Traditional Chinese medicine composition for conditioning intestinal flora as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for conditioning intestinal flora as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition comprises 18-30 parts of deer heart blood powder, 10-15 parts of ginseng, 4-10 parts of radix polygonati officinalis, 4-10 parts of spina date seeds, 18-25 parts of coix lacryma-jobi, 4-10 parts of citron, 8-15 parts of poria cocos and 8-15 parts of radix puerariae. The invention further provides a preparation method of the traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of products for resisting oxidation, increasing muscle holding power, promoting hair growth and blackening, delaying organ aging, reducing the occurrence risk of senile diseases and recovering intestinal rejuvenation. The traditional Chinese medicine composition adopts medicinal and edible raw materials, has no toxic or side effect and is easy to absorb.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for regulating intestinal flora, and a preparation method and application thereof. Background Art

[0002] Aging is a natural physiological process, the gradual deterioration of the structure and function of organisms over time. It is accompanied by a decline in physiological function, a weakening of cell regeneration, a decline in immune function, and changes in metabolic levels. The intestinal microbiota refers to the community of microorganisms living in the intestines, including bacteria, fungi, viruses, and archaea. The intestinal microbiota is closely related to human health and disease, playing a key role in regulating metabolism, immune function, and protecting the intestinal barrier. With age, the composition, function, and stability of the intestinal microbiota undergo significant changes, a phenomenon known as intestinal aging. The main characteristics of intestinal aging include decreased microbial diversity, an increase in pathogenic bacteria, and decreased microbial stability. Aging of the intestinal microbiota is closely associated with a variety of geriatric and chronic diseases, including immune aging, chronic inflammation, metabolic disorders, neurodegenerative diseases, and impaired intestinal barrier function.

[0003] Traditional Chinese medicine (TCM) has unique advantages in regulating human functions. Its multi-component, multi-target action characteristics are consistent with the complex ecosystem of the intestinal flora. Many Chinese medicines and their compound prescriptions can regulate the balance of the intestinal microecology with relatively few adverse reactions. However, many existing TCM products or prescriptions for regulating intestinal flora still have problems such as unreasonable formulations, unstable sources of medicinal materials, and non-standard quality control, and lack systematic scientific research and clinical verification. Therefore, it is of great practical significance to develop a TCM composition for regulating intestinal flora that is scientifically formulated, quality-controlled, safe and effective. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a traditional Chinese medicine composition for regulating intestinal flora, which has the effects of restoring the youthfulness of intestinal flora, anti-oxidation, increasing muscle grip, promoting hair growth and blackening, and delaying the aging of multiple organs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a traditional Chinese medicine composition for regulating intestinal flora. The composition comprises, by weight, 18 to 30 parts of deer heart blood powder, 10 to 15 parts of ginseng, 4 to 10 parts of polygonatum, 4 to 10 parts of spinach seeds, 18 to 25 parts of coix seeds, 4 to 10 parts of citron, 8 to 15 parts of poria cocos and 8 to 15 parts of kudzu root.

[0007] Preferably, the Chinese medicine composition for regulating intestinal flora includes, by weight, 30 parts of deer heart blood powder, 12 parts of ginseng, 6 parts of polygonatum, 6 parts of jujube seeds, 20 parts of coix seeds, 6 parts of citron, 10 parts of poria cocos and 10 parts of kudzu root.

[0008] Preferably, the deer heart blood powder is prepared by drying deer heart blood.

[0009] Preferably, the deer heart blood powder further comprises a protective agent.

[0010] The present invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition, comprising the following steps: mixing ginseng, polygonatum, jujube seed, coix seed, citron, poria and kudzu root, decocting the obtained mixture with water, and concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; mixing the medicinal material extract with deer heart blood powder, sterilizing, and obtaining the traditional Chinese medicine composition.

[0011] Preferably, the medicinal material extract is freeze-dried to obtain a freeze-dried medicinal material extract powder, which is then mixed with deer heart blood powder.

[0012] Preferably, the amount of water added for decocting is 8 to 10 times the mass of the mixture.

[0013] Preferably, the decoction is performed twice, with each decoction lasting 1 to 2 hours.

[0014] The present invention also provides the use of the above-mentioned Chinese medicine composition in any of the following applications:

[0015] (1) Preparation of antioxidant products;

[0016] (2) preparing drugs to increase muscle grip;

[0017] (3) preparing a drug for promoting hair growth and / or hair darkening;

[0018] (4) Preparation of drugs for delaying organ aging;

[0019] (5) Preparation of drugs to reduce the risk of geriatric diseases;

[0020] (6) Prepare products for regulating intestinal flora.

[0021] Preferably, the dosage form of the product or the medicine includes capsules, tablets, granules, pills, and oral liquids.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0023] The present invention uses Chinese medicinal and edible herbs as raw materials. The resulting compounded composition has the effects of restoring the youthfulness of intestinal flora, resisting oxidation, increasing muscle grip, promoting hair growth and blackening, and delaying the aging of multiple organs, ultimately achieving the effect of delaying aging. The compound composition of the present invention has no toxic side effects, is easily absorbed, and can be used to delay aging, resist oxidative damage, and reduce the risk of geriatric diseases. The compound composition disclosed in the present invention can be used as a dietary supplement or health food raw material for resisting oxidative damage and delaying aging, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Abdominal fat status of male mice in Experimental Example 1;

[0025] Figure 2 : HE staining results of liver tissue in Experimental Example 1;

[0026] Figure 3 : Experimental Example 1 kidney tissue HE staining results;

[0027] Figure 4 : Pathological HE staining results of spleen tissue in Experimental Example 1;

[0028] Figure 5 : Pathological HE staining results of testicular tissue in Experimental Example 1;

[0029] Figure 6 : Experimental Example 1 liver tissue immunohistochemical staining results;

[0030] Figure 7 : Test results of GSH and MDA contents in liver tissue of Experimental Example 1;

[0031] Figure 8 : Western blot test results of liver tissue in Experimental Example 1;

[0032] Figure 9 : Results of bacterial β diversity (PCoA and NMDS) of Experimental Example 1;

[0033] Figure 10 : Species distribution histogram of the mean relative abundance of intestinal flora in each group at the genus and species levels in Experimental Example 1;

[0034] Figure 11 : Experimental results of test example 2: grip force measurement;

[0035] Figure 12 : Experimental Example 2 Hair growth phenotype and HE staining results of back skin;

[0036] Figure 13 : HE staining results of ovarian tissue in Experimental Example 2;

[0037] Figure 14: HE staining results of liver tissue in Experimental Example 2;

[0038] Figure 15 : Experimental Example 2 kidney tissue HE staining results;

[0039] Figure 16 : Experimental Example 2 liver tissue immunohistochemical staining results;

[0040] Figure 17 : Test results of GSH and MDA contents in liver tissue of Experimental Example 2;

[0041] Figure 18 : Western blot test results of liver tissue in Experimental Example 2;

[0042] Figure 19 : Results of bacterial β diversity (PCoA and NMDS) of Experimental Example 2;

[0043] Figure 20 : Species distribution histogram of the mean relative abundance of intestinal flora in each group at the genus and species levels in Experimental Example 2;

[0044] Figure 21 : Experimental Example 3 Hair growth phenotype;

[0045] Figure 22 : Results of immunoblotting of liver tissue in Experimental Example 3. DETAILED DESCRIPTION

[0046] The present invention provides a traditional Chinese medicine composition for regulating intestinal flora, which comprises, by weight, 18-30 parts of deer heart and blood powder, 10-15 parts of ginseng, 4-10 parts of polygonatum, 4-10 parts of spinach seeds, 18-25 parts of coix seeds, 4-10 parts of citron, 8-15 parts of poria and 8-15 parts of kudzu root; preferably, the traditional Chinese medicine composition for regulating intestinal flora comprises, by weight, 20-25 parts of deer heart and blood powder, 10-15 parts of ginseng, 6-10 parts of polygonatum, 6-10 parts of spinach seeds, 20-25 parts of coix seeds, 6-10 parts of citron, 10-15 parts of poria and 10-15 parts of kudzu root. More preferably, the Chinese medicinal composition for regulating intestinal flora comprises, by weight, 30 parts of deer heart blood powder, 12 parts of ginseng, 6 parts of polygonatum, 6 parts of jujube seeds, 20 parts of coix seeds, 6 parts of citron, 10 parts of poria cocos and 10 parts of kudzu root.

[0047] Deer heart blood is drawn from the heart of a slaughtered sika deer or red deer. Deer heart blood powder is made from this blood. Modern clinical research indicates that deer heart blood can treat palpitations, insomnia, forgetfulness, falls, and anti-aging. It can also be used to treat rheumatism and rheumatoid arthritis.

[0048] Ginseng is the root of Panax ginseng (Camey), a plant of the Araliaceae family. It has a sweet, slightly bitter flavor and a slightly warm nature. It enters the lung, spleen, and heart meridians. It has the effects of tonifying vital energy, tonifying the spleen and lungs, promoting fluid production, and calming the mind and improving intelligence. It is primarily used to treat qi deficiency with impatience, weak pulse, spleen deficiency, sinking of qi in the middle, wheezing and coughing due to lung deficiency, shortness of breath and fatigue, thirst due to loss of fluid, polydipsia due to deficiency-heat, insomnia and forgetfulness, palpitations, blood deficiency, chlorosis, impotence, and cold uterus.

[0049] Polygonatum odoratum (Mill.) Druce is the rhizome of Polygonatum odoratum (Mill.) Druce, a plant of the Liliaceae family. It is sweet and neutral in nature, entering the Lung and Stomach meridians. It nourishes yin and moistens dryness, promotes fluid production and quenches thirst. It is primarily used to treat dry-heat cough, thirst due to loss of fluid, yin deficiency caused by external infection, headache, and fever.

[0050] Ziziphus jujuba seeds are the mature seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Huex H.F. Chou, a plant of the Rhamnaceae family. They are neutral in nature, sweet and sour in flavor. They enter the Liver, Gallbladder, and Heart meridians. They nourish the liver, calm the mind, astringe sweat, and promote fluid production. They belong to the category of tranquilizers, a type of heart-nourishing and tranquilizing medicine. They are used to treat insomnia, palpitations, excessive dreams, excessive sweating due to physical weakness, and thirst caused by loss of body fluids.

[0051] Job's tears, the mature seed kernel of Coix lacryma-jobi L. var. mayuen (Roman.) Stapf, a plant of the Poaceae family, is cool in nature, with a sweet, bland flavor. It enters the spleen, stomach, and lung meridians. It strengthens the spleen and eliminates dampness, relieves numbness and stops diarrhea, and clears heat and discharges pus. It belongs to the diuretic and stranguria-relieving category of diuretics. It is used to treat edema caused by water retention, dysuria, stranguria, beriberi-induced edema, spleen deficiency diarrhea, dampness-induced stiffness and muscle spasms, difficulty flexing and extending muscles, or weakness, lung abscesses, coughing up pus and sputum, and intestinal abscesses.

[0052] Citron is the mature fruit of Citrus wilsonii Tanaka. (also known as Citrus medic L.) in the Rutaceae family. It is warm in nature, with a pungent, bitter, and sour flavor. It enters the Liver, Spleen, and Lung meridians. It soothes the liver, regulates Qi, relieves fullness, and resolves phlegm. It is a Qi-regulating herb used to treat liver-stomach Qi stagnation, chest and rib pain, abdominal distension, vomiting, belching, and cough with excessive phlegm.

[0053] Poria cocos, the sclerotium of the Polyporaceae fungus Poria cocos (Schw.) Wolf., is neutral in nature, with a sweet and bland taste. It enters the Heart, Lung, Spleen, and Kidney meridians. It promotes diuresis and eliminates dampness, strengthens the spleen, and calms the mind. It belongs to the diuretic and anti-edema category of diuretics and dampness-eliminating drugs. It is used to treat edema, oliguria, phlegm and fluid retention, dizziness and palpitations, spleen deficiency, poor appetite, loose stools and diarrhea, restlessness, palpitations, and insomnia.

[0054] Pueraria root is the dried root of Pueraria lobata (Willd.) Ohwi, a plant of the genus Pueraria in the Leguminosae family. It has a sweet and pungent flavor and a cooling nature. It enters the spleen and stomach meridians. It relieves muscle tension, reduces fever, clears rashes, promotes fluid production and quenches thirst, and promotes yang and relieves diarrhea. It is primarily used to treat superficial symptoms of fever, stiff neck and back pain, measles that has not cleared up, thirst caused by febrile illness, polydipsia due to yin deficiency, febrile diarrhea due to heatiness, and diarrhea due to spleen deficiency.

[0055] The present invention uses deer heart blood powder and ginseng as main medicines (monarch medicines), which can greatly replenish deficiency, restore pulse and strengthen deficiency, benefit essence and blood, and strengthen tendons and bones; Polygonatum odoratum and Ziziphus jujuba seeds are auxiliary medicines, which can nourish yin and moisten dryness, promote body fluid and quench thirst, and calm the mind and tranquilize the nerves; Coix lachryma-jobi, citron, Poria cocos and Pueraria root can invigorate the spleen and eliminate dampness, regulate qi and harmonize the middle, and soothe the liver and relieve depression. The whole prescription has the effects of replenishing qi and blood, calming the mind and tranquilizing the nerves, invigorating the spleen and eliminating dampness, and soothing the liver and relieving depression. It is suitable for people with deficiency syndromes such as deficiency of both yin and yang, deficiency of both qi and blood, deficiency of spleen and stomach qi and blood, and can be used for people with deficiency syndromes caused by aging or disease.

[0056] The deer heart blood powder of the present invention is prepared by drying deer heart blood. Preferably, the deer heart blood powder also includes a protective agent, which includes mannitol, trehalose, sucrose, etc.; the drying method includes freeze drying and hot air drying. As an optional embodiment, the present invention uses a freeze dryer to dry the deer heart blood stock solution to prepare the deer heart blood powder; or the deer heart blood stock solution is mixed with the protective agent to form a uniform liquid, and dried to prepare the deer heart blood powder. The protective agent and deer heart blood are preferably mixed in a mass (g) / volume (mL) ratio in the range of 1% to 3% to form a uniform liquid. The freeze drying of the present invention is preferably placed at -40°C for freeze drying; the hot air drying of the present invention is preferably sprayed into a drying tower at a certain pressure (such as 2 to 3MPa), and the inlet air temperature is controlled at 180 to 200°C, so that the deer heart blood is quickly dried into powder.

[0057] A preparation method of the traditional Chinese medicine composition of the present invention comprises the following steps: mixing ginseng, polygonatum, jujube seed, coix seed, citron, poria and kudzu root, decocting the mixture with water, and concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; mixing the medicinal material extract with deer heart blood powder, and sterilizing to obtain the traditional Chinese medicine composition (i.e., a compound medicine).

[0058] Another method for preparing the traditional Chinese medicine composition of the present invention comprises the following steps: mixing ginseng, polygonatum, jujube seed, coix seed, citron, poria, and kudzu root, decocting the mixture with water, and concentrating the decoction to 1 / 3 of its original volume to obtain a medicinal extract; freeze-drying the medicinal extract to obtain a freeze-dried powder, which is then mixed with deer heart blood powder and sterilized to obtain the compound medicine of the traditional Chinese medicine composition. The medicinal extract of the present invention is freeze-dried at -40°C.

[0059] The amount of water added for the decoction according to the present invention is 8 to 10 times the mass of the mixture, preferably 9 times. The number of decoctions according to the present invention is preferably 2 times, more preferably 1 to 2 hours each time. As an optional embodiment, the present invention mixes ginseng, polygonatum, spinach seed, coix seed, citron, poria and kudzu root in proportion, decocts the mixture with water, filters to obtain a decoction, adds an equal amount of water to the filter residue for decoction again, filters again to obtain a decoction, and combines the decoctions. The present invention concentrates the decoction to 1 / 3 of its original volume, which can make the interaction between the effective ingredients more stable and reduce the denaturation or precipitation of the components due to excessive concentration.

[0060] The present invention does not limit the specific sterilization method.

[0061] The present invention also provides the use of the above-mentioned Chinese medicine composition in any of the following applications:

[0062] (1) Preparing an antioxidant product; the Chinese medicine composition significantly increases GSH content and reduces MDA content.

[0063] (2) Preparation of drugs for increasing muscle grip.

[0064] (3) Preparing a drug for promoting hair growth and / or hair darkening; the Chinese medicine composition improves senile hair loss, increases the number of hair follicles, darkens hair, and promotes dark hair.

[0065] (4) Preparation of drugs for delaying organ aging; the organs include liver, kidney, spleen, testicles, and ovaries.

[0066] (5) Preparation of drugs for reducing the risk of geriatric diseases; the Chinese medicine composition of the present invention reduces abdominal fat accumulation, reduces p53 and p16 in liver tissue INK4A The expression of glycoproteins can effectively prevent or reduce the occurrence of geriatric diseases. Geriatric diseases, also known as elderly diseases, refer to age-related and unique diseases suffered by people in old age. As the structure and function of various cell organs and tissues in the elderly age, their adaptability and resistance decrease with age, leading to increased morbidity. Geriatric diseases described in the present invention include tumors, hypertension and coronary heart disease, chronic bronchitis and pneumonia, gallbladder disease, benign prostatic hyperplasia, femoral fractures, and diabetes.

[0067] (6) Preparing a product for regulating intestinal flora; the Chinese medicine composition increases the relative abundance of beneficial bacteria Muribaculaceae, Desulfovibrio, Faecalibaculum rodentium, Bifidobacteriumpseudolongum and Parabacteroides.

[0068] The dosage forms of the product or the drug of the present invention include capsules, tablets, granules, pills, and oral liquids. The dosage forms of the product or the drug of the present invention are not limited thereto, and other achievable dosage forms are within the scope of protection of the present invention. The product of the present invention includes food, health products, or medicines. The product or drug of the present invention further includes acceptable carrier excipients to ensure that the prepared preparation is convenient for preparation and clinical application. Further preferably, the excipients of the present invention include, but are not limited to, at least one of a diluent, a wetting agent, an adhesive, a lubricant, a colorant, and a coating agent.

[0069] As an optional embodiment, the present invention accurately mixes the processed botanical extract, deer heart blood powder, and excipients (such as maltodextrin, microcrystalline cellulose, magnesium stearate, etc.) in accordance with the formula requirements. A V-shaped mixer is required during the mixing process to ensure that the raw materials and excipients are evenly distributed; the mixed raw materials are granulated using a one-step granulation and drying device; the prepared granules are dried to remove excess moisture (the temperature must be controlled below 80°C during the drying process to avoid deformation or cracking of the granules, and a hot air circulation oven is used for drying); the dried granules are sterilized to kill the microorganisms and bacteria therein to obtain granules. As another optional embodiment, the present invention fills the sterilized granules into capsule shells; hard capsule filling equipment is required during the filling process, and the dosage and weight difference of the capsules must be controlled to ensure the accuracy and consistency of the product.

[0070] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0071] The experimental data were processed using SPSS 21.0 software and expressed as x ± s. P < 0.05 indicated a significant difference, P < 0.01 indicated a relatively significant difference, and P < 0.001 indicated an extremely significant difference.

[0072] In the following examples, unless otherwise specified, all methods are conventional.

[0073] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0074] Example 1

[0075] A traditional Chinese medicine composition for regulating intestinal flora: 30 parts of deer heart blood powder, 12 parts of ginseng, 6 parts of polygonatum, 6 parts of jujube seeds, 20 parts of coix seeds, 6 parts of citron, 10 parts of poria cocos, and 10 parts of kudzu root. The deer heart blood stock solution is freeze-dried in a freeze dryer (-40°C) to produce the deer heart blood powder.

[0076] The preparation method comprises the following steps: mixing ginseng, polygonatum, spinach seed, coix seed, citron, poria and kudzu root, adding 8 to 10 times the weight of water to the mixture and decocting the mixture, filtering to obtain a decoction, adding an equal amount of water to the filter residue and decocting the mixture again, filtering again to obtain a decoction, and combining the decoctions; concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; mixing the medicinal material extract with deer heart blood powder, and sterilizing the mixture to obtain the compound medicine of the traditional Chinese medicine composition.

[0077] Example 2

[0078] A traditional Chinese medicine composition for regulating intestinal flora: 25 parts of deer heart blood powder, 15 parts of ginseng, 10 parts of polygonatum, 10 parts of jujube seeds, 25 parts of coix seeds, 10 parts of citron, 15 parts of poria cocos, and 15 parts of kudzu root. The deer heart blood stock solution is freeze-dried in a freeze dryer (-40°C) to produce the deer heart blood powder.

[0079] The preparation method comprises the following steps: mixing ginseng, polygonatum, spinach seed, coix seed, citron, poria and kudzu root, adding 8 to 10 times the weight of water to the mixture, decocting the mixture, filtering to obtain a decoction, adding an equal amount of water to the filter residue for decocting again, filtering again to obtain a decoction, and combining the decoctions; concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; freeze-drying the medicinal material extract at -40°C to obtain a freeze-dried medicinal material extract powder, mixing the obtained freeze-dried medicinal material extract powder with deer heart blood powder, and sterilizing the mixture to obtain the compound medicine of the traditional Chinese medicine composition.

[0080] Example 3

[0081] A traditional Chinese medicine composition for regulating intestinal flora: 20 parts of deer heart blood powder, 10 parts of ginseng, 6 parts of polygonatum, 6 parts of jujube seeds, 20 parts of coix seeds, 6 parts of citron, 10 parts of poria, and 10 parts of kudzu root. A protective agent (mannitol) is mixed with the deer heart blood at a mass (g) / volume (mL) ratio of 1% to form a uniform liquid. The liquid is then sprayed into a drying tower at a constant pressure (2-3 MPa), with the inlet air temperature controlled at 180-200°C, to rapidly dry the deer heart blood into a powder.

[0082] The preparation method comprises the following steps: mixing ginseng, polygonatum, spinach seed, coix seed, citron, poria and kudzu root, adding 8 to 10 times the weight of water to the mixture and decocting the mixture, filtering to obtain a decoction, adding an equal amount of water to the filter residue and decocting the mixture again, filtering again to obtain a decoction, and combining the decoctions; concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; mixing the medicinal material extract with deer heart blood powder, and sterilizing the mixture to obtain the compound medicine of the traditional Chinese medicine composition.

[0083] Example 4

[0084] A traditional Chinese medicine composition for regulating intestinal flora: 22 parts of deer heart blood powder, 14 parts of ginseng, 8 parts of polygonatum, 8 parts of jujube seeds, 22 parts of coix seeds, 7 parts of citron, 13 parts of poria cocos, and 13 parts of kudzu root. A protective agent (trehalose) is mixed with the deer heart blood at a mass (g) / volume (mL) ratio of 3% to form a uniform liquid. The liquid is then sprayed into a drying tower at a constant pressure (2-3 MPa), with the inlet air temperature controlled at 180-200°C, to rapidly dry the deer heart blood into a powder.

[0085] The preparation method comprises the following steps: mixing ginseng, polygonatum, spinach seed, coix seed, citron, poria and kudzu root, adding 8 to 10 times the weight of water to the mixture, decocting the mixture, filtering to obtain a decoction, adding an equal amount of water to the filter residue for decocting again, filtering again to obtain a decoction, and combining the decoctions; concentrating the decoction to 1 / 3 of the original volume to obtain a medicinal material extract; freeze-drying the medicinal material extract at -40°C to obtain a freeze-dried medicinal material extract powder, mixing the obtained freeze-dried medicinal material extract powder with deer heart blood powder, and sterilizing the mixture to obtain the compound medicine of the traditional Chinese medicine composition.

[0086] Test Example 1

[0087] 1. Experimental groups and drug administration:

[0088] Drug preparation: NMN (β-nicotinamide mononucleotide) was purchased from Aladdin and prepared in PBS at 52 mg / ml;

[0089] Coenzyme Q10 was purchased from Aladdin and prepared in PBS at 13.125 mg / ml. Shake well each time.

[0090] Compound high-dose group: the compound drug of Example 2 was prepared with PBS at 17.5 mg / ml;

[0091] Compound low-dose group: the compound drug of Example 2 was prepared with PBS at 8.75 mg / ml;

[0092] Ten-month-old male mice were divided into a normal control group (gavage with an equal amount of PBS), an NMN group (300 mg / kg), a CoQ10 group (75 mg / kg), a high-dose compound group (100 mg / kg), and a low-dose compound group (50 mg / kg), with eight mice in each group. Gavage was performed every other day for three months. The livers, kidneys, spleens, and testicles of each group were collected for relevant tests. Intestinal contents of each group were collected for 16S microbial analysis.

[0093] 2. Result detection

[0094] (1) After the experiment, the mouse abdomen was dissected and the results were as follows Figure 1 The results showed that the high-dose and low-dose compound groups had significantly lower abdominal white fat compared to the control group, Q10 group, and NMN group.

[0095] (2) Histopathology and immunohistochemistry: Tissue specimens were immediately fixed with 4% paraformaldehyde (pH 7.4) for 24 h to maintain morphological integrity. After fixation, the samples were dehydrated (70% to 100% graded ethanol), cleared (xylene), and paraffin-infiltrated (60°C). The embedded tissues were cut into 3 μm thick sections using a rotary microtome (Leica RM2235). To detect antigens, immunostaining was performed using a two-step polymer method (Dako, K8002), first quenching endogenous peroxidase activity with 3% H2O2, and then incubating with the primary antibody. Finally, the sections counterstained with hematoxylin were imaged under a bright-field microscope.

[0096] The results of pathological HE staining of liver tissue are as follows Figure 2 As shown. The results showed that in the liver tissue of the elderly male control mice, the hepatic lobule structure was difficult to distinguish, the hepatic cord structure was disordered, the hepatocytes were loose, the morphology was irregular, the size was reduced, and the boundaries were blurred. There was a large amount of fatty degeneration, the cytoplasm was lightly stained and loose, and round vacuoles were visible; some cell nuclei were condensed. Compared with the elderly control group, the morphology of the Q10, NMN, low-dose compound group and high-dose group was still intact. The hepatocytes were arranged regularly near the central vein, and the cells were arranged scattered away from the central vein. The hepatic cords were clearer. Compared with the other groups, the cytoplasm of the low-dose and high-dose compound groups was more uniform, with less fatty degeneration and smaller round vacuoles; the cell nuclei were clear and there was no obvious condensation, indicating that the low-dose and high-dose compound groups both had the effect of delaying liver aging.

[0097] The results of pathological HE staining of renal tissue are as follows Figure 3 The results showed that the number of glomeruli in the male control group was significantly reduced and sparsely distributed. The lumen of most renal tubules was enlarged, and the distance between tubules increased. The number of glomeruli per unit field of view in the Q10, NMN, and low- and high-dose compound groups was greater than that in the naturally aged control group. The renal tubules had narrow lumens and a regular shape, indicating that both the low- and high-dose compound groups had the effect of delaying renal aging.

[0098] The pathological HE staining results of spleen tissue are as follows Figure 4 The results showed that compared with the Q10 group, NMN group, low-dose and high-dose compound groups, the spleen tissue structure of the male control group was disordered, the boundary between the white pulp and red pulp was unclear, the white pulp area was significantly reduced, the red pulp area was expanded, congested, and had increased fibers, and the number of lymphocytes was reduced, indicating that both the low-dose and high-dose compound groups had the effect of delaying spleen aging.

[0099] The results of pathological HE staining of testicular tissue are as follows Figure 5The results showed that in male control mice, the gaps between seminiferous tubules were enlarged, and the number of spermatogenic cells decreased and their arrangement was disordered. The Q10, NMN, and low- and high-dose compound groups improved the gaps between seminiferous tubules and increased the number of spermatogenic cells in aged mice. The thickness of the spermatogenic epithelium in the naturally aged control mice decreased, while the Q10, NMN, and low- and high-dose compound groups increased both, with the low- and high-dose compound groups showing the most significant improvement.

[0100] Immunohistochemical staining results of liver tissue Figure 6 The results showed that the Q10 group, NMN group, low-dose compound group and high-dose compound group were significantly decreased compared with the male control group, indicating that the low-dose compound group and high-dose compound group can effectively reduce the immune inflammatory factor IL6 and thus achieve the effect of delaying aging.

[0101] (3) Evaluation of antioxidant effect in liver tissue: Reduced glutathione (GSH) and malondialdehyde (MDA) were used as evaluation indicators, and the reduced glutathione (GSH) assay kit (Nanjing Jiancheng / A006-2-1) and malondialdehyde (MDA) assay kit (Nanjing Jiancheng / A003-1-2) were used.

[0102] The results of GSH and MDA content in liver tissue were as follows Figure 7 The results showed that compared with the control group, the GSH levels of the NMN group, the low-dose compound group and the high-dose compound group were significantly increased; compared with the control group, the MDA levels of the Q10 group, the NMN group, the low-dose compound group and the high-dose compound group were significantly decreased, indicating that both the low-dose compound group and the high-dose compound group had antioxidant effects.

[0103] (4) Western blot: Extract total protein from cells or tissues, and determine the protein concentration using the BCA method. Mix equal amounts of protein samples with loading buffer, boil and denature, and separate the proteins by SDS-PAGE gel electrophoresis. Subsequently, transfer the separated proteins to a PVDF membrane, and block the membrane with 5% skim milk to block nonspecific binding. Add the primary antibody and incubate overnight. After washing, add the horseradish peroxidase (HRP)-labeled secondary antibody and incubate for 1 hour. After washing again, use ECL chemiluminescence reagent to develop, and detect the target protein bands using an imaging system.

[0104] Liver tissue was immunoblotted to detect senescence markers p53 and p16 INK4A The results are as follows Figure 8 The results showed that the Q10 group, NMN group, low-dose compound group and high-dose compound group could reduce the expression of p53 and p16INK4A in liver tissue, indicating that the compound group can effectively delay liver aging.

[0105] (5) 16S microbial sequencing and simple analysis methods: Total DNA was extracted using the QIAamp DNA stool mini kit (Qiagen, Germany). The purity and concentration of the metagenomic DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Construction and sequencing: After extracting the total DNA of the sample, primers were designed based on the conserved regions, and sequencing adapters were added to the ends of the primers. The forward primer 5'-ACTCCTACGGGAGGCAGCA-3' (SEQ ID No. 1) and the reverse primer 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID No. 2) were used to amplify the microbial 16S rRNA gene. After PCR amplification, the product was purified, quantified, and homogenized to form a sequencing library. The constructed library was first quality checked, and the qualified library was sequenced using Illumina HiSeq 2500. Raw image data files obtained through high-throughput sequencing (Illumina HiSeq and other sequencing platforms) were converted to raw sequencing reads through base calling analysis. The results were stored in the FASTQ (fq) file format, which contains sequence information and corresponding sequencing quality information. Microbial composition and structure analysis was performed using the QIIME2 platform and the R analysis toolkit. Beta diversity analysis was used to assess differences in microbial composition and structure between groups. Bray-Curtis distances between samples were first calculated, followed by principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS).

[0106] The PCoA and NMDS analysis results of β diversity are as follows Figure 9 The results showed that there were differences in the microbial structure among the groups, indicating that the compound Chinese medicine combination has a certain microbial regulatory function.

[0107] The top 12 species in relative abundance at the genus and species levels, and the species distribution bar graphs of the average relative abundance of intestinal flora in each group at the genus and species levels are shown in the figure below. Figure 10As shown. The results showed that the low-dose and high-dose groups of the compound jointly increased the average relative abundance of Muribaculaceae, Bifidobacterium, Faecalibaculum and Bacteroidales bacterium. At the same time, at the species level, the average relative abundance of Muribaculaceae, Desulfovibrio, Bifidobacterium pseudolongum, and Faecalibaculum rodentium in the low-dose and high-dose groups of the compound were higher than those in the control group. In summary, the Chinese medicine composition can regulate the intestinal flora of the aging intestine of male mice by increasing the relative abundance of the beneficial bacteria Muribaculaceae and Bifidobacterium pseudolongum, thereby playing a role in delaying aging.

[0108] Test Example 2

[0109] 1. Experimental groups and drug administration:

[0110] Drug preparation: NMN (β-nicotinamide mononucleotide) was purchased from Aladdin and prepared in PBS at 52 mg / ml;

[0111] Coenzyme Q10 was purchased from Aladdin and prepared in PBS at 13.125 mg / ml. Shake well each time.

[0112] Compound high-dose group: the compound drug of Example 2 was prepared with PBS at 17.5 mg / ml;

[0113] Compound low-dose group: the compound drug of Example 2 was prepared with PBS at 8.75 mg / ml;

[0114] Fourteen-month-old female mice were divided into a normal control group (gavage with an equal amount of PBS), an NMN group (300 mg / kg), a CoQ10 group (75 mg / kg), a high-dose compound group (100 mg / kg), and a low-dose compound group (50 mg / kg), with eight mice in each group. Gavage was performed every other day for three months. Skin, liver, kidney, and ovary samples were collected from each group for relevant tests. Intestinal contents were collected from each group for 16S microbial analysis.

[0115] 2. Result detection

[0116] (1) At the end of the experiment, the mouse gripping strength test was conducted. The mouse gripping strength test requires special equipment to accurately measure its gripping strength. The Sansbio rat gripping strength tester (model SA415) was used to perform the experimental operation. The experimental results are shown in the figure below. Figure 11The results showed that compared with the normal control group, the other groups of mice had stronger grip strength, indicating that the low-dose and high-dose groups of the compound can effectively improve the muscle strength of elderly female mice.

[0117] (2) After the experiment, the hair loss phenomenon of each group of mice was observed and HE staining of the back skin was performed. The method was the same as that of step 2 (2) of experimental example 1. The results are as follows: Figure 12 As shown. Hair growth phenotype results showed that compared with the natural aging control group, the elderly female mice in each group experienced severe hair loss, and the natural aging control group had more severe hair loss, indicating that each treatment group can effectively improve senile hair loss. Pathological HE staining of skin tissue showed that compared with the control group, the Q10 group, NMN group, low-dose compound group, and high-dose compound group had more hair follicles, indicating that the low-dose compound group and high-dose compound group can both delay senile hair loss.

[0118] (3) Histopathology and immunohistochemistry: The method is the same as that of step 2 (2) of Experimental Example 1.

[0119] Pathological HE staining results of ovarian tissue are as follows Figure 13 As shown, the results showed that compared with the naturally aging control group, the number of follicles in the ovaries of elderly female mice in each group was more and the hemosiderin deposition was very small, while significant hemosiderin deposition was observed in the naturally aging control group, indicating that each medication group can effectively improve ovarian aging and the effects of the low-dose compound group and the high-dose compound group are better, indicating that the low-dose compound group and the high-dose compound group can effectively delay ovarian aging.

[0120] The results of pathological HE staining of liver tissue are as follows Figure 14 Results showed that in the liver tissue of female control mice, the hepatic lobule structure was difficult to discern, the hepatic cord structure was disorganized, and the hepatocytes were loosely spaced, irregular in shape, smaller in size, and had blurred borders. There was extensive fatty degeneration, and the cytoplasm was lightly stained and loose, with round vacuoles visible. Some nuclei were pyknotic. Compared with the elderly control group, the morphology of the Q10, NMN, and low- and high-dose compound groups remained intact. Hepatocytes were regularly arranged near the central vein, but scattered farther from the central vein, with clearer hepatic cords. The low- and high-dose compound groups had more uniform cytoplasm than the other groups, with less fatty degeneration and smaller round vacuoles visible. The nuclei were clearer, with no obvious pyknotic, indicating that the compound has the effect of delaying liver aging and effectively reducing hepatic fat production.

[0121] The results of pathological HE staining of renal tissue are as follows Figure 15As shown, the results showed that the number of glomeruli in the female control group was significantly reduced and sparsely distributed. The lumen of most renal tubules was enlarged and the distance between renal tubules increased. The number of glomeruli per unit field of view in the Q10 group, NMN group, low-dose and high-dose compound groups was greater than that in the natural aging control group, and the renal tubules were narrow and neat in shape, indicating that it has the effect of delaying kidney aging.

[0122] Immunohistochemical staining results of liver tissue Figure 16 As shown, the results of immunohistochemical staining of IL6 in the liver tissue of female mice showed that the Q10 group, NMN group, low-dose and high-dose compound groups were significantly decreased compared with the male control group, indicating that it can effectively reduce the secretion of immune inflammatory factors, thereby achieving the effect of delaying aging.

[0123] (4) Evaluation of antioxidant effect in liver tissue: Reduced glutathione (GSH) and malondialdehyde (MDA) were used as evaluation indicators, and the reduced glutathione (GSH) assay kit (Nanjing Jiancheng / A006-2-1) and malondialdehyde (MDA) assay kit (Nanjing Jiancheng / A003-1-2) were used.

[0124] The results of GSH and MDA content in liver tissue were as follows Figure 17 As shown, the results showed that compared with the control group, the GSH of the Q10 group, the low-dose compound group and the high-dose compound group was significantly increased; compared with the control group, the MDA of the Q10 group, the NMN group, the low-dose compound group and the high-dose compound group was significantly decreased, indicating that the low-dose compound group and the high-dose compound group have antioxidant effects.

[0125] (5) Western blot: The method is the same as that in step 2 (4) of Experimental Example 1.

[0126] The results of immunoblotting of liver tissue were as follows Figure 18 The results showed that the Q10 group, NMN group, low-dose compound group and high-dose compound group could reduce the p16 INK4A The expression of liver function was significantly decreased, and the effects of low-dose and high-dose compound were better, indicating that the low-dose and high-dose compound groups can effectively delay liver aging.

[0127] (6) 16S microbial sequencing and simple analysis method: The method is the same as that of step 2 (5) of Experimental Example 1.

[0128] The PCoA and NMDS analysis results of β diversity are as follows Figure 19 As shown, the results showed that there were differences in the microbial structure among the groups, indicating that the compound Chinese medicine combination has a certain microbial regulatory function.

[0129] At the genus and species levels, the species distribution histograms of the average relative abundance of intestinal flora in each group are shown in Figure 2. Figure 20 As shown in the figure, the top 12 species in relative abundance at the genus and species levels were compared. The results showed that compared with the control group, the low-dose and high-dose compound groups increased the average relative abundance of Muribaculum and Parabacteroides. At the same time, at the species level, the average relative abundance of Muribaculumintestinale and Bacteroidalesbacterium in the low-dose and high-dose compound groups was higher than that in the control group.

[0130] Test Example 3

[0131] Twelve-month-old female mice were divided into a normal control group (Control) and a microbiota transplantation (FMT) group, with six mice in each group. Antibiotic treatment was performed before microbiota transplantation: a high-dose antibiotic cocktail (ABH) containing ampicillin (1 mg / mL), vancomycin (5 mg / mL), neomycin sulfonate (10 mg / mL), and metronidazole (10 mg / mL) was prepared in sterile ddH2O. A low-dose antibiotic cocktail (ABL) was prepared by diluting the ABH solution 10-fold in ddH2O. The antibiotic cocktail was freshly prepared on the day of treatment and administered orally twice daily, 250 μl each time, for 7 days.

[0132] Fecal suspension preparation: In an anaerobic workstation (80% N2, 10% CO2, and 10% H2), 0.5 g of mouse feces (feces from 8-month-old female mice that had been gavage-treated with 150 mg / kg of the compound for 45 days and feces from 8-month-old female mice) was added to 25 ml of sterile Ringer's buffer (9 g / L sodium chloride, 0.4 g / L potassium chloride, 0.25 g / L anhydrous calcium chloride, and 0.05% (w / v) L-cysteine). The mixture was vortexed for 5 minutes and allowed to stand. The supernatant was transferred to a clean centrifuge tube and an equal volume of 20% (w / v) sterile skim milk powder was added. The prepared fecal suspension was aliquoted according to the gavage volume (0.2 ml per mouse) and stored at -80°C. The normal control group received feces from 8-month-old female mice, while the microbiota transplantation group received feces from 8-month-old female mice that had been gavage-treated with 150 mg / kg of the compound for 45 days. Bacteria were transplanted three times a week and samples were collected for testing after three months.

[0133] After the experiment, the hair loss phenomenon of mice in each group was observed. Figure 21 As shown, the results showed that compared with the control group, the microbiota transplantation group could effectively improve senile hair loss and make the hair black, indicating that the intestinal microbiota regulated by the Chinese medicine composition can delay senile hair loss and promote black hair.

[0134] Western blot: The method is the same as that of step 2 (4) of Experimental Example 1. The liver tissue was detected by immunoblotting for the aging marker p16.INK4A , the results are as follows Figure 22 The results showed that compared with the control group, the microbiota transplantation group could reduce the aging marker p16 in liver tissue. INK4A This indicates that the intestinal flora regulated by the Chinese medicine composition can effectively delay liver aging.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for regulating intestinal flora, characterized in that: Calculated by weight, the composition comprises 18-30 parts of deer heart blood powder, 10-15 parts of ginseng, 4-10 parts of polygonatum, 4-10 parts of jujube seeds, 18-25 parts of coix seeds, 4-10 parts of citron, 8-15 parts of poria cocos and 8-15 parts of kudzu root.

2. The Chinese medicine composition according to claim 1, characterized in that The invention comprises, by weight, 30 parts of deer heart blood powder, 12 parts of ginseng, 6 parts of polygonatum, 6 parts of spinach seeds, 20 parts of coix seeds, 6 parts of citron, 10 parts of poria cocos and 10 parts of kudzu root.

3. The Chinese medicine composition according to claim 1, characterized in that The deer heart blood powder is prepared by drying deer heart blood.

4. The Chinese medicine composition according to claim 3, characterized in that The deer heart blood powder also includes a protective agent.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: Ginseng, polygonatum, jujube seeds, coix seeds, citron, poria and kudzu root are mixed, the obtained mixture is decocted with water, and the decoction is concentrated to 1 / 3 of the original volume to obtain a medicinal material extract; the medicinal material extract is mixed with deer heart blood powder and sterilized to obtain the traditional Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that The medicinal material extract is freeze-dried to obtain a medicinal material extract freeze-dried powder, which is then mixed with deer heart blood powder.

7. The preparation method according to claim 5, characterized in that The amount of water added for the decoction is 8 to 10 times the mass of the mixture.

8. The preparation method according to claim 5, characterized in that The decoction is performed twice, with each decoction lasting 1 to 2 hours.

9. The Chinese medicine composition according to any one of claims 1 to 4 is used in any of the following applications: (1) Preparation of antioxidant products; (2) preparing drugs to increase muscle grip; (3) preparing a drug for promoting hair growth and / or hair darkening; (4) Preparation of drugs for delaying organ aging; (5) Preparation of drugs to reduce the risk of geriatric diseases; (6) Prepare products for regulating intestinal flora.

10. The use according to claim 9, characterized in that The dosage forms of the product or the medicine include capsules, tablets, granules, pills, and oral liquids.

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