CN112889967A

CN112889967A - Preparation method of Pu-Er ripe tea extract and application of Pu-Er ripe tea extract in intestinal drug health-care products

Info

Publication number: CN112889967A
Application number: CN202110197445.3A
Authority: CN
Inventors: 侯艳; 肖蓉; 张智芳; 何飞
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2021-06-04

Abstract

The invention discloses a preparation method of a cooked puerh tea extract and application thereof in intestinal drug health care products, belonging to the field of pharmacy and health care food, wherein cooked puerh tea is ground and sieved by a sieve of 20-30 meshes; adding boiling water into tea powder, leaching for 20-40 minutes in a boiling water bath environment, filtering while hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate at the temperature of less than or equal to 70 ℃ under reduced pressure until the tea is: the volume of the concentrated solution is 1:5-1:10, sterilizing the concentrated solution in conical flask at 110-130 deg.C for 15-40min, and storing the obtained tea soup in refrigerator at 2-10 deg.C. The cooked Pu-Er tea extract can promote the growth of beneficial bacteria in intestinal tract, inhibit harmful bacteria, and restore the microbial balance in intestinal tract, and can be used in intestinal tract medicine.

Description

Preparation method of Pu-Er ripe tea extract and application of Pu-Er ripe tea extract in intestinal drug health-care products

Technical Field

The invention belongs to the field of pharmacy and health-care food, and relates to a preparation method of a Pu-Er ripe tea extract and application of the Pu-Er ripe tea extract in intestinal drug health-care products.

Background

In recent years, studies on the influence of intestinal microorganisms on cancer and tumor immunotherapy have been continuously published in the journal of science, and the immunotherapy that humans hopefully fight against cancer is actually determined by intestinal microorganisms. The intestinal tract is an important place for human digestion and absorption and is also the largest immune organ. The number of microorganisms in the intestinal tract of human body is up to 10¹⁴The mass of the biological bacteria is about 1.2kg, which is 10 times of the number of human cells, wherein 90-99% of the biological bacteria are obligate anaerobes, and 1-10% of the biological bacteria are aerobic bacteria, mainly comprise bacteria, archaea and eukaryotes, and play an extremely important role in aspects of nutrient metabolism, human body self-development, immunity, disease generation and the like. Human intestinal microorganisms mainly colonize the colon, about 10¹²cfu/mL, jejunum, ileum and duodenum, wherein different types of intestinal microorganisms are combined according to a certain proportion and are in interdependence and mutual coordination dynamic balance with a host, human age factors, diet types, environment, intestinal tract dynamic abnormality, psychological factors, application of antibiotics and the like can all influence intestinal flora, and the intestinal normal flora can promote metabolic function, synthesize nutrient substances required by a human body, protect intestinal mucosa, resist immune inflammation, resist tumors and the like; the dysbacteriosis of intestinal tract causes various diseases of host, such as inflammatory bowel disease, colorectal tumor, obesity, diabetes, metabolic syndrome, etc. Intestinal microorganisms affect human health from many aspects, and are closely related to obesity, diabetes, Parkinson's disease, inflammatory bowel disease, autism and the like. However, because the flora imbalance is caused by factors such as antibiotic abuse, intestinal canal dysfunction, age increase and the like, normal flora is reduced, pathogenic bacteria breed in large quantities, and a host is pathogenic, therefore, in order to better prevent and treat adverse consequences caused by the intestinal canal microorganism imbalance, a natural drink is searched, which can enrich the diversity of the intestinal canal flora, improve the intestinal canal microecology, enhance the immunity of the organism and obviously improve the cure rate of cancer and tumor rehabilitation.

Pu' er tea is considered as a beverage with health care efficacy all the time. Scientific research shows that the Pu' er tea contains tea polyphenol, tea pigment, protein, vitamins, fat, saccharides, mineral substances and other components, and has certain health care and disease prevention effects on human bodies. Pu 'er tea belongs to post-fermentation tea, and a large number of researches prove that the Pu' er tea has various health-care effects of losing weight, reducing fat, resisting tumors, reducing blood sugar and the like, and the occurrence of the diseases is closely related to intestinal microorganisms. The health care effect of Pu 'er tea is attributed to the fact that the Pu' er tea contains various bioactive components such as polyphenol, flavonoid, theabrownin, coffee and the like (Houyan, Shaobanhang and the like, 2009; Tu, Chen et al 2016, Roda, Marinelo et al 2019). After drinking, the components are decomposed by small intestine microorganisms in the gastrointestinal tract to form new metabolites, and the Pu' er tea regulates the intestinal microbiota through the components and the metabolites thereof (Lee, Jenner et al, 2006). The inhibition effect of Pu' er tea water on intestinal pathogenic bacteria vibrio parahaemolyticus, escherichia coli and salmonella is researched by Luxiaoqing and the like (2009); monatin and the like (2017) research that the Pu' er tea can promote the isolated intestinal canal of a mouse to automatically contract and relieve gastrointestinal diseases; huangfengjie et al (2019) found that theabrownin in Pu' er tea can reduce hypercholesterolemia by regulating intestinal microorganisms and bile acid metabolism. However, the research on the prevention and treatment of enteritis and intestinal dysbacteriosis of Pu 'er tea is few at present, and the action mechanism of the Pu' er tea is not clear. The research on the regulation of intestinal microorganisms is rarely reported at home and abroad.

Disclosure of Invention

The invention provides a preparation method of a Pu-Er ripe tea extract and application thereof in intestinal drug health care products, which solves the problems that normal flora is reduced, pathogenic bacteria are propagated in large quantities and a host is pathogenic due to flora imbalance caused by factors such as antibiotic abuse, intestinal dysfunction, age increase and the like in the prior art.

The technical scheme adopted by the invention is that the preparation method of the Pu-Er ripe tea extract comprises the following steps: grinding cooked Pu-Er tea, and sieving with 20-30 mesh sieve; adding boiling water into tea powder, leaching for 20-40 minutes in a boiling water bath environment, filtering while hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate at the temperature of less than or equal to 70 ℃ under reduced pressure until the tea is: the volume of the concentrated solution is 1:5-1:10, sterilizing the concentrated solution in conical flask at 110-130 deg.C for 15-40min, and storing the obtained tea soup in refrigerator at 2-10 deg.C.

Further, grinding the pu-er ripe tea, and sieving the ground pu-er ripe tea with a 20-mesh sieve; adding boiling water into tea powder, leaching for 30 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate at the temperature of less than or equal to 60 ℃ under reduced pressure until the weight of the tea leaves is as follows: the volume of the concentrated solution is 1:10, sterilizing the concentrated solution in conical flask at 121 deg.C for 20min, and storing the obtained tea soup in refrigerator at 4-8 deg.C.

The invention adopts another technical scheme that the cooked puerh tea extract is applied to intestinal drugs, and the cooked puerh tea extract can increase the relative abundance of mouse intestinal firmicutes, bacteroides, lactobacillus, bacteroides, clostridium XlVa and Scutellaria, and reduce the relative abundance of proteobacteria and paracoccus.

Furthermore, the Pu-Er ripe tea aqueous extract can increase the intestinal probiotics lactobacillus and bifidobacterium of mice with dysbacteriosis and inhibit the growth of intestinal conditional pathogenic bacteria enterobacter and enterococcus.

The invention adopts another technical proposal that the Pu-Er ripe tea extract is applied to the intestinal health care products.

The invention has the beneficial effects that: the embodiment of the invention has at least the following purposes:

the Pu-Er ripe tea extract can increase the body weight and food intake of mice with dysbacteriosis caused by antibiotics; while restoring platelet count, mean platelet volume, leukocyte count, lymphocyte count, monocyte count, erythrocyte count and hemoglobin in the blood to normal ranges.

The Pu-Er ripe tea extract has a good prevention effect on the jejunal histopathological changes of mice with dysbacteriosis, can better recover the gruenhagen's cavity under the damaged villus epithelium of the jejunum of the mice, reduces epithelial cell shedding and gland damage, and prompts that low, medium and high doses of Pu-Er ripe tea have the effect of protecting intestinal mucosa, wherein the low dose effect of the Pu-Er tea is the best.

After the combined antibiotic is used, the number of intestinal lactobacilli and bifidobacteria in the mice is obviously reduced (P is less than 0.05), and the enterobacteria and enterococci are not detected; after the Pu ' er ripe tea aqueous extract is dried, the number of lactobacillus and bifidobacterium in a low, medium and high dose group of Pu ' er tea is obviously higher than that in a model group (P is less than 0.05), the number of lactobacillus and bifidobacterium in the low, medium and high dose group of Pu ' er tea can be recovered to the level at the end of an adaptation period, and the number of enterobacteria and enterococcus in the low, medium and high dose group of Pu ' er tea is obviously lower than that in a natural recovery group and the level at the end of the adaptation period (P is less than 0.05), which shows that the Pu ' er ripe tea after gastric lavage can increase the number of beneficial bacteria in intestinal canals of mice with dysbacteriosis, inhibit the growth of the enterobacteria and the enterococcus, and has the best dose effect in the Pu.

The high-throughput sequencing result of the mouse fecal microorganism Illumina Hiseq shows that the dilution curve tends to be flat under the similarity level of 0.97, and the composition of the mouse fecal microorganism community can be basically reflected. The stomach-perfused Pu-erh cooked tea aqueous extract can obviously improve the number of OTU (over the air) of the flora abundance of mice with dysbacteriosis and Shannon and Simpson indexes of the diversity of the flora of the mouse intestinal tract.

Species clustering analysis results show that the water extract of the stomach-perfused Pu-Er ripe tea can increase beneficial flora F firmicutes, bacteroides, lactobacillus, bacteroides, clostridium XlVa and paracoccus of mice intestinal tracts, reduce the relative abundance of harmful flora proteobacteria and paracoccus, and show that the Pu-Er ripe tea can promote the growth of beneficial flora in the intestinal tracts, inhibit harmful flora and restore the intestinal microbial balance.

The Pu-Er ripe tea extract has protective effect on intestinal mucosa of mice with dysbacteriosis, can increase intestinal probiotic lactobacillus and bifidobacterium of the mice with dysbacteriosis, inhibit growth of intestinal pathogenic bacteria enterobacter and enterococcus, and improve intestinal flora abundance and flora diversity of the mice, thereby promoting intestinal microecological balance.

The cooked Pu-Er tea extract can be used in intestinal tract medicine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the small intestine of each group of mice;

FIG. 2(A) is a view showing the result of microscopic observation of the blank control group 0d small intestine tissue;

FIG. 2(B) is a view showing the microscopic observation results of the small intestine tissue of model group 7 d;

FIG. 2(C) is a view showing the microscopic observation results of the small intestine tissue in the model group 21 d;

FIG. 2(D) is a drawing showing the microscopic observation results of the tissue of the small intestine of the natural recovery group 21D;

FIG. 2(E) is a drawing showing the result of microscopic observation of the small intestine tissue of the positive control group 21 d;

FIG. 2(F) is a view showing the observation result of a low-dose group of Pu' er ripe tea on the tissue of small intestine for 21d under a microscope;

FIG. 2(G) is a microscope observation result of the tissue of small intestine of a dose group of 21d in Pu-Er ripe tea;

FIG. 2(H) is a microscope observation result of the tissue of the small intestine of the Pu-Er ripe tea high-dose group for 21 d.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: grinding Pu-Er ripe tea, and sieving with 20-30 mesh sieve; adding boiling water into tea powder, leaching for 20-40 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate under reduced pressure (less than or equal to 70 ℃) to tea leaves (weight): concentrating to obtain concentrated solution (volume) of 1:5-1:10, sterilizing in conical flask at 110-130 deg.C for 15-40min, and storing the obtained tea soup in refrigerator at 2-10 deg.C.

The conventional ingredients were measured as shown in Table 1 below

TABLE 1

Example 2: grinding cooked Pu-Er tea, and sieving with 30 mesh sieve; adding boiling water into tea powder, leaching for 20 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate under reduced pressure (less than or equal to 60 ℃) to tea leaves (weight): concentrating the filtrate at a volume of 1:5, sterilizing the concentrated solution in conical flask at 110 deg.C for 15min, and storing the obtained tea soup in refrigerator at 2-4 deg.C.

The conventional ingredients were measured as shown in Table 2 below

TABLE 2

Example 3: grinding cooked Pu-Er tea, and sieving with 20 mesh sieve; adding boiling water into tea powder, leaching for 40 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate under reduced pressure (less than or equal to 60 ℃) to tea leaves (weight): concentrating the filtrate at volume ratio of 1:8, sterilizing the concentrated solution in conical flask at 130 deg.C for 40min, and storing the obtained tea soup in refrigerator at 4-10 deg.C.

The conventional ingredients were measured as shown in Table 3 below

TABLE 3

Example 4: grinding cooked Pu-Er tea, and sieving with 20 mesh sieve; adding boiling water into tea powder, leaching for 30 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate under reduced pressure (less than or equal to 60 ℃) to tea leaves (weight): concentrating the filtrate at a volume of 1:10, sterilizing the concentrated solution in conical flask at 121 deg.C for 20min, and storing the obtained tea soup in refrigerator at 4-8 deg.C.

The conventional ingredients were measured as shown in Table 4 below

TABLE 4

Example 5 experiment of Pu-Er ripe tea extract on growth index of mice in dysbacteriosis model

(1) SPF-grade BALB/c female mice (20 ± 2g) of 105, 8 weeks old were selected and provided by the experimental animal technology ltd, viton, beijing [ batch No.: SCXK (Jing) 2012-. The cage and the padding should be comfortable, sanitary, nontoxic and safe. The padding is changed every 2 days, the drinking water bottles are periodically changed with water for disinfection, the feeding room is regularly disinfected by white vinegar or disinfectant, and the room is regularly ventilated, so that a good feeding environment is maintained.

(2) Mice with unqualified body weight were removed after 7 days of feeding. Randomly extracting 13 mice as blank groups according to similar body weights, and performing continuous gavage for 7 days by using the rest gavage clindamycin and ampicillin (1.88g/kg & bw +1.56g/kg & bw) to establish a dysbacteriosis mouse model.

(3) After successful flora modeling, the dysbacteriosis mice were randomly divided into 6 groups of 13 mice each. Respectively as follows: flora imbalance model group, natural recovery group, positive drug control group, and low, medium, and high dose groups of Pu-Er ripe tea (low, medium, and high doses are 1.0g/kg · bw, 1.5g/kg · bw, and 2.0g/kg · bw, respectively).

(4) According to the average body weight of each group of test animals, the prepared test samples are respectively subjected to oral gavage according to the designed dosage and the optimal gavage volume calculated according to the 0.1ml/10g · bw. In the modeling period, a blank control group is perfused with distilled water with the same volume as the perfused stomach, and the rest groups are perfused with antibiotics with corresponding doses; the experimental mouse grouping and gavage dose design of the Pu-Er ripe dried tea is shown in Table 5, wherein distilled water, a flora imbalance model group, a positive drug control group and a drug group with corresponding dose of low, medium and high dose groups of Pu-Er ripe tea obtained in example 1 are subjected to oral gavage respectively, and the distilled water, the flora imbalance model group, the positive drug control group and the drug group with corresponding dose are subjected to gastric gavage respectively.

TABLE 5

The experimental observation period is 21 days, the activity of the mice is observed every day, the weight of the mice is weighed every 3 days, the intragastric dose is adjusted according to the weight, and the food intake is recorded.

(5) Results

A) The results of the influence of the Pu-Er ripe tea extract on the body weight of the mice with dysbacteriosis are shown in Table 6

TABLE 6

Note: the post-molding body weight change rate (%) (post-molding body weight-initial body weight)/post-molding body weight 100, and the post-intervention body weight change rate (%) (final body weight-post-molding body weight)/final body weight 100.

Note: the difference between lower case letters in the same column indicates significant difference (P < 0.05), and the difference between upper case letters in the same row indicates significant difference (P < 0.05). "- - - -" indicates no detection. The following tables are the same.

As can be seen from Table 6, after the gavage of the antibiotics, the mice in the model group had increased weight loss and a greater rate of change of weight compared with the mice in the blank group, wherein the weight loss of the Pu-Er ripe tea at a high dose, the dose of the Pu-Er ripe tea at a medium dose, and the positive control group had significant differences (P < 0.05). After the prognosis of the cooked puerh tea, the weight average of mice in each test group is increased, the weights of a high-dose puerh tea group and a positive control group are greatly increased, the weight change rate is larger, but the weight change rate is not different from that of a model group (P is more than 0.05), the weight of a mouse in the model group is increased, and the mouse has self-regulation effect along with the prolongation of the gavage time.

B) The effect of Pu-Er ripe tea extract on average food intake of dysbacteriosis mice is shown in Table 7

TABLE 7

Group of	Modeling average food intake (g/only/day)	Average food intake (g/only/day) in intervention period of Pu' er ripe tea
			Blank control group	6.06±0.20^a	5.70±0.66^ab
Model set	4.91±0.13^b	6.76±1.45^a
			Natural recovery group	4.94±0.92^b	5.76±0.74^ab
Positive control group	5.17±0.31^ab	5.82±0.22^ab
			Pu-Er ripe teaLow dose group	4.50±0.77^b	5.64±0.15^ab
Dosage group in Pu-Er ripe tea	4.95±0.58^b	5.27±0.51^b
			High-dose Pu-Er ripe tea group	5.06±0.33^b	5.76±0.15^ab

As can be seen from Table 7, compared with the blank control group, the intragastric antibiotic reduces the daily food intake of the mice (P is less than 0.05), and after the Pu' er tea leaves are ripe, the daily food intake of the mice is increased, and has no significant difference with the blank group (P is more than 0.05).

Example 6 comparison of Pu-Er ripe tea extract to mouse viscera with dysbacteriosis

(4) According to the average body weight of each group of test animals, the prepared test samples are respectively subjected to oral gavage according to the designed dosage and the optimal gavage volume calculated according to the 0.1ml/10g · bw. In the modeling period, a blank control group is perfused with distilled water with the same volume as the perfused stomach, and the rest groups are perfused with antibiotics with corresponding doses; according to the prediction of the cooked puerh tea, distilled water, a flora imbalance model group, a positive medicine control group and a medicine group with corresponding dosage of oral gavage are respectively taken from a blank control group and a natural recovery group, wherein the distilled water, the flora imbalance model group, the positive medicine control group and the low, medium and high dosage groups of the puerh cooked tea in the embodiment 1, and the grouping and gavage dosage design of test mice are the same as the embodiment 5.

On the 21 st day of the Pu' er ripe tea for gastric lavage, a patient is fasted for 12 hours without water prohibition, the mouse is sacrificed, the heart, the liver, the spleen, the lung and the kidney are picked up, the ratio of the weight of the viscera to the weight of the body is calculated, and the ratio of the viscera to the weight of the body is organ weight (mg)/weight (g) after fasting.

(5) Results

The influence of Pu-Er ripe tea extract on the viscera ratio of mice with dysbacteriosis is shown in Table 8.

TABLE 8

As can be seen from Table 8, there was no significant difference in heart/body, liver/body, kidney/body, and spleen/body (P > 0.05) among the mice in each test group; compared with a positive control group, the lung/body of a dose group in the Pu-Er ripe tea is remarkably increased by 14.60 percent (P is less than 0.05).

Example 7 experiment of Pu-Er ripe tea extract on peripheral hemogram of flora-imbalanced mice

On the 0 th day, the 7 th day and the 21 st day of the stomach-perfused Pu-erh ripe tea, 20 mu L of tail vein blood is taken from each mouse, and the peripheral hemogram of the mouse is measured by a BC-2800vet full-automatic blood cell analyzer.

(5) Results

A) Influence of Pu' er ripe tea extract on blood platelet of mice with dysbacteriosis

1) The results of the effect of Pu' er ripe tea extract on the number of platelets and the average platelet volume of mice with dysbacteriosis are shown in Table 9.

TABLE 9

As can be seen from Table 9, the differences between PLT and MPV were not significant (P > 0.05) in the mice of each experimental group before modeling; after antibiotic modeling, compared with a blank group, the number of PLTs (P is less than 0.05) of other groups of test mice is remarkably reduced, and the volume of MPV (P is less than 0.05) of a part of groups is increased; compared with the blank group, the prognosis of the Pu-Er ripe tea dried bean curd is that the difference of the PLT number and the MPV volume of the Pu-Er ripe tea (low, medium and high) dosage group is not significant (P is more than 0.05), which indicates that the Pu-Er ripe tea increases the generation of blood platelets, changes the blood coagulation function to a certain extent and reduces the abnormal volume of the blood platelets.

2) The results of the effect of Pu' er ripe tea extract on the distribution width of platelets and the platelet aggregation in mice with dysbacteriosis are shown in Table 10.

Watch 10

As can be seen from Table 10, the differences between PDW and PCT in the mice of each test group were not significant (P > 0.05) before modeling; after antibiotic modeling, compared with a blank group, the distribution width of partial group PDW platelets is increased, the difference is obvious (P is less than 0.05), and the PCT difference of each test group mouse is not obvious (P is more than 0.05); after the Pu 'er tea is ripe, the difference between PDW and PCT of mice in each test group is not obvious (P is more than 0.05), which shows that the antibiotic increases the PDW of the mice, the volume and the size of platelets are different, and the Pu' er tea increases the uniformity of the platelets of the mice.

B) Influence of Pu-Er ripe tea extract on dysbacteriosis of mouse leukocyte

1) The influence of Pu-Er ripe tea extract on the white blood cell number and lymph number of dysbacteriosis mice is shown in Table 11.

TABLE 11

As can be seen from Table 11, the difference of the WBC and Lymph # numbers of the mice in each test group is not significant (P is more than 0.05) before modeling; after antibiotic modeling, the WBC and Lymph # numbers of the mice in the model group are obviously reduced compared with those in the blank group (P is less than 0.05); compared with the blank group, the numbers of WBC and Lymph # of the Pu-erh ripe tea stem prognosis has no significant difference (P is more than 0.05), which shows that the Pu-erh ripe tea can increase the numbers of WBC and Lymph # of mice, enhance the immunity of the organism and reduce the inflammation.

2) The results of the effect of Pu-Er ripe tea extract on the monocyte number and neutrophil number of dysbacteriosis mice are shown in Table 12.

TABLE 12

As can be seen from Table 12, the differences in the Mon # and Gran # numbers were not significant (P > 0.05) for each experimental group before modeling; after the antibiotic modeling, the number of Mon # of mice in a test group of the gavage antibiotic is reduced to different degrees, the difference between a natural recovery group and a low-dose group is obvious compared with that of a blank group (P is less than 0.05), and the difference between the number of Gran # of the mice in each test group is not obvious (P is more than 0.05); after the prognosis of the cooked puerh tea, the number of Mon # in the puerh cooked tea (low, medium and high) dosage group mice is increased, the difference with the blank group is not significant (P is more than 0.05), and the number of Gran # in the puerh cooked tea (low and medium) dosage group is obviously reduced (P is less than 0.05) compared with the blank group, which shows that the puerh cooked tea can increase the number of Mon # in the mice, increase the hematopoietic function of the mice and strengthen the defense system of the organism.

C) Influence of Pu-Er ripe tea extract on red blood cells of mice with dysbacteriosis

1) The results of the effect of Pu' er ripe tea extract on the number of red blood cells and the average volume of red blood cells in mice with dysbacteriosis are shown in Table 13.

Watch 13

As can be seen from Table 13, the difference between the number of red blood cells and the mean volume of red blood cells in each test group was not significant (P > 0.05) before modeling; after modeling, the number of red blood cells and the average volume of red blood cells of mice in the model group have no significant difference compared with the blank group (P is more than 0.05); after the Pu-Er ripe tea stem is used for prognosis, the average red blood cell volume difference of mice in each test group is not significant (P is more than 0.05), the red blood cell number of a dose group in the Pu-Er ripe tea is remarkably increased (P is less than 0.05) compared with that of a model group, and the red blood cell number of a model group mouse is remarkably reduced (P is less than 0.05) compared with that of a blank group, so that the Pu-Er ripe tea can increase the red blood cell number of the mice and increase the blood carrying function of the mice.

2) The results of the effect of Pu' er ripe tea extract on the hemoglobin and mean corpuscular hemoglobin concentration of the mice with dysbacteriosis are shown in Table 14.

TABLE 14

As can be seen from Table 14, the hemoglobin and mean corpuscular hemoglobin concentration of the mice of each test group were not significantly different (P > 0.05) before modeling; after antibiotic modeling, the hemoglobin concentration of the mice and the hemoglobin concentration of the average red blood cells are not obviously different from those of a blank group (P is more than 0.05); after the Pu-Er ripe tea is filled with stomach, compared with the blank group, the hemoglobin of the dose group in the Pu-Er ripe tea is increased (P is less than 0.05); the difference of the average erythrocyte hemoglobin concentration of each test group mouse is not obvious (P is more than 0.05), which indicates that the Pu-erh ripe tea can increase the hemoglobin of the mouse and enhance O₂、CO₂Transported in vivo.

Example 8 dynamic change test of Pu-Er ripe tea extract on small intestine tissue pathology of mice with dysbacteriosis

On test days 0, 7 and 14, 3 mice were selected from each group, sacrificed after fasting for 12 hours, and the small intestine of the mice was aseptically taken from the clean bench, and the operation procedure was as follows:

the contents of the small intestine were rinsed with PBS; placing part of jejunum tissue with a thickness of 3-5mm in an embedding box; fixing in 10% neutral formaldehyde solution (for use in preparation);

fixing: the volume of the fixing liquid is 10-20 times of that of the specimen, otherwise, the dyeing is affected due to insufficient fixation; the container for fixing the specimen is preferably such that the specimen is not deformed;

paraffin embedding: during embedding, the tissue is pressed to the bottom of the embedding mold fully to ensure that the expected observation section of the tissue is at the same level; pathological section;

bleaching the sheet: the temperature of the floating sheet is selected to be 10 ℃ lower than the melting point of the wax block. The wax strips can generate small bubbles with different numbers due to the over-high temperature;

baking slices: oven drying at 37 deg.C for 1 day or oven drying at 75 deg.C for 20 min;

dyeing: HE staining (hematoxylin-eosin staining) for 1 min;

sealing: drying the slices by blowing or baking, otherwise, observing under a mirror with a layer of white fog, and causing the cell outline to be unclear; the bubble-free sealing is performed; and carrying out microscope routine pathological analysis.

(5) Results

As shown in fig. 1, the intestine of each group of mice is shown, and the intestine of the mice is directly observed by naked eyes, so that the intestine of the mice in the blank group is light yellow, and the tube wall is thick; the mouse in the model group has congestion and swelling of intestinal canal wall, thin and thick canal wall and lymph nodes. After the Pu 'er tea is ripe, the small intestine appearance of each dose of the Pu' er tea and the positive control group is improved compared with that of the model group, wherein the small intestine tissue appearance of the Pu 'er tea low dose group is between the model group and the positive control group, and the small intestine tissue appearance of the Pu' er tea medium and high dose groups is closest to that of the blank group.

The results of evaluation of the cooked puerh tea extract on the pathological dynamic changes of the small intestine tissues and the improvement of the small intestine tissues of the mice are shown in Table 15.

Watch 15

Note: 0 minute: normal villi and glands; 1 minute: the subcutaneous Gruenhagen's cavity begins to form on top of the partial villi; and 2, dividing: subcutaneous Gruenhagen's lumen formation, mild gland damage; and 3, dividing: enlargement of the subepithelial space, engorgement of capillary vessels; and 4, dividing: moderate separation of epithelium from lamina propria, gland damage; and 5, dividing: part of the top fluff falls off; 6 min: the villus is obviously shed, and the blood capillaries are dilated; 7, dividing: the villi of the lamina propria falls off, the gland is obviously damaged; 8 min: the lamina propria begins to digest and decompose; 9 min: bleeding and ulceration.

As can be seen from Table 15, the Chiu's score for improvement of small intestinal mucosa, the determination of changes in small intestinal histology, diagnostic criteria and results.

After modeling: the blank group is scored to be 1.00 +/-1.00, the model group is scored to be 6.00 +/-1.00, the blank group and the model group have obvious difference (P is less than 0.05), and the antibiotic has damage to the small intestinal mucosa of the mouse and has obvious modeling effect.

Dry prognosis: after the prognosis of the Pu' er tea dried meat, except for a blank group, the small intestines of mice of all groups have pathological changes with different degrees. Compared with the model group, the Chiu's score of the low, medium and high dose Pu-erh tea group is obviously reduced (P is less than 0.05), which shows that the Pu-erh ripe tea has protective effect on the intestinal tissue damage of the diarrhea model mice. Compared with a natural recovery group, Chiu's scores of the low-dose and medium-dose Pu' er tea groups are remarkably reduced (P is less than 0.05), Chiu's scores of the low-dose, medium-dose and high-dose Pu' er tea groups are remarkably increased (P is less than 0.05) compared with a blank group, and Chiu's scores of the low-dose, medium-dose and high-dose Pu' er tea groups are not remarkably different (P is more than 0.05) compared with a positive control group.

The results of microscopic observation of the small intestine tissue at each stage of the experiment are shown in FIGS. 2(A) to (H), in which the blank control group 0 d: the mucosa structure is basically complete, the local villus subepithelial space is formed, and the capillary vessel congestion and epithelial desquamation do not exist.

Fig. (B) shows a model group 21 d: high edema of villi, obvious shedding of epithelial cells, obvious gland injury, formation and enlargement of gruenhagen's cavity, separation of epithelium from lamina propria, telangiectasia and hyperemia. Mucosal damage was close to or slightly lighter than post-intervention model groups.

Fig. (C) shows a model group 21 d: high edema of villi, obvious shedding of epithelial cells, severe gland injury, formation and enlargement of gruenhagen's lumen, separation of epithelium from lamina propria, telangiectasia and hyperemia. The group had the most severe damage to the small intestinal mucosa.

Fig. (D) shows the spontaneous recovery group 21D: the mucosal structure is basically intact, the villi are slightly edematous, the subcutaneous space on part of villi is enlarged, and the gruenhagen's cavity is formed, but the epithelium and the lamina propria are not obviously separated in most cases. In some cases, capillary congestion was observed, a few scattered epithelial cells were observed in the intestinal lumen, and the gland damage was more severe than in the blank group but less severe than in the model group and the positive control group.

Panel (E) is positive drug control group 21 d: moderate edema of villi, enlarged space under villous epithelium, formation and enlargement of gruenhagen's lumen, separation of part of epithelium from lamina propria, congestion of capillary vessels, and exfoliation of a small number of epithelial cells. The gland damage was slightly more severe or near the recovery group, but lighter than the model group.

The graph (F) shows that the Pu' er tea low-dose group is 21 d: the mucosa structure is basically complete, no obvious edema exists in the mucosa, part of villus epithelial and subcutaneous gruenhagen's cavities begin to form, part of subepithelial gaps are enlarged, and part of cases have local capillary congestion. This group is lighter than the recovery group.

The graph (G) shows that the dosage group in the Pu' er tea is 21 d: the recovery group is approached. Lesions were similar to recovery and high dose groups.

The graph (H) shows that the Pu' er tea high-dose group is 21 d: mild edema of villi, enlarged space under villous epithelium, formation of gruenhagen's lumen, congestion of local capillaries, and a few exfoliated epithelia in the intestinal lumen of individual cases. This group is close to the recovery group.

Example 9 experiment of adjusting intestinal microorganisms in disordered mice by Pu-Er ripe tea extract

Selecting 3 mice in each group on 0 day, 7 days and 21 days after the adaptation period is finished, immediately placing the mice on a super-clean workbench after cervical vertebra is removed and sacrificed, wiping the abdomen of the mice with alcohol cotton for disinfection, then cutting the abdomen of the mice with a scalpel, aseptically taking 0.5g of cecal contents of the mice, and placing the mice in an autoclave conical flask filled with glass beads. 4.5mL of sterile physiological saline was added, and the contents were homogenized by shaking on a shaker for 15 min. Then inoculating, culturing, separating and identifying the main microorganisms (lactobacillus, bifidobacterium, enterococcus and enterobacter) in the 4.

(5) Results

(A) Influence of modeling period and Pu' er ripe dried tea prediction on activity state and fecal dilution

And (3) an antibiotic modeling period: the blank mice normally drink water, actively move and have bright hair color in the whole molding process. On the 4 th day of the model building period of the model group mice, the excrement is wet, softened and yellow, and the activity and the hair color are normal; on day 6, the model group mice had mild diarrhea, slightly raised abdomen, and aversion to cold; on the 7 th day of modeling, mice in the model group are listened, averted to cold, curled into a ball, raised in abdomen, sparse and upright in hair, reduced in activity, lightened in body mass, reddish around the anus, diarrhea, anatomical observation, congestion of small intestine, enlargement of cecum, gas, watery cecum content, severe diarrhea death of 3 mice, and the modeling condition is the same as the research result of Zeng ao and the like, and the result proves that the intragastric clindamycin hydrochloride capsule and the ampicillin capsule are combined with antibiotics to successfully establish a dysbacteriosis model.

Prediction of cooked Pu' er tea: on the 5 th day, the diarrhea degree of the Pu' er tea (low, medium and high dose) group and the positive control group is better, and the diarrhea degree of the model group and the natural recovery group is reduced; on day 9, the Pu' er tea (low, medium and high dose) group and the positive control group mice normally move, the feces are wet, the particles are slightly large and dark brown, the tea pigment is possibly incompletely metabolized and is deposited in the feces, the model group mice have moderate diarrhea, the natural recovery group mice have mild diarrhea, and the natural recovery group mice can have regulating effect; on day 14, the model group mice had moderate diarrhea and 1 dead, and the remaining groups of mice were active normally and stools were essentially normal.

(B) The results of the effect of Pu-Er ripe tea on the intestinal flora of dysbacteriosis mouse with lactobacillus are shown in Table 16.

TABLE 16

As can be seen from Table 16, after the model period ended, the number of lactobacilli in mice in the model group was significantly reduced (P < 0.05) by the gavage antibiotics compared with the blank control group, and the dysbacteriosis model was successfully established; after the intervention period, the number of the lactobacilli is obviously increased (P is less than 0.05) in the Pu ' er tea (low, medium and high) dose group and the positive control group compared with the model group, the lactobacillus number difference is not obvious (P is more than 0.05) in the Pu ' er tea (low and medium) dose group and the positive control group compared with the blank control group, and the lactobacillus number difference is not obvious (P is more than 0.05) in the Pu ' er tea (low and medium) dose group compared with the positive control group.

Compared with the end of the model period, the number of lactobacillus in the Pu' er tea (low, medium and high) dose group and the positive control group is obviously increased (P is less than 0.05); compared with the end of the adaptation period, the end of the expected drying period has no obvious difference (P is more than 0.05) between the number of the lactobacillus in the Pu ' er tea in the dose group and the number of the lactobacillus in the positive control group, and the number of the lactobacillus in the mice with dysbacteriosis is close to the end and the blank group of the adaptation period, so that the Pu ' er ripe tea has a certain recovery and adjustment effect on the number of the lactobacillus of the intestinal beneficial bacteria of the mice with dysbacteriosis caused by antibiotics, and the effect of the dose group in the Pu ' er tea is the.

(C) The results of the effect of Pu' er ripe tea on bifidobacteria in intestinal flora of mice with dysbacteriosis are shown in Table 17.

TABLE 17

As can be seen from Table 17, after the model period ended, compared with the blank control group, the gavage antibiotic significantly reduced the number of bifidobacteria in the mice in the model group (P < 0.05), and the dysbacteriosis model was successfully established; after the intervention period, the number of bifidobacteria is obviously increased (P is less than 0.05) in the Pu ' er tea (low, medium and high) dosage group compared with the model group, the difference of the number of bifidobacteria is not significant (P is less than 0.05) in the Pu ' er tea (low, medium and high) dosage group compared with the positive control group, and the difference of the number of bifidobacteria is not significant (P is more than 0.05) in the Pu ' er tea (medium and high) dosage group compared with the blank control group.

Compared with the end of the model period, the number of bifidobacteria in a Pu 'er tea (low, medium and high) dose group and a positive control group is remarkably increased (P is less than 0.05), and the number of bifidobacteria in the Pu' er tea (low and medium) dose group is not remarkably different from the number of bifidobacteria in the adaptation period (P is more than 0.05) only, so that the Pu 'er ripe tea has a certain recovery and adjustment effect on the number of beneficial bacteria bifidobacteria in intestinal tracts of mice with dysbacteriosis caused by antibiotics, and the low and medium dose effects of the Pu' er tea are best and can be recovered to the level at the end of the adaptation period.

(D) The results of the effect of Pu-Er ripe tea on intestinal flora of mice with dysbacteriosis are shown in Table 18.

Watch 18

As can be seen from Table 18, after the model period is over, the enterobacter enteric canal of the mice in the model group is not detected by the gavage antibiotic, and the establishment of the dysbacteriosis model of the intestinal canal of the mice is successful; after the intervention period is finished, the increase amplitude of the enterobacter in the Pu 'er tea (low, medium and high) dose group and the positive control group is obviously lower than that in the natural recovery group (P is less than 0.05), and the enterobacter quantity in the Pu' er tea (low, medium and high) dose group is obviously lower than that in the blank group (P is less than 0.05).

After the intervention period is finished, the number of enterobacteria in (low, medium and high) Pu 'er tea dose groups is obviously lower than that of enterobacteria at the end of the adaptation period (P is less than 0.05), but the number of enterobacteria in the natural recovery groups is not obviously different from that at the end of the adaptation period (P is more than 0.05), which shows that the Pu' er ripe tea can inhibit the growth of conditional pathogenic enterobacteria and has the effect similar to that of bacillus subtilis in a positive control group.

(E) The results of the effect of Pu' er ripe tea on the intestinal bacteria enterococcus in the dysbacteriosis mice are shown in Table 19.

TABLE 19 (Unit: lgCFU/g)

As can be seen from Table 19, after the model period ended, the enterococcus in the mice in the model group was not detected by the gavage antibiotics, and the establishment of the dysbacteriosis model of the intestinal flora of the mice was successful; after the intervention period is finished, the enterococcus increasing amplitude of the Pu 'er tea (low, medium and high) dose group and the positive control group is obviously lower than that of the natural recovery group (P is less than 0.05), and the enterococcus number of the Pu' er tea (low, medium and high) dose group is obviously lower than that of the blank group (P is less than 0.05).

After the intervention period is finished, the quantity of enterococcus in the Pu 'er tea (low, medium and high) dose groups is obviously lower than that of the enterococcus in the adaptation period (P is less than 0.05), which shows that the Pu' er ripe tea can inhibit the growth of the conditional pathogenic bacteria enterococcus, and the effect is similar to that of the positive control group bacillus subtilis.

Example 10 microbial diversity assay of Pu-Er ripe tea extract on feces of disordered mice

After the intervention period of Pu' er ripe tea is finished, the sterilized padding is replaced on the sampling day for independent feeding, the excrement of the mouse is aseptically taken to be more than 0.5g, and the excrement of each mouse is collected and put into a sterilized EP tube for preservation at minus 80 ℃. Then extracting the genome DNA of the fecal microorganism, and synthesizing a specific primer with barcode according to a specified sequencing region by taking the sample genome DNA as a template. The 16S rDNA amplification selection region is a V3-V4 region, universal primers used are 341F and 806R, after the PCR product is successfully amplified, 2% agarose gel is used for detection, and the gel is cut and recovered by using an Axy Prep DNA gel recovery kit. After successfully amplifying the PCR product, detecting the PCR product by using 2% agarose gel, and cutting and recovering the PCR product by using an Axy Prep DNA gel recovery kit. And detecting and quantifying the purified PCR product library, and then mixing according to the sequencing quantity requirement of each sample in a corresponding proportion.

The experiment was divided into 7 groups, and 4 mice per group were randomly selected and numbered: blank control group (K2-1, K2-2, K2-3, K2-4); model group (M1-2, M1-3, M2-2); the natural recovery group (H1-1, H1-2, H1-3, H2-1); a positive control group (Y1-1, Y1-2, Y1-3, Y2-3); the Pu' er tea low dose group (D1-2, D1-2, D1-3, D2-1); the Pu' er tea medium dosage group (Z1-1, Z1-2, Z1-3 and Z3-1); high-dose Pu' er tea group (G1-1, G1-2, G1-3, G2-1).

(5) Results

Results of analysis of mouse intestinal flora lefse (lda effect size) by cooked puerh tea extract on main microorganisms with significantly higher abundance in different groups than in other groups are shown in table 20.

Watch 20

As can be seen from table 20, the abundance of proteobacteria of the model group is significantly higher than that of other groups, the proteobacteria often cause severe diarrhea and septicemia of animals, chronic intestinal inflammatory disease crohn with unknown etiology, Kaakoush N O and other studies, and the antibiotics significantly increase the proportion of the proteobacteria; the abundance of lactobacillus in the Pu ' er tea and lactobacillus in the Pu ' er tea low-dose group of lactobacillus family and lactobacillus is obviously higher than that of other groups, and the Pu ' er ripe tea can inhibit the growth of harmful microorganisms and promote the growth of beneficial microorganisms.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A preparation method of a cooked puerh tea extract is characterized in that cooked puerh tea is ground and sieved by a sieve with 20 to 30 meshes; adding boiling water into tea powder, leaching for 20-40 minutes in a boiling water bath environment, filtering while hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate at the temperature of less than or equal to 70 ℃ under reduced pressure until the tea is: the volume of the concentrated solution is 1:5-1:10, sterilizing the concentrated solution in conical flask at 110-130 deg.C for 15-40min, and storing the obtained tea soup in refrigerator at 2-10 deg.C.

2. The method for preparing a ripe pu-er tea extract according to claim 1, wherein ripe pu-er tea is ground and sieved by a 20-mesh sieve; adding boiling water into tea powder, leaching for 30 minutes in a boiling water bath environment, filtering while the tea powder is hot, washing residues for 2-3 times by using a small amount of boiling water, and filtering to obtain filtrate; then concentrating the filtrate at the temperature of less than or equal to 60 ℃ under reduced pressure until the weight of the tea leaves is as follows: the volume of the concentrated solution is 1:10, sterilizing the concentrated solution in conical flask at 121 deg.C for 20min, and storing the obtained tea soup in refrigerator at 4-8 deg.C.

3. The use of ripe puer tea extract according to claim 1 in enteral medicine, wherein the ripe puer tea aqueous extract can increase the relative abundance of mouse intestinal firmicutes, bacteroidetes, lactobacilli, bacteroides, clostridia XlVa, and cladosporium, and decrease the relative abundance of proteobacteria and paracoccus.

4. The use of ripe Pu-Er tea extract according to claim 3 in enteral medicine, wherein the ripe Pu-Er tea aqueous extract can increase the intestinal probiotic lactobacilli and bifidobacteria of mice with dysbacteriosis and inhibit the growth of intestinal conditionally pathogenic enterobacteria and enterococci.

5. The use of the ripe Pu-Er tea extract according to claim 1 in intestinal health products.