Use of castanea mollissima blume total bract alcohol extract in preparation of blood sugar lowering drugs

Abstract

This invention discloses the application of chestnut total burdock ethanol extract in the preparation of hypoglycemic drugs. In this invention, the chestnut total burdock ethanol extract with hypoglycemic effect is obtained by reflux extraction of chestnut total burdock with 70% ethanol. This extract can significantly inhibit the activity of α-glucosidase and has a significant inhibitory effect on postprandial blood glucose levels in T2DM model mice, exhibiting a significant hypoglycemic effect in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of chestnut total husk alcohol extract in the preparation of hypoglycemic drugs. Background Technology

[0002] Diabetes mellitus is a metabolic disorder that has attracted worldwide attention, incurring a heavy medical burden and impacting patients' quality of life. Type 2 diabetes mellitus (T2DM) accounts for 90% of all cases (Journal of Hainan Medical College, 2023, 29(7): 481-487). Currently, the management of T2DM mainly focuses on controlling blood glucose to prevent the deterioration of diabetes and the occurrence of other complications. Increasing evidence suggests that postprandial blood glucose 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, 16(10): e0258771). Therefore, one of the main strategies for treating T2DM is to reduce postprandial blood glucose levels. Alpha-glucosidase is a key enzyme in the body's digestion and absorption of carbohydrates. When its activity is inhibited, the body's glucose absorption is reduced, thereby lowering blood glucose and aiding in the treatment of diabetes. However, commonly used alpha-glucosidase inhibitors (acarbose, voglibose) can produce certain side effects with long-term use. Therefore, screening and researching natural products with α-glucosidase inhibitory activity has become a hot topic (Food Science and Human Wellness, 2014, 3:136-174; Biomedicine & Pharmacotherapy, 2020, 131: 110708; Asian Pacific Journal of Tropical Biomedicine, 2015, 5(9): 748-755).

[0003] Chestnut (Castanea mollissima Blume) belongs to the genus Castanea in the family Fagaceae and is a deciduous tree. It has high nutritional value, being rich in nutrients, containing abundant protein, starch, and trace elements. Plants in the Castanea genus have been reported to possess various pharmacological activities, such as antibacterial, antioxidant, anti-inflammatory, enzyme-inhibiting, and antitumor effects. Figure 1As shown, the chestnut involucre (commonly known as the chestnut hairy ball) is the spiny bract on the outside of the chestnut fruit, spherical in shape, 3-5 cm in diameter, with sharp, hairy spines on the outside. As a traditional Chinese medicine, the chestnut involucre has the effects of clearing heat and resolving phlegm, and stopping bleeding. It is often used for erysipelas, scrofula, whooping cough, hematochezia, and epistaxis. In addition, different parts of the chestnut also have medicinal value: chestnut root can promote qi circulation and relieve pain, invigorate blood and regulate menstruation; chestnut flower slightly promotes saliva production, resolves phlegm and relieves cough, clears heat and resolves phlegm, and stops bleeding; chestnut endocarp can disperse phlegm and lower qi, and nourish the skin; chestnut kernel can invigorate qi and strengthen the spleen, tonify the kidneys and strengthen muscles, invigorate blood and reduce swelling, and stop bleeding (Shi Xiaoxiao. Zouping Traditional Chinese Medicine Records [M]. Beijing: Traditional Chinese Medicine Ancient Books Publishing House, 2015, 67). However, current research has not reported any related studies on the hypoglycemic activity of the chestnut involucre, which may hinder its development and utilization. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the total spores of chestnut in the preparation of hypoglycemic drugs, thus opening up new uses for the total spores of chestnut and providing a new option for the preparation of hypoglycemic drugs.

[0005] This invention also discovered that the total spore extract of chestnut inhibited the activity of α-glucosidase and improved the fasting blood glucose level in T2DM model mice. The chemical components of the total spore extract of chestnut were also identified.

[0006] The technical solution adopted in this invention is as follows:

[0007] The total husk extract of chestnut was prepared by the following method:

[0008] Fresh chestnut husks (such as...) Figure 1 Wash and cut the raw materials into pieces, then grind them in a high-speed universal grinder. Mix the ground raw materials with 70% ethanol at a material-to-liquid ratio of 1:3 to 1:8 (unit: g / mL), and extract twice by heating and reflux (2-4 h each time). Combine the two extracts, cool to room temperature, and filter. The filtrate is concentrated under reduced pressure and freeze-dried under vacuum to obtain a dry powder of chestnut total scutellarin extract.

[0009] The chemical composition of the total spore extract of chestnut was determined by UHPLC-Q-Orbitriap MS (see details). Figure 2 (and Table 1), in which the main compound types are phenols, flavonoids and triterpenoids; through experimental research, this invention found that the total spore extract of chestnut significantly inhibited the activity of α-glucosidase and was more effective than the positive control drug acarbose; and improved the fasting blood glucose level in T2DM model mice.

[0010] By adopting the above technical solution, this invention has for the first time discovered a new use of chestnut total spore alcohol extract in the treatment of diabetes-related diseases, providing a new option for the preparation of hypoglycemic drugs and having important application value in the pharmaceutical industry. Attached Figure Description

[0011] Figure 1 This is a real photo of the chestnut involucre.

[0012] Figure 2 The UHPLC-Q-Orbitriap MS chromatogram of the total bract alcohol extract of chestnut;

[0013] Figure 3 The graph shows the inhibition rate of α-glucosidase by the total husk extract (EE) of chestnut.

[0014] Figure 4 The effect of chestnut total husk alcohol extract (EE) on fasting blood glucose levels in T2DM model mice. Detailed Implementation

[0015] An embodiment of the invention: Fresh chestnut husks (harvested in Xianfeng County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province; identified by Professor Hu Guoxiong of Guizhou University) were pulverized. The pulverized material was weighed, and 70% ethanol (at a material-to-liquid ratio of 1:5) was added. The mixture was heated and refluxed twice (2 hours each time). The two extracts were combined, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure and freeze-dried under vacuum to obtain an 11% (w / w, based on the fresh weight of the fruit) ethanol extract.

[0016] High-performance liquid chromatography-mass spectrometry (UHPLC-Q-Orbitriap MS) was used to identify the chemical components of the ethanol extract of chestnut total husks, combined with relevant databases. The HPLC system used was a Dionex Ultimate 3000 RSLC, and the chromatographic column was an ACE Ultracore 2.5 Super C18 column (1.9 μm, 2.1 mm inner diameter, 100 mm length). The mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile containing 0.1% formic acid, with an injection volume of 5 µL, a flow rate of 0.3 mL / min, and a column temperature controlled at 40℃. The gradient elution program was set as follows: acetonitrile content was 5% from 0 to 2 minutes; from 2 to 42 minutes, the acetonitrile content increased from 5% to 95%; from 42 to 47 minutes, the acetonitrile content was maintained at 95%; then from 47 to 47.1 minutes, the acetonitrile content decreased from 95% to 5%; finally, from 47.1 to 50 minutes, the acetonitrile content was maintained at 5% until the end.

[0017] The mass spectrometer was a Thermo Scientific Q Exactive Focus, equipped with HESI-II. Ion source parameters: S-Lens was 60; Probe Heater Temperature and Capillary Temperature were 350℃ and 320℃, respectively; Sweep Gas, AUX Gas, and Sheath Gas were set to 0 arb, 10 arb, and 35 arb, respectively; Spray Voltage was 3.0 kV (+) / 2.5 kV (-). The mass spectrometry acquisition mode was Full MS-ddMS², i.e., a data-dependent secondary scan triggered after a first-stage full scan. Relevant scan parameters were set as follows: NCE (Stepped NCE) was set to 20, 40, and 60 eV; Full MS and MS / MS resolutions were set to 70,000 and 17,500, respectively; the scan range covered m / z 100 to 1500. The target value for automatic gain control (AGC) is set to 1×10 in both Level 1 and Level 2 modes. 6 and 2×10 5 The maximum ion residence time (Maximum IT) for primary and secondary mass spectrometry was set to 100 ms and 50 ms, respectively. The minimum AGC target value was 8 × 10⁻⁶. 3 The intensity threshold is 1.6 × 10⁻⁶. 5 The isolation width was 1.5 m / z; the dynamic exclusion time was 5 s; the cycle count was 3 times; and the mass spectrometry data were acquired in profile mode. Compound identification was completed by referring to relevant literature and comparing with databases such as mzVault and mzCloud.

[0018] Chemical composition is shown in Table 1 and Figure 2 As shown, 39 compounds were identified from the total bromine extract of chestnut, mainly phenols, flavonoids and triterpenoids.

[0019]

[0020]

[0021]

[0022] Pharmacological Example 1: Inhibitory Activity Against α-Glucosidase

[0023] α-glucosidase activity was detected using 96-well plates with 4-nitrophenol-α-D-glucopyranoside as the substrate. First, 30 μL of the sample solution was added to each well, followed by 60 μL of PBS buffer (pH 6.8) and α-glucosidase (10 μL, 0.8 U / mL), and incubated at 37°C for 15 minutes. Then, 10 μL of 4-nitrophenol-α-D-glucopyranoside substrate (1 mmol / L) was added, and incubation continued at 37°C for another 15 minutes. After the reaction was complete, sodium carbonate solution (80 μL, 0.2 mol / L) was added to terminate the reaction. The absorbance (A1) was measured at 405 nm using a microplate reader. Simultaneously, a blank control group (A2: chestnut total extract + PBS + PBS + substrate + sodium carbonate), a negative control group (A3: PBS + PBS + enzyme + substrate + sodium carbonate), and a blank control group (A4: PBS + PBS + PBS + substrate + sodium carbonate) were set up. The stock solution samples were prepared into six gradient concentrations using a half-dilution method and the operations were performed simultaneously. Finally, the inhibition rate of the samples against α-glucosidase was calculated according to the following formula, and the results are expressed as acarbose equivalents (ACEs).

[0024] α-glucosidase inhibition rate (%) = (1 - )×100

[0025] As shown in Table 2 and Figure 3, for α-glucosidase, the total extract of chestnut (EE, IC50) 50 = 0.39 ± 0.01 µg / mL) showed stronger performance than the positive control drug acarbose (IC50). 50 The enzyme inhibitory activity was 212.31 ± 18.70 µg / mL, with an acarbose equivalent of 542.52 ± 53.63 g ACEs / g Extract.

[0026]

[0027] Pharmacological Example 2: Effect on postprandial blood glucose levels in T2DM model mice (in vivo hypoglycemic experiment)

[0028] Male ICR mice were purchased from Changsha Tianqin Biotechnology Co., Ltd., with an average weight of 22 ± 2 g. They were randomly divided into six groups of 10 mice each. During the experiment, mice had free access to water and were fed a high-fat diet for 4 weeks. Then, streptozotocin (150 mg / kg) was administered via a single intravenous injection via the tail vein to establish the model. The control group was fed a normal diet for 4 weeks and received an equal volume of blank solvent, with all other procedures performed the same. After streptozotocin modeling, mice were divided into a blank control group (Control), a model group (Model), an acarbose group (50 mg / kg), a high-dose group (200 mg / kg), a medium-dose group (100 mg / kg), and a low-dose group (50 mg / kg). Mice in each group were administered the drug once daily by gavage for 4 weeks. The model group and the blank control group were administered the corresponding physiological saline, while the drug administration groups were administered the corresponding concentration of chestnut total spore extract solution. The positive control group was administered the corresponding dose of acarbose solution by gavage. During the experiment, the postprandial blood glucose level of the experimental mice was monitored regularly. Fasting blood glucose was measured once a week after fasting for 12 hours. The mice were gavaged for 4 consecutive weeks. After fasting overnight, fasting blood glucose level was measured with a blood glucose meter.

[0029] As shown in Table 3 and Figure 4 As shown in Table 3, the total spore extract of chestnut effectively reduced fasting blood glucose levels in streptozotocin-induced diabetic mice in a time- and dose-dependent manner. Table 3 shows that under adequate feed and water conditions, the fasting blood glucose levels in the control group mice remained relatively stable, while the model group mice remained in a hyperglycemic state (P<0.001 vs. control group). Blood glucose levels in T2DM mice began to gradually decrease after 7 days of acarbose administration (P<0.05 vs. model group), and the inhibitory effect significantly increased after one week (P<0.01 vs. model group). 200 mg / kg of the total spore extract of chestnut effectively suppressed postprandial blood glucose on day 7 of administration (P<0.05 vs. model group), and from day 14 onwards, concentrations of 200, 100, and 50 mg / kg of the total spore extract of chestnut all exhibited significant hypoglycemic effects. These results indicate that the total spore extract of chestnut can effectively reduce fasting blood glucose levels in streptozotocin-induced diabetic mice.

[0030]

[0031] Based on the above pharmacological examples, the total spores of chestnut significantly inhibited the activity of α-glucosidase and had a significant inhibitory effect on blood glucose levels in T2DM model mice, demonstrating significant hypoglycemic effects both in vitro and in vivo.

Claims

1. Application of chestnut total husk alcohol extract in the preparation of hypoglycemic drugs.

2. Use according to claim 1, characterized in that: The total spore extract of chestnut was prepared by the following method: Take fresh chestnut spores, wash, cut into pieces, and crush them; mix the crushed raw material with 70% ethanol at a material-to-liquid ratio of 1:3 to 1:8 (unit: g / mL), heat and reflux to extract twice, each time for 2 to 4 hours, combine the two extracts, cool to room temperature, and filter; concentrate the filtrate under reduced pressure and freeze dry under vacuum to obtain chestnut spore fruit alcohol extract powder.

3. Use according to claim 1, characterized in that: Application of chestnut total spore alcohol extract in the preparation of inhibitors of α-glucosidase activity.

4. Use according to claim 1, characterized in that: Application of chestnut total spore alcohol extract in the preparation of drugs to improve streptozotocin-induced type 2 diabetes in ICR mice.

5. Use of the Chinese chestnut involucre according to claim 1 for the preparation of a hypoglycemic medicament, characterized in that, A drug formulated into tablets, capsules, suppositories, drops, or ointments by combining chestnut total burdock extract with a pharmaceutically acceptable carrier.

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