A triterpenoid compound, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical technology, and discloses a triterpenoid compound, its preparation method, and its applications. The structural formula of the triterpenoid compound is shown in formula (1). This triterpenoid compound can activate SIRT1, promote the deacetylation of heat shock factor-1, upregulate the expression of heat shock proteins, and reduce cardiomyocyte apoptosis. It can serve as a lead compound for the development of drugs to treat MIRI and has great potential application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a triterpenoid compound, its preparation method, and its application. Background Technology

[0002] Ischemic heart disease (IHD) is a leading cause of death worldwide, with approximately 9 million deaths registered in 2019. IHD often occurs after coronary artery stenosis or blockage, and reperfusion can reduce myocardial damage; however, reperfusion can cause further damage, known as myocardial ischemia / reperfusion injury (MIRI). MIRI can lead to irreversible damage to cardiomyocytes, resulting in impaired cardiac structure and function, and is a significant cause of myocardial fibrosis and heart failure.

[0003] SIRT1 is a highly conserved nicotinamide adenine dinucleotide (NAD1) + SIRT1 is a histone deacetylase dependent on MIRI. It participates in regulating various pathophysiological processes, including cell proliferation, survival, differentiation, autophagy, and oxidative stress. Therapeutic activation of SIRT1 protects the heart and cardiomyocytes from pathology-related stress, particularly MIRI. SIRT1 exerts its cardioprotective effect by regulating FOXO3α to inhibit MIRI-mediated apoptosis. NAD+ can restore SIRT1 activity, reduce the acetylation level of the tumor suppressor gene p53, and attenuate MIRI-induced apoptosis in H9C2 cells. The SIRT1 agonist resveratrol restores SIRT1 activity and NAD+ levels through an adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism, activates heat shock factor-1 deacetylation, upregulates heat shock protein expression, and reduces cardiomyocyte apoptosis. SIRT1 plays a very important role in the development and progression of myocardial infarction and may be a key target for the prevention and treatment of MIRI.

[0004] Natural products are the most common source of drugs, and compounds from microorganisms constitute a significant portion of these. Aspergillus fungi exhibit the ability to produce a rich variety of bioactive molecules, such as alkaloids, butenolides, phenenthelacetones, terpenoids, cytochalasin, p-terphenyl, xanthones, sterols, anthraquinones, and diphenyl ether derivatives. These molecules have attracted considerable attention in the pharmaceutical and various industrial fields. Therefore, the discovery and acquisition of compounds with SIRT1 agonist activity and capable of preventing and treating MIRI from Aspergillus secondary metabolites is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and to provide a triterpenoid compound, its preparation method and application.

[0006] To achieve the above objectives, the first aspect of the present invention provides a triterpenoid compound, the structural formula of which is shown in formula (1);

[0007]

[0008] A second aspect of the present invention provides a method for preparing the triterpenoid compounds described above, comprising the following steps:

[0009] (1) Aspergillus fungi were inoculated onto potato dextrose agar medium and then cultured at a constant temperature to obtain seed culture medium;

[0010] (2) Cut the seed culture medium into pieces and inoculate them into rice culture medium for fermentation culture;

[0011] (3) The fermentation product obtained in step (2) is extracted with ethanol and concentrated under reduced pressure to obtain the total extract;

[0012] (4) The total extract is mixed with water, then extracted with ethyl acetate and concentrated under reduced pressure to obtain ethyl acetate extract;

[0013] (5) The ethyl acetate extract was separated by chromatography to obtain the triterpenoid compound shown in formula (1).

[0014] Preferably, the Aspergillus fungus is Aspergillus sp. TJ507, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231079.

[0015] Preferably, in step (1), the conditions for constant temperature culture include: a temperature of 25-30°C and a time of 30-40 days.

[0016] Preferably, in step (2), the fermentation conditions include a temperature of 25-30°C and a time of 30-40 days.

[0017] Preferably, the ethanol extraction is performed 5-10 times.

[0018] Preferably, in step (4), the ethyl acetate extraction is performed 4-6 times.

[0019] Preferably, the specific process of step (5) includes:

[0020] S1: The ethyl acetate extract was subjected to column chromatography, and a gradient elution was performed using petroleum ether-ethyl acetate with a volume ratio of 20:1-0:1 to obtain 6 fractions.

[0021] S2: Component 3 was purified by reversed-phase silica column chromatography, normal-phase silica column chromatography, gel chromatography and high performance liquid chromatography to obtain the triterpenoid compound shown in formula (1).

[0022] Preferably, the gel chromatography conditions are isocratic elution with dichloromethane-methanol at a volume ratio of 1:1.

[0023] Preferably, the reversed-phase silica column chromatography conditions are gradient elution with methanol-water at a volume ratio of 20:80-100:0.

[0024] Preferably, the conditions for normal-phase silica gel column chromatography are gradient elution with petroleum ether-ethyl acetate-methanol in a volume ratio of 20:1:0-1:1:1.

[0025] Preferably, the high performance liquid chromatography conditions are isocratic elution with acetonitrile-water at a volume ratio of 88:12.

[0026] A third aspect of the present invention provides the use of the triterpenoid compounds described above in the preparation of medicaments for treating myocardial ischemia-reperfusion injury.

[0027] This invention provides a triterpenoid compound of formula (1) and a method for isolating and purifying this triterpenoid compound from the fungus *Aspergillus* sp. TJ507. This triterpenoid compound can activate SIRT1, promote the deacetylation of heat shock factor-1, upregulate the expression of heat shock proteins, and reduce cardiomyocyte apoptosis. It can serve as a lead compound for the development of drugs to treat MIRI and has significant potential application value. Attached Figure Description

[0028] Figure 1 This is a single-crystal diffraction pattern of the compound prepared in Example 1;

[0029] Figure 2 This is a graph showing the results of testing the effect of aspergillus A on the agonistic activity of SIRT1 using SIRT1 activity quantitative fluorescence detection in Test Example 1;

[0030] Figure 3 This is the result of the CCK8 assay used in Test Example 2 to test the protective effect of aspergillus acid A on cardiomyocytes.

[0031] Figure 4 This is a graph showing the results of the assessment of the protective effect of aspergillus A on cardiomyocytes by specifically identifying the mitochondrial membrane potential of cardiomyocytes using fluorescent probe dyes in Test Example 3. Detailed Implementation

[0032] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] The first aspect of the present invention provides a triterpenoid compound, the structural formula of which is shown in formula (1);

[0035]

[0036] In this invention, the triterpenoid compound represented by formula (1) is named aspergillus ketone A, which is a secondary metabolite of Aspergillus fungi.

[0037] A second aspect of the present invention provides a method for preparing the triterpenoid compounds described above, comprising the following steps:

[0038] (1) Aspergillus fungi were inoculated onto potato dextrose agar medium and then cultured at a constant temperature to obtain seed culture medium;

[0039] (2) Cut the seed culture medium into pieces and inoculate them into rice culture medium for fermentation culture;

[0040] (3) The fermentation product obtained in step (2) is extracted with ethanol and concentrated under reduced pressure to obtain the total extract;

[0041] (4) The total extract is mixed with water, then extracted with ethyl acetate and concentrated under reduced pressure to obtain ethyl acetate extract;

[0042] (5) The ethyl acetate extract was separated by chromatography to obtain the triterpenoid compound shown in formula (1).

[0043] In this invention, the Aspergillus fungus is Aspergillus sp. TJ507. This strain was obtained from the soil of a St. John's wort plantation and is currently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231079, deposit address: Wuhan University, Wuhan, China, deposit date: June 21, 2023, and classified as Aspergillus sp. TJ507.

[0044] In a preferred embodiment, in step (1), the conditions for constant temperature culture include: a temperature of 25-30°C and a time of 30-40 days; specifically, the temperature for constant temperature culture can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, and the time for constant temperature culture can be 30 days, 32 days, 35 days, 38 days or 40 days.

[0045] In a preferred embodiment, in step (2), the fermentation conditions include: a temperature of 25-30°C and a time of 30-40 days; specifically, the fermentation temperature can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, and the fermentation time can be 30 days, 32 days, 35 days, 38 days or 40 days.

[0046] In a preferred embodiment, the ethanol extraction is performed 5-10 times.

[0047] In a preferred embodiment, the ethyl acetate extraction is performed 4-6 times.

[0048] According to some specific embodiments of the present invention, in step (4), the ethyl acetate extraction process includes: dissolving the total extract in distilled water, adding ethyl acetate for extraction, and drying the ethyl acetate layer using a rotary evaporator to obtain ethyl acetate extract.

[0049] Preferably, the specific process of step (5) includes:

[0050] S1: The ethyl acetate extract was subjected to column chromatography, and a gradient elution was performed using petroleum ether-ethyl acetate with a volume ratio of 20:1-0:1 to obtain 6 fractions.

[0051] S2: Component 3 was purified by reversed-phase silica column chromatography, normal-phase silica column chromatography, gel chromatography and high performance liquid chromatography to obtain the triterpenoid compound shown in formula (1).

[0052] More preferably, the gel chromatography conditions are isocratic elution with dichloromethane-methanol at a volume ratio of 1:1.

[0053] More preferably, the reversed-phase silica column chromatography conditions are gradient elution with methanol-water at a volume ratio of 20:80-100:0.

[0054] More preferably, the conditions for normal-phase silica gel column chromatography are gradient elution with petroleum ether-ethyl acetate-methanol in a volume ratio of 20:1:0-1:1:1.

[0055] More preferably, the high-performance liquid chromatography (HPLC) conditions are isocratic elution with acetonitrile-water at a volume ratio of 88:12.

[0056] A third aspect of the present invention provides the use of the triterpenoid compounds described above in the preparation of medicaments for treating myocardial ischemia-reperfusion injury.

[0057] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0058] The Aspergillus fungus used in the following examples is Aspergillus sp. TJ507, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231079.

[0059] Example 1

[0060] Preparation and Identification of Aspergillus A

[0061] 1. Preparation of Aspergillus A:

[0062] (1) Aspergillus fungi were inoculated onto potato dextrose agar medium and then cultured at 28°C for 35 days to obtain seed culture medium.

[0063] (2) Cut the seed culture medium into pieces and inoculate it into rice culture medium, and ferment it at 28°C for 35 days;

[0064] (3) The fermentation product obtained in step (2) was extracted with industrial alcohol seven times, and the industrial alcohol was recovered by vacuum concentration at 50°C to obtain the total extract.

[0065] (4) Dissolve the total extract in distilled water, extract with ethyl acetate 5 times, and dry the ethyl acetate layer by rotary evaporator to obtain 750g of ethyl acetate extract.

[0066] (5) The ethyl acetate extract was subjected to column chromatography. The sample was mixed with 200-300 mesh silica gel and packed into the column by dry method. The column was eluted with a gradient of petroleum ether-ethyl acetate (20:1-0:1). The TLC was detected and the same components were combined to obtain a total of 6 components with polarity from small to large, which were denoted as components 1-6.

[0067] Fraction 3 was purified by reversed-phase silica gel column chromatography with gradient elution of methanol-water at a volume ratio of 20:80-100:0 to obtain 7 fractions, denoted as fractions 3.1-3.7.

[0068] Fraction 3.5 was purified by gel chromatography and eluted isocratically with dichloromethane-methanol at a volume ratio of 1:1 to obtain four fractions, denoted as fractions 3.5.1-3.5.4.

[0069] Fraction 3.5.2 was purified by normal silica gel chromatography using a gradient elution of petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:1 to obtain 8 fractions, denoted as 3.5.2.1-3.5.2.8.

[0070] Component 3.5.8.2 was purified by high performance liquid chromatography and isocratic elution with acetonitrile-water at a volume ratio of 88:12 to obtain aspergillus A (3.2 mg).

[0071] 2. Structural identification of aspergillus A

[0072] The prepared compound was analyzed using nuclear magnetic resonance (NMR), mass spectrometry (MS), optical rotation, infrared spectroscopy, ultraviolet spectroscopy, circular dichroism (ECD), and X-ray single-crystal diffraction to determine its structure. The results are as follows:

[0073] Aspergillus A: colorless crystals, mp 270.0-271.3; (c 2.9,MeOH); UV(MeOH)λ max (logε)=204(3.90)nm;IR(ν max ): 3440, 2969, 2936, 2854, 1695, 1631, 1456cm -1 ;ECD(MeOH)λ max (Δε)219(+6.7); HRESIMS m / z463.3606[M+Na]+(calcd for C 30 H 48 The absolute configuration of aspergillus acetonide A was determined by X-ray single-crystal diffraction. Crystal structure (single-crystal diffraction pattern) is shown below. Figure 1 As shown, the NMR data are described in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] Note: 'a' indicates that the test solvent is CDCl3 and the test instrument is a 400M nuclear magnetic resonance spectrometer.

[0078] Test Example 1

[0079] SIRT1 deacetylation was determined using a fluorescent group quantitative assay.

[0080] The purified SIRT1 protein was incubated at 200 nM per well with different concentrations of the compound aspergillus acetonide A at room temperature for 30 min. Then, a reaction solution containing 75 μM NAD+ and 25 μM peptide substrate (Ac-Arg-His-Lys-Lys(Ac)-AMC) was added, and the reaction was carried out at 37 °C for 1 h. The reaction was terminated by adding a stop solution (50 mM Tris-HCl, 100 mM NaCl, 6000 Units Trypsin, 4 mM nicotinamide). The fluorescence intensity of the AMC group was detected using a microplate reader at an excitation wavelength of 355 nm and an absorption wavelength of 460 nm. The results are as follows: Figure 2 As shown.

[0081] Depend on Figure 2 It can be seen that, without the action of the compound, the expression level of AMC is about 50. The compound aspergillus acetonide A can significantly stimulate the activity of SIRT1. At a concentration of 10 μM, the expression level of AMC is as high as 170, indicating that the compound aspergillus acetonide A is a good SIRT1 agonist.

[0082] Test Example 2

[0083] The protective effect of aspergillus A on cardiomyocytes was tested using the CCK8 kit.

[0084] 100 μL of H9C2 cells (5000 cells per well) were cultured in 96-well plates. After incubation for 24 h, the cells were pre-protected with different concentrations of the compound aspergillin A for 24 h. The culture medium was then replaced with glucose-free and serum-free DMEM, and the cells were placed in an anoxic chamber. The gas inside the chamber was replaced with a mixture of 95% N2 and 5% CO2 to ensure the oxygen concentration was below 0.3%. After 5 h of anoxic incubation, the culture medium was replaced with complete DMEM with high glucose, and the cells were reoxygenated for 2 h. Afterward, 10 μL of CCK8 (TargetMol) was added to each well, and the cells were incubated at 37°C for 40 min. The absorbance was measured at 450 nm using a Thermo Scientific microplate reader. The results are as follows: Figure 3 As shown.

[0085] Depend on Figure 3 It can be seen that the cell damage in the model group has reached 50%, and the cell survival rate reaches 60%-70% under the protection of the compound aspergillin A, indicating that the compound aspergillin A has a certain protective effect on cardiomyocytes.

[0086] Test Example 3

[0087] Fluorescent probe dyes were used to specifically identify the mitochondrial membrane potential of cardiomyocytes.

[0088] H9C2 cells (200,000 per well) were seeded in 6-well plates, pre-protected with aspergillin A, and then subjected to hypoxia-reoxygenation injury. Mitochondrial membrane potential was then stained using JC-1 (Solarbio) and TMRE (Beyotime). The results are as follows: Figure 4 As shown.

[0089] Depend on Figure 4 It can be seen that under hypoxia-reoxygenation, mitochondrial membrane potential decreases, red fluorescence of JC-1 and TMRE decreases, green fluorescence of JC-1 increases, and the compound aspergillusone A can improve the loss of mitochondrial membrane potential.

[0090] Based on the above test results, it can be seen that the compound aspergillus A isolated from the fungus Aspergillus has good SIRT1 agonist activity and good inhibitory effect on MIRI, and can be used as a lead compound for the development of anti-MIRI drugs.

[0091] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A triterpenoid compound, characterized in that, The structural formula of the triterpenoid compound is shown in formula (1); Equation (1).

2. A method for preparing the triterpenoid compound of claim 1, characterized in that, Includes the following steps: (1) Aspergillus fungi were inoculated onto potato dextrose agar medium and then cultured at a constant temperature to obtain seed culture medium; (2) Cut the seed culture medium into pieces and inoculate them into rice culture medium for fermentation culture; (3) The fermentation product obtained in step (2) is extracted with ethanol and concentrated under reduced pressure to obtain the total extract; (4) The total extract is mixed with water, then extracted with ethyl acetate and concentrated under reduced pressure to obtain ethyl acetate extract; (5) The ethyl acetate extract was separated by column chromatography to obtain the triterpenoid compound shown in formula (1); The Aspergillus fungus is Aspergillus sp. TJ507, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20231079; In step (1), the conditions for constant temperature culture include: a temperature of 25-30℃ and a time of 30-40 days; In step (2), the fermentation conditions include: a temperature of 25-30°C and a time of 30-40 days; The specific process of step (5) includes: S1: The ethyl acetate extract was subjected to column chromatography, and a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 20:1–0:1 was used to obtain 6 fractions. S2: Component 3 was purified by reversed-phase silica column chromatography, normal-phase silica column chromatography, gel chromatography and high performance liquid chromatography to obtain the triterpenoid compound shown in formula (1); The conditions for the gel chromatography were isocratic elution with dichloromethane-methanol at a volume ratio of 1:

1. The reversed-phase silica column chromatography conditions were as follows: gradient elution with methanol-water at a volume ratio of 20:80-100:

0. The normal-phase silica column chromatography conditions were as follows: gradient elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:

1. The high-performance liquid chromatography (HPLC) conditions were set as follows: isocratic elution with acetonitrile-water at a volume ratio of 88:

12.

3. The method according to claim 2, characterized in that, The ethanol extraction is performed 5-10 times.

4. The method according to claim 2, characterized in that, In step (4), the ethyl acetate extraction is performed 4-6 times.

5. The use of the triterpenoid compound of claim 1 in the preparation of a medicament for treating myocardial ischemia-reperfusion injury.

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