EVALUATION OF ANTI-HIV-1 ACTIVITY FROM CESTRUM NOCTURNUM AERIAL PARTS AND PHYTOCHEMICAL ANALYSIS

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1 EVALUATION OF ANTI-HIV-1 ACTIVITY FROM CESTRUM NOCTURNUM AERIAL PARTS AND PHYTOCHEMICAL ANALYSIS 1 KHALED RASHED, 2,3 XING-JIE ZHANG, 2,3 MENG-TING LUO, 2 YONG-TANG ZHENG 1 National Research Centre, Pharmacognosy Department, Dokki, Giza, Egypt. 2 Key Laboratory of Animal Models and Human Diseases Mechanisms of Chinese, Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Laboratory of Molecular, Kunming, Yunnan, China. 3 Graduate School of the Chinese Academy of Sciences, Beijing, China. ABSTRACT This study deals with evaluation of HIV-1 activity from Cestrum nocturnum aerial parts extracts and investigation of phytoconstituents in the plant extracts. Different extracts from Cestrum nocturnum aerial parts, methanol 80%, ethyl acetate, butanol and aqueous extracts were tested for their anti-hiv-1 activity using the syncytia assay. All the extracts were weak active as anti-hiv-1 agents. Phytochemical analysis of Cestrum nocturnum extracts have shown the presence of different phytocomponents, saponins, triterpenes and/or sterols flavonoids, tannins and coumarins. The results confirm that all extracts from Cestrum nocturnum aerial parts have a weak anti-hiv-1 activity. KEYWORDS: Cestrum nocturnum, aerial parts, cytotoxicity, anti-hiv-1 activity, Phytoconstituents. INTRODUCTION Acquired immunodeficiency syndrome (AIDS) is a clinical syndrome that is the result of infection with human immunodeficiency virus (HIV), which causes profound immunosuppression. HIV-1 is the cause of the world epidemic and is mostly commonly referred as HIV. The herbal medicines are frequently used as an alternate therapy for inhibitory effects on HIV replication. Medicinal plants and their products may be explored as a source of new anti-hiv-1 agents. In addition, herbal medicines have some advantages such as fewer side effects, better tolerance, relatively less expensive and freely available (Mlinaric et al. 2000). Cestrum nocturnum L. is a garden shrub from Solanaceae family, and its flowers exude a special sweet fragrance at night, the main reason for the folk names, Night Cestrum, Lady of the Night and Night Blooming Jasmine (Roig, 1988). It is widely naturalized in tropical and subtropical regions throughout the world, including Australia, southern China and the southernmost United States. In traditional medicine, leaves of C. nocturnum have been used for their pharmacological significance in burns and swellings. It is also used for treating epilepsy and as stupefying charm medicine in West Indian Islands (Perez-Saad and Buznego, 2008). The volatile oil is known to be mosquito-repellent and hence C. nocturnum is used to prevent malaria in several African 1

2 Nations (Mimaki et al., 2006). Pharmacological studies on the plant proved that the leaves have significant analgesic and bactericidal activity (Huang et al., 2006). Local anesthetic effect, inhibitory effect on central nerve system and cardiac arrhythmic effect of plant are also documented (Zeng et al., 2002). Furthermore, n-butanol and polysaccharides extracts from C. nocturnum had obvious in vivo effects on tumor inhibition (Zhong et al., 2008). Some phytochemical studies on leaves from C. nocturnum proved the presence of a calcinogenic glycoside (Mello, 2003) and other glycosides such as nocturnoside A and nocturnoside B (Ahmad et al., 1995), phenol glucosides (cesternosides A and B) (Sahai et al., 1994), flavonol glycosides and steroidal saponins and glycosides (Mimaki, 2001; Mimaki et al., 2002). The aim of the present study, evaluation of HIV-1 activity from Cestrum nocturnum aerial parts extracts, methanol 80%, ethyl acetate, butanol and aqueous extracts, as well, investigation of phytoconstituents in these extracts. Materials and methods Plant Material The aerial parts of Cestrum nocturnum were collected from the Agricultural Research Centre, Giza, Egypt in May 2011 during flowering and identified by Dr. Mohammed El-Gebaly, Department of Botany, National Research Centre (NRC) and by Mrs. Tereez Labib Consultant of Plant Taxonomy at the Ministry of Agriculture and director of Orman botanical garden, Giza, Egypt. A voucher specimen is deposited in the herbarium of Agricultural Research Centre, Giza, Egypt. Chemicals AZT (3 -azido-3 -deoxythymidine) was purchased from Sigma. All phenolic compounds were dissolved in DMSO. AZT was dissolved in RPMI-1640 and stored at -20. Reagents HEPES (N-2 (2-Hydroxyothyl) piperazine-n'-(2-ethanesufonic acid), MTT (3,(4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide), DMF (N, N'- Dimethyl formamine), Penicillin, Streptomycin sulfate, Glutamine were purchased from Sigma; 2-ME (2- Mercaptoethanol) was purchased from Bio-Rad. RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco. Cells and virus C8166 cells and HIV-1 IIIB were kindly donated by Medical Research Council, AIDS Regent Project. The cells were maintained at 37 in 5% CO 2 in RPMI-1640 medium supplemented with 10% heat-inactivating FBS (Gibco). HIV-1 IIIB was prepared from the supernatants of H9/HIV- 1 IIIB cells. The 50% HIV-1 tissue culture infectious dose (TCID 50 ) in C8166 cells was determined and calculated by Reed and Muench, Virus stocks were stored in small aliquots at

3 Preparation of the extracts Finely ground aerial parts from Cestrum nocturnum 640 g were extracted with methanol 80% by maceration. The extract was concentrated to dryness at 50 о C to yield 49 g of crude extract. 80% extract was defatted with n-hexane and then fractionated with ethyl acetate and butanol. The combined organic layer of each partition was evaporated to dryness using rotary evapoator to yield 8.5 g of ethyl acetate extract, 12 g of butanol extract and the aqueous extract was 16.5 g. Each extract was tested for the presence of the phytochemicals according to the following standard tests, Molisch 's test for carbohydrates, Shinoda test for flavonoids, forth test for saponins, Salkowski 's for terpenes and sterols, FeCl 3 and Mayer's reagents for detecting of tannins and alkaloids, respectively (Sofowra 1993, Trease and Evans 1989, Harborne 1973). Results: The results of cytotoxicity and anti-hiv-1 activity from Cestrum nocturnum aerial parts extracts are shown in table 1, table 2 and table 3, and the results of the phytoconstituents from Cestrum nocturnum aerial parts extracts are shown in table 4. Table 1 Cytotoxicity of Cestrum nocturnum aerial parts extracts in C8166 cell Extracts Concentration (µg/ml) Cell viability ±SD CC 50 (µg/ml) 80% Ethyl acetate Butanol Aqueous ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.20 > >1000 >200 3

4 AZT ± ± ± ± ± ± ± ± ± ± Anti-HIV activity of the extracts Table 2 Anti-HIV activity of Cestrum nocturnum aerial parts extracts in C8166 cell Extracts Concentration (µg/ml) Inhibition ± SD EC 50 (µg/ml) 80% Ethyl acetate Butanol Aqueous AZT ± ± ± ± ± ± ± ± ± ± ± ± ± Table 3. The summary of cytotoxicity and anti-hiv-1 activities of Cestrum nocturnum aerial parts extracts Extracts Experiment Method CC 50 EC 50 Therapeutic (µg/ml) (µg/ml) index (TI) Cytotoxicity assay MTT >200 > % Inhibition of syncytium CPE

5 Ethyl Cytotoxicity assay MTT acetate Inhibition of syncytium CPE Butanol Cytotoxicity assay MTT >1000 >2.99 Inhibition of syncytium CPE Aqueous Cytotoxicity assay MTT >200 >3.39 Inhibition of syncytium CPE AZT Cytotoxicity assay MTT Inhibition of syncytium CPE ng/ml Table 4: Phytochemical Analysis of the cestrum nocturnum aerial parts extracts Constituents 80% Ethyl acetate Butanol Aqueous Triterpenes and /or Sterols (Quassinoids) Carbohydrates and/or glycosides Flavonoids Coumarins Alkaloids and/or nitrogenous compounds Tannins Saponins (+) presence of constituents, (-) absence of constituents DISCUSSION Plant substances continue to serve as good sources of drugs for the world population and several plant-based drugs are in extensive clinical use. Medicinal plants are considered safer, and cheaper compared to synthetic drugs that have adverse effects. Cytotoxicity of Cestrum nocturnum aerial parts extracts, methanol 80%, ethyl acetate, butanol, and aqueous was carried out by using MTT colormetric methods. The results showed that Cestrum nocturnum extracts were minimal toxic and showed weak anti-hiv-1 activity. Ethyl acetate fraction from methanol (80%) extract of Cestrum nocturnum had a lowest cytotoxic effect, it was significantly different from that of the other extracts (Table 1). The anti-hiv-1 activity assay was performed by synctia. 80% ethyl acetate, butanol, and aqueous extracts showed weak anti-hiv-1 activity and their therapeutic index (TI) value were lower than 10 (table 2, table 3) in which the significant results from the 5

6 plant extracts as anti-hiv-1 agents are measured by their values of TI which should be more than 10 and less than 100 with comparison with AZT. The phytochemical analysis results confirmed the presence of the following different phytochemicals in the extracts, saponins, tannins, flavonoids, triterpenes and/or sterols and carbohydrates, and (Table 4) CONCLUSION In this research paper, we extracted Cestrum nocturnum aerial parts with methanol 80% and fractionated it with ethyl acetate and butanol. 80%, ethyl acetate, butanol and aqueous extracts were tested for their cytotoxicity and anti-hiv-1 activities, as well were phytochemically screened and the results showed that all the extracts were less active as anti- HIV-1 agent and the extracts have different phytoconstituents, saponins, triterpenes and/or sterols, flavonoids, tannins, and carbohydrates. Conflict of interest There is no conflict of interest associated with the authors of this paper. REFERENCES Ahmad VU, Baqai FT, Ahmad RZ (1995). A spirostanol. Naturforsch 50, Bauer J, Rojas R, Bustamante B Antimicrobial activity of selected Peruvian medicinal plants. Journal of Ethnopharmacology 88, Harborne J.B Phytochemical Methods. Chapman and Hall. Ltd., London. pp Huang LG, Zhang XC, Xiao H, Ye HY, Zeng J (2006). Analgesic effect of Cestrum nocturnum L. extract on mice. Chin. J. Clin. Rehab 10, Mello JRB (2003). Calcinosis-Calcinogenic plants (Review). Toxicon. 41, Mimaki Y, Ntonifor N N, Ngufor CA, Kimbi HK, Oben BO Traditional use of mosquito repellent to protect human against mosquito and other insect bites in rual community of Cameroon. East African Medicinal Journal 83, Mimaki Y, Watanabe K, Ando Y, Sakuma C, Sashida Y, Furuya S, Sakagami H (2001). Flavonol Glycosides and Steriodal Saponins from the leaves of Cestrum nocturnum and their cytotoxicity. J. Nat. Prod. 64, Mimaki Y, Watanabe K, Sakauma C, Sashida Y (2002). Steriodal Glycosides from the Leaves of Cestrum nocturnum J. Nat. Prod. 65, Mlinaric A, Kreft S, Umek A. Screening of selected plants extracts for in-vitro inhibitory activity on HIV reverse transcriptase. Pharmazie 2000, 55: Perez-Saad H, Buznego M.T Behavioral and antiepileptic effects of acute administration of the extract of the plant Cestrum nocturnum L. (lady of the night). Epilesy and Behaviour 12,

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