Preparation and application of heptadecanoic acid with hypoglycemic effect

Abstract

The present invention discloses a preparation method and application of heptadecanoic acid with hypoglycemic effect, belonging to the field of biology and medicine. The present invention uses Grifola frondosa as a raw material, and obtains heptadecanoic acid by two-phase solvent extraction and silica gel column chromatography separation. The present invention has the advantages of simple method, high product purity and high yield. After testing, the purity of the heptadecanoic acid prepared by the present invention is greater than 95%, the yield is 3.25%, and its inhibitory ability (IC 50 value is 106.91 μg / mL) is stronger than acarbose (IC 50 value is 216.42 μg / mL), which can be used as a potential active ingredient for lowering blood sugar and is conducive to industrial application.

Description

Technical Field

[0001] The invention belongs to the field of biology and medicine, and specifically relates to a preparation method and application of heptadecanoic acid with blood sugar lowering effect. Background Art

[0002] According to data released by the International Diabetes Federation, the number of diabetes patients worldwide reached 537 million in 2021, of which type 2 diabetes accounted for more than 90%. Type 2 diabetes is a chronic metabolic disorder characterized by high blood sugar levels. Persistent high blood sugar can cause a variety of complications that endanger health. Controlling and lowering blood sugar levels is the only way to treat diabetes. Although conventional drugs currently used to treat diabetes can effectively control blood sugar levels, their side effects on the patient's intestines cannot be underestimated, and they are expensive, which brings a great burden to patients. The absorption of carbohydrates in the human body depends on α-glucosidase. Inhibiting the activity of α-glucosidase can slow down the production and absorption of glucose, reduce postprandial blood sugar peaks, and adjust blood sugar levels. Therefore, it is urgent to find a safe and effective α-glucosidase inhibitor.

[0003] Grifola frondosa Grifola frondosa ) belongs to the Polyporaceae family, the genus Grifola frondosa, also known as "Maitake mushroom", is a rare medicinal and edible fungus. It has a fresh and fragrant smell, crisp and tender meat, rich in nutrients, rich in protein, amino acids and vitamins. In addition to its rich nutritional value, Grifola frondosa also has high medicinal value, such as lowering blood sugar and blood lipids, improving lipid metabolism, regulating immune function, anti-tumor, anti-oxidation, etc. At present, there are few studies on the chemical composition of Grifola frondosa, and there are no reports on the preparation of heptadecanoic acid in Grifola frondosa and its hypoglycemic activity. Summary of the invention

[0004] In order to overcome the deficiencies and shortcomings of the prior art, the object of the present invention is to provide a method for preparing heptadecanoic acid with simple procedures, high purity, high yield and hypoglycemic effect, which is of great significance for developing new drugs and improving the commercial value of Grifola frondosa.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing heptadecanoic acid having a hypoglycemic effect comprises the following steps:

[0007] (1) extracting the coarse powder of Grifola frondosa with 70 vol% ethanol solution, concentrating the extract, extracting with ethyl acetate, concentrating the extract and drying it to obtain a Grifola frondosa extract;

[0008] (2) Using silica gel column chromatography, the extract of Grifola frondosa was dissolved in a small amount of dichloromethane for loading, and dichloromethane and methanol were used as eluents for elution. The Rf The eluted fraction with a pH value of 0.79 was concentrated and dried to obtain heptadecanoic acid.

[0009] Furthermore, the mass volume ratio of the Grifola frondosa crude powder to the ethanol solution is 1:10-1:30 g / mL.

[0010] Furthermore, the extraction adopts ultrasonic extraction method, and its ultrasonic frequency is 20-40KHZ, the temperature is 40-60°C, the time is 1-3h, and the number of times is 2-4 times.

[0011] Furthermore, the silica gel used in the silica gel column chromatography has a mesh size of 200-400 meshes, the mass ratio of the silica gel to the Grifola frondosa extract is 80:1-120:1, and the volume ratio of dichloromethane to methanol in the eluent is 10:1.

[0012] Furthermore, the developing solvent of the thin layer chromatography is prepared by mixing dichloromethane and methanol in a volume ratio of 7:1.

[0013] Furthermore, the concentration in the operation adopts reduced pressure concentration at a temperature of 40-60° C., and the drying adopts vacuum freeze drying.

[0014] Furthermore, the purity of the obtained heptadecanoic acid is greater than 95%.

[0015] The prepared heptadecanoic acid has good blood sugar lowering function and can be used to prepare medicines or health products with blood sugar lowering effect.

[0016] The beneficial effects of the present invention are:

[0017] (1) The present invention adopts a separation and purification technology combining solvent extraction and column chromatography, which is a preparation method with low cost, simple procedure, high sample yield and suitable for industrial production.

[0018] (2) The heptadecanoic acid prepared by the present invention has high purity, good hypoglycemic effect, and the inhibitory ability of α-glucosidase (IC 50 value is 106.91 μg / mL) is stronger than acarbose (IC 50 value is 216.42μg / mL), which can be used as a potential hypoglycemic drug, and also provide theoretical support for improving the commercial value of Grifola frondosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the nuclear magnetic resonance spectrum of heptadecanoic acid prepared in Example, wherein A is 1 H-NMR spectrum, B is ten 13 C-NMR spectrum;

[0020] Figure 2This is a purity analysis chart of heptadecanoic acid prepared in Example;

[0021] Figure 3 This is a graph showing the inhibition of the activity of heptadecanoic acid prepared in the example on α-glucosidase. DETAILED DESCRIPTION

[0022] A method for preparing heptadecanoic acid having a hypoglycemic effect comprises the following steps:

[0023] (1) Take the coarse powder of Grifola frondosa, add 70 vol% ethanol solution at a mass volume ratio of 1:10-1:30 g / mL for ultrasonic extraction, the ultrasonic frequency is 20-40KHZ, the temperature is 40-60°C, the time is 1-3h, the number of times is 2-4 times, then concentrate the extract, extract it with ethyl acetate for multiple times, then combine the extracts, concentrate under reduced pressure at 40-60°C, and freeze-dry in vacuum to obtain Grifola frondosa extract;

[0024] (2) A silica gel chromatography column (column volume of 250-350 mL) was prepared using silica gel with a mesh size of 200-400 and a mass ratio of 1:80-1:120 to the Grifola frondosa extract. The Grifola frondosa extract was then dissolved in a small amount of dichloromethane for loading, and eluted with dichloromethane / methanol = 1:10 (v / v) as an eluent. The column was identified by thin layer chromatography (dichloromethane and methanol were mixed in a volume ratio of 7:1 as a developing solvent) and R f The eluted fraction with a pH value of 0.79 was concentrated under reduced pressure at 40-60°C and freeze-dried in vacuum to obtain heptadecanoic acid.

[0025] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Example

[0026] A method for preparing heptadecanoic acid having a hypoglycemic effect comprises the following steps:

[0027] (1) Take 100 g of Grifola frondosa crude powder, add 1.5 L of 70 vol % ethanol solution, and ultrasonicate twice at an ultrasonic frequency of 25 kHz and a temperature of 55 °C for 1 h each time. After the extract is concentrated, it is extracted with ethyl acetate three times, 500 mL each time. The extracts are combined, concentrated under reduced pressure at 45 °C, and then freeze-dried in vacuum to obtain 32.53 g of Grifola frondosa extract.

[0028] (2) Take 100 g of 200-300 mesh silica gel and load it into the chromatographic column. Add 1 g of Grifola frondosa extract dissolved in dichloromethane and elute with 300 mL of dichloromethane / methanol mixture (10:1, v / v) as eluent, ensuring the flow rate is 1 mL / min. Simultaneously, dichloromethane and methanol (7:1, v / v) are used as developing solvents. The eluate is identified by thin layer chromatography and the R f The eluted fraction with a value of 0.79 was concentrated under reduced pressure at 45 °C and then freeze-dried in vacuum to obtain 0.10 g of the product, which was heptadecanoic acid ( ), with a purity greater than 95% and a yield of approximately 3.25%.

[0029] Figure 1 is the NMR spectrum of the obtained product. As shown in the figure, 1 H-NMR DMSO- d 6,400 MHz) δ H : 2.15(2H, t, J = 7.6 Hz), 1.46 (2H, m), 1.22 (26H, m), 0.83 (3H, t, J = 7.2 Hz). 13 C-NMR (DMSO- d 6,100 MHz) δ C : 174.4 (C-1), 33.7 (C-2), 31.5 (C-3), 29.3 (C-4), 29.3 (C-5), 29.3 (C-6), 29.3 (C-7), 29.3 (C-8), 29.3 (C-9 ), 29.3 (C-10), 29.1 (C-11), 28.9 (C-12), 28.8 (C-13), 28.7 (C-14), 24.6 (C-15), 22.3 (C-16), 13.9 (C-17). The above data are basically consistent with the literature report (Sun Dongdong, Li Xiang, Chen Jianwei, et al. Study on the chemical components of Radix Isatidis [J]. West China Journal of Pharmaceutical Sciences, 2007, 22 (5): 487-488). Therefore, it was identified as heptadecanoic acid with a molecular formula of C 17 H 34 O2.

[0030] Application Examples

[0031] Phosphate buffer (0.1mmol / L, pH6.8) was used to prepare different concentration gradients of heptadecanoic acid solutions (62.5, 125, 250, 500, 1000 μg / ml), 30 μL was taken and placed in a 96-well plate, 30 μL of α-glucosidase (0.2U / mL) was added, and the plate was left to stand at 37°C for 10 min, followed by 30 μL of substrate P-NPG (5mmol / L), incubated at 37°C for 15 min, and finally 90 μL of Na2CO3 solution (0.1mol / L) was added to terminate the reaction, the absorbance was measured at 405 nm, and the inhibition rate of α-glucosidase was calculated (the inhibition rate calculation formula is as follows). The same concentration of acarbose was used as a positive control, the sample solution was replaced by phosphate buffer as a control group, and α-glucosidase was replaced by phosphate buffer as a background group.

[0032] , where A1 is the absorbance of the experimental group; A2 is the absorbance of the background group; A3 is the absorbance of the control group.

[0033] Figure 3 The figure shows the inhibition of α-glucosidase activity by heptadecanoic acid. It can be seen from the figure that heptadecanoic acid has a good inhibitory effect on α-glucosidase, and the inhibitory effect increases in a dose-dependent manner. After calculation, the IC 50 The value is 106.91 μg / mL, and the IC of acarbose 50 The value was 216.42 μg / mL, indicating that the inhibitory effect of heptadecanoic acid on α-glucosidase was stronger than that of acarbose, proving that it has a better hypoglycemic effect.

[0034] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing heptadecanoic acid having a hypoglycemic effect, characterized in that: The following steps are involved: (1) extracting the coarse powder of Grifola frondosa with 70 vol% ethanol solution, concentrating the extract, extracting with ethyl acetate, concentrating the extract and drying it to obtain a Grifola frondosa extract; (2) Using silica gel column chromatography, the extract of Grifola frondosa was dissolved in a small amount of dichloromethane for loading, and dichloromethane and methanol were used as eluents for elution. The R f The eluted fraction with a value of 0.79 is concentrated and dried to obtain heptadecanoic acid; The silica gel used in the silica gel column chromatography has a mesh size of 200-400 meshes, a mass ratio of silica gel to Grifola frondosa extract of 80:1-120:1, and a volume ratio of dichloromethane to methanol in the eluent of 10:1; The purity of the obtained heptadecanoic acid is greater than 95%.

2. The method for preparing heptadecanoic acid having a hypoglycemic effect according to claim 1, characterized in that: The mass volume ratio of the Grifola frondosa crude powder to the ethanol solution used is 1:10-1:30 g / mL.

3. The method for preparing heptadecanoic acid having a hypoglycemic effect according to claim 1, characterized in that: The extraction adopts ultrasonic extraction method, and its ultrasonic frequency is 20-40KHZ, the temperature is 40-60°C, the time is 1-3h, and the number of times is 2-4 times.

4. The method for preparing heptadecanoic acid having a hypoglycemic effect according to claim 1, characterized in that: The developing solvent of the thin layer chromatography is prepared by mixing dichloromethane and methanol in a volume ratio of 7:1.

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