BIOLOGICAL ACTIVITIES OF Limonia crenulata (Roxb.)

A THESIS SUBMITTED TO BHARATHIDASAN UNIVERSITY FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY IN BOTANY

By Mrs.S.MERINAL, M.Sc., M.Phil., B.Ed.,

DEPARTMENT OF BOTANY KUNTHAVAI NAACCHIYAAR GOVERNMENT ARTS COLLEGE FOR WOMEN (AUTONOMOUS) (AFFILIATED TO BHARATHIDASAN UNIVERSITY) THANJAVUR-613 007 TAMILNADU, INDIA.

JUNE 2011

KUNTHAVAI NAACCHIYAAR GOVERNMENT ARTS COLLEGE FOR WOMEN (AUTONOMOUS)

THANJAVUR-613 007 TAMILNADU, INDIA. [Affiliated to Bharathidasan University, Tiruchirapalli]

Date: Dr. G. VIJI STELLA BAI, M.Sc., M.Phil., Ph.D., Head of the Department & Controller of the Examinations

CERTIFICATE

This is to certify that the thesis entitled “BIOLOGICAL ACTIVITIES OF Limonia crenulata (Roxb.)” submitted to Bharathidasan University, Tiruchirapalli, for the award of the degree of DOCTOR OF PHILOSOPHY in BOTANY embodies the result of the bonafide research work carried out by Mrs.S.MERINAL, under my guidance and supervision in the Department of Botany, Kunthavai Naacchiyaar Government Arts College for Women (Autonomous), Thanjavur District, Tamil Nadu, India. I further certify that no part of this thesis has been submitted anywhere else for the award of any degree, diploma, associateship, fellowship or other similar titles to any candidate.

(G. VIJI STELLA BAI) RESEARCH ADVISER

Mrs.S.MERINAL, M.Sc., M.Phil., B.Ed., Research Scholar, Department of Botany, Kunthavai Naacchiyaar Government Arts College for Women (Autonomous), (Affiliated to Bharathidasan University, Tiruchirapalli). Thanjavur-613 007, Tamilnadu, India.

DECLARATION

I do hereby declare that this work has been originally carried out by me under the supervision of Dr. G. VIJI STELLA BAI, M.Sc., M.Phil., Ph.D., Head Department of Botany & Controller of the Examinations, Kunthavai Naacchiyaar Government Arts College for Women (Autonomous), Thanjavur district, Tamil Nadu, India. affiliated to Bharathidasan University, Tiruchirapalli – 620 024 and this work has not been submitted elsewhere for any other degree.

June 2011 Thanjavur

(S.Merinal)

ACKNOWLEDGEMENT

First of all my innumerable thanks to Almighty God for His blessings and guidance at every stages of my life. All respects for God for enlighting our souls with the essence of faith in lord and showering all His abundant blessings upon us and enriched me with knowledge and wisdom to complete this thesis in a successful manner.

It is a pleasure to convey my gratitude to people who rendered contribution in assorted ways to this research.

In the first place I would like to express my deepest thanks to my Guide, Dr.G. Viji Stella Bai, M.Sc., M.Phil., Ph.D, Head and Controller of the examinations, Department of Botany, Kunthavai Naacchiyaar Government Arts College for Women (Autonomous), Thanjavur for initially accepted me as a Ph.D candidate and later on for her guidance, helpful encouragement and continuous support during all stages of research and during the preparation of thesis. Her ability to probe beneath the text is a true gift and her insights have strengthened this study significantly. I will always be thankful for her knowledge and deep concern on me. It has been an honour to work with her. She built confidence in me .she showed me different ways to approach a research problem and the need to be persistent to accomplish any goal. I am very thankful for her timely help and valuable suggestions enthusiasm, unfailing interest throughout the period of my research work. Her constructive ideas and encouragement made my thesis as a profound and full-fledged one. I am very fortunate to have her guidance throughout my work.

I am thankful to Mrs.R.Thaiyanayaki, M.Sc., Former Principal, and also to the current principal in charge Dr.Anbu, M.Sc., M.Phil., Ph.D., Head, Department of Zoology, Kunthavai Naacchiyaar Government Arts College Women (Autonomous), Thanjavur, for admitting me and also providing necessary facilities to carry out this research successfully.

I sincerely thankful to the Botany Department staff members, Kunthavai Naacchiyaar Government Arts College Women (Autonomous), Thanjavur for their kind support during my research period.

It is my privilege expresses my deep sense of gratitude to Dr.B.Muthukumar, M.Sc., M.Phil., B.Ed., Ph.D., Associate Professor, Department of Botany, National College, Trichirappalli, as a doctoral committee member for his constant encouragement, valuable guidance at every stage of my work.

I am extremely thankful to Dr.A.Panneerselvam, M.Sc., M.Phil., B.Ed., Ph.D., Associate Professor, Department of Botany & Microbiology, A.V.V.M. Sri Pushpam College, Poondi, Thanjavur, his stupendous persuasiveness, supervision and crucial contribution, which made him a backbone of this research and so to this thesis.

I extend my sincere thanks to Dr.Jagadeesan, M.Sc., Ph.D., Professor and Head, Department of Environmental and Herbal Science, Tamil University, Thanjavur for providing me constant guidance, help and encouragement when I approached him during the course of research.

I am immensely thankful to Dr. M. Prabakaran, M.Sc., Ph.D., Chairman and Mr.V.Thennarasu, M.Sc., (Ph.D.,) Manager of Sri Gowri Biotech Research Academy for admitting me and also providing necessary facilities during the period of my research work.

I express sincere heartfelt gratitude to Ms.K.Uma Maheswsari, M.C.A., Sri Gowri Biotech Research Academy, Thanjavur for constructive criticisms, valuable suggestions, crucial contribution and encouragement in successfully carrying out this research work.

I extend my sincere thanks to Mr.R.Manikandan, M.B.A., Mr. R.Bharathidasan, M.Sc., M.Phil., B.Ed., (Ph.D)., Ms.K.Subha, M.Sc., M.Phil., Ms.S.Deepa, M.Sc., M.Phil., Ms.R.Kanireka, M.Sc., Ms. S.Tamil Selvi, M.Sc., M.Phil., B.Ed. , and Mr.R.Karthikeyan, Sri Gowri Biotech Research Academy, Thanjavur, for the timely and valuable help during the research period.

I deem it a great privilege to express my gratitude to Prof. Dr.R.Senthamarai, M.Pharm., Ph.D., Principal, Periyar College of Pharmaceutical Sciences for Girls, Trichy for her valuable ideas and remarks during the research work on pharmacology, pharmacognocy and pharmaceutics.

I would like to thank Prof.Dr.A.M.Ismali, M.Pharm., Ph.D., Principal Periyar College of Pharmaceutical Sciences for Girls, Trichy for his immense support and guidance for the completion of this work.

I record my sincere thanks to Dr.S.Karpagam, M.Pharm., Ph.D., Dr.T.Sri Vijayakiruba, M.Pharm.,Ph.D., Mrs.Umadevi (Ph.D.,), Mr.K.A.S. Mohammed Shafeeq, M.Pharm., (Ph.D.,) and Mr.T.N.K.Suriyaprakash, Periyar College of Pharmaceutical Sciences for Girls, Trichy for their motivation, enthusiasm, diligent upshot, valuable ideas and remarks during the research work on pharmacology, pharmacognosy and pharmaceutics.

I owe my special thanks with bounteous pleasure to Dr.J.Ebenaser, M.Sc., M.Phil., Ph.D., Head, Department of Zoology, Government Arts College, Ooty, or his encouragement throughout the period of my study.

I am highly thankful to Mr.S.Kumaravel, M.Sc., Scientist, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Government of India, Thanjavur for his contribution on GC-MS and HPLC analysis and interpretation.

I am grateful to Dr.Rajamanikam, M.Sc., M.Phil., Ph.D., and Dr.Subhashini, M.Sc., M.Phil., Ph.D., SASTRA University for given me an opportunity to carry out Physico – chemical and phyto chemical analysis.

With deepest love and appreciation, I would like to thank my family that their constant inspiration and guidance kept me focused and motivated. I am grateful to my father Mr. S. Sargunam, Rt.H.M for giving me the education I ever dreamed. I have to express my gratitude for my mother Mrs. C. Athisayamany, Rt.H.M in words, whose unconditional love has been my greatest strength. They taught me the value of hard work and importance of moral. I have express my gratitude my beloved sister Mrs. Christinal, my brother Mr.S.Jason and their family.

I thank my mother in law Mrs.R.Helen Rajaiah Rt.H.M for her support and blessings and I would like to thank my brother in laws Mr.R.Ebenezer and Mr.R.Samson Jeyasingh and their family members for constant encouragement. I express my sincere thanks to my husband Er.Prince Johnson, who is behind in all my success. I record my thanks for the constant love, support and education of I ever dreamed. I am very pleased with our sons Auqustine Prince & Alwyn Prince. They are genuinely acknowledged for their understanding, endless patience and encouragement which have made me to complete this work as a successful one. I would like express my gratitude to Ms.Grace for her timely help and care.

I would like to express my appreciation to Dr.M.Boominathan, M.Sc., M.Phil., Ph.D., Ms.S.Kalaiselvi, M.Sc., M.Phil., (Ph.D.,), Mr.R.Mahalingam, M.Sc., M.Phil., B.Ed., (Ph.D.,), Mr.L.Prince, M.Sc., M.Phil., (Ph.D.,), Mrs.Manimekalai, M.Sc., M.Phil., (Ph.D.,), Ms.Gomathi, M.Sc., M.Phil., (Ph.D.,), Mrs.P.Abirami, M.Sc., M.Phil., (Ph.D.,), Mrs.S.Devasundari, M.Sc., M.Phil., (Ph.D.,) and Ms.R.Prakashveni Valeshwari, M.Sc., M.Phil., (Ph.D.,) for their constant support and encouragement.

I extend my heartfelt thanks to all my friends for their encouragement leading to my success.

Finally my sincere thanks to all those who have helped me in various ways to make this project as a full- fledged one.

By

S.Merinal CONTENTS Page No. 1. INTRODUCTION 1 2. OBJECTIVES OF THE STUDY 24 3. REVIEW OF LITERATURE 26 3.1. Medicinal 26 3.2. Qualitative phytochemical analysis 29 3.3. Quantitative phytochemical analysis 35 3.4. Physico – chemical characters 35 3.5. Elemental analysis 37 3.6. Microbial analysis 38 3.7. High Power Thin Layer Chromatography (HPTLC) analysis 39 3.8. Gas Chromatography-Mass Spectrometry (GC – MS) analysis 41 3.9. High-Performance Liquid Chromatography (HPLC) analysis 45 3.10. Fourier Transform - Infra Red (FT- IR) spectroscopy analysis 47 3.11. Ultra Violet (UV) - Visible spectroscopy analysis 49 3.12. Antimicrobial acitivity 49 3.13. Antioxidant activity 64 3.14. Anti-inflammatory activity 71 3.15. Antiulcer activity 76 3.16. Antidiarrhoeal activity 82 3.17. Volatile oil 85 3.18. GC –MS analysis of volatile oil 86 3.19. Acute toxicity study 91 3.20. Herbal capsules 93 3.21. Herbal syrup 94 4. MATERIALS AND METHODS 95 4.1. Collection of materials 95 4.1.1. Anatomical analysis 98 4.1.2. Preparation of powder 99 4.1.3. Preparation of extracts 99 4.2. Qualitative phytochemical tests 100 4.2.1. Test for alkaloids 100 4.2.2. Test for carbohydrates 101 4.2.3. Test for glycosides 101 4.2.4. Test for sterols 102 4.2.5. Test for fixed oils and fats 102 4.2.6. Test for saponins 102 4.2.7. Test for proteins and amino acids 102 4.2.8. Test for flavonoids 103 4.3. Quantitative estimation of phytoconstituents 103 4.3.1. Estimation of total phenol 104 4.3.2. Estimation of total tannins 104 4.3.3. Estimation of proteins 104 4.3.4. Estimation of vitamin C 105 4.3.5. Estimation of vitamin E 105 4.3.6. Estimation of total carbohydrates 106 4.3.7. Estimation of total alkaloid 107 4.3.8. Estimation of tannins 109 4.3.9. Estimation of bitters 109 4.3.10. Estimation of flavonoids 110 4.3.11. Estimation of anthracene containing glycosides 110 4.3.12. Estimation of saponins 111 4.4. Physico - chemical characters 111 4.4.1. Total ash 111 4.4.2. Water insoluble ash 112 4.4.3. Acid insoluble ash 112 4.4.4. Determination of alcohol soluble extractive 113 4.4.5. Determination of water soluble extractive 113 4.4.6. Determination of crude fibre content 114

4.5. Elemental analysis by Atomic Absorption Spectrophotometer 114 4.5.1. Fe, Cu, Mn, Zn, Mg, Mo, Al, Ni, Li and V metals analysis 118 4.5.2. Na, K and Ca metals analyses 119 4.6. Microbial analysis 119 4.6.1. Principle 119 4.6.2. Procedure 119 4.7. High Performance Thin Layer Chromatography (HPTLC) analysis 120 4.7.1. Chromatographic conditions 120 4.8. Gas chromatography -Mass Spectrometry (GC – MS) analysis 121 4.8.1. Plant sample extraction 121 4.9. High Performance Liquid Chromatography (HPLC) analysis 123 4.9.1. Sample preparation 123 4.9.2. Analysis 123 4.10. Fourier Transform - Infra Red (FT-IR) spectroscopy analysis 124 4.11. Ultraviolet (UV) – Visible spectroscopy analysis 125 4.12. Antimicrobial activity 125 4.12.1. Preparation of nutrient agar medium 126 4.12.2. Preparation of potato dextrose agar medium 126 4.13. Antioxidant activity 127 4.14. Anti-inflammatory activity 128 4.15. Antiulcer activity 129 4.15.1. Total acidity 131 4.15.2. Free acidity 131 4.15.3. Ulcer index 131 4.16. Antidiarrhoeal activity 131 4.17. Extraction of volatile oil 132 4.18. GC – MS analysis of volatile oil 132 4.19. Acute toxicity studies 133 4.20. Herbal capsule preparation 134 4.20.1. Method of preparation 134 4.20.2. Procedure 135 4.20.3. Evaluation of capsules 135 4.21. Herbal syrup preparation 136 4.21.1. Preparation of suspension 137 4.21.2. Determination of viscosity 138 5. RESULTS 139 5.1. Medicinal plant 139 5.1.1. Morphological description 139 5.1.2. Propagation 141 5.1.3. Transverse section of leaf 142 5.1.4. Transverse section of stem 144 5.1.5. Transverse section of root 146 5.2. Qualitative phytochemical analysis 148 5.3. Quantitative phytochemical analysis 149 5.4. Physico -Chemical characters 151 5.5. Elemental analysis 152 5.6. Microbial analysis 153 5.7. High Power Thin Layer Chromatography (HPTLC) analysis 153 5.8. Gas Chromatography -Mass Spectrometry (GC – MS) analysis 158 5.9. High Performance Liquid Chromatography (HPLC) analysis 176 5.10. Fourier Transform - Infra Red (FT- IR) spectroscopy analysis 177 5.11. Ultraviolet (UV) - Visible spectroscopy analysis 182 5.12. Antimicrobial activity 184 5.13. Antioxidant activity 196 5.14. Anti-inflammatory activity 198 5.15. Antiulcer activity 201 5.16. Antidiarrhoeal activity 204 5.17. Volatile oil 208 5.18. GC –MS analysis of volatile oil 208 5.19. Acute toxicity study 222 5.20. Herbal capsule 225 5.21. Herbal syrup 226

6. DISCUSSION 228 6.1. Medicinal plants 228 6.2. Qualitative phytochemical analysis 230 6.3. Quantitative phytochemical analysis 231 6.4. Physico - chemical characters 233 6.5. Elemental analysis 235 6.6. Microbial analysis 238 6.7. High Power Thin Layer Chromatography (HPTLC) analysis 239 6.8. Gas Chromatography-Mass Spectrometry (GC – MS) analysis 240 6.9. High-Performance Liquid Chromatography (HPLC) analysis 241 6.10. Fourier Transform - Infra Red spectroscopy (FT- IR) analysis 243 6.11. Ultra Violet (UV) – Visible spectroscopy analysis 244 6.12. Antimicrobial activity 245 6.13. Antioxidant activity 247 6.14. Anti-inflammatory activity 249 6.15. Antiulcer activity 251 6.16. Antidiarrhoeal activity 254 6.17. Volatile oil 257 6.18. GC –MS analysis of volatile oil 258 6.19. Acute toxicity study 260 6.20. Herbal capsule 263 6.21. Herbal syrup 266 7. SUMMARY AND CONCLUSION 267 8. BIBLIOGRAPHY i-liii ABBREVIATIONS

% Percentage °C Degree Celsius µg Microgram µl Microlitre µm Micrometre 13C NMR Carbon-13 Nuclear Magnetic Resonance 1H NMR Hydrogen Nuclear Magnetic Resonance AAS Atomic Absorption Spectroscopy ABMS Atom Bombardment Mass Spectrometry ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) AIDS Acquired Immune Deficiency Syndrome ASP American Society of Pharmacognosy ATCC American Type Culture Collection ATP Adenosine triphosphate BC Before Christ BHA Butylated hydroxyanisole BSA Bovine Serum Albumin CAM Complementary or Alternative Medicine CAMP Conservation, Assessment and Monitoring Plan CAT Catalase cc Cubic centimetre Cf Californium CFC Common Fund for Commodities CL Chemiluminescence cm Centimetre COX-1 Cyclooxygenase-1 COX-2 Cyclooxygenase-2 CPCSEA Committee for the Purpose of Control and Supervision of Experiments on Animals Cps Centipoise CZE Capillary Zone Electrophoresis DCM Dichloromethane DHEA Dihydro eplandrosterone DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DNPH 2,4-Dinitrophenylhydrazine DPPH 2, 2-Diphenyl-1picrylhydrazyl

EC50 Half maximal effective concentration

ED50 Effective dose, 50% EMB Eosin Methylene Blue Agar EPEC Enteropathogenic Escherichia coli ESI-MS Electrospray Ionization Mass Spectrometry EtOH Ethyl alcohol eV Electron volts EXIM Export – Import FRAP Ferrous Reducing Antioxidant Pathway FT-IR Fourier Transform - Infra Red g(or) gm Gram GC – MS Gas chromatography -Mass Spectrometry GLC Gas Liquid Chromatography

H2O2 Hydrogen peroxide

H2SO4 Sulphuric acid HCL Hallow cathode Lamp HCl Hydrochloric acid

HCO3 Bicarbonate HIV Human Immunodeficiency Virus HNO3 Nitric acid HPLC High Performance Liquid Chromatography HPTLC High Performance Thin Layer Chromatography Hrs Hours

IC50 Half maximal inhibitory concentration I.P. Indian Pharmacopoeia IPR Intellectual Property Rights ISM &H Indian Systems of Medicine and Homeopathy KBr Potassium bromide Kg Kilogram

KMnO4 Potassium permanganate

LD50 Lethal dose, 50 % LPO Lipid Per oxidation LSIMS Liquid Secondary Ion Mass Spectrometry M Molarity MDA Malonyl Dialdehyde mg Milligram

MgSo4 Magnesium sulfate MHS-15 Mercury Hydride System-15 MIC Minimal Inhibitory Concentration mins Minutes ml Millilitre mm/s millimetre/seconds MMC Minimal Microbicidal Concentration MPs Medicinal plants N Normality NA Nutrient Agar NAA Neutron Activation Analysis NaOH Sodium hydroxide NIST National Institute of Standard and nm Nanometre NMR Nuclear Magnetic Resonance NMRS Nuclear Magnetic Resonance Spectroscopy NO Nitric acid NSAIA Non -Streoidal Antiinflammatory Agent OECD Organization for Economic Co-operation and Development p.o Per oral PDA Potato Dextrose Agar pH Power of hydrogen ppm parts per million PUD Peptic Ulcer Disease Q.S. Quantity Sufficient R&D Research & Development Rf Retention frequency Roxb. Roxburgh rpm rotation per minute RSA Radical Scavenging Activity

Rt Retention time SCR Standard Certified Reference SD Standard Deviation SEM Standard Error Mean SOD Superoxide Dismutase SPF Sun Protection Factor SSA Salmonella Shigella Agar SSD Silver sulphadiazine TCA Trichloro Acetic acid TCM Traditional Chinese Medicine TEAC Trolox Equivalent Antioxidant Capacity TLC Thin Layer Chromatography TM Traditional Medicine TMS Tandem Mass Spectroscopy TPC Total Phenolic Content UV Ultra Violet v/v Volume/volume w/v Weight/ volume w/w Weight/weight WHO World Health Organization

1. INTRODUCTION

Medicinal plants (MPs) played a significant role in various ancient traditional systems of medication such as Ayurvedic, Siddha and Unanic in India. Herbal medicine is the oldest and still the most widely used system of medicine in the world today. It is used in all societies and is common to all cultures. According to World Health

Organization (WHO), Herbal Medicine is defined as plant derived material or preparation, which contains raw or processed ingredients from one or more plants, with therapeutic values (Chapman and Chomchalow, 2004).

The WHO estimates that a minimum of 20,000 plant taxa has recorded medicinal uses. It is estimated that up to 70,000 plants species are used in folk medicine and a majority of these species are found in the Asia-Pacific region (Batugal, 2004). Interest in the exploitation of medicinal and aromatic plants as pharmaceuticals, herbal remedies, flavourings, perfumes and cosmetics, and other natural products has greatly increased in the recent years (Anonymous 1994; Ayensu 1996; Salleh et al. 1997; Kumar et al. 2000).

As with many other economic plants that are still being collected from the wild and exploited by humans unsustainably, threats to genetic diversity and species survival have also increased in the case of medicinal plants as a result of habitat destruction, over- exploitation, land use changes and other pressures (Arora and Engels 1993). In India alone, less than 10 % of the medicinal plants traded in the country are cultivated, about

90% are collected from the wild, very often in a destructive and unsustainable manner

(Natesh, 2000).

Biological activities of Limonia crenulata (Roxb.) 1

The number of organizations conducting research and other activities related to the use of medicinal and aromatic plants is large and increasing (Ayensu 1996; Sharma et al., 2002). Botanic gardens are particularly well distributed network of institutions with experience and expertise in this area. Work on conservation of this resource, particularly at the level of intra-specific genetic diversity, has not kept pace with advances in other areas, such as pharmacognosy and documentation of indigenous knowledge (Leaman et al., 1999; Kshirsagar and Singh 2001). The medicinal plants have been used by humans from the pre-historical times. Studies have pointed out that many drugs that are used in commerce have come from folk-use and use of plants by indigenous cultures

(Anonymous, 1994).

Of the 2,50,000 higher plant species on earth, more than 80,000 are medicinal.

India is one of the world’s 12 biodiversity centres with the presence of over 45000 different plant species. India’s diversity is unmatched due to the presence of 16 different agro-climatic zones, 10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of specific species). Of these, about 15000-20000 plants have good medicinal value.

However, only 7000-7500 species are used for their medicinal values by traditional communities. In India, drugs of herbal origin have been used in traditional systems of medicines such as Unani and Ayurveda since ancient times. The Ayurveda system of medicine uses about 700 species, Unani 700, Siddha 600, Amchi 600 and modern medicine around 30 species. The drugs are derived either from the whole plant or from different organs, like leaves, stem, bark, root, flower, seed, etc. Some drugs are prepared from excretory plant product such as gum, resins and latex. Even the Allopathic system

Biological activities of Limonia crenulata (Roxb.) 2

of medicine has adopted a number of plant-derived drugs which form an important segment of the modern pharmacopoeia. Some important chemical intermediates needed for manufacturing the modern drugs are also obtained from plants (Eg. diosgenin, solasodine, and ionone). Not only, that plant-derived drug offers a stable market world wide, but also plants continue to be an important source for new drugs (Joy et al., 1998).

Medicinal plants, as a group, comprise approximately 8000 species and account for about 50% of all the higher species in India. A large number of the country's rural population depends on medicinal plants for treating various illnesses.

About 1.5 million practitioners of the Indian Systems of Medicine and Homeopathy (ISM

&H) use medicinal plants for preventive, promotive and curative applications.

Furthermore, there are 7,843 registered ISM pharmacies and 851 of homoeopathy as well as a number of unlicensed small-scale units. Besides meeting national demands, India caters to 12% of the global herbal trade. In recent years, trade in herbal-based products has quantum leaped, particularly in the volume of plant material traded within and outside the country. Estimates by the EXIM (Export – Import) Bank put medicinal plants related international trade at US$ 60 billion per year and still growing at a rate of 7% annually (Maiti, 2004).

India is blessed with two mega centres of biodiversity such as the Hindustan

Centre of Origin and the Central Asia Centre of Origin. This biodiversity is mainly distributed in Western Ghat, North Eastern India and the Himalayan Region. India has about 141 endemic genera of 5,150 species belonging to 47 families of higher plants.

Among the different endemic species, 2,532 species are distributed in Himalayas, 1,788

Biological activities of Limonia crenulata (Roxb.) 3

species in the peninsular region and 185 species in the Andaman and Nicobar Islands.

About 43,000 plant species are eferred to Indian folklore but only about 1,700 plant species have actually been documented in old literature (Maiti, 2004).

Over three quarters of the world population relies mainly on plants and plant extracts for health care. More than 30% of the entire plant species, at one time or other, were used for medicinal purposes. It is estimated that world market for plant derived drugs may account for about Rs. 2,00,000 crores. Presently, Indian contribution is less than Rs. 2000 crores. Indian export of raw drugs has steadily grown at 26% to Rs. 165 crores in 1994 - 1995 from Rs. 130 crores in 1991-1992. The annual production of medicinal and aromatic plant’s raw material is worth about Rs.200 crores. This is likely to touch US $1150 by the year 2000 and US $5 trillion by 2050 (Joy et al., 1998).

Over 7000 species belonging mainly to the families Fabaceae, Euphorbiaceae,

Asteraceae, Poaceae, Cucurbitaceae, Apiaceae, Convolvulaceae, Malvaceae, and Solanaceae have been used from ancient times by various indigenous peoples in the country. This number corresponds to more than 25% of the world's known medicinal plants, estimated to be at around 30,000 species. Analyses of these plants show that they include all the major life forms (i.e., trees, shrubs, climbers and herbs), with the proportion of ferns and lichens being much smaller compared to flowering plants (Maiti,

2004).

Biological activities of Limonia crenulata (Roxb.) 4

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their use in traditional medicine. Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. For most of the developing world, the main issue of public health is still the acute need for basic health care, which is sadly lacking even at the most elementary level. This is true in both the rapidly growing cities and in the rural areas. The WHO indicates that more than half of the world's population does not have access to adequate health care services. This is due to the fact that poor people neither have access to nor could afford the present health care services. Therefore, innovative alternative approaches are needed to address this problem.

The use of plants for treating various diseases predates human history and forms the origin of much of the modern medicine. Long before the advent of modern medicine, herbs were the mainstream remedies for nearly all ailments. People commonly diagnosed their own illness, prepared and prescribed their own herbal medicines, or bought them from the local apothecaries.

Using plants as medicine provides significant advantages for treating many conditions. The therapeutic activity of a plant is due to its complex nature with different parts of the plant providing certain therapeutic effects.

Today we are witnessing a great deal of public interest in the use of herbal remedies. The WHO estimated that 80% of the earth's inhabitants rely on traditional

Biological activities of Limonia crenulata (Roxb.) 5

medicine for their primary health care needs and most of this therapy involves the use of plant extracts or their active components (Batugal, 2004).

Herbal medicines are being used increasingly as dietary supplements to fight or prevent common maladies like cancer, heart attacks and depression. When added to foods as supplements, herbs have also been termed as neutraceuticals. Herbal remedies are unpurified plant extracts containing several constituents, which often work together synergistically.

Health practices, approaches knowledge's and benefits incorporating plant, animal and mineral based medicines. Applied singularly or in combination to treat, diagnose and prevent illness or maintain well-being. In Africa upto 80% of the populations use traditional medicines for primary health care. In industrialized countries, adaptations of traditional medicine re-termed Complementary or Alternative Medicine (CAM). In

United States, 158 millions of the adult population use complementary medicine, US$ 17 billion was spent on traditional remedies in 2000. In the United Kingdom, annual expenditure an alternative medicine was US $ 230 million. The global market for herbal medicines currently stands at over US $ 60 billion annual and is growing steadily 25% of modern medicines are made from plants first use traditionally.

Over one third of the population in developing countries lack access to essential medicines. The provision of safe effective traditional medicine therapies could became a critical tool to increase access to health care.

Biological activities of Limonia crenulata (Roxb.) 6

70 countries have a national regulation on herbal medicines but the legislative control of medicinal plants has not evolved around a structured model. This is because medicinal products or herbs are defined differently in different countries and diverse approaches have been adopted with regard to licensing, dispensing, manufacturing and trading (WHO, 2008).

Indian sub continent is a rich source of plant and animal wealth, which is due to it's varied geographical and agroclimatic regions. Beside it is varied bio-diversity, it has a diverse cultural heritage too. Though at present Indian health care delivery consists of both modern system of medicines and traditional system of medicine like Ayurveda,

Siddha, Unani and unorganized system like Folk-medicine have been flourishing well.

Ayurveda and Siddha are of Indian origin and accounted for about 60% health care delivery in general and 75% of rural Indian population depends on these traditional systems.

These two systems of medicine used plants, minerals, metals and animals as a source of drugs, plants being the major source. It is estimated that roughly 1,550 plant species in Ayurveda and 1,200 plant species in Siddha have been used for drug preparation (Jain, 1991; Krishnakumar and Sureshkumar, 1995). In Indian Folk medicine use of about 7,500 plant species are recorded as medical plants (Anonymous, 1996).

Biological activities of Limonia crenulata (Roxb.) 7

Homeopathy system of medicine is a newer one which has been developed in 18th century by Samuel Hahnemann a German Physician and Chemist. These systems based on the concept of providing and prove (Treatment and Patient) that is the choice of drug for treatment depends on symptoms and the clinical condition of the patient (Kokate,

1994).

Nowadays peoples are attracted towards modern system of medicine because of sudden relief from illness, availability etc. But the trend has been changing now, because of side effect, low cost and more efficacy than the modern medicine consequently does it has been a demand for herbs and their products.

The major problem in quality control of herbal drug is lack of Pharmacological standards for all medicinal plants used in phyto drug making. Recently published Indian herbal pharmacopeias contain only a few (32) medicinal plants against about 500 drugs

Indian medicine manufactures are used the plants by their products. It is ironical that even botanical identity of many herbs used in medicinal plant is known in different vernacular names in different parts of India.

India is one of the 12 mega biodiversity centers of the world. The country is divided into 10 biogeography zones, and 25 hot spots representing all the major ecosystems. India is recognized as a country rich in biodiversity because of its tropical location, varied physical features and climate. These factors altogether have resulted in a diversity of ecological habitats like forests, grass land, wet lands, marine and desert ecosystems which harbors immense biodiversity. Indian biodiversity represents 7 percent

Biological activities of Limonia crenulata (Roxb.) 8

of world flora and 6.5 percent of world fauna. In general, nearly 45,000 plant species and

81,000 animal species constitute India's biodiversity. The Indian flora Limonia crenulata

(Roxb.) is a well known medicinal plant. All parts of this plant are medicinally useful.

Literature in Indian traditional medicinal systems like Ayurveda, Siddha, Unani and

Indian folk medicine has potential information on its therapeutic uses. Leaf, fruits and roots of this plant is used to treat dyspepsia, diarrhoea, fever and diabetes.

Traditional medicines (TM) have been defined by WHO as "diverse health practices, approaches, knowledge and beliefs incorporating plant, animal and or mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to maintain well being, as well as to treat, diagnose or prevent illness".

The advantages of traditional medicine include its widespread accessibility and relative cheapness, when most people in low income countries pay for medicines out of their own pockets. The government of China and India, amongst others, provides governmental support to strengthen training, research and the use of "traditional" medicine in their national health care strategies and a number of African countries are considering how to integrate traditional medicine into "main stream" health care. The possibilities for expanding such initiatives need to be examined. Apart from medical use, the production, sale and export of traditional medicine is an important component in some economies. China for instance, exports over 600 million dollars of traditional medicine products annually. Chinese health authorities have recently launched a nation wide program to build up Traditional Chinese Medicine (TCM) hospitals, each

Biological activities of Limonia crenulata (Roxb.) 9

specializing in the treatment of a particular condition, such as different types of cancers, heart and vascular diseases and hepatitis (Hepeng, 2004.)

Efficacy traditional medicine of is one of the most debated issues. There are philosophical, cultural, technical, methodological and practical aspects involved in efficacy or otherwise have been published in prestigious international scientific journals.

For instance, the efficacy of acupuncture in relieving pain and nausea has been well demonstrated and is now acknowledge world wide. For herbal medicines, some of the best-known evidence of efficacy of an herbal product includes Artemisia annua for the treatment of malaria, St. John's wort for the management of mild to moderate depression.

Patients usually experience fewer side effects than when treated with antidepressants such as amitriptyline. Such findings have inspired research worldwide to establish the efficacy of other extensively used TM. Many plant extracts have a variety of pharmacological effects, including anti inflammatory vasodilatory, antimicrobial, anticonvulsant, sedative and antipyretic effects. However, very few randomized controlled studies have been carried out to investigate the practice and treatment delivery of herbal practitioners in their every day work. Regarding no medication therapies, the 1999 British Medical

Journal (BMJ) series on CAM commented that randomized controlled trials have provided good evidence that both hypnologic and relaxation techniques can reduce anxiety and prevent panic disorders and insomnia. It has also shown hypnosis to be of value in treating asthma and irritable bowel syndrome. Yoga is to be of benefit in asthma and in helping elderly people to reduce their fear of falls. Besides these limitations, there

Biological activities of Limonia crenulata (Roxb.) 10

has been an enormous research that has been and is underway in many institutions globally. Many of the findings do substantiate the traditional claims (Patwardhan, 2005).

The major advantage of herbal medicines over modern medicine is the substantial absence of serious side effects of herbs compared with pharmaceutical medicines. Herbs have traditionally been used in cooking as well as in the healing arts.

Pharmaceutical medicines are their copies of herbal "active ingredient" are completely new molecules that do not occur in nature. People also tend to believe that natural products are inherently better than synthetic drugs. The natural drugs somehow contain the 'vital force' that is going to improve their health. The promise is of alternative and effective treatment for chronic disorders. The risk is borne out of the great-unknown effects of herbs on the human body. People who use herbal medicines for self-diagnosed ailments run potential health risks. The risks involve people of all ages (Litvinenko, et al., 1992).

Nowadays traditional medicines and practices form an integral part of

Complementary and Alternative Medicine (CAM). Although their efficacy and mechanisms of action have not been tested scientifically in most cases, these simple medicinal preparations often mediate beneficial responses due to their active chemical constituents (Park and Pezzuto, 2002).

Biological activities of Limonia crenulata (Roxb.) 11

The use of plant compounds for pharmaceutical purposes has gradually increased in the world. Various medicinal plants have been used as a source of medicine for years in daily life to treat diseases. Over 50% of all modern clinical drugs are of natural product origin (Stuffness and Dourous, 1982).

Ethnopharmacology provides an alternative approach for the discovery of antibacterial agents, namely the study of medicinal plants with a history of traditional use as a potential source of substances with significant pharmacological and biological activities (Pushpangadan, 1995).

Phytoconstituents are the natural bioactive compounds found in plants. These phytoconstituents work with nutrients and fibers to form an integrated part of defense system against various diseases and stress conditions. Phytochemicals are basically divided into two groups, i.e. primary and secondary constituents; according to their functions in plant metabolism. Primary constituents comprises common sugars, amino acid, proteins and chlorophyll while secondary constituents consists of alkaloids, terpenoids, saponins, phenolic compounds, flavonoids, tannins, etc.

Pharmacognosy means knowledge of drugs which is mainly concerned with naturally occurring substances this term was introduced by Seydler in 1815 in his work entitled ‘Analecta pharmacognostica' (Evans, 1983). In other words it is an applied science that deals with biological, biochemical and economical features of natural drugs and their constituents (Tyler et al., 1981). Natural drugs are originated either from plant

Biological activities of Limonia crenulata (Roxb.) 12

or animal origin. In the earlier days, only the morphological characters were used to identify the drugs. In the beginning of the present century, pharmacognosy had developed on the botanical side which is concerned with the description and identification of drugs both in whole and powdered states. Therapeutic efficacy of herbal drugs depends upon the quality and quantity of the active compounds that they contain. Pharmacognosy is the simultaneous application of various specific disciplines with the object of acquiring knowledge of drugs from every point of view.

Many higher plants are major sources of natural products used as pharmaceutical agrochemicals, flavour and fragrance ingredients, food additives and pesticides. The search for new plant derived chemicals should be a priority in current and future efforts towards sustainable conservation and rational utilization of biodiversity. Secondary metabolism in a plant plays a major role in the survival of the plant in its environment.

Attraction of pollinators, defense against predators and diseases are examples of the roles of secondary metabolites. Moreover, numerous plant secondary metabolites such as alkaloids, anthocyanins, flavonoids, tannins, fixed oil, resin, vitamin C, vitamin E, carbohydrate, protein and phytosterols have found commercial application as drug, dye, flavour, fragrance, insecticide etc. Such fine chemicals are extracted and purified from plant materials. The extraction is done by using different solvents. The crude extracts are further purified and the chemical structures of the purified material can then be analysed.

Techniques for further chemical analysis include chromatography, bioautography, radioimmunino assay, various methods of structure identification and newer tools such as fast Atom Bombardment Mass Spectrometry (ABMS), Tandem Mass Spectroscopy

Biological activities of Limonia crenulata (Roxb.) 13

(TMS), High Performance Liquid Chromatography (HPLC), Capillary Zone

Electrophoresis (CZE), Nuclear Magnetic Resonance Spectroscopy (NMRS), and X-ray

Crystallography (Cowan, 1999).

It has been well known since ancient times that plants and spices have antimicrobial activity. There has been a considerable interest to use plants and spices for the elimination of microorganisms because of increasing antibiotic resistance of microorganisms. The phytochemical analysis can be carried out to determine the presence of carbohydrates, glycosides, proteins, amino acids, phytosterols, saponins, flavonoids, alkaloids and tannins using various solvents. These compounds are taken for characterization that can be done by different methods. Laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro. More of these compounds should be subject to animal and human studies to determine their effectiveness in whole-organism systems, including in particular toxicity studies as well as an examination of their effects on beneficial normal microbiota. It would be advantageous to standardize methods of extraction and in vitro testing so that the search could be more systematic and interpretation of results would be facilitated.

Heterocyclic nitrogen compounds are called alkaloids. The first medically useful example of an alkaloid was morphine, isolated in 1805 from the Opium poppy, (Papaver somniferum). Solamargine, a glycoalkaloid from the berries of Solanum khasianum and other alkaloids may be useful against Human Immunodeficiency Virus (HIV) infection as

Biological activities of Limonia crenulata (Roxb.) 14

well as intestinal infections associated with Acquired immune deficiency syndrome

(AIDS). Berberine is an important representative of the alkaloid group. It is potentially effective against trypanosomes and plasmodia. The mechanism of action of highly aromatic plant quaternary alkaloids such as berberine and harmane is attributed to their ability to intercalate with DNA.

Some of the simplest bioactive phytochemicals consist of a single substituted phenolic ring. Cinnamic and caffeic acids are common representatives of a wide group of phenyl propane derived compounds which are in the highest oxidation state. The common herbs Tarragon and Thyme both contain caffeic acid, which is effective against viruses, bacteria and fungi. Phenolic compounds possessing a C3 side chain at a lower level of oxidation state and containing no oxygen are classified as essential oils and often cited as antimicrobial as well.

Flavones are phenolic structures containing one carbonyl group. The addition of a

3-hydroxyl group yields a flavonol. Flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit linked to an aromatic ring. Since they are known to be synthesized by plants in response to microbial infection, it should not be surprising that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms. Lipophilic flavonoids may also disrupt microbial membranes.

Tannin is a general descriptive name for a group of polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution, a property known as

Biological activities of Limonia crenulata (Roxb.) 15

astringency. Their molecular weights range from 500 to 3,000 and they are found in almost every plant part bark, wood, leaves, fruits and roots. They are divided into two groups, hydrolyzable and condensed tannins. Many human physiological activities, such as stimulation of phagocytic cells, host mediated tumour activity and a wide range of anti-infective actions, have been assigned to tannins. Condensed tannins have been determined to bind cell walls of luminal bacteria, preventing growth and protease activity.

The fragrance of plants is carried in the so called quinta essential, or essential oil fraction. These oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. They are called terpenes, their general chemical structure is C10S16 and they occur as diterpenes, triterpenes and tetraterpenes as well as hemiterpenes and sesquiterpenes. When the compounds contain additional elements, usually oxygen, they are termed terpenoids. Terpenes or terpenoids are active against bacteria, fungi and virus.

Papaya yields a milky sap, often called latex, which is a complex mixture of chemicals. Chief among them is papain, a well known proteolytic enzyme. An alkaloid, taparin, is also present. Terpenoids are also present and may contribute to its antimicrobial properties. Propolis is an extract of the balsam of various trees; it is often called bee glue, since honeybees gather it from the trees. Its chemical composition is very complex, like the latex, terpenoids are present as well as flavonoids, benzoic acids, substituted phenolic acids and esters (Amoros et al., 1992).

Biological activities of Limonia crenulata (Roxb.) 16

Pharmacognosy is the study of medicines derived from natural sources, including plants. The American Society of Pharmacognosy (ASP) as "the study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources."

It has been estimated that in developed countries such as United States, plant drugs constitute as much as 25% of the total drugs, while in fast developing countries such as China and India, the contribution is as much as 80%. Thus, the economic importance of medicinal plants is much more to countries such as India than to rest of the world. These countries provide two third of the plants used in modern system of medicine and the health care system of rural population depend on indigenous systems of medicine.

Research interest has focused on various herbs that possess hypoglycemic, anti platelet, antitumor, or immune - stimulating properties that may by useful adjuncts in helping reduce the risk of various diseases. In different herbs a wide variety of active phytochemical, including the flavanoids, terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols, curcumins, and phthalides have been identified (Tyler, 1981).

Traditional knowledge of medicinal plants has always been a valuable guide in the quest for new medicines. In spite of the advent of modern high therapeutic drug

Biological activities of Limonia crenulata (Roxb.) 17

discovery and screening techniques, traditional knowledge systems have given clues to the discovery of valuable drugs (Buenz et al., 2004). Traditional medicine offers distinct advantages even in modern times because they are often cheaper, locally available, and easily consumable as raw or simple medicinal preparations. Their role is still more predominant in developing countries.

Nature can be considered as the ultimate chemist as natural products offer us with an abundant source of novel chemo-types, pharmocophores or lead structures, which could be directly used or derived into readymade drugs. Plants and microbes offer us with a lucrative diversity for screening of the novel bioactivities. Thus nature remains as the potential source of organic structures of unparalleled diversity.

A number of enhancements can be made to the basic method of thin layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements.

Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. GC - MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification.

Biological activities of Limonia crenulata (Roxb.) 18

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.

The pharmacological industries have produced a number of new antibiotics in the last three decades resistance to these drugs by microorganisms has increased. In general bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents. Such a fact is cause for concern, because of the number of patients in hospitals who have suppressed immunity, and due to new bacterial strains which are multiresistant. The problem of microbial resistance is growing and the outlook for the use of antimicrobial drugs in the future is still uncertain. Therefore actions must be taken to reduce this problem, for example, to control the use of antibiotic, develop research to better understand the genetic mechanisms of resistance, and to continue studies to develop new drugs. The use of plant extracts and phytochemicals both with known antimicrobial properties can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to prove such efficiency. Many plants have been used because of their antimicrobial traits, which are due to compounds synthesized in the secondary metabolism of the plant.

Plant generally produces many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs. Plant

Biological activities of Limonia crenulata (Roxb.) 19

products still remain the principle source of pharmaceutical agent used in traditional medicine (Ibrahim, 1997; Ogundipe and Akinbiya, 1998). Since the introduction of antibiotics there has been tremendous increase in the resistance of diverse bacterial pathogens (Cohen, 1992; Gold and Moellering, 1996). Plant derived products have been used for medicinal purposes for centuries.

Since ancient times, plants have been model source of medicines as they are a reservoir of chemical agents with therapeutic properties. The general population is increasingly using herbal medicines as dietary supplements to relieve and treat many different human disorders. It has been well known since ancient times that plants and spices have antimicrobial activity (Ayres et al., 1980; Zaika, 1988).

Plants have been used as folk remedies and ethno botanical literature has described the usage of plant extracts, infusions and powders for centuries for diseases now known to be of bacterial origin. There is an increasing need for search of new compounds with antibacterial activity as the treatment of bacterial infections with the available antibacterial drugs is often unsatisfactory due to the problem of bacterial resistance coupled with the problem of bacterial latency and conflicting efficacy in recurrent infection in immunocompromised patients (Vlietinck and Vanden Berghe,

1991).

A number of compounds extracted from various species of higher plants have shown antibacterial activity (Fukuchi, et al., 1989). There has been a considerable

Biological activities of Limonia crenulata (Roxb.) 20

interest to use plants for the elimination of microorganisms because of increasing antibiotic resistance of microorganisms (Kunin, 1993; Finch, 1998; Smid and Gorris,

1999).

The problem of microbial resistance is growing and the outlook for the use of

antimicrobial drugs in the future is still uncertain. Therefore, there is a need for the

development of new drugs to reduce this problem. In general bacteria and fungi have the

genetic ability to transmit and acquire resistance to drugs, which are utilized as

therapeutic agents (Cohen, 1992).

Anti-inflammatory refers to the property of a substance or treatment that reduces inflammation. Anti-inflammatory drugs make up about half of analgesics, remedying pain by reducing inflammation as opposed to opioids which affect the central nervous system.

A recent review of references indicates that the antiulcerogenic effects of many taxa of medicinal plants have been assessed worldwide (Yesilada and Gurbuz, 2003). It is also reported that many plant species have been used to alleviate gastric symptoms such as gastric pain, etc.

Diarrhoeal diseases are one of the leading causes of morbidity and mortality in developing countries and are responsible for the death of millions of people each year..

Despite immense technological advancement in modern medicine, many people in the

Biological activities of Limonia crenulata (Roxb.) 21

developing countries still rely on the healing practices and medicinal plants for their daily health care needs. Therefore, the WHO encouraged studies for the treatment and prevention of diarrhoeal diseases depending on traditional medical practices.

Diarrhoea is an alteration in normal bowel movement and is characterized by an increase in the water content, volume, or frequency of stools. Plants have long been a very important source of new drugs. Many plant species have been screened for substances with therapeutic activity. Medicinal plants are a promising source of antidiarrhoeal drugs with this reason, international organizations including the studies and research on Diarrhoea.

World Health Organization (WHO) has encouraged studies pertaining to the treatment and prevention of diarrhoeal diseases using traditional medical practices.

In the development of medicine from plants, four important steps are involved:

identification of suitable plants to investigate

isolation and identification of single chemical entities from the chosen plant

identification of the pharmacological actions of those entities

conduct of controlled clinical trials

Herbal medicine is increasingly being validated by scientific investigation which seeks to understand the active chemistry of the plant. Many modern pharmaceuticals have been modeled on, or derived from chemicals found in plants.

Biological activities of Limonia crenulata (Roxb.) 22

The present need for development of new therapeutic agents from natural sources had made the researchers to refresh and uncoil the literatures. Development of suitable methods for effective isolation of bioactive compounds and reliable analytical techniques are research perspectives.

In recent years, medicinal plants have attracted global interest as they constitute a rich treasure of improved cultural information and are sources of natural products, which provide health security to millions in rural communities. There is a growing tendency all over the world to shift from synthetic to natural based projects including medicinal plants. The first half of the twentieth century was a period of tremendous advancements in medicine as causes of diseases were uncovered and new miracle drugs were isolated and synthesized. Initially, many modern medicines were isolated products from traditional plant derived extracts.

In the present scenario of emergence of multiple drug resistance to human pathogenic organism, search for now, safe and effective therapeutical agents from other source including plants are urgently needed. Anti-microbial agents from plants are plentiful in many countries, especially in India, where thousands of tribal communities still use medicinal plants today to cure. It is a necessity from the scientific point of view to establish a rational relationship between chemical, biological and therapeutical activities of medicinal plants.

Biological activities of Limonia crenulata (Roxb.) 23

Realizing the benefit of herbal and medicinal plants and to improve the quality and knowledge on the medicinal plants, the pharmacognostical studies with pharmacological experimentations are essential. Nature has provided a complete storehouse of remedies to cure all ailments of mankind and the history herbal medicine is as old as human civilization. , India has made tremendous progress in agro-technology, process technology, standardization, quality control, research and development etc.

Screening of medicinal plant for antimicrobial, antioxidant, anti - inflammatory,

antiulcer and antidiarrhoea for finding potential new compounds for therapeutic use.

There is a lack of scientific studies on Limonia crenulata (Roxb.).

Biological activities of Limonia crenulata (Roxb.) 24

2. OBJECTIVES OF THE STUDY

The present work was undertaken with the following objectives.

v To study the anatomical structures of root, stem and leaves of Limonia crenulata

(Roxb.)

v To prepare the leaf extracts using different solvents for further studies.

v To study the qualitative and quantitative phytochemical analysis of leaf extract of

Limonia crenulata (Roxb.)

v To analyse the physico - chemical characters of leaves of Limonia crenulata

(Roxb.)

v To study the elemental analysis of leaves of Limonia crenulata (Roxb.) by

atomic absorption spectrophotometer.

v To study the microbial analysis - (Enumeration of microorganisms) from leaves of

Limonia crenulata (Roxb.) by standard plate count method.

v To analyse the bioactive compounds using HPTLC & GC-MS of leaves of

Limonia crenulata (Roxb.)

v To elute the fraction of bioactive compounds using HPLC.

v To analyse the functional group of bioactive compounds using FT-IR & UV -

spectrometer.

v To screen the antimicrobial potential of leaf extract of Limonia crenulata (Roxb.)

using disc diffusion method.

v To evaluate the antioxidant properties of Limonia crenulata (Roxb.) by DPPH

method.

Biological activities of Limonia crenulata (Roxb.) 25

v To find out the anti-inflammatory, antidiarrhoeal and antiulcer activity of leaf

extract of Limonia crenulata (Roxb.) v To extract the volatile oil and GC –MS analysis of volatile oil v To evaluate the acute toxicity level of Limonia crenulata (Roxb.) v Preparation and evaluation of herbal capsules and syrup using leaf powder of

Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 26

3. REVIEW OF LITERATURE

3.1. Medicinal plants

World literature on patents related to Medicinal and Aromatic Plants claimed the period 1980-1992 have been analysed. The study was based on literature survey from various patent disseminating services and from the Medicinal and Aromatic Plants publications. The study reveals that nearly 750 plants have been patented for Medicinal and Aromatic activities for various processes by twelve countries. Japan is the major contributor of plant derived patents, followed by USA, USSR (now Commonwealth of

Independent States, CIS), UK, France, Germany, Canada and Romania. India has less than twenty five patents. Plants related patent information for facilitating better professional search has been briefly discussed. PID, New Delhi (Doreswamy and Jain,

1993).

Aegle marmelos (L.) Correa commonly known as Bael or Bilva belonging to the family Rutaceae has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. Although this plant is native to northern India it is also widely found throughout the Indian peninsula and in Ceylon, Burma, Thailand and Indo-

China. The decoction of the root and root bark is useful in intermittent fever, hypo-chondriasis, melancholia, and palpitation of the heart. The leaves and bark have been used in medicated enema. The leaves are also used in diabetes mellitus. The greatest medicinal value, however, has been attributed to its fruit and the unripe fruit is said to be an excellent remedy for diarrhoea and is especially useful in chronic diarrhoeas

Biological activities of Limonia crenulata (Roxb.) 27

(Nadkarni, 1954). The root is an important ingredient of the 'Dasmula' (ten roots) recipe

(Chopra, 1982).

There are more than 650 species of medicinal plants available in the forests of the

Kerala state. Fire, grazing, and collection of produce by the village folk poses serious threat to the medicinal and aromatic plant wealth of the state. Ways and means of protection, propagation and production of sufficient quantity of this plant wealth have been described. A thorough inventory of the medicinal and aromatic plant is an urgent necessity (Basha and Nair, 1995).

Members of Anacardiaceae, , Cneoraceae, Meliaceae, Ptaeroxylaceae,

Rutaceae, and Simaroubaceae were analysed cladistically to evaluate the familial and sub familial circumscriptions of Rutaceae. Taxa representing all subfamilies and tribes were sampled. The analysis shows that Rutaceae are paraphyletic, with Spathelia and

Dictyoloma (Rutaceae), Harrisonia (Simaroubaceae), Cneorum (Cneoraceae), and

Ptaeroxylon (Ptaeroxylaceae) forming a sister clade to all other Rutaceae.

Circumscription of Rutaceae to include all of these taxa is recommended. This analysis indicates that Simaroubaceae and Meliaceae are the out groups closest to Rutaceae.

Correlation of the molecular phylogenies with biochemical data indicates that chemotaxonomic information is more reliable than fruit type as an indicator of familial and sub familial circumscriptions. The sub familial classification needs revision none of the subfamilies of more than one genus is monophyletic (Mark et al., 1999).

Biological activities of Limonia crenulata (Roxb.) 28

Limonia acidissima L., syn. Feronia limonia is a moderate sized deciduous tree grown throughout India. The fruits are woody, rough and used as a substitute for bael in diarrhoea and dysentery. The fruits are used for tumors, asthma, wounds, cardiac debility and hepatitis. About 75 medicinal plants including L. were collected, identified botanically, arranged alphabetically along with their family names, local names for the purposes of Ethnobotanical Survey in Rural and Tribal people of Indo Nepal border. It describes the method of application of their part of plant and medicinal importance (Rajan, 2000).

India has a rich tradition in the use of medicinal plant to develop drugs from plants. Nowadays herbal drugs are prescribed widely even when their biologically active compounds are unknown because of their effectiveness, minimal side effects in clinical experience and relatively low cost (Valiathan, 1998). Last decade witnessed an increase in the investigations on plants as a sources of human disease management as well as various phytochemical constituents (Pegnyemb et al., 2001; Baser et al., 2002; Bezerra et al., 2002 ; Chalchat et al., 2002, Ciccio and Segrini, 2002; Ghosh and Bhattacharya, 2002 and Mounnissamy et al., 2002).

The leaves of Aegle marmelos (L.) were used in indigenous system of medicine as astringent, laxative, expectorant, in the treatment of various gastro-intestinal affections

(dysentery and piles), ophthalmia, deafness, inflammations, cataract, diabetes, diarrhoea, dysentery, heart palpitation, and asthmatic complications (Ghose,1980; Kirtikar and

Basu,1993) The leaves are also reported in the treatment of abscess, backache, diabetes,

Biological activities of Limonia crenulata (Roxb.) 29

eye disorders, fever, heat in abdomen, jaundice, vomiting, wounds and cuts (Jain, 1991), while contraceptive property of the drug was also been claimed (Bhattacharya, 1982)

Fresh aqueous and alcoholic leaf extracts were reported to have cardiotonic effects in mammals (Haravey, 1968; Nadkarni, 2000) said to possess hypoglycemic and antihyperglycemic activity (Paulose et al., 1993; Karunanayake et al., 1984; Gireesh et al., 2008) anti spermatogenic activity (Sur et al., 1999), antioxidant activity (Sabu and

Ramadasan, 2004) and anticancer effect (Jagetia et al., 2005) .

Extensive field studies were undertaken in order to study the utilization of wild medicinal plants which has resulted in the collection of 50 species belonging to 31 families. The plant name, family, vernacular name and ethno-botanical uses have been enumerated (Rajith and Ramachandran, 2010).

3.2. Qualitative phytochemical analysis

Ghosh et al. (1982) found that Limonia acidissima L. fruits contain flavonoids, glycosides, saponins and tannins. Krishnaveni et al. (2004) reported that phytochemical screening of various extracts of dried leaves of Cassia fistula showed the presence of phytosterols, flavonoids, glycosides, triterpenoids, alkaloids, saponins, tannins and steroids.

Berberine is an isoquinoline alkaloid found in the roots of Coptis japonica and cortex of Phellondendron amurense. This antibacterial alkaloid has been identified from a number of cell cultures, notably those of Coptis japonica (Sato and Yamada, 1984).

Biological activities of Limonia crenulata (Roxb.) 30

Phytochemical screening of fifty-one medicinal plants, which are used in indigenous systems of medicine as well as by local inhabitants either as single drugs or in combinations, for the cure of various ailments. The study carried out so far, revealed the presence of alkaloids in thirty-one plants of flavonoids in twenty eight glycosides in thirty four, saponins in thirty four, sterols in thirty seven and terpenoids in thirty three plants

(Agarwal et al., 1989).

Phytochemicals exert their antimicrobial activity through different mechanisms, for example tannins act by iron deprivation, hydrogen bounding or non-specific interactions with vital proteins such as enzymes (Scalbert, 1991).

Khan et al. (1993) explained the antibacterial activity exhibited by ethanol extract of leaves and stems of Withania coagulans has been attributed to the presence of polar components viz., salts, alkaloids, glycosides, saponins, polyols, resins and amino acids.

Nishibe (1994) investigated the bioactive phenolic compounds in traditional medicines. Arctigenin, matairesinol, trachelogenin and nortrachelogenin from Caulis trachelospermi, mauritianin from Herba catharanthi, acteoside and Plantaginin from

Plantago herb, Suspensaside, Forsythiaside and (+) Pinoresino gluccoside from Forsythia fruit and (+) Syringa resinol α-D glucoside from Eleutherococcus were isolated. The compounds isolated from these showed anti-tumerous activity, anti-inflammatory effects, anti-nephretic and immunosuppressive effects.

Biological activities of Limonia crenulata (Roxb.) 31

Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavonoids and amino acids. Taxol, a complex diterpene alkaloid found in the bark of the Taxus tree, is one of the most promising anticancer agents known due to its unique mode of action on the microtubular cell system

(Jordan and Wilson, 1995).

The secondary metabolites flavonoid, phenol and phenolic glycosides unsaturated lactones, sulphur compounds saponins, cyanogenic glycosides and glyconsinolates produced by plant have known antifungal activity (Osbournce, 1996).

Sener et al. (1998) studied the biological activities of some Turkish medicinal plants as a resource of new chemistry for public health and plant protection. A systematical approach to the discovery of drugs from these plants had resulted in the identification of active compounds representing a wide range of structures, including alkaloids, terpenoids and phenolic compounds. Fifty five organosoluble extracts prepared from Turkish medicinal plants were investigated for their biological activities against insects, nematodes, plant pathogens and brine shrimp in addition to their biological activities such as antimalarial, anticholinergic, analgesic and antiplatelet activities.

Assays for antimicrobial activity yielded 13 extracts with antibacterial activity and 4 with fungicidal activity.

Biological activities of Limonia crenulata (Roxb.) 32

Ahmad and Beg (2001) studied the effect of ethanolic extracts of 45 Indian medicinal plants for their antimicrobial activity against certain drug resistant bacteria and yeast. Of these, 40 plant extracts showed varied levels of antimicrobial activity against more bacteria and overall, broad spectrum antimicrobial activity was observed in 12 plants. Qualitative phytochemical tests, TLC and TLC bioautography of certain active extracts demonstrated the presence of common phytocompounds in the plant extracts including phenols, tannins and flavonoids as active constituents.

Mojab et al. (2003) studied a phytochemical screening of fifty five Iranian plants belonging to 21 families were carried out. A qualitative phytochemical analysis was performed for the presence of alkaloids, tannins, saponins and flavonoids. The medicinal uses of these plants are also reported.

Krasteva et al. (2004) studied that the phytochemical analysis of ethyl acetate extract from Astragalus corniculatus (Bieb.) and brain antihypoxic activity. Dry ethyl acetate extract containing flavonoids was obtained from above ground parts of Astragalus corniculatus (Bieb.). Seven flavonoids were isolated and identified as rutin, hyperoside, iso quercitrin, narcissin, quercetin, kaempferol and isorhamnetin for the first time. The extract was investigated for antihypoxic activity. Antihypoxic activity was especially pronounced in the model of circulatory hypoxia. This effect was attributed to the presence of flavonoids in the extract. The ethyl acetate extract was chromatographed on a cellulose column, using a 0.95% ethanol linear gradient. Seventy fractions were collected and analysed by TLC on silica gel. Identical fractions were put together and

Biological activities of Limonia crenulata (Roxb.) 33

rechromatographed and further purified by column chromatography. Five flavonol glycosides and three flavonol glycones were isolated.

Nalawade et al. (2006) observed the antimicrobial activity of the Spinach leaf extracts were investigated for presence of various chemical groups and antimicrobial activity. Qualitative chemical investigations showed presence of tannis, phenols, sugars, flavonoids, coumarins and sterols. Amongst all the extracts tested methanolic extract exhibited maximum inhibitory activity against all the bacteria culture used, compared with other extracts.

The bioactive chemical constituents to evaluate the antimicrobial activity of the ethanolic extract of traditionally used right medicinal plants of Nepal. A qualitative phytochemical analysis was performed for the detection of alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and reducing sugar. The highest yield of ethanolic extract was found in Azadiracta indica (29.08%). Ocimum sanctum contained all the chemicals except flavonoids and reducing sugar hormone the Colquhounia coccina locked alkaloids and reducing sugar. The antimicrobial activities of these plants extract were also observed. The extract of Rhododendron setosum and the essential oil of

Eucalyptus globules were most effective against Escherichia coli and Staphylococcus aureus respectively. But the extracts of Azadiracta indica and Elshotlzia feucticosa were found to be most effective against Klebsiella species (Chhetri et al., 2008).

Biological activities of Limonia crenulata (Roxb.) 34

From ancient days to recent civilization, human beings depend on nature for running their life smoothly from day to day. Plants remain a vital source of drugs and nowadays much emphasis have been given to nutraceuticals. Various parts of the plant have astringent, constipating, tonic for liver and lung, diuretic, carminative, and cardiotonic traditional uses. Various important phytoconstituents like alkaloids, phenolic compounds, triterpenoids, coumarins, tannins, steroids etc. have been isolated from

Kavith (Feronia limonia). But only few pharmacological activities like antimicrobial, antiviral, antitumour and antifungal activity have been scientifically reported. From enormous traditional uses documented in various traditional system of medicine and presence of vital phytoconstituents make Kavith (Feronia limonia) an important plant to be studied scientifically to prove various traditional uses. In present review we explore

Kaivtha’s description, traditional medicinal uses, and phytoconstituents and investigated pharmacological activities in various parts of the plant to show potential ethnopharmacological importance of the plant (Qureshi Absar et al., 2010).

Venkata et al. (2010) reported that 84 methanolic extracts prepared from the 54

Indian plants belonging to 33 different families collected from the forest located in

Eastern Ghats of India. A qualitative preliminary phytochemical screening was performed on aforesaid extracts for the presence of alkaloids, flavonoids, steroids and terpenoids.

Each analysis was carried out in triplicate, which resulted a total of 22, 19, 37 and 30 plant species were found to give positive results for alkaloids (41%), flavonoids (35%), steroids (69%) and terpenoids (56%), respectively.

Biological activities of Limonia crenulata (Roxb.) 35

Achras sapota (Linn.) belonging of Sapindaceae family and can be widely found in the world. The phytochemical study was carried by Monalisha et al., (2010) on the methanolic extraction of the modified stem of Achras sapota (Linn.) by standard method.

The principal constituents of Achras sapota (Linn.) include alkaloid, steroid, flavonoid, saponin, reducing sugar, tannin, amino acid, protein, anthraquinone glycoside, deoxy sugar, phenolic compound. The main biological activity was found as an anti-oxidant.

3.3. Quantitative phytochemical analysis

Amount of Vitamin C in dry powder of Limonia acidissima L. was determined.

Earlier workers observed that fresh juice of the plant contains more vitamin C content than that of dry powder (Anonymous, 1950).

The abundance of vitamin C (Ascorbic acid) in Limonia acidissima L serves to protect H + carrier system and thus helps in tissue oxidation. Antioxidant along with hyaluronic acid maintains capillary tone by keeping the endothelium intact. Along with proline, ascorbic acid enhances collagen synthesis. Ascorbic acid increases Fe2+ absorption, which in turn increases H6 formation. Blood loss due to ulceration will be compensated by H6formation. Vitamin C also initiates the maturation of red and white blood cells (Rathan, 1986; Rama Rao, 1989).

3.4. Physico – chemical characters

Biological activities of Limonia crenulata (Roxb.) 36

Mammen et al. (2010) analysed the various parameters such as ash analysis, extractive values and moisture content for three plants Aerva lanata, Hedyotis corymbosa and Leptadenia reticulata. Each plant was collected during summer, monsoon and winter to study the effect of change of season on the proximate analysis values. Similarly the analysis was carried out for samples collected from Gujarat, Maharashtra and Kerala, to study the effect of geographical variation in the plants. Interestingly, the values were found to be change with season and region of collection of these plants. The results indicates that the importance of best time and place of collection for the plant.

Mathur et al., (2010) studied macroscopic, microscopic and preliminary phytochemical investigation of leaves of Amaranthus spinosus which includes leaf constants, physiochemical parameters like ash values, extractive values and moisture content. The total ash, acid insoluble ash, water soluble ash values and sulfated ash were observed to be 6.33%, 3.60%, 2.44% and 0.80% w/w respectively. Alcohol soluble and water soluble extractive values of the leaves were observed to be 6.40%, 3.30%, respectively.

Naringi crenulata stem wood is a traditional cosmetics in Southeast Asia.

Physico – chemical analysis of Naringi crenulata showed moisture content and loss on drying were 6.125% ± 0.653 and 7.564% ± 1.146, respectively. Total and acid insoluble ash contents were 1.198% ± 0.515 and 0.035% ± 0.077, respectively. Extractive values of

95% ethyl alcohol, ethyl acetate and H2O were 0.165% ± 0.058, 0.036% ± 0.008 and

0.533% ± 0.117, respectively (Kanlayavattanakul et al., 2009).

Biological activities of Limonia crenulata (Roxb.) 37

Pectic polysaccharides have been isolated from the fruits of Naringi crenulata by extraction with water. The water extract contains large amount of protein. The polymers present in the water extract are fractionated by graded precipitation with ethanol, anion exchange chromatography, and size exclusion chromatography. Characterization of the sub fractions obtained from various chemical and physico-chemical methods of analysis reveals that the water extract contain pectic polymers substituted to various degrees with side chains comprising mainly of terminal, 1,4-, 1,6-, 1,3,6-linked galactose, together with lesser amounts of 1,2,4- and 1,3-linked galactose residues. Arabinose residues are terminal, 1,5-, 1,3,5-linked. These polymers contain acetyl groups and give viscous solution in water (Mondal Saroj et al., 2003).

3.5. Elemental analysis

Analysis of twenty medicinal plants i.e. Aganosma dichotoma, Ferula foetida,

Citrullus colocynthis, Desmotrichum firmbriatum, Tinospora cordifolia, Tylophora indica, Vetiveria zizanoides, Clerodendron serratum, Mallotus philippinensis, Eclipta alba, Celastrus paniculata, Chloroxylon swietenia, Commiphora mukul, Viola odorata,

Santaloides minus, Onosma bracteatum, Plumbago indica, Madhuca longifolia,

Tephrosia purpurea and Gloriosa superba for their mineral elements i.e. Na, Mg,

A1,K,Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Ag, Cd, Sr and Ce was carried out, using

Atomic Absorption Spectrophotometer, Inductively Coupled Plasma and Flame

Photometer. Mineral elements Na, Mg, K, Ca, Cr, Mn, Fe, Co and Zn were found to be common in all the medicinal plants analysed (Saily, 1994).

Biological activities of Limonia crenulata (Roxb.) 38

Rajurkar and Damame (1997) reported that the elemental analysis of some herbal plants used in the Ayurveda for curing of cardiovascular diseases has been performed using the techniques of neutron activation analysis and Atomic Absorption Spectroscopy

(AAS). The concentration of elements Mn, Na, K and Cl has been estimated by NAA using a 252Cf neutron source and a high purity germanium detector coupled to a multichannel analyser, while the elements Ca, Cr, Co, Cu, Fe, Pb, Zn, Ni, Cd and Hg were analysed by AAS using a Perkin Elmer 3100 instrument. The elements such as Na,

Mg, K, Ca, Mn, N and Cl were detected in tested plants. Solanum trilobatum (Linn.)

(Solanaceae-herbs) is an important medicinal plant. It contain rich amount of calcium, iron, phosphorus, carbohydrates, fat, crude fibre and minerals in the leaves (Jawahar et al.,2004).

3.6. Microbial analysis

Idu et al. (2010) evaluated the microbial load on 17 randomly selected plant samples from 60 ethnobotanically collected medicinal plants from five local markets in

Abeokuta, Ogun State, Nigeria. The pour plate method was used to cultivate serially diluted portions of the medicinal plant samples investigated. Enumeration of bacteria was carried out on nutrient agar while that of fungi was effected on Sabouraud agar. The identified microbial isolates include 12 bacterial and 6 fungal genera. The mean heterotrophic bacteria counts of the different herbal samples ranged from 1.3 × 105 cfu/g

(Cnestis ferruginea) to 6.7 × 106 cfu/g (Daniellia oliveri), while total fungal propagule counts ranged from 0.0 × 101 cfu/g ( superba, Cola gigantea, Rauwolfia

Biological activities of Limonia crenulata (Roxb.) 39

vomitoria, Zingiber officinale and Argemone mexicana) to 7.1 × 106 cfu/g (Nesogordonia papaverifera). The synopsis and frequency (prevalence rate) of microbial species isolation showed that Bacillus spp. (82.4 %) and Mucor sp. (47.1 %) had the highest prevalence rates among bacteria and fungi, respectively. The results emphasized the need for constant quality assessment of herbal drugs on sale in order to ensure the production of therapeutic products suitable for human consumption.

Minea et al. (2004) studied fresh Salvia officinalis had 1x104 microorganisms/g, and these microorganisms were identified as bacteria and moulds (Rhizopus, Mucor and

Penicilium). After irradiation with 0.2kGy, the bacteria were decreased at 1x103/g and moulds were destroyed. No microorganisms were survived after irradiation with 0.5 kGy.

Microbial load for Salvia officinalis and Calendula Officinalis have a high microbial load

(1x105 microorganisms/g). 1 kGy irradiation reduced the diameter of colonies and the number of microorganisms at 1x103/g . Microbiological contamination of medicinal herbs was a serious problem in the production of therapeutical preparations.

3.7. High Power Thin Layer Chromatography (HPTLC) analysis

A method for the estimation of quinine (Qn), cinchonine (Cn), and cinchonidine

(Cnd) and a new method based on fluorescence enhancement and detection and quantification of quinidine (Qnd) from Cinchona stem bark and its formulations, using

HPTLC has been reported. Standard solutions of Qn, Qnd, Cn and Cnd were applied on precoated HPTLC plates and developed with chloroform/diethylamine (9.6:104 v/v). The plates were scanned and quantified at 226 nm for Qn, Cn, Cnd and for Qnd at 366 nm in

Biological activities of Limonia crenulata (Roxb.) 40

fluorescene and reflectance mode (< K400 filter). The stem bark of Cinchona officinalis and some herbal were allowed for homeopathic formulations which was evaluated for their individual alkaloid content applying the developed method (Ravishankara et al.,

2001).

A simple sensitive and precise HPTLC method of analysis of trans - resveratrol in

Polygonum cuspidatum (Polygonaceae ) root extracts and in dosage forms was developed and validated. The separation was carried out on a TLC aluminium plates precoated with silica gel 60F – 254 as the stationary phase, eluted with chloroform, ethyl acetate and formic acid (2.5:1:0.1) as mobile phase. Densitometric analysis of trans-resveratrol was carried out in the absorbance mode at 313 nm. This system was found to give compact spot for trans - resveratrol (Rf value of 0.40 plus or minus 0.03). A good linear regression relationship was made between peak areas and the concentrations coefficient 0.9989.

The limit of detection and quantification was found to be 9 and 27 ng/ spot. The method was validated for precision and recovery. The spike recoveries were within 99.85 to

100.70 percent. The method can be applied for identification and quantitative determination of trans-resveratrol in herbal extracts and dosage forms (Babu et al., 2005).

Spectrophotometric study of Aegle marmelos (L.) showed presence of a compound with overlapping spectrum with the marker standard Umbelliferone - a coumarin glycoside which was further confirmed by the TLC and HPTLC studies of the ethanolic extract (Joshi et al., 2009).

Biological activities of Limonia crenulata (Roxb.) 41

An HPTLC method was developed for the quantitative estimation of gallic acid, rutin and quercetin from aqueous and ethanolic extract of Eruca sativa, precoated

HPTLC silica gel 60 F254 as stationary phase and mobile phase for gallic acid, toluene: ethyl acetate: formic acid (7:5:1) and mobile phase for quercetin and rutin, ethyl acetate: glacial acetic acid: formic acid: water (100:11:11:25). Detection and quantification were performed densitometrically at λ 280nm for gallic acid, 280 nm quercetin and 366nm for rutin. The standard Rf values of gallic acid, quercetin and rutin are 0.35±0.01, 0.98±0.01 and 0.34±0.02 respectively. The total peak areas of the standards gallic acid, quercetin and rutin and the corresponding peak areas of extracts were compared and the gallic acid, quercetin and rutin content were estimated to be 356.1, 4591.0 and 1277.1 (Sajeeth et al.,

2010).

3.8. Gas Chromatography-Mass Spectrometry (GC – MS) analysis

A new indole alkaloid, crenulatine, was isolated from the stems of Limonia crenulata (Roxb.). The below structure was identified by spectral means (Niu et al.,

2001).

Biological activities of Limonia crenulata (Roxb.) 42

Nayar et al. (1971) reported that the weak base 4-methoxy-1-methyl-2- quinolone(I). Sitosterol, 4-methoxy-1-methy-2-quinolone and four coumarins, one of which is a 1,2-epoxide, were isolated from the petrol extract of the root bark of

Hesperethusa crenulata (Nayar and Bhan, 1972).

Das and Thakur, (1989) reported that the leaf cutin of Limonia acidissima L. was found to comprise n-alkanoic (C12), α,ω-alkanedioic (C3---C16), hydroxyalkanoic (C3-

C16), dihydroxy alkanoic (C4---C20), hydroxy α,ω-alkanedioic (C14---C16) and aromatic acids, together with p-hydroxy benzaldehyde and heptadecane diol. The main constituents were 9, 16- and 10, 16-dihydroxyhexadecanoic acids (ca 30%), 10, 20- dihydroxyicosanaic acid (ca 10%) and 7-hydroxyhexadecane-1, 16-dioic acid (ca15%).

Lin and Hsu (1999) extracted tannin and related compounds from Terminalia catappa and Terminalia parviflora and isolated one novel complex type tannin, catappin

A, together with two phenolcarboxylic acids, two phenol glucoside gallates, seven tannins, one other hydrolysable tannin, four flavon-3-ols and two complex type tannins from the bark of T. catappa. Their structures were elucidated on the basis of chemical and spectroscopic evidence and tested for antibacterial and antifungal activity and found to have very strong activity.

Three simple coumarins scoparone, limettin and psoralen have been isolated as major components from the leaves of Euodia borbonica var. borbonica (Rutaceae) together with xanthoxylin, a common phenolic compound in Rutaceae family. Their

Biological activities of Limonia crenulata (Roxb.) 43

structures were elucidated through GC - MS and NMR studies. A minor furocoumarin, bergapten, was also detected in the extracts (Valenciennes et al., 1999).

Acetone extract of Limonia acidissima L. dried leaves afforded a potent mosquito larvicide’s identified as n-hexadecanoic acid and found to be effective against fourth instars larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegupti with LC

50, 129.24, 79.58 and 57.23 ppm respectively (Abdul Rahuman et al., 2000).

The chemical constituents of the essential oil from Plectranthus japonicas have been identified by using GC, GC-MS and spectral analysis. The oil was screened for antifungal activity against Aspergillus niger, Alternaria alternate, Penicillium citrinum,

Rhizopus nigricans and Trichoderma viride (Mathpal et al., 2002).

The taxonomic interest in the Neoraputia stimulated an investigation of N. paraensis searching for alkaloids. Fractions were monitored by Hydrogen Nuclear

Magnetic Resonance (1H NMR) and Electrospray Ionization Mass Spectrometry (ESI-

MS) and only those which showed features of anthranilate alkaloids and flavonoids absent in the previous investigations were examined. Stems afforded the alkaloids flindersine, skimmianine, 8-methoxyflindersine and dictamnine; leaves yielded 3',4',7,8- tetramethoxy-5,6-(2",2"-dimethylpyrano)-flavone, 3', 4', 5, 7, 8-penta-methoxyflavone, 5- hydroxy-3', 4', 6, 7- tetramethoxy flavone, 3',4'-methylenedioxy- 5, 6, 7- trimethoxyflavone & 5-hydroxy-3', 4'-methylenedioxy -6,7-dimethoxyflavone. A number of flavonoids isolated from N. paraensis, N. magnifica, Murraya paniculata, Citrus

Biological activities of Limonia crenulata (Roxb.) 44

sinensis graft (Rutaceae) and Lonchocarpus montanus (Leguminosae) were evaluated for their ability to inhibit the enzymatic activity of the protein glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi. Highly oxygenated flavones and isoflavone were the most actives (Valéria et al., 2003).

Skrzypek and Wysokinska (2003) studied the sterols and triterpenes in cell culture of Hyssopus officinalis L. Cell suspension cultures from hypocotyls derived callus of

Hyssopus officinalis were found to produce two sterols, i.e.,sitosterol and stigmasterol as well as several known pentacyclic triterpenes with an oleanene. The triterpenes were identified as oleanolic acid, ursolic acid, 2, 3 dihydroxyolean 12-en-28-oic acid, 2, 3- dihydroxyrus-12-en-28-oic acid, 2, 3, 24-trihydroxyolean-12-en-28-oic acid and 2, 3 24- trihydroxyurs-12-en-oic acid.

Major constituents of fruit pulp of Tamarindus indicus (Pino et al., 2004) were found to be Hexadecanoic acid; 27.4 % of this acid in the roots of Salivia hypolecuca

(Bigdeli et al., 2005), 31.9% in essential oil of Astragalus microcephaus (Rezaee et al.,

2006), palmitic acid 82.5% in Carisssa opaca flower (Rai et al., 2006) was estimated by

GC-MS combination of haxadecanoic acid, oleic and linoleic acid was observed in Coix lacryma – jobi L.(Numata et al., 1994) Salavadora persica (Hosamani and

Pattanashettar,, 2002) and Malvastrum coromandelianun (Hosamani et al., 2004).

The secondary metabolism in the leaves of Piper cernuum produces cinnamic and dihydrocinnamic acid derivatives and the lignin cubebin. In the case of P. crassinervium

Biological activities of Limonia crenulata (Roxb.) 45

flavonoids and prenylated hydroquinones were characterized as major compounds. The cell cultures showed the production of the phenylthylamines, dopamine and tyramine in

P. cernuum, while in P. crassinervium four alkamides were isolated as major compounds, including the new 2,3,4-trimethoxy-N-methyl-aristolaclam and 3-hydroxy-2-methoxy-N- methyl aristolactam (Danelutte et al., 2005).

Simonsen et al. (2006) reported that 2-Methoxyjuglone was isolated from the leaves of Lomatia hirsuta and found to be active against the pathogenic fungus Candida albicans. Cinnamic acid and Vanillic acid were identified as major constituents in the tea by GC-MS. The tea was found not to be toxic against Artemia salina. The presence of phenolic acids with antimicrobial properties supports the traditional use.

Central Council for Research in Ayurveda and Siddha reported that petrol extracts of leaf in Limonia crenulata (Roxb.) contains the phytoconstituents such as xanthotoxin and sitosterol, which is used for analysis of crude drugs or herbal formulations (Patra et al., 2010).

3.9. High-Performance Liquid Chromatography (HPLC) analysis

The biogenesis, structural diversity and distribution of simple, furano and pyranocoumarins in the Rutaceae is reviewed. The potential value of these compounds as taxonomic markers and their possible functions are discussed. The distribution of simple

Biological activities of Limonia crenulata (Roxb.) 46

cinnamic acid precursors of coumarins in the family is also reviewed (Alexander and

Waterman, 1978).

Kajita et al. (1997) reported the structural characterization of modified Lignin in

Transgenic Tobacco plants in which the activity of 4-coumarate: Coenzyme A Ligase is depressed. Transgenic tobacco plants in which the activity of 4-cocumarate: coenzyme

A ligase is very low, contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas- chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic and sinapic, which were bound to the cell walls, were apparently increased as a result of down regulation of the expression of gene for 4-coumarate: coenzyme A ligase. Their data indicated that the behaviour of some of the incorporated hydroxyl cinnamic acids resembles lignin monomers in the brownish tissue and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Two coumarins, auraptene and marmin were isolated from roots of Aegle marmelos (Rutaceae). The isolation process involved extraction with various solvents and separation using chromatography techniques. Antimicrobial activity and cytotoxic tests of the crude extracts of the roots and the isolated compounds against T-cell lymphoblastic leukemia cells were carried out and found to be very weak effect (Riyanto et al., 2002).

Biological activities of Limonia crenulata (Roxb.) 47

Studies on certain chemical constituents in the leaves of Ficus elastica Roxb. and their biological activities were reported by Abdalla et al., (2002). The phytochemical screening of the leaves indicated the presence of four compounds emodin, sucrose, morin and rutin. The bioactivity screening showed that the crude extract and pure isolated compounds possessed antibacterial activity on Bacillus cereus and Pseudomonas aeruginosa.

Olszewska and Wolbis (2002) isolated two new flavonol glycosides, quercetin

3-o-(2-o--D-glucopyranosyl)-a-L-arabinofuranoside and kaempferol 3-o-(2-o-E-p- coumaroyl)-a-L-arabinofuranoside-7-o-a-L-rhamnopyranoside from the leaves of

Prunus spinosa using HPLC. The known compounds, kaempferol, quercetin and their 3- arabinofuranosides, kaempferol 7-rhamnopyranoside, kaempferol 3,7- dirhamnopyranoside and kaempferol 3-arabinofuranoside 7-rhamnopyranoside were also identified.

From the flowers of Ficaria verna Huds. (Ranunculaceae), two flavonol triglycosides were isolated and their structures were elucidated by microscopic analysis

(HPLC, UV, NMR, MS) as 3-o-(-L-rhamnopyranosyl-(1®6)--D-gluco-pyranosyl)-7-o-(-

D-glucopyranosyl)-quercetin and 3-o-(-L-rhamnopyranosy- (1) D-glucopyranosyl) -7-o

(D-glucopyranosyl) -Kaempferol. In addition the structures were determined by using homo- and heteronuclear 2D NMR techniques (Tomczyk and Gudej, 2002).

Biological activities of Limonia crenulata (Roxb.) 48

The phytochemical studies on Terminalia catappa bark and leaves demonstrate the presence of tannins and flavonoid glycosides. Among them, gallic acid, corilagin, ellagic acid and rutin showed in vitro antibacterial activity (Thiem and Goslinska, 2004).

Six flavonoids, ciz., quercetin, 3 - O - methyl kaempferol, quercetin, kaempferol -

3 O - alpha - L - arabinofuranoside, rutin and neobudofficide and four sterols, namely, campesterol, stigmasterol, beta - sitosterol and stigmontanol were isolated by

Neretina et al.,(2004) from the aerial parts of Hedysarum setigerum.

Pharmacognostic specification of Naringi crenulata stem wood, traditional cosmetics in Southeast Asia was done by studying on twelve wood samples from different sources. The powdered stem wood had a sweet natural fragrance but tasteless.

Stem wood fibers were predominately found with large amount of longitudinal cells in addition to high lignin content in cell wall. Wood parenchyma contained starch granules and calcium oxalate crystals with oil globules thoroughly distributed. Alkaloids and coumarin tests were positive. HPLC chromatograms of twelve wood samples were similar in patterns but diverse in quantity. Arbutin content was 0.750% ± 0.414 of the crude extract weight (Kanlayavattanakul et al., 2009).

3.10. Fourier Transform - Infra Red (FT- IR) spectroscopy analysis

Maoela et al. (2009) repored the FT-IR absorption spectra of catechin and ethyl acetate extracts of Coprinus mellei and C. quadrifidus confirming the presences of catechin in the plant extracts. The spectra show the characteristic absorption regions for

Biological activities of Limonia crenulata (Roxb.) 49

O-H group (3400 – 3100 cm-1), C = C group around 1600 cm-1, as well as C – O group (1150 – 1010 cm-1).

Komal Kumar and Devi Prasad (2010) analysed the Fourier Transform Infrared technique to understand the composition, chemical structure and discrimination of biomolecules in medicinal plants of Tephrosia tinctoria and Atylosia albicans. IR spectrum in mid infrared region (4000–400cm–1) was used for discriminating and indentifying various functional groups present in two different species of medicinal plants belonging to the family Leguminosae. Presence of C=O, C–H, C=C and C–O, C–C, C–O were identified. These bonding structures were responsible for the presence of alkyl groups, methyl groups, alcohols, ethers, esters, carboxylic acid, anhydrides and deoxyribose. The results showed that Tephrosia tinctoria and Atylosia albicans are rich in phenolic compounds.

The FT-IR analysis of ethanol extract in G.kollimalayanum was confirmed the presence of the carboxylic acid and Alkenes-CH2; CH3 Aromatic stretching which shows major peaks at 1019.87 and 2922.33cm (Ramachandran and Viswan, 2011).

Sebnem et al. (2006) isolated the secondary metabolites from Phlomis syrica and found out their antioxidant activities. An iridoid glucoside, lamiide, 4-phenylethanoid glycosides, acteoside, -OH acteoside, leucosceptoside A and S amioside, a caffeic acid ester, chlorogenic acid, 2 flavone glucosides, leuteolin-7-o-glucopyranoside and chrysoeriol-7-o-glucopyranoside and a flavanone aglycone, naringenin were isolated

Biological activities of Limonia crenulata (Roxb.) 50

from the aerial parts of Phlomis syriaca. The structures of the isolated compounds were elucidated by means of spectroscopic (UV, IR, 1D and 2D-NMR and Fast Atom

Bombardment Mass Spectrometry (FAB-MS) methods.

3.11. Ultra Violet (UV) - Visible spectroscopy analysis

The UV- Visible spectroscopy analysis of ethanol extract in G.kollimalayanum was evaluated. The peak value were 413.77, 469.15, 664.43 and its electron transition due to OH group (Ramachandran and Viswan, 2011).

Maoela et al. (2009) repored the the UV-vis absorption spectra of Coprinus mellei and C. quadrifidus overlap with that of catechin, which confirms the presences of the catechin. The UV-vis absorption spectra of C. mellei and C. quadrifidus show two absorption bands, a strong one at 218 nm (Band II) and a weak one at 282 nm (Band I). In general terms the band II absorption may be considered as having originated from the A ring benzoyl system and band I from the B ring cinnamoyl system.

Mishra et al. (2010) reported that a new anthraquinone, 1-methyl-2-(3′-methyl- but-2′-enyloxy)-anthraquinone (1) has been isolated from seeds of Aegle marmelos (L.) and was characterized on the basis of spectral analysis (UV, IR, 1H NMR, 13C NMR, 2D

NMR and mass spectroscopy).

3.12. Antimicrobial acitivity

Biological activities of Limonia crenulata (Roxb.) 51

Padmaja and Thangasamy (1993) observed a pharmacological screening, substantial antibacterial, antifungal and antihelminthic activities of some medicinal plants. The hexane and ethyl acetate extracts of the root of Uveria hanun wall and

Uvaria hookeri (Kins.) showed maximum activity. Chromatographic fascination of these extracts led to the isolation of the triterpene, glutinole, taraxerol, b-sitosterol.

Saxena et al. (1994) studied the antimicrobial activity of the methanol extract and isolated constituents of Rhus glahra (Anacardiaceae), a species used in folk medicine by

North American native people was evaluated against 11 microorganisms, including gram- positive and gram-negative bacteria. The extract was subsequently fractionated and monitored by bioassays leading to the isolation of three antibacterial compounds, the methyl ester of 3, 4, 5 trihydroxy benzoic acid (methyl gallate), 4-methoxy and 3, 5 dihydroxybenzoic acid and gallic acid.

Hessa et al. (1995) studied antimicrobial activity of some species of medicinal plants. Vochysia divergens Pohl (Vochysiaceae) is a tree commonly found in wet soils of

Pantanal of Mato Grosso, Brazil and used in folk medicine against infections and asthma.

From the extracts of the stem bark b-sitosterol betulinic acid and sericic acid were isolated.

One hundred crude extracts obtained from various plant parts 59 species representing mostly the plant families Scrophudariacea and Acanthaceae have been investigated for their antimicrobial activity. Plants were selected using ethanobotanical

Biological activities of Limonia crenulata (Roxb.) 52

and chemotaxonomic information. Growth inhibition using agar diffusion assays was determined against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Growth inhibitory activity against one or more of the microbial species was detected in over 40% of the samples (Meurer-Grimes et al., 1996).

From the Indian traditional medicines, 78 plants were selected on the basis of their use in the treatment of infectious diseases. Different concentrations of 80% ethanolic extracts were tested, using the agar dilution method, against four bacteria such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using the agar well diffusion method and against two fungi Candida albicans and Aspergillus niger. In the lowest tested concentration of 1.6 mg/ml, 10% of the plant extracts were active; 44% in a concentration of 6.25 mg/ml and 90% of the plant extracts were active against at least two bacteria in a concentration of 25 mg/ml. Only 13% of the plant extracts were active against at least one fungus in a concentration of 50 mg/ml

(Valsaraj et al., 1997).

Ethanol extract of 109 plants reported to be used in the traditional medicine of

Baja California sur Mexicol were tested for antimicrobial activity against Staphylococcus aureus, Bacillus substilis, Streptococcus faecalis, Escherichia coli and Candida albicans of these, and 64 were active against one or more test organisms (Dimayaga et al., 1998).

A comparative study on the antimicrobial properties of extracts from medicinal plants obtained by two different methods was carried out. The screening of the

Biological activities of Limonia crenulata (Roxb.) 53

antimicrobial activity of extracts from six plants was conducted by a disc diffusion test against gram-positive and gram-negative and fungal organisms. The most active extracts were assayed for the minimum inhibitory concentration and submitted to phytochemical screening by thin-layer chromatography and bioautography. The results obtained indicated that the diethyl ether extracts were the most efficient antimicrobial compounds.

Bioautography showed that the antimicrobial activity was probably due to flavonoids and terpenes (Nostro, 2000).

Agarwal and Sudhir Singh (2000) isolated rhein, physeion, aloe-emodin and chrosophanol from Rheun emodi rhizomes and exhibited antifungal activities against

Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.

Iswar Singh and Ved Pal Singh (2000) studied the antimicrobial activity of aqueous and organic solution extracts of 50 plants belonging to 27 families of seed plants which were screened for antifungal activity against Aspergillus flavus and Aspergillus niger using the agar well diffusion method. The results showed most of the plants have antimicrobial activity.

Perumalsamy and Ignacimuthu (2000) studied the antibacterial properties of medicinal plants. A series of 30 Indian folklore medicinal plants used by tribal to treat infections were screened for antibacterial properties at 10 mg/ml concentration by using disc diffusion method against Bacillus substilis, E. coli and Klebsiella aerogens.

Biological activities of Limonia crenulata (Roxb.) 54

Iwalokun et al. (2001) studied on three Nigerian medicinal plants and investigated their activities against multidrug-resistant Shigella species isolated from patients with bacilliary dysentery in Lagos. Decoctions of Ocimum gratissimum and concoction of

O. gratissimum and Terminalia avicennoides at crude concentration of 3000 µg/ml markedly inhibited the growth of all isolates tested. Minimum inhibitory concentration and maximum bactericidal concentration revealed at higher Shigellocidal property of

Momordica balsamina. The results suggested that aqueous extracts of O. gratissimum and

T. avicennoides as decoctions and concoctions could be useful in the treatment of

Shigellosis and should be clinically evaluated specially in Nigerian region.

The aqueous extract of Limonia acidissima L. has antimicrobial activity against tested bacterial strain. Volatile oil in Limonia acidissima had also been found to have antimicrobial activity and anti helmintic activity (Garg, 2001).

Gnanamani et al. (2003) investigated antibacterial activity of crude alcoholic extract of Datura alba and Celosia argentica leaves were studied against pathogens isolated from infected burn patients. The disc diffusion method showed significant zone of lysis against all the pathogens studied and the results were comparable to the conventional antibiotic cream namely silver sulphadiazine (SSD). On comparing the efficiency of the two extracts of Datura alba exhibited more than 50% increase in antibacterial activity compared to Celosia argentica.

Biological activities of Limonia crenulata (Roxb.) 55

Alangium saliifolium is an ethnomedicinal plant used in folkfore as a medicine.

The leaves were extracted with water, ethanol and chloroform and each extract was evaluated for antibacterial activity against pathogenic strains of Escherichia coli, Proteus vulgaris, Bacillus subtilis, Enterobacter faecalis, Serratia marcescens and Klebsiella pneumoniae by using disc diffusion method. The zones of inhibition were recorded and compared with standard drugs i.e. chloramphenicol. Ethanolic extract showed the high degree of inhibition when compared with chloroform and aquous extracts (Natarajan et al., 2003).

Alam et al. (2003) synthesized and studied the antibacterial activity of

Pongaglabol was tested for antibacterial effects against Shigella dysenteriae, Salmonella typhi, Streptococcus B-haemotyticus and Staphylococcus aureus.

Somchit et al. (2003) screened the crude ethanol and water extract of leaves and barks from Cassia alata and they were tested in vitro against fungi, yeast and bacteria.

Results showed that the water extracts exhibited higher antibacterial activity than the ethanol extract from leaves (inhibition zones of 11-14 and 9-11 mm respectively).

Escheretia coli showed resistance to all types of extracts.

The methanol extracts of 306 plants of 52 families obtained from northeast of Iran

(Khorasan province) were tested for antimicrobial activity (in vitro) using the cylinder plate assay method. Activity against Escherichia coli, Klebsiella pneumoniae,

Salmonella typhi, Pseudomonas aeruginosa, Morganella morgani, Bacillus subtilis,

Biological activities of Limonia crenulata (Roxb.) 56

Staphylococcus aureus and Candida albicans was showed significant results (Frazly et al., 2003).

Kartal et al. (2003) studied the antimicrobial activity of two propolis samples from Kazan and Marmaris regions in Turkey. They were tested with four different ethanolic extracts (30, 50, 70 and 96% ethanol) of each sample against seven gram- positive, four gram-negative bacteria and one fungus culture. The activity was found to be mainly due to caffeic acid and its esters.

Matu and Staden (2003) investigated antimicrobial activities of plant species in

Kenya. Aqueous, hexane and methanol extracts of 12 plant species traditionally used in

Kenya for the treatment of ailments of infections and inflammatory nature were screened for in vitro antibacterial activities.

Rani and Khullar (2004) screened some important plants in Ayurvedic system of traditional medicines in India to treat enteric diseases. Fifty four plant extract (methanol and aqueous) were assayed for their activity against multi-drug resistant Salmonella typhi. Strong antibacterial activity was shown by the methanol extracts of Aegle marmelos, Punica granatum, Myristica fragrans, Terminalia chebula, Acacia catechu,

Solanum nigrum, Carum copticum, Apium graveolens, Ocimum sanctum and Butea monosperma etc.

Biological activities of Limonia crenulata (Roxb.) 57

Yogamoorthi and Srikala (2004) studied that the anti pathogenic bacterial properties of skin secretion (Mucus) of Narcine timelei. The fresh mucus extract was tested by the disc method against common pathogenic bacterial species namely

Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus faecalis, and Vibrio cholerae.

Triterpenes of a hispidissima were investigated and found to be active against selected bacteria and fungi. Beta amycin demonstrated the maximum activity against

Escherichia coli. Four new diterpenes were isolated from the leaves of Myrospermum frutescens as minor constituents. 6-Beta-1, 8-diacetoxycassan-13, 15 dinent, chargreslactone, chasresnone derivative products were obtained to test their activities against Chags’s disease. The hexane extract of the rhizome of Curcuma longa exhibited significant activity against gram-positive and insignificant activity against gram-negative bacteria assay as compared to standard antibiotics (Torres et al., 2004).

Kavitha et al. (2004) reported that alkaloids from the ethanolic extract of

Holarrhena antidysentrica seeds were evaluated for their antibacterial activity against clinical isolated enteropathogenic Escheritia coli and their antidiarrhoeal activity on castor oil induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of Enteropathogenic Escherichia coli

(EPEC) was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy.

The loss of plasmid DNA and suppression of high molecular weight proteins were

Biological activities of Limonia crenulata (Roxb.) 58

observed on alkaloids treatment. The results suggest the usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.

Adamu et al. (2005) investigated the preliminary antimicrobial activity of the aqueous extracts of the 84 medicinal plants. Among the 84 plants, 75 exhibited antimicrobial activity against several tested organisms at the concentrations of 200 mg/ml. The extracts were found to show strong activity against Proteus mirabilis,

Pseudomonas aeruginosa, Staphylococcus aureus and E. coli.

De Boer et al. (2005) reported that 29 plants used for medicinal purposes and 41 plants used for non-medicinal purposes in Tanzania. Six medicinally used plants were selected for bioactivity analysis. Extracts of Candida albicans, Aspergillus fumigatus,

Fusarium culmorum, Staphylococcus aureus, Pseudomonas syringae and Erwinia amylovora. All plants showed activity against several test organisms.

Kloucex et al. (2005) investigated the antimicrobial activity of 7 ethanol extracts of Brunfelsia grandiflora, Caesalpinia spinosa, Dracontium loretense, Equisetum giganteum. Terminalia catapa, Phyllanthus amarus and Piper aduncum. Among the plants tested, Phyllanthus amarus and Terminalia catappa showed the most promising antibacterial properties with minimum inhibitory concentration ranging from 0.25 to 16 mg/ml. The extract isolated from the aerial part of Piper aduncum was more active against gram - positive than gram - negative bacteria.

Biological activities of Limonia crenulata (Roxb.) 59

Phongpaichit et al. (2005) obtained 36 extracts from 10 plant species. They were screened for their antifungal activity against Candida albicans, Cryptococcus neoformans and Microsporum gypseum. The chloroform extracts of Alpinia galanga and Boesbergia pandurata showed strong antifungal activity against C. neoformans and M. gypseum, but weak activity against Candida albicans, Alpinia galanga and Boesbergia pandurata are excellent candidates for the development of a remedy from opportunistic fungal infections in AIDS patients.

Twenty five selected plants belonging to 19 families were collected from different localities of the island Soqutra dried, and extracted with the solvents chloroform, methanol and hot water to yield 80 extracts. The extracts were tested for their antimicrobial activity against several gram - positive and gram - negative bacteria and against one yeast species using agar diffusion method. Antibacterial activity was demonstrated especially against gram - positive bacteria including multi resistant

Staphylococcus strains. The greatest activity was exhibited by the methanolic extracts of

Boswellia elongata, B. ameero, Buxus hidebranchi, Commiphora parnifolia, Jatropha unicostata (Ramzi et al., 2005).

Meryem (2005) reported that the methanol extract of Verbascum georgicum

Bentham was investigated for its in vitro antimicrobial properties. A total 143 microorganisms belonging to 56 bacteria and four fungi and a yeast species were studied using the disk diffusion method and microdilution assays. The results indicated that the methanol extract of V. georgicum had an inhibitory effect on the growth of all Candida

Biological activities of Limonia crenulata (Roxb.) 60

albicans isolates and 17 strains in 10 different species of bacteria. Thus the results suggested that V. georgicum extract possesses compounds with antimicrobial properties that might be utilized for developing new drugs.

Santhi and Alagesaboopathi (2005) explained the antibacterial activity of the aqueous, ethanol and chloroform extracts from the leaves of Andrographis lineate Nees were determined by using the agar disc diffusion method against Staphylococcus aureus,

Shigella dysentriae, Salmonella typhi and Vibrio cholerae. Ethanolic leaf extract showed high antibacterial effect against V. cholerae, and S. dysentriae. The antibacterial activity of ethanol extract was found to be higher than that of distilled water extract.

Akgul and Saglikogul (2005) investigated the antibacterial activity of the methanolic extract and its fractions of aerial parts of Anthemis tinctoria (Asteraceae) against gram positive Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis

(ATCC 29212) and gram negative Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The activity was concentrated mainly in the dichloromethane

(DCM) and hexane fractions of crude methanolic extracts. The 5mg of DCM extracts per disk produced 15-16 mm of inhibition zone against E. faecalis and P. aeruginosa, however, no activity was found against E. faecalis and E. coli. The hexane fraction showed activity against S. aureus, P. aeruginosa and E .faecalis.

Manikandan et al. (2006) studied antibacterial activity of Aristolochia bracteata

Retz (Aristolochiaceae), a common annul herb, widely distributed in India and widely

Biological activities of Limonia crenulata (Roxb.) 61

used in indigenous system of medicine. The leaves were extracted with petroleum ether, chloroform and alcohol. The concentrated crude leaf extracts were tested against Bacillus subtilis, Lactobacillus plantarum, Escherichia coli, Staphylococcus aureus,

Streptococcus faecalis and Pseudomonas aeruginosa. Alcoholic extract showed significant antibacterial activity when compared to other extracts.

The ethanolic extracts of the leaves and flowers of Cleome viscosa and roots of

Gmelina asiatica were tested for antimicrobial activity. The two plants exhibited a broad spectrum of antimicrobial activity, particularly significative against E. coli, Proteus vulgaris, Pseudomonas aeruginosa. The leaf extract of C. viscosa showed moderate activity against pathogenic fungi (Sudhakar et al., 2006).

Pereira (2006) reported the antimicrobial activity of Indigofera suffruticosa.

Various organic and aqueous extracts of leaves of I. suffrueticosa Mill (Fabaceae) obtained by infusion and maceration were screened for their antibacterial and antifungal activities. The extracts were tested against five different species of human pathogenic bacteria and 17 fungal strains by the agar solid diffusion method. Most of the extracts were devoid of antifungal and antibacterial activities, except the aqueous extracts of the leaves by infusion, showed inhibitory activity against the gram-positive bacteria

Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 5000 g ml-1.

The ethanolic extract of Teclea afzelii together with three alkaloids identified as

Kokusaginine (1), Maculine (2), Kolbisine (4) and a common terpenoid, Lupeol (3),

Biological activities of Limonia crenulata (Roxb.) 62

isolated from the stem bark of Teclea afzelii were tested for their antimicrobial activity against Gram-positive and negative bacteria, fungi and Mycobacterium smegmatis. Agar diffusion assay was used for the determination of the sensitivity of test organisms to the samples. The micro-dilution method was used to determine the Minimal Inhibition

Concentration (MIC) and the Minimal Microbicidal Concentration (MMC). The results of the diffusion test showed that only compound 1 was active on all the tested micro- organisms, whilst the inhibition effect of the crude extract and that of compounds 2 and 4 was observed on 87.5% of the tested microbial species. The lowest MIC value

(19.53 µg/ml) for the crude extract was obtained on Escherichia coli, Bacillus subtilis and

Microsporum audorium. The corresponding value for the tested compounds (2.44 µg/ml) was recorded with compound 2 on B. subtilis. The crude extract, compounds 2 and 3 showed moderate activity against M. smegmatis. The overall results provide promising basis for the use of the crude extract as well as the isolated alkaloids in the treatment of specific microbial infections (Kuete et al., 2008).

The antimicrobial activity of methanolic extracts of some medicinal plants against

Escherichia coli, Salmonella typhimurum, Staphylococcus aureus and Enterococcus sp.

The methanolic extract of Caryophyllus aromaticus presented the highest anti S. aureus activity and was effective against all bacterial strains tested (Ushimalu et al., 2007).

The in vitro evaluation of antibacterial and antifungal activity was carried out by the well agar diffusion method on a panel of gram - positive and gram - negative bacteria such as Pseudomonas. aeruginosa, Escherichia coli, Streptococcus faecalis,

Biological activities of Limonia crenulata (Roxb.) 63

Salmonella cholera, Proteus mirabilis, Morganella norganii and two group of fungi

(filamentous, yeast). The results showed that 12 of 17 extracts demonstrated antibacterial activity against the pathogenic bacteria tested. The growth inhibition holes were ranged from 8.00 to 32.33. Among them extracts of Solanum aculeasteum (Solanaceae) and

Syngicum guinensis (Myctaceae) showed and higher antibacterial activity. For the antifungal activity growth inhibition holes varied from 8.00 to 17.55 mostly against

Geotrichum candidum and Penicillium species. The extracts Solanum acculeasteum demonstrated antibacterial and antifungal activity (Pieme et al., 2008).

Antimicrobial activity of the leaf and root extracts of Indigofera tinctoria (Linn.),

Wrightia tinctoria (Br.) and Rungia repens against human pathogenic bacteria such as

Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and pathogenic fungi such as Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus. Ethanolic leaf and root extracts were prepared and based on the susceptibility of the test organisms were determined. It was found that ethanolic leaf and root extracts were prepared and based on the susceptibility of the test organisms were determined. It was found that ethanolic extracts showed high inhibition zone those control experiments (Madhavan and Saritha,

2008).

The extracts of Acacia arabica, Nymphaea lotus, Sphaeranthus hirtus, Emblica officinalis, Ginchorium intybus and Cardus marianum were tested in vitro against bacterial species and fungal species by well diffusion method and micro dilution methods. The patterns of inhibition varied with the plant extracts, the solvent used for

Biological activities of Limonia crenulata (Roxb.) 64

extraction and the organisms tested. Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa were the most inhibited microorganisms. The extract of Sphaeranthus hirtus was the most active against multi drug resistant Pseudomonas aeruginosa and

Enterohemoerrhagic E. coli 0157. The ethanolic extract of S. hirtus exhibited a higher effect than the hot water extract. These plants extracts were analysed for elemental composition (Ammara Hassan et al., 2009).

Patil et al. (2009) was reported that Aegle marmelos (L.) (Rutaceae) possess a number of medicinal properties including antidiarrhoeal, antimicrobial, antifungal and activities. The antiaflatoxigenic effects of ethanolic extract of the leaves of Aegle marmelos (L.) were studied on common aflatoxigenic fungal species. Aegle marmelos

(L.) exhibited antifungal and antiaflatoxigenic activity at a concentration range of 0.5 to

2 mg/ml. The shake flask method was used to evaluate the antifungal and antiaflatoxigenic activity. The extract showed varied levels of antifungal and antiaflatoxigenic activity against the test fungi. Preliminary phytochemical tests of ethanolic extracts demonstrated the presence of major phytochemicals like phenols, tannins, flavonoids and alkaloids as major constituents.

Sumathi and Parvathi (2010) analysed the antimicrobial activity of the extracts of

Andrographis paniculata Nees; Phyllanthus niruri Linn; Terminalia bellerica Roxb.;

Terminalia chebula Retz.; and Vitex negundo Linn., against four gram - negative and one gram - positive bacteria. The results showed that the minimum inhibitory concentration

(MIC) of P. niruri leaf extract was 50 µg/ml against Salmonella typhi and

Biological activities of Limonia crenulata (Roxb.) 65

Staphylococcus aureus, where as, the MICs of T. bellerica fruit extract against

Escherichia coli and S. aureus were 50 and 200 µg/ml respectively. However, the leaf extracts of the Andrographis paniculata, T. chebula and V. negundo have not shown any antimicrobial activity in the tested concentrations.

Venkatesan and Karrunakaran (2010) reported Aegle marmelos, Solanum nigrum and Cassia fistula were extracted by soxhlet extraction method. Three plant materials were subjected to preliminary phytochemical screening activity against gram - negative organism of Escherichia coli and gram - positive organism of Staphylococcus aureus and they were compared with control drug Penicillin at different concentrations at 0.5, 1.0,

1.5, 2.0, and 2.5 mg/ml by disc diffusion method. At higher concentration of 2.5mg/ml

Cassia fistula exhibits maximum zone of inhibition of about 30.9 mm against

Staphylococcus aureus, and was considered as susceptible. The zone of inhibition was not found in Aegle marmelos and Solanum nigrum and considered as resistant. In case of

Escherichia coli, Solanum nigrum exhibits maximum zone of inhibition of about 30.1mm such zones were not found in Aegle marmelos and Cassia fistula and were considered as resistant and control drug penicillin shows less activity compared to the plant extract

Aegle marmelos, Solanum nigrum and Cassia fistula.

3.13. Antioxidant activity

Naringi crenulata (Roxb.) Nicolson belongs to the family of Rutaceae. Its stem had been claimed to cure prickly rash on the skin and Burmese people used as a whitening agent. Previous studies of N. crenulata found to exhibit antityrosinase, anti- inflammatory and antioxidative activities and active substances are umbelliferone and scopoletin. The aim of this special project is to develop N. crenulata to be a

Biological activities of Limonia crenulata (Roxb.) 66

sunscreen lotion from standardized alcoholic extract. The experiment was carried on the percentage of alcohol for the suitable extraction and the result showed that 70% ethanol was the suitable by evaluating the percentage of active compound (Thin

Layer Chromatography) and antioxidative activity (2, 2-Diphenyl-1picrylhydrazyl -

DPPH method). The lotion preparation was prepared from 1% alcoholic extract. The lotion contained 0.28 milligram% and 0.51 milligram% of umbelliferone and scopoletin, respectively. Its antioxidative activity was showed at the concentration of

23.32 microgram/milligram and sunscreen effective activity (Sun Protection Factor -

SPF) was 1.25 (Pensri and Kaewprakan, 2005).

The antioxidant activities of methanol and ethyl ether extracts obtained from the leaves of Thymus zygis, collected during the flowering or non - flowering period, were evaluated and compared by Soares et al. (1997). The results showed that the methanolic extracts are more potent as scavengers of peroxyl and superoxide radicals than the ethyl ether extracts. Apparently, there is a relationship between antioxidant potency and the total phenolic groups content in each extract.

A novel flavonoid diglycoside, 5, 6, 7, 4' - tetrahydroxyflavone 3 - O - rutinoside, and a previously known compound, kaempferol 3 - O - neohesperidoside were isolated from an ethyl acetate extract of Daphyniphyllum calycinum leaves showed significant antioxidant activity was reported by Gamez et al., (1998).

Biological activities of Limonia crenulata (Roxb.) 67

The in vitro antioxidant and free radical scavenging properties of bark extracts of

Anadenanthera macrocarpa (Benth.) (Fabaceae), Astronium uruncleuva (Engl.)

(Anacardiaceae), Mimosa verrucosa (Benth.) (Fabaceae) and Sideroxylon obtusifolium

T.D. (Penn.) were determined by monitoring the intensity of luminal-enhance

Chemiluminescence (CL), using 2, 2’azo bis (2-amidinopropane) as a peroxyl radical source (Desmarchelier et al., 1999).

Aquino et al. (2001) examined the methanolic extract of the leaves of

Anthurium versicolor and isolated two main fractions. Both the extract and the fractions were assayed for their radical - scavenging activity by means of an in vitro test (bleaching of the stable 1, 1 - dipheny 1 - 2 - picrylhydrazy 1-2 radical) and reported to have a significant radical - scavenging effect.

Methanol extracts, prepared separately from the roots, stems and leaves of four traditional Zulu medicinal plants (Rhoicissus digitata, R. rhomboidea, R. tomentosa and

R. tridentata), were tested for their antioxidant activity. The extracts of R.rhomboidea and R. tridentata inhibiter the activities of the 1, 1' - dipheny 1 - 2 picryhydrazyl free radical, zanthine oxidase and also prevented the production of thiobarbituric acid reactive substances and free radical mediated Deoxyribonucleic acid (DNA) sugar damage. The extracts have a strong chelating effect on Fe++ ions. R. digitata and R. tomentosa extracts, however, possessed some prooxidative properties at high concentrations (Opoku et al., 2002).

Biological activities of Limonia crenulata (Roxb.) 68

The antioxidant properties of six medicinal herbs used in the traditional

Paraguayan medicine were studied using free radical generating systems by Velazquez et al., (2003). The methanol extracts from Asristolochia giberti, Cecropia pachystachya,

Eugenia uniflora, Piper fulvescens, Schinus weinmannifolia and Schinus terebinthifolia protect against enzymatic and non - enzymatic lipid peroxidation in microsomal membranes of rat. C. pachystachys, E. uniflora and S. terebinthifolia showed the highest scavenging activity on the superoxide and 2, 2 dipheny 1 - 1 - picrylhydazyl

(DPPH) radicals.

The antioxidant activity of the different extracts and fractions of a aerial parts of

Otostegia persia (Burm) Boiss were evaluated using betacarotene bleaching and lipid peroxidation method. The inhibitory activity of the plant extracts on the peroxidation of linoleicacid was measured by ferric thiocyanate method in comparison to methanolic extracts of green tea, Ginkgo biloba, Vitamin E and BHA as positive control. Methanolic extract of the plant exhibited strong antioxidant activity (Shrififar et al., 2003).

Kim et al. (2003) suggested that the n-hexane and butanol fractions of Artemisia apiacea have significant free radicals scavenging effect. This was confirmed through

DPPH free radical scavenging activity.

Bajpai et al. (2005) indentified promising sources of antioxidants, from the leaves, bark and fruits of Terminalia arjuna, Terminalia bellerica, T. chebula and

Terminalia muelleri. The leaves and fruits of Phyllanthus emblica and the seeds of

Biological activities of Limonia crenulata (Roxb.) 69

Syzygium cumini were also found to have high total phenolic contents (72.0 - 167.2 mg/g) and high antioxidant activity (69.6 - 90.6%).

The phenolic extracts of the dried 11 Algerian medicinal plants have been performed using 70% ethanol. The antioxidant activity measurement, expressed as

Trolox Equivalent Antioxidant Capacity (TEAC), ranged from 9.40 to 33.06mM Trolox equivalent (Djeridane et al., 2006).

Antioxidant activity of methanol extract and ethyl acetate extract of root bark of firing tree (Chionanthus virginicus L.) were evaluated. Total antioxidant activity was measured according to ferric thiocynate method methanol extract and ethyl acetate extract showed 69.4, 79.3, 72.3 and 83.7% inhibition on lipid peroxidation of linoleic acid emulsion, respectively at the 10 and 20g/ml concentrations (Gulcin et al., 2007).

Umamaheswari et al. (2007) evaluated the antiulcer and antioxidant activities of

70% ethanolic extract of leaves of Jasminum grandiflorum L. They suggested that

Jasminum grandiflorum possess potential antiulcer activity, which may be attributed to its antioxidant activity. The in vitro antioxidant activities of the methanol extract of

Paullinia pinnata leaves were evaluated using different testing systems by Jimoh et al.,

(2007). The results showed that P. pinnata possessed strong scavenging activity and moderate reducing power. The total phenol, flavonoid and proanthocyanidin contents of the extracts were very close to those reported for most medicinal plants and showed good correlation with its antioxidant activities.

Biological activities of Limonia crenulata (Roxb.) 70

Four extracts of Enicostemma axillare were examined for in vitro antioxidant activity using nine different methods. In the 2,2'-azino-bis 3-ethylbenzthiazoline-6- sulphonic acid (ABTS) method. All the four extracts of E. axillare showed potent antioxidant activity with half maximal inhibitory concentration (IC50) values, ranging from 13.26 to 24.36 g/ml. All extracts showed moderate antioxidant capacity using the phosphomolibdenum method (Jaishree et al., 2008).

Adeolu et al. (2008) were studied the antibacterial antioxidant activities and phenolic contents of methanol extracts of the leaves and stems of Calpurnia aurea using in vitro standard method. The antioxidant activities were determined by DPPH and

Ferrous Reducing Antioxidant Pathway (FRAP) method which showed significant result.

Nickavar et al. (2008) evaluated the antioxidant and free radical scavenging properties and determine the phenolic content of the ethanol extract from five Mentha species. M. piperita exhibited the strong activity as a DPPH scavenger. All the extracts were active in ABTS+ assay and no significant difference was observed in this assay.

The total phenolic content of extract was determined by Folin-ciocalteu method and M. pipertia showed the highest Total Phenolic Content (TPC).

Elango and Chithra (2009) studied that effect of Limonia acidissima L.

(Rutaceae) on blood glucose levels and antioxidant enzyme levels in Alloxan induced diabetic rats. Alloxan (120 mg/kg, i.p) induced diabetic rats were treated with Limonia

Biological activities of Limonia crenulata (Roxb.) 71

acidissima L. methanolic extract at a dose levels of 200 and 400 mg/kg for 21 days.

Glucose level was measured in blood serum and antioxidant enzyme levels viz.

Superoxide Dismutase (SOD), Catalase (CAT) and Lipid Per oxidation (LPO) were measured in pancreatic homogenate, methanolic extract of fruit pulp of Limonia acidissima L. significantly (P<0.01) lowered the Alloxan induced hyperglycemia. It also produced a significant (P<0.01) decrease in peroxidation products viz. Malonyl

Dialdehyde (MDA) in blood serum. The activity of antioxidant enzymes such as SOD,

CAT was found to be higher in the blood serum of diabetic animals treated with the

Limonia acidissima L. extract. This confirms the antihyperglycemic and antioxidant activities of Limonia acidissima L. in Alloxan induced diabetic rats.

Dhruti et al.(2009) studied that the antioxidant activity of the methanolic and aqueous extracts of Martynia annua (Linn.) leaves were evaluated by several in vitro systems of assay namely such as reducing power assay, DPPH radical-scavenging activity, nitric oxide scavenging activity, H2O2 radical scavenging activity, superoxide radical scavenging assay, hydroxyl radical-scavenging activity, and total antioxidant capacity. Total phenolic content was measured by Folin–Ciocalteau reagent. The antioxidant property depends upon concentration and increased with increasing amount of the extract. The free radical scavenging and antioxidant activities may be attributed to the presence of phenolic and flavonoid compounds present in the extract. The results showed that the methanolic extract exhibited higher antioxidant activity than the aqueous extract. Chlorogenic acid is reported in this plant and a TLC densitometric method was developed for the quantification of chlorogenic acid.

Biological activities of Limonia crenulata (Roxb.) 72

Mahesh Kumar (2010) studied that in vitro antioxidant activity of methanolic extract of aerial parts of Salvia splendens was determined by DPPH free radical scavenging, hydrogen peroxide scavenging and superoxide anion scavenging assays.

Ascorbic acid and butylated hydroxyl anisole were used as standard antioxidants for the analysis. All the analysis was made with the use of UV–Visible spectrophotometer

(Schimadzu uv-vis 1700). The methanolic extract of aerial parts of Salvia splendens had shown very significant DPPH (1, 1, Diphenyl-2-picryl-hydrazyl) radical scavenging, hydrogen peroxide scavenging, and superoxide anion scavenging activity compared to standard antioxidants. The IC50 values of methanolic extract in DPPH radical scavenging, hydrogen peroxide scavenging and superoxide anion scavenging assays are found to be

460 µg/ml, 358 µg/ml and 527 µg /ml respectively.

The fruits of Terminalia chebula, Terminalia bellerica and Emblica officinalis are important herbal raw materials containing polyphenols. They form the major constituents of widely used Ayurvedic formulations like Triphala churna. The extracts of these materials were standardized with respect to their total polyphenol contents as determined by Prussian blue method using gum acacia and phosphoric acid as stabilizers. The antioxidant activities were determined by DPPH (1,1-diphenyl-2-picryl-hydrazyl) method and inhibition of lipid peroxide formation induced by Fe2+-ascorbate system. They were found to strongly correlate with total polyphenol contents. The half maximal effective concentration (EC50) value (µg/ml) for free radical scavenging activity by DPPH method and IC50 value (µg/ml) for lipid peroxidation inhibitory activity along with the total

Biological activities of Limonia crenulata (Roxb.) 73

polyphenol contents can be used as quality control parameters for standardization of herbal raw materials containing polyphenols as major phytoconstituents (Hazra et al.,

2010).

3.14. Anti-inflammatory activity

Gossypin is a bioflavonoid obtained from Hibiscus vitifolius flower. The anti- inflammatory activity of gossypin has been studied in comparison with the standard non steroid anti-inflammatory agent phenyl butazone against various experimental models of inflammation and increased vascular permeability. The migration of leucocytes and the formation of pleural exudates were also significantly reduced after pretreatment with gossypin in carageenin and turpentine induced rats (Palmer and Ghos, 1978).

Alcoholic extract of Teeburb has been found to produce significant anti- inflammatory activity at 2nd and 3rd hours of administration on carrageenin induced rat paw oedema. Phenylbutazone has been found to be having significantly higher anti- inflammatory activity than Tueeburb suggesting similar mechanism of action of these drugs (Sharma and Srivastava, 1991).

Petroleum ether extract of Ricinus communis exhibited significant anti- inflammatory activity against induced rats paw arthritis. Drug was safe upto a dose of mg/kg per oral (p.o). and at a dose 150 mg/kg p.o. exhibited no significant analgesic activity in rats (Banerjee et al., 1991).

Biological activities of Limonia crenulata (Roxb.) 74

Triterpenoids are one of the most abundant class of compounds in plants. It has frequently been suggested that triterpenoids play an defensive role against pathogens and herbivores. They also have several interesting pharmacological activities that include anti-inflammatory (Recio et al., 1995), antibacterial (Cantrell et al., 2001), antiviral and cytotoxic properties.

Methanolic extract of dried leaves of Alstonia macrophylla and its fractions were investigated for its anti-inflammatory activity. The extract was concentrations of 200 and

50 mg kg-1, p.o. showed the dextran - induced granuloma (Chronic model) in rats. Anti- inflammatory activity of the tested extract and its fractions was comparable with that of the standard drug indomethacin (10 mg kg-1) (Arunachalam et al., 2002).

Petrovic et al. (2003) reported that oral administration of the chloroform extract from Tanacetum larvatum (Griseb. ex apnt) Kanitz caused a dose dependent anti- inflammatory effect in the carrageenan - induced rat paw oedema test and the results showed statistical significance at a dose of 50 mg/kg.

Patil et al. (2003) investigated the ethanolic crude extract of Anacardium occidentale. leaves and its five different crude fractions for anti-inflammatory activity in albino rats (300 mg/kg). Ethanolic extract and butanone fraction exhibited significant anti-inflammatory activity when compared with control and standard drug diclofenac sodium (100 mg/kg).

Biological activities of Limonia crenulata (Roxb.) 75

Crude ethanolic extract of Pergularia extensa leaves was fractioned with petroleuum ether, solvent ether, ethyl acetate, butanol and butanone. The ethanolic extract and various fractions were investigated for anti-inflammatory activity in rats at a dose of

100 mg/kg intraperitoneally. Ethanol extract and it butanol fraction exhibited significant anti-inflammatory activity when compared with respective controls and were comparable with that of standard drug aspirin (Hukkeri et al., 2003).

Vishnoil et al. (2003) reported that the methanol and petroleum ether extracts of

Abies webbiana leaves exhibited significant anti-inflammatory activity against carrageenan induced rat hind paw oedema, the percentage protection was found to be effective as comparable to that standard drug diclofenac sodium.

Steroids, steroidal saponins, flavonoids, triterpenoidal and carbohydrates were detected in different extracts of the stem of Neptunea oleracea. The alcoholic extract was found to possess significant anti-inflammatory activity (Lakshmayya Joshi et al., 2003).

The anti-inflammatory effect of the methanol extract of the leaves of Bambusa arundinacea against carrageenin induced as well as immunologically induced paw edema and also its antiulcer activity in albino rats have been studied and found to be significant when compared to the standard drugs. The combination of methanol extract and phenylbutayone (Non - Streoidal Anti-inflammatory Agent, NSAIA) has also been studied and found to have the most potent anti-inflammatory activity experimentally with least toxic (no ulcerogenic) activity (Muniappan and Sundararaj, 2003).

Biological activities of Limonia crenulata (Roxb.) 76

Various extracts of Indigofera aspalathoides were tested for anti-inflammatory activity using carrageenan induced paw edema assay, to isolate bioactive compounds from active extracts and to screen the isolated compounds for in vitro COX-1/COX-2 inhibitory activity. The flavonoid rich fraction was obtained from ethyl acetate extract.

Repeated chromatography followed by crystallization of flavonoid rich fraction afforded the isolation of four compound viz., 4,2,4 – trihydroxyflavone and lutedin. The compounds, butein and 7,3,4 – trihydroxy flavone were tested in vitro for COX 1/COX 2 inhibitory activity. Both the compounds were COX-1 selective and the butein (28.4 micro M) was found to be potent than 7,3,4-trihydroxy flavone (35.7 micro M). Both the compounds showed moderate inhibitory activity towards COX-2. The observed percentage inhibition value at 100 micro g/ml where 37.2 and 30.3 percent for butein and

7,3,4-trihydroxy flavone, respectively (Brahmbhatt et al., 2004).

The intra peritoneal administration of the methanol extract of Oenothera rose at a dose of 400 mg/kg produced a high reduction of hind - paw edema was reported by

Meckes et al (2004). They also demonstrated a moderate inhibition of edema formation with the methanol extracts of Astianthus viminalis, Brickellia paniculate, Chamaedorea tepejilote and Justicia spicigera.

The antiociceptive, anti-inflammatory effect, and acute toxicity of the aqueous extract from leaves of Pimenta racemosa have been investigated by Garcia et al. (2004).

The aqueous extract (125 and 250 mg/kg) significantly and in a dose - dependant manner

Biological activities of Limonia crenulata (Roxb.) 77

reduced the nociception induced by the acetic acid intraperitoneal injection (P less than

0.001) and also reduced the carrageenan - induced paw in rat at 1, 3 and 5hrs (P< 0.001).

Hibiscus species have been used as folk remedy for the treatment of skin diseases, as an anti-inflammatory agent, antiseptic and carminative some compounds isolated from the sepsis, such as flavonoids, phenolic acids, are considered responsible for these activities (Vasudeva and Sharma, 2008).

Gupta1, et al., (2010) investigated the various extracts of leaves of Bryophyllum pinnatum in chemically induced inflammation rodents model. The extracts/ inhibited formaldehyde induced paw edema in rats. These inhibitions were statistically significant

(p<0.05-0.01, 0.001) as compared to control. Methanolic extract showed highest activity.

Modi et al., (2010) analysed the anti-inflammatory activity of the water extract of

Argyreia nervosa. Inflammatory diseases including different types of rheumatic diseases are very common throughout the world. Argyreia nervosa is used as a folk medicine for the treatment of inflammation in India. The plant Argyreia nervosa possesses a significant anti-inflammatory activity as evidenced in carrageenan induced paw edema method, which supports the folkloric claim of the anti-inflammatory activity of the plant.

3.15. Antiulcer activity

Peptic ulcer is a lesion of gastric or duodenal mucosa occurring at a site. The mucosal epithelium were exposed to aggressive factors (gastric acid & pepsin) and

Biological activities of Limonia crenulata (Roxb.) 78

mucosal defensive factors (blood flow, mucus, bicarbonate - HCO3 secretion, etc) (Sun,

1974).

Ulcer is a benign lesion of the gastric or duodenal mucosa, which occurs at a site where the mucosal epithelium is exposed to acid and pepsin stress, smoking, nutritional deficiencies and ingestion of nonsteroidal anti-inflammatory drugs can all increase the incidence of gastric ulcer (Belaiche et al.,2002). The mechanism of formation of peptic ulcer (Baron et al., 1980; Piper and Stiel, 1986), its treatment (Sung et al., 1995; Soll,

1996) and the action of the antiulcer and aspects of their adverse reactions and the recurrence of the ulcer have been reviewed (Ariyoshi et al., 1986).

Okwari et al., (2000) studied the effect of an aqueous extract of the leaves of

Dombeya bucetteri on gastric acid secretion and ethanol - induced gastric mucosal damage in rats. Pretreatment with the extract also reduced the extent of gastric mucosal damage induced by oral ethanol (75%), but had no effect no mucus secretion. It is suggested that the consumption of an extract of the leaves of D. buettneri may be beneficial in the prevention and treatment of peptic ulcer disease.

The antiulcerogenic effects of ethanol extract of the bark from Vocanga atricana were studied by Jannet et al. (2000) using albino rats. The effects of the extract on the volume of gastric juice, gastric pH, acid output, mucus production and peptic activity were recorded. Oral administration of the extract (500 - 750 mg.kg) inhibited the formation of gastric lesions induced by hydrochloric - HCI or ethanol (40-63%

Biological activities of Limonia crenulata (Roxb.) 79

inhibition) was also studied. The extract significantly reduced gastric lesion formation in pylorus ligated rats.

The 10% ethanol extract of the aerial of Calligonum comosam at different concentrations (100, 200 and 400 mg/kg) produced a significant and dose dependent inhibition to the acute gastric ulcers induced by phenylbutazome indomethacin 0.2 N sodium hydroxide (NaOH) and 80% ethanol (Liu et al.,2002).

The effect of alcoholic extract of Nigella sativa in rats was investigated by

Rajkapoor et al., (2002) to evaluate the antiulcer activity by using two models, i.e, pyloric ligation and aspirin induced gastric ulcer. The results indicated that the alcoholic extract significantly (P<0.001) decreased the volume of gastric acid secretion, free acidity, total acidity and ulcer index with respect to control.

Eclipta alba caused a significant reduction in ulcer index in all animal models and a significant inhibition was also observed in aspirin - induced gastric ulceration and secretion in pylorous ligated rats (Venkatesan et al., 2002).

The ulcer protective potential of methanolic extract of Emblica officinalis was assessed in different acute gastric ulcer models in rats induced by aspirin, ethanol, cold restraint stress and Pylorus ligation healing effect in chronic gastric ulcers induced by acetic acid in rats. Emblica officinalis extract, 10-50 mg/kg administered orally, twice daily for 5 days showed dose-dependent ulcer protective effects in all the above acute

Biological activities of Limonia crenulata (Roxb.) 80

models and significant ulcer healing effect in dose of 20mg/kg after 5 and 10 days treatment. The gastric mucosal factors showed that it significantly decreased the offensive factors like acid and pepsin and increased the defensive factors like mucin secretion, cellular mucus and life span of mucosal cells. Emblica officinalis extract showed significant antioxidant effects in stressed animals and did not have any effect on cell proliferation in terms of DNA micro g/mg protein or glandular weight (Sairam et al.,

2002).

Aqueous extract of the leaves of the Limonia acidissima L., was administered orally for seven to fifteen days of the carbon tetra chloride CC14 administered rats. The results showed the extracts have antiulcer activity when compare to normal group and standard drug Liv52 administered rats (Karmat et al., 2003).

Oral administration of butanol fraction of Gynostoma pentaphyllum (GPB) at 200 and 400 mg/kg weight significantly inhibited gastric ulcer induced by indomethacin,

HCl/EtOH and water immersion restraint stress in rats. In ethanol induced ulcerated rats, gastric wall mucus and hexosamine content were markedly preserved by GPB pretreatment. The findings indicated that the butanol fraction of G. pentaphyllum possesses gastric protective potential related to the preservation of gastric mucus synthesis and secretion (Rujjanawate et al., 2004).

Ocimum sanctum (OS) is known to possess various therapeutic properties.

Dharmani et al., (2004) evaluated its antiulcerogenic activity in aspirin, alcohol and pyloric ligation (PL) induced gastric ulcer models in Sprague Dailey rats, histamine

Biological activities of Limonia crenulata (Roxb.) 81

induced duodenal ulcer in guinea pigs and ulcer healing activity in acetic acid induce chronic ulcer model. The result indicated that O. sanctum not only decreased the incidence of ulcers but also enhanced the healing of ulcers.

The decoctions were collected from the aerial parts of Malva niglecta (Kallr.)

(Malvaceae), leaves of Potentilla reptans L. (Rosaceae) fruits of Runex patientia L.

(Polygonaceae), aerial parts of Siderites caesarea Daman (Lamiacceae) and flowers of

Verbasaem cheicanthi Boits var. (Scrophulariaceae) showed significant gastric protection against the ethanol induced gastric ulcer model in rats. Furthermore, healing effects were also confirmed through histopathological examination (Gurbuz et al., 2005).

Peptic Ulcer Disease (PUD) is a serious gastrointestinal disorder that requires a well targeted therapeutic strategy. A number of drugs including proton pump inhibitors and H2 receptor antagonists are available for the treatment of peptic ulcer, but clinical evaluation of these drugs has shown incidence of relapses, side effects, and drug interactions. This has been the rationale for the development of new antiulcer drugs and the search for novel molecules has been extended to herbal drugs that offer better protection and decreased relapse. Drugs of plant origin are gaining popularity and are being investigated for a number of disorders, including peptic ulcer. The antiulcerogenic and ulcer healing property of Ocimum sanctum, Allophylus serratus, Desmodium gagenticum, Azadirachta indica, Hemidesmus racemosus, Asparagus racemosus and

Musa sapientum were evaluated . The above plants reported that they have ulcerhealing activity. Ayurvedic knowledge supported by modern science is necessary to isolate,

Biological activities of Limonia crenulata (Roxb.) 82

characterise, and standardise the active constituents from herbal sources for antiulcer activity (Dharmani and Palit, 2006).

The bark of Anogeisus latifolia () has been reported to be used in the treatment of various disorders including stomach and slain disease. Govindarajan et al. (2006) studied the anti - ulcer potential and antimicrobial activity of the 50% aqueous alcoholic extract in order to validate ethnobotanical claims. A. latifolia extracts possessed gastro protective activity as evidenced by its significant inhibition in the formation of ulcers induced by physical and chemical agents.

Andrade et al. (2007) evaluated the antiulcerogenic property of the hydroalcoholic extract of aerial parts of Maytenus robusta. The effects of the extract on gastric content volume, pH and total acidity in the ethanol induced ulcer model showed that the extract significantly reduced the lesion index such as 75.1 ± 8.9 ± 7.4 and 75.5 ± 5.37 when treated with 50, 250 and 500 mg/kg of M. robusta respectively. They also observed significant inhibition in lesion index in the indomethacin induced ulcer model. Regarding the model of gastric secretion, a reduction in gastric juice and total acidity were observed, as well as an increase in gastric pH.

The Plectranthus barbatus (Lamiaceae) aqueous extract and isolated compounds were assayed in vivo in pylorus ligated mice, and in vitro on acid secretion measured as

(14 C-aminopyrine (14C-AP) accumulation in rabbit gastric glands and gastric H+, K+, adenosine triphosphate - ATPase preparations injected into the duodenal lumen, the

Biological activities of Limonia crenulata (Roxb.) 83

aqueous extract of the plant leaves (0.5 and 1.0g / mg) decreased the volume (62 and 76 percent) and total acidity (23 and 50 percent) of gastric acid secretion in pylorus ligated mice showing antiulcer activity (Schultz et al., 2007).

Ashok Kumar et al., (2010) reported the antiulcer activity of aqueous extract of

Physalis minima was investigated in experimental animal (rats). Gastric ulcers were induced by oral administration of 1ml ethanol 80%, the animals were divided in to six groups; in each group contain six animals. Administered aqueous extract of Physalis minima in two dose 100 mg & 200 mg/ kg body weight by oral route before one hour to administration of ethanol. The extract at doses of 100 and 200 mg/kg significantly (P<

0.05) showed an antiulcer effect characterized by reduction of acid volume (AV), free acidity (FA), total acidity (TA), total protein (TP), ulcer index (UI), Lipidperoxidation

(LPO), and increasing rate of pH, Glutathione (GLU), and Catalase (CAT) when compared to the control group. The results suggest that the extract acts to produce significant ulcer protective property.

Bhalke et al., (2010) investigated the antiulcer effect of ethanolic extract of leaves of S. grandiflora using different models of gastric ulceration in rats. Acute gastric ulceration in rats was produced by oral administration of various noxious chemicals including aspirin or ethanol or indomethacin. Gastric total acid output was estimated in the pylorus ligated rats. Gastric tissue was also examined histologically. The ethanolic extract of leaves of S. grandiflora was administered in the dose of 400 mg/kg orally in all experiments. Omeprazole, ranitidine, misoprostol were used as a reference drug. The

Biological activities of Limonia crenulata (Roxb.) 84

ethanolic extract of leaves of S. grandiflora at the dose of 400 mg/kg produced a significant reduction in the ulcer index.

3.16. Antidiarrhoeal activity

The effectiveness of Aegle marmelos fruit in diarrhoea and dysentery has resulted in its entry into the British Pharmacopoeia. This plant was also called as a Rasayana

(Pandeya et al., 1983). The pathogenesis of infectious diarrhoea has been widely studied. Enteric pathogens have evolved a remarkable array of virulence traits that enable them to colonize the intestinal tract. These organisms colonize and disrupt intestinal function to cause mal-absorption or diarrhoea by mechanisms that involve microbial adherence and localized effacement of the epithelium, production of toxin and direct epithelial cell invasion (Guerrant et al., 1999). The traditional use of A. marmelos unripe fruit has an antidiarrhoeal which was also reported. A. marmelos is effective in chronic cases of diarrhoea due to the presence of large quantities of mucilage, which act as a demulcent. Additionally, A. marmelos has been shown to be effective in experimental models of irritable bowel syndrome and physiological diarrhoea (Shoba and Thomas,

2001; Jagtap et al., 2004; Dhuley, 2003).

Diarrhoea is one of the main causes of morbidity and mortality in children under age of 5 years. In view of this problem, the WHO has a Diarrhoea Disease Control

Program, which includes studies of traditional medical practices together with the evaluation of health education and prevention approaches. A review of the last 7 years

Biological activities of Limonia crenulata (Roxb.) 85

about the studies of extracts of plants used to combat diarrhoea in different countries has been done (Gutierrez et al., 2007).

The antidiarrhoeal effect of ethanolic extract of the dried fruit pulp of

Aegle marmelos (L.) was studied on various intestinal pathogens. It showed excellent activity against Shigella boydii, S. sonnei and S. flexneri whereas the activity was found to be moderate against S. dysenteriae. The minimum inhibitory concentration against the strains of Shigella was recorded between 250 to 500 mg/ml. Preliminary phytochemical tests of extract demonstrated the presence of common phytochemicals including phenols, tannins and flavonoids as major active constituents (Joshi et al., 2009).

Aegle marmelos (L.) Correa has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. However, despite its traditional usage as an antidiarrhoeal there is limited information regarding its mode of action in infectious forms of diarrhoea. The hot aqueous extract (decoction) from dried unripe fruit pulps of

A. marmelos and effect on various aspects of pathogenicity of infectious diarrhoea

(Brijesh et al., 2009).

Meite et al. (2009) studied the ethyl acetate extract of Morinda morindoides

(Baker) Milne-Redh (Rubiaceae) properties against experimental diarrhoea induced by castor oil in albino Wistar rats. The ethyl acetate extract of Morinda morindoides (250,

500, and 1000 mg/kg body weight) was administered orally to three groups of rats (five animals per group) in order to evaluate the activity of the extract against castor oil- induced diarrhoea model in rat. Two other groups received normal saline (5 mg/kg) and

Biological activities of Limonia crenulata (Roxb.) 86

loperamide (5 mg/kg) as positive control. The effect of the extract on intestinal transit and castor oil-induced intestinal fluid accumulation (enteropooling) was assessed. At oral doses of 250, 500, and 1000 mg/kg body weight, the plant extract showed pronounced and dose-dependent antidiarrhoeal activity. The protective role of the extract at 1000 mg/kg was comparable to that of the reference drug, loperamide (5 mg/kg). The extract

(1000 mg/kg) produced a decrease in intestinal transit comparable to atropine (5 mg/kg), and significantly (p < 0.01) inhibited castor oil-induced enteropooling. No mortality and visible signs of general weakness were observed in the rats following the extract administration of up to a dose of 6000 mg/kg. The results showed that the extract of M. morindoides has a significant anti-diarrhoeal activity which supports its use in traditional herbal medicine practice.

Senthilkumar et al. (2010) reported that the antidiarrhoeal activity and gastrointestinal motility reducing activity of alcoholic and aqueous extract of bark of

Limonia acidissima L., was evaluated at two dose levels. Both the extracts showed significant antidiarrhoeal activity and reduced the mean weight of faeces and reduced the gastrointestinal motility significantly.

Akter et al. (2010) was scientifically evaluated hydromethanol extract of

Curcuma alimatifolia leaves for its antidiarrhoeal and antioxidant properties.

Antidiarrhoeal property was studied using castor oil and MgSO4 induced diarrhoeal models and charcoal induced gastrointestinal motility test in swiss albino mice. In all of these experimental models the extract, at higher dose (500 mg/kg body weight), exhibited

Biological activities of Limonia crenulata (Roxb.) 87

significant (p <0.05) antidiarrhoeal property compared to the control. The extract was found to possess high amount of phenols and flavonoids, expressed as gallic acid and quercetin equivalents respectively, that indicate the usefulness of C. alismatifolia leaves in diarrhoeal disease and other disorders linked to free radical-mediated oxidative stress.

The bioassay guided fractionation of the n-hexane extract of the seeds of

Murraya koenigii Spreng (Rutaceae) resulted in the isolation of three bioactive carbazole alkaloids, kurryam (I), koenimbine (II) and koenine (III). Of the three compounds (I) and

(II) exhibited significant inhibitory activity against castor oil-induced diarrhoea and

Prostaglandin E2 -induced enteropooling in rats. The compounds also produced a significant reduction in gastrointestinal motility in the charcoal meal test in Wistar rats

(Mandal et al., 2010).

3.17. Volatile oil

The effects of the time of year and the time of day of harvesting on essential oil concentrations, yield, and percentage of the principle component in Lemon verbena were reported by Vogel et al. (1999). Maximum essential oil concentration was found in young leaves in October with 0.95 ml/100 g dry weight, decreasing to values of 0.09 to 0.20 ml/100 g dry weight from December to April. Essential oil yield increased from October to March, reaching a maximum of 27.3 l/ha. Citral content was highest in November (up to 64%), than decreased to reach a minimum in December (34%). From February values maintained stable (52–45%). In November largest leaf areas were found with 9.2

Biological activities of Limonia crenulata (Roxb.) 88

cm2/leaf. No differences in essential oil concentration between different times of day of harvesting could be found.

The amount and composition of the essential oil from leaves of Hypericum androsaemum (Linn.) cultivated in Arouca (Portugal) were determined in six samples harvested during 1 year at intervals of 2 months. The seasonally dependent essential oil content ranged from 0.7 mg/g biomass dry weight in September to 3.4 mg/g in February.

The oil contained more than 80 compounds, 70 of which (constituting 88–93% of the total oil) were identified by GC and GC-MS. An approximation of the absolute quantification of each compound and compound class was performed using a GC method with an internal standard. The relative and the absolute content of each compound and compound class changed during the year. At the end of the winter and in the spring, the essential oil was dominated by sesquiterpene hydrocarbons and accumulated a high number of intermediate to long chain n-alkanes and 1-alkenes. In September, the essential oil contained the lowest levels of sesquiterpene hydrocarbons (43%) and the highest levels of 1-octene and 2-hexenal (38%). In February, the essential oil had the highest level of sesquiterpene hydrocarbons (73%) and the highest diversity of intermediate to long chain n-alkanes and 1-alkenes (Guedes et al., 2004). The total amount of essential oil obtained was 22 percent (w/w), which is higher than any species of the genus Pistacia (Delazar et al., 2004).

3.18. GC –MS analysis of volatile oil

Biological activities of Limonia crenulata (Roxb.) 89

Essential oils of nine cultivars of ginger (Zingiber officinale) were evaluated by gas chromatography. The selection of these cultivars is based on the yield of essential oils and gingerol content in oleoresin. The levels of 13 identified and 6 unidentified compounds of ginger oil are presented. The role of beta-sesquiphellandrene, zingiberene and curcumene in ginger aroma is described by Gopalam and Ratnambal (1989).

The essential oil of Cedrus deodara obtained in a yield 2.1 percent hydro distilling its saw dust, GC-MS analysis of its oil revealed the presence of 23 compounds out of which 7-compounds are uncharacterized sesquiterpenoids (Nigam et al., 1990).

The essential oil, obtained from the shade-dried leaves of Buddleia asiatica by hydrodistillation in a yield of 0.3 percent, was analysed by chromatographic spectroscopic and chemical methods. Eighteen components including various monoterpenoids and sesquiterpenoids were characterized and measured by area normalization. The oil was found rich in beta-caryophyllene oxide (21.7 percent), citronellol (16.7 percent) remained uncharacterized. The oil has been reported to possess good in vitro antifungal, antibacterial and anthelmintic activity (Garg and Dengre, 1992).

The essential oil of Mammillaria bombycina was produced by hydro distillation from fresh leaves. The oil was analysed by GC - MS and 40 components were identified, which constituted 93.7 percent of the oil. The major components of the oil were decanal

(12.5 Percent), 11-dodecenal (8.1 percent) and dodecanal (26.5 percent) (Choudhury and

Leclercq, 1995).

Biological activities of Limonia crenulata (Roxb.) 90

The cultivation of Eucalyptus globules and E. citriodora at about 7000 feet height in the two hills has attained considerable economic importance, former as the source of 1,8-cineole rich essential oil and the latter for citronellal rich essential oil for perfumery and fragrance industry. The leaves are used for the production of essential oil about 80 percent of the country’s total production of about 500 tons of E. globules oil is from Palni and Nilgiri hills of Tamilnadu. E. citriodora was cultivated in a limited area in Nilgiris (Kakaraparthi and Sushil Kumar, 1999).

Qualitative and quantitative variations of the essential oil from leaves of Limonia acidissima L., collected from the different regions was observed by the use of GC-MS.

The oils contains rich amount of methyl chavicol (68.3%), Linalool, caryophyllene, cis- anethole, p-methoxy phenyl-2-propanone, elemicine, 3,4-dimethoxy benzaldehyde, 3- dimethoxy cinnamic aldehyde and p-methoxy cinnamic alcohol are first reported in the oily (Garg, 2003).

The essential oil of the aerial parts of Achillea albicaulis was analysed by

Capillary GC - MS. The major constituents were 1, 8 - cinede (10.1%), camphoir (9.2%), germacrene D (7.8%), piperitone (6.2%), alpha - pinene (5.9%) and artemisia ketone

(5.7%) (Feizaakhsh et al., 2003). Bello et al., (2003) studied the chemical composition of the leaf oil of Psidium salutare by GC-MS. Thirty - four compounds were identified with caryophyllence oxide (39.8%) and turmerone (17.3%) as the major ones.

Biological activities of Limonia crenulata (Roxb.) 91

Agarwal and Rangari (2003) phytochemically investigated and evaluated the anti- inflammatory and anti-arthritic activities of essential oil of Strobilanthus ixiocephala

(Benth.) Column chromatographic fractionation of essential oil obtained by hydrodistillation from the flowering tops of S. ixiocephala resulted in the isolation of b- caryophyllene, fenchyl acetate, T-cadinol and a new sesquiterpene alcohol for which a name ixiocephol has been proposed. The GC-MS analysis of the essential oil has also revealed the presence of various monoterpenoids and sesquiterpenoids. The b- caryophyllene and fenchyl acetate were identified by Co-TLC with authentic samples whereas T-cadinol and ixiocephol were structurally elucidated by UV, IR, 1H NMR, 13C

NMR and mass spectral data.

Asadipour et al. (2003) investigated the volatile oil obtained from the aerial parts of Cymbopogon oliveri grown in Kerman Province, Iran by GC and GC-MS. Fifteen components were detected, representing 93.5% of the total oil, piperitone (61.10%), beta

- caryophyllence (14.4%), delta - O - carene (6.5%) and beta - eudesomol (4.2%) were the major constituents of the oil.

The volatiles of the fruit of Limonia acidissima L. were analysed by GC and

GC - MS. Character Impact odorants of the fruit were systematically characterized by

Aroma Extract Dilution Analysis (AEDA) with GC –Olfactometry (GC-O). A total of 75 compounds were identified including 28 esters, 11 alcohols, one acetal, 10 ketones, four lactones, one heterocyclic, four aliphatic hydrocarbons, one of furan and five acids.

However, only 44 volatiles were identified by GC-O. Among these, compounds, with the

Biological activities of Limonia crenulata (Roxb.) 92

most impact were ethyl butyrate (Fruit, Sweet, Banana like) and methyl valeric acid, 1- octen-3-01, Pentyl isobutyrate, 2-ethylhexanoic acid, ethyl octanoate, gamma- decalactone, 2,3-pentanedione, 3-octanone, 5-methyl-3-heptanone, 9-methyl-5- imdecene and (E)-2-hexenyl butyrate seem to contribute to Wood apple fruit flavor

(Apriyantono and Kumara, 2004).

The volatile compounds, obtained by hydro distillation of the aerial parts of

Rosmarinus officinalis, were analysed by GC - MS. Thirty compounds were characterized representing 98.2% of the essential oil with 1, 8 - cineole (29.5%), 2 - ethyl

1-4, 5 - dimethylphenol (12.0%) and camphor (11.5%) as the major components (Touafek et al., 2004).

The volatile components of the aerial parts of Ruta graveolens and Haplophyllum suaveolens, as well as leaves of Zanthoxylum limoncello, Z. panamense and Z. setulosum have been studied by GC - MS analysis. The biggest amount of aliphatic fatty acids was found in Z. panamense (17.2%), followed by R. graveolens (16.5%), Z. limoncello

(14.7%), H. suaveolens (9.2%) and Z. setulosum (5.3%). Pentanoic, hexanoic, octanoic and nonanoic acids are common compounds in the volatiles of the five species (Ivanovaa et al., 2004).

The oleoresin of Pistacia atlantica var. mutica growing in different regions of

Iran, is a popular, naturally occurring chewing gum and has been used traditionally in the treatment of peptic ulcer. The GC - MS analysis of the essential oil, obtained from steam

Biological activities of Limonia crenulata (Roxb.) 93

distillation of the oleoresin terpenoids, showed alpha pinene was the major constituent

(70%) followed by limonene oxide (9%) and myrienol (5.31%) (Delazar et al., 2004).

Cestrum diurnum is a single or multistemed shrub that is also known as Day

Jasmine. The essential oil of the mature leaves of C. diurnum was analysed by Gas Liquid

Chromatography (GLC) and Gas Liquid Chromatography – Mass Spectrometry (GLC-

MS) and altogether 14 components were detected. The main constituents were palmitic acid, stearic acid and oleic acid (Bhattacharjee et al., 2005).

Nurettin et al. (2006) reported that the essential oil of air-dried Minuartia meyeri

Bornm. (Caryophyllaceae) was analysed by GC-MS and fifty-two components were identified in the oil. The main components in the essential oil of M. meyeri were nonacosane (6.2%), 6,10,14-trimethyl-2-pentadecanone (5.1%), nonanal (4.6%), and α caryophyllene (2.9%).

Rai et al. (2006) studied the highly fragrant essential oil of the flowers of Carissa opaca (Apocynaceae). A total of 20 compounds accounting for 99.5 percent of the oil, were identified. The main component was palmitic acid (82.5 percent). Other major compounds were benzyl salicylate (6.0 percent), benzyl benzoate (4.6 percent) and (E,E)- alpha-farnesene (3.5 percent). Flowers on system distillation gave an essential oil (yield-

0.02%) rich (-90%) in ionones in which β-ionone predominated (Sukhdev, 2006).

Jasim Unddin and Nemal (2007) was identified the essential oil of the fresh leaves of Lantana camera growing in Dehra Dun was by GC and GC - MS. The major

Biological activities of Limonia crenulata (Roxb.) 94

constituents identified in the oil included β-caryophyllinee (23.3%), hemulene (11.5%) germacrene D (10.9%) davanone (7.3%) and γ-curcmene (6.3%).

Comparative analysis of essential oils from the leaves of Aegle marmelos and

Limonia acidissima L. was carried out with the help of GC-MS. Aegle marmelos oil contains 16 compounds including alpha phellandrene (35.7%), dilimonene (29%), subinene (16.7%), and alpha-pinene (6.9%). The Limonia acidissima L., has been contain 26 compounds including methyl chavicol (74.6%) and anethole (20%)

(Chowdhury and Yusuf, 2007).

3.19. Acute toxicity study

LD50 of > 1250 mg/kg body weight, no change in the behaviour and physiological activity was recorded (at this dose) in the acute oral toxicity test in mice with the ethanolic extract of A. marmelos (L.) dried fruit pulp (Joshi et al., 2009).

Mondal et al. (2009) investigated the acute and sub-acute toxicity of the methanolic extract of Cleome rutidosperma (family: Capparidaceae) root, Neolamarckia cadamba (family: Rubiaceae) and Spondias pinnata (family: Anacardiaceae) bark. In the acute toxicity study, the extracts were administered orally at dose ranging from 100-3000 mg/kg p.o. and the animals were observed continuously for the first 4 hours for any behavioral changes and they were then kept under observation up to 14 days after drug administration to find out the mortality if any. However, there was no mortality in any of the above plants extract at 3000 mg/kg dose. In sub-acute toxicity study was tested at the

Biological activities of Limonia crenulata (Roxb.) 95

dose of 600 mg/kg p.o. once daily for 14 days. The results concluded that the methanol extract of C. rutidosperma root, N. cadamba and S. pinnata bark at doses of 600 mg/kg, p.o. is nontoxic since no marked changes in haematological and biochemical parameters were observed.

Baghel et al. (2011) evaluated the acute toxicity study of Coccinia indica roots.

Rats were orally administrated single dose of 100, 500 and 1000mg/kg of aqueous extract of Coccinia indica. Mortality, signs of toxicity, body weight, food consumption and gross findings were observed for 07 days post treatment of Coccinia indica extract. In addition, no significant differences were noticed in the body and organ weights between the control and treated groups. These results state that aqueous extract of Coccinia indica is toxicologically safe by oral administration.

Sikarwar Mukesh et al. (2009) analysed the pharmacognostic, phytochemical parameters and acute toxicity of Crateva nurvala stem bark (family: Capparidaceae). The acute toxicity study of plant extract was also carried out in female albino rats

(50mg to 5000 mg/kg body weight) as per OECD guidelines. In the acute toxicity study, oral administration of 5g/kg of Crateva nurvala stem bark extracts produced neither mortality nor changes in behaviour or any other physiological activities.

3.20. Herbal capsules

Biological activities of Limonia crenulata (Roxb.) 96

The pharmacognostical study of leaf of Desmodium triflorum was conducted with a view to help in correct botanical identification of the drug, including leaf in powdered form. Diagnostic features of leaf are described. NSL, New Delhi (Deokule, 1992).

Herbal drug technology is used for converting botanical materials into medicines, where standardization and quality control with proper integration of modern scientific techniques and traditional knowledge is important. Herbal medicines are gaining more and more attention all over the world, due to their long historical clinical practice and less side effects. This paper reviewed the traditional methods in the quality control of herbal medicines, including, the traditional chromatographic methods and comprehensive methods, such as fingerprint and multi-component quantification are emphasized; hyphenated techniques, like HPLC, GC-MS. In a few word, the analysis and quality control of herbal medicines are moving towards an integrative and comprehensive direction, in order to better address the inherent holistic nature of herbal medicines (Patra et al., 2010).

Wangthong et al. (2010) studied that the stem bark powder of Hesperethusa crenulata or Thanaka has been used on the face by Myanmar women for more than a thousand years as a skin care regiment.

3.21. Herbal syrup

Contributions of plants are numerous in every sector of human life. They help to grow human body and also protect human beings from sickness due to their extensive use as medicine. A large number of plants are used as medicinal agents (Ghani, 1990). In

Biological activities of Limonia crenulata (Roxb.) 97

Bangladesh, about two hundred fifty plant species are used as medicinal agents. It has now been established that plants which naturally synthesize and accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils, minerals and vitamins possess medicinal properties (Ghani, 1998). Most important concept underlying antimicrobial therapy is selective toxicity, i.e., Selective inhibition of the growth of microorganism without damage to the host (Warren and Ernest, 1998). Natural products are known to play an important role in both drug discovery and chemical biology (Holetz et al., 2002).

Biological activities of Limonia crenulata (Roxb.) 98

4. MATERIALS AND METHODS

4.1. Collection of plant materials

Scientific classification of Limonia crenulata (Roxb.)

Division : Angiosperms

Class : Dicotyledons

Sub Class : Polypetalae

Series : Disciflorae

Order :

Family : Rutaceae

Genus : Limonia

Species : crenulata (Roxb.)

The selected medicinal plant was Limonia crenulata (Roxb.) was collected from

Keezanatham, Ariyalur (Dt.), Tamilnadu, India (Fig.1; Plate - 1). Collected plant leaves were carefully examined and identified with the help of regional Floras Gamble, 1967;

Matthew, 1983; Nair and Henry, 1983. The botanical identity was authenticated by

Dr. M.Jegadeesan, Professor and Head, Dept. of Environmental and Herbal Sciences,

Tamil University, Thanjavur. Specimen was further confirmed with reference to

Herbarium sheets available in the Rabinat Herbarium, St. Joseph’s College,

Thiruchirappalli, Tamilnadu, India. A voucher specimen has been placed in the

Department Herbarium for future reference.

Biological activities of Limonia crenulata (Roxb.) 99

Biological activities of Limonia crenulata (Roxb.) 100

Biological activities of Limonia crenulata (Roxb.) 101

Anatomy, preparation of extracts, phytochemical analysis, physico – chemical analysis, elemental analysis (AAS), microbial analysis, HPTLC were studied in

SASTRA University, Thanjavur.

GC – MS analysis of leaf extracts, GC – MS analysis of volatile oil and HPLC were evaluated in Indian Institute of Crop Processing Technology, Ministry of Food

Processing Industries, Government of India, Thanjavur.

Antimicrobial and antioxidant activities were investigated in Sri Gowri Biotech

Research Academy, Thanjavur.

The following research works were examined in Periyar College of

Pharmaceutical Sciences for Girls, Trichy – 21. Pharmacological experiments involving animals were get approved and executed. Anti-inflammatory, antiulcer, antidiarrhoeal activities and acute toxicity studies were analysed. In department of pharmacognosy extraction of volatile oil was prepared. Capsule and syrup preparation were investigated in the Pharmaceutical Department of the same premises.

4.1.1. Anatomical analysis (Krishnamoorthy, 1988)

Free hand section of fresh leaves, stem and root of the study plant parts were taken. The parts were stained with safranin and destaining was done. Sections were mounted on slides using glycerin and covered with cover glass and margins were sealed with DPX and observed under Nikon phase contrast microscope (Nikon, Japan).

Biological activities of Limonia crenulata (Roxb.) 102

4.1.2. Preparation of powder (Horborne, 1973)

The necessary plant leaves were collected and dried under shade. These dried leaves were mechanically powdered, sieved using 80 meshes and stored in an airtight container. These powdered materials were used for further physico – chemical and phytochemical analysis.

Chemicals

Analytical laboratory grade chemicals were used for the studies, which were purchased from the following manufacturers.

v S.D.Fine –Chem Ltd, Biosar.

v Fischer inorganics &Aromatics, Madras.

v Loba – Chemie Pvt. Ltd., Mumbai.

v Qualigenes fine chemicals, Mumbai.

v Sigma chemical company, U.S.A.

v Ecoline diagnostic Kit, Mumbai.

4.1.3. Preparation of extracts (Anonymous, 1966)

Various extracts were prepared according to the methodology of Indian

Pharmacopoeia (Anonymous, 1966) were subjected to our entire studies. The shade dried plants leaves were allowed to pulverization to get coarse powder. The coarse powder material was subjected to Soxhlet extraction separately and successively with acetone, chloroform, benzene and methanol extracts. The aqueous extraction achieved through the percolation method. These extracts were concentrated to dryness in flash evaporator

Biological activities of Limonia crenulata (Roxb.) 103

under reduced pressure and controlled temperature (40-50ºC). The all extracts put in air tight containers stored in a refrigerator.

Pharmacognostical study

Any scientific evaluation procedure component was subjected to clear authentification and identification. In this research, scientific authentification was carried out based on macroscopical as well as microscopical character observation through pharmacognostical studies. The procedures recommended in Indian Pharmacopoeia

(Anonymous, 1996) were followed for the determination of the following tests.

4.2. Qualitative phytochemical tests (Horborne, 1973; Trease and Evans, 1983)

Hydro alcoholic extracts was subjected to qualitative photochemical tests.

4.2.1. Test for alkaloids

A small portion of the extracts were stirred with a few drops of dilute hydrochloric acid and filtered and the filtrate was used for following tests.

Dragendroff’s reagent

An orange precipitate obtained exhibited the presence of alkaloids.

Hager’s reagent

A yellow precipitate formed it indicated the presence of alkaloids.

Biological activities of Limonia crenulata (Roxb.) 104

Wagner’s reagent

A brown precipitate obtained showed the presence of alkaloids.

4.2.2. Test for carbohydrates

A small quantity of extracts were dissolved separately in 5 ml of distilled water and filtered. The filtrate was subjected to Molisch’s test to detect the presence of carbohydrates.

Molisch’s test

Filtrate was treated with 2 – 3 drops of 1 % alcoholic naphthol solution and 2 ml of concentrated sulphuric acid was added along the sides of the test tube. The formation of purple colour showed the presence of carbohydrates.

4.2.3. Test for glycosides

Another portion of the extract was hydrolysed with hydrochloric acid for few hours in a water bath and the hydrolysate was subjected to Legal’s and Borntrager’s tests to detect the presence of different glycosides.

Legal’s test

To the hydrolysate, 1 ml pyridine and few drops of sodium nitroprusside solution were added and then it was made alkaline with sodium hydroxide solution. The formation of purple colour indicated the presence of glycosides.

Biological activities of Limonia crenulata (Roxb.) 105

Borntrager’s test

Hydrolysate was treated with chloroform and the chloroform layer was separated, to this equal quantity of dilute ammonia solution was added. The formation of yellow colour showed the presence of glycosides.

4.2.4. Test for sterols

Liberman Burchard test: 1 g of the extract was dissolved in few drops of dry acetic acid. 3 ml of acetic anhydride was added followed by few drops of concentrated sulphuric acid. The formation of green colour provided the presence of sterols.

4.2.5. Test for fixed oils and fats

All quantity of the various extracts was separately pressed between two filter papers. An oily stain obtained showed the presence of fixed oils.

Few drops of 0.5 N alcoholic potassium hydroxide was added to a small quantity of various extracts along with a drop of phenolphthalein. The mixture was heated on a water bath for 1 – 2 hours. The formation of yellow colouration indicated the presence of fixed oils.

4.2.6. Test for saponins

Biological activities of Limonia crenulata (Roxb.) 106

The extracts were diluted with 20 ml of distilled water and they were agitated in a graduated cylinder for 15 minutes. The absence of the formation of foam showed the non existence of saponins.

4.2.7. Test for proteins and amino acids

Small quantities of various extracts were dissolved in a few ml of water and treated with

Ninhydrin reagent

A purple colouration obtained showed the presence of amino acids.

Biuret reagent

A violet colouration obtained showed the presence of proteins.

Millon’s reagent

A yellow precipitate formed showed the presence of aromatic amino acids.

4.2.8. Test for flavonoids

The aqueous extracts were tested for the presence of flavonoids using aqueous sodium hydroxide solution. An increased in the intensity of yellow colour showed the presence of flavonoids.

4.3. Quantitative estimation of phytoconstituents

Biological activities of Limonia crenulata (Roxb.) 107

The quality refers to the intrinsic value of the drug i.e, the amount of medicinal principles present. The active constituents were carbohydrates, glycosides, tannins, flavonoids, phenolic compounds, alkaloids, proteins and vitamins. The biological activity of a plant was influenced by the presence of various phytoconstituents. Natural antioxidants such as Vitamin C and Vitamin E directly influence the activity. Certain phytoconstituents such as phenols, tannins, carbohydrates, proteins, Vitamin C and

Vitamin E were known to act synergistically. Hence, it had been quantified in the plants extract.

4.3.1. Estimation of total phenolics by Folin –Ciocalteau method (Bray et al., 1954)

Different concentration of samples were taken and made up to 3 ml with water.

0.5 ml Folin-Ciocalteau reagent was added to each tube and kept for 3 minutes, to each tube 2 ml of 20 % sodium carbonate solution was added and mixed thoroughly. The test tubes were kept in boiling for 1 min. and cooled down. The absorbance was measured at

650 nm against blank and standard graph was plotted.

4.3.2. Estimation of total tannins by Folin – Danis method (Anonymous 1980 -

AOAC)

Different concentration of samples were taken and made up to 3 ml with water.

0.5 ml Folin-Danis reagent was added to each tube and kept for 3 min, to each tube 2 ml of 20% sodium carbonate solution was added and mixed thoroughly. The test tubes were kept in boiling for 1 min and cooled down. The absorbance was measured at 650 nm against blank and standard graph was plotted.

Biological activities of Limonia crenulata (Roxb.) 108

4.3.3. Estimation of proteins by Folin –Ciocalteau method (Bray et al., 1954)

Extraction: 20 mg of the sample was homogenized in 5 ml of phosphate buffer at pH 7.0. The extract was centrifuged at 10000 rpm for 15 min. and the supernatant was collected. Total protein of the extract was precipitated by adding equal volumes of 5 % ice cold trichloro acetic acid (TCA) and centrifuged at 12000 rpm for 10 mins.

Estimation: 0.1 ml of the sample was made up to 1 ml with distilled water. 4.5 ml of Lowry’s reagent was added, shaked well and allowed to stand for 10 min. To this 0.5 ml of Folin – Ciocalteau reagent was added mixed well and kept at room temperature for

20 min. A standard solution of bovine serum albumin (BSA) at concentration of 20 – 100 mg of blank was treated in a similar manner and a brown colour developed was read at

620 nm.

4.3.4. Estimation of vitamin C (2,4-Dinitrophenylhydrazine - DNPH method;

Sarojini, and Nittala 1999)

Extraction: 50 mg of the powdered sample was mixed with 6 ml of 5 % TCA.

The contents were shaked well and centrifuged to get a protein free extract of the vitamin.

Estimation: Take 2 ml of the protein free vitamin extract, 1 drop of the indophenol reagent was added then 0.5 ml DNPH Thiourea mix reagent was added and the mixer was incubated at 60°C for 1 hour. The tubes were then cooled in an ice water bath and 2.5 ml of 85 % sulphuric acid was added to each tube. Red colour was

Biological activities of Limonia crenulata (Roxb.) 109

developed and measured at 540 nm. After 30 minutes a standard graph was prepared by taking standard ascorbic acid at a concentration of 100 mg/ml.

4.3.5. Estimation of vitamin E (Dipyridyl method; Jayasree et al., 1985).

Extraction: 100 mg of the sample was taken and homogenized in 10 ml of acetone and extracted. The acetone was concentrated and mixed well with 10 ml of petroleum ether and 10 ml of water in a separating funnel. The upper organic layer was separated and 10 ml of ethanol and 5 ml of 1N potassium hydroxide was added and saponified. 10 ml of petroleum ether and water was added to the saponified solution, shaked well in a separating funnel. The upper organic layer was collected and concentrated. The concentrated was dissolved in 1 ml of ethanol and used for estimation.

Estimation: 0.2 ml of the extract was made up to 1 ml with ethanol. 1 ml of α,α dipyridyl reagent was added followed by 0.5 ml of ferric chloride solution. The colour was read exactly after 15 min. at 520 nm. Simultaneously a standard graph was prepared and the amount of Tocopherol was calculated.

4.3.6. Estimation of total carbohydrates (Phenol –Sulphuric acid method; Dubois, et al., 1956)

Extraction: 50 mg of sample was ground well with 2-3 ml of 5 % TCA. To the de-proteinized supernatant 10ml of 45 % ethanol was added to precipitate the polysaccharides. After setting it to stand overnight in cold, the tube was centrifuged for

Biological activities of Limonia crenulata (Roxb.) 110

10 minutes at 4000 rpm. The dried precipitate was analysed for total carbohydrate by dissolved in 2ml of 1N NaOH.

Estimation: 0.1ml of the sample was made up to 1ml with water. 1ml of 5% phenol and 5ml of concentrated sulphuric acid was added. The mixture was mixed thoroughly with a glass rod. The solution was allowed to stand for 10 minutes at room temperature and its optical density was read at 490 nm in a spectrophotometer and standard graph was prepared by using different concentration of D-Glucose ranging from 10 to 100µg/ml.

4.3.7. Estimation of total alkaloid

50 gm. of the sample dissolved in 50 ml of 90 % v/v alcohol by refluxing on a water bath for half an hour. Filtered and the filter was washed thoroughly to take minimum quantity of soluble matter (refluxtion was repeated if dissolution was not complete). The alcoholic extract was concentrated to a thick paste. 50 ml of petroleum ether 60oC- 80oC was added and refluxed for half an hour. The petroleum ether soluble portion was discarded by filtering through an extraction thimble. The thimble was transferred to a soxhlet extractor and refluxed with chloroform for half an hour and soluble portion was discarded. The same treatment was done with carbon tetrachloride and ethyl acetate and respective soluble portion were discarded. The residue was dissolved in 10 ml methanol and poured drop wise into 50 ml of acetone with constant stirring. The precipitate was collected and dried at 105oC to constant weight and weighed.

Biological activities of Limonia crenulata (Roxb.) 111

Method of estimation of total alkaloids

1. Weighed accurately about 10 gm of the extract in a 150 ml conical flask.

Added 100 ml mixture of 4 volume of solvent ether and 1 volume of alcohol and added

5 ml of diluted ammonia solution and shaked frequently during one hour.

2. Decant and filtered the clear solution through cotton into a separator and washed the residue with further 100 ml of ether alcohol mixture in 5 lots of 20 ml each

(Added gum Tragacanth powder to stimulate the stratification).

3. To get the total ether alcohol solution was added to 30 ml of 1 N Sulphuric acid to added make it acidic to litmus. Shacked well and allowed separating. Lower layer was left and continue the extraction first with 25 ml and then with successive quantities each of volume of alcohol until complete extraction of the alkaloids was effected.

4. First washed with 10 ml of acid solution and then with two successive quantities each 5 ml of chloroform washed with each chloroform extract with the 20 ml. of acid alcohol mixture contained in another separator.

5. The chloroform transferred the acid liquid from second separator to the first separator make alkaline with dilute ammonia solution and added 5 ml. excess.

Biological activities of Limonia crenulata (Roxb.) 112

6. Shaked first with 25 ml. then with successive quantities each of 20 ml of chloroform until complete extraction of the alkaloids was effected wash each chloroform extract with 10 ml of water contained in the second separator and filter through cotton into a 150 ml. conical flask.

7. Allowed the chloroform to evaporate on a water bath, added 2 ml of alcohol and continue the evaporation on the water bath, repeat the process using further 2 ml of alcohol and dry the residue on the water bath for 5 minutes.

The residue was dried at 105oC to constant weight and calculated the content of total ‘Alkaloids’.

4.3.8. Estimation of tannins

4 gms of extract and 50 ml of water heated on a water bath for 30 minutes with frequent shaking. The liquid was allowed to settle and supernatant solution was filtered into 250 ml standard flask. Hot water extraction was continued till filtrate gives negative reaction for tannins (Test with Ammo. Ferric Alum). The combined filtrate cooled and made up to 250 ml with water.

Take 25 ml of the solution, added 750 ml of water and 25 ml of Indigo Sulphonic acid. The contents titrated against N/10 KMnO4 to a golden yellow end point. A blank was carried out simultaneously titrating 25 ml of Indigo sulphonic acid.

Each ml of N/10 KMnO4 = 0.004157 gm of Tannins.

Biological activities of Limonia crenulata (Roxb.) 113

Preparation of Indigo sulphonic acid

1 gm Indigo Caramine dissolved in 25 ml of H2SO4. Further 25 ml of H2SO4 was added and diluted 1000 ml with water. The acid solution to be poured, cautiously into water.

4.3.9. Estimation of bitters

5 gms extract of the sample with boiling water containing 0.5 gm of calcium carbonate and extracted with boiling water till the last portion of the extract was not having of bitterness concentrate in vacuum and dissolved the residue thrice on the filter with 10 ml portions of hot alcohol was removed. The alcohol from the filtrate takes up the residue repeatedly with 25, 20, 15 and 10ml of hot water. The aqueous extract was shaked repeatedly with 25, 20, 15ml and added 10 ml of ethyl acetate. Collect the ethyl acetate which was evaporated, dried and weighed.

4.3.10. Estimation of flavonoids

10 gms of the sample was shaked with 60 ml. of methanol for 1 hour and allowed to stand for overnight. Filtered and residue washed twice with 20 ml portion of methanol.

Filtrate was washed and concentrated to 10 ml. The concentrated solution was added drop wise continuously shaked into 100ml of ether. After the addition the contents were shaked vigorously for 10 minutes and allowed to stand for 10 minutes to settle. Filtered and filtrate was evaporated to dryness and dried to constant weight and weighed.

4.3.11. Estimation of anthracene containing glycosides

Biological activities of Limonia crenulata (Roxb.) 114

Weighed about 2.5 gm and added 15 ml of hot distilled water. Kept the flask

now on a water bath till extract was dissolved.

Added 25 ml of 80 % v/v alcohol, shaked well and added 50 ml of 95 % v/v

alcohol, allow settling.

Filtered on a filter paper and washed with 80 % v/v alcohol until washings

were colourless. Transfer filtrate and washed to dish and evaporated the

alcohol to syrup mass. Transferred to a 50 ml stoppered cylinder, washed

with distilled water up to 30 cc.

Added 3 cc of 10 % v/v sulphuric acid, slowly with constant, shaking.

Allowed to stand overnight at room temperatures.

Decanted supernatant liquid into a filter paper; washed the pot 2 or 3 times

with cold water was passed to wash each time through filter paper.

Dissolved the residue in the cylinder and filter it using filter paper. A little

dilute alcohol (45 %) was added 2 or 3 drops of ammonia (10 %) to neutralize

the acid.

Evaporated to dryness in a tared beaker and dried to a constant weight at

105oC. Increase in weight of beaker represents crude glycyrrhizin.

4.3.12. Estimation of saponins

5 gms of the sample dissolved in 50 ml of 90 % v/v alcohol by refluxing on a water bath for half an hour. Filtered and the filter was washed thoroughly to take maximum quantity of soluble matter. The alcoholic extract was concentrated to a thick paste. 50 ml of petroleum ether at 40oC to 60oC was added and refluxed for half an hour.

Biological activities of Limonia crenulata (Roxb.) 115

The petroleum ether soluble portion was discarded by filtering through an extraction thimble. The thimble was transferred to a soxhelt extractor and refluxed with chloroform for half an hour and soluble portion was discarded. The same treatment was done with carbon tetrachloride and ethyl acetate and respective soluble portions were discarded.

The residue was dissolved in 10 ml of methanol and poured drop wise into 50 ml of acetone with constant stirring. The precipitate was collected, dried at 105ºC to constant weight and weighed.

4.4. Physico - chemical characters

4.4.1. Total ash

5gms of the shade dried plant materials were taken in a pre cleaned, pre- weighed silica crucible and maintained in a muffle furnace at 600°C for 6 hours. The crucibles are then, taken out and, cooled at room temperature and weighed. The percentage of ash was obtained with reference to the air-dried sample.

Difference % of total ash = X 100 Weight of sample taken

4.4.2. Water insoluble ash

The total ash was boiled with 25 ml of water and filtered through an ash less filter paper (Whatmann-41). It was followed by washing with hot water. The filter paper was ignited in the silica crucible, cooled and the water insoluble matter was weighed. The water soluble ash was calculated by subtracting the water insoluble matter from the total ash.

Biological activities of Limonia crenulata (Roxb.) 116

Difference % of water insoluble ash = X 100 Weight of the sample

4.4.3. Acid insoluble ash

The obtained total ash was boiled for five minutes with 25 ml of 2 M hydrochloric acid and filtered through an ash less filter paper. The filter paper was ignited in the silica crucible, cooled and acid insoluble ash was weighed.

Difference % of acid insoluble ash = X 100 Weight of ash taken initially

4.4.4. Determination of alcohol soluble extractive

About 2 gms of the powder was macerated with 100 ml of alcohol of the specified strength in a closed flask for 24 hrs. The extract was frequently shaked during first 6 hrs and allowed to stand for 18 hrs. It was filtered rapidly taking precautions against loss of alcohol and 10 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish. Dried at 105ºC and weighed. The percentage of alcohol soluble extractive was calculated with reference to the air dried powder.

Difference X 100 X100 % of alcohol soluble extractive = Weight of sample taken X 10

Biological activities of Limonia crenulata (Roxb.) 117

4.4.5. Determination of water soluble extractive

About 1 gm of the powder was macerated with 100 ml of distilled water in a closed flask for 24 hrs shaked frequently during 6 hrs and allowed to stand for 18 hrs. It was filtered rapidly and 10 ml of the filtrate was evaporated to dryness in a tared flat bottomed shallow dish, dried at 105ºC and weighed. The percentage of water soluble extractive was calculated with reference to the air dried powder.

Difference X 100 X 100 % of water soluble extractive = Weight of sample X10

4.4.6. Determination of crude fibre content by Dutch process

About 2 gms of drug was weighed accurately and transferred to a porcelain dish.

50 ml of 10 % nitric acid was added and boiled for 30 seconds with constant stirring and filtered through fine mesh cotton cloth. The residue was washed with 5 ml of boiled water. The material from the cloth was collected in a porcelain dish and boiled with 50 ml of 2.5 % caustic soda.

Then the liquid was filtered by using the fine mesh cotton cloth. The residue was washed with 100 ml of boiling water. Then the fiber was collected in a dried, weighed crucible. The crucible was then placed at 105oC for 2 hours. Then placed in a desiccator and cooled. The cooled crucibles were weighed.

Biological activities of Limonia crenulata (Roxb.) 118

4.5. Elemental analysis by Atomic Absorption Spectrophotometer (Sondhi and

Agarwal, 1995)

Principle

Atomic absorption is the process that occurs when a ground state atom absorbs energy in the form of light to a specific wavelength and was elevated to an excited state.

The amount of light energy absorbed at this wavelength increases as the number of atoms of the selected element in the light path increases. The relationship between the amount of light absorbed and the concentration of analysis present in known standards can be used to determine unknown sample concentration by measuring the amount of light, they absorb.

The absorption of light was proportional to the concentration of free atoms in the flame. It was given by Lambart-beer law.

Absorbance = log 10 I0/It = k.c.l

Where,

Io = intensity of incident radiation emitted by the light source.

It = intensity of transmitted radiation.

c = concentration of sample (free atoms).

k = constant

l = path length

Biological activities of Limonia crenulata (Roxb.) 119

Sample preparation

The plant leaves were cleaned and dried under shade, that the samples were dried in an oven at 40-50ºC till a constant weight is obtained. The dried samples were then, ground and powdered with pestle and mortar. Samples were labeled and stored in pre- cleaned polyethylene bottles for further analysis.

Reagents and apparatus

All the reagents such as HNO3 and H2O2 used in the study were purchased from

MERCK (Analytical Grade). De-ionized water was used for all analytical work and all the glassware’s, polyethylene bottles, pipette tips and others were washed with 1 % HCl, rinsed with de-ionized water before preparing standards, reagents and samples.

Sample preparation-Ash method

The collected medicinal plant parts were cleaned and dried under shade. The dried part was then, ground to fine powder which was used to ashing. Necessary precautions were taken at every step to avoid metallic contamination in any form. Pre-cleaned silica crucibles are kept in muffle furnace maintained at 600o C. Until the weight of the crucible reaches a constant level, the crucible was kept in the furnace. Powdered plant material (5gm) are taken in the silica crucible and maintained in a muffle furnace at 600o

C for 6 hrs. The crucible are then taken out and cooled at room temperature by keeping it in a desiccator and then, the ash values were measured. Then, the ash was dissolved in

100 ml of 5 % HCl. The dissolved ash solutions were filtered through Whatman filter paper No.40 and stored in tightly capped plastic bottles. The prepared solutions were

Biological activities of Limonia crenulata (Roxb.) 120

directly used for the determination of various elements by using flame photometry and

AAS.

Digestion of samples (Sample preparation - acid digestion)

A multiwave 3000 micro oven system (from Anton paar, USA) with 16 position teflon vessels with capping was being used here. The digestion vessels were provided with a controlled pressure, temperature and release valve. Before use, all Teflon vessels were soaked with 10 % HNO3. The system was initially programmed by giving gradual rise of 20 %, 40 %, and 50 % power for 5, 15 and 20 minutes, respectively for the due warming up. The powder samples were being used without any further treatment for sample preparation. 0.2 gms of sample was weighed into the Teflon vessels followed by digestion mixture of HNO3 and H2O2 in the ratio of 3:1. According to the nature of samples, the ratio was being applied.

The resulting solution after microwave digestion was filtered through Whatman #

40 filter paper (if necessary) and diluted to 50 ml with de-ionized water. A sample blank containing only acid mixture was prepared at the same time. The method of standard addition was generally adapted to calibrate the instrument before going for the observation of the samples.

Determination of metals

All the atomic measurements were carried out with PerkinElmer model

400/HGA900/AS800 coupled with Mercury Hydride System-15 (MHS-15) and Flame

Biological activities of Limonia crenulata (Roxb.) 121

Photometer. The lamps of Hallow Cathode Lamp (HCL) for Fe, Cu, Mn, Zn, Mg, Mo etc., analysis were used as a light source to provide specific wavelength of the elements to be determined and high purity (99.999 %) Acetylene and Nitrous oxide were used to provide constant thermal energy for atomization process and argon gas used for carrier gas purging purposes for Graphite furnace.

Calibration of instruments

More than three working standard solution of elements to be determined were prepared, covering the concentration range as recommended by the manufacturer of the instrument for the elements to be determined. Before the analysis of samples, the instruments were calibrated with prepared working standard solution. The calibration curve was obtained for concentration versus absorbance data statistically analysed.

Calibration of the instrument was repeated periodically during operations and blanks were carried with each set of 10 samples or any one of the prepared working standard for every 10 samples to check the instrument drift and to validate analytical procedures and performance. Reagent blank readings were taken and necessary correction made during the calculation of concentration of various elements.

Standard Certified Reference (SCR) of National Institute of Standard and

Technology (NIST) is used for day-to-day for the evaluation of methods of analysis or test and for long-term quality assurance of measurements. A reagent blank reading was taken and necessary corrections were made during the calculation of concentration of various elements (The Japan Society for Analytical Chemistry).

Biological activities of Limonia crenulata (Roxb.) 122

4.5.1. Fe, Cu, Mn, Zn, Mg, Mo, Al, Ni, Li and V metals analysis (Flame

AAS/Graphite furnace) (Sondhi and Agarwal, 1995)

After calibrating the instrument with prepared working standard, the digested liquid samples solution was subjected to analysis of Fe, Cu, Mn, Zn, Ni Mg, Mo, Al, Ni,

Li and V , by AAS flame/Graphite furnace with specific instrumental conditions were given by instruments manufacturer. Introduce the solution into flame, recorded the reading, using the mean of the three readings and quantified the concentration of the metals in the given samples against the standard calibration curve obtained from concentration versus absorbance of the prepared known concentration on the day of the analysis.

Volume make up X volume make X Dilution factor ppm in sample = Weight of the sample

4.5.2. Na, K, & Ca Metals analyses by flame photometer - Systronics 125 model

After calibrating the instrument with prepared working standard, the digested liquid sample’s solution was subjected to analysis of Na, K, and Ca by Flame Photometer with specific instrumental conditions as given by instruments manufacturer. Introduced the solution into flame, recorded the reading, using the mean of the three readings and quantify the concentration of the metals in the given samples against the standard calibration curve obtained from Concentration versus Absorbance of the prepared known concentration on the day of the analysis.

Biological activities of Limonia crenulata (Roxb.) 123

Volume make up X volume make X Dilution factor ppm in sample -ppm = Weight of the sample

4.6. Microbial analysis (James and Sherman, 2002)

Enumerations of microorganisms from plant material by standard plate count method.

4.6.1. Principle

Materials containing microorganisms was cultured. Each viable organism was developed into a colony. Hence the number of colonies were appeared on a plate represents the number of living organisms presented in the given sample.

4.6.2. Procedure

Plant material samples obtained and thoroughly mixed to make composite sample for microbial analysis. 10gms of appropriate plant material weighed and 100ml of sterile distilled water in a sterilized conical flask were prepared for serial dilution. The flasks were kept in a mechanical shaker for five minutes to obtain uniform suspension of microorganisms. The dilution was 1-10 or 10-1. From this 1 ml of dilution from 10-1 sample was taken and transferred into 9ml, this was 10-2 dilution. The procedure was repeated up to 10-6 dilution. Transferred 1ml of serial dilution from10-1 to 10-6 into sterilized petri plates for enumerating bacteria, fungi and severe pathogens such as

Salmonella sp., Shigella sp. and E. coli. Two replications were maintained for each dilution, for each group of microorganisms. The medium such as Nutrient Agar (NA),

Biological activities of Limonia crenulata (Roxb.) 124

Potato Dextrose Agar (PDA), Salmonella Shigella Agar (SSA) and Eosin Methylene Blue

Agar (EMB) were added to the sterilized petri-plate with one ml sample and rotating the plate clock wise and anti clock to get a uniform distribution of microbial cells.

The medium was allowed to set and the plates were incubated in inverted position at 370C for about 1-2 days for bacteria and 3-5 days for fungi. The colonies were counted on the plates with the aid of colony counter. The numbers of colonies were observed from the two plates which were kept for replication. The total numbers of population were enumerated individually for fungi and bacteria by taking the average of the two dilutions employed and expressed 1gm of plant material.

4.7. High Performance Thin Layer Chromatography (HPTLC) analysis of Limonia crenulata (Roxb.)

4.7.1. Chromatographic Conditions

Chromatography was performed on a 10*3 cm pre-coated HPTLC Silica gel 60F254 plate. The plates were washed by methanol and activated at 60ºC for 5 minutes prior to chromatography. Samples were applied on to the plate as 6 mm band using CAMAG

Linomat V applicator. The slit dimension was kept at 5 mm, 0.45 mm and 20 mm/s scanning speed was employed. The mobile phase was chosen after trial and error and 10 ml of mobile phase was used per chromatography. Linear ascending development was carried out in 10 cm *10 cm twin glass chamber saturated with the mobile phase. The mixtures were spotted using CAMAG Linomat V applicator.

Biological activities of Limonia crenulata (Roxb.) 125

Stationary phase : Silica gel GF 254

Mobile phase : Chloroform: Ethyl acetate: Formic Acid

(5:4:1)

Scanning wavelength : 254 nm

Applied volume : 5, 10 & 15 µl

Development mode : Ascending mode

The plates were developed in the chosen mobile phase and were photo documented at 254 nm and 366 nm. The chromatogram of developed plate was recorded at 254 nm.

4.8. Gas Chromatography - Mass Spectrometry (GC – MS) Analysis of Limonia crenulata (Roxb.) (Ivanova et al., 2002)

4.8.1. Plant sample extraction

20 gms powdered plant material was soaked in 50ml of absolute alcohol overnight and then filtered through whatman filter paper No.41 along with 2gm sodium sulphate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with sodium sulphate was wetted with absolute alcohol. The filtrate was concentrated by bubbling nitrogen gas into the solution and reduce the volume to

1ml.The extract contains both polar and non-polar phytocomponents of the plant material used.

Biological activities of Limonia crenulata (Roxb.) 126

The GC –MS analysis was carried out using a Clarus 500 Perkin – Elmer (Auto system XL) Gas chromatograph equipped and coupled to a mass detector Turbo mass gold – Perkin Elmer Turbo mass 5.1spectrometer with an Elite - I (100% Dimethyl poly siloxane), 30mx 0.25 mm ID x 1 µm df capillary column. The instrument was set to an initial temperature of 110ºC and maintained at this temperature 2 mins. At the end of this period the oven temperature was rose up to 280 ºC at the rate of an increase of 5ºC/min and maintained for 9 mins. Injection port temperature was ensured as 250 ºC and Helium flow rate as 1ml/min. The ionization voltage was 70eV. The samples (1µl) were injected in split mode as 10:1. Mass spectral scan range was set as 45 – 450 (m/z).

The time at which each component eluted from the GC column was termed as

Retention time (RT). The eluted component was detected in the Mass detector. The spectrum of the unknown components were compared with the spectrum of the known components stored in the NIST library and ascertains the name, molecular weight and structure of the components of the test materials in GC-MS study.

4.9. High Performance Liquid Chromatography (HPLC) of Limonia crenulata

(Roxb.) (Singh et al., 2002)

Biological activities of Limonia crenulata (Roxb.) 127

High performance liquid chromatography is a chromatographic technique that can separate a mixture of compounds, to identify, quantify and purify the individual components of the plant extracts.

4.9.1. Sample preparation for the analysis of flavonoids

The plant extracts were analysed through High Performance Liquid

Chromatography (HPLC) as per the method of Singh et al. (2002). One gram of leaf powder was macerated and suspended in 5 ml ethanol: water (80:20; v/v). The collected samples were subjected to ultrasonication (Branson Sonifier, Danbury, CT, USA) for 15 min at 4°C followed by centrifugation at 12,500 x g for 15 min. The clear supernatant was subjected to charcoal treatment for the removal of pigments. The residue was re- extracted twice with the same extracting solution and the supernatant was pooled prior to evaporation under vacuum (Buchi Rotavapor Re Type, Labco, India; Ambala Cantt.

India). Dried extracts were resuspended in 1.0 ml HPLC grade methanol by vortexing and filtered through ultra membrane filter (pore size 0.45 µm: Millipore) before HPLC analysis.

4.9.2. Analysis

Quantitative analysis of the sample was performed according to the method of

Singh et al. (2002). The HPLC system (Shimadzu Corporation, Kyoto, Japan) was equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable Shimadzu

SPD-10 AVP UV-VIS detector and a Rheodyne Model 7725 injector with a loop size of

20 µl. The peak area was calculated with a Winchrom integrator. Reverse-phase

Biological activities of Limonia crenulata (Roxb.) 128

chromatographic analysis was carried out in isocratic conditions using a C-18 reverse phase column (250 x 4.6 mm i.e., particle size 5 µm, Luna 5µ C-18 (2); phenomenex,

Torrance, CA, USA) at 25°C. Running conditions included: injection volume 20µl; mobile phase, methanol: 0.4% acetic acid (80: 20 v/v); flow rate 1 ml/min; and detection at 280 nm. Samples were filtered through an ultra membrane filter (pore size 0.45 µm; E-

Merck, Darmstadt, Germany) prior to injection in the sample loop.

Gallic acid, caffeic acid, rutin and quercetin were used as standards. Flavonoids present in sample were identified by comparing chromatographic peaks with the retention time (Rt) of individual standards and further confirmed by co-injection with isolated standards. The amount of each flavonoid expressed as micrograms per gram of fresh weight unless otherwise stated.

4.10. Fourier Transform - Infra Red (FT-IR) spectroscopy analysis of Limonia crenulata (Roxb.)

A small quantity of leaf powder was collected, grinded adequately and was pressed to tablet with KBr method. Infrared spectrum of sample product was recorded in the range of 400 – 4000 cm-1 using Perkin Elmer Rx1 infrared scanner.

4.11. Ultraviolet (UV) – Visible spectroscopy analysis of Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 129

The fractionated sample was dissolved in acetonitrile. Its optical behavior was examined by Ultraviolet-Visible spectrum instrument model Lambda 35 and found that the crystal was transparent in the region between 380–900 nm.

4.12. Antimicrobial activity of Limonia crenulata (Roxb.) (Perez et al., 1990)

Antimicrobial activity was screened by agar well diffusion method (Perez et al.,

1990). The leaf extracts were tested for antimicrobial activity against and Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Shigella shigae, Salmonella typhi and Klebsiella aerogenes) and fungi such as (Aspergillus niger and Candida albicans).

The microorganisms were collected from Microbial Germ plasm Culture

Collection Unit at Sri Gowri Biotech Research Academy, Thanjavur and maintained in the laboratory by periodic subculture.

Five leaves extracts (Ethanol, Methanol, Alcohol, Benzene and Aqueous) were prepared separately at different concentrations such as 100µg/ml 150µg/ml and 250µg/ml by using dimethyl sulphoxide as solvent (DMSO). Ciprofloxacin (2µg/ml) and fluconazole (10µg/ml) were used as positive control (standard) for bacteria and fungi.

DMSO was used as negative control.

4.12.1. Preparation of nutrient agar medium

Biological activities of Limonia crenulata (Roxb.) 130

Beef extract - 3 gms

Peptone - 5 gms

Sodium chloride - 5 gms

Agar - 15 gms

Distilled water - 1000 ml

pH - 7

All the ingredients were weighed and put into the conical flask containing 1000 ml distilled water. The flask was sterilized by using an autoclave at 121°C for 20 min at15 lbs pressure.

4.12.2. Preparation of potato dextrose agar medium

Potato - 200 gms

Dextrose - 20 gms

Agar - 15 gms

Distilled water - 1000 ml

pH - 5.6

The potato tubers were peeled and weighed for about 200 gms. The tubers were chopped into small pieces with the help of a sterile knife. The chopped potatoes were transferred into a conical flask containing about 1000 ml of distilled water. The contents were boiled for 20 minutes. The supernatant was decanted and filtered through muslin cloth and the filtrate was collected. To this filtrate dextrose and agar were added and shaked well to dissolve the ingredients and made up to 1000 ml by addition of distilled

Biological activities of Limonia crenulata (Roxb.) 131

water. Finally, the medium was autoclaved at121ºC for 20 mins at 15 lbs pressure.

Streptomycin sulphate (50µg/ml) was added and mixed well to prevent the bacterial contamination. The nutrient agar medium and potato dextrose agar medium were poured into the sterile petri plates and allowed to solidify. The test bacteria and fungal cultures were evenly spreaded over the media by sterile cotton swabs. Then wells (6 mm) were made in the medium using sterile cork borer. 100 µl of each concentration of extracts were transferred into the separate wells. The standard antibiotic and solvents were used as controls. Then the plates were incubated at 37ºC for 24 hrs and 27ºC for 48 – 72 hrs for bacteria and fungi respectively. After the incubation the plates were observed for formation of clear inhibition zone around the well indicated the presence of antimicrobial activity. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well.

4.13. Antioxidant activity of Limonia crenulata (Roxb.) (Koleva et al., 2002)

The scavenging activity of the plant extract was determined by DPPH (2, 2-

Diphenyl-1picrylhydrazyl) method. The plant extract solution (0.2 ml) was mixed with equal volume of ethanol 0.001 M DPPH methanol solution (0.025ml) was added to the solution and absorbance at 550 nm was measured after 30 min. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the equation.

Biological activities of Limonia crenulata (Roxb.) 132

AT

RSA % =1- X 100

AO

Where AT is the absorbance of the test solution at 30 min. time and AO is the absorbance at zero time (initial absorbance).

4.14. Anti-inflammatory activity of Limonia crenulata (Roxb.) (Winter et al., 1962;

Vinegar et al., 1969)

Carrageenan induced hind paw edema was determined according to the method of

Winter et al., 1962 and Vinegar et al., 1969. Albino rates of either sex weighed 150-200 gms were divided into groups of six animals each the dosage of the drugs administered to the different groups were as follows group 1-control, group 2 to 6 – plant extracts, group

7- indomethacin (10 mg/kg ) all the drugs were administered orally.

After one hour of the administration of the drugs, dose 0.1ml of 1% w/v

carrageenan solution in normal saline was injected into the sub plantar tissue of the left

hind paw of the rats and right hind paw served as the control. The paw volume of the

rats were measured in the digital plethysmography (Ugo Basile, Italy), at the end of 0

min,60 min and 120 min., 180min the % increase in paw edema of the treated group was

compared with that of the drugs under investigation were calculated based upon the

percentage inhibition of the inflammation.

Biological activities of Limonia crenulata (Roxb.) 133

Percentage inhibition =

Control (increase in paw volume in 3rd hour)-Test (Increase in paw volume in 3rd hour) X 100 Control (Increase in paw volume in 3rd hour)

4.15. Antiulcer activity of Limonia crenulata (Roxb.) (Shay et al., 1945)

Albino rats of either sex weighing about 100-130 g were divided into groups of 6 each. Pregnancy was excluded. The animals were deprived of food for 24 hours before the commencement of the experiment, but water was allowed adlibitum. The drugs were given orally 2 hours prior to pylorus ligation which was carried out according to the technique of Shay et al., (1945).

Test drug

Group 1 vehicle 5 ml/kg (5% w/v Acacia)

Group 2 Famotidine 20 mg/kg

Group 3 Test sample A 200 mg/kg

Group4 Test sample B 200 mg/kg

Group 5 Test sample C 200 mg/kg

Group 6 Test sample D 200 mg/kg

Group 7 Test sample E 200 mg/kg

5% w/v Acacia mucilage was used as a vehicle at a dose of 5 ml/kg. The solvent control received equal volume of Acacia mucilage. The animals were sacrificed 6 hours

Biological activities of Limonia crenulata (Roxb.) 134

after pylorus ligation for observation of gastric lesion as described by Gupta et al.,

(1985).

Denuded epithelium - 10

Petechial and frank hemorrhages - 20

One or two ulcers - 30

Multiple ulcers - 40

Perforated ulcer - 50

The gastric juice was collected, centrifuged and its pH and volume were measured. Free and total acidity were estimated titrimetrically with 0.01N NaOH using methyl orange phenolphthalein as indicators.

Pipette 1 ml of filtered gastric contents into a small beaker, added 2-3 drops of methyl orange and titrated with 0.01N NaOH until all trace of the red colour disappears and the colour was yellowish orange. Observed the volume of alkali added. Then added

2-3 drops of phenolphthalein was added and continue titration until a definite red tinge reappears. Again read the burette and obtain the total volume of alkali added. If a yellow colour was obtained on adding methyl orange no free acid was present. Added the phenolphthalein and titrate the combined acid. The data concerning the pH, volume, acid secretion of gastric juice and ulcer index were analysed by student‘t’ test.

Biological activities of Limonia crenulata (Roxb.) 135

4.15.1. Total acidity

A volume of 2 ml. diluted gastric juice was titrated with 0.01 N sodium hydroxide run from a micro burette using phenolphthalein as indicator and the acidity was expressed as mg. HCl/100g body weight of rat.

4.15.2. Free acidity

It was determined in similar manner using topfer’s reagent as indicator and sodium hydroxide was run until canary yellow colour was observed.

4.15.3. Ulcer index

The method of Anderson and Soman (1965) was used for scoring the ulcer index.

Control mean ulcer index - Test mean ulcer index % of ulcer index = X 100 Control mean ulcer index

4.16. Antidiarrhoeal activity of Limonia crenulata (Roxb.) (Mujumdar et al., 2001)

Effect of drugs on epsom salt induced diarrhoea was measured by Mujumdar et al., 2001.

Swiss albino mice of either sex weighed between 20-25 gms were used for this experiment. They were housed in polypropylene cages in an air-conditioned area at

25±2ºC with 10:14 hour’s light and dark cycle. Animals were fasted for 24 hours before

Biological activities of Limonia crenulata (Roxb.) 136

study with free access to water. For each treatment at least six animals were used. Drugs were administrated 45 minutes before cathartics (MgSo4 2g/kg p.o.) immediately after cathartic agent challenge animals were kept in polypropylene cages lined with filter paper at the bottom. Animals were observed for parameter such as time of occurrence of diarrhoea, number of total defections up to 4 hrs. In case of normal defecations, the numbers of defecations were noted up to 4 hrs after drug administration.

4.17. Extraction of volatile oil from Limonia crenulata (Roxb.)

The determination of volatile oil in leaf was made by distilling the drug with a mixture of water and glycerin. The distillate collected in a graduated tube of Clevenger’s apparatus in which the aqueous portion of the distillate was automatically separated and returned to the distilling flask. The volume of oil collected and measured. The content of the volatile oil expressed as percentage v/w.

4.18. GC – MS analysis of volatile oil from Limonia crenulata (Roxb.)

The GC –MS analysis was carried out using a Clarus 500 Perkin – Elmer (Auto system XL) Gas chromatograph equipped and coupled to a mass detector Turbo mass gold – Perkin Elmer Turbo mass 5.1 spectrometer with an Elite-5 MS column (5%

Diphenyl, 95% Dimethyl poly siloxane) 30x0.25mmx0.25µm df capillary column. The instrument was set to an initial temperature of 110ºC and maintained at this temperature 2 min. Next the temperature was maintained at 200ºC at the rate of an increase of

10ºC/min. At the end of this period the oven temperature was rose up to 280 ºC at the rate of an increase of 5 ºC/min and maintained for 9 mins. Injection port temperature was

Biological activities of Limonia crenulata (Roxb.) 137

ensured as 250ºC and Helium flow rate as 1ml/min. The ionization voltage was 70 eV.

The samples (2µl) were injected in split mode as 10:1. Mass spectral scan range was set as 45 – 450 (m/z).

The time at which each component eluted from the GC column was termed as

Retention Time (RT). The eluted component was detected in the mass detector. The spectrum of the unknown components were compared with the spectrum of the known components stored in the NIST library and ascertains the name, molecular weight and structure of the components of the test materials in GC-MS study.

4.19. Acute toxicity studies of plant extract of Limonia crenulata (Roxb.) (Miller and Trainter, 1944)

The determination of ED50 (the effective in producing excepted response in 50% of animal group) values help in ascertaining potency of a drug in terms of reference standard. The calculation of ED50 value was done when a drug shows graded response in relation to dose. But when the response if quanta or none, the ED50 values become LD50

(the dose lethal to 50% of the animal group). The ED50 and LD50 values were important for knowing the safety of a drug. The ratio between LD50 and ED50 represents therapeutic index which is an index of safety of the drug. Greater the therapeutic index, safer was the drug.

LD50 was calculated by finding the least tolerated (smaller) dose (100% mortally) and most tolerated (highest) dose (0% mortality) by hit and trial method. Once these 2 doses are determined, at least 5 doses were selected in between the least tolerated dose

Biological activities of Limonia crenulata (Roxb.) 138

and the mortality due to these doses was observed. Generally mice were used for the purposes and each dose group normally should consist of 10 animals. LD50 of a new drug can be determined by either oral or any one of the parental routes of administration.

Swiss albino mice were fasted overnight, weighted and divided into 12 groups, each group consisting of ten animals. Test sample was administered separately in various doses by oral route (1000-2500 mg/kg) of test compounds. After administration of the compounds, the animals were observed continuously for the first two hours for death due to acute toxicity.

The results of LD50 study were analysed using Miller and Trainter method (1944).

The correction factor was applied to 0 and 100% mortality group. The percentage mortality values were converted to probit values by reading corresponding probit units from the probit table. The probit were plotted against log doses. LD50 values were read as the dose that correspounds to probit 5.

4.20. Herbal capsule preparation of Limonia crenulata (Roxb.) (Banker and Rhodes,

2002)

Formulations of capsules were presented in Table 1.

Table 1. Formulation of capsules

S. No Ingredients For 1 Capsule (g) For 50 Capsules (g)

1. Herbal Powder 0.430 21.5

Biological activities of Limonia crenulata (Roxb.) 139

4.20.1. Method of preparation

i. Capsule equipment name : MAC Lab. Equipments ii. Address : MAC Scientific Works, P.B.No.2151, Delhi - 7 iii. Capacity of equipment : 100 capsules / operation iv. Capsule size : ‘0’sized (Range:’000’,’00’,’0’,’1’,’2’,’3’,’4’,’5’) v. Type : Hard Gelatine Capsules vi. Colour : Double coloured 4.20.2. Procedure

The powdered herbal drug was filled into the empty hard gelatine capsule. There were different types of hard gelatine capsules were available in the market. For our present study, we choose ‘0’ sized capsule because this type of capsule was very widely used in the pharmaceutical industry.

First ‘0’ sized empty hard gelatine capsules were taken the capsule filling machine. The body and the cap of the capsules were separated and the calculated quantity of the herbal drug was filled into the body of the capsules. Then the body and the cap of the capsules were joined and ejected from the capsule machine. The average weight of the capsule was checked for consistency. The filled and ejected capsules were polished using satin cloth for shiny appearance and finishing.

4.20.3. Evaluation of capsules

The weight variation test was carried out according to the Indian Pharmacopoeia specifications (Table 2).

1. Procedure followed : As per Indian Pharmacopoeia

2. Specifications

Biological activities of Limonia crenulata (Roxb.) 140

Table 2. Weight Variation Test

Weight of the capsules Deviation permitted as per the I.P. specifications < 300 mg ±10 % > 300 mg ±7.5 %

4.21. Herbal syrup preparation of Limonia crenulata (Roxb.) (Aulton, 2002)

Herbal syrup was prepared under the specifications of Indian Pharmacopoeia. The formulations of suspensions and use of various ingredients were listed in Table 3 & 4.

Table 3. Formulations of Suspensions

S.No. Ingredients For 150 ml

1. Herbal Powder 2.5 g

2. Sodium Carboxyl Methyl Cellulose 5 %

3. Glycerin 1 ml

4. Sodium benzoate 0.1 g

5. Sodium chloride 0.5 g

6. Volatile oil of Limonia crenulata Q.S.

7. Art Caramel 0.1 g

8. De-mineralized water Q.S.

Biological activities of Limonia crenulata (Roxb.) 141

Table 4.Use of various ingredients

S.No Ingredients Use Remarks

1 Sodium Carboxyl Suspending To distribute the herbal powder uniformly in Methyl Cellulose agent the aqueous medium and to increase the viscosity of the preparation which will reduce the sedimentation rate 2 Glycerin Wetting agent To wet the herbal powder to improve dispersion 3 Sodium Preservative To preserve the prepared suspension for benzoate improved longevity 4 Sodium Flocculating To convert the dispersed phase particles into chloride agent agglomerates so that re-dispersion will be easy 5 Volatile oil of Flavouring To mask the odour and to improve the Limonia crenulata agent organoleptic properties (Roxb.) Art Caramel Colouring To mask the colour and to improve the agent organoleptic properties

4.21.1. Preparation of suspension

Sodium carboxyl methyl cellulose was dispersed in 50 ml of hot water in a beaker, mixed continuously for two hours and left overnight for swelling. The other

Biological activities of Limonia crenulata (Roxb.) 142

ingredients like glycerine, sodium benzoate and sodium chloride were dissolved in 50 ml of water.

The sodium carboxyl methyl cellulose dispersion was taken into a stirrer setting the speed of 100 rpm. The other ingredients dissolved in water was added and stirred for

15 minutes. To this aqueous dispersion the powdered herbal drug was added slowly allowing the powder to disperse completely. It was now finally made upto the volume of

150 ml. After the powder was completely added, the suspension was left stirring for 6 hours.

The suspension was left overnight for setting to the room temperature and then flavouring agent was added and mixed thoroughly. The suspension was evaluated for viscosity.

4.21.2. Determination of viscosity

i. Instrument : Brookfield Viscometer

ii. Model : LV-II

iii. Spindle : S-52

iv. Speed : 30 rpm

v. Temperature : 28°C

Biological activities of Limonia crenulata (Roxb.) 143

5. RESULTS

5.1. Medicinal plant

Plant is properly identified with the help of anatomical characters. In Limonia crenulata (Roxb.) transverse section of root, stem and leaves were studied to identify its internal characters. These characteristics give the different results between the others species of the Rutaceae. Thus it concluded as a different species.

5.1.1. Morphological description of Limonia crenulata (Roxb.)

Limonia crenulata (Roxb.) is a medium deciduous aromatic shrub like tree, armed with strong. The leaflets are simple and entire. The leaflets are ovate and contain lot of dotted glands. The margins are entire and symmetrical base. The apex of the leaf is bland, petiole is short and foliar surface smooth and the midrib is more prominent on abaxial side. The leaves are dark green in colour and aromatic in smell and slight bitter in taste. The lamina is completely divided into two separate segments called leaflets. The leaflets 3-7 are arranged in pairs. The leaves are arranged in imparipinnately and shapes of the leaves are oblong (Plate 2). The outer surface and inner surface both are glabrous and has a thin brittle texture. The lamina is dorsiventral (distinct upper and lower surface) histologically. The leaves contain anomocytic stomata with non galndular simple trichomes on both the surface of epidermis.

Biological activities of Limonia crenulata (Roxb.) 144

Biological activities of Limonia crenulata (Roxb.) 145

5.1.2. Propagation

Limonia crenulata (Roxb.) is propagated by seeds, cuttings and layering. It can be planted under almost all conditions of soil and climate (Plate 3).

Biological activities of Limonia crenulata (Roxb.) 146

5.1.3. Transverse section of leaf

T.S. of leaf containing, upper and lower epidermis, spongy parenchyma, palisade cells. The upper and lower epidermis are single cell layered with cuticle, the epidermal cells are thick cell walled in outer side with lignified cell walls. Some of the palisade cells were containing Idioplasts. Spongy parenchyma and palisade cells, the centre containing vascular bundles and some place in the entire leaf blade also containing xylem elements.

The vascular bundles containing, xylem, phloem and some pith regions in the mid rib.

The phloem was containing phloem fibers. The spongy parenchyma cells are present near to the lower epidermis but the palisade cells are present near to the upper epidermis. The oil secretory canals or cavities are present in the leaf blade in both sides of the veins. The secretory canals are surrounded by small cells (Plate 4).

Biological activities of Limonia crenulata (Roxb.) 147

Biological activities of Limonia crenulata (Roxb.) 148

5.1.4. Transverse section of stem T.S. of stem contains epidermis, cortex, vascular bundles and pith.

Epidermis It is single cell layered with tangential thumble shaped paranchymatous cells, the outer cell wall is lignified thick cell wall (Plate 5).

Cortex Several cell layered with paranchymatous polygonal shaped cells. The cells towards outer is thick and towards inner is thin cells. In cortex some places containing oil secretory cavities. Some of the paranchymatous cells are pitted.

Vascular bundle The vascular bundles containing xylem , phloem and cambium , the phloem tissue is several cell layered, in some places phloem fibers are present, the phloem fibers are thick cell walled polygonal cells with lignified cell wall, the cell lumen is small and polygonal shaped distributed scattered in phloem and these are 2-3 cell layered.

Xylem Xylam is endarch in nature, protoxylem in centre and metaxylem towards outer. The xylem contains vessel elements, xylem fibres and trachieary elements.

Pith This regions also present in the centre of the stem, the pith cells are paranchymatous with thin cell walled ovoid shaped cells.

Biological activities of Limonia crenulata (Roxb.) 149

Biological activities of Limonia crenulata (Roxb.) 150

5.1.5. Transverse section of root

T.S. of root contains outer epidermis, cortex, endodermis and vascular bundles.

The epidermis is single cell layered and the outer cell wall is very thick. In some places the epidermal cells are detached from the outer cortex. Endodermis is single cell layered, lies between outer cortex and cortex. The cortex cells are paranchymatous which has thin cell wall arranged in several cell layers. Cells are large in size in towards outer but in inner side cells are smaller but all the cells are spherical shape. Some of the cortex cells containing oil globules. Some times the cortex cells containing prism shaped calcium crystals (Plate 6).

Biological activities of Limonia crenulata (Roxb.) 151

Biological activities of Limonia crenulata (Roxb.) 152

5.2. Qualitative phytochemical analysis of Limonia crenulata (Roxb.)

The result of qualitative phytochemical are presented (Table 5). The qualitative phytochemical analysis of hydroalcoholic extract of Limonia crenulata (Roxb.) revealed that the preliminary identification of bioactive compounds such as alkaloids, carbohydrate, protein & amino acids, glycosides, flavonoids, fixed oil and fats are present except phytosterol and saponins which could make the plant useful in treating different ailments and having potential for providing useful drug for human use.

Table 5. Qualitative phytochemical analysis of hydroalcoholic extract of

Limonia crenulata (Roxb.)

S.No Hydroalcoholic Extract Result

1 Alkaloids +

2 Carbohydrate +

3 Phytosterol -

4 Protein & Amino Acid +

5 Glycosides +

6 Flavonoids +

7 Saponins -

8 Fixed oil & fats +

Biological activities of Limonia crenulata (Roxb.) 153

5.3. Quantitative phytochemical analysis of Limonia crenulata (Roxb.)

The total amount of medicinal principles present in Limonia crenulata (Roxb.),

the active constituents are carbohydrates (7.80±2.3 mg/ 100gm), tannins (2.52 ± 0.3),

phenolic compounds (13.20±0.1mg/ 100gm), proteins (32.0 ± 1.5 µg/100gm), vitamin C

(0.33 ± 0.2 µg/100gm) and vitamin E (0.45 ± 0.2 µg/100gm) (Table 6). Natural

antioxidants such as vitamin C and vitamin E directly influence the biological activity.

Table 6. Quantitative estimation of phyto-constituents on Limonia crenulata (Roxb.)

(values are mean±SD)

S.No. Total Total Total Vitamin C Vitamin E Total

Phenolics Tannins Proteins (µg/100g) (µg/100gm) Carbohydrate

(mg/100 gm) (mg /100gm) (µg/100gm) (mg/100gm)

1. 13.20±0.1 2.52±0.3 32.0±1.5 0.33±0.2 0.45±0.2 7.80±2.3

Biological activities of Limonia crenulata (Roxb.) 154

The total amount of phytocompounds present in Limonia crenulata (Roxb.) were glycosides (47.77%), alkaloids (40.73%), flavonoids (43.18%), tannins (41.81%), fixed oil (3.23%), resins (40.80%) and vitamin C (4.14%) (Table 7). This is because of the pharmacological activity of this plant is used to trace the particular compound.

Table 7. Quantitative estimation of phyto-constituents of Limonia crenulata (Roxb.)

S.No Name of the compounds % of the compound

1 Glycosides 47.77%

2 Alkaloids 40.73 %

3 Flavonoids 43.18 %

4 Tannins 41.81 %

5 Fixed oil 3.23 %

6 Resins 40.85 %

8 Bitters -

9 Vitamin C 4.1414 %

Biological activities of Limonia crenulata (Roxb.) 155

5.4. Physico -Chemical characters of Limonia crenulata (Roxb.)

Ash usually represents the inorganic part of the plant. Dried leaf powder of

Limonia crenulata (Roxb.) was studied for parameters like total ash, water soluble ash,

acid insoluble ash, crude fibre content, water soluble extract and ethanol soluble extract.

Results revealed that ethanol soluble extract had maximum percentage 13.15 % and acid

insoluble ash had minimum percentage 1.57 % (Table 8).

Table 8. Physico chemical characteristic of ash

S.No Raw Total Ash Water Acid Crude Water Ethanol drug (%) Insoluble Insoluble Fibre Soluble Soluble Ash Ash Content Extract Extract (%) (% ) (%) (%) (%)

1 Limonia 4.42 2.50 1.57 2.87 6.83 13.15 crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 156

5.5. Elemental analysis of Limonia crenulata (Roxb.) by Atomic Absorption

Spectrophotometer

Limonia crenulata (Roxb.) leaf powder was subjected to elemental analysis for the presence of sodium, calcium, potassium, lithium, iron, copper, manganese, nickel, zinc, cobalt, aluminum, vanadium and molybdenum (Table 9). The amount of calcium

(78.05 mg/l) and potassium (140.30 mg/l) were higher in Limonia crenulata (Roxb.).

The content of manganese, nickel and molybdenum were found to be less in Limonia crenulata (Roxb.).

Table 9. Elemental analysis of Limonia crenulata (Roxb.) by Atomic Absorption Spectrophotometer S.No Name of Elements Concentration of the metals (mg/l)

1 Na (Sodium) 1.86

2 Ca (Calcium) 78.05

3 K (Potassium) 140.30

4 Li (Lithium) 0.24

5 Fe (Iron) 1.98

6 Cu (Copper) 0.38

7 Mn (Manganese) 0.20

8 Ni (Nickel) 0.11

9 Zn (Zinc) 1.37

10 Co (Cobalt) 0.15

11 Al (Aluminum) 1.51

12 V (Vanadium) 0.88 13 Mo (Molybdenum) 0.22

Biological activities of Limonia crenulata (Roxb.) 157

5.6. Microbial analysis of Limonia crenulata (Roxb.)

The presence of microorganisms and bacterial count of the plant leaf material have been tabulated (Table 10). The total heterotrophic bacterial count was 112 X 104

(Cells in sample/g). Salmonella sp., Shigella sp., and Enterobacter sp. were absent. It revealed that the presence of pathogenic microorganisms in Limonia crenulata (Roxb.) within WHO limit.

Table 10. Microbial analysis of Limonia crenulata (Roxb.)

S.No Name of the WHO Limit Cells in Interference Microbes Sample/g

1. Escherichia coli 102 10 X 102 Within Limit

2. Salmonella sp. Absence - Within Limit

3. Shigella sp. Absence - Within Limit

4. Enterobacter sp. 104 - Within Limit

5. Total Heterotrophic 107 112 X 104 Within Limit Bacteria

6. Yeast &Mould 104 19 x 101 Within Limit

5.7. High Power Thin Layer Chromatography (HPTLC) finger printing for Limonia crenulata (Roxb.)

A densitometry HPTLC analysis was performed for the development of characteristic finger profile for alcoholic and aqueous extracts of Limonia crenulata

Biological activities of Limonia crenulata (Roxb.) 158

(Roxb.) (Fig. 2; Plate 7) which may be used as markers for quality evaluations and standardization of the drug.

Fig 2. HPTLC-3D display of Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 159

HPTLC fingerprint of alcoholic extract of Limonia crenulata (Roxb.) contains

maximum 8 compounds displayed at 5 µl in Chloroform: Ethyl acetate: Formic Acid

(5:4:1) ratio. HPTLC fingerprint of alcoholic extract in Limonia crenulata (Roxb.)

shows 8 peaks of Rf values such as 0.06,0.16,0.23,0.31,0.38,0.53,0.77,and 0.93 were

found to have greater area such as 173.1,655.0,1043.5,352.5,4326.5,1831.7,4685.6, and

448.3. The peak corresponding to the Rf value 0.77 has maximum peak area of 4685.6

(Table 11; Fig 3).

Table 11. HPTLC analysis of Limonia crenulata (Roxb.) at 5µl

Peaks 1 2 3 4 5 6 7 8

Rf 0.06 0.16 0.23 0.31 0.38 0.53 0.77 0.93

Area 173.1 655.0 1043.5 352.5 4326.5 1831.7 4685.6 448.3

Biological activities of Limonia crenulata (Roxb.) 160

Fig 3. Peaks display at 5 µl

HPTLC fingerprint of aqueous extract of Limonia crenulata (Roxb.) shows

maximum 8 compounds displayed at 10 µl in Chloroform: Ethyl acetate: Formic Acid

(5:4:1) ratio. Peaks of at Rf values such as 0.14, 0.22, 0.29, 0.33, 0.37, 0.53, 0.77 and 0.81

were found to have greater area such as 1018.1, 1887.7, 699.0, 1100.2, 6616.7, 1514.9,

3543.4 and 1179.1. The peak corresponding to the Rf value 0.77 has maximum peak area

of 3543.4 (Table 12 & Fig 4).

Table 12. HPTLC analysis of Limonia crenulata (Roxb.) at 10µl

Peaks 1 2 3 4 5 6 7 8

Rf 0.14 0.22 0.29 0.33 0.37 0.53 0.77 0.81

Area 1018.1 1887.7 699.0 1100.2 6616.7 1514.9 3543.4 1179.1

Biological activities of Limonia crenulata (Roxb.) 161

Fig 4. Peaks display at 10µl

HPTLC fingerprint of aqueous extract of Limonia crenulata (Roxb.) shows

maximum 10 compounds display at 15 µl in Chloroform: Ethyl acetate: Formic Acid

(5:4:1) ratio. Peaks of at Rf values such as 0.06, 0.08, 0.13, 0.21,0.30,0.37,0.52,0.59,

0.76 and 0.92 were found to have greater area such as 81.9, 91.1, 1499.3, 2162.5, 874.0,

11421.7, 1180.8, 543.0, 3512.1 and 369.4. The peak corresponding to the Rf value 0.37

has maximum peak area of 11421.7 (Table 13 & Fig 5).

Table 13. HPTLC analysis of Limonia crenulata (Roxb.) at 15µl

Peaks 1 2 3 4 5 6 7 8 9 10

Rf 0.06 0.08 0.13 0.21 0.30 0.37 0.52 0.59 0.76 0.92

Area 81.9 91.1 1499.3 2162.5 874.0 11421.7 1180.8 543.0 3512.1 369.4

Biological activities of Limonia crenulata (Roxb.) 162

Fig 5. Peaks display at 15 µl

5.8. Gas Chromatography -Mass Spectrometry (GC – MS) analysis of Limonia crenulata (Roxb.)

Gas Chromatography and Mass Spectrometry (GC-MS) studies were carried out in the methanol leaf extract of Limonia crenulata (Roxb.). This possessed totally 13 compounds were tabulated. The presence of chemical constituents with their molecular formula, weight, retention time, structure and percentage are presented (Table 14, 15 &

16 and Fig 6, 7 & 8). The GC-MS profile revealed that the presence of palmitic acid as major constituent along with a fragrance compound, palmitic acid, aromatic acid, aromatic compound, diterpene alcohol, ether compound, flavonoid fraction, aldehyde, aromatic compound, sesquiterpene, sesquiterpene oxide and ketone.

Biological activities of Limonia crenulata (Roxb.) 163

Table 14. GC – MS analysis of Limonia crenulata (Roxb.)

S. No RT Name of the Molecular MW Peak Compound Nature Activity compound Formula Area % 1 4.10 Glycerin C3H8O3 92 0.26 Alcohol Preservative, Antimicrobial 2 5.93 Propane, 1,1,3- C9H20O3 176 0.80 Ether compound No activity reported triethoxy- 3 7.58 4H-Pyran-4-one, 2,3- C6H8O4 144 0.66 Flavonoid fraction Antimicrobial dihydro-3,5- dihydroxy-6-methyl-

4 10.03 Benzeneacetic acid C8H8O2 136 4.43 Aromatic acid Antimicrobial

5 12.77 Benzaldehyde, 4- C7H6O2 122 4.59 Aldehyde Anticancer,Antimicrobial hydroxy- 6 13.47 Benzene, 1,2- C11H14O2 178 3.89 Aromatic compound No activity reported dimethoxy-4-(2- propenyl)- 7 14.80 Caryophyllene C15H24 204 1.71 Sesquiterpene Anti-tumor, Analgesic, Antibacterial, Anti-inflammatory, Sedative, Fungicide, Anti ulcer, Pesticide, Gastro protective Larvicide, Mosquitocide, Anti acne 8 18.59 Caryophyllene oxide C15H24O 220 1.13 Sesquiterpene oxide Anti-tumor, Analgesic, Antibacterial, Anti-inflammatory, Sedative, Fungicide, Anti ulcer, Pesticide, Gastro protective Mosquitocide, Anti acne

Biological activities of Limonia crenulata (Roxb.) 164

Fig 6. GC – MS analysis of Limonia crenulata (Roxb.)

(Time 259 – 2259)

Limonia crenulata extract PPRC TANJORE, 4-DEC-2007 + 13:14:31 Medicinal plant analysis182 Scan EI+ 8.78 TIC 100 3.65e9

% 12.77 13.47 10.03 14.80 15.53 4.10 5.93 7.58 18.59 21.95 0 Time 2.59 4.59 6.59 8.59 10.59 12.59 14.59 16.59 18.59 20.59 22.59

Retention Time

Name of the compound - Glycerin

61 100

43

OH

50 HO OH

74 0 40 100 160 220 280 (mainlib) Glycerin

Name: Glycerin Formula: C3H8O3 MW: 92 CAS#: 56-81-5 NIST#: 118748 ID#: 25690 DB: mainlib Other DBs: Fine, TSCA, RTECS, USP, HODOC, NIH, EINECS, IRDB Contributor: NIST Mass Spectrometry Data Center, 1990. 10 largest peaks: 61 999 | 43 777 | 44 433 | 31 387 | 29 304 | 15 220 | 60 130 | 42 102 | 27 68 | 28 68 |

Biological activities of Limonia crenulata (Roxb.) 165

Synonyms: 1.1,2,3-Propanetriol 2.Glycerol 3.Glycerine 4.Glyceritol 5.Glycyl alcohol 6.Glyrol 7.Glysanin 8.Osmoglyn 9.Propanetriol 10.Trihydroxypropane

Name of the compound - Propane, 1,1,3-triethoxy-

59 100 47 103

87 O 50 O O

131

0 40 110 180 250 320 (mainlib) Propane, 1,1,3-triethoxy-

Name: Propane, 1,1,3-triethoxy- Formula: C9H20O3 MW: 176 CAS#: 7789-92-6 NIST#: 108265 ID#: 24770 DB: mainlib Other DBs: Fine, RTECS, HODOC, NIH, EINECS Contributor: Chuck Anderson, Aldrich Chemical Co. 10 largest peaks: 59 999 | 47 847 | 103 809 | 87 638 | 75 557 | 31 333 | 45 314 | 29 309 | 57 213 | 58 134 | Synonyms: 1.á-Ethoxypropionaldehyde diethyl acetal 2.3-Ethoxypropionaldehyde diethyl acetal 3.Propionaldehyde, 3-ethoxy-, diethyl acetal

Biological activities of Limonia crenulata (Roxb.) 166

4.1,1,3-Triethoxypropane 5.Propane, 1,3,3-triethoxy- 6.1,3,3-Triethoxypropane

Name of the compound - 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-

43 100

O

144HO OH 50 101

73 O

0 40 130 220 310 400 (replib) 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-

Name: 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- Formula: C6H8O4 MW: 144 CAS#: 28564-83-2 NIST#: 108691 ID#: 1841 DB: replib Other DBs: RTECS Contributor: Philip Morris R&D 10 largest peaks: 43 999 | 44 742 | 144 563 | 101 402 | 45 264 | 73 249 | 72 182 | 55 177 | 42 48 | 58 43 | Synonyms: 1.3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one #

Biological activities of Limonia crenulata (Roxb.) 167

Name of the compound – Benzeneacetic acid

91 100 O OH

50 136

65 46 0 40 100 160 220 280 (replib) Benzeneacetic acid

Name: Benzeneacetic acid Formula: C8H8O2 MW: 136 CAS#: 103-82-2 NIST#: 109188 ID#: 11589 DB: replib Other DBs: Fine, TSCA, RTECS, EPA, HODOC, NIH, EINECS, IRDB Contributor: Philip Morris R&D 10 largest peaks: 91 999 | 136 406 | 92 164 | 65 147 | 39 63 | 137 37 | 63 36 | 46 36 | 51 34 | 89 32 | Synonyms: 1.Acetic acid, phenyl- 2.à-Toluic acid 3.Benzenacetic acid 4.Phenylacetic acid 5.ê-Phenylacetic acid 6.Phenylethanoic acid 7.Kyselina fenyloctova 8.2-Phenylacetic acid

Biological activities of Limonia crenulata (Roxb.) 168

Name of the compound – Benzaldehyde, 4-hydroxy-

121 100

O 50 65 93 HO

0 40 130 220 310 400 (mainlib) Benzaldehyde, 4-hydroxy-

Name: Benzaldehyde, 4-hydroxy- Formula: C7H6O2 MW: 122 CAS#: 123-08-0 NIST#: 135511 ID#: 74329 DB: mainlib Other DBs: Fine, TSCA, RTECS, EPA, HODOC, NIH, EINECS, IRDB Contributor: NIST Mass Spectrometry Data Center, 1994 10 largest peaks: 121 999 | 122 813 | 65 482 | 93 460 | 39 432 | 63 155 | 38 145 | 29 125 | 62 103 | 66 101 | Synonyms: 1.Benzaldehyde, p-hydroxy- 2.p-Formylphenol 3.p-Hydroxybenzaldehyde 4.p-Oxybenzaldehyde 5.4-Formylphenol 6.4-Hydroxybenzaldehyde 7.Parahydroxybenzaldehyde 8.USAF m-6 9.4-Hydroxybenzenecarbonal 10.Benzaldehyde, 4-hydroxy

Biological activities of Limonia crenulata (Roxb.) 169

Name of the compound – Benzene, 1,2-dimethoxy-4-(2-propenyl)-

178 100

O

O 91 50 147

0 40 130 220 310 400 (replib) Benzene, 1,2-dimethoxy-4-(2-propenyl)-

Name: Benzene, 1,2-dimethoxy-4-(2-propenyl)- Formula: C11H14O2 MW: 178 CAS#: 93-15-2 NIST#: 113174 ID#: 22315 DB: replib Other DBs: Fine, TSCA, RTECS, HODOC, NIH, EINECS, IRDB Contributor: NIST Mass Spectrometry Data Center, 1990. 10 largest peaks: 178 999 | 91 560 | 103 439 | 107 392 | 147 388 | 163 356 | 77 307 | 79 240 | 65 234 | 51 204 | Synonyms: 1.Benzene, 4-allyl-1,2-dimethoxy- 2.Ent 21040 3.Eugenol methyl ether 4.Eugenyl methyl ether 5.Methyl eugenol ether 6.Methyleugenol 7.O-Methyleugenol 8.Veratrole methyl ether 9.1-(3,4-Dimethoxyphenyl)-2-propene 10.1-Allyl-3,4-dimethoxybenzene

Biological activities of Limonia crenulata (Roxb.) 170

Name of the compound – Caryophyllene

41 100 69

133

50 55

161 189

0 40 130 220 310 400 (replib) Caryophyllene

Name: Caryophyllene Formula: C15H24 MW: 204 CAS#: 87-44-5 NIST#: 69482 ID#: 1122 DB: replib Other DBs: Fine, TSCA, RTECS, HODOC, NIH, EINECS Contributor: N.W. DAVIES, UNIV. OF TASMANIA, HOBART, TASMANIA, AUSTRALIA 10 largest peaks: 41 999 | 69 976 | 93 937 | 133 646 | 79 614 | 91 551 | 55 432 | 81 389 | 107 389 | 105 372 | Synonyms: 1.Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-, [1R-(1R*,4E,9S*)]- 2.Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-, (E)-(1R,9S)-(-)- 3.á-Caryophyllen 4.á-Caryophyllene 5.trans-Caryophyllene 6.L-Caryophyllene 7.Bicyclo(7.2.0)undec-4-ene, 8-methylene-4,11,11-trimethyl-, (E)-(1R,9S)-(-)- 8.Caryophyllene ,à + á mixt. 9.8-Methylene-4,11,11-(trimethyl)bicyclo(7.2.0)undec-4-ene 10.4,11,11-Trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene #

Biological activities of Limonia crenulata (Roxb.) 171

Name of the compound – Caryophyllene oxide

43 100 79

O

50

220 0 40 200 360 (mainlib) Caryophyllene oxide

Name: Caryophyllene oxide Formula: C15H24O MW: 220 CAS#: 1139-30-6 NIST#: 156329 ID#: 5639 DB: mainlib Other DBs: Fine, TSCA, RTECS, EINECS Contributor: Chemical Concepts 10 largest peaks: 43 999 | 41 927 | 79 885 | 93 661 | 91 573 | 95 420 | 69 407 | 55 393 | 67 377 | 81 373 | Synonyms: 1.5-Oxatricyclo[8.2.0.0(4,6)-]dodecane, 4,12,12-trimethyl-9-methylene-, [1R- (1R*,4R*,6R*,10S*)]- 2.5-Oxatricyclo(8.2.0.0(sup 4,6))dodecane, 4,12,12-trimethyl-9-methylene-, (1R,4R,6R,10S)- 3.Caryophylene oxide 4.Caryophyllene epoxide 5.(-)-á-Caryophyllene epoxide 6.á-Caryophyllene oxide 7.Epoxycaryophyllene 8.(-)-Epoxydihydrocaryophyllene 9.4,11,11-Trimethyl-8-methylene-5-oxatricyclo(8.2.0.0(4,6))dodecane 10.(-)-5-Oxatricyclo[8.2.0.0(4,6)]dodecane,4,12,12-trimethyl-9-methylene-, [1R- (1R*,4R*,6R*,10S*)]- 11.5-)xatricyclo[8.2.0.0(4,6))dodecane, 6,,11,11-trimethyl-2-methylene, [1R- (1R*,4R*,6R*,10S*)]-

Biological activities of Limonia crenulata (Roxb.) 172

Table 15. GC – MS analysis of Limonia crenulata (Roxb.)

(Retention Time 300 – 4300)

S.No RT Name of the Molecular MW Peak Compound Activity compound Formula Area Nature % 1 8.78 Estragole C10H12O 148 14.55 Fragrance Antiviral, antibacterial, compound Insecticide, Fragrance

2 10.03 Benzene acetic acid C8H8O 136 4.43 Aromatic acid Antimicrobial

3 13.47 Benzene, 1,2- C11H14O2 178 3.89 Aromatic acid No activity reported dimethoxy-4- (propenyl)- 4 26.75 n-hexadecanoic acid C16H32O2 256 32.14 Palmitic acid Antioxidant, Pesticide, Hypocholesterolemic, Lubricant, Antiandrogenic, Flavor, Hemolytic.

5 28.31 : 7H-Furo[3,2- C12H8O4 216 16.66 Aromatic No activity reported g][1]benzopyran-7- compound one, 4-methoxy-

6 29.53 Phytol C20H40O 296 10.89 Diterpene Antimicrobial Anti inflammatory Anticancer

Biological activities of Limonia crenulata (Roxb.) 173

Fig 7. GC – MS analysis of Limonia crenulata (Roxb.)

(Time 300 – 4300)

Limonia crenulata extract PPRC TANJORE, 4-DEC-2007 + 13:14:31 Medicinal plant analysis182 Scan EI+ 1.37 TIC 100 1.78e10

% 1.47 8.78 26.75 28.3129.53 44.29 10.03 13.47 0 Time 3.00 8.00 13.00 18.00 23.00 28.00 33.00 38.00 43.00

Biological activities of Limonia crenulata (Roxb.) 174

Name of the compound – Estragole

148 100

50 77 121 91 51 133 O

0 40 100 160 220 280 (mainlib) Estragole

Name: Estragole Formula: C10H12O MW: 148 CAS#: 140-67-0 NIST#: 113167 ID#: 94865 DB: mainlib Other DBs: Fine, TSCA, RTECS, EPA, HODOC, NIH, EINECS, IRDB Contributor: NIST Mass Spectrometry Data Center, 1990. 10 largest peaks: 148 999 | 147 599 | 77 438 | 121 432 | 117 418 | 91 305 | 105 300 | 115 274 | 78 267 | 133 250 | Synonyms: 1.Tarragon 2.Anisole, p-allyl- 3.Chavicol, O-methyl- 4.p-Allylanisole 5.p-Methoxyallylbenzene 6.Chavicol methyl ether 7.Esdragol 8.Esdragole 9.Esdragon 10.Estragol

Biological activities of Limonia crenulata (Roxb.) 175

Table 16. GC – MS analysis of Limonia crenulata (Roxb.)

S.No RT Name of the compound Molecular MW Peak Compound Activity Formula Area % Nature

1 23.95 Z,Z-6,28- C37H70O 530 4.11 Ketone No activity reported Heptatriactontadien-2- one

2 26.75 n-Hexadecanoic acid C16H32O2 256 32.14 Palmitic acid Antioxidant, Pesticide, Hypocholesterolemic, Lubricant, Antiandrogenic, Flavor, Hemolytic.

3 28.31 7H-Furo[3,2- C12H8O4 216 16.66 Aromatic No activity reported g][1]benzopyran-7-one, compound 4-methoxy-

4 29.53 Phytol C20H40O 296 10.89 Diterpene Antimicrobial Anti inflammatory Anticancer

Biological activities of Limonia crenulata (Roxb.) 176

Fig 8 . GC – MS analysis of Limonia crenulata (Roxb.)

(Time 2427 – 4427)

Limonia crenulata extract PPRC TANJORE, 4-DEC-2007 + 13:14:31 Medicinal plant analysis182 Scan EI+ 28.31 TIC 100 26.75 3.32e9 29.53 30.26 44.29 38.66 % 30.68 32.21 26.97 34.03 23.95 24.86 0 Time 24.27 26.27 28.27 30.27 32.27 34.27 36.27 38.27 40.27 42.27 44.27

Name of the compound - Z,Z-6,28-Heptatriactontadien-2-one

69 100 55

83 O

95

50 109

135 472 530 180 207 231250 273 305 345 373 403 431 0 50 90 130 170 210 250 290 330 370 410 450 490 530 (mainlib) Z,Z-6,28-Heptatriactontadien-2-one

Name: Z,Z-6,28-Heptatriactontadien-2-one Formula: C37H70O MW: 530 CAS#: 133530-21-9 NIST#: 111390 ID#: 28701 DB: mainlib Other DBs: None Contributor: Y. Murata, LBC, NIDDK, NIH, Bethesda, MD 20892 10 largest peaks: 69 999 | 55 965 | 67 862 | 83 791 | 81 725 | 71 674 | 57 673 | 95 632 | 97 560 | 68 559 | Synonyms: 1.Z,Z-6,28-Heptatriacontadien-2-one 2.(6Z,28Z)-6,28-Heptatriacontadien-2-one #

Biological activities of Limonia crenulata (Roxb.) 177

Name of the compound - n-Hexadecanoic acid

43 100 73

O OH 50

129 256 213 157 0 40 130 220 310 400 (mainlib) n-Hexadecanoic acid

Name: n-Hexadecanoic acid Formula: C16H32O2 MW: 256 CAS#: 57-10-3 NIST#: 151973 ID#: 8185 DB: mainlib Other DBs: Fine, TSCA, RTECS, EPA, HODOC, NIH, EINECS, IRDB Contributor: Chemical Concepts 10 largest peaks: 43 999 | 73 905 | 60 838 | 41 749 | 57 634 | 55 616 | 29 414 | 69 310 | 71 285 | 61 218 | Synonyms: 1.Hexadecanoic acid 2.n-Hexadecoic acid 3.Palmitic acid 4.Pentadecanecarboxylic acid 5.1-Pentadecanecarboxylic acid 6.Cetylic acid 7.Emersol 140 8.Emersol 143 9.Hexadecylic acid 10.Hydrofol

Biological activities of Limonia crenulata (Roxb.) 178

Name of the compound - 7H-Furo[3,2-g][1]benzopyran-7-one, 4-methoxy-

173216 100

O

50 145 O O O 89 51

0 40 130 220 310 400 (mainlib) 7H-Furo[3,2-g][1]benzopyran-7-one, 4-methoxy-

Name: 7H-Furo[3,2-g][1]benzopyran-7-one, 4-methoxy- Formula: C12H8O4 MW: 216 CAS#: 484-20-8 NIST#: 290666 ID#: 129900 DB: mainlib Other DBs: Fine, RTECS, NIH, EINECS Contributor: NIST Mass Spectrometry Data Center, 1998. 10 largest peaks: 216 999 | 173 984 | 145 478 | 201 354 | 89 256 | 51 206 | 188 168 | 63 167 | 217 136 | 174 130 | Synonyms: 1.Bergaptan 2.Bergapten 3.Bergaptene 4.Heraclin 5.Majudin 6.5-Methoxypsoralen 7.6-Hydroxy-4-methoxy-5-benzofuranacrylic acid, ç-lactone 8.5-Methoxy-6,7-furanocoumarin 9.4-Methoxy-7H-furo(3,2-g)(1)benzopyran-7-one 10.5-MOP

Biological activities of Limonia crenulata (Roxb.) 179

Name of the compound - Phytol

71 100

43 HO

50

196 278 0 40 140 240 340 440 (replib) Phytol

Name: Phytol Formula: C20H40O MW: 296 CAS#: 150-86-7 NIST#: 157813 ID#: 8058 DB: replib Other DBs: Fine, TSCA, RTECS, HODOC, EINECS Contributor: Chemical Concepts 10 largest peaks: 71 999 | 43 786 | 57 662 | 41 651 | 55 590 | 69 514 | 81 470 | 68 437 | 82 361 | 95 324 | Synonyms: 1.2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]- 2.trans-Phytol 3.3,7,11,15-Tetramethyl-2-hexadecen-1-ol 4.(2E)-3,7,11,15-Tetramethyl-2-hexadecen-1-ol #

Biological activities of Limonia crenulata (Roxb.) 180

5.9. High Performance Liquid Chromatography (HPLC) analysis of Limonia

crenulata (Roxb.)

High-performance liquid chromatography (HPLC) has been employed to analyse

several components in a medicinal preparation composed of several crude drugs. HPLC

analysis was carried for the methanol extracts of Limonia crenulata (Roxb.). Results

showed that the content of compounds such as Gallic acid (0.145), Caffeic acid (0.399),

Rutin (3.776) and Quercetin (0.416) were present at the concentration of µg/ml sample

(wet basis) (Fig 9 & Table 17).

Table 17. HPLC analysis of Limonia crenulata (Roxb.)

Detector A (280nm)

Retention Area Height Concentration µg/ml Compound Name

S.No Time sample (wet basis)

1 5.492 824,008 42,111 0.145 Gallic acid

2 9.533 695,472 110,910 0.399 Caffeic acid

3 10.533 16,294,065 1,816,623 3.776 Rutin

4 12.358 556897 41,458 0.416 Quercetin

Biological activities of Limonia crenulata (Roxb.) 181

Fig 9. HPLC analysis of Limonia crenulata (Roxb.)

3.5 Detector A (280nm) 3.5 commercial 191 plant extract 13.8.10 FRC 20ul a commercial 191 plant extract 13.8.10 FRC 20ul a Retention Time 3.0 3.0 Name

2.5 2.5

2.0 2.0

1.5 1.5 Volts Volts Caffeic acid acid Caffeic

1.0 1.0 Quercetin

9.533 Ferulic acid acid Ferulic

Gallic acid Gallic

0.5 0.5 12.358 23.767 Rutin 5.492

0.0 0.0 10.533

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes

5.10. Fourier Transform - Infra Red (FT-IR) spectroscopy analysis of Limonia crenulata (Roxb.)

The functional groups of isolated phytocompounds were detected by using FT-IR spectroscopy analysis. The IR spectrum of the isolated phytocompounds (Gallic acid,

Caffeic acid, Rutin and Quercetin) were recorded from Limonia crenulata (Roxb.) using

Perkin-Elmer Paragon 580 B FT-IR spectrophotometer (Table 18, 19, 20 & 21; Fig 10,

11, 12 & 13).

Biological activities of Limonia crenulata (Roxb.) 182

Table . 18. Detection of functional groups of Gallic acid

from Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

S.No Wave number cm-1 Wave number cm-1 Functional group assignment Reference 1 3405.11 3419 Polymeric O-H stretch

2 1638.47 1656 C=O stretch

3 1391.34 1410-1310 Phenol or tertiary alcohol, O-H bend

4 1276.98 1277 C-O stretch

5 1016.91 1026 C C-C C-C stretch

Fig 10. Detection of functional groups of Gallic acid

from Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

Biological activities of Limonia crenulata (Roxb.) 183

FTIR SPECTRUM Date: 24-8-2010 Spectrum Name: GA.sp Instrument Model: Spectrum RXI 100.0

90 1276.98 1016.91

80 2076.13 1391.34

70

60

50 %T 660.46

40

30

20 1638.47

10

3405.11 0.0 4000.0 3000 2000 1500 1000 400.0 cm-1 GA.pk

GA.sp 3601 4000.00 400.00 7.99 99.99 4.00 %T 5 1.00

REF 4000 99.43 2000 89.62 600 3405.11 7.99 2076.13 86.23 1638.47 24.56 1391.34 87.20 1276.98 94.43 1016.91 93.90 660.46 53.24

Biological activities of Limonia crenulata (Roxb.) 184

Table 19. Detection of functional groups of Caffeic acid from

Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

S.No Wave number cm-1 Wave number cm-1 Functional group assignment Reference 1 3401.97 3419 Polymeric O-H stretch 2 1639.67 1656 C=O stretch 3. 1387.69 1410-1310 Phenol or tertiary alcohol, O-H bend 4 1276.64 1277 C-O stretch 5 1016.25 1026 C-C stretch

Fig 11. Detection of functional groups of Caffeic acid from Limonia crenulata

(Roxb.) using FT-IR spectroscopy analysis

FTIR SPECTRUM Date: 24-8-2010 Spectrum Name: Caffeic acid .sp Instrument Model: Spectrum RXI 100.0

90 1276.64

80 2076.69 1387.69 1016.25 70

60 661.41

50 %T

40

30 1639.67

20

10

3401.97 0.0 4000.0 3000 2000 1500 1000 400.0 cm-1 Caffeic acid .pk

CAFFEI~1.SP 3601 4000.00 400.00 7.99 100.00 4.00 %T 5 1.00

REF 4000 99.94 2000 93.03 600 3401.97 7.99 2076.69 89.86 1639.67 34.91 1387.69 84.77 1276.64 95.20 1016.25 80.99 661.41 62.82

Biological activities of Limonia crenulata (Roxb.) 185

Table 20. Detection of functional groups of Rutin

from Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

S.No Wave number cm-1 Wave number cm-1 Functional group assignment Reference 1 3399.34, 3468.07 3419 Polymeric O-H stretch

2 1638.43 1656 C=O stretch 3 1385.59 1410-1310 Phenol or tertiary alcohol, O-H bend 4 1275.36 1277 C-O stretch 5 1016.37 1026 CC-C C-C stretch

Fig12.Detection of functional groups of Rutin from Limonia crenulata (Roxb.) using

FT-IR spectroscopy analysis

FTIR SPECTRUM Date: 24-8-2010 Spectrum Name: Rutin.sp Instrument Model: Spectrum RXI 100.0

90 1275.36 1117.67

2074.48 80 1016.37

1385.59 70 1413.58

60

656.42 50 %T

40

30

1638.43 3399.34 20

10

3468.07 0.0 4000.0 3000 2000 1500 1000 400.0 cm-1 Rutin.pk

Rutin.sp 3601 4000.00 400.00 8.18 99.57 4.00 %T 5 1.00

REF 4000 99.37 2000 92.23 600 3468.07 8.78 3399.34 8.18 2074.48 89.19 1638.43 30.05 1413.58 83.64 1385.59 84.04 1275.36 95.01 1117.67 96.04 1016.37 90.89 656.42 59.96

Biological activities of Limonia crenulata (Roxb.) 186

Table 21. Detection of functional groups of Quercetin

from Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

S.No Wave number cm-1 Wave number cm-1 Functional group assignment Reference 1 3403.41 3419 Polymeric O-H stretch 2 2954.61 2955 C-H group in aromatic ring 3 1646.70 1656 C=O stretch 4 1410.57 1410-1310 Phenol or tertiary alcohol, O-H bend 5 1110.51 1163 C=C stretch 6 1020.67 1026 C-C stretch

Fig.13. Detection of functional groups of Quercetin

from Limonia crenulata (Roxb.) using FT-IR spectroscopy analysis

FTIR SPECTRUM Date: 24-8-2010 Spectrum Name: Quercerin.sp Instrument Model: Spectrum RXI 100.0

3949.25 90 2520.76

80 2122.32

70 1110.51

60 1410.57

50 %T 672.01 2840.26 40

2954.61 30 1646.70

20 1020.67

10

3403.41 0.0 4000.0 3000 2000 1500 1000 400.0 cm-1 Quercerin.pk

QUERCE~1.SP 3601 4000.00 400.00 8.02 100.00 4.00 %T 5 1.00

REF 4000 99.62 2000 91.53 600 3949.25 97.87 3403.41 8.02 2954.61 37.22 2840.26 51.89 2520.76 94.78 2122.32 85.48 1646.70 36.27 1410.57 61.64 1110.51 79.96 1020.67 23.89 672.01 52.71

Biological activities of Limonia crenulata (Roxb.) 187

5.11. Ultra Violet (UV) - Visible spectroscopy analysis of Limonia crenulata (Roxb.)

The functional group of isolated phytocompounds such as gallic acid, caffeic acid,

rutin and quercetin were analysed with the help of Lambda 35 model UV – visible

spectrophotometer (Table 22; Fig 14, 15, 16 & 17).

Table 22: UV - Visible spectroscopy analysis of isolated phytocompounds from

Limonia crenulata (Roxb.)

S.No Test Sample λmax Absorbance

1 Gallic acid 211.93 0.436

3 Caffeic acid 209.96 0.786

4 Rutin 213.49 2.901

5 Quercetin 209.98 0.210

Fig 14. Detection of functional groups of Gallic acid from Limonia crenulata (Roxb.) using UV - Visible spectroscopy analysis

Date: 8/24/2010 UV spectrum Spectrum Name: GA.SP

0.600

0.55

0.50 211.93,0.43607

0.45

0.40

0.35

0.30 A

0.25

0.20

0.15

0.10

0.05

0.00 -0.020 200.0 250 300 350 400 450 500 550 600.0 nm Instrument Model: Lambda 35

Biological activities of Limonia crenulata (Roxb.) 188

Fig.15. Detection of functional groups of Caffeic acid from Limonia crenulata

(Roxb.) using UV - Visible spectroscopy analysis

Date: 8/24/2010 UV spectrum Spectrum Name: CAFF-ACI.SP

1.000

0.95

0.90 209.96,0.78602 0.85

0.80

0.75

0.70

0.65

0.60

0.55

0.50 A 0.45

0.40

0.35

0.30

0.25

0.20 259.23,0.11738 0.15

0.10

0.05

-0.020 200.0 250 300 350 400 450 500 550 600.0 nm Instrument Model: Lambda 35

Fig. 16. Detection of functional groups of Rutin from Limonia crenulata (Roxb.)

using

UV- Visible spectroscopy analysis

Date: 8/24/2010 UV spectrum Spectrum Name: RUTIN.SP

3.50 3.4

3.2 213.49,2.9008

3.0

2.8 219.89,2.5391

2.6

2.4

2.2

2.0

1.8 A 1.6

1.4

1.2 270.01,0.86623 335.21,0.77808

1.0

0.8

0.6

0.4

0.2

-0.02 210.0 250 300 350 400 450 500 550 600.0 nm Instrument Model: Lambda 35

Biological activities of Limonia crenulata (Roxb.) 189

Fig.17. Detection of functional groups of Quercetin from Limonia crenulata (Roxb.)

using UV - Visible spectroscopy analysis

Date: 8/24/2010 UV spectrum Spectrum Name: QUER.SP

0.300

0.28

0.26

0.24 209.98,0.21011

0.22

0.20

0.18

0.16

A 0.14

0.12

0.10

0.08

0.06

0.04

338.03,0.0071657 0.02

0.000 200.0 250 300 350 400 450 500 550 600.0 nm Instrument Model: Lambda 35

5.12. Antimicrobial activity of Limonia crenulata (Roxb.)

The antimicrobial compound was extracted from the leaves of Limonia crenulata

(Roxb.) using benzene, chloroform, ethanol, acetone and aqueous. The antimicrobial activity was performed by disc diffusion method. The present investigation proved that the Limonia crenulata (Roxb.) showed very promising result in antimicrobial activity. This plant is very efficient to control all the six strains of bacteria and two strains of fungi at 250 µg concentration. Remarkable antimicrobial activity was seen with increase in concentration of the extracts.

Benzene extract of leaf showed significant antimicrobial activity against the selected microbes (Inhibition zone ranging 8-22 mm). The benzene leaf extract has maximum activity against Salmonella typhi (22 mm), moderate activity against Sheigella

Biological activities of Limonia crenulata (Roxb.) 190

shigae (14 mm), Proteus vulgaris (16 mm), Escherichia coli (18 mm), Aspergillus niger

(18 mm) and least activity against Candida albicans (15 mm) at concentration 250µg

(Table 23 & Plate 8).

Table 23. Antimicrobial activity of benzene leaf extract of Limonia crenulata

(Roxb.) in different concentrations

Name of the Zone of inhibition (diameter in mm)

S.No Microorganisms 100 µg/ml 150 µg/ml 250 µg/ml

1. Pseudomonas aeruginosa 10 13 16 2. Sheigella shigae 8 11 14 3. Proteus vulgaris 8 12 16 4. Salmonella typhi 9 18 22 5. Escherichia coli 8 16 18 6. Klebsiella aerogenes 12 14 20 7. Aspergillus niger 8 15 18 8. Candida albicans 8 12 15

Biological activities of Limonia crenulata (Roxb.) 191

Biological activities of Limonia crenulata (Roxb.) 192

The chloroform leaf extract showed very promising antimicrobial activity against the selected microbes (Inhibition zone ranging of 10-25mm). The chloroform leaf extract has highest antimicrobial activity against Sheigella shigae (25mm), Candida albicans

(25mm) Klebsiella aerogenes (22mm), Escherichia coli (18mm) and least against

Pseudomonas aeruginosa (17mm) at concentration 250µg (Table 24 & Plate 9).

Table 24. Antimicrobial activity of chloroform leaf extract of Limonia crenulata

(Roxb.) in different concentrations

Zone of inhibition (diameter in mm) S.No Name of the Microorganisms 100 µg/ml 150 µg/ml 250 µg/ml 1. Pseudomonas aeruginosa 10 12 17 2. Sheigella shigae 12 20 25 3. Proteus vulgaris 12 15 20 4. Salmonella typhi 10 20 23 5. Escherichia coli 10 15 18 6. Klebsiella aerogenes 12 20 22 7. Aspergillus niger 14 18 20 8. Candida albicans 18 20 25

Biological activities of Limonia crenulata (Roxb.) 193

Biological activities of Limonia crenulata (Roxb.) 194

In ethanol leaf extract of Limonia crenulata (Roxb.) showed significant

antimicrobial activity against Pseudomonas aeruginosa (26 mm), Shigella shigae (25

mm), Salmonella typhi (22 mm), Proteus vulgaris (20 mm), Escherichia coli (18 mm)

and Klebsiella aerogenes (18 mm) at concentration of 250µg. It has least activity on

Proteus vulgaris (9 mm) at concentration of 100µg (Table 25 & Plate 10).

Table 25. Antimicrobial activity of ethanol leaf extract of Limonia crenulata

(Roxb.) in different concentrations

Zone of inhibition (diameter in mm) S.No Name of the Microorganisms 100 µg/ml 150 µg/ml 250 µg/ml 1. Pseudomonas aeruginosa 18 20 26 2. Sheigella shigae 11 16 25 3. Proteus vulgaris 9 15 20 4. Salmonella typhi 10 17 22 5. Escherichia coli 10 12 18 6. Klebsiella aerogenes 10 15 18 7. Aspergillus niger 10 12 16 8. Candida albicans 15 18 21

Biological activities of Limonia crenulata (Roxb.) 195

Biological activities of Limonia crenulata (Roxb.) 196

The acetone leaf extract showed very promising antimicrobial activity. The

maximum zone of inhibition was observed against Pseudomonas aeruginosa (28 mm) at

250 µg. This is the highest value among all the other values. This extract inhibits the least

against the growth of Candida albicans and Salmonella typhi. The zone of inhibition was

ranging between 8-28mm (Table 26 & Plate 11).

Table 26. Antimicrobial activity of acetone leaf extract of Limonia crenulata (Roxb.) in different concentrations

Zone of inhibition (diameter in mm) S.No Name of the Microorganisms 100 µg/ml 150 µg/ml 250 µg/ml 1. Pseudomonas aeruginosa 12 20 28 2. Sheigella shigae 10 15 18 3. Proteus vulgaris 14 16 22 4. Salmonella typhi 8 12 18 5. Escherichia coli 12 18 20 6. Klebsiella aerogenes 12 16 20 7. Aspergillus niger - - - 8. Candida albicans 10 12 18

Biological activities of Limonia crenulata (Roxb.) 197

Biological activities of Limonia crenulata (Roxb.) 198

Aqueous extract of leaf showed the inhibition zone diameter ranging from 8-

20mm against microbes (Table 27 & Plate 12).

Table 27. Antimicrobial activity of aqueous leaf extract of Limonia crenulata (Roxb.) in different concentrations Zone of inhibition (diameter in mm) S.No Name of the Microorganisms 100 µg/ml 150 µg/ml 250 µg/ml

1. Pseudomonas aeruginosa 10 12 16 2. Sheigella shigae 12 14 16 3. Proteus vulgaris 10 12 18 4. Salmonella typhi 12 12 15 5. Escherichia coli 8 10 16 6. Klebsiella aerogenes 10 17 20 7. Aspergillus niger 8 10 12 8. Candida albicans 10 14 16

Biological activities of Limonia crenulata (Roxb.) 199

Biological activities of Limonia crenulata (Roxb.) 200

The antibiotic sensitivity test using standard antibiotics ciprofloxacin (2 mg/disc)

and fluconazole (10 mg/disc) were tested against both bacteria and fungi studied. The

results of antibiotic sensitivity test were presented in table 28. All the antibiotics used

were exhibited higher antimicrobial activity.

Table 28. Effect of antibiotics on microbes (Positive control)

S.No Name of the Microorganisms Zone of inhibition (diameter in mm)

Ciprofloxacin Fluconazole 2 mg/disc 10 mg/disc 1. Pseudomonas aeruginosa 25 - 2. Shigella shigae 22 - 3. Proteus vulgaris 23 - 4. Salmonella typhi 23 - 5. Escherichia coli 25 - 6. Klebsiella aerogenes 28 - 7. Aspergillus niger - 22 8. Candida albicans - 20

The result of antimicrobial effect of five solvents revealed no activity against

bacteria and fungi. (Table 29).

Biological activities of Limonia crenulata (Roxb.) 201

Table 29. Effect of solvents on microbes (Negative control)

S. No Name of the Benzene Chloroform Ethanol Acetone Aqueous Microorganisms

1. Pseudomonas aeruginosa - - - - - 2. Shigella shigae - - - - - 3. Proteus vulgaris - - - - - 4. Salmonella typhi - - - - - 5. Escherichia coli - - - - - 6. Klebsiella aerogenes - - - - - 7. Aspergillus niger - - - - - 8. Candida albicans - - - - -

5.13. Antioxidant activity of Limonia crenulata (Roxb.)

Many aromatic plants have been known to support various biological activities

such as antimicrobial and antioxidant properties. The radical scavenging effects

(percentage of quenched radicals) were determined for Limonia crenulata (Roxb.) leaf

extracts. The leaf extracts or their constituents when mixed with DPPH decolorized due

to hydrogen donating ability. All the tested samples (ethanol, methanol and aqueous

extracts) revealed scavenging effects on DPPH (70 to 93%) as shown in Fig. 18.

Antioxidants are believed to neutralize the free radicals in lipid chains by

contributing a hydrogen atom usually from a phenolic hydroxyl group, which in turn

converts phenolic groups into stable free radicals that do not initiate or propagate further

oxidation of lipids.

Biological activities of Limonia crenulata (Roxb.) 202

The ethanol extract of Limonia crenulata (Roxb.) was found to act as strong free radical scavengers ( 83.69% ) in comparison with commercial antioxidants ascorbic acid and selenium as indicated by DPPH assays (Table 30).

Table 30. Antioxidant activity of Limonia crenulata (Roxb.)

% of inhibition

Concentration Leaf extracts Standard of leaf powder (mg/ml) Ethanol Methanol Water Ascorbic acid Selenium Standard Standard 5 83.69 77.89 70.59 66 64

Fig.18. Antioxidant activity of Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 203

5.14. Anti-inflammatory activity of Limonia crenulata (Roxb.)

Leaf extract of Limonia crenulata (Roxb.) treated groups significantly reduced the

swelling in the injected paw as compared with control group. The percentage inhibition

of paw volume of alcoholic extracts of Limonia crenulata (Roxb.) was 50.95% found to

be significant as that of the standard drug Indomethacin treated group which was 63.80

%. The chloroform, aqueous, benzene and acetone extracts of Limonia crenulata (Roxb.)

were found to be 40.44%, 27.33%, 29.05%, and 37.25% of paw volume inhibition

respectively (Table 31; Fig. 19; Plate 13).

Table 31. Anti-inflammatory activity of Limonia crenulata (Roxb.)

Percentage of Dose mg.kg per. Paw volume increase Treatment inhibition S.No oral after 3hr (ml) (%) 1 Control 5 ml/kg 121.61 ± 10.56 - 2 Alcoholic extract 200 59.64 ± 5.2 50.95*

3 Chloroform extract 200 72.42 ±5.8 40.44*

4 Aqueous extract 200 88.37 ± 6.6 27.33

5 Benzene extract 200 86.30 ± 6.4 29.05

6 Acetone extract 200 76.30 ± 8.4 37.25*

7 Indomethacin 10 40.4 ± 4.62 63.80 (Standard) *P < values are expressed as ± SEM Number of animals using are 6 in each group.

Biological activities of Limonia crenulata (Roxb.) 204

Fig. 19. Anti-inflammatory activity of Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 205

Biological activities of Limonia crenulata (Roxb.) 206

5.15. Antiulcer activity of Limonia crenulata (Roxb.)

The antiulcer activity of different extracts of Limonia crenulata (Roxb.) against

Acacia mucilage induced ulcer models of rats. The effect of the alcohol, chloroform,

aqueous, benzene and acetone extracts (200 mg/kg) and Famotidine (20 mg/kg) on

volume of gastric juice, pH, total and free acidity and ulcer index were shown in Table

32 & Fig.20.

Table 32. Antiulcer activity of Limonia crenulata (Roxb.)

S.No Groups Volume of pH Total acidity Free acidity Ulcer index gastric juice 1. Control 2.8± 0.5 1.3 ± 0.06 98 ± 7.4 77 ± 6.5 35.4 ± 3.3 2. Alcoholic 0.62**+ 0.03 3.6* +0.23 38.5* + 2.8 33.9**+ 4.8 12.9** + 3.1 extract (200mg/kg) 3. Chloroform 0.79* + 0.04 3.9** ± 0.18 47.3* ± 2.8 33.4* ± 2.2 21.7* ± 1.3 extract(200g/kg) 4. Aqueous extract 0.66** ± 0.05 4.1** ± 0.13 30.3** ± 2.5 28.4** ± 0.7 18.8** ± 0.9 (200 mg/kg) 5. Benzene extract 0.62** ± 0.05 3.8** ± 0.11 39.5** ± 1.9 39.2** ± 0.9 15.8 ± 1.8 (200 mg/kg) 6. Acetone extract 0.72** ± 0.15 3.9** ± 0.21 38.5 ± 1.7 39.6** ± 0.82 17.8 ± 2.9 (200 mg/kg) 7. Famotidine 0.50** ± 0.02 4.10** ± 0.18 29.3** ± 1.0 16.2** ± 0.9 10.5** ± 0.8 (20 mg/kg) *P< 0.01 Vs Control, **P< 0.001Vs control by student ‘t’ test Values are SEM

Biological activities of Limonia crenulata (Roxb.) 207

Fig. 20. Antiulcer activity of Limonia crenulata (Roxb.)

The alcohol, chloroform, aqueous, benzene and acetone extracts of Limonia crenulata (Roxb.) produced significant effect when compared with the control. The alcohol extracts exhibited more antiulcer activity compared with other extracts (Plate 14

& 15).

Biological activities of Limonia crenulata (Roxb.) 208

Biological activities of Limonia crenulata (Roxb.) 209

Biological activities of Limonia crenulata (Roxb.) 210

5.16. Antidiarrhoeal activity of Limonia crenulata (Roxb.)

The extract at the doses of 200 mg/kg, produced a dose dependent decrease in the number of faecal matters passed by the animals in Epsom salt induced diarrhoeal model

(Table 33; Fig 21; Plate 16). Maximum percentage of inhibition (89.88%) was observed in alcoholic extract of L. crenulata (Roxb.), at significant (p<0.001) followed by chloroform extract (89.00%), acetone extract (86.99%), benzene extract (85.06%) and aqueous extract (51.85%).

The alcoholic leaf extracts of Limonia crenulata (Roxb.) at dose of 200 mg / kg showed significant (p<0.001) reduction in the number of faecal pellets produced during four hours when compared to that of untreated control rats; the activity was similar to that of Diphenoxylate (5 mg / kg), the standard antidiarrhoeal agent. The alcoholic leaf extracts of Limonia crenulata (Roxb.) showed the challenging results (89.88%) when compared with the standard drug Diphenoxylate (93.80%). Significant control over the diarrhoea was also observed in all the tested extracts especially in alcoholic extract. This result provided scientific basis for the traditional use of the Limonia crenulata (Roxb.) for diarrhoea.

Biological activities of Limonia crenulata (Roxb.) 211

Table 33. Antidiarrhoeal activity of Limonia crenulata (Roxb.)

Time (min) Total Total Total S.No Groups occurrence of number of number of weight of Percentage diarrhoea faeces wet faeces faeces (mg)

Control 1. 45.20±2.98 28.2±0.98 25.7±0.82 203.62±3.86 - (MgSo42gm/kg)

MgSo4 + Alcoholic 2. extract (200 96.20±2.86* 7.4±0.68* 2.92±0.48* 20.6±3.22* 89.88 mg/kg)

MgSo4 + Chloroform 3. 102.48±2.28* 10.32±0.88* 3.46±0.62* 22.32±2.02* 89.00 extract (200mg/kg)

MgSo4 + Water 4. extract 128.02±2.24* 22.20±0.82* 8.26±0.62* 98.04±3.82* 51.85 (200mg/kg)

MgSo4 + Acetone 5. extract (200 98.84±3.34* 18.02±0.82* 5.32±0.38* 26.48±2.08* 86.99 mg/kg)

MgSo4 + Benzene 6. extract (200 106.42±2.18* 16.82±0.86* 5.24±0.58* 30.42±2.18* 85.06 mg/kg)

MgSo4+ 7. Diphenoxylate 140.6±1.32* 5.82±047* 2.02±0.18* 12.62±1.82* 93.80 (5mg/kg)

N=6; P*<0.001 Vs control, Results are expressed as mean ± SEM

Biological activities of Limonia crenulata (Roxb.) 212

Fig. 21. Antidiarrhoeal activity of Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 213

Biological activities of Limonia crenulata (Roxb.) 214

5.17. Volatile oil from Limonia crenulata (Roxb.)

The volatile oil obtained by steam distillation of the fresh leaves of Limonia crenulata (Roxb.). 1.00ml. of volatile oil isolated from 1kg fresh leaves. It was screened by GC –MS analysis.

5.18. GC –MS analysis of volatile oil from Limonia crenulata (Roxb.)

The volatile oil of Limonia crenulata (Roxb.) was analysed by GC-MS and 19 components were identified in the oil (Table 34; Fig 22). The main components in the volatile oil of Limonia crenulata (Roxb.) were anisole (34.82%), amino compound

(28.29%) and eugenol compound (12.45%).

Biological activities of Limonia crenulata (Roxb.) 215

Table 34. Activity of Phyto Components identified in Volatile oil extract of Limonia crenulata (Roxb.) using GC - MS

study

Peak Molecular Compound No. RT Name of the compound MW Area Activity Formula Nature % 1. 2.47 á-Pinene C10H16 136 3.46 Anti-tumor, Analgesic, Antibacterial, Anti-inflammatory, Sedative,Fungicide, Monoterpene Hypocholesterolemic, Insecticide, Insectifuge Chemopreventive, Pesticide,Antiacne, Nematicide 2. 4.97 Methanimidamide, N,N-dimethyl-N'- C9H12N2 148 28.29 Amino Antimicrobial phenyl- compound 3. 5.34 Anisole, p-allyl- C10H12O 148 34.82 Anisole Antioxidant compound 4. 5.68 2-Cyclohexen-1-one, 2-methyl-5-(1- C10H14O 150 1.34 Ketone Anticancer methylethenyl)-, (S)- compound [Synonyms: (+)-Carvone] 5. 5.95 Benzene, 1-methoxy-4-(1-propenyl)- C10H12O 148 1.07 Anisole Antioxidant [Synonyms: Anisole, p-propenyl-] compound 6. 7.41 Benzene, 1,2-dimethoxy-4-(2-propenyl)- C11H14O2 178 12.45 Eugenol Insecticide compound [Synonyms: Eugenol methyl ether] compound 7. 7.59 Caryophyllene C15H24 204 7.46 Sesquiterpene Anti-tumor, Analgesic Antibacterial, Biological activities of Limonia crenulata (Roxb.) 216

Anti-inflammatory Sedative, Fungicide 8. 7.93 à-Caryophyllene C15H24 204 1.30 Sesquiterpene -do- [Synonyms: Humulene] 9. 8.28 à-Farnesene C15H24 204 0.47 Sesquiterpene -do-

10. 8.82 E-11,13-Tetradecadienal C14H24O 208 1.36 Aldehyde Antimicrobial

11. 9.27 3-Methoxycinnamaldehyde C10H10O2 162 1.01 Aldehyde Antimicrobial

12. 9.44 Caryophyllene oxide C15H24O 220 2.71 Terpene oxide Anti-tumor, Analgesic Antibacterial, Anti-inflammatory Sedative, Fungicide 13. 10.01 Tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9- C15H24O 220 0.64 Alcoholic Antimicrobial ol, 4,4-dimethyl- compound 14. 10.72 Tetradecanal C14H28O 212 1.70 Aldehyde Antimicrobial

15. 11.77 Hexadecanal C16H32O 240 0.08 Aldehyde Antimicrobial

16. 12.05 2-Pentadecanone, 6,10,14-trimethyl- C18H36O 268 0.10 Ketone No activity reported compound 17. 12.72 cis,cis,cis-7,10,13-Hexadecatrienal C16H26O 234 0.56 Aldehyde Antimicrobial

18. 12.99 Octadecanal C18H36O 268 0.08 Aldehyde Antimicrobial

19. 15.56 Phytol C20H40O 296 1.08 Diterpene Antimicrobial Anti-inflammatory Anticancer,Diuretic **Source: Dr.Duke’s Phytochemical and Ethnobotanical Databases

Biological activities of Limonia crenulata (Roxb.) 217

Fig . 22. GC –MS analysis of Volatile oil from Limonia crenulata (Roxb.)

Biological activities of Limonia crenulata (Roxb.) 218

43 100 57 O

82

50 29 69 96

109 18 124 137 152 166 180 194 208 222 250 0 10 30 50 70 90 110 130 150 170 190 210 230 250 270 (mainlib) Octadecanal

Name: Octadecanal Formula: C18H36O MW: 268 CAS#: 638-66-4 NIST#: 36188 ID#: 5279 DB: mainlib Other DBs: None Contributor: R.T.Holman,University of Minnesota 10 largest peaks: 43 999 | 41 860 | 57 800 | 55 740 | 82 630 | 69 430 | 29 420 | 96 400 | 83 400 | 68 390 | Synonyms: 1.Stearaldehyde 2.Octadecyl aldehyde 3.Stearyl aldehyde 4.n-Octadecanal

Biological activities of Limonia crenulata (Roxb.) 219

57 100 82 43 O

68 96 50 29

110 124 138 168 152 184 194 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 (mainlib) Tetradecanal

Name: Tetradecanal Formula: C14H28O MW: 212 CAS#: 124-25-4 NIST#: 235587 ID#: 20845 DB: mainlib Other DBs: None Contributor: Japan AIST/NIMC Database- Spectrum MS-IW-3684 10 largest peaks: 57 999 | 82 862 | 43 806 | 55 710 | 41 673 | 68 551 | 96 515 | 69 509 | 83 472 | 71 439 | Synonyms: 1.Myristaldehyde 2.Myristylaldehyde 3.Tetradecylaldehyde 4.n-Tetradecanal 5.Aldehyde C-14 6.Aldehyde C-14, myristic 7.C-14 Aldehyde, myristic 8.Myristic aldehyde 9.1-Tetradecanal 10.1-Tetradecyl aldehyde

Biological activities of Limonia crenulata (Roxb.) 220

148 100

50 77 121 91 105 133 51 63 39 O 89 15 27 55 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 (mainlib) Anisole, p-allyl-

Name: Anisole, p-allyl- Formula: C10H12O MW: 148 CAS#: 140-67-0 NIST#: 113167 ID#: 86935 DB: mainlib Other DBs: None Contributor: NIST Mass Spectrometry Data Center, 1990. 10 largest peaks: 148 999 | 147 599 | 77 438 | 121 432 | 117 418 | 91 305 | 105 300 | 115 274 | 78 267 | 133 250 | Synonyms: 1.p-Allylanisole 2.p-Methoxyallylbenzene 3.Chavicol methyl ether 4.Chavicol, O-methyl- 5.Esdragol 6.Esdragole 7.Esdragon 8.Estragol 9.Estragole 10.Isoanethole 11.Methyl chavicol 12.Tarragon 13.1-Allyl-4-methoxybenzene 14.3-(p-Methoxyphenyl)propene

Biological activities of Limonia crenulata (Roxb.) 221

82 100

50 54 39 93 108 O 41 79 91 27 51 58 67 135 150 115 122 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 (mainlib) 2-Cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-, (S)-

Name: 2-Cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-, (S)- Formula: C10H14O MW: 150 CAS#: 2244-16-8 NIST#: 114678 ID#: 38570 DB: mainlib Other DBs: None Contributor: NIST Mass Spectrometry Data Center, 1990. 10 largest peaks: 82 999 | 54 457 | 39 318 | 93 312 | 108 258 | 53 203 | 107 190 | 41 175 | 79 166 | 91 148 | Synonyms: 1.p-Mentha-6,8-dien-2-one, (S)-(+)- 2.(+)-Carvone 3.(S)-(+)-Carvone 4.(S)-Carvone 5.Carvone, (+)- 6.D-(+)-Carvone 7.D-Carvone 8.d-p-Mentha-6,8(9)-dien-2-one 9.d-1-Methyl-4-isopropenyl-6-cyclohexen-2-one 10.l-Carvone

Biological activities of Limonia crenulata (Roxb.) 222

162 100 O 131

50 91 119 O 65 108 77 89 103 51 147 41 53 67 87 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 (mainlib) 3-Methoxycinnamaldehyde

Name: 3-Methoxycinnamaldehyde Formula: C10H10O2 MW: 162 CAS#: 56578-36-0 NIST#: 71689 ID#: 94291 DB: mainlib Other DBs: None Contributor: G.S. KING, CHEM. PATHOL. DEP., QUEEN CHARLOTTE'S HOSP., LONDON, 10 largest peaks: 162 999 | 161 700 | 131 660 | 91 352 | 119 328 | 133 312 | 134 276 | 65 248 | 108 248 | 63 212 | Synonyms: 1.(2E)-3-(3-Methoxyphenyl)-2-propenal #

Biological activities of Limonia crenulata (Roxb.) 223

68 100 O 54 81

50 41 95 208 109 123 135 151 165 179 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 (mainlib) E-11,13-Tetradecadienal

Name: E-11,13-Tetradecadienal Formula: C14H24O MW: 208 CAS#: 111876-44-9 NIST#: 130768 ID#: 26323 DB: mainlib Other DBs: None Contributor: J. Klune, Insect Chem. Ecol. Lab., USDA, Beltsville, MD 20705 10 largest peaks: 68 999 | 67 742 | 54 739 | 81 643 | 82 491 | 41 403 | 55 401 | 95 365 | 208 309 | 69 252 | Synonyms: 1.(11E)-11,13-Tetradecadienal

Biological activities of Limonia crenulata (Roxb.) 224

79 100 67 41

55 93 50 O

108 98 121 135 147 162 172 234 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 (mainlib) cis,cis,cis-7,10,13-Hexadecatrienal

Name: cis,cis,cis-7,10,13-Hexadecatrienal Formula: C16H26O MW: 234 CAS#: 56797-43-4 NIST#: 293049 ID#: 36574 DB: mainlib Other DBs: None Contributor: Th.R.Kemp. 10 largest peaks: 79 999 | 67 878 | 41 743 | 55 518 | 93 500 | 95 476 | 80 448 | 81 410 | 91 351 | 43 292 | Synonyms: 1.(7Z,10Z,13Z)-7,10,13-Hexadecatrienal #

Biological activities of Limonia crenulata (Roxb.) 225

43 100 79

93

50 O 55 69 109 27 121 135 149 161 177 220 187 205 0 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 (mainlib) Caryophyllene oxide

Name: Caryophyllene oxide Formula: C15H24O MW: 220 CAS#: 1139-30-6 NIST#: 156329 ID#: 5414 DB: mainlib Other DBs: None Contributor: Chemical Concepts 10 largest peaks: 43 999 | 41 927 | 79 885 | 93 661 | 91 573 | 95 420 | 69 407 | 55 393 | 67 377 | 81 373 | Synonyms: 1.5-Oxatricyclo[8.2.0.0(4,6)-]dodecane,4,12,12-trimethyl-9-methylene-,[1R- (1R*,4R*,6R*,10S*)]- 2.5-Oxatricyclo(8.2.0.0(sup 4,6))dodecane, 4,12,12-trimethyl-9-methylene-, (1R,4R,6R,10S)- 3.Caryophylene oxide 4.Caryophyllene epoxide 5.(-)-á-Caryophyllene epoxide 6.á-Caryophyllene oxide 7.Epoxycaryophyllene 8.(-)-Epoxydihydrocaryophyllene 9.4,11,11-Trimethyl-8-methylene-5-oxatricyclo(8.2.0.0(4,6))dodecane 10.(-)-5-Oxatricyclo[8.2.0.0(4,6)]dodecane,4,12,12-trimethyl-9-methylene-,[1R- (1R*,4R*,6R*,10S*)]-

Biological activities of Limonia crenulata (Roxb.) 226

178 100

O

O

50

163 147 91 103 151 41 51 65 77 115 135 27 46 57 0 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 (mainlib) Benzene, 1,2-dimethoxy-4-(2-propenyl)-

Name: Benzene, 1,2-dimethoxy-4-(2-propenyl)- Formula: C11H14O2 MW: 178 CAS#: 93-15-2 NIST#: 107171 ID#: 102024 DB: mainlib Other DBs: None Contributor: N.W. Davies, Centr. Sci. Lab., Univ. Tasmania, Hobart, Australia 10 largest peaks: 178 999 | 163 283 | 147 238 | 103 141 | 91 139 | 107 131 | 179 122 | 151 102 | 41 85 | 77 77 | Synonyms: 1.Benzene, 4-allyl-1,2-dimethoxy- 2.Ent 21040 3.Eugenol methyl ether 4.Eugenyl methyl ether 5.Methyl eugenol ether 6.Methyleugenol 7.O-Methyleugenol 8.Veratrole methyl ether 9.1-(3,4-Dimethoxyphenyl)-2-propene 10.1-Allyl-3,4-dimethoxybenzene

Biological activities of Limonia crenulata (Roxb.) 227

44 100 77 N 148 N 42 106 50 51

15 133 30 39 120 18 57 63 91 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 (mainlib) Methanimidamide, N,N-dimethyl-N'-phenyl-

Name: Methanimidamide, N,N-dimethyl-N'-phenyl- Formula: C9H12N2 MW: 148 CAS#: 1783-25-1 NIST#: 135328 ID#: 13357 DB: mainlib Other DBs: None Contributor: NIST Mass Spectrometry Data Center, 1994 10 largest peaks: 44 999 | 77 909 | 148 686 | 106 519 | 42 497 | 51 470 | 147 453 | 104 373 | 133 306 | 15 276 | Synonyms: 1.Formamidine, N,N-dimethyl-N'-phenyl- 2.N,N-Dimethyl-N'-phenylformamidine 3.N'-Phenyl-N,N-dimethylformamidine 4.N,N-Dimethyl-N'-phenylimidoformamide #

Biological activities of Limonia crenulata (Roxb.) 228

5.19. Acute toxicity study of Limonia crenulata (Roxb.)

The results of LD50 study were analysed using Miller and Trainter method (Table

35). The ED50 and LD50 values are important for knowing the safety of a drug. The ratio between LD50 and ED50 represents the therapeutic index which is an index of safety of the drug. Greater the therapeutic index, safer is the drug. The alcoholic extract of Limonia crenulata (Roxb.) was non-toxic up to a dose of 2500 mg/kg (Table 36; Plate 17).

Table 35. Acute toxicity studies by Miller and Trainter method. Behavioral parameters – Irwin test on test sample

S.No Symptoms Observation

1 Death - 2 Convulsions - 3 Tremor (Shiver) -

4 Straub tail - 5 Sedation - 6 Excitation - 7 Jumping - 8 Abnormal gait (step) -

9 Motor in-coordination -

10 Altered muscle tone -

11 Akincsia - 12 Catalepsy - 13 Loss of traction (grip) -

14 Lass of balance - 15 Fore paw treading -

Biological activities of Limonia crenulata (Roxb.) 229

16 Writhing - 17 Stereotypy - 18 Head movements - 19 Scratching - 20 Altered respiration - 21 Aggression (Violent behavior) - 22 Altered fear - 23 Altered reactivity to touch - 24 Loss of righting reflex - 25 Loss of corneal reflex - 26 Analgesia - 27 Defecation/Diarrhoea - 28 Salivation Normal 29 Lacrimation Normal 30 Body temperature Normal 31 Myosis/Mydriasis Normal

Table 36. LD50 value of Limonia crenulata (Roxb.)

Test compounds LD50 value (mg/kg)

Leaf extract 2500

Biological activities of Limonia crenulata (Roxb.) 230

Biological activities of Limonia crenulata (Roxb.) 231

5.20. Herbal capsule of Limonia crenulata (Roxb.)

The weight variation test performed for the prepared herbal capsules passes the test as per the specifications of Indian Pharmacopoeia (Table 37; Plate 18).

Table 37. Result of weight variation test for individual capsules

S.No. Weight of the capsule % of Deviation Result

1. 0.44 0.0007 PASS

2. 0.43 0.0000 PASS

3. 0.45 0.0015 PASS

4. 0.43 0.0000 PASS

5. 0.41 -0.0015 PASS

6. 0.41 -0.0015 PASS

7. 0.43 0.0000 PASS

8. 0.42 -0.0008 PASS

9. 0.43 0.0000 PASS

10. 0.43 0.0000 PASS

11. 0.43 0.0000 PASS

12. 0.43 0.0000 PASS

13. 0.43 0.0000 PASS

14. 0.44 0.0007 PASS

15. 0.42 -0.0008 PASS

16. 0.42 -0.0008 PASS

17. 0.42 -0.0008 PASS

18. 0.45 0.0015 PASS

19. 0.44 0.0007 PASS

20. 0.44 0.0007 PASS The weight variation test performed for the prepared herbal capsules passes the test as per the specifications of I.P.

Biological activities of Limonia crenulata (Roxb.) 232

Weight of 20 filled capsules =8.61 g

Weight of one filled capsule =8.61 / 20= 0.4305 g

As per I.P. standards, deviation permitted for 0.4305 g (≈431 mg) is ±7.5 %

Range for ±7.5% =0. 399 g to 0.463 g

5.21. Herbal syrup of Limonia crenulata (Roxb.)

The herbal syrup of Limonia crenulata (Roxb.) was prepared according to the

Indian Pharmacopoeia. Viscosity of herbal syrup was evaluated (Table 38; Plate 18).

Table 38. Viscosity of syrup

S.No. Viscosity in centipoises (in cps)

1. 24 2. 24

3. 24 Average 24 cps Viscosity

Biological activities of Limonia crenulata (Roxb.) 233

Biological activities of Limonia crenulata (Roxb.) 234

6. DISCUSSION

6.1. Medicinal plants

Medicinal plants (MPs) continue to play a significant role in the people welfare as they have been for several millennia. Since the medieval time plants played an important role in the life of human as the major source of food, as well as maintenance and improvement of health by elimination of the disease causing microbes. The World Health

Organization (WHO) estimated that about 80 percent of the world's population still relies on plant based medicines for their primary health care (Khalil et al., 2007). Various medicinal plants have been used for years in daily life to treat diseases all over the world.

The relatively lower incidence of adverse reactions to plant preparations compared to modern conventional pharmaceuticals, coupled with their reduced cost, is encouraging both the consuming public and national health care institutions to consider plant medicines as an alternatives to synthetic drugs (Nair et al., 2005). Recently various modern procedures and techniques were used for the determination of biological activity of plant extract and bioassay techniques. (Ahmad et al., 2002; Zafer et al., 2002). The plants have also been used as source of medicine. Higher plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient times (Farombi, 2003). Over 50 percent of all modern clinical drugs are natural product origin and natural products play an important role in drug development programmes in pharmaceutical industry (Baker et al., 1995).

Biological activities of Limonia crenulata (Roxb.) 235

Many studies made tremendous contribution for the therapeutic usage of medicinal plants. Large number of plants belonging to different families has been studied for their therapeutic usages (Bowers, 1976; Coredell, 1981; Stufffness and Crodell, 1987,

Mukhtar et al., 2002).

Rutaceae is a large family comprising 160 genera and 1,650 species largely distributed in the tropical and subtropical parts of the world (Jones, 1995). Limonia acidissima L. syn. Feronia elephantum Correa is a multipurpose tree species belonging to

Rutaceae family. Different parts of this tree, fruits, seeds, and leaves, have been reported to possess many medicinal properties and are widely used in folk medicine. The fruits and leaves are prescribed for vomiting, dysentery, indigestion and slight bowel affections in children (Kirtikar and Basu, 1935).

Limonia crenulata (Roxb.) is an Indian medicinal plant. All parts of the tree are medicinally useful. Literature in Indian traditional medical systems like Ayureda, Siddha and Unani were prescribed this as an Indian folk medicine which has much potential information on its therapeutic uses. In Ayurveda Limonia crenulata (Roxb.) is used as a folk medicine for renitent fever, puerperal fever, lightening of skin, diarrhoea, ulcer, inflammation, skin irritation, dyspepsia, diabetes and many other diseases. With this background, the present study plant Limonia crenulata (Roxb.) was undertaken to analyse phytochemical, pharmacological, pharmacognostical and pharmaceutics studies.

Biological activities of Limonia crenulata (Roxb.) 236

Morphological and anatomical observations

Microscopic and macroscopic characters are distinct and showed various morphological and anatomical characters. As the species is an untouched specimen most of the data’s are newly identified and checked with the similar genus.

6.2. Qualitative phytochemical analysis

Knowledge of the chemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but such information may be the value in disclosing new sources of such economic materials as tannins, oils, gums, precursors for the synthesis of complex chemical substances, etc. In addition, the knowledge of the chemical constituents of plants would further be valuable in discovering the actual value of folkloric remedies (Farnsworth, 1966).

Recently Attarde et al., (2011) reported that preliminary phytochemical analysis of methanolic leaf extracts of Limonia acidissima L. contains good amount of polyphenolic compounds. Khyade and Vaikos (2009) exhibited that the phytochemical screening of Wrightia tinctoria is considered to be very effective because of phytochemicals such as lipid, saponins, alkaloids, phenol, steroid, flavonoid and some other chemical constituents.

In the present investigation revealed that the qualitative phytochemical analysis of hydroalcoholic extract of Limonia crenulata (Roxb.) has preliminary identification of

Biological activities of Limonia crenulata (Roxb.) 237

bioactive compounds such as alkaloids, carbohydrate, protein & aminoacids, glycosides, flavonoids, fixed oil and fats were present except phytosterol and saponins.

Similarly the phytochemical studies were investigated in leaf of Limonia acidissima L revealed that the presences of wide range of phytoconstituents like sterols, terpenoids, glycoside, flavonoids, polyphenols, coumarin, and carbohydrates (Parial et al., 2010).

6.3. Quantitative phytochemical analysis

Secondary metabolites are produced by plants which play certain biological and ecological roles towards combating other plants, animals, insects and man (Mann, 1987).

Among the secondary metabolites flavonoids are the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants (Jeremy,

2008). The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Preliminary research indicates that flavonoids may modify allergens, viruses, carcinogens, and so may be biological "response modifiers". In vitro studies of flavonoids have displayed antiallergic, anti-inflammatory, antimicrobial (Cushnie and Lamb, 2005) and anticancer activities (De Sousa et al., 2007). Flavonoids (both flavons and flavonols) are most commonly known for their antioxidant activity. Additionally, at high experimental

Biological activities of Limonia crenulata (Roxb.) 238

concentrations that would not exist in vivo, the antioxidant abilities of flavonoids in vitro are stronger than those of vitamin C and E (Manashi et al., 1999). Hence many of secondary metabolites have been shown to present interesting biological and pharmacological activities and are used as chemotherapeutic agents for centuries to treat a variety of diseases or serve as the starting point in the development of modern medicines (Verpoorte, 1998).

All human beings require a number of complex organic or inorganic compounds in diet to meet the need for their activities. Evidently, crude tannins were isolated from leaves of Naringi crenulata exhibited antihelmintic activity. It contains many phytochemical constituents (Ramalingam et al., 2010). When it compared with the present study the total amount of medicinal principles present in Limonia crenulata

(Roxb.), the active constituents are carbohydrates (7.80±2.3 mg/ 100gm), tannins (2.52 ±

0.3), phenolic compounds (13.20±0.1mg/ 100gm) , proteins (32.0 ± 1.5 µg/100gm), vitamin C (0.33 ± 0.2 µg/100gm) and vitamin E (0.45 ± 0.2 µg/100gm). Natural antioxidants such as vitamin C and vitamin E directly influence the biological activity.

Similarly methyl extract of Limonia acidissima L. contains rich amount of total polyphenolics (1.2 mg/gm), total flavonoids (0.7 mg/gm) and total flavonols

(0.85mg/gm) (Attarde et al., 2011). The important constituents of diet are carbohydrates, fats, proteins, vitamins, minerals and water (Indrayan et al., 2005).

Biological activities of Limonia crenulata (Roxb.) 239

The total amount of compounds present in Limonia crenulata (Roxb.) were glycosides (47.77%), flavonoids (43.18%) and tannins (41.81%),Thus from the data it is evident that the study plant could make useful in treating different ailments and having potential for providing useful drug for human use. This is because of the pharmacological activity of this plant is used to trace the particular compound. The strong presence of glycosides, flavonoids and tannins are responsible for the biological activities of this plant. They are known to show medicinal potential and physiological activities

(Sofowara, 1993).

6.4. Physico - chemical characters

However the present analytical studies of Limonia crenulata (Roxb.) were carried out with a view to evolve standard and to validate its ethnobotanical uses. The ash of any material is composed of their non-volatile inorganic components. Inorganic elements play an important role in physiological process involved in human health. Controlled incineration of crude drugs results in the ash residue consisting of an inorganic material.

This value varies within fairly wide limits and is therefore an important parameter for the purpose of evaluation of crude drugs. In certain drugs, the percentage variation of the weight of ash from sample is very small and any marked difference indicates a change in quantity. More direct contamination, by sand of earth, is immediately detected by the ash value. According to Mukherijee, (2002) the ash values can be determined by three different methods to measure the total ash, the acid insoluble ash and the water soluble ash. Crude fibre consists of the material other than ash which cannot be dissolved in

Biological activities of Limonia crenulata (Roxb.) 240

water and cannot be digested by boiling with sulphuric acid or with sodium hydroxide which represents the more important resistant part of the plant cells as well as some less resistant cell wall component like cellulose and pectin. This work was established that total ash value, water soluble ash, acid insoluble ash and crude fibre content of Limonia crenulata (Roxb.) were found to be 4.42% , 2.50% , 1.57 % and 2.87% respectively.

The earlier studies of Vermani et al., (2010) that the physico - chemical analysis of ash of some medicinal plants growing in Uttarakhand results were shown as the percentage ash value was highest in acid followed by alcohol and water. Recently in the same parameters were evaluated in Gymnema sylvestre. The total ash for aerial parts was found to be

8.22% of which, acid insoluble ash was 1.08% and water soluble ash was 3.39%

(Kalidass and Mohan, 2010).

However the ash analysis method determines the amount of active constituents in a given amount of medicinal plant material when extracted with solvents. It is employed to that material for which no chemical or biological assay method exists. As mentioned in different official books the determination of water soluble and alcohol soluble extractives, is used as a means of evaluating crude drugs which are not readily estimated by other means.

Hence the extraction of any plant with a particular solvent yields a solution containing different phytoconstituents. The composition of these phytoconstituents in that particular solvent depends upon the nature of the plant and solvent used. The use of

Biological activities of Limonia crenulata (Roxb.) 241

a single solvent can be the means of providing preliminary information on the quality of a particulars plant sample. For example, in a plant where the extraction procedure for the constituents commences with water as the solvent any subsequent aqueous extraction on the re-dried residue will give a very low yield a soluble matter. Thus the present study revealed that water and ethanol soluble extract of Limonia crenulata (Roxb.) were found to be 6.83% and 13.15% respectively. Similarly Kalidass and Mohan (2010) reported that the Gymnema sylvestre extraction values were found to be 18.21% and 20.19% for water and alcohol respectively.

6.5. Elemental analysis

This analysis was carried out to understand the importance of elements. Plants are the rich source of all the elements essential for human beings. There is a relationship between the element content of the plant and its nutritional status. Some elements are essential for growth, structure formation, reproduction or as components of biologically active molecules while others have some other beneficial effects (Newwall et al., 1996).

Qualitative or quantitative determination of mineral elements present in plants is important because the concentration and type of minerals present must often be stipulated on the label of a food. The quality of many foods depends on the concentration and type of minerals which they contains, also play a very significant role against a variety of degenerative diseases and processes, they may also prevent and reduce injury from environmental pollutants and enhance the ability to work and learn, some minerals are

Biological activities of Limonia crenulata (Roxb.) 242

essential to a healthy diet (e.g. calcium, phosphorus, potassium and sodium) where as some can be toxic (e.g. lead, mercury, cadmium and aluminium). It is clear that mineral nutrition is important to maintain good health and because of that determination of As,

Ca, Fe, Mg, Na, K, Zn, Ni, Co etc. have been added to Ayurvedic Pharmacopoeia of

India (Anonymous, 1999). The study plant also consist of essential mineral nutrients.

From ancient times, Swarna bhasma (gold ash) has been used in several clinical manifestations including loss of memory, defective eyesight, infertility, overall body weakness and incidence of early aging. It was supported with Mitra et al., (2002) who stated the qualitative analyses indicated that Swarna bhasma contains not only gold but also several microelements Fe, Al, Cu, Zn, Co, Mg, Ca, As, Pb etc.

Potassium is important as diuretic and it takes part in ionic balance of the human body and maintains tissue excitability. Potassium is the principal intracellular cation and also consider as a very important constituent of the extracellular fluids. Potassium ions are concerned with the transmission of electrical impulse in the nerve cells and in maintaining the fluid balance of the body.

Analysis were made to confirm the activity of elements with this background

Venkataraman and Gopal Krishnan (2002) reported maximum concentration of Ca, Fe and K in nine plants traditionally used for jaundice and concluded that high concentration of K in the medicinal plants could be related to the diuretic action of drugs

Biological activities of Limonia crenulata (Roxb.) 243

prepared from medicinal plants. Calcium imparts strength and rigidity to bones and teeth.

Calcium ions are also needed in neuromuscular transmission, in excitability of nerves for normal excitability of heart, in clotting of blood and promoting muscular contraction. It also acts as an activator of the enzymes phospholipase, arginine kinase, adenosine triphosphatase and adenyl kinase.

Manganese is essential for haemoglobin formation but excess is harmful. Zinc is an essential component of a number of enzymes present in animal tissue including alcohol dehydrogenase, alkaline phosphatase, carbonic anhydrase and procarboxypeptidase, is also essential for the normal growth and reproduction and helps in the process of tissue repair and wound healing. Zinc deficiency causes growth retardation and skin lesions (Chatterjee and Shinde, 1995).

Some elements are essential for all the biological activities especially few elements are having high beneficial effects. Which was encomposed by Kar et al. (1999) that the inorganic parts (containing K, Zn, Ca traces of Cr etc) of Tinospora cordifolia

(stem) showed more pronounced action of glucose-tolerance factor than their corresponding organic parts.

Nickel aids the synthesis of haemoglobin in the bone marrow. Perhaps Iron is the most well known in biological system. It performs a wide range of biological functions.

Many of these functions are connected with oxidation-reduction and processes by which

Biological activities of Limonia crenulata (Roxb.) 244

energy is conserved in the body. However it forms an integral part of cytochromes, haemoglobin, myoglobin, etalloflavo proteins and certain enzymes such as catalase and peroxidases. Thus, iron is absolutely essential for transport of oxygen to the tissue and for operation of oxidation systems within the tissue cells, without which life would cease within a few seconds. Iron deficiency causes anemia.

The present study revealed that the amount of calcium (78.05 mg/l) and potassium (140.30 mg/l) were higher in Limonia crenulata (Roxb.). The content of iron was 1.98 mg/l, manganese (0.20 mg/l), nickel (0.11 mg/l ) and molybdenum (0.22 mg/l) were found to be less in Limonia crenulata (Roxb.). Our study accordance with the Vermani et al.,

(2010) that the analysis of eight mineral elements of plant’s ash by AAS of Cassia fistula, Tinospora cordifolia, Quercus infectoria and Cedrela toona showed maximum concentration of potassium (K) in comparison to other elements. Butea monosperma showed maximum results with magnesium. Calcium is present in all plants in good quantity it was highest in B. monosperma and lowest in C. toona. Iron concentration was found maximum in Q. infectoria and minimum in C. toona. Phosphorus concentration was found to be highest in T. cordifolia and lowest in C. toona.

6.6. Microbial analysis

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Microbial contamination usually medicinal plants containing bacteria and molds are coming from soil and atmosphere. Analysis of the limits of Escherichia coli and molds clearly throws light towards the harvesting and production practices. The substance known as aflatoxin will produce serious side effects if consumed along with the crude drugs. The microbial data will relate to the product safety and quality inputs of microbiology are essential in maintaining the integrity of products whilst protecting consumers safety.

The microbial analysis of Limonia crenulata (Roxb.) revealed the presence of pathogenic microorganisms within WHO limit. The total heterotrophic bacterial count was 112 X 104 (Cells in sample/g). Salmonella sp., Shigella sp., and Enterobacter sp. were absent. Indeed the similar work was carried out by Shrivastava and Leelavathi

(2010) in Catunaregum spinosa Thunb. Reports were stated that the total bacterial count

(9 X 103), total fungal count (8 X 104), Escherichia coli (7 X 103) and Salmonella was absent.

6.7. High Power Thin Layer Chromatography (HPTLC) analysis

HPTLC is a reliable method for quantification of a nanogram level even when present in complexes formulation. HPTLC finger print analysis is used for rapid identity check, for monitoring purity of drugs, for detection of adulterants, for determining whether a material is derived from defined botanical species, also to know whether the constituents are clearly characterized (Sethi, 1996).

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The recent study revealed that Feronia limonia (Linn.) (Rutaceae) have gained traditional therapeutic importance owing to their high essential oil and coumarins content.

A simple, sensitive and accurate High Performance Thin Layer Chromatographic

(HPTLC) method has been developed for the estimation of marmesin in the methanolic extract of stem bark of Feronia limonia (Linn.). The calibration curve was linear in the concentration range of 20 – 100 ng spot–1. This method was validated for precision, repeatability and accuracy. This technique has been applied, for the first time, for the estimation of marmesin. Therefore it holds potential for detection, monitoring and quantification of marmesin in Feronia Limonia (Linn.) and its related formulation Jain et al., (2010).

Based on the results obtained from Limonia crenulata (Roxb.) in the HPTLC fingerprint, it has been shown that the alcoholic extract of contains eight compounds displayed at 5 µl, 10 µl and 10 compounds displayed at 15 µl.

6.8. Gas Chromatography-Mass Spectrometry (GC – MS) analysis

Gas Chromatography - Mass Spectroscopy (GC-MS) plays a key role in the analysis of unknown components of plant origin. Generally, the plant materials are highly complexes, which make GC-MS well suited for their analysis because of its high sensitivity and selectivity. GC - MS ionizes compounds and measures their mass numbers. It provides additional information on structure of these profiles.

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The over all evaluation of the compounds present in the plant extract were analysed by using GC – MS. Alcohol was used as a solvent for the separation of bioactive compounds present in the plant leaves. Totally 13 compounds were identified in Limonia crenulata (Roxb.). The results indicated the presence of benzene acetic acid, benzaldehyde, 4-hydroxy-, benzene, 1, 2 – dimethoxy – 4 - (2 - propenyl) -, n - hexadecanoic acid, estragole, 7H – Furo (3,2-g) (1) benzopyran-7-one, 4-methoxy-, phytol and 4H - pyran --4-one, 2,3 dihydro-3,5 dihydroxy -6methyl -. These compounds contains the following properties such as antibacterial, antifungal, antioxidant, antiulcer, antiviral, anticancer, anti-inflammatory, fungicide, pesticide, anti acne, larvicide, gastro protective, flavour, hemolytic, preservative and analgesic (Dr. Duke's Photochemical and

Ethobotanical database). Among the identified phytochemicals, n- Hexadecanoic acid have the property of antioxidant antimicrobial and larvicidal activities (Bodoprost and

Rosemeyer, 2007; Falodun, et al., 2009). The phenolic constituents of the extracts of

Mentha spicata namely phytol was reported for its antimicrobial and antiviral activities, strong antioxidant and antitumor action (Mckay and Blumberg, 2006).

6.9. High-Performance Liquid Chromatography (HPLC) analysis

HPLC, is a chromatographic technique that can separate a mixture of compounds and used to identify, quantify and purify the individual components of the mixture. It has been developed and validated for stability, linearity, accuracy and precision.

Biological activities of Limonia crenulata (Roxb.) 248

Previous works were supported that the presence of bioactive compounds such as

Gallic acid (GA), catechin (CA), rutin (RU), ellagic acid (EA) and quercetin (QU) are phenolic compounds. Structurally they have phenolic groups which serve as a source of readily available hydrogen atoms such that the subsequent radicals produced can be delocalized over the phenolic structure (Robards et al., 1999; Nikolic, 2006). The interest in these compounds is due to their pharmacological activity as radical scavengers (Azzi et al., 2004; Baydar et al., 2007). They have been proved to have potential preventive and therapeutic effects in many diseases, where the oxidative stress has been implicated, including cardiovascular diseases, cancer, neurodegenerative disorders and in aging (Wu et al., 2006; Duthie et al., 2000; Myhrstad et al., 2002, Sun and Chen, 1998).

Interestingly many authors were reported the phenolics are in food, cosmetic and pharmaceutical industries, as substitutes for synthetic antioxidants. These five phenolics widely distribute in the plant kingdom (Justesen and Knuthsen, 2001; Soong and Barlow

2004; Rizzo et al., 2006.).

Samee and Vorarat (2007) studied that gallic acid (GA), catechin (CA), rutin

(RU), ellagic acid (EA) and quercetin (QU) were analysed simultaneously by HPLC with

UV detection at 280 nm. Calibration curves were found to be linear with ranges of 2.62 -

21.00, 10.85 - 86.80, 10.00 - 80.00, 10.05 - 80.40 and 10.05 - 80.40 mg.ml-1 for GA, CA,

RU, EA and QU respectively. The contents of GA, CA, RU, EA and QU in the flower extracts of Michelia alba, Caesalpinia pulcherrima and Nelumbo nucifera were successfully determined at 5.0, 6.8, 9.2, 9.8 and 11.3 mins respectively with satisfactory reproducibility and recovery.

Biological activities of Limonia crenulata (Roxb.) 249

HPLC procedure provided excellent identification and quantification of four phenolic compounds presented in Limonia crenulata (Roxb.). This method was used to isolate the compounds such as Gallic acid (0.145 µg/ml), Caffeic acid (0.399 µg/ml),

Rutin (3.776 µg/ml) and Quercetin (0.416 µg/ml). The experimental results indicated that

Limonia crenulata (Roxb.) extracts were contained an especially high concentration of rutin (3.776µg/ml). Since the phenolic compounds have been of interest of health benefits, the present analytical study could be a potential application to identify and quantify the phenolic compounds in plant extracts.

Similarly, a new indole alkaloid, crenulatine (1), along with twenty known compounds such as four alkaloids, four coumarins, two flavonones, three tetranortriterpenoids, one triterpenoid, three steroids, two lignans and two aromatic compounds were isolated from Limonia crenulata (Roxb.) (Niu et al., (2001).

The observations of Chatterjee et al. (1980) insisted a new coumarin, 7 – phenylacetoxycoumarin, has been isolated from Limonia crenulata (Roxb.) along with luvangetin, xanthotoxin, umbelliferone and limonin. This is the first report a coumarin phenol ester from a natural source. Michael (2003) reported that the isolation, structure determination synthesis and biological activity of quinoline, quinazoline and acridone alkaloids from plant, microbial and animal sources. The integriquinolone compound isolated from Limonia crenulata (Roxb.).

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6.10. Fourier Transform - Infra Red spectroscopy (FT- IR) analysis

IR Spectroscopy is a valuable tool in the determination of the structure of organic molecules and in establishing the identity of the sample. IR is used for the identification of functional groups like OH, NH CO and C=C, CH and NO2 in the molecules. The absence of such characteristic bands is definite proof of absence of such groups.

The FT –IR and UV spectrum were used to identify the functional group of the active components based on the Peak value in the region of infrared radiation. The ethanol extract of Gymnema kollimalayanum was passed into the FT - IR, the functional groups of the components were separated based on its peak ratio and the same was passed into UV spectroscopy for the electron transition of compounds. The results of FT - IR analysis was confirmed the presence of the carboxylic acid and Alkenes-CH2; CH3

Aromatic stretching which shows major peaks at 1019.87 and 2922.33cm (Natarajan et al., 2011) .

IR spectrum analysis of gallic acid, caffeic acid, rutin and quercetin isolated from Limonia crenulata (Roxb.) were recorded using Perkin-Elmer Paragon 580 B FT-IR spectrophotometer. The IR results indicated the following functional groups were present in gallic acid, such as polymeric O-H stretch, C=O stretch, phenol or tertiary alcohol, O-H bend, C-O stretch and CC-C C-C stretch. The functional group of caffeic acid were polymeric O-H stretch, C=O stretch, phenol or tertiary alcohol, O-H bend, C-O

Biological activities of Limonia crenulata (Roxb.) 251

stretch and C-C stretch. In rutin organic molecules such as polymeric O-H stretch, C=O stretch, phenol or tertiary alcohol, O-H bend, C-O stretch and CC-C C-C stretch. The spectrum showed polymeric O-H stretch, C-H group in aromatic ring, C=O stretch, phenol or tertiary alcohol, O-H bend, C=C stretch and C-C stretch were present in quercetin.

6.11. Ultra Violet (UV) – Visible spectroscopy analysis

The technique of UV spectroscopy is one of the most frequently method employed in plant drug analysis. It involves the measurement of the amount of ultraviolet (190-380 nm) radiation absorbed by a substance in solution. Ultraviolet - visible absorption spectra provide a useful source of supporting evidence in the elucidation of structures of organic compounds. Moreover, selective absorption also serves as an identifying fingerprint for a particular structure in many cases.

The value of UV - visible spectra in identifying unknown constituents is indicative of the compounds. The absorbance of the extract of Limonia crenulata (Roxb.) recorded using lambda 35 model UV-Visible spectrophotometer. The UV results were supported that the functional group of isolated compounds such as gallic acid, caffeic acid, rutin and quercetin.

6.12. Antimicrobial activity

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There has been an increasing incidence of multiple resistance in human pathogenic microorganisms in recent years, largely due to the indiscriminate use of commercial antimicrobial drugs commonly employed in the treatment of diseases. The number of resistant strains of microbial pathogens is growing, ever since penicillin resistance and multi resistance Pneumococci were reported (Meurer-Grimes et al., 1996;

Elloff, 1998). This situation, coupled with the under desirable side effects of certain antibiotics and the emergence of previously uncommon infections are a serious problem

(Marchese and Shito, 2001; Poole, 2001). This has forced scientists to search for new antimicrobial substance from various sources like the medicinal plants.

The screening of plant extracts and plant products for antimicrobial activity has shown that higher plants represent a potential source of novel antibiotic protypes

(Meurer-Grimes et al., 1996; Rabe and Van Staden, 1997; Afolayan, 2003). The presence of antibacterial and antifungal substances in the higher plants is well established (Fridous et al., 1990; Didry et al., 1998; Javed and Ali, 2002; Belboukhari and Cheriti, 2005).

The present investigation involving Limonia crenulata (Roxb.) also lends credence to the above observations. The leaf extracts showed significant antimicrobial activity against gram - positive bacteria as well as fungi. The different plant extracts differ significantly in their activity against tested microorganisms. These differences may be attributed the fact that the occurrence of different antimicrobial compounds with different solvents. The benzene leaf extract of Limonia crenulata (Roxb.) exhibited

Biological activities of Limonia crenulata (Roxb.) 253

maximum antimicrobial activity against Salmonella typhi (22 mm) and least activity against Sheigella shigae (14 mm) at 250 µg/ml. Sheigella shigae (25 mm) and Candida albicans (25 mm) were strongly inhibited by chloroform leaf extract and minimum activity was recorded in Pseudomonas aeruginosa (17 mm). The significant antimicrobial activity was observed against Pseudomonas aeruginosa (26 mm) and less activity was showed against Aspergillus niger (16 mm) in ethanol leaf extract. In the acetone leaf extract showed very promising antimicrobial activity against Pseudomonas aeruginosa

(28 mm) and no activity was observed against Aspergillus niger. In aqueous extract showed minimum and maximum antimicrobial activity against Aspergillus niger (12 mm) and Klebsiella aerogenes (20 mm) respectively. So that the present study concluded that

Limonia crenulata (Roxb.) had effective antimicrobial activity against pathogenic bacteria and fungi species. GC-MS analyses revealed the occurrence of more number of antimicrobial compounds in the leaf extracts.

Suresh et al., (2009) evaluated that methanolic extracts of Aegle marmelos (L.) have great potential as antimicrobial agent against both gram - positive and gram - negative organisms such as Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Salmonella typhi and Staphylococcus aureus.

6.13. Antioxidant activity

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Antioxidant means “Against oxidation” antioxidants work to protect lipids from peroxidation by free radicals. Antioxidants are effective because, they are willing to give up their own electrons to free radicals.

When a free radical gains the electron from an antioxidant it no longer needs to attack the cell and the chain reaction of oxidation is broken. After donating an electron an antioxidant becomes a free radical by definition. Antioxidants in this state are not harmful because they have the ability to accommodate the change in electrons without becoming reactive. The human body has an elaborate antioxidant defense system. Antioxidants are manufactured within the body and can also be extracted from the food humans eat such as fruits, vegetables, seeds, nuts, meats, and oil. There are two lines of antioxidant defense within the cell. The first line, found in the fat soluble cellular membrane consists of vitamin E, β – carotene, and Co – enzyme Q of these, vitamin E is considered the most potent chain breaking antioxidant within the membrane of the cell. Inside the cell water soluble antioxidant scavengers are present (Kaczmarski et al., 1999).

The antioxidants may be enzymatic or non – enzymatic, super oxide dismutase, glutathione peroxidases, catalase and peroxidases are some examples which come under enzymatically potential antioxidants. In the non-enzymatic category some of the known and documented antioxidants are vitamin C, vitamin E, vitamin A, carotenoids, uricacid, ubiquinone and synthetic compounds like melatonin, Dihydro eplandrosterone (DHEA)

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etc. (Naik, 2003). The antioxidant activity has been described for several triterpenes among other related compounds (Andrikopoulos et al., 2003).

The ethyl acetate extract of Pereskia grandifolia (Haw.) (Cactaceae), played a considerable role in antioxidant activity by DPPH method. This is the first report on the antioxidant activities on leaves of P. grandifolia (Sim et al., 2010).

However, hydro ethanolic extracts of Agle marmelos and Trigonella foenum was found to be more active in DPPH scavenging ability in comparison to the methanol and aqueous extract which contains higher levels of phenols. (Vijaya et al., 2002). Recently

Attarde et al., (2011) reported that petroleum ether, chloroform and methanolic extract of leaves of Limonia acidissima L. have potential antioxidant ability using DPPH method.

The present study to evaluate the in-vitro antioxidant activity of ethanol, methanol and aqueous leaf extracts of Limonia crenulata (Roxb.) by DPPH method. The DPPH radical scavenging activity of ethanol extracts showed a challenging result (83.69%), methanol and aqueous extracts were found to be 77.89% and 70.59% respectively.

Similar work was done by Sathishkumar et al., (2010) that the ethanol and acetone extracts of Polyalthia longifolia leaves showed significant DPPH radical scavenging activity were found to be 87.92% and 92.84% respectively.

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6.14. Anti-inflammatory activity

There is an increasing interest in the study of anti - inflammation. Anti - inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, and extravasations of fluid, cell immigration, tissue breakdown and repair (Vane and Bolting, 1995). It is also known that anti-inflammatory effects can be elicited by a variety of chemical agents and that there is little correlation between their pharmacological activity and chemical structure (Sertie et al., 1990).

Inflammation is a complex in the pathogenesis of inflammatory diseases (Conner and Grisham, 1996). Also, it is well known that inflammation sites present a high concentration of free radicals and oxidants, which play an important role in different inflammation process. Therefore antioxidant compounds can be helpful to avoid this process (Salvemini et al., 1996).

The previous investigation suggested that carrageenan induced paw edema is the most widely used primary test for the screening of new anti-inflammatory agents (Di

Rosa et al., 1971; Winter et al., 1962). The mechanism of action of carrageenan induced paw oedema is described as biphasic. The development of edema in the paw of the rat after the injection of carageenan is due to release of histamine, serotonin and prostaglandin like substances. Hence the initial phase seen at the 1st hour is attributed to the release of histamine and serotonin. The second phase is related to the release of prostaglandins like substance in 3rd hour. (Brooks and Day, 1991; Vinegar et al., 1969).

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Winter et al., (1992) reported that the second phase of edema is sensitive to drugs like hydrocortisone, phenylbutazone and indomethacin. Perhaps this associated with the complexity of the inflammatory process, makes the use of different experimental models essential when conducting pharmacological trials.

In Indian system of medicine, certain medicinal plants are claimed to provide relief of pain and inflammation. The claimed therapeutic reputation has to be verified in a scientific manner. Plants which belong to Rutaceae family are rich in flavonoids and bio flavonoids are known for their anti-inflammatory and antioxidant activities. Hence, in this study anti-inflammatory properties of Limonia crenulata (Roxb.) leaf extracts were tested using a number of experimental rat models representing different phases of inflammation.

The carrageenan paw inflammation has been accepted as a useful diagnostic tool for investigation of systemic anti-inflammatory activity of drugs. The percentage inhibition of paw volume of alcoholic extract of Limonia crenulata (Roxb.) was found to be significant (50.95%) as that of the standard drug Indomethacin treated group which was

63.80 %. The chloroform, aqueous, benzene and acetone extracts of Limonia crenulata

(Roxb.) were found to be 40.44%, 27.33%, 29.05%, and 37.25% of paw volume inhibition respectively. The results of exhibited that the study plant has reasonable anti- inflammatory activity.

Presence of terpenes, glycosides and sterols in plants has been found to exert active anti-inflammatory effects (Chawla et al., 1987). The more pronounced activity of

Biological activities of Limonia crenulata (Roxb.) 258

these plants may be due to the presence of certain polar constituents such as flavonoids and glycosides (Katith et al., 1996). The results of phytochemical analysis of Limonia crenulata (Roxb.) revealed the presence of flavonoids, alkaloids, glycoside and sterols.

Thus it has been taken into consideration which has made tremendous contribution of anti

- inflammatory activity.

Similar work was carried out by Rao, (2003) insisted the significant anti- inflammatory activity at a dose level of 200 mg/kg body weight by ethanolic extract of A. marmelos fruits. It contains several biological activities such as anti-inflammatory, analgesic and antibacterial effect (Ponnapalli, 2005). Trichodesma indicum, belonging to

Boraginaceae showed anti-inflammatory activity with different models (Periyanayagam et al.,2006).

The therapeutic use of Cassia fistula bark was treated for inflammatory conditions by practitioners of Ayurvedic system of medicine. The bark extracts of Cassia fistula possess significant anti-inflammatory effect in the acute and chronic anti-inflammatory model of inflammation in rats. The presence of alkaloid and flavonoids in Cassia fistula may be responsible for the anti-inflammatory and antioxidant effects (Yadava and

Verma, 2003; Gupta et al., 1989).

6.15. Antiulcer activity

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Gastric ulceration has been attributed to various causes such as stress, hormones, drugs, alcohols, smoking and ingestion of certain foods (McGuigan, 1991). Plant extract are some of the most attractive sources of new drugs and have shown promising results for the treatment of gastric ulcer . Plant extracts are more of the most attractive sources of new drugs and have shown promising results for the treatment of gastric ulcers. Several medicinal plants and herbs have been used to treat gastric intestinal disorders, including gastric ulcers (Adami et al., 1964; Best et al., 1984; Ageel et al., 1987; Goel et al., 1990;

Disi et al., 1998; Alkofahi and Atta, 1999).

A significant reduction in gastric ulcer in experimental animals was reported by many workers with ethanolic extracts of different plants such as Hemidesmus indicus

(Anoop and Jagadeesan, 2003), Triumfetta rhomboidea (Pradhan et al., 2003), Mimusops elengi (Shah et al., 2003), Carallum arabica (Zakaria et al., 2003), Solanum variable

(Antonio et al., 2004), Allophylus serratus (Dharmani et al., 2005), Terminalia pallida

(Gupta et al., 2005), Commiphora opobalsamum (Howiriny et al., 2005) and Kaempferia parviflora (Rujjanawate et al., 2005).

The present investigation was carried out to evaluate the antiulcer activity of different extracts of Limonia crenulata (Roxb.) against Acacia mucilage induced ulcer models of rats. The effect of the alcoholic, chloroform, aqueous, acetone and benzene extracts of Limonia crenulata (Roxb.) (200 mg/kg) and Famotidine (20 mg/kg) on gastric secretary volume, pH, total acidity, free acidity and gastric ulcers were evaluated.

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The result of present investigation revealed that the antiulcer activity of the leaf extracts of Limonia crenulata (Roxb.) has moderate effect when compared with the control. The plant extracts reduced gastric secretary volume, acidity and ulceration in pylorus legated rats. It can be used to discover bioactive natural products that may be serve as feeds for the development new pharmaceutical products.

Many investigations have suggested that the antiulcer effect is by the decrease in the aggressive factors like pepsin and protein and an increase in the resistance actor like pH, hexose hexoseamine, fucose and acid protection against experimental ulcers may be due to the effect of protogandins in the parietal cells (Takeuchi and Nobubara, 1985;

Lauritsen and Rask Madsen, 1986; Sumangala et al., (1998), as prostaglandins enhance the mucosal resistance, perhaps by increase the secretion of mucous and bicarbonates

(Hogan et al., 1994), strengthening the mucosal barrier, decreasing the gastric motility

(Szabo, 1984), increasing the release of endogenous mediators (Olsen et al., 1984) scavenging the free radicals (Szabo, 1984) decreasing the release of endogenous amines

(Whittle et al., 1985), stimulation of cellular growth and repair (Goal et al., 1985; Eugene and Jacobson, 1990). The antiulcer activity of plant extracts was not only related to local neutralization of gastric content, but also that it was effective after absorption of extracts indicating a systemic effect. This effect is also indicative of antihistaminic activity

(Andrade et al., 2007).

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Ahmed et al. (2010) observed the significant antiulcer activity of

Cassia auriculata leaf extract against pylorous ligation induced gastric ulcer. The methanolic leaf extract of Cassia auriculata at dose of 300 mg/kg p.o. markedly decrease the incidence of ulcers in pyloric ligated rats. In pyloric ligated rats, there was an increase in the gastric volume, free acidity, total acidity and ulcerative index as compared to the control group. The methanolic leaf extract of Cassia auriculata at dose of, 300 mg/kg showed significant reduction in the above parameters which was comparable to the standard drug famotidine (10 mg/kg). Cassia auriculata extract showed protection index

79.4%, whereas standard drug famotidine showed protection index 90.7%.

Pasquale et al. (1995) have reported that plant drugs containing saponin, terpenoids or amino acid have antiulcer activity. Presence of beta sitosterol in the samples may also enhance the antiulcer activity (Malini and Vanithakumari, 1989). It is well known fact that many flavonoids display antisecretary and cytoprotective properties in different experimental models of gastric ulcer (Zayachkivska et al., 2005.) Indeed flavonoids and tannins are one of the most important phytochemical compounds with antiulcer and gastroprotective activities (Alarcon de la Lastra et al., 1994; Borrelli and

Izzo, 2000; Gonzalez et al., 2000). In the present study plant also contains flavonoids and tannins.

6.16. Antidiarrhoeal activity

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Diarrhoea is considered as a consequence of altered motility and fluid accumulation. It has long been recognized as one of the most important health problems in the developing countries (Snyder and Merson, 1982). Diarrhoea is the second leading cause of death among children under five globally. Nearly one in five child deaths about

1.5 million each year is due to diarrhoea. The objective of WHO/UNICEF report was to focus attention on the prevention and management of diarrhoeal diseases as central to improving child survival.Secretory diarrhoea is the most dangerous symptoms of gastro intestinal problems and is associated with excessive defecation and stool outputs, the stools being of abnormally loose consistency (Park, 2000).

Diarrhoea results from an imbalance between the absorptive and secretory mechanisms in the intestinal tract accompanied by hurry resulting in an excess loss of fluid in the faeces. In some diarrhoea the secretory component predominates while other diarrhoea is characterized by hypermotility (Chitme et al., 2004). The magnesium sulphate (MgSO4) induced diarrhoea is acknowledged to be by osmotic properties and cholecystolinin production. It has been reported to induce diarrhoea by increasing the volume of intestinal content through prevention of reabsorption of water. It has also been reported that it promotes the liberation of cholecystokinin from the duodenal mucosa, which increases the secretion and motility of small intestine and thereby prevents the reabsorption of sodium chloride and water. Numerous reports have been available regarding magnesium sulphate (MgSO4) induced diarrhoea (Mujumdar et al., 2000 and

2001; Bajod, 2001; Galvez et al., 1993; Zavala et al., 1998).

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The antidiarrhoeal activity of the extract may also be due to denature proteins forming protein tannates which make intestinal mucosa more resistant and reduce secretion. In addition to other previous mechanisms to explain the diarrhoeal effect, recently nitric oxide has been claimed to contribute to the diarrhoea (Mascolo et al.,

1994). Antidiarrhoeal effect may be attributed, at least in part, to nitric oxide scavenging activity of the extract.

The aim of the therapy in diarrhoea is to treat the patient promptly to reduce the loss of electrolytes and water. There are several potent antidiarrhoeal drugs in the modern system of medicine however on prolonged use they do have some adverse effect (Galvez,

1993). For this reason uses of herbal medicines have increased as they are devoid of any adverse side effects. A range of medicinal plants with antidiarrhoeal properties is widely used by traditional healers. However, the effectiveness of many of these antidiarrhoeal traditional medicines has not been scientifically evaluated.

An integrated approach was made to prepare medication after the thorough observation of antidiarrhoeal activity in epsom salt (MgSO4) induced diarrhoea. The antidiarrhoeal activity of alcohol, chloroform, aqueous, acetone and benzene extracts of

Limonia crenulata (Roxb.) were found to be 89.88%, 89.00%, 51.85%, 86.99% and 85

.06% respectively. From the above results it can be concluded that the alcoholic leaf extracts of Limonia crenulata (Roxb.) showed the challenging result (89.88%) when compared with the standard drug Diphenoxylate (93.80%). The different parameters

Biological activities of Limonia crenulata (Roxb.) 264

enables in drawing out important conclusions regarding the antidiarrhoeal activity. The use of Limonia crenulata (Roxb.) leaf extracts as antidiarrhoeal drug as justified in folk medicine indicates that the people in rural area, where they are more aware of decoction procedures of plants than the electrolyte therapy or conventional antidiarrhoeal drug can very well use it. Similar results have been obtained in extract of Aegle marmelos which cause dose dependent decrease in the numbers of faecal matter in caster oil induced diarrhoea (Amresh et al., 2003).

It is indeed worthy that the flavonoids have been reported to inhibit intestinal motility and secretion (Discarlo et al., 1993), they may presumably exert antidiarrhoeal action (Rao et al., 1997). Earlier reports suggested that antidiarrhoeal properties of medicinal plants might be attributed to tannins, alkaloids, saponins, flavonoids, sterols and reducing sugars (Longanga et al., 2000). In the study plant also contains alkaloids, flavonoids, tannins, and vitamin C. These constituents may mediate the antidiarrhoeal property of the extract. Flavonoids, present in the plant extract were reported to inhibit release of autacoids and prostaglandins, thereby may inhibit motility and secretion (Veiga et al., 2001).

6.17. Volatile oil

Many investigations were suggested that essential oils are natural mixtures of terpenes or terpenoids, most of which are obtained from aromatic medicinal plants. The chemical composition of essential oil differs in each species or subspecies and is

Biological activities of Limonia crenulata (Roxb.) 265

characteristic for the species in question (Tunalier et al., 2002; Sefidkon et al., 2004;

Adams, 1995).

Syamasundar et al. (2010) found that the Limonnia acidissima L. leaf collected from Karanthai malai from Tamil Nadu State gave the highest yield of essential oil

(0.5%). All the other four samples from the central part of the Western Ghats and nearby plains, which are in the Karnataka State region, gave from 0.3 to 0.2% as the oil yield. In the present investigation revealed that the volume of volatile oil isolated from leaves of

Limonia crenulata (Roxb.) was 1.00 ml/ kg.

6.18. GC –MS analysis of volatile oil

One of the most popular methods of studying essential oil composition is Gas

Chromatography - Mass Spectrometry (GC-MS), which allows the identification of the specific natural compounds found in an essential oil by comparing their relative retention times indicates and their mass spectra (Yayli et al., 2005).

The composition of essential oil has been investigated by GC - MS analysis from different parts of various plants such as aerial parts of Sinapis alba (Sefidkon et al.,

2002b), Thymus persicus (Sefidkon et al., 2002a), Lantana xenica (Juliani et al., 2003), leaves of Zanthoxylum procerum (Vila et al., 2002), Agastache scrophulariaefolia

(Lognay et al., 2002), roots of Acorus calamus (Ozcan et al., 2002), rhizome of Rhodiola rosea (Rohloff, 2002) and seeds of Myristica fragrns (Simpson and Jackson, 2002).

Biological activities of Limonia crenulata (Roxb.) 266

Nor Azah et al. (2010) reported that the leaf oil of Zanthoxyllum acanthopodium was characterized by the presence of 1, 8-cineole (34.6%) and limonene (30.8%) and the other main constituents were -terpineol (6.2%), α - pinene (5.3%) and citronellal (3.9%).

Limonene (58.5%) was the most abundant compound in the leaf oil of Tetractomia tetranda and other chemical components such as α - pinene (5.8%), caryophyllene

(3.9%), terpinolene (3.3%) and safrole (2.5%).

The present study revealed the major chemical constituents of volatile oil of

Limonia crenulata (Roxb.) were anisole, p – allyl- (34.82%), methanimidamide, N,N – dimethyl – N’ phenyl (28.29%) and eugenol methyl ether (12.45%) . The other important minor constituents are α-pinene, caryophyllene oxide and caryophyllene.

The similar work was evaluated by Syamasundar et al. (2010) that the Limonnia acidissima L. leaf collected from Karanthai malai from Tamil Nadu State showed that methyl chavicol is the major constituent (91.2%) and the other important minor constituents were α-pinene, myrcene, limonene, linalool, anisaldehyde, and p- methoxycinnamaldehyde. Interestingly, the leaves collected from Bangalore and Mysore showed a low percentage of oil, and β -pinene as the major compound. All the other four samples from the central part of the Western Ghats and nearby plains, which are in the

Karnataka State region β –pinene as the major compound, ranging from 63.9–76.9%.

The other major compounds that contribute to these samples are β –pinene (4.7–6.1%),

Biological activities of Limonia crenulata (Roxb.) 267

sabinene (4.4–6.0%), and limonene (4.5–6.2%). The other important minor constituents are camphene, myrcene, E- ocimene, linalool, -terpineol, methyl chavicol, - caryophyllene, -cadinene, and glubulol.

Similarly there are numerous reports emphasis that the presence of active phytoconstituents such as methyl chavicol (72.7%) and anethole (26.2%) (Bhati and

Deshpande, 1949) trans-anethole (4.7%), methyl eugenol (3.6%), and anisaldehyde

(4.4%), methyl chavicol (68.3%) (Garg, 2003), trans-anethole (10.9%), methyl chavicol

(27.2%), and thymol (24.4%) (Ahmad et al., 1989) in volatile oil of Limonnia acidissima

L.

6.19. Acute toxicity studies

The definition "toxic" is ultimately a matter of viewpoint. Traditionally, herbs and herbal products have been considered to be non toxic and have been used by the general public and traditional medicine practitioner’s world wide to treat range of ailments. The active ingredients of plant extracts are chemicals that are similar to those in purified medications and they have the same potential to cause serious side effects. While the literature documents severe toxicity of herbs and herbal products it has not been recognized. Two kinds of side effects have been reported for herbal medicines. The first considered to be intrinsic to herbal drugs themselves, is mainly related to predictable

Biological activities of Limonia crenulata (Roxb.) 268

toxicity due to toxic constituents of the herbal ingredients and over dosage and second is allergy.

For several reasons it would be difficult to establish absolute safety standards for herbal preparations based solely on epidemiological studies. First, the types of studies would be costly, second, there is little published data in countries where the major use of medicinal plant occurs and thus general standards based on limited numbers of reports would have limited meaning. Third, the exact identification of the products implicated in side effects claimed for medicinal plants is usually lacking. In spite of these inadequacies there are a number of general comments that can be made with regard to avoiding potential serious side effects from herbal medicines.

Perhaps the major problem with regard to the safety of herbal medicines is related to the manufacturing practice, including contamination, substitution, incorrect preparation and dosage, intentional addition of unnatural toxic substances, interaction involving synthetic prescriptions, drugs and herbal medicines either intentional or unintentional mislabeling and presence mislabeling and presence of natural toxic contaminants. Many ordinary foods contain constituents that could be regarded as poisonous. Alpha gladin produced by gluten in wheat, oats and rye, the cyanogenetic glycosides in many fruit skin and seeds, thiocyanates of the Brassica vegetables and lections of many pulses including soya and red kidney bean are such examples. Cyanogenetic glycosides present in the kernel of many fruits can undergo gastric hydrolysis, resulting in the release of hydrogen

Biological activities of Limonia crenulata (Roxb.) 269

cyanide, viscotoxin which are constituents of mistletoe are both cytotoxic and cardiotoxic.

In these context herbs can be broadly classified in to three major categories

The food herbs: Medicines such as pepper, mint, ginger, garlic, lemon, betel leaves etc., are gentle in action have low toxicity and are unlikely to cause any adverse response. They can be consumed in substantial quantities over long period of time without any acute or chronic toxicity. However they may bring about allergic reactions in certain individuals.

The Medicinal herbs: These are not daily "tonics" and need to be used with greater knowledge for specific conditions (with a medical diagnosis) and usually for a limited period. They have a greater potential for adverse reaction and in some cases drug interaction. They include comfrey, ephedrine, gingko, ginseng, kava kava, senna etc.

The poisonous herbs: They have a strong potential for either acute or chronic toxicity and should only be prescribed by trained clinicians who understand their toxicology and appropriate use. Aconite, Belladonna, Datura, Digitalis, and Male fern are some examples (Ahmad and Aquil, 2006).

Before going for the preparation of any medication obviously the standardization is very important. To check the toxicity level of any plant the ED50 and LD50 values are

Biological activities of Limonia crenulata (Roxb.) 270

important for knowing the safety of a drug. The ratio between LD50 and ED50 represents the therapeutic index which is an index of safety of the drug. Greater the therapeutic index, safer is the drug.

The acute oral toxicity was carried out to determine the acute toxicity of single oral administration of the extract, in a dose of 2000 mg/kg. as per OECD, revised draft guidelines 423, received from Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment,

Government of India (OECD, 2001).

In the present study investigation was made to check weather the plant has any strong potential for either acute or chronic toxicity .With that motive Limonia crenulata

(Roxb.) underwent with the following strategies such as death, convulsion, tremor

(shiver), straub tail, sedation, excitation, jumping, abnormal gait (step), motor in co ordination, altered muscle tone, akincsia, catalepsy, loss of traction (grip), loss of balance, writhing, fore paw treading, stereotypy, head movement, scratching, altered respiration, aggression(violent behaviour), altered fear, altered reactivity to touch, loss of righting reflex, loss of corneal reflex, analgesia, defecation/diarrhoea, salivation, lacrimation, body temperature and myosis / mydriasis were analysed. After administration of the extract, the animals were observed individually for 4 hrs and thereafter 14 days to check mortality and their behavioral pattern. Result shows that there is no significant respond for toxicity. The extracts were found to be safe at the tested

Biological activities of Limonia crenulata (Roxb.) 271

dose level of 2500 mg/kg body weight indicating the high margin of safety of these extracts. Thus we conclude the extract of Limonia crenulata (Roxb.) leaves have a high margin of drug safety.

6.20. Herbal capsule

According to United Nations Development project report in 1994 the annual value of medicinal plants derived from developing countries is about 32 billion. Theoretically there is the possibility of discovering 328 new modern drugs lying hidden in nearly

3,25,000 species found in tropical rainforest. There are 47 major modern plant based drugs on the world market and predicted 328 more potential drugs have an estimated value 147 billion. India is ranked among the major exporters of medicinal plants and vegetative sap extracts (Atal and Kapur, 1982).

Musthaba et al. (2010) evaluated greater global interest in non synthetic, natural medicines derived from plant sources due to better tolerance and minimum adverse drug reactions as compared to synthetic medicines. Herbal products are also commonly used by the patients with certain chronic medical conditions, including breast cancer, liver disease, human immunodeficiency, asthma and rheumatological disorders. WHO estimates that about three quarters of the world's population currently uses herbs and other forms of traditional medicines for the treatment of various diseases. The herbs are formulated in different modern dosage forms, such as Tablets, Capsules, Topical cream,

Gel, Ointment and even some novel drug delivery forms, like extended release, sustained

Biological activities of Limonia crenulata (Roxb.) 272

release, and micro encapsules dosage forms. Patenting of herbal formulations has increased over the past few years and scientific evidence of therapeutic activity has been reported by performing various in vitro and in vivo experiments.

The only drawback in the promotion of these herbal medicines is the lack of standardization. Also in some areas confusion in nomenclature and controversial botanical identification exist. Hence development of quality control and standardization of plant drugs with adoption of good manufacturing practices and validation of claims of therapeutic efficacy are the challenges in the years ahead if the revival of global interest obvious today in these drugs is to be strengthened in future.

Due to the continuous increase of medical costs many countries provide support to encourage scientific research alternative therapies. Furthermore, the putative efficacy of medicinal herbs relies on empirical or anecdotal data and tradition of use, which frequently cannot satisfy the requirements of evidence based medicine. Thus, the steps back by the analysis of traditional herbal medicine and new drug development from herbs are started recently with high effort.

Few study had been made on Aegle marmelos was considered as one of the effective medicine for the treatment of non-specific diarrhoea. Therefore, its efficacy for the treatment of dysentery was tested in 25 patients, all the patients were treated with powdered unripe fruits of A. marmelos 5gms thrice daily for 21 days (Singh et al., 2000).

Biological activities of Limonia crenulata (Roxb.) 273

Drug from A. marmelos plays a vital role in diabetes in pregnant women and nursing mother (Bombarde and Bombarde, 1995). A. marmelos used as insertion of foreign body in vagina for reduction of vault is common practice in rural India. It also helps in stopping foul smell discharge from vagina (Karea et al., 1998).

In view of the diverse medicinal applications of Limonia crenulata (Roxb.) the present investigation dealt with the preparation and evaluation of capsule. Thus Limonia crenulata (Roxb.) have great potential use as phytomedicine in terms of pharmacognostical, phytochemical and pharmacological aspects. The present study plant having strong biological activities and biochemical compounds which may be responsible for arresting the diarrhoea. The pharmacological studies showed outstanding results when compared with the standard drug. There is a need to discover bioactive natural product that serves as a drug for the development of new pharmaceuticals. Development of phytomedicine is relatively inexpensive and less time consuming moreover it is suitable to our economic conditions. Thus the present prepared capsule would be highly beneficial for all range of people without any side effect.

6.21. Herbal syrup

India can emerge as the major country and play the lead role in production of standardized, therapeutically effective ayurvedic formulation. India needs to explore the medicinally important plants. This can be achieved only if the herbal products are

Biological activities of Limonia crenulata (Roxb.) 274

evaluated and analysed using sophisticated modern techniques of standardization such as

UV-visible, TLC, HPLC, HPTLC, GC-MS, spectrofluorimetric and other methods.

For the purpose of research work on standardization of herbal formulations and neutraceuticals a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of utmost importance.

The subject of herbal drug standardization is massively wide and deep. There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function. The herbal syrup of Limonia crenulata (Roxb.) has great potential to heal the diarrhoea. Our investigation is optimistic for the development of safe drug which has great therapeutic potential to control diarrhoea.

Biological activities of Limonia crenulata (Roxb.) 275

7. SUMMARY AND CONCLUSION

Plants are the basic source of knowledge of modern medicine. The relatively lower incidence of adverse reactions to plant preparations, compared to modern conventional pharmaceuticals, coupled with their reduced cost is encouraging both the consuming public and national health care institutions to consider plant medicines as alternative to synthetic drugs. Nowadays herbal drugs are prescribed widely even when their biologically active compounds are unknown because of their effectiveness and minimal side effect in clinical experience large numbers of plants belonging to different families have been studied for their therapeutic properties.

Our investigation aimed to scientifically explore the important medicinal uses of study plant. Nowadays there is an increase in research interest to identify new medications from plant parts. These criteria promoted us to formulate a new phytomedicine from the study plant with strict scientific protocol.

Limonia crenulata (Roxb.) belongs to Rutaceae have many medicinal properties, which was not studied for their pharmacological activities and hence the present investigation encompasses Pharmacognostical, Pharmacological and Pharmaceutical studies on leaf extract of Limonia crenulata (Roxb.).

Biological activities of Limonia crenulata (Roxb.) 276

v Qualitative phytochemical analysis confirms the presence of carbohydrate,

alkaloids, protein, amino acids, glycosides, flavonoids, fixed oil and fats except

saponin and phytosterol in hydroalchoholic extract.

v Quantitative phytochemical analysis revealed that the plant has rich amount of

glycosides (47.7739%), flavonoids (43.1895%) and tannins (41.8186%).

v Percentage of total ash, water insoluble ash and acid insoluble ash values were

4.42, 2.50 & 1.57 respectively.

v The elemental analysis of ash showed the maximum concentration of potassium

(140.30) and calcium (78.05) were found compared with other elements.

v The total heterotrophic bacterial count was 112 X 104 (Cells in Sample/g).

Salmonella sp., Shigella sp., and Enterobacter sp. were absent. It revealed that the

presence of pathogenic microorganisms in Limonia crenulata (Roxb.) within

WHO limit.

v HPTLC results showed the plant extracts contains maximum 10 compounds. Peak

at Rf value of 0.37 and 0.76 is found to have greater area such as (11421.7 &

3512.0) among the observed peaks. This area was directly proportional to quantity

of the compound present in the plant extract.

Biological activities of Limonia crenulata (Roxb.) 277

v GC - MS study on the ethanolic extract of the plant revealed that the following

active compounds viz., terpenes (Phytol, Caryophyllene, Caryophyllene oxide),

phenolic compounds (Eugenol, 2-Methoxy-4-vinyl Phenol), flavonoid fraction (

4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-), alkaloid ( Pyridine,

1,2,3,6-tetrahydro-1-methyl-4-phenyl-), antibacterial compounds (Glycerin,

Estragole, Benzene acetic acid, Benzaldehyde-4-hydroxy) and fatty acids

(Palmitic acid , Dodecanoic acid).

v HPLC study on the ethanolic extract of the plant revealed that the presence of four

phenolic compounds such as Gallic acid, Caffeic acid, Rutin and Quercetin.

Segregation of flavonoid fractions in HPLC by fractional collector and detect the

functional groups using FT – IR spectroscopy and UV-Visible spectroscopy

analysis.

v The benzene leaf extract of Limonia crenulata (Roxb.) exhibited maximum

antimicrobial activity against Salmonella typhi (22 mm). Sheigella shigae (25

mm) and Candida albicans (25 mm) were strongly inhibited by chloroform leaf

extract. The significant antimicrobial activity was observed against

Pseudomonas aeruginosa (26 mm) in ethanol leaf extract. In the acetone leaf

extract showed very promising antimicrobial activity against Pseudomonas

aeruginosa (28 mm). Aqueous extract showed maximum antimicrobial activity

against Klebsiella aerogenes (20 mm) at 250 µg/ml.

Biological activities of Limonia crenulata (Roxb.) 278

v Antioxidant activity of the plant extract revealed that the inhibition by 83.69% in

the DPPH method. Control ascorbic acid shows the antioxidant activity inhibition

of 66%. v Anti-inflammatory activity of leaf extracts of Limonia crenulata (Roxb.) revealed

that maximum in ethanol extracts (50.95%) followed by chloroform (40.44%),

acetone (37.25%), benzene (29.05%) and aqueous extract (27.33%) when

compared to standard drug Indomethacin (63.80%) against carragenan induced

paw edema in albino rats. This has moderate effect when compared with the

standard drug.

v Antiulcer studies of albino rats with the plant extracts showed positive results of

antiulcer activity. It showed moderate results when compared with the standard

drug of Famotidine. Ethanolic extract showed an ulcer index of 12.9 against

control of 35.4. However the ulcer index of other extracts was chloroform (21.7),

aqueous extract (18.8), acetone (17.8), benzene (15.8) and standard drug of

Famotidine (10.5). Limonia crenulata (Roxb.) have antiulcer activity by reducing

the volume of gastric juice, total acidity, free acidity, ulcer lesion and ulcer index

and by rising pH.

v The administration of epsom salt induced Diarrhoea with in albino mice is

eliminated by giving the ethanolic extract of the plant and brought down to 88.9%

followed by chloroform extract 89%. The standard drug administration of

Biological activities of Limonia crenulata (Roxb.) 279

Diphenoxylate brought down the diarrhoea to the tune of 93.8%. When it

compared with standard drug it showed challenging result thus it have great

potential to use as phytomedicine. So this plant can be used to discover bioactive

natural products that may serve as feeds for the development of new

pharmaceuticals.

v Volatile oil isolated from the fresh leaves was 1.00 ml/ kg which is used as the

aromatic substrate in the syrup preparation.

v In GC – MS analysis showed the volatile oil contains maximum 19 compounds.

The major chemical constituents are p – allyl- (34.82%), methanimidamide, N,N –

dimethyl – N’ phenyl (28.29%) and eugenol methyl ether (12.45%).

v Acute toxicity results (LD50 value) of plant extract of Limonia crenulata (Roxb.)

was 2500 mg/kg.

v Herbal capsule and herbal syrup were also prepared to arrest the diarrhoea. This

present development of new therapeutic agent capsule and syrup have maximum

pharmacological efficacy. The prepared phytomedicine for diarrhoea has high

therapeutic usage.

Biological activities of Limonia crenulata (Roxb.) 280

The overall investigations can be concluded that the Indian System of Medicine and Folk medicines on the therapeutic activities of Limonia crenulata (Roxb.) have been scientifically validated.

Distinguished characteristic features were observed in morphological studies, anatomical studies. From the results of our study plant its clear that the qualitative analysis, quantitative analysis, physico – chemical analysis, elemental analysis and microbial analysis were helped to get meaningful conclusion.

HPTLC analysis, GC –MS analysis, GC – MS analysis of volatile oil, HPLC analysis, FT- IR analysis and UV – visible spectrum analysis were showed accurate results of biological compounds it possess. Further fractionated compound activities flash more light in the pharmacological efficacy. Flavonoid fractions such as Gallic acid,

Caffeic acid, Rutin and Quercetin are having potential biological activity. Thus the present preparations of bioactive natural product recommended for therapeutic uses.

Pharmacological studies such as antimicrobial activity, antioxidant activity, anti- inflammatory, antiulcer activity, antidiarrhoeal activity, extraction of volatile oil, acute toxicity study, preparation of herbal capsule and syrup were undertaken on the leaf extract of Limonia crenulata (Roxb.) established their efficacy.

Biological activities of Limonia crenulata (Roxb.) 281

Besides the above findings, it would be more appropriate to enhance further research on clinical application for improving the plant based drug industry and the development of new drugs of herbal origin. The further clinical research would be continued in my Post Doctoral Work.

The present research plant possesse different bioactive compounds and its further activity guided fractionations would flash more lights to the new up coming research scholars. It paves a way for the emerging biological studies. If the research may attracts the global interest obviously these capsule and syrup are to be strengthened in future with pattern of this phytomedicine.

Biological activities of Limonia crenulata (Roxb.) 282

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