Shimana Nasrin
Pharmacy Discipline, Life Science School, Khulna University, Khulna-9208, Banglades
Md. Golam Hossain*
Pharmacy Discipline, Life Science School, Khulna University, Khulna-9208, Banglades
Spilanthes calva, Antioxidant, Antidiarrheal, Analgesic.
Khulna University, Bangladesh
Development of Host and Medicinal Plants
Medicinal Plants
Collection and Identification:The plant S. calva was collected from Khulna University, Bangladesh, during the month of 14th December, 2009 on the day time. The plant was identified by the experts of Bangladesh National Herbarium, Mirpur, Dhaka (Accession no: DACB -34992) and a voucher specimen was also deposited there.Preparation of plant extract: The collected plant parts were separated from undesirable materials or plants and air dried for three weeks. The plant parts were ground into a fine powder by using grinder (Capacitor start motor, Wuhu motor factory, China). The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. A glass jar with plastic cover was taken and washed thoroughly. The jar was rinsed with ethanol and dried. Then,150gm of the dried powder was taken in the jar. After that 95% ethanol (750 ml) was poured into the jar up to 1 inch height above the sample surface as it can sufficiently cover the sample surface. Aluminum foil was used to resist the entrance of air into the jar. This process was performed for 8 days. The jar was shaken and stirred several times during the process to get better extraction. After the extraction process,the extract was filtered by a piece of clean, white cotton material.Then,it was filtered through Whatman filter paper. The filtrate was collected in a beaker. The filtrate (ethanolic extracts) obtained was evaporated by rotary evaporator and after that,it was kept under ceiling fan to evaporate the ethanol completely. It rendered a gummy concentrate (5.2 gm) of greenish black color. The gummy concentrate was designated as crude extract of ethanol. The yield value was almost 3.46%.2.3.Antidiarrheal activity test: To prepare suspension of the test samples at the doses of 250 and 500 mg/kg per body weight, 250 and 500 mg of ethanolic extract were measured respectively. The extract was triturated in unidirectional manner by the addition of small amount of tween-80. After proper mixing of extract and tween-80, the distilled water was slowly added. The final volume of the suspensions was made 10.0 ml. For the preparation of standard drug,3 mg of Loperamide was taken and triturated in unidirectional manner by the addition of small amount of tween-80. After proper mixing, distilled water was slowly added up to the final volume 10ml.The amount to be administered to get desired concentration= (Body weight of mice × 0.01) ml. For the test,fresh mice were randomly chosen from the animal laboratory of Khulna University, Khulna, Bangladesh and divided into four groups having five mice in each. Of the experimental groups, group-Ior the control received only distilled water. Group-II or the positive control received standard anti-motility drug, Loperamide at a dose of 3 mg/kg-body weight as oral suspension. The test groups were treated with suspension of ethanolic extracts of S. calva DC at the oral dose of 250 mg/kg-body weight and 500 mg/kg-body weight. The mice were fed with the samples for 40 minutes (for proper absorption) prior to the oral administration of castor oil. After that,the mice were fed castor oil at a dose of 0.5 per ml. Individual animal of each group were placed in separate cages having adsorbent paper beneath and examined for the latent period and after that,diarrheal symptom was observed for every four hours. Number of stools or any fluid material that stained the adsorbent paper were counted at each successive hour during the 4-hour period and were noted for each mouse. At the beginning of each hour, new papers were placed for the old ones. In vitro Anti-oxidant Activity test (Qualitative and Quantitative analysis): Qualitative test: A small amount of ethanolic extract of the plant was dissolved in ethanol and diluted suitably and was applied on the two different TLC plate by spotter. A small amount of ascorbic acid was also dissolved in ethanol and diluted suitably and was applied on the former TLC plate by spotter at the same way. The plate was then kept in a TLC jar containing a solvent system. About 5 minutes later, spotted TLC plate was kept into the jar dipping into the solvent system. When the solvent system reached at the desired level then,it was removed from TLC jar and was subjected for air drying. At first, two chromatograms were developed into the solvent system of n-hexane and Acetone (2:1). This is a non-polar solvent system. After drying,chromatograms were observed under ultraviolet (UV) detector at longer wavelength (306 nm) and shorter wavelength (256 nm) and colored spots were marked by pencil.Spotwhich was observed into longer wavelength was manifested by the symbol ( ) and spot which was observed into shorter wavelength was evident by the symbol < >. Then,one of the chromatograms were taken and 0.02 % DPPH solution of ethanol was sprayed on it by a spray gun. Yellow color was formed on the chromatogram. Other chromatogram was treated with 10% H2SO4 at the same way. Then,the plate was heated on hot plate and was developed. Then,the sample was compared with standard. Another two chromatograms were developed into the solvent system of Chloroform, Methanol and Water (40:10:1) and also two chromatogram were developed using the solvent system Chloroform and Methanol (5:1). This is polar solvent system. Then, by the similar procedure described above was applied to compare the sample with standard. Quantitative analysis 2.5.1 DPPH radical scavenging assay: At first, 6 test tubes were taken to make aliquots of 6 conc. (1, 5, 10, 50, 100, 500 μg/ml). Plant extract and ascorbic acid were weighed 2 times and dissolved in ethanol to make the required concentrations by dilution technique. Here ascorbic acid was taken as ‘+’ve control. DPPH was weighed and dissolved in ethanol to make 0.004% (w/v) solution. To dissolve homogeneously,magnetic stirrer was used. After making the desired concentrations,1 ml plant extracts or standard of different concentration solution were taken into different test tubes and 3 ml of reagent solution was added into each of the test tubes. The test tubes were kept for 30 minutes in at room temperature in the light to complete the reactions. DPPH was also taken as blank in a test tube. After 30 minutes, absorbances of each test tube were determined by UV spectrophotometer at 517 nm. IC50 was determined from % inhibition vs concentration graph. The formula used for % inhibition ratio was % inhibition = (Blank OD-Sample OD/ Blank OD) × 100.
Biosci Bioeng Commun 2015; 1: 11-17
Journal