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Objective: To establish a sensitive, accurate and reliable LC-MS-MS analysis method for the nine major components (berberine, berberrubine, jatrorrhizine, palmatine, baicalin, wogonin, oroxylin A, geniposide and gardenin B) in Huanglian Jiedu Decoction (HJD) in rat hippocampus, cortex, striatum, and cerebrospinal fluid, and to study the distribution characteristics of the components in brain tissues of rats. Methods: The mass spectrometry detection conditions and chromatographic analysis conditions of nine main components and internal standards (clarithromycin and chloramphenicol) was optimized, and then the methodological investigations were performed. After HJD was administrated for 1 h, the samples of cerebrospinal fluid, hippocampus, cortex and striatum tissues were collected, and the contents of nine components in these samples were measured. Results: Berberine, jatrorrhizine, palmatine, baicalin, wogonin, oroxylin A and gardenin B showed a good linear relationship in the range of 1-250 ng/mL (r2 > 0.99). Berberrubine had a good linear relationship in the range of 1-500 ng/mL (r2 = 0.991 2), and geniposide had a good linear relationship in the range of 5-500 ng/mL, respectively (r2 = 0.999 5). The intra-day and inter-day precision of each component was less than 12.94%, the accuracy was from -12.71% to 6.91%; The extraction recovery rate was from 88.02% to 106.7%; The matrix effect was from 88.92% to 105.10%; And the short-term, freeze-thaw cycle and long-term stability were less than 12.51%. The content of each component in the hippocampus, cortex and striatum was berberine > baicalin > geniposide > berberrubine > palmatine > wogonin > jatrorrhizine > oroxylin A > gardenin B; And the content of each component in cerebrospinal fluid was geniposide > berberine > berberrubine > palmatine > baicalin> gardenin B > jatrorrhizine > wogonin > oroxylin A. Conclusion: The method can be used for the simultaneous detection of the concentration of nine active components in brain tissues of rats, and successfully applied to the study of brain tissue distribution, which provides a reference for HJD to treat brain diseases.
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Objective To determine the chemical composition of the integral processing and traditional processing of Phellodendri Chinensis Cortex (PCC), and discuss the advantages of the integral processing of PCC. Methods The content of chlorogenic acid, phellodendrine chloride, magnoflorine, jatrorrhizine hydrochloride, berberrubine, palmatine hydrochloride, berberine hydrochloride, limonin, and obacunone in the product integral processing and traditional processing PCC was determined by HPLC. Results Comparing the total of the mass fractions of the above index components, the content of the raw PCC and the salt PCC processed by the traditional methods was 3.956 9% and 4.525 0%, and the content of the raw PCC and the salt PCC processed by the integral methods was 6.074 2% and 8.942 2%, respectively. It can be drawn that the integral method surpasses the traditional method by contrast with the content of effective components. Conclusion The quality of the integral processing of PCC is good, and its operating procedures are simple and worth promoting.
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Objective To develop a sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to evaluate the pharmacokinetic behavior of berberrubine (BRB) and its glucuronide (BRBG) in rats. Methods BRB, BRBG and tetrahydroberberine (THB, internal standard) were isolated by liquid-liquid extraction in rat biological samples. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C18 (2.1 mm × 50 mm, 3.5-Micron) with a gradient mobile phases primarily containing acetonitrile, water with 0.1% formic acid and 5 mm ammonium acetate. The analytes were monitored by MS/MS in positive electrospray ionization mode. Herein, the feasibility of new developed method was validated with respect to specificity, linearity, precision, accuracy, stability, extraction efficiency and matrix effect. The appropriate method was used for the pharmacokinetic study in rats. Results The new developed method could be applied to the pharmacokinetic study of BRB in rats. BRB and BRBG showed good linearity over the ranges of 2-1000 ng/mL and 5-2000 ng/mL, respectively, and precision was no more than 15%. The accuracy, specificity and stability could be acceptable. Conclusion The new method is sensitive and reproducible. In pharmacokinetic study, BRB showed nonlinear elimination property. Meanwhile, BRB was rapidly absorbed and widely distributed in various tissues with the highest exposure of BRB in kidney and liver. The absolute bioavailability of BRB was determined to be 8.2% and at the dose of 40 mg/kg, a total of 44% BRB was excreted in urine and feces.
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Objective: To study the metabolic characteristics of berberine using the pooled human liver microsomes and recombinant human cytochrome enzymes P450 (CYP) isozymes, to identify CYP isozymes responsible for berberine metabolism and its contribution, and to determine the structures of metabolism. Methods: Pooled human liver microsomes were incubated with berberine (20, 100, 200, 400, 600, 800, and 1 200 ng/mL). The Michaelis-Menten parameters (Km), maximum velocity (Vmax), and clearance (CLint) of pooled liver microsomes were initially estimated by analyzing Lineweave-Brurk plot. Various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of berberine and the certain concentration of berberine was incubated with recombinant human CYP isozymes (CYP3A4, CYP1A2, CYP2D6, and CYP2C9). The concentration of berberine and metabolites in the incubation pool was determined by UPLC method. The P450 isozymes were ranked with the method of total normalized rate (TNR) and the related metabolites were identified by LC-MS/MS. Results: The Vmax, Km, and CLint of berberine in pooled human liver microsomes were 1.51 nmol·mg-1·h-1, 2.69 nmol/mL, and 0.56 mL·mg-1·h-1, respectively. Quinidine (the specific inhibitor of CYP2D6) and Furafylline (the specific inhibitor of CYP1A2) could significantly inhibit the berberine metabolism, and the other CYP inhibitors had no significant effect on the metabolism of berberine. CYP2D6 and CYP1A2 were responsible for 75.253 9% and 23.323 6% of the berberine metabolite M1 (demethyleneberberine), and responsible for 46.893 8% and 8.679 5% of M2 (thalifendine or berberrubine). The major metabolic pathway of berberine in pooled human liver microsomes incubation system is O-demethylated, demethyleneberberine, thalifendine, or berberrubine could be generated in vitro. Conclusion: Bererine is metabolized by CYP2D6 and CYP1A2 in human liver, the metabolites of berberine are demethyleneberberine and thalifendine or berberrubine.
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AIM:To research the chemical modification from berberine hydrochloride to berberrubine in Cortex Phellodendri before and after processing.METHODS:Berberrubine was isolated by column chromatography and the contents of berberine hydrochloride and berberrubine in different processed Cortex Phellodendri were determined by HPLC.RESULTS:With increacing processing temperature and time,part of berberine hydrochloride became berberrubine in processed Cortex Phellodendrin,and berberrubine content decreased under the condition of high temperature operation or overtime processing.CONCLUSION:The experimental data shows that the processed Cortex phellodendri has the maximum average content of berberrubine as processing temperature is maintained as 180℃ for 40 min.
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AIM:To research the chemical modification from berberine hydrochloride to berberrubine in Cortex Phellodendri before and after processing.METHODS:Berberrubine was isolated by column chromatography and the contents of berberine hydrochloride and berberrubine in different processed Cortex Phellodendri were determined by HPLC.RESULTS:With increacing processing temperature and time,part of berberine hydrochloride became berberrubine in processed Cortex Phellodendrin,and berberrubine content decreased under the condition of high temperature operation or overtime processing.CONCLUSION:The experimental data shows that the processed Cortex phellodendri has the maximum average content of berberrubine as processing temperature is maintained as 180 ?C for 40 min.