KR20110059090A - Use of leifsonia sp. gal45 for preparation of protopanaxatriol - Google Patents

Abstract

PURPOSE: A Leifsonia sp. GAL45 strain for preparing protopanaxatriol is provided to convert ginsenoside ingredients contained in ginseng or red ginseng into protopanaxatriol. CONSTITUTION: A method for preparing protopanazatriol comprises: a step of reacting a culture supernatant of Leifsonia sp. GAL45(deposit number KCTC18183P) with ginseng extract; and a step of collecting protopanaxatriol from the reactant. The ginseng extract is obtained from Panax ginseng, Panax notoginseng, Panax quinquefolia, Panax japonica, Panzx trifolia, Panax pseudoginseng, or Panax vietnamensis.

Description

Use of Leifsonia genus AL45 for the production of ProtoPanaxatriol {Use of Leifsonia sp. GAL45 for preparation of protopanaxatriol}

The present invention is Leifsonia sp. Use of GAL45 , ie Leifsonia sp. The present invention relates to a method for producing a protowave paratrile using GAL45.

Ginseng is Panax ginseng CA Meyer, which has been used as a medicinal herb in Korea and China for a long time. It is a perennial herb that belongs botanically to Araliaceae and Panax .

The components of ginseng are 60 ~ 70%, nitrogen compounds 12 ~ 16%, fat soluble components 1 ~ 2%, and ginseng saponins, ginseng proteins, polysaccharides, etc. Contains unique ingredients Saponin refers to a substance consisting of several cyclic compounds in non-sugar glycosides of the glycoside in the plant family. Saponins contained in ginseng have different structures and different pharmacological effects when compared to saponins in other plants. Specifically named ginsenoside. Looking at the known pharmacological effects of ginsenosides, it was found that there are anti-cancer effects, central nervous system inhibitory effect, liver function promotion and hangover removal effect, antioxidant activity, immune enhancement.

Ginsenosides are classified into protopanaxadiol (PD), protopanaxatriol (PT), and oleanane (oleanane) according to the characteristics of the chemical structure, and the structure of each compound is revealed. Among them, Korean ginseng has a relatively high content of ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1.

One of ginsenosides, protopananaxtriol, is a component known to be useful for anti-diabetic by activation of peroxysome proliferator-activated receptor γ (PPARγ). However, protopanax triol is present only in trace amounts in ginseng, and despite its usefulness, it is not easy to obtain a large amount to use as an active ingredient. It is superior to the main ingredients of ginseng in terms of pharmacological efficacy, but in order to obtain trace components such as Rg3, Rh2, and Compound K, which are rarely present in ginseng, almost no ginsenosides are present in ginseng. Research into the conversion and preparation of ginsenosides has been conducted, but there have been no proposals for obtaining a high yield of protopananax triol.

The main ginsenosides or ginseng saponin extract (irradiated saponin) contained in a large amount of ginseng, to provide a method for producing a high yield of protopananatriol rarely present in ginseng.

To this end, the present invention is Leifsonia sp. Uses of GAL45 , ie Leifsonia sp. Provided is a method for preparing a protowave paratriol using GAL45.

Leifsonia sp. Using GAL45, it is possible to convert ginsenoside components contained in ginseng or red ginseng into protopanaxanatriol. Therefore, it is possible to produce the protopananac triol in high yield, so that it can be utilized as an active ingredient of food or medicine.

It is a kind of ginsenoside containing a small amount of protofanaxatriol in ginseng, and has a structure represented by the following Chemical Formula 1.

[Formula 1]

The present inventors endeavored to find a strain capable of converting other ginsenosides contained in a large amount of probeponin or ginseng into protopanaxanatriol in order to obtain a higher content of protopanaxanatriol having an antidiabetic effect.

As a result, a strain having protopanaxanatriol converting ability was selected and isolated from the soil in which ginseng is grown, which was named GAL45. Analysis of the gene sequence of 16S rRNA to identify the GAL45, showing the highest sequence similarity of Leifsonia shinshuensis JCM 1059 ^T and 98.8%, so that the strain of the present invention is named Leifsonia sp. GAL45, 2009 On November 19, 19, it was deposited with the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC18183P.

As can be seen in the Examples below, Leifsonia sp. GAL45 has been shown to have a very good ability to convert ginsenosides such as probeponin, Re, Rg1, Rg2, Rh1, F1 into protopanaxanatriol.

Therefore, the present invention provides Leifsonia sp. Uses of GAL45 and Leifsonia sp. Provided is a method for preparing a protowave paratriol using GAL45.

In one embodiment, the present invention provides Leifsonia sp. Reacting the culture supernatant of GAL45 (Accession No. KCTC18183P) with ginseng extract; And it provides a method for producing a protopananax triol comprising the step of obtaining a protopoanax triol from the reaction.

In another embodiment, the present invention provides Leifsonia sp. Culturing GAL45 (Accession No. KCTC18183P) in a medium containing ginseng extract; And it provides a method for producing a protopananax triol comprising the step of obtaining a protopoanax triol from the culture.

Leifsonia sp. The substrate that GAL45 uses to produce ProtoPanaxtriol can be used in the form of ginseng extract. In the present invention, the ginseng extract is saponin, specific ginsenosides or mixtures thereof; Or a saponin fraction, a fraction comprising a particular ginsenoside, or a mixture thereof. In addition, the ginseng extract may include a solvent such as water, a buffer solution, an organic solvent, and the like. In the present invention, the ginseng extract used for the preparation of the protopanaxanatriol is a plant of the genus Panax which is known to contain saponin, but is not limited thereto, for example, Korean ginseng (Panax ginseng), Jeonchisam ( Panax notoginseng, Panax quinquefolia, Panax japonica, Panax japonica, Panax pseudoginseng, Panax viginnamensis, Panax vietnamensis, Panax enegatior, Panax It may be an extract of one or more plants selected from the group consisting of Panax wangianus, Panax bipinratifidus and Panax angustifolium.

The ginseng extract may be one containing irradiated saponin or ginsenosides. Preferably, the ginseng extract may include a protopanastriol-based ginsenoside. In one embodiment, the ginseng extract may include one or more ginsenosides selected from Re, Rg1, Rg2, Rh1, and F1. Methods of preparing ginseng extracts or specific ginseng extract fractions comprising saponins or specific ginsenosides are well known in the art, and saponins or specific ginsenosides may also be obtained commercially and used. As can be seen in the following examples, it is much better to convert the protopanaxtriol-based ginsenosides to the protopanaxanatriol as compared to the case of using the irradiated saponin extract.

Leifsonia sp. In the method for producing a protopanax triol using the culture supernatant of GAL45 , the culture supernatant is Leifsonia sp. GAL45 may be induced in a culture containing ginseng extract and centrifuged and concentrated. Induction culture may further increase the production of protopananac triol. Leifsonia sp. In the case of preparing a protopanaxatriol using the culture supernatant of GAL45 , Leifsonia sp. The extraction and purification process is more advantageous than when inoculating GAL45 directly on the medium.

Leifsonia sp. Culture supernatant of GAL45 or Leifsonia sp. In the method for producing a proto-paranatrial triol using GAL45, the conditions of the reaction or the culture are not particularly limited. Just Leifsonia sp. In consideration of the reactivity of GAL45 or the enzyme contained therein, the reaction or culturing is preferably performed at 10 ° C to 40 ° C, preferably at room temperature to 35 ° C. On the other hand, the reaction or incubation time, Leifsonia sp. When using the GAL45 culture supernatant, about 1 to 24 hours, preferably 4 to 12 hours, Leifsonia sp. When using GAL45 directly may be 12 to 60 hours, preferably 24 to 48 hours.

Leifsonia sp. Culture supernatant of GAL45 or Leifsonia sp. In the method for preparing a protopanaxtriol using GAL45, the step of obtaining protopananaxtriol from the reaction or culture is carried out by extracting the protopanaxanatriol from the reactant or culture using the saturated butanol. Can be.

By using the method as described above, it is possible to obtain a large amount of protopanaxatriol, which can be usefully used as an active ingredient of medicine or food.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

<Example 1> Selection of strains having ginsenoside converting ability among strains having beta glucosidase and beta galactosidase activity from ginseng soil

(1) Extraction of Irradiation

In order to proceed with the experiment for screening strains, the irradiated saponin was extracted from the red ginseng concentrate. The red ginseng concentrate was dissolved in distilled water, treated three times with ethylether to remove the fat-soluble component, and the saturated butanol layer obtained by treating three times with saturated butanol was concentrated under reduced pressure to obtain irradiated ponin.

(2) Strain Selection and Isolation with Ginsenoside Conversion Capability

Strains with betaglucosidase and beta galactosidase activity were isolated by Esculin and X-gal plate assays. To test saponin conversion, 96well was added R2A broth and sterilized irradiated saponin at a concentration of 1000 ppm. After incubation at 30 ° C for 48 hours, 200 μl of the supernatant of saponin dissolved in the culture supernatant was collected and centrifuged to extract saponin of ethyl ether and saturated butanol using shibata method in a 1.5 ml tube. HPLC analysis was performed by dissolving in acetonitrile. As a result, strains having ginsenoside converting ability were selected and separated and named as GAL45.

(3) genus Leifsonia Leifsonia  sp.) Identification of GAL45 strain

Gene sequences of 16S rRNA were analyzed to identify strains capable of converting ProtoPanaxatriol. First, the colonies obtained by smearing the strains on an R2A plate were suspended in sterile water, and then bathed at 100 ° C. for 10 minutes, and centrifugation was used to remove the precipitate to obtain a supernatant. PCR was used to amplify the gene using the template (template), and pBact 27F primer (5'-AGAGTTTGATCMTGGCTCAG-3 ') and pUniv 1492R (5'-GGYTACCTTGTTACGACTT-3') were used. This PCR amplified gene was confirmed by electrophoresis, DNA was isolated from the agarose using a gel extraction kit (QIAGEN, Germany). As a result of analyzing the nucleotide sequence of the isolated sample by Solgent Co., Ltd., it was confirmed that the 16S rRNA gene of the strain had the nucleotide sequence of SEQ ID NO: 1. Comparative analysis of the sequences in the Eztaxon database showed the highest sequence similarity of Leifsonia shinshuensis JCM 1059 ^T with 98.8%. Therefore, the strain of the present invention Leifsonia sp. It was named GAL45, and it was deposited on November 18, 2009 by Korea Institute of Bioscience and Biotechnology, and was assigned accession number KCTC18183P.

Example 2 Preparation of Enzyme Solution Induced by Irradiation

A liquid R2A medium to which the probeponin was added was used as the enzyme solution. 250 ml of 100 ml R2A liquid medium was made in the flask and sterilized irradiated solution was added at a concentration of 1000 ppm. Leifsonia sp. GAL45 strains were inoculated into the R2A liquid medium containing the irradiated phononine and cultured at 30 ° C. After 72 hours, a 30 ml sample of the culture supernatant was taken via centrifugation. The culture supernatant of 30ml was concentrated to 1ml by ultrafiltration (Ultrafiltration). Concentrated 1 ml culture supernatant was used as enzyme.

<Example 3> Protopananac Triol Preparation

ProtoPanaxtriol-based ginsenosides Re, Rg1, Rg2, Rh1, F1, and ginseng saponin extract (irradiated ponin) were prepared as substrates respectively. 50mM Tris-Hcl (pH 7.5) buffer Using the culture supernatant concentrated in Example 2 on the ginsenoside substrate dissolved in the solution as an enzyme was sufficiently enzymatic reaction in a 30 ℃ incubator for 12 to 24 hours. After the titration time of the enzyme reaction, the finished product was analyzed by HPLC. As a result, as shown in FIGS. 1-6 and Table 1, it was possible to obtain ProtoPanaxtriol in high yield (in the HPLC chromatogram of FIGS. 1 to 6, separately from the substrate and ProtoPanaxtriol, Unmarked peaks are the peaks of the substances included in the preparation of the enzyme solution). Leifsonia sp. When GAL45 was inoculated into a medium containing a ProtoPanaxatriol Ginsenoside Re, Rg1, Rg2, Rh1, F1, and Ginseng Saponin extract (irradiated ponin), respectively, as a substrate (within 5% of error range) The result was obtained.

Table 1 Production Yield of Protoparnaxatriol According to Substrate

Substrate Product (g) ^* Yield (%) Ginseng Saponin Extract 0.109 10.9 Re 0.763 76.3 Rg1 0.693 69.3 Rg2 0.844 84.4 Rh1 0.819707 82.0 F1 0.726467 72.6

^* The amount of product (protopanaxatriol) when 1 g of substrate is used

1 is an HPLC chromatogram showing the results of conversion of ginseng saponin extract (irradiated ponin) to protopanaxatriol according to the method of Example 3. FIG.

FIG. 2 is an HPLC chromatogram showing the results of converting ProtoPanaxtriol Ginsenoside Re into ProtoPanaxtriol according to the method of Example 3.

FIG. 3 is an HPLC chromatogram showing the results of converting ProtoPanaxtriol Ginsenoside Rg1 into ProtoPanaxtriol according to the method of Example 3.

FIG. 4 is an HPLC chromatogram showing the results of converting ProtoPanaxtriol Ginsenoside Rg2 into ProtoPanaxtriol according to the method of Example 3.

FIG. 5 is an HPLC chromatogram showing the results of converting ProtoPanaxtriol Ginsenoside Rh1 into ProtoPanaxtriol according to the method of Example 3.

FIG. 6 is an HPLC chromatogram showing the results of converting ProtoPanaxtriol Ginsenoside F1 into ProtoPanax Triol according to the method of Example 3.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Use of Leifsonia sp. GAL45 for preparation of protopanaxatriol <130> P091128 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1383 <212> DNA <213> Leifsonia sp. GAL45 <220> <221> rRNA (222) (1) .. (1383) <400> 1 cgaacgatga acctggagct tgctctaggg gattagtggc gaacgggtga gtaacacgtg 60 agtaacctgc ccttgactct gggataacct ccggaaacgg aagctaatac cggatatgac 120 gcacggaggc atctcctgtg cgtggaaaga atttcggtca aggatggact cgcggcctat 180 caggtagttg gtgaggtaac ggcccaccaa gcctacgacg ggtagccggc ctgagagggt 240 gaccggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 300 tattgcacaa tgggcgaaag cctgatgcag caacgccgcg tgagggacga cggccttcgg 360 gttgtaaacc tcttttagta gggaagaagc gaaagtgacg gtacctgcag aaaaagcacc 420 ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttgt ccggaattat 480 tgggcgtaaa gagctcgtag gcggtctgtc gcgtctgctg tgaaatcccg aggctcaacc 540 tcgggcctgc agtgggtacg ggcagactag agtgcggtag gggagattgg aattcctggt 600 gtagcggtgg aatgcgcaga tatcaggagg aacaccgatg gcgaaggcag atctctgggc 660 cgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 720 ccacgcccgt aaacgttggg cgctagatgt ggggaccatt ccacggtttc cgtgtcgcag 780 ctaacgcatt aagcgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt 840 gacgggggcc cgcacaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct 900 taccaaggct tgacatatac gagaacgggc tagaaatagt caactctttg gacactcgta 960 aacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020 cgagcgcaac cctcgttcta tgttgccagc gcgtaatggc gggaactcat aggagactgc 1080 cggggtcaac tcggaggaag gtggggatga cgtcaaatca tcatgcccct tatgtcttgg 1140 gcttcacgca tgctacaatg gccggtacaa agggctgcaa taccgtaagg tggagcgaat 1200 cccaaaaagc cggtctcagt tcggattgag gtctgcaact cgacctcatg aagtcggagt 1260 cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc 1320 gcccgtcaag tcatgaaagt cggtaacacc cgaagccggt ggcctaaccc ttgtggaagg 1380 agc 1383  

Claims (9)

Leifsonia sp. Reacting the culture supernatant of GAL45 (Accession No. KCTC18183P) with ginseng extract; And Method for producing a protopananax triol comprising the step of obtaining a protopoanax triol from the reactants. The method of claim 1, The ginseng extracts include Panax ginseng, Panax notoginseng, Panax quinquefolia, Panax japonica, Panxx trifolia, Himalaya ginseng, Panax pseudoginseng, and Panax vietnamensis. ), An extract of one or more plants selected from the group consisting of Panax enegatior, Panax wangianus, Panax bipinratifidus, and Panax angustifolium. Method for Producing Protowave Natriol. The method of claim 1, The ginseng extract is a method for producing a protopa Naksa triol containing irradiated saponin or ginsenosides. The method of claim 1, The ginseng extract is a method for producing a protopanaxatriol that comprises at least one ginsenoside selected from Re, Rg1, Rg2, Rh1 and F1. The method of claim 1, The culture supernatant is Leifsonia sp. Method of producing a protopananax triol is induction culture of GAL45 in a ginseng extract-containing medium, the medium is centrifuged and concentrated. Leifsonia sp. Culturing GAL45 (Accession No. KCTC18183P) in a medium containing ginseng extract; And Method for producing a protopananax triol comprising the step of obtaining a protopoanax triol from the culture. The method of claim 6, The ginseng extracts include Panax ginseng, Panax notoginseng, Panax quinquefolia, Panax japonica, Panxx trifolia, Himalaya ginseng, Panax pseudoginseng, and Panax vietnamensis. ), An extract of one or more plants selected from the group consisting of Panax enegatior, Panax wangianus, Panax bipinratifidus, and Panax angustifolium. Method for Producing Protowave Natriol. The method of claim 6, The ginseng extract is a method of producing a protopananax triol containing irradiated phonoponin or ginsenosides. The method of claim 6, The ginseng extract is a method for producing a protopanaxatriol that comprises at least one ginsenoside selected from Re, Rg1, Rg2, Rh1 and F1.

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