A ganoderma lucidum triterpenoid compound, and a preparation method and application thereof

Abstract

The application discloses a norchizhiol triterpenoid compound, a preparation method and application thereof. The norchizhiol triterpenoid compound norchizhiol A can be quickly prepared from Ganoderma lucidum. The test proves that the norchizhiol A can inhibit the accumulation of triglyceride in the differentiation process of 3T3L1 preadipocytes. The norchizhiol A can also effectively reduce the weight increase and liver coefficient of high-fat-diet-induced obese mice, regulate the blood lipid level of the obese mice, and significantly intervene and improve the lipoprotein distribution of the body. The compound can be used for preparing anti-obesity and lipid-lowering medicines.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and in particular to a Ganoderma lucidum triterpenoid compound, its preparation method, and its application. Background Technology

[0002] Reishi mushroom is a traditional and precious Chinese medicinal herb with high medicinal value. It has a good regulatory effect on the human nervous, immune, cardiovascular, and other systems, and is widely used in health foods, pharmaceuticals, and cosmetics. Reishi is rich in various chemical components, including reishi polysaccharides, reishi triterpenoids, amino acids, proteins, and sterols. Reishi triterpenoids are one of the key active ingredients in reishi. These triterpenoids have complex chemical structures, are diverse, and are highly lipid-soluble, possessing effects such as antioxidant activity, liver protection, anti-tumor activity, anti-inflammation, immune enhancement, and lowering blood lipids and blood sugar. In recent years, neutral triterpenoids have become a hot topic in reishi research.

[0003] Obesity has become a significant public health problem worldwide. Globally, the prevalence of obesity is on the rise. It is estimated that by 2050, the global obesity rate will increase to 60% among adult men, 50% among adult women, and 25% among children under 16. Obesity is closely linked to a variety of chronic diseases, including diabetes, cardiovascular disease, myocardial infarction, hypertension, and hyperlipidemia. These diseases not only reduce patients' quality of life and seriously threaten their health but also increase the risk of premature death and place a huge burden on healthcare systems.

[0004] Obesity results from an imbalance between energy intake and expenditure. Excess energy is stored in adipocytes as triglycerides, causing adipocytes to gradually enlarge. When adipocytes reach a certain size, new preadipocytes proliferate to prepare for triglyceride storage, ultimately leading to continuous enlargement of adipose tissue and weight gain. Currently, inhibiting adipocyte proliferation and preadipocyte proliferation and differentiation is an important strategy in combating obesity. Fat accumulation is a key step in the development of obesity; controlling this step can effectively control the progression of obesity, and the core process of obesity pathophysiology is excessive fat deposition. 3T3-L1 preadipocytes, which induce fat accumulation and differentiate into mature adipocytes in vitro, are currently the internationally recognized cell line for studying the fat deposition process and are widely used in research on the lipid-lowering and anti-obesity mechanisms of drugs. Diet-induced obesity mouse models are also common animal models for obesity research. By feeding mice a high-calorie, high-fat diet for a long period, mimicking unhealthy human eating habits, obesity is induced. This model can better reflect the role of dietary factors in the development of obesity. Using obese mouse models, scientists have gained a deeper understanding of the physiological and pathological mechanisms of obesity. In drug development, obese mouse models play a crucial role. New weight-loss drugs are typically tested on obese mice before entering clinical trials to assess their efficacy and safety. Summary of the Invention

[0005] The purpose of this invention is to address the technical deficiencies in the prior art by providing a Ganoderma lucidum triterpenoid compound.

[0006] Another object of the present invention is to provide a method for preparing the aforementioned Ganoderma lucidum triterpenoid compound.

[0007] Another object of the present invention is to provide the application of the aforementioned Ganoderma lucidum triterpenoid compound.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] A Ganoderma lucidum triterpenoid compound, the structural formula of which is shown in formula (Ⅰ):

[0010] .

[0011] Equation (I)

[0012] The Ganoderma lucidum triterpenoid compound is a neutral Ganoderma lucidum triterpenoid.

[0013] Another aspect of the present invention includes a method for preparing the aforementioned Ganoderma lucidum triterpenoid compound, comprising the following steps:

[0014] Step 1: Soak the pulverized Ganoderma lucidum in ethanol. Ganoderma lucidum (Leyss. ex Fr.)Karst, obtained Ganoderma lucidum extract;

[0015] Step 2: The Ganoderma lucidum extract obtained in Step 1 is suspended in an acidic salt solution, extracted with ethyl acetate, and the solvent is recovered to obtain the ethyl acetate extract;

[0016] Step 3: Using dichloromethane-methanol solution as the mobile phase, the ethyl acetate extract obtained in Step 2 was separated by silica gel column chromatography. The fractions were combined according to TLC analysis to obtain 5 fractions Fr.1~Fr.5. Fraction Fr.2 was purified by ODS column elution using methanol-water gradient elution to obtain 7 fractions Fr.2.1~Fr.2.7. Fr.2.4 was purified by Sephadex LH20 gel electrophoresis and then separated by preparative liquid chromatography using acetonitrile-water solution as the mobile phase to obtain the Ganoderma lucidum triterpenoid compounds.

[0017] In the above technical solution, in step 1, the ethanol concentration is 75-95 v / v.

[0018] In the above technical solution, in step 2, the acidic salt aqueous solution is a saturated aqueous solution of NaHCO3 or KHCO3.

[0019] In the above technical solution, in step 3, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 100:0 ~ 0:100.

[0020] In the above technical solution, in step 3, the volume ratio of methanol to water in the methanol-water solution is 20:80~100:0.

[0021] In the above technical solution, in step 3, when Fr.2.4 is purified by Sephadex LH20 gel, the mobile phase used is a methanol-chloroform solution or acetone, and the volume ratio of methanol to chloroform in the methanol-chloroform solution is 100:0~20:80.

[0022] In the above technical solution, in step 3, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 30:70~80:20.

[0023] Another aspect of the present invention includes the use of the aforementioned Ganoderma lucidum triterpenoid compounds in the preparation of lipid-lowering or anti-obesity drugs.

[0024] Another aspect of the present invention includes a lipid-lowering or anti-obesity medicament comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, said active ingredient comprising the aforementioned Ganoderma lucidum triterpenoid compound.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This invention discloses a method for preparing norchizhiol A from Ganoderma lucidum, which can obtain a large amount of Ganoderma lucidum triterpenoids simply, effectively and rapidly.

[0027] 2. This invention discloses the inhibition of 3T3 by the Ganoderma lucidum triterpenoid compound norchizhiol A. The compound norchizhiol A, a triterpenoid compound from Ganoderma lucidum, exhibits activity in the accumulation of triglycerides during L1 preadipocyte differentiation, effectively regulates blood lipid levels in obese mice, and significantly improves lipoprotein distribution in mice. It can be used to prepare pharmaceuticals for anti-obesity, lipid-lowering, and hypoglycemic purposes. Attached Figure Description

[0028] Figure 1 The proton NMR spectrum of the compound;

[0029] Figure 2 Carbon NMR spectra of the compound;

[0030] Figure 3 HSQC spectra of compounds;

[0031] Figure 4 HMBC spectrum of compound;

[0032] Figure 5 compound 1 H- 1 H COSY spectrum;

[0033] Figure 6 NOESY spectrum of compound;

[0034] Figure 7 Compound inhibits 3T3 Results of L1 preadipocyte proliferation;

[0035] Figure 8 Compound inhibits 3T3 L1 preadipocyte differentiation dose-response curve;

[0036] Figure 9 The effects of different concentrations of the compound on weight gain in obese mice;

[0037] Figure 10 The effect of different concentrations of the compound on the liver coefficient in obese mice;

[0038] Figure 11 The effects of different concentrations of the compound on total cholesterol in obese mice;

[0039] Figure 12 Effects of different concentrations of the compound on triglycerides in obese mice;

[0040] Figure 13 Effects of different concentrations of the compound on low-density lipoprotein cholesterol in obese mice;

[0041] Figure 14 Effects of different concentrations of the compound on high-density lipoprotein cholesterol in obese mice. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0043] Example 1

[0044] The preparation method of Ganoderma lucidum triterpenoids includes the following steps:

[0045] Step 1, Extraction of Ganoderma lucidum: Ganoderma lucidum (Leyss. ex Fr.) Karst was pulverized using a grinder and soaked in 15 times its volume of 95% ethanol for 7 days each time, for a total of 4 soaks. The extracts were combined, the ethanol was recovered under reduced pressure, and the extract was concentrated to obtain the extract paste.

[0046] Step 2, Preparation of the neutral fraction: Dissolve the extract obtained in Step 1 in a saturated sodium bicarbonate aqueous solution, extract with 1-2 times the volume of ethyl acetate, extract a total of 3 times, combine the ethyl acetate extracts, recover the ethyl acetate under reduced pressure, concentrate, and obtain the neutral fraction.

[0047] Step 3, separation of compounds: The neutral fraction obtained in Step 2 was separated by silica gel column chromatography using dichloromethane-methanol (100:0 ~ 0:100, V / V) as the mobile phase. The fractions were combined according to TLC analysis to obtain 5 fractions (Fr.1~Fr.5). Fr.2 was purified by ODS column chromatography using a methanol-water gradient elution (20:80~100:0, V / V) to obtain 7 fractions (Fr.2.1~Fr.2.7). Fr.2.4 was purified by Sephadex LH-20 column chromatography using methanol as the mobile phase. The Fr.2.4 fraction was then separated by preparative liquid chromatography using acetonitrile-water (45:55, V / V) as the mobile phase to obtain the compounds.

[0048] The structural formula of Ganoderma lucidum triterpenoids is shown in formula (1):

[0049]

[0050] Equation (I)

[0051] Ganoderma lucidum triterpenoids are white powders. HRESIMS molecular ion peaks. m / z 441.3000 [M + Na] +(calcd. for C 26 H 42 O4Na, 441.2981), combined 1 H-NMR spectrum and 13 C1-NMR spectrum, molecular formula is C1 26 H 42 O4. Based on infrared and ultraviolet spectroscopy, it is inferred that the compound contains hydroxyl groups (3420 cm⁻¹). -1 )and α , β -unsaturated ketones (1688 cm) -1 and 257nm). 1 ¹H NMR and ¹³C NMR combined with HSQC spectroscopy showed that the compound contained a 26-carbon signal, including 6 methyl carbons, 8 methylene carbons (one of which is oxymethylene), and 5 methine carbons (including 2 oxymethylene). δ C 77.9 and 71.9), 4 sps 3 The NMR signal is similar to that of ganosineniol A, but it lacks an oxygen-containing methine and has an additional methylene group. H-7 ( δ H 2.96 and 2.82) and C-8 ( δ C 166.1) and C-9 ( δ C 139.4) HMBC related signals, and H-7 ( δ H 2.96 and 2.82) and H-6 ( δ H 1.49) The COSY correlation signal indicates that the 7-position is a methylene group. H-23 ( δ H 4.53) and H-22 ( δ H The COSY correlation signal of 1.91 indicates that there is one more methylene group on the side chain at C17 than in ganosineniol A. H-3 ( δ H 3.19) and H-28 ( δ H 1.03) and H-5 ( δ H 0.87), and H-18 ( δ H 0.83) and H-15 ( δ H4.33) The key ROESY related signals are inferred to be 3-OH and 15-OH, respectively. β -and α - Orientation. Based on the biosynthetic relationships of Ganoderma lucidum triterpenes, compound 3 is inferred. S 10 S ,13R,14 R 15 S 17 R Configuration. The structure of the compound was identified as (3) S 10 S ,13R,14 R 15 S 17 R )-3,15-dihydroxy-17-(4-hydroxybutan-2-yl)-4,4,10,13,14-pentamethyl-1,2,3,4,5,6,7,10,12,13,14,15,16,17-tetradecahydro-11H-cyclopenta[ a ]phenanthren-11-one, commonly known as norchizhiolA.

[0052] Example 2

[0053] Ganoderma lucidum triterpenoids inhibit preadipocyte 3T3 Verification of L1 proliferation.

[0054] 3T3 L1 cells were cultured in complete medium (90% DMEM high-glucose medium, 10% fetal bovine serum) and incubated in an incubator until the logarithmic growth phase. After digestion, the cells were seeded into 96-well plates at a density of 5 × 10⁶ cells / well. 3 / well. Cells were incubated in a CO2 incubator at 37℃ for 24 h. After cell adhesion, the culture medium was replaced with DMEM complete medium containing different concentrations of the compound, resulting in final concentrations of norchizhiol A of 20, 40, 80, 160, and 320 μg / mL, respectively. The control group received DMEM complete medium without the compound, while the blank group received an equal volume of complete medium (excluding preadipocytes 3T3). L1 and compounds). Each group had 3 replicates. After 48 h, the culture medium was discarded, and 100 μL of the prepared MTT reagent was added. The mixture was incubated in an incubator for 4 h, and the absorbance (OD value) of each group was measured at 450 nm. The survival rate was calculated. The results are as follows. Figure 7 As shown, the compound at concentrations below 160 μg / mL has an effect on preadipocyte 3T3 cells. L1 did not show significant inhibitory effect on proliferation, and at a concentration of 320 μg / mL, it did not inhibit the proliferation of preadipocytes 3T3. L1 has a weak inhibitory effect on proliferation.

[0055] Example 3

[0056] Ganoderma lucidum triterpenoids inhibit 3T3 Validation of L1 preadipocyte differentiation activity

[0057] The test compound was prepared as a 1 mg / mL stock solution using DMSO, and then diluted to different concentrations for testing. 0.1% DMSO was used as a blank control, and berberine as a positive control. Adipocytes in the logarithmic growth phase (3T3 cells) were used... L1 cells were seeded in 48-well plates. After complete confluence, induction differentiation medium I (DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL insulin, 1 μM dexamethasone, and 0.5 mM 3...) was added. Isobutyl 1 Methylxanthine and 10 ng / mL biotin were added simultaneously, along with a certain concentration of the compound; the blank control did not contain the compound. After culturing for 48 h, the cells were cultured for another 72 h in induction differentiation medium II (DMEM complete medium containing 10 μg / mL insulin), and the test compound was added. The medium was removed, the cells were washed twice with PBS, and fixed with 10% formalin for 30 min. The fixative was discarded, and the cells were stained with Oil Red O for 15 min. The morphology of adipocytes and lipid droplets were observed under an inverted microscope. The stained cells were treated with isopropanol, and the OD value at 490 nm was measured to quantify the degree of differentiation.

[0058] like Figure 8 As shown, compound norchizhiol A inhibited 3T3 to varying degrees at concentrations ranging from 10 to 160 μg / mL. L1 cells differentiated and showed a dose-response relationship, with an IC50 value of 32.9 μg / mL. The positive control drug, berberine, had an IC50 value of 47.9 μg / mL. Compared with the positive control drug, the compound norchizhiol A inhibited 3T3... L1 preadipocyte differentiation activity.

[0059] Example 4

[0060] Experiment on the improvement of obese mice by Ganoderma lucidum triterpenoids

[0061] Four-week-old male C57BL / 6J mice were housed in an animal facility at 25 ℃ with a 12-hour light-dark cycle for 7 days of acclimatization. They were then randomly divided into four groups: normal control (CK), model group (MODEL), low-dose treatment group (GL), and high-dose treatment group (GH), with eight mice in each group. The CK group was fed a basal diet, while the MODEL, GL, and GH groups were fed a high-fat diet. After 16 weeks of feeding, the average body weight of mice in the model and treatment groups exceeded that of the normal control group by 20%. The CK and MODEL groups received intraperitoneal injections of 5 mL / kg of physiological saline once daily, the GL group received intraperitoneal injections of 2 mg / kg of norchizhiol A solution once daily, and the GH group received intraperitoneal injections of 20 mg / kg of norchizhiol A solution once daily. The intervention period for all groups was 8 weeks.

[0062] Mice were weighed weekly, and their water intake and feed consumption were recorded. After the last intervention, mice were fasted for 12 hours and weighed. Mice were euthanized, and their livers were dissected and collected from each group; their weight was measured, and liver indexes were calculated. After 8 weeks of norchizhiol A intervention, the weight gain of mice in each group was as follows: Figure 9 As shown, the weight gain of mice in the MODEL group was 14.20 g, which was significantly higher than that of mice in the CK group (P < 0.05). 0.01). Compared with the MODEL group, the weight gain of mice in the GL group was slightly lower (12.51±0.75 g), but the difference was not statistically significant (P < 0.01). The weight gain of mice in the GH group (7.22 ± 0.45 g) was significantly lower than that in the MODEL group (P < 0.05). 0.01). For example... Figure 10 As shown, there was a highly significant difference in liver index between the MODEL group (6.53%) and the CK group (3.93%) (P < 0.05). 0.01). Compared with MODEL mice, the liver index of GL group mice decreased (5.98%), which was statistically significant (P < 0.01). The liver index of the GH group mice (3.94%) was significantly lower than that of the MODEL group (P < 0.05). 0.01). After intervention with the compound norchizhiol A, the weight gain and liver fat in obese mice were inhibited to varying degrees. In particular, the effect of high concentrations of the compound norchizhiol A was similar to that of the blank control group, with no significant difference (P < 0.01). (0.05). The compound norchizhiol A can effectively reduce the weight gain and liver coefficient in high-fat diet-induced obese mice.

[0063] After 8 weeks of treatment with the compound norchizhiol A, mice in each group were fasted for 12 hours. Following anesthesia with sodium pentobarbital, blood was collected by enucleation and placed in EP tubes containing anticoagulant. After standing for 1 hour, the tubes were centrifuged at 3000 r / min for 10 min, and the supernatant was collected. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemical analyzer. Figure 11 , 12 As shown in Figures 13 and 14, compared with the CK group, the levels of TC, TG, and LDL-C in the MODEL group were significantly increased (P < 0.05). 0.05), HDL-C levels decreased significantly (P < 0.05). 0.05); In the MODEL group, serum TC increased by 0.99-fold, TG increased by 1.25-fold, LDL-C increased by 1.59-fold, and HDL-C decreased by 0.89-fold. Compared with the MODEL group, serum TC levels in the GL and GH groups were significantly lower (P < 0.05). (0.05), representing decreases of 16.76% and 46.91% respectively. From Figure 12 It was found that the serum TG levels in the GL and GH groups were reduced by 19.61% and 54.31% respectively compared to the CK group. Figure 13 and 14 It was found that the serum LDL-C levels in the GL and GH groups were reduced by 10.30% and 58.39% respectively compared with the CK group. Furthermore, the HDL-C levels in both the GL and GH groups were significantly increased compared with the CK group (P < 0.05). The levels of 0.05 and 23.61% and 83.26%, respectively, were increased. This indicates that different doses of norchizhiol A can effectively regulate blood lipid levels in obese mice in a dose-dependent manner. Among them, high doses of norchizhiol A can significantly intervene to improve the distribution of lipoproteins in the body.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Ganoderma lucidum triterpenoid compound, characterized in that, The structural formula is shown in equation (Ⅰ): 。 Equation (Ⅰ) 2. The method for preparing the Ganoderma lucidum triterpenoid compound as described in claim 1, characterized in that, Includes the following steps: Step 1: Soak the pulverized Ganoderma lucidum in ethanol. Ganoderma lucidum (Leyss. ex Fr.) Karst, obtained Ganoderma lucidum extract; Step 2: The Ganoderma lucidum extract obtained in Step 1 is suspended in an acidic salt solution, extracted with ethyl acetate, and the solvent is recovered to obtain the ethyl acetate extract; Step 3: Using dichloromethane-methanol solution as the mobile phase, the ethyl acetate extract obtained in Step 2 was separated by silica gel column chromatography. The fractions were combined according to TLC analysis to obtain five fractions Fr.1–Fr.

5. Fraction Fr.2 was purified using an ODS column and eluted with a methanol-water gradient to obtain seven fractions Fr.2.1–Fr.2.

7. Fr.2.4 was purified by Sephadex LH20 gel electrophoresis and then separated by preparative liquid chromatography, eluting with acetonitrile-water solution as the mobile phase to obtain the Ganoderma lucidum triterpenoid compounds. In step 1, the ethanol concentration is 75-95 v / v % %. In step 3, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 100:0 ~ 0:100; In step 3, the volume ratio of methanol to water in the methanol-water solution is 20:80~100:

0. In step 3, when Fr.2.4 is purified by Sephadex LH20 gel, the mobile phase used is a methanol-chloroform solution or acetone, and the volume ratio of methanol to chloroform in the methanol-chloroform solution is 100:0~20:

80. In step 3, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 30:70~80:

20.

3. The preparation method according to claim 2, characterized in that, In step 2, the acidic salt aqueous solution is a saturated aqueous solution of NaHCO3 or KHCO3.

4. The use of the Ganoderma lucidum triterpenoid compound as described in claim 1 in the preparation of lipid-lowering or anti-obesity drugs.

5. A lipid-lowering or anti-obesity drug, characterized in that, It includes an active ingredient and a pharmaceutically acceptable excipient or carrier, said active ingredient including the Ganoderma lucidum triterpenoid compound as described in claim 1.

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