Research progress on berberine with a special focus on its oral bioavailability

Fitoterapia Volume 109, March 2016, Pages 274-282 Review Author links open overlay panelChang-ShunLiuYu-RongZhengYing-FengZhangXiao-YingLong Guangdong Pharmaceutical University, Guangzhou, Guangdong Province 510006, China Received 2 November 2015, Revised 26 January 2016, Accepted 1 February 2016, Available online 2 February 2016. crossmark-logo https://doi.org/10.1016/j.fitote.2016.02.001 Get rights and content Abstract The natural product berberine (BBR) has become a potential drug in the treatment of diabetes, hyperlipidemia, and cancer. However, the oral delivery of BBR is challenged by its poor bioavailability. It is necessary to improve the oral bioavailability of BBR before it can be used in many clinical applications. Understanding the pharmacokinetic characteristics of BBR will enable the development of suitable formulas that have improved oral bioavailability. The key considerations for BBR are how to enhance the drug absorption and to avoid the intestinal first-pass effect. This review summarizes the pharmacological activities of BBR and analyzes the factors that lead to its poor oral bioavailability. In particular, the therapeutic potential of BBR in new indications from the aspect of oral bioavailability is discussed. In conclusion, BBR is a promising drug candidate for metabolic disorders and cancer but faces considerable challenges due to its poor oral bioavailability. Graphical abstract Unlabelled figure Download high-res image (211KB)Download full-size image Keywords Berberine Pharmacological activities Oral bioavailability Absorption First-pass effect Pharmacokinetics 1. Introduction Berberine (BBR), a protoberberine alkaloid (Fig. 1), is present in several plant species such as Coptis (Coptis chinensis and Coptis japonica Makino) and Berberis (Berberis vulgaris and Berberis croatica Horvat), which are common in the Eastern hemisphere [1]. Clinically, plants containing BBR have been used for centuries in many prescriptions to treat dysentery [2], diarrhea [3], stomatitis [4], and hepatitis [5] via its antiprotozoal, antimicrobial, and anti-inflammatory properties. BBR has been extensively used as a nonprescription drug to treat diarrhea caused by different sources since the 1950s in China [2]. Chemical structures of berberine (BBR) and its metabolites, berberrubine (M1),… Download high-res image (278KB)Download full-size image Fig. 1. Chemical structures of berberine (BBR) and its metabolites, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3), and jatrorrhizine (M4). In recent years, numerous studies have indicated that BBR may have many positive effects on some major medical pathologies, such as regulation of lipid and glucose metabolism [6,7], suppression of tumor cell proliferation [8], and induction of apoptosis [9]. However, the oral bioavailability of BBR appears to be very poor (below 1%) [10,11], indicating that such medical efficacy may never be obtained by patients taking BBR as a medical treatment. Therefore, the first purpose of this review was to summarize the effect of BBR on diabetes, hyperlipidemia, and cancer as well as to discuss its therapeutic potential for these diseases. In addition, we aimed to elaborate the pharmacokinetic characteristics of BBR and to discuss strategies to improve its oral bioavailability. 2. Pharmacological activities 2.1. Current medical uses 2.1.1. Antidiarrheal activity BBR has been used as a nonprescription drug for diarrhea [12] and has shown a good efficiency in the clinic. A total of 132 patients with diarrhea-predominant irritable bowel syndrome were randomized for treatment with BBR (400 mg/twice daily) or placebo for 8 weeks. The patients treated with BBR had a reduction of diarrhea frequency, abdominal pain frequency, and urgent need for defecation frequency. These results were significantly more pronounced in the BBR group than in the placebo group. Furthermore, BBR was well tolerated [13]. The antidiarrheal property of BBR may be associated with the following mechanisms: (1) BBR inhibits the intestinal secretory response of bacterial enterotoxins. The secretion of water and electrolytes stimulated by cholera toxin and the related toxins of Escherichia coli has been found to be one of the major factors that causes diarrhea, while BBR has been shown to reverse this secretion [14]. (2) BBR regulates intestinal motility. Intestinal motility dysfunction is an important pathological characteristic of diarrhea. In humans, BBR has been shown to reduce intestinal smooth muscle contraction and to delay intestinal transit time [15,16]. (3) BBR restores intestinal barrier function in disease states. In Crohn's disease, epithelial barrier dysfunction leads to leak-flux diarrhea, but the damage of intestinal epithelial tight junctions could be ameliorated by BBR (100 μM) treatment, and such amelioration is related to inhibition of proinflammatory cytokines [17]. 2.1.2. Antimicrobial activity BBR has been found to display broad-spectrum antibacterial activities against Staphylococcus epidermis, E. coli, etc. [18–20]. For example, BBR inhibited oral pathogens in an in vitro tooth model, and the minimum inhibitory concentration (MIC) values against Fusobacterium nucleatum, Prevotella intermedia, and Enterococcus faecalis were 31.25 μg/mL, 3.80 μg/mL, and 500 μg/mL, respectively [21]. In another in vitro study, the antimicrobial effect of BBR was evaluated against 17 microorganisms based on the half maximal inhibitory concentration (IC50), MIC, minimum microbicidal concentration (MMC), and minimum microbistatic concentration (MMS). The results showed that the IC50, MIC, MMC, and MMS values of BBR for the most sensitive Staphylococcus aureus strain were 14.6, 212, 250, and > 250 mg/L, respectively; while those values for Trichoderma viride (original green strain) were 809, 1345, 3000, and > 3000 mg/L, respectively [22]. The values against other microorganisms, including E. coli, Pseudomonas aeruginosa, and Bacillus subtilis, fell in between [22]. The role of BBR as an antibacterial agent may be due to its inhibitory effect on enzymatic and/or endotoxic (e.g., lipopolysaccharide; LPS) activities of bacteria. For example, BBR can inhibit the activity of the bacterial surface protein sortase [23], a transpeptidase that mediates covalent binding between Gram-positive bacterial surface proteins and cell walls [24]. In addition, BBR has been shown to be a high-affinity LPS antagonist and decrease the interaction of LPS with specific receptors on host immune cells; therefore, it can be used to treat LPS-induced diseases [25]. BBR acts topically inside the gastrointestinal (GI) lumen when it is used as an antibacterial agent. Based on its IC50 values (14.6–809 mg/L) for antibacterial activity, the solubility of BBR (2000 mg/L) in aqueous solution at 37 °C fully meets the effective therapeutic levels [26]. 2.2. Potential therapeutic applications 2.2.1. Antidiabetic effect Based on decades of basic and clinical studies, BBR is a strong candidate for the treatment of type 2 diabetes mellitus (T2DM) [6,27,28]. In the study by Yin et al., 36 T2DM patients were randomly treated with BBR or metformin (0.5 g t.i.d.) for 3 months. The hemoglobin A1c (HbA1c) level in the BBR-treated group was significantly decreased (from 9.47% to 7.48%), and this effect was comparable with that of metformin. Moreover, the fasting and postprandial blood glucose levels and plasma triglyceride (TG) levels were decreased by 36%, 44%, and 21%, respectively, which were lower than those of metformin [29]. Similarly, the effects of BBR in T2DM patients reported by Zhang et al. also support the above-mentioned results [30]. The antidiabetic effect of BBR may be associated with regulation of the insulin signaling pathway. BBR can not only reversibly inhibit the expression of insulin, but it can also increase the sensitivity of insulin receptor (InsR) and activate the insulin signaling pathway, thus improving insulin resistance and effectively improving glucose utility. Gene analysis has revealed that BBR causes a reversible concentration-dependent inhibition of the insulin promoter Ins2 in mouse NIT-1 islet cells, which inhibits insulin gene expression, resulting in the reduction of insulin and its mRNA levels [31]. Such inhibition can improve insulin resistance and glucose intolerance, thus protecting islet cells in T2DM patients. Furthermore, BBR (1–15 μg/mL) has been shown to increase InsR mRNA and protein expression levels in a dose- and time-dependent manner in human liver cells. BBR-enhanced InsR expression occurs mainly through protein kinase C-dependent activation of its promoter. Similar results also have been obtained in T2DM rats. Moreover, BBR has been shown to elevate InsR mRNA levels as well as protein kinase C activity in the liver, to lower fasting blood glucose and fasting serum insulin levels, and to increase insulin sensitivity [32]. In addition, BBR has been shown to promote the expression of glucose transporter protein 1 (GLUT1), which increases glucose uptake. Such an increase may partly be due to activation of adenosine monophosphate-activated protein kinase (AMPK). In L929 fibroblast cells, a cell line that only expresses GLUT1, BBR (10–100 μM)-activated glucose uptake increased the maximum stimulation by five-fold. Significant activation was observed within 5 min and reached the maximum by 30 min. These results were partly related to BBR acutely activating the glucose transport activity of GLUT1 [33]. In 3T3-L1 adipocytes, the phosphorylation of AMPK and glucose uptake were significantly increased in the presence of BBR cultivation for 2 h [34]; and after 6 h, the level of GLUT1 expression was distinctly elevated [35]. These results suggest that BBR may activate the AMPK signaling pathway, enhance GLUT1 expression, and then increase glucose uptake. Moreover, BBR-activated AMPK may also suppress mitochondrial function, reduce oxygen consumption, and elevate the adenosine monophosphate/adenosine triphosphate ratio, thus inhibiting gluconeogenesis and improving glycolysis. In diabetic rat liver tissues, BBR treatment downregulated protein expression of the key gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) via the AMPK signaling pathway. Thus, BBR might inhibit gluconeogenesis in the liver and reduce the blood glucose level [36,37]. In H4IIE hepatocytes, BBR (10 μM) increased glucose consumption by 61.6% in an AMPK-independent manner; meanwhile, a rise in lactic acid production was observed, indicating that BBR might improve glucose metabolism through stimulation of glycolysis [38]. Recently, BBR has been reported to exert antidiabetic actions through modulation of gut microbiota. In T2DM rats, BBR was shown to revert the effect on the high-fat diet-induced structural changes of gut microbiota. Sixty out of 134 operational taxonomic units that showed close associations with changes of obese phenotypes were significantly decreased by BBR, while those bacteria that could produce short-chain fatty acids and provide health-relevant functions for the host [39] were markedly increased. Similar results were also obtained by metformin [40]. These results suggest that shifting the gut microbiota structure improves GI health and eventually mediates the antidiabetic effect on the host, which may be shared mechanisms of BBR [41,42] and metformin [43,44]. Currently, BBR seems to be safe in the treatment of T2DM patients. However, in a clinical study of patients treated with BBR, 34.5% of patients suffered from transient GI adverse effects, such as diarrhea, constipation, flatulence, and abdominal pain [29]. Despite the fact that these adverse effects disappeared within a few days upon discontinuation of the drug or reduction of the dose, these adverse effects may have been due to the high oral dose (0.5 g/day) or gut microbe inhibition because of the antidiarrheal activity of BBR. 2.2.2. Antihyperlipidemic action The first study of the use of BBR (15 mg/kg, i.v.) in the treatment of cardiovascular diseases was reported in the 1980s [45]. In this paper, BBR was shown to improve cardiac dysfunction in patients with severe congestive heart failure. Since then, beneficial effects of BBR on cardiovascular diseases, especially hyperlipidemia [46], have been documented. The excessive production of plasma lipids, especially cholesterol, has been strongly associated with an increased risk of developing cardiovascular disease in humans. Therefore, increasing the expression of low-density lipoprotein receptors (LDLRs) regulates human plasma LDL cholesterol (LDL-C) homeostasis, which is an effective method for preventing and treating cardiovascular disease [47,48]. Based on a clinical trial, in vivo animal experiments, and in vitro cellular experiments, BBR has been scientifically proven to be a safe and effective cholesterol-lowering drug. For instance, 32 hypercholesterolemic patients administered orally with BBR (0.5 g, twice a day) for 3 months were found to have serum cholesterol levels reduced by 29%, TG levels reduced by 35%, and LDL-C levels reduced by 25%. The role of BBR in lowering lipid levels is related to downregulation of LDL-C via upregulating LDLR expression. In animal models, BBR (100 mg/kg, twice a day) was given orally to hyperlipidemic hamsters for 10 days, and serum cholesterol and LDL-C levels were reduced by 40% and 42%, respectively [49]. However, in human hepatoma cells, LDLR mRNA and protein levels were increased by 3.5-fold and 2.6-fold, respectively [49]. In fact, BBR may have dual actions on LDLR metabolism. First, BBR can upregulate LDLR expression by a post-transcriptional mechanism that stabilizes its mRNA [49–51]. Second, BBR has been shown to inhibit the expression of proprotein convertase subtilisin/kexin type 9 through ubiquitination and degradation of hepatocyte nuclear factor 1α, which in turn directly increases the protein abundance of LDLR at the post-translational level [52,53]. Additionally, BBR has been shown to reduce blood cholesterol levels by inhibiting cholesterol absorption and promoting its excretion. Treatment with BBR (50–150 mg/kg) in atherogenic rats for 8 weeks reduced plasma total cholesterol (TC) and non-high-density lipoprotein cholesterol (non-HDL-C) levels by 29–33% and 31–41%, respectively. On the contrary, the fractional dietary cholesterol absorption rate was decreased by 40–51% [54], which showed a strong correlation between plasma TC or non-HDL-C levels and cholesterol absorption rates. The mechanism may be related to the decrease of enterocyte cholesterol uptake and secretion [54,55]. In another study, hyperlipidemic hamsters with a large amount of cholesterol accumulation in their livers demonstrated great reductions in the serum TC, TG, and LDL-C levels after treatment with BBR (50 or 100 mg/kg), gradual decreases in the liver cholesterol levels at both doses, and increased bile cholesterol levels at the same time [56]. These data suggest that the promotion of cholesterol excretion from the liver into the bile may be another pathway for the lipid-lowering effect of BBR. Thus, the antihyperlipidemic mechanism of BBR is distinct from those of statins, which inhibit 3-hydroxy-3-methyl glutaryl coenzyme A reductase levels to decrease intracellular cholesterol biosynthesis [57]. Furthermore, compared to simvastatin therapy, which has been shown to maximally reduce LDL-C to 60% [58], the effect of BBR seems to be moderate. Until now, few studies have elaborated the mechanism of lipid lowering by BBR. The above-mentioned pathway involved in the regulation of cholesterol metabolism seems feasible for BBR treatment, but the high drug pharmacological dose (7.5 μg/mL) used in in vitro settings [49] may not translate to any meaningful effect in vivo. It is speculated that the antihyperlipidemic mechanism of BBR may be involved in other cell pathways; therefore, further investigation is necessary. 2.2.3. Anticancer property The role of BBR in cancer treatment has been reported in numerous studies. BBR may exert anticancer effects at various stages of cancer development including proliferation, growth, and metastasis. BBR seems to be more active in inhibiting tumor cell proliferation through upregulation of reactive oxygen species production, while it shows minor cytotoxicity to normal cells. In one study, BBR (10–40 μM) significantly inhibited human liver cancer cell line HepG2 proliferation in a dose-dependent manner. In addition, the cell viability was reduced by approximately 40% after BBR (40 μM) treatment. Simultaneously, reactive oxygen species generation was dose-dependently increased compared with non-BBR-treated cells. In contrast, no marked cytotoxic effects were observed in normal liver cells under the same conditions [59]. Similar results were demonstrated in mouse colon tumor cells. BBR markedly inhibited tumor cell proliferation with an IC50 of less than 50 μM, whereas it had little or no effect on primary cultured normal colon epithelial cells [60]. Moreover, BBR induces apoptosis to suppress tumor growth. In He′s report, treatment of human cholangiocarcinoma cells (QBC939) with BBR (10, 40, or 80 μM) for 48 h resulted in cell death of 21%, 38%, and 58%, respectively (P < 0.05). BBR-inhibited growth was associated with an increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL [61]. Additionally, BBR suppresses tumor metastasis via inhibiting transferases including urokinase-type plasminogen activator and matrix metalloproteinases. There is increasing evidence that these two transferases play an important role in tumor metastasis and angiogenesis. In vitro, BBR (62.5–125 μM) greatly inhibited the migration and invasion of human SCC-4 tongue squamous cancer cells with an inhibition ratio of 42–58%, and the mechanism was through downregulation of urokinase-type plasminogen activator as well as matrix metalloproteinase-2 and -9 expression [62]. Although BBR shows some effects as an anticancer agent, significant biological effects in vitro were only seen at concentrations of greater than 10 μM, which are thousands of times higher than those normally attainable following oral ingestion of BBR (4.0 nM) in humans [63]. Therefore, further in vivo studies are required to determine whether BBR could be an effective chemotherapeutic agent for the treatment of cancer. 3. Basic pharmacokinetic characteristics 3.1. Pharmacokinetics in animals BBR can be absorbed from the GI tract. For example, the maximum concentration (Cm) of BBR in plasma was 4 ng/mL after the oral administration of 100 mg/kg BBR in rats [64]. In another study, the Cm of BBR was 16.74 ng/mL at 15 min after the oral administration of 25 mg/kg BBR in rats, and the plasma concentration decreased rapidly within 12 h, but the very low plasma concentration was maintained for 36 h [65]. Recently, the absorption mechanism of BBR was elaborated by a conversion–absorption–reversion process that took place entirely in the intestinal environment. Gut microbiota could convert BBR into its absorbable form of dihydroberberine, which has an intestinal absorption rate five-fold higher than that of BBR in rats. This conversion was performed by nitroreductases of the gut microbiota. After entering intestinal wall tissues, dihydroberberine is reverted immediately to BBR through oxidization [66]. Although BBR was extensively distributed to the liver, kidneys, muscle, lungs, brain, heart, pancreas, and fat (in descending order) following the oral administration of 200 mg/kg BBR, it was predominantly distributed in the liver. The total area under the curve (AUC) of BBR in the above organs was 1355.5 h·ng/mL, but the AUC in the liver was 728.6 h·ng/mL, which was greater than that in the blood (86.37 h·ng/mL) [67]. Similar results were also obtained in another investigation [10]. The distribution of BBR in the liver may provide evidence for its cholesterol-, TG-, and glucose-lowering activities. In rats, BBR is metabolized in the liver by cytochrome P450 isoforms (CYPs) through phase I oxidative demethylation followed by phase II glucuronidation. Four phase I metabolites (berberrubine (M1), thalifendine (M2), demethyleneberberine (M3), and jatrorrhizine (M4), Fig. 1) and their respective glucuronide conjugates were found in most tissues, and M1 was the main metabolite in plasma [63,64,68]. In the liver, the AUC value of the phase I metabolites was 2103.5 h·ng/mL, which accounted for approximately three times that of BBR and about 90% of the total metabolites [67]. CYP2D6, CYP1A2, and CYP3A4 seemed to be the dominant CYPs transforming BBR into its main metabolites [68]. Some of these metabolites like M1 and M2 remained active on BBR's targets (InsR, LDLR, and AMPK) in the liver but with a reduced potency [69,70]. In rats, BBR is mainly excreted by the hepatobiliary system and kidneys in the form of metabolites [67]. Eleven BBR metabolites were observed in mouse urine and feces, and most of these metabolites were demethylated products [64,68,69]. Tan et al. have reported that unchanged BBR in the urine accounted for only 0.036% of the oral dose given in rats [71]. Chen and Chang supported that only 4.93% and 0.5% of a dose of 2 mg/kg BBR were eliminated from the urine and bile after an i.v. bolus administration [72]. The conjugates were hydrolyzed to free metabolites for reabsorption via the enterohepatic circulation, while conjugate conversion to the free form was restricted in the pseudo germ-free rats treated with antibiotics [70,73]. 3.2. Pharmacokinetics in humans There are relatively few studies of the pharmacokinetics of BBR in humans. In one study, 20 volunteers were treated orally with 400 mg of BBR, and the mean Cm and AUC values were 0.4 ng/mL and 9.2 h·ng/mL, respectively, [74]. Recently, the oral pharmacokinetics of BBR in humans was studied systematically. After a single oral dose of 500 mg in 10 volunteers, the Cm values of BBR and the two metabolites M2 and M4 were 0.07 ± 0.01, 0.14 ± 0.01, and 0.13 ± 0.02 nM, respectively. For BBR and M2, a plateau was reached at 1 h after BBR administration, but the plateau was delayed to 2 h for M4. These plateaus persisted for up to 24 h after ingestion. In contrast, the Cm of M1 was almost 10 times higher (1.4 ± 0.3 nM at 4 h) after the administration of BBR, with a slow decrease until the residual concentration of 0.15 ± 0.02 nM was reached after 24 h. After chronic administration (15 mg/kg) for 3 months in 12 subjects, the plasma levels of BBR and its metabolites were considerably higher than those after acute administration. The maximum steady-state concentrations were 4.0 ± 2.0, 6.7 ± 3.0, 1.7 ± 0.3, and 5.6 ± 2.0 nM for BBR, M1, M2, and M4, respectively. This finding can be explained by the fact that the daily dose in the chronic administration study was almost twice that of the single dose used in the acute administration study. A possible bioaccumulation during chronic administration could also be responsible for these values. A high intersubject variability in plasma levels was observed in the chronic administration study compared to the acute single-dose pharmacokinetic study. Once again, M1 was the main compound present in the plasma [63]. These results indicate that the pharmacokinetic characteristics of BBR in humans are consistent with those observed in animal studies. According to the pharmacokinetics of BBR in humans, the drug plasma level (4.0 nM) after chronic administration may reach the effective therapeutic concentration of hypoglycemic action (≥ 2.69 nM for insulin resistance improvement [32]), but the danger of adverse GI effects induced by a high dose (0.9 g/d) should be taken into consideration. 4. Barriers of oral bioavailability Oral bioavailability can be affected by many factors, such as the drug physicochemical properties (e.g., solubility, permeability, and stability in the GI tract), dosage form (e.g., drug dispersion degree and dissolution) as well as physiological factors (e.g., efflux and presystemic metabolism in the gut wall and liver). It has been reported that the absolute bioavailability of BBR after oral administration in rats is below 1% (0.36% in Ref. [10] and 0.68% in Ref. [11]). The low oral bioavailability of BBR may be due to its poor absorption (56%) and the first-pass effect in the intestine (43.5%) as well as in the liver (0.14%) [10]. Furthermore, the poor absorption of BBR may be attributable to self-aggregation, poor permeability, P-glycoprotein (P-gp)-mediated efflux, and hepatobiliary re-excretion (Fig. 2) [10,73]. The fate of BBR in vivo after oral administration Download high-res image (298KB)Download full-size image Fig. 2. The fate of BBR in vivo after oral administration. Totally 56% of drug was not absorbed in GI tract due to the self-aggregation (A), poor permeability (B), P-gp-mediated efflux (C) and hepatobiliary re-excretion (D), and the 43.5% was metabolized in the intestine. Only 0.5% of total given dose entered the portal vein and 28.2% of this fraction (0.14%) was metabolized in the liver. Thus, the absolute bioavailability of BBR is merely 0.36%. Firstly, drug self-aggregation can decrease the solubility of BBR in the GI tract, limiting its oral absorption. Under physiological conditions, BBR is mostly in the ionized form, so it easily self-aggregates in the acidic environment of the stomach and upper small intestine [63]. This phenomenon can be explained by in vitro solubility studies. In pH gradient tests, the pH-dependent solubility of BBR was shown in aqueous solution at 37 °C. The maximum solubility (9.69 ± 0.37 mM) in phosphate buffer (pH 7.0) was pH-dependently decreased, and the solubility at pH 1.2 (HCl) was nearly 20-fold lower than that at pH 7.0 [26,63]. Secondly, drugs are generally considered to be well absorbed in rats when the effective permeability coefficient (Peff) is greater than 0.2 × 10− 4 cm/s [75]; however, the Peff of BBR (0.178 × 10− 4 cm/s) across the rat intestinal mucous membrane confirmed its low permeability [10]. Moreover, the octanol–water partition coefficient of BBR is approximately − 1.5, and its aqueous solubility is about 2 mg/mL [26,63]. According to the biopharmaceutical classification system (BCS), BBR can be classified as a class III drug [11,76], which means that it has a high solubility and low permeability. Thirdly, P-gp is expressed in the apical membrane of the epithelial layer of the gut wall, where it can actively transport certain compounds in the blood-to-lumen direction and limit their transport in the absorptive direction. In addition, BBR has been confirmed to be a P-gp substrate [77,78]. In a Caco-2 cell monolayer model, the P-gp inhibitor cyclosporine A (CsA) decreased the efflux rate of BBR from 24.28 to 0.79 [79]. Similarly, in in vitro Ussing-type chambers, the rate of BBR from serosal-to-mucosal (BL–AP) transport across the rat ileum was three times greater than that in the reverse direction (AP–BL); however, this transport was significantly decreased by CsA. Moreover, in an in situ recirculating perfusion model, compared to the BBR control (2.5%), the amounts of BBR that crossed the ileum were increased to 14.8% and 17.2%, respectively, in the presence of the P-gp inhibitors CsA and verapamil [80]. Finally, due to the existence of the hepatoenteral circulation process, the re-excretion of BBR might also decrease its oral absorption. Although only about 7.8 × 10− 5% of BBR was excreted through the bile (24 h) [81] and considering the very small amount of the oral dose (0.5%) that entered the portal vein, the amount of excretion, especially the re-excretion, in the hepatobiliary system should not be overlooked [10,73]. After the injection of radioiodinated BBR, two peaks on the concentration–time curve of the gallbladder were found; the first peak (Cm of 0.10% ID/g) at 15 min was significantly lower than the second peak (0.71% ID/g) at 2 h. Furthermore, the Cm (6.23% ID/g) of BBR in the small intestine exactly coincided between the two peaks [82]. Moreover, this in vivo behavior and the pharmacokinetics were similar to those of orally administered BBR [81]. On the other hand, the mechanism of the first-pass effect of BBR in the small intestine is unknown and awaits further investigation. Among the influencing reasons (e.g., degradation due to pH, intestinal fluids [83], enzymes [84], and intestinal flora [70]), it is supposed that the intestinal metabolism of BBR may mainly be affected by the enzymes. Because BBR is metabolized by CYP2D6 and CYP3A4 in the liver after oral absorption [68], the intestinal metabolism of BBR might also be associated with these two enzymes. Like in the liver, CYPs are the main enzymes for phase I oxidative metabolism of drugs in the intestine, and 80% of drugs are metabolized by CYPs [85]. The liver CYP content is about 430 pmol/mg, and CYP3A and CYP2D6 account for 40% and 2%, respectively. The CYP content in the small intestine is 22–180 pmol/mg (mainly as CYP3A4) and CYP2D6 is 0.7%. Although the CYP content in the intestine is much lower than that in the liver, many studies have found that the drug extraction rate in the small intestine is close to that of the liver, and even exceeds that of the liver [86,87]. 5. Approaches to improve the oral bioavailability As mentioned previously, the potential therapeutic uses of BBR are restricted by its low oral bioavailability, mainly due to its poor absorption and intestinal first-pass effect, indicating that bioavailability enhancement seems to be an effective method to solve the problem. However, possibly due to the unclear mechanism of intestinal metabolism of BBR, there are no reports describing bioavailability improvement by reducing the intestinal first-pass metabolism. Nevertheless, considering the feasibility to enhance the oral absorption of BBR, some studies have explored the use of permeation enhancers [88], P-gp inhibitors [11], and microparticle delivery systems [89]. 5.1. Improvements in permeation with enhancers The use of intestinal absorption promoters is one approach that increases the oral bioavailability of drugs with a low permeability, and a large number of well-known substances have been shown to alter intestinal permeability including spices, peptide-based promoters, surfactants, and polymers [90]. Sodium caprate is an anionic surfactant that has been shown to be a safe and effective permeability enhancer [91]. In Lv's papers, the oral administration of BBR at 100 mg/kg with the addition of 50 mg/kg sodium caprate resulted in an increase of the AUC of BBR by 28%, compared to administration without sodium caprate. Moreover, the area under the glucose curve was decreased by 22.5%, suggesting that its antidiabetic effect was enhanced by improving the uptake of BBR [92,93]. In addition, Meng et al. have developed an amorphous solid dispersion of BBR with sodium caprate that is referred to as the Huang-Gui Solid Dispersion (HGSD) preparation. In rats, the AUC of HGSD is 3.81-fold greater than that of commercial BBR tablets. After HGSD (100 mg/kg) treatment, the fasting blood glucose level (4.97 ± 0.13 mM) was decreased to almost the same level as controls (4.21 ± 0.28 mM), thus showing a superior hypoglycemic effect compared to pure BBR (100 mg/kg; 7.84 ± 0.52 mM), BBR tablets (100 mg/kg; 7.96 ± 0.53 mM), and metformin (300 mg/kg; 7.54 ± 0.36 mM) [94]. It has been reported previously that sodium caprate enhances the membrane permeation of BBR by enlarging the tight junctions in the intestinal epithelium [91]. Recently, another mechanism proposes that the permeation of sodium caprate with BBR is related to the formation of BBR–caprate salts, resulting in increased lipophilicity of BBR [88]. In Fan's paper, the AUC of BBR (100 mg/kg) was incredibly increased by 41.1-fold after the addition of sodium caprate (100 mg/kg), compared to BBR alone. Another absorption enhancer, sodium deoxycholate, also significantly increased the AUC of BBR by 35.3-fold by the same mechanism. Generally, the oral administration of anionic surfactants is considered unsafe, but co-administration of sodium caprate caused no specific damage to the intact intestine, according to intestinal histological examination [88,92]. Indeed, Maher et al. have provided numerous evidence that sodium caprate results in only transient, reversible, mild, and rapid damage to the intestinal epithelium. Moreover, sodium caprate has been approved by the FDA as a direct food additive for human consumption and is used as an excipient in human medical preparations in Sweden (Doktacillin®) and Japan (Doktacillin®) [95]. In contrast to anionic surfactants, chitosan is a cationic polysaccharide from the shells of shrimps and crabs that displays a dose-dependent enhancement of BBR absorption. In rats, the AUC values of BBR were increased by 1.9-, 2.2-, and 2.5-fold when chitosan was present in formulations at 0.5%, 1.5%, and 3.0%, respectively [76]. Chitosan exerts absorption enhancing activity for BBR in two ways. Firstly, chitosan can interact with the anionic components of the glycoproteins on the epithelial cell surface, regulate tight junctions, and then enhance the drug paracellular permeability [96,97]. Secondly, chitosan, a high molecular weight polymer, can exert mucoadhesive properties and increase drug retention at the mucosal surface [98,99]. 5.2. Improvements in permeation via P-gp inhibitors As mentioned above, the efflux of P-gp is a major absorption barrier for many drugs; therefore, the use of P-gp inhibitors is a common strategy to prevent it. However, clinical trials studying the concomitant use of first-generation (e.g., CsA, quinindium) or second-generation (e.g., dexverapamil, PSC 833) P-gp inhibitors have been disappointing [100,101]. For example, CsA suppresses the body immune system and causes medical complications [102]. Therefore, the ideal P-gp inhibitor should be more effective and have a high safety profile [100]. In this sense, some pharmaceutical excipients have been proposed, such as surfactants (e.g., pluronic derivatives and PEGylated vitamin E derivatives), solubilizing agents (e.g., cyclodextrins), and cosolvents (e.g., polyethylene glycols) [100,103,104]. These excipients offer a number of advantages, including their well-known use in the formulation of parenteral and enteral medicines, safety, and regulatory acceptance [104]. Moreover, some natural compounds with P-gp inhibitory function, like silymarin [105] and tetrandrine (Tet) [106,107], may also be good candidates for BBR. The compound d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble form of vitamin E that is comprised of a hydrophilic polar head and a lipophilic alkyl tail with amphiphilic properties, a relatively low critical micelle concentration of 0.02 wt.%, and a hydrophile–lipophile balance value of 13.2 [108]. It effectively inhibits the efflux of several P-gp substrates, such as CsA, doxorubicin, and paclitaxel [11,109–113]. Using the combination of TPGS and the phospholipid complex of BBR, the relative oral bioavailability of BBR was 322.66% of that of BBR alone in rats as P-gp excretion was inhibited and the liposolubility of BBR was improved [114]. Compared to the BBR control, the oral administration of BBR containing 2.5% TPGS showed Cm and AUC values of BBR in rats that were increased by approximately 1.9- and 2.90-fold, respectively (P < 0.05), and these increases did not damage the epithelia or the intact villi structures [11]. Silymarin, an extract from Silybum marianum, has been used as a P-gp inhibitor in many clinical studies of BBR conducted in patients with metabolic disorders, and the clinical efficacy of the fixed combination of BBR with silymarin on lipid- and glucose-lowering demonstrated good results [115–118]. For example, 69 T2DM patients received either a standardized extract of Berberis aristata alone (1 g/day of BBR) or plus S. marianum (Berberol®, 1 g/day of BBR and 0.21 g/day of silymarin). Both treatments similarly decreased the fasting glucose, TC, LDL-C, TG, and liver enzyme levels; whereas the HbA1c values were significantly reduced by 12.35% in the Berberol® group (P < 0.05 vs. the B. aristata alone group; 7.18%) [119]. The better efficacy of the fixed combination may be related to the increase of BBR absorption by silymarin, which inhibited the P-gp-mediated excretion. Tet is another natural product that potentiates the hypoglycemic efficacy of BBR due to its P-gp inhibition. In vitro, Tet significantly inhibited the efflux (from 74.6% to 46.5%) and increased the uptake of BBR in Caco-2 intestinal cells. Interestingly, this efflux inhibition was translated into an improved bioavailability in vivo. When BBR was administered orally alone (100 mg/kg) or plus Tet (10, 20 mg/kg) in mice, the Cm value (increased by 0.36–0.62-fold) and AUC0–24 h value (increased by 0.33–0.61-fold) of BBR were significantly enhanced in a Tet dose-dependent manner. Furthermore, these improved pharmacokinetics by Tet (10 mg/kg/d) significantly decreased the fasting blood glucose level by 55.2% in diabetic KK-Ay mice, compared to BBR alone (20.6%), thus demonstrating the greatly enhanced hypoglycemic efficacy of BBR with the addition of Tet [120]. In addition, chemical modification is another method to overcome the efflux of BBR by using P-gp as a target. In Shan's study, the BBR analog pseudoberberine (IMB-Y53) was synthesized and shown to have a low affinity to P-gp [121–123]. In Caco-2 cells, IMB-Y53 was retained for a significantly longer period of time than BBR and was not affected by the P-gp inhibitor Tet. Administered at an equal dose in rats (200 mg/kg), the Cm and AUC values of IMB-Y53 were 1.61- and 2.27-fold of those of BBR, respectively, indicating an improved bioavailability. Importantly, IMB-Y53 also could stimulate glucose utility in cultured cells with a degree similar to that of BBR. Moreover, compared to BBR, which decreased blood glucose levels by 19.9% and 22.1% in KK-Ay (100 mg/kg) and db/db (300 mg/kg) diabetic mice, respectively, IMB-Y53 exhibited a higher glucose-lowering efficacy of 35.7% and 39.7%, respectively, when administered at equal doses [121]. 5.3. Improvements in absorption with lipid microparticle delivery systems Lipid microparticle drug delivery systems (LMDDSs) such as nano/microemulsions, micelles, liposomes, and solid lipid nanoparticles (SLNs) are the most popular and prospective strategies used in pharmaceutical technology. The unique properties of LMDDSs, which contain physiochemical diversity, biocompatibility, and ability to enhance oral bioavailability, have made them attractive carriers for oral drug delivery systems [124]. LMDDSs increase oral absorption through multiple mechanisms such as improving drug solubility and permeability in the GI tract, increasing endocytosis of encapsulated drug across the intestinal epithelia in intact microparticles, and enhancing drug transportation from the GI tract to the lymphatic system. The self-microemulsifying drug delivery system (SMEDDS), which is an isotropic mixture of oils, surfactants, cosurfactants, and drug, can disperse in the GI lumen to form microemulsions upon dilution with water or GI fluids. After the oral administration of BBR–SMEDDS or BBR commercial tablets at dose of 25 mg/kg, the Cm (236.6 ± 7.9 ng/mL) and AUC (709.6 ± 42.3 min·ng/mL) values of BBR–SMEDDS in rats were higher than those of the commercial tablet by 163.4% and 154.2%, respectively, and the relative bioavailability was 242% [89]. The enhancement of absorption might be related to the improved properties, such as the increased solubility and dissolution rate of BBR in the GI tract, as well as spontaneously forming the small emulsion droplet, reducing the interfacial surface tension, and enhancing the penetration of BBR to the epithelial cells. SLNs are derived from physiologically compatible lipids (e.g., lecithin and TG) and represent a safe and effective alternative in comparison to conventional nanoparticles [125]. Pharmacokinetic studies in rats have shown that BBR–SLNs increased the Cm and AUC values of BBR in rats up to 44.65 μg/L and 113.57 h·μg/L, respectively, compared to 11.06 μg/L and 56.48 h·μg/L, respectively, for BBR alone at a dose of 50 mg/kg. This absorption promotion effect directly enhanced the antidiabetic effect of BBR in db/db diabetic mice by improving the insulin sensibility, promoting islet function, and protecting against islet regeneration [126]. Moreover, an anhydrous reverse micelle (ARM) delivery system was also found to enhance the oral bioavailability and antidiabetic efficacy of BBR. An in vivo study in diabetic mice administered BBR (100 mg/kg via gavage) showed that the average blood glucose level decreased by 57% in the ARM group and that there was no blood glucose level reduction in the BBR solution group. The AUC and Cm values of the ARM group were 2.4-fold and 2.1-fold high than those of the BBR solution group, respectively [127]. In brief, improving the oral bioavailability of BBR seems to enhance its hypoglycemic effect. However, this effect is dose-dependent and a low dose (25 mg/kg) of BBR did not significantly improve the antidiabetic efficacy. Moreover, the safety of pharmaceutical excipients is another important issue that needs to be considered. 6. Prospects Despite the fact that BBR has been shown to be safe in the majority of human subjects studied in the short-term and chronically, it is mainly used as an antidiarrheal agent and only needs to act topically inside the GI lumen. In other words, in clinical practice, the long-term safety is based on little or no absorption of BBR. However, some transient GI adverse effects have been observed after a high-dose administration of BBR for the treatment of T2MD patients. Therefore, the safety of BBR should be prudently considered when it is extensively absorbed and chronically used. Improving the oral bioavailability of BBR could effectively enhance the antidiabetic activity of this agent. However, altering the epithelial transport properties of the intestine as a whole in order to enhance the uptake of BBR is likely to cause some problems in people, not the least of which would be an impaired ability of the gut immune system to recognize toxins from nutrients. Indeed, considering the therapeutic potential of BBR in new indications and the great compliance of oral delivery, a better approach is needed to improve the oral bioavailability, enhance the pharmacological effects, and reduce the oral dose of BBR, thus minimizing the adverse effects. Although current studies have attempted to enhance the hypoglycemic activity through increasing absorption, the extent of bioavailability enhancement has been limited and the oral dose is still high. It is expected that the oral bioavailability of BBR could be dramatically improved when absorption is increased and first-pass metabolism is avoided simultaneously. For example, the use of BBR with suitable excipients that have both the role of permeability improvement and P-gp efflux inhibition could lead to the development of some new formulas that can wrap BBR to avoid interference with the gut microbes and reduce the intestinal first-pass effect, which would be better solutions to improve the oral bioavailability of BBR. By enhancing the bioavailability via intactly absorbing the wrapped BBR, the adverse GI effects would be markedly lightened or disappear when the effective oral dose is decreased to the minimum. In conclusion, BBR is a highly promising drug candidate to treat diabetes, hyperlipidemia, and cancer. However, this drug faces considerable challenges to improve its oral bioavailability. Acknowledgments This work was supported by the National Natural Science Foundation of China (81573353) and the Medjaden Academy & Research Foundation for Young Scientists (Grant No. MJA20160107). References [1] I. Kosalec, B. Gregurek, D. Kremer, M. 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