Process for extracting flavone from honeysuckle by using semi-bionic enzyme extraction method

Abstract

The invention relates to the technical field of plant extraction, and provides a process for extracting flavone from honeysuckle by using a semi-bionic enzyme extraction method, which comprises the following steps: S1, drying and crushing honeysuckle to obtain honeysuckle powder; s2, dispersing the honeysuckle flower powder in water, adding pepsin, adjusting the pH value to 2.0-4.0, performing enzymolysis for 4-5 hours, and performing centrifugation to obtain first supernate and first residues; s3, immobilized double enzymes are added into the first residues, then the pH is adjusted to 8.0-8.5, enzymolysis is conducted for 5-7 h, and second supernate and second residues are obtained; s4, mixing the first supernate and the second supernate, and performing enzyme deactivation, concentration and vacuum drying to obtain honeysuckle flavone; the immobilized double enzymes comprise the following raw materials: a cellulase solution and a trypsin solution in a volume ratio of 1: (1-5). By means of the technical scheme, the problem that in the prior art, the extraction rate of flavone in honeysuckle is low is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant medicine, in particular, to a process for extracting flavones from honeysuckle by semi-bionic enzyme extraction method. BACKGROUND

[0002] Honeysuckle is the dried flower buds or the just opened flowers of Lonicera japonica Thunb. It tastes sweet and is cold in nature, and belongs to stomach, lung and heart channels. It is not only a traditional Chinese medicine widely used in clinical practice, but also a representative substance included in the category of "medicinal and edible homology", and has both medicinal efficacy and edible safety. In clinical practice, it is widely used for preventing and treating upper respiratory tract infection, hyperlipidemia and some epidemic diseases. From the perspective of medicinal material basis, honeysuckle contains rich chemical components, among which flavones are the main medicinal components. These compounds have the biological activities of lowering blood sugar, lowering blood lipid, inhibiting bacteria, relieving pain, and resisting viruses, and are widely used in the medical and food industries, and have development value. However, there are still challenges such as limitations of extraction methods and efficiency problems. Future research should further optimize the extraction process and improve the extraction efficiency to promote the efficient use of honeysuckle resources. SUMMARY

[0003] The present application provides a process for extracting flavones from honeysuckle by semi-bionic enzyme extraction method, which solves the problem of low extraction rate of flavones in honeysuckle in related technologies.

[0004] The technical scheme of the present application is as follows: The present application provides a process for extracting flavones from honeysuckle by semi-bionic enzyme extraction method, which includes the following steps: S1, drying and crushing honeysuckle to obtain honeysuckle powder; S2, dispersing the honeysuckle powder in water, adding pepsin, adjusting the pH to 2.0-4.0, and enzymolysis for 4-5 hours, then centrifuging to obtain the first supernatant and the first residue; S3, adding immobilized double enzymes to the first residue, adjusting the pH to 8.0-8.5, and enzymolysis for 5-7 hours to obtain the second supernatant and the second residue; S4, mixing the first supernatant and the second supernatant, inactivating the enzyme, concentrating, and vacuum drying to obtain honeysuckle flavones; The raw materials of the immobilized double enzymes include cellulase liquid and trypsin liquid in a volume ratio of 1:1-5.

[0005] As a further technical scheme, the preparation method of the immobilized double enzymes (cellulase and trypsin) includes the following steps: A1, preparing 1 mg / mL cellulase liquid and 1 mg / mL trypsin liquid with pH=6.8 phosphate buffer, then mixing the two enzyme liquids to obtain a mixed enzyme liquid; A2, the mixed enzyme solution is added to the sodium alginate solution, stirred, and left to obtain the ammonium alginate solution of the double enzyme solution; A3, the ammonium alginate solution of the double enzyme solution obtained in A2 is added dropwise into the CaCl2 fixing solution to form gel-like small balls, the immobilized double enzyme gel is filtered out, left to harden in a refrigerator at 4 DEG C for 0.5-1 h, washed twice with a NaCl solution, washed three times with distilled water, dried, and the immobilized double enzyme is obtained.

[0006] As a further technical solution, the volume ratio of the cellulase solution and the trypsin solution in the mixed enzyme solution is 1:2-3.

[0007] In the application, when the volume ratio of the cellulase solution and the trypsin solution is 1:2-3, the flavone extraction rate is further improved.

[0008] As a further technical solution, the mass fraction of the sodium alginate solution is 2%-5%.

[0009] As a further technical solution, the volume ratio of the mixed enzyme solution and the sodium alginate solution is 1:1-3.

[0010] As a further technical solution, the temperature of the shaking table is 60-70 DEG C, and the time is 1.5-2 h.

[0011] As a further technical solution, the mass fraction of the CaCl2 fixing solution is 3%-5%.

[0012] As a further technical solution, the mass fraction of the NaCl solution is 4%.

[0013] As a further technical solution, the drying temperature is 40-50 DEG C.

[0014] As a further technical solution, the enzyme activity of the pepsin in S2 is 3000 U / g.

[0015] As a further technical solution, the adjusting solution in S2 uses 1 mol / L HCl solution to adjust the pH.

[0016] As a further technical solution, the enzyme activity of the cellulase in S3 is 30000 U / g.

[0017] As a further technical solution, the enzyme activity of the trypsin in S3 is 100000 U / g.

[0018] As a further technical solution, the adjusting solution in S3 uses 10 wt% NaOH solution to adjust the pH.

[0019] As a further technical solution, the temperature when the pepsin and the immobilized double enzyme are added is 32-42℃.

[0020] As a further technical solution, the temperature for enzyme inactivation is 80-85℃, and the time is 15-30 min.

[0021] The working principle and beneficial effects of the present application are as follows: In the present application, pepsin and immobilized double enzyme (cellulase and trypsin) are used as core extraction enzymes. The cellulase hydrolyzes cellulose in the cell wall of honeysuckle, destroys the dense structure of the cell wall, and thus promotes the dissolution of flavonoids and improves the extraction rate of flavonoids. The addition of pepsin and trypsin makes the extraction process closer to the transport and absorption process of drugs in the human gastrointestinal tract. The immobilization technology improves the stability and cycle life of the enzyme. Through the combination of multi-enzyme synergistic effect and immobilization technology, the extraction rate of flavonoids is further improved. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are involved in the scope of protection of the present application.

[0023] Embodiment 1 The preparation method of the immobilized double enzyme (cellulase and trypsin) includes the following steps: A 1 mg / mL cellulase solution and a 1 mg / mL trypsin solution are prepared using a phosphate buffer with a pH of 6.8, and then the two enzyme solutions are mixed to obtain a mixed enzyme solution, wherein the volume ratio of the cellulase solution to the trypsin solution is 1:1. The mixed enzyme solution is added to a 3% sodium alginate solution, and the mixed enzyme solution and the sodium alginate solution are mixed uniformly at a volume ratio of 1:1.5. After stirring for 40 min and standing for 2 h, a sodium alginate solution of double enzyme is obtained. The obtained sodium alginate solution of double enzyme is added dropwise into a 4% CaCl2 fixing solution to form gel-like small balls, wherein the volume ratio of the sodium alginate solution of double enzyme to the CaCl2 fixing solution is 1:5. The immobilized double enzyme gel is filtered out, placed in a 4℃ refrigerator for 1 h of hardening, washed twice with a 4% NaCl solution, and then rinsed three times with distilled water. After drying at 45℃, the immobilized double enzyme is obtained. The process for extracting flavonoids from honeysuckle includes the following preparation processes: The cleaned honeysuckle is dried and then crushed into honeysuckle powder for standby use. 100g of honeysuckle powder was dispersed in ultrapure water with a substrate concentration of 1g / 30mL. Pepsin was added with an enzyme-substrate ratio of 10000U / g and a pepsin addition amount of 25mg. The pH was adjusted to 2.5 with 1mol / L HCl solution. The mixture was enzymatically hydrolyzed at 35℃ for 5h and centrifuged to obtain the first supernatant and the first residue. Ultrapure water was added to the first residue, the substrate concentration was 1g / 30mL, and immobilized dual enzymes (cellulase and trypsin) were added. The enzyme-substrate ratio of the immobilized dual enzymes was 10000U / g, and the amount of immobilized dual enzymes added was 280mg. The pH was then adjusted to 8.0 with 10wt% NaOH solution, and enzymatic hydrolysis was carried out at 35℃ for 7h to obtain the second supernatant and the second residue. The first and second supernatants were mixed, and the enzymes were inactivated by boiling at 85°C for 25 minutes. The mixture was then concentrated and dried under vacuum to obtain honeysuckle flavonoids.

[0024] Example 2 The method for preparing immobilized dual enzymes (cellulase and trypsin) includes the following steps: Prepare 1 mg / mL cellulase solution and 1 mg / mL trypsin solution using phosphate buffer at pH 6.8, and then mix the two enzyme solutions to obtain a mixed enzyme solution, wherein the volume ratio of cellulase solution to trypsin solution is 1:5. Add the mixed enzyme solution to a 3% sodium alginate solution, mix well, and the volume ratio of the mixed enzyme solution to the sodium alginate solution is 1:1.5. Stir for 40 minutes and let stand for 2 hours to obtain the sodium alginate solution of the dual enzyme solution. The sodium alginate solution of the obtained dual enzyme solution was added dropwise to a 4% CaCl2 fixative to form gel-like spheres. The volume ratio of sodium alginate solution to CaCl2 fixative was 1:5. The immobilized dual enzyme gel was filtered out and placed in a 4°C refrigerator for 1 hour to harden. It was then washed twice with a 4% NaCl solution, rinsed three times with distilled water, and dried at 45°C to obtain the immobilized dual enzyme. The process for extracting flavonoids from honeysuckle includes the following preparation steps: Dry the cleaned honeysuckle flowers, then grind them into honeysuckle powder for later use; 100g of honeysuckle powder was dispersed in ultrapure water with a substrate concentration of 1g / 30mL. Pepsin was added with an enzyme-substrate ratio of 10000U / g and a pepsin addition amount of 25mg. The pH was adjusted to 2.5 with 1mol / L HCl solution. The mixture was enzymatically hydrolyzed at 35℃ for 5h and centrifuged to obtain the first supernatant and the first residue. Ultrapure water was added to the first residue, the substrate concentration was 1g / 30mL, and immobilized dual enzymes (cellulase and trypsin) were added. The enzyme-substrate ratio of the immobilized dual enzymes was 10000U / g, and the amount of immobilized dual enzymes added was 280mg. The pH was then adjusted to 8.0 with 10wt% NaOH solution, and enzymatic hydrolysis was carried out at 35℃ for 7h to obtain the second supernatant and the second residue. The first and second supernatants were mixed, and the enzymes were inactivated by boiling at 85°C for 25 minutes. The mixture was then concentrated and dried under vacuum to obtain honeysuckle flavonoids.

[0025] Example 3 The method for preparing immobilized dual enzymes (cellulase and trypsin) includes the following steps: Prepare 1 mg / mL cellulase solution and 1 mg / mL trypsin solution using phosphate buffer at pH 6.8, and then mix the two enzyme solutions to obtain a mixed enzyme solution, wherein the volume ratio of cellulase solution to trypsin solution is 1:4. Add the mixed enzyme solution to a 3% sodium alginate solution, mix well, and the volume ratio of the mixed enzyme solution to the sodium alginate solution is 1:1.5. Stir for 40 minutes and let stand for 2 hours to obtain the sodium alginate solution of the dual enzyme solution. The sodium alginate solution of the obtained dual enzyme solution was added dropwise to a 4% CaCl2 fixative to form gel-like spheres. The volume ratio of sodium alginate solution to CaCl2 fixative was 1:5. The immobilized dual enzyme gel was filtered out and placed in a 4°C refrigerator for 1 hour to harden. It was then washed twice with a 4% NaCl solution, rinsed three times with distilled water, and dried at 45°C to obtain the immobilized dual enzyme. The process for extracting flavonoids from honeysuckle includes the following preparation steps: Dry the cleaned honeysuckle flowers, then grind them into honeysuckle powder for later use; 100g of honeysuckle powder was dispersed in ultrapure water with a substrate concentration of 1g / 30mL. Pepsin was added with an enzyme-substrate ratio of 10000U / g and a pepsin addition amount of 25mg. The pH was adjusted to 2.5 with 1mol / L HCl solution. The mixture was enzymatically hydrolyzed at 35℃ for 5h and centrifuged to obtain the first supernatant and the first residue. Ultrapure water was added to the first residue, the substrate concentration was 1g / 30mL, and immobilized dual enzymes (cellulase and trypsin) were added. The enzyme-substrate ratio of the immobilized dual enzymes was 10000U / g, and the amount of immobilized dual enzymes added was 280mg. The pH was then adjusted to 8.0 with 10wt% NaOH solution, and enzymatic hydrolysis was carried out at 35℃ for 7h to obtain the second supernatant and the second residue. The first and second supernatants were mixed, and the enzymes were inactivated by boiling at 85°C for 25 minutes. The mixture was then concentrated and dried under vacuum to obtain honeysuckle flavonoids.

[0026] Example 4 The difference between this embodiment and Example 3 is that the volume ratio of cellulase solution to trypsin solution is 1:1.5.

[0027] Example 5 The difference between this embodiment and Example 3 is that the volume ratio of cellulase solution to trypsin solution is 1:2.

[0028] Example 6 The difference between this embodiment and Example 3 is that the volume ratio of cellulase solution to trypsin solution is 1:3.

[0029] Comparative Example 1 The difference between this embodiment and Embodiment 3 is that cellulase solution is not added.

[0030] Comparative Example 2 The difference between this comparative example and Example 3 is that trypsin solution is not added.

[0031] Comparative Example 3 The difference between this comparative example and Example 3 is that no curing treatment is performed; cellulase solution and trypsin solution are added directly.

[0032] The content and extraction rate of flavonoids obtained in Examples 1-6 and Comparative Examples 1-3 were calculated: The calculation results are shown in Table 1: Table 1. Calculation results of flavonoid content and extraction rate obtained from Examples 1-6 and Comparative Examples 1-3.

[0033] 1. Compared with Comparative Examples 1-3, the flavonoid content extracted from honeysuckle in Examples 1-6 was significantly higher than that in Comparative Examples 1-3, and the extraction rate was significantly improved. This indicates that the combined use of cellulase and trypsin, and their immobilization treatment, improved the extraction rate of flavonoids from honeysuckle.

[0034] 2. Comparing Examples 1 to 6, Examples 5 and 6 extracted flavonoids with higher content, indicating that when the volume ratio of cellulase solution to trypsin solution is 1:2 to 3, the flavonoid extraction rate is further improved.

[0035] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for extracting flavonoids from honeysuckle by semi-bionic enzyme extraction method, characterized in that, The method comprises the following steps: S1, drying honeysuckle, crushing, to get honeysuckle powder; S2, the honeysuckle powder is dispersed in water, and pepsin is added, the pH is adjusted to 2.0~4.0, and enzymolysis is carried out for 4~5h, and then centrifugation is carried out, to obtain a first supernatant and a first residue; S3, the first residue is added with immobilized double enzymes, and the pH is adjusted to 8.0~8.5, and enzymolysis is carried out for 5~7h, to obtain a second supernatant and a second residue; S4, the first supernatant and the second supernatant are mixed, enzyme is inactivated, concentrated, vacuum dried, to obtain honeysuckle flavones; The raw material of the immobilized double enzymes comprises cellulase liquid and trypsin liquid in a volume ratio of 1:1~5.

2. The process for extracting flavonoids from honeysuckle by using semi-bionic enzyme extraction method according to claim 1, characterized in that, The preparation method of the immobilized double enzymes comprises the following steps: A1, 0.5~1.5mg / mL cellulase liquid and 0.5~1.5mg / mL trypsin liquid are prepared by using phosphate buffer, and then the two kinds of enzyme liquids are mixed, to obtain mixed enzyme liquid; A2, the mixed enzyme liquid is added into sodium alginate solution, stirred, and then placed to obtain ammonium alginate solution of double enzyme liquid; A3, the ammonium alginate solution of double enzyme liquid is added dropwise into CaCl2 fixing liquid, to form gel-like small balls, and then the immobilized double enzyme gel is filtered out, placed in a 4℃ refrigerator, and hardened for 0.5~1h, washed with NaCl solution for 2 times, washed with distilled water for 3 times, and dried, to obtain immobilized double enzymes.

3. The process for extracting flavonoids from honeysuckle using a semi-bionic enzyme extraction method according to claim 2, characterized in that, The pH of the phosphate buffer is 6.8; The volume ratio of cellulase liquid to trypsin liquid in the mixed enzyme liquid is 1:2~3; The mass fraction of the sodium alginate solution is 2%~5%; The volume ratio of the mixed enzyme liquid to sodium alginate solution is 1:1~3; The temperature of the shaking table is 60~70℃, and the time is 1.5~2h; The mass fraction of the CaCl2 fixing liquid is 3%~5%; The mass fraction of the NaCl solution is 4%; The drying temperature is 40~50℃.

4. The process for extracting flavonoids from honeysuckle using a semi-bionic enzyme extraction method according to claim 1, characterized in that, In step S2, the pepsin enzyme activity is 3000U / g.

5. The process for extracting flavonoids from honeysuckle according to claim 1, characterized in that, In step S2, the pH is adjusted by using 1~3mol / L HCl solution.

6. The process as claimed in claim 1, wherein the process for extraction of flavonoids from Honeysuckle by semi-bionic enzyme extraction method, wherein the process further comprises of the steps of: In the cellulase liquid, the cellulase enzyme activity is 30000~40000U / g.

7. The process for extracting flavonoids from honeysuckle using a semi-bionic enzyme extraction method according to claim 1, characterized in that, In the trypsin liquid, the trypsin enzyme activity is 100000~200000U / g.

8. The process as claimed in claim 1, wherein the process for extraction of flavonoids from Honeysuckle by semi-bionic enzyme extraction method, wherein the process further comprises of the steps of: In step S3, when the pH is adjusted, 8wt%~12wt% NaOH solution is used.

9. The process as claimed in claim 1, wherein the process for extraction of flavonoids from Honeysuckle by semi-bionic enzyme extraction method, wherein the process further comprises of the steps of: The temperature when the pepsin and the immobilized double enzymes are added is independently 32~42℃.

10. The process for extracting flavonoids from honeysuckle using a semi-bionic enzyme extraction method according to claim 1, characterized in that, The temperature for inactivating the enzyme is 80~85℃, and the time is 15~30min.

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