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ORIGINAL
ARTICLE
Assessment Of Bioactivity Of Bangladeshi Medicinal Plants Using Brine Shrimp
Lethality Assay
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M. Hasanur Rahman, Md. Mumit Hossain, Mohammed Rahmatullah
Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh.
Firoz Ahmed, Fatema Islam, Nusratun Nahar, Mithil Ahmed, Aynal Haque Rana, K.M. Hasanur Rahman, Md. Mumit Hossain, Mohammed Rahmatullah: Assessment Of Bioactivity Of Bangladeshi Medicinal Plants Using Brine Shrimp Lethality Assay
ABSTRACT
Twenty six Bangladeshi medicinal plants used in traditional medicines were evaluated for brine shrimp lethality toxicity. Different solvent extracts of Trachyspermum ammi, Cissampelos pareira, Vetiveria zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba, Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides, Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia, Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria were used in the study. Of the 26 plants tested, 20 plants (76.9%) were toxic to brine shrimp (LC50 < 30 microg/ml). Among the extracts screened, the ethanolic extract of Spinacia oleracea leaves and methanolic extract of Amaranthus viridis whole plants had the highest toxicity to brine shrimp (LC50 = 0.06 microg/ml). The drug vincristine sulfate was considered as reference standard.
Key words: Cytotoxicity, brine shrimp, Bangladesh, medicinal plants
Introduction
A large percentage of the population of developing countries depends on traditional medicines for their primary health-care needs (FAO, 2004). Bangladesh has a rich heritage of herbal medicines among the South Asian countries. The poor and ethnic peoples of Bangladesh rely on medicinal plants as prescribed by traditional medicinal practitioners for treatment against various diseases. Although a large number of medicinal plants are used in the traditional medicinal system of Bangladesh, the majority of these plants have not yet undergone chemical, pharmacological and toxicological studies to investigate their bioactivity (Ghani, 2003). Traditional records and ecological diversity indicate that Bangladeshi plants represent an exciting resource for possible lead compounds in drug design and development (Uddin et al., 2011).
Twenty six Bangladeshi medicinal plants (Trachyspermum ammi, Cissampelos pareira, Vetiveria zizanioides, Cassia angustifolia, Woodfordia fruticosa, Cinnamomum tamala, Neolomarckia cadamba, Amaranthus viridis, Amaranthus tricolor, Brassica juncea, Brassica oleracea, Raphanus sativus, Curcuma longa, Curcuma zedoaria, Elettaria cardamomum, Ficus religiosa, Ficus benghalensis, Prunus cerasoides, Chenopodium album, Spinacia oleracea, Symplocos racemosa, Terminalia chebula, Tinospora cordifolia, Cyperus rotundus, Pterocarpus santalinus, and Lagenaria siceraria) were collected from various regions of Bangladesh following accounts of their medicinal uses (Ghani, 2003; Yusuf et al., 1994) or when our ongoing ethnomedicinal surveys (Nawaz et al.,2009; Rahmatullah et al.,2009a-c; Chowdhury et al., 2010; Hasan et al., 2010; Hossan et al., 2010; Mollik et al., 2010a,b; Rahmatullah et al.,2010a-g; Haque et al., 2011; Islam et al., 2011; Jahan et al., 2011; Rahmatullah et al., 2011a,b; Sarker et al., 2011; Das et al., 2012; Rahmatullah et al., 2012a-d) indicated that they were used by traditional medicinal practitioners for treatment of various ailments. Extracts of various plant parts were screened for their cytotoxic activities.
et al. (1981) and Sleet and Brendel (1983). We used this assay for determination of the cytotoxic potential of the 26 Bangladeshi medicinal plants in the present study.
Materials and Methods
Plant materials:
The plant samples were collected from various regions of Bangladesh based on their ethnomedicinal uses. To determine whether a plant has any ethnomedicinal use, surveys were conducted among folk and tribal medicinal practitioners as described before in the Introduction. All plants were identified at the Bangladesh National Herbarium at Dhaka where voucher specimens were also deposited. The various plant parts were cut into small pieces, air-dried under shade and grounded using a laboratory mill and blender.
Extraction:
A known amount of dried powdered sample (usually 100g) was extracted by maceration with methanol or chloroform at a ratio of plant material to solvent of 1:5. Collected extracts were then filtered and concentrated in vacuo at 40oC. The solvent-free extracts were kept refrigerated at 4oC and used for cytotoxicity assays.
Toxicity testing against brine shrimp:
Hatching shrimp. Brine shrimp eggs, Artemia salina leach were hatched in artificial seawater prepared by dissolving 38g of sea salt in 1L of distilled water. The pH of the solution was adjusted to 8.5. After 48h incubation at room temperature (26-30oC) under constant aeration, the larvae (nauplii) were attracted to one side of the vessel with a light source and collected with a pipette. Nauplii were separated from eggs by aliquoting them three times in small beakers containing seawater.
Brine shrimp assay. The bioactivity of the extracts was monitored by the brine shrimp lethality test (Meyer
et al., 1982). Samples were dissolved in dimethylsulfoxide (DMSO) and diluted with artificial sea salt water so that final DMSO concentration did not exceed 0.05%. 50 l of 2000 g of the plant extract was placed in one sample tube and a two-fold dilution carried out down the column of sample tubes. The last sample tube was left with sea salt water and DMSO only to serve as the drug-free control. The total volume was adjusted to 5 ml with sea salt water. 100 l of suspension of nauplii containing 10 larvae was added into each tube and incubated for 24h. The tubes were then examined under a magnifying glass and the number of dead nauplii in each tube counted. Experiments were conducted with control (vehicle treated), and different concentrations of the test substances in a set of three tubes per dose. Vincristine sulfate was used as a positive control in all experiments.
Statistical analysis:
Lethality assays were evaluated by Finney computer statistical program to determine the LC50 values and 95% confidence intervals. All data were expressed as mean (Microg/ml) (Zhao et al., 1992; McLaughlin, 1991).
Results and Discussion
In the present study, DMSO was used as the solvent and as negative control. This is in accordance with previous report that brine shrimp nauplii can tolerate up to 11% of DMSO (Sam, 1993). Further studies are being conducted to isolate and purify the bioactive constituents for further evaluation in human cell line cultures for cytotoxic effects.
Table 1: The LC50 values of different Bangladeshi medicinal plant extracts against Artemia salina. Serial
number
Scientific name of plant Family Part extracted Solvent used for extraction
Apiaceae Fruit Ethanol 11.11
2 Cissampelos pareira L. Menispermaceae Root Ethanol 40.10 3 Vetiveria zizinioides (L.)
Nash.
Poaceae Root Ethanol 67.48
4 Cassia angustifolia Vahl.
Fabaceae Leaf Ethanol 109.35
5 Woodfordia fruticosa (L.) Kurz.
Myrtaceae Flower Ethanol 140.89
6 Cinnamomum tamala (Ham.) Nees & Eberm.
Lauraceae Leaf Ethanol 142.50
7 Neolomarckia cadamba (Roxb.) Bosser
Rubiaceae Leaf Methanol 36.72
8 Amaranthus viridis L. Amaranthaceae Whole plant Methanol 0.06 9 Amaranthus tricolor L. Amaranthaceae Whole plant Methanol 0.36 10 Brassica juncea (L.)
Czern.
Cruciferae Leaf Ethanol 17.04
11 Brassica oleracea L. Cruciferae Leaf Ethanol 7.81 12 Raphanus sativus L. Cruciferae Leaf Ethanol 0.13 13 Curcuma longa L. Zingiberaceae Rhizome Ethanol 3.88 14 Curcuma zedoaria 17 Ficus benghalensis L. Moraceae Bark Ethanol 6.82 18 Prunus cerasoides D.
Don.
Rosaceae Stem Ethanol 0.75
19 Chenopodium album L. Chenopodiaceae Whole plant Methanol 0.07 20 Spinacia oleracea L. Chenopodiaceae Leaf Ethanol 0.06 21 Symplocos racemosa
Menispermaceae Stem Ethanol 15.29
24 Cyperus rotundus L. Cyperaceae Root Ethanol 1.03 25 Pterocarpus santalinus
L.
Fabaceae Stem Ethanol 22.03
26 Lagenaria siceraria
The results are presented as LC50 values (microg/ml).
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