Preliminary Cytotoxicity and Antimicrobial Investigation of Anthocaphalus chinensis

The methanol extract of the stem bark of Anthocephalus chinensis as well as its n-hexane, carbon tetrachloride, chloroform and aqueous soluble fractions were subjected to brine shrimp lethality bioassay and microbiological investigation. In the brine shrimp lethality bioassay, the aqueous soluble partitionate of the methanolic extract revealed the highest cytotoxicity having LC50 of 1.19 μg/ml while in case of antimicrobial screening, the chloroform soluble materials demonstrated moderate inhibition of growth of test organisms.


Materials and Methods
The stem bark of A. chinensis was collected from Boalmari, Faridpur and was identified at the Bangladesh National Herbarium, Mirpur, Dhaka (Accession no: DACB 31749).The bark was cut into small pieces, dried at room temperature for about 20 days and then ground to a coarse powder.
The powdered bark (350 g) of A. chinensis was soaked into 1.2 L of methanol for 10 days and then filtered through a cotton plug followed by Whatman filter paper no. 1.The extract was concentrated with a rotary evaporator.A portion (5.0 g) of the concentrated methanol extract (MSBE) was fractionated by the modified Kupchan partitioning method [9] into n-hexane, carbon tetrachloride, chloroform and aqueous soluble fractions.Subsequent evaporation of solvents afforded n-hexane (HXSP, 0.61 g), carbon tetrachloride (CTSP, 0.63 g), chloroform (CFSP, 0.92 g) and aqueous soluble materials (AQSP, 2.84 g).
For cytotoxicity screening, DMSO solutions of the plant extracts were applied against Artemia salina in a 1-day in vivo assay [10,11] using the established protocol.For the experiment, 1 mg of each of the Kupchan fractions was dissolved in DMSO and solutions of varying concentrations such as 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 µg/ml were obtained by serial dilution technique.The median lethal concentration LC 50 of the test samples after 24 hours of exposure was obtained by a plot of percentage of the shrimps killed against the logarithm of the sample concentration.
The antimicrobial activity of the extractives was determined by the disc diffusion method [12][13][14].The samples were dissolved separately in solvent (methanol or chloroform) and applied to sterile discs at a concentration of 400 µg/disc and carefully dried to evaporate the residual solvent.

Results and Discussion
Following the procedure of Meyer [10], the lethality of the MSBE, HXSP, CTSP, CFSP and AQSP partitionates of the methanolic extract of A. chinensis was investigated against brine shrimp.It was found (Table 1) that the degree of lethality was directly proportional to the concentration of the extract ranging from the lowest concentration (0.78 µg/ml) to the highest concentration (400 µg/ ml).Maximum mortality was seen at 400 µg/ ml, whereas least mortality was observed at 0.78125 µg/ ml.
The LC 50 obtained from the best-fit line slope were 0.23, 7.24, 5.25, 3.71, 3.50 and 1.19 µg/ml for Standard VS (vincristine sulfate), MSBE, HXSP, CTSP, CFSP and AQSP, respectively.For antimicrobial screening, the zones of inhibition produced by the chloroform soluble partitionate and aqueous soluble fraction of the methanolic extract of A. chinensis ranged from 8-12 mm and 8-11 mm, respectively at a concentration of 400 µg/disc (Table 2).The chloroform soluble extractive of the methanolic extract showed moderate inhibitory activity against S. paratyphi and S. typhi having the zone size 12 mm and 11 mm, respectively.This fraction also showed mild activity against S. lutea and V. mimicus (10 mm each).At the same time, the aqueous soluble fraction demonstrated mild inhibitory activity against S. typhi (11 mm), S. paratyphi and S. lutea (10 mm each).The methanolic extract of A. chinensis as well as its n-hexane and carbon tetrachloride soluble materials did not exhibit noticeable inhibition of growth of any microbe (data not shown).In case of fungal strains, the growth of S. cerevaceae was mildly inhibited by the chloroform soluble material.In comparison with positive control, the cytotoxicity exhibited by the extractives was promising.These bioactivities exhibited by the plant extractives substantiate the folk uses of the plant in several diseases.

Table 1 .
LC 50 data of test samples of A. chinensis.

Table 2 .
Antimicrobial activity of the A. chinensis extractives (400 µg/disc) and kanamycin (30 µg/disc).Diameter of zone of inhibition< 8 mm was considered inactive.Thus, the results of MSBE, HXSP, and CTSP were not shown in table.