Composition for regulating intestinal flora as well as preparation method and application of composition

Abstract

The invention relates to a composition for regulating intestinal flora as well as a preparation method and application of the composition. The composition is prepared from the following raw materials: fried rhizoma atractylodis, pericarpium citri reticulatae, honey-fried licorice root, cortex magnoliae officinalis (processed with ginger), polyporus umbellatus, poria cocos (peeled), fried rhizoma atractylodis macrocephalae, rhizoma alismatis, cortex cinnamomi, rhizoma zingiberis and fructus mume. Through the synergistic effect of the raw materials, the effects of resisting inflammation, resisting oxidation, regulating intestinal flora, improving intestinal immunity, protecting intestinal barriers and the like are achieved.

Description

Technical Field

[0001] The present invention relates to the field of veterinary medicine, and in particular to a composition for regulating intestinal flora, and a preparation method and application thereof. Background Art

[0002] As an important component of the intestinal system of animals, the intestinal flora plays a vital role in the development of the animal intestine and the maturation of immune function. Different intestinal microorganisms have different effects on host health. Recent studies have shown that colonic microbiota dysbiosis is associated with intestinal immune dysfunction during active diarrhea in newborn rhesus monkeys. In addition, a study on newborn mice with diarrhea phenotype further showed that intestinal microbiota dysbiosis can trigger bacterial translocation and colonic barrier dysfunction.

[0003] Lactobacillus is a beneficial bacteria. It can relieve inflammation, prevent pathogen invasion and Clostridium difficile infection, activate the phagocytosis of macrophages, and enhance immunity. The pharmacological effects of lactobacillus include relieving intestinal inflammation, systemic inflammation, improving immunity, and preventing the invasion of foreign pathogens or toxic substances. Therefore, the increase in the abundance of lactobacillus is a valuable research direction, which means that the increase of lactobacillus has a beneficial effect on physical health. Summary of the invention

[0004] In view of the above problems, the present invention provides a composition with the functions of regulating intestinal flora, enhancing immunity and being antiviral. The composition can regulate intestinal flora, promote the growth of beneficial bacteria, improve the host's nonspecific immunity, and enhance the host's anti-diarrhea ability, so as to achieve a safe and effective treatment of gastrointestinal diseases and overcome the above defects.

[0005] The invention point of the present invention is to provide a composition for regulating intestinal flora, and its raw materials include fried atractylodes, tangerine peel, roasted licorice, Magnolia officinalis (processed with ginger), Polyporus, Poria (peeled), fried Atractylodes macrocephala, Alisma orientalis, cinnamon bark, dried ginger and black plum.

[0006] Furthermore, the above raw material components are matched in proportion by weight: 100-200 parts of stir-fried Atractylodes lancea, 100-200 parts of dried tangerine peel, 50-100 parts of roasted licorice, 100-200 parts of Magnolia officinalis (processed with ginger), 50-150 parts of Polyporus umbellatus, 100-200 parts of Poria cocos (peeled), 100-200 parts of stir-fried Atractylodes macrocephala, 50-150 parts of Alisma orientalis, 30-60 parts of cinnamon, 50-150 parts of dried ginger, and 50-150 parts of ebony.

[0007] Furthermore, the composition meets at least one of the following indicators: atractylodes lactone I ≥ 0.008 mg / g, atractylodes lactone II ≥ 0.036 mg / g, atractylodes lactone III ≥ 0.102 mg / g, magnolol ≥ 0.614 mg / g, magnolol ≥ 0.432 mg / g, and 23-acetyl alismatol B ≥ 0.034 mg / g.

[0008] Another inventive point of the present invention is to provide a method for preparing a composition for regulating intestinal flora, the specific steps of which include:

[0009] Step 1): Take stir-fried Atractylodes lancea, dried orange peel, roasted Licorice root, Magnolia officinalis (processed with ginger), Polyporus umbellatus, Poria cocos (peeled), stir-fried Atractylodes macrocephala, Alisma orientalis, cinnamon bark, dried ginger, and black plum according to weight proportions, and coarsely grind them;

[0010] Step 2): taking half of the amount of each component in the raw material of the composition in step 1), adding water and boiling for three times, filtering, combining the decoction, concentrating and drying to obtain a dry extract, and crushing the dry extract to obtain a fine extract powder;

[0011] Step 3): Take the remaining half of each component in the composition raw material in step 1), crush and sterilize, and mix with the extract powder prepared in step 2) to obtain the composition for regulating intestinal flora.

[0012] Furthermore, the decoction in step 2) includes three decoctions: adding 8-12 times the volume of water during the first decoction, soaking for 45-60 minutes, and then decoct for 2 hours; adding 6-10 times the volume of water during the second decoction, decoct for 1.5 hours; adding 4-6 times the volume of water during the third decoction, and decoct for 1 hour.

[0013] Furthermore, step 2) further comprises sterilizing the dry extract at 121° C. for 20 to 30 minutes before crushing, the relative density of the dry extract is 1.00 to 1.1 (g / mL), and the sterilization method comprises ultraviolet radiation sterilization and high-pressure steam sterilization.

[0014] Another inventive point of the present invention is to provide a composition for regulating intestinal flora for use in preparing drugs for treating diarrheal diseases, anti-inflammatory, anti-oxidation, regulating intestinal flora and enhancing intestinal immunity.

[0015] Furthermore, the diarrheal diseases include diarrhea in lambs and calves, diarrhea and dysentery in laying hens and waterfowl, ulcerative colitis, chronic enteritis, diarrhea and intestinal inflammation caused by irritable bowel syndrome.

[0016] Furthermore, the regulating of intestinal flora includes regulating the structure of intestinal flora and enriching beneficial bacteria; the enhancing of intestinal immunity includes promoting the differentiation of Treg cells, inhibiting the differentiation of Th17 cells, and regulating the balance of Treg / Th17.

[0017] The present invention also provides a drug for regulating intestinal flora, which comprises the composition described in any of the above paragraphs or the composition prepared by the preparation method.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention produces anti-inflammatory, anti-oxidative, intestinal flora regulating, intestinal immunity enhancing and intestinal barrier protective effects through the synergistic effect between the raw materials. It has a simple formula and significant therapeutic effect. It can replace the use of antibiotics and avoid the side effects of chemical drugs on livestock and poultry, and can bring greater social and economic effects.

[0020] The present invention relieves intestinal inflammation by regulating the levels of inflammatory factors and oxidative stress; the present invention can also regulate intestinal nonspecific immunity and intestinal flora, thereby increasing the diversity and richness of intestinal flora, increasing the content of tryptophan metabolites, and restoring the abundance and proportion of intestinal flora to normal proportions, thereby improving and treating diarrheal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Perianal feces diagram of rats;

[0022] Figure 2 The weight change trend of rats;

[0023] Figure 3 Rank-Abundance curve and Coverage curve of rat intestine;

[0024] Figure 4 Rat intestinal alpha diversity map, A. ace index; B. Chao index; C. Shannon index; D. Sobs index;

[0025] Figure 5 PCA analysis results of rat intestine;

[0026] Figure 6 Analysis of differences in the composition of rat intestinal flora, (A) phylum-level flora analysis; (B) genus-level flora analysis;

[0027] Figure 7 Analysis of tryptophan metabolites in rat intestine: (A) Indoleacrylic acid, (B) L-Tryptophan, (C) Indole-3-carboxylic acid, (D) 3-Indole-glyoxylic acid, (E) Indole-3-acetic acid, (F) Lactic acid;

[0028] Figure 8 Schematic diagram of Treg / Th17 balance in rat mesenteric lymph nodes, (A) the proportion of Treg cells in mesenteric lymph nodes; (B) the proportion of Th17 cells in mesenteric lymph nodes;

[0029] Fig. 9 H&E staining of rat colon;

[0030] Fig.10 Transmission electron micrograph of rat colon;

[0031] Fig.11 Blood biochemical parameters of calves after administration;

[0032] Fig.12 Calf intestinal Rank-Abundance curve and Shannon curve;

[0033] Fig.13 Alpha diversity analysis of calf intestine;

[0034] Fig.14 Analysis chart of PCA results of calf intestine;

[0035] Fig.15 Analysis of differences in the composition of calf intestinal flora, (A) phylum level flora analysis, (B) genus level flora analysis.

[0036] Specific implementation method

[0037] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0038] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0039] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0040] Example 1

[0041] This embodiment provides a composition for regulating intestinal flora, the raw materials of which include: stir-fried Atractylodes lancea, dried tangerine peel, roasted Licorice root, Magnolia officinalis (processed with ginger), Polyporus umbellatus, Poria cocos (peeled), stir-fried Atractylodes macrocephala, Alisma orientalis, cinnamon bark, dried ginger, and black plum.

[0042] The raw materials of the composition of the present invention include Atractylodes macrocephala, Atractylodes macrocephala and Magnolia officinalis as the main drugs, which warm the stomach and strengthen the spleen; Polyporus umbellatus, Poria cocos and Rhizoma Alismatis as the assistant drugs, which guide water downward, promote urination and solidify the stool; Tangerine peel, Cinnamon bark, Dry ginger and Prunus mume as the adjuvant drugs, which regulate Qi and strengthen the spleen, warm Yang and transform Qi, warm Qi and dispel cold, astringe the intestine and stop diarrhea; Licorice is the guiding drug, which harmonizes the middle and reconciles the drugs; the whole prescription is used together to exert the effects of warming and promoting stomach Yang, penetrating dampness and stopping diarrhea. This prescription can play the role of anti-inflammatory, anti-oxidation, regulating intestinal flora and balancing intestinal immunity in the treatment of patients with eczema, functional dyspepsia, ulcerative colitis, acute non-bacterial infectious diarrhea in children, antibiotic-related diarrhea and autumn diarrhea in children, and has significant clinical effect.

[0043] In this embodiment, by adding a certain amount of stir-fried Atractylodes lancea, dried tangerine peel, roasted Licorice root, Magnolia bark (prepared with ginger) and a certain amount of Polyporus umbellatus, Poria cocos (peeled), stir-fried Atractylodes macrocephala, Alisma orientalis, and especially adding cinnamon, dried ginger and black plum, the composition has an excellent effect of acting together, which significantly increases the metabolism of tryptophan in the intestine, and the immune balance significantly reverses the imbalance of intestinal immune Treg / Th17, and regulates the balance of intestinal immunity by regulating the balance of Treg / Th17, ultimately forming an intestinal immune balance with no significant difference from normal conditions.

[0044] As a further preferred embodiment, the raw material components are matched by weight: 100-200 parts of stir-fried Atractylodes lancea, 100-200 parts of dried tangerine peel, 50-100 parts of roasted licorice, 100-200 parts of Magnolia officinalis (processed with ginger), 50-150 parts of Poria cocos, 100-200 parts of stir-fried Atractylodes macrocephala, 50-150 parts of Alisma orientalis, 30-60 parts of cinnamon, 50-150 parts of dried ginger, and 50-150 parts of ebony. The raw material components can be further preferably proportioned by weight as follows: 115-175 parts of stir-fried atractylodes, 115-175 parts of dried tangerine peel, 65-95 parts of roasted licorice root, 115-175 parts of magnolia bark (processed with ginger), 65-130 parts of umbelliferae, 115-175 parts of tuckahoe (peeled), 115-175 parts of stir-fried atractylodes, 65-130 parts of oriental rhizome, 35-55 parts of cinnamon bark, 60-140 parts of dried ginger, and 60-145 parts of ebony. For example, the raw materials and weight portions of the composition can be: ① 150 parts of stir-fried atractylodes, 150 parts of dried tangerine peel, 60 parts of roasted licorice root, 150 parts of magnolia bark (processed with ginger), 100 parts of umbelliferae, 15 parts of tuckahoe (peeled), 150 parts of stir-fried atractylodes, 100 parts of oriental rhizome, 50 parts of cinnamon bark, 100 parts of dried ginger, and 100 parts of ebony. ② 100 parts of stir-fried Atractylodes lancea, 100 parts of dried tangerine peel, 50 parts of roasted licorice, 100 parts of Magnolia officinalis (processed with ginger), 50 parts of Polyporus, 100 parts of Poria (peeled), 100 parts of stir-fried Atractylodes lancea, 50 parts of Rhizoma Alismatis, 30 parts of cinnamon, 50 parts of dried ginger, and 50 parts of black plum. ③ 200 parts of stir-fried Atractylodes lancea, 200 parts of dried tangerine peel, 100 parts of roasted licorice, 200 parts of Magnolia officinalis (processed with ginger), 150 parts of Polyporus, 200 parts of Poria (peeled), 200 parts of stir-fried Atractylodes lancea, 150 parts of Rhizoma Alismatis, 60 parts of cinnamon, 150 parts of dried ginger, and 150 parts of black plum.

[0045] The following further descriptions can be made for each of the above raw materials and their weight portions:

[0046] Fried Atractylodes: Also known as bran fried Atractylodes, pungent, bitter, warm. Return to the spleen, stomach, liver meridians. Drying dampness and invigorating the spleen, dispelling wind and cold, improving eyesight. Used for abdominal distension, diarrhea, edema, rheumatic pain, cold, night blindness. Preferably, the stir-fried Atractylodes can be taken as: for example, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 parts by weight or any value between any two values.

[0047] Tangerine peel: functions of regulating qi, regulating the middle, drying dampness, and resolving phlegm, treating chest and abdominal distension, loss of appetite, vomiting and hiccups, and coughing with sputum. Preferably, the tangerine peel can be prepared in a weight ratio of, for example, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 parts or any value between any two values.

[0048] Licorice root: sweet and flat in nature. It enters the heart, lung, spleen and stomach meridians, and has the effects of tonifying the spleen and stomach, replenishing qi and restoring pulse. It is used for spleen and stomach weakness, fatigue, palpitations, and irregular pulse. Preferably, the proportion of licorice root by weight can be: for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 parts or any value between any two values.

[0049] Magnolia officinalis: bitter, warm in nature, warms the middle and lowers the qi, dries dampness, eliminates phlegm, treats chest and abdominal distension and pain, nausea and vomiting, indigestion, treats cold air for many years, thunder in the abdomen, removes water accumulation, breaks up old blood, digests water and food, soothes stomach qi, and relieves pain. Preferably, the ratio of Magnolia officinalis by weight can be: for example, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 parts or any value between any two values.

[0050] Polyporus: sweet, light and flat. It enters the kidney and bladder meridians. It mainly has diuretic, immune function enhancement, anti-tumor, anti-liver damage, antibacterial and other effects. Preferably, the proportion of Polyporus by weight can be: for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 parts or any value between any two values.

[0051] Poria: sweet, light, neutral in nature. It enters the heart, lung, spleen, and kidney meridians. It has the effects of promoting diuresis and removing dampness, strengthening the spleen and calming the mind. Preferably, the proportion of Poria by weight can be, for example, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 parts or any value between any two values.

[0052] Fried Atractylodes macrocephala: invigorating the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. It is mainly used to treat spleen deficiency, fatigue, poor appetite and abdominal distension, loose stools, water retention, dysuria, edema, spontaneous sweating due to qi deficiency, and restless fetus. Preferably, the fried Atractylodes macrocephala can be prepared in a weight ratio of, for example, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 parts or any value between any two values.

[0053] Rhizoma Alismatis: Nature and flavor: sweet, cold. Meridians: enter kidney and bladder meridians. Efficacy: diuresis, dampness elimination, heat dissipation. Treats dysuria, edema, vomiting, diarrhea, phlegm, beriberi, gonorrhea, hematuria. Preferably, the Rhizoma Alismatis can be prepared in a weight ratio of, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 parts or any value between any two values.

[0054] Cinnamon: spicy, sweet, very hot. It enters the kidney, spleen, heart, and liver meridians. It has the effects of replenishing fire and yang, guiding fire back to the origin, dispelling cold and relieving pain, and warming and unblocking the meridians. Preferably, the cinnamon can be prepared in a weight ratio of, for example, 30, 35, 40, 45, 50, 55, 60 parts or any value between any two values.

[0055] Dried ginger: spicy, hot in nature. It enters the spleen, stomach, kidney, heart and lung meridians. It has the effects of warming the middle and dispelling cold, restoring yang and unblocking meridians, warming the lungs and eliminating phlegm. Preferably, the dried ginger can be prepared in a weight ratio of, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 parts or any value between any two values.

[0056] Plum: sour, astringent, neutral in nature. Enters the liver, spleen, lung, and large intestine meridians. Has the effects of astringing the lungs, astringing the intestines, promoting the production of body fluids, and calming ascaris. Preferably, the weight ratio of Plum can be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 parts or any value between any two values.

[0057] The raw materials involved in the present invention are all natural Chinese herbal medicines, which are all Chinese medicines included in the 2020 edition of the Chinese Pharmacopoeia, and meet the quality requirements of the Pharmacopoeia. They are easy to obtain, simple to prepare, have a wide source of medicine, and low cost. The present invention follows the principles of Chinese medicine prescriptions, and various component medicines are decocted in water for oral administration.

[0058] The above-mentioned raw materials can be native drugs or processed products of native drugs.

[0059] As a further preferred embodiment, the traditional Chinese medicine preparation meets at least one of the following indicators: atractylodes lactone I ≥ 0.008 mg / g, atractylodes lactone II ≥ 0.036 mg / g, atractylodes lactone III ≥ 0.102 mg / g, magnolol ≥ 0.614 mg / g, magnolol ≥ 0.432 mg / g, and 23-acetyl alismatol B ≥ 0.034 mg / g.

[0060] The present invention measures the content of effective components of the composition, proposes the quality standard of the effective components of the composition of the present invention, and provides a theoretical basis for anti-inflammatory, anti-oxidative regulation of intestinal flora and intestinal immunity.

[0061] Example 2

[0062] This embodiment provides a method for preparing the composition for regulating intestinal flora according to embodiment 1, and the specific steps are as follows:

[0063] Step 1): Take stir-fried Atractylodes lancea, dried orange peel, roasted licorice, Magnolia officinalis (prepared with ginger), Polyporus, Poria (peeled), stir-fried Atractylodes macrocephala, Rhizoma Alismatis, Cinnamon bark, dried ginger, and Prunus mume according to weight ratio, and coarsely grind them; Step 2): Take half of the amount of each component in the raw material of the composition in step 1), add water and decoct three times, filter, combine the decoction, concentrate and dry to obtain a dry extract, grind the dry extract to obtain a fine extract powder. Step 3): Take the remaining half of the amount of each component in the raw material of the composition in step 1), grind and sterilize, and mix with the fine extract powder prepared in step 2) to obtain the composition for regulating intestinal flora.

[0064] As a further preferred embodiment, step 2) also includes adding 8 to 12 times the amount of water during the first decoction, soaking for 45 to 60 minutes, and then decocting for 2 hours; adding 6 to 10 times the amount of water during the second decoction, decocting for 1.5 hours; adding 4 to 6 times the amount of water during the third decoction, decocting for 1 hour.

[0065] Step 2) further comprises sterilizing the dry extract at 121° C. for 20 to 30 minutes, wherein the relative density of the dry extract is 1.00 to 1.1 (g / ml), and the sterilization method comprises ultraviolet radiation sterilization and high-pressure steam sterilization.

[0066] As a further preferred embodiment, the method further comprises step 4), wherein step 4) is to add pharmaceutically acceptable excipients and prepare a Chinese medicine preparation of a composition for regulating intestinal flora by conventional Chinese medicine preparation methods.

[0067] Preferably, the auxiliary materials include preservatives, disintegrants, wetting agents, diluents, binders, lubricants, flavoring agents, and the Chinese medicine preparations include at least one of liquid preparations, semisolid preparations, solid preparations and gaseous preparations.

[0068] Chinese medicine preparations include mixtures, oral liquids, alcoholic preparations, tinctures, injections, decoctions, fluid extracts, extracts, syrups, pills, powders, granules, tablets, capsules, aerosols, and sprays.

[0069] The route of administration can be supplied in bulk or unit dosage form depending on the expected route of administration because of the compositions provided herein. For example, for oral, buccal and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, soft capsules and caplets can be acceptable as solid dosage forms, and emulsions, syrups, elixirs, suspensions and solutions can be acceptable as liquid dosage forms. For injection, emulsions and suspensions can be acceptable as liquid dosage forms, and powders suitable for reconstitution with suitable solutions can be acceptable as solid dosage forms. For inhalation, solutions, sprays, dry powders and aerosols can be acceptable dosage forms. For local (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, emulsifiable pastes, lotions, gels, solutions and patches can be acceptable dosage forms.

[0070] Example 3

[0071] On the basis of Example 1, the above composition for regulating intestinal flora has a good effect.

[0072] This experiment provides a composition for regulating intestinal flora as described above for use in preparing drugs for treating diarrheal diseases, anti-inflammatory, anti-oxidation, regulating intestinal flora and enhancing intestinal immunity.

[0073] As a further preferred embodiment, the diarrheal diseases include diarrhea in lambs and calves, diarrhea and dysentery in laying hens and waterfowl, ulcerative colitis, chronic enteritis, diarrhea and intestinal inflammation caused by irritable bowel syndrome.

[0074] As a further preferred embodiment, the regulating intestinal flora includes regulating the intestinal flora structure and enriching beneficial bacteria; the enhancing intestinal immunity includes promoting the differentiation of Treg cells, inhibiting the differentiation of Th17 cells, and regulating the balance of Treg / Th17.

[0075] Tryptophan is an essential amino acid for mammals. It has the function of changing the composition and metabolism of intestinal flora and improving the intestinal mucosal barrier, and plays an important role in maintaining intestinal homeostasis. It is metabolized into kynurenine, 5-HT and its derivatives in host cells, and participates in various physiological processes such as neuronal function, body immunity and intestinal homeostasis. Intestinal microorganisms can recycle free tryptophan that is not decomposed by the host for protein synthesis, and produce many microbial metabolites such as aromatic hydrocarbon compounds and indole compounds. It is an important component of the substance for mutual dialogue between intestinal microbiota and host. According to the different ways of decomposition and metabolism in the body, tryptophan metabolites can be divided into endogenous tryptophan metabolites and microbial tryptophan metabolites, mainly including kynurenine, kynurenic acid, serotonin, indole, indole-3-acetic acid, indolepropionic acid, 5-methylindole and tryptamine, which play an important regulatory role in feeding regulation, immune response, anti-stress and anti-oxidation, intestinal barrier homeostasis, especially immune barrier. In addition, changes in tryptophan metabolism can in turn affect the composition and function of intestinal microorganisms and affect host physiology. When the body is in a disease state, the tryptophan metabolic pathway will undergo significant changes, participating in the occurrence or development of the disease.

[0076] The present invention significantly improves the abundance of lactobacilli and increases the content of tryptophan metabolites, and can be used for anti-inflammation, anti-oxidation, regulating intestinal flora and enhancing intestinal immunity.

[0077] A drug for regulating intestinal flora, comprising the above-mentioned composition or the above-prepared composition.

[0078] Example 4

[0079] According to the content of this application, the composition of Example 1 was prepared, and the specific description is as follows:

[0080] Test Example 1

[0081] Composition 1 for regulating intestinal flora: its raw material ratio: 150g of fried Atractylodes lancea, 150g of dried tangerine peel, 60g of roasted licorice, 150g of Magnolia officinalis (processed with ginger), 100g of Poria cocos, 150g of Poria cocos (peeled), 150g of fried Atractylodes macrocephala, 100g of Alisma orientalis, 50g of cinnamon bark, 100g of dried ginger, and 100g of black plum.

[0082] The preparation method thereof comprises the following specific steps:

[0083] Step 1): according to the weight ratio of the raw material components in composition 1, take the raw materials of stir-fried atractylodes, dried orange peel, roasted licorice, Magnolia officinalis (made with ginger), Polyporus, Poria (peeled), stir-fried Atractylodes macrocephala, Rhizoma Alismatis, cinnamon bark, dried ginger, and ebony, and coarsely grind them;

[0084] Step 2): take half of the components in the raw material of composition 1 in step 1), add water and boil three times, add 10 times the amount of water for the first boiling, soak for 45 minutes, boil for 2 hours, and filter; add 8 times the amount of water for the second boiling, boil for 1.5 hours, and filter; add 6 times the amount of water for the third boiling, boil for 1 hour, and filter; combine the decoctions, concentrate and dry to obtain a dry extract, so that the relative density of the dry extract is 1.00 (g / ml), then sterilize the dry extract by ultraviolet radiation at 121°C for 30 minutes, and finally crush the dry extract to obtain a fine extract powder.

[0085] Step 3): Take the remaining half of each component in the raw material of composition 1 in step 1), crush it, sterilize it by ultraviolet radiation, and mix it with the extract powder prepared in step 2) to obtain composition 1. The composition 1 meets the following indicators: atractylodes lactone I ≥ 0.008 mg / g, atractylodes lactone II ≥ 0.036 mg / g, atractylodes lactone III ≥ 0.102 mg / g, magnolol ≥ 0.614 mg / g, honokiol ≥ 0.432 mg / g, and 23-acetyl alismatol B ≥ 0.034 mg / g.

[0086] Test Example 2

[0087] Composition 2 for regulating intestinal flora, raw material ratio: fried Atractylodes 100g, dried tangerine peel 100g, roasted licorice 50g, Magnolia officinalis (processed with ginger) 100g, Poria 50g, Poria (peeled) 100g, fried Atractylodes 100g, Alisma orientalis 50g, cinnamon bark, dried ginger 50g, and black plum 50g.

[0088] The preparation method is similar to that of Test Example 1, except that in step 3), the relative density of the dry extract is 1.10 (g / ml), and the other conditions are the same as those of Test Example 1. The composition 2 meets the following indicators: magnolol ≥ 0.614 mg / g, and honokiol ≥ 0.432 mg / g.

[0089] Test Example 3

[0090] Composition 3 for regulating intestinal flora, raw material ratio: fried Atractylodes 200g, dried tangerine peel 200g, roasted licorice 100g, Magnolia officinalis (processed with ginger) 200g, Poria 150g, Poria (peeled) 200g, fried Atractylodes 200g, Alisma Orientalis 150g, cinnamon bark 60g, dried ginger 150g, and black plum 150g.

[0091] The preparation method is similar to that of Test Example 1, wherein the composition 3 meets the following indicators: 23-acetyl alismatol B ≥ 0.034 mg / g.

[0092] Comparative Example 1

[0093] The comparative composition 1 for regulating intestinal flora, the raw material ratio: 150g of fried Atractylodes lancea, 150g of dried tangerine peel, 60g of roasted licorice, 150g of Magnolia officinalis (processed with ginger), 100g of Poria cocos, 150g of Poria cocos (peeled), 150g of fried Atractylodes macrocephala, 100g of Alisma orientalis, and 60g of cinnamon.

[0094] The preparation method is the same as steps 1) to 3) of Experimental Example 1, except that the raw materials lack the components of dried ginger and black plum.

[0095] Comparative Example 2

[0096] Comparative composition 2 for regulating intestinal flora, raw material ratio: fried Atractylodes 150g, dried tangerine peel 150g, roasted licorice 60g, Magnolia officinalis (processed with ginger) 150g, Poria 100g, Poria (peeled) 150g, fried Atractylodes 150g, Alisma orientalis 100g, cinnamon 60g, ginger 100g, and jujube 100g.

[0097] The preparation method is the same as steps 1) to 3) of Experimental Example 1, except that the raw material components of this comparative example include ginger and jujube.

[0098] Comparative Example 3

[0099] The comparative composition 3 for regulating intestinal flora is made of Huoxiang Zhengqi Tablets, which are purchased from Tongrentang in Haidian District, Beijing. It has some of the same raw material components as the composition of the present application, namely, Atractylodes lancea, dried orange peel, Magnolia officinalis (prepared with ginger), Poria cocos, etc.

[0100] Crush Huoxiang Zhengqi tablets and dissolve them in distilled water until no obvious particles remain. Calculate the human clinical equivalent dose and convert it into a rat dose for administration.

[0101] Comparative Example 4

[0102] The comparative composition 4 for regulating intestinal flora, the raw material components are arranged in proportions by weight (the same as the raw materials of composition 1 in Test Example 1): 150g of stir-fried Atractylodes lancea, 150g of dried tangerine peel, 60g of roasted licorice, 150g of Magnolia officinalis (processed with ginger), 100g of Poria cocos, 150g of Poria cocos (peeled), 150g of stir-fried Atractylodes macrocephala, 100g of Alisma orientalis, 50g of cinnamon, 100g of dried ginger, and 100g of black plum.

[0103] The preparation method is different from that of Test Example 1, and the specific steps are as follows:

[0104] Step 1): according to the weight ratio of the raw material components in composition 4, take the raw materials of stir-fried atractylodes, dried tangerine peel, roasted licorice, Magnolia officinalis (made with ginger), Polyporus, Poria (peeled), stir-fried Atractylodes macrocephala, Rhizoma Alismatis, cinnamon bark, dried ginger, and ebony, and coarsely grind them;

[0105] Step 2): Take all the raw materials of composition 4 in step 1), add water and boil three times, add 10 times the amount of water for the first boiling, soak for 45 minutes, boil for 2 hours, and filter; add 8 times the amount of water for the second boiling, boil for 1.5 hours, and filter; add 6 times the amount of water for the third boiling, boil for 1 hour, and filter; combine the decoctions, concentrate and dry to obtain a dry extract, so that the relative density of the dry extract is 1.00 (g / ml), then sterilize the dry extract by ultraviolet radiation at 121°C for 30 minutes, and finally crush the dry extract to obtain extract fine powder to obtain comparative composition 4.

[0106] Example 5

[0107] Animals: 30 SPF Wistar rats, half male and half female, 8 weeks old, weighing 180±20g, purchased from Beijing Sibeifu Biological Co., Ltd., laboratory animal qualification certificate number: SCKX (Beijing) 2019-0010. The room temperature was adjusted at 20-25℃, the humidity was 45%±5%, 12h light and 12h dark environment, standard diet, free water supply. The experiment was carried out after 7 days of adaptation.

[0108] Reagents: sodium pentobarbital, product number: P3761, Sigma, USA; anhydrous ethanol, analytical grade, batch number 20221010, Tianjin Beilian Fine Chemicals Development Co., Ltd.; formaldehyde, analytical grade, Tianjin Damao Chemical Reagent Factory; rat IL-10, IL-6, TNF-α, IFN-γ, D-AO, D-LA ELISA kits, Shanghai ELISA Co., Ltd.; SOD, T-AOC, MDA, CAT kits, Nanjing Jiancheng Biological Co., Ltd.

[0109] Instruments: electronic balance, Shanghai Precision Scientific Instrument Co., Ltd.; AXTG16G desktop high-speed centrifuge, Shanghai Zhaodi Biotechnology Co., Ltd.; QL-901 vortex instrument, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; desktop low-speed high-performance refrigerated centrifuge, Changsha Xiangyi Centrifuge Instrument Co., Ltd.; constant temperature water bath, Jintan Hengfeng Instrument Factory; KQ-300DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; KDM-1000 temperature-adjustable electric heating mantle, Jiangsu Jerel Electric Co., Ltd.; portable pressure steam sterilizer, Guangzhou Kangmai Medical Equipment Co., Ltd.; Leica slicer, Germany Leica; 0.054 DP-71 microscope system, Olympus, Japan; Leica slicer, Leica, Germany; 0.054 DP-71 microscope system, Olympus, Japan; LRH-70 biochemical incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.; iMark microplate reader, BIO-RAD, USA; Mindray BC5300Vet five-category animal blood cell analyzer, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.; LG-R-80 blood viscosity meter, Beijing Zhongqinshidi Scientific Instrument Co., Ltd.; Erba XL-640 fully automatic biochemical analyzer, Erba, Germany.

[0110] 1. Test methods

[0111] Animal Grouping and Model Establishment Rats were weighed and randomly divided into 9 groups, with 8 rats in each group, namely blank control group, model control group, composition group 1, composition group 2, composition group 3, comparative composition group 1, comparative composition group 2, comparative composition group 3, and comparative composition group 4. The rats in the model control group, composition group 1, composition group 2, composition group 3, comparative composition group 1, comparative composition group 2, comparative composition group 3, and comparative composition group 4 were given sufficient high-fat feed once a day, and were free to drink water for 7 consecutive days. From the 8th day to the 21st day, they swam in ice water every day, were restrained for 4 hours, and were gavaged with 3g / kg of senna leaves. Starting from the 22nd day, the modeling of the model control group was completed and distilled water was gavaged. The gavage drugs and dosages of other groups are shown in Table 1. After the experiment, rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30mg / kg), and blood was collected from the abdominal aorta. 1ml was collected in EDTA-K2 anticoagulant tubes, 1ml was collected in heparin sodium anticoagulant tubes, and the rest of the blood was collected in ordinary vacuum blood collection tubes (standing in a 37°C incubator for 2h, centrifuged at 4000rpm to separate serum, and stored at -20°C for use). The cecal contents and colon feces were collected and quickly placed in liquid nitrogen and transferred to -80 for storage. The 2 cm ileum and 2 cm colon starting from the ileocecal region were quickly cut and immersed in a 4% paraformaldehyde solution at room temperature for 15 days.

[0112] Table 1 Drugs and dosages given orally to rats in each group

[0113] Group Name of drug administered by gavage Dosage Blank control group - - Model control group Distilled water - Composition 1 set Composition 1 2.16 g / kg per day for 7 days Composition 2 sets Composition 2 2.16 g / kg per day for 7 days Composition 3 groups Composition 3 2.16 g / kg per day for 7 days Comparative composition 1 group Comparative composition 1 2.16 g / kg per day for 7 consecutive days Comparative composition group 2 Comparative composition 2 2.16 g / kg per day for 7 days Comparative composition 3 (Huoxiang Zhengqi Tablets) group Huoxiang Zhengqi Tablets 4.2 g / kg per day for 7 days Comparative composition 4 groups Comparative composition 4 2.16 g / kg per day for 7 days

[0114] 2. Testing of various performance indicators of chronic stress model

[0115] 2.1 General condition observation The general condition of rats was observed every day, including mental state, activity status, growth and posture, coat, appetite, feces and urine.

[0116] Before the experiment, all rats were in good spirits, responsive, lively and active, with soft, white and shiny fur, normal appetite, hard black stools, and light yellow urine. After the experiment, there were no obvious abnormalities in the blank control group, and their weight gradually increased. The rats in the model control group had loose stools and anal stickiness, especially after forced swimming and restraint stress and gavage of senna leaves. The rats showed depression, fear of cold, rough and messy fur, loss of appetite, and watery and shapeless loose stools. Composition 1 group mainly alleviated the diarrhea of ​​the rats in the model control group, with a significantly reduced water content in the feces, shiny and soft fur, and the feces of each group were as follows: Figure 1 As shown (the blank control group is control, the model control group is model, the comparative composition group 1 is ZHW1, the comparative composition group 2 is ZHW2, the comparative composition group 3 is HXZQ, and the composition group 1 is ZY1).

[0117] Weight changes such as Figure 2 As shown in the figure (the blank control group is CON, the model control group is MOD, and the composition group 1 is ZY1), during the modeling period, the body weight of the rats in the blank control group gradually increased. In the high-fat diet stage from day 1 to day 7, the body weight of the model control group and the composition group gradually increased overall; the body weight gradually decreased during the chronic stress stage; the body weight gradually increased after drug administration, and the increasing trend of the composition group was greater than that of the model control group.

[0118] 2.2 Diarrhea Index

[0119] Diarrhea index determination Diarrhea index (DI) = loose stool rate × loose stool grade, loose stool rate = number of loose stools per rat / total number of stools. The loose stool grade is divided into four levels according to the size of the stain on the filter paper: grade 1 is a stain diameter of <1 cm, grade 2 is a stain diameter of 1-1.9 cm, grade 3 is a stain diameter of <2-3 cm, and grade 4 is a stain diameter of >3 cm, as shown in Table 2.

[0120] Table 2 Diarrhea index of chronic stress rats ( n=8)

[0121]

[0122]

[0123] Note: Compared with the blank control group, the model control group had *P<0.5, **P<0.01, ***P<0.01; compared with the combination group, #P<0.05, ##P<0.01.

[0124] As shown in Table 2, during the modeling period from day 1 to day 21, the diarrhea indexes of the model control group were significantly greater than those of the blank control group (P<0.05); the diarrhea indexes of the composition group were significantly greater than those of the blank control group (P<0.05) when compared with the blank control group from day 1 to day 21; there was no statistical difference between the composition group and the blank control group from day 22 to day 28 after administration (P>0.05); the diarrhea indexes of the composition group were significantly lower than those of the model control group when compared with the model control group from day 22 to 28 (P<0.05).

[0125] 2.3 Routine blood test indicators

[0126] Blood routine test: EDTA-K2 anticoagulated blood was used for blood routine test using a fully automatic blood analyzer.

[0127] Table 3 Blood routine indexes of rats in each group ( n=8)

[0128]

[0129]

[0130] Note: Compared with the blank control group, the model control group had *P<0.5, **P<0.01, ***P<0.001; compared with the combination group, the combination group had #P<0.05, ##P<0.01, ###P<0.001.

[0131] Table 3 shows that the white blood cell count, neutrophil count, red blood cell count, hematocrit and hemoglobin concentration in the routine blood test were significantly higher in the model control group than in the blank control group (P<0.05), while those in the combination group were significantly lower than those in the model control group (P<0.05), indicating that the animals in the model control group had inflammatory and cold reactions, which were significantly alleviated after treatment in the combination group.

[0132] 2.4 Blood viscosity index

[0133] Table 4 Blood routine indexes of rats in each group ( n=8)

[0134] Group <![CDATA[200s -1 (mpa·S)]]> <![CDATA[30s -1 (mpa·S)]]> <![CDATA[5s -1 (mpa·S)]]> <![CDATA[1s -1 (mpa·S)]]> Blank control group 3.55±0.52 4.67±0.35 9.63±1.82 10.58±1.37 Model control group <![CDATA[6.19±0.53 ** ]]> <![CDATA[8.15±0.68 ** ]]> <![CDATA[14.35±2.35 ** ]]> <![CDATA[15.35±0.58 ** ]]> Composition 1 set <![CDATA[3.89±0.89 ### ]]> <![CDATA[5.21±1.73 ## ]]> <![CDATA[8.78±3.18 ## ]]> <![CDATA[9.45±1.98 ## ]]> Composition 2 sets <![CDATA[5.25±1.67 ## ]]> 7.55±1.67 12.84±2.88 13.01±0.65 Composition 3 groups <![CDATA[4.23±1.45 ## ]]> <![CDATA[6.89±0.98 # ]]> <![CDATA[11.47±3.67 # ]]> <![CDATA[12.45±1.45 # ]]>

[0135] Note: Compared with the blank control group, the model control group, **P<0.01; compared with the combination group, # P<0.05,## P<0.01, ### P<0.001.

[0136] Table 4 shows that 200s -1 , 30s -1 、5s -1 , 1s -1 At several shear rates, the blood viscosity of the model control group was significantly higher than that of the blank control group (P<0.01); at the three shear rates of 200s-1, 30s-1, and 1s-1, the blood viscosity of the combination group was extremely significantly lower than that of the model control group (P<0.01), and at the shear rate of 5s1, the blood viscosity of the combination group was significantly lower than that of the model control group (P<0.05). This indicates that the blood viscosity of the rats in the model control group increased, and the combination group has a significant effect in alleviating the blood viscosity of chronically stressed animals.

[0137] 2.5 Blood biochemical indicators

[0138] Blood biochemical test The collected serum was tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), total cholesterol (TC), and triglyceride (TG) using an automatic biochemical analyzer.

[0139] Table 5 Blood biochemical indexes of rats in each group ( n=8)

[0140] Group ALT(U / L) AST(U / L) GGT(U / L) TG (mmol / L) TCH(mmol / L) Blank control group 42.89±5.43 123.01±15.37 1.58±0.38 0.48±0.19 1.39±0.27 Model control group <![CDATA[58.92±8.93 ** ]]> <![CDATA[177±31.79 ** ]]> <![CDATA[2.79±0.25 * ]]> <![CDATA[0.79±0.21 ** ]]> <![CDATA[1.80±0.17 * <!-- 10 -->]]> Composition 1 set <![CDATA[41.91±7.61 ## ]]> <![CDATA[144.10±12.39 # ]]> <![CDATA[1.58±0.12 # ]]> <![CDATA[0.49±0.11 ## ]]> <![CDATA[1.41±0.42 # ]]> Composition 2 sets <![CDATA[50.34±8.22 # ]]> <![CDATA[156.74±8.46 # ]]> <![CDATA[1.89±0.74 # ]]> <![CDATA[0.59±0.15 # ]]> 1.78±0.24 Composition 3 groups 51.09+3.44 168.17±15.67 <![CDATA[2.43±0.69 # ]]> 0.62±0.25 <![CDATA[1.65±0.55 # ]]>

[0141] Note: Compared with the blank control group, the model control group had *P<0.05, **P<0.01; compared with the model control group, the combination group had # P<0.05, ## P<0.01.

[0142] Table 5 shows that the ALT and AST in the model control group were significantly higher than those in the blank control group (P<0.01), and the combination group was significantly lower than those in the model control group (P<0.05). This indicates that the blood transaminase activity of the chronic stress model animals is increased, and the combination can reduce the transaminase activity. The GGT in the model control group was significantly higher than that in the blank control group (P<0.05), and the combination group was significantly lower than that in the model control group (P<0.05). This indicates that the blood GGT activity of the chronic stress model animals is increased, and the combination can reduce its activity. The TG in the model control group was significantly higher than that in the blank control group (P<0.01), and the combination group was significantly lower than that in the model control group (P<0.01); the TCH in the model control group was significantly higher than that in the blank control group (P<0.05), and the combination group was significantly lower than that in the model control group (P<0.05). This indicates that the blood lipids of the chronic stress model animals are increased, and the combination can reduce their blood lipids.

[0143] 2.6 Serum cytokine indicators

[0144] Serum cytokine detection Serum stored at −20°C was taken and thawed at room temperature, and IL-10, IL-1β, TNF-α, DAO, and D-LA were determined according to the instructions of the ELISA kit.

[0145] Table 6 Serum cytokines of rats in each group ( n=10)

[0146]

[0147]

[0148] Note: Compared with the blank control group, the model control group, **P<0.01; compared with the combination group, # P<0.05, ## P<0.01, ### P<0.01.

[0149] Table 6 shows that the IL-1β in the model control group was significantly higher than that in the blank control group (P<0.01), and the combination group was significantly lower than that in the model control group (P<0.01); the IL-10 in the model control group was significantly lower than that in the blank control group (P<0.01), and the combination group was significantly higher than that in the model control group (P<0.05); the TNF-α in the model control group was significantly higher than that in the blank control group (P<0.01), and the combination group was significantly lower than that in the model control group (P<0.05). This indicates that the inflammatory response of the chronic stress model animals increased, and the inflammatory response decreased after the combination treatment. The DAO in the model control group was significantly higher than that in the blank control group (P<0.01), and the combination group was significantly lower than that in the model control group (P<0.01); the D-LA in the model control group was significantly higher than that in the blank control group (P<0.01), and the combination group was significantly lower than that in the model control group (P<0.05), indicating that the intestinal barrier of the chronic stress model animals was damaged and recovered after the combination treatment.

[0150] 3. Analysis of intestinal flora diversity

[0151] 16S rRNA gene amplicon sequencing was used to analyze the microbial community composition. Fresh fecal samples from five to six rats in each group were collected for high-throughput sequencing. Total microbial DNA was isolated from fecal samples using a bacterial DNA isolation kit (Omega Bio-tek, Norcross, GA, USA). Universal primers (338F: 5'-ACTCCTACGGGAGGCAGCAG-3'; 806R: 5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3-V4 region of microbial DNA. The purified amplicons were sequenced on the Illumina MiSeq PE300 platform to assess their abundance and diversity. The obtained sequence data were selected for statistical analysis and visualization. All sequence data were optimized and analyzed on the bioinformatics analysis cloud platform (https: / / cloud.majorbio.com).

[0152] Rarefaction curve sequencing depth was used for analysis, and mothur[8] software (http: / / www.mothur.org / wiki / Calculators) was used to calculate the alpha diversity index Chao 1, Shannon index, etc., and the Wilcoxocontrol rank sum test was used to analyze the inter-group differences in alpha diversity.

[0153] Data statistics and analysis Data were expressed as mean ± standard deviation, the software used was SPSS24.0, and the mean statistical analysis was performed using one-way analysis of variance. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant.

[0154] 3.1 OTU data analysis

[0155] like Figure 3 As shown in A and 3B (the blank control group is control, the model control group is model, the comparative composition group 1 is ZHW1, the comparative composition group 2 is ZHW2, the comparative composition group 3 is HXZQ, the comparative composition 4 is ZHW4, and the composition group 1 is ZY1), when the sequencing depth is small, the OUT data changes dramatically, and the OUT data increases significantly with the increase of sequencing depth. When the depth reaches 30,000 reads, the dilution curve has reached saturation, indicating that the sequencing concentration has reached the depth of sequencing amount. The current sequencing depth can only increase species abundance but not diversity.

[0156] like Figure 4As shown (the blank control group is Con, the model control group is Mod, and the composition 1 group is ZY1), the number of OUTs reflecting Alpha diversity at the 97% similarity level was calculated according to the Bayesian algorithm. The results showed that the number of OUTs in the model control group was significantly lower than that in the blank control group, and the number of OUTs in the composition 1 group (ZY1) was significantly higher than that in the model control group, indicating that the stress model caused a significant decrease in the diversity of intestinal flora, and the administration of the composition significantly increased the diversity of the flora, and the intestinal flora of the rats in the composition administration group and the normal group were similar at the OUT level.

[0157] 3.2 PCA results analysis

[0158] like Figure 5 As shown in the figure (the blank control group is control, the model control group is model, the comparative composition 1 group is ZHW1, the comparative composition 2 group is ZHW2, the comparative composition 3 group is HXZQ, the comparative composition 4 is ZHW4, and the composition 1 group is ZY1), PCA analysis of the seven groups of data revealed obvious clustering, among which the blank control group and the combination 1 group had similar cluster distances and had more overlaps, while the model control group was far away from the blank control group and the combination 1 group, and the comparative composition group was also far away from the blank control group and the combination 1 group, indicating that after the intervention of the composition 1, the microbiota of the rats in the combination 1 group and the blank control group were more similar.

[0159] 3.3 Analysis of differences in bacterial community composition

[0160] like Figure 6 As shown in A, the dominant bacterial groups at the phylum level are:

[0161] Firmicutes: blank control group (control) 86.20%, model control group (model) 73.56%, comparative composition group 1 (ZHW1) 85.91%, comparative composition group 2 (ZHW2) 83.56%, comparative composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 85.91.22%, comparative composition 4 (ZHW4) 87.60%, composition group 1 (ZY1) 92.25%;

[0162] Actinomycetes: blank control group (control) 12.81%, model control group (model) 25.63%, comparative composition 1 group (ZHW1) 13.39%, comparative composition 2 group (ZHW2) 16%, comparative composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 12.68%, comparative composition 4 (ZHW4) 11.62%, composition 1 group (ZY1) 7.38%;

[0163] The proportion of Firmicutes in the model control group decreased significantly, while the proportion of Firmicutes in the combination groups recovered after administration, among which the Firmicutes in combination group 1 (ZY1) increased more significantly after administration; in addition, the proportion of Actinobacteria in the model control group increased significantly, while the proportion of Actinobacteria recovered to the level that was not significantly different from that in the blank group after administration of the control combination, and the proportion of Actinobacteria decreased significantly after administration of combination group 1 (ZY1).

[0164] like Figure 6 As shown in B, the dominant bacterial communities at the genus level are:

[0165] Lactobacillus: blank control group (control) 22.02%, model control group (model) 3.90%, comparison composition group 1 (ZHW1) 10.44%, comparison composition group 2 (ZHW2) 12.32%, comparison composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 11.64%, comparison composition 4 (ZHW4) 18.02%, combination group 1 (ZY1) 25.22%.

[0166] Kurthia: blank control group (control) 0%, model control group (model) 39.79%, comparison composition group 1 (ZHW1) 35.73%, comparison composition group 2 (ZHW2) 0%, comparison composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 0%, comparison composition 4 (ZHW4) 0.02%, combination group 1 (ZY1) 0.02%.

[0167] Romboutsia: blank control group (control) 23.07%, model control group (model) 4.33%, comparative composition group 1 (ZHW1) 3.79%, comparative composition group 2 (ZHW2) 21.21%, comparative composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 13.05%, comparative composition 4 (ZHW4) 13.23%, composition group 1 (ZY1) 18.94%.

[0168] Blautia: blank control group (control) 4.29%, model control group (model) 3.73%, comparative composition group 1 (ZHW1) 3.85%, comparative composition group 2 (ZHW2) 12.96%, comparative composition 3 (Huoxiang Zhengqi Tablets, HXZQ) 16.25%, comparative composition 4 (ZHW4) 10.82%, composition group 1 (ZY1) 7.56%.

[0169] Lactobacillus is a beneficial bacteria in the intestine. The number of Lactobacillus in the model control group rats was significantly reduced. The composition and the comparison composition reversed the intestinal flora imbalance after administration. Among them, composition 1 (ZY1) significantly increased the abundance of Lactobacillus, changed the composition of the intestinal flora of rats, affected the balance of the intestinal flora, and made the intestinal flora closer to that of the blank group.

[0170] 3.4 Differential analysis of tryptophan metabolites

[0171] like Figure 7 As shown (blank control group is control, model control group is model, comparative composition 1 group is ZHW1, comparative composition 2 group is ZHW2, comparative composition 4, comparative composition 3 group is HXZQ, composition 1 group is ZY1, *p<0.05, **p<0.01, ***p<0.001, compared with blank group; #p<0.05,

[0172] ##p<0.01,###p<0.001, compared with the model control group; $ p<0.05, $$ p<0.01, $$$ p<0.001, compared with ZHW3; ns: no difference in comparison), after analyzing the results, it was found that the tryptophan metabolites Indoleacrylic acid, L-Tryptophan, Indole-3-carboxylic acid, 3-Indoleglyoxyliac acid, Indole-3-acetic acid, Lactic acid, among which the composition 1 group was significantly higher than the model control group p<0.001, and at the same time, it was significantly higher than the control composition group 1, group 2, group 3, group 4, p<0.001. This shows that composition 1 can significantly increase the content of tryptophan metabolites.

[0173] 3.5 Intestinal immune Treg / Th17 balance

[0174] like Figure 8 As shown (Note: *p<0.05, **p<0.01, ***p<0.001, compared with the blank group; #p<0.05, ##p<0.01, ###p<0.001, compared with the model control group; $ p<0.05, $$ p<0.01, $$$p<0.001, compared with ZHW3; ns: no difference. ), the mesenteric lymph nodes of the model rats inhibited the differentiation of Treg cells and promoted the differentiation of Th17 cells. After administration of the composition, the differentiation level of Treg cells was significantly increased, and the differentiation level of Th17 cells was significantly inhibited. Among them, composition 1 (ZY1) significantly increased the differentiation level of Treg cells and significantly inhibited the differentiation level of Th17 cells, and formed significant differences with the comparison composition 1 (ZHW1), the comparison composition 2 (ZHW2), the comparison composition 3 (HXZQ) and the comparison composition 4 (ZHW4). It shows that the administration of composition 1 (ZY1) can significantly reverse the imbalance of intestinal immune Treg / Th17 caused by the model, and adjust the balance of intestinal immunity by adjusting the balance of Treg / Th17, and finally form an intestinal immune balance and there is no significant difference with the blank control group.

[0175] The present invention alleviates intestinal inflammation by regulating the levels of inflammatory factors and oxidative stress; the present invention can also regulate intestinal nonspecific immunity and intestinal flora, thereby increasing the diversity and richness of intestinal flora and restoring the abundance and proportion of intestinal flora to normal proportions, thereby improving and treating diarrheal diseases.

[0176] 4. Analysis of intestinal barrier protection results

[0177] After the experiment, the rats' colons were stained with H&E. Fig. 9 The results showed that the intestinal epithelial cells of the blank control group (Control) rats were neatly arranged, the goblet cells were normally distributed, the crypt structure was normal, the intermediate glands were clear, there was no inflammatory cell infiltration, no congestion, and no edema; the colon epithelial shedding of the model group (Model) rats was incomplete, there was inflammatory infiltration and edema, the structure of the colon glands was severely damaged, the epithelial cells were degenerated and necrotic, and the goblet cells were reduced; the colon epithelial cell structure of the rats in the combination 1 (ZY1) group was complete, neatly arranged, there was no inflammatory cell infiltration, no congestion, no edema, and the colon was closest to the blank control group. The colons of rats in other groups (ZHW1, ZHW2, HXZQ, ZHW4) all had varying degrees of epithelial cell shedding, inflammatory cell infiltration, and severe structural damage to the colon glands.

[0178] Transmission electron microscopy results of the colon Fig.10The results showed that the blank control group (Control) had normal colon structure, intact villi, and intact tight junctions between cells; compared with the blank control group, the model control group (Model) had disordered structure, missing villi, and broken tight junctions between cells; the colon epithelial cells of rats in the composition 1 (ZY1) group had normal colon structure, intact villi, and intact tight junctions between cells; the colons of rats in the other groups (ZHW1, ZHW2, HXZQ, and ZHW4) all had varying degrees of microvillus breakage and loss.

[0179] Example 6

[0180] Case: In September 2023, more than 120 3-7 day old calves suddenly fell ill in the calf house of a cattle farm in Beijing. The sick cows showed anorexia, weight loss, loose stools, and anal paste. Some calves had rough and messy hair, and some calves were dehydrated. Autopsy found that the intestines were bloated, and yellow pleural effusion or pericardial effusion appeared. According to the incidence, clinical symptoms and pathological changes, samples were collected for kit detection and diagnosed as bovine viral diarrhea. 30 calves with similar weight and typical symptoms were selected for clinical trials to observe the clinical efficacy of the composition of the present invention. The 30 calves were randomly divided into 3 groups, each with 10 calves. The first group (ZY1) was fed with Composition 1 prepared in Example 4, and Composition 1 was orally administered at 2g / kg; the second group (MOD) was used as the control group and no medication was given; the third group (JPZX) was used as the positive medication group and was given the commercially available anti-cold and dampness diarrhea Chinese medicine Jianpi Zhixie San, which was orally administered according to the instructions. All calves in the test group were raised separately in isolation pens. The calves were fed continuously for 7 days, and the clinical changes and cure rates were observed and recorded. At the same time, blood was collected from the jugular vein to separate serum, and the calf blood biochemical indicators were measured. At the same time, feces were collected to analyze the changes in intestinal flora.

[0181] Efficacy evaluation criteria: Cured: normal mental and physical strength, normal eating and drinking, normal bowel movement frequency, and normal appearance of feces; Ineffective: clinical symptoms have not improved or may even worsen.

[0182] Calculation method: mortality rate = number of dead animals / number of experimental animals × 100%, cure rate = number of cured animals / number of experimental animals × 100%, inefficiency rate = number of animals with symptoms at the end / number of experimental animals × 100%.

[0183] Test results

[0184] 1. Clinical efficacy test results show that the composition 1 prepared in Example 4 of the present invention has a good therapeutic effect on calf diarrhea, reaching a cure rate of 90%. Compared with the control group, the cure rate after administration of the composition is significantly improved. The results are shown in Table 7.

[0185] Table 7 Clinical changes and cure rates of experimental calves

[0186] Group Test calves (head) Cured calves Cure rate (%) 1 10 9 90 2 10 3 30 3 10 8 80

[0187] like Fig.11 As shown, blood biochemical results showed that administration significantly alleviated electrolyte imbalance and protected the liver and kidney damage of calves.

[0188] like Fig.12 and Fig.13 As shown, the administration of Composition 1 significantly increased the diversity of the flora, and the intestinal flora of calves in the Composition 1 administration group and the normal group were similar at the OUT level.

[0189] like Fig.14 As shown, it indicates that after the intervention of composition 1, there is a great similarity between the intestinal flora of the composition 1 group and the normal calves.

[0190] like Fig.15 As shown, composition 1 significantly increased the abundance of lactobacilli, changed the composition of the intestinal flora of calves, affected the balance of the intestinal flora, and made the intestinal flora closer to that of normal calves.

[0191] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A composition for regulating intestinal flora, characterized in that: Ingredients include: Stir-fried Atractylodes lancea, tangerine peel, roasted licorice, Magnolia officinalis (processed with ginger), Poria cocos, Poria cocos (peeled), stir-fried Atractylodes macrocephala, Alisma orientalis, cinnamon bark, dried ginger, and black plum.

2. The composition for regulating intestinal flora according to claim 1, characterized in that: The raw material components are matched by weight: 100-200 parts of stir-fried atractylodes, 100-200 parts of dried tangerine peel, 50-100 parts of roasted licorice, 100-200 parts of Magnolia officinalis (processed with ginger), 50-150 parts of Polyporus umbellatus, 100-200 parts of Poria cocos (peeled), 100-200 parts of stir-fried Atractylodes macrocephala, 50-150 parts of Alisma orientalis, 30-60 parts of cinnamon bark, 50-150 parts of dried ginger, and 50-150 parts of ebony.

3. The composition for regulating intestinal flora according to claim 1, characterized in that: The composition meets at least one of the following indicators: atractylodes lactone I≥0.008 mg / g, atractylodes lactone II≥0.036 mg / g, atractylodes lactone III≥0.102 mg / g, magnolol≥0.614 mg / g, honokiol≥0.432 mg / g, and 23-acetyl alismatol B≥0.034 mg / g.

4. A method for preparing a composition for regulating intestinal flora according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: Step 1): Take stir-fried Atractylodes lancea, dried orange peel, roasted Licorice root, Magnolia officinalis (processed with ginger), Polyporus umbellatus, Poria cocos (peeled), stir-fried Atractylodes macrocephala, Alisma orientalis, cinnamon bark, dried ginger, and black plum according to weight proportions, and coarsely grind them; Step 2): taking half of the amount of each component in the raw material of the composition in step 1), adding water to boil, filtering, combining the boiled liquid, concentrating and drying to obtain a dry extract, and crushing the dry extract to obtain a fine extract powder; Step 3): Take the remaining half of each component in the composition raw material in step 1), crush and sterilize, and mix with the extract powder prepared in step 2) to obtain the composition for regulating intestinal flora.

5. The preparation method according to claim 4, characterized in that: The decoction in step 2) includes three decoctions: adding 8-12 times the volume of water in the first decoction, soaking for 45-60 minutes, and then decoct for 1.5-2 hours; adding 6-10 times the volume of water in the second decoction, decoct for 1-1.5 hours; adding 4-6 times the volume of water in the third decoction, and decoct for 0.5-1 hour.

6. The preparation method according to claim 4, characterized in that: The dry extract in step 2) is sterilized at 121° C. for 20 to 30 minutes before being crushed, and the relative density of the dry extract is 1.0-1.1 (g / mL). The sterilization method includes ultraviolet radiation sterilization and high-pressure steam sterilization.

7. Use of the composition for regulating intestinal flora according to any one of claims 1 to 3 as a drug for treating diarrheal diseases, anti-inflammatory, anti-oxidation, regulating intestinal flora, enhancing intestinal immunity and protecting intestinal barrier.

8. The use according to claim 7, characterized in that: The diarrheal diseases include diarrhea in lambs and calves, diarrhea and dysentery in laying hens and waterfowl, ulcerative colitis, chronic enteritis, diarrhea caused by irritable bowel syndrome and intestinal inflammation.

9. The use according to claim 7, characterized in that: The regulating of intestinal flora includes regulating the structure of intestinal flora and enriching beneficial bacteria; the enhancing of intestinal immunity includes promoting the differentiation of Treg cells, inhibiting the differentiation of Th17 cells, and regulating the balance of Treg / Th17.

10. A drug for regulating intestinal flora, characterized in that: It comprises the composition according to any one of claims 1 to 4 or the composition prepared according to claim 5.

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