CN113143996B

CN113143996B - Application of mulberry extract in preparation of composition for treating intestinal inflammation of animals

Info

Publication number: CN113143996B
Application number: CN202110269805.6A
Authority: CN
Inventors: 刘玉玲; 申竹芳; 刘率男; 曹慧; 刘志华; 刘泉; 李彩娜; 雷蕾; 陈艳敏; 朱向阳; 邹媛媛
Original assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wuhebao Pharmaceutical Co ltd
Current assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wuhebao Pharmaceutical Co ltd
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2022-10-04
Anticipated expiration: 2041-03-12
Also published as: CN113143996A

Abstract

The invention relates to application of a mulberry extract in preparing a composition for treating intestinal inflammation of animals, which is characterized in that the mulberry extract contains alkaloid, polysaccharide, amino acid and flavone, preferably, the mulberry extract contains the following components in percentage by weight: 3-99% of alkaloid, 0.2-70% of polysaccharide, 0-10% of flavone, 0-50% of amino acid and 0-25% of other components. The mulberry extract can improve the intestinal inflammation state of a diabetic mouse, recover the integrity of intestinal mucosa, regulate the macrophage state and the immune barrier function in the intestinal mucosa, has high safety and good treatment effect on the intestinal inflammation.

Description

Application of mulberry extract in preparation of composition for treating animal intestinal inflammation

Technical Field

The invention relates to application of mulberry extract in preparing a composition for treating intestinal inflammation of animals.

Background

The intestinal inflammation is a defense response of the intestinal tract to stimulation, and the chronic inflammation of the intestinal tract can influence the number of endocrine cells in the intestinal tract and the secretion of intestinal hormone, influence a nerve regulation signal path of a brain-intestinal axis, or participate in the occurrence and development of insulin resistance and type 2 diabetes and the like by changing the permeability of an intestinal barrier. A great deal of research finds that inflammatory factors such as IL-1 beta, TNF-alpha, IFN-gamma and the like can participate in mediating the intestinal mucosa barrier dysfunction in various diseases and destroy the intercellular tight connection; inhibiting inflammatory factors can reduce cell permeability and protect intestinal barrier function. The MAPK/NF-kB pathway is an important pathway of intestinal inflammatory reaction. The MAPK pathway activity is enhanced in intestinal inflammation reaction models such as DSS colitis and inflammatory bowel disease. The nuclear factor-kB (NF-kB) can participate in the occurrence and development processes of various intestinal inflammatory diseases, and MAPK can phosphorylate TATA binding protein in an NF-kB complex, so that the transcriptional activity of NF-kB is regulated, and the transcription of inflammatory reaction is influenced. NF-kB can initiate inflammatory cascade reaction by regulating nuclear transcription factors of various inflammatory genes, plays a very important role in pathogenesis of ulcerative colitis, can relieve intestinal inflammatory reaction by inhibiting the activity of NF-kB and MAPK, improves systemic inflammatory state and immune response of ulcerative colitis, and plays a role in treating ulcerative colitis. Activation of the MAPK pathway leads to increased intestinal permeability, rearrangement of the intercellular tight junction proteins ZO-1 and occludin, and decreased mRNA expression of ZO-1 and Myosin Light Chain Kinase (MLCK).

At present, the medicines for treating intestinal inflammation mainly comprise small molecular medicines such as 5-aminosalicylic acid, glucocorticoid, anti-tumor necrosis factor-alpha medicine, anti-adhesion preparation, proinflammatory factor inhibitor, JAK kinase inhibitor and the like, and auxiliary treatment methods such as supplement, probiotics, enteral nutrition and the like, but the methods have poor treatment effect on a large part of patients or have serious side effects, so that the patients cannot tolerate the medicines. Because natural medicines have incomparable safety, effectiveness and characteristics of multiple points compared with chemical medicines, more and more researches are focused on the development of the natural medicines, and researches show that various plants such as rhizoma kaempferiae, orange peel, grape, cowberry fruit, purple carrot, astragalus, codonopsis pilosula, coptis chinensis, caulis sinomenii and the like contain components for improving intestinal inflammation. In particular, studies have shown that flavonoids and polysaccharides have certain effects of improving intestinal inflammation, for example, IGY and the like found that Genistein inhibits the overproduction of TNF- α and IL-6 in macrophages and the activation of NF- κ B after phosphorylation of AMPK (Genistein supressses LPS-induced inhibition of NF- κ B fol "locking AMP kinase activity in RAW264.7 macrophages J. PLos One,2012,7 (12): e 53101.); the codonopsis pilosula polysaccharide with different doses is given to mice with intestinal inflammation (mucositis caused by tumor chemotherapy) caused by 5-fluorouracil by the Wedner and the like, and the codonopsis pilosula polysaccharide is found to be capable of inhibiting the increase of TNF-alpha, IL-1 beta and IL-6 levels caused by the 5-fluorouracil, relieving inflammation, inhibiting the atrophy of intestinal villi and the increase of depth values of crypts and playing a role in protecting intestinal villi and crypts (the experimental study of the Wedner and the like, the codonopsis pilosula polysaccharide for improving the 5-fluorouracil induced intestinal mucositis, liaoning traditional Chinese medicine J); wujunxian et al found that fucoidan can reduce symptoms of intestinal edema, intestinal atrophy, splenomegaly, inflammatory cell infiltration and submucosal edema of mice with inflammatory bowel disease (induced by dextran sodium sulfate DSS) and protect colon structure by inhibiting expression of proinflammatory factors IL-1 beta, IL-13, TNF alpha, IL-6, IFN-gamma and chemotactic factors MIP-1, MIP-2 and MCP-1, promoting expression of anti-inflammatory factor IL-10 and intestinal epithelial tight junction protein ZO-1 (Wujunxian et al, action and mechanism of fucoidan on inflammatory bowel disease, nanjing university of medicine, traditional Chinese medicine report).

Alkaloids are organic compounds containing nitrogen bases, and the research reports on the influence of the alkaloids on intestinal inflammation are mostly found in berberine, matrine and sinomenine. For example, Y an et al have demonstrated that Berberine can reduce the levels of TNF- α, IFN-7 and IL-17, pro-inflammatory factors of colonic epithelial cells and macrophages in DSS-induced colitis mice, and can alleviate disruption of the intestinal mucosal barrier function and reduce apoptosis (Berberine proteins recovery of peptides and inhibitors of inflammation reactions in colominic molecules and epithelial cells in DSS treated mice); the chunkongdong and the like find that the matrine can obviously reduce inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-alpha, IL-1 beta and the like excessively expressed by ulcerative colitis mucous membranes, and improve inflammatory cell infiltration, edema and fibrosis degree of focus parts (the influence of the matrine on the intestinal mucosal cytokines and free radicals of rats with ulcerative colitis, chinese medical biotechnology); liulian et al found that sinomenine can reduce the activity of mononuclear/macrophage N F-kappa B by inhibiting the expression of proinflammatory factors IL-1, IL-6, TNF-alpha and IL-1 beta, promote the expression of anti-inflammatory factor IL-10, and play a role in protecting the intestinal mucosa immune system (influence of sinomenine on the gene expression of human peripheral blood mononuclear cell IL-1B and IL-8 cytokines. J. China J. Immunol.).

Moraceae plants are regarded as valuable materials for both medicine and food since the past because of high nutritional value and medicinal value, and are recorded in traditional Chinese medicine books and prescriptions for treating diseases such as mulberry twigs, white mulberry root-bark, mulberry leaves and mulberries, for example, the records of mulberry leaf juice decoction for tea, thirst elimination, fried decocted tea and thirst quenching are recorded as early as the compendium of materia Medica, and chemical components of the mulberry leaf juice mainly comprise flavonoids, polysaccharides, alkaloids and the like, so that the mulberry alkaloid is widely used for preparing medicines for reducing blood sugar, reducing blood fat, resisting viruses, regulating immunity and the like, wherein the mulberry alkaloid is a polyhydroxy alkaloid composition containing 1-deoxynojirimycin (1-DNJ), buckwheat alkali, 1, 4-dideoxy-1, 4-imino-D-arabitol and the like, and DNJ extract or mulberry alkaloid is not reported or reported in research on intestinal inflammation, and is not reported or applied to improvement of intestinal inflammation by DNJ monomers or compositions.

Disclosure of Invention

The inventor of the invention has made intensive studies and firstly discovers that the mulberry extract can improve and treat intestinal inflammation in a mouse model, and on the basis, the invention is completed and provides the application of the mulberry extract in preparing the composition for treating the intestinal inflammation of animals.

In one embodiment of the present invention, the mulberry extract contains alkaloids, polysaccharides, amino acids and flavones. Preferably, the content of each component by weight based on the mulberry extract is

More preferably, the weight content of each component based on the mulberry extract is

Further preferably, the weight content of each component based on the mulberry extract is

In one embodiment, the preparation of the mulberry extract comprises the steps of: preparing a crude extract; optionally, separation on a cationic resin and/or an anionic resin; optionally, carrying out alcohol precipitation treatment on the resin effluent; and optionally, concentrating and drying. Preferably, the preparation of the mulberry extract comprises the steps of: step 1): preparing a crude extract; step 2): separation on a cationic resin and/or optionally an anionic resin; optional step 3): carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2); optional step 4) concentration and drying treatment.

In one embodiment, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux, the solvent amount is 3-20 times of the original medicinal materials, extracting repeatedly for 1-3 times, mixing extractive solutions, concentrating, eluting with cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, concentrating the eluate, eluting with anion exchange resin, collecting unadsorbed part, adding ethanol, precipitating to remove impurities, centrifuging, and concentrating the clear solution under reduced pressure or spray drying or freeze drying to obtain extract.

In one embodiment, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux, repeatedly extracting for 1-3 times, mixing extractive solutions, concentrating, eluting with cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, concentrating eluate, eluting with anion exchange resin, collecting unadsorbed part, and concentrating under reduced pressure or spray drying or freeze drying to obtain extract.

In one embodiment, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux for 1-3 times with solvent amount 3-20 times of the original medicinal materials, mixing extractive solutions, concentrating, subjecting to cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, concentrating eluate under reduced pressure or spray drying or freeze drying to obtain extract.

The animal species of the "animal" of the present invention is not particularly limited, and may be any animal having an intestinal tract, preferably a mammal, more preferably a rat, a mouse and a human, and most preferably a human.

In one embodiment of the invention, the animal is a diabetic animal.

In one embodiment of the present invention, the intestinal inflammation according to the present invention is diabetes-induced intestinal inflammation.

In one embodiment of the invention, the intestinal inflammation comprises an intestinal tissue inflammatory response.

In one embodiment of the present invention, the inflammatory response of intestinal tissue comprises: increased expression levels of proinflammatory factors and chemokines, macrophage activation, and activation of gut inflammation-related signaling pathways. In one embodiment, the composition is for use in reducing the expression levels of proinflammatory factors and chemokines in the intestinal tissue, optionally the proinflammatory factors are selected from one of the following: TNF alpha, IL-1 beta, IL-5, IL-1a, IL-6, IL-12b; optionally, the chemokine is selected from one of the following: MCP1, ccl4, ccl5, and CXCl1.

In one embodiment of the invention, the composition is for use in reducing the expression level of pro-inflammatory factors and chemokines in the intestinal tissue, optionally the pro-inflammatory factor is TNF α; optionally, the chemokine is MCP1.

In one embodiment of the invention, the composition is for reducing the serum proinflammatory factor level and the serum chemokine level, optionally, the serum proinflammatory factor is selected from one of the following: interleukin 1 beta (IL-1 beta), interleukin 5 (IL-5), interleukin 1a (IL-1 a), interleukin 6 (IL-6), interleukin 12b (IL-12 b); optionally, the serum chemokine is selected from one of the following: chemokine C-C-motif ligand 1 (Ccl 1), chemokine C-C-motif ligand 4 (Ccl 4), chemokine C-C-motif ligand 5 (Ccl 5), and CXC chemokine ligand 1 (CXCl 1).

In one embodiment of the invention, the composition is used to reduce the expression of a macrophage marker, optionally, the macrophage marker is selected from the group consisting of F4/80 and CD11c.

In one embodiment of the invention, the composition is used to inhibit the transcriptional activity of the transcription factor NF- κ B. The transcription factor NF-kB is a multi-directional transcription factor and is involved in the expression of various molecular genes related to inflammation and immune response.

In one embodiment of the invention, the composition is used to increase the level of a serum anti-inflammatory factor, optionally selected from interleukin 10 (IL-10) and interleukin 13 (IL-13).

In one embodiment of the invention, the intestinal inflammation comprises intestinal mucosal injury, and optionally, the composition is used for increasing the expression level of zonulin ZO-1. The tight junction protein 1 (ZO-1) is related to the integrity of intestinal mucosa, and is one of the important components forming the tight junction, and the expression down regulation or activity reduction of the tight junction protein can influence the formation of intercellular tight junction, prevent the intestinal mucosa from playing an important defense barrier function, and increase the risk of enterogenous infection caused by harmful bacteria and toxin penetrating the intestinal tract and entering blood.

In one embodiment of the invention, the intestinal tissue is ileal tissue.

In one embodiment of the invention, the composition comprises a pharmaceutical composition or nutraceutical composition.

In one embodiment, the composition is used to modulate short chain fatty acid SCFA levels. Preferably, the short chain fatty acid SCFA is acetic acid, propionic acid, butyric acid, isobutyric acid (propionic acid-2-methyl), valeric acid, isovaleric acid (butyric acid-3-methyl), hexanoic acid and isocaproic acid (valeric acid-4-methyl). The SCFAs regulate mucosal production, provide energy to epithelial cells, and play a role in mucosal immune function. In one embodiment, the application is to increase the concentration of acetic acid and propionic acid in feces. In one embodiment, the composition is used to reduce the concentration of butyric acid, isobutyric acid (propionic acid-2-methyl), valeric acid, isovaleric acid (butyric acid-3-methyl), hexanoic acid, and isocaproic acid (valeric acid-4-methyl) in feces.

In one embodiment, the composition is for increasing SCFA transporter levels in the mouse ileum. Preferably, the SCFA transporters are monocarboxylate transporter-1 (MCT-1) and sodium-coupled monocarboxylate transporter (SLC 5 A8). The SCFA transporter is a transport system that functions in the uptake of SCFA cells in the gut, serves as an entry for SCFA in epithelial cells, is responsible for intercellular transfer, and is a determinant of gut function.

In one embodiment, the composition is used to reduce serum endotoxin levels in an animal.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The carrier is an inactive component which has no toxic action on human body and accords with the medication route or the administration mode. The carrier may be a solid or liquid vehicle. Solid excipients, for example, including microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropylcellulose, crospovidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients include, for example, water, ethanol, syrup, and glycerol.

Preferably, the pharmaceutical composition is in a form for oral administration; further preferably, the medicament is a tablet, a capsule, an oral solution, an oral emulsion, a pill, a granule, a syrup, and a powder.

Compared with the prior art, the invention has the beneficial effects that:

the mulberry extract has obvious effect of improving the intestinal inflammation, has definite components, controllable quality and high safety, is suitable for preparing novel medicaments for treating the intestinal inflammation, expands the medicament selection range of patients and provides a new variety for screening the medicaments for treating the intestinal inflammation.

Drawings

FIG. 1 shows the results of long-term administration of SZ-A from mulberry extract in KKAy mice: ileal tissue HE staining results (a) and ileal tissue inflammatory status scoring results (B-E); immunofluorescent staining results (F) of the macrophage maturation and activation marker F4/80 and of the polarization marker CD11c in ileal tissues; ileal tissue inflammation marker (F4/80, MCP1 and TNF alpha) mRNA expression difference results (G); the results of the protein expression difference of ileum tissue macrophage marker CD11c, inflammatory factor MCP1 and intestinal tight junction protein ZO-1 (H) and the expression of ileum tissue inflammatory pathway signaling protein phosphorylation NF-kB (I). * P <0.5, # P <0.01, # P <0.001, compared to the DM group.

FIG. 2 is a graph of changes in the composition of SCFA in feces, including (A) acetate, (B) propionate, (C) butyrate, (D) propionate-2-methyl, (E) valeric acid, (F) butyrate-3-methyl, (G) hexanoic acid, (H) pentanoic acid-4-methyl, and (I) changes in the abundance of MCT1 and SLC5A8 proteins in ileum tissue, following chronic administration of SZ-A in KKAY mice. * P <0.5, # P <0.01, # P <0.001, compared to the DM group.

FIG. 3 shows the results of long-term administration of SZ-A as mulberry extract to KKAy mice: serum endotoxin, inflammatory factor and chemokine levels, (a) serum endotoxin levels (B-L) serum inflammatory factor and chemokine levels. * P <0.5, # P <0.01, # P <0.001, compared to the DM group.

Detailed Description

The invention is explained in more detail below with reference to the figures and examples. The features and advantages of the present invention will become more apparent from the exemplary descriptions.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The contents of the components according to the present invention were measured according to a known method (see methods described in patent publication Nos. CN111077247A and CN 110393738A).

Example 1 preparation of Mulberry extract 1

Pulverizing fresh ramulus Mori (ramulus Mori 11 # with serrate Morus), adding 4000L water, extracting under reflux for 2 hr, mixing extractive solutions, and filtering to remove insoluble substances to obtain crude extractive solution. And (3) carrying out heat concentration on the crude extract until the solid content reaches 4%, and keeping the temperature at 50 ℃ to be used as a sample loading solution of a cation resin column.

Loading 150kg of D113 type macroporous weakly acidic styrene series cationic resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of an eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid through an anion column.

Loading 62.5kg of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; the activation is completed. And (4) loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 870L.

And centrifuging the collected liquid to remove impurities, concentrating the collected liquid by a counter ion permeable membrane, transferring the concentrated liquid with the specific gravity of 1.25 into an alcohol precipitation tank, and adding 25L of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 500rpm of a stirring paddle. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain extract.

Concentrating the effluent under reduced pressure to obtain ramulus Mori extract containing alkaloid 52%, polysaccharide 22%, flavone 0.8%, and amino acids 20%.

Example 2 preparation of Mulberry extract 2

Pulverizing fresh ramulus Mori (Morus alba L. Ex Fr.2) 10kg, adding 150L water, adding into the pulverized ramulus Mori 2L water, decocting for 3 hr each time, mixing extractive solutions, and filtering to remove insoluble substances. The extract was concentrated by heating until the solid content reached 8%, and transferred to an alcohol precipitation tank, to which 2367.9g of absolute ethanol (3L) was added under a stirring paddle of 300 rpm. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, and taking the supernatant as the sample solution of the cation resin column. The cationic resin was activated in the same manner as in example 1, using 002SC type strongly acidic styrene type cationic resin 5kg packed in a column. And (3) loading the extract subjected to concentration and alcohol precipitation, eluting with 100L 5mol/L potassium chloride at an elution speed of 5BV/h, detecting the effluent with 20% silicotungstic acid, starting to collect when white precipitates are generated, stopping collecting when the collected liquid reaches 25L, and directly purifying the collected liquid through an anion column.

The anion resin was activated in the same manner as in example 3 by using 10kg of type 711 strongly basic styrene anion resin packed in a column. And (4) loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 15L. The collected solution was re-loaded onto the cationic resin and re-separated twice with the cationic resin and the anionic resin in this order as described above.

And (3) centrifuging the collected liquid obtained after the three column separations, removing impurities, concentrating by a reverse ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, wherein the specific gravity of the concentrated liquid is 1.25, and adding 125g of absolute ethyl alcohol into the alcohol precipitation tank under the condition that the stirring paddle is 1000 rpm. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain extract. In addition, fresh cortex Mori and folium Mori (SANTOU No. 2) are extracted by the same method and parameters as above.

The obtained ramulus Mori extract contains alkaloid 98%, polysaccharide 0.2%, flavone 0.05%, and amino acids 0.

The obtained cortex Mori extract contains alkaloid 95%, polysaccharide 2%, flavone 0.1%, and amino acids 1%.

In the obtained mulberry leaf extract, the content of alkaloid is 90%, the content of polysaccharide is 4%, the content of flavone is 0.1%, and the content of amino acid is 3%.

Example 3 preparation of Mulberry extract 3

Pulverizing fresh ramulus Mori (ramulus Mori in Guangdong), adding 11500L water, heating and reflux-extracting for 2 hr, mixing extractive solutions, and filtering to remove insoluble substances to obtain crude extractive solution. The crude extract is subjected to centrifugation to remove impurities, and then is concentrated by a counter ion permeable membrane until the solid content reaches 1 percent, and the concentrated extract is used as a sample loading solution of a cation resin column.

The cation resin was activated by the method of preparation example 1 using 300kg of D001 type macroporous strongly acidic styrene cation resin packed in a column. And (3) eluting the concentrated crude extract with 5000L 0.04mol/L ammonium nitrate at the elution speed of 5BV/h, detecting the effluent with 20% silicotungstic acid, starting to collect the extract when a white precipitate is generated, and stopping collecting the extract when the volume of the extract reaches 1000L.

And (4) concentrating the collected liquid obtained after the separation of the cation column by using a nanofiltration membrane, and concentrating under reduced pressure to obtain extract.

The obtained ramulus Mori extract contains alkaloid 15%, polysaccharide 20%, flavone 7%, and amino acids 45%.

Example 4 preparation of Mulberry extract 4

Taking 333kg of dry ramulus Mori (Yue Mulberry No. 11), pulverizing, adding 4000L of water, extracting by heating reflux method twice, each reflux for 1h, mixing extractive solutions, filtering, and concentrating the extractive solution to 1 kg/L crude drug amount.

Loading 150kg of D113 type macroporous weakly acidic phenyl propene cation resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid through an anion column.

Loading 125kg of D218 macroporous strongly basic acrylic acid series anionic resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of an eluate is 9.0; and (4) completing activation. And loading the collected cation resin eluent to anion resin, and collecting the effluent with the pH value of more than 8 until the effluent reaches 870L.

And (3) filtering the collected liquid obtained after the anion column separation by using a microfiltration membrane to remove impurities, concentrating by using a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, wherein the specific gravity of the concentrated liquid is 1.1, and adding 15kg of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 400rpm of a stirring paddle. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain ramulus Mori extract. Sample content: the content of alkaloid is 80%, the content of polysaccharide is 5%, the content of flavone is 0.1%, and the content of amino acid is 4%.

Example 5 preparation of Mulberry extract 5

Taking 400kg of dry ramulus Mori (Yue Mulberry No. 11), pulverizing, adding 4000L of water, extracting by heating reflux method twice, refluxing for 1 hr each time, mixing extractive solutions, filtering, and concentrating the extractive solution to 1kg crude drug amount/L.

Loading 62.5kg of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of an eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of an eluate is 9.0; and (4) completing activation. Loading the collected concentrated extract solution to anion resin, and collecting effluent liquid.

And (3) filtering the collected liquid obtained after the anion column separation by using a microfiltration membrane to remove impurities, concentrating by using a reverse ion permeable membrane, and further concentrating and drying under reduced pressure to obtain the mulberry twig extract. Sample content: the content of alkaloid is 3%, the content of polysaccharide is 70%, the content of flavone is 10%, and the content of amino acid is 10%.

Example 6 preparation of Mulberry extract 6

Pulverizing fresh ramulus Mori (ramulus Mori 11) 1500kg, adding 6000L water, extracting under reflux for 2 hr, mixing extractive solutions, and filtering to remove insoluble substances to obtain crude extractive solution. And (3) carrying out heat concentration on the crude extract until the solid content reaches 4%, and keeping the temperature at 50 ℃ to be used as a sample loading solution of a cation resin column.

Loading 100kg of D113 type macroporous weakly acidic phenyl propene cation resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid through an anion column.

Loading 62.5kg of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; the activation is completed. And loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 870L. Concentrating the effluent under reduced pressure to obtain ramulus Mori extract, wherein the alkaloid content is 30%, polysaccharide content is 35%, flavone content is 2%, and amino acid content is 25%.

Example 7 preparation of Mulberry extract 7

Loading 100kg of D113 type macroporous weakly acidic phenyl propene cation resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be greater than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid through an anion column.

Loading the column with 62.5kg of D218 macroporous strongly basic acrylic acid series anionic resin, and washing with 1.5mol/L sodium hydroxide solution until the pH of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; and (4) completing activation. And (4) loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 870L. Concentrating the effluent under reduced pressure to obtain ramulus Mori extract, wherein the alkaloid content is 40%, polysaccharide content is 25%, flavone content is 0.5%, and amino acid content is 25%.

Example 8 preparation of Mulberry extract 8

Taking 333kg of dry ramulus Mori (Yue Mulberry No. 11), pulverizing, adding 4000L of water, extracting by heating reflux method twice, refluxing for 1 hr each time, mixing extractive solutions, filtering, and concentrating the extractive solution to 1kg crude drug amount/L.

Loading 150kg of D113 type macroporous weakly acidic styrene series cationic resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid through an anion column.

Loading 62.5kg of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; and (4) completing activation. And (3) loading the collected cation resin eluent to anion resin, and collecting the effluent with the pH value of more than 8 until the effluent reaches 870L.

And (3) filtering the collected liquid obtained after the separation of the anion column by using a microfiltration membrane to remove impurities, concentrating by using a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, and adding 15kg of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 400rpm of a stirring paddle, wherein the specific gravity of the concentrated liquid is 1.1. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain ramulus Mori extract. Sample content: the content of alkaloid is 63%, the content of polysaccharide is 23%, the content of flavone is 1%, and the content of amino acid is 5%.

Example 9 animal testing

Female KKAy mice of 12 weeks old are selected, fed with high-fat feed, after the weight of the animals is increased, the animals with higher blood sugar rising percentage at 30min are selected according to the results of oral glucose tolerance experiments, non-fasting blood sugar, serum triglyceride and cholesterol are combined, and the weight of the animals is divided into two dose groups of DM group, SZ-A100 mg/kg/day and 200 mg/kg/day (by total alkaloid weight), the mulberry extract SZ-A1 of the example 8 is administrated for one day, and the DM group is administrated with distilled water in equal amount for about 6 weeks continuously.

TABLE 1 spontaneous type 2 diabetes KKAY mouse cohort data

Compare with DM, data with

Denotes, n =8; DM is a KKAy model control group, SZ-A is an administration group (calculated by total alkaloids in mulberry extract, 100mg/kg and 200mg/kg respectively).

Pathological change of intestinal tissue and detection of related protein expression

The mice were sacrificed about 6 weeks after administration and ileal tissues were fixedly embedded for observation of the effect of SZ-a on its pathomorphological changes and differences in protein expression, thereby evaluating the effect of SZ-a on the intestinal tract. The main evaluation methods and indexes comprise HE staining and inflammatory scoring, positive expression rates of immunofluorescent marker macrophage markers F4/80 and CD11c, real-time PCR investigation of inflammatory markers F4/80, expression of TNF alpha and MCP1, western blot investigation of expression levels of monocyte chemotactic protein-1 (MCP-1) and intercellular tight junction protein (ZO-1), and expression level of immunohistochemical marker inflammatory pathway key factor phosphorylation NF-kappa B p 65.

As shown by HE staining and inflammatory scoring in figure 1, compact inflammatory cell infiltrates appeared in DM groups, mucosa and submucosal tissues, crypts showed typical shortening, focal crypts disappeared and surface epithelial cells destroyed (figure 1A), with each group given a microscopic total score and three characteristic (inflammation, degree of inflammation and crypt damage) scores (figures 1B-E). Compared with the DM group, the inflammatory fraction of the SZ-A group is obviously reduced, wherein the microscopic total fraction, the crypt injury fraction and the inflammatory fraction of the high-dose group are obviously reduced, which indicates that the inflammatory injury state of the ileum tissue of the KKAY mouse can be obviously improved and the integrity of intestinal mucosa can be restored after the SZ-A is administrated for a long time.

The results of immunofluorescence labeling macrophage markers F4/80 and CD11c showed that there was no significant difference in the positive expression rate of macrophage marker F4/80 in the ileum tissues of SZ-A group compared to DM group, but the M1 type marker CD11c was significantly reduced (FIG. 1F).

The Real-time PCR results show that compared with the DM group, both doses of SZ-A can significantly reduce the gene expression levels of proinflammatory factors TNF-alpha, chemotactic factors MCP-1 and macrophage activation marker F4/80 in ileum tissues (FIG. 1G); western blot results show that both SZ-A dose groups can significantly reduce the expression level of macrophage polarization marker CD11c protein compared with DM group, wherein high dose can significantly up-regulate the expression of intestinal mucosa integrity-related factor Claudin ZO-1 (FIG. 1H); immunohistochemistry results showed that both dose groups of SZ-a were able to significantly reduce the positive expression area of phosphorylated NF- κ B p65 in ileal tissues, i.e. inhibit the transcriptional activity of the transcription factor NF- κ B, compared to the DM group (fig. 1I). These results are consistent with the aforementioned HE staining observations, demonstrating that the extract of the invention is able to inhibit the expression of inflammatory factors, restoring the integrity of the intestinal mucosa.

The results show that the mulberry extract can play roles in inhibiting intestinal inflammation and repairing intestinal mucosa injury by inhibiting the expression of proinflammatory factor TNF-alpha and chemotactic factor MCP-1, promoting the expression of factor ZO-1 related to the integrity of intestinal mucosa and inhibiting the transcriptional activity of nuclear transcription factor NF-kappa B.

Fecal Short Chain Fatty Acid (SCFA) analysis

Mice were sacrificed about 6 weeks after administration, and intestinal contents (fecal samples) in the ileum were collected, rapidly frozen in liquid nitrogen, and stored at-80 ℃. SCFA were detected in these samples by gas chromatography-mass spectrometer (GS-MS, agilent Technologies inc. CA, USA) and quantified using Masshunter quantification software.

Short chain fatty acid SCFA produced by intestinal bacteria fermentation regulate mucosal production, provide energy to epithelial cells and play a role in mucosal immune function, thus allowing for quantitative detection of fecal SCFA concentrations.

The results showed that the concentration of acetic acid and propionic acid was increased and butyric acid, isobutyric acid (propionic acid-2-methyl), valeric acid, isovaleric acid (butyric acid-3-methyl), hexanoic acid and isocaproic acid (valeric acid-4-methyl) were decreased in the SZ-a treated group compared to the DM group (fig. 2A-H).

Several transport systems play a role in intestinal SCFA cellular uptake, including monocarboxylate transporter-1 (MCT-1) and sodium-coupled monocarboxylate transporter (SLC 5 A8). The transporter, which serves as an entry site for SCFA in epithelial cells, is responsible for intercellular transfer and is a determinant of intestinal function. The expression levels of MCT-1 and SLC5A8 proteins in ileal tissues were further examined and the results showed that the levels of MCT-1 and SLC5A8 proteins were significantly elevated in the ileum of mice in the SZ-A treated group compared to the DM group (FIG. 2I).

Cytokine and chemokine detection in serum

Mice were sacrificed about 6 weeks after dosing and blood was collected. Serum was prepared by centrifugation at 4000rpm and stored at-80 ℃. The endotoxin concentration was measured by ELISA kit, and the concentrations of interleukin 1 β (IL-1 β), interleukin 5 (IL-5), interleukin 10 (IL-10), interleukin 13 (IL-13), interleukin 1a (IL-1 a), interleukin 6 (IL-6), interleukin 12b (IL-12 b), chemokine C-C-motif ligand 1 (Ccl 1), chemokine C-C-motif ligand 4 (Ccl 4), chemokine C-C-motif ligand 5 (Ccl 5) and CXC chemokine ligand 1 (CXCl 1) were measured by Luminex liquid suspension chip assay (Wayen Biotechnologies, china, shanghai).

Intestinal injury not only leads to increased intestinal permeability, but also leads to bacterial products entering the systemic circulation, causing chronic low-grade inflammation. Thus, considering the effect of SZ-a in reducing intestinal inflammation, cytokine and chemokine levels were further examined.

The results show that SZ-a significantly decreased serum endotoxin, inflammatory cytokines, and chemokine levels and significantly increased serum anti-inflammatory cytokines IL-10 and IL-13 levels in mice compared to the DM group (fig. 3A-L).

Antibody for Western blot and Real-time PCR primer

The antibodies used for Western blot detection were: anti-CD 11C (97585, CST, USA) from Proteitech Group Inc., anti-MCP 1 (ARG 56590, arigo biolaborories Crop, taiwan, china), anti-ALC 5A8 (21433-1-AP), anti-MCT 1 (20139-1-AP), claudin ZO-1 from Aplygen Technologies Inc (61-7300, invitrogen, USA), beta-actin antibody (C1313) and secondary antibodies. Protein levels were normalized to β -actin levels.

The invention uses the conventional method to carry out quantitative Real-time PCR, and the primers used are shown in the following table 2.

TABLE 2 Real-time PCR primers

Example 10 in vitro assay

Influence of mulberry extract SZ-A on macrophage inflammation

1. Culturing RAW264.7 macrophages

RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, placed in a CO2 incubator at 37 ℃ and 5% CO ₂ Culturing under the conditions of (1).

2. Cell processing

The cells were cultured at 20X 10 ⁴ The cells were plated at density of one/ml in 6-well plates and cultured overnight. The blank group was serum-free DMEM medium, and the model group was LPS (0.01 ug/ml) stimulated. The SZ-A prepared in each example was used for treatment at a dose of 62ug/ml, and 30Min was pretreated first, followed by LPS. The stimulation was maintained for 6h. And sucking the supernatant, and detecting the protein content of IL-6 and TNFa secreted by the supernatant by an ELISA kit.

The experimental results are as follows: the levels of inflammatory factors secreted by the control group supernatant and the protein of the inflammatory factors are low, and the expression of IL6 and TNFa is increased after LPS stimulation (p is less than 0.05). After 62mg/ml SZ-A intervention, IL6, TNFa expression was down-regulated compared to the model group (P < 0.05).

The specific results are shown in Table 3.

TABLE 3 Effect of Mulberry extract prepared in examples 1 to 7 on IL6 and TNFa protein levels

Effect of mouse peritoneal macrophage (RAW 264.7) activation on injury of rat ileum epithelial cells (IEC-18)The experimental method comprises the following steps:

1. rat ileum epithelial IEC-18 cell culture: IEC-18 cells were cultured in DMEM high-glucose medium containing 5% fetal bovine serum, 0.1U/ml bovine insulin, 37 degrees Celsius, 5% CO2 and 95% humidity carbon dioxide incubator.

2. Cell co-culture

IEC-18 cells at 2X 10 ⁵ The cells were inoculated in a Transwell plate chamber at a density of one cell/ml, the solution was changed every other day, the resistance was recorded every day, and after 21-24 days of culture, a polar epithelial-like monolayer was formed. One day before co-culture, RAW264.7 cells were cultured at 1X 10 ⁵ One/ml was inoculated in 6-well plates and incubated overnight. The medium was changed to IEC-18 medium, and IEC-18 cell chambers were placed in 6-well plates containing RAW264.7 cells and incubated for 3h. Subsequently, 10ug/ml LPS was used for 12 hours, IEC-18 cells were harvested, RNA was extracted, and the expression of mRNA of Claudin ZO-1 was quantitatively determined. Collecting protein and detecting protein expression. The drug treated group was treated with 62ug/ml of SZ-A.

3 RT-PCR and western blot detection of expression of zon-1 protein

The Transwell chambers seeded with RAW264.7 cells or not seeded with cells were inserted into a 6-well plate pre-loaded with IEC-18 cells, and all cells were replaced with IEC-18 cell medium, and after co-culture incubation for 3h. Subsequently, 10ug/ml LPS was used for 12 hours, IEC-18 cells were harvested, RNA was extracted, and mRNA expression of Claudin ZO-1 was quantitatively determined. Collecting protein and detecting protein expression. The drug treated group was treated with 62ug/ml of SZ-A.

The experimental results are as follows:

in the absence of LPS stimulation, RAW264.7 cells had no effect on the mRNA expression of IEC-18 cell Claudin ZO-1. LPS can obviously inhibit the expression of ZO-1mRNA in IEC-18 cells, and RAW264.7 cells show a stronger effect of inhibiting the expression of ZO-1mRNA under the stimulation of LPS. The addition of SZ-A can increase the expression level of ZO-1, as shown in Table 4.

TABLE 4 Effect of Mulberry extracts prepared from examples 1 to 7 on the ZO-1 protein level

SZ-A	Expression level of ZO-1 (treatment group/model group)
		Example 1 ramulus Mori extract	150％
Example 2 ramulus Mori extract	140％
		Example 3 ramulus Mori extract	120％
Example 4 ramulus Mori extract	150％
		Example 5 ramulus Mori extract	120％
Example 6 ramulus Mori extract	140％
		Example 7 ramulus Mori extract	140％

The present invention has been described above in connection with preferred embodiments, which are merely exemplary and illustrative. On the basis of the above, the invention can be subjected to various substitutions and modifications, and the substitutions and the modifications are all within the protection scope of the invention.

Claims

1. Use of a mulberry extract for the preparation of a composition for the treatment of intestinal inflammation in an animal, said mulberry extract being prepared by the steps of: step 1): preparing a crude extract, and extracting with water; step 2): separation on a cationic resin and/or optionally an anionic resin; optional step 3): carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2); optional step 4) concentration and drying treatment;

the mulberry extract contains alkaloid, polysaccharide, amino acid and flavone, and the weight content of each component based on the mulberry extract is

30 to 99 percent of alkaloid,

0.2 to 35 percent of polysaccharide,

0 to 2 percent of flavone,

0 to 30 percent of amino acid,

0-20% of other components;

the intestinal inflammation is diabetes-induced intestinal inflammation.

2. The use of claim 1, wherein the intestinal inflammation comprises an intestinal tissue inflammatory response.

3. The use according to claim 2, wherein the inflammatory response of intestinal tissue comprises: increased expression levels of proinflammatory factors and chemokines, macrophage activation, and activation of gut inflammation-related signaling pathways.

4. The use according to claim 1, wherein said composition is for reducing the expression levels of pro-inflammatory and chemotactic factors in said intestinal tissue.

5. The use according to claim 4, wherein the proinflammatory factor is selected from one of: TNF alpha, IL-1 beta, IL-5, IL-1a, IL-6, IL-12b.

6. The use according to claim 4, wherein the chemokine is selected from one of the following: MCP1, ccl4, ccl5, and CXCl1.

7. The use according to claim 1, wherein the composition is for reducing the expression of a macrophage marker selected from the group consisting of F4/80 and CD11c.

8. The use according to claim 1, wherein the composition is for inhibiting the transcriptional activity of the transcription factor NF- κ B.

9. The use according to claim 1, wherein said composition is for increasing the level of a serum anti-inflammatory factor selected from the group consisting of IL-10 and IL-13.

10. The use of claim 1, wherein the intestinal inflammation comprises intestinal mucosa

And (4) damaging.

11. The use according to claim 1, wherein the composition is used for increasing the expression level of the compact protein ZO-1.

12. The use according to claim 2, wherein the intestinal tissue is ileal tissue.

13. The use according to any one of claims 1 to 12, wherein the composition comprises a pharmaceutical composition.

14. The use of claim 13, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

15. The use of claim 13, wherein the pharmaceutical composition is administered orally

The preparation is prepared.

16. Use according to claim 13, wherein the pharmaceutical composition is a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup and powder.