Chemicals and reagents

BBR was obtained from J&K Scientific Ltd (Beijing, China). Tetrahydropalmatine, as an internal standard, was purchased from the National Institute for Food and Drug Control (Beijing, China). Thalifendine (M1), berberrubine (M2), demethyleneberberine (M3), jatrorrhizine (M4) and dihydroberberine (dhBBR, M17) were obtained from Chengdu Herb Purity Co., Ltd (Chengdu, China). Rutin was purchased from the National Institute for Food and Drug Control (Beijing, China). The purity of the above-mentioned standards was more than 98% (HPLC). HPLC-grade acetonitrile was obtained from J&K Scientific Ltd (Beijing, China). All the other chemicals and reagents were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China) and were of HPLC-grade purity.

Ticlopidine, ketoconazole, β-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose 6-phosphate (G-6-P) and D-glucose-6-phosphate dehydrogenase (G-6-PDH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Furafylline, diethyldithiocarbamate (DDC) and sulfaphenazole were obtained from Santa Cruz Biotechnology Co. Ltd. (Shanghai, China). Quercetin, nootkatone and quinidine were purchased from J&K Scientific Ltd. (Beijing, China).

Nitroreductase (≥90%, recombinant, expressed in Escherichia coli), monoamine oxidase-B (recombinant, expressed in baculovirus-infected BTI insect cells) and deprenyl (a monoamine oxidase-B inhibitor) were purchased from Sigma-Aldrich Co. (Shanghai, China). 2-Iodosobenzoic (2-IBA, a nitroreductase inhibitor) and pargyline (another monoamine oxidase-B inhibitor) were obtained from J&K Scientific Ltd. (Beijing, China). Human intestinal microsomes (HIMs) were obtained from the Research Institute for Liver Diseases (Shanghai, China).

Animals

Male Sprague–Dawley (SD) rats (180–200 g) were supplied by the Institute of Laboratory Animal Science, Chinese Academy Medical Sciences (Beijing, China). The animals were housed in cage racks, with a 12-h light/12-h dark cycle (light on from 8:00 AM to 8:00 PM) at ambient temperature (22 °C–24 °C) and 45% relative humidity. The rats were fasted for 12 h before the experiments but during the study had free access to food and water. The research was conducted in accordance with the institutional guidelines and ethics and approved by the Laboratories Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.

Instruments

A liquid chromatography instrument coupled to an ion trap time-of-flight mass spectrometer (LC/MSn-IT-TOF) from Shimadzu Corporation (Kyoto, Japan) was used to identify the chemical structures of BBR and its possible metabolites. Liquid chromatography with tandem mass spectrometry (LC–MS/MS 8040 or 8050, Shimadzu Corporation, Kyoto, Japan) and gas chromatography with mass spectrometry (GC-MS QP2010, Shimadzu Cooperation, Kyoto, Japan) were used for analysis and quantification of BBR and its metabolites in biological samples.

Metabolizing BBR by large intestinal bacteria in vitro

The method was reported previously33. Briefly, colon contents from six rats were pooled and 5 g of the sample was transferred into a flask containing the anaerobic medium (100 ml). After thorough mixing, the cultures (which contained intestinal bacteria and anaerobic medium) were pre-incubated under anaerobic conditions with a N 2 atmosphere at 37 °C for 60 min. Rutin (10 μl, 1.0 mg/ml in methanol) was used as a positive reference for intestinal metabolism.

Ten microliters of BBR at different concentrations was added into the fresh human or rat intestinal bacteria cultures (990 μl), with methanol (10 μl) as the negative control. The final concentrations of BBR in the incubation system were 100, 10 and 5 μg/ml. The cultures were incubated at 37 °C for 72 h in the presence or absence of human or rat intestinal bacteria.

After termination of the reaction with acetonitrile (1 ml), 50 μl of the internal standard (tetrahydropalmatine, 2.0 mg/ml in methanol) was added into the BBR samples, which were then mixed for 30 sec and centrifuged at 7,500 g for 15 min. The supernatant was dried under nitrogen flow at room temperature and the residue was dissolved in 100 μl of methanol and centrifuged at 7,500 g for 15 min. The metabolites were analyzed by LC/MSn-IT-TOF using a previously reported method34.

GCMS-QP2010 was also used to analyze the BBR metabolites with low polarity. The GC column was an Alltech capillary column (ATTM-1701, 30 m × 0.25 mm × 0.25 μm) operated in the splitless mode. The helium carrier flow was 39.7 cm/s under a column head pressure of 68.1 kPa. The oven temperature was initially 50 °C for 2 min, gradually increased to 260 °C at a rate of 8 °C/min and maintained for 25 min. The injector and detector temperatures were set to 280 °C and 230 °C, respectively. The mass spectra were recorded at a scan range of m/z 40 to 800. Structure identification of possible metabolites was based on matching with standard mass spectra available in the Shimadzu GC-MS library.

Quantification analysis of BBR metabolites modified by the gut microbiota in vitro and in vivo

A working solution of BBR was prepared at a series of concentrations by diluting the stock solution (10 mg/ml) with methanol to generate the standard curves. LC-MS/MS 8040 was used to quantify BBR and its metabolites transformed by intestinal bacteria15.

For the determination of dhBBR obtained after incubation with intestinal bacteria in vitro, a working solution of dhBBR was prepared at concentrations of 500, 200, 100, 50, 20, 10 and 1 μg/ml by diluting the stock solution (1 mg/ml) with methanol, which were used to generate the standard curves of dhBBR. Samples from the in vitro intestinal bacteria incubation mixture were injected into the GC-MS instrument.

For the analysis of the in vivo intestinal metabolites of BBR, six SD rats were orally treated with BBR (200 mg/kg) and their feces were collected 0, 6, 12, 24, 36, 48 and 72 h after treatment. The method used for the preparation of the feces was reported previously34. An aliquot of 1 μl was injected into the GC-MS instrument. The level of dhBBR in feces was measured and calculated based on the standard curve obtained with the in vitro intestinal bacteria incubation mixture.

To prepare the human samples, we transferred fresh feces (5 g per person) from healthy volunteers (age from 20–25, two males and two females) into a flask containing the anaerobic medium (100 mL)33. After thorough mixing, the bacteria were cultured under anaerobic conditions with a N 2 atmosphere at 37 °C for 60 min. The culture solution of intestinal bacteria was then ready for analysis.

BBR metabolism in the rat small intestine bacteria

The small intestine contents from ten sacrificed SD rats were obtained and mixed with anaerobic medium to prepare the cultures. BBR was added into the incubation at final concentrations of 100, 50 and 10 μg/ml and the reaction was continued for 6, 12 or 24 h. Levels of BBR, dhBBR and other BBR metabolites were determinedusing the LC-MS/MS 8040 and GC-MS instruments according to the above-mentioned method.

Distribution of BBR, dhBBR and other metabolites in the small intestine

Three segments of small intestine (duodenum, jejunum and ileum) were collected 0, 6, 12, 24, 36 and 48 h after the oral administration of BBR in SD rats. The small intestine tissues were washed thoroughly with saline and dried. After being weighed, the samples were homogenized with mixed solution (ethanol: water, 1:1) at a ratio of 1:2 [w(g)/v(ml)]. The samples were centrifuged at 7,500 g for 10 min and the supernatant was collected and evaporated to dryness in a water bath at 40 °C using a rotary evaporator. The residues of the tissue extraction were dissolved in methanol (250 μl) and vortex-mixed for 5 min. The tissue solution was then centrifuged (at 14,000 g for 5 min) and the supernatant was passed through a 0.22-μm filter. An aliquot of 1 μl of the supernatant was injected into the GC-MS instrument and a 10-μl aliquot was used for LC-MS/MS analysis. The level of dhBBR was calculated based on the standard curve and BBR and other metabolites was quantified using the method described above.

BBR metabolism in the in vitro culture of 14 intestinal facultative anaerobes

Ten intestinal facultative anaerobes (clinical isolates)—Staphylococcus aureus (S. aureus) 08-43, Enterococcus faecium (E. faecium) 13-01, Enterococcus faecalis (E. faecalis) 13-01, Enterobacter cloacae (E. cloacae) 13-12, Escherichia coli (E. coli) 06-05, Staphylococcus epidermidis (S. epidermidis) 12-12, Pseudomonas aeruginosa (Ps. aeruginosa) 13-10, Klebsiella pneumonia (K. pneumoniae) 13-14, Proteus mirabilis (P. mirabilis) 13-01 and Acinetobacter baumannii (A. baumannii) 13-02—were collected from gastrointestinal specimens from patients from hospitals in Beijing between 2006 and 2013. The specimens were identified in the hospitals using the VITEK 2-COMPACT system (bioMerieux, Marcy l’Etoile, France). Lactobacillus casei (L. casei ATCC 334), Lactobacillus acidophilus (L. acidophilus ATCC 4356), Bifidobacterium longum (B. longum ATCC 15707) and Bifidobacterium breve (B. breve ATCC 15700) were purchased from the Microbial Culture Collection Center in Guangdong, China. The bacteria were transferred into a flask containing anaerobic medium. BBR was incubated with the 14 facultative anaerobes at a final concentration of 50 μg/ml at 37 °C for 0, 12, 24, 48 and 72 h. BBR and dhBBR in the culture were analyzed quantitatively using GC-MS and LC-MS/MS 8040.

Determination of nitroreductase

The detection of nitroreductase was performed using the Human Nitroreductase ELISA kit purchased from Beijing Luyuan Dade Biological Science and Technology Co., Ltd (Beijing, China) according to the manufacturer’s guidelines.

Pseudo germ-free (PGF) rats and BBR absorption

Male SD rats (180–200 g) were orally treated with cefadroxil (100 mg/kg), terramycin (300 mg/kg) and erythromycin (300 mg/kg) twice a day for 3 days and pharmacokinetic examination was performed 2 days after final administration. The colon contents of the rats were collected on the first and third days after the final treatment with antibiotics and the germ-free status was confirmed by culturing fecal samples aerobically on a nutrient agar culture medium. Fecal samples from non-antibiotic-exposed rats served as control samples.

Before oral administration of a single dose of BBR (200 mg/kg), the PGF rats were fasted overnight with free access to water. Blood samples were collected from the posterior orbital venous plexus into a heparinized tube at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h post-BBR treatment and then subjected to the procedure described above. Fecal samples were also collected at 0, 6, 12, 24, 36 and 48 h post-BBR treatment. The samples were stored at −20 °C for further analysis.

Anti-bacterial test

Seventeen bacterial strains (see above) were used in the susceptibility test for BBR and dhBBR. The bacterial strains, which belong to different species of bacteria, were collected and identified as described above. The minimum inhibitory concentrations (MICs) of BBR and dhBBR after 24 h were determined through the broth microdilution method according to the CLSI guidelines35. BBR and dhBBR were tested at a concentration range of 8 to 512 μg/ml. The MIC was defined as the lowest concentration of an agent that prevents turbidity. The experiment was repeated three times. All of the bacterial strains tested in this study (S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922, S. aureus 08-43, S. epidermidis 12-12, E. faecium 13-01, E. faecalis 13-01, E. coli 06-05, K. pneumoniae 13-14, Ps. aeruginosa 13-10, A. baumannii 13-02, E. cloacae 13-12 and P. mirabilis 13-01) were facultative anaerobes, of which S. aureus ATCC 29213, E. faecalis ATCC 29212 and E. coli ATCC 25922 were standard strains from ATCC (USA) and served as quality controls. L. casei ATCC 334, L. acidophilus ATCC 4356, B. longum ATCC 15707 and B. breve ATCC 15700 were also tested in this anti-bacterial experiment. Ampicillin was used as the positive control.

Molecular docking between BBR and nitroreductase

AutoDock Vina v.1.0.2 software was used to perform the molecular docking of BBR onto nitroreductase, whose crystal structures are available in the Protein Data Bank17. The docking parameters were set to the default values. The grid boxes were 20 Å × 20 Å × 20 Å, encompassing the active site cavities. The binding modes of BBR to enzymes were chosen to further optimize the docking conformation according to their binding free energy, distances from conserved water molecules and the flavin mononucleotide (FMN). The simulation results were visualized using the PyMOL Molecular Graphics System Version 1.3 (Schrödinger LLC, New York, NY, USA) and Discovery Studio Visualizer (Accelrys, Inc., San Diego, CA, USA).

Nitroreductase-mediated BBR reduction

BBR was incubated with nitroreductase in intestinal bacteria cultures. The reaction mixture consisted of BBR (50 μg/ml), an NADPH-regenerating system and nitroreductase (5 μg/ml) in a final volume of 1 ml under the protection of N 2 . After incubation for 0, 2, 4, 6 and 12 h at 37 °C, the reaction was terminated by the addition of 1 ml of ice-cold acetonitrile. The samples were then extracted with 1 ml of ethyl acetate after adding 1 ml of 0.5 M sodium hydroxide solution. The organic phase was evaporated to dryness under a nitrogen flow in a water bath at 40 °C. The residue was dissolved with 200 μl of the mobile phase for further analysis. 2-Iodosobenzoic acid (2-IBA), a specific inhibitor of nitroreductase, was used to verify the role of bacterial nitroreductase.

Absorption of BBR and dhBBR in Caco-2 cells

A Caco-2 cell assay was conducted using a method reported previously36. Stock solutions of dhBBR and BBR were prepared in DMSO at 1 mM and these were then diluted with HBSS buffer (10 mM HEPES, pH 7.4) to a final concentration of 5 μM for both samples. To determine the rate of drug transport in the apical-to-basolateral direction, 200 μl of the compound working solution was added to the filter well (apical compartment) and 800 μl of HBSS was added to the receiver plate (basolateral compartment). Accordingly, to determine the rate of drug transport in the basolateral-to-apical direction, 800 μl of the compound working solution was added to each well of the receiver plate and 200 μl of HBSS was added to the filter well. The plates were incubated for 30 min at 37 °C with shaking at 150 rpm on a rotary shaker. At the end of the transport period, aliquots of 50 μl were removed directly from the apical and basolateral wells and transferred to wells of new plates. Four volumes of cold methanol containing internal standards were added into each well. The samples were centrifuged at 16,000 g for 15 min. Aliquots of 200 μl of the supernatant were used for the LC-MS/MS analysis.

Absorption of dhBBR in vivo

Five SD rats were fasted for 12 h and then orally administered 200 mg/kg dhBBR. Blood samples (0.5 ml) were obtained from the posterior orbital venous plexus into a heparinized tube at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h and the samples were then centrifuged at 2,000 g for 5 min. The plasma (100 μl) was precipitated with 100 μl of acetonitrile after addition of 10 μl of the internal standard. BBR served as a control in the study. The plasma concentrations of BBR and dhBBR were determined using LC-MS/MS 8040 and LC-MS/MS 8050.

Drug effect on hepatic LDLR gene expression

The HepG2 human hepatoma cell line was obtained from the American Tissue Culture Collection (ATCC, USA) and grown in Eagle’s Minimum Essential Medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) at 37 °C. For the drug efficacy test, HepG2 cells were cultured for 24 h and then treated with BBR or dhBBR for 12 h. The compounds were freshly prepared in medium prior to use.

The total cellular RNAs were isolated with the SV Total RNA Isolation System (Promega, Madison, WI, USA) following the vendor’s instructions. The total RNAs were reversely transcribed into cDNAs and quantitative real-time PCR was performed using a 2-Step RT-qPCR System (Promega, Madison, WI, USA), with GAPDH as an internal control. The normalized LDLR mRNA expression levels were plotted as fold levels compared with the untreated control. The following primers were used: LDLR forward, aggacggctacagctaccc; LDLR reverse, ctccaggcagatgttcacg; GAPDH forward, agccacatcgctcagacac; and GAPDH reverse, gcccaatacgaccaaatcc.

Reversion from dhBBR to BBR

The rat duodenum, jejunum and ileum were collected separately, washed thoroughly with saline and dried. After being weighed, the tissues were homogenized with saline at a ratio of 1: 2 [w (g) /v (ml)]. DhBBR was dissolved in a mixture of methanol and DMSO at a ratio of 1:1 with a final concentration of 5 mg/ml. Ten microliters of dhBBR (5 mg/ml) was mixed with 1 ml of the homogenates. The samples were then centrifuged at 14,000 g for 5 min and the supernatants were collected and mixed with an equal volume of ice-cold acetonitrile to terminate the reaction. The samples were centrifuged at 14,000 g for 5 min and the supernatant was used for the HPLC analysis.

Transformation of dhBBR in pooled human intestinal microsomes (HIMs)

The typical incubation mixture contained 0.2 mg of HIMs, 100 mM phosphate buffer (pH 7.4), 100 μM dhBBR (dissolved in DMSO) and an NADPH-regenerating system [at a final concentration of 3.3 mM glucose-6-phosphate (G-6-P), 1.3 mM NADP+, 0.4 unit/ml glucose-6-phosphate dehydrogenase (G-6-PDH) and 3.3 mM MgCl 2 ] in a final volume of 200 μl. After pre-incubation of HIMs with dhBBR for 2 min at 37 °C, the reaction was initiated by the addition of the NADPH-regenerating system. The mixture was incubated at 37 °C with exposure to air for 2, 5 and 10 min and was terminated by the addition of 200 μl of ice-cold acetonitrile. Then, 20 μl of the internal standard was added to the mixture and the mixture was centrifuged at 14,000 g for 5 min. Fifty microliters of the supernatant was injected into the HPLC instrument for analysis. The control samples were incubated under the same conditions but without HIMs.

Oxidization of dhBBR by monoamine oxidase B (MAO-B)

The reaction system consisted of dhBBR (100 μM), MAO-B (0.2 mg/ml) and phosphate-buffered saline (PBS, pH = 7.4) in a final volume of 200 μl. The MAO-B was pre-incubated via centrifugation at 850 rpm at 37 °C for 5 min and this step was followed by the addition of dhBBR. The reaction was terminated after 30 min by adding an equal volume of ice-cold acetonitrile and centrifuging at 14,000 g for 5 min. Ten microliters of the supernatant was injected into the HPLC instrument for analysis. Selective MAO-B inhibitors (deprenyl, 100 μM or pargyline, 1 μM)37,38 were used in the tests.

Effect of vitamin C on dhBBR-to-BBR transformation

The rats (n = 3) were sacrificed to obtain fresh small intestine homogenate. The homogenate was then boiled for 2 min to inactivate all of the enzymes. After pre-incubation at 37 °C for 2 min, 5 μl of dhBBR (10 mM, dissolved in DMSO) was added into the inactivated homogenate and fresh homogenate (active one) was used as a reference. The sample was mixed thoroughly and centrifuged at 16,000 g for 5 min. The supernatant was transferred into new tubes, treated with 500 μl of acetonitrile and then centrifuged. Twenty-microliter aliquots were used for the HPLC analysis.

To examine the oxidative reaction, 100 μl of vitamin C was pre-incubated with the inactivated homogenate; the final concentrations of vitamin C were 95, 9.5 and 0.95 mM. The superoxide anion in the sample was detected with an O 2 − ELISA kit (Beijing Biolab Co., Ltd., Beijing, China; lot: 201404) following the manufacturer’s instructions.

Gut microbiota modulates the therapeutic effects of BBR on hyperglycemia and hyperlipidemia

KK-Ay mice with type 2 diabetes (female, 12 ± 1 weeks of age) were purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (Beijing, China). The mice were maintained at room temperature (23 ± 2 °C) with 50% moisture and exposed to a 12-h light/12-h dark cycle and food and water were provided ad libitum. Antibiotics at the doses described above were orally administered to KK-Ay mice for 3 days before the BBR treatment course and the pseudo germ-free (PGF) state of the experimental mice was confirmed (see above).

Four groups (n = 6 for each group) were used in the experiment: Group1, PGF KK-Ay mice orally treated with antibiotics (qd, at the dosage described above) plus BBR (200 mg/kg, qd) for 2 weeks; Group 2, untreated KK-Ay mice orally treated with BBR (200 mg/kg, qd) for 2 weeks; Group 3, PGF KK-Ay mice orally treated with antibiotics (qd, at a dosage identical to that for Group 1) for 2 weeks; Group 4, untreated KK-Ay mice. The antibiotics were administered in the morning and BBR was administered in the afternoon. All the animals were fed a high-fat diet. The germ-free state of Groups 1 and 3 was confirmed on days 7 and 14 of the treatment course using the same bacterial analytical method described above. All of the mice were euthanized on day 14 and blood samples were taken to measure the fasting serum glucose (Glu), triglyceride (TG) and cholesterol (CHO) using Glu, TG and CHO reagent kits (Biosino Bio-Technology and Science INC, Beijing China), respectively. C57BL/6J mice were used as a wild-type reference. The body weights of the mice before, during and after treatments were recorded for 14 days.

Blood samples of the mice in Groups 1 and 2 were collected into a heparinized tube 30 min after the oral administration of BBR. The samples were taken at days 1, 3, 6, 10 and 14 of the BBR treatment course. The samples were centrifuged at 10,000 g for 5 min and the plasma was collected and stored at −80 °C before analysis.

Effect of ions on the dhBBR-to-BBR transformation

dhBBR was added into the reaction solution containing FeCl 3 , CuCl 2 , ZnCl 2 and NaCl (at concentrations of 0.033, 0.333 and 3.33 mM, respectively). Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 10 mM), a chelating agent for metal ions, was used in the test.

Statistical analysis

The statistical analyses were conducted using two-way ANOVA and Student’s t-test with GraphPad Prism Version 5 (GraphPad Software, CA, USA). The data are expressed as the means ± standard deviation and p values less than 0.05 were considered statistically significant.