阅读说明：本技术 Cysestermerol A及其在改善糖尿病方面的应用 (Cysestermerol A and application thereof in improving diabetes ) 是由 王峰 张丽 李碧君 鄢琼芳 王淑美 于 2019-08-29 设计创作，主要内容包括：本发明属于医药技术领域,具体公开了一种化合物Cysestermerol A及其在改善糖尿病方面的应用。本发明从狗牙根中提取得到一种新型化合物Cysestermerol A,该化合物可改善IR-HepG2细胞胰岛素抵抗状态,促进胰岛素抵抗细胞对葡萄糖的消耗,还能抑制α-葡萄糖苷酶的活性,从而可有效降低血糖水平,为研究开发新的降血糖药物提供了新的选择和途径,具有很好的应用前景。(The invention belongs to the technical field of medicines, and particularly discloses a compound Cysestermerol A and application thereof in improving diabetes. The novel compound Cysestermerol A extracted from bermuda grass can improve the insulin resistance state of IR-HepG2 cells, promote the consumption of glucose by the insulin resistance cells and inhibit the activity of alpha-glucosidase, thereby effectively reducing the blood sugar level, providing a new choice and way for researching and developing new blood sugar-reducing drugs and having good application prospect.)

1. A Cysestermerol A compound of Cynodon dactylon is characterized in that the structural formula is as follows:

2. use of cynodon dactylon for the preparation of the compound Cysestermerol a according to claim 1.

3. Use of the Cysestermerol A according to claim 1 for the preparation of a medicament for ameliorating diabetes.

4. Use of the Cysestermerol A according to claim 1 for the preparation of a medicament for improving insulin resistance.

5. The use of the Cysestermerol A of claim 1 as or in the preparation of an insulin sensitiser.

6. Use of the Cysestermerol A of claim 1 in the preparation of a medicament for inhibiting α -glucosidase activity.

7. A medicament for treating diabetes or lowering blood glucose comprising the cystermerol a of claim 1.

8. The medicament of claim 7, further comprising insulin.

9. The pharmaceutical composition of claim 7 or 8, further comprising a pharmaceutically acceptable carrier, and being formulated into different dosage forms.

Technical Field

The invention belongs to the technical field of medicines. More particularly, relates to a compound Cysestermerol A of cynodon dactylon and application thereof in improving diabetes.

Background

Diabetes is a lifelong metabolic disease characterized by elevated blood sugar levels and diabetes mellitus, which is a disorder of sugar, fat and protein metabolism caused by multiple causes. Worldwide diabetic patients exceed 4 hundred million people, and epidemiological investigation results in 2014 show that Chinese diabetic patients reach 1.4 hundred million people, so that the prevention and treatment of diabetes are imminent.

At present, the diabetes mellitus can be treated by oral medicines and insulin, but the medicines have obvious side effects, and can cause a plurality of organ injuries and even death of patients after long-term administration. Among them, adverse reactions include hypoglycemia, cardiovascular side reactions, gastrointestinal side reactions, lactic acidosis, hepatotoxicity and the like, so that clinical application thereof is increasingly restricted. Therefore, a new hypoglycemic drug or insulin sensitizer which is convenient to use, has high, stable and lasting effect and small toxicity and side effect and can delay the occurrence and development of complications is urgently needed by diabetics.

In addition, the Chinese herbal medicine is a cultural treasure accumulated by Chinese nationalities for thousands of years, and compared with western medicines, the Chinese herbal medicine has the advantages of rich medicine sources, low price, small side effect, multi-link integral treatment and the like. The bermuda grass is widely distributed in China, and researches in recent years show that the bermuda grass has pharmacological effects of resisting bacteria, promoting urination, reducing blood sugar and the like, but the research on specific active ingredients of the bermuda grass is very few, so that the bermuda grass needs to be researched and developed into a novel natural blood sugar reducing medicament by taking the traditional Chinese medicine theory and the modern pharmacological pharmacodynamics as guidance.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides application of Cysestermerol A serving as a cynodon dactylon compound in preparation of a medicine or a preparation for improving diabetes.

Another object of the present invention is to provide Cysestermerol A, a compound of Cynodon dactylon.

Another object of the present invention is to provide the use of Cysestermerol A in the preparation of a medicament or formulation for improving insulin resistance.

The invention also aims to provide the application of Cysestermerol A in serving as or preparing an insulin sensitizer.

The invention also aims to provide the application of Cysestermerol A in preparing a medicament or preparation for inhibiting the activity of alpha-glucosidase.

Another object of the present invention is to provide a medicament for treating diabetes or lowering blood sugar.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention extracts a new compound Cysestermerol A which has the functions of reducing blood sugar and improving insulin resistance so as to improve diabetes mellitus from bermuda grass, and the structural formula of the compound is shown as follows:

the research of the invention discovers that an insulin resistant cell model IR-HepG2 is established by inducing HepG2 cells with high-concentration insulin, a glucose oxidase method (GOD-POD) is used for detecting the influence of Cystermerol A on the consumption of glucose in IR-HepG2 cells, compared with the IR-HepG2 cells in a model group, the consumption of the glucose by the IR-HepG2 cells added with Cystermerol A (the concentration is more than or equal to 3.125 mu mol/L) is obviously increased, and the consumption of the glucose by the IR-HepG2 cells added with the Cystermerol A dose group is increased in a dose-dependent manner, so that the Cystermerol A can promote the consumption of the glucose by the IR-HepG2 cells and improve the resistant state of the HepG2 cells on the insulin. Namely, the experimental result shows that Cysestermerol A can obviously increase the glucose consumption of insulin resistant cells, and the Cysestermerol A can promote the glucose absorption of the cells in the form of an insulin sensitizer and improve the insulin resistant state.

The invention further researches and discovers that the inhibition rate of Cysestermerol A on alpha-glucosidase is measured by taking 4-nitrobenzene-beta-D-glucopyranoside (PNPG) as a substrate, the inhibition rate of Cysestermerol A with the mass concentration of 0-250 mg/L on the alpha-glucosidase activity is rapidly increased along with the increase of the mass concentration, when the mass concentration is higher than 250mg/L, the inhibition rate is slowly increased, and the half inhibition concentration is 99.58 mg/L. Therefore, the Cysestermerol A can inhibit the activity of alpha-glucosidase and slow down the digestion and decomposition speed of starch substances in the intestinal tract into glucose, thereby reducing the absorption of the glucose in the intestinal tract and better reducing the postprandial blood sugar.

Therefore, the invention requests to protect the application of Cysestermerol A in preparing a medicament or preparation for improving diabetes.

The invention also claims a medicine for treating diabetes or reducing blood sugar.

The invention also claims the application of Cysestermerol A in preparing the medicine or preparation for improving insulin resistance.

The invention also claims the application of Cysestermerol A in serving as or preparing an insulin sensitizer.

The invention also claims application of Cysestermerol A in preparation of a medicine or preparation for inhibiting activity of alpha-glucosidase.

In addition, the application of Bermuda grass in preparing the Cysestermerol A compound is also within the protection scope of the invention. In addition, the compound Cysestermerol A of the present invention can also be synthesized by chemical means.

As an alternative, the method for extracting and preparing Cysestermerol A from bermuda grass comprises the following steps:

s1, extracting cynodon dactylon with 95% ethanol water solution to obtain extract, and sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol to obtain an ethyl acetate extract;

s2, loading the ethyl acetate extract into a silica gel chromatographic column (200-300 meshes), CH₂Cl₂the/Acetone gradient elution gives 5 fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5;

s3.Fr.5(1:1) obtaining Cysestermerol A through MCI gel CHP 20P column chromatography, Sephadex LH-20 gel column chromatography and semi-preparative chromatography.

Compared with the prior art, the invention has the following beneficial effects:

1. the Cysestermerol A provided by the invention can improve the insulin resistance state of the IR-HepG2 cells, promote the glucose consumption of the insulin resistance cells and inhibit the activity of alpha-glucosidase, thereby effectively reducing the blood sugar level.

2. The Cysestermerol A provided by the invention lays a foundation for the material foundation research of the cynodon dactylon in the aspect of reducing blood sugar, and provides a new idea for developing a natural novel insulin sensitizer.

Drawings

FIG. 1 is a 1H-1H COSY and HMBC related signal diagram (A) and a key NOESY related signal diagram (B) of cystermerol A;

FIG. 2 is an experimental and calculated ECD spectrum of cystermerol A;

FIG. 3 is a graph of the effect of Cysestermerol A on cell proliferation of HepG 2;

FIG. 4 is a graph of the effect of Cysestermerol A and rosiglitazone on glucose consumption by IR-HepG2 cells;

FIG. 5 is a graph of the effect of Cysestermerol A and acarbose on inhibition of alpha-glucosidase activity.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Research shows that the compound preparation can inhibit the degradation of starch substances and the absorption of glucose by the intestinal tract of an organism and can well reduce the postprandial blood sugar level; and improve the state of body cells resisting insulin, enhance the sensitivity to glucose, promote the consumption of blood sugar and well reduce the blood sugar level. In recent years, an insulin-resistant HepG2(IR-HepG2) cell model is established by using insulin-induced HepG2 cells, and a diabetes drug screening research is carried out, so that a better research result is obtained. Alpha-glucosidase is an important role in the intestinal tract for carbohydrate digestion and absorption, and inhibitors of this class of enzymes are currently used in the treatment of type 2 diabetes.

Therefore, the research carries out preliminary research on improving insulin resistance of bermuda grass by establishing a HepG2 cell insulin resistance model induced by high-concentration insulin in vitro, discusses the influence of the novel compound Cysestermerol A extracted from the bermuda grass on the glucose consumption of IR-HepG2 cells and the inhibition effect of the activity of alpha-glucosidase in vitro, aims to provide data for basic research of substances in the aspect of reducing blood sugar of the bermuda grass and provides a research basis for developing novel insulin sensitizers.

Sources of materials used in the following examples:

HepG2 cells were purchased from shanghai cell institute of chinese academy of sciences; rosiglitazone was purchased from Sigma, usa; south america brazil fetal calf serum (NBCS) was purchased from GIBCO, usa; DMEM high-glucose medium was purchased from GIBCO, usa; penicillin/streptomycin stock solution and trypsin were purchased from the institute of biomedical engineering of the Chinese medical academy; insulin was purchased from Sigma; glucose test kit (BestBio) purchased from Nanjing institute of bioengineering; phosphate Buffered Saline (PBS) was purchased from seimer feishale (suzhou) instruments ltd; cell Counting Kit-8(CCK-8) Cell proliferation-toxicity test Kit was purchased from Dongli chemical technology, Inc.; dimethyl sulfoxide (DMSO) was purchased from Amresco corporation; 96-well cell culture plates were purchased from NEST, USA; the water used for the reagents prepared in the experiment is double distilled water; 75% alcohol for the experiments was purchased from Shandong Lierkang GmbH; absolute ethanol (analytical grade) for the experiments was purchased from remote chemical reagents ltd, tianjin.

Carbon dioxide cell incubator (SERIES 8000 WJ): therno Scientific; clean bench (SW-CJ-1F): sujing group Suzhou Antai air technology; inverted phase contrast microscopy (CDX 41): shanghai division, Olympus (China) Co., Ltd.; full-automatic autoclave sterilizer (MJ-78A): guangzhou Feidi Biotech, Inc.; digital display constant temperature water bath (DK-S22): shanghai sperm macro laboratory Equipment Co., Ltd; -80 ℃ ultra low temperature refrigerator (DW-HW 328): midicinal water caltrop; electric heating constant temperature drying oven (DHG-9077A): shanghai sperm macro laboratory Equipment Co., Ltd; one-tenth-ten-thousandth analytical balance (S125D): sidorist scientific instruments (Beijing) Inc.; full-automatic enzyme labeling instrument (ELX 800): BicTek Instruments; low speed bench centrifuge (TDL-80-2B): shanghai' an pavilion scientific instrument factory; low temperature high speed centrifuge (TG16MW2-16 KL): sigma, USA; freeze dryer (FD-1C-50): beijing Bo Yi kang laboratory instruments GmbH; cryogenic refrigerator (BCD-216 SMK): fertile and beautiful Rongshida refrigerator Co., Ltd; micro-analysis type ultrapure water machine (WP-UP-WF-10): water treatment facilities, Szechwa Volter, Inc.

The statistical treatment used was: statistical analysis was performed using SPSS24.0 statistical software. Independent sample t test is adopted for the comparison of the measurement data between two groups, and a one-factor ANOVA analysis of variance method is adopted for the comparison of three groups (or more than three groups). The results are expressed asWhen P is present<At 0.05, the difference is significant, P<0.01 indicates that the difference is very significant, and both have certain statistical significance.