Computational Study Into the Oxidative Ring-Closure Mechanism During the Biosynthesis of Deoxypodophyllotoxin.

The nonheme iron dioxygenase deoxypodophyllotoxin synthase performs an oxidative ring-closure reaction as part of natural product synthesis in plants. How the enzyme enables the oxidative ring-closure reaction of (-)-yatein and avoids substrate hydroxylation remains unknown. To gain insight into the reaction mechanism and understand the details of the pathways leading to products and by-products we performed a comprehensive computational study. The work shows that substrate is bound tightly into the substrate binding pocket with the C7'-H bond closest to the iron(IV)-oxo species. The reaction proceeds through a radical mechanism starting with hydrogen atom abstraction from the C7'-H position followed by ring-closure and a final hydrogen transfer to form iron(II)-water and deoxypodophyllotoxin. Alternative mechanisms including substrate hydroxylation and an electron transfer pathway were explored but found to be higher in energy. The mechanism is guided by electrostatic perturbations of charged residues in the second-coordination sphere that prevent alternative pathways.

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Structures of L-proline trans-hydroxylase reveal the catalytic specificity and provide deeper insight into AKG-dependent hydroxylation.

L-Proline hydroxylase is a member of the non-heme Fe2+/α-ketoglutarate (AKG)-dependent hydroxylase family that catalyzes the reaction from L-proline to hydroxy-L-proline, which is widely used in drug synthesis, biochemistry, food supplementation and cosmetic industries. Here, the first crystal structure of L-proline trans-hydroxylase and its complexes with substrate and product are reported, which reveal the structural basis of trans-cis proline hydroxylation selectivity. Structure comparison with other AKG-dependent hydroxylases identifies conserved amino acid residues, which may serve as signatures of in-line or off-line AKG binding modes in the AKG-dependent enzyme family.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Potential herb-drug interaction risk of thymoquinone and phenytoin.

Thymoquinone is a main bioactive compound of Nigella sativa L. (N.sativa), which has been used for clinical studies in the treatment of seizures due to its beneficial neuroprotective activity and antiepileptic effects. It has been evidenced that thymoquinone may inhibit the activity of cytochrome P450 2C9 (CYP2C9). However, little is known about the effect of thymoquinone or N.sativa on the pharmacokinetic behavior of phenytoin, a second-line drug widely used in the management of status epilepticus. In this study, we systematically investigated the risk of the potential pharmacokinetic drug interaction between thymoquinone and phenytoin. The inhibitory effect of thymoquinone on phenytoin hydroxylation activity by CYP2C9 was determined using UPLC-MS/MS by measuring the formation rates for p-hydroxyphenytoin (p-HPPH). The potential for drug-interaction between thymoquinone and phenytoin was quantitatively predicted by using in vitro-in vivo extrapolation (IVIVE). Our data demonstrated that thymoquinone displayed effective inhibition against phenytoin hydroxylation activity. Enzyme kinetic studies showed that thymoquinone exerted a competitive inhibition against phenytoin hydroxylation with a Ki value of 4.45 ± 0.51 µM. The quantitative prediction from IVIVE suggested that the co-administration of thymoquinone (>18 mg/day) or thymoquinone-containing herbs (N.sativa > 1 g/day or N.sativa oil >1 g/day) might result in a clinically significant herb-drug interactions. Additional caution should be taken when thymoquinone or thymoquinone-containing herbs are co-administered with phenytoin, which may induce unexpected potential herb-drug interactions via the inhibition of CYP2C9.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Analysis of Fatty Acid Esters of Hydroxyl Fatty Acid in Nut Oils and Other Plant Oils.

Oils and lipids are common food components and efficient sources of energy. Both the quantity and the quality of oils and lipids are important with regard to health and disease. Fatty acid ester of hydroxy fatty acid (FAHFA) is a novel lipid class that was discovered as an endogenous lipid; FAHFAs have shown anti-diabetic effects in a mammalian system. We analyzed the overall FAHFA composition in nut oils and other common oils: almond (raw, roasted), walnut, peanut, olive, palm, soybean, and rapeseed oils. We developed a method of liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC-ESI/MS/MS) for a comprehensive target analysis of FAHFAs. The analysis revealed wide variation in the FAHFA profiles (15 compounds and 62 peaks). For 7-11 compounds of FAHFA, a total level of 8-29 pmol/mg oil was detected in nuts oils; for 11 compounds, 4.9 pmol/mg oil was detected in olive oil, and for 4-9 compounds, < 2 pmol/mg oil was detected in palm, soy, and rapeseed oils. The major FAHFAs were FAHFA 36:3, FAHFA 36:2, and FAHFA 36:4 in nut oil, FAHFA 36:2, FAHFA 34:1, and FAHFA 36:1 in olive oil, and FAHFA 32:1, FAHFA 34:0, FAHFA 36:0, and FAHFA 36:1 in all of the common oils. The composition of FAHFAs in nut oils is mainly unsaturated fatty acids, whereas those in olive oil are unsaturated fatty acids and saturated fatty acids. The composition of FAHFAs in common oils was mainly saturated fats. This is the first report to demonstrate the quality and quantity of the FAHFAs in the nut oils. Nuts have been described to be a great source of many nutrients and to be beneficial for our health. Our present findings comprise additional evidence that the intake of nuts in daily diets may prevent metabolic and inflammatory-based diseases.

Rubber Seed Oil-Based UV-Curable Polyurethane Acrylate Resins for Digital Light Processing (DLP) 3D Printing.

Novel UV-curable polyurethane acrylate (PUA) resins were developed from rubber seed oil (RSO). Firstly, hydroxylated rubber seed oil (HRSO) was prepared via an alcoholysis reaction of RSO with glycerol, and then HRSO was reacted with isophorone diisocyanate (IPDI) and hydroxyethyl acrylate (HEA) to produce the RSO-based PUA (RSO-PUA) oligomer. FT-IR and 1H NMR spectra collectively revealed that the obtained RSO-PUA was successfully synthesized, and the calculated C=C functionality of oligomer was 2.27 per fatty acid. Subsequently, a series of UV-curable resins were prepared and their ultimate properties, as well as UV-curing kinetics, were investigated. Notably, the UV-cured materials with 40% trimethylolpropane triacrylate (TMPTA) displayed a tensile strength of 11.7 MPa, an adhesion of 2 grade, a pencil hardness of 3H, a flexibility of 2 mm, and a glass transition temperature up to 109.4 °C. Finally, the optimal resin was used for digital light processing (DLP) 3D printing. The critical exposure energy of RSO-PUA (15.20 mJ/cm2) was lower than a commercial resin. In general, this work offered a simple method to prepare woody plant oil-based high-performance PUA resins that could be applied in the 3D printing industry.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Comparative profiling and natural variation of polymethoxylated flavones in various citrus germplasms.

Citrus fruits are the main dietary source of polymethoxylated flavones (PMFs) with significant effects on consumer health. In this study, eleven main PMFs were evaluated in the fruit flavedo or leaves of 116 citrus accessions via UPLC-DAD-ESI-QTOF-MS/MS combined with HPLC-DAD analysis, which revealed significant species-specific and spatiotemporal characteristics. All Citrus reticulata and their natural or artificial hybrids were found to have detectable PMFs, especially in the fruit flavedo of the wild or early-cultivated mandarins at early fruit development stages. However, PMFs were not detected in citrons, pummelos, kumquats, trifoliata oranges, papedas, Chinese box oranges and 'Mangshanyegan'. The results enlightened that PMF accumulation only in mandarins and mandarin hybrids is a phenotype inherited from mandarin ancestors. This study provides a comprehensive PMF profile in various citrus germplasms and will benefit future functional citrus breeding practices aimed at designing plants rich in total or specific PMFs for health benefits.

[Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin].

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

CYP82AR Subfamily Proteins Catalyze C-1' Hydroxylations of Deoxyshikonin in the Biosynthesis of Shikonin and Alkannin.

Shikonin and S-enantiomer alkannin are important naphthoquinone derivatives present in many Boraginaceae species. We report that cytochrome P450 monooxygenases (CYPs) from a new CYP82AR subfamily catalyzed hydroxylations of deoxyshikonin at C-1' position of isoprenoid side chain. Two homologues were discovered from each species of the four Boraginaceae plants. One CYP preferred converting deoxyshikonin into shikonin, and the other stereoselectively hydroxylated deoxyshikonin into alkannin. The discovery might be a general feature of shikonin/alkannin-producing Boraginaceae plants.

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

Pectin-Based Formulations for Controlled Release of an Ellagic Acid Salt with High Solubility Profile in Physiological Media.

Among bioactive phytochemicals, ellagic acid (EA) is one of the most controversial because its high antioxidant and cancer-preventing effects are strongly inhibited by low gastrointestinal absorption and rapid excretion. Strategies toward an increase of solubility in water and bioavailability, while preserving its structural integrity and warranting its controlled release at the physiological targets, are therefore largely pursued. In this work, EA lysine salt at 1:4 molar ratio (EALYS), exhibiting a more than 400 times increase of water solubility with respect to literature reports, was incorporated at 10% in low methoxylated (LM) and high methoxylated (HM) pectin films. The release of EA in PBS at pH 7.4 from both film preparations was comparable and reached 15% of the loaded compound over 2 h. Under simulated gastric conditions, release of EA from HM and LM pectin films was minimal at gastric pH, whereas higher concentrations-up to 300 µM, corresponding to ca. 50% of the overall content-were obtained in the case of the HM pectin film after 2 h incubation at the slightly alkaline pH of small intestine environment, with the enzyme and bile salt components enhancing the release. EALYS pectin films showed a good prebiotic activity as evaluated by determination of short chain fatty acids (SCFAs) levels following microbial fermentation, with a low but significant increase of the effects produced by the pectins themselves. Overall, these results highlight pectin films loaded with EALYS salt as a promising formulation to improve administration and controlled release of the compound.

Enzymatic hydroxylation of L-pipecolic acid by L-proline cis-4-hydroxylases and isomers separation.

OBJECTIVES: Establish a complete and efficient method for the preparation of cis-5-hydroxy-L-pipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. RESULTS: For non-heme Fe(II)/α-KG-dependent dioxygenases, α-ketoglutarate (α-KG) has great influence on the stability of Fe(II) ions, which is also the basic of the hydroxylation reaction to the substrate. L-pipecolic acids (L-Pip) was converted to cis-5HPA by whole-cell catalysis in water, which can reduce the loss of Fe(II) ions. 120 mM L-Pip can be transformed to 93% via cell and Fe(II) ions continuous supplementation under the reaction system optimization (the molar ratio of ascorbic acid/FeSO4·7H2O and α-KG/L-Pip were 8:1 and 1:1, respectively). After the catalytic reaction, the amino protection strategy was adopted to improve the resolution of isomer products on silica gel chromatography, and the amino protected cis-5HPA was obtained with a yield of 86.7%. CONCLUSIONS: We established a method which is promising to be used for cis-5HPA largescale preparation. It also provides a suitable reference for this type of enzyme-catalyzed reaction and the hydroxy pipecolic acid isomers separation.

Dietary isoflavone-induced, estrogen receptor-ß-mediated proliferation of Caco-2 cells is modulated by gallic acid.

Dietary isoflavones and their biotransformation products (from food fermentation) are estrogen mimics which activate estrogen receptors (ER)α and ERß. In silico molecular modelling is used to determine theoretical binding energies of genistein, daidzein and hydroxylated biotransformation products, and to investigate structure-binding energy relationships with ERß. Results suggest that ligand hydroxyl arrangement determines binding energy and influences binding affinity. Caco-2 cells (ERß expressing) are used to study the proliferative effect of genistein, daidzein and their hydroxylated biotransformation products. Isoflavones/biotransformation products showed weaker enhancement of Caco-2 proliferation than 17ß-estradiol. The EC50s of isoflavones/biotransformation products agreed with in silico-predicted binding affinity order. Hydroxylated biotransformation products studied showed greater Caco-2 proliferative effects than the parent isoflavones except 8-hydroxygenistein, probably due to unfavourable ERß interactions caused by 8-hydroxygenistein's extra hydroxyl. Caco-2 pre-treatment with UDP-glucose dehydrogenase inhibitor gallic acid promoted genistein/8-hydroxygenistein-mediated proliferation. This is probably due to a reduced isoflavone glucuronidation to form low estrogenicity glucuronides. Findings are discussed in the context of dietary isoflavones/gallic acid and effects on proliferation of ERß-expressing gut cancer cells.

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4ß-hydroxycholesterol (4ßOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4ßOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4ßOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism.

BACKGROUND: Styrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions. PURPOSE: This study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered. STUDY DESIGN: The inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated. METHODS: The inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis. RESULTS: In vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6ß-hydroxylation. CONCLUSION: Styrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.

PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line.

Polyhydroxylated naphthoquinones (PHNQs), known as spinochromes that can be extracted from sea urchins, are bioactive compounds reported to have medicinal properties and antioxidant activity. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that pure echinochrome A exhibited a cytotoxic effect on Saos-2 cells in a dose-dependent manner within the test concentration range (15.625-65.5 µg/mL). The PHNQ extract from New Zealand sea urchin Evechinus chloroticus did not induce any cytotoxicity within the same concentration range after 21 days of incubation. Adding calcium chloride (CaCl2) with echinochrome A increased the number of viable cells, but when CaCl2 was added with the PHNQs, cell viability decreased. The effect of PHNQs extracted on mineralized nodule formation in Saos-2 cells was investigated using xylenol orange and von Kossa staining methods. Echinochrome A decreased the mineralized nodule formation significantly (p < 0.05), while nodule formation was not affected in the PHNQ treatment group. A significant (p < 0.05) increase in mineralization was observed in the presence of PHNQs (62.5 µg/mL) supplemented with 1.5 mM CaCl2. In conclusion, the results indicate that PHNQs have the potential to improve the formation of bone mineral phase in vitro, and future research in an animal model is warranted.

Characterization of hydrocoptisonine metabolites in human liver microsomes using a high-resolution quadrupole-orbitrap mass spectrometer.

Hydrocoptisonine is a new compound that has been isolated from the rhizomes of Coptis chinensis, which belongs to the Ranunculaceae family of Chinese medicines. Although studies on C. chinensis have been reported, the metabolic pathway of hydrocoptisonine in human liver microsomes (HLMs) remains unelucidated. We identified 13 metabolites in HLMs, including six Phase I metabolites and seven glucuronide conjugates, using a high-resolution quadrupole-orbitrap mass spectrometer. The major metabolic pathway was the O-demethylation and mono-hydroxylation of hydrocoptisonine in HLMs. Notably, M3 metabolite was O-demethylated in dioxolane structures (cyclohexa-3,5-diene-1,2-dione), which was mediated by cytochrome P450 1A2. The locations of hydroxylation and hydroxyl-glucuronidation were identified by analyzing the signature fragments generated as a result of tandem mass spectrometry, indicating hydroxylation at an aliphatic chain or aromatic ring. We determined whether the hydroxylation and glucuronidation occurred in an aromatic moiety (M5 and M12) or an aliphatic moiety (M6 and M13), respectively, based on signature fragments of the metabolites.