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)RESEARCH ARTICLE 30Antiviral Activity and Phytochemical Analysis ofAilanthus Excelsa Roxb Bark* Khaled Rashed 1 , Ataa Said 1 , Mohammed Ahmed 21 Pharmacognosy Department, 2 Virology Department, National Research Centre, Dokki, Giza, Egypt.*Corresponding author: Dr. Khaled Nabih Zaki Rashed, Tel: 01003642233E-mail: khalednabih2015@yahoo.co.uk(Received March 20, 2013; Accepted April 25, 2013)Abstract— Resistance to current anti-herpetic drugs has beenincreasingly reported, so there is a need for discovering newantiviral agents, in particular from natural origin. The aim of thepresent study is to investigate chloroform and methanol (70%)extracts from Ailanthus excelsa bark for their antiviral activityagainst Herpes Simplex virus type 1 (HSV-1) using Plaquereduction assay. The results has shown that chloroform extract ofAilanthus excelsa has a significant anti-viral activity againstherpes Simplex virus type 1 in-vitro by 82.6% at concentration of50 µg, while methanol 70% extract was less active. Phytochemicalanalysis of both Chloroform and methanol extracts of Ailanthusexcelsa has shown that it has interesting bioactive compoundsinclude quassinoids (highly oxygenated triterpenes) and alkaloidsand further phytochemical analysis from bio-active chloroformextract resulted the isolation of the major alkaloid compound,canthin-6-one. In conclusion, Ailanthus excelsa extracts may bean appropriate candidate for further development of anti-HSV-1infection.Index Terms— Ailanthus excelsa, antiviral activity, quassinoids,alkaloids, canthin-6-one.VI. INTRODUCTIONiral infections, particularly the infections caused by herpessimplex virus (HSV), represent one of the most seriouspublic health concerns globally because of theirdevastating impact. Herpes simplex virus type 1 (HSV-1) is acommonly occurring human pathogen worldwide. There is anurgent need to discover and develop new agents for themanagement of HSV-1 infection. Since the ancient times,natural products has served as a major source of drugs. Aboutfifty percent of todays pharmaceutical drugs are derived fromnatural origin [1]. Also it is well established that naturalproducts are an excellent source of chemical compounds with awide variety of biological activities including antiviral andanticancer properties [2]. In our searching for new antiviralsubstances from plants, in particular from Simaroubaceaefamily, A. excelsa Roxb is a tree of rapid growth and is calledtree of heaven, leaves appear in March-April, 30-90 cm long,pinnate, the flowers, small in size, yellow in colour and*Corresponding author.arranged in panicles and the fruits are formed soon afterflowering. The fruits ripen in May-June, just before the onset ofmonsoon. A. excelsa was investigated previously to proveantibacterial [3], antifungal [4], Antifertility [5] and anticancer[6]. A. excelsa is used in treatment of skin erpution and for thecure of wounds, The bark is bitter, astringent, anthelmentic,febrifuge, appetizer, bitter tonic, taste bud stimulant, It is usefulin diarrhea, amoebic dysentery, chronic giardiasis, dyspepsia,abdominal spasm anorectal disease, haemorrhoids, fistula,fissures, ulcerative colitis as mentioned in traditional medicine[7] From chemical point of view the plant is a rich source ofalkaloids [8, 9] proteins [10], quassinoids [11, 12] andflavonoids were isolated from leaves [13]. In the present study,we investigated antiviral activity of chloroform and methanolextracts from A. excelsa bark against Herpes Simplex virus type1 (HSV-1) in-vitro and also determined the major compound inthe bioactive chloroform extract.II. MATERIALS AND METHODSPlant MaterialA. excelsa bark was collected from Zoo garden, Giza, Egypt inMay 2010. The plant was identified by Dr. Mohamed El-Gabaly, Professor of Taxonomy, National Research Centre. Avoucher specimen no. 13241 is deposited in the herbarium ofZoo garden, Giza, Egypt.Preparation of extracts and isolation of the major compoundof chloroform extractThe Air dried powered bark of A. excelsa 400 g wassuccessively extracted with Chloroform and methanol70% bymaceration for 24 hours and each extract was concentratedunder reduced pressure to give 15 g and 23 g of crude extracts,respectively. Each extract of A. excelsa was phytochemicallyscreened according to the following described by (Connolly etal. 1970) [14] for sterols and/or triterpenes (quassinoids); Wolfet al. 1962) [15] for carbohydrates and saponins; Harbone1973) [16] for flavonoids and alkaloids; Farnsworth 1966)[17] for coumarins; Geissman 1962 for tannins [18]Confirmation of the presence of quassinoids and alkaloidswere detected by Thin layer chromatography (TLC) byspraying with specific reagents. Chromatographic separationof chloroform extract on silical gel column chromatography(0.06-0.2 mm) eluted with chloroform: methanol (99:1)

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

JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE)32quassinoids and Spraying with draggendror ' s reagent resulted inthe formation of an orange colour specific for alkaloids. TheHSV-1 activity of chloroform extract may be due to thepresence of alkaloids (canthin-6-one) and quassinoids whichpresent in considerable amounts in the extract [8], [9], [11], [4]and this is in agreement with the activity of alkaloids isolatedfrom Tripterygium hypoglaucum against herpes simplex virustype1 [21] Also the antiviral activity of alkaloids was confirmedTable 1from Fumaria and Corydalis species [22] as well Quassinoidshave shown anti-HSV-1 [23] Chloroform extract of A. excelsabark has considerable amounts of alkaoids and quassionoidsand the major component is the alkaloid is canthin-6-one whilemethanol has the same bioactive chemical compounds but inless amount, so Chloroform extract has higher antiviral activitythan methanol.Anti-HSV-1 bioassay in Vero cell line by plaque reduction assay of A. excelsa extractsHSV-1ExtractConcentrationInitial virus count(PFU/ml)Virus count(PFU/ml)% of Virus reductionChloroform 20 µg 1.96 x 10 7 1.22 x 10 7 66.550 µg 1.96 x 10 7 1.2 x 10 7 82.6Methanol 20 µg 1.96 x 10 7 1.08 x 10 7 44.950 µg 1.96 x 10 7 0.94 x 10 7 52Table 2Results of Phytochemical analysis of A. excelsa extractsChemical Constituents Chloroform extract Methanol extractCarbohydrates and/or glycosides - +Tanninsa. Condensed tanninsb. Hydrolysable tannins----Alkaloids and/or nitrogenous bases ++ +Flavonoids - -Sterols and/or triterpenes (Quassinoids) + + +Saponins - -Coumarins - -+ denotes the presence of the constituents- denotes the absence of the constituents

JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE)33VI. CONCLUSIONAilanthus excelsa bark Chloroform extract can be as a promisingsource as antiviral agent and this is due to the presence of theinteresting bioactive phytoconstituents as quassinoids andalkaloids (canthin-6-one ) and it may be appropriate for furthertherapeutic studies against herpes viruses.Conflict of interestThere is no conflict of interest associated with the authors of thispaper.REFERENCES[1] Clark, A. M. (1996). Natural Products as a Resource for New Drugs.Pharmaceutical Research 13: 1133-1141.[2] Cragg, G. M. and Newman, D. J. (2005). Plants as a source of anticanceragents. Journal of Ethanopharmacology 100: 72-79.[3] Shrimali, M., Jain, D. C., Darokar, M. P., Sharma, R. P. (2001).Antibacterial activity of Ailanthus excelsa (Roxb.). Phytotherapy Research15:165-166.[4] Joshi, B. C., Pandey, A., Sharma, R. P, Khare, A. (2003b). Quassinoidsfrom Ailanthus excelsa Phytochemistry 62: 579-584.[5] Dhanasekaran, S., Suresh, B., Sethuraman, M., Rajan, S. and Dubey, R.,(1993). Antifertility activity of Ailanthus excelsa Linn in female albino rats.Indian Journal of Experimental Biology 31:384-391.[6] Ogura, M., Cordell, G. A., Kinghorn, A. D., Farnsworth, N. R. (1977).Potential anticancer agents VI. Constituents of Ailanthus excelsa(Simaroubaceae). Lioydia 40: 579-584.[7] Asolkar, L. V., Kakkar, K. K., Chakre O. J. (1992) “ Glossary of IndianMedicinal Plants with Active Principles ” , C.S.I.R., New Delhi, Part-I, pp. 34-58.[8] Cordell GA, Ogura M, Farnsworth NR. (1978). Alkaloids constituents ofAilanthus excelsa. Lioydia 41, 166- 168.[9] Said A., Rashed K., Tokuda H., Huefner A.(2012). Antitumor activity ofAilanthus excelsa Roxb. stem bark fractions and of canthin-6-one. IUFSJournal of Biology 71(1): 112-121.[10] Nag A, Matai S. (1994). Ailanthus excelsa Roxb. (Simaroubaceae), apromising source of leaf protein. Journal of Agriculture and Food Chemistry,35: 1115-1117.[11] Khan, S. A, Shamsuddin, K. M. (1980). Glaucarubol from Ailanthusexcelsa. Phytochemistry 19:2484-2485.[12] Joshi, BC., Pendey, A, Chaurasia L., Pal M., Sharma R. P., Khare A.(2003a). Antifungal activity of the stem bark of Ailanthus excelsa.Fitotherpia 74:689-691.[13] Said A, Tundis R, Hawas WU, El-Kousy S, Rashed K, Menichini F,Bonesi M, Huefner A, Monica RL, Menichini F. (2010). In vitro Antioxidantand antiproliferative activities of flavonoids from Ailanthus excelsa (Roxb)(Simaroubaceae) leaves. Z. Naturforsch, 65c, 180-186.[14] Connolly J. D, Overton K. H, Polonsky J. (1970). The chemistry andbiochemistry of the linonoids and quassinoids. In: Reinhold l, Liwashitz Y(eds) Progress in phytochemistry. Wiley, London, pp 385-412.[15] Wolf, H. H, Swinyard E. A, Goodman L. S. (1962). Anticonvulsantproperties of some N-substituted hydanoins. J Phrama Sci 51:74-76.[16] Harbone, J. B. (1973). Phytochemical methods. Chapman & Hall,London, pp.40-107.[17] Farnsworth, N. R (1966). Biological and phytochemical screening ofplants. Journal of Pharmceutical Sciences 55, 225-276.[18] Geissman, T.A. (1962). The Chemistry of flavonoids compounds.Pergamon, London, pp.126-147.[19] Arisawa, M., Hayashi K, Nikaido T, Koike K, Fujita D, Nunomura N,Tanaka, M, Sasaki T. (1997). International J. of Pharmacology 35 (1):6-14.[20] Husing, G. D, Melnick J. L. (1957). Morphologic characteristics ofplaques produced on monkey kidney monolayer cultures by enteric viruses(poliomyelitis, Coxsackie, and echo groups. Journal of Immunology78:128-135.[21] Ren Z, Zhang C. H, Wang L. J, Cui Y. X, Qi R. B, Yang C. R, Zhang Y.J, Wei X.Y, Lu D. X, Wang Y. F. (2010). In vitro anti-viral activity of thetotal alkaloids from Tripterygium hypoglaucum against herpes simplex virustype 1. Virologica sinica 25:107-114.[22] IIkay O, Berrin Ozcelik, Taner K, Bilge S. (2007). Antiviral activity ofalkaloids isolated from Fumaria and Corydalis species. Z. Naturforsch, 62c,19-26.[23] Nugraha, A.S, Keller P.A. (2011). Revealing indigenous Indonesiantraditional medicine: anti-infective agents. Natural Product Communications6 (12):1953-1966.