Mulberry leaf flavone as well as preparation method and application thereof

Abstract

The invention discloses mulberry leaf flavone as well as a preparation method and application thereof, and belongs to the technical field of natural product extraction and preparation. Flavone is released through a microwave-assisted enzyme method, flavone is dissolved through a deep eutectic solvent, the reaction rate is increased through microwave treatment, meanwhile, the catalytic activity of enzyme can be enhanced, and the enzymatic reaction speed and yield are effectively increased; the deep-eutectic solvent contains a large amount of hydroxyl groups and amino groups, the hydroxyl groups and the amino groups can easily form hydrogen bonds with flavone, the solubility of the flavone in the deep-eutectic solvent can be increased, and meanwhile the total extraction amount of the flavone is increased under the dual auxiliary effects of microwaves and enzyme; by adopting a resin separation method, high-purity mulberry leaf flavone, mulberry leaf alkaloid and mulberry leaf polysaccharide can be obtained. The mulberry leaf flavone and the mulberry leaf alkaloid which are prepared and separated by the method can be conveniently and rapidly proportioned according to the requirements, and the mulberry leaf flavone and the mulberry leaf alkaloid can be widely applied to the aspect of hypoglycemic food.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and preparation of natural products, and particularly relates to a mulberry leaf flavonoid, a preparation method thereof and an application thereof. Background Art

[0002] Mulberry leaf is one of the substances that are both food and medicine announced by the National Health Commission. It was first recorded in "Shennong Ben Cao Jing". It tastes sweet, bitter and cold, and has the effects of dispelling wind and heat, moistening the lungs and relieving dryness, clearing the liver and improving eyesight. Flavonoid compounds are one of the main active ingredients of mulberry leaves. Mulberry leaves contain various flavonoid components such as quercetin, astragalin, rutin, icariside II, and salidroside. Research shows that mulberry leaf flavonoids have a wide range of pharmacological activities such as antioxidant, hypoglycemic, hypolipidemic, anti-tumor, and antibacterial.

[0003] The extraction methods of mulberry leaf flavonoids include: solvent method, ultrasonic-assisted method, enzymatic hydrolysis method, microwave-assisted method, supercritical CO2 extraction method, etc. These single extraction methods all have advantages and disadvantages to varying degrees. For example, in the solvent method, the consumption of organic solvents is large and it is easy to remain; the enzymatic hydrolysis method has high requirements for enzyme reaction conditions and a long extraction time; however, the actual effect of the ultrasonic-assisted extraction method is easily restricted by two factors: the thickness of the container wall and the placement of the container itself, and the noise generated when the ultrasonic extractor works is relatively large, so it is difficult to apply in large-scale industrial production; the supercritical CO2 extraction method has high requirements for equipment and high costs. The patent application with the publication number CN 101869605 A discloses an extraction and separation method of a mulberry leaf flavonoid and alkaloid complex, which is a method integrating acid water extraction - solvent extraction - column chromatography separation. Acidified deionized water is used for extraction; ethyl acetate is used for extraction; the mulberry leaf flavonoid is adsorbed and separated by macroporous resin D101 to obtain the flavonoid in the mulberry leaf composite extract; this method introduces impurities by acid water extraction and may cause environmental pollution.

[0004] Therefore, improving the extraction and purification efficiency of the effective extract of mulberry leaf flavonoids and increasing the purity of mulberry leaf flavonoids have positive significance for the further development and utilization of mulberry leaves and promoting the in-depth development of mulberry leaf resources. Summary of the Invention

[0005] The first object of the present invention is to provide a preparation method of a mulberry leaf flavonoid to improve the purity of the mulberry leaf flavonoid; the second object of the present invention is to provide a mulberry leaf flavonoid with a high flavonoid content; the third object of the present invention is to provide an application of a mulberry leaf flavonoid in hypoglycemic foods.

[0006] The present invention provides a preparation method of a mulberry leaf flavonoid, and the mulberry leaf flavonoid is prepared by the following steps:

[0007] S1. Preparation of mulberry leaf flavonoid extract: Add dry mulberry leaf powder to deionized water, add an enzyme mixture consisting of cellulase, hemicellulase, papain, and pectinase, adjust the system to be weakly acidic, perform microwave treatment and heat with stirring; then add a deep eutectic solvent, continue microwave treatment and heat with stirring, cool after boiling water bath, centrifuge to obtain the supernatant, and filter to obtain the mulberry leaf flavonoid extract;

[0008] S2. Purification of the mulberry leaf flavonoid extract: Dissolve the mulberry leaf flavonoid extract in an ethanol aqueous solution, adsorb it through a cation exchange resin to obtain effluent A; let effluent A flow through macroporous resin a for adsorption to obtain the adsorbed macroporous resin a; elute the adsorbed macroporous resin with an alkaline solution to obtain eluate A; acidify eluate A and then adsorb it through macroporous resin b to obtain the adsorbed macroporous resin b, elute the adsorbed macroporous resin b with an ethanol aqueous solution to obtain eluate B, and concentrate and dry eluate B to obtain mulberry leaf flavonoid.

[0009] Further, the particle size of the dry mulberry leaf powder is 40 - 60 mesh;

[0010] The pH value of the weakly acidic solution is 4 - 5;

[0011] The power of the microwave treatment is 300W - 400W;

[0012] The conditions for heating and stirring are stirring at 45℃ - 55℃ for 15 - 30 min;

[0013] The time of the boiling water bath is 10 - 20 min; the pore size of the filter membrane for filtration is 0.25 - 0.45 μm.

[0014] Further, the deep eutectic solvent is prepared by the following steps:

[0015] Choline chloride, glucose, and absolute ethanol are heated and stirred at a molar ratio of 1:1:1 under a water bath condition of 55 - 65℃ until the solution becomes clear, and then cooled to room temperature to obtain the deep eutectic solvent.

[0016] Further, the mass ratio of the cellulase, hemicellulase, papain, and pectinase is (1 - 2):(1 - 2):(0.5 - 1):(3 - 4), and the addition amount of the mixed enzyme is 3% - 8% of the mass of the dry mulberry leaves; the dosage ratio of the dry mulberry leaf powder, deionized water, and deep eutectic solvent is 1 g:(10 - 15) mL:(30 - 45) mL.

[0017] Further, the purification of the mulberry leaf flavonoid extract includes the following steps:

[0018] The mulberry leaf flavonoids extract is dissolved in an ethanol aqueous solution, and is adsorbed on a cation exchange resin to obtain an adsorbed cation exchange resin and an effluent A; the effluent A is passed through a macroporous resin a to obtain an adsorbed macroporous resin a and an effluent B; the adsorbed macroporous resin a is eluted with water and a sodium carbonate aqueous solution in sequence, and the eluate of the sodium carbonate aqueous solution is collected to obtain an eluate A; the eluate A is adjusted to be acidic, and is passed through a macroporous resin b for adsorption, and the macroporous resin b is eluted with water and an ethanol aqueous solution in sequence, and the eluate of the ethanol aqueous solution is collected to obtain an eluate B; the eluate B is concentrated and dried to obtain mulberry leaf flavonoids.

[0019] Furthermore, the mass ratio of the macroporous resin a, macroporous resin b and dry mulberry leaf powder is (1-2):(1-2):(0.8-1.5), the sample loading flow rate is 10-15 mL / min; the concentration of the ethanol aqueous solution is 50%-75%; and the pH value of the acidic sodium carbonate water eluent is adjusted to 4-5.

[0020] Furthermore, the adsorbed cation exchange resin is sequentially eluted with 50% to 75% ethanol aqueous solution, water and 0.5 to 0.8 mol / L ammonia water, and the ammonia eluate is collected; the ammonia eluate is then passed through an anion exchange resin, and the effluent is collected, concentrated and dried to obtain mulberry leaf alkaloids, with a mulberry leaf alkaloid content of 60% to 70%; the mass ratio of the cation exchange resin, the anion exchange resin and the dry mulberry leaf powder is (3 to 4): (3 to 4): (4 to 6), and the sample flow rate is 3 to 6 mL / min.

[0021] Furthermore, the effluent B enters a container filled with macroporous anion exchange resin to obtain effluent C, which is evaporated under reduced pressure and spray-dried to obtain mulberry leaf polysaccharide with a mulberry leaf polysaccharide content of 50% to 60%; the mass ratio of the macroporous anion exchange resin to dry mulberry leaf powder is (2 to 3): (3 to 4), and the sample loading flow rate is 20 to 30 mL / min.

[0022] Another embodiment of the present invention provides a mulberry leaf flavonoid prepared by the above-mentioned preparation method of mulberry leaf flavonoid, wherein the flavonoid content in the mulberry leaf flavonoid is 70% to 80%.

[0023] Another embodiment of the present invention provides the use of the mulberry leaf flavonoids in the preparation of blood sugar-lowering foods.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention provides a method for preparing mulberry leaf flavonoids. The method of the present invention first uses microwave-assisted enzymatic method to release flavonoids, and then uses deep eutectic solvents to dissolve flavonoids, avoiding the influence of organic solvents on enzyme activity and maximizing the extraction amount of flavonoids. The mixed enzymes can not only effectively destroy the cell wall of mulberry leaves through enzymatic hydrolysis, increase the release of flavonoids, but also break the binding between proteins and flavonoids, thereby increasing the extraction amount of flavonoids; in addition, microwave treatment can enhance the catalytic activity of enzymes while increasing the reaction rate, effectively improving the enzymatic reaction rate and yield; deep eutectic solvents contain a large number of hydroxyl and amino groups, which are easy to form hydrogen bonds with flavonoids, increasing the solubility of flavonoids in deep eutectic solvents. At the same time, under the dual assistance of microwave and enzyme, the dissolution amount of flavonoids increases, thereby increasing the total extraction amount of flavonoids.

[0026] (2) The resin separation method adopted by the present invention is that after macroporous resin adsorbs flavonoids, weak base solution is first used to change the charge of flavonoids, resulting in repulsion with the resin and elution. Impurities such as terpenoids, lignans and pigments are still adsorbed by the macroporous resin and removed. The weak base eluate is acidified and then adsorbed by the macroporous resin again, and high-purity mulberry leaf flavonoids can be obtained by ethanol-water elution; chemical adsorption of mulberry leaf alkaloids by cation exchange resin and removal of acidic impurities by anion exchange resin can obtain high-purity mulberry leaf alkaloids; in addition, cationic impurities in the effluent are removed by macroporous anion exchange resin, and high-purity mulberry leaf polysaccharides can be obtained after drying. The method for preparing mulberry leaf flavonoids provided by the present invention can prepare separated mulberry leaf flavonoids and mulberry leaf alkaloids, and can conveniently mix the mulberry leaf flavonoids and mulberry leaf alkaloids in the required ratio, and is more widely applied to hypoglycemic foods. Specific embodiments

[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0028] Example 1

[0029] This example provides a mulberry leaf flavonoid and its preparation method and application, which are prepared through the following steps:

[0030] S1. The mulberry leaves were dried at 60°C, crushed, and sieved through a 60-mesh sieve to obtain dry mulberry leaf powder. 100g of dry mulberry leaf powder was added to 1L of deionized water, and 5g of mixed enzymes (cellulase, hemicellulase, papain, and pectinase were mixed in a mass ratio of 2:1:0.5:4) were added. The pH was adjusted to 5, and the mixture was microwaved at 400W at 50°C and stirred for 15min. 3L of a low eutectic solvent (choline chloride, glucose, and anhydrous ethanol were heated and stirred at a molar ratio of 1:1:1 in a 60°C water bath until the solution was clear and cooled to room temperature) was added. The mixture was microwaved at 400W at 50°C for 15min, then cooled in a boiling water bath for 15min. The supernatant was centrifuged and filtered through a 0.45μm filter membrane to obtain a mulberry leaf flavonoid extract.

[0031] S2, the mulberry leaf flavonoids extract was dissolved in 5L 50% ethanol aqueous solution and passed through 75g cation exchange resin at a sample flow rate of 4mL / min to obtain the adsorbed cation exchange resin and effluent A; the adsorbed cation exchange resin was washed with 100mL 50% ethanol aqueous solution, 200mL water and 300mL The elution was carried out with 0.5 mol / L ammonia water, and the ammonia water eluate was collected; the ammonia water eluate was passed through 75 g of anion exchange resin at a sample flow rate of 4 mL / min, the effluent was collected, concentrated and dried to obtain mulberry leaf alkaloids; the effluent A was used to recover ethanol and then passed through 125 g of macroporous resin a at a sample flow rate of 10 mL / min to obtain the adsorbed macroporous resin and effluent B; the effluent B entered a column filled with 135 g of macroporous anion exchange resin at a sample flow rate of 20 mL / min to obtain effluent C, which was evaporated under reduced pressure and spray-dried to obtain mulberry leaf polysaccharides; the adsorbed macroporous resin was washed with 400 mL of water, 200 mL of The solution was eluted with a sodium carbonate aqueous solution having a pH of 9, and the eluate of the sodium carbonate aqueous solution was collected to obtain an eluate A; the pH of the eluate A was adjusted to 4, and the solution was passed through 125 g of a macroporous resin b at a sample flow rate of 10 mL / min; the solution was eluted with 200 mL of water and 400 mL of a 70% ethanol aqueous solution in sequence, and the eluate of the 70% ethanol aqueous solution was collected to obtain an eluate B; the eluate B was concentrated and dried to obtain mulberry leaf flavonoids.

[0032] The mulberry leaf flavonoids are used in foods for lowering blood sugar.

[0033] Example 2

[0034] Compared with Example 1, the difference between this example and Example 1 is that "100 g of dry mulberry leaf powder is added to 1 L of deionized water" in S1 is changed to "100 g of dry mulberry leaf powder is added to 1.5 L of deionized water".

[0035] The remaining raw materials and preparation process remain the same as in Example 1.

[0036] Example 3

[0037] Compared with Example 1, the difference in this example is that in S2, “75 g of cation exchange resin, 75 g of anion exchange resin” and “125 g of macroporous resin a, 125 g of macroporous resin b” are changed to “125 g of cation exchange resin, 125 g of anion exchange resin” and “150 g of macroporous resin a, 150 g of macroporous resin b”.

[0038] The remaining raw materials and the preparation process are the same as those in Example 1.

[0039] Example 4

[0040] Compared with Example 1, the difference in this example is that in S1, “5 g of mixed enzymes” is changed to “8 g of mixed enzymes”.

[0041] The remaining raw materials and the preparation process are the same as those in Example 1.

[0042] Example 5

[0043] Compared with Example 1, the difference in this example is that in S1, “5 g of mixed enzymes” is changed to “3 g of mixed enzymes”.

[0044] The remaining raw materials and the preparation process are the same as those in Example 1.

[0045] Example 6

[0046] Compared with Example 1, the difference in this example is that in S1, “cellulase, hemicellulase, papain and pectinase are mixed in a mass ratio of 2:1:0.5:4” is changed to “cellulase, hemicellulase, papain and pectinase are mixed in a mass ratio of 1:1:0.5:3”.

[0047] The remaining raw materials and the preparation process are the same as those in Example 1.

[0048] Example 7

[0049] Compared with Example 1, the difference in this example is that in S1, “continuous microwave treatment at 400 W for 15 min” is changed to “continuous microwave treatment at 400 W for 30 min”.

[0050] The remaining raw materials and the preparation process are the same as those in Example 1.

[0051] Example 8

[0052] Compared with Example 1, the difference in this example is that in S2, “loading flow rate is 4 mL / min” and “loading flow rate is 10 mL / min” are changed to “loading flow rate is 6 mL / min” and “loading flow rate is 15 mL / min” in S2.

[0053] The remaining raw materials and the preparation process are the same as those in Example 1.

[0054] Comparative Example 1

[0055] Compared with Example 1, the difference in this comparative example is that microwave treatment is not used in S1. The specific implementation steps are as follows:

[0056] S1. Dry mulberry leaves at 60°C, crush them, and pass through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, add 5 g of a mixed enzyme (cellulase, hemicellulase, papain, and pectinase are mixed in a mass ratio of 2:1:0.5:4), adjust the pH to 5, and heat and stir at 50°C for 0.5 h; then add 3 L of a deep eutectic solvent (prepared by heating and stirring choline chloride, glucose, and absolute ethanol in a molar ratio of 1:1:1 in a 60°C water bath until the solution is clear, and cooling to room temperature), heat and stir at 50°C for 0.5 h, cool after a 15-min boiling water bath, centrifuge to obtain the supernatant, and filter through a 0.45-μm filter membrane to obtain a mulberry leaf flavonoid extract;

[0057] The remaining raw materials and the preparation process are the same as those in Example 1.

[0058] Comparative Example 2

[0059] Compared with Example 1, the difference in this comparative example is that the mixed enzyme is not used for decomposition in S1. The specific implementation steps are as follows:

[0060] S1. Dry mulberry leaves at 60°C, crush them, and pass through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, perform microwave treatment at 400 W at 50°C, and stir for 0.5 h; then add 3 L of a deep eutectic solvent (prepared by heating and stirring choline chloride, glucose, and absolute ethanol in a molar ratio of 1:1:1 in a 60°C water bath until the solution is clear, and cooling to room temperature), continue microwave treatment at 400 W for 0.5 h, centrifuge to obtain the supernatant, and filter through a 0.45-μm filter membrane to obtain a mulberry leaf flavonoid extract;

[0061] The remaining raw materials and the preparation process are the same as those in Example 1.

[0062] Comparative Example 3

[0063] Compared with Example 1, the difference in this comparative example is that the deep eutectic solvent in S1 is replaced with an equal volume of 70% ethanol aqueous solution. The specific implementation steps are as follows:

[0064] S1. Dry the mulberry leaves at 60 °C, crush them, and sieve them through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, add 5 g of a mixed enzyme (cellulase, hemicellulase, papain, and pectinase mixed in a mass ratio of 2:1:0.5:4), adjust the pH to 5, perform microwave treatment at 400 W at 50 °C, and stir for 0.5 h; then add 3 L of 70% ethanol aqueous solution, continuously perform microwave treatment at 400 W for 0.5 h, cool after boiling water bath for 15 min, centrifuge to obtain the supernatant, and filter through a 0.45-μm filter membrane to obtain the mulberry leaf flavonoid extract;

[0065] The remaining raw materials and the preparation process are the same as those in Example 1.

[0066] Comparative Example 4

[0067] Compared with Example 1, the difference in this comparative example is that in S1, neither microwave nor the mixed enzyme is used. The specific implementation steps are as follows:

[0068] S1. Dry the mulberry leaves at 60 °C, crush them, and sieve them through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, heat and stir at 50 °C for 0.5 h; then add 3 L of a deep eutectic solvent (prepared by heating and stirring choline chloride, glucose, and absolute ethanol in a molar ratio of 1:1:1 in a 60 °C water bath until the solution is clear and cooling to room temperature), heat and stir for 0.5 h, centrifuge to obtain the supernatant, and filter through a 0.45-μm filter membrane to obtain the mulberry leaf flavonoid extract;

[0069] The remaining raw materials and the preparation process are the same as those in Example 1.

[0070] Comparative Example 5

[0071] Compared with Example 1, the difference in this comparative example is that in S1, neither microwave nor the deep eutectic solvent is used and the deep eutectic solvent is replaced with deionized water of the same volume. The specific implementation steps are as follows:

[0072] S1. Dry the mulberry leaves at 60 °C, crush them, and sieve them through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, add 5 g of a mixed enzyme (cellulase, hemicellulase, papain, and pectinase mixed in a mass ratio of 2:1:0.5:4), adjust the pH to 5, stir at 50 °C for 15 min; then add 3 L of deionized water, continuously heat and stir for 15 min, cool after boiling water bath for 15 min, centrifuge to obtain the supernatant, and filter through a 0.45-μm filter membrane to obtain the mulberry leaf flavonoid extract;

[0073] The remaining raw materials and the preparation process are the same as those in Example 1.

[0074] Comparative Example 6

[0075] In this comparative example, compared with Example 1, the difference is that in S1, "adding 100 g of dry mulberry leaf powder to 1 L of deionized water" is changed to "adding 100 g of dry mulberry leaf powder to 1 L of 70% ethanol aqueous solution";

[0076] The remaining raw materials and the preparation process are the same as those in Example 1.

[0077] Comparative Example 7

[0078] In this comparative example, compared with Example 1, the difference is that in S1, "adding 5 g of mixed enzyme (cellulase, hemicellulase, papain and pectin are mixed according to the mass ratio of 2:1:0.5:4)" is changed to "adding 5 g of mixed enzyme (cellulase and pectin are mixed according to the mass ratio of 2:4)".

[0079] The remaining raw materials and the preparation process are the same as those in Example 1.

[0080] Comparative Example 8

[0081] In this comparative example, compared with Example 1, the difference is that the step of "microwave treatment at 400 W for 15 min and stirring at 50°C" in S1 is removed, and the microwave treatment time is extended to 30 min. The specific implementation steps are as follows:

[0082] S1. Dry the mulberry leaves at 60°C, crush them, and pass through a 60-mesh sieve to obtain dry mulberry leaf powder. Add 100 g of dry mulberry leaf powder to 1 L of deionized water, add 5 g of mixed enzyme (cellulase, hemicellulase, papain and pectin are mixed according to the mass ratio of 2:1:0.5:4), adjust the pH to 5, then add 3 L of deep eutectic solvent (prepared by heating and stirring choline chloride, glucose and absolute ethanol in a molar ratio of 1:1:1 in a water bath at 60°C until the solution is clear and cooling to room temperature), perform microwave treatment at 400 W for 30 min and stir; after boiling water bath for 15 min, cool, centrifuge to take the supernatant, and filter through a 0.45-μm filter membrane to obtain mulberry leaf flavonoid extract;

[0083] The remaining raw materials and the preparation process are the same as those in Example 1.

[0084] Comparative Example 9

[0085] In this comparative example, compared with Example 1, the difference is that the macroporous resin in S2 is not eluted with an alkaline solution. The specific implementation steps are as follows:

[0086] S2. Dissolve the mulberry leaf flavonoid extract in 5 L of 50% ethanol aqueous solution, pass it through 75 g of cation exchange resin with a loading flow rate of 4 mL / min to obtain the adsorbed cation exchange resin and effluent A; the adsorbed cation exchange resin is eluted successively with 100 mL of 50% ethanol aqueous solution, 200 mL of water, and 300 mL of 0.5 mol / L ammonia water, and the ammonia water eluate is collected; the ammonia water eluate then flows through 75 g of anion exchange resin with a loading flow rate of 4 mL / min, and the effluent is collected, concentrated and dried to obtain mulberry leaf alkaloids; after recovering ethanol from effluent A, it flows through 125 g of macroporous resin a with a loading flow rate of 10 mL / min; the adsorbed macroporous resin and effluent B are obtained; effluent B enters a column filled with 135 g of macroporous anion exchange resin with a loading flow rate of 20 mL / min to obtain effluent C, and effluent C is evaporated under reduced pressure and spray-dried to obtain mulberry leaf polysaccharides; the adsorbed macroporous resin is eluted successively with 200 mL of water and 400 mL of 70% ethanol aqueous solution with a loading flow rate of 10 mL / min; the eluate of 70% ethanol aqueous solution is collected, concentrated and dried to obtain mulberry leaf flavonoids.

[0087] The remaining raw materials and preparation process are the same as those in Example 1.

[0088] Weigh the mulberry leaf flavonoids, mulberry leaf alkaloids and mulberry leaf polysaccharides prepared in Examples 1 - 8 and Comparative Examples 1 - 9; according to the spectrophotometry method (instrument model used: UV-1100D / V-1100D ultraviolet / visible spectrophotometer), detect the contents of the mulberry leaf flavonoids, mulberry leaf alkaloids and mulberry leaf polysaccharides prepared in Examples 1 - 8 and Comparative Examples 1 - 9.

[0089] The results are shown in Table 1:

[0090] Table 1

[0091]

[0092] As can be seen from Table 1, 2.36 g of mulberry leaf flavonoids are prepared according to the method provided in Example 1, with a flavonoid content of 78.8%, 1.66 g of mulberry leaf alkaloids are obtained, with an alkaloid content of 65.4%, and 6.12 g of mulberry leaf polysaccharides are obtained, with a polysaccharide content of 57.7%; compared with Example 1, Examples 2 - 8 only differ in the adjustment of preparation conditions and raw materials within a reasonable range, and the difference is not significant compared with Example 1.

[0093] Compared with Example 1, in Comparative Example 1, without microwave treatment, the dissolution efficiency of flavonoids becomes slower, resulting in a decrease in the quality of the finally prepared product; when comparing Comparative Example 2, Comparative Example 7, Examples 4 to 6 with Example 1, in the mixed enzyme, cellulase, hemicellulase and pectinase can not only effectively destroy the mulberry leaf cell wall through enzymatic hydrolysis, but papain can also break the binding between proteins and flavonoid compounds, thereby increasing the extraction amount of flavonoids. Therefore, the extraction amount of flavonoids decreases after the enzyme is not added; when comparing Comparative Example 4, Comparative Examples 1 to 2 with Example 1, from the results, the simultaneous microwave treatment and enzymatic decomposition have a better effect than the single-condition treatment, with a high extraction amount and content of flavonoids, indicating that microwave not only speeds up the reaction rate but also promotes the extraction of flavonoids by enzymatic decomposition; when comparing Comparative Example 1, Comparative Example 3, Comparative Example 5 with Example 1, the eutectic solvent used contains a large number of hydroxyl and amino groups, which are easy to form hydrogen bonds with flavonoids, thereby increasing the solubility of flavonoids in the eutectic solvent and improving the extraction amount, and microwave treatment can improve the deficiency of the high viscosity and low reaction efficiency of the eutectic solvent; when comparing Comparative Example 6 with Example 1, although ethanol has a stronger solubility for flavonoids than water, ethanol will affect the activity of the mixed enzyme and instead reduce the extraction amount of flavonoids. The method of the present invention uses microwave-assisted enzymatic release of flavonoids first and then uses a eutectic solvent to dissolve flavonoids to maximize the extraction amount of flavonoids; when comparing Comparative Example 8 with Example 1, not only does ethanol affect the enzyme activity, but the eutectic solvent with a large viscosity also affects the amount of flavonoids released by enzymatic hydrolysis and reduces the reaction rate; when comparing Comparative Example 9 with Example 1, after the macroporous resin adsorbs flavonoids, a weak base solution is first used to elute the flavonoids, and after acidification, the flavonoids are adsorbed again by the macroporous resin, which can remove impurities such as terpenoids, lignans and pigments, thereby obtaining high-purity mulberry leaf flavonoids.

[0094] To explore the application of mulberry leaf flavonoids in blood glucose regulation, a diabetic mouse model was established. After one week of adaptive feeding, the mice were fasted for 24 h and then injected with alloxan saline solution (50 mg / kg) via the tail vein. After 72 h, the injection was repeated once with the same dose. One week later, after fasting for 4 - 5 h, blood was taken from the tail vein to measure the blood glucose level. Mice with a blood glucose concentration ≥ 11.1 mmol / L were considered as diabetic mouse models. Until 35 diabetic mice were obtained and evenly divided into 7 groups;

[0095] The mice were grouped and fed. Group A was fed 0.2 mL / kg / d of normal saline per meal; Group B was fed 100 mg / kg / d of mulberry leaf flavonoids; Group C was fed 100 mg / kg / d of mulberry leaf alkaloids; Group D was fed a flavonoid and alkaloid composition (mass ratio 1:2) at 100 mg / kg / d; Group E was fed a flavonoid and alkaloid combination (mass ratio 1:3) at 100 mg / kg / d; Group F was fed a flavonoid and alkaloid combination (mass ratio 1:3) at 200 mg / kg / d; Group G was fed acarbose at 10 mg / kg / d. One hour after the feeding ended, blood was taken from the tail vertebrae of the mice to measure blood glucose. The results are shown in Table 2:

[0096] Table 2

[0097]

[0098] In summary, the mulberry leaf flavonoids provided by the present invention have good effects in reducing blood glucose. Moreover, the separated mulberry leaf flavonoids and mulberry leaf alkaloids prepared by the preparation method of a mulberry leaf flavonoid provided by the present invention can be proportioned more scientifically and effectively, and have a wider application in blood glucose-lowering foods.

[0099] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0100] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of mulberry leaf flavonoids, characterized in that, The mulberry leaf flavonoids are prepared by the following steps: S1. Prepare the mulberry leaf flavonoid extract: Add dry mulberry leaf powder into deionized water, add an enzyme mixture composed of cellulase, hemicellulase, papain and pectinase, adjust the system to be weakly acidic, perform microwave treatment and heat with stirring; then add a deep eutectic solvent, continuously perform microwave treatment and heat with stirring, cool after boiling water bath, centrifuge to obtain the supernatant, and filter to obtain the mulberry leaf flavonoid extract; S2. Purify the mulberry leaf flavonoid extract: Dissolve the mulberry leaf flavonoid extract in an ethanol aqueous solution, adsorb it through a cation exchange resin to obtain effluent A; let effluent A flow through macroporous resin a for adsorption to obtain the adsorbed macroporous resin a; elute the adsorbed macroporous resin with an alkaline solution to obtain eluate A; acidify eluate A and then adsorb it through macroporous resin b to obtain the adsorbed macroporous resin b, elute the adsorbed macroporous resin b with an ethanol aqueous solution to obtain eluate B, and concentrate and dry eluate B to obtain the mulberry leaf flavonoids.

2. The preparation method of mulberry leaf flavonoids according to claim 1, characterized in that, The particle size of the dry mulberry leaf powder is 40 - 60 mesh; The pH value of the weak acid is 4 - 5; The power of the microwave treatment is 300W - 400W; The conditions for heating and stirring are stirring at 45°C - 55°C for 15 - 30 min; The time of the boiling water bath is 10 - 20 min; the pore size of the filter membrane for filtration is 0.25 - 0.45 μm.

3. A method for preparing mulberry leaf flavonoids according to claim 1, characterized in that The deep eutectic solvent is prepared by the following steps: Choline chloride, glucose and absolute ethanol are heated and stirred at a molar ratio of 1:1:1 under a water bath condition of 55 - 65°C until the solution is clear, and then cooled to room temperature to obtain the deep eutectic solvent.

4. A method for preparing mulberry leaf flavonoids according to claim 1, wherein, The mass ratio of the cellulase, hemicellulase, papain and pectinase is (1 - 2):(1 - 2):(0.5 - 1):(3 - 4), and the addition amount of the mixed enzyme is 3% - 8% of the mass of the dry mulberry leaves; the dosage ratio of the dry mulberry leaf powder, deionized water and deep eutectic solvent is 1 g:(10 - 15) mL:(30 - 45) mL.

5. A method for preparing mulberry leaf flavonoids according to claim 1, characterized in that, The purification of the mulberry leaf flavonoid extract includes the following steps: Dissolve the mulberry leaf flavonoid extract in an ethanol aqueous solution, adsorb it through a cation exchange resin to obtain the adsorbed cation exchange resin and effluent A; let effluent A flow through macroporous resin a to obtain the adsorbed macroporous resin a and effluent B; the adsorbed macroporous resin a is eluted successively with water and a sodium carbonate aqueous solution, and the eluate of the sodium carbonate aqueous solution is collected to obtain eluate A; adjust eluate A to be acidic and let it flow through macroporous resin b for adsorption, and macroporous resin b is eluted successively with water and an ethanol aqueous solution, and the eluate of the ethanol aqueous solution is collected to obtain eluate B, and concentrate and dry eluate B to obtain the mulberry leaf flavonoids.

6. The preparation method of mulberry leaf flavonoids according to claim 5, characterized in that, The mass ratio of the macroporous resin a, macroporous resin b and dry mulberry leaf powder is (1 - 2):(1 - 2):(0.8 - 1.5), and the sample loading flow rate is 10 - 15 mL / min; the concentration of the ethanol aqueous solution is 50% - 75%; the pH value for adjusting the acidity of the sodium carbonate washing solution is 4 - 5.

7. The preparation method of a mulberry leaf flavonoid according to claim 5, characterized in that, The adsorbed cation exchange resin is eluted successively with 50%-75% ethanol aqueous solution, water and 0.5-0.8 mol / L ammonia water, and the ammonia water eluate is collected; the ammonia water eluate then flows through the anion exchange resin, the effluent is collected, concentrated and dried to obtain mulberry leaf alkaloids, and the content of mulberry leaf alkaloids is 60%-70%; the mass ratio of the cation exchange resin, the anion exchange resin and the dry mulberry leaf powder is (3-4):(3-4):(4-6), and the sample loading flow rate is 3-6 mL / min.

8. The preparation method of mulberry leaf flavonoids according to claim 5, characterized in that, The effluent B enters the macroporous anion exchange resin column to obtain the effluent C, the effluent C is evaporated under reduced pressure and spray-dried to obtain mulberry leaf polysaccharides, and the content of mulberry leaf polysaccharides is 50%-60%; the mass ratio of the macroporous anion exchange resin and the dry mulberry leaf powder is (2-3):(3-4), and the sample loading flow rate is 20-30 mL / min.

9. A mulberry leaf flavonoid, characterized in that, Prepared by the preparation method according to any one of claims 1-8, the flavonoid content in the mulberry leaf flavonoids is 70%-80%.

10. Use of the mulberry leaf flavonoids as claimed in claim 9 in the preparation of hypoglycemic foods.

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