Use of a dogwood extract in the preparation of a preparation for modulating the intestinal flora and / or improving the intestinal barrier function

Abstract

The application belongs to the technical field of biological medicine, and specifically provides application of a Prunus humilis Bge. extract in preparation of a preparation for regulating intestinal flora and / or improving intestinal barrier function. In particular, the application is in increasing abundance of Lachnospiraceae_NK4A136_group, Clostridia_UCG-014 and Ruminococcaceae; reducing abundance of Alistipes, Escherichia-Shigella and Bilophila; inhibiting ROS / OPN / αvβ3 / PI3K / Akt / NF-κB pathway; and reducing expression levels of TNF-α, IL-1β and IL-6 inflammatory factors. The new application of the Prunus humilis Bge. extract provides a new method for regulating intestinal flora and improving intestinal barrier function, and has important significance for deep development and utilization of Prunus humilis Bge. resources and industrial upgrading.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Cerasus humilis (Bge.) Sok. extract in preparation of a preparation for regulating intestinal flora and / or improving intestinal barrier function. BACKGROUND

[0002] The intestine is not only an important place for digestion and absorption, but also the largest immune organ of the organism. Damage and dysfunction of the intestinal mucosal barrier is an important mechanism for the occurrence of ulcerative colitis. The intestinal mucosal barrier is the first line of defense against the invasion of symbiotic bacteria and intestinal pathogens in the host. The intestinal mucosal barrier is mainly composed of mechanical, immune, biological and chemical barriers. The interaction of each part of the intestinal mucosal barrier will affect the function of the intestinal mucosal barrier, leading to intestinal diseases and even damage to the function of the organism. Intestinal microorganisms, including bacteria, fungi, viruses and other microorganisms, are an important secondary organ system for the host. Intestinal microbial imbalance has been proven to be an important factor in inducing intestinal inflammation in inflammatory bowel disease.

[0003] Cerasus humilis (Bge.) Sok. is a small shrub of Rosaceae Prunus, and its seeds are traditional Chinese medicine Yuliiren, which has the effects of moistening the intestines, relieving constipation, and promoting water excretion. Its fruits are a unique third-generation small fruit in China, with a sweet and sour taste and a slightly astringent taste, mainly due to the rich polyphenols and proanthocyanidins (PACs) in them. PACs are natural polyphenol compounds polymerized from flavan-3-ol and its derivatives, and have strong antioxidant capacity. The PACs content of Cerasus humilis is significantly higher than that of sweet cherries, sour cherries and other fruits of the same genus, and even higher than that of some known PACs-rich fruits (such as cranberries, blueberries, etc.), which is an excellent raw material for industrial extraction. Studies have found that Cerasus humilis PACs have antioxidant capacity, can protect against carbon tetrachloride-induced liver damage in mice, and can also play a role in preventing diabetes by reducing the content of fast-digestible starch in potato starch, increasing the content of slow-digestible starch, and inhibiting alpha-amylase activity. However, there is no related report on the effect of Cerasus humilis extract on intestinal barrier function and intestinal microorganisms. SUMMARY

[0004] The purpose of the present application is to provide a new use of Cerasus humilis extract and promote the deep development and utilization of Cerasus humilis resources.

[0005] To this end, the present application provides a use of Cerasus humilis extract in the preparation of a preparation for regulating intestinal flora and / or improving intestinal barrier function.

[0006] Specifically, the above-mentioned regulating intestinal flora includes increasing the abundance of Lachnospiraceae_NK4A136_group, Clostridia_UCG-014 and Ruminococcaceae.

[0007] Specifically, the above-mentioned regulating intestinal flora includes reducing the abundance of Alistipes, Escherichia-Shigella and Bilophila.

[0008] Specifically, the above-mentioned improving intestinal barrier function includes one or more of the following items:

[0009] (1) inhibiting the ROS / OPN / αvβ3 / PI3K / Akt / NF-κB pathway;

[0010] (2) reducing the expression level of TNF-α, IL-1β and IL-6 inflammatory factors.

[0011] Specifically, the above-mentioned Cerasus humilis extract includes Cerasus humilis proanthocyanidin.

[0012] Specifically, the above-mentioned preparation method of Cerasus humilis extract includes: after the Cerasus humilis pulp is crushed, an alcohol solution is added for extraction, the extraction liquid is filtered, the alcohol in the filtrate is removed, and the Cerasus humilis extract is obtained.

[0013] Specifically, the above-mentioned alcohol solution includes an ethanol aqueous solution with a volume fraction of 10-70%; the liquid-to-material ratio is (5-45):1; the extraction temperature is 30-80℃, and the extraction time is 1-5h.

[0014] Specifically, the above-mentioned preparation method further includes: after the alcohol in the filtrate is removed, the filtrate is added to a macroporous adsorption resin, and the eluate is collected after adsorption and elution.

[0015] Specifically, the above-mentioned preparation method further includes: adding a gelatin solution to the eluate from which the alcohol is removed to obtain a precipitate; after the precipitate is dissolved by refluxing with an acetone solution, the acetone is removed, and then ethyl acetate is used for extraction, and the ethyl acetate layer is concentrated under reduced pressure and freeze-dried to obtain the purified Cerasus humilis extract.

[0016] Specifically, the above-mentioned preparation includes health food, medicine and feed.

[0017] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0018] The new use of the Cerasus humilis extract provided by the present application provides a new method for regulating intestinal flora and improving intestinal barrier function, and has important significance for the deep development and utilization of Cerasus humilis resources and the upgrading of the industry.

[0019] The present application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a graph of the optimization experiment results in Example 2; wherein A is the adsorption kinetics curve of the macroporous adsorption resin to PACs; B is the adsorption breakthrough curve of the sample solution at different flow rates on AB-8 resin; C is the PACs adsorption capacity of AB-8 resin when the sample solution at different flow rates reaches the breakthrough point; D is the dynamic elution curve of the eluent of different concentrations of ethanol; E is the PACs elution rate of the eluent of different concentrations of ethanol; F is the PACs elution rate at different elution flow rates.

[0021] Figure 2 is PACs of Cerasus humilis obtained by different purification treatments in Example 3; wherein, A is Cerasus humilis crude extract, B is macroporous resin purification; C is gelatin precipitation method purification.

[0022] Figure 3 is the OTU wayon chart of the intestinal flora of the colon of each group of mice in Example 5.

[0023] Figure 4 is the change of the alpha diversity index of the intestinal flora of the colon of each group of mice in Example 5.

[0024] Figure 5 is the beta diversity of the intestinal flora of each group of mice in Example 5.

[0025] Figure 6 is the column chart of the distribution of the phylum level of the intestinal flora of each group of mice in Example 5 (A) and the F / B value (B).

[0026] Figure 7 is the column chart of the distribution of the genus level of the intestinal flora of each group of mice in Example 5.

[0027] Figure 8 is the biomarker analysis of the relative abundance of species of the phylum level of C group / M group (A) and M group / YH group (B) in Example 5.

[0028] Figure 9 is the analysis of the relative abundance of species of the genus level of C group / M group in Example 5.

[0029] Figure 10 is the analysis of the relative abundance of species of the genus level of M group / YH group in Example 5.

[0030] Figure 11 is the taxonomic cladogram and LEfSe analysis LDA discriminant analysis chart of C group and M group in Example 5.

[0031] Figure 12 is the taxonomic cladogram and LEfSe analysis LDA discriminant analysis chart of M group and YH group in Example 5.

[0032] Figure 13is a heatmap of the correlation between the genus level gut microbiota and UC disease-related indicators in Example 5.

[0033] Figure 14 is a heatmap of the correlation between the genus level gut microbiota and inflammatory factors in Example 5.

[0034] Figure 15 is a heatmap of the correlation between the genus level gut microbiota and intestinal tight junction proteins in Example 5.

[0035] Figure 16 is a heatmap of the correlation between the genus level gut microbiota and oxidative stress-related indicators in Example 5.

[0036] Figure 17 is a heatmap of the correlation between the genus level gut microbiota and SCFAs in Example 5.

[0037] Figure 18 is a correlation network diagram of the difference between the YH group and the M group in Example 5 and the UC-related indicators.

[0038] Figure 19 is the RT-qPCR result in Example 6.

[0039] Figure 20 is the effect of Prunus humilis PACs on the expression levels of OPN / β3 / PI3K / Akt / NF-κB pathway proteins in mouse colon in Example 6. DETAILED DESCRIPTION

[0040] The technical solutions in the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Although the representative embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present application without departing from the scope of the present application. Therefore, the scope of the present application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0041] The present application provides the use of Prunus humilis extract in the preparation of a preparation for regulating gut microbiota and / or improving intestinal barrier function.

[0042] Among them, regulating gut microbiota includes increasing the abundance of Lachnospiraceae_NK4A136_group, Clostridia_UCG-014 and Ruminococcaceae genera; and reducing the abundance of Alistipes, Escherichia-Shigella and Bilophila genera.

[0043] Improving intestinal barrier function includes one or more of the following items:

[0044] (1) inhibiting ROS / OPN / αvβ3 / PI3K / Akt / NF-κB pathway;

[0045] (2) reducing the expression levels of TNF-α, IL-1β and IL-6 inflammatory factors.

[0046] The Cerasus humilis extract includes Cerasus humilis PACs, and is preferably prepared by the following steps:

[0047] (1) selecting Cerasus humilis mature fruits with similar size, no damage and no pests and diseases, removing branches, leaves and kernels, and crushing the pulp with a juicer.

[0048] (2) adding an alcohol solution to the pulp for extraction, filtering the extraction solution, removing alcohol from the filtrate by rotary evaporation to obtain a Cerasus humilis crude extract which can be used as a macroporous resin loading solution.

[0049] Preferably, the alcohol solution is an ethanol aqueous solution with a volume fraction of 10-70%, and more preferably 20-60%; the liquid-to-material ratio is preferably (5-45) : 1, and more preferably (15-35) : 1; the extraction temperature is preferably 30-80°C, and the extraction time is 1-5h, preferably 3-5h.

[0050] (3) adding the macroporous resin loading solution to a macroporous adsorption resin for adsorption, fully washing off impurities such as sugars and acids with deionized water, collecting the eluate after elution with an alcohol solution, and removing alcohol by rotary evaporation to obtain a purified Cerasus humilis extract.

[0051] Preferably, the loading solution is loaded at a speed of 1-3BV / h for 4-7BV, and more preferably at a speed of 2BV / h for 5BV; and the elution is preferably performed with 20-80% ethanol at a flow rate of 1-3BV / h, and the first 2.5BV eluate is collected.

[0052] (4) After the macroporous resin purification by rotary evaporation to remove ethanol, 5% gelatin aqueous solution is added to the PACs solution while stirring until the precipitation is complete, and then centrifugation is performed to obtain the precipitate. The PACs in the precipitate are dissolved by refluxing with 50-80% acetone aqueous solution. The obtained acetone solution is concentrated under reduced pressure to obtain a solid powder, which is dissolved in water and then extracted with ethyl acetate. The ethyl acetate layer is concentrated under reduced pressure and freeze-dried to obtain a high-purity Cerasus humilis extract PACs.

[0053] The application effect of the Cerasus humilis extract is studied by specific examples as follows.

[0054] Example 1:

[0055] The present example provides a preparation method of a Cerasus humilis extract, including the following steps:

[0056] (1) Select the size of the similar, no damage, no disease and insect pests of mature fruit of Prunus humilis Batal, branch, leaf, core, fruit pulp with juicer broken.

[0057] (2) To the fruit pulp added ethanol aqueous solution extraction, the filtrate was filtered, the filtrate was removed by rotary evaporation, and the Prunus humilis Batal crude extract was obtained.

[0058] Ten groups of Prunus humilis Batal extracts were prepared by the above method, and the liquid material ratio (mL / g), extraction temperature (℃), extraction time (h) and PACs extraction rate (mg / 100g FW) of each group were shown in Table 1.

[0059] PACs extraction rate determination method: precisely take 50 μL of the sample solution diluted appropriately in a 96-well plate, add 0.1% DMAC (ethanol: water: concentrated hydrochloric acid = 6:1:1 acidified ethanol) 200 μL, stand for 20 min, then measure the absorbance at 640 nm wavelength. The standard curve regression equation of proanthocyanidin B1 is Y=7.223X+0.0024 (R 2 =0.9999), the linear range is 0.0100-0.300 mg / mL.

[0060] PACs extraction amount = [(C x V) / m] x 100g;

[0061] In the formula: C (mg / mL) is the PACs concentration of the extract; V (mL) is the volume of the extract; m (g) is the mass of Prunus humilis Batal pulp.

[0062] Table 1 Prunus humilis Batal extraction conditions and extraction rate

[0063] No. Extraction temperature Liquid-to-material ratio Extraction time PACs extraction rate 1 60 15 3 616.0±9.0 2 60 25 4 669.8±11.4 3 60 35 5 673.0±3.0 4 70 15 4 745.0±10.1 5 70 25 5 746.1±2.4 6 70 35 3 623.4±7.7 7 80 15 5 795.8±17.9 8 80 25 3 834.3±12.3 9 80 35 4 859.6±1.9 10 80 25 4 883.0±18.0

[0064] Example 2:

[0065] This example is based on Example 1 to optimize the preparation method of Prunus humilis Batal extract, the specific steps are as follows. The results are expressed as mean ± SD values, and single factor ANOVA test is performed by SPSS23.0 statistical software, and P<0.05 is considered to be significantly different. GraphPad Prism9.5 software is used for drawing.

[0066] 1. Preparation of loading solution

[0067] Using the same method as the 10th group in Table 1, the ethanol was removed by rotary evaporation under reduced pressure at 60℃ to prepare Prunus humilis Batal crude extract as macroporous resin loading solution (Prunus humilis Batal PACs loading solution).

[0068] 2. Resin pretreatment

[0069] The macroporous resin was immersed in 95% ethanol and stirred with a glass rod to expel the air bubbles, and soaked for 24 h. An appropriate amount of fully swollen macroporous resin was taken in a glass chromatographic column, and 95% ethanol was continuously washed at a flow rate of 3 bed volumes (BV) / h until 1 part of ethanol in 3 parts of water did not produce white turbidity. Deionized water was passed through the resin layer at a flow rate of 6-8 BV / h until there was no alcohol smell.

[0070] 3. Screening of macroporous adsorption resins

[0071] Determination of adsorption capacity and desorption rate of PACs: 1.00 g of each of the 13 pretreated macroporous resins was weighed into a 250 mL ground stoppered flask, and 60 mL of Chinese dwarf cherry PACs sample solution was added, with 3 replicates for each resin, and a blank sample solution without resin was set. The flasks were placed in a 25°C constant temperature shaker and shaken at a shaking frequency of 120 r / min for 24 h. After sufficient adsorption, the solution was filtered with a 500 mesh nylon filter cloth, and the equilibrium concentration of PACs in the filtrate was determined. The macroporous adsorption resin washed with deionized water was loaded into a 100 mL ground stoppered flask, 50 mL of 95% ethanol was added, and it was placed in a 25°C constant temperature shaker and shaken at a shaking frequency of 120 r / min for 24 h. After sufficient desorption, the solution was filtered, and the concentration of PACs in the filtrate was determined. The adsorption capacity (mg / g) and desorption rate (%) of each resin were calculated according to the following formula.

[0072] Adsorption rate Q = [(C0-C e ) x V] / W;

[0073] C0represents the concentration of the blank sample solution, mg / mL; C e represents the equilibrium concentration, mg / mL; V represents the volume of the sample solution, 60 mL; and W represents the weight of the resin, g.

[0074] Desorption rate D = [(C x V) / W] x 100%;

[0075] C represents the concentration of PACs in the desorption solution, mg / mL; V represents the volume of the desorption solution, 50 mL; Q represents the adsorption capacity, mg / g; and W represents the weight of the resin, g.

[0076] Determination of adsorption rate of PACs: The adsorption rate of several resins selected according to the adsorption capacity and desorption rate was determined. 2.00 g of each of the selected macroporous resins was accurately weighed into a 250 mL ground stoppered flask, 100 mL of Chinese dwarf cherry PACs sample solution with a certain concentration was added, and it was placed in a 25°C constant temperature shaker and shaken, with a shaking frequency of 120 r / min. Within 4 h, 0.4 mL of the solution was taken every hour and the concentration of PACs was determined.

[0077] The parameters and adsorption and desorption effects of different types of macroporous adsorption resins are shown in Table 2.

[0078] Table 2 Parameters of different macroporous resins and adsorption capacity, desorption rate and desorption capacity

[0079]

[0080] From Table 2, it can be seen that different types of macroporous adsorption resins have different adsorption and desorption capacities for Prunus humilis PACs due to the differences in their physical parameters such as polarity, specific surface area and average pore size. Among the 13 macroporous adsorption resins investigated, the top three resins in terms of adsorption capacity (mg / g) were HPD400 (47.77), AB-8 (46.20) and HPD826 (45.83), and the top three resins in terms of desorption rate (%) were NKA-2 (97.04), DM130 (96.12) and DA201 (96.10). Considering the adsorption capacity and desorption capacity of the resins, the resin with the largest desorption capacity (mg / g) was HPD826 (43.89), followed by AB-8 (43.36), which are more suitable for purifying Prunus humilis PACs. In addition, considering the purification efficiency, the resins should also have a faster adsorption speed, so the adsorption speed of HPD826 and AB-8 resins was investigated, and the results are shown in Figure 1 A.

[0081] From Figure 1 A, it can be seen that the adsorption rates of AB-8 and HPD826 resins were similar before 1 h, and the adsorption capacity of AB-8 resin was greater than that of HPD826 resin after 1 h. Both resins reached adsorption equilibrium after 4 h. Considering the adsorption-desorption capacity and adsorption rate of the 13 macroporous adsorption resins for Prunus humilis PACs, AB-8 is the best macroporous resin for purifying Prunus humilis PACs.

[0082] 4. Effect of sample solution flow rate on adsorption

[0083] Weigh 3 g of pretreated and dried AB-8 macroporous adsorption resin, soak in 95% ethanol for 24 h, and then load into a chromatography column with a size of 1.0 cm x 30 cm using wet method, with a height of about 10 cm and a packing volume of about 8 mL. Sample solution with a PACs concentration of about 6 mg / mL was loaded at flow rates of 1, 2 and 3 BV / h, respectively. The effluent was collected in test tubes, with 0.5 BV per tube, and the PACs concentration (mg / mL) of the effluent was determined. When the PACs concentration of the effluent reached 10% of the concentration before loading, it was considered that the breakthrough point of adsorption had been reached, and the PACs adsorption capacity (mg / g) at the breakthrough point was calculated, and the results are shown in Figure 1 B-1C.

[0084] From Figure 1 B, it can be seen that as the flow rate of the sample solution increases, the adsorption breakthrough curve becomes steeper and the breakthrough point is earlier. When the flow rate was 1, 2 and 3 BV / h, the breakthrough point was 7, 5 and 4 BV, respectively. FromFigure 1 It can be seen that with the increase of sample flow rate, the PACs adsorption capacity of the resin gradually decreases when reaching the breakthrough point. When the sample flow rate is 1 BV / h, the PACs adsorption capacity is the largest, but it takes 7 h to reach the breakthrough point, which is low in working efficiency. Considering the adsorption effect and working efficiency, the sample flow rate of 2 BV / h is preferred. At this rate, it takes only 2.5 h to reach the breakthrough point after 5 BV of sample is loaded.

[0085] 5. Effect of ethanol concentration of eluent on elution effect

[0086] The resin was loaded, and the sample with a PACs concentration of about 6 mg / mL was loaded at a flow rate of 2 BV / h. The resin column was washed with 30 BV of deionized water at a flow rate of 10 BV / h until the effluent was colorless, and then eluted with 20%, 40%, 60% and 80% ethanol aqueous solution at a flow rate of 2 BV / h, respectively. The eluent was collected in test tubes, 0.5 BV per tube, and 10 tubes were collected. The PACs concentration of each tube of eluent was determined, and the results are shown in Table 1 and Fig. D-1E. Figure 1

[0087] It can be seen that within the range of 20% to 80% ethanol concentration, the PACs concentration peak in the eluent is advanced and increased with the increase of the ethanol concentration of the eluent. Figure 4 It can be seen that within the range of 20% to 60% ethanol concentration, the PACs desorption rate is significantly increased with the increase of the ethanol concentration of the eluent (P<0.05). When 60% ethanol is used for elution, the desorption rate is 101.4%, and there is no significant increase in the desorption rate when the ethanol concentration of the eluent is further increased. It is preferred to use 60% ethanol aqueous solution to elute PACs from AB-8 resin. Figure 5 6. Effect of elution flow rate on elution effect

[0088] 60% ethanol aqueous solution was used for elution at a flow rate of 1, 2 and 3 BV / h, respectively. The eluent was collected in test tubes, 0.5 BV per tube, and 10 tubes were collected. The PACs concentration of each tube of eluent was determined, and the results are shown in Table 2 and Fig. F.

[0089] Figure 1 It can be seen from Fig. F that with the increase of the eluent volume, the PACs elution rate increases. Compared with a higher flow rate, a slower elution flow rate requires less eluent volume to elute the same amount of PACs, which can shorten the subsequent time for rotary evaporation to concentrate the eluent. Therefore, it is preferred to elute PACs at a flow rate of 1 BV / h. At this flow rate, the elution rate of the first 2.5 BV of effluent can reach 99.61%.

[0090] Figure 1

[0091] ​​​​The purity of PACs in the collected freeze-dried powder of effluent was 47.52%, which was a deep red loose powder with a bitter and astringent taste. According to the process, the PACs of Prunus humilis Bunge were purified, and it only took 8 hours from loading to desorption; the purification process did not involve toxic reagents, and the obtained PACs had a high purity, which could be used as a functional additive in health food and cosmetics.

[0092] Example 3

[0093] In this example, the preparation method of Prunus humilis Bunge extract was optimized based on Example 2, and the specific steps were as follows.

[0094] In the PACs solution obtained by rotary evaporation to remove ethanol under the optimal conditions in Example 2, 5% gelatin aqueous solution was added while stirring until the precipitation was complete, and then the precipitation was obtained by centrifugation at a speed of 8000 r / min for 10 min. The PACs in the precipitation were dissolved by refluxing with 50% acetone aqueous solution. The obtained acetone solution was concentrated under reduced pressure to obtain a solid powder, which was dissolved in water and then extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure and freeze-dried to obtain a PACs powder.

[0095] After purification by macroporous adsorption resin, the PACs of Prunus humilis Bunge were a deep red powder Figure 2 B) with a purity of 47.52%, and the color of PACs substances was generally colorless or brown. Therefore, it was speculated that the freeze-dried powder also contained anthocyanins and other polyphenols. PACs were tannin compounds, so the PACs purified by macroporous resin were further refined by taking advantage of the property that tannin could form a precipitate with gelatin. The freeze-dried powder after gelatin precipitation was a red-brown powder Figure 2 C) with a bitter and astringent taste, and the purity of PACs was 98.14%. Since this purification method required toxic reagents such as acetone and ethyl acetate, and the operation was complex, but the obtained PACs had a high purity, and the obtained PACs could be used for pharmacological activity research to exclude the interference of other polyphenols.

[0096] Table 3 Purity and properties of Prunus humilis Bunge PACs before and after purification

[0097]

[0098] Example 4

[0099] In this example, a Prunus humilis Bunge extract was prepared by the following steps:

[0100] (1) After the Prunus humilis Bunge pulp was crushed, 40% ethanol was added at a solid-liquid ratio of 25 mL / g, and the mixture was extracted in a water bath at 80°C for 4 h. After the ethanol was removed from the filtrate by rotary evaporation, a macroporous resin loading solution was obtained. The freeze-dried powder of the loading solution was a crude PACs extract of Prunus humilis Bunge.

[0101] (2) 6 mg / mL PACs sample solution was loaded at a speed of 2 BV / h for 5 BV, and deionized water was used to elute the impurities such as sugar and acid. 60% ethanol was used to elute at a flow rate of 1 BV / h, and the first 2.5 BV of eluent was collected. The ethanol was removed by rotary evaporation, and the PACs with a purity of 47.52% were obtained by freeze-drying.

[0102] (3) After removing the ethanol by rotary evaporation, 5% gelatin aqueous solution was added to the PACs solution purified by macroporous resin while stirring until the precipitation was complete. Then, the precipitate was obtained by centrifugation at a speed of 8000 r / min for 10 min. The PACs in the precipitate were dissolved in 50% acetone water by reflux. The obtained acetone solution was concentrated under reduced pressure to obtain a solid powder. The powder was dissolved in water and then extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure and freeze-dried to obtain the PACs with a purity of 98.14% from the Prunus humilis Bunge extract.

[0103] Example 5

[0104] This example is based on the Prunus humilis Bunge extract (PACs) prepared in Example 4 to study its effect on intestinal flora. All mice were raised in the specific pathogen-free animal room of Beijing University of Chinese Medicine, and the experimental unit was licensed under license number SYXK (Jing) 2020-0033.

[0105] I. Preparation of ulcerative colitis (UC) mouse model

[0106] C57BL / 6 male mice were allowed to freely drink 3% DSS aqueous solution (g / mL) for 8 consecutive days to prepare the UC mouse model. The 3% DSS solution in the drinking bottle of the mice was replaced every two days.

[0107] II. Experimental grouping and treatment

[0108] 56 mice were randomly divided into 7 groups after adaptive feeding for 7 days: normal group (C), model group (M), positive drug group (P, 200 mg / kg), low-dose Yali PACs prevention group (YL, 20 mg / kg), high-dose Yali PACs prevention group (YH, 40 mg / kg), low-dose Yali PACs treatment group (ZL, 20 mg / kg), and high-dose Yali PACs treatment group (ZH, 40 mg / kg), with 8 mice in each group. From 14 days before modeling, the Yali PACs prevention groups were given Yali PACs by gavage for prevention until the end of modeling, for a total of 22 days of administration. The remaining groups were given deionized water by gavage every day. After 14 days, the normal group freely drank distilled water, and the model group, positive drug group, Yali PACs prevention group, and treatment group freely drank 3% DSS solution for 8 days to induce the construction of the UC model. The positive drug group and Yali PACs treatment group were given continuous gavage from the first day after modeling, and the blank group was given deionized water by gavage.

[0109] The body weight of the mice was measured every day during the experiment. The stool condition and hematochezia of the mice were observed, the body weight loss rate was calculated, and the disease activity index score was performed.

[0110] After the experiment, the colon was dissected, and the colon contents were squeezed into a ribonucleic acid (RNA) enzyme-free centrifuge tube. The short-chain fatty acid (SCFA) content of the colon contents was detected by gas chromatography.

[0111] Immunofluorescence sections of colon tight junction proteins were prepared.

[0112] The total protein content of the colon tissue homogenate supernatant was determined using a total protein quantification kit from Nanjing Jiancheng Biological Engineering Institute.

[0113] The TNF-α, IL-1β, and IL-6 contents in the serum and colon tissue were determined using an ELISA kit from Wuhan Aibotek Biological Technology Co., Ltd.

[0114] Three, high-throughput sequencing

[0115] The 16S rDNA high-throughput sequencing technology was used to sequence and annotate the species of the colon content flora of the normal group, model group, and high-dose Yali PACs prevention and treatment groups. The flora diversity and different flora between groups were analyzed. The correlation between the intestinal flora and UC disease-related indicators was analyzed to clarify the correlation between the flora and the disease and to mine the key flora of Yali PACs regulating the intestine.

[0116] Four, sequencing results

[0117] 1. OTU abundance

[0118] To study the species composition of each sample, the OTUs of all samples were clustered after flattening the data, and then the sequences of the OTUs were annotated. According to statistical analysis, the total number of OTUs of normal mice was 493, and the number of OTUs in the four groups was the highest, as shown in Table 4 and Figure 3

[0119] Table 4 OTU analysis of colon intestinal flora of mice in each group

[0120] Group Number of OTUs Control 493 Model 359 YH 304 ZH 268

[0121] 2. Alpha diversity

[0122] Alpha diversity includes Chao, ACE, Shannon, Simpson, InvShannon, Fisher and PD index, etc., which are used to measure the richness and diversity of microbial community. It can be seen from Figure 4 that compared with the normal group, the model group has lower Observedspecies, Chao, ACE, Shannon, InvShannon, Fisher and PD index (P<0.05), indicating that DSS modeling can reduce the richness and diversity of intestinal flora of mice. Compared with the model group, the Observedspecies, Chao, ACE, Shannon and Fisher index of intestinal flora of YH group mice were significantly increased (P<0.05), and the Shannon index of intestinal flora of ZH group mice was significantly increased (P<0.05), indicating that the richness and diversity of intestinal microbial community of mice after intervention of Prunus humilis PACs are higher, and Prunus humilis PACs can better resist the changes of microbial community caused by DSS.

[0123] 3. Beta diversity

[0124] According to the unweighted-unifrac algorithm, PCoA method was used to analyze the beta diversity of mouse flora at OTU level. It can be seen from Figure 5 that the samples of C group, M group, YH group and ZH group are clustered, indicating that the microbial composition of samples in each group is similar, and there is a certain difference between samples of each group. In the horizontal coordinate direction, the Control group is far away from the other three groups, indicating that the flora of normal mice has unique characteristics, and there is a large difference between the flora of mice after DSS intervention. In the vertical coordinate direction, the YH group sample is far away from the M group, and the ZH group is close to the M group, indicating that preventive administration of Prunus humilis PACs has a certain intervention effect on the flora of DSS mice, and the therapeutic administration of Prunus humilis PACs has a relatively small effect on the flora, which is consistent with the result of alpha diversity.

[0125] 4. Differential flora analysis ​

[0126] 4.1 Door level, genus level flora composition

[0127] The intestinal flora of mice in different groups was divided into 13 bacterial phyla. More than 80% of the bacteria in the flora of mice in different treatment groups were divided into Firmicutes and Bacteroidetes, Bacteroidetes was the main dominant phylum, followed by Firmicutes Figure 6 ). An increase or decrease in the ratio of Firmicutes and Bacteroidetes abundance F / B was considered to be a flora ecological imbalance, and a decrease in the F / B ratio was associated with diseases such as inflammatory bowel disease. The results of this study showed that the F / B ratio in the M group was lower than in the C group (P<0.001); the F / B ratio in the YH group was higher than in the M group (P<0.01). The results showed that Prunus humilis PACs could improve the flora structure of mice at the door level, and had a significant improvement effect on the Firmicutes and Bacteroidetes of mice.

[0128] It can be seen from Figure 7 that Muribaculaceae, Lachnospiraceae_NK4A136_group, Akkermansia, Alistipes, Clostridia_UCG-014, etc. are the main flora at the genus level, and the abundance of flora at the genus level in each group is different.

[0129] 4.2 Differential flora analysis based on random forest method

[0130] In this study, the random forest method was used to screen the door level classification characteristics (biomarker) of each group, and the characteristics were sorted according to the importance (MeanDecreaseGini value) in the figure. The horizontal axis is the MeanDecreaseGini value, which is used to evaluate the species classification of the model, and the larger the MeanDecreaseGini value, the greater the importance of the variable; the vertical axis is the biomarker, which is arranged in descending order according to the size of the MeanDecreaseGini coefficient. The results, as shown in Figure 8 , compared with the M group, Firmicutes (Firmicutes) was significantly enriched in the C group, Deferribacterota (Deferribacterota) and Bacteroidetes (Bacteroidetes) were significantly enriched in the M group; compared with the YH group, Proteobacteria (Proteobacteria) and Bacteroidetes (Bacteroidetes) were significantly enriched in the M group, and Firmicutes (Firmicutes) was significantly enriched in the C group.

[0131] Further, the random forest method was used to screen the genus level biomarker of each group, and the resultsFigure 9 and Figure 10 Lactobacillus, Lachnospiraceae_UCG-001, A2, Prevotellaceae_NK3B31_group, Roseburia, Alloprevotella, Muribaculum, Lachnospiraceae_NK4A136_group were significantly enriched in C group; Mucispirillum, Parasutterella, Bacteroides were significantly enriched in M group. Escherichia-Shigella, Bilophila, Alistipe were significantly enriched in M group, Lachnospiraceae_NK4A136_group, Clostridia_UCG-014, Ruminococcaceae were significantly enriched in YH group.

[0132] 4.3 Differential microbiota analysis based on LEfSe

[0133] LEfSe analysis is an analysis tool for discovering and interpreting biomarkers (taxa, pathways, genes) in high-dimensional data, which can find species with significant differences in abundance between groups (i.e. biomarker). The analysis results of C group and M group are shown in Figure 11 Muribaculaceae, Lachnospiraceae_NK4A136_group were significantly enriched in C group (P<0.05 and linear discriminant analysis (LDA) score>4.0), Bacteroides, Parasutterella, etc. were significantly enriched in M group (P<0.05 and LDA score>4.0). The analysis results of M group and YH group are shown in Figure 12 Alistipes was significantly enriched in M group (P<0.05 and LDA score>4.0), Clostridia_UCG-014, Lachnospiraceae_NK4A136_group were significantly enriched in YH group (P<0.05 and LDA score>4.0).

[0134] 5. Correlation analysis between intestinal flora and intestinal barrier function

[0135] 5.1 Correlation analysis between intestinal flora and UC disease-related indicators

[0136] The correlation between the abundance of bacterial genera and the UC disease-related indicators, the levels of inflammatory factors, the oxidative stress indicators, the levels of colon tight junction proteins, and the contents of SCFAs in the colon contents was observed.

[0137] Spearman correlation analysis was performed on the abundance of intestinal flora at the genus level and the UC disease-related indicators, and a heatmap was drawn, as shown in Figure 13 A total of 48 genera were significantly correlated with the UC disease-related indicators (P < 0.05), of which 11 genera were significantly correlated with the weight loss rate, 28 genera were significantly correlated with the disease activity index, 19 genera were significantly correlated with the colon length, 14 genera were significantly correlated with the colon wall weight, 23 genera were significantly correlated with the colon damage score, 6 genera were significantly correlated with the thymus index, and 13 genera were significantly correlated with the spleen index. Lachnospiraceae_FCS020_group was significantly correlated with all the above seven UC disease-related indicators. This genus was significantly negatively correlated with the weight loss rate, the disease activity index, the colon wall weight, the colon damage score, and the spleen index, and was significantly positively correlated with the colon length and the thymus index.

[0138] Among the 48 genera, Lachnospiraceae_NK4A136_group, which was significantly enriched in the YH group compared with the M group, was significantly negatively correlated with the weight loss rate, the disease activity index, the colon damage score, and the spleen index, and was significantly positively correlated with the colon length. Ruminococcaceae, which was significantly enriched in the YH group, was significantly negatively correlated with the weight loss rate. Alistipes, which had a significantly reduced abundance in the YH group, was significantly negatively correlated with the colon wall weight. The results showed that the Prunus humilis PACs can play a role in intervening UC by increasing the abundance of beneficial bacteria such as Lachnospiraceae_NK4A136_group and Ruminococcaceae, and reducing the abundance of harmful bacteria such as Alistipes.

[0139] 5.2 Correlation analysis between intestinal flora and the levels of inflammatory factors

[0140] Spearman correlation analysis was performed on the abundance of intestinal flora at the genus level and the levels of colon and serum inflammatory factors, and a heatmap was drawn, as shown in Figure 14A total of 47 genera were significantly correlated with the levels of inflammatory factors (P < 0.05), and 26, 13, 21, 23, 26, and 30 genera were significantly correlated with the levels of colon TNF-a, colon IL-1b, colon IL-6, serum TNF-a, serum IL-1b, and serum IL-6, respectively. Five genera were significantly correlated with the levels of the above six inflammatory factors, among which Lactobacillus, Butyricicoccus, Lachnospiraceae_FCS020_group, and [Eubacterium]_ventriosum_group genera were significantly negatively correlated with the levels of inflammatory factors, and were potential beneficial bacteria; Christensenellaceae_R-7_group genus was significantly positively correlated with the levels of inflammatory factors, and was a potential harmful bacteria.

[0141] Among the 47 genera, the abundance of Lachnospiraceae_NK4A136_group genus, which was significantly enriched in YH group compared with M group, was significantly negatively correlated with the levels of colon TNF-a, colon IL-1b, serum TNF-a, serum IL-1b, and serum IL-6 (P < 0.05), and was a key bacterial group for regulating the levels of inflammatory factors in Euonymus PACs.

[0142] 5.3 Correlation analysis between intestinal flora and the levels of intestinal tight junction proteins

[0143] Spearman correlation analysis was performed on the genus-level intestinal flora abundance and the levels of intestinal tight junction proteins, and a heatmap was generated, as shown in Figure 15 A total of 35 genera were significantly correlated with the levels of tight junction proteins (P < 0.05), and 23, 16, and 10 genera were significantly correlated with the levels of colon Claudin-1, Occludin, and ZO-1, respectively. Three genera were significantly correlated with the levels of the above three tight junction proteins, among which ASF356 and Lachnospiraceae_FCS020_group genera were significantly positively correlated with the levels of Claudin-1, Occludin, and ZO-1, and were potential beneficial bacteria; Ruminiclostridium genus was significantly negatively correlated with the levels of Claudin-1, Occludin, and ZO-1, and was a potential harmful bacteria.

[0144] Compared with M group, Lachnospiraceae_NK4A136_group significantly positively correlated with claudin-1, Clostridia_UCG-014 significantly positively correlated with ZO-1, Ruminococcaceae significantly positively correlated with Occludin, Escherichia-Shigella significantly negatively correlated with Occludin in YH group. Therefore, Prunus humilis PACs may improve the level of intestinal tight junction proteins by regulating the abundance of Lachnospiraceae_NK4A136_group, Clostridia_UCG-014, Ruminococcaceae and Escherichia-Shigella, and then interfere with the UC process.

[0145] 5.4 Correlation analysis between intestinal flora and oxidative stress level

[0146] Spearman correlation analysis was performed between the abundance of genus-level intestinal flora and the oxidative stress indicators of colon and serum, and a heatmap was made, as shown in Figure 16 A total of 42 genera were significantly correlated with oxidative stress indicators (P<0.05), and 9, 14, 23, 20, 28, and 25 genera were significantly correlated with colon SOD activity, colon GSH, colon MDA, serum SOD activity, serum GSH, and serum MDA, respectively. Two genera were significantly correlated with all the above six oxidative stress indicators. Among them, Lachnospiraceae_FCS020_group was significantly positively correlated with colon and serum SOD activity and GSH, and significantly negatively correlated with colon and serum MDA, which was a potential beneficial bacteria; Faecalibacterium was significantly negatively correlated with colon and serum SOD activity and GSH, and significantly positively correlated with colon and serum MDA, which was a potential harmful bacteria.

[0147] Compared with M group, Lachnospiraceae_NK4A136_group significantly positively correlated with serum GSH and significantly negatively correlated with serum MDA in YH group; Alistipes significantly positively correlated with serum MDA and Bilophila significantly negatively correlated with colon SOD activity in YH group. Therefore, the regulation of Prunus humilis PACs on the level of oxidative stress in the body may be related to the regulation of the abundance of Lachnospiraceae_NK4A136_group, Alistipes, and Bilophila.

[0148] 5.5 Correlation analysis between intestinal flora and SCFAs

[0149] Spearman correlation analysis was performed between the abundance of genera and the content of SCFAs in colon contents, and the results were shown in the heat map as Figure 17 A total of 26 genera were significantly correlated with SCFAs (P < 0.05), and 11, 8, 3, 20, 1, 9, and 13 genera were significantly correlated with the content of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and total SCFAs, respectively. The most genera were significantly correlated with butyric acid, indicating that the content of butyric acid was greatly affected by the microbiota. Among the 20 significantly correlated genera, 12 genera were significantly positively correlated with the content of butyric acid, and 8 genera were significantly negatively correlated with the content of butyric acid.

[0150] Compared with group M, Lachnospiraceae_NK4A136_group, which was significantly enriched in group YH, was significantly positively correlated with the content of acetic acid and propionic acid, and Escherichia-Shigella, which was significantly reduced in group YH, was significantly negatively correlated with the content of valeric acid and total SCFAs. Therefore, the regulation of SCFAs content in the intestinal contents by Prunus humilis PACs may be related to the regulation of the abundance of Lachnospiraceae_NK4A136_group and Escherichia-Shigella.

[0151] 5.6 Correlation analysis between key intestinal flora and indicators of intestinal barrier function

[0152] The abundance of Lachnospiraceae_NK4A136_group, Clostridia_UCG-014, and Ruminococcaceae was significantly increased in group YH, and the abundance of Alistipes, Escherichia-Shigella, and Bilophila was reduced in group YH. The network diagram of the 6 differentially expressed genera in group YH and the UC-related indicators significantly correlated with them was shown in Figure 18Lachnospiraceae_NK4A136_group was significantly correlated with 15 UC indicators, mainly negative correlation (weight loss rate, disease activity index, colon damage index, spleen index, colon and serum inflammatory factor levels, serum MDA level), and a small part of positive correlation (colon length, Claudin-1 protein level, propionic acid content, acetic acid content, serum GSH level). Ruminococcaceae was significantly correlated with 2 UC indicators, significantly negatively correlated with weight loss rate, and significantly positively correlated with Occludin protein level. Alistipes was significantly positively correlated with 2 UC indicators, including colon wall weight and serum MDA level. Clostridia_UCG-014 was significantly positively correlated with ZO-1 protein level. Escherichia-Shigella was significantly negatively correlated with 4 UC indicators, including Occludin protein level, acetic acid content, valeric acid content and total SCFAs content. Bilophila was significantly negatively correlated with colon SOD activity. The above results show that Lachnospiraceae_NK4A136_group, Clostridia_UCG-014 and Ruminococcaceae, which have significantly increased in abundance after intervention of Prunus humilis PACs, may be potential beneficial bacteria, and Alistipes, Escherichia-Shigella and Bilophila, which have significantly decreased in abundance after intervention of Prunus humilis PACs, may be potential harmful bacteria. Lachnospiraceae_NK4A136_group is significantly correlated with many UC indicators, and may play an important role in the process of Prunus humilis PACs intervention in UC.

[0153] Example 6

[0154] Based on the animal experiment of Example 5, the effect of Prunus humilis extract (Prunus humilis PACs) on the key signal pathways for improving intestinal barrier function was studied.

[0155] 1. RT-qPCR

[0156] Extract the total RNA of the colon tissue of each group, use Nanodrop2000 to detect the RNA concentration and purity, and dilute to make the final concentration 200 ng / μL.

[0157] Using total RNA as a template, use a reverse transcription kit to synthesize cDNA, configure a reverse transcription reaction solution, and perform reverse transcription reaction.

[0158] The full-length sequence of the mRNA of the target gene was obtained from the NCBI (national center for biotechnology information), and the primer sequence was designed by using the primer and probe design software Primer 5.0. The primer sequence has specificity after Blast analysis. The primers used are shown in Table 5. The primers were synthesized by Wuhan Saivier Biotechnology Co., Ltd.

[0159] Table 5 primer information

[0160]

[0161] Take 0.1 mL of PCR reaction plate, prepare the reaction system, and prepare 3 tubes for each reverse transcription product. After mixing, immediately place it in a fluorescence quantitative PCR instrument, and react under the conditions shown in Table 6. Use 2 -ΔΔCT method to analyze the relative expression amount of the gene.

[0162] qPCR reaction system: 7.5 μL of 2x SYBR Green qPCR Master Mix (None ROX), 1.5 μL of 2.5 μM gene primer (upstream + downstream), 2 μL of reverse transcription product (cDNA), 4 μL of Water Nuclease-Free.

[0163] Table 6 qPCR reaction conditions

[0164]

[0165] 2, Western blot experiment

[0166] 2.1 Protein extraction

[0167] According to the method of the protein extraction kit, the protein of the mouse colon tissue was extracted. Take 5 mL of radioimmunoassay precipitation solution, add 50 μL of 100 mmol / L benzyl sulfonyl fluoride solution, and then add 100 μL of 50x phosphatase inhibitor mixture, mix well to obtain the protein extraction solution. Take the mouse colon on ice, take about 20 mg of colon tissue and put it in a 2 mL centrifuge tube, add 200 μL of protein extraction solution to each tube, put the steel ball in the ball mill and grind the tissue, after sufficient lysis, centrifuge at 4°C, 12000 r / min for 10 min, collect the supernatant into a pre-cooled new centrifuge tube, and store at -80°C for standby.

[0168] 2.2 Sample preparation

[0169] The protein concentration of the protein extract was determined according to the operation instruction of the bicinchoninic acid protein assay kit, and the protein concentration of each sample was diluted to 2 mg / mL with normal saline. After dilution, 4x protein loading buffer was added to the protein extract at a ratio of 3:1, mixed well, and heated in boiling water for 5 min. After cooling, centrifuge at 12000 r / min for 5 min, and store the supernatant at -80°C for standby.

[0170] 2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0171] The glass plate containing the separation gel and the concentration gel was fixed on the electrode holder and placed in the electrophoresis tank. The electrophoresis buffer solution was added to the electrode holder until it reached the upper edge of the glass plate, and the comb was slowly pulled out vertically. 5 μL of standard pre-stained protein molecular marker was added to the first loading hole on the left side and filled to 20 μL with 1x protein loading buffer, and then each protein sample was added to the loading hole in turn. Cover the electrophoresis tank cover, connect the power supply, and adjust the voltage to 120 V when the bromophenol blue band enters the separation gel. Stop electrophoresis when the bromophenol blue approaches the bottom of the glass plate.

[0172] 2.4 Transmembrane

[0173] 0.45 μm polyvinylidene fluoride (PVDF) membrane was activated by soaking in anhydrous methanol for 30 s. The gel after electrophoresis was taken out, the concentration gel at the top and the bromophenol blue at the bottom were cut off, and the clamp was installed in order in the transfer buffer: clamp negative - sponge - filter paper - separation gel - PVDF membrane - filter paper - sponge - clamp positive, there should be no air bubbles between each layer, and the transmembrane clamp was tightly clamped and installed in the electrophoresis tank filled with transfer buffer, and the transmembrane was carried out at 240 mA constant current for 1 h under ice water bath condition.

[0174] 2.5 Antibody incubation

[0175] After transmembrane, the PVDF membrane was taken out and placed in a 5% skim milk solution prepared with tris-buffered saline with tween-20 (TBST) added, and incubated at room temperature for 2 h. Discard the blocking solution, add the primary antibody solution prepared with the blocking solution, and incubate at 4°C overnight. The next day, the primary antibody was recovered, the PVDF membrane was washed with TBST solution 5 times, each time for 5 min at room temperature. Discard the rinse solution, add the secondary antibody solution prepared with the blocking solution, and incubate at room temperature for 1 h. Then wash the PVDF membrane with TBST 5 times, each time for 5 min.

[0176] 2.6 Development

[0177] Mix equal volume of Enhance Chemiluminescence Substrate A and B, add to the developing box, clamp the membrane to touch the filter paper at one corner to absorb the excess liquid, then immerse the membrane into the luminescence solution, and immediately expose to the gel imager to take the photo.

[0178] 2.7 Image analysis

[0179] The ratio of the target protein band to the internal reference protein band was calculated by using ImageJ software to analyze the protein bands, and the relative expression of the target protein was obtained.

[0180] 3. Results and analysis

[0181] 3.1 mRNA expression level of OPN / β3 / PI3K / Akt / NF-κB pathway

[0182] The mRNA expression levels of eight key genes in the extracellular matrix-receptor interaction pathway, including osteopontin (osteopontin / secreted phosphoprotein 1, Spp1) and integrin β3 (integrin β3, Itgb3), the PI3K-AKT signaling pathway, including PI3K (phosphoinositide-3-kinase regulatory subunit 1, Pik3r1) and Akt (Akt1), and the NF-κB pathway, including p65 (Rela), TNF-α (Tnf), IL-1β (Il1b), and IL-6 (Il6), were detected to verify the results of transcriptomics. As shown in Figure 19 , the expression of these genes was regulated by C. cormuta PACs.

[0183] 3.2 Protein expression level of OPN / β3 / PI3K / Akt / NF-κB pathway

[0184] The protein expression levels of OPN / β3 / PI3K / Akt / NF-κB pathway in mouse colon tissues were detected, and the results are shown in Figure 20Compared with group C, the expression levels of OPN and β3 proteins in the colon of UC mice were increased (P < 0.001), and the protein phosphorylation levels in PI3K-Akt and NF-κB pathways were increased to different degrees (p-PI3K / PI3K, p-Akt / Akt, p-IκBα / IκBα, p-p65 / p65). Compared with group M, the expression levels of OPN and β3 proteins in the colon of YH group mice were decreased (P < 0.001), and the levels of p-PI3K / PI3K, p-Akt / Akt, p-IκBα / IκBα and p-p65 / p65 were decreased (P < 0.05, 0.001, 0.05, 0.01). The above results showed that Prunus humilis PACs can inhibit the activation of the OPN / β3 / PI3K / Akt / NF-κB pathway in the colon of mice.

[0185] The above examples are only illustrative of the present application, and do not constitute a limitation on the scope of protection of the present application. Any design identical or similar to the present application falls within the scope of protection of the present application.

Claims

1. The use of *Prunus armeniaca* extract in the preparation of formulations for regulating gut microbiota and / or improving intestinal barrier function; the method for preparing the *Prunus armeniaca* extract includes: After crushing the pulp of the European plum, it is extracted with a 10-70% ethanol aqueous solution. The extract is filtered, the ethanol is removed from the filtrate, and the extract is added to a macroporous adsorption resin. After adsorption and elution, the eluent is collected. Gelatin solution is added to the eluent after removing the ethanol to obtain a precipitate. After dissolving the precipitate by reflux with acetone solution, the acetone was removed, and then extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure and freeze-dried to obtain the purified Prunus cerasifera extract.

2. The application as described in claim 1, characterized in that: The regulation of gut microbiota includes increasing Lachnospiraceae_NK4A136_group , Clostridia_UCG-014 , Ruminococcaceae Fungal abundance.

3. The application as described in claim 1, characterized in that: The regulation of gut microbiota includes reducing Alistipes , Escherichia-Shigella and Bilophila Fungal abundance.

4. The application as described in claim 1, characterized in that, The improvement of intestinal barrier function includes one or more of the following: (1) Inhibits the ROS / OPN / αvβ3 / PI3K / Akt / NF-κB pathway; (2) Reduce the expression levels of inflammatory factors such as TNF-α, IL-1β and IL-6.

5. The application as described in claim 1, characterized in that: The Prunus cerasifera extract includes Prunus cerasifera proanthocyanidins.

6. The application as described in claim 1, characterized in that: The alcohol solution comprises an aqueous ethanol solution with a volume fraction of 10-70%; the liquid-to-solid ratio is (5-45):1; the extraction temperature is 30-80℃; and the extraction time is 1-5 h.

7. The application as described in claim 1, characterized in that: The preparations include health foods, pharmaceuticals, and animal feed.

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