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WO2009079921A1 - Saponines triterpénoïdes de cucurbitane, composition pharmaceutique de celles-ci, leur préparation et leur utilisation - Google Patents

Saponines triterpénoïdes de cucurbitane, composition pharmaceutique de celles-ci, leur préparation et leur utilisation Download PDF

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Publication number
WO2009079921A1
WO2009079921A1 PCT/CN2008/001955 CN2008001955W WO2009079921A1 WO 2009079921 A1 WO2009079921 A1 WO 2009079921A1 CN 2008001955 W CN2008001955 W CN 2008001955W WO 2009079921 A1 WO2009079921 A1 WO 2009079921A1
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WIPO (PCT)
Prior art keywords
compound
hydrogen
methanol
hydroxyl group
glucopyranosyl
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English (en)
Chinese (zh)
Inventor
Yang Ye
David E. James
Ying Leng
Minjia Tan
Jiming Ye
Xiao Liu
Changqiang Ke
Ying Feng
Tong Chen
Xiqiang Li
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Garvan Institute of Medical Research
Shanghai Institute of Materia Medica of CAS
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Garvan Institute of Medical Research
Shanghai Institute of Materia Medica of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • 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)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • 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

Definitions

  • the invention belongs to the field of medicinal chemistry, and more particularly to a cucurbitane-type triterpenoid saponin compound extracted from bitter gourd, a pharmaceutical composition thereof, a preparation method thereof and application thereof in preventing/treating diabetes, obesity and hyperlipemia .
  • T2D type II diabetes patients
  • insulin sensitizers which are drugs for treating diabetes by improving insulin resistance
  • AMPK adenosine monophosphate-activated protein kinase activators
  • T2D type II diabetes patients
  • PPARs peroxisome proliferator-activated receptors
  • AMPK adenosine monophosphate-activated protein kinase activators
  • the two commonly used drugs are thiazolidinediones (TZDs) and biguanides.
  • weight-loss drugs approved for long-term use on the market: one is orlistat, which is a specific gastrointestinal lipase inhibitor, but it has a very common gastrointestinal side reaction; the second is Xibuqu An inhibitor of monoamine reuptake, but Will raise blood pressure and speed up heart rate. Therefore, finding safe and effective weight-loss drugs is an urgent problem to be solved.
  • Momorafca L. is a cucurbitaceae plant of the genus Cucurbitaceae.
  • folks in China have been widely used in the treatment of diseases such as heat-clearing and detoxification, eyesight and detoxification, stomach and thirst, laxation, and deworming.
  • the chemical constituents of bitter gourd include triterpenoid saponins, cerebral glycosides, proteins and the like. The report on the hypoglycemic effect of bitter gourd is very extensive.
  • Chinese patent application CN1520838A "Bitter melon extract plant insulin; new use of bitter melon saponin” is complicated in the production process, and the obtained extract has many components, and the composition of the extract is not clear.
  • Chinese patent CN1209155C "Preparation of bitter melon products containing hypoglycemic active ingredients” described in the bitter melon products made of bitter gourd juice, the composition of which is extremely complex, and the polyphenol oxidase and protein electrophoresis detection provided by the patent Methods such as thin layer chromatography are not sufficient to explain the active ingredients and to perform corresponding quality control.
  • It is still another object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of the above-described triterpenoid saponin compound.
  • Still another object of the present invention is to provide a process for the preparation and separation of the above-mentioned trisaponin compound and its pharmaceutical composition from bitter gourd.
  • a further object of the present invention is to provide a use of the above triterpenoid saponin compound and a pharmaceutical composition thereof for the preparation of a medicament for preventing/treating diabetes, obesity and hyperlipemia.
  • the above-mentioned cucurane-type triterpenoid saponin compound is a muscle and fat cell glucose uptake promoter, an insulin sensitizer, a glucose transporter 4 (GLUT4) film promoter, a monophosphate in the preparation of a medicament for preventing and treating diabetes.
  • GLUT4 glucose transporter 4
  • the above-mentioned cucurane-type triterpenoid saponin compound is an adenosine monophosphate-activated protein kinase activator or a fatty acid oxidative metabolism promoter in the preparation of a medicament for preventing and treating obesity.
  • the above-mentioned cucurane-type triterpenoid saponin compound is a lipid triglyceride and free fatty acid level lowering agent or a lipid tolerance improving agent in a state of disorder of lipid metabolism in the preparation of a medicament for preventing and treating hyperlipemia.
  • the present invention provides a class of triterpenoid saponins extracted from bitter gourd having the activity of preventing/treating diabetes, obesity and hyperlipidemia, which has the structure shown in the following structural formula I:
  • R 2 is a hydroxyl group, hydrogen or ⁇ -D-glucopyranooxy group
  • R 3 is a hydroxyl group or hydrogen
  • R 4 is hydroxy, hydrogen, ⁇ -D-glucopyranosyloxy or ⁇ -D-allopyranosyloxy; R 5 is hydroxy or hydrogen;
  • R 6 is hydroxy, ⁇ -D-glucopyranoxy or hydrogen
  • R 7 is a methyl group or an aldehyde group
  • C5 and C6, C6 and C7, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form a double bond
  • R 3 , R 4 , and R 5 are a hydroxyl group, the chirality of C22, C23, and C24 is S, S, and configuration;
  • R 4 and R 5 are a hydroxyl group and R 3 is hydrogen
  • the chirality of C23 and C24 is 8, S configuration; when R 3 is a hydroxyl group and R 4 is a ⁇ -D-glucopyranoseoxy group.
  • C22 and C23 are in the S, S configuration;
  • C5 and C19 may be linked by an oxygen bridge
  • R 2 and R 6 are a hydroxyl group
  • R 3 , R 4 , and R 5 are hydrogen
  • C 5 and C 6 , C 23 and C 24 form a double bond
  • R 7 is not an aldehyde group
  • R 2 , R 5 , R 6 are hydrogen
  • R 3 is a hydroxyl group
  • R 7 is a methyl group
  • C6 and C7, C24 and C25 form a double bond
  • R 4 is not a ⁇ -D-allopyranosyloxy group.
  • the present invention provides a pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipemia, which comprises a therapeutically effective amount of one or more of triterpenoid saponins isolated from bitter gourd represented by the above formula I.
  • the composition may further comprise the known compound soy-cerebroside I, momor-cerebroside, tris, 3, 7,25-tris:-decorane-5,23 (E -diene- 19-aldehyde, 22 (S), 23 (R), 24 (R), 25-tetrahydroxy cucurbit-5- ling, triammonium momordicoside A, momordicoside B, momordicoside M or momordicoside N ( One or more of Magnetic Resonance In Chemistry, 2007, 63, 615-621), their specific structural formulas are:
  • the present invention provides the above-mentioned pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipemia, wherein the compound is derived from an extract of an active part of bitter gourd, and the extract is obtained by the following steps (preparation flow chart is shown in Fig. 1):
  • the above triterpenoid saponins are used for preparing glucose uptake in muscle and fat cells, promoting membrane transport of glucose transporter 4 (GLUT4), increasing adenosine monophosphate-activated protein kinase (AMPK) activity, promoting fatty acid oxidative metabolism, and improving insulin resistance. Glucose tolerance, lowering plasma triglyceride and free fatty acid levels or improving the use of lipid tolerance drugs in disorders of lipid metabolism.
  • the above triterpenoid saponins have a function of promoting glucose uptake and insulin sensitization in muscle and fat cells, have stable hypoglycemic activity in both type I and type II diabetes, and are capable of preventing/treating obesity and hyperlipidemia.
  • the above-described cucurane-type triterpenoid saponin compound of the present invention and a pharmaceutical composition thereof are a muscle and fat cell glucose uptake promoter, a membrane agonist for promoting glucose transporter 4 (GLUT4), and adenosine monophosphate-activating protein kinase (AMPK) Activator, a drug that promotes oxidative metabolism of fatty acids, improves glucose tolerance under insulin resistance, lowers plasma triglyceride and free fatty acid levels, and improves lipid tolerance in a state of disorder of lipid metabolism.
  • a muscle and fat cell glucose uptake promoter a membrane agonist for promoting glucose transporter 4 (GLUT4)
  • AMPK adenosine monophosphate-activating protein kinase
  • KG8 stimulated glucose uptake in both short-term and long-term fat cells, indicating that KG8 can increase insulin sensitivity.
  • type 1 diabetes model alloxan-induced diabetic mice, KG8 had a significant hypoglycemic effect at the 25 mg/kg administration level.
  • Ob/Ob mouse diabetes model KG8 can stably reduce blood glucose in mice for a long time.
  • the experiment proves that KG8 has obvious insulin sensitization effect and has good hypoglycemic effect on type I and type II diabetes.
  • KG8 and Compound F significantly improved glucose intolerance in a mouse glucose tolerance test in an insulin resistant state.
  • the compound significantly promoted the membrane of glucose transporter 4 (GLUT4) in muscle and fat cells.
  • AMPK adenosine monophosphate-activated protein kinase
  • compounds A and F significantly enhanced AMPK kinase activity.
  • Compound F significantly promoted glucose utilization in mice.
  • the compound of the present invention has an activity of promoting glucose uptake and insulin sensitization of muscle and fat cells to lower blood sugar.
  • KG8 and Compound F can significantly promote the acute fatty acid oxidative metabolism and overall energy consumption in mice and have the effect of treating obesity.
  • KG8 can significantly improve the lipid tolerance of DIO mice and reduce plasma triglyceride and free fatty acid levels.
  • KG8 has a therapeutic effect on diabetes in Ob/Ob mice, and KG8 can significantly reduce plasma and liver triglyceride levels in mice.
  • the composition of the compound of the present invention is capable of treating hyperlipidemia by lowering the levels of triglycerides and free fatty acids.
  • Figure 1 is a flow chart for the preparation of KG8.
  • Figure 2 is a flow chart for the separation of compounds A - I.
  • Figure 3 is a graph showing the effect of KG8 on glucose uptake in 3T3 - L1 cells.
  • Figure 4 is a graph showing the effect of KG8 on plasma triglycerides in Ob/Ob mice.
  • Figure 5 is a graph showing the effect of KG8 on hepatic triglyceride in Ob/Ob mice.
  • Figure 6 is an experimental diagram of KG8 improving glucose tolerance in insulin resistance.
  • Figure 7 is a graph showing the effect of KG8 on fatty acid oxidation metabolism in HF mice.
  • Figure 8 is a graph showing the effect of KG8 on lipid tolerance in DIO mice.
  • Figure 9 is a graph showing the effect of KG8 on plasma triglycerides in DIO mice.
  • Figure 10 is a graph showing the effect of KG8 on plasma free fatty acids in DIO mice.
  • Figure 11 is a graph of the effect of KG8 on oral glucose tolerance in DIO mice (plasma glucose concentration).
  • Figure 12 is a graph of the effect of KG8 on oral glucose tolerance in DIO mice (plasma insulin concentration).
  • Figure 13 is a graph of the effect of KG8 on insulin tolerance in DIO mice (plasma glucose concentration).
  • Figure 14 is a graph of the effect of KG8 on insulin tolerance in DIO mice (percentage of blood glucose reduction).
  • Figure 15 is a graph showing the membrane activity test of the compound on glucose transporter 4 (GLUT4).
  • Figure 16 is a graph showing the activity of adenosine monophosphate-activated protein kinase (AMPK) of compounds A and F.
  • Figure 17 is a graph showing the acute effect of Compound F on energy expenditure and fatty acid oxidation metabolism in mice.
  • AMPK adenosine monophosphate-activated protein kinase
  • Figure 18 is a graph showing the utilization of Compound F against glucose in mice.
  • the present invention provides a triterpenoid saponin compound isolated from bitter gourd and a pharmaceutical composition thereof, a preparation method thereof, and use in the prevention and treatment of diabetes, obesity and hyperlipemia.
  • the prevention/treatment of diabetes, obesity and hyperlipidemia means promoting glucose absorption, resistance and reduction of fasting and postprandial hyperglycemia, insulin-like action and insulin sensitization of muscle and fat cells, Promotes the membrane of glucose transporter 4 ( GLUT4 ), increases adenosine monophosphate-activated protein kinase (AMPK ) activity, promotes fatty acid oxidative metabolism, improves glucose tolerance under insulin resistance, lowers plasma triglyceride and free fatty acid levels, and Improve lipid tolerance in a state of lipid metabolism disorder.
  • GLUT4 glucose transporter 4
  • AMPK adenosine monophosphate-activated protein kinase
  • the present invention provides a class of triterpenoid saponins which are extracted from bitter gourd and which have preventive/treating diabetes, obesity and hyperlipemia activity. It will be readily understood by those skilled in the art that the present invention relates to compounds having asymmetric carbon atoms (chiral centers) or double bonds, as well as racemates containing these compounds, diastereomers, enantiomers, geometries of these compounds isomer.
  • the triterpenoid saponin compound of the present invention has a structure represented by the following structural formula I:
  • ⁇ -D-glucopyranosyl is hydrogen, ⁇ -D-glucopyranosyl, ⁇ -D-allopyranosyl, ⁇ -D-glucopyranosyl (1 ⁇ 6) _ ⁇ -D-glucopyranosyl or ⁇ -D-xylopyranosyl ( 1 ⁇ 4) - [ ⁇ -D-glucopyranosyl ( 1 ⁇ 6) ] - ⁇ -D-glucopyranosyl;
  • R 2 is a hydroxyl group, hydrogen or ⁇ -D-glucopyranooxy group
  • R 3 is hydroxy or hydrogen
  • R 4 is hydroxy, hydrogen, ⁇ -D-glucopyranosyloxy or ⁇ -D-allopyranosyloxy; R 5 is hydroxy or hydrogen;
  • R 6 is hydroxy, ⁇ -D-glucopyranoxy or hydrogen
  • R 7 is a methyl group or an aldehyde group
  • C5 and C6, C6 and C7, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form a double bond
  • R 3 , R 4 , and R 5 are hydroxyl groups, the chirality of C22, C23, and C24 is the S, S, and R configurations;
  • R 4 and R 5 are a hydroxyl group and R 3 is hydrogen
  • the chirality of C23 and C24 is 8, S configuration; when R 3 is a hydroxyl group and R 4 is a ⁇ -D-glucopyranoseoxy group.
  • C22 and C23 are in the S, S configuration;
  • C5 and C19 can be linked by an oxygen bridge
  • R 2 and R 6 are a hydroxyl group
  • R 3 , R 4 , and R 5 are hydrogen
  • C 5 and C 6 , C 23 and C 24 form a double bond
  • R 7 is not an aldehyde group
  • R 2 , R 5 , R 6 are hydrogen
  • R 3 is a hydroxyl group
  • R 7 is a methyl group
  • C6 and C7, C24 and C25 form a double bond
  • R 4 is not a ⁇ -D-allopyranosyloxy group.
  • the compound of the present invention is a natural product.
  • the compound of formula I is a compound having the formula la:
  • Ri is ⁇ -D-glucopyranosyl or ⁇ -D-allopyranosyl or hydrogen;
  • R 3 is a hydroxyl group or hydrogen
  • R 4 is hydroxy, hydrogen, ⁇ -D-glucopyranosyloxy or ⁇ -D-allopyranosyloxy;
  • R 5 is a hydroxyl group or hydrogen
  • R 6 is hydroxy, ⁇ - D - glucopyranosyloxy or hydrogen
  • C22 and C23, C23 and C24, C24 and C25, C25 and C27 form a double bond; and in the case where R 3 , R 4 and R 5 are hydroxyl groups, the chirality of C22, C23, C24 is S , S, R configuration; in the case where R 4 and R 5 are hydroxyl groups and R 3 is hydrogen, the chirality of C23 and C24 is 8, S configuration; and when it is ⁇ -D-glucopyranosyl or ⁇ -D-allopyranosyl, R 5 and R 6 are hydrogen, R 3 is a hydroxyl group, and when C24 and C25 form a double bond, R 4 is not ⁇ -D-allopyranosyloxy.
  • the compound of formula I is a compound having the formula lb:
  • R 2 is hydroxy, hydrogen or a ⁇ -D- glucopyranosyl group
  • R 3 is a hydroxyl group or hydrogen
  • R 4 is hydroxy, hydrogen or ⁇ -D-glucopyranoseoxy
  • R 5 is a hydroxyl group or hydrogen
  • R 6 is hydroxy, ⁇ -D-glucopyranoxy or hydrogen
  • R 7 is a methyl group or an aldehyde group
  • C22 and C23, C23 and C24, C24 and C25, C25 and C27 form a double bond; and in the case where R 3 , R 4 and R 5 are hydroxyl groups, the chirality of C22, C23, C24 is S , S, R configuration; and in the case where R 3 is a hydroxyl group and R 4 is a ⁇ -D-glucopyranoseoxy group, C22 and C23 are in the S, S configuration;
  • Ri is hydrogen, ⁇ -D-glucopyranosyl (1 ⁇ 6)- ⁇ -D-glucopyranosyl or ⁇ -D-xylopanosyl (1 ⁇ 4)-[ ⁇ -D - glucopyranosyl ( 1 ⁇ 6 ) ] - ⁇ - D - glucopyranosyl
  • R 2 is hydrogen, and R 3 , R 4 , R 5 , and R 6 are hydroxy groups, and R 7 is not a methyl group;
  • R 2 and R 6 are a hydroxyl group
  • R 3 , R 4 and R 5 are hydrogen
  • C23 and C24 form a double bond
  • R 7 is not an aldehyde group
  • R is ⁇ - D -xypyranosyl ( 1 ⁇ 4 ) - [ ⁇ - D - Portuguese
  • R 2 is hydrogen
  • R 6 is hydrogen
  • R 7 is a sulfhydryl group
  • the active site extract KG8 was subjected to column chromatography using 100-200 mesh silica gel, followed by chloroform:methanol in a volume ratio of 40:3:1, 20:3:1, 10:3:1 and 65:35:10:
  • the lower layer of water is eluted by TLC plate.
  • the volume ratio of the developing agent to the volume ratio of 10:1, 6:1, 4:1 of chloroform:methanol is 10:3:1, 65:35: 10 chloroform: methanol: water lower layer liquid, 5% sulfuric acid-vanillin color development, according to the TLC plate, the similar components were combined and concentrated, according to the Rf value between 0.6 and 0.2 from large to small 1- 9 nine components;
  • the fraction 3 was subjected to silica gel column chromatography, eluted with chloroform:methanol in a volume ratio of 10:1, and detected by a TLC plate.
  • the developing solvent was chloroform: methanol, 5% sulfuric acid-vanillaldehyde in a volume ratio of 8:1. Coloring, the eluate according to the spot on the TLC plate showing Rf value of about 0.4 is combined and concentrated to obtain the compound I;
  • the fraction 4 was chromatographed on a MCI column in a volume ratio of 50:50 to 90:10 methanol:water eluent, and the obtained 70:30 methanol:water eluent fraction was subjected to silica gel column chromatography.
  • the elution was carried out by using a volume ratio of 9:1 chloroform:methanol, and the developing agent was a chloroform:methanol with a volume ratio of 5:1, and 5% sulfuric acid-vanillin was developed, and the Rf value was displayed according to the TLC plate. Speckles approximately equal to 0.3 and 0.4 respectively
  • the eluate is combined and concentrated to obtain Compound A and Compound B;
  • Fraction 6 was chromatographed on a MCI column using a methanol:water eluent gradient of 30:70 to 80:20 to give a 60:40 methanol:water eluent fraction for RP _ 18 column. Chromatography, volume ratio
  • Fraction 7 was chromatographed on a MCI column using a methanol:water eluent gradient of 30:70 to 80:20 to give a 50:50 methanol:water eluent fraction and a RP-18 column. Chromatography, volume ratio
  • Fraction 8 was chromatographed on a MCI column using a methanol:water eluent gradient of 30:70 to 70:30 to give a 40:60 methanol:water eluent fraction and a RP-18 column. Chromatography, volume ratio
  • Fraction 9 was chromatographed on a MCI column, eluting with a methanol:water eluent gradient of 20:80 to 60:40.
  • the 40:60 methanol:water eluent fraction was subjected to silica gel column chromatography. , chloroform with a volume ratio of 10:3:1: methanol: liquid eluted in the underwater layer, detected by TLC plate, the developing agent is a chloroform: 65:45:1 by volume: methanol: underwater liquid, 5% sulfuric acid - pandan
  • the aldehyde color will be displayed according to the TLC plate.
  • the present invention provides a pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipidemia, which comprises a therapeutically effective amount of one or more of the triterpenoid saponin compounds isolated from bitter gourd represented by the above structural formula I .
  • the composition may further comprise a known compound, soya-cerebroside I, momor-cerebroside, three, which is extracted from bitter gourd.
  • composition may further comprise pharmaceutically conventional excipients such as various solvents which are commonly used in pharmacy.
  • the composition can be prepared by a pharmaceutically conventional preparation method.
  • the present invention provides the pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipidemia as described above, wherein the compound is derived from an extract of the active part of Momordica charantia.
  • the bitter gourd plant is a Cucurbitaceae Momordica plant, or more specifically a Momordica charantia.
  • the bitter gourd active site extract may comprise a natural product of triterpenoid saponin, such as a compound of formula Ia, lb, soy-cerebroside I, momordica
  • momor-cerebroside triterpenoid 3,7,25-trihydroxy cucurbitane-5,23 ( )-dien-19-aldehyde, 22 ( S ) , 23 ( R ) , 24 ( R ) , 25-four a mixture of any one or more of hydroxy valerane-5-ene, saponin momordicoside A, momordicoside B, momordicoside M and momordicoside N, more specifically any one or more of the compounds described in Table 1. mixture.
  • TLC Thin layer chromatography
  • the ratio of solvents involved is a volume ratio.
  • aqueous solution was then liquid-liquid partitioned with n-butanol (20 L) to obtain 800 g of n-butanol.
  • 800 g of the n-butanol fraction was mixed with 500 g of AB _ 8 macroporous resin (from Tianjin Bone Glue Factory), and the sample-mixed resin was placed on a chromatography column containing 3 kg of AB-8 resin.
  • the fractions were respectively eluted with 12 L of pure water, 30:70 alcohol/pure water (volume ratio), and 95:5 alcohol/pure water (volume ratio) to obtain KG6 600g, KG7 60g, and KG8 80g, respectively.
  • Example 2 Example 2
  • Figure 2 illustrates the separation of compound A _ I by chromatography.
  • Example 1 80 g of the active site extract KG8 obtained in the above Example 1 was subjected to column chromatography using 2 kg of 100-200 mesh silica gel, and the volume ratios were 40:3:1, 20:3:1, 10:3:1 and 65: 35: 10 chloroform: methanol: The lower layer of water is eluted with 10L of water, and the concentration per 500ml is one minute.
  • the volume of the liquid or volume ratio of chloroform:methanol can be 10:1, 6:1, 4:1 by the TLC plate.
  • the fraction 3 was subjected to silica gel column chromatography and eluted with a volume ratio of 10:1 of chloroform:methanol 1 L, and the concentration per 20 ml was one minute, and the developing solvent was a chlorine ratio of 8:1 by a TLC plate.
  • methanol, 5% sulfuric acid-vanillin coloring the eluate according to the spot on the TLC plate showing Rf value of about 0.4 is combined and concentrated to obtain a compound I 9mg;
  • Fraction 4 was chromatographed on a MCI column in a volume ratio of 50:50 to 90:10 methanol: 1 L of water eluent, and the obtained 70:30 methanol: water eluent component was further subjected to silica gel column chromatography.
  • Fraction 5 was eluted by MCI column chromatography with a volume ratio of 30:70 to 80:20 in methanol:water eluent, 1 L gradient, and the obtained 60:40 methanol:water eluent fraction was subjected to silica gel column. Chromatography, volume ratio of 20:3:1 chloroform:methanol: 1.5L of aqueous layer liquid elution, one cent per 20ml concentration, detected by TLC plate, the developing agent is chloroform with a volume ratio of 4:1: Methanol, 5% sulfuric acid-vanillin coloring, the eluate according to the spot on the TLC plate showing Rf value of about 0.3, respectively, is combined and concentrated to obtain the compound momordicoside M 230mg;
  • Fraction 6 was chromatographed on a MCI column in a volume ratio of 30:70 to 80:20 in methanol:water eluent to a gradient of 1 L each, and the resulting 60:40 methanol:water eluent fraction was subjected to RP- 18 column chromatography, volume ratio of 40:60-60:40 methanol: water 500ml gradient elution, by TLC plate detection, the developer is a volume ratio of 10:3:1 chloroform: methanol: underwater layer liquid, 5% sulfuric acid-vanillin color development, will be based on TLC board An eluate showing spots with an Rf value of approximately 0.4 was combined and concentrated to obtain a compound momordicoside N 21 mg;
  • Fraction 7 was chromatographed on a MCI column in a volume gradient of 30:70 to 80:20 in a 1 L gradient of methanol:water eluent.
  • the 50:50 methanol:water eluent fraction was then subjected to RP- 18 column chromatography, volume ratio of 40:60 ⁇ 60:40 methanol: water gradient of 500ml each, a concentration of 20ml per 20ml, detected by TLC plate, the volume ratio of the developer is 8:3:1
  • Chloroform: methanol: aqueous layer liquid, 5% sulfuric acid-vanillin coloring the eluate according to the spot on the TLC plate showing Rf value of about 0.4 is combined and concentrated to obtain compound C 25mg;
  • Fraction 8 was chromatographed on a MCI column in a volume gradient of 30:70 to 70:30 in a 1 L gradient of methanol:water eluent.
  • the 40:60 methanol:water eluent fraction was then subjected to RP- 18 column chromatography, volume ratio of 30:70 ⁇ 60:40 methanol: water gradient of 500ml each, a concentration of 20ml per 20ml, detected by TLC plate, the volume ratio of the developer is 8:3:1
  • Chloroform: methanol: aqueous layer liquid, 5% sulfuric acid-vanillin coloring the eluate according to the spot on the TLC plate showing Rf value of about 0.3 is combined and concentrated to obtain compound H 30mg;
  • Fraction 9 was chromatographed on a MCI column in a volume ratio of 20:80 to 60:40 in methanol:water eluent to a gradient of 1 L each, and the resulting 40:60 methanol:water eluent fraction was passed through a silica gel column.
  • the compound A was hydrolyzed in a 0.5 M aqueous solution of hydrochloric acid at room temperature for 7 days, and the obtained reactant was subjected to preparative thin layer chromatography, and the developing solvent was chloroform:methanol 6:1, and the Rf value of 0.5-0.6 was collected and washed with chloroform:methanol 8:2. Deprotection and concentration gave compound D 10 mg.
  • VU VU nn VU VU nn .
  • 3T3-L1 cells were completely differentiated into adipocytes, they were cultured for 16 hours in serum-free DMEM containing 0.5% BSA, and then added to KG8 (final concentration 2 ( ⁇ g/ml) for 4 days.
  • the blank control group was added with an equal volume of DMS0.
  • the cells were washed twice with I xPBS pre-warmed at 37 ° C, and added Krebs buffer containing no or insulin-containing 0.5% BSA (NaCl 140 mM, KC1 5 mM, MgS0 4 2.5 mM, CaCl 2 I mM, HEPES 20 mM, pH 7.4), after incubation at 37 ° C for 40 minutes, add 2 - [1,2-3H(N )]-deoxy-D-glucose solution (final concentration 0.5 ⁇ / ⁇ 1), and act at 37 °C for 5 minutes.
  • BSA NaCl 140 mM, KC1 5 mM, MgS0 4 2.5 mM, CaCl 2 I mM, HEPES 20 mM, pH 7.4
  • the reaction was terminated by washing 3 times with ice bath in I xPBS, and then 0.15 ml of 0.1% Triton lysed cells were added, and the cells were flash-counted, and the CPM value was corrected by the amount of protein to calculate the glucose uptake of 3T3-L1 cells.
  • mice ICR male mice (30-35 g) were injected into the prepared alloxan solution 60 mg/kg via the tail vein. After 7 days, fasting for 6 hours, blood was taken from the eyelids, and plasma was separated and determined by glucose oxidase method. blood sugar. Mice with predicted blood glucose levels of l l. l-30.0 mmol/L were selected as experimental diabetic mice. The selected diabetic mice were fasted for 6 hours and fasting blood glucose was measured, 8 in each group. The mice in the administration group were orally administered with 25 mg/kg of KG8, the positive control group was given 150 mg/kg of metformin hydrochloride, and the blank control group was given 1% CMC. Each group of mice was continuously administered for 21 days, and the fasting blood glucose of the mice was measured on the 7th, 14th, and 21st days after administration.
  • Ob/Ob mice showed a significant decrease in blood glucose levels after administration of 50 mg/kg KG8 compared with before administration; after 21 days of administration, mouse plasma (Fig. 4) and liver (Fig. 5) triglyceride levels were compared with the control group. Compared to the significant drop.
  • Dosage Pre-dose blood glucose Post-dose blood glucose (mmol/1) mg/kg (mmol/1) 7 days 14 days 21 days
  • KG8 improves glucose tolerance in insulin resistance (Fig. 6) C57BL/6J male mice (8-10 weeks old), fed with high fat diet for 8 weeks, induced insulin resistance in mice (expressed glucose intolerance) ). In the last two weeks, high-fat diets were added to KG8 (100 mg/kg daily) or the hypoglycemic agent metformin (Met, 200 mg/kg daily). By measuring fasting 5-7 small Glucose tolerance (intraperitoneal injection of 2.0 g/kg glucose) was used to evaluate insulin sensitivity.
  • Normal control group Glucose-tolerant normal mice fed with normal diet (sensitive to insulin).
  • High-fat model group Control mice with impaired glucose tolerance with high-fat diet (no drug added).
  • High-fat metformin group Mice with high-fat diet added with metformin.
  • High-fat KG8 group High-fat diet mice added to KG8.
  • Test Example 5
  • KG8 in high-fat diet mice can affect fatty acid oxidative metabolism in mice by reducing respiratory exchange rate (RER).
  • RER respiratory exchange rate
  • the mice were placed in metabolic cages for 24 hours starting at 10 am.
  • KG8 100 mg/kg was added to the food 2 weeks before the measurement.
  • the food itself is used as a solvent control (Veh).
  • DIO mice were divided into 4 groups according to body weight and blood glucose, and KG8 (50, 100 mg/kg), metformin (Met, 250 mg/kg) or model control (1% CMC) were administered daily for 32 days.
  • KG8 50, 100 mg/kg
  • metformin Metal, 250 mg/kg
  • model control 1% CMC
  • mice were orally administered with a lipid tolerance test (oral long-chain fatty acid 10 ml/kg); 32 days after the administration, the plasma triglyceride and free fatty acid levels were measured after 6 hours of fasting.
  • lipid tolerance test oral long-chain fatty acid 10 ml/kg
  • the insulin tolerance test (intraperitoneal injection of 0.5 U/kg) was performed for 6 hours after fasting; on the 28th day after administration, the mice were orally administered for 6 hours after the glucose tolerance test (orally 2.5). g/kg glucose), in order to evaluate the improvement of KG8 on insulin resistance.
  • Compound F is used for glucose in mice (Fig. 18)
  • mice The acute effect of Compound F on blood glucose in normal diet mice.
  • the mice were intraperitoneally injected with Compound F or physiological saline (control group) for 60 minutes, then the mice were injected with glucose, and then blood glucose was measured at a given time.
  • compound F can significantly promote glucose utilization in glucose-tolerant mice.

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Abstract

La présente invention concerne le domaine pharmaceutique et décrit des saponines triterpénoïdes de cucurbitane extraites de Momordica charantia, une composition pharmaceutique qui les contient et leur préparation. L'invention concerne également l'utilisation de tels composés dans la préparation de médicaments pour le traitement du diabète, de l'obésité et de l'hyperlipémie. Les saponines décrites dans l'invention sont représentées par la formule I.
PCT/CN2008/001955 2007-11-30 2008-12-01 Saponines triterpénoïdes de cucurbitane, composition pharmaceutique de celles-ci, leur préparation et leur utilisation Ceased WO2009079921A1 (fr)

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WO2011107494A1 (fr) 2010-03-03 2011-09-09 Sanofi Nouveaux dérivés aromatiques de glycoside, médicaments contenants ces composés, et leur utilisation
WO2011161030A1 (fr) 2010-06-21 2011-12-29 Sanofi Dérivés de méthoxyphényle à substitution hétérocyclique par un groupe oxo, leur procédé de production et leur utilisation comme modulateurs du récepteur gpr40
WO2012004270A1 (fr) 2010-07-05 2012-01-12 Sanofi Dérivés 1,3-propanedioxyde à substitution spirocyclique, procédé de préparation et utilisation comme médicament
WO2012004269A1 (fr) 2010-07-05 2012-01-12 Sanofi Dérivés d'acide ( 2 -aryloxy -acétylamino) - phényl - propionique, procédé de production et utilisation comme médicament
WO2012010413A1 (fr) 2010-07-05 2012-01-26 Sanofi Acides hydroxy-phényl-hexiniques substitués par aryloxy-alkylène, procédé de production et utilisation comme médicament
WO2013037390A1 (fr) 2011-09-12 2013-03-21 Sanofi Dérivés amides d'acide 6-(4-hydroxyphényl)-3-styryl-1h-pyrazolo[3,4-b]pyridine-4-carboxylique en tant qu'inhibiteurs de kinase
WO2013045413A1 (fr) 2011-09-27 2013-04-04 Sanofi Dérivés d'amide d'acide 6-(4-hydroxyphényl)-3-alkyl-1h-pyrazolo[3,4-b] pyridine-4-carboxylique utilisés comme inhibiteurs de kinase
CN115724900A (zh) * 2022-12-14 2023-03-03 广西师范大学 达玛烷型三萜皂苷类化合物及其制备方法和在制备降血糖药物中的应用

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CN109265510A (zh) * 2018-11-06 2019-01-25 广东省生物工程研究所（广州甘蔗糖业研究所） 一种四环三萜类化合物及其制备方法
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011107494A1 (fr) 2010-03-03 2011-09-09 Sanofi Nouveaux dérivés aromatiques de glycoside, médicaments contenants ces composés, et leur utilisation
WO2011161030A1 (fr) 2010-06-21 2011-12-29 Sanofi Dérivés de méthoxyphényle à substitution hétérocyclique par un groupe oxo, leur procédé de production et leur utilisation comme modulateurs du récepteur gpr40
WO2012004270A1 (fr) 2010-07-05 2012-01-12 Sanofi Dérivés 1,3-propanedioxyde à substitution spirocyclique, procédé de préparation et utilisation comme médicament
WO2012004269A1 (fr) 2010-07-05 2012-01-12 Sanofi Dérivés d'acide ( 2 -aryloxy -acétylamino) - phényl - propionique, procédé de production et utilisation comme médicament
WO2012010413A1 (fr) 2010-07-05 2012-01-26 Sanofi Acides hydroxy-phényl-hexiniques substitués par aryloxy-alkylène, procédé de production et utilisation comme médicament
WO2013037390A1 (fr) 2011-09-12 2013-03-21 Sanofi Dérivés amides d'acide 6-(4-hydroxyphényl)-3-styryl-1h-pyrazolo[3,4-b]pyridine-4-carboxylique en tant qu'inhibiteurs de kinase
WO2013045413A1 (fr) 2011-09-27 2013-04-04 Sanofi Dérivés d'amide d'acide 6-(4-hydroxyphényl)-3-alkyl-1h-pyrazolo[3,4-b] pyridine-4-carboxylique utilisés comme inhibiteurs de kinase
CN115724900A (zh) * 2022-12-14 2023-03-03 广西师范大学 达玛烷型三萜皂苷类化合物及其制备方法和在制备降血糖药物中的应用
CN115724900B (zh) * 2022-12-14 2024-06-07 广西师范大学 达玛烷型三萜皂苷类化合物及其制备方法和在制备降血糖药物中的应用

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