WO2022188851A1 - 桑提取物在制备减轻动物体重的药物中的应用 - Google Patents

桑提取物在制备减轻动物体重的药物中的应用 Download PDF

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WO2022188851A1
WO2022188851A1 PCT/CN2022/080278 CN2022080278W WO2022188851A1 WO 2022188851 A1 WO2022188851 A1 WO 2022188851A1 CN 2022080278 W CN2022080278 W CN 2022080278W WO 2022188851 A1 WO2022188851 A1 WO 2022188851A1
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extract
mulberry
animal
content
weight
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PCT/CN2022/080278
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English (en)
French (fr)
Inventor
刘玉玲
申竹芳
刘率男
陈艳敏
刘志华
刘泉
李彩娜
雷蕾
朱向阳
曹慧
廉春放
孙倩雯
邹媛媛
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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
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Priority claimed from CN202110272018.7A external-priority patent/CN113143997A/zh
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
Priority to CA3211708A priority Critical patent/CA3211708A1/en
Priority to AU2022233818A priority patent/AU2022233818A1/en
Priority to KR1020237032601A priority patent/KR20230154038A/ko
Priority to JP2023553607A priority patent/JP2024509199A/ja
Priority to EP22766373.9A priority patent/EP4306122A1/en
Priority to US18/550,120 priority patent/US20240156889A1/en
Publication of WO2022188851A1 publication Critical patent/WO2022188851A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Definitions

  • the present invention relates to the application of mulberry extract in preparing medicine for reducing animal body weight.
  • the causes of obesity include an increase in the size of adipocytes and an increase in the number of adipocytes, wherein the increase in the number of adipocytes is caused by the differentiation of preadipocytes into mature adipocytes, and the differentiation of preadipocytes into mature adipocytes occurs in a series of Activation and expression of transcription factors are accomplished.
  • the data shows that the number of diabetic patients ranks first in the world, of which the proportion of overweight people is 41.0% and the proportion of obese people is 24.3%.
  • mulberry plants Because of its high nutritional value and medicinal value, mulberry plants have been regarded as precious materials for both medicine and food since ancient times, and mulberry branches, mulberry white bark, mulberry leaves and For example, in the Compendium of Materia Medica, there are records of "mulberry leaf juice frying in place of tea, which can quench thirst” and “decoction when cooked, and substitute tea for quenching thirst".
  • the chemical constituents of Moraceae plants mainly include flavonoids, polysaccharides, alkaloids, etc., and are now widely used in the preparation of hypoglycemic, lipid-lowering, antiviral, and immunomodulatory drugs.
  • patent application CN1631246A discloses the weight loss and lipid reduction effect of mulberry twig extract, which is embodied in that mulberry twig ethanol extract can reduce the body weight of NIH mice and reduce blood pressure.
  • the level of triglyceride and cholesterol, but its administration method and dose-effect relationship are not clear;
  • patent application CN102370708A discloses the application of Chinese herbal medicine mulberry leaf water extract or alcohol extract in the preparation of weight-loss and lipid-lowering drugs, and records the mulberry extract The application of weight loss and lipid lowering.
  • the KKAy mouse is a type 2 diabetes animal model mouse that is transformed into a mutant gene (ay) on the basis of the KK mouse. Its onset is induced by environmental factors on the basis of genetic susceptibility. The performance of diabetes is very similar, and its individual differences are small, and the experiment has good repeatability. It is an ideal animal model of spontaneous type 2 diabetes.
  • the clinical manifestations of KKAy mice are polydipsia, polyuria, and obesity. They are characterized by hyperglycemia, high insulin resistance, pancreatic islet insufficiency, liver disease, and kidney disease. The study showed that the body weight, liver fat and epididymal fat weight of KKAy mice were significantly higher than those of normal mice. There is no research report on the effect of mulberry-related extracts on the body weight of KKAy mice.
  • the inventor's research found that the mulberry extract of the present invention has a significant effect on the weight loss of animals, and based on this, the present invention provides an application of the mulberry extract in preparing a medicine for reducing animal body weight.
  • the mulberry extract contains alkaloids, polysaccharides, amino acids and flavonoids.
  • the weight content of each component is
  • the weight content of each component is
  • the weight content of each component is
  • the preparation of the mulberry extract comprises the following steps: preparing a crude extract; optionally, separating by cation resin and/or anion resin; optionally, performing alcohol precipitation on the resin effluent; And optionally, a concentrated drying process.
  • the preparation of the mulberry extract includes the following steps: step 1): preparing a crude extract; step 2): separating by cation resin and/or optional anion resin; optional step 3): for step 2 ) of the resin effluent to alcohol precipitation treatment; optional step 4) concentration and drying treatment.
  • the mulberry extract is prepared according to the following steps: pulverizing mulberry branch, mulberry leaf or mulberry white bark, heating and refluxing extraction with water and/or alcohol solution or acid water, and the solvent amount is 3-3% of the original medicinal material. 20 times, repeat the extraction for 1-3 times, combine the extracts, concentrate, apply cation exchange resin, distilled water to wash away the impurities that are not adsorbed, elute with 0.2-3N ammonia water, concentrate the eluent on anion exchange resin, and collect the non-adsorbed impurities. Part, add ethanol, remove impurities by precipitation, centrifuge, and concentrate the clear liquid under reduced pressure or spray drying or freeze drying to obtain an extract.
  • the mulberry extract is prepared according to the following steps: pulverizing mulberry branch, mulberry leaf or mulberry white bark, heating and refluxing extraction with water and/or alcohol solution or acid water, and the solvent amount is 3-3% of the original medicinal material. 20 times, repeat the extraction for 1-3 times, combine the extracts, concentrate, apply cation exchange resin, distilled water to wash away the impurities that are not adsorbed, elute with 0.2-3N ammonia water, concentrate the eluent on anion exchange resin, and collect the non-adsorbed impurities. Part, concentrated under reduced pressure or spray-dried or freeze-dried to obtain an extract.
  • the mulberry extract is prepared according to the following steps: pulverizing mulberry branch, mulberry leaf or mulberry white bark, heating and refluxing extraction with water and/or alcohol solution or acid water, and the solvent amount is 3-3% of the original medicinal material. 20 times, repeat the extraction 1-3 times, combine the extracts, concentrate, apply cation exchange resin, distilled water to wash off the impurities that are not adsorbed, eluted with 0.2-3N ammonia water, and the eluent is concentrated under reduced pressure or spray-dried or freeze-dried, get the extract.
  • 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, most preferably humans.
  • the animal is a diabetic animal.
  • the body mass index BMI of the diabetic animal is greater than or equal to 28.
  • the animal is a healthy animal.
  • the medicament reduces the body weight of the animal by reducing fat accumulation in cells.
  • the cells are liver cells and/or epididymal adipocytes. More preferably, the cells are liver cells.
  • the medicament further includes a pharmaceutically acceptable carrier.
  • the carrier is an inactive ingredient that is in line with the route of administration or the mode of administration and has no toxic effect on the human body.
  • the carrier can be a solid or liquid excipient.
  • Solid excipients include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, 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 glycerin.
  • the drug is in the form of oral administration; further preferably, the drug is in the form of tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups and powders.
  • the mulberry extract of the present invention can not only control the weight gain of spontaneous diabetic obese model mice, but also has clear ingredients, controllable quality, no adverse reactions, high safety and low cost, and provides a new choice for diabetic obese people.
  • the development and screening of slimming drugs provides new varieties.
  • Figure 1 shows the effect of long-term administration of mulberry extract on the body weight of KKAy mice in Example 9: (A) body weight of each group before and after administration, (B) body weight change before and after administration. **P ⁇ 0.01, ***P ⁇ 0.001, compared with DM group.
  • FIG. 2 is a graph showing the changes in body weight of individuals after 8 weeks of clinical administration in Example 11.
  • FIG. 2 is a graph showing the changes in body weight of individuals after 8 weeks of clinical administration in Example 11.
  • FIG. 3 is a graph showing the changes in body weight of individuals after 24 weeks of clinical administration in Example 11.
  • FIG. 4 is a graph showing the change in body weight of C57 mice fed with high-fat by gavage in Example 13.
  • FIG. 5 is a graph showing the body weight change of high-fat-fed C57 mice administered by injection in Example 13.
  • Figure 6 is the effect of different doses of SZ-A on palmitic acid-induced hepatic triglyceride and cholesterol accumulation.
  • SZ-A represents the mulberry branch extract SZ-A treatment group
  • MET represents the metformin positive control group
  • PA represents the palmitic acid stimulated insulin resistance model group
  • BSA represents the HepG2 cell normal control group (0.25%BSA treatment). **P ⁇ 0.01, compared with normal control group.
  • the content of the components involved in the present invention is detected according to the published method (refer to the methods described in the patents with publication numbers CN111077247A and CN110393738A).
  • Use D218 type macroporous strong basic acrylic anion resin 62.5kg to pack the column, use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 870L.
  • the collected liquid was centrifuged to remove impurities and then concentrated by reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid was 1.25. It was transferred to the alcohol precipitation tank, and 25 L of absolute ethanol was added at 500 rpm of the stirring paddle. After the addition of ethanol, the stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain the extract.
  • the effluent is concentrated under reduced pressure to obtain a mulberry branch extract extract, wherein 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%.
  • the anion resin was activated according to the method of Example 3 using 10 kg of 711 type strongly basic styrene-based anion resin packed into a column.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 15L.
  • the collected liquid was reloaded to the cation resin, and the cation resin and the anion resin were used to separate two more times in sequence according to the above method.
  • the collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated by a reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank, and 125 g of anhydrous ethanol was added at 1000 rpm of the stirring paddle. After the addition of ethanol, the stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain the extract.
  • fresh mulberry white bark and mulberry leaf (Sangteyou No. 2) were extracted, and the extraction method and parameters were the same as the above-mentioned methods.
  • the content of alkaloids is 98%
  • the content of polysaccharides is 0.2%
  • the content of flavonoids is 0.05%
  • the content of amino acids is 0.
  • the content of alkaloids is 95%
  • the content of polysaccharides is 2%
  • the content of flavonoids is 0.1%
  • the content of amino acids is 1%.
  • the content of alkaloids is 90%
  • the content of polysaccharides is 4%
  • the content of flavonoids is 0.1%
  • the content of amino acids is 3%.
  • the collected solution obtained after separation by the cation column is concentrated by nanofiltration membrane, and concentrated under reduced pressure to obtain the extract.
  • the content of alkaloids is 15%
  • the content of polysaccharides is 20%
  • the content of flavonoids is 7%
  • the content of amino acids is 45%.
  • Use D218 type macroporous strong basic acrylic anion resin 125kg to pack the column use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5 ; 1.5mol/L sodium hydroxide solution was washed until the pH of the eluate was 9.0; the activation was completed.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent with pH greater than 8 was collected until the effluent reached 870L.
  • the collected liquid obtained after the separation of the anion column is subjected to microfiltration membrane filtration to remove impurities and then concentrated with a reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid is 1.1, which is transferred to the alcohol precipitation tank, and anhydrous ethanol is added under the stirring paddle at 400 rpm. 15kg. After the addition of ethanol, the stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain the extract of the mulberry branch extract.
  • Sample content the content of alkaloids is 80%, the content of polysaccharides is 5%, the content of flavonoids is 0.1%, and the content of amino acids is 4%.
  • Use D218 type macroporous strong basic acrylic anion resin 62.5kg to pack the column, use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation.
  • the collected extract concentrate was loaded onto an anion resin and the effluent was collected.
  • the collected solution obtained after the anion column separation is filtered through a microfiltration membrane to remove impurities, then concentrated by a reverse ion osmosis membrane, and further concentrated and dried under reduced pressure to obtain the 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%.
  • Use D218 type macroporous strong basic acrylic anion resin 62.5kg to pack the column, use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 870L.
  • the effluent is concentrated under reduced pressure to obtain a mulberry branch extract extract, wherein the content of alkaloids is 30%, the content of polysaccharides is 35%, the content of flavonoids is 2%, and the content of amino acids is 25%.
  • Use D218 type macroporous strong basic acrylic anion resin 62.5kg to pack the column, use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent was collected until the effluent reached 870L.
  • the effluent is concentrated under reduced pressure to obtain a mulberry branch extract extract, wherein the content of alkaloids is 40%, the content of polysaccharides is 25%, the content of flavonoids is 0.5%, and the content of amino acids is 25%.
  • Use D218 type macroporous strong basic acrylic anion resin 62.5kg to pack the column, use 1.5mol/L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5mol/L hydrochloric acid solution to wash until the pH of the eluate is 3.5; wash with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation.
  • the collected cation resin eluate was loaded onto the anion resin, and the effluent with pH greater than 8 was collected until the effluent reached 870L.
  • the collected liquid obtained after the separation of the anion column is subjected to microfiltration membrane filtration to remove impurities and then concentrated with a reverse ion osmosis membrane.
  • the specific gravity of the concentrated liquid is 1.1, which is transferred to the alcohol precipitation tank, and anhydrous ethanol is added under the stirring paddle at 400 rpm. 15kg. After the addition of ethanol, the stirring was stopped, and the alcohol was precipitated for 24 hours. The supernatant was taken and concentrated under reduced pressure to obtain the extract of the mulberry branch extract.
  • Sample content the content of alkaloids is 63%, the content of polysaccharides is 23%, the content of flavonoids is 1%, and the content of amino acids is 5%.
  • Example 9 The effect of mulberry extract on the body weight of spontaneously type 2 diabetic KKAy mice
  • 12-week-old female KKAy mice were selected and fed with high-fat diet for 3 weeks. According to random blood glucose, fasting blood glucose and body weight, they were divided into 3 groups (DM group, SZ-A 160 (mg mulberry extract/kg), SZ-A 320 (mg mulberry extract/kg)), 8 mice in each group, the mulberry extract of Example 8 was administered by gavage once a day for about 6 weeks, and the changes in the body weight of mice before and after administration were recorded, As shown in Table 1, Table 2 and Figure 1.
  • the average weight of mice in each group was about 43 g.
  • the weight of mice in two dose groups of mulberry extract decreased significantly, and the weight of mice in SZ-A160 dose group decreased by 2.0g compared with DM group, and the weight loss rate was 4.2%;
  • SZ-A320 Compared with the DM group the mice in the dose group lost 4.1 g of body weight, and the weight loss rate was 8.6%.
  • the weight growth rate of the mice in the two dose groups of SZ-A was also significantly reduced.
  • the average weight of the mice in the DM group increased by 4.4g, the average increase in the SZ-A160 dose group by 2.6g, and the average increase in the SZ-A320 dose group.
  • the average increase was 0.1g, indicating that the mulberry extract had a significant effect on controlling the weight gain of KKAy mice.
  • DM is the KKAy model control group
  • SZ-A is the administration group. **P ⁇ 0.01, ***P ⁇ 0.001 compared with DM group
  • mulberry branch extract of Example 1 Take the mulberry branch extract of Example 1, add appropriate amount of auxiliary materials, mix well, add water to make soft material, granulate and dry, add magnesium stearate, mix well, and press into tablets to obtain a mulberry branch extract preparation, each tablet containing total Alkaloid 50mg.
  • Example 1 64-year-old, 75.0 kg, 168 cm tall male, suffering from type 2 diabetes for 12 months, taking acarbose for 2 months and then stopping the drug, 8 months later, the tablet containing Example 1 was taken orally, three times a day , 2 tablets at a time for 24 weeks, weight loss of 7 kg.
  • Mulberry twig extract reduces weight in diabetic patients
  • a randomized, double-blind, double-dummy, parallel-controlled, multicenter, non-inferiority study design was adopted.
  • the main exclusion criteria were: 1) a-glucosidase inhibitor allergy or intolerance; 2) change in FBS level between the first and second follow-up visits >2.5mmol/L (>45mg/dL); 3) with History of severe diabetic complications (proliferative stage of diabetic retinopathy, stage V of diabetic nephropathy, diabetic ketoacidosis, diabetic hyperosmolar coma, diabetic lactic acidosis); 4) Combination therapy with drugs affecting glucose metabolism, such as antidiabetic traditional Chinese medicine or Glucocorticoids; 5) Hyperlipidemia with a history of irregular lipid-lowering drug intake; 6) Chronic gastrointestinal dysfunction, marked digestion and absorption disorders or endocrine diseases such as hyperthyroidism, hypercortisolism and acromegaly 7) Severe heart disease, myocardial infarction, unstable angina, chronic cardiac insufficiency or poor blood pressure control; 8) Impaired liver or kidney function; 9) Pregnancy.
  • the experimental group was orally administered tablets (the preparation method of the tablet was: the extract of Example 1, adding an appropriate amount of auxiliary materials, mixing uniformly, adding water to make soft materials, granulating and drying, adding magnesium stearate, mixing uniformly, and pressing into tablets to obtain mulberry branches. Extract preparation, each tablet contains 50 mg of total alkaloids), the initial dose is 1 tablet each time, 3 times a day; after 4 weeks, it is gradually increased to 2 tablets each time, 3 times a day; the control group takes acarbose orally, 1 tablet each time, 3 times a day.
  • Mulberry branch extract reduces weight in healthy people
  • non-diabetic people There are 17 non-diabetic people, 11 males and 6 females, all healthy people, normal diet during taking the medicine, taking the medicine for a period of 0.7 to 8 months, the dose is 50 or 100 mg/time, and the frequency of taking the medicine is 2- 3 times/day.
  • Weight loss judgment criteria (1) Excellent effect, the percentage of weight loss is greater than or equal to 10%; (2) Significantly effective, the percentage of weight loss is greater than or equal to 5% and less than 10%; (3) Effective, the percentage of weight loss is greater than or equal to 3% and less than 5% ; (4) Invalid, the percentage of body weight loss is less than 3%.
  • the total effective rate (the number of markedly effective cases + the number of effective cases)/the number of cases*100%.
  • TID means: Take the medicine three times a day
  • BID means: Take the medicine twice a day
  • mice Thirty 6-week-old healthy male C57 mice were randomly divided into normal group, model group and SZ-A group, with 10 mice in each group.
  • the normal group mice were given basal diet, and the model group and SZ-A group were given high-fat diet.
  • the mice in each group were given the corresponding drugs by gavage every day for 6 consecutive weeks. Dose of solvent, and monitor mouse body weight during drug treatment. After dosing, all mice were fasted for 12 hours and weighed.
  • the results are shown in Figure 4.
  • the average weight of mice in the model group was 50.3 g, and the average weight of mice in the administration group was 43.7 g.
  • SZ-A could significantly reduce the weight of mice on a high-fat diet.
  • the third group carries out intraperitoneal injection according to body weight by total alkaloids of mulberry branch 200mg/kg, and the time of injection administration is 6 weeks.
  • the first group and the second group were injected with normal saline according to body weight. Dosing once a day.
  • the results are shown in Figure 5.
  • the average weight of the model group was 50.6 g, and the average weight of the administration group was 42.8 g. SZ-A injection could significantly reduce obesity caused by high-fat diet.
  • the cell density was adjusted to 2 ⁇ 10 5 cells/ml with DMEM (high glucose) complete medium containing 10% FBS, and 2 ml/well was transferred to a 6-well plate.
  • DMEM high glucose
  • model cells were randomly divided into SZ-A group (50ug/ml, 25ug/ml, 12.5ug/ml mulberry branch extract SZ-A of Example 1), metformin group (200umol/l MET) and palmitic acid stimulation model group (PA), and set HepG2 cells as normal control group (0.25%BSA). Except for the normal control group (0.25%BSA) and model group (PA), other groups were added with corresponding drugs and palmitic acid to replicate the palmitic acid stimulation model. Each group was set up with 3 replicate wells.
  • test results are shown in Figure 6. Too much fat accumulation in the liver can easily lead to fatty liver. Stimulation of HepG2 liver cells with PA palmitate can mimic fatty liver formation. Adding different doses of SZ-A, it can be seen that SZ-A inhibits the accumulation of hepatic triglyceride and cholesterol caused by palmitic acid. The effect of SZ-A is comparable to that of MET.
  • model cells were treated with the mulberry extract (dose of 25ug/ml) prepared in Examples 2-7, and triglyceride and total cholesterol in the cells were measured respectively.
  • the results showed that compared with the PA group, the accumulation of triglyceride and cholesterol in the liver caused by palmitic acid was reduced to a certain extent after treatment with the mulberry extract.
  • the specific results are shown in Table 3 below.
  • mulberry extract has a good weight control effect.
  • Long-term administration of mulberry extract can inhibit the weight gain of mice and humans, inhibit the weight gain of type 2 diabetic mice and diabetic patients; and has an inhibitory effect on the accumulation of visceral fat.

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Abstract

一种桑提取物在制备减轻动物体重的药物中的应用，所述桑提取物含有3-99％生物碱、0.2-70％多糖、0-50％氨基酸、0-10％黄酮和0-25％其它组分。该桑提取物能够控制体重增长，对2型糖尿病合并肥胖症具有良好的治疗效果。

Description

桑提取物在制备减轻动物体重的药物中的应用 技术领域
本发明涉及桑提取物在制备减轻动物体重的药物中的应用。
背景技术
随着生活水平的提高，肥胖和超重现象正在全世界范围内以惊人的速度增长。流行病学研究表明，肥胖和超重是糖尿病、心血管疾病、癌症和过早死亡的重要危险因素。肥胖形成的原因包括脂肪细胞体积的增大和脂肪细胞数目的增多，其中脂肪细胞数量的增加由前体脂肪细胞分化为成熟脂肪细胞造成的，而前体脂肪细胞分化为成熟脂肪细胞是在一系列转录因子的激活与表达下完成的。数据显示，糖尿病患者人数居全球首位，其中超重人群比例为41.0％，肥胖人群比例为24.3％。与单纯肥胖患者相比，2型糖尿病合并肥胖患者减重并维持体重更加困难(中国2型糖尿病合并肥胖综合管理专家共识，中华内分泌代谢杂志,2016,32(08):623-627.)。
桑科植物因具有较高的营养价值和药用价值，自古以来就被认作是药食两用的珍贵材料，在历代中医药典籍、处方中多有用桑枝、桑白皮、桑叶和桑椹等治疗疾病的记载，如早在《本草纲目》中便有“桑叶汁煎代茗，能止消渴”，“炙熟煎饮，代茶止渴”的记载。桑科植物的化学成分主要包括黄酮类化合物、多糖类化合物、生物碱等，如今被广泛用于降糖、降脂、抗病毒、免疫调节等药物的制备。
现有技术中有一些有关桑提取物减肥降脂的报道，如专利申请CN1631246A公开了桑枝提取物的减肥降脂作用，具体表现为桑枝乙醇提取物能够减轻NIH小鼠的体重，降低血液甘油三酯和胆固醇的水平，但是其给药方法、量效关系均不明确；专利申请CN102370708A公开了中草药桑叶水提物或醇提物在制备减肥降脂药物中的应用，记载了桑提取物减肥降脂的应用。
KKAy小鼠是在KK小鼠的基础上转入突变基因(ay)而成的一种2型糖尿病动物模型鼠，其发病是在遗传易感的基础上加环境因素而诱发，和人类2型糖尿病的表现极为相似，且其个体差异小，实验重复性好，是一种比较理想的自发性2型糖尿病动物模型。KKAy小鼠临床表现为多饮多食多尿并伴有肥胖，具有高血糖、高度胰岛素抵抗、胰岛功能不足、肝脏病变、肾脏病变等特征。研究表明，KKAy小鼠的体重、肝脏脂肪和附睾脂肪重量明显高于正常小鼠。目前还没有成分明确的桑相关提取物对KKAy小鼠体重影响的研究报道。
发明内容
发明人研究发现，本发明的桑提取物对动物的体重减轻具有显著影响，基于此，本发明提供了一种桑提取物在制备减轻动物体重的药物中的应用。
在本发明的一种实施方式中，所述桑提取物含有生物碱，多糖，氨基酸和黄酮。优选地，基于所述桑提取物，各组分重量含量为
Figure PCTCN2022080278-appb-000001
更优选地，基于所述桑提取物，各组分重量含量为
Figure PCTCN2022080278-appb-000002
Figure PCTCN2022080278-appb-000003
进一步优选地，基于所述桑提取物，各组分重量含量为
Figure PCTCN2022080278-appb-000004
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。
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。
在本发明的一种实施方式中，所述动物为糖尿病动物。优选地，所述糖尿病动物的身体质量指标BMI≥28。
优选地，所述动物为健康动物。
在本发明的一种实施方式中，所述药物通过减少细胞中脂肪累积减轻动物体重。优选地，所述细胞为肝脏细胞和/或附睾脂肪细胞。更优选地，所述细胞为肝脏细胞。
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。
本发明的桑提取物不仅能够控制自发性糖尿病肥胖模型小鼠的体重增长，同时成分明确、质量可控、无不良反应、安全性高、成本低廉，为糖尿病肥胖人群提供了新的选择，为减肥药的开发和筛选提供了新的品种。
附图说明
图1是实施例9中桑提取物长期给药对KKAy小鼠体重的影响：(A)给药前及给药后各组体重，(B)给药前后的体重变化。**P<0.01,***P<0.001，与DM组相比。
图2是实施例11中临床给药8周后个体体重变化图。
图3是实施例11中临床给药24周后个体体重变化图。
图4是实施例13中灌胃给药高脂喂养C57小鼠体重变化图。
图5是实施例13中注射给药高脂喂养C57小鼠体重变化图。
图6是不同剂量SZ-A对棕榈酸引起的肝脏甘油三酯和胆固醇积累的影响。SZ-A表示桑枝提取物SZ-A处理组，MET为二甲双胍阳性对照组，PA表示棕榈酸刺激胰岛素抵抗模型组，BSA表示HepG2细胞正常对照组(0.25％BSA处理)。**P<0.01,与正常对照组相比。
具体实施方式
以下通过附图和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。
在这里专用的词“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)。
实施例1桑提取物的制备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，减压浓缩得提取物浸膏。
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52％，多糖的含量为22％，黄酮的含量为0.8％，氨基酸的含量为20％。
实施例2桑提取物的制备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，按照实施例3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。
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％。
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％。
实施例3桑提取物的制备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％。
实施例4桑提取物的制备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％。
实施例5桑提取物的制备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％。
实施例6桑提取物的制备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％。
实施例7桑提取物的制备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％。
实施例8桑提取物的制备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％。
实施例9桑提取物对自发性2型糖尿病KKAy小鼠体重的影响
选择12周龄的雌性KKAy小鼠，高脂饲料喂养3周后，根据随机血糖、空腹血糖及体重等指标，平均分成3组(DM组，SZ-A 160(mg桑提取物/kg)，SZ-A 320(mg桑提取物/kg))，每组8只，每日灌胃给予实施例8的桑提取物一次，连续给药约6周，并记录给药前后小鼠体重变化，如表1、表2和图1所示。
表1自发性2型糖尿病KKAy小鼠分组数据
Figure PCTCN2022080278-appb-000005
给予桑提取物之前，各组小鼠的体重平均值均为43g左右。给予桑提取物约6周后，桑提取物两个剂量组的小鼠体重明显减轻，SZ-A160剂量组的小鼠体重较DM组减轻了2.0g，体重减轻率为4.2％；SZ-A320剂量组的小鼠体重较DM组减轻了4.1g，体重减轻率为8.6％。在给药期间，SZ-A两个剂量组的小鼠体重增长速度也显著降低，DM组小鼠的体重平均增长了4.4g，SZ-A160剂量组平均增长了2.6g，SZ-A320剂量组平均增长了0.1g，说明桑提取物具有显著的控制KKAy小鼠体重增长的作用。
表2自发性2型糖尿病KKAy小鼠体重变化
Figure PCTCN2022080278-appb-000006
与DM比较，数据以`X±SD表示，n＝8；DM为KKAy模型对照组，SZ-A为给药组。**P<0.01,***P<0.001与DM组相比
实施例10临床试验1
取实施例1的桑枝提取物，加入适量辅料，混合均匀，加水制软材，制粒干燥，加入硬脂酸镁，混合均匀，压片，即得桑枝提取物制剂，每片含总生物碱50mg。
临床病例及治疗：
(1)43岁，84.2公斤，身高170cm的中国男性，患有2型糖尿病3个月，口服含实施例1桑提取物的片剂，一日三次，一次两片，口服24周，体重减轻10.2公斤。
(2)65岁，68.3公斤，身高173cm男性，患有2型糖尿病7个月，合并高脂血症，口 服含实施例1的片剂，一日三次，一次两片，持续24周，体重减轻6.3公斤。
(3)64岁，75.0公斤，身高168cm男性，患有2型糖尿病12个月，服药阿卡波糖两个月后停药，8个月后口服含实施例1的片剂，一日三次，一次两片，持续24周，体重减轻7公斤。
(4)48岁，81.0公斤，身高164cm女性，患有2型糖尿病8个月，合并高脂血症，口服含实施例1的片剂，一日三次，一次两片，持续24周，体重减轻7公斤。
实施例11临床试验2
1、实验目的：
桑枝提取物减轻糖尿病患者体重
2、实验方法
2.1实验分组
采用随机、双盲双模拟、阿卡波糖片平行对照、多中心、非劣性研究设计。纳入年龄18-70岁、19kg/m 2≤BMI≤30kg/m 2、7％≤HbA1C≤10％，血糖控制不佳的2型糖尿病受试者600例，随机分成桑枝总生物碱片组(SZ-A，N＝360)和阿卡波糖(拜唐苹
Figure PCTCN2022080278-appb-000007
)组(CON，N＝240)。均签署了知情同意书。
主要排除标准为：1)a-glucosidase抑制剂过敏或不耐受；2)第一次随访和第二次随访之间的FBS水平变化>2.5mmol/L(>45mg/dL)；3)有严重糖尿病并发症史(糖尿病视网膜病变的增殖阶段，糖尿病肾病V期，糖尿病酮症酸中毒，糖尿病高渗昏迷、糖尿病性乳酸酸中毒)；4)药物联合治疗影响葡萄糖代谢，比如抗糖尿病中药或糖皮质激素；5)高脂血症伴有不规则降脂药物摄入史；6)慢性胃肠功能障碍，明显消化和吸收障碍或内分泌疾病比如甲状腺机能亢进，高皮质醇症和肢端肥大症；7)严重心脏病、心肌梗塞、不稳定性心绞痛、慢性心功能不全或者血压控制不佳；8)肝功能或肾功能受损；9)怀孕。
2.2给药方法
实验组口服给药片(药片的制备方法为：实施例1提取物，加入适量辅料，混合均匀，加水制软材，制粒干燥，加入硬脂酸镁，混合均匀，压片，即得桑枝提取物制剂，每片含总生物碱50mg)，起始剂量每次1片，一日3次；4周后递加至每次2片，一日3次；对照组口服阿卡波糖，每次给药1片，每天3次。
3、实验数据检测与处理
3.1检测指标
(1)给药第1周-24周，每天观察并记录各组的身高、体重及增重。
3.2统计学分析
采用SPSS 20.0软件进行数据处理，组间差异采用T-检验。
4、实验结果
给药8周、24周后，各组二型糖尿病人的不同BMI分组实验数据结果如表3、4所示，以及图2图和3所示。
给药8周后，BMI≥28kg/m 2的患者中，实验组与对照组相比，体重显著减轻，平均变化量分别为-0.503kg和0.210kg(P＝0.016)；给药24周后，BMI≥28kg/m 2的患者中，实验组与对照组相比，体重显著减轻，平均变化量分别为-1.228kg和-0.182kg(P＝0.022)；其他BMI分组，体重变化没有统计学意义。
表3：8周独立样本检验分析
  BMI≥18kg/m 2 BMI≥24kg/m 2 BMI≥28kg/m 2
P 0.719 0.714 0.016
表4：24周独立样本检验分析
  BMI≥18kg/m 2 BMI≥24kg/m 2 BMI≥28kg/m 2
P 0.664 0.84 0.022
说明BMI≥28kg/m 2的糖尿病人使用桑枝提取物后不仅能够治疗2型糖尿病，还能够达到减重目的。
实施例12临床试验例3
1、实验目的
桑枝提取物减轻健康人体重
2、实验方法
2.1实验仪器与材料
体重计。
2.2实验设计
非糖尿病人群，共17例，男性为11例，女性为6例，均为健康人群，服药期间正常饮食，服药周期为0.7到8个月，剂量为50或100mg/次，用药次数为2-3次/天。
2.3检测指标
在进行实验前和实验结束时，检测以下身体指标：体重，身高。
减重判断标准：(1)优效，体重下降百分数大于等于10％；(2)显效，体重下降百分数大于等于5％小于10％；(3)有效，体重下降百分数大于等于3％小于5％；(4)无效，体重下降百分数小于3％。
3、实验结果
优效的为2例，显效的为9例，有效的为5例，无效的为1例，总有效率为94％。此外各个受试者服药后无不良反应，表明该药对非糖尿病人减重作用的效果显著。受试者信息和实验结果见下表5。
总有效率＝(显效例数+有效例数)/例数*100％。
TID表示：每日服药三次
BID表示：每日服药两次
表5
Figure PCTCN2022080278-appb-000008
实施例13、高脂喂养C57小鼠体重减轻实验
1、灌胃给药
随机将30只6周龄健康的雄性C57小鼠分为正常组、模型组、SZ-A组，每组各10只。其中正常组小鼠给予基础饲料喂养，模型组和SZ-A组给予高脂饮食。饲养14周后，每天对各组小鼠给予相应的药物灌胃，连续给药6周，SZ-A组按桑枝总生物碱400mg/kg/d灌胃，正常组和模型组灌胃相应剂量的溶剂，药物治疗期间，监测小鼠体重。给药结束后，所有小鼠禁食12小时后，称体重。
结果如图4所示，模型组小鼠平均体重为50.3g，给药组小鼠平均体重为43.7g，SZ-A可显著降低高脂饮食小鼠的体重。
2、注射给药
(1)动物建模:30只6周龄C57小鼠随机分为三组，第一组喂养正常饮食、第二组和第三组喂养高脂饮食，持续喂养14周后。
(2)给药处理:第三组根据体重按桑枝总生物碱200mg/kg进行腹腔注射，注射给药的时间为6周。第一组和第二组分别按体重注射生理盐水。每天给药一次。
(3)体重测量：在给药处理后，用天平测量小鼠体重并记录。
结果如图5所示，模型组平均体重为50.6g，给药组平均体重为42.8g，注射SZ-A可显著降低高脂饮食引起的肥胖。
实施例14体外细胞试验
1.处于对数生长期的HepG2细胞消化后，用含10％FBS的DMEM(高糖)完全培养基调整细胞密度为2x10 5个/ml，2ml/孔转入6孔板。
2.培养24h后，将模型细胞随机分为SZ-A组(50ug/ml，25ug/ml，12.5ug/ml实施例1的桑枝提取物SZ-A)、二甲双胍组(200umol/l MET)和棕榈酸刺激模型组(PA)，同时设HepG2细胞做为正常对照组(0.25％BSA)。除正常对照组(0.25％BSA)和模型组外(PA)，其它各组分别加入相应的药物及棕榈酸，复制棕榈酸刺激模型，各组设置3个复孔。
3.培养24h后，加入预热PBS洗一次后，6孔板每孔加入400ul含有1％PMSF Triton x-100裂解液。置于冰上或4℃裂解1h移液器轻轻吹打，使裂解液和细胞充分接触。裂解好的液体不离心，涡旋后，直接测定10ul甘油三酯和10ul总胆固醇。
试验结果见图6，肝脏的脂肪积累过多容易形成脂肪肝。用棕榈酸PA刺激HepG2肝脏细胞，可以模拟脂肪肝形成。加入不同剂量SZ-A，可以看到SZ-A抑制棕榈酸引起的肝脏甘油三酯和胆固醇的积累。SZ-A作用效果和MET相当。
参照以上方法，用实施例2-7中制备的桑提取物(剂量为25ug/ml)处理模型细胞， 分别对细胞中的甘油三酯和总胆固醇进行测定。结果显示，与PA组相比，经桑提取物处理后，棕榈酸引起的肝脏甘油三酯和胆固醇的积累均有一定程度的降低。具体结果见下表3。
表3由实施例1至7制备的桑提取物对甘油三酯和胆固醇水平的影响
Figure PCTCN2022080278-appb-000009
上述实施例表明，桑提取物具有良好的体重控制作用。长期给予桑提取物能够抑制小鼠和人体体重增长、抑制2型糖尿病小鼠和糖尿病患者的体重增长；而且对脏器脂肪积累具有抑制作用。
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。

Claims (7)

  1. 桑提取物在制备减轻动物体重的药物中的应用，其特征在于，所述桑提取物含有生物碱，多糖，氨基酸和黄酮，基于所述桑提取物，各组分重量含量为
    Figure PCTCN2022080278-appb-100001
    更优选地，基于所述桑提取物，各组分重量含量为
    Figure PCTCN2022080278-appb-100002
  2. 根据权利要求1所述的应用，其特征在于，所述动物为哺乳动物，包括人和啮齿类动物。
  3. 根据权利要求1或2所述的应用，其特征在于，所述动物为糖尿病动物。
  4. 根据权利要求1或2所述的应用，其特征在于，所述动物为健康动物。
  5. 根据权利要求1所述的应用，其特征在于，所述药物还包括药学上可接受的载体。
  6. 根据权利要求1所述的应用，其特征在于，所述药物为口服给药剂型；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。
  7. 根据权利要求3所述的应用，其特征在于，所述糖尿病动物的BMI≥28。
PCT/CN2022/080278 2021-03-12 2022-03-11 桑提取物在制备减轻动物体重的药物中的应用 WO2022188851A1 (zh)

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