A fermented traditional Chinese medicine for treating hyperlipidemia, its preparation method and application

Abstract

This invention discloses a fermented traditional Chinese medicine for treating hyperlipidemia, its preparation method, and its application, relating to the field of traditional Chinese medicine. This fermented traditional Chinese medicine is prepared using Bacillus sp. JQ.Y-1, which was deposited on June 24, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2021761. The fermented traditional Chinese medicine provided by this invention offers a new alternative drug for the treatment of hyperlipidemia of the dampness-obstruction type in the middle jiao (middle burner). It has a significant lipid-lowering effect, is safe and reliable, can regulate intestinal flora, protect the liver and kidneys, and has no side effects.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a fermented traditional Chinese medicine for treating hyperlipidemia, its preparation method, and its application. Background Technology

[0002] Hyperlipidemia is a major health problem worldwide and one of the most common metabolic diseases threatening human health. Its main clinical features are elevated levels of total cholesterol (T-CHO), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), or decreased levels of high-density lipoprotein cholesterol (HDL-C). It is a major contributing factor to diseases such as atherosclerosis, fatty liver, pancreatitis, and coronary heart disease. Currently, the main treatments for hyperlipidemia are fibrates and statins, but these have single targets and long-term use can easily cause side effects such as myotoxicity. Therefore, it is essential to seek drugs that can effectively treat hyperlipidemia.

[0003] Traditional Chinese medicine (TCM) fermentation refers to the process of using microorganisms and enzymes to catalyze and decompose herbs, thereby enhancing their efficacy, reducing toxicity, or producing new medicinal effects. Most herbs develop a yellowish-white mycelial coat at the end of fermentation. Studies have confirmed that fermented TCM can treat hyperlipidemia. However, because current technologies often rely on natural fermentation environments with complex microbial communities, the fermentation process can be uncontrollable, the fermentation endpoint is difficult to standardize, and the quality of fermentation cannot be guaranteed. Summary of the Invention

[0004] The purpose of this invention is to provide a fermented traditional Chinese medicine for treating hyperlipidemia, its preparation method, and its application, in order to solve the problems existing in the prior art. The fermented traditional Chinese medicine provided by this invention offers a new alternative drug for the treatment of hyperlipidemia of the dampness obstructing the middle jiao type. It has a significant lipid-lowering effect, is safe and reliable, can regulate intestinal flora, protect the liver and kidneys, and has no side effects.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Bacillus sp. JQ.Y-1, which was deposited on June 24, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M2021761.

[0007] The present invention also provides the application of the above-mentioned Bacillus in improving the efficacy of traditional Chinese medicine compositions.

[0008] Furthermore, the traditional Chinese medicine composition, by weight, comprises the following components: 5-20 parts of patchouli, 5-20 parts of magnolia bark, 1-10 parts of poria cocos, 1-10 parts of tangerine peel, 1-10 parts of alisma plantago-aquatica, 1-10 parts of atractylodes macrocephala, 2-9 parts of atractylodes lancea, 4-18 parts of hawthorn, 1-8 parts of lotus leaf, 5-20 parts of coix seed, and 1-5 parts of licorice.

[0009] The present invention also provides a fermented traditional Chinese medicine for treating hyperlipidemia of the dampness obstructing the middle jiao type. The fermented traditional Chinese medicine is obtained by inoculating the above-mentioned Bacillus subtilis with the following traditional Chinese medicine raw materials in parts by weight: 5-20 parts of patchouli, 5-20 parts of magnolia bark, 1-10 parts of poria cocos, 1-10 parts of tangerine peel, 1-10 parts of alisma plantago-aquatica, 1-10 parts of atractylodes macrocephala, 2-9 parts of atractylodes lancea, 4-18 parts of hawthorn, 1-8 parts of lotus leaf, 5-20 parts of coix seed, and 1-5 parts of licorice.

[0010] Furthermore, the preparation method of the fermented traditional Chinese medicine specifically includes the following steps:

[0011] (1) After crushing the Chinese herbal raw materials, add wheat bran and mix well, then mix evenly with flour paste to make a soft material;

[0012] (2) The Bacillus spp. of claim 1 is inoculated into the soft material, mixed evenly, pressed into blocks, and then fermented and dried to obtain the fermented Chinese medicine.

[0013] Further, in step (1), the flour paste is made by mixing flour and water in a mass ratio of 1:(10-20).

[0014] Furthermore, in step (2), the humidity of the fermentation culture is 60-90%, the temperature is 25-40℃, and the fermentation time is 24-72h.

[0015] The present invention also provides a fermented traditional Chinese medicine preparation for treating hyperlipidemia of the dampness obstructing the middle jiao type. The effective components of the fermented traditional Chinese medicine preparation are obtained by extracting the above-mentioned fermented traditional Chinese medicine using a solvent.

[0016] Furthermore, the solvent is water.

[0017] Furthermore, the temperature for extracting the above-mentioned fermented Chinese medicine using water is 60–100℃.

[0018] The present invention discloses the following technical effects:

[0019] This invention relates to a fermented traditional Chinese medicine that primarily focuses on dispelling dampness and strengthening the spleen, supplemented by regulating qi and harmonizing the stomach. By promoting qi circulation and resolving dampness, it achieves the effect of dispelling dampness and strengthening the spleen, and can be used to treat dampness obstructing the middle jiao syndrome. Symptoms often include abdominal distension, especially after eating, heaviness and fatigue, poor appetite, a thick, white, greasy tongue coating, and a slow pulse, similar to hyperlipidemia in modern medicine. This invention offers a new approach for clinical medication.

[0020] Among them, the formula contains: Patchouli, which is pungent, warm, and aromatic, which dispels turbidity, eliminates foulness, and harmonizes the middle jiao, so that when dampness is removed, the spleen can function properly, and when the spleen is healthy, dampness can be transformed; Magnolia officinalis, which is pungent, warm, and dispersing, is good at promoting qi circulation and relieving fullness, and when the spleen qi is in good circulation, dampness is transformed. Moreover, it is bitter and dry in nature, so it can dry dampness. Both of these are the chief herbs. Poria cocos strengthens the spleen and drains dampness to eliminate phlegm; Citrus reticulata peel is pungent, warm, and unblocks, regulates qi and harmonizes the stomach, dries dampness and awakens the spleen, regulates qi and transforms phlegm, so that qi flows smoothly and phlegm disappears. It can transform accumulated phlegm and prevent the source of phlegm production. It is the assistant herb; Alisma plantago-aquatica has a descending nature and drains turbidity. Alisma plantago-aquatica is added to assist Poria cocos in promoting diuresis, while Atractylodes macrocephala is retained to strengthen the spleen; Atractylodes lancea harmonizes the stomach and eliminates dampness; Crataegus pinnatifida transforms turbidity and lowers lipids, and is suitable for symptoms of cold-dampness obstruction, epigastric fullness, and poor appetite; Nelumbo nucifera leaf clears summer heat and transforms dampness, and raises clear yang, which is especially suitable for spleen deficiency and diarrhea; Coix lacryma-jobi is bland, drains dampness and strengthens the spleen, so that dampness is removed from the lower jiao. All of these herbs are the adjuvant herbs. Licorice acts as a harmonizing agent for the other herbs, both tonifying and harmonizing, thus eliminating dampness without harming the spleen and stomach, achieving a state of balance. The combined effects of these herbs internalize dampness and turbidity, promote the smooth flow of Qi, harmonize the spleen and stomach, and allow the clear Qi to ascend and the turbid Qi to descend, thereby alleviating various symptoms.

[0021] This invention provides a fermented traditional Chinese medicine for treating hyperlipidemia and describes its preparation process. It offers a new alternative drug for the treatment of hyperlipidemia of the dampness obstructing the middle jiao type. It has a significant lipid-lowering effect, is safe and reliable, can regulate intestinal flora, protect the liver and kidneys, and has no side effects.

[0022] This invention incorporates bacterial strains into the natural fermentation process, which, compared to traditional natural fermentation methods, further ensures the stability of the fermentation process, improves the efficacy of this fermented traditional Chinese medicine in treating hyperlipidemia, and provides basic research data for clinical application. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The colony morphology (A) and Gram staining results (B) of strain JQ.Y-1 are shown.

[0025] Figure 2 Oil Red O staining results for rat livers in each group (×200);

[0026] Figure 3 Immunohistochemical images of AMPK, p-AMPK, LKB1 and HMGCR proteins in liver tissue of rats in each group (×200);

[0027] Figure 4The results of IOD values ​​for the expression of AMPK(A), p-AMPK(B), LKB1(C), and HMGCR(D) proteins in the liver tissue of rats in each group were measured. n = 10, × 10 4 Compared with the normal group: * p < 0.05 ** p < 0.01; compared with the model group: # p < 0.05 ## p < 0.01;

[0028] Figure 5 The effect of this study on PCoA in hyperlipidemic rats was analyzed; where CON: normal group (control), POS: positive control group, HLP: model group (hyperlipemia), NF: naturally fermented traditional Chinese medicine group, and SF: strain fermented traditional Chinese medicine group.

[0029] Figure 6 The effects of the normal group, positive drug group, model group, high-dose naturally fermented group, and high-dose fermented bacterial strain group on hyperlipidemic rats were analyzed at the genus level.

[0030] Figure 7 The effects of normal group, positive drug group, model group, high-dose natural fermentation group, and high-dose bacterial fermentation group on the relative abundance of genus levels in hyperlipidemic rats were investigated. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] The patchouli, magnolia bark, poria cocos, tangerine peel, alisma plantago-aquatica, atractylodes macrocephala, atractylodes lancea, hawthorn, lotus leaf, coix seed, and licorice used in the following examples all comply with the relevant provisions of each item in the main text of Part I of the Chinese Pharmacopoeia (2020 edition).

[0037] Example 1: Isolation, Identification, Preservation and Culture of Strains

[0038] 1. Separation

[0039] A strain of bacteria, designated JQ.Y-1, was isolated, purified, and cultured from a naturally fermented traditional Chinese medicine used to treat hyperlipidemia.

[0040] 2. Identification

[0041] Morphological identification: such as Figure 1 As shown, the Gram staining results of strain JQ.Y-1 indicate that the strain is a Gram-positive bacterium. The colonies are round, yellowish-white, granular, slightly wrinkled, rough and moist, easy to pick up, and appear spherical under a microscope.

[0042] Physiological and biochemical identification: The oxidase and catalase tests of strain JQ.Y-1 were positive, and the VP test was positive, indicating that it can hydrolyze starch.

[0043] Molecular biological identification:

[0044] Sequencing analysis was performed by Sangon Biotech (Shanghai) Co., Ltd., and the 16S rDNA sequencing results of JQ.Y-1 are as follows:

[0045]

[0046] JQ.Y-1 was identified as belonging to the genus Bacillus sp.

[0047] 3. Preservation

[0048] JQ.Y-1 was deposited at the China Center for Type Culture Collection (CCTCC) on June 24, 2021, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2021761.

[0049] 4. Cultivation

[0050] JQ.Y-1 was inoculated into LB liquid medium and cultured at 37℃ for 48 h to obtain fermentation broth. The fermentation broth was centrifuged, the supernatant was removed, and the bacterial sludge was resuspended in sterile physiological saline to prepare 10% concentrations of different solutions. 5 ~10 10 CFU / mL bacterial suspension.

[0051] Example 2

[0052] Ingredients: 9g of patchouli, 9g of magnolia bark, 5g of poria cocos, 6g of dried tangerine peel, 6g of alisma plantago-aquatica, 6g of atractylodes macrocephala, 6g of lancea, 9g of hawthorn, 6g of lotus leaf, 9g of coix seed, 3g of licorice root, 14.8g of wheat bran, and 7.4g of flour.

[0053] (1) Powdering: Weigh the above raw materials separately, and grind the patchouli, magnolia bark, poria cocos, tangerine peel, alisma plantago-aquatica, atractylodes macrocephala, atractylodes lancea, hawthorn, lotus leaf, coix seed and licorice powder into fine powder of 80 mesh. Pass the wheat bran through a 30 mesh sieve, and then mix them evenly to make medicinal powder.

[0054] (2) Soft material preparation, mixing and pressing into blocks: Add flour to 148g water to make a thin paste, boil it, and while it is hot, knead it evenly with the medicinal powder obtained in step (1), knead it into a ball by hand, and it will fall apart when thrown to make soft material.

[0055] (3) Inoculation and fermentation: 0.6 mL of the strain prepared in Example 1 with a concentration of 10 was added to the soft material obtained in step (2). 8 The bacterial suspension at CFU / mL was mixed evenly, pressed into blocks, and shaped into bricks. These blocks were then placed in a constant temperature and humidity incubator at 30℃ and 80% relative humidity for 36 hours of fermentation. After fermentation, the blocks were dried in a 60℃ oven to obtain the fermented traditional Chinese medicine. During fermentation, gaps were maintained between the blocks. At the end of fermentation, white mycelium should be present without black spores.

[0056] (4) Preparation: The fermented Chinese medicine prepared in step (3) is extracted three times with 5 times its weight of water, each time at 100℃ for 30 min, cooled and filtered, and the three filtrates are combined and collected for later use to obtain a fermented Chinese medicine preparation for the treatment of hyperlipidemia.

[0057] Example 3

[0058] Same as Example 2, except that the raw materials are: 5g of patchouli, 20g of magnolia bark, 1g of poria cocos, 10g of tangerine peel, 1g of alisma plantago-aquatica, 10g of atractylodes macrocephala, 2g of atractylodes lancea, 18g of hawthorn, 1g of lotus leaf, 20g of coix seed, 5g of licorice, 25g of wheat bran, and 3g of flour.

[0059] (1) Pulverization: Weigh the above raw materials separately, and pulverize the patchouli, magnolia bark, poria cocos, tangerine peel, alisma plantago-aquatica, atractylodes macrocephala, atractylodes lancea, hawthorn, lotus leaf, coix seed and licorice into fine powder of 120 mesh. Pass the wheat bran through a 10 mesh sieve, and then mix them evenly to make medicinal powder.

[0060] (2) Preparation of soft material: Add flour to 84g of water to make a thin paste, boil it, and while it is hot, knead it evenly with the medicinal powder obtained in step (1), knead it into a ball by hand, and it will fall apart when thrown to make soft material.

[0061] (3) Inoculation and fermentation: Add 1 mL of the 10-concentration strain prepared in Example 1 to the soft material obtained in step (2). 5 The bacterial suspension (CFU / mL) was mixed evenly, pressed into blocks, and shaped into bricks. These blocks were then placed in a constant temperature and humidity incubator at 25℃ and 60% relative humidity for 24 hours of fermentation. After fermentation, the blocks were dried in an oven at 80℃ to obtain the fermented traditional Chinese medicine. During fermentation, gaps were maintained between the blocks. At the end of fermentation, white mycelium should be present without black spores.

[0062] (4) Preparation: The fermented Chinese medicine prepared in step (3) is extracted three times with 5 times its weight of water, each time at 80°C for 30 minutes. After cooling and filtration, the three filtrates are combined and collected for later use to obtain a fermented Chinese medicine preparation for the treatment of hyperlipidemia.

[0063] Example 4

[0064] Same as Example 2, except that the raw materials are: 20g of patchouli, 5g of magnolia bark, 10g of poria cocos, 1g of tangerine peel, 10g of alisma plantago-aquatica, 1g of atractylodes macrocephala, 9g of atractylodes lancea, 4g of hawthorn, 8g of lotus leaf, 5g of coix seed, 1g of licorice, 10g of wheat bran, and 15g of flour.

[0065] (1) Pulverization: Weigh the above raw materials separately, and pulverize the patchouli, magnolia bark, poria cocos, tangerine peel, alisma plantago-aquatica, atractylodes macrocephala, atractylodes lancea, hawthorn, lotus leaf, coix seed and licorice into fine powder of 120 mesh. Pass the wheat bran through a 30 mesh sieve, and then mix them evenly to make medicinal powder.

[0066] (2) Preparation of soft material: Add flour to 84g of water to make a thin paste, boil it, and while it is hot, knead it evenly with the medicinal powder obtained in step (1), knead it into a ball by hand, and it will fall apart when thrown to make soft material.

[0067] (3) Inoculation of strains and fermentation: Add 0.8 mL of the bacterial suspension with a concentration of 10 10 CFU / mL prepared in Example 1 to the soft material obtained in step (2), mix evenly, press into blocks, shape into brick-like blocks, place in a constant temperature and humidity incubator at 40°C and 90% relative humidity, ferment for 72 h and then take out, dry in an oven at 70°C to obtain fermented traditional Chinese medicine. During the fermentation process, keep gaps between the blocks. At the end of fermentation, white hyphae should be distributed and there should be no black spores.

[0068] (4) Preparation of preparation: Extract the fermented traditional Chinese medicine prepared in step (3) three times with 5 times its weight of water at 60°C for 30 min each time, cool and filter, combine and collect the filtrates of the three extractions, store for later use to obtain a fermented traditional Chinese medicine preparation for treating hyperlipidemia.

[0069] Comparative Example 1

[0070] Same as Example 2, the only difference is that the bacterial suspension prepared in Example 1 is not added.

[0071] Effect verification

[0072] The therapeutic effect of the present invention is demonstrated by the following specific pharmacodynamic experiments of Example 1.

[0073] 1. Animals

[0074] Species: SPF-grade SD rats

[0075] Source: Purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., animal production license: SCXK(Shandong)20180003

[0076] Gender: Male

[0077] Body weight: 180 - 220 g

[0078] Feeding conditions: During the experiment, the environmental temperature of the animal breeding room is 20 - 25°C, the relative humidity is 55 - 60%, with normal lighting. The rats drink water and eat freely, and the drinking water for the rats is all ultrapure water for laboratory use.

[0079] Approved by the Experimental Animal Ethics Committee of Henan University of Traditional Chinese Medicine, approval number: DWLLGZR202202211

[0080] 2. Experimental method

[0081] Seventy SD rats were divided into seven groups: a normal control group, a model group, a positive control group, a naturally fermented low-to-high dose group, and a bacterial fermented low-to-high dose group. The normal control group was given a standard maintenance diet, while the other groups were given a high-fat diet for 6 weeks to establish a hyperlipidemia (HLP) model. After successful model establishment, the drugs were administered to each group according to Table 1, once daily for 6 weeks.

[0082] Table 1

[0083] Group Dosage normal group 10 mL / kg of distilled water Model group 10 mL / kg of distilled water Positive drug group Simvastatin tablets are administered at a dose of 20 mg / kg. Natural fermentation low-dose group <![CDATA[The fermented traditional Chinese medicine preparation obtained in Comparative Example 1 was administered at a dose of 1600 mg·kg -1 dose]]> High-dose group of natural fermentation <![CDATA[The fermented traditional Chinese medicine preparation obtained in Comparative Example 1 was administered at a dose of 8000 mg·kg -1 > Low-dose fermentation group of strains <![CDATA[The fermented traditional Chinese medicine preparation obtained in Example 2 was administered at a dose of 1600 mg·kg -1 > High-dose fermentation group of strains <![CDATA[The fermented traditional Chinese medicine preparation obtained in Example 2 was administered at a dose of 8000 mg·kg -1 >

[0084] 3. Pharmacological studies

[0085] ① Rat body weight gain

[0086] During the modeling period, the body weight of rats was measured every 7 days. Rats with excessively low body weight were housed separately to reduce individual differences caused by uneven intake of high-fat diets. During the drug administration period, the body weight of rats was measured every 7 days, the gavage dosage was adjusted, and the effects of different drugs on changes in rat body weight were observed. Intergroup statistical analysis was performed. During the drug administration period, the body weight of the model group was significantly higher than that of the normal group (P < 0.01). At week 6 of drug administration, the body weight of all drug administration groups was significantly lower than that of the model group (P < 0.01). Compared with the normal group, the body weight of rats in the positive control group was lower (P < 0.05), and the body weight of rats in the high-dose fermented product group was significantly lower (P < 0.01).

[0087] ② Blood lipid levels and liver and kidney function levels in rats

[0088] Compared with the control group, the serum HDL-C level of rats in the model group was significantly decreased (P<0.01), while the levels of other indicators were significantly increased (P<0.01). TC detection results showed that, compared with the model group, the TC levels in the positive drug group, the naturally fermented group, and the high-dose fermented group were significantly lower (P < 0.01 or P < 0.05); TG, LDL-C, and AST detection results showed that, compared with the model group, the TG, LDL-C, and AST levels in each treatment group were significantly lower (P < 0.01); HDL-C results showed that, compared with the model group, the HDL-C levels in the positive drug group and the high-dose fermented group were significantly higher (P < 0.01 or P < 0.05); ALT results showed that, compared with the model group, the ALT levels in the positive drug group, the naturally fermented group, and the high-dose fermented group were significantly lower (P < 0.01); BUN and Cr results showed that the positive drug group, the high-dose naturally fermented traditional Chinese medicine group, and the low- and high-dose fermented traditional Chinese medicine groups significantly improved renal index levels (P < 0.01 or P < 0.05).

[0089] ③ Pathological changes in rat liver tissue

[0090] In the normal group, the intercellular spaces of hepatocytes were clear, with only a very small number of orange-red lipid droplets observed. In the model group, orange-red areas were visible throughout the hepatocytes, with diffuse lipid droplets, damaged cell nuclei, and significant lipid deposition in the liver. Compared with the model group, the positive control group and the low and high dose groups of the fermented traditional Chinese medicine strain showed a significant reduction in orange-red lipid droplets and lipid deposition. The high dose group of the fermented traditional Chinese medicine strain showed an IOD (integrated optical density) value close to that of the normal group. Figure 2 .

[0091] ④ Expression of AMPK, p-AMPK, LKB1 and HMGCR proteins in rats

[0092] Compared with the normal group, the IOD values ​​of AMPK, p-AMPK, and LKB1 proteins in the liver tissue of rats in the model group were significantly decreased (P < 0.01), while the IOD value of HMGCR protein was significantly increased (P < 0.01). Compared with the model group, the IOD values ​​of AMPK, p-AMPK, and LKB1 proteins in the liver tissue of rats in all treatment groups were increased, with the positive control group and the high-dose group of fermented traditional Chinese medicine being significantly increased (P < 0.01). Compared with the model group, the IOD value of HMGCR protein in the liver tissue of rats in all treatment groups was significantly decreased (P < 0.01 or P < 0.05). The immunohistochemical results of AMPK, p-AMPK, LKB1, and HMGCR proteins in the liver tissue of rats in each group are shown below. Figure 3 The IOD value measurement results are shown in [link to relevant documentation]. Figure 4 .

[0093] Since the serum levels, liver pathological sections, and immunohistochemical results all showed that the high-dose group had a stronger regulatory effect on hyperlipidemic rats than the low-dose group, and considering that the regulatory effect of each low-dose group may not have been prominent due to the short intervention time, this gut microbiota study did not focus on the naturally fermented low-dose group or the strain-fermented low-dose group. The following gut microbiota sequencing results are only for the fecal microbiota of rats in the naturally fermented high-dose group and the strain-fermented high-dose group.

[0094] ⑤ Analysis of Beta diversity in rat gut microbiota

[0095] Beta diversity refers to the species differences between different environmental communities. Principal component analysis (PCA) can extract the two coordinate axes that best reflect the differences between samples, thus representing the differences in multidimensional data on a two-dimensional coordinate graph. For example... Figure 5 As shown, the sum of PCA1 and PCA2 on the two axes is 63.26%, indicating that the analysis is relatively reliable. There are significant differences between the normal group and the model group. The bacterial flora of each drug-treated group showed a regression compared with the model group. In particular, the bacterial flora of the high-dose fermentation group of the strain had a very high similarity with the normal group.

[0096] ⑥ Analysis of differences in gut microbiota at the genus level in rats

[0097] At the genus level, the composition and distribution of the gut microbiota in each group of rats are as follows: Figure 6 and Figure 7 As shown, the most abundant bacterial genera in the gut microbiota of normal rats included *Ligilactobacillus* (15.55%), *Firmicutes_unclassified* (8.22%), and *Lachnospiraceae_NK4A136_group* (7.64%). In the model group, the dominant bacterial genera included *Akkermansia* (17.61%), *Ligilactobacillus* (11.71%), and *Lactobacillus* (7.94%). Compared to the normal group, the levels of beneficial bacteria such as *Ligilactobacillus*, *Firmicutes_unclassified*, *Lachnospiraceae_NK4A136_group*, and *Clostridia_UCG-014_unclassified* in the feces of model rats decreased, while these levels rebounded somewhat in the naturally fermented high-dose group and the bacterial strain fermented high-dose group. The grouped bar chart and heatmap showed that naturally fermented Chinese medicine and bacterial fermented Chinese medicine clustered together with the normal group, indicating that bacterial fermented Chinese medicine has an ameliorative effect on intestinal flora disorder in hyperlipidemic rats, and the proportion of bacterial flora in rats with bacterial fermented Chinese medicine is most similar to that in the normal group.

[0098] In summary, the fermented traditional Chinese medicine (TCM) produced by this strain has a good therapeutic effect on hyperlipidemic rats, can restore the body weight of hyperlipidemic rats, can significantly reduce the levels of four lipid markers in the serum of hyperlipidemic rats, can significantly improve lipid deposition in liver tissue, reduce the degree of hepatocyte steatosis, and improve liver and kidney function. Immunohistochemical results showed that, compared with the model group, the high-dose group of the fermented TCM produced by this strain had a large amount of AMPK, p-AMPK, and LKB1 protein deposition in the liver tissue, and a small amount of HMGCR protein deposition, indicating that the fermented TCM produced by this strain can enhance the transduction of the LKB1 / AMPK signaling pathway. Gut microbiota sequencing results showed that the fermented TCM produced by this strain can regulate the gut microbiota structure of hyperlipidemic rats, increase the relative abundance of Firmicutes, Ligilactobacillus, and Lachnospiraceae_NK4A136_group, and restore the normal microbiota structure.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fermented traditional Chinese medicine for treating hyperlipidemia of dampness blocking middle energizer type, characterized in that, The fermented traditional Chinese medicine is obtained by inoculating Bacillus sp. into the following traditional Chinese medicine raw materials and then fermenting them; The traditional Chinese medicine raw materials are as follows: Pogostemonis Herba 5-20 parts, Magnoliae Officinalis Cortex 5-20 parts, Poria 1-10 parts, Citri Reticulatae Pericarpium 1-10 parts, Alismatis Rhizoma 1-10 parts, Atractylodis Macrocephalae Rhizoma 1-10 parts, Atractylodis Rhizoma 2-9 parts, Crataegi Fructus 4-18 parts, Nelumbinis Folium 1-8 parts, Coicis Semen 5-20 parts, and Glycyrrhizae Radix et Rhizoma 1-5 parts; The preservation number of the Bacillus sp. is CCTCC NO: M2021761; The preparation method of the fermented traditional Chinese medicine specifically comprises the following steps: (1) After the traditional Chinese medicine raw materials are crushed, wheat bran is added and mixed, and then the mixture is mixed with flour paste to form soft materials; (2) The soft materials are inoculated with the Bacillus sp. and mixed uniformly, and then the mixture is pressed into a block, and then fermented, dried, and the fermented traditional Chinese medicine is obtained; In step (1), the flour paste is prepared by mixing flour and water at a mass ratio of 1: (10-20); In step (2), the humidity of the fermentation culture is 60-90%, the temperature is 25-40℃, and the fermentation time is 24-72h.

2. A fermented traditional Chinese medicine preparation for treating hyperlipidemia of dampness blocking middle energizer type, characterized in that, The effective component of the fermented traditional Chinese medicine preparation is obtained by extracting the fermented traditional Chinese medicine of claim 1 by using a solvent; The solvent is water, and the extraction temperature is 60-100℃.

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