• Biomolecules & Therapeutics
• /
• v.4 no.3
• /
• pp.280-284
• /
• 1996

Licorice has been widely used in combination with other herbs or synthetic drugs for various disorders. In an effort to study the effect of licorice roots (Glycyrrhizae Radix, GR) and glycyrrhizin on the hepatic glucuronidation, we have previously found that the pretreatment of GR or glycyrrhizin for 6 days resulted in a marked increase in the enzymatic activity of 3-methylcholanthrene (3-MC)-inducible hepatic UDP-glucuronosyltransferase (UGT) isozyme that has high affinity toward phenolic substrates (p-nitrophenol form, UGTIA) in Sprague-Dawley rats. As an approach to elucidate the mechanism for the enzyme activation by licorice in rat liver, we examined the levels of hepatocellular mRNAs for UGTIA upon the treatment of GR or glycyrrhizin. The hepatic mRNAs were extracted from Sprague-Dawley rats and Wistar rats after the treatment of the methanol extract of GR (1 g/kg, p.o.), glycyrrhizin (23 mg/kg, p.o.) for 6 days, or 3-MC (40 mg/kg, i.p.) for 3 days. Using the UGT1A1 CDNA as a probe, we found that the mRNAs for the enzyme were induced by 3-MC treatment while those were influenced neither by GR nor by glycyrrhizin in both strains of rats. These results indicate that the activation of rat liver UGTI A by licorice and glycyrrhizin was not due to the induction of mRNAs for the enzyme.

• The Journal of Korean Medicine
• /
• v.38 no.2
• /
• pp.15-30
• /
• 2017

Objectives: Hwangryunhaedok-tang (HHT; Huanglianjiedu-tang, Orengedoku-to), a traditional herbal formula, is used for treating inflammation, hypertension, gastritis, liver dysfunction, cerebrovascular diseases, dermatitis and dementia. The objective of this study was to assess the sub-acute toxicity of HHT in Sprague-Dawley (SD) rats, and its effect on the activities of human microsomal cytochrome P450s (CYP450s) and UDP-glucuronosyltransferases (UGTs). Methods: Male and female SD rats were orally administered HHT once daily at doses of 0, 500, 1000 and 2000 mg/kg for 4 weeks. We analyzed mortality, clinical observations, body weight, food consumption, organ weights, urinalysis, hematology, serum biochemistry, and histopathology. The activities of major human CYP450s (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) and UGTs (UGT1A1, UGT1A4, and UGT2B7) were assessed using in vitro fluorescence- and luminescence-based enzyme assays, respectively. Results: No toxicologically significant changes related to the repeated administration of HHT were observed in both male and female SD rats. The no observed adverse effect level (NOAEL) value was more than 2000 mg/kg/day for both sexes. HHT inhibited the activities of human microsomal CYP1A2, CYP2C19, CYP2D6, and CYP2E1, whereas it weakly inhibited the activities of CYP2B6, CYP2C9, CYP3A4, and UGT1A1. In addition, HHT negligibly inhibited the activities of human microsomal UGT1A4 and UGT2B7 with $IC_{50}$ values in excess of $1000{\mu}g/mL$. Conclusions: Our findings indicate that HHT may be safe for repeated administration up to 4 weeks. In addition, these findings provide information on the safety and effectiveness of HHT when co-administered with conventional drugs.

• Journal of Ginseng Research
• /
• v.41 no.3
• /
• pp.419-427
• /
• 2017

Background: Glycosylation of natural compounds increases the diversity of secondary metabolites. Glycosylation steps are implicated not only in plant growth and development, but also in plant defense responses. Although the activities of uridine-dependent glycosyltransferases (UGTs) have long been recognized, and genes encoding them in several higher plants have been identified, the specific functions of UGTs in planta remain largely unknown. Methods: Spatial and temporal patterns of gene expression were analyzed by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and GUS histochemical assay. In planta transformation in heterologous Arabidopsis was generated by floral dipping using Agrobacterium tumefaciens (C58C1). Protein localization was analyzed by confocal microscopy via fluorescent protein tagging. Results: PgUGT72AL1 was highly expressed in the rhizome, upper root, and youngest leaf compared with the other organs. GUS staining of the promoter: GUS fusion revealed high expression in different organs, including axillary leaf branch. Overexpression of PgUGT72AL1 resulted in a fused organ in the axillary leaf branch. Conclusion: PgUGT72AL1, which is phylogenetically close to PgUGT71A27, is involved in the production of ginsenoside compound K. Considering that compound K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of ginsenosides in ginseng plant growth and development. The organ fusion phenotype could be caused by the defective growth of cells in the boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, and requires further analysis.

• Clinical and Experimental Pediatrics
• /
• v.51 no.2
• /
• pp.150-155
• /
• 2008

Purpose : It has been known that breast milk cause prolonged unconjugated hyperbilirubinemia. UGT1A1 is a important gene of uridine diphosphate glucuronosyltransferase (UGT) which has a major role of bilirubin metabolism. These findings suggest that there is a relationship between UGT1A1 gene mutation and prolonged jaundice of breast feeding infant. The aim of study was to investigate whether a polymorphism of the UGT1A1 gene exist in prolonged hyperbilirubinemia of breast milk feeding Korean infant. Methods : The genomic DNA was isolated from 50 full term Korean neonates, who had greater than a 10 mg/dL of serem bilirubin after 2 weeks of birth with no significant cause, and the other genomic DNA was isolated from 162 full term Korean neonates of the control population. Both group fed breast milk. We performed direct sequencing of TATA box and Gly71Arg polymorphism of the UGT1A1 gene. Results : Two of the 50 neonates with hyperbilirubinemia had AA polymorphism, and 40 had GA polymorphism. Five of the 129 neonates of the control group had AA polymorphism, and 4 had GA polymorphism. The allele frequency of G>A polymorphism in the hyperbilirubinemia group was 44.0%; it was significantly higher than 5.4% of the control group. TATA box polymorpism was not different both group significantly. Conclusion : Our result indicated that Gly71Arg polymorphism is associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean, while TATA box polymorphism is not associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.5
• /
• pp.657-662
• /
• 2022

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.

• The Journal of Korean Medicine
• /
• v.42 no.4
• /
• pp.10-24
• /
• 2021

Objectives: Yongdamsagan-tang (YST) and Paljung-san (PJS) in traditional medicine and finasteride in modern medicine are used to treat benign prostatic hyperplasia (BPH). In recent, the use of combination herbal remedies with conventional drugs has been increasing. Therefore, we investigated the anti-inflammatory effects of these drugs to treat BPH and the influence of herbal formulas on finasteride metabolism. Methods: The inhibitory effects of the herbal formulas and finasteride on the production of inflammatory mediators and cytokines were determined in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Additionally, the influence of herbal formulas on activities of human drug metabolizing enzymes (DMEs) was assessed using human microsomal enzymes. Results: We observed that YST, PJS and finasteride inhibited the production of nitric oxide (NO), prostaglandin E₂ (PGE₂) and interleukin-6 (IL-6) in RAW 264.7 cells. The half maximal inhibitory concentration (IC₅₀) of YST on PGE₂ production was calculated to be below 25 ㎍/mL. YST inhibited the activity of uridine diphosphate-glucuronosyltransterase (UGT) 1A4 with an IC₅₀ value of 49.35 ㎍/mL. The activities of cytochrome P450 (CYP) 1A2, CYP2B6, CYP2C19, CYP3A4, and UGT1A1 were inhibited by PJS (IC₅₀ < 100 ㎍/mL, each). Although PJS and YST inhibited the activities of CYP3A4 and UGT1A4, respectively, these formulas may not influence the metabolism of finasteride because the IC₅₀ values of herbal formulas on DMEs are too high to affect metabolism. Conclusions: Our results suggest that the combination of finasteride and YST or PJS might not influence their drug metabolism and that the drugs may have synergistic effects against BPH.

• Journal of Applied Biological Chemistry
• /
• v.64 no.3
• /
• pp.309-316
• /
• 2021

Kaempferol 3-O-galactoside (Trifolin), a member of the flavonol group, has been reported to have anticancer effects against promyelocytic leukemia, histocytic lymphoma, skin melanoma and lung cancer. Trifolin has been extracted and used from several plants, but the extraction process is complicated and the final yield is low. Biotransformation is an alternative tool to produce high value-added chemicals from inexpensive compounds. To synthesis trifolin from naringenin, three genes (PeFLS and OsUGE-PhUGT) were introduced into Escherichia coli, respectively. In order to synthesis trifolin from naringenin, a co-culture fermentation system was established by optimizing the cell concentration, biotransformation temperature and medium, isopropyl-β-D-thiogalactoside (IPTG) concentration, substrate supply concentration, and recombinant protein induction time. The established optimal conditions for trifolin production were a 3:1 ratio of BL-UGTE to BL-FLS, induction of recombinant protein at 25 ℃ for 4 h after addition of 2.0 mM IPTG, biotransformation at 30 ℃, and supply of 300 μM naringenin. Through the optimized co-culture fermentation system, trifolin was biosynthesized up to 67.3 mg/L.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.11
• /
• pp.1859-1864
• /
• 2018

Synthesis of flavonoid glycoside is difficult due to diverse hydroxy groups in flavonoids and sugars. As such, enzymatic synthesis or biotransformation is an approach to solve this problem. In this report, we used stepwise biotransformation to synthesize two quercetin bisglycosides (quercetin 3-O-glucuronic acid 7-O-rhamnoside [Q-GR] and quercetin 3-O-arabinose 7-O-rhamnoside [Q-AR]) because quercetin O-rhamnosides contain antiviral activity. Two sequential enzymatic reactions were required to synthesize these flavonoid glycosides. We first synthesized quercetin 3-O-glucuronic acid [Q-G], and quercetin 3-O-arabinose [Q-A] from quercetin using E. coli harboring specific uridine diphopsphate glycosyltransferase (UGT) and genes for UDP-glucuronic acid and UDP-arabinose, respectively. With each quercetin 3-O-glycoside, rhamnosylation using E. coli harboring UGT and the gene for UDP-rhamnose was conducted. This approach resulted in the production of 44.8 mg/l Q-GR and 45.1 mg/l Q-AR. This stepwise synthesis could be applicable to synthesize various natural product derivatives in case that the final yield of product was low due to the multistep reaction in one cell or when sequential synthesis is necessary in order to reduce the synthesis of byproducts.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.8
• /
• pp.3371-3376
• /
• 2015

Pigmented rice bran has been suggested to be a valuable source of beneficial phytochemicals. We investigated genotoxic and anti-genotoxic effects of purple rice bran extract (PRBE) in rats using a liver micronucleus assay. Purple rice bran was extracted with methanol, obtaining large amounts of phenolic compounds, including anthocyanins and small amounts of gamma-oryzanol. The experimental protocols were divided into two sets. Male rats were divided into three groups. Group 1 was a negative control, while Groups 2 and 3 were fed with 100 and 500 mg/kg bw of PRBE, respectively, for 28 days. PRBE had no effect on micronucleus formation or xenobiotic metabolizing enzymes in rat liver. Experiments concerning the effect of PRBE on $AFB_1$ showed that PRBE significantly lessened the amount of micronucleated hepatocytes in $AFB_1$ treated rats. Furthermore, it modulated metabolic activation of $AFB_1$ metabolism in the liver by suppressing activity and protein expression of CYP1A2, CYP3A and CYP 450 reductase, and enhancing phase II enzymes including GST and UGT. Overall, purple rice bran extract was not genotoxic in rats. It exhibited anti-genotoxicity by modulation some xenobiotic enzymes active in $AFB_1$ metabolism.

• Journal of Ginseng Research
• /
• v.43 no.1
• /
• pp.116-124
• /
• 2019

Background: Ginsenosides are known as the principal pharmacological active constituents in Panax medicinal plants such as Asian ginseng, American ginseng, and Notoginseng. Some ginsenosides, especially the 20(R) isomers, are found in trace amounts in natural sources and are difficult to chemically synthesize. The present study provides an approach to produce such trace ginsenosides applying biotransformation through Escherichia coli modified with relevant genes. Methods: Seven uridine diphosphate glycosyltransferase (UGT) genes originating from Panax notoginseng, Medicago sativa, and Bacillus subtilis were synthesized or cloned and constructed into pETM6, an ePathBrick vector, which were then introduced into E. coli BL21star (DE3) separately. 20(R)-Protopanaxadiol (PPD), 20(R)-protopanaxatriol (PPT), and 20(R)-type ginsenosides were used as substrates for biotransformation with recombinant E. coli modified with those UGT genes. Results: E. coli engineered with $GT95^{syn}$ selectively transfers a glucose moiety to the C20 hydroxyl of 20(R)-PPD and 20(R)-PPT to produce 20(R)-CK and 20(R)-F1, respectively. GTK1- and GTC1-modified E. coli glycosylated the C3-OH of 20(R)-PPD to form 20(R)-Rh2. Moreover, E. coli containing $p2GT95^{syn}K1$, a recreated two-step glycosylation pathway via the ePathBrich, implemented the successive glycosylation at C20-OH and C3-OH of 20(R)-PPD and yielded 20(R)-F2 in the biotransformation broth. Conclusion: This study demonstrates that rare 20(R)-ginsenosides can be produced through E. coli engineered with UTG genes.