TWI337867B - Compound and its composition - Google Patents

Description

MODIFICATION OF THE EMBODIMENT OF THE INVENTION EMBODIMENT OF THE INVENTION The present invention relates to a compound composition and a preparation method thereof, particularly to reducing cellular insulin resistance for treating the second type. A compound of diabetes, a composition thereof, and a method of preparing the same. [Prior Art] According to statistics, the number of people with type 2 diabetes worldwide is as high as 3.5 billion to 1.6 13⁄4. This type of diabetes is also known as adult type diabetes, which is more common in middle-aged people. Studies have found that insulin binds to receptors on the cell surface, increases cell membrane permeability, and induces cells to absorb glucose. This type of diabetes is mainly caused by decreased sensitivity of body cells to insulin, ie, insulin resistance. The imbalance in the metabolic response of the body to insulin regulation, so insulin supplementation can not improve glucose metabolism, so this type of diabetes is also known as non-insulin-dependent diabetes mdlitus (Gerich, 2001). At present, many drugs have been developed for the treatment of diabetes, such as sulfonylurea, biguanide or TZD [thiazolidione]. Triterpenoids extracted from bitter gourd have also been shown to reduce blood sugar. Blood sugar in diabetic mice (jjarinantenaina et al, 2006) 'eg 5 厶, 19-epoxy-3 β, 25-dihydroxycucurbita-6, 23 〇?)-diene and 3,7 /5 ,25-trihydr〇Xycucurbita_5,23 (^_dien_19_al. However, most diterpenoids have not found similar functions. In addition, the mechanism for lowering blood glucose in type 2 diabetes is complicated. It is not yet confirmed that the two compounds have a reduction of 1337867. ^Insulin resistance is light. Xuxiang research refers to the premature cause of diabetes, so the type: not only for U = already suffering from type 2 diabetes, insulin resistance has not developed but not developed into diabetes T avoids 1 2 positive ^Pre-clockwise coffee pool & W, which deteriorates to diabetes, the main object of the present invention is to provide a compound and a conjugate thereof, a pot

The compound c_rb.5,23(E)_diene, 7p,25 sheb isolated from bitter gourd and confirmed by liver cells that the compound does have a cake (10) island-like Wei, which restores the ability of cells to absorb glucose, and The composition is tested to provide the present invention with the effect of lowering blood glucose concentration. "

According to the preparation method of the compound of the present invention and the composition thereof, the extract of the bitter gourd is extracted by an organic solvent and the extract is purified by column chromatography to obtain the compound. The compound has a structure as shown, which restores the ability of cells to absorb glucose, and thus can be used as an active ingredient of a medicament for the treatment of type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will become more apparent from the <RTIgt

Cucurbita-5,23(;E)-diene-3p,7p,25-triol (hereinafter referred to as the compound revised replacement on November 25, 1999), as shown in the following formula I:

Formula I

And /, the salt that can be tolerated in music. The pharmaceutically acceptable salt is selected from the group consisting of, but not limited to, salts of sodium, potassium, lithium, calcium, magnesium, zinc, and iron. The compound is extracted from bitter gourd in purified form or directly obtained by organic synthesis. The extraction process utilizes the following methods or utilizes other techniques and techniques known in the art to provide fresh extraction and purification techniques. The bitter gourd tissue is obtained from the cucumber plant, and the bitter gourd jj ' is extracted by an organic solvent, and the organic solvent may be selected as a non-polar organic shop, a polar organic: a back or a mixture thereof to obtain a chelating compound, and is selected as a bitter gourd plant. A part, such as leaves and stems, and: a non-polar solvent is a white phase, π is not two and root 4, the benzene, dimethyl 4 is not limited to carbon tetrachloride, two gas, a stupid, an armor _ ϋ 5 hectares' · the polar solvent system, selected from but one ether i alcohol, isopropyl alcohol, propylene steel, 2-butyl, ethyl acetate, acetaminophen, furan, gas, pyrrole, dimethyl A One (four) literary amines and their mixtures. The use of ", - methyl H-dimethylformamidine is preferably selected from the group consisting of: / or bitter melon - towel organic solvent grinding treatment of the compound is preferably processed into a fine film or other such solvent The surface area of contact Λ σ extraction procedure is to first place the bitter gourd tissue in a container containing 1337867 99 Π 25 曰 modified replacement or f polar solvent f non-polar solvent, the small number of immersion is extracted by its scale device. The liquid _ &gt; is condensed, for example, by volatilization, and the polar or non-polar concentrate of the compound is volatilized to obtain the compound containing the polar or non-polar concentrate and then mechanized. Is an extract containing the compound at a higher concentration == raw, and the polar or non-polar concentrate can be separated and purified by crystal (for example, or preferably by column chromatography), for example, Shi Xisheng column Chromatography and six-phase phase chromatography [Ηριχ]#. Column chromatography When the compound is purified from the polar or non-polar concentrate, it is subjected to several times of chromatography to obtain the compound of appropriate purity, preferably After the silicone Column chromatography to obtain a number of distinctions, one of which distinguishes one of the compounds of higher purity. The distinction is repurified to obtain a higher purity of the compound. The selection of the extract from the bitter gourd tissue is carried out by purification or separation directly from the organically synthesized compound. Since the compound has the effect of lowering blood sugar, it can be used for medical diabetes m, especially for type 2 diabetes. The insulin-resistant 'visitage has the function of reducing the metabolism of glucose by the isletous heterogeneous cells, and can be used for preparing various compositions such as drugs or foods, or for academic research, such as the action mechanism and activity of hypoglycemic agents. The compound is used in the form of purified _g/ml, and the compound or the composition containing the compound can be modified through various routes - 8 - 1337867 99 U 25 25 Replacement of ' _ — % \ 施 ' ” includes but is not limited to oral and injection. The injection method is selected from - but not limited to intravenous, intraperitoneal, subcutaneous For injection and intramuscular injection, the preferred route of administration is oral. The composition may further comprise other hypoglycemic agents such as insulin, sulfonylurea, biguanide, TZD [thiazolidione], adrenal gland. P3-adrenergic receptor agonist or glycolysis inhibitor [α. -g^osidase], etc. Sulfonamide contains acetohexamide, chlorpropamide, and doramic acid. Amine • Wazamide, dobutamine, glyburide, glyryzide, gyidazide Ming [buformin], TZD contains troglitazone; adrenal receptor stimulants include CL-316, 243, etc.; glycolytic antispasmodic inhibitors include acarbose and migralide [migiat〇1] . The compound can also be administered to a pharmaceutically acceptable salt in a therapeutically effective amount, such as a salt of sodium, potassium, lithium, calcium, magnesium, zinc, iron. Referring to Figure 1, the compound is represented by the following preferred examples: a stem of 18 kg of dried bitter gourd, processed into a fine piece, and subjected to extraction filtration step 101: soaking three times with 30 liters of methanol each time. When soaking. · One week. The soaking liquid is subjected to hydrazine, concentrated and volatilized to obtain a decyl alcohol thick liquid to obtain *crude extract 102. The partitioning extraction step (10) is carried out, and the methanol thick liquid is added to water to 1 liter to suspend the ocean, and firstly extracted and extracted with 1 liter of acetic acid (EtOAc) to obtain 386 g of ethyl acetate layer, and the obtained extract is The first organic solution_extract 104' is further subjected to extraction with n-liter n-butanol (n_Bu〇H) ~9~1337867, and the last county layer is the aqueous layer extract 105. The first separation step 106 is carried out. · The acetic acid is applied to the layer, and the layer is added with lion gel. The 7 gum of the rhyme is added to the first mL, n-hexane n-hexane: EtOAc] n-hexane: EtOAc N-hexane: EtOAc] n-hexane: EtOAc] n-hexane: EtOAc: EtOAc: EtOAc: EtOAc Pipe column 1σ7 is filled with 6 kg of broken glue for fixing =, with a mixture of hexose, ethyl acetate and methanol as the mobile phase 'biliary mixing _ _ as the mixing difficulty, according to the extraction of 11 distinctions, respectively, to distinguish 1 to Distinguish Chuan District i = [4000 mL, 49:1 [4000 mL, 19:1 [4000 mL, 9:1 [4000 mL, 17:3 [4000 mL, 8:2 [4000 mL, 7:3 [3000 mL] , 5:5 [3000 mL, 4:6 [3000 mL, 2:8, the difference between 2, 3, 4, 4, 5, 6, 6, 7, 8, 9, 9, 10 n-he :Et〇Ac] and the extract obtained by distinguishing n[ _ ◦ 2 222 2 8 is the second organic soluble extract (10). Separation step 1〇9: Separation of the column 铯仃 £ £ £ £ £ , , 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 The mixture of methane and ethyl acetate was extracted by ^ ^ ^ phase to obtain 6 distinctions. The JH2Cl2: Xie °] is a moving knife. It is 8-8 to the liver, and each area is 500 mL. There is a trip to the third organic solvent to extract the extract m. Carry out the first: age, m knife, step 112: distinguish 8DiX high-performance liquid, 10, November 25, 1999, replace the phase chromatography method gas, and transfer to the third chromatography column 113 (250 x 10mm) Lichrosorb Silica Ming 160 (5//111) is a stationary phase. It is extracted with a mobile phase of n-hexane and acetone mixture [7:3 n_hexane:acetone] at a flow rate of 2 mL/min and purified in 25.4 minutes. 7 mg of this compound, the extract obtained was the fourth organic solvent layer extract 114. Please refer to Figures 2 to 4 and Table 1. The purified amorphous white powder of this compound was subjected to nuclear magnetic resonance spectrometry [dept (135., 90°) η (:-ΝΜί〇Η-ΝΜίυ, mass spectrometer [EI- MS], infrared spectrometer, ultraviolet spectrometer, polarimeter and X_ray crystal diffraction analyzer for structural analysis' results show that the compound is Cucurbita-5,23(E)-diene-3, 7 3,25-trio molecular formula c30H48O2 ; optical rotation [to +13.5 (c 0.4, CHC13); infrared spectrum IR (KBr) % 3550, 3020, 2945, 2872, 1703, 1654, 1455, 1377, 1270, 1036, 973, 734 cm · 1; EIMS w/z 458 [M]+ (1), 440 (14), 422 (35), 407 (18), 404 (21), 389 (100), 187 (30), 171 (36), 157 ( 29), 133 (37), 109 (50), 81 (35); high-resolution mass spectrometry HREIMS m/z 440.3649 (calcd for C30H48O2 440.3656); nuclear magnetic resonance spectra are shown in Table 1. Table 1. Nuclear Magnetic Resonance Spectroscopy] taru ^HNMRDataforlClOOMHz and 400 MHz in CDC13) position 1 20.9 1.52 m, 1.56 m 2 28.7 1.74 m, 1.94 m 3 76.7 3.53 br s 4 41.5 5 146.8 6 122.5 5.80 d (6.4) 7 68,2 3.92 d (6.4)

2-28 dd(l〇4 6 m L44 m, 1.62 m 1·48 m, 1.62 m 1.30 m, 1.34 m 132 m, l.88m 1.48 m 0.88 s 1.04 s 1.48 m 0.85 d (6.4) 1.70 m, 2.14 m 5.56 rti 5.56 m 1.28 s 1.28 s 1.01 s 1.18s 0.66 s The eye is shown in Figure 5 and Table 2. The effect of insulin resistance, σ is reduced to assess the cell to 1 + _ blood glucose concentration. i. Cell culture is analyzed by 纟—(4) (4) The evaluation of the amount of force is the change of the _ ^ (4) sugar molecules, which reflects the absorption capacity of the swimming Φ. The first, second, third and second tests of the control group are prepared separately, and X is performed. The cell was evaluated for glucose uptake. In the control group, the small gas liver cell strain was cultured in a multi-well plate, centrifuged to attach the cells, the medium was aspirated, and fresh medium was added, respectively, in 〇, 1, 2 The medium was taken at 3, 4 and 5 hours 'analyzed by the colorimetric method using a glucose quantitative kit (Biosystems, Barcelona, Spain) - 12 1337867 November 25, 1999 corrected replacement of the glucose concentration change. The preparation is in the preparation process of the control group. Plus human G.1 // M Insulin treatment gland cells' respectively square, 2,3,4 Bu 5 hours and the change in the medium taken sugar concentration analysis.

In the preparation of the control group, the liver cell line FL83B was cultured in a multi-well plate, and the cells were treated with g/ml of tumor necrosis factor "[TNF-α] for 5 hours. After the resistance, the cells were attached by centrifugation, and the medium containing TNF_a was added and then the fresh medium was added to the medium to add 〇1#M insulin, and the medium was taken for analysis of glucose at 2, 3, 4, and 5 hours, respectively. The concentration of the experimental group was determined in the control group and the medium was taken at G 2, 3, 4 and 5 hours to analyze the glucose concentration change.

The results of the evaluation of glucose uptake by the cells are shown in Table 2 below. In control group 1, cells that were not treated with TNF-a absorbed glucose slowly, and only 5 hours decreased the glucose concentration in the medium by about 16 bf. In the control group 2, the same cells were added with O.lyM insulin to induce a decrease in glucose concentration in the medium by 2.6 mM. In the control group III, insulin-resistant cells produced by TNF-alpha treatment were similar to those induced by insulin-free. In the experimental group, the insulin-resistant cells in the right group were added with 1 〇 #g/ml of the compound and the concentration of glucose in the medium after 0.1//M was reduced by 3 22 sec. The results of the experiment show that the compound has the effect of reducing insulin resistance, and the cells that originally lost insulin sensitivity can be re-infected with insulin. - 13337867 99] Modified on January 25, to restore the glucose absorption capacity of the cells. . Table 2, sugar absorption evaluation results House jjg concentration QnM) time (hr) and · 6362 control · 纟 and = three | control group three experimental group 6.150 2

5.775JJ%sms 4J62 6.309 6.005 5.552 5.232 6.557 '6ΛΪ2K090 .6.299 5.694 4.2433ΜΪ 5.157 Τ58Τ

Although the present invention has been disclosed by the above-described preferred embodiments, it is not intended to be a preferred embodiment of the invention, and it is to be understood by those skilled in the art that the various modifications and modifications of the above-mentioned implementations are still within the scope of the invention. The technical scope of the present invention is protected by the scope of the invention as defined in the appended claims.

-14 - November 25, 1999 Revision Replacement [Simplified description of the drawings] Fig. 1 is a flow chart showing the process of the compound of the preferred embodiment of the present invention. Figure 2: 1H-NMR spectrum of the compound of the preferred embodiment of the invention. Figure 3: The spectrum of the compound of the preferred embodiment of the invention. Figure 4: EI-MS mass spectrum of the compound of the preferred embodiment of the invention. Fig. 5 is a graph showing the results of changes in the glucose concentration of the cell culture medium in the cell evaluation of glucose absorption in the preferred embodiment of the present invention. [Main component symbol description] 101 extraction filtration step 103 distribution extraction step 105 aqueous layer extract 107 first chromatography column 109 second separation step 111 second organic solvent layer extract 113 third chromatography column 102 crude extract 104 first organic solvent layer extract 106 first separation step 108 second organic shaft layer extract 110 second chromatography column 112 third separation step 114 fourth organic solvent layer extract

Claims (1)

Mountain / 867 'Application for Patent Park: November 25, 1999, amending the replacement of this compound, which has the structure shown in the following formula I: Formula I And pharmaceutically acceptable salts thereof. 2. A use according to the compound of claim 1 for the preparation of a medicament for reducing cellular insulin resistance. 3. The use of a compound as described in claim 1 for the preparation of a medicament for reducing jk sugar. 4. The use of the compound as described in claim 1 is for the preparation of a medicament for the treatment of type 2 diabetes, and the preparation method of the compound as described in claim 1 of the patent application includes : f take the filtration step, soak the bitter gourd with T alcohol, and then filter and concentrate = piece-methanol concentrated liquid, the methanol thick liquid system contains a crude extract; The human water and the acetic acid B were extracted by means of extraction, and the ethyl acetate δ layer was obtained. The acetic acid layer contained the first organic extract, and the acetyl alcohol was then extracted with n-butyl = row-- Alcohol layer and water layer, the water layer belongs to the stroke; -· 16 9 , 10 , 11 , ~ the first separation step , the Chiang - chromatography column is filled with a mixture = acid = _ Organic solution _ extract; 仃 technology, transfer - distinguish it contains ~ second separation step camp separation, and the human-chromatographic column for the above distinction, δ liquid for scouring, to obtain a distinction between the system 3 First organic granule layer extract; high second separation step to obtain the difference Subjected to column chromatography third sub-column two exit ㈣- = Μ comprising a fourth layer __ extract. The method of preparation according to item 5, wherein the mixing of the third separation step (four) is a mixture of hexane and _. The preparation method according to the fifth aspect of the invention, wherein the second component is a mixture of two gas and acetic acid ethyl acetate. The preparation method according to item 5 of the invention of the invention, wherein the mixture of the first and second steps is a mixture of hexanyl acetate, acetic acid, and sterol. The preparation method described in claim 5, wherein the extraction step is extracted from the stem portion of bitter gourd. According to the financial method described in the patent application (4), wherein the weight ratio of sterol to bitter gourd in the extraction filtration step is 3:4. The preparation method according to claim 5, wherein the bitter melon is soaked in sterol three times for one week in the extraction filtration step. A pharmaceutical composition for reducing the resistance of sirins, which comprises the compound described in claim 1 of the patent application as an active ingredient. —17 — 12, 1337867 . ' '; January 11th, 2010 曰 amended replacement page ": \\^- • ---------1--- 13, a pharmacy for lowering blood sugar A composition comprising the compound according to claim 1 as an active ingredient. 14. A pharmaceutical composition for treating type 2 diabetes, comprising the compound of claim 1 as an active ingredient. S — 18 — 1337867 November 25, 1999 Revision and Replacement VII. Designation of Representative Representatives (1) The representative representative of the case is: (1). (b) The symbol of the representative figure is briefly described: 101 extraction filtration step 103 distribution extraction step 105 aqueous layer extract 107 first chromatography column 109 second separation step 111 third organic solvent layer extract 113 third chromatography Column 102 crude extract 104 first organic solvent layer extract 106 first separation step 108 second organic solvent layer extract 110 second chromatography column 112 third separation step 114 fourth organic solvent layer extract VIII, the case If there is a chemical formula, please reveal the chemical formula that best shows the characteristics of the invention: —4——