CN113616744A

CN113616744A - Preparation method and application of millet whole grain flavone active component

Info

Publication number: CN113616744A
Application number: CN202111019237.0A
Authority: CN
Inventors: 剌晓琴; 刘一志; 李卓玉; 张立超; 李汉卿
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-11-09

Abstract

The invention discloses a preparation method and application of a millet whole grain flavone active component. Pulverizing whole semen Setariae, defatting, ultrasonic extracting with 60% ethanol, mixing filtrates, concentrating, vacuum freeze drying to obtain powder, adding ultrapure water for redissolving, centrifuging to remove impurities, collecting supernatant, adjusting pH to 5.0, and filtering with 0.45 μm ultrafiltration membrane to obtain semen Setariae whole grain flavone crude extract; the crude extract is enriched by D101 type macroporous resin, and is purified by 10 percent and 30 percent ethanol, and then is eluted by 50 percent ethanol to obtain the millet whole grain flavone active component MWGF-50. In vivo and in vitro activity experiments show that: the MWGF-50 can obviously improve the injury of the gastric mucosa epithelial cells induced by alcohol and relieve the acute alcoholic gastric mucosa injury of rats, and has no obvious toxic or side effect on the gastric epithelial cells GES-1 and main organs of the rats. The MWGF-50 has simple preparation process and convenient large-scale production, and can be used for preparing functional foods, health products, medicines and the like for preventing and treating diseases related to alcoholic gastric mucosa injury.

Description

Preparation method and application of millet whole grain flavone active component

Technical Field

The invention relates to preparation of millet whole grain active components, belongs to the technical field of natural product extraction, and particularly belongs to a preparation method and application of millet whole grain flavone active components.

Background

China is a big country for producing and consuming traditional white spirit, and people who drink the white spirit are in the forefront of the world. However, alcohol has a great effect of damaging gastric mucosa, so that the number of the disease related to gastric mucosa damage in China is high. Generally, the gastric mucosa has certain self-healing capacity, and early mild injury does not need medicine intervention, so that the injury of the gastric mucosa caused by drinking is difficult to attract attention of people. However, the gastric mucosa can be repeatedly damaged by long-term drinking, and gradually develops superficial gastritis, gastric erosion, gastric ulcer and even canceration, thereby seriously threatening the physical and mental health of residents in China and bringing great economic burden to families and society. If the components with the functions of nourishing and protecting the stomach in the food can be deeply excavated to guide drinking people to perform targeted dietary intervention in daily life, the further development of diseases related to gastric mucosa injury can be prevented. However, the current dietary nutritional intervention approaches associated with gastric mucosal damage are relatively lacking.

Millet (Setaria italica), also known as millet, belongs to the family annual grass of the family Setaria. Throughout the past, the medical works have the theory of homology of medicine and food, as early as recorded in the compendium of materia medica, millet is named as porridge and food, benefits the dantian, tonifies deficiency and impairment, promotes urination and treats regurgitation and dysentery. The millet is also considered by folks to have good nutritional and health-care effects on patients who are just cured of illness and people who are weak in spleen and stomach, such as pregnant and lying-in women. However, the scientific connotation of the stomach nourishing and protecting efficacy of millet is still lack of research and discussion. Modern nutriology and medical research shows that millet is rich in balanced basic nutrients such as amino acids, fatty acids, dietary fibers, vitamins and the like required by human bodies, is rich in functional active substances such as polyphenol, flavone, active peptide and the like, and has various health care and medical functions such as oxidation resistance, tumor resistance, immunoregulation and the like. Therefore, the active ingredients with the effect of relieving gastric mucosa injury in the foxtail millet are screened, the application of the foxtail millet in the aspects of medicines or health-care foods and the like is facilitated, and the added value of the foxtail millet product is improved.

The flavone is a natural compound with a 2-phenyl chromone structure, and has a plurality of effects of resisting oxidation, resisting cancer, relieving cardiovascular and cerebrovascular diseases and the like. As one of the bioactive substances in the millet, the flavone is distributed in the millet and the millet bran and has high content. However, the preparation of millet whole grain flavone and the application of millet whole grain flavone in the aspect of gastric mucosa protection are not reported yet at present.

Disclosure of Invention

The invention aims to provide a preparation method of a millet whole grain flavone active component and application of the active component in preparing alcoholic gastric mucosa injury protection medicines and functional foods.

The invention provides a preparation method of millet whole grain flavone active components, which comprises the following steps:

s1, crushing the whole millet grains and sieving the whole millet grains by a sieve with 50-80 meshes;

s2, mixing the millet whole grain powder obtained in the S1 according to the weight ratio of 1: putting the mixture into petroleum ether with the boiling range of 60-90 ℃ in a weight-volume ratio of 18-25, stirring for 1h at room temperature, and performing suction filtration;

s3, repeating S2 twice to completely remove lipid components in the whole millet grains, and drying filter residues in a constant-temperature oven at 60 ℃ for about 4 hours to obtain defatted whole millet grain powder;

s4, mixing the defatted whole millet flour of S3 according to the weight ratio of 1: ultrasonic extracting with 55-65% ethanol 15-25 at 45-55 deg.C for 40-50min for 2 times, filtering, mixing the extractive solutions of 2 times, concentrating to 1/5-1/4 of the volume of the original extractive solution, and vacuum freeze drying to obtain lyophilized powder;

s5, redissolving the freeze-dried powder obtained in the S4 by using ultrapure water, centrifuging twice to completely remove impurities such as protein, polysaccharide and the like, then sucking supernatant, adjusting the pH value to 5.0, and further filtering by using a 0.45-micron ultrafiltration membrane to obtain a flavone crude extract, which is named as MWGF;

s6, loading the activated D101 type macroporous resin into a column by a wet method, adding the flavone crude extract obtained in the S5 into the resin column at the flow rate of 5mL/min, and then gradually eluting the macroporous resin by ultrapure water and ethanol with the volume fraction of 10% and 30% in a gradient manner to remove impurities; eluting with 50% ethanol, collecting the eluate, and rotary evaporating at 45-55 deg.C for concentration to obtain active component of semen Setariae whole grain flavone, named as MWGF-50;

s7, adopting sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method to measure MWGF-50 content.

In vivo and in vitro experimental tests prove that the MWGF-50 has good relieving effect on gastric mucosa injury caused by alcohol, and the MWGF-50 has no obvious toxic or side effect on cells and main organs of rats. The MTT method is adopted to detect the cytotoxicity of MWGF-50 on gastric epithelial cells GES-1 and the protective effect on GES-1 under the action of alcohol, and the results show that: after the MWGF-50 with the concentration range of 0-30 mu g/mL respectively acts on the GES-1 cells for 24 hours, 48 hours and 72 hours, the activity of the cells is not obviously inhibited; and when the action concentration of the MWGF-50 reaches 10 mu g/mL, the GES-1 cell death caused by alcohol can be obviously inhibited. Establishing a Wistar rat acute alcoholic gastric mucosa injury model in a mode of intragastric 75% alcohol for 4h, and observing rat stomach tissues through anatomy, wherein the result shows that: the MWGF-50 can effectively relieve the congestion and the injury of the gastric mucosa of the rat caused by the alcohol, and the MWGF-50 with the concentration of 10mg/Kg has no obvious toxic or side effect on the main viscera of the whole body of the rat.

Compared with the prior art, the invention has the beneficial effects that:

the MWGF-50 prepared by the method has the advantages of safe and wide source, simple extraction process and easy large-scale production; can obviously improve the damage of the gastric mucosa caused by alcohol, and has larger development prospect and application value, such as: in the aspect of medicine, the compound can be used for preparing an auxiliary medicine with the gastric mucosa protection function; in the aspect of health products, the health oral liquid with the function of protecting gastric mucosa can be prepared.

Based on the advantages of rich millet resources in China, the invention utilizes the extracted millet whole grain flavone to research and develop novel millet whole grain food and health care products with nutrition and health care functions for people with alcoholic gastric mucosa injury, thereby realizing the high-efficiency utilization of millet resources and the sustainable development of production.

Drawings

FIG. 1 is a photograph showing a sample of MWGF-50 extract

FIG. 2 is a bar graph of the effect of MWGF-50 on GES-1 cell viability

FIG. 3 is a bar graph of the effect of alcohol on GES-1 cell viability

FIG. 4 is a bar graph of the protective effect of MWGF-50 on alcohol-induced GES-1 in gastric epithelial cells

FIG. 5 is a photograph of an HE stained paraffin section showing the effect of MWGF-50 on heart, liver, spleen, lung and kidney of Wistar rat

FIG. 6 is a photograph of a stomach tissue showing the protective effect of MWGF-50 on alcohol-induced injury of the gastric mucosa of Wistar rats

Detailed Description

Example 1

Preparation of MWGF-50:

s1, crushing the whole millet grains and sieving the whole millet grains with a 50-mesh sieve;

s2, mixing the millet whole grain powder obtained in the S1 according to the weight ratio of 1: 20 (W/V is 1 g: 20mL) is placed in petroleum ether with a boiling range of 60-90 ℃ and stirred for 1h at room temperature, and then is filtered;

s3, repeating S2 twice to completely remove lipid components in the whole millet grains, and drying filter residues in a constant-temperature oven at 60 ℃ for 4 hours to obtain defatted whole millet grain powder;

s4, weighing 50g of defatted whole millet flour in S3, placing the defatted whole millet flour in 60% ethanol solution according to the weight-to-volume ratio (W/V is 1 g: 20mL) for ultrasonic extraction for 2 times, each time for 45min, the ultrasonic power is 350W, the reaction temperature is 50 ℃, performing suction filtration, combining 2 times of about 2L of extracting solution, concentrating the extracting solution to 400mL by using a rotary evaporator, freezing the extracting solution at-80 ℃ overnight, and placing the extracting solution in a freeze dryer for 3d to obtain about 5g of freeze-dried powder;

s5, re-dissolving 5g of freeze-dried powder obtained in S4 by using 100mL of ultrapure water, centrifuging twice at 12,000rpm for 15min each time to remove impurities such as protein and polysaccharide, then sucking supernatant, adjusting the pH value to 5.0, and further filtering by using a 0.45-micron ultrafiltration membrane to obtain the millet whole grain flavone crude extract, which is named as MWGF;

s6, taking about 70g of D101 type macroporous resin, placing the resin in 95% ethanol solution, stirring for 30min at room temperature, and enabling the resin to be fully contacted with ethanol. After standing for 24h, the resin was washed with a large amount of distilled water until free of an alcoholic smell. Then 5% hydrochloric acid is used for soaking the resin for 3-4h, and distilled water is used for washing the resin to be neutral. Then continuously soaking the mixture for 3 to 4 hours by using a 5 percent NaOH solution, washing the mixture to be neutral by using distilled water, and storing the mixture for later use at 4 ℃;

s7, filling the activated D101 type macroporous resin in the S6 into a column by a wet method, adding all the crude flavone extracting solution obtained in the S5 into the resin column at the flow rate of 5mL/min, eluting the macroporous resin by 300mL of 200mL of ultrapure water, 10% of ethanol with volume fraction and 30% of ethanol respectively in sequence, and removing impurities, wherein the flow rate is set to be 5 mL/min; eluting with 300mL of 50% ethanol at flow rate of 5mL/min, collecting the eluate, and rotary evaporating at 60 deg.C to volume of 4mL to obtain active component of semen Setariae whole grain flavone, named as MWGF-50, which is yellow brown (shown in figure 1);

s8, adopting sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method to determine the MWGF-50 flavone content obtained in S7. Taking 0.20mg/mL rutin as a standard substance, accurately sucking 0.20mg/mL rutin standard solution 0, 0.05, 0.10, 0.20, 0.30, 0.40 and 0.50mL, placing in 4.00mL EP tubes, then respectively adding 5% sodium nitrite solution 0.10mL into each tube, shaking up and standing for 6 min; then 0.10mL of 10 percent aluminum nitrate solution is added, and the mixture is shaken up and kept stand for 6 min; and finally, adding 0.50mL of 4% sodium hydroxide solution, diluting to 2.5mL with 50% ethanol, shaking up, standing for 10min, and measuring the absorbance value at 510 nm. And then taking the absorbance value as a horizontal coordinate and the rutin content as a vertical coordinate to obtain a standard curve linear regression equation between the absorbance (x) and the rutin content (y) as follows: y 214.86x-1.67, R²0.999. As a result, it was found that 50g of the defatted whole millet flour had an MWGF-50 flavone content of about 6.24 mg.

Example 2

Collecting stomach epithelial cell GES-1 in logarithmic growth phase, making into cell suspension, adjusting cell density to 3000/100 μ L/well by counting, inoculating into 96-well plate, and culturing at 37 deg.C with 5% CO₂Cell culture chamber ofAfter the culture at night, discarding the old culture medium, adding MWGF-50 with different doses to make the final concentrations respectively 0, 1, 5, 10, 15, 20 and 30 mug/mL, setting 5 multiple wells for each concentration, and continuing to incubate for 24h, 48h and 72 h; old medium was discarded, washed 2 times with PBS, and each well was replaced with 200. mu.L of fresh complete medium, and 20. mu.L of 5.0mg/mL MTT solution was added and incubation continued for 4 h. The culture medium was discarded, 100. mu.L of DMSO was added to each well, the mixture was shaken and mixed for 10min on a shaker, and the absorbance of each well was measured at a wavelength of 570 nm. The effect of MWGF-50 on the viability of GES-1 cells at different concentrations over time was calculated, using the control group as 100% viability (as shown in FIG. 2).

The effect of different concentrations (0, 100, 200, 400, 800, 1000mM) of alcohol on GES-1 cells after 6h and 12h on their cell viability was determined in the same manner as described above (see FIG. 3).

The same method as described above was used to further measure the effect of MWGF-50 on GES-1 cells at different concentrations (0, 1, 5, 10, 15, 20, 30. mu.g/mL) for 48h, and the effect of MWGF-50 on the reduction in alcohol-induced GES-1 cell viability after further treatment with 1000mM alcohol for 12h (FIG. 4).

The result of the in vitro gastric epithelial cell activity test of the millet whole grain flavone active component MWGF-50 obtained by the invention shows that: the MWGF-50 has obvious improvement effect on the activity reduction of gastric epithelial cells caused by alcohol and has no obvious toxic or side effect on the cells.

Example 3

Male Wistar rats are fed adaptively for 1 week in an animal room with relative humidity of 50% -60% and temperature of 23-25 ℃ and are given normal diet. One week later, rats were randomly divided into a control group, a single MWGF-50 gavage group, a model group, an MWGF-50 intervention group, and a rebamipide positive drug group, 10 per group. The gavage treatment was performed at 9 am every day, wherein rats in the control group and the model group were administered with physiological saline (1mL/100g), the MWGF-50 group alone was administered with 10mg/Kg of MWGF-50, the MWGF-50 intervention group was administered with MWGF-50 at different doses (10 mg/Kg at the high dose and 5mg/Kg at the low dose), the positive drug group was administered with rebamipide (30 mg/Kg at the last 6 days), and all rats in the groups were continuously administered with gavage for 3 weeks. Rats were fasted without water deprivation the last night of dosing. The following day, the model group and MWGF-50 intervention group were gazed with 75% alcohol (dose 10 mL/Kg). After 4h, rats were sacrificed by cervical dislocation after anesthesia with 10% chloral hydrate.

The main organs of the rats in the control group and the single MWGF-50 gastric lavage group were obtained by dissection, and the toxic effects on the heart, liver, spleen, lung and kidney of the rats after MWGF-50 gastric lavage were examined (as shown in FIG. 5).

The stomach was opened and removed, the stomach was cut along the greater curvature of the stomach, the contents of the gastric mucosa were washed clean with pre-cooled physiological saline and then blotted dry with filter paper, and the damage of the gastric mucosa was recorded by photographing (as shown in fig. 6).

The results of the in vivo experiments on the active component MWGF-50 of the millet whole grain flavone, which is obtained by the invention, show that: the rats in the control group and the rats subjected to single intragastric administration of MWGF-50 have smooth gastric mucosa surfaces, complete fold regularity and more mucus, and do not have erosion and bleeding; the model group rats have gastrorrhoea and edema of gastric mucosa, and can be seen with various linear or circular erosions with different sizes, flat and shallow plica and even defects, and less mucus on the surface of mucosa; after the MWGF-50 is used for dry preparation, the bleeding and erosion degree of the gastric mucosa of the rat is obviously reduced compared with a model group, the plica of the mucosa is more complete and smooth, more mucus is contained, the effect is best with high dosage, and the effect is similar to that of the positive medicament rebamipide. HE staining of rat major organs showed: compared with the control group, the MWGF-50 has no obvious influence on the tissue structure and color of the heart, the liver, the spleen, the lung and the kidney of the rat, and shows that the MWGF-50 has no toxic or side effect on the main organs of the rat.

Claims

1. A preparation method of millet whole grain flavone active components is characterized by comprising the following steps:

s5, redissolving the freeze-dried powder obtained in the S4 by using ultrapure water, centrifuging twice to remove impurities, then sucking supernatant liquid, adjusting the pH value to 5.0, and further filtering by using a 0.45-micron ultrafiltration membrane to obtain a flavone crude extract;

s6, loading the activated D101 type macroporous resin into a column by a wet method, adding the flavone crude extract obtained in the S5 into the resin column at the flow rate of 5mL/min, and then gradually eluting the macroporous resin by ultrapure water and ethanol with the volume fraction of 10% and 30% in a gradient manner to remove impurities; eluting with 50% ethanol, collecting the eluate, and rotary evaporating at 45-55 deg.C for concentration to obtain semen Setariae whole grain flavone active component.

2. The method according to claim 1, wherein the freeze-drying conditions are freezing at-80 ℃ overnight and then freezing in a vacuum freeze-dryer for 3d at S4.

3. The method according to claim 1, wherein in S5, the centrifugation speed is 12,000rpm and the centrifugation time is 15 min.

4. The method according to claim 1, wherein in S6, the elution volume of the ultrapure water is 2 times of the sample volume of the crude flavone extract, and the elution volume of the ethanol is 3 times of the sample volume of the crude flavone extract.

5. The method of claim 1, wherein in S6, the elution flow rate of the coarse extract of millet whole grain flavonoids in the D101 macroporous resin is 5 mL/min.

6. The millet whole grain flavone active ingredient prepared according to any one of claims 1 to 5.

7. The use of the active component of millet whole grain flavonoids according to claim 6 in the preparation of drugs for the protection of alcoholic gastric mucosa injury.

8. The use of the active component of millet whole grain flavonoids according to claim 6 in the preparation of alcoholic gastric mucosa injury protection health food.