Oleanane-type triterpene derivative with 21-28 lactone ring and double bond substitution, preparation method and application thereof

Abstract

The present invention belongs to the field of medical technology and discloses an oleanane-type triterpene derivative having a 21-28 lactone ring and double bond substitution, a preparation method, and an application thereof. The oleanane-type triterpene derivative is 3-carbonyl-oleanane-1,9(11),12-triene-21β,28-lactone and has a structure of Formula I: In vitro anti-tumor cell experiments and in vitro anti-neuroinflammatory experiments have confirmed that the oleanane-type triterpene derivative of Formula I provided by the present invention has significant anti-tumor activity and anti-neuroinflammatory activity, can be used as an active ingredient in anti-tumor drugs and anti-neuroinflammatory drugs, and has a wide range of uses.

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-type triterpenes are the most widely occurring 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. Oleanolic acid is the most well-known of these. Pharmacological studies have shown that oleanane-type triterpenes have multiple biological activities, including anti-inflammatory, anti-tumor, antibacterial, and antiviral. Oleanolic acid has been clinically used to treat acute viral hepatitis with good efficacy. Oleanane-type triterpenes are also important intermediates in the development of new drugs. Typically, a series of derivatives are designed and synthesized using oleanane-type triterpenes as raw materials using conventional chemical synthesis methods. Some derivatives have shown good anti-cancer and antiviral activities, demonstrating the important role of oleanane-type triterpenes in the development of new drugs.

[0003] Oleanolic acid, an oxidation product of the 3-hydroxyl group of oleanolic acid, is also found in nature, but its content is generally lower than that of oleanolic acid. Oleanolic acid is present in high concentrations in the traditional Chinese medicine jujube, where it is one of its main active ingredients. Studies have shown that oleanolic acid exhibits anti-inflammatory, anti-cancer, and hypoglycemic activities, and is also an important raw material for the chemical synthesis of derivatives. However, research on its structural modification and transformation using microbial transformation techniques is relatively limited.

[0004] Microbial transformation is a simple and effective technology for modifying the structure of natural compounds. It can introduce functional groups regioselectively and stereoselectively, and 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 that can be directly used for 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 to chemical synthesis, microbial transformation methods are highly random, and the results cannot be as predictable or purposefully designed. Furthermore, even when similar substrates are transformed by the same or different bacterial strains, the products obtained can vary significantly. Furthermore, the same substrate transformed by different bacterial strains can also yield significantly different products. Therefore, selecting the appropriate bacterial strain is crucial 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 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 culture, and removing mycelium to obtain a fermentation broth, wherein the microorganism is Aspergillus minsclerotigenes;

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

[0013] 3) treating the transformed 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 the structural 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 transformant was separated by ODS-C18 column chromatography, and gradient elution was performed using a methanol-water two-phase system. The fractions were collected, analyzed by HPLC, and then combined 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 a methanol to water volume ratio 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 and 12.0×250 mm was used. The mobile phase was an acetonitrile-water solution with a volume ratio of acetonitrile to water of 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 a 21-28 lactone ring and a double bond, the preparation method, and the use thereof provided in the embodiments of the present invention have at least one or a part of the following advantages:

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

[0027] (2) 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 taken in conjunction with the accompanying drawings, in which:

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

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

[0031] Figure 3 This is the HSQC 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;

[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 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. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be further described in detail below through 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 limiting 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 structure 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 the China Center for Culture Collection of Microorganisms, Chinese Academy of Sciences (CGMCC) and stored in a 4°C refrigerator on a solid slant medium using potato culture medium.

[0038] 1) Fermentation, conversion and extraction

[0039] Aspergillus minsclerotigenes CGMCC 3.86, stored on the slant of a test tube, was inoculated into a 250 mL Erlenmeyer flask (filled with 100 mL of potato broth) as a seed culture. After shaking on a shaker at 160 rpm and 26°C for 24 hours, 1 mL of the seed culture was pipetted using a sterile pipette and added to 20 1000 mL shake flasks (filled with 400 mL of potato broth). After shaking for 24 hours, 20 mg of oleanolic acid (0.50 mL of a 40 mg / mL ethanol solution) was added to each shake flask, for a total of 400 mg of substrate. Transformation was continued under the same conditions for 7 days. The fermentation broth was filtered to remove the mycelium, and the filtrate was extracted three times with equal volumes of ethyl acetate. The extract was then concentrated to dryness under reduced pressure to yield approximately 0.97 g of a crude transformant extract.

[0040] 2) ODS-C18 column chromatography separation

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

[0042] 3) Reverse-phase high performance liquid chromatography purification

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

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

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

[0046] The conversion product obtained in step 3) was 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, 600 MHz) and C NMR spectrum (CDCl3, 150 MHz) 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 passaging procedure, centrifuge and discard the culture medium, add 4 mL of complete culture medium, and gently pipette to evenly distribute the cells. After trypan blue staining, count the cells and then add 90 μL of a 4×10 4 / mL of cell fluid, the cells were evenly seeded 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-A value of the experimental group / A value of the control group) × 100%]. The experiment was repeated 3 times. SPSS11.5 software was used to make a regression equation to calculate the half-maximal 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 value 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 in 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 assay: 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 Cells were seeded in 96-well culture plates at 50 μL / mL. 80 μL of cell suspension was added to each well of the drug-treated and control groups, with triplicate wells per group. The blank control group received only DMEM (80 μL per well, with triplicate wells per well). The 96-well culture plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. 10 μL of MTT was added to each well of the 96-well plate. Following addition of MTT, the cells were incubated for an additional 4 hours. After 4 hours, the supernatant was aspirated and 100 μL of DMSO was added to each well. The absorbance was then measured at 570-630 nm on a microplate 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 of PBS. 700 μL of trypsin was added to dislodge the cells from the dish. 1 mL of fresh culture medium was added to terminate the digestion. The cells were then gently pipetted and collected in a 15 mL centrifuge tube. 2 mL of culture medium was added and the above procedure was repeated once. The centrifuge tube was then centrifuged in a low-speed centrifuge for 3 minutes 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 into 96-well plates at a density of 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 a concentration gradient method. After adding 10 μL of 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 another 10 μL of LPS was added to the drug group to ensure 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-maximal inhibitory concentration (IC50) of each test sample for inhibiting NO release. 50 ).

[0066] 2) Experimental results

[0067] Based on the test results of the MTT assay and the Griess assay, the effect of the compound of formula I of the present invention on the 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] NO, a key marker of inflammation, is increasingly being used as a detection indicator in inflammatory models. In this experiment, Griess reagent was used to measure NO levels. The results showed that the compound represented by Formula I exhibited no significant cytotoxicity against BV-2 cells. It also significantly reduced LPS-induced NO release in BV-2 cells, demonstrating excellent anti-neuroinflammatory activity. Its anti-neuroinflammatory activity was significantly superior to that of the positive control, L-NMMA, suggesting its potential as an active ingredient in anti-neuroinflammatory drugs.

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

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

[0073] (2) 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 according to claim 1, characterized in that: The preparation method comprises: using Aspergillus minsclerotigenes to carry out biotransformation on oleanolic acid and then performing separation and purification to obtain the oleanane-type triterpene derivative; The preparation method comprises the following steps: 1) fermenting and culturing a microorganism, adding oleanolic acid, continuing transformation culture, and removing mycelium to obtain a fermentation broth, wherein the microorganism is Aspergillus minsclerotigenes; 2) extracting the fermentation broth with an organic solvent to obtain a transformed product; the organic solvent is ethyl acetate; 3) treating the transformed 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 the structural formula I; Wherein, the reverse phase silica gel column chromatography treatment is specifically: The transformant was separated by ODS-C18 column chromatography, and gradient elution was performed using a methanol-water two-phase system. The fractions were collected, analyzed by HPLC, and then combined to obtain different components.

3. The preparation method according to claim 2, 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.

4. The preparation method according to claim 3, characterized in that The reversed-phase high performance liquid chromatography treatment is specifically: A YMC ODS A chromatographic column with a specification of 5 μm and 12.0×250 mm was used. The mobile phase was an acetonitrile-water solution with a volume ratio of acetonitrile to water of 65:

35. The flow rate was 3.0 mL / min and the detection wavelength was 203 nm.

5. Use of the oleanane-type triterpene derivative according to claim 1 as an active ingredient in the preparation of an anti-tumor drug, wherein the tumor comprises one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

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

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