Production Of Aglycones Research Articles

A three-step “ping-pong” mechanism of a GH20 β-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily

β-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-β-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 μM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s−1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s−1, which is rate-limiting. The result clearly indicated a three-step “ping-pong” mechanism for VhGlcNAcase.

A review on applications of β-glucosidase in food, brewery, pharmaceutical and cosmetic industries

β-glucosidases hydrolyse glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides via catalytic mechanisms. It is very well known that the β-glucosidase enzyme is used in biorefineries for cellulose degradation, where β-glucosidases is the rate-limiting enzyme for the final glucose production from cellobiose. The β-glucosidase enzyme is used as a catalyst in other industrial sectors, including pharmaceuticals, breweries, dairy, and food processing. With the aid of β-glucosidase enzymes, cyanogenic glycosides and plant glycosides are transformed into sugar moiety and aglycones. These aglycone compounds are employed as aromatic compounds in the food processing and brewing industries. They are also used as medications and dietary supplements based on their pharmacological qualities. Applications of aglycones and the microbiological sources of β-glucosidase in aglycone production have been discussed in this review.

The enhanced production and secretion of high-value cajaninstilbene acid and flavonoid aglycones in Cajanus cajan (Linn.) Millsp. cell suspension cultures elicited by Aspergillus oryzae Y-29

For the first time, Cajanus cajan cell suspension cultures (CCSCs) were elicited by living GRAS (Generally Recognized as Safe) fungi to enhance the production of bioactive phenolic compounds. Under the inoculum size of mycelia (0.5 %), the optimal fungus Aspergillus oryzae Y-29 (AO Y-29) could achieve the enhanced production of cajaninstilbene acid (5.79-fold increase) in 49-day-old CCSCs at 8 h. Also, AO Y-29 could promote the production of flavonoid aglycones including luteolin (2.25-fold increase), genistein (2.04-fold increase), apigenin (1.96-fold increase), isorhamnetin (1.43-fold increase), and biochanin A (6.48-fold increase) in CCSCs at 12 h. Moreover, the levels of genistein and cajaninstilbene acid in culture media after fungal elicitation increased by 3.58- and 18.02-fold over control, respectively. AO Y-29 mycelia was found to trigger the oxidative stress thus activating the expression of pathogenesis-, biosynthesis-, and ABC transport-related genes, which contributed to the accumulation and secretion of flavonoid aglycones and cajaninstilbene acid in CCSCs. Furthermore, ABCB11 was likely to be correlated with the transport of genistein and cajaninstilbene acid, which was important for systematic metabolic engineering to increase their productivity. Overall, the elicitation of CCSCs with AO Y-29 was attractive for obtaining high yields of valuable flavonoid aglycones and cajaninstilbene acid in pharmaceutical/nutraceutical fields.

Enhancement of Antioxidant Activities in Black Soy Milk through Isoflavone Aglycone Production during Indigenous Lactic Acid Bacteria Fermentation

Black soybeans contain high antioxidant compounds such as isoflavone but mainly in glucoside form, with low antioxidant activities. Fermentation by lactic acid bacteria (LAB) can enhance the antioxidant properties, but its ability is strain-dependent. This study aims to study the ability of Indonesian indigenous LAB, Lactiplantibacillus plantarum WGK 4, Streptococcus thermophilus Dad 11, and Lactiplantibacillus plantarum Dad 13, to enhance the antioxidant properties during black soy milk fermentation. Fermentation was carried out at 37 °C for 24 h. Viable cell, acid production, Folin–Ciocalteu assay, antioxidant activity (DPPH), isoflavone aglycone daidzein and genistein, and β-glucosidase activity were measured every six hours. All LAB strains could grow well during the fermentation of black soy milk. Lactiplantibacillus plantarum WGK 4 produced the highest acid (1.50%). All three LAB strains could enhance antioxidant activity (DPPH) from 24.90% to 31.22–38.20%, followed by increased isoflavone aglycone. All strains could increase daidzein and genistein content, ranging from 61% to 107% and 81% to 132%, respectively. All three Indonesian indigenous LAB enhanced antioxidant properties of black soy milk relatively at the same level and potentially could be used as a starter culture of black soy milk fermentation.

Analysis of phenolic compounds in pickled chayote and their effects on antioxidant activities and cell protection

Although pickled chayote is a favorite Chinese fermented vegetable with high nutritional values, little is known about its bioactive compounds and antioxidant activities. In the work, changes of phenolic components and antioxidant activities in pickled chayote during 90 days’ natural fermentation period were investigated. The results revealed a total of 15 phenolics were positively or tentatively recognized and quantified in pickled chayote. Among these phenolics, flavonoid aglycones (i.e., luteolin, apigenin, diosmetin, and isorhamnetin) significantly increased during fermentation (p < 0.05). In addition, total phenolic content, total flavonoid content, and antioxidant activities (ABTS and DPPH assays) were detected with the highest values in pickled chayote at the late stage of fermentation (56 – 90 days). Furthermore, pickled chayote could protect HepG-2 cells against H2O2-caused oxidative damage by the reduction of reactive oxygen species generation and the promotion of innate antioxidant defense system (p < 0.05). Correlation analysis showed that 8 phenolic compounds were highly associated with the increase of extracellular and intracellular antioxidant activities. Besides, Lactobacillus alimentarius and Lactobacillus futsaii could increase the content of apigenin derivatives and Pichia membranifaciens could promote the production of flavonoid aglycones. In general, fermentation is an effective approach to enhance antioxidant potentials of pickled chayote and thereby increasing its functional values.

Production of Daidzein and Genistein from Seed and Root Extracts of Korean Wild Soybean (Glycine soja) by Thermostable β-Galactosidase from Thermoproteus uzoniensis

Isoflavone glycosides are commonly biotransformed into isoflavone aglycones due to the superior biological activities of the latter. Wild soybeans contain a higher isoflavone content than domesticated soybeans due to their high level of genetic diversity. In this study, we cloned and characterized a thermostable β-galactosidase from the extreme thermophile Thermoproteus uzoniensis for potential application in isoflavone conversion in Korean wild soybeans. The purified recombinant enzyme exhibited a maximum specific activity of 1103 μmol/min/mg at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme exhibited the highest activity for p-nitrophenyl (pNP)-β-D-galactopyranoside among aryl glycosides and it hydrolyzed isoflavone glycosides in the order genistin &gt; daidzin &gt; ononin &gt; glycitin. The enzyme completely hydrolyzed 2.77 mM daidzin and 3.85 mM genistin in the seed extract of wild soybean after 80 and 70 min with productivities of 1.86 and 3.30 mM/h, respectively, and 9.89 mM daidzin and 1.67 mM genistin in the root extract after 180 and 30 min, with the highest productivities of 3.30 and 3.36 mM/h, respectively, compared to other glycosidases. Our results will contribute to the industrial production of isoflavone aglycone using wild soybean and this is the first report on the enzymatic production of isoflavone aglycones from isoflavone glycosides in wild soybeans.

Limonoid detection and profile in callus culture of sweet orange

Plant tissue culture has emerged as an important tool to produce bioactive compounds from various plant species, including the sustainable production of limonoids that are receiving considerable attention due to the benefits associated with human health such as anticancer activities. The purpose of the present study was to evaluate the capacity of limonoids aglycone production from callus culture from sweet orange cv. Pera (Citrus sinensis) seeds and identify the compounds produced in this cell line. Callus induction occurred in Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic (2,4-D), malt extract, agar and coconut water. For the analysis and identification of the limonoids, CG-MS-EI ion-positive mode and UPLC-QTOF-ESI were used operating in positive and negative mode. An intense peak corresponding to limonin appeared in the callus extracts at a retention time of 58.1 min. in CG-MS-EI and four major limonoids aglycone by positive ion mode UPLC-QTOF-ESI: limonin, nomilin, deacetylnomilin, and nomilinic acid. The culture medium was efficient at the bioproduction of limonoids aglycone in callus cultures of C. sinensis seeds. Therefore, data obtained from UPLC-QTOF-ESI proved its importance at identifying new compounds that benefit human health, and may assist future work in the identification of known or new limonoids in Citrus species and related genera.

Bioanalysis of doxorubicin aglycone metabolites in human plasma samples\u2013implications for doxorubicin drug monitoring

The widespread clinical use of the cytostatic doxorubicin together with the induction of chronic cardiomyopathy necessitates the conduct of further pharmacokinetic trials. Novel analytical technologies suitable for point-of-care applications can facilitate drug level analyses but might be prone to interferences from structurally similar compounds. Besides the alcohol metabolite doxorubicinol, aglycone metabolites of doxorubicin might affect its determination in plasma. To evaluate their analytical relevance, a validated HPLC method for the quantification of doxorubicin, doxorubicinol and four aglycones was used. The degradation pattern of doxorubicin in plasma under long-term storage was analysed with respect to the formation of aglycone products. In addition, overall 50 clinical samples obtained within the EPOC-MS-001-Doxo trial were analysed. Substantial degradation of doxorubicin in plasma occurred within a storage period of one year, but this did not lead to the formation of aglycones. In clinical samples, 7-deoxydoxorubicinolone was the major aglycone detectable in 35/50 samples and a concentration range of 1.0–12.7 µg L−1. If at all, the other aglycones were only determined in very low concentrations. Therefore, analytical interferences from aglycones seem to be unlikely with the exception of 7-deoxydoxorubicinolone whose concentration accounted for up to 65% of the doxorubicin concentration in the clinical samples analysed.

Production of flavonoid and lignan aglycones from flaxseed and soy extracts by Bifidobacterium strains

SummaryFlaxseed and soybean are an important source of phytochemical compounds, mainly lignans and isoflavones, but also of flavones, flavanones and flavonols. These compounds appear mainly glycosylated in plant food and are converted into aglycones by the intestinal microbiota, increasing their bioavailability. In this work, we analyse the ability of Bifidobacterium strains (n = 25) to produce lignan and flavonoids aglycones from flaxseed and soybean extracts. Most of the Bifidobacterium strains increased the concentrations of secoisolariciresinol, daidzein, genistein, naringenin, eriodyctiol, luteolin and apigenin. Moreover, Bifidobacterium pseudocatenulatum and Bifidobacterium breve strains showed high production of herbacetin, increased the kaempferol concentration and produced quercetin and quercetagetin. B. pseudocatenulatum INIA P815 and B. breve INIA P367 produced 32.37 ± 2.81 and 28.64 ± 3.36 mg respectively of herbacetin g−1 of lignan extract. Bifidobacterium strains transformed the glycosides of a wide range of flavonoids into their aglycones, increasing the antioxidant activity and improving their bioavailability.

Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme

(1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale should consider enzyme specificity and cost-effectiveness. (2) Methods: In this study, we evaluated the most effective method for the enzymatic conversion of flavonoids from GT extract. One enzyme derived from Aspergillus niger (molecular weight 80–90 kDa) was ultimately selected, showing two distinct but simultaneous activities: intense glycoside hydrolase activity via deglycosylation and weak tannin acyl hydrolase activity via degalloylation. (3) Results: The optimum conditions for producing flavonol aglycones were pH 4.0 and 50 °C. Myricetin glycosides were cleaved 3.7–7.0 times faster than kaempferol glycosides. Flavonol aglycones were produced effectively by both enzymatic and hydrochloride treatment in a time-course reaction. Enzymatic treatment retained 80% (w/w) catechins, whereas 70% (w/w) of catechins disappeared by hydrochloride treatment. (4) Conclusions: This enzymatic process offers an effective method of conditionally producing flavonol aglycones and de-galloylated catechins from conversion of food-grade enzyme.

Oleuropein Aglycone Production and Formulation by Integrated Membrane Process

In this work, for the first time, the integration of a biocatalytic membrane reactor (BMR) and a membrane emulsification (ME) process, has been studied. The production and formulation of an importa...

Effect of Enzymatic Treatment of Chrysanthemum indicum Linné Extracts on Lipid Accumulation and Adipogenesis in High-Fat-Diet-Induced Obese Male Mice.

Enzyme treatment of the foods and herbs has been used to improve the absorption rate the efficiency of plant extracts by converting the glycosides of the plant into aglycones. In this study, we examined the obesity-inhibitory effect of Chrysanthemum indicum Linné (CI) treated with enzymes such as viscozyme and tannase, which are highly efficient in converting glycosides to aglycones and then compared with untreated CI extract. The enzyme-treated CI ethanol extract (CIVT) was administered orally at various doses for 7 weeks in the high fat diet (HFD)-fed male mice. CIVT administration reduced the body weights, the food efficiency and the serum levels of lipid metabolism-related biomarkers, such as triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and leptin in the dose-dependent manner but not those high-density lipoprotein cholesterol (HDL-c) and adiponectin. CIVT also reduced considerably the total lipid amount in the liver and the size of adipocytes in the epididymal white adipose tissue (eWAT). CIVT effectively downregulated the adipogenesis-related transcription factors such as peroxisome proliferation activated receptor (PPAR)-γ and CCAAT/enhancer binding protein-α (C/EBP-α) but up-regulated PPAR-α, in the liver and eWAT. In addition, when compared to the enzyme-untreated CI 50% ethanol extract (CIEE), CIVT enhanced the reduction of body weight and lipid accumulation. Moreover, the viscozyme and tannase treatment of CI increased the flavonoid contents of the aglycone form. Therefore, our results support that the enzymatic treatment induced the production of aglycones for potentially suppressing the adipogenesis and lipid accumulation in HFD-fed mice. It suggests that CIVT might be an effective candidate for attenuating the over-weight and its related diseases.

Assessment of Genistein and Daidzein Production By Some Local Fungal and Bacterial Isolates

TWENTY-THREE different bacterial and fungal isolates were grown and screened for their capability to transform soy glycosides to their aglycone forms with higher titer of antioxidant activity compared to unfermented soy flour. Most of the bacterial isolates showed higher amounts of daidzein than of genistein, which are the aglycone products of daidzin and genistin. After fermentation of soybean flour using bacterial and fungal isolates, the content of isoflavone aglycones varied from 0.0 to 431.89 μg/g compared to unfermented autoclaved soybean flour. Extracellular β-glucosidase activity was ranged from 1.22 to 11.56 mU/mL and 0.3-534.3 U/mL for bacterial and fungal isolates, respectively, while, bacterial cell-bound β-glucosidase ranged from 44.72 to 128.89 mU/mL. Most of the bacterial isolates more efficiently transformed daidzin and genistin into the aglycones than fungal isolates. Among the tested bacterial isolates, the most potent one was selected, characterized according to the morphological and 16S rDNA sequence analysis and identified as Bacillus licheniformis NRC24.

Acceleration of Aglycone Isoflavone and γ-Aminobutyric Acid Production from Doenjang Using Whole-Cell Biocatalysis Accompanied by Protease Treatment.

Recently, soybean isoflavone aglycones (i.e., daidzein and genistein) and γ-aminobutyric acid (GABA) have begun to receive considerable consumer attention owing to their potential as nutraceuticals. To produce these ingredients, multiple microorganisms and their enzymes are commonly used for catalysis in the nutraceutical industry. In this work, we introduce a novel fermentation process that uses whole-cell biocatalysis to accelerate GABA and isoflavone aglycone production in doenjang (a traditional Korean soybean paste). Microbial enzymes transform soybean isoflavone glycosides (i.e., daidzin and genistin) and monosodium glutamate into soybean isoflavone aglycones and GABA. Lactobacillus brevis GABA 100 and Aspergillus oryzae KACC 40250 significantly reduced the production time with the aid of a protease. The resulting levels of GABA and daidzein were higher, and genistein production resembled the levels in traditional doenjang fermented for over a year. Concentrations of GABA, daidzein, and genistein were measured as 7,162, 60, and 59 μg/g, respectively on the seventh day of fermentation. Our results demonstrate that the administration of whole-cell L. brevis GABA 100 and A. oryzae KACC 40250 paired with a protease treatment is an effective method to accelerate GABA, daidzein, and genistein production in doenjang.

Metabolism of Quercetin and Naringenin by Food-Grade Fungal Inoculum, Rhizopus azygosporus Yuan et Jong (ATCC 48108).

Rhizopus azygosporus Yuan et Jong (ATCC 48108), a starter culture for fermented soybean tempeh, produces β-glucosidases that cleave flavonoid glycosides into aglycones during fermentation. However, recent data suggest that fermentation of a flavonoid glycoside-rich extract with this strain did not result in the production of aglycones. Thus, in this paper, flavonoid metabolism of this strain was investigated. Incubation of flavonoid aglycones, naringenin and quercetin, with R.azygosporus resulted in the production of flavonoid glucosyl-, hydroxyl-, and sulfo-conjugated derivatives. Naringenin was completely metabolized within 96 h into eriodictyol sulfate and eriodictyol glucoside, whereas quercetin was partially metabolized into quercetin glucoside, diglucoside, sulfate, and glucosyl-sulfate. Most of these metabolites were found to be excreted by the fungi into the culture medium. Toxicity analysis revealed that incubation with both quercetin and naringenin did not exert inhibitory effects on fungal growth. This study presents an interesting mechanism of fungal detoxification of flavonoids in foods.

Elucidation of the fragmentation pathways of a complex 3,7-O-glycosyl flavonol by CID, HCD, and PQD on an LTQ-Orbitrap Velos Pro hybrid mass spectrometer

The present study was designed to systematically investigate the ESI-MSn behavior of a complex 3, 7-O-glycosyl flavonol, kaempferol 3-O-α-L-[2,3-di-O-β-D-(6-E-p-coumaroyl)glucopyranosyl]-rhamnopyranosyl-7-O-α-L-rhamnopyranoside (KO) isolated from Epimedium wushanense, and to address the elimination priority among different glycosylation sites and different sugars/substituents. The direct-infusion ESI-MSn experiment of KO was performed on a hybrid LTQ-Orbitrap Velos Pro mass spectrometer in both negative and positive ion modes by three different fragmentation mechanisms (CID, HCD, and PQD). The CID, HCD, and PQD analyses of KO exhibited remarkable discrimination in respect of the scan range, richness, and distribution of product ions through the entire spectra. KO experienced different fragmentation pathways between two ionization modes: the negative mode CID of KO eliminated the glycosyl portions (priority: 7-sugar > 3-substituent and terminal substituents > inner sugar) and produced aglycone product ions at m/z 284.03/285.04; however, abundant sodium-adduct B32 together with subsequent i,jX30 cleavages were found characteristic for the positive mode CID-MSn. The fragmentation pathways by CID for KO were proposed by analyzing the high accuracy ESI-MSn data. Complementary structural information of KO regarding the aglycone and glycosyl portions was obtained by analyzing the ESI-MSn data in both ionization modes. In conclusion, the LTQ-Orbitrap method facilitates highly reliable qualitative analysis of bioactive flavonoids with three alternative fragmentation modes.

Changes in soymilk during fermentation with kefir culture: oligosaccharides hydrolysis and isoflavone aglycone production

The objective of this study was to evaluate the changes in oligosaccharides and isoflavone aglycone content in soymilk during fermentation with commercial kefir culture. Soymilk was fermented with kefir culture at 25 °C for 30 h. The counts of lactic acid bacteria, Lactococcus lactis, Leuconostoc sp and yeasts; measurements of pH, acidity, α-galactosidase and β-glucosidase activity, sugar and isoflavone contents were performed at the intervals of time. In the fermented soymilk, the lactic acid bacteria counts increased from 7.6 log to 9.1 CFU g−1, pH reached to 4.9 and lactic acid reached 0.34 g 100 g− 1. The α-galactosidase was produced (0.016 AU g−1) with 100% raffinose and 92% stachyose hydrolysis being observed after the depletion of galactose, glucose and sucrose. Kefir culture produced β-glucosidase (0.0164 AU g−1), resulting in 100% bioconversion of glycitin and daidzin and 89% bioconversion of genistin into the corresponding aglycones. The fermented soymilk presented 1.67 μmol g−1 of daidzein, 0.28 μmol g−1 of glicitein and 1.67 μmol g −1 of genistein.

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily.

Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones.

Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2)

BackgroundThe roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng. MethodsWe constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. ResultOverexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides (Rg1, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, Rb2, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds. ConclusionThe production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

Screening of six UGT enzyme activities in human liver microsomes using liquid chromatography/triple quadrupole mass spectrometry.

Uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes are essential for the clearance of many drugs; however, altered UGT activity is a potential cause of adverse drug-drug interactions (DDI). The early detection of potential DDI is an important aspect of drug discovery that has led to the development of new screening methods for drug interactions. We developed a screening method for the simultaneous evaluation of six human liver UGT enzyme activites using in vitro cocktail incubation and tandem mass spectrometry. The two in vitro cocktail doses were developed to minimize drug interactions among substrates. The method is based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Electrospray ionization (ESI) in both positive and negative modes was used to quantify the metabolites and the diagnostic loss of the glucuronosyl moiety to form the aglycone product was estimated using the selected reaction monitoring (SRM) mode. The method was validated by comparing inhibition data obtained from the incubation of each individual probe substrate alone with data from the cocktail method. The intra- and inter-day accuracy and precision data for the six UGT metabolites ranged from 92.2 to 100.3% and less than 15.2%, respectively. The IC(50) values showed no significant differences between individual and cocktail incubations. As a screening technique for inhibitory interactions of these six human liver UGT enzymes, this method will be useful for advancing mechanistic understanding of drug interactions.