Protective role of berberine and Coptischinensis extract on T2MD rats and associated islet Rin­5f cells.

The aim of the present study was to compare the different effects of berberine (Ber) and Coptischinensis extract (CCE) on a rat model of type 2 diabetes mellitus (T2DM), and the islet Rin­5f cell line was used to examine the differences between Ber and CCE and the underlying mechanisms. CCE was extracted and purified prior to analysis. Male Sprague­Dawley rats were provided with a high­fat diet to induce insulin resistance prior to injecting with streptozotocinto establish the T2DM model, the T2DM rats were treated with Ber and CCE, and blood samples and pancreatic tissues were obtained and compared to examine T2DM metabolic syndromes among the groups of rats, which included healthy rats, model rats, and model rats treated with Ber and CCE at different doses between 0 and 8 weeks. The protective effects of Ber and CCE on the Rin­5f islet cell line were also evaluated. The effects on Rin­5f cell proliferation and cell cycle, glucose­stimulated insulin release test (GSIS), the anti­apoptotic effects caused by fat induction, and protein expression levels of poly ADP­ribose polymerase (PARP­1) were evaluated. The results showed that the content of the prepared CCE was 96.07% for five alkaloids. When it was used for treatment of the T2DM rats, compared with Ber, metformin and rosiglitazone, the fasting blood glucose, glucosylated serum protein (GSP) and glucose infusion rate indicesin the fasting rats were ameliorated, compared with those in the T2MD rats, with no significant differences between treatment with Ber or CCE and metformin or rosiglitazone. The indices of mean optical density and fasting ß­cell function index (FBCI) were different following treatment with Ber and CCE, compared with those in the model rats, which may have stimulated the pancreatic secretion of insulin. When Ber and CCE were used to examine the protective effects on Rin­5F cells, it was found that the Rin­5f cell GSIS, cell cycle, lipotoxic islet cell proliferation and protein expression of PARP­1 were altered and improved, which may have protected pancreatic islet ß­cells by improving islet ß­cell proliferation and the protein expression of PARP­1. CCE and Ber exerted similar effects when used for the treatment of T2MD rats, and may have stimulated the pancreatic secretion of insulin through the protective effect on islet ß­cells via improving islet ß­cell proliferation and the protein expression of PARP­1.

Poor permeability and absorption affect the activity of four alkaloids from Coptis.

Coptidis rhizoma (Coptis) and its alkaloids exert various pharmacological functions in cells and tissues; however, the oral absorption of these alkaloids requires further elucidation. The present study aimed to examine the mechanism underlying the poor absorption of alkaloids, including berberine (BER), coptisine (COP), palmatine (PAL) and jatrorrhizine (JAT). An ultra­performance liquid chromatography (UPLC) method was validated for the determination of BER, COP, PAL and JAT in the above experimental medium. In addition, the apparent oil­water partition coefficient (Po/w); apparent permeability coefficient (Papp), determined using a parallel artificial membrane permeability assay (PAMPA) plate; membrane retention coefficient (R %); and effect of P­glycoprotein (P­gp) inhibitor on the Papp of the four alkaloids were investigated. The intestinal absorption rate constant (Ka) and absorption percentage (A %) of the four alkaloids were also determined. The results of the present study demonstrated that the Po/w of the four alkaloids in 0.1 mol·l­1 HCl medium was significantly higher (P<0.01), compared with those of the alkaloids in phosphate buffer (pH 7.4). The Papp of BER was 1.0­1.2x10­6 cm·s­1, determined using a PAMPA plate, and the Papp of BER, COP, PAL and JAT decreased sequentially. The concentrations of the four alkaloids on the apical­to­basolateral (AP­BL) surface and the basolateral­to­apical (BL­AP) surface increased in a linear manner, with increasing concentrations between 10 and 100 µmol. In addition, the transportation of BER on the BL­AP surface was significantly faster (P<0.01), compared with that on the AP­BL surface and, following the addition of verpamil (a P­gp inhibitor), the Papp (AP­BL) of the four alkaloids increased, whereas the Papp (BL­AP) was significantly decreased (P<0.01). The rat intestinal perfusion experiment demonstrated that the four alkaloids were poorly absorbed; however, the Ka of BER was significantly higher, compared with the three other alkaloids. Furthermore, the A % and Ka provided evidence that the absorption of BER was increased in the jejunum, compared with in the ileum. In conclusion, the four alkaloids from Coptis appeared to be poorly absorbed, determined using a shake flask, pre­coated PAMPA plates, a Caco­2 cell monolayer model and intestinal perfusion; however, absorption was higher in the jejunum than in the ileum. Among the four alkaloids, the permeability of BER was markedly higher than the others, and P­gp efflux had a significant effect on the absorption of those alkaloids.

In vitro studies of berberine metabolism and its effect of enzyme induction on HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberine (BER) and BER-original herbal medicines have a variety of pharmacological functions and have been widely used in clinical. However, its effect of enzyme induction on cytochrome P450 (CYP) in human hepatocytes is unknown. MATERIAL AND METHOD: Metabolism of berberine and its effect on main metabolic enzymes in HepG2 cell in vitro was investigated. Cocktail probe drugs, mRNA expression and protein expression were used to evaluate the metabolism potency. Meanwhile, an UPLC-MS/MS method was validated for the analysis of BER and four probe drugs in HepG2 cell. RESULT: BER significantly increased the metabolism of midazolam, phenacetin and tolbutamide by inducing the CYP1A2 and 3A4 enzyme in a dose-dependent manner, the mRNA and protein expression of CYP1A2 and 3A4 were increased by berberine at 1000ng·mL(-1). The activity of CYP1A2 and 3A4 could be induced by BER more than 500ng·mL(-1) in HepG2 cell, which was confirmed by the increase of its mRNA and protein expression. CONCLUSION: BER increases the metabolism of cocktail drugs such as midazolam, phenacetin and tolbutamide by increasing the mRNA and protein expression of CYP1A2 and 3A4.

Effects of Kaixin Jieyu Decoction () on behavior, monoamine neurotransmitter levels, and serotonin receptor subtype expression in the brain of a rat depression model.

OBJECTIVE: To determine the mechanisms underlying the anti-depressant effects of Kaixin Jieyu Decoction (, KJD) by investigating the effects of KJD on behavior, monoamine neurotransmitter levels, and serotonin (5-HT) receptor subtype expression in the brain in a rat model of depression. METHODS: The rat depression model was established using chronic unpredictable mild stress (CUMS). Forty-eight Sprague Dawley rats were randomly divided into control, depression model (CUMS), CUMS+KJD (7.7 g/kg(-1)·d(-1) of crude drug), and CUMS+fluoxetine (2.4 mg/kg(-1)·d(-1)) groups (n=12 in each group), and the treatments lasted for 21 days. We regularly evaluated body weight, sucrose consumption, and horizontal and vertical activity scores in open-field tests. The content of the monoamine neurotransmitters 5-HT, norepinephrine (NE), and dopamine (DA) and the DA metabolite homovanillic acid in the cerebral cortex, and 5-HT1A and 5-HT2A receptor mRNA in the cerebral cortex and the hippocampus, were determined respectively by high-performance liquid chromatography-coularray electrochemical detector and real-time polymerase chain reaction. RESULTS: Compared with the control group, CUMS rats showed a variety of depression-like behavioral changes, including a significant reduction in body weight, sucrose consumption, and horizontal and vertical activity scores in open-field tests (P<0.05 or P<0.01), and a significant decrease in 5-HT and NE levels and 5-HT2A receptor mRNA expression. In contrast, they showed a significant increase in 5-HT1A receptor mRNA expression in the cerebral cortex. In the hippocampus, 5-HT1A receptor mRNA expression was lower whereas 5-HT2A receptor mRNA expression was higher than in the control group (P<0.05 or P<0.01). Treatment with KJD or fluoxetine partially attenuated these changes (P<0.05 or P<0.01). CONCLUSION: KJD could normalize the levels of 5-HT and NE and adjust the balance of 5-HT1A and 5-HT2A receptor expression in rat cerebrum, and this may be one of mechanisms of antidepressant effects of KJD.

[CYP450 enzyme inhibition of berberine in pooled human liver microsomes by cocktail probe drugs].

OBJECTIVE: To investigate the effect of CYP450 enzyme inhibition of berberine in pooled human liver microsomes by cocktail probe drugs. METHOD: Cocktail probe drugs method has been established, an LC-MS/MS analytical method has been established to determine the five probes of midazolam, phenacetin, dextromethorphan, tolbutamide, chlorzoxazone and the internal standard was benzhydramine to evaluate the effect of CYP450 activity following administration of berberine in pooled human liver microsomes. RESULT: Compared with control group, the pharmacokinetics of midazolam, phenacetin and tolbutamide were no significant differences, but the pharmacokinetics of chlorzoxazone was significantly decreased. There were no significant differences for the pharmacokinetics of dextromethorphan when the concentration of berberine was 50 microg x L(-1). The pharmacokinetics of dextromethorphan was significantly decreased when the concentration of berberine was exceed 200 microg x L(-1). CONCLUSION: Berberine has no influence on the activities of CYP3A4, CYP1A2 and CYP2C19 below 2 000 microg x L(-1), but can inhibit the activity of CYP2E1 and CYP2D6 in concentration-dependent.

[Species differentiation and quality assessment of Ziziphus jujuba var. spinosa based on the HPLC fingerprint and quantitative analysis].

OBJECTIVE: To establish an HPLC method of a characteristical chemical fingerprint analysis in combination with simultaneous determination of four bioactive components for species differentiation and quality assessment of Ziziphus jujuba var. spinosa. METHODS: The chromatographic separation was performed on an Agilent TC-C18 BDS (250 mm x 4.6 mm, 5 microm) column. The mobile phase consisted of acetonitrile and water in a linear gradient elution procedure. The evaporator tube temperature of ELSD was set at 110.5 degrees C with the nebulizing gas flow rate of 3.1 mL/min and the flow rate of mobile phase was 1.0 mL/min. The column was maintained at 30 degrees C. The injection volume was 20 microL. RESULTS: HPLC methodology for both chemical fingerprint analysis and quantitative determination of four active ingredients were validated, respectively. According to the contents of the four ingredients and the chemical fingerprints of Ziziphus jujuba var. spinosa using principal component analysis, Ziziphus jujuba var. spinosa was different from the fake derived from the seeds of Ziziphus mauritiana. CONCLUSION: The developed HPLC method is exclusive and repetitive for the species identification and quality evaluation of Ziziphus jujuba var. spinosa.

[Determination of effective components in different positions of Panax notoginseng by HPLC].

OBJECTIVE: A quantitative method was developed by gradient elution for the determination of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in different positions of Panax Notoginseng by HPLC. The content of 4 kinds saponins in different positions of Panax Notoginseng were compared. METHODS: The different positions of Panax notoginseng (including root, rhizome, branch root, leaf, flower) were extracted with methanol. The HPLC condition was as following: Kromasil C18 column (250 mm x 4.6 mm, 5 microm), acetonitrile and water linearity gradient elution, flow rate at 1.0 mL/min, column temperature at 25 degrees C, wavelength 203 nm. RESULTS: The linear ranges of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 were 4.4-440 microg/mL, 4.32-1080 microg/mL, 4.24-212 microg/mL and 4.48-1120 microg/mL, respectively. The RSD (n=5) of average contents of intra-day and inter-day of 4 kinds saponins were 0.46%, 0.24%, 0.77%, 0.68% and 1.64%, 0.69%, 0.52%, 0.65%, respectively. The average recoveries were (102.93 +/- 1.22)%, (103.18 +/- 0.49)%, (103.20 +/- 1.58)%, (103.86 +/- 0.39)%, respectively. The content of 4 kinds saponins in different position of Panax notoginseng was: rhizome > root > branch root > flower > leaf; the content of 4 kinds saponin in the root of Panax notoginseng was: 80 pieces in 500 g >60 pieces in 500 g >20 pieces in 500 g >40 pieces in 500 g >100 pieces in 500 g. CONCLUSION: This method is simple, sensitive, accurate and repeat, and is suitable in determination of the content of 4 kinds saponins in different positions of Panax notoginseng.