Preparation of ethyl acetate extract of Desmodium gangeticum root
The whole plant of Desmodium gangeticum was authenticated by Prof James Joseph. The voucher specimen A/C no. 3908 was retained in our laboratory for future reference.
The roots were dried under shade and ground to a powder (100 g) which was extracted by ethyl acetate (60-80°C) in a Soxhlet apparatus for 72 hours. The extract was concentrated under vacuum and dried at room temperature. The brownish extract (8.8 g) was resinous. Various qualitative tests [12] were performed on the extract to confirm the chemical constituents, namely triterpenoids, tannins, phenolic compounds and glycosides. All chemicals used were of analytical grade.
Experimental animals
Adult albino Wistar male rats (weighing 250-280 g) were obtained from King Institute of Preventive Medicine, Chennai, India. They were fed on commercial rat chow (Hindustan Lever, India) and had free access to water. Handling of the animals was approved by the Indian Ministry of Social Justices and Empowerment. The experimental protocol was approved by the institutional ethics committee.
Heart preparation
Isolated rat heart model was prepared according to Döring [13]. The rats were anesthetized at a dosage of 40 mg per kg body weight of sodium thiopentenone. After an intravenous injection of heparin (300 units), the heart was rapidly excised via a midsternal thoracotomy and arrested in ice cold Krebs-Henseleit (KH) buffer containing 118 mM/L NaCl, 4.7 mM/L KCl, 1.2 mM/L MgSO4, 1.2 mM/L KH2PO4, 1.8 mM/L CaCl2, 25 mM/L NaHCO3 and 11 mM/L C6H12O6. The heart was attached to a Lagendorff apparatus via an aorta for retrograde perfusion with KH buffer maintained at 37°C and pH7.4 and saturated with a gas mixture of 95 ml O2 and 5 ml CO2. The coronary perfusion pressure was maintained at 80 mmHg. The left ventricular pressure developed with the ventricle filled with Krebs solution was recorded with a pressure transducer, which in turn was connected to a device amplifier and chart recorder. This left ventricular pressure was an indication of the mechanical performance of the heart. Coronary flow was measured simply by collecting the perfusate draining from the heart in a graduated cylinder for a defined time. The heart rate was measured by counting the number of contractions (obtained from the left ventricular pressure recorder) per minute.
Experimental protocol
Rats were divided into three groups. In the normal/control group (Group 1), hearts were perfused for 90 minutes with KH buffer and used for the biochemical analysis. In the reperfusion group (Group 2), the 30-minute ischemic hearts (n = 6 in each subgroup) were subjected to 15 minutes of reperfusion (Subgroup 2.1), 30 minutes of reperfusion (Subgroup 2.2) or 45 minutes of reperfusion (Subgroup 2.3). All animals in the treatment group (Group 3) were pretreated orally (through a ball-tipped classic steel 15-16 gauge hypodermic needle) with Desmodium gangeticum at a dose of 100 mg per kg body weight for 30 days and then divided into three subgroups. In Subgroup 3.1, rat hearts (n = 6) were perfused for 90 minutes with KH buffer and used for the biochemical analysis. In Subgroup 3.2, rat hearts (n = 6) were subjected to 30 minutes of global ischemia after equilibration, followed by 30 minutes of reperfusion. In Subgroup 3.3, rat hearts (n = 6) were subjected to 30 minutes of global ischemia after equilibration, followed by 45 minutes of reperfusion.
Biochemical assays
Thiobarbituric acid-reactive substances (TBARS) were measured [14] as a marker of lipid peroxidation. The endogenous antioxidants, superoxide dismutases (SOD) Cu-Zn SOD and Mn SOD [15, 16], catalase [17] and glutathione peroxidase [18] were estimated in a UV-1601 Shimadzu spectrophotometer (Shimadzu, USA). Protein concentration was measured with Folin phenol reagent according to Lowry et al[19].
In vitro antioxidant activity
Determination of superoxide radical scavenging activity
Superoxide scavenging was determined by the nitroblue tetrazolium reduction method [20]. The reaction mixture consisted of ethylenediaminetetraacetic acid (EDTA; 6 μM), sodium cyanide (3 μg), riboflavin (2 μM), nitroblue tetrazolium (50 μM), various concentrations of Desmodium gangeticum extracts (5-50 μg/ml) and phosphate buffer (67 mM, pH7.8) in a final volume of 3 ml. The tubes were uniformly illuminated with an incandescent visible light for 15 minutes, and the optical density was measured at 530 nm before and after the illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes.
Determination of hydroxyl radical scavenging activity
The scavenging capacity for hydroxyl radical was measured according to a modified method of Halliwell et al. [21]. Stock solutions of EDTA (1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM) and deoxyribose (10 mM) were prepared in distilled deionized water. The assay was performed by adding 0.1 ml EDTA, 0.01 ml of FeCl3, 0.1 ml of H2O2, 0.36 ml of deoxyribose, 1.0 ml of Desmodium gangeticum extract (10-100 μg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH7.4) and 0.1 ml of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1 hour. A 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10 g/100 g TCA and 1.0 ml of 0.5 g/100 g TBA (in 0.025 M NaOH containing 0.025 g/100 g TBA) to develop the pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as percentage inhibition of deoxyribose degradation.
Lipid peroxide scavenging activity
A 5 ml reaction mixture containing rat liver homogenate (0.1 ml, 25 g/100 ml) in Tris-HCl buffer (40 mM, pH7.0), KCl (30 mM), ferrous iron (0.16 mM) and ascorbic acid (0.06 mM) was incubated for 1 hour at 37°C in the presence or absence of Desmodium gangeticum extract (20-180 μg/ml). The lipid peroxidation was measured by TBARS formation [14]. Of this incubation mixture, 0.4 ml was treated with sodium dodecyl sulphate (8.1 g/100 ml, 0.2 ml), TBA (0.8 g/100 g, 1.5 ml) and acetic acid (20 ml/100 ml, 1.5 ml, pH3.5). The total volume was then made up to 4 ml by adding distilled water and kept in a water bath at 100°C for 1 hour. After cooling, 1 ml of distilled water and 5 ml of a mixture of n-butanol and pyridine (15:1 v/v) was added. The mixture was centrifuged at 5000 × g for10 minutes and remixed. The absorbance of the organic layer was measured at 532 nm. The percentage inhibition of lipid peroxidation was determined by comparing results of the test compounds with those of controls and tubes not treated with the extracts.
Diphenylpicrylhydrazyl radical scavenging activity
The free radical scavenging activity of the Desmodium gangeticum extract and butylated hydroxyl toluene was measured with the stable radical diphenylpicrylhydrazyl (DPPH) [22] in terms of hydrogen-donating or radical-scavenging activity. A 0.1 mM solution of DPPH in ethanol was prepared, and 1.0 ml of this solution was added to 3.0 ml of extract solution in water at different concentrations (10-100 μg/ml). After 30 minutes, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The antioxidant activity of the extract was expressed as IC50, which was defined as the concentration (in μg/ml) of extract that inhibits the formation of DPPH radicals by 50%.
Nitric oxide scavenging
Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide (NO), which interacts with oxygen to produce nitrite ions that can be estimated by use of Griess reagent [23, 24]. Scavengers of NO compete with oxygen, leading to reduced production of NO. Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with 3.0 ml of various concentrations (10-320 μg/ml) of Desmodium gangeticum extract dissolved and incubated at 25°C for 150 minutes. The samples were then reacted with Greiss reagent (1 g/100 ml sulphanilamide, 2 ml/100 ml H3PO4, and 0.1 g/100 ml napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite also treated with Griess reagent.
Gas chromatography-mass spectrometry (GC-MS) analysis
All GC-MS analyses were conducted with a PerkinElmer Clarus 500 gas chromatograph (Perkin Elmer, USA). The chromatographic conditions were as follows. Elite-1 (100 g/100 ml dimethylpolysiloxane) column was used. Helium was used as the carrier gas with a flow rate of 1 ml per minute. Desmodium gangeticum aqueous root extract (1 ml) was injected into the system in splitless mode at 250°C. The column oven temperature was maintained at 110°C for 2 minutes, then programmed at 75°C to 200°C for 1 minute and increased to 280°C by sequential increment of 5°C per minute.
Statistical analysis
All data are presented as mean ± SD. Results were analyzed by one-way analysis of variance with SPSS software 12.00 (IBM, USA), followed by Duncan's multiple range test. P < 0.05 was considered statistically significant. Linear regression analysis was used to calculate IC50 values.
