Centella asiatica acid microbial transformation derivatives and preparation methods and applications thereof

Abstract

The invention discloses a Centella asiatica microbial transformation derivative and a preparation method and application thereof. By using the enzyme system of microorganisms, hydroxyl groups are introduced into the non-chemical reaction active sites 7, 20, 21 and 22 of the parent nucleus of Centella asiatica, so that the 20 and 28 positions form lactones, and two novel Centella asiatica microbial transformation derivatives, 2α, 3β, 7β, 23-tetrahydroxy-20(21)-double bond ursane-28β-carboxylic acid and 2α, 3β, 7β, 23-tetrahydroxy-21(22)-double bond ursane-20β, 28β-lactone, are obtained, which can be used as pharmaceutical intermediates. It is confirmed by in vitro anti-tumor cell tests that the two Centella asiatica microbial transformation derivatives have significant anti-tumor activity.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an asiatic acid microbial transformation derivative and a preparation method and application thereof. Background Art

[0002] Asiatic acid, also known as Asiatic acid, is an ursane-type pentacyclic triterpene acid extracted from the Umbelliferae plant Centella asiatica. In the 1970s, after researchers discovered that Asiatic acid has the effect of promoting skin wound repair, the structural modification and pharmacological activity of Asiatic acid and its derivatives gradually became the focus of attention. However, these compounds have the problems of poor solubility and low bioavailability, which are relatively limited in clinical application. Chemical modification is a common method to solve such problems. Specifically, Asiatic acid can be structurally modified by chemical modification in order to obtain derivatives with stronger solubility and biological activity. However, the structure of pentacyclic triterpene compounds is special, and the parent nucleus lacks chemically reactive groups. The sites for structural modification of Asiatic acid using organic chemical methods are mainly the two original chemically reactive groups in Asiatic acid - the hydroxyl groups at positions 2, 3 and 23 on the A ring and the carboxyl group at position 28 on the D / E ring. Most of the CH bonds on the parent nucleus of Centella asiatica acid are non-chemically reactive sites, and it is difficult to modify the parent nucleus structure using conventional chemical reaction methods to obtain derivatives modified with chemically reactive groups such as hydroxyl and carbonyl groups on the parent nucleus. Therefore, the diversity of the reaction sites and derivatives of the chemical structure modification of Centella asiatica acid is severely limited.

[0003] Microbial transformation is the process of using enzymes in microbial cells to modify the structure of substrates and transform them into other compounds. The special intracellular or extracellular enzymes produced by fermentative fungi, actinomycetes and other microorganisms during their growth can not only catalyze their own substances, but also catalyze specific chemical reactions for foreign compounds, such as hydrolysis, oxidation, reduction, cleavage, skeleton rearrangement, etc., thus becoming a powerful tool for structural modification and transformation of complex natural products. Our research group has long been engaged in the research of microbial transformation of natural active ingredients, especially the microbial transformation of natural active triterpenes. The transformation of common triterpenes from natural sources (including dammarane-type, cycloartane-type, ursane-type, oleanane-type triterpenes and lupetane-type, etc.) was studied using a variety of microorganisms. It was found that the microbial enzyme system can selectively catalyze multiple non-chemical reaction active sites on the triterpene mother nucleus, thereby obtaining derivatives with chemically active groups such as hydroxyl and carbonyl groups on the mother nucleus (Journal of natural products 2021, 84: 2664-2674; Phytochemistry 2021, 182: 112608; Natural Product Research 2021, 35(16): 2685-2690; Phytochemistry 2019, 166: 112076; Planta Medica 2019, 85(1): 56-61). On the one hand, microbial transformation products can obtain derivatives with stronger biological activity that can be directly used in drug research and development. On the other hand, the newly introduced chemically active groups on the parent nucleus after microbial transformation increase the sites for chemical modification and transformation, solving the problem of few reaction sites for the organic chemical preparation of triterpenoid derivatives.

[0004] Compared with organic chemistry methods, microbial transformation cannot predict or determine which microorganism has the ability to transform from theory or the fermentation method itself, nor can it predict or determine the structure of the transformation product. It can only be confirmed after the compound is prepared and then identified. Studies have shown that even if the structure of the lead compound is not much different, the structure of the derivative obtained by selective catalysis of the microbial enzyme system will be very different. Even for the same lead compound, the structure of the derivative obtained after catalysis by different microbial enzymes will be significantly different, which is also the diversity of microbial transformation. Therefore, as a method for modifying the structure of compounds, the product structure of microbial transformation is characterized by diversity and uncertainty, and can only be finally determined after preparation and structural confirmation. Therefore, microbial transformation cannot design compounds first and then reverse synthesize them like organic chemistry. At the same time, there are many types of microorganisms in nature, and selecting suitable microbial strains is the key to structural modification and transformation of Centella asiatica using microbial transformation.

[0005] In addition, there are many kinds of microorganisms in nature, and the transformation capabilities of different types of microorganisms vary greatly. As a complete compound structure, the drug molecule only has differences in carbonyl, hydroxyl and methyl groups, and the pharmacological activity it exhibits will also be significantly different. Before the corresponding compound is obtained and pharmacological tests are carried out, its effect cannot be accurately predicted. Therefore, the screening and selection of microbial strains are crucial for discovering compounds with better activity. For example, Xu et al. found that the product (4a) of asiatic acid (4) after microbial transformation did not show anti-tumor activity (RSC Advances 2017, 7: 20754).

[0006] Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a microbial transformation derivative of Centella asiatica acid and a preparation method and application thereof. Compared with Centella asiatica acid, the Centella asiatica acid derivative has more sites that can be chemically modified and transformed and more significant anti-tumor activity, can be used to prepare pharmaceutical intermediates or anti-tumor drugs, and has a wide range of medical uses.

[0008] The present invention provides a Centella asiatica acid microbial transformation derivative, wherein the Centella asiatica acid microbial transformation derivative is 2 α ,3 β ,7 β ,23-Tetrahydroxy-20(21)-Double-bond Ursane-28 β - carboxylic acid having the structure of formula I:

[0009]

[0010] The present invention provides a Centella asiatica acid microbial transformation derivative, wherein the Centella asiatica acid microbial transformation derivative is 2 α ,3 β ,7 β ,23-Tetrahydroxy-21(22)-Double-bond Ursane-20 β ,28 β - lactone, having the structure of formula II:

[0011]

[0012] The present invention also provides a method for preparing a microbial transformation derivative of asiatic acid represented by formula I or II. The reaction scheme of the preparation process is shown in the following formula, wherein the preparation method comprises the following steps:

[0013] 1) fermenting and culturing a microorganism, adding asiatic acid to a culture medium, then performing transformation culture, and removing mycelium to obtain a fermentation liquid, wherein the microorganism is a strain of the genus Cunninghamella;

[0014] 2) extracting the fermentation broth to obtain a transformant;

[0015] 3) purifying the transformation product by reverse phase silica gel column chromatography, wherein the reverse phase silica gel column purification adopts a methanol-water two-phase system gradient elution, and collecting and merging the components;

[0016] 4) Purify the components by reverse phase high performance liquid chromatography to obtain 2 α ,3 β ,7 β ,23-Tetrahydroxy-20(21)-Double-bond Ursane-28 β -carboxylic acid and 2 α ,3 β ,7 β ,23-Tetrahydroxy-21(22)-Double-bond Ursane-20 β ,28 β -lactone.

[0017]

[0018] Preferably, the microorganism is Cunninghamella echinulata CGMCC3.970.

[0019] Preferably, in step 1), the transformation culture temperature is 28° C., the shaking speed is 140 rpm, and the culture time is 72 hours.

[0020] Preferably, in step 2), the extraction solvent is dichloromethane.

[0021] Preferably, in step 3), the preferred gradient elution conditions are methanol: water 20:80-40:60-60:40-80:20-100:0.

[0022] The present invention also provides a pharmaceutical intermediate, which is a Centella asiatica microbial transformation derivative shown in Formula I or Formula II, or a Centella asiatica microbial transformation derivative prepared by the above-mentioned preparation method.

[0023] Application of the asiatic acid microbial transformation derivatives shown in formula I or II or the asiatic acid microbial transformation derivatives prepared by the above preparation method in anti-tumor drugs.

[0024] Preferably, the tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

[0025] Compared with the prior art, the present invention utilizes microbial transformation technology to successfully carry out structural modification at positions 7, 20, 21 and 22 of the non-chemically reactive sites on the parent nucleus, introduces an active reactive group hydroxyl group, and further forms lactone at positions 20 and 28, thereby obtaining a microbial transformation derivative of asiatic acid (compound formula I and formula II) with more sites for chemical modification and transformation, which can be used as an important pharmaceutical intermediate. The in vitro anti-tumor cell test confirmed that the microbial transformation derivative obtained by the present invention has more significant anti-tumor activity, can be used as an active ingredient of anti-tumor drugs, and has a wide range of uses. DETAILED DESCRIPTION

[0026] In order to further illustrate the present invention, the novel Centella asiatica derivatives and the preparation method thereof provided by the present invention are described in detail below in conjunction with examples.

[0027] Example 1: Preparation of compounds of formula I and formula II

[0028] The present invention adopts a microbial transformation method, uses asiatic acid as a raw material, and prepares the compound of the present invention through fermentation, extraction, separation and other steps. The strain of Cunninghamella can be purchased from the Microbiological Culture Collection Management Center of the Chinese Academy of Sciences (CGMCC), and a potato culture medium is selected and stored in a refrigerator at 4°C on a solid slant culture medium.

[0029] Taking Cunninghamella echinulata CGMCC 3.970 as an example, the process for preparing the compounds of formula I and formula II is as follows:

[0030] 1) Fermentation, transformation and extraction

[0031] Cunninghamella echinulata CGMCC 3.970 was inoculated into two 250 mL triangular flasks (containing 100 mL potato culture medium) as seed liquid. After 1 day of shaking culture on a shaker at 140 rpm and 28 ° C, 1 mL of seed liquid was taken with a sterile pipette and added to 20 1000 mL shake flasks (containing 400 mL potato culture medium) when the mycelium was in a vigorous growth period. After 1 day of shaking culture, 20 mg of asiatic acid (0.2 mL, 100 mg / mL ethanol solution) was added to each shake flask, and a total of 400 mg of substrate was used. The transformation was continued for 3 days under the same conditions, the fermentation broth was filtered, the mycelium was filtered out, and the filtrate was extracted 3 times with an equal volume of dichloromethane. The extract was concentrated to dryness under reduced pressure to obtain a crude extract of about 0.49 g of the transformant.

[0032] 2) Reverse phase silica gel column chromatography separation

[0033] The crude extract was separated by reverse phase silica gel column chromatography. The gradient elution was methanol: water (20:80, 40:60, 60:40, 80:20, 100:0). The fractions were collected and combined after HPLC analysis to obtain combined components AE.

[0034] 3) Reverse-phase HPLC purification

[0035] The combined fraction E was purified by reverse phase high performance liquid chromatography. The preparation conditions were semi-preparative chromatography column YMCODSA-5μm, 10.0×250mm, acetonitrile-water (45:55, V / V), flow rate 3.0mL / min, detection wavelength 203nm. The conversion products with the structural formula of formula I and formula II were obtained, and their mass spectrum and spectral data are shown below.

[0036] Compound Ⅰ: 2α,3β,7β,23-tetrahydroxyurs-20(21)-en-28-oic acid; optical rotation (c=0.1,MeOH); Main absorption peak of infrared spectrum (KBr)ν max :3462,3035,2947,1723,1381,1217,1051cm -1 ; High resolution mass spectrum m / z505.3529[M+H] + (calcd.505.3524for C 30 H 49 O 6 ); the H NMR and C NMR data are shown in Table 1.

[0037] Compound II: 2α,3β,7β,23-tetrahydroxyurs-21(22)-en-28,20β-lactone; optical rotation (c=0.1,MeOH); Main absorption peak of infrared spectrum (KBr)ν max :3511,3033,2934,1703,1428,1226,1069cm -1 ; High resolution mass spectrum m / z503.3367[M+H] + (calcd.503.3367forC 30 H 47 O 6 ); the H NMR and C NMR data are shown in Table 1.

[0038] Table 1. Attribution of H-NMR and C-NMR data of compounds Ⅰ and Ⅱ (deuterated methanol)

[0039]

[0040] The above results show that the structure of the obtained compound is correct.

[0041] Example 2: Antitumor activity of compounds I and II

[0042] 1) Experimental Materials

[0043] Instruments and reagents: CO 2 Incubator (Jouan IGO150); microplate reader (Bio-TEK ELx800); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotechnology Research Institute), RPMI 1640 medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.).

[0044] The tumor cell lines used for the test: Hela cells (human cervical cancer cells), K562 cells (human leukemia cells), K562 / ADR cells (human leukemia resistant cells), SH-SY5Y cells (human neuroblastoma cells), Du-145 (human prostate cancer cells), HePG2 cells (human liver cancer cells), MCF-7 cells (human breast cancer cells), CT26 cells (colon cancer cells), were purchased from the Institute of Oncology, Chinese Academy of Medical Sciences.

[0045] Test samples: Asiatic acid and compounds I and II synthesized in Example 1, with a purity of more than 95%; at the same time, cisplatin was selected as a positive control drug, and each compound was dissolved in DMSO and then diluted.

[0046] 2) Experimental methods

[0047] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50 Value: Take tumor cells in the logarithmic growth phase and adjust the cell concentration to 5×10 5 / mL, inoculated in 96-well culture plates, drug treatment group and cell control group added 100μL cell suspension per well, each group set up 3 replicates, blank control group only added RPM I 1640 full culture medium, 100μL per well, set up 3 replicates. 96-well culture plates were placed at 37℃, 5% CO 2After 24 hours of incubation in the incubator, different concentrations of the test samples were added to make the final concentration of 0.1-100 μM, and the culture was continued for 72 hours. The absorbance (A) value at 570 nm was measured on a microplate reader according to the MTT method, and the inhibition rate was calculated [inhibition rate = (1-A value of the experimental group / A value of the control group) × 100%]. The experiment was repeated 3 times. SPSS 11.5 software was used to make a regression equation to calculate the half-maximal inhibitory concentration (IC) of each test sample on tumor cells for 72 hours. 50 ).

[0048] 3) Experimental results

[0049] According to the MTT test results, calculate the IC of the test sample for the above cells 50 The results are shown in Table 2.

[0050] Table 2. In vitro cytotoxic activity screening results of test samples

[0051]

[0052] The results show that the compounds I and II of the present invention have good anti-tumor activity and can be used as active ingredients of anti-tumor drugs.

[0053] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A Centella asiatica acid microbial transformation derivative, characterized in that: The asiatic acid microbial transformation derivative is 2α, 3β, 7β, 23-tetrahydroxy-20(21)-double bond ursane-28β-carboxylic acid, having a structure of formula I:

2. A Centella asiatica acid microbial transformation derivative, characterized in that: The asiatic acid microbial transformation derivative is 2α,3β,7β,23-tetrahydroxy-21(22)-double bond ursane-20β,28β-lactone, having a structure of formula II:

3. A method for preparing a derivative of Centella asiatica acid transformed by microbial transformation according to claim 1 or 2, characterized in that: The steps include: 1) fermenting and culturing a microorganism, adding asiatic acid to a culture medium, then performing transformation culture, removing mycelium to obtain a fermentation liquid, wherein the microorganism is Cunninghamella echinulata CGMCC 3.970; the transformation culture temperature is 28° C., the shaking speed is 140 rpm, and the culture time is 72 hours; 2) extracting the fermentation broth to obtain a transformant; 3) purifying the transformation product by reverse phase silica gel column chromatography, wherein the reverse phase silica gel column purification adopts a methanol-water two-phase system gradient elution, and collecting and merging the components; 4) Purifying the components by reverse phase high performance liquid chromatography to obtain 2α,3β,7β,23-tetrahydroxy-20(21)-double bond ursane-28β-carboxylic acid and 2α,3β,7β,23-tetrahydroxy-21(22)-double bond ursane-20β,28β-lactone.

4. Use of the asiatic acid microbial transformation derivative according to claim 1 or 2 or the asiatic acid microbial transformation derivative prepared by the preparation method according to claim 3 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

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