Method for synchronously extracting rumex hanus protein and flavone through one-pot method

Abstract

The invention provides a method for synchronously extracting rumex hanus protein and flavone by a one-pot method, which adopts a low-temperature plasma combined deep eutectic solvent (DES) method to perform efficient extraction, realizes a serial mechanism of physical wall breaking and chemical capture, optimizes the formula of the DES and the process conditions of the low-temperature plasma, ensures that the synergistic interaction effect is obvious, and has the advantages of simple process, low cost and high efficiency. According to the method, the extraction time is greatly shortened, the process is integrated and simplified, the activity of the rumex hanus protein and flavone can be better maintained, the yield can be remarkably increased, a direction is provided for green and efficient extraction of plant active ingredients, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of plant protein and flavonoid extraction technology, and more specifically, to a one-pot method for simultaneous extraction of plant protein and flavonoids from leafy greens. Background Technology

[0002] Rumex patientia L. × Rumex tianschanicus A. LOS, scientifically known as Rumex patientia L. × Rumex tianschanicus A. LOS, is also called protein grass due to its relatively high crude protein content, reaching 30%-45%, and is hailed as a "nutritional plant" that is high in protein, high in yield, and high in efficiency. According to Document No. 9 issued by the National Health Commission in 2021, Rumex patientia has been officially included in the new food ingredient classification, thus attracting widespread attention to research on the development of Rumex patientia foods.

[0003] Edible grass, as a high-quality plant for medicinal and nutritional purposes, is rich in nutrients, including protein, dietary fiber, vitamins, and minerals. In addition, it contains abundant flavonoids, carotene, SOD enzymes, and other bioactive substances. These components have excellent physiological regulatory effects, accelerating metabolism and enhancing the immune system. Edible grass protein and flavonoids can be used as food additives in various food processing techniques and products.

[0004] Traditional methods for extracting plant proteins and flavonoids, such as organic solvent precipitation, alkaline solution precipitation, heating, ultrafiltration, salting out, and enzymatic methods, typically require stepwise steps. These processes are cumbersome, time-consuming, consume large amounts of solvent, and cause significant pollution. Furthermore, they can only extract plant proteins or flavonoids, not both simultaneously. In recent years, extraction technologies based on eutectic solvents (DES) have shown significant advantages in the extraction of plant active ingredients, providing a new strategy for addressing this issue. However, there is a lack of systematic and in-depth research reports on the specific application of this technology to the efficient and green integrated development of plant resources, and its application potential in the deep processing of plant resources remains to be further explored and validated.

[0005] Therefore, there is an urgent need to find a simple, low-cost, mild, and environmentally friendly one-pot method for simultaneously extracting leafwort protein and its flavonoids. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a one-pot method for the simultaneous extraction of plant protein and flavonoids. It employs a combination of low-temperature plasma and DES (Distilled Extraction System) for efficient extraction, achieving a tandem mechanism of physical cell disruption and chemical capture. The DES formulation and low-temperature plasma process conditions have been optimized, resulting in a significant synergistic effect, greatly shortening the extraction time, and simplifying the process integration. This method not only better preserves the activity of plant protein and flavonoids but also significantly improves the yield, providing a direction for the green and efficient extraction of plant active ingredients and showing broad application prospects.

[0007] On one hand, the present invention provides a one-pot method for simultaneous extraction of physalis protein and flavonoids, wherein physalis and DES aqueous solution are mixed and subjected to low-temperature plasma extraction; wherein the DES includes a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond acceptor is one of choline chloride and an amino acid, and the hydrogen bond donor is any one or more of lactic acid, glucose, glycerol, 1,4-butanediol, polyethylene glycol, urea, n-propanol, and propylene glycol.

[0008] Low-temperature plasma is a novel non-thermal extraction technology that contains a variety of active ingredients, such as reactive oxygen species, reactive nitrogen species, charged particles, electrons, and photons. It has strong biological activity and oxidizing properties. High-energy particles bombard biological cells, causing etching on their surfaces.

[0009] This invention utilizes low-temperature plasma combined with DES to create a unique one-pot method for the simultaneous extraction of plant protein and flavonoids from leafwort. Using low-temperature plasma as a pilot step, the dense plant cell walls are disrupted through physicochemical action, opening a pathway for DES to rapidly and unimpededly penetrate the cells and fully contact the target components. Compared to extraction methods using DES alone, this method increases the contact area between protein and DES, improves the rate at which DES dissolves protein, and significantly shortens the extraction time. It features low temperature, rapid extraction with no residue, simple operation, high efficiency, and environmental friendliness. Furthermore, it significantly increases the yield of leafwort protein and flavonoids, exhibiting a clear synergistic effect. Suitable for large-scale industrial production, this method has significant application potential. The entire process can be carried out at low or room temperature, perfectly avoiding the heat-sensitive protein denaturation and flavonoid degradation caused by traditional thermal extraction methods, thus better preserving their natural activity and meeting the requirements of green chemistry and sustainable development.

[0010] Meanwhile, the present invention also found that the leafy grass protein extracted by this method has better solubility. The reason may be that plasma treatment causes specific and controllable modification of the leafy grass protein, thereby improving its solubility and making it more conducive to subsequent applications.

[0011] Furthermore, the DES also includes a protective agent, which includes any one or more of vitamin C, vitamin E, L-cysteine, dithiothreitol, and propyl gallate.

[0012] Since plasma generation is accompanied by the production of a large amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS), these highly reactive substances may oxidize proteins (such as causing side chain amino acid oxidation, cross-linking, and aggregation) and degrade flavonoids (such as destroying their phenolic hydroxyl structure, causing them to lose their antioxidant activity). Therefore, it is necessary to add a protective agent to DES to ensure the activity of the extracted leafwort protein and flavonoids at the same time.

[0013] In some embodiments, the protective agent includes vitamin C, vitamin E, and L-cysteine.

[0014] Studies have shown that different protectants have different protective effects on leafy green protein and flavonoids. Protectants prepared with vitamin C, vitamin E and L-cysteine ​​can achieve synergistic effect and provide comprehensive protection. They not only have excellent protective performance on leafy green protein, but also have a good protective effect on flavonoids, with excellent dual protection effect.

[0015] In some embodiments, the mass ratio of vitamin C, vitamin E, and L-cysteine ​​is (0.1~1):(0.05~0.5):(0.1~1).

[0016] In some embodiments, the mass ratio of vitamin C, vitamin E, and L-cysteine ​​is 0.5:0.1:0.5.

[0017] Furthermore, the hydrogen bond acceptor is selected from choline chloride, and the hydrogen bond donor is selected from glycerol and lactic acid.

[0018] This invention utilizes a mixture of lactic acid and glycerol to create a novel hydrogen bond donor that combines the properties of both. The acidity of lactic acid facilitates the hydrolysis of the cell walls of edible grasses, opening channels for the dissolution of internal components. Glycerol provides a relatively stable solvent environment, preventing the degradation of extracted proteins and flavonoids. Furthermore, pure glycerol has a high viscosity, which is not conducive to mass transfer. The addition of lactic acid can effectively reduce the overall viscosity, improving the mobile phase and permeability of the DES. At the same time, the pH of the DES can be precisely controlled by adjusting the ratio of lactic acid to glycerol, keeping the DES in a weakly acidic environment, which is more conducive to the simultaneous extraction of edible grass proteins and flavonoids, and also makes the protective agents (such as vitamin C) in the DES more stable.

[0019] Furthermore, the molar ratio of hydrogen bond acceptor to hydrogen bond donor in the DES is 1:(1-5); the volume ratio of glycerol to lactic acid in the hydrogen bond donor is 1:(0.5~2).

[0020] Furthermore, the edible grass is edible grass powder, and the edible grass powder is mixed with DES at a solid-liquid ratio of 1g:(5-50)mL.

[0021] Furthermore, the discharge form of the low-temperature plasma is selected from any one or more of glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge.

[0022] Furthermore, the working voltage of the low-temperature plasma is 15-45V, and the processing time is 1-4min; the working gas of the low-temperature plasma is selected from any one or more of argon, helium, and air.

[0023] In some embodiments, the cryogenic plasma operates at a voltage of 35V.

[0024] Furthermore, it also includes adding an antisolvent to the crude extract obtained from the extraction process to precipitate the phytoestrogen protein, thereby achieving solid-liquid separation of the phytoestrogen protein and flavonoids.

[0025] In some embodiments, the method includes the following steps: (1) Mix the leafy grass powder and DES, and perform low-temperature plasma extraction to obtain crude extract; (2) Add antisolvent to crude extract, centrifuge to obtain precipitate and supernatant; (3) The precipitate phase was purified to obtain edible grass protein; the supernatant was purified to obtain edible grass flavonoids.

[0026] In some methods, the antisolvent in step (2) is water. By adding deionized water to disrupt the hydrogen bond network of DES, its solubility is drastically reduced, and the large difference in solubility of phytoesin and flavonoids in the DES-water mixture is used to separate phytoesin and flavonoids.

[0027] In some methods, the purification of the precipitate phase in step (3) includes dialysis and ultrafiltration; the purification of the supernatant includes macroporous resin adsorption purification.

[0028] In some methods, after the supernatant is treated with macroporous resin to extract flavonoids, the DES in it can be recycled.

[0029] On the other hand, the present invention provides a leafy grass protein, which is prepared by the method described above.

[0030] In another aspect, the present invention provides a leafwort flavonoid, which is prepared by the method described above.

[0031] The use of a composition for preparing a reagent for the one-pot simultaneous extraction of physalis protein and flavonoids is characterized in that the composition comprises choline chloride, lactic acid, glycerol, and a protective agent for preparing DES; the one-pot simultaneous extraction of physalis protein and flavonoids employs low-temperature plasma combined with DES extraction; the protective agent comprises any one or more of vitamin C, vitamin E, L-cysteine, dithiothreitol, and propyl gallate; and the DES is required to simultaneously extract physalis protein and flavonoids with the assistance of low-temperature plasma.

[0032] The present invention has the following beneficial effects: 1. A novel one-pot method for simultaneous extraction of plant protein and flavonoids is provided. The method uses low-temperature plasma combined with DES for extraction, which increases the contact area between protein and DES, improves the rate of protein dissolution by DES, and significantly shortens the extraction time. It features low temperature, rapid and residue-free extraction, simple operation, high efficiency and environmental protection, and significantly improves the yield of plant protein and flavonoids, with a significant synergistic effect. 2. By adding a protectant to DES and screening a suitable protectant formulation, the activity of the extracted leafwort protein and flavonoids can be effectively protected. 3. Hydrogen bond donors in DES were screened to create a weakly acidic environment for DES, which has a synergistic effect and is more conducive to the simultaneous extraction of leafwort protein and flavonoids. 4. The DES formulation and low-temperature plasma process conditions were optimized, resulting in a significant synergistic effect, simplified process integration, and better preservation of the activity of leafy green protein and flavonoids. Attached Figure Description

[0033] Figure 1 This is a flowchart of the one-pot simultaneous extraction of leafwort protein and flavonoids in Example 1; Figure 2 A photograph of the leafwort protein powder prepared in Example 1; Figure 3 This is a photograph of the flavonoids prepared in Example 1. Detailed Implementation

[0034] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0035] Example 1: Simultaneous extraction of leafwort protein and flavonoids in a one-pot process The one-pot simultaneous extraction process of leafwort protein and flavonoids provided in this embodiment is as follows: Figure 1 The specific steps are as follows: 1. DES configuration 1) Prepare basic DES Choline chloride (purity ≥98%), glycerol (purity ≥99%), and lactic acid (analytical grade) were mixed in a molar ratio of 1:2:1 and placed in a round-bottom flask. A stir bar was placed in the flask containing the mixture, and the flask was placed in a preheated oil bath. A magnetic stirrer was connected, and stirring and heating were started. The temperature was set at 60°C, and stirring was continued for 2 hours. The mixture gradually changed from a turbid, opaque slurry to a homogeneous, transparent, colorless liquid, which yielded the basic DES.

[0036] 2) Add a protective agent First, add 0.5% L-cysteine ​​to the basic DES and stir continuously for 15 minutes until it is completely dissolved; then add 0.5% vitamin C and stir continuously for 15 minutes until it is completely dissolved; finally, add 0.1% vitamin E and stir continuously at 40~50℃ for 1 hour to obtain DES.

[0037] 2. Low-temperature plasma-assisted DES extraction Dried edible grass powder (purchased from Henan Jundao Edible Grass Park Operation and Management Co., Ltd.) was added to DES. The solid-liquid ratio of dried edible grass powder to DES was 1:30 (g / mL). The mixture was mixed evenly, ensuring that the surface of the mixture was as flat and exposed as possible. The mixture was then placed in a dielectric barrier discharge low-temperature plasma reaction chamber and treated at a high voltage of 40 kV for 2 minutes to obtain a crude extract.

[0038] 3. Add antisolvent (deionized water) Under continuous magnetic stirring, deionized water was slowly added dropwise to the crude extract. As water was added, the solution gradually became turbid, indicating that the leafwort protein began to denature and precipitate. The volume ratio of deionized water to crude extract was 1:2. After the addition was complete, stirring was continued for 20 minutes to ensure complete precipitation. The mixture was then centrifuged at 8000 rpm for 15 minutes, and the precipitate and supernatant were collected separately.

[0039] 4. Purification of leafy green protein The precipitate phase was washed with a small amount of water (e.g., 20 mL of water to wash 1.0 g of precipitate phase) to remove residual DES and water-soluble impurities. Then, it was reconstituted with phosphate-buffered saline (PBS) at pH 7.4, with a PBS to precipitate phase ratio of 15:1 (mL / g) to obtain a protein solution. The protein solution was then placed in dialysis buffer and dialyzed with 20 times its volume of deionized water for 20–48 hours, changing the buffer every 2 hours to obtain a purified protein solution. This purified protein solution was then freeze-dried to obtain leafwort protein powder (Figure 2).

[0040] Purification of flavonoids from edible leaves The supernatant was diluted with 1 part deionized water and then passed through a pre-treated macroporous resin column (AB-8) at a flow rate of 2 BV / h. Flavonoids were specifically adsorbed onto the resin, while impurities were washed away with the effluent. The resin column was then rinsed with a 70%-80% ethanol aqueous solution as the eluent to elute the flavonoids. The ethanol eluent containing flavonoids was collected, and the ethanol was removed by rotary evaporation at a low temperature (<40°C) to obtain an aqueous flavonoid solution. This solution was then freeze-dried to obtain a brownish-yellow flavonoid powder (Figure 3).

[0041] Example 2: Effects of different extraction methods on the extraction efficiency of plant protein and flavonoids from *Echinochloa crus-galli* This embodiment extracts physalis protein and flavonoids according to the method provided in Example 1. During the extraction process using DES, the effects of different physical fields, such as low-temperature plasma and ultrasound, on the extraction efficiency were compared. The ultrasound-combined DES extraction method involved uniformly mixing physalis powder with DES and placing the mixture in an ultrasound chamber. The ultrasound temperature was set at 25°C, and the ultrasound time was 15 minutes. The effects of different extraction methods on the extraction yield of physalis protein and flavonoids were investigated.

[0042] The extraction yield of *Gynostemma pentaphyllum* protein was determined as follows: first, the total protein mass in the raw material was determined by the BCA method; then, the purified protein solution obtained after extraction was used to determine the total protein mass by the BCA method, which is the amount of protein obtained after extraction; the extraction yield of *Gynostemma pentaphyllum* protein = (amount of protein obtained after extraction / total protein mass in the raw material) × 100%. The extraction yield of *Gynostemma pentaphyllum* flavonoids was determined as follows: first, the total flavonoid mass in the raw material was determined by the aluminum nitrate colorimetric method; then, the purified flavonoid aqueous solution was used to determine the total flavonoid mass by the aluminum nitrate colorimetric method, which is the amount of flavonoids obtained after extraction; the extraction yield of *Gynostemma pentaphyllum* flavonoids = (mass of flavonoids obtained after extraction / mass of total flavonoids in the raw material) × 100%. The test results are shown in Table 1.

[0043] Table 1. Effects of different auxiliary methods on the extraction of protein and flavonoids from leafwort. As shown in Table 1, the extraction yields of phytoestrogen and flavonoids were lower than those obtained by DES extraction alone. Ultrasonic or low-temperature plasma-assisted extraction significantly improved the extraction effect of DES. In particular, the extraction yields of phytoestrogen and flavonoids were the highest when low-temperature plasma was combined with a eutectic solvent. This may be because under low-temperature plasma treatment, phytoestrogen and flavonoids can be more effectively dissolved from plant cells without being destroyed.

[0044] Example 3: Screening of hydrogen bond acceptors In this embodiment, leafwort protein and flavonoids were extracted according to the method provided in Example 1. Different types of hydrogen bond acceptors, as shown in Table 2, were used for the DES, and glycerol was used as the hydrogen bond donor. The molar ratio of hydrogen bond acceptor to hydrogen bond donor was 1:2. The effect of DES prepared using different hydrogen bond acceptors on the extraction yield of leafwort protein and flavonoids was investigated. The test results are shown in Table 2.

[0045] Table 2. Effects of different hydrogen bond donors on the extraction of protein and flavonoids from leafwort. As shown in Table 1, different hydrogen bond acceptors have a certain impact on the extraction rates of physalis protein and physalis flavonoids. Some are more conducive to the extraction of flavonoids (such as choline chloride and L-aspartic acid), while others are more conducive to the extraction of physalis protein (such as choline chloride and L-proline). Choline chloride is the most preferred option, which maximizes the yield of both physalis protein and flavonoids.

[0046] Example 4: Screening of hydrogen bond donors This embodiment extracts physalis protein and flavonoids according to the method provided in Example 1. The hydrogen bond donors for the DES are different types provided in Table 3, and the hydrogen bond acceptor is choline chloride. The effect of DES prepared with different hydrogen bond donors on the extraction yield of physalis protein and flavonoids was investigated. The test results are shown in Table 3.

[0047] Table 3. Effects of different hydrogen bond donors on the extraction of physalis protein and flavonoids According to Table 3, different hydrogen bond donors have a certain impact on the extraction rate of physalis protein and physalis flavonoids. Moreover, when glycerol is combined with another hydrogen bond donor, the effect can be further improved. In particular, the combination of glycerol and lactic acid can significantly improve the extraction effect of physalis protein and physalis flavonoids.

[0048] Example 5: Effect of adding a protective agent to DES Adding a preservative can significantly improve the extraction rate of physalis protein and physalis flavonoids, with different preservatives resulting in different extraction effects. This example extracts physalis protein and flavonoids according to the method provided in Example 1, using preservatives as listed in Table 4 to prepare eutectic solvents at the same proportions as in Example 1. The effect of different preservatives used in the preparation of DES on the extraction yield of physalis protein and flavonoids was investigated. The results are shown in Table 4.

[0049] Table 4. Effects of different preservatives on the extraction of protein and flavonoids from leafwort. According to Table 4, compared with the case without the addition of a protectant, the addition of a protectant significantly improves the extraction rates of physalis protein and physalis flavonoids. The optimal protectant is L-cysteine ​​+ vitamin C + vitamin E, which can exert a better synergistic effect, maximizing the extraction rates of both physalis protein and physalis flavonoids. It not only provides excellent protection for physalis protein but also has a good protective effect on flavonoids, exhibiting an excellent dual protection effect.

[0050] Example 6: The effect of the working voltage of low-temperature plasma on the extraction effect This embodiment extracts physalis protein and flavonoids according to the method provided in Example 1. The working voltage of the low-temperature plasma is as shown in Table 5. The effect of using different working voltages for the low-temperature plasma on the extraction yield of physalis protein and flavonoids was investigated. The test results are shown in Table 5.

[0051] Table 5. Effect of low-temperature plasma working voltage on the extraction of protein and flavonoids from leafwort. According to Table 5, when the plasma working voltage is selected at 35KV, the extraction effect of phytoesophageal protein and flavonoids is the highest, with the extraction rate of phytoesophageal protein reaching 92% and the extraction rate of flavonoids reaching 30%.

[0052] While the present invention has been disclosed above, it is not limited thereto. Its applications in medicine can be expanded accordingly. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for simultaneous one-pot extraction of leafwort protein and flavonoids, characterized in that, The leafy grass and DES aqueous solution were mixed and extracted by low-temperature plasma. The DES included a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond acceptor was one of choline chloride, urea, and amino acids. The hydrogen bond donor was any one or more of lactic acid, glucose, glycerol, 1,4-butanediol, polyethylene glycol, urea, n-propanol, and propylene glycol.

2. The method as described in claim 1, characterized in that, The DES also includes a protective agent, which includes any one or more of vitamin C, vitamin E, L-cysteine, dithiothreitol, and propyl gallate.

3. The method as described in claim 2, characterized in that, The hydrogen bond acceptor is choline chloride, and the hydrogen bond donors are glycerol and lactic acid.

4. The method as described in claim 3, characterized in that, The molar ratio of hydrogen bond acceptor to hydrogen bond donor in the DES is 1:(1-5).

5. The method as described in claim 1, characterized in that, The edible grass is edible grass powder, and the edible grass powder is mixed with DES at a solid-liquid ratio of 1g:(5-50)mL.

6. The method as described in claim 1, characterized in that, The discharge mode of the low-temperature plasma is selected from any one or more of the following: glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge.

7. The method as described in claim 6, characterized in that, The working voltage of the low-temperature plasma is 15-45V, and the processing time is 1-4 minutes; the working gas of the low-temperature plasma is selected from any one or more of argon, helium, and air.

8. The method as described in claim 1, characterized in that, It also includes adding an antisolvent to the crude extract obtained from the extraction process to precipitate the phytoestrogen protein, thereby achieving solid-liquid separation of the phytoestrogen protein and flavonoids.

9. A type of leafwort protein or leafwort flavonoid, characterized in that, Prepared using the method described in any one of claims 1 to 8.

10. The use of a composition for preparing a reagent for the one-pot simultaneous extraction of leafwort protein and flavonoids, characterized in that, The composition includes choline chloride, lactic acid, glycerol, and a protectant for preparing DES; the one-pot method for simultaneous extraction of leafwort protein and flavonoids uses low-temperature plasma combined with DES for extraction; the protectant includes any one or more of vitamin C, vitamin E, L-cysteine, dithiothreitol, and propyl gallate; the DES is required to simultaneously extract leafwort protein and flavonoids with the assistance of low-temperature plasma.

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