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Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 μM) and demonstrated the effects of BBR (10, 50, and 100 μM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV., (© 2024 John Wiley & Sons Ltd.)

Background: Berberine is the main bioactive constituent of Coptis chinensis, a quaternary ammonium alkaloid. While berberine's cardiovascular benefits are well-documented, its impact on thrombosis remains not fully understood., Purpose: This study investigates the potential of intestinal microbiota as a novel target for preventing thrombosis, with a focus on berberine, a natural compound known for its effectiveness in managing cardiovascular conditions., Methods: Intraperitoneal injection of carrageenan induces the secretion of chemical mediators such as histamine and serotonin from mast cells to promote thrombosis. This model can directly and visually observe the progression of thrombosis in a time-dependent manner. Thrombosis was induced by intravenous injection of 1 % carrageenan solution (20 mg/kg) to all mice except the vehicle control group. Quantitative analysis of gut microbiota metabolites through LC/MS. Then, the gut microbiota of mice was analyzed using 16S rRNA sequencing to assess the changes. Finally, the effects of gut microbiota on thrombosis were explored by fecal microbiota transplantation., Results: Our research shows that berberine inhibits thrombosis by altering intestinal microbiota composition and related metabolites. Notably, berberine curtails the biosynthesis of phenylacetylglycine, a thrombosis-promoting coproduct of the host-intestinal microbiota, by promoting phenylacetic acid degradation. This research underscores the significance of phenylacetylglycine as a thrombosis-promoting risk factor, as evidenced by the ability of intraperitoneal phenylacetylglycine injection to reverse berberine's efficacy. Fecal microbiota transplantation experiment confirms the crucial role of intestinal microbiota in thrombus formation., Conclusion: Initiating our investigation from the perspective of the gut microbiota, we have, for the first time, unveiled that berberine inhibits thrombus formation by promoting the degradation of phenylacetic acid, consequently suppressing the biosynthesis of PAG. This discovery further substantiates the intricate interplay between the gut microbiota and thrombosis. Our study advances the understanding that intestinal microbiota plays a crucial role in thrombosis development and highlights berberine-mediated intestinal microbiota modulation as a promising therapeutic approach for thrombosis prevention., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Metformin (MTF) and berberine (BBR) share several therapeutic benefits in treating metabolic-related disorders. However, as the two agents have very different chemical structure and bioavailability in oral route, the goal of this study is to learn their characteristics in treating metabolic disorders. The therapeutic efficacy of BBR and MTF was systemically investigated in the high fat diet feeding hamsters and/or ApoE (-/-) mice; in parallel, gut microbiota related mechanisms were studied for both agents. We discovered that, although both two drugs had almost identical effects on reducing fatty liver, inflammation and atherosclerosis, BBR appeared to be superior over MTF in alleviating hyperlipidemia and obesity, but MTF was more effective than BBR for the control of blood glucose. Association analysis revealed that the modulation of intestinal microenvironment played a crucial role in the pharmacodynamics of both drugs, in which their respective superiority on the regulation of gut microbiota composition and intestinal bile acids might contribute to their own merits on lowering glucose or lipids. This study shows that BBR may be a good alternative for MTF in treating diabetic patients, especially for those complicated with dyslipidemia and obesity., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The phenylalanine-tyrosine-dopa-dopamine pathway provides dopamine to the brain. In this process, tyrosine hydroxylase (TH) is the rate-limiting enzyme that hydroxylates tyrosine and generates levodopa (L-dopa) with tetrahydrobiopterin (BH 4 ) as a coenzyme. Here, we show that oral berberine (BBR) might supply H • through dihydroberberine (reduced BBR produced by bacterial nitroreductase) and promote the production of BH 4 from dihydrobiopterin; the increased BH 4 enhances TH activity, which accelerates the production of L-dopa by the gut bacteria. Oral BBR acts in a way similar to vitamins. The L-dopa produced by the intestinal bacteria enters the brain through the circulation and is transformed to dopamine. To verify the gut-brain dialog activated by BBR's effect, Enterococcus faecalis or Enterococcus faecium was transplanted into Parkinson's disease (PD) mice. The bacteria significantly increased brain dopamine and ameliorated PD manifestation in mice; additionally, combination of BBR with bacteria showed better therapeutic effect than that with bacteria alone. Moreover, 2,4,6-trimethyl-pyranylium tetrafluoroborate (TMP-TFB)-derivatized matrix-assisted laser desorption mass spectrometry (MALDI-MS) imaging of dopamine identified elevated striatal dopamine levels in mouse brains with oral Enterococcus, and BBR strengthened the imaging intensity of brain dopamine. These results demonstrated that BBR was an agonist of TH in Enterococcus and could lead to the production of L-dopa in the gut. Furthermore, a study of 28 patients with hyperlipidemia confirmed that oral BBR increased blood/fecal L-dopa by the intestinal bacteria. Hence, BBR might improve the brain function by upregulating the biosynthesis of L-dopa in the gut microbiota through a vitamin-like effect.

Berberine (BBR) is a multi-target drug (MTD) that has proven effective in the treatment of metabolism-related chronic diseases (CDs). However, the mode of action (MOA) of BBR remains to be clarified. At a cellular level, the inhibitory effect of BBR on mitochondrial enzymes is probably responsible for many of its biological activities, including the activation of low-density lipoprotein receptor (LDLR), AMP-activated protein kinase (AMPK) and insulin receptor (InsR); these biological activities contribute to ameliorate peripheral blood metabolic profiles, e.g. by reducing plasma lipids and glucose levels, thus improving signs and symptoms of metabolic disorders. In this perspective, BBR acts as a targeted therapy. However, it also exerts pleiotropic systemic activities on some root causes of CDs that include antioxidant / anti-inflammatory effects and modifications of gut microbiota composition and metabolism, which may also contribute to its disease-modifying effects. After reviewing the different MOA of BBR, here we propose that BBR acts through a drug-cloud (dCloud) mechanism, as different to a drug-target effect. The dCloud here is defined as a group of terminal molecular events induced by the drug (or/and related metabolites), as well as the network connections among them. In this scenario, the therapeutic efficacy of BBR is the result of its dCloud effect acting on symptoms/signs as well as on root causes of the diseases. The dCloud concept is applicable to other established MTDs, such as aspirin, metformin, statins as well as to nutrient starvation, thus providing a novel instrument for the design of effective therapies against multifactorial metabolism-related CDs., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study is designed to investigate the effects of berberine (BBR) on galectin-3 (Gal-3) and the relationships to its suppressive activities on adipocyte differentiation, proliferation and adiposity. Our results showed that BBR greatly suppressed the differentiation and proliferation of mouse primary preadipocytes isolated from epididymal white adipose tissue (eWAT), during which the expression level of Gal-3 was down-regulated significantly. Overexpression of Gal-3 totally abolished the suppressive activities of BBR on Gal-3 expression, preadipocyte differentiation and proliferation. BBR reduced Gal-3 promoter activity, destabilized its mRNA and inhibited firefly luciferase activity of a recombinant plasmid containing the Gal-3 3' untranslated region (UTR). Furthermore, BBR up-regulated microRNA (miRNA) let-7d expression and the suppressive activity on Gal-3 3'UTR was abolished by point mutation on the let-7d binding site. In mice fed a high-fat diet (HFD), BBR up-regulated let-7d and down-regulated Gal-3 expression in eWAT; it also suppressed adipocyte differentiation and proliferation and reduced adiposity greatly. In summary, our study proves that BBR inhibits the differentiation and proliferation of adipocytes through down-regulating Gal-3, which is closely associated with its anti-obesity effect. Our results may support the future clinical application of BBR for the treatment of obesity or related diseases.

Short-chain fatty acids (SCFAs) are a major group of endogenous metabolites generated by the gut microbiota and have been reported to play an important role in physical health, such as improving energy metabolism. Here, using 2-bromoacetophenone as the derivatization reagent (BP, 10 mg/mL, 40 °C for 20 min), a sensitive liquid chromatography-tandem mass spectrometric method was established for the quantitative determination of seven short-chain fatty acids in plasma and feces. The analyses were performed on a C 18 column in positive multiple reaction monitoring mode. Specificity, linearity, accuracy, precision, recovery, and stability were observed within the quantitative limits of biological sample analysis. The established method has largely improved the sensitivity by 200- to 2000-fold than that in gas chromatography (GC). Especially for butyrate, the lower quantitative limit of 1 ng/mL, 1600-fold higher in sensitivity than that of GC (1.6 μg/mL), ensured the accurate determination of its low level in blood or feces (88 ± 29 ng/mL in blood, 176 ± 18 μg/g in feces). Then, the validated method was applied for therapeutic studies of berberine in hyperlipidemia hamsters in vivo and screening of 13 compounds (including five metabolites of berberine and eight typical isoquinoline alkaloids) in vitro. After berberine treatment (oral, 200 mg/kg, 2 weeks) to hyperlipidemia hamsters, the levels of butyrate were significantly upregulated in blood (77 ± 10 ng/mL vs. 117 ± 13 ng/mL, *P < 0.05) and feces (132 ± 11 μg/g vs. 547 ± 57 μg/g, ***P < 0.001), which further verified butyrate as an active endogenous metabolite in coordination with berberine to lower the blood lipids. Additionally, the berberine metabolites (M1, M2, M3), as well as two isoquinoline alkaloids (tetrandrine and dauricine), could also obviously induce the production of SCFAs (butyrate, etc.) in gut microbiota. In total, we have successfully established a new derivative LC-MS/MS method for the targeted quantitative determination of seven SCFAs in biological samples. Graphical abstract.

Cardiovascular and metabolic disease (CMD) remains a main cause of premature death worldwide. Berberine (BBR), a lipid-lowering botanic compound with diversified potency against metabolic disorders, is a promising candidate for ameliorating CMD. The liver is the target of BBR so that liver-site accumulation could be important for fulfilling its therapeutic effect. In this study a rational designed micelle (CTA-Mic) consisting of α-tocopheryl hydrophobic core and on-site detachable polyethylene glycol-thiol shell is developed for effective liver deposition of BBR. The bio-distribution analysis proves that the accumulation of BBR in liver is increased by 248.8% assisted by micelles. Up-regulation of a range of energy-related genes is detectable in the HepG2 cells and in vivo. In the high fat diet-fed mice, BBR-CTA-Mic intervention remarkably improves metabolic profiles and reduces the formation of aortic arch plaque. Our results provide proof-of-concept for a liver-targeting strategy to ameliorate CMD using natural medicines facilitated by Nano-technology.

Berberine is a Chinese herbal medicine extracted from rhizoma coptidis that functions to improve insulin resistance, hyperlipidemia, hepatosteatosis and inflammation. Berberine can modify the activity of cell metabolism and signaling pathways by regulating expression of genes. However, the roles and effects of differential microRNA (miRNA) expression induced by berberine treatment are largely unexplored. It is believed that miRNAs expression modified by berberine contributes to its therapeutic effects to diseases such as hepatosteatosis and non-alcoholic fatty liver disease. By identifying novel miRNAs and their putative gene targets associated with abnormal hepatic lipid deposition, the underlying mechanism of these diseases could be established and effective therapies against the diseases could be developed. Here, we used the immortalized hepatocyte cell line MIHA as a model to study the effect of berberine on global miRNA expression profile of hepatocytes. Global miRNA expression levels were measured in berberine-treated MIHA cells by quantitative reverse transcription PCR miRNA panel, and the potential berberine regulated miRNAs were then validated in MIHA and HepG2 cells. MicroRNA-373 (MiR-373) was consistently upregulated in both cell lines upon berberine treatments. Gene expression microarray showed that berberine upregulated Early Growth Response 1 (EGR1) level which functioned to transactivate miR-373 expression. Subsequently, we showed that upregulation of miR-373 depleted its target gene AKT serine/threonine kinase 1 (AKT1) mRNA level, which led to the inhibition of AKT-mTOR-S6K signaling pathway in hepatocytes that was critical in the development of hepatosteatosis. Study of the therapeutic effect of manipulating miR-373 against abnormal lipid deposition in liver is warranted., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-dioxygenase 1 (IDO1), twenty-five new berberine (BBR) derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-γ-induced IDO1 promoter activities. Structure-activity relationship (SAR) indicated that large volume groups at the 9-position might be beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with inhibition rate of 71-90% compared with BBR. Their effects on IDO1 expression were further confirmed by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed that both of them inhibited IFN-γ-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation., (Copyright © 2017 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Twenty-three new berberine (BBR) analogues defined on substituents of ring D were synthesized and evaluated for their activity for suppression of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation. Structure-activity relationship (SAR) analysis indicated that suitable tertiary/quaternary carbon substitutions at the 9-position or rigid fragment at position 10 might be beneficial for enhancing their anti-inflammatory potency. Among them, compounds 2d , 2e , 2i and 2j exhibited satisfactory inhibitory potency against NF-κB activation, with an inhibitory rate of around 90% (5 μM), much better than BBR. A preliminary mechanism study revealed that all of them could inhibit TNF-α-induced NF-κB activation via impairing IκB kinase (IKK) phosphorylation as well as cytokines interleukin (IL)-6 and IL-8 induced by TNF-α. Therefore, the results provided powerful information on further structural modifications and development of BBR derivatives into a new class of anti-inflammatory candidates for the treatment of inflammatory diseases., Competing Interests: The authors declare no conflict of interest.

Short-chain fatty acids (SCFAs) of intestine microbial have caught accumulating attention for their beneficial effects on human health. Botanic compounds with low bioavailability such as berberine (BBR) and resveratrol might interact with intestinal microbial ecosystem and promote gut bacteria to produce SCFA, which contribute to their biological effects. In the present study, a comprehensive assay system was built to detect SCFAs production in intestinal bacteria, in which stringent anaerobic culture was applied for in vitro bacterial fermentation, followed by direct-injection GC detection (chemical detection) in combination with real time polymerase chain reaction (RT-PCR, biological detection). BBR was used as positive reference. The direct injection GC method was calibrated and successfully applied to analyze the concentration of SCFAs in gut microbiota and BBR was proved to be effective in the dose- and time-dependent up-regulation of SCFAs production. As compared to the saline group, the concentration of acetic acid, propionate acid and butyric acid (the main SCFAs in gut microbiota) were increased by 17.7%, 11.1% and 30.5%, respectively, after incubating intestinal bacteria with 20μg/mL BBR for 24h. The increase reached to 34.9%, 22.4% and 51.6%, respectively when the BBR was 50μg/mL. Additionally, consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) were designed for the detection of acetate kinase (ACK), Methylmalonyl-CoA decarboxylase (MMD) and butyryl-CoA: acetate-CoA transferase (BUT), as they are the key enzymes in the synthetic pathway for acetic acid, propionate acid and butyric acid, respectively. After 24hr's incubation, BBR was shown to promote the gene expression of ACK, MMD and BUT significantly (86.5%, 27.2% and 60.4%, respectively, with 20μg/mL BBR; 130.2%, 84.2% and 98.4%, respectively, with 50μg/mL BBR), showing a solid biological support for the chemical detection. This comprehensive assay system might be useful in identifying SCFAs promoting agents with information on their mechanism., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nitroreductases (NRs) are bacterial enzymes that reduce nitro-containing compounds. We have previously reported that NR of intestinal bacteria is a key factor promoting berberine (BBR) intestinal absorption. We show here that feeding hamsters with high fat diet (HFD) caused an increase in blood lipids and NR activity in the intestine. The elevation of fecal NR by HFD was due to the increase in either the fraction of NR-producing bacteria or their activity in the intestine. When given orally, BBR bioavailability in the HFD-fed hamsters was higher than that in those fed with normal chow (by +72%, * P <0.05). BBR (100 mg/kg/day, orally) decreased blood lipids in the HFD-fed hamsters (** P <0.01) but not in those fed with normal diet. Clinical studies indicated that patients with hyperlipidemia had higher fecal NR activity than that in the healthy individuals (** P <0.01). Similarly, after oral administration, the blood level of BBR in hyperlipidemic patients was higher than that in healthy individuals (* P <0.05). Correlation analysis revealed a positive relationship between blood BBR and fecal NR activity ( r =0.703). Thus, the fecal NR activity might serve as a biomarker in the personalized treatment of hyperlipidemia using BBR., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: Berberine (BBR) clinically lowers blood lipid and glucose levels via multi-target mechanisms. One of the possible mechanisms is related to its effect on the short chain fatty acids (SCFAs) of the gut microbiota. The goal of this study is to investigate the therapeutic effect and mode of action of BBR working through SCFAs of the gut microbiota (especially, butyrate)., Methods: Gas chromatography (GC) was used to detect butyrate and other SCFAs chemically. The effect of BBR on butyrate production was investigated in vitro as well as in several animal systems. Microarrays were used to analyze the composition change in the intestinal bacteria community after treatment with BBR. BBR-induced change in the energy production and gene regulation of intestinal bacteria was examined in order to elucidate the underlying molecular mechanisms., Results: We show that oral administration of BBR in animals promoted the gut microbiota to produce butyrate, which then enters the blood and reduces blood lipid and glucose levels. Incubating gut bacterial strains in vitro with BBR increased butyrate production. Orally treating animals directly with butyrate reduced blood lipid and glucose levels through a mechanism different from that of BBR. Intraperitoneal BBR administration did not increase butyrate but reduced blood lipid and glucose levels, suggesting that BBR has two modes of action: the direct effect of the circulated BBR and the indirect effect working through butyrate of the gut microbiota. Pre-treating animals orally with antibiotics abolished the effect of BBR on butyrate. A mechanism study showed that BBR (given orally) modified mice intestinal bacterial composition by increasing the abundance of butyrate-producing bacteria. Furthermore, BBR suppressed bacterial ATP production and NADH levels, resulting in increased butyryl-CoA and, eventually, butyrate production via upregulating phosphotransbutyrylase/butyrate kinase and butyryl-CoA:acetate-CoA transferase in bacteria., Conclusion: Promotion of butyrate (etc) production in gut microbiota might be one of the important mechanisms of BBR in regulating energy metabolism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The mangiferin-berberine (MB) salt was synthesized by ionic bonding of mangiferin (M) and berberine (B) at an equal molecular ratio. This study aimed to investigate the activities of MB salt in modulating lipid and glucose metabolisms in HepG2 cells. After 24 h treatment of the studying compounds, cellular AMP-activated protein kinase α (AMPKα)/acetyl-CoA carboxylase (ACC) protein levels and carnitine palmitoyltransferase (CPT) 1 activities, intracellular lipid contents, mRNA expression levels of target genes, glucose consumption, and glucose production amounts were determined. Compound C (CC) was used in the blocking experiments. Our results showed that MB salt increased p-AMPKα (Thr172)/p-ACC (Ser79) levels and CPT1 activity and suppressed oleic acid- (OA-) induced lipid accumulation and upregulation of lipogenic genes potently in HepG2 cells. The above activities of MB salt were AMPK dependent and were superior to those of M or B when administered at an equal molar concentration. MB salt enhanced basal and insulin-stimulated glucose consumption and suppressed gluconeogenesis more potently than M or B alone. The inhibiting activity of MB salt on cellular gluconeogenesis was AMPK dependent. Our results may support MB salt as a new kind of agent for the development of novel lipid or glucose-lowering drugs in the future.

Background: Berberine (BBR), as a new medicine for hyperlipidemia, can reduce the blood lipids in patients. Mechanistic studies have shown that BBR activates the extracellular-signal regulated kinase pathway by stabilizing low-density-lipoprotein receptor mRNA. However, aside from inhibiting the intestinal absorption of cholesterol, the effects of BBR on other metabolic pathways of cholesterol have not been reported. This study aimed to investigate the action of BBR on the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters., Methods: Golden hamsters were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia, followed by oral treatment with 50 and 100 mg/kg/day of BBR or 10 and 30 mg/kg/day of lovastatin for 10 days, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), transaminases, and total bile acid in the serum, liver, bile and feces were measured using an enzyme-linked immunosorbent assay. The cholesterol (as well as coprostanol) levels in the liver, bile and feces were determined by gas chromatography-mass spectrometry., Results: The HFD hamsters showed significantly hyperlipidemic characteristics compared with the normal hamsters. Treatment with BBR for 10 days reduced the serum TC, TG and LDL-C levels in HFD hamsters by 44-70, 34-51 and 47-71%, respectively, and this effect was both dose- and time-dependent. Initially, a large amount of cholesterol accumulated in the hyperlipidemic hamster livers. After BBR treatment, reductions in the liver cholesterol were observed by day 3 and became significant by day 7 at both doses (P < 0.001). Meanwhile, bile cholesterol was elevated by day 3 and significantly increased at day 10 (P < 0.001). BBR promoted cholesterol excretion from the liver into the bile in hyperlipidemic hamsters but not in normal hamsters, and these results provide a link between the cholesterol-lowering effect of BBR with cholesterol excretion into the bile., Conclusions: We conclude that BBR significantly promoted the excretion of cholesterol from the liver to the bile in hyperlipidemic hamsters, which led to large decreases in the serum TC, TG and LDL-C levels. Additionally, compared with lovastatin, the BBR treatment produced no obvious side effects on the liver function.

Objectives: A randomized, parallel controlled, open-label clinical trial was conducted to evaluate the effect of a botanic compound berberine (BBR) on NAFLD., Methods: A randomized, parallel controlled, open-label clinical trial was conducted in three medical centers (NIH Registration number: NCT00633282). A total of 184 eligible patients with NAFLD were enrolled and randomly received (i) lifestyle intervention (LSI), (ii) LSI plus pioglitazone (PGZ) 15mg qd, and (iii) LSI plus BBR 0.5g tid, respectively, for 16 weeks. Hepatic fat content (HFC), serum glucose and lipid profiles, liver enzymes and serum and urine BBR concentrations were assessed before and after treatment. We also analyzed hepatic BBR content and expression of genes related to glucose and lipid metabolism in an animal model of NAFLD treated with BBR., Results: As compared with LSI, BBR treatment plus LSI resulted in a significant reduction of HFC (52.7% vs 36.4%, p = 0.008), paralleled with better improvement in body weight, HOMA-IR, and serum lipid profiles (all p<0.05). BBR was more effective than PGZ 15mg qd in reducing body weight and improving lipid profile. BBR-related adverse events were mild and mainly occurred in digestive system. Serum and urine BBR concentrations were 6.99ng/ml and 79.2ng/ml, respectively, in the BBR-treated subjects. Animal experiments showed that BBR located favorably in the liver and altered hepatic metabolism-related gene expression., Conclusion: BBR ameliorates NAFLD and related metabolic disorders. The therapeutic effect of BBR on NAFLD may involve a direct regulation of hepatic lipid metabolism., Trial Registration: ClinicalTrials.gov NCT00633282.

The gut microbiota is important in the pathogenesis of energy-metabolism related diseases. We focused on the interaction between intestinal bacteria and orally administered chemical drugs. Oral administration of berberine (BBR) effectively treats patients with metabolic disorders. However, because BBR exhibits poor solubility, its absorption mechanism remains unknown. Here, we show that the gut microbiota converts BBR into its absorbable form of dihydroberberine (dhBBR), which has an intestinal absorption rate 5-fold that of BBR in animals. The reduction of BBR to dhBBR was performed by nitroreductases of the gut microbiota. DhBBR was unstable in solution and reverted to BBR in intestine tissues via oxidization. Heat inactivation of intestinal homogenate did not inhibit dhBBR oxidization, suggesting the process a non-enzymatic reaction. The diminution of intestinal bacteria via orally treating KK-Ay mice with antibiotics decreased the BBR-to-dhBBR conversion and blood BBR; accordingly, the lipid- and glucose-lowering efficacy of BBR was reduced. Conclusively, the gut microbiota reduces BBR into its absorbable form of dhBBR, which then oxidizes back to BBR after absorption in intestine tissues and enters the blood. Thus, interaction(s) between the gut microbiota and orally administrated drugs may modify the structure and function of chemicals and be important in drug investigation.

Our previous studies showed that berberine (BBR) increases liver low-density lipoprotein (LDL) receptor expression in an extracellular signal-regulated kinase (ERK)-dependent manner. This study was designed to explore the upstream cellular signaling molecules recruited by BBR to activate the ERK mitogen-activated protein kinase (MAPK) cascade. Chemical inhibitors such as GW5074, manumycin A, and compound C or specific small interfering RNAs (siRNAs) were used in the blocking experiments; Western blot was used to determine the phosphorylation of kinases; real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression level of LDL receptor mRNA. Our results indicate that BBR increases p-Raf-1 (ser338) level time and dose dependently in HL-7702 cells, but has no influence on Ras activity; the stimulating activities of BBR on Raf-1 signaling and LDL receptor expression can be blocked by GW5074 completely, but not by manumycin A, a Ras inhibitor. BBR activates hepatic Raf-1 signaling and up-regulates LDL receptor expression in a rat model of hyperlipidemia with no impact on liver Ras activity. Importantly, our results show that the stimulating activities of BBR on hepatic Raf-1 signaling and LDL receptor expression are totally blocked by compound C, a selective inhibitor of AMP-activated protein kinase (AMPK), and also by silencing its expression with siRNA. Taken together, our results demonstrate for the first time that BBR up-regulates LDL receptor expression through Ras-independent, but AMPK-dependent Raf-1 activation in liver cells. Our study will help to elucidate the molecular pharmacology of BBR and provide new scientific evidence for its clinical application.

Berberine (BBR) has been confirmed to show extensive bioactivities for the treatments of diabetes and hypercholesterolemia in clinic. However, there are few pharmacokinetic studies to elucidate the excretions of BBR and its metabolites. Our research studied the excretions of BBR and its metabolites in rats after oral administration (200 mg/kg). Metabolites in bile, urine, and feces were detected by liquid chromatography coupled to ion trap time-of-flight mass spectrometry; meanwhile, a validated liquid chromatography coupled with tandem mass spectrometry method was developed for their quantifications. Sixteen metabolites, including 10 Phase I and six Phase II metabolites were identified and clarified after dosing in vivo. Total recovered rate of BBR was 22.83% (19.07% of prototype and 3.76% of its metabolites) with 9.2 × 10(-6) % in bile (24 h), 0.0939% in urine (48 h), and 22.74% in feces (48 h), respectively. 83% of BBR was excreted as thalifendine (M1) from bile, whereas thalifendine (M1) and berberrubine (M2) were the major metabolites occupying 78% of urine excretion. Most of BBR and its metabolites were found in feces containing 84% of prototype. In summary, we provided excretion profiles of BBR and its metabolites after oral administration in rats in vivo., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR's plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR's level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR's metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC (0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR's pharmacological effects on human diseases in clinic.

Objective: Cellular efflux transporters, especially P-glycoprotein (P-gp), impel berberine (BBR) out of cells, and therefore reduce bioavailability of the compound. This study was designed to overcome efflux of BBR using P-gp as a target., Materials/methods: Molecular docking study was done to identify BBR analogues that were with low affinity to P-gp. Flow cytometry was used to determine cellular efflux of chemicals. Pharmacokinetic study was performed in Wistar rats, following oral administration of the study compounds. The efficacies of chemicals on glucose homeostasis were determined both in cultured cells and diabetic KK-Ay and db/db mice., Results: In the molecular docking study, we found that among BBR analogues pseudo-berberine (IMB-Y53) has low affinity to P-gp. IMB-Y53 was retained in Caco-2, HL-7702 and C2C12 cells for a significantly longer period of time than BBR did. P-gp inhibitor tetrandrine (Tet) abolished the efflux of BBR at different extent depending on the expression level of P-gp; however, Tet had no impact on IMB-Y53 efflux. BBR increased P-gp expression dose-dependently in intestinal and liver cells; IMB-Y53 also up-regulated P-gp but at a much lower level as compared with BBR. Administered at equal dose in rats, the maximum plasma concentration (C(max)) and area under concentration-time curve (AUC(0-24)) of IMB-Y53 were 1.61 and 2.27-fold of those of BBR, respectively, indicating an improved bioavailability. IMB-Y53 stimulated glucose utility in cultured cells with a degree similar to that of BBR, but exhibited enhanced glucose-lowering efficacy in KK-Ay and db/db diabetic mice., Conclusions: These results suggest that overcoming cellular efflux especially P-gp's function improves bioavailability and hypoglycemic effect of BBR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study was designed to improve the absorption and hypoglycemic efficacy of berberine (BBR), which is a substrate of P-glycoprotein (P-gp), by combination with a P-gp inhibitor tetrandrine (Tet). Flow cytometry and LC-MS/MS were used to determine the cellular efflux or retention of chemicals. Pharmacokinetic study was performed in ICR mice following oral administration of the study compounds. The hypoglycemic efficacies of the compounds were evaluated in diabetic KK-Ay mice. In the in vitro experiments, Tet significantly inhibited the efflux and increased the uptake of P-gp substrates rhodamine-123 as well as BBR in MCF7/DOX cells and Caco-2 intestinal cells. Meanwhile, Tet greatly reduced the expression of P-gp in Caco-2 cells. The inhibition of BBR efflux by Tet was translated into improved pharmacokinetics in vivo. When co-administered, Tet dose-dependently increased the average maximum concentration (C(max)) and area under concentration-time curve (AUC₀₋₂₄) of BBR in mice. Tet itself had no impact on glucose metabolism. However, it greatly potentiated the hypoglycemic efficacy of BBR in diabetic KK-Ay mice. In addition, we found that Tet had moderate inhibitory effect on the catalytic activity of CYP3A4, which played a role in the bio-transformation of BBR, and this may also take part in the improvement of the pharmacokinetics of BBR. In summary, combination with P-gp inhibitors such as Tet can improve the pharmacokinetics and hypoglycemic efficacy of BBR greatly; this implicates a feasible strategy for exploring the therapeutic effects of BBR and other pharmaceuticals which are substrates of P-gp.

Currently there is no approved medicine for the treatment of metabolic syndrome. A series of new derivatives of berberine (BBR) or pseudoberberine (1) was synthesized and evaluated for their activity on AMP-activated protein kinase (AMPK) activation and up-regulatory low-density-lipoprotein receptor (LDLR) gene expression, respectively. In addition, the four major metabolites of BBR in vivo were also examined for their activity on AMPK in order to further understand the chemical mechanisms responsible for its glucose-lowering efficacy. Among those BBR analogues, compound 1 exhibited the potential effect on AMPK activation and LDLR up-regulation as compared with BBR. The results suggested that compound 1 might be a multiple-target agent for the treatment of metabolic syndrome, and thus was selected as a promising drug candidate for further development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Twenty-eight new 13-n-octylberberine derivatives were synthesized and evaluated for their activities against drug-susceptible Mycobacterium tuberculosis (M. tuberculosis) strain H(37)Rv. Among these compounds, compound 16e was the most effective anti-tubercular agent with a MIC value of 0.125 μg/mL. Importantly, compound 16e exhibited more potent effect against rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains than both RIF and INH, suggesting a new mechanism of action. Therefore, it has been selected as a drug candidate for further investigation, or as a chemical probe for identifying protein target and studying tuberculosis biology. We consider 13-n-octylberberine analogs to be a promising novel class of antituberculars against multi-drug-resistant (MDR) strains of M. tuberculosis., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Berberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways., Methods: BBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay., Results: In rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR., Conclusions: Four major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.

Scavenger receptor CD36 could bind and endocytose oxLDL into macrophages which were then differentiated into foam cells that constitute the atherosclerotic lesion core, and was considered to be a potential target to treat atherosclerosis. In the establishment of the compound library of berberine (BBR, 1) analogues, we discovered that 13-hexylberberine (2) showed an antagonistic activity against CD36. Taking 2 as the lead compound, 21 derivatives were synthesized and their antagonistic activities were evaluated via an ELISA-like high-throughput screening (HTS) model. The primary structure-activity relationships were studied. It was indicated that the introduction of suitable groups at the 2- and 3-position of the aromatic ring A or at the 9-position of the aromatic ring D could enhance the activity. Among the 21 studied compounds, 7g bearing a benzyloxyl group at the 9-position provided a highest CD36 antagonistic activity with the IC50 value of 7.7 micromol L(-1). Besides, its antagonistic activity was further verified with Sf9 insect cell HTS model. So berberine analogues are a new family of CD36 receptor antagonists and worthy to be studied further.

In order to enhance oral bioavailability of berberine (BBR) for its cholesterol-lowering efficacy in vivo, a series of ester or ether prodrugs of berberrubine (M1), which is an active metabolite of BBR after first-pass metabolism, were designed, semi-synthesized, and evaluated. Among these M1 prodrugs, compound 5g possessing palmitate at the 9-position showed a moderate LogP value and esterase hydrolysis rate for releasing M1 in blood. Its cholesterol-lowering efficacy in vivo was evaluated in hyperlipidemic SD rats. Compound 5g (100mg/kg/d) reduced blood CHO and LDL-c by 35.8% and 45.5%, respectively, similar to that by BBR. It also exhibited a good safety in rats with no side-effect on liver and kidney function. Therefore, the design of M1 prodrug appears to be an effective strategy to improve pharmacokinetic feature of BBR for its lipid-lowering efficacy in vivo., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Our previous work demonstrated that berberine (BBR) increases insulin receptor (InsR) expression and improves glucose utility both in vitro and in animal models. Here, we study the InsR-up-regulating and glucose-lowering activities of BBR in humans. Our results showed that BBR increased InsR messenger RNA and protein expression in a variety of human cell lines, including CEM, HCT-116, SW1990, HT1080, 293T, and hepatitis B virus-transfected human liver cells. Accordingly, insulin-stimulated phosphorylations of InsR beta-subunit and Akt were increased after BBR treatment in cultured cells. In the clinical study, BBR significantly lowered fasting blood glucose (FBG), hemoglobin A(1c), triglyceride, and insulin levels in patients with type 2 diabetes mellitus (T2DM). The FBG- and hemoglobin A(1c)-lowering efficacies of BBR were similar to those of metformin and rosiglitazone. In the BBR-treated patients, the percentages of peripheral blood lymphocytes that express InsR were significantly elevated after therapy. Berberine also lowered FBG effectively in chronic hepatitis B and hepatitis C patients with T2DM or impaired fasting glucose. Liver function was improved greatly in these patients by showing reduction of liver enzymes. Our results confirmed the activity of BBR on InsR in humans and its relationship with the glucose-lowering effect. Together with our previous report, we strongly suggest BBR as an ideal medicine for T2DM with a mechanism different from metformin and rosiglitazone., (Copyright 2010 Elsevier Inc. All rights reserved.)

Berberine (BBR) is a natural compound with up-regulating activity on both low-density-lipoprotein receptor (LDLR) and insulin receptor (InsR). This one-drug-multiple-target characteristic might be suitable for the treatment of metabolic syndrome. In searching for up-regulators effective for both LDLR and InsR expression, the structure-activity relationship (SAR) analysis for BBR analogues was done. Fourteen BBR analogues were designed, synthesized and biologically evaluated. SAR analysis revealed that appropriate modifications on the phenyl ring A or D of BBR might retain the up-regulatory activities on the expression of both LDLR and InsR. Among these compounds, compound 13a bearing 9-methoxy and 10-hydroxyl on the ring D showed promising activities on either LDLR or InsR gene expression. The 10-hydroxyl of 13a could be an arm to connect proper chemical groups for optimizing drug-bioavailability in vivo. Thus, 13a could be considered to be a parent compound to make pro-drugs for either blood lipids or glucose.

Twenty-nine derivatives of berberine (1) or pseudoberberine (2) were designed, semisynthesized, and evaluated for their up-regulatory activity on the low-density-lipoprotein receptor (LDLR) expression. SAR analysis revealed that (i) the methylenedioxy group at the 2- and 3-position is an essential element to keep the activity, (ii) the 7-position quaternary ammonium and planar structure of the compound are activity-required, and (iii) addition of electron-donating groups at the 7- or 13-position reduced the activity. Of the compound 1 analogues, compound 2 exhibited an increased activity on LDLR expression compared to 1. In the hyperlipidemic rats, compound 2 (100 (mg/kg)/day) reduced blood CHO and LDL-c by 42.6% and 49.4%, respectively, more efficient than 1 did (p < 0.01 for both). The results were confirmed in the hyperlipidemic mice. LD(50) of 2 in mice was over 5000 mg/kg (oral). We consider compound 2 a promising cholesterol-lowering drug candidate.

Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

Berberine (BBR, 1) is a novel cholesterol-lowering agent that up-regulates low-density-lipoprotein receptor (LDLR) expression through a mechanism different from that of statins. Because of the unique mode of action and good safety record, BBR provoked our interest to do structure modification at different domains for its cholesterol-lowering activity. Nineteen BBR analogues with substituents on the benzene ring D were synthesized in the present study. The analysis of structure-activity relationship (SAR) indicated that the two methoxyl groups in an ortho-distribution on this benzene ring afforded a good activity. Among the 19 analogues, compound 8j bearing a methoxyl at both 10- and 11-position showed an increased LDLR up-regulatory activity in respect to BBR, and therefore has been selected as a promising cholesterol-lowering drug candidate for further evaluation.

We have identified berberine (BBR) as a novel cholesterol-lowering drug acting through stabilization of the low-density lipoprotein receptor (LDLR) messenger RNA. Because the mechanism differs from that of statins, it is of great interest to examine the lipid-lowering activity of BBR in combination with statins. Our results showed that combination of BBR with simvastatin (SIMVA) increased the LDLR gene expression to a level significantly higher than that in monotherapies. In the treatment of food-induced hyperlipidemic rats, combination of BBR (90 mg/[kg d], oral) with SIMVA (6 mg/[kg d], oral) reduced serum LDL cholesterol by 46.2%, which was more effective than that of the SIMVA (28.3%) or BBR (26.8%) monotherapy (P < .01 for both) and similar to that of SIMVA at 12 mg/(kg d) (43.4%). More effective reduction of serum triglyceride was also achieved with the combination as compared with either monotherapy. Combination of BBR with SIMVA up-regulated the LDLR messenger RNA in rat livers to a level about 1.6-fold higher than the monotherapies did. Significant reduction of liver fat storage and improved liver histology were found after the combination therapy. The therapeutic efficacy of the combination was then evaluated in 63 hypercholesterolemic patients. As compared with monotherapies, the combination showed an improved lipid-lowering effect with 31.8% reduction of serum LDL cholesterol (P < .05 vs BBR alone, P < .01 vs SIMVA alone). Similar efficacies were observed in the reduction of total cholesterol as well as triglyceride in the patients. Our results display the rationale, effectiveness, and safety of the combination therapy for hyperlipidemia using BBR and SIMVA. It could be a new regimen for hypercholesterolemia.

Objective: Our recent studies identified berberine (BBR) as a novel cholesterol-lowering drug that upregulates low-density lipoprotein (LDL) receptor expression through mRNA stabilization. Here, we investigated mechanisms underlying regulatory effects of BBR on LDL receptor (LDLR) messenger., Methods and Results: We show that the extracellular signal-regulated kinase (ERK) signaling pathway is used primarily by BBR to attenuate the decay of LDLR mRNA in HepG2 cells. Using different reporter constructs, we demonstrate that BBR affects LDLR mRNA stability entirely through 3' untranslated region (UTR) in an ERK-dependent manner, and this stabilizing effect is more prominent in liver-derived cells than nonhepatic cell lines. In contrast to BBR, the mRNA stabilizing effect of bile acid chenodeoxycholic acid is mediated through the LDLR coding sequence, whereas the 5'UTR, 3'UTR, and the coding sequence of LDLR mRNA are all implicated in the action of phorbol 12-myristate 13-acetate. By performing UV cross-linking and SDS-PAGE, we identify 2 cytoplasmic proteins of 52 and 42 kDa that specifically bind to the LDLR 3'UTR in BBR-inducible and ERK-dependent manners., Conclusions: These new findings demonstrate that the BBR-induced stabilization of LDLR mRNA is mediated by the ERK signaling pathway through interactions of cis-regulatory sequences of 3'UTR and mRNA binding proteins that are downstream effectors of this signaling cascade.

We identify berberine (BBR), a compound isolated from a Chinese herb, as a new cholesterol-lowering drug. Oral administration of BBR in 32 hypercholesterolemic patients for 3 months reduced serum cholesterol by 29%, triglycerides by 35% and LDL-cholesterol by 25%. Treatment of hyperlipidemic hamsters with BBR reduced serum cholesterol by 40% and LDL-cholesterol by 42%, with a 3.5-fold increase in hepatic LDLR mRNA and a 2.6-fold increase in hepatic LDLR protein. Using human hepatoma cells, we show that BBR upregulates LDLR expression independent of sterol regulatory element binding proteins, but dependent on ERK activation. BBR elevates LDLR expression through a post-transcriptional mechanism that stabilizes the mRNA. Using a heterologous system with luciferase as a reporter, we further identify the 5' proximal section of the LDLR mRNA 3' untranslated region responsible for the regulatory effect of BBR. These findings show BBR as a new hypolipidemic drug with a mechanism of action different from that of statin drugs.

Nonalcoholic fatty liver disease (NAFLD), ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), is a liver disease worldwide without approved therapeutic drugs. Anti-inflammatory and hepatoprotective drug bicyclol and multi-pharmacological active drug berberine, respectively, have shown beneficial effects on NAFLD in murine nutritional models and patients, though the therapeutic mechanisms remain to be illustrated. Here, we investigated the combined effects of bicyclol and berberine on mouse steatosis induced by Western diet (WD), and NASH induced by WD/CCl4. The combined use of these was rather safe and better reduced the levels of transaminase in serum and triglycerides and cholesterol in the liver than their respective monotherapy, accompanied with more significantly attenuating hepatic inflammation, steatosis, and ballooning in mice with steatosis and NASH. The combined therapy also significantly inhibited fibrogenesis, characterized by the decreased hepatic collagen deposition and fibrotic surface. As per mechanism, bicyclol enhanced lipolysis and β-oxidation through restoring the p62-Nrf2-CES2 signaling axis and p62-Nrf2-PPARα signaling axis, respectively, while berberine suppressed de novo lipogenesis through downregulating the expression of acetyl-CoA carboxylase and fatty acid synthetase, along with enrichment of lipid metabolism-related Bacteroidaceae (family) and Bacteroides (genus). Of note, the combined use of bicyclol and berberine did not influence each other but enhanced the overall therapeutic role in the amelioration of NAFLD. Conclusion : Combined use of bicyclol and berberine might be a new available strategy to treat NAFLD. [ABSTRACT FROM AUTHOR]

Berberine (BBR) is an isoquinoline alkaloid extracted from Coptis chinensis that improves diabetes, hyperlipidemia and inflammation. Due to the low oral bioavailability of BBR, its mechanism of action is closely related to the gut microbiota. This study focused on the CYP51 enzyme of intestinal bacteria to elucidate a new mechanism of BBR transformation by demethylation in the gut microbiota through multiple analytical techniques. First, the docking of BBR and CYP51 was performed; then, the pharmacokinetics of BBR was determined in ICR mice in vivo, and the metabolism of BBR in the liver, kidney, gut microbiota and single bacterial strains was examined in vitro. Moreover, 16S rRNA analysis of ICR mouse feces indicated the relationship between BBR and the gut microbiota. Finally, recombinant E. coli containing cyp51 gene was constructed and the CYP51 enzyme lysate was induced to express. The metabolic characteristics of BBR were analyzed in the CYP51 enzyme lysate system. The results showed that CYP51 in the gut microbiota could bind stably with BBR, and the addition of voriconazole (a specific inhibitor of CYP51) slowed down the metabolism of BBR, which prevented the production of the demethylated metabolites thalifendine and berberrubine. This study demonstrated that CYP51 promoted the demethylation of BBR and enhanced its intestinal absorption, providing a new method for studying the metabolic transformation mechanism of isoquinoline alkaloids in vivo. [Display omitted] • The demethylation metabolism of natural drugs difficult to absorb through the gut microbiota was first reported. • Six different methods were presented to explain the metabolic mechanism of natural isoquinoline alkaloids. • The findings provided a new idea for studying the mechanism of drug metabolism of gut microbiota. [ABSTRACT FROM AUTHOR]

Epilepsy is a neuronal dysfunction syndrome characterized by transient and diffusely abnormal discharges of neurons in the brain. Previous studies have shown that mutations in the syntaxin 1b (stx1b) gene cause a familial, fever-associated epilepsy syndrome. It is unclear as to whether the stx1b gene also correlates with other stimulations such as flashing and/or mediates the effects of antiepileptic drugs. In this study, we found that the expression of stx1b was present mainly in the brain and was negatively correlated with seizures in a pentylenetetrazole (PTZ)-induced seizure zebrafish model. The transcription of stx1b was inhibited by PTZ but rescued by valproate, a broad-spectrum epilepsy treatment drug. In the PTZ–seizure zebrafish model, stx1b knockdown aggravated larvae hyperexcitatory swimming and prompted abnormal trajectory movements, particularly under lighting stimulation; at the same time, the expression levels of the neuronal activity marker gene c-fos increased significantly in the brain. In contrast, stx1b overexpression attenuated seizures and decreased c-fos expression levels following PTZ-induced seizures in larvae. Thus, we speculated that a deficiency of stx1b gene expression may be related with the onset occurrence of clinical seizures, particularly photosensitive seizures. In addition, we found that berberine (BBR) reduced larvae hyperexcitatory locomotion and abnormal movement trajectory in a concentration-dependent manner, slowed down excessive photosensitive seizure-like swimming, and assisted in the recovery of the expression levels of STX1B. Under the downregulation of STX1B, BBR's roles were limited: specifically, it only slightly regulated the levels of the two genes stx1b and c-fos and the hyperexcitatory motion of zebrafish in dark conditions and had no effect on the overexcited swimming behavior seen in conjunction with lighting stimulation. These findings further demonstrate that STX1B protein levels are negatively correlated with a seizure and can decrease the sensitivity of the photosensitive response in a PTZ-induced seizure zebrafish larvae; furthermore, STX1B may partially mediate the anticonvulsant effect of BBR. Additional investigation regarding the relationship between STX1B, BBR, and seizures could provide new cues for the development of novel anticonvulsant drugs. [ABSTRACT FROM AUTHOR]

Berberine (BBR) is considered a multi-target drug that has significant advantages. In contrast to its significant pharmacological effects in clinic, the plasma level of BBR is very low. Our previous work revealed that dihydroberberine (dhBBR) could be an absorbable form of BBR in the intestine, and butyrate is an active metabolite that is generated by gut bacteria in rats. In this study, for the first time we describe gut microbiota-regulated pharmacokinetics in beagle dogs after oral administration of BBR by single (50 mg/kg) or multiple doses (50 mg/kg/d) for 7 days. GC-MS, GC, LC-MS/MS, and LC/MSn-IT-TOF were used to detect dhBBR, butyrate and BBR as well as its Phase I and II metabolites, respectively. The results showed that dhBBR was not detected in dog plasma but was excreted in small amounts in the feces of dogs examined on days 3 and 7. Butyrate was generated by gut bacteria and increased by 1.3- and 1.2-fold in plasma or feces, respectively, after 7 days of BBR treatment compared to the levels before treatment. Changes of intestinal bacterial composition were analyzed by 16S rRNA genes analysis. The results presented that dogs treated with BBR for 7 days increased both the abundance of the butyrate- and the nitroreductases- producing bacteria. We also identified chemical structures of the Phase I and II metabolites and analyzed their contents in beagle dogs. Eleven metabolites were detected in plasma and feces after BBR oral administration (50 mg/kg) to dogs, including 8 metabolites of Phase I and III metabolites of Phase II. The pharmacokinetic profile indicated that the concentration of BBR in plasma was low, with a Cmax value of 36.88 ± 23.45 ng/mL. The relative content of glucuronic acid conjugates (M11) was higher than those of other metabolites (M1, M2, M12, and M14) in plasma. BBR was detected in feces, with high excreted amounts on day 3 (2625.04 ± 1726.94µg/g) and day 7 (2793.43 ± 488.10µg/g). In summary, this is the first study to describe gut microbiota-regulated pharmacokinetics in beagle dogs after oral administration of BBR, which is beneficial for discovery of drugs with poor absorption but good therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Gut dysbiosis has been found to be involved in the pathogenesis of energy metabolic disorders and might be a new strategy for these ailments. Berberine (BBR), a botanical medicine, shows therapeutic efficacy in patients with metabolic diseases. Numerous reports have shown BBR's modulating effect on gut microbiota, opening a new avenue to understand BBR's mechanism. In this study, a colon‐specific delivery system, BBR‐CS/PT‐NP, is investigated by the assembly of pH/gut microflora dual stimuli‐responsive nanoparticles for enhancing the interaction between BBR and gut microbiota. After oral administration, the delivering system remains stable in the stomach and small intestine, followed by a burst release of BBR after reaching the colon segment rich in intestinal bacteria. The enzymes produced by bacteria degrade the nanoparticle, causing direct exposure of BBR to gut microbiota. In the high fat diet‐induced obese hamsters, BBR‐CS/PT‐NP intervention inhibits weight‐gain and fat deposition, decreases plasma lipids and glucose levels, improves inflammation condition and insulin resistance, alleviates hepatic steatosis, at a level significantly higher than the pure BBR does. The mechanisms might be attributable to the enhanced interaction between BBR and the gut flora. The results provide a novel proof‐of‐concept for drug delivery targeting gut microbiota to ameliorate metabolic disorders. Gut dysbiosis is involved in the pathogenesis of energy metabolic disorders and represents a new strategy for treating these ailments. In this study, a pH/gut microflora dual stimuli‐responsive system, berberine (BBR)‐CS/PT‐NP with colon‐homing and microbiota‐targeting characteristics, is investigated to enhance the interaction between BBR and the gut microbiota. It provides a novel proof‐of‐concept for drug delivery targeting gut microbiota to ameliorate metabolic diseases. [ABSTRACT FROM AUTHOR]

Abstract: A series of new 1,13-cycloprotoberberine derivatives defined through variations at the 9-position were designed, synthesized and evaluated for their cytotoxicities in human HepG2 (hepatoma), HT1080 (fibrosarcoma) and HCT116 (colon cancer) cells. The preliminary structure−activity relationship (SAR) revealed that the replacement of 9-methoxyl with an ester moiety might significantly enhance the antiproliferative activity in vitro. Notably, compound 7f demonstrated equipotent cytotoxicity activity against breast cancer MCF-7 (parent) and doxorubicin (DOX)-resistant MCF-7 (MCF-7/ADrR) cells, indicating a mode of action different from that of DOX. Further mechanism study showed that 7f significantly inhibited activity of DNA topoisomerase I (Top I) and Top II. G2/M phase arrest and tumor cell growth reduction was observed thereafter. Thus, we consider cycloprotoberberine analogues to be a new family of promising antitumor agents with an advantage of inhibiting drug-resistant cancer cells. [Copyright &y& Elsevier]

Abstract: Our previous studies proved that berberine (BBR) up-regulates the insulin receptor (InsR) gene by stimulating its promoter and calphostin C blocks this effect. Here, the present study was designed to discover the specific kinase isoform(s) used by berberine. In the blocking experiment, we found that Gö6976, a kinase inhibitor that potently inhibit PKCμ/protein kinase D 1 (PKD1), effectively and specifically reduced the activity of BBR on InsR. PKD1/PKCμ is a member of the PKD family that also covers PKD2 and PKD3/PKCν with high homology. The role of PKD1 in InsR expression was also proved by using another PKD-activator oligomycin. In the RNA interference experiment, we found that the effects of BBR on InsR expression and on cellular glucose consumption were partially eliminated by silencing any one of the three PKDs and were totally abolished by silencing all of them. BBR enhanced the PKD1 catalytic activity, but not its expression. Along with BBR treatment, PKD1 ser916 autophosphorylation was increased time- and dose-dependently, indicating an activation of PKD1 by BBR. BBR also induces PKD1 translocation from cytosol-to-plasma membrane, further verifying the activation of PKD1. These results suggest that the PKD family is involved in the transcriptional regulation of the InsR gene; we consider it to be a potential new target to discover drugs for sugar-related disorders in the future. [ABSTRACT FROM AUTHOR]