The works Lannea coromandelica native to Bangladesh and its root bark is reported to be used in the traditional medical specialty for the intervention of ulcers, sprains, contusions, tegument diseases, and dysentery. The root bark is has acerb belongings and used in odontalgia every bit good as in tooth powder. However really few plants has been reported so far in footings of phytochemical and pharmacological activity of this works. In the present survey, the works has been tested for antibacterial, antioxidant and cytotoxic activity. The ethanol infusion of L. coromandelica root bark showed antibacterial activity against a broad scope of Gram positive and Gram negative bacteriums in disc diffusion and agar good diffusion check. The minimal repressive concentration ( MIC ) against these bacteriums were found to be between 125 to 500 ?g/ml. In DPPH check, the infusion showed good extremist scavenging activity with IC50 value of 15 ?g/ml. Entire phenolic content was found to be g combining weight of Gallic acid/g works stuff. The works infusion besides showed toxicity against brine runt nauplii and the LC50 was determined to be ?g/ml. In the acute toxicity trial 66 and 33 % of the trial animate beings died during the first H of experiment for groups that received 4000 and 2000 mg/kg dosage. No mortality was observed at the dosage of 1000 mg/kg. The above information demonstrates that the works has some of import biological activities with sufficient authority. Therefore, we suggest the works L. coromandelica would be a good campaigner in hunt for drug lead.
Cardinal words: L. coromandelica ; antibacterial ; MIC ; DPPH ; Folin Ciocalteu ‘s reagaent ; brine shrimp deadliness
The works Lannea coromandelica ( Houtt. ) Merr ( Anacardiaceae ) is a deciduous tree with midst, milky to gray bark. The bark feels smooth and flakes off in little pieces when prohibitionist. The works is known as Bhadi, jial bhandi in Bangladesh and is distributed throughout the state. It is besides found in tropical moist and dry deciduous woods 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 normally of little dimension in Bangladesh but is said to turn larger in more favorable clime. The bark has astringent belongings and is used in the intervention of ulcers, sprains, contusions, tegument diseases, and dysentery. Decoction of the bark is applied in odontalgia while the powdery signifier of the bark is used as tooth powder. The foliages are used to bring around elephantiasis and in the poached signifier used to handle puffinesss and strivings ( Sivarajan. V.V. and et Al ) .
In a old survey, the root bark of L. coromandelica showed zoosporocidal activity ( MIC 0.1 ?g/mL and its polyflavonoid tannic acids were credited for the activity ( Islam et al. , 2002 ) . Previous phytochemical studies indicated the presence of ?-sitosterol, physcion, and physcion anthranol B, dihydroflavonols in the root bark, flavonols and ellagic acid in the foliages, leucocyanidin together with some leucodelphinidin in the duramen ( Islam and Tahara, 2002 ; Subramanian and Nair, 1971 ) . In our present survey, the ethnol infusion of the root bark of L. coromandelica was subjected to phytochemical analysis to cognize the major categories of compounds present. The infusion was besides tested for a figure of biological activities.
Materials and methods
Plant stuff: Stem bark of Lannea coromandelica ( Houtt. ) Merr. was collected from Maheshkhali, Cox ‘s bazaar, Bangladesh in May ‘ 2010. The works parts were identified by the experts at Bangladesh National Herbarium, Dhaka, where a verifier specimen ( DACB 35242 ) has been submitted for future mention.
Extraction: The shed dried works stuffs were grinded into all right pulverization and soaked in ethyl alcohol for three yearss with occasional shaking. The infusion was filtered and dried utilizing rotary vacuity evaporator. The bark and the foliages yielded 27 and 23 % infusion, severally.
Animal: Swiss albino mice purchased from Animal Resources Section of International Centre for Diarrhoeal Disease Research, Bangladesh ( ICDDR, B ) , Dhaka, Bangladesh. The animate beings were fed with tap H2O and ICDDR, B formulated nutrient ad libitum and kept for one hebdomad prior to experiment to acclimatized with the research lab status.
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,
Antibacterial activity trial by disc diffusion check: Sterile blank phonograph records were impregnated with the trial extracts at the concentrations of 250 and 500 ?g/disc. Sample incorporating phonograph record, antibiotic incorporating standard phonograph record ( Kanamycin, ( BBL, Cocksville, USA ) , control phonograph record were placed on alimentary agar media seeded with trial being. The Petri dishes were so incubated at 37 oC for 16 h. The zone of suppression was measured utilizing a digital slide callipers ( Bauer et al. , 1966 ;
Cruickshank, 1968 ) .
Antibacterial activity trial by agar good diffusion checks:
Determination of Minimum Inhibitory Concentration ( MIC ) : Minimal repressive concentration was determined by broth macrodilution check ( Andrews, 2001 ; Sarker et al. , ) . In brief, selected bacterial strains were cultured on food agar media at 37 oC for nightlong. The bacterial settlement was suspended in unfertile 0.9 % NaCl solution in such a manner to acquire an optical density of 0.1 at 620 nanometers ( 1 – 108 CFU/ml ) . Aliquot of 100 ?l of this bacterial suspension was so assorted with 10 milliliters of Mueller Hinton stock to acquire the inoculant ( 1 – 106 CFU/ml ) . The infusions were assorted with Mueller Hinton stock with the aid of DMSO to acquire a concentration of 2 mg/ml ( DMSO concentration & A ; lt ; 5 % ) . The infusion was so serially diluted in unfertile capped tubings and so inoculants was added to each tubing to acquire a starting concentration of 1000 ?g/ml of infusion in the first good. The same process was besides followed for standard antibiotic Rocephin. The tubings were so incubated for 18 h. MIC values were recorded as the lowest concentration with no bacterial growing. The MIC values were farther confirmed with the add-on of resazurin ( 0.01 % in unfertile distilled H2O ) and farther incubation for 5 proceedingss. Pink colour or stain of resazurin indicated bacterial growing.
In vitro Antioxidant activity trial: Antioxidant activity of the infusion was determined utilizing stable free extremist DPPH ( 2,2-Dipheenyl-1-picrylhydrazyl ) harmonizing to Takao et Al ( 1994 ) with some alteration ( Sarker et al, 2003 ) .
Qualitative analysis: Tender loving care home bases were developed with solvent systems of different mutual oppositions to decide non-polar, average polar and polar compounds. The home bases were sprayed with 0.02 % DPPH in ethyl alcohol. Bleaching of DPPH ( yellow on violet background ) by the single-minded sets was observed for 30 min and was noted.
Quantitative analysis: Stock solution of the works infusion was prepared in ethyl alcohol. Aliquots from the stock solution were diluted in ethyl alcohol to acquire concentrations of 1, 5, 10, 50, 100 and 500 ?g/ml. Diluted solutions ( 1 milliliter ) were assorted with 2 milliliters DPPH solution ( 0.004 % in ethyl alcohol ) and kept for 30 min to finish any reaction that occur. The optical density was recorded at 517 nanometer. Ascorbic acid was used as the criterion in this experiment. Percent suppression was calculated utilizing the expression ( 1 – A1/A0 ) – 100, where A0 is the concentration of control and A1 is the optical density of sample/standard. IC50 was determined from % suppression V concentration secret plan ( Ref ) .
Determination of entire phenolic content
Dried works stuff was weighed and 0.5 of it was assorted with 50 milliliters of 80 aqueous methyl alcohol. It was sonicated for 20 min. Two milliliter of the infusion was centrifuged for 15 min at 4000 revolutions per minute.
Entire phenolic content was determined by Folin Ciocalteu ‘s reagent ( Ref ) . In brief, Gallic acerb solution in methyl alcohol was prepared to acquire concentrations of 20, 40, 60, 80 and 100 mg/ml. From each of the concentrations, every bit good as from the infusion, 1 milliliter was transferred in 25 milliliters volumetric flasks incorporating 9 milliliter distilled H2O. Folin Ciocalteu ‘s reagent ( 1 milliliter ) was added to each volumetric flask and was mixed by agitating. After 5 min, 10 milliliter of 7 % Na2CO3 was added to it and the volume was adjusted to 25 milliliters by adding distilled H2O. Keeping for 25 min at room temperature, optical density was measured at 750 nanometers against space. Blank was prepared by following all the above stairss except the add-on of Gallic acid. Standard curve was prepared utilizing the optical density of assorted Gallic acid concentrations. Gallic acerb content of the infusion was determined from the standard curve and expressed as milligram Gallic acid equivalent/100 g dried works stuff.
Analgesic activity was tested utilizing acetic acid wrestling in mice ( Koster et al, 1959 ) . The animate beings were orally administered with the infusions at the doses of 250 and 500 mg/kg of organic structure weight. After 30 min of the extract disposal, the animate beings were injected ( i.p ) with 0.7 % of acetic acid to bring on wrestling. After 5 min of acetic acid disposal, the writhing was counted for 15 min. Diclofenac Na was used as the positive control in this experiment.
Hatching runt: Artificial sea H2O was prepared by fade outing 20 g of NaCl and 18 g of table salt in one liter of distilled H2O and filtered off to acquire a clear solution. A rectangular armored combat vehicle was divided in to two unequal compartments by a porous centrifuge. The larger compartment was darkened while the smaller 1 was kept lighted. The eggs of Artemia Salina were hatched at room temperature ( 25-30 oC ) for 24 h. The larvae ( nauplii ) were attracted by the visible radiation and moved to the smaller compartment through the holes. They were so collected by a pipette.
Brine runt deadliness bio-assay: Cytotoxicity of the infusions was tested by seawater runt deadliness trial ( Meyer et al. , 1982 ) . Samples were dissolved in DMSO and so transferred to phials to acquire concentrations of 160, 80, 40, 20 and 10 ?g/ml in 5 ml unreal sea H2O with 10 naupliis in each phial. The concentration of DMSO did non transcend 0.01 % in any of the vial. Control vials contain DMSO in unreal sea H2O at the same concentration as in trial phials. After 24 h incubation at room temperature ( 25-30 oC ) , figure of feasible naupliis were counted utilizing a magnifying glass.
Acute toxicity trial
Trial animate beings ( n=6 ) were fed with infusions at the doses of 62.5, 125, 250, 500, 1,000, 2,000 and 4,000 mg/kg organic structure weight while the control group received normal saline. Mortality of the animate beings were observed for 24 H and recorded ( Lorke, 1983 ) .
Consequences of antibacterial activity trial
Antibacterial sensitiveness trial was carried out by agar good diffusion and disc diffusion check. In both trials, the root bark of L. coromandelica showed