The effects of Coptidis Rhizoma extract on a hypercholesterolemic animal model.
The serum cholesterol (total, free, esterified, low density lipoprotein (LDL) and oxidized LDL) levels of rats fed a diet containing, by weight, 1% cholesterol and 0.5% cholic acid increased, as compared with those of rats fed a normal diet. The levels, especially of total cholesterol, LDL and oxidized LDL, were reduced significantly in a dose-dependent manner, in rats given Coptidis Rhizoma extract orally at doses of 50 and 100 mg/kg body wt./day for 30 days. These results indicate that Coptidis Rhizoma extract is effective in reducing the pathological damage caused by hypercholesterolemia, through lowering of serum cholesterol levels. In addition, Coptidis Rhizoma extract reduced the level of liver cholesterol, but it did not reduce that of fecal cholesterol, suggesting that the cholesterol level-lowering effect resulted from the reduction of cholesterol synthesis, not the enhancement of its excretion. Furthermore, the serum thiobarbituric acid-reactive substance level decreased after oral administration of Coptidis Rhizoma extract, indicating that Coptidis Rhizoma could prevent hypercholesterolemic disease through reducing lipid peroxidation. This study demonstrates that Coptidis Rhizoma may be a useful therapy for hypercholesterolemia through reducing oxidative stress and cholesterol levels.
Key words: Coptidis Rhizoma, hypercholesterolemia, oxidized LDL, thiobarbituric acid-reactive substance, rat
Cardiovascular disease, including atherosclerosis, is the most common cause of mortality and morbidity worldwide (Krieger, 1998). Although several factors, such as life style, a diet high in saturated fat and cholesterol, family history, age, hypertension and diabetes mellitus, have been reported to cause heart failure (The expert panel, 1988, 1993; Schaefer et al., 1995), high levels of cholesterol, particularly LDL cholesterol, in the blood are mainly responsible for it (Castelli et al., 1986; Gordon and Rifkind, 1989; Krieger, 1998). When the sum of the cholesterol synthesized and obtained from the diet exceeds the amount required for the synthesis of membranes, bile salts and steroids, pathological accumulations of cholesterol in blood vessels can develop in humans, resulting in the obstruction of blood vessels as a result of atherosclerosis. Therefore, an increased cholesterol level is a significant predictor of the development of coronary artery disease (CAD).
Reducing cholesterol levels lowers the frequency of CAD and improves pathological damage in persons with heart failure. Although numerous trials of diet- or drug-based cholesterol-reduction have provided compelling evidence that reducing cholesterol levels decrease the incidence of CAD (Levine et al., 1995; Kwiterovich, 1997; Brown, 2001), the therapeutic or improving effects of Chinese traditional medicines, including Coptidis Rhizoma, against pathological conditions caused by hypercholesterolemia have rarely been studied.
Coptidis Rhizoma is prescribed as a crude drug in Oren-gedoku-to (Huang-Lian-Jie-Du-Tang) and San'o-shashin-to (San-Huang-Xie-Xin-Tang), which are folk medicines used widely for the treatment of hypertension, hemoptysis, hematemesis, melena, cerebral hemorrhage, gastrointestinal disorders and inflammatory disease. In view of these uses, Coptidis Rhizoma itself would also be expected to show activity against hypercholesterolemia. Fortunately, a direct relationship between LDL cholesterol level and atherosclerosis has been established in animal models. Animals consuming diets high in saturated fat and cholesterol develop elevated LDL cholesterol levels and atherosclerosis and intimal lesions that progress from fatty streaks to ulcerated plaques resembling those of human atherosclerosis. These animal studies also support the idea that lowering total and LDL cholesterol concentrations reduces the incidence of CAD events (Ross, 1993; Schaefer et al., 1995). Based on these studies, we investigated the effects of Co ptidis Rhizoma on hypercholesterolemia using a hypercholesterolemic animal model.
* Materials and Methods
Preparation of Coptidis Rhizoma extract
The rhizome of Coptidis Rhizoma (Coptis japonica Makino), grown in China and supplied by Uchida Wakan-yaku Co. Ltd., Tokyo, Japan, was finely powdered and extracted using distilled water at 100[degrees]C for 1 h (rhizoma:water = 1:10, w/v). After removal of the insoluble matter by filtration, the filtrate was concentrated in vacuo and lyophilized to yield a residue. The yield was 19.7%, by weight, of the original material and was composed of 20.8% berberine, 6.1% coptisine and 5.2% palmatine. A high-performance liquid chromatogram of the extract is shown in Fig. 1.
Hypercholesterolemic animal model
Male Wistar strain rats (Japan SLC Inc., Hamamatsu, Japan) weighing about 180 g each were kept under a conventional light regimen with a dark night at room temperature (about 23[degrees]C) and humidity (about 60%). Hypercholesterolemia was induced by feeding the rats a diet containing, by weight, 18% casein, 1% cholesterol and 0.5% cholic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan). The rats were divided into 4 groups, avoiding any intergroup differences in body weight. The normal group was given a basal diet (cholesterol- and cholic acid-free) and the control and treated groups (Coptidis Rhizoma extract) were fed the cholesterol diet according to a pair-feeding schedule for the experimental period. Coptidis Rhizoma extract was dissolved in water and given orally to rats at a dose of 50 or 100 mg/kg body wt./day for 30 days using a stomach tube. Six hours after the last dose, the rats were decapitated, their blood was collected and serum for the measurement of cholesterol levels was obtained immed iately by centrifugation. Each liver was removed, dried on tissue paper, weighed and stored at -80[degrees]C until analysis. The feces of each rat were collected for the final 2 days of the experimental period.
Measurement of cholesterol levels
Serum total and free cholesterol levels were determined using commercial kits (Cholesterol E-Test Wako and Free Cholesterol E-Test Wako, respectively, obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan). Esterified cholesterol levels were calculated by subtracting free cholesterol levels from total cholesterol levels. Serum LDL cholesterol levels were determined according to the method of Noma et al. (1978, 1979). The liver and feces of each rat were homogenized, the total cholesterol was extracted with a mixture of chloroform and methanol (2:1, v/v) and the amounts of total cholesterol were determined using the Wako kit described above.
LDL preparation and oxidation
LDL was isolated from serum using density-gradient ultracentrifugation, as described by Havel et al. (1955), and dialysis against two changes of 150 mM NaCl (pH 7.4) for 48 h at 4[degrees]C. Before oxidation, LDL was diluted with 150 mM NaCl (pH 7.4) to a concentration of 300 [micro]g protein/ml. LDL protein levels were determined using the method of Lowry et al. (1951). Oxidation of LDL was carried out by incubation with freshly prepared 20 mM Cu[SO.sub.4] at 37 [degrees]C for 4 h in a shaking water bath, immediately after which the extent of lipid peroxidation was determined by measuring the amount of thiobarbituric acid (TBA)-reactive substance formed (Ohkawa et al., 1979).
Determination of serum TBA-reactive substance levels The serum TBA-reactive substance levels were measured using the method of Naito and Yamanaka (1978).
Data are presented as means [+ or -] S.E. Differences among groups were analyzed using the Dunnett's test and those at p < 0.05 were accepted as significant.
Figure 2 shows the serum cholesterol profiles of rats fed the 1% cholesterol and 0.5% cholic acid diet, together with Coptidis Rhizoma extract at an oral dose of 50 or 100 mg/kg body wt./day for 30 days. The rats fed a high cholesterol diet had markedly higher serum levels of total, free, esterified and LDL cholesterol relative to those fed a normal diet. The serum cholesterol levels of rats given Coptidis Rhizoma extract were, however, significantly reduced as compared with those of the hypercholesterolemic control rats and the serum cholesterol level-reducing activity increased more as the oral dose administered increased. In particular, the administration of Coptidis Rhizoma extract at a dose of 100 mg/kg body wt./day for 30 days reduced the total and LDL cholesterol levels by 20%.
We also investigated the effect of Coptidis Rhizoma extract on oxidized LDL (Table 1). As compared to normal rats, the oxidized LDL level was markedly elevated in hypercholesterolemic rats. The administration of Coptidis Rhizoma extract, however, reduced the oxidized LDL level. After oral doses of 50 and 100 mg, the oxidized LDL level declined from 1.66 to 1.02 (39% decrease, p < 0.001) and 0.89 nmol/ml (46% decrease, p < 0.001), respectively.
The total cholesterol levels of the liver and feces of rats fed the 1% cholesterol and 0.5% cholic acid diet were significantly higher than those of rats fed the normal diet (Table 2). The cholesterol levels of the liver of rats given Coptidis Rhizoma extract at oral doses of 50 and 100 mg/kg body wt./day were effectively reduced by 9% and 24%, respectively, whereas the total cholesterol levels of their feces did not show significant changes after the administration of Coptidis Rhizoma extract at either oral dosage.
The effects of Coptidis Rhizoma extract on lipid peroxidation in rats fed a hypercholesterolemic diet are presented in Table 3. The oral administration of Coptidis Rhizoma extract at doses of 50 and 100 mg/kg body wt./day for 30 days significantly reduced the serum TBA-reactive substance level from 2.35 to 2.17 and 1.99 nmol/ml, respectively.
Unregulated cholesterol levels lead to serious pathological conditions. It is widely understood that cholesterols, especially LDL cholesterol and its oxidized derivatives, play an important role in the pathogenesis of atherosclerotic conditions (Gordon and Rifkind, 1989; Krieger, 1998). Normally, most cholesterol serves as a structural element in the walls of cells, whereas much of the rest is in transit through the blood or functions as the starting material for the synthesis of bile acids in the liver, steroid hormones in endocrine cells, or vitamin D in the skin. Increased cholesterol concentrations in plasma are, however, a cause of coronary atherosclerosis and increase risk of CAD (Kannel and Castelli, 1979; Stamler et al., 1986; Gordon and Rifkind, 1989; Anderson et al., 1991; Berliner et al., 1995). In addition, the relationship between LDL cholesterol and CAD has been established in many observational epidemiological studies and in numerous clinical trials of lipid-lowering interventions through diet, drugs and surgery (Ballantyne, 1998; Criqui and Golomb, 1998). Several studies have indicated that diet treatment or drug therapy that lowers LDL cholesterol levels can reduce subsequent CAD-associated morbidity and mortality (Levine et al., 1995; Kwiterovich, 1997). Based on the evidence, great strides have been made in CAD risk reduction worldwide, by lowering LDL cholesterol through diet or drug therapy.
In the present study, we investigated the effects on hypercholesterolemia of Coptidis Rhizoma, which contains bioactive alkaloids, such as berberine and coptisine, known to have blood pressure-lowering activity. While the rats fed the hypercholesterolemic diet showed high serum concentrations of cholesterol as compared to rats given the normal diet, oral administration of Coptidis Rhizoma extract reduced the high levels of cholesterol (Fig. 2). As shown in the results, the decrease of total serum cholesterol induced by Coptidis Rhizoma extract was ascribed to the decreases of both free and esterified cholesterol levels. A small fraction of the cholesterol in the liver is incorporated into the membranes of hepatocytes, but most of it is exported. Esterified cholesterol is one of the exported forms. It is formed in the liver through the action of acyl-CoA-cholesterol acyl transferase, which catalyzes the conversion of cholesterol into a more hydrophobic form that is transported in secreted lipoprotein particles to other tissues that use cholesterol or is stored in the liver. Thus, the reduction of esterified cholesterol level indicates that the cholesterol was used for the synthesis of vital molecules in tissues, including the liver.
The serum LDL cholesterol level was significantly reduced by Coptidis Rhizoma extract (Fig. 2). When there is excess LDL in the blood, it is deposited in the blood vessel walls and becomes a major component of atherosclerotic plaque lesions. Therefore, serum LDL cholesterol concentration should be used as the basis for initiating and monitoring treatment of patients with elevated blood cholesterol (The expert panel, 1988, 1993; Schaefer et al., 1995). Our results indicate that Coptidis Rhizoma is a potential therapeutic agent for hypercholesterolemia because of its reduction of LDL cholesterol levels. Experimental animals which consumed high dietary levels of cholesterol developed elevated LDL cholesterol levels and atherosclerosis (Ross, 1993). Ross (1993) also reported a relationship between LDL cholesterol and atherosclerosis, and suggested the pathological process could be reversed by reducing the serum LDL cholesterol level. In view of our present results with a hypercholesterolemic rat model, we would e xpect Coptidis Rhizoma to reduce CAD, including atherosclerosis, through lowering serum levels of cholesterol.
Oxidized LDL exerts a multitude of potentially atherogenic effects in vivo and in vitro (Steinberg et al., 1989; Witztum and Steinberg, 1991; Ramirez-Tortosa et al., 1999) and it could initiate and promote atherosclerosis. The link between oxidized LDL and atherogenesis explains the rationale for using antioxidants to preserve endothelial cell function and to prevent atherosclerotic plaque formation (Ribeiro Jorge et al., 1998). Coptidis Rhizoma extract reduced oxidized LDL levels elevated by consuming a hypercholesterolemic diet (Table 1), which suggests that Coptidis Rhizoma would prevent hypercholesterolemic atherosclerosis induced by oxidized LDL.
We also measured the levels of liver and fecal total cholesterol to investigate whether the cholesterol-lowering effect of Coptidis Rhizoma extract was due to the reduction of cholesterol synthesis or the enhancement of its excretion (Table 2). While Coptidis Rhizoma extract reduced the level of liver cholesterol, it did not reduce that of fecal cholesterol. This result indicates that the serum cholesterol level-lowering effect resulted from the reduction of cholesterol synthesis, not the stimulation of cholesterol excretion.
Hypercholesterolemic atherosclerosis is associated with an increase in the level of the lipid peroxidation product malondialdehyde, which is an index of the level of oxygen free radicals (Prasad and Kalra, 1993; Prasad, 1999). A decrease in lipid peroxidation leads to a reduction in arterial wall cholesterol content. Therefore, reduction of atherosclerosis caused by hypercholesterolemia was associated with a decrease in lipid peroxidation, as the serum TBA-reactive substance level decreased after the administration of Coptidis Rhizoma extract (Table 3). On the basis of this result, we hypothesize that Coptidis Rhizoma would prevent the development of hypercholesterolemic atherosclerosis by decreasing lipid peroxidation.
Some herbs have recently attracted a great deal of attention as alternative therapies for various pathological conditions. Chinese traditional herbal medicines and their mixtures showing beneficial effects against human diseases have been developed as a result of clinical experience accumulated over time and have been used widely for the treatment of a variety of inflammatory conditions, cardiovascular disorders and other ailments (Ozaki, 1995; lizuka et al., 2000). Among these herbal medicines, Coptidis Rhizoma has long been used for treating gastroenteritis and diarrhea (Fukutake et al., 1998), and has attracted much attention as an anti-tumor agent (Iizuka et al., 2000). Moreover, Coptidis Rhizoma is prescribed as a crude drug in folk medicines, such as Oren-gedoku-to and San'o-shashin-to, for several vascular disorders. The results of our present study support the hypothesis that the Coptidis Rhizoma would itself be useful as an alternative therapy for hypercholesterolemia. Our results also suggest that C optidis Rhizoma would be effective in preventing hypercholesterolemic atherosclerosis and lowering the relative risk of CAD through decreases in lipid peroxidation and cholesterol levels.
Herbal extracts consist of various ingredients and their biological activity is usually not attributable to a single moiety. Although it remains unclear which of the components of Coptidis Rhizoma exhibit cholesterol level-lowering activity, the alkaloids present in Coptidis Rhizoma (berberine, coptisine, jateorrhizine and palmatine) were considered to be its active constituents (Otsuka et al., 1974; Peng et al., 1997; Schmeller et al., 1997). In particular, berberine is an alkaloid present in numerous plants of the genus Coptis and shows a wide range of pharmacological and biological activities, including lowering blood pressure and antisecretory, antiplatelet, anti-cerebral ischemic, vasodilatory, antiarrhythmic, anti-inflammatory, anti-microbial, anti-cancer and anti-neoplastic properties (Lin et al., 1999a, b; Wu et al., 1999). Based on the results of our present investigation, further studies on the active component(s) of Coptidis Rhizoma and the mechanism(s) of its protective effect against hypercholest erolemia are needed.
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Table 1 Effects of Coptidis Rhizoma extract on serum oxidized LDL levels in hypercholesterolemic rats. Group Dose (mg/kg Oxidized LDL body wt./day) (nmol/ml) Normal rats -- 0.15 [+ or -] 0.02 Hypercholesterolemic rats Control -- 1.66 [+ or -] 0.17 (a) Coptidis Rhizoma extract 50 1.02 [+ or -] 0.08 (a,b) Coptidis Rhizoma extract 100 0.89 [+ or -] 0.04 (a,b) Statistical significance: (a) p < 0.001 vs. normal rats (b) p < 0.001 vs. hypercholesterolemic control rats. Table 2 Effects of Coptidis Rhizoma extract on liver and fecal total cholesterol levels in hypercholesterolemic rats. Group Dose (mg/kg Liver body wt./day) (mg/g tissue) Normal rats - 5.39 [+ or -] 0.24 Hypercholesterolemic rats Control - 24.08 [+ or -] 1.02 (a) Coptidis Rhizoma extract 50 21.94 [+ or -] 0.96 (a,b) Coptidis Rhizoma extract 100 18.33 [+ or -] 0.66 (a,c) Group Feces (mg/g) Normal rats 2.49 [+ or -] 0.12 Hypercholesterolemic rats Control 16.72 [+ or -] 1.83 (a) Coptidis Rhizoma extract 17.48 [+ or -] 1.05 (a) Coptidis Rhizoma extract 16.84 [+ or -] 0.52 (a) Statistical significance: (a)p < 0 vs. normal rats, (b)p < 0.01, (c)p < 0.001 vs. hypercholesterolemic control rats. Table 3 Effects of Coptidis Rhizoma extract on serum thiobarbituric acid-reactive substance levels in hypercholesterolemic rats. Group Dose TBA-reactive (mg/kg substance body wt./day) (nmol/ml) Normal rats - 1.72 [+ or -] 0.10 Hypercholesterolemic rats Control - 2.35 [+ or -] 0.12 (b) Coptidis Rhizoma extract 50 2.17 [+ or -] 0.07 (b,c) Coptidis Rhizoma extract 100 1.99 [+ or -] 0.10 (a,d) Statistical significance: (a)p < 0.01, (b)p < 0.001 vs. normal rats, (c)p < 0.05, (d)p < 0.001 vs. hypercholesterolemic control rats.
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