The protective effect of coptisine on experimental atherosclerosis ApoE-/- mice is mediated by MAPK/NF-κB-dependent pathway.

Coptisine is one of main bioactive compounds extracted from the traditional Chinese herbal medicine Rhizoma Coptidis. It is reported that coptisine can attenuate obesity-related inflammation and oxidant damage in Syrian golden hamsters. Therefore，coptisine may exhibit beneficial effects for the treatment of atherosclerosis (AS) due to its hypolipidemic and anti-inflammation activities. The present study investigated the anti-atherosclerotic and anti-inflammatory properties of coptisine using apoE-/- mice as AS model. The atherosclerotic plaque area of aorta, serum lipid profile and the expression of inflammatory cytokines were determined. After coptisine treatment, the serum level of TC, TG and LDL-C decreased; the serum level of IL-6, IL-1ß and TNF-α were decreased; the mRNA levels of NF-κBp65, VCAM-1, ICAM-1, IL-6 and IL-1ß in both aorta and liver were down-regulated; the p-p38 and p-JNK1/2 protein expression level were decreased. Coptisine decreased atherosclerotic plaque area significantly through both anti-inflammation and lipid lowering effect. The anti-inflammatory effect of coptisine is achieved through inhibiting activation of MAPK signaling pathways and NF-κB nuclear translocation. Therefore，the combined anti-inflammation and lipid lowering effect of coptisine attributed the decreased atherosclerotic plaque area in coptisine treated apoE-/- mice. The results of this study will afford a novel application for coptisine in the treatment of atherosclerosis and other chronic inflammatory disease.

Activation of Akt and JNK/Nrf2/NQO1 pathway contributes to the protective effect of coptisine against AAPH-induced oxidative stress.

Coptisine (COP) is one of the main active constituents of Coptidis Rhizoma. Previous studies have clarified that COP possesses antioxidant activity, but its defensive effects against pathological characteristics accompanied by oxidative damage in animal models and antioxidant mechanism are still unclear. Therefore, our purpose was to confirm the antioxidant activity of COP and explore its mechanism of action. We first detected the effects of COP on intracellular reactive oxygen species (ROS), heart beating rate, lipid peroxidation and cell death in zebrafish model with AAPH-induced oxidative stress. The results showed that COP of 10µg/mL significantly reduced ROS production, the increase of heart beating rate, lipid peroxidation and cell death by 41.3%, 24.5%, 26.5% and 30.0%, respectively. In addition, COP of 0.8µg/mL also decreased ROS, increased glutathione (GSH) content and elevated activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) by 40.1%, 19.8%, 18.3% and 49.3%, respectively in HepG2 cells. Further assays were carried out to explore the mRNA expression in zebrafish and protein expression of key factors in HepG2 cells. We demonstrated that COP up-regulated phase II antioxidant enzymes NAD(P)H/quinone oxidoreductase 1 (NQO1) through activating the nuclear factor erythroid-2 related factor 2 (Nrf2). Moreover, as the upstream signalings of Nrf2, the protein kinase B (Akt) and c-Jun NH2-terminal kinase (JNK) signalings were also induced by COP. And up-regulating Nrf2-mediated NQO1 expression of COP was in Akt and JNK-dependent manner. Taken together, COP exerted its antioxidant activity against AAPH-induced toxicity involving in activating Akt and JNK/Nrf2/NQO1 pathway.

Comparative effect of berberine and its derivative 8-cetylberberine on attenuating atherosclerosis in ApoE-/- mice.

Berberine (BBR), one of the main bioactive compounds in Rhizoma coptidis, has multiple pharmacological activities. It has been reported that 8-cetylberberine (8-BBR-C16) has increased anti-microbial property in vivo and a higher bioavailability in hamsters. Therefore, in the present study, we used apolipoprotein E-deficient mice (ApoE-/-) as an atherosclerosis model to investigate the anti-atherosclerosis effects of 8-BBR-C16. After 12weeks of treatment, the atherosclerotic plaque area of the aorta, serum lipid profile, the plasma redox state and the expression of inflammatory cytokines in ApoE-/- mice were determined. Both BBR and 8-BBR-C16 significantly decreased the atherosclerotic plaque area by suppressing inflammatory and oxidative markers in ApoE-/- mice. Treatment with BBR or 8-BBR-C16, decreased serum levels of IL-1ß and TNF-α as well as mRNA levels of NF-κBp65, i-NOS, ICAM-1, IL-6 in the aorta. In addition, the expression of NF-κB p65 protein decreased in the nucleus, whereas IκBα levels increased in the cytosol. The anti-inflammatory and anti-oxidative effect of BBR and 8-BBR-C16 attributed to inhibition of the translocation of NF-κB to the nucleus. Since the dosage of BBR used was 10 fold higher than that of 8-cetylberberine, we conclude that 8-BBR-C16 is more efficient in treating atherosclerosis in ApoE-/- mice.

Rhizoma Coptidis alkaloids alleviate hyperlipidemia in B6 mice by modulating gut microbiota and bile acid pathways.

It is hypothesized that Rhizoma Coptidis (RC) alkaloids exert their hypolipidemic effects primarily by targeting the gastrointestinal tract and liver. Thus, this study was conducted to evaluate the antihyperlipidemic mechanisms of RC alkaloids (at a daily dose of 140mg/kg for 35days) in high-fat and high-cholesterol induced hyperlipidemic B6 mice. After treatment, serum lipid parameters were determined, the expression of lipid metabolism related genes and pathways such as the sterol regulatory element binding proteins (SREBPs) and bile acid signaling in mice were also investigated. Meanwhile, Illumina sequencing was used to investigate the differences in gut microbiota of B6 mice. The results indicated that RC alkaloids reduced the body weight gain and serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), total bile acids (TBA) and lipopolysaccharide of B6 mice. Liver fat deposition and epididymal adipose cell size were also deceased in therapy group. RC alkaloids feeding significantly promoted the abundance of Sporobacter termitidis, Alcaligenes faecalis, Akkermansia muciniphila in the gut of mice, whereas, the abundance of Escherichia coli, Desulfovibrio C21_c20, Parabacteroides distasonis was suppressed. The observed antihyperlipidemic effects of RC alkaloids can also be attributed to their action as agonists of FXR and TGR5, activators for SREBP2, LDLR, UCP2 and CYP7A1, inhibitors of HMGCR, TXNIP, TLR4 and JNK. Therefore, this study expands current knowledge on hypolipidemic mechanisms of RC alkaloids and presents new evidence supporting a key role for RC alkaloids as regulators of lipid homeostasis by modulation gut microbiota and hepatic lipid metabolism.

Antihyperglycemia and Antihyperlipidemia Effect of Protoberberine Alkaloids From Rhizoma Coptidis in HepG2 Cell and Diabetic KK-Ay Mice.

Preclinical Research Rhizoma Coptidis (RC), the root of Coptis chinensis Franch, a species in the genus Coptis (family Ranunculaceae), has been commonly prescribed for the treatment of diabetes in Chinese traditional herbal medicine applications. The present study is focused on the assessment of the antihyperglycemia and antidiabetic hyperlipidemia effect of five protoberberine alkaloids, berberine (BBR), coptisine (COP), palmatine (PAL), epiberberine (EPI), and jatrorrhizine (JAT), separated from R. Coptidis in hepatocellular carcinoma HepG2 cells and diabetic KK-Ay mice. Protoberberine alkaloids are effective in modulating hyperglycemia and hyperlipidemia. After adding BBR and COP to culture medium, glucose consumption of HepG2 cells was increased. In KK-Ay mice assays, suppressed fasting blood glucose level and ameliorated glucose tolerance were observed after BBR/COP administration. After treated with berberine and coptisine, in the same dose of 5 µg/mL, the glucose consumption of HepG2 cells were promoted and, respectively, reached 96.1% and 17.6%. Body weight, food consumption, water intake, and urinary output of KK-Ay mice were reduced after treated with EPI. Serum total cholesterol and triglyceride of mice were decreased after treated with palmatine and jatrorrhizine. Serum high-density lipoprotein cholesterol of mice was increased after palmatine, jatrorrhizine, and berberine administrated. Moreover, hepatomegaly was attenuated in JTR-treated mice. Suggested that these protoberberine alkaloids from R. Coptidis have potential curative effect for diabetes. Drug Dev Res 77 : 163-170, 2016. © 2016 Wiley Periodicals, Inc.

Hypolipidemic Effects of Alkaloids from Rhizoma Coptidis in Diet-Induced Hyperlipidemic Hamsters.

This study was conducted to evaluate the antihyperlipidemic activity of five major alkaloids in Rhizoma Coptidis using high-fat- and high-cholesterol-induced hyperlipidemic hamsters. Hyperlipidemic hamsters were treated with coptisine, berberine, jatrorrhizine, palmatine, epiberberine, and total Rhizoma Coptidis alkaloids with a dose of 46.7 mg/kg × day for 140 days. Serum total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total bile acids were examined after alkaloid treatment. The results showed that all therapy agents prevented body weight gain, reduced the serum total cholesterol, and increased the high-density lipoprotein cholesterol of hamsters. Berberine, jatrorrhizine, and total Rhizoma Coptidis alkaloids decreased the triglyceride level in hyperlipidemic hamsters, while coptisine, jatrorrhizine, palmatine, and total Rhizoma Coptidis alkaloids significantly suppressed the elevation of the low-density lipoprotein cholesterol level. The fecal excretion of bile acids was significantly elevated by berberine, coptisine, jatrorrhizine, palmatine, total Rhizoma Coptidis alkaloids, and orlistat. Notably, total Rhizoma Coptidis alkaloids possess a much stronger lipid-lowering effect than the pure Rhizoma Coptidis alkaloids. Quantitative reverse transcription-polymerase chain reaction analyses revealed that Rhizoma Coptidis alkaloids could retard the synthesis of cholesterol by downregulating the mRNA expression of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and accelerate the clearance of lipids by upregulating the low-density lipoprotein receptor, cholesterol 7α-hydroxylase, and uncoupling protein-2 expression. These findings highlight the critical role of Rhizoma Coptidis alkaloids in hyperlipidemia treatment. Thus, they need to be considered in future therapeutic approaches.

Purification of α-glucosidase from mouse intestine by countercurrent chromatography coupled with a reverse micelle solvent system.

Countercurrent chromatography coupled with a reverse micelle solvent was applied to separate α-glucosidase, which is stable at pH 6.0-8.8, 15-50°C. The separation conditions are as follows: stationary phase: pH 4.0 Tris-HCl buffer phase containing 50 mM Tris-HCl and 50 mM KCl; mobile phase A: isooctane containing 50 mM anionic surfactant sodium di(2-ethylhexyl)sulfosuccinate; mobile phase B: 50 mM Tris-HCl buffer containing 500 mM KCl (pH 8.0); In total, 25 mL (23.9 mg) crude enzyme was injected through the injection valve, the enzymatic reaction and sodium dodecylsulfate polyacrylamide gel electrophoresis results imply that the activity of purified α-glucosidase is 6.63-fold higher than that of the crude enzyme. Therefore, countercurrent chromatography coupled with a reverse micelle solvent is capable for protein separation and enrichment.

The Hypoglycemic and Synergistic Effect of Loganin, Morroniside, and Ursolic Acid Isolated from the Fruits of Cornus officinalis.

Hypoglycemic activity-guided separation of ethanol extracts from the fruits of Cornus officinalis Sieb. et Zucc (CO) led to the isolation of loganin, morroniside, and ursolic acid. The antidiabetic capacity of CO extracts and related compounds was further investigated in diabetes mellitus mice. The results suggested that both CO extracts and pure compounds could ameliorate diabetes-associated damages and complications. Oral administration of loganin and morroniside decreased fasting blood glucose levels in diabetes mellitus mice. Ursolic acid exhibited the highest reactive oxygen species scavenging activity and α-glucosidase inhibitory activity. Notably, we noticed an interesting synergistic effect between loganin and ursolic acid. Given these favorable hypoglycemic properties, C. officinalis, a food and medicinal plant in China, may be used as a valuable food supplement for the treatment of diabetes mellitus.

Epiberberine reduces serum cholesterol in diet-induced dyslipidemia Syrian golden hamsters via network pathways involving cholesterol metabolism.

This study aimed to evaluate the cholesterol-lowering effect of epiberberine in dyslipidemia Syrian golden hamsters induced by high fat and high cholesterol (HFHC) diet and its regulation mechanism on some key genes involved in cholesterol metabolism. Hamsters were divided into six groups: normal control group (NC), HFHC group, simvastatin (Sim) and three doses of epiberberine group. The body weight, organs weight and serum lipid levels, as well as total cholesterol (TC) and total bile acids (TBA) levels in liver and feces were determined. Furthermore, the antidyslipidemia effect of epiberberine on key genes involved in cholesterol biosynthesis, uptake, conversion and elimination such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), low density lipoprotein receptor (LDL receptor), 7-alpha-hydroxylase (CYP7A1) and apical sodium dependent bile acid transporter (ASBT) were investigated. The results showed that epiberberine at high dosage significantly reduced serum TC, low density lipoprotein cholesterol (LDL-c) and TBA levels by 20.2%, 22.3% and 43.8%, respectively, and increased TBA and TC levels in feces. Epiberberine inhibited HMGCR mRNA and protein expressions and slightly reduced the protein level of ASBT, as well as dramatically up-regulated mRNA and protein expressions of CYP7A1 and LDL receptor. These findings suggested that the antidyslipidemia effects of epiberberine can be achieved via inhibiting the synthesis of cholesterol, promoting the uptake and conversion of TC in liver and increasing the excretion of TC and TBA in feces. Thus, epiberberine should be considered as one of the promising natural drugs for the treatment of dyslipidemia.

[Study on membrane injury mechanism of total alkaloids and berberine from Coptidis Rhizoma on Aeromonas hydrophila].

To explore the antibacterial activity and mechanism of total alkaloids and berberine from Coptidis Rhizoma on Aeromonas hydrophila, and determine the effect of total alkaloids and berberine from Coptidis Rhizoma on minimum inhibitory concentrations, permeability and fluidity of cell membrane, conformation of membrane proteins and virulence factors of A. hydrophila. The results showed that both total alkaloids and berberine from Coptidis Rhizoma had antibacterial activities on A. hydrophila, with minimum inhibitory concentrations of 62.5 and 125 mg · L(-1), respectively. Total alkaloids and berberine from Coptidis Rhizoma could increase the fluidity of membrane, change the conformation of membrane porteins and increase the permeability of bacteria membrane by 24.52% and 19.66%, respectively. Besides, total alkaloids and berberine from Coptidis Rhizoma significantly decreased the hemolysis of exotoxin and the mRNA expressions of aerA and hlyA (P < 0.05, P < 0.01), the secretion of endotoxin and the mRNA expression of LpxC (P < 0.05, P < 0.01). The results suggested that the antibacterial activity of total alkaloids and berberine from Coptidis Rhizoma on A. hydrophila may be related to the bacteria membrane injury. They inhibited the bacterial growth by increasing membrane lipid fluidity and changing conformation of membrane proteins, and reduced the secretion of virulence factors of A. hydrophila to weaken the pathogenicity.

Coptisine attenuates obesity-related inflammation through LPS/TLR-4-mediated signaling pathway in Syrian golden hamsters.

It is known that obesity resulted from consumption of diets high in fat and calories and associated with a chronic low-grade inflammation. Because the fat, sterol and bile acid metabolism of male Syrian golden hamster are more similar to that of human, in the present study, high fat and high cholesterol (HFHC) induced obese hamsters were used to evaluate the anti-inflammation and hypolipidemic role of coptisine. The results showed that body weight, plasma lipid levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-c), very low density lipoprotein-cholesterol (VLDL-c), ApoB and pro-inflammatory cytokines including TNF-α, IL-6 and lipopolysaccharide (LPS) were significantly altered in hamsters fed with HFHC diet. A strong correlation was observed between the LPS level in serum and the level of LBP and pro-inflammatory cytokines. Coptisine from the concentrations of 60 to 700 mg/L dose-dependently inhibited Enterobacter cloacae growth, which can easily induce obesity and insulin resistance. The results of endotoxin neutralization assay suggest that coptisine is capable of reducing the LPS content under inflammation status. Real time RT-PCR analyses revealed that coptisine suppressed TLR-4 in visceral fat of hamsters and decreased CD14 expression in livers of hamsters. These encouraging findings make the development of coptisine a good candidate for preventing obesity-related diseases through the LPS/TLR-4-mediated signaling pathway.

Hypolipidemic Effect and Mechanism of Palmatine from Coptis chinensis in Hamsters Fed High-Fat diet.

Palmatine (PAL) is one of the main alkaloids in Coptis chinensis. The present aim was to investigate the hypolipidemic effect and mechanism of palmatine in hamsters fed with high-fat diet (HFD). PAL treatment decreased serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels, as well as increased fecal excretion of TC and total bile acids (TBA) in hyperlipidemic hamsters. Furthermore, PAL treatment up-regulated low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein expression and down-regulated apical sodium-dependent bile salt transporter (ASBT) mRNA and protein expression. These results demonstrated that PAL as a potential natural cholesterol lowering agent works by up-regulating LDLR and CYP7A1 mRNA and protein expression, down-regulating ASBT mRNA and protein expression, as well as enhancing fecal excretion of TC and TBA. The findings in our study suggest that palmatine could be a potential natural agent for treating hyperlipidemia.

The safety and anti-hypercholesterolemic effect of coptisine in Syrian golden hamsters.

Current work was conducted to evaluate the cholesterol-lowering effect of coptisine extracted from Rhizoma coptidis in Syrian golden hamsters. The safety results indicated that coptisine was a safe and low-toxic compound. Coptisine showed a beneficial effect in the abnormal serum lipid levels induced by a high-fat and high-cholesterol diet (HFHC): at a concentration of 70.05 mg/kg, coptisine significantly led to a decrease in total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-c) levels by 26.70, 15.38, and 22.22 %, respectively, and high-density lipoprotein cholesterol (HDL-c) was increased by 41.74 % in serum of hamsters (p < 0.01). In addition, total bile acid (TBA) levels in feces of hamsters were elevated after coptisine administration. Further investigation has suggested that the mRNA and protein expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) in the liver of hamsters was down-regulated by high-dosage coptisine treatment (p < 0.05); mRNA and protein expression of low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1) were dramatically up-regulated by coptisine administration. The apical sodium-dependent bile salt transporter expression was down-regulated in the coptisine-treated animals, but showed no significant differences from the HFHC groups. Taken together, our results demonstrate that a high dosage of coptisine could inhibit cholesterol synthesis via suppressing the HMGCR expression and promoting the use and excretion of cholesterol via up-regulating LDLR and CYP7A1 expression. These findings suggest a critical role for coptisine in anti- hypercholesterolemia, and thus it needs to be considered as a potential natural cholesterol lowering agent.

[Effect of different parts, harvesting time and processing technologies on alkaloids content of Coptis chinensis adventitious root].

OBJECTIVE: To investigate the effect of different parts, harvesting time and processing technologies on alkaloids content of Coptis chinensis adventitious root. METHODS: The content of alkaloids were analyzed by HPLC. RESULTS: The content of total alkaloids in adventitious root harvested in different time was ranged from 2.5% to 2.9%, in which that of berberine and coptisine were the highest, reaching to 1%, and that of palmatine was only 0.1%. It suggested there was no significant difference of total alkaloids at different harvesting time. Nevertheless, the difference of the alkaloids content from different parts was much significant. The content of total alkaloid of adventitious root near to rhizome was about 4%, 2 times higher than that away from rhizome (only 2%). In addition, different processing technologies would affect alkaloids content obviously. There was hardly loss of alkaloids when the fresh adventitious root was washed with water, but it would decrease alkaloids content when the dried adventitious root was washed. CONCLUSION: Medicine value of Coptis chinensis adventitious root near to rhizome is higher than that away from rhizome. And fresh Coptis chinensis adventitious root can be washed with water.

The antihypercholesterolemic effect of jatrorrhizine isolated from Rhizoma Coptidis.

Current work was conducted to evaluate the safety and antihypercholesterolemic activity of jatrorrhizine extracted from Rhizoma Coptidis (RC) and its potential mechanism on regulating cholesterol metabolism. It was found that the LD50 of jatrorrhizine in mice was more than 5,500 mg/kg and there were no influences on clinical signs, organ weight changes, urinalysis and hematological parameters, gross necropsy and histological alterations in jatrorrhizine-treated rats during the 3-month period, compared to the control group. Jatrorrhizine showed a strong lipid-lowering effect in a dose-dependent manner. Oral administration of 70.05 mg/kg of jatrorrhizine on Mesocricetus auratus (Syrian golden hamsters) exhibited significant decrease in TC, TG, and LDL-c levels by 20%, 43%, and 19%, respectively, and increase in HDL-c and total bile acids (TBA) content in feces (p<0.01), compared to high-fat and high-cholesterol (HFHC) group. Besides, jatrorrhizine dose-dependently slowed the rate of weight gain. The results of qRT-PCR, western blotting and ELISA revealed that jatrorrhizine significantly up-regulated the mRNA and protein expression of LDLR and CYP7A1, but exhibited no significant effect on mRNA and protein expression of HMGR and ASBT in hamsters. In conclusion, jatrorrhizine was a safe and potential antihypercholesterolemic agent from RC which could improve the utilization and excretion of cholesterol by up-regulating the mRNA and protein expression of LDLR and CYP7A1.

[Comparative study of pharmacokinetics and tissue distribution of 8-cetylberberine and berberine in rats].

The concentrations of berberine (BBR) and 8-cetylberberine (8-BBR-C16) in rat plasma and tissue were determined by RP-HPLC. Both the plasma pharmacokinetics characteristic and tissue distribution differences of BBR and 8-BBR-C16 were compared to provide experimental data for the mechanism research and further drug development. After the oral administrations of BBR and 8-BBR-C16 at the dose of 80 mg x kg(-1) for rats, the pharmacokinetics result showed that compared with BBR, the C(max) and AUC(0-t), of 8-BBR-C16 increased by 2.8 times and 12.9 times respectively, t1/2 extended from 3.61 h to 11.90 h. The tissue distribution result showed that compared with BBR, the concentration of 8-BBR-C16 in various organizations increased and the retention time extended remarkably. The maximum concentration was achieved in lung and the highest concentration in it was 3 731.82 ng x g(-1). After being derived, the C(max) in plasma and bioavailability of 8-BBR-C16 increased remarkably and the circulation time in vivo extended. The drug concentration in tissue increased remarkably, and the distribution ratio changed too, with strong targeting selection in lung.