Dietary fiber-isochlorogenic acid compound as well as preparation method and application thereof

Abstract

The invention provides a dietary fiber-isochlorogenic acid compound as well as a preparation method and application thereof, and relates to the technical field of food processing. The Chinese cabbage outer leaf soluble dietary fiber is extracted by adopting an ultrasonic-assisted deep-eutectic solvent method, a hydrogen bond donor of the deep-eutectic solvent is lactic acid, and a hydrogen bond receptor is one of choline chloride, glycine and alanine. Meanwhile, a Chinese cabbage outer leaf soluble dietary fiber-stevia rebaudiana isochlorogenic acid compound is prepared, and the protective effect of the compound on the intestinal barrier of mice is explored. Results show that the Chinese cabbage outer leaf soluble dietary fiber-stevia rebaudiana isochlorogenic acid compound can protect completeness of intestinal villi of mice, reduce permeability of intestinal barriers, maintain immune organs and intestinal immune barrier functions of the mice, maintain completeness of intestinal mechanical barriers and improve intestinal flora structures; humoral immunity, cellular immunity and non-specific immunity functions are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and in particular relates to a dietary fiber-isochlorogenic acid complex and a preparation method and application thereof. Background Art

[0002] Chinese cabbage (Brassica rapa var.glabra Regel) is a plant of the genus Brassica in the family Cruciferae. It has a very long history of cultivation and is the largest vegetable crop in terms of planting area and yield. However, Chinese cabbage processed products are mainly clean cabbage, sauerkraut, spicy cabbage, etc., with low comprehensive utilization rate, and a large amount of waste such as cabbage outer leaves will be generated during the processing. Relevant studies have shown that Chinese cabbage is rich in dietary fiber, but there are few reports on the extraction method of soluble dietary fiber (SDF) from Chinese cabbage, and there is even less research on its health benefits.

[0003] Traditional SDF extraction mainly adopts water extraction, acid extraction and alkali extraction. Water extraction is simple to operate and low in cost, but the extraction time is long and the extraction rate is low. Acid-base extraction is often used to extract SDF containing acidic groups or acidic SDF, which can promote the dissolution of SDF and shorten the extraction time, but it is easy to destroy the glycosidic bond and spatial structure of SDF. At present, the auxiliary extraction methods based on traditional solvents include enzyme-assisted extraction, ultrasound-assisted extraction, microwave-assisted extraction, subcritical extraction and supercritical extraction. These auxiliary methods have significantly improved the extraction efficiency, but are also accompanied by the degradation of SDF and higher extraction costs. With the continuous deepening of SDF research, efficient, safe and green SDF extraction methods are important prerequisites 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, which usually require a large amount of solvent. The extraction process is prone to waste of solvents and has certain safety hazards and environmental pollution problems. In recent years, deep 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 temperature and inappropriate acidity and alkalinity during the extraction process, and excellent extraction effect and degradability.

[0004] Stevia, also known as stevia and sweet grass, is a perennial herb of the genus Stevia in the Asteraceae family. Stevia is the main source of stevioside. The stevia residue industrial by-product after stevioside is extracted contains rich isochlorogenic acid.

[0005] Therefore, further exploring the nutritional functions of Chinese cabbage and developing functional composite products are of great significance to solving the problem of high yield and low value of Chinese cabbage. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a dietary fiber-isochlorogenic acid complex and a preparation method thereof, wherein the complex prepared by the preparation method can protect the intestinal barrier and improve the intestinal flora, while having the functions of improving humoral immunity, cellular immunity and nonspecific immunity; the 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] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0010] The Chinese cabbage outer leaf powder is mixed with a low eutectic solvent, and the supernatant is collected after ultrasonication and centrifugation; the supernatant is subjected to alcohol precipitation to obtain a precipitate; the precipitate is redissolved in water to obtain the Chinese cabbage outer leaf soluble dietary fiber;

[0011] The stevia residue is mixed with an ethanol aqueous solution, subjected to ultrasonic extraction for 40 to 60 minutes, then subjected to reflux extraction for 0.5 to 2 hours, filtered, and concentrated under reduced pressure to obtain an extract to obtain stevia isochlorogenic acid;

[0012] The soluble dietary fiber from the outer leaves of Chinese cabbage and the isochlorogenic acid from stevia are mixed in a mass ratio of 1:2 to 6 to obtain a dietary fiber-isochlorogenic acid complex;

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

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

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

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

[0017] Preferably, the Chinese cabbage outer leaf soluble dietary fiber and stevia isochlorogenic acid are mixed and stirred at room temperature for 3 to 5 hours, and then dialyzed for 40 to 50 hours to equilibrium.

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

[0019] The present invention also provides an application of the dietary fiber-isochlorogenic acid complex in the preparation of a product for protecting the intestinal barrier.

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

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

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

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

[0024] The present invention provides a dietary fiber-isochlorogenic acid complex and a preparation method and application thereof. The extraction effect of soluble dietary fiber (SDF) from Chinese cabbage outer leaves is evaluated by using different types of deep eutectic solvents (DES), and the process of SDF from Chinese cabbage outer leaves is clarified by single factor experiment. A composite formula of soluble dietary fiber from Chinese cabbage outer leaves and stevia isochlorogenic acid is prepared, and the protective effect of the composite formula on the intestinal barrier of mice is explored. The results show that ultrasound-assisted DES can significantly increase the yield of SDF in Chinese cabbage outer leaves. Compared with the hot water method and ultrasound-assisted enzyme method, the yield of ultrasound-assisted DES method is increased by 6.27 times and 4.98 times, respectively, and the time is shortened by 1.4 times. At the same time, the present invention further finds that the composite formula of soluble dietary fiber from outer leaves of Chinese cabbage and stevia isochlorogenic acid can protect the integrity of the intestinal villi of mice, reduce the permeability of the intestinal barrier, maintain the immune organs and intestinal immune barrier function of 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 the humoral immunity, cellular immunity, and nonspecific immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a single-factor test for ultrasound-assisted DES extraction of SDF;

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

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

[0028] Figure 4 Immunoglobulin A (sIgA), β-defensin (β-DF) and lysozyme (LZM) in mice under different treatment groups. In the figure, compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, *P < 0.05;

[0029] Figure 5 The effect of different treatment groups on the levels of short-chain fatty acids in the intestinal contents of mice, the same letters in the figure indicate no significant difference, and the different letters indicate significant difference (P < 0.05);

[0030] Figure 6 The effect of different treatment groups on the expression of tight junction protein ZO-1. In the figure, compared with the negative control group, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01;

[0031] Figure 7 The effect of different treatment groups on the expression of tight junction protein Claudin-1. In the figure, compared with the negative control group, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01;

[0032] Figure 8 The effect of different treatment groups on the expression of tight junction protein Occludin. In the figure, compared with the negative control group, ## represents P < 0.01; compared with the model group, ** represents P < 0.01;

[0033] Fig. 9 The results of Alpha diversity of mouse cecal intestinal flora. In the figure, * represents P < 0.05 compared with the model group;

[0034] Fig.10 This is the PCoA analysis of differences in mouse cecal microbial communities;

[0035] Fig.11 Clustered stacked bar chart showing differences in the phylum level of mouse intestinal flora;

[0036] Fig.12The changes of four representative microorganisms in the intestinal microbial community structure of mice, A is Desulfobacteria, B is Actinobacteria, C is Proteobacteria, and D is Firmicutes; In the figure, compared with the negative control group, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01;

[0037] Fig.13 It is a clustered stacked bar chart of the differences in the genus level of the mouse intestinal flora;

[0038] Fig.14 The changes of 6 representative microorganisms in the intestinal microbial community structure of mice. In the figure, compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01;

[0039] Fig.15 The results of the expression of IgA, IgM and IgG immunoglobulins in mouse serum. In the figure, ## represents P < 0.01 compared with the negative control group; ** represents P < 0.01 compared with the model group;

[0040] Fig.16 The results of IL-4 expression in mouse serum. In the figure, ## represents P < 0.01 compared with the negative control group; ** represents P < 0.01 compared with the model group;

[0041] Fig.17 The results of the expression of IL-2, INF-γ and IL-1β in mouse serum. In the figure, ## represents P < 0.01 compared with the negative control group; ** represents P < 0.01 compared with the model group;

[0042] Fig.18 The results of mouse serum complement C3 and C4 expression. In the figure, compared with the negative control group, # represents P < 0.05; compared with the model group, ** represents P < 0.01. DETAILED DESCRIPTION

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

[0044] The Chinese cabbage outer leaf powder is mixed with a low eutectic solvent, and the supernatant is collected after ultrasonication and centrifugation; the supernatant is subjected to alcohol precipitation to obtain a precipitate; the precipitate is redissolved in water to obtain the Chinese cabbage outer leaf soluble dietary fiber;

[0045] The stevia residue is mixed with an ethanol aqueous solution, subjected to ultrasonic extraction for 40 to 60 minutes, then subjected to reflux extraction for 0.5 to 2 hours, filtered, and concentrated under reduced pressure to obtain an extract to obtain stevia isochlorogenic acid;

[0046] The soluble dietary fiber from the outer leaves of Chinese cabbage and the isochlorogenic acid from stevia are mixed in a mass ratio of 1:2 to 6 to obtain a dietary fiber-isochlorogenic acid complex;

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

[0048] In the present invention, the preparation process of the soluble dietary fiber of the outer leaves of Chinese cabbage more preferably includes pretreatment of raw materials, preparation of a low eutectic solvent (DES) and extraction of soluble dietary fiber (SDF). In the present invention, the pretreatment of the raw materials is preferably: the outer leaves of Chinese cabbage are washed with water until there is no sand on the surface, chopped, the surface water is drained, placed in an oven at 45-55°C for drying, crushed by a high-speed pulverizer, and passed through an 80-100 mesh sieve. Soak in 95% ethanol aqueous solution and 70% ethanol aqueous solution for 12h in sequence to remove pigments and low molecular weight sugars, place in an oven at 45-55°C for drying, obtain Chinese cabbage outer leaf powder, put into a sealed bag, and place at -20°C for standby use.

[0049] In the present invention, the preparation of the low eutectic solvent (DES) is preferably as follows: the hydrogen bond donor and the hydrogen bond acceptor are mixed in a molar ratio of 1 to 2: 1 to 3, placed in a conical flask, distilled water is added, stirred in a water bath at 80°C until dissolved, and cooled to room temperature to obtain DES. In the present invention, the water content of the DES is preferably 30% to 40%. The hydrogen bond donor in the low eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine and alanine; the hydrogen bond donor and the hydrogen bond acceptor are more preferably mixed in a molar ratio of 1: 3. Due to differences in polarity, solubility, viscosity, surface tension and physical chemistry of different DES, the intermolecular interaction strength is different, so that the extraction efficiency of the target extract is different. The present invention can improve the yield of soluble dietary fiber in the outer leaves of Chinese cabbage by adjusting the type and dosage ratio of hydrogen bond donors and hydrogen bond acceptors.

[0050] In the present invention, the extraction of the soluble dietary fiber (SDF) is preferably: adding the Chinese cabbage outer leaf powder to the above-mentioned DES at a mass volume ratio of 1g: 30-50mL and mixing evenly, ultrasonicating at 70-80℃ for 40-55min, centrifuging at 4000rpm for 10-20min, collecting the supernatant, adding four times the volume of 95% ethanol, alcohol precipitation for 10-15h, and re-dissolving the precipitate in water to obtain Chinese cabbage outer leaf soluble dietary fiber. In the present invention, the Chinese cabbage outer leaf powder and the low eutectic solvent are more preferably mixed at a mass volume ratio of 1g: 40mL; the ultrasonic conditions of the Chinese cabbage outer leaf powder and the low eutectic solvent are more preferably 80℃ ultrasonic 40min; the centrifugation time is more preferably 15min; the alcohol precipitation time is more preferably 12h. The present invention adopts the ultrasound-assisted low eutectic solvent method and optimizes the extraction conditions to improve the extraction rate and extraction efficiency of Chinese cabbage soluble dietary fiber. The experimental results show that compared with the hot water method and ultrasound-assisted enzyme method, the yield of the ultrasound-assisted DES method increased by 6.27 times and 4.98 times respectively, and the time was shortened by 1.4 times.

[0051] In the present invention, ultrasound-assisted ethanol extraction is preferably used to extract isochlorogenic acid (SICA) from stevia residue. The extraction process is more preferably to mix stevia residue with ethanol aqueous solution at a mass volume ratio of 1g:30mL, ultrasonically extract for 50min, then reflux extract for 1h, filter, and concentrate under reduced pressure to obtain stevia isochlorogenic acid. The content of total isochlorogenic acid in the stevia isochlorogenic acid extracted by the present invention is greater than 90%, wherein the content of isochlorogenic acid A is 24%, the content of isochlorogenic acid B is 30%, and the content of isochlorogenic acid C is 40%.

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

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

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

[0055] The present invention also provides a dietary fiber-isochlorogenic acid complex, or a use of the dietary fiber-isochlorogenic acid complex obtained by the preparation method in preparing a product for regulating intestinal flora.

[0056] The present invention also provides a use of the dietary fiber-isochlorogenic acid complex, or the dietary fiber-isochlorogenic acid complex obtained by the preparation method, in the preparation of an immunity enhancing product.

[0057] In the present invention, the immunity includes one or more of humoral immunity, cellular immunity and non-specific immunity.

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

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0060] Example 1

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

[0062] (1) Pretreatment of raw materials

[0063] Wash the outer leaves of Chinese cabbage with water until there is no sand on the surface, chop them, drain the surface water, dry them in an oven at 50°C, grind them with a high-speed grinder, and pass them through an 80-mesh sieve. Soak them in 95% ethanol aqueous solution and 70% ethanol aqueous solution for 12 hours to remove pigments and low-molecular-weight sugars, dry them in an oven at 50°C, and obtain Chinese cabbage outer leaf powder, put them into a sealed bag, and store them at -20°C for 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 (100 g), placed in a 250 mL conical flask, added with 67 mL of distilled water, stirred in a water bath at 80°C until dissolved, and cooled to room temperature to obtain DES with a water content of 40%.

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

[0067] The Chinese cabbage outer leaf powder and the above-mentioned DES were mixed evenly at a mass volume ratio of 1g:30mL, ultrasonicated at 80℃ for 50min, centrifuged at 4000rpm for 15min, the supernatant was collected, four times the volume of 95% ethanol was added, and the alcohol precipitation was carried out for 12h. The precipitate was re-dissolved in water to obtain the Chinese cabbage outer leaf soluble dietary fiber.

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

[0069] The stevia residue was mixed with a 65% ethanol aqueous solution, subjected to ultrasonic extraction for 50 min at an ultrasonic power of 600 W, then subjected to reflux extraction for 1 h, filtered, and concentrated under reduced pressure to obtain an extract 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 Chinese cabbage soluble dietary fiber prepared in step (3) and the stevia isochlorogenic acid prepared in step (4) were mixed in a mass ratio of 1:4, stirred for reaction for 4 hours, and then dialyzed for 48 hours using a 1000Da dialysis bag until equilibrium was obtained to obtain a dietary fiber-isochlorogenic acid complex.

[0072] Example 2

[0073] The difference from Example 1 is that the hydrogen bond acceptor in step (2) is glycine, and the other steps remain unchanged.

[0074] Example 3

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

[0076] Example 4

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

[0078] Example 5

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

[0080] Example 6

[0081] The difference from Example 1 is that the ultrasonic time in step (3) is 40 minutes, and the other steps remain unchanged.

[0082] Example 7

[0083] The difference from Example 1 is that the ultrasonic time in step (3) is 55 min, and the other steps remain unchanged.

[0084] Example 8

[0085] The difference from Example 1 is that the ultrasonic temperature in step (3) is 70° C., and the other steps remain unchanged.

[0086] Example 9

[0087] The difference from Example 1 is that the ultrasonic temperature in step (3) is 75° C., and the other steps remain unchanged.

[0088] Example 10

[0089] The difference from Example 1 is that the water content of DES in step (2) is 30%, and the other steps remain unchanged.

[0090] Embodiment 11

[0091] The difference from Example 1 is that the water content of DES in step (2) is 35%, and the other steps remain unchanged.

[0092] Comparative Example 1

[0093] The soluble dietary fiber from the outer leaves of Chinese cabbage is extracted by hot water method, and the specific steps are as follows: the outer leaves of Chinese cabbage powder is obtained according to step (1) of Example 1, and is mixed with distilled water in a certain proportion, and the mixture is placed in a water bath at 80°C for 120 minutes, and the reaction mixture is centrifuged at 4000r / min for 20 minutes, and the supernatant is collected. 4 times the volume of 95% ethanol aqueous solution is added, and the mixture is precipitated for 12 hours. The precipitate is collected and freeze-dried in a vacuum at -50°C for 24 hours.

[0094] Comparative Example 2

[0095] The soluble dietary fiber of the outer leaves of Chinese cabbage is extracted by ultrasound-assisted enzyme method, and the specific steps are as follows: the outer leaves of Chinese cabbage powder is obtained according to step (1) of Example 1, and is evenly mixed with distilled water in a certain proportion, ultrasonicated at room temperature for 30 minutes, 1 mol / L hydrochloric acid is added to adjust the pH to 5.0, 5% (w / w mass ratio) of cellulase is added, and the mixture is water bathed at 55°C for 120 minutes, and the reaction mixture is centrifuged at 4000r / min for 20 minutes, and the supernatant is collected. 4 times the volume of 95% ethanol aqueous solution is added, and the alcohol precipitation is carried out for 12 hours. The precipitate is collected and vacuum freeze-dried at -50°C for 24 hours.

[0096] Example 12

[0097] In this example, the effect of low eutectic solvents on the yield of soluble dietary fiber from Chinese cabbage outer leaves was studied by selecting hydrogen bond donors and hydrogen bond acceptors of different low eutectic solvents, and the low eutectic solvent system was optimized. The method for extracting soluble dietary fiber from Chinese cabbage outer leaves is the same as steps (1) to (3) of Example 1, except that the hydrogen bond donors and hydrogen bond acceptors of the low eutectic solvents and the ratio between the two are different from Example 1. The content of SDF in Chinese cabbage outer leaves was determined by the phenol-sulfuric acid method using the yield of SDF as an indicator. The type of low eutectic solvents, the ratio relationship and the yield of SDF are shown in Table 1.

[0098]

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

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

[0101] serial number Hydrogen bond donor Hydrogen bond acceptor Molar Ratio Water content (%) Yield (%) DES1 Glycerol Betaine 3:1 40 1.37 DES2 Malic Acid Betaine 3:1 40 2.15 DES3 Citric acid Betaine 3:1 40 2.17 DES4 Levulinic acid Betaine 3:1 40 1.52 DES5 lactic acid Betaine 3:1 40 1.26 DES6 Urea Betaine 3:1 40 2.50 DES7 Malonic acid Betaine 3:1 40 2.07 DES8 Isopropyl alcohol Betaine 3:1 40 1.24 DES10 Ethylene glycol Choline chloride 2:1 40 1.16 DES11 Glycerol Choline chloride 2:1 40 0.90 DES12 Isopropyl alcohol Choline chloride 3:1 40 0.73 DES13 oxalic acid Choline chloride 1:1 40 1.81 DES14 Citric acid Choline chloride 1:2 40 2.59 DES15 1,4-Butanediol Choline chloride 4:1 40 1.29 DES16 Levulinic acid Choline chloride 4:1 40 1.75 DES17 Urea Choline chloride 3:1 40 2.26 DES18 Malic Acid Choline chloride 1.5:1 40 3.14 DES19 lactic acid Choline chloride 2:1 40 3.53 DES20 Malonic acid Choline chloride 2:1 40 2.06 DES21 Methyl urea Choline chloride 1:1 40 1.44 DES22 acetic acid Choline chloride 1:1 40 1.50 DES23 Propionic acid Choline chloride 1:2 40 1.95 DES24 Glutaric acid Choline chloride 1:1 40 1.74 DES25 lactic acid Glycine 1:3 40 4.40 DES26 lactic acid Proline 1:3 40 1.71 DES27 lactic acid Alanine 1:3 40 4.74

[0102] Since different DES have differences in polarity, solubility, viscosity, surface tension and physical chemistry, the intermolecular interaction strength is different, which makes the extraction efficiency of the target extract different. As shown in Table 1, different types of DES solvents have significant differences in the SDF yield of Chinese cabbage outer leaves. Among them, the DES system composed of DES27 (lactic acid / alanine) has the highest SDF yield of 4.74%, which is significantly higher than other DES systems. Therefore, lactic acid and alanine were determined for subsequent process optimization experiments.

[0103] Embodiment 13

[0104] This example sets up a single factor experiment on the ultrasonic time (20min, 30min, 40min, 50min, 60min), water content (30%, 35%, 40%, 45%, 50%), solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50g / mL), and ultrasonic temperature (50℃, 60℃, 70℃, 80℃, 90℃) of the dietary fiber of the outer leaves of Chinese cabbage to study the effect of ultrasonic-assisted DES on the SDF yield and optimize the extraction process. The extraction method of soluble dietary fiber from the outer leaves of Chinese cabbage is the same as steps (1) to (3) of Example 1, and the difference from Example 1 is the change of ultrasonic time, water content, solid-liquid ratio and ultrasonic temperature. The determination and calculation of SDF yield are the same as Example 12.

[0105] Through single factor experiment, the process of ultrasonic-assisted DES extraction of SDF was optimized. The research results are as follows Figure 1As shown in the figure, with the gradual increase of ultrasonic time, the yield of SDF showed a trend of gradually increasing and then decreasing. When the ultrasonic time was 50min, the highest SDF rate was 12.13%. With the gradual increase of water content, the yield of SDF showed a trend of gradually decreasing. Since DES is difficult to synthesize when the water content is lower than 30%, the highest SDF yield was 13.30% when the water content was 30%. With the gradual increase of the solid-liquid ratio, the yield of SDF showed a trend of gradually increasing and then decreasing. When the solid-liquid ratio was 1g:30mL, the highest SDF yield was 13.35%. With the gradual increase of ultrasonic temperature, the yield of SDF showed a trend of gradually increasing and then decreasing. When the ultrasonic temperature was 80℃, the highest SDF rate was 13.45%. The results show that the optimal process conditions are ultrasonic time of 50min, water content of 30%, solid-liquid ratio of 1g:30mL, and ultrasonic temperature of 80℃.

[0106] Embodiment 14

[0107] This example studies and compares the effects of the optimal extraction process determined in Example 13, Comparative Example 1 and Comparative Example 2 on the yield of soluble dietary fiber from Chinese cabbage outer leaves. The determination and calculation of SDF yield are 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] Extraction method Yield (%) Time (min) Hot water method <![CDATA[1.85±0.02 c ]]> 120 Ultrasound-assisted enzyme method <![CDATA[2.25±0.06 b ]]> 120 Ultrasound-assisted DES 13.45±0.12a 50

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

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

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

[0113] 1. Experimental method: Establishment of intestinal barrier injury model in mice: Male ICR mice, with a body weight range of 20 g ± 2 g, were adaptively fed for 3 days and then randomly divided into a negative control group, a model group, a low, medium, and high (125, 250, 500 mg / kg·bw) intervention group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex (SDF-SICA) prepared in Example 10, and a low, medium, and high (125, 250, 500 mg / kg·bw) intervention group of Chinese cabbage soluble dietary fiber (SDF) extracted according to the optimal extraction process determined in Example 13, with 10 mice in each group, and gavage was continued for 21 days. From the 19th day onwards, except for the negative control group, the mice in the model group, the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex intervention group and the Chinese cabbage soluble dietary fiber intervention group were intraperitoneally injected with 50 mg / kg·bw of cyclophosphamide saline solution, and the mice in the negative control group were injected with an equal volume of saline, once a day 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 the 21st day, the mice were fasted for 24 hours and given drinking water. After the experiment, blood samples were collected from the mice, and serum was taken to detect D-amino acid oxidase (DAO), D-lactic acid (D-LA) and lipopolysaccharide (LPS) in the serum. All the methods and steps were carried out according to the instructions of the kit for detecting DAO, D-LA and LPS. The mice were killed by cervical dislocation, and the thymus and spleen of the mice were removed, weighed and recorded, and the immune organ index was calculated. Part of the intestine was fixed in tissue fixative, and the supernatant was taken. Biochemical indicators such as immunoglobulin A (sIgA), β-defensin (β-DF) and lysozyme (LZM) were determined strictly according to the instructions of the kit, and the morphological observation and cell count of the sections were performed. The ileum tissue was taken and the protein imprints of Occludin, Claudin-1 and ZO-1 were detected using a chemiluminescence detection system. Gas chromatography was used to determine the content of short-chain fatty acids in cecal contents. High-throughput sequencing of 16SrDNA of intestinal flora was used to determine the alpha diversity, beta diversity, and microbial changes at the phylum and genus levels of cecal intestinal flora.

[0116] (2) Evaluation of the protective effect of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on the immune function of mice: ELISA kits were used to measure the levels of immunoglobulins, inflammatory factors and complement in mouse serum.

[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 figure, the villi of the jejunum and ileum of the mice in the negative control group were neatly arranged and elongated, with small gaps between the villi. The villi of the jejunum and ileum of the mice in the model group were irregular in shape, the villi of the small intestine were loose and shortened, and some villi broke and fell off, indicating that the model was successfully established.

[0120] Compared with the model group, the improvement in the low-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was milder; the villus morphology of the medium- and high-dose groups of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was greatly improved, the villi were long and closely arranged, the phenomenon of villus breakage and shedding was reduced, and the villus gap became smaller. After treatment with the same dose of Chinese cabbage soluble dietary fiber, the villus structure of the jejunum and ileum of mice did not show significant improvement. Therefore, cyclophosphamide has a destructive effect on the villus morphology of the mouse intestine, and the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the damage of cyclophosphamide to the villus structure of the small intestine and protect the intestinal barrier.

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

[0122] Villus length / μm Crypt depth / μm Villus length / crypt depth ratio (V / C) Negative control group 407.78±23.95 104.93±7.93 3.92±0.46 Model Group <![CDATA[296.19±43.13 # ]]> 117.42±13.10 2.57±0.54# Low dose group (SDF) 312.27±24.16 106.32±7.32 2.94±0.54 Medium dose group (SDF) 319.90±23.34 113.14±5.69 2.83±0.32 High dose group (SDF) 325.19±45.32 116.52±8.32 2.79±0.28 Low-dose group (SDF-SICA) <![CDATA[376.93±31.41 * ]]> 126.97±9.59 2.97±0.41 Medium dose group (SDF-SICA) <![CDATA[409.90±18.17 ** ]]> 117.61±6.98 <![CDATA[3.45±0.12 * ]]> High-dose group (SDF-SICA) <![CDATA[461.56±57.64 ** ]]> 106.36±10.43 <![CDATA[4.34±0.40 ** ]]>

[0123] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents 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] Villus length / μm Crypt depth / μm Villus length / crypt depth ratio (V / C) Negative control group 317.75±16.66 131.44±7.07 2.42±0.11 Model Group <![CDATA[125.13±19.64 ## ]]> 92.36±9.12# <![CDATA[1.34±0.14 ## ]]> Low dose group (SDF) 137.02±3.52 96.23±6.38 1.42±0.14 Medium dose group (SDF) 136.25±4.13 101.19±3.23 1.35±0.25 High dose group (SDF) 143.53±14.32 92.56±4.74 1.55±0.12 Low-dose group (SDF-SICA) <![CDATA[213.10±4.37 ** ]]> 106.40±7.54 <![CDATA[2.01±0.15 * ]]> Medium dose group (SDF-SICA) <![CDATA[266.61±26.82 ** ]]> 131.39±6.94* <![CDATA[2.05±0.27 * ]]> High-dose group (SDF-SICA) <![CDATA[201.75±21.12 ** ]]> 102.88±5.19 <![CDATA[1.97±0.23 * ]]>

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

[0127] According to the results in Tables 3 and 4, compared with the negative control group, the length of jejunal villi and V / C in the model group were significantly reduced (P<0.05), and the length of ileal villi and V / C were significantly and extremely reduced (P<0.01). Due to the early intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex, the length of jejunal villi in the medium and high dose groups increased significantly (P<0.05), and the length of ileal villi increased extremely significantly (P<0.01). The V / C of the jejunal high dose group and the V / C of the ileal medium and high dose group increased extremely significantly (P<0.01). This shows that the intervention of 250 and 500 mg / kg·bw of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex protects the villi structure of the small intestine of mice well.

[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 of mice in different treatment groups

[0130]

[0131]

[0132] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01.

[0133] Diamine oxidase, D-lactic acid and lipopolysaccharide are important indicators for judging intestinal barrier damage. The level of diamine oxidase in serum can specifically reflect the integrity of the intestinal wall tissue structure, while D-lactic acid and lipopolysaccharide can reflect the damage to the intestine and changes in permeability.

[0134] According to the results in Table 5, the negative control group of D-LA and DAO in the serum indexes of mice showed extremely significant differences with the model group, and the negative control group of LPS showed significant differences with the model group. The low-dose group of serum LPS content showed significant differences compared with the model group, and the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed extremely significant differences compared with the model group; the low-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed significant differences compared with the model group, and the medium-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex showed extremely significant differences compared with the model group; the serum DAO content of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex intervention group showed significant differences with the model group. After the same dose of Chinese cabbage soluble dietary fiber was treated, there was no significant improvement in the serum D-amino acid oxidase (DAO), D-lactate (D-LA) and lipopolysaccharide (LPS) of mice. This shows that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can protect mucosal epithelial cells and reduce intestinal damage.

[0135] (3) Protective effect on intestinal immune barrier

[0136] According to the results in Table 6, compared with the negative control group, the relative mass of immune organs in the model group decreased significantly (P < 0.01). Compared with the model group, the relative mass of thymus and spleen in the group treated with Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex in advance increased, among which the low-dose group increased significantly (P < 0.05), and the thymus index in the high-dose group increased extremely significantly (P < 0.01). After treatment with the same dose of Chinese cabbage soluble dietary fiber, the thymus index and spleen index of mice did not improve significantly. This indicates that Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can reduce the damage of cyclophosphamide to immune organs.

[0137] Table 6 Mouse immune organ index (unit: mg / g)

[0138]

[0139]

[0140] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01.

[0141] Table 7 Changes in the number of intestinal intraepithelial lymphocytes in mice in different treatment groups

[0142] Jejunum (unit) Ileum (unit) Negative control group 12.27±1.43 5.17±0.64 Model Group <![CDATA[7.24±1.26 ## ]]> <![CDATA[3.23±0.79 ## ]]> Low dose group (SDF) 7.45±1.21 3.54±1.62 Medium dose group (SDF) 8.09±1.16 4.16±1.19 High dose group (SDF) 8.55±0.97 4.45±1.31 Low-dose group (SDF-SICA) <![CDATA[8.96±1.78 * ]]> 4.17±1.22 Medium dose group (SDF-SICA) <![CDATA[9.78±1.09 * ]]> <![CDATA[5.22±1.05 * ]]> High-dose group (SDF-SICA) <![CDATA[13.13±0.54 ** ]]> <![CDATA[6.93±1.11 ** ]]>

[0143] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01.

[0144] The changes in the number of intraepithelial lymphocytes in the intestines 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 extremely significantly reduced (P < 0.01). Compared with the model group, the number of intraepithelial lymphocytes in the jejunum and ileum of the medium-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P < 0.05), and the number of intraepithelial lymphocytes in the jejunum and ileum of the high-dose group was extremely significantly increased (P < 0.01). After treatment with the same dose of Chinese cabbage soluble dietary fiber, the number of lymphocytes in the jejunum and ileum of mice did not improve significantly.

[0145] Table 8 Changes in the number of intestinal goblet cells in mice in different treatment groups

[0146] Jejunum (unit) Ileum (unit) Negative control group 13.47±2.46 7.65±1.99 Model Group <![CDATA[6.38±1.26 ## ]]> <![CDATA[5.35±0.76 ## ]]> Low dose group (SDF) 7.34±0.28 5.38±1.56 Medium dose group (SDF) 7.89±0.98 5.86±0.75 High dose group (SDF) 8.42±1.74 6.21±0.59 Low-dose group (SDF-SICA) 8.29±0.78 5.74±1.44 Medium dose group (SDF-SICA) <![CDATA[10.18±1.09 * ]]> <![CDATA[6.86±1.39 * ]]> High-dose group (SDF-SICA) <![CDATA[13.13±0.84 ** ]]> <![CDATA[7.93±1.09 ** ]]>

[0147] Note: Compared with the negative control group, # represents P < 0.05, ## represents P < 0.01; compared with the model group, * represents P < 0.05, ** represents P < 0.01.

[0148] The changes in the number of intestinal goblet cells in each group of mice are shown in Table 8. Compared with the negative control group, the number of goblet cells in the jejunum and ileum of the model group mice was extremely significantly reduced (P < 0.01). Compared with the model group, the number of goblet cells in the jejunum and ileum of the medium-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P < 0.05), and the number of goblet cells in the jejunum and ileum of the high-dose group was significantly increased (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 improve significantly. This shows that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex formula can restore the damage caused by cyclophosphamide to intestinal immune cells and protect the intestinal immune barrier.

[0149] like Figure 4 As shown, the levels of sIgA (secretory immunoglobulin A), β-DF (β-defensin) and LZM (lysozyme) in the mouse small intestinal tissue homogenate model group were significantly lower than those in the negative control group (P < 0.05), indicating that the cyclophosphamide intestinal injury model was successfully established; the levels of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex in different treatment groups were significantly increased compared with the model group (P < 0.05).

[0150] sIgA, β-DF and LZM are all important components of intestinal immunity in mice. The above three indicators in the high-dose group were significantly upregulated compared with the model group (P < 0.05), indicating that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex formula has a good protective effect on the small intestinal immune barrier.

[0151] (4) Effects on short-chain fatty acids in intestinal contents

[0152] Effects of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on the levels of short-chain fatty acids (SCFAs) in the cecum of mice injured by cyclophosphamide Figure 5 As shown. Compared with the negative control group, the SCFAs in the model group decreased (p<0.05, p<0.01). Due to the early intervention of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex in each dose group, the levels of multiple short-chain fatty acids and total short-chain fatty acids in the medium and high dose groups increased significantly or extremely significantly (p<0.05, p<0.01). This shows 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.

[0153] SCFAs are metabolites of dietary fiber used by beneficial intestinal bacteria. The low energy produced has health benefits for colon cells. In addition, short-chain fatty acids can promote the secretion of the mucus layer and provide nutrition for intestinal epithelial cells, thereby improving the physical barrier capacity; they can affect the pH value of the intestine to regulate the chemical barrier, and they can also provide nutrients for immune cells, promote the secretion of mucin, lubricate the intestine, and reduce the adhesion of pathogenic bacteria to the intestinal mucosa, thereby enhancing the immune barrier.

[0154] (5) Protective effect of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on intestinal physical barrier

[0155] Western blot was used to detect the protein expression levels of ZO-1, Claudin-1 and Occludin. Figures 6 to 8 As shown in the figure, compared with the negative control group, the expression of ZO-1 protein in the small intestine of the model group mice was significantly reduced (P < 0.01); compared with the model group, the protein expression levels of the low-dose and medium-dose groups of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex were significantly increased (P < 0.01), which indicates that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex activated the expression of ZO-1 protein in the small intestine tissue of mice ( Figure 6 ); Compared with the negative control group, the expression levels of Claudin-1 and Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex in the small intestine of the model group mice were significantly increased (P < 0.05), and the expression levels of high-dose group proteins were extremely significantly increased (P < 0.01) ( Figure 7 ). The expression of occludin protein in the model group was significantly reduced (P<0.01); the protein expression level in the medium-dose and high-dose groups of the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P<0.01) ( Figure 8 ).

[0156] The intestinal physical barrier function is closely related to the tight junctions of intestinal epithelial cells, which are complexes composed of multiple proteins, including occludin, ZO protein, claudin, etc. The results of this experiment showed that after cyclophosphamide modeling, the occludin and claudin-1 proteins in the mouse intestine were significantly downregulated, indicating that the permeability of the mouse intestine increased and the physical barrier was damaged. The Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can significantly upregulate the expression of intestinal occludin and claudin-1 proteins, slow down the trend of increased intestinal permeability, and protect the intestinal physical barrier from the damage caused by cyclophosphamide to the intestine.

[0157] (6) Impact on intestinal flora

[0158] Alpha diversity indicates the species abundance and species diversity of a sample. The present invention analyzed the effects of CSF on the Shannon index, Simpson index and Chao index of the intestinal microbiota in the cecum of mice at the OTU level. The richness of the microbial community was positively correlated with the above indexes. Fig. 9 Compared with the negative group, the Shannon, Simpson and Chao indexes of the model group were all down-regulated; compared with the model group, the Shannon index, Simpson index and Chao index of the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex all rebounded, among which the Simpson index and Shannon index rebounded significantly (p < 0.05). Therefore, the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the Alpha diversity of intestinal flora to a certain extent, and has the tendency to inhibit the decrease in intestinal flora richness caused by cyclophosphamide.

[0159] The principal coordinate analysis (PCoA) method was used to analyze the β-diversity of the mouse cecal microbial intestinal flora. Fig.10 As shown in the figure. Each point in the figure represents a mouse sample, and the distance between points indicates the degree of difference. The closer the distance between samples on the coordinate graph, the higher the similarity. Fig.10 It was found that compared with the negative control group, the sample points in the model group were relatively far away and scattered, indicating that the cecal intestinal flora of the mice in the model group had undergone significant changes. Compared with the model group, the sample points in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex were relatively clustered and close to the scattered points of the negative control group, indicating that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex formula treatment can significantly reverse the intestinal flora disorder of mice caused by cyclophosphamide.

[0160] At the phylum level, the maximum abundance of intestinal flora in different groups of mice was ranked. Fig.11 The results showed that the abundance of Firmicutes and Desulfobacteria was the highest in each group of mice, and the composition of the dominant phyla of intestinal flora at the phylum level was similar between the negative control group and the high-dose group, while the model group was quite different from the other two groups.

[0161] Depend on Fig.12 The results showed that compared with the negative control group, the relative abundance of Desulfobacteria in the model group increased significantly (P<0.01). Compared with the model group, the relative abundance of Desulfobacteria in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex decreased significantly (P<0.01), and there was no significant difference with the negative control group ( Fig.12A). Compared with the negative control group, the relative abundance of Actinobacteria in the model group decreased slightly, but there was no significant difference; 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) ( Fig.12 B). Compared with the negative control group, the relative abundance of Proteobacteria in the model group decreased slightly, but there was no significant difference; 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) ( Fig.12 C). Compared with the negative control group, the relative abundance of Firmicutes in the model group 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 increased, and the relative abundance was similar to that of the NC group, but there was no significant difference among the three groups ( Fig.12 D).

[0162] Fig.13 The top 30 species with the highest abundance at the genus level in the intestinal flora of each group of mice are shown, among which Desulfovibrio and Akkermansia have the highest abundance. As can be seen from the figure, in terms of the composition of the dominant bacterial phyla of the intestinal flora at the genus level, the negative control group and the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex are relatively similar, and the model group is quite different from the other two groups.

[0163] according to Fig.14The results showed that compared with the negative control group, the relative abundance of the intestinal harmful bacteria Desulfovibrio in the model group increased significantly (P < 0.01), and compared with the model group, the relative abundance of the intestinal harmful bacteria Desulfovibrio in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex decreased significantly (P < 0.01), and there was no significant difference with the negative control group. Compared with the negative control group, the relative abundance of Akkermansia in the model group decreased significantly (P < 0.01), and compared with the model group, the relative abundance of Akkermansia in the high-dose group of 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 decreased significantly (P<0.05), and the relative abundance of Lactobacillus in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.05) compared with the model group, and there was no significant difference between the two groups. Compared with the negative control group, the relative abundance of Lachnospira in the model group decreased significantly (P<0.05), and the relative abundance of Lachnospira in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.05) compared with the model group, and there was no significant difference between the two groups. Compared with the negative control group, the relative abundance of Helicobacter in the model group increased significantly (P<0.05), and the relative abundance of Lachnospira in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex decreased significantly (P<0.01) compared with the model group. Compared with the negative control group, the relative abundance of Clostridium in the model group increased significantly (P<0.01). Compared with the model group, the relative abundance of Clostridium in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex decreased significantly (P<0.05), and there was no significant difference between the high-dose group and the negative control group.

[0164] (7) Protective effect of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex on immune function of mice

[0165] 1) Results of mouse serum immunoglobulin expression

[0166] The ELISA kit method was used to determine the levels of IgA, IgM and IgG in mouse serum. Fig.15As shown. Compared with the negative control group, the IgA in the serum of the mice in the model group was extremely significantly decreased (P<0.01), and the IgA in the serum of the mice in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was extremely significantly increased compared with the model group (P<0.01). Compared with the negative control group, there was no significant difference in the IgM in the serum of the mice in the model group, and the IgM in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was extremely significantly increased compared with the model group (P<0.01). Compared with the negative control group, the IgG in the serum of the mice in the model group was extremely significantly decreased (P<0.01), and the IgG in the serum of the mice in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was extremely significantly increased compared with the model group (P<0.01). The above results show that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex can improve the decrease in the humoral immune function of mice caused by cyclophosphamide.

[0167] 2) Expression results of inflammatory factors in mouse serum

[0168] The results of anti-inflammatory factor IL-4 are as follows Fig.16 Compared with the negative control group, the IL-4 level in the serum of the mice in the model group was significantly decreased (P<0.01). Compared with the model group, the IL-4 level in the serum of the mice in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex was significantly increased (P<0.01).

[0169] The results of proinflammatory cytokines IL-2, INF-γ and IL-1β are as follows Fig.17 As shown. Compared with the negative control group, the IL-2 in the serum of the model group mice was significantly increased (P<0.01), and compared with the model group, the IL-2 in the serum of the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex mice was significantly decreased (P<0.01). Compared with the negative control group, the INF-γ in the serum of the model group mice was significantly increased (P<0.01), and compared with the model group, the INF-γ in the serum of the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex mice was significantly decreased (P<0.01). Compared with the negative control group, there was no significant difference in the IL-1β in the serum of the model group mice, and compared with the model group, the IL-1β in the serum of the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex mice was significantly decreased (P<0.01).

[0170] Complement results Fig.18As shown. Compared with the negative control group, there was no significant difference in the C3 content in the serum of the mice in the model group. Compared with the model group, the C3 content in the serum of the mice in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.01). Compared with the negative control group, the C4 content in the serum of the mice in the model group decreased significantly (P<0.05). Compared with the model group, the C4 content in the serum of the mice in the high-dose group of Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex increased significantly (P<0.01).

[0171] The above results indicate that the Chinese cabbage soluble dietary fiber-stevia isochlorogenic acid complex formula can improve the decreased cellular immune function of mice caused by cyclophosphamide.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a dietary fiber-isochlorogenic acid complex, characterized in that: The steps include: The Chinese cabbage outer leaf powder is mixed with a low eutectic solvent, and the supernatant is collected after ultrasonication and centrifugation; the supernatant is subjected to alcohol precipitation to obtain a precipitate; the precipitate is redissolved in water to obtain the Chinese cabbage outer leaf soluble dietary fiber; The stevia residue is mixed with an ethanol aqueous solution, subjected to ultrasonic extraction for 40 to 60 minutes, then subjected to reflux extraction for 0.5 to 2 hours, filtered, and concentrated under reduced pressure to obtain an extract to obtain stevia isochlorogenic acid; The soluble dietary fiber from the outer leaves of Chinese cabbage and the isochlorogenic acid from stevia are mixed in a mass ratio of 1:2 to 6 to obtain a dietary fiber-isochlorogenic acid complex; The hydrogen bond donor in the low eutectic solvent is lactic acid, and the hydrogen bond acceptor is one of choline chloride, glycine and alanine; the hydrogen bond donor and the hydrogen bond acceptor are mixed in a molar ratio of 1-2:1-3.

2. The preparation method according to claim 1, characterized in that: The Chinese cabbage outer leaf powder is mixed with a low eutectic solvent at a mass volume ratio of 1 g:30-50 mL.

3. The preparation method according to claim 1, characterized in that: The ultrasonic conditions of the Chinese cabbage outer leaf powder and the low eutectic solvent are 70-80° C. for 40-55 minutes.

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

5. The preparation method according to claim 1, characterized in that: The Chinese cabbage outer leaf soluble dietary fiber and stevia isochlorogenic acid are mixed and reacted at room temperature for 3 to 5 hours with stirring, and then dialyzed for 40 to 50 hours to equilibrium after the stirring is completed.

6. The dietary fiber-isochlorogenic acid complex prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the dietary fiber-isochlorogenic acid complex according to claim 6 in the preparation of a product for protecting the intestinal barrier.

8. Use of the dietary fiber-isochlorogenic acid complex according to claim 6 in the preparation of a product for regulating intestinal flora.

9. Use of the dietary fiber-isochlorogenic acid complex according to claim 6 in the preparation of an immune-enhancing product.

10. The use according to claim 9, characterized in that: The immunity includes one or more of humoral immunity, cellular immunity and non-specific immunity.

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