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

Abstract

The invention relates to the field of biological medicine technology or health food, in particular to application of cholesterol sulfate in lowering cholesterol and / or lowering hyperlipidemia. The research finds that the cholesterol sulfate has a wide cholesterol regulation function, can assist in regulating endogenous synthesis, exogenous uptake, excretion, esterification and other processes of cholesterol, is beneficial to maintaining healthy plasma triglyceride, cholesterol and low-density lipoprotein levels, and further provides a new health-care support for people with high fat risk. The cholesterol sulfate can be used as an effective supplement of existing lipid-regulating medicines or health-care foods, and is expected to become an innovative health-care food component with an auxiliary lipid-lowering function.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology or health food, and in particular to the application of cholesterol sulfate in reducing cholesterol and / or hyperlipidemia. Background Art

[0002] Hyperlipidemia refers to a common metabolic disease in which elevated levels of plasma triglycerides or cholesterol and low-density lipoprotein cholesterol (LDL-C) cause abnormal lipid metabolism and endothelial cell loss. It is a major risk factor for many cardiovascular and cerebrovascular diseases, such as atherosclerotic cardiovascular disease. At present, with the development of the economy, the dietary structure and living habits have undergone major changes, the average life expectancy of Chinese people has gradually increased, and the incidence of hyperlipidemia has continued to grow, seriously endangering human health. Although cholesterol metabolic homeostasis has been widely studied in the past century, there is increasing evidence that cholesterol metabolic disorders are closely related to cardiovascular and cerebrovascular diseases and other diseases. Past studies have deepened the understanding of cholesterol metabolism under physiological and pathological conditions, and also provided new targets and strategies for the treatment of cholesterol-related diseases. At present, HMGCR inhibitors—statins—have been widely used to treat cardiovascular diseases. However, the efficacy of statins is limited by the compensatory increase of HMGCR protein. Although ezetimibe and PCSK9 inhibitors can further reduce LDL-C levels in patients with hyperlipidemia taking statins, cholesterol reduction requires further exploration of more effective and safer methods that can reduce side effects.

[0003] Bacteroides is an important genus of intestinal cornerstone bacteria, accounting for more than 40% of the total. In a study published in Nature Microbiology in August 2022, researchers discovered for the first time that Bacteroides intestinal bacteria are specific species that metabolize cholesterol by sulfonation, and can be responsible for converting cholesterol into cholesterol sulfate (CHS) through cholesterol sulfonate transferase (SULT2B1b), clarifying the mechanism of intestinal flora involved in cholesterol metabolism. Cholesterol sulfate is widely distributed in the human body, such as skin, adrenal glands, liver, lungs, brain, and endometrium. The concentration of cholesterol sulfate in human plasma reaches 134-322μg / mL. In a study published in Nature Commun. in July 2022, researchers discovered for the first time the mechanism of action of cholesterol sulfate against ulcerative colitis. Cholesterol sulfate activates sterol regulatory element binding protein 2 in colonic epithelial cells by binding to Niemann-Pick C2 protein, promoting cholesterol biosynthesis, and thus alleviating ulcerative colitis. The feedback regulation of cholesterol sulfate on host cholesterol effects remains unclear. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of cholesterol sulfate in lowering cholesterol and / or reducing hyperlipidemia.

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

[0006] In a first aspect, the present invention provides the use of cholesterol sulfate as an active ingredient in the preparation of a product for lowering cholesterol and / or reducing hyperlipidemia.

[0007] The present invention has been found through research that cholesterol sulfate has a wide range of cholesterol regulating functions, and can assist in regulating the endogenous synthesis, exogenous uptake, excretion and esterification of cholesterol, helping to maintain healthy plasma triglycerides, cholesterol and low-density lipoprotein cholesterol (LDL-C) levels, thereby providing new health care support for people at risk of high fat. Cholesterol sulfate can be used as an effective supplement to existing lipid-regulating drugs or health foods, and is expected to become a class of innovative health food ingredients with auxiliary lipid-lowering functions.

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

[0009]

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

[0011] Preferably, the product includes medicine, health care product or food.

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

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

[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] The present invention prepares an inclusion compound by compounding cyclodextrin and cholesterol sulfate, thereby increasing the water solubility of cholesterol sulfate and thus improving its utilization rate.

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

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

[0019] S2. Stirring solution A, slowly adding cholesterol sulfate to carry out inclusion reaction to obtain inclusion solution;

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

[0021] Preferably, the cyclodextrin includes at least one of 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 ethanol is 95%.

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

[0025] Preferably, in step S2, solution A is placed at 40°C-60°C with magnetic stirring.

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

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

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

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

[0030] More preferably, the conditions of the inclusion reaction are: temperature 50° C., time 2 h.

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

[0032] Preferably, the product includes medicine, health care product or food.

[0033] Preferably, the product further comprises auxiliary materials and / or acceptable carriers that meet the standards.

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

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

[0036] Preferably, the carrier comprises at least one of a lubricating component, a disintegrating component, a solubilizing component, a filling component, and a binding component.

[0037] Preferably, the lubricating component includes but is not limited to magnesium stearate and paraffin.

[0038] Preferably, the disintegrating component includes but is not limited to at least one of sodium carboxymethyl starch, microcrystalline cellulose, cross-linked polyvinylpyrrolidone and pregelatinized starch.

[0039] Preferably, the solubilizing component includes but is not limited to polyvinyl pyrrolidone.

[0040] Preferably, the filling component includes but is 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 slurry and starch.

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

[0043] Preferably, the active ingredient having 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 assist in improving 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 form, semisolid dosage form, liquid dosage form, syrup, 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, micropellets, dropping pills, microcapsules and microspheres.

[0047] Preferably, the liquid dosage form includes but is not limited to at least one of an oral solution and a liposome.

[0048] Preferably, the product is administered orally, including but not limited to at least one of soft candies, biscuits, jelly, tablets, capsules, teas and granules.

[0049] The beneficial effects of the present invention are:

[0050] The present invention has been found through research that cholesterol sulfate has a wide range of cholesterol regulating functions, which can assist in regulating the endogenous synthesis, exogenous uptake, excretion and esterification of cholesterol, and help maintain healthy plasma triglyceride, cholesterol and low-density lipoprotein levels, thereby providing new health care support for people at risk of high fat. Cholesterol sulfate can be used as an effective supplement to existing lipid-regulating drugs or health foods, and is expected to become a class of innovative health food ingredients with auxiliary lipid-lowering functions.

[0051] The present invention also provides a composition including cholesterol sulfate and a water-soluble inclusion complex containing cholesterol sulfate, which can be prepared into a health product or food, can effectively assist in lowering the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The figures show the effect of cholesterol sulfate on blood lipid levels in P407-induced mice; Figure A shows the results of serum TG determination; Figure B shows the results of serum TC determination; Figure C shows the results of serum LDL-C determination; Figure D shows the results of serum HDL-C determination; and Figure E shows the results of liver TC determination.

[0053] Figure 2 This is a diagram showing the effect of cholesterol sulfate on P407-induced lipid accumulation in mouse liver.

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

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

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

[0057] Figure 6 This is a graph showing the effect of cholesterol sulfate on the mRNA and protein of cholesterol synthesis-related indicators (SREBP2, INSIG1, HMGCR, SM, SCAP) in mice induced by P407.

[0058] Figure 7This is a graph showing the effect of cholesterol sulfate on P407-induced mouse SREBP2 protein.

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

[0060] Fig. 9 This is a graph showing the effect of cholesterol sulfate on the mRNA and protein of cholesterol transport-related indicators (LXR, ABCG5, SRB1, LDLR) in mice induced by P407.

[0061] Fig.10 This figure shows the effect of cholesterol sulfate on cholesterol levels in mice with hypercholesterolemia induced by a high cholesterol diet.

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

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

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

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

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

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

[0068] Fig.17 The dissolution test results of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (cholesterol sulfate-hydroxypropyl-β-cyclodextrin) in artificial gastric juice. DETAILED DESCRIPTION

[0069] In order to better illustrate the purpose, technical solutions 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 the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0071] In order to better understand the essence of the present invention, the pharmacological experiments and results of cholesterol sulfate on P407-induced hyperlipidemia mice, high cholesterol diet-induced hypercholesterolemia mice, and dexamethasone- and fructose-induced hypercholesterolemia rats will be combined to illustrate its role in preparing drugs and health foods for treating or preventing hyperlipidemia and hypercholesterolemia.

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

[0073] 1. Experimental methods

[0074] Sixty SPF male C57BL / 6j mice (22-24g) were randomly divided into 6 groups: normal group, P407 group, atorvastatin group (AVT group, 10mg / kg), ezetimibe group (EZ group, 10mg / kg), low-dose cholesterol sulfate group (CHS-L, 100mg / kg), and high-dose cholesterol sulfate group (CHS-H, 200mg / kg), with 10 mice in each group. All C57BL6j mice were intraperitoneally injected with P407 400mg / kg daily to induce hyperlipidemia model, and the corresponding drugs were given for treatment 1h after modeling. The normal group and P407 group were given the corresponding drug solvent. The drug administration lasted for 6 days. On the seventh day, all mice were killed after blood was drawn from the orbital vein and samples were taken for detection.

[0075] Observation indicators:

[0076] (Ⅰ) Blood 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) mRNA and protein expression of intestinal cholesterol absorption-related indicators;

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

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

[0081] (VI) Expression of hepatic cholesterol transporter mRNA and protein.

[0082] 2. Experimental results

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

[0084] like Figure 1 As shown in the figure, compared with the normal group, the levels of serum TG, TC, serum LDL-C and liver TC in the P407 group mice were significantly increased (P<0.01), while the level of serum HDL-C was significantly decreased, indicating that the hyperlipidemia model was successfully established. Compared with the P407 group, the levels of serum TG, TC, serum LDL-C and liver TC in the AVT group, EZ group and low- and high-dose cholesterol sulfate groups were significantly decreased. It is worth noting that the level of serum HDL-C did not change significantly after treatment with cholesterol sulfate, but showed an increasing trend after treatment with AVT, suggesting that the lipid-lowering mechanism of cholesterol sulfate may be different from that of statins.

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

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

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

[0088] like Figure 3 As shown in the figure, the results showed that compared with the normal group, P407 reduced the cholesterol content in the large intestine of mice; compared with the P407 group, the positive drug EZ and cholesterol sulfate administration could significantly increase the cholesterol content in the large intestine of mice, suggesting that cholesterol sulfate may play a cholesterol-lowering role by promoting cholesterol excretion. The positive drug AVT could not play a cholesterol-lowering role by promoting cholesterol excretion, while the positive drug EZ could play a cholesterol-lowering role by promoting cholesterol excretion.

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

[0090] like Figure 4 As shown in the figure, the results showed that compared with the normal group, P407 modeling could increase the level of liver TBA, suggesting that P407 may feedback inhibit the synthesis of liver TBA; compared with the P407 group, after the administration of the positive drug AVT, the level of liver TBA decreased, the level of bile TBA increased, but the level of feces TBA had no effect, suggesting that AVT may promote the enterohepatic circulation of TBA; while the positive drug EZ increased the levels of TBA in both bile and feces, and had the effect of promoting the excretion of TBA from bile and feces. After the administration of cholesterol sulfate, the level of liver TBA decreased, and the level of bile acid in bile and large intestine contents was significantly increased, suggesting that cholesterol sulfate may promote the metabolism of cholesterol into bile acid and promote the excretion of bile acid, thereby playing a lipid-lowering role.

[0091] (2.5) Effects on mRNA and protein of cholesterol absorption in the intestine of mice

[0092] like Figure 5 As shown in the results, compared with the blank group, the mRNA of NPC1L1 and ACAT2 in the small intestine of mice in the P407 group had no significant effect. Compared with the P407 group, the positive drug EZ could significantly down-regulate the mRNA expression of NPC1L1 in the small intestine of mice, and had no significant effect on the mRNA and protein expression of ACAT2; however, the administration of cholesterol sulfate could significantly down-regulate the mRNA and protein expression of NPC1L1 and ACAT2 at the same time, suggesting that cholesterol sulfate may play a role in lowering cholesterol by inhibiting cholesterol absorption and esterification, reducing cholesterol absorption, while the positive drug EZ only inhibited cholesterol absorption.

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

[0094] like Figure 6 , 7 As shown in the figure, the results showed that compared with the blank group, the P407 group significantly upregulated the mRNA expression levels of SREBP2, HMGCR, SM, and SCAP in the liver, downregulated the mRNA expression level of INSIG1, and reduced the transfer of SREBP2 into the nucleus. Compared with the P407 group, the positive drug AVT significantly downregulated the mRNA expression levels of SREBP2, HMGCR, and SCAP in the liver, and promoted the transfer of SREBP2 into the nucleus. The administration of cholesterol sulfate can significantly downregulate the mRNA expression levels of SREBP2, HMGCR, SM, and SCAP in the liver, upregulate the mRNA expression level of INSIG1, and promote the transfer of SREBP2 into the nucleus; indicating that cholesterol sulfate, like the positive drug AVT, can inhibit liver cholesterol synthesis.

[0095] Unlike the positive drug AVT, which is an HMGCR inhibitor, the administration of cholesterol sulfate 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 transfer of SREBP2 to the cell nucleus.

[0096] (2.7) Effects on mRNA and protein of cholesterol and bile acid metabolism in the liver

[0097] like Figure 8As shown in the figure, the results showed that compared with the blank group, the P407 group significantly downregulated the mRNA and protein expression levels of CYP7A1 in the mouse liver. Compared with the P407 group, the positive drug AVT had no significant effect on the mRNA and protein expression levels of CYP7A1, while the administration of cholesterol sulfate significantly upregulated the mRNA and protein expression levels of CYP7A1 in the mouse liver, suggesting that cholesterol sulfate may play a cholesterol-lowering role by promoting the metabolism of cholesterol into bile acid.

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

[0099] like Fig. 9 As shown in the results, compared with the blank group, P407 modeling can significantly downregulate the mRNA expression levels of liver ABCG5, SRB1, and LDLR, but has no significant effect on the mRNA expression level of LXR, suggesting that P407 prevents cholesterol in the blood circulation from entering the liver and reduces its excretion into the bile. Compared with the P407 group, the positive drug AVT can downregulate the mRNA expression levels of liver ABCG5, SRB1, and LDLR and the LDLR protein level, suggesting that it promotes cholesterol in the blood circulation to enter the liver and increase its excretion into the bile; cholesterol sulfate administration can significantly upregulate the mRNA expression levels of liver ABCG5, SRB1, LDLR, and LXR and the LDLR protein level, suggesting that cholesterol sulfate may promote cholesterol in the blood circulation to enter the liver and increase its excretion into the bile.

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

[0101] 1. Experimental methods

[0102] Thirty SPF male Kunming mice were randomly divided into a blank group (N group, n = 6) and an experimental group. The model group was fed with a high cholesterol diet (73% basal diet, 20% fat and 7% cholesterol) for 14 days to establish the model. On the 7th day, the mice were fasted for 12 hours but not water, and the levels of TC, LDL-C and HDL-C in the serum were measured by blood sampling. The high cholesterol mouse model was established with the standard of TC ≥ 2.0 mmol / L and LDL-C ≥ 1.49 mmol / L in the serum. Then the experimental group was randomly divided into 4 groups (model group (HC, n = 6), atorvastatin group (AVT group, n = 6), cholesterol sulfate group: low-dose group (cholesterol sulfate treatment dose of 150 mg / kg, n = 6) and high-dose group (cholesterol sulfate treatment dose of 300 mg / kg, n = 6)). After 7 days of administration, the daily body weight changes were recorded, and the mice were fasted but not watered for 12 hours. Blood was collected by removing the eyeballs to determine the levels of serum TC, LDL-C, HDL-C, and TBA, and the levels of TC and TBA in the liver and feces were also determined.

[0103] 2. Experimental results

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

[0105] The experimental results are as follows Fig.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 could reduce the body weight, serum TC levels, LDL-C levels, and liver TC levels of high cholesterol mice, and the cholesterol sulfate group increased the fecal TC level and promoted the excretion of cholesterol from the 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] The experimental results are as follows Fig.11 As shown in the figure, compared with the normal group, the HC group had increased TBA levels in serum and liver, but significantly decreased TBA levels in feces, suggesting that increasing the host's exogenous cholesterol intake can adaptively promote the bile acid metabolism of cholesterol, but reduce bile acid excretion. For mice fed a high-cholesterol diet, cholesterol sulfate can significantly reduce serum TBA, upregulate liver TBA levels, and significantly increase fecal TBA content, suggesting that cholesterol sulfate can inhibit the reabsorption of bile acid and promote its 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 male SD rats were randomly divided into a blank 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 injected with dexamethasone (DEX, 250 μg / kg, once a day) by intraperitoneal injection, and freely drank 30% fructose solution for 14 days. The other groups were given drugs according to the corresponding groups at the same time as the modeling, and the drugs were given once a day for a total of 14 days. During the experiment, the body weight, food intake and water intake of the rats were measured and recorded every day, and blood was collected from the orbital venous plexus every week to detect the changes in serum TG and TC. After the experiment, all animals were fasted but not watered for 12 hours, and blood was collected from the abdominal aorta to measure the levels of serum TC, LDL-C, HDL-C, and TBA in the rats. The levels of TC and TBA in the liver and feces were also measured. Figure 12-15shown.

[0110] like Figure 12-13 As shown, compared with those of group N, the serum TC and LDL-C levels and liver TC level of rats in group M were significantly increased; compared with group M, both the AVT group and the CHS group could reduce the serum TC, LDL-C levels and liver TC and levels of rats, indicating that cholesterol sulfate can significantly alleviate dexamethasone-fructose-induced hypercholesterolemia in mice.

[0111] like Fig.14 As shown in the figure, compared with the normal group, the serum TBA level in the M group increased, but the TBA level in the liver and feces decreased significantly, indicating that dexamethasone and fructose can increase the level of bile acid metabolism, but inhibit bile acid excretion. In dexamethasone-fructose-induced hypercholesterolemia rats, CHS administration can significantly downregulate serum TBA levels and significantly upregulate fecal TBA levels, indicating that cholesterol sulfate can inhibit the reabsorption of bile acid and promote its excretion, thereby reducing host cholesterol levels.

[0112] like Fig.15 As shown in the results, compared with the normal group, the expression of CYP7A1, NTCP1 and BESP proteins in the liver of rats in group M was significantly decreased, and the level of ASBT protein in the small intestine was significantly increased; compared with group M, the CHS group could significantly up-regulate the levels of CYP7A1, NTCP1 and BESP proteins in the liver of rats, and down-regulate the level of ASBT protein in the small intestine, indicating that cholesterol sulfate promotes the metabolism of cholesterol into bile acid, while promoting the excretion of bile acid and improving bile acid metabolism.

[0113] Example 4: Cholesterol sulfate acute toxicity test

[0114] Experimental animals and methods:

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

[0116] After administration, the mice were given free access to food and water and were observed for 14 consecutive days. The weight changes, food intake and other daily living conditions of the mice and the death conditions were recorded every two days. In addition, within 12 hours after administration, the toxic reactions, number of deaths and death time of each group of mice were observed and recorded every 3 hours. After 12 hours of administration, the above 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 test

[0120]

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

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

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

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

[0125] S2. Place solution A at 40°C to 60°C with magnetic stirring, slowly add cholesterol sulfate to carry out inclusion reaction, and obtain inclusion solution;

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

[0127] Among 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 solubility of cholesterol sulfate.

[0128] In order to optimize the preparation conditions, the present invention conducted the following research:

[0129] 1. Inclusion ratio exploration: The inclusion ratio of cholesterol sulfate and hydroxypropyl-β-cyclodextrin was determined by the phase solubility method. Take excess cholesterol sulfate, add a series of hydroxypropyl-β-cyclodextrin of different concentrations, heat and stir at 37°C for 48 hours, and place at 37°C for 12 hours after stirring to reach dissolution equilibrium. The sample was then centrifuged, the supernatant was taken, and the insoluble components were removed by a 0.22μm microporous filter membrane. The concentration of cholesterol sulfate in the sample was calculated by high performance liquid chromatography, and a phase solubility diagram was drawn.

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

[0131] 3. Result determination:

[0132] (3.1) Cholesterol sulfate analysis method:

[0133] Chromatographic conditions: chromatographic 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 easily soluble in methanol, and cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex is soluble in water and methanol, the present invention calculates the inclusion rate of the inclusion complex by the content of water-soluble and methanol-soluble cholesterol sulfate.

[0135] According to formula 1 and formula 2, the inclusion rate and yield are calculated:

[0136] Inclusion rate = 1-(water-soluble cholesterol sulfate content / total cholesterol sulfate content) × 100% - Formula 1

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

[0138] (3.3) Determination of the solubility of cholesterol sulfate: 40 mg of cholesterol sulfate, cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex and physical mixture (molar ratio 1:1, equivalent to 40 mg of cholesterol sulfate, take the two and mix them directly in a mortar) were put into gelatin capsules as samples for testing, and the drug dissolution was tested according to the second method (paddle method) of Appendix 160 of Part I of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The dissolution medium was 500 mL of artificial gastric juice, the temperature was 37±0.3℃, the speed was 100r / min, and 1 mL was quantitatively sampled at 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min and 120 min, respectively, and 1 mL of the same temperature medium was supplemented at the same time. After the sample was filtered through a 0.22 μm microporous membrane, the content of cholesterol sulfate in the sample was detected by high performance liquid chromatography, and the dissolution was calculated using the corresponding dissolution medium as a blank. The experiment was repeated 3 times and the average value was taken.

[0139] Table 3 Orthogonal experimental factor level table

[0140]

[0141] Table 4 Orthogonal experimental design results

[0142]

[0143]

[0144] Table 5: Determination of the yield and inclusion rate of cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex

[0145]

[0146] The results of phase solubility experiments are shown in Fig.16 As shown, the obtained phase solubility curve has a good linear relationship (R2 = 0.9958), indicating that the cholesterol sulfate and hydroxypropyl-β-cyclodextrin molecules can be combined in a molar ratio of 1:1. From the results of the orthogonal experiment and range analysis in Table 4, it can be seen that the factors affecting the inclusion of each component are A>B>C in order, and the best ratio can be selected as A1, B2, and C2, that is, the optimal preparation conditions of the cholesterol sulfate cyclodextrin inclusion complex are: the molar ratio of cholesterol sulfate to hydroxypropyl-β-cyclodextrin = 1:1, reaction conditions: 50℃, 2h. The average inclusion rate of the optimal product is 95.35±1.77% (Table 5).

[0147] The dissolution curve of the optimal product is as follows: Fig.17As shown. Cholesterol sulfate API is insoluble in artificial gastric juice. At 120 min, the solubility of cholesterol sulfate-hydroxypropyl-β-cyclodextrin physical mixture in artificial gastric juice and artificial intestinal juice was 14.03%, respectively. Compared with cholesterol sulfate API and cholesterol sulfate-hydroxypropyl-β-cyclodextrin physical mixture, cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex can reach a solubility of nearly 73% in artificial gastric juice at 45 min, indicating that cholesterol sulfate-hydroxypropyl-β-cyclodextrin inclusion complex can significantly improve the solubility of cholesterol sulfate.

[0148] Example 6: Preparation of pharmaceutical composition

[0149] Example 1: (Tablets) Take 1000g of cholesterol sulfate, add 250g of cross-linked polyvinylpyrrolidone, 500g of starch, and 250g of microcrystalline cellulose, mix well, use 200g of 5% (w / v) starch slurry as a binder, wet granulate, dry, add 10g of magnesium stearate and mix well, and press into 10,000 tablets containing 100mg of cholesterol sulfate per tablet according to conventional methods, and each tablet has a net weight of 0.2g. Oral, twice a day, 1-2 tablets each time; take for 30 consecutive days for the treatment of hyperlipidemia. The results show that the above tablets have a good effect of lowering serum triglycerides, cholesterol, and low-density lipoprotein (LDL) levels.

[0150] Example 2: (Tablets) Weigh 500g of cholesterol sulfate, add 1000g of alpha-cyclodextrin, and prepare 10,000 tablets containing 50mg of cholesterol sulfate per tablet according to conventional methods with appropriate pharmaceutical excipients. Orally take 1-3 tablets twice a day for 30 consecutive days for the treatment of hyperlipidemia. The results show that the above tablets have a good effect of lowering plasma triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0151] Example 3: (Tablets) Take 400g of cholesterol sulfate, add 480g of lactose and 754g of starch and mix evenly, use 350g of 7% (w / v) starch slurry as a binder, wet granulate, dry, add 16g of magnesium stearate and mix evenly, press into 10,000 tablets containing 40mg of cholesterol sulfate per tablet, and each tablet has a net weight of 0.2g. Oral, 3 times a day, 4 tablets each time; take for 30 consecutive days, for the treatment of hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, lowering triglycerides or lowering density lipoprotein lipids, reducing body weight, and lowering transaminase.

[0152] 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, 3 times a day, 4 tablets each time; continuous administration for 30 days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, triglycerides or low-density lipoprotein lipids, reducing body weight, and reducing transaminase.

[0153] 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, 3 times a day, 4 tablets each time; continuous administration for 30 days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, triglycerides or low-density lipoprotein lipids, reducing body weight, and reducing transaminase.

[0154] 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, once a day, 1 tablet each time; take for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, triglycerides or low-density lipoprotein lipids, reducing body weight, and reducing transaminase.

[0155] 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, once a day, 1 tablet each time; take for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, lowering triglycerides or lowering high-density lipoprotein lipids, reducing body weight, and lowering transaminase.

[0156] 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, 2 times a day, 1 tablet each time; take for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above tablets have good effects of lowering blood cholesterol, reducing high-density lipoprotein lipids, reducing body weight, and reducing transaminase.

[0157] 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, 3 times a day, 4 capsules each time; continuous administration for 30 days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above capsules have good effects of lowering blood cholesterol, triglycerides or low-density lipoprotein lipids, reducing body weight, and reducing transaminase.

[0158] 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, 3 times a day, 4 capsules each time; take for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above capsules have good effects of lowering blood cholesterol, lowering triglycerides or lowering high-density lipoprotein lipids, reducing body weight, and lowering transaminase.

[0159] Example 11: (Capsules) Weigh 1000g of cholesterol sulfate and 500g of nicotinic acid, add appropriate pharmaceutical excipients, and prepare capsules containing 100mg of cholesterol sulfate per capsule according to conventional methods. Oral administration, 3 times a day, 4 capsules each time; take for 30 consecutive days. 3 times a day, 4 capsules each time; take for 30 consecutive days. Used to treat hyperlipidemia, obesity, and fatty liver. The results show that the above capsules have good effects of lowering blood cholesterol, lowering triglycerides or lowering high-density lipoprotein lipids, reducing body weight, and lowering transaminase.

[0160] Example 12: (Tablets) Weigh 1000 g of cholesterol sulfate, add 3350 g of hydroxypropyl-β-cyclodextrin and 2000 mL of ethanol, heat at 50°C, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 225 mg / g of cholesterol sulfate).

[0161] Weigh 2200g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex and prepare 10,000 tablets containing 50mg of cholesterol sulfate in each tablet according to conventional methods. Oral administration, 2 times a day, 1-3 tablets each time; continuous administration for 30 days, for the treatment of metabolic diseases represented by fatty liver. The results show that the tablets have a good effect of lowering serum triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0162] Example 13: (Capsules) Weigh 500g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 capsules containing 50mg of cholesterol sulfate per capsule according to conventional methods. Take orally, twice a day, 3 capsules each time; take for 30 consecutive days, for the treatment of hyperlipidemia. The results show that the capsules have a good effect of lowering serum cholesterol and low-density lipoprotein (LDL) levels.

[0163] Example 14: (Capsules) Weigh 200g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 capsules containing 20mg of cholesterol sulfate per capsule according to conventional methods. Take orally, twice a day, 3 capsules each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by fatty liver. The results show that the capsules have good effects of lowering blood lipids and cholesterol.

[0164] Example 15: (Capsules) Weigh 1000g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare 10,000 sustained-release capsules containing 100mg of cholesterol sulfate per capsule according to conventional methods. Take orally, twice a day, 3 capsules each time; take for 30 consecutive days, for the treatment of hypercholesterolemia. The results show that the above-mentioned sustained-release capsules have a good cholesterol-lowering effect.

[0165] Example 16: (Capsules) Weigh 1000 g of cholesterol sulfate, add 3350 g of hydroxypropyl-β-cyclodextrin and 2000 mL of ethanol, heat at 50°C, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 225 mg / g of cholesterol sulfate).

[0166] Weigh 2200g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex and prepare 10,000 capsules containing 50mg of cholesterol sulfate in each capsule according to conventional methods. Oral administration, 2 times a day, 1-3 capsules each time; take for 30 consecutive days for the treatment of hypercholesterolemia. The results show that the capsule has a good effect of lowering serum triglyceride, cholesterol, and low-density lipoprotein (LDL) levels.

[0167] Example 17: (Drop pills) Take 300g of cholesterol sulfate, add 900g of PEG4000 as a matrix, and use liquid paraffin as a coolant; make drop pills containing 10mg of cholesterol sulfate per pill, and each pill has a net weight of 30mg. Take orally, twice a day, 10 pills each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by fatty liver. The results show that the above-mentioned drop pills have good effects in lowering triglycerides and cholesterol.

[0168] Example 18: (Drop pills) Weigh 1000g of cholesterol sulfate, add appropriate pharmaceutical excipients, and prepare drop pills containing 30mg of cholesterol sulfate per pill according to conventional methods. Oral administration, 2 times a day, 3-6 pills each time; take for 30 consecutive days for the treatment of hypercholesterolemia. The results show that the above-mentioned drop pills have a good effect of lowering cholesterol and low-density lipoprotein (LDL) levels.

[0169] Example 19: (Soft capsule) Weigh 100g of cholesterol sulfate and compress 1000 soft capsules with a specification of 100mg cholesterol sulfate per capsule, with a net weight of 500mg per capsule, and liquid paraffin as a lubricant. Oral administration, 3 times a day, 1 capsule each time; continuous administration for 30 days. For the treatment of hypercholesterolemia, the results show that the soft capsule has the effect of lowering cholesterol and low-density lipoprotein (LDL) levels.

[0170] Example 20: (Soft capsule) Weigh 200g of cholesterol sulfate, add appropriate excipients, and prepare soft capsules containing 200mg of cholesterol sulfate in accordance with conventional methods. Oral administration, 3 times a day, 1 capsule each time; continuous administration for 30 days. Used to treat metabolic diseases represented by fatty liver. The results show that the soft capsules have good triglyceride and cholesterol lowering effects.

[0171] Example 21: (Soft capsule) Weigh 100g of cholesterol sulfate, add appropriate excipients, and prepare soft capsules containing 100mg of cholesterol sulfate in accordance with conventional methods. Take orally, 3 times a day, 1 capsule each time; take for 30 consecutive days. Used for the treatment of hyperlipidemia. The results show that the soft capsule has a good effect of lowering triglycerides and cholesterol.

[0172] 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. Take orally, twice a day, 3 tablets each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by fatty liver. The results show that the above tablets have good effects of lowering blood sugar and blood lipids.

[0173] 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 containing 50mg of cholesterol sulfate. Oral administration, 2 times a day, 3 capsules each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by diabetes and fatty liver. The results show that the capsules have good effects of lowering blood sugar, triglycerides and cholesterol.

[0174] 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 containing 50mg of cholesterol sulfate. Oral administration, 2 times a day, 1-3 capsules each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by diabetes and fatty liver. The results show that the above capsules have good effects of lowering blood sugar, triglycerides and cholesterol.

[0175] Example 25: (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 containing 50mg of cholesterol sulfate. Oral administration, 2 times a day, 1-3 capsules each time; take for 30 consecutive days, for the treatment of metabolic diseases represented by diabetes and fatty liver. The results show that the above capsules have good effects of lowering blood sugar, triglycerides and cholesterol.

[0176] Example 26: (Capsules) Take 1000g of cholesterol sulfate and 300g of dapagliflozin, add appropriate amounts of pharmaceutical excipients such as lactose, starch, micro-powder silica gel, polyvinyl alcohol pyrrolidone, mix evenly, granulate, dry, add 16g of magnesium stearate and mix evenly, and make 10,000 capsules (each containing 100mg of cholesterol sulfate and 30mg of dapagliflozin respectively). Oral, 3 times a day, 3 capsules each time; take for 30 consecutive days. For the treatment of diabetes and hyperlipidemia. The results showed that the above capsules have good effects in lowering blood sugar and blood lipids.

[0177] Example 27: (Capsules) Take 500g of cholesterol sulfate and 100g of fenofibrate, add appropriate amounts of pharmaceutical excipients, and prepare 10,000 capsules (each containing 50mg of cholesterol sulfate and 10mg of fenofibrate) according to conventional methods. Take orally, 3 times a day, 2 capsules each time; take for 30 consecutive days. Used for the treatment of hyperlipidemia. The results show that the above capsules have good triglyceride and cholesterol lowering effects.

[0178] Example 28: (Capsules) Take 500g of cholesterol sulfate and 100g of probucol, add appropriate amounts of pharmaceutical excipients, and prepare 10,000 capsules (each containing 50mg of cholesterol sulfate and 10mg of probucol) according to conventional methods. Oral administration, 3 times a day, 2 capsules each time; take for 30 consecutive days. Used for the treatment of hyperlipidemia. The results show that the above capsules have good effects of lowering blood sugar and blood lipids.

[0179] Example 29: (Capsules) Take 500g of cholesterol sulfate and 100g of rosuvastatin, add appropriate amounts of pharmaceutical excipients, and prepare 10,000 capsules (each containing 50mg of cholesterol sulfate and 10mg of rosuvastatin) according to conventional methods. Oral administration, 3 times a day, 1 capsule each time; take for 30 consecutive days. For the treatment of hyperlipidemia. The results show that the above capsules have good triglyceride and cholesterol lowering effects.

[0180] Example 30: (Tablets) Weigh 2200g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 500g of cholesterol sulfate) and 100g of rosuvastatin, add appropriate excipients, and prepare 10,000 tablets (each containing 50mg of cholesterol sulfate and 10mg of rosuvastatin) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; continuous administration for 30 days. Used to treat fatty liver and hyperlipidemia. The results show that the above tablets have a good lipid-lowering effect.

[0181] Example 7: Preparation of health food composition

[0182] Example 31: (Soft candy) Weigh 50g of cholesterol sulfate, add appropriate food raw materials, and prepare soft candy according to conventional methods, each containing 5mg of cholesterol sulfate. Oral administration, 2-10 times a day, 1-3 capsules each time; take for 30 consecutive days, for food auxiliary treatment of obesity and hyperlipidemia. The results show that the soft candy has good effects of controlling weight, lowering triglycerides and cholesterol.

[0183] Example 32: (Soft candy) Weigh 220g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion compound (containing 50g of cholesterol sulfate) and add appropriate food raw materials to prepare soft candy according to conventional methods, each containing 5mg of cholesterol sulfate. Oral administration, 2-10 times a day, 1-3 capsules each time; take for 30 consecutive days, for food auxiliary treatment of obesity and hyperlipidemia. The results show that the soft candy has a good effect of lowering triglycerides and cholesterol.

[0184] Example 33: (Biscuits) Weigh 220g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 50g of cholesterol sulfate) and add appropriate high dietary fiber, and prepare biscuits according to the conventional method, with 50g of cholesterol sulfate 5mg per 50g. Used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above biscuits have good effects on controlling weight, lowering triglycerides and lowering cholesterol.

[0185] Example 34: (Gel) Weigh 220g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 50g of cholesterol sulfate) and add appropriate gel, water-soluble dietary fiber and other food raw materials to prepare a gel according to the conventional method, containing 5mg of cholesterol sulfate per 100g. Used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above gel has good effects on controlling weight, lowering triglycerides and cholesterol.

[0186] Example 35: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of stachyose, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in controlling weight, lowering triglycerides and lowering cholesterol.

[0187] Example 36: (Capsules) Weigh 500g of cholesterol sulfate and 1000g of phytosterols, add appropriate excipients, and prepare 10,000 tablets (each containing 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; continuous administration for 30 days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the capsules have good effects in controlling weight, lowering triglycerides, and lowering cholesterol.

[0188] Example 37: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of betasitosterol, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in controlling weight, lowering triglycerides and lowering cholesterol.

[0189] Example 38: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of stigmasterol, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in controlling weight, lowering triglycerides and lowering cholesterol.

[0190] Example 39: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of Ganoderma lucidum extract, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in controlling weight and lowering cholesterol.

[0191] Example 40: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of Rehmannia glutinosa extract, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in controlling weight and lowering cholesterol.

[0192] Example 41: (Tablets) Weigh 500g of cholesterol sulfate and 1000g of ginseng, add appropriate excipients, and prepare 10,000 tablets (each tablet contains 50mg of cholesterol sulfate) according to conventional methods. Oral administration, 3 times a day, 1 tablet each time; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the above tablets have good effects in lowering triglycerides and cholesterol.

[0193] Example 42: (Tea) Weigh 500g of cholesterol sulfate and 1000g of tea leaves, and prepare 10,000 bags of teabags (each bag contains 50mg of cholesterol sulfate) according to conventional methods. Drink with boiling water; take for 30 consecutive days. Used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the teabag has a good effect of lowering triglycerides and cholesterol.

[0194] Example 43: (Tea) Weigh 500g of cholesterol sulfate and 1000g of Dendrobium officinale, and prepare 10,000 bags of tea (each bag contains 50mg of cholesterol sulfate) according to the conventional method. Drink with boiling water; take for 30 days. It is used as a food supplement for the treatment of obesity and hyperlipidemia. The results show that the tea bag has good effects on controlling weight, lowering triglycerides and cholesterol.

[0195] Example 8: Preparation of cyclodextrin pharmaceutical composition

[0196] Example 44: (Powder) Weigh 467 g of cholesterol sulfate, add 1140 g of β-cyclodextrin and 2000 mL of ethanol, heat at 50°C, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate β-cyclodextrin inclusion complex (containing 290 mg / g of cholesterol sulfate).

[0197] Weigh 345g of cholesterol sulfate β-cyclodextrin inclusion complex (containing 100g of cholesterol sulfate) and prepare 10000g of granules containing 10mg of cholesterol sulfate per gram according to conventional methods. Take 3-5g orally, 1-3 capsules each time; take for 30 consecutive days for the treatment of hypercholesterolemia. The results show that the capsule has a good effect of lowering serum triglycerides, cholesterol, and low-density lipoprotein (LDL) levels.

[0198] Example 45: (Powder) Weigh 467 g of cholesterol sulfate, add 973 g of α-cyclodextrin and 2000 mL of ethanol, heat at 50°C, stir at 600 r / min for 2 h, recover the ethanol, and dry to obtain cholesterol sulfate alpha-cyclodextrin inclusion complex (containing 320 mg / g of cholesterol sulfate).

[0199] Weigh 313g of cholesterol sulfate α-cyclodextrin inclusion complex (containing 100g of cholesterol sulfate) and prepare 10000g of granules containing 10mg of cholesterol sulfate per gram according to conventional methods. Take 3-5g orally, 1-3 capsules each time; take for 30 consecutive days for the treatment of hypercholesterolemia. The results show that the capsule has a good effect of lowering serum triglycerides, cholesterol, and low-density lipoprotein (LDL) levels.

[0200] Example 46: (Oral liquid) Weigh 220g of cholesterol sulfate hydroxypropyl-β-cyclodextrin inclusion complex (containing 50g of cholesterol sulfate) and add water-soluble dietary fiber and other food raw materials to prepare an oral liquid according to conventional methods, containing 50mg of cholesterol sulfate per 100g. It is used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the oral liquid has good effects of controlling weight, lowering triglycerides and lowering cholesterol.

[0201] Example 47: (Oral liquid) Weigh 320g of γ-cyclodextrin inclusion compound (containing 50g of cholesterol sulfate) and add appropriate oral liquid, water-soluble dietary fiber and other food raw materials to prepare oral liquid according to conventional methods, containing 5mg of cholesterol sulfate per 100g. It is used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above oral liquid has good effects of controlling weight, lowering triglycerides and lowering cholesterol.

[0202] Example 48: (Oral liquid) Weigh 350g of sulfobutyl-β-cyclodextrin inclusion compound (containing 50g of cholesterol sulfate) and add appropriate oral liquid, water-soluble dietary fiber and other food raw materials to prepare oral liquid according to conventional methods, containing 5mg of cholesterol sulfate per 100g. It is used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above oral liquid has good effects of controlling weight, lowering triglycerides and lowering cholesterol.

[0203] Example 49: (Oral liquid) Weigh 270g of hydroxyethyl-β-cyclodextrin inclusion compound (containing 50g of cholesterol sulfate) and add appropriate oral liquid, water-soluble dietary fiber and other food raw materials to prepare oral liquid according to conventional methods, containing 10mg of cholesterol sulfate per 100g. It is used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above oral liquid has good effects of controlling weight, lowering triglycerides and lowering cholesterol.

[0204] Example 50: (Oral liquid) Weigh 250g of methyl-β-cyclodextrin inclusion compound (containing 50g of cholesterol sulfate) and add appropriate oral liquid, water-soluble dietary fiber and other food raw materials to prepare oral liquid according to conventional methods, containing 10mg of cholesterol sulfate per 100g. It is used as a food auxiliary treatment for obesity and hyperlipidemia. The results show that the above oral liquid has good effects of controlling weight, lowering triglycerides and lowering cholesterol.

[0205] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The use of cholesterol sulfate as an active ingredient in the preparation of products for lowering cholesterol and / or reducing hyperlipidemia.

2. The use according to claim 1, characterized in that The products include medicines, health products or foods.

3. The use according to claim 1, characterized in that The product comprises at least one of cholesterol sulfate, a salt formed by cholesterol sulfate and an acceptable base, and a water-soluble inclusion compound containing cholesterol sulfate.

4. The use according to claim 3, characterized in that The water-soluble inclusion compound is an inclusion compound formed by cyclodextrin and cholesterol sulfate.

5. The use according to claim 4, characterized in that The preparation method of the water-soluble inclusion compound comprises the following steps: S1. Add ethanol to cyclodextrin and heat to dissolve to obtain solution A; S2. Stirring solution A, slowly adding cholesterol sulfate to carry out inclusion reaction to obtain inclusion solution; S3. The inclusion solution obtained in step S2 is continuously stirred, ethanol is removed, and the solution is dried to obtain the water-soluble inclusion complex.

6. The use according to claim 5, characterized in that The cyclodextrin includes at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

7. The use according to claim 5, characterized in that The molar ratio of the cyclodextrin in step S1 to the cholesterol sulfate in step S2 is cyclodextrin:cholesterol sulfate=(1-3):

1.

8. The use according to claim 5, characterized in that The conditions of the inclusion reaction are: temperature 40-60° C., time 1-3 h.

9. A product for lowering cholesterol and / or reducing hyperlipidemia, characterized in that: It comprises at least one of cholesterol sulfate, a salt formed by cholesterol sulfate and an acceptable base, and a water-soluble inclusion compound containing cholesterol sulfate.

10. The product according to claim 9, characterized in that The product may also include auxiliary materials and / or acceptable carriers that meet the standards.

Images