Tylophora indica: Phytochemical, Biotechnological and Pharmacological Approach

Table of Contents

TABLES AND FIGURES

PREFACE

BRIEF BIO-SKETCH OF PROF. ABHAY CHOWDHARY

BRIEF BIO-SKETCH OF DR PRATIBHA CHATURVEDI

ACKNOWLEDGEMENT

Introduction

1. About Experimental Plant

2. Biochemical studies
2.1 Phytochemical variation in Tylophora indica (Asclepeadaceae) leaves collected from different regions

3. Pharmacological Studies
3.1 Anti-inflammatory activity

4. Biotechnological studies
4.1Tissue culture of Tylophora indica
4.2 Study of organogenesis in Tylophora indica
4.3 Molecular characterization of Tylophora indica regenerated plants in vitro by RAPD and ISSR analysis
4.4 Pharmacological potential of Tylophora indica (antamul) callus

5. Secondary metabolite production from Tylophora indica tissue culture
5.1 Bio regulation of Tylophorin in Tylophora indica callus
5.2 Optimization for Enhancement of Tylophorin in Tylophora indica (Asclepeadaceae) Callus
5.3 Bio enhancement of Kaempferol in Tylophora indica (Asclepediaceae) by Static and Suspension culture
5.4 Bio regulation of Stigmasterol in Tylophora indica (Asclepeadaceae) in vitro tissue culture
5.5 Enhancement of production of Kaempferol from Tylophora indica by culture immobilization

6.  Microbiological studies
6.1 Identification of Kaempferol in endophytic fungi extracts of Tylophora indica (Asclepeadaceae) and its Biological assays
6.2 Evaluation of anti-bacterial potential of chromatographic fraction of Tylophora indica

References

Dedicated

To

My parents

TABLES AND FIGURES

Table 2.1.1 Depicts the Tylophorin and Stigmasterol content (%) of Tylophora indica leaves collected from different regions

Table 3.1.1 The anti- inflammatory %activity of Tylophorin from Tylophora indica leaves and comparison with DFS a standard compound

Table 4.1.1 Effect of various compounds on Growth of Tylophora Table 4.1.2 General appearance, growth, color and consistency of static cultures of Tylophora indica

Table 4.2.1 Showing the effect of different hormone on bud formation

Table 4.2.2 Showing the effect of cytokinin on shoot formation

Table 4.3.1 PCR reaction mixtures for RAPD and ISSR analysis

Table 4.3.2 RAPD and ISSR PCR conditions

Table 4.3.3 Depicts the RAPD analysis of Tylophora indica regenerated plants using different Primers

Table 4.3.4 Depicts the ISSR analysis of Tylophora indica regenerated plants using different Primers

Table 4.4.1 CC50of Callus (Aqueous Extract)of Tylophora indica

Table 4.4.2 Showing the antioxidant activity of Tylophora indica callus

Table 5.1.1 Showing the  Tylophorin content  increased  by feeding  the  callus  by  different concentration of phenylalanine of Tylophora indica

Table 5.1.2 Showing the effect of Zenk media on production of Tylophorin content in Tylophora indica

Table 5.1.3 Showing the effect of different concentration  of BAP on Tylophorin production

Table 5.2.1 Showing the effect of different compound on Tylophorin production in Tylophora indica callus

Table 5.3.1 Showing the effect  of  different compound on Tylophorin production in callus of Tylophora indica

Table 5.4.1 HPTLC analysis parameters for Stigmasterol analysis.

Table 5.4.2 Stigmasterol analysis of 2,4,6 week old callus of Tylophora indica grown in Zenk Media

Table 5.4.3 Showing the effect of different sugars on Stigmasterol production

Table 5.5.1 Enhancement of Kaempferol by immobilize culture in Tylophora indica

Table 6.1.1 Depicts Kaempferol content obtained from endophytic fungi and the anti bacterial assay of endophytic fungi isolated fromTylophora indicastem and leaf by Plate method at 500µg/ml .300 µg/ml and 200 µg/ml did not shown any inhibition.

Table 6.2.1 Showing the Bacterial growth inhibition of Tylophorin rich fraction  showing the growth inhibition of bacteria

Figure 2.1.1 Showing the HPTLC analysis of Tylophorin from methanol : ethanol (8;2) leaf extract and Tylophorin STD (A-Finger printing-Chromatogram of leaf C-Std Tylophorin at 254nm

Figure 2.1.2 Showing the HPTLC analysis of ethanolic extract of Tylophora indica leaves of different area with std Stigmasterol(Chromatogram A-Leaves ethanolic extract ,B- Std),C- HPTLC Fingerprinting at 254nm

Figure 2.1.3 Depicts the Tylophorin and Stigmasterol content (%) of leaves of Tylophora indica collected f r o m different regions

Figure 3.1.1 Standard of Tylophorin (254nm),B-Fingerprinting of High Performance Liquid Chromatography of methanol: ethanol( 8:2) extract of Tylophora indica leaves,C-HPTLC Chromatogram of methanol:  ethanol(  8:2)  extract  of Tylophora indica leaves  ,D-Standard  E-Infra  Red spectral studies of isolated Tylophorin with standard

Figure 3.1.2 The graphical Presentation of anti-inflammatory activity of Tylophorin as compared to standard compound DFS

Figure 3.2.1 Graph showing the cytotoxicity of Leaves(methanolic extract) of Tylophora indica: 30 mg/ml

Figure 3.2.2 Showing the anti-influenza activity of methanolic leaf extractof Tylophora indica by Hemagglutination assay

Figure 3.2.3 Photograph Showing the HPTLC fingerprinting of Tylophora indica A for alkaloid at 254 nm, B for flavonoids at 480 nm

Figure 4.1.1 Developmental stages of Callus (A-D) initiated from leaf explants in Tylophora indica

Figure 4.2.1 Showing the organogenesis in Tylophora indica callus (A-B)

Figure 4.3.1 The DNA fingerprinting of 1(a,b,c,d,e,f)for RAPD and 2(a, b, c, d, e) for ISSR analysis .Fig 1(a) RKAT-1, 1(b): RKAT-2 , 1(c):RKAT 3, 1(d):RKAT 4primer 1(e):RKAT 5 primer. Well 1:100bp ladder. Well 2-Mumbai. Well 3 -Anand. Well  4: Sample from Jaipur.  Well 5-10:Samples from regenerated micro shoots Fig 1f):RKAT  6 Fig 1(g): RKAT-7 primer. Well 1:100bp ladder. Well 2:Mumbai. Well 3:Sample Fig.2(a) Primer A-98;2(b)HB-09 :well 1:marker100bp , sample  1:control  sample from Mumbai,Sample 2:sample from Anand, sample 3:sample from Jaipur. Sample 4-9(well 5-10):regenerated  micro shoots Fig.2(c)Primer HB-10;1(d) Primer HB-11;1(e) Primer HB-13:well 1:marker 100bp , sample 1:control sample from Mumbai,Sample 2:sample from Anand, sample 3:sample from Jaipur. Sample4-9(well 5-10):regenerated micro shoots

Figure 4.4.1 CC50 of  Callus (Aqueous Extract) of Tylophora indica :

Figure 4.4.2 CC50 of Callus (Methanolic Extract) of Tylophora indica:

Figure 4.4.3 Showing the antioxidant activity of Tylophora indica callus

Figure 5.1.1 Showing the Tylophorin content increased by feeding the callus by different concentration of phenylalanine of Tylophora indica mean value ±S.E of three replicate

Figure.5.1.2 Showing the  effect of  Zenk mediaon production of Tylophorin content in Tylophora indica

Figure 5.1.3 Showing the effect of different concentration of BAP on content  of Tylophorin alkaloid in Tylophora indica tissue culture

Figure 5.1.4 Showing the HPTLC analysis at 254 nm of leaves(A) with standard reference compound of Tylophorin (B)

Figure.5.2.1 Showing the HPTLC analysis of methanolic leaf extract (A) and standard compound (B) of Tylophorin

Figure.5.2.2 Showing the graphical presentation (A and B ) of HPTLC analysis difference in a period of one month of Tylophorin standard at 430 nm .The peak area of both the sample depicts the reduction in Tylophorin accumulation

Figure 5.2.3 Effect of different compounds on Tylophorin production

Figure 5.3.1 Showing the biosynthetic pathway of Kaempferol

Figure.5.4.1 HPTLC analysis A (2 week old callus), B (4 weeks old callus), C (6week old callus) grown in Zenk media incorporated with different compounds for Stigmasterol biosynthetic regulation.

Figure 5.4.2 Effect of different compounds on biosynthetic regulation ofStigmasterol in Tylophora indica callus

Figure 5.4.3 Showing the HPTLC fingerprinting of synergistic effect of growth hormone onStigmasterol biosynthesis in Tylophora indica callus(I-Two wks old,II-Four wks old, III- Three wks old

Figure 5.5.1 A- Depicts the method  of Calcium alaginate beads formation;B-Beads in culture

Figure 5.5.2 Depicts the High Performance Thin Layer Chromatographic analysis of  induced samples with standard compound of Kaempferol in immobilized culture of Tylophora indica at 254nm

Figure 5.5.3 Enhancement of Kaempferol by immobilized culture of Tylophora indca

Figure 6.1.1 Isolation of Kaempferol from endophytic fungi of Tylophora indica (a MTT assay , b- Fusarium chlamydosporum grown with red pigment isolated from stem of T.indica, c- HPTLC analysis Fingerprinting of methanolic and  aqueous extracts  of Fusarium chlamydosporum for Kaempferol estimation with standard compound d-HPTLC Chromatogram of methanolic extract, e HPTLC Chromatogram of aqueous extract of Fusarium chlamydosporum, f- with standard Kaempferol

Figure 6.2.1 Showing the Co -TLC of  third fraction of column chromatography at 254 nm

Figure 6.2 2 Showing the Column chromatography of methanolic fraction of Tylophora indica

PREFACE

Present book pronounces the classification, taxonomic description, medicinal use, habitat, distribution of Tylophora indica,an important indigenous plant use for many deseases like asthama, inflammation, anticanceous, immunomodulatory.This contain the efficient protocols of indirect organogenesis and various strategies of secondary metabolites enhancement (Tylophorin, Stigmasterol and Kaempferol) in vitro static, suspension and immobilize culture of Tylophora indica.In addition to this, the book also covers the aspects related to the study of antioxidant activity, cytotoxicity ,antiinfluenza and anti-inflammatory activity of Tylophora indica.Apart from this, the present book includes the determination of Kaempferol in Fusarium clamydosporum,a endophytic fungi identified and isolated from Tylophora indica stem for the first time. The molecular characterization of regenerated micro shoots of Tylophora indica was carried out by using the RAPD and ISSR analysis

The Book divided in to separate topics and subtopics, which covers the various aspects like Biochemistry, Biotechnology, Pharmacological, Molecular biology and Microbiology

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BRIEF BIO-SKETCH OF PROF. ABHAY CHOWDHARY

Dr. Abhay Chowdhary (Born on 5th March 1955) is the Director of Prestigious Haffkine Institute for Training, Research and Testing, Mumbai, India. He was also the Director of AIDS Research and Control (ARCON) center, Mumbai. He is board certified in Microbiology and Virology from India. MBBS 1975, MD Micro 1980, DHA 1990, DM Virology 1993, FIMSA 2006.Dr Chowdhary has an experience of over 31 years as a graduate and post-graduate teacher & examiner for University of Mumbai, Maharashtra University of Health Sciences, Rajiv Gandhi Karnataka UHS, NTR Andhra UHS, Dr MGR TN UHS, Sri Ramchandra University, Goa University and various other Indian universities as well as Kathmandu University, Nepal. He also serves as an advisor and consultant in the field of Laboratory Medicine, HIV/AIDS and Healthcare safety. He has been invited as a guest faculty for the First International Conference of American Society of Microbiology (ASM) in Beijing, China on ‘Emerging Technologies in Diagnosis of Infectious Diseases and Detection of Pathogenic Microbes’ held in April 2008.Prof Chowdhary was an expert team member for Assessment of National Reference Laboratories (NRLs) under National AIDS Control Organization, Government of India along with Expert Teams from CDC, USA and WHO in July 2008,in addition to that ,he serves on editorial boards and is reviewer for several peer-reviewed journals in India. His expertise and special interests include Medical Virology, Clinical Microbiology, Parasitology, Immunology and Infectious Diseases. He is the recipient of Fogarty fellowship for Training at Johns Hopkins University, USA and the Fellowship by International Medical Sciences Academy. Dr Abhay Chowdhary is a Certified Expert Technical Assessor for NABL. (National Accreditation Board for Testing & Calibration Laboratories).Dr Abhay Chowdhary was the Secretary(2007-2009) and President(2009-2010) of Indian Association of Medical Microbiologists (IAMM) and now he is the Secretary of Infectious Diseases Society of India (IDSI). Recently he has been conferred the “Dr H I Jhala Award” for outstanding contribution in field of Medical Microbiology by Indian Association of Medical Microbiologists 2012.He has published above 80 research papers in peer reviewed journals and 4 books of National and international repute

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BRIEF BIO-SKETCH OF DR PRATIBHA CHATURVEDI

Dr Pratibha Chaturvedi (Born on 28 March 1965) is Ph d from Rajasthan University in the year 1991.She has work experience of over 10 years in the field of Plant Tissue culture and secondary metabolites production of medicinal plants. She has to her credit two books on Medicinal plants, more than 24 Research papers published in various research journals of national and international repute and has also presented Papers in various Seminars. Her work has been awarded by two reputed organizations

A women scientist awardee (Department of Science and Technology, Ministry of Science and Technology, New delhi) has main interest in Plant Biotechnology, Natural  Products,  Analytical  plant  Biochemistry,  Proteomics,  In  vitro enhancement of Secondary metabolites, Plant Physiology etc. A life member of Indian Science congress, Dr Chaturvedi is recognized teacher of Mumbai University and Tibdewal University, Rajasthan for Ph d degree. She is also a member of editorial board in an international journal

ACKNOWLEDGEMENT

I take this opportunity to express my sincere gratitude to my research supervisor Prof. Dr. Abhay Chowdhary  (Director,Haffkine  Institute,Mumbai)for their  benevolent supervision  of  my research work. Prof.Chowdhary have been a source of constant encouragement and inspiration, their profound knowledge, prolific guidance and constructive criticism throughout the course of my research work, have contributed tremendously in shaping the whole work into viable and successful endeavor. My thanks are due to Prof. Pushpa Khanna my Phd guide, Dr. O.P.Sharma, and Dr. H.C. Chaturvedi (Sr. Scientist NBRI, Luknow) for their constant encouragement. Thanks are due to Dr Sarala Mennon, J.J. hospital , Byculla for identification of endophyte isolated from experimental plant

My gratitude is also due to Shri Dinesh Malekar C.E.O. Haffkine Institute, Dr. Shashikant Vaidya Assistant Director, Haffkine Institute, A word of appreciation is due to my husband Mr.Anil Chaturvedi for his constant co-operation and sincere help without which this work was not possible .Pillars of inspiration and strength are my parents Shri D.G. Chaturvedi and late Mrs. Krishna Chaturvedi, my father in law Mr. R.N. Chaturvedi, mother in law Mrs. Kasturi Chaturvedi and the all family members including my kids Ankit and Vedika who have taken keen interest in the progress of my research. I also take this opportunity to thanks to Shri Sandeepan Mukherjee, Dr Ritwik Dahake,Dr Saroj Bapna and my all friends of Haffkine Institute who have always encouraged me in my academic pursuit and taking care of me during the period of this work. Last but not the least; thanks are due to the Department of Science & Technology, Government of India, and New Delhi for granting funds under the Women Scientist Scholarship Scheme

Pratibha Chaturvedi

Introduction

Tylophora indica (family Asclepeadaceae) is experimental plant that has been used in the present study .The leaves and roots of Tylophora indica have emetic, cathartic, laxative, expectorant, diaphoretic and purgative properties. It has also been used for the treatment of allergies, cold, dysentery, hay fever and arthritis. It has reputation as an alternative and as a blood purifier, often used in rheumatism and syphilitic rheumatism. Root or leaf powder is used in diarrhea, dysentery and intermittent fever. Dried leaves are emetic diaphoretic and expectorant. It is regarded as one of the best indigenous substitute for ipecacuanha. It is traditionally used as a folk remedy in certain regions of India for the treatment of bronchial asthma, inflammation, bronchitis, allergies, rheumatism and dermatitis. It also seems to be a good remedy in traditional medicine as anti-psoriasis, seborrhea, anaphylactic and leucopenia. The experimental plant of Tylophora indica was procured from Kelkar farm house, Mulund,Mumbai ,India (The latitude coordinate of Mumbai is 18 degree 58' 30 North and longitude coordinate is 72 degree 49' 32" East) in the month of April.It has many secondary metabolite viz Tylophorin,Kaempferol and Stigmasterol. Among them Tylophorin, an antiasthmatic and anti-cancerous phenanthro indolizidine alkaloid is the main constituent of Tylophora indica. The experiments related to Phytochemical studies, Biotechnological studies, Pharmacological studies,BioEnhancement of Chief secondary metabolites and Microbiological studies were carried out which have given the significant results

The Book covers the various aspects and devided into following chapters

1. About Experimental plant
2. BioChemical studies
3. Pharmacological studies
4. Biotechnological studies
5. Enhancement of Secondary metabolites in tissue culture
6. Microbiological studies

Book is sufficient to illustrate a broad spectrum of various protocols that have been used in all experiments , can be followed by reader easil

1. About Experimental Plant

Tylophora is a family of slender climbing perennial plants which has about 60 species from various parts of the world.This name has been derived from two ancient greek words – ‘Tylos’ meaning “knot” and ‘phoros’ meaning “bearing”. It was earlier placed in Asclepiadaceae which has now been sunk into Apocyanaceace .The book is divided into several parts as follows

1. Classification Kingdom-Plantae Order-Gentiales Family-Apocynaceae

Subfamily-Asclepeadaceae

Genus-Tylophor,Species-indica

2. Vernacular names

Bengali- Antamul; Hindi- Antmool, Janglipikvan;, Kannada- Adumuttada, Nepala; Malayalam- Vallippala; Marathi- Kharaki-rasna, Anthamul, Pitmari; Oriya- Mendi, Mulini; Tamil- Koorinja, Peyppalainadu; Telugu- Verripala, Kukka-pala

3. Taxonomic description

Perennial, small, slender, much branched pubescent twining or climbing herbs or under shrubs; sap yellowish,. found in the sub-himalayan tract from Uttar Pradesh to Meghalaya and in the central and peninsular India, ascending up to 1,260 m. Rootstock 2.5-5 cm. thick, roots long, fleshy, with longitudinally fissured light brown, corky bark;; lLeaves 6.0-10.5 x 3.8-6.0 cm,  ovate-oblong to elliptic-oblong, acute to acuminate, cordate at base, thick, pubescent beneath when young, glabrous above; petioles up to 12 mm long.; flowers minute, 1-1.5 cm across, in 2 to 3-flowered fascicles in axillary umbellate cymes.; Calyx divided nearly to the base, densely hairy outside; segments lanceolate, acute. Corolla  greenish-yellow or greenish - purple; lobes oblong, acute. Fruit a follicle, .up to 7 x 1 cm,; ovoid-lancelets, tapering at apex forming fine micro, finally striate, glabrous. Seeds 0.6-0.8 x 0.3-0.4 cm, broadly ovate or ovate- oblong, flat, brown, dark colored in center; coma 2.0-2.5 cm long.(Arnold et al.,2003;Aly et al.,2010;Scragg etal.,1986;Diplock etal.,1998;Abad etal.,2000;Ajibade etal.2010)

4. Medicinal use

Its antitumor, immune modulatory, antioxidant, antiasthmatic, smooth  muscle relaxant, anti-histaminic, hypotensive activities are scientifically proven. In Ayurveda, the plant has been used in treatment of asthama, dermatitis and ruematism.Other reported activities include cytotoxic effect (Ali et al., 1989) immune modulatory activity (Anonymus,1976) anti cancerous and anti-amoebic activity (Antonio et al.,2001)

5. Habitat

Found in the plains, forests, and hilly slopes and outskirts of the forest .Forms dense patches in the forest in moist and humid conditions in open hill slopes and narrow valleys, also cultivated for its medicinal uses. The plant shows stunted growth in the areas with lesser rainfall

6. Distribution

The plant inhabits up to an elevation of 1,260 m in the sub-Himalayan tract and in the central and peninsular India. It also occur with in Eastern, North-East and Central India, Bengal and, parts of South India .Except throughout plains of India, it also harbor in Ceylon, Malay island and Borneo

7. Propagation

Tylophora indica is conventionally propagated through the seeds. The seeds show good germination percentage, but fruit set is rare. Seeds start germination in 10 days and the germination will complete in 3 weeks. After germination the 3 months old plantlets are ready to

transplanting in the field but the transplantation should be done in rainy season and plan distance should also  be  maintained.  The annual rainfall required for Tylophora plant is 1000 -1500mm.The plant prefers partial shade condition of the forest and soil rich in humus. It needs the support of host for climbing to a sunny location. For its cultivation, loamy soil to clay and supplemented with farmyard manure, ambient conditions of temperatures and sunlight are desirable

8. Phyto constituent

Tylophora indica plant has been reported to contain 0.3-0.4% of alkaloids viz Tylophorine, Tylophorinine, Tylophorinidine, Septicine, Isotylocrebrine, Tylophoricine, sterols, flavonoids, wax, resins and tannins Actually, the major constituent of T ylophora is Tylophorine, responsible for a strong inflammatory action. The active constituents of Tylophora indica are phenanthro indolizidine, alkaloids. Recently some rare alkaloids namely tyloindicinesAB,C,D,E,F,G,H,andJ,desmethylTylophorine,desmethylTylophorinine,isotylocrebrine,anhydrousTylophorinine,a nhydrous-dehydroTylophorinine, γ-fagarine,skimmianine, 14-hydroxyiso tylocrebrine, 4,6- desmethylisodroxy-o-MethylTylophorinindine have been reported. Presence of tannin, saponin and terpenoid in aqueous extract revealed more pharmacological activity

2.Biochemical studies

2.1 Phytochemical variation in Tylophora indica (Asclepeadaceae) leaves collected from different regions

Genetic diversity plays an important role in plant conservation and their survival in adverse conditions. Many environmental factors such as precipitation, mean temperature, soil, wind speed, low and high temperature extremes, duration of snow-cover, length of the vegetation period, and the intensity of radiation under clear sky conditions have been reported to differ between low and high altitude sites(Korner 1999).Moreover, study on phytochemicals of wild populations of plants at different altitudes were performed, and it is not conclusive whether the observed variations are a response of individual plants to environmental factors  related to altitude or a genetic adaptation of the populations growing at different altitudes to their specific environment (Ruhland et al., 2000,Zidorn 2001a,2005b)

Plants may produce as much as 100,000 small molecules(Dixon 2001) .which include primary metabolites that are present essentially in all plants resulting from primary metabolic activities, but the secondary metabolites that are specific to certain plant species, are produced in small quantities, have geographical impact on their production and are generally produced in a particular plant part Zhilin etal,2007).These compounds are the result of the secondary metabolic pathways that take place in certain plant species. For plants, little effects have been attributed to these potential substances like, defense against microorganisms, insects and herbivores. While some of them give plants their odors and pigments many of them are responsible for plant flavors but for humans, at instances, they become lifesaving drugs (Griffin et al., 2000).The Chemotaxonomy and geographical distribution of tropane alkaloid has been studied in Datura sps (Griffin2000)

Tylophora indica (Burm. f.) Merill. (Asclepiadaceae) commonly known as “Ant mool” is an important medicinal plant, traditionally used as a folk remedy in treatment of bronchial asthma, bronchitis, rheumatism, allergies and inflammation. The roots and leaves contain 0.2 to0.46%therapeutically important alkaloids Tylophorine, Tylophorinine and tylophrinidine. Major alkaloid Tylophorine has immunosuppressive, anti-inflammatory, anti-tumor (Donald et      1968) stimulant of adrenal cortex (Upupa et al., 1991) and anti-amoebic (Bhutani et al., 1985) properties. Prior to this, Gujrati et al., (2007) reported the hepatoprotective activity of alcoholic and aqueous extracts of leaves of T. indica (Kumar 2007) . Tylophorine and its analogs are phenanthro indolizidine alkaloids, many of which have been isolated from plants of the family Asclepiadaceae, including members of the genus Tylophora that are native to India and South east Asia. Phytochemical screening of the methanolic leaf extract of the plant revealed the presence of tannins, phytosterols (Stigmasterol), saponins, flavonoids, carbohydrates and alkaloids (Tylophorin)Bharti et al., 2010.The main aim of the present work was to examine the content of Stigmasterol and Tylophorin from Tylophora indica plant that has been collected from six different region of India(Jaipur, Anand, Navsari, Bharatpur, Mumbai, Nagpur) and to find out the best source of both the natural products among them .Leaves were selected as the experimental material in the present study because this part of Tylophora indica has been used as medicine for asthama ,inflammation, from ancient time. Stigmasterol is used as a precursor in the manufacture of semisynthetic progesterone, Sundaram etal.,1977, a valuable human hormone that plays an important physiological role in the regulatory and tissue rebuilding mechanisms related to estrogen effects, as well as acting as an intermediate in the biosynthesis of androgens, estrogen  and  corticoids. It is  also used as precursor of Vitamin D3(Kametani,1987).The Upjohn company used Stigmasterol as the starting raw material for the synthesis of cortisone.(Hogg,1992) Research has indicated that Stigmasterol may be useful in prevention of certain cancers, including ovarian, prostate, breast, and colon cancers. Studies have also indicated that a diet high in phyto sterols may inhibit the absorption of cholesterol and lower serum cholesterol levels by competing for intestinal absorption. Studies with laboratory animals fed Stigmasterol found that both cholesterol and sitosterol absorption decreased 23% and 30%, respectively, over a 6-week period. It was demonstrated that it inhibits several pro- inflammatory and matrix degradation mediators typically involved in osteoarthritis induced cartilage degradation. (Gabey et al., 2010)

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C₂₄H₂₇NO₄Tylophorin

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C₂₉H₄₈O Stigmasterol

Material and Methods

The plant material was collected from various geographical region of India such as Anand, Nagpur, Bharatpur, Mumbai, Navsari and Jaipur in the month of March. Powdered (100g) leaves of Tylophora indica was defatted exhaustively separately with petroleum ether (60‐80oc). The residue was extracted with ethanol for Stigmasterol estimation while in ethanol: methanol (8:2) for Tylophorin. The Thin Layer Chromatographic analysis with the reference compounds of Stigmasterol and Tylophorin were carried out separately along with their respective reference compound .TLC chamber was saturated with solvent system (Toluene: Diethyl amine: Ethyl acetate; 14:2:2) for 15 min. prior to use .0.1mm thick silica del plates were used as stationary phase and Toluene: Di ethylamine: Ethyl acetate (14:2:2) was used as mobile phase. The confirmation of alkaloid was done by spraying the developed chromatogram with Dragendorf’s reagent which gave the brick red colored five spot. The Tylophorin was further confirmed by running TLC plate with standard Tylophorin (Allexis Co. New Delhi).A very small amount of methanol was poured into the dried extract in the Petri plates and this was further used for TLC analysis. Further confirmation of presence of Tylophorin was carried out by using Co-TLC with standard Tylophorin (Rf value0.59). The developed chromatograms were observed in UV light at 254nm (Fig. 2), which gave bright yellow color. The further confirmation of Tylophorin was done by using HPTLC .In the case of analysis of Stigmasterol. TLC was used in the same way (solvent system -Hexane: acetone; 8:2). Developed plates were sprayed with  5% of sulphuric acid which gave a characteristic grey color (Rf-0.91) and suggested the presence of Stigmasterol in the leaves of Tylophora indica. The samples were subjected to the HPTLC analysis for quantitative estimation

HPTLC (High Performance Thin Layer Chromatography) analysis

HPTLC analysis was carried out in Anchrom Test Lab Pvt. Ltd using silica gel plates

(60F254Manufacturer E. MERCK KGaA),Sample application was carried out on CAMAG Linomat 5 Instrument (CAMAG Linomat 5 "Linomat5_080222" S/N 080222 ).Inert gas was used as spray gas. Sample solvent type was methanol. Dosage speed was 150nl/s and syringe size was 100µl and the analysis wave length was 430 nm. Toluene: Ethyl acetate: Diethyl amine (14:2:2) was used as mobile phase and Hexane: acetone (8:2) was used as mobile phase for Stigmasterol analysis

Results and Discussions

The plants of Tylophora indica were collected from different regions of India (Jaipur, Anand, Navsari, Bharatpur, Mumbai, and Nagpur) in the month of March. The mature leaves were collected and shade dried, powdered and subjected for methanol: ethanol (8:2) for Tylophorin and ethanol for Stigmasterol. The cold extraction was chosen for the present investigation. As mentioned above Tylophorin is the chief constituent of Tylophora indica, which is active principal for its anti-cancerous, anti-inflammatory and anti-asthmatic activity, whereas Stigmasterol is the precursor of many hormones

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Figure 2.1.1 Showing the HPTLC analysis of Tylophorin from methanol : ethanol (8;2) leaf extract and Tylophorin STD (A-Finger printing-Chromatogram of leaf C-Std Tylophorin at 254nm

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Figure2.1.2 Showing the HPTLC analysis of ethanolic extract of Tylophora indica leaves of different  area  with  std  Stigmasterol(  Chromatogram A-Leaves  ethanolic  extract  ,B- STD),C- HPTLC Fingerprinting at 254nm

To find out the better source of these compounds, the plants of Tylophora indica were collected from different regions (Jaipur, Anand, Navsari, Bharatpur, Mumbai, Nagpur) in the month of March,. The leaves were dried, powdered and subjected for their Tylophorin and Stigmasterol content analysis separately. The quantitative analysis of both the compound was carried out with the help of HPTLC analysis with their respective standard compound separately. The Quantitative estimation of both compounds was calculated by using their peak area. . In literature, evaluation of ()-(S)-Tylophorine [DCB-3500 (NSC-717335)] and its analog DCB-3503 (NSC-716802) for antitumor activity at the National Cancer Institute has been carried out It showed a fairly uniform and potent growth-inhibitory activity (GI518M) in 60 cell lines (Li et al.,2001,Rao et al.,1971,1997,2000Strawrk et al., 2002,Ganguli 2002,Komatsu et al., 2001).Genetic expression at the time of secondary metabolites production in plant cell is governed by many factors and geographical distribution Thus a variation in Tylophorin as well as Stigmasterol content have been examined among all the samples used. It was observed that Tylophorin content was maximum in  plant collected from Mumbai region, whereas in the case of Stigmasterol it was highest in Bharatpur region’s samples(Table1).So it can be concluded that Tylophora indica collected from Mumbai region is good for Tylophorin production while the same plant collected from Bharatpur region is well suited for the Stigmasterol production Tylophora indica has been well examined biotechnologically as well as phytochemically (Chaturvedi et al,2011,2011a,b,c,d,e,f,2012,2012a,b). Hence, it can be concluded that Tylophora indica grown in these two regions are favorable for the production of their respective natural product synthesis. These results can be helpful for the pharma industries as also for the cryopreservation of the elite germplasm

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Table 2.1.1 Depicts the Tylophorin and Stigmasterol content (%) of Tylophora indica leaves collected from different regions mean ± S.E .of three replicates

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Figure2.1.3. Depicts the Tylophorin and Stigmasterol content (%) of leaves of Tylophora indica Collectedfrom different region

3. Pharmacological Studies

3.1 Anti-inflammatory activity

Inflammation is a condition when a body reacts to any infection, injury or irritation, environmental changes or malignancy, but sometimes some internal inflammation can result giving rise to fever and other discomforts. The inflammatory response involves the activation of white blood cells that start releasing some chemicals such as cytokines and prostaglandin. ( Scher et al., 2009). Prostaglandins are produced within the body's cells by the enzyme cyclooxygenase (COX). There are two COX enzymes, COX-1 and COX-2, produce prostaglandins that promote inflammation, pain, and fever. However, only COX-1 produces prostaglandins that support platelets and protect the stomach. No steroidal anti-inflammatory drugs (NSAIDs) block the COX enzymes and reduce prostaglandins throughout the body. As a consequence, ongoing inflammation, pain, and fever are reduced (Abad et al., 2000).Anti-inflammatory refers to the property of a substance or treatment that educes inflammation. Despite the progress made in the medical science chronic inflammation is still considered as major health problem thus this area need new drugs and further research. Many herbs have been evaluated for the anti-inflammatory   activity including Tylophora indica, which is a perennial plant, belongs to family Asclepeadaceac. The plant has been explored biotechnologically as well as phytochemically (Chaturvedi et al.,2011,2011a,b,c,d,e,f,2012,2012a) This climber plant is reported to have many pharmacological important activity(Nilesh et al., 2011).Anti-inflammatory activity of different plant alkaloids has been  reviewed  extensively(Wang  et al.,2010). These natural products are most common in flowering plants, and usually in the Papaveraceae (poppies), Papilonaceae (lupins), Ranunculaceae (aconites), and Solanaceae (tobacco and potatoes) families (Lewis et .,1989). Isoquinoline, indole and diterpene alkaloids were the most alkaloids that were extensively examined by many scientist. They were effective on different assays including carrageenan induced paw edema, adjuvant-induced arthritis and acetic acid induced vascular permeability tests (Luo etal.1991,Zang et al., 1982). Tylophora alkaloids originate from various plants of the Asclepiadaceae family, , are native of India and Southeast Asia(Saito et al., 1982) .They have antitumor,(Li et al.,2001,Donaldson etal.,1968,Rao  et al.,  2000,Staerk  et  al.,  2002)anti-inflammatory,Gaoetal.,2004)antiarthritis,(Yang et al., 2006)and anti-lupus activity in vivo.(You et al.,2006).Tylophorin from Ficus septica from china has been evaluated anti-inflammatory activity,70% anti-inflammatory activity  was  reported  in  this  case  at  the  concentration  of3-10  µg/ml(Ravi Kumar  et  al., 22011),Some scientist have carried out the study of anti-inflammatory effect of Tylophora indica plant extract on carrageenan induced paw edema model ,They have worked on ethanolic extract of the plant ,but did not isolated the bioactive compound that responsible for the ant- inflammatory activity, so it was highly essential to find the Indian source of Tylophorin. To fulfill this need Tylophora indica leaves from Maharashtra region was examines for this activity. Hence, in the present study, the isolation, identification and characterization of Tylophorin were carried out qualitatively from Tylophora indica by using different techniques such as Thin Layer Chromatography (TLC), Preparative Thin Layer Chromatography (PTLC),High Performance Liquid Chromatography (HPTLC) and Infra-Red Spectral studies (IR)along with the standard reference compound of Tylophorin .The isolated and characterized compound was used for anti-inflammatory assay

Material and Methods

The experimental plant of Tylophora indica was procured from Kelkar farm house, Mulund, Mumbai (The latitude coordinate of Mumbai is 18 degree 58' 30 North and longitude coordinate is 72 degree 49' 32" East) in the month of April and maintained in Haffkine Institute campus till full flourish growth was achieved. The mature leaves (20g) of climber plant were collected in the month of April. The fresh paste was prepared and then subjected for cold extraction for  48  hours  at  room  temperature with solvent methanol: ethanol (8:2).The extract was then decanted into petri plates and the extract was allowed to evaporate and dried, which was now ready for further experimental work

Qualitative method

The Thin Layer Chromatography(TLC) was used to identify the Tylophorin alkaloid with that of the reference compound .TLC chamber was saturated with solvent system Toluene: Di ethylamine: Ethyl acetate (14:2:2) for 15 min. prior to use .0.1mm thick silica del plates were used as stationary phase and Toluene: Diethyl amine: Ethyl acetate (14:2:2) was used as mobile phase. The confirmation of alkaloid was done by spraying the developed chromatogram with Dragendorf’s reagent which gave the brick red colored five spot. The Tylophorin was further confirmed by running TLC plate with standard Tylophorin (Allexis Co. New Delhi).A very small amount of methanol was poured into the dried extract in the Petri plates and this was further used for TLC analysis. Further confirmation of presence of Tylophorin was carried out by using Co- TLC along with standard Tylophorin (Rf value0.59).When developed plates were sprayed with Dragendorf’s reagent brick red color was suggest the presence of alkaloid. The developed chromatograms were observed in UV light at 254nm (Fig 2), which gave bright yellow color. The further confirmation of Tylophorin was done by using HPTLC and IR spectral studies with reference compound of the same. (Fig. 2)

Preparative Thin Layer Chromatography (PTLC)

The isolation of Tylophorine was carried out by Preparative TLC method. After being separated out on the TLC plate, Tylophorine was purified by scraping off the silica from the plate and dissolving it in ethanol. This filtrate was then collected and allowed to evaporate, which left behind pale yellow crystal of Tylophorine. This was done repeatedly two times to procure the pure compound. The compound was then subjected for anti-inflammatory activity by using the mentioned method

High Performance Liquid Chromatography (HPTLC ) Analysis

HPTLC analysis was carried out in Anchrom Test Lab Pvt. Ltd using silica gel plates (60F254Manufacturer E. MERCK KGaA),Sample application was carried out on CAMAG Linomat 5Instrument (CAMAG Linomat 5 "Linomat5_080222" S/N 080222).Inert gas was used as spray gas. Sample solvent type was methanol. Dosage speed was 150nl/s and syringe size was 100µl and the analysis wave length was 430 nm. Toluene: Ethyl acetate: Diethyl amine (14:2:2) was used as mobile phase

Anti-inflammatory test of Tylophorine:

The extraction, isolation, and characterization of Tylophorin alkaloid from Tylophora indica leaves were carried out and it was evaluated for anti-inflammatory activity using the HRBC mambrane method Principle The plant kingdom consists of about 400.000 plant species and is a huge reservoir of bioactive molecules, many of which have yet to be explored for various pharmaceutical applications (Ali, M. and Bhutani, K.K.1989). Asthma is a frightening condition which can seriously impede one’s ability to breathe. An asthma attack is described as an allergic reaction of the respiratory tract leading to a drastic narrowing of air passages, triggered by a variety of air pollutants commonly described as allergens. The antioxidant activity of Tylophora indica leaves has been evaluated ( Gore, et al. 1979) Antioxidant compounds in food play an important role as a health protecting factor. Scientific evidence suggests that antioxidants reduce risk for chronic disease. The main ability of an antioxidant compound is its ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a variety of sources; these free radicals may oxidize nucleic acids, proteins, lipids, or DNA and can initiate degenerative disease. Antioxidant compounds scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanism that lead to degenerative disease. These are vital substances which possesses the ability to protect the body from damage caused by free radical induced oxidative stress (Ta-Hsien etal.,2006), they protect the human body against free radical that may cause pathological conditions such as ischemia, anemia, asthma, arthritis, inflammation, neuro-degenertion, Parkinson’s disease, mongolism, ageing process (Gupta, et al., 2011), Medicinally bioactive products are accumulated after certain age of maturity. To meet the ever increasing demand on medicinal plants the natural resources are not sufficient, an effective alternate method is to develop tissue in vitro which can yield the same phytoconstituents in larger quantities. Natural sources of plants may have contaminations by bacteria, virus, fungi, insects, etc. Extraction and isolation of alkaloids from the plants are so lengthy and expensive processes. By obtaining phytochemicals from in vitro cultured callus tissue, we can eliminate all these disadvantages. Micro propagation by plant tissue culture becomes helpful for conservation of plants. We can enhance the production of these secondary metabolites in callus. The relation between antioxidant activity and asthma has been evaluated by some scientist. They found out that to cure asthma antioxidant medicine should be taken (Gordon et al., 1995 Colin et al., 1995) Therefore the evaluation of cytotoxicity and antioxidant activity of an anti-asthmatic plant is very essential study and this study is also very important to find out an alternative and convenient source of these plant to conserve our green resources. The tissue culture studies for callus induction was carried out by many scientist, but their media was different as was used in the present study (Faisal et al.,2005 , Rao ,et al.,1970). The antioxidant activity was not reported so far in callus of Tylophora indica.The present study pertains the cytotoxity and antioxidant activities of six weeks old callus of anti-asthmatic plant Tylophora indica for the first time . The cytotoxicity examination were carried out on V cells using the MTT assay.(Mosmann,.1983)and the DPPH method was used to examined the antioxidant activity of Tylophora indica callus. ( Khurana et al.,2010) The lysosomal enzyme is produced during the process of inflammation ,which in turn create a variety of disorder. The extra cellular activity of this enzyme is said to be related to the acute or chronic inflammation. The non-steroidal drugs Diclo Fenac Sodium (DFS) acts either by inhibiting this lysosomal enzyme or by stabilizing the lysosomal membrane. The hypo saline used causes the hemolysis of RBC as its concentration increased .This causes the membrane components to leak out. Which is then measured spectro photo metrically at 560nm? Thus increased concentration of hypo saline increases the OD.(as increased concentration of hemolysis increases the extent of hemolysis.) DFS (standard compound) stabilizes the membrane, thereby reducing the hemolysis. Thus with increment in the concentration of DFS membrane is stabilized and membrane components are prevented from leaking. Thus  as the concentration. of DFS increases the OD decreases thereby decreasing the effect of hypo tonicity caused by hypo saline.% Hemolysis= (OD of drug treated sample/OD of control)*100.The ability of the test sample to stabilize the membrane or to prevent hemolysis is compared with that of standard

Anti-inflammatory activity of Tylophorin

Protocol

Step 1- RBC solution

Alsevers solution (3ml) + Blood (3ml)

Centrifuge at 3000rpm for 10 min

Discard the supernatant and wash the pallet with iso-saline thrice

Dilute 1ml of pallet with 9 ml of iso-saline i.e.  10% v/v

STEP-2: Sample (Tylophorine  and DFS  Dilutions)

The dilutions (10mg/ml, 1mg/ml, 0.1mg/ml,.01mg/ml,.001mg/ml) of Tylophorin and DFS( Standard compound) were made using DMSO separately. All the samples for further study were prepared (0.5ml sample + 1 ml Phosphate buffer + 2 ml Hypo saline +0.5mlRBC Solution) separately. Here positive control (1ml Phosphate buffer: + 2ml Hypo saline +0.5 mlRBC solution+ 0.5 ml DMSO) and Blank (2ml Phosphate buffer + 4mlHyposaline +2ml DMSO) were used STEP 3: All the samples were prepared as stated earlier and kept for 30 min. at 37oc following  by centrifugation at 300rpm for 20 min .After the completion of the process the OD of the supernatant were taken at 560 nm separately. On the completion of the experiment % hemolysis and %anti- inflammatory activity of samples were calculated by using their respective formula . The process of making Alsevers solution, Phosphate Buffer Hypo saline and Isosaline are as follows. Alsevers solution (Add 50mlDextrose - 1gm, Sodium citrate - 0.4gm,Citric acid – 0.025gm,Sodium chloride - 0.21gm and dissolve in distilled water to make up the volume up to 50ml. Phosphate buffer (A-0.2 M of NaH2PO4Dissolve 4.792gm of NaH2PO4 in 200ml of distilled water,B-0.2 M of Na2HPO4Dissolve 5.678gm of Na2HPO4 in 200ml of distilled water and mix 32ml of ‘A’ with 168ml of ‘B’ and make C solution. Take 150ml of ‘C’ adjusts the pH to 7.5 and make up the volume to 200ml using distilled water.) Hypo saline Dissolve 0.9gm of NaCl in 250ml of distilled water. Isosaline Dissolve 2.125gm of NaCl in 250ml of distilled water

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Figure.3.1.1 A- Standard of Tylophorin (254nm),B-Fingerprinting of High Performance Liquid Chromatography of methanol: ethanol( 8:2) extract of Tylophora indica leaves,C-HPTLC Chromatogram of methanol:  ethanol(  8:2)  extract  of Tylophora indica leaves  ,D-Standard  E-Infra  Redspectral studies of isolated Tylophorin with standard.

Results and Discussion

Tylophorin from Tylophora indica leaves extract exhibit the significant anti-inflammatory effect as compared to DFS.(Table1,Figure 2)

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Table 3.1.1.The anti-inflammatory %activity of Tylophorin from Tylophora indica leaves and comparison with DFS a standard compound SD ± mean of three replicates significant p value p>0.01(Chi square method

The main objective of the study was to find out the Indian herbal source for anti-inflammatory activity. The potentiality of Tylophora indica as anti-inflammatory agent was confirmed significantly. Tylophorine was extracted, from Tylophora indica leaves by using methanol: ethanol (8:2) solvent at room temperature. The TLC chromatogram were developed with standard compound of Tylophorin in Toluene: Di ethylamine: Ethyl acetate (14:2:2) mobile phase. On spraying with Dragendorf’s reagent, brick red color was appeared, which confirm the presence of Tylophorin alkaloid (Rf value 0.59) qualitatively. For Further confirmation of presence of Tylophorin HPTLC and IR spectral studies was done. The cytotoxicity of methanolic extract of Tylophora indica leaves has been evaluated by MTT assay and LC50 30mg/ml was obtained in this experiment. The anti-inflammatory activity of Tylophorine was compared with standard anti-inflammatory drug DFS. From the graph (Fig. 2) we can see the activity of Tylophorine at the correspondent dilution was far better than the DFS. Increase concentration of Tylophorine gradually brings down hemolysis activity denoted by OD. When compared with standard DFS (24.62%) ,Tylophorin has given better results(45.14%) at the concentration of 10mg/ml. From this study, it can be concluded that the Tylophorine of Tylophora indica of Maharashtra origin acts as an anti-inflammatory agent significantly and in 2future it might be used in the pharma industries. Asclepeadaceae was evaluated for anti-inflammatory activity It was established that phenanthro indolizidine alkaloid inhibit the activation of COX-II

promoter activity alpha functional group of Tylophorin is involved in anti-inflammatory activity and hence it acts as an anti-inflammatory agent. In this experiment scientists have reported around 70% inhibition at 9-10 µ M (activity at 393mg/ml;Mol.wt.0f Tylophorin is 393.48) concentration .In this case Tylophorin exhibited potent suppression of nitric oxide production which is twenty times lower than our results (10mg/mL:45.14%).The study showed the enthusiastic and considerable results now it can be said that the Indian herbal source of Tylophorin is much more efficient to combat inflammatory disorder

3.2 In-vitro Anti - influenza activity of Tylophora indica (Asclepeadaceae) Leaves.

Influenza viruses are ubiquitous, they have been around for hundreds of years, and are likely to remain with us for a long time (Cannell et al., 2008) They produce significant annual morbidity and mortality throughout the world, and the occasional pandemic with potentially devastating consequences for human and animal health and the global economy. Some innovative molecular approaches have been suggested, based on cellular signaling pathways utilized by the virus for its replication (Ludwig S 2009 Suzuki Y 2009.) Herbal remedies, including traditional Chinese Medicine (TCM) have also been. suggested as alternatives (Song etal., 2005). These generally may be safer. than chemical drugs, and are less likely to encounter resistant viruses, because of their multivalent functions. Several hundred plant and herb species that have potential as novel antiviral agents have been studied, with surprisingly little overlap. A wide variety of active phytochemicals, including flavonoids, terpenoids, lignans, sulphides, polyphenolics, coumarins, saponins, compounds, alkaloids, polyamines, thiophenes, proteins and peptides have been identified. Some volatile essential oils of commonly used culinary herbs, spices and herbal teas have also exhibited a high level of antiviral activity .Several of these phytochemicals have complementary and overlapping mechanisms of action, including antiviral effects by either inhibiting the formation of viral DNA or RNA or inhibiting the activity of viral reproduction.).If viral enzymes could be neutralized, viral replication would not take place. The proteolytic processing of viral poly protein precursors by viral proteins. Virus Designing specific inhibitors for each of viral protease is thus a desirable objective, so the basic requirement behind the use of herbs as an antiviral agents are to disrupt the replication cycle, to stimulate as also to support the immune system (Mike Adams 2010 ) Every plant on our planet manufactures at least one anti-viral medicine in its own cells (Xiaoyan et al.,., 2006).The development of viral resistance towards antiviral agents enhances the need for new effective compounds against viral infections. Medicinal plants have a variety of chemical constituents, which have the ability to inhibit the replication cycle of various types of DNA or RNA viruses. Compounds from natural sources are of interest as possible sources to control viral infection (James B. Hudson 2009). In this context various research groups  in  Asia,  Far  East,  Europe  and  America  have  given  particular  attention  to  develop antiviral agents from their native traditional plant medicines. Some typical examples of such medicines and their antiviral activities are shown in Table 1.(Wang et al., ,2006 )

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Keeping all the above facts Tylophora indica was selected for anti- influenza evaluation. . Tylophora indica (Asclepediacaeae) is a creeper plant and is used as an anti - asthamatic medicine in India pharmacopeia, Plant is having Tylophorin as a main constituent, which is reported as an anti asthamatic and anti-inflammatory agent. Secondary metabolites from many plant species have been reported for the anti -influenza activity (Wang X, Jia W, Zhao A, Wang X 2006 Nagai T, Miyaichi Y, Tomimori T, Suzuki Y, Yamada H 2008Liu AL, Liu B, Qin HL, Lee SM, Wang YT, Du GH 1992 Xiaoyan Wang ,Wei JiaAihua Zhao 2006). Some scientist have worked on the synthesis and development of anti TMV drugs, which is a derivative of phenanthrene nucleous like Tylophorin, , the main constituent of Tylophora indica. No work has been documented on anti-influenza activity of Tylophora indica till date

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