Antiviral Activity and Phytochemical Analysis of Ailanthus Excelsa ...

JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE)31resulted in the isolation of major compound of the chloroformextract, canthin-6-one, detection of this compound wasdetected by sparing with dragendroff reagent which is specificfor alkaloids on TLC in which the compound gave red-orangespot.Preparation of the extracts for bioassayExtracts were dissolved as 100 mg in 1 ml of 10% DMSO inwater. The final concentration was 100 µg/ µl (Stock solution).The dissolved solutions were sterilized by addition ofantibiotic antimycotic mixture [19] Sterility test were carriedout in nutrient agar.Cell CultureAfrican green monkey kidney-derived cells (VERO) wereused. The cells were propagated in Hanks ٫ Minimum essentialmedium, MEM supplemented with 10% Foetal bovine serum,1% antibiotic-antimycotic mixture. The pH was adjusted at7.2-7.4 by 7.5% sodium bicarbonate solution. The mixture wassterilized by filtration through 0.2 µm pore size nitrocellulosemembrane.VirusesHerpes Simplex virus type 1 was obtained from EnvironmentalVirology Lab., Department of Water Pollution Research,National Research Centre. Antiviral assay was carried byPlaque reduction assay [20].Plaque reduction assayA 6-well plate was cultivated with Vero cell culture (10 5 cell/ml) and incubated for 2 days at 37 C. HSV-1 was diluted togive 10 4 PFU/ml final concentration and mixed with the plantextract at 100 mg in 1 ml of 10% DMSO in water andincubated overnight at 4C. Growth medium was removedfrom the multiwell plate virus-compound mixture wasinoculated (100 µg/well). After 1 hr contact time, the inoculumwas aspirated and 3 ml of Minimal Essential Medium (MEM)with 1% agarose was overlaid the cell sheets. The plates wereleft to solidify and incubated at 37C until the development ofvirus plaques. Cell sheets were fixed in 10% formalinesolution for 2 hrs, and stained with crystal violet stain. Controlvirus and cells were treated identically without chemicalcompound. Virus plaques were counted and the percentages ofreduction were calculated [20].III. RESULTS AND DISCUSSIONRESULTSThe Results of antiviral activity of Ailanthus excelsa stem barkextracts are included in table 1, it has shown that chloroformextract is more potent than methanol extract as anti-HSV-1agent, where chloroform extract showed virus reduction by82.6, while methanol extract showed virus reduction by 52 atthe concentration of 50 µg. Phytochemical analysis of theextracts are included in table 2 which prove that each extracthas interesting bio-active compounds, quassinoids, andalkaloids and the major alkaloid compound, canthin-6-one wasisolated from the bioactive chloroform extract and thechemical structure of the compound was identified by 1 H-NMR, 13 C-NMR and MSStructure elucidation of the alkaloid compound, canthin-6-one:Canthin-6-one: 1 H NMR (400 MHz, CDCl 3 ): H 6.98 (d, J =9.9 Hz, H-5), 7.50 (t, J = 7.6 Hz, H-10), 7.68 (t, J = 7.6 Hz, H-9), 7.95 (d, J = 4.8 Hz, H-1), 8.01 (d, J = 9.9 Hz, H-4), 8.09(d, J = 7.6 Hz, H-11), 8.64 (d, J = 8.1 Hz, H-8), 8.79 (d, J =4.8 Hz, H-2). 13 C NMR (100 MHz, CDCl 3 ): c 116.49 (C-1),117.30 (C-8), 122.74 (C-11), 124.37 (C-11a), 125.72 (C-10),128.95 (C-5), 130.47 (C-11b), 130.95 (C-9), 132.07 (C-11c),136.09 (C-3a), 139.49 (C-4), 139.49 (C-7a), 145.76 (C-2) and159.59 (C-6).EI-MS: m/z 220.10911811a7aON11b11c5Figure 1. Canthin-6-oneDISCUSSIONThe present study for the first time, proved antiviral activity of A.67excelsa bark extracts. The results has shown that chloroform hasa good antiviral activity than methanol extract (Table 1) in adose dependent mannar. Chloroform extract of A. excelsa stembark at concentration 20 µg has shown an inhibition for the virusby 66.5%, and at concentration of 50 µg, chloroform fraction ofA. excelsa stem bark has a significant anti-viral activity byinhibition for the virus by 82.6%. Methanol extract atconcentration 20 µg has shown an inhibition for the virus by44.9%, and at concentration of 50 µg, it has shown inhibition ofthe virus by 52%. The anti-HSV-1 properties of A. excelsaextracts in this research could be due to multiple differentcomponents in these extracts. The phytochemicalcharacterization of extracts and the identification of thebioactive compounds are now needed. Phytochemical analysisof the extracts has shown that both extracts have interestingbioactive compounds include quassinoids and alkaloids (Table2). Chloroform extract contained canthin-6-one as the majorcomponent and the higher activity of chloroform extract isprobably due to the higher concentration of these bioactivecompounds in the chloroform extract, while methanol is lessactive due to its low concentrations of these bioactivecompounds. Analysis of both extracts by TLC has proved manyspots for chloroform extract and less number of spots formethanol extract and these spots were detected under ultraviolet(UV) light. Spraying these spots with vanillin-sulphuric acidreagent followed by heating for 5 min until the appearance ofpurplish-blue colours specific for13a4N23