Astragalus mongholicus fermentation liquor as well as preparation method and application thereof

Abstract

The invention provides astragalus mongholicus fermentation liquor and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine ferment.The preparation method of the astragalus mongholicus fermentation liquor comprises the steps that a culture medium containing astragalus mongholicus alcohol extract is inoculated with lactobacillus reuteri for fermentation and concentration, and the astragalus mongholicus fermentation liquor is obtained; wherein the main component of the radix astragali alcohol extract is flavonoids which do not contain polysaccharide; the content of formononetin in the astragalus mongholicus fermentation liquor is more than two times that of the astragalus mongholicus alcohol extract; the astragalus mongholicus fermentation liquor obtained by the method disclosed by the invention can be used for remarkably relieving the overall symptoms of ulcerative colitis model mice, including inhibiting weight loss, diarrhea and hemafecia symptoms, improving colonic mucosa inflammatory cell infiltration, goblet cell disappearance and crypt structure damage, reducing the content of colonic tissue inflammatory cytokines, and improving the ulcerative colitis model mice. And ulcerative colitis mouse crus gastrocnemius atrophy can be relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine fermentation, in particular to an astragalus fermentation liquid and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC), a chronic, recurrent inflammatory bowel disease, faces a dual dilemma in clinical treatment: limited efficacy and long-term safety risks. Furthermore, more than one-third of patients experience sarcopenia. Existing drugs, such as aminosalicylic acid preparations, glucocorticoids, and biologics, while widely used, commonly suffer from adverse reactions such as single patient targets, uneven response rates, high rates of relapse after discontinuation, and potential infections. Safer and more effective alternative therapies are particularly needed for patients with moderate to severe UC and associated sarcopenia. Traditional Chinese medicine (TCM) has a long history of treating UC. Its symptomatic treatment can improve colon inflammation at multiple targets and alleviate sarcopenia in a targeted manner. It has the advantages of significant efficacy and minimal side effects. Discovering therapeutic drugs for UC from TCM is an important approach to developing UC treatments.

[0003] The traditional Chinese medicine (TCM) Astragalus membranaceus has the effects of tonifying qi and raising yang, promoting diuresis and reducing swelling, promoting fluid production and nourishing blood, promoting circulation and relieving numbness, expelling toxins and pus, and promoting tissue regeneration. The persistent or recurrent diarrhea, bloody and mucous stools, accompanied by abdominal pain, tenesmus, and muscle atrophy associated with UC are associated with TCM syndromes such as spleen and stomach qi deficiency and qi stagnation and blood stasis, corresponding to the effects and indications of Astragalus membranaceus, suggesting its therapeutic potential for UC. The main components of Astragalus membranaceus include polysaccharides and small-molecule compounds, saponins and flavonoids. Astragalus polysaccharides comprise approximately 1%-5% and have been reported to regulate intestinal immunity. Small-molecule compounds, saponins and flavonoids, exhibit anti-inflammatory and intestinal mucosal barrier repair activities, but their content is extremely low: total saponin content is approximately 0.1%-0.5%, and total flavonoid content is approximately 0.5%-2%. Formononetin and its aglycone, formononetin, are the main flavonoid components. Formononetin has strong anti-inflammatory activity, but its content is only approximately 0.02%, resulting in an extremely low effective therapeutic concentration in colonic lesions after oral administration. Therefore, it is urgent to develop new technical means to improve the overall efficacy of Astragalus small molecule component groups in the treatment of UC and promote their clinical application.

[0004] Biofermentation technology offers a new approach to overcoming the aforementioned bottlenecks in the production of active ingredients in botanical medicines. The fermentation process can promote the metabolic transformation of active substances. However, there is currently no technology for the targeted fermentation of small-molecule components of Astragalus (especially formononetin) by microorganisms, and even less research on the enhanced efficacy of fermented small-molecule components of Astragalus in the treatment of UC. Summary of the Invention

[0005] The present invention constructs for the first time the technical chain of "alcohol extraction and enrichment - strain fermentation - efficacy enhancement" of Astragalus, and provides an Astragalus fermentation liquid obtained by fermentation of Lactobacillus reuteri with the efficacy of alleviating UC, as well as its preparation method and application.

[0006] To achieve the above-mentioned purposes of the application, in a first aspect, the application provides a preparation method of a radix astragali fermentation liquor, comprising the following steps: Inoculating Lactobacillus reuteri into a culture medium containing a radix astragali ethanol extract for fermentation and concentration to obtain the radix astragali fermentation liquor. In the application, the main component of the radix astragali ethanol extract is flavonoids without polysaccharides, and the content of formononetin in the radix astragali fermentation liquor is more than twice that in the radix astragali ethanol extract.

[0007] Optionally, the Lactobacillus reuteri is preserved in the Henan Provincial Industrial Microorganism Engineering Technology Research Center on April 8, 2022, and the preservation number is BNCC192190.

[0008] Optionally, the total viable count of the Lactobacillus reuteri is 2-3×10 8-10 CFU / mL, the fermentation temperature is 31-45℃, and the fermentation time is 18-30 hours.

[0009] Optionally, the preparation method of the radix astragali ethanol extract comprises the following steps: (1) After adding water to radix astragali decoction pieces, decocting, and filtering, a residue and a first medicinal liquid are obtained; water is added to the residue, and the residue is decocted to obtain a second medicinal liquid; (2) The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain a radix astragali decoction; (3) Ethanol is added to the radix astragali decoction, and the mixture is allowed to stand and filter to obtain a precipitate part and a first supernatant part; (4) The precipitate part is redissolved in ethanol, and the mixture is allowed to stand and filter to obtain a second supernatant part; (5) The first supernatant part and the second supernatant part are combined and concentrated to obtain the radix astragali ethanol extract.

[0010] Optionally, in steps (3) and (4), the concentration of the ethanol is 75-85%.

[0011] Optionally, in step (5), when the radix astragali decoction pieces are 1.4-1.6 g, the volume of the radix astragali ethanol extract is 1 mL.

[0012] Optionally, in step (2), when the radix astragali decoction pieces are 1.4-1.6 g, the volume of the radix astragali decoction is 1 mL.

[0013] Optionally, in step (1), the volume ratio of the radix astragali decoction pieces to water is 1:8-11, the decoction temperature is 95-98℃, the decoction time is 0.5-1 hour, and the number of filtration is 1-3 times.

[0014] Optionally, in steps (3) and (4), the number of filtration is 1-3 times.

[0015] In a second aspect, the present invention provides an Astragalus fermentation broth obtained by the preparation method described in the first aspect.

[0016] In a third aspect, the present invention provides a pharmaceutical preparation comprising an effective dose of the Astragalus fermentation broth described in the second aspect.

[0017] Optionally, the pharmaceutical preparation is an enteric-coated capsule, and the preparation method is as follows: weigh the astragalus fermentation broth described in the second aspect in proportion, add excipients, crush after drying, add sweeteners and wetting agents, granulate, dry, and granulate, and fill the enteric-coated capsule shell mainly prepared from hydroxypropyl methylcellulose and enteric polymer acrylic resin to make enteric-coated capsules.

[0018] Optionally, the pharmaceutical preparation is a granule, and the preparation method is as follows: weigh the Astragalus fermentation broth described in the second aspect according to proportion, add excipients, grind after drying, add sweeteners and wetting agents, granulate, dry, and granulate to prepare granules.

[0019] In a fourth aspect, the present invention further provides use of the Astragalus fermentation broth described in the second aspect or the pharmaceutical preparation described in the third aspect in the preparation of a drug for alleviating ulcerative colitis.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing an astragalus fermentation broth. The astragalus fermentation broth obtained by the preparation method has a significant effect in alleviating ulcerative colitis in mice. Specifically, the astragalus fermentation broth obtained by the method can significantly alleviate the overall symptoms of ulcerative colitis model mice, including inhibiting weight loss, diarrhea, and bloody stools in the model mice, improving colonic mucosal inflammatory cell infiltration, goblet cell disappearance, and crypt structural destruction, reducing the concentration of inflammatory cytokines in colonic tissue, and alleviating calf gastrocnemius muscle atrophy in mice with ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The changes of formononetin content in Astragalus ethanol extracts before and after fermentation by different lactic acid bacteria; Figure 2 The body weight changes of mice during pharmacodynamic evaluation; Figure 3 The changes in pathological scores of mice during pharmacodynamic evaluation; Figure 4 Comparison of colon length between groups of mice during pharmacodynamic evaluation; Figure 5 Comparison between groups of HE staining of mouse colon pathological sections during pharmacodynamic evaluation; Figure 6 is the concentration of TNF-α in mouse colon tissue during pharmacodynamic evaluation; Figure 7 The concentration of IL-1β in the colon tissue of the mice is used for pharmacodynamic evaluation; Figure 8 The HE staining of the gastrocnemius muscle tissue sections of the mice is compared among groups for pharmacodynamic evaluation. DETAILED DESCRIPTION

[0022] The application provides a preparation method of a radix astragali fermentation liquor, which comprises the following steps: (1) Radix astragali decoction pieces are added with water, decocted, filtered to obtain dregs and a first medicinal liquid; water is added to the dregs to decoct to obtain a second medicinal liquid; (2) The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain a radix astragali decoction liquid; (3) Ethanol is added to the radix astragali decoction liquid, and then the mixture is left to stand and filtered to obtain a precipitate part and a first supernatant part; (4) The precipitate part is redissolved with ethanol, left to stand and filtered to obtain a second supernatant part; (5) The first supernatant part and the second supernatant part are combined and concentrated to obtain a radix astragali ethanol extract; (6) Lactobacillus reuteri is inoculated into a culture medium containing the radix astragali ethanol extract to perform fermentation, and then the mixture is concentrated to obtain a radix astragali fermentation liquor.

[0023] In the application, in step (1), the volume ratio of the radix astragali decoction pieces to water is 1:9-11, and is further preferably 1:10; the decoction temperature is 96-98 ℃, and is further preferably 97 ℃; the decoction time is 0.5-1 hour, and is further preferably 0.75 hour; and the filtration times are 1-3 times, and are further preferably 2 times.

[0024] In the application, in steps (2) and (3), the concentration of ethanol in the radix astragali decoction liquid is 75-85%, and is further preferably 80%; and the filtration times are 1-3 times, and are further preferably 2 times.

[0025] In the application, in step (2), the radix astragali decoction pieces are 1.4-1.6 g, and are further preferably 1.5 g; and the volume of the radix astragali decoction liquid is 1 mL.

[0026] In the application, in step (5), the radix astragali decoction pieces are 1.4-1.6 g, and are further preferably 1.5 g; and the volume of the radix astragali ethanol extract is 1 mL.

[0027] In the application, in step (6), the total viable count of the Lactobacillus reuteri is 2-3×10 8-10 CFU / mL, and is further preferably 2.5×10 8-10CFU / mL; the inoculation amount of the strain is 9-11% of the volume of the Astragalus alcohol extract, further preferably 10%; the fermentation temperature is 31-45°C, further preferably 36-39°C, and the fermentation time is 18-30 hours, further preferably 22-26 hours.

[0028] Screening of fermentation conditions of alcohol extract of Astragalus membranaceus The orthogonal experiment of the step (6) of the preparation method of the Astragalus fermentation liquor is carried out by setting three factors, i.e., bacterial inoculation amount, fermentation temperature and fermentation time, each with three levels, using an L9 (3 3 ) orthogonal table (shown in Table 1) to design 9 groups of experiments, inoculating the corresponding strains into the sterilized Astragalus alcohol extract medium (Astragalus alcohol extract:MRS medium = 1:1) according to the orthogonal table, and culturing at the specified temperature for the corresponding time. After the fermentation is completed, the active ingredient formononetin of the prepared Astragalus fermentation liquor is detected by high performance liquid chromatography, and the results are shown in Table 2. The optimal combination is A2B2C2: 2.5x10 8-10 CFU / mL inoculation amount, 36-39°C temperature and 22-26h time, so that the metabolic product accumulation of the bacterial population reaches a peak. After repeated verification, the formononetin content under this condition is increased by 15%-30% compared with other combinations, and the optimal scheme of the Astragalus alcohol extract fermentation condition is finally established.

[0029] Table 1. Orthogonal experiment design table for Lactobacillus reuteri fermentation of Astragalus alcohol extract

[0030] Table 2. Results of orthogonal experiment for Lactobacillus reuteri fermentation of Astragalus alcohol extract

[0031] Screening of fermentation bacteria of alcohol extract of Astragalus membranaceus The fermentation bacteria in the step (6) of the preparation method of the Astragalus fermentation liquor are screened. 2.5x10 8-10 CFU / mL of Lactobacillus johnsonii or Lactobacillus murinus or Lactobacillus reuteri is inoculated into the medium containing the Astragalus alcohol extract (Astragalus alcohol extract:MRS medium = 1:1) for fermentation, the inoculation amount of the strain is 10% of the volume of the Astragalus alcohol extract, the fermentation temperature is 38°C, and the fermentation time is 24 hours, to obtain the Astragalus fermentation liquor.

[0032] The experimental results are shown in Table 2. Figure 1As shown, the fermentation efficiency of the three fermentation strains follows the order of Lactobacillus reuteri > Lactobacillus johnsonii > Lactobacillus murinus. Specifically, the content of the active ingredient formononetin in the astragalus alcohol extract increased after fermentation with the different strains, and the increase followed the order of Lactobacillus reuteri > Lactobacillus johnsonii > Lactobacillus murinus. The formononetin content in the Lactobacillus reuteri fermentation group increased significantly more than in the other two groups, followed by the Lactobacillus johnsonii group, and the Lactobacillus murinus group had the smallest increase. This indicates that Lactobacillus reuteri can more efficiently promote the conversion and accumulation of formononetin.

[0033] Example 1 A method for preparing an astragalus fermentation broth comprises the following steps: Add 10 times the amount of water to the Astragalus slices, decoct for 0.75 hours, filter, and obtain the residue and the first liquid. Add 10 times the amount of water to the residue and boil for 0.75 hours to obtain the second medicinal solution; The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain an astragalus decoction; Add ethanol to the Astragalus membranaceus decoction to adjust the ethanol concentration to 80%, let it stand, and filter to obtain a precipitate and a first supernatant; Add water to the precipitate to redissolve it, add ethanol to adjust the ethanol concentration to 80%, let it stand, and filter to obtain the precipitate and the second supernatant; The first supernatant and the second supernatant were combined and concentrated to obtain an astragalus ethanol extract; 2.5×10 9 CFU / mL of Lactobacillus reuteri (deposited in Henan Industrial Microbial Strain Engineering Technology Research Center on April 8, 2022, with the deposit number BNCC192190) was inoculated into a culture medium containing astragalus alcohol extract (astragalus alcohol extract: MRS culture medium = 1:1) for fermentation. The inoculation amount of the strain was 10% of the volume of the astragalus alcohol extract, the fermentation temperature was 38°C, the fermentation time was 24 hours, and the fermentation liquid was concentrated to obtain astragalus fermentation liquid A.

[0034] Example 2 A method for preparing an astragalus fermentation broth comprises the following steps: Add 9 times the amount of water to the Astragalus slices, boil for 0.5 hours, filter, and obtain the residue and the first liquid. Add 9 times the amount of water to the medicinal residue and boil for 0.5 hours to obtain the second medicinal solution; The first medicinal solution and the second medicinal solution are combined and concentrated to obtain an astragalus water extract; Add ethanol to the Astragalus membranaceus aqueous extract to adjust the ethanol concentration to 75%, let it stand, and filter to obtain a precipitate and a first supernatant; Add water to the precipitate to redissolve it, add ethanol to adjust the ethanol concentration to 75%, let it stand, and filter to obtain the precipitate and the second supernatant; The first supernatant and the second supernatant were combined and concentrated to obtain an astragalus ethanol extract; 2×10 9 Lactobacillus reuteri with a CFU / mL was inoculated into a culture medium containing an alcohol extract of Astragalus (Astragalus alcohol extract: MRS medium = 1:1) for fermentation. The inoculation amount of the strain was 9% of the volume of the aqueous extract of Astragalus. The fermentation temperature was 37°C, the fermentation time was 20 hours, and the fermentation liquid was concentrated to obtain Astragalus fermentation liquid A'.

[0035] Example 3 A method for preparing an astragalus fermentation broth comprises the following steps: Add 11 times the amount of water to the Astragalus slices, boil for 1 hour, filter, and obtain the residue and the first liquid. Add 11 times the amount of water to the residue and boil for 1 hour to obtain the second medicinal solution; The first medicinal solution and the second medicinal solution are combined and concentrated to obtain an astragalus water extract; Add ethanol to the Astragalus membranaceus aqueous extract to adjust the ethanol concentration to 85%, let it stand, and filter to obtain a precipitate and a first supernatant; Add water to the precipitate to redissolve it, add ethanol to adjust the ethanol concentration to 85%, let it stand, and filter to obtain the precipitate and the second supernatant; The first supernatant and the second supernatant were combined and concentrated to obtain an astragalus water extract; 3×10 9 Lactobacillus reuteri with a CFU / mL was inoculated into a culture medium containing an alcohol extract of Astragalus (Astragalus alcohol extract: MRS medium = 1:1) for fermentation. The inoculation amount of the strain was 11% of the volume of the aqueous extract of Astragalus. The fermentation temperature was 39°C, the fermentation time was 30 hours, and the fermentation liquid was concentrated to obtain Astragalus fermentation liquid A''.

[0036] Comparative Example 1 A method for preparing an astragalus decoction comprises the following steps: Add 10 times the amount of water to the Astragalus slices, decoct for 0.75 hours, filter, and obtain the residue and the first liquid. Add 10 times the amount of water to the residue and boil for 0.75 hours to obtain the second medicinal solution; The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain the Astragalus membranaceus decoction.

[0037] Comparative Example 2 A method for preparing an astragalus fermentation broth comprises the following steps: Add 10 times the amount of water to the Astragalus slices, decoct for 0.75 hours, filter, and obtain the residue and the first liquid. Add 10 times the amount of water to the residue and boil for 0.75 hours to obtain the second medicinal solution; The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain an astragalus decoction; 2.5×10 8-10 Lactobacillus reuteri with a CFU / mL concentration was inoculated into a culture medium containing astragalus decoction (astragalus decoction: MRS medium = 1:1) for fermentation. The inoculation amount of the strain was 10% of the volume of the astragalus decoction. The fermentation temperature was 38°C, the fermentation time was 24 hours, and the fermentation liquid was concentrated to obtain astragalus fermentation liquid B.

[0038] Comparative Example 3 A method for preparing an astragalus ethanol extract comprises the following steps: Add 10 times the amount of water to the Astragalus slices, decoct for 0.75 hours, filter, and obtain the residue and the first liquid. Add 10 times the amount of water to the residue and boil for 0.75 hours to obtain the second medicinal solution; The first medicinal liquid and the second medicinal liquid are combined and concentrated to obtain an astragalus decoction; Add ethanol to the Astragalus membranaceus decoction to adjust the ethanol concentration to 80%, let it stand, and filter to obtain a precipitate and a first supernatant; Add water to the precipitate to redissolve it, add ethanol to adjust the ethanol concentration to 80%, let it stand, and filter to obtain the precipitate and the second supernatant; The first supernatant portion and the second supernatant portion were combined and concentrated to obtain the astragalus ethanol extract.

[0039] Efficacy trials Animal experiments were approved by the Animal Ethics Committee of Southern Medical University (Approval No. L202312007, Date: December 14, 2023). Male Balb / c mice (20 ± 2 g) were purchased from the Laboratory Animal Center of Southern Medical University and acclimated under standard laboratory conditions for 7 days prior to the experiment. Forty-two male Balb / c mice were randomly placed into eight cages and housed for 7 days to allow them to acclimate. After the acclimation period, mice in each group were gavage-treated on days 8 to 14 (preventive period) and 15 to 17 (treatment period). From day 8 to 14 (modeling period), mice were given free access to 3% dextran sulfate sodium salt (DSS). Feces were collected from each group on days 15 to 17 (treatment period). On day 17, mice were fasted for 24 hours, anesthetized with an intraperitoneal injection of 1% sodium pentobarbital, and then euthanized by cervical dislocation. Plasma and colon tissues of mice were collected for determination of various indicators. The specific groups are as follows (Table 3): (1) Normal group: During the entire experimental period, mice were fed with normal feed and drinking water. Each mouse was gavaged with 0.2 mL of normal saline per day. (2) Model group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of normal saline by gavage every day; (3) Mesalazine group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of mesalazine solution by gavage every day, with the dose range converted from human to mouse body surface area (0.3 g / kg); (4) Huangqi water decoction group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of Huangqi water decoction by gavage every day; (5) Huangqi water decoction fermentation group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of Huangqi fermentation liquid B by gavage every day; (6) Huangqi ethanol extract group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of Huangqi ethanol extract by gavage every day; (7) Huangqi ethanol extract fermentation group: During the whole experimental period, the mice were supplied with normal feed and drinking water. During the modeling period, the mice were replaced with 3% DSS solution, and each mouse was given 0.2 mL of Huangqi fermentation liquid A by gavage every day.

[0040] Table 3. Experimental grouping and drug liquid control table

[0041] Pharmacodynamic evaluation According to the body weight change of the mice during the experiment, as well as the disease activity index (DAI) score according to the body weight loss, diarrhea and degree of hematochezia of the mice, the length of the colon of the mice was measured and histopathological analysis was performed, and the above indexes were used to evaluate the efficacy.

[0042] In the comprehensive efficacy evaluation of ulcerative colitis model mice, the results were as follows: Figures 2-4As shown in the figure: the normal group always maintained the optimal physiological state, and its stable weight gain trend, no diarrhea and bloody stools, and intact colon structure constituted a healthy benchmark; the model group showed a typical pathological process, manifested as continuous weight loss, significant diarrhea and bloody stools, and these symptoms were evaluated by the disease activity index score that comprehensively evaluated weight loss, stool characteristics, and bloody stools. At the same time, the colon length was shortened, which clearly verified the success of the model; the positive control drug mesalazine group showed a stable treatment effect, with weight restored to a near-healthy level, the disease activity index score dropped to a slightly abnormal range, and the degree of colon shortening significantly improved; the efficacy of Astragalus fermentation liquid A was similar to that of mesalazine. There was no substantial difference between the two groups in terms of weight recovery dynamics, control of core symptoms of the disease activity index including weight fluctuation, diarrhea frequency and severity of bloody stools, and colon mucosal protection, and all of them were significantly better than other traditional intervention groups; the astragalus water decoction group, the astragalus water decoction fermentation group and the astragalus alcohol extract group all showed certain anti-UC activity, but the efficacy was not as good as the astragalus alcohol extract fermentation group; the astragalus water decoction fermentation group and the astragalus alcohol extract group did not completely eliminate the symptoms of diarrhea and bloody stools, and had limited effect on improving colon damage; the astragalus water decoction group had poor effects on weight recovery, control of comprehensive symptoms of the disease activity index, especially control of bloody stools, and colon protection.

[0043] In the results of hematoxylin-eosin staining (HE staining) of colon tissue of UC model mice, Figure 5 As shown, green arrows indicate mucosal damage or inflammatory cell infiltration. The model group exhibited typical pathological changes, including extensive mucosal necrosis with deep ulceration, severe glandular destruction in the lamina propria, a significant decrease or even disappearance of goblet cells, extensive distortion or loss of crypt architecture, extensive neutrophil and lymphocyte infiltration throughout the lamina propria, and submucosal congestion and edema, consistent with successful UC modeling. The mesalazine group and the Astragalus alcohol extract fermentation group showed comparable efficacy. The mucosal epithelium remained largely intact, with only minor localized glandular destruction. The number of goblet cells approached normal levels, the crypt architecture was slightly distorted but continuous, and a small number of inflammatory cells were occasionally observed in the submucosal layer, demonstrating a significant mucosal protective effect. The Astragalus decoction fermentation and Astragalus alcohol extract groups showed less improvement than the Astragalus alcohol extract fermentation group. Erosions were observed in some areas of the mucosa, with a moderate number of residual goblet cells. Moderate inflammatory cell infiltration was observed from the submucosal layer to the muscularis, suggesting limited repair of colonic injury. The Astragalus decoction group showed the weakest efficacy, with extensive crypt destruction and uneven depth of the remaining crypts, as well as extensive inflammatory cell infiltration in the submucosal layer extending into the muscularis. These findings suggest that the Astragalus alcohol extract fermentation group almost completely reversed the pathological lesions, including crypt destruction, goblet cell loss, and inflammatory infiltration, observed in the intestines of mice with UC, significantly outperforming the other groups.

[0044] The levels of inflammatory factors TNF-α (MM-0132M1, purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.) and IL-1β (MM-0905M1, purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.) in colon tissue were detected by ELISA kits. The results were as follows: Figures 6-7 As shown in the results, compared with the normal group, the concentrations of inflammatory factors TNF-α and IL-1β in the colon tissue of the model group were significantly increased, indicating an inflammatory response in the mucosal tissue. The mesalazine group and the fermentation group of the astragalus alcohol extract could significantly reduce the levels of TNF-α and IL-1β in the colon mucosal tissue, and the reduction was significantly better than that of the astragalus alcohol extract group, the fermentation group of the astragalus decoction and the astragalus decoction group. This result was similar to the result of hematoxylin-eosin staining.

[0045] This study also found that HE staining Figure 8 As shown, compared with the normal group, the calf gastrocnemius muscles of mice with ulcerative colitis showed significant atrophy, with characteristics such as thinning of muscle fibers or "angular" or irregular shrinkage of some muscle fibers, reduced sarcoplasm, and blurred striations. Administration of the fermented Astragalus alcohol extract significantly improved these symptoms. Combined with the improvement in weight loss after administration, this suggests that the fermented Astragalus extract of this application has an alleviating effect on skeletal muscle atrophy in mice with ulcerative colitis.

[0046] This study showed that the fermentation process of Astragalus ethanol extract increased the content of active ingredients and thus improved the overall anti-UC efficacy of traditional Chinese medicine. This method is highly effective and safe.

[0047] Application Example 1 An enteric-coated capsule comprises the following steps: The Astragalus fermentation broth A prepared according to Example 1 was concentrated to an extract with a relative density of 1.5 g / mL, dextrin was added in a 4-fold weight ratio, vacuum-dried at 80° C., and pulverized to pass through a 65-mesh sieve to obtain a dry powder. Powdered sugar was then added in a 2-fold weight ratio, and a soft material was prepared using 95% ethanol as a wetting agent. The material was granulated through a 16-mesh sieve, dried, and sized. The granules were then filled into enteric capsule shells mainly prepared from hydropropyl methylcellulose and an enteric polymer acrylic resin to prepare enteric capsules.

[0048] Application Example 2 A granule preparation comprising the following steps: The Astragalus fermentation broth A prepared according to Example 1 was concentrated to an extract with a relative density of 1.5 g / mL, dextrin was added 4 times by weight, vacuum dried at 80° C., and pulverized to pass through a 65-mesh sieve to obtain a dry powder. Then, 2 times by weight of powdered sugar was added, and a soft material was prepared using 95% ethanol as a wetting agent. The powder was granulated through a 16-mesh sieve, dried, and sized to prepare granules.

[0049] This specific implementation manner is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the specific implementation manner as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing an astragalus fermentation broth, characterized in that: The steps include: inoculating Lactobacillus reuteri into a culture medium containing an alcohol extract of Astragalus membranaceus for fermentation and concentration to obtain an Astragalus membranaceus fermentation liquid; The main components of the astragalus alcohol extract are flavonoids without polysaccharides; the content of formononetin in the astragalus fermentation liquid is more than twice that of the astragalus alcohol extract.

2. The preparation method according to claim 1, characterized in that The Lactobacillus reuteri was deposited in the Henan Industrial Microbiology Strain Engineering Technology Research Center on April 8, 2022, with the deposit number BNCC192190.

3. The preparation method according to claim 1, characterized in that The total viable count of Lactobacillus reuteri is 2-3×10 8-10 CFU / mL; the fermentation temperature is 31-45°C, and the fermentation time is 18-30 hours.

4. The preparation method according to claim 1, characterized in that The preparation method of the astragalus ethanol extract comprises the following steps: (1) After adding water to the Astragalus slices, decocting and filtering, a residue and a first liquid are obtained; water is added to the residue, decocting, and a second liquid is obtained; (2) combining and concentrating the first medicinal liquid and the second medicinal liquid to obtain an astragalus decoction; (3) adding ethanol to the astragalus decoction, allowing it to stand, and filtering to obtain a precipitate and a first supernatant; (4) Redissolving the precipitated portion in ethanol, allowing it to stand, and filtering to obtain a second supernatant portion; (5) The first supernatant fraction and the second supernatant fraction are combined and concentrated to obtain an astragalus ethanol extract.

5. The preparation method according to claim 4, characterized in that In steps (3) and (4), the concentration of ethanol is 75-85%.

6. The preparation method according to claim 4, characterized in that In the step (5), when the amount of the Astragalus slices is 1.4-1.6 g, the volume of the Astragalus alcohol extract is 1 mL.

7. The preparation method according to claim 4, characterized in that In the step (2), when the amount of the Astragalus slices is 1.4-1.6 g, the volume of the Astragalus decoction is 1 mL.

8. An astragalus fermentation broth obtained by the preparation method according to any one of claims 1 to 7.

9. A pharmaceutical preparation, characterized in that: A fermented astragalus broth comprising an effective dose of the astragalus radix fermented liquid according to claim 8.

10. Use of the astragalus fermentation broth according to claim 8 or the pharmaceutical preparation according to claim 9 in preparing a drug for treating or preventing ulcerative colitis.

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