CN117659112A - Dammarane type triterpene saponin oxidation derivative and preparation method and application thereof - Google Patents
Dammarane type triterpene saponin oxidation derivative and preparation method and application thereof Download PDFInfo
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- CN117659112A CN117659112A CN202311664697.8A CN202311664697A CN117659112A CN 117659112 A CN117659112 A CN 117659112A CN 202311664697 A CN202311664697 A CN 202311664697A CN 117659112 A CN117659112 A CN 117659112A
- Authority
- CN
- China
- Prior art keywords
- dammarane
- triterpene saponin
- saponin
- type triterpene
- derivative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000007254 oxidation reaction Methods 0.000 title abstract description 43
- 230000003647 oxidation Effects 0.000 title abstract description 26
- 241000233866 Fungi Species 0.000 claims abstract description 32
- 150000007949 saponins Chemical class 0.000 claims abstract description 26
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- 150000008130 triterpenoid saponins Chemical class 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 34
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
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Abstract
The invention discloses a dammarane type triterpene saponin oxidation derivative, a preparation method and application thereof. The invention prepares a series of rare oxidized derivatives by directionally oxidizing dammarane type triterpene saponins through fungi, and the preparation method of the invention can be used as a powerful means for preparing the triterpene saponin series oxidized derivatives and excavating novel compounds. In addition, the hydroxylation modification of the dammarane type saponin increases the bioactivity of the repair of the myocardial injury of the saponin, and can be used as a medicament for treating myocardial injury related diseases.
Description
Technical Field
The invention relates to the field of natural product structure modification, in particular to a dammarane type triterpene saponin oxidation derivative, a preparation method and application thereof.
Background
The Ginseng radix (Panaxginseng C.A. Meyer) is plant of Panax of Araliaceae, and the pharmacological activity of triterpene saponin isolated from Ginseng radix mainly comprises neuroprotection, antiinflammatory, cardiovascular protection, anticancer, and antidiabetic. Triterpene saponins can be classified into dammarane type, octoterrane type and olean type according to the structural skeleton of sapogenin. Among them, dammarane-type triterpene saponins are considered to be the main pharmacologically active ingredient of triterpene saponins, and the pharmacological actions and potential molecular mechanisms of dammarane-type (DA) triterpene saponins are gradually elucidated.
Dammarane type triterpene saponin is one of the main representative types of triterpene saponin, and has pharmacological effects of resisting tumor, relieving inflammation, lowering blood sugar, etc. However, dammarane type triterpene saponins have the pharmaceutical defects of low natural content, low in vivo bioavailability, low water solubility and the like, and seriously prevent the application and development of the compounds in scientific research and medical treatment. The modification of the chemical component structure before the formation of the pharmaceutical preparation is an important content in the research of natural pharmaceutical chemistry, and how to modify the chemical structure of dammarane type triterpenoid saponins, so that the effects of improving the solubility and the absorption performance of the drug, regulating the pharmacokinetics, playing roles in synergism and toxicity reduction and expanding clinical indications are still to be further researched.
The biological conversion of the effective components in the traditional Chinese medicine is an effective means for digging and reforming new medicines, the biological enzyme system in the system is quite rich and various, and the structural modification of the traditional Chinese medicine active components which is difficult to realize by a physical or chemical method can be completed. The active substances of the natural medicines are good lead compounds in medicine research, but the prototype compounds of the active substances are not necessarily capable of meeting the pharmaceutical requirements of the preparation, so that the active substances need to be subjected to structural modification so as to better play the role of the medicines in practical clinical application.
Disclosure of Invention
In order to solve at least part of the technical problems in the prior art, the inventor obtains a series of high-activity dammarane type triterpenoid saponin oxidation derivatives with novel structures through a great deal of researches. The invention utilizes microorganisms to carry out structural modification on the series dammarane type triterpenoid saponins, and finds that partial conversion products of the dammarane type triterpenoid saponins have higher bioavailability and pharmacological activity compared with prototype saponins, and can be used as prodrugs in various fields of myocardial injury resistance, cancer resistance, inflammation resistance and the like. Meanwhile, the invention optimizes the whole bioconversion condition, develops the separation and purification of main derivatives after the biological conversion of dammarane type triterpenoid saponin, and clarifies the pharmacological action mechanism. The physical, chemical properties and biological activity of the triterpene saponin after the microbial structure modification are changed to a certain extent. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided an oxygenated derivative of dammarane-type triterpene saponins having the structure represented by the following formula I:
wherein,
r1, R2, R3 are the same or different and are each independently selected from mono-, oligo-or H;
r4 and R5 are hydroxyl.
Preferably, the monosaccharide is selected from at least one of beta-D-glucopyranosyl, arabinopyranosyl, arabinofuranosyl, xylopyranosyl, alpha-L-rhamnopyranosyl; the oligosaccharide is at least one of cellobiose group, glc-Glc, glc-Ara-Xyl, glc-Glc-Xyl, glc-Ara, glc-Xyl, glc-Glc-Ac and Glc-Rha.
In certain embodiments, the dammarane-type triterpene saponin oxidized derivatives according to the present invention, wherein the derivatives comprise at least one of the following compounds:
in a second aspect of the invention, a pharmaceutical composition is provided, comprising an oxidized derivative of dammarane-type triterpenoid saponins according to the invention.
In a third aspect of the present invention, there is provided a method for producing an oxidized derivative of dammarane-type triterpene saponin, comprising the step of introducing a hydroxyl group into the dammarane-type triterpene saponin structure in the presence of a fungus or an oxidase derived from the fungus, using the dammarane-type triterpene saponin as a substrate.
In certain embodiments, the method of preparing an oxidized derivative of dammarane-type triterpene saponin according to the present invention, wherein the dammarane-type triterpene saponin oxidized derivative has hydroxyl groups at the C-24 and C-25 positions of the aglycone mother core skeleton structure.
In certain embodiments, the process for preparing an oxidized derivative of dammarane-type triterpene saponin according to the present invention, wherein the dammarane-type triterpene saponin is selected from the group consisting of ginsenoside Rh 2 、Rg 3 、Rb 2 、Rb 1 、Rd、Rc、F 2 、F 1 、Rg 2 、Rg 1 、Rh 1 Re, rf and notoginsenoside R 1 And R is 2 At least one of them.
In certain embodiments, the process for the preparation of an oxidized derivative of dammarane-type triterpene saponin according to the present invention, wherein the fungus is derived from the genus mucor. Preferably Mucor Spinosus or Mucor plus.
In certain embodiments, the method for preparing the dammarane-type triterpene saponin oxidation derivative according to the present invention, wherein the dammarane-type triterpene saponin is used as a substrate by an oxidase of the fungus, and a hydroxyl group is introduced through an oxidation reaction.
In certain embodiments, the method of preparing an oxidized derivative of dammarane-type triterpene saponin according to the present invention, wherein the oxidase is selected from at least one of cytochrome P450 (ONT.10421.5), NADH flavin oxidoreductase/NADH oxidase family protein (ONT.5329.1), and Cupredoxin (ONT.2555.5), or a combination thereof.
In a fourth aspect, the invention provides the use of an oxidized derivative of dammarane-type triterpenoid saponin according to the invention for the preparation of a medicament for ameliorating, preventing or treating a disease.
The beneficial technical effects of the invention include:
(1) The invention takes the oxidation reaction of the triterpenoid saponin skeleton as an entry point to carry out bioconversion strain screening and biocatalysis system construction, and provides powerful support for enriching the structural diversity of saponin substances and the large-scale development of rare triterpenoid saponins.
(2) The condition of fungus MS to convert dammarane type triterpenoid saponin is examined, and the bioconversion efficiency of fungus MS is improved by systematically optimizing the composition of a culture medium, the time of adding a substrate and the concentration of the substrate. Through analysis of time-course experiments, a bioconversion way of the dammarane type triterpenoid saponin oxidation reaction is constructed.
(3) The triterpene saponin oxidation derivative is prepared and qualitatively analyzed by utilizing solid phase extraction, liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy, 5 rare triterpene saponin oxidation derivative monomers are found, separated and purified, and the reaction specificity of the biological catalytic system is defined.
(4) The antioxidant capacity, cytotoxicity, activity of resisting myocardial injury and molecular action mechanism of the dammarane type triterpene saponin oxidation derivative are researched. Provides a new medicine source for treating diseases such as heart ischemia reperfusion injury, heart failure, angina and the like, provides a new thought for structural modification of dammarane type saponin, and promotes the creation of new medicines of natural products and the development of new dosage forms of active ingredients of traditional Chinese medicines.
(5) By utilizing differential transcriptome analysis and bioinformatics means, potential oxidase for catalyzing the oxidation reaction of triterpenoid saponin is found, and the molecular catalysis mechanism is clarified. The chemical analysis of the bioconversion process is combined with transcriptome sequencing, so that the method is used for discovering triterpenoid saponin oxidase, and a new idea is provided for excavating other functional proteins.
(6) The mature biological conversion method is established for directionally oxidizing and modifying the dammarane type triterpenoid saponin, and the method is high-efficiency, exclusive, economical and feasible, and breaks through the restriction of the traditional chemical method. Lays a foundation for researching the structural modification of the series of compounds in future.
Drawings
FIGS. 1-2 show TLC analysis results of TLC detection results of ginsenoside Re catalytic conversion of different strains and ginsenoside conversion of fungus MS.
FIG. 3 is a schematic illustration of ginsenoside F 2 HPLC-MS chromatograms of the samples after conversion.
FIG. 4 is a view exemplarily showing ginsenoside Rg 2 HPLC-MS chromatogram of (c).
FIG. 5 schematically shows the substrate Rg 2 And corresponding conversion derivative content change curves.
FIG. 6 is an exemplary illustration of ginsenoside Rg 2 And (5) measuring the purity of the derivative and determining a liquid quality map.
FIG. 7 is an exemplary illustration of ginsenoside Rg 2 Oxidation of derivatives 13 C-NMR spectrum.
FIG. 8 is a volcanic chart of myocardial cell differential genes before and after drug administration.
FIG. 9 (R), 25-dihydroxyRg 3 Effects on expression of RAS pathway related proteins in cardiomyocytes.
FIG. 10 shows the gene expression of 7 oxidase enzymes and ginsenoside Rg 3 And the content change curve of the oxidized derivatives thereof.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Any intermediate product and desired compound product obtained in the synthesis process of the present invention can be determined by known means by those skilled in the art, including, but not limited to, high Performance Liquid Chromatography (HPLC), mass Spectrometry (MS), or gas chromatography-mass spectrometry (GC-MS). And further by, for example 1 H、 13 C and various two-dimensional Nuclear Magnetic Resonance (NMR) techniques are used for characterizing the molecular structure of any compound in the preparation process.
The term "isomer" refers to compounds of the same chemical structure but of different arrangements, wherein R, S, as defined according to Cahn-Ingold-Prelog (CIP) rules, represents the absolute configuration of the asymmetric carbon atoms in the compound.
The term "group" refers to any portion of a compound.
Unless otherwise indicated, the configurations, element symbols, short lines, wedge solid lines, wedge broken lines, and the like in the structural formulae shown herein have meanings commonly understood in the art.
Dammarane type triterpene saponin oxidation derivative
The invention prepares a dammarane type triterpene saponin oxidation derivative (sometimes referred to as a rare triterpene saponin derivative) through intensive research, wherein the dammarane type triterpene saponin oxidation derivative is a compound with hydroxyl introduced, and particularly hydroxyl is simultaneously introduced at 24 th and 25 th positions of dammarane type triterpene saponin, and hydroxylation generated through directional modification has important significance for new medicine excavation. More importantly, the directional modification is accomplished by bioconversion, i.e., modification of the structure of the triterpenoid saponin directly by an organism, particularly by an enzyme produced by the growth and metabolism of the fungus organism itself, which has the advantage that no change in the structure of the triterpenoid saponin is caused by the production of acidic or basic substances during the process.
In the present invention, when a monosaccharide or oligosaccharide is attached to the aglycone of the dammarane-type triterpene saponin, particularly at the C-6 position, the monosaccharide or oligosaccharide is attached to the backbone of the dammarane-type triterpene saponin through a glycosidic bond.
Preparation method
The invention also provides a preparation method of the dammarane type triterpene saponin oxidation derivative, and in a specific embodiment, the preparation method comprises the following steps:
(1) Culturing fungi in a culture solution containing dammarane type triterpenoid saponin as a substrate;
(2) Separating and purifying the dammarane type triterpene saponin oxidation derivative, wherein the substrate is transformed by directionally modifying oxidase generated in the mycelium growth and metabolism process of the fungus, so that hydroxyl groups are simultaneously introduced at the C-24 and C-25 positions of the dammarane type triterpene saponin.
In the step (1), the fungus is a fungus of the genus Mucor, preferably Mucor Spinosus or Mucor pleurotus, and the strain is CICC.NO.40243, and the preservation time is 11 months and 25 days in 2005.
The invention also optimizes the conditions in the preparation process, thereby determining the optimum culture medium composition, optimum substrate concentration and other parameters in the biological transformation process, and the prepared dammarane type triterpene saponin oxidation derivative has obviously improved conversion rate and purity. The culture medium (medium) used for transformation includes: 1-10g/L of potato soaked powder, 1-50g/L of glucose, 1-20g/L of peptone and 0.1-5g/L of sodium chloride. Preferably, the culture medium for transformation comprises: 2-8g/L of potato soaked powder, 10-40g/L of glucose, 5-15g/L of peptone and 0.2-2g/L of sodium chloride. Also preferably, the culture solution for transformation includes: 3-7g/L of potato soaked powder, 15-25g/L of glucose, 8-12g/L of peptone and 0.5-1.5g/L of sodium chloride.
The invention researches the concentration of the substrate, and discovers that the too high or too low concentration of the substrate is unfavorable for the preparation of oxidized derivatives, the too high concentration of the substrate can cause excessive substrate, can influence the selectivity and purity of reaction products, and the too low concentration of the substrate can cause slow reaction rate and long reaction period, thereby being unfavorable for the improvement of production efficiency. Preferably, the substrate concentration is 10-400mg/L, and still more preferably 50-250mg/L, e.g.50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250mg/L.
In the step (1), the reaction conditions such as temperature, pH, rotation speed, and incubation time are not particularly limited and may be adjusted as needed.
Use of the same
The invention also provides application of the dammarane type triterpenoid saponin oxidation derivative in preparing medicines for improving, preventing or treating diseases, and particularly in treating myocardial injury related diseases.
Example 1
This example shows microbial excavation mediating the oxidation reaction of the dammarane-type triterpenoid saponin backbone, comprising the steps of:
1. experimental method
1. Rejuvenation of strain, passage and preparation of culture medium
The used strains include: cordyceps militaris (Cordyceps militaris, CICC. No.14015, 10 th month of preservation), curvularia lunata (Tolypocladium inflatum, CICC. No.2595, 9 th month of preservation 2013, 4 th month of preservation), cordyceps (Ophiocordyceps sinensis, CICC. No.14092, 11 th month of preservation 2013), aghanomyces spinosa (Cunninghamella echinulata, CICC. No.40267, 25 th month of 2005), ganoderma lucidum (Ganoderma lingzhi, CICC. No.50004, 26 th month of preservation 2006), ganoderma sinensis (Ganoderma sinense, CICC. No.14049, 10 th month of preservation 2006), mucor (Mucor plus, CICC. No.40243, 11 th month of original preservation 2005), all of which are provided by the China industry microbiological culture Collection center (CICC).
Rejuvenating and passaging the strain, preparing seed solution, measuring spore number of seven fungi in logarithmic growth phase (2-3 days) to obtain spore concentration of 1×10 7 The spores/mL is transferred to a constant temperature and humidity shaking table to be cultured to the logarithmic phase (fungus, curvularia longifolia and Mucor echinoderm 1-2 days and other strains 3-4 days) for standby.
The transformation medium was prepared as follows: according to the proportion of 3g/L of potato soaked powder and 20g/L of glucose, the potato soaked powder and the glucose are dissolved in conical shake flasks, 100mL of each flask is sealed by a sterile breathable sealing film in 250mL shake flasks, and the mixture is sterilized by high-pressure steam.
2. Transformed strain screening
The ginsenoside Re is precisely weighed to be prepared into a substrate solution of 5 mg/mL. And (3) respectively taking 1mL of substrate solution, adding the substrate solution into the transformation culture mediums of different strains, culturing for 6 days, stopping the reaction, simultaneously extracting, combining the extracts, drying under reduced pressure, and adding chromatographic methanol for dissolving for later use.
3. Substrate range preliminary screening of optimal strain
Precisely weighing ginsenoside Re and Rg 1 、Rh 1 、Rg 2 、Rb 1 、Rc、Rg 3 、F 2 、Rh 2 And CK, dissolved to 1mg/mL with ethanol solution. 1mL of substrate solution is respectively added into the transformation culture medium of the optimal strain for 6 days.
4. TLC detection method and HPLC-MS analysis
By using silica gel GF 254 TLC detection was performed on the thin layer plates. Chromatographic conditions for HPLC-MS analysis: the column model was ZORBAX series SB-Aq (4.6 mm. Times.250 mm), mobile phase B was acetonitrile and A was 0.1% formic acid/water. Mass spectrometry conditions: the sample was subjected to a full scan (scan) using Shimadzu HPLC-QQQ-MS under Negative ion mode (Negative) to obtain excimer ion peak and additive ion peak information.
2. Experimental results
1. Bacterial screening results
Analysis of 7 fungi transformed dammarane humansAs can be seen from TLC thin layer diagram of ginsenoside Re, the conversion effect of fungus MS in all the screened strains is very obvious in unit time, and other strains are hardly converted. The product band of the ginsenoside Re converted by the fungus MS appears in a direction with lower specific shift value (Rf), and the oxidation reaction of the product occurs in consideration of the fact that normal phase thin layer chromatography is adopted, and more polar hydroxyl or other groups are newly added in the structure, so that the polarity of the compound is increased, and the retention time is prolonged. The oxidation reaction is different from the general glycosyl hydrolysis reaction, has unique biological significance, so that fungus MS is selected as a transformation strain in the following experiments to continue the transformation experiment, the result is shown in figure 1, wherein, in the left graph, 1-mixed label (Re, rg) 1 、Rh 1 ) The method comprises the steps of carrying out a first treatment on the surface of the 2-strain G; 3-strain LZ; 4-strain N; 5-strain NG; 6-strain P; 7-strain DX; 8-strain MS; in the right panel, 1-Rg 1 (day 0); 2-Rg 1 (end point); 3-Rg 2 (day 0); 4-Rg 2 (end point); 5-Rg 3 (day 0); 6-Rg 3 (end point); 7-Rh 1 (day 0); 8-Rh 1 (end point).
2. TLC detection result of fungus MS converting different ginsenosides
In the left panel of FIG. 2, 1-Re (day 0); 2-Re (endpoint); 3-Rb 1 (day 0); 4-Rb 1 (end point); 5-Rc (day 0); 6-Rc (end point); 7-F 2 (day 0); 8-F 2 (end point); in the right diagram of FIG. 2, 1-Rh 2 (day 0); 2-Rh 2 (end point); 3-CK (day 0); 4-CK (endpoint). As can be seen from the right graph of FIG. 1 and FIG. 2, ginsenoside Rg 1 The sample bands of Rc and CK are not changed obviously, which indicates that the conversion effect of fungus MS is poor; while ginsenoside F 2 、Rb 1 、Re、Rg 2 、Rg 3 、Rh 1 And Rh 2 The sample bands of (a) are changed to different degrees, wherein, ginsenoside Rg 2 ，Rg 3 ，Rh 1 And Rh 2 In the low Rf region, new sample bands are generated, and oxidation increases the polarity of the compound, so that the new sample bands are oxidized derivatives. In addition, ginsenoside Rb 1 Re and F 2 Sample bands were generated in both the high Rf and low Rf regions,the product of the high Rf region is therefore the secondary glycoside after deglycosylation, while the low Rf region is the primary glycoside or an oxidized derivative of the secondary glycoside.
3. HPLC-MS detection result of fungus MS converting different ginsenosides
The invention relates to seven kinds of saponins F 2 、Rb 1 、Re、Rg 2 、Rg 3 、Rh 1 And Rh 2 The result is subjected to liquid chromatography-mass spectrometry analysis, and the conversion path of the dammarane type triterpenoid saponin converted by the fungus MS is primarily presumed through the chemical structure of the substrate and the mass-to-charge ratio of the product. The following is an exemplary illustration of ginsenoside F 2 The converted samples were analyzed by HPLC-MS and the results are shown in FIG. 3.
According to TLC and LC-MS analysis, most saponins generate conversion products with larger polarity after being converted by fungus MS, and the conversion products are analyzed to be oxidized derivatives according to the change of mass-to-charge ratio; after conversion of some saponins by fungal MS, the oxidized derivatives were detected, along with some secondary glycosides generated by hydrolysis of the glycosidic bond, indicating that the reaction is selective for the structure of the saponins: some saponins with structures can only undergo oxidation reaction, some saponins can undergo oxidation reaction and hydrolysis reaction at the same time, and some saponins cannot undergo any obvious reaction. The mechanism leading to the above structural selection differences may be related to the steric structure of the oxidase catalyzing the reaction, requiring that the catalytic enzyme be mined and the functional domain structure be elucidated before further determination can be made. Meanwhile, some of the conversion products of the saponins have 2-3 isomers, and the homologs are caused by different hydroxyl linking positions in consideration of a plurality of oxidation sites on the DA-type saponin framework.
Example 2
This example shows the condition optimization of fungal MS bioconversion of dammarane type triterpenoids, as follows.
1. Experimental method
1. Optimization of transformation Medium
Different types of basal media include Sabouraud medium, potato Dextrose Agar (potato dextrose agar), malt Extract Agar (malt agar), czapek-Dox medium, and the like.
2. Optimization of induction factors of fungal MS catalytic system
Preparing an inducer stock solution by using the optimized basic culture medium, and respectively adding different inducer MgSO with the concentration of 1g/L 4 ·7H 2 O、KH 2 PO 4 、NaCl、FeSO 4 ·7H 2 O、CoCl 2 、MnSO 4 ·H 2 O、FeCl 3 ·6H 2 O and ZnSO 4 ·7H 2 O。
3. Substrate concentration screening
In the experiment, five concentrations (50 mg/L, 100mg/L, 150mg/L, 200mg/L and 250 mg/L) of ginsenoside Rg are respectively added into the liquid culture medium 2 As a substrate.
4. Conversion time course experiment of series dammarane type triterpenoid saponins
The fungus MS conversion experiment is carried out, the substrate addition day is 0 day, 0.5mL is sampled at the same time point every day, and the relative quantitative analysis of the saponin conversion condition is carried out by adopting an HPLC-QQQ-MS method. Calculating the relative content of dammarane type triterpenoid saponin substrate and corresponding oxidized derivative, drawing content change curve, and analyzing five transformation paths of dammarane type saponin.
2. Experimental results
1. Carbon source and nitrogen source selection for fungal MS transformation media
The variety and proportion of different carbon sources can obviously influence the expression quantity and biomass of key enzymes of a fungus catalytic substrate, thereby influencing the conversion efficiency of the fungus MS to convert the saponins.
TABLE 1 different carbon sources vs. substrate Rg 2 Effects of the relative content of (2)
In order to increase the rate of the conversion reaction, the nitrogen source species also significantly affected the microbial growth and metabolism, and the results are shown in Table 2.
TABLE 2 different nitrogen sources versus substrate Rg 2 Is (n=3)
2. Substrate concentration screening results
The invention discovers that the growth of strains and the activity of invertase are directly influenced by the concentration of the dammarane type triterpenoid saponin as a substrate, the excessive concentration of the substrate can cause excessive substrate, the selectivity and purity of a reaction product can be influenced, and the low concentration of the substrate can cause slow reaction rate and long reaction period, thereby being unfavorable for the improvement of production efficiency. The concentration of the triterpenoid saponin is regulated, so that the reaction is carried out at the optimal concentration, the conversion rate of the triterpenoid saponin is improved, and the activity of oxidase is fully exerted. The concentration of saponin added to the system thus directly determines the efficiency of synthesis, secretion and conversion of the key catalytic enzyme.
With Rg 2 For example, the concentration was 50mg/L with a conversion of 90.6% + -2.9, 100mg/L with a conversion of 91.2% + -1.5, 150mg/L with a conversion of 92.2% + -3.2, 200mg/L with a conversion of 80.2% + -3.1, and 250mg/L with a conversion of 75.2% + -3.5.
3. Time course experiment for catalyzing five dammarane type triterpenoid saponin by using fungi MS
In the Negative mode, where M/z [ M+COOH] - 830 is substrate ginsenoside Rg 2 The method comprises the steps of carrying out a first treatment on the surface of the Analysis of ginsenoside Rg by liquid chromatography-mass spectrometry 2 The result of time course experiment in the transformation process shows that Rg 2 2 major metabolites were produced, approximately 18+16Da higher than the substrate saponins, substrate at t 1 Peak at 31.719min, conversion product at t 2 ＝26.578min、t 3 Peak was seen for = 29.694 min. Combining the above experimental contents, the products were judged to be two hydroxylated Rg 2 FIG. 4 shows liquid ginsenoside Rg 2 HPLC-MS and substrate Rg of (E) 2 And corresponding analytical junction of the conversion derivative content profileAnd (5) fruits. FIG. 5 shows the substrate Rg 2 And corresponding conversion derivative content change curves.
The structural characteristics of the dammarane type saponin selected by the invention are different, and the experimental results are analyzed and summarized to find that when the aglycone C-20 is connected with glycosyl, fungus MS can not directly oxidize the triterpenoid saponin, and the oxidation reaction can be carried out only after the glycosyl at the C-20 is hydrolyzed. This is because steric hindrance may reduce the reaction rate and limit the reaction path. The absence of separate hydration (+18Da) or hydroxylation (+16Da) reactions in the product from the liquid assay indicated that the two oxidation reactions were performed simultaneously (+34 Da) and that little by-product (or difficult to detect) was produced other than the oxidized product in the experiment, simply because the high concentration of glucose in the medium inhibited hydrolysis of the glycosidic linkages in the saponins, concentrating the oxidation reaction of the bioconversion metabolites towards sapogenins. The biocatalysis system of the invention can be used as a saponin oxidation modification product to lay a foundation for large-scale preparation and development.
Example 3
This example shows the catalytic preparation and structural identification of dammarane type triterpene saponin derivatives.
1. NMR analysis of derivatives
Dissolving by using chromatographic methanol, analyzing the purity of the product by using HPLC-MS, selecting a sample with the peak area of the target product being more than or equal to 90% by using a peak area normalization method, evaporating the methanol in a water bath at 73 ℃, standing for 12 hours at 73 ℃ in a vacuum drying oven, dissolving the sample to be detected by using deuterated pyridine, and transferring the sample solution into a nuclear magnetic tube by using a pipette for standby.
After the instrument is calibrated, an internal standard TMS is added, and the separated and purified target compound is subjected to a Bruce 500MHz superconducting Fourier nuclear magnetic resonance spectrometer 13 C-NMR analysis, wherein the chemical structure of the derivative is determined by comparison with databases such as pubchem, chemspider and scibinder; for compounds not recorded in the database, 2D-NMR analysis is carried out, and the three-dimensional structure of the compound is finally determined by referring to substrate chemical structure information and mass spectrum multistage fragmentation rules through HSQC, HMBC and other modes.
2. Experimental results
1. Purity measurement result of target derivative
In the invention, F 2 The separation purity is 98%, rh 2 The separation purity is 99%, rh 1 The separation purity is 91%, rg 2 The separation purity is 96%, rg 3 The separation purity of the derivative (1) is 96%, rg 3 The isolation purity of the derivative (2) was 95%. FIG. 6 shows only exemplary ginsenoside Rg 2 And (5) measuring the purity of the derivative and determining a liquid quality map.
2. Structure identification results of target derivatives
Triterpene saponin oxidation derivative 13 The C-NMR data and patterns are shown in the following Table and FIG. 7. The table below and FIG. 7 show only exemplary ginsenoside Rg 2 Oxidation of derivatives 13 C-NMR data and spectra.
2.1 ginsenoside Rg 2 Oxidized derivatives
TABLE 3 ginsenoside Rg 2 Oxidation of derivatives 13 C-NMR data
Substrate-binding ginsenoside Rg 2 The structural characteristics and the results of the liquid chromatography-mass spectrometry analysis show that the ginsenoside Rg 2 There are two structural types of oxidized derivatives: one is to insert hydroxy groups at the C24 and C25 positions after opening the C24-C25 double bond to form C24, C25-dihydroxyl Rg 2 The method comprises the steps of carrying out a first treatment on the surface of the Another type is the addition of a hydroxyl group at other positions after hydration of the C24-C25 double bond. Introducing the structure into a scanner database for searching to obtain nuclear magnetic carbon spectrum data of different types of derivatives, comparing the nuclear magnetic carbon spectrum data with the derivative nuclear magnetic carbon spectrum data extracted in the text, and finally determining ginsenoside Rg 2 The conversion product of (C) 24, C25-dihydroxyRg. The transformation route is as follows: rg 2 24, 25-dihydroxyRg 2 The method is specifically as follows:
2.2 ginsenoside Rh 1 Oxidized derivatives
Substrate-binding ginsenoside Rh 1 The structural characteristics and the results of the liquid chromatography-mass spectrometry analysis show that the ginsenoside Rh 1 There are two structural types of oxidized derivatives: one is to insert hydroxy groups at the C24 and C25 positions after opening the C24-C25 double bond to form C24, C25-dihydroxyRh 1 The method comprises the steps of carrying out a first treatment on the surface of the Another type is the addition of a hydroxyl group at other positions after hydration of the C24-C25 double bond. Introducing the structure into a scanner database for searching to obtain nuclear magnetic carbon spectrum data of different types of derivatives, comparing the nuclear magnetic carbon spectrum data with the derivative nuclear magnetic carbon spectrum data extracted in the text, and finally determining ginsenoside Rh 1 The conversion product of (C) 24, C25-dihydroxyRh 1 . The transformation route is as follows: rh (rhodium) 1 24, 25-dihydroxyRh 1 The method is specifically as follows:
2.3 ginsenoside F 2 Oxidized derivatives
Substrate-binding ginsenoside F 2 The structural characteristics and the results of the liquid chromatography-mass spectrometry analysis show that the substrate ginsenoside F 2 Removing 20-position glycosyl under the action of glycosidase, and then ginsenoside F under the action of oxidase 2 There are two structural types of oxidized derivatives: one is to insert hydroxy groups at the C24 and C25 positions after opening the C24-C25 double bond to form C24, C25-dihydroxyRh 2 The method comprises the steps of carrying out a first treatment on the surface of the Another type is the addition of a hydroxyl group at other positions after hydration of the C24-C25 double bond. Introducing the structure into a scanner database for searching to obtain nuclear magnetic carbon spectrum data of different types of derivatives, comparing the nuclear magnetic carbon spectrum data with the derivative nuclear magnetic carbon spectrum data extracted in the text, and finally determining ginsenoside F 2 The conversion product of (C) 24, C25-dihydroxyRh 2 . The transformation route is as follows: f (F) 2 →Rh 2 24, 25-dihydroxyRh 2 The method is specifically as follows:
2.4 ginsenoside Rh 2 Oxidized derivatives
Substrate-binding ginsenoside Rh 2 The structural characteristics and the results of the liquid chromatography-mass spectrometry analysis show that the ginsenoside Rh 2 There are two structural types of oxidized derivatives: one is to insert hydroxy groups at the C24 and C25 positions after opening the C24-C25 double bond to form C24, C25-dihydroxyRh 2 The method comprises the steps of carrying out a first treatment on the surface of the Another type is the addition of a hydroxyl group at other positions after hydration of the C24-C25 double bond. Introducing the structure into a scanner database for searching to obtain nuclear magnetic carbon spectrum data of different types of derivatives, comparing the nuclear magnetic carbon spectrum data with the derivative nuclear magnetic carbon spectrum data extracted in the text, and finally determining ginsenoside Rh 2 The conversion product of (C) 24, C25-dihydroxyRh 2 . The transformation route is as follows: rh (rhodium) 2 24, 25-dihydroxyRh 2 The method is specifically as follows:
2.5 ginsenoside Rg 3 Oxidized derivatives
Substrate-binding ginsenoside Rg 3 The structural characteristics and the results of the liquid chromatography-mass spectrometry analysis show that the ginsenoside Rg 3 There are two structural types of oxidized derivatives: one is to insert hydroxy groups at the C24 and C25 positions after opening the C24-C25 double bond to form C24, C25-dihydroxyl Rg 3 The method comprises the steps of carrying out a first treatment on the surface of the Another type is the addition of a hydroxyl group at other positions after hydration of the C24-C25 double bond. Introducing the structure into a scanner database for searching to obtain nuclear magnetic carbon spectrum data of different types of derivatives, comparing the nuclear magnetic carbon spectrum data with the derivative nuclear magnetic carbon spectrum data extracted in the text, and finally determining ginsenoside Rg 3 Is converted into two mutually isomeric C24, C25-dihydroxyRg 3 . The following transformation paths are as follows: rg 3 24 (S/R), 25-dihydroxyRg 3 The method is specifically as follows:
example 4
The experimental example is a study on the myocardial injury resistance of dammarane type triterpene saponin oxidation derivatives, and the cell strain used is AC16 human myocardial cells (BNCC 339980) purchased from Henan province industrial microorganism strain engineering technology research center.
1. Experimental method
10 kinds of triterpenoid saponins are respectively prepared into solutions with different concentrations. The effect of different concentrations of 10 triterpenoid saponins on the scavenging ability level of DPPH free radical and on the activity of superoxide dismutase (SOD) was measured, and the toxicity of AC16 human myocardial cells was evaluated. In addition, the DOX-induced AC16 human cardiomyocyte viability was determined.
The invention also screens the differential genes in the gene by transcriptome sequencing and clarifies the occurrence mechanism and potential drug targets of myocardial injury.
2. Experimental results
The result of the clearance rate of triterpenoid saponin and derivative thereof to DPPH free radical shows that ginsenoside Rg 3 And derivatives and Rh thereof 1 The derivative has stronger scavenging rate to DPPH free radical. 24 (S), 25-dihydroxyl Rg 3 For example, the DPPH radical scavenging rate was 69.42% + -2.34 at 100 μg/mL.
The activity of the triterpenoid saponins with different concentrations on superoxide dismutase (SOD) is measured, and experimental results show that the activity of the superoxide dismutase (SOD) increases with the increase of the concentration of 10 triterpenoid saponins samples. 24 (S), 25-dihydroxyl Rg 3 For example, the activity of SOD was 64.11.+ -. 1.30 at 100. Mu.g/mL.
The results showed that ten saponins were non-toxic to AC16 human cardiomyocytes in the range of 25-100. Mu.g/mL. Even Rg 3 、Rg 3 Derivatives, rh 1 And Rh 1 After the derivative treatment, the AC16 human heart muscle is treatedThe cells have a certain proliferation effect. With 24, 25-dihydroxyRh 1 For example, the viability of AC16 human cardiomyocytes was 115.01% + -1.45 at 100 μg/mL.
By adding 24 (R), 25-dihydroxyRg 3 And comparing the expression profile of the cultured AC16 human myocardial cells with the DOX model group, identifying the differential genes of the cultured AC16 human myocardial cells, obtaining 1098 differential genes, and forming a differential gene volcanic map. Fold change>log 2 (1.5) and pvalue<0.05, see FIG. 8, the left dot of the left vertical dashed line represents the down-regulation, the right dot of the right vertical dashed line represents the up-regulation, and the remaining dots represent no significant difference.
Annotating and analyzing the enrichment result obtained by gene enrichment, and displaying 24 (R), 25-dihydroxyRg 3 The MAPK signal pathway may play a key role in resisting DOX-induced myocardial injury by alleviating oxidative stress injury, inhibiting apoptosis and promoting the growth mechanism of myocardial cells.
The invention annotates the KEGG database-based Rg 3 Differential genes of the derivative group and DOX model, and 5 pathway enrichment was found. RAS signal paths with high correlation with myocardial damage resistance are selected for analysis. On the way, rg 3 The derivatives are involved in the regulation of key genes Akt/PKB, JNK, ERK and the like on a PI3K-Akt signal pathway and a MAPK signal pathway related to myocardial damage, and up-regulate the level of ETS (one of downstream products of the MAPK signal pathway with higher correlation with myocardial damage resistance) in the downstream products. Finally, rg 3 The role of the derivatives is also related to affecting the expression level of the NF- κb gene and to regulating inflammatory responses, cells and survival related biological processes.
Each group showed varying degrees of change after 24 hours of saponin administration treatment, with 100. Mu.g/mLRg 3 The p-Akt expression of the derivative was increased by 45.82%. In addition, PI3K expression was reduced by 66.63%, p-ERK expression was reduced by 51.78%, p-38 expression was reduced by 65.54%, and NF- κB expression was reduced by 53.65%.
The results in conclusion show that Rg 3 The derivatives regulate the expression of downstream NF- κB through PI3K/Akt pathway and MAPK pathway, therebyInhibition of Doxorubicin (DOX) -induced apoptosis of cardiomyocytes is shown in figure 9.
Example 5
This example is a fungal MS oxidase dig based on transcriptomics technology, revealing the molecular mechanism by which dammarane-type triterpenoid saponin oxidase acts.
1. Experimental method
Mycelia of 0,1,2,4,6,8 days were selected as samples for transcriptome analysis, and full-length transcriptome sequencing experimental procedures were performed according to standard protocols (supplied by ONT corporation). The newly obtained transcript sequences were annotated using different analytical methods (GO, KEGG, etc.), BLAST aligned using COG, KOG, swissprot, NR, pfam, KEGG and GO databases, and their functional information was obtained. GO and KEGG enrichment assays were performed on the screened differentially expressed transcripts, from which the first 30 classifications of GO enrichment assays and the first 20 pathways of KEGG enrichment assays were screened as final results.
In addition, ginsenoside Rg in transformation medium at different transcriptome sampling time points 3 And its oxidized derivative is relatively quantitatively analyzed, and a content change curve is drawn; meanwhile, the change condition of the differential expression oxidase transcripts in the blank control group vs different substrate induction time groups at different conversion times is drawn into corresponding curves, and chemical content change and oxidase transcript expression change curves are compared and analyzed to screen potential oxidase genes.
2. Experimental results
Searching target enzyme transcripts in a differential transcript database, and after comprehensive comparison analysis of homologous enzyme functions, locking enzymes potentially participating in triterpenoid saponin skeleton oxidation reaction into 7 of the following: catase/peroxidase HPI, cytochrome P450, cytochrome P450 78A3,NADH:flavin oxidoreductase/NADH oxidase family protein, O-methylsterigmatocystin oxidoreductase, CORD and CS domain protein, cupredoxin. The gene expression of the 7 enzymes is plotted into a curve, and the curve is matched with ginsenoside Rg 3 And the content change curves of the oxidized derivatives thereof were subjected to comparative analysis, and the results are shown in FIG. 10.
Transcript Nr database annotation: ont.2552.2: catanase/peroxidase HPI, ONT.10421.5: cytochrome P450, ont.3403: cytochrome P450 78A3, ONT.5329.1: NADH flavin oxidoreductase/NADH oxidase family protein, ONT.4469.1: o-methylsterigmatocystin oxidoreductase, push, ONT.9107.2: CORD and CS domain protein, ont.2555.5: cupredoxin
As a result, it was found that ginsenoside Rg 3 The content of the oxidized derivative of (2) rises slowly in the first 2 days of conversion, the content rises sharply from the 4 th day, the slope of the reaction curve reaches the maximum value after the 6 th day, and the reaction gradually becomes gentle from the 8 th day. Curve Rate and Rg by fitting analysis 3 The enzyme genes with more consistent oxidation derivative content change curves are respectively: cytochrome P450 (ONT.10421.5), NADH flavin oxidoreductase/NADH oxidase family protein (ONT.5329.1) and Cupredoxin (ONT.2555.5). The time of highest abundance of gene transcripts of the three enzymes is concentrated on the 4 th day, and then the expression level gradually begins to decline, because a lag process exists from the gene expression to the protein synthesis to the secretion and activity of the enzymes, and thus the expression level of the 3 enzymes changes with Rg 3 The synthesis trend of the oxidized derivatives was consistent. Further through enzyme functional analysis, cytochrome P450 (ONT.10421.5) corresponds to 3-hydroxyphenylacrylate 6-hydroxyase [ EC:1.14.13.63 ]]. The invention discovers that the enzyme can catalyze 3-hydroxyphenylacetic acid to generate 1, 4-dihydroxyphenylacetic acid, NADH is needed for the reaction, and the reason that the expression trend of NADH: flavin oxidoreductase/NADH oxidase family protein (ONT.5329.1) is similar to that of the enzyme is explained, and the two are probably enzyme clusters which cooperatively play the role of catalyzing the oxidation reaction of triterpenoid saponin; another enzyme, cupredoxin (ont.2555.5), mainly plays a role in copper ion mediated electron transport, which is involved in the oxidation reaction of triterpenoid saponins according to its function.
In summary, this group of enzymes, screened by differential transcriptome analysis, was the first reported to be involved in the oxidation of triterpenoid saponins. The invention screens out 3 transcripts related to the oxidation reaction of the triterpenoid saponin, which correspond to one key oxidase and two coenzymes possibly participating in the oxidation reaction process respectively.
While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments without departing from the scope or spirit of the invention. The scope of the invention is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. An oxygenated derivative of dammarane type triterpenoid saponins is characterized by having a structure shown in the following formula I:
wherein,
r1, R2, R3 are the same or different and are each independently selected from mono-, oligo-or H;
r4 and R5 are hydroxyl.
2. The dammarane-type triterpene saponin oxidized derivative according to claim 1, characterized in that it comprises at least one of the following compounds:
3. a pharmaceutical composition comprising the dammarane-type triterpene saponin oxidized derivative according to claim 1 or 2.
4. A process for producing an oxidized derivative of dammarane-type triterpene saponin as defined in claim 1 or 2, characterized by comprising the step of introducing a hydroxyl group into the dammarane-type triterpene saponin structure in the presence of a fungus or an oxidase derived from the fungus, using a dammarane-type triterpene saponin as a substrate.
5. The method for producing an oxidized derivative of dammarane type triterpene saponin according to claim 4, wherein the culture solution for the directional transformation comprises 1 to 10g/L potato starch, 1 to 50g/L glucose, 1 to 20g/L peptone, and 0.1 to 5g/L sodium chloride.
6. The method for producing an oxidized derivative of dammarane-type triterpene saponin according to claim 5, wherein the dammarane-type triterpene saponin is selected from the group consisting of ginsenoside Rh 2 、Rg 3 、Rb 2 、Rb 1 、Rd、Rc、F 2 、F 1 、Rg 2 、Rg 1 、Rh 1 Re, rf and notoginsenoside R 1 And R is 2 At least one of them.
7. The method for producing an oxidized derivative of dammarane type triterpene saponin as defined in claim 6, wherein the fungus is derived from the genus mucor.
8. The method for producing an oxidized derivative of dammarane type triterpene saponin according to claim 7, wherein the concentration of the substrate is 10 to 400mg/L.
9. The method for producing an oxidized derivative of dammarane type triterpene saponin according to claim 8, wherein the oxidase is at least one selected from the group consisting of cytochrome P450 (ONT.10421.5), NADH flavin oxidoreductase/NADH oxidase family protein (ONT.5329.1) and Cupredoxin (ONT.2555.5), or a combination thereof.
10. Use of an oxidized derivative of dammarane type triterpenoid saponin according to claim 1 or 2 for the preparation of a medicament for ameliorating, preventing or treating a disease.
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