Triterpenoids in Ganoderma leukotrichum as well as preparation method and application of triterpenoids

Abstract

The invention relates to the technical field of medicines, and provides a triterpenoid compound in ganoderma leukemia as well as a preparation method and application of the triterpenoid compound. Adding pure methanol according to a solid-liquid ratio of 1: 10, extracting at 60 DEG C for 2h (hour, hour), filtering to obtain a methanol extracting solution, repeating for three times, and combining filtrates; concentrating the filtrate to 1 / 5 of the original volume at 50 DEG C, and adding distilled water with the volume fraction of 2 / 3 until the methanol concentration is 60% to obtain a preparation raw material; the preparation raw materials are subjected to coarse separation in a C18 column. The invention proves that the DM (12-O-Deacetuleuccontextin M) has antioxidant activity, can inhibit the activity of alpha-glucosidase, cholesterol esterase and pancreatic lipase, and shows a multi-target lipid-lowering potential. In a high glucose induced nematode model, the DM can be used for effectively reducing lipid accumulation and lowering the level of TG (triglyceride) and NEFA (non-esterified fatty acid), and the DM can be used for effectively reducing the level of TG (triglyceride) and NEFA (non-esterified fatty acid).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a triterpenoid compound in Ganoderma lucidum with white flesh, its preparation method, and its application. Background Technology

[0002] With changes in modern lifestyles and dietary structures, the global prevalence of hyperlipidemia continues to rise, becoming a key risk factor for a series of cardiovascular and cerebrovascular metabolic diseases, including atherosclerosis, coronary heart disease, and non-alcoholic fatty liver disease, seriously threatening human health. Currently, while commonly used lipid-lowering drugs such as statins are highly effective, long-term use may be accompanied by side effects such as liver damage and muscle pain. Therefore, exploring novel, highly effective, and low-toxicity lipid-lowering active ingredients from natural products has become a research hotspot in the fields of pharmaceutical and food science.

[0003] Reishi mushroom, a traditional macrofungus used for both food and medicine, enjoys the reputation of "immortal herb" in Asian countries, and its medicinal value has been recorded in classical medical books such as the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). Modern pharmacological research shows that the health benefits of reishi mushroom are mainly attributed to its rich content of polysaccharides, triterpenes, and peptides. Among them, reishi triterpenes are important secondary metabolites that exert the pharmacological effects of reishi mushroom, and have been proven to have various biological activities such as anti-tumor, immunomodulatory, antibacterial and antiviral, hepatoprotective, and lipid and blood sugar regulation.

[0004] However, Ganoderma triterpenes are a complex mixture, with significant differences in activity among different components. In-depth research into their specific physiological functions is crucial for elucidating the pharmacodynamic material basis of Ganoderma. This study obtained a novel triterpenoid component from the fruiting body of white-fleshed Ganoderma lucidum through systematic solvent extraction and purification using modern chromatographic techniques. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a triterpenoid compound in Ganoderma lucidum with white flesh, its preparation method, and its application.

[0006] The present invention adopts the following technical solution.

[0007] A triterpenoid compound comprising the following structural formula: .

[0008] A method for preparing the above-mentioned triterpenoid compound, comprising: Weigh out the crushed white-fleshed Ganoderma lucidum; Add pure methanol at a material-to-liquid ratio of 1:10 and extract at 60℃ for 2 hours. Filter to obtain methanol extract, repeat three times, and combine the filtrates. The filtrate was concentrated to 1 / 5 of its original volume at 50°C, and 2 / 3 of the volume of distilled water was added to make the methanol concentration 60%, thus obtaining the raw material for preparation. The raw materials were subjected to crude separation on a C18 column at a wavelength of 250 nm. The mobile phase was 40% acetonitrile containing 0.05% phosphoric acid by volume, and the flow rate was 250 mL / min to obtain crude Ganoderma triterpenes. The crude Ganoderma lucidum triterpenes were refined using a C18 column at a wavelength of 250 nm (nanometre). The mobile phase was 65% methanol containing 0.05% phosphoric acid by volume, and the flow rate was 250 mL / min. Collect the qualified segments, and then perform concentration, enrichment, and deacidification in sequence. 90% of the methanol is extracted and concentrated to dryness. Ganoderma triterpenoids were obtained.

[0009] The above-mentioned triterpenoids are used in the preparation of lipid-lowering drugs.

[0010] A pharmaceutical composition comprising the above-mentioned triterpenoid compounds.

[0011] The present invention has the following beneficial effects: 1. In vitro experiments have demonstrated that DM has antioxidant activity and can inhibit the activity of α-glucosidase, cholesterol esterase and pancreatic lipase, showing multi-target lipid-lowering potential.

[0012] 2. In a high-glucose-induced nematode model, DM effectively reduced lipid accumulation, lowered TG (triacylglycerol) and NEFA (non-esterified fatty acid) levels, improved exercise and stress resistance, and prolonged lifespan. Its lipid-lowering mechanism involves multi-pathway synergistic regulation: including upregulation of nhr-49 and fatty acid β-oxidation genes to promote lipolysis; downregulation of sbp-1 / mdt-15 and fat-5 / 6 / 7 to inhibit lipid synthesis; regulation of akt-1 / daf-16 / sod-3 to enhance antioxidant capacity; and activation of AMPK (aak-2) and 5-HT signaling (tph-1 / mod-1) to regulate energy balance. Attached Figure Description

[0013] Figure 1 The diagram shows the structure of a Ganoderma lucidum triterpenoid compound according to the present invention, wherein a is the molecular structure of DM; b is the molecular structure of Leucocontextin E; and c is the molecular structure of Leucocontextin M.

[0014] Figure 2This is a schematic diagram of the in vitro antioxidant activity of the Ganoderma lucidum triterpenoid compounds of the present invention, wherein a represents the free radical scavenging ability of the positive control Trolox ABTS; b represents the free radical scavenging ability of DM ABTS; c represents the free radical scavenging ability of the positive control Trolox DPPH; and d represents the free radical scavenging ability of DM DPPH.

[0015] Figure 3 This diagram illustrates the enzyme inhibitory activity of the Ganoderma lucidum triterpenoid compounds of the present invention, wherein a represents the inhibitory activity of acarbose (positive control) against α-glucosidase; b represents the inhibitory activity of DM against α-glucosidase; c represents the inhibitory activity of orlistat (positive control) against cholesterol esterase; d represents the inhibitory activity of DM against cholesterol esterase; e represents the inhibitory activity of orlistat (positive control) against pancreatic lipase; and f represents the inhibitory activity of DM against pancreatic lipase.

[0016] Figure 4 This is a schematic diagram of the basic physiological indicators of the nematode of the present invention, wherein a is body length; b is body width; c is body curvature; d is head swaying; and e is egg production.

[0017] Figure 5 This diagram illustrates the lifespan, stress resistance, and red percentage of Oil Red O staining in nematodes according to the present invention. In the diagram, a represents the lifespan survival curve; b represents the heat stress survival curve; c represents the oxidative stress survival curve; and d represents the percentage of red staining in Oil Red O.

[0018] Figure 6 This is a schematic diagram of nematode oil red O staining according to the present invention.

[0019] Figure 7 This is a schematic diagram of the lipid metabolites and enzyme activities of the present invention, wherein a is triglyceride; b is free fatty acid; c is malondialdehyde; d is catalase; and e is total superoxide dismutase.

[0020] Figure 8 This is a schematic diagram of the expression levels of lipid metabolism-related genes in nematodes according to the present invention, wherein a represents the nuclear receptor signaling pathway; and b represents the SREBP signaling pathway.

[0021] Figure 9 This is a schematic diagram of the expression levels of lipid metabolism-related genes in nematodes according to the present invention, where a represents the insulin signaling pathway; b represents the 5-hydroxytryptamine signaling pathway and the AMPK signaling pathway. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] This study obtained a novel triterpenoid component from the fruiting body of Ganoderma lucidum using systematic solvent extraction and modern chromatographic techniques. Caenorhabditis elegans, as a classic model organism, has been widely used in the study of lipid metabolism, the mechanism of obesity and related diseases, and drug screening due to its clear genetic background, short lifespan, ease of culture, and high conservation of metabolic pathways with mammals.

[0024] Based on the above background, this application mainly introduces three aspects: 1) the isolation and structural identification of a novel triterpenoid (12-O-Deacetylleucocontextin M, DM) from Ganoderma lucidum; 2) the evaluation of the in vitro antioxidant, hypoglycemic, and lipid-lowering bioactivity of Ganoderma lucidum triterpenoid DM; and 3) the in-depth investigation of the effects of Ganoderma lucidum triterpenoid DM on lipid metabolism and its potential mechanism of action using a high-fat diet-induced Caenorhabditis elegans model. The results of this study will provide a solid scientific basis for the development of Ganoderma lucidum triterpenoid DM as a potential lipid-lowering functional factor or drug lead compound.

[0025] The main experimental materials, reagents, and instruments used in this invention are as follows: White-fleshed Ganoderma lucidum; wild-type N2 *C. elegans* and uracil-deficient *Escherichia coli* OP50 (*E. coli* OP50) were kindly provided by Sichuan Normal University; anhydrous ethanol, chloroform, and calcium carbonate were from Chengdu Kelong Chemical Reagent Factory; phenol, sulfuric acid, phenolic acid, and hydrochloric acid were from Chengdu Kelong Chemical Co., Ltd.; sodium hypochlorite was from Tianjin Jindong Tianzheng Fine Chemical Reagent Factory; peptone, agar powder, sodium chloride, cholesterol, glucose, magnesium sulfate, and calcium chloride were from Beijing Aoboxing Biotechnology Co., Ltd.; α-glucosidase, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were also provided. ABTS), water-soluble vitamin E (Trolox), acarbose, orlistat, cholesterol esterase, sodium taurocholate, 4-nitrophenyl butyrate, Oil Red O, Shanghai Yuanye Biotechnology Co., Ltd.; BCA, SOD, CAT, NEFA, MDA, TG reagent kits, Nanjing Jiancheng Biotechnology Institute; Trizol reagent, Sangon Biotech (Shanghai) Co., Ltd.; HiScript III All-in-one RT SuperMix Perfect for qPCR, Taq Pro Universal SYBR Vazyn qPCR Master Mix, Nanjing Nuowei Biotechnology Co., Ltd.

[0026] OLYMPUS U-RFLT50 fluorescence microscope (Olympus Corporation, Japan); 3000 FA multi-functional microplate reader (Shanghai Flash Spectrum Co., Ltd.); SW-CJ-IBH ultra-clean workbench (Sujing Antai Group Co., Ltd.); BSP-150 biochemical incubator; LDZF-75KB-Ⅱ vertical autoclave; Thermo / ST16R high-speed refrigerated centrifuge (Thermo Scientific Corporation, USA); MyiQ2 real-time quantitative PCR machine (Beijing Yuanye Bole Technology Development Co., Ltd.).

[0027] Example 1 (1) Extraction of Ganoderma triterpenes The extraction method of Ganoderma lucidum triterpenes involves accurately weighing 4500g of pulverized white-fleshed Ganoderma lucidum, adding pure methanol at a material-to-liquid ratio of 1:10, and extracting at 60℃ for 2 hours. The methanol extract is obtained by filtration, and the extraction is repeated three times. The filtrates are combined. The extract is concentrated to 1 / 5 of its volume at 50℃, and 2 / 3 of the volume of distilled water is added to make the methanol concentration 60%, thus obtaining the raw material. The raw material is then subjected to coarse separation on a C18 column at a wavelength of 250nm, with a mobile phase of 40% acetonitrile containing 0.05% phosphoric acid and a flow rate of 250mL / min to obtain crude Ganoderma lucidum triterpenes. The Ganoderma lucidum triterpenes are then subjected to fine separation on a C18 column at a wavelength of 250nm, with a mobile phase of 65% methanol containing 0.05% phosphoric acid and a flow rate of 250mL / min. The qualified fraction is collected, concentrated, enriched, deacidified, eluted with 90% methanol, concentrated and evaporated to dryness, yielding 96mg of Ganoderma lucidum triterpenoid compound 1.

[0028] (2) Identification of Ganoderma triterpenes Identification was performed using a comprehensive spectroscopic approach, including mass spectrometry (MS) and one-dimensional and two-dimensional nuclear magnetic resonance (NMR) techniques. MS analysis: Electrospray ionization mass spectrometry (ESI-MS) was used in negative ion mode. The sample was dissolved in methanol and directly injected for analysis. NMR analysis: MHz NMR spectrometer was used. The sample was dissolved in deuterated dimethyl sulfoxide (DMSO-d6), and ¹H-NMR and ¹³C-NMR spectra were acquired at room temperature. Chemical shifts (δ) are expressed in ppm and calibrated with reference to the residual solvent peak (DMSO-d6: ¹H δ 2.50 ppm; ¹³C δ 39.52 ppm). All two-dimensional NMR experiments were performed using standard pulse sequences. Based on the obtained C and H spectra, the structure was finally identified using NMR databases and with reference to its physicochemical properties.

[0029] (3) Appraisal results ESI-MS analysis showed that compound 1 has the molecular formula C1. 30The presence of a molecular ion peak ((MH)- = 527 ppm) at position H48O8 in negative mode indicates a molecular weight of 528. Based on comparison with structural spectra in the literature, it is preliminarily identified as Leucocontextin E. Chemical shifts and the assignment of two-dimensional signals confirm that both compounds have the same planar structure, and the shifts in their tetracyclic systems are essentially identical, indicating consistent chirality of their tetracyclic systems. Table 1 shows that the main difference lies in the chemical shifts at positions 25, 26, and 27 in the cited literature, which are δC 133.4, 177.8, and 13.5 ppm. However, in compound 1, the shifts at positions 25, 26, and 27 are δC 129.0, 171.7, and 12.5 ppm, presumably due to the (E) configuration of the double bond at positions Δ25 and 26 in compound 1. In the literature, compounds with the (E) configuration of the double bond at positions Δ25 and 26 have chemical shifts at positions 25 and 27 of approximately 129.0, 172.0, and 12.5 ppm, respectively. Conversely, if the double bond is in the (Z) configuration, the shifts at these positions are approximately 133.4, 177.8, and 13.5 ppm. Therefore, compound 1 has the (E) configuration of the double bond at positions Δ25 and 26, and its structure is as follows: Figure 1 As shown in Figure a. To further determine the configuration of the double bond at positions Δ25 and Δ26 of compound 1, the NMR data of the acetylated product at position 12, Leucocontextin M, was also included for comparison. The main difference between compound 1 and Leucocontextin M lies in the altered chemical shifts of the carbon atoms at positions 11, 12, 13, and 18 due to the acetylation of the hydroxyl group at position 12. Conversely, since the configuration of the double bond at position 24 is identical in both compounds, the chemical shifts near all double bonds are also largely consistent.

[0030] In summary, this further confirms that the double bond of compound -1 has an (E) configuration, and it is named 12-O-Deacetylleucocontextin M, abbreviated as Ganoderma triterpenoid DM, with the structure shown below. Figure 1 As shown, the molecular formula is C 30 H 48 O8 has a molecular weight of 528.63.

[0031] Table 1. NMR Assignments of Ganoderma lucidum Triterpenoids DM Example 2 In this embodiment, the Ganoderma triterpenoids (DM) extracted and identified in Example 1 were used for in vitro lipid-lowering activity assays.

[0032] (1) ABTS free radical scavenging ability Take 15 μL of the Ganoderma lucidum triterpenoid solution obtained in Example 1 at different concentrations, add 185 μL of ABTS working solution, and react at room temperature in the dark for 30 min. After the reaction, measure the absorbance at a wavelength of 734 nm, using Trolox as a positive control and distilled water as a blank control. (2) DPPH free radical scavenging ability Take 10 μL of the Ganoderma lucidum triterpenoid solution obtained in Example 1 at different concentrations, add 190 μL of DPPH working solution, react at room temperature in the dark for 30 min, and measure the absorbance at a wavelength of 520 nm. Trolox was used as a positive control and distilled water was used as a blank control. (3) α-glucosidase inhibitory activity Take 20 μL of the Ganoderma lucidum triterpenoid solution obtained in Example 1 at different concentrations, add 50 μL of 1 U / mL α-glucosidase, mix and incubate at 37℃ in the dark for 10 min, then add 50 μL of 5 mmol / L pNPG, incubate in a water bath at 37℃ in the dark for 30 min, and stop the reaction by adding 50 μL of 0.2 mol / L Na2CO3. Immediately measure the absorbance at 405 nm. The positive control is acarbose, and the blank control is distilled water.

[0033] (4) Cholesterol esterase inhibitory activity Take 50 μL of the Ganoderma lucidum triterpenoid solution obtained in Example 1 at different concentrations, add 50 μL of 1U / mL cholesterol esterase, mix, and incubate at 37℃ in the dark for 10 min. Add 100 μL of 20 μmol / L PNPB, and react in a water bath at 37℃ in the dark for 15 min. Immediately measure the absorbance at 405 nm. Orlistat was used as the positive control, and distilled water was used as the blank control.

[0034] (5) Pancreatic lipase inhibitory activity Take 50 μL of the Ganoderma lucidum triterpenoid solution obtained in Example 1 at different concentrations, add 50 μL of 0.1 mg / mL pancreatic lipase and mix. Incubate at 37℃ in the dark for 10 min, then add 100 μL of 0.25 mg / mL PNPP and react in a water bath at 37℃ in the dark for 20 min. Immediately measure the absorbance at a wavelength of 405 nm. The positive control is orlistat, and the blank control is distilled water.

[0035] (6) Results of in vitro lipid-lowering activity assay Ganoderma lucidum triterpenoid DM showed significant antioxidant activity in in vitro experiments. Figure 2 ) and lipid-lowering enzyme inhibitory activity ( Figure 3The effect of Ganoderma lucidum triterpenoids DM was positively correlated with dosage, suggesting that it has certain in vitro lipid-lowering potential. In the ABTS and DPPH free radical scavenging experiments, the IC50 values ​​of Ganoderma lucidum triterpenoids DM were 5.59±0.62 mg / mL and 10.21±0.53 mg / mL, respectively. Although these values ​​were lower than the positive control Trolox (IC50 values ​​were 27.16±0.16 μg / mL and 57.20±4.79 μg / mL, respectively), their clear dose-dependent effect indicates that they have reliable free radical scavenging ability. Multiple studies have indicated that the antioxidant capacity of natural products is closely related to their lipid-lowering effects. For example, Wang et al. reported that Ganoderma lucidum polysaccharides can improve lipid metabolism disorders by reducing oxidative stress. The results of this study are consistent with this, suggesting that Ganoderma lucidum triterpenoids DM may protect hepatocyte and vascular endothelial function and maintain lipoprotein homeostasis through a similar mechanism. The experimental results of the in vitro antioxidant activity of Ganoderma lucidum triterpenoids are as follows: Figure 2 As shown, a) Trolox ABTS free radical scavenging ability (positive control); b) DM ABTS free radical scavenging ability; c) Trolox DPPH free radical scavenging ability (positive control); d) DM DPPH free radical scavenging ability.

[0036] Alpha-glucosidase catalyzes the breakdown of carbohydrates into glucose, promoting elevated blood glucose levels. Prolonged hyperglycemia can induce insulin resistance, thereby inhibiting adipose tissue catabolism and ultimately leading to pathological changes such as obesity and metabolic syndrome. The IC50 of Ganoderma lucidum triterpenoid DM against α-glucosidase is 3.17±0.41 mg / mL, which is significantly higher than that of acarbose (0.15±0.02 μg / mL), but still falls within the range of natural products exhibiting some inhibitory activity. For example, other fungal triterpenoid components still show significant α-glucosidase inhibitory activity at the milligram level.

[0037] Cholesterol is an essential component of cell membranes; however, excessive intake can raise blood cholesterol levels, thereby increasing the risk of cardiovascular disease. Cholesterol esterases play a crucial regulatory role in cholesterol absorption by catalyzing the hydrolysis of cholesterol esters and promoting their transport in small intestinal epithelial cells, thus being considered potential targets for lipid metabolism intervention. On the other hand, pancreatic lipase (PL) is a key enzyme responsible for the digestion of dietary fat, hydrolyzing triglycerides in the small intestine into more easily absorbed free fatty acids and monoacylglycerols. Studies have shown that PL is involved in approximately 50%–70% of dietary fat breakdown and plays a vital regulatory role in lipid absorption and energy balance, thus becoming an important target in weight management strategies. Using specific inhibitors to reduce PL activity can effectively inhibit the digestion and absorption of dietary fat, thereby helping to reduce total energy intake. Therefore, targeted inhibition of PL activity is widely considered an important strategy for treating obesity and related metabolic diseases. The IC50 values ​​of DM against cholesterol esterase and pancreatic lipase were 1.69±0.14 mg / mL and 1.65±0.23 mg / mL, respectively. Although these values ​​were lower than those of orlistat (IC50 values ​​of 13.37±1.08 μg / mL and 34.80±4.03 μg / mL, respectively), its synergistic inhibitory ability against multiple targets still has significant physiological implications. In particular, its inhibitory ability against pancreatic lipase is on the same order of magnitude as that of some previously reported plant extracts (such as tea polyphenols and garlic saponins), suggesting that it may exert a comprehensive lipid-lowering effect by inhibiting dietary fat hydrolysis and cholesterol absorption.

[0038] In summary, Ganoderma lucidum triterpenoid DM exhibits multi-target, dose-dependent lipid-lowering activity in vitro, with its mechanism of action involving multiple pathways such as anti-oxidative stress and inhibition of glycolipid digestive enzyme activity. Although its absolute activity is still lower than that of synthetic drugs, the multi-component synergistic effect and low toxicity of natural products make it a potential candidate for use in functional foods and as an adjunct to pharmaceuticals. To further evaluate its in vivo efficacy, this study will verify the lipid-lowering activity of Ganoderma lucidum triterpenoid DM in a high-lipid-induced Caenorhabditis elegans model. This model has been widely used for the preliminary evaluation of the lipid metabolism regulatory effects of natural products due to its conserved lipid metabolism pathways, high throughput, and ease of gene intervention. Through analysis of in vivo physiological indicators, lipid accumulation, and lipid metabolism gene expression, the lipid-lowering effect and mechanism of action of Ganoderma lucidum triterpenoid DM can be further clarified.

[0039] Example 3 In this embodiment, the Ganoderma triterpenoids (DM) extracted and identified in Example 1 were used to determine their effects on lipid metabolism in a high-lipid Caenorhabditis elegans model.

[0040] (1) Cultivation of nematodes and experimental grouping.

[0041] ① OP50 culture, Cryopreserved *Escherichia coli* OP50 strain was streaked onto LB solid medium for purification, and single colonies were obtained after incubation at 37°C. A single purified colony was picked and inoculated into 5 mL of LB liquid medium, and cultured on a shaker at 37°C and 180 rpm for 12–18 h until the absorbance at 600 nm was 1.0. This culture was then used as food for nematodes and inoculated onto NGM medium.

[0042] ②Nematode synchronization Once a large number of eggs and pregnant adults are present on the NGM agar plates, wash the eggs and adults in the culture medium with M9 buffer and collect them into sterile EP tubes. Centrifuge at 4000 rpm for 2 min, discard the supernatant, and retain 100 μL of the solution containing obvious nematode precipitate. Add 1 mL of lysis buffer (double-distilled water:sodium hypochlorite:sodium hydroxide = 3.5:1:0.5) to the sterile EP tube, gently shake for 3-5 min, and after the nematode corpses are completely lysed, centrifuge at 4000 rpm for 1 min. Aspirate the supernatant, add 1 mL of M9 buffer, mix well, and centrifuge at 4000 rpm for 1 min. Repeat the above operation at least three times. Collect the resuspended precipitate containing eggs and inoculate it into fresh NGM medium to complete synchronization.

[0043] ③ Experimental grouping The experiment was divided into a normal group (normal culture medium, NC), a high-fat model group (same OP50 as the NC group, CM), a positive control group (orlistat concentration of 50 μg / mL in OP50 culture medium, OST), an L group (Ganoderma lucidum triterpenoid concentration of 500 μg / mL in OP50 culture medium, L), an M group (Ganoderma lucidum triterpenoid concentration of 1000 μg / mL in OP50, M), and an H group (Ganoderma lucidum triterpenoid concentration of 2000 μg / mL in OP50 culture medium, H). Except for the NC group, all groups were conducted in a high-glucose culture medium (containing 0.1 mol / L glucose).

[0044] (2) Body length, body width, locomotion, oviposition rate and lifespan of nematodes ① Body length / body width measurement Synchronized nematodes were picked up with a nematode picking needle and placed on the culture medium of each group. After culturing for 72 h, the nematodes were transferred to empty plates with the nematode picking needle and imaged under a fluorescence microscope. The body length and body width of the nematodes were quantified using ImageJ software. Ten nematodes were measured in each group.

[0045] ②Analysis of athletic ability Using a picking needle, synchronized nematodes were picked onto culture media of each group. After culturing for 72 hours, the number of head thashes of the nematodes within 1 minute was recorded. The nematodes were then picked onto new NGM culture media plates without food, and 1 drop of M9 buffer was added. The number of head thashes of the nematodes within 1 minute was recorded and counted under a microscope.

[0046] ③ Egg production Use a nematode-picking needle to pick up the synchronized nematodes onto each group's culture medium (2 nematodes / plate, 5 plates per group). After they enter the reproductive period, transfer them to a new culture medium to allow them to lay eggs. Perform the plate transfer operation every day and record the number of eggs laid that day until the end of the reproductive period.

[0047] ④ Lifetime determination Using a nematode-picking needle, collect synchronized nematodes onto the culture medium of each group, with 30 nematodes per group and three replicates per group. The day of nematode collection is recorded as day 0, and the plates are rotated every 24 hours. Observe the nematode status and record the number of surviving, lost, and dead nematodes until all nematodes on all plates in each group have died, at which point the experiment ends.

[0048] (3) Comparison of nematode resistance ① Heat stress Using a nematode-picking needle, collect synchronized nematodes onto the culture medium of each group. After culturing for 3 days, transfer the nematodes to a new culture medium. Each group contains 30 nematodes, and each group is tested in triplicate. Incubate at a constant temperature of 35℃, observing the nematode status and recording the number of surviving, lost, and dead nematodes every 1 hour until all nematodes on all plates in each group have died, at which point the experiment is concluded.

[0049] ②Oxidative stress Dissolve the sterilized NGM medium by heating, cool to 50°C, add 3‰ hydrogen peroxide, mix well, pour into plates, and wait for solidification. Transfer nematodes of different concentrations to an oxidative stress incubator without OP50 coating, place in a 20°C constant temperature incubator, and observe nematode mortality every 0.5 h, using the same criteria as in the lifespan experiment, until all nematodes die, at which point the experiment ends.

[0050] (4) Oil Red O staining Synchronized nematodes were picked up with a picking needle and placed on culture media of each group. After culturing for 3 days, they were washed three times with M9 buffer (static precipitation method) to remove E. coli. 1 mL of M9 buffer and 50 μL of 10% formaldehyde were added for fixation. After mixing, the mixture was frozen at -80℃ for 20 minutes and then thawed at room temperature. This process was repeated three times (the last thawing was done slowly on ice). Pre-cooled M9 buffer was used for washing three times to remove formaldehyde. After cooling, the nematodes were dehydrated with 1,2-propanediol for 5 min (8000 rpm, centrifuged for 2 min). 1.5 mL of 0.5% filtered Oil Red O staining solution was added, and the mixture was stained with shaking at 37℃ and 220 rpm for 4 h (microscopic observation was possible). The nematodes were then destained with 98% and 85% 1,2-propanediol for 5 min each, followed by two final washes with M9 buffer. 2% agarose slides were prepared and placed upside down under a fluorescence microscope to observe the lipid droplets (which appeared red). ImageJ software was used to analyze the percentage of Oil Red O positive area (red area / total nematode area).

[0051] (5) Assay of lipid metabolites and enzyme activities in nematodes The synchronized nematodes were picked up with a picking needle and placed on the culture medium of each group. After culturing for 3 days, the washed-off nematodes were crushed with a tissue grinder. The total protein was extracted by BCA method after tissue grinding (BCA kit, Nanjing Jiancheng Bioengineering Institute). The TG, NEFA, MDA, CAT, SOD and other indicators of the nematodes were measured according to the kit instructions.

[0052] (6) Determination of expression of lipid metabolism-related genes Synchronized nematodes were picked up with a picking needle and placed on culture media for 3 days. After washing with M9, the nematode samples were collected, 1 mL of Trizol reagent was added, and the samples were pulverized using a tissue homogenizer to extract total RNA. The RNA was then reverse transcribed into cDNA and subjected to SYBR Green RT-qPCR. Gene sequences were retrieved from the NCBI database, and primers were designed using Primer5 software (see Table 2). qPCR conditions: 95℃ pre-denaturation for 30 s, followed by 40 cycles of 95℃ for 10 s and 60℃ for 30 s. Melting curve analysis was performed at 65℃-95℃. Act-1 was used as an internal reference gene, and relative gene expression was measured using 2... -ΔΔCt Calculations are performed. Simultaneously, a sequence listing file of primer sequences for nematode lipid metabolism-related genes should be submitted.

[0053] Table 2 Primer sequences for lipid metabolism-related genes in nematodes (7) Effects of DM on lipid metabolism in Caenorhabditis elegans (high-lipid Caenorhabditis elegans) ① Basic physiological indicators of nematodes A long-term high-sugar diet can induce lipid accumulation in *C. elegans*, and is therefore often used to construct high-fat models. For example... Figure 4As shown in a and b, although the high-sugar diet did not significantly affect the body length of nematodes (P>0.05), it significantly increased their body width (P<0.05). After intervention with orlistat and DM, the body width of nematodes was significantly reduced compared with the CM group (P<0.05).

[0054] A high-sugar diet also led to a decrease in the motility of nematodes. Compared with the NC group, the CM group showed a significant reduction in both body bending frequency and head wagging frequency (P<0.05). After treatment with orlistat and different concentrations of DM, the motility behavior of nematodes was significantly improved compared with the CM group (P<0.05), with the low-dose DM (L group) showing the most significant effect, increasing body bending and head wagging frequencies by 80.67% and 64.23%, respectively.

[0055] To investigate whether the lipid-lowering effect was related to reproductive energy consumption, we examined changes in egg production. For example... Figure 4 As shown in e, there was no significant difference in daily oviposition rate between the orlistat and DM-treated nematodes and the CM group (P>0.05), indicating that the lipid-reducing effect of DM is independent of reproduction-related energy allocation.

[0056] In addition, a high-sugar diet significantly shortens the lifespan of nematodes, such as Figure 5 As shown in a, the mean lifespan of the CM group was 8.70 ± 0.70 days, which was 23.28% shorter than that of the NC group (11.34 ± 0.69 days) (P<0.05). Orlistat (OST) and all dose groups of DM significantly prolonged lifespan (P<0.05), reaching 10.80 ± 0.56 days, 14.15 ± 0.45 days, 13.42 ± 0.53 days, and 13.27 ± 0.27 days, respectively, representing increases of 19.44%, 62.64%, 52.25%, and 52.53% compared to the CM group.

[0057] In summary, Ganoderma lucidum triterpenoid DM can effectively antagonize metabolic disorders in nematodes induced by a high-sugar diet. It not only specifically reduces lipid accumulation but also comprehensively improves the resulting decline in motor function and shortened lifespan, among other health problems, and these beneficial effects are independent of the energy expenditure associated with reproduction. Low-dose DM showed the best effect in improving motor function, while all doses exhibited highly significant and powerful effects in extending lifespan.

[0058] ② Nematode resistance like Figure 5As shown in b and c, a high-sugar diet-induced lipid metabolism disorder significantly reduced the stress resistance of nematodes. Compared with the NC group, the survival time of nematodes in the CM group was significantly shortened under heat stress and oxidative stress conditions (P<0.05), with the mean survival time decreasing from 6.54 ± 0.57 h and 2.68 ± 0.08 h to 5.11 ± 0.35 h and 2.26 ± 0.10 h, respectively, representing reductions of 21.87% and 15.67%. This result suggests that excessive lipid accumulation may reduce the body's stress resistance by promoting oxidative damage and weakening cellular stress response.

[0059] After intervention with orlistat and different concentrations of DM, the stress resistance of nematodes in all treatment groups was significantly improved (P<0.05). The low-dose DM (L group) showed the most significant effect, increasing the mean survival time under heat stress to 6.11 ± 0.54 h and 2.76 ± 0.21 h under oxidative stress, respectively, representing increases of 19.57% and 22.12% compared to the CM group. This improvement in stress resistance may be closely related to DM's regulation of lipid metabolism, reduction of lipotoxicity, and enhancement of antioxidant defense. Studies have shown that lipid metabolism and stress response are mutually regulated through shared pathways such as the insulin / IGF-1 signaling pathway, and improved lipid homeostasis helps the body cope with environmental stress.

[0060] ③Oil Red O staining Oil Red O, a fat-soluble azo dye, specifically binds to neutral lipids and cholesterol esters, allowing for qualitative assessment of lipid accumulation levels in nematodes through staining depth and distribution. Utilizing the transparent nature of *C. elegans*, the distribution of red lipid droplets within the worm can be directly observed after staining with Oil Red O. Figure 5 d and Figure 6 The results showed that the CM group had the deepest overall staining and the highest proportion of red areas in the nematodes, indicating that the high-sugar diet successfully induced lipid deposition. After intervention with orlistat and different concentrations of DM, the red color in the nematodes significantly lightened and the proportion of lipid droplets decreased, indicating that lipid accumulation was effectively alleviated. Compared with the CM group, the proportion of red areas in the OST, L, M, and H groups decreased by 32.29% (P<0.05), 46.26% (P<0.05), 33.88% (P<0.05), and 33.16% (P<0.05), respectively, with the L group showing the most significant improvement. This confirms that DM can effectively inhibit lipid accumulation induced by a high-sugar diet. The reduction in lipid droplets not only reduces the risk of lipotoxicity but may also enhance cellular stress response by improving cell membrane fluidity and energy supply.

[0061] ④ Lipid metabolites and enzyme activity Triglycerides (TG) are the main lipid form for energy storage in organisms, and changes in their levels can effectively reflect the body's lipid metabolism status. For example... Figure 7As shown in a, compared with the NC group, the TG content of nematodes in the high-sugar diet CM group was significantly increased (P<0.05), indicating successful modeling. After intervention with orlistat and different doses of DM, the TG levels in all groups were significantly lower than those in the CM group (P<0.05), with the L group showing the largest decrease, reaching 79.66%. To further evaluate the effect of DM on lipid metabolism, we measured the content of free fatty acids (NFFA) and the lipid peroxidation product malondialdehyde (MDA). Figure 7 As shown in b and c, the levels of NFFA and MDA in the CM group were significantly higher than those in the NC group (P<0.05), indicating that a high-sugar diet not only causes lipid accumulation but also leads to oxidative damage. After drug intervention, the levels of NFFA and MDA were significantly reduced (P<0.05). The L group showed the largest decrease in NFFA (62.5% lower than the CM group); orlistat and DM at all doses showed significant effects on MDA clearance, with decreases of 68.35%, 66.35%, and 67.40% in the OST, L, and M groups, respectively (P<0.05).

[0062] Obesity is often accompanied by oxidative stress, leading to dysfunction of the antioxidant system. To investigate whether diabetes mellitus (DM) synergistically regulates antioxidant capacity, we measured the activities of catalase (CAT) and superoxide dismutase (SOD). The results showed that... Figure 7 As shown in d and e, a high-sugar diet significantly inhibited the activities of CAT and SOD in nematodes (P<0.05), while orlistat and DM treatment significantly reversed this trend (P<0.05). Among them, the M group showed the most significant increase in CAT activity, increasing by 116.17% compared to the CM group; the L group showed the largest increase in SOD activity, reaching 252.48%. These results indicate that DM significantly enhances the body's antioxidant defense capacity while improving lipid metabolism.

[0063] ⑤ Expression levels of lipid metabolism-related genes The nematode nhr-49, a functional homolog of PPAR-α, directly activates the expression of key fatty acid β-oxidation enzyme genes (cpt-1, acs-2, ech-1.1) by forming a regulatory complex with mdt-15. Figure 8 As shown in Figure a, compared to the NC group, the expression levels of nhr-49, cpt-1, acs-2, and ech-1.1 were significantly downregulated in the CM group (P<0.05). After intervention with different doses of DM, except for the M and H groups where cpt-1 showed no significant change (P>0.05), the expression levels of all genes in each treatment group were significantly upregulated (P<0.05). Therefore, DM enhances the energy metabolism level of nematodes and effectively reduces lipid accumulation by activating the nhr-49 pathway and its downstream key genes for fatty acid β-oxidation (cpt-1, acs-2, ech-1.1), which provides a molecular explanation for the aforementioned decrease in triglycerides.

[0064] In nematodes, sbp-1 (a homolog of mammalian SREBP) and mdt-15 are key factors regulating lipid metabolism. sbp-1 dominates the synthesis of fatty acids and triglycerides, while mdt-15, as a key effector molecule in the SREBP pathway, maintains fatty acid homeostasis by regulating the expression of metabolic genes. Studies have shown that downregulating the expression of either gene can significantly reduce fat storage in nematodes by inhibiting anabolic pathways. Figure 8 As shown in b, compared to the NC group, the expression levels of sbp-1 and mdt-15 genes in the CM group of nematodes were significantly upregulated (P<0.05). After intervention with different doses of DM, the expression levels of sbp-1 and mdt-15 genes in the L, M, and H groups were significantly downregulated (P<0.05). In nematodes, sbp-1 affects lipid metabolism by regulating the expression of fatty acid desaturase genes (fat-5, fat-6, and fat-7). These genes encode homologous proteins of mammalian SCD1, catalyzing the rate-limiting step in the conversion of saturated fatty acids (C16:0 / C18:0) to monounsaturated fatty acids (C16:1n-7 / C18:1n-9), a process that is an important therapeutic target for metabolic diseases. Furthermore, the nuclear hormone receptor nhr-80 also maintains lipid homeostasis by regulating these desaturases. Figure 8 As shown in b, compared to the NC group, the expression levels of fat-5, fat-6, fat-7, and nhr-80 genes in the CM group were significantly upregulated (P<0.05). After intervention with different doses of DM, the expression levels of fat-5, fat-6, and fat-7 genes in the L, M, and H groups were significantly downregulated (P<0.05), but the expression level of nhr-80 did not change significantly (P>0.05). This transcriptional evidence confirms that DM can reduce lipid production by inhibiting the lipid synthesis pathway, which is consistent with the results of Oil Red O staining showing a reduction in lipid droplets and decreased levels of TG and NFFA.

[0065] In nematodes, the insulin / IGF-1 signaling pathway (IIS) regulates lipid metabolism via the daf-2 receptor and its downstream FOXO transcription factor daf-16. Glucose promotes lipid accumulation by activating the IIS pathway and subsequently inhibiting daf-16 activity. Figure 9As shown in a, a high-sugar diet significantly upregulated the expression level of the daf-2 gene in nematodes (P<0.05) and downregulated the expression level of the daf-16 gene (P<0.05). After intervention with different doses of DM, the expression level of the daf-2 gene in the L, M, and H groups was not significantly different from that in the CM group (P>0.05); the expression level of the daf-16 gene was significantly upregulated compared with the CM group (P<0.05). The key kinase akt-1 in the nematode IIS pathway (homologically related to mammalian akt) regulates metabolism and aging by phosphorylating and inhibiting the transcription factor daf-16. A high-fat diet can promote akt-1 activation, thereby inhibiting daf-16, ultimately leading to lipid accumulation and accelerated aging. The target gene sod-3, which is activated by daf-16, alleviates oxidative stress by encoding superoxide dismutase, thereby combating aging. Compared to the NC group, the AKT-1 gene expression was significantly upregulated (P<0.05) and the SOD-3 gene expression was significantly downregulated (P<0.05) in the CM group. After intervention with different concentrations of DM, the AKT-1 gene expression was significantly downregulated in the L and M groups (P<0.05), while the gene expression in the H group was not significantly downregulated (P>0.05). Meanwhile, the SOD-3 gene expression was significantly upregulated in the L, M, and H groups (P<0.05). This indicates that DM enhances antioxidant capacity by regulating the IIS pathway, providing a molecular-level explanation for how DM improves the body's stress resistance and increases survival rate under stress.

[0066] Nematode 5-HT synthesis is neuron-specific (NSM, etc., depend on tph-1 catalysis), and its defects lead to lipid accumulation; signal transduction is mediated by mod-1 ion channel receptors, resulting in neuronal hyperpolarization. Figure 9 As shown in b, a high-sugar diet downregulates the expression levels of tph-1 and mod-1 genes. After intervention with different concentrations of DM, the expression levels of tph-1 and mod-1 genes in the L, M, and H groups were significantly upregulated compared to the CM group (P<0.05), suggesting that DM may participate in energy balance regulation through neural modulation.

[0067] In *Caenorhabditis elegans*, AMPK activity mediated by the aak-2 gene (encoding a catalytic subunit homologous to mammalian AMPKα2) is a central hub of energy metabolism. It maintains energy homeostasis by inhibiting lipid synthesis and promoting fatty acid β-oxidation and glycolysis; its loss of function directly leads to metabolic disorders and shortened lifespan. Figure 9 As shown in b, a high-sugar diet significantly downregulated the expression level of the aak-2 gene (P<0.05). After intervention with different concentrations of DM, the expression level of the aak-2 gene was significantly upregulated in groups L and M (P<0.05), while the expression level of the aak-2 gene in group H was not significantly upregulated (P>0.05). This indicates that DM can promote the reprogramming of energy sensing and metabolic homeostasis, and further synergistically promote lipid breakdown and energy consumption.

[0068] In summary, DM regulates lipid metabolism through multiple pathways. On the one hand, it activates the nhr-49-mediated fatty acid oxidation and AMPK energy sensing pathway, and on the other hand, it inhibits the sbp-1 / mdt-15-dominated lipogenesis pathway. Simultaneously, it regulates the IIS / daf-16 antioxidant signaling and 5-HT neural regulation system, thereby improving lipid homeostasis, enhancing antioxidant capacity, and prolonging lifespan. This is consistent with the aforementioned physiological, biochemical, and staining results, which together elucidate the lipid-lowering mechanism of DM.

[0069] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0070] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A triterpenoid compound, characterized in that, The structure of the compound is shown below: 。 2. A method for preparing the triterpenoid compound according to claim 1, characterized in that: Weigh out the crushed white-fleshed Ganoderma lucidum; Add pure methanol at a material-to-liquid ratio of 1:10 and extract at 60℃ for 2 hours. Filter to obtain methanol extract. Repeat three times and combine the filtrates. The filtrate was concentrated to 1 / 5 of its original volume at 50°C, and 2 / 3 of the volume of distilled water was added to make the methanol concentration 60%, thus obtaining the raw material for preparation. The raw materials were subjected to crude separation on a C18 column at a wavelength of 250 nm. The mobile phase was 40% acetonitrile containing 0.05% phosphoric acid by volume, and the flow rate was 250 mL / min, to obtain crude Ganoderma triterpenes. The crude Ganoderma lucidum triterpenes were refined using a C18 column at a wavelength of 250 nm. The mobile phase was 65% methanol containing 0.05% phosphoric acid by volume, and the flow rate was 250 mL / min. Collect the qualified segments, and then perform concentration, enrichment, and deacidification in sequence. 90% of the methanol is extracted and concentrated to dryness. Ganoderma triterpenoids were obtained.

3. The application of the triterpenoid compound according to claim 1, characterized in that, The application of the triterpenoids in the preparation of lipid-lowering drugs.

4. A pharmaceutical composition, characterized in that, It includes the triterpenoid compound as described in claim 1.

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