CN113599467A

CN113599467A - Tibetan red yeast, Tibetan medicine red yeast extract, preparation method and application

Info

Publication number: CN113599467A
Application number: CN202111006433.4A
Authority: CN
Inventors: 胡学民
Original assignee: Tibet Yuewang Drugstore Ecological Tibetan Medicine Technology Co ltd
Current assignee: Tibet Yuewang Drugstore Ecological Tibetan Medicine Technology Co ltd
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2021-11-05

Abstract

The invention discloses a saffron koji and a preparation method thereof, wherein the preparation method comprises (1) mixing highland barley, auxiliary materials and nutrient solution, and then curing to obtain a fermentation substrate; the nutrient solution comprises the following components in percentage by weight: sucrose, glutamic acid, histidine, calcium nitrate, sodium nitrate, monopotassium phosphate, n-octanoic acid, ascorbic acid, EDTA-disodium and water; (2) inoculating the monascus strain seed liquid into the fermentation substrate, and then fermenting to obtain a fermented product; (3) drying the fermented product to obtain Tibetan red yeast rice; the preparation method has good process stability, small component difference among batches and stable quality; the invention optimizes the nutrient solution, and the fermentation and drying processes, improves the content of lovastatin in the Tibetan red yeast rice and can effectively improve the content of acid lovastatin.

Description

Tibetan red yeast, Tibetan medicine red yeast extract, preparation method and application

Technical Field

The invention relates to the technical field of fermentation, and particularly relates to Tibetan red yeast, a Tibetan medicine red yeast extract, a preparation method and application.

Background

Red rice is a common lipid-lowering medicine and is prepared by fermenting Monascus purpureus. The currently clinically used monascus medicines are 2 types, namely the traditional Chinese medicine monascus and the Tibetan medicine Tibetan monascus. The traditional Chinese medicine red yeast rice is prepared by fermenting the stem rice serving as a matrix, is also called red yeast rice, and clinically uses red yeast rice decoction pieces and patent medicines thereof, namely a blood fat recovery capsule and a zhibituo capsule; the Tibetan medicine Tibetan Monascus (original name: highland barley Monascus) is prepared by fermenting kernels of special crops in Tibetan areas of China as a matrix and is clinically used.

The active ingredient of red rice for reducing blood fat of the traditional Chinese medicine is lovastatin ingredients, the standard of the lovastatin ingredients is attached to the 'blood fat recovery capsule' in the 'Chinese pharmacopoeia', but the research on the lovastatin ingredients in the red rice of Tibetan is less.

Disclosure of Invention

In view of the above, the application provides the Tibetan red yeast rice and the Tibetan medicine red yeast rice extract, the preparation method and the application. The application optimizes the nutrient solution, and the fermentation and drying processes, and can effectively improve the content of acid lovastatin while improving the content of lovastatin in the Tibetan red yeast rice. The Tibetan red koji extract can obviously improve blood fat indexes, inhibit the rising of a weight curve of a hyperlipemia model, and simultaneously can reduce liver indexes and inhibit the formation of fat deposition.

In order to solve the technical problems, the technical scheme provided by the application is a preparation method of the safranine yeast, which comprises the following steps:

(1) highland barley, auxiliary materials and nutrient solution according to the weight ratio (40-100): (5-60): (20-45) mixing and curing to obtain a fermentation substrate; the nutrient solution comprises the following components in percentage by weight: 0.1-1 part of cane sugar, 0.2-1 part of glutamic acid, 0.01-0.26 part of histidine, 0.01-0.15 part of calcium nitrate, 0.01-0.15 part of sodium nitrate, 0.01-0.05 part of monopotassium phosphate, 0.01-0.05 part of n-octanoic acid, 0.1-0.5 part of ascorbic acid, 0.00001-0.00005 part of EDTA-disodium and 96-98.07 part of water;

(2) inoculating the monascus strain seed liquid into the fermentation substrate, and then fermenting to obtain a fermented product;

(3) drying the fermented product.

Preferably, the highland barley, the auxiliary materials and the nutrient solution are 88: 12: 40.

preferably, the auxiliary materials consist of bran and soybean meal, and the weight ratio of the bran to the soybean meal is (5-12): (2-5).

Preferably, the auxiliary materials consist of bran and soybean meal, and the weight ratio of the bran to the soybean meal is 10: 2.

preferably, the nutrient solution consists of 0.41 percent of sucrose, 0.76 percent of glutamic acid, 0.26 percent of histidine, 0.09 percent of calcium nitrate, 0.03 percent of sodium nitrate, 0.01 percent of monopotassium phosphate, 0.01 percent of n-octanoic acid, 0.34 percent of ascorbic acid, 0.00001 percent of EDTA-disodium and 98.07 percent of water in percentage by mass.

Preferably, the highland barley is subjected to deslagging and crushing treatment.

Preferably, the mixing process of the highland barley, the auxiliary materials and the nutrient solution is to mix the highland barley, the auxiliary materials and the nutrient solution for 5 to 7 minutes. The materials are uniformly mixed, are kneaded into balls by hands, and are dispersed by light pressure.

Preferably, the curing condition is 121 ℃ and the curing time is 30 minutes.

Preferably, the method further comprises: mixing semen Avenae Nudae, adjuvants, and nutrient solution at a certain weight ratio, and bottling

360-440 g/bottle to obtain the bottled material; aging the bottled materials to obtain the fermentation substrate.

Preferably, before the monascus strain seed liquid is inoculated to the fermentation substrate in the step (2), the fermentation substrate is cooled to 25-45 ℃.

Preferably, the preparation process of the monascus strain seed liquid comprises the following steps:

inoculating Monascus strain to slant culture medium, culturing at 30 deg.C for 6-7 days, and eluting with water to obtain spore suspension;

inoculating the spore suspension into a fermentation tank, and culturing at 30 deg.C for 2-3d to obtain red rice strain seed liquid.

inoculating Monascus strain to slant culture medium, culturing at 30 deg.C for 6-7 days, and eluting with 300ml sterile water to obtain spore suspension;

inoculating the spore suspension liquid into a fermentation tank, and culturing at 30 deg.C for 2-3d to obtain red rice strain seed liquid.

Preferably, the Monascus species is Monascus pilosus (strain: YWG-1), a fungus of the family Aspergillus.

Preferably, the fermentation process in the step (2) specifically comprises:

high-temperature fermentation: inoculating the monascus seed liquid to the fermentation substrate, and fermenting for 6 days at the temperature of 27-29 ℃ and the relative humidity of 60% -65%;

gradient cooling fermentation: the temperature is reduced to 23 ℃ in a gradient way at the speed of 2-3 ℃/day, and the relative humidity is kept unchanged for carrying out gradient temperature reduction fermentation;

low-temperature fermentation: fermenting at low temperature of 21-23 deg.C and relative humidity of 60-65% for 7-8 days to obtain fermented product.

Preferably, the inoculation amount of the monascus strain seed liquid in the step (2) is 0.114-0.139 mL/g.

Preferably, the fermentation is finished when the total lovastatin content in the fermentation product in the step (2) is more than or equal to 0.6 percent.

Preferably, in the high-temperature fermentation process, after the monascus strain seed liquid is inoculated to the fermentation substrate, the first shake flask is carried out for 48 hours, and then the shake flask is carried out once every 24 hours for 4 times.

Preferably, the flask is shaken 1 time in the gradient cooling fermentation process.

Preferably, the high-temperature fermentation process is performed with shaking once every 72 hours, and the shaking time is not more than 4 times.

Preferably, the step (3) specifically includes: drying the fermented product at 60 deg.C, and stopping drying when the water content of the red rice is less than 10%.

The invention also provides application of the Tibetan monascus in preparation of blood fat reducing medicines and foods.

The invention also provides a preparation method of the Tibetan red koji extract, which comprises the following steps:

(A) pulverizing the Tibetan red rice, and sequentially performing stuffy moistening and percolation extraction to obtain percolate;

(B) and concentrating and drying the percolate to obtain the Tibetan red koji extract.

Preferably, the (a) is specifically: pulverizing the Tibetan red rice of claim 7, and sequentially performing moistening with ethanol and percolating extraction with ethanol to obtain percolate;

preferably, the ethanol in step (a) is 70% vol ethanol.

Preferably, ethanol is adopted for moistening in the step (A) for 12 hours, wherein the ethanol moistening is 0.8 time of 70% vol ethanol moistening.

Preferably, the step (4) is specifically: taking 14kg of the Tibetan red yeast rice, crushing the Tibetan red yeast rice by using a grinding groove, sieving by using a No. 3-4 sieve, adding 0.8 time of 70% vol ethanol, carrying out stuffiness moistening for 12h, adding into a percolation barrel, carrying out percolation extraction by using the 70% vol ethanol to obtain a percolate, and collecting 150L of the percolate.

Preferably, the concentration in the step (B) is reduced pressure concentration, and the reduced pressure concentration condition is 60-80 ℃ and the rotating speed is 30-60 r/min.

Preferably, the drying treatment in the step (B) is freeze drying, and the freeze drying condition is that the temperature is minus 10 ℃ to minus 50 ℃ and the vacuum degree is 1.3 to 13 pa.

The invention provides a Tibetan red yeast extract which is prepared by the preparation method of the Tibetan red yeast extract.

The invention provides an application of the Tibetan monascus extract in preparation of blood fat reducing medicines and health-care foods.

Preferably, the unit dose of the hypolipidemic drug and the health food contains 0.1g to 10g of the Tibetan red koji extract. .

At present, most chemical drugs used for clinically treating hyperlipidemia comprise statins, fibrates, nicotinic acid drugs, resin drugs, antioxidants, ezetimibe drugs and the like, and the action mechanism of reducing blood fat mainly comprises three ways, namely inhibiting synthesis and absorption of endogenous and exogenous lipids and promoting in-vivo lipid transportation, metabolism and excretion. The exogenous lipid absorption is inhibited by reducing the absorption of cholesterol in intestinal tract by influencing the absorption of cholesterol in small intestinal tissues, such as ezetimibe, which belongs to a cholesterol absorption inhibitor; inhibiting endogenous lipid synthesis comprises reducing cholesterol synthesis by inhibiting activity of 3-hydroxy-3-methyl glutamyl coenzyme A (HMG-Co A) reductase and its mRNA expression level, and reducing fatty acid synthesis by inhibiting acetyl coenzyme A transferase (ACC) and Fatty Acid Synthase (FAS); the promotion of lipid transport, metabolism and excretion in vivo is mainly achieved by promoting the expression of low density lipoprotein receptor (LDL-R), and the promotion of fatty acid excretion is mainly achieved by enhancing the activity of cholesterol 7 a-hydroxylase (CYP7a-1), etc.

Most of the commonly used lipid-lowering drugs in clinic have adverse reactions, and although the drugs can obtain better curative effect in a short period, the drugs can cause different degrees of harm to human bodies after being used for a long time. The adverse reaction of statins is mainly manifested as liver function damage, the degree of damage to the liver function after long-term administration is still to be observed, and the muscle toxicity is rare; fibrate drugs are mainly caused by incomplete absorption in vivo, and can cause gastrointestinal symptoms such as nausea, stomach discomfort, inappetence and the like, and in addition, symptoms such as headache, dizziness, insomnia, skin pruritus, urticaria, rash, alopecia, hyposexuality, liver and kidney function change and the like can also occur; the nicotinic acid drugs can cause adverse reactions such as skin itch, erythema, hot flashes, headache and the like caused by skin vasodilatation, and can also cause gastrointestinal side effects such as nausea, diarrhea and the like; the resin medicine does not work for any type of hypertriglyceridemia, gastrointestinal reactions such as abdominal distension, gas production, constipation and the like also occur, and simultaneously, the resin medicine can influence the absorption of thyroxine, digoxin, warfarin, folic acid and other fat-soluble vitamins, so that a patient who takes the resin medicine for a long time needs to supplement folic acid, vitamin A, D, K and calcium at irregular time, which brings inconvenience to the patient and increases the mental and economic burden of the patient; the antioxidant drugs can reduce blood fat and HDL-C in blood of patients; ezetimibe, a kind of medicine, generally causes skin symptoms such as rash, urticaria and the like and thrombocytopenic purpura, also causes musculoskeletal damage, has no effect on non-dietary hypercholesterolemia patients, and also causes the increase of blood cholesterol; the purpose of controlling blood fat can be achieved by taking the deep sea fish oil omega 3 fatty acid, but the symptoms of vision reduction, even bleeding and the like can be caused by taking the deep sea fish oil omega 3 fatty acid for a long time. Therefore, the development of drugs for the prevention and treatment of hyperlipidemia is of great importance, especially in developing countries where the incidence of hyperlipidemia and cardiovascular and cerebrovascular diseases is increasing.

Compared with the prior art, the detailed description of the application is as follows:

the invention provides a safranine yeast and a preparation method thereof, and the preparation method has the advantages of good process stability, small component difference among batches and stable quality. The invention optimizes the nutrient solution, and the fermentation and drying processes, improves the content of lovastatin in the Tibetan red yeast rice and can effectively improve the content of acid lovastatin.

The HPLC fingerprint comparison analysis of the Tibetan red yeast and similar products such as Xuezhikang capsules and zhibituo tablets in the market shows that the Tibetan red yeast decoction pieces are greatly different from other products in components, and 11 different components are preliminarily identified, wherein the different components mainly comprise nucleoside components and monacolin components, and lovastatin is one of the main components.

The invention provides a Tibetan red koji extract, a preparation method and application thereof, wherein the Tibetan red koji extract can obviously improve blood fat indexes, can inhibit the weight curve rise of a hyperlipemia model, enables the weight curve to be consistent with a blank group weight curve, and can reduce liver index and inhibit fat deposition formation.

The Tibetan red koji extract of the invention may contain other components which are helpful for reducing blood fat besides lovastatin and beta-glucan.

The Tibetan red koji extract group can inhibit the weight gain of a golden hamster hyperlipidemia model, so that the weight change curve of the golden hamster is consistent with that of a blank group, and the abdominal fat and epididymal fat deposition are reduced.

The Tibetan red yeast extract (containing lovastatin equivalent to 10mg daily dose) has better lipid-lowering effect, the effect is better than that of a lovastatin group (containing lovastatin equivalent to 20mg daily dose), and the weight loss, abdominal fat reduction and epididymis fat deposition are not obviously different from that of the lovastatin group, but the lovastatin group has a reduction trend, so that the Tibetan red yeast extract contains the lovastatin lipid-lowering drugs and possibly other substances which can assist in reducing blood fat, reducing weight and reducing fat deposition, or beta-glucan contained in the Tibetan red yeast has a systematic effect on the lovastatin.

The Tibetan red koji extract group has different components from the Tibetan red koji water decoction, and has components which influence the mental state of a mouse and quickly reduce the weight.

Drawings

FIG. 1 shows a peak matching fingerprint in 3.1 of effect example 1;

FIG. 2 shows the comparison fingerprint in 3.1 of effect example 1;

FIG. 3 is a full spectrum peak matching graph of the comparison fingerprint of the 3.2 Zhongzhixuekang capsule sample and the saffron decoction pieces of the effective example 1;

FIG. 4 is a full spectrum peak matching graph of the comparison fingerprint of zhibituo sample and saffron decoction piece in 3.3 of effect example 1;

FIG. 5 is a full spectrum peak matching graph of the comparison fingerprint spectra of other red rice samples and saffron red rice decoction pieces in 3.4 of the effect example 1;

FIG. 6 is a graph showing the common characteristic fingerprint peaks in the comparison fingerprint of the Tibetan medicated leaven decoction pieces in 3.6 of effect example 1;

FIG. 7 is a graph showing the matching of the full spectrum peaks of the fingerprints of 4 groups of red yeast rice products in 3.7 of effect example 1;

FIG. 8 is a table of matching data of "peak-peak area" of 4 groups of red yeast rice products in 3.7 of effect example 1;

FIG. 9 is a diagram showing principal component analysis in 3.7 of effect example 1;

FIG. 10 is a dendrogram of cluster analysis in 3.7 of Effect example 1;

FIG. 11 is a chart showing a heat map analysis of 3.7 Tibetan red koji decoction pieces, Zhixuekang capsules, zhibituo tablets and other red koji samples of the effect example 1;

FIG. 12 is a diagram showing the difference between the Zanghongqu decoction pieces and Xuezhikang capsules in 3.7 of effective example 1;

FIG. 13 is a thermal map analysis chart of the 3.7 Zhonghongqu decoction pieces and Xuezhikang capsules of effect example 1;

FIG. 14 is a graph showing a change in body weight in 3.1 of effect example 2;

FIG. 15 is a graph showing the effect of 3.2 Zhonghongqu in effect example 2 on serum TC in a hyperlipidemia model;

FIG. 16 is a graph showing the effect of 3.2 of effective example 2 on serum TG in a hyperlipidemia model;

FIG. 17 is a graph showing the effect of 3.2 Zhonghong koji on the HDL-C in the hyperlipidemia model serum;

FIG. 18 is a graph showing the effect of 3.2 Zhonghong koji on serum LDL-C in a hyperlipemia model in example 2;

FIG. 19 is a graph showing the effect of the medicated leaven Tibetan red in 3.3 of effect example 2 on the liver index of a hyperlipidemia model;

FIG. 20 is a graph showing the effect of the extract of the Tibetan medicated leaven in 3.4 of Effect example 2 on abdominal fat;

FIG. 21 is a graph showing the effect of the extract of the Tibetan medicated leaven in 3.4 of effect example 2 on epididymal fat;

FIG. 22 is a graph showing the effect (x200) of the Tibetan red koji extract on the pathological morphology of the liver tissue of golden hamster;

fig. 23 is a flowchart of metabolomics detection in 2.4 of effect example 2;

FIG. 24 is a PCA analysis chart of a liver metabolite of golden hamster in 3 of effect example 2;

FIG. 25 is a graph showing the clustering analysis of liver metabolites of golden hamster in 3 of effect example 2;

FIG. 26 is a KEGG compound classification diagram in 3 of Effect example 2;

fig. 27 is a HMDB compound classification chart in 3 of effect example 2;

FIG. 28 is a KEGG functional pathway diagram in effect example 2, 3;

FIG. 29 is a graph of the KEGG pathway enrichment results in 3 of Effect example 2;

FIG. 30 is a diagram showing a PPAR signal pathway in Effect example 2, example 3;

FIG. 31 is a graph showing the effect of the extract of the safranine in 3 of effect example 2 on PPAR α, CYP7A1 and CPT-1 proteins;

FIG. 32 is a PPAR α signal pathway in Effect example 2, example 4.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

A method for preparing Tibetan Monascus purpureus comprises:

(3) drying the fermented product.

Preferably, the curing condition is 121 ℃ and the curing time is 30 minutes.

Preferably, the fermentation process in the step (2) specifically comprises:

preferably, the ethanol in step (a) is 70% vol ethanol.

Preferably, the unit dose of the hypolipidemic drug and the health food contains 0.1g to 10g of the Tibetan red koji extract.

The content of the methanol and ethanol solution in the effect example of the invention is calculated by volume percentage.

The Monascus strain is Monascus pilosus (strain: YWG-1) (commercially available)

Example 1

Preparing a seed solution:

(1) inoculating Monascus strain with good growth state on the slant of eggplant-shaped bottle, culturing at 30 deg.C for 6-7d (specific operation reference: standard operation procedure for subculturing and purifying strain XZYW-SOP-001-01), and eluting with 300ml sterile water to obtain spore suspension.

(2) Inoculating spore suspension into a fermentation tank according to the inoculation amount of about 1 wt%, dissolving oxygen at 30 deg.C and 100% and 140r/min, and culturing for 2-3d to obtain seed solution.

The preparation method of the Tibetan red yeast rice comprises the following steps:

(1a) raw material treatment: removing impurities, removing residues, removing mildew, pulverizing, sieving with 20 mesh sieve

(1b) Preparing a culture medium: preparing a culture medium, and preparing the culture medium according to the proportion of 88 wt% of highland barley, 10 wt% of bran and 2 wt% of soybean meal.

TABLE 1 culture Medium formulation

(1c) Preparing a nutrient solution: the nutrient solution is prepared according to the following formula and is weighed by calculation.

TABLE 2 nutrient solution formula

(1d) Mixing material

Adding the nutrient solution into the dry culture medium according to the proportion of 40 wt% to obtain the prepared material.

Adding the prepared materials into a mixer for mixing for 5-7 minutes to ensure that the highland barley, the bran, the bean pulp and the nutrient solution are uniformly mixed, kneading into a dough by hand, and dispersing by light pressure.

(1e) Bottling: after the stirring is finished, bottling is carried out according to the filling amount of each bottle being controlled at 400 g/bottle (+ -10%), and the bottled material is obtained. In this process, QA (quality assurance) is required to randomly spot check the bottling volume every 15 minutes to ensure the bottling volume.

(1f) Curing: curing the bottled materials to obtain a fermentation substrate; the curing temperature is 121 ℃, and the curing time is 30 minutes. And immediately taking out the fermentation substrate to an inoculation room after the curing is finished, scattering and cooling.

(2a) Inoculation: and (3) cooling the fermentation substrate to 25-45 ℃, inoculating the monascus strain seed liquid, wherein the inoculation amount in each bottle is 50ml, and immediately shaking the bottle after inoculation.

(2b) Fermentation of

low-temperature fermentation: fermenting at low temperature of 21-23 deg.C and relative humidity of 60-65% for 7-8 days to obtain fermented product, and stopping fermentation when total lovastatin content in the fermented product is not less than 0.6%.

The fermentation parameters of the fermentation process are shown in Table 3

TABLE 3 fermentation parameters

(3a) And (3) bottle inverting: and (4) after the fermentation is finished, inverting the bottle to remove the polluted and caked fermented product.

(3b) And (3) drying: drying the fermented product at 60 deg.C for 1-2 times, and stopping drying when the water content of the saffron is less than 10%.

(3c) Receiving: when the water content of the stored red yeast rice is less than or equal to 10 percent, the collection is arranged immediately, the yeast collection time cannot be prolonged, and the storage is dried excessively. And (4) subpackaging the dried saffron koji with a clean plastic bag, weighing and marking.

Preparing the Tibetan red rice extract:

(4) taking 14kg of the Tibetan red yeast rice, crushing the Tibetan red yeast rice by using a grinding groove, sieving by using a No. 3 sieve, adding 0.8 time of 70% vol ethanol (the volume ratio of the ethanol to the Tibetan red yeast rice is 1:0.8), moistening for 12 hours, adding into a percolation cylinder, percolating and extracting by using 20 times of 70% vol ethanol to obtain percolate, and collecting 150L of the percolate;

(5) collecting percolate, concentrating under reduced pressure in large rotary evaporator at 60-80 deg.C and rotation speed of 30-60 r/min to 10000ml to obtain total extract concentrate, and freeze drying at-25 deg.C and vacuum degree of 1.3-13pa for 120min to obtain the final product.

Comparative example 1

The comparative example differs from example 1 only in the culture medium formulation, nutrient solution formulation and fermentation parameters, as specified in the following table.

TABLE 4 culture Medium formulation

TABLE 5 nutrient solution formula

TABLE 6 fermentation parameters

Effect example 1

1. Test instruments, materials and reagents

1.1 KQ-300DE type numerical control ultrasonic cleaner (power 300W, adjustable frequency, Kunshan city ultrasonic apparatus Co., Ltd.) of a test instrument; Milli-Q ultra pure water instruments (Millipop, USA); an electric heating constant temperature air-blast drying oven (Shanghai Xinmiao medical instrument manufacturing Co., Ltd.); an alcohol meter; BT25S model electronic analytical balance (beijing sidoris instruments systems ltd); model R-210 rotary thin film evaporator (BUCHI, Switzerland); LC-20ATXR Shimadzu liquid chromatograph (Shimadzu instruments, Japan); AB sicex TOF5600+ mass spectrometer (ibobesi instruments, usa); disposable sterile syringes (with needle) santa aurora medical products gmbh; disposable 0.22, 0.45 μm filter membrane Guangzhou city Borui scientific instruments Inc.; a YMC 18 column (japan YMC technologies);

1.2 test material fingerprint establishment 14 batches of Tibetan red yeast oral medicinal materials are used, wherein the Tibetan red yeast is provided by Tibetan moon king biotechnology limited and is prepared by the preparation method of Tibetan red yeast rice in the embodiment 1; in addition, 12 samples of the commercial Xuezhikang, zhibituo and Chinese medicine red yeast rice are collected.

Sample information is shown in table 7.

TABLE 7 saffron red koji decoction pieces and Chinese medicinal red koji sample information

1.3 preparation of test reagent samples methanol was used as analytical grade and purchased from Xiong chemical Co., Ltd. The methanol used for liquid chromatography was purchased as chromatographic pure (batch: 654655-.

2 method

2.1 chromatographic conditions: the chromatographic column is YMC C18 chromatographic column (specification of 150mm × 4.6mm,3 μm), column temperature of 30 deg.C, flow rate of 1mL/min, detection wavelength of 250nm, and sample injection amount of 10 μ L. The mobile phase is a pure water (A) -methanol (B) system, and the gradient elution procedure is as follows: 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.

2.2 Mass Spectrometry conditions: the ion source is an electrospray ionization source (ESI) in a negative ion mode; the mass scanning range m/z is 50-1200; spraying voltage: 4500V, atomizing gas temperature: 550 ℃, air curtain gas: 170.64kPa, atomizing gas and auxiliary gas: 350.25 kPa; declustering voltage (DP): -100V; the TOF/MS primary pre-scanning and triggering secondary scanning TOF/MS/MS ion accumulation time is respectively 500 MS and 200MS, CE collision energy is 35eV, CES collision energy is superposed to be (35 +/-10) eV, the triggering secondary method is IDA, the triggering secondary condition is Multiple Mass Defect (MMDF) and Dynamic Background Subtraction (DBS), and the secondary scanning is performed with priority.

2.3 preparation of test solution: respectively taking about 0.5g of each batch of medicinal materials (screened by a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 30% methanol, sealing the plug, weighing, ultrasonically treating (power 140W, frequency 42kHz) for 1.5 hours, cooling to 15-35 ℃, weighing again, complementing the lost weight with methanol, centrifuging the extract at high speed, taking the supernatant, filtering through a 0.45 mu ml filter head, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition.

2.4 HPLC fingerprint methodology investigation of the Tibetan Red Yeast medicinal materials

2.4.1 investigation of fingerprint chromatogram conditions the abundance of chromatogram peak information presented by the fingerprint and the extraction rate of main effective component lovastatin are taken as investigation indexes, and finally the elution conditions, column temperature and detection wavelength of the chromatogram mobile phase are determined. Under the condition, chromatographic peaks from large polarity to small polarity can be better separated and presented.

2.4.2 the preparation method of the test solution considers the abundance of chromatographic peak information presented by a fingerprint and the extraction rate of main effective component lovastatin as research indexes, considers the solvent selection (30%, 60%, 100% methanol and 30%, 60%, 100% ethanol) and the ultrasonic time (0.5h, 1h, 1.5h, 2h, 2.5h) of the preparation method, and finally determines the preparation method of the test solution, which can more comprehensively reflect the components from small polarity to large polarity in the Tibetan red yeast.

2.4.3 precision test taking reference medicinal material sample solution, continuously injecting sample for 6 times, analyzing according to 2.1 conditions, taking lovastatin peak as reference peak, and calculating relative peak area and retention time of each 6 common chromatographic peaks. As a result, the relative peak areas RSD are all less than 2.0%, and the relative retention time RSD is all less than 1%, which indicates that the precision of the instrument is good.

2.4.4 repeatability test the same batch of medicinal materials were taken, 6 parts were prepared in parallel according to the method under item 2.2, analyzed according to the 2.1 conditions, the relative peak area and retention time of each 6 common chromatographic peaks were calculated using the lovastatin peak as the reference peak. As a result, the relative peak areas RSD are all less than 3.0%, and the relative retention time RSD is all less than 1%.

2.4.5 stability test the same batch of test solution of medicinal material is sampled at intervals, the sampling interval is two hours each time, the sampling is carried out at 0 h, 2h, 4h, 6 h and 8h … … 48h respectively, the analysis is carried out according to 2.1 conditions, lovastatin is taken as a reference peak, the relative peak area and the retention time of each 6 common chromatographic peaks are calculated, and the relative peak area and the relative retention time of each common peak are calculated. As a result, the RSD of the common peak is less than 3.0% in 24h, and the RSD of the common peak is less than 1% in relative retention time, which indicates that the sample is stable in 24 h.

2.5 the data processing and analysis adopts fingerprint spectrum software of Chinese pharmacopoeia 2012 edition to analyze the fingerprint spectrum. Performing mass spectrum analysis on the common peak through Peakview 1.2 software of ABCIEX company, and performing structure identification on the characteristic peak through a database and literature reference; a "component-peak area" data sheet for each sample was obtained by fingerprinting analysis and was imported into MetabioAnalyst on-line analysis software for principal component analysis (pca) and heatmap analysis (heatmap).

3 results and discussion

HPLC fingerprint analysis of 3.114 batches of Tibetan Hongqu decoction pieces

Under the condition of 2.1, taking 14 batches of saffron koji decoction piece samples (saffron koji serial numbers are 1-14), taking 2 samples from each batch of samples, automatically integrating chromatographic peaks with peak heights of more than 1000 within 2-38 min in chromatographic data of 28 samples, selecting a sample chromatogram with the batch number of 20080201 as a reference chromatogram by a median generation method, selecting the chromatographic data with the time width of 0.1min, introducing the chromatographic data of 28 samples into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), performing multi-point correction by comparing and selecting 6 common characteristic component peaks as marker peaks, performing full spectrum peak matching to obtain a peak matching fingerprint (figure 1), and generating a comparison fingerprint (figure 2) by software. Similarity data of samples of each batch are obtained by software similarity analysis, and the results are shown in table 8.

FIG. 1 shows the fingerprint analysis and full spectrum peak matching of 28 batches of the Tibetan red koji decoction pieces (R: comparison fingerprint; S1-S28: Tibetan red koji decoction piece samples)

FIG. 2 shows the comparison fingerprint of the Tibetan Hongqu decoction pieces (No. 1-10 peak is the common characteristic fingerprint peak, No. 8 peak is the reference peak S)

TABLE 828 set of samples of Tibetan medicated leaven decoction pieces and the similarity analysis result of the comparison chart

The result shows that the similarity between the reference fingerprint of the Tibetan red koji decoction pieces established in the experiment and 28 samples is more than 0.95. The quality stability of the samples of the Tibetan red yeast rice decoction pieces in each batch for the test is good, and the difference among the batches is small.

3.2 fingerprint comparison analysis of Tibetan Hongqu decoction pieces and Chinese patent medicine Liaoxuekang capsule

Detecting 6 batches of Xuezhikang capsules under the condition of 2.1, taking 2 samples from each batch of samples, introducing the obtained chromatographic data and the saffron koji decoction piece comparison fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and calculating the similarity, wherein the result is shown in a figure 3 and a table 9.

The result shows that the similarity of each sample of the Xuezhikang capsule and the comparison fingerprint of the Tibetan red koji decoction pieces is 0.85-0.88, and the Xuezhikang capsule and the Tibetan red koji decoction pieces have certain difference in component composition.

FIG. 3 shows the full spectrum peak matching between the Zhaohuankang capsule sample and the Tibetan red koji decoction piece (R: Tibetan red koji comparison fingerprint;

S1-S12: xuezhikang capsule sample)

TABLE 9 similarity analysis result of fingerprint comparison between XUEZHIKANG Capsule and ZANGHONGQU decoction pieces

3.3 fingerprint comparison analysis of the Tibetan Hongqu decoction pieces and the zhibituo tablet sample

Detecting 1 batch of Chinese patent medicine zhibituo tablets under the condition of 2.1, taking 6 samples, introducing the obtained chromatographic data and the saffron koji decoction piece comparison fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and calculating the similarity, wherein the result is shown in a figure 4 and a table 10.

FIG. 4 shows the full spectrum peak matching between zhibituo sample and saffron decoction piece comparison fingerprint (R: saffron decoction piece comparison fingerprint; S1-S6: zhibituo sample)

TABLE 10 analysis of the degree of similarity between zhibituo sample and saffron decoction pieces by reference to fingerprint

The result shows that the similarity of the zhibituo tablets and the saffron medicated leaven decoction pieces in comparison with the fingerprint is about 0.95, and the difference of the component composition of the zhibituo tablets and the saffron medicated leaven decoction pieces is smaller.

3.4 fingerprint comparison analysis of Tibetan red koji decoction pieces and other Chinese medicinal red koji samples

Under the condition of 2.1, 5 batches of other traditional Chinese medicine red yeast rice samples are detected, 10 samples are counted (2 samples are taken from each batch of samples), the obtained chromatographic data and the reference fingerprints of the Tibetan red yeast rice slices are led into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), the similarity is calculated, and the result is shown in a figure 5 and a table 11.

FIG. 5 full spectrum peak matching graph of comparison fingerprint of other red rice samples and the Tibetan red rice pieces (R: Tibetan red rice pieces comparison fingerprint; S1-S10: other red rice samples)

TABLE 11 comparison of the similarity between the other Monascus samples and the Tibetan Monascus decoction pieces with the fingerprint

The result shows that the similarity of the sample of5 manufacturers used in the research and the reference fingerprint of the saffron koji decoction piece is at least 0.33 and at most 0.81, which shows that the traditional Chinese medicine red yeast rice sample of the 5 manufacturers has great difference with the saffron koji decoction piece in component composition and the quality stability of each batch of samples is poor.

3.5 discussion:

the fingerprint analysis of the samples of the Tibetan red yeast decoction pieces and other traditional Chinese medicine red yeast rice products shows that the quality and the stability of the Tibetan red yeast rice decoction piece products can be better reflected by the prepared Tibetan red yeast rice decoction pieces by contrasting the fingerprint, and the difference between the Tibetan red yeast rice decoction pieces and other traditional red yeast rice products which are clinically and commonly used, such as Xuezhikang capsules, zhibituo tablets and the like can be identified. The results show that it is possible to display,the zhibituo tablet is similar to the Tibetan red koji decoction piece in the component compositionThe similarity is about 0.95; the Xuezhikang capsule has great difference with the Tibetan red koji decoction pieces in the component composition, and the difference is between 0.85 and 0.88; the red yeast rice samples of5 manufacturers have obvious component difference with the saffron red yeast decoction pieces of the invention, the similarity is between 0.33 and 0.81,reflecting the poor quality consistency of the red yeast rice decoction pieces produced by different manufacturers in the market.

3.6 identification of component peaks in the fingerprint of the Tibetan Red koji by UHPLC-MS/MS method

Database search is carried out through ultraviolet of chromatographic peaks and mass spectrum fragment information, preliminary identification is carried out on 21 common characteristic peaks in the comparison fingerprint of the Tibetan red yeast decoction pieces through literature comparison, 20 components are identified in total, the number of each peak is shown in figure 6 (figure 6 is the common characteristic fingerprint peak in the comparison fingerprint of the Tibetan red yeast decoction pieces), and the related information of the mass spectrum of each compound identification result is shown in table 12.

Among the identified components, the components represented by the

peaks

2, 3 and 4 are all nucleoside substances, the components represented by the

peaks

13, 14 and 15 are all monascus pigment substances, the peak 18 is the main active component lovastatin (Monacolin K) of the Tibetan red koji decoction pieces, and the peak 10 is the main toxic component citrinin (Cirinin); the other 14 chromatographic peaks represent the components of the Moraxellin (monacolins) class. The results show that the Tibetan red koji decoction pieces contain various moracoline (statin) components, and lovastatin, namely Monacolin K is a representative component with higher content in the components. In addition, the Tibetan red koji decoction pieces also contain citrinin which is a commonly common toxic component in red yeast products.

TABLE 12 Mass Spectrometry identification information Table of ingredients in the Tibetan Hongqu decoction pieces

3.7 comparative analysis of chemical components of Tibetan Monascus decoction pieces and other red yeast products such as Xuezhikang

3.7.1 dividing all red yeast products into four groups, i.e. saffron decoction pieces, Xuezhikang capsules, zhibituo tablets and other red yeast (Chinese medicine red yeast), taking 6 samples from each group, analyzing by the fingerprint method, introducing the data into a fingerprint software system, performing full spectrum peak matching (see figure 7) after multipoint correction, and extracting a matching data table of 'chromatogram peak-peak area' of each sample (figure 8). This table data was formatted and uploaded to the MetabioAnalyst on-line analysis software System for principal component analysis (pca), system number analysis, and heatmap analysis. The result shows that 130 chromatographic peaks are extracted by the fingerprint software system.

FIG. 74 shows fingerprint spectrum full-spectrum peak matching graphs of red rice products (S1-S6: Zanghongqu decoction piece samples ZHQ-1-ZHQ-6; S7-S12: Xuezhikang capsule samples XZK-1-XZK-6; S13-S18: zhibituo tablet samples ZBT-1-ZBT-6; S19-S24: other red rice (Chinese medicine) decoction piece samples OTH-1-OTH 6)

FIG. 84 is a table of matching data of "Peak-Peak area" for Red Rice products

3.7.2 based on the data in the chromatogram peak-peak area table, four groups of red yeast rice samples were statistically analyzed. The analysis of the main components (figure 9) (Scores plot between the selected PCs.) shows that 4 groups of samples of the Tibetan red yeast rice decoction pieces, the Xuezhikang capsules, the zhibituo tablets and other red yeast rice (Chinese medicine red yeast rice) are respectively clustered together, and the polymerization degree of the first 3 groups of samples is higher. The fingerprint analysis shows that the components of 2 samples in the other red yeast sample groups have larger difference with other samples, and the red yeast sample groups hardly contain lovastatin components contained in other red yeast. In addition, the relation between the components of the Tibetan red yeast decoction pieces, the lipid-containing capsules, the zhibituo tablets and other red yeast samples can be seen from a hierarchical Clustering analysis dendrogram (figure 10) (Clustering utilization of the arboreal, and Clustering utilization of the atlas) and a heat map analysis (Clustering utilization of the as-hot map) (figure 11), and the components of the Tibetan red yeast decoction pieces are sequentially sorted into the zhibituo tablets, the lipid-containing capsules and other red yeast samples according to the similarity degree, so that the similarity analysis result is consistent with the similarity analysis result in the fingerprint. In the other sample groups, 2 samples (OTH-5 and OTH-6) were individually pooled together, indicating that they were significantly different in composition from the other red yeast samples. From the fingerprint analysis results, the 2 samples do not contain the characteristic active ingredient lovastatin in the red yeast rice product.

3.7.3 statistical analysis was performed on 2 groups of samples of the saffron medicated leaven pieces and the Xuezhikang capsules based on the data of the chromatogram peak-peak area table. Based on the difference components of the top45, by heat map analysis (figure 13) (Clustering result as heat map top45 (distance measurement using average), and Clustering algorithm using average), the fingerprint spectrums of the Tibetan red koji decoction pieces and the Xuezhikang capsules are preliminarily determined to be respectively the dominant components of the No. 1 to No. 6 peaks in the fingerprint spectrums of the Tibetan red koji decoction pieces and the No. I to No. V peaks in the Xuezhikang capsules, and can be used as the components with obvious difference in the Tibetan red koji decoction pieces and the Xuezhikang capsules (figure 12).

The 11 components were structurally characterized by high resolution mass spectrometry. The results of the preliminary identification are shown in Table 13.

FIG. 12: the different components of the Tibetan red koji decoction pieces and the Xuezhikang capsules (A: Tibetan red koji 1-6; B: Xuezhikang I-V)

TABLE 13 identification information table of difference components of Tibetan Hongqu decoction pieces and Xuezhikang capsules

4 conclusion

4.1 the analysis result is established to show that the similarity of each batch of samples of the Tibetan red yeast rice and the reference fingerprint spectrum is more than 0.95, which shows that the Tibetan red yeast rice decoction pieces have good process stability, small component difference among batches and stable quality.

4.2 the standard fingerprint spectrum prepared by the invention can effectively distinguish the saffron koji decoction pieces from similar products in the market such as Xuezhikang capsules (the similarity is lower than 0.85), but the degree of distinguishing the saffron decoction pieces from the zhibituo tablets of another product is not very high (the similarity is about 0.95), but the number of collected zhibituo samples is small.

4.3 the chromatographic peak in the standard fingerprint of the Tibetan red koji decoction pieces is preliminarily identified by the liquid phase high resolution mass spectrometry, and 21 main components are identified.

4.4 through the comparison and analysis of HPLC finger prints of the Tibetan red koji decoction pieces and similar products such as Xuezhikang capsules and zhibituo tablets in the market, the great difference between the Tibetan red koji decoction pieces and other products is shown, and 11 different components are preliminarily identified, wherein the different components mainly comprise nucleoside components and monacolin components, and lovastatin is one of the main components.

Effect example 2

Research on lipid-lowering activity of Tibetan medicated leaven

(1) Taking male golden hamster as experimental object, continuously feeding high-fat feed for 2 weeks after blood fat is increased, continuously feeding the high-fat feed for 4 weeks and simultaneously feeding Tibetan Monascus purpureus extract, detecting the blood fat reducing effect and mechanism of the Tibetan Monascus purpureus, and observing whether the Tibetan Monascus purpureus can inhibit the progress of hyperlipidemia to non-alcoholic fatty liver.

(2) The experiment set up 7 groups: blank group (Normal), Model group (Model), safranine yeast low-dose group (ZHQ-low), safranine yeast high-dose group (ZHQ-high), blood fat control group (XZK), Lovastatin positive control group (Lovastatin), and Lovastatin + beta-glucan group control group (Lovastatin + beta G). The control feed is given to the blank group, the model group, the low and high dose group of the Tibetan red koji extract, the blood fat recovery group, the Lovastatin control group and the Lovastatin + beta-glucan control group (Lovastatin + beta G) are given to the high fat feed, and the six groups are given enough drinking water. Performing intragastric administration on the low-dose group and the high-dose group of the Tibetan red rice extract by the original drug amount of 0.42g/kg and 0.84g/kg every day; the lovastatin control group is infused with lovastatin (1.87mg/kg) aqueous solution every day, and the lovastatin + beta-glucan group is infused with lovastatin (1.87mg/kg) + beta-glucan (0.017g/kg) aqueous solution every day; the normal group and the model group were infused with distilled water every day, and the volume of the infused stomach was 10 mL/kg. Recording body weight every 3 days; finally, the clear Total Cholesterol (TC) and Triglyceride (TG) values were determined by orbital bleeding.

(3) Pharmacodynamic index detection of the Tibetan red koji for reducing blood fat at the end of the experiment

1) Drawing a curve table of weight change of each group, and observing the influence of the Tibetan red yeast rice on the weight of the hyperlipoidemia golden yellow hamster (detected)

2) Weighing the weight of liver, kidney, abdominal fat and epididymal fat of each group, calculating the organ coefficient, and observing the influence of the Tibetan red rice on each organ coefficient.

3) Serum TC, TG, high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) were measured, and the influence of the dirty red yeast rice on blood lipid was observed.

4) And (3) observing the liver tissue structure by hematoxylin-eosin (HE) staining, observing the formation condition of liver fat drops, and judging whether the Tibetan red yeast rice can inhibit or delay the formation of the non-alcoholic fatty liver.

(4) Research on mechanism of Tibetan monascus for reducing blood fat

1) The influence of the Tibetan red yeast rice on liver metabonomics of the golden hamster hyperlipidemia model is detected, so that the metabolic pathway for detecting the influence of the blood lipid reducing effect of the Tibetan red yeast rice is analyzed.

2) Mechanism research in cholesterol lowering: protein blotting (Western-Blot) measures the level of protein expression associated with cholesterol metabolism in liver tissues.

3) Mechanism research in triglyceride reduction: protein blotting (Western-Blot) measures the expression level of egg proteins associated with fatty acid metabolism in liver tissues.

Pharmacodynamic study on blood fat reducing effect of Tibetan red yeast

1. Experimental Material

1.1 Experimental drugs

Water decoction of saffron: the usage and dosage of the safranine yeast (Tibet Yueyu Wang dynasty ecological Tibetan medicine science and technology Limited company) prepared in the embodiment 1 of the invention are as follows: oral administration: decocting soup, 6-12g, soaking in boiled water: 3g each time, 2-4 times daily, or according to the prescription.

The test is carried out by calculating the human dose of 9 g/day and the adult weight of 70kg, and converting the dose into golden hamster dose of 0.84g/kg (equivalent to the human dose of 9 g/day) according to the body surface area method, wherein the dose is taken as a high dose group of the Tibetan red koji extract, and the dose is taken as a low dose group of the Tibetan red koji extract of 0.42g/kg (equivalent to the human dose of 4.5 g/day).

Preparing the Tibetan red yeast rice extract: example 1 was prepared.

Blood fat recovery capsules: beijing Daweixin Biotech, Inc., specifications: 0.3 g/12 pellets/cassette; the usage and dosage are as follows: orally administered 2 granules at a time, 2 times a day, in the morning and at night; the granule is administered to patients with mild or moderate degree 2 granules a day after supper. In this study, the dose of human as a positive control drug was 1.2 g/day (calculated according to the lovastatin content specified in the pharmacopoeia 2015 edition, 1.2g of the Xuezhikang capsules contained 10mg of lovastatin), and the dose of the adult was calculated as 70kg by body surface area method, which was converted to 0.11g/kg of golden hamster (this dose corresponds to 10mg of lovastatin daily administered to the adult).

Lovastatin capsules: yangzhijiang pharmaceutical industry group Limited, Specification: 20 mg; the usage and dosage are as follows: the dose is generally 20mg daily and is taken once at dinner. In this study, the dose of the positive control drug, which is 20 mg/day for human and 70kg for adult, was 1.87mg/kg in mice with golden yellow mice converted by the body surface area method (this dose corresponds to 20mg of lovastatin daily for adults).

Beta-glucan: ningbo Qiancao Biotech Co., Ltd., purity 70%. The content of beta-glucan in the Tibetan red yeast rice product is not less than 1.5 percent, the average content is about 2 percent, 9g of Tibetan red yeast rice contains 0.18g of beta-glucan, and the dosage of the beta-glucan converted into golden hamster by a body surface area method is 0.017g/kg

1.2 feed

The high-fat high-fructose high-cholesterol molding feed, the control feed and the hamster maintenance feed are purchased from Nantong Temilion feed science and technology Limited, and the production license number is as follows: suzhou feed (2019) 06092.

1.3 Experimental animals

56 male SPF-grade golden yellow rats (110-. The indoor temperature is 21-24 ℃, and the day and night cycle is 12h, so that the golden hamster can eat and drink water freely.

2 method of experiment

2.1 preparation of gastric juice

0.011g/ml Lixuekang intragastric liquid: 0.55g of the ground Xuezhikang is weighed, distilled water is added to a constant volume of 50ml, and the mixture is heated in a water bath at 37 ℃ and stirred until the Xuezhikang is completely dissolved.

0.187mg/ml lovastatin gastric juice: weighing 18.7mg of lovastatin, adding distilled water to 100ml, heating in water bath at 37 deg.C, and stirring to dissolve completely.

0.0168g/ml safranine yeast high-dose gastric perfusion fluid: grinding the Tibetan red rice extract, weighing 0.84g, adding distilled water to a constant volume of 50ml, heating in a water bath at 37 ℃, and stirring until the extract is completely dissolved. The low dose group was diluted one-fold on the high dose basis.

Lovastatin + β -glucan gastric lavage fluid: the lovastatin concentration is 0.187mg/ml, and the beta-glucan concentration is 0.0017 g/ml. 0.12g of beta-glucan extract containing 70% beta-glucan was added to a volume of 50ml with a lovastatin-containing gastric juice of 0.187mg/ml, heated in a water bath at 37 ℃ and stirred until completely dissolved.

2.2 Molding and grouping

After the adaptive feeding of56 male SPF-level golden yellow hamster (110-. After 2 weeks, fasting is carried out for 12 hours without water prohibition, blood is taken from the inner canthus, after standing on ice for 2 hours, serum is centrifugally separated at 4 ℃ and 3500r/min for 15 min. Measuring TC and TG content in serum, dividing model mice into 7 groups according to TC and TG content and body weight, wherein each group comprises 8 mice, namely a model group, a blood fat recovery control group (0.11g/kg), a lovastatin control group (1.87mg/kg), a Tibetan Monascus low dose group (0.112g/kg), a Tibetan Monascus high dose group (0.224g/kg), a lovastatin + beta glucan group (1.87mg/kg +0.017g/kg), performing intragastric administration for 1 time every day, and performing intragastric administration on the model group and a blank group by using distilled water, wherein the intragastric administration volume is 10 mL/kg. The gavage dose and concentration for each group are shown in table 14. Except for the blank group, the control feed is fed, and other groups are fed with the high-fat high-fructose high-cholesterol molding feed. Water was freely available during dosing. After drug intervention, weighing was performed every 3 days, and the weight change of each group of golden hamster was recorded. After two weeks of administration, fasting is carried out for 12 hours without water prohibition, blood is taken from the inner canthus, serum is separated, and serum biochemical indexes are measured. After administration for four weeks, fasting is carried out for 12 hours without water prohibition, blood is taken from the inner canthus after weighing, serum biochemical indexes are measured, anesthesia is carried out by ether, then cervical vertebra is taken off for death, 3-5 grains of excrement in the large intestine are taken and placed in a freezing tube, and liquid nitrogen is frozen for microbial diversity detection. Taking livers, photographing and weighing, fixing the same part of liver tissue by using 4% paraformaldehyde for pathological histology examination, taking 4 parts of liver tissue from each liver, freezing and storing the liver tissue at minus 80 ℃, and respectively using the liver tissue for Western Blot, PCR, liver metabonomics detection and liver tissue biochemical index detection, wherein the liver tissue detected by each index is the same part of liver tissue.

TABLE 14 dosing schedules for each group of mice

The lovastatin is the main active ingredient for reducing blood fat in the Tibetan red yeast rice and the Xuezhikang, and the dosage of each group is converted into the equivalent dosage of the lovastatin for objective comparison according to different lovastatin contents in different samples.

2.3 measurement of body weight index of organ

After the administration is finished, fasting is not forbidden for 12h, the body is weighed, diethyl ether is used for anesthesia, then cervical vertebra is removed, and the liver, abdominal fat and epididymal fat are taken and weighed respectively. Organ index and body fat rate were calculated.

Organ index ═ organ weight/body weight 100%

Body fat rate ═ fat weight/body weight ═ 100%

2.4 serum index determination

Blood is taken from inner canthus, after standing for 2h on ice, upper serum is taken out and subpackaged after centrifugation for 15min at 4 ℃, 3500r/min and-20 ℃ to avoid repeated freeze thawing and re-melting at room temperature. Measuring triglyceride TG, total cholesterol TC, low density lipoprotein cholesterol LDL-C, high density lipoprotein cholesterol HDL-C, glutamic-oxaloacetic transaminase AST, glutamic-pyruvic transaminase ALT, superoxide dismutase SOD and malondialdehyde MDA according to the reagent specification

2.5 liver index determination

Assay of TG, TC, SOD and MDA: 100mg of right leaf tissue of the golden hamster liver is taken into a centrifuge tube, and 900 mu L of precooled physiological saline is added for homogenate. Centrifuging at 10000r/min at 4 deg.C for 20min, separating supernatant, and determining indexes of liver tissue according to the instruction.

2.6 histopathological Observation of the liver

And (3) carrying out HE (human epinastine) staining and oil red staining on liver tissues, observing the size and the number of fatty bubbles in the liver, and grading the degree of liver steatosis by adopting a semi-quantitative method. And calculating the comprehensive score of the liver fatty lesion.

Grading standard of hepatic steatosis

Level 1: less than 30% of the hepatocytes in the lobules of the liver are fatty

And 2, stage: fatty degeneration of 30-50% of liver cells in liver lobule

And 3, level: fatty degeneration of 50-75% of liver cells in liver lobule

4, level: greater than 75% of the hepatocytes in the lobules of the liver are fatty

2.7 data analysis

Statistical processing was performed using graphPad Prism 6.0 software, experimental data expressed as mean ± SD, and One-Way analysis of variance (One-Way ANOVA) was performed, with P <0.01 or P <0.05 representing differences of statistical significance.

3 results

3.1 Effect of the Tibetan Red koji extract on the body weight of hyperlipoidemia golden yellow hamster

During the experiment, the blank group had good mental status, smooth fur and sensitive movement. The skin hair of the model group was dull, the response was slightly retarded, and the activity decreased. Compared with the model group, the blood fat-reducing health-care food has the advantages that the low-dose group, the high-dose group, the lovastatin group and the lovastatin + beta-glucan group of the Tibetan red koji extract are improved in mental state, and the activity is increased. Compared with the blank group, the golden hamster of the model group has rapid weight increase, the weight of the Xuezhikang group is obviously lower than that of the blank group 4 weeks after administration, the weight increase trends of the Tibetan red koji high-dose group, the lovastatin group and the lovastatin + beta-glucan group are consistent with those of the blank group, and the weight of the golden hamster is obviously reduced compared with the model group without obvious difference with the blank group.

The body weight change curves of the groups are shown in FIG. 14, and the body weight of the Model group (Model) mice was significantly increased (P <0.05) since day 13 after the start of the administration, as compared with that of the blank group (Normal). Compared with the model group, the body weights of the Xuezhikang group (XZK) and the safranine yeast extract high-dose group (ZHQ-high) are remarkably reduced (P <0.05) at the 10 th day of administration, the trend is consistent and continues to 28 days after administration, and the body weights are continuously lower than that of the blank group, which shows that the Xuezhikang group (XZK) and the safranine yeast extract high-dose group can inhibit the weight increase of the golden hamster model, but the Xuranine yeast extract high-dose group and the blank group are consistent in weight change, and the Xuezhikang group is remarkably reduced (P <0.05) compared with the blank group. The weight of the low dose group of the Tibetan red koji extract (ZHQ-low) is not consistently reduced significantly compared with the model group. The weight average of the Lovastatin group (Lovastatin) and the Lovastatin + beta-glucan group (Lovastatin + beta G) is obviously lower than that of the model group on the 13 th day after administration, and the weight change trend of the Lovastatin group and the Lovastatin + beta-glucan group is consistent with that of the blank group.

FIG. 14 influence of Tibetan Red Rice on the change of weight curve of hyperlipidemia model

(in contrast to the blank group,^###，P<0.001，^#，P<0.05; in comparison to the set of models,

*，P<0.05，**，P<0.01，***，P<0.001)

3.2 Effect of the Tibetan Red koji extract on the blood lipid of golden hamster

3.2.1 the Tibetan Monascus purpureus extract significantly reduced serum cholesterol (TC) levels

Compared with a blank group (Normal), the serum TC content of a Model group (Model) is obviously increased (P <0.001), compared with a Model group, the blood fat recovery group (XZK), the Lovastatin group (Lovastatin), the Lovastatin + beta-glucan group (Lovastatin + beta G), the low-dose group (ZHQ-low) and the high-dose group (ZHQ-high) of the Tibetan monascus have the tendency of reducing the serum TC, but the blood fat recovery group and the Lovastatin group have no obvious difference in reducing the TC, and the low-dose group, the high-dose group and the Lovastatin + beta-glucan group of the Tibetan monascus can obviously reduce the serum TC content (P < 0.05).

It is noted that the combination of lovastatin and beta-glucan can significantly reduce serum TC, indicating that beta-glucan helps lovastatin to reduce TC (i.e., there is a synergistic effect), and that the amount of lovastatin in the low and high dose groups of safranine is less than or equal to the amount of lovastatin in Xuezhikang, but it can significantly reduce TC, suggesting that monascus may also contain components other than lovastatin that can reduce TC, or due to the synergistic effect of beta-glucan in monascus on lovastatin to reduce TC. (FIG. 15)

Table 15 influence of Tibetan monascus on serum TC of hyperlipidemia model (n ═ 8, mean ± SD)

Group of	TC(μmol/L)
		Normal	2378.64±70.36
Model	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*

FIG. 15 Effect of Tibetan Red Rice on serum TC of hyperlipemia model

(in contrast to the blank group,^###，P<0.001; compared with the model group<0.05)

3.2.2 Tibetan Monascus purpureus extract significantly reduced serum Triglyceride (TG) levels

Compared with a blank group (Normal), the serum TG content of a Model group (Model) is obviously increased (P <0.001), and compared with the Model group, the low-dose group (ZHQ-low) of the Tibetan monascus purpureus obviously reduces the serum TG level (P <0.05), the Xuezukang group (XZK), the Lovastatin group (Lovastatin), the Lovastatin + beta-glucan group (Lovastatin + beta G) and the high-dose group (ZHQ-high) of the Tibetan red koji obviously reduce the serum TG (P < 0.001). (FIG. 16) serum TG levels were lowest in the Xuezhikang group and the high dose group of safranine, and the low dose group and the high dose group of safranine were dose-dependent in lowering TG levels.

Table 16 effect of Tibetan red rice on serum TG of hyperlipidemia model (mean ± SD, n ═ 8)

FIG. 16 Effect of Tibetan Red Rice on serum TG of hyperlipidemia model

(in contrast to the blank group,^###，P<0.001; compared with the model group<0.05,***P<0.001)

3.2.3 the saffron extract significantly increased serum high density lipoprotein (HDL-C) levels

The serum HDL-C content of the Model group (Model) is significantly reduced (P <0.001) compared with that of the blank group (Normal); compared with the model group, the Xuezukang group (XZK), the Lovastatin + beta-glucan group (Lovastatin + beta G), the low-dose group (ZHQ-low) and the high-dose group (ZHQ-high) of the Tibetan Monascus purpureus went all obviously increased the serum HDL-C level (P <0.05 and P <0.01), although the serum HDL-C level of the Lovastatin group was increased, the serum HDL-C level was not obviously different from that of the model group (P >0.05), and the combination of the Lovastatin and the beta-glucan can obviously increase the serum HDL-C level, as shown in FIG. 3. This result shows that β -glucan in combination with lovastatin contributes to raising serum HDL-C levels. In addition, the high-dose group (containing lovastatin equivalent to 10mg of daily dosage of a human) of the Tibetan red yeast rice can obviously improve the serum HDL-C level (P is less than 0.01), while the group (containing lovastatin equivalent to 20mg of daily dosage of the human) of the lovastatin can not obviously improve the serum HDL-C level (P is more than 0.05), and the Tibetan red yeast rice is prompted to contain other components capable of improving the serum HDL-C level. (FIG. 17)

TABLE 17 influence of Tibetan Red Rice on the serum HDL-C of hyperlipidemia model (mean. + -. SD, n ═ 8)

Group of	HDL(mmol/L)
		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**

FIG. 17 influence of Tibetan Red Rice on serum HDL-C of hyperlipidemia model

(in contrast to the blank group,^###，P<0.001; compared with the model group<0.05,**P<0.01)

3.2.4 saffron extract significantly reduced serum low density lipoprotein (LDL-C) levels

The LDL-C content in the serum of the Model group (Model) is obviously increased (P <0.001) compared with that of the blank group (Normal); compared with the model group, the blood fat recovery group (XZK), the Lovastatin + beta-glucan group (Lovastatin + beta G), the Tibetan monascus low dose group (ZHQ-low) and the Tibetan monascus high dose group (ZHQ-high) all have the tendency of reducing the LDL-C level of serum, but only the Tibetan monascus high dose group and the Lovastatin + beta-glucan group can obviously reduce the LDL-C level of the serum (P <0.05, P <0.01), and the blood fat recovery group, the Lovastatin group and the Tibetan monascus low dose group can not obviously reduce the LDL-C of the serum (P > 0.05). Lovastatin, Lovastatin + β -glucan groups (Lovastatin + β G) all contained the same amount of Lovastatin, but the Lovastatin + β G group was more effective in lowering LDL-C, suggesting that β -glucan contributes to lowering serum LDL-C when Lovastatin is used in combination with β -glucan. The high-dose group and the Xuezhikang group of the Tibetan Monascus purpureus can reduce the LDL-C level of serum, but the LDL-C level of the Tibetan Monascus purpureus is obviously different from the LDL-C level of the Tibetan Monascus purpureus, and the lovastatin content in the two groups is always the same, which indicates that the Tibetan Monascus purpureus may also contain other components capable of reducing the LDL-C level of the serum or is caused by the synergistic effect of beta-glucan contained in the Tibetan Monascus purpureus on the LDL-C reduction of lovastatin. (FIG. 18)

TABLE 18 influence of Tibetan Red Rice on serum LDL-C of hyperlipidemia model (mean. + -. SD, n ═ 8)

FIG. 18 Effect of Tibetan Monascus on serum LDL-C of hyperlipidemia model

3.3 Effect of the Tibetan Red koji extract on the hyperlipidemia model liver

The high-dose group and the blood lipid recovery group of the Tibetan monascus extract can obviously inhibit the weight increase of the hyperlipidemic golden-yellow hamster and inhibit the hepatomegaly (P <0.05 and P <0.01), as shown in Table 8. After each group of animals was dissected, the appearance and morphology of the liver was visually observed: the blank group has no abnormal change of liver, red brown liver, flexible texture, good elasticity, smooth capsule and thin edge. The liver of the model group is light yellow in appearance, the volume is increased, the surface is changed like sand grains, the elasticity is poor, the capsule is tense, and the edge is blunt. The liver color, texture and elasticity of the administration group were changed compared with those of the model group. The model group is light red when viewed from the outside of the liver, the volume is increased, the model group is supposed to be possibly developed into fatty liver from hyperlipidemia, further pathological observation and further verification are needed for further pathological section, the appearance color of the liver of the Tibetan red koji group extract group is improved compared with that of the model group, and the liver volume is reduced.

The high-dose group and the blood lipid-recovering group of the Tibetan red rice extract can obviously reduce the liver index. The liver index was significantly increased (P <0.001) in the Model group (Model) compared to the blank group (Normal); compared with the model group, the liver indexes of the blood fat recovery group (XZK), the Lovastatin group, the Lovastatin + beta-glucan group (Lovastatin + beta G), the low-dose group (ZHQ-L) and the high-dose group (ZHQ-H) of the Tibetan red rice are all reduced, wherein the liver indexes of the blood fat recovery group and the Tibetan red rice high-dose group can be obviously reduced (P is less than 0.05). (FIG. 19, watch 19)

FIG. 19 influence of Tibetan Red Rice on liver index of hyperlipidemia model

TABLE 19 influence of Tibetan Red Rice on body weight, liver coefficient (mean + -SD, n ═ 8)

(note: in comparison to the blank group,^###，P<0.001; compared with the model group<0.05,**P<0.01。)

3.4 Effect of Tibetan Monascus purpureus extract on abdominal fat and epididymal fat of hyperlipidemia model

The Tibetan medicated leaven extract can not only reduce weight, but also inhibit abdominal fat and epididymal fat deposition (tables 20 and 21, fig. 20 and 21). Compared with the blank group, the abdominal fat weight of the model group was significantly increased (P <0.05) (fig. 20, table 16), and the epididymal fat weight was not significantly changed (P >0.05), but increased (fig. 21, table 17). The abdominal fat index and epididymis fat index of the blood fat recovery group and the Tibetan red koji extract high-dose group of the administration group are lower than those of the model group, but have no significant significance. The lipid-lowering preparation may have the effects of losing weight and inhibiting fat deposition while reducing the lipid, but the administration time may be short, and the effects of losing weight and reducing the lipid are not obviously different.

Table 20 influence of Tibetan red rice on body weight, abdominal fat factor (mean ± SD, n ═ 8)

Note: in comparison to the blank set, the data is,^#，P<0.05; compared with the model group<0.05,**P<0.01

Table 21 influence of Tibetan red rice on body weight, epididymal fat, and epididymal fat coefficient (mean ± SD, n ═ 8)

Note: in comparison to the blank set, the data is,^#，P<0.05; compared with the model group<0.05,**P<0.01。

FIG. 20 Effect of the Tibetan Red koji extract on the abdominal fat of the hyperlipidemic golden hamster

FIG. 21 the effect of the Tibetan medicated leaven extract on the epididymal fat of the hyperlipoidemia golden yellow hamster

3.5 Effect of the Tibetan Red koji extract on the histopathological morphology of the liver of golden hamster

HE staining results showed: the liver tissues of a blank group (Normal) golden hamster are uniformly colored, the morphological structure of liver cells is Normal, and the liver cell steatosis is not seen. A large amount of lipid drop vacuoles with different sizes can be seen in liver tissues in liver lobules of golden hamster of a high-fat Model group (Model), and the liver lobules are structurally disordered; the blood fat recovery group (XZK) and the Lovastatin group (Lovastatin) golden hamster can show slight hepatic cell steatosis, the liver cells have a scattered vacuole phenomenon, the hepatic cells are arranged neatly compared with the model group, and the degree of the steatosis is greatly improved compared with the model group; the Lovastatin + beta-glucan group (Lovastatin + beta G), the low-dose group (ZHQ-L) and the high-dose group (ZHQ-H) of the Tibetan monascus purpureus have obviously improved degrees of hepatic tissue steatosis and hepatic cell swelling compared with the model group, and the number of hepatic cells is relatively increased and tends to normal cells. The low and high dose groups of the Tibetan monascus, the lovastatin + beta glucan group and the positive drug control group (the blood fat recovery group and the lovastatin group) have no great difference in pathological changes and have good effect of inhibiting the fatty degeneration of liver cells, so the low and high dose groups of the Tibetan monascus have good effect of inhibiting the formation of fatty liver of the hyperlipidemic golden hamster.

FIG. 22 the effect of the Tibetan red koji extract on the histopathological morphology of the liver of golden hamster (x200)

4 conclusion

When golden yellow hamster is fed with high-fat and high-cholesterol feed, a stable hyperlipidemia model is successfully formed in a short time. The high-fat feed is fed to approximate the diet state of human, and the model accords with the formation process of human hyperlipidemia. In addition, the extrahepatic synthesis proportion of the golden hamster is about 85 percent, the intrahepatic synthesis is less, and about 10 percent of human endogenous cholesterol is synthesized intrahepatically, so the hyperlipidemia formation characteristics and the sensitivity to statins of the golden hamster are consistent with that of clinical human hyperlipidemia.

Because of the characteristic of less intrahepatic synthesis of cholesterol in human, it is presumed that the statin lipid-lowering drugs for clinical application have limitations because the statin lipid-lowering drugs have a competitive inhibition effect on HMG-CoA reductase, which is a rate-limiting enzyme in the process of synthesizing cholesterol by hepatocytes, and HMG-CoA reductase catalyzes the conversion of HMG-CoA into Mevalonate (MVA), which is an intermediate product, during the synthesis of cholesterol. However, HMG-CoA reductase does not participate in the extrahepatic synthesis of cholesterol, so statins have less effect on extrahepatic synthesis of cholesterol. The experimental result shows that the independent application of the lovastatin does not show a good effect of reducing the serum cholesterol, while the lovastatin + beta glucan group, the high and low dose group of the safranine koji and the high dose group of the safranine koji have obvious lipid-reducing effect but only reduce 11 percent of the cholesterol.

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

the experimental result shows that the lovastatin hardly has lipid-lowering effect, the model group and the administration group are both continuously fed with high-fat high-cholesterol feed and continuously take exogenous cholesterol, and the lipid-lowering effect of the cholesterol is mainly to inhibit the synthesis of the cholesterol in the liver.

② the lovastatin plus beta glucan group, the high and low dose of the Tibetan red koji and the high dose of the Tibetan red koji have obvious lipid-lowering effect, but only reduce about 11 percent of cholesterol, compared with the lovastatin group but have no cholesterol-lowering effect, the Tibetan red koji contains exogenous cholesterol-lowering components presumably, and also can play a role of beta-glucan.

The following conclusions can also be drawn from the results section: the Tibetan red koji extract can obviously improve the blood lipid index of the golden hamster hyperlipidemia model, can inhibit the weight curve rise of the hyperlipidemia model to be consistent with the blank group weight curve, and can reduce the liver index and inhibit the formation of fat deposition. The Tibetan red koji extract has the following advantages in the aspect of reducing blood fat:

firstly, the safranine yeast definitely contains lovastatin and beta-glucan, and the research result shows that the beta-glucan is beneficial to reducing TC and LDL-C in serum and increasing the HDL-C level in serum;

② the low and high dose group of the Tibetan red yeast can still improve the indexes of TC, TG, HDL-C and LDL-C of serum under the condition that the content of lovastatin is relatively lower than that of the lovastatin group, and the high dose group of the Tibetan red yeast rice has better effect on reducing TC and LDL-C than the blood fat recovery and lovastatin group, and no other components which are helpful for reducing blood fat are supposed to be contained in the Tibetan red yeast rice.

And thirdly, the Tibetan red koji high-dose group can inhibit the weight gain of the golden hamster hyperlipidemia model and reduce the abdominal fat and epididymal fat deposition.

And fourthly, the low and high dosage groups of the Tibetan medicated leaven can inhibit the formation of the fatty liver of the hyperlipoidemia golden yellow hamster.

Metabonomics research of blood fat reducing effect of Tibetan red yeast

1. Material

The same as 'pharmacodynamics research of blood fat reducing effect of Tibetan red yeast 1. experimental materials'.

The liver tissue samples of each group, 6 samples in each group, were taken and then rapidly frozen with liquid nitrogen for use.

2. Method of producing a composite material

2.1 preparation of gastric juice

In the same way, pharmacodynamic research on blood fat reducing effect of Yizang red yeast 2.1 preparation of gastric juice "

2.2 Molding and grouping

The pharmacodynamics research of the hypolipidemic action of the Tibetan medicine Zanghong koji 2.2 "

2.3 Metabonomics detection background

All small molecule metabolites in the LC-MS platform sample are simultaneously detected and analyzed, and the difference of metabolic spectra is mined by comparison among groups, so that the difference metabolites and metabolic pathways are found out.

2.4 Metabonomics detection procedure

Sample pretreatment, metabolite extraction

LG/MS analysis information extraction, namely denoising and smoothing, base line correction and overlapping peak identification

Data preprocessing, normalization, data conversion and standardization

Pattern recognition multivariate statistical analysis (PCA, PLS-DA, OPLS-DA)

Differential metabolite analysis, multiple algorithm VIP value analysis, correlation analysis and statistics

Functional analysis-analysis of the KEG pathway

FIG. 23 metabolomics detection scheme

2.5 software information for analysis

TABLE 22 software information for analysis

3 results

3.1 liver metabolite PCA analysis and Cluster analysis results

The method is characterized in that the liver metabolites of golden hamster are subjected to PCA analysis, after a sample is subjected to dimensionality reduction analysis, relative coordinate points are arranged on principal components p1 and p2, the distance between the coordinate points represents the aggregation and dispersion degree among the samples, a normal control group, a model group and other administration groups have a separation trend among the groups, and no abnormal point appears at a 95% confidence level. There was an overlap between the administration groups, such as: the Lovastatin + beta-glucan group (Lovastatin + beta G), the Tibetan monascus low dose group (ZHQ-L) and the high dose group (ZHQ-H) are greatly overlapped, the Xuezuk group (XZK) and the Lovastatin group (Lovastatin) are more overlapped, and the overlapping of the groups indicates that the metabolites of the Lovastatin group and the Tibetan monascus low dose group have similarity (figure 24). The trend can be also obviously seen in metabolite clustering analysis (figure 2.3), the Lovastatin + beta G group, the ZHQ-L group and the ZHQ-H group are clustered together, the XZK group and the Lovastatin group are clustered together, and the clustered groups show that the metabolite expression patterns are close to each other. The five administration groups all contain Lovastatin, except that the Lovastatin + beta G group contains beta-glucan group, the ZHQ-L group and the ZHQ-H group are Tibetan red koji extract group and also contain beta-glucan group; the combination of the XZK group and the Lovastatin group may be related to their Lovastatin content. It was therefore speculated that the division of the five administered groups into two clusters might be related to β -glucan.

FIG. 24 analysis of liver metabolites of golden hamster by PCA

Note: PCA score plot. After the samples are subjected to dimension reduction analysis, relative coordinate points exist on the principal components p1 and p2, the distance of each coordinate point represents the aggregation and dispersion degree among the samples, the closer the distance is, the higher the similarity among the samples is, and the farther the distance is, the greater the difference among the samples is. The separation trend among groups in the experimental model and whether abnormal points appear can be observed through PCA analysis, and the variation degree among groups and in groups is reflected on the original data. Confidence ellipses indicate that the set of "true" samples are distributed within this region at 95% confidence; exceeding this region may be considered a potentially anomalous sample.

FIG. 25 clustering analysis of liver metabolites of golden hamster

Note: each column in the graph represents a sample, each row represents a metabolite, the color in the graph represents the relative expression amount of the metabolite in the group of samples, and the specific expression amount changes according to the trend shown in the numerical label under the color bar at the lower right. The left side is a dendrogram of metabolite clustering, the right side is the name of the metabolite, and the closer the two metabolite branches are, the closer the expression quantity of the two metabolite branches is; the upper part is a sample clustering dendrogram, the lower part is a sample name, and the closer the two sample branches are, the closer the expression patterns of all metabolites of the two samples are, namely the closer the change trend of the metabolite expression is.

3.2 KEGG Compound Classification and HMDB Compound Classification of metabolites

KEGG Compound is a collection of small molecules, biopolymers, and other chemicals associated with biological systems. The following categories of metabolites were found by KEGG compound classification of different groups of golden hamster liver metabolites (fig. 26): peptides, Vitamins and Cofactors, Nucleic acids, Lipids, hormons and transporters (Hormones and delivery media) Carbohydrates, where lipid metabolism is largely influenced by lipid abundance. In addition, metabolites were aligned to the HMDB 4.0 database to obtain the taxonomic information and statistical mapping of metabolites (27), which also showed that lipid and lipid molecules (Lipids and lipid-like molecules) accounted for 55.30%.

FIG. 26 KEGG Compound Classification

Note: the ordinate is the KEGG compound classification, the abscissa is the number of the compounds annotated to the class, and the color of the bar indicates that the compound belongs to the class of the first class classification of the compound

FIG. 27 HMDB Compound Classification

Note: the names of the selected HMDB levels (Superclass, Class or Subclas) and the percentage of metabolites are shown in order from high to low, depending on the number of metabolites. The different colors in each pie chart represent different HMDB classes, with the areas representing the relative proportions of metabolites in the class.

3.3 KEGG functional pathway for metabolites

KEGGPATHWAY the database is a collection of artificially drawn metabolic pathways that mainly describe the information of molecular interactions, physiological and biochemical reactions, and relationships among gene products. According to the information of the metabolite ratio to the KEGG compound ID, the metabolic pathway information in which the metabolite participates can be obtained, so that the influence of the metabolite on the biological metabolic process can be evaluated. The metabolites of this experiment were found to be more compounds than the lipidbetacolism pathway, with 66 lipid metabolism-related products (figure 28).

The enrichment degree of the metabolites through KEGG pathway enrichment is found in a pathway of TOP 20 (figure 29), wherein the pathway with high enrichment rate and significance is PPARSIGNALINGpathway, the PPARSIGNALINGpathpathway is related to lipid metabolism and oxidation (figure 30), the red point in figure 12 indicates that substances involved in the PPARSIGNALINGpathway are eicosanoids (eicosanoids), which are also called eicosanoids (eicosanoids), and the eicosanoids are shown to be involved in metabolic pathways influenced by the PPARalpha and the PPARgamma, influence lipid metabolism, including cholesterol metabolism, fatty acid oxidation and the like.

FIG. 28 KEGG functional pathway

Note: the ordinate is the secondary classification of the KEGG metabolic pathway and the abscissa is the number of compounds annotated to the pathway. KEGG metabolic pathways may be divided into 7 major classes: metabolism (Metabolism), Genetic Information Processing (Genetic Information Processing), Environmental Information Processing (Environmental Information Processing), Cellular Processes (Cellular Processes), biological Systems (organic Systems), Human Diseases (Human Diseases), drug development (drug development). The colors of the bars represent different metabolic pathway classes.

FIG. 29 KEGG pathway enrichment results

Note: the abscissa represents the pathname and the ordinate represents the enrichment ratio, and the larger the ratio, the larger the degree of enrichment. The column color gradient indicates the significance of the enrichment, the darker the default color, the more significant the enrichment of the KEGGterm, with the label of Pvalue or FDR <0.001 and the label of Pvalue or FDR < 0.01.

FIG. 30 PPAR signaling pathway

3.4 Effect of drugs on proteins in PPAR signaling pathway

From the functional pathway enrichment results of metabolites, it is known that substances participating in PPAR signaling pathway are eicosanoids (eicosanoids), which are also called eicosanoids, participating in PPAR α and PPAR γ affected metabolic pathways, affecting lipid metabolism, including cholesterol metabolism and fatty acid oxidation, and the Western Blot verification is carried out on proteins related to cholesterol metabolism and fatty acid oxidation. For example, the expression of PPAR alpha, CYP7A1 and CPT1A on PPAR signaling pathway in liver tissue is detected.

The Western Blot detection result shows that compared with the model group, the low and high dose group, the lovastatin group and the lovastatin + beta glucan group of the Tibetan monascus can all up-regulate the expression of PPAR alpha and CPT-1 (figure 31), thereby promoting lipid oxidation, regulating the utilization and storage of lipid and balancing lipid metabolism. Meanwhile, compared with a model group, the low and high dose groups of the Tibetan monascus, the lovastatin group and the lovastatin + beta glucan group can all up-regulate CYP7A1 expression (figure 32), and the CYP7A1 is a speed-limiting step enzyme for catalyzing cholesterol catabolism and bile acid biosynthesis and is important for cholesterol homeostasis.

FIG. 31 Effect of the saffron extract on PPAR α, CYP7A1 and CPT-1 proteins

4 summary of the invention

Analysis of metabolites of various groups of livers shows that lipid metabolites are more, and the pathway which is enriched by KEGG pathway and has the highest enrichment degree in TOP 20 is PPAR signaling pathway which is related to lipid metabolism and oxidation, and PPAR alpha, CPT-1 and CYP7A1 in the PPAR signaling pathway affect fatty acid oxidation and cholesterol metabolism.

PPAR alpha is a subtype of Peroxisome Proliferator Activated Receptors (PPARs), and when the expression of PPAR-alpha gene is increased, the expression of peroxisome beta-oxidation rate-limiting enzyme ACOX-1 is enhanced, the activity of cytochrome P450 enzyme is directly enhanced to promote microsome omega-oxidation, and the utilization of lipid by liver is promoted. Meanwhile, PPAR alpha regulates carnitine palmitoyl transferase-1 (CPT-1) outside a mitochondrial membrane to control the transport of fatty acid on the mitochondrial membrane and catalyze the speed-limiting step of beta-oxidation, so that the utilization and storage of lipid are regulated, and the lipid metabolism is in balance. CYP7a1 catalyzes the rate-limiting step in cholesterol catabolism and bile acid biosynthesis by introducing a hydrophilic moiety at the 7-position of cholesterol, which is important for cholesterol homeostasis. It has now been found that monascus hainanensis can up-regulate PPAR α expression, which may promote fatty acid metabolism and cholesterol excretion by regulating ACSL, CPT-1, ACOX1, CYP7a1 (fig. 32). It has been demonstrated that safranine yeast can up-regulate CPT-1 and CYP7A 1.

FIG. 32 PPAR α signaling pathway

From the current results, it can be seen that the Tibetan red yeast extract can promote fatty acid metabolism by up-regulating PPAR alpha, further up-regulating CPT1, and up-regulating CYP7A1 can promote cholesterol metabolism to play a role in reducing blood fat, but lovastatin and Xuezhikang also have a regulating effect on CPT-1 and CYP7A1, and the advantages and disadvantages of the Tibetan red yeast extract, Xuezhikang and lovastatin cannot be distinguished by the mechanism of reducing blood fat. However, it is speculated from the drug effect that other lipid-lowering mechanisms may exist in the Tibetan red rice.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. A preparation method of Tibetan red yeast rice is characterized by comprising the following steps:

(3) drying the fermented product.

2. The method of claim 1, wherein the highland barley, the auxiliary materials and the nutrient solution are mixed according to a weight ratio of 88: 12: 40; the auxiliary materials comprise bran and soybean meal, and the weight ratio of the bran to the soybean meal is 10: 2.

3. the method according to claim 1, wherein the nutrient solution consists of, in mass percent, 0.41% of sucrose, 0.76% of glutamic acid, 0.26% of histidine, 0.09% of calcium nitrate, 0.03% of sodium nitrate, 0.01% of monopotassium phosphate, 0.01% of caprylic acid, 0.34% of ascorbic acid, 0.00001% of EDTA-disodium, and 98.07% of water.

4. The method according to claim 1, wherein the fermentation process in step (2) comprises:

5. The method according to claim 7, wherein in the high-temperature fermentation process, the fermentation substrate is shaken for the first time 48 hours after being inoculated with the monascus strain seed solution, and is shaken for 4 times every 24 hours; shaking the flask for 1 time in the gradient cooling fermentation process; in the high-temperature fermentation process, the flask is shaken once every 72 hours, and the flask shaking is not more than 4 times.

6. The method according to claim 1, wherein the step (3) comprises in particular: drying the fermented product at 60 deg.C, and stopping drying when the water content of the red rice is less than 10%.

7. A safranine koji obtained by the production method according to any one of claims 1 to 9.

8. The use of the safranine koji as claimed in claim 7 in preparing blood lipid lowering medicine and food.

9. A preparation method of a Tibetan red koji extract is characterized by comprising the following steps:

(A) pulverizing the Tibetan red rice of claim 7, and sequentially performing stuffy moistening and percolation extraction to obtain percolate;

10. A Tibetan red koji extract which is prepared by the preparation method of the Tibetan red koji extract of claim 8.

11. The use of the Tibetan red koji extract as claimed in claim 9 in the preparation of hypolipidemic drugs and foods.