KR101836419B1 - Gramisterol Glucoside Compound, Method of Preparing the Same and Pharmaceutical Composition Comprising the Same - Google Patents

Abstract

The present invention relates to a glycomerol glucoside compound, a process for producing the same, and a pharmaceutical composition containing the same. The sterol glycoside- derived enzyme derived from microorganisms of Micromonospora rhodorangea according to the present invention is expressed in Escherichia coli Glomeric-3- O- β-glucoside enzymatically biosynthesized using a novel recombinant sterol glycosyltransferase (MrSGT1) was found to be more potent than glamisterol, which is the original compound, in the mouse murine adenocarcinoma tumor cell line WEHI-3 The anti-tumor effect is relatively improved, the skin whitening activity similar to that of arbutin, which is a whitening ingredient, in the melanin cell line, and the effect of improving some wrinkles of skin through the activity of inhibiting elastase enzyme, It can be used as medicine.

Description

Gramisterol Glucoside Compound, Method of Preparing the Same and Pharmaceutical Composition Comprising the Same,

The invention that lamina sterol glucoside compound, a preparation method thereof, and relates to a pharmaceutical composition comprising the same, and more particularly, to micro-mono spokes la also ditch Jia (Micromonospora rhodorangea), wherein the blood cancer sterol dangjeon of Streptomyces origin is used for the enzyme To a method for producing the same, and a pharmaceutical composition containing the same.

Plant-derived phytosterol is an essential component of plant cell membranes and is structurally similar to cholesterol biosynthesized in animals. Recently, a variety of biological activities have been reported and have attracted attention in the health functional foods, cosmetics and pharmaceutical industries. Representative phytosterols include beta-sitosterol, campesterol, and stigmasterol, and are contained in many plant oils and vegetable oils (Ostlund et al., Annu . Rev. Nutr . 22 : 533-549, 2002). Although the cholesterol lowering effect and the angiogenic effect of phytosterol have been reported and studied since the 1950's, the effect on the cardiovascular diseases such as angina pectoris, myocardial infarction and cerebrovascular disease closely related to the cholesterol concentration in the body and its mechanism have been scientifically proven There is no bar yet (Weingartner et al., Eur . Heart J. 30 (4): 404-409, 2009).

The development of pharmacological activity through the structural modification of phytosterol and the development of a drug candidate are recognized as key technologies for the development of health functional foods as well as improved new drugs. One of them is the hydroxyl group (especially C-3 position) There is an enzymatic production method using a sterol glycosyltransferase (SGT) capable of transferring a sugar molecule. The specific covalent bonding of such a specific compound can further improve its solubility and its absorbability at the time of bioavailability compared to the original substance, and thus can be used for the improvement of formulations of the functional food and medicine, the development of new formulations, and the prodrug have.

SGT, a sterol glycoside enzyme, is classified into the first family (family 1) among 94 kinds of glycosyltransferases in the CAZY (Carbohydrate Active enZYmes) database. Most of the SGT-encoding genes, plant-based and (Madina et al, Biochim Biophys Acta 1774: 392-402, 2007; Warnecke et al, plant Molecular Biol 35:....... 597-603, 1997; Potocka et al, Acta Biochim . Polonica 55: 127-134,2008; Debolt et al., Plant Physiol . 151: 78-87, 2009). Sterol glucoside (SG), which is biosynthesized through the enzymatic reaction of sterol glycoside, is distributed in microorganisms and some animals in addition to plants. These sugar moieties include triterpenoids, steroidal alkaloids and the like, most of which are structures in which a sugar is bonded to a hydroxyl group located at carbon number 3 in the steroid structure.

Gramisterol (GR), which is known to be present in the cereal bran, vegetable oil, fruit kernel oil or sugarcane shell of grapes as one of the phytosterols, 4-methylsterol (4-methylsterol) in which the methyl group is added to the carbon atom of the structure (Jeong et al., Lipids 10: 634-640, 1975; Willuhn et al., Planta Med . 46: 99-104, 1982; Feong et al., J. Sep. Sci. 37: 1308-1314, 2014; Suttiarporn et al., Nutrients 7: 1672-1687, 2015). In recent years, anti-tumor pharmacological activity of GR derived from rice bran of Thai rice bran has been reported in a tumor cell line associated with myelogenous leukemia and a mouse model of blood cancer. (Somitara et al ., PLoS One 11: e0146869, 2016). Immune enhancing activity has also been reported in the same experimental group. However, there has been no report of a GR derivative to which a sugar has been added to the 3-position hydroxyl group including GRAMSTERYL GLYCOSIDE (hereinafter referred to as GRG) to which a sugar has been added to the GR. Likewise, The preparation of sugar-bearing compounds is not known to date.

The present inventors have found that, in addition to the main phytosterol as well as the phytosterol derived from seaweed, fucosterol is added to a sugar-added phos- pherol through a sugar-added phytosterol (Korean Patent Application No. 10-2015-0104324 entitled "Method for producing sterol glucoside using sterol- Has succeeded in the conversion of phytosterol derivatives. For this purpose, to the phytosterol as a substrate dangjeon the discovery of micro mono spokes la also ditch Jia (Micromonospora rhodorangea) transferase per actinomycetes-derived novel (GT), enabling the reaction, the biosynthesis of the assignment via an enzymatic reaction phytosterol, and further new Compared with the original compound fucosterol, fucosiderol glucoside, which is a derivative of one sugar moiety, has been found to have anti-cancer and anti-diabetic complications.

Accordingly, the present inventors have found that GRAM is a biosynthesis of GRAM as a glycomerol derivative to which sugar is added by utilizing a recombinant glycosyltransferase (MrSGT1) derived from Micromonospora rhodorangea actinomycetes, As a result, by comparing the in vitro antitumor activity against the hematological tumor cell line WEHI-3, the inhibitory activity against melanin biosynthesis in human melanoma cell line and the inhibitory activity against elastase, The present inventors have confirmed that it is possible to improve the skin whitening effect and to improve the skin wrinkle while having tumor activity, and thus the present invention has been completed.

It is an object of the present invention to provide a method for biosynthesizing glycosaminoglycan derivatives, which can be used for the treatment of hematologic malignancies while providing pharmacological activity as a medicament for blood cancer-related cell lines, and a method for biosynthesizing recombinant glycosyltransferase (MrSGT1) It is an object of the present invention to provide such

In order to accomplish the above object, there is provided a pharmaceutical composition comprising the glamisterol glucoside compound represented by the general formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof:

[Chemical Formula 1]

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.

The present invention also provides a pharmaceutical composition comprising: (a) reacting glamisterol and a sugar donor in the presence of a sterol glycosyltransferase (MrSGT1) represented by the amino acid sequence of SEQ ID NO: 4 to produce glamisterol 3- O -glucoside ; Provides a process for the preparation of glucoside - the lamina sterol 3- O represented by the following formula (1) comprising the step of recovering the glucoside - and (b) the synthesis of the 3- O lamina sterols.

The present invention also provides a pharmaceutical composition for preventing or treating blood cancer, comprising as an effective ingredient, a lamysterol glucoside compound represented by the formula (1), an isomer thereof, or a pharmaceutically acceptable solvate, hydrate or prodrug thereof .

The present invention also provides a cosmetic composition for skin whitening or wrinkle improvement comprising as an active ingredient a lamellistol glucoside compound represented by the formula (1), an isomer thereof, or a pharmaceutically acceptable solvate, hydrate or prodrug thereof .

The glycomerol glucoside enzymatically biosynthesized by using the recombinant sterol glycosyltransferase (MrSGT1) in which the sterol glycosyltransferase derived from microorganisms of Micromonospora rhodorangea is expressed in E. coli according to the present invention, And exhibits an antitumor effect as a cell line subject, it is useful as a medicament for the prevention or treatment of a blood cancer-related disease. In addition, it exhibits an inhibitory activity against melanin biosynthesis in human melanoma cell line and an enzyme inhibitory activity against elastase, thereby being useful as a cosmetic composition for skin whitening and skin wrinkle improvement.

Brief Description of the Drawings Fig. 1 is a schematic diagram showing an enzymatic reaction of the pertussis transferase MrSGT1. Fig.
FIG. 2 is a chromatogram showing the analytical result of a high-pressure liquid chromatography-electrospray ionization-mass spectrometry instrument of a grammasterol glucoside compound through a glycosidase reaction (A: B: Recombinant glycoprotein MrSGT1, substrate GRAMSterol, and UDP-Glc were reacted with each other to obtain a mixture of ethyl acetate extract ).
FIG. 3 is a graph showing antitumor activity against mice-derived blood cancer tumor cell line WEHI-3 in the treatment with lamivudine glucoside compound and a comparative gramicosterol.
FIG. 4 is a graph comparing melanin biosynthesis inhibitory activities in human melanoma cell lines with invitro of the ramisterol glucoside compound and arbutin, a positive control.
5 is a graph comparing the degree of inhibition of elastase enzyme activity of erucic sterol glucoside compound and ascorbic acid, which is a positive control, with ascorbic acid.

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.

The present invention separates the GT-encoding gene from the genome of the microorganism monospora rhodorania strain, and produces a recombinant expression vector and a transformed E. coli containing the GT-encoding gene. Then, the expression of the sterol-conjugated enzyme MrSGT1 Uridine, a sterol (GR) and a nucleotidyl sugar, was reacted with uridine diphosphate glucose (UDP-Glc) as a sugar donor to biosynthesize a sugar-added GRG compound. Next, the structure was confirmed by analyzing mass spectrometer and nuclear magnetic resonance spectrometry instrument.

Accordingly, one aspect of the present invention relates to the gramicosterol glucoside compound represented by formula (I), an isomer thereof, or a pharmaceutically acceptable solvate, hydrate or prodrug thereof.

[Chemical Formula 1]

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a glycerol derivative and a sugar donor which are reacted with each other in the presence of a sterol glycosyltransferase (MrSGT1) represented by the amino acid sequence of SEQ ID NO: O -glucoside; Relates to a process for the preparation of glucoside - the lamina sterol 3- O represented by the following formula (1) comprising the step of recovering the glucoside - and (b) the lamina sterol 3- O The above synthesis.

In the present invention, the sterol glucoside represented by the general formula (1) is preferably glamysterol-3- O- beta -glucoside, but the present invention is not limited thereto.

The compound of formula (I) according to the present invention may be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, lactic acid, maleic acid, pumaric acid, gluconic acid, Succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid and the like can be used. Sulfuric acid may be preferably used.

The compounds of formula (I) according to the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

In addition, the compound of Formula 1 according to the present invention may be prepared in crystalline form or amorphous form, and may be arbitrarily hydrated or solvated when the compound of Formula 1 is prepared in crystalline form.

In the present invention, the saccharide donor is preferably UDP-glucose (Glc), UDP-galactose (Gal) or TDP-2'-deoxy-Glc, but is not limited thereto. In addition, examples of the sugar added include monosaccharides such as glucose, mannose, galactose, allose, altrose, idose, talose, ), Fructose, arabonose or xylose, and the amino sugars may be N-acetyl-galactosamine, N-acetyl-glucosamine acetyl-neuraminic acid, glucosamine or galactosamine, and in the case of di-saccharide, sucrose, ), Cellobiose, maltose, lactose, trehalose, gentiobiose or melibiose, and in the case of tri-saccharide, raffinose but are not limited to, raffinose or gentianose.

The step (b) may be carried out using a reversed phase C18 stationary phase, mobile phase mixture of methanol: acetonitrile: water: formic acid 45: 30 to 50:10 to 20: 0.1 to 0.5 (v / v / v / v) Preferably a reverse phase C18 stationary phase, methanol: acetonitrile: water: formic acid 45: 40: 14.8: 0.2 (v) / v / v / v) mixed liquid mobile phase and 15-16.6 min retention time conditions.

In order to confirm the biochemical activity of the glycomylated glucoside derivative (GRG) to which sugar has been added, the present inventors examined in vitro the antitumor activity of the original compound GR and its derivative GRG, , And the blood cancer antitumor activity of the GRG derivative to which glucose was enzymatically added was confirmed.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating blood cancer, comprising as an active ingredient a lamellar sterol glucoside compound represented by the general formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof ≪ / RTI >

The present invention relates to a method for inhibiting the biotin-melanin biosynthesis inhibition activity of a glomeric glucoside derivative (GRG) to which a sugar has been added and a melanin biosynthesis inhibitory activity against albumin which is a whitening component of a GRG derivative to which glucose is enzymatically added Or skin whitening activity.

Accordingly, the present invention relates to a cosmetic composition for improving skin whitening activity comprising, as an effective ingredient, the lamysterol glucoside compound represented by the general formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof .

The present invention also relates to a method for inhibiting the enzyme-inhibiting activity of glabrider glucoside derivative (GRG) to which melanoma cell line has been added, Wrinkle improving activity.

Accordingly, the present invention provides, in a further aspect, a cosmetic composition for improving skin wrinkles comprising the glamisterol glucoside compound represented by the general formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof as an active ingredient .

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

The term "prophylactic " used in the present invention means any action that inhibits or delays a cancerous disease by the administration of a pharmaceutical composition comprising the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof. The term "treatment ", as used herein, refers to any treatment that improves or alleviates the symptoms of a blood cancer disease by administering a pharmaceutical composition comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof it means.

The compound of formula (I) or a pharmaceutically acceptable salt thereof of the present invention exhibits antitumor activity against a blood cancer-associated tumor cell line and can be useful for the prevention or treatment of a related cancer disease.

The pharmaceutical compositions of the present invention may be formulated into oral or parenteral administration forms according to standard pharmaceutical practice. These formulations may contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, adjuvant or diluent.

When the composition of the present invention is used as a medicine, a pharmaceutical composition containing at least one compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally or parenterally But are not limited thereto.

The formulations for oral administration may be, for example, tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like, (E.g., silica, talc, magnesium salt of stearic acid, calcium salt of stearic acid, and / or polyethylene glycol), such as sodium chloride, dextrose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine have. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt The same disintegrating or boiling mixture and / or absorbing agent, coloring agent, flavoring agent, and sweetening agent.

Formulations for parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. In this case, in order to formulate the composition for parenteral administration, the pharmaceutical composition of the present invention contains at least one compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof, which is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, Which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and / or contain preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances and may be mixed, granulated Or a coating method.

The dose of the compound of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. In general, when referring to an adult patient weighing 70 kg, Day, preferably 0.01 to 500 mg / day, and may be administered once to several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist.

The present invention also relates to a sterol glycosyltransferase (MrSGT1) represented by the amino acid sequence of SEQ ID NO: 4.

The present invention relates to a polynucleotide encoding said enzyme.

In the present invention, the polynucleotide may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 3, wherein the polynucleotide is derived from a strain of Micromonospora rhodorangea .

The present invention relates to an expression vector comprising the polynucleotide.

The present invention relates to the polynucleotide or a recombinant microorganism into which the expression vector is introduced.

In the present invention, the microorganism is preferably Escherichia coli, but is not limited thereto.

The present invention relates to a method for producing a recombinant sterol-conjugated enzyme (MrSGT1) comprising culturing the microorganism to induce expression of a sterol glycosyltransferase and recovering the enzyme.

As used herein, "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the 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. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several to several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector, (C) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using a calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982). As a vector used for overexpression of the gene according to the present invention, an expression vector known in the art can be used. The framework vectors that may be used in the methods of the present invention include, but are not limited to, various vectors capable of transforming into E. coli selected from the group consisting of pT7, pET / Rb, pGEX, pET28a, pET-22b Can be used.

A nucleotide sequence is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide, and the promoter or enhancer is a sequence Or the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site is arranged to facilitate translation, Lt; / RTI > sequence. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host. Preferably the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector should further comprise a useful expression marker in the eukaryotic expression host.

The host cells transformed with the recombinant vectors described above constitute 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.

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.

It will be apparent to those skilled in the art that the gene has the same effect as that of introducing the recombinant vector into the host cell by inserting the gene into the genome chromosome of the host cell.

In the present invention, the gene may be inserted into the chromosome of the host cell by a commonly known gene manipulation method. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector , A poxvirus vector, a lentiviral vector, or a nonviral vector.

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.

[Example]

Example  One: Micro Monospore Rhodolajia  From the dielectric SGT  Isolation of the coding gene

In order to isolate the sterol glycosyltransferase from the microorganism Monospora rhodorans, first, the genome isolated from the microorganism Monospora rhodorans strain was used as a template, The polymerase chain reaction (PCR) was carried out using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, which were prepared based on the nucleotide sequence, as follows. The N-terminal primer is the sequence of SEQ ID NO: 1 below and the C-terminal primer is the sequence of SEQ ID NO: 2 below.

[SEQ ID NO: 1] 5'-GG CATATG ATCCACGCGCACGACTTCCGGATG-3 '( Nde I restriction site)

[SEQ ID NO: 2] 5'-CG CTCGAG ATTCGGCCTGCGCCTCCCACGTCCA-3 '( Xho I restriction site)

The genomic DNA of the strain and the primers were mixed with Taq DNA polymerase to perform a total of 32 cycles of PCR (initial inactivation was performed at 98 for 5 min, 52.8 to 69.3 for 72, Termination after reaction). PCR product was purified and ligated to pGEMR-T easy vector (pGEMR-T easy vector, Promega, Madison, Wis., USA) and then ligated to E. coli XL1 blue (XL1 blue, Stratagene, La Jolla, For 45 seconds and then transformed. After isolating and purifying the T-iso vector from the selected transformed E. coli, the nucleotide sequence thereof (SEQ ID NO: 3) and the amino acid sequence deduced therefrom (SEQ ID NO: 4) were determined. The nucleotide sequence of the entire ORF is 1185 bp, and the translated protein is composed of 394 amino acids (total 41.4 kDa).

Example  2: Recombinant sterol in E. coli The party  enzyme MrSGT1's  Expression

The MrGT1 DNA fragment obtained by treating the T-isovector of Example 1 with Nde I and Xho I restriction enzymes was subjected to restriction enzyme digestion with pET-28 (a +) (Novagen, Madison, WI, USA ), And then transformed into E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA). At this time, 50 ppm of kanamycin antibiotics was used for selection of transformants.

The recombinant Escherichia coli was inoculated in the 1% by volume ratio to the LB medium (Luria Bertani) supplemented with the above-mentioned antibiotic and 1 M sorbitol and 2.5 mM betaine, and then cultured at 37. D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo., USA) having a final concentration of 0.5 mM was added to adjust the optical density to 0.6 to 0.8 When growth was confirmed, the protein expression was induced and the recombinant E. coli strain was further cultured for 20 to 24 hours. After the culture, the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and then the cells were dissolved in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) Sonication was performed. After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected separately and analyzed by 12% SDS-PAGE to confirm the expression level of the recombinant sterol-conjugated MrSGT1.

The purified supernatant was diluted with a Talon-metal affinity resin (Clontech, Mountain View, CA, USA) equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) to purify the expressed recombinant protein. USA) and cultured for one hour at 4 ° C. After refrigerated centrifugation at 2000 rpm for 5 minutes, the resin was introduced into a disposable column and washed with phosphate buffer solution containing 20 mM imidazole in a volume of 10 times the resin. Finally, the recombinant sterol-conjugated enzyme MrSGT1 bound to the resin was purified with 3 ml of phosphate buffer containing 180 mM imidazole.

Example  3: MrSGT1  By recombinant enzyme reaction Graham sterol  Biosynthesis, separation and purification of glucoside compounds

50 mM of glamisterol (Leancare Ltd., Flintshire, UK) used as a substrate was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of 1 mM was adjusted with a reaction buffer (50 mM phosphate buffer, 10 mM magnesium chloride) After the dilution, 30 μM of the enzyme MrSGT1 and 2 mM of UDP-glc per nucleic acid were added to the enzyme, and the enzyme reaction was carried out at 37 for 2 hours (FIG. 1). After the reaction, an equal amount of ethyl acetate was added to stop the reaction. The reaction mixture was centrifuged at 3000 rpm for 5 minutes, and only the upper organic solvent layer was collected and vacuum dried. High pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC) analysis was performed to confirm the biosynthesis of the target compound in the extract. (Acquity CSH C18 (Waters, 50 x 1.0 mm, v / v / v) as a mobile phase at a rate of 150 μl / min in methanol: acetonitrile: water: formic acid 45:40:14.8: 1.7 μm; Milford, Mass., USA) (FIG. 2).

On the other hand, the following Combi Flash Rf medium pressure liquid chromatography (MPLC) system was used for the isolation and purification of the desired glycomicosterol compound from the crude extract. To an MPLC system in which a mixed liquid of methanol: acetonitrile: water: formic acid 45: 40: 14.8: 0.2 (v / v / v / v) was flowed to a mobile phase at a rate of 8 ml per minute in a reverse-phase C18 cartridge, After injecting the crude extract dissolved in 5 ml of mobile phase, the peak detected on the chromatogram was automatically collected. The individual fractions were further concentrated under reduced pressure, and then analyzed for their mass spectra using an ion trap mass spectrometer (LCQ ion-trap mass spectrometer, ThermoFinnigan, San Jose, Calif., USA) to isolate the desired glycerol sterols. Gramicosterol used as the substrate was aliquoted at a retention time of about 20.4 minutes on the MPLC system and the desired glucuronidereol compound was detected at 15-16.6 minutes, which is a faster retention time than the substrate. The above glucose-addition compound GRAMISSTEROL GLUCOSIDE (GRG; GRAMIS STEREO 3- O -GLUCOSIDE) was finally purified to 4.3 mg.

NMR samples were prepared by dissolving the compound in 200 μl of CD ₃ OD and then leaving the solvent in 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, Mo.). ¹³ C NMR spectra were acquired at 298 K using a Varian INOVA 500 spectrophotometer and chemical shifts were recorded in ppm using TMS as an internal standard. All NMR data were calculated using Mnova Suite 5.3.2 software and compared to the GRAMIS sterol using the ¹³ C-NMR spectrum of the glucose addition glyceryl stearate (GRAMIS sterol 3- O -glucoside) as substrate One).

location Graham sterol Gramic sterol-3- O -? - glucoside δ _c δ _c One 36.9 37.2 2 30.1 28.3 3 76.4 90.3 4 41.3 39.6 5 52.1 52.3 6 31.7 31.8 7 117.5 117.5 8 139.4 139.4 9 50.0 50.0 10 42.7 42.6 11 21.2 21.2 12 39.6 39.6 13 45.3 45.2 14 56.4 56.5 15 23.8 23.7 16 27.7 27.6 17 56.7 56.8 18 14.2 14.2 19 14.6 14.7 20 36.3 36.3 21 19.6 19.6 22 34.1 34.1 23 31.4 31.4 24 156.8 156.7 25 33.9 34.0 26 & 27 21.7 21.7 28 105.3 105.3 29 13.2 13.5 One' 110.2 2' 74.3 3 ' 76.8 4' 71.6 5 ' 81.4 6 ' 62.3

Example  4: Graham sterol  Identification of Antitumor Activity of Glucoside Compounds in Mouse Pancreatic Cancer Tumor Cell Lines

In order to test the antitumor activity of the GRAMIC sterol 3- O -glucoside compound produced in Example 3, WEHI-3, a tumor cell line of mouse myelogenous leukemia, was tested for its antitumor activity by MTT method Respectively. As a comparative group, the tumor cell line was used in the same concentration gradient as that of the tumor cell line in which the anticancer activity was already reported.

First, WEHI-3 (ATCC, Rockville, USA), which is a murine canine monocytic hematological cancer cell line, was activated. Each cell line was cultured in RPMI 1640 medium containing 100 ppm streptomycin, 10 ppm penicillin, 1% glutamine and 10% fetal bovine serum in a 5% carbon dioxide atmosphere at 37. WEHI-3 cells on the first day of culturing were dispensed into a 96-well microplate at a level of 1.5 x 10 ⁵ per well and the cells were treated with the comparative group (GRAMIS STEROL, GR) and the compound of Example 3 (GRAMISSTEROL 3- O- Glucoside, and GRG) were dissolved in 10% DMSO to final levels of 0.25, 0.5, 1, 2, 4, and 8 μM, respectively. After incubation, the supernatant was replaced with MTT solution, color reaction was carried out at 37 for 4 hours, and the IC ₅₀ (50% cell growth) for WEHI-3 cell line was quantified by quantifying the degree of color development with a final microplate reader The concentration of the compound causing the inhibition) was measured.

As a result, as shown in FIG. 3, the comparative group, GRAMIS sterol, showed antitumor activity according to the concentration gradient with respect to the blood cancer cell line WEHI-3. The IC ₅₀ of graham sterol was about 2.61 μM when cultured for 24 hours. On the other hand, the treated group, gravimetrol 3- O -glucoside, exhibited an IC ₅₀ value of about 2.17 μM lower than that of the original substance gravimetrol in the above-described blood cancer tumor cell line. As a result, it was confirmed that the gravidosterol 3 - O - glucoside compound, which is a novel derivative of glyoxylated glyoxylate, exhibits an improved antitumor activity over that of glyoxylate. This difference is due to the addition of a saccharide to the chemical structure of the relatively hydrophobic gravimetrol, which results in hydrophilicity lower than that of the original material, ie, gravimeterol 3 - O - Glucoside is expected to improve the permeability of WEHI-3 cells.

Example  5: Graham sterol  The human glucoside compound Melanoma  Confirmation of Melanin Inhibitory Activity on Cell Lines

In order to test the melanin production inhibitory activity of the gramicosterol 3- O -glucoside compound produced in Example 3, a B16 / F10 melanoma cell line was used. That is, DMEM medium containing 10% FBS was dispensed at a density of 2000 cells / well in a 96-well plate, and then cultured at 37 ° C in a concentration of 5% carbon dioxide for 24 hours. After incubation, the medium was replaced with DMEM medium containing 2 μM α-melanocyte stimulating hormone and 2 mM theophylline, respectively, and the cells were sequentially seeded at the final concentrations of 50, 100, 300 and 500 μM (DMSO dissolution) Diluted gravimerol 3- O -glucoside was treated three times daily for three days. After incubation for 5 days, the medium was first removed, treated with 0.25% trypsin-EDTA (Sigma) solution to recover melanoma cells, and then the supernatant was removed by refrigerated centrifugation at 10,000 rpm for 10 minutes. The recovered cells were dried at 55 ° C, and 100 μl of 1 N sodium hydroxide solution was added thereto to dissolve intracellular melanin. The cells were diluted with phosphate buffer solution and the absorbance was measured with a final 475 nm microplate reader (Molecular Devices). As a control, DMSO was used as a positive control, and arbutin at a level of 300 μM (DMSO dissolution) was used as a positive control. Melanin biosynthesis inhibitory activity was represented by a bar graph in FIG. 4 in terms of the following formula 1.

[Equation 1]

As a result, as shown in FIG. 4, melanin biosynthesis inhibitory activity of melanoma cell line was about 43% in the positive control group arbutin, and the lamysterol 3- O -glucoside compound showed four different concentrations (50, 100, 300 and 500 μM), the inhibitory activity was inhibited in a range of about 16 to 56% in a concentration-dependent manner. In particular, it was confirmed that at 300 μM, which is the same as that of positive control arbutin, melanin biosynthesis inhibitory activity (about 43%) similar to arbutin, which is a conventional whitening agent, was found.

Example  6: Graham sterol  Of the glucoside compound Elastase  Confirm inhibitory activity

The wrinkle-reducing effect was evaluated by comparing the elastase inhibitory activity of the lamastinol 3- O -glucoside compound produced in Example 3 above. Elastase is in the elastase (Sigma) derived from porcine pancreas, the substrate is N-succinyl- [L-alanine- alanine-alanine] - p -nitroanilide (Sigma) after 20 minutes reaction at 25 using, 405nm Absorbance was measured with a microplate reader (Molecular Devices). As positive control group, ursolic acid (Sigma) and ascorbic acid were used as negative control and DMSO for sample dissolution was used as a negative control group. Elastase inhibition was expressed as the concentration of compound required to reduce the absorbance by 50% relative to the negative control (IC ₅₀ ).

As a result, as shown in Fig. 5, the positive control group, ascorbic acid, showed an IC ₅₀ of about 0.09 mM while ascorbic acid was about 4.96 mM. Gramicosterol 3- O -glucoside compounds showed an IC ₅₀ of about 0.22 mM. Glamisterol 3- O -glucoside, which is a sugar-transferred polysaccharide, exhibits a wrinkle-reducing effect to some extent by exhibiting a higher inhibitory activity of elastase than ascorbic acid, while being lower than a positive control group of ascorbic acid having stronger elastase inhibitory activity .

As shown in the above results, it is possible to improve the existing compounds through structural modification by the sugar transfer of existing pharmacologically active compounds. Such improvements can be achieved by prodrug substitution of existing drugs, a new dosage form, The potential for the use of pharmacological activity as an advanced drug substance was confirmed.

Thus, the compounds produced in the above Examples were formulated as follows.

Formulation example  1: Tablet (direct pressurization)

After 5.0 mg of the compound was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone (USNF), and 0.1 mg of magnesium stearate were mixed and pressed to prepare tablets.

Formulation example  2: Tablet (wet assembly)

After 5.0 mg of the compound was sieved, 16.0 mg of lactose and 4.0 mg of starch were mixed. After 800.3 mg of polysorbate was dissolved in pure water, an appropriate amount of this solution was added to make it finely pulverized. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The mixture was pressurized to prepare tablets.

Formulation example  3: Powder and Capsule

5.0 mg of the compound was sieved, followed by mixing with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

Formulation example  4: Injection

180 mg of mannitol, 26 mg of Na ₂ HPO _{4 12} H ₂ O and 2974 mg of distilled water were added to 100 mg of the active ingredient to prepare an injection.

As shown in the above results, it is possible to improve the existing compound through transformation of the structure-modified derivative by sugar transfer of the existing pharmacologically active compound, and as such, as a substitute prodrugation and a new dosage form of the existing drug The possibility of application as a raw material for pharmaceuticals and medicines including the applicability of the present invention can be confirmed.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the invention will be defined by the claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Sterol Glucosides Using Sterol          Glucosyltransferase <130> P15-B198 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> P1 <400> 1 ggcatatgat ccacgcgcac gacttccgga tg 32 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> P2 <400> 2 cgctcgagat tcggcctgcg cctcccacgt cca 33 <210> 3 <211> 1185 <212> DNA <213> Micromonospora rhodorangea <400> 3 atgcgcatcc tcatcgccac cgccggctcg cggggcgacg tggccccgta caccggcctc 60 ggcgcggccc tgcggcgcgc ggggtacgac gtggccgtcg ccacgaccga cacgttcgcc 120 cggatggtcc gggacgccgg cctggggttc cgccggctcc cggccgacac ccgcggccac 180 gcgggcgtgc gctccaaccg cgaggtcgtg cgcgcggccg gcgttcaccg cggaactggg 240 ccagggcttc gccgacgcgc cgtctcggag ggcacggacc tcctgctgct gtcgacgacc 300 accgcgccgc tggggtggct catcggcgag gcgacgggca tccggtccct cggggtctac 360 ctccagccca ccgcccccac cgccgacttc ccgccggtcg tcaccggcgc gcgctccctg 420 gggcggctcg gcaaccgcac ggccggccgc ctggccctgc gcatggccga ccggatctac 480 gccgaggccg tcgcgggcct gcgggagcgg ctgtgcctgc cgccgctgtc cgcggccgcg 540 atgcgggcgc ggcaggagcg ggccggctgg cccgtcctgc acggcttcgg cacggccctc 600 gtcccgcggc cggccgactg gcgccccggc ctcgaggtcg tggggccgtg gtggccgcac 660 ccgaggccg gcgagcgcct gccctcggaa ctcgaggact tcctggacgc gggaccgcgg 720 ccggtcctgg tgggcttcgg cagcatggcc gccggggacg gcgagcggct cagcgaactg 780 gcggtgcgcg cgctgcgccg cgccggcctc cgcggcatcc tccagtccgg caacgcctgc 840 ctcgcggcgg agggcgacga cgtcctgacg gtcggcgacg tcccccacgc cctcctgttc 900 ccccggctcg cggcggtggt gcaccacgcg cgccggcacc agcgccgcga ccctgcgggc 960 ggccgtcccc tcggtggcgg tcccggtgac cgccgaccag agccgttctg ggcgggccgg 1020 ctggcgcgga tcggggccgc ccccgccccg gtccccttca ccacgctgac cgcggacggg 1080 ctcgccggcg ccctgggccg cgtcgtcggc gacgaggcgt acgcgcgggc ggccgagaag 1140 gccgcccggc acctggccgc ggaggacggc gccgcgcgcg tgtga 1185 <210> 4 <211> 394 <212> PRT <213> Micromonospora rhodorangea <400> 4 Met Arg Ile Leu Ile Ala Thr Ala Gly Ser Arg Gly Asp Val Ala Pro   1 5 10 15 Tyr Thr Gly Leu Gly Ala Ala Leu Arg Arg Ala Gly Tyr Asp Val Ala              20 25 30 Val Ala Thr Thr Asp Thr Phe Ala Arg Met Val Arg Asp Ala Gly Leu          35 40 45 Gly Phe Arg Arg Leu Pro Ala Asp Thr Arg Gly His Ala Gly Val Arg      50 55 60 Ser Asn Arg Glu Val Val Arg Ala Ala Gly Val His Arg Gly Thr Gly  65 70 75 80 Pro Gly Leu Arg Arg Ala Val Ser Glu Gly Thr Asp Leu Leu Leu                  85 90 95 Leu Ser Thr Thr Ala Pro Leu Gly Trp Leu Ile Gly Glu Ala Thr             100 105 110 Gly Ile Arg Ser Leu Gly Val Tyr Leu Gln Pro Thr Ala Pro Thr Ala         115 120 125 Asp Phe Pro Pro Val Val Thr Gly Ala Arg Ser Leu Gly Arg Leu Gly     130 135 140 Asn Arg Thr Ala Gly Arg Leu Ala Leu Arg Met Ala Asp Arg Ile Tyr 145 150 155 160 Ala Glu Ala Val Ala Gly Leu Arg Glu Arg Leu Cys Leu Pro Pro Leu                 165 170 175 Ser Ala Ala Met Arg Ala Arg Gln Glu Arg Ala Gly Trp Pro Val             180 185 190 Leu His Gly Phe Gly Thr Ala Leu Val Pro Arg Pro Ala Asp Trp Arg         195 200 205 Pro Gly Leu Glu Val Val Gly Pro Trp Trp Pro His His Hisp Ala Gly     210 215 220 Glu Arg Leu Pro Ser Glu Leu Glu Asp Phe Leu Asp Ala Gly Pro Arg 225 230 235 240 Pro Val Leu Val Gly Phe Gly Ser Met Ala Ala Gly Asp Gly Glu Arg                 245 250 255 Leu Ser Glu Leu Ala Val Arg Ala Leu Arg Arg Ala Gly Leu Arg Gly             260 265 270 Ile Leu Gln Ser Gly Asn Ala Cys Leu Ala Ala Glu Gly Asp Asp Val         275 280 285 Leu Thr Val Gly Asp Val Pro His Ala Leu Leu Phe Pro Arg Leu Ala     290 295 300 Ala Val Val His His Ala Arg Arg His Gln Arg Arg Asp Pro Ala Gly 305 310 315 320 Gly Arg Pro Pro Leu Gly Gly Gly Pro Gly Asp Arg Arg Pro Glu Pro Phe                 325 330 335 Trp Ala Gly Arg Leu Ala Arg Ile Gly Ala Ala Pro Ala Pro Val Pro             340 345 350 Phe Thr Thr Leu Thr Ala Asp Gly Leu Ala Gly Ala Leu Gly Arg Val         355 360 365 Val Gly Asp Glu Ala Tyr Ala Arg Ala Ala Glu Lys Ala Ala Arg His     370 375 380 Leu Ala Gla Asp Gly Ala Ala Arg Val 385 390

Claims (11)

The grammicolide glucoside compound represented by the formula (1) or a pharmaceutically acceptable solvate or hydrate thereof:
[Chemical Formula 1]

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.
The grammysterol glucoside compound or a pharmaceutically acceptable solvate or hydrate thereof according to claim 1, wherein the glamisterol glucoside compound is glamisterol-3- O- beta -glucoside.
A process for producing glamisterol 3- O -glucoside represented by the following formula 1, comprising the steps of:
(a) synthesizing glamisterol 3- O -glucoside represented by the following formula 1 by reacting glamisterol and a sugar donor in the presence of a sterol glycosyltransferase (MrSGT1) represented by the amino acid sequence of SEQ ID NO: 4; And
(b) recovering said synthesized glamisterol 3- O -glucoside:
(I)

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.
4. The method according to claim 3, wherein the sugar donor is UDP-glucose, UDP-galactose or TDP-2'-deoxyglucose.
4. The method of claim 3, wherein the lamina sterol 3-O- glucoside compound is prepared characterized in that the lamina of sterol -3- O -β- glucoside.
The method according to claim 3, wherein the step (b) is carried out using a reversed phase C18 stationary phase, a mobile phase mixture of methanol: acetonitrile: water: formic acid 45: 30 to 50:10 to 20: 0.1 to 0.5 (v / v / v / v) Characterized in that a medium pressure liquid chromatography (MPLC) is used for 10 to 20 minutes of holding time.
A pharmaceutical composition for prevention or treatment of blood cancer, comprising as an effective ingredient, a grammysterol glucoside compound represented by the general formula (1) of claim 1 or a pharmaceutically acceptable solvate or hydrate thereof:
(I)

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.
8. The pharmaceutical composition according to claim 7, wherein the glamisterol glucoside compound is glamisterol-3- O- beta -glucoside.
8. A pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
A cosmetic composition for skin whitening or wrinkle improvement comprising the grammysterol glucoside compound represented by the general formula (1) of claim 1 or a pharmaceutically acceptable solvate or hydrate thereof as an active ingredient:
(I)

In Formula 1, R ₁ is glucose, 2-deoxy-glucose, or galactose.
11. The cosmetic composition according to claim 10, wherein the glamisterol glucoside compound is glamisterol-3- O- beta -glucoside.

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