KR20150045247A - Method for Preparing Resveratrol Glucoside - Google Patents

Abstract

The present invention relates to a manufacturing method of resveratrol glucoside, which makes resveratrol with sugar donors under UDP glycosyltransferase and is used for mass-producing a cost-effective resveratrol glucoside, thereby being widely used in medical fields of industrial sizes like drug.

Description

Methods for Preparing Resveratrol Glucoside < RTI ID = 0.0 >

The present invention relates to a method for producing resveratrol glycoside from resveratrol, and more particularly to a method for preparing resveratrol glycoside, which comprises reacting resveratrol with a sugar donor in the presence of UDP-glycosyltransferase.

Resveratrol is a phytoalexin that is naturally produced by other plants, especially grapes, peanuts, blueberries and hops. It is known that Botrytis cinerea ) derived microbial infection, damage, ultraviolet radiation and other stressors (Roldan, A. et al., J. Agric . Food Chem . 51: 1464-8, 2003; Roggero, JP et al., Sci . Aliments , 15: 411-22, 1995). This is the same as that of the transformer (Fig. 1) including the glucoside, dimer (pallidol, 3 in Fig. 1), trimer (grandiphenyl, grandiphenol C, 4 in Fig. 1; 1) and cis (2 in FIG. 1) arrangement (Shan, H. et al., Biochem . Biophys . Res . Commun . , 379: 283-7, 2009). However, trans-resveratrol contributes most of the biological activity. In addition, other post-modified resveratrol derivatives with potential biological activity have been found to be effective in the treatment of various diseases such as phthalostilbene (12 in FIG. 1), trans-polyhedin (trans-phytase, 13 in FIG. 1) Gt; 1, < / RTI > 6) and astrin (14 in Figure 1). The hydrolyzed resveratrol derivative 4,4'-dihydroxy-trans-stilbene (7 in FIG. 1) and 3,3 ', 3', 5-tetrahydroxy- , 4,4 ', 5-tetrahydroxy-trans-stilbene (9 in FIG. 1), 3,3', 4,4 ', 5,5'-hexahydroxy- 11) and 3,3 ', 4', 5,5 ', - pentahydroxy-trans-stilbene (10 in FIG. 1) have been successfully synthesized and their structure- Studies have been completed (Coppa, T. et al., J. Med . Food , 14: 1173-80, 2011).

Since the popularity of the "French paradox", extensive research into resveratrol has been performed and the potential for various diseases including type II diabetes, obesity, arteriosclerosis, Alzheimer's disease, cardiovascular diseases (hypertension and ischemic diseases) Biological activity was revealed (Szkudelski, T. et al., J. Pharmacol ., 552: 176-81, 2006; Jang, M. et al., JM Science , 275: 218-20, 1997). Similar to other antioxidants, resveratrol neutralizes self-generated free radical species such as active oxygen species and active nitrogen species in the body, and prevents radicals from damaging organs. Thus, the biological activity of most resveratrol contributes to its intrinsic radical scavenging activity. In addition, resveratrol can be used to detect various intracellular targets including receptors (other cell membrane boundaries or intracellular), signal molecules (silentin and 5'-adenosine monophosphate-activated kinase signal pathways), transcriptional and DNA- I have. Based on a database on the US National Institutes of Health website, a series of clinical studies are under way involving resveratrol as a lead compound. However, more information and research on the safety, pharmacokinetics, pharmacodynamics, and clinical efficacy of resveratrol are essential to the use of resveratrol as a drug for certain diseases (Patel, KR et al., Acad . Sci ., 1215: 161-9, 2011).

Recently, the importance of resveratrol has been demonstrated as a source of youth developed as a drug for the prolongation of human life that activates SIRT1 (Quideau, S. et al., Chem . Int . Ed. Engl . , 51: 6824-6, 2012; Hubbard, BP et al., Science , 339: 1216-9, 2013). SIRT1 protein prolongs the lifespan of yeast, larvae, flies, fish and mice (Knutson, MD et al, Nutr Rev, 66:... 591-6, 2008). SIRT1 upregulators reduce cellular oxidative stress in diabetic environments (Yun, JM et al., Nutr . Biochem . , 23: 699-705, 2012).

Most biologically active natural products (NPs) are sugar-complexes (calicheamicin, daunomycin, streptomycin, vancomycin, digitotoxin and amphotericin). The NP sugar moiety is important for particular properties, such as recognition of RNA by streptomycin, cell membrane recognition by amphotericin, and recognition of other target molecules in the cell, such as inhibiting or promoting target enzyme activity . Therefore, the engineering of sugar moieties in NP glucosides has the potential to create novel therapies to be used as future drugs. In addition, the glycosylation pattern of modified NP glucosides (C- / O- / N-) may improve the efficacy of the drug.

The concept of glycorandomization has been proposed within the last decade to diversify NPs. Some small molecules have been successfully glycosylated by chemical enzymes or chemical synthesis approaches (neo-glycosylation). For example, digoxin, novobiocin, vancomycin, colchicine, and 4-methylumbelliferone are glycosylated by the addition of other types of sugars that produce a large number of specific sugar-related NPs. Some glyco-randomized products exhibit enhanced biological activity compared to the parent molecule (Langenhan, JM et al., Proc . Natl . Acad . Sci . USA , 102: 12305-10, 2005). In most studies, glycosyltransferases (e. G., OleD and variants thereof) are engineered to enhance activity against donor and acceptor substrates. Based on these studies, there is a need for studies on glycosyltransferase (GT) with broad donor and acceptor matrix flexibility.

Although the anti-proliferative and anti-inflammatory activity of resveratrol has been reported in many studies, the efficacy of resveratrol and its glycosylated derivatives on the development of the immune response has not been widely known (Gao, X. et al., Biochem . Pharmacol, 62: 1299-1308, 2001 ; Gao, X. et al, Biochem Pharmacol, 66:.... 2427-35, 2003).

Accordingly, the present inventors have made intensive efforts to develop a method for producing a large amount of resveratrol glycoside. As a result, they have found that a method for preparing resveratrol glycoside, which comprises reacting resveratrol and a saccharide donor in the presence of UDP-glycosyltransferase , Confirming that the conversion rate from resveratrol to resveratrol glucoside is effectively increased, and the present invention is completed.

It is an object of the present invention to provide a method for preparing resveratrol glycoside, which comprises reacting resveratrol with a sugar donor in the presence of UDP-glycosyltransferase.

In order to achieve the above object, the present invention provides a method for preparing resveratrol glycoside, which comprises reacting resveratrol with a sugar donor in the presence of UDP-glycosyltransferase.

The resveratrol glucoside is produced in a large amount by using the resveratrol glycoside production method wherein resveratrol and sugar donor are reacted in the presence of UDP-glycosyltransferase according to the present invention, , Alzheimer's disease, and cardiovascular disease treatments.

Figure 1 shows the structure of polyhydroxylated, methoxylated or glycosylated resveratrol derivatives and resveratrol and its polymers. Resveratrol derivatives are those isolated from natural sources or chemically or enzymatically synthesized.
Figure 2 shows the results of SDS-PAGE analysis of the purified protein of Example 2-1.
Figure 3 shows the results of a high performance liquid chromatography-photodiode assay (HPLC-PDA) of Example 2-1.
4 shows the HPLC-PDA-HRQTOF-ESI / MS spectrum of resveratrol glucoside of Example 2-1.
Figure 5 is an embodiment 2-1 of the (E) -1- (3-β -D- glucopyranosyl-5-hydroxyphenyl) -2- (4-hydroxyphenyl) ethene A of (P _Ⅰ) ¹ ) H NMR, B) C ¹³ -NMR, C) ¹ H- ¹ H COZY NMR, D) close-up view of ¹ H- ¹ H COSY NMR of the sugar moiety, E) ROESY NMR, F) G) HSQC, H) Proximity of HSQC, I) HMBC result.
Figure 6 is an embodiment 2-1 of the (E) -1- (3,5- dihydroxy phenol) -2- (4-β-D- glucopyranosyl oxyphenyl) A) ¹ of ethene (P _Ⅱ) parts per ^{H-NMR, B) C 13} -NMR, C) 1 H- 1 H COSY NMR, D) surface-up (close view) of ¹ H- ¹ H NMR COSY of the sugar moiety, E) ROESY NMR, F) , G) HSQC, H) HMBC results.
Figure 7 A of Example 2-1 (E) -1- (3,5-Bis -β-D- glucopyranosyl-oxy) -2- (4-hydroxy phenol) ethene (P _Ⅲ)) ¹ H-NMR, B) ¹ H- ¹ H COSY NMR, C) close view of ¹ H- ¹ H COSY NMR of the sugar moiety, D) ROESY NMR, E) proximity of the ROESY NMR of the sugar moiety , F) HSQC, G) HMBC, and H) HMBC.
Figure 8 shows the identified products derived from the glycosylation of resveratrol using YjiC of Example 2-1.
Figure 9 shows the structure of different flavonoids used for glycosylation for receptor substrate studies of Example 2-2.
FIG. 10 shows HPLC-PDA analysis results of the reaction mixture of UDP-α-D-glucoside and YjiC of each of the substrates, which is the picetine, naringenin and daidze of Example 2-2 and the respective substrates.
FIG. 11 shows HPLC-PDA-HRQTOF-ESI / MS spectra of the A) naringin glucoside of Example 2-2, B) picetin glucoside, and C) dideoxyne glucoside.
12 shows the conversion of other substrates by YjiC in Example 2-2 by HPLC-PDA chromatogram analysis.
Figure 13 compares the retention time with resveratrol glucoside analogues identified by reaction and NMR of resveratrol and UDP-glycosyltransferase ( YjiC ) using other NDP-sugar donor substrates of Example 2-3 And shows the predicted structure based on the above.
14 shows the result of thin film chromatography (TLC) analysis of the reaction mixture of Example 2-3.
FIG. 15 is a graph showing the changes in the concentration of resveratrol 3-O-beta-D-galactosidase of Example 2-3, A) resveratrol glucoside, B) resveratrol 4'-O-beta -D- galactoside, C) resveratrol 2- L-laminoside, E) resveratrol 4'-O-? -D-biosaminoside F) resveratrol 3-O-? -L-fucoside G) HPLC-PDA-HRQTOF-ESI / MS Lt; / RTI >
Fig. 16 is a graph showing the results of the comparison between the resveratrol of Example 2-3 and the YjiC promoting glycosylation of other NDP- And the distribution of the resulting products.
Figure 17 shows the desaturization of resveratrol 3-O- [beta] -D-glucoside by YjiC in the presence of excess amounts of UDP of Examples 2-4.
Figure 18 shows a superimposed ribbon diagram of YjiC with crystal structures of OleD and CalG4 of Examples 2-5.
Fig. 19 is a graph showing the relationship between the YjiC The binding of the critical amino acids around the resveratrol and resveratrol of the active site is shown in the molecular docking and binding mode of UDP with resveratrol and YjiC protein.
20 shows an image of a selected YjiC amino acid in the resveratrol docking model of Example 2-5.
Figure 21 shows the effect of different concentrations of resveratrol and its glycosylated derivatives on mouse macrophage RAW 264.7 cells of Example 3, showing (A) cytotoxicity, (B) secretion of interleukin-6 (C) shows nitrogen monoxide production.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, resveratrol and sugar donor were reacted in the presence of UDP-glycosyltransferase to prepare resveratrol glycoside. As a result, it was confirmed that the conversion rate of resveratrol to resveratrol glycoside was improved.

In the present invention, glycosylation refers to a reaction in which a glycosyl group is transferred to another compound. In most cases, a sugar nucleotide is used as a substrate, and a glycosyltransferase specific for each is catalyzed . In other words, glucosylation refers to a reaction that converts various sugars to other compounds.

Accordingly, in one aspect, the present invention relates to a method for preparing resveratrol glycoside, which comprises reacting resveratrol and a sugar donor in the presence of UDP-glycosyltransferase.

In the present invention, the UDP-glycosyltransferase is a recombinant microorganism into which the gene of the enzyme is introduced.

In the present invention, the gene is YjiC .

In the present invention, the microorganism is characterized by being E. coli .

In the present invention, the UDP-glycosyltransferase is a bacillus licheniformis licheniformis ).

In the present invention, the donor sugar is UDP-D- glucose, TDP-D- rainy samin, UDP-D- galactose, TDP-D-2- deoxy-glucose, UDP- N - acetylglucosamine, UDP-D- gluconic But is not limited to, any one selected from the group consisting of chronic acid, TDP-L-rhamnose, and GDP-L-fucose.

In the present invention, resveratrol glucoside may be used in combination with (E) -resveratrol-3-O- beta -D-glucoside, (E) , 5-O-? -D-glucoside, 5-O-? -D-glucoside or (E) -resveratrol-3,5,4'-O-? -D-triglycoside. But is not limited thereto.

The introduction of the gene can be carried out by a commonly known gene manipulation method. For example, a physical method such as a method using a vector such as a virus, a non-viral method using a synthetic phospholipid or a synthetic cationic polymer, or an electrotransfer method in which a gene is introduced by applying temporary electrical stimulation to the cell membrane can be used , But is not limited thereto.

As used herein, the term " vector " means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art.

A host cell transformed or transfected with the vector constitutes another aspect of the present invention. As used herein, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. This includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate , Lipofectamine, and dry / inhibition-mediated transformation methods, and the like. The host cell of the invention may be a prokaryotic or eukaryotic cell. In addition, a host having high efficiency of introduction of DNA and high efficiency of expression of the introduced DNA is usually used. Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera prolipida (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting the expression control sequence, a number of factors must be considered. For example, the relative strength of the sequence, controllability and compatibility with the DNA sequences of the present invention should be considered in relation to particularly possible secondary structures. The single cell host may be selected from a selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to fold the protein correctly, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention And ease of purification. Within the scope of these variables, one skilled in the art can select various vector / expression control sequences / host combinations that can express the DNA sequences of the invention in fermentation or in large animal cultures. A binding method, a panning method, a film emulsion method, or the like can be applied as a screening method for cloning cDNA of NSP protein by expression cloning.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example  1: Preparation of recombinant microorganism and enzyme reaction experiment and analysis

UDP-D-glucuronic acid, UDP- N -acetylglucosamine, daidzein, naringenin and pisetin (Sigma-Aldrich, St. Louis, Mo., USA), TDP-L-laminose, GDP-L-fucose, TDP-2-deoxyglucose and TDP-D-biosamine (GeneChem, Daejeon, Korea) were used. All chemicals and reagents were of high chemical grade.

High resolution MS spectra were obtained in cationic mode on ACQUITY (UPLC, Waters Corp., Milford, MA, USA) coupled with SYNAPT G2-S (Waters Corp., Milford, Mass., USA). Compounds were characterized by ¹ H-NMR, ¹³ C-NMR and two-dimensional NMR, COZY, NOESY, ROESY, HSQC and HMBC with 900 MHz Bruker BioSpin NMR in methanol-d ₄ (Sigma). The multiplicity is expressed as s (single line), d (doublet), t (triplet), q (quartet), qn (double line), m (multiplet) and br (broad). The chemical shifts are expressed in ppm and the coupling constants J in Hz.

1-1: Preparation of recombinant microorganisms

Expression and purification of YjiC (Pandey, RP et al., Appl . Environ. Microbiol . , 79: 3516-21, 2013).

YjiC PCR was carried out using primer pairs of YjiC- F (SEQ ID NO: 1) and YjiC- R (SEQ ID NO: 2) using Bacillus licheniformis total DNA as a template and YjiC The gene DNA was amplified:

YjiC F-5'- GGATCC ATGGGACATAAACATATCGCG-3 'BamHI and YjiC R-5'- CTCGAG TTATTTTACTCCTGCGGGTGCTAA-3 ' XhoI ( underlined part represents a restriction site).

After cutting the fragment of 1,191 base YjiC from pGEM®-T vector with BamHI / XhoI, it was cloned in the same site and the pET28a (+) to make a recombinant expression vector pET28-YjiC. pET28- YjiC was transformed into E. coli BL21 (DE3) using a heat shock transformation method.

50 μL of E. coli BL21 (DE3) containing pET28- YjiC was incubated overnight in 50 mL of LB medium and incubated at 37 ° C. until the absorbance at 600 nm (OD ₆₀₀ ) reached 0.5-0.7 Respectively. The culture was shaken for 20 hours and continued to incubate at 20 ° C, and then 0.5 mM isopropyl -? - D-thiogalactopyranoside (IPTG) was injected. Cells were obtained by centrifugation at 3,000 rpm for 10 min, then rinsed twice with 100 mM Tris-Cl (pH 8.0) and resuspended in 1 mL of the same buffer. The cells were sonicated to dissolve and the lysates were purified by centrifugation at 12,000 rpm for 30 minutes at 4 ° C.

1 mL of Ni-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Valencia, Calif., USA) was mixed with the purified lysate and purified according to the manufacturer's instructions. The fragment containing the purified protein was evaluated by 12% SDS-PAGE and concentrated using Amicon Ultra-15 (Millipore, 10K NMWL centrifuge). The purified protein was stored in a buffer containing 100 mM Tris-Cl (pH 8.0) and 20% [v / v] glycerol until use. Protein concentration was confirmed using a Bradford assay kit (Bio-Rad, Hercules, Calif., USA).

1-2: Typical laboratory-scale ( lab - scale ) Glycosyltransferase  Enzyme reaction

YjiC The reactions were carried out in a reaction mixture of 100 μl volume containing 100 mM Tris-Cl (pH 8.0), 4 mM NDP-sugar, 2 mM resveratrol, 10 mM MgCl ₂ and 50 μg of moderately diluted enzyme. The reaction mixture was incubated at 37 [deg.] C for 3 hours. An enzyme-free assay mixture was used as a control. 400 [mu] l of cooled methanol was added to quench and mixed by vortexing for several minutes. The aliquot was then centrifuged at 12,000 rpm to remove the protein, and the reaction product after dilution was directly observed with HPLC-PDA.

1-3: Resveratrol UDP Production scale by -D-glucose ( preparative scale ) reaction

UDP-D- glucose (~ 122 mg) of the purified enzyme YjiC (reaction mixture in a final concentration of 500 ㎍ / ml) final concentration of 20 mM as a donor, 5 mM of resveratrol as a receptor (dissolved in DMSO, 11.4 mg) and 10 It was analyzed using mM of _{MgCl 2 · H Tris-HCl (} pH 8.0) buffer at 100 mM containing O _2. The reaction was carried out at 30 DEG C for 12 hours in a final volume of 10 ml and was stopped by the addition of three volumes of chilled methanol. The reaction mixture was then vortexed and centrifuged at 12,000 rpm for 30 min at 4 < 0 > C to remove the denatured protein. The supernatant was concentrated by evaporation and lyophilization. Finally, the compound was dissolved in 2 mL of HPLC grade methanol and subjected to preparative HPLC purification (Shimadzu, Tokyo, Japan). The purified compound was dried, lyophilized, dissolved in methanol-d ₄ and further analyzed by NMR analysis.

1-4: Desolvation  Reaction array

A desaturase array was performed using resveratrol 3-O- [beta] -D-glucoside as a substrate. Of 100 mM Tris-HCl (pH 8.0 ), it was used a reaction mixture containing the appropriately diluted enzyme in 10 mM UDP, 10 mM of MgCl _2, and 50 ㎍ of. The reaction mixture was incubated at 37 ° C. 10 占 퐇 of reaction samples were taken and prepared as in Example 1-2.

1 -5: Analytical method

Reverse phase HPLC-PDA analysis PDA (308 nm) associated with the C _18; a (YMC-Pack ODS-AQ 4.6 X 150 mm, 5 ㎛) column, 1 mL / min for 25 minutes at a flow rate, H ₂ O (0.1% Trifluoroacetic acid buffer) and 100% acetonitrile (ACN). The concentrations of ACN were as follows: 20% (0-5 min), 50% (5-10 min), 70% (10-15 min), 90% (15-20 min), 10% minute). Purification of the compound was carried out in a C ₁₈ (YMC-Pack ODS-AQ; 150 x 20 mm ID, 10 탆) column connected to a UV detector (308 nm) , 40% (5-10 min), 40% (10-15 min), 90% (15-25 min), 90% (25-30 min), 10% (30-35 min) Program.

Example  2: Resveratrol Glycosylation  And Desolvation

2-1: Resveratrol Glycosylation

Functionally expressed E. coli BL21 (DE3) -derived YjiC protein in soluble materials was purified for use in in vitro reaction assays (Wu, CZ et al., Appl . Environ . Microbiol. , 78: 7680-6, 2012 ; Fig. 2). YjiC (50 ㎍), UDP- D- glucose (5.0 mM) at 37 ℃ for up to 3 hours in a pH 8.0 in 100 mM Tris-HCL buffer, MgCl ₂ (10.0 mM) and resveratrol (dissolved in DMSO, 2.0 mM) Lt; / RTI > Analysis of the cooled methanol quench reaction samples revealed the presence of four different glucosides through a high performance liquid chromatography-photodiode assay detector system (HPLC-PDA; Shimadzu, Japan) (Fig. 3). Analysis of the same reaction mixture by HPLC-PDA combined with high-resolution quantitative Flight Time Electrospray Mass Spectrometry (HRQTOF-ESI / MS) revealed a precise mass of [P _Ⅰ or P _Ⅱ ] + H ⁺ ~ 391.1384 as the monoglucoside of resveratrol (P _I and P _II : 13.32 min and 12.9 min retention time, respectively). Similarly, another prominent peak at 11.4 min (P _Ⅲ ) is the two glucose bound to resveratrol, and the precise mass of [P _Ⅲ ] + Na ⁺ ~ 575.1755 was observed. Peak (P _Ⅳ) left of 10.1 minutes and the tree glucose of ^{_{resveratrol, [P Ⅳ] + Na +}} ~ 737.2266 was (Figure 4, Tables 1 and 2).

HPLC - PDA - HRQTOF - ESI / MS  analysis compound Name of compound Ionic and ionic Calculated m / z The observed m / z
Narinenin

Monoglucoside C ₂₁ H ₂₃ O ₁₀ [M + glucose] + H ⁺
C ₂₁ H ₂₂ NaO ₁₀ [M + glucose] + Na ⁺ 435.1291

435.1111 435.1286

457.1080
Diglycoside C ₂₇ H ₃₃ O ₁₅ [M + 2xglucose] + H ⁺
C ₂₇ H ₃₂ NaO ₁₅ [M + 2xglucose] + Na ⁺ 597.1819

619.1639 597.1801

619.1631 Aglycone C ₁₅ H ₁₃ O ₅ [M +] + H ⁺ 273.0763 273.0755

Physetine

Monoglucoside C ₂₁ H ₂₁ O ₁₁ [M + glucose] + H ⁺ 449.1084 449.1083 The diglucoside (a) C ₂₇ H ₃₁ O ₁₆ [M + 2xglucose] + H ⁺ 611.1612 611.1616 The diglycoside (b) C ₂₇ H ₃₁ O ₁₆ [M + 2xglucose] + H ⁺ 611.1612 611.1613 Triglycoside C ₃₃ H ₄₁ O ₂₁ [M + 3xglucose] + H ⁺ 773.2140 773.2134 Aglycone C ₂₅ H ₁₁ O ₆ [M +] + H ⁺ 287.0556 287.0551
Daid Jane

The monoglucoside (a) C ₂₁ H ₂₁ O ₉ [M + glucose] + H ⁺
C ₂₁ H ₂₀ KO ₉ [M + glucose] + K ⁺ 417.1186
455.0744 417.1187
455.0743 The monoglucoside (b) C ₂₁ H ₂₁ O ₉ [M + glucose] + H ⁺
C ₂₁ H ₂₀ KO ₉ [M + glucose] + K ⁺ 417.1186
455.0744 417.1190
455.0748 Diglycoside C ₂₇ H ₃₁ O ₁₄ [M + 2xglucose] + H ⁺
C ₂₇ H ₃₀ KO ₁₄ [M + 2xglucose] + K ⁺ 579.1714
617.1273 579.1717
617.1274 Aglycone C ₁₅ H ₁₁ O ₄ [M +] + H ⁺ 255.0657 255.0667

HPLC - PDA  Analysis
compound Retention time
Conversion Rate (%) Resveratrol glucoside 1h 2h 3h   Resveratrol 3-O- [beta] -D-glucoside 13.3 19.36 12.35 9.3   Resveratrol 4'-O- [beta] -D-glucoside 12.6 12.2 16.8 18.63   Resveratrol 3,5-O- [beta] -D-di-glucoside 11.4 23.12 42.7 52.79   Resveratrol 3,5,4'-O- [beta] -D-tri-glucoside 10.1 1.3 5.9 9.2 Resveratrol 4'-O- beta -D-galactoside 12.6 13.2 29.8 45.14 Resveratrol 3-O- [beta] -D-2-deoxyglucoside 13.8 9.3 24.65 36.11 Resveratrol 3,5-O- [beta] -D-di-2-deoxyglucoside 13.1 2.89 5.36 8.5 Resveratrol 3-O- [beta] -L-lamboside 13.8 3.98 15.6 21.6 Resveratrol 4'-O- beta -D-biosaminoside 12.6 17.62 42.34 49.1 Resveratrol 3-O-β-L-fucosid 13.8 4.6 11.5 15.4 Resveratrol N -acetylglycosaminoside ND - - Resveratrol glucuronoside ND - - Resveratrol control (control) 15.15 0 0 0

The purified as donor substrate YjiC (500 ㎍ / mL) and 20 mM (~ 122ng) UDP- D- glucose, 5 mM as a receptor (dissolved in DMSO, 11.4 mg) of resveratrol, and 10 mM MgCl _{2 ·} H ₂ O in the Was performed with a 100 mM Tris-HCl (pH 8.0) buffer containing 10 mM Tris-HCl buffer (pH 7.0) in a 10 mL reaction volume. The product was purified as mentioned above and was characterized by other nuclear magnetic resonance (NMR) analyzes including correlation with ¹ H-NMR, ¹³ C-NMR and two-dimensional NMR, correlation spectroscopy (COSY), nuclear over- , Rotation-frame NOE spectroscopy (ROESY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) (FIGS. 5-7). The purified P _I product was analyzed with (E) -1- (3 -? - D-glucopyranosyloxy-5-hydroxyphenyl) -2- (4-hydroxyphenyl) -β-D-glucoside) [1.81 mg, 465 μM, ~9.3%], already confirmed from enzymatic reactions as well as other plant materials. Likewise, the _PII product was obtained from (E) -1- (3,5-dihydroxyphenyl) -2- (4-β-D-glucopyranosyloxyphenyl) ethene (generic name: resveratrol 4'- β-D- glucoside) [3.63 mg, 931.5μM ,, ~ 18.6%] was confirmed, P _ⅲ product is (E) -1- (3,5-Bis -β-D- glucopyranosyl oxyphenyl) (14.58 mg, 2639 [mu] M, ~ 52.8%], which was previously confirmed by another group, with (2- (4-hydroxyphenyl) ethene (generic name: resveratrol 3,5- di-O-? -D-glucoside) will be. P _Ⅳ tree glucoside product is (E) -1- (3,5-Bis -β-D- glucopyranosyl-oxy) -2-a (4-β-D- glucopyranosyl oxyphenyl) ethene ( Resveratrol 3,5,4'-tri-O-? -D-glucoside) [3.28 mg, 460 μM, ~ 9.2%] was confirmed only by mass spectrometry. The lack of mass spectra of the two glucose moieties attached to one another confirms that glucose is attached to three different hydroxyl positions and is not linked to the sugar on a sugar chain. All useful hydroxyl groups of resveratrol produce the production of (E) -1- (3,5-Bis-? - D-glucopyranosyloxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) (Fig. 8). The purity of each product was greater than 95% and the conversion of resveratrol to glucoside was 89.92%. The enzyme exhibited relatively flexibility for all hydroxyl positions of resveratrol. YjiC showed high flexibility when phloretin was a receptor molecule. Thorson's group reported on the flexibility of OleD and its variants (ASP, AIP, TDP16 and 3-1-H12) with resveratrol producing two monoglucosides and two diglucosides. However, no resveratrol triglycoside has been reported. Likewise, a time-dependent study of the reaction indicates that the diglucoside is produced as a major product over time. The concentration of (E) -1- (3 -? - D-glucopyranosyloxy-5-hydroxyphenyl) -2- (4-hydroxyphenyl) ethene decreased with increasing incubation time.

2-2: Flavonoids and Isoflavonoid  have YjiC  Receptor substrate availability

To demonstrate YjiC receptor substrate availability, a lab-scale reaction with other flavonoid species such as, for example, flavanone (naringenin), flavonol (picetin) and isoflavonoid (daidzein) (Fig. 9). All reaction products were analyzed by HPLC-PDA according to HRQTOF-ESI / MS. Various products were observed by HPLC-PDA analysis (Fig. 10). However, by precise mass spectrometry, various glycosylation of all flavonoids produced by YjiC could be observed. [D1] or [D2] + H ⁺ ~ 417.1187) and one diglucoside (D3; [D3] H ⁺ ~ 579.1717). Likewise, two glycosylated products of flavanone (one monoglucoside; N1; [N1] + H ⁺ ~ 435.1286) and one diglucoside (N2; [N2] + H ⁺ ~ 597.1801) , Flavone ((monoglucoside; F1; [F1] + H ⁺ ~ 449.1083), (F2 and F3; diglucoside; [F1] or [F2] + H ⁺ ~ 611.1616) and (triglycoside; F4 ; [F4] + H ⁺ ~ 773.2134)) to observe the four products (Fig. 11). These results show that YjiC accepts other polyphenols including flavonoids, isoflavonoids and stilbenes as receptors for glycosylation and non-regiospecifically transfers them from UDP-D-glucose to other hydroxyl groups Could know. Substrate conversion analysis shows that YjiC can equally promote (> 90%) the glycosylation reaction for all three different flavonoid species including stilbene (Fig. 12).

2-3: Donor substrate availability

Because YjiC has flexibility for different hydroxyl groups and various acceptor matrix flexibility for flavonoid groups, resveratrol as a model receptor substrate has demonstrated donor substrate flexibility for other NDP-sugars. Both NDP-D-sugar and NDP-L-sugar were used for the glycosyltransferase activity of YjiC (Figure 13). Among the NDP-D-sugars, four different kinds of sugar donors were used in comparison with UDP-D-glucose. (UDP-D-galactose), C-6 position-modified glucose (UDP-D-2 deoxyglucose and UDP- N -acetylglucosamine) D-gluconic acid) and C-4 and C-5 position-modified sugars (TDP-D- biosamine ) were used to demonstrate YjiC activity against various NDP-D-sugars. Likewise, two different NDP-L-sugars (TDP-L-lambda nose and GDP-L-fucose) were used under the same conditions in the laboratory-scale reaction.

Preliminary analysis of the laboratory-scale reaction mixture by thin-film chromatography on normal-phase silica plates (F ₂₅₄ , Merck, Darmstadt, Germany) confirmed the presence of additional points rather than standard resveratrol at lower t _R than aglycon 14). The reaction mixture was analyzed by HPLC-PDA, indicating other sugars conjugated with resveratrol at 308 nm and further confirmed by HPLC-PDA combined with HRQTOF-ESI / MS in positive mode (Figure 15 and Table 3 ). When using different sugars, we could get quite different results.

HPLC - PDA - HRQTOF - ESI / MS  analysis compound Name of compound Ionic and ionic Calculated m / z The observed m / z



Glucoside Resveratrol 3-O-? -D-glucoside (P _I ) or resveratrol 4'-O-? -D-glucoside (P _II ) C ₂₀ H ₂₃ O ₈ [M + glucose] + H ⁺ 391.139293 391.1384 Resveratrol 3,5-O- [beta] -D-di-glucoside ( _PIII ) C ₂₆ H ₃₂ NaO ₁₃ [M + 2xglucose] + Na ⁺ 575.174061 575.1755 Resveratrol 3,5,4'-O-β-D- tri-glucoside (P _Ⅳ) C ₃₂ H ₄₂ NaO ₁₈ [M + 3xglucose] + Na ⁺ 737.226885 737.2266

Galactoside
Resveratrol 4'-O- beta -D-galactoside C ₂₀ H ₂₃ O ₈ [M + galactose] + H ⁺
C ₂₀ H ₂₂ NaO ₈ [M + galactose] + Na ⁺ 391.139293
413.121238 391.1402
413.1229

2-deoxyglucoside Resveratrol 3-O- [beta] -D-2-deoxyglucoside C ₂₀ H ₂₂ NaO ₇ [M + 2-deoxyglucose] + Na ⁺ 397.126323 397.1267 Resveratrol 3,5-O- [beta] -D-di-2-deoxyglucoside C ₂₆ H ₃₂ NaO ₁₁ [M + 2x (2-deoxyglucose)] + Na ⁺ 543.184232 543.1867 Lamboside Resveratrol 3-O- [beta] -L-lamboside C ₂₀ H ₂₃ O ₇ [M + rhamnose] + H ⁺ 375.144378 375.1462

Biosaminoside

Resveratrol 4'-O- beta -D-biosaminoside C ₂₀ H ₂₄ NO ₆ [M + viosamine] + H ⁺
C ₂₀ H ₂₃ NNaO ₆ [M + viosamine] + Na ⁺ 374.160363
396.142307 374.1599
396.1412
Foucault
Resveratrol 3-O- [beta] -L-fucosid C ₂₀ H ₂₃ O ₇ [M + fucose] + H ⁺
C ₂₀ H ₂₃ NaO ₇ [M + fucose] + Na ⁺ 375.144378
397.126323 375.1446
397.1263 N-acetylglucosaminoside Resveratrol N-acetylglucosaminoside ND ND ND Glucuronoside Resveratrol glucuronide ND ND ND Resveratrol Aglycone C ₁₄ H ₁₃ O ₃ 229.086469 229.0861

The conversion rate of the substrate as well as the product formation number varied depending on the type of NDP-sugar used in the reaction mixture (FIG. 16). All NDP-D-sugars used in the reaction, except for TDP-2-deoxy-D-glucose, showed a single product with one sugar moiety attached to resveratrol. Due to the availability of excess NDP-sugars and the difficulty of enzyme synthesis, all products were presumably depicted by HPLC-PDA and high resolution precision mass spectrometry. In addition, t _R of the glyco-randomized product was compared to the glycosylated product of resveratrol, which was structurally explained by NMR analysis. Resveratrol 3-O- β-D-2- deoxy-glucoside _{(t R: 13.8 min, [} M + Na] + ~ 397.1267) and resveratrol 3,5-O- β-D-di -2- deoxy glucosyl The seed (t _R : 13.1 min, [M + Na] ⁺ ~ 543.1867) has a low t _R when compared to the respective resveratrol monoglucoside and diglucoside due to deoxy sugar binding. Similarly, resveratrol 4'-O-β-D- galactoside _{(t R: 12.6 min, [} M + Na] + ~ 413.1229) and (t _R resveratrol 4'-O-β-D- non Osaka unexposed seed: 12.6 min, [M + Na] ⁺ ~ 396.1412) has t _R similar to resveratrol 4'-O-? -D-glucoside. The two products were also produced in two independent reaction mixtures. However, no product was found when UDP-D-glucuronic acid and UDP- N -acetylglucosamine were used as sugar donors. Likewise, HPLC-PDA and HRQTOF-ESI / MS when two independent NDP-L-sugars (TDP-L-lambda and GDP-L-fucose) were used to induce independent glycosyltransferase reactions the mono-glycosylated product by analysis resveratrol 3-O-β-L- ramno seed _{(t R: 13.8 min, [} M + H] + ~ 375.1462) and resveratrol 3-O-β-L- fucose seed (t _R : 13.8 min, [M + H] ⁺ ~ 375.1446) (Fig. 15).

The percent conversion of resveratrol was determined using different saccharide donors at different reaction time intervals (Fig. 16). Similar product formation patterns were observed in all the NDP-sugars used in the reaction. The highest conversion of resveratrol to resveratrol glucoside was followed by TDP-D-biosamines (~ 49%), UDP-D-galactose (~ 45%) and TDP-2-deoxy-D-glucose UDP-D-glucose (~ 90%). The TDP-L-laminose and GDP-L-glucose reactions showed relatively low resveratrol conversion rates of 21.6% and 15.4%, respectively. The product formation rate was increased from 1 hour to 3 hours. However, with increasing incubation time, unexpected results were seen in resveratrol 3-O- [beta] -D-glucoside. The concentration of resveratrol 3-O- [beta] -D-glucoside was higher after 1 hour incubation and decreased spontaneously after 2 hours and 3 hours incubation. The decrease in the initially formed product appears to be based on two reasons. The first may be the thermodynamic instability of the product. Second, resveratrol and UDP-D-glucose were synthesized by the reverse of the glycosylation reaction by YjiC as in the case of floretine (Pandey, RP et al., Appl . Environ Microbiol , 79: 3516-21, 2013) Lt; / RTI > Resveratrol 3-O- [beta] -D-glucoside used the same enzyme and was converted to one of the diglycosides or triglycosides.

2-4: Resveratrol  Glucoside Desolvation

In order to demonstrate the instability of resveratrol 3-O-? -D-glucoside in the reaction mixture, resveratrol 3-O-? -D-glucoside derived glucose was converted to UDP-D-glucose and resveratrol agglutinin by the inverse of the glycosylation reaction. A desolvation reaction, which is carried out in UDP, was carried out to form a ricon. HPLC-PDA analysis of the dehydroxylation reaction mixture at different time intervals indicates the reaction state. The compound resveratrol 3-O- [beta] -D-glucoside slowly decreased to 20% of the initial amount within 1 hour after incubation at 37 [deg.] C. Resveratrol was accumulated, increasing the concentration in the reaction mixture (Figure 17). After 12 h and 24 h incubation, the reaction mixture was incubated with spontaneous (E) -1- (3,5-dihydroxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) Ethene and (E) -1- (3,5-Bis-? -D-glucopyranosyloxyphenyl) -2- (4-hydroxyphenyl) ethene. The UDP-D-glucose and resveratrol formed after desalting of resveratrol 3-O- [beta] -D-glucoside can be used by YjiC to produce a newly formed product in the dehydroxylation reaction mixture. However, no triglycoside (E) -1- (3,5-Bis-? -D-glucopyranosyloxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) , Which contributes to the deficiency of excess UDP-D-glucose in the reaction mixture. Through this, the enzyme can desaturate resveratrol glucoside, thus producing UDP-D-glucose and resveratrol, which is eventually used by the same enzymes to produce other products (FIG. 17). Thus, the product formed is thermodynamically more stable than resveratrol 3-O- [beta] -D-glucoside. (E) -1- (3,5-dihydroxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) ethene and -β-D-glucopyranosyloxyphenyl) -2- (4-hydroxyphenyl) ethene is relatively higher than that of resveratrol 3-O-β-D-glucoside HPLC-PDA analysis (Figure 3). Conversely, resveratrol 3-O- beta -D-glucoside after continuous glycosylation at the C-5 hydroxyl position of resveratrol 3-O- beta -D-glucoside is another thermodynamically more stable (E) -1- 3,5-Bis- [beta] -D-glucopyranosyloxyphenyl) -2- (4-hydroxyphenyl) ethene.

2-5: Prediction of Essential Amino Acids for Reaction Catalysts

Oleandomycin Glycosyltransferase (OleD) (PDB database no. 2IYA) derived from the characterized Streptomyces antibiotics and calicheamicin glycosyltransferase derived from Micro Monospore Echinospora (PDB database no. 3IA7) were selected as templates based on high similarity and homology to the YjiC structural model using Accelrys Discovery Studio 3.1 software (Accelrys, San Diego, USA) (Fig. 18). PSI-BLAST (Comparison Matrix, BLOSUM 62; E-threshold, 10) using ExPASy server (2010) was used to find the homogeneity of YjiC . Both templates OleD and CalG4 were selected based on their high similarity to YjiC. Adjust the amino acid sequence of a YjiC OleD and CalG4, and using the built homologous model of Discovery Studio 3.1 (build homology model) was used to build the model, and finally created a model by MODELLER. For the YjiC model, the UDP ligand was obtained from OleD (2IYA) and placed in a similar position as in the template OleD. Final YjiC The model was made using molecular dynamics after active site optimization and was used for docking resveratrol with UDP as an orientation ligand such as OleD (Figure 19). For the valid model of YjiC, resveratrol was docked using LigandFit (Discovery Studio). Twenty-five different poses were made (Monte Carlo Trails) to select the optimal resveratrol binding mode to produce different forms of resveratrol in the YjiC protein. in The silico studies have shown remarkable results with respect to important amino acids present around the YjiC active site. The amino acid residues facing the substrate bound to the pocket were His16, Phe111, Ser144, Ile145 and His298 (Fig. 20). The amino acids His16 and His298 are important in the glycosyltransferase activity of enzymes including OleD His19, VvGTl His20 and MurG His19 (Zhou, M. et al., Synthesis , 8: 1301-6, 2006; hu, YN et al, Proc Natl Acad Sci USA, 100:..... 845-9, 2003). Histidine serves as a Bronsted base for activating the oxygen of the polyphenol hydroxyl group for nucleophilic attack at the C1 position of the sugar of the NDP-sugar donor. His350 and Ser355 interact with the? -Phosphate of UDP of VvGTl and the oxygen of? -Phosphate (Offen, W. et al., EMBO J. , 25: 1396-1405, 2006). Similarly, His293 and Thr298 of GtfA interact with similar oxygen of UDP phosphate (Mulichak, AM et al., Proc . Natl . Acad . Sci . USA , 100: 9238-43, 2003). We found His 298 adjacent to? -Phosphate of YjiC UDP. His298 residue was also adjacent to the C-4 'hydroxyl group of resveratrol (Figures 19 and 20). In addition, His16 is another important amino acid that has proven to play an important role in most glycosyltransferase proteins, which is adjacent to the C-3 hydroxyl position of resveratrol. However, substrate resveratrol has two binding modes, similar to resveratrol written by OleD (head-to-tail precedence). These results can be seen in glycosylation at the 3,5 and 4 'hydroxyl positions of resveratrol. These results are consistent with the in vitro response to the discovery of the production of resveratrol diglycoside and triglycoside. Other essential amino acids in the donor substrate bound to the pocket include His14, Thr234, Asn302, Ser303 and Glu306 adjacent to the UDP ligand (Figure 18). Detailed studies of protein structure and mutagenesis studies are essential to reveal the mode of activity of the correct amino acid and bacterial glycosyltransferases.

Example  3: Resveratrol  And objection Glycosylated  Effect of derivatives

3-1: Cell viability of mouse macrophages

The effect of naturally occurring resveratrol and its glycosylated derivatives on cell viability was evaluated by measuring the activity of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide in rat macrophage RAW 264.7 cells MTT) microculture tetrazolium viability assay. Resveratrol showed significant cytotoxicity against RAW 264.7 cells (Fig. 21A). However, resveratrol, a glycosylated derivative of resveratrol, 3-O- beta -D-glucoside and (E) -1- (3,5-dihydroxyphenyl) -2- (4-β-D-glucopyranosyl -Oxyphenyl) ethene did not decrease cell viability up to 100 / / ml, whereas (E) -1- (3,5-dihydroxyphenyl) -2- (4 -? - D-glucopyranosyloxy Phenyl) ethene showed no detectable levels of cytotoxicity at 150 [mu] g / ml. These results show that resveratrol glycosylation at one of the 3 or 4 ' positions obliterates resveratrol toxicity to macrophages and that glycosylation at the 4 ' position is more effective than 3-position in reducing resveratrol cytotoxicity Could know. Improved cell viability of resveratrol will increase the water solubility of resveratrol glucoside, thereby reducing cell membrane permeability compared to aglycone.

3-2: RAW  By 264.7 cells IL -6 secretion

Resveratrol modulates lymphocyte proliferation at low concentrations (<12 μM) (Feng, YH et al., Acta Pharmacol Sin, 23: 893-7, 2002) IL-6 production of the immunological response because the association that it throttles (Diehl, S. et al, Immunol , 39:. 531-6, 2002), resveratrol and its glycosides We tested whether the misfolded derivative could stimulate IL-6 secretion. Enzyme binding immunoabsorption analysis showed that resveratrol at low concentrations (50 and 75 μg / ml) slightly stimulated IL-6 secretion compared to the control (DMSO treated cells), but at relatively high concentrations (100 and 150 μg / ml) Of resveratrol significantly reduced IL-6 secretion by dose. In contrast, both resveratrol 3-O- beta -D-glucoside and (E) -1- (3,5-dihydroxyphenyl) -2- (4-β-D-glucopyranosyloxyphenyl) -6 secretion significantly (Fig. 21B). However, IL-6 secretion pattern was very different depending on the position of glycosylation. Treatment of (E) -1- (3,5-dihydroxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) ethene slightly reduced the concentration of Il-6, while resveratrol 3- O- [beta] -D-glucoside dramatically induced IL-6 secretion in a dose-dependent manner (Fig. 20). These results demonstrate that glycosylation of resveratrol clearly controls IL-6 production in macrophages compared to aglycone resveratrol and that 3-position glycosylation of resveratrol exhibits more effective immunostimulatory activity than 4 ' -position glycosylation Could know. However, the glycosylated resveratrol derivative resveratrol 3-O- beta -D-glucoside and (E) -1- (3,5-dihydroxyphenyl) -2- (4-β-D-glucopyranosyloxy Phenyl) ethene significantly increased IL-6 secretion by dose, suggesting that these glucoside derivatives lose immunostimulatory activity through upregulation of IL-6 secretion while losing the natural anti-inflammatory activity of resveratrol .

3-3: RAW  264.7 intracellular NO  production

NO is an important cellular signaling molecule involved in many physiological and pathological processes and is produced by activated macrophage-induced nitric oxide synthase (iNOS), which exhibits immunomodulatory effects (Detmers, PA et al., J. Immunol ., 165: 3430-5, 2000). Therefore, the effect of resveratrol and its glucoside on NO production was examined using RAW 264.7 cells in the presence of different concentrations of the compound. Relatively low concentrations (50 [mu] g / ml) resveratrol aglycon are more effective in NO production than glycosylated derivatives (Fig. 21C). However, the NO concentration significantly decreases with increasing concentrations of resveratrol (75-150 [mu] g / ml), which is similar to the results of IL-6 secretion. Given the effect of resveratrol on cell viability, a dose-dependent decrease in IL-6 and NO concentrations may be due to cytotoxicity to RAW 264.7 cells. However, glycosylated derivatives are more effective for NO production compared to relatively high concentrations (75-150 [mu] g / ml) resveratrol (Fig. 21). (E) -1- (3,5-dihydroxyphenyl) -2- (4 -? - D-glucopyranosyloxyphenyl) ethene was administered at a concentration of 75-100 占 퐂 / -D-glucoside, although the effect on NO and IL-6 concentrations is comparable to that of resveratrol, the effect is reversed at 150 μg / ml, which is somewhat different from the result of IL-6 secretion different. These results demonstrate that glycosylation of resveratrol apparently modulates NO and IL-6 in macrophages compared to resveratrol aglycon. Significant increases in dose of NO production by resveratrol glucoside means that the immunostimulatory activity is obtained earlier by upregulating NO production while losing the natural anti-inflammatory activity of resveratrol.

It has been shown that glycosylation of resveratrol eliminates the cytotoxicity of resveratrol and that glycosylated resveratrol derivatives have a clear effect on the production of NO and IL-6 in macrophages compared to resveratrol aglycon, demonstrating a beneficial effect on early immune stimulation. Although the novel resveratrol analog has not been proven, it has been found that it has improved biological activity when combined with various sugars.

Experimental data are expressed as mean ± SEM (standard mean error). Unpaired Student's t-tests were compared between groups. A p-value less than 0.05 was considered statistically valid.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> SUNMOON UNIVERSITY INDUSTRY- UNIVERSITY COOPERATION FOUNDATION <120> Preparing Resveratrol Glucoside <130> P13-B241 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> YjiC-F <400> 1 ggatccatgg gacataaaca tatcgcg 27 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YjiC-R <400> 2 ctcgagttat tttactcctg cgggtgctaa 30

Claims (7)

Wherein the resveratrol is reacted with the sugar donor in the presence of UDP-glycosyltransferase.
The method of claim 1, wherein the UDP-glycosyltransferase is a recombinant microorganism into which the gene of the enzyme is introduced.
2. The method according to claim 1, wherein the gene is YjiC .
The method of claim 1, wherein the microorganism is E. coli .
According to claim 1, UDP- glycosyl transferase dehydratase is Bacillus piece you miss formate (Bacillus licheniformis . &lt; / RTI &gt;
The method of claim 1, wherein the sugar donor UDP-D- glucose, TDP-D- rainy samin, UDP-D- galactose, TDP-D-2- deoxy-glucose, UDP- N - acetylglucosamine, UDP-D- gluconic Wherein the resveratrol glucoside is any one selected from the group consisting of citric acid, gluconic acid, gluconic acid, gluconic acid, gluconic acid, gluconic acid, gluconic acid, gluconic acid,
The method of claim 1, wherein the resveratrol glucoside is selected from the group consisting of (E) -resveratrol-3-O- beta -D-glucoside, (E) 3,5-O-? -D-glucoside, 3,5-O-? -D-glucoside, and 3,5-O-? -D-glucoside. Seed production method.

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