| Cardiovascular diseases are the leading cause of both death and disability around the world, and hyperlipidaemia is a long-known risk factor for cardiovascular disease. For these reasons, finding methods for early prevention and control of hyperlipidaemia is crucial. Hyperlipidaemia refers to abnormal levels in serum levels, such as low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglyceride (TG). Most current hypolipidemic drugs are expensive and have potential side effects, so researches are increasingly focusing on natural alternative medicines that reduce blood lipid levels. Coptis chinensis (Ranunculaceae) is a natural herb that is widely used in China for prevention and treatment of infectious diseases. The main active compounds in Coptis chinensis are alkaloids. The major alkaloids in Coptis chinensis are berberine, palmatine, coptisine, and epiberberine. Among those main alkaloids, the hypolipidemic effect and mechanism of berberine have been verified. In our previous studies, we found that the oral LD50 of palmatine, epiberberine, coptisine and berberine in Kunming mice was 1533mg/kg, 1360mg/kg,852mg/kg, and 712mg/kg, respectively. According to the results from previous studies, we could conclude that palmatine was the safest alkaloid monomer in Coptis chinensis. However, it is still not clear whether palmatine has the hypolipidemic effect similar to that of berberine. In order to clarify the hypolipidemic effect and mechanism of palmatine and support for its further utilization, we compared the major alkaloid contents in rhizoma and fibrous root of Coptis chinensis. And we isolated palmatine monomer from the rhizoma of Coptis chinensis, and studied the hypolipidemic effect and mechanism of palmatine in hyperlipidaemia hamsters induced by high fat diet. The methods and results are follows:1. Comparison of major alkaloid contents in rhizoma and fibrous root of Coptis chinensisHigh performance liquid chromatography was applied for quantitative analysis of major alkaloid contents (including berberine, palmatine, coptisine, as well as epiberberine) in rhizoma and fibrous root of Coptis chinensis.The results showed that berberine, palmatine, coptisine, and epiberberine existed in the rhizoma as well as fibrous root of Coptis chinensis, however, the compositions of major alkaloids in the rhizoma as well as fibrous root of Coptis chinensis were different. The content of berberine, palmatine, coptisine, and epiberberine in the rhizoma of Coptis chinensis was 5.61%,1.48%,1.74% and 0.72%, respectively. The content of berberine, palmatine, coptisine, and epiberberine in the fibrous root of Coptis chinensis was 1.20%,0.08%,1.28%, and 0.53%. According above results, although the kind of alkaloids in the rhizoma and fibrous root of Coptis chinensis was similar, the content of total alkaloids in the rhizoma of Coptis chinensis was higher than that in the fibrous root of Coptis chinensis. Furthermore, the distribution of alkaloids in the fibrous root and rhizoma of Coptis chinensis was also different. In the rhizoma of Coptis chinensis, the content of berberine was the highest, and the content of epiberberine was the lowest. In contrast, in the rhizoma of Coptis chinensis, the content of coptisine was the highest, and the content of palmatine was the lowest.2. Preparation of palmatine from the rhizoma of Coptis chinensisA sample of rhizoma of Coptis chinensis (10kg) was dried and extracted by cold-soaked extraction. The cold-soaked extraction conditions were as follows:ethanol concentration 70%, extracting time 24h, ratio of solvent to sample 1:10, and extracting for three times. These extracted solutions were combined and then the solvent was recycled to obtain the residue, and the yield of residue was calculated as the yield of ethanol extract. The crude extract of palmatine from the rhizoma of Coptis chinensis was obtained by the high-speed counter-current chromatography. The conditions for separating palmatine from ethanol extract by high-speed counter-current chromatography were as follows:chloroform-rnethanol-0.2mol/L HC1 (4:1.5:2, v/v/v), mobile phase flow rate was 2.0mL/min, detection wavelength was at 254nm, host speed at 800r/min, constant temperature circulating temperature is at 25℃. High-speed counter-current chromatography separation of effluent collected division,6 min/tube. The collected effluent was monitored by thin layer chromatograph (TLC), and the expansion system used in this experiment was cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethyla-mine (3:3.5:1:1.5:0.5:1, v/v/v/v/v/v). The detection wavelength in TLC was 345nm. Effluents containing palmatine were combined, concentrated under vacuum, and dried resulting in the crude extract of palmatine. Then the crude extract of palmatine was further purified by Sephadex LH-20 column chromatography, and the yield of palmatine was calculated. The molecular weight of palmatine was determined by mass spectrometry. The chemical structure of palmatine was elucidated on the basis of spectroscopy data. High performance liquid chromatography was applied to analyze the purity of palmatine prepared from Coptis chinensis.The results showed that the yield of ethanol extract of Coptis chinensis was 36%, and the the yield of palmatine from the ethanol extract by high-speed counter-current chromatography and Sephadex LH-20 column chromatography was 4%. The purity of palmatine prepared from the rhizoma of Coptis chinensis was up to 95%.3. Hypolipidemic effect of palmatine in vivoMale hamster was chosen as the animal model in the study of hypolipidemic effect of palmatine. Whether the hyperlipidaemia golden hamster model was build successfully or not was judged according to the serum levels of lipids. Hyperlipidaemia golden hamster model was induced by high fat diet for 4 weeks. After the hyperlipidaemia golden hamster model had been induced successfully, the hyperlipidaemia hamsters were then divided into model group, palmatine groups (low-dose:23.35mg/kg; middle-dose:46.70mg/kg; high-dose:70.05mg/kg), as well as simvastatine positive control group (1.2mg/kg). Each group had 10 hamsters. In addition, ten normal hamsters fed with normal regular diet were chosen as the normal control group. All drugs were intragastrically to hamsters fed with high fat diet for 4 weeks. In addition, the feces from hamsters in all groups were collected for 3 consecutive days for quantitative analysis of content of TC and TBA in feces.The results showed that serum levels of TC, TG, LDL-C and HDL-C in hamsters were significantly increased by high fat diet for 4 weeks in hamsters(P<0.01), indicating hyperlipidemia golden hamster model had been successfully established. Compared with the normal control group, the serum levels of TC, TQ LDL-C, and HDL-C were increased by 2.4-fold,4.3-fold,2.2-fold, and 2.0-fold, respectively in the model control group. Palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) could significantly decrease serum levels of TC, TG and LDL-C in hyperlipidaemia hamsters (P<0.01 or 0.05). However, palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) showed no effect on serum level of HDL-C in hyperlipidaemia hamsters (P>0.05). Positive control simvastatine also could significanltly decrease serum levels of TC, TG and LDL-C in hyperlipidaemia hamsters (P<0.01). However, simvastatine showed no effect serum level of HDL-C in hyperlipidaemia hamsters (P>0.05). Palmatine at high-dose (70.05mg/kg) decreased serum levels of TC, TG, and LDL-C in hyperlipidaemia hamsters by 41%,64%, and 48%, respectively. The results from quantitative analysis of feces from hamsters showed that high fat diet could result in significant fecal excretion of TC and TBA in hamster. Compared with the normal control group, fecal excretion of TC and TAB in model group was increased by 66% and 58%, respectively. Compared with the model control group, palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) could significantly enhanced fecal excretion of TC and TBA (P<0.01 or 0.05). Palmatine at high-dose (70.05mg/kg) enhanced fecal excretion of TC and TBA by 41% and 70%, respectively. In contrast, simvastatine showed no significant effect on the fecal excretion of TC and TBA in hyperlipidaemia hamsters (P>0.05). So palmatine not only significantly improved abnormal serum level of lipids but also enhanced fecal excretion of TC and TBA in hyperlipidaemia hamsters.4. Hypolipidemic mechanism of palmatine in vivoThe hypolipidemic mechanism of palmatine was studied by detecting the messenger ribonucleic acid (mRNA) and protein expression of hepatic 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR), hepatic low-density lipoprotein receptor, (LDLR), hepatic cholesterol 7a-hydroxylase (CYP7A1), and ileal apical Na+-bile acid transproter (ASBT) in hyperlipidaemia hamsters.The results from real-time quantitative polymerase chain reaction (qRT-PCR) showed that hepatic HMGR mRNA expression was not affected significanlty by high fat diet in hamsters (P>0.05). However, hepatic LDLR mRNA and CYP7A1 mRNA expression were significantly decreased by 70%(P<0.01) and 67%(P<0.01), respectively. In contrast, ileal ASBT mRNA expression was significantly increased by 2.0-fold (P<0.01). After treatment by palmatine for 4 weeks, palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) showed no significant effect on hepatic HMGR mRNA expression compared with the model control group (P> 0.05). Palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) increased hepatic LDLR mRNA expression significantly (P< 0.01 or 0.05), and palmatine at high-dose (70.05mg/kg) significantly increased LDLR mRNA expression by 2.3-fold compared with the model control group. Palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) increased hepatic CYP7A1 mRNA expression significantly (P<0.01 or 0.05), and palmatine at high-dose (70.05mg/kg) significantly increased CYP7A1 mRNA expression by 2.0-fold compared with the model control group. Palmatine at middle-dose (46.70mg/kg) and high-dose (70.05mg/kg) decreased ileal ASBT mRNA expression significantly (P< 0.01 or 0.05), and palmatine at high-dose (70.05mg/kg) significantly decreased ileal ASBT mRNA expression by 44% compared with the model control group. Results form qRT-PCR also showed that simvastatine could significantly inhibit hepatic HMGR mRNA expression by 55%(P<0.01) and increase hepatic LDLR mRNA expression by 2.0-fold (P<0.01) compared with the model control group. However, simvastatine showed no effect on the CYP7A1 mRNA and ASBT mRNA expression in hyperlipidaemia hamsters (P>0.05).According the results from western blot, that hepatic HMGR protein expression was not affected significantly by high fat diet in hamsters (P>0.05). However, hepatic LDLR protein and CYP7A1 protein expression were significantly decreased by 54% (P<0.01) and 50%(P<0.01), respectively. In contrast, ileal ASBT protein expression was significantly increased by 2.4-fold (P<0.01). After treatment by palmatine for 4 weeks, palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) showed no significant effect on hepatic HMGR protein expression compared with the model control group (P>0.05). Palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) increased hepatic LDLR protein expression significantly (P<0.01 or 0.05), and palmatine at high-dose (70.05mg/kg) significantly increased LDLR protein expression by 1.7-fold compared with the model control group. Palmatine at low-dose (23.35mg/kg), middle-dose (46.70mg/kg), and high-dose (70.05mg/kg) increased hepatic CYP7A1 protein expression significantly (P<0.01 or 0.05), and palmatine at high-dose (70.05mg/kg) significantly increased CYP7A1 protein expression by 1.8-fold compared with the model control group. Palmatine at middle-dose (46.70mg/kg) and high-dose (70.05mg/kg) decreased ileal ASBT protein expression significantly (P<0.01), and palmatine at high-dose (70.05mg/kg) significantly decreased ileal ASBT protein expression by 49% compared with the model control group. Results form western blot also showed that simvastatine could significantly inhibit hepatic HMGR protein expression by 33% and increase hepatic LDLR protein expression by 1.5-fold compared with the model control group. However, simvastatine showed no effect on the CYP7A1 protein and ASBT protein expression in hyperlipidaemia hamsters. |
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