2.1 Plant Material Mussaenda roxburghii was collected from a local area (Bhatiary) of Chittagong district, Bangladesh and authenticated by the Botanist Dr. Shaikh Bokhtear Uddin, Assistant professor, Department of Botany, University of Chittagong, Bangladesh.
2.2 Preparation of Extract The leaf was indirectly sun-dried and ground. The ground (300 g) was soaked insufficient amount of methanol for one week at room temperature with occasional shaking and stirring then filtered through a cotton plug followed by Whitman filter paper No. 1. The solvent was evaporated under vacuum at room temperature to yield semisolid. The extract was then preserved in a refrigerator till further use.
2.3 Reagents The chemicals used were Bovine serum albumin (BSA), Diclofenac sodium, Sodium dihydrogen phosphate, Disodium hydrogen phosphate, Sodium Chloride, Dextrose, sodium citrate, citric acid, were purchased from Sigma-Aldrich. Commercially available lyophilized Streptokinase vial of 15 00000 I.U. was purchased from Durakinase, Dongkook Phama. Co. Ltd, South Korea. Absolute methanol (99.50%) and vincristine sulfate (VS) were purchased from Sigma-Aldrich, Munich, Germany. All chemicals in this investigation were of analytical reagent grade.
2.4 Inhibition of Protein DenaturationDiclofenac sodium was used as standard for the inhibition of protein denaturation. The test solution (0.5 ml) contains 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of methanolic extract of Mussaenda roxburghii. The control solution (0.5 ml) contains 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of distilled water. Product control (0.5 ml) contains 0.45 ml of distilled water and 0.05 ml of methanol extract of Mussaenda roxburghii. Standard solution (0.5 ml) contains 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of diclofenac sodium. Six concentrations (31.25, 62.5, 125, 250, 500, and 1000 μg/ml) of methanol extract of Mussaenda roxburghii and diclofenac sodium (standard) were taken, respectively. All the solutions were adjusted to pH 6.3 using 1 N HCI. Samples were kept in the incubator at 37°C for 20 min and the temperature was increased to keep the samples at 57°C for 3 min. After cooling, 2.5 ml of phosphate buffer was added to the previous solutions. The absorbance was measured using a UV-Visible spectrophotometer at 416 nm. The control clearly shows 100% protein denaturation. The results were compared with diclofenac sodium. The percentage inhibition of protein denaturation of different concentrations is tabulated. The percentage inhibition of protein denaturation can be calculated as: % inhibition = [100 - (OD of test solution - OD of product control)] ×100 Where OD = optical density. The control represents 100% protein denaturation. The results were compared with diclofenac sodium.
2.5.1 The human red blood cell (HRBC)
membrane stabilization method
In this method human red blood cell membrane
was used for the hypotonicity induced membrane
lysis. 2 ml blood was drawn from Healthy
volunteer (human) who had not taken any
NSAIDs for prior to the experiment. Then the
blood was mixed with equal volume of Alsever
solution (2%
dextrose, 0.8% sodium citrate, 0.5%
citric acid and
0.42% NaCl) and centrifuged at
3,000 rpm. Isosaline was used for the washing of
the packed cells. A 10% v/v
suspension was
made and kept at 4ºC undistributed before use.
Six concentrations (31.25, 62.5, 125, 250, 500,
1000
μ
g/ml) of extracts were used and blood
control (distilled water instead of hypo saline to
produce 100% hemolysis) were separately mixed
with 1 ml (0.15 M) of sodium phosphate buffer,
2 ml of hyposaline and 0.5 ml of 10% HRBC
the suspension was added to the prepared. When drugs
were omitted in blood control, erythrocyte
the suspension was absent in drug control. All the
assay mixture were kept in an incubator at 37ºC for
30 min and centrifuged at 3000 rpm for 20 min
and hemoglobin content of the supernatant solution
was estimated spectrophotometrically at 560 nm
[16].
2.5.1 The human red blood cell (HRBC) membrane stabilization method: In this method human red blood cell membrane was used for the hypotonicity induced membrane lysis. 2 ml blood was drawn from Healthy volunteer (human) who had not taken any NSAIDs for prior to the experiment. Then the blood was mixed with equal volume of Alsever solution (2%dextrose, 0.8% sodium citrate, 0.5% citric acid and0.42% NaCl) and centrifuged at 3,000 rpm. Isosaline was used for the washing of the packed cells. A 10% v/vsuspension was made and kept at 4ºC undistributed before use. Six concentrations (31.25, 62.5, 125, 250, 500, 1000 μg/ml) of extracts were used and blood control (distilled water instead of hypo saline to produce 100% hemolysis) were separately mixed with 1 ml (0.15 M) of sodium phosphate buffer, 2 ml of hyposaline and 0.5 ml of 10% HRBC suspension was added to prepared. When drugs were omitted in blood control, erythrocyte suspension was absent in drug control. All the assay mixture were kept in incubator at 37ºC for 30 min and centrifuged at 3000 rpm for 20 min and hemoglobin content of supernant solution was estimated spectrophotometrically at 560 nm.
2.6 Anti-cancer Screening Anticancer screening was performed by brine shrimp lethality bioassay which is used for screening bioactive compounds for the evaluation of cytotoxicity of the methanol extract. In this experiment, Artemia salina, a simple zoological organism was used as a convenient monitor for the experiment. The eggs of the brine shrimp were collected from an aquarium shop (Dhaka, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) for 48 hrs. to develop into larval shrimp called nauplii. Meyer Method was performed upon the brine shrimp for the evaluation of cytotoxic assay. The test samples (extract) were prepared by dissolving them in DMSO (not more than 50 μL in 5 mL solution) plus seawater (3.8% NaCl in water) to attain concentrations of 10, 50, 100, 150, 200 and 300 μg/ml. A vial containing 50 μL DMSO diluted to 5 mL was used as a control. Standard vincristine sulfate was used as a positive control. Mature shrimps were placed into each of the experimental vials. After 24 h, the vials were observed using a magnifying glass, and the number of surviving nauplii in each vial was counted. From these data, the percentage of lethality of the brine shrimp nauplii was calculated for each concentration using the following formula.