阅读说明：本技术 一种甲基丁香酚在制备保护胰腺的药物中的应用 (Application of methyl eugenol in preparing medicament for protecting pancreas ) 是由 宫念樵 于 2021-08-26 设计创作，主要内容包括：本发明提供了一种甲基丁香酚在制备保护胰腺的药物中的应用,包括含有甲基丁香酚的药物组合物以及甲基丁香酚的各种药学上可接受的药物制剂,对保护胰腺内分泌腺细胞或改善胰腺内分泌腺细胞具有作用,本发明对进一步开发与应用具有临床实际意义与价值。(The invention provides an application of methyl eugenol in preparing a medicament for protecting pancreas, which comprises a medicinal composition containing the methyl eugenol and various pharmaceutically acceptable medicinal preparations of the methyl eugenol, has an effect on protecting pancreatic endocrine gland cells or improving the pancreatic endocrine gland cells, and has clinical practical significance and value for further development and application.)

1. Application of methyl eugenol in preparing medicine for protecting pancreas is provided.

2. Use according to claim 1, characterized in that: the administration dose of the medicine is not less than 3 mg/kg/day.

3. Use according to claim 1, characterized in that: the protecting pancreas is protecting pancreatic endocrine gland cells or improving pancreatic endocrine gland cell function.

4. Application of a pharmaceutical composition containing methyl eugenol in preparing a medicament for protecting pancreas is provided.

5. Use according to claim 4, characterized in that: the protecting pancreas is protecting pancreatic endocrine gland cells or improving pancreatic endocrine gland cell function.

6. A medicinal preparation for protecting pancreas comprises injection, oral preparation, powder for injection, gel, capsule and patch; the content of methyl eugenol in the preparation is more than 80%.

7. The pancreatic protective pharmaceutical formulation of claim 6, wherein: the protecting pancreas is protecting pancreatic endocrine gland cells or improving pancreatic endocrine gland cell function.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of methyl eugenol in preparing a medicine for protecting pancreas.

Background

Asarum herb belongs to Aristolochiaceae, is a traditional Chinese medicine in China, is widely used for wind-cold type common cold, headache, toothache, nasal obstruction, watery nasal discharge, phlegm retention, cough and asthma and the like, Methyl Eugenol (ME) is used as a main component of the asarum volatile oil, and researches show that ME has various pharmacological effects of analgesia, anesthesia, antianaphylaxis, anti-inflammation, anti-tumor and the like.

The pancreas is an organ composed of endocrine gland tissues (islets of langerhans) called islets of pancreas and exocrine gland tissues that secrete digestive enzymes. Beta cells, alpha cells, delta cells, pancreatic polypeptide cells, etc., are present in pancreatic islets, which greatly affect the control and metabolism of blood glucose. Among them, beta cells play a particularly important role as cells for producing insulin.

The natural compound Methyl Eugenol (ME), a substance structurally similar to eugenol, is an alkenylbenzene compound, has been detected in 450 various plants, many of which are used as a source of food, essential oils or drugs. The ten years of history that ME is used as a main component of a traditional Chinese medicine formula for tissue repair and wound healing has reported that the main target organ and metabolic site of rodent metabolic energy are livers. However, to date, no reports or descriptions of the preservation of pancreas by ME have been reported or documented in the prior art.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a use of methyl eugenol in preparing a medicament for protecting pancreas.

The purpose of the invention is realized by the following technical scheme:

one of the purposes of the invention is the application of methyl eugenol in preparing medicaments for protecting pancreas.

Preferably, the drug is administered at a dose of not less than 3 mg/kg/day.

The second purpose of the invention is to provide the application of the pharmaceutical composition containing methyl eugenol in preparing the pancreatic protection medicine.

The invention also aims to provide a pancreatic protection pharmaceutical preparation, which comprises an injection, an oral preparation, a powder injection, a gel, a capsule and a patch; the content of methyl eugenol in the preparation is more than 80%.

The protecting pancreas is protecting pancreatic endocrine gland cells or improving pancreatic endocrine gland cell function.

The invention has the outstanding effects that:

the invention provides application of methyl eugenol in preparing medicaments for protecting pancreas, which comprises a medicinal composition containing the methyl eugenol and various pharmaceutically acceptable medicinal preparations of the methyl eugenol, has an effect on protecting pancreatic endocrine gland cells or improving the pancreatic endocrine gland cells, and has clinical practical significance and value for further development and application.

Drawings

FIG. 1 is a microscopic enlarged view of HE-stained sections of a Normal group (Normal group), a hypoxia/reoxygenation group (H/R group), a hypoxia/reoxygenation + DMSO solvent group (H/R + DMSO group), and a hypoxia/reoxygenation + methyleugenol group (H/R + Me group) according to the present invention;

FIG. 2 is a graph showing the data for measuring the amount of insulin secretion in the Normal group (Normal group), the hypoxia/reoxygenation group (H/R group), the hypoxia/reoxygenation + DMSO solvent group (H/R + DMSO group), and the hypoxia/reoxygenation + methyleugenol group (H/R + Me group) according to the present invention;

FIG. 3 is a fluorescence image of Min6 cell apoptosis detected by flow cytometry;

FIG. 4 is a graph showing the data of the measurement of Min6 apoptosis rate by flow cytometry.

Detailed Description

In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided, but the technical solutions of the present invention are not to be construed as limiting the implementable scope of the present invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1 methyl eugenol improves islet cell viability

Male C57BL/6J mice (6-8 weeks old, 20-22 g body weight, Beijing HFK Bioscience) were selected for the experiments. These mice were divided into Normal group (Normal group), hypoxia/reoxygenation group (H/R group), hypoxia/reoxygenation + DMSO solvent group (H/R + DMSO group), hypoxia/reoxygenation + methyleugenol group (H/R + Me group). The pancreas of the 4 groups of mice is fixed by Bonn's fluid, dehydrated, transparent and waxed, then embedded by paraffin, sliced, the thickness is 4.5-5.5 μm, the pancreas slices are heated and fixed by an oven at 30-45 ℃, the pancreas slices are subjected to HE staining and aldehyde red-recovery staining, dehydrated, transparent and sliced, observed under a microscope and photographed and recorded, as shown in figure 1, compared with the Normal cultured islet cells of the Normal group, the number of the red-infected cells in the islet cells of the H/R group is increased and the cell death is increased after the anoxia/reoxygenation treatment, while compared with the islet cells of the H/R group and the H/R + DMSO group, the number of the red-infected cells in the islet cells of the H/R + Me group is decreased and the cell death is reduced after the methyl eugenol treatment.

Example 2 function of methyl eugenol to improve insulin secretion

Male C57BL/6J mice (6-8 weeks old, 20-22 g weight, Beijing HFK Bioscience) were selected for the experiment, the abdomen was sacrificed by cervical dislocation, common bile duct was clamped by an artery clamp into the duodenum end, and the liver was turned up to free the common bile duct. The pancreas was fully distended by perfusing approximately 3ml of a pre-cooled Liberase RI enzyme working solution (0.153mg/ml) through common bile duct. Intact pancreas was isolated blunt and digested in a 37 ℃ water bath for 30 min. After digestion was complete, the digestion was stopped by vigorous shaking on a vortex shaker for 10s and addition of pre-cooled PBS. The digestate was washed twice with PBS, 15s at 1000rpm each time. 4ml of 25% (w/v) Ficoll is added to the tissue sediment, the mixture is shaken and mixed evenly, and then 2ml of 23%, 21% and 11% Ficoll are added in turn carefully to form a density gradient separation solution with clear interface. The tube was placed in a bucket-type rotor and centrifuged at 1000rpm for 10min to obtain purified islet cells, which were cultured overnight in RPMI 1640 medium (containing 11mM D-glucose) containing 10% fetal bovine serum under an environment of 95% air + 5% CO 2. The samples were picked one by one with a small-gauge tip, placed in low-sugar DMEM culture medium (containing 5.5mM D-glucose), and randomly divided into four groups, i.e., Normal group (Normal group), anoxic/reoxygenation group (H/R group), anoxic/reoxygenation + DMSO solvent group (H/R + DMSO group), and anoxic/reoxygenation + methyleugenol group (H/R + Me group). The amount of Me was 40. mu.g/ml and the incubation time was 24 h. After the experiment, the islets were picked out and placed in Eppendorf tubes, and 3 islets with uniform size and diameter of about 150 μm were collected in one tube, to which 1ml of low-sugar DMEM culture solution had been added. The tube was placed in a CO2 incubator for 1h, briefly centrifuged, the supernatant taken and the insulin release measured using a mouse insulin ELISA kit. As shown in fig. 2, the insulin secretion amounts of the hypoxia/reoxygenation group (H/R group) and the hypoxia/reoxygenation + DMSO solvent group (H/R + DMSO group) were significantly decreased, and the insulin secretion amounts of the hypoxia/reoxygenation + methyleugenol group (H/R + Me group) were significantly increased, as compared with the insulin secretion amount of the Normal group (Normal group). Thus, it was found that treatment with methyl eugenol improved insulin secretion from islet cells treated with hypoxia/reoxygenation.

Example 3 Methyleugenol reduces the apoptosis rate of Min6 cells

Recovering and culturing MIN6 cells: taking out the cells in a refrigerator at the temperature of minus 80 ℃, centrifuging the cells to remove the upper frozen stock solution, adding a 1640 culture medium containing 10 percent fetal calf serum and 1 percent double antibody to resuspend the cells, culturing the cells in an incubator at the temperature of 37 ℃ and 5 percent CO2 with the relative humidity of 95 percent; and (3) replacing the fresh culture medium every 24-36 h according to the growth condition of the cells, and carrying out passage or cryopreservation when the cells grow to 75% -85%.

Well-grown Min6 cells were uniformly inoculated into a 96-well plate, 100 μ L of complete medium (medium containing 10% fetal bovine serum) was added at 5 × 105/mL, and after 24 hours of culture, the original medium was removed and divided into a Normal group (Normal group), an anoxic/reoxygenation group (H/R group), an anoxic/reoxygenation + DMSO solvent group (H/R + DMSO group), and an anoxic/reoxygenation + methyleugenol group (H/R + Me group). Cell apoptosis was detected by flow cytometry using phospholipid binding protein V (Annexin V) and Propidium Iodide (PI) double staining, as shown in FIGS. 3 and 4, and the results showed that Min6 cell apoptosis was significantly increased in the H/R group and the H/R + DMSO group compared with that in the Normal group; after methyl eugenol is applied, the total apoptosis rate of the H/R + Me group Min6 cells is reduced, which shows that the ME pretreatment can improve the H/R damage of HK-2 cells and inhibit the Min6 cell apoptosis.

Example 4

This example provides a pharmaceutical composition containing methyl eugenol for protecting the pancreas. The pharmaceutical composition can also be prepared into acceptable pharmaceutical preparations, which comprise injections, oral preparations, powder injections, gels, capsules and patches; the content of methyl eugenol in the medicinal preparation is more than 80%.