Application of traditional Chinese medicine formula SFMF combined with lactobacillus reuteri in preparation of medicine for treating fatty liver disease

Abstract

The invention provides an application of a traditional Chinese medicine formula SFMF formula and lactobacillus reuteri in preparing a medicine for treating fatty liver diseases. The traditional Chinese medicine formula SFMF formula is a composition formed by semen ziziphi spinosae, poria with hostwood, radix ophiopogonis and light wheat. By adopting the technical scheme of the invention, the liver index and groin fat weight of LR08 and SFMF prescriptions are obviously reduced compared with those of an M group (Plt; 0.05), which prompts that the medicine has a certain compensatory effect on the damage of the liver metabolism function of the mouse. The LR08 and the SFMF can effectively reduce liver lipid accumulation of NASH mice and improve liver fatty degeneration of NAFLD mice, and the LR08 and the SFMF are combined to have an obvious treatment effect on hepatic tissue pathological damage of a mouse NASH model.

Description

Technical Field

[0001] The invention relates to application of a traditional Chinese medicine formula and Lactobacillus reuteri in pharmaceutical manufacturing, and belongs to the technical field of biopharmaceuticals. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease closely related to hypertension, hyperlipidemia and diabetes. In recent years, the incidence of NAFLD has been on the rise, and its global prevalence has risen to 29.38% by 2021. However, the main treatment for NAFLD is to change the patient's lifestyle, intervene in diet and exercise, and even perform bariatric surgery to ultimately achieve the goal of weight loss. Western medicine still lacks ideal and targeted specific drugs.

[0003] At present, more and more studies have shown that the occurrence of NAFLD is closely related to the ecological imbalance of intestinal microbiota. Probiotics are a general term for active beneficial microorganisms that colonize in the human intestine and reproductive system and can produce definite health effects, thereby improving the balance of the host microecology and playing a beneficial role. Among them, Lactobacillus reuteri ( Lactobacillus reuteri , L. reuteri) is a common probiotic that often colonizes the human gastrointestinal tract. It has anti-inflammatory, antibacterial, immunity-enhancing, and intestinal barrier-enhancing effects, and it shows good application potential in the treatment of some diseases such as obesity, intestinal infection, and systemic lupus erythematosus.

[0004] Based on this, the present invention provides a prescription of four Chinese medicinal herbs with a specific ratio, namely, Semen Ziziphi Spinosae (S), Poria (F), Ophiopogon japonicus (M), and Triticum chuanxiong (F) (referred to as "SFMF prescription") combined with Lactobacillus reuteri (LR08) on NAFLD and its related mechanism of action, providing a new idea for the clinical prevention and treatment of metabolic diseases including NAFLD. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a use of a Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of a drug for treating fatty liver disease. The Chinese medicine formula SFMF is a composition formed by spiny jujube seeds, poria, ophiopogon japonicus, and floating wheat.

[0006] The mass ratio of Chinese jujube seed, poria, ophiopogon and floating wheat is 1-4:1-4:1-4:1-4.

[0007] The mass ratio of Chinese jujube seed, poria, ophiopogon japonicus and floating wheat is 4:2:1:4.

[0008] The preparation method of the Chinese medicine formula SFMF combined with Lactobacillus reuteri includes the following steps: (1) Mix the Chinese date seeds, poria, ophiopogon japonicus and wheat and dissolve them in PBS to make a 0.5-5 g / ml solution for later use; (2) After activating Lactobacillus reuteri, adjust the concentration to a bacterial solution with OD600nm = 0.05-0.5 using 2X sugar-free MRS broth; (3) The Chinese medicine solution of step (1) and the bacterial solution of step (2) are mixed in a volume ratio of 1:3-10, and cultured at 30-40° C. for 12-24 hours under anaerobic conditions to obtain a preparation of the Chinese medicine formula SFMF combined with Lactobacillus reuteri.

[0009] Application of the Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of drugs providing cholesterol scavenging ability.

[0010] Application of the traditional Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of drugs for reducing ALT and CHO levels.

[0011] Improvement of the preparation of the traditional Chinese medicine formula SFMF combined with Lactobacillus reuteri Lipid accumulation in the liver Application in medicine.

[0012] It can be seen from the technical solution of the present invention that LR08 and SFMF square The liver index and inguinal fat weight of the NASH group were significantly lower than those of the M group (P<0.05), indicating that the drug has a certain compensatory effect on the damage to the liver metabolic function of mice. LR08 and SFMF can effectively reduce the liver lipid accumulation of NASH mice and improve the liver fatty degeneration of NAFLD mice. The combination of the two has a significant therapeutic effect on the liver tissue pathological damage of the NASH model in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The growth curve and standard curve of LR08. Note: A: growth curve of LR08 B: standard curve of LR08.

[0014] Note: A: Growth curve of LR08 B: Standard curve of LR08.

[0015] Figure 2 Orthogonal experiments were performed to screen the prebiotic and antioxidant activities of TCM formulas.

[0016] Note: A: prebiotic activity B: DPPH free radical scavenging rate.

[0017] Figure 3 Orthogonal experiment was used to screen the lipid-lowering activity of TCM formula.

[0018] Note: A: cholesterol-lowering activity; B: BSH enzyme activity detection (1) MRS medium (2) BSH medium; C: BSH relative activity.

[0019] Figure 4 The average daily food intake, body weight, inguinal fat and liver index of mice.

[0020] Note: A: average daily food intake of mice; B: body weight of mice; C: liver index of mice; D: inguinal fat weight of mice; N: normal group; M: model group; LR: LR08; SFMF: Chinese medicine formula group; Combine: SFMF formula + LR08 synbiotic group.

[0021] Figure 5 Represents the ALT and CHO content in mice.

[0022] Note: A: ALT content in mouse serum; B: CHO content in mouse serum; N: normal group; M: model group; LR: LR08; SFMF: Chinese medicine formula group; Combine: SFMF formula + LR08 synbiotic group.

[0023] Figure 6 HE staining and Oil Red O staining of mouse liver.

[0024] Note: A: HE staining of mouse liver; B: Oil red O staining of mouse liver; N: normal group; M: model group; LR: LR08; SFMF: Chinese medicine formula group; Combine: SFMF formula + LR08 synbiotic group. DETAILED DESCRIPTION

[0025] Example 1 Experimental Materials and Methods Total cholesterol (TC / TCH) assay kit (single reagent GPO-PAP method) (A111-1-1), alanine aminotransferase (ALT / GPT) test kit (Reitman method) (C009-2-1), and Oil Red O staining solution (D027-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0026] MRS broth, MRS agar, beef powder, (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), cholesterol, L-cysteine, sodium acetate, diammonium citrate (Shanghai McLean Biochemical Technology Co., Ltd.), trypsin, yeast powder (UK Oxoid Company), Tween-80 (Nanjing Chemical Reagent Co., Ltd.), (Shanghai McLean Biochemical Technology Co., Ltd.), sodium chloride, magnesium sulfate, manganese sulfate (Sinopharm Chemical Reagent Co., Ltd.), ox bile salt (Beijing Solebold Technology Co., Ltd.), sodium taurodeoxycholate, sodium thioglycolate (Shanghai Yuanye Biotechnology Co., Ltd.), dipotassium hydrogen phosphate (Tianjin Tianli Chemical Reagent Co., Ltd.), calcium chloride (Guangdong Xilong Chemical Co., Ltd.), DSS (Yishen Biotechnology (Shanghai) Co., Ltd.).

[0027] The instruments and equipment used include: dry constant temperature box, electric constant temperature incubator, dry heat disinfection box: Shanghai Fuma Experimental Equipment Co., Ltd., China; full-wavelength microplate reader (Thermo Electron), optical microscope (Olympus BX53 biological microscope), vortex oscillator mixer (Vortex-2 Genie), ultrapure water instrument (Cascade), analytical balance (AL-104), clean bench (VS-1300L-U), magnetic stirrer (79-3 type, Shanghai Luxi Analytical Instrument Factory), vacuum freeze dryer (Shanghai Yetuo Technology Co., Ltd.), tissue dehydrator, ultrathin slicer, slice spreader, slice baking machine (German Leica Company), HE stainer (Shenzhen Dakowei Medical Technology Co., Ltd.), frozen slicer (Shenyang Longshou Electronic Instrument Co., Ltd.) 1.1 Gram staining The probiotic strain Lactobacillus plantarum LR08 (deposit number CGMCC No. 1.12733) required for the experiment was provided by the Institute of Infection and Inflammatory Injury of Three Gorges University.

[0028] Lactobacillus reuteri is the Lactobacillus reuteri LR08 strain, the preservation unit is the General Microbiology Center of China Culture Collection Administration, the preservation number is CGMCC No.1.12733, the preservation date is July 20, 2020, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The probiotic strains preserved in glycerol were taken out from the -80℃ refrigerator, transferred to MRS liquid culture medium at 1% (V / V), cultured anaerobically at 37℃ for 12h for initial activation, and then separated by streaking on MRS agar medium. Single clones were picked for preliminary identification such as Gram staining before use.

[0029] Use a pipette to add 20μL of LR08 bacterial solution to a clean glass slide, use a sterile inoculation loop to spread the bacterial solution evenly, and dry it with fire to fix it; primary staining: add an appropriate amount of oxalic acid to the fixed bacterial surface with crystal violet staining solution, dye it for 10s, and then gently rinse it with running water until the outflowing liquid is colorless; mordanting: add iodine solution, mordanting for about 10s, and then wash it with water; decolorization: tilt the glass slide slightly, add 95% alcohol to the bacterial surface for decolorization for about 25 seconds, and then gently wash it with water until the outflowing liquid is colorless. Restaining: After the glass slide is dry, add safranin solution to the smear for restaining for 10s, wash it with water, dry it with absorbent paper, and dry it in the air. Microscopic examination: observe with an oil microscope after drying. The bacteria that are stained red are Gram-negative bacteria (G-), and those that are stained blue-purple are Gram-positive bacteria (G+).

[0030] 1.2 Probiotics and growth curve and standard curve detection Growth curve and standard curve: The activated LR08 strain was inoculated into MRS medium at 1% (V / V) and cultured anaerobically at 37°C. One tube was taken out at 3, 6, 9, 12, and 24 hours, 300ul of bacterial solution was centrifuged and the precipitate was washed twice, and resuspended with an equal volume of PBS. 100ul of bacterial solution was taken to measure the OD value of the bacterial solution at a wavelength of 600nm. The experiment was repeated three times in parallel, and the growth curve was drawn with the time point as the horizontal axis and OD600 as the vertical axis. According to the growth curve results, the standard bacterial solution in the logarithmic growth phase was diluted in different proportions to make its OD600 between 0.2 and 0.9. Six appropriate dilution plates were selected for colony counting (the total number of colonies was between 30 and 200). The experiment was repeated three times in parallel and the absorbance OD was used as the final value. 600 A standard curve of the correlation between the number of LR08 bacteria and absorbance was established with the number of viable bacteria (cfu) counted on the plate as the abscissa and the number of viable bacteria (cfu) counted on the plate as the ordinate.

[0031] 1.3 Orthogonal experimental design of Chinese medicine compound ratio The formula granules of Suanzaoren, Poria, Ophiopogon and Fumai (Beijing Kangrentang Pharmaceutical Co., Ltd.) were purchased from the Pharmacy Department of Renhe Hospital Affiliated to Three Gorges University. According to the content of Suanzaoren, Poria, Ophiopogon and Fumai Chinese medicine granules, the corresponding granules were weighed and dissolved in PBS solution to make a concentration of 1g / ml (raw drug), sterilized at 121℃, and packaged on a sterile operating table, and stored at -20℃ for later use.

[0032] Four experimental factors, namely, spina jujuba seed (A), poria (B), ophiopogon (C), and floating wheat (D), were selected, and an orthogonal experimental design was carried out with three levels (concentrations) of each factor (see Tables 1 and 2 for details). The experiment was repeated three times in parallel, and the optimized formula was obtained by the comprehensive score of prebiotic effect, antioxidant activity, and lipid-lowering ability.

[0033] Take 9 sterile EP tubes, dilute the Chinese medicine stock solution according to Table 1 and Table 2, and then prepare it orthogonally for immediate use.

[0034] Table 1 Influencing factors of orthogonal experiment

[0035] Table 2 Orthogonal test number

[0036] Prebiotic activity detection: The second generation of activated LR08 bacteria was centrifuged (3673g*5min), washed once with sterile PBS, and the concentration was adjusted to OD600nm=0.1 with 2X sugar-free MRS broth. The orthogonal Chinese medicine was mixed with LR08 bacterial solution at a ratio of 1:4 (v / v), and anaerobic culture was performed at 37℃ for 24 hours to measure OD 600 , calculate the prebiotic activity, where the experimental group ( ), control group ( ) (with an equal volume of PBS instead of drugs), and set up corresponding blank groups ( and ) (Replace LR08 bacterial solution with an equal volume of 2X sugar-free MRS).

[0037] Calculate the prebiotic activity (%) using the formula =

[0038] Antioxidant activity detection: 1,1-diphenyl-2-picrylhydrazyl (DPPH) can be used to quantitatively determine the antioxidant capacity. The same orthogonal test and bacterial culture as the above prebiotic activity detection were performed. The fermentation broth was centrifuged (3673 g / 5 min) and the supernatant was taken and mixed with an equal volume of 0.1 mg / mL DPPH solution. The mixture was incubated at room temperature in the dark for 30 min and the assay was performed. (A) The blank group was tested by replacing DPPH solution with an equal volume of anhydrous ethanol. ( ), the control group used an equal volume of distilled water instead of sample supernatant ( , and the DPPH free radical scavenging rate was calculated.

[0039] Calculate the DPPH free radical scavenging rate (%) using the formula =

[0040] Cholesterol content determination: MRS high cholesterol medium (MRS-CHO) was prepared according to the literature: ox bile salt (final mass concentration was 3 mg / mL) and cholesterol (final mass concentration was 0.1 mg / mL) were added to the MRS liquid medium. After the concentration of LR08 bacteria was adjusted to OD600nm=0.1 with MRS-CHO medium, the orthogonal Chinese medicine was mixed with the LR08 bacterial solution at a ratio of 1:4 ( ), the control group received an equal volume of PBS instead of drugs ( ), 2 blank groups ( and ) is to replace the LR08 bacterial solution with an equal volume of MRS-CHO medium, and culture at 37℃ for 24 hours. The fermentation liquid is centrifuged (3673g / min for 10min) and the supernatant is taken. The COD-PAP method is used to measure the absorbance of the supernatant at 505nm and the cholesterol clearance rate of the probiotics is calculated according to the formula.

[0041] The cholesterol clearance rate of probiotics is calculated by the formula (%) =

[0042] =

[0043] Qualitative detection of bile salt hydrolase activity Add 3 g / L sodium taurocholate, 2 g / L sodium thioglycolate, and 0.37 g / L CaCl2 to MRS agar medium to prepare BSH medium, sterilize at 121℃ for 15 min, pour into a sterile plate, and evenly place 6 mm sterile filter paper on the plate after solidification. Inoculate 10 μL of the fermentation liquid after orthogonal experimental design on the sterile filter paper, and culture anaerobically at 37℃ for 72 h. Observe the characteristics of opaque halo or opaque granular white colonies around the filter paper to indicate bile salt hydrolase activity. Pure MRS medium was used as a negative control.

[0044] Experimental animals and treatment 50 SPF Kunming mice, weighing (20±2) g, half male and half female, were provided by the Experimental Animal Center of Three Gorges University. They were allowed to eat and drink freely, and the ambient temperature was controlled at 20-25℃, with natural light. They were adaptively raised for three days before the experiment.

[0045] The mice were randomly divided into five groups, with 10 mice in each group: normal control group (Normal, N), high fat feed (HFD) model group (Model, M), Lactobacillus reuteri group (LR08, LR), Ziziphus jujuba, Poria, Ophiopogon japonicus, and No. 5 floating wheat compound prescription group (SFMF), and probiotics + No. 5 compound prescription combined administration group (Combine), a total of five groups.

[0046] During the experiment, the N group was fed with ordinary feed, and the other groups were fed with HFD (basic feed: lard: fructose: sodium cholate: cholesterol: salt: H2O = 0.43: 0.2: 0.2: 0.02: 0.04: 0.01: 0.1, mixed and fixed in proportion, irradiated and sterilized for use), and fed with 200μL 3% DSS gavage to establish a NAFLD progression model.

[0047] After 10 days of feeding, different groups were given 200 μL of the corresponding solution by gavage at 12:00 noon every day, and mice in groups N and M were given an equal volume of normal saline; at 18:00 in the afternoon, group N was given 200 μL of normal saline, and the remaining groups were given an equal volume of 3% DSS by gavage. The feeding intervention lasted for 28 days. During the entire experiment, the general condition and weight changes of the mice were continuously monitored, and the weight changes and daily average diet and water intake of each mouse were recorded. The SFMF dosage for mice was calculated based on the body surface area ratio of a 60 kg body weight human (adult: mouse = 1:9.1). The daily dosage of SFMF for adults is 12 g / day. Based on the conversion and orthogonal experimental results, the daily dosage of SFMF for mice was 36.4 mg (13.2 mg of Ziziphus jujuba seeds + 6.6 mg of Poria cocos + 3.3 mg of Ophiopogon japonicus + 13.2 mg of Triticum tiliaceum) / 20 g of mouse weight.

[0048] After culturing SFMF 5# and LR08 bacterial solution at a ratio of 1:4 for 24 h, the supernatant was taken and freeze-concentrated in a freeze dryer for 30 h to the desired concentration. The LR group was freeze-concentrated in the same way, and the TCM group was adjusted to the desired concentration with PBS after high pressure. The prepared drugs were packaged in a sterile clean bench and stored in a -20℃ refrigerator for later use.

[0049] Serological index detection The mice were fasted but not watered for 12 h before the end of the experiment. The mice were weighed and then killed by cervical dislocation. Blood was collected from the eyeballs of mice in each group and centrifuged at low temperature (517 g / 15 min) to absorb the upper serum and detect the serum ALT and CHO levels.

[0050] Liver and spleen index and inguinal fat measurement The mouse liver, spleen, epididymal fat and other visceral tissues were completely removed, cleaned with physiological saline, weighed separately, and the liver index was calculated according to the following formula. Take an appropriate amount of liver tissue, fix a portion in 4% paraformaldehyde, and wait for later paraffin sectioning; a portion of fresh tissue was wrapped in tin foil and stored in a -80℃ ultra-low temperature refrigerator for frozen sectioning, then stained with Oil Red O and observed under a light microscope; the remaining portion was frozen in a 1.5 mL EP tube at -80℃ for subsequent analysis of liver biochemical indicators.

[0051] Liver index (%) is calculated by the formula = *100; Spleen index (%) = *100.

[0052] Liver histopathological analysis Weigh them separately, and calculate the liver index according to the following formula. Take an appropriate amount of liver tissue, fix a part of it in 4% paraformaldehyde, and perform gradient dehydration, paraffin embedding, sectioning and hematoxylin-eosin (HE) staining according to routine procedures after 24 hours, and perform liver tissue pathological analysis under light microscopy; a part of fresh tissue is frozen sectioned, then stained with Oil Red O and observed under light microscopy; the rest is frozen at -80℃ for analysis of liver biochemical indicators.

[0053] Statistical analysis The experimental data were analyzed using GraphPad 8.0, and the data were expressed as x±s. The data between groups were analyzed by one-way analysis of variance, and P < 0.05 was considered statistically significant.

[0054] Experimental results and analysis Growth curve and standard curve of probiotics With time as the horizontal axis, OD600 As the vertical axis, the growth curve of LR08 can be obtained. According to the different growth rates of bacteria, the growth curve is divided into four stages: slow phase, logarithmic phase, stable phase and decay phase. In the late logarithmic growth phase, the bacteria are most active and have the largest number of live bacteria (reference). Figure 1 A shows that LR08 enters the logarithmic phase after 3 hours and begins to multiply rapidly, which lasts until 9 hours. At 9-12 hours, OD600 The growth rate gradually leveled off and reached a maximum value. Therefore, 12 h was determined as the optimal harvest time for LR08 in this experiment. Figure 1B shows the OD600 The linear relationship between CFU and OD600 By counting the plate, the linear relationship is: y = 28.984x - 3.5618, = 0.9992.

[0055] SFMF compound prebiotic activity results The results of the in vitro prebiotic test showed that different drug concentration ratios can be used as prebiotics to promote the growth of LR08 bacteria. Figure 2 A It can be seen that the effect of promoting the growth of LR08 bacteria in the orthogonal combination is 5>6>1>9>2>8>7>3>4, that is, formula No. 5 (Spiny Ziziphus jujuba seed: Poria: Ophiopogon japonicus: Floating wheat = 4:2:1:4) has the strongest prebiotic activity.

[0056] Effect of SFMF compound on the antioxidant activity of LR08 The DPPH free radical scavenging ability experiment is usually used to evaluate the antioxidant capacity of probiotics. The results show that the antioxidant capacity of LR08 bacteria affected by different drug concentration ratios is 2>1>9>5>3>7>8>4>6, that is, the antioxidant activity of formula No. 2 (sour jujube seeds: poria: ophiopogon: floating wheat = 2:1:1:1) is the highest ( Figure 2 B).

[0057] Based on the above experiments, especially the results of prebiotic activity, four combinations of SFMF prescriptions with strong antioxidant and prebiotic activities, namely, combinations 1, 5, 6, and 9, were selected for subsequent in vitro lipid-lowering experiments.

[0058] Lipid-lowering experiment The results of cholesterol content determination showed that the four selected Chinese medicine prescriptions all had certain lipid-lowering activity and could improve the cholesterol clearance ability of LR08, and the effects were 5>1>6>9 ( Figure 3 A).

[0059] Bile salt hydrolase can hydrolyze the bound bile salts in the culture medium to form free bile salts. Under slightly acidic conditions, free bile salts are more easily The presence of BSH in the strain makes it more tolerant to bile salts, which also helps to lower the host's blood cholesterol level. Figure 3 .B Opaque granular white colonies were observed around LR08, indicating that it had BSH enzyme activity, which was consistent with previous studies; and the orthogonal fermentation broths 1, 5, 6, and 9 could improve the BSH enzyme activity of LR08 to a certain extent.

[0060] Based on the above in vitro experimental results, the Chinese medicine formula No. 5 was selected, i.e., Ziziphus jujuba seed: Poria: Ophiopogon japonicus: Triticum tiliaceus = 4:2:1:4, for subsequent in vivo animal experiments.

[0061] Observation of general signs and visceral indexes of mice During the experiment, the general condition of the mice was observed. The mice fed a high-fat diet gradually developed greasy hair, weight gain, increased food intake, and decreased activity, while the hair of the mice in the drug intervention group was smoother and more shiny than that in the M group. Figure 4 As shown, the food intake of mice in each group was different. Compared with the M group, the SFMF group and the Combine group improved the increased food intake of mice to varying degrees, and the difference was statistically significant.

[0062] The weight of mice was continuously monitored during the experiment, and the results showed that the weight of mice in group M increased significantly, and the difference was statistically significant compared with group N ( P <0.01). The body weight of mice in the experimental group was lower than that in the M group, and the difference in the Combine group was significant ( P <0.05) Figure 4 .B).

[0063] The significance of liver index and inguinal fat (literature). Analysis of liver index and inguinal fat showed that the liver index and inguinal fat weight of group M were significantly higher than those of group N (P<0.05), and the difference was statistically significant. The liver index and inguinal fat weight of the Combine group were significantly lower than those of the M group (P<0.05), indicating that the drug has a certain compensatory effect on the damage to the liver metabolic function of mice. ( Figure 4 B, C, D).

[0064] Mouse liver serum biochemical indexes ALT and CHO are the main markers of liver damage and lipid metabolism disorder in NALFD, respectively. Compared with the N group, the ALT and CHO levels in the M group were significantly increased (P<0.01), and the difference was statistically significant. Compared with the M group, the ALT and CHO levels in the experimental group decreased, among which the CHO level in the SFMF group was significantly lower than that in the M group (P<0.05); the ALT and CHO levels in the Combine group were significantly lower than those in the M group (P<0.01). Figure 3 ).

[0065] Observation of liver specimens by HE staining and Oil Red O staining HE staining of liver pathological sections of mice in each group showed that the liver lobules of mice in group N were clear and the hepatocyte morphology was intact. The liver tissue of mice in group M showed obvious fatty degeneration and the liver cells were The cells are of different sizes, the hepatic cords are disordered, and the hepatic lobule structure disappears. Accompanied by scattered inflammatory cell infiltration and ballooning degeneration. Experimental group mice The above pathological changes were significantly alleviated. The number of fat vacuoles and inflammatory cell infiltration in hepatocytes were significant Reduced, the contours of the liver cords and liver lobules are restored ( Figure 6 A).

[0066] observe Figure 6 B shows that compared with group N, Oil red O staining area of ​​liver in model group mice Densely distributed fat of varying sizes droplets and lipid droplet flake fusion, This indicates that there is obvious lipid accumulation in the liver. The oil red O staining area of ​​the liver of mice in each treatment group is reduced. The formula can effectively reduce liver lipid accumulation in NASH mice and improve liver lipid in NAFLD mice. The combination of the two has a significant therapeutic effect on the pathological damage of liver tissue in the NASH mouse model.

Claims

1. Use of the Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of a drug for treating fatty liver disease, characterized in that: The traditional Chinese medicine formula SFMF is a composition formed by spiny jujube seeds, poria, ophiopogon japonicus and floating wheat.

2. The use according to claim 1, characterized in that The mass ratio of Chinese jujube seed, poria, ophiopogon and floating wheat is 1-4:1-4:1-4:1-4.

3. The use according to claim 2, characterized in that: The mass ratio of Chinese jujube seed, poria, ophiopogon japonicus and floating wheat is 4:2:1:

4.

4. The use according to claim 1, characterized in that The preparation method of the Chinese medicine formula SFMF combined with Lactobacillus reuteri includes the following steps: (1) Mix the Chinese date seeds, poria, ophiopogon japonicus and wheat and dissolve them in PBS to make a 0.5-5 g / ml solution for later use; (2) After activating Lactobacillus reuteri, adjust the concentration to a bacterial solution with OD600nm = 0.05-0.5 using 2X sugar-free MRS broth; (3) The Chinese medicine solution of step (1) and the bacterial solution of step (2) are mixed in a volume ratio of 1:3-10, and cultured at 30-40° C. for 12-24 hours under anaerobic conditions to obtain a preparation of the Chinese medicine formula SFMF combined with Lactobacillus reuteri.

5. The use according to claim 1, characterized in that: Application of the Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of drugs providing cholesterol scavenging ability.

6. The use according to claim 1, characterized in that: Application of the traditional Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of drugs for reducing ALT and CHO levels.

7. The use according to claim 1, characterized in that Application of the traditional Chinese medicine formula SFMF combined with Lactobacillus reuteri in the preparation of drugs for improving liver lipid accumulation.

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