CN117618515A - Traditional Chinese medicine probiotic composition for controlling weight and reducing hepatic steatosis - Google Patents
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Abstract
The invention provides a traditional Chinese medicine probiotics composition for controlling weight and reducing liver fat transformation, which comprises traditional Chinese medicine raw materials including lotus leaves, poria cocos, coix seeds, chinese dates, mulberries, peach kernels, snakegourd fruits, hawthorns, fructus momordicae, pagodatree flowers, radish seeds, kudzuvine roots and cassia seeds, and probiotics including Bifidobacterium animalis, lactobacillusseri and Lactobacillus paracasei. The traditional Chinese medicine probiotic composition can maintain weight, protect liver, improve intestinal microorganisms, enhance substance and energy metabolism and improve human health level.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a traditional Chinese medicine for controlling weight and reducing hepatic steatosis and a traditional Chinese medicine probiotic composition.
Background
Obesity is a chronic metabolic disease that is caused by excessive accumulation and abnormal distribution of fat in the body due to imbalance in energy intake and consumption [ Zhang C, et al front in Pharmacology,2021,12:615895]. Over 2 million adults worldwide are overweight or obese, and prevalence continues to rise, becoming a global public health problem [ Aaseth J, et al biomedicine & Pharmacotherapy,2021,140:111789]. Obesity increases the risk of heart disease, diabetes, digestive system diseases and cancer, the first considered to be a disease [ PichEM-E, et al circulation Research 2020,126 (11): 1477-1500]. Currently, two types of drugs have been clinically proven to contribute to weight loss. One is acting on the central nervous system, such as cloxacillin, but these drugs have side effects including insomnia, constipation and anxiety [ Fan Q, et al front in Pharmacology,2021,12:696603]. The other is acting on the non-central nervous system, e.g. orlistat, with the same side effects [ Krentz A J, et al diabetes, obenity & Metabolism,2016,18 (6): 558-570]. According to modern medicine, the pathogenesis of obesity is complex, involving dietary habits, genetic factors, and environmental factors [ Lin X, et al front in Endocrinology,2021,12:706978]. Obesity causes fatty liver, liver steatosis, liver injury, inflammation, etc., and thus it is necessary to treat and prevent liver injury and lesions while controlling body weight. In addition, more and more studies have shown that intestinal microorganisms as a key environmental factor may lead to the development and progression of obesity [ Cheng Z, et al, front in Endocrinology,2022,13:1025706].
The human intestinal microorganism works in coordination with the host, and has important physiological functions in nutrition and substance metabolism, formation of intestinal mucosa epithelial biological barrier, immunoregulation, anti-tumor and the like [ Jia Q, et al journal of Cellular and Molecular Medicine,2019,23 (4): 2314-2323]. However, dietary habits or environmental factors can influence the composition of intestinal microorganisms, such as a high fat and high protein diet, altering the colonic structure of lactase bacteria in the intestinal mucosa, reducing the abundance of key lactase bacteria [ Qiao B, et al front in Cellular and Infection Microbiology,2022,12:1096202]. Dysbiosis of gut microorganisms may be involved in the pathogenesis of obesity by a variety of mechanisms including disruption of energy homeostasis, lipid synthesis and storage, central regulation of appetite and eating behavior, and chronic low-grade inflammation [ Liu B-N, et al world Journal of Gastroenterology,2021,27 (25): 3837-3850], and thus, improving gut dysbiosis is considered a critical direction for the treatment of obesity.
The history of the traditional Chinese medicine treatment on obesity is long, the "yellow emperor Nei Jing" systematically records obesity, for example, the "Lingqiu Wei Qi Jiujingjingjing" divides obesity into "fat, paste and meat". The theory of Su Wen-Qi Bing Lun indicates that obesity is caused by the thick taste of the food and paste, and its pathogenesis mainly includes phlegm-dampness, qi deficiency and yang deficiency. The traditional Chinese medicine has definite curative effect on obesity, has few toxic and side effects, and can promote or inhibit the growth and proliferation of certain microorganisms by interaction of the active ingredients of the traditional Chinese medicine with intestinal microorganisms when entering the gastrointestinal tract, and the intestinal microorganisms degrade the traditional Chinese medicine ingredients, so that the absorption and conversion of the traditional Chinese medicine are promoted, and the efficacy is improved or reduced. For example, secondary metabolites produced by intestinal microorganisms react with certain bioactive components of traditional Chinese medicine to form new compounds, changing the pharmaceutical properties of traditional Chinese medicine [ Gong X, et al pharmaceutical Research 2020,157:104824]. Polysaccharide, protein and the like of the traditional Chinese medicine are degraded into oligosaccharide, monosaccharide, polypeptide, amino acid and the like through microorganisms to promote the absorption and conversion of the traditional Chinese medicine, and improve the curative effect [ Qia J, et al Nature,2007,448 (7150):126-128 ].
Disclosure of Invention
The first object of the invention is to provide a traditional Chinese medicine probiotic composition for controlling weight according to the theory of traditional Chinese medicine and modern biology.
The second object of the invention is to provide a traditional Chinese medicine probiotic composition capable of improving liver steatosis and reducing liver fat according to the theory of traditional Chinese medicine and modern biology.
The third aim of the invention is to provide an oral preparation containing the traditional Chinese medicine probiotic composition and a preparation method thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly discloses a traditional Chinese medicine composition for controlling weight and reducing hepatic steatosis, which is characterized by comprising the following raw materials in parts by weight: 1-50 parts of lotus leaf, 1-50 parts of poria cocos, 1-50 parts of coix seed, 1-50 parts of jujube, 1-50 parts of mulberry, 1-50 parts of peach seed, 1-50 parts of snakegourd fruit, 1-50 parts of hawthorn, 1-50 parts of fructus momordicae, 1-50 parts of pagodatree flower, 1-50 parts of radish seed, 1-50 parts of kudzuvine root and 1-50 parts of cassia seed.
The invention further discloses a traditional Chinese medicine probiotic composition for controlling weight and reducing liver fat transformation, which comprises an extracting solution prepared from the traditional Chinese medicine composition and probiotics.
In the Chinese medicinal probiotic composition, the probiotics comprise B.animalis, L.gaseri and L.paramasei, and the content of B.animalis in the Chinese medicinal probiotic composition is 1-10X10% 8 CFU/(g solid matter of Chinese medicinal extractive solution), L.gaseri content is 1-10X10 8 CFU/(g solid matter of Chinese medicinal extractive solution), L.Paracasei content is 1-10X10 8 CFU/(g solids of Chinese medicinal extract).
As a preferred scheme, the traditional Chinese medicine probiotic composition comprises the following raw materials in parts by mass: 7 parts of lotus leaf, 6 parts of poria cocos, 5 parts of coix seed, 10 parts of jujube, 4 parts of mulberry, 8 parts of peach seed, 6 parts of semen trichosanthis, 6 parts of hawthorn, 7 parts of fructus momordicae, 8 parts of sophora flower, 7 parts of radish seed, 3 parts of kudzuvine root and 9 parts of cassia seed. The probiotics used include: b.animalis, content of 1×10 9 CFU/(g solids of the herbal extract); l.gaseri in an amount of 1X 10 9 CFU/(g solids of the herbal extract); paracasei, content 1×10 9 CFU/(g solids of Chinese medicinal extract).
The invention also discloses an oral preparation which is prepared from the traditional Chinese medicine composition or the traditional Chinese medicine probiotics. The oral preparation can be oral liquid, dry powder, capsule, granule or tablet, and the preparation method of the oral preparation is as follows.
Preparing a traditional Chinese medicine extracting solution: mixing the raw materials in the traditional Chinese medicine composition uniformly according to a proportion, adding 1-20 times of water, soaking for 30-240 minutes (preferably 180 minutes), and decocting and extracting at 80-100 ℃ for 30-120 minutes (preferably 40 minutes at 100 ℃) to obtain an extracting solution; repeating the extraction for 2-5 times (preferably 2 times), mixing filtrates, and concentrating to density of 1.00-1.50g/cm 3 Is preferably a liquid having a liquid density of 1.00 to 1.15g/cm 3 )。
Preparation of composite probiotics: the MRS culture medium and the TPY culture medium are conventional microorganism culture medium, and can be understood and implemented by those skilled in the art, L.gaseri and L.paracasei are respectively streaked and inoculated on the MRS solid culture medium, B.animalis is streaked and inoculated on the TPY solid culture medium, the culture is carried out in an anaerobic box at 37 ℃ for 24-48 hours, single bacterial colony is selected and inoculated on the corresponding liquid culture medium, and the culture is carried out at the anaerobic temperature of 37 ℃ for 24-48 hours, and the expansion culture is carried out according to the requirement. Centrifuging the probiotic fermentation liquid at 4000-10000rpm for 10-20 min, washing thallus with sterile deionized water equal to the fermentation liquid in volume, and re-suspending with sterile physiological saline or Chinese medicinal liquid.
Preparation of oral liquid preparation: feeding the above prepared part of Chinese medicinal extractive solution and fungus solutionMixing, and mixing to obtain thallus density of 1-10X10 9 CFU/mL (preferably 1X 10) 9 CFU/mL), freeze-drying to obtain the freeze-dried bacterial powder. Filling the rest Chinese medicinal extractive solution under aseptic condition, wherein the volume of each bottle is 10-500mL (preferably 60 mL), and adding lyophilized powder to the probiotic amount of 2-10×10 per dose 10 CFU (preferably 2X 10) 10 CFU)。
Preparation of solid preparation: mixing the above prepared part of Chinese medicinal extractive solution with bacterial liquid, and mixing to obtain bacterial density of 1-10×10 9 CFU/mL (preferably 1X 10) 9 CFU/mL), freeze-drying, and preparing freeze-dried bacterial powder. And freeze-drying or spray-drying the rest Chinese medicinal extractive solution to obtain Chinese medicinal powder. Mixing the traditional Chinese medicine powder with the freeze-dried fungus powder, and integrating the particle size to obtain the oral preparation. Or mixing the powder with lyophilized powder, adding appropriate binder, wetting agent, and other adjuvants, making into soft mass, sieving, making into wet granule, drying, granulating, tabletting, and encapsulating, and making into granule, tablet, capsule, etc. 2-20g (preferably 5 g) of each dose in the solid preparation, and the content of probiotics is 2-10 multiplied by 10 10 CFU, preferably 2X 10 10 CFU。
The composition comprises the following components in parts by weight: the lotus leaf bitter leaves dry dampness and cool in nature and clear heat, can help spleen and remove dampness, has light smell and ascending and dispersing, can complement spleen and stomach, can effectively disperse wind heat of upper jiao, ascending and assisting clear yang, and nourish middle warmer and upper body. Poria has effects of promoting urination, removing dampness, invigorating dryness, invigorating spleen and stomach, regulating stomach and invigorating qi. Coix seed is beneficial to intestines and stomach, eliminates edema and enables people to feel comfortable and benefit qi after long-term eating. Jujube calms stomach qi, nourishes spleen qi, supplements middle-jiao and supplements qi. Lotus leaf, poria cocos, coix seed and jujube are combined together to tonify spleen and qi, raise clear and lower turbid, and are all monarch drugs. Mulberry, fructus Mori, fructus Mori, and fructus Mori can nourish yin, enrich blood, promote salivation and moisten dryness, and be used for constipation due to intestinal dryness. Semen Persicae has effects of loosening bowel to relieve constipation, and can be used for treating constipation due to intestinal dryness. Semen Trichosanthis has effects of moistening lung, resolving phlegm, and relieving constipation. Hawthorn has the effects of strengthening spleen, promoting qi circulation, promoting digestion, removing food retention, eliminating turbid pathogen and reducing blood lipid. Momordica grosvenori has effects of clearing heat and detoxicating, loosening bowel to relieve constipation, moistening lung and relieving cough. The mulberry, peach kernel, snakegourd fruit, hawthorn and momordica grosvenori are combined to dredge intestines and stomach together, and the effect of preventing the excessive absorption of energy by the digestive system is achieved, so that the mulberry, peach kernel, snakegourd fruit, hawthorn and momordica grosvenori are ministerial drugs. The pagodatree flower has the effects of cooling blood, stopping bleeding, clearing liver fire, purging fire and improving microcirculation of human bodies. Radish seed, semen Raphani has the effects of resolving phlegm and removing food retention, lowering qi and relieving asthma, and eliminating abdominal distention. Sophora flower and radish seed are used as adjuvant drugs. Pueraria lobata and semen Cassiae are used as guiding drugs for moistening the upper and lower aspects and promoting the production of body fluid to quench thirst. Meanwhile, the composition probiotics B.animalis helps to control the whole body fat amount, the trunk fat amount, the waistline, the energy intake and the like, and L.gaseri promotes metabolism of a body, suppresses appetite, and L.paracasei regulates intestinal tracts and enhances immunity.
The traditional Chinese medicine probiotic composition is prepared from traditional Chinese medicine extracts and probiotics by optimizing the proportion: the adopted traditional Chinese medicine has the functions of reducing liver fat transformation, has the effects of protecting liver, and has synergistic effect with probiotics, so that the proliferation of the probiotics is promoted, the survival rate of the probiotics is improved, and the prebiotics are replaced; the probiotics degrade the Chinese medicinal extract and the like, promote the absorption of active ingredients by human bodies, increase satiety, reduce appetite and strengthen metabolism, thereby balancing weight and maintaining the health level of human bodies. The traditional Chinese medicine probiotics composition can maintain weight, protect liver, improve intestinal microorganisms, enhance substances and energy metabolism and improve human health level, and can be used for preparing medicines for controlling weight and/or reducing liver fat changes, such as preventing and/or treating fatty liver, alcoholic liver and the like.
Drawings
Fig. 1 is a schematic diagram of a process flow of preparing an oral preparation from the traditional Chinese medicine probiotic composition.
Fig. 2 shows the weight change of mice in each group in the pharmacodynamic experiment according to the embodiment of the present invention, and the probiotic model group of traditional Chinese medicine maintains the weight of mice relatively constant.
Fig. 3 shows the differences between the low density lipoprotein (fig. 3 (a)) and the high density lipoprotein (fig. 3 (b)) in each group of model mice in the efficacy test of the present invention, and the number indicates the degree of the differences.
Fig. 4 shows the amounts of ALT (fig. 4 (a)) and AST (fig. 4 (b)) in each group in the efficacy experiment of the present invention, which indicates significant differences between groups, and the amounts thereof indicate the degree of the differences.
Fig. 5 is a diagram of liver pathological section of each group of mice in the pharmacodynamic experiment according to the embodiment of the present invention, wherein: a is orlistat group; b is a traditional Chinese medicine probiotics group; c is a traditional Chinese medicine group; d is a probiotic group; e is a model group; f is a control group.
Fig. 6 shows a comparison of the respective abilities and nutrition provided to the body by each group in the pharmacodynamic test according to the embodiment of the present invention, wherein (a) is glycosylated hemoglobin (HbA 1 c), (b) is glycemic (Glu), and the number indicates the degree of the difference.
FIG. 7 is a graph showing the richness index of intestinal microorganisms of mice in each group, wherein each box in the graph represents a group, the horizontal line inside the box is the average value, and the horizontal lines at the upper end and the lower end of the box are the upper quartile and the lower quartile; lower case letters above boxes represent the results of the inter-group difference significance test (p < 0.05).
Fig. 8 shows the results of PCA analysis (Principal Component Analysis) in the efficacy test of the present invention.
FIG. 9 is a graph of the difference bubbles between groups of microorganism species in the efficacy experiment of the present invention, where the analysis is based on the abundance top30 species, and if the detected species is less than 30 species, all the detected species are shown.
Figure 10 shows the results of a test of the growth of three probiotics promoted by the traditional Chinese medicine in the efficacy experiment of the embodiment of the invention.
Fig. 11 shows the test results of the protective effect of the traditional Chinese medicine in promoting the freeze-drying of three probiotics in the efficacy experiment of the embodiment of the invention.
Detailed Description
In order to more clearly clarify the effects and advantages of the present invention, a detailed description of a specific embodiment of the invention will be given below with reference to examples. The following is merely illustrative and explanatory of the principles of the invention, as various modifications and additions may be made to the specific embodiments described, by those skilled in the art, or similar arrangements may be substituted, without departing from the principles of the invention or beyond the scope of the appended claims.
The probiotics used in the invention are derived from commercial probiotics, and have no specific brand requirements.
EXAMPLE 1 liquid oral formulation
The traditional Chinese medicine raw materials of the embodiment are as follows: 70g of lotus leaf, 60g of poria cocos, 50g of coix seed, 100g of jujube, 40g of mulberry, 80g of peach seed, 60g of semen trichosanthis, 60g of hawthorn, 70g of momordica grosvenori, 80g of sophora flower, 70g of radish seed, 30g of kudzuvine root and 90g of cassia seed.
The extract of the traditional Chinese medicine composition of the embodiment is prepared according to the following steps:
(1) The raw materials of the traditional Chinese medicine composition are uniformly mixed according to the proportion, 15L of water is added, and the mixture is soaked for 180 minutes.
(2) Boiling with strong fire, timing after boiling, boiling with small fire for 40 min, filtering with 100 mesh sieve, and collecting filtrate.
(3) Adding 15L of water into the Chinese medicinal residues in the step (2), boiling with strong fire, starting timing after boiling, boiling with small fire for 40 minutes, filtering with a 100-mesh filter screen, and keeping a filtrate.
(4) Mixing the filtrates, decocting with strong fire, concentrating, and concentrating to 1.00-1.15g/mL.
The strain preparation in this example was as follows:
(1) L.gaseri and L.paralasei were streaked onto MRS solid medium, and B.animals was streaked onto TPY solid medium, and cultured in an anaerobic tank at 37℃for 48 hours.
(2) Single colony is selected and inoculated in a corresponding liquid culture medium, and the culture is performed for 24 hours at the temperature of 37 ℃ in an anaerobic way, and the expansion culture is performed according to the requirement.
(3) And (3) respectively centrifuging the cultured probiotic liquid at 4000rpm and 20 ℃ for 10 minutes.
(4) Discarding supernatant, washing probiotics with sterile deionized water equal to the volume of fermentation broth, centrifuging under the same condition as step (3), discarding supernatant, and re-suspending the probiotics with part of Chinese medicinal extract.
(5) The OD600 value is measured by a visible spectrophotometer, the required concentration is regulated, then freeze-drying is carried out, and the viable count of the freeze-dried powder is required to be 2-10 multiplied by 10 10 CFU/(g lyophilized powder).
Sterilizing and aseptically packaging the extracted Chinese medicinal liquid60mL of the medicine is prepared into oral preparations. Adding lyophilized powder before administration, wherein each dosage ensures viable count of more than 2×10 10 CFU。
EXAMPLE 2 Freeze-dried solid oral preparation
The traditional Chinese medicine raw materials of the embodiment are as follows: 70g of lotus leaf, 60g of poria cocos, 50g of coix seed, 100g of jujube, 40g of mulberry, 80g of peach seed, 60g of semen trichosanthis, 60g of hawthorn, 70g of momordica grosvenori, 80g of sophora flower, 70g of radish seed, 30g of kudzuvine root and 90g of cassia seed.
The extract of the traditional Chinese medicine composition of the embodiment is prepared according to the following steps:
(1) The raw materials of the traditional Chinese medicine composition are uniformly mixed according to the proportion, 15L of water is added, and the mixture is soaked for 180 minutes.
(2) Boiling with strong fire, timing after boiling, boiling with small fire for 40 min, filtering with 100 mesh sieve, and collecting filtrate.
(3) Adding 15L of water into the Chinese medicinal residues in the step (2), boiling with strong fire, starting timing after boiling, boiling with small fire for 40 minutes, filtering with a 100-mesh filter screen, and keeping a filtrate.
(4) Mixing the filtrates, decocting with strong fire, concentrating, and concentrating to 1.00-1.15g/mL.
The strain preparation in this example was as follows:
(1) L.gaseri and L.paralasei were streaked onto MRS solid medium, and B.animals was streaked onto TPY solid medium, and cultured in an anaerobic tank at 37℃for 48 hours.
(2) Single colony is selected and inoculated in a corresponding liquid culture medium, and the culture is performed for 24 hours at the temperature of 37 ℃ in an anaerobic way, and the expansion culture is performed according to the requirement.
(3) And respectively taking the cultured probiotic liquid for centrifugation, wherein the centrifugation conditions are 4000rpm and 20 ℃, and the centrifugation is carried out for 10 minutes.
(4) Discarding supernatant, washing probiotics with sterile deionized water equal to the volume of fermentation broth, centrifuging under the same condition as step (3), discarding supernatant, and re-suspending the probiotics with Chinese medicinal extract.
(5) The OD600 value is measured by a visible spectrophotometer, the required concentration is adjusted, then freeze-drying is carried out, and the viable count of the freeze-dried powder is required to be 5-10 multiplied by 10 10 CFU/(g jelly)Dry powder) at a dose of 3g, with a probiotic content of greater than 2×10 10 CFU。
EXAMPLE 3 other solid oral formulations
The traditional Chinese medicine raw materials of the embodiment are as follows: 70g of lotus leaf, 60g of poria cocos, 50g of coix seed, 100g of jujube, 40g of mulberry, 80g of peach seed, 60g of semen trichosanthis, 60g of hawthorn, 70g of momordica grosvenori, 80g of sophora flower, 70g of radish seed, 30g of kudzuvine root and 90g of cassia seed.
As shown in fig. 1, the extract of the traditional Chinese medicine composition of this embodiment is prepared according to the following steps:
(1) The raw materials of the traditional Chinese medicine composition are uniformly mixed according to the proportion, 15L of water is added, and the mixture is soaked for 180 minutes.
(2) Boiling with strong fire, timing after boiling, boiling with small fire for 40 min, filtering with 100 mesh sieve, and collecting filtrate.
(3) Adding 15L of water into the Chinese medicinal residues in the step (2), boiling with strong fire, starting timing after boiling, boiling with small fire for 40 minutes, filtering with a 100-mesh filter screen, and keeping a filtrate.
(4) Mixing the filtrates, decocting with strong fire, concentrating to 1.00-1.15g/mL to obtain the final product.
(5) Spray drying part of the Chinese medicinal extractive solution, and making into Chinese medicinal powder.
The strain preparation in this example was as follows:
(1) L.gaseri and L.paralasei were streaked onto MRS solid medium, and B.animals was streaked onto TPY solid medium, and cultured in an anaerobic tank at 37℃for 48 hours.
(2) Single colony is selected and inoculated in a corresponding liquid culture medium, and the culture is performed for 24 hours at the temperature of 37 ℃ in an anaerobic way, and the expansion culture is performed according to the requirement.
(3) And respectively taking the cultured probiotic liquid for centrifugation, wherein the centrifugation conditions are 4000rpm and 20 ℃, and the centrifugation is carried out for 10 minutes.
(4) Discarding supernatant, washing probiotics with sterile deionized water equal to the volume of fermentation broth, centrifuging under the same condition as step (3), discarding supernatant, and re-suspending the probiotics with part of Chinese medicinal extract.
(5) The OD600 value was measured by a visible spectrophotometer, adjusted to the desired concentration, and then lyophilized.
Mixing the Chinese medicinal powder with lyophilized powder, adding appropriate binder, wetting agent, and other adjuvants, making into soft mass, sieving, making into wet granule, drying, granulating, tabletting, and encapsulating, and making into solid dosage forms such as granule, tablet, and capsule. The solid preparation has a probiotic content of more than 2×10 and each dose is 5g 10 CFU。
Experiment of drug efficacy
1 Experimental materials
1.1 Experimental drugs and reagents
Mixture of traditional Chinese medicine concentrated solution and probiotics, orlistat, physiological saline and matching reagent of full-automatic biochemical analyzer: blood lipid TG, total cholesterol TC, high density lipoprotein HDL-C and low density lipoprotein LDL-C content, glutamic pyruvic transaminase ALT, glutamic oxaloacetic transaminase AST, glycosylated hemoglobin HbA1C, blood glucose Glu (Shenzhen Lei Du life sciences Co., ltd.), formalin (Shanghai bioengineering Co., ltd.), PBS buffer, xylene (Shanghai bioengineering Co., ltd.), absolute ethanol (Shanghai bioengineering Co., ltd.), hematoxylin reagent (Shanghai bioengineering Co., ltd.), eosin reagent (Shanghai bioengineering Co., ltd.), ethanol hydrochloride, resin sealing tablet (Shanghai bioengineering Co., ltd.).
1.2 laboratory apparatus
Anaerobic working table (Chongqing river snow science, inc.), ultra clean working table, full-automatic biochemical analyzer (Shenzhen Lei Du life science, inc.), fume hood, electromagnetic oven, desk type centrifuge (Thermo Fisher Scientific), low temperature centrifuge (Thermo Fisher Scientific), electrothermal constant temperature incubator (Shanghai-Heng), ultraviolet spectrophotometer, full-automatic dehydrator (Leica), paraffin embedding machine (Leica), refrigerating table (Leica), paraffin slicer (Leica), tablet spreading machine (Leica), refrigerating slicer (Leica), liquid nitrogen tank (Thermo Fisher Scientific), 80 ℃ ultra low refrigerator (Thermo Fisher Scientific), 4 ℃ refrigerator (Thermo Fisher Scientific), electronic balance (Sartorius).
1.3 laboratory animals
70C 57BL/6J male mice, 13-15g in weight, 3 weeks old, purchased from Beijing Vietnam Lihua laboratory animal technology Co., ltd, produced license number SCXK (Beijing) 2021-0006, and fed to the national center for comprehensive fertilizer science center laboratory animal platform, animal feeding license SYXK (Anhui) 2020-006. The raising environment temperature is 22-24 ℃, and the feed and the water are taken freely.
2 Experimental methods
2.1 obese mice were modeled
After adaptively feeding C57BL/6 mice for 1 week, the mice are randomly divided into a control group and an experimental group, the mice in the control group are fed with common feed, the mice in the experimental group are fed with 60% high-fat feed, and the rest feeding conditions are kept consistent. Molding success standard: body weight increased by 20% relative to control, i.e. obesity = (experimental group mouse body weight-control group mouse body weight)/control group mouse body weight.
2.2 grouping and administration
The mice with successful obesity modeling are randomly divided into 5 groups, namely orlistat groups (20 mg/kg), and the traditional Chinese medicine probiotic composition (traditional Chinese medicine amount is 12.6g/kg, and probiotic amount is 2×10) 9 CFU/kg), chinese medicinal composition (Chinese medicinal composition 12.6 g/kg), and probiotic composition (probiotic composition 2×10) 9 CFU/kg), model group (equal volume of normal saline), and control group (normal feed fed mice) for a total of 6 groups of 8-12 mice each, administered by intragastric administration once daily at am for 7 weeks.
2.3 serological index detection
The mice were collected from the eyes, and the serum was examined for blood lipid TG, cholesterol CHO, high density lipoprotein HDL-C/low density lipoprotein LDL-C, glutamic-pyruvic transaminase ALT, glutamic-pyruvic transaminase AST, glycosylated hemoglobin HbA1C, and glycemic Glu using a full-automatic bioanalyzer.
2.4 detection of intestinal microbial diversity in mice
2.4.1 sample collection and sequencing
The mouse cecum is taken, quick frozen by liquid nitrogen, and sent to Shanghai Ling En biotechnology limited company for three generations of sequencing by dry ice protection, wherein the sequencing platform is PacBIO sequence II (U.S. PacBio Seque Pacific Biosciences), and the sequencing method is carried out according to the standard flow of the sequencing company.
2.4.2 data analysis
Quality control was performed on the raw sequence obtained by sequencing using smrtrink (V9), resulting in high quality of the sub-reads. When amplicon analysis was performed, OTU clustering based on 98.65% similarity, an international default standard for full length Strain levels.
2.5 probiotic CFU colony assay
The samples are subjected to ten-fold ratio dilution, proper dilution is selected, the samples are plated, the solid culture medium is TPY culture medium, the samples are subjected to anaerobic culture at 37 ℃ for 48 hours, then colony numbers are counted, the number of probiotics CFU in the samples is calculated, and each sample is independently repeated for 3 times.
2.6 statistical treatment analysis
Statistical analysis is carried out on the detection result by using Graphpad language, single-factor and double-factor variance analysis is adopted, and p is smaller than 0.05 and has statistical difference.
3 results of experiments
3.1 traditional Chinese medicine and traditional Chinese medicine probiotic composition for maintaining physical health level
3.1.1 traditional Chinese medicine probiotic composition for reducing weight of obese mice
After induction of 60% of high-fat feed for 12 weeks, the success rate of obesity reaches 95% (standard obesity degree of modeling success > 20%), the weight change of mice after 7 weeks of grouped administration is shown in figure 2, and the weight change trend of mice in traditional Chinese medicine group and model group is similar and is a slow rising trend; the weight change trend of the mice of the traditional Chinese medicine probiotics composition group, the probiotics group and the orlistat group is similar, wherein the weight of the mice of the traditional Chinese medicine probiotics composition group and the probiotics group shows earlier trend of decrease, but the weight of the mice of the traditional Chinese medicine probiotics composition group and the probiotics group is not obviously decreased as compared with the weight of the mice of the orlistat group, and in addition, the weight of the mice of the traditional Chinese medicine probiotics composition group fluctuates less after 28 days, and the weight basically maintains unchanged. Therefore, the traditional Chinese medicine probiotic composition can not only provide proper nutrition for organisms, but also maintain the body weight at a stable level.
3.1.2 traditional Chinese medicine probiotic composition reducing "bad" Density lipoprotein increasing "good" Density lipoprotein
Low density lipoprotein (LDL-density lipoprotein) is the primary carrier for transporting endogenous cholesterol, which is degraded and transformed by binding to low density lipoprotein receptor (LDL-R) on its cell membrane, and is the primary carrier for transporting cholesterol to extrahepatic tissues. The concentration of the protein is obviously positively correlated with the incidence of coronary heart disease, and is also an important index for evaluating the risk factors of the occurrence of the coronary heart disease of individuals, and the protein is called as bad density lipoprotein. As shown in fig. 3, LDL values of the groups were significantly higher than that of the control group except the traditional Chinese medicine probiotic group. High density lipoprotein (HDL-density lipoprotein) is one of serum proteins, and is a complex lipoprotein composed of lipids and proteins and their carried regulatory factors. Carrying cholesterol in surrounding tissues, reconverting it to bile acids or directly excreting it from the intestine, angiography demonstrates that high density lipoprotein cholesterol levels are significantly inversely related to the extent of arterial lumen stenosis, known as "good" density lipoproteins. The increase of the high-density lipoprotein has positive effects on preventing atherosclerosis and coronary heart disease, but can be seen in diseases such as chronic hepatitis, primary biliary cholangitis and the like, so that the high-density lipoprotein is higher and further combined with liver function examination: such as determining the presence or absence of abnormal levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and the like in blood, helping to understand liver function, to examine liver disease, and to biochemically examine: such as determining total bilirubin, bound bilirubin, alkaline phosphatase, and blood and urine amylase levels to aid in determining whether the patient is cholangitis. In conclusion, the lipoprotein of the mice treated by the traditional Chinese medicine probiotics is maintained at a relatively stable level.
3.1.3 traditional Chinese medicine probiotic composition for repairing liver injury of obese mice
ALT is widely present in tissue cells, with the highest hepatocyte content, and is involved in the transamination process from glutamate to alanine. ALT has two isoenzymes, sALT and mALT, present in the liver cytoplasm and mitochondria, respectively. ALT is released into blood in large quantity during acute stage of various viral hepatitis and toxic liver cell necrosis in medicine, so that it is an important index for diagnosing viral hepatitis and toxic hepatitis. AST also has 2 isozymes in the liver, one of which is present in the cytoplasm (sAST) and is readily released into the blood; the other exists in mitochondria (mAST). When light and medium hepatocellular damage occurs at different time after the hepatocellular damage, sALT and sAST firstly enter blood, mainly the increase of sALT, and AST is increased with smaller amplitude of ALT or is short in time although the amplitude is larger. If the mitochondria are severely compromised by hepatocellular damage, mAST and mALT will enter the blood, with the elevation of transaminases being dominated by mAST due to the lower mALT content. The model group shown in fig. 4 has highest ALT and AST values, and is remarkably higher than that of a control group and a traditional Chinese medicine probiotic group (p < 0.001), the traditional Chinese medicine probiotic group has no biological difference with the control group and the traditional Chinese medicine group, is remarkably lower than that of the probiotic group (p < 0.001), the AST is remarkably lower than that of the orlistat group, the traditional Chinese medicine group has no remarkable difference with the control group, the probiotic group has remarkably higher content than that of the control group, the ALT of the orlistat group is remarkably lower than that of the model group, the orlistat group and the other groups have no remarkable difference except the traditional Chinese medicine probiotic group, so that the traditional Chinese medicine group and the traditional Chinese medicine probiotic group can have a reduced effect on the elevation of ALT and AST caused by obesity, and the probiotic group has no such function, and the traditional Chinese medicine probiotic group may have a repairing function on liver injury caused by obesity, and the probiotic cannot repair liver injury. Combined with HDL and LDL index analysis, obesity may cause liver injury, and traditional Chinese medicine or traditional Chinese medicine probiotics combination can reduce liver injury, and maintain stability of various indexes of organism.
3.1.4 traditional Chinese medicine probiotic composition and traditional Chinese medicine for improving liver disease state of mice
Orlistat group (fig. 5A): compared with the model group, the liver lobule structure is more disordered, the hepatic chordae arrangement is irregular, the manifold area is blurred, and the liver lobule structure has more mild fibrosis and inflammatory response. The liver cells are different in size, the nuclei are unevenly distributed, the cytoplasmic lightening or cavitation is realized, the fat degeneration range is obviously reduced compared with a model group, the fat drop quantity is reduced, the cellular edema degree is reduced, the nuclei are extruded to one side, the severe fat transformation is obvious, and the lymphocyte and the neutrophil infiltration are realized around the nuclei. Liver sinus endothelial cell proliferation, thickening, astrocyte activation, proliferation, kupffer cell proliferation.
Traditional Chinese medicine probiotics group (fig. 5B): compared with the model group, the liver lobule structure tends to be normal, the hepatic chordae arrangement is more regular, the catchment area is clearer, and the fibrosis and inflammatory reaction are reduced. The liver cells are uniform in size, the nuclei are distributed regularly, the cytoplasm is clear, and the phenomena of lipid drop accumulation, nuclear shrinkage, edema and the like are reduced. Lymphocyte and neutrophil infiltration is also reduced. The phenomena of hyperplasia, thickening, astrocyte activation, hyperplasia, kupffer cell increase and the like of the liver sinus endothelial cells are also improved.
Traditional Chinese medicine group (fig. 5C): compared with the model group, the liver lobule structure tends to be normal, the hepatic chordae arrangement is regular, the catchment area is clear, and the fibrosis and inflammatory reaction are reduced. The liver cells are uniform in size, the nuclei are distributed regularly, the cytoplasm is clear, the range of fatty degeneration is obviously reduced compared with that of a model group, the number of lipid drops is reduced, and the degree of cell edema is reduced. Some hepatocytes exhibit nuclear contractility (black dot), and lymphocyte and neutrophil infiltration is reduced. The phenomena of hyperplasia, thickening, astrocyte activation, hyperplasia, kupffer cell increase and the like of the liver sinus endothelial cells are also improved.
Probiotic group (fig. 5D): compared with the model group, the liver lobule structure is slightly recovered to be normal, the hepatic chordae arrangement rule is improved, and the liver lobule structure has lighter fibrosis and inflammatory response. The liver cells are slightly uniform in size, the nuclei are distributed uniformly, the fat transformation range is larger compared with the traditional Chinese medicine group and the traditional Chinese medicine probiotics group, the fat drops are more, and the cell edema is not obviously reduced compared with the orlistat group. Liver sinus endothelial cell proliferation, thickening, astrocyte activation, proliferation, kupffer cell proliferation
Model set (fig. 5E): the liver lobular structure is disordered, the hepatic chordae are arranged irregularly, the manifold area is blurred, and mild fibrosis and inflammatory reaction are caused. The liver cells are different in size, uneven in nuclear distribution, light in cytoplasmic staining or cavitation, accumulated in a large amount of lipid droplets, nuclear shrinkage (black spots) of part of the liver cells occurs, lymphocyte and neutrophil infiltration is performed around part of the liver cells, and part of cell oedema is obvious. Liver sinus endothelial cell proliferation, thickening, astrocyte activation, proliferation, kupffer cell proliferation.
Control group (fig. 5F): the liver lobule has complete structure, the hepatic chordae are orderly arranged, the catchment area is clear and visible, and no obvious pathological changes such as fibrosis, inflammation, necrosis, cholestasis and the like exist. The liver cells are uniform in size, regular in nuclear distribution, clear in cytoplasm, and free from obvious phenomena of lipid drop accumulation, nuclear shrinkage, edema and the like. Liver sinus endothelial cells, astrocytes, kupffer cells, etc.
The results of the pathological sections show that the traditional Chinese medicine group and the traditional Chinese medicine probiotics group can reduce the accumulation of lipid drops of the liver, the protection effect of the independent probiotics on the liver is not obvious, the orlistat has no protection effect on the liver, and the traditional Chinese medicine compound extract has the protection effect on the liver by combining the detection of biochemical AST and ALT, so that the protection effect can be cooperated by adding probiotics in the traditional Chinese medicine extraction.
3.1.5 effects of traditional Chinese medicine probiotic composition on blood glucose in mice
Glycosylated hemoglobin (HbA 1 c) is the product of the combination of hemoglobin in red blood cells and carbohydrates (mainly glucose) in serum by a non-enzymatic reaction. The non-enzymatic reaction forming the glycosylated hemoglobin has the characteristics of being continuous, slow and irreversible, so that the content of the glycosylated hemoglobin is determined by the past blood glucose concentration rather than the instant blood glucose concentration, and is irrelevant to factors such as whether the patient is empty or not, whether insulin is injected or not, whether the hypoglycemic agent is taken or not before detection. It is generally believed that the glycosylated hemoglobin concentration effectively reflects the average blood glucose level over the past 8-12 weeks. Glucose in blood is called blood glucose (Glu), and a normal human body needs a lot of sugar every day to provide energy, so that power is provided for normal operation of various tissues and organs, and the instant blood glucose level of the organism is reflected. As can be seen from fig. 6, the traditional Chinese medicine probiotic composition provides the organism with the corresponding ability and nutrition, and although slightly higher than the probiotic group, it does not cause hyperglycemia of the organism, and has no significant difference from the control group.
3.2 traditional Chinese medicine probiotic composition for improving intestinal microbial flora
3.2.1 within-group Alpha diversity analysis
The diversity of a single sample (Alpha diversity) may reflect the abundance and diversity of the microbial community within the sample. The alpha diversity index for reflecting the sample microorganism mainly comprises the Observed features, chao 1, ACE, shannon, simpson, pielou_J and Pd_faith, wherein the Observed features, chao 1 and ACE are richness indexes, shannon and Simpson are diversity indexes, pielou_J is uniformity index and Pd_faith is evolutionary diversity index.
According to the results of the Observed specs richness index (fig. 7), compared with the control group, obesity (model group) causes the intestinal microorganisms of the mice to change obviously, and under the action of the weight-losing drug orlistat (orlistat group), the intestinal microorganisms of the mice are obviously different from the control group (p < 0.05), namely the original richness is not restored. After the probiotics are taken (probiotics group), the intestinal microorganism diversity is recovered, and the average abundance is higher than that of the Yu Aoli span group, which is lower than that of the control group, but is not obvious. The intestinal diversity of mice with the traditional Chinese medicine group and the traditional Chinese medicine probiotics group is obviously higher than that of the orlistat group (p is less than 0.05), and the richness is obviously improved. The average richness of the intestinal microorganisms of the mice in the traditional Chinese medicine probiotics group is higher than that in the control group and the model group, and the traditional Chinese medicine group is higher than that in the model group and is close to the control group, but no obvious difference exists. This suggests that obesity can reduce the diversity of mice intestinal microorganisms, consistent with existing studies. Orlistat can reduce the weight of mice, but can not increase the richness of intestinal microorganisms, and traditional Chinese medicine probiotics groups and traditional Chinese medicine groups can improve the richness of intestinal microorganisms, but further research is needed.
Other indicators of richness and diversity characterizing the mouse intestinal microflora, such as the richness index (Chao 1, ACE), diversity index (Shannon and Simpson), uniformity index (Pielou_J), evolutionary diversity index (Pd_faith) are similar to the Observed species richness index.
3.2.2 analysis of microbial Beta diversity between different groups
In Beta diversity research, PCA analysis (Principal Component Analysis), i.e., principal component analysis, can reflect the differences and distances between samples by analyzing OTU (97% similarity) composition of different samples, and PCA uses variance decomposition to reflect differences of multiple sets of data on a two-dimensional graph, e.g., the more similar the sample composition, the closer the distances reflected in the PCA plot. The results of fig. 8 show that the distance between the traditional Chinese medicine probiotics group and the control group microbiome is the shortest, which shows that the difference of the microbial diversity of the traditional Chinese medicine probiotics group and the control group is the smallest, and the model group and the orlistat group have the largest difference of the microbial diversity compared with the control group.
3.2.3 differential species analysis
According to the 16SRNA sequencing analysis results (fig. 9), the first 30 species with greater variation between groups included mainly Roseburia intestinalis, roseburia hominis, romboutsia ilealis, muribaculum sp002492595, faecalibaculum robustum. Roseburia intestinalis, roseburia hominis belong to the genus Roche and account for 2-31% of the total number of healthy intestinal bacteria, producing short chain fatty acids, especially butyric acid, affecting colonic motility, with anti-inflammatory properties. Roseburia intestinalis is one of the key bacteria for degrading dietary fiber xylan in human intestinal tracts [ Maria Louise Leth, et al Nat Microbiol,2018,3:570-580], colonizing Luo Sibai Rayleigh bacteria in mice, and can reduce inflammation markers, improve atherosclerosis under the influence of diet rich in plant polysaccharide [ KA, et al Nat Microbiol,2018,3 (12): 1461-1471], interfere with alcoholic fatty liver mice, interfere with and improve alcoholic fatty liver in mice, and strengthen intestinal barrier [ Seo B, et al cell Host microbe.2020,27 (1): 25-40.e6]. However, studies have shown that Roseburia intestinalis might trigger antiphospholipid syndrome (APS) in susceptible individuals, suggesting that the "good" and "bad" intestinal bacteria are not invariable [ Ruff W, et al cell Host micro.2019, 26 (1): 100-113.e8].
Muribaculum sp002492595 was significantly higher in both the traditional Chinese medicine probiotic group and the control group than in the model group, the probiotic group and the orlistat group. The abundance of the strain in the traditional Chinese medicine group is obviously higher than that of the probiotics group and the orlistat group, but the difference between the strain and the model group is not obvious, which indicates that the traditional Chinese medicine can promote the propagation of Muribaculum sp002492595, and the abundance of Muribaculum sp002492595 cannot be changed by the orlistat and the probiotics. Studies have shown that the intestinal beneficial bacteria abundance and diversity are relatively reduced in western diet mice, the number of Muribaculum intestinale involved in carbohydrate metabolism is significantly reduced, while the number of Muribaculum intestinale in standard diet and sports mice is increased, which bacteria and their family may affect their host energy harvesting [ Monica P.McNamara, et al J Exp Biol,2021,224 (4): jeb239699 ]
The abundance of intestinal microorganisms Faecalibaculum redentium (Frod) is reduced after being treated by orlistat, traditional Chinese medicine probiotics and traditional Chinese medicine administration, and the Frod abundance is not obviously different from that of a control. The model group has obvious difference from the control group, and after the stomach is irrigated by probiotics, the abundance of Frod is still obvious in difference from the control group. This shows that traditional Chinese medicine and orlistat can significantly inhibit proliferation of Frod, while probiotics lavage can not inhibit proliferation of Frod in the intestinal tract of mice. The remarkable increase of Frod in mice fed with high fat diet and sugar, the segmented filamentous bacteria (Segmented Filamentous Bacteria, SFB) and Frod were colonized in the intestinal tract of the sterile mice by the intragastric method, frod remarkably inhibited colonization of SFB, which was inversely related to Frod. SFB can induce the production of Th17 cells in the intestinal tract of mice [ II Ivanov, et al front immunol.2019 (22), 10:2750], and the lipid content of intestinal epithelial cells and liver of Th17 normal mice is obviously reduced, and the lipid level in feces is increased. High fat diets lead to increased Frod and inhibit SFB colonization, resulting in reduced Th17 cells and increased risk of metabolic disease [ Kawano Y, et al cell.2022,185 (19): 3501-3519.e20].
3.3 traditional Chinese medicine extracts promote the growth of probiotics
Bacterial solutions of freshly cultured b.animalis, l.gaseri and l.paramasei were centrifuged, bacterial pellet washed with sterile PBS without oxygen, and bacterial pellet resuspended with sterile PBS added. Taking 100 mu L of bacterial liquid, respectively inoculating 900 mu L of traditional Chinese medicine extracting solution (the traditional Chinese medicine extracting solution is subjected to sterile filtration in advance) with the total volume of 1mL, and culturing the mixed liquid of traditional Chinese medicine probiotics in an anaerobic box at 37 ℃. And (3) taking 100 mu L of bacterial liquid, performing multiple dilution, and simultaneously taking proper dilution multiple to coat a TPY solid culture medium, and measuring the number of CFU in 100 mu L of bacterial liquid.
Culturing the mixed solution of the traditional Chinese medicine probiotics for 48 hours, taking 100 mu L of culture solution, performing double ratio dilution, performing the operation steps with the inoculated bacterial solution, and calculating the CFU number in 1mL of traditional Chinese medicine probiotics. The above experiment was independently repeated 3 times.
The results show (FIG. 10) that the traditional Chinese medicine is able to promote the growth of three probiotics, B.animalis, L.gaseri and L.Paracasei.
3.4 protective effect of Chinese medicinal extract on freeze-drying of probiotics
Bacterial solutions of fresh cultures B.animalis, L.gaseri and L.paramasei were centrifuged at 100mL each, bacterial pellet was washed with sterile PBS without oxygen, and bacterial suspensions were resuspended by adding 30mL of sterile PBS without oxygen, respectively. Taking 100 mu L of bacterial liquid, performing multiple dilution, and simultaneously taking proper dilution multiple to coat a TPY solid culture medium, and respectively measuring and calculating the number of CFU in 30mL of bacterial liquid.
And respectively adding 30mL of bacterial liquid into 100mL of traditional Chinese medicine extract, uniformly mixing, then placing the mixture at the temperature of minus 80 ℃ for freezing overnight, then placing the mixture into a vacuum freeze dryer for freeze drying, collecting solid, weighing, and measuring the number of the probiotics CFU in the solid under the same measuring conditions.
The results show (FIG. 11) that the traditional Chinese medicine has protective effect on the lyophilization of three probiotics of B.animal, L.gaseri and L.Paracasei.
Claims (10)
1. The traditional Chinese medicine composition for controlling weight and reducing hepatic steatosis is characterized by comprising the following raw materials in parts by weight: 1-50 parts of lotus leaf, 1-50 parts of poria cocos, 1-50 parts of coix seed, 1-50 parts of jujube, 1-50 parts of mulberry, 1-50 parts of peach seed, 1-50 parts of snakegourd fruit, 1-50 parts of hawthorn, 1-50 parts of fructus momordicae, 1-50 parts of pagodatree flower, 1-50 parts of radish seed, 1-50 parts of kudzuvine root and 1-50 parts of cassia seed.
2. A traditional Chinese medicine probiotics composition for controlling weight and reducing hepatic steatosis is characterized in that: the traditional Chinese medicine probiotics composition comprises an extracting solution prepared from the traditional Chinese medicine composition of claim 1 and probiotics.
3. The traditional Chinese medicine probiotic composition for controlling body weight and reducing hepatic steatosis according to claim 2, wherein: the probiotics include Bifidobacterium animalis (b.animals), lactobacillus gasseri (l.gaseri) and lactobacillussParacasei (l.Paracasei).
4. A traditional Chinese medicine probiotic composition for controlling body weight and reducing hepatic steatosis according to claim 3, characterized in that: in the Chinese medicinal probiotic composition, the content of B.animalis is 1-10X10 8 CFU/(g solid matter of Chinese medicinal extractive solution), L.gaseri content is 1-10X10 8 CFU/(g solid matter of Chinese medicinal extractive solution), L.Paracasei content is 1-10X10 8 CFU/(g solids of Chinese medicinal extract).
5. Use of a traditional Chinese medicine composition or a traditional Chinese medicine probiotic composition according to any one of claims 1 to 4 for preparing a medicament for controlling body weight and/or reducing hepatic steatosis.
6. An oral formulation characterized by: the oral preparation is prepared from the traditional Chinese medicine composition or the traditional Chinese medicine probiotic composition according to any one of claims 1 to 4.
7. The oral formulation of claim 6, wherein: the oral preparation is oral liquid, dry powder, capsule, granule or tablet.
8. A method of preparing the oral formulation of claim 6 or 7, comprising the steps of:
step 1, uniformly mixing the raw materials of the traditional Chinese medicine composition according to a proportion, adding 1-20 times of water, soaking, decocting, filtering, concentrating to obtain a traditional Chinese medicine extract, sterilizing and canning to obtain a traditional Chinese medicine oral liquid;
step 2, activating, culturing, washing and counting probiotics to obtain a bacterial liquid containing probiotics with the required number of living bacteria, and mixing and freeze-drying the bacterial liquid with part of the traditional Chinese medicine extract prepared in the step 1 to obtain bacterial powder;
and step 3, uniformly mixing the rest of the traditional Chinese medicine extract in the step 1 with the fungus powder in the step 2 before use to obtain the traditional Chinese medicine probiotics oral liquid.
9. A method of preparing the oral formulation of claim 6 or 7, comprising the steps of:
step 1, uniformly mixing the raw materials of the traditional Chinese medicine according to a proportion, adding 1-20 times of water by weight, soaking, decocting, filtering and concentrating to obtain a traditional Chinese medicine extract;
step 2, activating, culturing, washing and counting probiotics to obtain a bacterial liquid containing the probiotics with the required number of living bacteria;
and step 3, uniformly mixing the traditional Chinese medicine extract in the step 1 with the bacterial liquid in the step 2, freeze-drying until the water content is not more than 5%, grinding into particles, and sieving to obtain the oral preparation.
10. A method of preparing the oral formulation of claim 6 or 7, comprising the steps of:
step 1, uniformly mixing the raw materials of the traditional Chinese medicine according to a proportion, adding 1-20 times of water by weight, soaking, decocting, filtering and concentrating to obtain a traditional Chinese medicine extract;
step 2, activating, culturing, washing and counting probiotics to obtain a bacterial liquid containing the probiotics with the required number of living bacteria;
step 3, adding a part of the traditional Chinese medicine extract in the step 1 into the bacterial liquid in the step 2, uniformly mixing, and freeze-drying to obtain freeze-dried bacterial powder;
step 4, freeze-drying or spray-drying the residual traditional Chinese medicine extract in the step 1 to obtain traditional Chinese medicine powder;
step 5, mixing the freeze-dried bacterial powder in the step 3 with the traditional Chinese medicine powder in the step 4, and integrating the particle size to obtain an oral preparation;
or mixing the lyophilized powder obtained in step 3 with the solid particles obtained in step 4, adding adjuvants to obtain soft material, sieving to obtain wet granule, drying, granulating, tabletting and/or encapsulating to obtain oral preparation in the form of granule, tablet or capsule.
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