阅读说明：本技术 一种黑莓提取物的制备方法及其应用 (Preparation method and application of blackberry extract ) 是由 肖婷 郭正红 骆光佳 张道芳 于 2021-09-14 设计创作，主要内容包括：本发明涉及一种黑莓提取物的制备方法及其应用,所述提取物是将黑莓鲜果冷冻干燥后用甲醇进行提取,再进行浓缩干燥或经聚酰胺树脂纯化后浓缩干燥即得。提取物有效剂量为25-100mg/kg/d,该提取物能够降低酒精性肝损伤的危害,显著降低小鼠血清中AST、ALT、SOD、MDA的水平,同时黑莓提取物能够显著改善酒精诱导的小鼠肝组织肝炎、肝坏死、脂肪肝、肝纤维化等肝损伤,黑莓提取物可以很好地应用于抗酒精性肝损伤的药物、功能饮料及保健食品中。(The invention relates to a preparation method and application of a blackberry extract, wherein the blackberry extract is prepared by freeze-drying fresh blackberries, extracting with methanol, and then concentrating and drying or concentrating and drying after purifying by polyamide resin. The effective dose of the extract is 25-100mg/kg/d, the extract can reduce the harm of alcoholic liver injury, and remarkably reduce the levels of AST, ALT, SOD and MDA in mouse serum, meanwhile, the blackberry extract can remarkably improve liver injury such as alcohol-induced liver tissue hepatitis, liver necrosis, fatty liver, hepatic fibrosis and the like of mice, and the blackberry extract can be well applied to medicines, functional beverages and health-care foods for resisting alcoholic liver injury.)

1. A preparation method of a blackberry extract is characterized by comprising the following steps:

(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 45-52h under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;

(2) precisely weighing a proper amount of freeze-dried powder, adding 60-80% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:4-10g/mL, extracting for 3 times by adopting any one of an ultrasonic method, a reflux method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.1-1.5 for later use;

(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.

2. The method of claim 1, comprising the steps of:

(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;

(2) precisely weighing a proper amount of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 3 times by adopting any one of a reflux method, an ultrasonic method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;

(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.

3. The method according to any one of claims 1 or 2, wherein the ultrasonic method, the reflux method, the dipping method in step 2); wherein the ultrasonic method: the temperature is 40 ℃, the power is 300W, and the time is 90 min; the reflux method comprises the following steps: extracting at 35 deg.C for 90 min; the impregnation method comprises the following steps: the leaching time is 3 h/time.

4. The method according to any one of claims 1 or 2, wherein step 3) further comprises: taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 5-10, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extract complex solution by polyamide chromatography with 30-60 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting eluent until the eluent is colorless, concentrating under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.

5. The blackberry extract prepared by the preparation method according to any one of claims 1 or 2, and is characterized in that the blackberry extract is applied to the preparation of the drug for treating alcoholic liver injury.

6. The blackberry extract of claim 5, wherein the blackberry extract is used for preparing the drug for treating alcoholic fatty liver, liver fibrosis, liver cirrhosis, liver ascites and hepatitis.

7. The blackberry extract according to claim 5, wherein the blackberry extract can be made into granules, tablets, capsules, oral liquids, syrups, mixtures, pills or powders with pharmaceutically acceptable excipients.

8. The extract as claimed in claim 5, wherein the total effective dose of the blackberry extract in resisting alcoholic liver injury is 25-100 mg/kg/d.

9. The blackberry extract of claim 5, wherein the blackberry extract is used in the preparation of health food and functional beverage thereof.

10. The blackberry extract of claim 5, wherein the blackberry extract has a major component of blackberry anthocyanin.

Technical Field

The invention relates to the field of medicines and foods, in particular to a preparation method and application of a blackberry extract.

Background

Alcohol abuse and dependence are important public health problems worldwide. With the continuous improvement of the living standard of people, alcohol consumption people are increased year by year in China, and the damage to nerves and a digestive system caused by alcoholism is increased day by day. The liver is a main organ in the alcohol metabolism process, and long-term drinking can cause Alcoholic liver injury (ALD), such as fatty liver, Alcoholic hepatitis, hepatic fibrosis and liver cirrhosis, and simultaneously can increase the incidence of liver cancer. According to the report of the World Health Organization (WHO), 20 hundred million people in the world drink alcohol, wherein over 750 million people have the phenomenon of alcohol abuse, and excessive drinking is a main cause of liver diseases. Alcoholic liver disease is the second largest type of liver disease in China after infectious liver disease, and the number of patients is increasing at an alarming rate in a state of being younger. Liver disease has become the fifth leading fatal disease worldwide following heart disease and stroke. In recent years, natural products with the advantages of low toxic and side effects, multiple ways and multiple targets are concerned, various food-borne natural products are reported to have the effect of protecting alcoholic liver injury, and the important significance is achieved in finding a medicine, a functional beverage or a health-care food which has definite curative effect and strong activity and is used for resisting alcoholic liver injury.

Blackberry (Blackberry) is a rosaceous rubus plant, named raspberry, and native north america, and is a new fruit which has rapid world development in recent years and integrates nutrition and health care. The fresh fruits are soft and juicy, have unique flavor and rich nutritive value; the blackberry has very high medicinal value, has the effects of regulating metabolic function, delaying senility, eliminating fatigue, improving immunity and the like, particularly has the effects of reducing cholesterol content, preventing and treating heart diseases and resisting cancers (reducing toxic and side effects caused by chemotherapy), and is known as purple life fruit.

Application number CN201711145154.X, invention name is a preparation method and application of novel blackberry powder, and discloses a preparation method: cleaning, namely cleaning the harvested blackberries by using a cleaning agent; crushing and pulping: primarily pressing the cleaned blackberries, then spraying high-pressure steam, rapidly cooling the blackberries to below 5 ℃, and pulping to obtain a first blackberry stock solution; mixing and stirring: adding a composite stabilizer into the blackberry stock solution, uniformly mixing, and then crushing by using a high-speed crusher to obtain a second blackberry stock solution; biological fermentation: fermenting the second blackberry stock solution with yeast to obtain a third blackberry stock solution; and (3) sterile treatment: performing high-temperature instantaneous sterilization, and then cooling to 30-40 ℃ to obtain a fourth blackberry stock solution; and (3) freeze-drying treatment: freeze-drying and pulverizing the fourth blackberry stock solution with a vacuum freeze-drying machine, and sieving with a 100-mesh sieve to obtain novel blackberry powder; also discloses application of the blackberry powder food.

Technical problem with application No. cn201711145154. x: 1) the blackberry stock solution is extracted for four times, so that the operation is complex, the cost is high, and the industrial production is not easy to realize; 2) the biological fermentation easily damages blackberry cells and influences the original components and quality of the blackberry; the effective components of the blackberry are easy to damage by adopting high-temperature instantaneous sterilization; 4) only the application of the blackberry powder food is mentioned, and the application of the blackberry extract in resisting alcoholic liver injury is not mentioned.

The application number CN201510433616.2 is the name of a preparation method of a blackberry extract, and discloses a preparation method of a blackberry extract, wherein the blackberry cleaned is placed in a homogenizer, and an ethanol solution with the weight 5-10 times of that of the blackberry is added for homogenizing and extraction; placing the filtrate after homogenate extraction in an ultrasonic device for ultrasonic extraction; standing the filtrate after ultrasonic extraction at 2-8 ℃, and centrifuging; and (3) performing molecular distillation on the centrifuged filtrate, and separating under the conditions that the feeding speed is 100-200 g/h, the film scraping speed is 300-500 r/min, the distillation temperature is 40-50 ℃, and the distillation pressure is 100-200 Pa to obtain the blackberry extract. The extraction method is a pure physical method, the homogenate extraction method realizes the crushing and extraction of the materials in one step, the ultrasonic extraction method can go deep into the inside of the cells of the materials, and the extraction rate of effective components is greatly improved; can be carried out under mild conditions, thereby avoiding the damage of effective components.

The technical problem of application No. CN201510433616.2 is: 1) directly placing the blackberries into a homogenizer, adding 5-10 times of ethanol solution for extraction, and removing impurities without peracid, so that the phenomena of more impurities, incomplete extraction, low extract yield and the like are easily caused; 2) the preparation of the extract by molecular distillation is costly.

Application number CN201510018395.2, the name of the invention is a blackberry extract and application thereof in preparing liver cell oxidative damage inhibitor, and discloses a blackberry extract, which comprises the following components by mass percent: 58.3 to 72.5 percent of cyanidin-3-glucoside; cyanidin-3-xyloside 11.6% -15.8%; cyanidin-3-oxalyl glucoside 9.8% -14.6%; 3.1 to 6.4 percent of cyanidin-3-malonyl glucoside; 2.7 to 4.1 percent of delphinidin-3-xylose and 0.3 to 0.8 percent of cyanidin-3-arabinoside. The extraction steps are as follows: and (2) carrying out enzymolysis on the fresh blackberry fruits by pepsin and trypsin in sequence, carrying out centrifugal separation to obtain a supernatant, filtering the supernatant by an ultrafiltration membrane, intercepting components with the molecular weight of more than 10kDa, and carrying out vacuum freeze drying on the filtrate to obtain the blackberry extract. The invention also discloses application of the blackberry extract in preparation of an inhibitor for oxidative damage of liver cells.

The technical problem of application No. CN201510018395.2 is: 1) the extraction method is complex, pepsin, trypsin, ultrafiltration membrane filtration and the like are required for extraction, and the cost is high; 2) the extract comprises the following main components: cyanidin-3-glucoside, cyanidin-3-xyloside, cyanidin-3-oxalylglucoside, cyanidin-3-malonylglucoside, delphinidin-3-xyloside and cyanidin-3-arabinoside, with no mention of blackberry anthocyanins in the extract; 3) the extract is mentioned for application in an inhibitor of acrylamide-induced oxidative damage of liver cells, but is not directed to application in an anti-alcoholic liver injury medicament.

Aiming at the problems, aiming at solving the problem of liver injury caused by alcoholism in the current alcohol consumer group, the invention group combines the technical problems existing in the blackberry extraction, and obtains the preparation method of the blackberry extract by a large amount of experimental researches on the influence of an acidifier, an extractant, ultrasonic time, methanol volume fraction, ultrasonic temperature, ultrasonic time and ultrasonic power on the extraction rate of the blackberry anthocyanin in the extraction.

Disclosure of Invention

The invention aims to provide a preparation method of a blackberry extract.

The invention also aims to provide application of the blackberry extract in preparing a medicament for treating alcoholic liver injury.

The preparation method of the blackberry extract comprises the following steps:

(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 45-52h at the freeze drying parameter of-50 ℃, the vacuum degree of 60Pa and the tray temperature of 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;

(2) precisely weighing a proper amount of freeze-dried powder, adding 60-80% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:4-10g/mL, extracting for 3 times by adopting any one of an ultrasonic method, a reflux method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.1-1.5 for later use;

(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.

It is preferable that

The preparation method of the blackberry extract comprises the following steps:

(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;

(2) precisely weighing a proper amount of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 3 times by adopting any one of a reflux method, an ultrasonic method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;

(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.

The ultrasonic method, the reflux method and the dipping method in the step 2) of the invention; wherein the ultrasonic method: the temperature is 40 ℃, the power is 300W, and the time is 90 min; the reflux method comprises the following steps: extracting at 35 deg.C for 90 min; the impregnation method comprises the following steps: the leaching time is 3 h/time.

The step 3) of the invention can also be:

taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 5-10, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extract complex solution by polyamide chromatography with 30-60 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting eluent until the eluent is colorless, concentrating under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.

The blackberry extract disclosed by the invention is applied to the preparation of medicines for treating alcoholic liver injury.

The blackberry extract is applied to the preparation of medicines for treating alcoholic fatty liver, hepatic fibrosis, liver cirrhosis, hepatic ascites and hepatitis.

The medicine can be added with pharmaceutically acceptable auxiliary materials to be prepared into granules, tablets, capsules, oral liquid, syrup, mixture, pills or powder.

The total effective dose of the blackberry extract in resisting alcoholic liver injury is 25-100 mg/kg/d.

The blackberry extract contains blackberry anthocyanin as main ingredient.

The blackberry extract disclosed by the invention is applied to the preparation of health-care food and functional beverage thereof.

The invention has the beneficial effects that:

1. the blackberry extract obtained by the preparation method can effectively reduce the harm of alcoholic liver injury, and compared with a model group, the blackberry extract can remarkably reduce liver index of a mouse and the levels of AST, ALT, SOD and MDA in serum, and H & E staining shows that the blackberry extract can improve cell morphological changes such as tissue vacuolization, edema, inflammation, cell necrosis and the like caused by the alcoholic liver injury of the mouse; the red oil O staining shows that the blackberry extract can improve fat deposition caused by alcoholic liver injury of mice and reduce the area of fat drop halo; the sirius red staining shows that the blackberry extract can improve the extension of collagen fiber in alcoholic liver injury and weaken the degree of hepatic fibrosis caused by alcohol.

2. The parameters of the preparation method of the invention pass through the establishment and analysis experiments of a response surface model, and the Design-Expert 8.0 software is adopted to carry out variance analysis on a regression model to obtain a multivariate quadratic equation model of anthocyanin extraction rate to methanol volume fraction (A), feed-liquid ratio (B), ultrasonic time (C), ultrasonic power (D) and ultrasonic temperature (E), and finally obtain the blackberry anthocyanin extraction rate and factor variables: y (anthocyanin extraction ratio) is 2775.40+46.8A +17.08B +0.16C +0.27D +12.64CE +0.16DE +1356.10a2+668.75B2+597.06C2+772.99D2+1341.82E2, and it can be seen from the analysis of variance table 3 that the significance level P of the model is less than 0.0001, indicating that the regression variance model is significant. In addition, kinetic analysis of blackberry extract degradation was also performed.

3. According to the three-dimensional graph and the contour map of the response surface of the blackberry anthocyanin content of each factor in the experimental result, the trend that the response surface is open downwards and the extraction rate of anthocyanin increases and then decreases along with the change of each factor is shown, and the model has a stable point with the maximum value. Finally, according to the F value and the P value, the regression model can be well fitted with the measured value, so that the model can predict and analyze the extraction process result of the blackberry anthocyanin. According to the steep degree of the response surface, the influence of various factors on the extraction amount of the blackberry anthocyanin is shown in the following sequence: ultrasonic temperature (E) > methanol volume fraction (A) > material-liquid ratio (B) > ultrasonic power (C) > ultrasonic time (D). According to the obtained model, the optimal technological parameters for extracting the blackberry anthocyanin can be predicted to be as follows: 70 percent of methanol acidified by formic acid, the ratio of material to liquid is 1:8, the ultrasonic time is 90min, the ultrasonic temperature is 40 ℃, the ultrasonic power is 300W, and the extraction rate is about 52.00 percent.

4. According to the invention, through determination of the optimal conditions and verification of a regression model, the optimal extraction scheme of the result system model optimization is that the ultrasonic power is 299.81W, the ultrasonic temperature is 37.69 ℃, the methanol volume fraction is 70.99%, the material-liquid ratio is 1:9.58, the ultrasonic time is 89.90min, and the extraction efficiency model value is 52.2467%.

5. According to the invention, through the degradation kinetics analysis of the blackberry anthocyanin, the result shows that the degradation of the blackberry anthocyanin accords with the first-order reaction of the kinetics, and the degradation constant K is increased along with the rise of the temperature and the pH value; t1/2 is reduced along with the increase of temperature and pH value, the activation energy is reduced along with the increase of pH value, the thermal stability is highest when the pH value is 2, which indicates that the degradation energy of the blackberry anthocyanin is the highest when the pH value is approximate to 2, otherwise, the degradation energy is the most stable and the blackberry anthocyanin is not easy to decompose. The experimental result provides a certain experimental basis and theoretical basis for the extraction and the prevention of decomposition of the blackberry anthocyanin in the processes of production, transportation, storage, use and the like in the industries of food, medicine, cosmetics and the like.