WO2023186181A1 - 桑提取物在制备治疗溃疡性结肠炎的药物中的应用 - Google Patents

桑提取物在制备治疗溃疡性结肠炎的药物中的应用 Download PDF

Info

Publication number
WO2023186181A1
WO2023186181A1 PCT/CN2023/093572 CN2023093572W WO2023186181A1 WO 2023186181 A1 WO2023186181 A1 WO 2023186181A1 CN 2023093572 W CN2023093572 W CN 2023093572W WO 2023186181 A1 WO2023186181 A1 WO 2023186181A1
Authority
WO
WIPO (PCT)
Prior art keywords
ulcerative colitis
extract
mulberry
patients
mulberry extract
Prior art date
Application number
PCT/CN2023/093572
Other languages
English (en)
French (fr)
Inventor
刘玉玲
叶军
刘东东
王洪亮
王沫
李仁杰
杨艳芳
Original Assignee
北京五和博澳药业股份有限公司
广西五和博澳药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京五和博澳药业股份有限公司, 广西五和博澳药业有限公司 filed Critical 北京五和博澳药业股份有限公司
Publication of WO2023186181A1 publication Critical patent/WO2023186181A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • the invention belongs to the field of medicine, and specifically relates to the application of mulberry extract in preparing medicine for treating ulcerative colitis.
  • IBD Inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • the diabetic mice disclosed in CN113143996 A developed chronic low-grade inflammation of the whole intestine after high-fat feeding, and the inflammation of ulcerative colitis was limited to the large intestine (Inflammatory Bowel Disease Group of the Gastroenterology Branch of the Chinese Medical Association. Inflammatory Consensus opinion on the diagnosis and treatment of enteric diseases (2018, Beijing) [J].
  • Ulcerative colitis is different from chronic low-grade inflammation in that it can also present the following symptoms: 1. Blood in the stool, perianal abscess; 2. Systemic symptoms such as weight loss, fever, fatigue, anemia, etc.; 3.
  • Extraintestinal manifestations such as joints and skin , mucous membranes, eye lesions, etc.; in severe cases, intestinal perforation, intestinal obstruction, massive bleeding, etc., and even cancer may occur.
  • the course of ulcerative colitis is protracted and relapses, and it is still incurable.
  • aminosalicylic acid preparations are mainly used for treatment of mild UC. After treatment with sufficient amounts of aminosalicylic acid preparations (usually 2 to 4 weeks), symptoms are poorly controlled, especially those with more extensive lesions. Glucocorticoid treatment can be used. For those who are ineffective or dependent on hormones, immunosuppressive thiopurine drugs are used. If the above treatment methods still cannot alleviate the disease, anti-TNF ⁇ inhibitors will be administered.
  • IBD drugs are accompanied by serious side effects, including loss of immune tolerance and drug resistance, infectious complications, endocrine damage, and kidney and liver damage.
  • Current IBD treatment options have low efficacy and increased adverse reactions are major concerns in clinical treatment.
  • the purpose of the present invention is to provide new pharmaceutical uses of mulberry extract or its main active ingredients.
  • ulcerative colitis is embodied in at least one of the following aspects:
  • the present invention also protects the application of mulberry extract or its main active ingredient, which is at least one of the following (b1)-(b8):
  • the product is a drug or pharmaceutical preparation.
  • the mulberry extract is mulberry branch extract, mulberry bark extract and/or mulberry leaf extract.
  • the mulberry extract can also be provided in the form of commercially available mulberry branch total alkaloid tablets (National Drug Approval No. Z20200002).
  • Mulberry extract can be prepared by referring to the method recorded in CN 113143997 A.
  • the specific preparation method includes the following steps:
  • the method may also include the following steps:
  • the method may further include the following steps: concentrating and drying the resin effluent in step 2).
  • the mulberry extract acts on humans or mammals.
  • the Moraceae plants may be selected from the group consisting of Guangdong mulberry, Lu mulberry, white mulberry, Tentodon mulberry, Mountain mulberry or hybrid mulberry.
  • the hybrid mulberry is preferably Yuesang No. 11, Guisangyou 62 or Sangteyou 2.
  • Various parts such as leaves, roots, branches, bark, buds, stems and fruits of the plants can be used.
  • the mulberry extract mainly includes alkaloids, and also includes polysaccharides, flavonoids and amino acids.
  • the alkaloids include 1-deoxynojirimycin (1-deoxynojirimycin or DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG) , 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-ribitol (1,4-dideoxy-1,4-imino-D-arabinitol or DAB ), calystegin B2 (calysteginB2), calystegin C1 (calysteginC1), 2-oxo-( ⁇ -D-galactopyranosyl)-1-deoxynojirimycin (2-O-( ⁇ -D-galactopyranosyl)-1-deoxynojirimycin), 6-O-( ⁇ -D-glucopyranosyl)-1-deoxynojirimycin (6-O-( ⁇ -D-glucopyranosyl)
  • the weight percentage of DNJ is not less than 50% of the total alkaloids.
  • the weight content of each component is:
  • the weight content of each component of the mulberry extract is:
  • the preparation of the mulberry extract includes the following steps: preparing a crude extract; optionally, separating through cationic resin and/or anionic resin; optionally, performing alcohol precipitation treatment on the resin effluent; and optional Ground, concentrated and dried.
  • the preparation of the mulberry extract includes the following steps: step 1): prepare a crude extract; step 2): separate through cationic resin and/or optional anionic resin; optional step 3): perform step 2 ), the resin effluent is subjected to alcohol precipitation treatment; optional step 4) concentration and drying treatment.
  • the mulberry extract is prepared according to the following steps: pulverize mulberry branches, mulberry leaves or mulberry white bark, and extract by heating and refluxing with water and/or alcohol solution or acidic water.
  • the amount of solvent is 3-3 of the original medicinal material. 20 times, repeat the extraction 1-3 times, combine the extracts, concentrate, apply to cation exchange resin, wash away non-adsorbed impurities with distilled water, elute with 0.2-3N ammonia water, concentrate the eluate and apply to anion exchange resin, collect non-adsorbed impurities part, add ethanol, precipitate to remove impurities, centrifuge, and the clear liquid is concentrated under reduced pressure or spray-dried or freeze-dried to obtain the extract.
  • the mulberry extract is prepared according to the following steps: pulverize mulberry branches, mulberry leaves or mulberry white bark, and extract by heating and refluxing with water and/or alcohol solution or acidic water.
  • the amount of solvent is 3-3 of the original medicinal material. 20 times, repeat the extraction 1-3 times, combine the extracts, concentrate, apply to cation exchange resin, wash away non-adsorbed impurities with distilled water, elute with 0.2-3N ammonia water, concentrate the eluate and apply to anion exchange resin, collect non-adsorbed impurities Part of it is concentrated under reduced pressure or spray-dried or freeze-dried to obtain an extract.
  • the mulberry extract is prepared according to the following steps: pulverize mulberry branches, mulberry leaves or mulberry white bark, and extract by heating and refluxing with water and/or alcohol solution or acidic water.
  • the amount of solvent is 3-3 of the original medicinal material. 20 times, repeat the extraction 1-3 times, combine the extracts, concentrate, apply to cation exchange resin, wash away non-adsorbed impurities with distilled water, elute with 0.2-3N ammonia water, concentrate the eluent under reduced pressure or spray dry or freeze-dry. Get the extract.
  • the mulberry extract is prepared according to the following steps: pulverize mulberry branches, mulberry leaves or mulberry white bark, and extract by heating and refluxing with water.
  • the amount of solvent is 3-20 times of the original medicinal materials (preferably 4-15 times).
  • the extraction time is preferably 0.5-3h each time, and more preferably 1-2h each time
  • the extraction time is preferably 0.5-3h each time, and more preferably 1-2h each time
  • the extraction time is preferably 0.5-3h each time, and more preferably 1-2h each time
  • the anion exchange resin collects the non-adsorbed part (i.e., the anion resin effluent)
  • spray drying or freeze drying to obtain the extract.
  • the alkaline solution is washed until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5; preferably, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution; Preferably, the concentration of the alkaline solution is 0.5-4mol/L.
  • the acidic solution is used to wash until the pH of the eluate is 3.0-7.0, preferably 4.5-6.5.
  • the acidic solution is selected from the group consisting of hydrochloric acid solution (the concentration may be 1.5-2 mol/L), phosphoric acid solution, and disodium hydrogen phosphate-citrate buffer.
  • the cationic resin can be rinsed with 3-5 times the column volume of deionized water after the last acidic solution wash.
  • the cationic resin is type 732 strongly acidic styrene-based cation exchange resin or type 734 strongly acidic cation exchange resin.
  • Styrene-based cation exchange resin D001 macroporous strong acidic styrene-based cation exchange resin or D113 macroporous weakly acidic styrene-based cationic resin.
  • the weight ratio of the amount of the cationic resin to the plant raw materials is 1:2-20 (a more preferred weight ratio of the cationic resin to the plant raw materials is 1:2-7).
  • the loaded cationic resin is eluted with an eluent.
  • the concentration of the eluent is 0.5-2.5 mol/L.
  • the eluent flow rate is 5-10 BV/h, more preferably 5-6 BV/h. Detect the pH of the cation column effluent to collect the eluate when it is >7, and pass the collected liquid directly through the anion column for purification.
  • the alkaline solution is washed until the pH of the eluate is 8.0-9.5, preferably 8.5-9.5; preferably, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution; Preferably, the concentration of the alkaline solution is 0.5-4mol/L.
  • the acid solution is washed until the pH of the eluate is 3.0-7.0, preferably 3.5-4.5.
  • the acidic solution is selected from the group consisting of hydrochloric acid solution (the concentration may be 1.5-2 mol/L), phosphoric acid solution, and disodium hydrogen phosphate-citrate buffer.
  • the anion resin is type 717 strong basic styrene anion exchange resin, type D201 macroporous strong basic styrene anion exchange resin or type D218 macroporous strong basic acrylic anion exchange resin.
  • the weight ratio of the amount of the anionic resin to the plant raw materials is 1:1-32 (a more preferred weight ratio of the anionic resin to the plant raw materials is 1:5-16).
  • Collection begins when the liquid flows out of the anionic resin (it is preferred to collect the effluent with a pH greater than 8). Preferably, collection is stopped when the volume of the collection liquid reaches 0.1-5 times the weight of the plant raw materials.
  • the weight ratio of ethanol to plant raw materials used in the alcohol precipitation treatment is 1:20-300 (more preferably 1:20-50).
  • the stirring speed is 40-500rpm.
  • the time of alcohol precipitation treatment is 12-24h.
  • the anion resin effluent also includes the step of centrifuging the anionic resin effluent to remove impurities or filtering it with a microfiltration membrane, and then concentrating with a reverse ion permeation membrane.
  • the specific gravity of the concentrated liquid can be 1.1-1.25.
  • animal in the present invention is not particularly limited and can be any animal with intestinal organs, preferably mammals, more preferably rats, mice and humans, and most preferably humans.
  • the medicament also includes a pharmaceutically acceptable carrier.
  • the carrier is an inactive ingredient that is non-toxic to the human body and conforms to the route or mode of administration.
  • the carrier can be a solid or liquid excipient.
  • Solid excipients include 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, Examples include water, ethanol, syrup, and glycerol.
  • the medicine is in an oral dosage form; further preferably, the medicine is in the form of tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups and powders.
  • the present invention also provides a method for preventing and/or treating ulcerative colitis, which includes the following steps: administering the mulberry extract or its main active ingredient to a recipient animal or human to prevent and/or treat ulcerative colitis. .
  • the animal may be a mammal.
  • the mulberry extract of the present invention has the unique advantages of small toxic and side effects, mild and long-lasting effects, and multiple pathways and multiple targets. Therefore, mulberry extract, which has greatly improved drug safety, has great advantages in treating inflammatory bowel disease.
  • Figure 1 shows the colon length of mice in each treatment group (using the mulberry extract in Preparation Example 1), * means p ⁇ 0.05 compared with the model group, ** means p ⁇ 0.01 compared with the model group, *** means p ⁇ 0.001 compared with model group;
  • Figure 2 is a picture of the colon of mice in each treatment group (using the mulberry extract in Preparation Example 1);
  • Figure 3 is a graph showing the weight change trend of mice in each treatment group (using the mulberry extract in Preparation Example 1);
  • Figure 4 shows the disease activity index DAI score results of mice in each treatment group (using the mulberry extract in Preparation Example 1); *** indicates p ⁇ 0.001 compared with the model group;
  • Figure 5 shows the disease activity index DAI score results of mice treated with mulberry extract in each preparation example; *** indicates p ⁇ 0.001 compared with the model group;
  • Figure 6 shows the pathological picture results of mice in each treatment group (using the mulberry extract in Preparation Example 1).
  • exemplary here means “serving as an example, example, or illustrative.” Any embodiment described herein as “exemplary” is not necessarily to be construed as superior or superior to other embodiments.
  • Use 150kg D113 macroporous weakly acidic styrene-based cationic resin to pack the column use 2mol/L hydrochloric acid solution to wash until the eluate pH is 4.5; use 1mol/L sodium hydroxide solution to wash until the eluate pH is 8.5; 2mol /L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation.
  • the collected liquid is centrifuged to remove impurities and then concentrated by reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid is 1.25. Transfer it to the alcohol precipitation tank and add 25L of absolute ethanol at a stirring paddle of 500rpm. After the addition of ethanol, stop stirring and let the alcohol settle for 24 hours. Take the supernatant and concentrate it under reduced pressure to obtain mulberry branch extract.
  • the alkaloid content is 52%
  • the polysaccharide content is 22%
  • the flavonoid content is 0.8%
  • the amino acid content is 20%.
  • the content of 1-DNJ is 60%
  • FAG is 17%
  • DAB is 15%.
  • the collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated through a reverse ion permeation membrane.
  • the specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank and 125g of absolute ethanol was added at a stirring paddle of 1000 rpm. After the addition of ethanol, stop stirring and let the alcohol settle for 24 hours. Take the supernatant and concentrate it under reduced pressure to obtain mulberry branch extract. In addition, fresh mulberry bark and mulberry leaves (Sangteyou No. 2) were taken for extraction.
  • the extraction method and parameters were the same as the above method.
  • the alkaloid content is 98%
  • the polysaccharide content is 0.2%
  • the flavonoid content is 0.05%
  • the amino acid content is 0.
  • the content of 1-DNJ is 99%
  • FAG is 0.5%
  • DAB is 0.4%.
  • the alkaloid content is 95%
  • the polysaccharide content is 2%
  • the flavonoid content is 0.1%
  • the amino acid content is 1%.
  • the content of 1-DNJ is 96%
  • FAG is 1.5%
  • DAB is 1.4%.
  • the alkaloid content is 90%
  • the polysaccharide content is 4%
  • the flavonoid content is 0.1%
  • the amino acid content is 3%.
  • the content of 1-DNJ is 91%
  • FAG is 3.1%
  • DAB is 2.8%.
  • the collected liquid obtained after cation column separation is concentrated by nanofiltration membrane, and concentrated under reduced pressure to obtain an extract extract.
  • the alkaloid content is 15%
  • the polysaccharide content is 20%
  • the flavonoid content is 7%
  • the amino acid content is 45%.
  • the content of 1-DNJ is 55%
  • FAG is 23%
  • DAB is 10%.
  • Use 150kg D113 macroporous weakly acidic styrene-based cationic resin to pack the column use 2mol/L hydrochloric acid solution to wash until the eluate pH is 4.5; use 1mol/L sodium hydroxide solution to wash until the eluate pH is 8.5; 2mol /L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation.
  • the collected liquid obtained after anion column separation is filtered with a microfiltration membrane to remove impurities and then concentrated with a reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid is 1.1. Transfer it to the alcohol precipitation tank and add absolute ethanol at a stirring paddle of 400 rpm. 15kg. After the addition of ethanol, stop stirring and let the alcohol settle for 24 hours. Take the supernatant and concentrate it under reduced pressure to obtain mulberry branch extract.
  • the content of 1-DNJ is 75%
  • FAG is 12%
  • DAB is 10%.
  • the collected liquid obtained after anion column separation is filtered with a microfiltration membrane to remove impurities and then concentrated with a counter ion permeation membrane.
  • the liquid is further concentrated and dried under reduced pressure to obtain a mulberry branch extract extract.
  • Sample content the content of alkaloids is 3%, the content of polysaccharides is 70%, the content of flavonoids is 10%, and the content of amino acids is 10%.
  • the content of 1-DNJ is 68%, FAG is 17%, and DAB is 8%.
  • the effluent was concentrated under reduced pressure to obtain a mulberry branch extract, in which the alkaloid content was 30%, the polysaccharide content was 35%, the flavonoid content was 2%, and the amino acid content was 25%.
  • the alkaloids the content of 1-DNJ is 62%, FAG is 20%, and DAB is 13%.
  • the effluent was concentrated under reduced pressure to obtain a mulberry branch extract, in which the alkaloid content is 40%, the polysaccharide content is 25%, the flavonoid content is 0.5%, and the amino acid content is 25%.
  • the content of 1-DNJ is 57%, FAG is 24%, and DAB is 16%.
  • Use 150kg D113 macroporous weakly acidic styrene-based cationic resin to pack the column use 2mol/L hydrochloric acid solution to wash until the eluate pH is 4.5; use 1mol/L sodium hydroxide solution to wash until the eluate pH is 8.5; 2mol /L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete the activation.
  • the collected liquid obtained after anion column separation is filtered with a microfiltration membrane to remove impurities and then concentrated with a reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid is 1.1. Transfer it to the alcohol precipitation tank and add absolute ethanol at a stirring paddle of 400 rpm. 15kg. After the addition of ethanol, stop stirring and let the alcohol settle for 24 hours. Take the supernatant and concentrate it under reduced pressure to obtain mulberry branch extract.
  • the content of 1-DNJ is 61.9%
  • FAG is 16.6%
  • DAB is 11.1%.
  • mice aged 6-8 weeks were admitted to the animal room and kept adaptively for 3 days. After weighing, they were randomly divided into 8 groups (counted as 0 days), with 5-6 mice in each group.
  • the experimental groups are: 1 normal control group, 2 model group, 3 positive control group, and 4-8 groups of SZ-A with different dosages. See Table 1 for details.
  • 3% DSS preparation method 3g DSS (dextran sulfate sodium salt) is dissolved in 100mL sterile water;
  • the mulberry branch extract prepared in Preparation Example 1 was prepared in different concentrations and dissolved in 1% CMC solution;
  • Positive control drug Sulfasalazine enteric-coated tablets (National Drug Approval No. H31020557).
  • the main ingredient of this product is: sulfasalazine; its chemical name is: 5-[p-(2-pyridineaminesulfonyl)benzene]azosalicylic acid.
  • mice in the 8th SZ-A-5 (pretreatment) group were orally administered mulberry branch extract (see the table below for dosage) once a day. All groups had normal diet and drinking water.
  • mice in the 8th group Three days after the administration of the mice in the 8th group, except for the normal control group, all other groups began to drink 3% DSS (dextran sulfate sodium salt) freely for modeling and intragastric administration; among them, the mice in the positive control group were given The dosage is: 5-[p-(2-pyridineaminesulfonyl)benzene]azosalicylic acid 500mg/kg/d.
  • DSS dissulfate sodium salt
  • the dosage is: 5-[p-(2-pyridineaminesulfonyl)benzene]azosalicylic acid 500mg/kg/d.
  • mice in each group were administered daily.
  • the feces characteristics and blood in the stool of the mice were monitored daily, and the mouse disease activity index DAI score was calculated according to Table 2 below. The results are shown in Figures 1-3 and 4.
  • DAI weight loss score + stool character score + occult blood score; normal stool: formed stool; semi-liquid stool: pasty, semi-formed stool that does not adhere to the anus; loose stool: thin watery stool that can adhere to the anus Convenience.
  • Tissue samples 3 mm thick, dehydrated in graded alcohol of 70%, 80%, 95%, 100% for 30 minutes each, two bottles of xylene for 20 minutes each, two bottles of paraffin immersed in wax for 12 minutes each, embedded, and sectioned into 4 micron sections , grilled slices.
  • Hematoxylin eosin (HE) staining (1) Dewaxing, three bottles of xylene for 8 minutes each; two bottles of 100% alcohol for 8 minutes each; 90% alcohol, 80% alcohol, 60% alcohol 8 minutes each. (2) Hematoxylin staining for 4 minutes, rinse with running water; (3) Hydrochloric acid alcohol differentiation for 2-3 seconds, rinse with running water; (4) 0.5% ammonia solution for 20 seconds, rinse with running water, and observe under the microscope. (5) 0.5% eosin staining for 1 minute; (6) Differentiation of 80% alcohol and 90% alcohol for 3-5 seconds each; 95% alcohol for 5 minutes; three bottles of 100% alcohol for 5 minutes each; two bottles of xylene for 5 minutes each.
  • the mulberry branch extracts in each preparation example were compared with mouse disease activity index DAI scores.
  • the drugs were the mulberry branch extracts in preparation examples 1, 3, 4, 6 and 8, and the dosage was as follows: In terms of mulberry branch extract, the dosage is 200mg/kg/d. The results are shown in Figure 5.
  • the positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 can both reduce the disease activity index DAI in UC model mice.
  • Preparation Example 1 the content of alkaloids is 52%, the content of polysaccharides is 22%, the content of flavonoids is 0.8%, and the content of amino acids is 20%
  • Preparation Example 8 the content of alkaloids is 22%) (63%, polysaccharide content is 23%, flavonoid content is 1%, and amino acid content is 5%).
  • the specific mulberry extract provided can reduce the disease activity index of UC model mice, while the mulberry extract in other preparation examples The effect of drug treatment is not significant.
  • the present invention has confirmed through experiments that a specific mulberry extract can inhibit colon atrophy in UC model mice, inhibit weight loss in UC model mice, reduce disease activity index DAI in UC model mice, and cause colon tissue disease in mice after treatment with mulberry extract.
  • the physical condition has been significantly improved, the colon structure is relatively complete, the glands are arranged regularly, and the inflammatory cell infiltration is lighter.
  • mulberry extract or its main active ingredient can be used to prepare and treat ulcerative colitis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明公开了桑提取物的应用。该应用是桑提取物在制备治疗和/或预防溃疡性结肠炎产品中的应用。本发明通过实验证实，阳性药物组、SZ-A-1、SZ-A-2、SZ-A-3、SZ-A-4和SZ-A-5(pretreatment)均能显著抑制UC模型小鼠结肠萎缩，SZ-A-2、SZ-A-3和SZ-A-4组的效果与阳性药组相当，SZ-A-5(pretreatment)较阳性药抑制结肠萎缩的效果更好。本发明的桑提取物作为来源于天然植物的成分具有毒副作用小、作用温和持久的独特优势。因此用药安全性大大提升的桑提取物在溃疡性结肠炎方面具有巨大优势。

Description

桑提取物在制备治疗溃疡性结肠炎的药物中的应用 技术领域
本发明属于医药领域，具体涉及桑提取物在制备治疗溃疡性结肠炎的药物中的应用。
背景技术
炎症性肠病(IBD)是指各种原因引起的结肠炎症性病变的自身免疫性疾病，主要包括克罗恩病(CD)和溃疡性结肠炎(UC)，我国近20年来其确诊人数呈快速上升趋势，至今为止其发病机制尚不完全清楚，影响因素众多，如：环境因素，免疫反应因素，遗传因素，微生物因素等。炎症性肠病(IBD)不同于一般的肠道炎症(如CN113143996 A中的)。具有抗炎活性的药物或活性成分，往往可用于治疗肠道炎症，但对炎症性肠病(IBD)却束手无策。虽然关于炎症性肠病的治疗已经取得了很多进展，但其仍是不治之症，全球范围内仍没有特效药物。
CN113143996 A中公开的糖尿病小鼠经过高脂喂养后出现全身肠道的慢性低度炎症，溃疡性结肠炎的炎症只局限于大肠(中华医学会消化病学分会炎症性肠病学组.炎症性肠病诊断与治疗的共识意见(2018年，北京)[J].中华消化杂志,2018,38(5):292-311.DOI:10.3760/cma.j.issn.0254-1432.2018.05.002)。溃疡性结肠炎与慢性低度炎症不同的是还会出现如下症状：1.便血、肛周脓肿；2.全身症状如体重下降、发热、乏力、贫血等；3.肠外表现如关节、皮肤、粘膜、眼部病变等；严重者可出现肠穿孔、肠梗阻、大出血等，甚至癌变。此外，溃疡性结肠炎病程迁延，反复发作，目前仍无法治愈。
临床上，主要采用氨基水杨酸制剂进行治疗，用于轻度UC的治疗，足量氨基水杨酸制剂治疗后(一般2～4周)症状控制不佳者，尤其是病变较广泛者，可采用糖皮质激素治疗。对于激素无效或依赖者则采用免疫抑制剂硫嘌呤类药物。若以上治疗方式仍不能缓解，则对其施用anti-TNFα抑制剂。
而且目前的IBD药物还伴随着严重的副作用，包括丧失免疫耐受性和耐药性、感染并发症、内分泌损伤、肾脏肝脏损伤。目前IBD的治疗方案疗效低，不良反应升高是临床治疗主要关注的问题。
因此，研发一种安全有效的治疗IBD的药物已成为亟需解决的重大科学问题。
发明公开
本发明的目的是提供桑提取物或其主要活性成分的药物新用途。
本发明所提供的桑提取物或其主要活性成分的新用途为如下(a1)-(a4)中任一所述：
(a1)桑提取物在制备治疗溃疡性结肠炎产品中的应用；
(a2)桑提取物在制备预防溃疡性结肠炎产品中的应用；
(a3)桑提取物在治疗溃疡性结肠炎中的应用；
(a4)桑提取物在预防溃疡性结肠炎中的应用。
所述预防和/或治疗溃疡性结肠炎病体现在下述至少一方面：
1)抑制溃疡性结肠炎患者结肠萎缩；
2)抑制溃疡性结肠炎患者体重下降；
3)降低溃疡性结肠炎患者疾病活动指数DAI；
4)改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润。
本发明还保护桑提取物或其主要活性成分的应用，为如下(b1)-(b8)中的至少一种：
(b1)制备抑制溃疡性结肠炎患者结肠萎缩的产品；
(b2)制备抑制溃疡性结肠炎患者体重下降的产品；
(b3)制备降低溃疡性结肠炎患者疾病活动指数DAI的产品；
(b4)制备改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润的产品；
(b5)抑制溃疡性结肠炎患者结肠萎缩；
(b6)抑制溃疡性结肠炎患者体重下降；
(b7)降低溃疡性结肠炎患者疾病活动指数DAI；
(b8)改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润。
所述产品为药物或药物制剂。
所述桑提取物为桑枝提取物、桑白皮提取物和/或桑叶提取物。或，所述桑提取物也可以采用市售的桑枝总生物碱片(国药准字Z20200002)的形式提供。
桑提取物可参照CN 113143997 A中记载的方法进行制备，具体制备方法包括以下步骤：
1)制备桑科植物粗提液；
2)将所述粗提液经阳离子树脂和/或任选的阴离子树脂分离，得到所述桑提取物。
所述方法还可包括下述步骤：
3)对步骤2)的树脂流出液进行醇沉处理，收集上清液；
4)对所述上清液进行浓缩干燥处理。
所述方法还可包括下述步骤：将步骤2)的树脂流出液进行浓缩干燥处理。
所述桑提取物作用于人或哺乳动物。
所述桑科植物可选自广东桑、鲁桑、白桑、细齿桑、山桑或杂交桑，所述杂交桑优选为粤桑11号、桂桑优62号或桑特优2号。可以使用所述植物的叶、根、枝、皮、芽、茎、果实等各部位。
在本发明的一种实施方式中，所述桑提取物主要包括生物碱，还包括多糖、黄酮和氨基酸。
优选地，所述生物碱包含1-脱氧野尻霉素(1-deoxynojirimycin或DNJ)、N-甲基-1-脱氧野尻霉素(N-methly-1-deoxynojirimycin)、荞麦碱(fagomine或FAG)、3-表荞麦碱(3-epi-fagomine)、1,4-双脱氧-1,4-亚氨基-D-核糖醇(1,4-dideoxy-1，4-imino-D-arabinitol或DAB)、打碗花精B2(calysteginB2)、打碗花精C1(calysteginC1)、2-氧-(α-D-半乳吡喃糖基)-1-脱氧野尻霉素(2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin)、6-氧-(β-D-吡喃葡萄糖)-1-脱氧野尻霉素(6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin)、1，4-双脱氧-1，4-亚胺-(2-氧-β-D-吡喃葡萄糖)-D-阿拉伯糖醇(1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol)中的至少一种。
其中，DNJ的重量百分比不低于总生物碱的50％。
基于所述桑提取物，各组分重量含量为：
优选的，桑提取物，各组分重量含量为：
在一种实施方式中，所述桑提取物的制备包括以下步骤：制备粗提液；任选地，经阳离子树脂和/或阴离子树脂分离；任选地，对树脂流出液进行醇沉处理；以及任选 地，浓缩干燥处理。优选地，所述桑提取物的制备包括以下步骤：步骤1)：制备粗提液；步骤2)：经阳离子树脂和/或任选的阴离子树脂分离；任选的步骤3)：对步骤2)的树脂流出液进行醇沉处理；任选的步骤4)浓缩干燥处理。
在一种实施方式中，所述桑提取物按照以下步骤制备：将桑枝、桑叶或桑白皮粉碎，用水和/或醇溶液或酸水加热回流提取，溶剂量为原药材的3-20倍，重复提取1-3次，合并提取液，浓缩，上阳离子交换树脂，蒸馏水洗尽不吸附的杂质，用0.2-3N氨水洗脱，洗脱液浓缩上阴离子交换树脂，收集不吸附的部分，加入乙醇，沉淀去除杂质，离心，清液减压浓缩或喷雾干燥或冷冻干燥，得提取物。
在一种实施方式中，所述桑提取物按照以下步骤制备：将桑枝、桑叶或桑白皮粉碎，用水和/或醇溶液或酸水加热回流提取，溶剂量为原药材的3-20倍，重复提取1-3次，合并提取液，浓缩，上阳离子交换树脂，蒸馏水洗尽不吸附的杂质，用0.2-3N氨水洗脱，洗脱液浓缩上阴离子交换树脂，收集不吸附的部分，减压浓缩或喷雾干燥或冷冻干燥，得提取物。
在一种实施方式中，所述桑提取物按照以下步骤制备：将桑枝、桑叶或桑白皮粉碎，用水和/或醇溶液或酸水加热回流提取，溶剂量为原药材的3-20倍，重复提取1-3次，合并提取液，浓缩，上阳离子交换树脂，蒸馏水洗尽不吸附的杂质，用0.2-3N氨水洗脱，洗脱液减压浓缩或喷雾干燥或冷冻干燥，得提取物。
在一种实施方式中，所述桑提取物按照以下步骤制备：将桑枝、桑叶或桑白皮粉碎，用水加热回流提取，溶剂量为原药材的3-20倍(优选为4-15倍，更优选4-12倍)，重复提取1-3次(提取时间优选为每次0.5-3h，更有选为每次1-2h)，合并提取液，浓缩，上阳离子交换树脂，蒸馏水洗尽不吸附的杂质，用0.2-3N氨水洗脱，洗脱液上阴离子交换树脂，收集不吸附的部分(即阴离子树脂流出液)，加入乙醇，沉淀去除杂质，离心，清液减压浓缩或喷雾干燥或冷冻干燥，得提取物。
优选地，在阳离子树脂装柱后，按照酸性溶液洗、碱性溶液洗、酸性溶液洗的顺序进行活化。优选地，碱性溶液洗至洗出液pH为8.0-9.5，优选为8.5-9.5；优选地，所述碱性溶液选自氨水溶液、氢氧化钠溶液、氢氧化钾溶液或碳酸钠溶液；优选地，所述碱性溶液的浓度为0.5-4mol/L。优选地，酸性溶液洗至洗出液pH为3.0-7.0，优选为4.5-6.5。优选地，所述酸性溶液选自盐酸溶液(浓度可为1.5-2mol/L)、磷酸溶液、磷酸氢二钠-柠檬酸缓冲液。任选地，所述阳离子树脂经最后一次酸性溶液洗后还可以使用3-5倍柱体积的去离子水冲洗。
优选地，所述阳离子树脂为732型强酸性苯乙烯系阳离子交换树脂、734型强酸性 苯乙烯系阳离子交换树脂、D001型大孔强酸性苯乙烯系阳离子交换树脂或D113型大孔弱酸性苯丙烯系阳离子树脂。
优选地，所述阳离子树脂的用量与植物原材料投料重量比为1:2-20(更优选的所述阳离子树脂的用量与植物原材料投料重量比为1：2-7)。上样植物粗提液至阳离子树脂后，用洗脱剂对上样后的阳离子树脂进行洗脱，优选地，所述洗脱剂的浓度为0.5-2.5mol/L。优选地，洗脱剂流速为5-10BV/h，更优选为5-6BV/h。检测阳离子柱流出液的pH>7时收集洗脱液，将收集液直接过阴离子柱纯化。
优选地，在阴离子树脂装柱后，按照碱性溶液洗、酸性溶液洗、碱性溶液洗的顺序进行活化。优选地，碱性溶液洗至洗出液pH为8.0-9.5，优选为8.5-9.5；优选地，所述碱性溶液选自氨水溶液、氢氧化钠溶液、氢氧化钾溶液或碳酸钠溶液；优选地，所述碱性溶液的浓度为0.5-4mol/L。优选地，酸性溶液洗至洗出液pH为3.0-7.0，优选为3.5-4.5。优选地，所述酸性溶液选自盐酸溶液(浓度可为1.5-2mol/L)、磷酸溶液、磷酸氢二钠-柠檬酸缓冲液。
优选地，所述阴离子树脂为717型强碱性苯乙烯系阴离子交换树脂，D201型大孔强碱性苯乙烯系阴离子交换树脂或D218型大孔强碱性丙烯酸系阴离子交换树脂。优选地，所述阴离子树脂的用量与植物原材料投料重量比为1:1-32(更优选的所述阴离子树脂的用量与植物原材料投料重量比为1：5-16)。当液体流出阴离子树脂时开始收集(优选收集pH大于8的流出液)。优选地，当收集液的体积达到植物原材料投料重量的0.1-5倍时，停止收集。
优选地，醇沉处理所用的乙醇与植物原材料投料重量比为1:20-300(更优选为1：20-50)。醇沉处理中，搅拌速度为40-500rpm。醇沉处理的时间为12-24h。
进一步的，所述醇沉处理前，还包括将阴离子树脂流出液进行离心除杂或微滤膜过滤除杂，再进行反离子渗透膜浓缩的步骤。浓缩后的液体比重可为1.1-1.25。
本发明所述的“动物”所指的动物种类不受特别限制，可以为任何具有肠道器官的动物，优选哺乳动物，更优选大鼠、小鼠和人，最优选人。
优选地，所述药物还包括药学上可接受的载体。所述载体为符合用药途径或给药方式的对人体无毒害作用的非活性成分。所述载体可以是固体或液体辅料。固体辅料，例如，包括微晶纤维素、甘露醇、乳糖、预胶化淀粉，低取代羟丙基纤维素、交联聚维酮，羧甲基淀粉钠、阿斯巴甜、磷酸氢钙、乳酸钠、泊洛沙姆、十二烷基硫酸钠、羧甲基纤维素钠、明胶、黄原胶、聚维酮、淀粉、硬脂酸镁、羧甲基淀粉钠和滑石粉；液体辅料，例如，包括水、乙醇、糖浆和甘油。
优选地，所述药物为口服给药剂型；进一步优选地，所述药物为片剂、胶囊剂、口服溶液剂、口服乳剂、丸剂、颗粒剂、糖浆剂和散剂。
本发明还提供了一种预防和/或治疗溃疡性结肠炎的方法，包括如下步骤：给受体动物或人施用所述桑提取物或其主要活性成分以预防和/或治疗溃疡性结肠炎。
本发明中，所述动物可为哺乳动物。
本发明具有以下优点：
1、与目前临床主要应用药物严重的毒副作用相比，如：氨基水杨酸制剂可能会使患者出现过敏反应、黄疸、腹痛等；糖皮质激素毒副作用相较于氨基水杨酸制剂更大，本发明的桑提取物作为来源于天然植物的成分具有毒副作用小、作用温和持久、多通路多靶点的独特优势。因此用药安全性大大提升的桑提取物在治疗炎症性肠病方面具有巨大优势。
2、实验结果表明，特定的桑提取物能抑制UC模型小鼠结肠萎缩、抑制UC模型小鼠体重降低、降低UC模型小鼠疾病活动指数DAI，且经桑提取物治疗后小鼠结肠组织病理学得到明显的改善，结肠结构较为完整，腺体排列规则，炎性细胞浸润较轻。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为各处理组小鼠的结肠长度(采用制备例1中的桑提取物)，*表示与模型组相比p<0.05,**表示与模型组相比p<0.01，***表示与模型组相比p<0.001；
图2为各处理组小鼠的结肠图片(采用制备例1中的桑提取物)；
图3为各处理组小鼠的体重变化趋势图(采用制备例1中的桑提取物)；
图4为各处理组小鼠的疾病活动指数DAI评分结果(采用制备例1中的桑提取物)；***表示与模型组相比p<0.001；
图5为各制备例中的桑提取物处理后小鼠的疾病活动指数DAI评分结果；***表示与模型组相比p<0.001；
图6为各处理组小鼠病理图片结果(采用制备例1中的桑提取物)。
实施发明的最佳方式
以下通过实施例对本发明进一步详细说明。通过这些示例性说明，本发明的特点和优点将变得更为清楚明确。但本发明并不限于以下实施例。所述方法如无特 别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
在这里专业的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
此外，下面所描述的本发明不同实施方式中涉及的技术特征只要彼此之间未构成冲突就可以相互结合。
本发明涉及的组分含量根据已公开的方法进行检测(参考公开号为CN111077247A和CN110393738A的专利中记载的方法)。
一、桑提取物的制备实例
制备例1
取新鲜桑枝(细齿桑粤桑11号)1000kg，粉碎后，加4000L水，用加热回流法提取2h，合并提取液，过滤除去不溶物，得到粗提液。粗提液热浓缩至固形物质量百分含量达到4％，保温50℃作为阳离子树脂柱的上样液。
使用D113型大孔弱酸性苯丙烯系阳离子树脂150kg装柱，使用2mol/L的盐酸溶液洗至洗出液pH为4.5；使用1mol/L氢氧化钠溶液洗至洗出液pH为8.5；2mol/L的盐酸溶液洗至洗出液pH为4.5；再使用5倍柱体积的去离子水冲洗，完成活化。上样经浓缩处理的提取液，然后使用1000L 2.5mol/L氨水洗脱，洗脱速度为6BV/h，检测阳离子柱流出液的pH>7时收集洗脱液，当收集液达到900L时，停止收集，将收集液直接过阴离子柱纯化。
使用D218型大孔强碱性丙烯酸系阴离子树脂62.5kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集流出液至流出液达到870L结束。
收集液进行离心除杂再进行反离子渗透膜浓缩，浓缩后的液体比重为1.25，将其转移至醇沉罐内，搅拌桨500rpm下加入无水乙醇25L。乙醇加入完毕后停止搅拌，醇沉24h，取上清液，减压浓缩得桑枝提取物浸膏。
所述桑枝提取物浸膏中，生物碱的含量为52％，多糖的含量为22％，黄酮的含量为0.8％，氨基酸的含量为20％。在生物碱中，1-DNJ的含量为60％，FAG为17％，DAB为15％。
制备例2
取新鲜桑枝(桑特优2号)10kg，粉碎后，加150L水，分2次加入，每次用煎煮法提 取3h，合并提取液，过滤除去不溶物。提取液热浓缩至固形物质量百分含量达到8％，将其转移至醇沉罐，搅拌桨300rpm下加入2367.9g无水乙醇(3L)。乙醇加入完毕后停止搅拌，醇沉24h，取上清液作为阳离子树脂柱的上样液。使用002SC型强酸性苯乙烯系阳离子树脂5kg装柱，按照实施例1的方法对阳离子树脂进行活化。上样经浓缩醇沉处理的提取液，然后使用100L 5mol/L氯化钾洗脱，洗脱速度为5BV/h，用20％硅钨酸检测流出液，当有白色沉淀生成时开始收集，当收集液达到25L时，停止收集，将收集液直接过阴离子柱纯化。
使用711型强碱性苯乙烯系阴离子树脂10kg装柱，按照制备例1的方法对阴离子树脂进行活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集流出液至流出液达到15L结束。将收集液重新上样至阳离子树脂，按照上述方法依次用阳离子树脂和阴离子树脂再分离两次。
将经过三次柱分离后得到的收集液进行离心除杂再进行反离子渗透膜浓缩，浓缩后的液体比重为1.25，将其转移至醇沉罐内，搅拌桨1000rpm下加入无水乙醇125g。乙醇加入完毕后停止搅拌，醇沉24h，取上清液，减压浓缩得桑枝提取物浸膏。另外取新鲜桑白皮和桑叶(桑特优2号)进行提取，提取方法和参数与上述方法相同。
得到的桑枝提取物浸膏中，生物碱的含量为98％，多糖的含量为0.2％，黄酮的含量为0.05％，氨基酸的含量为0。在生物碱中，1-DNJ的含量为99％，FAG为0.5％，DAB为0.4％。
得到的桑白皮提取物中，生物碱的含量为95％，多糖的含量为2％，黄酮的含量为0.1％，氨基酸的含量为1％。在生物碱中，1-DNJ的含量为96％，FAG为1.5％，DAB为1.4％。
得到的桑叶提取物中，生物碱的含量为90％，多糖的含量为4％，黄酮的含量为0.1％，氨基酸的含量为3％。在生物碱中，1-DNJ的含量为91％，FAG为3.1％，DAB为2.8％。
制备例3
取新鲜桑枝(广东桑)1000kg，粉碎后，加11500L水，加热回流提取2h，合并提取液，过滤除去不溶物，得到粗提液。粗提液先经离心除杂，再用反离子渗透膜浓缩至固形物质量百分含量达到1％，作为阳离子树脂柱的上样液。
使用D001型大孔强酸性苯乙烯系阳离子树脂300kg装柱，按照制备例1的方法对阳离子树脂进行活化。上样经浓缩处理的粗提液，使用5000L 0.04mol/L硝酸铵洗脱，洗 脱速度为5BV/h，用20％硅钨酸检测流出液，当有白色沉淀生成时开始收集，当收集液达到1000L时，停止收集。
将经过阳离子柱分离后得到的收集液进行纳滤膜浓缩，减压浓缩得提取物浸膏。
得到的桑枝提取物中，生物碱的含量为15％，多糖的含量为20％，黄酮的含量为7％，氨基酸的含量为45％。在生物碱中，1-DNJ的含量为55％，FAG为23％，DAB为10％。
制备例4
取干桑枝(粤桑11号)333kg，粉碎后，加4000L水，加热回流法分两次提取，每次回流1h，合并提取液，过滤，将提取液浓缩至1kg生药量/L。
使用D113型大孔弱酸性苯丙烯系阳离子树脂150kg装柱，使用2mol/L的盐酸溶液洗至洗出液pH为4.5；使用1mol/L氢氧化钠溶液洗至洗出液pH为8.5；2mol/L的盐酸溶液洗至洗出液pH为4.5；再使用5倍柱体积的去离子水冲洗，完成活化。上样经浓缩处理的提取液，然后使用1000L 2.5mol/L氨水洗脱，洗脱速度为6BV/h，检测阳离子柱流出液的pH>7时收集洗脱液，当收集液达到900L时，停止收集，将收集液直接过阴离子柱纯化。
使用D218型大孔强碱性丙烯酸系阴离子树脂125kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集pH大于8的流出液至流出液达到870L结束。
将经过阴离子柱分离后得到的收集液进行微滤膜过滤除杂再用反离子渗透膜浓缩，浓缩后的液体比重为1.1，将其转移至醇沉罐内，搅拌桨400rpm下加入无水乙醇15kg。乙醇加入完毕后停止搅拌，醇沉24h，取上清液，减压浓缩得桑枝提取物浸膏。样品含量：生物碱的含量为80％，多糖的含量为5％，黄酮的含量为0.1％，氨基酸的含量为4％。在生物碱中，1-DNJ的含量为75％，FAG为12％，DAB为10％。
制备例5
取干桑枝(粤桑11号)400kg，粉碎后，加4000L水，加热回流法分两次提取，每次回流1h，合并提取液，过滤，将提取液浓缩至1kg生药量/L。
使用D218型大孔强碱性丙烯酸系阴离子树脂62.5kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的提取浓缩液上样至阴离子树脂， 收集流出液。
将经过阴离子柱分离后得到的收集液进行微滤膜过滤除杂再用反离子渗透膜浓缩，进一步减压浓缩干燥得桑枝提取物浸膏。样品含量：生物碱的含量为3％，多糖的含量为70％，黄酮的含量为10％，氨基酸的含量为10％。在生物碱中，1-DNJ的含量为68％，FAG为17％，DAB为8％。
制备例6
取新鲜桑枝(细齿桑粤桑11号)1500kg，粉碎后，加6000L水，用加热回流法提取2h，合并提取液，过滤除去不溶物，得到粗提液。粗提液热浓缩至固形物质量百分含量达到4％，保温50℃作为阳离子树脂柱的上样液。
使用D113型大孔弱酸性苯丙烯系阳离子树脂100kg装柱，使用2mol/L的盐酸溶液洗至洗出液pH为4.5；使用1mol/L氢氧化钠溶液洗至洗出液pH为8.5；2mol/L的盐酸溶液洗至洗出液pH为4.5；再使用5倍柱体积的去离子水冲洗，完成活化。上样经浓缩处理的提取液，然后使用1000L 2.5mol/L氨水洗脱，洗脱速度为6BV/h，检测阳离子柱流出液的pH>7时收集洗脱液，当收集液达到900L时，停止收集，将收集液直接过阴离子柱纯化。
使用D218型大孔强碱性丙烯酸系阴离子树脂62.5kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集流出液至流出液达到870L结束。将流出液减压浓缩得桑枝提取物浸膏，其中，生物碱的含量为30％，多糖的含量为35％，黄酮的含量为2％，氨基酸的含量为25％。在生物碱中，1-DNJ的含量为62％，FAG为20％，DAB为13％。
制备例7
取新鲜桑枝(细齿桑粤桑11号)1000kg，粉碎后，加4000L水，用加热回流法提取2h，合并提取液，过滤除去不溶物，得到粗提液。粗提液热浓缩至固形物质量百分含量达到4％，保温50℃作为阳离子树脂柱的上样液。
使用D113型大孔弱酸性苯丙烯系阳离子树脂100kg装柱，使用2mol/L的盐酸溶液洗至洗出液pH为4.5；使用1mol/L氢氧化钠溶液洗至洗出液pH为8.5；2mol/L的盐酸溶液洗至洗出液pH为4.5；再使用5倍柱体积的去离子水冲洗，完成活化。上样经浓缩处理的提取液，然后使用1000L 2.5mol/L氨水洗脱，洗脱速度为6BV/h，检测阳离子柱流 出液的pH>7时收集洗脱液，当收集液达到900L时，停止收集，将收集液直接过阴离子柱纯化。
使用D218型大孔强碱性丙烯酸系阴离子树脂62.5kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集流出液至流出液达到870L结束。将流出液减压浓缩得桑枝提取物浸膏，其中，生物碱的含量为40％，多糖的含量为25％，黄酮的含量为0.5％，氨基酸的含量为25％。在生物碱中，1-DNJ的含量为57％，FAG为24％，DAB为16％。
制备例8
取干桑枝(粤桑11号)333kg，粉碎后，加4000L水，加热回流法分两次提取，每次回流1h，合并提取液，过滤，将提取液浓缩至1kg生药量/L。
使用D113型大孔弱酸性苯丙烯系阳离子树脂150kg装柱，使用2mol/L的盐酸溶液洗至洗出液pH为4.5；使用1mol/L氢氧化钠溶液洗至洗出液pH为8.5；2mol/L的盐酸溶液洗至洗出液pH为4.5；再使用5倍柱体积的去离子水冲洗，完成活化。上样经浓缩处理的提取液，然后使用1000L 2.5mol/L氨水洗脱，洗脱速度为6BV/h，检测阳离子柱流出液的pH>7时收集洗脱液，当收集液达到900L时，停止收集，将收集液直接过阴离子柱纯化。
使用D218型大孔强碱性丙烯酸系阴离子树脂62.5kg装柱，使用1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；使用1.5mol/L的盐酸溶液洗至洗出液pH为3.5；1.5mol/L氢氧化钠溶液洗至洗出液pH为9.0；完成活化。将收集的阳离子树脂洗脱液上样至阴离子树脂，收集pH大于8的流出液至流出液达到870L结束。
将经过阴离子柱分离后得到的收集液进行微滤膜过滤除杂再用反离子渗透膜浓缩，浓缩后的液体比重为1.1，将其转移至醇沉罐内，搅拌桨400rpm下加入无水乙醇15kg。乙醇加入完毕后停止搅拌，醇沉24h，取上清液，减压浓缩得桑枝提取物浸膏。样品含量：生物碱的含量为63％，多糖的含量为23％，黄酮的含量为1％，氨基酸的含量为5％。在生物碱中，1-DNJ的含量为61.9％，FAG为16.6％，DAB为11.1％。
二、桑提取物的效果验证
实验例9：桑提取物预防和/或治疗炎症性肠病的药效学试验
一、实验方案
1.1实验动物
6-8周龄小鼠入驻动物房后进行适应性饲养3天，称重后，随机分为8组(计为0天)，每组5-6只。
实验分组分别为：1正常对照组、2模型组、3阳性对照组、4-8为不同给药剂量的SZ-A组。详见表1。
1.2实验药物
3％DSS配置方法：3g DSS(葡聚糖硫酸钠盐)溶解于100mL无菌水；
制备例1制备的桑枝提取物配制不同浓度，溶于1％CMC溶液；
阳性对照药：柳氮磺吡啶肠溶片(国药准字H31020557)。本品主要成份为：柳氮磺吡啶；其化学名为：5-[对-(2-吡啶胺磺酰基)苯]偶氮水杨酸。
1.3造模与给药
分组完成当天，对第8组SZ-A-5(pretreatment)组小鼠口服灌胃给药桑枝提取物(给药剂量见下表)，每天给药1次。所有组别均正常饮食饮水。
第8组小鼠给药3天后，除正常对照组外，其余各组均开始自由饮用3％DSS(葡聚糖硫酸钠盐)进行造模和灌胃给药；其中，阳性对照组的给药剂量为：5-[对-(2-吡啶胺磺酰基)苯]偶氮水杨酸500mg/kg/d。造模开始5天后，停止3％DSS饮用，所有组更换为正常饮水，给药正常进行；继续给药观察10天，脱颈椎处死小鼠；解剖获取小鼠结肠，测量结肠长度并拍照。给药期间，检测各组小鼠体重。造模及给药期间每日监测小鼠大便性状及便血情况，根据下表2进行小鼠疾病活动指数DAI评分。结果见图1-3以及图4。
表1实验分组及给药剂量
表2小鼠疾病活动指数DAI评分标准
注：DAI＝体重下降分数+粪便性状分数+隐血分数；正常大便：成形大便；半稀大便：不粘附于肛门的糊状、半成形大便；稀便：可粘附于肛门的稀水样便。
取结肠组织，进行H&E染色，具体包括如下步骤：
1、组织样本，取材3mm厚，梯度酒精脱水70％、80％、95％、100％各30分钟，二甲苯两瓶各20分钟，石蜡浸蜡两缸各12分钟，包埋，切片4微米，烤片。
2、苏木素伊红(hematoxylin eosin，HE)染色：(1)脱蜡，三瓶二甲苯每瓶脱蜡8分钟；两瓶100％酒精每瓶8分钟；90％酒精、80％酒精、60％酒精各8分钟。(2)苏木素染色4分钟，流水清洗；(3)盐酸酒精分化2-3秒，流水清洗；(4)0.5％氨水20秒，流水清洗，上镜观察。(5)0.5％伊红染色1分钟；(6)80％酒精、90％酒精各分化3-5秒；95％酒精5分钟；三瓶100％酒精各5分钟；两瓶二甲苯各5分钟。(7)中性树脂胶封固；光镜观察并显微照相。取各组新鲜结肠组织放入4％多聚甲醛中固定，48小时以后，置于不同浓度的酒精中，梯度脱水，随后置于二甲苯中透明。将已透明的组织置于石蜡中进行包埋。包埋好的蜡块固定于切片机上，进行切片。
1.4各个制备例中的桑枝提取物的疾病活动指数DAI评分比较
参考上述1.1-1.3实验步骤，各个制备例中的桑枝提取物进行小鼠疾病活动指数DAI评分比较，药物为制备例1、3、4、6和8中的桑枝提取物，用药剂量按桑枝提取物计，均为200mg/kg/d。结果见图5。
1.5实验结果
从图1-2中可以看出，与正常对照组相比，模型组结肠长度显著下降；与模型组相比，阳性药物组、SZ-A-1、SZ-A-2、SZ-A-3、SZ-A-4和SZ-A-5(pretreatment)均显著抑制UC模型小鼠结肠萎缩，SZ-A-2、SZ-A-3和SZ-A-4组的效果与阳性药组相当，SZ-A-5(pretreatment)较阳性药的效果更好。
从图3中可以看出，与正常对照组相比，模型组小鼠体重显著下降；与模型组相比，阳性药物组、SZ-A-1、SZ-A-2、SZ-A-3、SZ-A-4和SZ-A-5(pretreatment)均能抑制UC模型小鼠体重降低，且效果相当。
从图4中可以看出，与模型组相比，阳性药物组、SZ-A-1、SZ-A-2、SZ-A-3、SZ-A-4 和SZ-A-5(pretreatment)均能降低UC模型小鼠疾病活动指数DAI。
从图5中可以看出，制备例1(生物碱的含量为52％，多糖的含量为22％，黄酮的含量为0.8％，氨基酸的含量为20％)和制备例8(生物碱的含量为63％，多糖的含量为23％，黄酮的含量为1％，氨基酸的含量为5％)提供的特定的桑提取物能够降低UC模型小鼠疾病活动指数，而其他制备例中的桑提取物治疗效果不显著。
从图6可以看出，在光镜下观察UC模型小鼠组织切片，对照组小鼠结肠结构完整、清楚，腺体排列规则；而模型组中，连续饮用3％DSS的小鼠结肠组织存在结肠壁充血、水肿、黏膜层和黏膜下层出现严重的炎症细胞浸润，以及上皮细胞损伤脱落，杯状细胞缺失、隐窝肿胀破坏和较为严重的结肠溃疡情况；经桑提取物治疗后小鼠结肠组织病理学得到明显的改善，结肠结构较为完整，腺体排列规则，炎性细胞浸润较轻，且效果优于阳性对照组。说明桑提取物可以有效防治UC的发生，对炎症肠病有较好的防治效果。
以上结合了优选的实施方式对本发明进行了说明，不过这些实施方式仅是范例性的，仅起到说明性的作用。在此基础上，可以对本发明进行多种替换和改进，这些均落入本发明的保护范围内。
工业应用
本发明通过实验证实，特定的桑提取物能抑制UC模型小鼠结肠萎缩、抑制UC模型小鼠体重降低、降低UC模型小鼠疾病活动指数DAI，且经桑提取物治疗后小鼠结肠组织病理学得到明显的改善，结肠结构较为完整，腺体排列规则，炎性细胞浸润较轻。上述结果表述，可将桑提取物或其主要活性成分用于制备治疗溃疡性结肠炎。

Claims (10)

  1. 桑提取物或其主要活性成分的应用，为如下任一所述：
    (a1)桑提取物在制备治疗溃疡性结肠炎产品中的应用；
    (a2)桑提取物在制备预防溃疡性结肠炎产品中的应用；
    (a3)桑提取物在治疗溃疡性结肠炎中的应用；
    (a4)桑提取物在预防溃疡性结肠炎中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述预防和/或治疗溃疡性结肠炎体现在下述至少一方面：
    1)抑制溃疡性结肠炎患者结肠萎缩；
    2)抑制溃疡性结肠炎患者体重下降；
    3)降低溃疡性结肠炎患者疾病活动指数DAI；
    4)改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润。
  3. 桑提取物或其主要活性成分的应用，为如下任一所述：
    (b1)制备抑制溃疡性结肠炎患者结肠萎缩的产品；
    (b2)制备抑制溃疡性结肠炎患者体重下降的产品；
    (b3)制备降低溃疡性结肠炎患者疾病活动指数DAI的产品；
    (b4)制备改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润的产品；
    (b5)抑制溃疡性结肠炎患者结肠萎缩；
    (b6)抑制溃疡性结肠炎患者体重下降；
    (b7)降低溃疡性结肠炎患者疾病活动指数DAI；
    (b8)改善溃疡性结肠炎患者结肠结构、腺体排列及炎性细胞浸润。
  4. 根据权利要求1-3中任一所述的应用，其特征在于：所述桑提取物为桑枝提取物、桑白皮提取物和/或桑叶提取物。
  5. 根据权利要求1-4中任一所述的应用，其特征在于：所述产品为药物或药物制剂。
  6. 根据权利要求1-5中任一项所述的应用，其特征在于：
    所述桑提取物的制备方法包括以下步骤：
    1)制备桑科植物粗提液；
    2)将所述粗提液经阳离子树脂和/或任选的阴离子树脂分离，得到所述桑提取物。
  7. 根据权利要求5所述的应用，其特征在于：所述方法还包括下述步骤：
    3)对步骤2)的树脂流出液进行醇沉处理，收集上清液；
    4)对所述上清液进行浓缩干燥处理；
    或，所述还包括下述步骤：将步骤2)的树脂流出液进行浓缩干燥处理。
  8. 根据权利要求1-7中任一项所述的应用，其特征在于：所述桑提取物的主要活性成分包括：1-脱氧野尻霉素、N-甲基-1-脱氧野尻霉素、荞麦碱、3-表荞麦碱、1，4-双脱氧-1，4-亚氨基-D-核糖醇、打碗花精B2、打碗花精C1、2-氧-(α-D-半乳吡喃糖基)-1-脱氧野尻霉素、6-氧-(β-D-吡喃葡萄糖)-1-脱氧野尻霉素、1，4-双脱氧-1，4-亚胺-(2-氧-β-D-吡喃葡萄糖)-D-阿拉伯糖醇中的至少一种。
  9. 根据权利要求1-8中任一项所述的应用，其特征在于：所述桑提取物作用于人或哺乳动物。
  10. 一种预防和/或治疗溃疡性结肠炎的方法，包括如下步骤：给受体动物或人施用权利要求1-8中任一项所述的所述桑提取物或其主要活性成分以预防和/或治疗溃疡性结肠炎。
PCT/CN2023/093572 2022-04-01 2023-05-11 桑提取物在制备治疗溃疡性结肠炎的药物中的应用 WO2023186181A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210338779.2 2022-04-01
CN202210338779.2A CN114601865A (zh) 2022-04-01 2022-04-01 桑提取物在制备治疗溃疡性结肠炎的药物中的应用

Publications (1)

Publication Number Publication Date
WO2023186181A1 true WO2023186181A1 (zh) 2023-10-05

Family

ID=81867492

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/093572 WO2023186181A1 (zh) 2022-04-01 2023-05-11 桑提取物在制备治疗溃疡性结肠炎的药物中的应用

Country Status (2)

Country Link
CN (1) CN114601865A (zh)
WO (1) WO2023186181A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114601865A (zh) * 2022-04-01 2022-06-10 北京五和博澳药业股份有限公司 桑提取物在制备治疗溃疡性结肠炎的药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104857329A (zh) * 2015-05-06 2015-08-26 青岛市市立医院 一种治疗溃疡性结肠炎的灌肠液
KR20180042729A (ko) * 2016-10-18 2018-04-26 고려대학교 산학협력단 상지 주정 추출물을 함유하는 장질환을 예방 또는 치료하기 위한 조성물
KR20190125020A (ko) * 2018-04-27 2019-11-06 원광대학교산학협력단 뽕잎을 첨가한 대장염 개선 천연 조미료
CN113143996A (zh) * 2021-03-12 2021-07-23 北京五和博澳药业股份有限公司 桑提取物在制备治疗动物肠道炎症的组合物中的应用
CN114601865A (zh) * 2022-04-01 2022-06-10 北京五和博澳药业股份有限公司 桑提取物在制备治疗溃疡性结肠炎的药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104857329A (zh) * 2015-05-06 2015-08-26 青岛市市立医院 一种治疗溃疡性结肠炎的灌肠液
KR20180042729A (ko) * 2016-10-18 2018-04-26 고려대학교 산학협력단 상지 주정 추출물을 함유하는 장질환을 예방 또는 치료하기 위한 조성물
KR20190125020A (ko) * 2018-04-27 2019-11-06 원광대학교산학협력단 뽕잎을 첨가한 대장염 개선 천연 조미료
CN113143996A (zh) * 2021-03-12 2021-07-23 北京五和博澳药业股份有限公司 桑提取物在制备治疗动物肠道炎症的组合物中的应用
CN114601865A (zh) * 2022-04-01 2022-06-10 北京五和博澳药业股份有限公司 桑提取物在制备治疗溃疡性结肠炎的药物中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHENG SHUN HUA, CUI RU TAO: "Experimental Study on Ulcerative Colitis Treated by "Lung-intestine Nourishing Decoction"", SHANG HAI ZHONG YI YAO ZA ZHI : YUE KAN = SHANGHAI JOURNAL OF TRADITIONAL CHINESE MEDICINE, SHANG HAI ZHONG YI YAO DA XUE; SHANG HAI SHI ZHONG YI YAO XUE HUI ZHU BAN. SHANG HAI ZHONG YI YAO ZA ZHI SHE BIAN JI CHU BAN, CN, no. 06, 15 June 2000 (2000-06-15), CN , pages 10 - 12, XP009549442, ISSN: 1007-1334, DOI: 10.16305/j.1007-1334.2000.06.004 *

Also Published As

Publication number Publication date
CN114601865A (zh) 2022-06-10

Similar Documents

Publication Publication Date Title
WO2023186181A1 (zh) 桑提取物在制备治疗溃疡性结肠炎的药物中的应用
EP2040726A1 (en) Trachelospermi caulis extract composition for the treatment and prevention of inflammatory diseases
WO2007007993A1 (en) Pharmaceutical composition for the prevention and treatment of liver disease comprising a lonicera caerulea l. var. edulis extract
CN111568948A (zh) 桑提取物在制备改善胰岛功能药物中的应用
CN101234159A (zh) 一种具有协同作用的药物组合物
WO2021179902A1 (zh) 岩黄连及其制剂在制备治疗非酒精性脂肪性肝病的药物中的应用
JP2002538214A (ja) 心血管疾病治療用薬剤組成物及びその製造方法
CN109939153A (zh) 一种治疗肠炎的蒙药及其制备方法和制剂及制备方法
CN106963777B (zh) 一种“黄芩苷-小檗碱”复合物的制备方法及其应用
CN103301179B (zh) 杜仲木脂素提取物在制备用于防治高甘油三脂血症的药物中的应用
CN113940945A (zh) 鱼腥草多糖在制备防治炎症性肠病药物中的用途
CN111568949A (zh) 桑提取物在制备调节动物肠道菌群的药物中的用途
CN102293928A (zh) 治疗高尿酸血症及痛风性关节炎的中药组合物及其制备方法与应用
CN113209182B (zh) 一种用于治疗类风湿关节炎的中药组合物及其制备方法
CN112691114B (zh) 苦瓜多糖在制备用于治疗溃疡性结肠炎药物中的应用及其药物制剂
CN111407783B (zh) 赶黄草在制备治疗高蛋白尿药物中的应用
CN103933100A (zh) 功劳木总生物碱的制备及其在预防和治疗胃溃疡药物中的应用
CN113144015A (zh) 香连产物或其与抗生素联合在抗幽门螺杆菌药物中的应用
CN113209154A (zh) 鸡骨香提取物在制备预防和/或治疗溃疡性结肠炎药物中的应用
WO2024051650A1 (zh) 桑提取物在制备预防和/或治疗慢性肾病的药物中的应用
CN106138194B (zh) 一种治疗高尿酸血症的中药复方制剂及其制备方法
Wang et al. Protective effects of ginsenoside Rg1 on acute myocardial infarction
CN115671219B (zh) 一种治疗痛风的中药组合物及其制备方法和应用
CN110898170B (zh) 一种治疗代谢综合征的中药组合物及其制剂
CN101757460A (zh) 一种治疗痛风的中药制剂

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23778545

Country of ref document: EP

Kind code of ref document: A1