WO2021129166A1 - 红曲黄素c在制备减脂制品中的应用 - Google Patents

红曲黄素c在制备减脂制品中的应用 Download PDF

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WO2021129166A1
WO2021129166A1 PCT/CN2020/126114 CN2020126114W WO2021129166A1 WO 2021129166 A1 WO2021129166 A1 WO 2021129166A1 CN 2020126114 W CN2020126114 W CN 2020126114W WO 2021129166 A1 WO2021129166 A1 WO 2021129166A1
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fat
reducing
group
monascus
application
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PCT/CN2020/126114
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English (en)
French (fr)
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陈勉华
王路
张妍
王小璐
王玉荣
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天津科技大学
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Priority claimed from CN201911338798.XA external-priority patent/CN111304093B/zh
Priority claimed from CN201911340509.XA external-priority patent/CN110898053B/zh
Application filed by 天津科技大学 filed Critical 天津科技大学
Priority to JP2022536532A priority Critical patent/JP7330575B6/ja
Publication of WO2021129166A1 publication Critical patent/WO2021129166A1/zh
Priority to US17/372,347 priority patent/US20210330639A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the application relates to the use of monascus flavone C, and in particular to the application of monascus flavone C in the preparation of fat-reducing products.
  • lipids are mainly composed of cholesterol and triglycerides. They are not only used for the synthesis of sterol substances in the body, but also provide energy for the body's metabolism. Excessive lipid accumulation in the human body can cause hyperlipidemia. Hyperlipidemia (HLP) is a significant increase in human plasma total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels, high-density lipoprotein cholesterol (HLD-C) Low metabolic disease is a key factor in inducing cardiovascular diseases such as atherosclerosis and fatty liver.
  • HLP Hyperlipidemia
  • TC total cholesterol
  • TG triglycerides
  • LDL-C low-density lipoprotein cholesterol
  • HLD-C high-density lipoprotein cholesterol
  • Cholesterol is one of the main components of the cell membrane of living organisms, and it is also the precursor of all steroid hormones, which is essential for life activities.
  • the liver is the core organ for cholesterol metabolism, and it mainly relies on a variety of apolipoproteins and peripheral tissues for cholesterol transport. Under normal circumstances, the synthesis, absorption, and excretion of cholesterol remain stable. Excessive synthesis or absorption of cholesterol can lead to disorders of cholesterol metabolism and hyperlipidemia.
  • Triglycerides are formed by esterification of glycerol and fatty acids. They are the main components of blood lipids and are also important energy-supply substances for the body. Triglycerides are mainly produced by two pathways: the phosphatidic acid pathway and the monoglyceride pathway.
  • Hepatocytes mainly synthesize triglycerides through the phosphatidic acid pathway, then synthesize very low-density lipoproteins with apolipoproteins, cholesterol, etc., and transport them to extrahepatic tissues for storage or utilization through blood circulation.
  • the synthesis of triglycerides in liver cells increases, the secretion of very low-density lipoproteins decreases, and the ability to oxidize fatty acids decreases, the accumulation of triglycerides in the liver can result in the formation of non-alcoholic fatty liver (NAFLD).
  • NAFLD non-alcoholic fatty liver
  • the prevalence of NAFLD is closely related to obesity. Through dietary regulation, intervention and prevention from the early stage of NAFLD occurrence is currently an important link to prevent NAFLD from further deterioration.
  • the monoglyceride pathway is mainly carried out in the small intestine.
  • the fat ingested by the human body is initially digested by the oral cavity and stomach, and is hydrolyzed by pancreatic lipase into glycerol and fatty acids in the small intestine, and then absorbed by the body.
  • Excessive intake of triglycerides can lead to obesity.
  • Pancreatic lipase inhibitors can inhibit the activity of pancreatic lipase in the small intestine and reduce the absorption of lipids to a certain extent. It is one of the effective ways to treat obesity with drugs.
  • Monascus has been used in fermented food in China for thousands of years.
  • Lovastatin Monacolin K
  • Monascus pigments are secondary metabolites of monascus. There are currently more than 60 types of monascus pigments with clear and complete structural analysis data recorded in the literature.
  • Taiwan invention patent TW1437001B reported a monascuspiloin named Monascuspiloin (TW1437001B), which can inhibit 5 ⁇ -reductase activity, reduce the production of the male hormone dihydrotestosterone, and treat androgen disorders related diseases.
  • the pigment Monascuspiloin and Monascinol have the same structure and are the same compound.
  • the pigments Monascuspiloin and Monascinol have not been found to have the effects of reducing blood lipids, controlling weight gain, and inhibiting body fat accumulation.
  • the application provides the application of monascinol in the preparation of fat-reducing products.
  • the monascinol has the functions of significantly reducing blood fat, controlling weight gain, and inhibiting body fat accumulation.
  • Monascinol has the same structure as the pigments Monascuspiloin and Monascinol, and is the same substance.
  • Monascinol C Monascinol
  • MC Monascinol
  • the fat-reduced product is used to reduce triglycerides.
  • the reduced-fat product includes a reduced-fat functional food.
  • the fat-reducing functional food is used to prevent or improve the sub-health state related to obesity of the individual.
  • the fat-reducing product includes a fat-reducing drug.
  • the fat-reducing medicine is used to prevent or treat diseases related to abnormal lipid metabolism in an individual.
  • the disease associated with abnormal lipid metabolism in the individual includes one or more of fatty liver, cardiovascular disease, diabetes, and asthma.
  • the fatty liver includes non-alcoholic fatty liver.
  • the preparation form of the fat-reducing drug may be a hard capsule, a soft capsule, a tablet, a granule, a powder, a suspension, a syrup, an oral liquid or an injection.
  • the fat-reducing drugs may also include pharmaceutically acceptable excipients, excipients and the like.
  • the fat-reducing drug is a unit preparation.
  • the unit preparation contains 50-200 mg of monascoflavin C.
  • the above-mentioned amount of monascoflavin C can also be appropriately adjusted according to the different subjects of administration, as long as the prevention or treatment of diseases related to abnormal lipid metabolism can be achieved.
  • the amount of monascoflavin C in a unit preparation is the amount of the active ingredient in a single-administered medicament based on the body weight of an average adult.
  • the unit preparation is a preparation that satisfies the active ingredients required for one-time administration, and common unit preparations are, for example, a unit (tablet) tablet, a unit (needle) injection, or powder injection.
  • the amount of the drug required by the patient for one administration can be conveniently obtained by calculating the product of the patient's weight and the unit weight dose required for the patient's one administration.
  • the weight of an adult is 50-90 kg
  • the dosage can be determined by the equivalent dose conversion relationship between the unit weight dose of experimental animals and humans.
  • you can follow the guidance proposed by the FDA, SFDA and other drug regulatory agencies, or refer to (Huang Jihan et al., "Equivalent dose conversion between animals and between animals and humans in pharmacological trials", “Chinese Clinical Pharmacology and Therapeutics” , 2004 Sep; 9(9):1069-1072) to determine.
  • the human and hamster's body surface area conversion coefficient of 0.12 can be used to convert the dose of human and hamster.
  • This application provides a method for preventing or treating diseases related to abnormal lipid metabolism in an individual, using the above-mentioned fat-reducing drugs.
  • Monascus in the solution of the present application can be obtained by the method for obtaining Monascuspiloin described in Taiwan Invention Patent TW1437001 or the method for obtaining Monascinol described in JP2008-56618A.
  • Fermentation injecting the seed liquid into the solid fermentation medium for fermentation to obtain fermented rice, namely red yeast rice;
  • Extracting monascus flavone C After the red yeast rice is dried and crushed, it is extracted with ethanol, and the extract is separated by high performance liquid chromatography to obtain the monascus flavone C.
  • the source of the rice is not particularly limited, and the rice may be, for example, rice that is generally commercially available.
  • the monascus used may be the monascus described in Taiwan Invention Patent TW1437001 in 2014. It can also be deposited by the applicant at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on September 20, 2019. The address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No .18578 of Monascus.
  • CGMCC General Microbiology Center
  • the preparation of Monascus C can also be carried out according to the following steps:
  • the seed liquid is injected into the solid fermentation medium at a ratio of 1:5-10L/kg for fermentation to obtain fermented rice, namely red yeast rice;
  • the method for preparing monascoflavin C includes the following steps:
  • the monascus is activated by using an activation medium and then inoculated into a seed liquid medium for cultivation to prepare a seed liquid of monascus;
  • Fermentation filter the seed liquid of the monascus to remove the mycelium, and inject the seed liquid of the monascus from the mycelium into the solid fermentation medium at a ratio of 1:5-10L/kg Fermentation is carried out to obtain fermented rice, namely red yeast rice;
  • step 1) the ratio of rice to water during the soaking process is 1:1 kg/L, and the soaking process is carried out at room temperature for 4-24 hours.
  • the activation temperature of the monascus in step 2) is 25-35°C, and the activation culture is 2-5 days. Further, in the step 2), the activation medium is a wort slant medium.
  • the wort slant medium is a conventional medium in the field and can be purchased. It can also be prepared as follows: lightly pulverize barley malt grains, weigh 250.0g, add 1L of water, 60°C constant temperature water bath for 4h, filter, add water, dilute the sugar content to 12°Brix, add 3.0g agar, and hold at 121°C , Sterilize for 20min under 0.1MPa condition.
  • step 2) the activated Monascus is inoculated into the seed liquid medium and cultivated in a constant temperature shaker, the shaker temperature is controlled to be 25-35°C, and the shaker speed is 150-200r/min. The time is 24-48 hours.
  • the seed liquid culture medium is a conventional culture medium in the field and can be purchased. It can also be prepared according to the following formula.
  • the seed liquid culture medium includes, by weight, 6.0 parts of glucose, 2.0 parts of peptone, 1.0 part of NaNO 3 , 1.0 part of KH 2 PO 4 , 0.5 part of MgSO 4 , and 100 parts of water .
  • the temperature of fermentation in step 3 is 25-35°C, and the fermentation time is 10-25 days.
  • step 4 the extraction liquid is separated by high performance liquid chromatography to obtain the monascoflavin C, which further includes obtaining monascin (MS) from the extraction liquid.
  • MS and MC are present in the extract at the same time, and they can be separated from the extract by conventional technical means. For example, according to the difference in peak time of MS and MC, they can be separated in the separation process of high performance liquid chromatography. Collect MS and MC, which is achievable for those skilled in the art.
  • MS can be converted into MC by the method of formula 1; the method for preparing MS provided in this application may also include step 5) converting MS obtained from the extract into MC, so as to further increase the yield of MC,
  • the MS is further transformed into MC according to Formula 1, which can further increase the yield of MC produced by the Monascus fermentation of the present application.
  • Monascus Xanthin C has the functions of significantly lowering blood lipids, controlling weight gain, and inhibiting body fat accumulation; and has reduced cytotoxicity, and has higher safety in use as a fat-reducing product.
  • This application provides the application of monascus flavone C in the preparation of fat-reducing functional foods to prevent or improve the sub-health state of individuals related to obesity.
  • the individuals may, for example, include adults and adolescents. Sub-healthy people with weight management.
  • the application also provides the application of Monascus C in the preparation of fat-reducing products including fat-reducing drugs for the prevention or treatment of diseases related to abnormal lipid metabolism in individuals, including fatty liver, cardiovascular disease, diabetes and asthma ; It can also be used for the prevention and treatment of a series of diseases closely related to obesity such as diabetes, cardiovascular disease, asthma and osteoarthritis.
  • Figure 1A shows the condition of HepG2 cells before the addition of oleic acid
  • Figure 1B shows the lipid accumulation of HepG2 cells after the addition of oleic acid.
  • Figure 2 shows the effect of different doses of MC and MS on the survival rate of HepG2 cells modeled with oleic acid.
  • Figure 3 shows the effect of different MC administration concentrations on the TG content in HepG2 cells after oleic acid modeling.
  • Figure 4 shows the effect of MC and MS on TG content in HepG2 cells after oleic acid modeling.
  • Fig. 5A is a diagram of lipid accumulation in HepG2 cells of the oleic acid model
  • Fig. 5B is a diagram of lipid accumulation in HepG2 cells of MS group
  • Fig. 5C is a diagram of lipid accumulation in HepG2 cells of MC group.
  • Figure 6 shows a graph of the inhibitory activity of MC on pancrelipase as a function of concentration.
  • Figure 7A is a graph showing the results of inhibition rate of MS (Monascin) reactants in different order of addition
  • Figure 7B is a graph showing the results of inhibition rate of AK (Ankaflavin) reactants in different order of addition
  • Figure 7C is The results of the inhibition rate of MC (Monascinol C, Monascinol) reactants in different order of addition.
  • Figure 8 shows MC's Lineweaver–Burk curve.
  • Figures 9A-9C show the results of the liver tissue sections of each group of hamsters under a 100x light microscope; Figures 9a-9c show the results of the liver tissue sections of each group of hamsters under a 400x light microscope.
  • Figures 10A-10C show the results of adipose tissue slices of each group of hamsters under a 100x light microscope; Figures 10a-10c show the results of adipose tissue slices of each group of hamsters under a 400x light microscope.
  • Figure 11 shows the LCMS first-order positive ion spectrum of MC.
  • Figure 12 shows the 1 H-NMR spectrum of MC.
  • Figure 13 shows the 13 C-NMR spectrum of MC.
  • the HepG2 cells in the examples of this application were purchased from Xiehe Cell Resource Center (resource number: 3111C0001CCC000035).
  • the various reagents used in the experiment were all conventional reagents, all of which were commercially available.
  • Monascinol C (Monascinol, MC for short) can be prepared by the method in JP2008-56618A or TW1437001B.
  • Monascin (MS for short) can be prepared by the method in JP2008-56618A.
  • Monascinol C (Monascinol, MC for short) and Monascin (MS for short) can also be prepared by using the Monascus (Monascus sp) described in this application.
  • the Monascus sp was deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on September 20, 2019, and the deposit number is CGMCC No. 18578.
  • the raw material is ordinary commercially available rice, and the components in the culture medium are all commercially available reagents conventionally in the art.
  • HepG2 cells in good growth condition were seeded in a 6-well culture plate at 2*10 5 cells/mL, 1 mL per well. When the cell adhesion rate reaches 80%-90%, the culture medium is discarded. Set up the blank group and the model group. The blank group is added with complete medium, and the model group is added with 0.3mmol/L oleic acid induction solution diluted with complete medium. Add 2 mL per well. After 24 hours of culture, observe the cells by oil red O staining The accumulation of lipid droplets.
  • Example 2 The effect of monascoflavin C in different dosages on cell triglyceride content
  • Blank control group add 2mL complete medium
  • Oleic acid manufacturing module add 2mL of complete medium containing 0.3mmol/L oleic acid;
  • MC low-dose group Add 2 mL of complete medium containing oleic acid 0.3 mmol/L and 2 ⁇ g/mL monascusin C;
  • MC medium-dose group add 2 mL of complete medium containing oleic acid 0.3mmol/L and 4 ⁇ g/mL monascusin C;
  • MC high-dose group Add 2 mL of complete medium containing oleic acid 0.3 mmol/L and 8 ⁇ g/mL monascoflavin C.
  • the cells of each group were cultured for 24 hours, and the cells of each group were collected, and the triglyceride content in HepG2 cells of each group was determined using a triglyceride kit (Beijing Pulilai Gene Technology Co., Ltd.).
  • blank control group No treatment for normal culture of HepG2 cells; oleic acid model (0.3mmol/L oleic acid), no treatment for normal culture of HepG2 cells modeled by oleic acid; MC group: HepG2 cells modeled with oleic acid were administered with 2 ⁇ g/mL monascusin C or MC; MS group: HepG2 cells modeled with oleic acid were administered with 2 ⁇ g/mL monascusin MS.
  • Figure 5A is a diagram of lipid accumulation in HepG2 cells of the oleic acid-made module
  • Figure 5B is the intracellular lipid accumulation of HepG2 in the MS group.
  • Figure 5C is the intracellular lipid accumulation diagram of HepG2 cells in the MC group. It can be seen that, compared with the oleic acid-made model, the 2 ⁇ g/mL MS group and the MC group can reduce the lipid droplets in the cells. The color becomes lighter. This is consistent with the test result of TG.
  • Example 4 The inhibitory activity of MC on pancreatic lipase and the type of inhibition
  • 4-methylumbelliferone oleate (4-MUO) was used as the substrate and the fluorescence detection method was used to evaluate the inhibitory activity of MC on pancrelipase (type II, sourced from porcine pancreas).
  • Blank group no MC sample, only substrate, pancrelipase and Tris-HCL buffer;
  • Blank background group no MC sample and pancrelipase, only substrate and Tris-HCL buffer;
  • Sample background group no pancrelipase, only MC sample, substrate and Tris-HCL buffer;
  • Sample group No Tris-HCL buffer, only MC sample, substrate and pancrelipase.
  • Inhibition rate (%) [(A 1 –A 2 )–(A 3 –A 4 ]/(A 1 –A 2 ) ⁇ 100;
  • a 1 Fluorescence value of the blank group;
  • a 2 Fluorescence value of the blank background group
  • A3 fluorescence sample group
  • a 4 Sample set background fluorescence
  • the substances contained in each group were added to the wells of the black 96-well culture plate according to the ratio shown in Table 1. Three parallel experiments were set up in each group.
  • the concentration of 4-MUO was 0.1 mM, and the MC samples were dissolved in DMSO (setting 6 Different concentrations: 5, 10, 20, 40, 80, 160 ⁇ g/mL), the concentration of lipase solution is 1mg/mL, after 30min reaction at 25°C, add 100 ⁇ L 0.1M sodium citrate buffer solution to stop the reaction .
  • Lipase will catalyze the substrate 4-methylumbelliferone oleate (4-MUO) to produce the product 4-methylumbelliferone.
  • 4-methylumbelliferone has fluorescence intensity at excitation wavelength of 340nm and emission wavelength of 460nm. .
  • the lipase activity can be indirectly judged (that is, the higher the lipase activity, the more 4-methylumbelliferone is catalyzed and the stronger the fluorescence signal. Therefore, at the excitation wavelength of 360 ⁇ 40nm, the emission The product 4-methylumbelliferone was measured at a wavelength of 460 ⁇ 40nm.
  • the types of inhibition can be divided into two types: reversible inhibition and irreversible inhibition.
  • Irreversible inhibition usually uses a relatively strong covalent bond to bind to the group in the enzyme protein to inactivate the enzyme.
  • Reversible inhibition is divided into competitive inhibition, non-competitive inhibition and anti-competitive inhibition.
  • Studies have reported that the type of inhibition of MS and AK on pancreatic lipase is non-competitive inhibition. In this part of the experiment, MS and AK are used as controls.
  • the type of inhibition of MC is determined by changing the order of addition of reactants, and the intervention concentration of the three pigment inhibitors Both are 80 ⁇ g/mL. The results are shown in Figures 7A-7C.
  • Figure 7A shows the results of the inhibition rate of MS (Monascin) reactants in different addition orders;
  • Figure 7B shows the different addition orders of AK (Ankaflavin) reactants.
  • Figure 7C shows the results of the inhibition rate of MC (Monascinol) reactants in different order; it can be seen that the inhibition type of MC is different from that of MS and AK.
  • S substrate
  • I inhibitor
  • E enzyme.
  • Figure 8 shows the Lineweaver–Burk curve of MC, where 1/S is the reciprocal of the substrate concentration; 1/V is the reciprocal of the reaction rate, and the reaction rate is the fluorescence difference before and after the reaction divided by the time difference.
  • the basic process of drawing the Lineweaver-Burk curve experiment is: under the condition of a certain lipase concentration (1mg/mL), a series of different substrate concentrations are used to determine the enzymatic reaction rate.
  • the blank group contains no inhibitor MC (no MC sample, only different concentrations of substrate, pancrelipase and Tris-HCL buffer), and the MC group is added (160 ⁇ g/mL MC, different concentrations of substrate, pancrelipase And Tris-HCL buffer.
  • the double reciprocal mapping method shows that the two straight lines tend to be parallel, which is an anti-competitive inhibition in the reversible inhibition type. It shows that MC does not directly bind to the enzyme, but through the enzyme- The way the substrate complex binds reduces the catalytic reaction rate of pancrelipase.
  • hamsters Twenty-four male Syrian golden hamsters (Golden Syrian hamsters, referred to as hamsters) used in animal experiments were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., weighing 110-120 grams, and aged 8 weeks.
  • NOR Normal group
  • High-fat group Freely ingest chewing feed with high-fat ingredients, and give the same amount of CMC-Na solution to the MC group by gavage;
  • MC group Freely ingested chewing feed with high fat content, and gavage MC (dissolved in CMC-Na solution) at a dose of 20 mg/kg body weight.
  • Table 2 records the hamster liver, kidney, fat and other organ weight indicators associated with lipid metabolism. From the above experimental data, it can be seen that compared with the HFD group, the MC group can significantly inhibit (p ⁇ 0.05) the increase in body weight, liver weight and peripheral fat accumulation of hamsters caused by high-fat diet.
  • the collected serum samples used Cobas8000E602 automatic biochemical analyzer (Roche Diagnostics Co., Ltd., Germany) to measure TC and TG in the serum. The results are recorded in Table 3.
  • TG and TC contents in the liver and serum of hamsters in the HFD group were significantly higher than those in the NOR group (p ⁇ 0.05).
  • the content of TG and TC in the liver and serum of the hamsters in the MC group was significantly down-regulated (p ⁇ 0.05).
  • the average content of TG in the liver of the hamsters on a high-fat diet with MC intervention was compared with that in the NOR group. the same. It shows that MC has a significant inhibitory effect on lipid accumulation in serum and liver caused by high-fat diet, especially the inhibitory effect on TG accumulation in liver and serum. This is the result of cell experiment MC inhibiting TG accumulation in HepG2 cells Unanimous.
  • Figures 9A-9C show the results of the liver tissue sections of each group of hamsters under a 100x light microscope
  • Figure 9a-9c show the results of the liver tissue sections of each group of hamsters under a 400x light microscope
  • Figure 9A and Figures 9a is the NOR group
  • Fig. 9B and Fig. 9b are the HFD group
  • Fig. 9C and Fig. 9c are the MC group. It can be seen that compared with the hepatocytes in the HFD group that were enlarged due to lipid accumulation, the lipid accumulation in the MC group was significantly reduced, and the hepatocyte cord tended to be normal.
  • Figures 10A-10C show the results of adipose tissue slices of each group of hamsters under a 100x light microscope
  • Figures 10a-10c show the results of adipose tissue slices of each group of hamsters under a 400x light microscope
  • Figure 10A and Figure 10a It is the NOR group
  • Fig. 10B and Fig. 10b are the HFD group
  • Fig. 10C and Fig. 10c are the MC group.
  • the volume of adipose tissue cells around the epididymis and kidney of the HFD group was significantly larger than that of the NOR group, while the volume of the cells in the MC group was significantly less than that of the HFD group, and the cell volume was between that of the NOR group and the HFD group between.
  • MC can effectively prevent weight gain, peripheral fat accumulation, accumulation of blood and liver TC and TG caused by high-fat diet. It can be used to prevent and treat abnormal lipid metabolism caused by obesity and related metabolic syndromes (such as fatty liver, diabetes, asthma, osteoarthritis, etc.).
  • the red yeast rice enriched with MC obtained by fermentation of the monascus provided in this application, or MC or MS obtained by extraction and separation of the red yeast rice can be used to develop functional foods related to abnormal lipid metabolism and raw materials as medicines.
  • CGMCC General Microbiology Center
  • the raw material is ordinary commercially available rice, and the components in the culture medium are all commercially available reagents conventionally in the art.
  • monascus flavone C includes the following steps:
  • the slant medium used for activation is lightly crushed by barley malt kernels. Weigh 250.0g, add 1L of water in a 60°C constant temperature water bath for 4h, filter and add water to dilute the sugar content to 12°Brix, add 3.0g agar, and add 3.0g agar. Obtained by sterilizing for 20 min under the conditions of °C and 0.1MPa).
  • Seed liquid culture medium is through 6.0g glucose, 2.0g peptone, 1.0g NaNO 3 , 1.0g KH 2 PO 4 , 0.5g MgSO 4 dissolved in 100mL water and aliquoted into 250mL Erlenmeyer flasks, 8 layers of gauze It is sealed with kraft paper and sterilized at 121°C and 0.1MPa for 20 minutes.
  • the result of the ESI-MS measurement of monascoflavin C is shown in FIG. 11, the first-order positive ion peak m/z is 361.2000 [M+H] + , and the molecular formula of the compound is C 21 H 28 O 5 .
  • the 1 H-NMR results of Monascin C are shown in Figure 12, and the 13 C-NMR results of Monascin C are shown in Figure 13.
  • the results are the same as the structural characterization information of Monascinol described in the document JP2008-56618A.
  • the fermented product ie fermented red yeast rice
  • grind and sieve 200 mesh
  • a 70% ethanol solution with a material-to-liquid ratio of 1:20 Centrifuge at 3500r/min for 30min for 10min, dilute the supernatant appropriately and filter the extract through a 0.22 ⁇ m organic filter into a liquid phase vial for detection by HPLC.
  • the MC peak area detected in the sample, the sample dilution factor and the MC standard curve, the MC content in the fermentation product was calculated to be 8 mg/g.
  • This content is much higher than the MC content (0.31mg/g) in red yeast rice fermented with monascus used in Taiwan invention patent TW1437001B.
  • the MC-rich red yeast product fermented by the Monascus CGMCC No. 18578, such as fermented red yeast rice, can be used to develop functional foods related to abnormal lipid metabolism and raw materials for medicines.

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Abstract

红曲黄素C(Monascinol)在制备减脂制品中的应用。该减脂制品包括减脂功能性食品或减脂药品，减脂功能性食品用于预防或改善个体与肥胖相关的亚健康状态，所述减脂药品用于预防或治疗个体与脂代谢异常相关的疾病。红曲黄素C具有显著的降低血脂、控制体重增加、抑制体脂肪堆积的新功能；并具有降低的细胞毒性，作为减脂制品具有更高的使用安全性。

Description

红曲黄素C在制备减脂制品中的应用
本申请要求于2019年12月23日提交中国专利局、申请号为201911340509.X、申请名称为“红曲黄素C在制备减脂制品中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。本申请要求于2019年12月23日提交中国专利局、申请号为201911338798.X、申请名称为“一种制备红曲黄素C用的红曲菌及利用其制备红曲黄素C的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及红曲黄素C的用途，尤其涉及制备红曲黄素C在制备减脂制品中的应用。
背景技术
脂质作为细胞膜和脂蛋白的组成成分之一，主要由胆固醇和甘油三酯组成，不仅用于体内固醇类物质的合成，更能为人体提供新陈代谢所需的能量。人体内过多脂质积累会引发高脂血症。高脂血症(Hyperlipidemia,HLP)是人体血浆总胆固醇(TC)，甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)水平明显升高，高密度脂蛋白胆固醇(HLD-C)偏低的代谢疾病，是诱发动脉粥样硬化、脂肪肝等心血管疾病的关键因素。
胆固醇是生物体细胞膜的主要成分之一，同时也是所有类固醇激素的前体，对于生命活动至关重要。肝脏是胆固醇代谢的核心器官，主要依靠多种载脂蛋白与外围组织进行胆固醇的转运。正常情况下，胆固醇的合成、吸收以及排泄保持稳态，过量的胆固醇合成或吸收会导致胆固醇代谢紊乱引发高脂血症。甘油三酯由甘油和脂肪酸酯化而成，是血脂的主要成分，也是机体重要供能物质。甘油三酯主要由两种途径生成：磷脂酸途径和甘油一酯途径。肝细胞中主要通过磷脂酸途径合成甘油三酯，再与载脂蛋白、胆固醇等合成极低密度脂蛋白，通过血液循环运送到肝外组织储存或利用。当肝细胞中甘油三酯的合成增加、极低密度脂蛋白分泌减少及脂肪酸氧化能力降低时均可 导致肝脏中甘油三酯堆积，形成非酒精性脂肪肝(NAFLD)。NAFLD的流行与肥胖密切相关。通过饮食调节，从NAFLD发生的早期阶段介入预防是目前防止NAFLD进一步恶化的重要环节。甘油一酯途径主要在小肠中进行，人体摄入的脂肪经口腔和胃的初步消化，在小肠中被胰脂肪酶水解为甘油和脂肪酸，进而被人体吸收。过量甘油三酯摄入会导致肥胖，胰脂肪酶抑制剂可抑制小肠中胰脂肪酶活性，一定程度上减少脂类物质的吸收，是药物治疗肥胖的有效途径之一。
甘油三酯和胆固醇虽为人体脂类物质的重要组成成分，过度存储将导致肥胖、心血管疾病、糖尿病等发病风险飙升。肥胖引发的血脂过高可导致动脉硬化，增加心血管疾病的风险；肥胖人群更容易出现脂肪肝；在肥胖人群中，身体中过多的脂肪还会沉积在呼吸道中，影响呼吸系统功能，使哮喘患者的症状恶化。肥胖是撬动心血管疾病、糖尿病、哮喘、脂肪肝和骨关节炎癌症等发生的一个重要“支点”。
红曲菌(Monascus)在中国发酵食品中的应用已有上千年的历史。红曲菌发酵产物中发现的洛伐他汀(Monacolin K)，通过抑制内源性胆固醇合成起到降血脂的功效。红曲色素是红曲菌的次级代谢产物，目前文献记载的具有明确完整结构解析资料的红曲色素种类超过60种。潘子明教授等人研究发现，红曲素(Monascin：MS)和安卡红曲黄素(Ankaflavin：AK)通过抑制甘油三酯生成和促进脂肪酸β-氧化减轻了油酸诱导小鼠FL83B肝细胞的脂肪变性。方玉祥等研究发现MS和AK非竞争性抑制胰脂肪酶活性，具有潜在的抗肥胖功效。吕旭聪等发现红曲色素可改善高脂饮食Wistar大鼠肝脏中脂肪堆积，改善脂质代谢紊乱。MS和AK两种红曲黄色素具有预防机体脂肪积累，改善血脂异常的生理活性。
2008年日本发明专利JP2008-56618A首次报道了一种被命名为Monascinol的红曲黄色素具有抗炎、防癌功能。2014年台湾发明专利TW1437001B报道了一种被命名为Monascuspiloin的红曲黄色素(TW1437001B)，它能够抑制5α-还原酶活性，减少雄性激素二氢睾固酮的生成，治疗雄性激素失调相关病症。经比较，色素Monascuspiloin与Monascinol具有相同的结构，为同一种化合物。
尚未发现色素Monascuspiloin与Monascinol具有降低血脂、控制体重增 加、抑制体脂肪堆积功效。
发明内容
本申请提供红曲黄素C(Monascinol)在制备减脂制品的应用，该红曲黄素C具有显著的降低血脂、控制体重增加、抑制体脂肪堆积的功能。
在本申请的方案中，红曲黄素C(Monascinol)与色素Monascuspiloin和Monascinol具有相同的结构，为同一物质，在本申请中，申请人命名为红曲黄素C(Monascinol)，简称MC，申请人发现其具有显著的降低血脂、控制体重增加、抑制体脂肪堆积的功能。
本申请提供的红曲黄素C在制备减脂制品中的应用。
在本申请的一个具体实施方式中，所述减脂制品用于降低甘油三酯。
在本申请的一个具体实施方式中，所述减脂制品包括减脂功能性食品。
进一步的，所述减脂功能性食品用于预防或改善个体与肥胖相关的亚健康状态。
在本申请的另一个具体实施方式中，所述减脂制品包括减脂药品。
进一步的，所述减脂药品用于预防或治疗个体与脂代谢异常相关的疾病。
进一步的，所述个体与脂代谢异常相关的疾病包括脂肪肝、心血管疾病、糖尿病和哮喘中的一种或多种。
进一步的，所述脂肪肝包括非酒精性脂肪肝。
更进一步的，所述减脂药品的制剂形式可以为硬胶囊、软胶囊、片剂、颗粒剂、粉剂、悬浮液、糖浆、口服液或注射液。
根据选择剂型的不同，所述减脂药品还可以包括医药学上可接受的赋形剂，辅料等。
进一步的，在本申请的方案中，所述减脂药品为单位制剂。例如所述单位制剂中含有50-200mg的红曲黄素C。当然上述红曲黄素C的量也可以根据施用对象的不同适当调整，只要能实现预防或治疗个体与脂代谢异常相关的疾病即可。
本申请的方案中，单位制剂中的红曲黄素C的量是针对一般成人体重、单次施用的药剂中所述有效成分的量。所述单位制剂为满足一次给药所需有效成分的制剂，常见的单位制剂如一单位(片)片剂、一单位(针)针剂或 粉针剂等。患者一次施用所需的药物的量可以方便地通过计算患者的体重和该患者一次用药所需单位体重剂量的乘积得到。例如，在制备药物的过程中，一般认为成人体重为50-90kg，可以通过实验动物与人的单位体重剂量之间的等效剂量换算关系来确定用药量。例如，可以根据FDA、SFDA等药品管理机构提出的指导意见，也可参考(黄继汉等，“药理试验中动物间和动物与人体间的等效剂量换算”，《中国临床药理学与治疗学》,2004Sep；9(9):1069-1072)来确定。在本申请的实施方式中，可以使用按照人和仓鼠的体表面积折算系数0.12来换算人和仓鼠的剂量。
本申请提供一种用于预防或治疗个体与脂代谢异常相关的疾病的方法，使用上述的减脂药品。
本申请方案中的红曲黄素C可以利用2014年台湾发明专利TW1437001中记载的获得Monascuspiloin的方法或JP2008-56618A中记载的获得Monascinol的方法获得。
或者，可以利用以下制备红曲黄素C的方法获得，包括：
1)制备固态培养基：将大米与水浸泡后灭菌获得所述固态培养基；
2)制备种子液：活化所述红曲菌，将活化的红曲菌接种至种子液培养基中培养，获得种子液；
3)发酵：将所述种子液注入所述固态发酵培养基中进行发酵，以得到发酵后的大米即红曲米；
4)提取红曲黄素C：所述红曲米经干燥、粉碎后，使用乙醇进行提取，提取液再经高效液相色谱分离，以获得所述红曲黄素C。
在上述方法中，对于大米的来源没有特殊限定，所述大米例如可以是普通商购可获得的大米。
使用的红曲菌可以是2014年台湾发明专利TW1437001记载的红曲菌。也可以是申请人于2019年9月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，其地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.18578的红曲菌。
在使用CGMCC No.18578的红曲菌制备的情况下，为提高产量，也可以按照以下步骤进行红曲黄素C的制备：
1)制备固态培养基：将大米与水浸泡后灭菌获得所述固态培养基；
2)制备种子液：活化所述红曲菌，将活化的红曲菌接种至种子液培养基中培养，获得种子液；
3)发酵：将所述种子液按1：5-10L/kg的比例注入所述固态发酵培养基中进行发酵，以得到发酵后的大米即红曲米；
4)提取红曲黄素C：所述红曲米经干燥、粉碎后，使用50％-80％乙醇进行提取，提取中控制红曲米与乙醇的重量比为1：5-20，提取液再经高效液相色谱分离，以获得所述红曲黄素C。
在本申请的另一个具体实施方式中，所述制备红曲黄素C的方法包括以下步骤：
1)制备固态培养基：将所述大米用水浸泡后形成的湿米平铺在锥形瓶中，灭菌，得到固态发酵培养基；
2)制备种子液：将所述红曲菌使用活化培养基活化后接种至种子液培养基中培养，制成红曲菌的种子液；
3)发酵：过滤所述红曲菌的种子液以去除菌丝体，将去除菌丝体的所述红曲菌的种子液按比1：5-10L/kg注入所述固态发酵培养基中进行发酵，得到发酵后的大米即红曲米；
4)提取红曲黄素C：将发酵后得到的红曲米经干燥、粉碎后，使用50％-80％乙醇进行提取，提取中控制红曲米与乙醇的重量比为1：5-20，提取液再经高效液相色谱分离，以获得所述红曲黄素C。
进一步的，步骤1)浸泡过程中大米与水的比例为1:1kg/L，在室温下浸泡4-24h。
进一步的，步骤2)中所述红曲菌的活化温度为25-35℃，活化培养2-5天。进一步的，在所述步骤2)中，所述活化培养基为麦芽汁斜面培养基。
所述麦芽汁斜面培养基为本领域常规的培养基，可以购买获得。也可以按照以下制备获得：将大麦芽粒轻度粉碎，称取250.0g，加入1L水，60℃恒温水浴4h，过滤后加水，将糖度稀释至12°Brix，加入3.0g琼脂，于121℃、0.1MPa条件下灭菌20min。
更进一步的，步骤2)中将活化的红曲菌接种至种子液培养基中培养在恒温摇床中进行，控制摇床温度为25-35℃，摇床转速为150-200r/min， 培养时间为24-48小时。
所述种子液培养基为本领域常规的培养基，可以购买获得。也可以按照以下配方制备获得，所述种子液培养基包括，以重量计，6.0份葡萄糖、2.0份蛋白胨、1.0份NaNO 3、1.0份KH 2PO 4、0.5份MgSO 4，以及100份的水。例如将6.0g葡萄糖、2.0g蛋白胨、1.0g的NaNO 3、1.0g的KH 2PO 4、0.5g的MgSO 4溶于100mL水并分装至250mL锥形瓶中，8层纱布和牛皮纸封口，于121℃、0.1MPa条件下灭菌20min获得。
进一步的，步骤3)中发酵的温度为25-35℃，发酵时间为10-25天。
所述步骤4)，提取液再经高效液相色谱分离，以获得所述红曲黄素C的过程中，还包括从提取液获得红曲素(Monascin,简称MS)。申请人检测发现MS与MC同时存在于该提取液中，可以通过常规技术手段将二者从提取液中分离出来，例如根据MS与MC出峰时间的不同，在高效液相色谱分离过程中分别收集MS与MC，这对于本领域技术人员是可以实现的。
进一步，MS可以通过式1的方式转变为MC；本申请提供的制备MS的方法，也可以包括步骤5)将从提取液获得的MS转变为MC，以进一步提高MC的产量，
Figure PCTCN2020126114-appb-000001
该MS照式1进一步转化为MC，可进一步提高本申请红曲菌发酵生产MC的产量。
本申请方案具有以下优点：
1)首次发现红曲黄素C具有显著的降低血脂、控制体重增加、抑制体脂肪堆积的功能；并具有降低的细胞毒性，作为减脂制品具有更高的使用安全性。
2)本申请提供了红曲黄素C在制备减脂功能性食品中的应用，用于预防 或改善个体与肥胖相关的亚健康状态，所述个体例如可以是包括成年和青少年在内的需要体重管理的亚健康人群。
3)本申请还提供了红曲黄素C在制备减脂制品包括减脂药品中的应用，用于预防或治疗个体与脂代谢异常相关的疾病，包括脂肪肝、心血管疾病、糖尿病和哮喘；还可用于与肥胖密切相关的一系列疾病诸如糖尿病、心血管疾病、哮喘和骨关节炎等的预防和治疗。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据附图获得其他的附图。
图1A显示了油酸添加前HepG2细胞情况，图1B显示了油酸添加后HepG2细胞脂质堆积情况。
图2显示了MC和MS不同给药量对油酸造模后的HepG2细胞存活率的影响。
图3显示了不同MC给药浓度对油酸造模后的HepG2细胞内TG含量的影响。
图4显示了MC和MS对油酸造模后的HepG2细胞内TG含量的影响。
图5A为油酸造模组HepG2细胞内脂质堆积图；图5B为MS组HepG2细胞内脂质堆积图；图5C为MC组HepG2细胞内脂质堆积图。
图6显示了MC对胰脂肪酶的抑制活性随浓度的变化图。
图7A为MS(红曲素,Monascin)反应物添加顺序不同的抑制率结果图；图7B为AK(安卡红曲黄素,Ankaflavin)反应物添加顺序不同的抑制率结果图；图7C为MC(红曲黄素C,Monascinol)反应物添加顺序不同的抑制率结果图。
图8显示了MC的Lineweaver–Burk曲线。
图9A-图9C显示了各分组仓鼠的肝脏组织切片在100x光镜下的结果；图9a-图9c显示了各分组仓鼠的肝脏组织切片在400x光镜下的结果。
图10A-图10C显示了各分组仓鼠的脂肪组织切片在100x光镜下的结果；图10a-图10c显示了各分组仓鼠的脂肪组织切片在400x光镜下的结果。
图11显示了MC的LCMS一级正离子谱图。
图12显示了MC的 1H-NMR谱图。
图13显示了MC的 13C-NMR谱图。
具体实施方式
本申请实施例中的HepG2细胞购自协和细胞资源中心(资源编号：3111C0001CCC000035)，实验过程中使用的各种试剂均为常规试剂，均可商购获得。红曲黄素C(Monascinol，简称MC)可以采用JP2008-56618A或TW1437001B中方法制备。红曲素(Monascin,简称MS)可以采用JP2008-56618A中方法制备得到。红曲黄素C(Monascinol，简称MC)和红曲素(Monascin,简称MS)也可以使用本申请中记载的红曲菌(Monascus sp)制备得到。该红曲菌(Monascus sp)为2019年9月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏编号为CGMCC No.18578的红曲菌。原料为普通商购获得的大米，培养基中各成分均为本领域常规可以商购获得的试剂。
实施例1红曲黄素C的细胞毒性
1)油酸造模HepG2细胞
取生长状态良好的HepG2细胞以2*10 5个/mL，每孔1mL接种于6孔培养板内。待细胞贴壁率达到80％-90％时，弃去培养基。设置空白组与模型组，空白组加完全培养基，模型组加用完全培养基稀释的0.3mmol/L的油酸诱导液，每孔加2mL，培养24h后，通过油红O染色观察细胞中脂滴蓄积情况。
空白组细胞形态呈梭形，细胞边缘清晰，细胞内无明显脂滴积累；加入2mL 0.3mmol/L的油酸诱导液的贴壁HepG2细胞，在孵育培养24h后，细胞没有明显的增殖损伤，且与空白组相比细胞边缘可以看到有明显的红色脂滴环绕在细胞核周围，细胞轮廓较清晰，表明建模成功，参见图1，图1A-图1B显示了油酸添加前后对HepG2细胞脂质堆积的影响(倒置显微镜×400)。
2)红曲黄素C(Monascinol，简称MC)和红曲素(Monascin，简称MS)对油酸造模的HepG2细胞的细胞毒性实验
a.取生长状态良好的HepG2细胞以5*10 4个/mL，每孔100μL接种于96 孔培养板内。待细胞贴壁率达到80％-90％时，弃去培养基。
b.将MS和MC分别溶于二甲基亚砜(DMSO)中配置成5mg/mL的母液，将母液置于-20℃保存。取5mg/mL的母液用含油酸0.3mmol/L的完全培养基稀释成实验所需的MS样品浓度或MC样品浓度(2、4、6、8、10、12μg/mL)，保证每个样品中DMSO的终浓度为0.5％。
c.设置造模组与加药组，
第1天(弃去培养基后)，在造模组的HepG2细胞中添加含0.3mmol/L油酸的完全培养基200μL；在加药组的HepG2细胞中添加含0.3mmol/L油酸及上述不同浓度的MS或MC样品的完全培养基200μL。
24h后，按MTT实验方法检测各组HepG2细胞存活率。
d.结果如图2所示，当MS给药浓度达到10μg/mL以上时，细胞存活率仅为36.7％，开始出现细胞毒性，细胞大量死亡。MC给药浓度12μg/mL时细胞存活率为66.7％，比相同给药浓度MS的细胞毒性明显小。
实施例2红曲黄素C不同给药剂量对细胞甘油三酯含量的影响
按照以下进行实验：
a.选取长势良好的HepG2细胞按2*10 5个/mL，每孔1mL接种于6孔培养板中培养12h，待细胞贴壁后弃去培养基。
b.弃去培养基后，设置空白组，造模组，加药组。
空白对照组：加入2mL完全培养基；
油酸造模组：加入2mL含油酸0.3mmol/L的完全培养基；
MC低剂量组：加入2mL含油酸0.3mmol/L以及2μg/mL的红曲黄素C的完全培养基；
MC中剂量组：加入2mL含油酸0.3mmol/L以及4μg/mL的红曲黄素C的完全培养基；
MC高剂量组：加入2mL含油酸0.3mmol/L以及8μg/mL的红曲黄素C的完全培养基。
完成给药后，培养各组细胞24小时，收集各组细胞，利用甘油三酯试剂盒(北京普利莱基因技术有限公司)测定各组HepG2细胞中甘油三酯的含量。
c.结果如图3所示，不同字母表示与空白组比有显著性差异，P＜0.01。
从图3可以看出，与空白组相比，造模组中的TG(甘油三酯,Triglyceride) 含量显著上升(P＜0.01)，细胞内TG含量达到正常组的3.2倍，表明造模成功。与造模组相比，MC低、中、高三个剂量均能显著(P＜0.01)降低细胞中TG的含量。MC浓度在2μg/mL和4μg/mL时使细胞中TG含量分别下降38.9％，34.6％，浓度为8μg/mL时使细胞中TG含量下降51.3％。因此，MC有效降低HepG2细胞内TG含量。
实施例3红曲黄素C(Monascinol，简称MC)和红曲素(Monascin，简称MS)对油酸造模的HepG2细胞的甘油三酯含量的影响
按照以下进行实验：
首先获得正常HepG2细胞，并按照实施例1方法获得油酸造模后的HepG2细胞(油酸浓度0.3mmol/L)。
第1天，空白对照组：对HepG2细胞正常培养不做处理；油酸造模组(油酸浓度0.3mmol/L)，对油酸造模后的HepG2细胞正常培养不做处理；MC组：对油酸造模后的HepG2细胞给药2μg/mL的红曲黄素C即MC；MS组：对油酸造模后的HepG2细胞给药2μg/mL的红曲素MS。
完成给药后，继续培养各组细胞24小时，收集各组细胞，利用甘油三酯试剂盒(北京普利莱基因技术有限公司)测定各组HepG2细胞中甘油三酯的含量，结果如图4所示。
从图4可以看出，0.3mmol/L的油酸孵育HepG2细胞24h后，细胞中TG含量较空白组显著升高(P＜0.01)。相同浓度的MS和MC均使细胞中TG的含量显著下降(P＜0.01)。2μg/mL的MS和MC可使细胞中TG含量分别下降26.3％和33.3％，两者降脂效果无显著性差异(P>0.01)。
通过油红O染色进一步进行形态学观察，结果如图5A-5C(倒置显微镜×400)所示，图5A为油酸造模组HepG2细胞内脂质堆积图；图5B为MS组HepG2细胞内脂质堆积图；图5C为MC组HepG2细胞内脂质堆积图，可以看出，与油酸造模组相比，2μg/mL的MS组和MC组均可使细胞中的脂滴减少，颜色变浅。这与TG的检测结果一致。
实施例4MC对胰脂肪酶的抑制活性及抑制类型
实验以4-甲基伞形酮油酸酯(4-MUO)为底物，采用荧光检测法评价MC对胰脂肪酶(type II，来源猪胰腺)的抑制活性。
设置以下组(也可参见表1)：
空白组：不含MC样品，仅有底物、胰脂肪酶和Tris-HCL缓冲液；
空白背景组：不含MC样品和胰脂肪酶，仅有底物和Tris-HCL缓冲液；
样品背景组：不含胰脂肪酶，仅有MC样品、底物和Tris-HCL缓冲液；
样品组：不含Tris-HCL缓冲液，仅有MC样品、底物和胰脂肪酶。
表1 体外测定胰脂肪酶活力方法
Figure PCTCN2020126114-appb-000002
根据以下公式计算抑制率：
抑制率(％)＝[(A 1–A 2)–(A 3–A 4]/(A 1–A 2)×100；
A 1：空白组荧光值；A 2：空白背景组荧光值
A3：样品组荧光值；A 4：样品背景组荧光值
将各组含有的物质按照表1所示比例添加在黑色96孔培养板中的孔中，每组实验设三个平行，其中4-MUO的浓度为0.1mM，MC样品使用DMSO溶解(设置6个不同的浓度：5、10、20、40、80、160μg/mL)，脂肪酶溶液的浓度为1mg/mL，在25℃条件下反应30min后，加入100μL 0.1M柠檬酸钠缓冲溶液终止反应。脂肪酶会催化底物4-甲基伞形酮油酸酯(4-MUO)生成产物4-甲基伞形酮，4-甲基伞形酮在激发波长340nm，发射波长460nm下有荧光强度。通过检测荧光值的大小，间接判断脂肪酶活力(即脂肪酶活力越高，催化生成的4-甲基伞形酮越多，荧光信号也就越强。因此，在激发波长360±40nm，发射波长460±40nm条件下对产物4-甲基伞形酮进行测定。
将上述所述MC的6个浓度转化为对数浓度后，以对数浓度为横坐标，抑制率为纵坐标，在Origin85中绘制出图6所示的s型曲线，可以看出，MC对胰脂肪酶的抑制率随着浓度的增加而上升。进一步，基于该S型曲线后，利用本领域已知软件计算IC50值为75.8μg/mL。此部分结果表明MC对胰脂 肪酶有一定的抑制活性，通过抑制甘油一酯吸收途径减少甘油三酯经肠道摄取量，具有潜在的抗肥胖功效。
根据抑制剂与酶结合方式和特点不同，抑制类型可分为可逆抑制和不可逆抑制两种类型。不可逆抑制通常以比较牢固的共价键与酶蛋白中的基团结合使酶失活。可逆抑制分为竞争性抑制，非竞争性抑制和反竞争性抑制。研究已报道MS和AK对胰脂肪酶的抑制类型为非竞争性抑制，本部分实验以MS，AK为对照，通过改变反应物的添加顺序判断MC的抑制类型，三种色素抑制剂的干预浓度均为80μg/mL。结果如图7A-7C所示，图7A为MS(红曲素,Monascin)反应物添加顺序不同的抑制率结果图；图7B为AK(安卡红曲黄素,Ankaflavin)反应物添加顺序不同的抑制率结果图；图7C为MC(红曲黄素C,Monascinol)反应物添加顺序不同的抑制率结果图；可以看出MC的抑制类型与MS，AK的抑制类型不同。图7A-7C中，S：底物，I：抑制剂，E：酶。
以下根据Lineweaver－burk双倒数作图法的结果，来确定MC对胰脂肪酶的抑制类型。本领域已知，若两条直线相交于1/S轴，则抑制类型为非竞争性抑制；若两条直线相交1/V轴，则抑制类型为竞争性抑制；若两条直线平行，则抑制类型为反竞争性抑制。
图8显示了MC的Lineweaver–Burk曲线，其中，1/S为底物浓度倒数；1/V为反应速率倒数，反应速率为反应前后的荧光差值除以时间差值。绘制Lineweaver-Burk曲线实验的基本流程为：在脂肪酶浓度(1mg/mL)一定的条件下，以一系列不同的底物浓度进行酶促反应速率的测定。空白组不含抑制剂MC(不含MC样品，仅有不同浓度底物、胰脂肪酶和Tris-HCL缓冲液)，MC组是加入(160μg/mL的MC，不同浓度底物、胰脂肪酶和Tris-HCL缓冲液。从图8中可以看出，双倒数作图法显示两条直线趋向平行，属可逆抑制类型中的反竞争性抑制。说明MC不直接与酶结合，通过与酶-底物复合物结合的方式降低胰脂肪酶的催化反应速率。
实施例5MC动物实验数据
动物实验使用的24只雄性叙利亚金黄地鼠(Golden Syrian hamster，简称仓鼠)购自北京维通利华实验动物技术有限公司，体重110-120克，年龄8周。
预养一段时间后，随机分为3组，每组8只，按照以下进行分组和实验：
第1天开始每天：
正常组(NOR)：自由摄取基础饲料，并灌胃给与MC组等同量CMC-Na溶液；
高油脂组(HFD)：自由摄取具高油脂成分的咀嚼饲料，并灌胃给与MC组等同量CMC-Na溶液；
MC组：自由摄取具高油脂成分的咀嚼饲料，并且按照20mg/kg体重的剂量灌胃MC(溶解于CMC-Na溶液中)。
每周测量仓鼠的体重、摄食量。10周后，采集组织样本或血清样本并进行以下分析。
5.1 MC对仓鼠体重、肝、肾与脂肪组织重量的影响
表2记录了仓鼠肝脏、肾脏、脂肪等与脂代谢相关联的脏器重量指标。由以上实验数据可知，与HFD组相比，MC组可显著抑制(p＜0.05)仓鼠因高脂饮食导致的体重、肝重增加及外周脂肪的堆积。
表2
Figure PCTCN2020126114-appb-000003
5.2 MC对仓鼠肝脏、血清中TG(甘油三酯，Triglyceride)与TC(总胆固醇，Total Cholesterol)的影响
基于Folch法提取肝脏中的TC和TG，使用南京建成生物工程研究所生产的TC和TG检测试剂盒，按说明书提供的方法检测肝组织TC和TG含量。
采集的血清样本使用Cobas8000E602全自动生化分析仪(德国罗氏诊断有限公司)测量血清中的TC和TG。结果记录于表3。
由表3数据可知，HFD组仓鼠肝脏和血清中TG与TC含量与NOR组相比显著上升(p＜0.05)。相较于HFD组的仓鼠，MC组的仓鼠肝脏和血清中TG与TC的含量显著下调(p＜0.05)，特别值得注意的是MC干预的高脂饮食仓鼠肝脏中的TG平均含量与NOR组相同。说明MC对于因高脂饮食而导致的血清与肝脏中的脂质堆积有显著抑止效果，尤其对肝脏和血清中TG堆积的抑止效果更佳，这与细胞实验MC抑制HepG2细胞中TG堆积的结果一致。
表3 肝脏与血清中TC与TG含量
Figure PCTCN2020126114-appb-000004
5.3 仓鼠肝脏、脂肪组织病理切片分析
图9A-图9C显示了各分组仓鼠的肝脏组织切片在100x光镜下的结果,图9a-图9c显示了为各分组仓鼠的肝脏组织切片在400x光镜下的结果,其中图9A和图9a为NOR组，图9B和图9b为HFD组，图9C和图9c为MC组。可以看出，与HFD组因脂质堆积而增大的肝细胞相比，MC组细胞中脂质堆积显著减少，肝细胞索趋于正常。
图10A-图10C显示了各分组仓鼠的脂肪组织切片在100x光镜下的结果,图10a-图10c显示了各分组仓鼠的脂肪组织切片在400x光镜下的结果,其中图10A和图10a为NOR组，图10B和图10b为HFD组，图10C和图10c为MC组。可以看出，HFD组附睾周围脂肪组织细胞和肾周围脂肪组织细胞较NOR组体积明显增大，而MC组的细胞较HFD组体积明显减少，细胞体积大小介于NOR组与HFD组的细胞体积之间。
综合以上细胞实验、体外酶实验和动物实验，MC能够有效预防高脂饮食导致的体重增加、外周脂肪堆积、血液和肝脏TC和TG的堆积。可用于预防和治疗因肥胖引发的脂代谢异常及其相关的代谢综合症(如脂肪肝、糖尿病、哮喘、骨关节炎等)。经本申请提供的红曲菌发酵获得的富含该MC的红曲 米，或者由该红曲米提取分离获得的MC或者MS可用于开发调控脂代谢异常相关功能食品和作为药品的原料药。
实施例6 利用本申请提供的红曲菌制备红曲黄素C
红曲菌为2019年9月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏编号为CGMCC No.18578的红曲菌。原料为普通商购获得的大米，培养基中各成分均为本领域常规可以商购获得的试剂。
一、利用红曲菌CGMCCNo.18578制备红曲黄素C，包括以下步骤：
1)将大米和水按1:1的质量体积比(kg/L)在室温下浸泡4-24h，将水沥干后装入培养瓶中，在121℃、0.1MPa条件下灭菌20min，得到固态发酵培养基；
2)将保存于4℃冰箱的红曲菌CGMCCNo.18578转接入新鲜的麦芽汁斜面培养基上，30℃培养48h以活化该红曲菌；将灭菌后的无菌水在无菌操作下倒入活化好的菌种斜面中，接种环刮下孢子制成孢子悬浮液，转接至种子液培养基中，30℃，180r/min恒温摇床培养36h，以获得红曲菌的种子液。
其中活化用的斜面培养基为通过大麦芽粒轻度粉碎，称取250.0g，加入1L水，60℃恒温水浴4h，过滤后加水，将糖度稀释至12°Brix，加入3.0g琼脂，于121℃、0.1MPa条件下灭菌20min制备获得)。
种子液培养基为通过6.0g葡萄糖、2.0g蛋白胨、1.0g的NaNO 3、1.0g的KH 2PO 4、0.5g的MgSO 4溶于100mL水并分装至250mL锥形瓶中，8层纱布和牛皮纸封口，于121℃、0.1MPa条件下灭菌20min获得。
3)用双层纱布过滤所述红曲菌的种子液以去除菌丝体，将去除菌丝体的所述红曲菌的种子液按1：5-10L/kg的体积重量比注入所述固态发酵培养基中在30℃发酵20天，得到发酵后的大米即红曲米；
4)将发酵后的红曲米放置于烘箱60℃干燥、粉碎后，使用70％乙醇进行提取，红曲米与乙醇的重量比为1：5-20，经提取液再经高效液相色谱分离以获得所述红曲黄素C。
二、红曲菌CGMCCNo.18578制备得到的MC结构解析
ESI-MS测定所获得的红曲黄素C的结果如图11所示，其一级正离子峰m/z为361.2000[M+H] +，该化合物分子式为C 21H 28O 5。红曲黄素C的 1H-NMR 结果如图12所示，红曲黄素C的 13C-NMR结果如图13所示，该结果与文献JP2008-56618A记载Monascinol的结构表征信息相同。
三、红曲菌CGMCCNo.18578制备得到的MC含量测定
MC标准曲线的绘制：
将制备纯化的MC(HPLC纯度>95％)溶解于70％乙醇，配置成4mg/ml母液，经梯度稀释得到浓度分别为400、200、100、50、25μg/ml的MC标准工作液，美国安捷伦科技有限公司1260型HPLC检测绘制标准曲线，HPLC条件：色谱柱ZORBAX Eclipse Plus C18(5μm，4.6×250mm)；流动相乙腈-0.1％甲酸水60:40(V/V)，等度洗脱；二极管阵列检测器；检测波长390nm；柱温25℃；流速1mL/min；进样体20μL。以峰面积Y对浓度X进行线性回归，得到MC回归方程为Y＝31.63x+122，R 2＝0.998。
红曲发酵产物中MC含量的测定：
60℃烘干发酵产物(即发酵后的红曲米)，磨粉过筛(200目)后准确称取0.50g入10mL离心管中，料液比为1:20的70％乙醇溶液超声提取30min，3500r/min离心10min，将上清液适当稀释后经0.22μm有机滤器过滤提取液至液相小瓶中，HPLC检测。根据样品中检测到的MC峰面积和样品稀释倍数及MC标准曲线，计算得到发酵产物中MC含量为8mg/g。此含量远高于台湾发明专利TW1437001B中所用红曲菌发酵的红曲米中MC含量(0.31mg/g)。该红曲菌CGMCCNo.18578发酵的富含MC的红曲产物例如发酵后的红曲米，可以用于开发调控脂代谢异常相关功能食品和作为药品的原料药。

Claims (12)

  1. 红曲黄素C在制备减脂制品中的应用。
  2. 根据权利要求1所述的应用，其中，所述减脂制品用于降低甘油三酯。
  3. 根据权利要求1或2所述的应用，其中，所述减脂制品为减脂功能性食品。
  4. 根据权利要求3所述的应用，其中，所述减脂功能性食品用于预防或改善个体与肥胖相关的亚健康状态。
  5. 根据权利要求1或2所述的应用，其中，所述减脂制品为减脂药品。
  6. 根据权利要求5所述的应用，其中，所述减脂药品用于预防或治疗个体与脂代谢异常相关的疾病。
  7. 根据权利要求6所述的应用，其中，所述个体与脂代谢异常相关的疾病包括脂肪肝、心血管疾病、糖尿病和哮喘中的一种或多种。
  8. 根据权利要求7所述的应用，其中，所述脂肪肝包括非酒精性脂肪肝。
  9. 根据权利要求5-8任一项所述的应用，其中，所述减脂药品的制剂形式为硬胶囊、软胶囊、片剂、颗粒剂、粉剂、口服液或注射液。
  10. 根据权利要求5-9任一项所述的应用，其中，所述减脂药品为单位制剂。
  11. 根据权利要求10所述的应用，其中，所述单位制剂中含有50-200mg的红曲黄素C。
  12. 一种用于预防或治疗个体与脂代谢异常相关的疾病的方法，使用权利要求1-2或5-11任一项所述的红曲黄素C的应用。
PCT/CN2020/126114 2019-12-23 2020-11-03 红曲黄素c在制备减脂制品中的应用 WO2021129166A1 (zh)

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