Oleanane type triterpenoid derivative with 21-28 lactone rings and double-bond substitution, preparation method and application of oleanane type triterpenoid derivative

Abstract

The invention belongs to the technical field of medicines, and discloses an oleanane type triterpene derivative with 21-28 lactone rings and double bond substitution, and a preparation method and application of the oleanane type triterpene derivative. The oleanane triterpene derivative is 3-carbonyl-oleanane-1, 9 (11), 12-triene-21 beta, 28-lactone and has a structure shown in a formula I: # imgabs0. In-vitro anti-tumor cell experiments and in-vitro anti-neuroinflammation experiments prove that the oleanane triterpene derivative shown in the formula I provided by the invention has remarkable anti-tumor activity and anti-neuroinflammation activity, and the oleanane triterpene derivative can be used for preparing anti-neuroinflammation medicines. The compound can be used as an active ingredient of anti-tumor drugs and anti-neuroinflammation drugs, and has wide application.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution, a preparation method and an application thereof. Background Art

[0002] Oleanane triterpenes are the most widely existing pentacyclic triterpenoid compounds in nature. They are widely distributed in the roots, stems, leaves, flowers and fruits of plants in the form of free bodies and glycosides. Among them, oleanolic acid is the most well-known. Pharmacological studies have shown that oleanane triterpenes have multiple biological activities such as anti-inflammatory, anti-tumor, antibacterial and antiviral. Oleanolic acid has been clinically used to treat acute viral hepatitis with good efficacy. Oleanane triterpenes are also important intermediates for the development of new drugs. Typically, a series of derivatives are designed and synthesized using oleanane triterpenes as raw materials using conventional chemical synthesis methods. Some derivatives have shown good anti-cancer and antiviral activities, showing the important role of oleanane triterpenes in the development of new drugs.

[0003] Oleanolic acid is the 3-hydroxyl oxidation product of oleanolic acid. It is also distributed in nature, but its content is generally lower than that of oleanolic acid. Oleanolic acid is present in high levels in the traditional Chinese medicine jujube and is one of the main active ingredients of jujube. Studies have shown that oleanolic acid has biological activities such as anti-inflammatory, anti-cancer and hypoglycemic, and is also an important raw material for chemical synthesis of derivatives. However, there are relatively few studies on its structural modification and transformation using microbial transformation technology.

[0004] Microbial transformation is a simple and effective technology for modifying the structure of natural compounds. It can introduce functional groups regioselectively and stereoselectively. It has the advantages of high efficiency, economy, and environmental protection, which are difficult to achieve with conventional chemical modification. As an efficient modification tool, microbial transformation technology can modify the structure of natural active compounds with different skeletons and has been widely used in the structural modification of various compounds. On the one hand, the transformation products obtained by microbial transformation can obtain derivatives with stronger biological activity for direct use 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, thereby solving the problem of few reaction sites for the organic chemical preparation of derivatives of triterpenoid compounds.

[0005] However, compared with chemical synthesis, microbial transformation methods are highly random, and the results of transformation cannot be predicted like chemical synthesis, nor can they be purposefully designed. At the same time, even similar substrates can have very significant differences in the products obtained after catalytic transformation by the same strain or different strains. The same substrate can also have very significant differences in the transformation products obtained after transformation by different strains. Therefore, it is crucial to select the appropriate strain when using microbial transformation methods to modify the structure of compounds. Summary of the invention

[0006] In order to solve at least one aspect of the above-mentioned problems and defects existing in the prior art, the purpose of the embodiments of the present invention is to provide an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution, a preparation method and application thereof.

[0007] The first aspect of the present invention provides an oleanane triterpene derivative having a 21-28 lactone ring and double bond substitution, wherein the oleanane triterpene derivative is 3-carbonyl-oleanane-1,9(11),12-triene-21β,28-lactone, and the oleanane triterpene derivative has a structure of Formula I:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned oleanane-type triterpene derivatives, wherein the preparation method comprises: using Aspergillus minsclerotigenes to biotransform oleanolic acid and then separating and purifying it to obtain the oleanane-type triterpene derivatives.

[0010] In some embodiments of the present invention, the preparation method comprises the following steps:

[0011] 1) fermenting and culturing a microorganism, adding oleanolic acid, continuing transformation and culturing, removing mycelium to obtain a fermentation liquid, wherein the microorganism is Aspergillus minsclerotigenes;

[0012] 2) extracting the fermentation broth with an organic solvent to obtain a transformation product;

[0013] 3) treating the transformation product by reverse phase silica gel column chromatography to obtain different components;

[0014] 4) Selecting a component containing the target compound from the different components obtained in step 3) and subjecting it to reverse phase high performance liquid chromatography to obtain an oleanane-type triterpene derivative having a structural formula of formula I.

[0015] In some embodiments of the present invention, the organic solvent is ethyl acetate.

[0016] In some embodiments of the present invention, the reverse phase silica gel column chromatography treatment is specifically:

[0017] The transformation product was separated by ODS-C18 column chromatography, and gradient elution was performed using a methanol-water two-phase system. The fractions were collected and combined after HPLC analysis to obtain different components.

[0018] In some embodiments of the present invention, the gradient elution using a methanol-water two-phase system is specifically as follows:

[0019] Gradient elution was performed using a methanol-water two-phase system with volume ratios of methanol to water of 40:60, 60:40, 80:20 and 100:0, respectively.

[0020] In some embodiments of the present invention, the reverse phase high performance liquid chromatography treatment is specifically:

[0021] A YMC ODS A chromatographic column with a specification of 5 μm, 12.0×250 mm was used, the mobile phase was acetonitrile-water solution, the volume ratio of acetonitrile to water in the acetonitrile-water solution was 65:35, the flow rate was 3.0 mL / min, and the detection wavelength was 203 nm.

[0022] The third aspect of the present invention provides a use of the above-mentioned oleanane-type triterpene derivative as an active ingredient in the preparation of an anti-tumor drug.

[0023] In some embodiments of the present invention, the tumor comprises one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

[0024] A fourth aspect of the present invention provides a use of the above-mentioned oleanane-type triterpene derivative as an active ingredient in the preparation of an anti-neuroinflammatory drug.

[0025] The oleanane-type triterpene derivatives substituted with 21-28 lactone rings and double bonds, preparation methods and applications thereof provided in the embodiments of the present invention have at least one or a part of at least one of the following advantages:

[0026] (1) The present invention utilizes the catalytic effect of Aspergillus minsclerotigenes to regioselectively and stereoselectively introduce double bonds at the 1-position and 9(11)-position of the oleanane parent nucleus, and simultaneously forms lactones at the 21β and 28-positions, thereby obtaining an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution.

[0027] (2) The in vitro anti-tumor cell experiments and in vitro anti-neuroinflammatory experiments confirmed that the oleanane-type triterpene derivatives shown in Formula I provided by the present invention have significant anti-tumor activity and anti-neuroinflammatory activity, can be used as active ingredients of anti-tumor drugs and anti-neuroinflammatory drugs, and have a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of the preferred embodiments in conjunction with the accompanying drawings, in which:

[0029] Figure 1 The present invention is an embodiment of an oleanane triterpene derivative having a 21-28 lactone ring and double bond substitution 1 H-NMR spectrum;

[0030] Figure 2 The present invention is an embodiment of an oleanane triterpene derivative having a 21-28 lactone ring and double bond substitution 13 C-NMR spectrum;

[0031] Figure 3 This is a HSQC spectrum of an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution according to an embodiment of the present invention;

[0032] Figure 4 This is the HMBC spectrum of an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution according to one embodiment of the present invention;

[0033] Figure 5 This is a HR-ESIMS graph of an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution according to an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further specifically described below by examples and in conjunction with the accompanying drawings. In the specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation of the present invention.

[0035] Example 1: Preparation of oleanane-type triterpene derivative compounds having a 21-28 lactone ring and double bond substitution of formula I

[0036] The present invention adopts a microbial transformation method, takes oleanolic acid as a substrate, and prepares the compound of the present invention through the steps of fermentation, extraction, separation and structural identification: an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution.

[0037] Aspergillus minsclerotigenes CGMCC 3.86 was purchased from China Microbiological Culture Collection (CGMCC), Chinese Academy of Sciences, and stored in a 4°C refrigerator on a solid slant medium using potato medium.

[0038] 1) Fermentation, transformation and extraction

[0039] The Aspergillus minsclerotigenes CGMCC 3.86 stored on the test tube slope was inoculated into a 250mL conical flask (containing 100mL potato liquid culture medium) as seed liquid. After shaking culture on a shaker at 160rpm and 26°C for 24 hours, when the mycelium growth is in a vigorous stage, 1mL of seed liquid is taken with a sterile pipette and added to 20 1000mL shake flasks (containing 400mL potato liquid culture medium). After shaking culture for 24 hours, 20mg of oleanolic acid (0.50mL, 40mg / mL ethanol solution) was added to each shake flask, and 400mg of substrate was used in total. The transformation was continued for 7 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 ethyl acetate. The extract was concentrated to dryness under reduced pressure to obtain about 0.97g of the crude extract of the transformant.

[0040] 2) ODS-C18 column chromatography separation

[0041] The crude extract was separated by ODS-C18 column chromatography, with methanol:water gradient elution (40:60, 60:40, 80:20, 100:0); the fractions were collected, analyzed by HPLC and combined to obtain the combined components Fr.1-Fr.4.

[0042] 3) Reverse-phase HPLC purification

[0043] Component Fr.4 was selected from the combined components Fr.1-Fr.4 and purified by reverse phase high performance liquid chromatography. The preparation conditions were semi-preparative chromatography column YMC ODS A-5μm, 12.0×250mm, acetonitrile-water (65:35, V / V), flow rate of 3.0mL / min, and detection wavelength of 203nm to obtain the conversion product.

[0044] 4) Structural identification of the transformation product obtained in step 3)

[0045] The conversion product obtained in step 3) was subjected to nuclear magnetic resonance (NMR) spectrum testing. The measured spectra were 1H nuclear magnetic resonance spectrum (hydrogen spectrum, providing relevant information of hydrogen atoms in the compound structure. The results are shown in the attached Figure 1 ), 13C NMR spectrum (carbon spectrum, providing relevant information about carbon atoms in the compound structure, the results are shown in the attached Figure 2HSQC, provides direct information about the hydrogen-carbon in the compound. The results are shown in the attached Figure 3 The results are shown in the attached figure) and the heteronuclear multi-correlation spectrum (HMBC, which provides long-range information about hydrogen and carbon in the compound. Figure 4 The purpose is to determine the structure of the compound by analyzing the spectrum. The instrument used is Varian INOVA 600 MHz nuclear magnetic resonance instrument, and the test reagent used is deuterated chloroform.

[0046] The conversion product obtained in step 3) is subjected to high-resolution mass spectrometry to determine the molecular weight and molecular formula of the compound (the results are shown in the attached Figure 5 The instrument used was a Finnigan LCQDECA mass spectrometer.

[0047] Step 3) Mass spectrum and spectroscopic data of the conversion product obtained: optical rotation +43.7°(c=0.16,MeOH); High resolution mass spectrum: m / z=471.2874[M+Na] + (cal.471.2875for C 30 H 40 O3Na).

[0048] The H NMR and C NMR data of the conversion product obtained in step 3) are shown in Table 1.

[0049] Table 1 H NMR spectrum (CDCl3, 600MHz) and C NMR spectrum (CDCl3, 150MHz) data of the conversion products

[0050]

[0051]

[0052] The above results show that the structure of the conversion product obtained in step 3) is Formula I.

[0053]

[0054] Example 2: Antitumor activity of the compound of formula I (Compound I)

[0055] 1) Experimental methods

[0056] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50Value: Culture the tumor cells until they cover more than 90% of the bottom of the culture dish. Obtain the cell suspension according to the cell culture procedure. Discard the culture medium by centrifugation. Add 4 mL of complete culture medium and gently pipette to mix evenly. Count the cells after trypan blue staining. Then add 90 μL of a 4×10 4 / mL of cell fluid, the cells were evenly inoculated in a 96-well plate and incubated overnight. Different concentrations of the test samples were added to make the final concentration 0.1-100μM, and the culture was continued for 72h. The absorbance (A) value at 570nm was measured on a microplate reader according to the MTT method, and the inhibition rate was calculated [inhibition rate = (1-experimental group A value / control group A value) × 100%]. The experiment was repeated 3 times. SPSS11.5 software was used to make a regression equation to calculate the half inhibitory concentration (IC50) of each test sample on tumor cells for 72h 50 ).

[0057] 2) Experimental results

[0058] 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.

[0059] Table 2. In vitro cytotoxic activity screening results of test samples

[0060]

[0061] The results show that the compound of the present invention with the structure of formula I has good anti-tumor activity and can be used as an active ingredient of anti-tumor drugs. At the same time, the compound of the present invention with the structure of formula I can effectively kill human leukemia resistant cells.

[0062] Example 3: Anti-neuroinflammatory activity of compounds of formula I

[0063] 1) Experimental methods

[0064] The cell viability was determined by MTT method: BV-2 cells in the logarithmic growth phase were cultured in DMEM containing 10% calf serum and 1% penicillin-streptomycin double antibody solution, and the cell concentration was adjusted to 3×10 5 / mL, inoculated in 96-well culture plates, drug treatment group and cell control group added 80μL cell suspension per well, each group set up 3 replicates, blank control group only added DMEM full culture medium, 80μL per well, set up 3 replicates. After culturing the 96-well culture plate in a 37°C, 5% CO2 incubator for 24h, 10μL MTT was added to each well of the 96-well plate, cells were cultured for 4h after adding MTT, supernatant was discarded after 4h, and 100μL DMSO was added to each well. Then the absorbance value at 570-630nm was detected on an ELISA reader.

[0065] The Griess method was used to determine the effect of each test compound on LPS-induced NO release in BV-2 cells: the cell supernatant was aspirated and then washed twice with 2 mL PBS. 700 μL of trypsin digestion solution was added to remove the cells from the dish, and 1 mL of fresh culture medium was added to terminate the digestion. Then the cells were gently blown and collected in a 15 mL centrifuge tube, and 2 mL of culture medium was added and the above operation was repeated once. Then the centrifuge tube was placed in a low-speed centrifuge and centrifuged for 3 min at 800 rpm. The cell supernatant was aspirated and 4 mL of culture medium was added to the centrifuge tube. 3.0×10 5 The cells were seeded at a density of 100 μL / mL in a 96-well plate, with 180 μL per well. The plates were placed in a 37°C, 5% CO2 incubator for 24 hours. The test compounds were diluted to different concentrations using the concentration gradient method. After adding 10 μL of the compound to each well, the final concentrations in each well were ensured to be 1.25, 2.5, 5, 10, 20, and 40 μM. One hour after compound pretreatment, 10 μL of LPS was added to the non-drug group, and 10 μL of LPS was added to the drug group, ensuring that the concentration of LPS in each well was 500 ng / mL. After continuing to culture for 12 hours, the supernatant was taken and the level of NO in the culture medium was determined according to the instructions of the kit. The data were analyzed and processed using SPSS Statistics 25 software to calculate the half inhibitory concentration (IC50) of each test sample for inhibiting NO release. 50 ).

[0066] 2) Experimental results

[0067] According to the test results of MTT method and Griess method, the effect of the compound of formula I of the present invention on NO release of BV-2 cells induced by LPS was calculated. The results are shown in Table 3.

[0068] Table 3. Results of the test samples inhibiting LPS-induced NO release in BV-2 cells

[0069] Compound <![CDATA[IC 50 (μM)]]> Cell viability (%) L-NMMA 20.76±2.05 102.54±3.48 Compound Ⅰ 4.51±0.90 100.72±1.39

[0070] As an important expression quantity in the inflammatory process, NO is increasingly used as a detection indicator in inflammatory models. In this experiment, Griess reagent was used to detect the NO content. The results showed that the compound with the structure of formula I had no obvious cytotoxic effect on BV-2. At the same time, it can significantly reduce the release level of NO in BV-2 cells induced by LPS, and has good anti-neuroinflammatory activity. Its anti-neuroinflammatory activity is also significantly better than the positive control L-NMMA, and can be used as an active ingredient in anti-neuroinflammatory drugs.

[0071] The oleanane-type triterpene derivatives substituted with 21-28 lactone rings and double bonds, preparation methods and applications thereof provided in the embodiments of the present invention have at least one or a part of at least one of the following advantages:

[0072] (1) The present invention utilizes the catalytic effect of Aspergillus minsclerotigenes to regioselectively and stereoselectively introduce double bonds at the 1-position and 9(11)-position of the oleanane parent nucleus, and simultaneously forms lactones at the 21β and 28-positions, thereby obtaining an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution.

[0073] (2) The in vitro anti-tumor cell experiments and in vitro anti-neuroinflammatory experiments confirmed that the oleanane-type triterpene derivatives shown in Formula I provided by the present invention have significant anti-tumor activity and anti-neuroinflammatory activity, can be used as active ingredients of anti-tumor drugs and anti-neuroinflammatory drugs, and have a wide range of uses.

Claims

1. An oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution, characterized in that: The oleanane triterpene derivative is 3-carbonyl-oleanane-1,9(11),12-triene-21β,28-lactone, and the oleanane triterpene derivative has a structure of formula I:

2. A method for preparing an oleanane-type triterpene derivative as claimed in claim 1, characterized in that: The preparation method comprises the following steps: using Aspergillus minsclerotigenes to carry out biotransformation on oleanolic acid, followed by separation and purification, to obtain the oleanane-type triterpene derivative.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: 1) fermenting and culturing a microorganism, adding oleanolic acid, continuing transformation and culturing, removing mycelium to obtain a fermentation liquid, wherein the microorganism is Aspergillus minsclerotigenes; 2) extracting the fermentation broth with an organic solvent to obtain a transformation product; 3) treating the transformation product by reverse phase silica gel column chromatography to obtain different components; 4) Selecting a component containing the target compound from the different components obtained in step 3) and subjecting it to reverse phase high performance liquid chromatography to obtain an oleanane-type triterpene derivative having a structural formula of formula I.

4. The preparation method according to claim 3, characterized in that: The organic solvent is ethyl acetate.

5. The preparation method according to claim 3, characterized in that: The reverse phase silica gel column chromatography treatment is specifically: The transformation product was separated by ODS-C18 column chromatography, and gradient elution was performed using a methanol-water two-phase system. The fractions were collected and combined after HPLC analysis to obtain different components.

6. The preparation method according to claim 5, characterized in that: The gradient elution using a methanol-water two-phase system is specifically as follows: Gradient elution was performed using a methanol-water two-phase system with volume ratios of methanol to water of 40:60, 60:40, 80:20 and 100:0, respectively.

7. The preparation method according to claim 6, characterized in that: The reverse phase high performance liquid chromatography treatment is specifically: A YMC ODS A chromatographic column with a specification of 5 μm, 12.0×250 mm was used, the mobile phase was acetonitrile-water solution, the volume ratio of acetonitrile to water in the acetonitrile-water solution was 65:35, the flow rate was 3.0 mL / min, and the detection wavelength was 203 nm.

8. Use of the oleanane-type triterpene derivative as claimed in claim 1 as an active ingredient in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor includes one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

10. Use of the oleanane-type triterpene derivative as claimed in claim 1 as an active ingredient in the preparation of an anti-neuroinflammatory drug.

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