Deep eutectic solvent for extracting lotus leaf polyphenol and lotus leaf polyphenol extraction method

Abstract

The invention discloses a deep eutectic solvent for extracting lotus leaf polyphenol and a lotus leaf polyphenol extraction method. The deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond receptor comprises at least one of choline chloride, betaine and L-proline; the hydrogen bond donor comprises at least one of lactic acid, maltose, D-glucose, glycerol and urea. The deep eutectic solvent for extracting the lotus leaf polyphenol has the advantages of high lotus leaf polyphenol extraction efficiency, environment friendliness, high safety and the like. The method for extracting the lotus leaf polyphenol has the advantages of high lotus leaf polyphenol extraction efficiency, less degradation, short extraction time, small environmental influence, high application value and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction, and in particular relates to a deep eutectic solvent for extracting lotus leaf polyphenols and a lotus leaf polyphenol extraction method. Background Art

[0002] Polyphenol compounds in lotus leaves have multiple effects, including antioxidant, anti-inflammatory, antibacterial, hypolipidemic and hypoglycemic effects. Related studies have found that lotus leaf polyphenols have a strong scavenging effect on free radicals such as superoxide anions, hydroxyl radicals and hydrogen peroxide, and their antioxidant capacity is equivalent to that of vitamin C and vitamin E. It can also inhibit the production of inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and prostaglandin E2 (PGE2), thereby reducing inflammatory responses.

[0003] At present, polyphenol compounds are usually extracted from lotus leaves and other medicinal plants using various traditional organic solvents, such as n-hexane, ethyl acetate, chloroform, acetone and methanol. Although these organic solvents have good dissolution and extraction properties, their use conflicts with the concept of green development.

[0004] Deep eutectic solvents are environmentally friendly alternative solvents for extracting polyphenolic compounds. While reducing the environmental pollution caused by the use of traditional solvents, they also improve the efficiency of extracting polyphenolic substances with antioxidant, anti-inflammatory and anti-cancer biological activities from natural resources such as lotus leaves. Therefore, optimizing the extraction process of polyphenolic compounds by deep eutectic solvents is of great significance for reducing chemical waste and pollution and improving the extraction efficiency of polyphenolic compounds. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a deep eutectic solvent for extracting lotus leaf polyphenols and a method for extracting lotus leaf polyphenols. The deep eutectic solvent for extracting lotus leaf polyphenols of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, environmental friendliness and high safety. The method for extracting lotus leaf polyphenols of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, low environmental impact and high application value.

[0006] In the first aspect of the present invention, the present invention proposes a deep eutectic solvent for extracting lotus leaf polyphenols. According to an embodiment of the present invention, the deep eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor includes at least one of choline chloride, betaine and L-proline; the hydrogen bond donor includes at least one of lactic acid, maltose, D-glucose, glycerol and urea. The deep eutectic solvent for extracting lotus leaf polyphenols according to an embodiment of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, environmental friendliness and high safety.

[0007] According to an embodiment of the present invention, the deep eutectic solvent for extracting lotus leaf polyphenols may also have the following additional technical features:

[0008] According to an embodiment of the present invention, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes at least one of lactic acid, maltose, D-glucose and urea.

[0009] According to an embodiment of the present invention, the hydrogen bond acceptor includes betaine, and the hydrogen bond donor includes at least one of maltose, glycerol and urea.

[0010] According to an embodiment of the present invention, the hydrogen bond acceptor includes L-proline, and the hydrogen bond donor includes at least one of maltose, glycerol and urea.

[0011] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-5):(1-5).

[0012] According to an embodiment of the present invention, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes lactic acid.

[0013] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-5).

[0014] According to an embodiment of the present invention, the method for preparing the deep eutectic solvent comprises: mixing and heating the hydrogen bond acceptor and the hydrogen bond donor; the heating treatment is performed at a temperature of 60° C. to 100° C. and for a time of 1 min to 60 min.

[0015] In the second aspect of the present invention, the present invention proposes a method for extracting lotus leaf polyphenols. According to an embodiment of the present invention, the lotus leaf raw material is extracted and treated with the aforementioned deep eutectic solvent for extracting lotus leaf polyphenols to obtain an extract containing lotus leaf polyphenols. The method for extracting lotus leaf polyphenols according to an embodiment of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, low environmental impact and high application value.

[0016] According to an embodiment of the present invention, the above method for extracting lotus leaf polyphenols may also have the following additional technical features:

[0017] According to an embodiment of the present invention, the extraction process comprises: mixing the lotus leaf raw material and a deep eutectic solvent to obtain a mixed liquid; and ultrasonically treating the mixed liquid to obtain an extract containing lotus leaf polyphenols.

[0018] According to an embodiment of the present invention, the solid-liquid ratio of the lotus leaf raw material to the deep eutectic solvent is 1:(10-30), and the unit is g / mL.

[0019] According to an embodiment of the present invention, the lotus leaf raw material includes lotus leaf powder, and the particle size of the lotus leaf powder is no more than 1 mm.

[0020] According to an embodiment of the present invention, the temperature of the ultrasonic treatment is 25°C to 40°C.

[0021] According to an embodiment of the present invention, the power of the ultrasonic treatment is 240W to 700W.

[0022] According to an embodiment of the present invention, the frequency of the ultrasonic treatment is 20 kHz to 50 kHz.

[0023] According to an embodiment of the present invention, the ultrasonic treatment time is 30 min to 90 min.

[0024] In a third aspect of the present invention, the present invention provides a lotus leaf polyphenol extract. According to an embodiment of the present invention, the lotus leaf polyphenol extract is prepared by the aforementioned method. The lotus leaf polyphenol extract according to the embodiment of the present invention has a high lotus leaf polyphenol content, low potential risks to the environment and human health, and has antioxidant, anti-inflammatory, antibacterial, lipid-lowering and blood sugar-lowering effects.

[0025] In a fourth aspect of the present invention, the present invention provides a food, cosmetic or medicine. According to an embodiment of the present invention, the food, cosmetic or medicine comprises the lotus leaf polyphenol extract.

[0026] It should be noted that the characteristics and advantages described above for lotus leaf polyphenol extract are also applicable to this application and will not be repeated here.

[0027] In a fifth aspect of the present invention, the present invention proposes the use of the lotus leaf polyphenol extract in preparing a product. According to an embodiment of the present invention, the food, cosmetic or medicine has at least one of the following uses: anti-oxidation, anti-inflammatory, antibacterial, hypolipidemic and hypoglycemic.

[0028] It should be noted that the characteristics and advantages described above for lotus leaf polyphenol extract are also applicable to this application and will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0030] Figure 1 This is a standard curve diagram of the forin phenol colorimetry method in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0032] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0034] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0035] Terms and Definitions

[0036] In this article, the term “DES” refers to a deep eutectic solvent, which is a new type of green solvent formed by mixing a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA) in a certain proportion under certain conditions and can replace traditional organic solvents.

[0037] As used herein, the term "HBA" refers to a hydrogen bond acceptor, which is a molecule or ion capable of accepting hydrogen bonds, such as quaternary ammonium salts, metal chlorides, amides, etc., and is used in the present invention to prepare a deep eutectic solvent.

[0038] Herein, the term "HBD" refers to a hydrogen bond donor, which is a molecule or ion that can provide hydrogen bonds, such as organic acids, alcohols, amines, etc., and is used to prepare a deep eutectic solvent in the present invention.

[0039] In this article, the term "gallic acid" refers to a polyphenolic compound that is often used as a standard substance for the determination of polyphenol content.

[0040] Deep eutectic solvents

[0041] The present invention proposes a deep eutectic solvent for extracting lotus leaf polyphenols. According to an embodiment of the present invention, the deep eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor includes at least one of choline chloride, betaine and L-proline; the hydrogen bond donor includes at least one of lactic acid, maltose, D-glucose, glycerol and urea. The deep eutectic solvent for extracting lotus leaf polyphenols according to an embodiment of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, environmental friendliness and high safety.

[0042] Lotus leaf polyphenols are a type of secondary metabolites with a polyphenol structure, which are widely present in lotus leaves. The biological activity of lotus leaf polyphenols is closely related to its chemical structure, especially the number of hydrogen bonds and electron distribution characteristics within its molecules. According to the chemical properties of lotus leaf polyphenols, the inventors selected suitable hydrogen bond donors and hydrogen bond acceptors, and prepared deep eutectic solvents. In the deep eutectic solvent, based on the interaction between hydrogen bond donors and hydrogen bond acceptors, medium conditions with excellent solubility properties for polyphenol compounds are formed, achieving efficient and environmentally friendly extraction while maintaining the biological activity of lotus leaf polyphenol extracts.

[0043] According to an embodiment of the present invention, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes at least one of lactic acid, maltose, D-glucose and urea. Thus, the extraction efficiency of lotus leaf polyphenols can be further improved. In some preferred embodiments, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes lactic acid.

[0044] According to an embodiment of the present invention, the hydrogen bond acceptor includes betaine, and the hydrogen bond donor includes at least one of maltose, glycerol and urea. Thus, the extraction efficiency of lotus leaf polyphenols can be further improved.

[0045] According to an embodiment of the present invention, the hydrogen bond acceptor includes L-proline, and the hydrogen bond donor includes at least one of maltose, glycerol and urea. Thus, the extraction efficiency of lotus leaf polyphenols can be further improved.

[0046] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-5):(1-5). Thus, by optimizing the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor, the extraction efficiency of lotus leaf polyphenols can be further improved.

[0047] Illustratively, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.

[0048] According to an embodiment of the present invention, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes lactic acid. In some preferred embodiments, when the hydrogen bond acceptor is choline chloride and the hydrogen bond donor is lactic acid, the extraction efficiency of lotus leaf polyphenols is better.

[0049] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-5). Thus, when the hydrogen bond acceptor is choline chloride and the hydrogen bond donor is lactic acid, the extraction efficiency of lotus leaf polyphenols can be further improved by optimizing the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor.

[0050] Illustratively, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1, 1:2, 1:3, 1:4, 1:5, preferably 1:1 to 1:4, and more preferably 1:2.

[0051] In a specific embodiment, by adding water to the configured deep eutectic solvent, its viscosity can be significantly reduced and the fluidity of the deep eutectic solvent can be enhanced. The deep eutectic solvent can be mixed with water, and the water content of the mixed solution can be 0% to 50%, which can also be used for the extraction of lotus leaf polyphenols.

[0052] According to an embodiment of the present invention, the preparation method of the deep eutectic solvent comprises: mixing the hydrogen bond acceptor and the hydrogen bond donor and performing a heating treatment; the heating treatment temperature is 60°C to 100°C and the time is 1min to 60min. Thus, a stable deep eutectic solvent is prepared, which can be used for the extraction of lotus leaf polyphenols.

[0053] Illustratively, the temperature of the heat treatment is 60°C, 70°C, 80°C, 90°C, 100°C, preferably 80°C.

[0054] Illustratively, the heating treatment time is 1 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, preferably 60 min.

[0055] According to an embodiment of the present invention, when the hydrogen bond acceptor is choline chloride and the hydrogen bond donor is lactic acid, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2, thereby achieving better extraction efficiency of lotus leaf polyphenols.

[0056] method

[0057] The present invention proposes a method for extracting lotus leaf polyphenols. According to an embodiment of the present invention, the lotus leaf raw material is subjected to extraction treatment using the aforementioned deep eutectic solvent for extracting lotus leaf polyphenols to obtain an extract containing lotus leaf polyphenols. The method for extracting lotus leaf polyphenols according to an embodiment of the present invention has the advantages of high lotus leaf polyphenol extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, low environmental impact, and high application value.

[0058] According to an embodiment of the present invention, the extraction process includes: mixing the lotus leaf raw material and the deep eutectic solvent to obtain a mixed liquid; and ultrasonically treating the mixed liquid to obtain an extract containing lotus leaf polyphenols. Thus, the lotus leaf raw material is fully extracted, and the extraction efficiency of lotus leaf polyphenols is further improved.

[0059] According to an embodiment of the present invention, the solid-liquid ratio of the lotus leaf raw material to the deep eutectic solvent is 1:(10-30), in g / mL. Thus, the lotus leaf raw material and the deep eutectic solvent are fully mixed to further improve the extraction efficiency.

[0060] Illustratively, the solid-liquid ratio (in g / mL) of the lotus leaf raw material to the deep eutectic solvent is 1:10, 1:15, 1:20, 1:25, 1:30, preferably 1:30.

[0061] According to an embodiment of the present invention, the lotus leaf raw material includes lotus leaf powder, and the particle size of the lotus leaf powder is not greater than 1 mm, thereby facilitating full mixing so that the solvent can fully act on the lotus leaf and improve the extraction efficiency of lotus leaf polyphenols.

[0062] According to an embodiment of the present invention, the temperature of the ultrasonic treatment is 25° C. to 40° C. This helps to further improve the extraction efficiency of lotus leaf polyphenols and reduce the degradation of lotus leaf polyphenols.

[0063] Illustratively, the temperature of the ultrasonic treatment is 25°C, 30°C, 35°C, 40°C, preferably 30°C.

[0064] According to an embodiment of the present invention, the power of the ultrasonic treatment is 240 W to 700 W. Thus, the extraction efficiency of lotus leaf polyphenols can be further improved.

[0065] Illustratively, the power of the ultrasonic treatment is 240W, 300W, 350W, 400W, 450W, 500W, 550W, 600W, 650W, 700W, preferably 500W.

[0066] According to an embodiment of the present invention, the frequency of the ultrasonic treatment is 20 kHz to 50 kHz, thereby further improving the extraction efficiency of lotus leaf polyphenols.

[0067] Illustratively, the frequency of the ultrasonic treatment is 20 kHz, 25 kHz, 30 kHz, 35 kHz, 40 kHz, 45 kHz, 50 kHz, preferably 40 kHz.

[0068] According to an embodiment of the present invention, the ultrasonic treatment lasts for 30 minutes to 90 minutes, thereby further improving the extraction efficiency of lotus leaf polyphenols.

[0069] Exemplarily, the ultrasonic treatment time is 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, preferably 60 min.

[0070] According to an embodiment of the present invention, when the hydrogen bond acceptor of the deep eutectic solvent is choline chloride and the hydrogen bond donor is lactic acid, the hydrogen bond acceptor and the hydrogen bond donor are configured at a molar ratio of 1:2, and lotus leaf polyphenols are extracted under the conditions of a solid-liquid ratio of 1:30 (g:mL), an ultrasonic time of 60 min, an ultrasonic temperature of 30°C, an ultrasonic power of 500 W, and an ultrasonic frequency of 40 kHz. The lotus leaf polyphenol content in the obtained extract containing lotus leaf polyphenols is the highest, and the lotus leaf polyphenol extraction effect is better.

[0071] Lotus leaf polyphenol extract

[0072] The present invention provides a lotus leaf polyphenol extract. According to an embodiment of the present invention, the lotus leaf polyphenol extract is prepared by the aforementioned method. The lotus leaf polyphenol extract according to the embodiment of the present invention has a high lotus leaf polyphenol content, low potential risks to the environment and human health, and has antioxidant, anti-inflammatory, antibacterial, lipid-lowering and blood sugar-lowering effects.

[0073] Food, cosmetics and medicines

[0074] The present invention provides a food, cosmetic or medicine. According to an embodiment of the present invention, the food, cosmetic or medicine comprises the lotus leaf polyphenol extract.

[0075] It should be noted that the characteristics and advantages described above for lotus leaf polyphenol extract are also applicable to this application and will not be repeated here.

[0076] use

[0077] The present invention proposes the use of the lotus leaf polyphenol extract in preparing products. According to an embodiment of the present invention, the food, cosmetic or medicine has at least one of the following uses: anti-oxidation, anti-inflammatory, antibacterial, hypolipidemic and hypoglycemic.

[0078] It should be noted that the characteristics and advantages described above for lotus leaf polyphenol extract are also applicable to this use and will not be repeated here.

[0079] In the embodiment of the present invention, before extracting polyphenols from lotus leaves, a stable deep eutectic solvent must be prepared first. The preparation of the deep eutectic solvent requires two parts: hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). The specific steps of the naming and preparation method of the deep eutectic solvent are as follows:

[0080] Take hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs), mix them in a certain molar ratio, heat them at 80°C and stir them vigorously under magnetic stirring to form a uniform transparent liquid deep eutectic solvent, and name it the deep eutectic solvent according to the specific names and molar ratios of hydrogen bond acceptors and hydrogen bond donors. 氢键受体名称-氢键供体名称(氢键受体与氢键供体的摩尔比) After cooling, the deep eutectic solvent 氢键受体名称-氢键供体名称(氢键受体与氢键供体的摩尔比) Store at room temperature until needed.

[0081] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0082] Example 1: Feasibility analysis of determining lotus leaf polyphenol content by Folin phenol colorimetry and drawing of standard curve

[0083] Determining the content of lotus leaf polyphenol in lotus leaf polyphenol extract is of great significance for optimizing the extraction process, quality control, biological activity evaluation, product development, etc. The inventors chose to use the Folin phenol colorimetric method to determine the content of lotus leaf polyphenol in lotus leaf polyphenol extract. The specific operation steps are as follows:

[0084] 1. DES 氯化胆碱-乙酰丙酸(1:2) Taking the deep eutectic solvent extraction system as an example, the feasibility of applying the Folin phenol colorimetric method to the deep eutectic solvent extraction system to determine the content of lotus leaf polyphenols was explored.

[0085] (1) Literature review

[0086] Gallic acid has good chemical stability, similarity to the structure of lotus leaf polyphenols, high purity and easy detection, making it an ideal internal standard. The inventor consulted relevant literature and learned that when gallic acid is 0.1 mg, the absorbance is about 0.8, which meets the experimental requirements for exploring the feasibility.

[0087] (2) Preparation of Gallic Acid Solution

[0088] Accurately weigh 0.01g of gallic acid standard, dissolve it fully with distilled water, transfer it to a 10ml brown volumetric flask and make up to volume to obtain a gallic acid solution with a mass concentration of 1mg / mL. Take 1ml of the above 1mg / mL gallic acid solution and 9ml of water, mix well, and obtain a gallic acid solution with a mass concentration of 0.1mg / ml.

[0089] (3) Folin-Ciocalte reagent

[0090] The Folin phenol reagent used in the examples was purchased from Solarbio with the product number F8060.

[0091] (4) Preparation of deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2)

[0092] The deep eutectic solvent DES was carried out with choline chloride as hydrogen bond acceptor (HBAs) and levulinic acid as hydrogen bond donor (HBDs) at a molar ratio of choline chloride to levulinic acid of 1:2. 氯化胆碱-乙酰丙酸(1:2) Configuration.

[0093] (5) Preparation of control solution and determination of absorbance value

[0094] Take 1 ml of 0.1 mg / ml gallic acid solution, add 4 ml of distilled water and 1 ml of Folin phenol reagent, shake thoroughly and keep it away from light for 3 minutes, finally add 4 ml of 0.94 mol / L sodium carbonate solution and shake well, keep it in a constant temperature water bath at 50℃ for 5 minutes, and use an enzyme marker to measure the absorbance A1 at 765nm. Use distilled water without gallic acid as a blank control, and measure the absorbance A 1-0 At this time, the background of the absorbance value measured is distilled water.

[0095] (6) Preparation of 50-fold diluted DES aqueous solution and gallic acid-DES aqueous solution and determination of absorbance value

[0096] Take 1 ml of deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2) Mix with 49 ml of distilled water to obtain a 50-fold diluted DES solution. Accurately weigh 0.001 g of gallic acid standard and dissolve it in 10 ml of 50-fold diluted DES solution to obtain a 0.1 mg / ml gallic acid-DES solution. Use an ELISA reader to measure the absorbance A2 at 765 nm. Using the 50-fold diluted DES solution as a control, measure the absorbance A 2-0 At this time, the absorbance background is DES diluted 50 times.

[0097] (7) Preparation of pure DES aqueous solution and gallic acid-pure DES aqueous solution and determination of absorbance value

[0098] Accurately weigh 0.001g of gallic acid standard and dissolve it in 10ml of deep eutectic solvent DES. 氯化胆碱-乙酰丙酸(1:2) , and a gallic acid-pure DES solution with a mass concentration of 0.1 mg / ml was obtained. The absorbance A3 was measured at 765 nm using an ELISA reader, and the absorbance A was measured using the pure DES solution as a control. 3-0 At this time, the absorbance value background is pure DES.

[0099] Deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2) Taking the extraction system as an example, the feasibility of applying the Folin phenol colorimetric method to determine the content of lotus leaf polyphenols was explored, and the results are shown in Table 1.

[0100] Table 1 Deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2) The feasibility of using the Folin phenol colorimetric method to determine the content of lotus leaf polyphenols was explored using the extraction system as an example.

[0101]

[0102] The experimental results show that when the background of 0.1 mg gallic acid is DES diluted 50 times, the absorbance value measured by this method is reasonable and normal. This method for measuring polyphenol content is highly applicable and easy to implement, and can be used to study the extraction of polyphenols in lotus leaves by deep eutectic solvents.

[0103] 2. Drawing of the standard curve of Folin phenol colorimetry

[0104] Accurately weigh 0.1g of gallic acid, fully dissolve it in 50mL of distilled water, mix well, and then transfer it to a 100mL volumetric flask to make up to volume to obtain a gallic acid solution with a mass concentration of 1mg / mL. Take 0, 1.0, 2.0, 3.0, 4.0, and 5.0mL from it and transfer it to a 50mL brown volumetric flask to make up to volume. Take 1mL of each gallic acid standard solution of different mass concentrations and transfer it to a test tube, add 4.0mL of distilled water and 1mL of Folin phenol reagent respectively, shake well and let it stand for 3min, and finally add 4.0mL of sodium carbonate solution with a molar concentration of 0.94mol / L, shake well, and keep it in a constant temperature water bath at 50℃ for 5min. Use the standard solution without gallic acid as the blank control, and use an enzyme reader to measure the absorbance value at 765nm and record it.

[0105] The standard curve of Folin phenol colorimetry is shown in Figure 1 .

[0106] Example 2: Screening of deep eutectic solvents (DES)

[0107] 1. Preparation of deep eutectic solvent

[0108] Based on the results of literature review, the inventors preliminarily determined that the following deep eutectic solvent configuration can be used for the extraction of lotus leaf polyphenols. The systems involved in the deep eutectic solvent configuration are shown in Table 2.

[0109] Table 2 Configuration and name of deep eutectic solvents

[0110]

[0111]

[0112] 2. Pretreatment of lotus leaf raw materials

[0113] Put a sufficient amount of lotus leaves into an oven and dry them at 60°C for 24 hours. Use a grinder to crush the lotus leaves to obtain lotus leaf dry powder. Pass the lotus leaf dry powder through a molecular sieve with a pore diameter of 0.425 mm (40 mesh). The lotus leaf fine powder treated with the 40-mesh molecular sieve is packed in a sealed bag and sealed for later use.

[0114] 3. Extraction of lotus leaf polyphenols

[0115] Accurately weigh 19 portions of 0.50 g lotus leaf fine powder and soak them in 10 mL of candidate deep eutectic solvent respectively, and vortex them vigorously for 30 seconds until a uniform thick mixture is obtained. The uniform thick mixture is ultrasonicated for 30 minutes at a temperature of 30°C, an ultrasonic power of 140 W, and an ultrasonic frequency of 40 kHz to obtain a lotus leaf mixed solution. The lotus leaf mixed solution is centrifuged at a speed of 8800 rpm for 6 minutes, and the supernatant is collected to obtain a lotus leaf polyphenol extract.

[0116] 4. Determination of absorbance by Folin phenol colorimetry

[0117] Each group took 0.1mL of lotus leaf polyphenol extract, added 4.9mL of distilled water to obtain lotus leaf polyphenol extract samples, then added 1mL of Folin phenol reagent, shook thoroughly and let stand for 3 minutes, and finally added 4.0mL of sodium carbonate solution with a molar concentration of 0.94mol / L, shook well, and kept in a constant temperature water bath at 50℃ for 5 minutes to obtain the test solution. The blank control was added with distilled water, and the absorbance value was repeatedly measured 3 times at 765nm using an enzyme marker and recorded as A1, A2, and A3 respectively, and the average absorbance value was recorded as A 平均 The average absorbance value of the blank control group is recorded as A 平均(空白) .

[0118] 5. Calculation of total lotus leaf polyphenol content

[0119] Based on the average absorbance value A 平均 The absorbance values ​​of the blank control group were calculated according to A 平均-空白 =A 平均 -A 平均(空白) and the standard curve equation of Folin phenol colorimetry ( Figure 1 ), calculate the lotus leaf polyphenol content X1 in the test solution.

[0120] The lotus leaf polyphenol content in the lotus leaf polyphenol extract was calculated based on the following formula:

[0121] Total polyphenol content (mg / g) = (X1×V1×V2) / W

[0122] Wherein: X1 is the concentration of lotus leaf polyphenols in the test solution (mg / mL), V1 is the dilution multiple of the lotus leaf polyphenol extract, V2 is the total volume of the lotus leaf polyphenol extract (mL), and W is the dry mass of the raw material of lotus leaf fine powder (g).

[0123] The extraction results of lotus leaf polyphenols using different deep eutectic solvent extraction systems are shown in Table 3.

[0124] Table 3 Extraction results of lotus leaf polyphenols in different deep eutectic solvent extraction systems

[0125]

[0126]

[0127] The experimental results show:

[0128] Among the candidate deep eutectic solvents, when the hydrogen bond acceptor is choline chloride and the hydrogen bond donor is one of lactic acid, maltose, D-glucose, and urea, the total polyphenol extraction content is higher; when the hydrogen bond acceptor is betaine and the hydrogen bond donor is one of maltose, glycerol, and urea, the total polyphenol extraction content is higher; when the hydrogen bond acceptor is L-proline and the hydrogen bond donor is one of maltose, glycerol, and urea, the total polyphenol extraction content is higher. Among them, the selected deep eutectic solvent is DES 氯化胆碱-乳酸(1:2) When , the total polyphenol extraction content was the highest, which was 84.52060359 mg / g, which was much higher than the total polyphenol content level of lotus leaf polyphenols extracted using other deep eutectic solvent systems.

[0129] Example 3: Screening of methods for extracting lotus leaf polyphenols using deep eutectic solvents

[0130] In order to improve the extraction efficiency of lotus leaf polyphenols by deep eutectic solvent, the inventors used deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2) Taking the extraction system as an example, the means of assisting deep eutectic solvents in extracting lotus leaf polyphenols were screened. The specific operation steps are as follows:

[0131] 1. Pretreatment of lotus leaf raw materials

[0132] Put a sufficient amount of lotus leaves into an oven and dry them at 60°C for 24 hours. Use a grinder to crush the lotus leaves to obtain lotus leaf dry powder. Pass the lotus leaf dry powder through a molecular sieve with a pore diameter of 0.425 mm (40 mesh). The lotus leaf fine powder treated with the 40-mesh molecular sieve is packed in a sealed bag and sealed for later use.

[0133] 2. Prepare deep eutectic solvent DES 氯化胆碱-乙酰丙酸(1:2)

[0134] The deep eutectic solvent DES was carried out with choline chloride as hydrogen bond acceptor (HBAs) and levulinic acid as hydrogen bond donor (HBDs) at a molar ratio of choline chloride to levulinic acid of 1:2. 氯化胆碱-乙酰丙酸(1:2) Configuration.

[0135] 3. Setting up the processing group

[0136] Choosing ultra-high pressure and ultrasound for DES 氯化胆碱-乙酰丙酸(1:2) The extraction system was processed, and the specific parameter settings are shown in Table 4.

[0137] Treatment group 1: ultrahigh pressure (300 MPa, 10 min) + ultrasound (ultrasonic power: 500 W; ultrasonic frequency: 40 KHz; ultrasonic temperature 30°C; ultrasonic time 10 min).

[0138] Treatment group 2: Same as treatment group 1, except that no ultrasonic treatment was performed.

[0139] Treatment group 3: Same as treatment group 1, except that no ultrahigh pressure treatment was performed.

[0140] Treatment Group 4: None.

[0141] Table 4 Auxiliary means condition parameter settings

[0142]

[0143] 4. Determination of absorbance by Folin phenol colorimetry and calculation of total lotus leaf polyphenol content

[0144] The polyphenol content in the extract obtained from each treatment group was determined respectively. The specific determination and calculation methods refer to steps 4 to 5 of Example 2.

[0145] The measurement results are shown in Table 4.

[0146] Table 4 Effects of ultrasound and ultrahigh pressure assisted extraction on DES 氯化胆碱-乙酰丙酸(1:2) Effect of extraction system on polyphenol extraction efficiency

[0147]

[0148] The results showed that the absorbance values ​​were ranked as ultrasound only > ultrahigh pressure + ultrasound > none > ultrahigh pressure only.

[0149] The above results show that ultrahigh pressure may destroy the hydrogen bonds formed by components in the deep eutectic solvent, so it has no obvious improvement on the extraction efficiency of polyphenols, while ultrasound-assisted extraction significantly improves the extraction efficiency of polyphenols. Therefore, ultrasound-assisted deep eutectic solvent extraction system was selected to extract lotus leaf polyphenols.

[0150] Example 4: Ultrasound-assisted deep eutectic solvent extraction of lotus leaf polyphenols

[0151] 1. Preparation and processing of lotus leaves

[0152] Put a sufficient amount of lotus leaves into an oven and dry them at 60°C for 24 hours. Use a grinder to crush the lotus leaves to obtain lotus leaf dry powder. Pass the lotus leaf dry powder through a molecular sieve with a pore diameter of 0.425 mm (40 mesh). The lotus leaf fine powder treated with the 40-mesh molecular sieve is packed in a sealed bag and sealed for later use.

[0153] 2. Preparation of deep eutectic solvent

[0154] The deep eutectic solvent DES was carried out with choline chloride as hydrogen bond acceptor (HBAs) and lactic acid as hydrogen bond donor (HBDs) at a molar ratio of choline chloride to lactic acid of 1:1. 氯化胆碱-乳酸(1:1) Configuration.

[0155] 3. Extraction of lotus leaf polyphenols

[0156] Accurately weigh 0.5000g lotus leaf powder and add it to 10mL DES 氯化胆碱-乳酸(1:1) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, with a solid-liquid ratio (g: mL) of 1:20 (Note: solid-liquid ratio = lotus leaf fine powder raw material (g): deep eutectic solvent (mL)), extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0157] 4. Determination of absorbance by Folin phenol colorimetry and calculation of total lotus leaf polyphenol content

[0158] Determine the polyphenol content in the lotus leaf polyphenol extract containing lotus leaf polyphenol. For specific determination and calculation methods, refer to steps 4 to 5 of Example 2.

[0159] Examples 5-18: Optimization of Ultrasound-Assisted Deep Eutectic Solvent Extraction Conditions for Lotus Leaf Polyphenols

[0160] Based on the selection of choline chloride and lactic acid as HBA and HBD in the deep eutectic solvents for extracting lotus leaf polyphenols, the inventors used single factor experiments to further investigate the effects of the molar ratio of HBD and HBA, the solid-liquid ratio, and the extraction time on the extraction content level of lotus leaf polyphenols.

[0161] The differences between Examples 5 to 23 and Example 4 are shown in Table 5, wherein:

[0162] 1. Optimization of the molar ratio of HBD and HBA

[0163] In Examples 4 to 8, the inventors optimized the molar ratio of HBD to HBA. The specific difference between Examples 5 to 8 and Example 4 is step 2, as follows:

[0164] In Example 5, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is lactic acid, and the molar ratio of choline chloride to lactic acid is 1:2 to perform deep eutectic solvent DES. 氯化胆碱-乳酸(1:2) Configuration.

[0165] In Example 6, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is lactic acid, and the molar ratio of choline chloride to lactic acid is 1:3. 氯化胆碱-乳酸(1:3) Configuration.

[0166] In Example 7, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is lactic acid, and the molar ratio of choline chloride to lactic acid is 1:4, and a deep eutectic solvent DES is performed. 氯化胆碱-乳酸(1:4) Configuration.

[0167] In Example 8, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is lactic acid, and the molar ratio of choline chloride to lactic acid is 1:5, and a deep eutectic solvent DES is performed. 氯化胆碱-乳酸(1:5) Configuration.

[0168] 2. Optimization of material-liquid ratio

[0169] In Examples 9 to 13, the inventors optimized the material-liquid ratio. The specific difference between Examples 9 to 13 and Example 4 lies in step 3, as follows:

[0170] In Examples 9 to 13, the hydrogen bond acceptor (HBAs) was choline chloride, the hydrogen bond donor (HBDs) was lactic acid, and the molar ratio of choline chloride to lactic acid was 1:4, and deep eutectic solvent DES was performed. 氯化胆碱-乳酸(1:4) Configuration.

[0171] In Example 9, 1.0000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:10, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0172] In Example 10, 0.6667 g lotus leaf powder was accurately weighed and added with 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:15, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0173] In Example 11, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0174] In Example 12, 0.4000 g of lotus leaf powder was accurately weighed and added to 10 mL of DES 氯化胆碱-乳酸(1:4)In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:25, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0175] In Example 13, 0.3333 g of lotus leaf powder was accurately weighed and added to 10 mL of DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:30, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0176] 3. Optimization of extraction time

[0177] In Examples 14 to 18, the inventors optimized the ultrasonic extraction time. The specific differences between Examples 14 to 18 and Example 4 are step 2 and step 3, as follows:

[0178] In Examples 14 to 18, the hydrogen bond acceptor (HBAs) was choline chloride, the hydrogen bond donor (HBDs) was lactic acid, and the molar ratio of choline chloride to lactic acid was 1:4, and deep eutectic solvent DES was performed. 氯化胆碱-乳酸(1:4) Configuration.

[0179] In Example 14, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 20 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0180] In Example 15, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 30 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0181] In Example 16, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 40 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0182] In Example 17, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 50 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0183] In Example 18, 0.5000 g lotus leaf powder was accurately weighed and added to 10 mL DES 氯化胆碱-乳酸(1:4) In a deep eutectic solvent, under an ultrasonic-assisted extraction method with an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz, the solid-liquid ratio (g:mL) of lotus leaf fine powder and deep eutectic solvent was 1:20, and extraction was carried out at 30°C for 60 min to obtain a lotus leaf polyphenol extract containing lotus leaf polyphenols.

[0184] Table 5 Setting of extraction conditions for lotus leaf polyphenols by ultrasound-assisted deep eutectic solvent extraction

[0185]

[0186] In Examples 4 to 18, the results of the ultrasound-assisted deep eutectic solvent extraction of lotus leaf polyphenols are shown in Table 6.

[0187] Table 6 Ultrasound-assisted deep eutectic solvent extraction results of lotus leaf polyphenols

[0188]

[0189]

[0190] The results show:

[0191] (1) In Examples 4 to 8, under the same conditions of lotus leaf fine powder mass, lotus leaf polyphenol extraction system solid-liquid ratio, and extraction time, when the molar ratio of HBD to HBA was 1:1 to 1:4, the extraction efficiency of lotus leaf polyphenols was better than that of HBD to HBA at a molar ratio of 1:5, among which the extraction effect of the molar ratio of 1:2 was better, which was 143.1545 mg / g.

[0192] (2) In Examples 9 to 13, under the same molar ratio of HBD to HBA and extraction time, when the solid-liquid ratio was 1:20 to 1:30, the extraction efficiency of lotus leaf polyphenols was better than that when the solid-liquid ratio was 1:10 to 1:20, among which the extraction effect of the solid-liquid ratio of 1:30 was even better, which was 99.6730 mg / g.

[0193] (3) In Examples 14 to 18, when the mass of lotus leaf fine powder, the molar ratio of HBD to HBA, and the extraction time were the same and 60 min, the extraction efficiency of lotus leaf polyphenols was better than that of other extraction times, which was 135.6773 mg / g.

[0194] The examples given in the invention are not intended to limit the implementation methods of the invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here, and the obvious changes or modifications derived from them are still within the scope of protection of the invention. It can be understood that the above embodiments are exemplary and cannot be understood as limitations of the invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the invention.

Claims

1. A deep eutectic solvent for extracting lotus leaf polyphenols, characterized in that: The deep eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; The hydrogen bond acceptor comprises at least one of choline chloride, betaine and L-proline; The hydrogen bond donor includes at least one of lactic acid, maltose, D-glucose, glycerol and urea.

2. The deep eutectic solvent according to claim 1, characterized in that The hydrogen bond acceptor comprises choline chloride, and the hydrogen bond donor comprises at least one of lactic acid, maltose, D-glucose and urea; Optionally, the hydrogen bond acceptor comprises betaine, and the hydrogen bond donor comprises at least one of maltose, glycerol and urea; Optionally, the hydrogen bond acceptor comprises L-proline, and the hydrogen bond donor comprises at least one of maltose, glycerol and urea; Optionally, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-5):(1-5).

3. The deep eutectic solvent according to claim 1, characterized in that The hydrogen bond acceptor comprises choline chloride, and the hydrogen bond donor comprises lactic acid; Optionally, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-5).

4. The deep eutectic solvent according to claim 1, characterized in that The preparation method of the deep eutectic solvent comprises: The hydrogen bond acceptor and the hydrogen bond donor are mixed and heated; The temperature of the heating treatment is 60° C. to 100° C., and the time is 1 min to 60 min.

5. A method for extracting lotus leaf polyphenols, characterized in that: include: The deep eutectic solvent for extracting lotus leaf polyphenols according to any one of claims 1 to 4 is used to extract lotus leaf raw materials to obtain an extract containing lotus leaf polyphenols.

6. The method according to claim 5, characterized in that The extraction process comprises: The lotus leaf raw material and the deep eutectic solvent are mixed to obtain a mixed liquid; The mixed liquid is subjected to ultrasonic treatment to obtain an extract containing lotus leaf polyphenols.

7. The method according to claim 5, characterized in that The solid-liquid ratio of the lotus leaf raw material to the deep eutectic solvent is 1:(10-30), and the unit is g / mL.

8. The method according to claim 5, characterized in that The lotus leaf raw material includes lotus leaf powder, and the particle size of the lotus leaf powder is no more than 1 mm.

9. The method according to claim 6, characterized in that The temperature of the ultrasonic treatment is 25°C to 40°C; Optionally, the power of the ultrasonic treatment is 240W to 700W; Optionally, the frequency of the ultrasonic treatment is 20kHz to 50kHz; Optionally, the ultrasonic treatment time is 30 min to 90 min.

10. A lotus leaf polyphenol extract, characterized in that: The lotus leaf polyphenol extract is prepared by the method according to any one of claims 5 to 9.

11. A food, cosmetic or medicine, characterized in that: Including the lotus leaf polyphenol extract according to claim 10.

12. Use of the lotus leaf polyphenol extract according to claim 10 in preparing a product, characterized in that: The food, cosmetic or medicine has at least one of the following uses: anti-oxidation, anti-inflammatory, antibacterial, lipid-lowering and blood sugar-lowering.

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