Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B
Introduction
There is increasing interest in natural antioxidant products for use as medicines and food additives (Willcox et al., 2004). Vitamin C, vitamin E and carotenoids are some of these widely used natural antioxidants. Antioxidants played an important role in lowering oxidative stresses caused by reactive oxygen species (ROS). ROS including superoxide anion radical, hydroxyl radical and hydrogen peroxide are generated under physiological and pathological stresses in human body (Nordberg and Arner, 2001). Some severe chronic diseases, such as arthritis, cancer, diabetes, cardiovascular diseases, inflammations and neurological disorders are related to the imbalance of ROS formation and their elimination (Halliwell, 1996). Antioxidants protect against these complex diseases through scavenging free radicals and reducing hydrogen peroxide (Shahidi and Wanasundara, 1992). Therefore, the identification and study of novel compounds characterized with antioxidant activity from natural sources is an important strategy to improve human health condition and life quality.
Salvia miltiorrhiza (Danshen in Chinese) is one of the most widely used traditional herb medicines for the treatment of a variety of diseases, such as cardiovascular diseases, hepatitis, hepatocirrhosis, chronic renal failure and dysmenorrhea (Li, 1998). The formulae derived from this herb like Compound Danshen Dripping Pill, Danshen Pian and Danshen Injection have been developed and used in the clinic in China, Korea and Russia (Zhao et al., 2006). The pharmacologically active compounds of S. miltiorrhiza comprise two fractions: lipophilic diterpenoid tanshinones and water-soluble phenolic acids. In recent years research interests have been focused on phenolic acids. Total twenty-five phenolic acid compounds have been isolated and identified from this species (Jiang et al., 2005), of which danshensu (3-(3,4-dihydroxyphenyl)lactic acid) and salvianolic acid B (lithospermate acid B) (Fig. 1) have the highest contents, accounting for over 1% and 3–5% of total dried weight, respectively (Hu et al., 2005).
Effects of salvianolic acid B on oxidative stresses have been investigated extensively in different animal models. In cerebral and heart ischemia-reperfusion animal models, salvianolic acid B enhanced antioxidant defense, decreased malondialdehyde level, and attenuated brain and heart damage in mice and rabbit (Chen et al., 2000, Du et al., 2000). Salvianolic acid B restored activities of superoxide dismutase (SOD) and catalase, decreased hydroxyl radical level, and suppressed the progression of renal damage and failure induced by streptozotocin andcephaloridine in rats (Yokozawa et al., 1997, Lee et al., 2003). Administration of salvianolic acid B to Wistar rats decreased the urinary levels of creatol that was derived from oxidation degradation of creatinine (Yokozawa et al., 1995). These in vivo oxidative stress protective activities of salvianolic acid B were also supported by the results of in vitro cell culture experiments. Salvianolic acid B protected ventricular myocytes and aortic endothelial cells against necrosis inflicted by oxygen radicals generated from xanthine oxidase and inhibited the reduction of cytochrome c by superoxide radical anion (Fung et al., 1993). Furthermore, it was demonstrated that salvianolic acid B protected endothelial cells from membrane hypermeability and injury induced by inflammatory factors such as vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), and cholestantriol (Qui et al., 2001, Ren et al., 2003, Zhou et al., 2005). Salvianolic acid B suppressed lactate dehydrogenase leakage and malondialdehyde formation in the epithelial cells induced by cisplatin (Yokozawa et al., 1997) and inhibited intracellular ROS generation in cultured mesangial cells induced by high glucose and hydrogen peroxide (Lee et al., 2003). Salvianolic acid B also inhibited both NADPH-Vitamin C and iron-cysteine induced lipid peroxidation in rat brain, liver and kidney homogenates, microsomes and hepatocytes in vitro (Huang and Zhang, 1992, Liu et al., 1992, Wang et al., 2005). It showed a hepatoprotective effect on hepatocyte necrosis induced by carbon tetrachloride, d-galactosamine and lipopolysaccharide (Hase et al., 1997). Comparing to salvianolic acid B, danshensu was less studied. But, protective effects of danshensu in human umbilical vein endothelial cell against injuries induced by homocysteine and its inhibition of ROS production in human neroblastoma cells stimulated by 1-methyl-4-phenylpyridinium ion were reported recently (Chan et al., 2004, Wang and Xu, 2005).
Although the potential antioxidant capacity of salvianolic acid B and danshensu could be deduced from their protective effects against oxidative stresses in animal models and in cell cultures as mentioned above, the chemical mechanisms of their antioxidant activities in the molecular level were not well understood. Danshensu and salvianolic acid B are caffeic acid derivatives that occur in many plants as secondary metabolites. It is believed that the antioxidant activities of danshensu and salvianolic acid B are associated with their phenol functional group. But, little information is available about their antioxidant activities in chemical testing systems. Furthermore, to our knowledge, no research has been done to compare the antioxidant activities of salvianolic acid B and danshensu. Therefore, we conducted the present study to evaluate the antioxidant activities of danshensu and salvianolic acid B isolated from S. miltiorrhiza root in chemical testing assays. The antioxidant activities, including reducing power, chelating iron and scavenging reactive oxygen species of superoxide anion, and hydroxyl radicals, hydrogen peroxide (H2O2), 2,2′-azino-di-3-ethylbenzthiazoline sulphonate (ABTS), and DPPH, were investigated in seven different chemical assays. The differences of radical scavenging and antioxidant properties between danshensu and salvianolic acid B were compared with the same dose of vitamin C, a standard antioxidant commonly used in food and pharmaceutical industries. The protective effects of danshensu and salvianolic acid B were also evaluated against the human vascular endothelial cell damage induced by long hydrogen peroxide treatment.
Section snippets
Chemicals
2,2′-Azino-di-3-ethylbenzthiazoline sulphonate (ABTS), 2,2’-azobis (2-amidino-propane) dihydrochloride (AAPH), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), 1,1-diphenyl-2-picrylhydrazyl (DPPH), nicotinamide adenine dinucleotide (NADH), Nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), 1,10-phenanthroline, 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 30% hydrogen peroxide (H2O2), and vitamin C (l-ascorbic acid) were purchased from
Ferrous ion chelating activity
Chelating agent can interfere with the formation of ferrous and ferrozine complex and thus decrease the red color. The chelating activities of the salvianolic acid B and danshensu for ferrous ion were assessed. As shown in Fig. 2, salvianolic acid B and danshensu slightly inhibited the formation of the red-colored complex. At maximum concentration of 1.0 mg ml−1, salvianolic acid B and danshensu chelated merely 7.5% and 2.8% of ferrous ions, equivalent to approximately 1/2 and 1/5 of that of
Discussion
Phenolic acids are thought to play a positive role in the prevention of human diseases and food industry. Salvianolic acid B and danshensu are the most abundant active ingredients in S. miltiorrhiza. Increasing reports on the protective effects of salvianolic acid B and danshensu against oxidative stresses implied that they may be potential antioxidants. An antioxidant exerts its antioxidant activity through various mechanisms, among them are chelating of metal ferrous iron, decomposition of
Acknowledgements
This study was supported by National Natural Science Foundation of China (30371712), the National Fund for Distinguished Young Scholars (20425620), the Program of Introducing Talents of Discipline to Universities (No: B6006) and Tianjin Municipal Key Technologies R&D Program (023183011).
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