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A Survey on Phytochemical and Bioactivity of Plant Extracts from Malaysian Forest Reserves

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A Survey on Phytochemical and Bioactivity of Plant Extracts from Malaysian Forest Reserves Journal of Medicinal Plants Research Vol. 4(3), pp. 203-210, 4 February 2010 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875© 2010 Academic Journals Full Length Research Paper A survey on phytochemical and bioactivity of plant extracts from Malaysian forest reserves J. Nardiah Rizwana, I. Nazlina*, A. R. Mohd Razehar, A. Z. Siti Noraziah, C. Y. Ling, S. A. Shariffah Muzaimah, A. H. Farina, W. A. Yaacob, I. B. Ahmad and L. B. Din Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi 43600, Selangor, Malaysia. Accepted 30 December, 2009 Methanolic extracts of leaves from plants collected from two different forest reserves, namely, Gunung Stong, Dabong, Kelantan and Ulu Muda, Kedah in Malaysia were subjected to a study in phytochemical constituents screen, toxicity to brine shrimp, cytotoxicity to normal cells and antiviral activity. All the plant extracts contain saponins except for Cyrtandra cf. anisophyllea. Goniothalamus macrophyllus contains alkaloids, steroids/triterpenes and saponins. All the tested extracts were not toxic to the brine shrimp. In the cytotoxicity assay, the CTC50 values recorded for all the extracts ranged between 21.540 mg/L (Galearia fulva) to more than 1000 mg/L (Cinnamomum javanicum, Cryptocarya costata, Scaphochlamys biloba, Diospyros frutescens, Galearia maingayi and Urophyllum blumeanum). From the CTC50 values, ten of the extracts were considered toxic to Vero cells. In the antiviral test carried out using 0.1 CTC50 of the various extracts, results showed that antiviral activity is effective when the cells were treated with the extracts and inoculated with measles virus at the same time. From this study, it was postulated that the most effective route for the extracts in preventing the killing of Vero cells by (MV) was through inactivation of virus particle or prevention of viral entry into the cells. Keywords: Phytochemical constituent, toxicity to brine shrimp, cytotoxicity, antiviral activity. INTRODUCTION World Health Organisation (WHO) has defined medicinal is still resisting eradication, and there is no available plants as plants that contain properties or compounds therapeutic treatment. that can be use for therapeutic purposes or those that In Malaysia, 2,000 species from 14,500 flowering plants synthesize metabolites to produce useful drugs (WHO, have been reported to contain medicinal properties and 2008). The development of antiviral drugs is a many have been scientifically proven (Jaganath and Ng, complicated task because of the toxicity that can affect 2000). Plants that were shown to have antiviral properties both virus and the host cells. Plus, virus resistance to include (Cymbopogon nardus (L.) Rendle) and Datura antiviral drugs through mutation especially RNA viruses stramonium L. against bacteria, Newcastle disease virus such as measles virus (MV) makes it more difficult to and measle virus (Ahmad et al., 1993; Nurul Aini et al., produce new drugs (Kott et al., 1999). MV causes 2006), Chaetomium trilaterale Chivers towards poliovirus measles and every year almost one million children died (Ahmad and Romlah, 1993), Melastoma malabathricum of this disease (Rager-Zisman et al., 2003). MV is an against herpes simplex virus type-1 and measles virus enveloped, negative, single-stranded RNA virus (Nazlina et al., 2008). Plant-derived compounds offer belonging to the Paramyxoviridae family (Horikami and potential source of new antimicrobial, anticancer and anti- Moyer, 1995). Despite large vaccination campaigns, MV HIV agents among other pharmaceuticals (Gurib-Fakim et al., 2005). Malaysian is wealthy in her plant diversity and potential antiviral drugs are still waiting to be ex- plored. This study focuses on plants that were collected *Corresponding author. E-mail: [email protected]. in Gunung Stong, Kelantan and Ulu Muda, Kedah both 204 J. Med. Plant. Res. Kedah both states of Malaysia with the hope of exploring RESULTS new compounds for anti-measles therapy with low or no toxicity. The plant chemical content and the antiviral Phytochemical constituents mode of action of the plant extracts were determined in this study. All the plant extracts except for Cyrtandra anisophyllea cf. contain saponins at different concentrations (Table 2). In this study, only two plant extracts, that is, Urophyllum MATERIALS AND METHODS blumeanum and Goniothalamus macrophyllus contain alkaloids. Five extracts contain steroids/triterpenes at low Plant identification and preparation of extracts levels and G. macrophyllus contains all three chemical Leaves from various plants were collected from Gunung Stong, groups with moderate amounts of alkaloids and saponins. Dabong, Kelantan and Ulu Muda, Kedah in Malaysia. The list of On the contrary, C. anisophyllea does not contain any of plants’ names, plant family, their specimen voucher numbers and the chemicals tested. their traditional uses are shown in Table 1. Extracts were prepared from dried leaves soaked in methanol for at least seven consecutive days and later dried using a rotary evaporator (Buchi). The alkaloids, steroids/triterpenes and saponins contents were Toxicity to brine shrimp determined using simple qualitative methods of Sofowora (1984) and Harborne (1993). In this study, all 30 plant extracts (20 extracts from Gunung Stong and 10 extracts from Ulu Muda) were not toxic to brine shrimp with the LC values are more than Brine shrimp lethality bioassay 50 1000 mg/L (Table 2). The toxicity of the different extracts was tested by lethality to Artemia salina brine shrimp according to Meyer et al. (1982). Concentrations of 10, 100 and 1000 ppm of each active extract Cytotoxicity to Vero cells were tested. The number of dead larvae was recorded and used to calculate the 50% lethal concentration (LC50) and 95% confidence Cytotoxicity of the plant extracts to Vero cells were intervals were determined from the 24 h counts using the Finney screened before the evaluation of antiviral activity and probability analysis software. LC50 values greater than 1000 ppm were considered inactive and not toxic. recorded in Table 2. According to Marini et al. (1998), any plants that have CTC50 values less than 208 µg/ml are considered toxic to cells. In this study, six plants from Ulu Cytotoxicity test Muda were found to be cytotoxic, that is, Connarus cochinchinensis, Dioscorea esculenta, Euonymus wrayi, Cytotoxicity testing was done according to Marini et al. (1998) with modification. Plant extracts were dissolved in DMSO to prepare a Hodgsonia macrocarpa, Kopsia pauciflora and stock extract of 105 mg/L. Serial doubling dilutions were prepared in Lasianthus densifolius and four plant extracts from Dubelco’s Minimal Essential Medium (DMEM) with 2% foetal bovine Gunung Stong (Gluta elegans, Saurauia roxburghii, serum (FBS) (GIBCO). Diluted extracts (200 µl) were treated to 5 Galearia maingayi and Tabernaemontana corymbosa) confluent Vero cells (2.5 x 10 cells/ml) grown in 96-well microtitre have CTC lower than 208 µg/ml (Table 2). It is a known plate (Costar). After 48 h of incubation, the microtitre plates were 50 processed and stained with 2% Eosin B. The dye was then fact that G. elegans is a toxic plant and can cause edema dissolved with 100 µl cell lysis buffer and the amount of dye and inflammation if touched (Wiart, 2002) and this is retained in each well was measured with microplate reader confirmed in this study. The other plants extracts have (Labsystems Multiscan Multisoft, Finland) at 540 nm. The CTC50 higher CTC50 values. Some of the plants used in folklore values were calculated graphically from the optical density readout medicine showed higher values in CTC50 in this study from each well after normalisation against empty wells. CTC50 value confirming the safety of these plants for human use was presented as the percentage of survived cells compared to control cells. (Wiart, 2002). Antiviral assay Antiviral activity of the plant extracts MV from Edmonston strain at 1000 TCID50 (Serum Institute of India In the first treatment, cells were infected with virus for 24 Ltd.) was used in this study. Confluent Vero cells were treated with MV using three different treatments for detection of antiviral activity h and treated with plant extracts. Poikilospermum in each plant extracts according to Nurul Aini et al. (2006) that is, I. suaveolens and Psueduvaria macrophylla showed virus (V) were inoculated to the cells (C) 1 h before treatment with moderate antiviral activity, three plant extracts (Saurauia extracts (E), that is (V+C)+E; II. Cells were treated with extract 1 roxburghii, Tabernaemontana corymbosa and Kopsia day before inoculation with virus, that is (C+E)+V and III. Virus and pauciflora) have mild antiviral activity and the rest of extracts were added concomitantly to the cells, that is C + (V+E). extracts demonstrated very mild or no antiviral activity. For the antiviral tests, the extracts were diluted to 0.1 CTC50. Plates were developed according to the method discussed in the Extract were added to the cells a day before virus cytotoxicity testing. inoculation in the second treatment. No extract showed Rizwana et al. 205 Table 1. Plant name and family, specimen voucher number, their local names, traditional uses and plants’ origin. Specimen Plant name/ Family voucher Local name Traditional uses Origin number Firewood, fruit is edible Saurauia roxburghii SK557/03 Kelapong, Lengadir (Faridah Hanum and van de Gunung Stong, Kelantan Actinidiaceae Maesen, 1997) Gluta elegans Has antitumor, antifungal and
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