Cytotoxic, thrombolytic, membrane stabilizing and antioxidant activities of Hygrophila schulli

Materials and Methods

Plant materials

Healthy and disease free plants of H. schulli were collected from Avoynogor, Jessore, Bangladesh in March 2012. The exsiccated plant sample was identified by Bangladesh National Herbarium, Mirpur, Dhaka. A voucher specimen of the plant has been preserved for future reference (Accession number: DACB-39,532).

Extraction and fractionation

The fresh, exsiccated and shade dried plants were powdered using a suitable blender (Noka super blender, China). The powdered materials (300 g) were soxhlet extracted with methanol at a temperature not exceeding 40ºC. The methanol (crude) extract thus obtained was concentrated at 40ºC with a rotary evaporator (Bibby RE200, Sterilin Ltd., U.K) to get a greenish gummy concentrate of 27.9 g (percent of yield = 9.3%). The crude extract was then stored in a refrigerator at 4ºC. The crude extract (6.2 g) was further fractionated by modified Kupchan partitioning protocol (Venwagenen et al., 1993) to afford petroleum ether, chloroform, ethyl acetate and aqueous soluble materials.

Evaluation of cytotoxicity

Brine shrimp lethality bioassay (McLaughlin and Rogers, 1998; Meyer et al., 1982) technique was used to test cytotoxicity of the extracts. Anticancer drug vincristine sulfate (Techno Drugs Ltd, Bangladesh) was used as positive control and dimethyl sulfoxide (DMSO, BioReagent, for molecular biology; Sigma-Aldrich, India) was used as solvent and negative control. In this experiment, 4 mg of each test sample was dissolved in DMSO to obtain solutions of varying concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 μg/mL) by serial dilution technique. DMSO solutions were then applied against Artemia salina for 24 hours to calculate percent mortality.LC₅₀ and LC₉₀ values were determined from the analysis of concentration-mortality data.

Thrombolytic activity

In vitro thrombolytic activity was determined by following established protocol (Rahman et al., 2013). Lyophilized altepase (streptokinase) vial (Beacon Pharmaceutical Ltd) of 15,00,000 IU and distilled water were used as positive and negative control, respectively. Venous blood drawn from healthy volunteers was incubated in pre weighed sterile microcentrifuge tubes (1 mL/tube) at 37°C for 45 min. After clot formation, the serum was completely removed and clot weight was determined for each tube. 100 μL aqueous solution of    different partitionates along with the crude extract was added separately to each microcentrifuge tube containing pre-weighed clot. All the tubes were then incubated at 37°C for 90 min and observed for clot lyses. Percent of clot lyses were determined with the following equation:

% of clot lysis = (weight of released clot /clot weight) × 100

Membrane stabilizing activity

Membrane stabilizing activity in hypotonic solution- and heat-induced so;ution was studied by previously described method (Shinde et al., 1999). Absorbance values of the supernatants were measured at 540 nm using Shimadzu UV spectrophotometer.

Hypotonic solution-induced hemolysis

The extracts and standard acetyl salicylic acid were taken in different centrifuge tubes. In each tube 0.50 mL erythrocyte (RBC) suspension, 5 mL of hypotonic solution (50 mM NaCl) and 10 mM sodium phosphate buffer saline (pH 7.4) were taken. The mixtures were incubated at room temperature for 10 min and then centrifuged for 10 min at 3,000 rpm. After centrifugation the soluble supernatant of each tube was decanted and filtered. Then the absorbance for each tube was measured. The percentage inhibition of hemolysis or membrane stabilization was calculated using the following equation:

% Inhibition of hemolysis = 100 × {(OD₁- OD₂)/ OD₁}

Where, OD₁ = Optical density of hypotonic-buffered saline solution alone (control) and

OD₂ = Optical density of test sample in hypotonic solution.

Heat induced hemolysis: Aliquots of 5 mL isotonic buffer, 1.0 mg/mL of an extract and 30 µL erythrocyte suspension were taken in two different sets of centrifuge tubes. The same aliquot without the extract was taken in two other centrifuge tubes. The tubes were mixed gently by inversion. One pair of the tubes was incubated at 54ºC for 20 min in a water bath while the other was maintained at 0-5ºC in an ice bath. The reaction mixtures were centrifuged for 3 min at 1300 rpm and the absorbance of the supernatants were measured. The percentage inhibition or, acceleration of hemolysis in tests was calculated according to the following equation:

% Inhibition of hemolysis = 100 × [1 – (OD₂-OD₁/ OD₃-OD₁)]

Where, OD₁ = test sample unheated, OD₂ = test sample heated & OD₃ = control sample heated

Antioxidant activity

Antioxidant activity was assessed by the previously developed method (Brand-Williams et al., 1995). Tert-butyl-1-hydroxytoluene (BHT) (Jayson Pharmaceuticals Ltd., Bangladesh) was used as positive control. In this study, 2.0 mL of a methanol solution of the sample (extractives/control) at different concentrations (500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953 and 0.977 μg/mL) were mixed with 3.0 mL of a DPPH methanol solution (20 μg/mL). After 30 min reaction period at room temperature in dark place the absorbance was measured at 517 nm against methanol as blank with a UV-visible spectrophotometer-1240 (Shiamdzu, Japan). Solutions of varying concentrations of extractives and control were obtained from serial dilution of their respective mother solutions (1000 μg/mL). Inhibition of free radical DPPH in percent (1%) was calculated as follows:

(I%) = (1 – A_sample/A_blank) X 100

Where A_blank is the absorbance of the control reaction (containing all  reagents except the test material). IC₅₀ was calculated from the graph plotted inhibition percentage against extract concentration.