Differences in Metabolite Profiles of Dihydroberberine and Micellar Berberine in Caco-2 Cells and Humans-A Pilot Study.

We investigated the pharmacokinetic pathway of berberine and its metabolites in vitro, in Caco-2 cells, and in human participants following the administration of dihydroberberine (DHB) and micellar berberine (LipoMicel®, LMB) formulations. A pilot trial involving nine healthy volunteers was conducted over a 24 h period; blood samples were collected and subjected to Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analyses to quantify the concentrations of berberine and its metabolites. Pharmacokinetic correlations indicated that berberrubine and thalifendine follow distinct metabolic pathways. Additionally, jatrorrhizine sulfate appeared to undergo metabolism differently compared to the other sulfated metabolites. Moreover, berberrubine glucuronide likely has a unique metabolic pathway distinct from other glucuronides. The human trial revealed significantly higher blood concentrations of berberine metabolites in participants of the DHB treatment group compared to the LMB treatment group-except for berberrubine glucuronide, which was only detected in the LMB treatment group. Similarly, results from in vitro investigations showed significant differences in berberine metabolite profiles between DHB and LMB. Dihydroberberine, dihydroxy-berberrubine/thalifendine and jatrorrhizine sulfate were detected in LMB-treated cells, but not in DHB-treated cells; thalifendine and jatrorrhizine-glucuronide were detected in DHB-treated cells only. While DHB treatment provided higher blood concentrations of berberine and most berberine metabolites, both in vitro (Caco-2 cells) and in vivo human studies showed that treatment with LMB resulted in a higher proportion of unmetabolized berberine compared to DHB. These findings suggest potential clinical implications that merit further investigation in future large-scale trials.

Development of capillary-paper spray for small-molecule analysis in complex samples.

We develop a capillary-paper spray (CPS) ion source which allows for sample separation in the capillary and enables rapid and sensitive paper spray (PS) mass spectrometry (MS) analysis of biofluids. The CPS employs a glass capillary to load liquid analytes, vertically standing at the rear of the PS. To further reduce the matrix effect, a nitrocellulose filter membrane is placed between the glass tube and chromatography paper to absorb proteins and other macromolecules, which is beneficial for the detection of the small molecules. Compared with the normal PS method, the CPS method markedly improves spray stability and prolongs analysis duration, and also generates significantly better signal intensities during the analysis of drugs, thus indicating its potential for clinical use. As a proof of concept, quantitative analysis of drugs (metformin hydrochloride and berberine hydrochloride) in serum is performed.

A rapid LC-MS/MS method for simultaneous determination of berberine and irbesartan in rat plasma: Application to the drug-drug pharmacokinetic interaction study after oral administration in rats.

A sensitive, reproducible, and specific liquid chromatography tandem mass spectrometry method was developed and validated to simultaneously determine the concentration of berberine (BBR) and irbesartan in Sprague-Dawley rat plasma, and applied to study the pharmacokinetic drug-drug interaction (DDI) between BBR and irbesartan in rats. In this method, diphenhydramine was used as the internal standard, and the liquid-liquid extraction method using ethyl acetate as the extraction agent was used for sample preparation. After extraction, the prepared samples were run on an Agilent Welchrom C18 column with the mobile phase consisting of methanol-acetonitrile-water solution with 0.5% formic acid (45:50:5, v/v/v) at a flow rate of 0.8 mL·min-1 . The analytes BBR, irbesartan, and diphenhydramine (IS) were detected using multiple reactions monitoring mode, with the ion transitions being m/z 336.1 → m/z 320.0, m/z 429.1 → m/z 206.9, and m/z 256.2 → m/z 167.0, respectively. In the rats' plasma, BBR had good linearity in the range of 0.5-100 ng·mL-1 with the lower limit of quantitation of 0.5 ng·mL-1 , and the accuracy, intra-day, and inter-day precision were less than 12.33%. Irbesartan had good linearity in the range of 20-1200 ng·mL-1 with the lower limit of quantification of 20 ng·mL-1 , and the accuracy, intra-day, and inter-day precision were less than 13.55%. The validated method was verified to meet the determination requirements of biological samples. It was the first time to study the pharmacokinetics of DDI between BBR and irbesartan successfully, which would be necessary and beneficial to explore the clinical safety and efficacy of the combination of BBR and irbesartan in the treatment of diabetic nephropathy.

A highly fluorescent lanthanide metal-organic framework as dual-mode visual sensor for berberine hydrochloride and tetracycline.

A microscale highly fluorescent Eu metal-organic framework (Eu-MOF) was synthesized with terephthalic acid and 1H-1,2,4-triazole-3,5-diamine by one-pot hydrothermal method. And it was characterized by scanning electron microscope, Fourier transform infrared spectroscopy, powder X-ray diffraction, fluorescence spectroscopy, thermogravimetric analysis, and energy dispersive X-ray mapping. The prepared Eu-MOF has high quantum yield of 30.99%, excellent water dispersibility, good fluorescence stability, and favorable thermal stability. Based on the distinctly different fluorescence responses of different emission, the prepared Eu-MOF was used as dual-mode visual sensor for the sensitive detection of berberine hydrochloride and tetracycline. The limits of detection are 78 nM and 17 nM, respectively. The sensing mechanism was also discussed. Moreover, a filter paper sensor has been designed for sensing tetracycline with a notable fluorescence color change from blue to red. The prepared Eu-MOF is promising to be developed as a multi-mode luminescent sensor for visual detection in biochemical analysis. Graphical abstract Illustration of the synthesis of Eu-MOF and its sensing applications for berberine hydrochloride and tetracycline.

Different structures of berberine and five other protoberberine alkaloids that affect P-glycoprotein-mediated efflux capacity.

Berberine, berberrubine, thalifendine, demethyleneberberine, jatrorrhizine, and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences. The oral delivery of most PAs is hindered by their poor bioavailability, which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux. Meanwhile, P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates, and these changes might strongly affect the P-gp-binding specificity. To confirm whether these six compounds are substrates of P-gp, to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp, we conducted a series of in vivo, in vitro, and in silico assays. Here, we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo. The efflux capacity (net efflux) ranked as berberrubine > berberine > columbamine ~ jatrorrhizine > thalifendine > demethyleneberberine based on in vitro transport studies in Caco-2 monolayers. Using molecular dynamics simulation and molecular docking techniques, we determined the transport pathways of the six compounds and their binding affinities to P-gp. The results suggested that at the early binding stage, different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp, whereas electrostatic interactions are the main determinant at the late release stage. In addition to hydrophobic interactions, hydrogen bonds play an important role in discriminating the binding affinities.

Rapid Identification of Berberine Metabolites in Rat Plasma by UHPLC-Q-TOF-MS.

In this study, a reliable and rapid method based on ultra high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) technology and MetabolitePilotMT software was developed for berberine metabolites identification in rat plasma. The chemical structures of the metabolites and their product ions were tentatively characterized or identified according to the molecular weights detected and MS/MS data. In all, nine metabolites, including M1 (demethyleneberberine, C19H18NO4, m/z 324), M2 (glucuronic acid-conjugated demethyleneberberine, C25H26NO10, m/z 500), M3 (diglucuronide-conjugated demethyleneberberine, C31H34NO16, m/z 676), M4 (glucuronic acid-conjugated jatrorrhizine or glucuronic acid-conjugated columbamine, C26H28NO10, m/z 514), M5 (berberrubine or thalifendine, C19H16NO4, m/z 322), M6 (glucuronic acid-conjugated berberrubine or glucuronic acid-conjugated thalifendine, C25H24NO10, m/z 498), M7 (sulfite-conjugated berberrubine or sulfite-conjugated thalifendine, C19H16NO7S, m/z 402), M8 (dihydroxy berberrubine or dihydroxy thalifendine, C19H16NO6, m/z 354) and M9 (dihydroxy berberine, C20H18NO6, m/z 368) were tentatively characterized or identified. Several new deposition patterns and three new metabolites (M7, M8 and M9) are reported in this paper for the first time. This work not only provides significant insights into the understanding of the metabolic pathways of berberine, but also contributes in identifying potential active drug candidates from the metabolites.

Reno-protective effect of berberine.

OBJECTIVE: To assess the reno-protective effect of berberine on diclofenac-induced acute kidney injury in rats. METHODS: The experimental study was conducted at the College of Medicine, Mustansiriya University, Baghdad, Iraq, from January to March 2018, and comprised Sprague Dawley male rats which were divided into 3 equal groups. Group1 rats were treated with distilled water plus normal saline for 14 days, Group2 rats were treated with distilled water plus diclofenac for 14 days and Group3 rats were treated with berberine plus diclofenac for 14 days. Parameters measured were blood urea, serum creatinine, serum malondialdehyde, superoxide dismutase, glutathione reductase, neutrophil gelatinase associated lipocalin, kidney injury molecules-1, Interleukin-18and cystatin-c. Anthropometric measurements and estimated glomerular filtration rate were also noted. SPSS 20 was used for data analysis. RESULTS: Of the 30 rats, the three groups had 10(33.3%) each. Berberine reduced blood urea, serum creatinine, malondialdehyde, neutrophil gelatinase associated lipocalin, kidney injury molecules-1 and Interleukin-18 significantly compared to diclofenac-induced acute kidney injury (p<0.01 each). Berberine improved anti-oxidant capacity through significant elevation of superoxide dismutase and glutathione reductase sera levels (p<0.01 each). CONCLUSIONS: Berberine was found to be an effective agent in the attenuation of diclofenac-induced acute kidney injury through the modulation of pro-inflammatory and oxidative stress biomarkers.

In-Transit Electroextraction of Small-Molecule Pharmaceuticals from Blood.

An electrokinetic platform was developed for extracting small-molecule pharmaceuticals from a dried blood spot. Through the exclusion of liquid reagents and use of low field strength (6 V cm-1 ), the electroextraction of a drug from a dried blood spot, deposited on a polymer inclusion membrane (PIM), could be realised while in transit in the mail. In transit sample preparation provides a potential solution to in situ sample degradation and may accelerate the workflow upon arrival of a patient sample at the analytical facility. The electroextraction method was enabled through our discovery of the use of 15-20 µm thin PIMs as electrophoretic separation medium in absence of liquid reagents. Here, a PIM consisting of cellulose triacetate as polymer base, 2-nitrophenyl octyl ether as plasticizer and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide as carrier was used. The PIM, was packaged with two 12 V batteries to supply the separation voltage. A blood spot containing berberine chloride was deposited and dried before the applying the separation potential, allowing for the electroextraction while the packaged device was shipped in internal mail. Upon arrival in the analytical laboratory, the PIM was analysed using a fluorescence microscope with photon multiplier tube, quantifying the berberine extracted away from the sample matrix. This platform represents a new opportunity for processing clinical samples during transport to the laboratory, saving time and manual handling to accelerate the time to result.

In vitro assessment of the glucose-lowering effects of berberrubine-9-O-ß-D-glucuronide, an active metabolite of berberrubine.

Berberrubine (BRB) is the primary metabolite of berberine (BBR) that has shown a stronger glucose-lowering effect than BBR in vivo. On the other hand, BRB is quickly and extensively metabolized into berberrubine-9-O-ß-D-glucuronide (BRBG) in rats after oral administration. In this study we compared the pharmacokinetic properties of BRB and BRBG in rats, and explored the mechanisms underlying their glucose-lowering activities. C57BL/6 mice with HFD-induced hyperglycemia were administered BRB (50 mg·kg-1·d-1, ig) for 6 weeks, which caused greater reduction in the plasma glucose levels than those caused by BBR (120 mg·kg-1·d-1) or BRB (25 mg·kg-1·d-1). In addition, BRB dose-dependently decreased the activity of α-glucosidase in gut of the mice. After oral administration of BRB in rats, the exposures of BRBG in plasma at 3 different dosages (10, 40, 80 mg/kg) and in urine at different time intervals (0-4, 4-10, 10-24 h) were dramatically greater than those of BRB. In order to determine the effectiveness of BRBG in reducing glucose levels, we prepared BRBG from the urine pool of rats, and identified and confirmed it through LC-MS-IT-TOF and NMR spectra. In human normal liver cell line L-O2 in vitro, treatment with BRB or BRBG (5, 20, 50 µmol/L) increased glucose consumption, enhanced glycogenesis, stimulated the uptake of the glucose analog 2-NBDG, and modulated the mRNA levels of glucose-6-phosphatase and hexokinase. However, both BBR and BRB improved 2-NBDG uptake in insulin-resistant L-O2 cells, while BRBG has no effect. In conclusion, BRB exerts a stronger glucose-lowering effect than BBR in HFD-induced hyperglycemia mice. Although BRB significantly stimulated the insulin sensitivity and glycolysis in vitro, BRBG may have a greater contribution to the glucose-lowering effect because it has much greater system exposure than BRB after oral administration of BRB. The results suggest that BRBG is a potential agent for reducing glucose levels.

Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 µm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.

PEEK tube-based online solid-phase microextraction-high-performance liquid chromatography for the determination of yohimbine in rat plasma and its application in pharmacokinetics study.

Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R2 of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.

Effects of type 2 diabetes mellitus on the pharmacokinetics of berberine in rats.

CONTEXT: Berberine is an active alkaloid isolated from Rhizoma coptidis [Coptis chinensis Franch. (Ranunculaceae)] that is widely used for the treatment of diabetes, hyperlipidemia and hypertension. However, the pharmacokinetics of berberine in normal rats and type 2 diabetes mellitus (T2DM) model rats are not clear. OBJECTIVE: This study compares the pharmacokinetics of berberine between normal and T2DM model rats. MATERIALS AND METHODS: The T2DM model rats were fed with high fat diet for 4 weeks, induced by low-dose (30 mg/kg) streptozotocin for 72 h and validated by determining the peripheral blood glucose level. Rats were orally treated with berberine at a dose of 20 mg/kg and then berberine concentration in rat plasma was determined by employing a sensitive and rapid LC-MS/MS method. RESULTS: The significantly different pharmacokinetic behaviour of berberine was observed between normal and T2DM model rats. When compared with the normal group, Cmax, t1/2 and AUC(0-t) of berberine were significantly increased in the model group (17.35 ± 3.24 vs 34.41 ± 4.25 µg/L; 3.95 ± 1.27 vs 9.29 ± 2.75 h; 151.21 ± 23.96 vs 283.81 ± 53.92 µg/h/L, respectively). In addition, oral clearance of berberine was significantly decreased in the model group (134.73 ± 32.15 vs 62.55 ± 16.34 L/h/kg). DISCUSSION AND CONCLUSION: In T2DM model rats, the pharmacokinetic behaviour of berberine was significantly altered, which indicated that berberine dosage should be modified in T2DM patients.

Development and application of a UPLC-MS/MS method for simultaneous determination of fenofibric acid and berberine in rat plasma: application to the drug-drug pharmacokinetic interaction study of fenofibrate combined with berberine after oral administration in rats.

With the purpose of carrying out pharmacokinetic interaction studies ofnberberine (BBR) and fenofibrate (FBT), an UPLC-MS/MS method has been developed and validated. The analytes, BBR and fenofibric acid (FBA, metabolite of FBT) and the internal standard, tetrahydropalmatine, were extracted with dichloromethane-diethyl ether (3:2, v/v) and separated on an Agilent Eclipse XDB C18 column using a mobile phase composed of acetonitrile and water. With positive ion electrospray ionization, the analytes were monitored on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. Linear calibration curves were obtained over the concentration ranges of 0.1-100.0 ng/mL for BBR and 10.0-50,000.0 ng/mL for FBA. For BBR and FBA, the intra- and inter-day precisions were <11.5 and 11.9%, respectively. The accuracy was within 11.7% and 11.3%. The mean recoveries of BBR at three concentrations of 0.2, 20.0, 80.0 ng/mL were >85.6%, and those of FBA at three concentrations of 20.0, 2500.0, 40,000.0 ng/mL were >87.9%. Consequently, the proposed method was applied to the pharmacokinetic interaction study of FBT combined with BBR after oral administration in rats and was proved to be sensitive, specific and reliable to analyze BBR and FBA in biological samples simultaneously. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolite identification of seven active components of Huan-Nao-Yi-Cong-Fang in rat plasma using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry.

Huan-Nao-Yi-Cong-Fang (HNYCF) is a potential prescription in treating Alzheimer's disease. Seven constituents [ferulic acid (FA), 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (THSG), berberine hydrochloride (BHCl), emodin, ginsenoside Rg1 (Rg1), ginsenoside Re (Re) and ginsenoside Rb1 (Rb1)] have been used as quality chemical markers of HNYCF owing to their biological significance and high contents in crude plant materials. This study explored the metabolites of the seven bioactive components in rat plasma to give useful data for further study of the action mechanism of HNYCF. LC/MS-IT-TOF was used to simultaneously characterize the metabolites of the seven components. Using the combination of MetID Solution 1.0 software and accurate mass measurements, the metabolites of HNYCF were reliably characterized. Their structures were elucidated based on the accurate MS(2) spectra and comparisons of their changes in accurate molecular masses and fragment ions with those of parent compounds. A total of five parent active compounds (BHCl, emodin, Rg1, Rb1 and Re) and 10 metabolites were found from the rat plasma 2 h after oral administration of HNYCF dosage, of which two metabolites of emodin were observed for the first time. The proposed metabolic pathways of the bioactive components in the rat plasma are helpful for further studies on the pharmacokinetics and real active compound forms of this drug.

Effects of berberine on the pharmacokinetics of losartan and its metabolite EXP3174 in rats and its mechanism.

CONTEXT: Losartan and berberine (BBR) are often simultaneously used for the treatment of senile diabetic nephropathy in clinics. However, the potential herb-drug interaction between losartan and BBR is unknown. OBJECTIVE: This study investigates the influence of BBR on the pharmacokinetics of losartan and EXP3174, and investigates the effects of BBR on the metabolic stability of losartan. MATERIALS AND METHODS: The pharmacokinetic profiles losartan and EXP3174 of orally administered losartan (10 mg/kg) with and without pretreatment with BBR (20 mg/kg) within 24 h were determined in Sprague-Dawley rats. The inhibitory effects of BBR on the metabolic stability of losartan were investigated using rat liver microsomes. RESULTS: The Cmax (1.26 ± 0.37 versus 1.96 ± 0.45 mg/L) and the AUC(0-t) (8.25 ± 0.89 versus 12.70 ± 1.42 mg h/L) of losartan were significantly (p < 0.05) increased by BBR compared to the control, while the Cmax (0.97 ± 0.15 versus 0.77 ± 0.06 mg/L) of EXP3174 was significantly decreased compared to the control (p < 0.05). The Tmax of losartan was prolonged from 0.41 ± 0.12 to 0.52 ± 0.18 h, but the difference was not significant. However, the Tmax of EXP3174 was decreased significantly (p < 0.05) from 8.14 ± 0.36 to 3.33 ± 0.28 h. The metabolic stability of losartan was increased from 37.4 to 59.6 min. DISCUSSION AND CONCLUSION: We infer that BBR might increase the plasma concentration of losartan and decrease the concentration of EXP3174 through inhibiting the activity of CYP3A4 or CYP2C9.

Pharmacokinetics and brain distribution of tetrahydropalmatine and tetrahydroberberine after oral administration of DA-9701, a new botanical gastroprokinetic agent, in rats.

DA-9701, a new botanical gastroprokinetic agent, has potential for the management of delayed gastric emptying in Parkinson's disease if it has no central anti-dopaminergic activity. Therefore, we examined the pharmacokinetics of DA-9701 components having dopamine D2 receptor antagonizing activity, tetrahydropalmatine (THP) and tetrahydroberberine (THB), following various oral doses (80-328 mg/kg) of DA-9701. The distribution of THP and THB to the brain and/or other tissues was also evaluated after single or multiple oral administrations of DA-9701. Oral administration of DA-9701 yielded dose-proportional area under the plasma concentration-time curve (AUC0-8 h) and maximum plasma concentration (Cmax) values for THP and THB, indicating linear pharmacokinetics (except for THB at the lowest dose). THP and THB's large tissue-to-plasma concentration ratios indicated considerable tissue distribution. High concentrations of THP and THB in the stomach and small intestine suggest an explanation for DA-9701's potent gastroprokinetic activity. The maximum concentrations of THP and THB in brain following multiple oral DA-9701 for 7 d (150 mg/kg/d) was observed at 30 min after the last oral DA-9701 treatment: 131±67.7 ng/g for THP and 6.97±4.03 ng/g for THB. Although both THP and THB pass through the blood-brain barrier, as indicated by brain-to-plasma concentration ratios greater than unity (approximately 2-4), oral administration of DA-9701 at the effective dose in humans is not expected to lead to sufficient brain concentrations to exert central dopamine D2 receptor antagonism.

Pharmacokinetics and Brain Distribution and Metabolite Identification of Coptisine, a Protoberberine Alkaloid with Therapeutic Potential for CNS Disorders, in Rats.

Coptisine (COP), a protoberberine alkaloid (PBA) from Chinese medicinal plants (such as family Berberidaceae), may be useful for improving central nervous system disorders. However, its pharmacokinetics, disposition and metabolism are not well defined. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the analysis of COP in biological samples. To better understand its in vivo pharmacological activities, COP concentrations in rat plasma were determined after oral (50 mg/kg) and intravenous administration (10 mg/kg). For the brain distribution study, the concentration of COP in five different regions was examined after intravenous administration at 10 mg/kg. Pharmacokinetic parameters from the COP concentration-time profiles in plasma and brain, and the brain-to-plasma coefficient (Kp, brain) were calculated by non-compartmental analysis. The metabolites of COP in rats in vivo and in vitro (urine, bile, liver microsomes and intestinal bacteria incubation) were also identified. Seventeen metabolites, including 11 unconjugated metabolites formed by hydroxylation, hydrogenation, demethylation, dehydrogenation, demethylation, and 6 glucuronide and sulfate conjugates were identified for the first time. The results suggested that COP had low oral bioavailability of 8.9% and a short (plasma) half-life (T1/2=0.71 h) in rats. After intravenous administration, it quickly crossed the blood-brain barrier, accumulating at higher concentrations and then was slowly eliminated from different brain regions. Moreover, COP was transformed into metabolites through multiple metabolic pathways in vivo and in vitro. These results should help to promote further research on COP and contribute to clarifying the metabolic pathways of PBAs.

Short-term effects of a combined nutraceutical of insulin-sensitivity, lipid level and indexes of liver steatosis: a double-blind, randomized, cross-over clinical trial.

BACKGROUND: Overweight subjects easily develop alterations of the glucose and lipid metabolism and are exposed to an increased cardiometabolic risk. This condition is potentially reversible through the improvement of dietary and behavioural habits. However, a well-assembled nutraceutical would be a useful tool to better improve the metabolic parameters associated to overweight and insulin resistance. METHODS: To evaluate the effect of a combined nutraceutical containing berberine, chlorogenic acid and tocotrienols, we performed a double blind, cross-over designed trial versus placebo, in 40 overweight subjects with mixed hyperlipidaemia. After the first 8 weeks of treatment (or placebo), patients were asked to observe a 2-week washout period, and they were then assigned to the alternative treatment for a further period of 8 weeks. Clinical and laboratory data associated to hyperlipidaemia and insulin resistance have been obtained at the baseline, at the end of the first treatment period, after the washout, and again after the second treatment period. RESULTS: Both groups experienced a significant improvement of anthropometric and biochemical parameters versus baseline. However, total cholesterol, LDL cholesterol, triglycerides, non-HDL cholesterol, fasting insulin, HOMA-IR, GOT and Lipid Accumulation Product decreased more significantly in the nutraceutical group versus placebo. CONCLUSIONS: This combination seems to improve a large number of metabolic and liver parameters on the short-term in overweight subjects. Further studies are needed to confirm these observations on the middle- and long-term.

Comparative pharmacokinetics of active alkaloids after oral administration of Rhizoma Coptidis extract and Wuji Wan formulas in rat using a UPLC-MS/MS method.

Wuji Wan (WJW), containing Rhizoma Coptidis (Huanglian in Chinese, HL), Frutus Evodiae Rutaecarpae (Wuzhuyu, WZY) and Radix Paeoniae Alba (Baishao, BS), is a classical traditional Chinese medical formula employed in treating intestinal disorders. Berberine (BBR) and palmatine (PMT) are the major active alkaloids in HL and have analgesic and anti-microbial effects. A sensitive, specific and validated ultra-performance liquid chromatography-tandem mass spectrometric method was developed to investigate the pharmacokinetic profiles of BBR and PMT in rat plasma and in situ intestinal perfusion solution. In comparison with the pharmacokinetic parameters of BBR and PMT, t(1/2), C(max), T(max), AUC, CL and MRT after intragastric (i.g.) administration with HL extract alone, those remarkably changed after i.g. administration with WJW formulas 1 and 2 (herb proportions are 12:2:3 and 12:1:12). Particularly, the oral bioavailability of PMT in WJW formula 1 was significantly increased. In rat intestinal perfusion experiments, the apparent permeability coefficient value of PMT was (1.45 ± 0.72) × 10(-5) cm/s when perfusion with HL was performed, and the value was significantly increased to (3.92 ± 0.52) × 10(-5) cm/s on perfusion with WJW formula 1. These results indicate that the pharmacokinetic parameters and absorption of BBR and PMT are affected by the other herbs or ingredients from WJW formulas.

Berberine and its metabolites: relationship between physicochemical properties and plasma levels after administration to human subjects.

Berberine (1) is an alkaloid used widely in the treatment of several diseases. However, its physicochemical properties, pharmacokinetics, and metabolism remain unclear, and conflicting data have been reported. In this study, the main physicochemical properties of 1 and its metabolites were evaluated, including lipophilicity, solubility, pKa, and albumin binding. A sensitive HPLC-ESIMS/MS method was developed and validated to identify 1 and its main metabolites in human plasma. This method was used to quantify their levels in the plasma of healthy volunteers and hypercholesterolemic patients following a single dose and chronic administration, respectively. In both cases, berberrubine (2) was found to be the main metabolite. Surprisingly, 2 is more lipophilic than 1, which suggests that this compound tautomerizes to a highly conjugated, electroneutral quinoid structure. This was confirmed by NMR studies. These results indicate that the higher plasma concentration of 2 was a consequence of a more efficient intestinal absorption, suggesting that berberrubine is potentially more pharmacologically active than berberine.