Vol2 no3 5 by Tarig Khider

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE )

RESEARCH ARTICLE 30

Antiviral Activity and Phytochemical Analysis of Ailanthus Excelsa Roxb Bark *

Khaled Rashed 1, Ataa Said 1, Mohammed Ahmed2

Pharmacognosy Department, 2Virology Department, National Research Centre, Dokki, Giza, Egypt. *Corresponding author: Dr. Khaled Nabih Zaki Rashed, Tel: 01003642233 E-mail: khalednabih2015@yahoo.co.uk (Received March 20, 2013; Accepted April 25, 2013)

 Abstract— Resistance to current anti-herpetic drugs has been increasingly reported, so there is a need for discovering new antiviral agents, in particular from natural origin. The aim of the present study is to investigate chloroform and methanol (70%) extracts from Ailanthus excelsa bark for their antiviral activity against Herpes Simplex virus type 1 (HSV-1) using Plaque reduction assay. The results has shown that chloroform extract of Ailanthus excelsa has a significant anti-viral activity against herpes Simplex virus type 1 in-vitro by 82.6% at concentration of 50 µg, while methanol 70% extract was less active. Phytochemical analysis of both Chloroform and methanol extracts of Ailanthus excelsa has shown that it has interesting bioactive compounds include quassinoids (highly oxygenated triterpenes) and alkaloids and further phytochemical analysis from bio-active chloroform extract resulted the isolation of the major alkaloid compound, canthin-6-one. In conclusion, Ailanthus excelsa extracts may be an appropriate candidate for further development of anti-HSV-1 infection.

arranged in panicles and the fruits are formed soon after flowering. The fruits ripen in May-June, just before the onset of monsoon. A. excelsa was investigated previously to prove antibacterial [3], antifungal [4], Antifertility [5] and anticancer [6]. A. excelsa is used in treatment of skin erpution and for the cure of wounds, The bark is bitter, astringent, anthelmentic, febrifuge, appetizer, bitter tonic, taste bud stimulant, It is useful in 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 of alkaloids [8, 9] proteins [10], quassinoids [11, 12] and flavonoids were isolated from leaves [13]. In the present study, we investigated antiviral activity of chloroform and methanol extracts from A. excelsa bark against Herpes Simplex virus type 1 (HSV-1) in-vitro and also determined the major compound in the bioactive chloroform extract. II. MATERIALS AND METHODS

Index Terms— Ailanthus excelsa, antiviral activity, quassinoids, alkaloids, canthin-6-one.

I. INTRODUCTION iral infections, particularly the infections caused by herpes simplex virus (HSV), represent one of the most serious public health concerns globally because of their devastating impact. Herpes simplex virus type 1 (HSV-1) is a commonly occurring human pathogen worldwide. There is an urgent need to discover and develop new agents for the management of HSV-1 infection. Since the ancient times, natural products has served as a major source of drugs. About fifty percent of todays pharmaceutical drugs are derived from natural origin [1]. Also it is well established that natural products are an excellent source of chemical compounds with a wide variety of biological activities including antiviral and anticancer properties [2]. In our searching for new antiviral substances from plants, in particular from Simaroubaceae family, A. excelsa Roxb is a tree of rapid growth and is called tree of heaven, leaves appear in March-April, 30-90 cm long, pinnate, the flowers, small in size, yellow in colour and

*Corresponding author.

Plant Material A. excelsa bark was collected from Zoo garden, Giza, Egypt in May 2010. The plant was identified by Dr. Mohamed ElGabaly, Professor of Taxonomy, National Research Centre. A voucher specimen no. 13241 is deposited in the herbarium of Zoo garden, Giza, Egypt. Preparation of extracts and isolation of the major compound of chloroform extract The Air dried powered bark of A. excelsa 400 g was successively extracted with Chloroform and methanol70% by maceration for 24 hours and each extract was concentrated under reduced pressure to give 15 g and 23 g of crude extracts, respectively. Each extract of A. excelsa was phytochemically screened according to the following described by (Connolly et al. 1970) [14] for sterols and/or triterpenes (quassinoids); Wolf et al. 1962) [15] for carbohydrates and saponins; Harbone 1973) [16] for flavonoids and alkaloids; Farnsworth 1966) [17] for coumarins; Geissman 1962 for tannins [18] Confirmation of the presence of quassinoids and alkaloids were detected by Thin layer chromatography (TLC) by spraying with specific reagents. Chromatographic separation of chloroform extract on silical gel column chromatography (0.06-0.2 mm) eluted with chloroform: methanol (99:1)

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE ) 31 resulted in the isolation of major compound of the chloroform extract, canthin-6-one, detection of this compound was detected by sparing with dragendroff reagent which is specific for alkaloids on TLC in which the compound gave red-orange spot. Preparation of the extracts for bioassay Extracts were dissolved as 100 mg in 1 ml of 10% DMSO in water. The final concentration was 100 µg/ µl (Stock solution). The dissolved solutions were sterilized by addition of antibiotic antimycotic mixture [19] Sterility test were carried out in nutrient agar. Cell Culture African green monkey kidney-derived cells (VERO) were used. The cells were propagated in Hanks٫ Minimum essential medium, MEM supplemented with 10% Foetal bovine serum, 1% antibiotic-antimycotic mixture. The pH was adjusted at 7.2-7.4 by 7.5% sodium bicarbonate solution. The mixture was sterilized by filtration through 0.2 µm pore size nitrocellulose membrane.

isolated from the bioactive chloroform extract and the chemical structure of the compound was identified by 1HNMR, 13C-NMR and MS Structure elucidation of the alkaloid compound, canthin-6-one: Canthin-6-one: 1H NMR (400 MHz, CDCl3): 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, H9), 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). 13C NMR (100 MHz, CDCl3): 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) and 159.59 (C-6). EI-MS: m/z 220. 11

1 11a

Plaque reduction assay

The 5 A 6-well plate was cultivated with Vero cell culture (10 cell/ ml) and incubated for 2 days at 37 C. HSV-1 was diluted to give 104 PFU/ml final concentration and mixed with the plant extract at 100 mg in 1 ml of 10% DMSO in water and incubated overnight at 4C. Growth medium was removed from the multiwell plate virus-compound mixture was inoculated (100 µg/well). After 1 hr contact time, the inoculum was aspirated and 3 ml of Minimal Essential Medium (MEM) with 1% agarose was overlaid the cell sheets. The plates were left to solidify and incubated at 37C until the development of virus plaques. Cell sheets were fixed in 10% formaline solution for 2 hrs, and stained with crystal violet stain. Control virus and cells were treated identically without chemical compound. Virus plaques were counted and the percentages of reduction were calculated [20]. III. RESULTS AND DISCUSSION RESULTS The Results of antiviral activity of Ailanthus excelsa stem bark extracts are included in table 1, it has shown that chloroform extract is more potent than methanol extract as anti-HSV-1 agent, where chloroform extract showed virus reduction by 82.6, while methanol extract showed virus reduction by 52 at the concentration of 50 µg. Phytochemical analysis of the extracts are included in table 2 which prove that each extract has interesting bio-active compounds, quassinoids, and alkaloids and the major alkaloid compound, canthin-6-one was

7 7a

Viruses Herpes Simplex virus type 1 was obtained from Environmental Virology Lab., Department of Water Pollution Research, National Research Centre. Antiviral assay was carried by Plaque reduction assay [20].

11b

11c

4 5

Figure 1. Canthin-6-one DISCUSSION present study for the first time, proved antiviral activity of A. excelsa bark extracts. The results has shown that chloroform has a good antiviral activity than methanol extract (Table 1) in a dose dependent mannar. Chloroform extract of A. excelsa stem bark at concentration 20 µg has shown an inhibition for the virus by 66.5%, and at concentration of 50 µg, chloroform fraction of A. excelsa stem bark has a significant anti-viral activity by inhibition for the virus by 82.6%. Methanol extract at concentration 20 µg has shown an inhibition for the virus by 44.9%, and at concentration of 50 µg, it has shown inhibition of the virus by 52%. The anti-HSV-1 properties of A. excelsa extracts in this research could be due to multiple different components in these extracts. The phytochemical characterization of extracts and the identification of the bioactive compounds are now needed. Phytochemical analysis of the extracts has shown that both extracts have interesting bioactive compounds include quassinoids and alkaloids (Table 2). Chloroform extract contained canthin-6-one as the major component and the higher activity of chloroform extract is probably due to the higher concentration of these bioactive compounds in the chloroform extract, while methanol is less active due to its low concentrations of these bioactive compounds. Analysis of both extracts by TLC has proved many spots for chloroform extract and less number of spots for methanol extract and these spots were detected under ultraviolet (UV) light. Spraying these spots with vanillin-sulphuric acid reagent followed by heating for 5 min until the appearance of purplish-blue colours specific for

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE ) 32 quassinoids and Spraying with draggendror's reagent resulted in the formation of an orange colour specific for alkaloids. The HSV-1 activity of chloroform extract may be due to the presence of alkaloids (canthin-6-one) and quassinoids which present in considerable amounts in the extract [8], [9], [11], [4] and this is in agreement with the activity of alkaloids isolated from Tripterygium hypoglaucum against herpes simplex virus type1 [21] Also the antiviral activity of alkaloids was confirmed Table 1

from Fumaria and Corydalis species [22] as well Quassinoids have shown anti-HSV-1 [23] Chloroform extract of A. excelsa bark has considerable amounts of alkaoids and quassionoids and the major component is the alkaloid is canthin-6-one while methanol has the same bioactive chemical compounds but in less amount, so Chloroform extract has higher antiviral activity than methanol.

Anti-HSV-1 bioassay in Vero cell line by plaque reduction assay of A. excelsa extracts HSV-1 Extract

Concentration

Initial virus count (PFU/ml)

Virus count

% of Virus reduction

(PFU/ml) Chloroform

Methanol

20 µg

1.96 x 107

1.22 x 107

66.5

50 µg

1.96 x 107

1.2 x 107

82.6

20 µg

1.96 x 107

1.08 x 107

44.9

50 µg

1.96 x 107

0.94 x 107

Table 2 Results of Phytochemical analysis of A. excelsa extracts Chemical Constituents

Chloroform extract

Methanol extract

Carbohydrates and/or glycosides

Condensed tannins

Hydrolysable tannins

Alkaloids and/or nitrogenous bases

Flavonoids

Sterols and/or triterpenes (Quassinoids)

Saponins

Coumarins

Tannins a. b.

+ denotes the presence of the constituents - denotes the absence of the constituents

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(3), 30-33 ISSN:2325–4513(PRINT) ISSN 2325 - 453X (ONLINE ) 33 VI. CONCLUSION Ailanthus excelsa bark Chloroform extract can be as a promising source as antiviral agent and this is due to the presence of the interesting bioactive phytoconstituents as quassinoids and alkaloids (canthin-6-one ) and it may be appropriate for further therapeutic studies against herpes viruses. Conflict of interest There is no conflict of interest associated with the authors of this paper. 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 anticancer agents. 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 Research 15:165-166. [4] Joshi, B. C., Pandey, A., Sharma, R. P, Khare, A. (2003b). Quassinoids from 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 Indian Medicinal Plants with Active Principles”, C.S.I.R., New Delhi, Part-I, pp. 3458. [8] Cordell GA, Ogura M, Farnsworth NR. (1978). Alkaloids constituents of Ailanthus excelsa. Lioydia 41, 166- 168. [9] Said A., Rashed K., Tokuda H., Huefner A.(2012). Antitumor activity of Ailanthus excelsa Roxb. stem bark fractions and of canthin-6-one. IUFS Journal of Biology 71(1): 112-121.

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