CN110982869B - Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins 1 Is a method of (2) - Google Patents
Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins 1 Is a method of (2) Download PDFInfo
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Abstract
The invention discloses a method for preparing 20 percent of biological conversion total saponins of panax notoginsengR) Ginsenoside Rh 1 The method adopts aspergillus tubingensis to carry out bioconversion on the panax notoginseng saponins to prepare the panax notoginseng saponins of 20 #R) Ginsenoside Rh 1 The method comprises the steps of carrying out a first treatment on the surface of the 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 #R) Ginsenoside Rh 1 Is prepared in large quantities; the method is simple, efficient, safe, green and low in cost, and provides a new method for industrial production, food production and new medicine preparation.
Description
Technical Field
The invention relates to a method for preparing 20 percent of biological conversion total saponins of panax notoginsengR) Ginsenoside Rh 1 The method uses special extracellular or intracellular enzyme produced by microorganism as biocatalyst to selectively hydrolyze glycosyl side chain of main saponin with high content in total saponins of Notoginseng radix to prepare 20%R) Ginsenoside Rh 1 。
Background
Modern pharmacological research shows that triol type is 20%R) Ginsenoside Rh 1 Has wide biological activity, particularly has remarkable anti-tumor effect, 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, studies have shown that 20%R) Ginsenoside Rh 1 Can 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 human colorectal cancer cell SW480 induced by PMA by inhibiting MMP-9 expression. In addition, 20%R) Ginsenoside Rh 1 Has obvious effects in relieving renal tissue fibrosis, improving renal function, resisting inflammation, enhancing organism immunity, protecting nerve cells, protecting cardiovascular and cerebrovascular, reducing myocardial injury caused by ischemia and improving memory. Also, the study shows that 20%R) Ginsenoside Rh 1 Can inhibit adipogenic differentiation of adipocyte 3T3-L1 cells, and prevent obesity. In addition, 20%R) Ginsenoside Rh 1 Can obviously promote the synthesis of collagen of skin fibroblasts, effectively improve skin wrinkles, and has the effect of resisting skin aging. Because of the complex structure, chemical synthesis is not feasible, and the plant extract is obtained from plants such as pseudo-ginseng, gynostemma pentaphylla and the like, but because the content of the plant extract is low and plant resources are limited, a large amount of 20 percent of total saponins of pseudo-ginseng are obtainedR) Ginsenoside Rh 1 The method of (2) is simpler and more practical.
In order to obtain the rare ginsenoside with extremely high medicinal value, chemical and biotechnology engineering researchers at home and abroad begin from the last eighties of century, and the research targets of the structural modification engineering of the ginsenoside are mainly locked on the structural modification and the modification of glycosyl side chains connected with aglycone in panaxadiol type saponin and triol type saponin by utilizing different methods, so that the glycosyl side chains of the ginsenoside with higher content in medicinal materials are directionally converted into rare ginsenoside through hydrolysis reaction, and at present, the main methods for the structural modification and the modification of the glycosyl side chains of the ginsenoside are chemical methods and microbial conversion methods.
Aspergillus tubingensis (Fr.) KummerAspergillus tubingensis) Endophytes separated from eupatorium adenophorum, which are widely distributed in nature, can be separated from soil, cantaloupe, umbrella bacteria, cow dung and other resources. The fungus contains rich enzyme systems, so that the modification of the structure of natural products can be promoted, and polyurethane plastics can be efficiently degraded.
Disclosure of Invention
The invention provides a method for preparing 20 percent of biological conversion total saponins of panax notoginsengR) Ginsenoside Rh 1 The method utilizes aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 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 difficult purification in the existing fermentation process.
The technical scheme of the invention is as follows:
preparation of biological conversion notoginseng total saponin 20%R) Ginsenoside Rh 1 The method comprises the following specific steps:
(1) Taking Aspergillus tubingensis preserved at 4 ℃ inclined planeAspergillus tubingensis) Performing activation culture: inoculating the strain deposited on the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized culture medium; shaking culture for 3-5 d, controlling temperature at 25-28 deg.C and rotating speed at 150-170 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the seed liquid obtained in the step (1) into a sterilized culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.02-2.00% of the mass of the culture medium, and performing shake culture for 3-5 d to obtain a bacterial suspension;
(3) Taking the total saponins of pseudo-ginseng as 0.02-2.00% of the mass of the bacterial suspension in the step (2), mixing the total saponins of pseudo-ginseng with sterilized purified water according to the mass ratio of 1:2-1:4, and fully dissolving the total saponins of pseudo-ginseng by ultrasound; under aseptic condition, the notoginseng total saponin solution is filtered by an aseptic filter membrane, and the filtrate is added into the bacterial suspension in the step (2) under aseptic condition, and the shake cultivation is continued for 6-12 d;
(4) After the culture and fermentation are finished, breaking mycelium clusters by adopting an ultrasonic method, carrying out ultrasonic treatment for 10-30 min, carrying out vacuum suction filtration, separating fermentation liquor and mycelium, concentrating the fermentation liquor after suction filtration, respectively extracting the fermentation liquor and the mycelium with water saturated n-butanol for 3-4 times in equal volume, merging n-butanol phases, and carrying out vacuum evaporation concentration to obtain the fermented liquid rich in 20 #R) Ginsenoside Rh 1 Is a conversion product of (a).
The culture medium in the step (1) and the step (2) is PDB culture medium or Ma Dingshi culture medium, and the preparation method is as follows:
PDB medium: cutting 200g of fresh commercial 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, fixing the volume to 1L with purified water, adding 20g of glucose per liter, mixing uniformly, packaging and sterilizing for later use; medium sterilization conditions: 121 ℃, for 30min;
ma Dingshi medium: KH (KH) 2 PO 4 1.0g，MgSO 4 ·7H 2 0.5g of O, 5.0g of peptone, 10.0g of glucose, and purified water to 1L, mixing, packaging and sterilizing for later use, wherein the culture medium sterilization conditions are as follows: 121 ℃, for 30min.
The method can also adopt crude enzyme of the bacterium to carry out catalytic conversion in the conversion process, the bacterial suspension in the step (3) is replaced by the crude enzyme liquid of the aspergillus tubingensis, the conversion product is subjected to shaking table conversion for 1-2D, the conversion product is eluted by D101 macroporous resin sequentially by water, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution, 90% ethanol aqueous solution and pure ethanol to obtain four components A-D, and the component C is subjected to gradient elution sequentially by silica gel column chromatography by methylene dichloride/methanol with the volume ratio of 30:1, 20:1, 10:1 and 1:5 to obtain six subfractions C 1 -C 6 Subfraction C 3 The compound 20 is obtained by high pressure liquid phase preparation chromatography and water/acetonitrile gradient elution with the ratio of 100:5 to 64:36R) Ginsenoside Rh 1 。
The preparation method of the aspergillus tubingensis crude enzyme liquid comprises the following steps: 5000-12000 r.min of the bacterial suspension in the step (2) -1 Centrifuging for 10min, separating by alcohol precipitation to obtain crude enzyme, specifically by adding 2-4 times of absolute ethanol into supernatant, mixing, standing in ice bath for 30min, and separating out 10000-12000 r min after crude enzyme is sufficiently separated out -1 Centrifuging to obtain crude enzyme precipitate, adding phosphate buffer solution 0.1-3.0L in the proportion of 1g of crude enzyme, and vortex oscillating and dissolving the crude enzyme precipitate by using phosphate buffer solution with pH=6.0-7.0 to obtain Aspergillus tubingensis crude enzyme solution.
The Notoginseng radix total saponin is obtained by pulverizing main root of Notoginseng radix of Panax of Araliaceae, ultrasonic extracting with 70% methanol by volume fraction, filtering, concentrating the extractive solution, eluting with D101 macroporous adsorbent resin with 70% ethanol by volume fraction until colorless, and drying.
After the aspergillus tubingensis converts the panax notoginseng saponins, TLC detection shows that the original ginsenoside Rg with higher content in the panax notoginseng saponins 1 But the content is higher than 20%R) Ginsenoside Rh 1 According to the structure and content changes of the two, the ginsenoside Rg is estimated 1 Hydrolysis and desugarization are converted into 20 ℃ under the transformation of aspergillus tubingensisR) Ginsenoside Rh 1 The structural formula is as follows:
the invention mainly utilizes aspergillus tubingensisAspergillus tubingensis) The enzyme in (2) carries out bioconversion on the total saponins of panax notoginseng, the utilization of the total saponins of panax notoginseng is relatively specific, the conversion rate is higher, secondary metabolites generated in the fermentation process of fungi can be effectively avoided by a product enrichment mode of n-butanol extraction, hyphae and spores can be effectively separated by vacuum filtration, and the separation and enrichment of rare saponins are improved.
The method of the invention can prepare monomer rare ginsenoside, and can directly utilize the crude extract of the conversion product to develop the product, thereby achieving the processing purposes of simplicity, high efficiency, safety, green and low cost and providing new guarantee for industrial production, food production and new medicine preparation.
Detailed Description
The present invention will be described in further detail by way of examples, but the scope of the invention is not limited to the above description, and the methods in the examples are all conventional methods, and the reagents used are all conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.
Example 1
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 Is a method of (2)The method comprises the following steps of:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional PDB culture medium:
(1) PDB medium: cutting 200g of fresh commercial 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, fixing the volume of the purified water to 1L, adding 20g of glucose per liter, mixing, packaging and sterilizing for later use; medium sterilization conditions: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of the PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized PDB culture medium per flask under the aseptic environment, and the culture temperature is as follows: 26.5 ℃; shake cultivation for 5d; the rotation speed is 150 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the activated strain seed liquid in the step (1) to a sterile PDB culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.02 percent of the mass of the culture medium, and carrying out shake culture for 5 days to obtain a strain suspension;
(3) Taking the total saponins of panax notoginseng according to the mass ratio of 0.2% of the bacterial suspension in the step (2), mixing the total saponins of panax notoginseng with sterile purified water according to the mass ratio of 1:2, dissolving the total saponins of panax notoginseng with sterile purified water, soaking the total saponins of panax notoginseng for 15min, and fully dissolving the total saponins of panax notoginseng powder by ultrasound; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under aseptic conditions, and then the bacterial suspension is placed in a shaking table for continuous culture for 10d;
(5) After the culture and fermentation of the step (4), breaking mycelium clusters by adopting an ultrasonic method, carrying out ultrasonic treatment for 10min, then carrying out vacuum filtration to separate fermentation liquor and mycelium, properly concentrating the fermentation liquor after the vacuum filtration, respectively carrying out isovolumetric extraction on the fermentation liquor and the mycelium with water saturated n-butanol for 3 times, merging n-butanol phases, and carrying out vacuum evaporation concentration to obtain the 2-enriched food0(R) Ginsenoside Rh 1 Is prepared from Notoginseng radix total saponin.
Example 2
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 The method comprises the following steps:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional Ma Dingshi culture medium:
(1) preparation of Ma Dingshi culture medium: KH (KH) 2 PO 4 1.0g，MgSO 4 ·7H 2 O0.5 g, peptone 5.0g, glucose 10.0. 10.0g, adding purified water to a volume of 1L, and sterilizing the culture medium: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of a PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized Ma Dingshi culture medium per flask under the aseptic environment, and the culture temperature is as follows: 27.5 ℃; shake cultivation for 5d; the rotation speed is 170 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the activated strain seed liquid in the step (1) into a sterile Ma Dingshi culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.2% of the mass of the culture medium, and culturing for 5d by a shaking table to obtain a strain suspension;
(3) Taking the total saponins of panax notoginseng as 2% of the mass of the bacterial suspension in the step (2), mixing the total saponins of panax notoginseng with sterile purified water according to the mass ratio of 1:2, dissolving the total saponins of panax notoginseng with the sterile purified water, soaking the total saponins of panax notoginseng for 15min, and fully dissolving the total saponins of panax notoginseng powder by ultrasound; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under aseptic conditions, and then the bacterial suspension is placed in a shaking table for continuous culture for 10d;
(5) After the culture and fermentation of the step (4) are finished, breaking hyphae by adopting an ultrasonic methodPerforming ultrasonic treatment for 10min, vacuum filtering to separate fermentation broth and mycelium, concentrating the filtered fermentation broth, extracting the fermentation broth and mycelium with water saturated n-butanol for 3 times, mixing n-butanol phases, and concentrating under reduced pressure to obtain extract rich in 20%R) Ginsenoside Rh 1 Is prepared from Notoginseng radix total saponin.
Example 3
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 The method comprises the following steps:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional Ma Dingshi culture medium:
(1) preparation of Ma Dingshi culture medium: KH (KH) 2 PO 4 1.0g，MgSO 4 ·7H 2 O0.5 g, peptone 5.0g, glucose 10.0. 10.0g, adding purified water to a volume of 1L, and sterilizing the culture medium: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of a PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized Ma Dingshi culture medium per flask under the aseptic environment, and the culture temperature is as follows: 28 ℃; shake cultivation for 3d; the rotation speed is 170 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the activated strain seed liquid in the step (1) into a sterile Ma Dingshi culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.2% of the mass of the culture medium, and culturing for 5d by a shaking table to obtain a strain suspension;
(3) Taking the total saponins of panax notoginseng as 1% of the mass of the bacterial suspension in the step (2), mixing the total saponins of panax notoginseng with sterile purified water according to the mass ratio of 1:4, dissolving the total saponins of panax notoginseng with the sterile purified water, soaking the total saponins of panax notoginseng for 15min, and fully dissolving the total saponins of panax notoginseng powder by ultrasound; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the bacterial suspension fully cultured in the step (2) under aseptic conditions, and then the bacterial suspension is placed in a shaking table for continuous culture for 8d;
(5) After the culture and fermentation of the step (4), breaking mycelium clusters by adopting an ultrasonic method, carrying out ultrasonic treatment for 30min, then carrying out reduced pressure suction filtration to separate fermentation liquor and mycelium, properly concentrating the fermentation liquor after suction filtration, respectively carrying out equal volume extraction on the fermentation liquor and the mycelium by using water saturated n-butanol for 3 times, merging n-butanol phases, and carrying out reduced pressure evaporation concentration to obtain the fermentation liquor rich in 20 #R) Ginsenoside Rh 1 Is prepared from Notoginseng radix total saponin.
Example 4
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 The method comprises the following steps:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional PDB culture medium:
(1) PDB medium: cutting 200g of fresh commercial 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, fixing the volume of the purified water to 1L, adding 20g of glucose per liter, mixing, packaging and sterilizing for later use; medium sterilization conditions: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of the PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized PDB culture medium per flask under the aseptic environment, and the culture temperature is as follows: 25 ℃; shake cultivation for 3d; the rotation speed is 170 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the strain seed liquid after the activation in the step (1) into a sterile PDB culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.02 percent of the mass of the culture medium, culturing for 5 days by a shaking table, and when mycelium is fully distributed in the culture medium, preparing a strain suspension of 5000 r.min -1 Centrifuging, collecting supernatant, separating by alcohol precipitation to obtain crude enzyme, specifically by adding 2-4 times of absolute ethanol into supernatant, mixing, standing in ice bath for 30min, and separating out 10000 r.min after crude enzyme is sufficiently separated -1 Centrifugal separation to obtain crude enzyme precipitate, adding phosphate buffer solution 3L in a proportion of 1.0g crude enzyme, and using phosphate buffer solution with pH=6Vortex vibration to dissolve crude enzyme precipitate to obtain crude enzyme solution;
(3) Taking the total saponins of pseudo-ginseng as 0.2% of the mass of the crude enzyme liquid in the step (2), mixing the total saponins of pseudo-ginseng with sterile purified water according to the mass ratio of 1:4, dissolving the total saponins of pseudo-ginseng with the sterile purified water, soaking the total saponins of pseudo-ginseng for 15min, and fully dissolving the total saponins of pseudo-ginseng powder by ultrasonic; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the crude enzyme liquid fully cultured in the step (2) under aseptic conditions, the total saponins of panax notoginseng are added in an amount which is 0.2 percent of the mass of the crude enzyme liquid, and then the obtained product is placed in a shaking table to shake and convert for 1d;
(5) The obtained conversion product is eluted by D101 macroporous resin sequentially with water, 30% ethanol aqueous solution with volume fraction, 50% ethanol aqueous solution with volume fraction, 70% ethanol aqueous solution with volume fraction, 90% ethanol aqueous solution with volume fraction and pure ethanol to obtain four components A-D, and component C is eluted sequentially by silica gel column chromatography with dichloromethane/methanol with volume ratio of 30:1, 20:1, 10:1 and 1:5 to obtain six subfractions C 1 -C 6 Subfraction C 3 High pressure liquid phase preparation chromatography is carried out for 0 to 12min (volume ratio of 100:0 to 81:19 water/acetonitrile); gradient elution is carried out for 12-60 min (volume ratio of 81:19-64:36 water/acetonitrile) to obtain the compound 20 #R) Ginsenoside Rh 1 。
Example 5
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 The method comprises the following steps:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional PDB culture medium:
(1) PDB medium: cutting 200g of fresh commercial 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, fixing the volume of the purified water to 1L, adding 20g of glucose per liter, mixing, packaging and sterilizing for later use; medium sterilization conditions: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of the PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized PDB culture medium per flask under the aseptic environment, and the culture temperature is as follows: 28 ℃; shake cultivation for 4d; the rotation speed is 160 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the activated strain seed liquid in the step (1) into a sterile PDB culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 1% of the mass percentage of the culture medium, carrying out shake culture for 3d, and when mycelia are fully distributed in the culture medium, carrying out bacterial suspension 12000 r.min -1 Centrifuging, collecting supernatant, separating by alcohol precipitation to obtain crude enzyme, specifically by adding 2-4 times of absolute ethanol into supernatant, mixing, standing in ice bath for 30min, and separating out 11000 r.min after crude enzyme is fully separated out -1 Centrifugal separation is carried out to obtain crude enzyme sediment, and the crude enzyme sediment is dissolved by vortex vibration of phosphate buffer solution with pH=6 according to the proportion of adding 0.1L of phosphate buffer solution into 1.0g of crude enzyme to prepare crude enzyme solution;
(3) Taking the total saponins of pseudo-ginseng as 0.2% of the mass of the crude enzyme liquid in the step (2), mixing the total saponins of pseudo-ginseng with sterile purified water according to the mass ratio of 1:4, dissolving the total saponins of pseudo-ginseng with the sterile purified water, soaking the total saponins of pseudo-ginseng for 15min, and fully dissolving the total saponins of pseudo-ginseng powder by ultrasonic; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the crude enzyme liquid fully cultured in the step (2) under aseptic conditions, the total saponins of panax notoginseng are added in an amount which is 0.2 percent of the mass of the crude enzyme liquid, and then the obtained product is placed in a shaking table to shake and convert for 1d;
(5) The obtained conversion product is sequentially treated with water, 30 percent ethanol aqueous solution with volume fraction, 50 percent ethanol aqueous solution with volume fraction, 70 percent ethanol aqueous solution with volume fraction and D101 macroporous resinEluting with 90% ethanol water solution and pure ethanol to obtain four components A-D, subjecting component C to silica gel column chromatography, and sequentially gradient eluting with 30:1, 20:1, 10:1, and 1:5 dichloromethane/methanol to obtain six subfractions C 1 -C 6 Subfraction C 3 High pressure liquid phase preparation chromatography is carried out for 0 to 12min (volume ratio of 100:0 to 81:19 water/acetonitrile); gradient elution is carried out for 12-60 min (volume ratio of 81:19-64:36 water/acetonitrile) to obtain the compound 20 #R) Ginsenoside Rh 1 。
Example 6
Utilizing aspergillus tubingensisAspergillus tubingensis) Biological conversion of total saponins of pseudo-ginseng to prepare 20%R) Ginsenoside Rh 1 The method comprises the following steps:
(1) The aspergillus tubingensis is activated and cultured by adopting a conventional Ma Dingshi culture medium:
(1) preparation of Ma Dingshi culture medium: KH (KH) 2 PO 4 1.0g，MgSO 4 ·7H 2 O0.5 g, peptone 5.0g, glucose 10.0. 10.0g, adding purified water to a volume of 1L, and sterilizing the culture medium: 121 ℃, for 30min;
(2) the Aspergillus tubingensis preserved on the inclined surface of a PDB test tube at the temperature of 4 ℃ is taken for activation culture, and the strain is picked and inoculated from the inclined surface of the test tube into a 250mL conical flask containing 150mL of sterilized Ma Dingshi culture medium per flask under the aseptic environment, and the culture temperature is as follows: 26 ℃; shake cultivation for 5d; the rotation speed is 150 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the activated strain seed liquid in the step (1) into a sterile Ma Dingshi culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 2% of the mass percentage of the culture medium, performing shake culture for 4d, and when mycelia are fully distributed in the culture medium, preparing a bacterial suspension 6000 r.min -1 Centrifuging, collecting supernatant, separating by alcohol precipitation to obtain crude enzyme, specifically by adding 2-4 times of absolute ethanol into supernatant, mixing, standing in ice bath for 30min, and separating out 12000 r.min after crude enzyme is sufficiently separated out -1 Centrifugal separation is carried out to obtain crude enzyme sediment, and the crude enzyme sediment is dissolved by vortex vibration of phosphate buffer solution with pH=7 according to the proportion of adding 0.8L of phosphate buffer solution into 1.0g of crude enzyme to prepare crude enzyme solution;
(3) Taking the total saponins of pseudo-ginseng as 0.2% of the mass of the crude enzyme liquid in the step (2), mixing the total saponins of pseudo-ginseng with sterile purified water according to the mass ratio of 1:4, dissolving the total saponins of pseudo-ginseng with the sterile purified water, soaking the total saponins of pseudo-ginseng for 30min, and fully dissolving the total saponins of pseudo-ginseng powder by ultrasonic; pulverizing radix Notoginseng of Panax of Araliaceae, ultrasonic extracting with 70% methanol, filtering, concentrating, eluting with D101 macroporous adsorbent resin with 70% ethanol, and drying to obtain total saponins;
(4) Under aseptic conditions, the dissolved product in the step (3) is filtered through an aseptic filter membrane, the filtrate is added into the crude enzyme liquid fully cultured in the step (2) under aseptic conditions, the total saponins of panax notoginseng are added in an amount which is 0.1 percent of the mass of the crude enzyme liquid, and then the obtained product is placed in a shaking table to shake and convert for 2d;
(5) The obtained conversion product is eluted by D101 macroporous resin sequentially with water, 30% ethanol aqueous solution with volume fraction, 50% ethanol aqueous solution with volume fraction, 70% ethanol aqueous solution with volume fraction, 90% ethanol aqueous solution with volume fraction and pure ethanol to obtain four components A-D, and component C is eluted sequentially by silica gel column chromatography with dichloromethane/methanol with volume ratio of 30:1, 20:1, 10:1 and 1:5 to obtain six subfractions C 1 -C 6 Subfraction C 3 High pressure liquid phase preparation chromatography is carried out for 0 to 12min (volume ratio of 100:0 to 81:19 water/acetonitrile); gradient elution is carried out for 12-60 min (volume ratio of 81:19-64:36 water/acetonitrile) to obtain the compound 20 #R) Ginsenoside Rh 1 。
Determination of Compound 20 based on Nuclear magnetic resonance and Mass Spectrometry dataR) Ginsenoside Rh 1 The chemical structural formula is as follows:
the spectrum data are as follows: 20 (R)-Ginsenoside Rh 1 White amorphous powder with molecular formula of C 36 H 62 O 9 ，ESI-MS m/z 661 [M + Na] + Molecular weight 638.
1 H 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’)。
13 C NMR（150MH Z ，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 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins 1 Is characterized by comprising the following specific steps:
(1) Inoculating the aspergillus tubingensis strain preserved on the inclined surface of the test tube at the temperature of 4 ℃ into a sterilized culture medium containing 150 mL; shaking culture for 3-5 d, controlling temperature at 25-28 deg.C and rotating speed at 150-170 r.min -1 Obtaining seed liquid of the strain;
(2) Inoculating the seed liquid obtained in the step (1) into a sterilized culture medium for expansion culture, wherein the inoculum size of the seed liquid accounts for 0.02-2.00% of the mass of the culture medium, and performing shake culture for 3-5 d to obtain a bacterial suspension;
(3) Taking total saponins of pseudo-ginseng accounting for 0.02-2.00% of the mass of the bacterial suspension in the step (2), mixing the total saponins with sterilized purified water according to the mass ratio of 1:2-1:4, and fully dissolving by ultrasound; under aseptic condition, the notoginseng total saponin solution is filtered by an aseptic filter membrane, and the filtrate is added into the bacterial suspension in the step (2) under aseptic condition, and the shake cultivation is continued for 6-12 d;
after the culture fermentation is finished, the ultrasonic treatment is carried out for 10 to 30 minutes,vacuum filtering, separating fermentation liquor and mycelium, concentrating the fermentation liquor after vacuum filtering, respectively extracting the fermentation liquor and mycelium with water saturated n-butanol for 3-4 times in equal volume, mixing n-butanol phases, and concentrating by vacuum evaporation to obtain 20 (R) -ginsenoside Rh-enriched extract 1 Is a conversion product of (a).
2. Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins according to claim 1 1 The method is characterized in that the culture medium in the step (1) and the step (2) is PDB culture medium or Ma Dingshi culture medium, and the preparation method is as follows:
PDB medium: cutting 200g of fresh commercial potatoes into square potato blocks with side length of 1cm, adding 1000mL of purified water, heating to boil, filtering with 4 layers of gauze, fixing volume to 1L with purified water, adding 20g of glucose per liter, mixing, packaging and sterilizing for later use; medium sterilization conditions: 121 ℃, for 30min;
ma Dingshi medium: KH (KH) 2 PO 4 1.0g，MgSO 4 ·7H 2 0.5g of O, 5.0g of peptone, 10.0g of glucose, and purified water to 1L, mixing, packaging and sterilizing for later use, wherein the culture medium sterilization conditions are as follows: 121 ℃, for 30min.
3. Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins according to claim 1 1 The method is characterized in that the bacterial suspension in the step (3) is replaced by a aspergillus tubingensis crude enzyme liquid, the conversion product is transformed by a shaking table for 1-2D, the conversion product is sequentially eluted by D101 macroporous resin with water, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution, 90% ethanol aqueous solution and pure ethanol to obtain four components A-D, and the component C is sequentially eluted by silica gel column chromatography with dichloromethane/methanol with volume ratios of 30:1, 20:1, 10:1 and 1:5 to obtain six subfractions C 1 -C 6 Subfraction C 3 Performing high-pressure liquid phase preparation chromatography, and gradient eluting with 100:5-64:36 water/acetonitrile to obtain compound 20 (R) -ginsenoside Rh 1 。
4. A method according to claim 3Bioconversion of Panax notoginseng saponins to prepare 20 (R) -ginsenoside Rh 1 The method is characterized by comprising the following steps of: 5000-12000 r.min of the bacterial suspension in the step (2) -1 Centrifuging, collecting supernatant, separating with alcohol precipitation method to obtain crude enzyme, 10000-12000 r.min -1 Centrifugal separation is carried out to obtain crude enzyme precipitate, and phosphate buffer solution with pH=6.0-7.0 is added according to the proportion of 0.1-3.0L of 1g of crude enzyme, and vortex oscillation is carried out to dissolve the crude enzyme precipitate to obtain the Aspergillus tubingensis crude enzyme solution.
5. Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins according to claim 1 1 The method is characterized in that in the step (3), the total saponins of the pseudo-ginseng are prepared by crushing main roots of pseudo-ginseng of the genus Panax of the family Araliaceae, extracting with 70% methanol by volume fraction ultrasound, filtering and concentrating the extracting solution, eluting with 70% ethanol by volume fraction with D101 macroporous adsorption resin until the total saponins of the pseudo-ginseng are colorless, and drying.
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