Biological Activity Study on a Malvaceae Plant : Bombax ceiba

The work described in this paper details the biological investigation on Bombax ceiba, species of Malvaceae. The methanol crude extract of Bombax ceiba was fractionated with kupchan method and n-hexane, carbon tetrachloride, chloroform fraction were made for screening the antimicrobial and antitumor potentials using disc diffusion method and brine shrimp lethality bioassay respectively. An established antibiotic (kanamycin, 30μg/disc) and cytotoxic agent (vincristine sulphate) were used to compare the results. From the graphs the concentration of methanolic crude extract give LC50 (50% mortality) value of 3.90μg/ml. LC90 was also determined from the graph to establish the therapeutic index and the value was found 150.0μg/ml. The four fractions were assayed for antimicrobial screening and the carbon tetrachloride fraction showed most prominent zone of inhibition against a number of bacterial and fungal strains.

Bangladesh is a good storehouse of medicinal plants.Malvaceae is a family of flowering plants containing a number of genera, generally with five showy petals and five green sepals and numerous stamens that are fused at the base and form a tube around the pistil.Most species are herbs or shrubs but some species are trees.There are over 100 genera with close to 1,500 species in the family depending on the classification of some of the genera.In the broader APG circumscription, the Malvaceae are also a cladistically monophyletic group but take in a much larger number of genera, Bombacaceae is one of them that include a species named Bombax ceiba.They are native to tropical areas in western Africa, the Indian subcontinent, Southeast Asia.Common names for the genus include Silk Cotton Tree, Simal, Red Cotton Tree, Kapok [1][2][3][4][5][6][7][8][9][10][11].

Plant materials
Root sample of Bombax ceiba was collected from an ayurbadic shop of Dhaka new market in September 2007.It was then air-dried and powdered with crushing machine.Then the powdered material was successively extracted with methanol by using cold extraction process [18].The crude extract was then fractionated into n-hexane, carbon tetrachloride and chloroform by using kupchan partitioning method [19].

Method for cytotoxic study
4.0 mg of each fraction (n-hexane, carbon tetrachloride, chloroform and methanol) of Bombax ceiba were taken and dissolved in 200µl of pure dimethyl sulfoxide (DMSO) in two vials to get stock solutions of 400µg/ml.A series of solutions of different concentrations were prepared from the stock solution by serial dilution method and the concentrations were as: 400µg/ml, 200µg/ml, 100µg/ml, 50µg/ml, 25µg/ml, 12.5µg/ml, 6.25µg/ml, 3.125µg/ml, 1.5625µg/ml and 0.78125µg/ml.Then the samples were subjected to brine shrimp lethality bioassay [20,21] for cytotoxic studies.In each test tube, containing different concentrations of test sample, 10 brine shrimp nauplii (Artemia salina) were added.
Two control groups were used in cytotoxicity study, to validate the test method and results obtained due to the activity of the test agent.In the study vincristine sulphate was used as the positive control.Measured amount of the vincristine sulphate was dissolved in DMSO to get an initial concentration of 20 µg/ml and serial dilutions were made using DMSO to get 10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 µg/ml and 0.0390 µg/ml of concentration.30 µl of DMSO was added to each of three premarked glass vials containing 5 ml of simulated seawater and 10 shrimp nauplii to use as negative control groups.After 24 hours, the test tubes were observed and the numbers of survived nauplii in each test tube were counted and the results were noted.From this, the percentage of lethality of brine shrimp nauplii was calculated at each concentration for each sample.

Method for antimicrobial assay
Collected all fractions, i.e. n-hexane, carbon tetrachloride, chloroform and methanol extracts were tested for antimicrobial study by using standard disc diffusion method [22,23].In this study, 16 microorganisms were obtained from the Institute of Nutrition and Food Sciences (INFS), University of Dhaka, Bangladesh.Standard Kanamycin (30 µg/disc) and blank sterile filter paper disc (diameter, 6 mm) were used as positive and negative controls, respectively.Nutrient agar medium (DIFCO) was used in the present study for testing the sensitivity of the organisms to the test materials and to prepare fresh cultures.The sample discs, the standard antibiotic discs and the control discs were placed gently on the previously marked zones in the agar plates, pre-inoculated with test bacteria.The discs were then incubated on the plate aerobically at 37ºC for 24 hours.The diameter of inhibition zone around each disc was measured and recorded at the end of the incubation period.

Cytotoxic study
In the present bioactivity study, the crude methanolic extract showed positive results indicating that the test samples are biologically active.Plotting of log of concentration (log C) versus percent mortality (% mortality) for all test samples showed an approximate linear correlation (Fig. 1).From the graph, the median lethal concentration (LC 50 , the concentration at which 50% mortality of brine shrimp nauplii occurred) were determined but in few cases due to higher mortality rates LC 50 cannot be determined and for them LC 90 values were determined.The crude extract of Bombax ceiba showed significant cytotoxic activity against brine shrimp nauplii and LC 50 value was found to be 3.90µg/ml whereas the LC 90 (90% mortality rate) was calculated 150.00µg/ml to get the therapeutic index (Table 1).For the conformity of the result, the test was done for two times.An approximate linear correlation was observed when logarithm of concentration versus percentage of mortality was plotted and the values of LC 50 were calculated using Microsoft Excel 2000.All the values were compared with standard cytotoxic agent, vincristine sulphate for which LC 50 was found to be 0.625 µg/ml.

Antimicrobial study
From the study, the zones of inhibition produced by the methanol, n-hexane, chloroform and carbon tetrachloride extract were found to be 10 − 14 mm, 09 − 15 mm and 13 − 20 mm respectively (Table 2) at a concentration of 200 µg/disc in case of 09 bacterial strain and 02 fungal strain but the bacterial strain of B. subtilis, S. aureus, S. boydii, S. dysenteriae and one fungal strain (Saccharromyces cevevaceae) showed no sensitivity.The significant activity was found by hexane extract against Sarcina lutea (13mm) and Pseudomonas aeruginosa (12mm).The chloroform extract showed prominent activity against Vibrio mimicus (15mm); significant activity against Bacillus megaterium (12mm) and Vibrio parahemolyticus (12mm).The carbon tetrachloride extract showed prominent (zone of inhibition >15mm) activity against almost all bacterial strain.All the fractions of Bombax ceiba were also tested for antifungal activity against 03 fungi.The extracts had inhibitory effect against all the test pathogens in different degree.The chloroform extract and carbon tetra chloride extract showed profound activity against Aspergillus niger and Candida albicans, respectively.

Table 2 .
Antibacterial and antifungal activity of different extracts of Bombax ceiba.