CN101450963B - Gourd alkane type triterpene saponin compounds, medicament composition thereof as well as preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of medicinal chemistry, and specifically discloses a cucurbitane-type triterpene saponin compound extracted from bitter melon, a pharmaceutical composition thereof, a preparation method thereof, and an application in preparation of drugs for preventing/treating diabetes, obesity and hyperlipidemia. The structural formula of the saponins compound disclosed in the present invention is shown in formula I below.

Description

Cucurbitane-type triterpene saponin compound, its pharmaceutical composition, its preparation method and use

technical field

The invention belongs to the field of medicinal chemistry, and more specifically relates to a class of cucurbitane-type triterpene saponins extracted from bitter gourd, its pharmaceutical composition, its preparation method and its application in the prevention/treatment of diabetes, obesity and hyperlipidemia . the

Background technique

There are more than 150 million people with diabetes in the world, and it is expected to reach 300 million by 2025. Among them, mainly patients with type 2 diabetes (T2D). Since the main metabolic abnormality of type II diabetes is insulin resistance, insulin sensitizers, that is, drugs for the treatment of diabetes by improving insulin resistance, are one of the hotspots in current diabetes drug research. There are two pharmacological pathways to improve insulin resistance: one is through peroxisome proliferator-activated receptors (PPARs), or through the activation of protein kinase activator (AMPK) by adenosine monophosphate. The two most commonly used drugs are thiazolidinediones (TZDs) and biguanides. Although thiazolidinediones are widely used, they have many adverse reactions, such as obesity, fluid retention and heart failure. Metformin, a biguanide that does not cause obesity, is not a satisfactory treatment for diabetes because it primarily acts on the liver rather than muscle. Searching for more ideal insulin sensitizers is a hot spot in diabetes drug research in the world. the

At present, there are more than 1 billion overweight adults in the world, of which 300 million are obese patients, and the trend is rising sharply, which also leads to the incidence of obesity-related diseases (such as type II diabetes, heart disease, stroke and high blood pressure, etc.) of continuously rising. The main causes of overweight and obesity are high-fat and high-calorie diet, lack of exercise and accelerated urbanization. At present, there are only two weight-loss drugs approved for long-term use on the market: one is orlistat, which is a specific gastrointestinal lipase inhibitor, but it has very common gastrointestinal adverse reactions; the other is sibutril. It has been shown to be a monoamine reuptake inhibitor, but it increases blood pressure and heart rate. Therefore, finding safe and effective weight-loss drugs is an urgent problem to be solved at present. the

Bitter melon (Momordica charantia L.) is a plant of the genus Momordica cucurbitaceae. For a long time, folks in my country have widely used bitter gourd to clear away heat and detoxify, improve eyesight and detoxify, invigorate the stomach and quench thirst, laxative, and deworming. The chemical components in bitter gourd include triterpenoid saponins, cerebrosides, proteins, etc. There are extensive reports on the hypoglycemic effect of bitter gourd. the

International patent application WO0061619 reports that bitter gourd polypeptide K has the effects of lowering blood sugar, lowering blood fat, relieving pain and inflammation, lowering blood pressure and increasing immunity. WO9843484 reported that bitter gourd polypeptide MC6 and its derivatives have oral hypoglycemic effect. They all reported the activity of polypeptide compounds contained in bitter melon. the

Chinese patent CN1069534C "natural charantin hypoglycemic drug and its preparation method" does not specify the chemical composition of natural charantin hypoglycemic drug, and does not clarify its insulin-sensitizing effect; CN1098858C "extraction process of charantin" does not determine charantin structure, and its activity on type I and type II diabetes has not been clarified. The extract KGE obtained in the Chinese patent application CN1528785A "Resin Preparation Method of Bitter Melon Hypoglycemic Extract (KGE)" has complex components, and its chemical components have not been clarified. Chinese patent application CN1520838A "Bitter Melon Extract Phytoinsulin; New Use of Momordica Momordica Saponin" stated that the production process is complicated, and the obtained extract has many and miscellaneous components, and the composition of the extract is not clear at the same time. Chinese patent CN1209155C "Preparation of Bitter Gourd Products Containing Hypoglycemic Active Components" describes the bitter gourd products of different formulations made from bitter gourd juice, which contain extremely complex ingredients, and the polyphenol oxidase and protein electrophoresis detection provided in the patent , thin-layer chromatography and other methods are not enough to explain its active ingredients and carry out corresponding quality control. the

In summary, in the prior art, there is no specific compound, chemical structure and pharmacological data of cucurbitane-type triterpene saponins extracted from Momordica charantia that have the biological activity of preventing/treating diabetes, obesity and hyperlipidemia. the

Contents of the invention

Aiming at the above-mentioned defects in the prior art, an object of the present invention is to provide a class of cucurbitane-type triterpene saponins extracted from Momordica balsam pear which has the activity of preventing/treating diabetes, obesity and hyperlipidemia. the

Another object of the present invention is to provide a pharmaceutical composition containing a therapeutically effective amount of the above-mentioned triterpene saponins. the

Another object of the present invention is to provide a preparation method for extracting and isolating the above-mentioned triterpene saponins and pharmaceutical compositions thereof from Momordica charantia. the

Another object of the present invention is to provide the application of the above-mentioned triterpene saponin compound and its pharmaceutical composition in the preparation of drugs for the prevention/treatment of diabetes, obesity and hyperlipidemia. the

The above-mentioned cucurbitane-type triterpene saponins are used in the preparation of drugs for the prevention and treatment of diabetes as muscle and fat cell glucose uptake promoters, insulin sensitizers, glucose transporter 4 (GLUT4) membrane promoters, adenomonophosphate Glycoside-activated protein kinase activator, fatty acid oxidation metabolism enhancer or glucose tolerance improver in insulin resistant state. the

The above-mentioned cucurbitane-type triterpene saponins compound is an adenosine monophosphate-activated protein kinase activator or a fatty acid oxidation metabolism accelerator in the application of preparing medicines for preventing and treating obesity. the

The above-mentioned cucurbitane-type triterpene saponins compound is used as an agent for lowering plasma triglyceride and free fatty acid levels or an agent for improving lipid tolerance in the state of lipid metabolism disorder in the preparation of drugs for the prevention and treatment of hyperlipidemia. the

A class of triterpene saponins extracted from Momordica balsam pear with the activity of preventing/treating diabetes, obesity and hyperlipidemia provided by the present invention has the structure shown in the following structural formula I:

Wherein, R ₁ is hydrogen, β-D-glucopyranosyl, β-D-alopyranosyl, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl Glycosyl or β-D-xylopyranosyl (1→4)-[β-D-glucopyranosyl (1→6)]-β-D-glucopyranosyl;

R ₂ is hydroxyl, hydrogen or β-D-glucopyranosyloxy;

R ₃ is hydroxyl or hydrogen;

R ₄ is hydroxyl, hydrogen, β-D-glucopyranosyloxy or β-D-allopyranosyloxy;

R ₅ is hydroxyl or hydrogen;

R ₆ is hydroxyl, β-D-glucopyranosyloxy or hydrogen;

R ₇ is a methyl group or an aldehyde group;

Or alternatively, C5 and C6, C6 and C7, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form double bonds;

And when R ₃ , R ₄ , and R ₅ are hydroxyl groups, the chiralities of C22, C23, and C24 are S, R, and R configurations;

When R ₄ and R ₅ are hydroxyl and R ₃ is hydrogen, the chiralities of C23 and C24 are R and S configurations;

When R ₃ is hydroxyl and R ₄ is β-D-glucopyranosyloxy, C22 and C23 are S, R configurations;

In the case where R is _methyl , C5 and C19 can be connected by an oxygen bridge;

And when R ₁ is hydrogen, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl or β-D-xypyranosyl (1→4)-[β- D-glucopyranosyl (1→6)]-β-D-glucopyranosyl, R ₂ is hydrogen, R ₃ , R ₄ , R ₅ , R ₆ are hydroxyl, and C5 and C6 form a double bond , R ₇ is not methyl;

When R ₂ and R ₆ are hydroxyl, R ₁ , R ₃ , R ₄ and R ₅ are hydrogen, and C ₅ and C ₆ , C23 and C24 form double bonds, R ₇ is not an aldehyde group;

When R ₁ is β-D-glucopyranosyl or β-D-alopyranosyl, R ₂ , R ₅ , R ₆ are hydrogen, R ₃ is hydroxyl, R ₇ is methyl, and C6 and C7, C24 and C25 form a double bond, and when C5 and C19 are connected by an oxygen bridge, R ₄ is not β-D-alopyranosyloxy.

A pharmaceutical composition for the prevention/treatment of diabetes, obesity and hyperlipidemia provided by the present invention, the composition comprises one or more of the triterpene saponins isolated from Momordica charantia in a therapeutically effective amount shown in the above structural formula I kind. In addition, the composition may further comprise known compounds daidzein I (soya-cerebroside I), momor-cerebroside (momor-cerebroside), triterpene 3,7,25-trihydroxycucurbitane-5,23(E) -diene-19-al, 22(S), 23(R), 24(R), 25-tetrahydroxycucurbitane-5-ene, triterpenoid saponins momordicoside A, momordicoside B, momordicoside M or momordicoside N (Magnetic One or more in Resonance In Chemistry, 2007, 63, 615-621), their specific structural formulas are respectively:

Daidzein I Momordica glycosides

Triterpene 3,7,25-trihydroxycucurbitane-5,23(E)-diene-19-aldehyde Triterpene saponin momordicoside A

22(S), 23(R), 24(R), 25-tetrahydroxycucurbitane-5-ene Triterpene saponin momordicoside B

momordicoside M momordicoside N. the

The above-mentioned pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipidemia provided by the present invention, the compound is derived from the extract of the active part of bitter melon, and the extract is prepared through the following steps (the preparation flow chart is shown in Figure 1):

(a) Soak the bitter gourd sample with alcohol, water or a mixture thereof, and obtain the extract after filtering; wherein the alcohol is methanol or ethanol;

(b) evaporation of the solvent in the obtained extract to form a slurry;

(c) adding water to the slurry to obtain an aqueous suspension of the total extract;

(d) Liquid-liquid extraction of the above suspension with chloroform, dichloromethane or a mixture thereof to obtain an extract of chloroform, dichloromethane or a mixture thereof; then, further extract with n-butanol to obtain n-butanol extraction thing;

(e) After concentrating the n-butanol extract, perform macroporous resin chromatography, and elute with pure water, a mixture of alcohol/pure water with a volume ratio of 30:70, and a mixture of alcohol/pure water with a volume ratio of 95:5 ; Wherein said alcohol is methanol or ethanol;

(f) Evaporate to dryness the eluate obtained by eluting with a mixture of alcohol/pure water with a volume ratio of 95:5 to obtain the active site extract KG8. the

The active part extract KG8 of bitter melon obtained above can be separated by chromatography to obtain the triterpene saponins of the present invention. the

The above-mentioned triterpene saponins are effective in preparing muscle and fat cells for glucose uptake, promoting the upper membrane of glucose transporter 4 (GLUT4), increasing the activity of adenosine monophosphate-activated protein kinase (AMPK), promoting fatty acid oxidation metabolism, and improving insulin resistance. Glucose tolerance, reducing plasma triglyceride and free fatty acid levels or improving the application of lipid tolerance drugs in the state of lipid metabolism disorders. The above-mentioned triterpene saponins can promote glucose uptake and insulin sensitization in muscle and fat cells, have stable hypoglycemic activity for both type I and type II diabetes, and can prevent/treat obesity and hyperlipidemia. Therefore, the above-mentioned cucurbitane-type triterpene saponin compound and pharmaceutical composition thereof of the present invention are muscle and adipocyte glucose uptake accelerator, promote glucose transporter 4 (GLUT4) membrane agonist, adenosine monophosphate-activated protein kinase ( AMPK) activators, drugs that promote fatty acid oxidation metabolism, improve glucose tolerance in insulin resistance, reduce plasma triglyceride and free fatty acid levels, and improve lipid tolerance in lipid metabolism disorders. the

In the 3T3-L1 adipocyte experiment, KG8 can stimulate glucose uptake in adipocytes in both short-term and long-term, indicating that KG8 can increase insulin sensitivity. In type I diabetes model alloxan-induced diabetic mice, KG8 had obvious hypoglycemic effect at the administration level of 25mg/kg. In the Ob/Ob mouse diabetes model, KG8 can stably lower blood sugar in mice for a long time. Experiments have proved that KG8 has obvious insulin-sensitizing effect, and has a good hypoglycemic effect on both type I and type II diabetes. In the glucose tolerance experiment of mice under the state of insulin resistance, KG8 and compound F can significantly improve glucose intolerance. In the test of the activity of the compound on the glucose transporter 4 (GLUT4) membrane, the compound can significantly promote the glucose transporter 4 (GLUT4) membrane in muscle and fat cells. In the adenosine monophosphate-activated protein kinase (AMPK) activity test, compounds A and F can significantly enhance the AMPK kinase activity. In the glucose utilization experiment in mice, compound F can significantly promote glucose utilization in mice. The compound of the present invention has the activity of promoting glucose uptake and insulin sensitization in muscle and adipocytes to lower blood sugar. the

In the acute effects of energy consumption and fatty acid oxidation metabolism in mice, KG8 and compound F can significantly promote acute fatty acid oxidation metabolism and overall energy consumption in mice and have the effect of treating obesity. the

In the experiment on insulin sensitivity and lipid metabolism of high-fat diet-induced obese (DIO) mice, KG8 can significantly improve the lipid tolerance of DIO mice, and reduce the levels of plasma triglycerides and free fatty acids. The therapeutic effect of KG8 on diabetes in Ob/Ob mice, KG8 can significantly reduce the levels of plasma and liver triglycerides in mice. The composition of the compound of the present invention can treat hyperlipidemia by reducing triglyceride and free fatty acid levels. the

Description of drawings

Fig. 1 is the preparation flowchart of KG8. the

Fig. 2 is the separation scheme of compound A-I. the

Figure 3 is a graph showing the effect of KG8 on glucose uptake in 3T3-L1 cells. the

Fig. 4 is a graph showing the effect of KG8 on plasma triglyceride in Ob/Ob mice. the

Figure 5 is a graph showing the effect of KG8 on liver triglycerides in Ob/Ob mice. the

Fig. 6 is an experimental graph of glucose tolerance under the state of insulin resistance improved by KG8. the

Figure 7 is a graph showing the effect of KG8 on fatty acid oxidation metabolism in HF mice. the

Fig. 8 is a graph showing the effect of KG8 on lipid tolerance in DIO mice. the

Figure 9 is a graph showing the effect of KG8 on plasma triglyceride in DIO mice. the

Figure 10 is a graph showing the effect of KG8 on plasma free fatty acids in DIO mice. the

Fig. 11 is a graph showing the effect of KG8 on oral glucose tolerance (plasma glucose concentration) of DIO mice. the

Fig. 12 is a graph showing the effect of KG8 on oral glucose tolerance (plasma insulin concentration) of DIO mice. the

Fig. 13 is a graph showing the effect of KG8 on insulin tolerance (plasma glucose concentration) in DIO mice. the

Fig. 14 is a graph showing the effect of KG8 on insulin tolerance in DIO mice (percentage reduction in blood sugar). the

Figure 15 is a graph showing the activity of the compound on the glucose transporter 4 (GLUT4) membrane. the

Figure 16 is a diagram showing the activity of compounds A and F on adenosine monophosphate-activated protein kinase (AMPK). the

Figure 17 is a diagram showing the acute effects of compound F on energy consumption and fatty acid oxidation metabolism in mice. the

Figure 18 is a graph of Compound F's glucose utilization in mice. the

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but not as a limitation of the present invention. the

I. Summary

The invention provides a triterpene saponin compound isolated from bitter melon and its pharmaceutical composition, its preparation method and its application in the prevention and treatment of diabetes, obesity and hyperlipidemia. the

The activity of preventing/treating diabetes, obesity and hyperlipidemia in the present invention refers to the ability to promote glucose absorption in muscle and fat cells, resist and reduce fasting and postprandial high blood sugar concentration, have insulin analog-like effect and insulin sensitization effect, Promote glucose transporter 4 (GLUT4) on the membrane, increase adenosine monophosphate-activated protein kinase (AMPK) activity, promote fatty acid oxidative metabolism, improve glucose tolerance in insulin resistant states, reduce plasma triglyceride and free fatty acid levels and Improve lipid tolerance in the state of lipid metabolism disorder. the

II. Compounds

The invention provides a class of triterpene saponins extracted from bitter melon and having the activity of preventing/treating diabetes, obesity and hyperlipidemia. Those skilled in the art will easily understand that the present invention relates to some compounds possessing asymmetric carbon atoms (chiral centers) or double bonds, as well as racemates containing these compounds, diastereomers, enantiomers, geometric isomer. the

Triterpene saponins compound of the present invention has the structure shown in following structural formula I:

Wherein, R ₁ is hydrogen, β-D-glucopyranosyl, β-D-alopyranosyl, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl Glycosyl or β-D-xylopyranosyl (1→4)-[β-D-glucopyranosyl (1→6)]-β-D-glucopyranosyl;

R ₂ is hydroxyl, hydrogen or β-D-glucopyranosyloxy;

R ₃ is hydroxyl or hydrogen;

R ₄ is hydroxyl, hydrogen, β-D-glucopyranosyloxy or β-D-allopyranosyloxy;

R ₅ is hydroxyl or hydrogen;

R ₆ is hydroxyl, β-D-glucopyranosyloxy or hydrogen;

R ₇ is a methyl group or an aldehyde group;

Or alternatively, C5 and C6, C6 and C7, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form double bonds;

And when R ₃ , R ₄ , and R ₅ are hydroxyl groups, the chiralities of C22, C23, and C24 are S, R, and R configurations;

When R ₄ and R ₅ are hydroxyl and R ₃ is hydrogen, the chiralities of C23 and C24 are R and S configurations;

When R ₃ is hydroxyl and R ₄ is β-D-glucopyranosyloxy, C22 and C23 are S, R configurations;

In the case where R is _methyl , C5 and C19 can be connected by an oxygen bridge;

And when R ₁ is hydrogen, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl or β-D-xypyranosyl (1→4)-[β- D-glucopyranosyl (1→6)]-β-D-glucopyranosyl, R ₂ is hydrogen, R ₃ , R ₄ , R ₅ , R ₆ are hydroxyl, and C5 and C6 form a double bond , R ₇ is not methyl;

When R ₂ and R ₆ are hydroxyl, R ₁ , R ₃ , R ₄ and R ₅ are hydrogen, and C5 and C6, C23 and C24 form double bonds, R ₇ is not an aldehyde group;

When R ₁ is β-D-glucopyranosyl or β-D-alopyranosyl, R ₂ , R ₅ , R ₆ are hydrogen, R ₃ is hydroxyl, R ₇ is methyl, and C6 and C7, C24 and C25 form a double bond, and when C5 and C19 are connected by an oxygen bridge, R ₄ is not β-D-alopyranosyloxy.

The compound of the present invention is a natural product, and in a preferred embodiment, the compound of general formula I is a compound with the following structural formula Ia:

Wherein, R ₁ is β-D-glucopyranosyl or β-D-alopyranosyl or hydrogen;

R ₃ is hydroxyl or hydrogen;

R ₄ is hydroxyl, hydrogen, β-D-glucopyranosyloxy or β-D-allopyranosyloxy;

R ₅ is hydroxyl or hydrogen;

R ₆ is hydroxyl, β-D-glucopyranosyloxy or hydrogen;

Or alternatively, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form double bonds; and when R ₃ , R ₄ , and R ₅ are hydroxyl groups, the chirality of C22, C23, and C24 is S , R, R configuration; when R ₄ and R ₅ are hydroxyl and R ₃ is hydrogen, the chirality of C23 and C24 is R and S configuration; and when R ₁ is β-D-glucopyranose or β-D-alopyranosyl, R ₅ and R ₆ are hydrogen, R ₃ is hydroxyl, and when C24 and C25 form a double bond, R ₄ is not β-D-alopyranosyl .

In another preferred embodiment of the present invention, the compound of general formula I is the compound with following structural formula Ib:

Wherein, R ₁ is hydrogen, β-D-glucopyranosyl, β-D-alopyranosyl, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl Glycosyl or β-D-xylopyranosyl (1→4)-[β-D-glucopyranosyl (1→6)]-β-D-glucopyranosyl;

R ₂ is hydroxyl, hydrogen or β-D-glucopyranosyloxy;

R ₃ is hydroxyl or hydrogen;

R ₄ is hydroxyl, hydrogen or β-D-glucopyranosyloxy;

R ₅ is hydroxyl or hydrogen;

R ₆ is hydroxyl, β-D-glucopyranosyloxy or hydrogen;

R ₇ is a methyl group or an aldehyde group;

Or alternatively, C22 and C23, C23 and C24, C24 and C25, C25 and C27 form double bonds;

And when R ₃ , R ₄ , and R ₅ are hydroxyl groups, the chiralities of C22, C23, and C24 are S, R, and R configurations;

And when R ₃ is hydroxyl and R ₄ is β-D-glucopyranosyloxy, C22 and C23 are S, R configurations;

And when R ₁ is hydrogen, β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl or β-D-xypyranosyl (1→4)-[β- D-glucopyranosyl (1→6)]-β-D-glucopyranosyl, R ₂ is hydrogen, R ₃ , R ₄ , R ₅ , R ₆ are hydroxyl, R ₇ is not methyl ;

When R ₂ and R ₆ are hydroxyl, R ₁ , R ₃ , R ₄ and R ₅ are hydrogen, and C23 and C24 form a double bond, R ₇ is not an aldehyde group.

The most preferred specific compounds of the present invention, namely compounds A, B, C, D, F, G, H, I, are listed in Table 1 below. the

Table 1 Compound A, B, C, D, F, G, H, I structural formula

The preparation method of the above-mentioned compounds of the present invention is described in detail below. The above-mentioned bitter melon active part extract KG8 obtained above can be obtained after chromatographic separation. Above-mentioned compounds A, B, C, D, F, G, H, I and Compound momordicoside M, momordicoside N, the method comprises:

The active site extract KG8 was subjected to column chromatography with 100-200 mesh silica gel, followed by chloroform:methanol: The lower layer of water is eluted and detected by TLC plate. The developing solvent can be the lower layer of chloroform: methanol with a volume ratio of 10:1, 6:1, 4:1 or a volume ratio of 10:3:1, 65:35: 10 Chloroform: Methanol: The lower layer of water, 5% sulfuric acid-vanillin for color development, according to the TLC plate display, similar components were combined and concentrated, and 1- 9 nine components;

Component 3 was subjected to silica gel column chromatography, eluted with chloroform:methanol at a volume ratio of 10:1, and detected by a TLC plate. The developing solvent was chloroform:methanol at a volume ratio of 8:1, 5% sulfuric acid-vanillin For color development, the eluent of the spot with Rf value approximately equal to 0.4 according to the TLC plate was combined and concentrated to obtain compound I;

Component 4 was subjected to MCI column chromatography, and the volume ratio was 50:50 to 90:10 methanol:water eluent for gradient elution, and the resulting 70:30 methanol:water eluent fraction was subjected to silica gel column chromatography, Use a volume ratio of 9:1 chloroform:methanol for elution, and detect it through a TLC plate. The developer is 5:1 volume ratio of chloroform:methanol, 5% sulfuric acid-vanillin for color development, and the Rf value will be displayed according to the TLC plate The eluents of spots approximately equal to 0.3 and 0.4 respectively were combined and concentrated to obtain compound A and compound B;

Component 5 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol:water eluent gradient elution, and the obtained 60:40 methanol:water eluent fraction was subjected to silica gel column chromatography , the volume ratio is 20:3:1 chloroform:methanol:water layer liquid is eluted, detects by TLC plate, developing agent is the volume ratio is the chloroform:methanol of 4:1, 5% sulfuric acid-vanillin color develops, will According to the TLC plate, the eluents of the spots with Rf values approximately equal to 0.3 were combined and concentrated to obtain the compound momordicoside M;

Component 6 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol:water eluent gradient elution, and the obtained 60:40 methanol:water eluent fraction was then subjected to RP-18 column Chromatography, methanol: water gradient elution with a volume ratio of 40:60 to 60:40, detected by a TLC plate, the developing solvent was chloroform: methanol: a water bottom layer with a volume ratio of 10:3:1, 5% sulfuric acid- Vanillin color development, according to the TLC plate shows the eluate of the spot with an Rf value approximately equal to 0.4 combined and concentrated to obtain the compound momordicoside N;

Component 7 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol:water eluent gradient elution, and the obtained 50:50 methanol:water eluent fraction was then subjected to RP-18 column Chromatography, methanol: water gradient elution with a volume ratio of 40:60 to 60:40, detected by a TLC plate, the developing solvent was chloroform: methanol: a water layer liquid with a volume ratio of 8:3:1, 5% sulfuric acid- Vanillin color development, according to the TLC plate shows the eluent of the spot with an Rf value approximately equal to 0.4 combined and concentrated to obtain compound C;

Component 8 was chromatographed by MCI column, and the volume ratio was 30:70 to 70:30 methanol:water eluent gradient elution, and the obtained 40:60 methanol:water eluent fraction was then subjected to RP-18 column Chromatography, methanol: water gradient elution with a volume ratio of 30:70 to 60:40, detected by a TLC plate, the developing solvent was chloroform: methanol: a water bottom layer with a volume ratio of 8:3:1, 5% sulfuric acid- Vanillin is used for color development, and the eluent of the spot with an Rf value approximately equal to 0.3 according to the TLC plate is combined and concentrated to obtain compound H;

Component 9 was subjected to MCI column chromatography, and the volume ratio was 20:80 to 60:40 methanol:water eluent gradient elution, and the obtained 40:60 methanol:water eluent fraction was subjected to silica gel column chromatography , a volume ratio of 10:3:1 of chloroform:methanol:water layer liquid elution, detected by TLC plate, the developer is 65:45:1 volume ratio of chloroform:methanol:water layer liquid, 5% sulfuric acid-pandan For aldehyde color development, the eluents of the spots with Rf values approximately equal to 0.3 and 0.4 respectively according to the TLC plate were combined and concentrated to obtain compound F and compound G;

Compound A was hydrolyzed under 0.5M hydrochloric acid aqueous solution at room temperature for 7 days, and the resulting reactant was subjected to preparative thin-layer chromatography. The developer was chloroform:methanol 6:1, and the part with Rf value 0.5-0.6 was collected and washed with chloroform:methanol 8:2. Take off and concentrate to obtain compound D. the

III. Pharmaceutical composition

The present invention provides a pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipidemia, the composition comprising one or more of the triterpenoid saponins isolated from Momordica charantia in a therapeutically effective amount shown in the above structural formula I . In addition, the composition may further comprise known compounds extracted from bitter melon soya-cerebroside I, momor-cerebroside, triterpene 3,7,25-trihydroxycucurbitane-5 , 23(E)-diene-19-aldehyde, 22(S), 23(R), 24(R), 25-tetrahydroxycucurbitane-5-ene, triterpenoid saponins momordicoside A, momordicoside B, momordicoside M Or one or more of momordicoside N. In addition, the composition may also contain pharmaceutically conventional excipients, such as various pharmaceutically commonly used solvents. The composition can be prepared by conventional pharmaceutical preparation methods. the

In one embodiment of the present invention, the above-mentioned pharmaceutical composition for preventing/treating diabetes, obesity and hyperlipidemia provided by the present invention, said compound is derived from the active part extract of bitter melon. In the above preparation method, the bitter gourd plant is a plant of the genus Momordica of Cucurbitaceae, or more specifically Momordica charantia of the genus Momordica of the family Cucurbitaceae. the

The active part extract of bitter melon may include natural products of triterpene saponins, such as compounds of structural formula Ia, Ib, soya-cerebroside I, momor-cerebroside, triterpene 3,7 , 25-trihydroxycucurbitane-5, 23(E)-dien-19-aldehyde, 22(S), 23(R), 24(R), 25-tetrahydroxycucurbitane-5-ene, triterpene A mixture of any one or more compounds in saponins momordicoside A, momordicoside B, momordicoside M and momordicoside N, more specifically, a mixture of any one or more compounds in the compounds described in Table 1. the

specific embodiment

Instruments used: The optical rotation of the sample in methanol solution was measured with a Perkin-Elmer 341 polarimeter. ESI-MS (electrospray mass spectrometry) was determined with a Finnegan LCQ-DECA instrument. ¹ H and ¹³ CNMR nuclear magnetic resonance (measured in deuterated pyridine) were determined by Varian INOVA 500 and Bruker AM-400 nuclear magnetic resonance instruments. Chemical shifts δ (ppm) are internally referenced to TMS and coupling constants (J) are given in Hz.

Thin-layer chromatography (TLC) silica gel plates and silica gel (200-300 mesh) for column chromatography were produced by Qingdao Ocean Chemical Factory; TLC spots were sprayed with vanillin sulfate ethanol solution to develop color. the

In this article, unless otherwise specified, the ratios of solvents involved are volume ratios. the

Example 1

As shown in Figure 1, get fresh balsam pear 850kg, after low-temperature freeze-drying, weigh 85kg, pulverize, be soaked three times under the normal temperature of 90% ethanol aqueous solution with volume ratio, each 3 days, and the consumption of ethanol aqueous solution is 10 times of crude drug weight. The ethanol solutions obtained three times were combined, then distilled under reduced pressure and concentrated to obtain the total ethanol extract. The total ethanol extract was diluted with water (50L) and then partitioned with dichloromethane (20L) to obtain dichloromethane fraction and aqueous solution. Then its aqueous solution and n-butanol (20 L) were subjected to liquid-liquid partitioning to obtain 800 g of n-butanol. The 800g n-butanol part is mixed with 500g AB-8 type macroporous resin (from Tianjin Bone Glue Factory) as a sample, and the resin mixed with the sample is added to a chromatographic column equipped with 3kg AB-8 resin. Use 12L pure water, 30:70 alcohol/pure water (volume ratio), 95:5 alcohol/pure water (volume ratio) to elute respectively, and obtain parts KG6 600g, KG7 60g, KG8 80g respectively. the

Example 2

Figure 2 illustrates the separation of Compound A-I using chromatography. the

The active part extract KG8 80g that above-mentioned embodiment 1 obtains is carried out column chromatography with 2kg 100-200 mesh silica gel, and the volume ratio is 40:3:1, 20:3:1, 10:3:1 and 65: 35:10 chloroform:methanol:water, elute with 10L of the lower layer liquid, concentrate every 500ml into one fraction, detect by TLC plate, use chloroform:methanol with a volume ratio of 10:1, 6:1, 4:1 as developing solvent The lower layer of liquid or the lower layer of chloroform:methanol:water with a volume ratio of 10:3:1, 65:35:10, 5% sulfuric acid-vanillin for color development, according to the TLC plate display, similar components were combined and concentrated , according to the Rf value between 0.6 and 0.2, nine components from 1 to 9 are obtained from large to small;

Component 3 was subjected to silica gel column chromatography, eluted with 1 L of chloroform with a volume ratio of 10:1:methanol, and every 20 ml was concentrated as a fraction, and detected by a TLC plate. The developer was chloroform with a volume ratio of 8:1: Methanol, 5% sulfuric acid-vanillin for color development, the eluent of the spot showing an Rf value approximately equal to 0.4 according to the TLC plate was combined and concentrated to obtain 9 mg of compound I;

Component 4 was subjected to MCI column chromatography, and the volume ratio was 50:50-90:10 methanol:1 L of water eluent was used for gradient elution, and the obtained 70:30 methanol:water eluent was subjected to silica gel column layer Analysis, eluting with a volume ratio of 9:1 chloroform:methanol, every 10ml was concentrated as a fraction, detected by TLC plate, the developer was 5:1 volume ratio of chloroform:methanol, 5% sulfuric acid-vanillin For color development, the eluents showing spots with Rf values approximately equal to 0.3 and 0.4 according to the TLC plate were combined and concentrated to obtain 180 mg of compound A and 90 mg of compound B;

Component 5 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol: each 1 L of water eluent was gradiently eluted, and the obtained 60:40 methanol: water eluent fraction was then subjected to silica gel column Chromatography, the volume ratio is 20:3:1 chloroform:methanol:1.5L of the lower layer liquid is eluted, every 20ml is concentrated as a fraction, detected by TLC plate, the developing solvent is 4:1 volume ratio of chloroform:methanol , 5% sulfuric acid-vanillin for color development, according to the TLC plate, the eluents showing spots with Rf values approximately equal to 0.3 were combined and concentrated to obtain the compound momordicoside M 230mg;

Component 6 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol: each 1 L gradient elution of water eluent, and the obtained 60:40 methanol: water eluent fraction was subjected to RP- 18 column chromatography, methanol with a volume ratio of 40:60 to 60:40: each 500ml gradient elution of water, detected by a TLC plate, the developer is chloroform with a volume ratio of 10:3:1: methanol: the liquid in the lower layer of water, 5% sulfuric acid-vanillin was used for color development, and the eluents showing spots with an Rf value approximately equal to 0.4 according to the TLC plate were combined and concentrated to obtain the compound momordicoside N21mg;

Component 7 was chromatographed by MCI column, and the volume ratio was 30:70 to 80:20 methanol: each 1 L of water eluent was gradiently eluted, and the resulting 50:50 methanol: water eluent fraction was subjected to RP- 18 column chromatography, the volume ratio is 40:60 ~ 60:40 methanol: 500ml of water gradient elution, every 20ml concentration is a fraction, detected by TLC plate, the developing solvent is the volume ratio of 8:3:1 Chloroform: Methanol: Underwater liquid, 5% sulfuric acid-vanillin for color development, according to the TLC plate, the eluent of the spot with an Rf value approximately equal to 0.4 was combined and concentrated to obtain 25 mg of Compound C;

Component 8 was chromatographed by MCI column, and the volume ratio was 30:70 to 70:30 methanol: each 1 L gradient elution of water eluent, and the obtained 40:60 methanol: water eluent fraction was subjected to RP- 18 column chromatography, methanol with a volume ratio of 30:70 to 60:40: gradient elution of 500ml of water, each 20ml concentration is a fraction, detected by TLC plate, the developing solvent is a volume ratio of 8:3:1 Chloroform: Methanol: Underwater layer liquid, 5% sulfuric acid-vanillin for color development, according to the TLC plate, the eluent of the spot with an Rf value approximately equal to 0.3 was combined and concentrated to obtain 30 mg of compound H;

Component 9 was chromatographed by MCI column, and the volume ratio was 20:80 to 60:40 methanol: each 1 L gradient elution of water eluent, and the obtained 40:60 methanol: water eluent fraction was then subjected to silica gel column Chromatography, the volume ratio is 10:3:1 chloroform:methanol:500ml of the lower layer liquid is eluted, every 20ml is concentrated as a fraction, detected by TLC plate, the developer is chloroform:methanol with a volume ratio of 65:45:1 : Underwater layer liquid, 5% sulfuric acid-vanillin for color development, the eluents showing spots with Rf values approximately equal to 0.3 and 0.4 according to the TLC plate were combined and concentrated to obtain 60 mg of compound F and 110 mg of compound G;

Compound A was hydrolyzed under 0.5M hydrochloric acid aqueous solution at room temperature for 7 days, and the resulting reactant was subjected to preparative thin-layer chromatography. The developer was chloroform:methanol 6:1, and the part with Rf value 0.5-0.6 was collected and washed with chloroform:methanol 8:2. After stripping and concentrating, 10 mg of compound D was obtained. the

The characteristics of compounds A, B, C, D, F, G, H, and I are as follows:

Compound A amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-75$ (c0.094, MeOH); IR v _max (KBr) 3421, 2949, 2873, 1637, 1470, 1377, 1153, 1086, 1032cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and Table 3; HRESIMS m/ z 675.4064 (calcd for _C36H60O10Na [M+Na] ⁺ , _{675.4084 )} .

Compound B amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-76$ (c0.1600, MeOH); IR v _max (KBr) 3396, 3165, 2935, 2783, 1612, 1514, 1454, 1417, 1277, 1263, 1223, 1165, 1122, 1039, 814, 689cm ^-1 ; ¹ HNMR and ¹³ CNMR data in Tables 2 and 3; HRESIMS m/z 675.4113 (calcd for C ₃₆ H ₆₀ O ₁₀ Na[M+Na]+, 675.4084).

Compound C amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-64$ (c0.188, MeOH); IR v _max (KBr) 3415, 2928, 2874, 1643, 1466, 1377, 1155, 1082, 1032cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and Table 3; HRESIMS m/ z 6837.4632 (calcd for _C42H70O15Na [M+Na]+, _{837.4612 )} .

Compound D is an amorphous powder; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and Table 3; LRESIMS m/z 513 ([M+Na] ⁺ ).

Compound F amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-8$ (c0.2245, MeOH); IR v _max (KBr) 3406, 2933, 2873, 1646, 1551, 1452, 1383, 1306, 1076, 1038, 534cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and Table 3 ; HRESIMS m _/ z 1001.5253 (calcd for _C48H82O20Na [M+Na] ⁺ _, 1001.5297).

Compound G amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-1$ (c0.1385, MeOH); IR v _max (KBr) 3408, 2931, 2857, 1639, 1468, 1381, 1308, 1163, 1076, 1039cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and Table 3; HRESIMS m/z 1113.6025 (calcd for _C54H94O23Na [M+Na]+, _{1113.6048 )} .

Compound H amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=-5.0$ (c0.3425, MeOH); IR v _max (KBr) 3415, 2931, 2874, 1637, 1551, 1460, 1381, 1306, 1163, 1076, 1040, 534cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and _Table 3; HRESIMS m _/ z 1099.5618 (calcd for _C53H88O22Na [M+Na] ⁺ , 1099.5665).

Compound I amorphous powder; ${\left[\mathrm{\α}\right]}_{\mathrm{D.}}^{\mathrm{twenty\; three}}=+39.8$ (c0.3425, MeOH); IR v _max (KBr) 3404, 2928, 2875, 1709, 1662, 1454, 1385, 1306, 1076, 1041, 966, 534cm ^-1 ; ¹ HNMR and ¹³ CNMR data are shown in Table 2 and table 3. _HRESIMS m _/ z 639.3816 (calcd for _C36H56O8Na [M+Na] ⁺ , 639.3873).

Table 2 ¹³ CNMR spectra (C ₅ D ₅ N, ppm) of the compounds of the present invention

Table 3 ¹ HNMR spectrum of the compounds of the present invention (C ₅ D ₅ N, ppm)

Test Example

Test Example 1

Test the effect of KG8 on glucose uptake in 3T3-L1 cells (Figure 3) 

After 3T3-L1 cells were completely differentiated into adipocytes, they were cultured in serum-free DMEM containing 0.5% BSA for 16 hours, and treated with KG8 (final concentration 20 μg/ml) for 4 days. The blank control group was added with an equal volume of DMSO, and then treated with Wash cells twice with 1×PBS pre-warmed at 37°C, add 0.5% BSA Krebs buffer (NaCl 140mM, KCl 5mM, MgSO ₄ 2.5mM, CaCl ₂ 1mM, HEPES 20mM, pH 7.4) without or with insulin , after incubating at 37°C for 40 minutes, add 2-[1,2-3H(N)]-deoxy-D-glucose solution (final concentration: 0.5 μCi/ml), act at 37°C for 5 minutes, and use ice-bathed 1× Wash 3 times with PBS to stop the reaction, then add 0.15ml 0.1% Triton to lyse the cells, count by liquid scintillation, and calculate the glucose uptake of 3T3-L1 cells after correcting the CPM value with the amount of protein.

P＜0.05，

P＜0.01，

P＜0.001。  The results showed that after KG8 treated 3T3-L1 cells for 4 days (Figure 3), the glucose uptake of the cells at the basal level and stimulated by 1nM and 100nM insulin could be significantly increased. Data are expressed as mean±standard error (X±SE) (n=3). Compared with the blank control group under the corresponding conditions: ^* P<0.05, ^** P<0.01; compared with the glucose intake at the respective basal levels:

P<0.05,

P<0.01,

P<0.001.

Test Example 2

Effect of KG8 on fasting blood glucose in alloxan-induced diabetic mice (Table 4)

ICR male mice (30-35 grams) were injected with 60 mg/kg of the now prepared alloxan solution through the tail vein. After 7 days, fasted for 6 hours, blood was taken from the canthus of the eyes, and the plasma was separated. blood sugar. The mice whose predicted blood glucose level was 11.1-30.0mmol/L were selected as experimental diabetic mice. The fasting blood glucose was measured after the selected diabetic mice were fasted for 6 hours, 8 mice in each group. The mice in the administration group were orally administered 25 mg/kg of KG8, the positive control group was administered 150 mg/kg metformin hydrochloride, and the blank control group was administered 1% CMC. The mice in each group were administered continuously for 21 days, and the fasting blood glucose of the mice was measured on the 7th, 14th and 21st days after the administration. the

The results showed that after alloxan injection in ICR mice, the fasting blood glucose level of the mice increased significantly, indicating that the modeling was successful. The fasting blood glucose value of the KG8 group was significantly lower than that of the model control group, indicating that KG8 has the effect of lowering the blood glucose of the alloxan mouse model. the

Table 4 Effect of KG8 on fasting blood glucose in alloxan-induced diabetic mice

Note: Data are expressed as mean ± standard error (X ± SE) (n = 8). Compared with blank control group: ^* P<0.05, ^** P<0.01, ^*** P<0.001.

Test Example 3

The therapeutic effect of KG8 on diabetes in Ob/Ob mice (Table 5, Fig. 4, 5). the

After two weeks of adaptive feeding, Ob/Ob mice were weighed and random blood glucose was measured, and fasting blood glucose was measured after fasting for 5 hours. At the same time, considering the standard of random blood sugar, the Ob/Ob mice were divided into 3 groups, 8 mice in each group, half male and half male, and were given 50mg/kg KG8, 1% CMC and 150mg/kg metformin respectively. At the same time, littermates of C57 mice were used as normal controls. The mice in each group were administered continuously for 21 days, and the fasting blood glucose was measured on the 7th, 14th and 21st days after administration. On the last day after administration, blood was collected to determine the content of plasma triglyceride and liver triglyceride. Compared with Ob/Ob mice, ^* P<0.05, ^** P<0.01, ^*** P<0.001.

The results showed that after the Ob/Ob mice were given 50mg/kg KG8, the blood glucose level decreased significantly compared with that before the administration; after 21 days of administration, the levels of triglycerides in the plasma (Fig. 4) and liver (Fig. 5) of the mice were the same as those of the control group Significantly decreased compared to . the

Table 5 KG8 hypoglycemic effect on Ob/Ob mice

Note: Data are expressed as mean ± standard error (X ± SE) (n = 8). Compared with the blood glucose level before administration, ^* P<0.05, ^** P<0.01, ^*** P<0.001.

Test Example 4

KG8 improves the glucose tolerance test in the state of insulin resistance (Figure 6)

C57BL/6J male mice (8-10 weeks old) were fed a high-fat diet for 8 weeks to make the mice insulin resistant (exhibiting glucose intolerance). In the last two weeks, high-fat feed was added to KG8 (100 mg/kg per day) or metformin (Met, 200 mg/kg per day). Insulin sensitivity was evaluated by measuring glucose tolerance (2.0 g/kg glucose intraperitoneally) after a 5-7 hour fast. Normal control group: mice with normal glucose tolerance (insulin-sensitive) fed with normal diet. High-fat model group: control mice with impaired glucose tolerance fed with high-fat diet (no drug added). High-fat metformin group: high-fat diet mice added with metformin. High-fat KG8 group: high-fat diet mice added with KG8. The data are represented by mean±standard error (X±SE) (n=6-9/group), ^* P<0.05, ^** P<0.01. The experimental results show that: KG8 can significantly improve glucose intolerance, and thus can treat diabetes.

Test Example 5

Effect of KG8 on fatty acid oxidation metabolism in HF mice (Fig. 7). the

Long-term oral administration of KG8 to mice on a high-fat diet (HF) can affect fatty acid oxidation metabolism in mice by reducing the respiratory exchange rate (RER). Beginning at 10 AM sharp, mice were placed in metabolic cages for 24 hours. Two weeks before the assay, KG8 (100 mg/kg) was added to the diet. The food itself served as a solvent control (Veh). Data are presented as mean±standard error (X±SE) (n=5-6). Compared with KG8 solvent: ^* P<0.05, ^** P<0.01.

Experimental results: Figure A shows that KG8 can significantly promote fatty acid oxidation metabolism in mice; Figure B shows that KG8 can significantly promote fatty acid oxidation metabolism in mice during the day (10 o'clock to 18 o'clock). Experiments show that: KG8 can treat obesity diseases. the

Test Example 6

Effects of KG8 on insulin sensitivity and lipid metabolism in high-fat diet-induced obese (DIO) mice. (Figure 8, 9, 10, 11, 12, 13, 14)

C57BL/6J male mice (3-4 weeks old) were fed high-fat diet for 12-16 weeks to form a diet-induced obesity (DIO) mouse model, which simultaneously exhibited obvious insulin resistance and lipid metabolism disorders. DIO mice were divided into 4 groups according to body weight and blood sugar, and were given KG8 (50, 100 mg/kg), metformin (Met, 250 mg/kg) or model control (1% CMC) every day, respectively, for 32 consecutive days. On the 20th day after the administration, the mice were fasted overnight for an oral lipid tolerance test (10ml/kg of medium and long-chain fatty acids orally); 32 days after the administration, the plasma triglycerides, free Fatty acid levels were used to evaluate the regulatory effect of KG8 on lipid metabolism. On the 24th day after the administration, the mouse insulin tolerance test (intraperitoneal injection of insulin 0.5 U/kg) was carried out after 6 hours of fasting; on the 28th day after the administration, the oral glucose tolerance test of mice was carried out after the 6-hour fast (oral 2.5 U/kg). g/kg glucose), to evaluate the improvement effect of KG8 on insulin resistance. the

The experimental results showed that KG8 could significantly improve the lipid tolerance of DIO mice (Figure 8), and reduce the levels of plasma triglycerides (Figure 9) and free fatty acids (Figure 10). KG8 can significantly improve the oral glucose tolerance (Fig. 11, 12) and insulin tolerance (Fig. 13, 14) of DIO mice, suggesting that it can significantly improve the insulin resistance state of DIO mice, and thus can treat diabetes. Data are expressed as mean±standard error (X±SE) (n=9). Compared with the model control: ^* P<0.05, ^** P<0.01, ^*** P<0.001.

Test Example 7

Test of the translocation activity of the compound on the glucose transporter GLUT4 (Figure 15)

Effects of compounds on translocation of GLUT4 from the cytoplasm to the plasma membrane (PM). Figure 15A shows the effect on L6 muscle cells (n=3); Figure 15B shows the effect on 3T3-L adipocytes (n=3). Insulin group: 100 nM insulin (positive control). Cells were treated with 10 μM of each compound for 2 hours. Standard errors are in the histograms when no standard errors are indicated. Data are expressed as mean±standard error (X±SE) (n=3). Compared with the solvent control group: ^* P<0.05, ^** P<0.01.

Experiments show that the compound can promote the transfer of glucose transporter 4 (GLUT4) to the cell membrane, thereby allowing glucose to be taken up by the cell. the

Test Example 8

Compounds A and F were tested for the activity of adenosine monophosphate-activated protein kinase (AMPK) ( FIG. 16 ). Compound Effects on AMPK Pathway. 3T3-L1 adipocytes were incubated with 10 μM compound A, F, 1 mM 5-amino-4-imidazole carboxamide nucleotide (AICAR, positive control) or DMSO solvent control (Veh) for 60 min, and treated with 100 nM insulin for 2 min or 25 min , and then the proteins pAS160, pACC and pAMPK in the cell lysate were detected by the corresponding antibodies. Use the 14-3-3 total protein amount as the quality control of the sample amount. The results show that compounds A and F can significantly activate AMPK signaling pathway, and further have therapeutic effects on diabetes and obesity. the

Test Example 9

P＜0.01。  Compound F acutely affects energy consumption and fatty acid oxidation metabolism in mice ( FIG. 17 ). Compound F can affect the body's energy consumption (VO ₂ ) and fat oxidation (measured by reducing the respiratory exchange rate RER) after acute action on normal diet mice. The mice were placed in the metabolic cage at nine o'clock in the morning, and two hours later, the mice were subcutaneously injected with compound F (100 mg/kg), 5-amino-4-imidazole carboxamide nucleotide (AICAR) (250 mg/kg , positive control drug) and normal saline solvent control group. Data are presented as mean ± standard error (X ± SE) (n=6-8). Compared with the solvent control group: ^* P<0.05, ^** P<0.01; F compared with the solvent control group: P<0.05,

P<0.01.

Experiments show that compound F has the effect of treating obesity by promoting acute fatty acid oxidation metabolism and body energy consumption in mice. the

Test Example 10

Glucose Utilization of Compound F in Mouse (Figure 18)

Acute Effects of Compound F on Blood Glucose in Normal Chow Mice. After mice were intraperitoneally injected with compound F or normal saline (control group) for 60 minutes, glucose was injected into mice, and then blood glucose was measured at a given time. Data are expressed as mean±standard error (X±SE) (n=6). ^* P<0.05, ^** P<0.01.

Experiments show that compound F can significantly promote glucose utilization in mice with glucose tolerance. the

Claims (10)

1. one type of cucurbitane type triterpene saponin componds is characterized in that, described compound has the structure shown in the following structural formula Ia:

Wherein, described compound is the compd A with following substituting group and configuration:

R ₁Be β-D-A Luo pyrans glycosyl;

R ₃Be hydrogen;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be hydroxyl;

C23, the chirality of C24 is R, the S configuration;

Or described compound is the compd B with following substituting group and configuration:

R ₁Be β-D-glucopyanosyl base;

R ₃Be hydrogen;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be hydroxyl;

C23, the chirality of C24 is R, the S configuration;

Or described compound is the Compound C with following substituting group and configuration:

R ₁Be β-D-A Luo pyrans glycosyl;

R ₃Be hydrogen;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be β-D-glucopyanosyl oxygen base;

C23, the chirality of C24 is R, the S configuration;

Or described compound is the Compound D with following substituting group and configuration:

R ₁Be hydrogen;

R ₃Be hydrogen;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be hydroxyl;

C23, the chirality of C24 is R, the S configuration.

2. one type of cucurbitane type triterpene saponin componds is characterized in that, described compound has the structure shown in the following structural formula Ib:

Wherein, described compound is the compound F 17-hydroxy-corticosterone with following substituting group and configuration:

R ₁Be β-D-glucopyanosyl base (1 → 6)-β-D-glucopyanosyl base;

R ₂Be hydrogen;

R ₃Be hydroxyl;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be β-D-glucopyanosyl oxygen base;

R ₇Be methyl;

C22, C23, the chirality of C24 is S, R, R configuration;

Or described compound is the compound G with following substituting group and configuration:

R ₁Be β-D-xylopyranose base (1 → 4)-[β-D-glucopyanosyl base (1 → 6)]-β

-D-glucopyanosyl base;

R ₂Be hydrogen;

R ₃Be hydroxyl;

R ₄Be hydroxyl;

R ₅Be hydroxyl;

R ₆Be β-D-glucopyanosyl oxygen base;

R ₇Be methyl;

C22, C23, the chirality of C24 is S, R, R configuration;

Or described compound is the compound H with following substituting group, two key and configuration: R ₁Be β-D-xylopyranose base (1 → 4)-[β-D-glucopyanosyl base (1 → 6)]-β

-D-glucopyanosyl base;

R ₂Be hydrogen;

R ₃Be hydroxyl;

R ₄Be β-D-glucopyanosyl oxygen base;

R ₅Be hydrogen;

R ₆Be hydrogen;

R ₇Be methyl;

C24 and C25 form two keys;

C22 and C23 are S, the R configuration;

Or described compound is the compound I with following substituting group and two keys:

R ₁Be hydrogen;

R ₂Be β-D-glucopyanosyl oxygen base;

R ₃Be hydrogen;

R ₄Be hydrogen;

R ₅Be hydrogen;

R ₆Be hydrogen;

R ₇Be aldehyde radical;

C23 and C24 form two keys between C25 and the C27.

3. the preparation method of claim 1 or 2 said compounds is characterized in that this method comprises:

Soak the balsam pear sample with alcohol, water or its mixture, obtain extracting solution after the filtration, wherein said alcohol is methyl alcohol or ethanol; Solvent evaporation in the extracting solution that obtains is formed soup compound; Water is joined in this soup compound, obtain the aqueous suspension of general extractive; With chloroform, methylene dichloride or its mixture above-mentioned suspension is carried out liquid-liquid extraction, obtain the extract of chloroform, methylene dichloride or its mixture; Then, further extract, obtain n-butyl alcohol extract with propyl carbinol; After n-butyl alcohol extract concentrated, carry out the macroporous resin chromatography, using pure water, volume ratio respectively is that 30: 70 alcohol/pure water mixtures, volume ratios are that 95: 5 alcohol/pure water mixtures carry out wash-out, and wherein said alcohol is methyl alcohol or ethanol; It is dried that volume ratio is that alcohol/pure water mixture wash-out obtained in 95: 5 elutriant is evaporated to, reactive site extract KG8;

Reactive site extract KG8 is carried out column chromatography with 100-200 order silica gel; Using volume ratio successively is the chloroform of 40: 3: 1,20: 3: 1,10: 3: 1 and 65: 35: 10: methyl alcohol: lower floor's liquid wash-out of water; Detect through the TLC plate; Developping agent available volume ratio is 10: 1,6: 1,4: 1 a chloroform: lower floor's liquid of methyl alcohol or volume ratio are 10: 3: 1,65: 35: 10 chloroform: methyl alcohol: lower floor's liquid of water, and 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde colour developing shows according to the TLC plate; With similar component merging and concentrated, between 0.6～0.2, obtain nine components of 1-9 from big to small according to the Rf value;

Component 3 is carried out silica gel column chromatography; The use volume ratio is 10: 1 a chloroform: methanol-eluted fractions; Detect through the TLC plate, developping agent is that volume ratio is 8: 1 a chloroform: methyl alcohol, 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde colour developing; To show that elutriant that Rf value approximates 0.4 spot merges and concentrates according to the TLC plate, must compound I;

Component 4 is used the MCI column chromatography, and volume ratio is 50: 50～90: 10 methyl alcohol: gradient elution is carried out in the water elution agent, 70: 30 methyl alcohol of gained: the water elution fluid component carries out silica gel column chromatography again; Using volume ratio is 9: 1 chloroforms: methyl alcohol carries out wash-out; Detect through the TLC plate, developping agent is that volume ratio is 5: 1 a chloroform: methyl alcohol, 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde colour developing; To show that elutriant that Rf value approximates 0.3,0.4 spot respectively merges and concentrates according to the TLC plate, must compd A and compd B;

Component 7 is used the MCI column chromatography; Volume ratio is 30: 70～80: 20 a methyl alcohol: water elution agent gradient elution; 50: 50 methyl alcohol of gained: the water elution fluid component carries out the RP-18 column chromatography again, and volume ratio is 40: 60～60: 40 a methyl alcohol: the water gradient elution, detect through the TLC plate; Developping agent is that volume ratio is 8: 3: 1 a chloroform: methyl alcohol: subsurface layer liquid; The colour developing of 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde will show that elutriant that Rf value approximates 0.4 spot merges and concentrates, and gets Compound C according to the TLC plate;

Component 8 is used the MCI column chromatography; Volume ratio is 30: 70～70: 30 a methyl alcohol: water elution agent gradient elution; 40: 60 methyl alcohol of gained: the water elution fluid component carries out the RP-18 column chromatography again, and volume ratio is 30: 70～60: 40 a methyl alcohol: the water gradient elution, detect through the TLC plate; Developping agent is that volume ratio is 8: 3: 1 a chloroform: methyl alcohol: subsurface layer liquid; The colour developing of 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde will show that elutriant that Rf value approximates 0.3 spot merges and concentrates, and gets compound H according to the TLC plate;

Component 9 is used the MCI column chromatography; Volume ratio is 20: 80～60: 40 a methyl alcohol: water elution agent gradient elution; 40: 60 methyl alcohol of gained: the water elution fluid component carries out silica gel column chromatography again, and volume ratio is 10: 3: 1 a chloroform: methyl alcohol: subsurface layer liquid wash-out, detect through the TLC plate; Developping agent is 65: 45: 1 a chloroform of volume ratio: methyl alcohol: subsurface layer liquid; The colour developing of 5% sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde will show that elutriant that Rf value approximates 0.3,0.4 spot respectively merges and concentrates, and gets compound F 17-hydroxy-corticosterone and compound G according to the TLC plate;

With compd A hydrolysis under room temperature 0.5M aqueous hydrochloric acid, the gained reactant is prepared thin-layer chromatography, developping agent is a chloroform: methyl alcohol 6: 1, collect Rf value 0.5-0.6 part and use chloroform: 8: 2 wash-outs of methyl alcohol, the concentrated Compound D that promptly gets.

4. one kind has the active pharmaceutical composition of preventing/treating mellitus, obesity and hyperlipidemia, it is characterized in that, described compsn comprises the claim 1 of treating significant quantity or in the 2 described compounds one or more.

5. according to claim 4 have an active pharmaceutical composition of preventing/treating mellitus, obesity and hyperlipidemia, it is characterized in that described compound is from balsam pear reactive site extract, and this extract makes through following steps:

(a) soak the balsam pear sample with alcohol, water or its mixture, obtain extracting solution after the filtration; Wherein said alcohol is methyl alcohol or ethanol;

(b) solvent evaporation in the extracting solution that obtains is formed soup compound;

(c) water is joined in this soup compound, obtain the aqueous suspension of general extractive;

(d) with chloroform, methylene dichloride or its mixture above-mentioned suspension is carried out liquid-liquid extraction, obtain the extract of chloroform, methylene dichloride or its mixture; Then, further extract, obtain n-butyl alcohol extract with propyl carbinol;

(e) n-butyl alcohol extract is concentrated after, carry out the macroporous resin chromatography, using pure water, volume ratio respectively is that 30: 70 alcohol/pure water mixtures, volume ratios are that 95: 5 alcohol/pure water mixtures carry out wash-out; Wherein said alcohol is methyl alcohol or ethanol;

(f) it is dried volume ratio to be that alcohol/pure water mixture wash-out obtained in 95: 5 elutriant is evaporated to, and promptly gets reactive site extract KG8.

6. according to claim 5 have an active pharmaceutical composition of preventing/treating mellitus, obesity and hyperlipidemia, it is characterized in that described balsam pear is a Curcurbitaceae Momordica plant balsam pear (Momordica charantia).

7. claim 1 or the 2 described cucurbitane type triterpene saponin componds application in preparation preventing/treating mellitus, obesity and high blood cholesterol drug.

8. application according to claim 7; It is characterized in that described cucurbitane type triterpene saponin componds is the glucose tolerance activator under muscle and adipocyte glucose uptake promotor, euglycemic agent, membrane of glucose transporter 4 promotor, adenylic acid activated protein kinase activator, Fatty Acid Oxidation metabolic improver or the insulin resistant state in the application of preparation prevention and treatment diabetes medicament.

9. application according to claim 7 is characterized in that, described cucurbitane type triterpene saponin componds is adenylic acid activated protein kinase activator or Fatty Acid Oxidation metabolic improver in the application of preparation prevention and treatment antiobesity agents.

10. application according to claim 7; It is characterized in that described cucurbitane type triterpene saponin componds is the fat tolerance activator under plasma triglyceride and free fatty acid levels depressant or the disorders of lipid metabolism state in the application of preparation prevention and treatment high blood cholesterol drug.

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