Method for extracting total flavonoids from alfalfa

Abstract

The invention provides a method for extracting total flavonoids from alfalfa. The method comprises the following steps: 1) de-enzyming and drying; 2) crushing; 3) preparation of an aqueous two-phase system: taking 28-36% by mass of ethanol and 12-30% by mass of a (NH4) 2SO4 solution to prepare an ethanol-(NH4) 2SO4 aqueous two-phase system; 4) extraction: adding the alfalfa powder into an ethanol-(NH4) 2SO4 aqueous two-phase system, and performing ultrasonic extraction to obtain an alfalfa total flavone crude extract; 5) suction filtration; 6) concentration: performing vacuum centrifugal concentration on the crude extract at 60-70 DEG C to obtain a crude extract solid; 7) washing with water; and (8) secondary concentration: carrying out secondary vacuum centrifugal concentration on the crude extract solid at 35-45 DEG C to obtain a dry alfalfa total flavone solid. According to the method, the production cycle can be remarkably shortened, the yield can be increased, the alcohol phase is easy to recover, the system cost is low, and the efficiency, selectivity and economical efficiency are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of total flavonoid extraction, and specifically relates to a method for extracting total flavonoids from alfalfa. Background Art

[0002] As one of the leguminous forages, alfalfa is rich in secondary metabolites such as flavonoids, saponins, and polysaccharides. Among them, flavonoids have become the core target for the functional development of alfalfa due to their unique chemical structure and bioactive functions. The main structural types of alfalfa total flavonoids are: flavonols and their glycosides, isoflavonoids, isoflavanes, dihydroflavones, dihydroisoflavones, chalcones, and pterocarpans. Flavonoids have bioactive functions such as antioxidant, anti-inflammatory, antibacterial, anticancer, estrogen-like, and immune function enhancement, and have very important development and utilization value in the pharmaceutical field and livestock production. Therefore, based on the extraction and functional verification of alfalfa total flavonoids, the development of functional active products with low cost and high activity has high value in the pharmaceutical and livestock production.

[0003] At present, the traditional extraction methods of flavonoids generally have certain limitations: the solvent consumption of the maceration method is large and the recovery is difficult; the pH dependence of the alkali extraction and acid precipitation method is strong and it is easy to cause component degradation; there is a risk of local overheating in microwave-assisted extraction; enzyme-assisted extraction faces problems such as unstable enzyme activity, poor recyclability, and high cost; while supercritical fluid extraction has high selectivity but requires huge equipment investment and high energy consumption. These technical bottlenecks jointly restrict the popularization and implementation of industrial production.

[0004] Chinese Patent Application CN102697837A discloses a method for extracting and separating total flavonoids from Medicago sativa. Medicago sativa is crushed with a pulverizer, deionized water is added, and the pH is adjusted with an alkali solution, and the extraction is carried out several times to obtain a filtrate; the pH of the filtrate is adjusted with hydrochloric acid, and the precipitate is obtained by filtration, and the precipitate is rinsed with deionized water until the rinsing solution is colorless; the precipitate is dissolved in a 55-65% ethanol solution, the filtrate is collected after filtration, and the filtrate is concentrated under reduced pressure to a thick paste; the thick paste is dissolved in 60% ethanol, deionized water is added, the pH is adjusted with a dilute NaOH solution, and macroporous resin adsorption is carried out; the adsorbed macroporous resin is rinsed with deionized water until colorless, and then rinsed with 55-65% ethanol until colorless, and 89-92% ethanol solution is added for desorption, and the desorbed solution is concentrated and dried to obtain a yellowish-brown extract containing flavonoids. However, this patent uses multi-step acid-base adjustment (NaOH / HCl) for flavonoid extraction. High temperature (90-100°C) and strong acid and alkali treatment are likely to damage the thermosensitive flavonoid aglycone ring structure, and multiple acid-base conversions result in a long process flow (a total of 3 pH regulations are involved); in addition, the ethanol gradient switches frequently, which not only increases the solvent recovery cost, but also easily causes incomplete phase separation and impurity residues.

[0005] Chinese Patent Application CN105748570A discloses a method for ultrasonic-assisted extraction of total flavonoids from alfalfa, which includes the following steps: (1) drying and pulverizing alfalfa grass to obtain alfalfa grass powder; (2) adding an organic solvent (such as petroleum ether) to the alfalfa grass powder obtained in step (1), heating under reflux for 4 to 24 hours, filtering to remove the organic solvent to obtain alfalfa grass powder, and then drying the alfalfa grass powder; (3) mixing an ethanol solution with a volume fraction of 20 to 70% and the alfalfa grass powder obtained in step (2) according to a liquid-to-solid ratio of 20:1 to 70:1 mL:g, and extracting under the temperature of 30 to 80 °C with ultrasonic assistance to obtain an alfalfa total flavonoid extract. However, in this patent, petroleum ether is used for heating under reflux for 4 to 24 hours, which is too long. High temperature (the boiling point of petroleum ether is 40 to 60 °C) may cause degradation of some thermally unstable flavonoids (such as flavonoid glycosides), resulting in a low extraction yield. The risk of petroleum ether residue is high, it is difficult to recover, and the energy consumption for extraction and long-term high-temperature treatment is high. In step 2), the defatted alfalfa grass powder needs to be dried, but in step 3), it is immediately extracted with an ethanol solution (containing water), and drying is of no practical significance and instead increases energy consumption.

[0006] Chinese Patent Application CN108969566A discloses an extraction device and an extraction method for alfalfa total flavonoids, which mainly include the following steps: S1, cleaning; S2, draining; S3, pulping; S4, ultrasonic extraction; S5, filtering: after ultrasonic extraction ends, the material enters a filtering device for filtering; S6, microwave extraction; S7, collection: after microwave extraction ends, the alfalfa total flavonoid extract is obtained and enters a collection device (53) for collection. However, in this patent, after alfalfa is cleaned and drained, it is directly pulped without drying or pulverizing treatment. Fresh alfalfa has a high water content, and direct pulping will cause the extraction system to be overly diluted, reducing the solvent efficiency. In addition, ultrasonic extraction (S4) is carried out first and then microwave extraction (S6). However, ultrasound is usually used for cell disruption, and microwave promotes solvent penetration. After ultrasound, the material may have its cell structure damaged, resulting in a decrease in the microwave action efficiency; in the patent, the particle size difference in the pulping process will lead to inconsistent extraction rates of different particles. It is difficult for the active ingredients inside large particles to be fully released, and small particles may cause cell debris aggregation due to excessive fragmentation, hindering solvent penetration. Particle size unevenness may cause risks such as pipeline blockage or filter screen damage. And the extraction agent is an ethanol-kerosene-water mixed system, and kerosene increases the risks of flammability and explosion and the later separation cost. If high-temperature short-time blanching is adopted, it can effectively inhibit the activity of hydrolytic enzymes, reduce the hydrolysis of flavonoid glycoside components, that is, reduce the degradation of flavonoids, and thus retain flavonoid components.

[0007] In summary, the existing methods for extracting alfalfa total flavonoids generally have problems such as strong process destructiveness, high solvent toxicity, and high energy consumption, and it is difficult to balance extraction efficiency and the protection of component activity. These technical bottlenecks severely restrict the industrial production and application development of alfalfa flavonoids, and there is an urgent need to develop a greener, more efficient, and lower-cost extraction new process. Summary of the Invention

[0008] In view of the deficiencies of the prior art, the present invention provides a method for extracting total flavonoids from alfalfa to solve one or more technical problems in the prior art.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a method for extracting total flavonoids from alfalfa, comprising the following steps:

[0011] 1) Blanching and drying: Put fresh alfalfa into a constant temperature drying oven at 85-105°C for high-temperature blanching for 20-30 minutes, then adjust the temperature to 55-70°C and continue drying until constant weight;

[0012] 2) Crushing: Crush the dried alfalfa and pass it through a grading sieve of 0.2-0.4 mm to obtain alfalfa powder;

[0013] 3) Preparation of aqueous two-phase system: Based on the total mass of the aqueous two-phase system, take 28-36% by mass of ethanol and 12%-30% by mass of (NH4)2SO4 solution to prepare an ethanol-(NH4)2SO4 aqueous two-phase system;

[0014] 4) Extraction: Add the alfalfa powder to the ethanol-(NH4)2SO4 aqueous two-phase system for ultrasonic extraction. The extraction conditions are: ultrasonic power 100-500 W, ultrasonic time 0.1-3 h, ultrasonic temperature 20-90°C, liquid-solid ratio 30:1-100:1 g / g. After ultrasonic extraction, obtain a suspension of total flavonoids from alfalfa;

[0015] 5) Filtration: Use a filtration system to remove the filter residue from the suspension and separate to obtain a crude extract. The pore size of the microporous filter membrane used in the filtration system is 0.3-0.5 μm;

[0016] 6) Concentration: Concentrate the crude extract under vacuum centrifugation at 60-70°C to obtain a solid crude extract;

[0017] 7) Washing: Wash the solid crude extract with ultrapure water to dissolve the (NH4)2SO4 salt in the extract;

[0018] 8) Secondary concentration: Perform secondary vacuum centrifugation concentration on the solid crude extract at 35-45°C to obtain a solid of dried total flavonoids from alfalfa.

[0019] Preferably, single-factor variance analysis is used to optimize the composition of the aqueous two-phase system and the ultrasonic extraction parameters. Taking the yield of total flavonoids as an index, single-factor analysis is carried out on the concentrations of ethanol and (NH4)2SO4 solution, ultrasonic power, ultrasonic time, ultrasonic temperature, and liquid-solid ratio.

[0020] Preferably, in step 3), in the ethanol-(NH4)2SO4 system, the mass fractions of ethanol and (NH4)2SO4 are 30% and 16% respectively.

[0021] Preferably, in step 4), the ultrasonic power is 400 - 500 W, the ultrasonic time is 0.5 - 2.5 h, the ultrasonic temperature is 40 - 80 °C, and the liquid-to-solid ratio is 50:1 - 90:1 g / g.

[0022] Preferably, in step 4), the ultrasonic power is 500 W, the ultrasonic time is 1.5 h, the ultrasonic temperature is 70 °C, and the liquid-to-solid ratio is 70:1 g / g.

[0023] Preferably, the CCD response surface method is adopted to consider the influence of the interaction of factors on the total flavonoid yield, and the ultrasonic time, ultrasonic temperature and liquid-to-solid ratio are used as test factors, and the total flavonoid yield is used as the response value to optimize the ultrasonic extraction parameters.

[0024] Preferably, the optimal extraction conditions for ultrasonic-assisted aqueous two-phase system are: ultrasonic time 0.5 h, temperature 75 °C, liquid-to-solid ratio 70:1 g / g.

[0025] Preferably, the total flavonoid yield reaches 11.20 - 11.87 mg / g.

[0026] Preferably, after the crude extract is separated in step 5), it is left standing, and the obtained crude extract is poured into a separating funnel and left standing for 20 min.

[0027] The second aspect of the present invention provides a dried total flavonoid solid of alfalfa extracted according to the above method.

[0028] The beneficial effects of the present invention compared with the prior art are as follows: The present invention provides a method for extracting total flavonoids from alfalfa, which can significantly shorten the production cycle and increase the yield, and has significant improvements in efficiency, selectivity and economy. Specifically, at least one or more of the following effects can be achieved:

[0029] (1) Pretreatment drying optimizes the retention of activity: By drying the harvested alfalfa in an oven after blanching, the activity of hydrolytic enzymes is inhibited, the hydrolysis of flavonoid glycoside components is reduced, and the Maillard reaction caused by high-temperature treatment can promote the synthesis of flavonoid compounds, maximizing the retention of flavonoid content and antioxidant activity, providing high-quality raw materials for subsequent extraction, and further ensuring the quality of the final product.

[0030] (2) Alcohol / salt aqueous two-phase system: Utilizing the efficient partitioning mechanism brought about by the difference in the partition coefficient of the alcohol / salt aqueous two-phase system, by optimizing the concentration ratio of ethanol and salt, the difference in the partition coefficient of the target compound can be maximized, thereby more effectively achieving the selective separation and enrichment of the target compound.

[0031] (3) The ultrasonic-assisted aqueous two-phase extraction technology utilizes the cavitation effect of ultrasonic waves (combined with the efficient partitioning mechanism of aqueous two-phase extraction). By means of the mechanical vibration of ultrasonic waves, it destroys the plant cell wall, significantly enhancing the solvent permeability and the release efficiency of the target substance. This technology has the dual advantages of low-temperature rapid extraction and high-purity separation.

[0032] (4) The vacuum centrifugal concentration method is adopted to concentrate in gradients to protect heat-sensitive components. For the first concentration, the solvent is rapidly removed at a high temperature of 60 - 70 °C, reducing the processing time. For the second concentration, it is switched to a low temperature of 35 - 45 °C for deep concentration, which not only avoids the breakage of flavone ester bonds or the oxidation of phenolic hydroxyl groups caused by long-term high temperature but also shortens the overall process cycle, achieving the efficient recovery of total flavonoids.

[0033] (5) The composition of the aqueous two-phase system and ultrasonic parameters are simultaneously optimized by the response surface method to achieve the optimal extraction parameters. First, taking the yield of total flavonoids as the index, single-factor analyses are carried out on the concentrations of ethanol and (NH4)2SO4 solutions, ultrasonic power, ultrasonic time, ultrasonic temperature, and solid-liquid ratio. Then, using the central composite design (CCD), the best results obtained from the single-factor experiments are set as the central points of the response surface scheme. Taking ultrasonic time, ultrasonic temperature, and liquid-solid ratio as the experimental factors and the yield of total flavonoids as the response value, the extraction process parameters are optimized. The obtained data model is true, reliable, and has significant regularity, which can provide a theoretical reference for the extraction of total flavonoids from alfalfa.

[0034] (6) Given the above extraction methods and efficiency, the present invention can significantly shorten the production cycle and increase the output. Moreover, the alcohol phase is easy to recover, the system cost is low, and it has both environmental protection and industrialization potential. It has a significant improvement in efficiency, selectivity, and economy compared with the traditional ultrasonic extraction and single aqueous two-phase method.

[0035] It should be understood that the implementation of any embodiment of the present invention does not mean that multiple or all of the above beneficial effects need to be simultaneously achieved or reached. Description of the Drawings

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings described below are only exemplary. For those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained based on the provided drawings.

[0037] The structures, proportions, sizes, etc. shown in this specification are only used to match the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the implementation conditions of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope covered by the technical content disclosed in the present invention.

[0038] Figure 1 It is a process method flow chart provided by an embodiment of the present invention.

[0039] Figure 2-1 It is a schematic diagram showing the influence of the mass fraction of (NH4)2SO4 on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0040] Figure 2-2 It is a schematic diagram showing the influence of the mass fraction of (NH4)2SO4 on the yield of total flavonoids provided by an embodiment of the present invention.

[0041] Figure 2-3 It is a schematic diagram showing the influence of the mass fraction of ethanol on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0042] Figure 2-4 It is a schematic diagram showing the influence of the mass fraction of ethanol on the yield of total flavonoids provided by an embodiment of the present invention.

[0043] Figure 3-1 It is a schematic diagram showing the influence of ultrasonic power on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0044] Figure 3-2 It is a schematic diagram showing the influence of ultrasonic power on the yield of total flavonoids provided by an embodiment of the present invention.

[0045] Figure 3-3 It is a schematic diagram showing the influence of ultrasonic time on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0046] Figure 3-4 It is a schematic diagram showing the influence of ultrasonic time on the yield of total flavonoids provided by an embodiment of the present invention.

[0047] Figure 3-5 It is a schematic diagram showing the influence of ultrasonic temperature on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0048] Figure 3-6 It is a schematic diagram showing the influence of ultrasonic temperature on the yield of total flavonoids provided by an embodiment of the present invention.

[0049] Figure 3-7 It is a schematic diagram showing the influence of the liquid-to-material ratio on the extraction rate and distribution coefficient of total flavonoids provided by an embodiment of the present invention.

[0050] Figure 3-8 Schematic diagram of the influence of liquid-to-material ratio on the total flavonoid yield provided by the embodiments of the present invention.

[0051] Figure 4-1 Influence of interaction on the total flavonoid yield provided by the embodiments of the present invention (ultrasonic time and temperature).

[0052] Figure 4-2 Influence of interaction on the total flavonoid yield provided by the embodiments of the present invention (ultrasonic time and liquid-to-material ratio).

[0053] Figure 4-3 Influence of interaction on the total flavonoid yield provided by the embodiments of the present invention (ultrasonic temperature and liquid-to-material ratio).

[0054] Figure 5-1 Schematic diagram of the influence of drying method on the total flavonoid content and antioxidant activity of alfalfa provided by the embodiments of the present invention (DPPH free radical scavenging rate).

[0055] Figure 5-2 Schematic diagram of the influence of drying method on the total flavonoid content and antioxidant activity of alfalfa provided by the embodiments of the present invention (FRAP). Detailed implementation manners

[0056] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the embodiments of the present invention will be further described in detail below in conjunction with the embodiments and the drawings. Here, the illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not limit the present invention.

[0057] It should be understood that the terms "including / comprising", "consisting of...", or any other variant is intended to cover non-exclusive inclusion, so that a product, device, process or method including a series of elements not only includes those elements, but may also include other elements not explicitly listed when needed, or elements inherent to such product, device, process or method. Without further limitations, the elements defined by the statements "including / comprising...", "consisting of..." do not exclude the existence of additional identical elements in the product, device, process or method including the said elements.

[0058] Unless otherwise specified, the raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0059] The embodiments of the present invention provide a method for extracting total flavonoids from alfalfa, which includes the following steps:

[0060] 1) Blanching and drying: Put fresh alfalfa into a constant temperature drying oven at 85-105°C for high-temperature blanching for 20-30 minutes, and then adjust the temperature to 55-70°C and continue drying to constant weight;

[0061] The present invention uses the leguminous plant alfalfa as a material. Through high-temperature fixation by baking and drying, it can inhibit the activity of hydrolase, reduce the hydrolysis of flavonoid glycoside components, and the high temperature during oven drying can trigger the Maillard reaction, which can promote the synthesis of flavonoid compounds, maximizing the retention of the total flavonoid content and antioxidant activity.

[0062] 2) Crushing: Crush the dried alfalfa and pass it through a 0.2 - 0.4 mm grading sieve to obtain alfalfa powder.

[0063] In the present invention, the crushed alfalfa is passed through a 0.2 - 0.4 mm grading sieve because if the sieve pores are too fine, the particles are prone to agglomeration, forming a dense structure that hinders solvent penetration and solute diffusion, instead reducing the mass transfer efficiency. Moreover, the increased specific surface area exacerbates the thermal effect, resulting in the decomposition of active ingredients due to local overheating. While if the sieve pores are too large, it will reduce the contact area between the solvent and the sample, leading to a decrease in the diffusion rate of the target component from the inside of the sample particles to the solvent and a decline in the extraction efficiency; the cell structure inside the large particles is difficult to be fully penetrated by the solvent, which may cause some target components to remain in the unbroken cells and cannot be effectively dissolved. Through experimental research, a 0.2 - 0.4 mm grading sieve can meet the subsequent extraction requirements.

[0064] 3) Preparation of an aqueous two-phase system: Based on the total mass of the aqueous two-phase system, take 28 - 36% by mass of ethanol and 12% - 30% by mass of (NH4)2SO4 solution to prepare an ethanol-(NH4)2SO4 aqueous two-phase system.

[0065] The present invention first prepares an alcohol / salt aqueous two-phase system. The alcohol / salt aqueous two-phase system has a partition coefficient difference, that is, the concentration ratio of a substance in the two phases. Different substances have different partition coefficients in the same aqueous two-phase system. For example, flavonoids have higher solubility in the ethanol phase, while other impurities may have higher solubility in the salt phase. Using this efficient partitioning mechanism, by adjusting and optimizing the concentration ratio of ethanol and salt, the partition coefficient difference of the target compound can be maximized. Using this difference, flavonoids can be more inclined to distribute in a certain phase, and it can more effectively separate them from other components, realizing the selective separation and enrichment of the target compound.

[0066] 4) Extraction: Weigh 0.5 g of alfalfa powder, add it to the ethanol-(NH4)2SO4 aqueous two-phase system for ultrasonic extraction. The extraction conditions are: ultrasonic power 100 - 500 W, ultrasonic time 0.1 - 3 h, ultrasonic temperature 20 - 90 °C, liquid-to-solid ratio 30:1 - 100:1 g / g. After ultrasonic extraction, an alfalfa total flavonoid suspension is obtained.

[0067] Based on the alcohol / salt aqueous two-phase system, the present invention adopts an ultrasonic-assisted aqueous two-phase extraction technique. Utilizing the cavitation effect of ultrasonic waves, that is, when ultrasonic waves propagate in a liquid, regions of alternating high and low pressure are generated. When the pressure is lower than the vapor pressure of the liquid, bubbles are formed, and these bubbles rapidly collapse in the high-pressure phase, generating microjets and shock waves. Combining with the efficient partitioning mechanism of aqueous two-phase extraction, the plant cell walls are disrupted by the mechanical vibration of ultrasonic waves, significantly enhancing the solvent permeability and the release efficiency of the target substances.

[0068] 5) Vacuum filtration: Use a vacuum filtration system to remove the filter residue from the suspension to obtain a crude extract of total flavonoids from alfalfa. The pore size of the microporous filter membrane used in the vacuum filtration system is 0.3 - 0.5 μm;

[0069] During specific implementation, the vacuum filtration system needs to separately place disposable microporous filter membranes of different specifications.

[0070] Regarding the pore size of the microporous filter membrane, if the pore size is too large, the penetration rate of microorganisms or impurities will increase significantly; in chromatographic analysis, large-pore filter membranes may let protein aggregates pass through, resulting in peak area errors; impurity particles will clog equipment such as the sieve plate of the chromatographic column, shortening the instrument life. If the pore size is too fine, the filtration rate decreases, the time required for vacuum filtration is too long, and the too-fine pore size filter membrane is easily clogged and needs to be frequently replaced, resulting in increased costs. Through comprehensive analysis, the designed pore size of the microporous filter membrane in the present invention is 0.3 - 0.5 μm to meet the extraction requirements.

[0071] 6) Concentration: Concentrate the crude extract under vacuum centrifugation at 60 - 70 °C to obtain a solid crude extract;

[0072] 7) Washing with water: Wash the solid crude extract with ultrapure water to dissolve the (NH4)2SO4 salt in the extract;

[0073] 8) Secondary concentration: Perform secondary vacuum centrifugation concentration on the solid crude extract at 35 - 45 °C to obtain a dry solid of total flavonoids from alfalfa.

[0074] The purpose of the present invention is to obtain a solid of total flavonoids containing more flavonoids. The crude extract of total flavonoids from alfalfa is obtained through the previous extraction method, concentrated into a solid crude extract for the first time, then washed to remove impurities, and then concentrated to obtain a solid of total flavonoids from alfalfa with higher purity. The dried solid is the direct raw material for subsequent applications and can be directly used.

[0075] Example 1

[0076] A method for extracting total flavonoids from alfalfa, comprising the following steps:

[0077] Fixing color and drying: Put alfalfa into an oven at 105 °C for high-temperature fixing color for 30 min, adjust the temperature to 65 °C, and continue drying until constant weight;

[0078] Crushing: The dried alfalfa is crushed and passed through a 0.3 mm grading sieve to obtain alfalfa powder;

[0079] Preparation: Based on the total mass of the aqueous two-phase system, the mass fractions of (NH4)2SO4 are 12%, 14%, 16%, and 18% respectively, and the mass fraction of the ethanol solution is 34% to prepare an ethanol-(NH4)2SO4 aqueous two-phase system;

[0080] Extraction: Weigh 0.5 g of the alfalfa powder after blanching, drying and drying, add it to the aqueous two-phase system for ultrasonic extraction. The extraction conditions are: ultrasonic power 300 W, ultrasonic time 1.5 h, ultrasonic temperature 50 °C, liquid-solid ratio 50:1 g / g. After ultrasonic extraction, an alfalfa total flavonoid suspension is obtained;

[0081] Suction filtration: Use a suction filtration system to remove the filter residue and separate the crude extract. The pore size of the filter membrane is 0.45 μm. Pour the obtained crude extract into a separating funnel and let it stand for 20 min to ensure complete phase separation, and collect the upper-phase crude extract.

[0082] Concentration: Vacuum centrifugal concentration is carried out at 60 °C to obtain a crude extract solid.

[0083] Washing with water: Wash the crude extract solid with ultrapure water to dissolve the (NH4)2SO4 salt in the extract.

[0084] Secondary concentration: The remaining crude extract solid is subjected to secondary vacuum centrifugal concentration at 45 °C to obtain a dry alfalfa total flavonoid solid.

[0085] Example 2

[0086] This example is basically the same as Example 1, except that: the mass fractions of (NH4)2SO4 and ethanol solution in the preparation step are different.

[0087] Preparation of the aqueous two-phase system: The mass fraction of (NH4)2SO4 is 16%, and the mass fractions of the ethanol solution are 28%, 30%, 32%, and 36%.

[0088] Example 3

[0089] This example is basically the same as Example 1, except that: the mass fractions of (NH4)2SO4 and ethanol solution in the preparation step, and the ultrasonic power in the extraction step are different.

[0090] Preparation of the aqueous two-phase system: The mass fraction of (NH4)2SO4 is 16%, and the mass fraction of the ethanol solution is 30%.

[0091] Extraction: The ultrasonic powers are 100, 200, 400, and 500 W.

[0092] Example 4

[0093] This example is basically the same as Example 3, except that: the ultrasonic power and ultrasonic time in the extraction step are different.

[0094] Extraction: The ultrasonic power is 500 W, and the ultrasonic time is 0.5, 1, 1.5, 2, 2.5 h.

[0095] Example 5

[0096] This example is basically the same as Example 4, except that: the ultrasonic time and ultrasonic temperature in the extraction step are different.

[0097] Extraction: The ultrasonic time is 1.5 h, and the ultrasonic temperature is 40, 50, 60, 70, 80 °C.

[0098] Example 6

[0099] This example is basically the same as Example 5, except that: the ultrasonic temperature and liquid-to-solid ratio in the extraction step are different.

[0100] Extraction: The ultrasonic temperature is 50 °C, and the liquid-to-solid ratio is 50:1, 60:1, 70:1, 80:1, 90:1 (g / g).

[0101] Example 7

[0102] This example is basically the same as Example 6, except that: the ultrasonic time, temperature, and liquid-to-solid ratio in the extraction step are different.

[0103] According to the results of the single-factor experiment, three factors, namely ultrasonic time (A), ultrasonic temperature (B), and liquid-to-solid ratio (C), are selected. CCD is used to explore their effects on the response value of total flavonoid yield, and the optimal extraction process conditions are optimized by response surface. Each factor uses five levels (-2, -1, 0, +1, +2), and a total of 20 test points are taken, including 14 factorial points and 6 central points. The factors and levels of the response surface test design are shown in Table 1.

[0104] Table 1 Factors and levels of CCD response surface test design

[0105]

[0106] Example 8

[0107] This example is basically the same as Example 6, except that: the drying method is different in the blanching and drying step. The ultrasonic time, temperature, and liquid-to-solid ratio in the extraction step are different.

[0108] Fixing and drying: Fixing and drying, vacuum freeze-drying and natural drying are carried out respectively. Vacuum freeze-drying: Put alfalfa into a refrigerator at -80 °C for 24 h, take it out and place it in a freeze dryer, and freeze-dry it to constant weight under vacuum. Natural drying: Place alfalfa in a cool and ventilated place and dry it to constant weight.

[0109] Extraction conditions: The ultrasonic time is 0.5 h, the ultrasonic temperature is 75 °C, and the liquid-to-solid ratio is 70:1 g / g.

[0110] Comparative Example 1

[0111] This comparative example is basically the same as Example 8, except that: the concentration method is different, high-temperature concentration at 60 °C is adopted throughout the process, and there is no secondary concentration. The pretreatment adopts the drying method after fixing.

[0112] Comparative Example 2

[0113] This comparative example is basically the same as Comparative Example 1, except that: single low-temperature concentration at 45 °C, without a high-temperature stage.

[0114] Comparative Example 3

[0115] This comparative example is basically the same as Comparative Example 1, except that: the concentration method is different, gradient concentration is adopted. For the first concentration, the solvent is quickly removed at a high temperature of 80 °C to reduce the processing time; for the second concentration, it is switched to a low temperature of 20 °C for deep concentration.

[0116] Determination:

[0117] Determination of DPPH free radical scavenging rate:

[0118] Dilute the crude extract of total flavonoids by 10 times, prepare a 0.2 mmol / L DPPH solution with methanol, prepare it freshly and store it in the dark at 4 °C. Take 100 μL of the test solution into a 96-well plate, add 100 μL of DPPH solution to each well, mix well, and react in the dark at room temperature for 30 min. Use 100 μL of 30% ethanol as a blank control, and measure the OD value at 517 nm.

[0119] DPPH free radical scavenging rate (%) = [1 - (Ai - Aj) / A0)] × 100

[0120] In the formula: Ai is the OD value of the test solution added; Aj is the background value of the test solution; A0 is the control value without the test solution added.

[0121] Determination of ferric ion reducing antioxidant power (FRAP):

[0122] Take 1.0 mL of the test solution in a test tube, and successively add 1.0 mL of 0.2 mol / L phosphate buffer solution (pH 6.6), 1.0 mL of 1% potassium ferricyanide solution, and mix well by shaking. Heat in a water bath at 50 °C for 20 min, then add 1.0 mL of 10% trichloroacetic acid solution by mass, mix well by shaking, and centrifuge at a speed of 4000 r / min for 10 min. Take 0.5 mL of the supernatant, add 0.5 mL of deionized water and 0.1 mL of 0.1% FeCl3 solution by mass, mix well by shaking, heat in a water bath at 50 °C for 10 min again, and measure at a wavelength of 700 nm, using deionized water as the blank control.

[0123] Data analysis:

[0124] In order to obtain the optimal production process parameters, the present invention adopts the response surface analysis method, which is an experimental method developed by Box-Wilson in 1951 for optimizing and evaluating the levels of various experimental factors and their interactions through the analysis of function response surfaces and contour lines, and exploring the optimal operating conditions through optimization calculations.

[0125] Synchronously optimize the composition of the aqueous two-phase system and ultrasonic parameters by the response surface method to achieve the optimal extraction parameters. Specifically, taking the total flavonoid yield as the index, perform single-factor analysis on the concentrations of ethanol and (NH4)2SO4 solution, ultrasonic power, ultrasonic time, ultrasonic temperature, and solid-liquid ratio. Set the best results obtained from the single-factor experiments as the center point of the response surface scheme, and then use the central composite design CCD to explore the influence of the interaction of factors on the response value of the total flavonoid yield. Take ultrasonic time, ultrasonic temperature, and liquid-solid ratio as experimental factors, and the total flavonoid yield as the response value to optimize the extraction process parameters, and finally determine the optimal extraction conditions for ultrasonic-assisted aqueous two-phase extraction of total flavonoids from alfalfa. (1)

[0127] Measure the total flavonoid extraction rate, distribution coefficient, and yield in Example 1 and Example 2, and the measurement results are as Figure 2-1 、 2-2 、2-3, 2-4 shown.

[0128] As can be seen from the figure, in the ethanol-(NH4)2SO4 system, when the mass fractions of (NH4)2SO4 and ethanol are 16% and 30% respectively, the best mass fractions are reached. (2)

[0130] Measure the total flavonoid extraction rate, distribution coefficient, and yield in Example 3, Example 4, Example 5, and Example 6, and the measurement results are as Figure 3-1 、 3-2 、3-3, 3-4, 3-5, 3-6, 3-7, 3-8 shown.

[0131] The effects of ultrasonic power on the extraction rate, partition coefficient, and yield of total flavonoids are as Figure 3-1 , 3-2 shown. Within the power range achievable by the instrument, the extraction rate, partition coefficient, and yield of total flavonoids all increase with the increase of ultrasonic power, and the yield reaches the peak at 500 W.

[0132] The effects of ultrasonic time on the extraction rate, partition coefficient, and yield of total flavonoids are as Figure 3-3 , 3-4 shown. Within the range of 0.5 h to 2.5 h, as the extraction time prolongs, the yield of total flavonoids first increases and then decreases. At 1.5 h, the extraction rate, partition coefficient, and yield of total flavonoids all reach the maximum values.

[0133] The effects of ultrasonic temperature on the extraction rate, partition coefficient, and yield of total flavonoids are as Figure 3-5 , 3-6 shown. At 50 °C and 70 °C, the extraction rates of total flavonoids are significantly higher than those at other temperatures, and there is no significant difference in the extraction rates at these two temperatures. As the ultrasonic temperature increases, the yield of total flavonoids shows a trend of first increasing and then decreasing. When the extraction temperature reaches 70 °C, the yield reaches the peak.

[0134] The effects of liquid-to-solid ratio on the extraction rate, partition coefficient, and yield of total flavonoids are as Figure 3-7 , 3-8 shown. As the liquid-to-solid ratio increases, the extraction rate, partition coefficient, and yield of total flavonoids all show a trend of first increasing and then decreasing. When the liquid-to-solid ratio is 70:1 g / g, the extraction rate and yield of total flavonoids reach the maximum values.

[0135] Based on the results of the above single-factor experiments, the optimal extraction conditions for total flavonoids from alfalfa under single factors are determined as ultrasonic power of 500 W, ultrasonic time of 1.5 h, ultrasonic temperature of 70 °C, and liquid-to-solid ratio of 70:1 g / g.

[0136] Three factors, namely ultrasonic time, ultrasonic temperature, and liquid-to-solid ratio, are selected. The optimal results obtained from the above single-factor experiments are set as the central point of the response surface scheme, and the CCD is used to explore the influence of the interaction between factors on the response value, the yield of total flavonoids. The extraction rates, partition coefficients, and yields of total flavonoids in the 20 experiments of the three factors in Example 7 are continuously measured, and the measurement results are shown in Table 2. The analysis of variance of the CCD experimental design results is shown in Table 3.

[0137] Table 2 CCD experimental design scheme and results

[0138]

[0139]

[0140] Table 3 Analysis of variance of CCD experimental design results

[0141]

[0142] R 2 = 0.9983, Adjusted R 2 = 0.9967

[0143] Note: *, P < 0.05; ****, P < 0.0001; NS, P > 0.05.

[0144] The results in Table 2 show that when the ultrasonic time, ultrasonic temperature, and liquid-to-solid ratio = (-1, +1, +1), the total flavonoid yield is the highest, which is 10.20 mg / g. To describe the relationship between the total flavonoid yield and related parameters, multiple regression fitting was used, and the obtained regression equation model is as follows:

[0145] Y = 8.56 - 0.8805A - 0.2634B + 0.5039C - 0.7225AB + 0.05AC - 0.055BC - 0.1829A 2 + 0.3952B 2 - 0.2518C 2

[0146] In the formula, Y represents the total flavonoid yield, A represents the ultrasonic time, B represents the ultrasonic temperature, and C represents the liquid-to-solid ratio.

[0147] The statistical result analysis in Table 3 shows that the model reaches an extremely significant level (P < 0.0001). By using the determination coefficient R 2 to evaluate the goodness of fit of the analysis of variance, the closer R 2 is to 1, the better the model fitting effect. In this model, R 2 is 0.9983, indicating that the model fitting effect is good and has high reliability. In the present invention, the lack-of-fit term is not significant (F = 1.06, P = 0.4759 > 0.05), indicating that the model fits well, has high credibility, can effectively explain the experimental data, and can be used for prediction within the designed range.

[0148] Response surface plots of the interaction effects were drawn through the regression equation, as shown in Figure 4-1 、 4-2 、4 - 3. Using DesignExpert 13.0 to optimize the extraction condition parameters, based on the mathematical solution of the regression equation and combined with the intuitive verification of the response surface plot, the optimal extraction conditions were predicted, as shown in Table 4. Further verification of the feasibility of the optimal extraction conditions was carried out, and the results are shown in Table 5. The relative standard deviation (RSD) is less than 5%, further verifying the feasibility of the theoretical model, and it can predict the total flavonoid yield of alfalfa under different extraction conditions.

[0149] Table 4 Optimal extraction condition optimization scheme

[0150]

[0151] Table 5 Feasibility Verification of Optimal Extraction Conditions

[0152]

[0153] Finally, the optimal extraction conditions for ultrasonic-assisted aqueous two-phase extraction of total flavonoids from alfalfa were determined as follows: ultrasonic time 0.5 h, temperature 75 °C, liquid-to-solid ratio 70:1 g / g ， At this time, the yield of total flavonoids was 11.20 - 11.87 mg / g. (III)

[0155] The yields and antioxidant properties of the total flavonoids in Example 8 and Comparative Examples 1, 2, and 3 were measured, and the measurement results are as Figure 5-1 、 5-2 shown.

[0156] First of all, it should be noted that in the determination of DPPH radical scavenging rate, the flavonoid concentration was the concentration after being diluted 10 times.

[0157] After measurement, as Figure 5-1 、 5-2 shown, the total flavonoid content after oven drying treatment was the highest, reaching 10.04 mg / g, which was significantly higher than that after vacuum freeze drying (6.23 mg / g) and natural sun drying (5.91 mg / g) (P < 0.05) 。 Therefore, drying after blanching is the best pretreatment method to maximize the retention of total flavonoids in alfalfa.

[0158] In terms of antioxidant capacity, the DPPH scavenging rate and reducing power of alfalfa after oven drying treatment were significantly higher than those of the other two drying methods (P < 0.05). Therefore, drying after blanching is the best pretreatment method to maximize the retention of the antioxidant activity of total flavonoids in alfalfa. (IV)

[0160] The total flavonoid content and antioxidant properties of the alfalfa flavonoid powder product with the pretreatment of blanching and oven drying in Example 8 were compared with the alfalfa flavonoid powders obtained in Comparative Examples 1, 2, and 3. The specific results are shown in Table 6 for details.

[0161] Table 6 Measurement Results of Total Flavonoid Content and Antioxidant Properties

[0162]

[0163] In Comparative Example 1, a fixed single temperature was adopted for high-temperature concentration throughout the process. In Comparative Example 2, a fixed single temperature was adopted for low-temperature concentration throughout the process. In Comparative Example 3, the first concentration temperature was increased and the second concentration temperature was decreased compared with Example 8. The results in Table 2 show that, compared with Example 8, the total flavonoid purity, DPPH scavenging rate, and FRAP reducing power of Comparative Examples 1-3 all decreased. Through secondary concentration in the present invention, it can be seen that the total flavonoid purity and antioxidant capacity are significantly better than those of the comparative examples. Therefore, the comparative examples indicate that deviating from the gradient concentration parameters may lead to problems such as a decrease in total flavonoid purity and product stability, verifying the necessity of the gradient design.

[0164] In summary, the embodiments of the present invention disclose a pretreatment method after alfalfa harvesting and a method for extracting total flavonoids from alfalfa based on the response surface method. On the basis of aqueous two-phase extraction, ultrasonic assistance is combined, and the single-factor experiment method is used to explore the effects of ultrasonic power, ultrasonic temperature, ultrasonic time, and liquid-to-solid ratio on the extraction rate of total flavonoids from alfalfa. The extraction process is optimized by response surface, and finally the optimal extraction process parameters of total flavonoids from alfalfa are determined: ultrasonic power 500W, ultrasonic time 0.5h, ultrasonic temperature 75°C, and liquid-to-solid ratio 70:1 g / g. In addition, the present invention provides a method for treating alfalfa by oven drying to achieve a higher extraction rate of total flavonoids from alfalfa and maximize the antioxidant activity. As an important secondary metabolite of alfalfa, total flavonoids from alfalfa have functional characteristics such as antibacterial, antioxidant, promoting growth and development, and improving the quality of livestock products, providing a scientific basis for alfalfa feed processing and the development of high-value-added products.

[0165] The embodiments of the present invention have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other ordinary skilled persons in the technical field to understand the embodiments disclosed herein.

Claims

1. A method for extracting total flavonoids from alfalfa, characterized in that: The steps include: 1) Drying and drying: put the fresh alfalfa into a constant temperature drying oven at 85-105°C for 20-30 minutes, then adjust the temperature to 55-70°C and continue drying until constant weight; 2) Grinding: Grinding the dried alfalfa and passing it through a 0.2-0.4 mm grading sieve to obtain alfalfa powder; 3) preparing a two-phase aqueous system: taking the total mass of the two-phase aqueous system as a reference, taking 28% to 36% by mass of ethanol and 12% to 30% by mass of (NH4)2SO4 solution to prepare an ethanol-(NH4)2SO4 two-phase aqueous system; 4) Extraction: Add alfalfa powder to ethanol-(NH4)2SO4 aqueous two-phase system for ultrasonic extraction, extraction conditions: ultrasonic power 100-500W, ultrasonic time 0.1-3h, ultrasonic temperature 20-90°C, liquid-to-solid ratio 30:1-100:1g / g, and obtain alfalfa total flavonoid suspension after ultrasonic extraction; 5) Filtration: Use a filtration system to remove the filter residue from the suspension to separate and obtain a crude extract, wherein the pore size of the microporous filter membrane used in the filtration system is 0.3-0.5 μm; 6) Concentration: The crude extract is concentrated by vacuum centrifugation at 60-70°C to obtain a crude extract solid; 7) Water washing: Use ultrapure water to wash the crude extract solid to dissolve the (NH4)2SO4 salt in the extract; 8) Secondary concentration: The crude extract solid is subjected to secondary vacuum centrifugation concentration at 35-45° C. to obtain a dry alfalfa total flavonoid solid.

2. The method according to claim 1, characterized in that The composition of the two-phase solution and ultrasonic extraction parameters were optimized by one-way analysis of variance. The total flavonoid yield was used as an indicator to perform one-way analysis on the concentrations of ethanol and (NH4)2SO4 solution, ultrasonic power, ultrasonic time, ultrasonic temperature, and solid-liquid ratio.

3. The method according to claim 2, characterized in that In step 3), in the ethanol-(NH4)2SO4 system, the mass fractions of ethanol and (NH4)2SO4 are 30% and 16% respectively.

4. The method according to claim 2, characterized in that: In step 4), the ultrasonic power is 400-500 W, the ultrasonic time is 0.5-2.5 h, the ultrasonic temperature is 40-80° C., and the liquid-to-solid ratio is 50:1-90:1 g / g.

5. The method according to claim 4, characterized in that In step 4), the ultrasonic power is 500 W, the ultrasonic time is 1.5 h, the ultrasonic temperature is 70° C., and the liquid-to-solid ratio is 70:1 g / g.

6. The method according to any one of claims 2 to 5, characterized in that: The central composite design (CCD) response surface methodology was used to consider the effect of the interaction of factors on the total flavonoids yield. The ultrasonic extraction parameters were optimized with ultrasonic time, ultrasonic temperature and liquid-to-solid ratio as experimental factors and total flavonoids yield as the response value.

7. The method according to claim 6, characterized in that The optimal conditions for ultrasound-assisted aqueous two-phase extraction are: ultrasound time 0.5h, temperature 75℃, and liquid-to-solid ratio 70:1g / g.

8. The method according to claim 7, characterized in that The total flavonoid yield reached 11.20-11.87 mg / g.

9. The method according to claim 1, characterized in that: Step 5) After separation, the crude extract was allowed to stand, and the crude extract was poured into a separatory funnel and allowed to stand for 20 minutes.

10. A dried alfalfa total flavonoid solid extracted according to the method according to any one of claims 1 to 9.

Images