A method for extracting flavonoids from plants at low temperature assisted by directed enzyme and its application

Abstract

The present invention discloses a method and application of directional enzyme-assisted low-temperature extraction of flavonoids from plants, including mixing plant materials with combined enzymes, adding water as an extraction solvent, and performing enzymolysis at a temperature not exceeding 40°C for 4 to 8 hours under the protection of an inert gas; after the extraction, adding polyols or oils approved for use in food and cosmetics to the extract for dissolution, and stirring the extracts sufficiently; removing solid particles with a 300-mesh filter bag, filtering the supernatant with a 2μm microporous filter membrane, filtering the filtrate with an ultrafiltration membrane, collecting the filtrate, and obtaining plant flavonoids. The present invention adopts a combination of multiple enzymes, uses water as the only extraction solvent, uses an inert gas protection during the extraction process, and the extraction temperature does not exceed 40°C, thereby avoiding the defects of organic reagent pollution, oxidation of active ingredients, or high-temperature destruction of active ingredients caused by traditional extraction processes, and achieving green, gentle, and efficient extraction of plant active ingredients.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and in particular relates to a method for extracting flavonoids from plants at low temperature assisted by a directed enzyme and an application thereof. Background Art

[0002] Plant extracts are products made from plants as raw materials. According to the needs of the final product, they are extracted through a physical and chemical extraction and separation process to obtain and concentrate one or more active ingredients in the plant without changing the structure of the active ingredients.

[0003] Traditional extraction processes generally use organic reagents (usually methanol, ethanol, ethyl acetate, etc.) or composite solvents of water and organic reagents to extract plants, assisted by auxiliary materials and equipment such as enzymes, ionic liquids, ultrasound, microwaves, etc., and are carried out in a high temperature environment (>40°C) or air atmosphere. This method has problems such as high energy consumption, low environmental protection, and high equipment requirements.

[0004] Cellulose, lignin and other substances in plant materials have the characteristics of water absorption, protecting plant cell walls, and hindering the diffusion of active ingredients. They are key factors that hinder the plant extraction rate. By using targeted enzymes to enzymatically hydrolyze cellulose, lignin and other substances, it is beneficial to fully release the active ingredients of plants. Common plant active ingredients include flavonoids, phenols, alkaloids, triterpenes and other substances. These substances are unstable, mainly due to the high temperature and oxygen environment of the extraction process. In addition, in traditional extraction processes, enzyme inactivation generally uses high-temperature inactivation, which causes further loss of plant active ingredients. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for extracting flavonoids from plants by directional enzyme-assisted low-temperature extraction.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for extracting flavonoids from plants at low temperature with the assistance of directed enzymes, comprising:

[0009] The plant material is mixed with the combined enzyme, water is added as an extraction solvent, and enzymolysis is performed at a temperature not exceeding 40°C for 4 to 8 hours under the protection of an inert gas;

[0010] After the extraction is completed, add polyols or fats approved for food and cosmetics to the extract to dissolve and stir thoroughly;

[0011] The solid particles were removed by using a 300-mesh filter bag, the supernatant was filtered by using a 2-μm microporous filter membrane, the filtrate was filtered by using an ultrafiltration membrane, and the filtrate was collected to obtain plant flavonoids.

[0012] As a preferred embodiment of the method of the present invention, the combination enzyme includes a combination of two or more of β-glucanase, α-glucosidase, β-glucosidase, xylanase, glucomannanase, lignin peroxidase, laccase, manganese peroxidase and pectinase.

[0013] As a preferred embodiment of the method of the present invention, the plant materials include wood, weeping birch, lamp tree, multi-spiked essen bean tree, rosewood, European basswood, European larch, sandalwood, sugar pine, Siberian larch, roquercus robur, olive wood, guaiac wood, soap tree, rosewood, cypress leaves, North American holy grass, and astragalus.

[0014] As a preferred embodiment of the method of the present invention, the polyols include glycerol, 1,3-propylene glycol, dipropylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,2-pentanediol, 1,2-hexanediol, caprylyl glycol, and sorbitol.

[0015] As a preferred embodiment of the method of the present invention, the oil comprises caprylic capric triglyceride, octyl dodecanol, isononyl isononanoate, coconut oil-caprylate caprate, glyceryl tri(ethylhexanoate) ester) C12-15 alcohol benzoate, isopropyl myristate, squalane, olive oil, meadowfoam seed oil, camellia seed oil, jojoba oil, and marula oil.

[0016] As a preferred embodiment of the method of the present invention, the inert gas includes nitrogen, carbon dioxide, helium and argon.

[0017] As a preferred embodiment of the method of the present invention, the molecular weight range of the ultrafiltration membrane is 10000 Da-50000 Da.

[0018] As a preferred embodiment of the method of the present invention, the ratio of the polyol or oil approved for use in food and cosmetics to the plant material is 60-80g:20g.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for extracting flavonoids from plants using a directed enzyme-assisted low-temperature method.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of flavonoids in the preparation of cosmetics.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a method for directional enzyme-assisted low-temperature extraction of flavonoids in plants. By adopting a combination of multiple enzymes, water is used as the only extraction solvent, inert gas protection is used during the extraction process, and the extraction temperature does not exceed 40°C, the defects of organic reagent pollution, oxidation of active ingredients or high-temperature destruction caused by traditional extraction processes are avoided, and green, mild and efficient extraction of plant active ingredients is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 Graph showing the effect of combined enzymes on total flavonoid content and extraction rate in an embodiment of the present invention.

[0025] Figure 2 Graph showing the effect of temperature on total flavonoid content and extraction rate in an embodiment of the present invention.

[0026] Figure 3 Graph showing the effect of nitrogen protection on the total flavonoid content and extraction rate in an embodiment of the present invention.

[0027] Figure 4 Graph showing the effects of different polyols on the total flavonoid content and extraction rate in the examples of the present invention. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0031] In the embodiment of the present invention, the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined according to "SN / T 4592-2016 Determination of total flavonoids in exported food", and the total flavonoid extraction rate without polyol was calculated as follows:

[0032] ω(%)=(c÷10 6 ×300÷20)×100%

[0033] Where: ω—total flavonoid extraction rate, %;

[0034] c—total flavonoid content, ppm;

[0035] 300—total mass of the extract, g;

[0036] 20—Mass of guaiac wood powder, g.

[0037] The total flavonoid extraction rate of polyol extract is calculated as follows:

[0038] ω(%)=(c÷10 6 ×360÷20)×100%

[0039] Where: ω—total flavonoid extraction rate, %;

[0040] c—total flavonoid content, ppm;

[0041] 360—total mass of the extract, g;

[0042] 20—Mass of guaiac wood powder, g.

[0043] The raw materials in the present invention are all common commercially available products.

[0044] Example 1

[0045] Effect of combined enzymes on the extraction rate of total flavonoids from Guaiac wood:

[0046] (1) Accurately weigh 20 g of guaiac powder crushed by an industrial grinder, add 300 g of deionized water, place in a 500 mL stoppered conical flask, and shake well to fully soak the guaiac powder;

[0047] (2) The total amount of immobilized enzyme added was 0.1 g, and the mass ratio of each enzyme was 1:1. 0.02 g of β-glucanase, β-glucosidase, xylanase, lignin peroxidase, and laccase were accurately weighed and added to a stoppered conical flask as a sample group;

[0048] Among them, β-glucanase—enzyme activity 13w; β-glucosidase—enzyme activity 1000; xylanase—enzyme activity 28w; lignin peroxidase—enzyme activity 1w; laccase—enzyme activity 1w, enzyme activity unit: U / g, purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0049] The control group samples were used to reduce the types of enzymes in turn to compare the extraction rates with the sample group. The enzyme combination method is shown in Table 1;

[0050] (3) Add a magnetic stir bar to all samples and place them in a 40°C constant temperature magnetic stirring water bath for extraction for 4 h;

[0051] (4) After the extraction, solid particles were removed with a 300-mesh filter bag, the supernatant was filtered with a 2 μm microporous filter membrane, and the filtrate was filtered with a small ultrafiltration membrane system with a molecular weight of 50,000 Da, and the filtrate was collected;

[0052] According to “SN / T 4592-2016 Determination of total flavonoids in exported food”, the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined.

[0053] The results are as follows Figure 1 shown.

[0054] Table 1 Enzyme combination

[0055]

[0056]

[0057] The results show that under the combined extraction conditions of 5 enzymes co-existing, the extraction efficiency of total flavonoids in guaiac powder in the embodiment is the best; when the number of enzyme combinations is reduced to 3 or less, the extraction efficiency is significantly reduced.

[0058] Example 2

[0059] Effect of extraction temperature on the extraction rate of total flavonoids from Guaiac wood:

[0060] (1) Accurately weigh 20 g of guaiac powder crushed by an industrial grinder, add 300 g of deionized water, place in a 500 mL stoppered conical flask, and shake well to fully soak the guaiac powder;

[0061] (2) Accurately weigh 0.02 g of β-glucanase, β-glucosidase, xylanase, lignin peroxidase, and laccase respectively and add them into a stoppered conical flask;

[0062] A magnetic stirring bar was added to all samples and the samples were placed in a constant temperature magnetic stirring water bath for extraction for 4 h;

[0063] (3) All samples were subjected to different extraction temperatures (20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C) to compare the extraction rates;

[0064] (4) After the extraction, solid particles were removed with a 300-mesh filter bag, the supernatant was filtered with a 2 μm microporous filter membrane, and the filtrate was filtered with a small ultrafiltration membrane system with a molecular weight of 50,000 Da, and the filtrate was collected;

[0065] According to “SN / T 4592-2016 Determination of total flavonoids in exported food”, the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined.

[0066] The results are as follows Figure 2 As shown; the results show that the extraction efficiency of total flavonoids from Guaiac wood is low when the extraction is carried out at lower temperatures (20℃, 30℃), which is related to the low permeability of low-temperature water to wood fiber and the low activity of enzymes at low temperatures; when the extraction temperature is higher than 40℃, increasing the temperature within an appropriate range is beneficial to the improvement of enzyme activity, but is not conducive to the preservation of flavonoids; especially when the extraction temperature is increased to 80℃, the total flavonoid content decreases significantly, which is related to the high-temperature inactivation of the enzyme and the simultaneous destruction of flavonoids.

[0067] Example 3

[0068] Effect of nitrogen protection on the extraction rate of total flavonoids from Guaiac wood:

[0069] (1) Accurately weigh 20 g of guaiac powder crushed by an industrial grinder, add 300 g of deionized water, place in a 500 mL stoppered conical flask, and shake well to fully soak the guaiac powder;

[0070] (2) Accurately weigh 0.02 g of β-glucanase, β-glucosidase, xylanase, lignin peroxidase, and laccase respectively and add them into a stoppered conical flask;

[0071] A magnetic stirrer was added to the sample and nitrogen was introduced for 5 min, followed by immediate sealing with a stopper;

[0072] (3) The sample was placed in a 40°C constant temperature magnetic stirring water bath for extraction for 4 h;

[0073] (4) After the extraction, solid particles were removed with a 300-mesh filter bag, the supernatant was filtered with a 2 μm microporous filter membrane, and the filtrate was filtered with a small ultrafiltration membrane system with a molecular weight of 50,000 Da, and the filtrate was collected;

[0074] According to "SN / T 4592-2016 Determination of total flavonoids in exported food", the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined, and compared with sample 1# in Example 1.

[0075] The results are as follows Figure 3 As shown in the figure, the results show that extraction under nitrogen protection is beneficial to improving the extraction rate of total flavonoids. Flavonoids are a class of natural products with high antioxidant properties. They can be partially oxidized when extracted in an air environment. Inert gas protection and closed extraction are used during the extraction process, which is beneficial to the preservation of flavonoids.

[0076] Example 4

[0077] Effect of polyol content on the extraction rate of total flavonoids from Guaiac wood:

[0078] (1) Accurately weigh 20 g of guaiac powder crushed by an industrial grinder, add 300 g of deionized water, place in a 500 mL stoppered conical flask, and shake well to fully soak the guaiac powder;

[0079] (2) Accurately weigh 0.02 g of β-glucanase, β-glucosidase, xylanase, lignin peroxidase, and laccase respectively and add them into a stoppered conical flask;

[0080] A magnetic stirrer was added to the sample and nitrogen was introduced for 5 min, followed by immediate sealing with a stopper;

[0081] (3) The sample was placed in a 40°C constant temperature magnetic stirring water bath for extraction for 4 h;

[0082] (4) Add 60 g of glycerol, dipropylene glycol, and 1,3-butylene glycol to each sample and continue stirring for 5 min;

[0083] (5) After the extraction, solid particles were removed with a 300-mesh filter bag, the supernatant was filtered with a 2 μm microporous filter membrane, and the filtrate was filtered with a small ultrafiltration membrane system with a molecular weight of 50,000 Da, and the filtrate was collected;

[0084] According to “SN / T 4592-2016 Determination of total flavonoids in exported food”, the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined.

[0085] The results are as follows Figure 4 As shown in the figure, the results show that after adding polyols, the total flavonoid content and extraction rate of the extract are greatly improved; flavonoids are generally slightly soluble in water, and adding appropriate polyols after the extraction process is completed can help improve the solubility of flavonoids in the extract. Figure 4 It can be seen that both dipropylene glycol and 1,3-butylene glycol are helpful for the dissolution of flavonoids in the guaiac wood extract, among which dipropylene glycol is the best.

[0086] Example 5

[0087] Under the conditions of Example 4, in step (4): 30 g of dipropylene glycol and 30 g of 1,3-butanediol were added to each sample, and stirring was continued for 5 min; other conditions were the same as in Example 4.

[0088] According to “SN / T 4592-2016 Determination of total flavonoids in exported food”, the total flavonoid content of the filtrate was tested and the total flavonoid extraction rate was determined.

[0089] Test results: total flavonoid content 1216.42ppm, total flavonoid extraction rate 2.19%. The results show that the synergistic addition of dipropylene glycol and 1,3-butylene glycol can further improve the solubility of flavonoids in the extract.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for extracting flavonoids from plants by directed enzyme-assisted low-temperature extraction, characterized in that: include, The plant material is mixed with the combined enzyme, water is added as an extraction solvent, and enzymolysis is performed at a temperature not exceeding 40°C for 4 to 8 hours under the protection of an inert gas; After the extraction is completed, add polyols or fats approved for food and cosmetics to the extract to dissolve and stir thoroughly; The solid particles were removed by using a 300-mesh filter bag, the supernatant was filtered by using a 2-μm microporous filter membrane, the filtrate was filtered by using an ultrafiltration membrane, and the filtrate was collected to obtain plant flavonoids.

2. The method according to claim 1, characterized in that: The combination enzyme includes a combination of two or more of β-glucanase, α-glucosidase, β-glucosidase, xylanase, glucomannanase, lignin peroxidase, laccase, manganese peroxidase and pectinase.

3. The method according to claim 1 or 2, characterized in that: The plant materials include wood, weeping birch, lampstand, multi-spiked essenbergia, rosewood, European basswood, European larch, sandalwood, sugar pine, Siberian larch, oak, olive, guaiac, soapwood, rosewood, cypress leaves, North American sage, and astragalus.

4. The method according to claim 3, characterized in that: The polyols include glycerol, 1,3-propylene glycol, dipropylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,2-pentanediol, 1,2-hexanediol, caprylyl glycol, and sorbitol.

5. The method according to claim 1 or 4, characterized in that: The oils include caprylic capric triglyceride, octyldodecanol, isononyl isononanoate, coconut oil alcohol-caprylate caprate, glyceryl tri(ethylhexanoate) ester) C12-15 alcohol benzoate, isopropyl myristate, squalane, olive oil, meadowfoam seed oil, camellia seed oil, jojoba oil, and marula oil.

6. The method according to claim 5, characterized in that: The inert gas includes nitrogen, carbon dioxide, helium and argon.

7. The method according to claim 1 or 6, characterized in that: The molecular weight range of the ultrafiltration membrane is 10000Da-50000Da.

8. The method according to claim 7, characterized in that: The ratio of the polyol or oil to the plant material used in the food and cosmetics is 60-80g:20g.

9. Flavonoids obtained by the method according to any one of claims 1 to 8.

10. Use of the flavonoids according to claim 9 in the preparation of cosmetics.

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