Preparation method of aronia extract and application of aronia extract in protecting intestinal barrier function

Abstract

The invention discloses a preparation method of an aronia extract and an application of the aronia extract in protecting an intestinal barrier function, and belongs to the technical field of natural product extraction. The method comprises the following steps: mixing a hydrogen bond acceptor and a hydrogen bond donor according to a molar ratio of 1: (1-3), heating, and continuously stirring to obtain a natural eutectic solvent; dissolving an enzyme in the natural eutectic solvent to obtain an enzyme solution; mixing the enzyme solution with the aronia fruit powder, and performing ultrasonic extraction, enzyme deactivation and purification to obtain the aronia extract. According to the method, a hydrogen bond donor in a natural eutectic solvent is comprehensively utilized to improve the stability and solubility of anthocyanin in a system, and the good regulation and control capability and molecular complexing stability of hydrogen bond receptor natural quaternary ammonium salt; the method is superior to a traditional method in the aspects of extraction rate, environmental friendliness and raw material availability, the content of the extracted effective components is increased by nearly two times or more, and the method is suitable for high-value utilization of anthocyanin-rich natural resources represented by aronia.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a method for preparing cherifolia extract and its application in protecting intestinal barrier function. Background Technology

[0002] Anthocyanins are water-soluble compounds formed by anthocyanidins and one or more sugar molecules linked by glycosidic bonds. They are widely found in the fruits, petals, and leaves of fruits and vegetables. Anthocyanins belong to the flavonoid class of compounds and have various biological activities such as anti-oxidation, anti-inflammation, and regulation of intestinal microecology. They have been shown to enhance intestinal barrier function through mechanisms such as upregulating tight junction protein expression and inhibiting the production of inflammatory factors. Therefore, natural products rich in anthocyanins have become a core resource for the development of novel functional foods or nutritional supplements.

[0003] Currently, the content of cyanidin-3-O-galactoside (C3gal) in Aronia melanocarpa fruit is 1.0–3.6 g / 100g fresh weight, but about 50% exists in bound form (NEAs). Traditional organic solvent extraction can only obtain free form (EAs), resulting in a resource utilization rate of less than 50%.

[0004] In existing extraction technologies:

[0005] Strong acid / alkali hydrolysis method: It easily degrades active ingredients and requires extreme pH conditions (such as pH < 2 or > 10), and has high energy consumption;

[0006] Traditional DES extraction: Due to its high viscosity and excessive hydrophilicity, it is difficult to effectively break the cell wall-anthocyanin binding bond;

[0007] Enzymatic hydrolysis alone: ​​Pectinase or cellulase alone can only release less than 60% of the enzyme, and the cost is high.

[0008] Although there have been reports on the use of DES to extract flavonoids or polyphenols, such as Chinese patent CN111568934A which discloses a method for extracting total flavonoids from Moringa using a deep eutectic solvent, and Chinese patent CN105777696B which discloses a method for extracting anthocyanins using a deep eutectic solvent, these methods mostly focus on free or water-soluble compounds and are not suitable for the extraction of bound anthocyanins, thus failing to effectively extract anthocyanins from Aronia arguta. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the above-mentioned technical problems and provide a method for preparing a wild cherifolia extract that is convenient to promote and use and can effectively extract bound cyanidin glycosides from wild cherifolia, and its application in protecting the intestinal barrier function.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a wild cheriberry extract, wherein the extract is wild cheriberry anthocyanin, including cyanidin-3-O-galactoside.

[0011] In another aspect, the present invention provides the use of chokeberry extract in the preparation of foods, health products or pharmaceuticals that protect the intestinal barrier function.

[0012] Preferred methods include increasing TEER value, reducing permeability, and upregulating the expression of ZO-1, OCLN, and CLDN1 linker proteins to repair the epithelial barrier.

[0013] Another aspect of the present invention provides a method for preparing chokeberry extract, comprising the following steps:

[0014] Step 1: Mix hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:1 to 3, heat, and stir continuously to obtain a natural eutectic solvent;

[0015] Step 2: Dissolve the enzyme in the natural eutectic solvent obtained in Step 1 to obtain an enzymatic hydrolysis-hydrated natural eutectic solvent;

[0016] Step 3: Mix the enzymatic hydrolysis-hydrated natural eutectic solvent obtained in Step 2 with aronia berry powder, extract by ultrasonication, inactivate the enzyme, and purify to obtain aronia berry extract.

[0017] Preferably, the hydrogen bond acceptor in step 1 is at least one of betaine, choline chloride, carnitine salt, glycine, and sodium acetate;

[0018] The hydrogen bond donor is at least one of lactic acid, malic acid, citric acid, glycerol, urea, xylitol, sorbitol and ethylene glycol.

[0019] Preferably, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is citric acid, with a molar ratio of 1:1.

[0020] Preferably, in step 3, the mass ratio of the enzyme in the enzymatic hydrolysis-hydration natural eutectic solvent to the wild cherry fruit powder is 5% to 15%.

[0021] The ratio of wild cherry fruit powder to enzymatically hydrolyzed hydrated natural eutectic solvent is 1:20;

[0022] The extraction temperature is 30–50℃;

[0023] The extraction time is 60–180 min.

[0024] Preferably, in step 2, the enzyme is one or more of the following: complex cellulase, pectinase, protease, and α-amylase.

[0025] Preferably, the enzyme is a combination of pectinase and compound cellulase in a mass ratio of 1:1 or a combination of compound cellulase, pectinase and β-amylase in a mass ratio of 2:2:1.

[0026] The advantages of this invention compared to existing technologies are as follows: This invention enables the synergistic extraction of free and bound anthocyanins, improving extraction efficiency and resource utilization. By constructing an "enzymatic hydrolysis-hydration natural eutectic solvent (DES) synergistic extraction system," the hydrogen bond donors in the prepared natural eutectic solvent are utilized to increase the stability and solubility of anthocyanins in the system, as well as the good regulatory ability and molecular complexation stability of the natural quaternary ammonium salts of hydrogen bond acceptors. This allows for the simultaneous and efficient extraction of free and bound anthocyanins from wild cherifolia. The extraction rate, environmental friendliness, and raw material utilization are all superior to traditional methods. The content of the extracted effective components is increased by nearly 2 times, and the residual anthocyanin content in the residue is significantly reduced, which is conducive to the full utilization of resources and subsequent functional evaluation.

[0027] The extract obtained in this invention exhibits superior bioactivity in the in vitro Caco-2 intestinal barrier model, significantly increasing TEER value, reducing permeability, and upregulating the expression of key tight junction proteins such as ZO-1, OCLN, and CLDN1. It significantly repairs the damaged epithelial barrier under the stimulation of inflammatory factors and has a clear intestinal protective function, providing a new direction for the application and development of wild cheri in functional foods, nutritional interventions, and intestinal health products.

[0028] By innovatively integrating the dual strategies of "green extraction + structural release", it breaks through the viscosity bottleneck of traditional DES extraction. By adjusting the viscosity of DES through hydration, it significantly enhances its penetration and diffusion ability in plant tissues, making the extraction system have both "structural dissociation ability + high solubility". It forms an innovative closed loop in terms of food applicability, biosafety and viscosity control, and provides a new solution for achieving mild and low-energy extraction of bound active substances.

[0029] It can provide a new path for the extraction and application development of active ingredients in functional agricultural products, and is particularly suitable for the high-value utilization of natural resources rich in anthocyanins, such as wild cherries. Attached Figure Description

[0030] Figure 1 This is a high-performance liquid chromatogram of cyanidin-3-O-galactoside extracted and purified from wild chokeberry in an embodiment of the present invention; wherein peak a is cyanidin-3-O-galactoside (t = 22.763 min); peak b is cyanidin-3-O-glucoside (t = 24.966 min); peak c is cyanidin-3-O-arabinoside (t = 26.120 min); and peak d is cyanidin-3-O-xyloside (t = 34.180 min).

[0031] Figure 2 The total polyphenol content of the aronia berry extract in this embodiment of the invention was determined by the Folin-Ciocalteu method, and the results are expressed as mgGAE (Gallic Acid Equivalent) / g. The control group consisted of methanol extraction (MeOH) and water extraction (H2O), and the extracts numbered 1–18 corresponded to aronia berry extracts obtained by different enzymatic hydrolysis synergistic deep eutectic solvent (DES) systems, respectively.

[0032] Figure 3 The content of cyanidin-3-O-galactoside (C3gal) in the aronia berry extract obtained in the embodiments of the present invention was quantitatively analyzed by HPLC external standard method. The control group consisted of methanol extraction (MeOH) and water extraction (H2O), and the extracts were numbered 1–18, corresponding to the aronia berry extracts obtained after extraction by different enzymatic methods in conjunction with DES system.

[0033] Figure 4 The total bound polyphenol content of wild cherry pomace after acid hydrolysis in this embodiment of the invention was determined using the Folin-Ciocalteu method, and the results are expressed as mg GAE (Gallic Acid Equivalent) / g. The control group consisted of pomace obtained after methanol extraction (MeOH) and water extraction (H2O), with numbers 1–18 corresponding to pomace obtained after extraction using different enzymatic methods in conjunction with the DES system.

[0034] Figure 5 The content of bound cyanidin-3-O-galactoside (C3gal) was measured after acid hydrolysis of the extracted Aronia arguta pomace in this embodiment of the invention, and quantitative analysis was performed using HPLC external standard method. The control group consisted of pomace obtained after methanol extraction (MeOH) and water extraction (H2O), with numbers 1–18 corresponding to pomace obtained after extraction using different enzymatic methods in conjunction with DES systems.

[0035] Figure 6 This invention illustrates the effect of different extracts on the recovery of transmembrane resistance (TEER) in Caco-2 cells after inflammatory injury. TEER values ​​are expressed as a percentage of the initial value before treatment (%initial). The control group included a normal control (Control), an inflammation model group (IC), and a methanol extraction group (MeOH); numbers 2, 5, 8, 11, 14, and 17 correspond to Aronia arguta extracts obtained after extraction using different enzymatic methods in conjunction with DES systems.

[0036] Figure 7This study analyzes the effects of different extracts on the changes in Caco-2 cell barrier permeability induced by inflammatory factors in the embodiments of the present invention. (A) Cumulative throughput of Lucifer Yellow over 24 hours; (B) Apparent permeability coefficient (Papp) measurement results. The control group included normal control (Control), inflammation model group (IC), and methanol extraction group (MeOH); numbers 2, 5, 8, 11, 14, and 17 correspond to the wild cherries extracts obtained after extraction by different enzymatic methods combined with DES system;

[0037] Figure 8 This study investigated the regulatory effects of extracts obtained by different extraction methods on the mRNA and protein expression of tight junction proteins (ZO-1, OCLN, CLDN1) in Caco-2 cells under inflammatory stimulation. The control group included the inflammation model group (IC) and the methanol extraction group (MeOH); numbers 2, 5, 8, 11, 14, and 17 corresponded to wild cherries extracts obtained after extraction using different enzymatic methods in conjunction with the DES system. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings.

[0039] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0040] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0041] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0042] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0043] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0044] This invention provides a method for preparing aronia berry extract, comprising the following steps:

[0045] Step 1: Preparation of natural eutectic solvent

[0046] The hydrogen bond acceptor and hydrogen bond donor are mixed at a molar ratio of 1:(1-3), and stirred continuously at 600 rpm at 40-60°C until the solution is clear and homogeneous, thus obtaining a natural eutectic solvent.

[0047] The hydrogen bond acceptor includes, but is not limited to, at least one of naturally derived betaine (Bet), choline chloride (ChCl), carnitine salt, glycine, and sodium acetate; the hydrogen bond donor includes, but is not limited to, at least one of lactic acid, malic acid, citric acid, glycerol, urea, xylitol, sorbitol, and ethylene glycol; preferably, it is a eutectic mixture of betaine and citric acid; the molar ratio of hydrogen bond acceptor to hydrogen bond donor is preferably 1:1.

[0048] Step 2: Preparation of enzymatic hydrolysis-hydration natural eutectic solvent

[0049] With an enzyme / substrate (E / S) ratio of 5% to 15%, the enzyme is dissolved in the natural eutectic solvent obtained in step one to obtain an enzymatic hydrolysis-hydration natural eutectic solvent.

[0050] The enzymes include, but are not limited to, one or more of compound cellulase, pectinase, protease and α-amylase; preferably a combination of compound cellulase, pectinase and α-amylase, more preferably a mass ratio of compound cellulase, pectinase and α-amylase of 2:2:1; the enzyme / substrate (E / S) ratio is preferably 10%; the material ratio of wild cherry fruit powder to natural eutectic solvent is 1:(10-30), preferably 1:20.

[0051] Step 3: Preparation of Aronia berry extract

[0052] 1) Mix the enzymatic hydrolysis-hydration natural eutectic solvent obtained in step 2 with wild cherry fruit powder, and stir continuously for 90-180 min at 30-60℃, ultrasonic power 240W, and frequency 60kHz to obtain crude extract.

[0053] 2) Heat the crude extract in a 90°C water bath for 5 minutes to inactivate the enzyme; then centrifuge at 3000g at 4°C and collect the supernatant; then dilute the extract with pure water at a ratio of 1:(1-5) (preferably 1:4), and load the solution onto an AB-8 macroporous resin adsorption column. Wash away the eutectic solvent components with deionized water, and then elute with an acidified methanol aqueous solution (preferably 40% methanol aqueous solution containing 0.01% citric acid) at a volume of 5-8 times the column volume (preferably 6 times). Concentrate the collected eluent under nitrogen blowing and freeze-dry to obtain the wild cherry extract.

[0054] This invention also provides the application of the aronia berry extract obtained by the above method in foods, health products, or pharmaceuticals that protect intestinal barrier function, and the application of the above extract in foods, health products, or pharmaceuticals that alleviate intestinal barrier dysfunction caused by inflammation. Experimental results show that, compared with traditional methanol extraction, the cyanidin-3-galactoside extract obtained by DES solvent extraction has a significantly stronger protective effect against intestinal epithelial barrier dysfunction induced by inflammatory factors (IC) TNF-α, IL-1β, and IFN-γ, manifested as higher integrity and tightness of the Caco-2 cell monolayer barrier under inflammatory factor stimulation. Compared with the extract obtained by the traditional methanol extraction method, the TEER% value of the Caco-2 cell monolayer membrane induced by inflammatory factors after DES extract treatment recovered to 49.42±7.83%~70.69±4.02%, and the TEER% value of some DES extracts was significantly higher than that of the traditional methanol (50% v / v methanol aqueous solution containing 0.1% hydrochloric acid) extraction method, with a transmembrane resistance value 1.48~1.79 times that of the methanol method. Figure 1 The transmembrane transport of Lucifer Yellow fluorescent dye treated with DES extract decreased by 36.01±7.79% to 70.74±3.16%, significantly lower than that of LY transmembrane transport by methanol extraction (13.25±7.06%). Figure 2 Furthermore, compared with methanol extraction, DES extract treatment significantly increased the mRNA and protein expression levels of ZO-1, OCLN, and CLDN1 in the intestinal barrier tight junctions. Figure 3 Compared to the methanol method, certain DES extracts significantly increased the mRNA expression levels of tight junction proteins ZO-1, OCLN, and CLDN1 by 26.49%, 19.47%, and 56.95%, respectively; and significantly increased the protein expression levels by 32.78%, 12.37%, and 88.85%. These studies on intestinal barrier function based on the Caco-2 monolayer cell membrane demonstrate that, compared to traditional extraction methods, the natural eutectic solvent extracts prepared in this invention have enhanced protective effects on damaged intestinal barriers.

[0055] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0056] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0057] Example 1: Construction of an enzymatic hydrolysis-assisted natural eutectic solvent (DES) extraction system

[0058] 1.1 Experimental Materials

[0059] (1) Pretreatment of Aronia berries: After removing the leaves and stems from commercially available fresh aronia berries, vacuum freeze-dry them, then pulverize them through a 40-mesh sieve to obtain aronia berry powder. Store the powder in a sealed, light-proof container at -80℃ for later use.

[0060] (2) Enzyme preparations: pectinase (Sigma Aldrich P2401, derived from Rhizopus), compound cellulase (Viscozyme L, Sigma Aldrich V2010), protease (Sigma Aldrich P5380) and α-amylase (Sigma Aldrich P5380), pepsin (Roche 10108057001, derived from porcine gastric mucosa), trypsin (Sigma Aldrich P7545, derived from porcine pancreas), xylanase (Sigma Aldrich X2753, derived from Thermomyces lanuginosus).

[0061] (3) Preparation of natural eutectic solvent (NADES): Weigh a certain mass of hydrogen ion donors (citric acid, lactic acid, malic acid, and urea) and hydrogen ion acceptors (betaine, choline chloride, glycine, and sodium acetate) according to the molar ratio in the table below, mix them in a flask, stopper the flask, and magnetically stir at 600 rpm in a water bath at 40–60°C until a clear and transparent solution is obtained. After cooling to room temperature, vacuum dry at 80°C for 24 hours, and transfer to a desiccator for sealed storage. When ready to use, add pure water in a certain proportion to obtain the DES extraction solvent.

[0062] 1.2 Extraction System Construction Method

[0063] To investigate the synergistic extraction effect of enzymatic hydrolysis and DES under different enzyme addition methods, the following three system construction methods were designed:

[0064] (1) Method A: Enzymatic hydrolysis first, followed by the addition of DES. Dissolve each enzyme (1g) in distilled water (10mL) to prepare a 10% enzyme working solution. Add the enzyme working solution to aronia berry powder at a 1:1 ratio to make the E / S ratio 10%, mix well to form a uniform slurry, incubate in a 55℃ constant temperature shaker for 2 hours, and then heat in a 90℃ water bath for 5 minutes to inactivate the enzyme. After the slurry cools to room temperature, add NADES solvent to the slurry at a ratio of fruit powder to DES solvent of 1:20 and mix well. Extract in an ultrasonic water bath at 40℃ for 120 minutes, with an ultrasonic power of 240W and a frequency of 60kHz.

[0065] (2) Method B: The enzyme and fruit powder are dry-mixed, and then NADES is added for direct co-extraction. The selected enzyme is added directly to the lyophilized fruit powder at an enzyme / substrate ratio of 10% (E / S) and mixed thoroughly. The mixture is incubated in a 55°C shaker for 2 hours. Subsequently, it is heated in a 90°C water bath for 5 minutes to inactivate the enzyme. After cooling to room temperature, a slurry is obtained. DES solvent is added to the slurry at a ratio of fruit powder to DES solvent of 1:20 and mixed thoroughly. The mixture is then extracted in an ultrasonic water bath at 40°C for 120 minutes with an ultrasonic power of 240W and a frequency of 60kHz.

[0066] (3) Method C: Enzymes are pre-dissolved in NADES and directly extracted with fruit powder. The enzyme is dissolved in DES to form an enzyme solution system, then mixed with fruit powder at a material-to-liquid ratio of 1:20 and extracted at 40°C. Each enzyme (10% E / S) and DES solvent (fruit powder to solvent ratio 1:20) are added to the lyophilized fruit powder and mixed thoroughly. Extraction is performed in an ultrasonic water bath at 40°C for 120 min, with an ultrasonic power of 240 W and a frequency of 60 kHz, to achieve simultaneous enzymatic hydrolysis and solvent extraction. Afterwards, the enzymes are inactivated by heating in a 90°C water bath for 5 min.

[0067] 1.3 Collaborative Extraction Steps

[0068] Taking method C as an example, the specific steps are as follows:

[0069] (1) Weigh a certain amount of betaine and citric acid in a molar ratio of 1:1, and stir magnetically at 600 rpm for 1-2 hours in a water bath at 40-60℃ until a clear and homogeneous solution is formed.

[0070] (2) The freeze-dried wild cherry fruit powder and pectinase (10:1 mass ratio, the enzyme mass being 10% of the fruit powder mass) were mixed with the above NADES solution at a material-to-liquid ratio of 1:20; the mixture was extracted for 120 min by magnetic stirring in an ultrasonic water bath at 40°C with an ultrasonic power of 240W and a frequency of 60kHz.

[0071] (3) After extraction, heat in a 90℃ water bath for 5 min to terminate enzyme activity;

[0072] (4) Centrifuge at 4℃ and 3000g, and take the supernatant for total polyphenol content analysis.

[0073] (5) Dilute the supernatant with pure water at a ratio of 1:4, then load the sample onto an AB-8 macroporous resin adsorption column. Wash away the eutectic solvent components with deionized water, then elute with an acidified methanol aqueous solution (preferably a 40% methanol aqueous solution containing 0.01% citric acid) at a volume of 5 to 8 column volumes (preferably 6). Concentrate the collected eluent under nitrogen blowing, then freeze-dry to obtain a high-purity aroniaberry anthocyanin extract. Used for UPLC to determine the anthocyanin content.

[0074] 1.4 Preliminary Results Verification

[0075] The total polyphenol content was determined using the Folin-Ciocalteu method, with traditional methanol extraction (50% v / v methanol aqueous solution containing 0.1% hydrochloric acid, extracted in an ultrasonic water bath at 40℃ for 120 min, ultrasonic power 240W, frequency 60kHz) as a control. The total polyphenol content was 70.42 mg GAE (Gallic Acid Equivalent) / g. Freeze-dried arugula powder was subjected to enzymatic hydrolysis with different enzymes, including pectinase, complex cellulase, protease, α-amylase, pepsin, trypsin, and xylanase, in three different extraction systems (A, B, and C). The results are shown in Table 1. Method C (enzymatic hydrolysis and DES extraction were performed simultaneously) was superior to methods A and B in terms of extraction efficiency and total polyphenol content, and it was also convenient to operate and the system was stable, making it suitable as the basis for subsequent optimization experiments. During this process, the anthocyanin content obtained using pectinase extraction was the highest, followed by complex cellulase (see Table 1). Pepsin, trypsin, and xylanase were excluded from the core process of this invention because they could not significantly increase the total polyphenol content of aronia berries. Furthermore, the extract obtained by enzymatic hydrolysis combined with NADES extraction, analyzed by high-performance liquid chromatography, showed that the main component was cyanidin-3-O-galactoside (C3gal), which accounted for up to 85.69% of the total anthocyanin content. Figure 1 As shown in the figure. Furthermore, pretreatment with enzymes before extraction prolongs the contact time between the enzyme and the substrate, which may not only lead to the degradation of phenolic compounds but also release a large amount of protein. This protein may then form a complex with the phenolic substances, thereby inhibiting their effective release. The poor treatment effect of the pre-prepared enzyme solution indicates that the residual moisture in the lyophilized sample is sufficient to maintain enzymatic hydrolysis, and no additional large amount of water is needed. Therefore, the addition of excess water actually inhibits the extraction rate of anthocyanins.

[0076] Table 1. Total polyphenol content of wild cherries after treatment with different enzyme-deep eutectic extraction systems

[0077]

[0078] Note 1: The above values ​​are mean ± standard deviation (n=3), and the unit is mg GAE (Gallic Acid Equivalent) / g;

[0079] 2: The differences between the means of different superscript letters in the same row or column (ac indicates between rows, α-δ indicates between columns) are statistically significant (p≤0.05).

[0080] Example 2: Optimization of key parameters in an enzymatic hydrolysis-natural eutectic solvent (NADES) synergistic extraction system

[0081] 2.1 Parameter Optimization Design and Methods

[0082] 2.1.1 Enzyme Screening: Based on previous enzymatic hydrolysis experiments (Table 1), it was found that the total polyphenol content was significantly higher in enzymatic hydrolysis involving pectinase, compound cellulase, and α-amylase than in the control group, with the order being pectinase > compound cellulase > α-amylase. Therefore, pectinase was selected as the basic enzyme for compound hydrolysis studies to disrupt cell wall structure and release anthocyanins. A combined application experiment of pectinase and compound cellulase was conducted with a total enzyme dosage of 10% (E / S). The results are shown in Table 2 below. The two enzymes were used in a 1:1 mass ratio, demonstrating strong synergistic degradation ability. The total polyphenol content was 85.43 ± 0.13 mg GAE / g, and the cyanidin-3-O-galactoside (C3gal) content was 19.54 ± 0.20 μg / mg, significantly higher than other treatment groups.

[0083] Table 2. Effects of different enzyme combinations on the total polyphenol and cyanidin-3-O-galactoside content of cherifolia extract.

[0084]

[0085]

[0086] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0087] Based on the results in Table 2, the combined effect of pectinase and compound cellulase at a 1:1 mass ratio was further investigated with α-amylase. The results are shown in the table below:

[0088] Table 3. Effects of the ratio of three enzymes on the total polyphenols and cyanidin-3-O-galactoside content of cherifolia extract.

[0089]

[0090] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0091] As shown in the table above, the combination of compound cellulase, pectinase, and α-amylase in a 2:2:1 ratio resulted in significantly higher total polyphenol and cyanidin-3-O-galactoside contents compared to other combinations, demonstrating a strong synergistic ability to degrade cell walls and release active ingredients. Therefore, subsequent experiments will employ a 2:2:1 combination of compound cellulase, pectinase, and α-amylase for enzymatic hydrolysis.

[0092] 2.1.2 Enzyme / substrate ratio (E / S) optimization

[0093] The mass ratios of the three compound enzymes to the freeze-dried fruit powder were set to 5%, 10%, and 15%, respectively. The results are shown in Table 4. The results indicate that the extraction rate was optimal under the 10% E / S condition, which was used as a fixed parameter for subsequent experiments.

[0094] Table 4. Effects of different enzyme addition amounts on the total polyphenol and cyanidin-3-O-galactoside content of cherifolia extract.

[0095]

[0096] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0097] 2.1.3 NADES Formulation and Moisture Content Optimization

[0098] Using betaine (Bet), choline chloride (ChCl), glycine (Gly), and sodium acetate (SA) as hydrogen bond acceptors, and citric acid (Cit), lactic acid (Lac), malic acid (Mal), and urea (Urea) as hydrogen bond donors, in molar ratios ranging from 1:1 to 1:3, the effects of different NADES solvent formulations on the total polyphenol content in Aronia chinensis extract were investigated. The results are shown in Table 5.

[0099] Table 5. Effects of different natural eutectic solvent compositions and molar ratios on the total polyphenol content of Aronia rotundi extract.

[0100]

[0101] Note: Different superscript letters in the same row indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0102] Table 5 shows that the total polyphenol content of Aronia arguta extracts obtained with different hydrogen ion donor and acceptor compositions exhibited significant differences. Specifically, the total polyphenol content of ChCl:Urea, Cly:Lac, and SA:Lac compositions with molar ratios of 1:1 to 1:3 was lower than that of other groups. The remaining groups showed significant differences in the molar ratios of hydrogen ion acceptors and donors, with a molar ratio of 1:1 resulting in the highest total polyphenol content. Therefore, considering all factors, future research will focus on an acceptor:donor molar ratio of 1:1, selecting betaine (Bet) and choline chloride (ChCl) as hydrogen bond acceptors, and citric acid (Cit), lactic acid (Lac), malic acid (Mal), and urea (Urea) as hydrogen bond donors, to further investigate the optimal water content. Eighteen enzymatic hydrolysis-NADES synergistic extraction systems were constructed, numbered 1–18, as shown in Table 6.

[0103] Table 6 Enzymatic hydrolysis-NADES synergistic extraction system

[0104]

[0105]

[0106] 2.1.4. Optimization of Extraction Conditions

[0107] Under the above combined conditions, betaine and citric acid were selected as the NADES solvent, and the optimal conditions for material-liquid ratio, extraction temperature, and extraction time were screened in turn.

[0108] The solid-liquid ratio was set to 1:10, 1:20, and 1:30. At an extraction temperature of 30℃ and an extraction time of 60 min, the total polyphenol content is shown in Table 7. The results show that 1:20 is the optimal solid-liquid ratio.

[0109] Table 7. Effects of different material ratios on the total polyphenol and cyanidin-3-O-galactoside content of cherifolia extract.

[0110]

[0111] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0112] We continued with a material-to-liquid ratio of 1:20 and an extraction time of 60 min to explore the changes in total polyphenol content of wild cherries extract at different extraction temperatures of 30, 40, 50, and 60℃. The results are shown in Table 8.

[0113] Table 8. Effects of different extraction temperatures on the total polyphenol and cyanidin-3-O-galactoside content of Pineapple extract.

[0114]

[0115] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0116] Results at different extraction temperatures showed that appropriately increasing the temperature was beneficial for the release of total polyphenols and active substances in the extract. At an extraction temperature of 40℃, both the total polyphenol content and C3gal content were significantly higher than at 30℃. However, with continuous increase in temperature, the extraction efficiency decreased significantly, so the optimal extraction temperature was set at 40℃. Next, the effect of extraction time on the total polyphenol yield was studied at a material-to-liquid ratio of 1:20 and a temperature of 40℃. The results are shown in Table 9.

[0117] Table 9. Effects of different extraction temperatures on the total polyphenol and cyanidin-3-O-galactoside content of Pineapple extract.

[0118]

[0119] Note: Different superscript letters in the same column indicate that the difference between the means is statistically significant (p≤0.05), and the value is mean ± standard deviation (n=3).

[0120] Table 8 shows the effect of reaction time (60–180 min) on anthocyanin extraction rate. The results clearly indicate that the total polyphenol and cyanidin-3-O-galactoside contents reach their maximum values ​​at 90 min, while the extraction rate decreases significantly beyond this time (p≤0.05). This may be due to the inhibition of cellulase activity caused by prolonged treatment. Therefore, the optimal extraction time is selected as 90 min.

[0121] 2.2 Experimental Results and Analysis

[0122] 2.2.1 Total polyphenol extraction rate

[0123] like Figure 2 As shown, each NADES synergistic system significantly increased the total polyphenol content compared to traditional methanol (MeOH) and water (H2O) extraction. Different degrees of hydration resulted in varying effects on total polyphenol extraction; overall, 20% hydrated NADES was the optimal solution. Among them, extracts numbered 2 (Bet:Cit + 20% H2O) and 5 (Bet:Lac + 20% H2O) had the highest total polyphenol content, reaching approximately 150.03 ± 3.55 mg GAE / g and 125.11 ± 3.65 mg GAE / g, respectively.

[0124] 2.2.2 Changes in cyanidin-3-O-galactoside C3gal content

[0125] Figure 3 The results showed that different degrees of hydration had varying effects on the extraction of total polyphenols. Overall, 20% hydrated NADES was the optimal result. Samples 2 and 5 also showed the best performance in cyanidin-3-O-galactoside content, reaching 25.12±0.13 μg / mg and 21.89±0.16 μg / mg respectively, significantly higher than the MeOH group (16.61±0.63 μg / mg) and the H2O group (12.18±0.13 μg / mg), demonstrating good synergistic extraction ability.

[0126] 2.2.3 Analysis of Residual Anthocyanins (Bound Anthocyanins) in Fruit Pomace

[0127] Figure 4 and Figure 5 The results showed that after treatment with the Bet:Cit+20% H2O (number 2) and Bet:Lac+20% H2O (number 5) systems, the residual bound total polyphenols and C3gal content in the pomace of Aronia arugula were significantly lower than those in the traditional methanol and water extraction groups. This indicates that the system has a stronger degradation ability on the plant cell wall structure during the extraction process, which can promote the efficient release of phenols and anthocyanins bound to polysaccharides or proteins. This further verifies the enzymatic hydrolysis combined with natural eutectic solvent synergistic extraction system established in this invention. By enzymatically degrading cell wall polysaccharides and intercellular matrix structures, and under the action of NADES with high polar solubility, the system achieves effective release of bound anthocyanins, significantly improving the extraction efficiency of active ingredients. This system provides a novel extraction process with a clear mechanism, strong extraction ability, and the ability to replace traditional organic solvents, suitable for the green and efficient extraction of natural active ingredients.

[0128] 2.3 Conclusion

[0129] This embodiment systematically optimized the key parameters of the enzymatic hydrolysis-natural eutectic solvent (NADES) synergistic extraction system, clarifying that betaine (Bet) or choline chloride (ChCl) from natural sources are used as hydrogen bond acceptors, and citric acid (Cit), lactic acid (Lac), malic acid (Mal), or urea (Urea) are used as hydrogen bond donors, respectively, to form a NADES system with a hydration degree of 20%. Combined with an enzyme / substrate ratio of 10%, an extraction temperature of 40℃, an extraction time of 120 min, and a solid-liquid ratio of 1:20, it is possible to achieve highly efficient extraction of total polyphenols and cyanidin-3-O-galactoside (C3gal) from Aronia chinensis. Compared with the traditional extraction method of methanol extraction (1.91±0.38%), the cyanidin glycoside content is twice that of the traditional method, reaching 3.80±0.01%, with a purity of 98.42%. The synergistic extraction system established in this study not only has good extraction efficiency and selectivity, but also has advantages such as strong controllability and good repeatability, providing reliable technical support and process foundation for subsequent functional verification studies and its industrial application in food, nutrition or medicine.

[0130] Example 3: Protective effect of chokeberry extract obtained by enzymatic hydrolysis-NADES synergistic extraction on the barrier function of inflammatory Caco-2 cells.

[0131] To verify the bioactivity of the wild cherifolia extract obtained through the optimized extraction system in terms of intestinal barrier function, this embodiment selected the enzymatically hydrolyzed – 20% hydrated NADES synergistic extract as the research object, and established a Caco-2 cell monolayer model stimulated by inflammatory factors to simulate the state of intestinal barrier damage. A comprehensive evaluation was conducted using multiple indicators, including transmembrane resistance (TEER), Lucifer Yellow permeability measurement, apparent permeability coefficient (Papp), and mRNA and protein expression levels of tight junction proteins (ZO-1, OCLN, CLDN1), to systematically assess the potential function of different NADES extracts in protecting the intestinal epithelial barrier.

[0132] 3.1 Experimental Materials

[0133] Caco-2 cells (HTB-37) were purchased from the Shanghai Institute of Chinese Academy of Sciences; cytokines: tumor necrosis factor-α (TNF-α)

[0134] (PeproTech, 300-01A), Interleukin IL-1b (PeproTech, 200-01B), Interferon IFN-g (PeproTech, 300-02), Lipopolysaccharide LPS (Gibco); Lucifer Yellow (L453 Thermo Fisher Scientific); Occludin rabbit mAB E6B4R Cell Signaling Technology; ZO-1 rabbit mAB (D6L1E Cell Signaling Technology); Claudin-1 rabbit mAB (D3J7C, Cell Signaling Technology); Anti-rabbit IgG (Alexa Fluor 488 conjugate) (4412S,

[0135] Cell Signaling Technology).

[0136] 3.2 Experimental Methods

[0137] (1) Culture and differentiation of Caco-2 cells

[0138] Caco-2 cells were grown in DMEM medium containing 10% (v / v) FBS, 1% (v / v) MEMNEAA, and 1% (v / v) penicillin / streptomycin mixture. Cells were cultured in T-25 flasks in a CO2 incubator (37°C, 5% CO2). When cells reached 80-90% confluence (3-4 days), they were digested with 0.25% trypsin-EDTA digestion solution. Well-grown cells were seeded into transwell plates pre-loaded with 0.4 μM polycarbonate membranes. The plates were incubated in a CO2 incubator (37°C, 5% CO2) for 21 days to allow cell differentiation. The growth medium was changed every 2-3 days.

[0139] (2) Inflammatory factor (IC) induced model of damaged intestinal barrier

[0140] Caco-2 cells differentiated for 21 days in Transwell plates were co-cultured with inflammatory cytokines (ICs) to induce a damaged intestinal barrier model using a Caco-2 cell monolayer. The inflammatory cytokines placed in the lower layer of the Transwell membrane included: 50 ng / mL tumor necrosis factor-α (TNF-α), 25 ng / mL interleukin-1β (IL-1β), and 50 ng / mL interferon-gamma (IFN-γ); 10 μg / mL lipopolysaccharide (LPS) was placed in the upper layer to simulate a dysregulated gut microbiota. This IC-induced Caco-2 cell model mimicked inflammatory bowel disease (IBD) and reduced the tightness of the intestinal barrier.

[0141] (3) Measurement and analysis of transmembrane resistance (TEER)

[0142] Transepithelial electrical resistance (TEER) is an indicator of intestinal barrier integrity. After 21 days of differentiation, Caco-2 cells seeded on Transwell membranes were transferred to a biosafety cabinet and placed on a heated plate at 37°C before measurement. Electrodes of the Millicell ERS-2 transepithelial electrical resistance meter were vertically inserted into the upper and lower layers of the Transwell membrane, ensuring the electrodes were fully immersed in the culture medium and avoiding contact with cells. Each well was measured three times. Wells without cells were used as background blanks. TEER values ​​were calculated using the following formula:

[0143] TEER value (ohm·cm) 2 ) = (R measurement well - R blank well) × A

[0144] Where A represents the effective membrane area, and the effective membrane area of ​​the well plate used in this experiment is 0.33 cm². 2 For untreated Caco-2 cell monolayers, the initial TEER values ​​were 300-500 ohms·cm. 2 It is considered to be fully differentiated, and the intestinal barrier system model is valid.

[0145] (4) Analysis of the permeability of the fluorescent dye Lucifer yellow

[0146] 15 μg of Lucifer yellow dye was dissolved in HBSS / HEPES to prepare solutions with two concentration gradients. Fluorescence intensity was measured using a fluorescence microplate reader (excitation / emission = 428 / 520 nm). A linear regression was performed with fluorescence intensity on the x-axis and the amount of LY (μg) on ​​the y-axis. The standard curve is shown below, with fluorescence value on the x-axis and the amount of LY on the y-axis.

[0147] After linear regression fitting, the equation is obtained as: y = 0.985x - 1.0727. R = 0.9999.

[0148] After cell differentiation, different aronia berry extracts were added to the corresponding wells according to the experimental design. After 24 hours, the culture medium in the upper and lower chambers of the Transwell was aspirated, and the cells were washed with HBSS containing 5 mM HEPES. The Transwell was then transferred to a new 24-well plate. The upper chamber of the Transwell contained HBSS / HEPES containing 0.5 mg / mL Lucifer yellow (LY), and the lower chamber contained HBSS / HEPES. The plate was incubated on a shaker at 37°C with a speed of 150 RPM for 24 hours. Every 4 hours, HBSS in the lower chamber of the Transwell was aspirated to quantify the LY that had permeated into the lower layer. The same volume of fresh, preheated 37°C HBSS / HEPES was then placed in the lower chamber of the plate, and the plate was returned to the incubator until monitoring was completed. The LY in the HBSS / HEPES aspirated from the lower chamber was quantified using a microplate reader, and the fluorescence intensity was detected at excitation / emission wavelengths of 428 / 520 nm. The permeation amount (μg) of LY measured at each detection time point was obtained from the corresponding fluorescence intensity according to the standard curve in 4.3.7.1. The permeation amount of LY measured at each time point was accumulated, and the permeability coefficient P was calculated according to the following formula. app (apparent permeability coefficient, cm / s). Where dQ / dt (mol / s) is the permeation rate, i.e., the amount of LY passing through the Caco-2 cell monolayer membrane per unit time during a 24-hour monitoring period, and A is the effective membrane area (here, 0.33 cm²). 2 Co represents the initial LY concentration placed in the upper layer of the Transwell, in mol / mL.

[0149]

[0150] 3.3 TEER value recovery analysis

[0151] Under inflammatory stimulation, the TEER value of IC group cells significantly decreased to about 45% of the initial value, indicating that the barrier integrity was disrupted. Although the TEER value improved after treatment with methanol extract (MeOH), the recovery was limited. In contrast, extracts numbered 2 (Bet:Cit+20% H2O), 5 (Bet:Lac+20% H2O), and 8 (ChCl:Lac+20% H2O) significantly increased the TEER value (p<0.001–0.0001), recovering it to over 70%, indicating that they have significant barrier repair potential.

[0152] 3.4 Barrier permeability assessment

[0153] Results of Lucifer Yellow osmosis experiment ( Figure 7 (A) showed that the permeability of the IC in the inflammation model group increased rapidly, while groups 2, 5, and 8 significantly inhibited its permeability, and were superior to the MeOH group. Papp analysis results ( Figure 7 The results for group B) were consistent, indicating that these extracts effectively reduced cell monolayer permeability and had a good barrier protective effect. Extracts numbered 11, 14, and 17 also showed some effect, but their overall efficacy was slightly inferior to the aforementioned three groups.

[0154] 3.5 Regulation of tight junction protein expression

[0155] Inflammatory stimulation can significantly downregulate the mRNA and protein expression levels of ZO-1, OCLN, and CLDN1. Figure 8 The results showed that extracts 2, 5, and 8 significantly upregulated the expression of these three key tight junction proteins, with a recovery rate significantly higher than that in the MeOH group. Moreover, the mRNA and protein levels were highly consistent, suggesting that they play a positive role in maintaining the integrity of the barrier structure.

[0156] 3.6 Conclusion

[0157] In summary, the results indicate that Aronia arguta extract obtained through enzymatic hydrolysis of a 20% hydrated NADES system (especially Bet:Cit, Bet:Lac, and ChCl:Lac) significantly improved Caco-2 cell barrier dysfunction caused by inflammatory damage, manifested as restoration of TEER values, decreased permeability, and increased expression levels of tight junction proteins, far superior to traditional methanol extracts. This synergistic extract demonstrates excellent intestinal barrier protection, providing a scientific basis for its application in intestinal health functional foods or as an adjunct intervention for inflammatory bowel disease.

[0158] This invention employs a synergistic extraction strategy combining natural deep eutectic solvents (NADES) and enzymatic hydrolysis. A natural deep eutectic solvent is prepared by heating and stirring a mixture of hydrogen bond acceptors and hydrogen bond donors at a molar ratio of 1:(1-3). A specific enzyme is dissolved in the solvent to form an enzyme solution, which is then mixed with aronia berry powder, followed by ultrasonic treatment, enzyme inactivation, and purification to finally obtain aronia berry extract. UHPLC analysis showed that the main active ingredient in the extract was cyanidin-3-O-galactoside, with a content of 25.12±0.13 μg / mg, accounting for 98.42% of the total anthocyanins. This fully utilizes the ability of hydrogen bond donors in NADES to improve the stability and solubility of anthocyanins, as well as the synergistic advantages of hydrogen bond acceptors (such as natural quaternary ammonium salts) in molecular complexation regulation, significantly improving the simultaneous extraction efficiency of free and bound anthocyanins from aronia berry, with the extraction rate of bound anthocyanins reaching 85.48%. Compared to traditional organic solvent extraction methods, this technology offers significant advantages in extraction efficiency, environmental friendliness, and raw material utilization, with an effective component content nearly doubled. Further validation using a Caco-2 cell model stimulated by inflammatory factors showed that the extracted material, at physiological concentrations, significantly improved intestinal barrier dysfunction, manifested as increased transmembrane electrical resistance (TEER), decreased barrier permeability, and enhanced expression levels of tight junction proteins, demonstrating superior efficacy compared to traditional methanol extracts. This method is suitable for the efficient, green, and high-value utilization of anthocyanin-rich natural resources, such as Aronia japonica, and provides reliable scientific evidence and technical support for its application in intestinal health functional foods or adjunctive intervention products for inflammatory bowel disease.

[0159] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A Rubus corchorifolius extract, characterized by: The extract is a wild cherry flower anthocyanin, including cyanidin-3-O-galactoside.

2. Use of the wild cherry extract of claim 1 in the preparation of a food, health product or medicine for protecting the intestinal barrier function.

3. Use of a Rubus corchorifolius extract according to claim 2 for the preparation of a food, health food or pharmaceutical product for protecting the intestinal barrier function, characterized in that: Including improving TEER value, reducing permeability, and up-regulating the expression of ZO-1, OCLN, CLDN1 junction proteins, repairing the epithelial barrier.

4. A method of preparing the Aronia extract according to claim 1, characterized by: Comprising the following steps: Step 1, mix the hydrogen bond acceptor and the hydrogen bond donor at a molar ratio of 1:1~3, heat and continuously stir, to obtain a natural eutectic solvent; Step 2, dissolve the enzyme in the natural eutectic solvent obtained in step 1 to obtain an enzyme-hydrated natural eutectic solvent; Step 3, mix the enzyme-hydrated natural eutectic solvent obtained in step 2 with wild cherry fruit powder, ultrasonic extraction, enzyme inactivation, purification, to obtain wild cherry extract.

5. The method of claim 4, wherein the method of preparing a Rubus coreanus extract is characterized by: The hydrogen bond acceptor in step 1 is at least one of betaine, choline chloride, carnitine salt, glycine and sodium acetate; The hydrogen bond donor is at least one of lactic acid, malic acid, citric acid, glycerol, urea, xylitol, sorbitol and ethylene glycol.

6. The method of claim 5, wherein the preparation of the Aronia extract is characterized by: The hydrogen bond acceptor is betaine, and the hydrogen bond donor is citric acid, and the molar ratio is 1:

1.

7. A method of preparing an Aronia extract according to claim 4, characterized in that: The mass ratio of the enzyme in the enzyme-hydrated natural eutectic solvent in step 3 to the wild cherry fruit powder is 5%~15%; The solid-liquid ratio of wild cherry fruit powder to enzyme-hydrated natural eutectic solvent is 1:20; The extraction temperature is 30~50 °C; The extraction time is 60~180 min.

8. The method of claim 7, wherein the preparation of the Aronia extract is characterized by: In step 2, the enzyme is a combination of one or more of complex cellulase, pectinase, protease and alpha-amylase.

9. The method of claim 7, wherein the method is characterized by: The enzyme is a combination of pectinase and complex cellulase at a mass ratio of 1:1, or a combination of complex cellulase, pectinase and alpha-amylase at a mass ratio of 2:2:1.

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