Ferra extract with hypoglycemic activity and pharmaceutical composition and application thereof

Abstract

The invention provides a plant extract extracted from epicarp and seeds of Mesurea ferrea L., a preparation method of the plant extract, a pharmaceutical composition containing the extract and application of the pharmaceutical composition in preparation of a medicine for treating diabetes, and belongs to the technical field of medicines. A series of extracts, which are named as MY125, MY126, MY127, MY128, MY129, MY129 and MY131, are extracted and prepared from dried epicarp and seeds of the ferrum. Experimental research shows that except MY131, all extracts can remarkably promote the glucose uptake ability of 3T3-L1 fat cells, and it is indicated that the MY131 extract possibly has the insulin sensitization effect. Except MY129, other extracts show a remarkable inhibition effect (the inhibition rate gt is 77%) on alpha-glucosidase, and the inhibition effect is remarkably superior to that of positive control quercetin (the inhibition rate is 52.1%). Meanwhile, MY128, MY129 and MY130 also show pancreatic lipase inhibitory activity, can be used for developing novel drugs for treating or preventing diabetes mellitus and reducing blood sugar, and have important application prospects and market potential.

Description

Technical Field:

[0001] The present invention belongs to the field of pharmaceutical technology, specifically, to the field of botanical medicine technology, and more specifically, to an extract of Mesua ferrea, its pharmaceutical composition, its preparation method, and its application in the preparation of a drug for treating or preventing diabetes or reducing blood sugar. Background Art:

[0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia and is one of the important factors leading to human death globally. Complications of diabetes such as blindness, kidney failure, heart attacks, strokes, and lower limb amputations seriously threaten the quality of life and lifespan of patients. In addition, diabetes is also associated with an increased mortality rate of cardiovascular diseases related to hyperglycemia. Although existing anti-diabetic drugs relieve the symptoms of diabetic patients to a certain extent, their long-term use still has many limitations, such as inconvenient administration methods, difficulty in stably controlling blood sugar levels, and some drugs may cause relatively serious side effects. These limitations not only restrict the treatment effect but also may have an adverse impact on the long-term health and quality of life of patients. Therefore, developing new hypoglycemic drugs to overcome the deficiencies of existing treatment methods has become a crucial research direction in the field of anti-diabetic treatment.

[0003] Mesua ferrea L., also known as Mesua ferrea, Mesua ferrea, etc., is mainly distributed in southern and southeastern tropical Asia. Mesua ferrea has important medicinal value and is widely used in Uyghur medicine, Dai medicine, and the Ayurvedic system in India. Currently, the research on Mesua ferrea flowers is relatively extensive, and it has been found to have various pharmacological activities such as anti-cancer, antibacterial, anti-inflammatory, anti-convulsant activity, and immunomodulation. Currently, there is no report on the hypoglycemic activity of the outer pericarp and seeds of Mesua ferrea, and even less on the hypoglycemic activity of its extract. This indicates that the extract of the outer pericarp and seeds of Mesua ferrea has great potential and innovation in the development of hypoglycemic drugs. Summary of the Invention:

[0004] The object of the present invention is to provide an extract of Mesua ferrea, its preparation method, or its application in the preparation of a drug for treating or preventing diabetes or reducing blood sugar.

[0005] In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

[0006] Mesua ferrea extract with hypoglycemic activity, wherein the Mesua ferrea extract is extracted from the outer pericarp and seeds of Mesua ferrea, including Mesua ferrea seed extracts MY125, MY126, and MY127, and Mesua ferrea outer pericarp extracts MY128, MY129, MY130, and MY131. It is prepared by the following method: (a) Dry the Mesua ferrea seeds, crush them, and successively extract them with petroleum ether, ethyl acetate, and 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min. Filter and combine them respectively, recover the solvent, and obtain the Mesua ferrea seed extracts MY125, MY126, and MY127 as extracts; (b) Dry the outer pericarp of Mesua ferrea, crush it, and successively extract it with petroleum ether, ethyl acetate, and 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min. Filter and combine them respectively, recover the solvent, and obtain the Mesua ferrea outer pericarp extracts MY128, MY129, and MY130 as extracts; (c) Dry the outer pericarp of Mesua ferrea, crush it, extract it with 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min, filter and recover the solvent, and obtain the Mesua ferrea outer pericarp extract MY131 as an extract.

[0007] A pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned Mesua ferrea extracts MY125, MY126, MY127, MY128, MY129, MY129, and MY131, and a pharmaceutically acceptable carrier.

[0008] Use of the above-mentioned Mesua ferrea extract or pharmaceutical composition in the preparation of a drug for treating diabetes or a hypoglycemic drug.

[0009] A botanical drug for improving or treating diabetes, which contains a therapeutically effective amount of the above-mentioned Mesua ferrea extract and a pharmaceutically acceptable carrier.

[0010] The present invention also provides a preparation method of Mesua ferrea extract with hypoglycemic activity, wherein the Mesua ferrea extract is extracted from the outer pericarp and seeds of Mesua ferrea, including Mesua ferrea seed extracts MY125, MY126, and MY127, and Mesua ferrea outer pericarp extracts MY128, MY129, MY130, and MY131. The method includes the following steps: (a) Dry the Mesua ferrea seeds, crush them, and successively extract them with petroleum ether, ethyl acetate, and 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min. Filter and combine them respectively, recover the solvent, and obtain the Mesua ferrea seed extracts MY125, MY126, and MY127 as extracts; (b) Dry the outer pericarp of Mesua ferrea, crush it, and successively extract it with petroleum ether, ethyl acetate, and 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min. Filter and combine them respectively, recover the solvent, and obtain the Mesua ferrea outer pericarp extracts MY128, MY129, and MY130 as extracts; (c) Dry the outer pericarp of Mesua ferrea, crush it, extract it with 90% ethanol by ultrasonic extraction in a 40°C water bath for 30 min, filter and recover the solvent, and obtain the Mesua ferrea outer pericarp extract MY131 as an extract.

[0011] Also, a method for preparing a pharmaceutical composition, which first obtains the extracts MY125, MY126, MY127, MY128, MY129, MY129 and MY131 of Mesua ferrea L. by a method for preparing extracts, and then adds a pharmaceutically acceptable carrier.

[0012] In the present invention, the dried outer pericarp and seeds of Mesua ferrea L. are separately extracted with a solvent to prepare MY125, MY126, MY127, MY128, MY129, MY129 and MY131. The glucose uptake experiment of the extracts of Mesua ferrea L. on 3T3-L1 adipocytes shows that, except for MY131, all extracts can significantly promote the glucose uptake of 3T3-L1 adipocytes. The α-glucosidase inhibition experiment shows that, except for MY129, all extracts exhibit strong α-glucosidase inhibitory activity (the inhibition rate is greater than 77%), which is significantly better than the positive control quercetin; among them, the IC 50 values of MY125, MY126, MY127 and MY131 are 3.7 - 37.6 μg / mL, showing a good dose-dependent inhibitory effect. At the same time, the pancreatic lipase inhibition experiment shows that MY128, MY129 and MY130 have weak pancreatic lipase inhibitory activity, indicating that they may have potential effects on diseases related to lipid metabolism. The extracts of Mesua ferrea L. of the present invention show significant biological activities in multiple pharmacological models. These results indicate that the extracts of Mesua ferrea L. provide important candidate substances for the development of new drugs for treating or preventing diabetes and hypoglycemic health products, and have broad application prospects and market potential.

[0013] In the present invention, the mass percentage content of the extracts MY125, MY126, MY127, MY128, MY129, MY129 and MY131 of the present invention in the pharmaceutical composition is preferably 0.1 - 99%, more preferably 0.5 - 90%; the total mass percentage content of the pharmaceutically acceptable carrier and / or excipient in the pharmaceutical composition is preferably 1 - 99.9%, more preferably 10 - 99.5%.

[0014] The present invention has no special limitation on the pharmaceutically acceptable carrier or the excipient, and the pharmaceutically acceptable carrier or excipient well-known in the art can be used, specifically, one or more of solid, semi-solid or liquid diluents, fillers or pharmaceutical product adjuvants. In the present invention, the preparation types of the pharmaceutical composition preferably include liquid preparations and solid preparations; the liquid preparations preferably include injections, suspensions, emulsions, solutions or syrups; the solid preparations preferably include tablets, capsules, granules or instant granules. The present invention has no special limitation on the preparation method of the pharmaceutical composition, and the preparation method well-known in the art can be used.

[0015] In the present invention, the administration method of the pharmaceutical composition is preferably injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection or subcutaneous injection.

[0016] In the present invention, the pharmaceutical composition is preferably used in the form of a dosage per unit body weight. In the present invention, the pharmaceutical composition is preferably used in the form of a dosage per unit body weight. In the present invention, the dosage per unit body weight is preferably 0.5 to 20 mg / kg.

[0017] Compared with the prior art, the present invention has the following beneficial effects: for the first time, the extracts of the outer peel and seeds of the ironwood were used to promote the uptake of glucose by adipocytes, the α-glucosidase inhibitory activity and the pancreatic lipase inhibitory activity. The activity evaluation results showed that, except for MY131, all extracts could significantly promote the glucose uptake capacity of 3T3-L1 adipocytes; except for MY129, all extracts showed strong α-glucosidase inhibitory activity and the inhibition rate was greater than 77%; in addition, MY128, MY129 and MY130 also showed pancreatic lipase inhibitory activity. The ironwood and its extracts described in the present invention are used as raw materials to prepare drugs for the prevention and treatment of diabetes. On the basis of the prior art, any medicinal preparation is prepared by conventional methods of traditional Chinese medicine preparations. For example, the ironwood extract is made into a powder for oral administration; the extract is made into tablets, capsules, granules, and pills, but this does not limit the scope of protection of the present invention. Specific implementation method:

[0018] The following embodiments of the present invention are used to further illustrate the essential content of the present invention, but the present invention is not limited thereto.

[0019] Embodiment 1:

[0020] Preparation of MY125, MY126 and MY127.

[0021] Take 100.0 g of dried seed sample of Mesua ferrea L., crush it and use petroleum ether (1.0 L), ethyl acetate (1.0 L), and 90% ethanol (1.0 L) in sequence, extract it in a 40°C water bath ultrasonic manner for 30 min, filter it, and recover the solvent by reducing pressure at 60°C to obtain 23.3 g of petroleum ether extract (No. MY125), 11.0 g of ethyl acetate extract (No. MY126) and 3.0 g of ethanol extract (No. MY127).

[0022] Embodiment 2:

[0023] Preparation of MY128, MY129 and MY130.

[0024] Take 88.4 g of the dried outer pericarp sample of Mesua ferrea L., pulverize it, and successively extract it with petroleum ether (0.5 L), ethyl acetate (0.5 L), 90% ethanol (0.5 L) by ultrasonic extraction in a water bath at 40 °C for 30 min. Filter, and recover the solvents from the filtrates under reduced pressure at 60 °C to obtain 3.9 g of petroleum ether extract (No. MY128), 3.0 g of ethyl acetate extract (No. MY129), and 1.2 g of ethanol extract (No. MY130).

[0025] Example 3:

[0026] Take 71.2 g of the dried outer pericarp sample of Mesua ferrea L., pulverize it, extract it with 90% ethanol (0.5 L) by ultrasonic extraction in a water bath at 40 °C for 30 min, filter, and recover the solvent from the filtrate under reduced pressure at 60 °C to obtain 6.0 g of extract (No. MY131).

[0027] Example 4:

[0028] The activity of Mesua ferrea L. extract in promoting glucose uptake by adipocytes.

[0029] 1. Experimental materials

[0030] The adipocyte 3T3-L1 was purchased from ATCC in the United States, high-glucose and low-glucose DMEM media were purchased from Shanghai Sangon Biotech Co., Ltd., and the glucose detection kit was purchased from Shanghai Rongsheng Biotech Co., Ltd.

[0031] 2. Experimental methods

[0032] After inducing the differentiation of 3T3-L1 cells into adipocytes, digest the cells and inoculate them into a 96-well plate, and culture overnight. After washing the cells once with low-glucose medium (glucose concentration is 1800 mg / L), add 200 μL of low-glucose medium containing different drugs respectively. The control groups were incubated with DMSO, 100 nM insulin, and berberine respectively, and the test sample groups were incubated with a final concentration of 50 μg / mL. Each sample was set with 3 replicates. After incubating for 24 hours, aspirate 10 μL of cell culture medium, and determine the glucose concentration in the culture solution by the glucose oxidase-peroxidase method. At the same time, add 20 μL of MTS to the remaining cell culture medium, incubate at 37 °C for 2 hours, and measure the absorbance value at 492 nm to determine whether the compound is toxic to adipocytes.

[0033]

[0034] Glucose consumption rate (%) = [(initial glucose concentration - glucose concentration in experimental wells) / initial glucose concentration] × 100%

[0035] 3. Experimental results

[0036] The results of the in vitro glucose consumption activity test are shown in Table 1 and Table 2. The results showed that all extracts except MY131 significantly promoted the glucose uptake activity of adipocytes (p < 0.05). Among them, MY128 had the most obvious promoting effect (73.5% higher than the control group), followed by MY125 (43.9% higher than the control group). MY127 showed obvious cytotoxicity to 3T3-L1 cells at concentrations of 25 μg / mL and 50 μg / mL (cell survival rate < 90%), indicating that its application at high doses needs to be cautious.

[0037] Table 1 Glucose uptake activity of fat cells promoted by other extracts of Mesua ferrea Linn. except MY127

[0038]

[0039] Table 2 Glucose uptake activity of fat cells promoted by Mesua ferrea Linn. extract MY127

[0040]

[0041] Example 5:

[0042] Experiment on the inhibition of α-glucosidase by Mesua ferrea Linn. extract.

[0043] 1. Experimental materials

[0044] α-Glucosidase, substrate 4-Nitrophenyl α-D-glucopyranoside, and positive control quercetin were all purchased from Sigma.

[0045] 2. Experimental method

[0046] The samples (starting from a final concentration of 50 μg / mL and serially diluted), enzyme solution (final concentration 0.025 U / mL), buffer, and substrate (final concentration 1 mM) were added to a 96-well microplate in sequence, mixed well, and three replicates were set. At the same time, a blank control without drug and a quercetin positive control were set. Incubate at 37 °C for 50 min, and measure the OD value at 405 nm with a microplate reader, and calculate the inhibition rate of α-glucosidase activity.

[0047] Inhibition rate (%) = (1 - OD of experimental well 405nm / OD of blank well 405nm ) × 100%

[0048] IC 50 (50% concentration of inhibition) was calculated by the Reed & Muench method.

[0049] 3. Experimental results

[0050] The inhibition test results of α-glucosidase are shown in Table 3. The results show that except for MY129, the extracts of Mesua ferrea L. all exhibited good α-glucosidase inhibitory activity, which was significantly better than that of the positive control quercetin. Among them, MY131 had the best inhibitory activity, with an IC50 value of 3.7 μg / mL. The above results indicate that the extracts of Mesua ferrea L. have significant α-glucosidase inhibitory ability, further verifying its application value in the treatment of diabetes.

[0051] Table 3 IC of the extract of Mesua ferrea L. on the inhibition of α-glucosidase enzyme activity 50 value or inhibition rate

[0052]

[0053]

[0054] Example 6:

[0055] 1. Experimental materials

[0056] Porcine pancreatic lipase (PPL) was purchased from Aladdin Reagent Co., Ltd.; p-nitrophenyl butyrate (p-NPB), Tris, and Orlistat were purchased from Sigma Co., Ltd.; D-PBS was purchased from Hyclone Co., Ltd.; the HMG-CoA Reductase kit was purchased from Sigma-Aldrich.

[0057] 2. Experimental methods

[0058] On a 96-well microplate, the test compound was thoroughly mixed with the PPL solution, with 3 replicates set for each concentration, at 37 °C for 15 min; p-NPB was added and mixed evenly, at 37 °C for 15 min, and the OD value (400 nm / 630 nm) was measured with a microplate reader. The detection wavelength was 400 nm, and the reference wavelength was 630 nm. A blank control and an orlistat positive control were set up simultaneously in the experiment.

[0059] Calculation formula:

[0060] PPL activity inhibition rate (%) = (1 - OD value of the sample / OD value of the experimental control well) × 100%

[0061] 3. Experimental results

[0062] The PPL inhibition test results are shown in Table 4. The results show that the extracts of Mesua ferrea L. MY128, MY129, and MY130 had certain pancreatic lipase inhibitory activity at a concentration of 50 μg / mL.

[0063] Table 4 The effect of the extract of Mesua ferrea L. on PPL inhibition

[0064]

[0065] Formulation Examples

[0066] In the following formulation examples, conventional reagents are selected and formulation preparation is carried out according to existing conventional methods. This application example only demonstrates that at least one of the extracts of Mesua ferrea described in the present invention can be prepared into different formulations, and specific reagents and operations are not specifically defined::

[0067] 1. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is dissolved in a small amount of DMSO, and then injection water is added conventionally, followed by fine filtration and filling and sterilization to prepare an injection solution, and the concentration of the injection solution is 0.5 - 5 mg / mL.

[0068] 2. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is dissolved in a small amount of DMSO, then it is dissolved in sterile injection water, stirred to dissolve, filtered through a sterile suction filter funnel, then sterile fine filtered, dispensed into ampoules, freeze-dried at low temperature and then hermetically sealed aseptically to obtain a powder injection.

[0069] 3. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is added with an excipient in a weight ratio of 9:1 to prepare a powder.

[0070] 4. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is added with an excipient in a weight ratio of 5:1 to granulate and press into tablets.

[0071] 5. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is prepared into an oral liquid according to the conventional oral liquid preparation method.

[0072] 6. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is added with an excipient in a weight ratio of 5:1 to prepare a capsule.

[0073] 7. At least one of the extracts of the pericarp and seeds of Mesua ferrea of the present invention is added with an excipient in a weight ratio of 5:1 to prepare a granule.

Claims

1. An extract of the ironwood tree having hypoglycemic activity, characterized in that The extract is extracted from the outer peel and seeds of the ironwood tree, including ironwood seed extracts MY125, MY126, MY127, ironwood outer peel extracts MY128, MY129, MY130 and MY131; which are prepared by the following method: (a) the dried ironwood seeds are crushed and then extracted with petroleum ether, ethyl acetate and 90% ethanol in turn, and ultrasonically extracted in a water bath at 40°C for 30 min, and then combined and filtered, and the solvent is recovered to obtain extracts of ironwood seed extracts MY125, MY126 and MY127; (b) the dried outer peel of the ironwood tree is crushed and then extracted with petroleum ether, ethyl acetate and 90% ethanol in turn, and ultrasonically extracted in a water bath at 40°C for 30 min, and then combined and filtered, and the solvent is recovered to obtain extracts of ironwood outer peel extracts MY128, MY129 and MY130; (c) the dried outer peel of the ironwood tree is crushed and then extracted with 90% ethanol, and ultrasonically extracted in a water bath at 40°C for 30 min, and then combined and filtered, and the solvent is recovered to obtain extracts of ironwood outer peel extracts MY128, MY129 and MY130. ℃ water bath ultrasonic extraction for 30 minutes, filtration and recovery of the solvent, to obtain the extract of the ironwood peel extract MY131.

2. A pharmaceutical composition comprising a therapeutically effective amount of the ironwood extract with hypoglycemic activity according to claim 1, and a pharmaceutically acceptable carrier.

3. Use of the ironwood extract with hypoglycemic activity according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a drug for treating diabetes.

4. Use of the ironwood extract with hypoglycemic activity according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of hypoglycemic drugs.

5. A botanical medicine for improving or treating diabetes, comprising a therapeutically effective amount of the ironwood extract with hypoglycemic activity according to claim 1 and a pharmaceutically acceptable carrier.

6. A method for preparing a ferrum ferrum extract having hypoglycemic activity, characterized in that The method comprises the following steps: (a) drying the seeds of the ironwood tree, crushing them, sequentially extracting them with petroleum ether, ethyl acetate and 90% ethanol in a water bath at 40°C for 30 min, combining and filtering them respectively, recovering the solvent, and obtaining extracts of the ironwood seed extracts MY125, MY126 and MY127; (b) drying the outer peel of the ironwood tree, crushing them, sequentially extracting them with petroleum ether, ethyl acetate and 90% ethanol in a water bath at 40°C for 30 min, combining and filtering them respectively, recovering the solvent, and obtaining extracts of the ironwood outer peel extracts MY128, MY129 and MY130; (c) drying the outer peel of the ironwood tree, crushing them, sequentially extracting them with 90% ethanol in a water bath at 40°C for 30 min, min, filter and recover the solvent to obtain the extract of the ironwood peel extract MY131; the extract is extracted from the ironwood peel and seeds, including the ironwood seed extracts MY125, MY126, MY127, and the ironwood peel extracts MY128, MY129, MY130 and MY131.

7. The method for preparing the pharmaceutical composition according to claim 2, characterized in that: The method comprises the following steps: firstly obtaining the ironwood extracts MY125, MY126, MY127, MY128, MY129, MY130 and MY131 with hypoglycemic activity by the preparation method described in claim 6; and then adding a pharmaceutically acceptable carrier.

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