The plant Lannea coromandelica native to Bangladesh and its stem bark is reported to be used in the traditional medicine for the treatment of ulcers, sprains, bruises, skin diseases, and dysentery. The stem bark is has astringent property and used in toothache as well as in toothpowder. However very few works has been reported so far in terms of phytochemical and pharmacological activity of this plant. In the present study, the plant has been tested for antibacterial, antioxidant and cytotoxic activity. The ethanol extract of L. coromandelica stem bark showed antibacterial activity against a wide range of Gram positive and Gram negative bacteria in disc diffusion and agar well diffusion assay. The minimum inhibitory concentration (MIC) against these bacteria were found to be between 125 to 500 μg/ml. In DPPH assay, the extract showed good radical scavenging activity with IC50 value of 15 μg/ml. Total phenolic content was found to be g eq of gallic acid/g plant material. The plant extract also showed toxicity against brine shrimp nauplii and the LC50 was determined to be μg/ml. In the acute toxicity test 66 and 33% of the test animals died during the first h of experiment for groups that received 4000 and 2000 mg/kg dose. No mortality was observed at the dose of 1000 mg/kg. The above data demonstrates that the plant has some important biological activities with sufficient potency. Therefore, we suggest the plant L. coromandelica would be a good candidate in search for drug lead.
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Key words: L. coromandelica; antibacterial; MIC; DPPH; Folin Ciocalteu's reagaent; brine shrimp lethality
The plant Lannea coromandelica (Houtt.) Merr (Anacardiaceae) is a deciduous tree with thick, whitish to grey bark. The bark feels smooth and flakes off in small pieces when dry. The plant is known as Bhadi, jial bhandi in Bangladesh and is distributed throughout the country. It is also found in tropical moist and dry deciduous forests of Himalaya (Swat to Bhutan), Assam, Burma, Indo-China, Ceylon, Andaman Island, China and Malaysia ([Sivarajan. V.V. and et al, Benth Hall and et al, K.M. Matthew, Forestry Compendium]). The tree is usually of small dimension in Bangladesh but is said to grow larger in more favourable climate. The bark has astringent property and is used in the treatment of ulcers, sprains, bruises, skin diseases, and dysentery. Decoction of the bark is applied in toothache while the powdered form of the bark is used as toothpowder. The leaves are used to cure elephantiasis and in the boiled form used to treat swellings and pains (Sivarajan. V.V. and et al).
In a previous study, the stem bark of L. coromandelica showed zoosporocidal activity (MIC 0.1 μg/mL and its polyflavonoid tannins were credited for the activity (Islam et al., 2002). Previous phytochemical reports indicated the presence of β-sitosterol, physcion, and physcion anthranol B, dihydroflavonols in the stem bark, flavonols and ellagic acid in the leaves, leucocyanidin together with some leucodelphinidin in the heartwood (Islam and Tahara, 2002; Subramanian and Nair, 1971). In our present study, the ethnol extract of the stem bark of L. coromandelica was subjected to phytochemical analysis to know the major classes of compounds present. The extract was also tested for a number of biological activities.
Materials and methods
Plant material: Stem bark of Lannea coromandelica (Houtt.) Merr. was collected from Maheshkhali, Cox's bazar, Bangladesh in May' 2010. The plant parts were identified by the experts at Bangladesh National Herbarium, Dhaka, where a voucher specimen (DACB 35242) has been submitted for future reference.
Extraction: The shed dried plant materials were grinded into fine powder and soaked in ethanol for three days with occasional shaking. The extract was filtered and dried using rotary vacuum evaporator. The bark and the leaves yielded 27 and 23% extract, respectively.
Animal: Swiss albino mice purchased from Animal Resources Section of International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh. The animals were fed with tap water and ICDDR,B formulated food ad libitum and kept for one week prior to experiment to acclimatized with the laboratory condition.
Bacterial strains: Bacterial strains were collected from the Microbiology Laboratory of International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh. Test organisms include Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes,
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Antibacterial activity test by disc diffusion assay: Sterile blank discs were impregnated with the test extracts at the concentrations of 250 and 500 μg/disc. Sample containing discs, antibiotic containing standard discs (Kanamycin, (BBL, Cocksville, USA), control discs were placed on nutrient agar media seeded with test organism. The Petri dishes were then incubated at 37 oC for 16 h. The zone of inhibition was measured using a digital slide calipers (Bauer et al., 1966;
Antibacterial activity test by agar well diffusion assay:
Determination of Minimum Inhibitory Concentration (MIC): Minimum inhibitory concentration was determined by broth macrodilution assay (Andrews, 2001; Sarker et al., ). In brief, selected bacterial strains were cultured on nutrient agar media at 37 oC for overnight. The bacterial colony was suspended in sterile 0.9% NaCl solution in such a way to get an absorbance of 0.1 at 620 nm (1 - 108 CFU/ml). Aliquot of 100 μl of this bacterial suspension was then mixed with 10 ml of Mueller Hinton broth to get the inoculum (1 - 106 CFU/ml). The extracts were mixed with Mueller Hinton broth with the assistance of DMSO to get a concentration of 2 mg/ml (DMSO concentration < 5%). The extract was then serially diluted in sterile capped tubes and then inoculums was added to each tube to get a starting concentration of 1000 μg/ml of extract in the first well. The same procedure was also followed for standard antibiotic ceftriaxone. The tubes were then incubated for 18 h. MIC values were recorded as the lowest concentration with no bacterial growth. The MIC values were further confirmed with the addition of resazurin (0.01% in sterile distilled water) and further incubation for 5 minutes. Pink color or discolouration of resazurin indicated bacterial growth.
In vitro Antioxidant activity test: Antioxidant activity of the extract was determined using stable free radical DPPH (2,2-Dipheenyl-1-picrylhydrazyl) according to Takao et al (1994) with some modification (Sarker et al, 2003).
Qualitative analysis: TLC plates were developed with solvent systems of different polarities to resolve non-polar, medium polar and polar compounds. The plates were sprayed with 0.02% DPPH in ethanol. Bleaching of DPPH (yellow on purple background) by the resolved bands was observed for 30 min and was noted.
Quantitative analysis: Stock solution of the plant extract was prepared in ethanol. Aliquots from the stock solution were diluted in ethanol to get concentrations of 1, 5, 10, 50, 100 and 500 μg/ml. Diluted solutions (1 ml) were mixed with 2 ml DPPH solution (0.004% in ethanol) and kept for 30 min to complete any reaction that occur. The absorbance was recorded at 517 nm. Ascorbic acid was used as the standard in this experiment. Percent inhibition was calculated using the formula (1 - A1/A0) - 100, where A0 is the concentration of control and A1 is the absorbance of sample/standard. IC50 was determined from % inhibition vs concentration plot (Ref).
Determination of total phenolic content
Dried plant material was weighed and 0.5 of it was mixed with 50 ml of 80 aqueous methanol. It was sonicated for 20 min. Two ml of the extract was centrifuged for 15 min at 4000 rpm.
Total phenolic content was determined by Folin Ciocalteu's reagent (Ref). In brief, gallic acid solution in methanol was prepared to get concentrations of 20, 40, 60, 80 and 100 mg/ml. From each of the concentrations, as well as from the extract, 1 ml was transferred in 25 ml volumetric flasks containing 9 ml distilled water. Folin Ciocalteu's reagent (1 ml) was added to each volumetric flask and was mixed by shaking. After 5 min, 10 ml of 7% Na2CO3 was added to it and the volume was adjusted to 25 ml by adding distilled water. Keeping for 25 min at room temperature, absorbance was measured at 750 nm against blank. Blank was prepared by following all the above steps except the addition of gallic acid. Standard curve was prepared using the absorbance of various gallic acid concentrations. Gallic acid content of the extract was determined from the standard curve and expressed as mg gallic acid equivalent/100 g dried plant material.
Analgesic activity was tested using acetic acid writhing in mice (Koster et al, 1959). The animals were orally administered with the extracts at the doses of 250 and 500 mg/kg of body weight. After 30 min of the extract administration, the animals were injected (i.p) with 0.7% of acetic acid to induce writhing. After 5 min of acetic acid administration, the writhing was counted for 15 min. Diclofenac sodium was used as the positive control in this experiment.
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Hatching shrimp: Artificial sea water was prepared by dissolving 20 g of NaCl and 18 g of table salt in one litre of distilled water and filtered off to get a clear solution. A rectangular tank was divided in to two unequal compartments by a porous separator. The larger compartment was darkened while the smaller one was kept illuminated. The eggs of Artemia salina were hatched at room temperature (25-30 oC) for 24 h. The larvae (nauplii) were attracted by the light and moved to the smaller compartment through the holes. They were then collected by a pipette.
Brine shrimp lethality bioassay: Cytotoxicity of the extracts was tested by brine shrimp lethality test (Meyer et al., 1982). Samples were dissolved in DMSO and then transferred to vials to get concentrations of 160, 80, 40, 20 and 10 μg/ml in 5 ml artificial sea water with ten naupliis in each vial. The concentration of DMSO did not exceed 0.01% in any of the vial. Control vials contain DMSO in artificial sea water at the same concentration as in test vials. After 24 h incubation at room temperature (25-30 oC), number of viable naupliis were counted using a magnifying glass.
Acute toxicity test
Test animals (n=6) were fed with extracts at the doses of 62.5, 125, 250, 500, 1,000, 2,000 and 4,000 mg/kg body weight while the control group received normal saline. Mortality of the animals were observed for 24 h and recorded (Lorke, 1983).
Results of antibacterial activity test
Antibacterial sensitivity test was carried out by agar well diffusion and disc diffusion assay. In both tests, the stem bark of L. coromandelica showed