A new hexahydroindene compound and its use

Abstract

The application belongs to the field of compounds and particularly relates to a new hexahydroindene compound and application thereof. A specific technical scheme is as follows: a hexahydroindene compound is obtained by fermentation of monascus or obtained by fermentation of co-cultivation of monascus and aspergillus oryzae. A new hexahydroindene compound Macolaton is separated from secondary metabolites produced by monascus, the compound has obvious effects of reducing blood sugar, blood lipid and preventing and treating arteriosclerosis, and has great pharmaceutical potential. The application further provides a preparation method of the compound Macolaton, the compound Macolaton can be directly fermented by monascus, and preferably, the compound Macolaton is obtained by fermentation of co-cultivation of monascus and aspergillus oryzae. The co-cultivation fermentation can shorten the fermentation time and improve the yield of the compound Macolaton.

Description

Technical Field

[0001] This invention belongs to the field of compounds, specifically relating to a novel hexahydroindene compound and its applications. Background Technology

[0002] With improved living standards, metabolic diseases such as hyperglycemia, hyperlipidemia, and arteriosclerosis are becoming increasingly common. Hyperglycemia damages blood vessels, leading to complications and potentially cardiovascular disease. Hyperlipidemia deposits on blood vessel walls, forming plaques that cause arteriosclerosis, block blood vessels, and ultimately lead to heart disease or cerebrovascular disease. Arteriosclerosis can also cause insufficient blood supply to other organs. Therefore, developing compounds that lower blood sugar and lipids and eliminate arteriosclerotic plaques is of great significance.

[0003] Monascus spp. is a traditional food fermentation microorganism that produces a large number of different secondary metabolites, such as red pigment and γ-aminobutyric acid (GABA). Currently, there are no reports of metabolites produced by Monascus spp. that can simultaneously prevent and treat hyperglycemia, hyperlipidemia, and arteriosclerosis. Summary of the Invention

[0004] The purpose of this invention is to provide a novel hexahydroindene compound and its applications.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a hexahydroindene compound, the structural formula of which is:

[0006]

[0007] Accordingly, the hexahydroindene compound is prepared by fermentation with Monascus purpureus.

[0008] Preferably, it is obtained by co-culturing and fermenting Monascus purpureus and Aspergillus oryzae.

[0009] Preferably, during co-culture fermentation, Monascus purpureus and Aspergillus oryzae are inoculated and fermented at a volume ratio of 2:1.

[0010] Preferably, it is obtained by isolating and purifying mycelium obtained from fermentation.

[0011] Preferably, the separation and purification method includes the following steps:

[0012] (1) Add methanol to the mycelium, centrifuge to separate the liquid and solid, collect the liquid, and distill under reduced pressure to obtain a paste;

[0013] (2) Dissolve the paste in water, add petroleum ether and stir to extract, then let stand and separate the aqueous phase and petroleum ether phase, and collect the petroleum ether phase;

[0014] (3) Add NaHCO3 solution to the petroleum ether phase in step (2), stir and extract, let stand and separate the aqueous phase and the petroleum ether phase, and collect the petroleum ether phase.

[0015] (4) Add aminoacetic acid-hydrogen chloride buffer to the petroleum ether phase from step (3), stir and extract, and after standing, separate the aqueous phase and the petroleum ether phase, and collect the petroleum ether phase.

[0016] (5) Add KH2PO4 buffer to the petroleum ether phase in step (4), stir and extract, and after standing, separate the aqueous phase and the petroleum ether phase. Collect the petroleum ether phase as solution A.

[0017] (6) Add ion exchange resin to solution A, shake, and then filter to remove the ion exchange resin to obtain solution B;

[0018] (7) Add ZGSO107 resin to solution B and stir thoroughly to obtain solution C;

[0019] (8) Dehydrate solution C by rotary evaporation under reduced pressure to obtain a paste, and dissolve the paste in methanol;

[0020] (9) The effluent was separated and purified by a semi-preparative liquid chromatography pigment analyzer, and the effluent was collected and combined. The solvent and water were removed to obtain the desired hexahydroindene compound.

[0021] Preferably, in step (6), the ion exchange resin is pretreated with ethanol before being added to solution A; and / or, in step (7), the ZGSO107 resin is pretreated with methanol before being added to solution B.

[0022] Accordingly, the hexahydroindene compound or the paste or petroleum ether phase or hexahydroindene compound obtained by the preparation method are used in the preparation of hypoglycemic drugs.

[0023] Accordingly, the hexahydroindene compound or the paste or petroleum ether phase or hexahydroindene compound obtained by the preparation method are used in the preparation of lipid-lowering drugs.

[0024] Accordingly, the hexahydroindene compound or the paste or petroleum ether phase or hexahydroindene compound obtained by the preparation method is used in the preparation of drugs for eliminating vulnerable atherosclerotic plaques and preventing and treating arteriosclerosis.

[0025] The present invention has the following beneficial effects: The present invention has isolated a new hexahydroindene compound, Macolaton, from the secondary metabolites produced by Monascus purpureus. This compound has significant effects in lowering blood sugar and blood lipids and preventing and treating arteriosclerosis, and has great pharmaceutical potential.

[0026] This invention also provides a method for preparing the compound Macolaton, which can be directly fermented with Monascus purpureus, preferably using a co-culture fermentation of Monascus purpureus and Aspergillus oryzae. Co-culture fermentation can shorten the fermentation time and increase the yield of the compound Macolaton. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the process for isolating and purifying the compound Macolaton according to the present invention;

[0028] Figure 2 This is a schematic diagram of the proton NMR spectrum of the compound Macolaton.

[0029] Figure 3 This is a schematic diagram of the carbon NMR spectrum of the compound Macolaton.

[0030] Figure 4 A schematic diagram illustrating the effect of different mixed inoculation ratios of Monascus purpureus and Aspergillus oryzae on the yield of the compound Macolaton.

[0031] Figure 5 This is a schematic diagram illustrating the effect of the compound Macolaton on FBG values ​​in hyperglycemic mice.

[0032] Figure 6 This is a schematic diagram illustrating the effect of the compound Macolaton on GSP values ​​in hyperglycemic mice.

[0033] Figure 7 This is a schematic diagram illustrating the effect of the compound Macolaton on triglycerides in hyperlipidemic rats;

[0034] Figure 8 This is a schematic diagram illustrating the effect of the compound Macolaton on high-density lipoprotein in hyperlipidemic rats.

[0035] Figure 9 This is a schematic diagram illustrating the effect of the compound Macolaton on low-density lipoprotein in hyperlipidemic rats.

[0036] Figure 10 This is a schematic diagram illustrating the effect of the compound Macolaton on the ratio of atherosclerotic plaque to arterial lumen area. Detailed Implementation

[0037] This invention provides a novel hexahydroindene compound, Macolaton, with the following structural formula:

[0038]

[0039] The present invention also provides a method for preparing the compound Macolaton. The compound Macolaton is obtained by fermentation with Monascus purpureus.

[0040] The preferred method is to prepare the compound Macolaton by co-culturing Monascus purpureus and Aspergillus oryzae, which can increase the yield of the compound Macolaton and shorten the production cycle.

[0041] This invention also provides a method for isolating and purifying the compound Macolaton, with preferred isolation and purification methods as follows: Figure 1 As shown, the specific steps include the following:

[0042] Methanol was added to the mycelium and extracted at 40°C for more than 1 hour. The liquid and solid were separated by centrifugation. The solid was extracted again using the same method. All the liquid was then collected and most of the solvent was removed by vacuum distillation to obtain a paste.

[0043] Dissolve the paste in water, adjust the pH to 3, add petroleum ether and stir to extract. After standing, separate the aqueous and petroleum ether phases and collect the petroleum ether phase. Add NaHCO3 solution (pH=9) to the petroleum ether phase and stir thoroughly to extract. Some impurities enter the aqueous phase, while Macolaton remains in the petroleum ether phase. After standing, separate the aqueous and petroleum ether phases and collect the petroleum ether phase. Add glycine-hydrogen chloride buffer (pH=12) to the petroleum ether phase and stir to extract, allowing acidic impurities to dissolve into the aqueous phase. After standing, separate the aqueous and petroleum ether phases and collect the petroleum ether phase. Add KH2PO4 buffer (pH=6) to the petroleum ether phase and stir thoroughly to extract, allowing neutral impurities to dissolve into the aqueous phase, while Macolaton remains in the petroleum ether phase, obtaining solution A.

[0044] 10g of A500Plus ion exchange resin pretreated with ethanol was added to solution A. The mixture was shaken at 40°C to adsorb salts from solution A. The resin solid was then removed by filtration, yielding solution B. ZGSO107 resin pretreated with methanol was added to solution B, and the mixture was stirred thoroughly to remove lipid-soluble impurities such as amino acids and fatty acids, yielding solution C. Most of the solvent in solution C was removed by rotary evaporation under reduced pressure, followed by dehydration with anhydrous sodium sulfate to obtain a paste. The paste was then dissolved in methanol and purified using a semi-preparative liquid chromatography-chromosome dye analyzer. The eluent containing macroton was collected and combined, and the solvent and water were removed using a nitrogen evaporator to obtain an orange solid powder. The macroton purity in the powder obtained by the above method was >95%. Ethanol and methanol pretreatment can optimize the physical properties of the resin, remove impurities, activate exchange sites, and improve the exchange separation efficiency and effect.

[0045] The compound Macolaton can effectively lower blood sugar and blood lipids, and eliminate atherosclerotic plaques, and has potential pharmaceutical applications.

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0047] Example 1: Preparation, isolation, and purification of compound Macolaton

[0048] The purple Monascus purpureus strain ATCC 96218 was selected (it should be noted that ATCC 96218 is not the only strain that can be fermented to prepare the compound Macolaton; the inventors tested various commercially available Monascus strains, and all of them could be fermented to obtain the compound Macolaton using the method of this invention). Approximately 0.1 g of mycelium was inoculated into 50 mL of spore culture medium and cultured at 30°C for 5 days. The supernatant was discarded, and the mycelium was gently removed and placed in 10 mL of physiological saline. The mixture was shaken for 30 min, and the spores detached from the mycelium and dissolved into the physiological saline. The mycelium was then removed by filtration.

[0049] Spore culture medium preparation method: Add 10 g / L glucose, 2 g / L NH4HPO4, 0.5 g / L MgSO4, and 0.1 g / L CaCl2 to potato juice to a final volume of 1000 mL. Adjust the pH to 6.0 and sterilize at 121℃ for 20 min. Potato juice preparation method: Cut 200 g of potatoes into chunks and place them in 1.0 L of water. Boil for 20 minutes, then filter to obtain the clear liquid as potato juice.

[0050] Dilute the Monascus purpureus spore solution to 10 with sterile saline. 5 The mycelium was obtained by inoculating 2 mL of diluted spore solution into 50 mL of potato dextrose medium (PDB medium) and fermenting at 30 °C and 150 rpm for 20 days.

[0051] like Figure 1 As shown, 25 mL of 80% methanol was added to the mycelium obtained from fermentation in 0.5 g of PDB medium, and the mixture was extracted at 40 °C for 1 h. The liquid and solid were separated by centrifugation, and the solid was extracted again using the same method. All liquids were collected and most of the solvent was removed by vacuum distillation, yielding 130 mg of a paste containing Macolaton. The paste obtained at this time contained a large amount of red yeast rice pigment, which needed to be isolated and purified before the structure of the desired compound Macolaton could be identified.

[0052] The paste was further purified using a multi-stage separation and purification method. 130 mg of the paste containing Macolaton was completely dissolved in 15 mL of water, and the pH was adjusted to 3 with HCl. Then, 25 mL of petroleum ether was added and the mixture was stirred and extracted for 60 min. After standing, the aqueous and petroleum ether phases were separated. The petroleum ether phase was collected, and 25 mL of NaHCO3 solution (pH = 9) was added. The mixture was stirred and extracted for 60 min, allowing some impurities to enter the aqueous phase, while Macolaton remained in the petroleum ether phase. The aqueous and petroleum ether phases were separated after standing, and the petroleum ether phase was collected. 25 mL of glycine-hydrogen chloride buffer (pH = 12) was added to the petroleum ether phase, and the mixture was stirred and extracted for 60 min to dissolve the acidic impurities into the aqueous phase. After standing, the aqueous and petroleum ether phases were separated, and the petroleum ether phase was collected. Add 25 mL of KH2PO4 buffer (pH=6) to the petroleum ether phase, stir thoroughly and extract for 60 min to dissolve neutral impurities into the aqueous phase, while Macolaton remains in the petroleum ether phase. Allow to stand to separate the aqueous and petroleum ether phases. The petroleum ether phase at this point is solution A.

[0053] 10g of A500Plus ion exchange resin pretreated with ethanol was added to solution A, and the mixture was shaken at 40℃ for 10 min to adsorb salts from solution A. The resin solid was removed by filtration, yielding solution B. 5g of ZGSO107 resin pretreated with methanol was added to solution B, and the mixture was stirred thoroughly to remove lipid-soluble impurities such as amino acids and fatty acids, yielding solution B. Most of the solvent in solution B was removed by rotary evaporation under reduced pressure, and the solution was dehydrated with anhydrous sodium sulfate to obtain a paste. The paste was dissolved in 1mL of methanol and purified using a semi-preparative liquid chromatography-chromosome analyzer. The eluent containing macroton was collected and combined, and the solvent and water were removed by nitrogen blowing to obtain an orange solid powder. The sample was analyzed by HPLC to obtain a liquid chromatogram, and the macroton purity in the sample was calculated to be >95% based on the peak area.

[0054] The compound Macolaton was determined by nuclear magnetic resonance. 1 H and 13 Chemical shifts of C (as shown below) Figure 2 , Figure 3 As shown), the molecular structure of Macolaton was thus determined to be:

[0055]

[0056] Example 2: Production of compound Macolaton by co-culturing Monascus purpureus and Aspergillus oryzae

[0057] Monascus purpureus was cultured according to the method in Example 1, and the fermentation curve of the compound Macolaton was measured and plotted during the culture process. The results are as follows: Figure 4As shown in the figure, the metabolite Macolaton only appeared on day 12, reaching its maximum concentration of 0.8 μg / mL on day 18. This indicates that Macolaton is only produced in the later stages of Monascus fermentation, and its concentration is only about one-thousandth that of known Monascus pigments. The long fermentation time and low concentration obtained may be the reason why Macolaton has not been discovered until now. Furthermore, the fact that prolonged fermentation only yields low concentrations of Macolaton also hinders its widespread application.

[0058] The inventors discovered that co-culturing Monascus purpureus and Aspergillus oryzae can increase the yield of the compound Macolaton. The Monascus purpureus and Aspergillus oryzae were diluted to 10... 5 The spore solution was prepared at a concentration of 1 conidia / mL. Monascus purpureus spore solution and Aspergillus oryzae spore solution were mixed in four volume ratios: 1 mL:2 mL, 1 mL:1 mL, 2 mL:1 mL, and 2 mL:2 mL, and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of potato dextrose agar (PDB) medium. Fermentation was carried out at 30°C and 150 rpm for 16 days. The fermentation curve of the compound Macolaton was measured and plotted throughout the entire process.

[0059] The results are as follows Figure 4 As shown, Figure 4 The ratio refers to the volume ratio of Monascus purpureus to Aspergillus oryzae. Results showed that when the ratio was 2:1, Macolaton appeared on the second day of fermentation; after 14 days of fermentation, the Macolaton yield reached its maximum of 2.2 μg / mL, a 175% increase compared to the maximum yield from Monascus purpureus alone (0.8 μg / mL). Simultaneously, the fermentation period for maximum yield was reduced from 18 days to 14 days, shortening the production cycle by 22%.

[0060] Example 3: Demonstration of the blood sugar-lowering effect of the compound Macolaton

[0061] A type 2 diabetes mellitus (T2DM) mouse model was established. The animal experimental protocol was approved by the Ethics Committee of the Experimental Animal Center of Jiangnan University and conducted under the supervision of the Experimental Animal Center of Huazhong University of Science and Technology. Specific pathogen-free (SPF) grade male Kunming mice (3–4 weeks old) were purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. The animals were housed in the animal facility of the Experimental Animal Center of Huazhong University of Science and Technology, under constant temperature and humidity (24±2℃, 50%–70% humidity) and with 12-hour circulating lighting. A total of 40 mice with no significant differences were selected. After acclimatization, 8 mice were randomly selected as the control group and fed a standard diet; the other 32 mice underwent modeling treatment, with 8 mice in each group. After successful modeling, the mice were fed an experimental diet based on rodent-specific feed D12450J. Different amounts of the compound Macolaton were mixed with the basal diet to obtain the experimental diet. The feeding period was four weeks. The positive control group received metformin, a commonly used clinical hypoglycemic drug, mixed with the basal diet (150 mg / kg). The model group consisted of hyperglycemic model mice fed a normal diet without any treatment. The experimental groups are shown in Table 1. mg / kg refers to the amount of drug administered per kg of mouse body weight.

[0062] Table 1. Comparison of Grouping Schemes for Blood Glucose Lowering Experiments

[0063] 1 Blank group Unmodeled mice + normal diet 2 Model group Hyperglycemic model mice + normal diet 3 Positive control group Hyperglycemic mice + 150 mg / kg metformin + normal diet 4 High-dose group Hyperglycemic mice + 10 mg / kg Macolaton + normal diet 5 low-dose group Hyperglycemic mice + 1.0 mg / kg Macolaton + normal diet

[0064] First, mice in the model group, positive control group, high-dose group, and low-dose group were fed a high-sugar diet (75% basal diet and 25% sucrose) for four weeks to establish the model. During model establishment, mice were initially fasted but allowed free water for 12 hours, followed by intraperitoneal injection of streptozotocin (STZ) at a dose of 60 mg / kg. STZ solution was prepared by dissolving the substance in 0.1 mol / L citrate buffer (pH = 4.2) before injection. After injection, mice were fed a high-sugar diet for another 7 days to stabilize the model. The criterion for successful model establishment was a fasting blood glucose (FBG) value greater than 11.1 mmol / L.

[0065] After successful modeling, the dietary status of mice and FBG values ​​were monitored: Each group of mice was fed different diets according to Table 1 for 2 weeks, with free access to water. After 14 days, the mice were fasted for 12 hours but allowed free access to water, and blood was collected from the tail vein to record FBG values ​​and glycosylated serum protein (GSP) levels. The GSP detection kit was purchased from Nanjing Jiancheng Biotechnology Institute. FBG results are shown below. Figure 5 As shown. The changes in GSP values ​​are as follows. Figure 6 As shown.

[0066] Figure 5 The results showed that the FBG value of the control group mice remained relatively stable at 6.6 mmol / L, with good homogeneity between groups and normal blood glucose levels. The FBG value of the model group mice was significantly higher than that of the control group, reaching 19.4 mmol / L, indicating that the hyperglycemic model was successfully induced. After 4 weeks of feeding, the FBG of the model group mice reached 19.6 mmol / L, indicating that the FBG value of the T2DM hyperglycemic model mice remained at a high level without any treatment. In the positive control group mice, after 4 weeks of treatment with metformin, the PBG value decreased to 8.4 mmol / L. After 4 weeks of feeding with the low-dose Macolaton group, the FBG value of the mice decreased to 10.2 mmol / L, a decrease of 48.0% compared with the model group; after 4 weeks of feeding with the high-dose Macolaton group, the FBG value of the mice decreased to 6.7 mmol / L, almost the same as that of the control group mice, reaching the physiological value of normal mice, with a decrease of 65.8% compared with the model group. This demonstrates that the compound Macolaton has a significant hypoglycemic effect.

[0067] Glucose combines with serum proteins through a non-enzymatic reaction to form glycosylated serum protein (GSP), the amount of which is closely related to blood glucose levels. Detecting GSP values ​​can avoid errors in blood glucose values ​​caused by stress responses in mice and can more realistically reflect changes in blood glucose levels in hyperglycemic model mice. Figure 6 The results showed that the GSP content in the model group was significantly higher than that in the control group, with an increase of 94%. After 4 weeks of feeding with Macolaton feed, compared with the model group, the GSP content in the high-dose and low-dose groups decreased by 41% and 30%, respectively, proving that the compound Macolaton does indeed have a significant hypoglycemic effect.

[0068] Example 4: Demonstration of the lipid-lowering effect of compound Macolaton

[0069] The experimental rats were housed in the animal facility of the Experimental Animal Center of Huazhong University of Science and Technology under the following conditions: temperature 25±2℃, humidity 50%–70%, and 12 hours of light followed by 12 hours of darkness. The rats had free access to food and water throughout the experiment. The experimental design is shown in Table 2. Forty 8-week-old male Sprague-Dawley rats were acclimatized in the animal facility for one week, then fasted for 24 hours and weighed. They were then randomly divided into five groups according to their body weight: a blank control group, a model group, a positive control group, a high-dose Macolaton group, and a low-dose Macolaton group, with eight rats in each group (n=8). Throughout the experimental period, rats in the control group and model group received normal food and water, and were administered 200 μL of physiological saline by gavage. Rats in the high-dose Macolaton group were administered 200 μL of physiological saline containing 5 mg / kg Macolaton daily by gavage; rats in the low-dose Macolaton group were administered 200 μL of physiological saline containing 0.5 mg / kg Macolaton daily by gavage; and rats in the positive control group were administered 200 μL of physiological saline containing 10 mg / kg simvastatin daily by gavage. Water intake was stopped 2 hours before gavage. mg / kg refers to the amount of drug administered per kg of mouse body weight.

[0070] Table 2. Comparison of Grouping Schemes for Lipid-Lowering Experiments

[0071] 1 Blank group Unmodeled rats + normal diet 2 Model group Hyperlipidemia model rats + normal diet 3 Positive control group Hyperglycemic rats + 10 mg / kg simvastatin + normal diet 4 High-dose group Hyperglycemic rats + 10 mg / kg Macolaton + normal diet 5 low-dose group Hyperglycemic rats + 1.0 mg / kg Macolaton + normal diet

[0072] A high-fructose diet can lead to weight gain in animals, decrease serum high-density lipoprotein levels, and significantly increase triglyceride levels. An 8-week-old male Sprague-Dawley rat model of hyperlipidemia was established by adding 10% fructose to their drinking water. Except for the control group, all other rats were fed 10% fructose water for 12 weeks. This resulted in significant changes in fat metabolism and a significant increase in triglyceride and other indicators.

[0073] After successful modeling, each group of rats was fed different diets according to Table 2 for 2 weeks, with free access to water. After 2 weeks of treatment, the rats were fasted but allowed free water for 12 hours. Blood samples were collected via ocular sampling, centrifuged at 4℃ and 3000rpm for 10 minutes, and the supernatant plasma sample was collected and stored at -80℃ for later use. Rat plasma triglycerides were measured using a kit from Nanjing Jiancheng Technology Co., Ltd., according to the instructions.

[0074] The effects of Macolaton on triglyceride, high-density lipoprotein, and low-density lipoprotein levels in rats are as follows: Figure 7 , 8 As shown in Figures 9 and 9.

[0075] like Figure 7 , 8As shown in Figures 9 and 1, compared with the blank group, the model group rats showed significantly increased triglyceride levels, decreased high-density lipoprotein (HDL) levels, and significantly increased low-density lipoprotein (LDL) levels, indicating successful model establishment. Compared with the model group, the triglyceride levels in the high-dose Macolaton group were almost identical to those in the blank control group, indicating that a dose of 5 mg / kg Macolaton was sufficient to regulate triglyceride levels in hyperlipidemic model rats to normal levels, comparable to the treatment results of the clinical drug simvastatin (10 mg / kg). The triglyceride level in the low-dose Macolaton group was 0.9 mmol / L, a decrease of 18% compared to the model group. This demonstrates that the compound Macolaton can effectively increase HDL and decrease LDL levels. Based on these two indicators, the mechanism by which Macolaton reduces hyperlipidemia may be through regulating HDL levels to normal.

[0076] Example 5: Demonstration of the effect of compound Macolaton in eliminating vulnerable atherosclerotic plaques

[0077] All laboratory mice were housed in the animal facility of the Laboratory Animal Center at Huazhong University of Science and Technology. The temperature in the housing was controlled at 25±2℃, and the humidity at 50–70%, with alternating light and dark lighting every 12 hours. Animals had free access to food and water, and their bedding was changed daily. All handling of the mice in this study was strictly carried out in accordance with the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals" and was subject to full supervision.

[0078] ApoE- / - mice fed a high-fat diet developed numerous large atherosclerotic plaques, similar to the progression of atherosclerosis in humans. Mice in the model group were fed a high-fat diet with free access to food and water. The high-fat diet consisted of 0.5% cholesterol, 15% egg yolk powder, 5% lard, and 79.5% basal diet. After 12 weeks of continuous feeding, one mouse was randomly selected to confirm successful model establishment by observing the presence and morphology of atherosclerotic plaques.

[0079] Forty 8-week-old male ApoE- / - mice were randomly housed in cages, four mice per cage. They were acclimatized for one week and fed standard diet. Mice without abnormalities were randomly divided into a control group (n=8) and a model group (n=32). The control group was fed standard diet with free access to food and water. After confirming successful modeling in the model group, at week 13, the 32 model mice were randomly divided into four groups: model group, positive drug treatment group, high-dose Macolaton treatment group, and low-dose Macolaton treatment group, with eight mice in each group, as shown in Table 3.

[0080] Table 3. Comparison of Experimental Grouping Schemes for Eliminating Vulnerable Atherosclerotic Plaques

[0081] 1 Blank group Unmodeled mice + normal diet 2 Model group Mice with atherosclerotic plaques + normal diet 3 Positive control group Model mice + 5 mg / kg atorvastatin + normal diet 4 High-dose group Model mice + 10 mg / kg Macolaton + normal diet 5 low-dose group Model mice + 1.0 mg / kg Macolaton + normal diet

[0082] Intervention began at week 13 with gavage administration of atorvastatin at 5 mg / kg / day in the positive control group; 10 mg / kg / day and 1.0 mg / kg / day in the high-dose and low-dose Macolaton groups, respectively; and an equal volume of distilled water in the control and model groups. Administration was once daily via gavage, with free access to food and water, but fasting was required 2 hours prior to administration. The total intervention period was 12 weeks, ending at the end of week 24.

[0083] Twenty-four hours after the last drug intervention, mice in each group were anesthetized with ether and euthanized by cervical dislocation. The aorta was quickly dissected by blunt dissection of the thoracic cavity, and observed and photographed. The aorta was fixed in 4% paraformaldehyde solution. Subsequently, the tissue was dehydrated, cleared, and embedded in paraffin. The tissue was then cut into 4μm thick sections using a microtome. After drying at 50℃ on a slide for about 3 hours, the sections were stored at 4℃ for later use. The paraffin sections of the artery were baked for 10 minutes and then dewaxed with toluene and ethanol. Hematoxylin staining was performed for 5 minutes using an automated staining system. Subsequently, the sections were washed with water for 1 minute to remove excess stain, differentiated with 1% hydrochloric acid and ethanol for 5 seconds, washed with water for 10 minutes, stained with 1% eosin for 3 minutes, washed with water for 30 seconds to remove excess stain, dehydrated with ethanol solution, cleared with xylene, and fixed with neutral resin. The sections were observed and photographed using an optical microscope. The lumen and plaque areas were analyzed and statistically analyzed using the Image Pro Plus 6.0 computer image analysis system, and the percentage of plaque area to lumen area was calculated.

[0084] The quantitative evaluation results of the ratio of atherosclerotic plaque to arterial lumen area are as follows: Figure 10 The results showed that, compared with the control group, the ratio of arterial plaque to lumen area was significantly increased in the model group mice, indicating successful modeling. Compared with the model group, the ratio of plaque to lumen area was significantly decreased in the positive drug treatment group, indicating that the clinical drug atorvastatin can prevent and treat the formation of atherosclerotic plaques. The plaque to lumen area ratio in the high-dose Macolaton treatment group and the low-dose Macolaton treatment group was reduced by 46.7% and 22.2% respectively compared with the model group. This demonstrates that the compound Macolaton can effectively inhibit the increase of atherosclerotic plaque volume.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A hexahydroindene compound, characterized in that: The structural formula of the compound is: 。 2. The method for preparing the hexahydroindene compound according to claim 1, characterized in that: The method of separation and purification includes the following steps: It is obtained by isolating and purifying mycelia from Monascus purpureus fermentation, or by isolating and purifying mycelia from Monascus purpureus and Aspergillus oryzae co-culture fermentation. During co-culture fermentation, Monascus purpureus and Aspergillus oryzae are inoculated at a volume ratio of 2:1 before fermentation. (1) Add methanol to the mycelium, centrifuge to separate the liquid and solid, collect the liquid, and distill under reduced pressure to obtain a paste; (2) Dissolve the paste in water, add petroleum ether and stir to extract, then let stand and separate the aqueous phase and the petroleum ether phase, and collect the petroleum ether phase; (3) Add NaHCO3 solution to the petroleum ether phase from step (2), stir and extract, let stand and then separate the aqueous phase and the petroleum ether phase, and collect the petroleum ether phase; (4) Add glycine-hydrogen chloride buffer to the petroleum ether phase from step (3), stir and extract, and after standing, separate the aqueous phase and the petroleum ether phase, and collect the petroleum ether phase; (5) Add KH2PO4 buffer to the petroleum ether phase from step (4), stir and extract, let stand and then separate the aqueous phase and the petroleum ether phase. Collect the petroleum ether phase as solution A. (6) Add ion exchange resin to solution A, shake, and then filter to remove the ion exchange resin to obtain solution B; (7) Add ZGSO107 resin to solution B and stir thoroughly to obtain solution C; (8) Dehydrate solution C by rotary evaporation under reduced pressure to obtain a paste, and dissolve the paste in methanol; (9) The effluent was separated and purified by a semi-preparative liquid chromatography pigment analyzer, and the effluent was collected and combined. The solvent and water were removed to obtain the desired hexahydroindene compound.

3. The preparation method according to claim 2, characterized in that: In step (6), the ion exchange resin is pretreated with ethanol and then added to solution A; and / or; in step (7), the ZGSO107 resin is pretreated with methanol and then added to solution B.

4. The use of the hexahydroindene compound of claim 1 or the hexahydroindene compound obtained by the preparation method of claim 2 or 3 in the preparation of hypoglycemic drugs.

5. The use of the hexahydroindene compound of claim 1 or the hexahydroindene compound obtained by the preparation method of claim 2 or 3 in the preparation of lipid-lowering drugs.

6. The use of the hexahydroindene compound of claim 1 or the hexahydroindene compound obtained by the preparation method of claim 2 or 3 in the preparation of a drug for eliminating vulnerable atherosclerotic plaques and preventing and treating arteriosclerosis.

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