Application of mulberry extract in the production of drugs to treat ulcerative colitis

Abstract

The present invention discloses the application of mulberry extract, which is the application of mulberry extract in the manufacture of products for treating and / or preventing ulcerative colitis. The present invention has been demonstrated through experiments that the positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) can all significantly inhibit colon atrophy in UC model mice, and the effects of SZ-A-2, SZ-A-3 and SZ-A-4 groups are equivalent to the positive drug group, and the effect of SZ-A-5 (pretreatment) in inhibiting colon atrophy is higher than that of the positive drug. The mulberry extract of the present invention, as an ingredient derived from a natural plant, has the unique advantages of low toxic side effects, mild and long-lasting effects. Therefore, the mulberry extract, which greatly increases the safety of medication, has a great advantage against ulcerative colitis.

Description

[Technical field]

[0001] The present invention relates to the field of medicine, specifically to the application of mulberry extract in the preparation of medicaments for treating ulcerative colitis. [Background technology]

[0002] Inflammatory bowel disease (IBD) is an autoimmune disease that causes inflammatory lesions in the colon due to various factors, mainly including Crohn's disease (CD) and ulcerative colitis (UC). In Japan, the number of infected people has been increasing rapidly over the past 20 years, and the pathogenesis has not yet been fully elucidated, and there are many influencing factors, such as environmental factors, immune response factors, genetic predisposition, and microbial factors. Inflammatory bowel disease (IBD) is different from general enteritis (e.g., CN113143996A). Drugs or active ingredients with anti-inflammatory activity can often be used to treat enteritis, but they have no effect on inflammatory bowel disease (IBD). Although there has been great progress in the treatment of inflammatory bowel disease, it is still not possible to completely treat it, and there is no specific drug in the world.

[0003] CN113143996A discloses that mice suffering from diabetes develop mild chronic inflammation of the whole body intestine after high fat feeding, while the inflammation of ulcerative colitis is merely limited to the large intestine (Inflammatory Bowel Disease Group, Gastroenterology Division, Chinese Medical Association. Consensus Opinion on Diagnosis and Treatment of Inflammatory Bowel Disease (2018, Beijing) [J]. Chinese Journal of Digestive Diseases, 2018, 38(5): 292-311. DOI: 10.3760 / cma.j.issn.0254-1432.2018.05.002). Ulcerative colitis differs from mild chronic inflammation in that it causes the following symptoms: 1. Bloody stool, perianal abscess, 2. Systemic symptoms such as weight loss, fever, weakness, anemia, 3. Manifestations other than the intestine such as joints, skin, mucous membranes, and eyes, and in severe cases, intestinal perforation, intestinal obstruction, heavy bleeding, and even tumor formation. Ulcerative colitis also has a protracted disease course, recurrent attacks and remains incurable.

[0004] Clinically, treatment is mainly with aminosalicylic acid preparations, but when used to treat mild UC, even if sufficient aminosalicylic acid treatment is administered (generally for 2 to 4 weeks), symptoms are not well controlled, especially when the affected area is extensive, glucocorticoids can be used for treatment. If hormone ineffectiveness or dependence is present, azathioprine drugs, which are immunosuppressants, are used. If the above treatment methods do not alleviate symptoms, anti-TNFα inhibitors are administered.

[0005] In addition, current IBD drugs are also accompanied by serious side effects, including immune resistance and loss of drug tolerance, infectious complications, endocrine disorders, and kidney and liver damage. The problems of low therapeutic efficacy and increased adverse reactions in current IBD treatment methods are the main focus of clinical treatment.

[0006] Therefore, research and development of drugs to safely and effectively treat IBD has become a major scientific problem that urgently needs to be solved. Summary of the Invention

[0007] The present invention aims to provide a new medicinal use of mulberry extract or its main active ingredients.

[0008] The new uses of the mulberry extract or its main active ingredients provided by the present invention are: (a1) The use of mulberry extract in the manufacture of a product for treating ulcerative colitis; (a2) The use of mulberry extract in the manufacture of a product for preventing ulcerative colitis; (a3) The application of mulberry extract in the treatment of ulcerative colitis; (a4) The application of mulberry extract in the prevention of ulcerative colitis; It is one of the following.

[0009] The prevention and / or treatment of ulcerative colitis includes: 1) Suppression of colon atrophy in patients with ulcerative colitis; 2) Suppression of weight loss in patients with ulcerative colitis; 3) Reduction of the disease activity index DAI in patients with ulcerative colitis and 4) Improvement of colonic structure, glandular arrangement and inflammatory cell infiltration in patients with ulcerative colitis; is embodied in at least one of the following:

[0010] The present invention relates to (b1) the manufacture of a product for inhibiting colon atrophy in patients with ulcerative colitis; (b2) Manufacture of a product for inhibiting weight loss in patients with ulcerative colitis; and (b3) Manufacturing a product that reduces the disease activity index (DAI) of patients with ulcerative colitis; (b4) the manufacture of a product that improves colonic structure, glandular arrangement and inflammatory cell infiltration in patients with ulcerative colitis; (b5) Inhibition of colon atrophy in patients with ulcerative colitis; (b6) suppression of weight loss in patients with ulcerative colitis; (b7) Reduction of the disease activity index DAI in patients with ulcerative colitis; (b8) Improvement of colonic structure, glandular arrangement and inflammatory cell infiltration in patients with ulcerative colitis; The present invention further provides an application of at least one of the mulberry extract or its main active ingredients.

[0011] The product is a drug or a drug formulation.

[0012] The mulberry extract is a mulberry twig extract, a mulberry bark extract and / or a mulberry leaf extract, or the mulberry extract may be provided as a commercially available mulberry twig total alkaloids extract (national drug standard Z20200002).

[0013] The mulberry extract may be prepared with reference to the method described in CN113143997A, specifically, the preparation method includes: 1) preparing a crude extract of a mulberry plant; 2) separating the crude extract by a cation resin and / or an optional anion resin to obtain the mulberry extract.

[0014] The method comprises: 3) subjecting the resin effluent of step 2) to ethanol precipitation and collecting the supernatant; 4) the step of concentrating and drying the supernatant may be further included.

[0015] The method may further include a step of concentrating and drying the resin effluent of step 2).

[0016] The mulberry extract functions in humans or mammals.

[0017] The plant of the family Mulberry may be selected from Guangdong mulberry, Lu mulberry, White mulberry, Morus Serrata Roxb, Mountain mulberry or hybrid mulberry, and the hybrid mulberry is preferably Yue Sang No. 11, Gui Sang You No. 62 or Mulberry Special You No. 2. Any part of the plant, such as leaves, roots, branches, bark, buds, stems, fruits, etc., may be used.

[0018] In one embodiment of the present invention, the mulberry extract mainly contains alkaloids, and further contains polysaccharides, flavones and amino acids.

[0019] Preferably, the alkaloid is 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxy-(α- The compound contains at least one of 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-oxy-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0020] Of these, the weight percentage of DNJ is greater than 50% of the total alkaloids.

[0021] Based on the mulberry extract, the weight content of each component is as follows:

[0022] Alkaloids: 50-65%, Polysaccharide: 20~25%, Flavones: 0.5-1.5%, Amino acids: 3-20%, Other ingredients: 8-20%.

[0023] Preferably, the mulberry extract has the following weight contents of each component:

[0024] Alkaloids: 50-65%, Polysaccharide: 20~25%, Flavones: 0.5-1.5%, Amino acids: 5-20%, Other ingredients: 8-20%.

[0025] In one embodiment, the preparation of the mulberry extract comprises the steps of preparing a crude extract, optionally separating with a cation and / or anion resin, optionally precipitation of the resin effluent with ethanol, and optionally concentrating and drying.Preferably, the preparation of the mulberry extract comprises the steps of 1) preparing a crude extract, 2) separating with a cation and / or an optional anion resin, optionally precipitation of the resin effluent from step 2 with ethanol, and optionally 4) concentrating and drying.

[0026] In one embodiment, the mulberry extract is prepared according to the following steps: crush mulberry twigs, mulberry leaves or mulberry bark, and extract with water and / or alcohol solution or acidic water under heating under reflux, the amount of solvent is 3-20 times the amount of the original herb, and the extraction is repeated 1-3 times, the extract is combined and concentrated, and loaded onto a cation exchange resin, the unadsorbed impurities are washed with distilled water, eluted with 0.2-3N ammonia water, the eluate is concentrated and added to anion exchange resin, the unadsorbed part is collected, and then ethanol is added to precipitate to remove impurities, centrifuged, and the clear liquid is vacuum concentrated or spray-dried or freeze-dried to obtain the extract.

[0027] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry twigs, mulberry leaves or mulberry bark are crushed, and extracted under heating and reflux with water and / or alcohol solution or acidic water, the amount of solvent is 3-20 times the amount of the original herb, and the extraction is repeated 1-3 times, the extract is combined and concentrated, and loaded onto a cation exchange resin, the unadsorbed impurities are washed with distilled water, eluted with 0.2-3N ammonia water, the eluate is concentrated and added to an anion exchange resin, and the unadsorbed portion is collected and concentrated under reduced pressure or spray-dried or freeze-dried to obtain an extract.

[0028] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry twigs, mulberry leaves or mulberry bark are crushed, and extracted under heating and reflux with water and / or alcohol solution or acidic water, the amount of solvent is 3-20 times the amount of the original herb, and the extraction is repeated 1-3 times, the extracts are combined and concentrated, and loaded onto a cation exchange resin, the unadsorbed impurities are washed with distilled water, eluted with 0.2-3N ammonia water, and the eluate is concentrated under reduced pressure or spray-dried or freeze-dried to obtain the extract.

[0029] In one embodiment, the mulberry extract is prepared according to the following steps: mulberry twigs, mulberry leaves or mulberry bark are crushed, and extracted with water under reflux, with the amount of solvent being 3-20 times (preferably 4-15 times, more preferably 4-12 times) the amount of the original herb, and the extraction is repeated 1-3 times (preferably 0.5-3 hours each time, more preferably 1-2 hours each time), the extracts are combined and concentrated, and loaded onto a cation exchange resin, the unadsorbed impurities are washed with distilled water, and the eluate is eluted with 0.2-3N ammonia water, the eluate is added to anion exchange resin, the unadsorbed portion (i.e., anion exchange resin effluent) is collected, and ethanol is added to precipitate to remove impurities, centrifuged, and the clear liquid is vacuum concentrated or spray-dried or freeze-dried to obtain the extract.

[0030] Preferably, the cationic resin is placed in a column, and then activated by washing with an acidic solution, an alkaline solution, and an acidic solution in that order. Preferably, the cationic resin is washed with an alkaline solution until the pH of the eluate reaches 8.0 to 9.5, preferably 8.5 to 9.5, and preferably the alkaline solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution, and preferably the concentration of the alkaline solution is 0.5 to 4 mol / L. Preferably, the cationic resin is washed with an acidic solution until the pH of the eluate reaches 3.0 to 7.0, preferably 4.5 to 6.5. Preferably, the acidic solution is selected from a hydrochloric acid solution (which may have a concentration of 1.5 to 2 mol / L), a phosphoric acid solution, or a disodium hydrogen phosphate-citrate buffer solution. Optionally, the cationic resin may be washed one last time with an acidic solution, and then further washed with deionized water in an amount 3 to 5 times the column volume.

[0031] Preferably, the cation resin is a strongly acidic styrene-based cation exchange resin of type 732, a strongly acidic styrene-based cation exchange resin of type 734, a strongly acidic macroporous styrene-based cation exchange resin of type D001, or a weakly acidic macroporous allylbenzene-based cation exchange resin of type D113.

[0032] Preferably, the ratio of the amount of the cationic resin used to the weight of the plant raw material is 1:2-20 (more preferably, the ratio of the amount of the cationic resin used to the weight of the plant raw material is 1:2-7). After the crude plant extract is loaded onto the cationic resin, the loaded cationic resin is eluted with an eluent, preferably having a concentration of 0.5-2.5 mol / L. The flow rate of the eluent is preferably 5-10 BV / h, more preferably 5-6 BV / h. When it is detected that the pH of the effluent from the cationic column is >7, the eluent is collected, and the collected liquid is directly passed through an anionic column for purification.

[0033] Preferably, after placing the anion resin in a column, it is activated by washing with an alkaline solution, an acidic solution, and an alkaline solution in that order. Preferably, washing with an alkaline solution is performed until the pH of the eluate reaches 8.0 to 9.5, preferably 8.5 to 9.5, and preferably the alkaline solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution, and preferably the concentration of the alkaline solution is 0.5 to 4 mol / L. Preferably, washing with an acidic solution is performed until the pH of the eluate reaches 3.0 to 7.0, preferably 3.5 to 4.5. Preferably, the acidic solution is selected from a hydrochloric acid solution (which may have a concentration of 1.5 to 2 mol / L), a phosphoric acid solution, or a disodium hydrogen phosphate-citrate buffer solution.

[0034] Preferably, the anion resin is a 717-type strongly alkaline styrene-based anion exchange resin, a D201-type macroporous strongly alkaline styrene-based anion exchange resin, or a D218-type macroporous strongly alkaline acrylic acid-based anion exchange resin. Preferably, the ratio of the amount of the anion resin used to the weight of the plant raw material input is 1:1-32 (more preferably, the ratio of the amount of the anion resin used to the weight of the plant raw material input is 1:5-16). Collection is started when liquid flows out of the anion resin (preferably, the effluent with a pH greater than 8 is collected). Preferably, collection is stopped when the volume of the collected liquid becomes 0.1-5 times the weight of the plant raw material input.

[0035] Preferably, the weight ratio of ethanol to the plant raw material used in the ethanol precipitation treatment is 1:20 to 300 (more preferably, 1:20 to 50), and the stirring speed in the ethanol precipitation treatment is 40 to 500 rpm. The duration of the ethanol precipitation treatment is 12 to 24 hours.

[0036] Furthermore, the method further includes a step of centrifuging the anion resin effluent to remove impurities or filtering the same through a microfiltration membrane to remove impurities before the ethanol precipitation treatment, and then concentrating the liquid through a counterion permeable membrane. The specific gravity of the liquid after concentration may be 1.1 to 1.25.

[0037] The type of animal referred to in the present invention as "animal" is not particularly limited, and may be any animal having an intestinal organ, preferably a mammal, more preferably a rat, mouse, or human, and most preferably a human.

[0038] Preferably, the drug further comprises a pharma- ceutically acceptable carrier. The carrier is an inactive ingredient that is compatible with the route of administration or the mode of administration and has no toxic side effects on the human body. The carrier may be a solid or liquid excipient. Examples of solid excipients include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium starch glycolate, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium dodecyl sulfate, sodium carboxymethylcellulose, gelatin, xanthan gum, polyvinylpyrrolidone, starch, magnesium stearate, sodium starch glycolate and talc powder, and examples of liquid excipients include water, ethanol, syrup and glycerol.

[0039] Preferably, the medicament is in an oral dosage form, more preferably tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups and powders.

[0040] The present invention further provides a method for preventing and / or treating ulcerative colitis comprising the step of administering said mulberry extract or its main active components to an animal or human recipient to prevent and / or treat ulcerative colitis.

[0041] In the present invention, the animal may be a mammal. Effect of the Invention

[0042] The present invention has the following advantages: 1. Compared to the serious toxic side effects of the main drugs currently in clinical use, for example, aminosalicylic acid preparations that may cause allergic reactions, jaundice, abdominal pain, etc. in patients, glucocorticoids have more toxic side effects, while the mulberry extract of the present invention, as a natural plant-derived ingredient, has the unique advantages of low toxic side effects, mild and long-lasting effects, and multi-pathway and multi-target. Therefore, the mulberry extract, which greatly improves the safety of medication, has a high advantage in the treatment of inflammatory bowel disease. 2. The experimental results show that the specific mulberry extract can inhibit colon atrophy, weight loss, and reduce the disease activity index DAI in UC model mice. After treatment with mulberry extract, the pathology of the mice's colon tissue was obviously improved, the colon structure was relatively complete, the glandular arrangement was regular, and the inflammatory cell infiltration was mild. [Brief description of the drawings]

[0043] In order to more clearly describe the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings used to describe the specific embodiments or the prior art. Of course, the drawings described below are part of the embodiments of the present invention, and those skilled in the art can think of other drawings based on these drawings without creative efforts. [Figure 1] Colon length of mice in each treatment group (using mulberry extract of Preparation Example 1), where * indicates p<0.05 compared to the model group, ** indicates p<0.01 compared to the model group, and *** indicates p<0.001 compared to the model group. [Diagram 2] 1 shows images of the colons of mice in each treatment group (using the mulberry extract of Preparation Example 1). [Diagram 3] FIG. 1 is a graph showing the trend of changes in body weight of mice in each treatment group (using the mulberry extract of Preparation Example 1). [Figure 4] The results show the disease activity assessment index DAI for mice in each treatment group (using the mulberry extract of Preparation Example 1), with *** indicating p<0.001 compared to the model group. [Diagram 5] The results show the disease activity assessment index DAI of mice treated with mulberry extract in each preparation example, with *** indicating p<0.001 compared to the model group. [Figure 6] Pathological images of mice in each treatment group (using the mulberry extract of Preparation Example 1) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention will be described in more detail below with reference to examples. Through these illustrative descriptions, the features and advantages of the present invention will be more clearly and visibly. However, the present invention is not limited to the following examples. The above methods are all common methods unless otherwise specified. The above raw materials can be obtained from public commercial channels unless otherwise specified.

[0045] The technical term "exemplary" means "serving as an example, embodiment, or description." Any embodiment described as "exemplary" herein is not necessarily to be construed as superior or preferred over other embodiments.

[0046] Furthermore, the technical features according to different embodiments of the present invention described below may be combined as long as they are not inconsistent with each other.

[0047] The content of the components of the present invention is measured according to the disclosed methods (see the methods described in the patents with publication numbers CN111077247A and CN110393738A).

[0048] 1. Example of preparation of mulberry extract Preparation Example 1 Take 1000 kg of fresh mulberry twigs (Mollus serrata No. 11) and crush them. Then add 4000 L of water and extract for 2 hours by heating under reflux. Combine the extracts and filter out the insoluble matter to obtain a crude extract. Heat concentrate the crude extract until the mass percentage of solids is 4%, and keep the temperature at 50℃ to become the loading liquid for the cation resin column.

[0049] 150 kg of D113 type macroporous type weak acid allylbenzene-based cation resin is placed in the column, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, washed with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, and then washed with 5 times the column volume of deionized water to complete activation. The concentrated extract is loaded, and then eluted with 1000L 2.5 mol / L ammonia water. The elution rate is 6BV / h. When it is detected that the pH of the effluent from the cation column is >7, the eluate is collected. When the collected liquid reaches 900L, the collection is stopped, and the collected liquid is directly passed through the anion column for purification.

[0050] 62.5 kg of D218 type macroporous strong alkaline acrylic acid anion resin is placed in a column, washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, and washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0 to complete activation. The collected cation resin eluate is loaded onto the anion resin, and the effluent is collected until the volume is 870 L.

[0051] The collected liquid is centrifuged to remove impurities, then concentrated with a counterion permeation membrane to make the specific gravity of the concentrated liquid 1.25, then transferred to an ethanol precipitation tank and 25 L of absolute ethanol is added with a stirring paddle of 500 rpm. After the ethanol is completely added, the stirring is stopped and the ethanol precipitation process is carried out for 24 hours, and the supernatant is taken and concentrated under reduced pressure to obtain the mulberry branch extract.

[0052] The mulberry branch extract contains 52% alkaloids, 22% polysaccharides, 0.8% flavones, and 20% amino acids. The alkaloids are 60% 1-DNJ, 17% FAG, and 15% DAB.

[0053] Preparation Example 2 Take 10 kg of fresh mulberry twigs (Mulberry Special No. 2) and crush them. Then add 150 L of water in two batches and extract for 3 h each time by decoction method. Combine the extract and filter out the insoluble matter. Heat concentrate the extract until the mass percentage of solids is 8%, then transfer it to an ethanol precipitation tank and add 2367.9 g of absolute ethanol (3 L) with a stirring paddle of 300 rpm. After the ethanol is completely added, stop stirring and perform ethanol precipitation for 24 h, and take the supernatant as the loading liquid for the cation resin column. Put 5 kg of 002SC type strong acid styrene-based cation resin into the column and activate the cation resin according to the method of Example 1. The concentrated and ethanol precipitated extract is loaded, then eluted with 100L 5mol / L potassium chloride. The elution rate is 5BV / h. The effluent is detected with 20% tungstosilicic acid. Collection begins when a white precipitate forms. When the collected liquid reaches 25L, collection is stopped and the collected liquid is directly passed through an anion column for purification.

[0054] Place 10 kg of 711 type strong alkaline styrene-based anion resin into the column and activate the anion resin according to the method of Preparation Example 1. Load the collected cation resin eluate onto the anion resin and collect the effluent until it reaches 15 L. Load the collected eluate onto the cation resin again and separate twice again using the cation resin and anion resin in sequence according to the above method.

[0055] The collected liquid obtained after three separations in the column is centrifuged to remove impurities, then concentrated with a counterion permeation membrane to make the specific gravity of the concentrated liquid 1.25, then transferred to an ethanol precipitation tank and added with 125g of absolute ethanol at a stirring paddle of 1000 rpm. After complete addition of ethanol, the stirring is stopped, and the ethanol precipitation process is carried out for 24 hours, and the supernatant is taken and concentrated under reduced pressure to obtain the mulberry branch extract. In addition, fresh mulberry bark and mulberry leaves (Mulberry Special No. 2) are taken for extraction, and the extraction method and parameters are the same as those of the above method.

[0056] The obtained mulberry branch extract has an alkaloid content of 98%, a polysaccharide content of 0.2%, a flavone content of 0.05%, and an amino acid content of 0. In terms of alkaloids, the 1-DNJ content is 99%, FAG is 0.5%, and DAB is 0.4%.

[0057] The resulting mulberry bark extract contains 95% alkaloids, 2% polysaccharides, 0.1% flavones, and 1% amino acids. The alkaloid content is 96% 1-DNJ, 1.5% FAG, and 1.4% DAB.

[0058] The mulberry leaf extract obtained had an alkaloid content of 90%, polysaccharide content of 4%, flavone content of 0.1%, and amino acid content of 3%. Among the alkaloids, the 1-DNJ content was 91%, FAG was 3.1%, and DAB was 2.8%.

[0059] Preparation Example 3 Take 1000 kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500 L of water, heat and reflux for 2 hours, combine the extracts, filter out the insoluble matter, and obtain a crude extract. The crude extract is centrifuged to remove impurities, and then concentrated with a counterion permeation membrane until the solid mass percentage is 1%, which becomes the loading liquid for the cation resin column.

[0060] 300 kg of D001 type macroporous type strong acid styrene-based cation resin is placed in a column, and the cation resin is activated according to the method of Preparation Example 1. The concentrated crude extract is loaded and eluted with 5000L 0.04mol / L ammonium nitrate, the elution rate is 5BV / h, and the effluent is detected with 20% tungstosilicic acid. When a white precipitate is formed, collection is started, and when the collected liquid reaches 1000L, collection is stopped.

[0061] The collected liquid obtained after separation through a cation column is concentrated through a nanofiltration membrane and then concentrated under reduced pressure to obtain the extract.

[0062] The obtained mulberry branch extract has an alkaloid content of 15%, polysaccharide content of 20%, flavone content of 7%, and amino acid content of 45%. Among the alkaloids, the 1-DNJ content is 55%, FAG is 23%, and DAB is 10%.

[0063] Preparation Example 4 333 kg of dried mulberry twigs (Guangdong mulberry No. 11) were taken and crushed, after which 4000 L of water was added and extracted twice using the heating reflux method, refluxing for 1 hour each time. The extracts were combined and filtered, and then concentrated to 1 kg herbal medicine amount per L.

[0064] 150 kg of D113 type macroporous type weak acid allylbenzene-based cation resin is placed in the column, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, washed with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, and then washed with 5 times the column volume of deionized water to complete activation. The concentrated extract is loaded, and then eluted with 1000L 2.5 mol / L ammonia water. The elution rate is 6BV / h. When it is detected that the pH of the effluent from the cation column is >7, the eluate is collected. When the collected liquid reaches 900L, the collection is stopped, and the collected liquid is directly passed through the anion column for purification.

[0065] Put 125 kg of D218 type macroporous strong alkaline acrylic acid anion resin into the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, and wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0 to complete activation. Load the collected cationic resin eluate onto the anionic resin, and collect the effluent with a pH greater than 8 until it reaches 870 L.

[0066] The collected liquid obtained after separation in the anion column is filtered through a microfiltration membrane to remove impurities, and then concentrated through a counterion permeation membrane to make the specific gravity of the concentrated liquid 1.1, which is then transferred to an ethanol precipitation tank and 15 kg of anhydrous ethanol is added at a stirring paddle of 400 rpm. After complete addition of ethanol, the stirring is stopped and the ethanol precipitation process is carried out for 24 hours, the supernatant is taken and concentrated under reduced pressure to obtain the mulberry branch extract. The content of the sample is 80% alkaloid, 5% polysaccharide, 0.1% flavone, and 4% amino acid. In the alkaloids, the content of 1-DNJ is 75%, FAG is 12%, and DAB is 10%.

[0067] Preparation Example 5 Take 400 kg of dried mulberry twigs (Guangdong mulberry No. 11) and crush them. Then add 4000 L of water and extract them twice using the heating and refluxing method, refluxing for 1 hour each time. The extracts are combined and filtered, and concentrated until the extract reaches 1 kg of herb per L.

[0068] Put 62.5 kg of D218 type macroporous type strong alkaline acrylic acid-based anion resin into the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, and complete the activation. Load the collected extract concentrate onto the anion resin and collect the effluent.

[0069] The collected liquid obtained after separation through an anion column is filtered through a microfiltration membrane to remove impurities, then concentrated through a counterion permeation membrane, and then vacuum concentrated and dried to obtain the mulberry branch extract. The sample contents are 3% alkaloid, 70% polysaccharide, 10% flavone, and 10% amino acid. In terms of alkaloids, the 1-DNJ content is 68%, FAG is 17%, and DAB is 8%.

[0070] Preparation Example 6 Take 1500 kg of fresh mulberry twigs (Mollus serrata No. 11) and crush them. Then add 6000 L of water and extract for 2 hours by heating under reflux. The extracts are combined and insoluble matter is filtered off to obtain a crude extract. The crude extract is then thermally concentrated until the mass percentage of solids is 4%, and the temperature is maintained at 50℃ to become the loading liquid for the cation resin column.

[0071] 100 kg of D113 type macroporous type weak acid allylbenzene-based cation resin is placed in a column, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, washed with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, and then washed with 5 times the column volume of deionized water to complete activation. The concentrated extract is loaded, and then eluted with 1000L 2.5 mol / L ammonia water. The elution rate is 6BV / h. When it is detected that the pH of the effluent from the cation column is >7, the eluate is collected. When the collected liquid reaches 900L, the collection is stopped, and the collected liquid is directly passed through the anion column for purification.

[0072] 62.5 kg of D218 type macroporous type strong alkaline acrylic acid-based anion resin is placed in the column, washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, and washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0 to complete activation. The collected cationic resin eluate is loaded onto the anionic resin, and the effluent is collected until it reaches 870 L. The effluent is concentrated under reduced pressure to obtain the mulberry branch extract, in which the alkaloid content is 30%, the polysaccharide content is 35%, the flavone content is 2%, and the amino acid content is 25%. In the alkaloids, the 1-DNJ content is 62%, FAG is 20%, and DAB is 13%.

[0073] Preparation Example 7 Take 1000 kg of fresh mulberry twigs (Mollus serrata No. 11) and crush them. Then add 4000 L of water and extract for 2 hours by heating under reflux. Combine the extracts and filter out the insoluble matter to obtain a crude extract. Heat concentrate the crude extract until the mass percentage of solids is 4%, and keep the temperature at 50℃ to become the loading liquid for the cation resin column.

[0074] 100 kg of D113 type macroporous type weak acid allylbenzene-based cation resin is placed in a column, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, washed with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, and then washed with 5 times the column volume of deionized water to complete activation. The concentrated extract is loaded, and then eluted with 1000L 2.5 mol / L ammonia water. The elution rate is 6BV / h. When it is detected that the pH of the effluent of the cation column is >7, the eluate is collected. When the collected liquid reaches 900L, the collection is stopped and the collected liquid is directly passed through the anion column for purification.

[0075] 62.5 kg of D218 type macroporous type strong alkaline acrylic acid-based anion resin is placed in the column, washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, and washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0 to complete activation. The collected cationic resin eluate is loaded onto the anionic resin, and the effluent is collected until it reaches 870 L. The effluent is concentrated under reduced pressure to obtain the mulberry branch extract, in which the alkaloid content is 40%, the polysaccharide content is 25%, the flavone content is 0.5%, and the amino acid content is 25%. In the alkaloids, the 1-DNJ content is 57%, FAG is 24%, and DAB is 16%.

[0076] Preparation Example 8 333 kg of dried mulberry twigs (Guangdong mulberry No. 11) were taken and crushed, after which 4000 L of water was added and extracted twice using the heating reflux method, refluxing for 1 hour each time. The extracts were combined and filtered, and then concentrated to 1 kg herbal medicine amount per L.

[0077] 150 kg of D113 type macroporous type weak acid allylbenzene-based cation resin is placed in the column, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, washed with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5, and then washed with 5 times the column volume of deionized water to complete activation. The concentrated extract is loaded, and then eluted with 1000L 2.5 mol / L ammonia water. The elution rate is 6BV / h. When it is detected that the pH of the effluent from the cation column is >7, the eluate is collected. When the collected liquid reaches 900L, the collection is stopped, and the collected liquid is directly passed through the anion column for purification.

[0078] 62.5 kg of D218 type macroporous strong alkaline acrylic acid anion resin is placed in a column, washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0, washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5, and washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0 to complete activation. The collected cation resin eluate is loaded onto the anion resin, and the effluent with a pH greater than 8 is collected until the volume is 870 L.

[0079] The collected liquid obtained after separation in the anion column is filtered through a microfiltration membrane to remove impurities, and then concentrated through a counterion permeation membrane to make the specific gravity of the concentrated liquid 1.1, which is then transferred into an ethanol precipitation tank and 15 kg of anhydrous ethanol is added with a stirring paddle at 400 rpm. oAfter the ethanol is completely added, the stirring is stopped, and the ethanol precipitation process is carried out for 24 hours. The supernatant is taken and concentrated under reduced pressure to obtain the mulberry branch extract. The sample contents are 63% alkaloid, 23% polysaccharide, 1% flavone, and 5% amino acid. In terms of alkaloids, the 1-DNJ content is 61.9%, FAG is 16.6%, and DAB is 11.1%.

[0080] 2. Verification of the effects of mulberry extract Experimental Example 9: Pharmacological study of mulberry extract in the prevention and / or treatment of inflammatory bowel disease 1. Experimental Plan 1.1 Experimental animals Mice aged 6 to 8 weeks were placed in an animal house and then reared for three days for adaptation. After weighing, the mice were randomly divided into eight groups (total 0 days), with five to six mice per group. The experimental groups were divided into normal control group 1, model group 2, positive control group 3, and different doses of SZ-A group 4 to 8. See Table 1 for details.

[0081] 1.2 Experimental drugs 3% DSS preparation method: Dissolve 3g DSS (dextran sulfate sodium salt) in 100mL of sterile water. The mulberry branch extract prepared in Preparation Example 1 was mixed at different concentrations and dissolved in 1% CMC solution. The positive control drug is Azulfidine enteric-coated tablets (National Pharmaceutical Standards Code H31020557). The main ingredient of this product is Azulfidine, whose chemical name is 5-[p-(2-pyridineaminesulfonyl)benzene]azosalicylic acid.

[0082] 1.3 Modeling and administration On the day when grouping is completed, mice in group 8, SZ-A-5 (pretreatment), are orally and intragastrically injected with mulberry branch extract (see the table below for dosage) once a day. All groups are allowed to eat and drink normally.

[0083] After the 8th group of mice was administered for 3 days, all other groups, except for the normal control group, were allowed to drink 3% DSS (dextran sulfate sodium salt) ad libitum, and modeling and intragastric injection were performed, where the dose of 5-[p-(2-pyridinaminesulfonyl)benzene]azosalicylic acid was 500 mg / kg / d for the positive control group. Five days after the start of modeling, the drinking of 3% DSS was stopped, and all groups were replaced so that they could drink normally, and were administered normally. The mice were continuously administered and observed for 10 days, and the mice were killed by dislocating the cervical vertebrae, dissected to obtain the colon of the mouse, and the length of the colon was measured and photographed. During the administration period, the weight of each group of mice was measured. During the modeling and administration period, the properties of the mouse's stool and the state of bloody stool were monitored every day, and the disease activity evaluation index DAI of the mouse was evaluated based on Table 2 below. The results are shown in Figures 1 to 3 and 4. Table 1. Experimental groupings and doses [Table 1]

[0084] Table 2. Evaluation standards for the disease activity index DAI in mice [Table 2] Note: DAI = weight loss score + stool condition score + occult bleeding score; normal stool: formed stool; semi-loose stool: pasty semi-formed stool that does not stick to the anus; loose stool: watery stool that can stick to the anus.

[0085] Colon tissue was taken and stained with H&E, which specifically includes the following steps: 1. Take a 3 mm thick tissue sample, dehydrate it in gradient alcohols of 70%, 80%, 95%, and 100% for 30 minutes each, immerse it in two xylenes for 20 minutes each, infiltrate it in two paraffin bowls for 12 minutes each, embed it, slice it into 4 micrometers, and dry it at high temperature. 2. Hematoxylin and eosin (HE) staining: (1) Dewax each tube with 3 bottles of xylene for 8 minutes, 2 bottles of 100% alcohol for 8 minutes, 90% alcohol, 80% alcohol, and 60% alcohol for 8 minutes. (2) Stain with hematoxylin for 4 minutes, wash with running water. (3) Differentiate with hydrochloric acid alcohol for 2-3 seconds, wash with running water. (4) Treat with 0.5% ammonia water for 20 seconds, wash with running water, and observe under a microscope. (5) Stain with 0.5% eosin for 1 minute. (6) Differentiate with 80% alcohol and 90% alcohol for 3-5 seconds each, differentiate with 95% alcohol for 5 minutes, differentiate with 3 bottles of 100% alcohol for 5 minutes each, and differentiate with 2 bottles of xylene for 5 minutes each. (7) Seal with neutral resin adhesive, observe with an optical microscope, and take microphotographs. Fresh colon tissues from each set were fixed in 4% paraformaldehyde for 48 hours, dehydrated in different alcohol concentrations, and then cleared in xylene. The cleared tissues were embedded in paraffin. The embedded wax block was fixed on a slicer and sliced.

[0086] 1.4 Comparison of the disease activity index DAI of mulberry branch extract in each preparation example With reference to the above experimental steps 1.1 to 1.3, the mulberry branch extracts in each preparation example were evaluated and compared for the disease activity evaluation index DAI in mice, and the drugs were the mulberry branch extracts in preparation examples 1, 3, 4, 6 and 8, and the dosages calculated based on the mulberry branch extract were all 200 mg / kg / d. The results are shown in Figure 5.

[0087] 1.5 Experimental results As can be seen from Figures 1 and 2, compared with the normal control group, the colon length of the model group was significantly shortened; compared with the model group, the positive drug groups, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) all significantly inhibited colon atrophy in UC model mice; the effects of SZ-A-2, SZ-A-3 and SZ-A-4 groups were equivalent to those of the positive drug group, and the effect of SZ-A-5 (pretreatment) was higher than that of the positive drug.

[0088] As can be seen from Figure 3, the model group of mice lost a significant amount of body weight compared to the normal control group, but the positive drug groups SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) were all able to inhibit the weight loss of UC model mice and had comparable effects to the model group.

[0089] As can be seen from Figure 4, compared to the model group, the positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment), can all reduce the disease activity assessment index DAI in UC model mice.

[0090] As can be seen from FIG. 5, the specific mulberry extracts provided in Preparation Example 1 (alkaloid content 52%, polysaccharide content 22%, flavone content 0.8%, amino acid content 20%) and Preparation Example 8 (alkaloid content 63%, polysaccharide content 23%, flavone content 1%, amino acid content 5%) can reduce the disease activity evaluation indexes of UC model mice, while the mulberry extracts in other Preparation Examples do not have significant therapeutic effects.

[0091] As can be seen from Fig. 6, when observing the tissue slices of UC model mice under optical microscope, the colon structure of the mice in the control group is complete and clear, and the glandular arrangement is regular, but in the model group, the mice that continuously drink 3% DSS have colon wall congestion, edema, severe inflammatory cell infiltration in the mucosal layer and submucosa, epithelial cells are damaged and sloughed off, goblet cells are lost, crypts are swollen and destroyed, and colonic ulcers are severe. After being treated with mulberry extract, the pathology of the colonic tissue of the mice is obviously improved, the colon structure is complete, the glandular arrangement is regular, inflammatory cell infiltration is mild, and the effect is better than that of the positive control group. It is shown that mulberry extract can effectively prevent the occurrence of UC and has a high preventive effect on inflammatory bowel disease.

[0092] The above describes the present invention in combination with preferred embodiments, but these embodiments are merely illustrative and for the purpose of explanation, based on which various substitutions and improvements can be made to the present invention, all of which fall within the scope of protection of the present invention.

[0093] Industrial Applications Through experiments, the present invention demonstrates that certain mulberry extract can inhibit colon atrophy, weight loss, and reduce disease activity evaluation index DAI of UC model mice, and after treatment with mulberry extract, the pathology of mouse colon tissue is obviously improved, colon structure is complete, glandular arrangement is regular, and inflammatory cell infiltration is mild.The above results show that mulberry extract or its main active components can be used to prepare ulcerative colitis treatment drug.

Claims

1. A mulberry extract or its main active component, to be used for the treatment and / or prevention of ulcerative colitis in patients requiring treatment.

2. The treatment and / or prevention of the aforementioned ulcerative colitis is 1) Suppression of colonic atrophy in patients with ulcerative colitis, 2) Suppression of weight loss in patients with ulcerative colitis, 3) Reduction of the disease activity index DAI in patients with ulcerative colitis, 4) Improvement of colonic structure, glandular arrangement, and inflammatory cell infiltration in patients with ulcerative colitis, The mulberry extract or its main active ingredient according to claim 1, characterized in that it is embodied in at least one of the following.

3. The treatment and / or prevention of ulcerative colitis, (b1) Suppression of colon atrophy in patients with ulcerative colitis, (b2) Suppression of weight loss in patients with ulcerative colitis, (b3) Reduction of the disease activity index DAI in patients with ulcerative colitis, (b4) Improvement of colonic structure, glandular arrangement and inflammatory cell infiltration in patients with ulcerative colitis, The mulberry extract or its main active ingredient according to claim 1, having the effect of at least one of the following.

4. A mulberry extract or its main active component for reducing DAI, a disease activity assessment index for patients with ulcerative colitis.

5. The mulberry extract according to Claim 1 or its main active ingredient, characterized in that the mulberry extract is a mulberry branch extract, a mulberry bark extract and / or a mulberry leaf extract.

6. A pharmaceutical or pharmaceutical preparation for the treatment and / or prevention of ulcerative colitis, comprising mulberry extract or its main active ingredient.

7. The method for preparing the mulberry extract is: 1) A step of preparing a crude extract of a mulberry plant, 2) The step of separating the crude extract of the mulberry plant with a cationic resin and / or an optionally selected anionic resin to obtain the mulberry extract, A mulberry extract or its main active ingredient as described in claim 1, characterized in that

8. The preparation method is 3) The resin effluent from step 2) is subjected to ethanol precipitation treatment, and the supernatant liquid is collected. 4) The process further includes the step of performing a concentration and drying treatment on the supernatant liquid, Alternatively, the process further includes the step of concentrating and drying the resin effluent from step 2). The mulberry extract or its main active ingredient according to claim 7, characterized in that

9. The mulberry extract or its main active ingredient according to claim 1, characterized in that the main active ingredient of the mulberry extract is at least one of 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin, fagomine, 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol, callistegin B2, callistegin C1, 2-oxy-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-oxy-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-oxy-β-D-glucopyranosyl)-D-arabinitol.

10. The mulberry extract according to claim 1, or its main active ingredient, characterized in that the mulberry extract functions in humans or mammals.

11. With respect to the mulberry extract, The alkaloid content is 15-98% by weight. The polysaccharide content is 0.2 to 35% by weight. The flavonoid content is 0.05 to 7% by weight, and The amino acid content is 0-45% by weight. The mulberry extract or its main active ingredient according to claim 1, wherein the content is based on the total weight percentage of each component in the mulberry extract.

12. With respect to the mulberry extract, The alkaloid content is 30-98% by weight. The polysaccharide content is 0.2 to 35% by weight. The flavonoid content is 0.05 to 2% by weight, and The amino acid content is 0-25% by weight. The mulberry extract or its main active ingredient according to claim 1, wherein the content is based on the total weight percentage of each component in the mulberry extract.

13. With respect to the mulberry extract, The alkaloid content is 50-65% by weight. The polysaccharide content is 20-25% by weight. The flavonoid content is 0.5 to 1.5% by weight. The amino acid content is 3 to 20% by weight, and The content of other ingredients is 8-20%. Preferably, The alkaloid content is 50-65% by weight. The polysaccharide content is 20-25% by weight. The flavonoid content is 0.5 to 1.5% by weight. The amino acid content is 5-20% by weight, and The mulberry extract according to claim 1, or its main active ingredient, wherein the content of other components is 8 to 20% by weight.

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