standard curve, prepared using sugar (glucose) concentration from 10-100 µg/mL.

3.2.2c Estimation of Phenol (Bray and Thorpe, 1954)

Principle Phenols are aromatic compounds that possess one (or) more hydroxyl substituent bonded on to an aromatic ring. Total phenol estimation can be carried out with the Folin-Ciocalteu’s agent.

Phenols react with phosphomolybdic acid in Folin-Ciocalteu’s reagent in alkaline medium to produce blue coloured molybdenum complex.

Reagents a) 12% Sodium carbonate solution b) Folin’s phenol: Distilled water (1:2)

Materials and Methods

Procedure  To 1 mL of culture filtrate 2.5 mL of 12% sodium carbonate solution was added and shaken well.  Then 0.75 mL of Folin's phenol was added and mixed thoroughly.  The tubes were incubated for 1 hr at room temperature.  After incubation, optical density was measured spectrophotometrically at 725 nm.  Concentration of phenol was determined by computing optical density against the standard curve, prepared using standard phenol concentration from 10-100 µg/mL.

3.2.2d Estimation of Lignin (Modified Klason Lignin Assay) (Tappi, 1992)

Principle

Sulfuric acid (H2SO4) hydrolysis is adopted to solubilize cellulose, hemicellulose and protein leaving the lignin as residue.

Reagents Concentrated sulfuric acid

Procedure  100 mg of lignocellulosic residue was taken into a boiling tube.  To this 2 mL of concentrated sulfuric acid was added and incubated for one hour with occasional shaking.  After incubation, 56 mL of distilled water was added.  The suspension was autoclaved at 121°C and allowed to cool.  The acid insoluble material (Klason Lignin) was collected on a pre-weighed filter paper (Whatman No.1) (A) and washed several times with distilled water.  The filter paper was dried at 70-80°C for 48-72 hr and weighed (B).  The lignin content was calculated by using initial and final weight of the filter paper (B-A). The final percentage of lignin present in the sample was calculated by

Lignin (%) = Initial weight / final weight X 100

Materials and Methods

3.2.3 Biodegradation with using optimized conditions  Based on the biochemical analysis the optimum ratio and particle size was selected as 0.1:0.3 (fresh:dry weight ratio) and 1-2 mm for better degradation of P. juliflora.  Hence, further experiments were carried out with the optimized ratio in which the biodegradation experiment was performed with cyanobacterial biomass (O. laetevirens) treated with lignocellulosic waste of P. juliflora in ratios of 0.1:0.3.  Respective control was also maintained with culture (O. laetevirens) alone and with waste (P. juliflora) alone.  The experimental setup was incubated for 30 days under white fluorescent light of 1500 lux at 25±2ºC with 14/10 hr dark/light cycle.  After the incubation period, pellet and supernatant were separated and subjected to biochemical estimations to confirm the degradative ability of the selected cyanobacterial strain and the rate of degradation.

3.2.4 Microscopic observation of P. juliflora degradation Microscopic observation at different stages (1st, 5th, 15th and 30th) of P. juliflora degradation by O. laetevirens was observed under light microscope at 10X magnification.

3.2.5 Estimation of growth parameters

3.2.5a Estimation of Chlorophyll a

The protocol was followed as mentioned elsewhere in the thesis (See section 3.2.2a).

3.2.6 Colorimetric enzyme assay Lignolytic enzyme profile of O. laetevirens was studied colorimetrically by estimating the activity of laccase, polyphenol oxidase and manganese independent peroxidase. The respective enzymes activity was also studied at varying pH (4, 5, 6, 7, 8 and 9) and temperatures (25 and 35°C).

Materials and Methods

3.2.6a Laccase (Caramelo et al., 1999)

Reagents Sodium acetate buffer Guaiacol

Procedure  0.1 mL of enzyme sample was added to 0.9 mL of sodium acetate buffer containing 10 mM guaiacol.  Optical density was immediately taken at 470 nm

3.2.6b Polyphenol oxidase (Caramelo et al., 1999)

Reagents Sodium tartrate buffer O-catechol.

Sulfuric acid (H2SO4)

Procedure  0.1 mL of enzyme sample was added to 2 mL sodium tartrate buffer containing 0.15 M O-catechol.  OD was taken at 420 nm for 2 min. and the reaction was stopped by 0.5 mL of

5% sulfuric acid (H2SO4).

3.2.6c Manganese independent peroxidase (Caramelo et al., 1999)

Reagents Sodium tartrate buffer 2,6-dimethoxy phenol Hydrogen peroxide

Procedure  0.1 mL of enzyme sample was added to 2 mL sodium tartrate buffer containing 0.1 mM 2, 6-dimethoxy phenol.

Materials and Methods

 4 mM H2O2 was added and the optical density was immediately taken at 469 nm.

3.2.6d Hydrogen peroxide (Green and Hill, 1984)

Reagents A. Phenol B. 4-aminoantipyrine C. Potassium phosphate buffer pH 6.9 (0.1 M) D. Horseradish peroxidase E. Hydrogen peroxide The reagent solution (100 mL) was prepared using 0.234 g reagent A, 0.10 g reagent B and 1 mL of reagent C and contains 2x10-8 M reagent D.

Procedure  The reaction mixture (4 mL) was mixed with the peroxide sample (control O. laetevirens and O. laetevirens exposed to P. juliflora) and made up to 10 mL with double distilled water.  The change in absorbance at 505 nm was measured until a constant reading was obtained (approximately 5 min. at ambient temperature).  A 4 mL aliquot of the reagent solution made up to 10 mL with double distilled water served as reference.  The amount of hydrogen peroxide released by O. laetevirens was calculated from a standard curve prepared with varying amounts (1-10 μM) of standard hydrogen peroxide.  The results are expressed as μmol hydrogen peroxide per gram dry weight.

3.2.7 Analysis of biochemical parameters

3.2.7a Estimation of reducing sugar

The protocol was followed as mentioned elsewhere in the thesis (See section 3.2.2b).

Materials and Methods

3.2.7b Estimation of Phenol

The protocol was followed as mentioned elsewhere in the thesis (See section 3.2.2c).

3.2.7c Spectrum analysis (Viswajith, 2008)

The ability of the cyanobacterium Oscillatoria laetevirens to grow in the presence of P. juliflora at varying dry weight ratios were tested in ASN III medium under previously mentioned condition for 40 days. The supernatants were centrifuged and subjected to spectral analysis using a Jasco UV- 550 spectrophotometer (Japan) in the wavelength range from 200-800 nm.

3.2.7d Estimation of nitrate (Jenkins and Medsken, 1964)

Principle 2, 4 phenoldisulphonic acid produces yellow coloured 6-nitro-1, 2, 4 phenoldisulphonic acid, (an alkaline salt) with nitrate which can be spectrophotometrically read at 410 nm.

Reagents Standard nitrate solution

13.7 mg sodium nitrate (NaNO3) was dissolved in 100 mL distilled water -1 [Concentration of nitrate (NO3) 100 μmL ].

Brucine reagent In a beaker 50 mL of distilled water and 3 mL of concentrated HCl was taken and heated to boil. 1 g of brucine and 0.1 g sulphanilic acid was added and stirred. The solution was allowed to cool and made up to 100 mL.

Sulphuric acid 500 mL of concentrated sulphuric acid was carefully mixed with 100 mL distilled water.

Materials and Methods

Procedure  2 mL of sample was taken into a 100 mL beaker.  To this, 1 mL of brucine sulphanilic acid reagent and 10 mL sulphuric acid was added.  The content was stirred gently for 5 minutes.  Then the beaker was covered with watch glass and kept in dark for 10 minutes.  After the development of yellow colour, 10 mL of distilled water was added and incubated in dark for 30 minutes.  Absorbance of solution was measured at 410 nm.  The concentration of nitrate present in the sample was determined by extrapolating the optical density in standard curve (10-100 µg) and expressed in µg/mL.

3.2.7e Estimation of ammonia (Emmet, 1968)

Principle Ammonia reacts with phenol and alkaline hypochlorite to form indophenols blue. The reactions are catalyzed by the nitroprusside or ferrocyanide. The resulting absorbance is proportional to the concentration of ammonia and is measured spectrophotometrically at 640 nm.

Reagents Standard ammonia solution Standard solution of ammonium chloride (conc.10 μg/mL) was prepared by dissolving 3.1 mg of ammonium chloride in 100 mL distilled water.

Hypochlorite stock (1.6 N) 5.5% chlorine solution.

Alkaline stock 100g tri-sodium citrate and 5 g of sodium hydroxide was dissolved in 300 mL of distilled water and made up to 500 mL.

Materials and Methods

Nitroprusside reagent 1 g of sodium nitroprusside was dissolved in 50 mL distilled water and made up to 200 mL.

Oxidizing reagent Alkaline stock and hypochlorite solutions were mixed in a 4:1 ratio. This solution was prepared freshly.

Phenol reagent 100 g phenol was dissolved in 50 mL of 95% ethyl alcohol and made up to 1000 mL with distilled water.

Procedure  To 1 mL of sample, 0.4 mL of phenol reagent and 0.4 mL of nitroprusside reagent was added and mixed well.  Then 1 mL of the oxidizing reagent was added and tubes are stoppered immediately.  The content was vortexed and incubated for 1 hr at room temperature in the dark.  The absorbance was measured at 640 nm in a spectrophotometer.  Standard graph was prepared using different concentrations of ammonia (1 to 10 μg ammonium chloride).  Ammonia concentration was determined by plotting the optical density in a standard graph.

3.2.7f Estimation of protein (Lowry et al., 1951)

Principle Protein reacts with Folin-Ciocalteu’s reagent to give a coloured complex. The colour formation is due to the reaction of the alkaline copper with the protein at the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of the colour depends on the amount of these aromatic acids present and this may vary for different proteins.

Materials and Methods

Reagents Alkaline sodium carbonate solution 2 g of sodium carbonate was dissolved in 0.1 N sodium hydroxide.

5% Copper sulphate 10% Sodium potassium tartrate Copper sulphate - Sodium potassium tartrate solution One part of copper sulphate solution was mixed with one part of sodium potassium tartrate solution and eight parts of distilled water was added.

Alkaline reagent Prepared freshly by mixing 50 mL of alkaline sodium carbonate solution and 1 mL of copper sulphate - sodium potassium tartrate solution.

Folin-Ciocalteu’s reagent The reagent was diluted with equal amount of distilled water.

Trichloro acetic acid 10% (w/v) in distilled water.

Standard protein Bovine serum albumin (BSA) (100 μg/mL)

Procedure  100 mg of sample was ground with water in mortar and pestle.  The contents were centrifuged at 5000 rpm for 5 min.  0.1 mL of this solution was made up to 1 mL using distilled water.  4.5 mL of alkaline reagent was added and incubated for 3 min.  Then 0.5 mL of Folin - Ciocalteu’s phenol reagent was added and allowed to stand for 30 min.  Absorbance was read at 750 nm in JASCO UV-Vis spectrophotometer.  The amount of protein in each sample was calculated using a standard graph and expressed as μg/mL.

Materials and Methods

3.2.7g Estimation of lignin

The protocol was followed as mentioned elsewhere in the thesis (See section 3.2.2d).

3.2.7h Estimation of holocellulose (Tappi, 1992)

Principle Holocellulose fractions were obtained after acidic hydrolysis of polysaccharides or delignification with sodium chlorite and glacial acetic acid.

Reagents Sodium chlorite Glacial acetic acid

Procedure  Extractive free dust 5 gm (oven dry) was taken in Erlenmeyer flask (500 mL) containing distilled water (160 mL).  The dust was then treated with sodium chlorite (1.5g) and glacial acetic acid (0.5 mL) was added.  The process was repeated at least 5 times till the dust became white.  Dust thus obtained was filtered through G2 crucible, washed with distilled water followed by acetone washing.  The crucible was dried to constant weight in an oven at 105±3 °C. The holocellulose content was calculated as follows:

Holocellulose (%) = A x 100 W

A = oven dried weight of holocellulose W = oven dried weight of sample

Materials and Methods

3.2.8 Phytochemical analysis (Edeoga et al., 2005)

3.2.8a Alkaloids (Dragandorff’s Test)

To 1 mL of extract, 1 mL of dragandorff’s reagent (Potassium bismuth iodide solution) was added. Formation of an orange red precipitate indicates the presence of alkaloids.

3.2.8b Flavonoids

5 ml of diluted ammonia solution was added to the extract followed by addition of concentrated sulfuric acid. A yellow coloration observed in each extract indicates the presence of flavonoids.

3.2.8c Terpenoids (Puncal D test)

A few mL of puncal D reagent (Ammonium Heptamolybdate + Ceric sulphate in concentrated sulphuric acids) solution was added to the extract and heated. Formation of blue colour indicates the presence of terpenoids.

3.2.8d Saponin

A few mL of water was added to the extract and formation of frothing indicates the presence of saponin.

3.2.8e Steroids

A few mL of concentrated sulphuric acid was added to the extract and formation of green colour indicates the presence of steroids.

3.3. COMPOUND IDENTIFICATION 3.3.1 Preparation of extract (Sirmah, 2009)  The degraded P. juliflora by O. laetevirens was ground to fine powder, passed through a 115-mesh sieve and dried at 60 ºC before extraction.  Soxhlet extraction was done using ethanol in which 10 g of sample powder was extracted with 180 mL of the solvent for 15 hours at a rate of 10 to 12 cycles per

hour.

Materials and Methods

 After extraction, the solvent was evaporated under reduced pressure in a Buchi

rotavapor and the crude extract dried under vacuum in a desiccator over P2O5.  The mass of the remaining extract was measured and used for phytochemical analysis, compound identification, animal experiments and antioxidant testing.

3.3.1a Spectrum analysis

The protocol was followed as mentioned elsewhere in the thesis (See section 3.2.7e).

3.3.1b Thin Layer Chromatography (TLC) (Touchstone, 1992)

Principle In thin layer chromatography, the mobile phase is a liquid and the stationary phase is a solid absorbent. The solid phase, adsorbent was coated onto a solid support as a thin layer (about 0.25 mm thick). The mixture to be separated was dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom. A solvent or mixture of solvents called eluant is allowed to flow up the plate capillary action.

Requirements Precoated Silica gel TLC plates TLC solvent chamber Capillary tubes Iodine chamber Iodine Ethyl acetate Hexane

Loading Sample  The precoated silica gel aluminium plates (Merck, Germany) were cut into 8 cm x7 cm size.  A line was drawn lightly with a pencil about 1.5 cm from the bottom and 1.0 cm from the top.

Materials and Methods

 The capillary tubes (1 mm in diameter) were washed with chloroform for three times to avoid contamination on each time of spotting.  The extracts to be separated were applied as a small spot (1 to 2 mm diameter) using capillary tubes.

Development of Thin Layer Plates  The chamber used for development of the chromatogram (20 cm x 10 cm) was covered with a glass plate.  The developing solvent, Ethyl acetate : Hexane (4:6) poured into the chamber was saturated by lining with filter paper for 30 minutes prior to development.  The spotted plate was then placed in the chamber with the spotted end down and the solvent level should be below the spots.  The solvent was then slowly raised in the adsorbent by capillary action.

Visualization  When the solvent front was moved to within about 1 cm of the top end of the plate (after 30 minutes), the plate was removed from the developing chamber.  The position of the solvent front marked and the solvent was allowed to evaporate.  Then the plate was kept in iodine chamber and visualized.

Calculation The relationship between the distance travelled by the solvent front and the substance is usually expressed as Rf value.

R Value = distance travelled by solu te / distance travelled by solvent f

The Rf values are strongly depend upon the nature of the adsorbent and solvent.

3.3.1c High Performance Thin Layer Chromatography (HPTLC) - (Khakpour et al., 2005)

Principle High performance thin layer chromatography is a sophisticated and automated form of TLC which utilizes the conventional technique of TLC in a more optimized

Materials and Methods way which includes an optimized coating material with separation power, with a new method of feeding the mobile phase with competent data acquisition and processing system.

Steps Involved In HPTLC 1. Selection of chromatographic layer. 2. Sample and standard preparation. 3. Layer pre-washing. 4. Layer pre-conditioning. 5. Application of sample and standard. 6. Chromatographic development. 7. Detection of spots. 8. Scanning. 9. Documentation of chromatic plate.

20 µl of samples was applied on precoated silica gel aluminium plates (Merk, Germany). HPTLC was performed using a CAMAG HPTLC Spectrometer provided with a scanner II densitometer and Linomat IV applicator.

Analysis of the Sample The samples were applied on an HPTLC plate using an automatic applicator. The following parameters were used.

Scanning Parameters Plate size 10x10 mm Scanning speed 4.0 mm/s Lamp Tungsten Wavelength 400 nm Reflectance/ Transmission Reflectance Sensor Automatic Span 5 Monochromator band 10 nm width

Materials and Methods

Detection and visualization  Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length).  Non UV absorbing compounds were observed by dipping the plates in 0.1% iodine solution.

3.3.1d High Performance Liquid Chromatography (HPLC) (Sirmah, 2009)

Principle HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analysed and the column. The principle is to force the analyte through a column of the stationary phase by pumping a liquid (mobile phase) at high pressure through the column.

Standard (+)-Catechin (98% assay) was used as reference control and purchased from Sigma Alderich

Requirements HPLC instrument (UFLC) Sonicator, with temperature control (BRAUN) Volumetric flasks, appropriate sizes Syringes, 3-cc disposable with Luer-lok tip Filters, 0.45-μm Pipettes

Chemicals Water (deionized) HPLC grade 0.05% of trifluroacetic acid Methanol (HPLC grade)

Materials and Methods

Running conditions HPLC column Supercosil TM LC-18 column (250 mm x 4.6 mm i.d,) Column temperature 35°C Mobile phase Solvent A (water containing 0.05% of TFA) Solvent B (methanol (HPLC grade) containing 0.05% of TFA) Flow rate 1 mL/min. Injection volume 20 μl Detection wavelength 210 nm to 400 nm Running time 30 minutes Post running time 10 minutes Detector Waters 2996 photo diode array (PDA) detector

3.3.1e Gas Chromatography Mass Spectroscopy (GC-MS) (Sirmah, 2009)

Principle GC-MS composed of two major systems, a gas chromatograph and a mass spectrometer. In gas chromatography the chemical properties between different molecules in a mixture separate the molecule as the sample travels the column and the mass spectrometer breaks each molecule into an ionized fragment and detects them using their mass to charge ratio.

Requirements Instrument: JEOL GCMATE II GC-MS

Chemicals Anhydrous acetonitrile N,O-bis-trimethylsilyl Trimethylchlorosilane

Sample Preparation Test samples were analyzed as trimethyl derivatives using the following procedure.

Materials and Methods

 In a screw-capped vial, 1 mg of dry sample was dissolved in 0.5 mL of anhydrous acetonitrile (Acros Organics).  0.4 mL of N,O-bis-trimethylsilyl (trifluoroacetamide) containing 1% of trimethylchlorosilane (Acros Organics) was added.  The solution was sonicated for about 1 min and heated at 60°C for 60 min.  After evaporation of the solvent in a stream of dry nitrogen, the residue was diluted in 1 mL of anhydrous acetonitrile.

Running conditions Column 5% diphenyl / 95% dimethyl polysiloxane fused- silica capillary column (Elite-5ms, 60 m x 0.25 mm, 0.25 mm film thickness, Perkin Elmer Inc,) Injector Mode split Injection volume 1 μl GC Inlet Temperature 250°C Total Flow 20 mL/min Septum Purge 1.2 mL/min GC Oven Ramp 200°C constant for 4 min, 200°C to 330°C at a rate of 5°C/min and then constant for 330°C Ion source electron impact Ionization voltage 70 eV Scan Range 35 to m/z 700 a.m.u Solvent Delay 10 min

3.3.1f Fourier Transform Infrared Spectroscopy (FTIR) (Sirmah, 2009)

Principle The FTIR spectrum is a plot of infrared light absorbed by the sample as a function of wavelength or frequency. It is perhaps the most powerful tool for identifying types of chemical bonds (functional groups). Thus, by interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined.

Materials and Methods

Running conditions Model: PERKIN ELMER Spectrum one FTIR Scan Range MIR 450-4000 cm-1 Resolution 1.0 cm-1 Sampling Technique KBr

3.3.1g Nuclear Magnetic Resonance Spectroscopy (NMR) (Sirmah, 2009)

Principle NMR spectroscopy is the name given to a technique which exploits the magnetic properties of certain nuclei. It is one of the principal techniques used to obtain physical, chemical, electronic and structural information about molecules due to either the chemical shift, Zeeman effect or the Knight shift effect or a combination of both on the resonant frequencies of the nuclei present in the sample. It is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in liquid or solid state.

Requirements Instrument: Bruker AVIII 500 MHz NMR spectrometer

Chemicals methanol-D4

Running conditions Magnetic field 11.7 Tesla, Wide bore (89mm) Probes 5mm Broad Band inverse probe Solvent methanol-D4 Magnetic nuclei 1H

3.4. PHARMACOLOGICAL STUDIES 3.4.1 Acute toxicity study (Ecobichon, 1997)  Acute toxicity studies were performed according to Organisation for Economic Cooperation and Development guidelines (OECD).

Materials and Methods

 Male albino rats (Rattus norvegicus) (150-180g) were selected by random sampling technique and employed in this study.  Before experimentation the animals were fasted for four hours with free access to water only.  Twenty five male rats were divided into five groups, containing five animals each were tested for this study up to a period of 3 days.  The first group served as control and received 3 ml/kg normal saline orally. Group II, III and IV received graded doses (50, 100, 200, and 500 mg/kg) of P. juliflora wood extract to observe the mortality.

3.4.2 Subacute toxicity study (Ecobichon, 1997) Experimental animals  Twenty (20-month old) albino rats (140-180 g body weight) were used.  Animals were housed in tarsons poly propylene cages (8”x12”x8”) with metal grill tops.  The cages were kept in a well ventilated room with 28±2ºC temperature, humidity of 50-60% and regular 12 hours light/dark cycle.

Experimental group  The animals were separated into four groups.  Group I served as control received 3 ml/kg normal saline orally.  Group II was orally administered with O. laetevirens extract (200 mg/kg body weight).  Group III received Prosopis juliflora extract (200 mg/kg body weight).  Group IV was administered with degraded Prosopis juliflora with similar dose.  Extract were given orally using oral gavage needle for 30 days without anaesthesia using hand restraint.

Food and water consumption All animals were allowed to free access of water and fed with commercially available pellet feed (M/s. Hindustan Lever Ltd., Mumbai).

Materials and Methods

3.4.3 Morphological observation 3.4.3a Body weight

The change in their body weight was measured at an initial and final stage of the experimental period using a electronic balance (Mettler toledo).

3.4.4 Haematological and biochemical analysis by auto analyzer Anaesthesia (William, 1965) The rats were anaesthetised using the solvent diethyl ether.

Blood Collection  Blood was collected from the orbital sinus (retro orbital puncture technique) of rats.  Bleeding requires that the capillary tube was inserted with gentle rotation while directing the tube caudally and towards the midline.  1-1.5 mL of blood was collected in EDTA coated tubes for haematological analysis.  Serum was obtained by centrifugation of fresh blood samples without EDTA at 3000 rpm for 10 minutes and used for biochemical analysis.

Analysis The following biochemical and haematological analysis were measured using a fully automated analyzer (Erba Mannheim EM 360 clinical chemistry analyser, Mannheim, Germany). Commerical Erba kits (Erba Diagnostics Mannheim, Germany) were used for this analysis.

Haematology  Haemoglobin  Erythrocyte Sedimentation Rate (ESR)  Packed Cell Volume (PCV)  Red Blood Cells (RBC)  White Blood Cells (WBC)  Neutrophil  Lymphocyte  Eosinophil

Materials and Methods

Biochemical studies  Glucose  Protein  Albumin  Globulin

Lipid analysis  Cholesterol  Triglycerides (TGL)

Renal function tests  Creatinine  Urea  Uric acid

Liver function tests  Serum Glutamic Pyruvic Transaminase (SGPT)  Serum Glutamic Oxaloacetic Transaminase (SGOT)  Total bilirubin  Indirect bilirubin  Alkaline Phosphatase (ALP)

3.4.5 Sperm count (Gopalakrishnan et al., 1990) Reagents Trypan Blue Phosphate buffer saline

Procedure  The cauda epididymal duct on one side was exposed and incised.  The connective tissue capsule around the cauda epididymus was teased out and the duct was uncoiled.

Materials and Methods

 The semen that oozed out into the cavity block was quickly sucked into a capillary tube up to the 0.05 µl mark and transferred to an eppendorf tube.  The sample was diluted 200-250 times in physiological saline.  After thorough mixing by blowing air throw a blowpipe, the sperm suspension was used for analysis of count.  Then a drop of the diluted semen was transferred to an improved neubauer counting chamber and a cover glass was overlaid.  The counting chamber was observed under a research microscope at 400X magnification and sperm in the central core were counted.  The central square which has 25 large squares and volume of each of the 25 squares was 0.1 mL.  The sperm counts are counted using the following formula

Sperm count = (Number of sperm in 25 squares / 25) x 10 x dilution factor x 1000

3.4.6 Histopathology For the study of the structure and function of the organ, the histological studies are used in which tissue sections were made and observed under microscope. The steps involved are as follows.

3.4.6a Kidney (Mark et al., 2008)

Step 1: Fixation  The fixation of the tissue was done with Bouin’s fluid. Composition of Bouin’s fluid: Saturated Picric acid-70 mL 40% formaldehyde-25 mL Glacial acetic acid-5 mL  The duration of fixation was 24 hrs. After fixation the tissues were washed with tap water to remove picric acid.

Materials and Methods

Step 2: Dehydration  The dehydration was performed with the series of increasing concentrations of ethanol , i.e 30%, 50%, 70%, 90% and 100%.

Step 3: Clearing  Clearing was done with xylene and the time period was 5 to 10 min.  The process was repeated thrice followed by infiltration with paraffin.

Step 4: Impregnation  Tissues were then taken out of xylene and kept in molten embedding bath.  The bath temperature is 58-60˚C and the process was repeated three times with 20 minutes gap.

Step 5: Embedding  Tissues are embedded in fresh paraffin wax and the optimum melting point of which is 58-60˚C.  The glass plate used for embedding was smeared with glycerine.

Step 6: Section cutting Section cutting was done with a rotary microtome.  Excess paraffin must be removed by trimming.  0.5 cm of the tissue was used.  Block was attached to a heated object holder.  Additional support by extra wax was given along with the length of the block.  Before sectioning the screws were tightened.  Uniform section microtome knife was adjusted in a proper angle.  Care must be taken so that the paraffin block was contacted by knife alone but not the knife holder.  The thickness of the section was 5-10 µm.

Step 7: Flattening and spreading  The tissue was floated in warm water bath.  The section spread was detached from the knife by hair line brush.  The slides are coated with egg albumin and then they are kept at room temperature.

Materials and Methods

Step 8: Staining  Deparaffinization with xylene was carried out for 2 times with 10 min interval.  After rehydration with descending grades of alcohol the sample was stained with haemotoxylin for 15 min followed by washing in tap water and blowing for 10 min.  Again the destained sample was rinsed with distilled water and stained with eosin dye.  After staining, the sample was dehydrated with ascending grades of alcohol followed by clearing with xylene for two times in 5 min interval.

Step 9: Mounting  The slide was mounted with DPX and a micro cover slip was placed and observed under microscope.

3.4.6b Liver

The protocol was followed as mentioned elsewhere in the thesis (See section 3.4.6a)

3.4.7 Antioxidant properties (Rao et al., 2008) Principle An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols.

The free radical scavenging activity of the extract was analyzed by free radicals like 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The various methods used to measure antioxidant activity of food products can give varying results depending on the specific free radical being used as a reactant.

Materials and Methods

Reagents 1, 1-diphenyl-2-picrylhydrazyl (DPPH) Methanol

Methodology  The initial OD value of 0.1 ml of DPPH solution (0.1 mM DPPH in methanol) and 0.8 mL of methanol was taken at 517 nm in UV Spectrometer.  10 mg of the test sample was weighed and dissolved in 1 mL of 99% methanol with a final concentration of 10 mg/ml.  In this assay, 100 μl of test sample was mixed with DPPH solution and incubated for half an hour.  The absorbance of the solution was then measured at 517 nm by a UV Spectrometer.  The radical scavenging activity was represented as percentage inhibition of DPPH radicals.

DPPH inhibition ratio was expressed as a percentage after being calculated from the following equation:

% inhibition = 100 x (ac –ae / ac)

where ac is the absorbance of the control and ae the absorbance of the test extract.

Data and Statistical Analysis Only significant data were included in the tables and were presented with mean and standard error (±) of three replicates per treatment and repeated three times. Two factor analysis of variance (ANOVA) was used to assess the significance (p = 0.05) of the mean values of treatments and the differences were compared using Duncan’s Multiple Range Test.

RESULTS AND

DISCUSSION

Results and Discussion

4.1. GROWTH AND DEGRADATION 4.1.1 Screening of cyanobacteria Among the thirteen cyanobacteria screened, three strains such as Oscillatoria laetevirens BDU 20801, Phormidium valderianum BDU 20041, Lyngbya sp. BDU 141961 were showed a better colonization on P. juliflora. However, Oscillatoria laetevirens colonized the substrates and started degradation more rapidly when compared to other cyanobacterial species (Table-1). Considering the high growth rate and colonization on the selected lignocellulosic material, Oscillatoria laetevirens (Plate-1a) was selected for further studies.

The microscopic observation of the selected cyanobacterium showed the following morphological features. The culture possessed trichomes single or forming mat or spongy thallus without sheath, motile, showing typical oscillatory movements, terminal portion of the trichome cells hooked or spindle shaped hormogones present (Desikachary, 1951).

Order - Nostacales

Family - Oscillatoriaceae

Genera - Oscillatoria

Species - laetevirens

4.1.2 Optimization of growth conditions Optimization of O. laetevirens growth with different sized particle (100 µm- 1 mm, 1-2 mm, 2-3 mm) (Plate-2a) of P. juliflora with varying dry weight ratios (0.05, 0.1, 0.2, 0.3 and 0.4) were tested. The results showed better colonization and degradation of lignocellulosic material of a particle size between 1-2mm by the

Results and Discussion

O. laetevirens. Also, O. laetevirens and P. juliflora in 0.1:0.3 (wet:dry weight) ratio was found to be optimum for colonization of cyanobacterium as well as degradation of lignocellulosic material. Wood particles of P. juliflora with the size of 1-2 mm and 0.3 dry weight ratio enhanced the cyanobacterial colonization and growth, additionally it also facilitated the penetration of O. laetevirens and enzyme secretion for the degradation.

4.1.2a Biochemical analysis Chlorophyll a Chlorophyll a estimation was carried out to determine the biomass of O. laetevirens which was grown along with P. juliflora. Results indicated that O. laetevirens grown with P. juliflora with different particle size and ratio (Fig. 8) on 15th day showed a high amount of chlorophyll content when compared to the control (O. laetevirens alone). Furthermore, O. laetevirens grown with 1-2 mm particle size and 1:3 ratio of P. juliflora showed higher amount of chlorophyll a content when compared with other particle size (100 µm-1 mm and 2-3 mm) and ratio (0.05, 0.1, 0.2 and 0.4) and control O. laetevirens alone.

Chlorophyll a has been used to determine the biomass of cyanobacteria (Manoharan and Subramanian, 1992; Shashirekha et al., 1997; Ramirez et al., 2000 Vijayakumar et al., 2005; and Saswati et al., 2004). Chlorophyll a content in Oscillatoria pseudogeminata var. unigranulata was studied by Manoharan and Subramanian (1993) to monitor its growth response on exposure to ossein effluent. Malliga (1993) studied the variation in chlorophyll a content to determine the growth response of Azolla pinnata to various nitrogen sources like nitrogen free medium, sodium nitrate, ammonium chloride and ammonium nitrate.

Anabaena azollae ML2 grown in the presence of coir pith showed an increase in growth rate in terms of chlorophyll a was reported by Malliga et al. (1996). Evaluation of degradative efficiency of petroleum hydrocarbons by Oscillatoria agardhi and Anabaena sphaerica revealed increase in biomass when compared with a control culture (Gamila et al., 2003). Parikh and Madamwar, (2005) measured the growth of cyanobacteria during textile dye decolorization by measuring chlorophyll a.

Results and Discussion

Thus it can be concluded that the increased chlorophyll a content in test samples indicated that the presence of lignocellulosic waste P. juliflora did not inhibit the growth of O. laetevirens but, surprisingly O. laetevirens utilized the degraded lignocellulosic materials as a source of nutrients for its growth and other metabolic activities.

Reducing Sugar The presence of reducing sugars in the supernatant can provide supporting evidence for the lignolytic activity of O. laetevirens. Concentration of reducing sugar was considerably higher in O. laetevirens degraded lignocellulosic’s culture filtrate (0.1:0.3 of O. laetevirens with P. juliflora respectively) when compared with control cyanobacteria and control lignocellulosic alone (Fig. 9). This may be due to cleavage of complex cellulosic material to simple sugars by the cyanobacterial enzymes. The obtained results were similar with the previous studies of Shasirekha et al. (2001) who reported the release of reducing sugars and amino acids into media during the growth of Pleurotus florida on rice straw. Also the reducing sugar content was found to be higher in culture filtrate of degraded P. juliflora by a fresh water cyanobacterium Phormidium sp. (Prabha et al., 2005). Yu et al. (2009) reported that biological delignification enhances the sugars released during the enzymatic hydrolysis. Roy et al. (2001) and Sigoillot et al. (2002) confirmed the xylanase activity in Aeromonas caviae and Pycnoporus cinnabarinus by measuring the reducing sugars released into the medium. Similarly, Ojumu et al. (2003) mentioned that the presence of reducing sugar in the supernatant could confirm the presence of cellulase activity in Aspergillus flavus Linn. isolate NSPR 101.

Furthermore, the presence of reducing sugars and aromatic compounds in media of pulp and paper production was used to confirm the production of peroxidase and endoxylanase from Streptomyces (Tuncer et al., 2004). Kaya et al. (2000) confirmed the influence of lignin and its degradation products by enzymatic hydrolysis of xylan and by estimating the amount of reducing sugars in the media.

Phenol The release of phenol from control O. laetevirens and P. juliflora was much lower when compared to culture filtrate of degraded P. juliflora by O. laetevirens. A

Results and Discussion higher amount of phenol content was observed in degraded P. juliflora culture filtrate of 1-2 mm particle size and in ratio 0.1:0.3 (O. laetevirens with P. juliflora) when compared to other particle size (100 µm-1 mm and 2-3 mm) and ratio (0.05, 0.1, 0.2 and 0.4) on 15th day (Fig. 10). This result was in accordance with the biodegradation of P. juliflora in which phenol was quantified more in P. juliflora exposed to Phormidium sp. culture filtrate (Prabha et al., 2005).

Shashirekha et al. (1997) reported the ability of a marine cyanobacterium Phormidium valderianum to degrade phenol up to a concentration of 100 mg/ml. Wurster et al. (2003) reported the extracellular degradation of phenol by a cyanobacterium Synechococcus PCC 7002. Their investigations led to the identification of muconic acid as the major product of phenol transformation by Synechococcus PCC 7002 and demonstrated that the cleavage of the aromatic ring system by cyanobacteria for the first time. This pathway of phenol degradation seems to be similar to the well known ortho-fission of phenolic compounds by heterotrophic bacteria and yeasts, which give rise in cis, cis-muconic acid.

All the evidences clearly suggest and justify that the presence of phenol in the media supernatant can be due to the degradation of phenolic polymers from lignocellulosic material by Oscillatoria laetevirens.

Lignin Analysis of the lignin content in the control P. juliflora after 15 days of incubation revealed that there was no reduction in the lignin content. However, 1-2 mm particle size and 0.1:0.3 ratio of P. juliflora treated with O. laetevirens showed maximum reduction of lignin (26.8%) content when compared to other treatments (Fig. 11). Malliga et al. (1996) have reported that Anabena azollae while being used as a biofertilizer, exhibited lignolysis and released phenolic compounds which include profuse sporulation of the organism. This report gives the usefulness of coir waste as a carrier for cyanobacterial biofertilizers with supporting enzyme studies on lignin degrading ability of cyanobacteria and use of lignocellulosic coir waste as an excellent and inexpensive carrier for cyanobacterial biofertilizers.

Results and Discussion

Crawford et al. (1983) studied the lignin degrading ability of Streptomyces viridosporus and reported that it degraded 19.7% lignin in 8 weeks. Perestelo et al. (1994) studied the Kraft lignin degradation by Serratia marcescens and found only 15% degradation. Akin et al. (1995) also reported the delignification of Bermuda grass by white-rot fungi. The biodegradation of Bermuda grass stems was improved by 29- 32% using Ceriporiopsis subvermispora and 63-77% using Cyathus stercoreus after 6 weeks in the studies with Daedalea flavida and two of the Phlebia sp. Furthermore, degradation of intermediates and products of synthetic lignin by Phlebia tremallosa was observed by Gutierrez et al. (1996).

Kannan et al. (1989) studied the mechanism of degradation of paper mill sludge containing lignin and cellulose by Pleurotus sajor-caju. It degraded 78% lignin and 73% cellulose after 30 days of incubation. Investigations by Wurster et al. (2003) of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability to metabolize phenol under non-photosynthetic conditions up to 100 mg/L.

An investigation made by Wu et al. (2005) to explore the lignin degrading capacity of five white rot fungi Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22 reported that they were individually used to treat black liquor from pulp and paper mill. The treatment removed 71% of lignin and 48% of chemical oxygen demand (COD) from the waste water in 16 days. Anbuselvi and Rebecca (2009) reported that the selected three different cyanobacterial species such as Phormidium sp, Oscillatoria sp. and Anabaena azollae degrade high lignin containing coir waste efficiently and estimated that 89% of lignin and 92% of hemicelluloses were found to be reduced in 30 days.

4.1.3 Biodegradation of optimized P. juliflora The P. juliflora particle size 1-2 mm and the 0.1:0.3 (O. laetevirens and P. juliflora) were selected for further investigation

4.1.3a Microscopic view of degraded P. juliflora The mineralization of Prosopis juliflora by Oscillatoria laetevirens during different days of incubation was observed under microscope and it revealed that

Results and Discussion

O. laetevirens colonize the Prosopis wood particles and effectively degrade the intact cell wall by their lignolytic enzyme activity (Plate-3a-3d). The degradation of coir pith by Oscillatoria annae was observed under microscope during different stages showed lignin breakdown (Anandharaj, 2007).

Confocal laser scanning microscopy (CLSM) has been used to examine bacterial and fungal decay in wood (Kim and Singh, 1999). Furthermore, electron microscopy has been used to observe the biological deterioration of wood-based composites exposed to various kinds of molds and fungi (Chung et al., 1999). Electron microscopic and biochemical studies of lignocellulose degradation by wood-rotting fungi have shown that enzymes such as lignin peroxidases, manganese-dependent peroxidases, laccases and cellulases are too large to penetrate undegraded secondary wood cell walls. Degradation occurs by surface interaction between cell wall and enzymes, but initiation of decay at a distance from the fungal hyphae must involve diffusible low-molecular mass agents (Evans et al., 1994).

Thus the present study revealed that microscopic observation at 10th, 20th 30th and 40th days of degradation showed gradual degradation of lignocellulosic particles and high extent of O. laetevirens colonization with P. juliflora and it was merely higher in 30th and 40th day sample when compared with 10th and 20th day.

4.1.3b Growth parameters Chlorophyll a Chlorophyll a estimation was performed to find out the growth status of Oscillatoria laetevirens along with the substrate P. juliflora. The results showed significant increase of chlorophyll a in Oscillatoria laetevirens when grown with Prosopis juliflora (47.7 mg/g) in 30th day when compared with control O. laetevirens alone (24.4 mg/g) and other days samples (Fig. 12). Supporting evidence proved that the growth of a cyanobacterium namely Anabaena azollae ML2 with coir pith showed an increase in growth rate in terms of chlorophyll a (Malliga et al., 1996).

Another report showed that two isolated cyanobacterial strains, Oscillatoria agardhi and Anabaena sphaerica, were evaluated for their degradative efficiency of

Results and Discussion petroleum hydrocarbons. Both strains revealed high algal biomass when compared with control (Gamila et al., 2003). Increase of chlorophyll a in O. annae when grown with coir pith was reported by Anandharaj (2007). Similarly, the fresh water cyanobacterium Oscillatoria annae while grown with Lantana camara showed the highest growth rate followed by P. juliflora and coir pith (Viswajith, 2008).

4.1.3c Enzyme assay Lignolytic enzyme profile of O. laetevirens was studied for justifying its lignin degrading ability on P. juliflora. Optimization of temperature and pH of each lignolytic enzymes were performed colorimetrically. Laccase showed a maximum activity at temperature 35 °C and pH 7.0 (Fig. 13 & 14) whereas, polyphenol oxidase showed optimal activity at temperature 35 °C and pH 5.0 (Fig. 15 & 16). However, Manganese independent peroxidase showed optimum activity at 25 °C and pH 4.0 (Fig. 17&18).

It is showed that polyphenol oxidase activity is highly induced in Oscillatoria annae exposed to coir pith when compared to control (O. annae alone) (Anandharaj, 2007). Viswajith, (2008) revealed the presence of other manganese independent peroxidase, laccase, polyphenol oxidase, cellulolytic enzymes like endogluconase and xylanase in O. annae. Pointing (2001) reported that Lignin peroxidase (LiP), mangnese peroxidase (MnP), laccase and versatile peroxidase (VPs) are the major lignin modifying enzymes involved in lignin and xenobiotic degradation by wood rot fungi.

Both, lignin peroxidase and manganese-dependent peroxidase have been found in the extra-cellular filtrates of many white-rot fungi which can degrade wood cell walls (Kirk and Farrell, 1987; Waldner et al., 1988). Enzymes including polyphenol oxidases, laccases, H2O2 producing enzymes and quinone-reducing enzymes can degrade lignin (Blanchette, 1991). The white-rot fungus Phanerochaete chrysosporium produces lignin-degrading enzymes, lignin peroxidases and manganese-dependent peroxidases during secondary metabolism in response to carbon or nitrogen limitation (Boominathan and Reddy, 1992). The advantages of biological pretreatment include low energy requirement and mild environmental conditions.

Earlier studies showed that two freshwater cyanobacteria were able to metabolize and degrade phenol (Gibson, 1982). Further, Shashirekha et al. (1997)

Results and Discussion reported that a marine cyanobacterium Phormidium valderianum BDU 30501 was capable of degrading phenol through the activities of polyphenol oxidase and laccase which were found to be intracellular. The absence of detectable activity of these enzymes in control cultures establishes the inducible nature of these enzymes in this cyanobacterium.

Enzyme profile of fresh water cyanobacterium Oscillatoria annae on exposure to lignocellulosic material Lantana camara revealed the profund induction of both major and accessory lignolytic enzymes like laccase, polyphenol oxidase, peroxidase and esterase (Viswajith and Malliga, 2008).

Estimation of Hydrogen Peroxide It is revealed that O. laetevirens exposed to P. juliflora released the maximum amount (2.14 µM/g) of H2O2 when compared to control O. laetevirens (0.65 µM/g) alone (Fig. 19). Similar result was reported in Oscillatoria annae exposed to coir pith (Viswajith, 2008).

The possible involvement of hydrogen peroxide (H2O2) derived hydroxyl radical (OH) in lignin degradation by Phanerochaete chrysosporium was investigated by Forney et al. (1982). According to their study when P. chrysosporium was grown in low nitrogen medium (2.4 mM N), increase in specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. Faison and Kirk (1983) studied the relationship between the production of reduced oxygen species, hydrogen peroxide (H2O2), superoxide (O2), and hydroxyl radical (OH) and the oxidation of synthetic lignin to CO2 was studied in the cultures of white-rot fungus Phanerochaete chrysosporium. Also in-vitro studies showed that milled wood lignin was depolymerised by laccase in the presence of hydrogen peroxide (Evans, 1985).

The kinetics of the synthesis of H2O2 coincided with the appearance of the lignolytic system. Also, H2O2 production was markedly enhanced by growth under 100% O2 mimicking the increase in ligninolytic activity characteristic of cultures grown under elevated oxygen tension (Viswajith, 2008). These reports clearly indicate that increased release of H2O2 from test samples was due to the lignolytic activity of O. laetevirens.

Results and Discussion

4.1.3d Biochemical estimations Reducing sugar The presence of reducing sugars in the supernatant acted as supporting evidence for the lignolytic activity of Oscillatoria laetevirens. From the results it was observed that the concentration of sugar was considerably higher in Prosopis juliflora degraded culture filtrate in 30th day sample when compared with other days and with control cyanobacteria and Prosopis juliflora (Fig. 20). This might be due to cleavage of complex polymers to simple sugars by cyanobacterial enzymes. The obtained results coincide with the previous studies made by Anandharaj (2007) who reported the release of reducing sugars into the media during degradation of coir pith by Oscillatoria annae. Also, the concentration of reducing sugar was considerably higher in O. annae degraded Prosopis juliflora, Lantana camara and coir pith culture filtrates when compared with control cyanobacterium and control lignocellulosics of experiments carried out both in laboratory and field conditions (Viswajith, 2008). Hence, it can be concluded that the presence of reducing sugars in the supernatant acted as a confirmation of degradation of Prosopis juliflora by Oscillatoria laetevirens.

Phenol Degradation of Prosopis juliflora resulted in release of some organic compounds such as phenol which resulted in color changes of media from colorless to brown. The phenol content was considerably high in Prosopis juliflora treated cyanobacterial culture filtrate when compared to control Oscillatoria laetevirens and Prosopis juliflora alone (Fig. 21). The presence of phenolic compounds in the culture filtrate was further confirmed by UV spectrum analysis. The results obtained clearly indicated the degradation of lignocellulosic material of Prosopis juliflora containing lignin and holocellulose by Oscillatoria laetevirens. The degradation of lignocellulosic material was so far demonstrated by other microorganisms such as bacteria (Perestelo et al., 1994), actinomycetes (Ferraz and Duran, 1995) and fungi (Bhat and Narayan, 2003).

It was reported that the release of phenol content was considerably higher in coir pith treated cyanobacterial culture filtrate when compared to control O. annae in laboratory and field conditions. Also, O. annae grown with coir pith in urea medium

Results and Discussion released higher amounts of phenol when compared to BG11 and other minimal media like NPK and Complex (Anandharaj, 2007).

UV-Vis Spectrum analysis During the degradation of Prosopis juliflora by Oscillatoria laetevirens an appreciable amount of phenolic compounds were released into the surrounding medium that was studied by spectroscopic analysis (Fig. 22). In general the lignocellulose consists of 45% cellulose, 30% hemicellulose and 25% lignin. Lignin shows a maximum absorption at 270-310 nm wavelength. Thus, spectrophotometric detection of the media supernatant in the UV range of 270-310 nm indicates the degradation of lignin and the release of some interesting compounds into the medium. From the result it was confirmed that the selected strain Oscillatoria laetevirens has the ability to degrade the lignocellulosic waste of Prosopis juliflora and release compounds which may be of bioactive in nature.

The present finding correlates with the spectral analysis of culture filtrate of degraded coir pith by Oscillatoria annae which showed absorbance peak at 270 nm (Anandharaj, 2007). Also, Vendramani and Trugo (2004) studied the phenolic compounds in acerola fruit (Malpighia punicifolia L.) and reported that the absorbance in UV region denoted that the compound released is of aromatic nature. Thus, this report confirmed that the increase in the absorbance may be due to an increase in concentration of phenolic compound in the supernatant released as a result of degradation of Prosopis juliflora by Oscillatoria laetevirens.

Nitrate and Ammonia Nitrate and ammonia are the sources of nitrogen that are essential for plant growth. But, to some extent non-availability can be compensated by nitrogen fixing cyanobacteria through atmospheric nitrogen fixation. Quantifying nitrate and ammonia are valuable for assessing the potential of the cyanobacteria to fix nitrogen. In this investigation estimations of nitrate and ammonia were carried out to analyze the nitrogen fixing ability of the selected strain Oscillatoria laetevirens grown in ASN III media along with Prosopis juliflora (Fig. 23 & 24). The nitrate and ammonia present in the supernatants were analyzed and the obtained results showed that the amount of

Results and Discussion nitrate and ammonia were higher in Prosopis juliflora treated along with cyanobacteria samples when compared to control (cyanobacteria alone) in 30th day sample. The increased concentration of nitrate and ammonia depends on the availability of the nitrogen source during the culture growth. The control cyanobacterial sample exhibited a lesser amount of nitrate and ammonia when compared with test samples.

From the investigations, it is evident that cyanobacteria grown with Prosopis juliflora exhibited a high quantity of nitrate and ammonia released into the medium. This may be due to the fixation of atmospheric nitrogen by cyanobacteria under microaerophilic and dark conditions which were created by addition of P. juliflora. Stal and Krumbein (1985) reported about the fixation of nitrogen under aerobic conditions by the non-heterocystous cyanobacterium Oscillatoria sp. According to them Oscillatoria sp. while grown under alternating light-dark cycles, fixes nitrogen preferably in the dark. Similarly, providing light-dark condition in lab and natural light-dark condition in field during the degradation of coir pith by O. annae might have been resulted in nitrogen fixation and accumulation of nitrate and ammonia in the media (Anandharaj, 2007).

Protein The obtained results showed that the concentration of protein in P. juliflora treated O. laetevirens samples were higher when compared to control O. laetevirens and P. juliflora alone (Fig. 25). The increase in protein content of test samples may be due to the release of lignolytic enzymes into the medium which was required for the degradation of lignin present in P. juliflora. Also the increase in protein can be attributed to the de novo synthesis of phenol degrading enzymes and stress-related proteins (Bhagwat and Apte, 1989). Shashirekha et al. (1997) reported the growth of Phormidium valderianum 30501 during phenol degradation and observed that the test samples showed higher protein content on exposure to phenol.

Early reports showed that the heterocystic, filamentous cyanobacterium Calothrix sp. belonging to section IV exhibited variation in its protein content while grown in BG11 with varying concentration of nitrogen (Ramirez et al., 2000). This report clearly supported the difference in protein content exhibited by O. annae on

Results and Discussion exposure to various media like BG11, NPK, complex and urea. This could be due to the variation in the availability of nitrogen present in the media. Maximum protein content was observed in O. annae grown in urea supplemented media which was an easily available nitrogen source in its simplest form (Anandharaj, 2007). Viswajith (2008) showed that the protein was quantified higher in O. annae exposed to coir pith, P. juliflora and L. camara when compared to control cyanobacteria and lignocellulosic wastes alone.

Lignin In P. juliflora a wide range of lignin content (11.5-31%) was reported by Pasiecnik et al. (2001). While studying the effect of O. laetevirens on the degradation of P. juliflora, it was observed that the reduction of lignin content in control P. juliflora was negligible whereas, there was considerable decrease of lignin content in P. juliflora exposed to O. laetevirens samples. The maximum percent of lignin reduction (34.3%) was recorded in 30th day test sample when compared with other samples and control (Fig. 26). Supporting evidences showed that 27% of lignin was found to be reduced in coir pith treated with Oscillatoria annae when compared to control coir pith alone during minimum incubation of 7 days period (Anandharaj, 2007). A fresh water cyanobacterium Phormidium sp. was found to degrade 47% of lignin in P. juliflora and 22% lignin in coir pith within 30 days (Prabha et al., 2005; Malliga and Viswajith, 2007).

Previous studies with Daedalea flavida and two of the Phlebia sp. showed their capability of degrading lignin selectively when inoculated with wheat straw. The degradation of intermediates of synthetic lignin by Phlebis tremallosa was observed by Gutierraz et al. (1996). Arora et al. (2002) showed that Daedalea flavida and two of the Phlebia spp. (Phlebia radiata and Phlebia floridensis) have the capability of degrading lignin selectively when inoculated with wheat straw.

In a biodegradation study, the selected lignocellulosics showed highest lignin content in coir pith (37%) followed by Prosopis juliflora (23%) and Lantana camara (22%). However, Oscillatoria annae treated lignocellulosics showed maximum reduction of lignin content in L. camara (18.2%) followed by P. juliflora (17.4%) and

Results and Discussion coir pith (16.9%) after 30 days of incubation (Viswajith, 2008). Gupta et al. (2010) reported that Pycnoporus cinnabarinus degrades about 7.69-10.08% of lignin in P. juliflora and 6.89-7.31% in L. camara and eventually enhanced the holocellulose content by 2.90-3.97% and 4.25-4.61%, respectively.

Holocellulose Reports on the chemical wood composition of other Prosopis species says that 70% of the wood tissue of P. juliflora is composed of holocellulose (Pasiecnik et al., 2001; Scholz et al., 2005). Holocellulose estimation revealed feasible reduction of holocellulose content in control (P. juliflora alone) (0.8%) while O. laetevirens treated P. juliflora showed 45.3% of reduction (Fig. 27). Supporting evidences showed that maximum reduction of holocellulose was observed in O. annae treated L. camara (8.7%) followed by P. juliflora (8.3%) and coir pith (2.1%) which revealed that O. annae mediated lignin degradation is very much similar to the first group of wood rot fungi (Viswajith, 2008).

Basidiomycetes are the most potent degraders of holo-cellulose because many species grow on dead wood or litter, in environments rich in cellulose (Baldrian and Valaskova, 2008). Report showed that the six fungal isolates degrade lignin and holocellulose in mangium wood meal over 1 to 4 weeks. An increase in incubation time tended to decrease the amounts of lignin and holocellulose. Isolate 371 was found to be best at degrading lignin and holocellulose in mangium wood meal (Djarwanto and Tachibana, 2009).

4.1.3e Phytochemical analysis A phytochemical analysis revealed the presence of alkaloids, flavanoids, terpenoids, saponin and steroids in the control (P. juliflora alone) and test (O. laetevirens treated P. juliflora) samples (Table-2). The previous reports showed two new flavonoid glycosides, kaempferol 4′-methyl ether 3-O-β-D-galactopyranoside and retusin 7-O-neohesperidoside which is characterized from the stem bark of Prosopis juliflora (Ranjana and Misra, 1981). Similarly, plant growth inhibitory alkaloids were isolated from the extract of mesquite leaves by Nakano et al. (2004a).

Results and Discussion

Studies on phytochemical composition in ethanol extract of root and stem of Prosopis africana showed the presence of saponins, tannins, alkaloids, phenols and steroids. The presence of secondary metabolites in a significant amount in the investigated parts may have conferred antimicrobial activity. In this regard, the higher concentration of these phytochemicals in the stem extract may have been responsible for significantly higher inhibition exhibited by the stem extract on Candida albicans, when compared to root extracts (Kolapo et al., 2009). Sathiya and Muthuchelian, (2008); Sivakumar et al. (2009); Seetha Lakshmi et al. (2010) reported the presence of alkaloids, terpenoids and steroids in methanolic extracts of Prosopis juliflora.

4.2. COMPOUND IDENTIFICATION 4.2.1 Spectrum analysis The UV-visible absorption spectra and retension time of phenolic compound enable its identification using chromatographic peaks. The phenolic molecules can absorb light by their functional elements and the benzene ring system found in simple phenols absorbs in 260-280nm region (Waterman and Mole, 1994). The spectrum of ethanolic extract of different days of degraded P. juliflora extract confirmed absorption bands at 270nm and the peak indicates that the compound present in the extract was of aromatic in nature (Fig. 28). According to literature all the flavanols present are quite similar in UV absorption pattern, but appear at distinct retention times (Sirmah, 2009). Anandharaj (2007) reported that during degradation of coir pith by Oscillatoria annae an appreciable amount of phenolic compounds were released into the surrounding medium and it was determined by spectral analysis. From this it is evident that O. laetevirens degrades P. juliflora and releases phenolic compounds and other metabolites into the media.

4.2.2 Thin Layer Chromatography (TLC) Thin Layer Chromatography analysis of the ethanolic extract of P. juliflora alone and degraded P. juliflora samples showed the presence of major phenolic compounds in plates eluted with Hexane:Ethyl acetate (6:4) (Fig. 29). The retention factor (Rf) value of separated spots were measured and matched with standard phenols (Table-3) which showed the presence of 4- ethoxy benzoic acid (0.24), 3,4- diethoxy

Results and Discussion benzoic acid (0.62) and catechin (0.65) in both samples. Also a few other compounds at

Rf value of 0.60, 0.66, 0.74 were observed.

Nadeem (1992) reported the presence of juliprosin and isojuliprosine in the polar fraction of P. juliflora leaf extract. Later, catechin was isolated and identified as the main compound in the methanol extract of Prosopis alba trunk (Astudillo et al., 2000). Sivakumar et al. (2009) reported the presence of two alkaloids, which were identified as 3-oxo-juliprosopine and secojuliprosopinol from the methanolic extract of Prosopis juliflora bark.

4.2.3 High Performance Thin Layer Chromatography (HPTLC) Madan et al. (2010) used HPTLC method for the separation and identification of six different phenolic compounds of gallic acid, ferulic acid, syringic acid, catechin, protocatechuic acid and vanillin in a single analysis which was found to enable rapid, precise, accurate, simple and cost-effective analysis of these important phenolic compounds from different plant materials and herbal products.

The amount of phenolic compound present in the ethanolic extract of P. juliflora and degraded P. juliflora was shown in Fig. 30 and listed in Table-4. From the obtained results, the absorbance in UV region indicates the presence of phenolic compounds and this result coincides with TLC pattern which showed similar number of bands and presence of flavonoid like compounds.

Supporting evidences showed that HPTLC separation of catechin and epicatechin was achieved by multiple gradient development (MGD) with increasing concentrations of acetonitrile (from 20 to 22%) in the water-formic acid mixture and the UV detection at λ = 282 nm and λ = 500 nm were compared for estimation of catechin content (Olech et al., 2007). Furthermore, Dhalwal et al. (2008) reported that HPTLC quantification of Bergenia ciliata and Bergenia ligulata extract revealed the presence of high concentration of bergenin, catechin and gallic acid.

4.2.4 High Performance Liquid Chromatography (HPLC) The phenolic compounds present in each sample were identified by comparing chromatographic peaks with the retention time (Rt) of individual standard (Fig. 31).

Results and Discussion

The obtained results showed the presence of catechin (Rt = 9.8) like compound in

Prosopis juliflora (Rt = 9.9) and degraded Prosopis juliflora (Rt = 9.2). Similar results were obtained in HPLC analysis of crude acetonic extract of P. juliflora heartwood which indicated the presence of mesquitol as sole compound which corroborate with the present results (Sirmah et al., 2009).

Supporting evidence showed that catechin was identified as the main free radical scavenger from the exudate of Prosopis alba. HPLC analysis of the methanol soluble part of the exudate showed a mean peak with Rt = 24.80min, corresponding to catechin (Astudillo et al., 2000).

Carrillo et al. (2008) reported the presence of major compounds in acetone and water extracts of Prosopis laevigata were identified as (-)-epicatechin, (+)-catechin and taxifolin which are quantitatively determined by liquid chromatography (RP-HPLC- UV).

4.2.5 Gas Chromatography Mass Spectrometry (GC-MS) The mass spectrum showed the m/z ratio (mass to charge) at 292 for P. juliflora and 291 for degraded P. juliflora which match with the molecular mass of mesquitol which is 291. These results confirmed the presence of mesquitol like compounds in both the control and test samples (Fig. 32). Further confirmation and identification of the compound was done by FT-IR and NMR analysis.

GC-MS analysis of toluene or ethanol bark extractives of P. juliflora indicated the presence of numerous compounds among which 4-O-methylgallocatechin as the main component. Further, identifications of different compounds from bark extracts using National Institute of Standards and Technology (NIST) library and literature information indicate the presence of epicatechins, catechins, methylgallocatechins, gallocatechins, fatty acids and sugars (Sirmah, 2009). Also Tapia et al. (2000) reported catechins, as the main constituent from exudates of Prosopis flexuosa.

4.2.6 Fourier Transform Infrared Spectrometry (FTIR) FTIR spectrum for P. juliflora showed characteristic hydroxyl group absorption at 3405 cm-1. Fatty acids groups were identified by the presence of C-H vibrations

Results and Discussion between 2955 and 2838 and carbonyl bands at 1631 cm-1. Aromatic C=C skeletal vibrations at 1603, 1460 and 1361 cm-1 are typical of aromatic structure in flavanols. A strong absorption at 1019 cm-1 is characteristic of C-O vibrations in sugar units. Similarly, hydroxyl group absorption at 3396 cm-1, C-H vibration between 2948 and 2835, aromatic C=C skeletal vibration at 1599, 1456, 1364 cm-1 and C-O sugar unit at 1024 cm-1 were observed for degraded P. juliflora (Fig. 33).

Similar results were observed in FTIR spectrum of acetone extractives of P. juliflora heartwood spectrum, indicated hydroxyl group absorption at 3350 cm-1 and aromatic C=C skeletal vibrations at 1613, 1514 and 1475 cm-1 which resembles flavanol structure. Also FTIR spectrum of acetone extractives of P. juliflora leaves showed absorption bands at 3400 cm-1 characteristic of OH group and bands at 1119 and 1071 cm-1, which could be ascribed to sugars units. A strong absorption band at 2917 and 2849 cm-1 characteristic of C-H vibrations and 1728 cm-1 characteristic of C=O vibrations can be attributed to the presence of fatty acids in their free or esterified form. Also, bands at 1510 and 1450 cm-1 are ascribable to aromatic structures (Sirmah, 2009).

4.2.7 Nuclear Magnetic Resonance Spectroscopy (NMR) NMR spectrum showed a higher complexity indicating the presence of different families of product such as fat, sugar along with typical flavonol signal in control P. juliflora while spectrum of degraded P. juliflora extract showed the presence of main component of flavanol signal (Fig. 34). From the spectrum data it is revealed that mesquitol is the main component of ethanolic extract without any noticeable impurities in degraded P. juliflora.

Supporting evidence showed that high amounts (8%) and purity of the rare flavonoid (-)-mesquitol was identified as the major metabolite in heartwood extractives, while (+)-epicatechin, (+)-catechin, gallocatechins, methylgallocatechins, fatty acids and free sugar are present in the bark of P. juliflora (Sirmah et al., 2009). Pods contain important quantity of galactomanans, mannose, saturated and unsaturated fatty acids and free sugar. Leaves of P. juliflora contain alkaloids such as tryptamine, piperidine, phenethylamine and juliprosopine (Tapia et al., 2000).

Results and Discussion

Based on the results of TLC, HPTLC, HPLC, GC-MS, FTIR and NMR it is confirmed and concluded that the presence of mesquitol as major compound in the degraded P. juliflora as well as in extract of P. juliflora alone that which has been previously reported by Sirmah, (2009). Thus, from the accumulated data the structure of the compound identified was mesquitol (C15H15O6).

4.3. PHARMACOLOGICAL STUDIES 4.3.1 Experimental animal- Rattus norvegicus It is evidence that various vertebrate species have similar biochemical and physiological systems and are most closely related to humans genetically or evolutionarily. In the present study Rattus norvegicus was used for its sensitivity, acceptable results and to eliminate the chance of variation. Rats have been used in many experimental studies to understand their genetics, diseases and the effects of drugs (Krinke, 2000).

Laboratory rats are used more often in research every year than any other animal species. Mice, rats and hamsters make up over 90% of the animals used in biomedical research. In addition to having bodies that work similar to humans, these animals are small in size, easy to handle, relatively inexpensive to buy and keep and produce many offspring in a short period of time (Logan, 2001). Moreover, the rat has been made to fit the emerging metaphors through selective breeding and standardization laboratory rats are now potent symbols of scientific endeavor indeed they stand alongside the ubiquitous double helix as icons of the laboratory in modern western culture (Lynda, 2003).

4.3.1a Mode of drug administration Oral drug delivery is the most desirable and preferred method of administering therapeutic agents for their systemic effects. Recent research has opened many novel avenues for the more effective, sustained and rate-controlled oral delivery of both existing and new therapeutic agents, including peptide and protein drugs emerging from the biotechnology arena. Furthermore, the oral route offers an attractive approach of drug targeting at the specific sites within the gastrointestinal tract for the treatment of certain pathological conditions such as gastroesophageal reflux disorder,

Results and Discussion gastroduodenal ulcers, inflammatory bowel disease and stomach and colon cancers (Brahma and Kwon, 2006). Hence, oral drug delivery as a mode of administration was chosen for this study.

4.3.2 Acute toxicity study Acute toxicity study was crried out to evaluate the toxic characteristics of the P. juliflora extract. Shetty et al. (2007) reported that acute oral toxicity is usually an initial assessment of toxic manifestations and is one of the initial screening experiments performed with all compounds. According to the Organization for Economic Co-operation and Development (OECD) guidelines for acute oral toxicity, the given th LD50 dose of the ethanolic extract of P. juliflora (30 day sample) did not cause any mortality up to 200 mg/kg of body weight and was considered as safe (Table-5). Similar acute toxicity study revealed that the methanolic extract of P. juliflora bark was safe up to a dose level of 400 mg/kg of body weight (Sivakumar et al., 2009).

Quintas-Junior et al. (2004) in their preliminary study on the total alkaloid fraction of the pods of P. juliflora reported that they induced behavioral changes in rodents, suggesting some effect on the central nervous system. They also showed an acute toxicity (LD50) of 10.3 mg/kg intraperetonially and 637 mg/kg orally. Cytotoxic effects of the extract containing alkaloids on GL-15 (Glial cells of lineage) cell line as confirmed by the MTT (Methyl Thiazol Tetrazolium) test, LDH (Lactate Dehydrogenase) ctivity and Trypan blue staining. The cytotoxic effects of alkaloid extract from P. juliflora pods upon the viability were confirmed by the MTT test, LDH activity and Trypan blue staining (Hughes et al., 2005).

Lydia et al. (2010) reported the oral median lethal dose of the methanolic extract of Prosopis africana was found to be 3.808 g/kg in mice and > 5 g/kg in rats and the study results supports the folkloric claim of the use of P. africana in analgesic and anti-inflammatory activities.

4.3.3 Sub-acute toxicity study 4.3.3a Morphological observation- Body weight Individual animal weights were recorded shortly before the first treatment (0th day), and at study termination (30th day). The body weight of the control rats was found

Results and Discussion to be normal while ethanolic extract of O. laetevirens alone and P. juliflora alone treated male and female rats showed slightly decreased body weight. The ethanolic extract of degraded P. juliflora treated rats showed moderate increase in body weight (Fig. 35). The present study showed that there was no significant weight gain in both male and female rats at 200 mg/kg body weight.

Similar results were observed in animals administered with coir pith based cyanobacterial culture filtrate in which the experimental rat did not show any increase in body weight (Prabha et al., 2009). The aqueous leaf extract of Anogeissus leiocarpus containing alkaloids, flavonoids, tannins and saponins when administered orally (100 and 200 mg/kg) up to 28 days showed average body weight while a control group showed consistent increase in weight. Those group treated with highest dose (300 mg/kg) showed a decline in weight in 3rd and 4th week of the study (Agaie et al., 2007).

The efficacy of ethanolic extract of Cassia kleinii on streptozotocin-induced diabetic rats showed increased body weight in normal control while, diabetic rat treated with 200 mg/kg extract showed a significant prevention in the reduction of body weight (Babu et al., 2003).

The compound benzylcarbamothioethionate isolated from the ethanolic extract of the root bark of Moringa oleifera L. when administered to experimental rats did not show any significant changes in body weight of all the rats (Farjana et al., 2009).

4.3.4 Haematology 4.3.4a Haemoglobin (Hb) Adequate haemoglobin percent is needed for normal physiology of animals, which depends on the erythrocyte count. From the results, it was observed that haemoglobin content was normal in control and the extract of degraded P. juliflora treated male and female rats. The extract of P. juliflora alone and O. laetevirens alone treated rats showed slightly decreased haemoglobin content (Fig. 36). The variation in hemoglobin content may be correlated with the presence of phenolic compounds and it is suggested that intake of phenolic compounds should be as moderate as possible.

Results and Discussion

Ingestion of high dose of phenolic compounds has the tendency to cause anemia. Similar results were obtained in rats treated with culture filtrate of degraded coir pith by O. annae (Prabha et al., 2009).

Supporting evidence showed that prolonged administration of Artemisia judaica (0.5 and 1 g/kg body weight) containing flavonoid as the major component in diabetics induced rats did not show significant effect on hemoglobin content (Salwa et al., 2009). Similar results were observed in rats administered with ethanolic extract of Pothomorphe umbellata L. Miq. at 500 mg/kg body weight (Barros et al., 2005).

Ibrahim et al. (1997) reported that leaf extract of Anogeissus leiocarpus containing flavonoids have been shown to increase vascular integrity and also act as antihaemerrhagic (Dharmancida, 1991).

The effect of short term (15 days) alcoholic extract of Cassia kleinii (200 and 400 mg/kg) administration on the hematological and serum biochemical parameters of male mice did not result in any change in hemoglobin content, total leucocyte counts, serum urea, cholesterol, total lipid, glutamate pyruvate transaminase, glutamate oxalate transaminase and alkaline phosphatase (Babu et al., 2003).

The aqueous leaves extract of Anogeissus leiocarpus administered in rats did not show any significant changes on haemoglobin content in both control and extract treated rats. This may be an indication of the relative safety of the extract at the doses (100, 200 and 300 mg/kg) used in the study on haemopoetic system (Agaie et al., 2007).

Salwa et al. (2009) revealed the effect of prolonged administration of Artemisia judaica extracts on hematological parameters did not show any significant effect on hemoglobin concentration, packed cell volume percentage, erythrocytic count, total leucocytic count and differential leucocytic count in rats allover the period of the experiment.

Results and Discussion

4.3.4b Erythrocyte Sedimentation Rate (ESR) Robbins, (1976) reported that flavonoids exert an apparent regulatory action on erythrocyte aggregation and concentration which are two major factors affecting blood viscosity and flow. The methoxylated compounds exhibit a highly significant antiadhesive action on erythrocytes and inhibit erythrocyte sedimentation rate (ESR), while hydroxylated glycosides accelerate aggregation and ESR. The aggregating effect of flavonoids has been interpreted as a mechanism whereby erythrocyte concentration is reduced since clumped cells are sequestered and removed from circulation. Such action appears to be selective and similar to that of polylysine which preferentially aggregates old erythrocytes with less charge than young erythrocytes which are having higher negative charges (Obdulio, 1997).

The erythrocyte sedimentation rate was found to be normal in control and experimental males treated with extract of O. laetevirens, P. juliflora and degraded P. juliflora while it was slightly decreased in control and experiment female rats administered with ethanolic extract of O. laetevirens, P. juliflora and degraded P. juliflora (Fig. 37). Thus the alteration of erythrocyte sedimentation rate within male and female was insignificant and remained within the normal range.

Ratheesh et al. (2009) reported that arthritic rats exhibited a reduced RBC count, reduced haemoglobin level and an increased ESR. The treatment with methanolic extract of Ruta graveolens (20 mg/kg body weight) improved the RBC count, haemoglobin level and the ESR to a near normal level indicating the significant recovery from the anaemic condition. This may be due to sparing effect of the antioxidant defense system as the extract has quenched the increased generation of free radical by the presence of antioxidants.

The anti-arthritic activity of ethanolic extract of seeds of Moringa oleifera in adjuvant-induced arthritis rats showed normal level of rheumatoid factor value, erythrocyte sedimentation rate and histopathology studies in the entire tested group (Shailaja et al., 2007).

Results and Discussion

4.3.4c Packed Cell Volume (PCV) The hematocrit (Ht or HCT) or packed cell volume (PCV) or erythrocyte volume fraction (EVF) is the proportion of blood volume that is occupied by red blood cells. It is normally about 40-64% for male rat and 36-47% for female rat. From the obtained results, it was clear that the tested extracts in all rats had no significant effect on the packed cell volume in all experimental groups and this agreed with the finding that ethanolic extract of degraded P. juliflora caused no significant changes at concentration of 200 mg/kg body weight by oral administration for 30 days (Fig. 38).

The oral administration of ethanolic extract of Artemisia judaica containing flavonoids as a major component showed no significant changes in hematological parameters in rats after oral administration of the plant extract for 3 months (Salwa et al., 2009).

The effect of oral administration of aqueous extract of Pelargonium reniforme root containing flavonoid and tannins as the principal phenolic contents when administered orally showed significant increase in the level of PCV at 100 mg/kg body weight of rat while there was no significant changes observed at 200 and 400 mg/kg body weight when compared with control (Adewusi and Afolayan, 2009).

The methanol extract of the stem bark of Prosopis africana (at doses of 62.5, 125 and 250 mg/kg) evaluated for analgesic and anti-inflammatory activities using acetic acid-induced writhing assay and carrageenan-induced inflammation in rats significantly attenuated the writhing with the highest activity at 250 mg/kg (76.89%) comparable to that of piroxicam (83.16%) the standard agent used. Also the extract showed significant anti-inflammatory activity from the third hour (Lydia et al., 2010).

Ekor et al. (2010) revealed that alloxan-induced diabetes was associated with decreases in total WBC count and PCV. Both ethanolic and methanolic leaf extracts of Sida acuta significantly prevented the decrease in total WBC and PCV induced by alloxan both at 200 and 400 mg/kg.

Results and Discussion

4.3.4d Red Blood Cell (RBC) The red blood cell (RBC) count determines the total number of red cells (erythrocytes) in a sample of blood. An elevated RBC count may be caused by dehydration, hypoxia (decreased oxygen), or a disease called polycythemia. The RBC count is also decreased due to cancer, kidney diseases and excessive intravenous fluids. From the result, it was observed that RBC count was normal in control, extract of degraded P. juliflora treated male and female rats, while extract of P. juliflora alone and O. laetevirens alone showed reduction in RBC count (Fig. 39). The variation of RBC count may be due to uptake of higher phenolic compound which interfere with the haematological system of the experimental rats.

Supporting evidence showed that acute dosages of 0.5, 1.0, and 3 g/kg body weight and chronic dosage of 100 mg/kg/day of ethanolic extracts of Curcuma longa rhizomes caused poor weight gain, changes in the heart and lungs weights, and reduced levels of white and red blood cells (Qureshi et al., 1992). Prabha et al. (2009) reported that open access to 0.3% coir pith based phenolic culture filtrate to albino rat’s showed normal red blood cells count when compared with controls.

The arthritic rats exhibited a reduced RBC count which indicated the anaemic condition that is a common diagnostic feature with chronic arthritis. The treatment with methanolic extract of Ruta graveolens (20 mg/kg) improved the RBC count to a near normal level indicating the significant recovery from the anaemic condition (Ratheesh et al., 2009).

The chloroform soluble fraction of ethanolic extract of root bark of Moringa oleifera containing benzylcarbamothioethionate when administered to rat revealed that hematological parameters such as the total RBC (red blood cell), total WBC (white blood cell), differential count of WBC, platelet count, hemoglobin and ESR (erythrocytes sedimentation rate) remained unchanged in both experimental and control groups (Farjana et al., 2009).

4.3.4e White Blood Cell (WBC) Leucocytes are known to increase sharply when infection occurs, as one of the first line of defense of the body. The experimental study revealed that no significant variations in WBC count was recorded in all male rats administered with ethanolic

Results and Discussion extract of O. laetevirens, P. juliflora and O. laetevirens treated P. juliflora at 200 mg/kg body weight (Fig. 40). The control and experimental groups of female rats showed normal WBC count. A slight elevation in WBC count was recorded in degraded P. juliflora extract treated female rat. This increase in count may be due to antioxidant property of degraded P. juliflora which helps in getting rid of free radicals that make the body more susceptible to infectious diseases.

Supporting evidence showed that there were no significant changes in various haematological parameters such Hb, total count (TC) of RBC and WBC, differential count (DC) of WBC and platelet count compared to the control group, which indicates that test extracts of Cleome rutidosperma root, Neolamarckia cadamba and Spondias pinnatabark did not show any toxic effect on circulating red cells (Sumanta et al., 2009).

The extract of Pelargonium reniforme caused an increase in the level of the total WBC count and its differentials (neutrophils, monocytes, lymphocytes, eosinophils and basophils). This is an indication that the principal function of phagocytes has been enhanced (Jimoh et al., 2008). The observed changes in the levels of the WBC count and its differentials may provide a basis for the antibacterial, antifungal and antitubercular properties of P. reniforme (Mativandlela et al., 2006).

The anti-inflammatory and anti-oxidant effects of methanolic extract of Ruta graveolens L. in adjuvant induced arthritis rats revealed that there is a significant restoration of WBC count in extract treated groups when compared to indomethacin treated groups and normal control (Ratheesh et al., 2009).

The methanolic extract of Arthrospira platensis which is known to be rich in antioxidants such as phenolic acids, α-tocopherol, and β-carotene was examined for subchronic toxicities at doses of 6, 12 and 24 mg/kg body weight daily for 12 week showed normal white blood cells count in all treated groups and in control. Also no significant changes were observed in differential count (Hutadilok et al., 2010).

Results and Discussion

4.3.4f Neutrophil Neutrophils play a crucial role in the development and manifestation of inflammation and they are the major source of free radicals at the site of inflammation. Neutrophil derived free radical is known to cause inflammation and cytokines produced by neutrophils are also responsible for inflammation (Young et al., 1989). From the result obtain it is evident that neutrophil content was found to be normal in control and experimental male rats and showed slightly decrease in neutrophil level in control female rats when compared to experimental female rats (Fig. 41). This activity is related to the phenolic compounds present in the extract.

Muruganandan et al. (2005) reported that mangiferin on cyclophosphamide induced immunotoxicity and it is also evident that mangiferin exhibits an immunoprotective role mediated through the inhibition of reactive intermediate- induced oxidative stress in lymphocytes, neutrophils and macrophages.

Chunlaratthanaphorn et al. (2007) revealed that rats treated with water extract from root of Imperata cylindrica (1,200 mg/kg/day) showed significant difference in neutrophil, lymphocyte and eosinophil count from the control values, the alteration of hematological and white blood cell count values were insignificant and remained within the normal range.

Ethanolic leaf extracts of Sida acuta (400 mg/kg) against alloxan-induced diabetes rats significantly reversed the decrease in neutrophil count associated with alloxan toxicity. Effects produced on neutrophil count in the hyperglycaemic rats by other treatments were not significant when compared with the saline-treated alloxan diabetic rats (Ekor et al., 2010).

4.3.4g Lymphocyte WBC and indices relating to it such as lymphocytes usually shows increase in activity in response to toxic environment (Robins, 1974). In this study, WBC was not significantly altered while lymphocytes, the main effectors cells of the immune system (McKnight et al., 1999) showed slightly decrease in counts in P. juliflora and O. laetevirens alone treated male and female rats when compared to the controls while

Results and Discussion there is a marginal increase in experimental rats administered with degraded P. juliflora extract (200 mg/kg) in both male and female, thus suggesting that the extract only exerted minimal challenge on the immune system of the animals (Fig. 42).

Similar results were observed in rats treated with ethanolic extract of Sphenocentrum jollyanum at 50, 100 and 200 mg/kg body weight showing increased in serum lymphocyte count when compared to control (Mbaka et al., 2010).

It is reported that Artemisia afra caused no significant changes in hematological parameters in rats after oral administration of the aqueous plant extract for a period of 3 months (Mukinda and Syce, 2007). Similarly, methanolic extracts of Cleistocalyx nervosum which contained high levels of phenols, flavonoids and tannin when administered orally at 100 and 500 mg/kg for four weeks showed no significant changes in hemoglobin, hematocrit, platelets and white blood cells in rat male and female treatment groups (Sirinya et al., 2009).

Adewusi and Afolayan (2009) reported effects of graded doses of the aqueous extract of Pelargonium reniforme on haematological parameters of wistar rats showing a significant increase in lymphocyte counts in all the three doses 100, 200 and 400 mg/kg body weight, respectively. The observed changes in the levels of the WBC count and its differentials may provide a basis for the antibacterial, antifungal and antitubercular properties of P. reniforme (Mativandlela et al., 2006).

4.3.4h Eosinophils The obtained results revealed that eosinophil count were found to be average in control group and slightly decreased in rats treated with extracts of O. laetevirens and with degraded P. juliflora. A slight elevation of eosinophil counts was observed in extracts of P. juliflora alone treated rats which may be due to allergic reactions (Fig. 43).

Supporting evidence showed that apple condensed tannins (ACT) supplement in patients with atopic dermatitis (AD) at oral doses of 10 mg/kg per day for 8 weeks reduced the inflammation, lichenification, cracking, itching, sleep disturbance and

Results and Discussion peripheral blood eosinophil counts when compared with the control group. This may be due to anti-allergic effects described by previous reports (Takatsugu et al., 2000).

Besides, it is reported that when trans-resveratrol administered orally to male rats for 28 days at a dose of 20 mg/kg/day did not alter the normal levels of hematologic and biochemical tests (Emilia et al., 2002).

Kumarappan and Subhash (2007) revealed that the polyphenolic extract (PPE) of leaves of Ichnocarpus frutescens evaluated for antitumor activity in vivo against a Murine Ehrlich ascites carcinoma (EAC) model showed increase in eosinophil counts with EAC control; while PPE (100 mg/kg) treated animals restored the eosinophil level to its normal range compared to the control.

The anti-stress potential of ethanolic extracts of Lagenaria siceraria in rats showed elevated blood cell counts of neutrophils, eosinophils and monocytes in stress induced control rats while it has been significantly reduced by the L. siceraria ethanol extract in a dose dependant manner of 100-400 mg/kg treated groups (Lakshmi and Sudhakar, 2009).

4.3.5 Biochemical studies 4.3.5a Protein Albumin and globulin are proteins found in highest concentrations in the plasma. Albumin transports many small molecules in the blood and also prevents the fluid in the blood from leaking out into the tissues (Duncan et al., 1994). Since albumin is produced in the liver, increased serum albumin may indicate that the extract promotes positive functioning of the liver. An increase in globulin levels indicates the potential of immune response by increasing antibody production (Puri et al., 1993).

Experimental results revealed that serum protein was found to be normal in control and in ethanolic extracts of degraded P. juliflora treated male and female rats while ethanolic extracts of O. laetevirens alone and P. juliflora alone treated rats showed slightly decreased serum protein levels (Fig. 44). Similar results were observed

Results and Discussion in rats treated with 0.3% coir pith based cyanobacterial culture filtrate containing phenolic compounds (Prabha et al., 2009).

Barros et al. (2005) reported that the ethanolic root extract of Pothomorphe umbellata L. contains 6.5% of 4-nerolidilcatecol, a phenolic compound with high antioxidant activity when administered at 500mg/ kg body weight for a period of 40 days did not show any significant variations in total protein concentration in males and female rats.

The treatment with ethanolic extracts of Indigofera trita at 200 and 400 mg/kg body weight showed a significant and dose dependant increase in the levels of albumin and total protein in CCl4 intoxicated rats when compared to control rats suggesting the possibility of the extract to give protection against liver injury (Kumar et al., 2008).

Phenolic compounds extracted from the leaves of Myrtus communis when administered to streptozotocin induced diabetic rats at 800 mg/ kg body weight recorded a normal level of total proteins and showed a drastic decrease in albumin and a corresponding increase in globulin when compared to normal rats that indicates hepatic damage (Fahim et al., 2009).

4.3.5b Glucose Glucose is the primary energy source for somatic cells, being carried through the bloodstream and absorbed by the cells with the intervention of insulin. The results obtained indicate that the control group showed normal glucose levels. Extract of O. laetevirens alone, P. juliflora alone and degraded P. juliflora treated animals show a slight decrease in glucose levels in both male and female rats of the respective groups (Fig. 45). This may be due to hypoglycemic activity of the test compound. Supporting evidences are provided by hypoglycemic activity of 0.3% of coir pith based cyanobacterial culture filtrate in treated rats (Prabha et al., 2009).

Kateina et al. (2007) reported that the use of certain dietary polyphenolic compounds may alter glucose metabolism, thus decreasing the risk for type 2 diabetes. The effect of phenolic acids (caffeic, chlorogenic, rosmarinic, and ferulic) and extracts

Results and Discussion from Smallanthus sonchifolius and Prunella vulgaris on glucose production in rat hepatocytes showed that the phenolics at 500 µM and after 1 hr incubation lowered glucose production via both gluconeogenesis and glycogenolysis compared to metformin.

Similarly phenolic compounds extracted from Myrtus communis when administered at 800 mg/kg body weight have made a remarkable antihyperglycemic effect in experimental animal models (Fahim et al., 2009). Turan et al. (2010) investigated the effects of oral administration of extract of green tea (Camellia sinensis) and ginseng (American ginseng-Panax quinquefolium L.), given alone or together decreases blood glucose levels and increases the levels of serum insulin in the diabetic rats. And also reported that these effects have largely been attributed to the most prevalent polyphenol contained in green tea, the catechin or flavanol (-) epigallocatechin-3-gallate (Sato and Miyata, 2000; Dona et al., 2003).

Sokeng et al. (2005) reported the ethanol extract of Bridelia ndellensis had no hypoglycemic effect in type 1 diabetic rats and postprandial glucose load conditions in type 2 diabetic rats in fasting conditions. However, ethyl acetate and dichloromethane fractions significantly lowered blood glucose levels in type 2 diabetic rats when fed simultaneously with glucose. The active principles responsible for the antihyperglycaemic effect are concentrated in the ethyl acetate and dichloromethane fractions of the extract.

4.3.5c Albumin and Globulin Albumin is synthesized by the liver using dietary protein. Its presence in the plasma creates an osmotic force that maintains fluid volume within the vascular space. Experimental results showed that albumin content in control and degraded P. juliflora extract treated male and female rats were found to be normal. Subsequently the rats treated with O. laetevirens and P. juliflora showed a decrease in albumin content in both groups (Fig. 46). This may be due to chronic infections (parasites, some cases of viral and bacterial infection), liver disease (biliary cirrhosis, obstructive jaundice) or kidney dysfunction (nephrosis). Globulin content was found to be normal in males and females of all experimental and control groups.

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Results and Discussion

Supporting evidences showed that the reduction in plasma total protein and albumin levels were observed in diabetic rats and this is consistent with the results obtained by Tuvemo et al. (1997). The decrease in protein and albumin may be due to microproteinuria and albuminuria, which are important clinical markers of diabetic nephropathy (Mauer et al., 1981) or may be due to increased protein catabolism (Almdal and Vilstrup, 1988).

The levels of albumin and albumin/ globulin ratio in plasma were found to be normal in control rats while it was decreased in diabetic animals. This may be due to microproteinuria and albuminuria which are important clinical markers of diabetic nephropathy and/or may be due to increased protein catabolism. These lowered levels of plasma albumin and albumin/globulin ratio levels were significantly reverted in Helicteres isora (100, 200 mg/kg/p.o) treated diabetic rats due to proper protein metabolism (Kumar et al., 2007).

This observation was corroborated by the findings of Ismail et al. (1997) who reported that enhanced serum albumin content was reduced to normal levels after the administration of 1000 mg/kg root bark powder of Salacia oblongo and leaf powder of Azima tetracantha in inflamed rats. Reports also showed that the extract of Myrtus communis when administered to streptozotocin induced diabetic rats at 800 mg/kg recorded a normal level of total proteins but showed a drastic decrease in albumin and a corresponding increase in globulin indicating hepatic damage (Fahim et al., 2009).

4.3.6 Lipid Analysis 4.3.6a Cholesterol Cholesterol is insoluble in blood, but transported in the circulatory system bound to lipoproteins like low density lipoproteins (LDL) and high density lipoproteins (HDL) which carry cholesterol to the liver. Alterations in the concentration of major lipids like cholesterol, high-density lipoprotein cholesterol and triglycerides can give useful information on the lipid metabolism as well as on the predisposition of the animals to atheriosclerosis and its associated coronary heart diseases (Yakubu et al., 2008).

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Results and Discussion

From the results obtained it was renowed that the total cholesterol level in all the experimental male and female rats treated with extracts of O. laetevirens, P. juliflora and degraded P. juliflora were found to be at lower levels when compared with that of control rats (Fig. 47). This may be due to a high consumption of phenolic compounds that reduced cholesterol levels.

This result concurred with an early study made by Fahim et al. (2009) reporting phenolic compounds extracted from the leaves of Myrtus communis (800 mg/kg body weight) inducing diabetic rats to show a progressive decline in cholesterol levels towards normal while diabetic rats treated with 400 mg/kg showed only a moderate decline.

Flavonoids are known for their diverse biological activities including hypolipidemic activities. Ethanolic extracts of Clerodendron phlomoidis showed the presence of flavonoids and related phenolic compounds. Oral administration of ethanol extract of leaves of C. phlomoidis resulted in a significant reduction of serum lipid levels in rats with hyperlipidemia viz. triglyceride and total cholesterol (Dhanabal et al., 2008). Such dual property has also been reported from methanol extracts of Prunus dadidiana (Rosaceae) and its flavonoid constituent prunin (Choi and Yokozawa, 1991).

The effect of ethanolic extract of Cassia kleinii on streptozotocin-induced diabetic rats showed normal cholesterol level in control rats while diabetic control rats showed increased cholesterol level. Subsequently, rats treated with glibenclamide (500 μg/kg) as well as Cassia kleinii extract (200 mg/kg) showed significant reduction in cholesterol content to normal range (Babu et al., 2003).

The ethanolic extract of the roots of Pseudarthria viscida was evaluated for anti diabetic activity against alloxan induced diabetes in albino rats which showed significant activity as compare to standard glibenclamide. The result revealed that both ethanolic extract (100 mg/kg) and (200 mg/kg) has significantly reversed the diabetes- induced hyperlipidemia compared to standard drugs (Masirkar et al., 2008).

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Results and Discussion

4.3.6b Triglycerides (TGL) Reports showed that phenolic compounds, propenylbenzenes and some alkaloids decrease the cholesterol and triglycerides in rats (Argueta, 1994). This hypocholesterolemic effect might be due to their abilities to lower serum triglyceride and lower the magnitude of lipoprotein cholesterol levels as well as slowing the lipid peroxidation process and enhancing antioxidant enzyme activity (Ines et al., 2007). In the present study it was observed that there is no significant variation in triglycerides level of the control, O. laetevirens extract, P. juliflora extract and degraded P. juliflora extract treated rats (Fig. 48).

Antia, (2005) reported the effect of leaf extract of Catharanthus roseus Linn. When administered orally at 0.1, 0.5 and 1.0 mg/kg/day for seven consecutive days, they showed a significant decrease in total serum cholesterol, total triglycerides, LDL-cholesterol and VLDL-cholesterol of rats. However, the level of HDL-cholesterol was not altered by any of the doses given. The reduction in the level of LDL and VLDL could have resulted from the antioxidant effect of the fresh leaf juice of C. roseus, whose phytochemical component included flavonoids which are known for their antioxidant effects.

Murthy et al. (2009) reported that phenolic compounds or phytochemicals present in fruits and vegetables have possess anti-obesity and lipid-lowering effects. In vitro and in vivo effects of phenolic compounds on the induction of pre-adipocytic and adipocytic apoptosis and inhibition of adipocytic lipid accumulation were also reported (Chin and Gow, 2008).

The synergistic effect of silymarin and ethanolic extracts of Phyllanthus amarus against CCl4 induced hepatotoxicity rats showed decreased triglyceride levels in rats treated with silymarin and aqueous extracts of P. amarus when compared to control, silymarine alone ethanolic and aqueous plant extract alone treated rat (Yadav et al., 2008).

The effects of the oral administration of aqueous extracts of Pelargonium reniforme roots at 100, 200 and 400 mg/kg body weight for 21 days showed a decrease

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Results and Discussion in the level of triglycerides in rats treated with 100 and 200 mg/kg of the extract and a significant increase at 400 mg/kg (Adewusi and Afolayan, 2009). The increase in the serum triglyceride levels might be due to accelerated lipolysis and this implies depletion in the storage of fatty acids at this dose (Yakubu et al., 2008).

4.3.7 Renal function test Kidney function tests are a collective term for a variety of individual tests and procedures that can be done to evaluate how well kidneys are functioning.

4.3.7a Urea Urea is a product of the protein metabolism that is excreted in urine and its retention in the body may indicate renal damage (Rabo, 1998). From the result obtained it is evident that control and degraded P. juliflora treated rats showed normal levels of urea in the blood serum while O. laetevirens alone and P. juliflora alone treated animal groups showed slightly elevated levels of urea (Fig. 49). This could be due to either increased levels of protein being metabolized or due to a decrease in the body's ability to filter it out. It was reported that phenolic compounds and flavonoids have the ability to remove excess ammonia and urea and offer protection against hyperammonemia (Essa et al., 2006) which corroborates the present findings.

Supporting evidence showed that flavonoids containing leaf extract of A. leiocarpus caused a significant dose-dependent decrease in urea level in all treated groups when compared to the control (Agaie et al., 2007). Kumar et al. (2007) reported a significant increase in the level of plasma urea in diabetic rats when compared with control rats, whereas after the treatment of diabetic rats with the bark extract of Helicteres isora (100 and 200 mg/kg) the levels of urea significantly decreased.

Vijayalakshmi et al. (2000) revealed that acute (72 hr) and subacute (30 days) treatment of the extracts of Semecarpus anacardium nuts with different dosages (50, 100, 250 and 500 mg/kg body weight) showed increased levels of blood glucose, plasma urea, uric acid and creatinine in 500 mg/kg treated rats. Prabha et al. (2009)

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Results and Discussion reported that there was no significant change in serum urea in control and 0.3% coir pith based cyanobacterial culture filtrate administered rats.

Essa and Subramanian (2006) reported that the level of blood ammonia, urea, uric acid, non-protein nitrogen and creatinine were found to be higher in hyperammonemia induced rats while these levels were significantly restored to near normal upon the administration of alcoholic extract of Hibiscus sabdariffa (250 mg/kg body weight) leaves containing glycosides, anthocyanins, polyphenols and flavones as major components.

4.3.7b Uric acid The effect of oral administration of ethanolic extract of O. laetevirens, P. juliflora and degraded P. juliflora for 30 days on serum uric acid was recorded. The results showed no significant variations in extracts of O. laetevirens alone and P. juliflora alone treated groups when compared to control group (Fig. 50). On the contrary uric acid was found to be slightly decreased in serum of rats treated with extracts of degraded P. juliflora. From this it became evident that phenolic compounds alone do not exhibit any toxic effects in the tested animals.

Alderman and Redfern (2004) reported that elevated serum uric acid (hyperuricemia) can result from high intake of purine-rich foods, high fructose or impaired excretion by the kidneys. A slightly higher level of uric acid did not cause much harm, but extremely high levels of uric acid lead to the formation of crystals which accumulate in the joints and may lead to gout.

Aliquots of the crude juice of olive leaves (Kronakii cultivar) which contained 215 ppm of polyphenolic compounds were administered to rats daily for 6 weeks by stomach tube at 600, 1200 and 2400 ppm. The liver, kidney function tests and serum contents were measured to assess the safety limits of polyphenolic compounds and the data of the aforementioned measurements indicated that the administration did not cause any changes in liver and kidney functions (Farag et al., 2006).

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Results and Discussion

Mageid et al. (2009) reported the effect of water algae extracts of red algae (Asparagopsis taxiformis) and brown algae (Sargassum vulgare) on the concentration of serum urea and uric acid in hypercholesterolemic rats showed an increase in the urea and uric acid levels in the control group and rats administered with algae extracts resulted in significant decreases in urea and uric acid levels. Also, the total lipids and triglycerides level was reduced as a result of administrating the rats with algal water extract as polyphenols.

4.3.7c Creatinine Creatinine, a by-product of muscle energy metabolism that similar to urea is filtered from the blood by the kidneys and excreted into the urine. With normal kidney function, the amount of creatinine in the blood remains relatively constantly low but an elevated blood creatinine level is a more sensitive indicator of impaired kidney function (Fountain et al., 2007).

Serum levels of creatinine showed normal range in control male and female rats, while the female rats treated with Prosopis juliflora and Oscillatoria laetevirens showed slightly elevated levels of creatinine when compared with male rats. Rats treated with degraded P. juliflora showed a decrease in levels of serum creatinine (Fig. 51). Early reports showed that oral administration of phenolic compounds containing coir pith based culture filtrate in experimental animals did not show any significant variation in serum creatinine when compared with that of the control (Prabha et al., 2009).

Oral administration of catechin at 40 mg/kg, twice daily for 4 days in ischemia/ reperfusion (I/R) induced rats which showed impaired renal function, produced a significant reduction in the serum levels of creatinine and urea nitrogen. This may be due to free radical scavenging activity and antioxidant capacity of the catechin (Devinder et al., 2005).

Farag et al. (2006) reported that aliquots of concentrated olive leaf juice at 600, 1200 and 2400 ppm as polyphenols and butylated hydroxy toluene (BHT 200 ppm) when administered to rats daily for 6 weeks indicated that the administration of olive

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Results and Discussion leaf juice did not cause any changes in liver and kidney functions. On the contrary, BHT at 200 ppm induced a significant increase in the enzyme activities and the serum levels of uric acid, urea and creatinine.

Kumar et al. (2007) reported that diabetic hyperglycemia induces the elevation of plasma levels of urea, uric acid and creatinine, which are considered as reliable markers of renal dysfunction. Their experimental results showed a significant increase in the level of plasma urea and creatinine in the diabetic rats when compared with respective control rats, while after the treatment of streptozotocin (STZ) induced diabetic rats with the bark extract of Helicteres isora (100 and 200 mg/kg), the levels of urea, uric acid and creatinine were significantly decreased.

4.3.8 Liver function test 4.3.8a Serum Glutamic Pyruvic Transaminase (SGPT) Liver damage is associated with cellular necrosis, increased tissue lipid peroxidation and depletion in tissue GSH levels. In addition to that serum levels of many biochemical markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin, alkaline phosphatase can be elevated (Mascolo et al., 1998). From the obtained results it was found that SGPT level were normal in control and experimental rats, whereas they were elevated in rats treated with extracts of Oscillatoria laetevirens and P. juliflora alone which is showing liver toxicity (Fig. 52). This may be due to the toxic effect of the administered compound.

Supporting evidence showed that aqeous extracts of A. leiocarpus caused a significant dose-dependent increase in the serum level of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase in all treatment groups compared to the control. This indicates hepatocellular damage (Agaie et al., 2007). Woodman, (1988) reported that an increase in the activity of these enzymes in the plasma is often seen following liver damage and it is attributed to the loss of the enzyme from the damaged hepatocytes rather than its increased production.

Maheswari et al. (2008) reported that there is alteration in the levels of biochemical markers of hepatic damage like SGOT, SGPT, ALP and lipid peroxides in

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Results and Discussion rats of paracetamol induced hepatotoxicity. Treatment with methanolic extract of Orthosiphon stamineus leaves containing phenolic compound and flavanoids as major components (200 mg/kg) decreased the levels of lipid peroxidation and the elevated levels of above mentioned biochemical markers to near normal levels.

The level of ALP, SGOT, SGPT enzymes were found to be decreased in rats treated with coir pith based cyanobacterial culture filtrate when compared to control rats (Prabha et al., 2009). The protective effect of extracts on liver cells might exist due to the presence of flavonoids and their antioxidant effects (Sallie et al., 1991).

4.3.8b Serum Glutamic Oxaloacetic Transaminase (SGOT) The SGOT level was found to be normal in control and experimental male rats, whereas it was high in female rats treated with Oscillatoria laetivirens alone. This may be due to toxic effects of the administered compounds (Fig. 53).

Supporting evidences showed that hepatotoxicity induced rat by carbon tertrachloride showed extensive liver damage confirmed by the elevation of SGOT and SGPT. Pretreatment with the extract of Leucas aspera (400 mg/kg) significantly reduced the elevation in liver enzymes, thereby showing that Leucas aspera has a hepatoprotective nature (Mangathayaru et al., 2005).

Kumar et al. (2007) also reported an increase in the activities of aspartate transaminase and alanine transaminase in the liver of diabetic animals. The rise in the activity of alanine transaminase is due to hepatocellular damage and is usually accompanied with aspartate transaminase. Treatment with Helicteres isora (100 and 200 mg/kg) or tolbutamide (250 mg/kg) normalized these enzymes activities.

Induced hepatotoxicity in rats by CCl4 showed liver injury as indicated by the elevation of enzymes SGOT, SGPT and ALP. Treatment of animals with ethanolic and aqueous extracts of Phyllanthus amarus showed partial hepatoprotection, where as these extracts in combination with silymarin exhibited hepatoprotection as indicated by significant changes in various liver parameters (Yadav et al., 2008).

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Results and Discussion

Mohammed (2010) reported hepatoprotective activity of the ethanolic extract of the leaves of Spinacia oleracea L. (EESO) in hepatotoxicity induced rats by carbon tetrachloride (CCl4). Thus pretreatment of rats with EESO, at 250 and 500 mg/kg body weight for 21 consecutive days significantly prevented the CCl4 induced hepatic damage which is indicated by serum marker enzymes (SGOT, SGPT, ALP and GGT) and bilirubin levels.

4.3.8c Total and Indirect bilirubin Assessment of liver function can be made by estimating the activities of serum ALT, AST, ALP and bilirubin, which are originally present in higher concentrations in the cytoplasm. When there is hepatopathy, these enzymes leak into the bloodstream in conformity with the extent of liver damage (Nkosi et al., 2005). Bilirubin is one of the most useful clinical clues to the severity of necrosis, and its accumulation is a measure of binding, conjugation and excretory capacity of hepatocytes.

It is observed that total bilirubin and indirect bilirubin levels are normal in control and degraded P. juliflora treated rats, while extract of O. laetevirens alone and P. juliflora extract alone treated rats showed mildly elevated levels of bilirubin which may be due to toxic metabolites of the administered extracts (Fig. 54). Normal levels were observed in degraded P. juliflora treated rats, probably due to the antioxidant properties of the flavonoids present in the extract.

Supporting evidence showed that there is a significant increase in the total bilirubin contents, SGOT, SGPT and ALP activities in thioacetamide induced hepatic damage groups. Treatment with polyphenolic extracts of Silybum marianum and Chicorium intybus leads to a decrease in total bilirubin, SGOT, SGPT and ALP activities as compared with thioacetamide treated groups. This confirms that polyphenolic extracts have protective effects against hepatic cell injury induced by thioacetamide (Madani et al., 2008).

Agaie et al. (2007) reported that the aqueous leave extracts of Anogeissus leiocarpus administered to rats caused significantly lowered bilirubin levels in all treatment groups compared to the control. This may be attributed to the depressant

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Results and Discussion effect of the extracts. Odutola, (1992) observed that some depressant compounds are known to decrease bilirubin levels in the serum.

Bairwa et al. (2010) reported that administration of the ethyl acetate fraction of stem bark of Ceiba pentandra (400 mg/kg) containing tannin, C-glycoside, phenolic compounds, flavonoids, reducing sugar and triterpenes possesses a hepatoprotective potential in hepatotoxicity induced rats by paracetamol (3 g/kg), which showed a significant reduction in serum enzymes SGOT, ALP and the total bilirubin content.

4.3.8d Alkaline phosphatase (ALP) Increased levels of circulatory Alkaline phosphatase (ALP) may be due to the liver damage caused by the generation of free radicals. Reports showed that ammonium chloride treated rats showed a significant increase in the level of circulatory ammonia, urea, TBARS (thiobarbituric acid and reactive substances), HP (hydroperoxides) and liver marker enzymes such as AST (aspartate transaminase), ALT (alanine transaminase) and ALP (alkaline phosphatase). These changes were significantly decreased in rats treated with Hibiscus sabdariffa leaf extract (HSEt) and ammonium chloride which indicates that HSEt offers hepatoprotection by influencing the levels of lipid peroxidation products and liver markers in experimental hyperammonemia and this could be due to its free radical scavenging property and the presence of natural antioxidants (Essa et al., 2006) which upholded the present findings.

The untreated control and degraded P. juliflora rats showed normal ALP while extracts of O. laetevirens alone and P. juliflora alone treated rats showed slightly elevated ALP (Fig. 55). This may be due to toxic effects of metabolites present in the extract. A normal range of ALP in degraded P. juliflora treated rat indicates the antioxidant effect and scavenging activity of flavonoid compounds present in the extract. This coincides with the scavenging activity results.

Reports showed that the treatment with polyphenolic extracts of Silybum marianum and Chicorium intybus decreased the total bilirubin, SGOT, SGPT and ALP activities which were elevated in thioacetamide treated group. And this confirms that

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Results and Discussion polyphenolic extracts have protective effects against hepatic cell injury induced by thioacetamide (Pyo et al., 2004).

A remarkable elevation was observed in serum and tissue ALP, GPT and GOT activities in hepatotoxicity induced rats by CCl4 administration. Thus, oral administration of the extract (250 and 500 mg/kg body weight), significantly reduced

CCl4 induced hepatotoxicity in rats, as judged from the serum and tissue activity of marker enzymes (Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP)) (Sunilson et al., 2008). Similar intoxication with CCl4 (500 μL/kg, i.p) to vehicle control rats showed a significant increase in ALT, AST and ALP levels when compared to normal control rats. Anogeissus latifolia administered at 300 mg/kg produced a significant reduction in the above enzymes. Thus, the presence of rutin, quercetin and other antioxidants in Anogeissus latifolia may be the contributing factor towards its hepatoprotective activity and justifies the folkloric use of the plant in liver diseases (Pradeep et al., 2009).

4.3.9 Sperm count It is observed that sperm count was normal in control rats. Rats treated with O. laetevirens, P. juliflora extract showed decreased sperm counts, while animals treated with degraded P. juliflora extracts showed normal sperm counts (Fig. 56). Microscopic observations of sperms showed that higher deformed sperms were observed in ethanolic extracts of O. laetevirens alone, P. juliflora extract treated animals and it was found to be less in rats treated with ethanolic extracts of degraded P. juliflora.

Orisakwe et al. (2003) reported the subchronic effect of Hibiscus sabdariffa calyx aqueous extracts on the rat testes and showed that a significant decrease in epididymal sperm counts in rats treated with 4.6 g/kg when compared to 1.15, 2.30 g/kg treated and control. From this it is concluded that our results are consistent with the above reports in regards to a decrease in sperm counts. Similarly, rats treated with 400 mg/kg of Morinda lucida leaf extract for 13 weeks showed a significant reduction in sperm motility, viability and epididymal sperm counts when compared to control rats (Shittu et al., 2008).

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Results and Discussion

4.3.10 Histopathology 4.3.10a Kidney No pathological symptoms were observed upon treatment with ethanolic extract of degraded P. juliflora by O. laetevirens and control rats. While rats treated with 200 mg/kg extract of O. laetevirens alone and P. juliflora alone showed sclerosis in glomeruli (Plate-4). Early reports revealed that kidney of rats treated with phenolic compounds alone do not exhibit any toxic effects as evidenced by the absence of histological changes in the liver and kidney in non diabetic control rats. It is also revealed that phenolic compounds exhibit marked antidiabetic effects when administered at 800 mg/kg body weight (Fahim et al., 2009).

Microscopic examinations of kidney cells of rats administered with the phenolic compounds of olive leaf juice showed histological characters of control rats, whilst, the administration of BHT at 200 ppm severely damaged the rat kidney tissues (Farag et al., 2006).

Kumar et al. (2008) reported the antioxidant and hepatoprotective properties of

Indigofera trita (EIT) on CCl4 treated rats showed edema of lining epithelium of renal tubules and lumen of some of the tubules containing eosinophilic material. The treatment with ethanolic extracts of Indigofera trita (EIT) at 400 mg/kg body weight and vitamin E treatment showed normal renal tubules and minimal edema and focal lymphocytic collection in the interstitial tissue.

Histopathological study of rat kidneys treated with methanol extracts of Cleome rutidosperma root, Neolamarckia cadamba and Spondias pinnata bark at doses of 600 mg/kg upto 14 days showed normal size and shape of glomeruli, tubules, interstitium and blood vessels. Also acute tubular necrosis or glomerular changes were absent (Sumanta et al., 2009).

Hutadilok et al. (2010) reported that histopathological assessment of the internal organs did not produce any significant changes in heart, spleen and kidney tissues of all the wistar rats orally administered with methanol extract of Arthrospira (Spirulina) platensis at doses of 6, 12 and 24 mg/kg body weight daily for 12 weeks.

112

Results and Discussion

4.3.9b Liver The histopathological pattern of untreated control rats showed normal hepatocytic cells whereas rats treated with O. laetevirens and P. juliflora extracts at 200 mg/kg showed scattered individual cell necrosis due to hepatotoxicity of the administered phenolic compounds present in the extract. Interestingly, normal hepatocytes were observed in experimental rats (Plate-5). This revealed the hepatoprotective activity of the ethanolic extract of degraded P. juliflora. Early reports showed that high percentages of quercetin, rutin and gallic acid in the extract justifies potent antioxidant activities (Boyle et al., 2000). Thus, the reactive species-mediated hepatotoxicity can be effectively managed upon administration of agents possessing antioxidants (Labib et al., 2003), free radical scavenger (Sohn et al., 2003) and anti- lipid peroxidant (Gao and Zhou, 2005) properties.

Green tea has been found to provide protection to the liver against a variety of toxic insults. Catechins have been discovered to be powerful antioxidants, which is though to be at least in part responsible for green tea's hepatoprotective activity (Frank and Painter, 1999). Kumar et al. (2008) reported the histopathological profile of liver of CCl4 intoxicated rats showing early degenerative changes in hepatocyte cells. Liver of rats treated with ethanol extract of Indigofera trita (EIT) at 200 mg/kg showed changes of the hepatocytes in few areas along with focal lymphocytic collections and a few degenerated cells. The treatment at 400 mg/kg body weight of the extract and vitamin E treatment recovered the normal structure of liver cells.

Recent reports on the histopathology of liver and kidney revealed hydrophobic changes till the end of the trial as recorded in diabetic rats treated with 400 mg of phenolic compounds. Also reported, the phenolic compounds alone do not exhibit any toxic effects as evidenced by the absence of histological changes in the liver and kidney in non diabetic control rats (Fahim et al., 2009).

Multiple sections of the liver of rat treated with methanolic extracts of Cleome rutidosperma root, Neolamarckia cadamba and Spondias pinnata bark at doses of 600 mg/kg showed normal lobular architecture. Hepatocytes appear normal and are arranged in single cell cords radiating away from the central vein. No sign of non

113

Results and Discussion specific lobular hepatitis was observed at the tested dose and there was no evidence of bile stasis, granuloma, dysplasia or malignancy (Sumanta et al., 2009).

4.3.11 Antioxidant properties Research in the recent past has accumulated substantial evidences that revealed that enrichment of body systems with natural antioxidants may correct the vitiated homeostasis and can prevent the onset as well as treat diseases caused and/or fostered due to free-radical mediated oxidative stress. These developments accelerated the search for antioxidant principles that lead to the identification of natural resources, the isolation of active principles and further modification and refinement of active antioxidant molecules (Halliwell, 1994; Tiwari, 1999; Pietta, 2000).

Generally heartwood extractives showed higher antioxidant properties in comparison with bark and sapwood extractives. Hydrophilic extractives from the more polar solvents such as acetone and toluene/ethanol mixture gave higher antioxidant properties than lipophilic extractives, suggesting that flavanols such as (-)- mesquitol present in Prosopis juliflora extracts are responsible for the antioxidant activity (Wang et al., 2004; Haupt et al., 2003).

The free radical scavenging activity of the extract was analyzed by the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The radical scavenging activity was represented as percentage inhibition of DPPH radicals. From the obtained results the free radical scavenging activity was found to be higher in the extract of degraded Prosopis juliflora (77.4%) when compared with that of Prosopis juliflora (74.46%) alone (Fig. 57).

114

SUMMARY

AND

CONCLUSION

Summary and Conclusion

The scope of this research is to take benefit of Prosopis juliflora invasion to develop a potential new resource for the local population through the development of efficient utilization of P. juliflora wood.

This study was undertaken to estimate the lignolytic ability of cyanobacteria from the germplasm of National Facility for Marine Cyanobacteria for better degradation of the selected lignocellulosic waste P. juliflora. Among thirteen organisms screened, Oscillatoria laetevirens (BDU- 20801) showed better growth in the presence of lignocellulosic material of 1-2mm particle size and with the ratio of 1:3. Hence the present investigation was carried out to study the degradation of P. juliflora by O. laetevirens.

The lignin degrading ability of the cyanobacterial species Oscillatoria laetevirens on Prosopis juliflora was studied. The result showed significant increase in chlorophyll a content when O. laetevirens grown along with lignocellulosic waste. In order to determine the biochemistry of P. juliflora biodegradation, estimations for the presence of reducing sugar, phenols, protein, nitrate and ammonia were carried out. The presence of reducing sugar in the supernatant was influenced by lignolytic activity of O. laetevirens and this may be due to cleavage of complex polymers present in the lignocellulosic waste to simple polymers and sugars. Spectral analysis and phenol quantification of the media supernatant confirmed the degradation of P. juliflora. Increase in protein content, nitrate and ammonia in test samples showed an enhanced rate of metabolic activity during the degradative action of cyanobacteria on P. juliflora. The percentage of lignin and holocellulose content in test sample was found to be reduced during the process of degradation. It was estimated that 34.3% of the lignin and 45.3%

119

Summary and Conclusion of hollocellulose was found to be reduced in 30 days. Probably, the degradative ability of cyanobacteria was due to the strong oxidative activity and the low substrate specificity of their ligninolytic enzymes which was confirmed by colorimetric assay of laccase, polyphenol oxidase and manganese independent peroxidase activity. Also it was quantified that the rate of hydrogen peroxide was high in O. laetevirens grown with P. juliflora when compared with O. laetevirens alone.

Compound study by TLC revealed that a higher number of phenolic compounds were separated in plates eluted with Hexane: Ethyl acetate (6:4).

The Rf value of the test samples when matched with the Rf value of the standard which showed the presence of phenolic compounds like 3, 4 diethoxy benzoic acid, 4-ethoxybenzoic acid and catechol. This result was further confirmed by HPTLC analysis. Structural and physicochemical elucidation based on HPLC, GC-MS, FTIR and 1H NMR analysis clearly demonstrated the presence of mesquitol as the major compound without any noticeable impurities in the extract of degraded P. juliflora when compared with control P. juliflora alone.

DPPH assay is used extensively for screening antioxidants from natural products. 1 mg/ml of the P. juliflora and O. laetevirens treated P. juliflora extract showed 74.46% and 77.4% inhibition of DPPH (1,1– diphenyl–2-Picrylhydrazyl) radical respectively. Free radical scavenging is one of the known mechanisms whereby antioxidants inhibit lipid peroxidation or stop the enzymatic activities of micro-organisms.

Bioactive nature of the degraded P. juliflora on Rattus norvegicus was carried out. The results revealed that 200 mg/kg of P. juliflora, O. laetevirens and O. laetevirens treated P. juliflora extract showed normal body weight in all the control and experimental rat animals. No significant variations were observed in haematological (Haemoglobin, RBC, WBC, neutrophil, lymphocyte and eosinophil) and Biochemical parameters (Erythrocyte sedimentation rate (ESR), Packed cell volume (PCV), protein, albumin,

120

Summary and Conclusion globulin, glucose, cholesterol and triglyceride) studies. Renal function studies (Creatinine, Urea and Uric acid) revealed that the control and degraded P. juliflora treated animals showed a normal range while O. laetevirens alone and P. juliflora alone treated animals showed elevated levels. Similarly, liver function tests (SGPT, SGOT, Bilirubin and ALP) showed that the liver was affected in O. laetevirens alone and P. juliflora alone treated animals, while control and extract of degraded P. juliflora treated animals showed normal secretion of the liver enzymes. This was again confirmed by histopathological observation of the kidney and liver. Thus control and degraded P. juliflora treated animals showed normal histology of liver but O. laetevirens alone and P. juliflora alone treated animal liver section showed scattered individual cell necrosis. No specific abnormality was observed in animals treated with degraded P. juliflora, while O. laetevirens and P. juliflora alone administered animal kidney showed few sclerosis of glomeruli. Gravimetric analysis revealed that the sperm count was found to be normal in all experimental groups. From the above study it is confirmed that the important amount of mesquitol present in the ethanolic extract of degraded P. juliflora could therefore be of valuable interest as a potential source of antioxidants from a renewable origin.

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