TWI522108B - Compositions for treating diabetes - Google Patents

Description

Composition for treating diabetes

The present invention relates to a composition for treating diabetes comprising an extract derived from hydrazine, and in particular, the extract is obtained by extraction with an organic solvent. The invention also relates to a composition for the treatment of diabetes comprising a compound of formula (I).

茯苓 (Polyaceae) is a fungus on decaying pine trees. It has long been used as a traditional Chinese medicine and food. As mentioned previously, several triterpenes have been identified from sputum. See, for example, Phytochemistry , vol. 32, No. 5, pp. 1239-1244, 1993; and Phytochemistry , vol. 39, No. 5, pp. 1165-1169.

Diabetes is a serious chronic metabolic disease, and currently 3% of the world's population suffers from this disease. More than 90% of the diabetic population is diagnosed with type 2 diabetes. Diabetes is caused by defects in insulin production, defects in insulin action, or both. The drug for treating diabetes may be an insulin releasing agent (increasing the release of insulin), or an insulin sensitizer (promoting the action of insulin), or a Glucagon-like peptide-1 (GLP-1), etc. . However, there are not many clinical drugs available for diabetes, and available drugs often have adverse side effects such as reduced efficacy over time and low cost effectiveness.

Therefore, there is still a need for alternative agents for treating diabetes.

Based on bioactivity-directed fractionation and isolation (BDFI, see Figure 1), we demonstrate that the crude extract of ruthenium and chloroform are layered and the triterpenoids isolated therefrom are small in db/db. Mice exhibited hypoglycemic activity, and the ability of STZ mice to reduce blood glucose concentrations via increased insulin sensitizer activity was independent of the peroxisome proliferator-activated receptor-gamma (PPAR-[gamma]) pathway. Our results provide additional options for developing drugs that fight diabetes through different mechanisms.

Accordingly, in one aspect, the invention provides a composition for treating diabetes comprising an effective amount of an extract from sputum. Specifically, the hydrazine extract is obtained by extraction with an organic solvent. In a particular example, the organic solvent used is selected from the group consisting of alcohols, chloroform, ethyl acetate, and any combination thereof.

In another aspect, the present invention provides a composition for treating diabetes comprising an effective amount of a compound of formula (I):

Wherein, R ₁ is H, OH, keto group, OCH ₃ or OR, R is an alkyl group, alkenyl group, alkynyl group or mono-glycosylated; C _a -C _f, triterpene backbone, comprising C _a -C _d conjugated alkenyl group, C _a -C _b and / or C _d -C _e alkenyl, C _b -C _c alkenyl or C _c -C _f conjugated alkenyl group; R ₂ is COOH, COOCH ₃ or COOR, R Is an alkyl, alkenyl or alkynyl group; R ₃ is 2-(3-methyl-1-butyl) or 1-isopropenyl; R ₄ is CH ₃ or OCH ₃ ; R ₅ is H or C _c Forming an OO bridge; and R ₆ is H or OH.

Typically, compounds of formula (I) are isolated from hydrazine.

Specifically, the composition of the present invention is used as an insulin sensitizer. More specifically, this insulin sensitizer is independent of PPAR-γ. In a specific embodiment, the composition according to the invention further comprises an anti-diabetic agent known in the art, for example, a product from Bidens pilosa .

Also within the scope of the invention is an extract from medlar and a compound of formula (I) for use in the treatment of diabetes and in the preparation of a medicament for the treatment of diabetes. Specifically, this agent acts as an insulin sensitizer. More specifically, this insulin sensitizer is independent of PPAR-γ. In a specific embodiment, the pharmaceutical system according to the present invention is used in combination with an anti-diabetic agent known in the art, for example, a product from Xianfeng Grass.

The details of one or more specific embodiments of the invention are set forth below. Other features and advantages of the present invention will be apparent from the description of the appended claims appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning All documents mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials associated with the reference.

In this document, the singular forms "a", "the" Thus, for example, when reference is made to "a compound", it includes a plurality of such compounds and their equivalents known to those skilled in the art.

In one aspect, the invention provides a composition for treating diabetes comprising an effective amount of an extract derived from mash.

As is known in the art, diabetes includes both first and second types. The term "type 1 diabetes", "young-onset diabetes" or "insulin-dependent diabetes" as used herein refers to a disease characterized by too little or no insulin produced by the pancreas. The term "type 2 diabetes", "adult-onset diabetes" or "non-insulin-dependent diabetes" as used herein refers to a disease characterized by excessive glucose production even with insulin and insufficient glucose clearance. Blood sugar is maintained at an excessively high concentration.

The term "treatment" as used herein includes the prevention of a particular disorder or condition, or the alleviation of symptoms associated with a particular disorder or condition, and/or the prevention or elimination of such symptoms. For example, the term "treating diabetes" as used herein generally refers to reducing glucose concentration, increasing insulin sensitivity, or increasing glucose consumption.

As used herein, "effective amount" refers to the amount of each active agent that provides a therapeutic effect to an individual, either alone or in combination with one or more other active agents. As will be appreciated by those skilled in the art, effective amounts will vary with the route of administration, the use of excipients, and in conjunction with other active agents.

As used herein, "茯苓" encompasses the fungus itself and any products made therefrom, such as powders, granules, extracts, slurries, concentrates, and precipitates.茯苓 is commercially available. The extract of Lycium barbarum L. can be prepared by methods known in the art. Typically, dried mash is purchased from the local market in Taipei, Taiwan, ground into a powder, mixed with a solvent to form a suspension, and the supernatant is collected to obtain an extract of sputum. Additional steps for concentration, such as evaporation, or additional steps for purification, such as filtration, centrifugation, and chromatography, can be performed. In a specific embodiment, the extract from hydrazine is obtained by extraction with an organic solvent, specifically, the organic solvent is selected from the group consisting of alcohols, chloroform, ethyl acetate, and any combination thereof. "Alcohol" means any compound containing a hydroxyl group (OH) including, but not limited to, methanol and ethanol.

In another aspect, the present invention provides a composition for treating diabetes comprising a compound of formula (I):

Wherein R ₁ is H, OH, keto, OCH ₃ or OR, R is alkyl, alkenyl, alkynyl or monosaccharide; C _a- C _f is a triterpene backbone comprising C _a -C _d conjugated alkenyl group, C _a -C _b and / or C _d -C _e alkenyl, C _b -C _c alkenyl group or a C _c -C _f conjugated alkenyl group; R ₂ is COOH, COOCH ₃ or COOR, R Is an alkyl, alkenyl or alkynyl group; R ₃ is 2-(3-methyl-1-butyl) or 1-isopropenyl; R ₄ is CH ₃ or OCH ₃ ; R ₅ is H or C _c Forming an OO bridge; and R ₆ is H or OH.

The term "keto" refers to a Rx(CO)Ry group, wherein Rx and Ry are each independently alkyl, alkoxy, alkenyl, alkynyl or aryl, bonded to (CO) through a carbon-carbon bond. Group.

"Alkyl" is the term B means a straight or branched chain monovalent hydrocarbon, unless otherwise stated, contain 1 to 20 carbon atoms (e.g., C ₁ -C _8). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The term "alkenyl" refers to a straight or branched divalent hydrocarbon containing from 2 to 20 carbon atoms (eg, C ₂ -C ₁₀ ) and one or more double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, propenylene, allyl, and 1,4-butadienyl. The term "alkynyl" refers to a straight or branched chain monovalent or divalent hydrocarbon containing from 2 to 20 carbon atoms (eg, C ₂ -C ₁₀ ) and one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, ethynylene, 1-propynyl, 1- and 2-butynyl, and 1-methyl-2-butynyl. As used herein, the term "monosaccharide" refers to a saccharide unit (such as a six-carbon sugar or a five-carbon sugar) that can be linked via a glycosidic linkage (ester linkage), for example, a glucosyl group and an arabinose group. As used herein, the term "conjugated alkenyl", when taken alone or in combination, refers to an alkenyl group having at least two carbon-carbon double bonds, both of which are separated by a carbon-carbon single bond.

The triterpene compounds described herein having formula (I) include the compounds themselves as well as their salts, prodrugs and solvates (if appropriate). For example, a salt can be formed between an anion and a cationic group (e.g., an amine group) on the compound. Suitable anions include chlorine, bromine, iodine, sulfate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate, Glutamate, glucuronide, lactate, glutarate, and maleate. Similarly, salts can also be formed between the cation and an anion group (e.g., a carboxyl group) on the triterpene compound. Suitable cations include sodium ions, potassium ions, magnesium ions, calcium ions, and ammonium cations, for example, tetramethylammonium ions. Examples of prodrugs include esters and other pharmaceutically acceptable compounds which, when administered to an individual, provide an active triterpenoid compound. By solvate is meant a complex formed from a reactive triterpenoid compound with a pharmaceutically acceptable solvent.

In a particular embodiment, R ₁ is OH; R ₂ is COOH; the triterpene backbone comprises a C _a -C _d conjugated alkenyl group; and R ₃ is 2-(3-methyl-1-butyl); ₄ is CH ₃ ; R ₅ is H; and/or R ₆ is OH.

In another particular embodiment, R ₁ is OH; R ₂ is of COOH; triterpene backbone comprises a C _a -C _d conjugated alkenyl group; R ₃ is a 1-isopropenyl group; R ₄ is CH _3; R ₅ Is H; and/or R ₆ is H.

In a specific example, the specific triterpene compound of the present invention is selected from the group consisting of dehydrotumulosic acid (DHTuA), pachymic acid (PA), and dehydrotrametenolic acid (dehydrotrametenolic acid, Group of DHTA). The structure of DHTuA, PA and DHTA is as follows:

The triterpenoid compound used in the present invention may be in an isolated form, i.e., prepared by synthetic methods or by enrichment from a natural source such as hydrazine. By isolated triterpene compound is meant a preparation (by dry weight) containing at least 30%, preferably at least 40%, more preferably at least 50% of the compound. The purity of the isolated compound can be measured by, for example, column chromatography, mass spectrometry, high performance liquid chromatography (HPLC), NMR, or any other suitable method. Methods for isolating such compounds are well known in the art. See, for example, Phytochemistry , vol. 32, No. 5, pp. 1239-1244; and Phytochemistry , vol. 39, No. 5, pp. 1165-1169.

The triterpene compound can comprise a non-aromatic double bond and one or more centers of symmetry. Thus, triterpenoid compounds can exist in the following forms: racemates and racemic mixtures, single mirror phase isomers, single non-mirror isomers, mirror phase isomer mixtures, and cis or trans isomers. All isomeric forms as described above are contemplated.

To carry out the treatments described above, the extract of sputum or one or more triterpenoids described herein can be formulated into a composition with a physiologically acceptable carrier.

As used herein, "physiologically acceptable" means that the carrier is compatible with the active ingredient contained in the composition, preferably to stabilize the active ingredient, and is not deleterious to the individual to be treated. The carrier can act as a diluent, carrier, excipient or matrix for the active ingredient. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gum arabic gum, gelatin, calcium citrate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The composition may additionally comprise a lubricant, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, for example, methylbenzoic acid and propylhydroxybenzoic acid; sweeteners; And flavoring agents.

The compositions of the present invention can be administered orally, parenterally, by inhalation spray or in an implantable reservoir. Typically, the compositions of the invention are for oral administration.

Sterile injectable compositions (e.g., aqueous or oily suspensions) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable carriers and solvents, mannitol, water, Ringer's solution and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are conventionally employed as a solvent or suspension base (synthetic monoglycerin or diglycerol). Fatty acids, for example, oleic acid and its glyceride derivatives, and pharmaceutically acceptable natural oils, for example, olive oil or castor oil, especially in its polyoxyethylated form, are useful in the preparation of injectables. The above oil solution or suspension may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or a similar dispersing agent.

Oral compositions can be in any form suitable for oral administration, including, but not limited to, lozenges, capsules, emulsions, and aqueous suspensions, dispersions and solutions. Tablet carriers commonly used include lactose and corn starch. Lubricants such as magnesium stearate are also typically added to the tablet. For oral administration in capsule form, diluents which may be employed include lactose and dried corn starch. When the water-soluble suspending agent or emulsion is administered orally, the active ingredient can be suspended or dissolved in an oil phase in admixture with an emulsifying or suspending agent. Specific sweeteners, flavoring or coloring agents may be added if desired.

Inhaled compositions can be prepared according to techniques well known in the art of pharmaceutical formulation, and may utilize benzyl alcohol or other suitable preservatives, absorption enhancers that enhance bioavailability, fluorocarbons, and/or other agents known in the art. A solvent or dispersant formulated as a solution in saline.

As evidenced by the following examples, the extract comprising hydrazine or the composition of the compound of formula (I) of the present invention lowers blood glucose concentration and is therefore useful for treating diabetes. In particular, the compositions of the invention are useful as insulin sensitizers. More specifically, this insulin sensitizer is independent of PPAR-γ.

The term "insulin sensitizer" refers to a compound which exhibits hypoglycemic activity by enhancing the action of insulin in a target tissue. Insulin sensitizers are particularly useful for the treatment of insulin-resistant patients by allowing the body to exhibit a more normal response to insulin secretion, which inhibits pancreatic secretion of insulin and helps restore hormonal balance.

In one embodiment, the compositions of the present invention further comprise an anti-diabetic drug known in the art, for example, a product from Bidens pilosa .

Also within the scope of the invention is an extract from medlar and a compound of formula (I) for use in the treatment of diabetes and in the preparation of a medicament for the treatment of diabetes. Specifically, this agent acts as an insulin sensitizer. More specifically, this insulin sensitizer is independent of PPAR-γ. In a specific embodiment, the pharmaceutical system according to the present invention is used in combination with an anti-diabetic drug known in the art, for example, a product from Xianfeng Grass.

Without further elaboration, it is believed that the foregoing description may be Accordingly, the following specific examples are to be understood as merely illustrative, and in no way limit the. All documents cited herein are incorporated herein by reference in their entirety.

material

Dimethyl sulfoxide (DMSO), glimepiride (GLM), metformin (Met), methanol, ethyl acetate (EA), chloroform, cyanomethane (ACN), trifluoroacetic acid (TFA) And streptozocin (STZ) was purchased from Sigma-Aldrich (Missouri, USA). Tannin and insulin were purchased from Apin Chemical Co., Ltd. (Oxfordshire, England) and Novo Nordisk (New Jersey, USA). 3T3-L1 adipocytes were cultured at 37 ° C, 5% CO ₂ and DMEM medium (Invitrogen, California, USA) supplemented with 10% calf serum (BCS), 100 units/ml penicillin, 100 μg/ml of Streptomyces , 6 mM HEPES (Invitrogen, California, USA) and 2 mM L-glutamic acid (Invitrogen). pSGGAL-PPAR-γ, a chimeric protein that expresses a Gal-4 DNA binding domain linked to a PPAR-γ ligand binding domain, and a (UAS) 5-tk-LUC reporter construct comprising a gene linked to a cold light enzyme The upstream activated sequence pentamers of yeast are generously donated by Dr. Krister Bamberg (Asttricon, Moendal, Sweden).

Statistical Analysis

The results of three or more independent experiments are presented as mean ± standard error. Data were analyzed by ANOVA. Post-comparison comparisons were made using the Bonferroni method when it was appropriate to detect sources of significant differences. Differences in P values less than 0.05 were considered statistically significant.

Example 1: Preparation of Crude Extract, Layering and Preparation of Triterpenoid Compound

It was purchased by the local market in Taipei, Taiwan. It was appraised by the Taiwan Biotechnology Development Center. The evidence specimens are deposited in the Agricultural Biotechnology Research Center.

To prepare a crude methanol extract of hydrazine, the dried mash was ground to a powder. The cerium powder (5 kg) was extracted with methanol (1:10 w/v) three times for six days to yield a methanol extract of 7.5 纯度 purity. The crude methanol extract was dissolved in water (1 L) and extracted with chloroform (1 L x 5). After chloroform layering (0.82 Torr) was obtained, the remaining extract was further extracted with ethyl acetate (1 L×5) to give ethyl acetate layer (2.9 ‰) and water layer (1.8 ‰). Active chloroform by using a normal phase high performance liquid chromatography (HPLC) column (Phenomenex, Luna 5 μ silica, 250 × 10 mm) set to a flow rate of 5 ml/min and detected with 242 nm UV light. The layering was further layered into chloroform sub-layers 1 (0.10 ‰), 2 (0.11 ‰), 3 (0.11 ‰), 4 (0.12 ‰), 5 (0.10 ‰), and 6 (0.24 ‰). The solvent gradient for HPLC was ethyl acetate (A) and n-hexane (B): 0 to 60 minutes, 30% A; 60 to 90 minutes, 100% A. As published elsewhere ( J Pharm Biomed Anal 2009; 49:267-275), an RP-C 18 HPLC column (Phenomenex, Luna 5 μ) set at a flow rate of 1 ml/min and detected at 242 nm UV light C18, 250 x 4.6 mm) to describe the chemical characteristics of the six chloroform sub-layers. The solvent gradient for HPLC is water/0.05% TFA (C) and acetonitrile/0.05% TFA (D): 0 to 15 minutes, 5 to 15% D; 15 to 35 minutes, 5 to 20% D; 35 to 65 Minutes, 20 to 40% of D; 65 to 100 minutes, 40 to 100% of D; 100 to 110 minutes, 100% of D. A ruthenium compound was identified from a chloroform sub-layer using a RP HPLC column (Cosmosil, 5 μC18, 250 × 10 mm) with a flow rate of 5 ml/min and detection at 242 nm. The solvent gradient used in HPLC was water/0.05% TFA (E) and acetonitrile/0.05% TFA (F): 0 to 10 minutes, 30 to 52% F; 10 to 50 minutes, 51 to 100% F; 50 to 60 minutes, 100% F. Identification was obtained by comparing data from NMR and MS with previously published data ( J Pharm Biomed Anal 2009; 49:267-275; Phytochemistry 1993; 32:1239-1244; Phytochemistry 1995; 39:1165-1169) Hydrogen 16α-hydroxy perforate, dehydroabietic acid and citric acid.

Figure 1A shows the crude extract, layered, sub-layered and triterpene compound yields. The HPLC profile was determined as shown in Figure 1B. According to the results herein, chloroform sub-layers 4 and 6 are active, while in the active chloroform sub-layer 4, a triterpenoid compound, dehydrogenated 16α-hydroxy perforate, is identified. The other two triterpenoids, citric acid and dehydroabietic acid, were isolated from the non-active chloroform sub-layers 2 and 3.

Example 2: Biological Analysis 2.1 Animals

Animals were purchased from Jackson Laboratory (Maine, USA) and the National Experimental Research Laboratory Experimental Animal Center (Taiwan, Taipei), which were male C57BL/KsJ-db/db mice with point mutations in leptin receptors. And male C57BL/6J mice. All animals were housed and treated according to the guidelines of the Academia Sinica Animal Management Group.

2.2 hypoglycemic effect in type 2 diabetes model

Six to eight week old diabetic C57BL/KsJ mice (type 2) were fasted for 12 hours prior to the experiment and then allowed to free access to food and water for 2 hours. At time 0, the food is removed (water is available), and the blood sampled at this point in time belongs to the postprandial. After grouping, the mice were fed a tube carrier, metformin (an anti-diabetic drug known in the art), a crude extract, a layered, sub-stratified or isolated compound. The postprandial blood glucose concentration was monitored for 4 hours. Table 1 shows the results.

Table 1A shows the hypoglycemic effect of the crude extract of Lycium barbarum L. in db/db mice.

Note: Diabetic db/db mice were fed with water (Ctr), metformin (Met, 60 mg/kg body weight) and crude extract (CE, 10, 50, 100 mg/kg body weight). Blood glucose concentrations were measured before (0 hours) and after (1 to 4 hours) tube feeding.

As shown in Table 1A, diabetic db/db mice spontaneously developed hyperglycemia with a postprandial blood glucose concentration of approximately 350 to 400 mg/dL, whereas db/db mice receiving a dose of control vehicle, Its blood glucose concentration dropped to 165 mg/dL after 4 hours. As expected, formazan significantly reduced the blood glucose concentration of db/db mice. Unexpectedly, the crude extract of sputum also significantly reduced blood glucose concentration at a single dose of 50 or 100 mg/kg body weight.

Table 1B shows the hypoglycemic effect of sputum water, ethyl acetate and chloroform layered on db/db mice.

Note: Same as the procedure in Table 1A except that water (Ctr), metformin (Met, 60 mg/kg body weight), water layer (Water, 50 mg/kg body weight), ethyl acetate layer (EA, 50) Mg/kg body weight) and chloroform layer (Chl, 50 mg/kg body weight) were administered.

As shown in Table 1B, db/db mice receiving a dose of control vehicle reduced their blood glucose concentration to 164.5 mg/dL after 4 hours, and metformin effectively lowered blood glucose levels, and surprisingly, chloroform stratified It also significantly reduces blood sugar levels.

We further evaluated the hypoglycemic effect of chloroform sub-stratification from 1 to 6 with active chloroform stratification. Table 1C shows the results.

Note: Same as the procedure in Table 1A except that water (Ctr), metformin (Met, 60 mg/kg body weight) and six chloroform substrates (Chl-1 to Chl-6, 50 mg/kg body weight) Feeding.

As shown in Table 1C, one dose of metformin effectively reduced the blood glucose concentration of db/db mice compared to the control vehicle, while chloroform sub-stratifications 4 and 6 were larger than the other four sub-stratifications. Significantly lower blood glucose levels. The grading 4 (50 mg/kg body weight) hypoglycemic effect seems to last longer than the secondary stratification 6 (50 mg/kg body weight).

Finally, we evaluated the hypoglycemic effect of the triterpenoid compound obtained in Example 1 in db/db mice. Table 1D shows the results.

Note: Same as the procedure in Table 1A except that DMSO/water (Ctr), metformin (Met, 60 mg/kg body weight), dehydrogenated 16α-hydroxyporous acid (DHTuA, 1, 5, 10 mg/kg) Body weight), niacin (PA, 1, 5, 10 mg/kg body weight) and dehydroabietic acid (DHTA, 1, 5, 10 mg/kg body weight) were administered. All of the above data are expressed as mean ± standard error. A P value (*) of less than 0.05 was considered to be statistically significant. The number of experimental mice is indicated in brackets.

Similar to metformin, dehydrogenated 16α-hydroxy perforate showed significant hypoglycemic activity in db/db mice, while dehydrochaetinoic acid and citric acid also showed hypoglycemic activity.

2.3 hypoglycemic effect in the first type of diabetes model

In order to understand the anti-diabetic mechanism of the crude extract and the triterpenoid compound, we tested the hypoglycemic effect in STZ-treated mice (type 1) in which β-islet cells were destroyed. Five-week-old C57BL/6J mice were treated with intraperitoneal injection of streptozotocin (STZ, 200 mg/kg). Mice with a blood glucose concentration higher than 500 mg/dL and a serum insulin concentration of less than 0.18 ng/ml were selected, and then the control agent, metformin, and melmepiride were fed by tube irrigation (known in this field). An anti-diabetic drug), a crude extract and an isolated compound (time point - 1 hour). Sixty minutes after the tube was fed with the mice, the mice were intraperitoneally injected with 2.5 IU/kg of body weight of insulin. The blood glucose concentration was measured within 4 hours, and the mice were given free access to food and water at all time points. Blood glucose concentrations were measured using an Elite blood glucose tester (Bayer, PA, USA). This test modified from the previous study (Diabetes 2000; 49: 436-444; J Ethnopharmacol 2009; 122: 379-383). Table 2 shows the hypoglycemic effect of the crude extract of sputum and the triterpenoid compound in STZ-treated mice.

Note: For C57BL/6J mice that have received high doses of STZ, one dose of vehicle (DMSO/water, Ctr), formic acid (Met), Marlin (GLM), and crude extract of sputum are fed by tube irrigation. (CE), dehydrogenated 16α-hydroxy perforate (DHTuA), dehydroabietic acid (DHTA) and citric acid (PA). Blood glucose concentrations were determined before (-1 and 0 hours) and after (1 to 4 hours) before tube feeding and intraperitoneal injection of insulin (2.5 IU/kg body weight). A P value (*) of less than 0.05 was considered to be statistically significant.

As shown in Table 2, insulin administration reduced the blood glucose concentration of STZ mice, and as expected, Marse's pancreas (insulin release agent) did not show insulin-regulated blood glucose decline in STZ-treated mice, but metformin (insulin) The sensitizer) effectively enhances the insulin-regulated blood glucose drop. In the same manner as metformin, the crude extract of sputum enhances insulin-regulated blood glucose decline. Similar results were obtained with dehydrogenation of 16α-hydroxy perforate, dehydroabietic acid and, if any, citric acid. The above results indicate that the guanidine and triterpenoid compounds have an activity as an insulin sensitizer rather than an insulin release agent.

2.4 PPAR-γ reporter analysis

PPAR-γ plays a role in glucose metabolism, which is the target of insulin sensitizing drugs. Therefore, we further examined whether the crude extract and the triterpenoid compound regulate the activation of PPAR-γ. 3T3-L1 (10 ⁷ ) cells were treated with pSGGAL-PPAR-γ and (UAS)5-tk-LUC using a Bio-Rad electroporator (Hercules, Bio-Rad, Calif.) set at 260 V and 975 μF. Electroporation. Two hours after transfection, the cells were grouped and treated with DMSO, rosiglitazone (RSG), crude extract of sputum and triterpenoids for 24 hours. 10 μg of whole cell lysate was subjected to double luminescence enzyme assay as shown in published literature ( Mol Immunol 2009; 46:3218-3223). The PPAR-γ activity multiple was defined as the firefly/jellyfish luminescence enzyme ratio, normalized by the firefly/jelly chilling enzyme ratio of the control group. Figure 3 shows the results.

As expected, rosiglitazone (PPAR-gamma agonist) activates PPAR-[gamma]. However, the crude extract, dehydroabietic acid and citric acid did not activate PPAR-γ. All data suggest that sputum and triterpenoids are enhanced by insulin sensitivity to lower blood glucose concentrations, which is independent of PPAR-[gamma] (Figure 3).

Overall, experiments with db/db mice and STZ-treated mice showed that crude extracts of triterpenoids and triterpenoids significantly enhanced insulin sensitivity and thus reduced blood glucose concentrations in diabetic mouse models. This enhancement is independent of PPAR-γ activation.

Other specific examples

All features disclosed in this specification can be combined in any manner. Each feature disclosed in this specification can be substituted for an alternative feature that is the same, equivalent, or the like. Therefore, unless expressly stated otherwise, each feature disclosed is merely an equivalent of the one of the one of the

From the above description, those skilled in the art can readily clarify the essential features of the invention, and various modifications and changes can be made to the various uses and conditions without departing from the spirit and scope of the invention. Therefore, other specific examples are also in the scope of patent application.

To illustrate the invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the preferred embodiments shown.

In the schema:

Figure 1 shows a bioactive-directed fractionation and isolation (BDFI) study of sputum and its activity against diabetes.

Figure 2 shows the separation steps of crude extracts, fractions, sub-fractions and triterpenoids and their high-performance liquid chromatography (HPLC) spectra. Figure 2A is a flow chart showing the steps for preparing the crude extract, layered, sub-layered and triterpene compound, the yields of which are indicated in parentheses. Figure 2B shows the chemical extract of the crude extract and layered ruthenium obtained by UV detector after HPLC column, in which dehydrogenated 16α-hydroxy perforated acid (DHTuA, 1), 茯苓Acid (pachymic acid, PA, 2) and dehydrotrametenolic acid (DHTA, 3) were identified from sub-stratified Chl-4, Chl-2 and Chl-3, respectively.

Figure 3 shows the effect of crude extracts of deuterium, dehydrogenation of 16α-hydroxy perforate (DHTuA) and citrate (PA) PPAR-γ. In Fig. 3A, "N" indicates treatment with DMSO (negative control), "RSG" indicates treatment with rosiglitazone (1 μM), and "CE" indicates crude extract with sputum (0.063, 0.25 and 0.63 mg) /ml) processing. In Figure 3B, "N" indicates treatment with DMSO (negative control), "RSG" indicates treatment with rosiglitazone (1 μM), and DHTuA indicates dehydrogenation of 16α-hydroxy perforate (10, 40 or 100 μM) Treatment, while PA means treatment with citric acid (10, 40 or 100 μM). All data in this figure are expressed as mean ± standard error. A P value of less than 0.05 was considered statistically significant.

Claims (6)

Use of a methanol or ethanol extract of hydrazine or a compound of formula (I) for the preparation of a medicament for insulin sensitizers for diabetic patients, wherein the compound of formula (I) is: Wherein R ₁ is OH or a keto group; and C _a -C _f is a triterpene main chain comprising a C _a -C _d conjugated alkenyl group, C _a -C _b and/or C _d -C _ealkenyl , C _b -C _c alkenyl or C _c -C _f conjugated alkenyl group; R ₂ is COOH or COOR, R is an alkyl, alkenyl or alkynyl group; R ₃ is 2- (3-methyl-1-butyl Or 1-isopropenyl; R ₄ is CH ₃ or OCH ₃ ; R ₅ is H; and R ₆ is H or OH; and the compound of formula (I) is selected from dehydrogenated 16α-hydroxy perforate (dehydrotumulosic) Acid, DHTuA), pachymic acid (PA) and dehydrotrametenolic acid (DHTA). According to the use of the first aspect of the patent application, wherein the diabetic patient is a type 1 diabetes patient. The use according to the first aspect of the patent application, wherein the medicament is for oral administration. The use according to the first aspect of the patent application, wherein the compound of the formula (I) is citric acid or dehydroabietic acid. The use of any one of claims 1 to 4, wherein the medicament is further compatible with a product from Bidens pilosa for treating diabetes. Use of a methanol or ethanol extract of any of claims 1 to 4 or a combination of a compound of formula (I) with a product from Bidens pilosa for the treatment of diabetes The drug.