CN110982869A - Preparation of 20(R) -ginsenoside Rh by biotransformation of panax notoginseng saponins1Method (2) - Google Patents

Preparation of 20(R) -ginsenoside Rh by biotransformation of panax notoginseng saponins1Method (2) Download PDF

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CN110982869A
CN110982869A CN201911316533.XA CN201911316533A CN110982869A CN 110982869 A CN110982869 A CN 110982869A CN 201911316533 A CN201911316533 A CN 201911316533A CN 110982869 A CN110982869 A CN 110982869A
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杨晓艳
李瑞婷
崔秀明
曲媛
杨野
刘迪秋
王承潇
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Kunming University of Science and Technology
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Abstract

The invention discloses a method for preparing 20 (A) by biotransformation of panax notoginseng saponinsR) -ginsenoside Rh1The method adopts aspergillus tubingensis to carry out biotransformation on the panax notoginseng saponins to prepare 20: (R) -ginsenoside Rh1(ii) a The strain adopted by the invention is convenient and easy to obtain, the method is simple to operate, the product conversion yield is high, and the method can be used for 20: (III) ((III))R) -ginsenoside Rh1The mass preparation of (2); the method is simple, efficient, safe, green and low in cost, and provides a new method for industrial production, food production and new drug preparation.

Description

Preparation of 20 (by biotransformation of Panax notoginsenosides)R) -ginsenoside Rh1Method (2)
Technical Field
The invention relates to a method for preparing 20 (by biotransformation of panax notoginseng saponins)R) -ginsenoside Rh1The method uses special extracellular or intracellular enzyme produced by microorganism as biocatalyst to selectively hydrolyze glycosyl side chain of high-content main saponin in Panax notoginsenosides, and then 20 (A)R) -ginsenoside Rh1。
Background
Modern pharmacological studies have shown that triol form 20 (C: (R))R) -ginsenoside Rh1The compound has wide biological activity, particularly has obvious effect on resisting tumors, and has inhibition effect on the activities of human melanoma cells (A375-S2), human uterine cancer cells (HeLa cells), human fetal glioma cells (T98G cells), human breast cancer cells MDA-MB-231 and MCF-7 cells; also, it has been shown that 20: (R) -ginsenoside Rh1Can induce apoptosis of cervical cancer Hela cell line and leukemia K562 cell line, and has obvious anticancer effect, such as inhibiting migration and invasion of PMA-induced human colorectal cancer cell SW480 cell by inhibiting expression of MMP-9. Further, 20(R) -ginsenoside Rh1Has obvious effects on relieving renal tissue fibrosis, improving renal function, resisting inflammation, enhancing the immunologic function of an organism, protecting nerve cells, protecting heart and cerebral vessels, reducing myocardial damage caused by ischemia and improving memory. Further studies have shown that 20: (R) -ginsenoside Rh1Can inhibit adipogenic differentiation of fat cell 3T3-L1 cell, and prevent obesity. Furthermore, 20: (R) -ginsenoside Rh1Can obviously promote the synthesis of collagen of skin fibroblasts, effectively improve skin wrinkles and has the function of resisting skin aging. Because of complicated structure and infeasible chemical synthesis, the saponin is extracted from plants such as pseudo-ginseng, gynostemma pentaphylla and the like, but because the content of the plants is low and the plant resources are limited, a large amount of 20 (A) is obtained by utilizing abundant pseudo-ginseng total saponinsR) -ginsenoside Rh1The method is more convenient and feasible.
In order to obtain the rare ginsenoside with high medicinal value, from the eighties of the last century, researchers in chemical and biological technology at home and abroad start the engineering of structural modification of ginsenoside, and the research aim mainly locks on structural modification and modification of glycosyl side chains connected with aglycones in panaxadiol type saponin and triol type saponin by using different methods, so that glycosyl side chains of ginsenoside with high content in medicinal materials are directionally converted into rare ginsenoside through hydrolysis reaction.
Aspergillus tubingensis (A. tubingensis)Aspergillus tubingensis) The endophyte is separated from eupatorium adenophorum, is widely distributed in nature, and can be separated from resources such as soil, Hami melon, agaricus, cow dung and the like. The fungus contains rich enzyme system, and can promote the modification of natural product structure, so that the polyurethane plastic can be degraded efficiently.
Disclosure of Invention
The invention provides a method for preparing 20 (by biotransformation of panax notoginseng saponins)R) -ginsenoside Rh1The method of (1) using Aspergillus tubingensisAspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method is a green, efficient and specific fermentation method for preparing high-purity rare ginsenoside, and overcomes the defects of overlong period, unstable fermentation product and difficulty in purification in the existing fermentation process.
The technical solution of the invention is as follows:
preparation of 20 (by biotransformation of Panax notoginsenosides)R) -ginsenoside Rh1The method comprises the following specific steps:
(1) taking Aspergillus tubingensis preserved at 4 DEG CAspergillus tubingensis) Performing activation culture: inoculating the strain deposited on the tube slant into a 250mL Erlenmeyer flask containing 150mL of sterilized medium; culturing for 3-5 days by shaking, controlling the temperature at 25-28 ℃, and rotating at 150-170 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the seed solution obtained in the step (1) into a sterilized culture medium for expanded culture, wherein the inoculation amount of the seed solution accounts for 0.02-2.00% of the mass percent of the culture medium, and performing shake culture for 3-5 days to obtain a bacterial suspension;
(3) taking the panax notoginseng saponins which account for 0.02-2.00% of the mass of the bacterial suspension in the step (2), mixing the panax notoginseng saponins with sterilized purified water according to the mass ratio of 1: 2-1: 4, and carrying out ultrasonic full dissolution; under the aseptic condition, filtering the panax notoginseng saponins solution with an aseptic filter membrane, adding the filtrate into the bacterial suspension obtained in the step (2) under the aseptic condition, and continuing shake culture for 6-12 days;
（4）after the culture and fermentation in the step (3) are finished, crushing mycelium pellets by an ultrasonic method, carrying out ultrasonic treatment for 10-30 min, carrying out vacuum filtration, separating fermentation liquor and mycelium, concentrating the fermentation liquor after vacuum filtration, respectively carrying out isovolumetric extraction on the fermentation liquor and the mycelium for 3-4 times by using water saturated n-butyl alcohol, combining n-butyl alcohol phases, and carrying out vacuum evaporation concentration to obtain the polysaccharide extract rich in 20: (A)R) -ginsenoside Rh1The conversion product of (1).
The culture medium of the step (1) and the step (2) is a PDB culture medium or a martin culture medium, and the preparation method is as follows:
PDB culture medium: taking 200g of fresh commercially available potatoes, cutting the fresh commercially available potatoes into square potato blocks with the side length of 1cm, adding 1000mL of purified water, heating for 30-50 min until boiling, filtering with 4 layers of gauze, adding 20g of glucose per liter, mixing uniformly, subpackaging and sterilizing for later use, wherein the volume is constant to 1L by using the purified water; and (3) sterilizing conditions of the culture medium: 30min at 121 ℃;
martin's medium: KH (Perkin Elmer)2PO41.0g，MgSO4·7H20.5g of O, 5.0g of peptone, 10.0g of glucose and purified water, wherein the volume is fixed to 1L, the materials are uniformly mixed, subpackaged and sterilized for later use, and the sterilization conditions of the culture medium are as follows: 121 ℃ and 30 min.
The crude enzyme of the strain can be adopted for catalytic conversion in the conversion process, the strain suspension in the step (3) is replaced by crude enzyme liquid of aspergillus tubingensis, the strain is converted for 1-2D by a table concentrator, the converted product is eluted by D101 macroporous resin sequentially by water, ethanol aqueous solution with the volume fraction of 30%, ethanol aqueous solution with the volume fraction of 50%, ethanol aqueous solution with the volume fraction of 70%, ethanol aqueous solution with the volume fraction of 90% and pure ethanol to obtain four components A-D, and the component C is subjected to silica gel column chromatography and is eluted by dichloromethane/methanol with the volume ratio of 30:1, 20:1, 10:1 and 1:5 sequentially in a gradient manner to obtain six sub-components C1-C6Subfraction C3Performing high pressure liquid chromatography with water/acetonitrile gradient elution of 100: 5-64: 36 to obtain a compound 20: (R) -ginsenoside Rh1。
The preparation method of the crude enzyme liquid of the aspergillus tubingensis comprises the following steps: 5000-12000 r-min of the bacterial suspension in the step (2)-1Centrifuging for 10min, separating by an alcohol precipitation method to obtain crude enzyme, specifically, adding 2-4 times of absolute ethyl alcohol into supernate, mixing,placing in an ice bath for 30min, and after the crude enzyme is fully separated out, 10000-12000 r.min-1And (3) centrifuging to obtain a crude enzyme precipitate, adding 0.1-3.0L of phosphate buffer solution into 1g of crude enzyme, and dissolving the crude enzyme precipitate by vortex oscillation of the phosphate buffer solution with the pH = 6.0-7.0 to obtain a crude enzyme solution of the aspergillus tubingensis.
The Panax notoginsenosides are obtained by pulverizing main root of Panax notoginseng (Burk.) F.H.Chen of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying.
After the panax notoginseng saponins are converted by the aspergillus tubingensis, TLC detection shows that the ginsenoside Rg with higher content in the panax notoginseng saponins1Not found, but appeared in a higher content of 20: (R) -ginsenoside Rh1The ginsenoside Rg is presumed according to the structure and content change of the two120 (from hydrolytic desugarization under transformation with A.tubingensisR) -ginsenoside Rh1The structural formula is as follows:
Figure 702260DEST_PATH_IMAGE002
the invention mainly utilizes aspergillus tubingensis (A.tubingensis)Aspergillus tubingensis) The enzyme in the method carries out biotransformation on panax notoginseng saponins, the utilization of panax notoginseng saponins is relatively specific, the conversion rate is high, secondary metabolites generated in the fungus fermentation process can be effectively avoided through a product enrichment mode of n-butyl alcohol extraction, hypha and spores can be effectively separated through reduced pressure suction filtration, and the separation and enrichment of rare saponins are improved.
The method can be used for preparing the monomer rare ginsenoside, can also be used for developing products by directly utilizing the crude extract of the converted product, achieves the processing purposes of simplicity, high efficiency, safety, greenness and low cost, and provides new guarantee for industrial production, food production and new drug preparation.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the above-described contents, and the methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.
Example 1
Using Aspergillus tubingensis (Aspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional PDB culture medium:
① PDB culture medium is prepared by cutting 200g of fresh potato into square potato blocks with side length of 1cm, adding 1000mL of purified water, heating for 30-50 min to boil, filtering with 4 layers of gauze, adding 20g of glucose per liter, mixing, packaging, and sterilizing under 121 deg.C for 30 min;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized PDB culture medium under aseptic condition, culturing at 26.5 deg.C in shaking table for 5d, and rotating at 150r min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile PDB culture medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 0.02% of the mass of the culture medium, and performing shake culture for 5d to obtain a strain suspension;
(3) taking the panax notoginseng saponins accounting for 0.2% of the mass of the bacterial suspension in the step (2), mixing the panax notoginseng saponins with sterile purified water according to the mass ratio of 1:2, dissolving the panax notoginseng saponins with the sterile purified water, soaking the panax notoginseng saponins for 15min, and performing ultrasonic treatment to fully dissolve the panax notoginseng saponins powder; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, the lysate in the step (3) is filtered by an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under the aseptic condition, and then the bacterial suspension is placed on a shaking table for continuous culture for 10 d;
(5) after the culture and fermentation in the step (4) are finished, crushing mycelium pellets by an ultrasonic method, performing ultrasonic treatment for 10min, performing vacuum filtration to separate fermentation liquor and mycelium, properly concentrating the filtered fermentation liquor, performing isovolumetric extraction on the fermentation liquor and the mycelium for 3 times by respectively using water saturated n-butyl alcohol, combining n-butyl alcohol phases, and performing vacuum evaporation and concentration to obtain the product which is rich in 20 (C)R) -ginsenoside Rh1The panax notoginseng saponins.
Example 2
Using Aspergillus tubingensis (Aspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional martin culture medium:
① preparation of Martin's medium KH2PO41.0g，MgSO4·7H20.5g of O, 5.0g of peptone and 10.0g of glucose, adding purified water to a constant volume of 1L, and sterilizing the culture medium: 30min at 121 ℃;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized Martin's medium under aseptic condition, culturing at 27.5 deg.C, shaking table culturing for 5d, and rotating at 170 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile Martin medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 0.2% of the mass percent of the medium, and performing shake culture for 5d to obtain a strain suspension;
(3) taking the panax notoginseng saponins accounting for 2% of the mass of the bacterial suspension in the step (2), mixing the panax notoginseng saponins with sterile purified water according to the mass ratio of 1:2, dissolving the panax notoginseng saponins with the sterile purified water, soaking the panax notoginseng saponins for 15min, and performing ultrasonic treatment to fully dissolve the panax notoginseng saponins powder; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, the lysate in the step (3) is filtered by an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under the aseptic condition, and then the bacterial suspension is placed on a shaking table for continuous culture for 10 d;
(5) after the culture and fermentation in the step (4) are finished, crushing mycelium pellets by an ultrasonic method, performing ultrasonic treatment for 10min, performing vacuum filtration to separate fermentation liquor and mycelium, properly concentrating the filtered fermentation liquor, performing isovolumetric extraction on the fermentation liquor and the mycelium for 3 times by respectively using water saturated n-butyl alcohol, combining n-butyl alcohol phases, and performing vacuum evaporation and concentration to obtain the product which is rich in 20 (C)R) -ginsenoside Rh1The panax notoginseng saponins.
Example 3
Using Aspergillus tubingensis (Aspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional martin culture medium:
① preparation of Martin's medium KH2PO41.0g，MgSO4·7H20.5g of O, 5.0g of peptone and 10.0g of glucose, adding purified water to a constant volume of 1L, and sterilizing the culture medium: 30min at 121 ℃;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized Martin's medium under aseptic condition, culturing at 28 deg.C in shaking table for 3d, and rotating at 170 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile Martin medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 0.2% of the mass percent of the medium, and performing shake culture for 5d to obtain a strain suspension;
(3) taking the panax notoginseng saponins which are 1 percent of the mass of the bacterial suspension in the step (2), mixing the panax notoginseng saponins with sterile purified water according to the mass ratio of 1:4, dissolving the panax notoginseng saponins with the sterile purified water, soaking the panax notoginseng saponins for 15min, and performing ultrasonic treatment to fully dissolve the panax notoginseng saponins powder; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, the lysate in the step (3) is filtered by an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under the aseptic condition, and then the bacterial suspension is placed on a shaking table for continuous culture for 8 d;
(5) after the culture and fermentation in the step (4) are finished, crushing mycelium pellets by an ultrasonic method, performing ultrasonic treatment for 30min, performing vacuum filtration to separate fermentation liquor and mycelium, properly concentrating the filtered fermentation liquor, performing isovolumetric extraction on the fermentation liquor and the mycelium for 3 times by respectively using water saturated n-butyl alcohol, combining n-butyl alcohol phases, and performing vacuum evaporation and concentration to obtain the product which is rich in 20 (C)R) -ginsenoside Rh1The panax notoginseng saponins.
Example 4
Using Aspergillus tubingensis (Aspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional PDB culture medium:
① PDB culture medium is prepared by cutting 200g of fresh potato into square potato blocks with side length of 1cm, adding 1000mL of purified water, heating for 30-50 min to boil, filtering with 4 layers of gauze, adding 20g of glucose per liter, mixing, packaging, and sterilizing under 121 deg.C for 30 min;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized PDB culture medium under aseptic condition, culturing at 25 deg.C in shaking table for 3d, and rotating at 170 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile PDB culture medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 0.0 mass percent of the culture medium2 percent, shake culturing for 5 days, when the mycelium is covered with the culture medium, the bacterial suspension is 5000 r.min-1Centrifuging, taking supernate, and separating by adopting an alcohol precipitation method to obtain crude enzyme, wherein the specific operation is that 2-4 times of absolute ethyl alcohol is added into the supernate, the mixture is placed in an ice bath for 30min, and 10000 r.min is carried out after the crude enzyme is fully separated out-1Centrifuging to obtain a crude enzyme precipitate, adding 3L of phosphate buffer solution into 1.0g of crude enzyme, and dissolving the crude enzyme precipitate by vortex shaking with the phosphate buffer solution with pH =6 to obtain a crude enzyme solution;
(3) mixing Notoginseng radix total saponin 0.2% of the crude enzyme solution in step (2) with sterile purified water at a mass ratio of 1:4, dissolving Notoginseng radix total saponin with sterile purified water, soaking Notoginseng radix total saponin for 15min, and ultrasonic treating to dissolve Notoginseng radix total saponin powder completely; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, filtering the dissolved product in the step (3) by using an aseptic filter membrane, adding the filtrate into the crude enzyme liquid fully cultured in the step (2) under the aseptic condition, wherein the addition amount of the panax notoginseng saponins is 0.2 percent of the mass of the crude enzyme liquid, and then placing the mixture in a shaking table for shaking and converting for 1 d;
(5) eluting the obtained conversion product with D101 macroporous resin with water, 30% ethanol water solution by volume fraction, 50% ethanol water solution by volume fraction, 70% ethanol water solution by volume fraction, 90% ethanol water solution by volume fraction and pure ethanol to obtain four components A-D, and gradient eluting the component C with dichloromethane/methanol at volume ratio of 30:1, 20:1, 10:1 and 1:5 by silica gel column chromatography to obtain six sub-components C1-C6Subfraction C3Performing high pressure liquid phase preparative chromatography for 0-12 min (volume ratio of 100: 0-81: 19 water/acetonitrile); performing gradient elution for 12-60 min (the volume ratio is 81: 19-64: 36 water/acetonitrile) to obtain a compound 20: (R) -ginsenoside Rh1。
Example 5
Using Aspergillus tubingensis (Aspergillus tubingensis) BiotransformationPreparation of panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional PDB culture medium:
① PDB culture medium is prepared by cutting 200g of fresh potato into square potato blocks with side length of 1cm, adding 1000mL of purified water, heating for 30-50 min to boil, filtering with 4 layers of gauze, adding 20g of glucose per liter, mixing, packaging, and sterilizing under 121 deg.C for 30 min;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized PDB culture medium under aseptic condition, culturing at 28 deg.C in shaking table for 4d, and rotating at 160 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile PDB culture medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 1% of the mass percent of the culture medium, performing shake culture for 3d, and when the mycelium is fully distributed in the culture medium, performing 12000r & min on the strain suspension-1Centrifuging, taking supernatant, and separating by an alcohol precipitation method to obtain crude enzyme, wherein the specific operation is that 2-4 times of absolute ethyl alcohol is added into the supernatant for mixing, then the mixture is placed in an ice bath for 30min, and after the crude enzyme is fully separated out, 11000 r.min-1Centrifuging to obtain a crude enzyme precipitate, adding 0.1L phosphate buffer solution into 1.0g of crude enzyme, and dissolving the crude enzyme precipitate by vortex shaking with the phosphate buffer solution with pH =6 to obtain a crude enzyme solution;
(3) mixing Notoginseng radix total saponin 0.2% of the crude enzyme solution in step (2) with sterile purified water at a mass ratio of 1:4, dissolving Notoginseng radix total saponin with sterile purified water, soaking Notoginseng radix total saponin for 15min, and ultrasonic treating to dissolve Notoginseng radix total saponin powder completely; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, filtering the dissolved product in the step (3) by using an aseptic filter membrane, adding the filtrate into the crude enzyme liquid fully cultured in the step (2) under the aseptic condition, wherein the addition amount of the panax notoginseng saponins is 0.2 percent of the mass of the crude enzyme liquid, and then placing the mixture in a shaking table for shaking and converting for 1 d;
(5) eluting the obtained conversion product with D101 macroporous resin with water, 30% ethanol water solution by volume fraction, 50% ethanol water solution by volume fraction, 70% ethanol water solution by volume fraction, 90% ethanol water solution by volume fraction and pure ethanol to obtain four components A-D, and gradient eluting the component C with dichloromethane/methanol at volume ratio of 30:1, 20:1, 10:1 and 1:5 by silica gel column chromatography to obtain six sub-components C1-C6Subfraction C3Performing high pressure liquid phase preparative chromatography for 0-12 min (volume ratio of 100: 0-81: 19 water/acetonitrile); performing gradient elution for 12-60 min (the volume ratio is 81: 19-64: 36 water/acetonitrile) to obtain a compound 20: (R) -ginsenoside Rh1。
Example 6
Using Aspergillus tubingensis (Aspergillus tubingensis) Preparation of biotransformation panax notoginseng saponins 20R) -ginsenoside Rh1The method comprises the following steps:
(1) activating and culturing aspergillus tubingensis by adopting a conventional martin culture medium:
① preparation of Martin's medium KH2PO41.0g，MgSO4·7H20.5g of O, 5.0g of peptone and 10.0g of glucose, adding purified water to a constant volume of 1L, and sterilizing the culture medium: 30min at 121 ℃;
② activating and culturing Aspergillus tubingensis stored in PDB test tube slant at 4 deg.C, picking and inoculating the strain from the slant to 250mL conical flask containing 150mL sterilized Martin's medium under aseptic condition, culturing at 26 deg.C in shaking table for 5d, and rotating at 150r min-1Obtaining seed liquid of the strain;
(2) inoculating the activated strain seed liquid obtained in the step (1) into a sterile Martin medium for amplification culture, wherein the inoculation amount of the seed liquid accounts for 2% of the mass percent of the medium, performing shake culture for 4d, and when the mycelium is fully distributed with the medium, enabling the strain suspension to be 6000 r.min-1Centrifuging, taking outSeparating clear liquid by an alcohol precipitation method to obtain crude enzyme, specifically adding 2-4 times of absolute ethyl alcohol into the clear liquid, mixing, placing in an ice bath for 30min, and after the crude enzyme is fully precipitated, 12000 r.min-1Centrifuging to obtain a crude enzyme precipitate, adding 0.8L phosphate buffer solution into 1.0g of crude enzyme, and dissolving the crude enzyme precipitate by vortex shaking with the phosphate buffer solution with pH =7 to obtain a crude enzyme solution;
(3) mixing Notoginseng radix total saponin 0.2% of the crude enzyme solution in step (2) with sterile purified water at a mass ratio of 1:4, dissolving Notoginseng radix total saponin with sterile purified water, soaking Notoginseng radix total saponin for 30min, and ultrasonic treating to dissolve Notoginseng radix total saponin powder completely; the Notoginseng radix total saponin is prepared by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70 vol.% methanol, filtering the extractive solution, concentrating, eluting with D101 macroporous adsorbent resin with 70 vol.% ethanol until colorless, and drying to obtain Notoginseng radix total saponin;
(4) under the aseptic condition, filtering the dissolved product in the step (3) by using an aseptic filter membrane, adding the filtrate into the crude enzyme liquid fully cultured in the step (2) under the aseptic condition, wherein the addition amount of the panax notoginseng saponins is 0.1 percent of the mass of the crude enzyme liquid, and then placing the mixture in a shaking table for shaking and converting for 2 d;
(5) eluting the obtained conversion product with D101 macroporous resin with water, 30% ethanol water solution by volume fraction, 50% ethanol water solution by volume fraction, 70% ethanol water solution by volume fraction, 90% ethanol water solution by volume fraction and pure ethanol to obtain four components A-D, and gradient eluting the component C with dichloromethane/methanol at volume ratio of 30:1, 20:1, 10:1 and 1:5 by silica gel column chromatography to obtain six sub-components C1-C6Subfraction C3Performing high pressure liquid phase preparative chromatography for 0-12 min (volume ratio of 100: 0-81: 19 water/acetonitrile); performing gradient elution for 12-60 min (the volume ratio is 81: 19-64: 36 water/acetonitrile) to obtain a compound 20: (R) -ginsenoside Rh1。
Determination of Compound 20 based on NMR and Mass Spectrometry data (R) -ginsenoside Rh1The chemical structural formula is as follows:
Figure 593992DEST_PATH_IMAGE004
the spectral data are as follows: 20(R)-Ginsenoside Rh1White amorphous powder with molecular formula C36H62O9，ESI-MSm/z 661 [M + Na]+And molecular weight 638.
1H NMR(600 MHz，Pyridine-d 5)δ H：1.25 (3H, s, H-18)，1.06 (3H, s, H-19)，1.42 (3H, s, H-21)，1.68 (3H, s, H-26)，1.65 (3H, s, H-27)，2.08 (3H, s, H-28)，1.59 (3H, s, H-29)，0.89 (3H, s, H-30), 5.03 (1H, d，J=7.4 Hz，H-1’)。
13C NMR（150MHZ，Pyridine-d 5）δ C：39.5(t, C-1), 28.0(t, C-2), 78.7(d, C-3),40.4(s, C-4), 61.7(d, C-5),80.1(d, C-6), 45.3(t, C-7), 41.2(s, C-8), 50.3(d,C-9), 39.8(s, C-10), 32.3(t, C-11),71.0(d, C-12), 49.0(d, C-13), 51.4(s, C-14), 31.5(t, C-15), 26.7(t, C-16), 50.7(d, C-17), 17.5(q, C-18), 17.8(q, C-19), 73.1(s, C-20), 22.8(q, C-21), 43.5(t, C-22), 22.5(t, C-23), 124.7(d, C-24), 131.9 (s, C-25), 25.5(q, C-26), 17.8(q, C-27), 31.5(q, C-28), 16.5(q, C-29), 16.7(q, C-30), 106.4(d, C-1’), 75.5 (d, C-2’), 79.8(d, C-3’), 71.6(d, C-4’), 78.5(d, C-5’),62.5 (t, C-6’)。

Claims (5)

1. Preparation of 20 (by biotransformation of Panax notoginsenosides)R) -ginsenoside Rh1The method is characterized by comprising the following specific steps:
(1) inoculating the Aspergillus tubingensis strain preserved on a test tube inclined plane at 4 ℃ into a sterilized medium containing 150 mL; culturing for 3-5 days by shaking, controlling the temperature at 25-28 ℃, and rotating at 150-170 r.min-1Obtaining seed liquid of the strain;
(2) inoculating the seed solution obtained in the step (1) into a sterilized culture medium for expanded culture, wherein the inoculation amount of the seed solution accounts for 0.02-2.00% of the mass percent of the culture medium, and performing shake culture for 3-5 days to obtain a bacterial suspension;
(3) taking panax notoginseng saponins accounting for 0.02-2.00% of the mass of the bacterial suspension in the step (2), mixing the panax notoginseng saponins with sterilized purified water according to the mass ratio of 1: 2-1: 4, and carrying out ultrasonic full dissolution; under the aseptic condition, filtering the panax notoginseng saponins solution with an aseptic filter membrane, adding the filtrate into the bacterial suspension obtained in the step (2) under the aseptic condition, and continuing shake culture for 6-12 days;
(4) after the culture and fermentation in the step (3) are finished, performing ultrasonic treatment for 10-30 min, performing vacuum filtration, separating the fermentation liquor and the mycelia, concentrating the filtered fermentation liquor, extracting the fermentation liquor and the mycelia respectively with water saturated n-butyl alcohol for 3-4 times in equal volume, combining n-butyl alcohol phases, and performing vacuum evaporation and concentration to obtain the extract rich in 20 (C)R) -ginsenoside Rh1The conversion product of (1).
2. Preparation of bioconverted panax notoginseng saponins 20 (according to claim 1)R) -ginsenoside Rh1The method is characterized in that the culture medium in the step (1) and the step (2) is a PDB culture medium or a martin culture medium, and the preparation method is as follows:
PDB culture medium: taking 200g of fresh commercially available potatoes, cutting into square potato blocks with the side length of 1cm, adding 1000mL of purified water, heating to boil, filtering with 4 layers of gauze, adding 20g of glucose per liter, mixing uniformly, subpackaging and sterilizing for later use, wherein the volume of the purified water is 1L; and (3) sterilizing conditions of the culture medium: 30min at 121 ℃;
martin's medium: KH (Perkin Elmer)2PO41.0g，MgSO4·7H20.5g of O, 5.0g of peptone, 10.0g of glucose and purified water, wherein the volume is fixed to 1L, the materials are uniformly mixed, subpackaged and sterilized for later use, and the sterilization conditions of the culture medium are as follows: 121 ℃ and 30 min.
3. Preparation of bioconverted panax notoginseng saponins 20 (according to claim 1)R) -ginsenoside Rh1The method is characterized in that the bacterial suspension in the step (3) is replaced by crude enzyme liquid of the aspergillus tubingensis, the conversion is carried out for 1-2 days by a table concentrator, and the conversion product is eluted by D101 macroporous resin in turn by water, ethanol water solution with the volume fraction of 30%, ethanol water solution with the volume fraction of 50%, ethanol water solution with the volume fraction of 70%, ethanol water solution with the volume fraction of 90% and pure ethanol to obtain four components A-D, performing silica gel column chromatography on the component C, and performing gradient elution by using dichloromethane/methanol with volume ratios of 30:1, 20:1, 10:1 and 1:5 in sequence to obtain six sub-components C1-C6Subfraction C3Performing high pressure liquid chromatography with water/acetonitrile gradient elution of 100: 5-64: 36 to obtain a compound 20: (R) -ginsenoside Rh1。
4. Preparation of biologically converted Panax notoginsenosides of claim 3 (20)R) -ginsenoside Rh1The method is characterized in that the preparation method of the crude enzyme liquid of the aspergillus tubingensis comprises the following steps: 5000-12000 r-min of the bacterial suspension in the step (2)-1Centrifuging, taking supernatant, and separating by an alcohol precipitation method to obtain crude enzyme of 10000-12000 r.min-1And (3) carrying out centrifugal separation to obtain a crude enzyme precipitate, adding 0.1-3.0L of phosphate buffer solution into 1g of crude enzyme, and carrying out vortex oscillation on the crude enzyme precipitate by using the phosphate buffer solution with the pH = 6.0-7.0 to obtain a crude enzyme solution of the aspergillus tubingensis.
5. Preparation of bioconverted panax notoginseng saponins 20 (according to claim 1)R) -ginsenoside Rh1The method is characterized in that the panax notoginseng saponins in the step (3) are prepared by crushing main roots of panax notoginseng of panax of Araliaceae, ultrasonically extracting with methanol with volume fraction of 70%, filtering and concentrating the extract, eluting with D101 macroporous adsorption resin with volume fraction of 70% ethanol until colorless, and drying.
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