Application of secalonic acid compound in preparation of medicine or health care product for treating or relieving obesity and medicine for treating or relieving obesity

Abstract

The invention belongs to the technical field of biology, and discloses application of a secalonic acid compound in preparation of a medicine or a health care product for treating or relieving obesity and the medicine for treating or relieving obesity. The secalonic acid compound disclosed by the invention can effectively relieve leptin resistance, improve the sensitivity of an organism to leptin and enhance a signal transmission process after the leptin is combined with a leptin receptor, so that obesity and related abnormal glycolipid metabolism are improved, and the curative effect of the secalonic acid compound is improved. The invention has excellent potential of being applied as a medicine or a health care product for relieving and / or treating obesity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the use of ryegrassic acid compounds in the preparation of medicines or health products for treating or alleviating obesity, and to medicines for treating or alleviating obesity. Background Art

[0002] Obesity is a chronic metabolic disease caused by multiple factors, characterized by excessive body fat accumulation and / or abnormal fat distribution. Obesity is not only a direct manifestation of abnormal body fat accumulation but also a core contributor to metabolic disorders such as insulin resistance, hypertension, and non-alcoholic fatty liver disease. Approximately 80% of obese individuals experience at least one symptom of metabolic syndrome. This pathological condition is fundamentally rooted in an imbalance in energy metabolism, with underlying mechanisms involving adipose tissue dysfunction and abnormal hormone signaling, posing significant risks to human health.

[0003] Currently, GLP-1 receptor agonists such as semaglutide and lipase inhibitors such as orlistat are mainly used in clinical practice to treat obesity. Among them, although GLP-1 agonists can achieve a 15-20% weight loss by suppressing appetite, they are accompanied by up to 30% gastrointestinal side effects and rapid rebound after discontinuation of the drug; orlistat achieves weight loss by blocking fat absorption, but it may cause fat-soluble vitamin deficiency and steatorrhea, resulting in low patient compliance. More importantly, none of the existing obesity drugs address the core pathological link of obesity - leptin resistance. The failure of the leptin signal secreted by fat cells directly leads to appetite control and energy metabolism disorders, becoming a key bottleneck restricting the effectiveness of treatment.

[0004] The essence of leptin resistance lies in the fact that elevated leptin levels in obese patients are unable to effectively transmit signals, and the molecular mechanisms behind this process are multifaceted and complex. The high-fat diet in obese patients not only induces abnormal methylation in the promoter region of the hypothalamic leptin receptor (LEPR) gene, causing a sharp decrease in receptor protein expression by 50-70%, but also activates SOCS3 protein through chronic inflammation, blocking the phosphorylation process of the JAK2 / STAT3 signaling pathway, causing leptin to lose its ability to inhibit appetite-related neuropeptides such as NPY. Furthermore, obesity leads to degradation of the tight junction protein ZO-1, thereby disrupting the integrity of the blood-brain barrier. This reduces the efficiency of leptin transport to the central nervous system by over 60%, creating a vicious cycle of "hyperleptinemia, signaling impairment, and metabolic disorders."

[0005] Therefore, obtaining a leptin sensitizer that can effectively alleviate leptin resistance is of great significance for the treatment of obesity. Summary of the Invention

[0006] Based on the problem that existing obesity treatment drugs cannot effectively alleviate leptin resistance, the inventors of the present invention have discovered through extensive and in-depth research that ryegrassic acid compounds can effectively alleviate leptin resistance, increase the body's sensitivity to leptin, and enhance the signal transmission process after leptin binds to leptin receptors, thereby improving obesity and its related abnormal glucose and lipid metabolism. They have excellent potential for use as drugs or health products to alleviate and / or treat obesity.

[0007] Among them, secalin D can inhibit the differentiation process of adipocytes and reduce the accumulation of lipid droplets to reduce the formation of white adipose tissue, thereby reducing leptin secretion to alleviate hyperleptinemia, restore the sensitivity of the hypothalamus to leptin, enhance the leptin receptor post-signaling pathway, and improve the responsiveness of cells to leptin, so that leptin can better play the role of suppressing appetite and promoting energy consumption, ultimately achieving the effect of reducing weight. It can be used as a leptin sensitizer to achieve the effects of maintaining normal blood lipids and controlling body fat. The secalin D is a class of compounds with great potential for use as medicines or health products for alleviating and / or treating obesity. Based on this, the technical solution of the present invention is obtained.

[0008] The first object of the present invention is to provide a use of a rye ketone acid compound in the preparation of a drug or health product for treating or alleviating obesity. The structure of the rye ketone acid compound is shown in formula (1).

[0009]

[0010] In formula (1), R1 is a hydrogen atom or a methyl group, and R2 is a hydrogen atom or a methyl group.

[0011] Furthermore, the secalone acid compounds include one or more of secalone acid A, secalone acid B, secalone acid C, secalone acid D, secalone acid E, secalone acid F, secalone acid G, secalone acid J and secalone acid K.

[0012] The second object of the present invention is to provide the use of secalone D in the preparation of an adipocyte differentiation inhibitor.

[0013] The third object of the present invention is to provide the use of secalin D in the preparation of a drug for maintaining normal leptin levels.

[0014] The fourth object of the present invention is to provide the use of secalin D in the preparation of a leptin sensitizer.

[0015] A fifth object of the present invention is to provide a use of secalone D in the preparation of medicines or health products for maintaining normal blood sugar levels.

[0016] The sixth object of the present invention is to provide the use of ryegrassic acid D in the preparation of medicines or health products for maintaining normal blood lipid levels.

[0017] A seventh object of the present invention is to provide a drug for treating or alleviating obesity, wherein the drug comprises secalin D.

[0018] An eighth object of the present invention is to provide a method for preparing ryegrassic acid D, the preparation method specifically comprising: S1, fermenting and culturing Talaromyces stipitatus to obtain a fermentation culture solution; S2, purifying the fermentation culture solution to obtain ryegrassic acid D; wherein the deposit number of Talaromyces stipitatus is GDMCC 66111.

[0019] Furthermore, in step S1, the fermentation culture medium includes mannitol, maltose, monosodium glutamate, glucose, yeast extract, corn steep liquor, sodium dihydrogen phosphate and magnesium sulfate in a mass ratio of (1-3):(1-3):(0.5-1.5):(0.5-1.5):(0.1-0.5):(0.05-0.2):(0.01-0.1):(0.01-0.05).

[0020] Furthermore, in step S1, based on the total mass of the fermentation medium, the concentration of mannitol is 1.5wt% to 2.5wt%, the concentration of maltose is 1.5wt% to 2.5wt%, the concentration of monosodium glutamate is 0.8wt% to 1.2wt%, the concentration of glucose is 0.8wt% to 1.2wt%, the concentration of yeast extract is 0.2wt% to 0.4wt%, the concentration of corn steep liquor is 0.08wt% to 0.12wt%, the concentration of sodium dihydrogen phosphate is 0.02wt% to 0.07wt%, the concentration of magnesium sulfate is 0.02wt% to 0.04wt%, and seawater is used to make up the total mass to 100wt%.

[0021] Furthermore, in step S1, in the fermentation culture, the inoculation amount of the Talaromycesstipitatus is 1% to 3%.

[0022] Furthermore, the fermentation culture temperature is 25°C to 30°C, and the time is 14 days to 28 days.

[0023] Furthermore, in step S2, the purification process includes: taking the fermentation culture broth for crushing and ethyl acetate extraction to obtain a fermentation broth extract; taking the fermentation broth extract for extraction and chromatography purification to obtain the ryegrassic acid D.

[0024] Biological Deposits

[0025] The marine stalked basket fungus (Talaromyces stipitatus) provided by the present invention has a preservation date of April 8, 2025, a preservation number of GDMCC 66111, a preservation unit of Guangdong Provincial Microbiological Culture Collection Center, and a preservation address of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 HPLC spectrum of the yellow crystals provided in the preparation example of the present invention;

[0027] Figure 2 The yellow crystals provided in the preparation example of the present invention 1 H-NMR spectrum;

[0028] Figure 3 The yellow crystals provided in the preparation example of the present invention 13 C-NMR spectrum;

[0029] Figure 4 This is a graph showing the experimental results of the adipocyte differentiation inhibitory effect test of SAD provided in Example 1 of the present invention (scale bar: 100 μm);

[0030] Figure 5 Figure 2 is the experimental result of the test on the adipocyte differentiation inhibition effect of SAD provided in Example 1 of the present invention;

[0031] Figure 6 This is a graph showing the experimental results (weight change) of the appetite suppression test of SAD provided in Example 2 of the present invention;

[0032] Figure 7 Figure 2 (food intake) of the experimental results of the appetite suppression test for SAD provided in Example 2 of the present invention;

[0033] Figure 8 This is a graph showing the experimental results of the test on the improvement effect of SAD on obesity provided in Example 3 of the present invention (mouse weight);

[0034] Figure 9 Figure 2 (weight change rate) of the experimental results of the test on the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0035] Figure 10 Figure 3 (food intake) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0036] Figure 11 Figure 4 (blood glucose concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0037] Figure 12 Figure 5 (TC concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0038] Figure 13 Figure 6 (TG concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0039] Figure 14 Figure 7 (LDL-C concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0040] Figure 15 Figure 8 (HDL-C concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0041] Figure 16 Figure 9 (ALT concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0042] Figure 17 Figure 10 (AST concentration) is the experimental result of testing the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0043] Figure 18 Figure 11 (tissue photograph) shows the experimental results of the test on the improvement effect of SAD on obesity provided in Example 3 of the present invention;

[0044] Figure 19 Figure 12 shows the experimental results of the test on the improvement effect of SAD on obesity provided in Example 3 of the present invention (pathological section, scale is 50 μm);

[0045] Figure 20 This is one of the experimental results of the test on the enhanced leptin sensitivity of SAD provided in Example 4 of the present invention;

[0046] Figure 21 Figure 2 is the experimental result of the test on the enhanced leptin sensitivity of SAD provided in Example 4 of the present invention;

[0047] Figure 22 Figure 3 is the experimental result of the test on the effect of enhancing leptin sensitivity of SAD provided in Example 4 of the present invention;

[0048] Figure 23 Figure 4 is the experimental result of the test on the leptin sensitivity enhancement effect of SAD provided in Example 4 of the present invention;

[0049] Figure 24Figure 5 is the experimental result of the test on the enhanced leptin sensitivity of SAD provided in Example 4 of the present invention;

[0050] Figure 25 Figure 6 is the experimental result of the test on the enhanced leptin sensitivity of SAD provided in Example 4 of the present invention;

[0051] Figure 26 Figure 7 is the experimental result of the test on the enhanced leptin sensitivity of SAD provided in Example 4 of the present invention;

[0052] Figure 27 Figure 8 is the experimental result of the test on the leptin sensitivity enhancement effect of SAD provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0053] After extensive and in-depth research and numerous experiments, the inventors of the present invention have creatively discovered that secalin compounds have the biological activity of inhibiting adipocyte differentiation; they can reduce the formation of adipose tissue, reduce leptin secretion to reduce leptin levels in the body, increase the body's sensitivity to leptin, effectively alleviate leptin resistance, and restore the signal transmission process after leptin binds to the leptin receptor, so that leptin can better play its role in regulating the body's energy intake and utilization, ultimately achieving the effect of treating or alleviating obesity.

[0054] Based on the potential of secalone compounds in achieving leptin normalization and weight normalization in obese patients, the present invention first provides the use of secalone compounds in the preparation of drugs or health products for treating or alleviating obesity.

[0055] In the present invention, the secalone acid compound refers to a class of natural products produced by fungi (such as Penicillium), which has a dimeric dibenzopyrone skeleton structure. The structure of the secalone acid compound is specifically shown in formula (1):

[0056]

[0057] In formula (1), R1 is a hydrogen atom or a methyl group, and R2 is a hydrogen atom or a methyl group.

[0058] In some specific embodiments, specific examples of the secalone acid compounds include but are not limited to: one or more of secalone acid A, secalone acid B, secalone acid C, secalone acid D, secalone acid E, secalone acid F, secalone acid G, secalone acid J and secalone acid K. The structures of each substance are specifically shown in Table 1. Among them, using the same type of adipocytes under the same culture conditions as a control, the inhibition rate of rye ketoic acid A on the lipid droplet accumulation of adipocytes was 22%-27%, the inhibition rate of rye ketoic acid B on the lipid droplet accumulation of adipocytes was 25%-29%, the inhibition rate of rye ketoic acid C on the lipid droplet accumulation of adipocytes was 17%-26%, the inhibition rate of rye ketoic acid D on the lipid droplet accumulation of adipocytes was 35%-43%, the inhibition rate of rye ketoic acid E on the lipid droplet accumulation of adipocytes was 30%-36%, the inhibition rate of rye ketoic acid F on the lipid droplet accumulation of adipocytes was 32%-37%, the inhibition rate of rye ketoic acid G on the lipid droplet accumulation of adipocytes was 28%-34%, the inhibition rate of rye ketoic acid J on the lipid droplet accumulation of adipocytes was 26%-30%, and the inhibition rate of rye ketoic acid K on the lipid droplet accumulation of adipocytes was 32%-36%, showing excellent biological activity in inhibiting the formation and accumulation of lipid droplets.

[0059] Table 1.

[0060]

[0061]

[0062] In the present invention, the secalone acid compound is preferably secalone acid D. In this case, compared with other secalone acid compounds, secalone acid D has better effects of inhibiting adipocyte differentiation, reducing white adipose tissue formation, restoring the body's leptin level, increasing the body's leptin sensitivity, and achieving blood sugar, blood lipid, and body weight control, and has higher biosafety, and has better application prospects in treating or alleviating obesity.

[0063] Based on the excellent biological activity of secalone D, the present invention also provides the use of secalone D in the preparation of an adipocyte differentiation inhibitor.

[0064] In the present invention, the secalone D can reduce the formation of white adipose tissue by inhibiting the adipogenic differentiation of preadipocytes and reducing the amount of lipid droplet accumulation in cells.

[0065] Based on the excellent biological activity of secalin D, the present invention also provides the use of secalin D in the preparation of a drug for maintaining normal leptin levels.

[0066] In the present invention, the secalone acid D can improve the abnormal high leptin secretion state of the body to reduce the expression of leptin, so that the leptin level of the body drops back.

[0067] Based on the excellent biological activity of secalin D, the present invention also provides the use of secalin D in the preparation of a leptin sensitizer.

[0068] In the present invention, the secalone D can increase the body's sensitivity to leptin, so as to enhance the signal transmission process after leptin binds to the leptin receptor.

[0069] Based on the excellent biological activity of secalone D, the present invention also provides the use of secalone D in the preparation of medicines or health products for maintaining normal blood sugar levels.

[0070] In the present invention, the secalone D can improve and regulate the body's energy intake and utilization through the LEP / LEPR pathway to achieve blood sugar regulation.

[0071] Based on the excellent biological activity of secalone D, the present invention also provides the use of secalone D in the preparation of medicines or health products for maintaining normal blood lipid levels.

[0072] In the present invention, the secalone D can improve and regulate the body's energy intake and utilization through the LEP / LEPR pathway to achieve a blood lipid regulation effect.

[0073] Based on the excellent biological activity of secalin D, the present invention also provides a drug for treating or alleviating obesity. The drug specifically comprises secalin D.

[0074] In the present invention, the drug also includes a pharmaceutically acceptable excipient that has no antagonistic effect on secalin D. The pharmaceutically acceptable excipient refers to other ingredients in the drug preparation, in addition to the active ingredient, used to improve the drug preparation process, stability, safety, or efficacy. It is not directly involved in the treatment, but is crucial to the formation, absorption, release, and storage of SAD.

[0075] In some specific embodiments, specific examples of the pharmaceutically acceptable excipients include, but are not limited to, one or more of water, ethanol, microcrystalline cellulose, hypromellose, and propylene glycol.

[0076] In the present invention, specific examples of the dosage form of the drug include, but are not limited to, one or more of tablets, capsules, injections, granules, suspensions, and solutions.

[0077] Based on the above-mentioned secalone acid D, the present invention also provides a method for preparing secalone acid D. The preparation method specifically comprises: S1, fermenting and culturing Talaromyces stipitatus to obtain a fermentation broth; S2, purifying the fermentation broth to obtain the secalone acid D; wherein the deposit number of Talaromyces stipitatus is GDMCC 66111.

[0078] In the present invention, in step S1, the fermentation culture medium specifically includes mannitol, maltose, monosodium glutamate, glucose, yeast extract, corn steep liquor, sodium dihydrogen phosphate and magnesium sulfate in a mass ratio of (1-3):(1-3):(0.5-1.5):(0.5-1.5):(0.1-0.5):(0.05-0.2):(0.01-0.1):(0.01-0.05), and the mass ratio of each substance can be specifically 1:1:0.5:0.5:0.1:0.05:0.01:0.01, 2:1:1:1:0.3:0.2:0.1:0.05, 3:3:1.5:1.5:0.5:0.2:0.1:0.05 or any value therebetween.

[0079] In some specific embodiments, based on the total mass of the fermentation medium, the concentration of the mannitol is preferably 1.5wt% to 2.5wt%, specifically 1.5wt%, 1.8wt%, 1.9wt%, 2wt%, 2.3wt%, 2.5wt% or any value therebetween; the concentration of the maltose is preferably 1.5wt% to 2.5wt%, specifically 1.5wt%, 1.7wt%, 1.9wt%, 2.3wt%, 2.5wt% or any value therebetween; the concentration of the sodium glutamate is preferably 0.8wt% to 1.2wt%, specifically 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt% or any value therebetween; the concentration of the glucose is preferably 0.8wt% to 1.2wt%, specifically 0.8wt%, 0.85wt%, 0.93wt%, 1wt%, 1.05wt%, 1.2wt% or any value therebetween. The concentration of the yeast extract is preferably 0.2wt% to 0.4wt%, specifically 0.2wt%, 0.3wt%, 0.35wt%, 0.4wt% or any value therebetween; the concentration of the corn steep liquor is preferably 0.08wt% to 0.12wt%, specifically 0.08wt%, 0.09wt%, 0.1wt%, 0.11wt%, 0.12wt% or any value therebetween; the concentration of the sodium dihydrogen phosphate is preferably 0.02wt%. t% to 0.07wt%, specifically 0.02wt%, 0.03wt%, 0.05wt%, 0.06wt%, 0.07wt% or any value therebetween; the concentration of magnesium sulfate is preferably 0.02wt% to 0.04wt%, specifically 0.02wt%, 0.023wt%, 0.028wt%, 0.03wt%, 0.035wt%, 0.04 or any value therebetween; and the concentration is supplemented to 100wt% with seawater.

[0080] In the present invention, in step S1, in the fermentation culture, the inoculation amount of the marine stalked basket fungus (Talaromycesstipitatus) is preferably 1% to 3%, specifically 1%, 1.3%, 1.6%, 1.8%, 2%, 2.5%, 3% or any value therebetween.

[0081] In the present invention, in step S1, the fermentation culture conditions specifically include a temperature of preferably 25°C to 30°C, specifically 25°C, 25.6°C, 27°C, 28°C, 29°C, 30°C or any value therebetween; and a time of preferably 14d to 28d, specifically 14d, 15d, 18d, 20d, 21d, 28d or any value therebetween.

[0082] In the present invention, in step S1, the marine talaromyces is preferably activated on a plate culture medium having the same or similar nutritional components as the fermentation medium before fermentation culture until the colony grows to maturity.

[0083] In the present invention, in step S2, the purification process includes: taking the fermentation culture broth for crushing and ethyl acetate extraction to obtain a fermentation broth extract; taking the fermentation broth extract for extraction and chromatography purification to obtain the secalin D.

[0084] In the present invention, in step S2, the disruption refers to the process of destroying the cell structure to release the cell contents, which is a technical means commonly used in the prior art and is not particularly limited in the present invention.

[0085] In the present invention, in step S2, the ethyl acetate extraction treatment refers to a process of purifying secalin D by utilizing the difference in interaction force between ethyl acetate and water. It is a technical means commonly used in the prior art and is not particularly limited in the present invention.

[0086] In the present invention, in step S2, the extraction treatment refers to the process of purifying secalin D by utilizing the difference in solubility or partition coefficient of secalin D in two mutually immiscible (or slightly soluble) solvents. This is a technical means commonly used in the prior art and is not particularly limited in the present invention.

[0087] In the present invention, in step S2, the chromatographic purification refers to the process of utilizing the physical and chemical property differences between rye ketone D and other impurities to effectively separate rye ketone D from other impurities using a chromatographic column, thereby achieving the purification of rye ketone D. This is a technical means commonly used in the prior art and is not particularly limited in the present invention.

[0088] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0089] Preparation Example

[0090] This preparation example is used to illustrate the preparation of secalone acid D (SAD). The fermentation strain is Talaromyces stipitatus, which is deposited in the Guangdong Provincial Microbial Culture Collection Center with the accession number GDMCC 66111, and specifically includes:

[0091] 1. Fermentation culture of Talaromyces marinum: inoculate Talaromyces marinum into a fermentation medium at an inoculum volume of 2%, adjust the pH value of the culture solution to 7.5, culture at 25° C. for 21 days, and collect the fermentation culture fluid.

[0092] The fermentation medium includes 2wt% mannitol, 2wt% maltose, 1wt% monosodium glutamate, 1% glucose, 0.3wt% yeast extract, 0.1wt% corn steep liquor, 0.05wt% KH2PO4, 0.03wt% MgSO4·7H2O, and is made up to 100wt% with seawater.

[0093] 2. Purification of the fermentation broth: (1) Filter the fermentation broth through a Buchner funnel to obtain the fermentation broth and mycelium;

[0094] (2) The fermentation broth was mixed with ethyl acetate in a volume ratio of 1:1 and subjected to extraction treatment (the extraction treatment time was 24 h and the number of times was 3), and the extracts were combined to obtain a fermentation broth extract;

[0095] (3) The mycelium was homogenized and crushed at room temperature, and then ethyl acetate was added in a mixing ratio of 1:1 for ultrasonic extraction (the frequency of ultrasonic extraction was 70 kHz, the time was 2 h, and the number of times was 3), and the ethyl acetate was recovered to obtain a mycelium extract;

[0096] (4) After mixing the fermentation broth extract and mycelium extract, add 1000 mL of methanol-water solution (V 甲醇 :V 水 =9:1) for ultrasonic suspension and dissolution, followed by extraction with an equal volume of n-hexane (extraction time 20 min, number of times 3), collecting the methanol / water fraction to obtain a methanol-purified product;

[0097] (5) Add 1000 mL of methanol-water solution (V 甲醇 :V 水 =1:9) for ultrasonic suspension and dissolution, followed by adding an equal volume of dichloromethane for extraction (extraction times: 3), collecting the dichloromethane portion to obtain a dichloromethane purified product;

[0098] (6) A normal phase silica gel column (Qingdao Ocean Chemical, catalog number 20034365) was used in accordance with the instructions, and n-hexane-ethyl acetate with a volume ratio of 3:1, 2:1, and 1:1 was used as the eluent, and elution was performed at a rate of 5 mL / min for 5 column volumes. After component 6 was taken to recover the solvent, a Sephadex LH-20 column chromatography (Macklin, catalog number S822637) was used in accordance with the instructions, and methanol was used as the elution solvent at a rate of 0.2 mL / min. After collecting the eluate and performing solid-liquid separation, the precipitate was redissolved in acetone, and the yellow crystals were precipitated by standing. The yellow crystals were dried to obtain yellow crystals.

[0099] 3. Structural identification of yellow crystals: The structure of the obtained yellow crystals was identified by HPLC and NMR spectroscopy. The results are as follows: Figures 1 to 3 shown.

[0100] Depend on Figure 1 The results shown show that the peak time of yellow crystals is 11.613 min.

[0101] Depend on Figure 2 The results shown show that, 1 H-NMR (MeOD: 600MHz), δH: 7.48(2H,d,J=8.4Hz,H-4,4'), 6.65(2H,d,J=8.4Hz,H-3,3'), 3.95(2H,d,J=11.2Hz,H-13,13'), 3.5(3H,s,H -17,17'), 2.76(2H,dd,J=19.4Hz,6.5Hz,H-11), 2.44(2H,m,H-12,12'), 2.34(2H,dd,J=19.4Hz,8.5Hz,H-11'), 1.20(3H,s,H-15,15').

[0102] Depend on Figure 3 The results shown show that, 13 C-NMR (MeOD: 150MHz), δC: 186.7(C-8,8'), 178.3(C-10,10'), 170.2(C-16,1 6'), 159.0(C-2,2'), 158.6(C-6,6'), 140.3(C-4,4'), 117.5(C-5,5'), 107. 7(C-3,3'), 106.4(C-7,7'), 101.8(C-9,9'), 85.3(C-14,14'), 75.5(C-13,1 3'), 52.9(C-17,17'), 35.9(C-11,11'), 29.9(C-12,12'), 17.9(C-15,15').

[0103] comprehensive Figures 1 to 3 The results show that the yellow crystals prepared have the structural formula shown below, that is, the yellow crystals are SAD.

[0104]

[0105] Example 1

[0106] This example is used to illustrate the adipocyte differentiation inhibitory effect of SAD. The SAD prepared in the preparation example was used as an experimental reagent. The test specifically included:

[0107] (1) According to 2×10 4 3T3-L1 preadipocytes were seeded into 24-well plates and 400 μL / well of DMEM medium (Shanghai Yuanpei Biotechnology Co., Ltd., Catalog No. L120KJ) containing 10% fetal calf serum (Shanghai Fuheng Biotechnology Co., Ltd., Catalog No. FH-F100-901) and 1% penicillin-streptomycin (Xiamen Abikang Biotechnology Co., Ltd., Catalog No. ABK0005C) were added. The cells were cultured at 37°C and 5% CO2 until the cell confluence reached 100%. The cells were then grouped and treated as follows:

[0108] i. Blank control group: Add 100 μL / well of adipogenic differentiation medium and culture at 37°C, 5% CO2 for 3 days. Then, replace with maintenance medium (500 μL / well) and culture at 37°C, 5% CO2 for 7 days. The culture medium was changed every two days during the maintenance culture.

[0109] ii. Experimental group - low dose: In addition to the blank control group, cells were treated with SAD at a final concentration of 0.1 μM during differentiation and maintenance culture.

[0110] iii. Experimental group - medium dose: In addition to the blank control group, cells were treated with SAD at a final concentration of 0.3 μM during differentiation and maintenance culture.

[0111] iv. Experimental group - high dose: In addition to the blank control group, cells were treated with SAD at a final concentration of 1 μM during differentiation and maintenance culture.

[0112] The adipogenic differentiation medium was 10% FBS-DMEM culture medium containing 5 mmol / L IBMX, 10 μg / mL insulin and 1 μmol / L dexamethasone; the maintenance medium was 10% FBS-DMEM culture medium containing 10 μg / mL insulin.

[0113] (2) After the maintenance culture, the culture medium was discarded and the cells were washed twice with PBS. The cells were stained with Oil Red O staining kit (Beijing Solebow Technology Co., Ltd., catalog number G1262) according to the instructions. The stained cells were observed under a microscope and photographed. The lipid droplet accumulation was quantitatively analyzed. The results were as follows. Figure 4 and 5 shown.

[0114] Depend on Figure 4 and 5 The test results shown show that compared with the blank control group, treatment of cells with 0.3-1 μM SAD in differentiation culture and maintenance culture can effectively inhibit the adipogenic differentiation of 3T3-L1 precursor adipocytes and significantly reduce the amount of lipid droplet accumulation in the cells. It can inhibit the adipogenic differentiation of 3T3-L1 cells in a dose-dependent manner and significantly reduce the formation and accumulation of lipid droplets.

[0115] Example 2

[0116] This example is used to illustrate the appetite suppressant effect of SAD. The SAD prepared in the preparation example is used as an experimental reagent. The test specifically includes:

[0117] (1) SPF-grade C57BL / 6J mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used as experimental subjects. The mice were raised at 21-23°C with a 12-hour light-dark cycle and fed a standard diet (4% fat content) until they were 8 weeks old. The mice were randomly divided into a normal control group, an experimental group (low-dose SAD treatment, low-medium-dose SAD treatment, and high-dose SAD treatment), and a solvent control group, with 8 mice in each group. The following treatments were performed:

[0118] i. Normal control group: mice were fed a standard diet (4% fat content) at 21-23°C with a 12-hour light-dark cycle for 19 weeks;

[0119] ii. Experimental Group - Low Dose: Mice were maintained on a high-fat diet (60% fat) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. SAD was then administered daily by gavage at a dose of 1 mg / kg for 21 days, in addition to the high-fat diet.

[0120] iii. Experimental Group - Medium Dose: Mice were maintained on a high-fat diet (60% fat) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. SAD was then administered daily by gavage at a dose of 2 mg / kg for 21 days, in addition to the high-fat diet.

[0121] iv. Experimental Group - High Dose: Mice were maintained on a high-fat diet (60% fat) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. SAD was then administered daily by gavage at a dose of 4 mg / kg for 21 days, in addition to the high-fat diet.

[0122] v. Solvent control group: Mice were fed a high-fat diet (60% fat content) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. In addition to the high-fat diet, the mice were given a 0.5% sodium carboxymethylcellulose solution containing 0.5% DMSO by oral gavage daily for 21 days.

[0123] In the experimental group, SAD was diluted with 0.5% sodium carboxymethyl cellulose aqueous solution containing 0.5% DMSO before administration, and the administration volume of the experimental group and the solvent control group was the same.

[0124] (2) During the administration period, the weight changes and food intake of each group of mice were recorded regularly. The results are as follows: Figure 6 and 7 shown.

[0125] Depend on Figure 6 and 7 The test results shown show that on the 0th day of administration, the body weight of mice in the experimental group and the solvent control group was significantly higher than that in the normal control group, indicating that the high-fat diet-induced mouse model was successfully constructed; during the continuous administration period, the body weight of mice in the solvent control group was maintained at around 50 g, while the body weight of mice in the experimental group given 4 mg / kg SAD showed a downward trend, and when the dosage was 4 mg / kg, the body weight of the mice dropped to a level equivalent to that of the normal control group, and the food intake of the mice decreased significantly during the administration period; that is, SAD inhibited the appetite of mice in a dose-dependent manner, thereby achieving the effect of treating obesity.

[0126] Example 3

[0127] This example is used to illustrate the improvement effect of SAD on obesity and its safety. The SAD prepared in the preparation example was used as an experimental reagent. The test specifically included:

[0128] (1) SPF-grade C57BL / 6J mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used as experimental subjects. The mice were raised at 21-23°C with a 12-hour light-dark cycle and fed a standard diet (4% fat content) until they were 8 weeks old. The mice were randomly divided into an experimental group and a solvent control group, with 8 mice in each group, and the following treatments were performed:

[0129] i. Experimental group: Mice were fed a high-fat diet (60% fat content) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. SAD was then administered daily by gavage at a dose of 2 mg / kg for 14 days, in addition to the high-fat diet.

[0130] ii. Solvent control group: Mice were fed a high-fat diet (60% fat content) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. In addition to the high-fat diet, mice were administered a 0.5% sodium carboxymethylcellulose solution containing 0.5% DMSO by oral gavage daily for 14 days.

[0131] In the experimental group, SAD was diluted with 0.5% sodium carboxymethyl cellulose aqueous solution containing 0.5% DMSO before administration, and the administration volume of the experimental group and the solvent control group was the same.

[0132] (2) The weight changes and food intake of each group of mice were recorded regularly during the administration of the drug. The results are as follows: Figures 8-10 shown.

[0133] Depend on Figures 8-10 The test results shown show that, compared with the solvent control group, the body weight and food intake of mice decreased after 14 days of SAD treatment, indicating that SAD has the effect of reducing body weight and suppressing appetite.

[0134] (3) On the 10th day after administration, mice in the experimental group and the solvent control group were fasted for 6 hours and then intraperitoneally injected with 1 g / kg of 10% glucose solution. Blood was collected by tail cutting before injection (0 min) and at 15 min, 30 min, 60 min, 90 min, and 120 min after injection. Blood glucose was measured using a Roche blood glucose meter and blood glucose test strips. The results are as follows: Figure 11 shown.

[0135] As shown in the test results in Figure 11, compared with the solvent control group, the blood glucose concentration of mice treated with SAD was significantly reduced, indicating that SAD has the effect of lowering blood glucose.

[0136] (4) On the 14th day after administration, blood was collected from the orbits of the mice in the experimental group and the solvent control group to obtain mouse serum. The levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of each group of mice were detected using an automatic biochemical analyzer (Mindray, BS-240VET). The results are shown in Figure 4. Figures 12-17 shown.

[0137] Depend on Figures 12-17 The test results shown show that compared with the solvent control group, the blood lipid concentration of mice treated with SAD was significantly reduced, indicating that SAD has the effect of lowering blood lipids, and there was no significant difference in the ALT concentration and AST concentration of the experimental group mice, which represent indicators of liver function, indicating that SAD has no obvious effect on liver function.

[0138] (5) After the end of continuous drug administration, the mice were dissected to remove the inguinal adipose tissue (iWAT), epididymal adipose tissue (eWAT) and scapular brown adipose tissue (BAT), and photographed and pathologically sectioned. The methods and conditions used for pathological sectioning of mice in each group were consistent. The results are shown in Figure 2. Figure 18 and 19 shown.

[0139] Depend on Figure 18 and 19 The test results shown show that the white fat cells of the mice in the solvent control group became larger and expanded under the induction of a high-fat diet, while the white fat cells in the inguinal part of the mice treated with SAD were significantly reduced; the brown fat in the scapular region is heat-producing healthy fat in young mice. The HE staining results showed that the brown fat of the mice in the solvent control group was significantly whitened under the induction of a high-fat diet, and unhealthy fat droplets accumulated and expanded, while in the mice treated with SAD, the fat droplets in the brown fat cells were significantly reduced, thereby alleviating the course of brown fat whitening; the epididymal fat tissue of the mice in the dissolution control group became larger and expanded under the induction of high fat, while the white fat in the epididymis of the mice treated with SAD was significantly reduced, indicating that SAD has the effect of improving lipid metabolism function and enhancing the body's metabolic capacity.

[0140] Example 4

[0141] This example is used to illustrate the effect of SAD on enhancing leptin sensitivity. The SAD prepared in the preparation example was used as an experimental reagent. The test specifically included:

[0142] 1. Leptin secretion inhibition in SAD

[0143] (1) SPF-grade C57BL / 6J mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used as experimental subjects. The mice were raised at 21-23°C with a 12-hour light-dark cycle and fed a standard diet (4% fat content) until they were 8 weeks old. The mice were randomly divided into an experimental group and a solvent control group, with 8 mice in each group, and the following treatments were performed:

[0144] i. Experimental group: Mice were fed a high-fat diet (60% fat content) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. SAD was then administered daily by gavage at a dose of 2 mg / kg for 14 days, in addition to the high-fat diet.

[0145] ii. Solvent control group: Mice were fed a high-fat diet (60% fat content) at 21-23°C with a 12-hour light-dark cycle for 16 weeks. In addition to the high-fat diet, mice were administered a 0.5% sodium carboxymethylcellulose solution containing 0.5% DMSO by oral gavage daily for 14 days.

[0146] In the experimental group, SAD was diluted with 0.5% sodium carboxymethyl cellulose aqueous solution containing 0.5% DMSO before administration, and the administration volume of the experimental group and the solvent control group was the same.

[0147] (2) After the end of continuous drug administration, blood was collected from the orbits of mice in the experimental group and the solvent control group, and the mouse serum was obtained after centrifugation. The leptin concentration was detected using a leptin ELISA detection kit (Lianke Bio, product number EK297) according to the instructions. The results are as follows: Figure 20 shown.

[0148] Depend on Figure 20 The test results shown show that compared with the reagent control group, the serum leptin concentration of mice treated with SAD was significantly decreased, and the leptin level dropped, which is conducive to the restoration of leptin's normal regulatory ability on appetite and metabolism.

[0149] 2. The effect of SAD on enhancing leptin sensitivity depends on leptin

[0150] (1) SPF-grade Lep KO (ob / ob) male mice (purchased from Saiye (Suzhou) Biotechnology Co., Ltd.) were used as experimental subjects. The mice were raised at 21-23°C with a 12-h light-dark cycle and fed a standard diet (4% fat content) until they were 6 weeks old. The mice were randomly divided into an experimental group and a solvent control group, with 5 mice in each group, and the following treatments were performed:

[0151] i. Experimental group: Mice were maintained on a standard diet (4% fat content) at 21-23°C with a 12-hour light-dark cycle. In addition to the standard diet, SAD was administered daily by gavage at a dose of 2 mg / kg for 18 days.

[0152] ii. Solvent control group: Mice were maintained on a standard diet (4% fat content) at 21-23°C with a 12-hour light-dark cycle. In addition to the standard diet, mice were administered a 0.5% sodium carboxymethylcellulose solution containing 0.5% DMSO by oral gavage daily for 18 days.

[0153] In the experimental group, SAD was diluted with 0.5% sodium carboxymethyl cellulose aqueous solution containing 0.5% DMSO before administration, and the administration volume of the experimental group and the solvent control group was the same.

[0154] (2) During the administration period, the weight changes and food intake changes of each group of mice were recorded regularly. The results are as follows: Figure 21 and 22 shown.

[0155] Depend on Figure 21 and 22 The test results shown in the figure show that administration of 0.5% sodium carboxymethylcellulose aqueous solution containing 0.5% DMSO and SAD to Lep KO (ob / ob) mice had no significant effect on the body weight and food intake of the mice, indicating that the effect of SAD in enhancing leptin sensitivity is dependent on leptin.

[0156] (3) After the end of continuous drug administration, the mice were dissected and the inguinal adipose tissue (iWAT), epididymal adipose tissue (eWAT) and scapular brown adipose tissue (BAT) were taken out and weighed. The results are as follows: Figure 23 shown.

[0157] Depend on Figure 23 The test results shown in the figure show that the administration of 0.5% sodium carboxymethylcellulose aqueous solution containing 0.5% DMSO and SAD to Lep KO (ob / ob) mice had no significant effect on the proportion of adipose tissue in the mice, indicating that the effect of SAD in enhancing leptin sensitivity is dependent on leptin.

[0158] 3. The effect of SAD on enhancing leptin sensitivity depends on the leptin receptor

[0159] (1) SPF-grade LepR KO (db / db) male and female mice (kindly provided by the Institute of Ophthalmology, Xiamen University) were used as experimental subjects. The mice were raised at 21-23°C with a 12-h light-dark cycle and fed a standard diet (4% fat content) until they were 6 weeks old. The mice were randomly divided into an experimental group and a solvent control group, with 5 mice in each group, and the following treatments were performed:

[0160] i. Experimental Group: Mice were maintained on a standard diet (4% fat content) at 21-23°C with a 12-hour light-dark cycle. In addition to the standard diet, SAD was administered to the mice by oral gavage at a dose of 2 mg / kg daily for 22 days.

[0161] ii. Solvent control group: Mice were maintained on a standard diet (4% fat content) at 21-23°C with a 12-hour light-dark cycle. In addition to the standard diet, mice were administered a 0.5% sodium carboxymethylcellulose solution containing 0.5% DMSO by oral gavage daily for 22 days.

[0162] In the experimental group, SAD was diluted with 0.5% sodium carboxymethyl cellulose aqueous solution containing 0.5% DMSO before administration, and the administration volume of the experimental group and the solvent control group was the same.

[0163] (2) The weight changes of mice in each group were recorded regularly during the administration period. Figure 24 and 25 shown.

[0164] Depend on Figure 24 and 25 The test results shown in the figure show that administration of 0.5% sodium carboxymethylcellulose aqueous solution containing 0.5% DMSO and SAD to LepR KO (db / db) mice had no significant effect on the body weight of the mice, indicating that the effect of SAD in enhancing leptin sensitivity is dependent on the leptin receptor.

[0165] (3) After the end of continuous drug administration, the mice were dissected and the inguinal adipose tissue (iWAT), epididymal adipose tissue (eWAT) and scapular brown adipose tissue (BAT) were taken out and weighed. The results are as follows: Figure 26 and 27 shown.

[0166] Depend on Figure 26 and 27 The test results shown show that the administration of 0.5% sodium carboxymethylcellulose aqueous solution containing 0.5% DMSO and SAD to LepR KO (db / db) mice had no significant effect on the proportion of adipose tissue in the mice, indicating that the effect of SAD in enhancing leptin sensitivity is dependent on the leptin receptor.

[0167] Based on the analysis of the above experimental results, it can be seen that SAD can regulate blood leptin concentration to alleviate high-fat diet-induced hyperleptinemia by inhibiting the differentiation process of adipocytes, thereby improving the body's leptin sensitivity. It also regulates the signal transmission process after leptin binds to the leptin receptor, improves the cell's responsiveness to leptin, and further enhances the body's sensitivity to leptin, allowing leptin to better play its role in suppressing appetite and ultimately achieving the effect of reducing weight. It has excellent potential for use as a drug or health product to alleviate and / or treat obesity.

[0168] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. Use of a rye ketone acid compound in the preparation of a drug or health product for treating or alleviating obesity, characterized in that: The structure of the ryegrassic acid compound is shown in formula (1), In formula (1), R1 is a hydrogen atom or a methyl group, and R2 is a hydrogen atom or a methyl group.

2. The use of the ryegrassic acid compound according to claim 1 in the preparation of a medicine or health product for treating or alleviating obesity, characterized in that: The secalone acid compounds include one or more of secalone acid A, secalone acid B, secalone acid C, secalone acid D, secalone acid E, secalone acid F, secalone acid G, secalone acid J and secalone acid K.

3. Application of ryegrassic acid D in the preparation of adipocyte differentiation inhibitors.

4. Application of ryegrassic acid D in the preparation of drugs for maintaining normal leptin levels.

5. Application of ryegrassic acid D in the preparation of leptin sensitizer.

6. Use of ryegrassic acid D in the preparation of medicines or health products for maintaining normal blood sugar levels.

7. Use of ryegrassic acid D in the preparation of medicines or health products for maintaining normal blood lipid levels.

8. A drug for treating or alleviating obesity, characterized in that: The drug includes secalone D.

9. A method for preparing rye ketone D, characterized in that: The preparation method comprises: S1, fermenting and culturing Talaromyces stipitatus to obtain a fermentation culture solution; S2, purifying the fermentation culture solution to obtain the secalone acid D; The deposit number of the marine stipitatus Talaromyces is GDMCC 66111.

10. The method for preparing rye ketone D according to claim 9, characterized in that: In step S1, the fermentation culture medium includes mannitol, maltose, monosodium glutamate, glucose, yeast extract, corn steep liquor, sodium dihydrogen phosphate and magnesium sulfate in a mass ratio of (1-3):(1-3):(0.5-1.5):(0.5-1.5):(0.1-0.5):(0.05-0.2):(0.01-0.1):(0.01-0.05); Optionally, based on the total mass of the fermentation medium, the concentration of mannitol is 1.5 wt% to 2.5 wt%, the concentration of maltose is 1.5 wt% to 2.5 wt%, the concentration of monosodium glutamate is 0.8 wt% to 1.2 wt%, the concentration of glucose is 0.8 wt% to 1.2 wt%, the concentration of yeast extract is 0.2 wt% to 0.4 wt%, the concentration of corn steep liquor is 0.08 wt% to 0.12 wt%, the concentration of sodium dihydrogen phosphate is 0.02 wt% to 0.07 wt%, the concentration of magnesium sulfate is 0.02 wt% to 0.04 wt%, and the concentration is supplemented to 100 wt% with seawater; Optionally, in the fermentation culture, the inoculation amount of the marine stipitatus is 1% to 3%; Optionally, the fermentation culture temperature is 25-30°C and the time is 14-28 days; Optionally, in step S2, the purification treatment includes: taking the fermentation culture broth for crushing and ethyl acetate extraction treatment to obtain a fermentation broth extract; taking the fermentation broth extract for extraction treatment and chromatography purification to obtain the ryegrassic acid D.

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