CN114533750A - Application of pomegranate bark tannin in preparation of medicine for treating enterotoxigenic escherichia coli intestinal diseases - Google Patents

Abstract

The invention belongs to the technical field of intestinal bacterial infection medicines, and discloses application of pomegranate bark tannin in preparation of a medicine for treating enterotoxigenic escherichia coli intestinal diseases. The pomegranate bark tannin can obviously improve intestinal inflammation caused by enterotoxigenic escherichia coli infection, repair damaged intestinal barrier function, maintain intestinal homeostasis, improve the medicinal value of pomegranate bark, and can also be used as a substitute scheme for treating enterotoxigenic escherichia coli-induced intestinal injury by using a green and safe antibiotic.

Description

Application of pomegranate bark tannin in preparation of medicine for treating enterotoxigenic escherichia coli intestinal diseases

Technical Field

The invention belongs to the technical field of intestinal bacterial infection medicines, and particularly relates to application of pomegranate bark tannin in preparation of a medicine for treating enterotoxigenic escherichia coli intestinal diseases.

Background

Enterotoxigenic escherichia coli, which is the most common pathogen isolated from travelers with infectious diarrhea, is transmitted via the faecal-oral route, mainly through food, and is abundant in areas where clean water and sanitary conditions are still limited. Enterotoxigenic escherichia coli does not invade intestinal epithelial cells, but binds with specific receptors on the apical membrane of the intestinal epithelial cells through adhesins to resist the scouring action of intestinal peristalsis to colonize and propagate on the intestinal mucosa in a large quantity, and then secretes thermolabile enterotoxins (LT) and/or heat-resistant enterotoxins (ST), so that the homeostasis of intestinal mucosa renewal is broken, mainly expressed by the fact that the number of intestinal mucosa goblet cells is reduced, so that the intestinal mucosa is damaged, and intestinal diseases such as diarrhea are caused. Although the mortality rate of diarrheal diseases caused by enterotoxigenic escherichia coli has been significantly reduced since the introduction of oral rehydration therapy and other measures, even mild episodes of enterotoxigenic escherichia coli diarrheal attacks can cause persistent sequelae to young children and animals, including growth retardation and malnutrition, which are closely related to intestinal dysfunction due to impaired integrity of the intestinal mucosa. Although enterotoxigenic escherichia coli and other pathogens have been many times thought to be associated with these sequelae in children, the molecular events behind these intestinal diseases are still poorly understood.

Inactivated polyvalent vaccine and antibiotic drugs are mostly adopted for preventing and treating enterotoxigenic escherichia coli, but antibiotic abuse can cause intestinal flora disorder and immunity reduction inside an animal body, and can also cause the drug resistance of microorganisms to antibiotics. Nowadays, various countries in the world issue measures of resistance reduction and resistance prohibition, and the search for antibiotics substitute drugs for treating enterotoxigenic escherichia coli infectious intestinal diseases is reluctant. The pomegranate rind is the dry pericarp of deciduous shrub or small-tree pomegranate of punica of punicaceae, is native to the middle and Asia regions such as Afghanistan and Iran, and is planted in the south and north of China, wherein the areas of Anhui, Jiangsu, Henan and the like are large. In addition, pomegranate rind is known for its health benefits and broad antimicrobial activity as a byproduct of the juice processing industry. The tannin and anthocyanin in the pomegranate peel extract are probiotics, have synergistic effect in promoting the characteristics of the probiotics, can inhibit pathogens and stimulate the growth of beneficial microflora of human intestinal tracts, and are important natural additives in food. The pomegranate rind component is particularly rich in tannin, and the content of the tannin can reach 15-40%. Pomegranate bark tannin is a water-soluble polyphenol compound with the characteristic of precipitating protein, and has the advantages of resisting bacteria, diminishing inflammation, stopping diarrhea, preventing stress gastrointestinal injury, being safe and low in toxicity and the like. In contrast to antibiotics, pomegranate bark tannin is a natural substance with low toxicity and side effects, but it is not clear whether pomegranate bark tannin can be used for treating intestinal mucosa damage and growth retardation caused by enterotoxigenic escherichia coli.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide the application of pomegranate bark tannin in preparing a medicine for treating enterotoxigenic escherichia coli intestinal diseases.

The purpose of the invention is realized by the following technical scheme:

application of pomegranate bark tannin in preparing medicine for treating enterotoxigenic escherichia coli intestinal diseases.

The pomegranate bark tannin is prepared by the following method: fully crushing pomegranate rind, soaking and extracting by using ultrasonic-assisted ethanol, concentrating the obtained extracting solution under reduced pressure, and precipitating by using a gelatin solution; dissolving and extracting the precipitate with acetone solution, concentrating the obtained extractive solution to obtain jelly, dissolving in water for several times, concentrating, and drying to obtain pericarpium Granati tannin.

Preferably, the pomegranate bark tannin is prepared by the following method:

1) fully crushing the pomegranate rind by a crusher;

2) extraction: weighing the pomegranate peel powder obtained in the step (1), and mixing the raw materials according to a material-liquid ratio of 1 g: adding 25mL of ethanol solution with volume percentage concentration of 60%, soaking for more than 2h, placing in an ultrasonic instrument, performing ultrasonic-assisted extraction for 50min, performing suction filtration, collecting filtrate, and repeating for 3 times; mixing filtrates, concentrating under reduced pressure in a rotary evaporator, adding gelatin solution with volume percentage concentration of 3% until no precipitate is generated, filtering, and collecting precipitate;

3) enrichment: collecting the precipitate obtained in the step (2), and mixing the precipitate according to a feed-liquid ratio of 1 g: adding 2mL of acetone solution with volume percentage concentration of 60%, placing in a water bath kettle at 50 ℃, extracting for 5min under stirring, centrifuging, taking the supernatant, concentrating under reduced pressure to be colloid, repeatedly dissolving with hot water for many times to remove insoluble substances, collecting the final supernatant, concentrating, and freeze-drying to obtain pomegranate bark tannin.

The effective dose of pomegranate bark tannin in the medicine is 10-90 wt%.

The medicine also comprises pharmaceutically acceptable auxiliary materials.

The medicine is an oral preparation.

In order to verify that the pomegranate bark tannin can be applied to preparing the medicine for treating enterotoxigenic escherichia coli intestinal diseases, the invention firstly establishes an animal intestinal injury model induced by enterotoxigenic escherichia coli and verifies the treatment effect of the pomegranate bark tannin on the animal enterotoxigenic escherichia coli infection, taking a mouse as an example, and the main experimental steps are as follows:

1) establishing an enterotoxigenic escherichia coli induced mouse intestinal injury model: the experimental period is 7 days, enterotoxigenic escherichia coli liquid is quantitatively gavaged in the middle period of the experiment, and the survival condition of the mouse is observed;

2) giving a mouse substance quantitative pomegranate bark tannin solution once a day during the experiment period, and recording the growth performance every day during the experiment period;

3) the treatment effect of pomegranate bark tannin on enterotoxigenic escherichia coli-induced animal intestinal injury and the effect of pomegranate bark tannin on reducing apoptosis level and improving intestinal barrier function are evaluated through HE staining, AB-PAS staining, immunofluorescence, immunohistochemistry, ELISA and Western-blot.

The principle of the invention is as follows:

the surface of the intestinal villus epithelium is composed of intestinal epithelial cells, goblet cells, clusteric cells, and enteroendocrine cells that proliferate upward from progenitor cells in the crypts in which intestinal stem cells and Pan cells reside to the villus surface. Intestinal cells are the most abundant cells in intestinal epithelial cells and are also the primary site of nutrient absorption. At the ultrastructural level, the "brush border" of the intestine is usually formed by a dense arrangement of microvilli on the surface of the intestinal cells. Microvilli are extensions of the apical plasma membrane of intestinal cells surrounding the central core of actin microfilaments. These protrusions greatly increase the effective surface area of the luminal surface of the bowel, allowing for maximum absorption of nutrients. Thus, interfering with microvilli development may lead to malnutrition. And villi damage caused by enterotoxigenic escherichia coli infection is shortened, the villi-crypt structure of the intestinal tract is damaged, and the intestinal tract nutrition absorption disorder can be caused. Goblet cells produce a large amount of mucin, which can protect intestinal tract barriers, and enterotoxigenic escherichia coli infected can destroy the interaction of the goblet cells of the intestinal tract cells, destroy the barriers of the intestinal tract and the intestinal tract homeostasis.

Unlike stress gastrointestinal injury, enterotoxigenic escherichia coli causes intestinal mucosal injury not by stress factors such as pathogenic microbial factors, physical factors, chemical factors, mechanical factors and the like, but colonizes intestinal epithelial cell brush border membrane receptors by adhesins on pili, and then secretes LT and/or ST, causing intestinal diseases such as mucosal damage, barrier disruption, intestinal mucosal renewal disorder, secretory diarrhea and the like, and even death. Enterotoxigenic escherichia coli secretes one or two toxins that, in addition to causing diarrhea, affect a variety of cellular pathways that control the normal homeostasis and ultimate absorptive capacity of the small intestine.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a pomegranate bark tannin medicine for effectively relieving intestinal injury induced by enterotoxigenic escherichia coli, and research results show that pomegranate bark tannin can obviously improve damage and update disorder of intestinal mucosa caused by enterotoxigenic escherichia coli infection, and acts on proliferating cells to improve the expression level of the proliferating cells, so that the damage of intestinal villi is reduced, and the function of nutrition absorption is maintained; promoting secretion of mucin, protecting tight junction protein, maintaining intestinal epithelial tight junction structure, repairing damaged intestinal barrier function, maintaining intestinal steady state, and promoting organism growth. The pomegranate bark tannin is applied to the preparation of the medicine for treating enterotoxigenic escherichia coli intestinal diseases, so that the medicinal value of pomegranate bark can be improved, and the pomegranate bark tannin can also be used as a green and safe alternative scheme for treating enterotoxigenic escherichia coli induced intestinal injury and growth obstruction by using antibiotics.

Drawings

FIG. 1 is a design of an experimental procedure;

FIG. 2 is a graph of experimental body weight changes in mice, wherein (a) is the daily gain of the mice and (b) is the relative body weight change of the mice;

FIG. 3 is a mouse ileum tissue HE stain wherein (a) is a mouse ileum tissue HE stain (40 ×), (b) is a mouse ileum tissue HE stain (100 ×), (c) is a mouse ileum tissue HE stain (200 ×);

FIG. 4 is a graph of AB-PAS staining of mouse ileum tissues, wherein (a) is AB-PAS staining of mouse ileum tissues (40X), (b) is AB-PAS staining of mouse ileum tissues (100X), and (c) is AB-PAS staining of mouse ileum tissues (200X);

FIG. 5 is a graph of statistical analysis of mouse ileal villi and crypt, wherein (a) is the length of mouse ileal villi, (b) is the depth of mouse ileal crypt, and (c) is the ratio of the length of mouse ileal villi to the depth of crypt;

FIG. 6 shows mouse serum DAO viability;

fig. 7 is a graph of mouse ileum tissue immunohistochemical Ki67 target, wherein (a) is mouse ileum tissue immunohistochemical Ki67 target (40 ×), (b) is mouse ileum tissue immunohistochemical Ki67 target (100 ×), (c) is mouse ileum tissue immunohistochemical Ki67 target (200 ×);

figure 8 is a plot of the mouse ileum tissue immunohistochemical MUC2 target, wherein (a) is the mouse ileum tissue immunohistochemical MUC2 target (40 ×), (b) is the mouse ileum tissue immunohistochemical MUC2 target (100 ×), (c) is the mouse ileum tissue immunohistochemical MUC2 target (200 ×);

FIG. 9 is a plot of mouse ileum tissue immunohistochemical E-cadherin targets, wherein (a) is mouse ileum tissue immunohistochemical E-cadherin target (40 ×), (b) is mouse ileum tissue immunohistochemical E-cadherin target (100 ×), (c) is mouse ileum tissue immunohistochemical E-cadherin target (200 ×);

FIG. 10 is a drawing of a mouse ileum tissue immunohistochemical ZO-1 target wherein (a) is the mouse ileum tissue immunohistochemical ZO-1 target (40X), (b) is the mouse ileum tissue immunohistochemical ZO-1 target (100X), (c) is the mouse ileum tissue immunohistochemical ZO-1 target (200X);

FIG. 11 is a graph of the statistical analysis of the target of immunohistochemistry for mouse ileum tissues, wherein (a) is the level of Ki67 in mouse ileum tissue, (b) is the level of MuC2 in mouse ileum tissue, (c) is the level of MUC2 in mouse ileum tissue, (d) is the average optical density of E-cadherin in mouse ileum tissue, and (E) is the average optical density of ZO-1 in mouse ileum tissue;

FIG. 12 shows the average daily food intake of piglets;

FIG. 13 is the average daily gain of piglets;

FIG. 14 shows the piglet feed weight ratio;

FIG. 15 shows the rate of diarrhea in piglets;

FIG. 16 is piglet diarrhea index;

fig. 17 shows the activity of serum DAO in piglets.

Detailed Description

Example 1: extraction and enrichment of pomegranate bark tannin

The extraction and preparation of pomegranate bark tannin are completed by the following steps:

(1) sample preparation:

fully crushing the pomegranate rind by a crusher;

(2) extraction:

weighing the pomegranate peel powder obtained in the step (1), and mixing the raw materials according to a material-liquid ratio of 1 g: adding 25mL of ethanol solution with volume percentage concentration of 60%, soaking for more than 2h, placing in an ultrasonic instrument, performing ultrasonic-assisted extraction for 50min, performing suction filtration, collecting filtrate, and repeating for 3 times; mixing filtrates, concentrating under reduced pressure in a rotary evaporator, adding gelatin solution with volume percentage concentration of 3% until no precipitate is generated, filtering, and collecting precipitate;

(3) enrichment:

collecting the precipitate obtained in the step (2), and mixing the precipitate according to a feed-liquid ratio of 1 g: adding 2mL of acetone solution with volume percentage concentration of 60%, placing in a water bath kettle at 50 ℃, extracting for 5min under stirring, centrifuging, taking the supernatant, concentrating under reduced pressure to be colloid, repeatedly dissolving with hot water for many times to remove insoluble substances, collecting the final supernatant, concentrating, and freeze-drying to obtain pomegranate bark tannin.

Example 2: application of pomegranate bark tannin in treating enterotoxigenic escherichia coli-induced intestinal injury of mice

We first set up enterotoxigenic Escherichia coli (ETEC) infected mouse model, and the experimental flow is designed as shown in FIG. 1.

Pomegranate bark tannin (Pomegranate Peel Tannins, PPT) treats enterotoxigenic Escherichia coli-induced intestinal injury model of mice, and comprises the following specific steps:

1) taking C57BL/6 mice of 6 weeks old, randomly dividing into 4 treatment groups, wherein the treatment groups are Ctr group, PPT group, ETEC group and ETEC + PPT group respectively, and the number of the mice in each treatment group and the treatment method are as follows:

control (n ═ 6): gavage 100 μ L PBS daily;

PPT group (n ═ 6): gavage 100 μ L per day of pomegranate bark tannin obtained in example 1 at a concentration of 200mg/mL to give a final dose of 20mg/mouse of pomegranate bark tannin;

ETEC group (n ═ 6): gavage 100. mu.L PBS daily while gavage 5X 10 on day 5⁸CFU/mouse ETEC；

ETEC + PPT group (n ═ 6): gavage 100 μ L per day of pomegranate bark tannin obtained in example 1 at a concentration of 200mg/mL, and gavage 5X 10 on day 5⁸CFU/mouse ETEC。

2) In the experimental period, the weight data are weighed and recorded every day, and the molding condition and the mental state of the excrement of the mice are observed.

3) Starting from 24 hours after enterotoxigenic escherichia coli induction treatment, fresh excrement samples of each mouse are collected every day, placed on white paper integrally and marked, and then photographed and stored.

4) Mice were sacrificed on day 8 and blood and small intestine samples of the mice were collected.

5) The collected intestinal tract sample is placed in 4% paraformaldehyde for fixation for 12 hours, 60%, 75%, 90% and absolute ethyl alcohol are used for dewatering step by step, and finally the intestinal tract sample is placed in absolute ethyl alcohol for storage at 4 ℃, is embedded in paraffin, and is subjected to HE dyeing and AB-PAS dyeing on slices.

The experimental results are as follows:

1) daily weight gain of mice (fig. 2 (a)); rate of change in body weight ((b) in FIG. 2)

As shown in (a) in FIG. 2, in the experimental process, the initial body weights of the Ctr group, the PPT group, the ETEC group and the ETEC + PPT group are basically all 26-28 g, and no significant difference exists. The body weights of mice in the Ctr group and the ETEC + PPT group show significant difference (#, P <0.01) at the 5 th day, and then no difference exists, which indicates that the enterotoxigenic escherichia coli induction treatment is effective; and the Ctr group and the ETEC group showed significant differences (++++, P <0.001) at day 5, and thereafter all showed very significant differences (++++, P < 0.0001); whereas the ETEC group showed significant differences from the ETEC + PPT group at day 6 (×, P <0.001), and thereafter did not show significant differences (×, P < 0.0001).

As can be seen from (b) of fig. 2, the body weights of the mice of the Ctr group and the ETEC + PPT group showed significant differences (##, P <0.001) at day 5 and significant differences (#, P <0.05) at day six, with no difference thereafter; and the Ctr group and the ETEC group showed significant differences (++++, P <0.001) at day 5, and thereafter all showed very significant differences (++++, P < 0.0001); whereas the ETEC group showed significant differences from the ETEC + PPT group at day 6 (. x., P <0.01), and all thereafter showed very significant differences (. x., P < 0.0001).

2) On the basis, we adopted the ileal terminal tissue to carry out HE staining (figure 3) and AB-PAS staining observation (figure 4)

The ileum epithelial structure morphology is observed under a 100-fold optical microscope (figure 3 (b)), the villus morphological structure of ileum is seriously damaged after enterotoxigenic escherichia coli induction treatment, the integrity of villus-crypt tissue is seriously damaged, the crypt quantity and the goblet cell quantity are both obviously reduced, the ileum epithelial morphological structure is recovered to a certain degree after pomegranate bark tannin is added for treatment, the villus morphology is complete, the crypt quantity is increased, the goblet cell quantity is obviously increased, and the small intestine barrier function is obviously improved.

Also, we took the terminal ileum for fixed AB-PAS staining observations. The ileum epithelial structure form observed under a 100-fold optical microscope (figure 4 (b)) shows that the villus morphological structure of ileum is seriously damaged after enterotoxigenic escherichia coli induction treatment, and the mucin on villus and crypts is less secreted, while the ileum epithelial structure is recovered to a certain extent after pomegranate bark tannin treatment, the villus form is complete, the number of crypts is increased, and the mucin secretion is increased.

3) Statistics of villi and crypts for each set of sections (FIG. 5)

From fig. 5 (a), we found that the ileal villus length of the ETEC group was significantly shortened compared to the other three treatment groups, and that there was a significant difference (×, P <0.0001) between the ETEC + PPT group and the Ctr group, and it was determined that enterotoxigenic e. In addition, after statistical analysis, no significant difference in ileal villus length was observed between the PPT group and both the Ctr group and the ETEC + PPT group.

From (b) in fig. 5, we found that compared with the other three treatment groups, the ileal crypt depth of the ETEC group was significantly shortened, and significantly different from both the ETEC + PPT group and Ctr group (, P <0.05), and it could be judged that after enterotoxigenic e. In addition, no significant difference in ileal crypt depth was seen between the PPT and Ctr and ETEC + PPT groups.

From (c) in fig. 5, we found that the ileal villus length to crypt depth ratio of the ETEC group is significantly smaller than that of the other three treatment groups, and the ileal villus length to crypt depth ratio of the ETEC + PPT group and Ctr group are significantly different (P is less than 0.05), so that it can be judged that the enterotoxin-producing escherichia coli induced treatment seriously damages the intestinal epithelial structure of the mouse, seriously damages the ileal villus-crypt structure, and successfully models the enterotoxin-producing escherichia coli induced intestinal injury model. In addition, no significant difference in the ileal villus length to crypt depth was seen between the PPT and Ctr and ETEC + PPT groups.

As described above, the ileum villus length, crypt depth and ratio of villus length to crypt depth of the PPT group, the Ctr group and the ETEC + PPT group have no significant difference, so that the punica granatum tannin can be inferred to have no toxic effect on the intestinal tract of the mouse, and the punica granatum tannin has safety.

4) DAO activity (fig. 6)

Furthermore, the mice are subjected to orbital bleeding, and subjected to DAO kit determination after red blood cells are removed from whole blood, analysis shows that the serum DAO activity after enterotoxigenic escherichia coli induction treatment is remarkably increased, the ETEC group has remarkable difference (P is less than 0.05) with the Ctr group and the ETEC + PPT group, the serum DAO activity after pomegranate bark tannin treatment is remarkably lower than that of the ETEC group and is similar to the DAO activity value of the Ctr group, which indicates that the integrity of the mouse intestinal tract mechanical barrier is damaged after enterotoxigenic escherichia coli induction treatment, so that the intestinal tract barrier is damaged, and the damage to the intestinal tract barrier can be relieved by adding the pomegranate bark tannin.

5) Immunohistochemistry (FIGS. 7-11)

Four targets were analyzed by immunohistochemistry on mouse ileum tissues, Ki67 target (fig. 7), MUC2 target (fig. 8), E-cadherin target (fig. 9), ZO-1 target staining (fig. 10).

As can be seen from fig. 11, after the enterotoxin-producing escherichia coli is induced, the amount of Ki67 cells in the crypt is significantly reduced, and is significantly different from both Ctr group and ETEC + PPT group (P is 0.01), which indicates that the proliferation activity of small intestine cells is reduced after the enterotoxin-producing escherichia coli is induced, the mechanical barrier capacity of intestinal tract is greatly reduced, while the amount of Ki67 cells in the crypt of ileum tissue is significantly increased by pomegranate bark tannin treatment, and is not significantly different from both Ctr group and PPT group, which indicates that the pomegranate bark tannin can effectively alleviate intestinal injury caused by enterotoxin-producing escherichia coli.

Immunohistochemical analysis of the MUC2 target based on statistical analysis showed that the level of MUC2 in the villi after enterotoxigenic e.coli induction treatment was significantly different from Ctr group (, P <0.05) and from ETEC + PPT group (, P < 0.01); while the levels of MUC2 in crypts after enterotoxigenic e.coli induction treatment were significantly different from both Ctr and ETEC + PPT groups (. about.p < 0.05). The level of MUC2 secreted by ileum tissues is obviously reduced after induction treatment of enterotoxigenic escherichia coli, and MUC2 secreted by villi or crypts is greatly reduced, which indicates that intestinal goblet cells are reduced, while the level of ileum MUC2 is obviously improved by pomegranate bark tannin treatment, and has no obvious difference with both Ctr group and PPT group.

The average optical density of ileum tissue E-cadherin of mice subjected to enterotoxigenic Escherichia coli induction treatment is reduced, and the E-cadherin level of an ETEC group is remarkably different from that of a Ctr group (, P <0.05) and is remarkably different from that of an ETEC + PPT group (, P < 0.0001). And the reduction of the E-cadherin level can cause the reduction of the cell adhesion strength to promote inflammation to occur, and proves that the enterotoxigenic escherichia coli induced treatment can damage the intestinal epithelial structure of the ileum, reduce the cell adhesion strength, promote the generation of intestinal inflammation and damage the intestinal tract. The E-cadherin level of ileum tissues is remarkably improved by pomegranate bark tannin treatment, and the E-cadherin level is not remarkably different from those of a Ctr group and a PPT group.

The mice were induced by enterotoxigenic escherichia coli to reduce the average optical density of ZO-1 protein, and the ZO-1 protein level of the ETEC group was significantly different from the Ctr group (x, P <0.0001) and significantly different from the ETEC + PPT group (x, P < 0.0001). And the fact that the ZO-1 protein level is reduced means that the intestinal epithelial cell tight junction structure is damaged, so that the mechanical barrier function is damaged, proves that the enterotoxin-producing escherichia coli induced treatment causes the intestinal epithelial structure of ileum to be damaged, so that intestinal inflammation is caused, and the intestinal tract is damaged. The level of ZO-1 protein in ileum is obviously improved by pomegranate bark tannin treatment, and the level of ZO-1 protein in ileum is not obviously different from that in both Ctr group and PPT group.

Example 3: application of pomegranate bark tannin in treating enterotoxigenic escherichia coli-induced intestinal injury of piglets

Firstly, establishing a piglet intestinal injury model for treating enterotoxigenic escherichia coli induction by pomegranate peel tannin, which comprises the following specific steps:

1) selecting healthy (21 +/-2) day-old Du multiplied by long multiplied by big weaned piglets with average weight (7.48 +/-0.37) kg, randomly dividing the weaned piglets into 4 treatment groups according to the weight similarity principle, wherein the treatment groups are a Ctr group, a PPT group, an ETEC group and an ETEC + PPT group respectively, and the number of the piglets in each treatment group and the treatment method are as follows:

ctr group (n ═ 6): feeding basal diet, and feeding piglets with the same dosage of sterile culture solution;

PPT group (n ═ 6): feeding experimental diet which is prepared by adding 1000g/t of pomegranate rind tannin obtained in example 1 into basic diet, and feeding piglets with sterile culture solution with the same dosage;

ETEC group (n ═ 6): feeding basal diet, and irrigating 5 × 10 piglets after 5 th sky abdomen weighing⁸CFU enterotoxigenic Escherichia coli liquid;

ETEC + PPT group (n ═ 6): feeding experimental diet containing 1000g/t of pomegranate rind tannin obtained in example 1 to basal diet, and drenching each piglet with 5 × 10 of the feed after weighing in the 5 th sky belly⁸CFU enterotoxigenic Escherichia coli liquid.

2) The experimental period is 7 days, the piglets are fed in a single cage, and are respectively fed in two different areas according to whether enterotoxigenic escherichia coli is used for induction treatment, so that cross infection is prevented. The temperature of the animal house is controlled to be 25-28 ℃, and the relative humidity is 60-70%. The basic feed for the piglets is a corn-soybean meal type feed, and is prepared according to the nutritional needs of the weaned piglets of NRC (2012) 7-25 kg. During the experiment, the piglets freely drink water and are fed for 4 times every day, and the feeding amount is proper to slightly remain in the trough after the piglets eat the feed.

3) After enterotoxigenic escherichia coli induction treatment, the mental status of piglets is observed daily, the diarrhea condition of each group of piglets is recorded, and when the excrement score is more than or equal to 2, the diarrhea is defined as diarrhea, and the diarrhea score standard is shown in table 1. And calculating the diarrhea rate and the diarrhea index according to the observed diarrhea condition. The diarrhea rate is calculated as follows: the diarrhea rate (%) [ number of diarrhea heads in the test period/(number of piglets in the test × number of test days) ] × 100. The diarrhea index is calculated as follows: diarrhea index is total diarrhea score/(number of piglets tested × number of days tested).

4) After the experiment is finished, 10mL of fasting and anterior vena cava blood collection of piglets is weighed, serum is prepared and stored at the temperature of minus 20 ℃, and the DAO index of the serum to be detected is obtained.

The experimental results are as follows:

1) piglet growth performance (fig. 12-14)

As can be seen from fig. 12, the Average Daily Food Intake (ADFI) of all groups of piglets in the whole period of the experiment was not significantly different (P > 0.05). From fig. 13-14, it can be seen that the Average Daily Gain (ADG) of the piglets in the ETEC group was very significantly decreased (P <0.01) and the feed-to-weight ratio (feed/gain, F/G) was significantly increased (P <0.05) compared to the Ctr group. Compared to the ETEC + PPT group, the ilex ADG poles of the ETEC group were significantly reduced (, P <0.01) and the F/G poles were significantly reduced (, P < 0.01).

2) Piglet diarrhea rate (fig. 15-16)

From fig. 15-16, it can be seen that the diarrhea rate and diarrhea index of the piglets in ETEC group were significantly increased compared to Ctr group after enterotoxigenic e.coli induction treatment (x, P < 0.0001). Compared with the ETEC + PPT group, the piglet diarrhea rate and diarrhea index of the ETEC group are both increased remarkably (P < 0.0001). The pomegranate bark tannin can relieve diarrhea symptoms caused by enterotoxigenic escherichia coli induced treatment, wherein the diarrhea score standard is shown in table 1.

Table 1 is the diarrhea score criteria.

3) Piglet DAO vitality (fig. 17)

1) The DAO activity of the serum of the piglets is determined by a DAO activity kit, and analysis shows that the DAO activity of the serum of the piglets after enterotoxigenic escherichia coli induction treatment is remarkably increased, compared with the Ctr group, the DAO activity of the piglets in the ETEC group is remarkably increased (x, P is less than 0.001), and the DAO activity of the serum of the piglets after the pomegranate rind tannin treatment is remarkably reduced (x, P is less than 0.01). The enterotoxigenic escherichia coli is induced to cause intestinal injury, and the addition of pomegranate bark tannin can reduce the toxicity and the pathogenicity of the enterotoxigenic escherichia coli, so that the injury of the enterotoxigenic escherichia coli to an intestinal barrier is relieved.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (6)

1. Application of pomegranate bark tannin in preparing medicine for treating enterotoxigenic escherichia coli intestinal diseases.

2. Use according to claim 1, characterized in that: the pomegranate bark tannin is prepared by the following method: fully crushing pomegranate rind, soaking and extracting by using ultrasonic-assisted ethanol, concentrating the obtained extracting solution under reduced pressure, and precipitating by using a gelatin solution; dissolving and extracting the precipitate with acetone solution, concentrating the obtained extractive solution to obtain jelly, dissolving in water for several times, concentrating, and drying to obtain pericarpium Granati tannin.

3. Use according to claim 2, characterized in that: the pomegranate bark tannin is prepared by the following steps:

1) fully crushing the pomegranate rind by a crusher;

2) extraction: weighing the pomegranate peel powder obtained in the step (1), and mixing the raw materials according to a material-liquid ratio of 1 g: adding 25mL of ethanol solution with volume percentage concentration of 60%, soaking for more than 2h, placing in an ultrasonic instrument, performing ultrasonic-assisted extraction for 50min, performing suction filtration, collecting filtrate, and repeating for 3 times; mixing filtrates, concentrating under reduced pressure in a rotary evaporator, adding gelatin solution with volume percentage concentration of 3% until no precipitate is generated, filtering, and collecting precipitate;

3) enrichment: collecting the precipitate obtained in the step (2), and mixing the precipitate according to a feed-liquid ratio of 1 g: adding 2mL of acetone solution with volume percentage concentration of 60%, placing in a water bath kettle at 50 ℃, extracting for 5min under stirring, centrifuging, taking the supernatant, concentrating under reduced pressure to be colloid, repeatedly dissolving with hot water for many times to remove insoluble substances, collecting the final supernatant, concentrating, and freeze-drying to obtain pomegranate bark tannin.

4. Use according to claim 1, characterized in that: the effective dose of pomegranate bark tannin in the medicine is 10-90 wt%.

5. Use according to claim 1, characterized in that: the medicine also comprises pharmaceutically acceptable auxiliary materials.

6. Use according to claim 1, characterized in that: the medicine is an oral preparation.

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