A composition for modulating gut flora, and a method of preparing and using the same

Abstract

The present application relates to a kind of composition for adjusting intestinal flora and its preparation method and application, and the raw materials of the composition include: fried atractylodes, dried tangerine or orange peel, fried liquorice, magnolia officinalis (ginger preparation), polyporus umbellatus, poria cocos (skin removal), fried atractylodes, alisma orientalis, cassia bark, dried ginger, dark plum.The present application produces the effect of anti-inflammatory, antioxidant, regulating intestinal flora, improving intestinal immunity and intestinal barrier protection effect etc. through the synergistic effect between raw materials.

Description

TECHNICAL FIELD

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

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

[0003] Lactobacillus belongs to beneficial bacteria. Lactobacillus 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 external pathogens or toxic substances. Therefore, the increase in the abundance of Lactobacillus is a direction worth studying, which means that the increase in Lactobacillus has a beneficial effect on physical health. SUMMARY

[0004] In view of the above problems, the present application provides a composition for regulating intestinal flora, enhancing immunity and resisting viruses. The composition can regulate intestinal flora, promote the growth of beneficial bacteria, improve the non-specific immunity of the host, and enhance the anti-diarrhea ability of the host, so as to achieve a safe and effective composition for treating gastrointestinal diseases and overcome the above defects.

[0005] The application point of the present application is to provide a composition for regulating intestinal flora, which comprises fried atractylodes, dried tangerine or orange peel, fried licorice, magnolia officinalis (prepared with ginger), polyporus umbellatus, poria cocos (peeled), fried atractylodes, alisma orientalis, cassia, dried ginger and prunus mume.

[0006] Further, the above raw material components are matched in weight parts as follows: fried atractylodes 100-200 parts, dried tangerine or orange peel 100-200 parts, fried licorice 50-100 parts, magnolia officinalis (prepared with ginger) 100-200 parts, polyporus umbellatus 50-150 parts, poria cocos (peeled) 100-200 parts, fried atractylodes 100-200 parts, alisma orientalis 50-150 parts, cassia 30-60 parts, dried ginger 50-150 parts, and prunus mume 50-150 parts.

[0007] Further, the composition meets at least one of the following indexes: atractyloides I ≥ 0.008 mg / g, atractyloides II ≥ 0.036 mg / g, atractyloides III ≥ 0.102 mg / g, magnolol ≥ 0.614 mg / g, and honokiol ≥ 0.432 mg / g, 23-acetyl alismol B ≥ 0.034 mg / g.

[0008] Another aspect of the present application provides a preparation method of the composition for regulating intestinal flora, and the specific steps include:

[0009] Step 1): The composition raw materials are taken by weight ratio, and the components are coarsely crushed.

[0010] Step 2): Half of each component in the composition raw materials in step 1) is taken, and water is added for decoction three times, and the decoction liquid is filtered and combined, and then concentrated and dried to obtain dry extract, and the dry extract is crushed to obtain extract fine powder.

[0011] Step 3): The remaining half of each component in the composition raw materials in step 1) is taken, and after being crushed and sterilized, the extract fine powder prepared in step 2) is mixed to obtain the composition for regulating intestinal flora.

[0012] Further, the water decoction in step 2) includes three decoctions: 8-12 times the volume of water is added in the first decoction, soaked for 45-60 min, and then decocted for 2 h; 6-10 times the volume of water is added in the second decoction, and decocted for 1.5 h; 4-6 times the volume of water is added in the third decoction, and decocted for 1 h.

[0013] Further, the dry extract in step 2) is sterilized at 121℃ for 20-30 min before being crushed, the relative density of the dry extract is 1.00-1.1 (g / mL), and the sterilization method includes ultraviolet radiation sterilization and high-pressure steam sterilization.

[0014] Another aspect of the present application provides the composition for regulating intestinal flora as an application in the preparation of drugs for treating diarrhea diseases, anti-inflammatory, antioxidant, regulating intestinal flora and enhancing intestinal immunity.

[0015] Further, the diarrhea diseases include lamb and calf diarrhea, egg chicken and waterfowl diarrhea and dysentery, ulcerative colitis, chronic enteritis, diarrhea and intestinal inflammation caused by irritable bowel syndrome.

[0016] Further, the regulation of intestinal flora includes regulating the structure of intestinal flora and enriching beneficial bacteria; and the enhancement 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 application also provides a medicine for regulating intestinal flora, which comprises the composition described in any one of the above paragraphs or the composition prepared by the preparation method.

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

[0019] The application produces the effects of anti-inflammation, anti-oxidation, regulating intestinal flora, improving intestinal immunity and intestinal barrier protection through the synergistic effect between raw materials, has simple formula, remarkable curative effect, can replace the use of antibiotics and avoid the side effects of chemical drugs on livestock and poultry animals, and can bring greater social and economic effects.

[0020] The application can relieve intestinal inflammation by regulating the levels of inflammatory factors and oxidative stress, can regulate intestinal non-specific immunity, can regulate intestinal flora, can increase the diversity and richness of intestinal flora, can increase the content of tryptophan metabolites, and can restore the abundance and proportion of intestinal flora to normal proportion, thereby improving and playing a role in treating diarrhea diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Perianal feces graph of rats;

[0022] Figure 2 Trend graph of body weight change of rats;

[0023] Figure 3 Rank-Abundance curve graph and Coverage curve graph of intestinal flora of rats;

[0024] Figure 4 Intestinal flora alpha diversity graph of rats, A.ace index; B.Chao index; C.Shannon index; D.Sobs index;

[0025] Figure 5 PCA result analysis graph of intestinal flora of rats;

[0026] Figure 6 Differential analysis graph of intestinal flora composition of rats, (A) analysis of flora at the door level; (B) analysis of flora at the genus level;

[0027] Figure 7 Intestinal flora tryptophan metabolite analysis graph of rats: (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 of rat mesenteric lymph nodes, (A) The proportion of Treg cells in the mesenteric lymph nodes; (B) The proportion of Th17 cells in the mesenteric lymph nodes;

[0029] Figure 9 H&E staining diagram of rat colon;

[0030] Figure 10 Transmission electron microscopy diagram of rat colon;

[0031] Figure 11 Blood biochemical indexes of calf after administration;

[0032] Figure 12 Rank-Abundance curve and Shannon curve of calf intestinal tract;

[0033] Figure 13 Alpha diversity analysis diagram of calf intestinal tract;

[0034] Figure 14 PCA result analysis diagram of calf intestinal tract;

[0035] Figure 15 Differential analysis diagram of calf intestinal flora composition, (A) Analysis of flora at the phylum level, (B) Analysis of flora at the genus level.

[0036] DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, 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 and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the terms used herein in the specification are only for the purpose of describing specific embodiments of the present application, and are not intended to limit the present application. The reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be described herein.

[0039] In order to further understand the present application, the present application will be further described in detail below in combination with the best embodiments.

[0040] Example 1

[0041] The present embodiment provides a composition for regulating intestinal flora, the raw materials of which include: fried atractylodes, dried tangerine or orange peel, fried licorice, magnolia officinalis (prepared with ginger), polyporus umbellatus, poria cocos (peel), fried atractylodes, alisma orientale, cinnamomum cassia, dried ginger, fructus mume.

[0042] The composition of the present application is prepared from rhizoma atractylodis, rhizoma atractylodis macrocephalae, cortex magnoliae officinalis as the monarch drug, which can warm the stomach and invigorate the spleen; poria cocos, poria cocos, and rhizoma alismatis as the ministerial drug, which can guide water downward and benefit urination to solidify the stool; pericarpium citri reticulatae, cortex cinnamomi, zingiber officinale, and prunus mume as the auxiliary drug, which can regulate qi and invigorate the spleen, warm yang and qi, warm and dispel cold, and astringe the intestines to stop diarrhea; and glycyrrhiza as the ministerial drug, which can harmonize the center and regulate all drugs; the whole prescription can play the functions of warming and unblocking stomach yang, and the functions of stopping diarrhea by penetrating dampness with pungent and light drugs. The present application can play the roles of anti-inflammation, anti-oxidation, regulation of intestinal flora, and balance of intestinal immunity in the treatment of patients with eczema, functional dyspepsia, ulcerative colitis, acute non-bacterial infectious diarrhea in children, antibiotic-associated diarrhea, and autumn diarrhea in children, and the clinical effect is remarkable.

[0043] In the present embodiment, by mixing a certain amount of stir-fried rhizoma atractylodis, pericarpium citri reticulatae, glycyrrhiza, cortex magnoliae officinalis (prepared with ginger), and a certain amount of poria cocos (with skin removed), poria cocos, stir-fried rhizoma atractylodis macrocephalae, and rhizoma alismatis, and especially adding cortex cinnamomi and zingiber officinale, prunus mume, the composition has excellent effects, which significantly increases the amount of tryptophan metabolism in the intestinal tract, and the immune balance significantly reverses the imbalance of intestinal immune Treg / Th17, regulates the balance of intestinal immune Treg / Th17, and finally forms the intestinal immune balance without significant difference from the normal condition.

[0044] As a further preferred embodiment, the raw material components are proportioned by weight parts as follows: fried atractylodes 100-200 parts, dried tangerine or orange peel 100-200 parts, fried licorice 50-100 parts, magnolia officinalis (processed with ginger) 100-200 parts, polyporus 50-150 parts, poria (peeled) 100-200 parts, fried atractylodes rhizome 100-200 parts, alisma 50-150 parts, cassia bark 30-60 parts, dried ginger 50-150 parts, fructus mume 50-150 parts. The raw material components can be further preferably proportioned by weight parts as follows: fried atractylodes 115-175 parts, dried tangerine or orange peel 115-175 parts, fried licorice 65-95 parts, magnolia officinalis (processed with ginger) 115-175 parts, polyporus 65-130 parts, poria (peeled) 115-175 parts, fried atractylodes rhizome 115-175 parts, alisma 65-130 parts, cassia bark 35-55 parts, dried ginger 60-140 parts, fructus mume 60-145 parts. For example, the raw materials and weight parts of the composition can be as follows: 1) fried atractylodes 150 parts, dried tangerine or orange peel 150 parts, fried licorice 60 parts, magnolia officinalis (processed with ginger) 150 parts, polyporus 100 parts, poria (peeled) 15 parts, fried atractylodes rhizome 150 parts, alisma 100 parts, cassia bark 50 parts, dried ginger 100 parts, fructus mume 100 parts. 2) fried atractylodes 100 parts, dried tangerine or orange peel 100 parts, fried licorice 50 parts, magnolia officinalis (processed with ginger) 100 parts, polyporus 50 parts, poria (peeled) 100 parts, fried atractylodes rhizome 100 parts, alisma 50 parts, cassia bark 30 parts, dried ginger 50 parts, fructus mume 50 parts. 3) fried atractylodes 200 parts, dried tangerine or orange peel 200 parts, fried licorice 100 parts, magnolia officinalis (processed with ginger) 200 parts, polyporus 150 parts, poria (peeled) 200 parts, fried atractylodes rhizome 200 parts, alisma 150 parts, cassia bark 60 parts, dried ginger 150 parts, fructus mume 150 parts.

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

[0046] Fried atractylodes: also known as bran-fried atractylodes, pungent, bitter, warm. Returns to the spleen, stomach, liver meridian. Dry wet spleen, dispel wind and cold, eyesight. For abdominal distension, diarrhea, edema, rheumatism, colds, night blindness. Preferably, fried atractylodes can be taken by weight parts, 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.

[0047] Dried tangerine or orange peel: functions to regulate qi, regulate the center, dry dampness, and resolve phlegm, treating chest and abdominal distension, loss of appetite, vomiting, hiccup, cough and excessive phlegm. Preferably, dried tangerine or orange peel can be taken by weight parts, 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] Radix Glycyrrhizae: sweet, neutral. It is related to heart, lung, spleen and stomach meridians, and has the effects of tonifying spleen and stomach, and benefiting qi and restoring pulse. It is used for weakness of spleen and stomach, lassitude, palpitation, and pulse knot. Preferably, Radix Glycyrrhizae can be taken in the proportion of, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 parts or any value between any two numerical values.

[0049] Magnolia officinalis: bitter, warm. It can warm the middle and lower qi, dry dampness, and resolve phlegm. It is used for treating chest and abdominal distention and pain, vomiting, and undigested food. It is mainly used for treating accumulated cold and qi, abdominal thunder, and eliminating water and blood stasis. It can also digest food, regulate stomach qi, and relieve pain. Preferably, Magnolia officinalis can be taken in the proportion 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 numerical values.

[0050] Polyporus umbellatus: sweet, mild, and neutral. It is related to kidney and bladder meridians. It has the effects of diuresis, enhancing immune function, anti-tumor, anti-liver damage, and anti-bacterial. Preferably, Polyporus umbellatus can be taken in the proportion 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 numerical values.

[0051] Poria cocos: sweet and mild, and neutral. It is related to heart, lung, spleen, and kidney meridians. It has the effects of diuresis and dampness, and tonifying spleen and heart. Preferably, Poria cocos can be taken in the proportion 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 numerical values.

[0052] Fried Atractylodes: it can tonify spleen and stomach, dry dampness, and benefit water, and it is used for treating spleen deficiency, fatigue, food intake, abdominal distension, diarrhea, water retention, and other symptoms. Preferably, Fried Atractylodes can be taken in the proportion 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 numerical values.

[0053] Alisma orientalis (S. et S.) Juz. : Sweet, cold. Meridians: Enter the kidney and bladder channels. Functions: Diuretic, diuresis, heat, edema, swelling, vomiting, diarrhea, phlegm, foot disease, gonorrhea, hematuria. Preferably, Alisma orientalis (S. et S.) Juz. can be taken by weight parts, 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 numerical values.

[0054] Cinnamomum cassia Presl: Pungent, sweet, hot. Nerves: Kidney, spleen, heart, liver. Has the effect of supplementing fire and assisting yang, guiding fire to the origin, dispelling cold and relieving pain, and warming the meridians. Preferably, Cinnamomum cassia Presl can be taken by weight parts, for example: 30, 35, 40, 45, 50, 55, 60 parts or any value between any two numerical values.

[0055] Zingiber officinale Roscoe: Pungent, hot. Nerves: Spleen, stomach, kidney, heart, lung. Has the effect of warming the middle and dispelling cold, returning yang and unblocking the vessels, and warming the lungs and resolving fluid. Preferably, Zingiber officinale Roscoe can be taken by weight parts, 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 numerical values.

[0056] Fructus Mume: Sour, astringent, neutral. Nerves: Liver, spleen, lung, large intestine. Has the effect of astringing the lungs, astringing the intestines, producing saliva, and anchoring ascarids. Preferably, Fructus Mume can be taken by weight parts, 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 numerical values.

[0057] The raw materials involved in the present application are all natural Chinese herbal medicines, all of which are Chinese medicinal herbs collected in the Chinese Pharmacopoeia 2020 Edition Part One, and meet the quality requirements of the Pharmacopoeia. It is easy to obtain materials, simple to prepare, wide in drug sources, and low in cost. The present application follows the principles of traditional Chinese medicine prescription, and various component drugs are taken by water decoction.

[0058] The above raw materials can be used as crude drugs, or as processed products of crude drugs.

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

[0060] The present application determines the content of effective components of the composition, and provides the quality standard of the effective components of the composition, which provides a theoretical basis for anti-inflammatory, antioxidant, regulation of intestinal flora and intestinal immunity.

[0061] Embodiment 2

[0062] The present embodiment provides a preparation method of the intestinal flora regulating composition according to Embodiment 1, and the specific steps are as follows:

[0063] Step 1): Take the components of fried atractylodes, dried tangerine or orange peel, prepared licorice, prepared magnolia officinalis, polyporus umbellatus, poria cocos (peel), fried atractylodes rhizome, alisma orientale, cinnamomum cassia, dried ginger and fructus mume by weight ratio, and coarsely crush them; Step 2): Take half of the amount of each component in the composition raw materials in step 1), and decoct them with water for three times, filter and combine the decocting liquid, and concentrate and dry to obtain dry extract, and crush the dry extract to obtain extract fine powder; Step 3): Take the remaining half of the amount of each component in the composition raw materials in step 1), crush and sterilize them, and mix them with the extract fine powder prepared in step 2) to obtain the intestinal flora regulating composition.

[0064] As a further preferred embodiment, step 2) further comprises adding 8-12 times the amount of water in the first decoction, soaking for 45-60 min, and decocting for 2 h; adding 6-10 times the amount of water in the second decoction, and decocting for 1.5 h; and adding 4-6 times the amount of water in the third decoction, and decocting for 1 h.

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

[0066] As a further preferred embodiment, it further comprises step 4), and step 4) is adding pharmaceutically acceptable adjuvants to prepare the traditional Chinese medicine preparation of the intestinal flora regulating composition by conventional traditional Chinese medicine preparation methods.

[0067] Preferably, the adjuvants comprise preservatives, disintegrants, wetting agents, diluents, binders, lubricants, flavoring agents, and at least one of the traditional Chinese medicine preparations including liquid preparations, semi-solid preparations, solid preparations and gas preparations.

[0068] The traditional Chinese medicine preparations include mixtures, oral liquids, wine preparations, tinctures, injections, decocted extract preparations, flow extract preparations, extract preparations, syrup preparations, pill preparations, powder preparations, granule preparations, tablet preparations, capsule preparations, aerosol preparations and spray preparations.

[0069] The route of administration can depend on the intended route of administration of the composition provided herein can be supplied in bulk or unit dosage form. 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 administration, emulsions and suspensions can be acceptable as liquid dosage forms, and powders suitable for reconstitution with a suitable solution can be acceptable as solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols can be acceptable dosage forms. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches can be acceptable dosage forms.

[0070] Example 3

[0071] On the basis of Example 1, the above-mentioned composition for regulating intestinal flora has good effects.

[0072] The present experiment provides a composition for regulating intestinal flora according to the above-mentioned composition for regulating intestinal flora as an application in the preparation of drugs for treating diarrhea diseases, anti-inflammatory, antioxidant, regulating intestinal flora, and enhancing intestinal immunity,

[0073] As a further preferred embodiment, the diarrhea diseases include lamb and calf diarrhea, egg chicken and waterfowl diarrhea and dysentery, ulcerative colitis, chronic enteritis, diarrhea caused by irritable bowel syndrome, and intestinal inflammation.

[0074] As a further preferred embodiment, the regulating intestinal flora includes regulating the structure of intestinal flora, 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, which plays an important role in maintaining intestinal homeostasis by changing the composition and metabolism of intestinal flora and improving the function of intestinal mucosal barrier. It is metabolized into kynurenine, 5-HT and its derivatives in host cells, and is involved in various physiological processes such as neuronal function, immune and intestinal homeostasis. Intestinal microorganisms can recycle undecomposed free tryptophan in the host to synthesize proteins and produce many microbial metabolites such as aromatic hydrocarbons and indole-containing compounds, which are important components of the dialogue between intestinal microbiota and the host. According to the different modes of in vivo decomposition and metabolism, tryptophan metabolites can be divided into endogenous tryptophan metabolites and microbial tryptophan metabolites, mainly including kynurenine, kynurenic acid, serotonin, indole, indole-3-acetic acid, indole propionic acid, 5-methyl indole and tryptamine, which play an important regulatory role in feeding regulation, immune response, anti-stress and antioxidant, intestinal barrier homeostasis, especially immune barrier. In addition, the change of tryptophan metabolism will affect the composition and function of intestinal microorganisms and the physiology of the host. When the body is in a diseased state, the metabolic pathway of tryptophan will change greatly, which is involved in the occurrence or development of diseases.

[0076] And the present application significantly improves the abundance of lactobacillus and increases the content of tryptophan metabolites, which can be used for anti-inflammatory, antioxidant, regulation of intestinal flora and enhancement of intestinal immunity.

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

[0078] Example 4

[0079] According to the content of the present application, the composition of example 1 is prepared, which is specifically described as follows:

[0080] Test example 1

[0081] Composition 1 for regulating intestinal flora: the raw material ratio is as follows: fried atractylodes 150g, dried tangerine or orange peel 150g, fried licorice 60g, magnolia officinalis (ginger processed) 150g, polyporus umbellatus 100g, poria cocos (peel) 150g, fried atractylodes 150g, alisma 100g, cassia bark 50g, dried ginger 100g, fructus mume 100g.

[0082] The preparation method is as follows:

[0083] Step 1): according to the weight ratio of the raw material components in composition 1, the raw materials fried atractylodes, dried tangerine or orange peel, fried licorice, magnolia officinalis (ginger processed), polyporus umbellatus, poria cocos (peel), fried atractylodes, alisma, cassia bark, dried ginger and fructus mume are coarsely crushed;

[0084] Step 2): Take half of each component in the raw material of Composition 1 in Step 1), and decoct with water for three times. For the first time, add 10 times the amount of water, soak for 45 min, and then decoct for 2 h, and filter. For the second time, add 8 times the amount of water, decoct for 1.5 h, and filter. For the third time, add 6 times the amount of water, decoct for 1 h, and filter. Combine the decocted liquids, and concentrate to dryness to obtain a dry extract, so that the relative density of the dry extract is 1.00 (g / ml). Then sterilize the dry extract at 121°C for 30 min using ultraviolet radiation sterilization. Finally, crush the dry extract to obtain an extract fine powder.

[0085] Step 3): Take the remaining half of each component in the raw material of Composition 1 in Step 1), crush, and sterilize using ultraviolet radiation sterilization. Mix the extract fine powder obtained in Step 2) to obtain Composition 1. The Composition 1 meets the following indexes: atractylenolide I ≥ 0.008 mg / g, atractylenolide II ≥ 0.036 mg / g, atractylenolide III ≥ 0.102 mg / g, magnolol ≥ 0.614 mg / g, and honokiol ≥ 0.432 mg / g, and 23-acetylzeanols B ≥ 0.034 mg / g.

[0086] Test Example 2

[0087] Composition 2 for regulating intestinal flora, raw material ratio: fried atractylodes 100 g, dried tangerine or orange peel 100 g, fried licorice 50 g, magnolia officinalis (with ginger) 100 g, polyporus 50 g, poria (with skin removed) 100 g, fried atractylodes 100 g, alisma 50 g, cassia bark 30 g, dried ginger 50 g, and fructus mume 50 g.

[0088] The preparation method is similar to that in Test Example 1, and in Step 3), the relative density of the dry extract is 1.10 (g / ml), and the other conditions are the same as in Test Example 1. The Composition 2 meets the following indexes: 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 200 g, dried tangerine or orange peel 200 g, fried licorice 100 g, magnolia officinalis (with ginger) 200 g, polyporus 150 g, poria (with skin removed) 200 g, fried atractylodes 200 g, alisma 150 g, cassia bark 60 g, dried ginger 150 g, and fructus mume 150 g.

[0091] The preparation method is similar to that in Test Example 1, and in Step 3), the relative density of the dry extract is 1.10 (g / ml), and the other conditions are the same as in Test Example 1. The Composition 2 meets the following indexes: magnolol ≥ 0.614 mg / g, and honokiol ≥ 0.432 mg / g.

[0092] Comparative Example 1

[0093] Comparative composition 1 for regulating intestinal flora, raw material ratio: fried atractylodes 150g, dried tangerine or orange peel 150g, fried licorice 60g, fried magnolia officinalis (with ginger) 150g, polyporus 100g, tuckahoe (peel) 150g, fried atractylodes rhizome 150g, alisma 100g, cassia 60g.

[0094] The preparation method is the same as steps 1) -3) of Test Example 1, except that the raw materials lack the components dried ginger and Fructus mume.

[0095] Comparative Example 2

[0096] Comparative composition 2 for regulating intestinal flora, raw material ratio: fried atractylodes 150g, dried tangerine or orange peel 150g, fried licorice 60g, fried magnolia officinalis (with ginger) 150g, polyporus 100g, tuckahoe (peel) 150g, fried atractylodes rhizome 150g, alisma 100g, cassia 60g, ginger 100g, jujube 100g.

[0097] The preparation method is the same as steps 1) -3) of Test Example 1, except that the raw material components of the present comparative example are ginger and jujube.

[0098] Comparative Example 3

[0099] Comparative composition 3 for regulating intestinal flora, the raw material is Huoxiang Zhengqi tablet, which is purchased from Tong Ren Tang in Haidian District, Beijing. It has some of the same raw material components as the composition of the present application, which are atractylodes, dried tangerine or orange peel, fried magnolia officinalis (with ginger), tuckahoe, etc.

[0100] After crushing the Huoxiang Zhengqi tablet, it is dissolved in distilled water until there are no obvious particles. The human clinical equivalent dose is converted into the rat administration dose for administration.

[0101] Comparative Example 4

[0102] Comparative composition 4 for regulating intestinal flora, raw material components are proportioned by weight (the same as the raw materials of composition 1 of Test Example 1): fried atractylodes 150g, dried tangerine or orange peel 150g, fried licorice 60g, fried magnolia officinalis (with ginger) 150g, polyporus 100g, tuckahoe (peel) 150g, fried atractylodes rhizome 150g, alisma 100g, cassia 50g, dried ginger 100g, Fructus mume 100g.

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

[0104] Step 1): The raw materials fried atractylodes, dried tangerine or orange peel, fried licorice, fried magnolia officinalis (with ginger), polyporus, tuckahoe (peel), fried atractylodes rhizome, alisma, cassia, dried ginger, and Fructus mume are coarsely crushed according to the weight proportion of the raw material components in composition 4.

[0105] Step 2): Take all the raw materials of composition 4 in step 1), decoct with water three times, add 10 times the amount of water in the first decoction, soak for 45 min, then decoct for 2 h, filter; add 8 times the amount of water in the second decoction, decoct for 1.5 h, filter; add 6 times the amount of water in the third decoction, decoct for 1 h, filter; combine the decoction, concentrate and dry to obtain dry extract, so that the relative density of the dry extract is 1.00 (g / ml), then the dry extract is sterilized at 121 ℃ under ultraviolet radiation for 30 min, finally the dry extract is crushed 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 ± 20 g, purchased from Beijing Sbielf Biological Co., Ltd., experimental animal qualification certificate number: SCKX(Jing)2019-0010. The room temperature is adjusted at 20-25℃, the humidity is 45%±5%, the 12h light and 12h dark environment, the standard diet, free water. After 7 days of adaptation, the test was carried out.

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

[0109] Instruments: electronic balance, Shanghai Precision Scientific Instrument Co., Ltd.; AXTG16G table type high-speed centrifuge, Shanghai Zhaodi Biological Technology Co., Ltd.; QL-901 vortex instrument, Haimen Qisun Biotech Instrument Co., Ltd.; table type low-speed high-performance refrigerated centrifuge, Changsha Xiangyi Centrifuge Instrument Co., Ltd.; constant temperature water bath, Jintan Hengfeng Instrument Factory; KQ-300DE type numerical control ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; KDM-1000 type temperature control electric heating jacket, Jiangsu Jierui Electric Appliance Co., Ltd.; hand-held pressure steam sterilization pot, Guangzhou Kangmai Medical Instrument Co., Ltd.; Leica microtome, Leica, Germany; Olympus DP-71 microphotographic system, Olympus, Japan; Leica microtome, Leica, Germany; Olympus DP-71 microphotographic system, Olympus, Japan; LRH-70 biochemical incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.; iMark microplate reader, BIO-RAD, USA; Mindray BC5300Vet five-classification animal blood cell analyzer, Shenzhen Mindray Biomedical Electronics Co., Ltd.; LG-R-80 blood viscosity instrument, Beijing Zhongjin Shidi Scientific Instrument Co., Ltd.; Germany Erba XL-640 fully automatic biochemical analyzer, Germany Erba Company.

[0110] 1. Test method

[0111] Animal grouping and model establishment The rats were weighed and randomly divided into 9 groups, 8 rats in each group, namely blank control group, model control group, composition 1 group, composition 2 group, composition 3 group, comparative composition 1 group, comparative composition 2 group, comparative composition 3 group and comparative composition 4 group. The rats in the model control group, composition 1 group, composition 2 group, composition 3 group, comparative composition 1 group, comparative composition 2 group, comparative composition 3 group and comparative composition 4 group were given sufficient high-fat feed once a day, free water for 7 days, ice water swimming for 4 hours a day on the 8th day, 21st day, and senna leaf gavage 3g / kg. On the 22nd day, the model control group was ended, and distilled water was gavaged. The drug and dosage of other groups are shown in Table 1. After the end of the test, the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30mg / kg), and blood was collected from the abdominal aorta, 1ml was collected in an EDTA-K2 anticoagulant tube, 1ml was collected in a heparin sodium anticoagulant tube, and the rest of the blood was collected in a general vacuum blood collection tube (37℃ constant temperature box, 4000rpm centrifugal separation serum, -20℃ storage for standby). Collect the cecal contents and colon feces and quickly put them into liquid nitrogen, then transfer them to -80 for standby. Quickly cut 2cm ileum and 2cm colon from the ileocecal junction and place them in 4% paraformaldehyde solution for room temperature immersion for 15 days.

[0112] Table 1 Gavage drug and dosage of rats in each group

[0113] Group Intragastric medicine name Medicine dose Blank control group - - Model control group Distilled water - Composition 1 group Composition 1 2.16 g / kg per day, continuously for 7 days Composition 2 group Composition 2 2.16 g / kg per day, continuously for 7 days Composition 3 group Composition 3 2.16 g / kg per day, continuously for 7 days Comparative composition 1 group Comparative composition 1 2.16 g / kg per day, continuously for 7 days Comparative composition 2 group Comparative composition 2 2.16 g / kg per day, continuously for 7 days Comparative composition 3 (Huoxiang Zhengqi tablet) group Huoxiang Zhengqi tablet 4.2 g / kg per day, continuously for 7 days Comparative composition 4 group Comparative composition 4 2.16 g / kg per day, continuously for 7 days

[0114] 2. Performance index detection of chronic stress model

[0115] 2.1 General condition observation The general condition indexes of rats were observed every day, including mental state, activity state, growth posture, fur, food appetite, feces, and urine.

[0116] Before the experiment, all rats were in good mental state, sensitive to reaction, active, with smooth and white fur with luster, normal food appetite, black and hard feces, and light yellow urine. After the experiment, the blank control group had no obvious abnormal performance, and the body weight gradually increased. The model control group of rats had loose stool and anus, especially after forced swimming and restraint stress and intragastric administration of senna, the rats showed mental depression, cold, rough and messy fur, reduced appetite, loose stool, and no shape. Composition 1 group mainly reduced the diarrhea of the model control group of rats, and the water content of feces was significantly reduced, the fur was lustrous and smooth, and the feces of each group was as shown in Table 3 (the blank control group was control, the model control group was model, the comparative composition 1 group was ZHW1, the comparative composition 2 group was ZHW2, the comparative composition 3 group was HXZQ, and the composition 1 group was ZY1). Figure 1

[0117] The body weight change was as shown in Table 4 (the blank control group was CON, the model control group was MOD, and the composition 1 group was ZY1). During the modeling period, the body weight of the rats in the blank control group gradually increased. During the high-fat diet stage of the first to seventh day, the body weight of the model control group and the composition group gradually increased in general; during the chronic stress stage, the body weight gradually decreased; after administration, the body weight gradually increased, and the increasing trend of the composition group was greater than that of the model control group. Figure 2

[0118] 2.2 Diarrhea index

[0119] The diarrhea index (DI) = loose stool rate x loose stool level, and the loose stool rate = the number of loose stool of each rat / the total number of feces. The loose stool level is divided into four levels according to the size of the stain on the filter paper: 1 level for stain diameter <1 cm, 2 level for stain diameter 1-1.9 cm, 3 level for stain diameter <2-3 cm, and 4 level for stain diameter >3 cm, as shown in Table 2.

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

[0121]

[0122]

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

[0124] As shown in Table 2, during the modeling period of 1-21 days, the diarrhea index of the model control group was significantly greater than that of the blank control group (P<0.05); during the period of 1-21 days, the diarrhea index of the composition group was significantly greater than that of the blank control group (P<0.05); during the period of 22-28 days after administration, there was no statistically significant difference between the composition group and the blank control group (P>0.05); during the period of 22-28 days, the diarrhea index of the composition group was significantly less than that of the model control group (P<0.05).

[0125] 2.3 Blood routine index

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

[0127] Table 3 Blood routine index of each group of rats n=8

[0128]

[0129]

[0130] Note: the model control group compared with the blank control group, *P<0.5, **P<0.01, ***P<0.001; composition group compared with the model control group, #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 blood routine test were significantly greater in the model control group than in the blank control group (P<0.05), and significantly less in the composition group than in the model control group (P<0.05), indicating that the model control group animals had inflammatory and cold reactions, which were significantly alleviated after treatment in the composition group.

[0132] 2.4 Blood viscosity index

[0133] Table 4 Blood routine index of each group of rats n=8

[0134] Group 200s -1 (mpa·S) 30s -1 (mpa·S) 5s -1 (mpa·S) 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 6.19 ± 0.53 ** ]] 8.15 ± 0.68 ** ]] 14.35 ± 2.35 ** ]] 15.35 ± 0.58 ** ]] Composition 1 group 3.89 ± 0.89 ### ]] 5.21 ± 1.73 ## ]] 8.78 ± 3.18 ## ]] 9.45 ± 1.98 ## ]] Composition 2 group 5.25 ± 1.67 ## ]] 7.55±1.67 12.84±2.88 13.01±0.65 Composition 3 group 4.23 ± 1.45 ## ]] 6.89 ± 0.98 # ]] 11.47 ± 3.67 # ]] 12.45 ± 1.45 # ]]

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

[0136] Table 4 shows that in the 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 shear rates of 200 s⁻¹, 30 s⁻¹, and 1 s⁻¹, the blood viscosity of the combined composition group was extremely significantly lower than that of the model control group (P<0.01), and at a shear rate of 5 s⁻¹, the blood viscosity of the combined composition 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 was increased, and the combined composition group had a significant effect on alleviating the blood viscosity of rats under chronic stress.

[0137] 2.5 Blood biochemical indicators

[0138] Blood biochemistry tests will use a fully automated biochemical analyzer to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), total cholesterol (TC), and triglycerides (TG) in the collected serum.

[0139] Table 5. Blood biochemical indicators 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 58.92 ± 8.93 ** ]] 177 ± 31.79 ** ]] 2.79 ± 0.25 * ]] 0.79 ± 0.21 ** ]] 1.80 ± 0.17 * <!-- 10 -->]] Composition 1 group 41.91 ± 7.61 ## ]] 144.10 ± 12.39 # ]] 1.58 ± 0.12 # ]] 0.49 ± 0.11 ## ]] 1.41 ± 0.42 # ]] Composition 2 group 50.34 ± 8.22 # ]] 156.74 ± 8.46 # ]] 1.89 ± 0.74 # ]] 0.59 ± 0.15 # ]] 1.78±0.24 Composition 3 group 51.09+3.44 168.17±15.67 2.43 ± 0.69 # ]] 0.62±0.25 1.65 ± 0.55 # ]]

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

[0142] Table 5 shows that ALT and AST in the model control group were significantly higher than those in the blank control group (P<0.01), while those in the composition group were significantly lower than those in the model control group (P<0.05). This indicates that the blood transaminase activity was elevated in the chronic stress model animals, and the composition could reduce transaminase activity. GGT in the model control group was significantly higher than that in the blank control group (P<0.05), while those in the composition group were significantly lower than those in the model control group (P<0.05). This indicates that the blood GGT activity was elevated in the chronic stress model animals, and the composition could reduce its activity. TG in the model control group was significantly higher than that in the blank control group (P<0.01), while those in the composition group were significantly lower than those in the model control group (P<0.01). TCH in the model control group was significantly higher than that in the blank control group (P<0.05), while those in the composition group were significantly lower than those in the model control group (P<0.05). This indicates that the blood lipids were elevated in the chronic stress model animals, and the composition could reduce their blood lipids.

[0143] 2.6 Serum cytokine indicators

[0144] Serum cytokine detection: The serum stored at -20℃ was thawed at room temperature, and IL-10, IL-1β, TNF-α, DAO, D-LA were determined according to the ELISA kit instructions.

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

[0146]

[0147]

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

[0149] Table 6 shows that the IL-1β of the model control group was extremely significantly higher than that of the blank control group (P <0.01), and the composition group was extremely significantly lower than the model control group (P <0.01); the IL-10 of the model control group was extremely significantly lower than that of the blank control group (P <0.01), and the composition group was significantly higher than the model control group (P <0.05); the TNF-α of the model control group was extremely significantly higher than that of the blank control group (P <0.01), and the composition group was significantly lower than the model control group (P <0.05). It is shown that the inflammatory response of the chronic stress model animal is increased, and the inflammatory response is decreased after treatment with the composition. The DAO of the model control group was extremely significantly higher than that of the blank control group (P <0.01), and the composition group was extremely significantly lower than the model control group (P <0.01); the D-LA of the model control group was extremely significantly higher than that of the blank control group (P <0.01), and the composition group was significantly lower than the model control group (P <0.05), indicating that the intestinal barrier of the chronic stress model animal is damaged, and it is restored after treatment with the composition.

[0150] 3. Intestinal flora diversity analysis

[0151] Microbial community composition was analyzed by 16S rRNA gene amplicon sequencing. 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). The V3-V4 region of microbial DNA was amplified using universal primers (338F: 5'-ACTCCTACGGGAGGCAGCAG-3'; 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). Purified amplicons were sequenced on an 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 a bioinformatics analysis cloud platform (https: / / cloud.majorbio.com).

[0152] Sequencing depth was analyzed using a dilution curve. Alpha diversity indices such as Chao 1 and Shannon index were calculated using mothur [8] software (http: / / www.mothur.org / wiki / Calculators), and Wilxocontrol rank sum test was used for inter-group difference analysis of Alpha diversity.

[0153] Data statistics and analysis Data are expressed as mean ± standard deviation. The software used was SPSS 24.0, and the mean statistical analysis was performed using one-way ANOVA. P<0.05 was considered statistically significant, and P<0.01 was considered extremely significant.

[0154] 3.1 OTU data analysis

[0155] As shown in Figure 3 As shown in Figures 3A and 3B (blank control group is control, model control group is model, comparative composition 1 group is ZHW1, comparative composition 2 group is ZHW2, comparative composition 3 group is HXZQ, comparative composition 4 is ZHW4, and composition 1 group is ZY1), when the sequencing depth is small and the OUT data changes dramatically, the OUT data increases significantly with the increase of sequencing depth. When the depth reaches 30000 reads, the dilution curve has reached a saturated state, indicating that the sequencing concentration has reached the depth of the sequencing amount. The current sequencing depth can only increase the abundance of species, but not the diversity.

[0156] As shown in Figures 3A and 3B (blank control group is control, model control group is model, comparative composition 1 group is ZHW1, comparative composition 2 group is ZHW2, comparative composition 3 group is HXZQ, comparative composition 4 is ZHW4, and composition 1 group is ZY1), when the sequencing depth is small and the OUT data changes dramatically, the OUT data increases significantly with the increase of sequencing depth. When the depth reaches 30000 reads, the dilution curve has reached a saturated state, indicating that the sequencing concentration has reached the depth of the sequencing amount. The current sequencing depth can only increase the abundance of species, but not the diversity. Figure 4The OUT number reflecting Alpha diversity was calculated according to the Bayesian algorithm at a 97% similarity level, and the results showed that the OUT number of the model control group was significantly lower than that of the blank control group, and the OUT number of the composition 1 group (ZY1) was significantly higher than that of the model control group, indicating that the stress model significantly reduced the intestinal flora diversity, and the composition administration significantly increased the diversity of the flora, and the composition administration and the normal group of rats had high similarity in the OUT level of intestinal flora.

[0157] 3.2 PCA result analysis

[0158] As Figure 5 shown (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 found that obvious clustering occurred, in which the blank control group and the composition 1 group had a similar clustering distance and had more overlap, while the model control group was far away from the blank control group and the composition 1 group, and the comparative composition group was also far away from the blank control group and the composition 1 group, indicating that after the composition 1 intervention, the flora of the composition 1 group and the blank control group of rats had great similarity.

[0159] 3.3 Flora composition difference analysis

[0160] As Figure 6 A shows that the dominant flora at the door level is:

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

[0162] Actinobacteria: 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 tablet, 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 composition group returned to the level after administration, and the proportion of Firmicutes in the composition 1 group (ZY1) increased more significantly after administration; in addition, the proportion of Actinobacteria in the model control group increased significantly, and the proportion of Actinobacteria in the composition group returned to no significant difference with the blank group after administration, and the proportion of Actinobacteria in the composition 1 group (ZY1) decreased significantly after administration.

[0164] As shown in Figure 6 The dominant bacteria at the genus level are:

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

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

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

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

[0169] Lactobacillus is a beneficial bacterium in the intestine. The number of Lactobacillus was significantly reduced in the model control group rats. After administration of the combination and the control combination, the intestinal flora imbalance was reversed. Composition 1 (ZY1) significantly increased the abundance of Lactobacillus, changed the composition of the rat intestinal flora, affected the balance of the intestinal flora, and made the intestinal flora closer to the state of the blank group.

[0170] 3.4 Differential Analysis of Tryptophan Metabolites

[0171] like Figure 7 As shown (the blank control group is called control, the model control group is called model, the comparison composition 1 group is ZHW1, the comparison composition 2 group is ZHW2, the comparison composition 4, the comparison composition 3 group is HXZQ, the composition 1 group is ZY1, *p<0.05, **p<0.01, ***p<0.001, compared with the 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). Analysis of the results revealed that the tryptophan metabolites (Indoleacrylic acid, L-Tryptophan, Indole-3-carboxylic acid, 3-Indoleglyoxyliac acid, Indole-3-aceticacid, Lactic acid) were significantly higher in group 1 than in the model control group (p<0.001), and also significantly higher than in control groups 1, 2, 3, and 4 (p<0.001). This indicates 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 improved, and the differentiation level of Th17 cells was significantly inhibited. Among them, the composition 1 (ZY1) significantly improved the differentiation level of Treg cells and significantly inhibited the differentiation level of Th17 cells, and formed a significant difference compared with the comparative composition 1 (ZHW1), the comparative composition 2 (ZHW2), the comparative composition 3 (HXZQ) and the comparative composition 4 (ZHW4). It shows that the administration of the composition 1 (ZY1) can significantly reverse the imbalance of intestinal immune Treg / Th17 caused by the model, regulate the balance of intestinal immune Treg / Th17 to regulate the balance of intestinal immune, and finally form the balance of intestinal immune without significant difference with the blank control group.

[0175] The present application can alleviate intestinal inflammation by adjusting the levels of inflammatory factors and oxidative stress. The present application can also regulate intestinal non-specific immunity and regulate intestinal flora, increase the diversity and richness of intestinal flora, restore the richness and proportion of intestinal flora to normal proportion, thereby improving and playing a role in treating diarrhea diseases.

[0176] 4. Analysis of intestinal barrier protection results

[0177] After the experiment, the colon of the rat was subjected to H&E staining, and the results are shown in Figure 9 It is shown that the intestinal epithelial cells of the blank control group (Control) rat are arranged in order, the goblet cells are normally distributed, the crypt structure is normal, the intermediate glands are clear, there is no inflammatory cell infiltration, no congestion, and no edema; the colon epithelium of the model control group (Model) rat is not complete, there is inflammatory infiltration and edema, the structure of the colon glands is severely damaged, the epithelial cells are degenerated and necrotic, and the goblet cells are reduced; the colon epithelial cells of the composition 1 (ZY1) group rat are complete in structure and arranged in order, there is no inflammatory cell infiltration, no congestion, and no edema, and the colon is most similar to that of the blank control group. The colons of the rats in the other groups (ZHW1, ZHW2, HXZQ, ZHW4) all have different degrees of epithelial cell shedding, inflammatory cell infiltration, and severe destruction of the structure of the colon glands.

[0178] The results of the transmission electron microscopy of the colon are shown in Figure 10The blank control group (Control) showed normal colon structure, complete villi, and complete intercellular tight junction. Compared with the blank control group, the model control group (Model) showed disordered structure, villus loss, and broken intercellular tight junction. The colon epithelial cells of the rats in the composition 1 (ZY1) group showed normal colon structure, complete villi, and complete intercellular tight junction. The colons of the rats in the other groups (ZHW1, ZHW2, HXZQ, and ZHW4) showed different degrees of microvillus rupture and loss.

[0179] Example 6

[0180] Case: In September 2023, more than 120 3-7-day-old calves in a calf house of a cattle farm in Beijing suddenly got sick. The sick calves showed reduced appetite, weight loss, loose stool, and paste anus. Some calves had rough and disordered hair, and some calves had dehydration. Autopsy found that the intestinal tract was inflated, and there was yellow thoracic water or pericardial effusion. According to the incidence, clinical symptoms, and pathological changes, samples were collected for kit detection, and the disease was diagnosed as bovine viral diarrhea. Thirty calves with similar body weight and typical symptoms were selected for clinical trials to observe the clinical efficacy of the composition. The 30 calves were randomly divided into three groups, with 10 calves in each group. The first group (ZY1) was fed with the composition 1 prepared in Example 4, and the composition 1 was administered at 2 g / kg. The second group (MOD) was used as a control group without medication. The third group (JPZX) was given a commercially available anti-cold and damp diarrhea traditional Chinese medicine, Jianpi Zhixie Powder, according to the instructions. All test calves were raised separately in isolated stalls. The calves were fed for 7 consecutive days, and the clinical changes and cure rates of the test calves were observed and recorded. Blood serum was collected from the jugular vein, and blood biochemical indicators were measured. Feces were collected to analyze intestinal flora changes.

[0181] Therapeutic evaluation criteria: cured: normal spirit and physical strength, normal food and water intake, normal stool frequency, and normal stool appearance; ineffective: no improvement or even worsening of clinical symptoms.

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

[0183] Test results

[0184] 1. The clinical efficacy test results showed that the composition 1 prepared in Example 4 had good therapeutic effect on calf diarrhea, with a cure rate of 90%. Compared with the control group, the cure rate was significantly improved after administration of the composition. The results are shown in Table 7.

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

[0186] Group Test calf (head) Healed calf (head) Healing rate (%) 1 10 9 90 2 10 3 30 3 10 8 80

[0187] As shown in Figure 11 , blood biochemical results showed that the electrolyte disorder was significantly alleviated after administration, and the liver and kidney damage of the calf was protected.

[0188] As shown in Figure 12 and Figure 13 , the administration of composition 1 significantly increased the diversity of the flora, and the similarity of the intestinal flora of the composition 1 administration group and the normal calf at the OUT level.

[0189] As shown in Figure 14 , it is shown that after the intervention of composition 1, the intestinal flora of the composition 1 group has great similarity with the intestinal flora of the normal calf.

[0190] As shown in Figure 15 , composition 1 significantly increased the abundance of lactobacillus, changed the composition of the intestinal flora of the calf, affected the balance of the intestinal flora, and made the intestinal flora closer to the normal calf.

[0191] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition for regulating intestinal flora, characterized in that, The ingredients are made from stir-fried Atractylodes lancea, dried tangerine peel, prepared licorice root, ginger-processed Magnolia officinalis, Polyporus umbellatus, peeled Poria cocos, stir-fried Atractylodes macrocephala, Alisma plantago-aquatica, cinnamon, dried ginger, and dried plum. The ingredients are proportioned by weight as follows: stir-fried Atractylodes lancea 100-200 parts, dried tangerine peel 100-200 parts, prepared licorice root 50-100 parts, ginger-processed Magnolia officinalis 100-200 parts, Polyporus umbellatus 50-150 parts, peeled Poria cocos 100-200 parts, stir-fried Atractylodes macrocephala 100-200 parts, Alisma plantago-aquatica 50-150 parts, cinnamon 30-60 parts, dried ginger 50-150 parts, and dried plum 50-150 parts.

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

3. A method for preparing a composition for regulating intestinal flora according to any one of claims 1-2, characterized in that, The specific steps are as follows: Step 1): Take the following ingredients according to the weight ratio: stir-fried Atractylodes lancea, dried tangerine peel, prepared licorice root, ginger-processed Magnolia officinalis, Polyporus umbellatus, peeled Poria cocos, stir-fried Atractylodes macrocephala, Alisma plantago-aquatica, cinnamon, dried ginger, and dried plum, and coarsely grind them. Step 2): Take half the amount of each component in the raw materials of the composition in Step 1), add water and decoct, filter, combine the decoction, concentrate and dry to obtain dry extract, and pulverize the dry extract to obtain extract fine powder. Step 3): Take half of the remaining amount of each component in the raw materials of the composition in Step 1), pulverize and sterilize it, and mix it with the fine powder of the extract obtained in Step 2) to obtain the composition for regulating intestinal flora.

4. The preparation method according to claim 3, characterized in that, The decoction process in step 2) includes three decoctions: for the first decoction, add 8-12 times the volume of water, soak for 45-60 minutes, and then decoct for 1.5-2 hours; for the second decoction, add 6-10 times the volume of water and decoct for 1-1.5 hours; for the third decoction, add 4-6 times the volume of water and decoct for 0.5-1 hour.

5. The preparation method according to claim 3, characterized in that, The dry extract described in step 2) is sterilized at 121°C for 20-30 min before pulverization. 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.

6. The use of a composition for regulating intestinal flora according to any one of claims 1-2 in the preparation of a drug for treating diarrheal diseases and regulating intestinal flora.

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