Application of alcohol extract of pyrus calleryana decoction in preparation of medicine for reducing blood sugar and preparation method thereof

Abstract

The application discloses application of an alcohol extract of Pyrus calleryana decoction fruit in preparation of a blood sugar reducing medicine and a preparation method thereof. The alcohol extract of Pyrus calleryana decoction fruit with alpha-glucosidase inhibiting effect and blood sugar reducing effect is obtained by reflux extraction of the fruit of Pyrus calleryana decoction with 70% ethanol. The extract can significantly inhibit the activity of alpha-glucosidase, has a significant inhibiting effect on the postprandial blood sugar level of a T2DM model mouse, and has a significant blood sugar reducing effect in vivo.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to application of an alcohol extract of a Pyrus communis fruit in preparing anti-inflammatory medicines and a preparation method thereof. Background Art

[0002] Diabetes is a metabolic disorder that has attracted worldwide attention, has a heavy medical burden, and affects the quality of life of patients. Among them, type 2 diabetes mellitus (T2DM) accounts for 90% of patients (Qin Hailong et al., Experimental study on the inhibition of α-glucosidase by ethanol extract of soursop fruit in the treatment of type 2 diabetes, Journal of Hainan Medical College, 2023). At present, the management of T2DM mainly focuses on increasing exercise and controlling blood sugar to prevent the deterioration of diabetes and the occurrence of other complications. More and more evidence shows that postprandial blood sugar fluctuations are highly correlated with microvascular and macrovascular morbidity or cardiovascular mortality in T2DM patients, and postprandial hyperglycemia is an important factor in the development of T2DM and the occurrence of complications (Plos One, 2021, 16(10), e0258771). Therefore, one of the main strategies for treating T2DM is to lower postprandial blood sugar levels. α-glucosidase is a key enzyme for the body to digest and absorb carbohydrates. When its activity is inhibited, it will reduce the body's glucose absorption, thereby achieving the effect of lowering blood sugar, which helps to treat diabetes. However, the commonly used α-glucosidase inhibitors (acarbose, voglibose) can cause certain side effects when used for a long time. Therefore, screening and researching natural products with α-glucosidase inhibitory activity has become a hot topic (Curr Drug Saf, 2021, 16(2): 122-128; Biomed Pharmacother, 2020, 131, 110708; Curr Diabetes Rev, 2021, 17(4), 437-456).

[0003] Pyrus calleryana Decne is a deciduous tree in the genus Pyrus of the Rosaceae family and is native to my country. my country is rich in Pyrus plant resources, with a total of 14 species, and Pyrus calleryana Decne is one of the most widely distributed Pyrus plants. Pyrus plants have been reported to have a variety of pharmacological activities, such as antibacterial, anti-inflammatory, antifungal, antioxidant, and analgesic (Journal of Hebei University of Science and Technology 2013, 27(03), 37-42). Pear was first recorded in the "Ming Yi Bie Lu" (Famous Doctors' Prescriptions) and has a long history of medicinal use. It is cool in nature, sweet and slightly sour in taste, and enters the lung and stomach meridians. The fruit, peel, leaves, flowers, and roots can all be used as medicine, with effects such as moisturizing, promoting fluid production, clearing heat, and resolving phlegm. The roots and leaves of Pyrus communis have the effects of moistening the lungs and relieving coughs, clearing away heat and detoxifying, and are mainly used to treat dry coughs and acute conjunctivitis. The fruit has the effects of strengthening the stomach and digestion, astringing the intestines and stopping dysentery, clearing away heat and promoting the production of body fluids. The branches can stop diarrhea, promote qi circulation and soothe the stomach. The leaves and flowers can detoxify the toxicity of Rhizoma Corydalis and Rhizoma Lepidopterae (Jiangsu Agricultural Science, 2015, 43(03):162-165). However, there are currently no reports on the hypoglycemic activity of Pyrus communis fruit, which may hinder its development and utilization. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of an alcohol extract of Pyrus communis fruit in preparing a drug for lowering blood sugar and a preparation method thereof.

[0005] The present invention has discovered the use of an alcohol extract of Pyrus communis fruit in preparing α-glucosidase inhibitors and blood sugar-lowering drugs, thus developing new uses of Pyrus communis fruit and providing a new option for preparing blood sugar-lowering drugs.

[0006] The present invention also found that the alcohol extract of the Pyrus chinensis fruit inhibited the activity of α-glucosidase in vitro and improved the fasting blood sugar level in vivo, and identified the chemical components of the alcohol extract of the Pyrus chinensis fruit.

[0007] The technical solution adopted by the present invention is as follows: application of the alcohol extract of Pyrus communis fruit in preparing a medicine for lowering blood sugar.

[0008] The invention relates to an application of the ethanol extract of the Pyrus communis fruit in the preparation of a drug for inhibiting the activity of α-glucosidase.

[0009] Application of ethanol extract of Pyrus communis fruit in preparing medicine for improving streptozotocin-induced type 2 diabetes.

[0010] The alcohol extract of the pear fruit and a pharmaceutically acceptable carrier are prepared into a medicine in the form of tablets, capsules, suppositories, dropping pills or ointments.

[0011] The preparation method of the alcohol extract of the aralia pyrifolia fruit comprises the following steps: taking fresh aralia pyrifolia fruit, washing, cutting into pieces, and crushing the pieces; mixing the crushed raw material with 70% ethanol by mass at a material-liquid ratio of 1:3 to 1:8, with the unit being g / mL; performing heating and reflux extraction twice, each time for 2 to 4 hours; combining the two extracts, cooling to room temperature, and then filtering; and concentrating the filtrate under reduced pressure and freeze-drying the filtrate to obtain dry powder of the aralia pyrifolia fruit alcohol extract.

[0012] The present invention determined the chemical components of the alcohol extract of Pyrus communis fruit by UHPLC-Q-Orbitriap MS (see Figure 1 and Table 1), which are mainly rich in phenolic, flavonoid and triterpenoid compounds; the present invention found through experimental research that the alcohol extract of Pyrus communis fruit significantly inhibited the activity of α-glucosidase and the effect was stronger than that of the positive drug acarbose; and improved the fasting blood glucose level of T2DM model mice in vivo.

[0013] By adopting the above technical solution, the present invention has discovered for the first time a new use in treating diabetes-related diseases, providing a new option for preparing blood sugar-lowering drugs, and has important application value in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the UHPLC-Q-Orbitriap MS chromatogram of the alcohol extract of Pyrus communis fruit;

[0015] Figure 2 The study aimed to investigate the effect of ethanol extract (EE) from Pyrus communis fruit on fasting blood glucose levels in T2DM model mice. DETAILED DESCRIPTION

[0016] In an embodiment of the present invention, fresh Pyrus communis fruit (harvested from Shansen Reservoir, Yudu County, Ganzhou City, Jiangxi Province; identified by Professor Hu Guoxiong of Guizhou University) was pulverized. The pulverized medicinal material was weighed and extracted twice (2 hours each time) under heating and reflux in a solvent (70% ethanol). The combined extracts were cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and freeze-dried to yield an 11% (w / w, based on the fresh weight of the fruit) alcohol extract.

[0017] Quantitative analysis of the Pear extract was performed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-Q-Orbitriap MS) technology, and the chemical components of Pear WE and EE were identified using a database. The UHPLC conditions used in this experiment were: mobile phase: 0.1% formic acid in water and acetonitrile (containing 0.1% formic acid); column temperature: 40°C; injection volume: 5 μL; flow rate: 5 μL / s; column: ThermoFisher Hypersil GOLD aQ (100×2.1 mm, 1.9 μm); liquid chromatography system: Dionex Ultimate 3000RSLC (HPG). Mass spectrometry conditions were: HESI-II ion source; mass spectrometry system: Thermo Scientific Q ExactiveFocus. Other gradient elution conditions, ion source parameters, and mass spectrometry scan parameters are shown in Tables 2 to 4.

[0018] Chemical composition such as Figure 1 As shown in Table 1 , 37 compounds were identified in the alcohol extract of Pyrus communis fruit, among which phenols, flavonoids and triterpenes were rich.

[0019] Table 1 Chemical analysis results of pear ethanol extract

[0020]

[0021]

[0022]

[0023]

[0024] Table 2 Gradient elution conditions

[0025]

[0026] Table 3 Ion source parameter settings

[0027]

[0028] Table 4 Mass spectrometry scanning parameter settings

[0029]

[0030]

[0031] Pharmacological Example 1: Inhibitory activity against α-glucosidase

[0032] α-glucosidase activity was assayed in a 96-well plate using 4-nitrophenol-α-D-glucopyranoside as the substrate:

[0033] Take out the reagents and samples prepared in advance, add the sample solution (30 μL) to a 96-well plate, then add PBS (pH = 6.8) buffer (60 μL) and α-glucosidase (10 μL, 0.8 U / mL), incubate at 37°C for 15 minutes, then add the substrate 4-nitrophenol-α-D-pyranoglucopyranoside (10 μL, 1 mmol / L), incubate at 37°C for 15 minutes, and finally add sodium carbonate solution (80 μL, 0.2 mol / L) to terminate the reaction. Use a microplate reader to measure the absorbance value A1 at a wavelength of 405 nm. At the same time, the sample blank group A2 (sample to be tested + PBS + PBS + substrate + sodium carbonate), the negative group A3 (PBS + PBS + enzyme + substrate + sodium carbonate), and the blank group A4 (PBS + PBS + PBS + substrate + sodium carbonate) are operated simultaneously. In the experiment, the mother solution sample is diluted into 6 gradient concentrations using the half-dilution method and operated simultaneously. The α-glucosidase inhibition rate of the sample was calculated according to the following formula (1). Acarbose was used as the standard and the results were expressed as acarbose equivalents (ACEs).

[0034]

[0035] As shown in Table 5, for α-glucosidase, EE (IC 50 =0.49±0.08μg / mL) showed a stronger effect than the positive drug acarbose (IC 50 =299.56±22.81μg / mL) of enzyme inhibition activity, and the acarbose equivalent was 617.30±54.96gACEs / g Extract.

[0036] Table 5 Inhibitory activity of pear ethanol extract on α-glucosidase

[0037]

[0038] Note: IC 50 Indicates the sample concentration at which the inhibition rate reaches 50%; Positive control: acarbose Pharmacological Example 2: Effect on postprandial blood glucose levels in T2DM model mice (in vivo hypoglycemic experiment)

[0039] Male ICR mice were purchased from Changsha Tianqin Biotechnology Co., Ltd., with an average body weight of 22±2g. They were randomly divided into six groups, each with 10 mice. During the experiment, they had free access to water. After being fed a high-fat diet for 4 weeks, a single tail vein injection of 150mg / kg of streptozotocin was used to establish the model. The control group was fed a normal diet for 4 weeks and injected with an equal volume of blank solvent. All other procedures were the same. After streptozotocin establishment, the mice were divided into a blank control group (Control), a model group (Model), an acarbose group (50mg / kg), a high-dose group (200mg / kg), a medium-dose group (100mg / kg), and a low-dose group (50mg / kg). Each group of mice was gavage-administered once daily for a total of 4 weeks. The model and blank groups were gavaged with the corresponding saline solution, the drug-treated groups were gavaged with the corresponding dose concentration of the ethanol extract of Pyrus communis fruit, and the positive control group was gavaged with the corresponding dose of acarbose solution. During the experiment, the postprandial blood glucose levels of the experimental mice were monitored regularly. The fasting blood glucose levels were measured once a week after fasting for 12 hours. The fasting blood glucose levels were measured with a blood glucose meter after fasting overnight after oral gavage for 4 consecutive weeks.

[0040] As shown in Table 6 and Figure 2 As shown in Table 6, the ethanol extract of Pyrus communis fruit effectively reduced the fasting blood glucose levels of diabetic mice induced by streptozotocin in a time- and dose-dependent manner. As shown in Table 6, with adequate feed and water, the fasting blood glucose levels of the blank group mice fluctuated slightly and tended to be stable. However, the model group mice remained in a state of hyperglycemia (P < 0.001 vs. blank group). The blood glucose levels of T2DM mice began to gradually decrease after 7 days of acarbose administration (P < 0.05 vs. model group), and the inhibitory effect was significantly enhanced after one week (P < 0.01 vs. model group). A 200 mg / kg ethanol extract of Pyrus communis fruit also suppressed postprandial blood glucose levels on the 7th day of administration (P < 0.05 vs. model group), and starting from the 14th day, ethanol extracts of Pyrus communis fruit at concentrations of 200, 100, and 50 mg / kg all had a significant hypoglycemic effect. These results indicate that the ethanol extract of Pyrus communis fruit can effectively reduce the fasting blood glucose levels of diabetic mice induced by streptozotocin.

[0041] Table 6 Inhibitory effect on streptozotocin-induced type 2 diabetes in mice (Mean ± SD)

[0042]

[0043] Note: Compared with the blank group: ### P<0.001; compared with the model group: * P<0.05, ** P<0.01, *** P < 0.001, (-) means no.

[0044] Based on the above pharmacological examples, the alcohol extract of Pyrus communis fruit can significantly inhibit the activity of α-glucosidase in vitro, and has a significant inhibitory effect on the blood glucose level of T2DM model mice in vivo, and has a significant hypoglycemic effect both in vitro and in vivo.

Claims

1. The use of the alcohol extract of Pyrus communis fruit in the preparation of a drug for lowering blood sugar, characterized in that: The alcohol extract is obtained by extraction with 70% ethanol.

2. The use according to claim 1, characterized in that: The invention relates to an application of the ethanol extract of the Pyrus communis fruit in the preparation of a drug for inhibiting the activity of α-glucosidase.

3. The use according to claim 1, characterized in that: Application of ethanol extract of Pyrus communis fruit in preparing medicine for improving streptozotocin-induced type 2 diabetes.

4. The use according to claim 1, 2 or 3, characterized in that: The alcohol extract of the pear fruit and a pharmaceutically acceptable carrier are prepared into a medicine in the form of tablets, capsules, suppositories, dropping pills or ointments.

5. The use according to claim 1, characterized in that: The preparation method of the alcohol extract of the Pyrus nobilis fruit comprises the following steps: washing, cutting and crushing fresh Pyrus nobilis fruit; mixing the crushed raw material with 70% ethanol by mass at a material-liquid ratio of 1:3 to 1:8, with the unit being g / mL; performing heating and reflux extraction twice, each time for 2 to 4 hours; combining the two extracts, cooling to room temperature, and then filtering; and concentrating the filtrate under reduced pressure and freeze-drying the filtrate to obtain a dry powder of the Pyrus nobilis fruit alcohol extract.

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