Method for preparing rare saponin and rare saponin

Abstract

The invention discloses a method for synthesizing rare ginsenoside by converting natural ginsenoside Rb1 with endophytic fungi separated from root rot ginseng. The method comprises the following steps: obtaining a Fusarium redolics c271F from root rot ginseng by utilizing a fungus separation method, carrying out conversion synthesis on a zymophyte of the Fusarium redolics c271F by taking ginsenoside Rb1 as a raw material to obtain rare saponins, and identifying the converted rare saponins as F2, Rg3 and CK on the basis of an HPLC (High Performance Liquid Chromatography) method. The method disclosed by the invention is simple to operate, green and safe, relatively high in yield and relatively low in cost, and a new method is provided for industrial production of the rare ginsenoside.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for generating rare ginsenosides by fermenting and transforming endophytic fungi and the rare ginsenosides. Background Art

[0002] Ginseng (Panax ginseng CAMeyer) is a traditional Chinese medicinal material in my country. The Compendium of Materia Medica records that its medicinal part is the root, which is sweet, slightly cold, non-toxic, and is a top-grade tonic. The National Health Commission approved ginseng roots (5 years and below) as new resource food raw materials in Announcement No. 17 of 2012. Ginseng, as a Chinese medicinal material for both medicine and food, has broad development prospects. Ginseng saponins are the main active ingredients of ginseng, and there are many types of them. According to the structural type of saponins, they are mainly divided into three types: tetracyclic triterpenoid dammarane type, pentacyclic triterpenoid and oleanolic acid type. The latter two are extremely low in ginseng. Tetracyclic triterpenoid dammarane type ginsenosides can be divided into protopanaxadiol type and protopanaxtriol type according to the different structures of C-3, C-6, and C-20 on the tetracyclic mother nucleus. According to the different substitution positions of the chiral C (C-20), protopanaxadiol and protopanaxatriol ginsenosides are divided into 20(S) and 20(R) types.

[0003] So far, more than 50 kinds of ginsenosides have been isolated and identified, among which the content of ginsenoside monomer compounds Rg1, Rb1, Re, Rb2, etc. is relatively high, while the content of rare ginsenosides is relatively low. Rare ginsenosides are usually obtained by hydrolyzing natural ginsenosides that account for more than 95% of the total saponins and lose the coordinated sugar chain. For example, the protopanaxadiol ginsenosides Rb1 and Rd lose the C-3 and C-20 sugar chains after hydrolysis, and can form rare saponins such as intermediates Rg3, F2, Rh2 and Compound K (CK). The medicinal value, biological activity and human absorption rate of rare saponins are much higher than those of natural ginsenosides. Through the study of saponin activity, it was found that the relationship between the anti-tumor activity of ginsenosides and the sugar chain structure of saponins is: aglycone>monosaccharide>diosaccharide>trisaccharide>tetrasaccharide. Natural ginsenosides have structural characteristics such as large relative molecular mass, small lipid-water partition coefficient, and large topological polar surface area. Their bioavailability is low. After oral administration, they need to be metabolized by intestinal flora in the gastrointestinal tract into secondary saponin components such as ginsenoside Rg3 and CK before they can be absorbed by the blood and become active substances that truly exert their medicinal effects. In addition, ginsenosides F2, Rg3, and CK have high therapeutic effects in anti-cancer, improving sugar metabolism, resisting central nervous system diseases, and treating cardiovascular and cerebrovascular diseases. Therefore, it is of great research significance to modify and transform natural ginsenosides and seek to obtain rare ginsenosides economically and efficiently. Among the rare ginsenosides, Rg3 has multiple pharmacological activities such as anti-tumor, prevention and treatment of cardiovascular and cerebrovascular diseases and coronary heart disease, inhibition of cancer cell metastasis, liver protection, nerve protection, and immunity enhancement. However, Rg3 is not only extremely rare in natural ginseng, but also the carbon atom at the 20th position of Rg3 is a chiral carbon, and there are two optical isomers: 20(S)-Rg3 and 20(R)-Rg3. The physicochemical and pharmacological properties of the two are quite different, especially the water solubility and bioavailability of 20(S)-Rg3 are much higher than those of 20(R)-Rg3. Therefore, the development of a method for preparing 20(S)-Rg3 has important practical application value.

[0004] In recent years, the methods for converting common ginsenosides into rare ginsenosides have developed rapidly, mainly including heat treatment, acid hydrolysis, enzymatic hydrolysis and microbial fermentation. Among them, microbial conversion has high specificity, high conversion rate of target products, strong conformational selectivity, mild reaction conditions, safety and environmental protection. It is a popular research direction for ginsenoside conversion and has a high industrial prospect. Existing microbial methods for obtaining rare ginsenosides mostly utilize soil microorganisms, molds and anaerobic bacteria in the intestine.

[0005] The main enzyme in the fermentation process is β-glucosidase, but β-glucosidase from different microbial sources has different selectivity for glycosidic bonds at different positions in ginsenosides. When some microorganisms transform ginsenoside Rb1, their β-glucosidase is more selective for the C-20 glycoside, and hydrolyzes the outer 1 molecule of glucose to obtain the intermediate product ginsenoside Rd, such as Cordyceps sinensis, Trichoderma longifolia, Aspergillus niger J7, and Lactobacillus paracasei subspecies tenacious. When some strains transform ginsenoside Rb1, they are more selective for the C-3 glycoside, and obtain the intermediate product gypenosyl saponin XVII, such as Lactobacillus paradigestus LH4. After some microorganisms decompose ginsenoside Rb1 into ginsenoside Rd, they can continue to decompose the C-20 and C-3 glycosides of ginsenoside Rd. In this process, if the selectivity for the C-3 sugar group is higher, ginsenoside F2 is generated, such as Cordyceps sinensis, Schizophyllum, Aspergillus niger J7, etc.; if the selectivity for the C-20 sugar group is higher, the product ginsenoside Rg3 is generated, such as Trichoderma longifolia and Lactobacillus paracasei subsp. tenacious. Some microorganisms can continue to hydrolyze ginsenoside F2 and Rg3. For example, strains such as Schizophyllum and Aspergillus niger J7 can continue to hydrolyze ginsenoside F2 and catalyze it to produce rare ginsenoside CK. Lactobacillus paracasei subsp. tenacious MJM60396 can continue to hydrolyze one molecule of glucose at the C-3 position in ginsenoside Rg3 to obtain the final product ginsenoside Rh2. Although it has been reported that many kinds of microorganisms can decompose ginsenoside Rb1, there are not many microorganisms that can cut both the C-20 sugar group and the C-3 sugar group of ginsenoside Rb1 at the same time, and related research has not been reported.

[0006] Fusarium redolens (abbreviated as F.redolens) is a fungus in the family Caliciaceae, order Ascomycetes, class Imperfect Fungi. F.redolens is reported to cause various symptoms in plants, including wilt, seedling dampness, and root rot. The pathogenic F.redolens was first isolated from peas with symptoms of wilt and root rot. Subsequent studies have found that it is associated with wilt of Aleppo pine, Fusarium wilt of chickpea, and root rot of soybean, asparagus, ginseng, and American ginseng. As an endophytic fungus, F.redolens can produce a variety of bioactive substances. For example, the endophytic F. redolens isolated from Himalayan Taxus can produce the anticancer compound paclitaxel; the endophytic F. redolens Dzf2 isolated from the rhizomes of Dioscorea zingiberensis can produce the insecticide perillamycin; the F. redolens 6WBY3 isolated from Forsythia suspensa can produce steroidal alkaloids, peimisine and imperialine-3β-D-glucoside. Recently, it has been reported that the F. redolens isolated from American ginseng can degrade ginsenosides Rg1 and Rd, but there are no reports on its use to transform natural ginsenoside Rb1 into rare ginsenosides.

[0007] The invention utilizes the fungus separated from ginseng with root rot to transform ginsenosides, providing new ideas for the synthesis of rare saponins and the application of endophytic strain F. redolens. Summary of the invention

[0008] The object of the present invention is to provide a method for preparing rare saponins by transforming ginsenosides and the rare saponins prepared according to the method.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing rare saponins by transforming ginsenosides, which utilizes endophytic fungus fermentation to transform ginsenosides.

[0011] Preferably, the endophyte is Fusarium redolens c271F.

[0012] Preferably, Fusarium odoratum c271F is an endophytic fungus isolated from ginseng with root rot.

[0013] Preferably, the ginsenoside is natural ginsenoside Rb1.

[0014] In a second aspect, the present invention provides a rare saponin prepared according to the above method.

[0015] The beneficial effects of the present invention are as follows:

[0016] The fermentation bacteria F. redolens c271F in the present invention is an endophytic bacteria isolated from ginseng with root rot, and the enzymes of the fragrant Fusarium (F. redolens) c271F can hydrolyze both C-20 glycosides and C-3 glycosides. The fermentation bacteria is used to transform ginsenoside Rb1, and the rare saponin conversion rate is high. The conversion product has simple and concentrated components, which is convenient for component analysis and product separation, and is conducive to obtaining 4 kinds of high-purity and high-yield rare ginsenosides, and the production cost is low, which is conducive to industrial batch fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention is described below in conjunction with the accompanying drawings, but the present invention is not limited thereto. In the accompanying drawings:

[0018] Figure 1 The HPLC spectra of rare ginsenosides obtained after 7 days and 14 days of fermentation under the method provided by the present invention. A is the HPLC spectra of 6 kinds of rare ginsenoside standards. Peak 1 represents Rb1, peak 2 represents Rd, peak 3 represents F2, peak 4 represents 20(S)-Rg3, peak 5 represents CK, and peak 6 represents Rh2. B and C are peaks of the products of 7 days and 14 days of fermentation of ginsenoside Rb1 by using F.redolens c271F according to the method provided by the present invention, and the high performance liquid chromatography analysis is performed, wherein c271F represents F.redolens c271F, Rb1 represents ginsenoside Rb1, and 7d and 14d represent 7 days and 14 days of fermentation, respectively. D represents the high performance liquid chromatography peak of the product after 14 days of fermentation of F.redolens c271F without adding ginsenoside Rb1.

[0019] Figure 2 The transformation pathway for the production of rare ginsenosides by fermentation of ginsenoside Rb1 by Fusarium redolens c271F. DETAILED DESCRIPTION

[0020] In order to more clearly understand the present invention, the present invention is described in detail below by means of preferred embodiments in conjunction with the accompanying drawings, but the present invention is not limited thereby.

[0021] It should be noted that the scientific terms and test methods mentioned in the present invention but not explained or described in detail have the same meanings and contents as those understood by those skilled in the art.

[0022] The PCR enzyme used in the following preferred embodiments was purchased from Tiangen Biochemical Technology Co., Ltd., and the specific operations were carried out according to the instructions of the kit. The molecular biology operation method was mainly carried out according to the fourth edition of Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0023] Example 1

[0024] Materials and Methods

[0025] The sequencing of plasmids and DNA products was assisted by the Major Platform Center of the Chinese Academy of Agricultural Sciences. The biennial ginseng materials with root rot were mainly collected from the cultivation site of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences in the winter of 2022. The PDA solid medium contains 200g / L potatoes, 20g / L glucose, and 20g / L agar. The PDB liquid medium contains 200g / L potatoes and 20g / L glucose. In terms of mass volume percentage, the fermentation medium contains the following components: (NH4)2SO40.10%, KH2PO4 0.05%, K2HPO4 0.15%, NaCl 0.10% and MgSO4·7H2O 0.01%. For the fermentation medium, the pH is 5.5-6.5 and sterilized at 121°C for 20min.

[0026] Main experimental steps:

[0027] 1. Isolation and identification of strains

[0028] The steps for extraction, separation and smear culture of endophytic fungi of ginseng root rot are as follows:

[0029] 1) Material cleaning

[0030] Fresh medicinal materials washed with tap water were placed in 50 mL sterile centrifuge tubes. Each tube was soaked in 30 mL of 5% sodium hypochlorite solution (g / mL) (from Meiyuan Water Purification Materials Co., Ltd.) for about 25 minutes. Ultrasonic assistance was used during the period, and the centrifuge tubes were turned upside down intermittently. On a sterile operating table (Guangzhou Ruizhi Purification Equipment Co., Ltd.), rinsed with 70% ethanol for 30 seconds, and then washed with sterile water 3 times. During the period, tweezers that were cooled after burning were used to pick up and pass.

[0031] 2) Isolation of endophytic fungi

[0032] Use a sterile blade to cut the washed and disinfected fresh medicinal materials into small cubes with a side length of about 0.3 cm, take the interface part of the patient, and inoculate it on the PDA plate. Seal with a sterile sealing film and culture at 29°C for 7 days. Pick a typical single colony on the plate and inoculate it in the PDB culture medium, and culture it at 29°C and 190r / min for 20 hours. Dilute the obtained bacterial solution 100 times, inoculate it on the PDA culture plate, culture it at 29°C for 48 hours, and isolate pure colonies. Inoculate the pure cultured strain into the PDB culture medium, then add an equal volume of 30% glycerol (from Tianjin Beilian Fine Chemicals Development Co., Ltd.), and store it in a -20°C refrigerator. One of the strains of Fusarium aromaticum was named c271F.

[0033] 3) Identification of endophytic fungus c271F

[0034] Morphological identification results of c271F: c271F grows turbidly in PDB medium, and the bacteria are yellow-white after long-term storage. On PDA medium, the colonies are white, the hyphae are white or light yellow after long-term storage, and the sclerotia are yellow to orange. Molecular biological methods were used to further identify the strain. The main steps include: extracting the genomic DNA of the strain using the CTAB method, designing ITS primers (ITS1: 5'-TTCCGTAGGTGAACCTGCGG-3'; ITS2: 5'-TCCTCCGCTTATTGATATGC-3') based on the conserved region of the gene sequence, and PCR amplification. PCR uses the standard reaction system provided in the instructions for the purchased Taq enzyme, and the PCR amplification procedure is as follows:

[0035] Denaturation at 94°C for 5 min; 35 temperature cycles, each cycle including denaturation at 94°C for 30 s, denaturation at 50°C for 30 s, extension at 72°C for 50 s; insulation at 72°C for 10 min.

[0036] The amplified product was recovered and sent to the Major Platform Center of the Chinese Academy of Agricultural Sciences for sequencing. The obtained ITS sequence is shown in SEQ ID NO.1 in the sequence table. The obtained ITS sequence was compared with the nucleic acid sequence in the GeneBank database by BLAST program, and it was found that the ITS sequence of c271F had a homology of 99.81% with the ITS sequence of Fusarium redolensisolate FWN2. Based on the above results, strain c271F was identified as Fusarium redolens. The amplified ITS sequence is shown in SEQ NO.1 in the sequence table.

[0037] 4) Using ginseng endophyte F. redolens c271F to transform natural ginsenoside Rb1 into rare ginsenosides

[0038] The F. redolens c271F was inoculated into PDB medium, activated and cultured at 29°C for 48 hours, with a shaking speed of 150-180 r / min, and centrifuged at 4-10°C and 3000 rpm for 8-10 minutes. The bacterial precipitate was collected and then suspended with an equal volume of sterilized fermentation medium aqueous solution to obtain a c271F bacterial suspension. The addition amount of natural ginsenoside Rb1 in the fermentation medium is 0.1-0.2 g / L, and the inoculation amount of the fermentation bacteria is 8%-15%; the fermentation culture temperature is 25-29°C, the shaking speed is 120-140 r / min, and the total fermentation days are 10-14 days; after the fermentation is completed, an equal volume of water-saturated n-butanol (from Tianjin Beilian Fine Chemicals Development Co., Ltd.) is added to the fermentation liquid to terminate the reaction, and after extraction with water-saturated n-butanol for 2-3 times, centrifugation is carried out at 4°C and 8000 rpm for 5 minutes, the supernatant is collected and concentrated under reduced pressure to obtain a dry product containing rare ginsenosides.

[0039] 5) Detection of rare ginsenosides by high performance liquid chromatography (HPLC)

[0040] The ginsenoside mixed standard (containing Rb1, F2, 20(S)-Rg3, Rh2, CK) and the saponin product containing rare saponins prepared in step 2) of Example 1 were dissolved in methanol, filtered through a 0.22 μm microporous filter membrane (from Tianjin Jinteng Experimental Equipment Co., Ltd.), and used for HPLC chromatographic analysis. HPLC analysis method: The chromatographic column was Agilent TC-C18 (2) 250×4.6 mm, 5 μm chromatographic column, the injection volume was 10 μL, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 203 nm. The mobile phase was A: acetonitrile, B: 0.1% phosphoric acid aqueous solution, and the elution program was: 0-15 min 80% B; 15-19 min 80%-72% B; 19-23 min 72%-67% B; 23-50 min 67%-59% B; 50-78 min 59%-20% B; 78-83 min 20%-80% B; 83-85 min 80% B.

[0041] 6) Identification results and content

[0042] like Figure 1 As shown, by analyzing the ginsenoside components in the products before and after fermentation, it can be seen that by comparing the retention time with the ginsenoside standard, it is determined that the content of ginsenoside Rb1 (peak 1) after fermentation is reduced, and ginsenoside Rd (peak 2), rare ginsenoside F2 (peak 3), rare ginsenoside 20(S)-Rg3 (peak 4), rare ginsenoside CK (peak 5) and a very small amount of rare ginsenoside Rh2 (peak 6) are obtained by fermentation conversion. The content of each ginsenoside is shown in Table 1.

[0043] This proves that four rare ginsenosides were obtained by fermentation and transformation of ginsenoside extract by Fusarium redolens c271F.

[0044] Table 1. Changes in ginsenoside content before and after fermentation

[0045]

[0046] Industrial Applicability

[0047] The present invention utilizes an endophytic fungus Fusarium redolens c271F isolated from ginseng suffering from root rot, and finds that the strain can transform ginsenoside Rb1 into rare ginsenoside F2, rare ginsenoside 20(S)-Rg3, rare ginsenoside CK, and rare ginsenoside Rh2. In actual production, ginsenoside Rb1 can be transformed into rare ginsenosides by co-fermentation with ginsenosides, thereby improving the activity and utilization rate of ginsenosides.

[0048]

[0049]

Claims

1. A method for preparing rare saponins by transforming ginsenosides, which utilizes endophytic fungus fermentation to transform ginsenosides.

2. The method of claim 1, wherein the endophytic fungus is Fusarium redolens c271F.

3. The method according to claim 1, wherein the Fusarium odoratum c271F is an endophytic fungus isolated from ginseng with root rot. The method according to claim 1 , wherein the ginsenoside is natural ginsenoside Rb1.

5. A rare saponin prepared according to the method of any one of claims 1 to 4.

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