TWI445538B - Use of lanostane and poria extract in treating diabetic - Google Patents

Description

Use of lanosterone and anthraquinone extract for the treatment of diabetes

The present invention relates to the use of a sputum extract for the treatment of diabetes, and more particularly to the use of a compound extracted from sputum for the treatment of diabetes caused by insulin deficiency in the blood.

Diabetes is a common chronic disease common in adults (especially the elderly) in rich countries. Diabetes can be divided into two types, type 1 diabetes or insulin-dependent diabetes mellitus. The cause is that the immune response causes β-cell destruction of the pancreas and cannot produce insulin. There is no insulin in the blood. Insulin supplementation can be used to treat diabetes. Type 2 diabetes or non-insulin-dependent diabetes mellitus is unknown, but genetic problems are one of the important factors, and lifestyle effects such as obesity are also important factors.

Around 500 AD, the Tang Dynasty medical books prepared a variety of prescriptions for the treatment of diabetes, some of which contain sputum. In 2002, Japanese researcher M. Sato published that the hydrolyzed dehydrotrametenolic acid of the molting part can be applied to type 2 diabetes (Biol. Pharm. Bull. 2002, 25(1), 81-86). Treatment, the principle of action is to achieve therapeutic goals by increasing insulin sensitivity (ie, reducing the body's resistance to insulin). However, from its in vitro fat cell test, the effective concentration of hydrogenated dehydroabietic acid was 10 ^-5 M, and the effective dose of hydrogenated dehydroabietic acid was 110 mg/kg from the results of animal experiments in vivo. The effective dose in the human body is at least as high as more than 700 mg. The effective dose of 700 mg is equivalent to more than 10 times the amount of the currently used clinical use, so development into a clinical medication can cause difficulties.

An object of the present invention is to disclose a use of lanostere having the following chemical formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the preparation of a medicament for treating diabetes caused by insulin deficiency in a mammal , Wherein R ₁ is H or CH ₃ ; R ₂ is OCOCH ₃ , =O or OH; R ₃ is H or OH; and R ₄ is -C(=CH ₂ )-C(CH ₃ ) ₂ R _a , wherein R _a is H or OH, or -CH=C(CH ₃ )-R _b , wherein R _b is CH ₃ or CH ₂ OH; R ₅ is H or OH; and R ₆ is CH ₃ or CH ₂ OH.

The present invention also discloses the use of a cockroach extract as an active ingredient in the preparation of a medicament for treating diabetes caused by insulin deficiency in a mammal, wherein the cockroach extract comprises 1-60% by weight of said Wool decane (I) and substantially free of ring-opened lanostane. Preferably, the mash extract comprises from 5 to 35% by weight of lanostere (I).

Preferably, the lanostane (I) has the following chemical formula: More preferably, the lanostane (I) has the following chemical formula:

Preferably, the drug is in the form of an injection.

Preferably, the drug is in an oral dosage form.

Preferably, the diabetes is type 1 diabetes.

Preferably, the diabetes is type 2 diabetes.

Preferably, the mammal is a human.

The present invention proves that the active ingredient isolated from sputum can function as insulin: (1) increase gene expression (mRNA) of glucose transporter (GLUT4), (2) manufacture of glucose transporter (GLUT4), and (3) Translocating the transporter (GLUT4) from the cell to the fat (or muscle) cell membrane, (4) the glucose transporter can transport extracellular glucose into the cell, and (5) can be manufactured in the same way as the insulin function Triglycerides are stored in cells. Therefore, the active ingredient has successfully played the role of insulin, which can transport blood sugar to the cells and lower blood sugar. It can be used in the treatment of type 2 diabetes and type 2 diabetes caused by insufficient insulin in the blood. In addition, the most important thing is that the in vitro test of fat can produce glucose absorption at a concentration of 0.01 μM. Compared with the above-mentioned Japanese scholar Sato, it takes 10 ^-5 M to produce a effect, and the dose difference between the two is 1000 times.

A suitable method for preparing an active ingredient for treating diabetes caused by insulin deficiency in blood is disclosed in the present invention, for example, the method disclosed in EP 1535619 A1, which comprises extracting hydrazine by a conventional extraction method to obtain a crude extract, and then By chromatography, it is divided into a small lanostane-like site (with dichloromethane:methanol (96:4) as the extract) and a highly polar open-chain lanolinane (secolanostane) Dichloromethane: methanol (90:10 or 0:100) is the extract), wherein the position of the lanostane-like part is shown by the thin layer chromatography of tannin, that is, the developing solvent is dichloro For methane-methanol (96:4), the chromatographic value (Rf) is As for the open-chain spheranostane-like component, the chromatographic value is less than 0.1. The lanostane moiety can be further separated by a gel column chromatography in which several lanostane compounds are isolated using dichloromethane:methanol (97:3 to 95:5).

The present invention will be further described in detail below with reference to the embodiments, but without the invention.

Embodiment 1

In Yunnan, 26 kg of pupa, 260 liters containing 75% alcohol, heated three times, combined with alcohol extract, concentrated under reduced pressure to obtain 225.2 grams of extract. The extract was quantitatively analyzed to obtain 76.27 mg of lanostere per gram of extract, of which K1 (Pachymic acid) 33.4 gram, K1-1 (dehydropachymic acid) 9.59 gram. , K2-1 (tumulosic acid) 19.01 gram, K2-2 (dehydrotumulosic acid) 6.75 gram, K3 (poylporenic acid C) 5.06 mg, K4 (3-epidehydrotumulosic acid) 2.46 g.

Embodiment 2

Example 1 125 g of the alcohol extract was extracted 6 times with 1.3 liters of dichloromethane, and the dichloromethane extract was combined. After concentration, 22.26 g of the extract was obtained. The dichloromethane extract was dissolved in 95% hot alcohol, and the insoluble matter was filtered after being allowed to cool. The filtrate was added in a small amount of water until the alcohol content was 45%. At this time, a precipitate was formed, and the precipitate was obtained by centrifugation to obtain 17.4 g. Precipitate. Quantitative analysis revealed that each gram of precipitate contained 264.78 mg of lanostane, of which K1-1 159.7 mg, K1-2 56.96 mg, K2-1 24.43 mg, K2-2 8.8 mg, K3 9.84 mg, K4 5.05 mg. The precipitate was tested by silica gel thin layer chromatography to prove that it did not contain ring-opened lanostane.

Embodiment 3

After immersing 100 kg of medicinal herbs in 800 kg of water for 3 hours, the mixture was allowed to cool to 50 ° C, and the solution was adjusted to pH 11 with 5 N NaOH, and the solution was stirred for 3 hours. The liquid and solid were then separated by a centrifuge, and the solid was further added with 800 kg of water. The mixture was adjusted to pH 11 with NaOH as described above, stirred and centrifuged to remove the solid. The two liquids were combined, and the liquid was concentrated in vacuo to a 100 kg solution at 50 ° C, and then 3N HCl was added to pH 6.5 to give a precipitate. The precipitate was separated, washed with 40 L of H ₂ O, and then the precipitate was separated by a centrifuge, and sprayed with 8 L of water to obtain about 380 g of a powder. The powder was extracted three times with 4 L of alcohol, and the extracts were combined and concentrated to give 238.9 g of an alcohol extract. The extract was tested by silica gel thin layer chromatography (TLC) to prove that it did not contain ring-opened lanostane. The extract was further separated by HPLC, and the extract was obtained as a main component of K2 214 mg, K3 23 mg, K4 24 mg and a small amount of K1 4.52 mg per gram of the extract, i.e., the extract contained about 265 mg of lanostane per gram.

Or extract the powder with 4L of 50% alcohol aqueous solution, remove 50% alcohol aqueous solution and collect insoluble powder, and repeat three times to obtain 245.7g of 50% alcohol aqueous solution insoluble matter. The TLC method indicates that the insoluble matter does not contain open loop. The lanostere was further purified by HPLC to separate the insoluble matter, and the main component was K2 214 mg, K3 23 mg, K4 24 mg and a small amount of K1 4.52 mg per gram, that is, the extract contained about 261 mg of lanostane per gram.

Embodiment 4

After 30 kg of glutinous rice in Yunnan, it was ground into powder and extracted with 120 L of alcohol (concentration 95%) for 24 hours, and separated by filtration. The foregoing extraction and solid-liquid separation were repeated three times. The filtrate was combined and concentrated to give a dry extract 265.2 g. The dried extract was subjected to partition extraction using a two-phase extractant (hexane: 95% methanol = 1:1). The methanol layer was taken out and concentrated to give 246.9 g of dry solid. The dried solid was separated by a silica gel column chromatography, which was filled with 10-40 times the weight of the dry solid, which was purchased from Merck, Silica gel 60, 70-230 mesh. The mixture of dichloromethane/methanol was used as a solvent (eluent), and the mixture was extracted in a ratio of 96:4, 90:10, and 0:100, and the eluate was subjected to silica gel thin layer chromatography. (Thin Layer Chromatography) (UV light and iodine detection, the developing solution is dichloromethane: methanol = 96: 4) The components are detected, and the same components are combined.

The PCM fraction of the hydrazine extract of the present invention was obtained by subjecting a mixture of dichloromethane-methanol (96:4) to a gel column chromatography. The PCM part can clearly see 6 traces according to the above-mentioned silica gel thin layer chromatography. The combined chromatography of dichloromethane:methanol (90:10) and (0:100) gave 168 g of PCW fraction.

The PCM fraction was further chromatographed with dichloromethane:methanol (96.5:3.5) as a stripping agent (same as the above-mentioned ruthenium column), and further purified to obtain a lanostane-like component K1 (K1) -1 and K1-2), K2 (K2-1 and K2-2), K3, K4, K4a, K4b, K5, K6a and K6b. Detailed separation steps and identification analysis data can be found in EP 1535619 A1.

The above K1 to K6b compounds have the following structure:

The yields of the lanosterane compounds K1 to K6b separated from the PCM fraction are shown in the following table. The PCM fraction contains about 15% by weight of lanosterane compounds K1 to K6b.

Example 5: Preparation of capsules

A capsule containing the hydrazine extract PCM component prepared in Example 4 was prepared according to the following composition:

The cockroach extract PCM and sodium strontium aluminate were respectively sieved through a #80 mesh sieve, the potato starch was sieved through a #60 mesh sieve, and magnesium stearate was sieved through a #40 mesh sieve, and then placed. The mixer was stirred well and then filled into vacant capsules, each containing about 1.68 mg (0.42% by weight) of active ingredient K1-K6.

Embodiment 6

Prevention and treatment of type 1 diabetes by triterpenoids

The hydrazine extract subjected to the following cell experiments was either the one prepared in Example 2 or the pure compound shown in Fig. 1. They are dissolved in an alcohol: DMSO (9:1) solvent and the resulting solution is added to a culture dish where each well is only added to one thousandth of the final volume.

First, fat cell culture

3T3-L1 is a mouse preadipocyte, which is in the form of a spine. After 2-3 days of incorporation of the inducer, the cell type can be observed to be round, and there is oil droplet accumulation in the cell. As the number of days increases, the more complete the differentiation. Before the differentiation, the main glucose transporter is GLUT1, and the differentiated cell, the main glucose transporter is GLUT4. For example, the more the GLUT4 protein on the cell membrane, the faster the glucose in the blood is absorbed by the cell through the cell membrane. The greater the amount, the faster the blood sugar drops. This 3T3-L1 adipocyte has a complete system of insulin-activated glucose uptake, which can be used for the study of carbohydrate metabolism and insulin information pathway. In addition, the complete lipid modulating process can be observed, so the differentiated 3T3-L1 adipocytes are one. Representative and widely used model cell lines, because the real fat cells in human tissues are difficult to subculture, the academic community generally uses this model for various tests and evaluations.

(a) Fat cell culture:

3T3-L1 cells were cultured in DMEM (Dulbecco's minimal essential medium) and 37 ° C incubator (5% carbon dioxide, 95% air), which was supplemented with 10% calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin and 1% non-essential amino acid. After the cells were completely covered, containing added fat cell differentiation promoting agent [containing 0.5mM 3- isobutyl - 1 - methylxanthine (3-isobutyl-1-methylxanthane , IBMX), 1μM corticosteroids (dexamethasone), 10% of 10μg/mL insulin] FBS/DMEM culture medium was cultured for two days, replaced with 10% FBS/DMEM medium containing 10μg/mL insulin for two days, and then replaced with insulin-free medium every two days for culture 4- 6 days. At this time, nearly 90% of the 3T3-L1 cells form the form of adipocyte phenotype, and then the test can be performed. 3T3-L1 cells were washed with PBS solution before the test, and then cultured overnight in serum-free and insulin-free 0.2% BSA/DMEM medium to remove serum and insulin interference.

(b) Adipocyte culture to inhibit glucose transporter 4 gene expression (mRNA) experiments:

Infection of 3T3-L1 preadipocytes (shG4-30) with a viral vector carrying the glucose transporter 4 ribonucleic acid (TRCN0000043630 shRNA, Academia Sinica Genome Research Center, Taiwan) to establish long-term inhibition of glucose transporter 4 gene expression And test the fat cells after differentiation of the strain cells.

(c) Detection of fat transport in the glucose transporter 4 transporter protein translocation assay:

The glucose transporter 4 (HA-GLUT4-GFP, presented by Timothy E. McGraw, Well Gornell Medical College, New York, USA) with the influenza virus protein HA marker was transfected into 3T3 with Lipofectamine 2000 (Invitrogen, CA, USA). -L1 preadipocytes, and the fat cell strain that continuously expresses the glucose transporter 4 transporter with the influenza virus protein HA marker was detected by G418 screening, and the glucose transporter 4 transporter translocation test was performed after differentiation into adipocytes. Evaluation.

Second, 2-deoxyglucose absorption assessment

In the test of glucose uptake by 3T3-L1 adipocytes, 3T3-L1 preadipocytes were cultured on a 6-well culture plate, and the cells were overgrown with adipocyte differentiation promoter for differentiation stimulation. After -12 days of 3T3-L1 cells mature into adipocytes, they can be used for glucose uptake testing. The above fat cells were first placed in serum-free medium (containing 0.2% BSA/DMEM) overnight, and then cultured in serum-free cell culture medium containing different concentrations of triterpenoids for 2-6 hours, and then washed with PBS solution. Once, change to KRP buffer (20 mM HEPES, 137 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 2.5 mM CaCl ₂ , and 2 mM pyruvate, pH 7.4 and 0.2% BSA) After incubation at 37 ° C for 3 hours, [ ¹⁴ C] 2-deoxyglucose [2-deoxy-D-[ ¹⁴ C]-glucose (2-DG, Amersham Biosciences, Little Chalfont, 0.2 μCi/mL) was added. Bucks, UK)] and 0.2 ml glucose buffer without radioactive 0.1 mM 2DG were substituted for KRP buffer to initiate glucose uptake experiments. After 5 minutes, the cells were removed and washed with PBS to stop glucose uptake. The cells were lysed in 0.2 mL of 0.2% SDS and 10 μL of the cell-dissolved solution was transposed into a UniFilter disk containing a filter tray (Perkim-Elmer, Wellesley, MA, USA) and dried in a vacuum oven at 37 ° C, and 30 μL of the counting solution was added to the wells and analyzed using a microdisc liquid scintillation counter (TopCount, Packard NXT, Packard BioScience Company, Meriden, CT, USA). The amount of glucose accumulated in the cells was calculated and divided by the protein concentration, and the resulting uptake rate was expressed as nanomolar glucose (nmol/min/mg) per milligram of cellular protein per minute. Protein concentration can be determined using standard Bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL, USA) analysis. 0.2 μCi of L-[ ¹⁴ C]-glucose was added to measure the uptake of non-specific glucose and subtracted from each of the measured values to obtain a specific glucose uptake. It was observed whether 3T3-L1 adipocytes had an effect on the absorption of glucose under the action of different concentrations of triterpenoids.

3. Evaluation of glucose transporter 1 and 4 (GLUT1 & 4) transporters

In the test of the triterpenoids promoting the expression of glucose transporters in 3T3-L1 adipocytes, the 3T3-L1 adipocytes differentiated and matured in the same manner as described above were cultured in serum-free cell culture medium overnight, and then contained in different concentrations of triterpenoids. The serum-free cell culture solution of the compound was cultured for 24 hours, and then washed with PBS solution, followed by 0.2 ml of lysed cell fluid [1% NP-40, 150 mM NaCl, 0.1% SDS, 50 mM Tris-HCl pH 7.6, 10 mM EDTA, 0.5%. Deoxycholate, 1 mM PMSF, 1 mM Na ₃ VO ₄ , 10 mM NaF, 10 mM β-glycerophosphate (glycerophosphate), 10 μg/mL protein inhibitor and phosphatase inhibitor] (at 4 ° C temperature). Each sample was separated by SDS-10% polyacrylamide electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Western blotting with specific antibodies, glucose transporter 1 antibody (GLUT1, Abcam, Cambridge, MA), glucose transporter 4 antibody (GLUT4, R&D systems, Minneapolis, MN), and β-actin antibody (β -Actin, Chemicon, Temecula, CA, USA) to observe whether 3T3-L1 adipocytes have an effect on glucose transporter protein expression under the action of different concentrations of triterpenoids. Each sample (protein) was treated with chemical luminescence, exposed to X-ray film, and analyzed for content in software.

IV. Evaluation of gene expression of glucose transporter 1 and 4 (GLUT1&4) transporters

The expression of glucose transporter ribonucleic acid (mRNA) was evaluated by 3T3-L1 adipocytes under the action of different concentrations of triterpenoids using real-time quantitative polymerase chain reaction (Q-PCR). The fully differentiated 3T3-L1 adipocytes were administered with different concentrations of the triterpenoids for 24 hours. After removing the cell culture medium, total RNA was extracted with Trizol reagent (Invitrogen, Irvine, CA, USA), and 1 μg of RNA was used. The mRNA was reverse transcribed into cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Darmstadt, Germany). Specific primers were designed for glucose transporter 1, glucose transporter 4 and β-actin, respectively, and the gene to be tested was amplified by SYBR Green Q-PCR analysis (Applied Biosystems, Foster City, CA, USA). (GLUT1 and GLUT4) and the internal reference gene (β-actin), and the relative cause expression value was calculated by the ΔΔ C _T method using StepOne v2.0 software (Applied Biosystems) software.

V. Evaluation of the translocation of glucose transporter 4 transporter

(1) Analysis of translocation of glucose transporter 4 transporter (1)

As insulin promotes glucose uptake by adipocytes or muscle cells, the glucose transporter 4 (GLUT4) transporter plays an important role in translocation from the intracellular organelle to the plasma membrane (PM). A test for translocation of the glucose transporter 4 transporter of 3T3-L1 adipocytes to the plasma membrane by a triterpenoid compound was performed. The mature and mature 3T3-L1 adipocytes were cultured in serum-free cell culture medium overnight, and then cultured in serum-free cell culture medium containing different concentrations of triterpenoids for 2 hours, followed by high-speed centrifugation at different speeds (16,000 g to 200,000). g ), separating the plasma membrane (PM) fraction and the low density microsome (LDM) of the cell (Liu, LZ, etc.; Mol Biol. Cell 17, (5), 2322 -2330, 2006). Using Western blotting method to observe the specificity of glucose transporter 4, 3T3-L1 adipocytes were translocated from the LDM of the cells to the plasma membrane by different concentrations of triterpenoids. PM) Whether it has an impact.

(2) Analysis of translocation of glucose transporter 4 transporter (2)

3T3-L1 preadipocytes stably expressing HA-GLUT4-GFP protein were seeded in 96-well culture plates, and differentiated with differentiation promoters after they were overgrown (Govers, R. et al; Mol Cell Biol. 24(14), 6456 -6466, 2004). The fully differentiated 3T3-L1 adipocytes were administered to different concentrations of the triterpenoids for 2 hours, the cell culture medium was removed, and the cells were washed with ice PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, then washed 2-3 times with ice PBS, and then incubated with HA primary antibody (12CA5) for 2 hours. The cells were washed 2-3 times with ice PBS, and cells were incubated with a conjugated rhodamine-conjugated secondary antibody (Leinco, Ballwin, MO) for 1 hour. The cells were washed 2-3 times with ice PBS, and the excitation wavelengths of rhodamine and green fluorescent protein (GFP) were detected by a fluorescence immunoassay analyzer (POLARstar Galaxy; BMG Labtechnologies, Offenburg, Germany). Em. 480/Ex. 425 nm and Em. 576 nm/Ex. 550 nm), and the relative amount of the plasma membrane of HA-GLUT4-GFP translocation was evaluated by the ratio of the salt-based peach red pigment to the green fluorescent protein. Because only when HA-tagged GLUT4 is translocated to the plasma membrane, it is labeled by the salt-based peach pigment, so the ratio can be used to assess the translocation of the glucose transporter 4 transporter to the plasma membrane.

The effects of hexaglyceride accumulation and glycerol release

In the test of the accumulation of triglyceride and the release of glycerol in 3T3-L1 adipocytes by the triterpenoids, the mature and mature 3T3-L1 adipocytes were cultured overnight in serum-free cell culture medium, and then contained in different The serum-free cell culture medium of the concentration of the triterpenoid was cultured for 24 hours. The culture medium was collected and detected by glycerol assay kit (Randox Laboratories, Antrim, UK). The release of glycerol from 3T3-L1 adipocytes under different concentrations of triterpenoids was observed. Does it affect? Detection of triglyceride in adipocytes was carried out by Oil-Red O staining (Ramirez-Zacarias, JL et al; Histochemistry 97, (6), 493-497, 1992 ). Fatty oil droplets formed by intracellular lipid accumulation were stained, washed twice with 60% isopropanol, and then extracted with 100% isopropanol, and the absorbance at 490 nm was measured. The absorbance values of fat cells not given triterpenoids were compared to evaluate the effect of different concentrations of triterpenoids on the accumulation of triglycerides in adipocytes.

The results of the experiment indicate that the triterpenoids have the following four properties as insulin, and therefore have the ability to treat type 1 diabetes:

(a) The sputum component or extract in the adipocyte mode has the ability to promote the absorption of glucose from the cell into the cell:

The evaluation of the sputum extract (Example 2) on mature adipocytes, as shown in Fig. 2A, shows that the sputum extract has a significant effect of increasing glucose absorption, which promotes the absorption effect as the dose is increased. Glucose uptake was observed as with 100 nM insulin. Further, the pure compound of Example 2 was used as an experiment. As shown in Fig. 2B, after the pure compound was administered to the adipocytes for two hours, three of the compounds citrate (PA), citric acid (TA) and porous citrate C (PPA) significantly increased the sugar uptake to 165.89 at 0.01 μM, respectively. %, 142.5% and 147.9%. Among them, the degree of PA increase was the most significant, so the evaluation of subsequent tests was carried out with PA.

Figure 3A shows that PA increases the sugar absorption with the increase of the administration time, and the increase of the two hours is the most significant (increased by 165.89%). In addition, with the increase of the concentration of PA, the degree of promotion of sugar absorption is also increased. increases, when 1μMPA, increased to 209.84%, as shown in Figure 3 B.

As shown in the results of Fig. 4, the PA promoted the sugar absorption effect only for the differentiated fat cells, and the pre-adipocyte PA did not promote the glucose absorption effect. For example, when a glucose transporter transporter inhibitor (PT, phloretin) was administered to both pre-adipocytes and mature adipocytes, the ability of the two cells to promote absorption was significantly suppressed. According to the literature, the fat cells only have the glucose transporter-1 (GLUT1) transporter, while the mature fat cells are the glucose transporter-4 (GLUT4) transporter. Therefore, the role of PA should increase GLUT4 and increase sugar uptake.

(B) The triterpenoid compound PA significantly induced the expression of glucose transporter 4 transporter protein (GLUT4) and information ribonucleic acid (mRNA) in mature adipocytes.

The results in Figure 5 show that different doses of PA were administered to mature adipocytes. After 24 hours, the glucose transporter 1 and 4 transporters (GLUT1, 4) were analyzed by Western blotting to evaluate the expression of GLUT1 and GLUT4 transporters by PA. As a result, PA had an effect of promoting expression of the GLUT4 transporter (Fig. 5A), and it did not have an effect of promoting expression of the GLUT1 transporter (Fig. 5B).

Quantitative polymerase chain reaction (Q-PCR) and specific probes were used to observe the expression of glucose transporter ribonucleic acid (mRNA) in mature 3T3-L1 adipocytes at different concentrations. 6 Results showed that 1 μM PA promoted GLUT4 gene expression to 228%. PA is shown to regulate the ability to increase GLUT4 gene and protein expression. In addition, interfering message ribonucleic acid technology was used to establish 3T3-L1 adipocytes that continuously reduced the GLUT4 transporter (Fig. 7A), and the fat cells were evaluated for sugar uptake. As shown in Fig. 7B, all the different doses of PA could not promote the The sugar uptake of fat cells further confirms that PA promotes sugar uptake directly related to GLUT4 transporter.

(3) PA has the effect of promoting the transfer of GLUT4 transporter from intracellular to plasma membrane in mature adipocytes

One of the mechanisms by which insulin promotes sugar uptake is to cause a large amount of GLUT4 to be translocated from the intracellular organelles to the plasma membrane (PM) for sugar absorption. Therefore, the GLUT4 transporter translocation efficiency was evaluated using 3T3-L1 mature adipocytes with intact insulin-activated glucose uptake system. From the results of Fig. 8A, it was observed by the Western blotting method of the plasma membrane (PM) separated by the ultracentrifugation method that 0.01 μM PA significantly increased the amount of GLUT4 in the plasma membrane to 141%, and increased to 328% as the dose was increased to 1 μM. Using the 3T3-L1 adipocyte method and fluorescent assay, which continuously expressed HA-GLUT4-GFP protein, it was confirmed that PA promoted glucose uptake to increase the shift of GLUT4 transporter protein to the plasma membrane. Figure 8B shows a significant increase in the translocation of the GLUT4 transporter to the plasma membrane by a 2.71 fold at a PA dose of 1.0 [mu]M. The above results demonstrate that PA does have the ability to promote translocation of the GLUT4 transporter to the plasma membrane.

(4) PA has the ability to promote the accumulation of triglycerides in fat cells and reduce the ability of fat cells to release glycerol into cell culture fluid.

In addition to observing the effect of PA on sugar uptake and observing the effects of fat cells, we evaluated the synthesis (storage) and lipolysis (glycerol release) of adipocyte triglycerides. As shown in the results of Fig. 9, when different doses of PA 24 were administered, the accumulation of triglyceride was evaluated by the oil red staining method, and it was observed that the accumulation of triglyceride exceeded 137%, and the release of glycerol was also observed to be reduced to 70 in PA administration. %the following. It shows that PA has the ability to promote fat regeneration and inhibit fat breakdown.

Figure 1 shows the structure of a lanosterane-type triterpenoid obtained by separation and purification of hydrazine.

Figure 2A shows that the sputum extract promotes sugar uptake by 3T3-L1 adipocytes.

Figure 2B shows that insulin (0.1 μM) was applied for 30 minutes and the purified triterpenoids promoted sugar uptake at a 0.01 μM dose for two hours of anti-sugar test. Data are expressed as mean ± standard error (n = 6). *p<0.05, **p<0.01 compared with the control group (no administration).

Figure 3A shows that citric acid (PA) gave the highest absorption rate at a dose of 0.01 μM for two hours. Figure 3B shows that the increase in sugar absorption is increased with increasing doses of pachymic acid (PA). After 2 hours of PA treatment, the sugar test was added, and the data were expressed as mean ± standard error (n=6), *p<0.05, **p<0.01 compared with the control group (not administered).

Figure 4 shows that PA promotes sugar uptake only on differentiated mature adipocytes. For example, when immature and mature adipocytes were added to the PA for 2 hours, the addition or absence of the glucose transporter inhibitor (PT) significantly inhibited the glucose uptake of the two cells and also inhibited the glucose uptake promotion of PA. Data are expressed as mean ± standard error (n = 6). *p<0.05, **p<0.01 compared with the control group.

Figure 5A shows that PA does not have the ability to promote expression of the glucose transporter 1 (GLUT1) transporter. While Figure 5B shows that PA has the ability to promote the expression of the glucose transporter 4 (GLUT4) transporter. Differentiated mature cells were treated with different concentrations of PA for 24 hours, and then GLUT1 and GLUT4 transporters were analyzed, and the data were expressed as mean ± standard error (n = 3). *p<0.05, **p<0.01 was compared to the control group (no administration).

Figure 6 shows that PA has the ability to promote the expression of GLUT4 mRNA, which was treated with insulin (0.1 μM) and 0.01 μM PA for 24 hours, respectively, and then analyzed for expression of GLUT1 and GLUT4 mRNA, with mean ± standard error (n = 3) Representation. *p<0.05, **p<0.01 was compared with the control group.

Figure 7 shows that adipocyte PA, which inhibits the expression of GLUT4 mRNA, does not have the ability to promote sugar uptake. Mature adipocytes and adipocytes (shG4-30) that inhibit GLUT4 mRNA expression were treated with different concentrations of PA and insulin, and then analyzed for GLUT4 transporter (Fig. 7A) and its sugar uptake capacity (Fig. 7B). The standard error (n=6) is indicated. *p<0.05, **p<0.01 was compared with the control group.

Figure 8A, by analyzing the GLUT4 transporter of different cell membrane layers isolated by centrifugation, shows that PA has the ability to facilitate transport of the glucose transporter 4 transporter from intracellular organelles to the cell membrane. Figure 8B. Fluorescence analysis of the GLUT4 transporter of the cell membrane layer of intact cells, showing that PA has the ability to promote transport of GLUT4 from intracellular organelles to the cell membrane. Data are expressed as mean ± standard error (n = 3) (Fig. 8A), (n = 6) (Fig. 8B). *p<0.05, **p<0.01 was compared to the control group (no administration).

Figure 9 shows that PA has the ability to promote the synthesis of adipocyte triglycerides and inhibit the production of lipolysis (product glycerol). Mature adipocytes were treated with insulin (0.1 μM) and different concentrations of PA for 24 hours, and the data were expressed as mean ± standard error (n = 6). *p<0.05, **p<0.01 was compared with the control group.

Claims (5)

A use of citric acid or a pharmaceutically acceptable salt thereof having the following chemical formula as an active ingredient for preparing a first type diabetes drug for treating a mammal, and the drug is not combined with other drugs for treating type 1 diabetes Application, The use of the first aspect of the patent application, wherein the medicament is an injectable dosage form. The use of the first aspect of the patent application, wherein the drug is an oral dosage form. The use of the first aspect of the patent application, wherein the mammal is a human. The use of the first aspect of the patent application, wherein the other drug for treating type 1 diabetes is insulin.