Postulated Mechanisms of Action of Cardioprotective Drug Terminalia arjuna: Effect of Triterpenoidal Constituents on the Process of Respiratory Oxyburst

Terminalia arjuna is an important medicinal plant widely used in the preparation of Ayurvedic formulations used in cardioprotection. Only one sapogenin acid i.e. arjunolic acid has been shown to provide cardiac protection in induced necrotic rats but no other report for the biological activity of any of the isolated components is available. The present work is to rationalize all saponins for their biological effects using oxidative mechanisms as reactive oxygen species (ROS) implicated in many pathogenic processes including the cardiovascular system. Two sapogenin namely arjungenin and arjunic acid and their respective glucosides arjunetin and arjunglucoside II, isolated from the alcoholic extract of bark of Terminalia arjuna were tested for their action on the free radical scavenging action using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, effect on the superoxide release from PMN cells using NBT (Nitroblue tetrazolium) reduction assay, and hypochlorous acid release from human neutrophils using luminol enhanced chemiluminescence assay. Arjungenin was found to be most active as direct free radical scavenger and inhibitor for the hypochlorous acid production followed by its glucoside that was almost 50% active. INTRODUCTION The damage due to excess ROS during the oxidative stress is known to play an important role in the pathogenesis of various human diseases (Finkel and Holbrook, 2000). Reactive oxygen species (ROS) are implicated in many pathogenic processes including the cardiovascular system. The research in the cardiovascular disease implies that oxidants play an important role in the atherogenesis and in epidemiological studies these diseases are associated with lower plasma concentrations of antioxidant enzymes. The reperfusion injury in ischemic tissue is the result of radicals produced by enzyme xanthine oxidase and is reported to be reduced by agents that decrease the formation or scavenge free radicals (Halliwell, 1987). There are increasing evidences where these diseases exponentially increase with age. One common feature of many of these conditions is the recruitment of inflammatory cells that leads to oxidative stress (Finkel and Holbrook, 2000). Further, there is a growing body of evidence suggesting that antioxidants contribute to cardioprotection (Maxwell, 1995). Terminalia arjuna (TA) is an Ayurvedic medicine for over three centuries, primarily used as cardiac tonic. The bark is reported to contain tannins, triterpenoid saponins, flavonoids, gallic acid, ellagic acid, oligomeric proanthocyanidins, and phytosterols. In in vitro studies the aqueous extract of TA showed significant DPPH radical scavenging activity that was similar to L-ascorbic acid (Munasinghe et al., 2001). Further the bark is also reported to augments endogenous antioxidant compounds of rat heart like SOD, GSH and CAT and also prevents oxidative stress associated with IRI of the heart (Gauthaman et al., 2001). In a randomized controlled trial the bark powder had shown to exhibit significant hypocholesterolaemic effect (Gupta et al., 2001). In rabbits TA showed hypolipidemic activity and induced partial inhibition of rabbit atheroma (Shaila et al., 1998). In another clinical study, TA significantly reduced the anginal Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS 2005 116 frequency and provided symptomatic relief in coronary heart failures. There is only one report in which an isolated chemical constituent was ascribed for the activity. Arjunolic acid treatment has been shown to provide significant cardiac protection in isoproterenol induced myocardial necrosis in rats by preventing the decrease in the levels of superoxide dismutase, catalase, glutathione peroxidase, ceruloplasmin, α-tocopherol, reduced glutathione (GSH), ascorbic acid, lipid peroxide, MPO (Sumitra et al., 2001). The above reports confirm that TA is a cardioprotective drug having traditional origin. The present investigation aims to evaluate sapogenins and saponins, isolated form TA, for their action on the release of reactive oxygen species from the phagocytic cells. Sapogenins arjunic acid and arjungenin and their respective glucosides arjunetin and arjunglucoside II were isolated from the methanolic extract of bark of TA. These compounds were tested for direct free radical scavenging activity in the DPPH assay, their action on the superoxide release from the human PMN cells in the NBT reduction assay and hypochlorous acid production from human neutrophils measured in the luminol enhanced chemiluminescence assay. The results indicate that arjungein and its glucoside are moderately active in the above assays. MATERIALS AND METHODS Plant Material The dried bark of the Terminalia arjuna was purchased from local market at Chandigarh. The corresponding author identified the material and the voucher specimen is preserved in the Herbarium of our institute. Chemicals and Reagents Diphenyl picryl hydrazyl, luminol, Nitroblue tetrazolium were procured from Sigma chemicals, USA, and the solvents were obtained from Ranbaxy, India. Fresh human blood was collected from healthy human volunteers by venipuncture. Isolation of Constituents The dried and powdered bark was defatted with n-hexane and extracted with methanol. The methanolic extract on de-tannification with lead acetate was subjected to column chromatography on silica gel with chloroform-methanol mixtures. Two sapogenins (1 and 2) and two saponins (3 and 4) were obtained on repeated column chromatographies. The extensive chemical and spectral studies along with comparison with the literature spectral data confirmed their chemical structures to be arjunic acid (1) (Row et al., 1970a), arjungenin (2) (Honda et al., 1976a), arjunetin (3) (Row et al., 1970b) and arjunglucoside II (4) (Honda et al., 1976 b). DPPH Assay Assay was performed by the method of Bouchet and Viturro (Viturro et al., 1999; Bouchet et al., 1998). DPPH solution (95 μL) was incubated in dark with methanolic solutions of the test compounds (5 μL) in microtitre plate well. After 15 min. the absorption was measured at 515 nm. Ascorbic acid was used as a standard. The IC50 was calculated by Sigmaplot software. Chemiluminescence Assay The assay was performed and standardized according to the method prescribed in the manual of the luminometer. To cell suspension, luminol stock solution was added so as to attain the concentration of 0.5 mM. The cell suspension containing luminol was added to the microtitre plates containing test compounds in triplicate. After 5 min PMA (150 μL) was added to trigger the reaction. The micrototre plate was measured in kinetic mode for 90 min. during which each well was read for 740 ms. A curve of light intensity (RLU) was plotted against time and the area under curve (AUC) was calculated as total luminescence. The percent inhibition of luminescence was calculated as