KR101750263B1 - Method for Preparing Genkwanin Using Biotransformation of Apigenin - Google Patents

Abstract

The present invention relates to a method for producing genkyn and nin by biological conversion of apigenin, and more particularly, to a method for producing genkyn and nin by the action of an O -methyl transferase (hereinafter referred to as " (SAM synthetase) gene that synthesizes the SaOMT2 gene coding for O-methyltransferase and S-adenosine-L-methionine, which is a cofactor of the enzyme, is used And to a method for producing Genkwanin from Apigenin.
According to the invention, the 7-hydroxyl group (7-hydroxyl group) with O to forward methyl (methyl) of a flavonoid - SaOMT2 gene and a cofactor of the enzyme adenosine design S- methyl transferase encoding the enzyme (O-methyltransferase) Genkwanin is produced from Apigenin with a high conversion rate by using a microorganism variant into which a SAM synthetase gene that synthesizes L-methionine (S-adenosine-L-methionine) is introduced .

Description

[0001] The present invention relates to a method for preparing genkwanin by bioconversion of apigenin,

The present invention relates to a method for producing genkyn and nin by biological conversion of apigenin, and more particularly, to a method for producing genkyn and nin by the action of an O -methyl transferase (hereinafter referred to as " (SAM synthetase) gene that synthesizes the SaOMT2 gene coding for O-methyltransferase and S-adenosine-L-methionine, which is a cofactor of the enzyme, is used And to a method for producing Genkwanin from Apigenin.

Flavonoids are low-molecular polyphenolic compounds present in plants that act to protect plants from predators, pathogens, and ultraviolet radiation (Dixon et al ., Mol. Plant Pathol. , 3: 371, 2002) and plays an important role in plant growth and development (Frick et al ., Phytochem . , 56: 1, 2001). Further, radical scavenging, anti-inflammatory, antimutagenic, anti-HIV, anti-allergic, it has some biological activities, including anti-platelet, anti-oxidant, an anti-neurodegeneration activity shows a chronic disease prevention effect (Manach et al ., Curr . Opin . Lipidol. , 16: 77, 2005; Heo and Lee, J. Agric . Food Chem . , ≪ / RTI > 53: 1445, 2005; Heo and Lee, J. Agric . Food Chem ., 52: 7514, 2004; Mojzisova and Kuchta, Physiol . Res. , 50: 529 2001; Middleton et al ., Pharmacol . Rev. , 52: 673,2000).

The functional diversity exhibited by flavonoids is due to the modification of core structures generated by various hydroxylases such as glucosyltransferases, methyltransferases, prenyltransferases, and sulfotransferases in the hydroxyl group at the center of flavon ( Martens et al ., Phytochem ., 71: 1040, 2010; Prescott et < RTI ID = al . , Annual Rev Plant Physiol Plant Mol . Biol ., 47: 245,1996). The biological role of phenolic compounds is governed by the type and location of the functional group attached to the flavonoid complex and changes its reactivity, solubility, and ability to interact with other chemicals or protein structures (Buer et al ., J. Integr . Plant Biol ., 52: 98, 2010).

Methyltransferases play an important role after post-translational modification of natural products. O by Enzyme-methylation (O -methylation) are different O-methyl transferase is catalyzed by the enzyme (O -methyltransferases, OMTs), ether (methyl ether) derivatives and S- adenosyl-homocysteine (S-adenosyl-L methionine (AdoMet) is transferred to the specific hydroxyl group of the acceptor compound (Ibrahim et < RTI ID = 0.0 > al ., Plant Mol . Biol ., 36: 1, 1998; Joshi et al . , Plant , Mol. Biol . , ≪ / RTI > 37: 663,1998). The O - methylation of the flavonoids changes the chemical reactivity of the phenolic hydroxyl groups to produce antibacterial activity (Middleton et al . , Champman & Hall, London, 619 , 1994 (harborne, JB (Ed)) to increase the intracellular compartment, as well as the film and increase the absorption of the lipophilic compound by giving increasing the transport capacity to be (Wen et al . , Drug Metab . Dispos ., 34: 1786, 2006; Walle, Int . J. Mol . Sci . , 10: 5002, 2009).

Genkwanin is a non-glycosylated flavonoid with a genkwa flos ( Daphene Genkwa Sieb. Meat. Zucc), Rosemary ( Rosmarinus officinalis L.) and Cistus laurifolius L. ( Pharmacopoeia of People's Republic of China , P148, 2010; Altinier G. et al. , J Agric Food Chem. , 55: 1718-1723, 2007; Sadhu SK et al . , J Ethnopharmacol, 108: 371-378, 2006).

Previous studies have shown that genk and nin are able to inhibit the growth of 17β (3-kinase) through regulation of anti-bacterial, antiplasmodial, radical scavenging, chemopreventive and miR-101 / MKP- And inhibition of the activity of 17β-Hydroxysteroid dehydrogenase type 1 (Cottiglia F et al. , Phytomedicine , 8: 302-305, 2001; Martini ND et al . , J Ethnopharmacol , 93: 207-212, 2004; Kraft C et al . , Phytother Res. , 17: 123-128, 2003; Kim AR et al. , Phytother Res , 18: 1-7, 2004; Suh N et al ., Anticancer Res. , 15: 233-239, 1995; Brozic P et al . , Mol Cell Endocrinol , 301: 229-234, 2009; Gao Y et al . , PLOS one , 9: 96741, 2014).

In connection with the metabolism due to the low oral bioavailability (oral bioavailability) and instability of flavonoids in vivo (in vivo ) flavonoids (Wen X et al . , Drug Metab . Dispos , 34: 1786-1792, 2006). It is known that this is due to the radical conjugation of glucononation and sulfation of the free hydroxyl groups of most flavonoid compounds.

Methylation of the free hydroxyl group of the flavonoids promotes absorption by promoting the metabolic stability and membrane transport of the flavonoids to promote absorption and promote oral bioavailability (Walle T, Int . J. Mol . Sci. , 10: 5002-5019, 2009).

According to recent studies by the present inventors, 7-hydroxy-8-methoxyflavone, unlike the non-methylated 7,8-dihydroxyflavone, And showed antioxidant activity at concentrations that did not affect survival of endothelial cells (Koirala N et < RTI ID = 0.0 > al . , J Biotech , 184: 128-137, 2014a). In addition, methylated isoflavonoids, rhamnetin and sakuranetin, showed higher anticancer and anti-angiogenic actions than the unmethylated form, and the methylated forms showed higher metabolic stability, oral bioavailability oral bioavailability) and biological activity.

Several methylated flavonoids have been shown to be capable of inhibiting carcinogen activating enzymes, effecting multidrug resistance proteins (MRPs), fungicidal properties, and other pharmacological applications (Wen X et al. , Drug Metab . Dispos , 34: 1786-1792, 2006; Bernini R et al . , Molecule S , 16: 1418-1425, 2011; Middleton E. Jr et al . , Pharmacol Rev. , ≪ / RTI > 52: 673-751,2000; Harborne JB et al . , Phytochemistry , 55: 481-504, 2000).

On the other hand, although plants are natural sources of genk and nin, mass production using plant extraction method, tissue culture, and mass production using chemical synthesis methods have not yet been put to practical use.

E. coli or yeast ( Saccharomyces cerevisiae ) and the like are transformed to biosynthesize compounds (Lim EK et al ., Biotechnol Bioeng , 87: 623631, 2004; Trantas E et al ., Metab Eng , 11: 355-366, 2009), for example, by using PFNS-1 (flavone synthase) and POMT-7 (flavone-7- O -methyltransferase) (Jeon MY et < RTI ID = 0.0 > al . , J Microbiol Biotechnol ., 19: 491-494, 2009).

Under these technical backgrounds, the present inventors have made intensive efforts to develop a method for producing genk and nin through the bioconversion of apigenin using apigenin, which is generally used in the manufacture of complex flavonoids. As a result, group (7-hydroxyl group) of methyl O to forward (methyl) - S- design cofactor adenosine SaOMT2 gene and the enzyme-methyl transferase encoding the enzyme (O-methyltransferase) -L- methionine (S-adenosine-L- (Genkwanin) can be prepared from Apigenin at a high conversion rate by using a microorganism variant into which a SAM synthetase gene is synthesized. .

It is an object of the present invention to provide a novel O -methyl transferase (SaOMT2) capable of efficiently methylating a flavonoid and a SAM synthetic enzyme (SOM) which synthesizes S-adenosine-L-methionine The present invention provides a method for producing genk and nin from apigenin using a microorganism variant in which a gene encoding a SAM synthetase is introduced.

In order to achieve the above object, the present invention is (a) 7- hydroxyl groups (7-hydroxyl group) with O to forward methyl (methyl) of the flavonoid-methyl transferase (O-methyltransferase: SaOMT2) gene encoding the Adenosine-L-methionine synthetase (S-adenosine-L-methionine synthetase), which synthesizes S-adenosine-L-methionine Culturing the transformed microorganism variant in a medium containing Apigenin to convert Apigenin to Genkwanin; And (b) obtaining the transformed genome and genkwanin. The present invention also provides a method for producing genkwanin.

The present invention also provides a pharmaceutical composition for treating cancer comprising genkwanin and genkwanin as an active ingredient.

The present invention also provides a pharmaceutical composition for treating an angiogenesis-related disease, comprising Genkwanin and Genkwanin as an active ingredient.

According to the invention, the 7-hydroxyl group (7-hydroxyl group) with O to forward methyl (methyl) of a flavonoid - SaOMT2 gene and a cofactor of the enzyme adenosine design S- methyl transferase encoding the enzyme (O-methyltransferase) Genkwanin is produced from Apigenin with a high conversion rate by using a microorganism variant into which a SAM synthetase gene that synthesizes L-methionine (S-adenosine-L-methionine) is introduced .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a method for producing genkwanin in transformed microorganism variants. FIG.
FIG. 2 shows a chromatogram of the genkwan and Genkwanin produced by chromatography and spectroscopy.
Fig. 3 is data obtained by mass-analyzing genkwanin and Genkwanin by QTOF-ESI / MS.
FIG. 4 is a graph showing temperature, pH using a fermentor, and concentration of Apigenin and the amount of genkwanin production (production) depending on the incubation time while controlling the oxygen level dissolved in the TB medium .
5 is a graph showing the effect of Genkwanin on the growth of various cancer cells.
6 is in vitro (in the HUVECs (umbilical vein endothelial cells) vitro) illustrates the effect of Genk and Nin (Genkwanin) on blood vessel formation (angiogenesis), (A) depicts the effects of Genk and non on endothelial cell invasion (endothelial cell invasion), (B ) is of HUVECs This shows the effect of genk and nin on capillary tube formation.

In the present invention, a gene encoding a SAM synthetase, which synthesizes a SaOMT2 methyltransferase gene and S-adenosine-L-methionine, which is a cofactor of the enzyme, is introduced into a microorganism, At the fermentor scale, which can produce genkwanin and genkwanin at a high conversion rate from the genus Apigenin, Microorganism variants having a conversion efficiency of 85% (170 μM (48.28 mg / L) genckinin) were prepared.

Therefore, the invention in one aspect, the present invention is (a) 7- hydroxyl groups (7-hydroxyl group) with O to forward methyl (methyl) of the flavonoid-methyl transferase (O-methyltransferase: SaOMT2) the coding Adenosine-L-methionine synthetase (S-adenosine-L-methionine synthetase), which synthesizes S-adenosine-L-methionine , Transforming Apigenin into Genkwanin by culturing the transformed microorganism variant in a medium containing Apigenin; And (b) obtaining the converted genk and genkwanin. The present invention also relates to a method for producing genkwanin.

In the present invention, O - methyl transferase (O-methyltransferase: SaOMT2) a gene encoding the Streptomyces cis Abbe reumi subtilis (Streptomyces avermitilis , and the gene coding for S-adenosine-L-methionine synthetase is E. coli K12.

The O - methyl transferase which is SaOMT2 Bahia using the S- adenine -L- methionine production by the S- adenine -L- methionine biosynthetic pathway genin (O-methyltransferase) (Apigenin) in Genk and Nin (Genkwanin) And methylation.

In the present invention, the microorganism may be E. coli , but is not limited thereto.

In the present invention, Apigenin may be added at a concentration of 100 to 1000 μM.

As used herein, the term " isoflavonoid methoxides "refers to methylated compounds of isoflavonoids, and the term" Genkwanin ", which refers to the methylated compound of the isoflavonoid, Apigenin, ) "And" Apigenin methoxides "are not different from each other and are used in this specification.

In one embodiment of the present invention, the Escherichia coli mutant is S-adenine-L-methionine produced by an enzyme involved in S-adenine-L-methionine biosynthesis, and when Apigenin is added to the Escherichia coli mutant, Methyltransferase can finally produce genckinin through the process of converting apigenin to genkwanin using the S-adenine-L-methionine produced above as a methyl donor (Fig. 1) .

In one embodiment of the present invention, Escherichia coli mutants were reacted with Apigenin in order to confirm whether the Escherichia coli mutant of the present invention produced Genkwanin from Apigenin and analyzed by HPLC. As a result, As shown in Fig. 2, the residence time (t _R ) of apigenin standard material and genk and nin was 10.0 to 10.6 minutes and 11.6 to 11.8 minutes, and the conversion rate from apigenin to genk and nin .

Further, as shown in FIG. 3, the quantitative determination of the produced genk and nin was analyzed by the QTOF-ESI / MS method. As a result, the total m / z value of the separated compounds was 285.0845 in the case of genk and nin, It was confirmed that apigenin was genetically modified by genetic mutants.

In another embodiment of the present invention, the conversion yield of Genk and Nin was determined by adding Apigenin at 100 μM, 200 μM, 300 μM, 400 μM, 500 μM and 1000 μM in order to increase the yield of Genk and Nin from apigenin using E. coli mutants , And 200 μM of apigenin was added thereto. When the reaction was carried out for 48 hours, it was confirmed that Genck and Nin were produced at a concentration of 142 μM (data not shown). On the other hand, when Escherichia coli mutants were cultured using LB medium, TB medium and M9 medium and apigenin was reacted, the apigenin conversion yield was 78% when the Escherichia coli mutant was cultured in the TB medium And OD _600nm (cell density) was also highest in TB medium.

In another embodiment of the present invention, an attempt was made to analyze the production amount (yield) of genkwanin from Apigenin in a scale-up system using a fermenter according to the manufacture of Genkwanin. As a result, as shown in Fig. 4, when the Escherichia coli mutant was reacted with apigenin in a 3 L fermentation broth under conditions showing a high conversion yield from apigenin to genk and nin, the conversion from apigenin to genk and nin As a result, it was confirmed that apigenin at a concentration of 200 μM was converted to 85% genckinin after 48 hours and finally converted to genckinin at 170 μM (48.28 mg / L).

In another aspect, the present invention relates to a pharmaceutical composition for treating cancer comprising genkwanin and genkwanin as an active ingredient.

In the present invention, the type of cancer includes all kinds of cancer known in the prior art such as gastric cancer, liver cancer, brain tumor, skin cancer, breast cancer, lung cancer, colon cancer and the like. In addition, prevention or treatment of cancer means prevention of cancer growth and metastasis at the same time by inhibiting endothelial cell proliferation, and furthermore, cancer can be prevented. Preferably, the cancer is gastric cancer, liver cancer, melanoma, glioblastoma, but is not necessarily limited thereto.

In another embodiment of the present invention, there is provided a method of screening for the biological activity (efficacy) of Genkwanin in melanoma (B16F10), gastric cancer (AGS), liver cancer (HepG2), glioblastoma (U87MG) (HUVECs) treated with genk and nin showed that their cell growth was reduced as shown in FIGS. 5 and 6. In particular, Genk and Nin were found to inhibit cancer cell lines (B16F10, HepG2, U87MG ) Growth inhibition effect.

In another aspect, the present invention relates to a pharmaceutical composition for treating an angiogenesis-related disease, comprising Genkwanin and Genkwanin as an active ingredient.

In another embodiment of the present invention, chemoinvasion and capillary tube formation assays have been performed to analyze the biological activity of Genkwanin and Genkwanin. As a result, as shown in Fig. 6, Genkwanin and Genkwanin were found to efficiently inhibit VEGF-induced invasion. Especially at the same concentration, Genk and Nin showed higher anti-neovascular efficacy than Apigenin.

Based on the above results, it was confirmed that Genck and Nin are candidates for the prevention and treatment of cancer having anti - cancer and anti - neovascular action superior to apigenin.

The composition comprising Genkwanin of the present invention can be formulated or used in combination with medicines such as antihistamines, antiinflammatory agents, anticancer agents and antibiotics already used.

The term "treating ", as used herein, unless otherwise indicated, refers to reversing, alleviating, or progressing one or more symptoms of the disease or condition to which the term applies, ≪ / RTI > As used herein, the term "treatment" refers to an act of treating when "treating" is defined as above.

The term " pharmaceutical composition "or" pharmaceutical composition "refers to a mixture of other chemical components, such as diluents or carriers, with a compound containing Genkwanin of the present invention.

The term "physiologically acceptable" is defined as a carrier or diluent that does not impair the biological activity and properties of the compound.

The term "carrier" or "vehicle" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of an organism.

The term "diluent" is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also dilutes in water to which the compound is dissolved. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, since it mimics the salt state of the human solution. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compounds.

The compounds containing Genkwanin, as used herein, are administered as a pharmaceutical composition, either alone or in combination with other active ingredients, such as in a combination therapy with a suitable carrier or excipient, to a human patient .

Pharmaceutical compositions or pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount refers to an amount of a compound that is effective in prolonging the survival of an object to be treated or preventing, alleviating or alleviating the symptoms of the disease. The determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

A therapeutically effective amount of the compounds of the present invention containing Genkwanin can be determined early from cell culture assays. For example, a dose can be calculated in an animal model to obtain a circulating concentration range that includes IC ₅₀ (half maximal inhibitory concentration) or EC ₅₀ (half maximal effective concentration) determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

The dosage of genkwanin can be varied within the above range depending on the dosage form employed and the route of administration used. The exact estimate, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al ., 1975, "The Pharmacological Basis of Therapeutics ", Ch. 1 p. 1).

The preferred dosage of Genkwanin of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. Typically, the dose range of the composition to be administered to a patient can be from about 0.5 to 1000 mg / kg of the patient's body weight. However, in order to obtain the desired effect, it is preferable that the genk and nin of the present invention is administered at a daily dose of 0.0001 to 200 mg / kg, preferably 0.001 to 100 mg / kg. The administration may be carried out once a day, or in several divided doses orally. The dose is not intended to limit the scope of the invention in any way.

The Genkwanin of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, when provided as a mixture containing components other than genkwanin, the composition contains 0.001 to 99.9% by weight of genk and nin containing the aromatic essential oil based on the total weight of the composition, 0.1% by weight to 99% by weight, and more preferably 1% by weight to 50% by weight.

The pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The term " pharmaceutically acceptable salt "means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The terms "hydrate "," solvate ", and "isomer" The pharmaceutical salt may be prepared by dissolving a compound containing Genkwanin of the present invention in an organic solvent such as mineral acid such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p- With organic carboxylic acids such as acetic acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like . Also, by reacting a compound containing a nanden with nenin of the present invention with a base, an alkali metal salt such as an ammonium salt, sodium or potassium salt, a salt such as an alkaline earth metal salt such as calcium or magnesium salt, a dicyclohexylamine, N -Methyl-D-glucamine, tris (hydroxymethyl) methylamine, and amino acid salts such as arginine, lysine and the like.

The pharmaceutical composition or medicinal composition containing Genkwanin according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, Suppositories, and sterile injectable solutions. Examples of carriers, excipients and diluents that can be included in the composition containing genk and nin include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate ), Sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 60, cacao paper, laurin, glycerogelatin and the like can be used.

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  One: O - Methyltransferase ( SaOMT2 ) And SAM  Synthetic enzyme ( EcSAM synthetase ) ≪ / RTI > and recombinant microorganism production

1-1: O - Methyltransferase (SaOMT2)  The production of a recombinant vector containing the coding gene and SaOMT2 Production capacity  Fabrication of recombinant microorganisms

The O used in the embodiment-methyl transferase (SaOMT2, O-methyltransferase; GenBank Accession NP_823558) a gene encoding based on the known sequence Streptomyces cis Abbe reumi subtilis (Streptomyces avermitilis ) (Kim et al . , Agric . Food . Chem . , 54: 823-828, 2006). The aOMT2 fragment was obtained by PCR using primer pairs aOMT2-F '(SEQ ID NO: 1) and SaOMT2-R' (SEQ ID NO: 2) based on the above gene sequence.

[SEQ ID NO: 1] SaOMT2-F'-5'- GGATCC GATGAGCTGCCGCACCGGCA-3 'BamHI

[SEQ ID NO: 2] SaOMT2-R'-5'- GAATTC TCAGCCAACCGCGCGCAGTTCGA-3 'EcoRI

(The underlined part indicates the restriction enzyme site)

PCR using the above primer pair was carried out under the following conditions: the initial denaturation was 95 ° C and the denaturation step (95 ° C for 1 minute), the annealing step (60 ° C for 1 minute) and the extension step (72 ° C for 1 minute) Cycle and final extension step (72 < 0 > C, 10 min).

The obtained SaOMT2 fragment was cloned into a pRSF-Duet-1 vector (Novagen, USA) to prepare pRSF-Duet-SaOMT2, and E. coli M, a strain of Escherichia coli , was prepared by transforming the vector into Escherichia coli Next, SaOMT2 protein expression of the mutant was confirmed by SDS-PAGE.

1-2: SAM synthase ( EcSAM synthetase ) ≪ / RTI >

The gene encoding the SAM synthase (EcSAM synthetase; GenBank Accession No. K02129) used in this example was cloned from E. coli K12 based on a known sequence (Markham et al . , J. Biol . Chem . , 259: 14505-14507, 1984). The EcSAM synthetase was subjected to PCR under the same conditions as in Example 1-1 using primer pairs of EcSAM-F '(SEQ ID NO: 3) and EcSAM-R' (SEQ ID NO: 4) to generate a SAM synthase (EcSAM synthetase) gene DNA was amplified:

[SEQ ID NO: 3] EcSAM-F'-5'- GATATC ATGGCAAAACACCTTTTTACGTCC-3 'EcoRV

[SEQ ID NO: 4] EcSAM-R-5'- CTCGAG TTACTTCAGACCGGCAGCATC-3 'Xho I

(The underlined part indicates the restriction enzyme site)

The base sequence of the SAM synthetic enzyme (EcSAM synthetase) amplified was pGEM ^? -TT vector (Promega, Madison, Wis., USA) and cloned again into the NdeI / XhoI site of the E. coli expression vector pRSFDuet-1 (Novagen, USA) to produce a recombinant vector pRSF-Duet-SaOMT2-EcSAM . This vector was verified by restriction site mapping, PCR, and sequencing of PCR amplification products.

1-3: Recombination O - Methyltransferase ( SaOMT2 ) And SAM synthase ( EcSAM synthetase ) student Production of recombinant microorganisms having abundance

Recombinant microorganisms were constructed using a recombinant vector containing genes encoding SaOMT2 gene and EcSAM synthetase of Example 1-2 for the production of recombinant O -methyltransferase (SaOMT2) and SAM synthetase. The recombinant vector (pRSF-Duet-SaOMT2-EcSAM) having the DNA base sequence confirmed was transformed into Escherichia coli) was transformed with BL21 (DE3) was prepared in E. coli MS E. coli mutant (strains).

1-4: Recombination O - Methyltransferase ( SaOMT2 ) And SAM  Synthetic enzyme ( EcSAM synthetase ) student Substrate using recombinant microorganism having an ability ( Apigenin ( Apigenin ) Bioconversion biotransformation ) And process optimization

Badges and reagents

The culture medium for the E. coli BL21 (DE3) mutant containing pRSF-Duet-SaOMT2-EcSAM prepared in Example 1-3 was LB (Lactobacillus sp., Bacto, Sparks, MD, USA) 6 g Na ₂ HPO ₄ , 3 g K ₂ HPO ₄ , 0.5 g NaCl, 0.1% NH ₄ CL, 1% glucose, 1 mM MgSO ₄ .7H (Bacto, Sparks, MD, USA) or M9 minimal medium ₂ O, was added to a final concentration of 50μg / mL of kanamycin (kanamycin) to 100μM CaCl ₂₎ was prepared Bahia genin (Apigenin) (Genk and Nin (Genkwanin) from Sigma Aldrich, St. Louis, MO, USA) . In the case of using a 3L fermenter, 1% mannitol, 1% glucose, and 1% glycerol were added.

Optimal reaction conditions for producing Genkwanin from Apigenin using E. coli BL21 (DE3) mutant containing pRSF-Duet-SaOMT2-EcSAM prepared in Example 1-3 were established. E. coli mutants were inoculated in 3 mL of LB liquid medium containing antibiotics and cultured overnight at 37 DEG C and 220 rpm. A taking and the cultured microbial variant 200μL inoculated into LB liquid medium containing the antibiotic after 50mL, until the OD ₆₀₀ reaches a value of 0.6 and incubated at 37 ℃. IPTG (isopropyl-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM and incubated at 20 ° C for 3 hours. Apigenin was dissolved in 20% dimethyl sulfoxide (DMSO) and added at various concentrations to determine the optimal conditions for the bioconversion reaction, followed by shaking at 220 rpm for 60 hours at 20 ° C. During the reaction, 500 μL of the culture was taken every 12 hours, treated with twice the volume of ethyl acetate, and then dried and concentrated to evaporate the solvent. The concentrated material was used in the chromatographic analysis of Example 3 by dissolving in 500 [mu] L of methanol.

Apigenin was added to the medium containing E. coli BL21 (DE3) variants containing RSF-Duet-SaOMT2-EcSAM at 100 μM, 200 μM, 300 μM, 400 μM, 500 μM or 1000 μM, The conversion yield of Genkwanin and Genkwanin in the culture solution obtained as described above was confirmed.

As a result, it was confirmed that Genck and Nin at a concentration of 142 μM were produced (yield of 71% apigenin conversion) when Apigenin of 200 μM was added and reacted for 48 hours (data not shown).

On the other hand, when Escherichia coli mutants were cultured using LB medium, TB medium and M9 medium in order to compare production (production) of genk and nin depending on the kind of medium, the apigenin conversion rate in the TB medium was 78 %, And OD _600nm (cell density) was also highest in TB media.

Example  2: Using a fermenter Scale-up ( Scale - up ) System, apigenin ( Apigenin) Of Genkwanin  Produce

Genkwanin was prepared in a 3 L fermenter using the substrate concentration, the culture time and the medium conditions of Example 2.

Fermentation was performed in a 3 L volume on a BioTron (BioTron Ltd., Incheon, Suto-Gwon, South Korea) equipped with a 5 L volume vessel. Temperature, pH, and rotor speed were maintained at 25 ° C, 7.5, and 400 rpm, respectively.

E. coli BL21 (DE3) variants containing the pRSF-Duet-SaOMT2-EcSAM prepared in Example 1-3 were cultured in TB media containing 3% (w / v) glucose with Apigenin as Genkwainin Genkwanin). The dissolved oxygen of the medium was maintained at 70% or more. The cultured mutant was added so that the final concentration of L-lactose was 0.15 M when the absorbance was changed to 5 at a wavelength of 600 nm, and D-glucose was added every hour to maintain the cell density and growth (Koirala N et al ., J Ind Microbiol Biotechnol , 41 (11): 1647-1658, 2014).

Apigenin was added and the culture was harvested at intervals of 6 hours to confirm the bioconversion of apigenin by HPLC analysis of Example 3. Forty-eight hours after the reaction, the culture solution was obtained. Ethyl acetate was added to extract, dried, and then dissolved in methanol to separate genkwanin from the genkwanin by HPLC analysis.

As a result, as shown in Fig. 4, when 200 μM Apigenin was added to the culture medium, about 85% of apigenin was converted to Genkwanin during 48 hours reaction, and 170 μM (48.28 mg / L ) Of Genk and Nin were produced.

Example  3: Purification and analysis of Genkwanin

HPLC  And QTOF - ESI / MS Purification and Analysis of Genkwanin Using

The culture solution containing Genkwanin prepared using E. coli mutant in Example 1 was treated with twice the volume of ethyl acetate and then dried and concentrated to evaporate the solvent. Additional purification of the concentrate may be carried out with 20% ACN (0-5 min), 40% (5-10 min), 40% (10-15 min), 90% (15-25 min), 90% ) and 10% (30-35 minutes) the flow rate of a UV detector (270㎚) using 36 minutes binary program proceeds to 10mL / min is connected to C ₁₈ column (YMC-Pack ODS-AQ ( 250 x 20mm ID, 10μm) ). ≪ / RTI >

The purified cy n and nin were separated by high-performance liquid chromatography coupled with HPLC using a reversed phase C ₁₈ column (Mightysil RP-18 GP, 250 x 4.6 mm, kanto chemical, Japan) diode array, shimadzu, Japan; SPD-M20A Detector).

As a result, as shown in Fig. 2, HPLC-PDA analysis showed that the residence time (t _R ) of Apigenin standard material and Genkwanin was 10.0 to 10.6 min and 11.6 to 11.8 min, It was confirmed that the conversion rate from apigenin to genk and nin increases with the reaction time of the mutant.

As shown in FIG. 3, QTOF-ESI / MS (ACQUITY (UPLC, Waters Corp., USA) -SYNAPT G2-S (manufactured by Waters Corp., USA) was used for quantitative confirmation of the prepared genk and Genkwanin )]. As a result, the total m / z value of the isolated compound was 285.0845, confirming that Genck and Nin were methylated by Escherichia coli mutants.

Example  3: Biological activity analysis of Genkwanin and Genkwanin

Human skin melanoma, stomach cancer, liver cancer, brain cancer cell, and endothelial cell were used to evaluate the biological activity effect to confirm the pharmaceutical potential as a cancer-related chemotherapeutic agent of Genkwanin in this example (Genkwanin), which is a parent of Genk and Nin, and is currently known as a preventive and chemotherapeutic adjuvant of cancer, Apigenin as a comparative substance. We also evaluated the effects of genk and nin on angiogenic phenotypes, chemoinvasion and capillary tube formation, as anti-angiogenic agents for angiogenesis-dependent diseases such as cancer.

Human umbilical vein endothelial cells (HUVECs) were divided into 4 to 8 passages. Endothelial growth medium-2 (EGM2) supplemented with 10% FBS (Fetal bovine serum, Invitrogen, Grand Island, NY, USA) , Lonza, Walkersville, MD, USA). B16F10 (melanoma) and HepG2 (hepatoma) cells were cultured in Dulbecco's modified Eagles medium (Invitrogen) supplemented with 10% FBS. AGS (gastric carcinoma) and U87MG (glioblastoma) cells were cultured in RPMI 1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS and MEM (minimum essential medium, Invitrogen, Lt; / RTI > All cells were maintained at 37 ° C in a 5% CO ₂ humidified incubator.

(1) Cell growth assay

For cell growth analysis, cells were transplanted with 2 x 10 ³ cells per well in a 96-well culture dish (SPL Lifesciences, Gyeonggi, Korea), and then treated with apigenin and Genkwanin for 72 hours, The cells were treated at various concentrations of 100 [mu] M. Cell growth was measured by MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide colorimetric assay). Here, the MTT assay is a method for measuring the growth of cells by measuring the degree of change of the tetrazolium-based coloring reagent from yellow to purple by the redox enzyme present in the cell as absorbance.

As a result, as shown in Fig. 5, Genkwanin inhibited cell proliferation in all cancer cell lines with different ranges of cell growth inhibition in each cell line. In particular, Genck and Nin were superior to Apigenin in inhibiting growth of cancer cells (B16F10, HepG2, U87MG).

(2) Chemoinvasion  analysis

Invasive (invasiveness) Analysis of endothelial cells in HUVECs (human umbilical vein endothelial cells) is in vitro was confirmed by using a trans-well chamber system (Transwell chamber system with polycarbonate filter inserts with a pore size of 8.0 mm (Corning Costar, Acton, MA, USA)). The lower filter layer of the chamber system was coated with 10 μL gelatin (1 mg / mL) and the upper layer was coated with 10 μL Matrigel (3 mg / mL). Serum-starved 1 x 10 ⁵ HUVECs were placed in the upper chamber of the filter and incubated with 5 μM Apigenin or 5 μM Genck in the presence of VEGF (30 ng / mL) (proangiogenic factor) Nin (Genkwanin) was added. The chamber was incubated at 37 ° C. for 18 hours, then fixed with methanol, stained with hematoxylin and eosin, and then stained with 200 μg / Cell numbers were calculated.

As a result, as shown in Fig. 6A, Genkwanin and Genkwanin were found to efficiently inhibit VEGF-induced invasion. Especially at the same concentration, Genk and Nin showed higher anti-neovascular efficacy than Apigenin. Trypan blue treatment did not result in cytotoxicity (data not shown).

(3) microtubule formation ( capillary tube formation ) analysis

HUVECs treated with serum-starved (1 x 10 ⁵ cells) were inoculated on the surface of Matrigel (BD Biosciences, San Jose, CA, USA) and incubated with 5 μM Apigenin (Apigenin) or 5 [mu] M Genkwanin for 6-18 hours. The morphological changes and tube formation of the cells were observed under a 200 magnification microscope (Olympus, Center Valley, PA, USA). Quantification of the tube formation was done by calculating the total number of branched tubes in a randomly selected field under a microscope at 200 magnifications.

As a result, as shown in FIG. 6B, Genkwanin and Genkwanin were found to efficiently inhibit VEGF-induced tube formation. Especially at the same concentration, Genkwanin showed higher anti-angiogenic activity than Apigenin. Trypan blue treatment did not result in cytotoxicity (data not shown).

Statistical analysis

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 appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Sun Moon University <120> Method for Preparing Genkwanin Using Biotransformation of          Apigenin <130> P15-B106 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SaOMT2-F <400> 1 ggatccgatg agctgccgca ccggca 26 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SaOMT2-R <400> 2 gaattctcag ccaaccgcgc gcagttcga 29 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> EcSAM-F <400> 3 gatatcatgg caaaacacct ttttacgtcc 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> EcSAM-R <400> 4 ctcgagttac ttcagaccgg cagcatc 27

Claims (7)

A method for producing Genkwanin comprising the steps of:
a: (SaOMT2 O-methyltransferase) methyl transferase - (a) 7- hydroxyl groups (7-hydroxyl group) by Streptomyces cis Abbe reumi subtilis (Streptomyces avermitilis) O of origin to deliver methyl (methyl) of the flavonoid Adenosine-L-methionine synthetase derived from Escherichia coli K12, which synthesizes S-adenosyl-L-methionine, which is a coenzyme of the enzyme, culturing the transformed microorganism variant with a gene encoding a synthetase in a medium containing 100 to 200 μM of Apigenin to convert Apigenin to Genkwanin; And
(b) obtaining the converted genk and nin (Genkwanin).
delete delete The method according to claim 1, wherein the microorganism is E. coli .
delete delete delete

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