Preparation method of aronia melanocarpa extract

Abstract

The invention provides an aronia melanocarpa extract preparation method, which comprises: crushing aronia melanocarpa, screening to obtain fine powder, weighing the aronia melanocarpa powder, placing in a brown container, adding a DES extraction liquid according to a ratio of the aronia melanocarpa powder to the DES extraction liquid of 1: 40-50 g / ml, carrying out ultrasonic treatment for 30-40 min under the conditions of 200-240 W and 37 DEG C constant temperature, centrifuging the obtained extraction liquid for 10-20 min, filtering, washing, and drying to obtain the aronia melanocarpa extract. And separating supernate to obtain the aronia melanocarpa extract. The method disclosed by the invention is high in efficiency, high in yield, stable, difficult to volatilize, free of purification, non-toxic and strong in activity, and has relatively strong application feasibility in food industry. In addition, the aronia melanocarpa berry extract obtained by the method is particularly suitable for development of natural antioxidants.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine preparation, and particularly relates to a method for preparing an extract of Sorbus melanocarpa. Background Art

[0002] Aronia melanocarpa (blackberry for short) is also known as wild berry, immortal berry, blackberry, etc. It is native to the eastern part of North America. The edible part of Aronia melanocarpa is the fruit, a black berry that looks like a blueberry and has a sweet, sour and bitter taste. Existing studies have shown that Aronia melanocarpa is rich in anthocyanins, proanthocyanidins, flavonoids, phenolic acids and other active substances, which has exceeded berries such as blueberries and is one of the richest sources of polyphenols in plant raw materials. They can scavenge free radicals in the human body, resist oxidation, regulate human immune function, lower blood sugar and blood lipids, fight cancer, reduce inflammation and inhibit bacteria, etc., and can be used as a source of natural antioxidants.

[0003] At present, the main extraction methods for extracting polyphenols from Aronia nigra include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, etc. The organic solvents involved, such as methanol, ethanol, acetone, etc., have the disadvantages of strong toxicity, low yield, and low activity. The enzyme-assisted extraction method is easy to operate and has a high extraction efficiency, but the enzyme requires high environmental conditions and is easily inactivated if the operation is improper. Although the supercritical CO2 fluid extraction method has a high extraction efficiency and the activity of the obtained extract is relatively high, the instruments and extractants used are expensive, and the extraction cost is too high.

[0004] Therefore, finding a better extraction process for polyphenols in Aronia nigra is of great significance for the utilization and development of Aronia nigra resources. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for preparing an extract of Aronia nigromaculata.

[0006] The object of the present invention is achieved in the following ways:

[0007] A method for preparing an aronia nigra extract comprises the following steps: crushing and sieving black fruits to obtain fine powder, weighing the black fruit powder and placing it in a brown container, adding a deep eutectic solvent (DES) extract, wherein the black fruit powder and the DES extract are mixed in a ratio of 1:40-50 g / ml, ultrasonicating for 30-40 min under a constant temperature of 200-240 W and 37° C., centrifuging the obtained extract for 10-20 min, separating the supernatant, and obtaining the aronia nigra extract.

[0008] In the above method, the hydrogen bond network of DES is combined with the polyphenolic compounds to be extracted through hydrogen bonding. Because of its certain viscosity, the dissolution efficiency of DES is increased, making the two closely combined. At the same time, ultrasound assists in effective extraction in a short time, dissolves polyphenolic components faster, reduces energy consumption and solvent usage, and further increases the extraction amount of black fruit polyphenols.

[0009] The DES extract is a mixture of choline chloride and lactic acid in a molar ratio of 1:1-3, preferably the DES extract is a mixture of choline chloride and lactic acid in a molar ratio of 1:3.

[0010] As the proportion of HBD (lactic acid) in the DES of the present invention increases, the viscosity increases accordingly, which is beneficial to the dissolution of the target substance, but at the same time affects the diffusion rate of polyphenols in the system, resulting in a decrease in the extraction amount; therefore, the most preferred molar ratio of choline chloride to lactic acid in the DES system is 1:3.

[0011] In addition, the extraction solvent of the present invention is a (choline chloride: lactic acid) low eutectic solvent, which is an edible medicinal solvent and does not require purification. The low eutectic solvent extract obtained by the method can be applied to animal experiments and observe human metabolism without purification and only after simple dilution treatment. Lactic acid, as a natural organic acid, can replace traditional acidic organic solvent extraction in industrial applications to reduce costs, recycle, be non-toxic, environmentally friendly, and easy to operate.

[0012] Preferably, the water content of the DES extract system is 30-80%, preferably 80%. The extraction amount of polyphenols increases with the increase of the water content of the DES system of the present invention, but excessive increase of water content will cause the breakage of hydrogen bonds between molecules, thereby reducing the extraction amount.

[0013] Preferably, the ultrasonic power is 240 W. The increase in ultrasonic power will generate greater energy to increase the diffusion rate of DES and promote the dissolution of target substances, but when the ultrasonic power increases to a certain extent, the structure of polyphenols will be destroyed, and other non-target substances will precipitate, resulting in a decrease in the extraction amount.

[0014] Preferably, the ultrasonic time is 35-40 minutes. As the ultrasonic time increases, the cell wall of the black fruit is broken and more target components are dissolved. The extraction amount of polyphenols will increase within 40 minutes, but too long of an ultrasonic time will destroy the structure of the dissolved polyphenols and increase the precipitation of non-target substances, resulting in a decrease in the extraction amount of black fruit polyphenols.

[0015] Preferably, the black fruit is the black fruit dried at a constant temperature of 40-50°C.

[0016] Preferably, the centrifugal condition is 10000-12000 r / min.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention is based on the development and utilization of Aronia nigra resources, and uses ultrasound-assisted DES to extract Aronia nigra berry polyphenols. The Aronia nigra polyphenol extraction obtained by the method of the present invention is 50.61 mg / g (relative standard deviation is 0.13%). At the same time, the FRAP (iron ion reducing ability) and ABTS of the polyphenol mixture extracted by the low eutectic solvent (unpurified) and the traditional organic solvent (purified) were tested. .+ OH - , DPPH free radical scavenging ability and other indicators. Under a certain concentration range and the same detection conditions, the antioxidant capacity of the polyphenols extracted by the present invention is significantly higher than that of the polyphenols extracted and purified by traditional solvents. The inhibitory ability of the gastric and intestinal digestive juices of black fruit and blueberry extracted by DES on the activity of α-glucosidase and pancreatic lipase was tested in vitro. The results showed that both black fruit and blueberry extracted by DES had a certain α-glucosidase inhibitory ability and a strong pancreatic lipase inhibitory ability.

[0019] Compared with traditional organic solvent extraction, the aronia berry extract extracted by the low eutectic solvent of the present invention has high efficiency, high yield, stability, low volatility, no need for purification, no toxicity, strong activity, and strong feasibility for application in the food industry. At the same time, the aronia berry extract is suitable for subsequent development of natural antioxidants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the extraction solvent test.

[0021] Figure 2 The effect of DES molar ratio on the extraction amount.

[0022] Figure 3 The effect of DES water content on the extraction amount.

[0023] Figure 4 The effect of ultrasonic power on the extraction amount.

[0024] Figure 5 The effect of ultrasonic time on the extraction amount.

[0025] Figure 6 3D and plane diagrams of the interactions between the factors on the response surface.

[0026] Figure 7 This is a T-AOC (FRAP method) test.

[0027] Figure 8 For ABTS .+ OH - , DPPH free radical scavenging test

[0028] Fig. 9 To simulate the hypoglycemic activity of Aronia nigra extract and blueberry gastric and intestinal digestive juice in vitro.

[0029] Fig.10 To simulate the lipid-lowering activity of Aronia nigra extract and blueberry gastric and intestinal digestive juice in vitro. DETAILED DESCRIPTION

[0030] The present invention is further explained below by way of examples:

[0031] The fruits of Aronia nigra were collected from the flower garden of the College of Landscape and Horticulture of Jiangsu Vocational College of Agriculture and Animal Husbandry Science and Technology. Blueberries were purchased from Taizhou Suguo Supermarket. Choline chloride, 1,4-butanediol, lactic acid, glycerol, urea, anhydrous glucose, malic acid (analytical grade, Shandong Keyuan Biochemical Co., Ltd.); hydrochloric acid, sodium hydroxide, anhydrous ethanol, methanol, sodium carbonate, potassium dihydrogen phosphate, disodium hydrogen phosphate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); gallic acid standard (chromatographic grade, Chengdu Munster Biotechnology Co., Ltd.); α-glucosidase, pepsin, trypsin, pancreatic lipase, pig bile salt, 4-nitrophenylβ-D-glucopyranoside (4-Nitrophenylβ-Dglucopyranoside, pNPG), 4-nitrophenylpalmitate (4-nitrophenylpalmitate, pNPP), Folin phenol, AB-8 macroporous resin, total antioxidant capacity detection kit (FRAP microplate method), (ABTS microplate method) (Shanghai Yuanye Biotechnology Co., Ltd.); hydroxyl free radical test kit, DPPH free radical test kit (Nanjing Jiancheng Biotechnology Co., Ltd.). The water used in the experiment was produced by ultrapure water system.

[0032] Example 1

[0033] The black fruit dried at 40°C was taken out, crushed and sieved to obtain fine powder. 0.5 g of black fruit powder was weighed and placed in a 50 mL brown centrifuge tube, 20 mL of DES solution was added and ultrasonicated for 40 min at 240 W and 37°C, the obtained extract was centrifuged at 10000 r / min for 10 min, and the supernatant was separated to obtain the black fruit glandular costa aronia extract (black fruit DES extract).

[0034] The DES solution was prepared as follows: choline chloride was used as HBA, and lactic acid was mixed in a molar ratio of 1:3, the mixed solution had a water content of 80%, and magnetic stirring was performed at 80° C. to prepare a clear, turbid DES extract solution, which was then cooled to room temperature.

[0035] Example 2

[0036] The black fruit dried at 40°C was taken out, crushed and sieved to obtain fine powder. 0.5 g of black fruit powder was weighed and placed in a 50 mL brown centrifuge tube, 20 mL of DES solution was added and ultrasonicated for 35 min at 200 W and 37°C. The obtained extract was centrifuged at 10000 r / min for 10 min, and the supernatant was separated to obtain the black fruit glandular costa aronia extract (black fruit DES extract).

[0037] The DES solution was prepared as follows: choline chloride was used as HBA, and lactic acid was mixed in a molar ratio of 1:1, the mixed solution had a water content of 30%, and magnetic stirring was performed at 80° C. to prepare a clear, turbid DES extract solution, which was then cooled to room temperature.

[0038] Comparative Example 1

[0039] The black fruit dried at 40°C was taken out, crushed and sieved to obtain fine powder. 0.5 g of black fruit powder was weighed and placed in a 50 mL brown centrifuge tube, 20 mL of DES solution was added and ultrasonicated at 200 W and 37°C for 30 min. The obtained extract was centrifuged at 10000 r / min for 10 min, and the supernatant was separated to obtain the black fruit DES extract.

[0040] The DES solution was prepared as follows: choline chloride was used as HBA and 1,4-butanediol was mixed at a molar ratio of 1:1, the mixed solution contained 30% water, and magnetic stirring was performed at 80° C. to prepare a clear, turbid and viscous DES extract, which was then cooled to room temperature.

[0041] Comparative Example 2

[0042] The preparation method of black fruit DES extract is the same as that of comparative example 1, except that the preparation of DES solution is to mix choline chloride, HBA and malic acid in a molar ratio of 1:1, the mixed solution has a water content of 30%, and after magnetic stirring at 80° C., it is cooled to room temperature.

[0043] Comparative Example 3

[0044] The preparation method of black fruit DES extract is the same as that of comparative example 1, except that the preparation of DES solution is to mix choline chloride, HBA and urea in a molar ratio of 1:1, the mixed solution has a water content of 30%, is magnetically stirred at 80°C, and then cooled to room temperature.

[0045] Comparative Example 4

[0046] The preparation method of black fruit DES extract is the same as that of comparative example 1, except that the preparation of DES solution is to mix choline chloride, HBA and anhydrous glucose in a molar ratio of 1:1, the water content of the mixture is 30%, and after magnetic stirring at 80°C, it is cooled to room temperature.

[0047] Comparative Example 5

[0048] The preparation method of black fruit DES extract is the same as that of comparative example 1, except that the preparation of DES solution is to mix choline chloride, HBA and glycerol in a molar ratio of 1:1, the mixed solution has a water content of 30%, and after magnetic stirring at 80° C., it is cooled to room temperature.

[0049] Test Example 1

[0050] The experimental instruments and equipment used are as follows: AL-204 analytical balance (Mettler-Toledo Instrument Co., Ltd.); Milli-Q Direct-Q pure water / ultrapure water integrated system (Merck, Germany); BIOMATE3S UV-visible spectrophotometer (ThermoFisher, USA); KQ-400DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DF-101B digital display heat-collecting magnetic stirrer (Changzhou Jintan Experimental Instrument Factory); S25 vortex mixer Combiner (IKA, Germany); THZ-C-1 full-temperature oscillator (Suzhou Peiying Experimental Equipment Co., Ltd.); 5810R high-speed refrigerated centrifuge (eppendorf, Germany); LGJ-1FD vacuum freeze dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd.); INFINITEMNANO microplate reader (TECAN, Switzerland); glass chromatography column 35*300*29 (Beijing Ruida Henghui Technology Development Co., Ltd.); RE-5203 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory).

[0051] 1 Experimental methods

[0052] 1.1 Preparation and physicochemical properties of DES

[0053] Choline chloride was selected as HBA, and six kinds of HBDs including different types of alcohol groups (1,4-butanediol, glycerol), acid groups (lactic acid, malic acid), amino groups (urea) and sugar groups (glucose) were accurately weighed in a conical flask at a molar ratio of 1:1. The water content of the DES system was 30%. The mixture was magnetically stirred in a water bath at 70°C-80°C until a uniform, clear, colorless liquid was formed. The mixture was cooled to room temperature and set aside.

[0054] The PH, density and viscosity of 6 groups of DES systems were measured to gain a deeper understanding of their physical and chemical properties. At room temperature (25°C), the pH of DES was measured using a pH meter, the density of DES was measured using a density tester, and the viscosity was measured using a viscometer at 20 r / min.

[0055] 1.2 Extraction of polyphenols

[0056] Take out the black fruit after constant temperature drying at 40℃, crush and sieve to obtain fine powder. Weigh 0.5g of powder, put it in a 50mL brown centrifuge tube, add 20mL of DES, 80% methanol and 60% ethanol solution respectively, ultrasonicate for 30min at 37℃ constant temperature and 200W, centrifuge the extract at 10000r / min for 10min, separate the supernatant, and obtain black fruit DES extract, 80% methanol and 60% ethanol extract.

[0057] 1.3 Single Factor Experiment

[0058] Choline chloride-lactic acid (DES-2) was used as the extraction solvent. A single factor experiment was conducted on the basis of a molar ratio of 1:1, a water content of 30%, an ultrasonic time of 30 minutes, and an ultrasonic power of 200W. The effects of the molar ratio, water content, ultrasonic time, and ultrasonic power in the DES system on the polyphenol extraction were studied. At the same time, the black fruit polyphenols were extracted by 80% methanol and 60% ethanol to investigate the effects of different extraction solvents on the extraction of black fruit polyphenols. 1.4 Optimization of extraction process by Box-Behnken response surface method

[0059] Based on the results of the single-factor experiment, the Box-Behnken response surface method experimental design was optimized with 4 independent variables at 3 levels (see Table 1). Design-Expert13 software was used to design 29 groups of crossover experiments, including 24 groups of factorial point experiments and 5 groups of repeated zero point experiments. IBMSPSSStatistics22.0 software was used for variance significance analysis, and Origin2021 software was used for graphic fitting and drawing.

[0060] Table 1 Box-Behnken response surface method factors and levels

[0061]

[0062] 2 Results and analysis

[0063] 2.1 Physical and chemical properties of DES

[0064] The composition of DES has a significant effect on its inherent physicochemical properties, which directly leads to the extraction efficiency of polyphenol active ingredients in natural plants. The main functional groups involved in hydrogen bonding and molecular interactions are carboxyl, carbonyl, hydroxyl and amino groups. Choline chloride was used as HBD to form 6 different structures of DES with alcohols (1,4-butanediol, glycerol), organic acids (lactic acid, malic acid), sugars (glucose) and amino groups (urea). As shown in Table 2, the raw materials of different DES have different molecular organizations and stacking modes. Among them, DES-2 (PH 2.23) and DES-3 (PH 2.54) prepared with organic acids have the lowest pH. Existing studies have shown that the polyphenol components in black fruit berries are mainly proanthocyanidins and anthocyanidins, which are easily affected by pH and are stable in the most suitable pH range (2-3).

[0065] Table 2 Physicochemical properties of DES

[0066]

[0067]

[0068] 2.2 Optimal extraction solvent selection

[0069] like Figure 1 As shown in the data, among the six DES, DES-2 (choline chloride: lactic acid) had the highest polyphenol yield of (43.19±0.26) mg / g, followed by DES-5 (39.37±0.52) mg / g prepared from glycerol and DES-1 (37.17±0.53) mg / g prepared from 1,4-butanediol, while DES-6 (30.96±0.16) mg / g prepared from glucose had the lowest yield. The results showed that lactic acid, as HBD, had the most suitable pH (2-3) and the lowest viscosity and density, which were conducive to the dissolution of polyphenols. Ultrasonic-assisted extraction produced cavitation, which could dissolve polyphenols faster and significantly improve the extraction efficiency of polyphenols from Aronia nigra. The yield of polyphenols from Aronia nigra was significantly different from that from traditional organic solvents, with the yields from 80% methanol extraction and 60% ethanol extraction being (15.32±0.6) mg / g and (10.14±0.3) mg / g, respectively. The content of polyphenols from Aronia nigra extracted with DES was 4 to 5 times that from organic solvents.

[0070] 2.3 Effect of molar ratio on extraction yield

[0071] Depend on Figure 2 It can be seen that with the increase of the molar ratio of the DES system, the extraction amount of black fruit polyphenols first increased and then decreased.

[0072] 2.4 Effect of water content on extraction yield

[0073] Depend on Figure 3 It can be seen that with the increase of water content in the DES system, the amount of black fruit polyphenols extracted first increased and then decreased. When the water content was 80%, the amount of black fruit polyphenols extracted reached the highest value and then decreased rapidly, so the water content was selected to be 80%.

[0074] 2.5 Effect of ultrasonic power on extraction yield

[0075] Depend on Figure 4 It can be seen that with the increase of ultrasonic power, the amount of black fruit polyphenols extracted first increased and then decreased. When the ultrasonic power was 240W, the extraction amount reached the highest value and then gradually decreased.

[0076] 2.6 Effect of ultrasonic time on extraction yield

[0077] Depend on Figure 5 It can be seen that the amount of black fruit polyphenols extracted first increased and then decreased with the increase of ultrasonic time. The extraction amount reached the highest value when the ultrasonic time was 40 minutes, and then decreased rapidly.

[0078] 2.7 Box-Behnken response surface methodology experiment

[0079] 2.7.1 Response surface experimental design and result analysis

[0080] Based on the preliminary test results, the test used the DES system molar ratio (factor A), water content (factor B), ultrasonic power (factor C) and ultrasonic time (factor D) as independent variables, and AMP yield as the response value. The Design-Expert 13 software was used to design a response surface experiment with four factors and three levels. The results are shown in Table 3.

[0081] Table 3 Box-Behnken response surface method experimental design and results

[0082]

[0083]

[0084] IBM SPSS Statistics27 statistical software was used for variance significance analysis, and Design-Expert13 software was used for graphic fitting. The results are shown in Table 4. The experimental data were analyzed and the regression equation was obtained as Y=50.61-0.0214A-0.2029B+0.4090C+0.2403D-0.1051AB-0.2158AC+0.1857AD+0.0854BC+0.1856BD+0.2943CD-0.8070A 2 -0.4027B 2 -0.6248C 2 -0.6691D 2

[0085] As shown in Table 4, the significance test F value of this model is 29.02, which is extremely significant (P model <0.0001), and the lack of fit term is not significant (P = 0.1484> 0.05), indicating that the model is statistically significant.

[12] The coefficient of determination R of the equation 2 =0.9667, the model's Adj R 2 =0.9334, Pre R 2 =0.8245, indicating that the equation established based on the change of AMP extraction has a good fit to the experiment and a small experimental error. The model can be used to analyze and predict the extraction process of AMP. From the F value, it can be seen that the influence of each factor on the AMP yield is from large to small: ultrasonic power (C)> ultrasonic time (D)> water content (B)> molar ratio (A). Factors C and A 2 , B 2 , C 2 , D 2 The influence of factors B, D, AC, AD, BC, and BD was extremely significant (P<0.0001), and the influence of factors B, D, AC, AD, BC, and BD was significant (P<0.05), indicating that the model is reliable. As shown in Table 3, the optimal extraction process (choline chloride-lactic acid molar ratio 1:3, water content 80%, ultrasonic power 240W, ultrasonic time 40min) was obtained under this condition. Five groups of parallel verification tests were carried out, and the average AMP yield was measured to be 50.61 mg / g, and the relative error with the theoretical value of 50.75 mg / g was 0.14%, indicating that the model can better reflect the AMP extraction conditions.

[0086] Table 4 Results of variance analysis

[0087]

[0088]

[0089] 2.2.2 Response surface analysis

[0090] The slope of the response surface and the elliptical shape of the contour map can determine the degree of influence of the two factors on the response value. The steeper the slope of the response surface, the more significant the interaction between the two factors; the smoother the trend of the surface, the less significant the interaction between the two factors. The response surface graphs of molar ratio-ultrasonic power, molar ratio-ultrasonic time, and water content-ultrasonic time have good steepness, and the contour lines are elliptical, indicating that the interaction of the two factors has a significant effect on AMP yield (P < 0.05). Ultrasonic power-ultrasonic time has a very significant effect on AMP yield (P < 0.01).

[0091] Test Example 2 Antioxidant Activity Test

[0092] (1) Free radical inhibition ability test

[0093] Depend on Figure 7 It can be seen that in the FRAP test, the antioxidant substances in polyphenols can convert Fe 3+ Reduction to Fe 2+ . Refer to Fe 2+ The standard solution gradient was used to determine the iron ion reducing ability of polyphenols extracted by DES, 80% methanol, and 60% ethanol. The results showed that the reducing ability of polyphenols extracted by DES (4.237±0.07mmol / L) was significantly higher than that of polyphenols extracted by 80% methanol (2.832±0.02)mmol / L and polyphenols extracted by 60% ethanol (2.369±0.01)mmol / L (p<0.05), and the antioxidant capacity was 1.7 to 1.8 times that of other methods.

[0094] Depend on Figure 8 It can be seen that in the ABTS test, ABTS can be oxidized to blue-green free radical ABTS + ABTS in the presence of antioxidants + The effects of polyphenols extracted from DES, 80% methanol, and 60% ethanol (purified) on ABTS were measured. .+ The results showed that the polyphenols extracted from DES (100±4.04)% ABTS .+ The clearance rate was significantly higher than that of 80% methanol-extracted polyphenols (72.8±2.16)% and 60% ethanol-extracted polyphenols (62.3±1.09)% (p<0.05), and the antioxidant capacity was 1.3 to 1.6 times that of other methods.

[0095] In the hydroxyl radical inhibition test, the amount of H2O2 and the OH produced by the Fenton reaction .- When the electron acceptor is given, it will be colored red with Griess reagent, and its color is proportional to OH - The scavenging ability of polyphenols extracted from DES, 80% methanol, and 60% ethanol on hydroxyl free radicals was determined. .- The inhibitory capacity (96.77±3.56)% was significantly higher than that of 80% methanol-extracted polyphenols (86.53±2.98)% and 60% ethanol-extracted polyphenols (64.67±2.12)% (p<0.05), and the inhibitory capacity was 1.1 to 1.5 times that of other methods.

[0096] In the DPPH free radical scavenging ability test, the DPPH free radical has a single electron and a strong absorption at 517nm. Its solution is purple. When the scavenger is present, the color becomes lighter. Its color is proportional to the DPPH free radical scavenging ability. The scavenging ability of polyphenols extracted by DES, 80% methanol, and 60% ethanol (purified) on DPPH free radicals was determined. The results showed that the DPPH scavenging rate of polyphenols extracted by DES (100±2.99)% was significantly higher than that of polyphenols extracted by 80% methanol (94.34±1.56)% and polyphenols extracted by 60% ethanol (87.35±2.34)% (p<0.05), and the DPPH free radical scavenging ability was 1.06 to 1.15 times that of other methods.

[0097] (2) Effects of simulated aronia nigra extract and blueberry gastric and intestinal digestive juice on α-glucosidase activity in vitro

[0098] The inhibition rates of the Aronia nigra extract (prepared according to the method of Example 1) and the blueberry gastric and intestinal digestive juices on α-glucosidase are as follows: Fig. 9 As shown in the results, after in vitro simulated digestion, the inhibition rate of each digestive juice on α-glucosidase decreased. The inhibition rate of undigested juice of black fruit on α-glucosidase reached (76.4±1.2)%, which decreased to (15.1±0.4)% after gastric juice digestion, and then increased to (30.4±1.2)% after intestinal juice digestion (p<0.01). The inhibition rate of undigested juice of blueberry on α-glucosidase reached (68.2±0.9)%, which decreased to (5.9±0.2)% after gastric juice digestion, and then increased to (9.1±0.2)% after intestinal juice digestion (p<0.01). The inhibition rate of gastric digestive juice of black fruit on α-glucosidase was 2.56 times that of gastric digestive juice of blueberry, and the inhibition rate of intestinal digestive juice of black fruit on α-glucosidase was 3.34 times that of intestinal digestive juice of blueberry. Inhibiting the activity of α-glucosidase in the intestine can effectively slow down the absorption of glucose in the small intestine, thereby controlling blood sugar. The results showed that the intestinal fluid environment (weakly alkaline) is more conducive to the release of hypoglycemic active substances in black fruit and blueberry than the gastric fluid environment (acidic). Secondly, black fruit has a stronger ability to lower blood sugar than blueberry.

[0099] (3) Effects of in vitro simulated black fruit and blueberry gastric and intestinal digestive juices on pancreatic lipase activity

[0100] The inhibition rates of the Aronia nigra extract (prepared according to the method of Example 1) and blueberry gastric and intestinal digestive juices on pancreatic lipase are as follows: Fig.10As shown, the inhibition rate of pancreatic lipase in the undigested juice of black fruit reached (100±2.2)%, and it still reached (100±1.9)% after digestion with gastric juice, and then decreased to (92.7±2.9)% after digestion with intestinal juice (p<0.05). The inhibition rate of α-glucosidase in the undigested juice of blueberry reached (100±1.5)%, and decreased to (95.5±1.1)% after digestion with gastric juice, and then decreased to (89.3±1.7)% after digestion with intestinal juice (p<0.05). The undigested juice of black fruit and blueberry completely inhibited the activity of pancreatic lipase, and the inhibition rate of pancreatic lipase in the gastric digestive juice of black fruit was 1.05 times that of the gastric digestive juice of blueberry. The inhibition rate of pancreatic lipase in the intestinal digestive juice of black fruit was 1.04 times that of the intestinal digestive juice of blueberry. Lipase plays an important role in human lipid metabolism. Inhibiting the activity of lipase can effectively reduce the absorption of lipids by the human body, thereby controlling blood lipids. The results showed that both black fruit and blueberry have strong ability to inhibit lipase. The gastric fluid environment (acidic) is more conducive to the release of lipid-lowering active substances in black fruit and blueberry than the intestinal fluid environment (weakly alkaline). Secondly, black fruit has a stronger lipid-lowering ability than blueberry.

[0101] The content determination method in the above embodiments and comparative examples is:

[0102] 1. Determination of polyphenol extraction

[0103] The content of black fruit polyphenols was determined by the Folin phenol colorimetric method. Take 0.01g of gallic acid reference substance, accurately weigh it, fully dissolve it and make up to 100mL, to obtain a gallic acid reference substance solution with a mass concentration of 0.1mg / mL. Measure 0.2-1.0mL of gallic acid reference substance solution respectively, transfer it to a 10mL volumetric flask, add water to 2.0mL, add 0.5mL of Folin phenol solution respectively, mix and react for 6min, and finally add 1.5mL of 10% Na2CO3 solution and make up to volume. React at room temperature in the dark for 2h, and measure the absorbance at 765nm. Perform linear regression with the mass concentration (X, mg / mL) of the gallic acid reference substance as the horizontal coordinate and the absorbance (Y) as the vertical coordinate, and the regression equation Y=14.355X+0.0561(R 2 =0.9993) The mass concentration of gallic acid reference substance has a good linear relationship with the absorbance in the range of 0.002-0.01 mg / mL. The calculation formula of polyphenol content in Aronia nigromaculata is shown in formula (1).

[0104]

[0105] Wherein Y is the content of black fruit polyphenols, C is the sample mass concentration calculated from the standard curve, V is the fixed volume, D is the dilution factor, and m is the sampling amount (g).

[0106] 2. Purification of polyphenols by macroporous resin

[0107] Preparation of black fruit sample solution: Take 10g of black fruit dry powder and mix it with DES, 80% methanol and 60% ethanol at a ratio of 1:40 (w / v). Extract under the best extraction conditions. Centrifuge the extract at 10000r / min and 4℃ for 10min, take the supernatant and filter it for later use.

[0108] Polyphenols were purified by column chromatography. AB-8 macroporous resin activated with 1BV was mixed with 2BV sample solution and oscillated at a constant temperature of 20°C and 160r / min until the macroporous resin was completely adsorbed. Load the column with a column load of 1 / 2. The 2BV sample solution was passed through the column, and choline chloride, lactic acid and other impurities were washed away with 3BV pure water until the outflowing liquid was colorless and odorless. The polyphenols adsorbed in the macroporous resin column were eluted with 2BV of 75% ethanol until the outflowing liquid was transparent. The eluate was collected and vacuum-freezed and dried after removing water, methanol and ethanol by rotary evaporation at 40°C. The freeze-dried powder was stored at -20°C for later use.

[0109] The obtained crude polyphenols and the purified polyphenol freeze-dried powder were used to determine the polyphenol content according to formula (1), and the polyphenol purity was determined according to formula (2).

[0110]

[0111] Wherein, C1 is the polyphenol content (mg / g) in the crude extract / purified product extracted by DES, 60% ethanol and 80% methanol, and C is the total phenol content (mg / g).

[0112] 3. Antioxidant activity test

[0113] (1) Free radical inhibition ability test

[0114] The antioxidant capacity of polyphenols extracted from DES and polyphenols extracted from 80% methanol and 60% ethanol (after purification) was evaluated using total antioxidant capacity (T-AOC) detection kits (FRAP microplate method, ABTS microplate method); hydroxyl free radical test kit, DPPH free radical test kit.

[0115] The polyphenols extracted by the three methods were prepared into test solutions with the same concentration. FRAP, ABTS and DPPH working solutions were prepared according to the instructions.

[0116] FRAP method: prepare Fe according to the instructions 2+ Standard gradient. Mix the FRAP working solution (264 μL) and the polyphenol test solution (30 μL), place in a 37°C constant temperature water bath for 30 min, and measure the absorbance at 593 nm. 2+ The corresponding Fe on the standard curve 2+ The concentration can be used to determine the iron ion reducing ability of the polyphenol test solution.

[0117] ABTS method: Mix ABTS working solution (280 μL) and polyphenol test solution (7 μL), react at room temperature for 6 min, and measure the absorbance at 734 nm. Calculate ABTS according to the absorbance of the polyphenol test solution according to formula (3): .+ Free radical scavenging rate.

[0118]

[0119] Among them, A x A is the test group, y A0 is the blank group in which the sample solution is replaced by anhydrous ethanol.

[0120] Hydroxyl free radical test: Determined by colorimetry. Prepare 0.03% H 2 O 2 Standard application fluid; Fe 2+ Substrate application solution; salicylic acid substrate application solution; Griess colorimetric reagent. Preheat the prepared application solution in a 37°C water bath for 3 minutes. First add Fe 2+ The substrate application solution (0.2 mL), polyphenol test solution (0.2 mL), and salicylic acid substrate application solution (0.4 mL) were added and the timing was started at the same time. The mixture was quickly vortexed and reacted at 37°C for 1 min (accurately measured with a stopwatch). Then, the color developer was immediately added to terminate the reaction. The hydroxyl radical scavenging rate was calculated according to formula (4).

[0121]

[0122] Among them A y No Fe added 2+ The control group of substrate application solution, A x A is the sample measurement group, c To add H 2 O 2 Standard application liquid group, A0 is anhydrous ethanol instead of sample blank group

[0123] DPPH free radical test: According to the instructions, the polyphenol test solution (400 μL) and the DPPH working solution (600 μL) were mixed, kept at room temperature 25°C in the dark for 30 min, centrifuged at 4000 r / min for 5 min, and the absorbance value was measured at 517 nm. The DPPH free radical scavenging rate was calculated according to formula (3).

[0124] (2) In vitro simulation of gastrointestinal digestion

[0125] Simulated oral and gastric digestion: Take 1 mL of Aronia nigra extract and 1 mL of blueberry extract, mix them with 5 mL of water, and then add 1 mL of simulated saliva to obtain black fruit and blueberry gastric digestive juices.

[0126] Simulated intestinal digestion: After the gastric digestive fluid was cooled to room temperature, the pH was adjusted to 7.60, and 7.5 mL of simulated intestinal fluid was added. The subsequent steps were consistent with gastric digestion to obtain Aronia nigra extract and blueberry intestinal digestive fluid.

[0127] (3) In vitro simulated gastric and intestinal digestive fluid inhibition test on α-glucosidase activity

[0128] A certain amount of α-glucosidase was dissolved in pH 7.20 phosphate buffer. Phosphate buffer, Aronia nigra extract and blueberry gastric and intestinal digestive juice were respectively aspirated into a 96-well plate, α-glucosidase solution was added, mixed and reacted at 37°C for 10 minutes, then pNPG solution with pH 6.80 was added and mixed, and reacted at the same temperature for 10 minutes. Finally, 0.2 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance value was measured at a wavelength of 405 nm. The inhibition rate of Aronia nigra extract and blueberry gastric and intestinal digestive juice on α-glucosidase activity was calculated according to formula (5).

[0129]

[0130] Among them, A m A is the control group without enzyme addition. b For the sample group with enzyme added, A c The blank group was composed of ethanol instead of the sample.

[0131] (4) In vitro test of the inhibition of pancreatic lipase activity by simulated gastric and intestinal digestive juices

[0132] A certain amount of pancreatic lipase was dissolved in pH 8.0 Tris-HCl buffer, and the extract of Aronia nigra and the gastric and intestinal digestive juice of blueberry were mixed with the pancreatic enzyme solution, respectively, and reacted at 37°C for 10 minutes. Then 2 mmol / L pNPP solution was added and reacted at the same temperature for 10 minutes. After the reaction was completed, the reaction was terminated in a boiling water bath for 5 minutes, and the supernatant was centrifuged and its absorbance was measured at 405 nm. The inhibition rate of the gastric and intestinal digestive juices of black fruit and blueberry on the activity of pancreatic lipase was calculated according to formula (6).

[0133]

[0134] 4. Data Processing

[0135] All experiments were repeated three times in parallel, and IBM SPSS Statistics 22.0 software was used to analyze the data, Design-Expert 13 software to design the experiments, and Origin 2021 software to draw the graphs.

Claims

1. A method for preparing an extract of Aronia nigra, characterized in that The method comprises the following steps: The black fruit is crushed and sieved to obtain fine powder, the black fruit powder is weighed and placed in a brown container, and DES extract is added, the ratio of black fruit powder to DES extract is 1:40-50g / ml, and after ultrasonication at 200-240W and 37°C for 30-40min, the obtained extract is centrifuged for 10-20min, and the supernatant is separated to obtain the product.

2. The method for preparing the Aronia nigra extract according to claim 1, characterized in that The DES extract is a mixture of choline chloride and lactic acid in a molar ratio of 1:1-3, and has a water content of 30-80%.

3. The method for preparing the Aronia melanocarpa extract according to claim 2, characterized in that The DES extract is a mixture of choline chloride and lactic acid in a molar ratio of 1:3, and has a water content of 80%.

4. The method for preparing the Aronia melanocarpa extract according to claim 1, characterized in that The black fruit is the black fruit dried at a constant temperature of 40-50°C.

5. The method for preparing the Aronia melanocarpa extract according to claim 1, characterized in that The centrifugal condition is 10000-12000r / min.

6. The method for preparing the Aronia melanocarpa extract according to claim 1, characterized in that The ultrasonic time is 35-40 minutes.

7. The method for preparing the Aronia melanocarpa extract according to claim 6, characterized in that The ultrasonic time is 40 minutes.

8. The method for preparing the Aronia melanocarpa extract according to claim 1, characterized in that The ultrasonic power is 240W.

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