Dietary fiber-isochlorogenic acid complex, and preparation method and application thereof

Abstract

This invention provides a dietary fiber-isochlorogenic acid complex, its preparation method, and its applications, relating to the field of food processing technology. The invention employs an ultrasound-assisted eutectic solvent method to extract soluble dietary fiber from the outer leaves of Chinese cabbage. The hydrogen bond donor in the eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine, and alanine. Simultaneously, this invention prepares a soluble dietary fiber-stevia isochlorogenic acid complex from the outer leaves of Chinese cabbage and investigates its protective effect on the intestinal barrier in mice. The results show that the soluble dietary fiber-stevia isochlorogenic acid complex from the outer leaves of Chinese cabbage can protect the integrity of mouse intestinal villi, reduce intestinal barrier permeability, maintain the function of mouse immune organs and the intestinal immune barrier, maintain the integrity of the intestinal mechanical barrier, improve the intestinal flora structure, and enhance humoral immunity, cellular immunity, and non-specific immune function.

Description

Technical Field

[0001] This invention relates to a dietary fiber-isochlorogenic acid complex, its preparation method, and its application. Background Technology

[0002] Chinese cabbage (Brassica rapa var. glabra Regel) is a plant belonging to the Brassicaceae family and the Brassica genus. It has a very long history of cultivation and is the vegetable crop with the largest planting area and yield. However, processed Chinese cabbage products are mainly shredded cabbage, sauerkraut, and kimchi, resulting in low comprehensive utilization and generating a large amount of waste such as outer cabbage leaves during processing. While some studies have shown that Chinese cabbage is rich in dietary fiber, there are few reports on methods for extracting soluble dietary fiber (SDF) from it, and even fewer studies on its health benefits.

[0003] Traditional SDF extraction methods mainly employ water extraction, acid extraction, and alkali extraction. Water extraction is simple and low-cost, but it is time-consuming and has a low extraction rate. Acid-alkali extraction is often used for SDF containing acidic groups or acidic materials, promoting SDF dissolution and shortening extraction time, but it can easily damage the glycosidic bonds and spatial structure of SDF. Current solvent-assisted extraction methods include enzyme-assisted extraction, ultrasound-assisted extraction, microwave-assisted extraction, subcritical extraction, and supercritical extraction. These methods significantly improve extraction efficiency, but also lead to SDF degradation and higher extraction costs. As SDF research continues to deepen, efficient, safe, and environmentally friendly SDF extraction methods are crucial for its low energy consumption and high application value. Traditional solvent extraction methods mainly use water, hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide, and ethanol as solvents, typically requiring large amounts of solvent. The extraction process can easily lead to solvent waste and poses certain safety hazards and environmental pollution problems. In recent years, eutectic solvents (DES) have been widely studied due to their low production cost, simple synthesis method, ability to avoid adverse effects on the structure and activity of SDF caused by excessively high temperatures and unsuitable acidity or alkalinity during extraction, excellent extraction effect, and biodegradability.

[0004] Stevia, also known as stevia or sweet grass, is a perennial herbaceous plant belonging to the genus Stevia in the family Asteraceae. Stevia is the main source of stevia sugar production, and the industrial byproduct of stevia residue after stevia sugar extraction contains abundant isochlorogenic acid.

[0005] Therefore, further exploring the nutritional functions of Chinese cabbage and developing functional compound products is of great significance for solving the problem of high yield but low value of Chinese cabbage. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a dietary fiber-isochlorogenic acid complex and its preparation method. The complex prepared by this method can protect the intestinal barrier and improve the intestinal flora, while also enhancing humoral immunity, cellular immunity and non-specific immunity. This preparation method can improve the extraction rate of soluble dietary fiber from Chinese cabbage and shorten the extraction time.

[0007] Another object of the present invention is to provide an application of a dietary fiber-isochlorogenic acid complex.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention also provides a method for preparing the dietary fiber-isochlorogenic acid complex, comprising the following steps:

[0010] Chinese cabbage outer leaf powder was mixed with a eutectic solvent, and the supernatant was collected after sonication and centrifugation. The supernatant was subjected to alcohol precipitation to obtain a precipitate. The precipitate was redissolved with water to obtain soluble dietary fiber from Chinese cabbage outer leaves.

[0011] Stevia residue was mixed with an ethanol-water solution, extracted by ultrasound for 40-60 min, then refluxed for 0.5-2 h, filtered, and concentrated under reduced pressure to obtain stevia isochlorogenic acid.

[0012] Soluble dietary fiber from the outer leaves of Chinese cabbage was mixed with isochlorogenic acid from stevia at a mass ratio of 1:2~6 to obtain a dietary fiber-isochlorogenic acid complex.

[0013] The hydrogen bond donor in the eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine, and alanine; the hydrogen bond donor and hydrogen bond acceptor are mixed in a molar ratio of 1~2:1~3.

[0014] Preferably, the powder of outer leaves of Chinese cabbage is mixed with a eutectic solvent at a mass-volume ratio of 1g:30~50mL.

[0015] Preferably, the ultrasonic conditions for the outer leaf powder of Chinese cabbage and the eutectic solvent are 70-80℃ for 40-55 minutes.

[0016] Preferably, the ultrasonic power of the stevia residue and the ethanol aqueous solution is 500~600W.

[0017] Preferably, the soluble dietary fiber from the outer leaves of Chinese cabbage is mixed with stevia isochlorogenic acid and stirred at room temperature for 3-5 hours. After stirring, the mixture is dialyzed for 40-50 hours until equilibrium is reached.

[0018] The present invention also provides a dietary fiber-isochlorogenic acid complex prepared by the preparation method described above.

[0019] The present invention also provides the application of the dietary fiber-isochlorogenic acid complex in the preparation of products that protect the intestinal barrier.

[0020] The present invention also provides an application of the dietary fiber-isochlorogenic acid complex described above in the preparation of products for regulating intestinal flora.

[0021] The present invention also provides the application of the dietary fiber-isochlorogenic acid complex in the preparation of immune-enhancing products.

[0022] Preferably, the immunity includes one or more of humoral immunity, cellular immunity, and nonspecific immunity.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a dietary fiber-isochlorogenic acid complex, its preparation method, and its applications. The extraction efficiency of soluble dietary fiber (SDF) from the outer leaves of Chinese cabbage was evaluated using different types of eutectic solvents (DES), and the extraction process of SDF from Chinese cabbage outer leaves was clarified through single-factor experiments. A compound formulation of soluble dietary fiber from Chinese cabbage outer leaves and stevia isochlorogenic acid was prepared, and its protective effect on the intestinal barrier in mice was investigated. The results showed that ultrasound-assisted DES significantly improved the yield of SDF from the outer leaves of Chinese cabbage. Compared with the hot water method and the ultrasound-assisted enzymatic method, the yield of ultrasound-assisted DES increased by 6.27 times and 4.98 times, respectively, while the extraction time was shortened by 1.4 times. Meanwhile, this invention further demonstrates that the compound formulation of soluble dietary fiber from the outer leaves of Chinese cabbage and stevia isochlorogenic acid can protect the integrity of intestinal villi in mice, reduce intestinal barrier permeability, maintain the function of immune organs and intestinal immune barrier in mice, promote the expression of tight junction proteins such as occludin, claudin-1, and ZO-1, maintain the integrity of the intestinal mechanical barrier, improve the structure of intestinal flora, and enhance humoral immunity, cellular immunity, and non-specific immune function. Attached Figure Description

[0025] Figure 1 Single-factor experiment for ultrasound-assisted DES extraction of SDF;

[0026] Figure 2 HE staining of jejunal sections from mice in different treatment groups. In the figure, the top row from left to right is the negative control group, model group, low-dose SDF-SICA group, medium-dose SDF-SICA group, and high-dose SDF-SICA group. The bottom row from left to right is the low-dose SDF group, medium-dose SDF group, and high-dose SDF group.

[0027] Figure 3HE staining of ileal sections from mice in different treatment groups. In the figure, the top row from left to right is the negative control group, model group, low-dose SDF-SICA group, medium-dose SDF-SICA group, and high-dose SDF-SICA group. The bottom row from left to right is the low-dose SDF group, medium-dose SDF group, and high-dose SDF group.

[0028] Figure 4 The figures show the levels of immunoglobulin A (sIgA), β-defensin (β-DF), and lysozyme (LZM) in mice from different treatment groups. In the figure, compared to the negative control group, # represents P < 0.05, and ## represents P < 0.01; compared to the model group, P < 0.05;

[0029] Figure 5 To illustrate the effect of different treatment groups on the levels of short-chain fatty acids in the intestinal contents of mice, the same labeled letters in the figure indicate no significant difference, while different labeled letters indicate significant differences (P < 0.05).

[0030] Figure 6 To illustrate the effect of different treatment groups on the expression of the tight junction protein ZO-1, in the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, This means P < 0.05. This means P < 0.01;

[0031] Figure 7 To illustrate the effects of different treatment groups on the expression of the tight junction protein Claudin-1, in the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, This means P < 0.05. This means P < 0.01;

[0032] Figure 8 To illustrate the effect of different treatment groups on the expression of the tight junction protein Occludin, in the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, This means P < 0.01;

[0033] Figure 9 The figure shows the Alpha diversity results of the cecal gut microbiota in mice. Compared with the model group, This represents P < 0.05;

[0034] Figure 10 PCoA analysis of differences in mouse cecal microbiota;

[0035] Figure 11 Clustered stacked bar chart showing differences in the gut microbiota at the phylum level in mice;

[0036] Figure 12 The graph shows the changes in four representative microorganisms in the mouse gut microbiota. In the figure, A represents Dethiobacterium, B represents Actinobacteria, C represents Proteobacteria, and D represents Firmicutes. In the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, ... This means P < 0.05. This represents P < 0.01;

[0037] Figure 13 Clustered stacked bar chart showing differences in gut microbiota at the genus level in mice;

[0038] Figure 14 The figure shows the changes in the gut microbiota of six representative microorganisms in mice. Compared with the negative control group, # represents P<0.05, and ## represents P<0.01; compared with the model group, This means P < 0.05. This represents P < 0.01;

[0039] Figure 15 The figures show the expression levels of serum IgA, IgM, and IgG immunoglobulins in mice. In the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, This means P < 0.01;

[0040] Figure 16 The results show the serum IL-4 expression levels in mice. In the figure, compared with the negative control group, ## represents P < 0.01; compared with the model group, This means P < 0.01;

[0041] Figure 17 The figures show the serum expression levels of IL-2, INF-γ, and IL-1β in mice. In the figure, ## represents P < 0.01 compared to the negative control group; compared to the model group, This means P < 0.01;

[0042] Figure 18 The results show the expression levels of complement C3 and C4 in mouse serum. In the figure, # represents P < 0.05 compared with the negative control group; compared with the model group, This means P < 0.01. Detailed Implementation

[0043] The present invention also provides a method for preparing the dietary fiber-isochlorogenic acid complex, preferably comprising the following steps:

[0044] Chinese cabbage outer leaf powder was mixed with a eutectic solvent, and the supernatant was collected after sonication and centrifugation. The supernatant was subjected to alcohol precipitation to obtain a precipitate. The precipitate was redissolved with water to obtain soluble dietary fiber from Chinese cabbage outer leaves.

[0045] Stevia residue was mixed with an ethanol-water solution, extracted by ultrasound for 40-60 min, then refluxed for 0.5-2 h, filtered, and concentrated under reduced pressure to obtain stevia isochlorogenic acid.

[0046] Soluble dietary fiber from the outer leaves of Chinese cabbage was mixed with isochlorogenic acid from stevia at a mass ratio of 1:2~6 to obtain a dietary fiber-isochlorogenic acid complex.

[0047] The hydrogen bond donor in the eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine, and alanine; the hydrogen bond donor and hydrogen bond acceptor are mixed in a molar ratio of 1~2:1~3.

[0048] In this invention, the preparation process of the soluble dietary fiber from the outer leaves of Chinese cabbage more preferably includes the pretreatment of raw materials, the preparation of a eutectic solvent (DES), and the extraction of soluble dietary fiber (SDF). In this invention, the pretreatment of the raw materials is preferably as follows: washing the outer leaves of Chinese cabbage with water until the surface is free of mud and sand, chopping, draining the surface moisture, drying in an oven at 45-55°C, pulverizing with a high-speed pulverizer, and passing through an 80-100 mesh sieve. The leaves are then soaked sequentially in 95% ethanol aqueous solution and 70% ethanol aqueous solution for 12 hours to remove pigments and low molecular weight sugars, dried in an oven at 45-55°C, to obtain Chinese cabbage outer leaf powder, which is then placed in a sealed bag and stored at -20°C for later use.

[0049] In this invention, the preferred method for preparing the eutectic solvent (DES) is as follows: a hydrogen bond donor and a hydrogen bond acceptor are mixed in a molar ratio of 1-2:1-3, placed in an Erlenmeyer flask, distilled water is added, and the mixture is stirred in a water bath at 80°C until dissolved. The mixture is then cooled to room temperature to obtain DES. In this invention, the water content of the DES is preferably 30%-40%. The hydrogen bond donor in the eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine, and alanine. More preferably, the hydrogen bond donor and hydrogen bond acceptor are mixed in a molar ratio of 1:3. Because different DESs differ in polarity, solubility, viscosity, surface tension, and physicochemical properties, the strength of intermolecular interactions varies, resulting in different extraction efficiencies for the target extract. This invention, by adjusting the type and dosage ratio of the hydrogen bond donor and hydrogen bond acceptor, can improve the yield of soluble dietary fiber from the outer leaves of Chinese cabbage.

[0050] In this invention, the preferred extraction method for soluble dietary fiber (SDF) is as follows: Chinese cabbage outer leaf powder is added to the above-mentioned DES at a mass-to-volume ratio of 1g:30-50mL, mixed thoroughly, sonicated at 70-80℃ for 40-55min, centrifuged at 4000rpm for 10-20min, the supernatant is collected, four times the volume of 95% ethanol is added, and alcohol precipitation is performed for 10-15h. The precipitate is then redissolved in water to obtain soluble dietary fiber from Chinese cabbage outer leaves. In this invention, the Chinese cabbage outer leaf powder and the eutectic solvent are more preferably mixed at a mass-to-volume ratio of 1g:40mL; the sonication conditions for the Chinese cabbage outer leaf powder and the eutectic solvent are more preferably sonicated at 80℃ for 40min; the centrifugation time is more preferably 15min; and the alcohol precipitation time is more preferably 12h. This invention, by employing an ultrasound-assisted eutectic solvent method and optimizing the extraction conditions, can improve the extraction rate and efficiency of soluble dietary fiber from Chinese cabbage. Experimental results show that, compared with the hot water method and the ultrasound-assisted enzymatic method, the yield of the ultrasound-assisted DES method is increased by 6.27 times and 4.98 times, respectively, and the time is shortened by 1.4 times.

[0051] In this invention, isochlorogenic acid (SICA) in stevia residue is preferably extracted using ultrasound-assisted ethanol extraction. More preferably, the extraction process involves mixing stevia residue with an aqueous ethanol solution at a mass-to-volume ratio of 1 g:30 mL, ultrasonically extracting for 50 min, then refluxing for 1 h, filtering, and concentrating under reduced pressure to obtain a paste, thus obtaining stevia isochlorogenic acid. The total isochlorogenic acid content in the stevia isochlorogenic acid extracted by this invention is greater than 90%, wherein isochlorogenic acid A content is 24%, isochlorogenic acid B content is 30%, and isochlorogenic acid C content is 40%.

[0052] In this invention, the ultrasonic power of the stevia residue and the ethanol aqueous solution is preferably 500~600W.

[0053] In this invention, after preparing soluble dietary fiber from the outer leaves of Chinese cabbage and stevia isochlorogenic acid, the two are mixed and compounded at a mass ratio of 1:2 to 6, more preferably at a mass ratio of 1:4. The compounding process is further preferably as follows: in a phosphate buffer solution system at room temperature and pH 5, the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid are mixed at a mass ratio of 1:4, stirred for 3 to 5 hours, and then dialyzed using a 1000 Da dialysis bag for 40 to 50 hours until equilibrium is reached, completing the compounding process. In this invention, the soluble dietary fiber from the outer leaves of Chinese cabbage and stevia isochlorogenic acid are more preferably mixed in a phosphate buffer solution system at room temperature (25°C) and pH 5, stirred for 4 hours, and then dialyzed for 48 hours until equilibrium is reached. In this invention, the amount of stevia isochlorogenic acid bound per gram of soluble dietary fiber from Chinese cabbage is preferably 1850 mg.

[0054] The present invention also provides the application of the dietary fiber-isochlorogenic acid complex described above, or the dietary fiber-isochlorogenic acid complex obtained by the preparation method described above, in the preparation of products for protecting the intestinal barrier.

[0055] The present invention also provides the application of the dietary fiber-isochlorogenic acid complex described above, or the dietary fiber-isochlorogenic acid complex obtained by the preparation method described above, in the preparation of products for regulating intestinal flora.

[0056] The present invention also provides the application of the dietary fiber-isochlorogenic acid complex described above, or the dietary fiber-isochlorogenic acid complex obtained by the preparation method described above, in the preparation of immune-enhancing products.

[0057] In this invention, the immunity includes one or more of humoral immunity, cellular immunity, and nonspecific immunity.

[0058] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0060] Example 1

[0061] A method for preparing a dietary fiber-isochlorogenic acid complex, the specific steps of which are as follows:

[0062] (1) Pretreatment of raw materials

[0063] Wash the outer leaves of Chinese cabbage with water until the surface is free of mud and sand, chop them, drain the surface water, and dry them in an oven at 50°C. Then, pulverize them using a high-speed grinder and pass them through an 80-mesh sieve. Soak them successively in 95% ethanol aqueous solution and 70% ethanol aqueous solution for 12 hours to remove pigments and low molecular weight sugars. After drying them in an oven at 50°C, you will obtain Chinese cabbage outer leaf powder, which will be placed in a sealed bag and stored at -20°C for later use.

[0064] (2) Preparation of DES

[0065] Lactic acid (hydrogen bond donor) and alanine (hydrogen bond acceptor) were mixed in a molar ratio of 1:3 (100g) and placed in a 250mL Erlenmeyer flask. 67mL of distilled water was added, and the mixture was stirred in an 80℃ water bath until dissolved. After cooling to room temperature, DES with a water content of 40% was obtained.

[0066] (3) Extraction process of soluble dietary fiber from the outer leaves of Chinese cabbage

[0067] Mix the powdered outer leaves of Chinese cabbage with the above-mentioned DES at a mass-volume ratio of 1g:30mL, sonicate at 80℃ for 50min, centrifuge at 4000rpm for 15min, collect the supernatant, add four times the volume of 95% ethanol, precipitate for 12h, and redissolve the precipitate with water to obtain soluble dietary fiber from the outer leaves of Chinese cabbage.

[0068] (4) Extraction process of isochlorogenic acid from stevia

[0069] Stevia residue was mixed with 65% ethanol aqueous solution and ultrasonically extracted for 50 min at an ultrasonic power of 600 W, then refluxed for 1 h, filtered, and concentrated under reduced pressure to obtain stevia isochlorogenic acid.

[0070] (5) Preparation of dietary fiber-isochlorogenic acid complex

[0071] In a phosphate buffer solution system at room temperature of 25°C and pH 5, the soluble dietary fiber of Chinese cabbage prepared in step (3) and the isochlorogenic acid of stevia prepared in step (4) were mixed at a mass ratio of 1:4 and stirred for 4 hours. After the reaction was completed, the mixture was dialyzed with a 1000 Da dialysis bag for 48 hours until equilibrium was reached to obtain the dietary fiber-isochlorogenic acid complex.

[0072] Example 2

[0073] Unlike Example 1, in step (2), the hydrogen bond acceptor is glycine, while the other steps remain unchanged.

[0074] Example 3

[0075] Unlike Example 1, in step (2), the hydrogen bond acceptor is choline chloride, and lactic acid (hydrogen bond donor) and choline chloride (hydrogen bond acceptor) are mixed in a molar ratio of 2:1, while the other steps remain unchanged.

[0076] Example 4

[0077] Unlike Example 1, in step (3), the outer leaf powder of Chinese cabbage and DES are mixed evenly at a mass-volume ratio of 1g:40mL, while the other steps remain unchanged.

[0078] Example 5

[0079] Unlike Example 1, in step (3), the outer leaf powder of Chinese cabbage and DES are mixed evenly at a mass-volume ratio of 1g:50mL, while the other steps remain unchanged.

[0080] Example 6

[0081] Unlike Example 1, the ultrasound time in step (3) is 40 min, while the other steps remain unchanged.

[0082] Example 7

[0083] Unlike Example 1, the ultrasound time in step (3) is 55 min, while the other steps remain unchanged.

[0084] Example 8

[0085] Unlike Example 1, the ultrasonic temperature in step (3) is 70°C, while the other steps remain unchanged.

[0086] Example 9

[0087] Unlike Example 1, the ultrasonic temperature in step (3) is 75°C, while the other steps remain unchanged.

[0088] Example 10

[0089] Unlike Example 1, the water content of DES in step (2) is 30%, while the other steps remain unchanged.

[0090] Example 11

[0091] Unlike Example 1, the water content of DES in step (2) is 35%, while the other steps remain unchanged.

[0092] Comparative Example 1

[0093] Soluble dietary fiber from the outer leaves of Chinese cabbage was extracted using a hot water method. The specific steps are as follows: Chinese cabbage outer leaf powder was obtained according to step (1) of Example 1. This powder was mixed evenly with distilled water in a certain proportion and heated in a water bath at 80℃ for 120 min. The reaction mixture was then centrifuged at 4000 r / min for 20 min, and the supernatant was collected. Four times the volume of 95% ethanol aqueous solution was added, and the mixture was precipitated for 12 h. The precipitate was collected and freeze-dried under vacuum at -50℃ for 24 h.

[0094] Comparative Example 2

[0095] Soluble dietary fiber from the outer leaves of Chinese cabbage was extracted using an ultrasound-assisted enzymatic method. The specific steps are as follows: Chinese cabbage outer leaf powder was obtained according to step (1) of Example 1. This powder was mixed evenly with distilled water in a certain proportion and sonicated at room temperature for 30 min. 1 mol / L hydrochloric acid was added to adjust the pH to 5.0. 5% cellulase (w / w mass ratio) was added, and the mixture was incubated in a water bath at 55℃ for 120 min. The reaction mixture was centrifuged at 4000 r / min for 20 min, and the supernatant was collected. Four times the volume of 95% ethanol aqueous solution was added, and the mixture was precipitated for 12 h. The precipitate was collected and freeze-dried under vacuum at -50℃ for 24 h.

[0096] Example 12

[0097] This embodiment studies the effect of different eutectic solvents on the yield of soluble dietary fiber from the outer leaves of Chinese cabbage by selecting hydrogen bond donors and acceptors, and optimizes the eutectic solvent system. The extraction method of soluble dietary fiber from the outer leaves of Chinese cabbage is the same as steps (1) to (3) in Example 1. The difference from Example 1 is the hydrogen bond donors and acceptors of the eutectic solvent and their ratio. The content of SDF in the outer leaves of Chinese cabbage was determined by the phenol-sulfuric acid method with the yield of SDF as the indicator. The types of eutectic solvents, their ratios, and the yield of SDF are shown in Table 1.

[0098]

[0099] In the formula: C is the SDF concentration (mg / mL) calculated from the standard curve; V is the sample volume (mL); N is the dilution factor; and M is the mass of Chinese cabbage outer leaf powder (g).

[0100] Table 1. Types and yields of eutectic solvents

[0101]

[0102] Different DES solvents exhibit variations in polarity, solubility, viscosity, surface tension, and physicochemical properties, leading to differences in intermolecular interaction strength and consequently, extraction efficiencies for the target extract. Table 1 shows that different types of DES solvents significantly affect the SDF yield of outer leaves from Chinese cabbage. The DES system composed of DES 27 (lactic acid / alanine) achieved the highest SDF yield at 4.74%, significantly higher than other DES systems. Therefore, lactic acid and alanine were selected for subsequent process optimization experiments.

[0103] Example 13

[0104] This embodiment investigates the effects of ultrasound-assisted soluble dietary fiber (SDF) extraction on SDF yield by setting up single-factor experiments for ultrasound time (20 min, 30 min, 40 min, 50 min, 60 min), water content (30%, 35%, 40%, 45%, 50%), material-to-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 g / mL), and ultrasound temperature (50℃, 60℃, 70℃, 80℃, 90℃), thereby optimizing the extraction process. The extraction method for soluble dietary fiber from the outer leaves of Chinese cabbage is the same as steps (1) to (3) in Example 1, except for the changes in ultrasound time, water content, material-to-liquid ratio, and ultrasound temperature. The determination and calculation of SDF yield are the same as in Example 12.

[0105] The ultrasonic-assisted DES extraction process for SDF was optimized through single-factor experiments, and the results are as follows: Figure 1As shown, the yield of SDF gradually increased and then decreased with increasing ultrasonic time, reaching a maximum of 12.13% at an ultrasonic time of 50 min. The yield of SDF gradually decreased with increasing water content. Since DES is difficult to synthesize when the water content is below 30%, the highest yield of SDF (13.30%) was achieved at a water content of 30%. The yield of SDF gradually increased and then decreased with increasing solid-liquid ratio, reaching a maximum of 13.35% at a solid-liquid ratio of 1 g:30 mL. The yield of SDF gradually increased and then decreased with increasing ultrasonic temperature, reaching a maximum of 13.45% at an ultrasonic temperature of 80℃. The results indicate that the optimal process conditions are: ultrasonic time of 50 min, water content of 30%, solid-liquid ratio of 1 g:30 mL, and ultrasonic temperature of 80℃.

[0106] Example 14

[0107] This embodiment studies and compares the effects of three different extraction processes—the optimal extraction process determined in Example 13, Comparative Example 1, and Comparative Example 2—on the yield of soluble dietary fiber (SDF) from the outer leaves of Chinese cabbage. The determination and calculation of SDF yield were the same as in Example 12, and the results are shown in Table 2.

[0108] Table 2. Effect of different extraction methods on SDF yield

[0109]

[0110] Note: The same labeled letters indicate no significant difference, while different labeled letters indicate a significant difference (P < 0.05).

[0111] The results are shown in Table 2. Compared with the hot water method and the ultrasound-assisted enzymatic method, the yield of the ultrasound-assisted DES method was increased by 6.27 times and 4.98 times, respectively, and the preparation time was shortened by 1.4 times. These results indicate that ultrasound-assisted DES is an efficient method for preparing SDF from the outer leaves of Chinese cabbage.

[0112] Example 15: Protective effect of soluble dietary fiber from Chinese cabbage—stevia isochlorogenic acid complex—on intestinal barrier damage in mice.

[0113] 1. Experimental Methods: Establishment of a mouse intestinal barrier injury model: Male ICR mice, weighing between 20g and 2g, were acclimatized for 3 days and then randomly divided into a negative control group, a model group, and intervention groups containing low, medium, and high (125, 250, and 500 mg / kg·bw) of soluble dietary fiber-stevia isochlorogenic acid complex (SDF-SICA) prepared in Example 10, and intervention groups containing low, medium, and high (125, 250, and 500 mg / kg·bw) of soluble dietary fiber (SDF) extracted according to the optimal extraction process determined in Example 13. Ten mice were administered the solution to each group by gavage for 21 consecutive days. Starting from day 19, except for the negative control group, mice in the model group, the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex group intervention group, and the Chinese cabbage soluble dietary fiber intervention group were injected intraperitoneally with 50 mg / kg·bw cyclophosphamide saline solution, while mice in the negative control group were injected with an equal volume of saline solution. The injections were administered once daily for 3 days.

[0114] 2. Evaluation Method:

[0115] (1) Evaluation of the protective effect of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on the intestinal barrier of mice: After intraperitoneal injection on day 21, mice were fasted for 24 hours and given drinking water. After the experiment, blood samples were collected from mice, serum was taken, and D-amino acid oxidase (DAO), D-lactic acid (D-LA) and lipopolysaccharide (LPS) in serum were detected. All methods and steps were performed in accordance with the instructions of the kit for detecting DAO, D-LA and LPS. Mice were euthanized by cervical dislocation, and the thymus and spleen of mice were removed, weighed, recorded and calculated for immune organ indices. Part of the intestine was fixed in tissue fixation solution, and the supernatant was taken. Immunoglobulin A (sIgA), β-defensin (β-DF) and lysozyme (LZM) and other biochemical indicators were measured strictly in accordance with the kit instructions. Morphological observation and cell counting were performed on the sections. Ileal tissue was taken and Occludin, Claudin-1 and ZO-1 protein imprints were detected using a chemiluminescence detection system. The content of short-chain fatty acids in cecal contents was determined by gas chromatography. High-throughput 16S rDNA sequencing of gut microbiota was used to clarify the trends in alpha diversity, bata diversity, and microbial changes at the phylum and genus levels of the cecal gut microbiota.

[0116] (2) Evaluation of the protective effect of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on mouse immune function: The contents of immunoglobulins, inflammatory factors and complement in mouse serum were determined by ELISA kit.

[0117] 3. Results Analysis

[0118] (1) Protective effect on the morphology and structure of intestinal mucosal epithelium

[0119] like Figures 2-3 As shown, in the negative control group, the villi of the jejunum and ileum of mice were neatly arranged and long, with small gaps between the villi. In the model group, the villi of the jejunum and ileum of mice were irregular in shape, the villi of the small intestine were loose and short, and some villi broke off, indicating that the model was successfully established.

[0120] Compared with the model group, the low-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed milder improvement; the medium and high-dose groups showed greater improvement in villus morphology, with longer and more tightly packed villi, reduced villus breakage and shedding, and smaller intervillous spaces. Treatment with the same dose of Chinese cabbage soluble dietary fiber did not significantly improve the villus structure of the jejunum and ileum in mice. Therefore, cyclophosphamide has a destructive effect on the villus morphology of the mouse intestine, while the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the damage of cyclophosphamide to the small intestinal villus structure and protect the intestinal barrier.

[0121] Table 3. Effects of cyclophosphamide treatment on jejunal villus length, crypt depth, and their ratio in different treatment groups in mice.

[0122]

[0123] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0124] Table 4. Effects of cyclophosphamide treatment on ileal villus length, crypt depth, and their ratio in mice under different treatment groups.

[0125]

[0126] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0127] According to the results in Tables 3 and 4, compared with the negative control group, the jejunal villus length and V / C ratio were significantly decreased in the model group (P<0.05), and the ileal villus length and V / C ratio were significantly and extremely decreased (P<0.01). Due to the prior intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex, the villus length in the high-dose jejunal group was significantly increased (P<0.05), and the villus length in the ileum was extremely significantly increased (P<0.01). The V / C ratio in the high-dose jejunal group and the V / C ratio in the high-dose ileal group were extremely significantly increased (P<0.01). This indicates that the intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex at 250 and 500 mg / kg·bw effectively protected the villus structure of the mouse small intestine.

[0128] (2) Effects on diamine oxidase (DAO), D-lactic acid (D-LA) and lipopolysaccharide (LPS)

[0129] Table 5. Intestinal LPS, D-LA, and DAO levels in mice from different treatment groups.

[0130]

[0131] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0132] Diamine oxidase, D-lactic acid, and lipopolysaccharide are important indicators for assessing intestinal barrier damage. Serum diamine oxidase levels can specifically reflect the integrity of intestinal wall tissue structure, while D-lactic acid and lipopolysaccharide can reflect intestinal damage and changes in permeability.

[0133] As shown in Table 5, the serum D-LA and DAO levels in mice showed highly significant differences compared to the model group, while the LPS negative control group showed a significant difference compared to the model group. The low-dose group showed a significant difference in serum LPS levels compared to the model group, and the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed a highly significant difference compared to the model group. The low-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed a significant difference in serum D-LA levels compared to the model group, and the medium-dose group showed a highly significant difference compared to the model group. The intervention group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed a significant difference in serum DAO levels compared to the model group. Treatment with the same dose of Chinese cabbage soluble dietary fiber did not significantly improve serum D-amino acid oxidase (DAO), D-lactic acid (D-LA), and lipopolysaccharide (LPS) levels in mice. This indicates that the soluble dietary fiber in Chinese cabbage—stevia isochlorogenic acid complex—can protect mucosal epithelial cells and reduce intestinal damage.

[0134] (3) Protective effect on the intestinal immune barrier

[0135] As shown in Table 6, compared with the negative control group, the relative mass of immune organs in the model group was significantly decreased (P < 0.01). Compared with the model group, the relative mass of the thymus and spleen in the pre-intervention group with the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased, with a significant increase in the low-dose group (P < 0.05) and a highly significant increase in the thymus index in the high-dose group (P < 0.01). Treatment with the same dose of Chinese cabbage soluble dietary fiber did not significantly improve the thymus index and spleen index in mice. This indicates that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can alleviate the damage of cyclophosphamide to immune organs.

[0136] Table 6. Mouse Immune Organ Index (Unit: mg / g)

[0137]

[0138] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0139] Table 7. Changes in the number of intraepithelial lymphocytes in mice under different treatment groups.

[0140]

[0141] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0142] The changes in the number of intraepithelial lymphocytes in the intestinal epithelium of mice in each group are shown in Table 7. Compared with the negative control group, the number of intraepithelial lymphocytes in the model group was significantly decreased (P < 0.01). Compared with the model group, the number of intraepithelial lymphocytes in the jejunum and ileum was significantly increased in the medium-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex (P < 0.05), and the number of intraepithelial lymphocytes in the jejunum and ileum was extremely significantly increased in the high-dose group (P < 0.01). After treatment with the same dose of Chinese cabbage soluble dietary fiber, the number of jejunum and ileum lymphocytes in mice did not show significant improvement.

[0143] Table 8. Changes in the number of goblet cells in the intestines of mice in different treatment groups.

[0144]

[0145] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, This means P < 0.05. This means P < 0.01.

[0146] The changes in the number of goblet cells in the intestines of mice in each group are shown in Table 8. Compared with the negative control group, the number of goblet cells in the jejunum and ileum of mice in the model group was significantly decreased (P < 0.01). Compared with the model group, the number of goblet cells in the jejunum and ileum was significantly increased in the medium-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex (P < 0.05), and significantly increased in the jejunum and ileum in the high-dose group (P < 0.01). After treatment with the same dose of Chinese cabbage soluble dietary fiber, the number of goblet cells in the jejunum and ileum of mice did not show significant improvement. This indicates that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can restore the damage to intestinal immune cells caused by cyclophosphamide and protect the intestinal immune barrier.

[0147] like Figure 4 As shown, the levels of sIgA (secretory immunoglobulin A), β-DF (β-defensin), and LZM (lysozyme) in mouse small intestinal tissue homogenate were significantly lower in the model group compared with the negative control group (P < 0.05), indicating that the cyclophosphamide intestinal injury model was successfully established; the levels of soluble dietary fiber-stevia isochlorogenic acid complex in different treatment groups of Chinese cabbage were significantly higher than those in the model group (P < 0.05).

[0148] sIgA, β-DF, and LZM are all important components of intestinal immunity in mice. The high-dose group showed significant upregulation of these three indicators compared to the model group (P < 0.05), indicating that the compound formula of Chinese cabbage soluble dietary fiber and stevia isochlorogenic acid has a good protective effect on the small intestinal immune barrier.

[0149] (4) Effects on short-chain fatty acids in intestinal contents

[0150] The effect of the soluble dietary fiber from Chinese cabbage—stevia isochlorogenic acid complex—on the levels of short-chain fatty acids (SCFAs) in the cecal contents of mice damaged by cyclophosphamide, as follows: Figure 5 As shown in the figure. Compared with the negative control group, SCFAs in the model group were decreased (p<0.05, p<0.01). Due to the prior intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex in each dose group, the levels of various short-chain fatty acids and total short-chain fatty acids in the medium and high dose groups were significantly or extremely significantly increased (p<0.05, p<0.01). This indicates that the intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex formula can increase the production of short-chain fatty acids in the cecal contents of mice.

[0151] SCFAs are metabolic products of beneficial gut bacteria after utilizing dietary fiber. The low energy they produce is beneficial to colon cells. In addition, short-chain fatty acids can promote the secretion of mucus, provide nutrients to intestinal epithelial cells, thereby improving the physical barrier function; they can affect the pH of the intestine to regulate the chemical barrier; they can also provide nutrients to immune cells, promote the secretion of mucin, lubricate the intestine, reduce the adhesion of pathogenic bacteria to the intestinal mucosa, and thus enhance the immune barrier.

[0152] (5) The protective effect of the soluble dietary fiber of Chinese cabbage-stevia isochlorogenic acid complex on the intestinal physical barrier

[0153] The protein expression levels of ZO-1, Claudin-1, and Occludin were detected by Western blot, and the results are as follows: Figures 6-8 As shown, compared with the negative control group, the expression of ZO-1 protein in the small intestine of mice in the model group was significantly decreased (P < 0.01); compared with the model group, the protein expression levels in the low-dose and medium-dose groups of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex were significantly increased (P < 0.01), indicating that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex activated ZO-1 protein expression in the small intestinal tissue of mice. Figure 6 Compared with the negative control group, the expression levels of Claudin-1 and the medium-dose soluble dietary fiber-stevia isochlorogenic acid complex in the small intestine of model group mice were significantly increased (P < 0.05), and the expression levels in the high-dose group were extremely significantly increased (P < 0.01). Figure 7The expression of Occludin protein in the model group was significantly decreased (P < 0.01); the protein expression levels in the medium- and high-dose groups of the soluble dietary fiber-stevia isochlorogenic acid complex from Chinese cabbage were significantly increased (P < 0.01). Figure 8 ).

[0154] The intestinal physical barrier function is closely related to the tight junctions of intestinal epithelial cells, which are complexes composed of various proteins, including the occlusive proteins occludin, ZO protein, and claudin. Our results showed that after cyclophosphamide modeling, the expression of occludin and claudin-1 proteins in the mouse intestine was significantly downregulated, indicating increased intestinal permeability and impaired physical barrier function. The soluble dietary fiber from Chinese cabbage—stevia isochlorogenic acid complex—significantly upregulated the expression of occludin and claudin-1 proteins in the intestine, slowed the increase in intestinal permeability, and protected the intestinal physical barrier from the damaging effects of cyclophosphamide.

[0155] (6) Effects on gut microbiota

[0156] Alpha diversity represents the species abundance and diversity of a sample. This invention analyzed the effects of CSF on the Shannon, Simpson and Chao indices of the intestinal microbiota in the mouse cecum at the OTU level. Microbial community richness was positively correlated with the above indices. Figure 9 Compared with the negative control group, the model group showed a decrease in Shannon, Simpson, and Chao indices. Compared with the model group, the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed an increase in Shannon, Simpson, and Chao indices, with the Simpson and Shannon indices showing a significant increase (p < 0.05). Therefore, the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the alpha diversity of the gut microbiota to a certain extent and has the potential to inhibit the trend of decreased gut microbiota richness caused by cyclophosphamide.

[0157] Principal coordinate analysis (PCoA) was used to analyze the β-diversity of the mouse cecal gut microbiota. The results are as follows: Figure 10 As shown in the figure. Each point in the graph represents a mouse sample, and the distance between points indicates the degree of difference. Samples that are closer together on the coordinate graph are more similar. Figure 10It was found that, compared with the negative control group, the sample points in the model group were more distant and dispersed, indicating that the intestinal flora of the cecum in the model group mice underwent significant changes. Compared with the model group, the sample points in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex were more clustered and similar to those in the negative control group, indicating that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex treatment can significantly reverse the cyclophosphamide-induced intestinal flora disorder in mice.

[0158] At the phylum level, the maximum abundance of gut microbiota in different groups of mice was ranked. Figure 11 These were the top 30 species. The results showed that Firmicutes and Dethiobacterium were the most abundant in each group of mice. In terms of the composition of the dominant phyla of the gut microbiota at the phylum level, the negative control group and the high-dose group were similar, while the model group was significantly different from the other two groups.

[0159] Depend on Figure 12 It was found that, compared with the negative control group, the relative abundance of Dethiobacterium in the model group increased significantly (P<0.01), while the relative abundance of Dethiobacterium in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.01) compared with the model group, and there was no significant difference compared with the negative control group. Figure 12 A). Compared with the negative control group, the relative abundance of Actinobacteria in the model group decreased slightly, but the difference was not statistically significant; compared with the model group, the relative abundance of Actinobacteria in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.05). Figure 12 B). Compared with the negative control group, the relative abundance of Proteobacteria in the model group decreased slightly, but the difference was not statistically significant; compared with the model group, the relative abundance of Proteobacteria in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.01). Figure 12 C). Compared with the negative control group, the relative abundance of Firmicutes in the model group was decreased. Compared with the model group, the relative abundance of Firmicutes in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex rebounded, and the relative abundance was similar to that of the NC group. However, there was no significant difference among the three groups. Figure 12 D).

[0160] Figure 13 The figure shows the top 30 most abundant species in the gut microbiota of each group of mice at the genus level, with *Desulfovibrio* and *Akkermansia* being the most abundant. As can be seen from the figure, in terms of the composition of the dominant phyla in the gut microbiota at the genus level, the negative control group and the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex were quite similar, while the model group differed significantly from the other two groups.

[0161] according to Figure 14 The results showed that, compared with the negative control group, the relative abundance of *Desulfovibrio* spp., a harmful bacterium in the gut of mice in the model group increased significantly (P < 0.01). Compared with the model group, the relative abundance of *Desulfovibrio* spp., a harmful bacterium in the gut of mice in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex decreased significantly (P < 0.01), and there was no significant difference between the model group and the negative control group. Compared with the negative control group, the relative abundance of *Ackermania* spp. in the model group decreased significantly (P < 0.01). Compared with the model group, the relative abundance of *Ackermania* spp. in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex did not reverse, but there was no significant difference between the model group and the high-dose group. Compared with the negative control group, the relative abundance of *Lactobacillus* in the model group was significantly decreased (P<0.05). Compared with the model group, the relative abundance of *Lactobacillus* in the high-dose group of the soluble dietary fiber-stevia isochlorogenic acid complex from Chinese cabbage was significantly increased (P<0.05), and there was no significant difference compared with the negative control group. Compared with the negative control group, the relative abundance of *Helicobacter* in the model group was significantly decreased (P<0.05). Compared with the model group, the relative abundance of *Helicobacter* in the high-dose group of the soluble dietary fiber-stevia isochlorogenic acid complex from Chinese cabbage was significantly increased (P<0.05), and there was no significant difference compared with the negative control group. Compared with the negative control group, the relative abundance of *Helicobacter* in the model group was significantly increased (P<0.05). Compared with the model group, the relative abundance of *Helicobacter* in the high-dose group of the soluble dietary fiber-stevia isochlorogenic acid complex from Chinese cabbage was extremely significantly decreased (P<0.01). Compared with the negative control group, the relative abundance of Clostridium species in the model group was significantly increased (P<0.01). Compared with the model group, the relative abundance of Clostridium species in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly decreased (P<0.05), and there was no significant difference with the negative control group.

[0162] (7) Protective effect of soluble dietary fiber from Chinese cabbage-stevia isochlorogenic acid complex on immune function in mice

[0163] 1) Results of serum immunoglobulin expression levels in mice

[0164] The levels of IgA, IgM, and IgG in mouse serum were determined using an ELISA kit. The results are as follows: Figure 15As shown in the figure. Compared with the negative control group, the serum IgA level in the model group mice was significantly decreased (P<0.01). Compared with the model group, the serum IgA level in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P<0.01). Compared with the negative control group, there was no significant difference in serum IgM level in the model group mice. Compared with the model group, the serum IgM level in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P<0.01). Compared with the negative control group, the serum IgG level in the model group mice was significantly decreased (P<0.01). Compared with the model group, the serum IgG level in the high-dose group of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P<0.01). These results indicate that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the cyclophosphamide-induced decrease in humoral immune function in mice.

[0165] 2) Results of expression of inflammatory factors in mouse serum

[0166] The results of the anti-inflammatory factor IL-4 are as follows Figure 16 As shown in the figure. Compared with the negative control group, the serum IL-4 level in the model group mice was significantly decreased (P<0.01), and compared with the model group, the serum IL-4 level in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex mice was significantly increased (P<0.01).

[0167] The results of pro-inflammatory factors IL-2, INF-γ, and IL-1β are as follows: Figure 17 As shown in the figure. Compared with the negative control group, the serum IL-2 level in the model group mice was significantly increased (P<0.01). Compared with the model group, the serum IL-2 level in the high-dose group of mice treated with the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid complex was significantly decreased (P<0.01). Compared with the negative control group, the serum INF-γ level in the model group mice was significantly increased (P<0.01). Compared with the model group, the serum INF-γ level in the high-dose group of mice treated with the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid complex was significantly decreased (P<0.01). Compared with the negative control group, there was no significant difference in serum IL-1β level in the model group mice. Compared with the model group, the serum IL-1β level in the high-dose group of mice treated with the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid complex was significantly decreased (P<0.01).

[0168] The result of complement Figure 18As shown in the figure. Compared with the negative control group, there was no significant difference in serum C3 content in the model group mice. Compared with the model group, the serum C3 content in the high-dose group of mice containing the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid complex was significantly increased (P<0.01). Compared with the negative control group, the serum C4 content in the model group mice was significantly decreased (P<0.05). Compared with the model group, the serum C4 content in the high-dose group of mice containing the soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid complex was significantly increased (P<0.01).

[0169] The above results indicate that the compound formula of soluble dietary fiber from Chinese cabbage and stevia isochlorogenic acid can improve the reduction of cellular immune function in mice caused by cyclophosphamide.

[0170] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a dietary fiber-isochlorogenic acid complex, characterized in that, Includes the following steps: Chinese cabbage outer leaf powder was mixed with a eutectic solvent, and the supernatant was collected after sonication and centrifugation. The supernatant was subjected to alcohol precipitation to obtain a precipitate. The precipitate was redissolved with water to obtain soluble dietary fiber from Chinese cabbage outer leaves. Stevia residue was mixed with an ethanol-water solution, extracted by ultrasound for 40-60 min, then refluxed for 0.5-2 h, filtered, and concentrated under reduced pressure to obtain stevia isochlorogenic acid. Soluble dietary fiber from the outer leaves of Chinese cabbage was mixed with isochlorogenic acid from stevia at a mass ratio of 1:2~6 to obtain a dietary fiber-isochlorogenic acid complex. The hydrogen bond donor in the eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine, and alanine; the hydrogen bond donor and hydrogen bond acceptor are mixed in a molar ratio of 1~2:1~3. The powder of outer leaves of Chinese cabbage is mixed with a low eutectic solvent at a mass-volume ratio of 1g:30~50mL; The ultrasonic conditions for the outer leaf powder of Chinese cabbage and the eutectic solvent are: ultrasonication at 70-80℃ for 40-55 minutes. The soluble dietary fiber from the outer leaves of Chinese cabbage was mixed with stevia isochlorogenic acid and stirred at room temperature for 3-5 hours. After stirring, the mixture was dialyzed for 40-50 hours until equilibrium was reached.

2. The preparation method according to claim 1, characterized in that, The ultrasonic power of the stevia residue and ethanol aqueous solution is 500~600W.

3. The dietary fiber-isochlorogenic acid complex prepared by the preparation method according to claim 1 or 2.

4. The use of the dietary fiber-isochlorogenic acid complex according to claim 3 in the preparation of products that protect the intestinal barrier.

5. The application of the dietary fiber-isochlorogenic acid complex according to claim 3 in the preparation of products for regulating intestinal flora.

6. The application of the dietary fiber-isochlorogenic acid complex according to claim 3 in the preparation of immune-enhancing products.

7. The application according to claim 6, characterized in that, The immunity mentioned includes one or more of humoral immunity, cellular immunity, and nonspecific immunity.

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