Inhibitory Effects of Stephania tetrandra S. MOORE on Free Radical-Induced Lysis of Rat Red Blood Cells

Abstract

Stephania tetrandra (ST) has been used as an analgesic drug for arthritis for thousands of years in China. ST has been reported to have anti-inflammatory 1,2) and immunosuppressive 3) properties in vitro and in experimental animals. This study was performed to clarify the role of ST from an antioxidative point of view.In the present study, we examined the effects of ST on scavenging activities of reactive oxygen species (ROS) by means of electron spin resonance (ESR) method, and on red blood cell (RBC) hemolysis induced by an azo free-radical initiator in healthy volunteers. The ESR method was used for the measurement of O 2 Ϫ · and HO · scavenging activities of ST. RBC hemolysis was used for determination of the antioxidative effect of ST, and the influence of ST on RBCs, which have the most important role in hemorrheology.Furthermore, we hypothesized that ingestion of ST extract (ST.E) would further strengthen the antioxidative effect of plasma, which already has a function of reducing the free radical-induced tissue damage in cerebrovascular disease and ischemic heart disease, if the responsible compounds in ST could be absorbed and circulated in blood. Therefore, the present study examined the effect on plasma antioxidative activity after a gavage-dose of ST.E in rats. MATERIALS AND METHODS Preparation of Drugs ST.E was prepared fromStephania tetrandra (Chinese origin) purchased commercially (Uchida Wakanyaku Co. Ltd., Tokyo, Japan). The extract was obtained by boiling the herb in water for 50 min and then freeze-drying into a resultant powder. We obtained 10.67 g of extract from 100 g of raw material.This powder extract was dissolved in phosphate buffered saline (PBS, pHϭ7.4).Reagents Tetrandrine (TET), fangchinoline (FAN) and cyclanoline (CYC) were the products of Tsumura & Co. (Tokyo, Japan). Their contents in ST.E were 2%, 1% and 1%, respectively. The chemical structures of TET, FAN and CYC are illustrated in Fig. 1. The purity of each compound was examined by HPLC using a photodiode array detector.ESR Assay for O 2 ؊ · and HO · Scavenging Activities of ST.E Measurement of O 2 Ϫ · and HO · scavenging activities was performed as described previously 4) with slight modification. ST.E was diluted to 0.2, 2.0, and 20.0 mg/ml with PBS. O 2 Ϫ · and HO · scavenging activities of each sample (50 ml) were assessed by electron spin resonance (ESR) technique 5) and then IC 50 (Inhibition Concentration 50%) was calculated.In this experiment, O 2 Ϫ · was generated from a hypoxanthine (HPX)-xanthine oxidase (XOD) reaction system in PBS.6) Briefly, 15 ml of 9.2 M 5,5-dimethyl-1-pyrroline Noxide (DMPO) (LABOTEC, Tokyo, Japan), 50 ml of 2 mM HPX (Sigma, St. Louis, MO, U.S.A.), 35 ml of 5.5 mM diethylenetriamine-N,N,NЈ,NЉ,NЉ-pentaacetic dianhydride (DETA- Crude preparations of Stephania tetrandra S. MOORE (ST), a traditional herbal medicine, have been used safely for arthritis and silicosis in China. In this study, we demonstrated that ST in vitro protects red blood cells from 2,2-azo-bis (2-amidinopropane) dihydrochlorid...

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

“…Our results, using isolated rat liver mitochondria, showing tetrandrine protection from ROS and MPT induction, suggest that the compound may protect cells from oxidative stress-induced apoptosis, in quite agreement with other authors (Seow et al, 1988;Matsuno et al, 1990;Castranova, 1994;Ren et al, 1995;Hui et al, 1996;Shen et al, 1999aShen et al, ,b, 2001Koh et al, 2003;Liu et al, 2004;Sekiya et al, 2005). However, others showed that tetrandrine-induced apoptosis associated with oxidative stress and MPT induction in both cancer cell lines (Jin et al, 2002;Oh and Lee, 2003;Jang et al, 2004) and rat primary hepatocytes (Yan et al, 2006).…”

“…We used rat liver mitochondria as a common biological material used in our laboratory to investigate general effects of compounds, specially on the mitochondrial function, as this is a wellcharacterized sub-cellular fraction with a high degree of purity (90%) and easily obtained in large amounts (150-200 mg of protein per liver). Tetrandrine concentrations used in this study, expressed as nanomol tetrandrine per milligram mitochondrial protein and corresponding to a maximum of 80 lM for protective actions, are within the concentration range used by other authors for studies with tissues and cells (Cao, 1996;Ye et al, 2000;Koh et al, 2003;Sekiya et al, 2005).…”

“…Tetrandrine protects several types of cells from oxidative stress by acting as a ROS scavenger, namely neutrophils (Seow et al, 1988;Matsuno et al, 1990;Castranova, 1994;Hui et al, 1996;Shen et al, 1999aShen et al, ,b, 2001, cerebellar granule neurons (Koh et al, 2003), erythrocytes (Sekiya et al, 2005), and tissues from heart (Ren et al, 1995) and liver (Liu et al, 2004). Tetrandrine was shown to induce depression of H 2 O 2 production and inhibition of O 2 AE À radical generation (Seow et al, 1988), and also to efficiently react with .…”

“…Recent studies have shown that tetrandrine-induced apoptosis in Neuro 2a mouse neuroblastoma cells, rat glioma cells (C-6) (Jin et al, 2002), human hepatoblastoma (Hep G2) (Oh and Lee, 2003), human leukaemia cells (U937) (Jang et al, 2004), and rat primary hepatocytes (Yan et al, 2006) is associated with ROS generation and cytochrome c release, thus suggesting that mitochondria are primarily involved. However, these studies seem to be controversial with those published by others (Seow et al, 1988;Matsuno et al, 1990;Castranova, 1994;Hui et al, 1996;Shen et al, 1999aShen et al, ,b, 2001Koh et al, 2003;Sekiya et al, 2005;Ren et al, 1995;Liu et al, 2004) demonstrating that tetrandrine protects several types of cells from oxidative stress by acting as a ROS scavenger.…”

Fangchinoline Inhibits Breast Adenocarcinoma Proliferation by Inducing Apoptosis

“…16) Fangchinoline was reported to be an antioxidant. 17) However, there is little information in the literature on the cytotoxic effects of fangchinoline on human breast cancer cells. In the present study, we therefore investigated the antitumor effects of fangchinoline in breast cancer cells (Fig.…”