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

RESEARCH ARTICLE 30

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.

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.

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

Plant Material

A. excelsa bark was collected from Zoo garden, Giza, Egypt in

May 2010. The plant was identified by Dr. Mohamed El-

Gabaly, 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)

Antiviral Activity and Phytochemical Analysis of

Ailanthus Excelsa Roxb Bark

*Khaled Rashed

1, Ataa Said

1, Mohammed Ahmed

2

1Pharmacognosy 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)

V

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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.

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].

Plaque reduction assay

A 6-well plate was cultivated with Vero cell culture (105

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 4C. 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 37C 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

isolated from the bioactive chloroform extract and the

chemical structure of the compound was identified by 1H-

NMR, 13

C-NMR and MS

Structure elucidation of the alkaloid compound, canthin-6-one:

Canthin-6-one: 1

H 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, 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, 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.

NN

O

9

5

2

11c

11b11a

11

10

8

7a

6

7

4

3a3

1

Figure 1. Canthin-6-one

DISCUSSION

The 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

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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

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.

Table 1

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

Table 2

Results of Phytochemical analysis of A. excelsa extracts

+ denotes the presence of the constituents

- denotes the absence of the constituents

Extract

Concentration

HSV-1

Initial virus count

(PFU/ml)

Virus count

(PFU/ml)

% of Virus reduction

Chloroform 20 µg 1.96 x 107 1.22 x 10

7 66.5

50 µg 1.96 x 107 1.2 x 10

7 82.6

Methanol

20 µg 1.96 x 107 1.08 x 10

7 44.9

50 µg 1.96 x 107 0.94 x 10

7 52

Chemical Constituents Chloroform extract Methanol extract

Carbohydrates and/or glycosides - +

Tannins

a. Condensed tannins

b. Hydrolysable tannins

-

-

-

-

Alkaloids and/or nitrogenous bases ++ +

Flavonoids - -

Sterols and/or triterpenes (Quassinoids) + + +

Saponins - -

Coumarins - -

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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.

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