WO2015139603A1 - 连钱草提取物、制备方法及其用于降糖减肥降脂的用途 - Google Patents

连钱草提取物、制备方法及其用于降糖减肥降脂的用途 Download PDF

Info

Publication number
WO2015139603A1
WO2015139603A1 PCT/CN2015/074366 CN2015074366W WO2015139603A1 WO 2015139603 A1 WO2015139603 A1 WO 2015139603A1 CN 2015074366 W CN2015074366 W CN 2015074366W WO 2015139603 A1 WO2015139603 A1 WO 2015139603A1
Authority
WO
WIPO (PCT)
Prior art keywords
extract
compound
chinensis
pharmaceutically acceptable
pharmaceutical composition
Prior art date
Application number
PCT/CN2015/074366
Other languages
English (en)
French (fr)
Inventor
朱维良
王贺瑶
张勇
孙鹏
李波
徐志建
蒋华良
陈凯先
Original Assignee
中国科学院上海药物研究所
南京派乐兴医药科技有限公司
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 中国科学院上海药物研究所, 南京派乐兴医药科技有限公司 filed Critical 中国科学院上海药物研究所
Priority to JP2017500116A priority Critical patent/JP6651498B2/ja
Priority to US15/126,676 priority patent/US10966996B2/en
Priority to EP15765481.5A priority patent/EP3120847B1/en
Priority to CN201580011098.3A priority patent/CN106061477B/zh
Priority to CN201911100870.5A priority patent/CN111195257B/zh
Publication of WO2015139603A1 publication Critical patent/WO2015139603A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention belongs to the field of medical technology.
  • the invention discloses an extract of Liancao grass for lowering blood sugar, lowering blood fat, reducing weight and treating kidney disease, a preparation method thereof, the use thereof or the treatment thereof for preparing blood sugar lowering, blood fat reducing, weight loss, treating kidney disease medicine A method of disease, and a composition comprising the extract.
  • the invention also discloses compounds I and II for lowering blood sugar, lowering blood fat, losing weight, treating kidney disease, a preparation method thereof, use thereof for preparing hypoglycemic, hypolipidemic, weight loss, medicine for treating kidney diseases or treating the same with the same A method of disease, and a composition comprising the compound.
  • Diabetes mellitus is a systemic metabolic disorder characterized by defects in insulin secretion and/or inability of insulin to exert normal physiological effects. Modern medical research has found a clear epidemiological link between hyperlipidemia and diabetes. Adjusting blood lipids in overweight people can prevent hyperglycemia. Losing weight and regulating blood lipids in diabetic patients is the key to reducing mortality and disability in diabetic patients.
  • Glechoma longituba (Nakai) Kurp. also known as Huoxue Dan and Tongguxiao
  • Huoxue Dan and Tongguxiao is a dry whole grass of the genus Rhubarb, which is widely distributed in the temperate zone of Eurasia and cultivated in North and South America. In China, except the northwest and Inner Mongolia, there are distributions throughout the country.
  • Qiancao was first published in the "Compendium of Materia Medica", which is bitter, spicy, and cool. It has the effects of diuresis, detoxification, dilatation and swelling.
  • Qiancao is mainly used for the treatment of urinary calculi, hepatobiliary stones, damp heat jaundice and puff injury.
  • the Chinese Pharmacopoeia (2010 edition) uses the Glechoma longituba (Nakai) Kupr. as a source of herbal medicine.
  • the object of the present invention is to provide an extract of Liancao grass for lowering blood sugar, lowering blood fat, reducing weight and treating kidney disease, a preparation method thereof, and the use thereof or the treatment thereof for preparing blood sugar lowering, blood fat reducing, weight loss, medicine for treating kidney disease A method of the disease, and a composition comprising the extract.
  • the invention also aims to provide compounds I and II for lowering blood sugar, lowering blood fat, reducing weight, treating kidney disease, a preparation method thereof, and the use thereof or the treatment thereof for preparing blood sugar lowering, blood fat reducing, weight loss, medicine for treating kidney diseases A method of the disease, and a composition comprising the compound.
  • the invention provides a extract of Liancao grass, comprising:
  • the weight of both Compound I and Compound II is from 1% to 75%, preferably from 1% to 25%, from 20% to 60%, or from 50% to 75% by weight based on the total weight of the extract.
  • the extract of the present invention contains both Compound I and Compound II in an amount of not less than 1%, 2%, 3%, 4%, 5%, 6%, 7% of the total weight of the extract. 8%, 9%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40 %, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 99%.
  • the extract of the invention has a compound I and a compound II which comprise no more than 99%, 95%, 90%, 85%, 80%, 79%, 78 of the total weight of the extract. %, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%.
  • the total weight of the extract of the compound I and the compound II of the present invention is in the range of any combination of the foregoing weight contents, for example, 1%-99%, 1%- 25%, 25%-50%, 50%-75%, 75%-99%, 25%-99%, 25%-75%, 50%-99%, 1%-75%, 1%-25% 20%-60%, 50%-75%, etc.
  • the scope of the invention includes or does not include endpoint values.
  • the invention also provides a extract of Liancao grass, comprising:
  • the Lithocarp extract is obtained by a method comprising the following steps:
  • the present invention optionally comprises one of the extracts of the stalk
  • the content of the compound I is more than 0.6%, preferably more than 25%, more preferably more than 39%, and the content of the compound II is more than 0.6%, preferably more than 25%, more preferably more than 32%.
  • the invention also provides:
  • the invention comprises a compound I or a pharmaceutically acceptable salt thereof, and/or a compound II or a pharmaceutically acceptable salt thereof, and/or a compound I and II or both
  • the mixture of acceptable salts has a compound I content of greater than 0.6%, preferably greater than 25%, more preferably greater than 39%, and a compound II content greater than 0.6%, preferably greater than 25%, more preferably greater than 32%.
  • the invention comprises Compound 1, or a pharmaceutically acceptable salt thereof, and/or Compound II, or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or both, are pharmaceutically acceptable
  • the content of the compound I in the mixture of salts or the content of the compound II The amount is not less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 21%, 22%, 23 respectively.
  • the present invention contains Compound I or a pharmaceutically acceptable salt thereof, and/or Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II or both.
  • the content of the compound I or the content of the compound II in the mixture of acceptable salts is not more than 99%, 95%, 90%, 85%, 80%, 79%, 78%, 77%, 76%, 75%, respectively. 74%, 73%, 72%, 71%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 15%, 10%, 9 %, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%.
  • the invention comprises a compound I or a pharmaceutically acceptable salt thereof, and/or a compound II or a pharmaceutically acceptable salt thereof, and/or a compound I and II or both
  • the content of the compound I or the content of the compound II in the mixture of acceptable salts is respectively a range of any combination of the foregoing weight contents, for example, 1% to 99%, 1% to 25%, 25% to 50%, 50% - 75%, 75%-99%, 25%-99%, 25%-75%, 50%-99%, 1%-75%, 1%-25%, 20%-60%, 50%-75% and many more.
  • the scope of the invention includes or does not include endpoint values.
  • the invention provides a pharmaceutical composition comprising: an optional one of the extracts of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising: Compound I according to the present invention, or a pharmaceutically acceptable salt thereof, and/or Compound II or a pharmaceutically acceptable salt thereof, and/or a compound Mixtures I and II or any pharmaceutically acceptable salt of either, and a pharmaceutically acceptable carrier.
  • the compound I and the compound II containing the optional extract of the sage extract of the present invention comprise more than 50% by weight of the total weight of the active ingredient in the pharmaceutical composition, or The drug composition of the extract is substantially removed After the compounds I and II, the biological activity of the pharmaceutical composition such as DPP4 inhibitory activity is reduced by more than 50%.
  • the pharmaceutical composition containing the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or both In a pharmaceutical composition of a mixture of acceptable salts, the compound I and the compound II comprise more than 50% by weight of the total active ingredient in the pharmaceutical composition, or after substantially removing the compounds I and II from the pharmaceutical composition comprising the mixture
  • the biological activity of the pharmaceutical composition such as DPP4 inhibitory activity is reduced by more than 50%.
  • the content of caffeic acid and rosmarinic acid in any of the extracts of the invention is less than 0.5%, preferably less than 0.1%.
  • the invention comprises a compound I or a pharmaceutically acceptable salt thereof, and/or a compound II or a pharmaceutically acceptable salt thereof, and/or a compound I and II or both
  • the content of caffeic acid and rosmarinic acid in the mixture of acceptable salts is less than 0.5%, preferably less than 0.1%.
  • the present invention discloses an optional one of the extracts of C. chinensis having the effects of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease.
  • the invention comprises a compound I or a pharmaceutically acceptable salt thereof, and/or a compound II or a pharmaceutically acceptable salt thereof, and/or a compound I and II or both
  • a mixture of acceptable salts has the activity of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease.
  • the present invention provides an extract for lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease.
  • the present invention provides a compound I or a pharmaceutically acceptable salt thereof, and/or a compound II or a pharmaceutically acceptable salt thereof, for use in lowering blood sugar, lowering blood fat, reducing weight, treating kidney disease, and / or a mixture of compounds I and II or either pharmaceutically acceptable salt.
  • the present invention provides a method for lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing an optional one of the extracts of the present invention to a patient in need thereof.
  • the present invention also provides a method of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof And/or a mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the present invention provides the use of any of the extracts of the scutellariae in the preparation of a medicament for lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof.
  • the invention also provides a method for extracting Liancao grass, comprising the steps as follows:
  • the aqueous solution of step a) of the method of extracting Liancao in the present invention has a water content of greater than 40%, preferably greater than 80%, most preferably water.
  • the extraction of step A) in the extraction method of the sylvestris of the present invention is heated reflux extraction or ultrasonic extraction, preferably 2-5 times.
  • the aqueous extract of C. chinensis obtained in step a) of the method for extracting Liancao in the present invention may be optionally extracted with an organic solvent and the organic phase is discarded, and the treated Liancao grass is treated.
  • the aqueous extract is reserved for subsequent use, wherein the organic solvent is preferably ethyl acetate or dichloromethane.
  • the aqueous extract of C. chinensis obtained in step a) of the method for extracting Liancao in the present invention may be further refrigerated after optionally concentrating, wherein the concentration is preferably decompressed. Concentrated, the refrigerated is preferably chilled at 4-6 °C.
  • the alcoholic solution in step b) of the method of extracting stalks of the present invention is preferably an ethanol-water mixed system, more preferably 90% ethanol, most preferably 95% ethanol.
  • the volume of the alcoholic solution in step b) of the method of extracting the sylvestris of the present invention is 2-4 times the volume of the optionally concentrated aqueous extract of C. chinensis.
  • the precipitate obtained by separation in step c) of the method for extracting stalks of the present invention may also optionally be lyophilized.
  • the precipitate obtained by the separation in the step c) of the method for extracting the sylvestris of the present invention may be optionally purified by macroporous adsorption resin, and the purification by using the macroporous adsorption resin is as follows Implementation of the steps:
  • the aqueous solution in step i) of the invention has a water content of greater than 40%, preferably greater than 80%, most preferably water.
  • the macroporous resin in step ii) of the present invention is D-101, D-101-I, DA-201, DM-301, DM-130, AB-8, HPD-100, HPD. -300, HPD-400, HPD-600, HPD-826 or similar filler of these resins, preferably D-101.
  • the aqueous eluate of step iii) of the invention has a water content of greater than 90%, preferably greater than 95%, most preferably water.
  • the alcoholic eluate of step iv) of the invention is an ethanol-water mixed system, preferably 5-20% ethanol, most preferably 10-15% ethanol.
  • the present invention provides a preferred preparation process of the compound I, the compound II monomer, characterized in that after the extract of the L. chinensis obtained by the steps i)-iv) of the present invention, a polyamide, a silica gel, a gel or a reverse phase filler can also be used.
  • a polyamide, a silica gel, a gel or a reverse phase filler can also be used.
  • the reverse phase packing is rapidly separated and purified by column chromatography, and the purity of the monomer compound can reach over 96%.
  • the pharmaceutical composition of the present invention is a tablet, a hard capsule, a soft capsule, an enteric capsule, a microcapsule, a granule, a syrup, an injection, a granule, an emulsion, a suspension, a solution, and is used for oral or parenteral administration.
  • the form of a sustained release preparation of the drug is a tablet, a hard capsule, a soft capsule, an enteric capsule, a microcapsule, a granule, a syrup, an injection, a granule, an emulsion, a suspension, a solution, and is used for oral or parenteral administration.
  • the form of a sustained release preparation of the drug is a tablet, a hard capsule, a soft capsule, an enteric capsule, a microcapsule, a granule, a syrup, an injection, a granule, an emulsion, a suspension, a solution, and is used for oral or parenteral administration.
  • the pharmaceutically acceptable carrier of the present invention refers to a pharmaceutically acceptable carrier well known to those skilled in the art, and the pharmaceutically acceptable carrier of the present invention includes, but is not limited to, a filler, a wetting agent, a binder, a disintegrating agent. , lubricants, binders, glidants, taste masking agents, surfactants, preservatives, etc. Fillers include, but are not limited to, lactose, microcrystalline cellulose, Starch, powdered sugar, dextrin, mannitol, calcium sulfate, and the like.
  • wetting agents and binders include, but are not limited to, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, sucrose, polyvinylpyrrolidone, and the like.
  • Disintegrators include, but are not limited to, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, croscarmellose sodium, low substituted hydroxypropylcellulose, and the like.
  • Lubricants include, but are not limited to, magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, and the like.
  • Binders include, but are not limited to, gum arabic, alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose, glucose binders, dextrin, dextrose, ethylcellulose, gelatin, liquid glucose, guar Glue, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, magnesium aluminum silicate, maltodextrin, methyl cellulose, polymethacrylate, polyvinylpyrrolidone, pregelatinized starch , sodium alginate, sorbitol, starch, syrup and tragacanth.
  • Glidants include, but are not limited to, colloidal silica, powdered cellulose, magnesium trisilicate, silica, and talc.
  • Taste masking agents include, but are not limited to, aspartame, stevioside, fructose, glucose, syrup, honey, xylitol, mannitol, lactose, sorbitol, maltitol, glycyrrhizin.
  • Surfactants include, but are not limited to, Tween-80, poloxamer.
  • Preservatives include, but are not limited to, paraben, sodium benzoate, potassium sorbate, and the like.
  • the pharmaceutically acceptable salts of the present invention include, but are not limited to, salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., with formic acid, acetic acid, propionic acid, oxalic acid, malonic acid.
  • Acidic acid such as succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, etc.
  • acidic amino acids such as aspartic acid and glutamic acid.
  • a salt, or a salt formed with a base such as a salt of an inorganic base such as sodium or potassium, or a basic addition salt with a basic amino acid such as lysine, arginine or ornithine.
  • the blood-sweetening plants also include G. hederacea Linn., G. biondiana (Diels) CYWu et C. Chen, and G. sinograndis CYWu. The above varieties have similar effects as Lian Qiancao.
  • the dipeptidyl peptidase IV (DPP4) of the present invention is a transmembrane serine protease belonging to the family of prolyl oligopeptidases.
  • DPP4 is a new target for the treatment of type 2 diabetes; it is one of the enzymes that mainly promote the degradation and inactivation of glucagon-like peptide-1 (GLP-1) in vivo and in vitro.
  • GLP-1 glucagon-like peptide-1
  • It has been medically proven that DPP4 inhibitor is a new type of anti-diabetic drug. The clinical results show that the drugs have good hypoglycemic effects, and no adverse reactions such as common weight gain and hypoglycemia caused by insulin and sulfonylurea diabetes drugs have been found. Therefore, research on DPP4 inhibitors has become an antidiabetic drug. Research hotspots.
  • the present inventors have found that Compound I and Compound II can effectively inhibit DPP4 activity, and the inhibition rates of DPP4 at a concentration of 100 ⁇ M are 40.8% and 34.1%, respectively, thereby having a hypoglycemic effect.
  • the present invention provides an extract for lowering blood glucose.
  • the invention further provides Compound I or a pharmaceutically acceptable salt thereof, and/or Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II or II, for lowering blood glucose Any mixture of pharmaceutically acceptable salts.
  • the present invention provides a method of lowering blood glucose, the method comprising: providing an optional one of the extracts of the present invention to a patient in need thereof.
  • the present invention also provides a method of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof And/or a mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the invention provides the use of any of the extracts of the sylvestris in the preparation of a hypoglycemic medicament.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof Use in the preparation of a hypoglycemic drug.
  • the present invention also provides the use of any one of the extracts of Liriomyza sinensis in the preparation of a medicament for inhibiting DPP4 activity.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof Use in the preparation of a medicament for inhibiting DPP4 activity.
  • the inhibition of dipeptidyl peptidase IV activity of the invention refers to the treatment of diabetes disease.
  • Glucose oxidase is an oxidase supplemented by flavin adenine mononucleotide (FMN), which catalyzes the oxidation of glycolic acid to oxalic acid.
  • FMN flavin adenine mononucleotide
  • Overexpression of GOX promotes the production of oxalate, which increases the risk of kidney stones and nephritis. Therefore, the search for inhibitors of gluconate oxidase has become one of the important methods and ways to develop kidney stones and nephritis.
  • Compound I and Compound II have certain inhibitory activities against GOX, Compound I has an IC 50 value of 0.5 mM, and Compound II has an IC 50 value of 0.4 mM, and has potential pharmacological activity for treating kidney stones and nephritis. .
  • the present invention provides an extract for treating kidney disease.
  • the invention further provides Compound I or a pharmaceutically acceptable salt thereof, and/or Compound II, or a pharmaceutically acceptable salt thereof, and/or Compounds I and II or II, for use in the treatment of kidney disease Any mixture of pharmaceutically acceptable salts.
  • the invention provides a method of treating kidney disease, the method comprising: providing an optional one of the extracts of the invention of the invention to a patient in need thereof.
  • the present invention also provides a method of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof And/or a mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the present invention provides the use of any of the extracts of the sylvestris chinensis in the preparation of a medicament for the treatment of kidney disease.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof Use in the preparation of a medicament for treating kidney disease.
  • Triglycerides are the predominant form of energy storage in eukaryotes. In mammals, triglycerides are mainly synthesized in the small intestine, liver and fat cells. Triglyceride metabolism - the absorption or de novo synthesis of both disorders or disorders - is associated with the pathogenesis of a variety of diseases. For example, obesity, hyperlipidemia, and the like.
  • Pancreatic lipase is secreted by pancreatic acinar cells, enters the duodenum through the pancreatic duct, and hydrolyzes triglycerides into absorbable 2-monoacylglycerols and free fatty acids at the oil-water interface at the upper end of the duodenum and small intestine.
  • Pancreatic fat The enzyme is responsible for hydrolyzing 50% to 70% of dietary fat. Inhibition of pancreatic lipase activity can inhibit the digestion and absorption of intestinal triacylglycerol, lower blood triglyceride levels and reduce tissue fat accumulation, thereby reducing blood fat and reducing weight.
  • Diacylglycerol acyltransferase 1 (DGAT1, Diacylglycerol acyltransferase 1) catalyzes the final step of triacylglycerol synthesis: the transfer of the acyl-CoA acyl group to 2-monoacylglycerol to form triacylglycerol is the rate-limiting enzyme for the synthesis of triacylglycerol.
  • the synthesis of triglycerides from diacylglycerols can be reduced by inhibiting or reducing DGAT1 enzyme activity.
  • DGAT1 enzyme inhibitors can be used to treat diseases associated with abnormal metabolism of triglycerides, such as hypolipidemic and weight loss.
  • Compound I and Compound II have an effect of inhibiting the absorption of fat in food, thereby having the effects of lowering blood fat and losing weight.
  • the present inventors have found that the compounds I and II can effectively inhibit the activity of DGAT1 and pancreatic lipase, thereby exerting the effects of lowering blood fat and losing weight.
  • Compounds I and II inhibited DGAT1 by more than 50% at a concentration of 50 ⁇ M; at concentrations of 10 ⁇ M, compounds I and II still exhibited about 20% inhibition of DGAT1.
  • Compounds I and II also inhibited pancreatic lipase by more than 40% at the same concentration.
  • the present invention provides an extract for lowering blood fat.
  • the invention further provides Compound I or a pharmaceutically acceptable salt thereof, and/or Compound II, or a pharmaceutically acceptable salt thereof, and/or Compounds I and II or II, for lowering blood fat Any mixture of pharmaceutically acceptable salts.
  • the present invention provides a method of lowering blood fat, the method comprising: providing an optional one of the extracts of the present invention to a patient in need thereof.
  • the present invention also provides a method of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof And/or a mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the present invention provides the use of any of the extracts of the sylvestris in the preparation of a hypolipidemic drug.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof Use in the preparation of a drug for lowering blood fat.
  • the present invention provides an extract for weight loss.
  • the invention also provides a compound I or a pharmaceutically acceptable salt thereof, and/or a compound for weight loss A mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the present invention provides a method of losing weight, the method comprising: providing an optional one of the extracts of the present invention to a patient in need thereof.
  • the present invention also provides a method of lowering blood sugar, lowering blood fat, losing weight, and treating kidney disease, the method comprising: providing a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof And/or a mixture of Compound II or a pharmaceutically acceptable salt thereof, and/or Compounds I and II, or any pharmaceutically acceptable salt thereof.
  • the present invention provides the use of any of the extracts of the sylvestris in the preparation of a slimming drug.
  • the present invention also provides a mixture comprising the compound I or a pharmaceutically acceptable salt thereof, and/or the compound II or a pharmaceutically acceptable salt thereof, and/or the compound I and II or a pharmaceutically acceptable salt thereof Use in the preparation of drugs for weight loss.
  • the diabetes of the present invention includes type 1 diabetes, type 2 diabetes, other special types of diabetes, and gestational diabetes.
  • the hypolipidemic means of the present invention means lowering the level of triglycerides in the blood.
  • the treatment of kidney disease according to the present invention refers to the treatment of kidney stones, treatment of nephritis and the like.
  • Figure 1 shows the results of HPLC analysis of the main components of the extract of C. chinensis obtained by different preparation processes.
  • Figure 2 shows the results of HPLC analysis of the main components in the extraction process of Liancao extract and the literature.
  • Fig. 3 shows the preferred preparation process and HPLC analysis results of the compounds I and II in the extract of L. chinensis.
  • Figure 4 shows that LQC-H-1 extract of L. chinensis L. at 250 mg/kg and 500 mg/kg can significantly enhance acute oral glucose tolerance in mice and increase blood insulin levels in mice after oral glucose.
  • Figure 5 shows that L. chinensis extract LQC-H-2 can significantly enhance acute oral glucose tolerance in mice at 250 mg/kg and 500 mg/kg.
  • Figure 5 (B) is the area under the oral glucose tolerance curve corrected according to the calibration group. (AUC), *p ⁇ 0.05, **p ⁇ 0.01 compared with the control group.
  • Figure 6 shows that the crude polysaccharides in the ethanol extract and the alcohol precipitation solids did not enhance the acute oral glucose tolerance in mice.
  • Figure 6 (A) crude polysaccharide and Lishui grass water extract alcohol supernatant components failed to effectively enhance oral glucose tolerance in mice, the abscissa is the time after glucose administration, the ordinate is blood glucose concentration; (B) According to the area under the oral glucose tolerance curve corrected by the calibration group, there was no significant difference between the administration group and the control group.
  • Figure 7 shows that long-term administration of L. chinensis extract LQC-H-1 improves diabetic symptoms in spontaneous type 2 diabetic db/db mice.
  • the abscissa was the administration time, the ordinate was blood glucose, and the 250 mg/kg dose group was compared with the control group, #p ⁇ 0.05, 500 mg/kg dose group compared with the control group, *p ⁇ 0.05;
  • Figure 8 shows that the main active monomer compounds I and II in the extract of L. chinensis as effective DPP4 inhibitors derived from natural products can significantly enhance acute oral glucose tolerance in mice.
  • Figure 8 (C) is the area under the oral glucose tolerance curve (AUC) corrected by the calibration group, and the control group Compared with *p ⁇ 0.05, **p ⁇ 0.01, n.s. indicates that there was no significant difference between the compound 200 mg/kg and the sitagliptin 10 mg/kg dose group.
  • Figure 9 shows that the main active monomer compound I in the extract of C. chinensis can significantly enhance acute oral glucose tolerance in mice at a dose of 100 mg/kg.
  • Figure 10 shows that long-term administration of Compound I is effective in reducing blood glucose in db/db mice.
  • Figure 10 (A) Compound I decreased the blood glucose of mice after 2 weeks of administration, the effect is close to the positive drug sitagliptin;
  • Figure 10 (B) Compound I decreased the fasting blood glucose after 2 weeks of administration, the effect is The positive drug, sitagliptin, was close to *p ⁇ 0.05, **p ⁇ 0.01 compared with the control group.
  • FIG 11 shows that L. chinensis extract LQC-H-1 can reduce serum triglyceride levels in mice with acute hypertriglyceridemia.
  • LqC-H-1 can reduce serum triglyceride levels in mice with acute hypertriglyceridemia.
  • the concentration of LCQ90810mg/kg is positive control.
  • the model group is given 2g/kg olive oil for modeling. The group did not give olive oil modeling. **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 12 shows that the literature reported that Liancao 65% ethanol extract (LQC-65% ethanol extract) had no significant effect on acute glucose tolerance in mice.
  • Figure 12 (B) according to the calibration group corrected oral sugar tolerance curve area, compared with the control group Ratio, **p ⁇ 0.01, ns no significant difference.
  • Reagents 95% ethanol, distilled water, ethyl acetate, dichloromethane, methanol and other reagents are analytically pure (Zhongguo Group Chemical Reagent Co., Ltd.); D101 macroporous adsorption resin (Zhongguo Group Chemical Reagent Co., Ltd.), column chromatography silica gel (H series, Qingdao Ocean Silicone Desiccant plant), thin layer chromatography silica gel GF254 (Qingdao Ocean Silica Gel Desiccant Factory), MCI chromatography packing material (Japan, GEL CHP20P, 75-100 ⁇ ), ODS chromatography packing material (Japan, YMC*GEL, s 50 ⁇ m), Sephadex LH-20 chromatography packing (GE Healthcare, Sweden).
  • Rotary evaporator EYELA Rotary evaporator N1001, Tokyo physics and chemistry.
  • Freeze dryer Christ, ALPHA 1-2LD PLUS, Germany.
  • Black box UV transilluminator WFH-203B, Shanghai Jingke Industrial Co., Ltd.
  • HPLC High Performance Liquid Chromatography
  • This embodiment provides three methods for preparing the extract of C. chinensis.
  • the supernatant was concentrated under reduced pressure at 50 ° C using a rotary evaporator to obtain a supernatant sample 2.
  • the alcohol precipitation solid was removed in a vacuum drying oven at 50 ° C, then frozen in a -70 ° C refrigerator for 12 h, and freeze-dried for 48 h using a freeze drying equipment (Christ freeze dryer) to obtain a dry solid of the extract of Liquorice extract. Powder 19.2 g (LQC-H-2).
  • Example 2 Isolation and Identification of Principal Components in Alcohol Sedimentation Supernatant in Preparation Process of Liancao Grass Extract
  • Example 3 Analysis and detection of main components in Lienian extract by high performance liquid chromatography
  • Detection wavelength 190-400 nm, preferably 360 nm for content determination
  • the above extract (compounds I and II accounted for 26.55% of the total extract) can be separated again by using D101 macroporous adsorption resin 300g, and the residual protein and polysaccharide components are eluted with 400 ml of distilled water, and then eluted with 800 ml of distilled water to continue elution. Deliquoring.
  • the eluate was concentrated under reduced pressure at 50-55 ° C using a rotary evaporator to obtain an extract rich in Compound I and Compound II.
  • the contents of Compounds I and II were 29.70% and 26.91%, respectively, and the sum of the two accounts for 56.61% of the weight of the extract (see Figure 3).
  • the above extract (Compound I and II accounted for 56.61% of the total extract) can continue to be separated and purified by using 200 g of D101 macroporous adsorption resin, and the pigment is eluted with 200 ml of distilled water, and then eluted with 500 ml of distilled water to continue elution. Deliquoring.
  • the eluate was concentrated under reduced pressure at 50-55 ° C using a rotary evaporator to obtain an extract rich in Compound I and Compound II.
  • the contents of Compounds I and II reached 39.75% and 32.77%, respectively, and the sum of the two accounts for 72.52% of the weight of the extract (see Figure 3).
  • the above three kinds of extracts containing different ratios of compounds I and II obtained by macroporous resin can be dissolved in a small amount of distilled water, and rapidly purified by YMC*GEL ODS filler, respectively, using water and 10% methanol gradient elution.
  • the collected fractions were tested by HPLC. After merging the same components and concentrating under reduced pressure at 50 ° C under a rotary evaporator, a total of 296 mg of monomer compound I (purity of 98.65%, see Figure 3) and 258 mg of compound II (purity greater than 96.52%, see Figure 3) were obtained. .
  • Example 5 LQC-H-1 extract of L. chinensis significantly enhanced acute oral glucose tolerance in mice and increased blood insulin levels after oral glucose in mice
  • Oral glucose tolerance test (OGTT) method 8 weeks old male C57BL/KsJ mice (Shanghai Slack Laboratory Animals), 8 rats in each group, weighing 20 ⁇ 2g, kept according to SPF procedures. Fasted for 16 hours before. Before the start of the experiment, blood was collected from the tail vein of each group, and the blood glucose concentration was measured by Roche ACCU-CHEK blood glucose meter, which was recorded as 0 time point. The mice in each administration group were intragastrically administered with different concentrations of compounds, and the control mice were given an equal amount of distilled water at a dose of 10 ml/kg.
  • mice Half an hour after the administration, each group of mice was intragastrically administered with 5 g/kg of glucose, and blood glucose concentrations of the mice in each group were measured and measured after 20 minutes, 40 minutes, and 80 minutes of gavage.
  • the OGTT curve was recorded and corrected for the blood glucose change (calibration group) of mice not given glucose according to the OGTT curve.
  • Area under curve (AUC), one-way analysis of variance (ONE-WAY-ANOVA) was used to compare significant differences between groups (Note: The acute oral glucose tolerance test in all the following active examples except for special instructions, experimental methods Both are the same as the experimental method in Example 5).
  • ELISA enzyme-linked immunosorbent assay
  • Animal grouping control group; calibration group; LQC-H-1 low dose group (250 mg/kg); LQC-H-1 high dose group (500 mg/kg).
  • LQC-H-1 extract of L. chinensis L. at 250 mg/kg and 500 mg/kg significantly enhanced acute oral glucose tolerance in mice and increased blood insulin levels after oral glucose in mice (Fig. 4). This effect is concentration dependent, suggesting that the extract has the effect of promoting islet ⁇ -cell secretion of insulin and improving postprandial blood glucose.
  • Example 6 L. chinensis extract LQC-H-2 can also significantly enhance acute oral glucose tolerance in mice
  • the experimental method was the same as in Example 5, animal grouping: control group; calibration group; LQC-H-2 low dose group (250 mg/kg); LQC-H-2 high dose group (500 mg/kg).
  • LQC-H-2 extract of L. chinensis could significantly enhance acute oral glucose tolerance in mice at 250 mg/kg and 500 mg/kg (Fig. 5), demonstrating that the active part of L. chinensis obtained by the optimized extraction method remains It has the effect of improving postprandial blood sugar.
  • Example 7 The crude polysaccharides from the ethanol extract and the alcohol precipitation solids did not enhance the acute oral glucose tolerance in mice.
  • the experimental method was the same as in Example 5, animal grouping: control group; crude polysaccharide group (1 g/kg); supernatant group sample after alcohol precipitation (300 mg/kg).
  • Example 8 Long-term administration of L. chinensis extract LQC-H-1 improves diabetic symptoms in spontaneous type 2 diabetes db/db mice
  • mice 8 weeks old male db/db spontaneous obese type 2 diabetic mice (background C57BL/KsJ, Jackson University, USA), 8 rats in each group, initial body weight 30 ⁇ 2g, kept according to SPF operating procedures, before the experiment
  • the control group was given the same amount of distilled water; the LQC-H-1 was divided into the low-dose group (250 mg/kg), the high-dose group (500 mg/kg) was administered orally, and the positive drug used the insulin sensitizer.
  • Glycoside (10 mg/kg) was administered at the same time, and the same strain of normal blood glucose mice (db/m) was compared as a blank.
  • the administration volume was 10 ml/kg once a day for 4 weeks, and the body weight, blood sugar, and dietary changes of the mice were monitored weekly. After 4 weeks, OGTT of each group of mice was measured according to the method of Example 1 except that the drug was not acutely administered, and AUC was calculated. Serum was measured for serum insulin content, and the improvement of diabetic symptoms in spontaneous type 2 diabetic db/db mice was evaluated by long-term administration of LQC-H-1.
  • LQC-H-1 had no significant effect on the diet and body weight of mice after long-term administration in this experiment, while rosiglitazone caused weight gain in mice. It can be seen that long-term use of rosiglitazone causes side effects such as dyslipidemia. It can be concluded that the LQC-H-1 extract can not only achieve the hypoglycemic and lipid-lowering effects, but also the safety is higher than the positive drug rosiglitazone.
  • Example 9 Main active monomer compounds I and II in the extract of L. chinensis as an effective DPP4 inhibitor derived from natural products, the effect can be significantly increased alone. Acute oral glucose tolerance in strong mice
  • Inhibition rate% (RFU blank-RFU compound)/(RFU blank-RFU negative control)*100%
  • RFU compounds, RFU blanks, RFU negative controls indicate the difference between the fluorescence values of compound wells, blank wells and non-enzymatic negative control wells for 30 min and 0 min, respectively;
  • the effects of Compound I and Compound II on acute glucose tolerance in mice The OGTT experimental method was the same as that in Example 5. The mice were divided into: control group; calibration group; compound I low dose group (100 mg/kg); compound I high dose group (200 mg/kg); compound II low dose group (100 mg/kg). High dose group (200 mg/kg); positive drug group (Sitagliptin 10 mg/kg, sitagliptin, Merck, USA).
  • Example 10 The main active monomer compound I in the extract of L. chinensis improved glycemic control in spontaneous type 2 diabetic db/db mice.
  • mice 8 weeks old male db/db spontaneous obese type 2 diabetic mice (C57BL/KsJ, Jackson Laboratory, USA), 8 rats in each group, initial body weight 30 ⁇ 2g, reared according to SPF procedures, and tested for acute glucose tolerance in db/db mice according to the method of Example 1. Effects, animal grouping: control group; Compound I group (100 mg/kg); positive drug sitagliptin group (10 mg/kg).
  • mice were subjected to long-term administration experiments according to the following groups: control mice were given an equal amount of distilled water; Compound I dose was 50 mg/kg, and positive control group was treated with DPP4 inhibitor sitagliptin 10 mg. /kg administered by intragastric administration.
  • the administration volume was 10 ml/kg, once a day for 2 weeks, and the changes in body weight, blood glucose, and diet of the mice were monitored, and the improvement of the symptoms of diabetes in spontaneous type 2 diabetic db/db mice was evaluated by long-term administration of Compound I.
  • mice 6-week-old male C57BL/KsJ mice (Shanghai Slack Laboratory Animals, 8 rats in each group, weighing 20 ⁇ 2 g, were housed according to SPF procedures and fasted for 16 hours overnight before the experiment.
  • the mice in each administration group were intragastrically administered with different concentrations of compounds, and the control group and the blank group mice were given equal volume of water. After 30 minutes, the mice in the drug-administered group and the control group were given 2 g/kg of olive oil by gavage, and the blank group was small.
  • the rats gave an equal volume of water.
  • DGAT Inhibitory activity of enzyme
  • human DGAT1 obtained by expression purification of insect cells is pre-incubated with compound, and in the presence of coenzyme A, substrate diacylglycerol is added to initiate the reaction, and the formed thiol is combined with CPM.
  • the resulting fluorescent signal is calculated according to the following formula for the inhibitory activity of the compound on DGAT:
  • Inhibition rate % (OD blank - OD compound) / (OD blank - OD negative control) * 100%
  • the OD compound, the OD blank, and the OD negative control respectively indicate the fluorescence values of the compound well, the blank well, and the negative control well without the DGAT1 enzyme, and the concentration of the compound was determined as the IC 50 value when the inhibition rate (%) of the enzyme activity reached 50%.
  • pancreatic lipase substrate p-Nitrophenyl acetate (Sigma, USA) with phosphate buffer (25 mM, pH 6.8) Formulated to 1.35 mM; porcine pancreatic lipase (sigma) was formulated into 10 mg/ml with phosphate buffer; brominated enacetylated compounds were formulated in phosphate buffer to prepare solutions of different concentrations. Then, 50 ⁇ l of a 1.35 mM p-Nitrophenyl acetate (Sigma, USA) solution and 10 ⁇ l of different concentrations of the test compound were sequentially added to a 96-well plate, and previously incubated at 37 ° C for 5 minutes.
  • pancreatic lipase (EC 3.1.1.3, Sigma, USA) at a concentration of 10 mg/ml was added and mixed, and the reaction was continued at 37 ° C for 20 minutes, and the absorbance of each well was measured at 492 nm.
  • the inhibition rate (%) of the test sample to pancreatic lipase was calculated from the absorbance at 492 nm, and the concentration of the compound when the enzyme activity inhibition rate (%) reached 50% was determined as the IC50 value.
  • the inhibition rate (%) can be made according to the following formula:
  • Inhibition rate (%) [(A-B) - (C-D)] / (A-B) ⁇ 100
  • A, B, C, and D represent the absorbance of the blank well after the reaction, the absorbance of the blank well before the reaction, the absorbance of the sample well after the reaction, and the absorbance of the sample well before the reaction.
  • LQC-H-1 extract of Lianhuacao can reduce serum triglyceride levels in mice with acute hypertriglyceridemia at a dose of 500 mg/kg, reducing the effect Close to the DGAT inhibitor positive drug LCQ908 (Fig. 11);
  • Compounds I and II have significant DGAT inhibitory activity (see Table 2).
  • the results suggest that L. chinensis extract LQC-H-1 has a significant acute reduction of serum triglyceride, and its components may be DGAT1 and pancreatic lipase inhibitory activity (see Table 3). The main active ingredient.
  • Example 12 Inhibitory activity of compounds I and II on GOX
  • GOX is an oxidase supplemented by flavin adenine mononucleotide (FMN), which catalyzes the oxidation of glycolic acid to oxalic acid.
  • FMN flavin adenine mononucleotide
  • Overexpression of GOX promotes the production of oxalate, which increases the risk of kidney stones and nephritis.
  • Enzyme coupling method is to determine the rate of gluconic acid oxidase catalytic reaction by using peroxidase (POD) and its substrate to react with H2O2 to form colored product.
  • POD peroxidase
  • POD catalyzes the reaction of H2O2 with phenol and 4-aminoantipyrine to form brown red ⁇ Class of substances.
  • the measurement result calculating IC 50 values were fitted with Graphpad Prism 5.0, the software fitting results show: Compound I IC 50 of 0.5mM, Compound II IC 50 of 0.4mM, quercetin IC 50 of 21 ⁇ M. It is proved that Compound I and Compound II have certain inhibitory activity against gluconate oxidase, and have potential pharmacological activities for treating kidney stones and nephritis.
  • Example 13 Comparison of the activity of the water extraction and alcohol precipitation sample LQC-H-2 of the present invention with the prior art 65% ethanol ultrasonic extract (Yuan Chunling et al., Chinese Pharmacology and Clinical Medicine, 2008, 24(3), 57-58)
  • the experimental method was the same as in Example 5, animal grouping: control group; LQC-H-2 group (500 mg/kg); LQC-65% ethanol extract group (500 mg/kg);

Abstract

公开了一种连钱草提取物，特别是化合物Ⅰ和Ⅱ，及其制备方法和在制备降血糖、降血脂、减肥、治疗肾病药物中的用途或治疗上述疾病的方法，以及含有所述提取物的组合物。

Description

连钱草提取物、制备方法及其用于降糖减肥降脂的用途 技术领域
本发明属于医药技术领域。本发明公开了用于降血糖、降血脂、减肥、治疗肾病的连钱草提取物、其制备方法、其在制备降血糖、降血脂、减肥、治疗肾病药物中的用途或用其治疗所述疾病的方法，以及含有所述提取物的组合物。本发明还公开了用于降血糖、降血脂、减肥、治疗肾病的化合物I和II、其制备方法、其在制备降血糖、降血脂、减肥、治疗肾病药物中的用途或用其治疗所述疾病的方法，以及含有所述化合物的组合物。
背景技术
糖尿病(Diabetes mellitus，DM)是以胰岛素分泌缺陷和(或)胰岛素不能发挥正常生理作用所致高血糖为特征的全身性代谢紊乱疾病。现代医学研究发现，高血脂和糖尿病之间有明确的流行病学联系。对超重人群调节血脂可以预防高血糖。对糖尿病患者减轻体重和调节血脂，是减少糖尿病患者死亡率和致残率的关键。
连钱草(Glechoma longituba(Nakai)Kupr.)又名活血丹、透骨消，为唇形科活血丹属植物的干燥全草，广泛分布于欧亚大陆温带地区，南北美洲也有栽培。在我国，除西北、内蒙古以外，全国各地均有分布。连钱草始载于《本草纲目拾遗》，性味苦、辛、性凉，归肝、肾、膀胱经，具有利湿通淋、清热解毒、散瘀消肿的功效。连钱草在临床上主要用于治疗尿路结石，肝胆结石，湿热黄疸和跌扑损伤。中国药典(2010版)则将唇形科植物活血丹Glechoma longituba(Nakai)Kupr.作为连钱草药材的来源。
文献报道，连钱草的65％乙醇提取物中的总黄酮能降低链脲佐菌素诱导的糖尿病小鼠的血糖水平，并认为其降糖机制是抑制链脲佐菌素诱导的胰岛β-细胞损伤，从而增加了链脲佐菌素诱导的糖尿病 小鼠的β-细胞数量(袁春玲等，中药药理与临床，2008，24(3)，57-58)。杨念云等(中国药科大学学报，2005，36(3)，210-212)从连钱草的80％乙醇提取物中通过萃取和柱层析的方法得到10个连钱草黄酮类化合物；此后杨念云等(中国天然药物，2006，4(2)，98-100)从连钱草80％乙醇回流提取液正丁醇萃取部位中再次分离得到10个化合物，其中化合物II的得率仅占百万分之二左右。袁春玲等和杨念云等均没有报道连钱草提取物中具体的黄酮化合物的抗糖尿病活性。同时，袁春玲等和杨念云等报道的总黄酮成分中也不包含本发明所述的木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸](化合物I)。
日本学者曾对日本活血丹(Glechoma hederacea subsp.grandis)水煎液进行了动物体内急性降糖研究(Shinji I.，et al.Nippon Shokuhin Kagaku Kogaku Kaishi，2007，54(9)：412-414)。该文献仅推测其所得日本活血丹的提取液中多糖类化学成分为降糖主要活性组分。但《中国药典》(2010年版)未将日本活血丹作为中药连钱草的来源，且《中国植物志》(中国科学院中国植物志编辑委员会，中国植物志(第65卷第2分册)，北京：科学出版社，1977)中明确将日本活血丹Glechoma hederacea subsp.grandis)作为活血丹属单独的一个品种列出，表明日本活血丹与连钱草(Glechoma longituba(Nakai)Kupr.)不属于同一个品种。
发明内容
本发明的目的在于提供用于降血糖、降血脂、减肥、治疗肾病的连钱草提取物、其制备方法、其在制备降血糖、降血脂、减肥、治疗肾病药物中的用途或用其治疗所述疾病的方法，以及含有所述提取物的组合物。本发明的目的还在于提供用于降血糖、降血脂、减肥、治疗肾病的化合物I和II、其制备方法、其在制备降血糖、降血脂、减肥、治疗肾病药物中的用途或用其治疗所述疾病的方法，以及含有所述化合物的组合物。
本发明提供连钱草提取物，包含：
化合物I：木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000001
和
化合物II：芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000002
并且化合物I和化合物II二者重量占提取物总重量1％-75％，优选1％-25％、20％-60％、或者50％-75％。
在一个优选的方面，本发明连钱草提取物包含的化合物I和化合物II二者占提取物总重量不低于1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、35％、40％、45％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、65％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、85％、90％、95％、99％。在还有一个优选的方面，本发明连钱草提取物包含的化合物I和化合物II二者占提取物总重量不超过99％、95％、90％、85％、80％、79％、78％、77％、76％、75％、74％、73％、72％、71％、70％、65％、60％、59％、58％、57％、56％、55％、54％、53％、52％、51％、50％、45％、40％、35％、30％、29％、28％、27％、26％、25％、24％、 23％、22％、21％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％。在还有另一个优选方面，本发明连钱草提取物包含的化合物I和化合物II二者占提取物的总重量是前述重量含量任意组合的范围，例如，1％-99％、1％-25％、25％-50％、50％-75％、75％-99％、25％-99％、25％-75％、50％-99％、1％-75％、1％-25％、20％-60％、50％-75％等等。本发明所述的范围包含或者不包含端点值。
本发明还提供连钱草提取物，包含：
化合物I：木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000003
和
化合物II：芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000004
并且
所述连钱草提取物通过包含如下步骤的方法获得：
a)用水性溶液一次或多次提取连钱草，得到连钱草水性提取液，任选地浓缩所得提取液；
b)向所述任选浓缩的连钱草水性提取液中加入一定体积醇性溶液以产生沉淀；
c)分离步骤b)中产生的沉淀。
在本发明的一个方面，本发明任选一种所述连钱草提取物中化 合物I含量大于0.6％，优选大于25％，更优选大于39％，化合物II的含量大于0.6％，优选大于25％，更优选大于32％。
本发明还提供：
含化合物I：木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000005
或其药学可接受的盐；和/或
化合物II：芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
Figure PCTCN2015074366-appb-000006
或其药学可接受的盐；
和/或化合物I和II或二者任一药学上可接受盐的混合物。
在本发明的另一个方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物中化合物I含量大于0.6％，优选大于25％，更优选大于39％，化合物II的含量大于0.6％，优选大于25％，更优选大于32％。
在一个优选的方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物中化合物I的含量或者化合物II的含 量二者分别不低于1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、35％、40％、45％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、65％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、85％、90％、95％、99％。在还有一个优选的方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物中化合物I的含量或者化合物II的含量二者分别不超过99％、95％、90％、85％、80％、79％、78％、77％、76％、75％、74％、73％、72％、71％、70％、65％、60％、59％、58％、57％、56％、55％、54％、53％、52％、51％、50％、45％、40％、35％、30％、29％、28％、27％、26％、25％、24％、23％、22％、21％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％。在还有另一个优选方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物中化合物I的含量或者化合物II的含量二者分别是前述重量含量任意组合的范围，例如，1％-99％、1％-25％、25％-50％、50％-75％、75％-99％、25％-99％、25％-75％、50％-99％、1％-75％、1％-25％、20％-60％、50％-75％等等。本发明所述的范围包含或者不包含端点值。
在本发明的一个方面，本发明提供药物组合物，包含：本发明任选一种所述连钱草提取物，和药学上可接受载体。在本发明的另一个方面，本发明还提供药物组合物，包含：本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物，和药学上可接受载体。
在本发明的一个方面，含本发明任选一种所述连钱草提取物的药物组合物中化合物I和化合物II占药物组合物中活性成分总重量的50％以上，或者从包含所述提取物的药物组合物中基本上除去化 合物I和II后该药物组合物的生物活性如DPP4抑制活性降低50％以上。在本发明的另一个方面，含本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物的药物组合物中化合物I和化合物II占药物组合物中活性成分总重量的50％以上，或者从包含所述混合物的药物组合物中基本上除去化合物I和II后该药物组合物的生物活性如DPP4抑制活性降低50％以上。
在本发明的一个方面，本发明任选一种所述连钱草提取物中咖啡酸和迷迭香酸的含量低于0.5％，优选低于0.1％。在本发明的另一个方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物中咖啡酸和迷迭香酸的含量低于0.5％，优选低于0.1％。
在本发明的一个方面，本发明公开了任选一种所述连钱草提取物具有降血糖、降血脂、减肥、治疗肾病的活性。在本发明的另一个方面，本发明所述含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物具有降血糖、降血脂、减肥、治疗肾病的活性。
因此，本发明提供用于降血糖、降血脂、减肥、治疗肾病的提取物。在本发明的另一个方面，本发明还提供用于降血糖、降血脂、减肥、治疗肾病的含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。
可选择的，本发明提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供本发明任选一种所述连钱草提取物。在本发明的另一个方面，本发明还提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供含本发明所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。
或者还是可选择的，本发明提供任选一种所述连钱草提取物在制备降血糖、降血脂、减肥、治疗肾病的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备降血糖、降血脂、减肥、治疗肾病的药物中的用途。
本发明还提供连钱草的提取方法，包含如下所述步骤：
a)用水性溶液一次或多次提取连钱草，得到连钱草水性提取液，任选地浓缩所得提取液；
b)向所述任选浓缩的连钱草水性提取液中加入一定体积醇性溶液以产生沉淀；
c)分离步骤b)中产生的沉淀。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤a)所述水性溶液中水的含量大于40％，优选大于80％，最优选水。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤a)所述提取为加热回流提取或者超声提取，优选次数为2-5次。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤a)中所得连钱草水性提取液还可被任选地有机溶剂萃取并且弃去有机相，处理过的连钱草水性提取液留作后续操作使用，其中所述有机溶剂优选乙酸乙酯或二氯甲烷。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤a)中得到所述连钱草水性提取液在任选地浓缩之后还可被进一步冷藏，其中所述浓缩优选减压浓缩，所述冷藏优选4-6℃冷藏。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤b)中所述醇性溶液，优选乙醇-水混合体系，更优选90％的乙醇，最优选95％的乙醇。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤b)中醇性溶液的体积为任选浓缩的连钱草水性提取液体积的2-4倍。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤c)中分离获得的沉淀还可选择地被冷冻干燥。
在本发明的一个方面，本发明所述连钱草的提取方法中步骤c)中分离获得的沉淀还可被任择地用大孔吸附树脂纯化，所述用大孔吸附树脂纯化通过包含如下步骤的实现：
i)将步骤c)中分离获得的沉淀用水性溶剂溶解制成水性溶液，任选地除去残留的醇；
ii)将任选除去残留醇的水性溶液加到大孔树脂上；
iii)用水性洗脱液除掉蛋白质、多糖组分；
iv)用醇性洗脱液洗脱、浓缩所得洗脱液得到纯化的连钱草提取物。
在本发明的一个方面，本发明步骤i)中所述水性溶液中水的含量大于40％，优选大于80％，最优选水。
在本发明的一个方面，本发明步骤ii)中所述大孔树脂为D-101、D-101-I、DA-201、DM-301、DM-130、AB-8、HPD-100、HPD-300、HPD-400、HPD-600、HPD-826或这些树脂的类似填料，优选D-101。
在本发明的一个方面，本发明步骤iii)所述水性洗脱液中水的含量大于90％，优选大于95％，最优选水。
在本发明的一个方面，本发明步骤iv)所述醇性洗脱液为乙醇-水混合体系，优选5-20％的乙醇，最优选10-15％的乙醇。
本发明提供化合物I、化合物II单体的优选制备工艺，其特征在于经本发明步骤i)-iv)得到的连钱草提取物后，还可采用聚酰胺、硅胶、凝胶或反相填料，优选反相填料，通过柱层析方法快速实现分离纯化，单体化合物纯度可达96％以上。
本发明所述药物组合物是片剂、硬胶囊、软胶囊、肠溶胶囊、微囊剂、颗粒剂、糖浆剂、注射剂、颗粒剂、乳剂、悬浮液、溶液和用于口服或非口服给药的缓释制剂的形式。
本发明所述药学上可接受载体是指本领域技术人员熟知的药学上可接受的载体，本发明的药学上可接受载体包括但不限于：填充剂、润湿剂、黏合剂、崩解剂、润滑剂、粘合剂、助流剂、掩味剂、表面活性剂、防腐剂等。填充剂包括但不限于乳糖、微晶纤维素、 淀粉、糖粉、糊精、甘露醇、硫酸钙等。润湿剂与黏合剂包括但不限于羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、明胶、蔗糖、聚乙烯吡咯烷酮等。崩解剂包括但不限于羧甲基淀粉钠、交联聚乙烯吡咯垸酮、交联羧甲基纤维素钠、低取代羟丙基纤维素等。润滑剂包括但不限于硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇、月桂醇硫酸镁等。粘合剂包括但不限于阿拉伯胶、藻酸、羧甲基纤维素钙、羧甲基纤维素钠、葡萄糖结合剂、糊精、右旋糖、乙基纤维素、明胶、液体葡萄糖、瓜尔胶、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、硅酸铝镁、麦芽糖糊精、甲基纤维素、聚甲基丙烯酸酯、聚乙烯吡咯烷酮、预明胶化淀粉、藻酸钠、山梨醇、淀粉、糖浆和黄蓍胶。助流剂包括但不限于胶体二氧化硅、粉状纤维素、三硅酸镁、二氧化硅和滑石粉。掩味剂包括但不限于阿斯巴坦、甜菊苷、果糖、葡萄糖、糖浆、蜂蜜、木糖醇、甘露醇、乳糖、山 梨醇、麦芽糖醇、甘草甜素。表面活性剂包括但不限于吐温-80、泊洛沙姆。防腐剂包括但不限于尼泊金酯、苯甲酸钠、山梨酸钾等。
本发明所述药学上可接受的盐包括并不限于与盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸形成的盐，与甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、乙磺酸等有机酸和天冬氨酸、谷氨酸等酸性氨基酸的酸性加成盐，或与碱形成的盐，如钠、钾等无机碱的盐，或与赖氨酸、精氨酸、鸟氨酸等碱性氨基酸形成的碱性加成盐。
本发明所述连钱草(Glechoma longituba(Nakai)Kupr.)又名活血丹、透骨消，为唇形科活血丹属植物的干燥全草，并作为《中国药典》(2010版)中连钱草药材的唯一法定来源品种。活血丹属植物还包含欧活血丹(G.hederacea Linn.)、白透骨消(G.biondiana(Diels)C.Y.Wu et C.Chen)和大花活血丹(G.sinograndis C.Y.Wu)等品种，上述品种与连钱草具有相似功效。
本发明所述二肽基肽酶IV(Dipeptidyl peptidase-4，DPP4)是一种跨膜丝氨酸蛋白酶，属脯氨酰寡肽酶家族的一员。DPP4是治疗2型糖尿病的新靶标；它是体内、外主要促使胰高血糖素样肽-1(Glucagon-like peptide-1，GLP-1)降解、失活的关建酶之一。目前医学上已经证实DPP4抑制剂是一种新型的抗糖尿病药。临床结果显示该类药物具有良好的降糖效果，同时未发现如胰岛素类、磺酰脲类糖尿病药物所产生的常见体重增加和低血糖等不良反应，因此有关DPP4抑制剂研究已成为抗糖尿病药物研究的热点。
本发明发现，化合物I和化合物II能有效抑制DPP4活性，100μM浓度下对DPP4的抑制率分别为40.8％和34.1％，从而具有降血糖效果。
因此，本发明提供用于降血糖的提取物。在本发明的另一个方面，本发明还提供用于降血糖的含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。可选择的，本发明提供降血糖的方法，所述方法包括：向有需要的患者提供本发明任选一种所述连钱草提取物。在本发明的另一个方面，本发明还提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供含本发明所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。或者还是可选择的，本发明提供任选一种所述连钱草提取物在制备降血糖的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备降血糖的药物中的用途。本发明还提供任选一种所述连钱草提取物在制备抑制DPP4活性的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备抑制DPP4活性的药物中的用途。在本发明的一个方面，本发明所述抑制二肽基肽酶IV活性是指治疗糖尿 病。
葡萄糖酸氧化酶(Glucose oxidase，GOX)是以黄素腺嘌呤单核苷酸(FMN)为辅基的氧化酶，它催化乙醇酸氧化生成草酸。GOX的过度表达则会促使草酸盐的生成，从而增加肾结石及肾炎风险。因此，寻找葡萄糖酸氧化酶的抑制剂已成为开发具有治疗肾结石及肾炎的重要方法和途径之一。本发明还发现，化合物I和化合物II对GOX具有一定的抑制活性，化合物I的IC50值为0.5mM，化合物II的IC50值为0.4mM，具有潜在的治疗治疗肾结石及肾炎的药理活性。
因此，本发明提供用于治疗肾病的提取物。在本发明的另一个方面，本发明还提供用于治疗肾病的含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。可选择的，本发明提供治疗肾病的方法，所述方法包括：向有需要的患者提供本发明任选一种所述连钱草提取物。在本发明的另一个方面，本发明还提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供含本发明所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。或者还是可选择的，本发明提供任选一种所述连钱草提取物在制备治疗肾病的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备治疗肾病的药物中的用途。
甘油三酯(或称三酰甘油)是真核生物中能量贮存的主要形式。在哺乳动物中，甘油三酯主要在小肠，肝和脂肪细胞合成。甘油三酯代谢-即吸收以及从头合成两者的紊乱或者失调-与多种疾病的发病机理相关。例如，肥胖症、高血脂等。胰脂肪酶由胰腺腺泡细胞分泌，通过胰管进入十二指肠，在十二指肠和小肠上端油水界面上将甘油三酯水解成可供吸收的2-单酰甘油和游离脂肪酸。胰脂肪 酶负责水解50％～70％的饮食性脂肪。抑制胰脂肪酶的活性可以抑制肠道三酰甘油的消化吸收，降低血甘油三酯水平和减少组织脂肪聚集，从而具有降血脂、减肥的作用。二酰甘油酰基转移酶1(DGAT1，Diacylglycerol acyltransferase 1)催化三酰甘油合成最后一步：脂酰辅酶A酰基转移到2-单酰甘油形成三酰甘油，是三酰甘油合成的限速酶。通过抑制或降低DGAT1酶活性可以减少从二酰甘油合成甘油三酯。DGAT1酶抑制剂可用于治疗与甘油三酯异常代谢有关的疾病，例如具有降血脂、减肥的作用。
本发明还发现，化合物I、化合物II具有抑制食物中的脂肪吸收的作用，从而具有降血脂、减肥的效果。本发明发现化合物I和II能有效抑制DGAT1和胰脂肪酶的活性，从而发挥降血脂、减肥的作用。化合物I和II在50μM浓度时，对DGAT1的抑制率均超过50％；10μM浓度时，化合物I和II仍表现出对DGAT1约20％的抑制活性。化合物I和II在同样浓度下，对胰脂肪酶的抑制率也均高于40％。
因此，本发明提供用于降血脂的提取物。在本发明的另一个方面，本发明还提供用于降血脂的含化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。可选择的，本发明提供降血脂的方法，所述方法包括：向有需要的患者提供本发明任选一种所述连钱草提取物。在本发明的另一个方面，本发明还提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供含本发明所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。或者还是可选择的，本发明提供任选一种所述连钱草提取物在制备降血脂的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备降血脂的药物中的用途。
因此，本发明提供用于减肥的提取物。在本发明的另一个方面，本发明还提供用于减肥的含化合物I或其药学可接受的盐、和/或化 合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。可选择的，本发明提供减肥的方法，所述方法包括：向有需要的患者提供本发明任选一种所述连钱草提取物。在本发明的另一个方面，本发明还提供降血糖、降血脂、减肥、治疗肾病的方法，所述方法包括：向有需要的患者提供含本发明所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物。或者还是可选择的，本发明提供任选一种所述连钱草提取物在制备减肥的药物中的用途。本发明还提供含所述化合物I或其药学可接受的盐、和/或化合物II或其药学可接受的盐、和/或化合物I和II或二者任一药学上可接受盐的混合物在制备减肥的药物中的用途。
本发明所述糖尿病包括1型糖尿病、2型糖尿病、其它特殊类型糖尿病和妊娠期糖尿病。
本发明所述降血脂是指降低血液中的甘油三酯水平。
本发明所述治疗肾病是指治疗肾结石、治疗肾炎等。
下面，本发明将通过实施例展示本发明的有益效果。本领域技术人员会知道，这些实施例是示例性的，而不是限制性的。这些实施例不会以任何方式限制本发明的范围。
附图说明
图1显示不同制备工艺得到的连钱草提取物中主成分的HPLC分析结果。
图2显示连钱草提取物与文献报道的提取工艺中主成分的HPLC分析结果。
图3显示连钱草提取物中化合物I和II优选制备工艺及HPLC分析结果。
图4显示连钱草提取物LQC-H-1在250mg/kg及500mg/kg均能显著增强小鼠急性口服葡萄糖耐量，提高小鼠口服葡萄糖后的血胰岛素水平。注：图4(A)LQC-H-1增强小鼠急性口服糖耐量，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图4(B)根据校准组 校正的口服糖耐量曲线下面积(AUC)，与对照组相比*p＜0.05，***p＜0.001；图4(C)口服给予葡萄糖后20分钟血清胰岛素浓度，与对照组相比**p＜0.01。
图5显示连钱草提取物LQC-H-2在250mg/kg及500mg/kg均能显著增强小鼠急性口服葡萄糖耐量。注：图5(A)LQC-H-2增强小鼠急性口服糖耐量，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图5(B)根据校准组校正的口服糖耐量曲线下面积(AUC)，与对照组相比*p＜0.05，**p＜0.01。
图6显示连钱草水提醇沉上清液以及醇沉固体中粗多糖未增强小鼠急性口服葡萄糖耐量。注：图6(A)粗多糖和连钱草水提醇沉上清液组分未能有效增强小鼠口服糖耐受量，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图6(B)根据校准组校正的口服糖耐量曲线下面积，给药组与对照组相比差异无统计学意义。
图7显示连钱草提取物LQC-H-1长期给药改善自发2型糖尿病db/db小鼠糖尿病症状。注：图7(A)LQC-H-1对小鼠体重无显著影响，罗格列酮长期给药增加了小鼠体重，横坐标为给药时间，纵坐标为体重；图7(B)LQC-H-1降低db/db小鼠随时血糖，药效与阳性药罗格列酮类似，横坐标为给药时间，纵坐标为血糖，250mg/kg剂量组与对照组相比，#p＜0.05，500mg/kg剂量组与对照组相比，*p＜0.05；图7(C)LQC-H-1给药4周后增强小鼠急性口服糖耐量，效果与阳性药罗格列酮近似，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图7(D)各组口服糖耐量曲线下面积，与对照组相比**p＜0.01，***p＜0.001；图7(E)LQC-H-1给药4周后提高db/db小鼠血清胰岛素水平，与对照组相比，*p＜0.05。
图8显示连钱草提取物中主要活性单体化合物I和化合物II作为有效的来源于天然产物的DPP4抑制剂，单独作用能够显著增强小鼠急性口服糖耐量。注：图8(A)化合物I及化合物II对DPP4酶的体外抑制活性，Sitagliptin(西格列汀)为阳性参照；图8(B)化合物I及化合物II增强小鼠急性口服糖耐量，Sitagliptin(西格列汀)为阳性参照，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图8(C)根据校准组校正的口服糖耐量曲线下面积(AUC)，与对照组 相比*p＜0.05，**p＜0.01，n.s.表示化合物200mg/kg与西格列汀10mg/kg剂量组差异无统计学意义。
图9显示连钱草提取物中主要活性单体化合物I在100mg/kg剂量能显著增强小鼠急性口服葡萄糖耐量。注：图9(A)小鼠急性口服糖耐量实验，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图9(B)各组小鼠口服糖耐量曲线下面积，与对照组相比*p＜0.05，**p＜0.01。
图10显示化合物I长期给药能够有效降低db/db小鼠血糖。注：图10(A)化合物I给药2周后降低小鼠随时血糖，效果与阳性药西格列汀接近；图10(B)化合物I给药2周后降低小鼠空腹血糖，效果与阳性药西格列汀接近，与对照组相比*p＜0.05，**p＜0.01。
图11显示连钱草提取物LQC-H-1能够降低急性高甘油三酯血症小鼠血清甘油三酯水平。注：连钱草提取物LQC-H-1能够降低急性高甘油三酯血症小鼠血清甘油三酯水平，LCQ90810mg/kg浓度为阳性对照，模型组为造模给予2g/kg橄榄油，空白组为不给予橄榄油造模。**p＜0.01，***p＜0.001。
图12显示文献报道连钱草65％乙醇超声提取物(LQC-65％乙醇提取物)对小鼠急性糖耐量无显著影响。注：图12(A)小鼠急性口服糖耐量实验，横坐标为给予葡萄糖后时间，纵坐标为血糖浓度；图12(B)根据校准组校正的口服糖耐量曲线下面积，与对照组相比，**p＜0.01，n.s.＝无显著性差异。
具体实施方式
材料、试剂与仪器
材料连钱草：2012年8月购自于上海康桥中药饮片有限公司；批号：120713；生产日期：2012年7月16日。
试剂：95％乙醇、蒸馏水、乙酸乙酯、二氯甲烷、甲醇等试剂均为分析纯(国药集团化学试剂有限公司)；D101大孔吸附树脂(国药集团化学试剂有限公司)、柱层析硅胶(H系列，青岛海洋硅胶 干燥剂厂)，薄层层析硅胶GF254(青岛海洋硅胶干燥剂厂)，MCI层析填料(日本，GEL CHP20P，75-100μ)，ODS层析填料(日本，YMC*GEL，s 50μm)，Sephadex LH-20层析填料(瑞典，GE Healthcare)。
仪器：
旋转蒸发仪：EYELA Rotary evaporator N1001，东京理化。
冻干机：Christ，ALPHA 1-2LD PLUS，德国。
电子天平：BT 125D，赛多利斯科学仪器(北京)有限公司。
暗箱紫外透射仪：WFH-203B，上海精科实业有限公司。
超声仪：SK7200H(350W)，上海科导超声仪器有限公司。
ESI-MS：Finnigan LCQ-DECA型质谱仪测定。
NMR：Varian INOVA 400型核磁共振仪测定，TMS为内标。
高效液相色谱仪(HPLC)：Agilent 1260高效液相系统，DAD检测器。
实施例1：连钱草提取物的制备
本实施例提供三种方法制备连钱草全草提取物。
(1)取中药连钱草全草100g，加水回流提取两次，第一次加水量为药材重量的15倍量，浸泡药材15min后100℃加热回流1.5h，第二次加水量为药材重量的10倍量，100℃加热回流1.5h，过滤后合并提取液。采用旋转蒸发仪50-55℃下减压浓缩至400ml，采用乙酸乙酯等体积萃取5次，合并乙酸乙酯层萃取液。余下水层减压浓缩至190ml，放入冰箱4℃冷藏3-4h，随后取出加入三倍量的95％(v/v)乙醇溶液，充分搅拌后放入冰箱4℃冷藏12h，过滤。滤液与乙酸乙酯萃取液合并，减压浓缩后得到上清液样品1。醇沉固体采用减压干燥箱在50℃环境下除去残留乙醇溶液，得到醇沉浸膏，随后放入-70℃冰箱冷冻12h，采用冷冻干燥设备(Christ冻干机)冷冻干燥48h，得到连钱草提取物干燥固体粉末21.04g(LQC-H-1)。
(2)取中药连钱草全草100g，加水回流提取两次，第一次加水量为药材重量的15倍量，浸泡药材15min后100℃加热回流1.5h， 第二次加水量为药材重量的10倍量，100℃加热回流1.5h。过滤后合并提取液，采用旋转蒸发仪50-55℃下减压浓缩至200ml，放入冰箱4℃冷藏3-4h。随后取出加入三倍量的95％(v/v)乙醇溶液，充分搅拌后放入冰箱4℃冷藏12h，过滤。上清液采用旋转蒸发仪50℃下减压浓缩得到上清液样品2。醇沉固体采用减压干燥箱在50℃环境下除去残留乙醇溶液，随后放入-70℃冰箱冷冻12h，采用冷冻干燥设备(Christ冻干机)冷冻干燥48h，得到连钱草提取物干燥固体粉末19.2g(LQC-H-2)。
(3)取中药连钱草全草100g，加水超声提取两次。第一次加水量为药材重量的15倍量，浸泡药材15min后超声提取45min，过滤收集滤液；第二次加水量为药材重量的10倍量，超声提取45min。过滤后合并两次滤液，采用旋转蒸发仪50-55℃下减压浓缩至200ml，放入冰箱4℃冷藏3-4h。随后取出加入三倍量的95％(v/v)乙醇溶液，充分搅拌后放入冰箱4℃冷藏12h，过滤。上清液采用旋转蒸发仪50℃下减压浓缩得到上清液样品3。醇沉固体采用减压干燥箱在50℃环境下除去残留乙醇溶液，随后放入-70℃冰箱冷冻12h，采用冷冻干燥设备(Christ冻干机)冷冻干燥48h，得到连钱草提取物干燥固体粉末14.1g(LQC-H-3)。
实施例2：连钱草提取物制备工艺中，醇沉上清液中主成分的分离鉴定
取实施例1中上清液样品1的浓缩浸膏约200mg，加入少量蒸馏水溶解，采用MCI填料进行分离纯化，甲醇-水梯度洗脱(水，20％甲醇，40％甲醇，60％甲醇，100％甲醇)，经HPLC检测。选取40％甲醇和60％甲醇洗脱组分进行后续分离。40％洗脱组分采用旋转蒸发仪50℃下减压浓缩成浸膏，少量甲醇溶解后，采用ODS填料进行分离纯化，30％甲醇等度洗脱，纯化后得到主峰1(t＝13.04min，5.4mg)。60％洗脱组分采用旋转蒸发仪50℃下减压浓缩成浸膏，少量甲醇溶解后，采用ODS填料进行纯化，40％甲醇等度洗脱，洗脱液减压浓缩后采用Sephadex LH-20填料，20％甲醇等度洗脱，纯化得 到主峰2(t＝29.98min，2.4mg)。
上清液样品1中主峰1(t＝13.04min)为黄色无定形粉末(甲醇)，经ESI-MS测定，负离子m/z：179[M-H]-；1H NMR(CD3OD，400MHz)δ：7.35(1H，d，J＝15.8Hz，H-7)，7.01(1H，d，J＝2.0Hz，H-2)，6.88(1H，dd，J＝8.1，2.0Hz，H-6)，6.75(1H，d，J＝8.1Hz，H-5)，6.29(1H，d，J＝15.8Hz，H-8)，与文献(Wang M F，et al.J Agric Food Chem，2000，48：235-238.)报道一致，确定为咖啡酸。
主峰2(t＝29.98min)经ESI-MS测定，负离子m/z：359[M-H]-，719[2M-H]-；1H NMR(CD3OD，400MHz)δ：7.53(1H，d，J＝15.8Hz，H-7)，7.05(1H，d，J＝1.9Hz，H-2)，6.94(1H，dd，J＝8.2，1.9Hz，H-6)，6.79(1H，d，J＝8.2Hz，H-5)，6.78(1H，d，J＝1.8Hz，H-2’)，6.69(1H，d，J＝8.0Hz，H-5’)，6.65(1H，dd，J＝8.0，1.8Hz，H-6’)，6.28(1H，d，J＝15.8Hz，H-8)，5.11(1H，d，J＝9.3Hz，H-8’)，3.13(1H，d，J＝13.6Hz，H-7’(α))，2.97(1H，d，J＝13.6，9.3Hz，H-7’(β))；13C NMR(CD3OD，100MHz)δ：173.7(C-9’)，167.7(C-9)，148.2(C-4)，145.5(C-7)，145.4(C-3)，144.6(C-3’)，143.5(C-4’)，129.5(C-1’)，126.4(C-1)，121.6(C-6)，120.3(C-6’)，116.1(C-2’)，115.1(C-5)，114.8(C-5’)，114.0(C-8)，113.6(C-2)，与文献(Ha T J，et al.Food Chemistry，2012，135：1397-1403.)报道一致，为迷迭香酸。
实施例3：采用高效液相色谱仪对连钱提取物中主成分进行分析检测
采用高效液相高效液相色谱仪对连钱草提取物LQC-H-1、LQC-H-2、LQC-H-3以及上清液样品1、样品2和样品3的主成分进行比较分析，并对提取物中化合物I和化合物II进行含量测定，结果如图1所示，其色谱条件如下：
色谱柱型号：Agilent ZORBAX SB-C18，5μm，4.6×250mm
流动相：乙腈(A)-水(B，含0.2％冰醋酸)；流速：1ml/min
样品浓度：LQC-H-1(21mg/ml)，LQC-H-2(24mg/ml)，LQC-H-3(20mg/ml)，上清液样品1(7.16mg/ml)，样品2(7.20mg/ml)， 样品3(6.8mg/ml)。
使用现有技术方法(袁春玲等.中药药理与临床，2008，24(3)：57-58；杨念云等.中国药科大学学报，2005，36(3)：210-212)的对照组：65％乙醇超声提取物(16.2mg/ml)，80％乙醇回流提取物(16.5mg/ml)，主成分检测结果如图2所示。
进样量：10μl
检测波长：190-400nm，优选360nm进行含量测定
梯度洗脱条件见表1：
表1.HPLC梯度洗脱条件
Figure PCTCN2015074366-appb-000007
图1分析结果显示，三种工艺制备得到的连钱草提取物LQC-H-1、LQC-H-2和LQC-H-3中主峰高度一致。表明本发明的三种工艺均能高效的获取活性化合物I和化合物II。由于LQC-H-2工艺减少了乙酸乙酯试剂萃取流程，得率高，从生产方面考虑更加快捷、安全、节约成本，为制备连钱草提取物的优选工艺。此外，三种工艺得到的上清液样品1、样品2和样品3中主成分也高度一致，均为咖啡酸和迷迭香酸。这证明直接采用水提醇沉工艺即可达到乙 酸乙酯萃取的相同效果。
此外，图1分析结果则显示，采用水提醇沉工艺收集固体沉淀，即可高效获得黄酮类化合物。该沉淀很好的富集了化合物I和化合物II，并快速的除去了咖啡酸类化合物。通过外标法测定，其中化合物I和化合物II在三种提取物中百分比分别为：LQC-H-1中化合物I占0.87％，化合物II占0.81％；LQC-H-2中化合物I占0.79％，化合物II占0.75％；LQC-H-3中化合物I占0.50％，化合物II占0.59％。咖啡酸和迷迭香酸在固体中的含量均低于0.1％。
图2分析结果显示，在和本发明相同的测试条件下，现有技术(袁春玲等.中药药理与临床，2008，24(3)：57-58；杨念云等.中国药科大学学报，2005，36(3)：210-212)中记载的提取方法(65％乙醇超声提取和80％乙醇回流提取工艺)所获得的提取物中未见明显的化合物I和化合物II的HPLC吸收峰，表明现有文献报道方法并不能有效获得化合物I和化合物II。
实施例4：连钱草提取物中主要活性成分化合物I和化合物II的优选制备工艺及结构解析
取中药连钱草全草200g(已完成切断粉碎)，加水回流提取两次，第一次加水量为药材重量的15倍量，浸泡药材15min后100℃加热回流1.5h，第二次加水量为药材重量的10倍量，100℃加热回流1.5h。过滤后合并提取液，采用旋转蒸发仪50-55℃下减压浓缩至380ml，放入冰箱4℃冷藏3-4h。随后取出加入三倍量的95％(v/v)乙醇溶液，充分搅拌后放入冰箱4℃冷藏12h。过滤，收集醇沉固体。加水充分溶解醇沉固体后，采用旋转蒸发仪50℃下减压浓缩除去残留乙醇溶剂，得到水层浓缩液400ml。采用D101大孔吸附树脂1kg进行分离，用蒸馏水2400ml洗脱掉蛋白质、多糖组分；随后采用15％乙醇2000ml进行洗脱，收集洗脱液。采用旋转蒸发仪50-55℃下减压浓缩洗脱液得到富含化合物I和化合物II的浸膏。含量测定方法与实施例3中方法一致，化合物I和II在浸膏中的含量分别为13.78％和12.77％，两者总和占到浸膏重量的26.55％，咖啡酸和迷迭 香酸含量低于0.01％(见图3)。
上述浸膏(化合物I和II占总浸膏的26.55％)可采用D101大孔吸附树脂300g再次进行分离，用蒸馏水400ml洗脱残留蛋白质、多糖组分，随后采用蒸馏水800ml继续洗脱，收集洗脱液。采用旋转蒸发仪50-55℃下减压浓缩洗脱液得到富含化合物I和化合物II的浸膏。化合物I和II含量分别为29.70％和26.91％，两者总和占到浸膏重量的56.61％(见图3)。
上述浸膏(化合物I和II占总浸膏的56.61％)可继续采用D101大孔吸附树脂200g进行第三次分离纯化，用蒸馏水200ml洗脱掉色素，随后采用蒸馏水500ml继续洗脱，收集洗脱液。采用旋转蒸发仪50-55℃下减压浓缩洗脱液得到富含化合物I和化合物II的浸膏。化合物I和II含量分别达到39.75％和32.77％，两者总和占到浸膏重量的72.52％(见图3)。
上述采用大孔树脂纯化得到的三种含化合物I和II不同比例的浸膏，均可使用少量蒸馏水溶解，采用YMC*GEL ODS填料进行快速纯化，分别采用水、10％甲醇梯度洗脱，采用HPLC对收集流份进行检测。合并相同组分后50℃下经旋转蒸发仪减压浓缩，即可分别得到单体化合物I共296mg(纯度98.65％，见图3)，化合物II共258mg(纯度大于96.52％，见图3)。
化合物I为棕黄色无定形粉末(甲醇)。负离子ESI-MS m/z：637[M-H]-；正离子ESI-MS m/z：639[M+H]+。1H NMR(DMSO-d6+D2O，400MHz)δ：7.47(2H，d，J＝1.9Hz，H-2’)，7.43(1H，dd，J＝8.2，1.9Hz，H-6’)，6.96(1H，s，H-3)，6.89(1H，d，J＝8.2Hz，H-5’)，6.71(1H，d，J＝2.2Hz，H-8)，6.49(1H，d，J＝2.2Hz，H-6)，5.20(1H，d，J＝6.5Hz，H-1”)，4.57(1H，d，J＝6.3Hz，H-1”’)，3.20-4.1(m，hidden)。13C NMR(DMSO-d6+D2O，100MHz)δ：182.4(C-4)，172.9(C-6”’)，170.2(C-6”)，164.9(C-2)，163.2(C-7)，161.0(C-9)，157.2(C-5)，150.1(C-4’)，146.0(C-3’)，121.7(C-1’)，119.6(C-6’)，116.5(C-5’)，113.7(C-2’)，105.8(C-1”’)，104.0(C-10)，103.4(C-3)，100.3(C-6)，98.5(C-1”)， 96.2(C-8)，81.8(C-2”)，76.2(C-5”’)，75.8(C-3”’)，75.1(C-5”)，74.6(C-3”)，74.4(C-2”’)，72.2(C-4”)，71.6(C-4”’)。以上数据与文献(Berashili D T，et al.Chemistry of Natural Compound，2006，42(1)：106-107)报道数据一致。本发明首次从活血丹属植物中分离得到化合物I。
化合物II为棕黄色无定形粉末(甲醇)。负离子ESI-MS m/z：621[M-H]-；正离子ESI-MS m/z：645[M+Na]+。1H NMR(DMSO-d6+D2O，400MHz)δ：7.95(2H，d，J＝8.0Hz，H-2’，6’)，6.94(2H，d，J＝8.0Hz，H-3’，5’)，6.93(1H，hidden，H-3)，6.80(1H，d，J＝1.9Hz，H-8)，6.52(1H，d，J＝1.9Hz，H-6)，5.21(1H，d，J＝6.6Hz，H-1”)，4.57(1H，d，J＝6.4Hz，H-1”’)，3.20-4.10(m，hidden)。13C NMR(DMSO-d6+D2O，100MHz)δ：182.5(C-4)，172.7(C-6”’)，171.8(C-6”)，164.8(C-2)，163.2(C-7)，161.6(C-9)，161.1(C-4’)，157.3(C-5)，129.2(C-2’，6’)，121.4(C-1’)，116.5(C-3’，5’)，105.8(C-10)，103.7(C-1”’)，103.4(C-3)，100.3(C-6)，98.3(C-1”)，96.0(C-8)，81.5(C-2”)，76.4(C-5”’)，75.9(C-3”’)，75.1(C-5”)，74.9(C-3”)，74.4(C-2”’)，72.2(C-4”)，71.8(C-4”’)。以上数据与文献(陈泽乃，等.药学学报，1988，23(10)：789-791)报道数据一致。
实施例5：连钱草提取物LQC-H-1显著增强小鼠急性口服葡萄糖耐量，提高小鼠口服葡萄糖后的血胰岛素水平
口服糖耐量实验(Oral glucose tolerance test，OGTT)方法：8周龄雄性C57BL/KsJ小鼠(上海斯莱克实验动物公司)，每组8只，体重20±2g，按照SPF级操作规程饲养，实验前禁食16小时。实验开始前各组小鼠尾静脉采血，利用罗氏ACCU-CHEK血糖仪测定血糖浓度，记为0时间点。各给药组小鼠分别灌胃给予不同浓度化合物，对照组小鼠给予等量蒸馏水，给药体积10ml/kg。给药半小时后，各组小鼠灌胃给予5g/kg葡萄糖，并在灌胃20min，40min，80min后分别测量记录各组小鼠血糖浓度。记录OGTT曲线，并根据OGTT曲线，用未给予葡萄糖的小鼠血糖变化(校准组)校正计算曲线下 面积(areas under curve，AUC)，利用单因素方差分析(ONE-WAY-ANOVA)比较各组间显著性差异(注：以下所有活性实施例中急性口服糖耐量实验除特别注明外，实验方法均与实施例5中实验方法相同)。在小鼠口服给予葡萄糖20分钟后，尾静脉取血，利用酶联免疫吸附法(ELISA，试剂盒购买自Millipore，美国)测定小鼠血清胰岛素水平。
动物分组：对照组；校准组；LQC-H-1低剂量组(250mg/kg)；LQC-H-1高剂量组(500mg/kg)。
实验结果：连钱草提取物LQC-H-1在250mg/kg及500mg/kg均能显著增强小鼠急性口服葡萄糖耐量，提高小鼠口服葡萄糖后的血胰岛素水平(图4)。该作用有浓度依赖性，提示该提取物有促进胰岛β-细胞分泌胰岛素，改善餐后血糖的作用。
实施例6：连钱草提取物LQC-H-2同样能够显著增强小鼠急性口服葡萄糖耐量
实验方法同实施例5，动物分组：对照组；校准组；LQC-H-2低剂量组(250mg/kg)；LQC-H-2高剂量组(500mg/kg)。
实验结果：连钱草提取物LQC-H-2在250mg/kg及500mg/kg均能显著增强小鼠急性口服葡萄糖耐量(图5)，证明优化后的提取方法获得的连钱草活性部位仍具有改善餐后血糖的作用。
实施例7：连钱草水提醇沉上清液以及醇沉固体中粗多糖未增强小鼠急性口服葡萄糖耐量
实验方法同实施例5，动物分组：对照组；粗多糖组(1g/kg)；醇沉后上清液样品1组(300mg/kg)。
通过上述活性测试证明，连钱草水提醇沉上清液以及粗多糖组分并非降糖活性部位(图6)。采用实施例1的工艺即可有效去除连钱草提取物中无降糖活性的咖啡酸类组分，从而提高该提取物的降糖效果。同时证实了文献(Shinji I.，et al.Nippon Shokuhin Kagaku Kogaku Kaishi，2007，54(9)：412-414)中推测的水溶性食物纤维(主要为多糖类成分)为有效降糖的组分是欠妥的，水溶性组分中发挥降 糖活性的部位应该归属为化合物I、化合物II或其混合物。
实施例8：连钱草提取物LQC-H-1长期给药改善自发2型糖尿病db/db小鼠糖尿病症状
实验方法：8周龄雄性db/db自发肥胖2型糖尿病小鼠(背景C57BL/KsJ，美国Jackson实验室)，每组8只，初始体重30±2g，按照SPF级操作规程饲养，实验前分为3组：对照组小鼠给予等量蒸馏水；LQC-H-1分为低剂量组(250mg/kg)，高剂量组(500mg/kg)灌胃给药，阳性药使用胰岛素增敏剂罗格列酮(10mg/kg)同时给药，同品系血糖正常小鼠(db/m)作为空白比较。给药体积10ml/kg，每天1次，持续4周，每周监测小鼠体重、血糖、饮食变化。4周后，除不急性给予药物外，按照实施例1中方法，测定各组小鼠OGTT，计算AUC。取血清测定小鼠胰岛素含量，评价LQC-H-1长期给药对自发2型糖尿病db/db小鼠糖尿病症状的改善情况。
实验结果：阳性对照罗格列酮给药4周增加了db/db小鼠体重，可能与该药长期服用导致血脂增高有关，连钱草提取物LQC-H-1对db/db小鼠的体重和饮食无明显影响(图7A)，表明LQC-H-1无明显毒性；同时LQC-H-1在250mg/kg及500mg/kg降低了db/db小鼠随时血糖，降糖效果接近罗格列酮(图7B)，给药4周后，与阳性药的药效类似，连钱草提取物LQC-H-1能够显著改善db/db小鼠口服糖耐量(图7C，7D)。此外，对LQC-H-1给药组与对照组db/db相比，血清胰岛素水平提高(图7E)，提示LQC-H-1长期给药能够增加小鼠胰岛素水平，降低db/db小鼠血糖，改善2型糖尿病症状。
本实验中长期给药后LQC-H-1对小鼠饮食及体重无明显影响，而罗格列酮导致小鼠体重增加。可见罗格列酮长期服用导致血脂异常等副作用。从而可说明LQC-H-1提取物不仅能同时达到降糖降脂作用，而且安全性高于阳性药罗格列酮。
实施例9：连钱草提取物中主要活性单体化合物I和化合物II作为有效的来源于天然产物的DPP4抑制剂，单独作用能够显著增 强小鼠急性口服糖耐量
实验方法：(1)化合物I和化合物II在体外对DPP4酶的活性抑制测定：人结肠癌细胞株Caco-2的细胞裂解液作为DPP4酶的来源(Thomas，L.，et al，2008，JPET)。在96孔板100μL/孔的筛药体系中，H-Gly-Pro-AMC底物(AnaSpec)的终浓度为244μM，加入不同浓度的待测化合物37℃孵育30min后，在激发波长380nm/发射波长460nm检测荧光信号，根据检测获得的荧光吸收值计算受试样品对DPP4的酶活抑制率(％)。抑制率(％)根据下式计算：
抑制率％＝(RFU空白-RFU化合物)/(RFU空白-RFU阴性对照)*100％
RFU化合物，RFU空白，RFU阴性对照分别表示化合物孔、空白孔和不加酶的阴性对照孔30min与0min的荧光值之差；(2)化合物I和化合物II对小鼠急性糖耐量的作用：OGTT实验方法同实施例5，小鼠分为：对照组；校正组；化合物I低剂量组(100mg/kg)；化合物I高剂量组(200mg/kg)；化合物II低剂量组(100mg/kg)；化合物II高剂量组(200mg/kg)；阳性药组(Sitagliptin 10mg/kg，西格列汀，美国Merck公司)。
实验结果：(1)化合物I及化合物II对DPP4均有不同程度的抑制活性，100μM浓度下对DPP4的抑制率分别为40.82±3.26％和34.09±3.91％(图8A)，两化合物均为首次报道对DPP4具有抑制活性；(2)化合物I及化合物II在100mg/kg与200mg/kg均能显著增强小鼠急性口服葡萄糖耐量(图8B，8C)，其中化合物I活性较化合物II活性为好。化合物I及化合物II高剂量(200mg/kg)活性与上市化学药物西格列汀在使用浓度(10mg/kg)药效相当(图8B，8C)，能够作为DPP4的抑制剂而有效发挥降糖作用，控制餐后血糖，是连钱草提取物的主要降糖活性成分之一。
实施例10：连钱草提取物中主要活性单体化合物I改善自发2型糖尿病db/db小鼠血糖控制。
实验方法：8周龄雄性db/db自发肥胖2型糖尿病小鼠 (C57BL/KsJ，美国Jackson实验室)，每组8只，初始体重30±2g，按照SPF级操作规程饲养，按照实施例1中方法测试化合物I在db/db小鼠中对急性糖耐量的影响，动物分组：对照组；化合物I组(100mg/kg)；阳性药西格列汀组(10mg/kg)。
实验结果：连钱草提取物中主要活性单体化合物I在100mg/kg剂量能显著增强小鼠急性口服葡萄糖耐量，作用效果类似于化学上市药物西格列汀(图9)，提示化合物I作为来源于天然产物的DPP4抑制剂，同样能够显著增强自发2型糖尿病db/db小鼠急性糖耐量，可能是连钱草提取物LQC-H-1中在db/db小鼠中起降糖作用的主要活性成分之一。
急性实验结束后，各组小鼠按照以下分组进行长期给药实验：对照组小鼠给予等量蒸馏水；化合物I剂量为50mg/kg给药组，阳性对照组使用DPP4抑制剂西格列汀10mg/kg灌胃给药。给药体积10ml/kg，每天1次，持续2周，监测小鼠体重、血糖、饮食变化，评价化合物I长期给药对自发2型糖尿病db/db小鼠糖尿病症状的改善情况。
实验结果：化合物I对小鼠体重、饮食无明显影响，给药2周后，随时血糖及空腹血糖均显著下降(图10)。提示化合物I长期给药能够有效降低db/db小鼠血糖，改善2型糖尿病症状。
实施例11：连钱草提取物LQC-H-1急性降脂活性测试
实验方法：(1)在急性高甘油三酯血症小鼠模型上，评价连钱草提取物LQC-H-1对血清甘油三酯水平的影响：6周龄雄性C57BL/KsJ小鼠(上海斯莱克实验动物公司)，每组8只，体重20±2g，按照SPF级操作规程饲养，实验前禁食16小时过夜。各给药组小鼠灌胃给予不同浓度化合物，对照组及空白组小鼠给等体积水。30分钟后，给药组及对照组小鼠灌胃给予2g/kg橄榄油，空白组小 鼠给等体积水。2小时后，各组小鼠眼眶静脉取血，分离血清，测定血清中甘油三酯浓度；(2)连钱草提取物LQC-H-1中分离得到的单体化合物对二酰基甘油酰基转移酶(DGAT)的抑制活性：利用昆虫细胞表达纯化获得的人源性DGAT1与化合物共同预孵育，在辅酶A存在的情况下，加入底物二酰甘油启动反应，测定生成的巯基与CPM结合后产生的荧光信号，按照如下公式计算化合物对DGAT的抑制活性：
抑制率％＝(OD空白-OD化合物)/(OD空白-OD阴性对照)*100％
OD化合物，OD空白，OD阴性对照分别表示化合物孔、空白孔和不加DGAT1酶的阴性对照孔的荧光值，并将酶活性抑制率(％)达到50％时化合物的浓度定为IC50值；(3)连钱草提取物LQC-H-1中分离得到的单体化合物对胰脂肪酶的抑制活性：底物p-Nitrophenyl acetate(美国Sigma公司)用磷酸缓冲液(25mM，pH 6.8)配成1.35mM；猪胰脂肪酶(sigma公司)用磷酸缓冲液配成10mg/ml；溴化烯炔类化合物用磷酸缓冲液配制成不同浓度的溶液。然后在96孔板中依次加入50μl浓度为1.35mM的p-Nitrophenyl acetate(美国Sigma公司)溶液以及10μl不同浓度的受试化合物，预先在37℃孵育5分钟。最后加入50μl浓度为10mg/ml的胰脂肪酶(EC 3.1.1.3，美国Sigma公司)混匀，于37℃继续反应20分钟，在492nm下检测每孔的吸光度。根据在492nm下的吸光度计算受试样品对胰脂肪酶的抑制率(％)，并将酶活性抑制率(％)达到50％时化合物的浓度定为IC50值。抑制率(％)可以根据下式进行：
抑制率(％)＝[(A-B)-(C-D)]/(A-B)×100
上式中，A，B，C，D分别表示反应后空白孔的吸光度，反应前空白孔的吸光度，反应后样品孔的吸光度，反应前样品孔的吸光度。
实验结果：(1)连钱草提取物LQC-H-1在500mg/kg剂量剂量能够降低急性高甘油三酯血症小鼠血清甘油三酯水平，降低效果 接近DGAT抑制剂阳性药LCQ908(图11)；(2)连钱草提取物LQC-H-1中分离得到的单体化合物中：化合物I、II具有显著的DGAT抑制活性(见表2)。实验结果提示，连钱草提取物LQC-H-1具有显著的急性降低血清甘油三酯作用，其组分中DGAT1及胰脂肪酶抑制活性(见表3)化合物单体可能是其发挥作用的主要有效成分。
表2.单体化合物对hDGAT1的抑制活性
Figure PCTCN2015074366-appb-000008
表3.单体化合物对胰脂肪酶的抑制活性
Figure PCTCN2015074366-appb-000009
实施例12：化合物I和II对GOX的抑制活性
GOX是以黄素腺嘌呤单核苷酸(FMN)为辅基的氧化酶，它催化乙醇酸氧化生成草酸。GOX的过度表达则会促使草酸盐的生成，从而增加肾结石及肾炎风险。
实验原理：酶偶联法是利用过氧化酶(POD)及其底物与产生的H2O2反应后生成有色产物来确定葡萄糖酸氧化酶催化反应的速率。POD催化H2O2同苯酚和4-氨基安替比林，反应生成棕红色醌 类物质。
实验方法：按照下表分别加入试剂，当最后加入底物乙醇酸后，立即启动反应，用酶标仪检测反应的动力学过程，用Vmax即最大反应速度的快慢表示酶活性的大小。
总体系：200μL
表8.试剂配制方法及浓度
Figure PCTCN2015074366-appb-000010
实验结果：测定化合物I和II在如下浓度梯度(100μM～0.1μM，3倍稀释)下，葡萄糖酸氧化酶反应的最大速度和抑制率，实验结果如下(表9)：
表9.化合物I和化合物II对葡萄糖酸氧化酶的抑制活性测定结果
Figure PCTCN2015074366-appb-000011
Figure PCTCN2015074366-appb-000012
对测定结果用Graphpad Prism 5.0进行拟合并计算IC50值，软件拟合结果显示：化合物I的IC50为0.5mM，化合物II的IC50为0.4mM，槲皮素IC50为21μM。证明化合物I和化合物II对葡萄糖酸氧化酶具有一定的抑制活性，具有潜在的治疗肾结石及肾炎的药理活性。
实施例13：本发明水提醇沉样品LQC-H-2与现有技术中65％乙醇超声提取物(袁春玲等，中药药理与临床，2008，24(3)，57-58)活性比较
实验方法同实施例5，动物分组：对照组；LQC-H-2组(500mg/kg)；LQC-65％乙醇提取物组(500mg/kg)；不灌糖校准组。
实验结果：连钱草提取物LQC-H-2在500mg/kg剂量能显著增强小鼠急性口服葡萄糖耐量，而连钱草65％乙醇超声提取物在同等剂量下对小鼠急性糖耐量无显著作用(图12)，提示通过本专利提供的制备方法获得的LQC-H-2提取物活性成分有别于65％乙醇超声提取工艺获得的总提物，LQC-H-2能够有效改善小鼠急性糖耐量。
实验结论：按制备实施例1中连钱草提取工艺获得的提取物LQC-H-2能够显著增强小鼠急性口服葡萄糖耐量，而同等剂量的连钱草65％乙醇超声提取物(LQC-65％乙醇提取物)则对小鼠急性糖耐量无显著影响。

Claims (33)

  1. 连钱草提取物，包含：
    化合物I木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100001
    化合物II芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100002
    并且化合物I和化合物II二者重量占提取物总重量的1％-75％，优选1％-25％、20％-60％、或者50％-75％。
  2. 连钱草提取物，包含：
    化合物I木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100003
    化合物II芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100004
    其中所述连钱草提取物通过包含如下步骤的方法获得：
    a)用水性溶液一次或多次提取连钱草，得到连钱草水性提取液，任选地浓缩所得提取液；
    b)向所述任选浓缩的连钱草水性提取液中加入一定体积醇性溶液以产生沉淀；
    c)分离步骤b)中产生的沉淀。
  3. 根据权利要求1-2所述任一项的连钱草提取物，其中化合物I含量大于0.6％，优选大于25％，更优选大于39％，化合物II的含量大于0.6％，优选大于25％，更优选大于32％。
  4. 根据权利要求1-3任一项所述的连钱草提取物，其中咖啡酸和迷迭香酸的含量低于0.5％，优选低于0.1％。
  5. 根据权利要求2-4任一项所述的连钱草提取物，其中步骤a)所述水性溶液中水的含量大于40％，优选大于80％，最优选水。
  6. 根据权利要求2-5任一项所述的连钱草提取物，其中步骤a)所述提取为加热回流提取或者超声提取，优选次数为2-5次。
  7. 根据权利要求2-6任一项所述的连钱草提取物，其中步骤a)中所的连钱草水性提取液还可被任选地用有机溶剂萃取并且弃去有机相，处理过的连钱草水性提取液留作后续操作使用，其中所述有机溶剂优选乙酸乙酯或二氯甲烷。
  8. 根据权利要求2-7任一项所述的连钱草提取物，其中步骤a)中得到所述连钱草水性提取液在任选地浓缩之后还可被进一步冷 藏，其中所述浓缩优选减压浓缩，所述冷藏优选4-6℃冷藏。
  9. 根据权利要求2-8任一项所述的连钱草提取物，其中步骤b)中所述醇性溶液，优选乙醇-水混合体系，更优选90％的乙醇，最优选95％的乙醇。
  10. 根据权利要求2-9任一项所述的连钱草提取物，其中步骤b)中醇性溶液的体积为任选浓缩的连钱草水性提取液体积的2-4倍。
  11. 根据权利要求2-10任一项所述的连钱草提取物，其中步骤c)中分离获得的沉淀还可选择地被冷冻干燥。
  12. 根据权利要求2-11任一项所述的连钱草提取物，其中步骤c)中分离获得的沉淀还可任择地用大孔吸附树脂纯化，所述用大孔吸附树脂纯化通过包含如下步骤的实现：
    i)将步骤c)中分离获得的沉淀用水性溶剂溶解制成水性溶液，任选地除去残留的醇；
    ii)将任选除去残留醇的水性溶液加到大孔树脂上；
    iii)用水性洗脱液除掉蛋白质、多糖组分；
    iv)用醇性洗脱液洗脱、浓缩所得洗脱液得到纯化的连钱草提取物。
  13. 根据权利要求12所述的连钱草提取物，其中步骤i)所述水性溶液中水的含量大于40％，优选大于80％，最优选水。
  14. 根据权利要求12-13任一项所述的连钱草提取物，其中步骤ii)所述大孔树脂为D-101、D-101-I、DA-201、DM-301、DM-130、AB-8、HPD-100、HPD-300、HPD-400、HPD-600、HPD-826或这些树脂的类似填料，优选D-101。
  15. 根据权利要求12-14任一项所述的连钱草提取物，其中步骤iii)所述水性洗脱液中水的含量大于90％，优选大于95％，最优选水。
  16. 根据权利要求12-15任一项所述的连钱草提取物，其中步骤iv)所述醇性洗脱液为乙醇-水混合体系，优选5-20％的乙醇，最优选10-15％的乙醇。
  17. 权利要求1-16任一项所述的连钱草提取物在制备降血糖的药物中用途。
  18. 权利要求1-16任一项所述的连钱草提取物在制备降血脂的药物中用途。
  19. 权利要求1-16任一项所述的连钱草提取物在制备减肥的药物中用途。
  20. 权利要求1-16任一项所述的连钱草提取物在制备治疗肾病的药物中用途。
  21. 权利要求1-16任一项所述的连钱草提取物在制备抑制二肽基肽酶IV活性的药物中的用途。
  22. 根据权利要求21所述的用途，其中所述抑制二肽基肽酶IV活性是指治疗糖尿病。
  23. 药物组合物，包含权利要求1-16任一项所述的连钱草提取物，和药学上可接受载体。
  24. 权利要求23所述的药物组合物，其中化合物I和化合物II占药物组合物中活性成分总重量的50％以上，或者从药物组合物中基本上除去化合物I和II后该药物组合物的生物活性如DPP4抑制活性降低50％以上。
  25. 含化合物I木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100005
    或其药学可接受的盐；和/或
    化合物II芹菜素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]
    Figure PCTCN2015074366-appb-100006
    或其药学可接受的盐；
    和/或化合物I和II或二者任一药学上可接受盐的混合物。
  26. 权利要求25所述的混合物在制备降血糖的药物中用途。
  27. 权利要求25所述的混合物在制备降血脂的药物中用途。
  28. 权利要求25所述的混合物在制备减肥的药物中用途。
  29. 权利要求25所述的混合物在制备治疗肾病的药物中用途。
  30. 权利要求25所述的混合物在制备抑制二肽基肽酶IV活性的药物中的用途。
  31. 权利要求25所述的用途，其中所述抑制二肽基肽酶IV活性是指治疗糖尿病。
  32. 药物组合物，包含权利要求25所述的混合物，和药学上可接受载体。
  33. 权利要求32所述的药物组合物，其中化合物I和化合物II占药物组合物中活性成分总重量的50％以上，或者从药物组合物中基本上除去化合物I和II后该药物组合物的生物活性如DPP4抑制活性降低50％以上。
PCT/CN2015/074366 2014-03-17 2015-03-17 连钱草提取物、制备方法及其用于降糖减肥降脂的用途 WO2015139603A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2017500116A JP6651498B2 (ja) 2014-03-17 2015-03-17 Ｇｌｅｃｈｏｍａ ｌｏｎｇｉｔｕｂａ （Ｎａｋａｉ） Ｋｕｐｒ．抽出物、その調製方法および血糖の低下、体重の減量または血液脂質の低下におけるその使用
US15/126,676 US10966996B2 (en) 2014-03-17 2015-03-17 Glechoma longitube extract, preparation method for same, and use thereof in sugar reduction, weight loss, and lipid reduction
EP15765481.5A EP3120847B1 (en) 2014-03-17 2015-03-17 Glechoma longituba extract for the treatment of kidney diseases or diabetes mellitus
CN201580011098.3A CN106061477B (zh) 2014-03-17 2015-03-17 连钱草提取物、制备方法及其用于降糖减肥降脂的用途
CN201911100870.5A CN111195257B (zh) 2014-03-17 2015-03-17 连钱草提取物、制备方法及其用于降糖减肥降脂的用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410104913.8 2014-03-17
CN201410104913 2014-03-17

Publications (1)

Publication Number Publication Date
WO2015139603A1 true WO2015139603A1 (zh) 2015-09-24

Family

ID=54143758

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/074366 WO2015139603A1 (zh) 2014-03-17 2015-03-17 连钱草提取物、制备方法及其用于降糖减肥降脂的用途

Country Status (5)

Country Link
US (1) US10966996B2 (zh)
EP (1) EP3120847B1 (zh)
JP (1) JP6651498B2 (zh)
CN (2) CN106061477B (zh)
WO (1) WO2015139603A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109091495A (zh) * 2018-08-22 2018-12-28 江西中医药大学 连钱草多糖在制备抗补体药物中的用途及其制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106061477B (zh) * 2014-03-17 2020-07-21 中国科学院上海药物研究所 连钱草提取物、制备方法及其用于降糖减肥降脂的用途
KR102147101B1 (ko) * 2018-06-19 2020-08-25 한국과학기술연구원 금전초 추출물을 포함하는 신경세포 보호용 조성물

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100905437B1 (ko) * 2008-11-24 2009-07-02 (주)동양정밀 아토피성 피부 치료제 조성물 및 그 제조방법

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4376977B2 (ja) 1995-09-22 2009-12-02 日本製粉株式会社 リパーゼ阻害剤、食品添加物及び食品
US7378115B2 (en) 2004-08-18 2008-05-27 Biologic Health Solutions Pty Ltd. Herbal compositions for the prevention or treatment of urinary incontinence and overactive bladder
JP2007099635A (ja) 2005-09-30 2007-04-19 Tokai Univ α−グルコシダーゼ阻害剤
KR100793019B1 (ko) 2005-11-25 2008-01-08 보령메디앙스 주식회사 항산화 및 항염 효과를 갖는 싸리, 금전초 및 반지련혼합추출물을 함유하는 화장료 조성물
JP2008199973A (ja) 2007-02-21 2008-09-04 Yaegaki Hakko Giken Kk 機能性食品
JP2012171951A (ja) 2011-02-24 2012-09-10 Kao Corp ミネラロコルチコイド受容体阻害剤
KR101395213B1 (ko) 2012-02-02 2014-05-16 (주)모아캠 레몬버베나 유래 루테오닌­７­디글루쿠로니드 함유 추출물 및 이를 포함하는 기능성 화장료 조성물
CN102746416B (zh) * 2012-07-27 2014-11-05 河南中医学院 一种连钱草多糖的制备方法及应用
CN103113196B (zh) 2013-02-05 2014-08-06 浙江大学 一种连钱草酚及其制备方法和应用
CN103340991B (zh) 2013-07-28 2014-12-24 郑宏伟 一种用于护理糖尿病患者的口服液及制备方法
CN106061477B (zh) * 2014-03-17 2020-07-21 中国科学院上海药物研究所 连钱草提取物、制备方法及其用于降糖减肥降脂的用途

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100905437B1 (ko) * 2008-11-24 2009-07-02 (주)동양정밀 아토피성 피부 치료제 조성물 및 그 제조방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN, ZENAI ET AL.: "Spectral Analysis Of Clerodendrin, Isolated From Clerodendron Trichotomum Thumb", ACTA PHARMACEUTICA SINICA, vol. 23, no. 10, 31 December 1988 (1988-12-31), pages 789 - 791, XP008182181 *
D. T. BERASHVILI ET AL.: "Luteolin diglucuronide from perilla nankinensis", CHEMISTRY OF NATURAL COMPOUNDS, vol. 42, no. 1, 31 December 2006 (2006-12-31), pages 106 - 107, XP019220095 *
YANG, NIANYUN ET AL.: "Chemical Constituents Of Glechoma Longituba", CHINESE JOURNAL OF NATURAL MEDICINES, vol. 4, no. 2, 31 March 2006 (2006-03-31), pages 98 - 100, XP008182177 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109091495A (zh) * 2018-08-22 2018-12-28 江西中医药大学 连钱草多糖在制备抗补体药物中的用途及其制备方法
CN109091495B (zh) * 2018-08-22 2020-09-29 江西中医药大学 连钱草多糖在制备抗补体药物中的用途及其制备方法

Also Published As

Publication number Publication date
US10966996B2 (en) 2021-04-06
JP6651498B2 (ja) 2020-02-19
EP3120847B1 (en) 2023-11-08
CN106061477A (zh) 2016-10-26
US20170087173A1 (en) 2017-03-30
EP3120847A1 (en) 2017-01-25
CN111195257A (zh) 2020-05-26
EP3120847A4 (en) 2017-03-22
CN111195257B (zh) 2023-06-20
JP2017508809A (ja) 2017-03-30
CN106061477B (zh) 2020-07-21

Similar Documents

Publication Publication Date Title
CN102526165B (zh) 一种红景天有效部位、其制备方法、其药物组合物及用途
US8829167B2 (en) Foliamangiferosides, preparation method and use thereof
US6399579B1 (en) Compositions comprising icariside I and anhydroicaritin and methods for making the same
Ouassou et al. Inhibition of α-glucosidase, intestinal glucose absorption, and antidiabetic properties by Caralluma europaea
CN107949394B (zh) 黑豆叶提取物在制备用于预防或治疗代谢综合征的药物中的用途
EP2664361B1 (en) Extract of fraxinus excelsior seeds and therapeutic applications therefor
US6376682B1 (en) Compound with α-glucosidase inhibiting action and method for producing the same
WO2015139603A1 (zh) 连钱草提取物、制备方法及其用于降糖减肥降脂的用途
KR101509554B1 (ko) 골질환의 예방 및/또는 치료용 의약 조성물, 그 조성물을 함유하는 기능성 식품 또는 건강 식품, 그리고 그 조성물을유효 성분으로 하는 의약 제제
KR100979459B1 (ko) 근육세포에서 포도당 흡수를 증가시키는 데이츄 추출물과이로부터 분리한 4ｈ-크로멘-4-온 유도체
WO2004039759A1 (fr) Compose naturel servant dans le traitement de diabetes, sa preparation et son utilisation
KR101676297B1 (ko) 이당류 소화효소활성 억제에 의한 혈당강하용 조성물
JP2009137920A (ja) グリコーゲンホスホリラーゼ阻害剤
CN112592328B (zh) 草豆蔻中二芳基庚烷-查尔酮聚合物及其药物组合物与应用
KR102066966B1 (ko) 상백피로부터 분리된 화합물을 포함하는 당뇨병의 예방 또는 치료용 약학 조성물
JP2000086653A (ja) 二糖加水分解酵素阻害剤
CN113929698A (zh) 二芳基庚烷二聚体及其药物组合物与其制备方法和应用
Choi et al. α-Glucosidase inhibitiors from seed extract of paeonia lactiflora
KR100895500B1 (ko) 호노키올을 유효성분으로 함유하는 지방간 질환의 예방 및치료용 조성물
KR102069125B1 (ko) 강황 추출물을 포함하는 간손상 예방 및 치료용 약학 조성물
KR20190093849A (ko) 김네마 실베스트레 추출물 또는 이로부터 분리한 화합물을 유효성분으로 포함하는 당뇨병 예방 또는 치료용 조성물
WO2017220051A2 (zh) 盐酸苄丝肼的药物组合物及其降血糖的医药用途
KR102092729B1 (ko) 강황 추출물을 포함하는 간손상 예방 및 치료용 약학 조성물
KR20230174959A (ko) 아마도리 화합물을 고농도로 함유하는 홍삼 추출물을 포함하는 식후 혈당상승억제용 조성물
KR100846441B1 (ko) 스테롤 계 유도체를 함유하는 약제학적 조성물, 건강식품조성물, 화장품 조성물 및 파나소이드 엑스 핵 수용체 활성억제제 조성물

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: 15765481

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017500116

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 15126676

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2015765481

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2015765481

Country of ref document: EP