Application of cholesterol sulfate in lowering cholesterol and / or lowering hyperlipidemia

Abstract

The present application relates to the field of biological medicine technology or health care products, and particularly relates to application of cholesteryl sulfate in lowering cholesterol and / or lowering hyperlipidemia. The present application finds that cholesteryl sulfate has a wide range of cholesterol regulation functions, can assist in regulating the endogenous synthesis, exogenous intake, excretion and esterification of cholesterol, and is helpful to maintain healthy plasma triglyceride, cholesterol and low density lipoprotein levels, thereby providing new health support for high-fat risk population. Cholesteryl sulfate can be used as an effective supplement of existing lipid-lowering drugs or health care products, and is expected to become an innovative health care product ingredient with auxiliary lipid-lowering function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology or health products, and in particular to the application of cholesterol sulfate in lowering cholesterol and / or lowering hyperlipidemia. Background Technology

[0002] Hyperlipidemia is a common metabolic disease characterized by elevated levels of plasma triglycerides or cholesterol, and low-density lipoprotein cholesterol (LDL-C), leading to abnormal lipid metabolism and endothelial cell loss. It is a major risk factor for many cardiovascular and cerebrovascular diseases, including atherosclerotic cardiovascular disease. Currently, with economic development and significant changes in dietary structure and lifestyle, the average lifespan of Chinese people is gradually increasing, leading to a continuous rise in the incidence of hyperlipidemia, seriously endangering human health. Although cholesterol metabolic homeostasis has been extensively studied over the past century, increasing evidence suggests that cholesterol metabolism disorders are closely related to cardiovascular and cerebrovascular diseases and other illnesses. Past research has deepened our understanding of cholesterol metabolism under physiological and pathological conditions and provided new targets and strategies for the treatment of cholesterol-related diseases. Currently, HMGCR inhibitors—statins—are widely used to treat cardiovascular diseases. However, the efficacy of statins is limited by compensatory increases in HMGCR protein. Although ezetimibe and PCSK9 inhibitors can further reduce LDL-C levels in hyperlipidemic patients taking statins, further research is needed to find more effective and safer methods with fewer side effects to lower cholesterol.

[0003] Bacteroides is a crucial genus of gut bacteria, accounting for over 40% of the total. A study published in *Nature Microbiology* in August 2022 revealed for the first time that *Bacteroides* gut bacteria are specific species responsible for sulfonating cholesterol, converting it to cholesterol sulfate (CHS) via cholesterol sulfonate transferase (SULT2B1b), elucidating the mechanism by which gut microbiota participates in cholesterol metabolism. Cholesterol sulfate is widely distributed in the human body, including the skin, adrenal glands, liver, lungs, brain, and endometrium. The concentration of cholesterol sulfate in human plasma ranges from 134 to 322 μg / mL. A study published in *Nature Communications* in July 2022 discovered for the first time the mechanism of action of cholesterol sulfate against ulcerative colitis. Cholesterol sulfate binds to the Niemann-Pick C2 protein, activating sterol regulatory element-binding protein 2 in colonic epithelial cells, promoting cholesterol biosynthesis, and thus alleviating ulcerative colitis. The feedback regulation of host cholesterol effects by cholesterol sulfate remains unclear. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide the application of cholesterol sulfate in lowering cholesterol and / or lowering hyperlipidemia.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of cholesterol sulfate as an active ingredient in the preparation of products that lower cholesterol and / or lower hyperlipidemia.

[0007] This invention has revealed that cholesterol sulfate possesses broad-spectrum cholesterol-regulating functions, assisting in the regulation of endogenous cholesterol synthesis, exogenous uptake, excretion, and esterification processes. This helps maintain healthy plasma triglyceride, cholesterol, and low-density lipoprotein cholesterol (LDL-C) levels, thus providing new health support for individuals at high risk of lipid deficiency. Cholesterol sulfate can serve as an effective supplement to existing lipid-lowering drugs or health products, and holds promise as an innovative health product ingredient with auxiliary lipid-lowering functions.

[0008] The molecular formula of the cholesterol sulfate is: C 27 H 46 O4S; molecular weight 466.7; CAS No.: 1256-86-6; its structural formula is shown below:

[0009] .

[0010] The purity of the cholesterol sulfate is preferably not less than 90%; more preferably greater than 95%.

[0011] Preferably, the product includes medicines and health products.

[0012] Preferably, the product comprises at least one of cholesterol sulfate, a salt formed from cholesterol sulfate and an acceptable base, or a water-soluble inclusion complex containing cholesterol sulfate.

[0013] Preferably, the acceptable base includes at least one of organic bases, inorganic bases, and amino acids.

[0014] The salt formed by cholesterol sulfate and an acceptable base includes sodium cholesterol sulfate.

[0015] Preferably, the water-soluble inclusion complex is an inclusion complex formed by cyclodextrin and cholesterol sulfate.

[0016] This invention prepares an inclusion complex by combining cyclodextrin and cholesterol sulfate, which increases the water solubility of cholesterol sulfate and thus improves its utilization rate.

[0017] Preferably, the method for preparing the water-soluble inclusion complex includes the following steps:

[0018] S1. Add ethanol to cyclodextrin and heat to dissolve to obtain solution A;

[0019] S2. Stir solution A, slowly add cholesterol sulfate to carry out inclusion reaction, and obtain inclusion solution;

[0020] S3. The inclusion solution obtained in step S2 is continuously stirred to remove ethanol and dried to obtain the water-soluble inclusion complex.

[0021] Preferably, the cyclodextrin includes at least one selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

[0022] More preferably, the cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0023] Preferably, the concentration of the ethanol is 95%.

[0024] Preferably, in step S1, the solution is heated to 50°C and stirred to dissolve and prepare a saturated solution.

[0025] Preferably, in step S2, solution A is placed at 40℃-60℃ and magnetically stirred.

[0026] Preferably, in step S3, magnetic stirring is used, and stirring is continued for 1-3 hours.

[0027] Preferably, the molar ratio of cyclodextrin in step S1 to cholesterol sulfate in step S2 is cyclodextrin:cholesterol sulfate = (1-3):1.

[0028] More preferably, the molar ratio of cyclodextrin in step S1 to cholesterol sulfate in step S2 is cyclodextrin:cholesterol sulfate = 1:1.

[0029] Preferably, the inclusion reaction conditions are: temperature 40-60℃, time 1-3 h.

[0030] More preferably, the inclusion reaction is carried out under the following conditions: temperature 50°C and time 2 hours.

[0031] Secondly, the present invention provides a product for lowering cholesterol and / or lowering hyperlipidemia, comprising at least one of cholesterol sulfate, a salt formed by cholesterol sulfate and an acceptable base, and a water-soluble inclusion complex containing cholesterol sulfate.

[0032] Preferably, the product includes medicines and health products.

[0033] Preferably, the product further includes excipients and / or acceptable carriers conforming to standards.

[0034] Preferably, the carrier includes a pharmaceutically acceptable carrier or a carrier acceptable in health products.

[0035] Preferably, the carrier includes at least one of a filler, a binder, a cosolvent, an adsorbent carrier, an antioxidant, an adsorbent, an osmotic pressure regulator, and a pH regulator.

[0036] Preferably, the carrier includes at least one of a lubricating component, a disintegrant component, a solubilizing component, a filler component, and a binder component.

[0037] Preferably, the lubricating components include, but are not limited to, magnesium stearate and paraffin wax.

[0038] Preferably, the disintegrating components include, but are not limited to, at least one of sodium carboxymethyl starch, microcrystalline cellulose, crospovidone, and pregelatinized starch.

[0039] Preferably, the solubilizing agent includes, but is not limited to, polyvinylpyrrolidone.

[0040] Preferably, the filler components include, but are not limited to, at least one of polyethylene glycol 4000 and starch.

[0041] Preferably, the adhesive component includes, but is not limited to, at least one of cross-linked polyvinylpyrrolidone, dextrin, starch paste, and starch.

[0042] Preferably, the product further includes at least one effective active ingredient that has an auxiliary lipid-lowering effect.

[0043] Preferably, the active ingredient with the auxiliary lipid-lowering effect includes, but is not limited to, at least one of atorvastatin, ezetimibe, rosuvastatin, probucol, and fenofibrate.

[0044] Preferably, the product can be used to help improve health conditions related to hyperlipidemia and hypercholesterolemia, such as hyperlipidemia, obesity, and fatty liver.

[0045] Preferably, the dosage form of the product includes, but is not limited to, at least one of solid dosage forms, semi-solid dosage forms, liquid dosage forms, syrups, pellets, sustained-release preparations, sustained-release preparations, emulsions, and suspensions.

[0046] Preferably, the solid dosage form includes, but is not limited to, at least one of tablets, capsules, granules, orally disintegrating tablets, sustained-release tablets, patches, microcapsules, pellets, microcapsules, and microspheres.

[0047] Preferably, the liquid dosage form includes, but is not limited to, at least one of oral liquids and liposomes.

[0048] Preferably, the product is administered orally, including but not limited to at least one dosage form selected from gummies, biscuits, jelly, tablets, capsules, teas, and powders.

[0049] The beneficial effects of this invention are as follows:

[0050] This invention has revealed that cholesterol sulfate possesses broad-spectrum cholesterol-regulating functions, assisting in the regulation of endogenous cholesterol synthesis, exogenous uptake, excretion, and esterification processes. This helps maintain healthy plasma triglyceride, cholesterol, and low-density lipoprotein levels, thus providing new health support for individuals at high risk of lipid deficiency. Cholesterol sulfate can serve as an effective supplement to existing lipid-lowering drugs or health products, and is expected to become an innovative health product ingredient with auxiliary lipid-lowering functions.

[0051] This invention also provides a composition comprising cholesterol sulfate and a water-soluble inclusion complex containing cholesterol sulfate, which can be prepared into a health product that can effectively help lower cholesterol and triglyceride levels in the blood, help maintain healthy blood lipids or reduce the risk of hyperlipidemia, and can be used in daily health care to help people maintain healthy blood lipid levels. Attached Figure Description

[0052] Figure 1 Figure 1 shows the effect of cholesterol sulfate on blood lipid levels in P407-induced mice. Figure 2 shows the serum TG measurement results; Figure 3 shows the serum TC measurement results; Figure 4 shows the serum LDL-C measurement results; Figure 5 shows the serum HDL-C measurement results; and Figure 6 shows the liver TC measurement results.

[0053] Figure 2 The graph shows the effect of cholesterol sulfate on P407-induced lipid accumulation in mouse liver.

[0054] Figure 3 The results show the effect of cholesterol sulfate on P407-induced cholesterol excretion in mice; Figure A shows the results of TC measurement in the gallbladder; Figure B shows the results of TC measurement in the large intestine contents.

[0055] Figure 4 The results show the effect of cholesterol sulfate on bile acid levels in P407-induced mice; Figure A shows the serum TBA measurement results; Figure B shows the liver TBA measurement results; Figure C shows the gallbladder TBA measurement results; and Figure D shows the large intestine contents TBA measurement results.

[0056] Figure 5 The figure shows the effect of cholesterol sulfate on the mRNA and protein of P407-induced cholesterol intestinal absorption-related indicators (NPC1L1, ACAT2) in mice.

[0057] Figure 6 The figure shows the effect of cholesterol sulfate on the mRNA and protein of P407-induced cholesterol synthesis-related indicators (SREBP2, INSIG1, HMGCR, SM, SCAP) in mice.

[0058] Figure 7 The figure shows the effect of cholesterol sulfate on P407-induced SREBP2 protein in mice.

[0059] Figure 8 The figure shows the effect of cholesterol sulfate on P407-induced CYP7A1 mRNA and protein in mouse liver.

[0060] Figure 9 The figure shows the effect of cholesterol sulfate on the mRNA and protein of cholesterol transport-related indicators (LXR, ABCG5, SRB1, LDLR) in P407-induced mice.

[0061] Figure 10 The effect of cholesterol sulfate on cholesterol levels in mice with hypercholesterolemia induced by a high-cholesterol diet is shown in the figure.

[0062] Figure 11 The figure shows the effect of cholesterol sulfate on bile acid levels in mice with hypercholesterolemia induced by a high-cholesterol diet.

[0063] Figure 12 The figure shows the effect of cholesterol sulfate on serum lipid levels in rats with dexamethasone-fructose-induced hypercholesterolemia.

[0064] Figure 13 The figure shows the effect of cholesterol sulfate on liver lipid levels in rats with dexamethasone-fructose-induced hypercholesterolemia.

[0065] Figure 14 The figure shows the effect of cholesterol sulfate on bile acid levels in dexamethasone-fructose-induced hypercholesterolemia rats.

[0066] Figure 15 The figure shows the effect of cholesterol sulfate on the protein expression levels of bile acid metabolism markers (CYP7A1, BESP, NTCP1, ASBT) in dexamethasone-fructose-induced hypercholesterolemia rats.

[0067] Figure 16 This is a phase solubility diagram of cholesterol sulfate in hydroxypropyl-β-cyclodextrin.

[0068] Figure 17 The results show the dissolution rate of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (cholesterol sulfate-hydroxypropyl-β-cyclodextrin) in artificial gastric fluid. Detailed Implementation

[0069] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0070] Unless otherwise specified, the experimental methods used in this invention are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0071] To better understand the essence of this invention, the following will combine pharmacological experiments and results on P407-induced hyperlipidemia mice, high-cholesterol diet-induced hypercholesterolemia mice, and dexamethasone and fructose-induced hypercholesterolemia rats to illustrate its role in the preparation of drugs and health products for the treatment or prevention of hyperlipidemia and hypercholesterolemia.

[0072] Example 1: Effects of cholesterol sulfate on P407-induced hyperlipidemia in mice

[0073] 1. Experimental Methods

[0074] Sixty SPF-grade male C57BL / 6j mice (22-24g) were randomly divided into six groups: normal control group, P407 group, atorvastatin group (AVT group, 10 mg / kg), ezetimibe group (EZ group, 10 mg / kg), low-dose cholesterol sulfate group (CHS-L, 100 mg / kg), and high-dose cholesterol sulfate group (CHS-H, 200 mg / kg), with 10 mice in each group. All C57BL6j mice underwent daily intraperitoneal injection of P407 400 mg / kg to induce a hyperlipidemia model, and were treated with the corresponding drug 1 hour after model establishment. The normal control group and P407 group received the corresponding drug solvent. Treatment continued for 6 days. On the seventh day, all mice were sacrificed after blood was collected via the orbital vein for analysis.

[0075] Observation indicators:

[0076] (I) Lipid-related indicators in serum, liver, bile and feces: TG, TC, HDL-C, LDL-C, TBA;

[0077] (II) Pathological examination: liver HE staining and Oil Red O staining;

[0078] (III) Expression of intestinal cholesterol absorption-related indicators mRNA and protein;

[0079] (IV) Expression of hepatic cholesterol synthesis-related indicators mRNA and protein;

[0080] (V) Expression of hepatic cholesterol metabolism-related indicators mRNA and protein;

[0081] (VI) Expression of cholesterol transport mRNA and protein in the liver.

[0082] 2. Experimental Results

[0083] (2.1) Effects on cholesterol levels in mice

[0084] like Figure 1 As shown, compared with the normal group, the serum TG, TC, serum LDL-C and liver TC levels of mice in the P407 group were significantly increased ( P The serum HDL-C level was significantly lower than 0.01, indicating a successful establishment of the hyperlipidemia model. Compared with the P407 group, the serum TG, TC, serum LDL-C, and liver TC levels were significantly lower in the AVT group, EZ group, and low- and high-dose cholesterol sulfate groups. Notably, serum HDL-C levels did not change significantly after cholesterol sulfate treatment, but showed an increasing trend after AVT treatment, suggesting that the lipid-lowering mechanism of cholesterol sulfate may differ from that of statins.

[0085] (2.2) Effects on lipid accumulation in mouse liver

[0086] Oil Red O staining results as follows Figure 2 As shown, compared with the normal group, the area of ​​Oil Red staining positive in the P407 group mice was significantly increased; compared with the P407 group, administration of AVT, EZ and cholesterol sulfate could significantly reduce the area of ​​Oil Red staining positive in the liver, indicating that cholesterol sulfate has the effect of reducing lipid deposition in the liver.

[0087] (2.3) Effects on cholesterol excretion in mice

[0088] like Figure 3 As shown, the results indicated that, compared with the normal group, P407 reduced the cholesterol content in the large intestine contents of mice; compared with the P407 group, administration of the positive control drug EZ and cholesterol sulfate significantly increased the cholesterol content in the large intestine contents of mice, suggesting that cholesterol sulfate may exert its cholesterol-lowering effect by promoting cholesterol excretion. The positive control drug AVT did not lower cholesterol by promoting cholesterol excretion, while the positive control drug EZ did lower cholesterol by promoting cholesterol excretion.

[0089] (2.4) Effects on bile acid levels in mice

[0090] like Figure 4 As shown, the results indicated that, compared with the normal group, P407 modeling increased hepatic TBA levels, suggesting that P407 may feedback-inhibit hepatic TBA synthesis. Compared with the P407 group, after administration of the positive control drug AVT, hepatic TBA levels decreased, bile TBA levels increased, but fecal TBA levels were unaffected, suggesting that AVT may promote the enterohepatic circulation of TBA. The positive control drug EZ increased TBA levels in both bile and feces, indicating a TBA-promoting effect from bile and feces. After administration of cholesterol sulfate, hepatic TBA levels decreased, while bile acid levels in bile and large intestine contents significantly increased, suggesting that cholesterol sulfate may promote cholesterol metabolism to bile acids and promote bile acid excretion, thereby exerting a lipid-lowering effect.

[0091] (2.5) Effects on mRNA and protein of cholesterol absorption in mouse intestines

[0092] like Figure 5 As shown, the results indicated that, compared with the control group, the mRNA levels of NPC1L1 and ACAT2 in the small intestine of mice in the P407 group were not significantly affected. Compared with the P407 group, the positive control drug EZ significantly downregulated the mRNA expression of NPC1L1 in the small intestine of mice, but had no significant effect on the mRNA and protein expression of ACAT2; however, cholesterol sulfate administration significantly downregulated the mRNA and protein expression of both NPC1L1 and ACAT2, suggesting that cholesterol sulfate may reduce cholesterol absorption by inhibiting cholesterol absorption and esterification, thereby playing a role in lowering cholesterol, while the positive control drug EZ only inhibited cholesterol absorption.

[0093] (2.6) Effects on cholesterol synthesis mRNA in mouse liver

[0094] like Figure 6 , 7 As shown, the results indicated that, compared with the control group, the P407 group significantly upregulated the mRNA expression levels of hepatic SREBP2, HMGCR, SM, and SCAP, downregulated the mRNA expression level of INSIG1, and reduced the translocation of SREBP2 into the nucleus. Compared with the P407 group, the positive control drug AVT significantly downregulated the mRNA expression levels of hepatic SREBP2, HMGCR, and SCAP, and promoted the translocation of SREBP2 into the nucleus. Cholesterol sulfate administration significantly downregulated the mRNA expression levels of hepatic SREBP2, HMGCR, SM, and SCAP, upregulated the mRNA expression level of INSIG1, and promoted the translocation of SREBP2 into the nucleus; suggesting that cholesterol sulfate, like the positive control drug AVT, can inhibit hepatic cholesterol synthesis.

[0095] Unlike AVT, a positive control drug that acts as an HMGCR inhibitor, cholesterol sulfate administration has a more significant effect in downregulating the mRNA expression of SREBP2 in the liver. It can comprehensively downregulate the mRNA expression levels of SREBP2, HMGCR, SM, and SCAP, upregulate the mRNA expression level of INSIG1, and promote the translocation of SREBP2 into the cell nucleus.

[0096] (2.7) Effects on hepatic cholesterol and bile acid metabolism mRNA and protein

[0097] like Figure 8As shown, the results indicated that, compared with the control group, the P407 group significantly downregulated the mRNA and protein expression levels of CYP7A1 in mouse liver. Compared with the P407 group, the positive control drug AVT had no significant effect on the mRNA and protein expression levels of CYP7A1, while cholesterol sulfate administration significantly upregulated the mRNA and protein expression levels of CYP7A1 in mouse liver, suggesting that cholesterol sulfate may exert its cholesterol-lowering effect by promoting cholesterol metabolism to bile acids.

[0098] (2.8) Effects on cholesterol transport mRNA and protein in mice

[0099] like Figure 9 As shown, the results indicated that, compared with the control group, P407 modeling significantly downregulated the mRNA expression levels of ABCG5, SRB1, and LDLR in the liver, while having no significant effect on the mRNA expression level of LXR, suggesting that P407 prevents cholesterol from entering the liver from the bloodstream and reduces its excretion into bile. Compared with the P407 group, the positive control drug AVT downregulated the mRNA expression levels of ABCG5, SRB1, and LDLR in the liver and the LDLR protein level, suggesting that it promotes the entry of cholesterol from the bloodstream into the liver and increases its excretion into bile; cholesterol sulfate administration significantly upregulated the mRNA expression levels of ABCG5, SRB1, LDLR, and LXR in the liver and the LDLR protein level, suggesting that cholesterol sulfate may promote the entry of cholesterol from the bloodstream into the liver and increase its excretion into bile.

[0100] Example 2: Effects of cholesterol sulfate on mice with high-cholesterol diet-induced hypercholesterolemia

[0101] 1. Experimental Methods

[0102] Thirty SPF-grade male Kunming mice were randomly divided into a control group (N group, n = 6) and an experimental group. The model group was fed a high-cholesterol diet (73% basal diet, 20% fat, and 7% cholesterol) for 14 days to establish the high-cholesterol mouse model. On day 7, the mice were fasted for 12 hours but allowed free access to water. Serum TC, LDL-C, and HDL-C levels were measured. The high-cholesterol mouse model was established with serum TC ≥ 2.0 mmol / L and LDL-C ≥ 1.49 mmol / L. The experimental group was then randomly divided into four subgroups: the model group (HC, n = 6), the atorvastatin group (AVT group, n = 6), and the cholesterol sulfate group (low-dose group: cholesterol sulfate treatment dose of 150 mg / kg, n = 6) and the high-dose group (cholesterol sulfate treatment dose of 300 mg / kg, n = 6). Seven days after administration, daily weight changes were recorded. Mice were fasted for 12 hours but allowed free access to water. Serum TC, LDL-C, HDL-C, and TBA levels were measured by enucleation. TC and TBA levels in the liver and feces were also measured.

[0103] 2. Experimental Results

[0104] (2.1) Effects on cholesterol levels in mice

[0105] Experimental results are as follows Figure 10 As shown, compared with the normal group, the body weight, serum TC and LDL-C levels, and fecal TC levels of mice in the HC group were significantly increased. Compared with the HC group, both the AVT group and the cholesterol sulfate group reduced the body weight, serum TC and LDL-C levels, and liver TC levels of mice with high cholesterol. The cholesterol sulfate group increased fecal TC levels and promoted cholesterol excretion from feces. This suggests that cholesterol sulfate can significantly alleviate hypercholesterolemia induced by a high-cholesterol diet in mice.

[0106] (2.4) Effects on bile acid levels in mice

[0107] Experimental results are as follows Figure 11 As shown, compared with the normal group, the HC group had increased TBA levels in both serum and liver, but significantly decreased TBA levels in feces; suggesting that increasing the host's uptake of exogenous cholesterol can adaptively promote cholesterol bile acid metabolism, but reduce bile acid excretion. In mice fed a high-cholesterol diet, cholesterol sulfate significantly decreased serum TBA, upregulated liver TBA levels, and significantly increased fecal TBA content; suggesting that cholesterol sulfate can inhibit the reabsorption of bile acids and promote their excretion, thereby promoting the host's cholesterol metabolism into TBA.

[0108] Example 3: Effects of cholesterol sulfate on dexamethasone-fructose-induced hypercholesterolemia in mice

[0109] Sixty SPF-grade male SD rats were randomly divided into four groups: a control group (N group, n = 10), a model group (M group, n = 10), an atorvastatin group (AVT group, 10 mg / kg, n = 10), a low-dose cholesterol sulfate group (CHS-L group, 50 mg / kg, n = 10), a medium-dose cholesterol sulfate group (CHS-M group, 100 mg / kg, n = 10), and a high-dose cholesterol sulfate group (CHS-H group, 200 mg / kg, n = 10). The model group animals were intraperitoneally injected with dexamethasone (DEX, 250 μg / kg, once daily) and allowed free access to 30% fructose solution for 14 days to establish the model. The other groups received the corresponding medications once daily for 14 days simultaneously with model establishment. During the experiment, rat body weight, food intake, and water intake were measured and recorded daily, and serum TG and TC levels were measured weekly via blood collection from the orbital venous plexus. After the experiment, all animals were fasted but allowed free access to water for 12 hours. Serum TC, LDL-C, HDL-C, and TBA levels in rats were measured via blood collection from the abdominal aorta. Simultaneously, TC and TBA levels in the liver and feces were measured. The experimental results are as follows: Figure 12-15 As shown.

[0110] like Figure 12-13 As shown, compared with group N, group M rats showed significantly increased serum TC and LDL-C levels and liver TC levels; compared with group M, both AVT and CHS groups reduced serum TC and LDL-C levels and liver TC levels in rats; suggesting that cholesterol sulfate can significantly alleviate dexamethasone-fructose-induced hypercholesterolemia in mice.

[0111] like Figure 14 As shown, compared with the normal group, the M group had increased serum TBA levels, but significantly decreased TBA levels in the liver and feces; suggesting that dexamethasone and fructose can increase bile acid metabolism but simultaneously inhibit bile acid excretion. In dexamethasone-fructose-induced hypercholesterolemia rats, CHS administration significantly downregulated serum TBA levels and significantly upregulated fecal TBA levels, suggesting that cholesterol sulfate can inhibit bile acid reabsorption and promote its excretion, thereby reducing host cholesterol levels.

[0112] like Figure 15 As shown, compared with the normal group, the expression levels of CYP7A1, NTCP1 and BESP proteins in the liver of rats in group M were significantly reduced, while the level of ASBT protein in the small intestine was significantly increased. Compared with group M, group CHS significantly upregulated the levels of CYP7A1, NTCP1 and BESP proteins in the liver of rats and downregulated the level of ASBT protein in the small intestine, suggesting that cholesterol sulfate promotes cholesterol metabolism to bile acids and promotes bile acid excretion, thereby improving bile acid metabolism.

[0113] Example 4: Acute toxicity test of cholesterol sulfate

[0114] Experimental animals and methods:

[0115] The median lethal dose (LD50) of cholesterol sulfate was determined according to the People's Republic of China National Standard, "Chemicals: Acute Oral Toxicity Test Methods (GB / T21603–2008)". KM mice (provided by Guangdong Provincial Experimental Animal Center; hereinafter the same), SPF grade, weighing 20±2g, were randomly divided into six groups (n=10 per group, half male and half female). Mice in groups -1 to -5 were administered cholesterol sulfate via gavage at a single dose of 0.1mL / 10g body weight, with corresponding daily doses of 50.2 mg / kg, 162.0 mg / kg, 512.2 mg / kg, 1638.0 mg / kg, and 5243.6 mg / kg, respectively. The control group received an equal volume of 0.5% Tween 80.

[0116] After administration, mice had free access to food and water and were observed continuously for 14 days. Changes in body weight, food intake, and other daily living conditions, as well as mortality, were recorded every two days. Furthermore, within the first 12 hours after administration, toxic reactions, the number of deaths, and the time of death were observed and recorded every 3 hours in each group. After 12 hours of administration, these indicators were observed and recorded every 6 hours. The results are shown in Tables 1 and 2.

[0117] Table 1 Results of acute toxicity test of cholesterol sulfate in mice

[0118]

[0119] Table 2. Changes in body weight of mice in acute toxicity tests.

[0120]

[0121] The results are shown in Table 1-2. No obvious abnormalities or deaths were observed in any of the cholesterol sulfate dosage groups within 14 days. The results indicate that the maximum tolerated dose of cholesterol sulfate is greater than 5243.6 mg / kg, which is classified as a low-toxicity chemical.

[0122] Example 5: Preparation of water-soluble cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex

[0123] The method for preparing the water-soluble inclusion complex of the present invention includes the following steps:

[0124] S1. Add 95% ethanol to hydroxypropyl-β-cyclodextrin, heat to 50°C and stir until saturated to obtain solution A;

[0125] S2. Place solution A at 40℃~60℃ and stir magnetically. Slowly add cholesterol sulfate to carry out the inclusion reaction and obtain the inclusion solution.

[0126] S3. The inclusion solution obtained in step S2 is continuously magnetically stirred for 1-3 h, the ethanol is recovered, and the mixture is vacuum dried to obtain the water-soluble inclusion complex, which is called cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex.

[0127] In the above preparation conditions, the inclusion ratio of cholesterol sulfate to hydroxypropyl-β-cyclodextrin and the reaction conditions will affect the inclusion rate and the dissolution rate of cholesterol sulfate.

[0128] To optimize the preparation conditions, the present invention explored the following:

[0129] 1. Inclusion Ratio Investigation: The inclusion ratio of cholesterol sulfate to hydroxypropyl-β-cyclodextrin was determined by phase solubility method. Excess cholesterol sulfate was added to a series of different concentrations of hydroxypropyl-β-cyclodextrin, and the mixture was heated and stirred at 37℃ for 48 h. After complete stirring, the mixture was left at 37℃ for 12 h to reach dissolution equilibrium. The sample was then centrifuged, and the supernatant was filtered through a 0.22 μm microporous membrane to remove insoluble components. The concentration of cholesterol sulfate in the sample was calculated by high performance liquid chromatography (HPLC), and a phase solubility diagram was plotted.

[0130] 2. Preparation process optimization: In order to determine the optimal preparation conditions of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex, based on the fixed amount of cholesterol sulfate (5 g) and solvent (1000 mL ethanol) and magnetic stirring at 600 r / min, the feed ratio, reaction temperature and reaction time were selected as factors to be investigated. The optimal preparation conditions of cyclodextrin inclusion complex of cholesterol sulfate were screened by orthogonal experiment, and a three-factor, three-level orthogonal experiment was designed. The orthogonal experimental design of L9(33) is shown in Table 3. The orthogonal experiment was carried out according to the factor level table (Table 4), and the results and range analysis are shown in Table 4.

[0131] 3. Result determination:

[0132] (3.1) Sulfuric acid cholesterol analysis method:

[0133] Chromatographic conditions: Column: Waters Atlantis C18 (300 mm × 4.6 mm); Mobile phase: Acetonitrile (A) and 0.77 g / L ammonium acetate (B); 0-20 min: 60% A; Flow rate: 1.0 mL / min; Detection wavelength: 210 nm; Injection volume: 10 μL.

[0134] (3.2) Since cholesterol sulfate is insoluble in water but readily soluble in methanol, and the cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex is soluble in both water and methanol, this invention calculates the inclusion rate of the inclusion complex by measuring the content of cholesterol sulfate dissolved in water and methanol.

[0135] Calculate the inclusion rate and yield according to Equations 1 and 2:

[0136] Inclusion rate = 1 - (Water-soluble cholesterol sulfate content / Total cholesterol sulfate content) × 100% — Equation 1

[0137] Yield = (mass of inclusion compound / total feed amount) × 100% — Equation 2

[0138] (3.3) Determination of cholesterol sulfate dissolution: 40 mg of cholesterol sulfate, cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex, and a physical mixture (molar ratio 1:1, equivalent to 40 mg of cholesterol sulfate, both directly mixed in a mortar) were filled into gelatin capsules as samples for testing. The drug dissolution was tested according to Method II (paddle method) in Appendix 160 of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The dissolution medium was 500 mL of artificial gastric fluid at a temperature of 37±0.3℃ and a rotation speed of 100 r / min. Quantitative samples of 1 mL were taken at 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, and 120 min, and an equal volume of the same temperature medium was added simultaneously. The samples were filtered through a 0.22 μm microporous membrane, and the content of cholesterol sulfate in the samples was detected by high performance liquid chromatography. The dissolution rate was calculated using the corresponding dissolution medium as a blank. This experiment was repeated 3 times, and the average value was taken.

[0139] Table 3. Factor Level Table for Orthogonal Experiment

[0140]

[0141] Table 4 Results of Orthogonal Experimental Design

[0142]

[0143] Table 5: Determination of yield and inclusion rate of cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex

[0144]

[0145] The phase solubility experiment results are as follows Figure 16 As shown, the obtained phase solubility curves exhibit good linearity (R² = 0.9958), indicating that cholesterol sulfate and hydroxypropyl-β-cyclodextrin molecules can bind in a 1:1 molar ratio. Table 4 shows the results of orthogonal experiments and range analysis, indicating that the factors influencing inclusion complex formation are, in order of importance, A > B > C. The optimal ratios are A1, B2, and C2. Therefore, the optimal preparation conditions for the cholesterol sulfate cyclodextrin inclusion complex are: a molar ratio of cholesterol sulfate to hydroxypropyl-β-cyclodextrin of 1:1, and a reaction temperature of 50℃ for 2 h. The average inclusion rate of the optimal product is 95.35 ± 1.77% (Table 5).

[0146] The dissolution curve of the obtained optimal product is as follows: Figure 17 As shown, cholesterol sulfate raw material is insoluble in simulated gastric fluid. At 120 min, the solubility of the cholesterol sulfate-hydroxypropyl-β-cyclodextrin physical mixture in simulated gastric and intestinal fluids was 14.03%. Compared to both the cholesterol sulfate raw material and the cholesterol sulfate-hydroxypropyl-β-cyclodextrin physical mixture, the cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex achieved a solubility of nearly 73% in simulated gastric fluid at 45 min, indicating that the cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex can significantly improve the solubility of cholesterol sulfate.

[0147] Example 6: Preparation of the pharmaceutical composition

[0148] Example 1: (Tablets) Take 1000 g of cholesterol sulfate, add 250 g of crospovidone, 500 g of starch, and 250 g of microcrystalline cellulose, mix well, use 200 g of 5% (w / v) starch slurry as a binder, wet granulate, dry, add 10 g of magnesium stearate and mix well, compress into 10,000 tablets, each containing 100 mg of cholesterol sulfate, with a net weight of 0.2 g per tablet, using conventional methods. For oral administration, take 1-2 tablets twice daily for 30 days to treat hyperlipidemia. Results showed that the above tablets had a good effect on lowering serum triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0149] Example 2: (Tablets) Weigh 500 g of cholesterol sulfate, add 1000 g of alfacyclodextrin, and appropriate pharmaceutical excipients, and prepare 10,000 tablets, each containing 50 mg of cholesterol sulfate, using conventional methods. Oral administration: 1-3 tablets twice daily for 30 days, for the treatment of hyperlipidemia. Results showed that the above tablets had a good effect on lowering plasma triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0150] Example 3: (Tablets) Take 400 g of cholesterol sulfate, add 480 g of lactose and 754 g of starch, mix well, use 350 g of 7% (w / v) starch slurry as a binder, wet granulate, dry, add 16 g of magnesium stearate, mix well, and compress into 10,000 tablets, each containing 40 mg of cholesterol sulfate, with a net weight of 0.2 g per tablet. Oral administration: 4 tablets three times daily for 30 days, used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets had good effects in lowering blood cholesterol, triglycerides, or low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0151] Example 4: (Tablets) Weigh 500g of cholesterol sulfate, add appropriate excipients, and prepare tablets containing 50mg of cholesterol sulfate per tablet according to conventional methods. Oral administration: 4 tablets 3 times daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets have good effects in lowering blood cholesterol, triglycerides, or reducing low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0152] Example 5: (Tablets) Weigh 1000g of cholesterol sulfate, add appropriate excipients, and prepare tablets containing 100mg of cholesterol sulfate according to conventional methods. Oral administration: 4 tablets 3 times daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets have good effects in lowering blood cholesterol, triglycerides, or reducing low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0153] Example 6: (Tablets) Weigh 500g of cholesterol sulfate and 100g of fenofibrate, add appropriate pharmaceutical excipients, and prepare 10,000 tablets according to conventional methods. Oral administration: one tablet once daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets have good effects in lowering blood cholesterol, triglycerides, or reducing low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0154] Example 7: (Tablets) Weigh 500g of cholesterol sulfate and 100g of lovastatin, add appropriate pharmaceutical excipients, and prepare 10,000 tablets according to conventional methods. Oral administration: One tablet once daily for 30 days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets have good effects in lowering blood cholesterol, triglycerides, or reducing low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0155] Example 8: (Tablets) Weigh 500g of cholesterol sulfate and 500g of probucol, add appropriate pharmaceutical excipients, and prepare 10,000 tablets according to conventional methods. Oral administration: 1 tablet twice daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above tablets have good effects in lowering blood cholesterol, reducing low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0156] Example 9: (Capsules) Weigh 1000g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare capsules containing 100mg of cholesterol sulfate per capsule according to conventional methods. Oral administration: 4 capsules three times daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above capsules had good effects in lowering blood cholesterol, triglycerides, or low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0157] Example 10: (Capsules) Weigh 500g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare capsules containing 50mg of cholesterol sulfate per capsule according to conventional methods. Oral administration: 4 capsules three times daily for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above capsules had good effects in lowering blood cholesterol, triglycerides, or low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0158] Example 11: (Capsules) Weigh 1000g of cholesterol sulfate and 500g of niacin, add appropriate pharmaceutical excipients, and prepare capsules containing 100mg of cholesterol sulfate per capsule according to conventional methods. Oral administration: 4 capsules 3 times daily for 30 days. Used to treat hyperlipidemia, obesity, and fatty liver. Results showed that the above capsules had good effects in lowering blood cholesterol, triglycerides, or low-density lipoprotein lipids, reducing weight, and lowering transaminase levels.

[0159] Example 12: (Tablets) Weigh 1000g of cholesterol sulfate, add 3350g of hydroxypropyl-β-cyclodextrin and 2000mL of ethanol, heat at 50℃, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 225mg / g of cholesterol sulfate).

[0160] 2200g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate was weighed and formulated into 10,000 tablets, each containing 50mg of cholesterol sulfate, using conventional methods. The tablets are taken orally, 1-3 tablets twice daily for 30 days, for the treatment of metabolic diseases, particularly fatty liver. Results showed that the tablets had a good effect on lowering serum triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0161] Example 13: (Capsules) Weigh 500g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 capsules, each containing 50 mg of cholesterol sulfate, according to conventional methods. Oral administration: 3 capsules twice daily for 30 days, for the treatment of hyperlipidemia. Results showed that the above capsules had a good effect on lowering serum cholesterol and low-density lipoprotein (LDL) levels.

[0162] Example 14: (Capsules) Weigh 200g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 capsules, each containing 20mg of cholesterol sulfate, using conventional methods. Oral administration: 3 capsules twice daily for 30 days, used to treat metabolic diseases such as fatty liver. Results showed that the above capsules had good lipid-lowering and cholesterol-lowering effects.

[0163] Example 15: (Capsules) Weigh 1000g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 sustained-release capsules, each containing 100mg of cholesterol sulfate, using conventional methods. Oral administration: 3 capsules twice daily for 30 days, for the treatment of hypercholesterolemia. Results showed that the above sustained-release capsules had a good cholesterol-lowering effect.

[0164] Example 16: (Capsule) Weigh 1000g of cholesterol sulfate, add 3350g of hydroxypropyl-β-cyclodextrin and 2000mL of ethanol, heat at 50℃, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 225mg / g of cholesterol sulfate).

[0165] 2200g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate was weighed and prepared into 10,000 capsules, each containing 50mg of cholesterol sulfate, using conventional methods. The medication was taken orally, 1-3 capsules twice daily for 30 days, for the treatment of hypercholesterolemia. Results showed that the capsules had a good effect on lowering serum triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0166] Example 17: (Drop Pills) Take 300g of cholesterol sulfate, add 900g of PEG4000 as a matrix, and use liquid paraffin as a coolant; prepare drop pills containing 10mg of cholesterol sulfate per pill, with a net weight of 30mg per pill. Take orally, 10 pills twice daily for 30 days, for the treatment of metabolic diseases such as fatty liver. Results showed that the above drop pills had good effects in lowering triglycerides and cholesterol.

[0167] Example 18: (Drip Pills) Weigh 1000g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare into drip pills containing 30mg of cholesterol sulfate per pill according to conventional methods. Oral administration, 3-6 pills twice daily for 30 days, for the treatment of hypercholesterolemia. Results showed that the above-mentioned drip pills had a good effect on lowering cholesterol and low-density lipoprotein (LDL) levels.

[0168] Example 19: (Soft Capsules) Weigh 100g of cholesterol sulfate and press it into 1000 soft capsules, each containing 100mg of cholesterol sulfate. Each capsule has a net weight of 500mg. Liquid paraffin is used as a lubricant. Take one capsule orally three times daily for 30 days. This was used to treat hypercholesterolemia. Results showed that the soft capsules had the effect of lowering cholesterol and low-density lipoprotein (LDL) levels.

[0169] Example 20: (Soft Capsules) Weigh 200g of cholesterol sulfate, add appropriate excipients, and prepare soft capsules containing 200mg of cholesterol sulfate per capsule according to conventional methods. Oral administration: 1 capsule 3 times daily for 30 consecutive days. Used to treat metabolic diseases such as fatty liver. Results showed that the above soft capsules had good effects in lowering triglycerides and cholesterol.

[0170] Example 21: (Soft Capsules) Weigh 100g of cholesterol sulfate, add appropriate excipients, and prepare soft capsules containing 100mg of cholesterol sulfate per capsule according to conventional methods. Oral administration: 1 capsule 3 times daily for 30 consecutive days. Used to treat hyperlipidemia. Results showed that the above soft capsules had good effects in lowering triglycerides and cholesterol.

[0171] Example 22: (Tablets) Weigh 500g of cholesterol sulfate and 500g of metformin, add appropriate pharmaceutical excipients, and prepare 10,000 tablets according to conventional methods. Oral administration: 3 tablets twice daily for 30 consecutive days, used to treat metabolic diseases such as fatty liver. Results showed that the above tablets had good hypoglycemic and hypolipidemic effects.

[0172] Example 23: (Capsules) Weigh 500g of cholesterol sulfate and 500g of metformin, add appropriate pharmaceutical excipients, and prepare 10,000 capsules according to conventional methods. Each capsule contains 50mg of cholesterol sulfate. Oral administration: 3 capsules twice daily for 30 days. Used to treat metabolic diseases such as diabetes and fatty liver. Results showed that the above capsules had good effects in lowering blood sugar, triglycerides, and cholesterol.

[0173] Example 24: (Capsules) Weigh 2200g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 500g of cholesterol sulfate) and 500g of metformin, add appropriate pharmaceutical excipients, and prepare 10,000 capsules according to conventional methods. Each capsule contains 50mg of cholesterol sulfate. Oral administration: 1-3 capsules twice daily for 30 days. Used to treat metabolic diseases such as diabetes and fatty liver. Results showed that the above capsules had good effects in lowering blood sugar, triglycerides, and cholesterol.

[0174] Example 25: (Capsules) 2200 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 500 g of cholesterol sulfate) and 500 g of metformin were weighed, and appropriate pharmaceutical excipients were added. The mixture was then formulated into 10,000 capsules using conventional methods, each containing 50 mg of cholesterol sulfate. The dosage was 1-3 capsules twice daily for 30 days, used to treat metabolic diseases such as diabetes and fatty liver. Results showed that the capsules had good effects in lowering blood sugar, triglycerides, and cholesterol.

[0175] Example 26: (Capsules) Take 1000 g of cholesterol sulfate and 300 g of dapagliflozin, add appropriate amounts of pharmaceutical excipients such as lactose, starch, micronized silica gel, and polyvinylpyrrolidone, mix evenly, granulate, dry, add 16 g of magnesium stearate and mix well to make 10,000 capsules (each capsule contains 100 mg of cholesterol sulfate and 30 mg of dapagliflozin). Oral administration: 3 capsules three times daily for 30 days. Used to treat diabetes mellitus with hyperlipidemia. Results showed that the above capsules had good hypoglycemic and hypolipidemic effects.

[0176] Example 27: (Capsules) Take 500 g of cholesterol sulfate and 100 g of fenofibrate, add appropriate pharmaceutical excipients, and prepare 10,000 capsules (each containing 50 mg of cholesterol sulfate and 10 mg of fenofibrate) according to conventional methods. Oral administration: 2 capsules three times daily for 30 days. Used to treat hyperlipidemia. Results showed that the above capsules had good triglyceride and cholesterol-lowering effects.

[0177] Example 28: (Capsules) Take 500 g of cholesterol sulfate and 100 g of probucol, add appropriate pharmaceutical excipients, and prepare 10,000 capsules (each capsule containing 50 mg of cholesterol sulfate and 10 mg of probucol) according to conventional methods. Take orally, 2 capsules 3 times daily for 30 consecutive days. Used to treat hyperlipidemia. Results showed that the above capsules had good hypoglycemic and hypolipidemic effects.

[0178] Example 29: (Capsules) Take 500g of cholesterol sulfate and 100g of rosuvastatin, add appropriate pharmaceutical excipients, and prepare 10,000 capsules (each containing 50mg of cholesterol sulfate and 10mg of rosuvastatin) according to conventional methods. Take one capsule orally three times daily for 30 days. Used to treat hyperlipidemia. Results showed that the above capsules had good triglyceride and cholesterol-lowering effects.

[0179] Example 30: (Tablets) Weigh 2200 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 500 g of cholesterol sulfate) and 100 g of rosuvastatin, add appropriate excipients, and prepare into 10,000 tablets (each containing 50 mg of cholesterol sulfate and 10 mg of rosuvastatin) according to conventional methods. Oral administration: 1 tablet three times daily for 30 days. Used to treat fatty liver and hyperlipidemia. Results showed that the above tablets have a good lipid-lowering effect.

[0180] Example 7: Preparation of Health Product Composition

[0181] Example 31: (Gummy Candy) Weigh 50 g of cholesterol sulfate, add appropriate ingredients, and prepare gummy candies according to conventional methods. Each gummy contains 5 mg of cholesterol sulfate. Take orally, 2-10 times daily, 1-3 gummy candies each time; continue for 30 days for adjunctive treatment of obesity and hyperlipidemia. Results showed that the above-mentioned gummy candies had good effects on weight control, lowering triglycerides, and lowering cholesterol.

[0182] Example 32: (Gummy Candy) 220 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 50 g of cholesterol sulfate) was weighed and added to appropriate raw materials. It was then formulated into gummies using conventional methods, with each gummy containing 5 mg of cholesterol sulfate. Oral administration: 1-3 gummies, 2-10 times daily, for 30 consecutive days, as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above gummies had good effects in lowering triglycerides and cholesterol.

[0183] Example 33: (Biscuits) 220 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 50 g of cholesterol sulfate) was weighed out and mixed with an appropriate amount of dietary fiber. The mixture was then prepared into biscuits using standard methods. Each 50 g biscuit contained 5 mg of cholesterol sulfate. This was used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the biscuits had good effects on weight control, lowering triglycerides, and lowering cholesterol.

[0184] Example 34: (Gel) Weigh 220 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 50 g of cholesterol sulfate), add appropriate gelling agents, water-soluble dietary fiber, and other ingredients, and prepare a gel according to conventional methods. Each 100 g contains 5 mg of cholesterol sulfate. It is used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above gel has good effects on weight control, lowering triglycerides, and lowering cholesterol.

[0185] Example 35: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of stachyose, add appropriate excipients, and prepare into 10,000 tablets (each tablet containing 50 mg of cholesterol sulfate) using conventional methods. Oral administration: 1 tablet three times daily for 30 days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0186] Example 36: (Capsules) Weigh 500 g of cholesterol sulfate and 1000 g of phytosterols, add appropriate excipients, and prepare into 10,000 tablets (each containing 50 mg of cholesterol sulfate) using conventional methods. Oral administration: 1 tablet three times daily for 30 days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above capsules had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0187] Example 37: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of beta-sitosterol, add appropriate excipients, and prepare into 10,000 tablets (each tablet containing 50 mg of cholesterol sulfate) according to conventional methods. Oral administration: 1 tablet three times daily for 30 days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0188] Example 38: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of stigmasterol, add appropriate excipients, and prepare into 10,000 tablets (each tablet containing 50 mg of cholesterol sulfate) according to conventional methods. Oral administration: 1 tablet three times daily for 30 consecutive days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0189] Example 39: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of Ganoderma lucidum extract, add appropriate excipients, and prepare into 10,000 tablets (each tablet containing 50 mg of cholesterol sulfate) according to conventional methods. Oral administration: 1 tablet three times daily for 30 consecutive days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets had good effects on weight control and cholesterol reduction.

[0190] Example 40: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of Rehmannia glutinosa extract, add appropriate excipients, and prepare into 10,000 tablets (each containing 50 mg of cholesterol sulfate) using conventional methods. Oral administration: 1 tablet three times daily for 30 days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets had good effects on weight control and cholesterol reduction.

[0191] Example 41: (Tablets) Weigh 500 g of cholesterol sulfate and 1000 g of ginseng, add appropriate excipients, and prepare into 10,000 tablets (each tablet contains 50 mg of cholesterol sulfate) according to conventional methods. Oral administration: 1 tablet three times daily for 30 consecutive days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above tablets have good effects in lowering triglycerides and cholesterol.

[0192] Example 42: (Tea preparation) Weigh 500 g of cholesterol sulfate and 1000 g of tea leaves, and prepare 10,000 tea bags (each containing 50 mg of cholesterol sulfate) using standard methods. Steep in boiling water and drink for 30 consecutive days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above-mentioned tea bags had good effects in lowering triglycerides and cholesterol.

[0193] Example 43: (Tea Preparation) Weigh 500 g of cholesterol sulfate and 1000 g of Dendrobium officinale, and prepare 10,000 tea bags (each containing 50 mg of cholesterol sulfate) using standard methods. Steep in boiling water and drink for 30 consecutive days. Used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above-mentioned tea bags had good effects on weight control, lowering triglycerides, and lowering cholesterol.

[0194] Example 8: Preparation of cyclodextrin pharmaceutical compositions

[0195] Example 44: (Powder) Weigh 467g of cholesterol sulfate, add 1140g of β-cyclodextrin and 2000mL of ethanol, heat at 50℃, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate β-cyclodextrin inclusion complex (containing 290mg / g of cholesterol sulfate).

[0196] 345 g of cholesterol sulfate β-cyclodextrin inclusion complex (containing 100 g of cholesterol sulfate) was weighed and prepared into 10,000 g of granules containing 10 mg of cholesterol sulfate per g using conventional methods. The dosage is 3-5 g, 1-3 capsules each time, for 30 consecutive days, for the treatment of hypercholesterolemia. Results showed that the capsules had good effects in lowering serum triglycerides, cholesterol, and low-density lipoprotein (LDL) levels.

[0197] Example 45: (Powder) Weigh 467 g of cholesterol sulfate, add 973 g of α-cyclodextrin and 2000 mL of ethanol, heat at 50℃, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate α-cyclodextrin inclusion complex (containing 320 mg / g of cholesterol sulfate).

[0198] 313 g of cholesterol sulfate α-cyclodextrin inclusion complex (containing 100 g of cholesterol sulfate) was weighed and prepared into 10,000 g of granules containing 10 mg of cholesterol sulfate per g using conventional methods. The dosage is 3-5 g, 1-3 capsules each time, for 30 consecutive days, for the treatment of hypercholesterolemia. Results showed that the capsules had good effects in lowering serum triglycerides, cholesterol, and low-density lipoprotein (LDL) levels.

[0199] Example 46: (Oral Liquid) 220 g of hydroxypropyl-β-cyclodextrin inclusion complex of cholesterol sulfate (containing 50 g of cholesterol sulfate) was weighed and added to water-soluble dietary fiber and other ingredients. The mixture was prepared into an oral liquid using conventional methods, with each 100 g containing 50 mg of cholesterol sulfate. It was used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the oral liquid had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0200] Example 47: (Oral Liquid) 320 g of γ-cyclodextrin inclusion complex (containing 50 g of cholesterol sulfate) was weighed and added to appropriate oral liquid, water-soluble dietary fiber, and other raw materials. The mixture was prepared using conventional methods to produce an oral liquid containing 5 mg of cholesterol sulfate per 100 g. It was used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the oral liquid had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0201] Example 48: (Oral Liquid) Weigh 350 g of sulfobutyl-β-cyclodextrin inclusion complex (containing 50 g of cholesterol sulfate), add appropriate amounts of oral liquid, water-soluble dietary fiber, and other ingredients, and prepare an oral liquid using conventional methods. Each 100 g contains 5 mg of cholesterol sulfate. It is used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above oral liquid has good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0202] Example 49: (Oral Liquid) 270 g of hydroxyethyl-β-cyclodextrin inclusion complex (containing 50 g of cholesterol sulfate) was weighed and added to appropriate oral liquid, water-soluble dietary fiber, and other raw materials. The mixture was prepared using conventional methods to produce an oral liquid containing 10 mg of cholesterol sulfate per 100 g. It was used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the oral liquid had good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0203] Example 50: (Oral liquid) Weigh 250 g of methyl-β-cyclodextrin inclusion complex (containing 50 g of cholesterol sulfate), add appropriate amounts of oral liquid, water-soluble dietary fiber, and other ingredients, and prepare an oral liquid according to conventional methods. Each 100 g contains 10 mg of cholesterol sulfate. It is used as an adjunct treatment for obesity and hyperlipidemia. Results showed that the above oral liquid has good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of cholesteryl sulfate as an active ingredient in the preparation of a medicament for treating or preventing hyperlipemia and hypercholesteremia.

2. Use according to claim 1, wherein The medicament comprises at least one of cholesteryl sulfate, a salt of cholesteryl sulfate formed with an acceptable base, and a water-soluble inclusion complex containing cholesteryl sulfate.

3. Use according to claim 2, wherein the compound is ###0002### The water-soluble inclusion complex is an inclusion complex of cholesteryl sulfate and cyclodextrin.

4. The use according to claim 3, wherein the compound is ###0002### The preparation method of the water-soluble inclusion complex comprises the following steps: S1. adding ethanol to cyclodextrin, heating and dissolving to obtain solution A; S2. stirring solution A, slowly adding cholesteryl sulfate, and performing an inclusion reaction to obtain an inclusion solution; S3. continuously stirring the inclusion solution obtained in step S2, removing ethanol, and drying to obtain the water-soluble inclusion complex.

5. The use according to claim 4, wherein the compound is ###0002### The cyclodextrin comprises at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

6. The use according to claim 4, wherein the compound is ###0002### The molar ratio of the cyclodextrin in step S1 to the cholesteryl sulfate in step S2 is cyclodextrin: cholesteryl sulfate = (1-3):

1.

7. The use according to claim 4, wherein the compound is ###0002### The inclusion reaction is performed at a temperature of 40-60°C for 1-3 hours.

8. Use of cholesteryl sulfate as an active ingredient in the preparation of a health product for assisting in lowering cholesterol and / or lowering high blood lipids.

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