A method for extracting polysaccharide and polyphenol from litchi pulp based on ternary eutectic solvent

Abstract

The present application relates to a lychee pulp polysaccharide and polyphenol extraction method based on a ternary eutectic solvent, belonging to the technical field of natural product extraction. The method uses a chlorocholine-L-proline-lactic acid ternary food-grade eutectic solvent, combined with ultrasonic and microwave double-field synergistic extraction technology, to realize efficient synergistic extraction of lychee pulp polysaccharide and polyphenol under mild conditions. The extract is purified by gradient ethanol precipitation in steps to obtain high-purity polysaccharide and high-purity polyphenol. The viscosity of the ternary system is greatly reduced compared to the binary system, and the polysaccharide yield and polyphenol yield are high, and the extraction time is greatly shortened. All solvent components are food-grade safe raw materials, and the extract has no solvent residue risk, and can be widely used in the fields of food, cosmetics and health products.

Description

Technical Field

[0001] This invention belongs to the technical field of natural product extraction, and more specifically, relates to a method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent. Background Technology

[0002] Lychee pulp not only possesses a unique flavor and texture but is also rich in various bioactive components such as polysaccharides and polyphenols. Studies have shown that lychee pulp polysaccharides exhibit a variety of biological activities, including antioxidant, immunomodulatory, and antitumor effects; while lychee pulp polyphenols demonstrate significant antioxidant, anti-inflammatory, and skin-whitening effects. Therefore, the efficient extraction of polysaccharides and polyphenolic active ingredients from lychee pulp is of great significance.

[0003] Traditional methods for extracting active ingredients from litchi pulp mainly include hot water extraction, organic solvent extraction, ultrasound-assisted extraction, and enzyme-assisted extraction. Hot water extraction is the most traditional method, but its extraction rate is low (usually <8%), it is time-consuming (3-5 hours), and high temperatures can easily lead to the degradation of heat-sensitive active ingredients. While organic solvent extraction has improved extraction efficiency, it suffers from the problem of toxic residues, making it unsuitable for the food and cosmetic industries. Ultrasound-assisted extraction can increase the extraction rate to around 12-15%, but the single ultrasonic field has limited impact on cell wall damage, leaving considerable room for improvement in extraction efficiency. Enzyme-assisted extraction, while offering the advantage of mild conditions, involves expensive enzyme preparations and extremely stringent requirements for reaction conditions (pH, temperature, time), hindering large-scale industrial production.

[0004] Eutectic solvents (DES) are a class of eutectic mixtures formed by hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) through hydrogen bonding. Compared with traditional ionic liquids, DES have advantages such as wide availability of raw materials, low cost, simple synthesis, good biodegradability, and low toxicity, and are considered to be a new generation of green solvents.

[0005] In the prior art, Chinese patent application CN202510518925.3 discloses a one-step extraction method for polysaccharides and polyphenols from litchi pulp based on a eutectic solvent. This method uses a choline chloride-lactic acid (ChCl-LA) binary DES system. After one-step extraction, the supernatant is centrifuged to extract polyphenols, and the precipitate is washed with water to extract polysaccharides. However, this method has the following shortcomings: (1) The viscosity of the binary DES system is high (about 850 mPa·s), resulting in large mass transfer resistance, which limits the extraction efficiency and requires an extraction time of more than 60 minutes; (2) The polyphenol extraction selectivity is insufficient, and the polysaccharides and polyphenols interfere with each other during co-extraction, resulting in low purity of the two products, with the polysaccharide purity being only about 65%; (3) There is a lack of effective stepwise purification methods, and the product quality is difficult to meet the needs of high-end applications. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low extraction efficiency, insufficient product purity, and poor solvent safety of existing technologies for active ingredients from litchi pulp, and to provide a method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent.

[0007] The objective of this invention can be achieved through the following technical solutions: The first aspect of this invention provides a method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent, comprising the following steps: S1. Mix choline chloride, L-proline and lactic acid in a molar ratio of 1:(0.8-1.2):(1.5-2.5) and stir under heating conditions until a homogeneous and transparent liquid is formed to obtain a ternary eutectic solvent. Add water to adjust the water content to 20-40% (w / w). S2. Fresh lychee pulp is freeze-dried, pulverized, and sieved to obtain lychee pulp powder. S3. Mix the litchi pulp powder obtained in step S2 with the ternary eutectic solvent obtained in step S1 at a material-to-liquid ratio of 1:(20-40)g / mL, and extract for 20-40 minutes under the action of ultrasonic and microwave dual fields with an ultrasonic power of 200-350W, a microwave power of 100-250W, and an extraction temperature of 40-60℃ to obtain the extraction mixture. S4. Centrifuge the extract mixture obtained in step S3 and collect the supernatant; extract the residue again with water and combine the extracts. S5. Concentrate the combined extract under reduced pressure, add ethanol for gradient precipitation, centrifuge to collect the crude polysaccharide precipitate, take the supernatant and add ethanol to precipitate polyphenols, collect the crude polyphenol precipitate.

[0008] Furthermore, the heating conditions in step S1 are 60-70℃, the stirring rate is 300-500rpm, and the stirring time is 30-60min; the water content is 25-35% (w / w).

[0009] Furthermore, in step S2, the freeze-drying temperature is -40℃ to -60℃, and the drying time is 24-48h; the sieving is done through a 40-60 mesh sieve.

[0010] Furthermore, the material-to-liquid ratio in step S3 is 1:(25-35)g / mL.

[0011] Furthermore, the conditions for the dual-field action of ultrasound and microwave in step S3 are as follows: ultrasound power 200-300W, ultrasound frequency 20-40kHz; microwave power 100-200W; extraction temperature 45-55℃, extraction time 25-35min.

[0012] Furthermore, the centrifugation conditions described in step S4 are: rotation speed 4000-6000 rpm, centrifugation time 10-20 min; add water to the residue at a material-to-liquid ratio of 1:(15-25) g / mL, and extract again under the same conditions, for 2-3 extractions.

[0013] Furthermore, the specific steps of step S5 are as follows: S51. Concentrate the combined extracts under reduced pressure at 40-50℃ to 1 / 4-1 / 3 of the original volume to obtain a concentrated solution. S52. Add anhydrous ethanol to the concentrate to make the final ethanol concentration reach 55%-65% (v / v), and let it stand at 4℃ for 12-24h to precipitate polysaccharides. S53. Centrifuge to collect the polysaccharide precipitate, wash with anhydrous ethanol, and vacuum dry to obtain crude polysaccharide; S54. Collect the supernatant after centrifugation in step S53, and continue to add anhydrous ethanol to make the final ethanol concentration reach 80%-90% (v / v). Let it stand at 4℃ for 12-24h to precipitate polyphenols. S55. Centrifuge to collect the polyphenol precipitate, and vacuum dry to obtain crude polyphenol product.

[0014] Furthermore, in step S55, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 10-15 min.

[0015] A second aspect of the present invention provides a litchi pulp polysaccharide extracted by the above method.

[0016] The polysaccharides include glucose, galactose, arabinose, and rhamnose.

[0017] A third aspect of the present invention provides the use of the polysaccharide in the preparation of antioxidant foods, functional foods or cosmetics.

[0018] A fourth aspect of the present invention provides a litchi pulp polyphenol extracted by the above method.

[0019] The polyphenols include proanthocyanidins B2, epicatechin, quercetin, and anthocyanin compounds.

[0020] The fifth aspect of this invention provides the application of the lychee pulp polyphenols in the preparation of antioxidant agents, whitening cosmetics, or functional beverages.

[0021] The sixth aspect of the present invention provides a complex active extract of lychee pulp polysaccharide-polyphenol obtained by the above method.

[0022] The polysaccharide-polyphenol complex active extract of litchi pulp contains 60-75% (w / w) polysaccharide and 15-25% (w / w, based on gallic acid equivalent), with the remainder being water and trace minerals. The polysaccharide in this complex extract is mainly a heteropolysaccharide composed of glucose, galactose, arabinose and rhamnose, and the polyphenols mainly include proanthocyanidin B2, epicatechin, quercetin and anthocyanin compounds.

[0023] Furthermore, the DPPH free radical scavenging rate IC of this compound extract was [not specified]. 50 ≤30μg / mL, ABTS free radical scavenging rate IC50 50 ≤10μg / mL.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention innovatively employs a ternary DES system of choline chloride (ChCl), L-proline (L-Pro), and lactic acid (LA), wherein the introduction of L-proline as an amino acid hydrogen bond donor (HBD) endows the system with a unique synergistic effect. L-proline contains a pyrrolidine ring structure, and its secondary amine group can form an additional hydrogen bond network with the hydroxyl groups in polysaccharide molecules, significantly enhancing the solubility of DES for polysaccharides. At the same time, the pyrrolidine ring structure of L-proline can form an additional hydrogen bond network with the hydroxyl groups in polysaccharide molecules, significantly enhancing the solubility of DES for polysaccharides; and, as a hydrogen bond donor and acceptor, L-proline can effectively improve the solubility of the ternary DES for both polysaccharides and polyphenols. Furthermore, the introduction of L-proline breaks the strong hydrogen bond network between choline chloride and lactic acid in the binary DES, reducing the density of intermolecular forces and significantly lowering the viscosity of the system to 420-500 mPa·s (about 40-50% lower than the approximately 850 mPa·s of the binary ChCl-LA system). This greatly reduces mass transfer resistance, increases the solvent penetration rate into plant cells, and also facilitates subsequent centrifugation and filtration.

[0025] (2) This invention employs a dual-field synergistic extraction mechanism combining ultrasound and microwave. The cavitation effect of ultrasound generates numerous microbubbles in the extract. Upon collapse, these bubbles generate localized high temperatures and pressures (approximately 5000 K, 1000 atm) and intense shock waves, effectively disrupting the cell walls and cell membranes of the litchi pulp, forming microchannels, and creating favorable conditions for solvent penetration and solute release. The thermal effect of microwaves rapidly raises the temperature of polar molecules (water, DES, etc.) within the cells through dielectric heating, generating a temperature gradient and pressure difference from the inside out, further promoting the release of active substances such as polysaccharides and polyphenols from the cells to the outside. The synergistic effect of the two physical fields is far superior to the superposition effect of a single physical field, achieving a synergistic effect of "1+1>2".

[0026] (3) This invention employs a gradient ethanol precipitation stepwise purification mechanism, utilizing the difference in solubility of polysaccharides and polyphenols at different ethanol concentrations to achieve efficient stepwise recovery of the two types of substances. Polysaccharides precipitate at low ethanol concentrations (approximately 60% v / v) because their macromolecular structure causes a sharp decrease in solubility in an environment with reduced polarity; while polyphenols form strong hydrogen bonds with DES, maintaining their solubility at low ethanol concentrations; when the ethanol concentration is increased to 85%, the high concentration of ethanol disrupts the hydrogen bond interaction between DES and polyphenols, causing polyphenols to lose the solubilizing effect of DES and precipitate. This gradient precipitation strategy based on solubility differences avoids the product cross-contamination problem caused by the co-precipitation of polysaccharides and polyphenols in traditional methods, and significantly improves product quality.

[0027] (4) The three components of the ternary DES of the present invention—choline chloride (a vitamin B4 derivative), L-proline (a natural amino acid), and lactic acid (a food acidulant)—are all food-grade safe raw materials. The extraction process does not involve any toxic or harmful organic solvents, and there is no risk of solvent residue in the product. It can be directly applied to food, cosmetics, health products, and other fields.

[0028] (5) Although the ternary DES system of this invention introduces one more component (L-proline) compared to the binary DES system, the energy consumption and labor costs per unit yield are significantly reduced due to the substantial improvement in extraction efficiency and the significant reduction in extraction time. Simultaneously, the stepwise ethanol precipitation technology significantly improves the recovery rate of polysaccharides and polyphenols, reducing the loss of active substances. Furthermore, the DES solvent can be recovered and reused through vacuum distillation, further reducing raw material costs. In summary, the overall production cost of this invention is significantly lower than that of the binary DES method.

[0029] (6) Thanks to the mild extraction conditions (45-55℃) and the stabilizing effect of ternary DES on polyphenols, the polysaccharides and polyphenols extracted in this invention maintain high bioactivity. The antioxidant activity of both polysaccharides and polyphenols is superior to that of products extracted by traditional methods, and the tyrosinase inhibition rate of polyphenols is ≥60%, showing good potential for whitening applications. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments, but the scope of protection of this invention is not limited thereto. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, all reagents and materials used are commercially available.

[0031] Example 1 A method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent, the specific steps of which are as follows: S1. Weigh out 83.8 g (0.60 mol) of choline chloride (ChCl), 69.1 g (0.60 mol) of L-proline (L-Pro), and 108.1 g (1.20 mol) of lactic acid according to a molar ratio of 1:1:2. Mix the mixture and place it in a 70°C water bath. Stir at 400 rpm for 40 min to form a homogeneous and transparent liquid. Add 108 mL of deionized water to adjust the water content to 30% (w / w), stir evenly, and obtain approximately 370 mL of ternary DES solution (density approximately 1.20 g / mL). Cool to room temperature for later use. The viscosity of this ternary DES solution was measured to be approximately 450 mPa·s (25°C, rotational viscometer method).

[0032] S2. Take fresh lychee pulp, remove the pits, and place it in a freeze dryer. Dry it for 36 hours at -50℃ and a vacuum degree ≤20Pa. After drying, pulverize the lychee pulp with a high-speed grinder, pass it through a 50-mesh sieve, collect the powder, and seal it in a desiccator.

[0033] S3. Weigh 10g of lychee pulp powder and add it to 300mL of the ternary DES solution prepared in step S1 at a material-to-liquid ratio of 1:30 (g / mL). Place the solution in an ultrasonic and microwave synergistic extraction instrument. Set the ultrasonic power to 250W (frequency 30kHz), the microwave power to 150W, the extraction temperature to 50℃, and the extraction time to 30min.

[0034] S4. After extraction, centrifuge the extract at 5000 rpm for 15 min and collect the supernatant (approximately 275 mL for the first supernatant). Add 200 mL of deionized water to the residue at a material-to-liquid ratio of 1:20 (g / mL, based on the dry weight of the raw material), and extract again for 20 min under the same conditions. Centrifuge at 5000 rpm for 15 min and collect the supernatant (approximately 190 mL for the second supernatant). Combine the two supernatants, with a total volume of approximately 465 mL.

[0035] S5. Stepwise purification and recovery: S51. The combined extract was concentrated under reduced pressure at 45℃ and -0.08MPa to 1 / 3 of the original volume (approximately 155mL). S52. Slowly add anhydrous ethanol to the concentrate while stirring, until the final ethanol concentration reaches 60% (v / v), then transfer to a 4°C refrigerator and let stand for 18 hours to precipitate the polysaccharide. S53. Centrifuge at 5000 rpm for 12 min, collect the precipitate, wash twice with anhydrous ethanol, and dry in a vacuum drying oven at 50℃ until constant weight, yielding 5.42 g of crude polysaccharide. S54. Collect the supernatant from step S53, and continue to add anhydrous ethanol to achieve a final concentration of 85% (v / v). Let it stand at 4°C for 18 hours. S55. Centrifuge at 5000 rpm for 12 min, collect the precipitate, and vacuum dry to constant weight to obtain 0.48 g of crude polyphenol.

[0036] Example 2 This embodiment is basically the same as Embodiment 1, except that: In step S1 of this embodiment, during the preparation of ternary DES, the molar ratio of ChCl, L-Pro, and LA is 1:0.8:1.5. 83.8g (0.60mol) of choline chloride, 55.3g (0.48mol) of L-proline, and 67.6g (0.75mol) of lactic acid are weighed out, and 90mL of deionized water is added to adjust the water content to 25% (w / w), resulting in approximately 300mL of ternary DES solution (viscosity approximately 480mPa·s).

[0037] The extraction conditions in step S3 were as follows: material-to-liquid ratio 1:25 (g / mL), ultrasonic power 200W, microwave power 100W, extraction temperature 45℃, and extraction time 35min.

[0038] In step S5, polysaccharide precipitation is performed when the final ethanol concentration reaches 58% (v / v); and polyphenol precipitation is performed when the final ethanol concentration reaches 82% (v / v).

[0039] Example 3 This embodiment is basically the same as Embodiment 1, except that: In step S1 of this embodiment, during the preparation of ternary DES, the molar ratio of ChCl, L-Pro, and LA is 1:1.2:2.5. 83.8 g (0.60 mol) of choline chloride, 82.9 g (0.72 mol) of L-proline, and 112.6 g (1.25 mol) of lactic acid are weighed out, and 133 mL of deionized water is added to adjust the water content to 35% (w / w), resulting in approximately 420 mL of ternary DES solution (viscosity approximately 430 mPa·s).

[0040] The extraction conditions in step S3 were as follows: material-to-liquid ratio 1:35 (g / mL), ultrasonic power 300W, microwave power 200W, extraction temperature 55℃, and extraction time 25min.

[0041] In step S5, polysaccharides are precipitated when the final ethanol concentration reaches 62% (v / v), and polyphenols are precipitated when the final ethanol concentration reaches 88% (v / v).

[0042] Example 4 This embodiment is basically the same as Embodiment 1, except that: In step S1 of this embodiment, during the preparation of ternary DES, the molar ratio of ChCl, L-Pro, and LA is 1:1:2.2. 83.8 g (0.60 mol) of choline chloride, 69.1 g (0.60 mol) of L-proline, and 118.9 g (1.32 mol) of lactic acid are weighed and 100 mL of deionized water is added to adjust the water content to 28% (w / w), resulting in approximately 375 mL of ternary DES solution (viscosity approximately 460 mPa·s).

[0043] The extraction conditions in step S3 were as follows: material-to-liquid ratio 1:28 (g / mL), ultrasonic power 280W, microwave power 180W, extraction temperature 48℃, and extraction time 32min.

[0044] Example 5 This embodiment is basically the same as Embodiment 1, except that: In step S1 of this embodiment, during the preparation of ternary DES, the molar ratio of ChCl, L-Pro, and LA is 1:0.9:1.8. 83.8 g (0.60 mol) of choline chloride, 62.2 g (0.54 mol) of L-proline, and 97.3 g (1.08 mol) of lactic acid are weighed out, and 116 mL of deionized water is added to adjust the water content to 33% (w / w), resulting in approximately 365 mL of ternary DES solution (viscosity approximately 470 mPa·s).

[0045] The extraction conditions in step S3 were as follows: material-to-liquid ratio 1:32 (g / mL), ultrasonic power 220W, microwave power 130W, extraction temperature 52℃, and extraction time 28min.

[0046] Comparative Example 1 In this comparative example, step S1 is performed according to the method in CN202510518925.3. Choline chloride (ChCl) and lactic acid (LA) are mixed at a molar ratio of 1:2 and stirred in an 80°C water bath until a homogeneous and transparent liquid is obtained, resulting in a binary DES solution (water content 30% w / w, viscosity approximately 850 mPa·s).

[0047] Step S2 is the same as in Example 1; In step S3, add 10g of lychee pulp powder at a material-to-liquid ratio of 1:30 (g / mL) and extract by stirring at 50℃ for 60 minutes.

[0048] Step S4 is the same as in Example 1; after centrifugation of the extract, a supernatant (containing polyphenols) is obtained, and the residue is washed with deionized water to obtain crude polysaccharide.

[0049] Comparative Example 2 In step S1 of this comparative example, a binary DES solution (30% w / w water content, viscosity approximately 620 mPa·s) was prepared by mixing choline chloride (ChCl) and L-proline (L-Pro) at a molar ratio of 1:1. Steps S2-S5 are the same as in Example 1.

[0050] Comparative Example 3 In this comparative example, step S1 involves preparing a binary DES solution (30% w / w water content, viscosity approximately 550 mPa·s) by mixing L-proline (L-Pro) and lactic acid (LA) at a molar ratio of 1:2. Steps S2-S5 are the same as in Example 1.

[0051] Comparative Example 4 This comparative example uses the traditional hot water extraction method to extract polysaccharides and polyphenols: 10g of litchi pulp powder was weighed and added to 300mL of deionized water at a material-to-liquid ratio of 1:30. The mixture was heated and extracted in a 90℃ water bath for 4 hours. After filtration, the extract was concentrated under reduced pressure, and 4 times its volume of anhydrous ethanol was added to precipitate the polysaccharides. The polysaccharide precipitate was collected by centrifugation and vacuum dried to obtain crude polysaccharides. The supernatant was rotary evaporated to remove ethanol and then freeze-dried to obtain crude polyphenols.

[0052] Comparative Example 5 This comparative example uses pure ultrasound-assisted water extraction to extract polysaccharides and polyphenols: 10g of litchi pulp powder was weighed and added to 300mL of deionized water at a material-to-liquid ratio of 1:30. The mixture was placed in an ultrasonic cleaner with an ultrasonic power of 300W, a frequency of 40kHz, an extraction temperature of 50℃, and an extraction time of 60min. After centrifugation and filtration, crude polysaccharides and crude polyphenols were obtained separately using the method of Comparative Example 2.

[0053] Comparative Example 6 In this comparative example, step S1 uses the binary DES solution of Comparative Example 1, and subsequent steps S2-S5 are the same as in Example 1.

[0054] Comparative Example 7 Steps S1 and S2 of this comparative example are the same as in Example 1, but in step S3, a pure microwave-assisted extraction method (without ultrasound) is used: 10g of litchi pulp powder is weighed and added to 300mL of the ternary DES solution (viscosity approximately 450mPa·s) prepared in step S1 of Example 1 at a material-to-liquid ratio of 1:30 (g / mL), and placed in a microwave extractor. Only the microwave is turned on, with a power of 200W, an extraction temperature of 50℃, and an extraction time of 30min (without ultrasonic field). After extraction, solid-liquid separation and stepwise purification and recovery are performed according to steps S4 and S5 of Example 1 to obtain crude polysaccharide and crude polyphenol products, respectively.

[0055] Comparative Example 8 In step S1 of this comparative example, ternary DES was prepared according to a molar ratio of ChCl:L-Pro:LA = 1:0.3:3.5. Specifically, 83.8 g (0.60 mol) of choline chloride, 20.7 g (0.18 mol) of L-proline, and 189.2 g (2.10 mol) of lactic acid were weighed out, and 80 mL of deionized water was added to adjust the water content to approximately 22% (w / w), yielding approximately 385 mL of ternary DES solution (viscosity approximately 380 mPa·s). In step S3, 10 g of litchi pulp powder was weighed out and added to 300 mL of the above ternary DES solution at a material-to-liquid ratio of 1:30. Extraction was carried out for 30 min under ultrasonic power of 250 W, microwave power of 150 W, and temperature of 50 °C. Other components and steps were the same as in Example 1.

[0056] Comparative Example 9 In step S5 of this comparative example, a one-step ethanol precipitation method was used to collect polysaccharides and polyphenols (without stepwise purification and recovery): Specifically, after preparing the ternary DES solution and completing extraction and solid-liquid separation according to steps S1-S4 of Example 1, in step S5, the combined extract was concentrated under reduced pressure to approximately 155 mL, and then anhydrous ethanol was directly added to achieve a final concentration of 75% (v / v). The mixture was then allowed to stand at 4°C for 18 h. At this ethanol concentration, polysaccharides and polyphenols co-precipitated simultaneously. All precipitates were collected by centrifugation and vacuum dried to constant weight to obtain a crude polysaccharide-polyphenol mixture. This method did not achieve stepwise separation and purification of polysaccharides and polyphenols.

[0057] Effect of L-proline dosage on extraction efficiency To verify the effect of L-proline dosage on extraction efficiency, a DES solution was prepared according to the molar ratio of choline chloride (ChCl), L-proline (L-Pro), and lactic acid (LA) of 1:x:2 as shown in the table below, following the method in step S1 of Example 1. The polysaccharide yield, polyphenol yield, and viscosity of the DES solution were then measured. The effect of L-proline dosage on extraction efficiency is shown in Table 1 below.

[0058] Performance testing methods (1) Determination of polysaccharide content, yield and purity: The phenol-sulfuric acid method was adopted, referring to the spectrophotometric principle in GB / T 15672. 1.0 mL of sample solution was taken, 1.0 mL of 5% phenol solution was added, followed by 5.0 mL of concentrated sulfuric acid. After shaking well, the solution was left at room temperature for 20 min, and the absorbance was measured at a wavelength of 490 nm. A standard curve was prepared using anhydrous glucose as the standard (linear range 10-100 μg / mL, R0). 2 ≥0.999). Polysaccharide yield (%) = polysaccharide mass (g) / raw material dry weight (g) × 100%. Polysaccharide purity (%) = mass of polysaccharides in crude product / total mass of crude product × 100%.

[0059] (2) Determination of polyphenol content and yield: The Folin-Ciocalteu colorimetric method was adopted, referring to the spectrophotometric principle in GB / T 8313. Accurately pipette 0.5 mL of the sample solution, add 2.5 mL of Folin-Ciocalteu reagent (diluted 10 times), shake well, react at room temperature for 5 min, then add 2.0 mL of 7.5% Na2CO3 solution, react at room temperature in the dark for 2 h, and measure the absorbance at 760 nm. A standard curve was prepared using gallic acid as a standard (linear range 5-50 μg / mL, R0). 2 ≥0.999). Results are expressed as gallic acid equivalents (mg GAE / g dry weight). Polyphenol purity (%) = mass of polyphenols in crude product (calculated as GAE) / total mass of crude product × 100%.

[0060] (3) Determination of DPPH free radical scavenging rate: Refer to the principle of DPPH method in GB / T 38570. Prepare sample solutions of different concentrations, and mix 2.0 mL of each solution with 2.0 mL of DPPH ethanol solution (0.2 mmol / L). Incubate at room temperature in the dark for 30 min, and measure the absorbance at 517 nm. Use vitamin C as a positive control. Plot the scavenging rate against the concentration and calculate the IC50. 50 value.

[0061] (4) ABTS ＋ Free radical scavenging rate determination: ABTS was prepared by mixing an equal volume of 7 mmol / L LABTS solution with 2.45 mmol / L potassium persulfate solution and incubating at room temperature in the dark for 16 h. ＋ Working solution. Dilute with ethanol to a absorbance of 0.70 ± 0.02 at 734 nm before use. Take 200 μL of sample solution and 3.8 mL of ABTS. ＋ The working solutions were mixed and reacted at room temperature in the dark for 6 min. The absorbance was measured at 734 nm. The clearance rate was plotted against the concentration, and the IC50 was calculated. 50 Value. IC 50 The lower the value, the stronger the antioxidant activity.

[0062] (5) Tyrosinase inhibition rate determination: Referring to the mushroom tyrosinase inhibition experiment method, using L-DOPA as the substrate, 2.5 mL of phosphate buffer (pH 6.8), 0.5 mL of mushroom tyrosinase solution (200 U / mL), and 0.5 mL of sample solution were mixed and pre-incubated at 37℃ for 10 min. 0.5 mL of L-DOPA solution (2 mg / mL) was added to start the reaction, and the reaction was carried out at 37℃ for 10 min. The absorbance was measured at 475 nm. Kojic acid was used as a positive control. Tyrosinase inhibition rate (%) = [1 - (change in absorbance of sample group / change in absorbance of control group)] × 100%.

[0063] The test results are shown in Table 1-3.

[0064] Table 1

[0065] Table 2

[0066] Table 3

[0067] The test results in Table 1 show that there is a significant jump effect in the amount of L-proline used within the molar ratio range of 0.8-1.2: When the L-Pro molar ratio increased from 0.6 to 0.8, the polysaccharide yield jumped from 18.5% to 21.5% (an increase of 16.2%), and the polyphenol yield jumped from 15.2% to 18.6% (an increase of 22.4%). Within the range of 0.8-1.2, the extraction effect remained at a high plateau. Beyond 1.2, the extraction effect tended to stabilize or slightly decrease.

[0068] The above results indicate that there is an optimal range of L-proline dosage (molar ratio 0.8-1.2). Too little L-proline will not form an effective triple hydrogen bond network, while too much L-proline will lead to excessive complexity of intermolecular forces.

[0069] As shown in Tables 2 and 3, the polysaccharide and polyphenol yields of Examples 1-5 of this invention are significantly higher than those of Comparative Example 1 (binary DES method), Comparative Example 4 (hot water extraction method), and Comparative Example 5 (ultrasonic water extraction method). Among them, Example 3 has the highest polysaccharide and polyphenol yields. The polysaccharide and polyphenol purity of this invention are also significantly better than those of the comparative examples. In addition, the extraction time of the method of this invention (25-35 min) is significantly shorter than that of the traditional method (240 min) and the binary DES method (60 min), resulting in a significant improvement in production efficiency, which is beneficial for continuous industrial production.

[0070] As shown in Table 3, the polyphenol DPPH scavenging rate IC of Examples 1-5 of the present invention is 50 The IC50 value for ABTS clearance was between 11.5 and 14.2 μg / mL. 50 The values ​​were between 7.9 and 9.8 μg / mL, and the tyrosinase inhibition rate was between 62.8 and 67.5%, both significantly better than the control groups. This indicates that the method of the present invention effectively maintains the bioactivity of the active ingredients while extracting them efficiently, and the stepwise purification helps to improve the bioactivity of each component.

[0071] The results from Example 1 and Comparative Examples 1-3 show that the ternary ChCl-L-Pro-LA system is significantly superior to any binary system in terms of polysaccharide yield, polyphenol yield, product purity, and viscosity. This proves that the ternary system has a synergistic effect that cannot be replaced by any binary system.

[0072] The results of Comparative Examples 1 and 6 (ChCl-LA binary system + dual-field extraction) show that even with the combined ultrasonic and microwave dual-field extraction, the yields and purity of polysaccharides and polyphenols in the binary system without L-proline are significantly lower than those in the ternary system of this invention. This fully demonstrates the crucial role of L-proline in the ternary DES system—it not only enhances the solubility of polysaccharides by strengthening the hydrogen bond network, but also improves the stability and extraction selectivity of polyphenols by regulating the pH of the system through its zwitterionic properties.

[0073] The results of Comparative Example 7 (pure microwave-assisted extraction, without ultrasound) showed that even using the ternary DES system of this invention, the polysaccharide yield (19.6%) and polyphenol yield (15.8 mg GAE / g) were significantly lower than those of Example 1 when relying solely on the microwave field without the assistance of ultrasonic cavitation. This indicates that the synergistic effect of ultrasound and microwave dual fields is key to achieving efficient extraction, and both the physical disruption of the cell wall by ultrasonic cavitation and the osmotic pressure drive of microwaves are indispensable.

[0074] The results of Comparative Example 8 (ternary DES ratio exceeding the specified range) showed that when the amount of L-proline was too low (molar ratio 0.3, far below the lower limit of 0.8) and the amount of lactic acid was too high (molar ratio 3.5, far exceeding the upper limit of 2.5), the polysaccharide yield (14.8%) and the polyphenol yield (11.5 mg GAE / g) were both significantly reduced, even lower than those of the binary system in Comparative Example 4. This is because insufficient L-proline cannot form an effective triple hydrogen bond network to enhance the solubility of polysaccharides; while excessive lactic acid leads to an excessively low pH (pH < 3.5), causing structural degradation of polyphenols under excessively acidic conditions (especially anthocyanin ring-opening decomposition). Simultaneously, the excessively low viscosity (380 mPa·s) results in insufficient solvent residence time within cells, affecting mass transfer efficiency.

[0075] The results of Comparative Example 9 (without stepwise purification and recovery) showed that although the polysaccharide yield (24.1%) and polyphenol yield (20.2 mg GAE / g) were basically the same as those of Example 1, indicating that the extraction ability of the ternary DES system for active ingredients is not affected by the purification method, the purity of the product decreased: the polysaccharide purity decreased from 87.2% to 71.5%, and the polyphenol purity decreased from 80.6% to 58.8%. This fully demonstrates that the gradient ethanol precipitation stepwise purification technology of the present invention is the key to achieving high-purity products—co-precipitation of polysaccharides and polyphenols at a 75% ethanol concentration results in a mixture of products, which is difficult to meet the purity requirements of high-end applications.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent, characterized in that, Includes the following steps: S1. Mix choline chloride, L-proline and lactic acid in a molar ratio of 1:(0.8-1.2):(1.5-2.5) and stir under heating conditions until a homogeneous and transparent liquid is formed to obtain a ternary eutectic solvent. Add water to adjust the water content to 20-40% (w / w). S2. Fresh lychee pulp is freeze-dried, pulverized, and sieved to obtain lychee pulp powder. S3. Mix the litchi pulp powder obtained in step S2 with the ternary eutectic solvent obtained in step S1 at a material-to-liquid ratio of 1:(20-40)g / mL, and extract for 20-40 minutes under the action of ultrasonic and microwave dual fields with an ultrasonic power of 200-350W, a microwave power of 100-250W, and an extraction temperature of 40-60℃ to obtain the extraction mixture. S4. Centrifuge the extract mixture obtained in step S3 and collect the supernatant; extract the residue again with water and combine the extracts. S5. Concentrate the combined extract under reduced pressure, add ethanol for gradient precipitation, centrifuge to collect the crude polysaccharide precipitate, take the supernatant and add ethanol to precipitate polyphenols, collect the crude polyphenol precipitate.

2. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 1, characterized in that, The heating conditions in step S1 are 60-70℃, the stirring rate is 300-500rpm, and the stirring time is 30-60min; the water content is 25-35% (w / w).

3. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 1, characterized in that, The freeze-drying process in step S2 is carried out at a temperature of -40°C to -60°C for 24-48 hours; the sieving process is carried out through a 40-60 mesh sieve.

4. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 1, characterized in that, The material-to-liquid ratio mentioned in step S3 is 1:(25-35)g / mL; The conditions for the dual-field ultrasonic and microwave interaction are as follows: ultrasonic power 200-300W, ultrasonic frequency 20-40kHz; microwave power 100-200W; extraction temperature 45-55℃, extraction time 25-35min.

5. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 1, characterized in that, The centrifugation conditions described in step S4 are: rotation speed 4000-6000 rpm, centrifugation time 10-20 min; add water to the residue at a material-to-liquid ratio of 1:(15-25) g / mL, and extract again under the same conditions, for 2-3 extractions.

6. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 1, characterized in that, The specific steps of step S5 are as follows: S51. Concentrate the combined extracts under reduced pressure at 40-50℃ to 1 / 4-1 / 3 of the original volume to obtain a concentrated solution. S52. Add anhydrous ethanol to the concentrate to make the final ethanol concentration reach 55%-65% (v / v), and let it stand at 4℃ for 12-24h to precipitate polysaccharides. S53. Centrifuge to collect the polysaccharide precipitate, wash with anhydrous ethanol, and vacuum dry to obtain crude polysaccharide; S54. Collect the supernatant after centrifugation in step S53, and continue to add anhydrous ethanol to make the final ethanol concentration reach 80%-90% (v / v). Let it stand at 4℃ for 12-24h to precipitate polyphenols. S55. Centrifuge to collect the polyphenol precipitate, and vacuum dry to obtain crude polyphenol product.

7. The method for extracting polysaccharides and polyphenols from litchi pulp based on a ternary eutectic solvent according to claim 6, characterized in that, In step S55, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 10-15 min.

8. A litchi pulp polysaccharide or litchi pulp polyphenol or litchi pulp polysaccharide-polyphenol complex active extract obtained by the method according to any one of claims 1-7.

9. The application of the litchi pulp polysaccharide according to claim 8 in the preparation of antioxidant foods, functional foods or cosmetics.

10. The application of the litchi pulp polyphenols according to claim 8 in the preparation of antioxidants, whitening cosmetics or functional beverages.

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