CN114366760A - Application of Artemisia annua polyphenols in the preparation of medicaments for the treatment of diabetes and preparation method thereof - Google Patents

Abstract

The present invention relates to the technical field of marine biology, in particular to the application of Artemisia annua polyphenols in the preparation of medicines for the treatment of diabetes and a preparation method thereof. The present invention finds that Artemisia annua polyphenols have good hypoglycemic effect, and therefore provides the method for preparing medicines for the treatment of diabetes. Application in Artemisia annua, and further gave the extraction method of Artemisia japonica polyphenols.

Description

Application of Artemisia annua polyphenols in the preparation of medicaments for the treatment of diabetes and preparation method thereof

technical field

The invention relates to the technical field of marine biology, in particular to the application of Artemisia annua polyphenols in the preparation of medicines for treating diabetes and a preparation method thereof.

Background technique

The research and application of seaweed polyphenols need to be based on the extraction and separation technology of polyphenols. Therefore, the effective extraction and separation process and analysis methods of seaweed polyphenols are indispensable links in the research of polyphenols. The general production method of seaweed polyphenols uses seaweed as raw material, and adopts the solvent extraction method, that is, firstly, the seaweed powder is mixed with the extraction solvent, extracted at high temperature, and immediately after the extraction is completed, it is filtered while hot under reduced pressure, and extracted in this way for 2-3 times. The product is obtained by drying and pulverizing. This method will destroy the seaweed polyphenols by heating, and has the disadvantages of long time, high temperature, low extraction rate, low extract purity, etc., which is not conducive to industrial production.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a new marine plant extract, which can be used for the treatment and prevention of diabetes, in view of the deficiencies of the prior art, which is further purified by using a macroporous resin.

In order to realize the purpose of the present invention, the following technical means are specifically adopted:

Application of Artemisia annua polyphenols in the preparation of medicaments for the treatment of diabetes.

Application of Artemisia annua polyphenols in the preparation of medicines with inhibitory effect on α-glucosidase.

The preparation method of the aforementioned Artemisia japonica polyphenol, described Artemisia japonica polyphenol is prepared by the following method:

(1) Artemisia seeds are freeze-dried for 30 to 50 hours, pulverized to obtain Artemisia seeds powder;

(2) According to the g/ml ratio of material to liquid, adding an ethanol aqueous solution with a volume concentration of 40% to 90% to Artemisia japonica powder, mixing and soaking;

(3) Ultrasonic extraction of Artemisia japonica polyphenols, extraction temperature is 40～60℃;

(4) repeat step (3) for extraction 1 to 3 times, and combine the supernatant;

(5) Evaporating and concentrating the supernatant at 40-50° C. to obtain a crude extract of Artemisia japonica polyphenol.

Preferably, the ultrasonic power of the step (3) is 50-200 W, the ultrasonic frequency is 20-80 kHz, and the ultrasonic time is 40-60 min.

Preferably, in the step (5), the crude extract is further purified with a macroporous resin, and then vacuum freeze-dried to obtain the total polyphenols of Artemisia annua.

beneficial effect

1, the present invention selects ethanol as extracting solution when extracting Artemisia japonica polyphenols, which is not only conducive to the dissolution of Artemisia japonica polyphenols, and ethanol has a protective effect on Artemisia japonica Polyphenols, making Artemisia japonica Polyphenols not easy to be oxidized, so it is not necessary to Adding antioxidants in addition reduces the process and saves the cost;

2. Vacuum freeze-drying can not only maintain the quality stability of Artemisia japonica polyphenol products during the drying process, but also effectively remove the taste of residual organic solvents, and the drying and deodorizing effect is good;

3. Further purification by macroporous resin to improve the purity of Artemisia japonica polyphenols:

4. The extraction method of the present invention overcomes the adverse effects of traditional extraction of polyphenols such as long time, high temperature, and low extraction rate, is simpler and saves time, and the extracted Artemisia japonica polyphenol product has a higher content.

5. The hypoglycemic effect of Artemisia japonica polyphenols was verified by in vivo and in vitro methods.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The extraction experiment of Artemisia japonica polyphenols is as follows:

Example 1

(1) Fresh sea wormwood seeds are freeze-dried for 40 hours and cut into pieces. Then put it into a pulverizer and pass through a 60-mesh sieve to obtain Artemisia japonica powder;

(2) Accurately weigh a certain amount of Artemisia japonica powder, add a 50% ethanol aqueous solution with a volume concentration of 1:40 (w/v) according to the material-to-liquid ratio, and fully mix and soak;

(3) Extracting polyphenols with a numerically controlled ultrasonic cleaner, the ultrasonic power is 100W, the ultrasonic frequency is 40kHZ, the extraction time is 40～60min, and the extraction temperature is 40～60℃;

(4) Evaporating and concentrating the supernatant on a rotary evaporator at 40-50° C. to obtain Artemisia japonica polyphenol extract.

(5) The polyphenol content was determined by GB/T8313-2008 "Folin Phenol Reagent Colorimetric Method": 5.50mg/g dry seaweed.

Implementation Example 2

(1) Fresh sea wormwood seeds are freeze-dried for 40 hours and cut into pieces. Then put it into a pulverizer and pass through a 60-mesh sieve to obtain Artemisia japonica powder;

(2) Accurately weigh a certain amount of Artemisia japonica powder, add a 50% ethanol aqueous solution with a volume concentration of 1:40 (w/v) according to the material-to-liquid ratio, and fully mix and soak;

(3) Extract polyphenols with a numerically controlled ultrasonic cleaner, the ultrasonic power is 100W, the ultrasonic frequency is 40kHZ, the extraction time is 40min, and the extraction temperature is 60°C;

(4) Repeat the extraction once, and combine the supernatant;

(5) Evaporating and concentrating the supernatant on a rotary evaporator at 40-50° C., and then vacuum freeze-drying to obtain a crude extract of Artemisia japonica polyphenol.

(6) The polyphenol content was determined by GB/T8313-2008 "Folin Phenol Reagent Colorimetric Method": 6.24mg/g dry seaweed.

Implementation Example 3

(1) Fresh sea wormwood seeds are freeze-dried for 40 hours and cut into pieces. Then put it into a pulverizer and pass through a 60-mesh sieve to obtain Artemisia japonica powder;

(2) Accurately weigh a certain amount of Artemisia japonica powder, add a 50% ethanol aqueous solution with a volume concentration of 1:40 (w/v) according to the material-to-liquid ratio, and fully mix and soak;

(3) Extracting polyphenols with a numerically controlled ultrasonic cleaner, the ultrasonic power is 100W, the ultrasonic frequency is 40kHZ, the extraction time is 40min, and the extraction temperature is 60°C;

(4) repeat the extraction 2 times, merge the supernatant;

(5) Evaporating and concentrating the supernatant on a rotary evaporator at 40-50° C. to obtain Artemisia japonica polyphenol extract.

(6) The polyphenol content was determined by GB/T8313-2008 "Folin Phenol Reagent Colorimetric Method": 6.17mg/g dry seaweed.

Example 4

(1) Fresh sea wormwood seeds are freeze-dried for 40 hours and cut into pieces. Then put it into a pulverizer and pass through a 60-mesh sieve to obtain Artemisia japonica powder;

(2) Accurately weigh a certain amount of Artemisia japonica powder, add a 50% ethanol aqueous solution with a volume concentration of 1:35 (w/v) according to the material-to-liquid ratio, and fully mix and soak;

(3) Extracting polyphenols with a numerically controlled ultrasonic cleaner, the ultrasonic power is 100W, the ultrasonic frequency is 40kHZ, the extraction time is 40min, and the extraction temperature is 60°C;

(4) repeat the extraction 2 times, merge the supernatant;

(5) Evaporating and concentrating the supernatant on a rotary evaporator at 40-50° C. to obtain Artemisia japonica polyphenol extract.

(6) The polyphenol content was determined by GB/T8313-2008 "Folin Phenol Reagent Colorimetric Method": 7.57mg/g dry seaweed.

(7) Purify with LS-305A macroporous resin dynamic adsorption, the operation step is 0.5mg/ml Artemisia japonica polyphenol extract 50ml, the sample flow rate is 0.5BV/h, after the sample is finished, let stand for 1 hour, use 1BV water , 1BV 5% ethanol to remove impurities, and eluted with 2.5BV 50% ethanol. The eluate was vacuum freeze-dried to obtain Artemisia japonica polyphenol extract.

Example 5

Example 4 Experiment on the inhibition of α-glucosidase by the Artemisia japonica polyphenol extract obtained by extraction:

Alpha-glucosidase is an important carbohydrate hydrolase in human intestinal epithelial brush progenitor cells, which promotes the digestion and absorption of carbohydrates by hydrolyzing oligosaccharides or disaccharides into monosaccharides. The screening model used p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate to establish the reaction system.

The prepared samples (the polyphenol extract of Artemisia japonica and its positive reference substance were prepared separately) with the solution concentration of 0.1, 0.2, 0.4, 0.8, 1.0 mg/ml, add 50 μL of sample solution for each concentration, and set 3 parallels for each concentration, take 50 μL of each sample solution. Add 50 μL of 0.1 U/mL α-glucosidase and 120 μL of PBS buffer with pH 6.8 to the sample solution of the concentration, respectively, and mix well. After 10 min of reaction at 37 °C, 50 μL of 5.0 mmol/L PNPG was added respectively, and then added after 10 min of reaction at 37 °C. The reaction was terminated with 100 μL of 0.2 mol/L Na ₂ CO ₃ , and the absorbance was finally measured at 405 nm (A). Acarbose was used as the positive control substance, and the sample background control group (without enzyme) and blank control group (without sample) under the same system were set at the same time. The effects of different concentrations of the sample solution on the inhibitory activity of α-glucosidase were determined, and the inhibition rate was calculated. The Origin8.0 software was used to plot the inhibition rate against the concentration and fit the regression equation to obtain the polyphenols of Artemisia annua and Acarba respectively. 50% inhibitory mass concentration (IC ₅₀ ) of sugars.

Inhibition rate (%)=[A _blank- (A _sample -A _background )]*100%/A _blank

In the formula: A _blank is the absorbance value of the blank control group; A _sample is the absorbance value of Artemisia japonica polyphenol or acarbose group; A _background is the absorbance value of the sample background control group.

Experimental results:

The enzyme activity inhibition results of the sample group and the control group are shown in Table 1. Compared with the control group, Artemisia annua polyphenols also significantly inhibited the activity of α-glucosidase.

Table 1 Effects of Artemisia japonica extract and acarbose on α-glucosidase activity

sample Half inhibitory mass concentration (IC₅₀)/(mg·mL-1) Artemisia Seed Polyphenols 0.107 Acarbose 0.091

Example 6

The effect experiment of the Artemisia japonica polyphenol extract obtained by embodiment 4 on type 2 diabetic mice:

Experimental materials: male mice, mouse feed, Artemisia japonica polyphenols

experimental method:

Animal grouping: Healthy mice were randomly divided into 5 groups, including blank control group, model group, positive control group (acarbose), high-dose group of Artemisia annua polyphenols, and low-dose group of Artemisia annua polyphenols, 10 mice in each group .

Diabetes model establishment: After the mice were purchased, they were kept adaptively for 1 week, and after fasting for 12 hours, 10 healthy mice were randomly selected to be injected with normal saline intraperitoneally as a blank control group; the remaining healthy mice were intraperitoneally injected with 150 mg/kg The alloxan solution (currently used and prepared). After 72 hours, mice with a blood glucose value greater than or equal to 11.1 mmol/L were selected as successful model mice.

Administration: The blank control group and the model group were given normal saline; the positive control group was given acarbose tablet solution 25 mg/kg; the sample group was given different doses of Artemisia japonica extract suspension, 30 and 60 mg/kg, respectively. low-dose and high-dose groups. After 4 weeks of administration, the mice in each group were recorded to measure the fasting blood glucose.

Table 2 Effects of Artemisia japonica extract on fasting blood glucose of tetrahydropyrimidine model mice (`X±S, n=10)

Compared with the model group, the fasting blood glucose level of the animals in the blank group was significantly lower than that in the model group (***P<0.001), indicating that the hyperglycemia model was successfully established. Compared with the model group, the blood glucose of each administration group was significantly decreased after administration (***P<0.001), indicating that the hypoglycemic effect of each extract was obvious.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims (5)

1. The application of Artemisia annua polyphenols in the preparation of medicines for treating diabetes. 2. The application of Artemisia annua polyphenols in the preparation of medicines with inhibitory effect on α-glucosidase. 3. the preparation method of the artemisia seed polyphenol described in any one of claim 1～2, is characterized in that, described artemisia seed polyphenol is prepared by the following method: (1) Artemisia seeds are freeze-dried for 30 to 50 hours, pulverized to obtain Artemisia seeds powder; (2) According to the g/ml ratio of material to liquid, adding an ethanol aqueous solution with a volume concentration of 40% to 90% to Artemisia japonica powder, mixing and soaking; (3) Ultrasonic extraction of Artemisia japonica polyphenols, extraction temperature is 40～60℃; (4) repeat step (3) for extraction 1 to 3 times, and combine the supernatant; (5) Evaporating and concentrating the supernatant at 40-50° C. to obtain a crude extract of Artemisia japonica polyphenol. 4. The preparation method according to claim 3, wherein the ultrasonic power of the step (3) is 50-200W, the ultrasonic frequency is 20-80kHZ, and the ultrasonic time is 40-60min. 5. The preparation method according to claim 3, characterized in that, in the step (5), the crude extract is further purified with a macroporous resin, and then vacuum freeze-dried to obtain total polyphenols of Artemisia annua.