Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min−1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h−1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6β-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

Phlorizin is the most abundant glucoside of phloretin from the apple tree and its products. Phlorizin and its aglycone phloretin are currently considered health-beneficial polyphenols from apples useful in treating hyperglycemia and obesity. Recently, we showed that phloretin could be regioselectively hydroxylated to make 3-OH phloretin by Bacillus megaterium CYP102A1 and human P450 enzymes. The 3-OH phloretin has a potent inhibitory effect on differentiating 3T3-L1 preadipocytes into adipocytes and lipid accumulation. The glucoside of 3-OH phloretin would be a promising agent with increased bioavailability and water solubility compared with its aglycone. However, procedures to make 3-OH phlorizin, a glucoside of 3-OH phloretin, using chemical methods, are not currently available. Here, a biocatalytic strategy for the efficient synthesis of a possibly valuable hydroxylated product, 3-OH phlorizin, was developed via CYP102A1-catalyzed regioselective hydroxylation. The production of 3-OH phlorizin by CYP102A1 was confirmed by HPLC and LC–MS spectroscopy in addition to enzymatic removal of its glucose moiety for comparison to 3-OH phloretin. Taken together, in this study, we found a panel of mutants from B. megaterium CYP102A1 could catalyze regioselective hydroxylation of phlorizin to produce 3-OH phlorizin, a catechol product.

Oleanolic acid (OA) is a pentacyclic triterpenoid widely found in plants and foods as an aglycone of triterpenoid saponins or as a free acid. OA exhibits beneficial activities for humans, including antitumor, antivirus, and hepatoprotection properties without apparent toxicity. The metabolites produced by the cytochrome P450 (P450) enzymes are critical for the evaluation of the efficacy and safety of drugs. In this study, the potential metabolites of OA were investigated by P450-catalyzed oxidation reactions. Among the various tested human P450s, only human CYP3A4 was active for the hydroxylation of OA. The major metabolite was characterized by a set of analyses using HPLC, LC–MS, and NMR. It was found to be 4-epi-hederagenenin, a chiral product, by regioselective hydroxylation of the methyl group at the C-23 position. These results indicated that CYP3A4 can hydroxylate an OA substrate to make 4-epi-hederagenenin. Possible drug–food interactions are discussed.

Phloretin and its glycoside phlorizin have been reported to prevent obesity induced by high-fat diet (HFD), but the effect of 3-OH phloretin, a catechol metabolite of phloretin, has not been investigated. In this study, we investigated the anti-obesity effects of phloretin and 3-OH phloretin in HFD-fed mice. The body weight gain induced by HFD was more inhibited by administration of 3-OH phloretin than by phloretin. The increases in fat mass, white adipose tissue (WAT) weight, adipocyte size, and lipid accumulation by HFD were also remarkably inhibited by 3-OH phloretin and, to a lesser extent, by phloretin. The HFD-induced upregulation of chemokines and pro-inflammatory cytokines was suppressed by 3-OH phloretin, preventing M1 macrophages from infiltrating into WAT and thereby reducing WAT inflammation. 3-OH phloretin also showed a more potent effect than phloretin on suppressing the expression of adipogenesis regulator genes, such as PPARγ2, C/EBPα, FAS, and CD36. Fasting blood glucose and insulin levels increased by HFD were diminished by the administration of 3-OH phloretin, suggesting that 3-OH phloretin may alleviate obesity-induced insulin resistance. These findings suggested that 3-OH phloretin has the potential to be a natural bioactive compound that can be used in the prevention or treatment of obesity and insulin resistance.

Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), which is the rate-limiting enzyme in cholesterol biosynthesis. Statin therapy reduces morbidity and mortality in those who are at high risk of cardiovascular disease. Monacolin J is a statin compound, which is an intermediate in the lovastatin biosynthesis pathway, in the fungus Aspergillus terreus. It is also found in red yeast rice, which is made by culturing rice with the yeast Monascus purpureus. Monacolin J has a hydroxyl substituent at position C’-8 of monacolin L. Here, a new statin derivative from monacolin J was made through the catalysis of CYP102A1 from Bacillus megaterium. A set of CYP102A1 mutants of monacolin J hydroxylation with high catalytic activity was screened. The major hydroxylated product was C-6’a-hydroxymethyl monacolin J, whose structure was confirmed using LC–MS and NMR analysis. The C-6’a-hydroxymethyl monacolin J has never been reported before. It showed a greater ability to inhibit HMG-CoA reductase than the monacolin J substrate itself. Human liver microsomes and human CYP3A4 also showed the ability to catalyze monacolin J in producing the same product of the CYP102A1-catalyzed reaction. This result motivates a new strategy for the development of a lead for the enzymatic and chemical processes to develop statin drug candidates.