CN109234347B - Method for converting protopanaxatriol saponin to obtain C25-OH derivative - Google Patents
Method for converting protopanaxatriol saponin to obtain C25-OH derivative Download PDFInfo
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Abstract
The invention relates to a method for converting protopanaxatriol type saponin into a C25-OH derivative, belonging to a structure modification method of a natural product. Selecting a biological transformation strain: preparing a seed solution and a transformation culture medium of cordyceps sinensis, adding the seed solution into the sterilized transformation culture medium, sealing a sterile filter membrane, putting the sterilized transformation culture medium into a constant-temperature shaking table, culturing at constant temperature, adding a saponin substrate into the transformation culture medium, converting for 6-8 days, adding an n-butyl alcohol reagent with the same volume into a reaction system, stirring uniformly, and separating a target product from other byproducts to finally obtain a pure product of the C25-OH derivative of the PPT-type ginsenoside. The technology adopted by the invention is a biological conversion method, the catalytic system is edible fungi with higher biological safety and genetic stability, the specificity of the catalytic substrate is strong, the change of the structure belongs to directional conversion, the byproducts are less, the chemical background is simple and clear, and the large-scale preparation, development and application of the components are facilitated.
Description
Technical Field
The invention belongs to a structure modification method of a natural product, and particularly relates to a method for generating a C25-OH derivative from protopanaxatriol type saponin.
Background
Ginsenoside is the main active component of plants in Panax of Araliaceae, has wide biological activity on human cardiovascular system, cerebrovascular system, nervous system, immune system and endocrine system, and has great application and development value.
Protopanaxatriol (PPT) type saponin: the saponin which is formed by taking 20(R/S) -dammarane-3 beta, 6 beta, 12 beta, 20-tetraol as a structural mother nucleus and combining 6-position and 20-position glycosidic bonds with glycosyl is one of main active ingredients in the ginseng plant and has the following chemical structure:
C25-OH derivative refers to PPT type ginsenoside derivative with the following structural characteristics:
drug metabolism experiments show that after being orally taken into the body, ginsenoside is mainly influenced by gastric acid, intestinal microorganisms and liver metabolic enzymes, so that the chemical structure is changed to a certain degree, and the metabolized derivatives are the main material basis for the ginsenoside to play pharmacological action; the hydroxylation at the C25 position is a common reaction of in-vivo metabolism of dammarane type ginsenoside, researchers find through structure-activity relationship analysis that C25-OH significantly influences the pharmacological activity and bioavailability of the dammarane type ginsenoside, and particularly in the aspect of tumor resistance, some C25-OH derivatives are even dozens of times stronger than the activity of original compounds, and can be used as anti-tumor candidate drugs with great development prospects. However, these C25-OH dammarane-type ginsenosides have a very small content in nature, and most of the results reported so far are found from in vivo metabolites and processed products of ginseng plants, which are difficult to satisfy the needs of scientific research and pharmaceutical markets; although these derivatives can be obtained by chemically modifying a specific substrate, these methods generally have the problems of poor reaction specificity, more byproducts, low conversion efficiency, serious environmental pollution and the like, and limit the sustainable development of the research field. In contrast, the biocatalysis method has mild conditions, good structure and stereoselectivity and high product purity, can achieve extremely high conversion efficiency under proper conditions, and is an important means for modifying the structure of a natural product.
At present, no report exists for directionally catalyzing double bonds at C24-C25 positions of PPT type saponin to generate C25-OH derivatives by a biotransformation means, and the most similar experimental scheme to the method is as follows: zhaoyuqing and the like, crude ginsenoside substances such as total saponins of ginseng stems and leaves, or total saponins of ginseng roots, or total saponins of ginseng fruits, or total saponins of ginseng flowers and the like are subjected to mixed reaction by an acid hydrolysis method, and C25-OH derivatives are separated from complex reaction products; the reaction process involves a large amount of organic reagents, is not beneficial to amplification production, and the reaction system is a complex mixed system, so that the reaction result has certain randomness, poor specificity, a large number of byproducts, complex reaction product components, complex separation process, and does not belong to a high-efficiency and directional structure modification process.
Disclosure of Invention
The invention provides a method for converting protopanaxatriol saponin to generate a C25-OH derivative, which aims to solve the problems that strong acid and a large amount of organic reagents are involved in a reaction process during chemical preparation, the amplification production is not facilitated, a reaction system is a complex mixed system, a reaction result has certain randomness, the specificity is poor, a large number of byproducts are generated, the components of a reaction product are complex, and the separation process is complicated.
The technical scheme adopted by the invention is as follows: comprises the following steps:
(1) selecting a biological transformation strain: cordyceps Sinensis (Cordyceps Sinensis/Ophiocerdyceps Sinensis)
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
Preparing a PDA slant culture medium, inoculating the recovered or activated cordyceps sinensis to the prepared slant, putting the slant culture medium into a constant-temperature constant-humidity incubator, and culturing at 25-30 ℃ for 3-4 days, so that the slant culture medium can be taken out for use;
2) cultivation of seed liquid
Preparing a seed liquid culture medium, washing a cordyceps sinensis slope with the age of 3-4 days by using a sterile 0.9% sodium chloride aqueous solution, observing a blood globule counting plate under a microscope for reading, and diluting the washed spore liquid to 107Inoculating the spore suspension into a prepared seed liquid culture medium according to the volume ratio of 2.5-7.5%, culturing in a constant-temperature shaking table at the temperature of 25-30 ℃ at 150-180 r/min for 2-3 days, and stopping fermentation, wherein the spore suspension can be directly used or stored at the temperature of 4 ℃ for later use;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: 15-25 g/L;
nitrogen source: 2.5-7.5 g/L;
adding each solid reagent and distilled water into a container according to a certain proportion, stirring until all solids are dissolved, sterilizing for 15-25 minutes at 121 ℃, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product work-up
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 5-7.5% (V/V), sealing a sterile filter membrane, putting the sterile filter membrane into a constant-temperature shaking table, culturing at the constant temperature of 25-30 ℃ for 2-3 days, adding 0.5-1 g/L of saponin substrate into the transformation medium, after 6-8 days of transformation, adding an n-butyl alcohol reagent with the same volume into a reaction system, and carrying out ultrasonic treatment, oscillation or uniform stirring to stop the reaction;
the product treatment method comprises the following steps: extracting for 3 times by using n-butanol with the same volume, combining n-butanol solutions, recovering a solvent, weighing a solute, dissolving a sample by using a 20-25% chromatographic acetonitrile solution with the volume (ml/g) of 40-60 times, separating on a reversed-phase C18 preparation chromatographic column, isocratically eluting by using the 20-25% chromatographic acetonitrile solution under the chromatographic condition, and separating a target product from other byproducts to finally obtain the pure product of the C25-OH derivative of the PPT-type ginsenoside.
The formula and preparation method of the PDA slant culture medium in the step (2) of the invention are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled (the small pieces can be punctured by a glass rod), residues are filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating and the stirring are continuously carried out for uniform mixing, the glucose is slowly added after the agar is completely dissolved, the uniform stirring is carried out, the lost moisture is replenished while the stirring is carried out, the solution is distributed into each test tube when the solution is hot, the bottle mouth is plugged by a cotton plug, the test tube is put into a high-pressure steam sterilizer, the sterilization is carried out for 15 to 25 minutes at the temperature of 121 ℃, the test tube is taken out when the temperature in the sterilizer is reduced to about 50 ℃, and the test tube is paved into an inclined plane on a sterile operation platform.
The composition and preparation method of the seed liquid culture medium in the step (2) of the invention are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a proportion, stirring until all the solid is dissolved, subpackaging into clean erlenmeyer flasks according to a proper amount, sealing the erlenmeyer flasks with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 15-25 minutes, taking out, shaking uniformly, placing into a sterile inoculation chamber, and cooling for later use.
The carbon source in step (3) of the invention is one of maltose, glucose, polyxylose, fructose, soluble starch, lactose or sucrose.
In the step (3) of the present invention, the nitrogen source is ammonium sulfate.
In the step (3), 0.05-0.15 g/L of catalytic factor can be added.
The catalytic factor of the step (3) of the invention is MgSO4、KH2PO4Or NaH2PO4One kind of (1).
In the step (3), the saponin substrate is one or a mixture of more than one of ginsenoside Re, Rg1, Rf, Rh1, Rg2, F1 and Notogensenoside R1.
And (3) conversion result: through the steps, the prototype PPT saponin is almost completely converted, the proportion of the C25-OH derivative in the product is extremely high, the result can float in a certain interval according to different structures of the prototype PPT saponin, generally speaking, the conversion rate of the prototype PPT saponin is more than 90%, and the molar ratio of the C25-OH derivative in the converted product is more than 60%.
The invention has the advantages that: the invention provides an efficient, green and directional structure modification method, which adopts a biological conversion method as a technology, adopts a catalytic system which is edible fungi with higher biological safety and genetic stability, has strong specificity of catalytic substrates, belongs to directional conversion of structural change, has few byproducts and simple and clear chemical background, and is beneficial to large-scale preparation, development and application of the components.
Drawings
FIG. 1 is an analytical chart of the transformation process of ginsenoside Re;
FIG. 2 is a diagram of the biotransformation pathway of ginsenoside Re;
FIG. 3 is a graph showing the transformation curve of ginsenoside Re;
FIG. 4 shows ginsenoside Rg1Analysis chart of transformation process;
FIG. 5 shows ginsenoside Rg1A map of biotransformation pathways of (a);
FIG. 6 shows ginsenoside Rg1A transformation change curve graph.
Detailed Description
Example 1
Comprises the following steps:
(1) selecting a biological transformation strain: cordyceps Sinensis (Cordyceps Sinensis/Ophiocerdyceps Sinensis)
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
The formula and the preparation method of the PDA slant culture medium are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled (the small pieces can be punctured by a glass rod), residues are filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating, the stirring and the uniform mixing are continued, the glucose is slowly added after the agar is completely dissolved, the stirring is uniformly carried out, the loss water is replenished while the stirring is carried out, the solution is distributed into each test tube when the solution is hot, the bottle mouth is plugged by a cotton plug, the test tube is put into a high-pressure steam sterilizer, the sterilization is carried out for 15 minutes at the temperature of 121 ℃, the test tube is taken out when the temperature in the sterilizer is reduced to 50 ℃, and an inclined plane is paved on an aseptic operation platform;
inoculating the recovered or activated Cordyceps sinensis into the prepared slant, placing into a constant temperature and humidity incubator, culturing at 25 deg.C for 4 days, observing white hypha spreading on the whole slant, and taking out;
2) cultivation of seed liquid
The composition and preparation method of the seed liquid culture medium are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, subpackaging in a clean conical flask according to a proper amount, sealing the conical flask with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 15 minutes, taking out, shaking uniformly, placing into a sterile inoculation room, and cooling for later use;
dissolving with sterile 0.9% sodium chlorideWashing 4-day-old slant of Cordyceps sinensis, observing blood globule counting plate under microscope, and diluting the washed spore solution to 107Inoculating spore suspension into prepared seed liquid culture medium at volume ratio of 2.5%, culturing in constant temperature shaker at 25 deg.C at 150 rpm for 3 days, stopping fermentation, and directly using or storing at 4 deg.C for use;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: 15g/L of maltose, wherein the maltose is a mixture of maltose and maltose,
nitrogen source: 2.5g/L of ammonium sulfate;
adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, sterilizing at 121 ℃ for 15 minutes, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product work-up
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 5% (V/V), sealing a sterile filter membrane, putting into a constant-temperature shaking table, culturing at the constant temperature of 25 ℃ for 3 days, adding 0.5g/L of saponin substrate into the transformation medium, wherein the saponin substrate is ginsenoside Re, after 6 days of transformation, adding an equal volume of n-butyl alcohol reagent into a reaction system, and carrying out ultrasonic treatment, oscillation or uniform stirring to stop the reaction;
the product treatment method comprises the following steps: extracting with n-butanol of equal volume for 3 times, mixing n-butanol solutions, recovering solvent, weighing solute, dissolving sample with 40 times volume (ml/g) of 20% chromatographic acetonitrile solution, separating on reversed phase C18 preparative chromatographic column under 20% chromatographic acetonitrile solution isocratic elution, separating target product from other by-products, and finally obtaining pure C25-OH derivative of PPT type ginsenoside.
Example 2
Comprises the following steps:
(1) selecting a biological transformation strain: cordyceps Sinensis (Cordyceps Sinensis/Ophiocerdyceps Sinensis)
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
The formula and the preparation method of the PDA slant culture medium are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled (the small pieces can be punctured by a glass rod), residues are filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating, the stirring and the uniform mixing are continued, the glucose is slowly added after the agar is completely dissolved, the stirring is uniformly carried out, the loss water is replenished while the stirring is carried out, the solution is distributed into each test tube when the solution is hot, the bottle mouth is plugged by a cotton plug, the test tube is put into a high-pressure steam sterilizer, the sterilization is carried out for 20 minutes at the temperature of 121 ℃, the test tube is taken out when the temperature in the sterilizer is reduced to 50 ℃, and an inclined plane is paved on an aseptic operation platform;
inoculating the recovered or activated Cordyceps sinensis into the prepared slant, placing into a constant temperature and humidity incubator, culturing at 30 deg.C for 3 days, observing white hypha spreading on the whole slant, and taking out;
2) cultivation of seed liquid
The composition and preparation method of the seed liquid culture medium are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, subpackaging in a clean conical flask according to a proper amount, sealing the conical flask with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 25 minutes, taking out, shaking uniformly, placing into a sterile inoculation room, and cooling for later use;
washing Cordyceps 3 days old with sterile 0.9% sodium chloride water solution, observing with microscope, counting, and diluting the washed spore solution to 107Inoculating spore suspension into prepared seed liquid culture medium at volume ratio of 7.5%, culturing in constant temperature shaking table at 30 deg.C at 180 rpm for 2 days, stopping fermentation, and directly using or storing at 4 deg.C for use;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: the concentration of glucose is 25g/L,
nitrogen source: ammonium sulfate 7.5 g/L;
catalytic factor: MgSO (MgSO)40.15g/L；
Adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, sterilizing at 121 ℃ for 25 minutes, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product work-up
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 7.5% (V/V), sealing a sterile filter membrane, putting into a constant-temperature shaking table, culturing at the constant temperature of 30 ℃ for 2 days, adding 1g/L saponin substrate into the transformation medium, wherein the saponin substrate is a mixture of ginsenoside Rg1 and ginsenoside Rf, after 8 days of transformation, adding an n-butanol reagent with the same volume into a reaction system, and carrying out ultrasonic treatment, oscillation or uniform stirring to stop the reaction;
the product treatment method comprises the following steps: extracting with n-butanol of equal volume for 3 times, mixing n-butanol solutions, recovering solvent, weighing solute, dissolving sample with 25% chromatographic acetonitrile solution of 60 times volume (ml/g), separating on reversed phase C18 preparative chromatographic column, isocratic eluting with 25% chromatographic acetonitrile solution, separating target product from other by-products, and finally obtaining pure C25-OH derivative of PPT type ginsenoside.
Example 3
Comprises the following steps:
(1) selecting a biological transformation strain: cordyceps Sinensis (Cordyceps Sinensis/Ophiocerdyceps Sinensis)
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
The formula and the preparation method of the PDA slant culture medium are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled (the small pieces can be punctured by a glass rod), residues are filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating, the stirring and the uniform mixing are continued, the glucose is slowly added after the agar is completely dissolved, the stirring is uniformly carried out, the loss water is replenished while the stirring is carried out, the solution is distributed into each test tube when the solution is hot, the bottle mouth is plugged by a cotton plug, the test tube is put into a high-pressure steam sterilizer, the sterilization is carried out for 20 minutes at the temperature of 121 ℃, the test tube is taken out when the temperature in the sterilizer is reduced to 50 ℃, and an inclined plane is paved on an aseptic operation platform;
inoculating the recovered or activated Cordyceps sinensis into the prepared slant, placing into a constant temperature and humidity incubator, culturing at 27.5 deg.C for 3.5 days, observing white hypha spreading on the whole slant, and taking out;
2) cultivation of seed liquid
The composition and preparation method of the seed liquid culture medium are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, subpackaging in a clean conical flask according to a proper amount, sealing the conical flask with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 20 minutes, taking out, shaking uniformly, placing into a sterile inoculation room, and cooling for later use;
washing 3.5 days old slant of Cordyceps with sterile 0.9% sodium chloride water solution, observing with microscope, counting with counting plate, and diluting the washed spore solution to 107Inoculating spore suspension into prepared seed liquid culture medium at volume ratio of 5.0%, culturing in constant temperature shaker at 27.5 deg.C for 2.5 days at 165 rpm, stopping fermentation, and using directly or storing at 4 deg.C;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: 20g/L of soluble starch;
nitrogen source: 5.0g/L of ammonium sulfate;
catalytic factor: KH (Perkin Elmer)2PO40.05g/L；
Adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, sterilizing at 121 ℃ for 20 minutes, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product work-up
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 6.5% (V/V), sealing a sterile filter membrane, putting into a constant-temperature shaking table, culturing at the constant temperature of 27.5 ℃ for 2.5 days, adding 0.75g/L of saponin substrate into the transformation medium, wherein the saponin substrate is a mixture of ginsenoside Re, Rg1, Rf, Rh1, Rg2 and F1, converting for 7 days, adding an equal volume of n-butanol reagent into a reaction system, and carrying out ultrasonic treatment, oscillation or uniform stirring to stop the reaction;
the product treatment method comprises the following steps: extracting with n-butanol of equal volume for 3 times, mixing n-butanol solutions, recovering solvent, weighing solute, dissolving sample with 22.5% chromatographic acetonitrile solution with 50 times volume (ml/g), separating on reversed phase C18 preparative chromatographic column under 22.5% chromatographic acetonitrile solution isocratic elution, separating target product from other by-products, and finally obtaining pure product of PPT type ginsenoside C25-OH derivative.
Example 4
Comprises the following steps:
(1) selecting a biological transformation strain: cordyceps Sinensis (Cordyceps Sinensis/Ophiocerdyceps Sinensis)
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
The formula and the preparation method of the PDA slant culture medium are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled (the small pieces can be punctured by a glass rod), residues are filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating, the stirring and the uniform mixing are continued, the glucose is slowly added after the agar is completely dissolved, the stirring is uniformly carried out, the loss water is replenished while the stirring is carried out, the solution is distributed into each test tube when the solution is hot, the bottle mouth is plugged by a cotton plug, the test tube is put into a high-pressure steam sterilizer, the sterilization is carried out for 20 minutes at the temperature of 121 ℃, the test tube is taken out when the temperature in the sterilizer is reduced to 50 ℃, and an inclined plane is paved on an aseptic operation platform;
inoculating the recovered or activated Cordyceps sinensis into the prepared slant, placing into a constant temperature and humidity incubator, culturing at 27 deg.C for 4 days, observing white hypha spreading on the whole slant, and taking out;
2) cultivation of seed liquid
The composition and preparation method of the seed liquid culture medium are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a certain proportion, stirring until the solid is completely dissolved, subpackaging in a clean conical flask according to a proper amount, sealing the conical flask with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 20 minutes, taking out, shaking uniformly, placing into a sterile inoculation room, and cooling for later use;
washing 4-day old Cordyceps slant with sterile 0.9% sodium chloride water solution, observing blood globule counting plate under microscope, and diluting the washed spore solution to 10%7Inoculating spore suspension into prepared seed liquid culture medium at volume ratio of 5.0%, culturing in constant temperature shaking table at 165 rpm and 27 deg.C for 3 days, stopping fermentation, and directly using or storing at 4 deg.C for use;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: 20g/L of sucrose;
nitrogen source: 5.0g/L of ammonium sulfate;
catalytic factor: NaH2PO40.10g/L；
Adding each solid reagent and distilled water in a proper container according to a certain proportion, stirring until the solid is completely dissolved, sterilizing at 121 ℃ for 20 minutes, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product work-up
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 6% (V/V), sealing a sterile filter membrane, putting into a constant-temperature shaking table, culturing at the constant temperature of 27 ℃ for 3 days, adding 0.8g/L of saponin substrate into the transformation medium, wherein the saponin substrate is a mixture of ginsenoside Re, Rg1 and Notogynsenoside R1, after 7 days of transformation, adding an n-butanol reagent with the same volume into a reaction system, and carrying out ultrasonic treatment, oscillation or uniform stirring and reaction termination;
the product treatment method comprises the following steps: extracting with n-butanol of equal volume for 3 times, mixing n-butanol solutions, recovering solvent, weighing solute, dissolving sample with 23% chromatographic acetonitrile solution of 50 times volume (ml/g), separating on reversed phase C18 preparative chromatographic column, isocratic eluting with 23% chromatographic acetonitrile solution, separating target product from other by-products, and finally obtaining pure C25-OH derivative of PPT type ginsenoside.
And (3) conversion result: through the steps, the prototype PPT saponin is almost completely converted, the proportion of the C25-OH derivative in the product is extremely high, the result can float in a certain interval according to different structures of the prototype PPT saponin, generally speaking, the conversion rate of the prototype PPT saponin is more than 90%, and the molar ratio of the C25-OH derivative in the converted product is more than 60%.
The invention is further illustrated below by means of experimental examples.
Experimental examples structural modification of ginsenosides Re and Rg1
1. Conversion process and method for preparing test solution
Performing operation on an ultraclean workbench, scraping spores from the inclined plane of Cordyceps sinensis, inoculating into sterilized seed culture medium, sealing with sterile filter membrane, placing into a constant temperature shaking table, and culturing at 28 deg.C for 72 hr to obtain seed solution for converting protopanaxatriol saponin.
Two bottles of transformation medium were prepared, with the composition being 20g/L glucose, 5g/L ammonium sulfate. Sterilizing with high pressure steam at 121 deg.C for 20 min, taking out, and cooling. Respectively inoculating the seed solutions into 2 bottles of sterilized transformation medium according to the volume ratio of 5%, sealing with an aseptic filter membrane, placing into a constant temperature shaking table, and culturing at constant temperature of 28 ℃ to expand the biomass. After 2 days of culture, 0.5g/L of ginsenoside Re and Rg are respectively added into two bottles of transformation culture medium1. And absorbing a certain volume of conversion sample solution every 24 hours from the addition of the saponin substrate, and marking for later use.
Preparing an internal standard solution: protopanaxadiol type saponinRb1Weighing ginsenoside Rb as internal standard substance1And dissolving the standard substance in water to prepare an internal standard solution with a certain concentration. Adding a volume of Rb to the conversion sample to be tested1After the internal standard solution is fully and uniformly mixed, extracting the sample solution for 3 times by using water saturated n-butanol with the same volume, combining the extract liquor, recovering the solvent, dissolving by using chromatographic methanol with a certain volume, passing through a 0.22 micron filter membrane, and transferring into a liquid phase small bottle to be used as a test sample for later use.
2. Ginsenoside Re and Rg1Analysis of transformation Process
2.1 high Performance liquid-triple quadrupole Mass Spectrometry (HPLC-TQ-MS) parameter settings
The detection of liquid chromatography was carried out on a Shim-pack XR-ODS II HPLC PACKED COLUMN (3.0 mm. times.75 mm,2.2 μm) COLUMN set at 40 ℃; the mobile phase A is 1 per mill of formic acid water, and the mobile phase B is acetonitrile; the elution order is shown in Table 1 below, with the flow rate set at 0.2mL/min and the sample size set at 5 ul.
TABLE 1 chromatographic elution conditions
The mass spectrum detection is carried out on an Shimadzu 8040TQ mass spectrometer system, an ion source is a dual-channel ESI, a mass scanning range is set to be m/z 300-1300, a scanning mode is set to be an anion mode, and the optimized mass spectrum parameters are as follows: the flow rate of the drying gas was 9L/min, the temperature of the drying gas was 350 ℃, the pressure of the spray gas was 30psi, the capillary voltage was 4500V, the fragmentation voltage was 175V, and the cone voltage was 65V. The formula calculation parameters are set to C0-80, H0-150, O0-60, and other elements such as N, S, P and Cl are not taken into consideration.
The quantification of saponin substrate, conversion intermediates and final products was performed in the Multiple Reaction Monitoring (MRM) mode of HPLC-MS with the following parameters set:
Rb1:m/z 1107→945[M-H]-
Re:m/z 991→946[M-H]-
Rg2:m/z 829→783[M-H]-
Rf2:m/z 847→801[M-H]-
Rg1:m/z 845→799[M-H]-
Rh1:m/z:683→475[M-H]-
25OH-Rh1:m/z:701→493[M-H]-
2.2 ginsenoside Re transformation Process analysis results
And (3) detecting the sample solution after the conversion of the ginsenoside Re processed in the step (1) by HPLC-TQ-MS, and performing qualitative and relative quantitative analysis on the converted product. The detection map is shown in FIG. 1.
Based on the above maps, we can find that the ginsenoside Re is basically not detected when the ginsenoside Re is transformed to the 7 th day, which indicates that the transformation is relatively complete, and the main transformation products are 4, namely 20(S) -ginsenoside Rg220(R) -ginsenoside Rg220(S) -ginsenoside Rf220(R) -ginsenoside Rf2Wherein 20(S/R) -ginsenoside Rf2Is C25-OH derivative of protopanaxatriol saponin, and its specific conversion pathway is shown in FIG. 2:
in order to clarify the content change of ginsenoside Re and its transformation products during the reaction, a relative quantitative analysis was performed using a Multiple Reaction Monitor (MRM) mode of triple quadrupole mass spectrometry, and the results are shown in table 2 and fig. 3:
TABLE 2 relative content changes of ginsenoside Re and its conversion products
From the above table, we can find that in the present reaction system, the C25-OH derivative is the main conversion product of ginsenoside Re, and its content is more than 70% of all the conversion products.
2.3 ginsenoside Rg1Analytical results of transformation Process
Subjecting the ginsenoside Rg treated in step 1 to1Converted test solutionAnd detecting by HPLC-TQ-MS, and performing qualitative and relative quantitative analysis on the converted product. The detection profile is shown in FIG. 4.
By the map, the ginsenoside Rg can be found1After the conversion had ended, it was virtually undetectable, indicating that the conversion was relatively complete, with 4 major conversion products, 20(S) -ginsenoside Rh120(R) -ginsenoside Rh120(S) -25-OH-ginsenoside Rh120(R) -25-OH-ginsenoside Rh1Wherein 20(S/R) -25-OH-ginsenoside Rh1Is C25-OH derivative of protopanaxatriol saponin, and its specific conversion pathway is shown in FIG. 5:
to clarify ginsenoside Rg1And the content change of the conversion product thereof during the reaction, were relatively quantitatively analyzed using a Multiple reaction monitoring mode (MRM) of triple quadrupole mass spectrometry, and the results are shown in table 3 and fig. 6:
TABLE 3 ginsenoside Rg1And the relative content of the conversion products thereof
From the above table, it can be found that, in the present reaction system, the C25-OH derivative is ginsenoside Rg1The content of the main conversion product in the total conversion products exceeds 65%.
Claims (4)
1. A method for converting protopanaxatriol-type saponin to C25-OH derivatives, comprising the steps of:
(1) selecting a biological transformation strain: cordyceps sinensis (berk.) SaccCordyceps sinensis/Ophiocordyceps Sinensis）
(2) Seed liquid preparation of biotransformation strain
1) Slant culture of biotransformation strain
Preparing a PDA slant culture medium, inoculating the recovered or activated cordyceps sinensis to the prepared slant, putting the slant culture medium into a constant-temperature constant-humidity incubator, and culturing at 25-30 ℃ for 3-4 days, so that the slant culture medium can be taken out for use;
2) cultivation of seed liquid
Preparing a seed liquid culture medium, washing a cordyceps sinensis slope with the age of 3-4 days by using a sterile 0.9% sodium chloride aqueous solution, observing the reading of a blood globule counting plate under a microscope, and diluting the washed spore liquid to 107Inoculating the spore suspension into a prepared seed liquid culture medium according to the volume ratio of 2.5-7.5%, culturing in a constant-temperature shaking table at the temperature of 25-30 ℃ at 150-180 r/min for 2-3 days, and stopping fermentation, wherein the spore suspension can be directly used or stored at the temperature of 4 ℃ for later use;
(3) biotransformation process
1) Preparation of transformation Medium
Carbon source: 15-25 g/L;
nitrogen source: 2.5-7.5 g/L;
catalytic factor: 0.05-0.15 g/L;
wherein the nitrogen source is ammonium sulfate, and the catalytic factor is MgSO4、KH2PO4Or NaH2PO4One of (1);
adding each solid reagent and distilled water into a container according to a certain proportion, stirring until all solids are dissolved, sterilizing for 15-25 minutes at 121 ℃, taking out, shaking up, putting into an aseptic inoculation chamber, and cooling for later use;
2) bioconversion parameters and product handling
The biotransformation parameters are as follows: adding the seed solution into a sterilized transformation medium according to the inoculation amount of 5-7.5% V/V, sealing a sterile filter membrane, putting the sterile filter membrane into a constant-temperature shaking table, culturing at the constant temperature of 25-30 ℃ for 2-3 days, adding 0.5-1 g/L of saponin substrates into the transformation medium, wherein the saponin substrates are ginsenoside Re and Rg1, adding an n-butyl alcohol reagent with the same volume into a reaction system after 6-8 days of transformation, and carrying out ultrasonic treatment, oscillation or uniform stirring to terminate the reaction;
the product treatment method comprises the following steps: extracting for 3 times by using n-butanol with the same volume, combining n-butanol solutions, recovering a solvent, weighing a solute, dissolving a sample by using a 20-25% chromatographic acetonitrile solution with the volume of 40-60 times of ml/g, separating on a reversed-phase C18 preparation chromatographic column, isocratically eluting by using the 20-25% chromatographic acetonitrile solution under the chromatographic condition, and separating a target product from other byproducts to finally obtain a pure product of the C25-OH derivative of the PPT-type ginsenoside.
2. The method of claim 1, wherein the C25-OH derivative is prepared by converting protopanaxatriol-type saponin to the following compounds: the formula and preparation method of the PDA slant culture medium in the step (2) are as follows: 200g/L of potato, 20g/L of glucose and 24g/L of agar, wherein the pH value is natural, the potato is cleaned, peeled and cut into small pieces, the small pieces are put into a proper amount of water to be boiled, the residue is filtered by eight layers of filter cloth, the agar is slowly and uniformly added, the heating and the stirring are continuously carried out to be uniformly mixed, the glucose is slowly added after the agar is completely dissolved, the uniform stirring is carried out, the reduced water is replenished while the solution is stirred, the solution is subpackaged into each test tube while being hot, the bottle mouth is tightly closed by a cotton plug, the high-pressure steam sterilizer is put into the high-pressure steam sterilizer, the test tube is sterilized at the temperature of 121 ℃ for 15 to 25 minutes, when the temperature in the sterilizer is reduced to 50 ℃, the test tube is taken out, and the test tube is paved into an inclined plane on a sterile operation platform.
3. The method of claim 1, wherein the C25-OH derivative is prepared by converting protopanaxatriol-type saponin to the following compounds: the composition and preparation method of the seed liquid culture medium in the step (2) are as follows: 10g/L of peptone, 2g/L of yeast extract powder, 20g/L of glucose, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and natural pH; adding each solid reagent and distilled water into a container according to a proportion, stirring until all the solid is dissolved, subpackaging into clean erlenmeyer flasks according to a proper amount, sealing the erlenmeyer flasks with a sterile filter membrane, placing into a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 15-25 minutes, taking out, shaking uniformly, placing into a sterile inoculation chamber, and cooling for later use.
4. The method of claim 1, wherein the C25-OH derivative is prepared by converting protopanaxatriol-type saponin to the following compounds: the carbon source in the step (3) is one of maltose, glucose, polyxylose, fructose, soluble starch, lactose or sucrose.
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