Effects of CYP2C19 Genetic Polymorphisms on PK/PD Responses of Omeprazole in Korean Healthy Volunteers.

The aim of this study was to examine the effects of CYP2C19*2 and *3 genetic polymorphisms on omeprazole pharmacokinetic (PK) and pharmacodynamic (PD) responses. Twenty-four healthy Korean volunteers were enrolled and given 20 mg omeprazole orally once daily for 8 days. The genotypes of CYP2C19 single nucleotide polymorphisms (SNPs) (*2, *3, and *17) were screened. The plasma concentrations of omeprazole, omeprazole sulfone, and 5-hydroxy (5-OH) omeprazole were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The noncompartmental method was used for the determination of PK parameters. Change of mean pH and proportion (%) of time of gastric pH above 4.0 were estimated. The poor metabolizer (PM) group had the lowest metabolic ratio and exhibited the highest area under the curve (AUC) for omeprazole among the CYP2C19 phenotype groups. The PM group showed the greatest change of mean pH and the highest % time of gastric pH above 4.0. The relationship between AUC of omeprazole and % time of gastric pH above 4.0 was confirmed. The study demonstrates that CYP2C19*2 and *3 influence the PKs and PDs of omeprazole in Korean healthy volunteers.

Metabolism of ginsenoside Rb1 by human intestinal microflora and cloning of its metabolizing ß-D-glucosidase from Bifidobacterium longum H-1.

To understand the role of intestinal microflora in expressing the pharmacological effect of ginsenoside Rb1, the metabolic activity of ginsenoside Rb1 by 148 fecal specimens was measured and its metabolizing ß-glucosidase was cloned. The average activities for p-nitrophenyl-ß-D-glucopyranoside and ginsenoside Rb1 were 0.097±0.059 µmol/min/mg and 0.311±0.118 pmol/min/mg, respectively. These enzyme activities were not different between male and female, or between ages. A gene encoding ß-D-glucosidase (BglX) was cloned from Bifidobacterium longum H-1, which transformed ginsenoside Rb1 to compound K. The probe for cloning was synthesized from the genes encoding a ß-D-glucosidase of previously reported B. longum DJO10A. The sequences of the cloned gene revealed 2364 bp open reading frame (ORF) encoding a protein containing 787 amino acids (molecular weight of 95 kDa). The gene exhibited 99% homology (identities) to that of B. longum. The cloned gene was expressed under T7 promoter of the expression vector, pET-39b(+), in Escherichia coli BL21(DE3), and the expressed enzyme was purified by using HiTrap immobilized metal affinity chromatography (IMAC) HP. The enzyme potently biotransformed ginsenoside Rb1, loganin, arctiin and arbutin to ginsenoside Rd, loganetin, arctigenin and hydroquinone, respectively, but was not active in the case of hesperidin, and kakkalide. This is the first report on cloning and expression of ß-D-glucosidase from B. longum. Based on these findings, ginsenoside Rb1 may be metabolized to bioactive compound(s) by exo-ß-D-glucosidase(s) produced from the intestinal bacteria and its pharmacological effects may be dependent on intestinal bacterial exo-ß-D-glucosidase(s) activity.

Artemisia princeps Pamp. Essential oil and its constituents eucalyptol and α-terpineol ameliorate bacterial vaginosis and vulvovaginal candidiasis in mice by inhibiting bacterial growth and NF-κB activation.

To investigate the inhibitory effects of Artemisia princeps Pamp. (family Asteraceae) essential oil (APEO) and its main constituents against bacterial vaginosis and vulvovaginal candidiasis, their antimicrobial activities against Gardnerella vaginalis and Candida albicans in vitro and their anti-inflammatory effects against G. vaginalis-induced vaginosis and vulvovaginal candidiasis were examined in mice. APEO and its constituents eucalyptol and α-terpineol were found to inhibit microbe growths. α-Terpineol most potently inhibited the growths of G. vaginalis and C. albicans with MIC values of 0.06 and 0.125 % (v/v), respectively. The antimicrobial activity of α-terpineol was found to be comparable to that of clotrimazole. Intravaginal treatment with APEO, eucalyptol, or α-terpineol significantly decreased viable G. vaginalis and C. albicans numbers in the vaginal cavity and myeloperoxidase activity in mouse vaginal tissues compared with controls. These agents also inhibited the expressions of proinflammatory cytokines (IL-1 ß, IL-6, TNF- α), COX-2, iNOS, and the activation of NF- κB and increased expression of the anti-inflammatory cytokine IL-10. In addition, they inhibited the expressions of proinflammatory cytokines and the activation of NF- κB in lipopolysaccharide-stimulated peritoneal macrophages, and α-terpineol most potently inhibited the expressions of proinflammatory cytokines and NF- κB activation. Based on these findings, APEO and its constituents, particularly α-terpineol, ameliorate bacterial vaginosis and vulvovaginal candidiasis by inhibiting the growths of vaginal pathogens and the activation of NF- κB.

Anti-scratching behavioral effect of Lactobacillus plantarum PM008 isolated from kimchi in mice.

To isolate antipruritic lactic acid bacteria (LAB) from kimchi, a traditional Korean food, we investigated the interleukin (IL)-4 production-inhibitory effect in the colon of mice for previously isolated LAB. Orally administered Lactobacillus plantarum PM008 potently inhibited the expression of IgE-switching cytokine, IL-4, and of proinflammatory cytokines, IL-1ß and TNF-α, in the colon of mice. Its inhibitory effect was dependent on the dosage and administration period. When PM008 was orally administered to mice, the number of PM008 detected in the intestine and feces by polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) methods was dependent on the administration dosage and period. The number of PM008 attached in the intestine was gradually decreased with increasing time after completion of its oral administration. PM008 dose-dependently inhibited the scratching behavior induced by histamine or compound 48/80. PM008 treated at a dose of 1 × 10(10) CFU for 14 days inhibited the histamine- and compound 48/80-induced scratching behaviors by 32.8% and 48.6%, respectively. This inhibitory effect continued, although reduced, at 7 days after stopping the oral administration of PM008 attached in the intestine. Based on these findings, L. plantarum PM008 may improve pruritus by inhibiting IL-4 expression.

Cloning, overexpression, and characterization of recombinant heparinase III from Bacteroides stercoris HJ-15.

Recombinant heparinase III (rHepIII) from Bacteroides stercoris HJ-15 was cloned, expressed, and characterized. The full-length heparinase III gene from B. stercoris HJ-15 was identified by Southern blotting, and the sequence was deposited in GenBank. The heparinase III gene, which is 2,001-bp long, was cloned and overexpressed in Escherichia coli; highly active rHepIII was easily purified using only one step of immobilized Ni(2+) affinity column chromatography. Enzymatic properties and substrate specificities of rHepIII were assessed, and its kinetic constants were calculated. rHepIII was most active in 50 mM sodium phosphate buffer with 350 mM NaCl (pH 6.6) at 45 degrees C. Through amino acid modification studies and site-directed mutagenesis assay, cysteines and histidines were identified as crucial residues for enzymatic activity. Moreover, this enzyme digested not only heparan sulfate but also heparin and hyaluronic acid, and their degradation products were verified by strong anion exchange/high-performance liquid chromatography. These characteristics, including active residues and substrate specificities were interesting compared with those of existing heparinase III from other species. We anticipate that the convenience of purification and the characteristics of this enzyme will make it a powerful tool for studies of glycosaminoglycans and their lyases.

Anti-allergic effects of fermented Ixeris sonchifolia and its constituent in mice.

To evaluate the antiallergic effect of fermented Ixeris sonchifolia (IS, family Compositae), we prepared IS Kimchi, isolated Lactic acid bacteria (LAB) from it, fermented IS with these LAB, and investigated their antiallergic effects. IS Kimchi more potently inhibited the passive cutaneous anaphylaxis (PCA) reaction induced by an IgE-antigen complex as well as the scratching behavior induced by compound 48/80 or histamine than IS. When IS was fermented with LAB isolated from IS Kimchi, its antiallergic effects was also increased. Of LAB used for fermentation, Lactobacillus brevis more potently increased the antiallergic effects. Its main constituents, chlorogenic acid and luteolin potently inhibited PCA reaction induced by IgE-antigen complex as well as pruritus induced by compound 48/80 or histamine. These constituents inhibited the expression of proinflammatory and allergic cytokines, TNF-alpha and IL-4, and transcription factor, NF-kappaB, activation induced by IgE-antigen complex in RBL-2H3 cells, as well as the degranulation of RBL-2H3 cells induced by an IgE-antigen complex. Luteolin more potently inhibited these allergic reactions than chlorogenic acid. These findings suggest that antiallergic effect of IS can be increased by LAB fermentation and fermented IS might improve allergic reactions, such as pruritus, anaphylaxis, and inflammation.

Metabolism of rutin and poncirin by human intestinal microbiota and cloning of their metabolizing α-L-rhamnosidase from Bifidobacterium dentium.

To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-Lrhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni(2+)-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 µmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1-->6) bonds than (1-->2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

Clinical and psychological characteristics of propofol abusers in Korea: a survey of propofol abuse in 38, non-healthcare professionals.

BACKGROUND: The aim of this study is to investigate the characteristics of propofol abuse based on the results of a survey analysis of abusers among non-healthcare professionals in Korea. METHODS: Thirty-eight propofol abusers were questioned between October and December 2010, and were enrolled and voluntarily participated in a structured survey consisting of an interview and completing a previously prepared questionnaire. The questionnaire was divided into three distinct parts: part 1 dealt with the history of propofol abuse; part 2 highlighted the problems caused by propofol abuse; and part 3 enquired regarding demographics of abusers. RESULTS: Thirty-one (81.6%) of the 38 interviewees abused propofol for more than one year. During the last 12 months, 34 (89.0%) received propofol at two or three times a week. The minimum and maximum amounts of propofol (median, range) administered each time were 500 (100, 1000) and 2000 (500, 4000) mg, respectively. Stress relief and the maintenance of a sense of well-being were quoted the most important reasons for the first-time administration of propofol and its subsequent abuse, respectively. The majority of abusers (36.0, 97.3%) reported a sense of pleasure or euphoria at the time of their propofol injection. Withdrawal symptoms occurred in five abusers (13.2%). Thirteen (36.1%) reported disruptions in their work life. None of the respondents had previously admitted to and or reported abuse of any other controlled substances. CONCLUSIONS: These results provided reference data for the regulation of propofol in Korea as a controlled substance and may also be of interest to international agencies in other countries.

Protective effect of fermented red ginseng on a transient focal ischemic rats.

Red ginseng and fermented red ginseng were prepared, and their composition of ginsenosides and antiischemic effect were investigated. When ginseng was steamed at 98-100 degrees C for 4 h and dried for 5 h at 60 degrees C, and extracted with alcohol, its main components were ginsenoside Rg3> ginsenoside Rb1 > ginsenoside Rb2. When the ginseng was suspended in water and fermented for 5 days by previously cultured Bifidobacterium H-1 and freeze-dried (fermented red ginseng), its main components were compound K > ginsenoside Rg3 > or = ginsenoside Rh2. Orally administered red ginseng extract did not protect ischemia-reperfusion brain injury. However, fermented red ginseng significantly protected ischemica-reperfusion brain injury. These results suggest that ginsenoside Rh2 and compound K, which was found to be at a higher content in fermented red ginseng than red ginseng, may improve ischemic brain injury.

Expression of heparinase I of Bacteroides stercoris HJ-15 and its degradation tendency toward heparin-like glycosaminoglycans.

Recombinant heparinase I was cloned from Bacteroides stercoris HJ-15 (BSrhepI), overexpressed in Escherichia coli, and intensively characterized. The complete gene of BSrhepI was identified by Southern blotting, and was overexpressed as an inclusion body. The inclusion body was solubilized with 4 M guanidine-HCl, and the denatured BSrhepI was easily purified using Ni(2+)-affinity column chromatography. The purified but denatured enzyme was then successfully refolded by dialysis against 50 mM Tris-HCl (pH 7.0) containing 1mM DTT and CaCl(2). BSrhepI was most active in 50mM Tris-HCl buffer containing 300 mM NaCl, 10 mM CaCl(2), and 1 mM DTT (pH 7.0) at 37°C. This enzyme digested not only heparin, but also heparan sulfate. Through comparative HPLC-analysis of each degraded product of heparin and heparan sulfate by digestion with BSrhepI or flavobacterial heparinase I, we verified that BSrhepI has a broader spectrum of substrate specificities than other reported heparinases.

Cloning and characterization of ginsenoside Ra1-hydrolyzing beta-D-xylosidase from Bifidobacterium breve K-110.

beta-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The (His6)-tagged recombinant enzyme, designated as XlyBK- 110, was efficiently purified using Ni²âº-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK- 100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The Km and Vmax values toward p-nitrophenyl-beta-D-xylopyranoside (pNPX) were 1.45mM and 10.75 micromol/min/mg, respectively. This enzyme had pH and temperature optima at 6.0 and 45 degrees C, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-alpha-Larabinofuranoside, p-nitrophenyl-beta-D-glucopyranoside, or p-nitrophenyl-beta-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of beta-Dxylosidase- hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.

Development of fecal microbial enzyme mix for mutagenicity assay of natural products.

Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.

Lactobacillus johnsonii HY7042 ameliorates Gardnerella vaginalis-induced vaginosis by killing Gardnerella vaginalis and inhibiting NF-κB activation.

Hydrogen peroxide-producing lactic acid bacteria (LAB) were isolated from women's vaginas and their anti-inflammatory effects against Gardnerella vaginalis-induced vaginosis were examined in ß-estradiol-immunosuppressed mice. Oral and intravaginal treatment with five LABs significantly decreased viable G. vaginalis numbers in vaginal cavities and myeloperoxidase activity in mouse vaginal tissues. Of the LABs examined, Lactobacillus johnsonii HY7042 (LJ) most potently inhibited G. vaginalis-induced vaginosis. This LAB also inhibited the expressions of IL-1ß, IL-6, TNF-α, COX-2, and iNOS, and the activation of NF-κB in vaginal tissues, but increased IL-10 expression. Orally administered LJ (0.2×10(8) CFU/mouse) also inhibited the expression of TNF-α by 91.7% in ß-estradiol-immunosuppressed mice intraperitoneally injected with LPS. However, it increased IL-10 expression by 63.3% in these mice. Furthermore, LJ inhibited the expressions of the pro-inflammatory cytokines, TNF-α and IL-1ß, and the activation of NF-κB in lipopolysaccharide-stimulated peritoneal macrophages. LJ also killed G. vaginalis attached with and without HeLa cells. These findings suggest that LJ inhibits bacterial vaginosis by inhibiting the expressions of COX-2, iNOS, IL-1ß, and TNF-α by regulating NF-κB activation and by killing G. vaginalis, and that LJ could ameliorate bacterial vaginosis.

Berberine ameliorates TNBS-induced colitis by inhibiting lipid peroxidation, enterobacterial growth and NF-κB activation.

Berberine, which is a major constituent of the rhizome of Coptidis japonica (CJ), inhibits IL-8 production in colonic epithelial cells and improves 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. In our preliminary studies, berberine inhibited lipid peroxidation in liposomes prepared from l-α-phosphatidylcholine as well as TLR-4-linked NF-κB activation in HEK cells. Therefore, to clarify its anticolitic mechanism, we examined the inhibitory effects of berberine in TNBS-induced colitic C3H/HeN and C3H/HeJ mice. Its oral administration inhibited macroscopic score, body weight gain, colon shortening, myeloperoxidase activity, and lipid peroxidation in the colons of TNBS-treated C3H/HeN and C3H/HeJ mice. Berberine inhibited colonic expression of iNOS, COX-2, IL-1ß, IL-6, and TNF-α, but increased IL-10 expression in the colons of TNBS-treated C3H/HeN and C3H/HeJ mice. Berberine also inhibited NF-κB activation in TNBS-treated C3H/HeN and C3H/HeJ mice, and inhibited TLR-4 expression in C3H/HeN, but not C3H/HeJ, mice. Treating C3H/HeN and C3H/HeJ mice with berberine significantly reduced the number of Enterobacteriaceae induced by TNBS, but restored the number of Bifidobacteria reduced by TNBS. Furthermore, berberine potently inhibited LPS-induced inflammation in peritoneal macrophages mainly via NF-κB and weakly via MAPKs. Based on these findings, berberine may improve colitis by inhibiting lipid peroxidation, enterobacterial growth and NF-κB activation.

Dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid induce lipid peroxidation by the proliferation of intestinal gram-negative bacteria in mice.

UNLABELLED: ABSTRECT: BACKGROUND: To understand whether TLR-4-linked NF-kB activation negatively correlates with lipid peroxidation in colitic animal models, we caused colitis by the treatment with dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to C3H/HeJ (TLR-4-defective) and C3H/HeN (wild type) mice, investigated inflammatory markers, lipid peroxidation, proinflammatory cytokines and TLR-4-linked NF-kappaB activation, in colon and intestinal bacterial composition in vivo. METHODS: Orally administered DSS and intrarectally injected TNBS all caused severe inflammation, manifested by shortened colons in both mice. These agents increased intestinal myeloperoxidase activity and the expression of the proinflammatory cytokines, IL-1beta, TNF-alpha and IL-6, in the colon. RESULTS: DSS and TNBS induced the protein expression of TLR-4 and activated transcription factor NF-kappaB. However, these colitic agents did not express TLR-4 in C3H/HeJ mice. Of proinflammatory cytokines, IL-1beta was most potently expressed in C3H/HeN mice. IL-1beta potently induced NF-kappaB activation in CaCo-2 cells, but did not induce TLR-4 expression. DSS and TNBS increased lipid peroxide (malondialdehyde) and 4-hydroxy-2-nonenal content in the colon, but reduced glutathione content and superoxide dismutase and catalase activities. These colitic inducers increased the number of Enterobacteriaceae grown in DHL agar plates in both mice, although the number of anaerobes and bifidobacteria grown in GAM and BL agar plates was reduced. E. coli, K. pneumoniae and Proteus mirabilis isolated in DHL agar plates increased lipid peroxidation in liposomes prepared by L-alpha-phosphatidylcholine, but B. animalis and B. cholerium isolated from BL agar plates inhibited it. DISCUSSION: These findings suggest that DSS and TNBS may cause colitis by inducing lipid peroxidation and enterobacterial proliferation, which may deteriorate the colitis by regulating proinflammatory cytokines via TLR-4-linked NF-kappaB activation pathway.

Cloning, expression and purification of arylsulfate sulfotransferase from Eubacterium A-44.

A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively.

In vitro inhibitory effect of flavonoids on growth, infection and vacuolation of Helicobacter pylori.

Flavonoids, which are main constituents of herbal medicines, have been reported to inhibit the growth of Helicobacter pylori (HP). Therefore, to evaluate the anti-HP activity of some flavonoids (flavanols, flavones, flavonols and isoflavonoids), their effects on the growth and vacuolation of HP as well as the infective properties of HP against HeLa cells were investigated. Catechins, quercetin and naringenin weakly inhibited the growth of HP, but all tested compounds did not inhibit HP infection into KATO III cells and HP urease activity. Quercetin and naringenin inhibited HP VacA vacuolation in HeLa cells with IC (50) values of 0.046 and 0.36 mM, respectively. Quercetin also inhibited procaspase-3 activation to caspase-3 in HeLa cells induced by HP VacA toxin, which may induce cell death via the proteolytic activation of a cascade of caspases. However, quercetin did not affect Bax and Bcl-2 protein levels. Based on these findings, quercetin may improve gastric cell death by inhibiting apoptotic signaling by HP VacA toxin. Abbreviations. HP: Helicobacter pyloriBSA:bovine serum albumin ESL:enhanced chemiluminescence MIC:minimum inhibitory concentration MTT:methylthiazolyldiphenyl-tetrazolium bromide PBS:phosphate-buffered saline VacA:Vacuolating cytotoxin.