Triterpenoid actinidone as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural medicines, and particularly relates to a triterpenoid actinidone as well as a preparation method and application thereof. The triterpenoid provided by the invention is 3-keto-2 alpha, 24-dihydroxy-ursane-12-ene-28-acid, is named as actinidia chinensis ketone, and is obtained by separating from the root of Actinidia chinensis Planch (the traditional Chinese medicine Chinese actinidia chinensis root). Cell experiments show that the compound can effectively inhibit liver fat accumulation and relieve the liver inflammation level, has a remarkable relieving effect on NAFLD, and can prevent or treat NAFLD.

Description

Technical Field

[0001] The invention belongs to the field of chemical medicines, and specifically relates to a triterpene compound pyrrolone, a preparation method and an application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic-related fatty liver disease without a history of excessive drinking. Clinical pathological manifestations include fat accumulation in hepatocytes, liver inflammation and damage. According to the degree of progression of liver lesions, NAFLD can be divided into simple fatty liver, fatty liver hepatitis, fatty liver fibrosis and cirrhosis. According to statistics, the incidence of NAFLD has been increasing year by year, and nearly 30% of the global population suffers from NAFLD. The pathogenesis of NAFLD is relatively complex and is related to factors such as lifestyle, environment and genetics. At present, clinical treatment drugs for NAFLD are still very scarce. For NAFLD without obvious fibrotic lesions, lifestyle intervention is mainly adopted. Resmetirom is the only drug approved by the US FDA for the relief of fatty liver hepatitis, but it has side effects such as diarrhea, nausea, itching and vomiting. Therefore, the development of new, safe and effective anti-NAFLD drugs is still very urgent and important.

[0003] The traditional Chinese medicine "Tengli root", which is the root of Actinidia chinensis Planch, has traditional effects such as clearing away heat and dampness, dispelling wind and removing numbness, detoxifying, and reducing swelling. It is used to treat jaundice, digestive tract tumors, sores, furuncles, etc. Tengli root is rich in triterpenoid compounds. More than dozens of triterpenoid compounds have been reported, and the main structural types are ursane type and oleanane type. At present, the pharmacological function research of triterpenoid compounds in Tengli root mainly focuses on anti-tumor and anti-macrophage inflammation in vitro. There are few reports on the anti-NAFLD effect of Tengli root, and its anti-NAFLD effect is still unclear. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a triterpene compound pyrrolone, a preparation method and an application thereof.

[0005] In the first aspect of the present invention, a triterpene compound, pyrrolidone, is provided, and its structural formula is shown in formula (I):

[0006]

[0007] The second aspect of the present invention provides the use of the triterpenoid compound pyrrolone or its pharmaceutically acceptable salt derivatives as described above in the preparation of a functional drug for preventing or treating non-alcoholic fatty liver disease.

[0008] The third aspect of the present invention provides a pharmaceutical composition having the function of preventing or treating non-alcoholic fatty liver disease, which contains a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable pharmaceutical excipient; the active ingredient comprises the triterpene compound pyrrolidone as described above or a pharmaceutically acceptable salt derivative thereof.

[0009] It is further provided that the active ingredient also includes currently pharmaceutically acceptable lipid-lowering and liver-protecting drugs. The liver-protecting drugs include but are not limited to one or more combinations of silybin, ursodeoxycholic acid, oleanolic acid and resmetirom.

[0010] It is further provided that the pharmaceutical composition has the following preparation forms: injection, tablet, capsule, aerosol, suppository, film, pellet, ointment, controlled release or sustained release and nano preparation.

[0011] The "pharmaceutical excipients" mentioned in the present invention refer to conventional drug carriers in the pharmaceutical field, such as: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc., fillers such as starch, sucrose, etc.; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan and fatty acid glyceride, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol, etc. Coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate, etc.; in addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0012] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers and then made into the desired dosage form. The preparation forms of the drug include injection tablets, granules, capsules, solutions, emulsions, suspensions, sprays, aerosols, powder sprays, drops, pills and nano preparations. The present invention can be administered to patients who need such treatment in the form of a composition through gastrointestinal administration, injection, respiratory administration, skin administration, mucosal administration and cavity administration. When used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., and into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, elixirs, etc.; when used for parenteral administration, it can be made into solutions, water or oil suspensions for injection, etc.

[0013] In a fourth aspect of the present invention, a method for preparing the triterpenoid compound pyrone as described above is provided, wherein the triterpenoid compound pyrone is extracted from the root of Actinidia chinensis Planch (the traditional Chinese medicine "pyrone root").

[0014] The present invention extracts the root of Actinidia chinensis Planch (Chinese medicine "actinidia root") to obtain a compound with a 3-keto-2α, 24-dihydroxy-ursane-12-ene-28-acid structure. The compound has not been reported before and is a new compound named actichinone. Moreover, compared with other triterpenoid compounds with the same skeleton structure, the compound has a very significant anti-NAFLD effect, so it has a good application prospect for preparing drugs for preventing or treating NAFLD diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0016] Figure 1 Compound actichinone 1 H NMR spectrum;

[0017] Figure 2 Compound actichinone 13 C NMR spectrum;

[0018] Figure 3 HSQC spectrum of compound actichinone;

[0019] Figure 4 HMBC spectrum of compound actichinone;

[0020] Figure 5 COSY spectrum of the compound actichinone;

[0021] Figure 6 ROESY spectrum of compound actichinone;

[0022] Figure 7 HR-ESI-MS spectrum of compound actichinone;

[0023] Figure 8 The effect diagram of compound 1-19 on the inhibitory effect of oleic acid-induced triglyceride accumulation in mouse primary hepatocytes;

[0024] Fig. 9 Validation of the inhibitory effect of the compound actichinone (1) on oleic acid-induced fat accumulation in primary mouse hepatocytes.

[0025] Fig.10 Verification of the effect of the compound actichinone (1) on the fat metabolism of primary mouse hepatocytes induced by oleic acid.

[0026] Fig.11 A graph showing the inhibitory effect of the compound actichinone (1) on oleic acid-induced inflammation in mouse primary hepatocytes. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1 Preparation of compound actichinone:

[0029] (1) The dried root of Actinidia chinensis Planch (Chinese medicine "vine pear root") was taken and crushed into coarse powder. It was extracted by soaking in a mixed solvent of dichloromethane / methanol (v / v, 1:1) at room temperature for 3 times, each time for 4 hours. The extracts were combined and concentrated to dryness under reduced pressure to obtain 46.0g of crude extract. The crude extract was separated by silica gel column chromatography and gradient eluted with petroleum ether / dichloromethane (v / v, 9 / 1), dichloromethane / ethyl acetate (v / v, 5 / 1), pure ethyl acetate, ethyl acetate / methanol (v / v, 5 / 1), and pure methanol to obtain 5 components (Fr.AE). Component Fr.D was separated by Sephadex LH-20 column chromatography (dichloromethane / methanol, v / v, 1 / 1), and the eluted components were analyzed by TLC and the same components were combined to obtain 3 components (Fr.D1~D3). The component Fr.D2 was repeatedly separated and purified by silica gel column chromatography (dichloromethane / methanol, v / v, 30 / 1, 20 / 1, 10 / 1), reversed phase ODS column chromatography (methanol / water, v / v, 70 / 30, 80 / 20, 90 / 10), reversed phase preparative HPLC (methanol / water, v / v, 78 / 22, 85 / 15), etc. to obtain compound 1-19. The structural formula of compound 1-19 was determined by spectral data analysis as shown below:

[0030]

[0031] (2) Compound 1 was obtained by semi-preparative HPLC chromatography, and the specific separation conditions were as follows:

[0032] Instrument: Agilent 1100 liquid chromatograph with G1314A UV detector.

[0033] Chromatographic column: YMC C 18 Column (250×15 mm, 5 μm).

[0034] Mobile phase: methanol / water (v / v, 85 / 15); flow rate: 2.0 mL / min; detection wavelength: 210 nm.

[0035] The chromatographic peak with a retention time of 46.54 min was collected and concentrated to dryness under reduced pressure to obtain compound 1, which was named actichinone.

[0036] The NMR data and mass spectrometry data of compound actichinone are as follows:

[0037] 1 H NMR(Pyridine-d5,500MHz): δ2.53(1H,dd,J=12.5,6.0Hz,H-1a),1.44(1H,m,H-1b),5.01(1H,dd,J=12.5,6.5Hz,H-2),1.38(1H,m,H-5),1.60 (1H,m,H-6a),1.54(1H,m,H-6b),1.49(1H,m,H-7a),1.34(1H,m,H-7b) ,1.71(1H,m,H-9),1.98(1H,m,H-11a),1.96(1H,m,H-11b),5.45(1H,br s,H-12),2.31(1H,td,J=13.0,4.0Hz,H-15),1.18(1H,m,H-15),2.12(1H,td,J=13.0,4.0Hz,H-16a),2.00(1H,m, H-16b),2.63(1H,d,J=11.0Hz,H-18),1.45(1H,m,H-19),1.01(1H,m,H-20),1.47(1H,m,H-21a),1.39(1H,m,H-21 b),1.97(2H,m,H2-22),1.63(3H,s,H3-23),4.46(1H,d,J=11.0Hz,H-24a),3.83(1H,d,J=11.0Hz,H-24b),1.23(3 H,s,H3-25),1.05(3H,s,H3-26),1.16(3H,s,H3-27),0.98(3H,d,J=6.5Hz,H3-29),0.96(3H,d,J=6.5Hz,H3-30);

[0038] 13C NMR (Pyridine-d5, 150MHz): δ51.0(C-1),70.6(C-2),215.1(C-3),55.8(C-4),58.9(C-5),20.1(C-6),33. 7(C-7),40.1(C-8),47.9(C-9),38.0(C-10),24.0(C-11),125.1(C-12),139.4(C-13),42.5(C-14),28.7(C -15),24.9(C-16),48.0(C-17),53.5(C-18),39.5(C-19),39.4(C-20),31.1(C-21),37.4(C-22),20.5(C- 23),65.4(C-24),17.0(C-25),17.5(C-26),23.9(C-27),179.9(C-28),17.5(C-29),21.4(C-30); HR-ESIMS m / z 509.3239[M+Na] + (C 30 H 46 O5Na).

[0039] Structural analysis based on the above spectral data shows that the compound has a chemical structure as shown in formula (1).

[0040]

[0041] After searching, it was found that the compound actichinone (1) has not been reported before, so it can be identified as a new compound.

[0042] Example 2 Determination of the inhibitory effect of compound 1-19 on oleic acid-induced triglyceride accumulation in primary mouse hepatocytes:

[0043] First, the CCK8 method was used to determine the toxicity of each compound (50 μM concentration) to mouse primary hepatocytes (extracted from 7±1 week old C57BL / 6J male mice). 4 Each well was inoculated in a 96-well culture plate, with 100 μL of culture medium per well, and cultured in a 37°C incubator containing 5% CO2. When the cells are attached, the cells are grouped and treated as follows. Blank control group: 1 μL DMSO + 99 μL DMEM low-glucose culture medium was used to culture the cells for 24 hours; drug-treated group: 1 μL compound DMSO dilution + 99 μL DMEM low-glucose culture medium was used to treat the cells for 24 hours. After the treatment, 10 μL CCK8 reagent was added to each well, and after incubation at 37°C for 3 hours, the absorbance OD was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. 450Calculate cell survival rate (OD 450 Value / control group OD 450 The experiment was repeated three times.

[0044] Secondly, the inhibitory effect of each compound (10 μM concentration) on triglyceride (TG) accumulation in mouse primary hepatocytes induced by oleic acid (OA) was determined. Mouse primary hepatocytes were cultured at 5×10 4 Cells were inoculated into 96-well culture plates, with 100 μL DMEM low-glucose medium in each well, and cultured in a 37°C incubator containing 5% CO2. When the cells were attached, the compound was added to each well and incubated for 3 hours, followed by the addition of 1% bovine serum albumin (BSA) and 1mM OA for 24 hours to induce lipid accumulation. The experiment set up a blank group, a model OA group, and a drug-treated group. The triglyceride TG commercial kit was used to determine the TG level of each group according to the method specified in the kit. This experiment was repeated three times.

[0045] The experimental results are as follows Figure 8 As shown in A, the 19 compounds had no significant effect on the cell viability of mouse primary hepatocytes at a concentration of 50 μM, that is, there was no hepatotoxicity at a concentration of 50 μM. Figure 8 As shown in Figure B, seven compounds (1–3, 5–7, and 12) were able to effectively counteract OA-induced triglyceride (TG) accumulation at a concentration of 50 μM, among which compound actichinone (1) was the most effective, with very significant anti-hepatocyte lipid accumulation activity and was able to treat or alleviate NAFLD.

[0046] Compounds 1-19 are all triterpenoid compounds with similar carbon skeleton structures. The main difference lies in the changes in the substitution groups and combinations at positions such as C-2, C-3, C-23, and C-24. According to the results of this experiment, only 7 of the 19 triterpenoid compounds showed anti-hepatocyte lipid accumulation effects. It can be seen that not all triterpenoid compounds have anti-hepatocyte lipid accumulation effects, and the changes in these substitution groups and the different combinations have a significant effect on the activity level, and there is no obvious pattern to follow. Since there is no obvious pattern to follow, it is impossible to predict which combination is the optimal combination.

[0047] Among them, the most effective compound among compounds 1-19 is actichinone (1), whose C-3 is a keto group. In the prior art, triterpenes with a keto group at C-3 are relatively rare and rarely reported. Prior to this, the effect of C-3 keto substitution on the anti-NAFLD activity level of triterpenoid compounds remained unknown. The compounds with the most similar structures to compound actichinone (1) are compounds 2 and 5, the only difference being that the substituents at C-3 of compounds 2 and 5 are hydroxyl -OH. In comparison, the anti-NAFLD activity level of compound actichinone (1) is significantly improved.

[0048] Example 3 Determination of the inhibitory effect of compound actichinone (1) on oleic acid-induced fat accumulation in primary mouse hepatocytes

[0049] The cytotoxicity and anti-hepatocyte fat accumulation activity of the compound actichinone (1) in primary mouse hepatocytes were confirmed, and the dose dependence was investigated. The CCK8 cytotoxicity assay method and the TG test experimental method are as described in Example 2. In the CCK8 experiment, the compound was set at 5 concentration gradients (6.25, 12.5, 25, 50, 100 μM). In the TG test experiment, the compound was set at 4 concentration gradients (5, 10, 20, 40 μM), and the natural triterpenoid hepatoprotective drug oleanolic acid was used as a positive control (OLEA, 20 μM).

[0050] Oil red staining (ORO) was used to evaluate the effect of compound actichinone (1) on OA-induced lipid droplet formation in hepatocytes. After pretreatment of mouse primary hepatocytes with the compound (10, 20 μM) for 3 h, 1% bovine serum albumin (BSA) and 1 mM OA were added and cultured for 24 h to induce lipid accumulation. The cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min. The cells were then stained with freshly prepared oil red O staining solution for 25 min, and then washed with 60% isopropanol and distilled water in sequence until the interstitium was clear. Hematoxylin counterstaining was performed for 1-2 min, washed with distilled water, differentiated with 1% hydrochloric acid alcohol for a few seconds, backstained with alkaline water, washed with distilled water, and the distribution of red lipid droplets was observed under an optical microscope (400×). Oleanolic acid (OLEA, 20 μM) was used as a positive control in the experiment.

[0051] The experimental results are as follows Fig. 9 As shown in the results, the compound actichinone (1) had no significant effect on the viability of mouse primary hepatocytes at a concentration of 100 μM, that is, there was no hepatotoxicity at a concentration of 100 μM ( Fig. 9A). At 10, 20, and 40 μM concentrations, the compound was able to significantly reduce the level of triglyceride TG in hepatocytes induced by oleic acid OA in a dose-dependent manner, and the effect was significantly dependent on the positive control oleanolic acid (OLEA, 20 μM) ( Fig. 9 B). The results of the oil red ORO staining experiment showed that the compound significantly reduced the formation of lipid droplets in hepatocytes at concentrations of 10 and 20 μM. Compared with the model group, the area of ​​red lipid droplets was significantly reduced, and the effect of the compound at a concentration of 20 μM was significantly better than that of the positive control OLEA at the same concentration ( Fig. 9 C, D). These experimental results confirm that the compound actichinone (1) has a significant effect in preventing fat accumulation in liver cells, and its effect is better than that of the liver-protecting drug oleanolic acid.

[0052] Example 4 Determination of the promoting effect of compound actichinone (1) on oleic acid-induced fat metabolism in primary mouse hepatocytes

[0053] The effect of compound actichinone (1) on the expression level of genes related to fat metabolism in hepatocytes was tested by RT-PCR. As described in Example 2, cells were incubated with compounds (10, 20 μM) for 3 h, followed by the addition of 1 mM OA for 24 h to induce lipid accumulation. Cell RNA was extracted using Trizol reagent. Reverse transcription was performed using a kit. The mRNA expression levels of three fat synthesis-related enzymes, fatty acid synthase (FASN), sterol regulatory element binding protein 1c (SREBP-1c) and stearoyl-CoA desaturase (SCD), as well as the mRNA expression levels of two fatty acid oxidation-related enzymes, peroxisome proliferator-activated receptor α (PPAR-α) and carnitine palmitoyltransferase 1 (CPT-1), were detected in cells by RT-PCR. The primer sequences are shown in Table 1. The primers were synthesized by Shanghai Biotech.

[0054] Table 1 Primer sequences

[0055]

[0056] RT-PCR results ( Fig.10 ) found that the compound actichinone (1) at concentrations of 10 and 20 μM can significantly reduce the mRNA expression levels of three fat synthesis-related enzymes, FASN, SREBP-1c, and SCD, in oleic acid OA-induced mouse primary hepatocytes, and significantly increase the mRNA expression levels of two fatty acid oxidation-related enzymes, PPAR-α and CPT-1, showing a significant effect on promoting liver fat metabolism.

[0057] Example 5 Determination of the inhibitory effect of compound actichinone (1) on oleic acid-induced inflammation in primary mouse hepatocytes

[0058] The RT-PCR method was used to test the effect of the compound actichinone (1) on the expression level of genes related to inflammatory factors in hepatocytes caused by lipid accumulation. As described in Example 2, the cells were incubated with the compound (10, 20 μM) for 3 h, and then 1 mM OA was added and cultured for 24 h to induce lipid accumulation. Cell RNA was extracted using Trizol reagent. Reverse transcription was performed using a kit. The mRNA expression levels of three inflammatory factors, IL-1β, IL-6 and TNF-α, in cells were detected by RT-PCR. The primer sequences are shown in Table 1. The primers were synthesized by Shanghai Bioengineering.

[0059] Experimental results ( Fig.11 ) found that the compound actichinone (1) at concentrations of 10 and 20 μM can significantly reduce the mRNA expression levels of three inflammatory factors IL-1β, IL-6 and TNF-α in primary mouse hepatocytes induced by oleic acid OA, that is, it effectively reduced the level of hepatocyte inflammation caused by lipid accumulation and showed a significant anti-fatty hepatitis effect.

[0060] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A triterpene compound pyrrolidone, characterized in that: Its structural formula is shown in formula (I):

2. Use of the triterpenoid compound pyrrolone or its pharmaceutically acceptable salt derivative as claimed in claim 1 in the preparation of a functional drug for preventing or treating non-alcoholic fatty liver disease.

3. A pharmaceutical composition having the function of preventing or treating non-alcoholic fatty liver disease, characterized in that It contains a therapeutically effective amount of active ingredients and pharmaceutically acceptable pharmaceutical excipients; the active ingredients include the triterpene compound pyrrolidone as claimed in claim 1 or its pharmaceutically acceptable salt derivatives.

4. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to claim 3, characterized in that: The active ingredients also include currently pharmaceutically acceptable lipid-lowering and liver-protecting drugs.

5. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to claim 4, characterized in that: The liver protection drug is one or more combinations of silybin, ursodeoxycholic acid, oleanolic acid and resmetirom.

6. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition has the following preparation forms: injection, tablet, capsule, aerosol, suppository, film, pellet, ointment, controlled release or sustained release and nano preparation.

7. The method for preparing the triterpene compound pyrone according to claim 1, characterized in that: It is extracted from the root of Actinidia chinensis Planch tree.

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