Preparation method and application of high-water-solubility astragaloside compound
The high water-soluble astragaloside-chipsol complex was prepared by enzymatic addition and fermentation method, which solved the problems of low content of astragaloside and poor water solubility, and realized its in-food food and innovative applications in the field of health products, and enhance antioxidant and lowering glycemic effects.
Patent Information
- Application Number
- CN202411355630.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2024-12-31
AI Technical Summary
Astragalus membranaceus has low content and poor water solubility in Astragalus, which limits its application in the fields of food and health products. The traditional extraction process is complex and environmental pollution problems. The existing chemical modification methods improve water solubility but limit its application.
The two-step method of enzymatic fermentation and fermentation was used to extract astragaloside and compound it with chitinoligosaccharides. The highly water-soluble astragaloside-chitinoligosaccharide complex was prepared by spray drying, which improved its in the fields of food and health products. application.
It improves the content and purity of Astragalus membranaceus, significantly improves its water solubility, enhances its antioxidant and lowering glycemic effects, expands its application potential in the fields of food and health products, and avoids environmental pollution from physical and chemical methods.
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Figure CN119214294A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food processing, and particularly relates to a preparation method and application of a highly water-soluble astragaloside IV complex. Background Art
[0002] Radix Astragali is one of the commonly used Chinese medicines. It is the dried root of Astragalusmembranaceus (Fischer) Bunge and A.membranaceus (Fisch.) Bungevar.mongholicus (Bunge) PKHsiao, both of the leguminous plants. Astragalosides IV (ASI) is a saponin compound with multiple pharmacological activities isolated from Radix Astragali and is an indicator component for the quality identification of Radix Astragali preparations. Studies have shown that Astragalosides IV has a wide range of pharmacological effects in the cardiovascular, immune, nervous, digestive, urinary, circulatory and endocrine systems. It is reported that Astragalosides IV can reduce the increased permeability of the blood-brain barrier caused by cerebral ischemia-reperfusion; it has a protective effect on ischemic brain damage; it exerts anti-cancer activity by regulating the expression of oncogenes; it has a hypoglycemic effect on diabetic mice; it has strong anti-hepatitis B virus activity; it can effectively inhibit or reverse PS-induced liver fibrosis in mouse models; its Chinese medicine preparation has been developed as a myocardial protective agent for the treatment of angina pectoris in coronary heart disease. However, practice shows that the content of astragaloside IV in Astragalus is extremely low and is greatly affected by the origin and season, generally about 0.04%. It is difficult to extract and separate, and the traditional extraction process is relatively complicated. Therefore, developing a relatively simple method that can increase the content of astragaloside IV has positive significance for the application and development of Astragalus. Astragaloside IV has poor water solubility and low bioavailability, which limits its development and use in the fields of food and health products. In order to improve the water solubility of astragaloside IV, some researchers have increased its water solubility through chemical modification, such as improving its physicochemical properties through derivatization; there are also studies to improve bioavailability by preparing nanosuspensions, polymer micelles, etc. The above-mentioned methods of improving the water solubility of astragaloside IV through chemical methods limit its application in the food field. Therefore, it is necessary to provide a simple and efficient method to improve the water solubility of astragaloside IV while also expanding its development in the food field. Chitosan oligosaccharide is a natural polymer with abundant sources. It is the only positively charged alkaline aminopolysaccharide among natural sugars. It has multiple biological activities such as anti-oxidation, anti-bacterial, anti-diabetic, and anti-tumor. At present, there is no report on the research of using chitosan oligosaccharide to improve the water solubility of astragaloside IV. Therefore, the present invention uses chitosan oligosaccharide and astragaloside IV to prepare a highly water-soluble astragaloside IV complex. Summary of the invention
[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a preparation method and application of a highly water-soluble astragaloside IV complex. Astragaloside IV is prepared by a two-step method of enzymatic hydrolysis and fermentation using astragalus as raw material, thereby increasing the content of astragaloside IV; then, chitosan oligosaccharides are compounded with astragaloside IV to obtain an astragaloside IV-chitosan oligosaccharide complex, thereby increasing the water solubility of astragaloside IV, having antioxidant and hypoglycemic effects, and expanding the application of astragaloside IV in the fields of food, health care products, etc.
[0004] Technical solution: A highly water-soluble astragaloside IV complex, wherein the astragaloside IV complex is specifically an astragaloside IV-chitosan oligosaccharide complex. Furthermore, the preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take the astragalus powder, dissolve it in water, add cellulase, and perform enzymatic hydrolysis to obtain an enzymatic solution; S3. Glucose, peptone, dipotassium hydrogen phosphate and sodium acetate were added to the enzymatic solution, and the pH was adjusted to 7.0-7.2 to obtain a fermentation medium; S4. The single colony of Lactobacillus casei was inoculated into MRS liquid medium and cultured in a shaking incubator at 30-37°C and 80-100 r / min for 20-24 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 30-40%, mixed evenly, and fermented for 4-5 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Furthermore, in step S2, the solid-liquid ratio of astragalus coarse powder to water is 1:(10-15). Furthermore, in step S2, the amount of cellulase added is 0.5-0.7%; the enzymatic hydrolysis time is 50-60 minutes, and the enzymatic hydrolysis temperature is 50-55°C. Furthermore, in step S3, the amount of glucose added is 30-80 g / L, the amount of peptone added is 30-80 g / L, the amount of dipotassium hydrogen phosphate added is 0.4-0.6 g / L, and the amount of sodium acetate added is 0.8-1.2 g / L. Furthermore, in step S5, the inoculation amount of Lactobacillus casei seed liquid is 2-5%. Furthermore, a method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 5-8 parts of astragaloside IV and 3-4 parts of chitosan oligosaccharide, dissolve in deionized water, and stir evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 800-1000 r / min for 8-10 min and take the supernatant; Step 3: spray-dry the supernatant at 80-120° C. and 0.5-5 L / h to obtain the astragaloside IV complex. Furthermore, the astragaloside IV complex prepared by the preparation method can be used in food and health care products. Beneficial effects: 1. The present invention uses astragalus as a raw material, adds cellulase, and the enzymolysis solution obtained after enzymolysis can improve the leaching of effective ingredients in astragalus. The use of the astragalus enzymolysis solution is more conducive to the subsequent extraction of astragaloside IV in astragalus by lactobacillus casei during the fermentation process, thereby having a positive impact on the fermentation process and improving the quality of the fermented product; the present invention sequentially adopts a two-step method of cellulase enzymolysis and lactobacillus casei fermentation to prepare astragaloside IV, replacing the traditional physical and chemical method, avoiding the environmental pollution problem caused by the physical and chemical method, and the extraction method of the present invention is more conducive to improving the content and purity of astragaloside IV in astragalus; 2. The present invention adopts a spray drying method to prepare an astragaloside IV-chitosan oligosaccharide complex. Astragaloside IV and chitosan oligosaccharide form a stable complex through non-covalent interactions of hydrogen bonds or van der Waals forces, which significantly improves the solubility of astragaloside IV in water, greatly enhances the antioxidant effect of the complex, and also has a certain hypoglycemic effect, which provides a new idea for the innovation of astragaloside IV in the fields of food, health care products, etc. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The scavenging effect of DPPH free radicals by Example 10 and Comparative Example 3 is shown; Figure 2 This is a diagram showing the scavenging effect of Example 10 and Comparative Example 3 on ABTS free radicals; Figure 3 This is a graph showing the inhibitory effect of Example 10 and Comparative Example 3 on α-amylase activity; Figure 4 The figure is a graph showing the inhibitory effect of Example 10 and Comparative Example 3 on α-glucosidase activity. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:10, add 0.5% cellulase, and hydrolyze at 50°C for 50 minutes to obtain an enzymatic solution; S3. Add 30 g / L glucose, 30 g / L peptone, 0.4 g / L potassium hydrogen phosphate and 0.8 g / L sodium acetate to the hydrolyzate, adjust the pH to 7.0, and prepare a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid culture medium and cultured on a shaker at 30°C and 80 r / min for 20 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 2%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 30%, mixed evenly, and fermented for 4 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Example 2 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:11, add 0.5% cellulase, and hydrolyze at 50°C for 52 minutes to obtain an enzymatic solution; S3. Add 40 g / L glucose, 40 g / L peptone, 0.4 g / L potassium hydrogen phosphate and 0.9 g / L sodium acetate to the hydrolyzate, adjust the pH to 7.0, and prepare a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid culture medium and cultured in a shaking incubator at 32°C and 80 r / min for 20 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 2%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 32%, mixed evenly, and fermented for 4 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Example 3 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:12, add 0.6% cellulase, and hydrolyze at 52°C for 54 minutes to obtain an enzymatic solution; S3. Add 50 g / L glucose, 40 g / L peptone, 0.5 g / L potassium hydrogen phosphate and 1.0 g / L sodium acetate to the hydrolyzate, adjust the pH to 7.0, and prepare a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid culture medium and cultured in a shaking incubator at 32°C and 90 r / min for 22 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 3%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 34%, mixed evenly, and fermented for 4 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Example 4 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:13, add 0.6% cellulase, and hydrolyze at 54°C for 56 minutes to obtain an enzymatic solution; S3. Add 60 g / L glucose, 50 g / L peptone, 0.5 g / L potassium hydrogen phosphate and 1.1 g / L sodium acetate to the hydrolyzate, adjust the pH to 7.2, and prepare a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid medium and cultured in a shaking incubator at 34°C and 90 r / min for 22 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 4%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 36%, mixed evenly, and fermented for 5 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Example 5 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:14, add 0.7% cellulase, and hydrolyze at 55°C for 58 minutes to obtain an enzymatic solution; S3. Add 70 g / L glucose, 60 g / L peptone, 0.6 g / L potassium hydrogen phosphate and 1.2 g / L sodium acetate to the hydrolyzate, adjust the pH to 7.2, and prepare a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid culture medium and cultured in a shaking incubator at 36°C and 100 r / min for 24 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 5%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 38%, mixed evenly, and fermented for 5 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Example 6 The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:15, add 0.7% cellulase, and hydrolyze at 55°C for 60 minutes to obtain an enzymatic solution; S3. 80 g / L glucose, 80 g / L peptone, 0.6 g / L potassium hydrogen phosphate and 1.2 g / L sodium acetate were added to the hydrolyzate, and the pH was adjusted to 7.2 to obtain a fermentation medium; S4. A single colony of Lactobacillus casei was inoculated into MRS liquid culture medium and cultured in a shaking incubator at 37°C and 100 r / min for 24 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 5%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 40%, mixed evenly, and fermented for 5 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Comparative Example 1 The difference between this comparative example and Example 3 is that only the enzymatic method is used to prepare astragaloside IV, which is as follows: The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take astragalus powder, dissolve it in water, the solid-liquid ratio is 1:12, add 0.6% cellulase, and hydrolyze at 52°C for 54 minutes to obtain an enzymatic solution; S3. The enzymatic hydrolysate is soaked in ethanol and extracted with n-butanol, and the obtained extract is separated and purified by macroporous resin to obtain astragaloside IV. Comparative Example 2 The difference between this comparative example and Example 3 is that only the fermentation method is used to prepare astragaloside IV, which is as follows: The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. To 150 g / L astragalus powder was added 50 g / L glucose, 40 g / L peptone, 0.5 g / L potassium hydrogen phosphate and 1.0 g / L sodium acetate, the pH was adjusted to 7.0 to prepare a fermentation medium; S3. A single colony of Lactobacillus casei was inoculated into MRS liquid medium and cultured in a shaking incubator at 32°C and 90 r / min for 22 h to obtain a Lactobacillus casei seed solution; S4. The Lactobacillus casei seed solution was inoculated into the fermentation medium, the inoculum amount was 3%, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 34%, mixed evenly, and fermented for 4 days to obtain a fermentation broth; S5. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV. Performance Test: (1) Determination of astragaloside content and purity The content of astragaloside IV was determined by high performance liquid chromatography and external standard method. The results are shown in Table 1: Table 1 Content and purity of astragaloside IV prepared in Examples and Comparative Examples sample Astragaloside IV content (mg / g) Astragaloside IV purity (%) Example 1 0.387 98.1 Example 2 0.398 98.3 Example 3 0.442 98.5 Example 4 0.425 97.9 Example 5 0.416 98.2 Example 6 0.431 98.1 Comparative Example 1 0.268 96.7 Comparative Example 2 0.289 97.2 As shown in Table 1, the content of astragaloside IV prepared in Examples 1-6 is 0.387-0.442 mg / g, and the purity is 97.9-98.5%; Comparative Example 1 uses only the enzymatic method to prepare astragaloside IV, with a content of 0.268 mg / g and a purity of 96.7%; Comparative Example 2 uses only the fermentation method to prepare astragaloside IV, with a content of 0.289 mg / g and a purity of 97.2%. It can be seen that the content and purity of astragaloside IV prepared by the two-step method of cellulose enzymatic hydrolysis and Lactobacillus casei fermentation are improved, and the astragaloside IV prepared in Example 3 is used for the subsequent preparation of astragaloside IV complex. Example 7 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 5 parts of astragaloside IV and 3 parts of chitosan oligosaccharide, dissolve them in deionized water, and stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 800 r / min for 10 min and take the supernatant; Step 3: spray-dry the supernatant at 80° C. and 0.5 L / h to obtain the astragaloside IV complex. Example 8 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 5 parts of astragaloside IV and 3 parts of chitosan oligosaccharide, dissolve them in deionized water, and stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 800-1000 r / min for 8-10 min and take the supernatant; Step 3: spray-dry the supernatant at 90° C. and 1.0 L / h to obtain the astragaloside IV complex. Example 9 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 5 parts of astragaloside IV and 3.5 parts of chitosan oligosaccharide and dissolve them in deionized water, stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at a speed of 850 r / min for 10 minutes and take the supernatant; Step 3: spray-dry the supernatant at 90° C. and 1.5 L / h to obtain the astragaloside IV complex. Example 10 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 6 parts of astragaloside IV and 3.5 parts of chitosan oligosaccharide and dissolve them in deionized water, stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at a speed of 900 r / min for 9 minutes and take the supernatant; Step 3: spray-dry the supernatant at 100° C. and 2 L / h to obtain the astragaloside IV complex. Embodiment 11 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 6 parts of astragaloside IV and 3.8 parts of chitosan oligosaccharide and dissolve them in deionized water, stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at a speed of 900 r / min for 9 minutes and take the supernatant; Step 3: spray-dry the supernatant at 110° C. and 2.5 L / h to obtain the astragaloside IV complex. Example 12 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 7 parts of astragaloside IV and 3.8 parts of chitosan oligosaccharide and dissolve them in deionized water, stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at a speed of 1000 r / min for 8 minutes and take the supernatant; Step 3: spray-dry the supernatant at 110° C. and 3 L / h to obtain the astragaloside IV complex. Embodiment 13 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 7 parts of astragaloside IV and 4 parts of chitosan oligosaccharide, dissolve them in deionized water, and stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 1000 r / min for 8 min and take the supernatant; Step 3: spray-dry the supernatant at 120° C. and 4 L / h to obtain the astragaloside IV complex. Embodiment 14 A method for preparing a highly water-soluble astragaloside IV complex comprises the following steps: Step 1: Weigh 8 parts of astragaloside IV and 4 parts of chitosan oligosaccharide, dissolve them in deionized water, and stir them evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 1000 r / min for 8 min and take the supernatant; Step 3: spray-dry the supernatant at 120° C. and 5 L / h to obtain the astragaloside IV complex. Comparative Example 3 The difference between this comparative example and Example 10 is that chitosan oligosaccharide, i.e., astragaloside IV prepared in Example 3, is not added. Performance Test: (2) Solubility determination An excess amount of the astragaloside IV complexes prepared in Examples 7-14 and the astragaloside IV prepared in Comparative Example 3 were placed in a brown glass bottle, 5 mL of water was added, and the mixture was vortexed for one hour. The amount dissolved was the solubility of astragaloside IV. Table 2 Solubility of Astragaloside Ⅳ prepared in Examples and Comparative Examples sample Solubility (mg / mL) sample Solubility (mg / mL) Example 7 13.6 Example 12 13.5 Example 8 13.8 Embodiment 13 13.7 Example 9 14.1 Embodiment 14 14.0 Example 10 14.2 Comparative Example 3 8.6 Embodiment 11 13.9 As can be seen from Table 2, the solubility of the astragaloside complex prepared in Examples 7-14 is 13.5-14.2 mg / mL, while the solubility of astragaloside IV prepared in Comparative Example 3 without adding chitosan oligosaccharide is only 8.6 mg / mL, which confirms that the addition of chitosan oligosaccharide allows astragaloside IV to form a stable complex with chitosan oligosaccharide through non-covalent interactions such as hydrogen bonds or van der Waals forces, thereby significantly improving the solubility of astragaloside IV in water. (3) Antioxidant assay 1) DPPH free radical scavenging rate The astragaloside IV complex prepared in Example 10 and the astragaloside IV prepared in Comparative Example 3 were selected and prepared into sample solutions of 0.0024, 0.012, 0.024, 0.12, 0.24 and 1.2 mg / mL with 50% methanol. Accurately measure 1 mL of the above test solution, add 1 mL of 0.1 mmol / L DPPH solution respectively, shake well, react at room temperature in the dark for 30 minutes, take 200 μL, and measure the absorbance A1 at a wavelength of 517 nm; take an equal volume of 50% methanol to replace the DPPH solution, and measure the absorbance A2 at a wavelength of 517 nm; take an equal volume of 50% methanol to replace the sample solution, and measure the absorbance A3 at a wavelength of 517 nm; take the absorbance value of VC as the positive control, and calculate the clearance rate according to the following formula: DPPH free radical scavenging rate (%) = [1-(A1-A2) / A3] × 100% Depend on Figure 1 It can be seen that with the increase of the concentration of astragaloside IV complex, the scavenging rate of DPPH free radicals also gradually increased. When the concentration is above 0.024 mg / mL, the DPPH scavenging rate can reach more than 50%, and the antioxidant effect is due to astragaloside IV, indicating that the antioxidant activity of astragaloside IV complex is improved compared with astragaloside IV. 2) ABTS free radical scavenging rate Mix 7.4mmol / LABTS solution and 2.6mmol / LK2S2O8 solution in a ratio of 1:1, keep in the dark at room temperature for 16h, dilute with ethanol to an absorbance of 0.70±0.02 (at 734nm wavelength), and use it immediately after preparation. The astragaloside IV complex prepared in Example 10 and the astragaloside IV prepared in Comparative Example 3 were selected and prepared into sample solutions of 0.0024, 0.012, 0.024, 0.12, 0.24 and 1.2 mg / mL with 50% methanol. 0.1 mL of the above sample solutions were measured, 3.9 mL of ABTS solution was added to each, shaken well, and allowed to stand at room temperature in the dark for 6 min. 200 μL was taken and the absorbance A1 was measured at 734 nm absorption wavelength; an equal volume of anhydrous ethanol was taken to replace the ABTS working solution, and the absorbance A2 was measured at 734 nm wavelength; an equal volume of 50% methanol was taken to replace the sample solution, and the absorbance A3 was measured at 734 nm wavelength. The absorbance value of VC was used as the positive control, and the clearance rate was calculated according to the following formula: ABTS free radical scavenging rate (%) = [1-(A1-A2) / A3] × 100% Depend on Figure 2It can be seen that with the increase of the concentration of astragaloside IV complex, the scavenging effect on ABTS free radicals also gradually increases, and the scavenging rate of astragaloside IV complex is higher than that of astragaloside IV alone. The higher the scavenging rate, the stronger the antioxidant activity of the sample. It can be seen that the astragaloside IV-chitosan oligosaccharide complex prepared by the addition of chitosan oligosaccharides is beneficial to improving the antioxidant properties of astragaloside IV. (4) Hypoglycemic effect 1) α-amylase activity inhibition assay Take an appropriate amount of the astragaloside complex prepared in Example 10 and the astragaloside prepared in Comparative Example 3, dissolve and dilute with distilled water to form sample solutions with mass concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 mg / mL, add 40 μL of PBS solution, 40 μL of sample solution, and 20 μL of α-amylase solution to a 96-well plate, mix, react at 37° C. for 10 min, add 30 μL of soluble starch solution, mix, react at 37° C. for 10 min, add 20 μL of HCl solution to terminate the reaction, and finally add 120 μL of iodine solution, mix, and measure the absorbance A1 at a wavelength of 565 nm; take an equal volume of PBS solution instead of the starch solution, and measure the absorbance A2 at a wavelength of 565 nm; take an equal volume of PBS solution instead of the sample and α-amylase solution, and measure the absorbance A3 at a wavelength of 565 nm. Acarbose with a mass concentration of 20, 10, 5, 2.5, 1.25, and 0.625 mg / mL was used as a positive control. The test was repeated three times in parallel, and the average value was taken to calculate the inhibition rate of α-amylase activity according to the following formula: Inhibition rate (%) = [1-(A1-A2) / A3] × 100% Depend on Figure 3 It can be seen that acarbose did not show dose dependence, and the inhibition of astragaloside IV and astragaloside IV complex on α-amylase was concentration-dependent, with the astragaloside IV complex being more obvious. This shows that after astragaloside IV prepared by enzymatic and fermentation methods is combined with chitosan oligosaccharides, the complex can enhance the inhibitory effect on α-amylase, thereby achieving a hypoglycemic effect. 2) α-Glucosidase inhibition assay Take an appropriate amount of the astragaloside complex prepared in Example 10 and the astragaloside prepared in Comparative Example 3, dissolve and dilute with distilled water to form sample solutions with mass concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, add 145 μL of PBS solution, 15 μL of each sample solution, and 15 μL of α-glucosidase to a 96-well plate, mix, react at 37°C for 10 min, add 15 μL of PNPG, mix, react at 37°C for 20 min, then add 60 μL of Na2CO3, and measure the absorbance A1 at a wavelength of 405 nm; take an equal volume of PBS solution to replace α-glucosidase, and measure the absorbance A2 at a wavelength of 405 nm; take an equal volume of PBS solution to replace PNPG, and measure the absorbance A3 at a wavelength of 405 nm. Acarbose with a mass concentration of 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL was used as a positive control, and the results were repeated 3 times. The average value was taken and the α-glucosidase inhibition rate was calculated according to the following formula: Inhibition rate (%) = [1-(A1-A2) / A3] × 100% Depend on Figure 4 It can be seen that acarbose has the strongest inhibition rate on α-glucosidase, and the inhibitory effect of astragaloside IV complex is higher than that of astragaloside IV, indicating that the complex prepared by combining chitosan oligosaccharides and astragaloside IV can enhance the inhibitory effect on α-glucosidase and lower blood sugar by delaying the absorption of glucose. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.
Claims
1. A highly water-soluble astragaloside IV complex, characterized in that: The astragaloside IV complex is specifically an astragaloside IV-chitooligosaccharide complex.
2. A method for preparing a highly water-soluble astragaloside IV complex, characterized in that: The preparation steps of astragaloside IV are as follows: S1. Wash and crush Astragalus membranaceus as raw material to prepare Astragalus membranaceus coarse powder; S2. Take the astragalus powder, dissolve it in water, add cellulase, and perform enzymatic hydrolysis to obtain an enzymatic solution; S3. Glucose, peptone, dipotassium hydrogen phosphate and sodium acetate were added to the hydrolyzate, and the pH was adjusted to 7.0-7.2 to obtain a fermentation medium; S4. The single colony of Lactobacillus casei was inoculated into MRS liquid medium and cultured in a shaking incubator at 30-37°C and 80-100 r / min for 20-24 h to obtain a Lactobacillus casei seed solution; S5. The Lactobacillus casei seed solution was inoculated into the fermentation medium, and then an appropriate amount of sterile water was added to make the water content of the fermentation system 30-40%, mixed evenly, and fermented for 4-5 days to obtain a fermentation broth; S6. Soak the fermentation broth in ethanol and extract with n-butanol, and separate and purify the obtained extract with macroporous resin to obtain astragaloside IV.
3. The method for preparing a highly water-soluble astragaloside IV complex according to claim 2, characterized in that: In the step S2, the solid-liquid ratio of the astragalus coarse powder to water is 1:(10-15).
4. The method for preparing a highly water-soluble astragaloside IV complex according to claim 2, characterized in that: The amount of cellulase added in step S2 is 0.5-0.7%; the enzymolysis treatment time is 50-60 minutes, and the enzymolysis temperature is 50-55°C.
5. The method for preparing a highly water-soluble astragaloside IV complex according to claim 2, characterized in that: In step S3, the amount of glucose added is 30-80 g / L, the amount of peptone added is 30-80 g / L, the amount of dipotassium hydrogen phosphate added is 0.4-0.6 g / L, and the amount of sodium acetate added is 0.8-1.2 g / L.
6. The method for preparing a highly water-soluble astragaloside IV complex according to claim 2, characterized in that: In step S5, the inoculation amount of Lactobacillus casei seed liquid is 2-5%.
7. The method for preparing a highly water-soluble astragaloside IV complex according to claim 1, characterized in that: The following steps are involved: Step 1: Weigh 5-8 parts of astragaloside IV and 3-4 parts of chitosan oligosaccharide, dissolve in deionized water, and stir evenly to obtain a mixed solution; Step 2: Centrifuge the mixed solution at 800-1000 r / min for 8-10 min and take the supernatant; Step 3: spray-dry the supernatant at 80-120° C. and 0.5-5 L / h to obtain the astragaloside IV complex.
8. Use of the astragaloside IV complex prepared by the preparation method according to any one of claims 1 to 7 in food and health care products.