Monascus, monascus extract, preparation method and application

Abstract

The application discloses a Tibetan monascus and a preparation method thereof, and the preparation method comprises the following steps: (1) mixing highland barley, auxiliary materials and a nutrient solution, and then performing aging to obtain a fermentation matrix; the nutrient solution comprises sucrose, glutamic acid, histidine, calcium nitrate, sodium nitrate, potassium dihydrogen phosphate, n-octanoic acid, ascorbic acid, disodium EDTA and water in terms of the weight ratio; (2) inoculating the fermentation matrix with a monascus seed solution to perform fermentation, and obtaining a fermentation product; and (3) drying the fermentation product to obtain the Tibetan monascus; the preparation method has good process stability, small component difference between batches and stable quality; the nutrient solution, the fermentation process and the drying process are optimized, so that the content of lovastatin in the Tibetan monascus is improved, and the content of acid lovastatin is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, specifically to a Tibetan red yeast rice, a Tibetan medicine red yeast rice extract, its preparation method, and its application. Background Technology

[0002] Red yeast rice is a commonly used lipid-lowering drug, fermented by Monascus purpureus. Currently, there are two types of red yeast rice drugs used clinically: the traditional Chinese medicine "red yeast rice" and the Tibetan medicine "Tibetan red yeast rice." The traditional Chinese medicine "red yeast rice" is made from fermented rice grains and is also commonly known as "red yeast rice." Clinically, it is used in the form of red yeast rice slices and its prepared medicines, Xuezhikang capsules and Zhibituo capsules. The Tibetan medicine "Tibetan red yeast rice" (originally called: highland barley red yeast rice) is made from the kernels of highland barley, a specialty crop of the Tibetan region of my country, and is fermented. Clinically, it is used in the form of slices.

[0003] The active ingredient in the Chinese herbal medicine red yeast rice that lowers blood lipids is lovastatin. As a raw material, its standard is attached to the "Xuezhikang Capsules" entry in the Chinese Pharmacopoeia. However, there is relatively little research on the lovastatin component in red yeast rice. Summary of the Invention

[0004] In view of this, this application provides a Tibetan red yeast rice, a Tibetan medicine red yeast rice extract, a preparation method, and applications. This application optimizes the nutrient solution, as well as the fermentation and drying processes, to increase the lovastatin content in the Tibetan red yeast rice while effectively increasing the content of acidic lovastatin. The Tibetan red yeast rice extract of this application can significantly improve blood lipid indicators and inhibit the rise in body weight curve in hyperlipidemia models, while also reducing liver indices and inhibiting fat deposition.

[0005] To solve the above technical problems, the technical solution provided in this application is a method for preparing saffron yeast, including:

[0006] (1) Barley, auxiliary materials, and nutrient solution are mixed in a weight ratio of (40-100):(5-60):(20-45) and then matured to obtain a fermentation substrate; the nutrient solution comprises, by weight: 0.1-1 parts sucrose, 0.2-1 parts glutamic acid, 0.01-0.26 parts histidine, 0.01-0.15 parts calcium nitrate, 0.01-0.15 parts sodium nitrate, 0.01-0.05 parts potassium dihydrogen phosphate, 0.01-0.05 parts octanoic acid, 0.1-0.5 parts ascorbic acid, 0.00001-0.00005 parts disodium EDTA, and 96-98.07 parts water;

[0007] (2) Fermentation was carried out after inoculating the fermentation substrate with red yeast seed liquid to obtain fermentation product;

[0008] (3) Dry the fermented material.

[0009] Preferably, the weight ratio of barley, auxiliary materials, and nutrient solution is 88:12:40.

[0010] Preferably, the auxiliary material consists of wheat bran and soybean meal, with a weight ratio of wheat bran to soybean meal of (5-12):(2-5).

[0011] Preferably, the auxiliary material consists of wheat bran and soybean meal, with a weight ratio of wheat bran to soybean meal of 10:2.

[0012] Preferably, the nutrient solution, by mass percentage, consists of 0.41% sucrose, 0.76% glutamic acid, 0.26% histidine, 0.09% calcium nitrate, 0.03% sodium nitrate, 0.01% potassium dihydrogen phosphate, 0.01% octanoic acid, 0.34% ascorbic acid, 0.00001% disodium EDTA, and 98.07% water.

[0013] Preferably, the barley is treated by removing residue and crushing.

[0014] Preferably, the mixing process of barley, auxiliary materials, and nutrient solution involves mixing the barley, auxiliary materials, and nutrient solution for 5-7 minutes. This ensures that the materials are evenly mixed, can be formed into a ball by hand, and crumble easily when lightly pressed.

[0015] Preferably, the curing conditions are 121°C and the curing time is 30 minutes.

[0016] Preferably, the method further includes: mixing highland barley, auxiliary materials, and nutrient solution in a weight ratio and then bottling them, with each bottle containing a certain amount.

[0017] 360-440g / bottle, to obtain bottled material; bottled material is then matured to obtain fermentation substrate.

[0018] Preferably, in step (2), the fermentation substrate is cooled to 25-45°C before inoculating the red yeast seed liquid.

[0019] Preferably, the preparation process of the red yeast seed solution includes:

[0020] Red Monascus strain was inoculated onto slant culture medium and cultured at 30℃ for 6-7 days. The spores were then washed off with water to obtain a spore suspension.

[0021] The spore suspension was inoculated into a fermenter and cultured at 30°C for 2-3 days to obtain Monascus purpureus seed solution.

[0022] Preferably, the preparation process of the red yeast seed solution includes:

[0023] Red Monascus strain was inoculated onto slant culture medium and cultured at 30℃ for 6-7 days. The spores were then washed with 300 ml of sterile water to obtain a spore suspension.

[0024] The spore suspension was inoculated into a fermenter and cultured at 30°C for 2-3 days to obtain the red yeast seed solution.

[0025] Preferably, the red yeast strain is Monascus pilosus (strain: YWG-1), a fungus belonging to the Aspergillus family.

[0026] Preferably, the fermentation process in step (2) specifically includes:

[0027] High-temperature fermentation: After inoculating the fermentation substrate with red yeast seed liquid, fermentation is carried out for 6 days at 27-29℃ and relative humidity of 60%-65%.

[0028] Gradient cooling fermentation: Gradient cooling fermentation is carried out by gradually decreasing the temperature to 23℃ at a rate of 2-3℃ / day, while keeping the relative humidity constant.

[0029] Low-temperature fermentation: Fermentation is carried out at 21-23℃ and relative humidity of 60%-65% for 7-8 days to obtain fermented product.

[0030] Preferably, the inoculation amount of Monascus purpureus seed liquid in step (2) is 0.114 to 0.139 mL / g.

[0031] Preferably, fermentation is terminated when the total lovastatin content in the fermented product is ≥0.6% in (2).

[0032] Preferably, during the high-temperature fermentation process, the fermentation substrate is shaken for the first time 48 hours after being inoculated with red yeast seed liquid, and then shaken once every 24 hours, for a total of 4 shakes.

[0033] Preferably, the flask is shaken once during the gradient cooling fermentation process.

[0034] Preferably, the flask is shaken once every 72 hours during the high-temperature fermentation process, and the number of shakes is ≤4.

[0035] Preferably, step (3) specifically includes: drying the fermented material at a temperature of 60°C, and stopping the drying process when the moisture content of the saffron yeast is ≤10%.

[0036] Preferably, step (3) specifically includes: drying the fermented material at a temperature of 60°C, and stopping the drying process when the moisture content of the saffron yeast is ≤10%.

[0037] The present invention also provides the application of the above-mentioned saffron yeast in the preparation of lipid-lowering drugs and food.

[0038] This invention also provides a method for preparing saffron yeast extract, comprising:

[0039] (A) After crushing the above-mentioned saffron yeast, it was successively subjected to moistening and percolation extraction to obtain percolate;

[0040] (B) The percolate was concentrated and dried to obtain saffron extract.

[0041] Preferably, step (A) specifically involves: pulverizing the saffron yeast of claim 7 and then sequentially soaking it in ethanol and percolating it with ethanol to obtain a percolate;

[0042] Preferably, the ethanol in step (A) is 70% vol ethanol.

[0043] Preferably, in step (A), the ethanol soaking is carried out using 0.8 times 70% vol ethanol for 12 hours.

[0044] Preferably, step (4) specifically involves: taking 14 kg of the saffron, crushing the saffron with a grinding trough, passing it through a No. 3-4 sieve, adding 0.8 times the amount of 70% vol ethanol and soaking it for 12 hours, then adding it to a percolator and extracting it by percolation with 70% vol ethanol to obtain percolate, and collecting 150 L of percolate.

[0045] Preferably, step (B) concentration is vacuum concentration, with vacuum concentration conditions of 60-80℃ and rotation speed of 30-60 rpm.

[0046] Preferably, the drying process in step (B) is freeze drying, and the freeze drying conditions are a temperature of -10℃ to -50℃ and a vacuum degree of 1.3-13 Pa.

[0047] This invention provides a saffron extract, which is prepared by the above-described method for preparing saffron extract.

[0048] This invention provides an application of the above-mentioned saffron extract in the preparation of lipid-lowering drugs and health foods.

[0049] Preferably, the lipid-lowering drug or health food contains 0.1g to 10g of the saffron yeast extract per unit dose.

[0050] Currently, commonly used chemical drugs for the clinical treatment of hyperlipidemia include statins, fibrates, niacin, resins, antioxidants, and ezetimibe. Their lipid-lowering mechanisms mainly involve three pathways: inhibiting endogenous and exogenous lipid synthesis and absorption, and promoting lipid transport, metabolism, and excretion. Inhibition of exogenous lipid absorption primarily affects cholesterol absorption in the small intestine, reducing cholesterol absorption within the intestine; for example, ezetimibe is a cholesterol absorption inhibitor. Inhibition of endogenous lipid synthesis occurs through two main mechanisms: first, inhibiting the activity and mRNA expression level of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-Co A) reductase to reduce cholesterol synthesis; and second, inhibiting acetyl-CoA transferase (ACC) and fatty acid synthase (FAS) to reduce fatty acid synthesis. Promoting lipid transport, metabolism, and excretion mainly involves promoting low-density lipoprotein receptor (LDL-R) expression, while promoting fatty acid excretion primarily involves enhancing cholesterol 7α-hydroxylase (CYP7a-1) activity.

[0051] Most commonly used lipid-lowering drugs have adverse reactions. Although they may provide good short-term efficacy, long-term use can cause varying degrees of harm to the body. Statins primarily cause liver damage; the extent of liver damage from long-term use remains to be observed. Less common adverse reactions include myotoxicity. Fibrates, mainly due to incomplete absorption, can cause gastrointestinal symptoms such as nausea, stomach discomfort, and loss of appetite. Other possible side effects include headache, dizziness, insomnia, itchy skin, urticaria, rash, hair loss, decreased libido, and changes in liver and kidney function. Niacin can cause skin vasodilation leading to itching, erythema, hot flashes, and headaches, as well as gastrointestinal side effects such as nausea and diarrhea. Resin-based drugs are ineffective for any type of hypertriglyceridemia and can cause gastrointestinal side effects such as bloating, gas, and constipation. Furthermore, it can affect the absorption of thyroid hormones, digoxin, warfarin, folic acid, and other fat-soluble vitamins, requiring long-term patients to supplement with folic acid, vitamins A, D, K, and calcium intermittently. This causes inconvenience and increases the mental and financial burden on patients. Antioxidant drugs, while lowering blood lipids, can also lower HDL-C levels in the blood. Drugs like ezetimibe generally cause skin symptoms such as rashes and urticaria, as well as thrombocytopenic purpura, and can also cause musculoskeletal damage. They are ineffective for non-dietary hypercholesterolemia and can even raise its cholesterol levels. While taking deep-sea fish oil with omega-3 fatty acids can also control blood lipids, long-term use can lead to decreased vision and even bleeding. Therefore, developing drugs for the prevention and treatment of hyperlipidemia is of great significance, especially in developing countries where the incidence of hyperlipidemia and cardiovascular diseases is increasing.

[0052] The present application is described in detail below compared with the prior art:

[0053] This invention provides a method for preparing saffron yeast and its preparation. The preparation method exhibits good process stability, minimal batch-to-batch compositional variation, and consistent quality. This invention optimizes the nutrient solution, fermentation, and drying processes, effectively increasing both the lovastatin content and the acid lovastatin content in the saffron yeast.

[0054] Comparative analysis of the HPLC fingerprints of saffron yeast and similar products on the market, such as Xuezhikang capsules and Zhibituo tablets, showed that saffron yeast slices differed significantly from other products in terms of composition. Eleven different components were preliminarily identified, mainly nucleoside and monacolins, with lovastatin being one of the main components.

[0055] This invention provides a saffron extract, its preparation method, and its application. The saffron extract can significantly improve blood lipid indicators and inhibit the rise of the weight curve in a hyperlipidemia model, making it more consistent with the weight curve of the blank group. At the same time, it can reduce liver index and inhibit fat deposition formation.

[0056] In addition to lovastatin and β-glucan, the saffron yeast extract of this invention may contain other components that help lower blood lipids.

[0057] The saffron extract group of this invention can inhibit the weight gain of the golden hamster hyperlipidemia model, making its weight change curve consistent with the blank group, and reducing the deposition of abdominal fat and epididymal fat.

[0058] The saffron extract of this invention (containing lovastatin equivalent to a human daily dose of 10 mg) has a better lipid-lowering effect than the lovastatin group (containing lovastatin equivalent to a human daily dose of 20 mg). Although there was no significant difference in weight loss, reduction of abdominal fat, and reduction of epididymal fat deposition compared to the lovastatin group, there was a decreasing trend. This suggests that in addition to lovastatin, the saffron extract may contain other substances that can help lower blood lipids, reduce weight, and reduce fat deposition, or that the β-glucan contained in saffron may have a systemic effect on lovastatin.

[0059] The components in the saffron extract group of this invention differ from those in the saffron decoction, and include components that affect the mental state of mice and rapidly reduce their weight. Attached Figure Description

[0060] Figure 1 The peak-matching fingerprint spectrum in Example 1, section 3.1;

[0061] Figure 2 The fingerprint spectrum is used as a reference in 3.1 of Example 1;

[0062] Figure 3 The full-spectrum peak matching diagram of the fingerprint spectrum of Xuezhikang capsules and saffron yeast slices in Example 1, section 3.2 is shown.

[0063] Figure 4 The full-spectrum peak matching diagram of the fingerprint spectrum of the 3.3 medium-liposome sample of Example 1 and the control of saffron yeast slices;

[0064] Figure 5 This is a full-spectrum peak matching diagram of the fingerprint spectra of other red yeast rice samples and saffron red yeast rice slices in Example 1, section 3.4;

[0065] Figure 6 Example 1, section 3.6, shows the common characteristic fingerprint peaks in the fingerprint spectrum of saffron yeast slices as a control.

[0066] Figure 7 The full-spectrum peak matching diagrams of the fingerprint spectra of the four groups of red yeast rice products in Example 1, section 3.7 are shown.

[0067] Figure 8 The table shows the peak-to-peak area matching data for the four groups of red yeast rice products in Example 1, section 3.7.

[0068] Figure 9 The principal component analysis plot in section 3.7 of Example 1 is shown.

[0069] Figure 10 The example is the cluster analysis dendrogram in section 3.7 of Example 1;

[0070] Figure 11 The image shows a thermogram analysis of 3.7 of the efficacy example 1, containing saffron red yeast rice slices, Xuezhikang capsules, Zhibituo tablets, and other red yeast rice samples.

[0071] Figure 12 The diagram shows the differences in components between saffron yeast slices and Xuezhikang capsules in Example 1, section 3.7.

[0072] Figure 13 Thermographic analysis of saffron yeast slices and Xuezhikang capsules in Example 1, section 3.7;

[0073] Figure 14 The weight change curve in 3.1 of Example 2;

[0074] Figure 15 The effect of saffron yeast on serum TC in the hyperlipidemia model in Example 2, section 3.2 is shown in the figure.

[0075] Figure 16 The effect of saffron yeast on serum TG in the hyperlipidemia model in Example 2, section 3.2 is shown in the figure.

[0076] Figure 17 The effect of saffron yeast on serum HDL-C in the hyperlipidemia model in Example 3.2 is shown in the figure.

[0077] Figure 18 The effect of saffron yeast on serum LDL-C in the hyperlipidemia model in Example 2, section 3.2 is shown in the figure.

[0078] Figure 19 The effect of saffron yeast on liver index in the hyperlipidemia model in Example 2, section 3.3 is shown in the figure.

[0079] Figure 20 The effect of saffron extract on abdominal fat in Example 2, section 3.4 is shown in the figure.

[0080] Figure 21 The effect of saffron extract on epididymal fat in Example 2, section 3.4 is shown in the figure.

[0081] Figure 22 Effects of saffron yeast extract on the pathological morphology of liver tissue in golden hamsters (x200);

[0082] Figure 23 The flowchart for metabolomics detection in section 2.4 of Example 2 is shown.

[0083] Figure 24 PCA analysis of liver metabolites in golden hamsters in Example 2(3);

[0084] Figure 25 Cluster analysis diagram of liver metabolites of golden hamsters in Example 2, Part 3;

[0085] Figure 26 This is a classification diagram of the KEGG compounds in Example 2, Section 3;

[0086] Figure 27 This is a classification diagram of the HMDB compounds in Example 2, Section 3;

[0087] Figure 28 This is the KEGG functional pathway diagram in Example 2, Section 3;

[0088] Figure 29 The graph shows the KEGG pathway enrichment results in Example 2, Section 3.

[0089] Figure 30 This is a diagram of the PPAR signal path in Example 2, Section 3.

[0090] Figure 31 The effect of the saffron extract in Example 2(3) on PPARα, CYP7A1 and CPT-1 proteins is shown in the figure.

[0091] Figure 32 The diagram shows the PPARα signal path in Example 2. Detailed Implementation

[0092] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0093] A method for preparing saffron yeast, comprising:

[0094] (1) Barley, auxiliary materials, and nutrient solution are mixed in a weight ratio of (40-100):(5-60):(20-45) and then matured to obtain a fermentation substrate; the nutrient solution comprises, by weight: 0.1-1 parts sucrose, 0.2-1 parts glutamic acid, 0.01-0.26 parts histidine, 0.01-0.15 parts calcium nitrate, 0.01-0.15 parts sodium nitrate, 0.01-0.05 parts potassium dihydrogen phosphate, 0.01-0.05 parts octanoic acid, 0.1-0.5 parts ascorbic acid, 0.00001-0.00005 parts disodium EDTA, and 96-98.07 parts water;

[0095] (2) Fermentation was carried out after inoculating the fermentation substrate with red yeast seed liquid to obtain fermentation product;

[0096] (3) Dry the fermented material.

[0097] Preferably, the weight ratio of barley, auxiliary materials, and nutrient solution is 88:12:40.

[0098] Preferably, the auxiliary material consists of wheat bran and soybean meal, with a weight ratio of wheat bran to soybean meal of (5-12):(2-5).

[0099] Preferably, the auxiliary material consists of wheat bran and soybean meal, with a weight ratio of wheat bran to soybean meal of 10:2.

[0100] Preferably, the nutrient solution, by mass percentage, consists of 0.41% sucrose, 0.76% glutamic acid, 0.26% histidine, 0.09% calcium nitrate, 0.03% sodium nitrate, 0.01% potassium dihydrogen phosphate, 0.01% octanoic acid, 0.34% ascorbic acid, 0.00001% disodium EDTA, and 98.07% water.

[0101] Preferably, the barley is treated by removing residue and crushing.

[0102] Preferably, the mixing process of barley, auxiliary materials, and nutrient solution involves mixing the barley, auxiliary materials, and nutrient solution for 5-7 minutes. This ensures that the materials are evenly mixed, can be formed into a ball by hand, and crumble easily when lightly pressed.

[0103] Preferably, the curing conditions are 121°C and the curing time is 30 minutes.

[0104] Preferably, the method further includes: mixing highland barley, auxiliary materials, and nutrient solution in a weight ratio and then bottling them, with each bottle containing a certain amount.

[0105] 360-440g / bottle, to obtain bottled material; bottled material is then matured to obtain fermentation substrate.

[0106] Preferably, in step (2), the fermentation substrate is cooled to 25-45°C before inoculating the red yeast seed liquid.

[0107] Preferably, the preparation process of the red yeast seed solution includes:

[0108] Red Monascus strain was inoculated onto slant culture medium and cultured at 30℃ for 6-7 days. The spores were then washed off with water to obtain a spore suspension.

[0109] The spore suspension was inoculated into a fermenter and cultured at 30°C for 2-3 days to obtain Monascus purpureus seed solution.

[0110] Preferably, the preparation process of the red yeast seed solution includes:

[0111] Red Monascus strain was inoculated onto slant culture medium and cultured at 30℃ for 6-7 days. The spores were then washed with 300 ml of sterile water to obtain a spore suspension.

[0112] The spore suspension was inoculated into a fermenter and cultured at 30°C for 2-3 days to obtain the red yeast seed solution.

[0113] Preferably, the red yeast strain is Monascus pilosus (strain: YWG-1), a fungus belonging to the Aspergillus family.

[0114] Preferably, the fermentation process in step (2) specifically includes:

[0115] High-temperature fermentation: After inoculating the fermentation substrate with red yeast seed liquid, fermentation is carried out for 6 days at 27-29℃ and relative humidity of 60%-65%.

[0116] Gradient cooling fermentation: Gradient cooling fermentation is carried out by gradually decreasing the temperature to 23℃ at a rate of 2-3℃ / day, while keeping the relative humidity constant.

[0117] Low-temperature fermentation: Fermentation is carried out at 21-23℃ and relative humidity of 60%-65% for 7-8 days to obtain fermented product.

[0118] Preferably, the inoculation amount of Monascus purpureus seed liquid in step (2) is 0.114 to 0.139 mL / g.

[0119] Preferably, fermentation is terminated when the total lovastatin content in the fermented product is ≥0.6% in (2).

[0120] Preferably, during the high-temperature fermentation process, the fermentation substrate is shaken for the first time 48 hours after being inoculated with red yeast seed liquid, and then shaken once every 24 hours, for a total of 4 shakes.

[0121] Preferably, the flask is shaken once during the gradient cooling fermentation process.

[0122] Preferably, the flask is shaken once every 72 hours during the high-temperature fermentation process, and the number of shakes is ≤4.

[0123] Preferably, step (3) specifically includes: drying the fermented material at a temperature of 60°C, and stopping the drying process when the moisture content of the saffron yeast is ≤10%.

[0124] Preferably, step (3) specifically includes: drying the fermented material at a temperature of 60°C, and stopping the drying process when the moisture content of the saffron yeast is ≤10%.

[0125] The present invention also provides the application of the above-mentioned saffron yeast in the preparation of lipid-lowering drugs and food.

[0126] This invention also provides a method for preparing saffron yeast extract, comprising:

[0127] (A) After crushing the above-mentioned saffron yeast, it was successively subjected to moistening and percolation extraction to obtain percolate;

[0128] (B) The percolate was concentrated and dried to obtain saffron extract.

[0129] Preferably, step (A) specifically involves: pulverizing the saffron yeast of claim 7 and then sequentially soaking it in ethanol and percolating it with ethanol to obtain a percolate;

[0130] Preferably, the ethanol in step (A) is 70% vol ethanol.

[0131] Preferably, in step (A), the ethanol soaking is carried out using 0.8 times 70% vol ethanol for 12 hours.

[0132] Preferably, step (4) specifically involves: taking 14 kg of the saffron, crushing the saffron with a grinding trough, passing it through a No. 3-4 sieve, adding 0.8 times the amount of 70% vol ethanol and soaking it for 12 hours, then adding it to a percolator and extracting it by percolation with 70% vol ethanol to obtain percolate, and collecting 150 L of percolate.

[0133] Preferably, step (B) concentration is vacuum concentration, with vacuum concentration conditions of 60-80℃ and rotation speed of 30-60 rpm.

[0134] Preferably, the drying process in step (B) is freeze drying, and the freeze drying conditions are a temperature of -10℃ to -50℃ and a vacuum degree of 1.3-13 Pa.

[0135] This invention provides a saffron extract, which is prepared by the above-described method for preparing saffron extract.

[0136] This invention provides an application of the above-mentioned saffron extract in the preparation of lipid-lowering drugs and health foods.

[0137] Preferably, the lipid-lowering drug or health food contains 0.1g to 10g of the saffron extract per unit dose.

[0138] In the examples of the effects of this invention, the methanol and ethanol solutions are expressed as volume percentages.

[0139] The red yeast strain is *Monascus pilosus* (strain: YWG-1) (commercially available), a fungus belonging to the Aspergillus family.

[0140] Example 1

[0141] Preparation of seed solution:

[0142] (1) Inoculate the red yeast strain with good growth status onto the slant of the eggplant-shaped bottle and incubate at 30℃ for 6-7 days (for specific operation, refer to: Standard Operating Procedure for Subculture and Purification of Production Strains XZYW-SOP-001-01). Wash with 300ml of sterile water to prepare spore suspension.

[0143] (2) Inoculate the spore suspension into the fermenter at an inoculation rate of about 1 wt%, and culture at 30℃, dissolved oxygen 100%, and 140 r / min for 2-3 days to obtain the seed solution.

[0144] Red Monascus strain was inoculated onto slant culture medium and cultured at 30℃ for 6-7 days. The spores were then washed off with water to obtain a spore suspension.

[0145] The spore suspension was inoculated into a fermenter and cultured at 30°C for 2-3 days to obtain the red yeast seed solution.

[0146] Preparation method of saffron yeast:

[0147] (1a) Raw material processing: After removing impurities, residue, and mold from the barley raw material, it is screened, crushed, and passed through a 20-mesh sieve.

[0148] (1b) Preparation of culture medium: The culture medium is prepared according to the ratio of 88wt% barley, 10wt% wheat bran and 2wt% soybean meal.

[0149] Table 1 Culture substrate formulation

[0150]

[0151] (1c) Preparation of nutrient solution: The nutrient solution shall be prepared strictly according to the following formula and weighing.

[0152] Table 2 Nutrient Solution Formula

[0153]

[0154] (1d) Mixing

[0155] Nutrient solution was added to the dry culture medium at a ratio of 40 wt% to obtain the prepared material.

[0156] Add the prepared materials to the mixer and mix for 5-7 minutes to ensure that the barley, bran, soybean meal and nutrient solution are mixed evenly. The mixture should be able to be formed into a ball by hand and crumble easily when pressed lightly.

[0157] (1e) Bottling: After mixing, the material is bottled at a rate of 400g / bottle (±10%) to obtain the bottled material. During this process, QA (Quality Assurance) personnel need to randomly check the bottling quantity every 15 minutes to ensure the bottling quantity is accurate.

[0158] (1f) Maturation: The bottled material is matured to obtain the fermentation substrate; the maturation temperature is 121℃ and the time is 30 minutes. After maturation, the fermentation substrate is immediately taken out to the inoculation room, broken up and cooled.

[0159] (2a) Inoculation: Cool the fermentation substrate to 25-45℃ and inoculate with red yeast seed liquid. The inoculation volume is 50ml per bottle. Shake the bottle immediately after inoculation.

[0160] (2b) Fermentation

[0161] High-temperature fermentation: After inoculating the fermentation substrate with red yeast seed liquid, fermentation is carried out for 6 days at 27-29℃ and relative humidity of 60%-65%.

[0162] Gradient cooling fermentation: Gradient cooling fermentation is carried out by gradually decreasing the temperature to 23℃ at a rate of 2-3℃ / day, while keeping the relative humidity constant.

[0163] Low-temperature fermentation: Fermentation is carried out at 21-23℃ and 60%-65% relative humidity for 7-8 days to obtain fermented product. Fermentation is terminated when the total lovastatin content in the fermented product is ≥0.6%.

[0164] Fermentation parameters are shown in Table 3.

[0165] Table 3 Fermentation parameters

[0166]

[0167] (3a) Inverting the bottle: After fermentation is complete, invert the bottle to remove contaminated or clumped fermentation material.

[0168] (3b) Drying: Dry the fermented material at a temperature of 60°C. The fermented material can be turned 1-2 times during the drying process. Stop drying when the moisture content of the saffron yeast is ≤10%.

[0169] (3c) Receiving materials: When the moisture content of the saffron is ≤10%, the materials should be received immediately. The receiving time should not be extended, and the drying process should not be overdryed. The dried saffron should be packaged in clean plastic bags, weighed, and labeled.

[0170] Preparation of Saffron Yeast Extract:

[0171] (4) Take 14 kg of the above-mentioned saffron, crush the saffron with a mortar, pass it through a No. 3 sieve, add 0.8 times 70% vol ethanol (the volume ratio of ethanol to saffron is 1:0.8) and let it soak for 12 hours, then add it to a percolator and extract it by percolation with 20 times the amount of 70% vol ethanol to obtain percolate, and collect 150 L of percolate.

[0172] (5) While collecting the percolate, the percolate was placed in a large rotary evaporator for vacuum concentration. The vacuum concentration conditions were 60-80℃ and 30-60 rpm. The concentration was carried out until the volume was 10000ml to obtain the total extract concentrate. Then, it was freeze-dried at -25℃ and vacuum degree 1.3-13pa for 120min to obtain the saffron extract.

[0173] Comparative Example 1

[0174] The only differences between this comparative example and Example 1 are the culture medium formulation, nutrient solution formulation, and fermentation parameters, as detailed in the table below.

[0175] Table 4 Culture substrate formulation

[0176]

[0177] Table 5 Nutrient Solution Formula

[0178]

[0179] Table 6 Fermentation parameters

[0180]

[0181]

[0182] Example of effect 1

[0183] 1. Experimental instruments, materials and reagents

[0184] 1.1 Experimental Instruments: KQ-300DE CNC ultrasonic cleaner (300W power, adjustable frequency, Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water system (Milli-Q Corporation, USA); electric thermostatic drying oven (Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.); alcohol meter; BT25S electronic analytical balance (Beijing Sartorius Instrument Systems Co., Ltd.); R-210 rotary thin-film evaporator (BUCHI GmbH, Switzerland); LC-20ATXR Shimadzu liquid chromatograph (Shimadzu Instruments, Japan); ABsicex TOF5600+ mass spectrometer (Abercrombie & Fitch Instruments, USA); disposable sterile syringes (with needles) (Shengguang Medical Products Co., Ltd.); disposable 0.22 and 0.45 μm filter membranes (Guangzhou Wenrui Scientific Instrument Co., Ltd.); YMC C18 chromatographic column (YMC Technology, Japan).

[0185] 1.2 Establishment of fingerprint spectrum for experimental materials A total of 14 batches of saffron were used as the target medicinal material. The saffron was provided by Tibet Yuewang Biotechnology Co., Ltd., and all were prepared by the preparation method of saffron in Example 1. In addition, 12 batches of samples of commercially available Xuezhikang, Zhibituo and Chinese medicine saffron were collected.

[0186] Sample information is shown in Table 7.

[0187] Table 7 Information on Tibetan Red Yeast Rice Slices and Samples of Traditional Chinese Medicine Red Yeast Rice

[0188]

[0189]

[0190] 1.3 Test Reagents Methanol used in sample preparation was analytical grade and purchased from Xilong Chemical Co., Ltd. Methanol used in liquid chromatography was chromatographic grade (batch number: 654655-5456, Tiandi Company, USA), and water was ultrapure water produced by a Millipore water purification system in the laboratory.

[0191] 2 methods

[0192] 2.1 Chromatographic conditions: The chromatographic column was a YMC C18 column (150 mm × 4.6 mm, 3 μm), the column temperature was 30 ℃, the flow rate was 1 mL / min, the detection wavelength was 250 nm, and the injection volume was 10 μL. The mobile phase was a pure water (A)-methanol (B) system with a gradient elution program: 0–3 min, 5% methanol; 3–7 min, 5%–15% methanol; 7–10 min, 15%–15% methanol; 10–15 min, 15%–56% methanol; 15–25 min, 56%–77% methanol; 25–32 min, 77%–77% methanol; 32–36 min, 77%–100% methanol; 36–39 min, 100%–100% methanol; 39–40 min, 100%–5% methanol; 40–44 min, 5% methanol.

[0193] 2.2 Mass Spectrometry Conditions: The ion source was an electrospray ionization source (ESI) in negative ion mode; the mass scan range was m / z 50–1200; the spray voltage was -4500V, the nebulizer gas temperature was 550℃, the curtain gas pressure was 170.64kPa, and the nebulizer gas and auxiliary gas pressures were 350.25kPa; the declustering voltage (DP) was -100V; data were acquired using the TOF-MS-IDA-MS / MS method. The TOF / MS first-stage pre-scan and triggered second-stage scan ion accumulation times were 500 ms and 200 ms, respectively. The CE collision energy was 35 eV, and the CES collision energy superposition was (35±10) eV. The second-stage scan was triggered by IDA, and the triggering conditions were multiple mass defect (MMDF) and dynamic background subtraction (DBS). The second-stage scan was prioritized if the above conditions were met.

[0194] 2.3 Preparation of test solution: Take about 0.5g of each batch of medicinal material (passed through a No. 4 sieve), weigh accurately, place in a stoppered conical flask, add 25ml of 30% methanol accurately, stopper tightly, weigh, sonicate (power 140W, frequency 42kHz) for 1.5 hours, cool to 15-35℃, weigh again, replenish the lost weight with methanol, centrifuge at high speed to extract the liquid, take the supernatant and filter it through a 0.45μml filter, and take the filtrate.

[0195] 2.4 Investigation of HPLC fingerprinting method for Tibetan red yeast rice

[0196] 2.4.1 The chromatographic conditions for fingerprint spectroscopy were investigated using the richness of chromatographic peak information presented in the fingerprint spectroscopy and the extraction rate of the main active ingredient, lovastatin, as evaluation indicators. The elution conditions of the mobile phase, column temperature, and detection wavelength were ultimately determined. Under these conditions, chromatographic peaks from highly polar to less polar were well separated and presented.

[0197] 2.4.2 The preparation method of the test solution was investigated using the richness of chromatographic peak information presented by the fingerprint spectrum and the extraction rate of the main active ingredient lovastatin as evaluation indicators. The solvent selection (30%, 60%, 100% methanol and 30%, 60%, 100% ethanol) and ultrasonic time (0.5h, 1h, 1.5h, 2h, 2.5h) of the preparation method were investigated. Finally, the preparation method of the test solution was determined, which can comprehensively reflect the components of saffron from low polarity to high polarity.

[0198] 2.4.3 Precision Test: The reference medicinal material test solution was injected six times consecutively and analyzed according to the conditions in section 2.1. Using the lovastatin peak as the reference peak, the relative peak area and retention time of each of the six common chromatographic peaks were calculated. The results showed that the RSD of the relative peak area was less than 2.0%, and the RSD of the relative retention time was less than 1%, indicating good instrument precision.

[0199] 2.4.4 Repeatability Test: Using the same batch of medicinal materials, six parallel samples were prepared according to the method in section 2.2. Analyzed under the conditions in section 2.1, using the lovastatin peak as a reference peak, the relative peak area and retention time of each of the six common chromatographic peaks were calculated. The results showed that the RSD of the relative peak area was less than 3.0%, and the RSD of the relative retention time was less than 1%.

[0200] 2.4.5 Stability Test: The same batch of medicinal material solution was injected at intervals of two hours, at 0, 2, 4, 6, 8…48 hours. Analysis was performed according to the conditions in section 2.1, using lovastatin as the reference peak. The relative peak area and retention time of each of the six common chromatographic peaks were calculated. Results showed that the RSD of the relative peak area of ​​all common peaks was less than 3.0% within 24 hours, and the RSD of the relative retention time was less than 1%, indicating that the sample was stable within 24 hours.

[0201] 2.5 Data Processing and Analysis Fingerprint analysis was performed using fingerprinting software based on the 2012 edition of the Chinese Pharmacopoeia. Common peaks were analyzed by mass spectrometry using Peakview 1.2 software from ABSCIEX, and characteristic peaks were structurally identified using database and literature references. A "component-peak area" data table for each sample was obtained through fingerprint analysis, and this data table was imported into MetaboAnalyst online analysis software for principal component analysis (PCA) and heatmap analysis.

[0202] 3 Results and Discussion

[0203] 3.1 HPLC fingerprint analysis of 14 batches of saffron yeast slices

[0204] Under the conditions described in section 2.1, 14 batches of Tibetan red yeast rice slices (serial numbers 1-14) were collected, with two samples taken from each batch. For the chromatographic data of the 28 samples, peaks with retention times between 2 and 38 min and peak heights greater than 1000 were automatically integrated. Using the chromatogram of sample number 20080201 as the reference chromatogram, the median generation method was employed, with a time width of 0.1 min. The chromatographic data of the 28 samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version). Six common characteristic component peaks were selected as marker peaks for multi-point correction, and full-spectrum peak matching was performed to obtain the peak-matching fingerprint spectrum. Figure 1 The software generates a comparison fingerprint spectrum. Figure 2 Similarity data for each batch of samples was obtained through software similarity analysis, and the results are shown in Table 8.

[0205] Figure 1 Fingerprint analysis and full-spectrum peak matching diagrams for 28 batches of saffron yeast slices (R: control fingerprint; S1~S28: saffron yeast slice samples).

[0206] Figure 2 Fingerprint spectrum of saffron yeast slices (peaks 1-10 are common characteristic fingerprint peaks, peak 8 is set as reference peak S)

[0207] Table 8. Similarity analysis results between 28 batches of saffron yeast slices and control chromatograms

[0208]

[0209]

[0210] The results showed that the similarity between the established fingerprint chromatogram of saffron yeast slices and the 28 samples was all above 0.95. This indicates that the quality stability of each batch of saffron yeast slices used in the experiment was very good, with small differences between batches.

[0211] 3.2 Comparative analysis of fingerprint spectra of saffron yeast slices and Xuezhikang capsules (a traditional Chinese medicine)

[0212] Six batches of Xuezhikang capsules were tested under the conditions described in section 2.1. Two samples were taken from each batch. The obtained chromatographic data and the fingerprint chromatogram of saffron yeast slices were imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" (2012 version) to calculate the similarity. The results are shown in [Figure 2]. Figure 3 Table 9.

[0213] The results showed that the similarity of the fingerprint spectra of each sample of Xuezhikang capsules and Tibetan red yeast rice slices was between 0.85 and 0.88, indicating that there are certain differences in the composition of Xuezhikang capsules and Tibetan red yeast rice slices.

[0214] Figure 3Full-spectrum peak matching diagram of fingerprint chromatogram of Xuezhikang capsule sample and saffron yeast slices (R: fingerprint chromatogram of saffron yeast).

[0215] S1~S12: Samples of Xuezhikang Capsules)

[0216] Table 9. Similarity analysis results of fingerprint spectra between Xuezhikang capsules and Zanghongqu decoction pieces.

[0217]

[0218]

[0219] 3.3 Comparative Analysis of Fingerprint Chromatography between Saffron Yeast Rice Slices and the Traditional Chinese Medicine Lipitor Tablets

[0220] Under the conditions described in section 2.1, one batch of the traditional Chinese medicine Lipitor tablets was tested. Six samples were taken, and the obtained chromatographic data and the fingerprint chromatogram of saffron yeast slices were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) to calculate the similarity. The results are shown in [the table below]. Figure 4 See Table 10.

[0221] Figure 4 Full-spectrum peak matching diagram of fingerprint chromatograms of Lipitor sample and saffron yeast slices (R: fingerprint chromatogram of saffron yeast slices; S1~S6: Lipitor sample)

[0222] Table 10. Similarity analysis results of fingerprint spectra between Lipitor samples and Saffron Yeast Extract slices.

[0223]

[0224] The results showed that the similarity of the fingerprint spectra between Lipitor tablets and Tibetan red yeast rice slices was about 0.95, indicating that there was little difference in the composition of Lipitor tablets and Tibetan red yeast rice slices.

[0225] 3.4 Comparative analysis of fingerprint spectra of saffron yeast slices and other Chinese herbal yeast samples

[0226] Under the conditions described in section 2.1, 10 samples from 5 batches of other traditional Chinese medicine red yeast rice samples (2 samples from each batch) were tested. The obtained chromatographic data and the fingerprint chromatogram of saffron red yeast rice slices were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) to calculate the similarity. The results are shown in […]. Figure 5 See Table 11.

[0227] Figure 5 Full-spectrum peak matching diagrams of fingerprint spectra of other red yeast rice samples and saffron red yeast rice slices (R: fingerprint spectrum of saffron red yeast rice slices; S1~S10: other red yeast rice samples)

[0228] Table 11. Similarity analysis results of fingerprint spectra between other red yeast rice samples and saffron red yeast rice slices.

[0229]

[0230] The results showed that the fingerprint similarity between the samples from the five manufacturers used in this study and the control saffron red yeast rice slices ranged from a minimum of 0.33 to a maximum of 0.81, indicating that the composition of the red yeast rice samples from these five manufacturers differed significantly from that of saffron red yeast rice slices, and that the quality stability among different batches of samples was poor.

[0231] 3.5 Discussion:

[0232] The above fingerprint analysis of saffron red yeast rice slices and other samples of red yeast rice from traditional Chinese medicine shows that the fingerprint spectrum of saffron red yeast rice slices prepared in this invention can better reflect the quality and stability of the saffron red yeast rice slices product, and can identify the differences between saffron red yeast rice slices and other traditional red yeast rice products commonly used in clinical practice, such as Xuezhikang capsules and Zhibituo tablets. The results show that... The composition of Lipitor tablets is quite similar to that of saffron yeast slices. The similarity is around 0.95; the composition of Xuezhikang capsules differs significantly from that of Tibetan red yeast rice slices, with similarities ranging from 0.85 to 0.88; while the red yeast rice samples from the five manufacturers used in this invention show significant similarities to the Tibetan red yeast rice slices of this invention, with similarities ranging from 0.33 to 0.81, reflecting the poor quality consistency of red yeast rice slices produced by different manufacturers in the market.

[0233] 3.6 U HPLC-MS / MS method for the identification of component peaks in the fingerprint spectrum of saffron yeast

[0234] Preliminary identification was performed by searching databases and comparing literature using UV and mass spectrometry fragment information of chromatographic peaks and comparing the fingerprint chromatogram of saffron yeast slices with 21 common characteristic peaks. A total of 20 components were identified, and the peak numbers are shown below. Figure 6 ( Figure 6 Table 12 shows the common characteristic fingerprint peaks in the fingerprint spectrum of saffron yeast slices (as a reference). The mass spectrometry-related information of the identification results of each compound is shown in Table 12.

[0235] Of the identified components, peaks 2, 3, and 4 represent nucleoside compounds; peaks 13, 14, and 15 represent red yeast rice pigments; peak 18 represents lovastatin (Monacolin K), the main active ingredient in saffron red yeast rice slices; and peak 10 represents cirin, the main toxic component. The other 14 chromatographic peaks represent monacolins. The results indicate that saffron red yeast rice slices contain various monacolin (statin) compounds, with lovastatin (Monacolin K) being a representative component with relatively high content. Furthermore, saffron red yeast rice slices also contain cirin, a toxic component commonly found in red yeast rice products.

[0236] Table 12 Mass Spectrometry Identification Information of Components in Saffron Yeast Rice Slices

[0237]

[0238]

[0239]

[0240]

[0241] 3.7 Comparative Analysis of Chemical Composition of Tibetan Red Yeast Rice Slices and Other Red Yeast Rice Products such as Xuezhikang

[0242] 3.7.1 All red yeast rice products were divided into four groups: Tibetan red yeast rice slices, Xuezhikang capsules, Zhibituo tablets, and other red yeast rice (traditional Chinese medicine red yeast rice). Six samples were taken from each group. The aforementioned fingerprinting method was used for analysis. The data were imported into the fingerprinting software system, and after multi-point calibration, full-spectrum peak matching was performed (see...). Figure 7 Extract the "chromatographic peak-peak area" matching data table for each sample. Figure 8 The data in this table was formatted and uploaded to the MetaboAnalyst online analysis software system for principal component analysis (PCA), systematic number analysis, and heatmap analysis. The results showed that the fingerprinting software system extracted a total of 130 chromatographic peaks.

[0243] Figure 7 Full-spectrum peak matching diagrams of fingerprint spectra for 4 groups of red yeast rice products (S1~S6: Tibetan red yeast rice slices ZHQ-1~ZHQ-6; S7~S12: Xuezhikang capsules XZK-1~XZK-6; S13~S18: Lipitor tablets ZBT-1~ZBT-6; S19~S24: other red yeast rice (traditional Chinese medicine red yeast rice) slices OTH-1~OTH6)

[0244] Figure 8 Four sets of peak-to-peak area matching data tables for red yeast rice products

[0245] 3.7.2 Based on the data from the "Chromatographic Peak-Peak Area" table, statistical analysis was performed on the four groups of red yeast rice samples. Principal component analysis (…) Figure 9 (Scores plot between the selected PCs.) shows that the four groups of samples—Saffron red yeast rice slices, Xuezhikang capsules, Zhibituo tablets, and other red yeast rice (traditional Chinese medicine red yeast rice)—clustered together, with the first three groups showing higher aggregation degrees. From the aforementioned fingerprint analysis, it can be seen that two samples in the "other red yeast rice sample group" have significantly different compositions from the other samples, containing almost no lovastatin, a component present in all other red yeast rice samples. Furthermore, the hierarchical clustering dendrogram (…) Figure 10)(Clustering results shown asdendrogram(distancemeasure using euclidean,and clustering algorithmusing average).)And heat map analysis(Clustering result shown as heatmap)( Figure 11 The analysis reveals the close relationship between the components of saffron red yeast rice slices, Xuezhikang capsules, Zhibituo tablets, and other red yeast rice samples. The order of similarity with saffron red yeast rice slices, from most similar to Zhibituo tablets, Xuezhikang capsules, and other red yeast rice samples, is consistent with the similarity analysis results in the fingerprint chromatograms. Two samples (OTH-5 and OTH-6) clustered together separately in the other sample groups, indicating significant differences in their composition compared to other red yeast rice samples. The aforementioned fingerprint chromatogram analysis shows that these two samples do not contain lovastatin, the characteristic active ingredient in red yeast rice products.

[0246] 3.7.3 Based on the "Chromatographic Peak-Peak Area" table data, statistical analysis was performed on the two groups of samples: saffron yeast slices and Xuezhikang capsules. Based on the top 45 differentially expressed components, heatmap analysis was conducted (…). Figure 13 (Clustering result shown as heatmap top 45 (distance measure using euclidean, and clustering algorithm using average).) Combining the fingerprint spectra of saffron yeast slices and Xuezhikang capsules, peaks 1-6 in the fingerprint spectra of saffron yeast slices and peaks I-V in Xuezhikang capsules were preliminarily identified as their dominant components, and can be considered as the components with more significant differences between saffron yeast slices and Xuezhikang capsules (see...). Figure 12 ).

[0247] The structures of these 11 components were identified by high-resolution mass spectrometry. The preliminary identification results are shown in Table 13.

[0248] Figure 12 Differences in components between saffron yeast slices and Xuezhikang capsules (A: Saffron yeast 1-6; B: Xuezhikang I-V)

[0249] Table 13. Mass Spectrometry Identification Information of Differential Components Between Zanghongye Decoction Pieces and Xuezhikang Capsules

[0250]

[0251]

[0252] 4. Conclusion

[0253] 4.1 The analysis results showed that the similarity between the fingerprint spectra of each batch of saffron yeast and the control was above 0.95, indicating that the processing stability of saffron yeast slices was good, the composition difference between batches was small, and the quality was stable.

[0254] 4.2 The standard fingerprint spectrum prepared by this invention can effectively distinguish between saffron yeast slices and similar products on the market, such as Xuezhikang capsules (the similarity is less than 0.85), but the distinction with another product, Lipitor tablets, is not very high (the similarity is about 0.95), but the number of Lipitor samples collected is relatively small.

[0255] 4.3 The chromatographic peaks in the standard fingerprint spectrum of saffron yeast slices were preliminarily identified by liquid chromatography-high resolution mass spectrometry, and 21 major components were identified.

[0256] 4.4 Comparative analysis of HPLC fingerprints of saffron yeast slices with similar products on the market, such as Xuezhikang capsules and Zhibituo tablets, showed that saffron yeast slices differed significantly from other products in terms of composition. Eleven different components were preliminarily identified, mainly nucleoside and monacolins, with lovastatin being one of the main components.

[0257] Example 2

[0258] Research on the lipid-lowering activity of saffron yeast

[0259] (1) Male golden hamsters were used as experimental subjects. After their blood lipids increased after being fed a high-fat diet for 2 weeks, they were fed a high-fat diet for 4 weeks and were given saffron extract at the same time. At the end of the experiment, the lipid-lowering effect and mechanism were tested, and it was observed whether saffron would inhibit the progression of hyperlipidemia to non-alcoholic fatty liver.

[0260] (2) The experiment was set up with 7 groups: blank group (Normal), model group (Model), low-dose saffron extract group (ZHQ-low), high-dose saffron extract group (ZHQ-high), lipid-lowering control group (XZK), lovastatin positive control group (Lovastatin), and lovastatin + β-glucan control group (Lovastatin + βG) control group. The blank group was given control feed, while the model group, low-dose and high-dose saffron extract groups, lipid-lowering group, lovastatin control group, and lovastatin + β-glucan control group (Lovastatin + βG) control group were given high-fat feed. All six groups were given sufficient drinking water. The low-dose and high-dose groups of saffron yeast extract were administered by gavage daily at doses of 0.42 g / kg and 0.84 g / kg, respectively. The lovastatin control group was administered lovastatin (1.87 mg / kg) aqueous solution by gavage daily, while the lovastatin + β-glucan group was administered lovastatin (1.87 mg / kg) + β-glucan (0.017 g / kg) aqueous solution by gavage daily. The normal group and the model group were administered distilled water by gavage daily, with a gavage volume of 10 mL / kg. Body weight was recorded every 3 days. Finally, total cholesterol (TC) and triglyceride (TG) levels were measured by orbital blood sampling.

[0261] (3) At the end of the experiment, the pharmacodynamic indicators of saffron yeast in lowering blood lipids were tested.

[0262] 1) Plot the weight change curves for each group to observe the effect of saffron yeast on the weight of hyperlipidemic golden hamsters (already tested).

[0263] 2) Weigh the liver, kidney, abdominal fat, and epididymal fat of each group to calculate the organ coefficients and observe the effect of saffron on the organ coefficients.

[0264] 3) Measure serum TC, TG, high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) to observe the effect of dirty red yeast rice on blood lipids.

[0265] 4) Hematoxylin-eosin (HE) staining was used to observe the liver tissue structure and the formation of liver lipid droplets to determine whether saffron can inhibit or delay the formation of non-alcoholic fatty liver disease.

[0266] (4) Research on the mechanism of saffron yeast lowering blood lipids

[0267] 1) To investigate the effects of saffron yeast on liver metabolomics in a golden hamster hyperlipidemic model, in order to analyze the metabolic pathways affected by the lipid-lowering effect of saffron yeast.

[0268] 2) Mechanism studies on cholesterol reduction: Western blotting was used to determine the expression levels of proteins related to cholesterol metabolism in liver tissue.

[0269] 3) Mechanism study on triglyceride reduction: Western blotting was used to determine the expression level of protein related to fatty acid metabolism in liver tissue.

[0270] (I) Pharmacodynamic study on the lipid-lowering effect of saffron yeast

[0271] 1. Experimental Materials

[0272] 1.1 Experimental Drugs

[0273] Saffron decoction: The saffron prepared in Example 1 of this invention (Tibet Yuewang Pharmaceutical Ecological Tibetan Medicine Technology Co., Ltd.) is used as follows: For internal use: decoct 6-12g; for external use: steep in boiling water: 3g each time, 2-4 times a day, or as directed by a physician.

[0274] The experiment was based on a human dose of 9g / day and an adult weight of 70kg. The dose was converted to 0.84g / kg for golden hamsters using the body surface area method (equivalent to a human dose of 9g / day). This dose was used as the high-dose group of saffron extract, and the low-dose group of saffron extract was 0.42g / kg (equivalent to a human dose of 4.5g / day).

[0275] Preparation of saffron yeast extract: prepared in Example 1.

[0276] Xuezhikang Capsules: Beijing Beida Weixin Biotechnology Co., Ltd., Specification: 0.3g*12 capsules / box; Dosage and administration: Oral administration, 2 capsules each time, twice a day, after breakfast and dinner; for mild to moderate patients, 2 capsules a day, after dinner. This study used a positive control drug with a human dose of 1.2g / day (calculated according to the 2015 edition of the Pharmacopoeia, 1.2g of Xuezhikang capsules contains 10mg of lovastatin). Based on an adult weight of 70kg, the dose converted to golden hamster dosage using the body surface area method is 0.11g / kg (this dose is equivalent to an adult daily lovastatin dose of 10mg).

[0277] Lovastatin Capsules: Yangtze River Pharmaceutical Group Co., Ltd., Specification: 20mg; Dosage and Administration: The usual starting dose is 20mg daily, taken once with dinner. In this study, as a positive control, the human dose was calculated based on 20mg / day for an adult weighing 70kg. The dose converted to a golden hamster dose using the body surface area method was 1.87mg / kg (this dose is equivalent to an adult daily dose of 20mg lovastatin).

[0278] β-glucan: Ningbo Qiancao Biotechnology Co., Ltd., purity 70%. The saffron yeast product contains no less than 1.5% β-glucan, averaging around 2%. 9g of saffron yeast contains 0.18g of β-glucan, which, converted to the body surface area method, corresponds to a β-glucan dosage of 0.017g / kg in golden hamsters.

[0279] 1.2 Feed

[0280] High-fat, high-fructose and high-cholesterol modeling feed, control feed and hamster maintenance feed were all purchased from Nantong Trophy Feed Technology Co., Ltd., production license number: Su Feed License (2019) 06092.

[0281] 1.3 Experimental Animals

[0282] Fifty-six male SPF-grade golden hamsters (110-130 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the use license number SCXK (Jing) 2016-0011. The indoor temperature was 21-24°C, with a 12-hour light-dark cycle, and the golden hamsters were guaranteed free access to food and drinking water.

[0283] 2 Experimental Methods

[0284] 2.1 Preparation of Intragastric Administration Solution

[0285] Xuezhikang intragastric solution with a concentration of 0.011 g / ml: weigh 0.55 g of ground Xuezhikang, adjust the volume to 50 ml with distilled water, heat in a 37°C water bath and stir until completely dissolved.

[0286] Lovastatin intragastric solution with a concentration of 0.187 mg / ml: weigh 18.7 mg of lovastatin, adjust the volume to 100 ml with distilled water, heat in a 37°C water bath and stir until completely dissolved.

[0287] High-dose Tibetan red yeast rice intragastric solution with a concentration of 0.0168 g / ml: after grinding the Tibetan red yeast rice extract, weigh 0.84 g, adjust the volume to 50 ml with distilled water, heat in a 37°C water bath and stir until completely dissolved. The low-dose group was diluted twice on the basis of the high-dose group.

[0288] Lovastatin + β-glucan intragastric solution: the concentration of lovastatin is 0.187 mg / ml, and the concentration of β-glucan is 0.0017 g / ml. 0.12 g of β-glucan extract containing 70% β-glucan was adjusted to a constant volume of 50 ml with 0.187 mg / ml lovastatin intragastric solution, heated in a 37°C water bath and stirred until completely dissolved.

[0289] 2.2 Modeling and Grouping

[0290] Fifty-six male SPF-grade golden hamsters (110-130g) were acclimatized for one week and then randomly divided into a control group (n=8) fed with a control diet and a model group (n=40) fed with a model diet. The feeding period lasted two weeks. After two weeks, the hamsters were fasted but allowed free access to water for 12 hours. Blood was collected from the inner canthus of the eye, placed on ice for 2 hours, and then centrifuged at 4°C, 3500 rpm for 15 minutes to separate the serum. Serum total cholesterol (TC) and triglyceride (TG) levels were measured. Based on TC and TG levels and body weight, the model mice were divided into 7 groups of 8 mice each: model group, Xuezhikang control group (0.11 g / kg), lovastatin control group (1.87 mg / kg), low-dose saffron group (0.112 g / kg), high-dose saffron group (0.224 g / kg), and lovastatin + β-glucan group (1.87 mg / kg + 0.017 g / kg). Mice were administered the medication once daily by gavage. The model group and control group were administered distilled water by gavage at a volume of 10 mL / kg. The gavage dosage and concentration for each group are shown in Table 14. Except for the control group, which received a control diet, all other groups received a high-fat, high-fructose, and high-cholesterol modeling diet. Free access to water was provided during the drug administration period. After drug intervention, the mice were weighed every 3 days to record changes in body weight. Two weeks after drug administration, the mice were fasted for 12 hours but allowed free access to water. Blood was collected from the inner canthus of the eye, serum was separated, and serum biochemical indicators were measured. Four weeks after drug administration, patients were kept on a fasting schedule but allowed free access to water for 12 hours. After weighing, blood was drawn from the inner canthus of the eye for serum biochemical markers. The patients were then euthanized under ether anesthesia with cervical dislocation. Three to five fecal pellets were collected from the large intestine and placed in cryovials for liquid nitrogen storage for microbial diversity assessment. Liver tissue was harvested, photographed, and weighed. Tissue samples from the same location were fixed with 4% paraformaldehyde for histopathological examination. Additionally, four liver tissue samples from each liver were frozen at -80°C for Western blotting, PCR, liver metabolomics analysis, and liver biochemical marker detection, respectively. The liver tissue samples used for each marker detection were from the same location.

[0291] Table 14 Dosage of drugs administered to mice in each group

[0292]

[0293] Lovastatin is the main active ingredient for lowering lipids in Tibetan red yeast rice and Xuezhikang. Given the different lovastatin contents in different samples, the dosage of each group was converted to the equivalent lovastatin dose for objective comparison.

[0294] 2.3 Measurement of organ weight indicators, etc.

[0295] After administration of the drug, the patient was kept on a fasting schedule but allowed free access to water for 12 hours. After weighing, the patient was anesthetized with ether, euthanized by cervical dislocation, and the liver, abdominal fat, and epididymal fat were harvested and weighed separately. Organ indices and body fat percentage were calculated.

[0296] Organ Index = Organ Weight / Body Weight * 100%

[0297] Body fat percentage = (fat weight / body weight) * 100%

[0298] 2.4 Serum marker determination

[0299] Blood was collected from the inner canthus of the eye, incubated on ice for 2 hours, then centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant serum was aliquoted and stored at -20°C, avoiding repeated freeze-thaw cycles, and thawed at room temperature. Triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured according to the reagent instructions.

[0300] 2.5 Liver Indicator Measurement

[0301] Determination of TG, TC, SOD, and MDA: 100 mg of right lobe liver tissue from a golden hamster was placed in a centrifuge tube and homogenized with 900 μL of pre-cooled physiological saline. The mixture was then centrifuged at 10,000 r / min for 20 min at 4°C. The supernatant was separated, and the various parameters in the liver tissue were determined according to the manufacturer's instructions.

[0302] 2.6 Histopathological observation of liver tissue

[0303] Liver tissue was stained with hematoxylin and eosin (HE) and Oil Red stained to observe the size and number of fat vesicles in the liver. A semi-quantitative method was used to grade the degree of hepatic steatosis. A comprehensive score for hepatic steatosis was calculated.

[0304] Grading criteria for hepatic steatosis

[0305] Grade 1: <30% of hepatocytes within the liver lobules show fatty degeneration.

[0306] Grade 2: 30%-50% of hepatocytes within the liver lobules show fatty degeneration.

[0307] Grade 3: 50%-75% of hepatocytes within the liver lobules show fatty degeneration.

[0308] Grade 4: >75% of hepatocytes within the liver lobules show fatty degeneration.

[0309] 2.7 Data Analysis

[0310] Statistical analysis was performed using graphPad Prism 6.0 software. Experimental data are expressed as mean ± SD and one-way ANOVA was conducted. P < 0.01 or P < 0.05 were considered statistically significant.

[0311] 3 Results

[0312] 3.1 Effects of Saffron Yeast Extract on Body Weight of Hyperlipidemic Golden Hamsters

[0313] During the experiment, the control group showed good mental state, smooth fur, and agile movements. The model group showed dull fur, slightly sluggish reactions, and reduced activity. Compared with the model group, the mental state and activity levels of the low- and high-dose groups of Xuezhikang, Tibetan red yeast rice extract, lovastatin, and lovastatin + β-glucan were improved. Compared with the control group, the golden hamsters in the model group showed rapid weight gain. The weight of the Xuezhikang group was significantly lower than that of the control group 4 weeks after administration. The weight gain trends of the high-dose Tibetan red yeast rice group, lovastatin group, and lovastatin + β-glucan group were consistent with those of the control group, with no significant difference from the control group, but significantly lower than that of the model group.

[0314] The weight change curves for each group are shown below. Figure 14 Compared with the control group (Normal), the body weight of mice in the model group (Model) significantly increased from day 13 after the start of drug administration (P<0.05). Compared with the model group, the body weight of both the Xuezhikang group (XZK) and the high-dose saffron extract group (ZHQ-high) significantly decreased from day 10 after drug administration (P<0.05), and this trend continued consistently until day 28 after drug administration, with the body weight remaining lower than that of the control group. This indicates that the Xuezhikang group (XZK) and the high-dose saffron extract group can inhibit the body weight gain of the golden hamster model. However, the body weight change in the high-dose saffron extract group was consistent with that in the control group, while the body weight of the Xuezhikang group was significantly lower than that of the control group (P<0.05). The body weight of the low-dose saffron extract group (ZHQ-low) did not show a consistent and significant decrease compared with the model group. Both the lovastatin group and the lovastatin + β-glucan group had significantly lower body weights than the model group from day 13 after administration, consistent with the trend of body weight change in the blank group.

[0315] Figure 14 Effects of saffron yeast on changes in body weight curve in a hyperlipidemic model

[0316] (Compared to the blank group, ### P<0.001 # P<0.05; compared with the model group,

[0317] *, P<0.05, **, P<0.01, ***, P<0.001)

[0318] 3.2 Effects of Saffron Yeast Extract on Blood Lipids in Golden Hamsters

[0319] 3.2.1 Saffron yeast extract significantly reduced serum cholesterol (TC) levels.

[0320] Compared with the control group (Normal), the serum TC level in the model group (Model) was significantly increased (P<0.001). Compared with the model group, the Xuezhikang group (XZK), lovastatin group, lovastatin + β-glucan group (Lovastatin + βG), low-dose saffron group (ZHQ-low), and high-dose saffron group (ZHQ-high) all showed a trend of decreasing serum TC. However, there was no significant difference in the reduction of TC between the Xuezhikang group and the lovastatin group. The low-dose saffron group, the high-dose saffron group, and the lovastatin + β-glucan group all significantly reduced serum TC levels (P<0.05).

[0321] It is noteworthy that the combined use of lovastatin and β-glucan significantly reduced serum total cholesterol (TC) levels, indicating that β-glucan helps lovastatin lower TC (i.e., there is a synergistic effect). The amount of lovastatin in the low- and high-dose groups of saffron was lower than or equal to the lovastatin content in Xuezhikang (a traditional Chinese medicine), but it still significantly reduced TC levels, suggesting that saffron may contain other components besides lovastatin that can lower TC, or this may be due to the synergistic effect of β-glucan in saffron on lovastatin's TC-lowering effect. Figure 15 )

[0322] Table 15 Effects of saffron yeast on serum total cholesterol (TC) in a hyperlipidemic model (n=8, mean±SD)

[0323] Normal 2378.64±70.36 Model <![CDATA[3476.16±492.04 ## ]]> XZK 3343.09±101.21 Lovastatin 3491.57±224.76 Lovastatin+βG 3062.44±201.33* ZHQ-low 2976.59±175.70* ZHQ-high 3094.98±164.54*

[0324] Figure 15 Effects of saffron yeast on serum total cholesterol (TC) in a hyperlipidemic model

[0325] (Compared to the blank group, ### *P<0.001; compared with the model group, *P<0.05)

[0326] 3.2.2 Saffron yeast extract significantly reduced serum triglyceride (TG) levels.

[0327] Compared with the control group (Normal), the serum TG level in the model group was significantly increased (P<0.001). Compared with the model group, the low-dose saffron yeast group (ZHQ-low) significantly decreased serum TG levels (P<0.05). The Xuezhikang group (XZK), lovastatin group, lovastatin + β-glucan group, and high-dose saffron yeast group (ZHQ-high) all significantly decreased serum TG levels (P<0.001). Figure 16 The serum TG levels were lowest in the Xuezhikang group and the high-dose Tibetan red yeast rice group. The low-dose and high-dose Tibetan red yeast rice groups showed a dose-dependent effect in reducing TG levels.

[0328] Table 16 Effects of saffron yeast on serum TG in a hyperlipidemic model (n=8, mean±SD)

[0329]

[0330]

[0331] Figure 16 Effects of saffron yeast on serum triglycerides in a hyperlipidemic model

[0332] (Compared to the blank group, ### *P<0.001; compared with the model group, *P<0.05,***P<0.001)

[0333] 3.2.3 Saffron yeast extract significantly increased serum high-density lipoprotein (HDL-C) levels.

[0334] Compared with the control group (Normal), the serum HDL-C level in the model group (Model) was significantly decreased (P<0.001). Compared with the model group, the serum HDL-C levels in the Xuezhikang group (XZK), the lovastatin + β-glucan group (Lovastatin + βG), the low-dose saffron yeast group (ZHQ-low), and the high-dose group (ZHQ-high) were significantly increased (P<0.05, P<0.01). Although the serum HDL-C level in the lovastatin group was increased, there was no significant difference compared with the model group (P>0.05). However, the combination of lovastatin and β-glucan significantly increased the serum HDL-C level. Figure 3 This result indicates that β-glucan, when used in combination with lovastatin, helps increase serum HDL-C levels. Furthermore, the high-dose saffron yeast group (containing lovastatin equivalent to a human daily dose of 10 mg) significantly increased serum HDL-C levels (P<0.01), while the lovastatin group (containing lovastatin equivalent to a human daily dose of 20 mg) did not significantly increase serum HDL-C levels (P>0.05), suggesting that saffron yeast contains other components that can increase serum HDL-C levels. Figure 17 )

[0335] Table 17 Effects of saffron yeast on serum HDL-C in a hyperlipidemic model (n=8, mean±SD)

[0336] Normal 1.930±0.289 Model 1.079±0.264### XZK 1.737±0.510** Lovastatin 1.479±0.329 Lovastatin+βG 2.142±0.670** ZHQ-low 1.670±0.276* ZHQ-high 1.722±0.537**

[0337] Figure 17 Effects of saffron yeast on serum HDL-C in a hyperlipidemic model

[0338] (Compared to the blank group, ### *P<0.001; compared with the model group, *P<0.05,**P<0.01)

[0339] 3.2.4 Saffron yeast extract significantly reduced serum low-density lipoprotein (LDL-C) levels.

[0340] Compared with the control group (Normal), the serum LDL-C level in the model group (Model) was significantly increased (P<0.001). Compared with the model group, the Xuezhikang group (XZK), lovastatin, lovastatin + β-glucan group (Lovastatin + βG), low-dose saffron group (ZHQ-low), and high-dose saffron group (ZHQ-high) all showed a trend of decreasing serum LDL-C levels. However, only the high-dose saffron group and the lovastatin + β-glucan group significantly decreased serum LDL-C levels (P<0.05, P<0.01). The Xuezhikang group, lovastatin group, and low-dose saffron group did not significantly decrease serum LDL-C levels (P>0.05). Both lovastatin and the lovastatin + β-glucan group contained the same amount of lovastatin, but the lovastatin + β-glucan group showed a better effect in lowering LDL-C, suggesting that when lovastatin is used in combination with β-glucan, β-glucan helps to lower serum LDL-C. Both the high-dose saffron yeast group and the Xuezhikang group could lower serum LDL-C levels, but there was a significant difference in the reduction of LDL-C levels by saffron yeast. The amount of lovastatin in both groups was consistent, suggesting that saffron yeast may contain other components that can lower serum LDL-C levels, or that the synergistic effect of β-glucan in saffron yeast on the LDL-C lowering effect of lovastatin may be the cause. Figure 18 )

[0341] Table 18 Effects of saffron yeast on serum LDL-C in a hyperlipidemic model (n=8, mean±SD)

[0342]

[0343]

[0344] Figure 18 Effects of saffron yeast on serum LDL-C in a hyperlipidemic model

[0345] (Compared to the blank group, ### *P<0.001; compared with the model group, *P<0.05,**P<0.01)

[0346] 3.3 Effects of Saffron Yeast Extract on Liver in Hyperlipidemic Model

[0347] High-dose saffron extract and Xuezhikang group significantly inhibited weight gain in hyperlipidemic golden hamsters and suppressed liver enlargement (P<0.05, P<0.01), as shown in Table 8. After dissection, macroscopic observation of the liver morphology in each group showed: the liver in the blank group showed no abnormalities, was reddish-brown, firm, elastic, with a smooth capsule and thin edges. The liver in the model group was pale yellow, enlarged, with a granular surface, poor elasticity, a tense capsule, and blunt edges. Compared with the model group, the livers in the treated groups showed changes in color, texture, and elasticity. From the external appearance of the liver, the model group was pale red and enlarged, suggesting that the model group may have progressed from hyperlipidemia to fatty liver; further pathological examination of sections is needed for verification. The liver in the saffron extract group showed improved color and reduced volume compared to the model group.

[0348] The high-dose group of saffron red yeast rice extract and the Xuezhikang group significantly reduced liver indices. Compared with the control group (Normal), the liver indices of the model group (Model) were significantly increased (P<0.001); compared with the model group, the liver indices of the Xuezhikang group (XZK), lovastatin group, lovastatin + β-glucan group (Lovastatin + βG), low-dose group of saffron red yeast rice (ZHQ-L), and high-dose group (ZHQ-H) all showed a decreasing trend, among which the Xuezhikang group and the high-dose group of saffron red yeast rice significantly reduced liver indices (P<0.05). Figure 19 (Table 19)

[0349] Figure 19 Effects of saffron yeast on liver index in a hyperlipidemic model

[0350] (Compared to the blank group, ### *P<0.001; compared with the model group, *P<0.05)

[0351] Table 19 Effects of saffron yeast on body weight, liver weight, and liver coefficient (mean ± SD, n = 8)

[0352]

[0353] (Note: Compared with the blank group, ### *P<0.001; compared with the model group, *P<0.05, **P<0.01.

[0354] 3.4 Effects of Saffron Red Yeast Extract on Abdominal and Epididymal Fat in a Hyperlipidemic Model

[0355] Saffron yeast extract can not only reduce weight, but also inhibit the deposition of abdominal and epididymal fat (Tables 20, 21). Figure 20 , 21 Compared with the control group, the abdominal fat weight in the model group was significantly increased (P<0.05). Figure 20(Table 16) Although there was no significant change in epididymal fat weight (P>0.05), there was a trend of increasing ( Figure 21 (Table 17). The abdominal fat index and epididymal fat index of the treated groups (Xuezhikang group and high-dose Tibetan red yeast rice extract group) were lower than those of the model group, but the difference was not statistically significant. While it may lower lipids, it may also have a weight-loss and fat-deposition-inhibiting effect; however, the administration time may be relatively short, and its weight-loss and lipid-lowering effects have not yet shown significant differences.

[0356] Table 20 Effects of saffron yeast on body weight, abdominal fat, and abdominal fat coefficient (mean ± SD, n = 8)

[0357]

[0358] Note: Compared with the blank group, # * P < 0.05; Compared with the model group, * P < 0.05, ** P < 0.01

[0359] Table 21 Effects of saffron yeast on body weight, epididymal fat, and epididymal fat coefficient (mean ± SD, n = 8)

[0360]

[0361] Note: Compared with the blank group, # P<0.05; compared with the model group, *, P<0.05, **P<0.01.

[0362] Figure 20 Effects of Saffron Yeast Extract on Abdominal Fat in Hyperlipidemic Golden Hamsters

[0363] Figure 21 Effects of Saffron Yeast Extract on Epididymal Fat in Hyperlipidemic Golden Hamsters

[0364] 3.5 Effects of Saffron Yeast Extract on the Pathological Morphology of Golden Hamster Liver Tissue

[0365] HE staining results showed that: in the blank group (Normal), the liver tissue of golden hamsters was uniformly stained, the morphology and structure of hepatocytes were normal, and no hepatocyte steatosis was observed. In the high-fat model group (Model), a large number of lipid droplets and vacuoles of varying sizes were visible in the liver lobules, and the lobular structure was disordered. In the Xuezhikang (XZK) and Lovastatin groups, golden hamsters showed mild hepatocyte steatosis, with scattered vacuoles in the hepatocytes. The hepatocytes were more neatly arranged than those in the model group, and the degree of steatosis was significantly improved. In the Lovastatin + β-glucan group (Lovastatin + βG), the low-dose saffron group (ZHQ-L), and the high-dose saffron group (ZHQ-H), the degree of hepatocyte steatosis and hepatocyte swelling were significantly improved compared with the model group, and the number of hepatocytes was relatively increased, tending to be normal cells. There were no significant differences in pathological changes between the low-dose and high-dose saffron yeast groups, the lovastatin + β-glucan group, and the positive control group (Xuezhikang group and lovastatin group). All groups showed good inhibitory effects on hepatocyte steatosis. Therefore, both the low-dose and high-dose saffron yeast groups showed good inhibitory effects on the formation of fatty liver in hyperlipidemic golden hamsters.

[0366] Figure 22 Effects of saffron yeast extract on the pathological morphology of liver tissue in golden hamsters (x200)

[0367] 4. Conclusion

[0368] Feeding golden hamsters a high-fat, high-cholesterol diet successfully established a stable hyperlipidemia model in a relatively short period. The high-fat diet closely resembles the human diet, and this model conforms to the formation process of hyperlipidemia in humans. Furthermore, golden hamsters synthesize approximately 85% of their cholesterol extrahepatically, with relatively little synthesized in the liver. Since about 10% of endogenous cholesterol in humans is synthesized in the liver, this characteristic of hyperlipidemia formation in golden hamsters and their sensitivity to statins are consistent with clinically observed hyperlipidemia in humans.

[0369] Because of the limited amount of cholesterol synthesized in the human liver, it is speculated that the clinical use of statins has limitations. Statins competitively inhibit HMG-CoA reductase, the rate-limiting enzyme in hepatocyte cholesterol synthesis. In cholesterol synthesis, HMG-CoA reductase catalyzes the conversion of HMG-CoA into the intermediate mevalonic acid (MVA), a crucial step in endogenous cholesterol synthesis. Statins have a similar chemical structure to HMG-CoA and a much higher affinity for HMG-CoA reductase, thus competitively inhibiting this enzyme and lowering serum cholesterol levels. However, HMG-CoA reductase does not participate in extrahepatic cholesterol synthesis; therefore, statins have a relatively small impact on extrahepatic cholesterol synthesis. Experimental results showed that lovastatin alone did not show a good effect in lowering serum cholesterol, while the lovastatin + β-glucan group, the high and low dose groups of saffron and the high dose group of saffron, although they had significant lipid-lowering effects, only reduced cholesterol by 11%.

[0370] Two points can be analyzed from the cholesterol-lowering mechanism and the experimental results of serum cholesterol:

[0371] ① It is understandable that lovastatin had almost no lipid-lowering effect in the experimental results, because both the model group and the drug treatment group were continuously fed a high-fat, high-cholesterol diet and continuously ingested exogenous cholesterol. The lipid-lowering effect of cholesterol is mainly to inhibit the synthesis of cholesterol in the liver.

[0372] ② Although the lovastatin + β-glucan group, the high and low dose group of saffron and the high dose group of saffron had significant lipid-lowering effects, they only reduced cholesterol by about 11%. The control group using lovastatin alone did not have a cholesterol-lowering effect. It is speculated that saffron contains components that lower exogenous cholesterol, and it is also possible that β-glucan is playing a role.

[0373] Furthermore, the results section leads to the following conclusions: Saffron yeast extract significantly improved lipid indicators in a golden hamster hyperlipidemia model and inhibited the rise in the body weight curve of the hyperlipidemia model, making it more consistent with the body weight curve of the control group. It also reduced liver indices and inhibited fat deposition. Saffron yeast extract has the following advantages in lowering blood lipids:

[0374] ①Saffron yeast has been found to contain lovastatin and β-glucan. The results of this study show that β-glucan helps to reduce serum TC and LDL-C and increase serum HDL-C levels;

[0375] ②The low- and high-dose saffron yeast groups, despite having relatively lower levels of lovastatin than the lovastatin group, still improved serum TC, TG, HDL-C, and LDL-C levels. Furthermore, the high-dose saffron yeast group showed better results in lowering TC and LDL-C than the Xuezhikang and lovastatin groups, suggesting that saffron yeast may contain other components that help lower blood lipids but have not yet been discovered.

[0376] ③ The high-dose group of saffron yeast can inhibit the weight gain of the golden hamster hyperlipidemia model and reduce the deposition of abdominal fat and epididymal fat.

[0377] ④ Both low- and high-dose saffron yeast can inhibit the formation of fatty liver in hyperlipidemic golden hamsters.

[0378] (II) Metabolomics Study on the Lipid-Lowering Effect of Saffron Red Yeast Rice

[0379] 1. Materials

[0380] Same as "I. Pharmacodynamic study on the lipid-lowering effect of saffron yeast 1. Experimental materials".

[0381] Six liver tissue samples were collected from each group and immediately frozen in liquid nitrogen for later use.

[0382] 2. Methods

[0383] 2.1 Preparation of Gavage Solution

[0384] Same as "I. Pharmacodynamic Study of the Lipid-Lowering Effect of Saffron Red Yeast Rice 2.1 Preparation of Gavage Solution"

[0385] 2.2 Modeling and Grouping

[0386] Same as "Pharmacodynamic Study on the Lipid-Lowering Effect of Saffron Red Yeast Rice 2.2"

[0387] 2.3 Background of Metabolomics Detection

[0388] By using the LC-MS platform to simultaneously detect and analyze all small molecule metabolites in the sample, and through inter-group comparisons, we can uncover differences in metabolic profiles, identify differentially expressed metabolites, and metabolic pathways.

[0389] 2.4 Metabolomics Detection Process

[0390] Sample pretreatment: Metabolite extraction

[0391] LG / MS analysis information extraction: noise reduction and smoothing, baseline correction, and overlapping peak identification.

[0392] Data preprocessing: normalization, data transformation, standardization

[0393] Pattern Recognition: Multivariate Statistical Analysis (PCA, PLS-DA, OPLS-DA)

[0394] Differential metabolite analysis: VIP value analysis using multiple algorithms, correlation analysis, and statistics.

[0395] Functional analysis: KEG pathway analysis

[0396] Figure 23 Metabolomics Detection Process

[0397] 2.5 Software Information for Analysis

[0398] Table 22 Software Information Used for Analysis

[0399]

[0400] 3 Results

[0401] 3.1 Results of PCA and Cluster Analysis of Liver Metabolites

[0402] PCA analysis was performed on liver metabolites of golden hamsters. After dimensionality reduction analysis, relative coordinate points were found on principal components p1 and p2. The distance between these coordinate points represented the degree of clustering and dispersion among samples. The normal control group, model group, and other drug-treated groups all showed a tendency for separation between groups, with no outliers observed at a 95% confidence level. There was overlap between drug-treated groups, such as in the lovastatin + β-glucan group (Lovastatin + βG), the low-dose saffron yeast group (ZHQ-L), and the high-dose group (ZHQ-H), which showed significant overlap. The Xuezhikang group (XZK) and the lovastatin group also showed considerable overlap. This overlap between groups indicates similarity in their metabolites. Figure 24 This trend is also evident in metabolite cluster analysis. Figure 2 3) The Lovastatin+βG group, ZHQ-L group, and ZHQ-H group clustered together, while the XZK group clustered together with the Lovastatin group. These clusters suggest that their metabolite expression patterns are similar. All five treatment groups contained lovastatin. The differences were that the Lovastatin+βG group contained β-glucan, and the ZHQ-L and ZHQ-H groups were both Tibetan red yeast rice extract groups, also containing β-glucan. The clustering of the XZK group and the Lovastatin group may be related to their shared presence of lovastatin. Therefore, it is speculated that the five treatment groups forming two clusters may be related to β-glucan.

[0403] Figure 24 PCA analysis of liver metabolites in golden hamsters

[0404] Note: PCA score plot. After dimensionality reduction analysis, the samples have relative coordinate points on principal components p1 and p2. The distance between these coordinate points represents the degree of clustering and dispersion among the samples; the closer the distance, the higher the similarity between the samples, and the farther the distance, the greater the difference between the samples. PCA analysis can observe the separation trend between groups in the experimental model, as well as whether outliers appear, and reflects the variability between and within groups from the original data. The confidence ellipse represents the distribution of the "true" samples in this group within this area at a 95% confidence level; samples outside this area can be considered potentially outliers.

[0405] Figure 25 Cluster analysis of liver metabolites in golden hamsters

[0406] Note: In the figure, each column represents a sample, and each row represents a metabolite. The color in the figure indicates the relative expression level of the metabolite in that group of samples. For specific trends in expression levels, please see the numerical labels under the color bars in the lower right corner. The left side is a dendrogram of metabolite clusters, and the right side shows the names of the metabolites. The closer two metabolite branches are, the closer their expression levels are. The top is a dendrogram of sample clusters, and the bottom is the name of the sample. The closer two sample branches are, the closer the expression patterns of all metabolites in those two samples are, meaning the trends in metabolite expression levels are more similar.

[0407] 3.2 Classification of KEGG and HMDB compounds in metabolites

[0408] KEGG compounds are a collection of small molecules, biopolymers, and other chemical substances related to biological systems. The classification of KEGG compounds into different groups of golden hamster liver metabolites is described. Figure 26 The study identified the following metabolites: peptides, vitamins and cofactors, nucleic acids, lipids, hormones and transmitters, and carbohydrates, with lipids being the most prevalent, indicating a significant impact on lipid metabolism. Furthermore, the metabolites were compared with the HMDB 4.0 database to obtain classification information and statistically plotted (27). The plot also showed that lipids and lipid-like molecules accounted for 55.30%.

[0409] Figure 26 KEGG compound classification

[0410] Note: The vertical axis represents the KEGG compound classification, and the horizontal axis represents the number of compounds annotated to that category. The color of the bar indicates the primary classification category of the compound.

[0411] Figure 27 HMDB Compound Classification

[0412] Note: The names and percentages of the selected HMDB levels (Superclass, Class, or Subclass) are displayed in descending order of metabolite quantity. Different colors in each pie chart represent different HMDB categories, and their area indicates the relative proportion of metabolites within that category.

[0413] 3.3 KEGG functional pathway of metabolites

[0414] The KEGGPATHWAY database is a collection of manually mapped metabolic pathways, primarily describing information such as intermolecular interactions, physiological and biochemical reactions, and relationships between gene products. Based on the KEGG compound ID mapping of metabolites, information about the metabolic pathways in which the metabolites participate can be obtained, thereby evaluating their impact on biological metabolic processes. In this experiment, a relatively large number of compounds were mapped to the Lipidmetabolism pathway, including 66 lipid metabolism-related products. Figure 28 ).

[0415] KEGG pathway enrichment of metabolites revealed the top 20 enriched pathways. Figure 29 The pathway with high enrichment and significant activity is the PPAR signaling pathway, which is related to lipid metabolism and oxidation. Figure 30 ), Figure 12 The substance marked with a red dot is an eicosanoid, which participates in the PPAR signaling pathway. Eicosanoids are also called eicosanoids. As shown in the figure, eicosanoids participate in the metabolic pathways affected by PPARα and PPARγ, and affect lipid metabolism, including cholesterol metabolism and fatty acid oxidation.

[0416] Figure 28 KEGG functional pathway

[0417] Note: The vertical axis represents the secondary classification of KEGG metabolic pathways, and the horizontal axis represents the number of compounds annotated under that pathway. KEGG metabolic pathways can be divided into 7 major categories: Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, Human Diseases, and Drug Development. The color of the bars indicates different metabolic pathway categories.

[0418] Figure 29 KEGG pathway enrichment results

[0419] Note: The horizontal axis represents the pathway name, and the vertical axis represents the enrichment rate. The larger the ratio, the greater the degree of enrichment. The bar color gradient represents the significance of enrichment. By default, the darker the color, the more significant the enrichment of the KEGGterm. P-values ​​or FDRs < 0.001 are marked as ***, and P-values ​​or FDRs < 0.01 are marked as **.

[0420] Figure 30 PPAR signaling pathway

[0421] 3.4 Effects of drugs on proteins in the PPAR signaling pathway

[0422] The functional pathway enrichment results of metabolites revealed that eicosanoids, also known as eicosanoids, are involved in the PPAR signaling pathway. Eicosanoids participate in the metabolic pathways influenced by PPARα and PPARγ, affecting lipid metabolism, including cholesterol metabolism and fatty acid oxidation. We validated these effects using Western blotting, specifically by detecting the expression of PPARα, CYP7A1, and CPT1A in the PPAR signaling pathway of liver tissue.

[0423] Western blot analysis showed that, compared with the model group, the low-dose and high-dose saffron groups, the lovastatin group, and the lovastatin + β-glucan group all upregulated the expression of PPARα and CPT-1. Figure 31This promotes lipid oxidation, regulates lipid utilization and storage, and maintains lipid metabolism in balance. Meanwhile, compared with the model group, the low-dose and high-dose saffron groups, the lovastatin group, and the lovastatin + β-glucan group all upregulated CYP7A1 expression. Figure 32 CYP7A1 is the rate-limiting enzyme in the catalytic metabolism of cholesterol and the biosynthesis of bile acids, and is important for cholesterol homeostasis.

[0424] Figure 31 Effects of Saffron Red Yeast Rice Extract on PPARα, CYP7A1 and CPT-1 Proteins

[0425] 4. Summary

[0426] Analysis of liver metabolites in each group revealed a high amount of lipid metabolites. KEGG pathway enrichment of metabolites showed that the PPAR signaling pathway had the highest and most significant enrichment rate among the top 20 pathways. The PPAR signaling pathway is related to lipid metabolism and oxidation. The PPAR signaling pathway contains proteins such as PPARα, CPT-1, and CYP7A1 that affect fatty acid oxidation and cholesterol metabolism.

[0427] PPARα is a subtype of peroxisome proliferator-activated receptors (PPARs). When PPARα gene expression is elevated, the expression of the peroxisome β-oxidation rate-limiting enzyme ACOX-1 is enhanced, directly increasing the activity of cytochrome P450 enzymes, promoting microsomal ω-oxidation, and promoting hepatic lipid utilization. Simultaneously, PPARα regulates extramitochondrial carnitine palmitoyltransferase-1 (CPT-1), controlling fatty acid transport across the mitochondrial membrane and catalyzing the rate-limiting step of β-oxidation, thereby regulating lipid utilization and storage, and maintaining lipid metabolism at homeostasis. CYP7A1, by introducing a hydrophilic moiety at position 7 of cholesterol, catalyzes the rate-limiting steps in cholesterol catabolism and bile acid biosynthesis, and is important for cholesterol homeostasis. Currently, it has been found that saffron can upregulate PPARα expression, and PPARα may promote fatty acid metabolism and cholesterol excretion by regulating ACSL, CPT-1, ACOX1, and CYP7A1. Figure 32 It has been confirmed that saffron can upregulate CPT-1 and CYP7A1.

[0428] Figure 32 PPARα signaling pathway

[0429] Current results indicate that saffron red yeast rice extract lowers lipids by upregulating PPARα, which in turn upregulates CPT1 to promote fatty acid metabolism, and by upregulating CYP7A1 to promote cholesterol metabolism. However, lovastatin and Xuezhikang also regulate CPT-1 and CYP7A1. This lipid-lowering mechanism cannot distinguish the superiority of saffron red yeast rice extract over Xuezhikang and lovastatin. Based on its efficacy, it is speculated that saffron red yeast rice may possess other lipid-lowering mechanisms.

[0430] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing saffron yeast, characterized in that, include: (1) Barley, auxiliary materials and nutrient solution are mixed in a weight ratio of 88:12:40 and then matured to obtain a fermentation substrate; (2) After inoculating the fermentation substrate with red yeast seed liquid, fermentation is carried out. During fermentation, high temperature fermentation is carried out first, and then the temperature is gradually reduced to low temperature fermentation. Fermentation is continued during the gradual cooling process to obtain fermented product. (3) Dry the fermented product; The auxiliary material in step (1) consists of wheat bran and soybean meal, with a weight ratio of wheat bran to soybean meal of 10:

2. The nutrient solution in step (1) consists of the following components Composition: By weight, sucrose 0.1-1 parts, glutamic acid 0.2-1 parts, histidine 0.01-0.26 parts, calcium nitrate 0.01-0.15 parts, sodium nitrate 0.01-0.15 parts, potassium dihydrogen phosphate 0.01-0.05 parts, octanoic acid 0.01-0.05 parts, ascorbic acid 0.1-0.5 parts, disodium EDTA 0.00001-0.00005 parts, water 96-98.07 parts; The curing conditions in step (1) are 121°C and the curing time is 30 minutes; The fermentation process in step (2) specifically includes: High-temperature fermentation: After inoculating the fermentation substrate with red yeast seed liquid, fermentation is carried out for 6 days at 27-29℃ and relative humidity of 60%-65%. Gradient cooling fermentation: Gradient cooling fermentation is carried out by gradually decreasing the temperature to 23℃ at a rate of 2-3℃ / day, while keeping the relative humidity constant. Low-temperature fermentation: Fermentation is carried out at 21-23℃ and relative humidity of 60%-65% for 7-8 days to obtain fermented product; In the high-temperature fermentation process, the fermentation substrate is shaken for the first time 48 hours after inoculation with red yeast seed liquid, and then shaken once every 24 hours, for a total of 4 shakes; in the gradient cooling fermentation process, the flask is shaken once; in the low-temperature fermentation process, the flask is shaken once every 72 hours, for a total of ≤4 shakes.

2. The method according to claim 1, characterized in that, Step (3) specifically includes: drying the fermented material at a temperature of 60°C, and stopping the drying process when the moisture content of the saffron yeast is ≤10%.

3. A type of saffron yeast, characterized in that, It is obtained by the preparation method according to any one of claims 1 to 2.

4. The use of the saffron yeast of claim 3 in the preparation of lipid-lowering drugs.

5. A method for preparing saffron yeast extract, characterized in that, include: (A) The saffron yeast described in claim 3 is pulverized and then subjected to sequential soaking and percolation extraction to obtain percolate; (B) The percolate was concentrated and dried to obtain saffron extract.

6. A saffron yeast extract, characterized in that, It is prepared by the method for preparing saffron extract as described in claim 5.

7. The use of the saffron extract according to claim 6 in the preparation of lipid-lowering drugs.

Images