Red earthworm fermentation liquor and application thereof in preparation of blood fat reducing medicine

Abstract

The invention discloses a red earthworm fermentation broth and application thereof in preparation of blood fat reducing drugs, fresh earthworms, red yeast rice fermentation broth and natto powder are fermented in a specific proportion to obtain the red earthworm fermentation broth, and then the red earthworm fermentation broth is mixed with auxiliary materials to prepare a pharmaceutical composition. The pharmaceutical composition can significantly reduce total cholesterol, total triglyceride, low-density cholesterol and blood sugar, can also significantly reduce the ratio of glutamic oxalacetic transaminase to glutamic-pyruvic transaminase, and can effectively reduce hyperlipidemia and improve liver function impairment. Meanwhile, the pharmaceutical composition is safe, free of toxic and side effects, low in production cost and suitable for wide popularization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial pharmaceutical technology, in particular to a red gecko fermentation liquor and its application in the preparation of a blood lipid-lowering drug. BACKGROUND

[0002] Hyperlipidemia is a disease characterized by abnormal blood lipid metabolism, and its clinical features are elevated total cholesterol, total triglycerides, low-density lipoprotein cholesterol in the circulation, and decreased high-density lipoprotein. It is called dyslipidemia in modern medicine. It is the main factor leading to atherosclerosis and a risk factor for the development of cardiovascular and cerebrovascular diseases. It is insidious and mostly without clinical symptoms, hence the name "silent killer".

[0003] Hyperlipidemia can be divided into primary and secondary. The primary is related to congenital and genetic, caused by single gene defects or multiple gene defects, resulting in abnormal receptors, enzymes or apolipoproteins involved in lipoprotein transport and metabolism, or caused by environmental factors (diet, nutrition, drugs) and unknown mechanisms. Secondary is often associated with metabolic disorders (diabetes, hypertension, myxedema, hypothyroidism, obesity, liver and kidney disease, adrenal cortical hyperfunction), or related to other factors such as age, gender, season, alcohol consumption, smoking, diet, physical activity, mental stress, emotional activity, etc.

[0004] Western medicine is widely used in the clinical treatment of hyperlipidemia because of its rapid onset and definite effect. It is currently a very important drug for the prevention and treatment of hyperlipidemia and atherosclerotic diseases, and is the most widely used blood lipid-lowering drug in clinical practice. The chemical blood lipid-lowering drugs currently used in clinical practice mainly include statins, fibrates, niacin, and bile acid chelating agents. Among them, statins are the first-line drugs that are currently dominant in terms of efficacy and safety, and can effectively reduce low-density cholesterol and triglycerides in serum; fibrates have a significant effect on reducing triglyceride levels; slow-release niacin performs well in low-density cholesterol; and cholesterol absorption inhibitors and bile acid chelating agents mainly reduce low-density cholesterol. However, chemical blood lipid-lowering drugs have definite effects but most of them have adverse reactions, are too single and can easily cause organic lesions such as liver damage. At the same time, these drugs need to be taken for a long time, and blood lipids are likely to rebound after stopping medication, with a relatively high recurrence rate, and cannot achieve both treatment and prevention, so there are certain limitations in clinical application. Therefore, it is of great clinical significance to provide a traditional Chinese medicine product with obvious effect and no toxic side effects. Currently, traditional Chinese medicine has better effect on regulating blood lipids in patients with mild to moderate hyperlipidemia, but patients with severe hyperlipidemia still need to be treated with statin chemical drugs. Overall, the total effective rate of traditional Chinese medicine for lowering blood lipids is about 30%, so there is still a lot of room for improvement in the efficacy of traditional Chinese medicine for regulating blood lipids.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The present application aims to provide a red earthworm fermentation liquor and its application in the preparation of a blood lipid-lowering drug. The blood lipid-lowering drug prepared from the red earthworm fermentation liquor has a better effect than that of general traditional Chinese medicine, the raw material of the product is both medicine and food, thus avoiding intolerance, and the microbial enzymatic fermentation technology can be used to produce blood lipid-lowering active substances with high purity and good activity in a mild and efficient manner, which has a huge application prospect.

[0007] In a first aspect, the present application provides a red earthworm fermentation liquor, which is prepared from fresh earthworm, red koji fermentation liquor and natto powder, wherein the mass ratio of the fresh earthworm, the red koji fermentation liquor and the natto powder is 3:8-9:24-27. In a second aspect, the present application further provides the application of the red earthworm fermentation liquor in the preparation of a blood lipid-lowering drug.

[0008] In a third aspect, the present application further provides the application of the red earthworm fermentation liquor in the preparation of a drug for improving liver function.

[0009] In a fourth aspect, the present application further provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient. The active ingredient is a powder obtained by freeze-drying and crushing the red earthworm fermentation liquor.

[0010] The present application has the following beneficial effects: The present application obtains the red earthworm fermentation liquor by fermenting fresh earthworm, red koji fermentation liquor and natto powder in a specific ratio, and then mixes the red earthworm fermentation liquor with excipients to prepare a pharmaceutical composition. The pharmaceutical composition can significantly reduce total cholesterol, total triglyceride, low-density cholesterol and blood sugar, and can also significantly reduce the ratio between glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase. The pharmaceutical composition of the present application can effectively reduce high blood lipid and improve liver function damage. Meanwhile, the pharmaceutical composition of the present application is safe, has no toxic side effects, and has a low production cost, thus being suitable for wide promotion. DETAILED DESCRIPTION

[0011] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the reagents or instruments are not indicated by the manufacturer, they are all conventional products that can be purchased in the market.

[0012] The present application provides a red earthworm fermentation liquor, and the preparation method is as follows: S1. Prepare red koji powder into a red koji powder aqueous solution, sterilize by high-pressure steam, inoculate Bacillus subtilis, and obtain red koji fermentation liquor after fermentation.

[0013] Monascus, also known as red yeast rice, is a genus of ascomycete fungi in the family Monascaceae, commonly used as a colorant and flavoring agent in brewing, vinegar making, and making fermented bean curd. It can produce a variety of metabolites during fermentation, mainly including red yeast rice pigment, cholesterol synthesis inhibitor, blood pressure lowering substance, various enzymes, bacteriostatic substance, and other physiological active substances.

[0014] In some embodiments, the Bacillus subtilis is derived from the National Type Culture Collection (NTCC), and the specific strain is Bacillus subtilis (RIK1285 strain).

[0015] In the present application, the main effects of using the above-mentioned Bacillus subtilis to ferment red yeast rice powder are: (1) Improving nutritional value and digestibility: It can degrade the nutritional components in red yeast rice waste, including carbohydrates and proteins, and generate metabolites with strong antioxidant activity. The cellulase produced by Bacillus subtilis can destroy the cell structure of the raw material, releasing the effective components and thus improving the efficacy and nutritional value. In addition, other enzymes produced by them can also degrade the corresponding substrates in the raw material, further enhancing the effect and nutrition.

[0016] (2) Enhancing antioxidant activity: The microecological preparation prepared by Bacillus subtilis fermentation of red yeast rice waste has strong antioxidant activity, which can scavenge DPPH free radicals and hydroxyl free radicals.

[0017] Meanwhile, the reason for choosing Bacillus subtilis in the present application is that Bacillus subtilis itself is also a probiotic, which can improve intestinal health, regulate the ecological balance of the gastrointestinal tract, promote the growth of beneficial anaerobes, and indirectly inhibit the growth of other pathogenic bacteria. It can reduce inflammation and improve the integrity of the intestinal barrier, and has a positive effect on reducing inflammation and maintaining intestinal homeostasis in patients with high blood lipids. In addition, Bacillus subtilis can synthesize enzymes such as α-amylase, protease, lipase, and cellulase, which can work together with digestive enzymes in the digestive tract to promote the digestion and absorption of nutrients.

[0018] In some embodiments, 5-15% of Bacillus subtilis seed solution is inoculated in 100 g / L of red yeast rice aqueous solution, and the bacterial concentration of the seed solution is 5-10 lg CFU / mL.

[0019] In some embodiments, the fermentation conditions of red yeast rice powder are as follows: fermentation temperature 35-40℃, fermentation time 36-60h, red yeast rice powder concentration 75-125 g / L, and shaking speed 200-240 r / min.

[0020] S2. Fresh Pheretima is washed and chopped.

[0021] Earthworm, also known as earthworm, is one of the traditional Chinese medicinal materials. Earthworm protein is the extract of earthworm, which is one of the active ingredients of earthworm. At present, it has been found that earthworm protein plays an important role in cardiovascular and cerebrovascular system, endocrine system, nervous system and respiratory system.

[0022] S3. Take the natto powder and crush the wall.

[0023] Natto is a soy product made from soybeans fermented by Bacillus natto. It has a sticky texture, a strong odor, and a slightly sweet taste. Not only does it retain the nutritional value of soybeans, but it is also rich in vitamin K2 and improves the digestion and absorption of protein. More importantly, the fermentation process produces a variety of physiologically active substances, which have the health benefits of dissolving fibrin and other physiological functions in the body.

[0024] S4. Put the red yeast fermentation broth, fresh earthworm and natto powder prepared in the above steps into the reaction kettle according to the proportion, stir uniformly, and then constant temperature fermentation and continuous slow stirring.

[0025] In some embodiments, the mass ratio of fresh earthworm, red yeast fermentation broth and natto powder is 3:8-9:24-27. Preferably, the mass ratio of fresh earthworm, red yeast fermentation broth and natto powder is 3:8:24.

[0026] In the present application, the reason for adding the three raw materials in the above mass ratio is that the fermentation effect is the best under this condition, which can significantly improve the active ingredients in the fermentation broth, and at the same time, the production of toxic waste during fermentation is less. According to the principle of maximizing the use of raw materials, it is found that the utilization rate of raw materials is the highest under this ratio, and the economic effect and cost benefit are the best.

[0027] In some embodiments, the constant temperature fermentation conditions are: constant temperature fermentation at 37℃ for 2-4h.

[0028] In the present application, the purpose of mixing the three raw materials before fermentation is: (1) Single control of fermentation conditions to improve the yield of active ingredients: by first fermenting red yeast alone, the fermentation conditions such as temperature, pH value, moisture content can be more accurately controlled to optimize the metabolic activity of Bacillus subtilis and possibly improve the active ingredients in red yeast; (2) Reduce bacterial contamination and metabolic waste: separate fermentation of red yeast can better control the fermentation environment, reduce bacterial contamination and ensure the purity of the fermentation process. At the same time, Bacillus subtilis can decompose some harmful ingredients in the fermentation of red yeast powder in advance, improving safety; (3) Improve fermentation efficiency: first fermenting red yeast can make Bacillus subtilis grow and reproduce fully under suitable conditions, so that red yeast can adapt to the environment more quickly and improve the overall fermentation efficiency when mixed with other raw materials for subsequent fermentation.

[0029] S5. obtaining the fermented liquid of red earth dragon.

[0030] The fermented liquid of red earth dragon can be obtained by the above method. Animal experiments show that the product prepared by the fermented liquid of red earth dragon can significantly reduce total cholesterol, total triglyceride, low-density cholesterol and blood sugar, and can also significantly reduce the ratio between glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase. Therefore, the fermented liquid of red earth dragon obtained by the present application can be further prepared into a medicine or food for reducing blood lipids and improving liver function damage.

[0031] Based on this, the present application can also provide a pharmaceutical composition, and the active ingredient of the pharmaceutical composition is the powder obtained by freeze-drying and crushing the fermented liquid of red earth dragon.

[0032] In some embodiments, the composition of the present application can be prepared into a medicine suitable for topical administration by using techniques known to those skilled in the art.

[0033] In some embodiments, the medicine prepared by the composition of the present application can also include pharmaceutically acceptable adjuvants, such as: buffers, emulsifiers, suspending agents, decomposing agents, disintegrating agents, dispersing agents, binding agents, excipients, stabilizers, chelating agents, diluents, gelling agents, sweeteners, preservatives, wetting agents, lubricants, absorption retardants, liposomes, etc.

[0034] In some embodiments, the medicine prepared by the composition of the present application can be in the following dosage forms: tablets, emulsions, creams, powders, aerosols, sprays, emulsions, milk gruel, pastes, foams, drops, suspensions, ointments, bandages and oils.

[0035] In some embodiments, the adjuvants in the above-mentioned pharmaceutical composition include erythritol, malt dextrin and magnesium stearate.

[0036] In some embodiments, the dosage form of the above-mentioned pharmaceutical composition is tablets.

[0037] Based on this, the present application can also provide a food, and the active ingredient of the food is the powder obtained by freeze-drying and crushing the fermented liquid of red earth dragon.

[0038] In some embodiments, the food prepared by the composition of the present application can also include food acceptable adjuvants, such as: solvents, binders, fillers and sweeteners, etc.

[0039] In some embodiments, the food prepared by the composition of the present application can be in the following types: tablets, capsules, pills, granules, decoctions, pastes, lotions, oral liquids, drop pills or syrups.

[0040] For the above-mentioned medicine or food, the present application also provides a preparation method of red earth dragon tablets, and the steps are as follows: S1. The red dragon fermentation broth is freeze-dried, crushed, and sieved for use; S2. Take the erythritol, malt dextrin, and magnesium stearate and mix them with the freeze-dried product in a mixer; S3. Use a tablet press to make the tablet preparation; S4. Pick out the broken, cracked, and other unqualified tablets, and you can get the red dragon tablet.

[0041] For the preparation method of the above red dragon tablet, the addition amount of the auxiliary materials erythritol, malt dextrin, and magnesium stearate can refer to the usage amount of conventional tablets, and the present application does not limit this.

[0042] The present application will be further illustrated by specific examples, but it should be understood that these examples are only for more detailed illustration and should not be understood as limiting the present application in any form.

[0043] Example 1 The present example is a preparation method of red dragon fermentation broth, and the specific steps are as follows: 1. Take the red koji powder and mix it with deionized water to make a 100 g / L red koji powder solution, sterilize it at 121℃ for 20 min, inoculate it with 15% Bacillus subtilis seed liquid, the seed liquid has a bacterial concentration of 5 lg CFU / mL, the shaking bed speed is 220 r / min, and the fermentation time is 48 h to obtain the red koji fermentation broth; 2. Clean and rinse the fresh dragon, and coarsely cut and crush it; 3. Crush and break the cell wall of the natto; 4. Put the treated fresh dragon, red koji fermentation broth, and natto into a reaction kettle, the ratio of fresh dragon, red koji fermentation broth, and natto is 3:8:24, stir them evenly, and ferment them at 37℃ for 2-4 h with continuous slow stirring; 5. Obtain the red dragon fermentation broth.

[0044] Example 2 The present example is a preparation method of red dragon tablet, and the specific steps are as follows: (1) The red dragon fermentation broth obtained in Example 1 is freeze-dried, crushed, and sieved through an 80-mesh sieve for use; (2) Take the erythritol, malt dextrin, and magnesium stearate and mix them with the freeze-dried product in a mixer, add malt dextrin at a ratio of 8%, add magnesium stearate at an amount of 0.9%, and add erythritol at an amount of 30%, and mix for 45 min; (3) Use a tablet press to make the tablet preparation; (4) Pick out the broken, cracked, and other unqualified tablets, and you can get the red dragon tablet.

[0045] Comparative Example 1 The present comparative example is a preparation method of polypeptide earthworm protein tablet, i.e., the earthworm protein tablet candy disclosed in CN111357858A, which is different from example 1 in that earthworm protein, red yeast powder and natto powder are mixed in a ratio of 10:2:3, and mulberry, radix puerariae, corn oligopeptide powder and other mixed ingredients are added at the same time, without fermentation.

[0046] Experimental example 1 The red earthworm tablet obtained in example 2 was applied to a hyperlipidemia model mouse, and the blood lipid related changes after administration were detected, and the specific steps were as follows: 1. Experimental method 1.1 Animal grouping and administration The mice used in this experiment were SPF C57BL / 6N mice, 6-7 weeks old, body weight 19±1g, purchased from Beijing Yakang Biotechnology Co., Ltd. 18 female C57 / BL6N mice and 18 male C57 / BL6N mice, body weight 19g±1g. Randomly divided into 3 groups, half male and half female, 6 female mice and 6 male mice in each group, a total of 12. Grouping as follows: ① normal control group (normal saline gavage 0.3mL / d), ② high-fat diet group (normal saline gavage 0.3mL / d), ③ high-fat diet+red earthworm intervention group (0.208mg / g red earthworm tablet candy suspension gavage 0.3mL / d). After 7 days of environmental adaptability feeding, diet intervention and administration were performed according to the group, and the body weight of the mice was measured every 7 days for 4 weeks. After four weeks, 45 mg / kg sodium pentobarbital was injected intraperitoneally for anesthesia, and blood was taken from the eye fundus plexus, and the contents of TG, CHO and LDL-C were detected. If all three are significantly increased, it is considered to be a successful modeling. After confirming the success of the hyperlipidemia mouse model, it was used for subsequent research.

[0047] 1.2 TC determination The total cholesterol detection kit (batch number 03039773190) of Roche Diagnostics (Shanghai) Co., Ltd. was used for detection. In the experiment of mouse serum TC (total cholesterol) determination, first, the mouse blood sample was collected, and after coagulation at room temperature, the serum was obtained by centrifugation at 2000-3000 rpm for 20 minutes. Then, the TC determination kit was taken out from the refrigerator, all reagents were balanced at room temperature for 15-30 minutes, and the standard solution and washing solution were prepared according to the instructions. The standard wells, blank wells and sample wells were set on the enzyme-labeled coated plate, and different concentrations of standard solution, sample diluent and diluted serum sample were added respectively. Then, the enzyme-labeled plate was sealed with sealing film, and incubated at 37°C for 30 minutes to promote the antigen-antibody reaction. After incubation, the sealing film was removed, and the liquid in the well was discarded, and the enzyme-labeled plate was washed with washing solution for 3-5 times, and then dried after each washing. Then, enzyme-labeled reagent containing cholesterol-specific binding antibody was added to each well except the blank well, and incubation and washing were performed again. Then, color developing agent solution was added to each well, and color developing was performed at 37°C in the dark for 15 minutes. After color developing, stop solution was added to terminate the enzyme reaction and stabilize the color. Finally, the absorbance value of each well was measured at 450 nm wavelength using an enzyme-labeled instrument, and the standard curve was drawn according to the absorbance value of the standard, so as to calculate the TC concentration in the test serum sample. In the whole experiment process, the operation guide of the kit instruction manual must be strictly followed, and the experimental conditions must be strictly controlled to ensure the accuracy and reliability of the determination results.

[0048] 1.3 TG determination The triglyceride detection kit (batch number 20767107322) of Roche Diagnostics (Shanghai) Co., Ltd. was used for detection. First, the sample was properly treated, such as tissue sample needed to be homogenized in ice bath and then centrifuged to obtain supernatant, cell or bacterial sample needed to be collected and then centrifuged to discard supernatant, and then reagent was added to be ultrasonically broken and then centrifuged to obtain supernatant, while serum (serum) and other liquid samples could be directly detected. Next, the spectrophotometer was preheated for 30 minutes, the wavelength was adjusted to 420 nm, and distilled water was used for zero adjustment. Then, according to the instructions in the kit instruction manual, standard solution, test sample and GPO-PAP working solution were added in turn, mixed thoroughly, and then incubated in a 37°C water bath for 5 to 10 minutes. After incubation, the absorbance was measured at 500 nm wavelength using a spectrophotometer or an enzyme-labeled instrument, and the absorbance of standard tubes and each test tube was read after the blank tube was adjusted to zero. Finally, the concentration of TG in the sample was calculated according to the standard curve, and the experimental results were obtained.

[0049] 1.4 LDL-C determination The low-density lipoprotein cholesterol detection kit (batch number 07005717190) from Roche Diagnostics (Shanghai) Co., Ltd. was used for detection. First, the mouse serum samples were properly treated using the ELISA kit, and the supernatant was taken for detection after centrifugation if there was a precipitate. Then, the spectrophotometer or enzyme label instrument was preheated to 546 nm wavelength and zeroed with distilled water. Next, the cholesterol standard solution was diluted to prepare a series of standard solutions with different concentrations. In the 96-well plate, the sample, standard solution and reagent were added according to the kit instructions, and after the incubation and washing steps, the color developing agent was added and the color developing reaction was carried out. After the color developing was completed, the reaction was terminated by adding the stop solution, and the absorbance (OD value) of each well was measured at 450 nm wavelength. Finally, the concentration of mouse LDL-C in the sample was calculated according to the standard curve.

[0050] 1.5 HDL-C determination The high-density lipoprotein cholesterol detection kit (batch number 07528566190) from Roche Diagnostics (Shanghai) Co., Ltd. was used for detection. The experimental operation method for determining the high-density lipoprotein cholesterol (HDL-C) in mouse serum was as follows: first, the mouse blood was collected in a test tube without pyrogen and endotoxin, and centrifuged at 3000 rpm for 10 minutes to separate the serum. Next, the serum sample or standard was added to the microwell coated with HDL-C antibody, followed by the addition of HRP-labeled detection antibody. After incubation at 37°C and washing, the substrate TMB was used for color development, and TMB was catalyzed by HRP to become blue, and finally became yellow under the action of acid. Finally, the absorbance value (OD value) of the solution was measured at 450 nm wavelength using an enzyme label instrument, and the sample concentration was calculated by combining the standard curve. During the entire experiment, all reagents should be equilibrated to room temperature (20-25°C), and immediately stored at 2-8°C after use.

[0051] 1.6 Blood glucose determination The experimental operation method for determining the blood glucose of mice was as follows: first, the mice were fasted for 16 hours before the experiment but not deprived of water. On the day of the experiment, the body weight of the mice was measured, and the tail was marked with a serial number at the root to facilitate identification. Next, the tail tip of the mouse was cut off about 1-2 mm, and the tail was gently squeezed to collect blood. The fasting blood glucose value was measured using a blood glucose meter as the 0-minute baseline. Then, according to the body weight of the mice, the glucose solution was injected intraperitoneally or orally, 0.01 ml per gram of body weight, and the timing started from the end of injection or gavage. At 15, 30, 60, 90 and 120 minutes after injection, blood sampling and blood glucose determination were repeated, and the blood glucose values at each time point were recorded. After the experiment, the mice were given feed, and the experimental results were analyzed using Excel software. During the entire experiment, the operation should be as gentle as possible to avoid unnecessary stress and pain to the mice.

[0052] 1.7 ALT / AST determination The serum samples were collected from the mice, and the blood was taken on an empty stomach and separated into serum, which was stored at -20°C or -80°C to avoid repeated freeze-thawing. During the experiment, all reagents were pre-warmed to room temperature, and the serum samples and ALT or AST substrate solution were added to the test tubes, which were incubated in a 37°C water bath for a certain period of time. Then, 2,4-dinitrophenylhydrazine solution was added to terminate the reaction and form 2,4-dinitrophenylhydrazone with α-ketoglutaric acid. Under alkaline conditions, pyruvic acid generates a darker color. The absorbance value was measured at a wavelength of 520 nm by a spectrophotometer, and the standard curve was used to calculate the amount of pyruvic acid generated in the sample, thereby obtaining the activity units of ALT and AST, and calculating the ALT / AST ratio.

[0053] 2. Detection results and analysis In this experiment, both male and female mice showed a blood lipid abnormality pattern of decreased TG and increased TC under the induction of high-fat diet, which was consistent with the literature reports.

[0054] (1) Effect of Hongdilong tablets on the body weight of mice The body weight changes of the mice in each group were as follows: Table 1 Effect of Hongdilong tablets on the body weight of mice (mean ± SD, n = 6)

[0055] Note: Compared with the normal control group, #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001; compared with the high-fat diet group, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0056] The results showed that compared with the normal control group, the body weight of female and male mice in the high-fat diet group was significantly increased (p < 0.001 and p < 0.0001), indicating that the modeling was successful. Compared with the high-fat diet group, the body weight of female and male mice in the Hongdilong intervention group was significantly decreased (p < 0.01 and *p < 0.05), indicating that Hongdilong tablets can reverse the increase in body weight caused by high-fat diet, and the effect on female mice is more significant.

[0057] (2) Effect of Hongdilong tablets on the blood lipid of mice Take 18 female C57 / BL6N mice, 18 male C57 / BL6N mice, body weight 19g±1g. Randomly divided into 3 groups, half male and half female, 6 female mice and 6 male mice in each group, a total of 12. Grouping as follows: ① normal control group (normal saline gavage 0.3mL / d), ② high-fat diet group (normal saline gavage 0.3mL / d), ③ high-fat diet + red dragon intervention group (0.208mg / g red dragon tablet suspension gavage 0.3mL / d). After 7 days of environmental adaptability feeding, diet intervention and drug administration were carried out according to the group, and the body weight of mice was measured every 7 days for 4 weeks. 24h after the last administration, blood was taken by eyeball, and the blood sample was centrifuged at 3000rpm for 15min after standing for 2h, and the upper serum sample was taken for detection of the content of each index in the serum. The changes of blood lipids in each group of mice are shown in Table 2: Table 2 Effect of red dragon tablet on blood lipids in mice (mean±SD, n=6)

[0058] Note: compared with the normal control group #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; compared with the high-fat diet group *p<0.05, **p<0.01, ***p<0.001.

[0059] The results showed that: compared with the normal control group, diet intervention and drug intervention had no significant effect on liver damage index (ALT, AST, ALT / AST) in female mice, while in male mice, high-fat diet could cause significant increase of serum ALT and ALT / AST (p<0.01 and p<0.05), and red dragon intervention could significantly reverse the increase of ALT and reduce the liver damage caused by high-fat diet.

[0060] Compared with the normal control group, the TC of female and male mice in the high-fat diet group was significantly increased (female p<0.0001, male p<0.0001), indicating that the modeling was successful, and the red dragon intervention could significantly reduce the increase of TC caused by high-fat diet (female p<0.01, male p<0.05). In female mice, red dragon could also significantly reduce TG (p<0.01), indicating that red dragon tablet could improve blood lipids and reduce serum lipid accumulation in mice. Compared with the normal control group, LDL-C was significantly increased in the high-fat diet group (female p<0.05, male p<0.001), and red dragon intervention could significantly reverse the increase of LDL-C caused by high-fat diet (female p<0.001, male p<0.05).

[0061] Example 2 The red dragon tablet of Example 2 and the earthworm protein pressed candy of Comparative Example 1 were compared in terms of hypolipidemic effect under similar experimental conditions and dosages, and the experimental results are shown in Table 3: Table 3 Comparison of experimental results of Example 2 and Comparative Example 1

[0062] According to the comparison results of Table 1, it can be seen that the overall results of the red dragon tablet of Example 2 can significantly reduce the TC level compared with the control group; while the changes of TC and TG of Comparative Example 1 and its control group are not significant. It is proved that the red dragon tablet obtained by the preparation method of the present application has better effect on reducing blood lipids than the product obtained by the existing direct mixing method.

[0063] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A fermentation liquid from red earthworms, characterized in that, The raw materials for the fermentation liquid include fresh earthworms, red yeast rice fermentation liquid, and natto powder, and the mass ratio of the fresh earthworms, red yeast rice fermentation liquid, and natto powder is 3:8-9:24-27.

2. The red earthworm fermentation liquid according to claim 1, characterized in that, The preparation of the fermentation liquid includes: mixing fresh earthworm, red yeast rice fermentation liquid and natto powder evenly according to the proportion, fermenting under constant temperature and stirring slowly to obtain the red earthworm fermentation liquid.

3. The red earthworm fermentation liquid according to claim 2, characterized in that, The conditions for constant temperature fermentation are: 37℃, fermentation time 2-4 hours.

4. The red earthworm fermentation liquid according to claim 2, characterized in that, The red yeast rice fermentation broth is a fermentation broth obtained by inoculating red yeast rice powder solution with Bacillus subtilis and then fermenting it.

5. The red earthworm fermentation liquid according to claim 4, characterized in that, The strain of Bacillus subtilis is RIK1285.

6. The red earthworm fermentation liquid according to claim 5, characterized in that, The fermentation conditions for the red yeast rice powder are as follows: fermentation temperature 35-40℃, fermentation time 36-60 h, red yeast rice powder concentration 75-125 g / L, and the inoculation amount of Bacillus subtilis per 100 g / L of the red yeast rice powder is: Bacillus subtilis inoculation amount 5-15%, bacterial concentration 5-10 1 g CFU / mL, and shaking speed 200-240 r / min.

7. The use of the red earthworm fermentation broth as described in any one of claims 1-6 in the preparation of lipid-lowering drugs.

8. The use of the red earthworm fermentation broth as described in any one of claims 1-6 in the preparation of a drug for improving liver function.

9. A pharmaceutical composition, characterized in that, It contains an active ingredient and pharmaceutically acceptable excipients; the active ingredient is the powder obtained by freeze-drying and pulverizing the red earthworm fermentation liquid according to any one of claims 1-6.

10. The pharmaceutical composition according to claim 9, characterized in that, The excipients include erythritol, maltodextrin, and magnesium stearate.

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