A prickly pear dietary fiber combined with polyphenols exhibiting significant hypoglycemic activity, its extraction method, and its application.

Abstract

This invention discloses a prickly pear dietary fiber-bound polyphenol with significant hypoglycemic activity, its extraction method, and its applications. The extraction method mainly includes extraction of insoluble dietary fiber from prickly pear residue, removal of free phenols, high-pressure hydrothermal treatment, fungal fermentation, ethanol extraction, vacuum concentration, and vacuum freeze-drying. This extraction method is a green and efficient method for extracting prickly pear dietary fiber-bound polyphenols, with a significantly higher yield than the alkaline extraction method. The obtained prickly pear dietary fiber-bound polyphenols are characterized by high purity and strong activity, significantly inhibiting α-glucosidase activity, with significantly stronger inhibitory activity than acarbose. Furthermore, it can effectively improve hyperglycemia in type 2 diabetic mice, reduce fasting blood glucose levels, and improve glucose tolerance. Its hypoglycemic activity is superior to metformin and polyphenols obtained by alkaline extraction, and it can be developed into a potential hypoglycemic functional factor for application in the preparation of functional foods or drugs.

Description

Technical Field

[0001] This invention belongs to the field of functional food factor preparation technology, specifically relating to a prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity, its extraction method and application. Background Technology

[0002] Prickly pear, also known as prickly strawberry or Wenxian fruit, is the fruit of a perennial deciduous shrub belonging to the Rosaceae family. The fruit is typically golden yellow when ripe and comes in various shapes, including oval, round, and flattened-round, with numerous small thorns on its surface. Recorded in ancient Chinese texts such as the *Compendium of Materia Medica* and the *Dictionary of Traditional Chinese Medicine*, prickly pear is a plant used for both food and medicine, widely distributed in mountainous areas of southwestern my country at altitudes of 500-2500 meters. It is cultivated in Guizhou, Sichuan, Shaanxi, and Yunnan provinces. Prickly pear is rich in nutrients, containing abundant polysaccharides, dietary fiber, flavonoids, polyphenols, vitamin C, and superoxide dismutase (SOD), and has a history of being used both as food and medicine. In recent years, numerous studies have confirmed that prickly pear possesses various biological activities, including antioxidant, anti-atherosclerotic, hypoglycemic, anti-aging, and anti-tumor effects, attracting widespread attention. In 2021, the planting area in Guizhou Province expanded to 2.1 million mu (approximately 140,000 hectares), with a fresh fruit yield of 289,100 tons, producing 161,400 tons of prickly pear products, with a value exceeding 11.164 billion yuan. Most prickly pear products on the market, such as prickly pear juice, prickly pear wine, dried prickly pear, and prickly pear preserves, are made from juice or whole fruit. However, nearly 50% of the prickly pear pulp is produced after juicing. In actual production and processing, most of this pulp is discarded as waste, resulting in a huge waste of resources. Therefore, the intensive processing of prickly pear pulp and the development of functional foods are of great significance for the high-value utilization of prickly pear resources.

[0003] Polyphenols have been proven to possess potent antioxidant activity and have preventative and therapeutic effects on cardiovascular diseases, diabetes, cancer, and obesity, and are widely used in food, health products, pharmaceuticals, and cosmetics. As one of the important functional components of prickly pear, current research on prickly pear polyphenols has focused solely on free phenols, neglecting the existence of bound phenols. Polyphenols exist in both free and bound forms in prickly pear, and many studies have shown that the functional activity of bound phenols is far superior to that of free phenols. Our previous invention patent (CN202111229571.9) disclosed that prickly pear residue contains abundant bound polyphenols, mainly including gallic acid, ellagic acid, ferulic acid, hydroxybenzoic acid, catechins, epicatechin, gallocatechin, quercetin, and kaempferol-3-O-glucoside, which exhibit significant antioxidant activity. In vitro digestion and fermentation experiments showed that the bound polyphenols in the prickly pear residue dietary fiber had good probiotic effects. However, the total phenols released during digestion and fermentation were significantly lower than the bound phenols extracted by the alkali method, indicating that the bioavailability of the bound polyphenols in the prickly pear residue dietary fiber was low after oral administration, resulting in resource waste.

[0004] Conjugated polyphenols exist in the primary and secondary cell walls of plant tissues in the form of ester, ether, or glycosidic bonds with cellulose, proteins, lignin, etc., making them difficult to extract directly with organic solvents. Currently, the most commonly used methods for extracting conjugated polyphenols include alkaline methods, acid methods, and enzymatic methods. Alkaline methods are effective because hydrolysis under alkaline conditions can effectively break the ether and ester bonds between phenolic substances and cell wall materials. Acid hydrolysis mainly breaks glycosidic bonds, with less damage to ester bonds or leaving them intact. Although both of these methods can effectively extract conjugated polyphenols, the loss of phenolic extracts due to strong acids, strong alkalis, and high temperatures is not negligible and is environmentally unfriendly. Enzymatic methods are based on the fact that the food matrix is ​​mainly composed of cell wall materials such as cellulose, hemicellulose, and pectin. Enzymatic conditions are mild, thus avoiding the loss or degradation of phenolic substances due to extreme pH values ​​or high temperatures. However, they also suffer from low extraction rates and are not yet mature. Therefore, inventing a method for efficiently and environmentally friendly extraction of conjugated polyphenols from prickly pear pomace dietary fiber, improving the utilization rate and high-value application of prickly pear pomace resources, has significant theoretical and practical value. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method and application for the green and efficient extraction of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity.

[0006] To achieve the above-mentioned technical objectives, the present invention is implemented through the following technical solution:

[0007] A method for extracting prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity includes the following steps:

[0008] (1) Raw material pretreatment and degreasing: Using prickly pear residue as raw material, firstly, the prickly pear residue is washed, deseeded, dried, crushed and sieved to obtain prickly pear residue powder. Then, the prickly pear residue powder is mixed with n-hexane at a material-to-liquid ratio of 1:10 to 1:30 g / mL and degreased by shaking at room temperature for 1 to 5 hours. The residue is collected by centrifugation. The degreasing is repeated 3 to 5 times and then dried to obtain degreased prickly pear residue powder.

[0009] (2) Preparation of prickly pear dietary fiber: The defatted prickly pear residue powder from step (1) was mixed with HCl-KCl buffer solution with pH 1.5-2.0 at a material-to-liquid ratio of 1:10-1:20 g / mL. Pepsin was added at a ratio of 0.4-2% g / mL relative to the HCl-KCl buffer solution. The mixture was incubated in a water bath at 37-40℃ for 1-2 h. After the reaction was completed, the pH of the mixed solution was adjusted to 7.5-8.5. Trypsin was added at a ratio of 0.05-1% g / mL relative to the HCl-KCl buffer solution. The mixture was incubated in a water bath at 37-40℃ for 4-6 h. Then, MES buffer solution at a ratio of 10-20% relative to the volume of the HCl-KCl buffer solution was added, and the pH was adjusted to 5.5-6.5. Medium-temperature α-amylase was added at a ratio of 1.5-5% g / mL relative to the HCl-KCl buffer solution. The mixture was reacted at 37-50℃ for 16-24 h. The residue was collected by centrifugation, and prickly pear dietary fiber was obtained.

[0010] (3) Removal of free phenols: The prickly pear dietary fiber prepared in step (2) is mixed with an ethanol aqueous solution with a volume fraction of 70-95% at a mass-to-volume ratio of 1:15-1:25 g / mL, and extracted for 2-5 hours. The supernatant is discarded by centrifugation, and the residue is extracted 2-5 times. The precipitate is collected by centrifugation and freeze-dried to obtain prickly pear dietary fiber with free phenols removed.

[0011] (4) High pressure wet heat treatment: The prickly pear dietary fiber that has been deprived of free phenols in step (3) is mixed with distilled water at a mass-volume ratio of 1:5 to 1:10 g / mL until it is completely soaked. The mixture is sealed with a breathable sealing film and placed in a high pressure steam sterilizer for high pressure wet heat treatment. Then the sample is placed on a clean bench to cool and obtain a prickly pear dietary fiber solution for later use.

[0012] (5) Preparation of bacterial culture: The revived bacterial culture was evenly spread on PDA medium and placed in a biochemical incubator at 20-30℃ for 3-5 days to activate the bacterial strain. After activation, sterile distilled water was added to scrape off the spores, and the mycelia were filtered with sterile cotton. The bacterial culture was collected, and the spores were counted under an optical microscope using a hemocytometer. The bacterial culture concentration was then diluted to 2×10⁻⁶. 7 ~1×10 8 Spores / mL, thus obtaining spore culture;

[0013] (6) Fungal fermentation: The spore liquid prepared in step (5) is mixed with the prickly pear dietary fiber solution cooled after high pressure wet heat treatment in step (4) at a volume ratio of 1:10 to 1:20. After stirring evenly, the mixture is placed in a biochemical incubator at 25 to 35°C and fermented for 3 to 5 days to obtain the fermented prickly pear dietary fiber solution.

[0014] (7) Polyphenol extraction: The fermented prickly pear dietary fiber solution obtained in step (6) is mixed with an ethanol aqueous solution or anhydrous ethanol with a volume ratio of 1:15 to 1:25. The mixture is shaken and extracted for 1 to 2 hours in a shaker. The supernatant is collected by centrifugation. The residue is extracted 6 to 8 times. All supernatants are combined, concentrated under reduced pressure, and freeze-dried to obtain prickly pear dietary fiber bound polyphenols.

[0015] Preferably, the drying method in step (1) is drying in a blower oven; the drying temperature is 40-50℃, the drying time is 24-48h; and the sieving is through a 30-60 mesh sieve.

[0016] Preferably, the pepsin in step (2) has an enzyme activity of 3000-3500 NFU / g; the pancreatic enzyme has an enzyme activity of ≥4000 U / g; and the mesophilic α-amylase has an enzyme activity of 4000-10000 U / g.

[0017] Preferably, the centrifugation conditions in steps (1), (2), (3) and (7) are 4000-6000g and 5-15min.

[0018] Preferably, the conditions for the high-pressure damp heat treatment in step (4) are: temperature 115-121℃, time 15-30min.

[0019] Preferably, the bacterial strain in the bacterial solution in step (5) is Aspergillus niger GDMCC 3.576 or Aspergillus niger GDMCC 3.546, purchased from Guangdong Provincial Microbial Culture Collection Center.

[0020] Preferably, the temperature for vacuum concentration in step (7) is 40–50°C; and the freeze-drying time is 36–72 h.

[0021] The above extraction method yields prickly pear dietary fiber combined with polyphenols that have significant hypoglycemic activity.

[0022] Preferably, the prickly pear dietary fiber-bound polyphenols with significant hypoglycemic activity include ellagic acid 5.60–6.63 mg / g, hydroxybenzoic acid 4.10–6.95 mg / g, epigallocatechin 1.56–2.04 mg / g, quercetin 0.23–3.45 mg / g, and 3,4-dihydroxyphenylpropionic acid 1.20–1.40 mg / g.

[0023] The above-mentioned application of prickly pear dietary fiber combined with polyphenols, which has significant hypoglycemic activity, in the preparation of hypoglycemic drugs and foods.

[0024] The prickly pear dietary fiber combined with polyphenols described in this invention has significant α-glucosidase inhibitory activity, which can effectively improve hyperglycemia in type 2 diabetic mice, reduce fasting blood glucose levels, and improve glucose tolerance. It can be applied to the preparation of functional foods and medicines with hypoglycemic effects.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The method of the present invention uses high pressure wet heat pretreatment and fungal fermentation synergistic processing technology to treat the prickly pear dietary fiber, which changes the tight structure of the dietary fiber, reduces the crystallinity, and destroys some of the chemical bonds between the bound polyphenols and the dietary fiber, effectively promoting the release of bound polyphenols in the prickly pear dietary fiber. Compared with the alkali extraction method, the yield of bound polyphenols in the prickly pear dietary fiber obtained by the method of the present invention is significantly higher than that of the alkali extraction method, reaching more than 3 times. The preparation process is green and environmentally friendly, pollution-free, and suitable for industrial application.

[0027] (2) The prickly pear dietary fiber bound to polyphenols prepared by the method of this invention has a high polyphenol content, mainly including ellagic acid, hydroxybenzoic acid, epigallocatechin, quercetin, and 3,4-dihydroxyphenylpropionic acid. In vitro hypoglycemic activity studies have shown that the prickly pear dietary fiber bound to polyphenols has superior α-glucosidase inhibitory activity, which is significantly stronger than that of acarbose, reaching more than 65 times.

[0028] (3) The prickly pear dietary fiber combined with polyphenols prepared by the method of the present invention can effectively improve hyperglycemia in obese diabetic model mice, reduce fasting blood glucose levels, and improve glucose tolerance. Its hypoglycemic activity is superior to that of metformin and prickly pear dietary fiber combined with polyphenols prepared by alkali extraction. It can be applied to the preparation of functional foods and drugs with hypoglycemic effects. Attached Figure Description

[0029] Figure 1 The yield of prickly pear dietary fiber combined with polyphenols prepared by different extraction methods in Examples 1 and 2 of this invention.

[0030] Figure 2 Fasting blood glucose levels of mice in different experimental groups.

[0031] Figure 3 The changes in oral glucose tolerance in mice from different experimental groups.

[0032] Figure 4 The area under the oral glucose tolerance curve (AUC) is the value of mice in different experimental groups. Detailed Implementation

[0033] To better understand the present invention, the following description is based on embodiments, but the implementation of the present invention is not limited thereto.

[0034] Example 1: Extraction of dietary fiber and polyphenols from prickly pear using the method of the present invention:

[0035] (1) Raw material pretreatment and defatting: Using prickly pear residue as raw material, the prickly pear residue was first washed, seeds removed, and dried in a 45℃ forced-air drying oven for 24 hours. After pulverizing, it was passed through a 60-mesh sieve to obtain prickly pear residue powder. The dry prickly pear residue powder was mixed with n-hexane at a material-liquid mass-volume ratio of 1:10 g / mL. The mixture was shaken at room temperature for 2 hours, centrifuged at 5000g for 5 minutes, and the residue was collected. The operation was repeated 5 times. The residue was dried in a 45℃ forced-air drying oven for 48 hours to obtain defatted prickly pear residue powder.

[0036] (2) Preparation of prickly pear dietary fiber: Weigh 10g of defatted prickly pear residue powder from step (1) and mix it with 100mL of HCl-KCl buffer solution (pH 1.5). Add 400mg of pepsin (3000NFU / g) and incubate in a water bath at 37℃ for 1h. After the reaction is complete, adjust the pH to 7.5, add 50mg of pancreatin (4000U / g), and incubate again in a water bath at 37℃ for 4h. Then add 10mL of LME buffer solution (pH 6.5) and adjust the pH to 6. Add 3g of medium-temperature α-amylase (10000U / g) and react at 37℃ for 16h. Centrifuge and collect the residue.

[0037] (3) Removal of free phenols: The residue prepared in (2) was mixed with an ethanol aqueous solution with a volume fraction of 75% at a mass-to-volume ratio of 1:15 g / mL, and extracted for 2 h. The supernatant was discarded by centrifugation. The residue was extracted three times. The precipitate was collected by centrifugation at 4500 g for 5 min and then freeze-dried to prepare prickly pear dietary fiber with free phenols removed.

[0038] (4) High pressure wet heat treatment: Weigh 1g of prickly pear dietary fiber with free phenols removed, place it in a 150mL conical flask, add 9mL of distilled water until the sample is completely wetted, seal it with a breathable sealing film, and place it in a 121℃ high pressure steam sterilizer for high pressure wet heat treatment for 15min. After the treatment, place the sample on a clean bench to cool and wait for use.

[0039] (5) Preparation of bacterial culture: The revived bacterial culture (Aspergillus niger GDMCC 3.576) was evenly spread on PDA medium and placed in a biochemical incubator at 27℃ for 3 days to activate the strain. After activation, sterile distilled water was added to scrape out spores, and the mycelia were filtered through sterile cotton. The bacterial culture was collected, counted under an optical microscope using a hemocytometer, and the bacterial culture concentration was diluted to 2×10⁻⁶. 7 Spores / mL, thus obtaining spore culture;

[0040] (6) Fungal fermentation: Mix 0.5 mL of spore culture from step (5) with the sample solution cooled after high pressure wet heat treatment in step (4), stir evenly, and place in a 27℃ biochemical incubator for 3 days to obtain fermented prickly pear dietary fiber solution.

[0041] (7) Extraction: The fermented prickly pear dietary fiber solution obtained in step (6) was mixed with a 70% ethanol aqueous solution at a volume ratio of 1:15. The mixture was shaken thoroughly in a shaker for 1 hour. The supernatant was collected by centrifugation. The residue was repeated 6 times under the same conditions. All supernatants were combined, concentrated under reduced pressure, and freeze-dried to obtain prickly pear dietary fiber bound polyphenols. The yield of prickly pear dietary fiber bound polyphenols was calculated to be 3.37% (based on the dry weight of prickly pear dietary fiber).

[0042] Example 2: Alkaline extraction of dietary fiber and polyphenols from prickly pear

[0043] (1) Raw material pretreatment and defatting: Using prickly pear residue as raw material, the prickly pear residue was first washed, seeds removed, and dried in a 45℃ forced-air drying oven for 24 hours. After pulverizing, it was passed through a 60-mesh sieve to obtain prickly pear residue powder. The dry prickly pear residue powder was mixed with n-hexane at a material-liquid mass-volume ratio of 1:10 g / mL. The mixture was shaken at room temperature for 2 hours, centrifuged at 5000g for 5 minutes, and the residue was collected. The operation was repeated 5 times. The residue was dried in a 45℃ forced-air drying oven for 48 hours to obtain defatted prickly pear residue powder.

[0044] (2) Preparation of prickly pear dietary fiber: Weigh 10g of defatted prickly pear residue powder from step (1) and mix it with 100mL of HCl-KCl buffer solution (pH 1.5). Add 400mg of pepsin (3000NFU / g) and incubate in a water bath at 37℃ for 1h. After the reaction is complete, adjust the pH to 7.5, add 50mg of pancreatin (4000U / g), and incubate again in a water bath at 37℃ for 4h. Then add 10mL of LME buffer solution (pH 6.5) and adjust the pH to 6. Add 3g of medium-temperature α-amylase (10000U / g) and react at 37℃ for 16h. Centrifuge and collect the residue.

[0045] (3) Removal of free phenols: The residue prepared in (2) was mixed with an ethanol aqueous solution with a volume fraction of 75% at a mass-to-volume ratio of 1:15 g / mL, and extracted for 2 h. The supernatant was discarded by centrifugation. The residue was extracted three times. The precipitate was collected by centrifugation at 4500 g for 5 min and then freeze-dried to obtain prickly pear dietary fiber with free phenols removed.

[0046] (4) Alkaline extraction: Weigh 1g of prickly pear dietary fiber (free phenols removed) and mix it with 10mL of sodium hydroxide solution (4M). Stir slowly under light-proof, oxygen-free, and room temperature conditions for 4h. Then, adjust the pH to 2 with 6M hydrochloric acid and extract with ethyl acetate (1:1, v / v). Centrifuge the mixture at 4500g for 5min to separate the supernatant and precipitate. Repeat the above extraction operation 6 times. Collect the organic fraction, concentrate, and freeze-dry to obtain prickly pear dietary fiber bound polyphenols. The yield of prickly pear dietary fiber bound polyphenols was calculated to be 2.25% (based on the dry weight of prickly pear dietary fiber).

[0047] Example 3: Yield of polyphenols bound to prickly pear dietary fiber

[0048] The total phenolic content of polyphenols bound to prickly pear dietary fiber was determined using the Folin-Ciocalteu reagent method. 100 μL of sample solution (800 μg / mL), 800 μL of distilled water, and 100 μL of Folin-Ciocalteu reagent were mixed and thoroughly shaken. The mixture was incubated at room temperature for 6 min. Then, 1 mL of NaCO3 solution (7%) and 1 mL of distilled water were added, mixed, and incubated at room temperature in the dark for 90 min. Methanol solution was used instead of Folin-Ciocalteu reagent as a blank sample tube. After the reaction, the absorbance of each reaction solution was measured at 760 nm using a microplate reader. A standard curve was plotted using gallic acid, and the total phenolic content of the prickly pear dietary fiber bound to polyphenols was calculated. The total phenolic content was expressed as milligram gallic acid equivalents (GAE) per gram of prickly pear dietary fiber dry weight (DW), in mg GAE / g DW.

[0049] The yields of prickly pear dietary fiber bound to polyphenols obtained by the alkali extraction method (Example 2) and the method of the present invention (Example 1) are as follows: Figure 1 As shown in Example 2, the yield of polyphenols bound to prickly pear dietary fiber prepared by the alkali extraction method was 5.33 mg GAE / g DW, with a purity of 23.7%; while in Example 1, the yield of polyphenols bound to prickly pear dietary fiber prepared by the present invention was 17.22 mg GAE / g DW, with a purity of 51.1%, which is 223.21% higher than that of the alkali extraction method. This indicates that compared with the alkali extraction method, the method of the present invention can better promote the release of polyphenols bound to prickly pear dietary fiber.

[0050] Example 4: Analysis of the main components of prickly pear dietary fiber bound to polyphenols

[0051] The main phenolic compounds in the samples were determined and quantitatively analyzed using an Agilent 1260 high-performance liquid chromatograph (HPLC). HPLC conditions were as follows: a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm) was used; the column temperature was set to 30℃; the flow rate was 0.8 mL / min; the injection volume was 50 μL; and the detection wavelength was set to 285 nm. Two mobile phases were used: 0.1% formic acid in water (A) and acetonitrile (B). The gradient elution conditions were: 0 min, 5% B; 5 min, 8% B; 10 min, 15% B; 25 min, 35% B; 30 min, 50% B; 31–35 min, 100% B; 36–50 min, 5% B.

[0052] Sample pretreatment: Take an appropriate amount of sample, use chromatographic grade methanol as solvent, fully dissolve and prepare a solution with a concentration of 5 mg / mL, then filter through a 0.22 μm organic filter membrane into a brown liquid chromatography vial, and store at -80℃ for later use.

[0053] Preparation of polyphenol standards: Weigh an appropriate amount of the standard and dissolve it in chromatographic grade methanol to prepare various standard stock solutions with a concentration of 2 mg / mL. Based on the peak value of each standard under liquid phase conditions, take an appropriate amount from each standard, mix them, and dilute with methanol to obtain a mixed standard solution of standard concentration.

[0054] The content of various phenolic compounds in the sample is expressed as milligrams of standard phenolic compounds per gram of extract (mg / g).

[0055] Table 1. Composition and content (mg / g) of the main polyphenols bound to prickly pear dietary fiber.

[0056]

[0057] The composition and content of the main bound phenols in the samples prepared in Examples 1 and 2 are shown in Table 1. The bound polyphenols prepared by the alkaline extraction method in Example 2 mainly include gallic acid, ellagic acid, epicatechin, hydroxybenzoic acid, and gallocatechin, and also contain small amounts of epigallocatechin, catechin, ferulic acid, and quercetin. In contrast, the bound polyphenols prepared in Example 1 mainly include epigallocatechin, hydroxybenzoic acid, ellagic acid, 3,4-dihydroxyphenylpropionic acid, and quercetin, with significantly higher contents of epigallocatechin, p-hydroxybenzoic acid, quercetin, and 3,4-dihydroxyphenylpropionic acid than those in Example 1. These results indicate that the bound polyphenols of prickly pear dietary fiber in Examples 1 and 2 have different polyphenol compositions and contents.

[0058] Example 5: Assay of α-glucosidase inhibitory activity

[0059] Add 50 μL of sample solutions of different concentrations or acarbose to a 96-well microplate, then add 50 μL of α-glucosidase solution, mix well, and incubate at room temperature for 10 min. Next, add 50 μL of p-nitrophenyl-D-glucopyranoside (5.0 mM) to initiate the reaction. After incubation for 10 min, add 100 μL of Na₂CO₃ (0.2 M) to terminate the reaction. Measure the absorbance of the mixture at 405 nm using a microplate reader. Use distilled water instead of α-glucosidase in the reaction system as a sample blank, and no sample is added as a control group. The α-glucosidase inhibition rate is calculated using the formula:

[0060] Inhibition rate (%) = (1-(A) S -A B ) / (AA C ))×100

[0061] Among them, A S A and A' are the absorbance values ​​of the sample group and the unsampled group in the reaction system, respectively. C and A B These represent the absorbance values ​​of the control group and the blank group in the reaction system, respectively.

[0062] Type 2 diabetes is caused by insufficient insulin or insulin resistance. Currently, several hypoglycemic drugs are effective in regulating high blood sugar, such as acarbose; however, these drugs often have side effects, such as gastrointestinal problems, diarrhea, and liver problems. Studies have shown that natural polyphenols, as an effective food functional factor, can effectively inhibit the activity of carbohydrate hydrolases, specifically α-glucosidase, and have the potential to improve or treat type 2 diabetes.

[0063] Using acarbose as a positive control, the α-glucosidase inhibitory activity of prickly pear dietary fiber-bound polyphenols prepared by different extraction methods was compared, and the results are shown in Table 2. The prickly pear dietary fiber-bound polyphenols extracted in Example 1, the prickly pear dietary fiber-bound polyphenols extracted by the alkaline method in Example 2, and acarbose all exhibited good α-glucosidase inhibitory activity, with half-maximal inhibitory concentrations (IC50) of [missing information]. 50 The values ​​were 0.29, 6.03 and 19.04 μg / mL, respectively. The results showed that the prickly pear dietary fiber bound polyphenols extracted by the method of the present invention in Example 1 had the strongest α-glucosidase inhibitory activity, which was 20 times that of the prickly pear dietary fiber bound polyphenols extracted by the alkaline method in Example 2 and 65 times that of acarbose.

[0064] Table 2. α-Glucosidase inhibitory activity of prickly pear dietary fiber bound to polyphenols.

[0065]

[0066] Example 6: In vivo hypoglycemic activity experiment

[0067] C57BL / 6 mice were housed in a standard animal facility under 12-hour light cycle, 26±1℃, and 50–60% relative humidity, with free access to food and water. After one week of acclimatization, they were randomly divided into two groups: a normal control group (NC) and a high-fat diet-induced obesity model group. The NC group was fed a normal diet and administered saline via gavage daily; the model group was fed a high-fat diet (HFD) and administered saline via gavage daily. After four weeks of continuous modeling, the obese model group mice were fasted overnight and then injected intraperitoneally with streptozotocin (STZ) at a dose of 30 mg / kg / day in a volume of 1 mL; the normal control group received an equal volume of buffer solution. Mice with a fasting blood glucose (FBG) level higher than 11.1 mmol / L were used as a type 2 diabetes model. Blood glucose levels in the diabetic mouse model were monitored for one week to establish a stable hyperglycemic state. Diabetic mice were randomly divided into four groups: a diabetic model group (MC), a metformin experimental group (Met, 200 mg / kg / d), an alkaline extraction experimental group (the conjugated polyphenol of Example 2) (150 mg / kg / d), and the experimental group of this invention (the conjugated polyphenol of Example 1) (150 mg / kg / d), with six mice in each group. The experiment lasted for four weeks. The normal control group was given a normal diet, while the other three groups of diabetic mice were given a high-fat diet.

[0068] Example 7: Fasting blood glucose and oral glucose tolerance test

[0069] Following the in vivo hypoglycemic activity experiment in Example 4, after a 12-hour fast, fasting blood glucose (FBG) levels in the tail vein of mice were measured using a glucometer. For the oral glucose tolerance test (OGTT), mice that had been fasted overnight were orally administered 1 g / kg of glucose, and blood glucose levels in the tail vein were measured at 0, 30, 60, 90, and 120 minutes using a glucometer. The area under the curve (AUC) of blood glucose levels relative to time was calculated using Origin Pro 8.5 software.

[0070] Fasting blood glucose (FBG) tests can be used to evaluate the hypoglycemic effect of prickly pear residue dietary fiber combined with polyphenols. Results are as follows: Figure 2 As shown in the figure, compared with the normal control group, the fasting blood glucose level of mice in the diabetic model group was significantly increased, from 5.55 mmol / L to 17.57 mmol / L, indicating that the diabetic model mouse was successfully established. After intervention with metformin and prickly pear dietary fiber combined with polyphenols, the fasting blood glucose level of mice in the diabetic model group was significantly reduced, with 10.63 mmol / L in the metformin experimental group, 11.32 mmol / L in the alkali extraction experimental group, and 7.2 mmol / L in the experimental group of this invention. These results indicate that the prickly pear dietary fiber combined with polyphenols in the experimental group of this invention has the strongest hypoglycemic activity, which is stronger than that of the metformin and alkali extraction experimental group samples.

[0071] The oral glucose tolerance test (OGTT) was used to evaluate the regulatory effect of prickly pear dietary fiber combined with polyphenols on glucose tolerance disorders in diabetic mice. The effects of prickly pear dietary fiber combined with polyphenols on glucose tolerance and AUC in diabetic mice were as follows: Figure 3 and Figure 4 As shown in the figure, when a certain amount of glucose solution was administered by gavage, the blood glucose concentration of mice in each treatment group rose rapidly, reaching its maximum at 30 minutes. As time progressed, the blood glucose concentration of each group gradually decreased. At each time point, the blood glucose concentration of the diabetic model group was higher than that of the other treatment groups, while the blood glucose concentration of the normal group was the lowest.

[0072] Compared with diabetic model mice, the AUC values ​​of other treatment groups were significantly lower, indicating that metformin, the alkaline extraction experimental group, and the experimental group of this invention can all effectively regulate blood glucose levels, with the experimental group of this invention showing the lowest AUC value. These results indicate that the prickly pear dietary fiber combined with polyphenols prepared by this invention can significantly improve glucose tolerance and blood glucose levels in obese diabetic model mice, and its hypoglycemic activity is superior to that of metformin and the prickly pear dietary fiber combined with polyphenols prepared by the alkaline extraction method.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A method for extracting prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity, characterized in that, Includes the following steps: (1) Raw material pretreatment and degreasing: Using prickly pear residue as raw material, firstly, the prickly pear residue is washed, deseeded, dried, crushed and sieved to obtain prickly pear residue powder. Then, the prickly pear residue powder is mixed with n-hexane at a material-to-liquid ratio of 1:10~1:30 g / mL and degreased by shaking at room temperature for 1~5 h. The residue is collected by centrifugation. The degreasing is repeated 3~5 times and dried to obtain degreased prickly pear residue powder. (2) Preparation of prickly pear dietary fiber: The defatted prickly pear residue powder from step (1) was mixed with HCl-KCl buffer solution with pH 1.5-2.0 at a material-to-liquid ratio of 1:10-1:20 g / mL. Pepsin was added at a ratio of 0.4-2% g / mL relative to the HCl-KCl buffer solution. The mixture was incubated in a water bath at 37-40℃ for 1-2 h. After the reaction was completed, the pH of the mixed solution was adjusted to 7.5-8.

5. Trypsin was added at a ratio of 0.05-1% g / mL relative to the HCl-KCl buffer solution. The mixture was incubated in a water bath at 37-40℃ for 4-6 h. Then, MES buffer solution at a ratio of 10-20% relative to the volume of the HCl-KCl buffer solution was added, and the pH was adjusted to 5.5-6.

5. Medium-temperature α-amylase at a ratio of 1.5-5% g / mL relative to the HCl-KCl buffer solution was added. The mixture was reacted at 37-50℃ for 16-24 h. The residue was collected by centrifugation, and prickly pear dietary fiber was obtained. (3) Removal of free phenols: The prickly pear dietary fiber prepared in step (2) is mixed with an ethanol aqueous solution with a volume ratio of 1:15~1:25 g / mL and extracted for 2~5 h. The supernatant is discarded by centrifugation. The residue is extracted 2~5 times, the precipitate is collected by centrifugation, and the precipitate is freeze-dried to obtain prickly pear dietary fiber with free phenols removed. (4) High pressure wet heat treatment: The prickly pear dietary fiber that has been deprived of free phenols in step (3) is mixed with distilled water at a mass-volume ratio of 1:5~1:10 g / mL until it is completely soaked. The mixture is sealed with a breathable sealing film and placed in a high pressure steam sterilizer for high pressure wet heat treatment. Then the sample is placed on a clean bench to cool and obtain a prickly pear dietary fiber solution for later use. (5) Preparation of bacterial culture: The revived bacterial culture was evenly spread on PDA medium and placed in a biochemical incubator at 20-30℃ for 3-5 days to activate the bacterial strain; after activation, sterile distilled water was added to scrape out the spores, and the mycelia were filtered with sterile cotton. The bacterial culture was collected, and the spores were counted under an optical microscope using a hemocytometer. The bacterial culture concentration was then diluted to 2×10⁻⁶. 7 ~1×10 8 The spores / mL yielded the spore culture; the strain in the culture was Aspergillus niger GDMCC 3.576 or Aspergillus niger GDMCC 3.546, purchased from the Guangdong Provincial Microbial Culture Collection Center. (6) Fungal fermentation: The spore liquid prepared in step (5) is mixed with the prickly pear dietary fiber solution cooled after high pressure wet heat treatment in step (4) at a volume ratio of 1:10~1:

20. After stirring evenly, it is placed in a biochemical incubator at 25~35℃ and fermented for 3~5 days to obtain the fermented prickly pear dietary fiber solution. (7) Polyphenol extraction: The fermented prickly pear dietary fiber solution obtained in step (6) is mixed with 70-100% ethanol aqueous solution or anhydrous ethanol at a volume ratio of 1:15~1:

25. The mixture is shaken and extracted for 1-2 h in a shaker. The supernatant is collected by centrifugation. The residue is extracted 6-8 times. All supernatants are combined, concentrated under reduced pressure, and freeze-dried to obtain prickly pear dietary fiber bound polyphenols.

2. The extraction method of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity according to claim 1, characterized in that, The drying method in step (1) is drying in a blower oven; the drying temperature is 40~50 °C and the drying time is 24~48 h; the sieving is through a 30~60 mesh sieve.

3. The extraction method of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity according to claim 1, characterized in that, The pepsin activity described in step (2) is 3000~3500 NFU / g; the pancreatin activity is ≥4000 U / g; and the mesophilic α-amylase activity is 4000~10000 U / g.

4. The extraction method of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity according to claim 1, characterized in that, The centrifugation conditions described in steps (1), (2), (3) and (7) are 4000~6000 g and 5~15 min.

5. The extraction method of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity according to claim 1, characterized in that, The conditions for the high-pressure damp heat treatment in step (4) are: temperature 115~121 ℃, time 15~30 min.

6. The extraction method of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity according to claim 1, characterized in that, The temperature for vacuum concentration in step (7) is 40~50 ℃; the freeze-drying time is 36~72 h.

7. The prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity obtained by the extraction method according to any one of claims 1-6.

8. The prickly pear dietary fiber combined with polyphenols having significant hypoglycemic activity according to claim 7, characterized in that, The prickly pear dietary fiber-bound polyphenols with significant hypoglycemic activity include ellagic acid 5.60~6.63 mg / g, hydroxybenzoic acid 4.10~6.95 mg / g, epigallocatechin 1.56~2.04 mg / g, quercetin 0.23~3.45 mg / g and 3,4-dihydroxyphenylpropionic acid 1.20~1.40 mg / g.

9. The use of prickly pear dietary fiber combined with polyphenols with significant hypoglycemic activity as described in claim 7 or 8 in the preparation of hypoglycemic drugs and foods that assist in hypoglycemic action.

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