A method of increasing solubility and facilitating polysaccharide permeation

Abstract

The present application belongs to the field of chemistry and biotechnology, and particularly relates to a method for increasing the solubility of polysaccharides and promoting the penetration of polysaccharides. The method for increasing the solubility of polysaccharides and promoting the penetration of polysaccharides utilizes the special properties of a eutectic solvent, suspends polysaccharides in the eutectic solvent, and the eutectic solvent can better dissolve polysaccharides and promote the penetration of polysaccharides. According to the solubility test, the solubility of the eutectic solvent for polysaccharides and starch is higher than that of water solvent in varying degrees, and the eutectic solvent has a certain solubilization effect on poorly soluble polysaccharides and starch. In-vitro skin penetration test shows that, compared with polysaccharides which are almost unable to penetrate into the skin, the eutectic solvent can better promote the penetration of polysaccharides. The present application provides a method which can not only increase the solubility of macromolecular polysaccharides but also deliver polysaccharides to the deep layer of the skin to play a role without causing irritation and other adverse effects to the skin, and improves the solubility of polysaccharides, which is conducive to the more extensive application of polysaccharides.

Description

Technical Field

[0001] This invention belongs to the fields of chemistry and biotechnology, and specifically relates to a method for increasing the solubility of polysaccharides and promoting polysaccharide permeation. Background Technology

[0002] Polysaccharides are composed of multiple monosaccharide molecules linked by glycosidic bonds. They have long molecular chains, high degrees of polymerization, and many branches contain hydroxyl groups, making them prone to intermolecular hydrogen bonds. Due to the numerous intramolecular and intermolecular hydrogen bonds in their main molecular chains, they are difficult to dissolve in common solvents (such as water and most organic solvents). Furthermore, many active polysaccharides extracted from natural drugs need to be dissolved in water to be absorbed by the human body and thus express their biological activities. For example, Pleurotus sclerotium polysaccharide has immunomodulatory and antitumor activities, but this polysaccharide is almost insoluble in water and not easily absorbed by the human body; therefore, it is necessary to improve its solubility. Improved polysaccharide solubility means increased hydrophilicity and water retention. These improvements can endow polysaccharides with more diverse functions. In industrial applications, improved polysaccharide solubility also means reduced time and energy consumption for dissolution, reducing the cost of using polysaccharides and improving their ease of use.

[0003] Currently, methods to increase polysaccharide solubility mainly include high-pressure homogenization, co-solvent methods, degradation methods, and derivatization methods. However, the mechanical action of high-pressure homogenization may damage the structural properties of polysaccharides. The solubilization mechanism of co-solvents is not yet clear, so there are no clear rules for their selection, and selection is mostly based on experience. Ultrasonic degradation is simple and efficient, but the long chains of polysaccharides are damaged in the primary structure after treatment, resulting in an irreversible decrease in molecular weight, which has a significant impact on the gel properties of polysaccharides such as agar and carrageenan. The degree of polysaccharide degradation in acid is related to the strength of the acid and the reaction temperature. Acid treatment has a significant impact on the physicochemical properties of agar; excessively high acid concentrations and reaction temperatures will lead to excessively rapid degradation. The difficulty in controlling the temperature leads to a sharp decline in agar performance. In practical applications, acid degradation processes inevitably encounter problems such as reactor corrosion and difficulties in wastewater treatment. Furthermore, the degradation products have a wide molecular weight distribution, poor uniformity, and are difficult to separate. Oxidation processes are simple and effective, improving the solubility of polysaccharides while also enhancing the antibacterial and antioxidant properties of the materials. However, for most polysaccharides, there are few oxidative sites on their sugar units, and the degree of reaction between branches and the main chain is difficult to precisely control. The stability and repeatability of the oxidation reaction need further improvement. Derivatization methods often employ heterogeneous reaction systems. Due to the influence of diffusion and mass transfer, there are problems with uneven substitution during the reaction, resulting in a large range between the initial dissolution temperature and the complete dissolution temperature of polysaccharides, and a long dissolution time.

[0004] Furthermore, polysaccharides, as important bioactive substances, have wide applications in medicine, cosmetics, and other fields. However, their poor water solubility and low transdermal absorption efficiency limit their applications. The skin, as the body's first line of defense, suffers from low permeability, severely restricting the penetration of most drug molecules. The main permeation barrier is located in the outermost layer of the skin, the stratum corneum, which consists of keratin-rich cells embedded in multiple lipid bilayers. To penetrate the stratum corneum, drugs must traverse tortuous lipid pathways around keratin-rich cells or undergo repeated partitioning between the aqueous and lipid phases rich in keratin. Therefore, only drugs with optimal physicochemical properties (molecular weight less than 500 Da, high hydrophobicity, and sufficient solubility in both aqueous and non-aqueous solvents) can be passively transported through the stratum corneum. Currently, the main methods to enhance transdermal absorption are pharmaceutical approaches such as modifying dosage forms. These pharmaceutical methods primarily utilize micron or nanocarriers, including microemulsions and liposomes, to improve the ability of drugs to penetrate and be absorbed through the skin. Research and applications on whether it's possible to promote transdermal absorption of drug molecules by dissolving them in a solvent without altering the dosage form are still limited. With the expansion of applications and the deepening of research, there is an urgent need for a method that can both increase polysaccharide solubility and promote transdermal absorption. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for increasing the solubility and promoting the penetration of polysaccharides. This method utilizes the unique properties of eutectic solvents to suspend polysaccharides in them. Eutectic solvents can effectively dissolve polysaccharides and promote their penetration, solving the problem that polysaccharides are difficult to dissolve in conventional solvents (such as water and most organic solvents) due to their unique structure. This significantly improves the solubility characteristics of polysaccharides, facilitates structural analysis, and promotes their wider development and application.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for increasing the solubility and promoting the penetration of polysaccharides, wherein the method utilizes the special properties of eutectic solvents to suspend the polysaccharides in the eutectic solvent; the specific steps of the method are as follows: dispersing the polysaccharides in the eutectic solvent and vortexing to mix; allowing it to stand for several hours until it is completely dissolved; centrifuging and taking the supernatant; the supernatant is the mixture of the eutectic solvent and the polysaccharides.

[0008] Preferably, the polysaccharide is a variety of plant polysaccharides or a variety of starches, including but not limited to at least one of the following: grass jelly polysaccharide, dried tangerine peel polysaccharide, tea branch citrus polysaccharide, Yangchun sand polysaccharide, Dendrobium officinale polysaccharide, sweet potato starch, potato starch, corn starch, wheat starch, and cassava starch.

[0009] Preferably, the mass ratio of the polysaccharide to the eutectic solvent is 1:(1-60).

[0010] Preferably, the eutectic solvent is composed of a hydrogen bond acceptor, a hydrogen bond donor, and water; the hydrogen bond acceptor is at least one of L-proline, betaine, D-(+)-glucose, or citric acid; and the hydrogen bond donor is at least one of malic acid, lactic acid, β-alanine, glycerol, or L-proline.

[0011] The eutectic solvent described in this invention can effectively dissolve polysaccharides and promote their penetration. The mechanism of this penetration promotion may be that the acidic components in the eutectic solvent have an acidifying effect on the stratum corneum of the skin, thereby promoting the penetration of macromolecular polysaccharides and promoting the release and absorption of polysaccharide activity.

[0012] More preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-6):1, more preferably 1:1; the eutectic solvent has a water content of 17%-30% (w / w) by mass fraction.

[0013] More preferably, the hydrogen bond acceptor-hydrogen bond donor of the eutectic solvent is betaine-lactic acid, betaine-glycerol, L-proline-lactic acid, citric acid-proline, betaine-malic acid, L-proline-malic acid, or citric acid-alanine.

[0014] Preferably, the eutectic solvent is prepared by the following steps: taking appropriate amounts of hydrogen donor and hydrogen acceptor, mixing them in a certain molar ratio, heating and stirring at 100°C until a homogeneous, stable and transparent solution is formed, and then placing it in a desiccator for later use.

[0015] Preferably, the settling temperature is 40℃~90℃; the settling time is 8h~96h.

[0016] Preferably, the centrifugation frequency is 10,000 to 20,000 rpm, and the centrifugation time is 5 to 15 minutes.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The method described in this invention can both increase the solubility of macromolecular polysaccharides and deliver them to the deep layers of the skin to exert their effects, without causing adverse effects such as skin irritation. This improves the solubility characteristics of polysaccharides, which is conducive to the wider application of polysaccharides. It provides new ideas and strategies for the efficient delivery of macromolecular polysaccharides and their application in biomedicine and cosmetics. This is also a new application of eutectic solvents.

[0019] 2. Due to the unique structure of the skin, large-molecule drugs are difficult to absorb transdermally, limiting the application of large-molecule active ingredients. Polysaccharides, as large molecules, are almost impossible to absorb through the skin. This invention utilizes the special properties of eutectic solvents to suspend polysaccharides in them. In vitro transdermal experiments show that the eutectic solvent-polysaccharide complex significantly promotes polysaccharide penetration compared to polysaccharides themselves, which are almost impossible to penetrate the skin. The method described in this invention can deliver large-molecule polysaccharides to the deep layers of the skin to exert their effects without causing skin irritation or other adverse effects.

[0020] 3. The eutectic solvent used in this invention is simple to prepare, low in toxicity, biodegradable, biocompatible, recyclable, and natural and green. It solubilizes polysaccharides, a natural macromolecule with diverse functions, and promotes the transdermal absorption of polysaccharides. Some of these polysaccharides can be directly added to food as additives or formulations. It is an excellent substitute for traditional solvents and ionic solvents and has broad application prospects. Attached Figure Description

[0021] Figure 1 This is the standard curve for glucose.

[0022] Figure 2 The values ​​represent the solubility of *Gynostemma pentaphyllum* polysaccharides in various solvents, where W represents distilled water, CAAla represents citric acid-β-alanine, CAPro represents citric acid-L-proline, BetMA represents betaine-malic acid, BetGly represents betaine-glycerol, BetLA represents betaine-lactic acid, ProMA represents L-proline-malic acid, and ProLa represents L-proline-lactic acid.

[0023] Figure 3 The values ​​represent the solubility of tangerine peel polysaccharides in various solvents, where W represents distilled water, CAAla represents citric acid-β-alanine, CAPro represents citric acid-L-proline, BetMA represents betaine-malic acid, BetGly represents betaine-glycerol, BetLA represents betaine-lactic acid, ProMA represents L-proline-malic acid, and ProLa represents L-proline-lactic acid.

[0024] Figure 4 The results of the in vitro transdermal assay of mesona chinensis polysaccharide, Q m The values ​​represent the cumulative permeation per unit area; PPP represents the cumulative permeation per unit area of ​​the in vitro transdermal test 2 of *Gynostemma pentaphyllum* polysaccharide aqueous solution; and ProLa represents the cumulative permeation per unit area of ​​the in vitro transdermal test 2 of *Gynostemma pentaphyllum* polysaccharide dissolved in L-proline-lactic acid eutectic solvent. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0026] 1. The preparation method of the herb *Mesona chinensis* polysaccharide in Example 1 is as follows: 200g of dried *Mesona chinensis* powder was pulverized through a 20-mesh sieve and defatted with 95% ethanol (volume ratio). Then, it was extracted twice with 0.125M Na2CO3 solution and ultrasonic extraction assisted by 300W for 1h. The material-to-liquid ratio was 1:20 (mg / mL). The extracts were combined and anhydrous ethanol was added until the final ethanol concentration was 70%. The precipitate was allowed to stand in a refrigerator at 4℃ for 24h. After centrifugation, the precipitate was evaporated and redissolved in water. The precipitate was purified by dialysis in a 3500Da dialysis bag for 5 days. After concentrating the volume, it was freeze-dried to obtain *Mesona chinensis* polysaccharide.

[0027] Determining the solubility of *Gynostemma pentaphyllum* polysaccharide in water at room temperature: Weigh 100 mg of *Gynostemma pentaphyllum* polysaccharide, dissolve it in 10 ml of secondary water, stir at 25 °C for 30 min, centrifuge at 5000 r / min for 20 min, take 5 ml of the supernatant, freeze at -80 °C for more than 6 h, freeze-dry for 24 h to obtain dry powder, weigh it, calculate the solubility (mg / ml), perform the operation in parallel three times, and take the average value.

[0028] The weights of the freeze-dried powders were 7.7 mg, 9.9 mg, and 7.4 mg, respectively. The calculated average solubility was 1.67 mg / mL, or 0.00167 g / g, with an RSD of 0.16%. According to the 2020 Chinese Pharmacopoeia, substances with a solubility of less than 0.01 g / 100 g water at room temperature are called sparingly soluble substances; therefore, *Gynostemma pentaphyllum* polysaccharide is a sparingly soluble substance.

[0029] 2. The preparation method of tangerine peel polysaccharide in Example 2 is as follows: Tangerine peel is dried at a constant temperature and then pulverized, passed through a 40-mesh sieve to obtain coarse tangerine peel powder. 200g of coarse tangerine peel powder is added to 4L of 95% ethanol and extracted at 80℃ for 3 hours. After filtration, the residue is extracted three times to remove pigments and small molecules. Finally, the residue is dried to obtain defatted tangerine peel powder. 200g of defatted tangerine peel powder is added to 4L of deionized water and extracted at 95℃ for 3 hours. After filtration, the supernatant is collected, and the residue is extracted three times. The extracts are combined and concentrated under vacuum at 60℃ to a certain volume. Four times the volume of anhydrous ethanol is added to the concentrate, and after vigorous stirring, it is left to stand overnight in a refrigerator at 4℃. Centrifuge to collect the precipitate, redissolve it in 400 mL of deionized water, add 80 mL of Sevage reagent (chloroform: n-butanol = 4:1 (v / v)), shake thoroughly, and centrifuge (5000 rpm, 5 min). Combine the supernatants after centrifugation, continue adding Sevage reagent, shake, and centrifuge until no denatured proteins appear in the solution. Vacuum concentrate to remove organic reagents from the solution, and freeze-dry under vacuum to obtain crude polysaccharides from fruits of A. villosum (AVPs) for later use.

[0030] Example 1:

[0031] (1) Weigh out 7 portions of 0.01g each of grass jelly polysaccharide;

[0032] (2) Weigh 15g of the DES that has been prepared in proportion, add 4.5g of water to a 50mL EP tube to prepare a DES solvent with a water content of 30%; the selected DES combinations are: betaine-lactic acid (molar ratio 1:1), betaine-glycerol (molar ratio 1:1), L-proline-lactic acid (molar ratio 1:1), citric acid-proline (molar ratio 1:1), betaine-malic acid (molar ratio 1:1), L-proline-malic acid (molar ratio 1:1), citric acid-alanine (molar ratio 1:1);

[0033] (3) Weigh 1g of DES solvent with a water content of 30% and disperse the weighed grass polysaccharide in the DES solvent respectively;

[0034] (4) After vortex mixing, let stand in a 60℃ oven and check whether it has dissolved to saturation at 12h, 24h, 48h and 72h respectively;

[0035] (5) After reaching saturation, centrifuge at high speed to remove residue and collect filtrate. The centrifugation frequency is 15000 rpm and the centrifugation time is 10 min.

[0036] (6) Take 0.5g of the supernatant after centrifugation;

[0037] (7) Dilute the supernatant taken out by 100 times and determine its total sugar content.

[0038] Example 2

[0039] The difference between Example 1 and Example 2 is that the polysaccharide used in Example 1 is grass jelly polysaccharide, while the polysaccharide used in Example 2 is tangerine peel polysaccharide. All other conditions and operations are the same as in Example 1.

[0040] Examples 3-7

[0041] The difference from Example 1 is that Example 1 used polysaccharides, while Examples 3-7 used plant starches. Example 3 used sweet potato starch, Example 4 used potato starch, Example 5 used corn starch, Example 6 used wheat starch, and Example 7 used cassava starch. The DES solvent combinations used in Example 1 were betaine-lactic acid (molar ratio 1:1) and betaine-glycerol (molar ratio 1:1).

[0042] 1) L-proline-lactic acid (molar ratio 1:1), citric acid-L-proline (molar ratio 1:1), betaine-malic acid (molar ratio 1:1), L-proline-malic acid (molar ratio 1:1), citric acid-alanine (molar ratio 1:1); The DES solvent combinations used in Example 3 were: betaine-lactic acid (molar ratio 1:1), betaine-glycerol (molar ratio 1:1), L-proline-lactic acid (molar ratio 1:1), and L-proline-lactic acid (molar ratio 1:1).

[0043] 1) L-proline-malic acid (molar ratio 1:1); The DES solvent combinations used in Example 4 were betaine-lactic acid (molar ratio 1:1), betaine-glycerol (molar ratio 1:1), L-proline-lactic acid (molar ratio 1:1), and L-proline-malic acid (molar ratio 1:1); The DES solvent combinations used in Example 5 were betaine-lactic acid (molar ratio 1:1) and L-proline-lactic acid (molar ratio 1:1); The DES solvent combinations used in Example 6 were betaine-lactic acid (molar ratio 1:1), betaine-glycerol (molar ratio 1:1), and L-proline-lactic acid (molar ratio 1:1); The DES solvent combinations used in Example 7 were betaine-lactic acid (molar ratio 1:1) and L-proline-malic acid (molar ratio 1:1).

[0044] 1) L-proline-lactic acid (molar ratio 1:1). All other conditions and procedures are the same as in Example 1.

[0045] Comparative Example 1

[0046] The difference from Example 1 is that Example 1 used DES solvent, while Comparative Example 1 used secondary water solvent. All other conditions and operations were the same as in Example 1.

[0047] Comparative Example 2

[0048] The difference between Example 2 and Comparative Example 2 is that Example 2 used DES solvent, while Comparative Example 2 used secondary water. All other conditions and operations were the same as in Example 1.

[0049] Comparative Example 3

[0050] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses secondary water as the solvent, and the polysaccharides used in Comparative Example 3 are sweet potato starch, potato starch, corn starch, wheat starch, and cassava starch, respectively. All other conditions and operations are the same as in Example 1.

[0051] I. Solubility Experiment

[0052] In Examples 1-7 and Comparative Examples 1-3, the total sugar content after solvent dissolution was used as an indicator to represent the solvent's solubility. The phenol-sulfuric acid method was used to determine the total sugar content, and the specific procedure was as follows:

[0053] ①Preparation of D-glucose standard solution

[0054] Accurately weigh 0.100g of D-glucose, dissolve it in an appropriate amount of deionized water, and dilute to 1000mL in a volumetric flask to prepare a 0.1mg / mL D-glucose standard solution.

[0055] ② Plot the glucose standard curve

[0056] Accurately transfer 0.00 mL, 0.10 mL, 0.15 mL, 0.20 mL, 0.25 mL, 0.30 mL, 0.35 mL, 0.40 mL, and 0.50 mL of glucose standard solution into 2 mL centrifuge tubes. Add deionized water to bring the volume to 0.5 mL. Then, transfer 200 μL of each solution into a separate 2 mL centrifuge tube. Perform three replicates for each replicate. Add 0.2 mL of 6% phenol solution, shake well, and quickly add 1 mL of concentrated sulfuric acid. Shake well again and allow the reaction to cool for 30 min. Pipette 280 μL of the test solution into a 96-well plate (directly placed in a UV spectrophotometer) and measure the absorbance at 490 nm. Take the average of the three absorbance values ​​and calculate the glucose concentration. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. Figure 1 As shown, the regression equation was derived. Its standard equation is A = 6.9442C. Glu -0.0137, R 2 =0.9998, where: A - absorbance, Abs; C Glu -D-glucose concentration, mg / mL.

[0057] ③ Determination of the solubility of polysaccharides and starch in various solvents

[0058] The supernatant obtained after dissolution and saturation in Examples 1-7 and Comparative Examples 1-3 was diluted 100 times (with the corresponding dilution solvent as a blank control), and the above operation was repeated. The total sugar content was calculated by substituting the solution into the standard curve.

[0059] II. Osmosis Enhancement Experiment

[0060] In vitro transdermal test 1: After fully dissolving the *Gynostemma pentaphyllum* polysaccharide in the L-proline-malic acid eutectic solvent used in Example 1, a permeation enhancement test was conducted. The specific method was as follows:

[0061] (1) Preparation before the experiment: Take the treated mouse back skin out of the refrigerator and thaw it; moisten the receiving pool in physiological saline; turn on the intelligent drug transdermal diffusion test instrument TP3A in advance to make the water bath temperature reach 32±0.5℃.

[0062] (2) Diffusion chamber assembly: Add 18 mL of physiological saline to the receiving chamber. Fix the thawed mouse back skin between the supply and receiving chambers of the diffusion chamber (diffusion area is 5.31 cm2). Take 50 μL of a mixed solution of L-proline-malic acid eutectic solvent to fully dissolve the licorice polysaccharide and place it in the supply chamber. Place the diffusion chamber into the corresponding slot in the upper part of the water tank.

[0063] (3) Sample collection: Samples were taken at set times of 0, 2, 4, 6, 8, 10, 12, and 24 hours, i.e., 1 mL of liquid was aspirated from the supply chamber and 1 mL of physiological saline was added simultaneously to ensure that the liquid volume in the receiving pool remained constant. After 24 hours of sampling, the skin surface was washed with purified water three to five times, the skin was removed, the residual liquid on the surface was wiped dry, and then the skin was crushed by a crusher. After crushing, the skin was centrifuged, and the supernatant was collected to calculate the amount of skin residue.

[0064] (4) The sugar content of the collected samples was determined by the phenol-sulfuric acid method, and the cumulative permeation per unit area of ​​the mixed solution of L-proline-malic acid eutectic solvent fully dissolved in grass polysaccharide was calculated.

[0065] In vitro transdermal test 2: After fully dissolving the herb polysaccharide in the L-proline-lactic acid eutectic solvent used in Example 1, a permeation enhancement test was conducted. The specific test method and dosage were the same as in in vitro transdermal test 1, and will not be repeated here.

[0066] Test results:

[0067] The detection results of Example 1 and Comparative Example 1 are as follows: Figure 2 As shown. By Figure 2It was found that different DES solvents could increase the solubility of *Gynostemma pentaphyllum* polysaccharides by varying degrees, and all were higher than the solubility in water. The total sugar content after solvent dissolution was used as an indicator of solvent solubility. The total sugar content in water was 0.30 mg / mL, in betaine-lactic acid solvent it was 2.46 mg / mL, in betaine-glycerol solvent it was 2.06 mg / mL, in L-proline-lactic acid solvent it was 4.91 mg / mL, in citric acid-proline solvent it was 1.32 mg / mL, in betaine-malic acid solvent it was 1.36 mg / mL, in L-proline-malic acid solvent it was 3.51 mg / mL, and in citric acid-alanine solvent it was 1.01 mg / mL.

[0068] The detection results of Example 2 and Comparative Example 2 are as follows: Figure 3 As shown. By Figure 2 It can be seen that different DES solvents can increase the solubility of tangerine peel polysaccharides by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of tangerine peel polysaccharides in water is 0.99 mg / mL, in betaine-lactic acid solvent it is 3.47 mg / mL, in betaine-glycerol solvent it is 5.68 mg / mL, in L-proline-lactic acid solvent it is 4.12 mg / mL, in citric acid-proline solvent it is 2.48 mg / mL, in betaine-malic acid solvent it is 3.11 mg / mL, in L-proline-malic acid solvent it is 3.63 mg / mL, and in citric acid-alanine solvent it is 1.65 mg / mL.

[0069] The test results of Comparative Example 3 and Examples 3-7 are shown in Table 1.

[0070] Table 1. Total sugar content of starch after dissolving it in different solvents in Comparative Examples 3 and Examples 3-7.

[0071]

[0072] A comparison of Example 3 and Comparative Example 3 shows that different DES solvents can increase the solubility of sweet potato starch by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of sweet potato starch in water is 0.37 mg / mL, in betaine-lactic acid solvent it is 3.13 mg / mL, in betaine-glycerol solvent it is 0.71 mg / mL, in L-proline-lactic acid solvent it is 3.99 mg / mL, and in L-proline-malic acid solvent it is 0.38 mg / mL.

[0073] A comparison of Example 4 and Comparative Example 3 shows that different DES solvents can increase the solubility of sweet potato starch by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of sweet potato starch in water is 0.38 mg / mL, in betaine-lactic acid solvent it is 7.05 mg / mL, in betaine-glycerol solvent it is 0.43 mg / mL, in L-proline-lactic acid solvent it is 7.76 mg / mL, and in L-proline-malic acid solvent it is 0.65 mg / mL.

[0074] A comparison of Example 5 and Comparative Example 3 shows that different DES solvents can increase the solubility of corn starch by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of corn starch in water is 1.15 mg / mL, in betaine-lactic acid solvent it is 4.36 mg / mL, and in L-proline-lactic acid solvent it is 3.87 mg / mL.

[0075] A comparison of Example 6 and Comparative Example 3 shows that different DES solvents can increase the solubility of wheat starch by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of wheat starch in water is 0.96 mg / mL, in betaine-lactic acid solvent it is 3.28 mg / mL, in betaine-glycerol solvent it is 4.74 mg / mL, and in L-proline-lactic acid solvent it is 2.32 mg / mL.

[0076] A comparison of Example 7 and Comparative Example 3 shows that different DES solvents can increase the solubility of cassava starch by varying degrees, and all are higher than the solubility in water. The total sugar content after solvent dissolution is used as an indicator of solvent solubility. The total sugar content of cassava starch in water is 0.57 mg / mL, and the total sugar content in L-proline-malic acid solvent is 0.65 mg / mL. The total sugar content in betaine-lactic acid solvent is 9.25 mg / mL, and the total sugar content in L-proline-lactic acid solvent is 4.31 mg / mL.

[0077] Figure 4The results are as follows: PPP represents the cumulative permeation per unit area of ​​the *Mesona chinensis* polysaccharide aqueous solution in in vitro transdermal test 2; ProLa represents the cumulative permeation per unit area of ​​the *Mesona chinensis* polysaccharide dissolved in L-proline-lactic acid eutectic solvent in in vitro transdermal test 2. The results show that the transdermal effect of the *Mesona chinensis* polysaccharide aqueous solution is not significant from 0 to 24 hours, almost non-transdermal, and the cumulative average permeation per unit area after 24 hours is only 0.00843 g / cm³. 2 The L-proline-malic acid and L-proline-lactic acid eutectic solvents dissolved in *Mesona chinensis* polysaccharides continuously penetrated the skin from 0 to 12 hours, reaching the maximum cumulative penetration at 12 hours, after which penetration almost ceased, indicating saturation. These results suggest that although *Mesona chinensis* polysaccharides have a large molecular weight and are difficult to absorb transdermally, transdermal absorption can be promoted with the aid of supramolecular solvents.

[0078] In summary, the eutectic solvent described in this invention exhibits higher solubility for both polysaccharides and starch compared to water, and it also provides some solubilization for poorly soluble polysaccharides and starch. Compared to polysaccharides themselves, which are almost impossible to penetrate the skin, the eutectic solvent effectively promotes polysaccharide penetration. This invention provides a method that increases the solubility of macromolecular polysaccharides while delivering them deep into the skin to exert their effects, without causing skin irritation or other adverse effects. This improved polysaccharide solubility characteristics facilitate wider application of polysaccharides and provides new ideas and strategies for the efficient delivery of macromolecular polysaccharides and their application in biomedicine and cosmetics. This also represents a novel application of eutectic solvents.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of increasing the solubility of a polysaccharide and promoting polysaccharide permeation, characterized by, The method is to suspend polysaccharide in a deep eutectic solvent, and the specific steps are: dispersing polysaccharide in a deep eutectic solvent, vortex mixing; standing at 40-90 DEG C for 8-96 h until complete dissolution; centrifugation at 10,000-20,000 rpm for 5-15 min, and taking the supernatant; the supernatant is a mixture of the deep eutectic solvent and the polysaccharide; the mass ratio of the polysaccharide to the deep eutectic solvent is 1: (1-60) ; The polysaccharide is mesona polysaccharide or dried tangerine or orange peel polysaccharide, and the deep eutectic solvent is betaine-lactic acid, betaine-glycerol, L-proline-lactic acid, citric acid-proline, betaine-malic acid, L-proline-malic acid, or citric acid-alanine.

2. A method of increasing the solubility of a polysaccharide and promoting polysaccharide permeation, characterized by, The method is to suspend polysaccharide in a deep eutectic solvent, and the specific steps are: dispersing polysaccharide in a deep eutectic solvent, vortex mixing; standing at 40-90 DEG C for 8-96 h until complete dissolution; centrifugation at 10,000-20,000 rpm for 5-15 min, and taking the supernatant; the supernatant is a mixture of the deep eutectic solvent and the polysaccharide; the mass ratio of the polysaccharide to the deep eutectic solvent is 1: (1-60) ; the polysaccharide is sweet potato starch, potato starch or corn starch, and the deep eutectic solvent is betaine-lactic acid or L-proline-lactic acid.

3. A method of increasing the solubility of a polysaccharide and promoting polysaccharide permeation, characterized by, The method is to suspend polysaccharide in a deep eutectic solvent, and the specific steps are: dispersing polysaccharide in a deep eutectic solvent, vortex mixing; standing at 40-90 DEG C for 8-96 h until complete dissolution; centrifugation at 10,000-20,000 rpm for 5-15 min, and taking the supernatant; the supernatant is a mixture of the deep eutectic solvent and the polysaccharide; the mass ratio of the polysaccharide to the deep eutectic solvent is 1: (1-60) ; the polysaccharide is wheat starch, and the deep eutectic solvent is betaine-lactic acid, betaine-glycerol, or L-proline-lactic acid.

4. A method of increasing the solubility of a polysaccharide and promoting polysaccharide permeation, characterized by, The method is to suspend polysaccharide in a deep eutectic solvent, and the specific steps are: dispersing polysaccharide in a deep eutectic solvent, vortex mixing; standing at 40-90 DEG C for 8-96 h until complete dissolution; centrifugation at 10,000-20,000 rpm for 5-15 min, and taking the supernatant; the supernatant is a mixture of the deep eutectic solvent and the polysaccharide; the mass ratio of the polysaccharide to the deep eutectic solvent is 1: (1-60) ; the polysaccharide is cassava starch, and the deep eutectic solvent is betaine-lactic acid or L-proline-malic acid.

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