Method for extracting polygonum multiflorum glycoside by low-temperature solvent enzyme method and application of polygonum multiflorum glycoside

Abstract

The invention discloses a method for extracting polygonum multiflorum glycoside through a low-temperature solvent enzyme method and application of the method, and belongs to the technical field of bioactive substance extraction.The method is characterized by comprising the following steps that polygonum multiflorum powder is treated through an ultrasonic continuous flow cell crusher to obtain polygonum multiflorum wall-broken powder, chelate resin, glycosidase H and EDTA are added, stirring is conducted, and the polygonum multiflorum wall-broken powder is obtained; centrifuging and taking supernatant liquid to obtain reaction liquid; adding a diethyl ether solvent for extraction, concentrating until diethyl ether liquid is volatilized, and drying to obtain a polygonum multiflorum glycoside crude product; the method has the beneficial effects that the polygonum multiflorum powder is treated through the ultrasonic cell crusher, the content of the polygonum multiflorum glycoside in a system is greatly increased, water-insoluble contents are removed through water extraction, and the polygonum multiflorum glycoside is transferred to a diethyl ether phase to be separated from water-soluble substances by means of diethyl ether extraction, so that the purity of the polygonum multiflorum glycoside is improved. And certain polygonum multiflorum glycoside organisms can be adsorbed by virtue of the chelating resin, so that introduction of impurities is avoided.

Description

Technical field:

[0001] The invention belongs to the technical field of bioactive substance extraction, and more specifically relates to a method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzyme method and application thereof. Background technology:

[0002] Polygonum multiflorum is the dried root of Polygonum Multiflorum Thunb, a plant of the Polygonaceae family. It has the effects of detoxification, carbuncle elimination, anti-malaria, laxative and the like. Modern pharmacological studies have shown that Polygonum multiflorum has anti-tumor, lipid-lowering, anti-atherosclerotic, anti-aging, immunity-enhancing, anti-inflammatory and other activities. Therefore, Polygonum multiflorum has been widely used in the fields of medicine, health products, cosmetics, etc. in recent years. Chemical composition studies have shown that Polygonum multiflorum root tubers mainly contain active ingredients such as stilbene glycosides, polysaccharides, anthraquinones, flavonoids, and phospholipids. The main active ingredient of Polygonum multiflorum extract is Polygonum multiflorum glycosides, which are unique water-soluble bioactive ingredients in Polygonum multiflorum glycosides and are also an effective cosmetic skin care ingredient. Therefore, Polygonum multiflorum extract is widely used as a cosmetic additive for external use. Studies on the effects and mechanisms of action in hair growth and prevention of hair loss have shown that Polygonum multiflorum glycoside, the active ingredient in Polygonum multiflorum extract, can regulate the expression of genes such as 5α-reductase, alkaline phosphatase (ALPL), androgen receptor, DKK-1, etc. that are related to hair growth or prevention of hair loss, and has significant effects in promoting hair growth and preventing hair loss.

[0003] At present, the extraction method of Polygonum multiflorum glycosides mostly uses water as the reaction solvent for extraction, and combines the corresponding ultrasonic treatment technology and microwave treatment technology. The research on extraction technology is relatively mature. Diphenylethylene glycosides are unique water-soluble bioactive components in Polygonum multiflorum glycosides. Water can be used as a preferred solvent for the extraction of diphenylethylene glycosides. However, Polygonum multiflorum is a woody plant. A part of Polygonum multiflorum glycosides is in a free state between cells. This part can be easily extracted by water extraction. Another part is in a bound or free state in the cells of Polygonum multiflorum or on the cell membrane protein. This part of Polygonum multiflorum glycosides cannot be directly extracted by water extraction.

[0004] Some recent studies have also shown that Polygonum multiflorum powder can be processed by an ultrasonic cell disruptor, which can destroy the cell wall and release the cell contents by breaking the cells. Polygonum multiflorum glycosides can then be extracted by water extraction. However, as the cells are broken, other water-soluble active substances will also enter the aqueous solution, bringing unsolvable problems to subsequent refinement and resulting in extremely poor product purity.

[0005] It is crucial to obtain a method for extracting Polygonum multiflorum glycosides with high purity, high yield and simple operation process. Summary of the invention:

[0006] In order to solve the above problems and overcome the shortcomings of the prior art, the present invention provides a method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method and its application, which can effectively solve the problem that Polygonum multiflorum glycosides cannot be directly extracted by water extraction and the extraction rate is low.

[0007] The specific technical solution of the present invention to solve the above technical problem is: a method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method, characterized by comprising the following steps:

[0008] (1) treating Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:(7-9), adding phospholipase and chelating resin, mixing and stirring, while controlling the temperature of the container to 40-50° C., reacting for 1-2 hours, and obtaining a treated solution; removing the chelating resin, centrifuging and obtaining a supernatant to obtain a treated solution;

[0009] (2) adding a chelating resin to the treatment solution, adding endoglycosidase H, the pH of the treatment solution is 7.5-8.3, controlling the temperature of the container to 40-50° C., reacting for 0.5 h, adding 1% EDTA to react for 0.5-2 h, removing the chelating resin, and filtering the supernatant to obtain a reaction solution;

[0010] (3) adding ether solvent to the reaction solution, extracting at room temperature for 48 hours, and obtaining a supernatant;

[0011] (4) The supernatant is concentrated and dried to obtain a crude product of Polygonum multiflorum glycosides.

[0012] Furthermore, 1-5% phospholipase, 10-15% chelating resin, 1%-5% endoglycosidase H and 1-2% reaction aid are added according to the mass percentage of the treatment liquid, and the pH of the treatment liquid is 7.5-8.3.

[0013] Furthermore, the reaction aid is a metal chelating agent, and the metal chelating agent is EDTA.

[0014] Furthermore, the mass volume ratio of the Polygonum multiflorum powder to the ether is (45-55) g: (0.5-1.5) L.

[0015] Furthermore, the crude Polygonum multiflorum glycoside is subjected to a refining process to obtain refined Polygonum multiflorum glycoside.

[0016] Furthermore, the refining process:

[0017] (1) Add distilled water to the crude product of Polygonum multiflorum glycosides, shake well, perform ultrasonic pretreatment, cool, and adjust the solution pH to 5-8; add cellulase in an amount of 0.5-2.5%, perform enzymolysis at a temperature of 45-65° C. for 4-8 hours, remove, inactivate the enzyme at high temperature, and cool;

[0018] (2) placing the enzymatic hydrolysate into a macroporous resin, eluting with water as an eluent to obtain an eluate, and drying the eluate to obtain refined Polygonum multiflorum glycosides.

[0019] A method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzyme method to prepare Polygonum multiflorum glycosides.

[0020] The invention discloses an application of Polygonum multiflorum glycoside in preparing cosmetics for promoting hair growth and preventing hair loss.

[0021] The beneficial effects of the present invention are:

[0022] The invention creatively processes Polygonum multiflorum powder by an ultrasonic cell disruptor to release cell contents, greatly improving the content of Polygonum multiflorum glycosides in the system, capturing glycoproteins by means of chelating resins, removing water-insoluble contents by water extraction, avoiding the introduction of impurities, and transferring the hydrolyzed Polygonum multiflorum glycosides to an ether phase by means of ether extraction to separate them from water-soluble substances.

[0023] The present invention creatively adds endoglycosidase H into the cell contents of Polygonum multiflorum powder. Endoglycosidase H can release Polygonum multiflorum glycosides bound in glycoprotein captured by chelating resin, greatly improving the content of Polygonum multiflorum glycosides in the system, thereby cooperating with the addition of ether to denature water-soluble proteins and allow them to enter the water phase, effectively separating the product from impurities, and greatly improving the yield and purity of Polygonum multiflorum glycosides.

[0024] The present invention creatively adds EDTA in steps, thereby ensuring that the chelating resin preferentially captures the glycoprotein, further serving as a metal chelator to prevent metal ions from degrading Polygonum multiflorum glycosides, and also having a certain buffering effect on endoglycosidase H, thereby avoiding the generation of excessive hydrolysis. Description of the drawings:

[0025] Attached Figure 1 It is a mass spectrum (MS) of stilbene glycosides in the refined Polygonum multiflorum glycosides prepared by the present invention;

[0026] Attached Figure 2 This is the infrared absorption spectrum of diphenylethylene glycosides in the refined Polygonum multiflorum glycosides prepared by the present invention: Specific implementation method:

[0027] In the description of the present invention, specific details are only for a full understanding of the embodiments of the present invention, but those skilled in the art should know that the implementation of the present invention is not limited to these details. In addition, well-known structures and functions are not described or shown in detail to avoid blurring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0028] Specific implementation of the present invention:

[0029] In order to better understand the present invention, a specific embodiment is used for illustration. It is worth emphasizing that the effect of this embodiment is not substantially different from the various embodiments within the protection scope of the present invention, including the respective reagents and the content ratio of the reagents, and all of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here.

[0030] Embodiment 1:

[0031] (1) treating Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:7, adding 2% phospholipase and 10% chelating resin according to the mass of the solution, mixing and stirring, while controlling the temperature of the container to 40° C., reacting for 1 hour to obtain a treated solution, removing the chelating resin, and centrifuging to obtain a supernatant to obtain a treated solution;

[0032] Among them, the centrifuge preferably has a speed of 1000×g (Kraf); it can precipitate the macromolecular contents well.

[0033] (2) adding a chelating resin to the treatment solution, adding 2% endoglycosidase H according to the mass percentage of the treatment solution, the pH of the treatment solution is 7.5, controlling the temperature of the container to 40° C., reacting for 0.5 h, adding 1% EDTA to react for 0.5 h, removing the chelating resin, and filtering the supernatant to obtain a reaction solution;

[0034] (3) according to the mass volume ratio of Polygonum multiflorum powder to ether being 45 g:0.5 L, ether solvent was added to the reaction solution, and after continuous extraction at room temperature for 48 hours, a supernatant was obtained;

[0035] (4) concentrating the supernatant until the ether liquid is evaporated and drying to obtain a crude product of Polygonum multiflorum glycosides;

[0036] Embodiment 2:

[0037] (1) treating Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:8, adding 3% phospholipase and 12.5% ​​chelating resin according to the mass of the solution, mixing and stirring, while controlling the temperature of the container to 45° C., reacting for 1.5 hours to obtain a treated solution, removing the chelating resin, and centrifuging to obtain a supernatant to obtain a treated solution;

[0038] Among them, the centrifuge preferably has a speed of 1200×g (Kraf); it can precipitate the macromolecular contents well.

[0039] (2) adding a chelating resin to the treatment solution, adding 3% endoglycosidase H according to the mass percentage of the treatment solution, the pH of the treatment solution is 8.0, controlling the temperature of the container to 45° C., reacting for 0.5 h, then adding 1.5% EDTA, reacting for 1.0 h, removing the chelating resin, and filtering the supernatant to obtain a reaction solution;

[0040] (3) adding ether solvent to the reaction solution according to the mass volume ratio of Polygonum multiflorum powder to the ether being 50 g:1 L, and continuously extracting at room temperature for 48 hours to obtain a supernatant;

[0041] (4) concentrating the supernatant until the ether liquid is evaporated and drying to obtain a crude product of Polygonum multiflorum glycosides;

[0042] Embodiment 3:

[0043] (1) treating Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:9, adding 4% phospholipase and 15% chelating resin according to the mass of the solution, mixing and stirring, while controlling the temperature of the container to 50° C., reacting for 2 hours to obtain a treated solution, removing the chelating resin, and centrifuging to obtain a supernatant to obtain a treated solution;

[0044] Among them, the centrifuge preferably has a speed of 1500×g (Kraf); it can precipitate the macromolecular contents well.

[0045] (2) adding a chelating resin to the treatment solution, adding 4% endoglycosidase H according to the mass percentage of the treatment solution, the pH of the treatment solution is 8.3, controlling the temperature of the container to 50° C., reacting for 0.5 h, then adding 1.5% EDTA, reacting for 2.0 h, removing the chelating resin, and filtering the supernatant to obtain a reaction solution;

[0046] (3) adding ether solvent to the reaction solution according to the mass volume ratio of Polygonum multiflorum powder to the ether being 50 g:1.5 L, and continuously extracting at room temperature for 48 hours to obtain a supernatant;

[0047] (4) concentrating the supernatant until the ether liquid is evaporated and drying to obtain a crude product of Polygonum multiflorum glycosides;

[0048] In order to more intuitively demonstrate the process advantages of the present invention, the method of extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method of the present invention is compared with the method of using equivalent replacement by the same process.

[0049] Comparative Example 1:

[0050] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, the Polygonum multiflorum powder is directly extracted in a low-temperature ether solvent;

[0051] (1) soaking the Polygonum multiflorum powder in an ether solvent for 48 hours to obtain a supernatant;

[0052] (2) concentrating, precipitating, separating and drying the supernatant to obtain a crude product of Polygonum multiflorum glycosides;

[0053] Comparative Example 2:

[0054] The preparation method is the same as that of Example 3, except that in the preparation process of this comparative example, chelating resin and EDTA are added simultaneously; specifically

[0055] (1) treating the Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain a Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:9, adding 4% phospholipase according to the mass of the solution, mixing and stirring, while controlling the temperature of the container to 50° C., reacting for 2 hours, and centrifuging to obtain a supernatant to obtain a treated solution;

[0056] (2) adding 4% endoglycosidase H to the treatment solution according to the mass percentage of the treatment solution, the pH of the treatment solution is 8.3, the temperature of the container is controlled to 50° C., the reaction is carried out for 0.5 h, and then 15% chelating resin and 1.5% EDTA are added, the reaction is carried out for 2.0 h, the chelating resin is removed, and the supernatant is taken by centrifugation to obtain a reaction solution;

[0057] Comparative Example 3:

[0058] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, no regulating endoglycosidase H is added;

[0059] Comparative Example 4:

[0060] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, no EDTA was added for adjustment;

[0061] Comparative Example 5:

[0062] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, EDTA is replaced by polyphosphonic acid;

[0063] Table 1: Effects of different examples and comparative examples on the preparation of Polygonum multiflorum glycosides

[0064] Preprocessing Chelating resin Endoglycosidase H Reaction Auxiliary Agent Yield purity Example 1 + + + EDTA 12.3 85.4 Example 2 + + + EDTA 13.6 85.7 Example 3 + + + EDTA 12.8 85.5 Comparative Example 1 - - - - 4.5 87.2 Comparative Example 2 + + + EDTA 5.8 82.2 Comparative Example 3 + + - EDTA 5.6 83.8 Comparative Example 4 + + + - 15.4 56.3 Comparative Example 5 + + + Polyphosphonic acid 14.9 32.6

[0065] From the data analysis in Table 1, we can see that:

[0066] (1) Comparison between Examples 1-3 and Comparative Example 1:

[0067] The purity of the crude Polygonum multiflorum glycosides prepared in Examples 1-3 and Comparative Example 1 is not much different, but the yield of Comparative Example 1 is only 4.5%, while that of Examples 1-3 is as high as 13%.

[0068] This may be because these Polygonum multiflorum glycosides exist in the cell and on the cell membrane in a non-free form. Since the cell wall is not broken in Comparative Example 1, the cell contents are not released, and the yield of Polygonum multiflorum glycosides extracted by solvent is very low, but the purity difference is not much different from that of Examples 1-3;

[0069] (2) Comparison between Examples 1-3 and Comparative Example 2:

[0070] Although chelating resin and EDTA were added in Comparative Example 2, the yield of the prepared crude Polygonum multiflorum glycosides was extremely low, only 5.8%, due to defects in the addition method, which was not much different from Comparative Example 1, and the purity difference was not much different from Examples 1-3;

[0071] This may be due to the working principle of chelating resin: chelating resin has a certain adsorption capacity, chelating resin is to use Ni 2+ , Cu 2+ 、Zn 2+ 、Co 2+ Transition metal ions such as hydroxyl groups can bind to histidine, tryptophan, or cysteine ​​on the surface of proteins, thereby adsorbing glycoproteins.

[0072] However, due to the simultaneous addition of chelating resin and EDTA, transition metal ions preferentially chelate with EDTA, causing the chelating resin to lose its coordination ability with histidine, tryptophan, or cysteine ​​on the protein surface. The chelating resin is unable to capture and adsorb glycoproteins, and the glycoproteins are separated from the system during centrifugation, resulting in a low yield.

[0073] Therefore, the chelating resin combined with the step-wise addition of EDTA ensures that the chelating resin preferentially captures the glycoprotein, and further acts as a metal chelator to prevent metal ions from degrading Polygonum multiflorum glycosides. Among them, the fact that metal ions can degrade Polygonum multiflorum glycosides belongs to the prior art and will not be demonstrated here.

[0074] (3) Comparison between Examples 1-3 and Comparative Example 3:

[0075] In Comparative Example 3, although the cell contents were released and the glycoprotein was captured by the chelating resin, the Polygonum multiflorum glycosides bound to the glycoprotein captured by the chelating resin could not be released due to the absence of endoglycosidase H. As the chelating resin left the ether system,

[0076] The yield and purity of Polygonum multiflorum glycosides are not much different from those in Comparative Example 2; therefore, endoglycosidase H can release Polygonum multiflorum glycosides in a glycoprotein-bound state captured by the chelating resin, greatly increasing the content of Polygonum multiflorum glycosides in the system, thereby cooperating with the effects of the reaction aid and the chelating resin to greatly increase the yield and purity of Polygonum multiflorum glycosides.

[0077] (4) Comparison between Examples 1-3, Comparative Example 2 and Comparative Example 4:

[0078] The difference between Comparative Example 4 and the present invention is that no reaction aid is added, and the chelating resin can capture the glycoprotein at the binding site of the glycoprotein under the action of the metal, and release the Polygonum multiflorum glycosides bound to the glycoprotein captured by the chelating resin under the action of endoglycosidase H.

[0079] However, it can be seen from Comparative Example 4 and Comparative Example 2 that the yield of Comparative Example 4 is higher but the purity is reduced. This may be because Comparative Example 4 does not have EDTA and cannot chelate metal ions. The metal ions also degrade Polygonum multiflorum glycosides, which leads to a decrease in purity.

[0080] Therefore, EDTA as a metal chelator can chelate metal ions in the reaction solution, especially adding EDTA in steps, which ensures the provision of binding sites for the chelating resin and glycoprotein, and also avoids the degradation of Polygonum multiflorum glycosides by metal ions, which has a more positive effect on the preparation of Polygonum multiflorum glycosides by the present invention.

[0081] (5) Comparison of Examples 1-3 and Comparative Example 5:

[0082] Comparison between Examples 1-3 and Comparative Example 5 shows that both EDTA and polyphosphonic acid, as metal chelating agents, can chelate metal ions in the reaction solution to prevent the degradation of Polygonum multiflorum glycosides by metal ions in the later stage.

[0083] However, when comparing Examples 1-3 with Comparative Example 5, it can be seen that the yield of Comparative Example 5 is not much different, and the purity is only 32.6%, which indicates that EDTA not only acts as a metal chelating agent but also plays a role in chelating metal ions;

[0084] EDTA not only acts as a metal chelator but also has a certain buffering effect on endoglycosidase H, thus avoiding excessive hydrolysis.

[0085] In order to more intuitively demonstrate the process advantages of the present invention, the EDTA prepared by the present invention is used to play a certain buffering role on endoglycosidase H to further study Polygonum multiflorum glycosides.

[0086] Table 2: Effects of different metal chelators and enzymatic hydrolysis time on the preparation of Polygonum multiflorum glycosides

[0087]

[0088] It can be seen from the data in Table 2 that EDTA and polyphosphonic acid are both metal chelators, and can chelate metal ions in the reaction solution. Metal ions can degrade Polygonum multiflorum glycosides, and EDTA not only exists as a metal chelator but also has a certain buffering effect on endoglycosidase H, avoiding the occurrence of excessive hydrolysis.

[0089] Polyphosphonic acid, as a metal chelator, has little difference from EDTA within 1 hour. As time goes by, the degree of hydrolysis increases, resulting in an increase in yield and a significant decrease in purity. This is due to excessive hydrolysis by endoglycosidase H.

[0090] In order to more intuitively demonstrate the process advantages of the present invention, the crude Polygonum multiflorum glycosides prepared by the present invention can also be refined.

[0091] Embodiment 4:

[0092] (1) The crude product of Polygonum multiflorum glycoside obtained in Example 1 was added with distilled water at a material-liquid mass ratio of 1:8, and the mixture was shaken and then ultrasonically pretreated for 0.5 h. The solution was cooled and the pH value of the solution was adjusted to 5. Cellulase was added in an amount of 0.5%, and enzymolysis was performed at a temperature of 45° C. for 4 h. The mixture was taken out and the enzyme was inactivated at 85° C. for 10 min, and the solution was cooled.

[0093] (2) The above enzymatic hydrolysate is placed into a macroporous resin and eluted with water at a flow rate of 4 ml / min; the amount of eluent water is 500 ml, and the eluent is dried to obtain refined Polygonum multiflorum glycosides.

[0094] Embodiment 5:

[0095] (1) The crude product of Polygonum multiflorum glycosides obtained in Example 2 was added to distilled water at a material-liquid mass ratio of 1:8, and after being shaken, ultrasonic pretreatment was performed for 1 hour, and the solution was cooled to adjust the pH value of 6.5; cellulase was added in an amount of 1.5%, and enzymolysis was performed at a temperature of 55° C. for 6 hours, and the product was taken out and the enzyme was inactivated at 95° C. for 12 minutes, and the solution was cooled;

[0096] (2) The above enzymatic hydrolysate is placed into a macroporous resin and eluted with water at a flow rate of 6 ml / min; the amount of eluent water is 800 ml, and the eluent is dried to obtain refined Polygonum multiflorum glycosides.

[0097] Embodiment 6:

[0098] (1) Add the crude Polygonum multiflorum glycosides obtained in Example 2 to distilled water at a material-liquid mass ratio of 1:8, shake well, perform ultrasonic pretreatment, and perform ultrasonic treatment for 1.5 hours. Cool the solution and adjust the solution pH to 8; add cellulase in an amount of 2.5%, perform enzymolysis at 65° C. for 8 hours, remove the cellulase, and inactivate the cellulase at 105° C. for 15 minutes, and cool the solution;

[0099] (2) The above enzymatic hydrolysate is placed into a macroporous resin and eluted with water at a flow rate of 10 ml / min; the amount of eluent water is 1000 ml, and the eluent is dried to obtain refined Polygonum multiflorum glycosides.

[0100] Table 3: Effects of different examples and comparative examples on the preparation of Polygonum multiflorum glycosides

[0101] Preprocessing Chelating resin Endoglycosidase H Reaction Auxiliary Agent refined Yield purity Example 1 + + + EDTA - 12.3 85.4 Example 2 + + + EDTA - 13.6 85.7 Example 3 + + + EDTA - 12.8 85.5 Example 4 + + + EDTA + 6.8 99.3 Example 5 + + + EDTA + 7.2 98.7 Example 6 + + + EDTA + 6.5 99.5

[0102] As shown in Table 3, the purified Polygonum multiflorum glycosides can be obtained by cellulase, enzyme inactivation, macroporous resin adsorption and elution, which can further improve the purity of Polygonum multiflorum glycosides, but the yield is reduced.

[0103] In summary:

[0104] The invention creatively processes Polygonum multiflorum powder by an ultrasonic cell disruptor to release cell contents, greatly improving the content of Polygonum multiflorum glycosides in the system, capturing glycoproteins by means of chelating resins, removing water-insoluble contents by water extraction, avoiding the introduction of impurities, and transferring the hydrolyzed Polygonum multiflorum glycosides to an ether phase by means of ether extraction to separate them from water-soluble substances.

[0105] The present invention creatively adds endoglycosidase H into the cell contents of Polygonum multiflorum powder. Endoglycosidase H can release Polygonum multiflorum glycosides bound in glycoprotein captured by chelating resin, greatly improving the content of Polygonum multiflorum glycosides in the system, thereby cooperating with the addition of ether to denature water-soluble proteins and allow them to enter the water phase, effectively separating the product from impurities, and greatly improving the yield and purity of Polygonum multiflorum glycosides.

[0106] The present invention creatively adds EDTA in steps, thereby ensuring that the chelating resin preferentially captures the glycoprotein, further serving as a metal chelator to prevent metal ions from degrading Polygonum multiflorum glycosides, and also having a certain buffering effect on endoglycosidase H, thereby avoiding the generation of excessive hydrolysis.

Claims

1. A method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method, characterized in that The steps include: (1) treating Polygonum multiflorum powder with an ultrasonic continuous flow cell disruptor to obtain Polygonum multiflorum wall-broken powder, placing the Polygonum multiflorum wall-broken powder in water at a solid-liquid ratio of 1:(7-9), adding phospholipase and chelating resin, mixing and stirring, while controlling the temperature of the container to 40-50° C., reacting for 1-2 hours, and obtaining a treated solution; removing the chelating resin, centrifuging and obtaining a supernatant to obtain a treated solution; (2) adding a chelating resin to the treatment solution, adding endoglycosidase H, the pH of the treatment solution is 7.5-8.3, controlling the temperature of the container to 40-50° C., reacting for 0.5 h, adding 1% EDTA to react for 0.5-2 h, removing the chelating resin, and centrifuging to obtain a supernatant to obtain a reaction solution; (3) adding ether solvent to the reaction solution, extracting at room temperature to obtain a supernatant; (4) The supernatant is concentrated and dried to obtain a crude product of Polygonum multiflorum glycosides.

2. The method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to claim 1, characterized in that According to the mass percentage of the treated liquid, 1-5% phospholipase, 10-15% chelating resin, 1%-5% endoglycosidase H and 1-2% reaction aid are added.

3. The method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to claim 1, characterized in that The reaction aid is a metal chelating agent, and the metal chelating agent is EDTA.

4. The method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to claim 1, characterized in that The mass volume ratio of Polygonum multiflorum powder to ether is (45-55) g: (0.5-1.5) L.

5. The method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to claim 1, characterized in that The crude product of Polygonum multiflorum glycoside is subjected to a refining process to obtain refined Polygonum multiflorum glycoside.

6. The method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to claim 5, characterized in that The refining process: (1) Add distilled water to the crude product of Polygonum multiflorum glycosides, shake well, perform ultrasonic pretreatment, cool, and adjust the solution pH to 5-8; add cellulase in an amount of 0.5-2.5%, perform enzymolysis at a temperature of 45-65° C. for 4-8 hours, remove, inactivate the enzyme at high temperature, and cool; (2) placing the enzymatic hydrolysate into a macroporous resin, eluting with water as an eluent to obtain an eluate, and drying the eluate to obtain refined Polygonum multiflorum glycosides.

7. Polygonum multiflorum glycosides prepared by the method for extracting Polygonum multiflorum glycosides by low-temperature solvent enzymatic method according to any one of claims 1 to 6.

8. Use of the Polygonum multiflorum glycoside as claimed in claim 7 in the preparation of cosmetics for promoting hair growth and preventing hair loss.

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