In-vitro Anti Influenza Activity of the Tylophora Indica ( Asclepeadaceae )

1. In-vitro Anti Influenza Activity of the TylophoraIndica (Asclepeadaceae) PratibhaChaturvedi1, Soumen Roy2, Dr. Abhay Chowdhary1,2 1Department of Zoonosis, Haffkine Institute, Mumbai 400012 2Department of Virology, Haffkine Institute, Mumbai 400012 INTRODUCTION: Influenza viruses are ubiquitous, they have been around for hundreds of years, and are likely to remain with us for a long time. They produce significant annual morbidity and mortality throughout the world,. The current annual vaccines and effective antiviral drugs are not available sufficiently,due to antigenic shifts and drifts of influenza virus, long-lasting vaccine has not been developed so far. The use of herbal products is one of the best alternatives against influenza virus. OBJECTIVES: To screen In-vitro Anti Influenza Activity of the TylophoraIndica (Asclepeadaceae) MATERIALS AND METHODS: The plant material of Tylophoraindica was collected from the Kelkar farmhouse, Mulund, and was subjected to the methanolic cold extraction for the evaluation of anti influenza experiment. Cytotoxicity assay was carried out on MadinDarbyCannine Kidney (MDCK) cell line by using MTTassay prier to anti influenza examination..in vitro susceptibility assay: post exposure method was performed and cytopathic effect was observed (3-4 day) and the activity was confirmed by Hemagglutination assay using 0.75 % guinea pig and 0.5% fowl RBC’s RESULTS AND DISCUSSION: TylophoraIndica extract of 20mg/ml showed 3 fold reduction of the HA titre (1:8) as compared to the virus control titre of 1:64. Tylophoraindica extract of 20mg/ml showed 3 fold reduction of the HA titre (1:8) as compared to the virus control titre of 1:64 which indicates that the extract has an anti-influenza activity. This is the preliminary study in which the Tylophoraindicaextract showed anti-influenza activity at 20mg/ml in post exposure treatment for the first time. INTRODUCTION RESULTS • Methanolic extracts of TylophoraIndica (Leaves) were obtained using the Soxhlet method of extraction. • In-vitro Cytotoxic assay performed by MTT assay and the CC50of leaves (Methanolic Extract) of TylophoraIndicawas found to be30 mg/ml as compared to control DMSO which showed higher cytotoxicity (Figure 3). • TylophoraIndica extract of 20mg/ml showed 3 fold reduction of the HA titre (1:8) as compared to the virus control titre of 1:64. (Figure 4) • . • Influenza viruses are ubiquitous, they have been around for hundreds of years, and are likely to remain with us for a long time. 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. • Due to antigenic shifts and drifts of influenza virus, long-lasting vaccine has not been developed so far. The current annual vaccines and effective antiviral drugs are not available sufficiently. Even the vaccines and anti-influenza medicines available in the market have numerous drawbacks such as their side effects and increase drug resistance. • Need of the hour in order to deal with influenza is to develop a novel drug against Influenza which will be both effective and safe for human consumption. Tylophoraindica (Asclepediacaeae), one of the medicinal plant which is being widely used as a traditional folk medicine in allergies and respiratory diseases. • Tylophoraindica(Asclepediacaeae) is a creeper plant and is used as an antiasthamatic medicine in indian pharmacopeia, Plant is having tylophorin as a main constituent, which is reported as anti asthamatic and also anti-inflammatory agent. Secondary metabolites from many plant species have been evaluated for the anti infuenzaactivity [1]. • Some workers have also worked on the synthesis and development of anti TMV drugs, which is a derivative of phenanthrenenucleous like tylophorin,which is main constituent of Tylophoraindica. There is no documented evidence of plant leaves exhibiting anti-influenza activity. In the present study, Tylophoraindica leaves (methanolic extract) were screened for anti-influenza activity. • Tylophoraindica extract of 20mg/ml showed 3 fold reduction of the HA titre (1:8) as compared to the virus control titre of 1:64 which indicates that the extract has an anti-influenza activity. • This is the preliminary study in which the Tylophoraindicaextract showed anti-influenza activity at 20mg/ml in post exposure treatment for the first time. so it can be used as a local disinfectant against influenza. • Further research can be done in the field by purifying and isolating the bioactive component responsible for anti-influenza activity. Figure 3: Cytotoxicity of TylophoraIndica : CC50of leaves (Methanolic Extract) of TylophoraIndica: 30 mg/ml OBJECTIVES Key: : Button formation : Hemagglutination • To screen In-vitro Anti Influenza Activity of the TylophoraIndica (Asclepeadaceae). MATERIALS AND METHODS Figure 4:TylophoraIndica extract of 20mg/ml showed 3 fold reduction of the HA titre (1:8) as compared to the virus control titre of 1:64. DISCUSSION AND CONCLUSION • Plant material:Tylophoraindica (commonly known as Antamul), belonging to the Family Asclepiadaceae .It is a dark copper coloured delicate creeper found growing wild in the plains of India and other sub-tropical regions of the world • Extraction of active components –The Plant of Tylophoraindica was collected from the Kelkar farmhouse, Mulund, and the leaves of Tylophoraindica sample was extracted by Soxhlet'sexctraction method (1879) with methanol.[2] REFERENCES • Kailiang Wang, YannaHu, Yuxiu Liu, Na Mi, Zhijin Fan, Yu Liu, and Qingmin Wang 2010 Design, Synthesis, and Antiviral Evaluation of Phenanthrene-Based Tylophorine Derivatives as Potential Antiviral AgentsJ. Agric. Food Chem.58 (23), pp 12337–12342 against tmvphenanthrene-based tylophorine derivatives (PBTs) represent another new template for antiviral studies and could be considered for novel therapy against plant virus infection. • Tina Ehrman. “Standard Method for the Determination of Extractives in Biomass”. • Mossmann T 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65: 55-63. • MehrbodP.,MotamedN.,Tabatabaian M., SoleimaniEstyar R Amini E., Shahidi M., KheiriM.T, In Vitro Antiviral Effect of "Nanosilver" on Influenza Virus, DARUVol 17, No. 2 2009 • Hirst GK. The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J. Exp. Med 1942;75:47-64 Figure 1: Tylophoraindica Figure 2:Soxhlet apparatus & assembly • In-vitrocyto toxicity assay:Before using extracts,Cytotoxicity assay was carried out on Madin Darby Cannine Kidney (MDCK) cell line by using MTT assay prior to anti influenza examination.[3] • Anti influenza assay: in vitro anti influenza assay (post exposure method) was performed [4].The anti viral activity was confirmed by Hemagglutination assay using 0.75 % guinea pig RBC’s[5] ACKNOWLEDGEMENT Author are thankful to DST Woman Scientist Scholarship scheme for doing this research work.