A preparation method of a novel skin care wet wipes liquid

Abstract

The present invention relates to the technical field of skin care wet wipes liquid medicine, discloses a preparation method of a novel skin care wet wipes liquid medicine, comprising the following steps: pre-treating silkworm cocoons to obtain silk fibroin powder for standby use; crushing eucalyptus leaves into granules, treating with isododecane and glycerol, then dialyzing with water, and then freeze-drying to obtain eucalyptus leaf extract; methylating the eucalyptus leaf extract with methylase and S-adenosylmethionine to obtain solution I; glycosylation of solution I with sucrose solution and starch sucrase to obtain solution II; mixing silk fibroin powder, solution II, glycerol and water with a mass ratio of 7-21:7-10:5-8:67-75 to obtain skin care wet wipes liquid medicine. The wet wipes liquid medicine in the present invention not only has good skin care efficacy and bactericidal, antibacterial and anti-inflammatory effects, but also has higher stability of liquid medicine components, can be stored for a long time, and improves the shelf life of the wet wipes liquid medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of skin care wet wipes liquid medicine, and more specifically relates to a preparation method of a novel skin care wet wipes liquid medicine. Background Art

[0002] At present, China's wet wipes industry is in a period of rapid development. Generally speaking, wet wipes on the market can be divided into two categories: one is that they cannot disinfect but contain skin care ingredients to maintain the skin; the other is disinfecting wet wipes that can disinfect. However, the formulas used in skin care wet wipes are still relatively traditional. Although they can be used normally, they are easy to irritate the skin after repeated use, and there will be residues after use, causing skin itching or even allergies.

[0003] Silk fibroin is a natural moisture-regulating factor with excellent functions and is green, environmentally friendly and harmless. Silk fibroin is extracted from silk and contains nearly 20 different amino acids. Glycine (Gly), alanine (Ala) and serine (Ser) have the highest proportions, with a total content of nearly 80%. Leucine has a good effect on accelerating cell metabolism, serine can slow down skin aging, and tryptophan has a good absorption function for ultraviolet rays. The performance of these amino acids determines the four major characteristics of silk fibroin: good comfort, good water absorption and waterproofness, good glossiness, and good anti-ultraviolet effect. Applying silk fibroin to wet wipes can make it have the effects of moisturizing, whitening, anti-wrinkle, and sun protection. Therefore, silk fibroin will have a great application prospect in the beauty and whitening field of the skin care market.

[0004] Eucalyptus leaf extracts contain flavonoids, triterpenes and other substances, which have antibacterial, anti-inflammatory, insecticidal, anthelmintic and antipruritic effects. However, due to their inherent instability and poor water solubility, these substances are difficult to use in products with high water content. Even if they are used, the shelf life of the products is relatively short, which is not conducive to promotion and use.

[0005] Therefore, the research on silk fibroin and eucalyptus leaf extract is of great significance in terms of health, safety, multifunctionality and easy preservation of wet wipes, and plays an important role in promoting the industrial reform of green wet wipes. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing a novel skin care wet wipes liquid, by applying silk fibroin and eucalyptus leaf extract to the wet wipes liquid formula, thereby achieving green, safe and healthy wet wipes skin care.

[0007] To achieve the above object, the specific technical solution of the present invention is: a method for preparing a novel skin care wet wipes liquid, comprising the following steps:

[0008] (1) Pre-treating silkworm cocoons to obtain silk fibroin powder for later use;

[0009] (2) crushing eucalyptus leaves into particles, dissolving them in isododecane at 20-40° C. for 2-5 h, filtering them, impregnating them with glycerin, and filtering them to recover solid particles;

[0010] (3) dialyzing the solid particles with water, and then freeze-drying them to obtain a eucalyptus leaf extract;

[0011] (4) mixing the eucalyptus leaf extract with a 0.02-0.04 mol / ml methylase solution, adding a 0.05-0.08 mol / ml S-adenosylmethionine solution, mixing and reacting at 36-37° C. for 2-3 hours, and then performing an enzyme inactivation treatment to obtain a solution I;

[0012] (5) mixing solution I with 0.04-0.06 mol / ml amylosucrase solution and 0.08-0.12 mol / ml sucrose solution, reacting at 36-37° C. for 2-3 h, and then performing enzyme inactivation treatment to obtain solution II for standby use;

[0013] (6) Silk fibroin powder, solution II, glycerol, and water are mixed in a mass ratio of 7-21:7-10:5-8:67-75 to prepare a skin care wet wipes solution.

[0014] The present invention applies silk fibroin to wet wipes, which can make them have the effects of moisturizing, whitening, anti-wrinkle, sun protection, etc., but its dispersibility in the wet wipes solution is poor and cannot fully exert its advantages. The extract of eucalyptus leaves contains flavonoids, triterpenes and other substances, which have antibacterial, anti-inflammatory, antipruritic and other effects. Applying them to wet wipes can reduce skin irritation and improve the comfort of wet wipes products.

[0015] The present invention uses isomeric dodecane to extract eucalyptus leaves, which is nontoxic and harmless, has no effect on the human body and is non-irritating, and has a good dissolving effect on the oily substances and ester substances in the eucalyptus leaves. Glycerol is used to clean the eucalyptus leaf extract, and isomeric dodecane is easily soluble in glycerol, and glycerol is harmless and non-irritating to the human body, non-toxic, healthy and environmentally friendly. If the flavonoids in the main active ingredients of the eucalyptus leaf extract are methylated by methylating their hydroxyl groups with S-adenosylmethionine as a methyl donor, the ability of the flavonoids to bind to proteins can be strengthened after methylation (the hydrophobicity of the flavonoids is strengthened after methylation, and the hydrophobic effect plays an important role in the combination of flavonoids and proteins, and the polarity of the flavonoids is decreased due to methylation, and the ability of the compounds to enter the hydrophobic cavity of the protein is enhanced), and the silk fibroin and the extract are completely combined, so that the advantages of the two can be better brought into play. Next, glycosylation can increase the water solubility and stability of flavonoid compounds, and can improve their biological activity. The present invention makes the eucalyptus leaf extract glycosylated, which can make the flavonoid components in the eucalyptus leaves exist in a more stable form in the liquid medicine, better play its bactericidal, antibacterial and anti-inflammatory effects, and can make it better preserved in the wet wipes liquid medicine, and improve the shelf life of the wet wipes liquid medicine. Eucalyptus leaves also contain 1,8-cineole, which is not only anti-inflammatory and analgesic, but also can resist tuberculosis bacteria, and can also stimulate the nasal cavity to promote mucus secretion, so the prepared liquid medicine can also make the nasal cavity and bronchial tubes more unobstructed. In the present invention, the methylation and glycosylation of the eucalyptus leaf extract are only effective for the flavonoid components, and 1,8-cineole has basically no effect, which can maximize the functions of the various components of the eucalyptus leaf extract, so that the liquid medicine has more side effects.

[0016] Therefore, the wet wipes liquid medicine in the present invention not only has good skin care effects, is non-irritating to the skin, is healthy and environmentally friendly, but also has higher stability of the liquid medicine components and can be stored for a long time, thereby improving the shelf life of the wet wipes liquid medicine.

[0017] More preferably, in step (1), the pretreatment comprises: removing the outer cocoon from the silkworm cocoons and crushing them to obtain silkworm fragments; 2 CO 3 The silk was degummed by boiling in the solution for 50-70 minutes, and the degumming was repeated for 3 times. The degummed silk was collected, washed and dried, and the obtained silk was added to the calcium alcohol solution, boiled until the silk was completely dissolved, cooled to room temperature, and vacuum filtered. The filtrate was dialyzed to obtain a silk fibroin solution, which was freeze-dried to obtain a silk fibroin powder for later use.

[0018] More preferably, the silkworm fragments and Na 2 CO 3 The ratio of the solution is 1:30-100g / ml; the Na 2 CO 3The concentration of the solution is 0.1-0.8wt%. The ratio of the silk to the calcium alcohol solution is 1:30-60g / ml; the calcium alcohol solution is prepared by dissolving calcium chloride in a mixed solution of ethanol and water, wherein the molar ratio of calcium chloride, anhydrous ethanol and water is 1:2:8.

[0019] More preferably, in step (2), the ratio of eucalyptus leaves to isododecane is 1:40-80 g / ml.

[0020] More preferably, in step (2), the eucalyptus leaves are impregnated with glycerol by: firstly impregnating with glycerol at 30-50°C for 6-12h, with the ratio of eucalyptus leaves to glycerol being 1:50-100 g / ml; and then impregnating with glycerol at 30-50°C for 8-16h, with the ratio of eucalyptus leaves to glycerol being 1:70-120 g / ml.

[0021] Further preferably, in step (3), the ratio of the solid particles to water is 1:10-30 g / ml; the solid particles are dialyzed with water by: dissolving the solid particles with water, pouring them into a dialysis bag with a molecular weight cutoff of 1000-5000 and dialyzing them for 2-3 days, and changing the water every 2-4 hours.

[0022] More preferably, in step (3), the freeze-drying temperature is -30 to -15°C and the time is 2 to 3 days.

[0023] It is further preferred that in step (4), the mass ratio of the eucalyptus leaf extract to the methylase solution is not greater than 5:6; the mass ratio of the methylase solution to the eucalyptus leaf extract to the S-adenosylmethionine solution is 1:5. It is further preferred that the mass ratio of the methylase solution to the S-adenosylmethionine solution is 5:6:30. The solvent of the methylase is PBS buffer with a pH of 7.4. 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride, and 0.2g of potassium chloride are dissolved in deionized water and then fixed to a 1000ml volumetric flask. The enzyme can function well in the PBS solution.

[0024] If the mass ratio is greater than 5:6, the methylation degree is too high, resulting in a low subsequent glycosylation degree, and the active ingredients of the eucalyptus leaf extract cannot be well preserved, which will greatly shorten the shelf life of the liquid medicine. However, at the same time, due to the high degree of methylation, the protein binding ability is strong, resulting in stronger anti-aging and moisturizing abilities of the liquid medicine.

[0025] The degree of methylation is controlled at about 40%. By controlling the proportion of methylation and balancing the degree of methylation and glycosylation, the overall performance of the wet wipes liquid can be optimized.

[0026] It is further preferred that in step (5), the mass ratio of the eucalyptus leaf extract to the starch sucrase solution is not greater than 6:7; the mass ratio of the starch sucrase solution to the sucrose solution is 7:40. It is further preferred that the mass ratio of the eucalyptus leaf extract, the starch sucrase solution and the sucrose solution is 6:7:40. Wherein. The solvent of the starch sucrase is PBS buffer with a pH of 7.4. 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride are dissolved in deionized water and then fixed to a 1000ml volumetric flask. The enzyme can function well in the PBS solution.

[0027] If the mass ratio is greater than 6:7, the glycosylation degree is too high, which will lead to a decrease in the methylation degree, and the protein binding ability of the eucalyptus leaf extract will be poor, which will lead to poor anti-aging and moisturizing abilities of the liquid medicine. At the same time, due to the high degree of glycosylation, the eucalyptus leaf extract can exist more stably in the solution, and the storage time of the liquid medicine will be greatly extended.

[0028] Compared with the prior art, the present invention has the following advantages: by applying silk fibroin and eucalyptus leaf extract to the wet wipes liquid formula, it not only has good skin care effects and bactericidal, antibacterial and anti-inflammatory effects, but is also non-irritating to the skin, healthy and environmentally friendly, and the liquid ingredients are more stable and can be stored for a long time, thereby improving the shelf life of the wet wipes liquid. DETAILED DESCRIPTION

[0029] The technical content and effects of the present invention are further described in detail below in conjunction with the embodiments, but the present invention is not limited thereto.

[0030] Example 1

[0031] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0032] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3 The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0033] (2) Take 10.0 g of eucalyptus leaves and grind them into particles (average particle size is 0.6 mm) with a crusher. The particles are dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles are filtered out and immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After being filtered out and recovered, the solid particles are immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles are filtered out and used for later use.

[0034] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0035] (4) Blend the eucalyptus leaf extract with 0.02 mol / ml methylase solution (solvent is PBS buffer solution with a pH of 7.4, the same below), add 0.05 mol / ml S-adenosylmethionine solution (solvent is PBS buffer solution with a pH of 7.4, the same below), and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0036] (5) Solution I was mixed with 0.04 mol / ml amylosucrase (solvent was PBS buffer solution with a pH of 7.4, the same below) and 0.08 mol / ml sucrose solution (solvent was PBS buffer solution with a pH of 7.4, the same below) for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution, and amylosucrase solution was 6:7:40, and then the enzyme was inactivated to obtain solution II for use.

[0037] (6) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 11:7:7:75 to obtain a skin care wet wipes solution.

[0038] Example 2

[0039] (1) 15.0 g of silkworm cocoons were taken, the outer layer was peeled off, the cocoons were cut open, washed with deionized water, dried in an oven at 40° C., cut into small pieces of 3 mm in length and 2 mm in width after drying, washed again with deionized water, and dried at 40° C.;

[0040] The silkworm pieces were mixed with 0.5 wt% Na 2 CO 3The degumming was performed by boiling the silk in the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:40 g / ml, and boiled until the silk fibroin was completely dissolved. The silk was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 60 hours. The water was changed every 3 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -25°C for 60 hours to obtain silk fibroin powder for later use.

[0041] (2) 15.0 g of eucalyptus leaves were crushed into particles (average particle size of 0.7 mm) using a pulverizer. The particles were dissolved in isododecane at a ratio of 1:60 g / ml and dissolved at 30°C for 4 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:70 g / ml at 40°C for 10 h. After filtration, they were immersed in glycerol at a bath ratio of 1:100 g / ml at 40°C for 12 h. The solid particles were recovered by filtration for later use.

[0042] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:20 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 1500 and dialyze them for 60 hours, changing the water every 3 hours. After the dialysis, freeze-dry them at -25°C for 60 hours to obtain the eucalyptus leaf extract for use.

[0043] (4) Blend the eucalyptus leaf extract with 0.03 mol / ml methylase solution, add 0.06 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0044] (5) Solution I was mixed with 0.05 mol / ml amylosucrase solution and 0.09 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution and amylosucrase solution was 6:7:40, and then the enzyme was inactivated to obtain solution II for use.

[0045] (6) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 15:8:7:70 to obtain a skin care wet wipes solution.

[0046] Example 3

[0047] (1) 18.0 g of silkworm cocoons were taken, the outer layer was peeled off, the cocoons were cut open, washed with deionized water, dried in an oven at 43° C., cut into small pieces of 2 mm in length and 1 mm in width after drying, washed again with deionized water, and dried at 43° C.;

[0048] The silkworm pieces were mixed with 0.75 wt% Na 2 CO 3 The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:50 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 72 hours. The water was changed every 4 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -20°C for 72 hours to obtain a silk fibroin powder for later use.

[0049] (2) 18.0 g of eucalyptus leaves were crushed into particles (average particle size 0.8 mm) using a pulverizer. The particles were dissolved in isododecane at a ratio of 1:70 g / ml and dissolved at 40°C for 3 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:90 g / ml at 50°C for 12 h. After filtration, they were immersed in glycerol at a bath ratio of 1:120 g / ml at 50°C for 16 h. The solid particles were recovered by filtration for later use.

[0050] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:30 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 1000 and dialyze them for 72 hours, changing the water every 4 hours. After the dialysis, freeze-dry them at -30°C for 72 hours to obtain the eucalyptus leaf extract for use.

[0051] (4) Blend the eucalyptus leaf extract with 0.04 mol / ml methylase solution, add 0.08 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0052] (5) Solution I was mixed with 0.06 mol / ml amylosucrase solution and 0.12 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution and amylosucrase solution was 6:7:40, and then the enzyme was inactivated to obtain solution II for use.

[0053] (6) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 18:9:6:67 to obtain a skin care wet wipes solution.

[0054] Comparative Example 1 (the only difference from Example 1 is that no silk fibroin is added)

[0055] (1) Take 10.0 g of eucalyptus leaves and grind them into particles (average particle size 0.6 mm) with a crusher. The particles are dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles are filtered out and then immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After being filtered out and recovered, the solid particles are immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles are filtered out and then used for later use.

[0056] (2) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0057] (3) Blend the eucalyptus leaf extract with 0.02 mol / ml methylase solution, add 0.05 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0058] (4) Solution I was mixed with 0.04 mol / ml amylosucrase solution and 0.08 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution and amylosucrase solution was 6:7:40, and then the solution was inactivated to obtain solution II for use.

[0059] (5) Solution II, glycerin, and ultrapure water were mixed in a ratio of 7:7:86 to obtain a skin care wet wipes solution. Comparative Example 2 (the only difference from Example 1 is that no eucalyptus leaf extract was added)

[0060] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0061] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0062] (2) Silk fibroin powder, glycerol, and ultrapure water are mixed in a ratio of 11:7:82 to obtain a skin care wet wipes liquid.

[0063] Comparative Example 3 (the only difference from Example 1 is that the eucalyptus leaf extract is not methylated)

[0064] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0065] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3 The degumming was performed by boiling the silk in the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0066] (2) 10.0 g of eucalyptus leaves were crushed into particles (average particle size of 0.6 mm) using a crusher. The particles were dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After filtration, they were immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles were recovered by filtration for later use.

[0067] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0068] (4) Mix the eucalyptus leaf extract with 0.04 mol / ml amylosucrase solution and 0.08 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the sucrose solution and the amylosucrase solution is 6:7:40. Then, the mixture is inactivated to obtain solution II for later use.

[0069] (5) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 11:7:7:75 to obtain a skin care wet wipes solution.

[0070] Comparative Example 4 (the only difference from Example 1 is that the eucalyptus leaf extract is not glycosylated)

[0071] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0072] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3 The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0073] (2) 10.0 g of eucalyptus leaves were crushed into particles (average particle size of 0.6 mm) using a crusher. The particles were dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After filtration, they were immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles were recovered by filtration for later use.

[0074] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0075] (4) Blend the eucalyptus leaf extract with 0.02 mol / ml methylase solution, add 0.05 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0076] (5) Silk fibroin powder, solution I, glycerol, and ultrapure water are mixed in a ratio of 11:7:7:75 to obtain a skin care wet wipes solution.

[0077] Comparative Example 5 (the difference from Example 1 is that the ratio of eucalyptus leaf extract to methylase solution is 2:1)

[0078] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0079] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3 The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0080] (2) 10.0 g of eucalyptus leaves were crushed into particles (average particle size of 0.6 mm) using a crusher. The particles were dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After filtration, they were immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles were recovered by filtration for later use.

[0081] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0082] (4) Blend the eucalyptus leaf extract with 0.02 mol / ml methylase solution, add 0.05 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 8:4:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0083] (5) Solution I was mixed with 0.04 mol / ml amylosucrase and 0.08 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution, and amylosucrase solution was 6:7:40, and then the solution was inactivated to obtain solution II for use.

[0084] (6) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 11:7:7:75 to obtain a skin care wet wipes solution.

[0085] Comparative Example 6 (the difference from Example 1 is that the ratio of eucalyptus leaf extract to amylosucrase solution is 2:1)

[0086] (1) Take 10.0 g of silkworm cocoons, peel off the outer layer of the cocoons, cut them open, wash them with deionized water, dry them in an oven at 37°C, cut them into small pieces of 4 mm long and 3 mm wide, rinse them again with deionized water, and dry them at 37°C;

[0087] The silkworm pieces were mixed with 0.25 wt% Na 2 CO 3 The degumming was performed by boiling the solution for 50 minutes, and the degumming was repeated for 3 times. The degumming result was collected, washed and dried. The obtained silk was added to a calcium alcohol solution with a molar ratio of 1:2:8 of calcium chloride, anhydrous ethanol and ultrapure water at a bath ratio of 1:30 g / ml, and boiled until the silk fibroin was completely dissolved. The silk fibroin was cooled to room temperature and vacuum filtered. The filtrate was put into a dialysis bag and dialyzed with ultrapure water for 48 hours. The water was changed every 2 hours. After the dialysis, a silk fibroin solution was obtained. The solution was freeze-dried at -30°C for 48 hours to obtain silk fibroin powder for later use.

[0088] (2) 10.0 g of eucalyptus leaves were crushed into particles (average particle size of 0.6 mm) using a crusher. The particles were dissolved in isododecane at a ratio of 1:50 g / ml and dissolved at 20°C for 5 h. The solid particles were recovered by filtration. The solid particles were immersed in glycerol at a bath ratio of 1:50 g / ml at 30°C for 8 h. After filtration, they were immersed in glycerol at a bath ratio of 1:80 g / ml at 30°C for 8 h. The solid particles were recovered by filtration for later use.

[0089] (3) Dry the solid particles to remove the residual organic solvent, then dissolve them in ultrapure water at a ratio of 1:10 g / ml, pour them into a dialysis bag with a molecular weight cutoff of 2000 and dialyze them for 48 hours, changing the water every 2 hours. After the dialysis, freeze-dry them at -20°C for 48 hours to obtain the eucalyptus leaf extract for use.

[0090] (4) Blend the eucalyptus leaf extract with 0.02 mol / ml methylase solution, add 0.05 mol / ml S-adenosylmethionine solution, and mix and react at 37° C. for 2 h, wherein the mass ratio of the eucalyptus leaf extract, the methylase solution and the S-adenosylmethionine solution is 5:6:30, and then perform enzyme inactivation treatment to obtain solution I for use.

[0091] (5) Solution I was mixed with 0.04 mol / ml amylosucrase and 0.08 mol / ml sucrose solution for reaction at 37° C. for 2 h, wherein the mass ratio of eucalyptus leaf extract, sucrose solution, and amylosucrase solution was 2:1:10. The mixture was then inactivated to obtain solution II for later use.

[0092] (6) Silk fibroin powder, solution II, glycerol, and ultrapure water are mixed in a ratio of 11:7:7:75 to obtain a skin care wet wipes solution.

[0093] Performance Testing

[0094] (1) Ultraviolet absorbance: Use a UV-visible spectrophotometer to detect the UV resistance of the wet wipes solution. Select the working wavelength of 200-800 and perform the test in absorbance mode. Rinse the cuvette with deionized water, and then fill it with the reference solution (deionized water) and the wet wipes solution in turn to measure the absorbance of the wet wipes solution.

[0095] (2) Moisture content of non-woven fabric after being naturally placed for 8 hours: Weigh a piece of non-woven fabric, immerse it in the wet wipe solution until the non-woven fabric is fully absorbed, take out the non-woven fabric and weigh it. The difference between the two is the amount of absorbed solution M. Then place it at room temperature and weigh it after 8 hours. Subtract the weight of the non-woven fabric from the obtained weight and calculate the content of the preserved solution as m. Then the moisture retention rate = m / M.

[0096] (3) Survival rate of bacteria in culture dishes: Take a culture dish, disinfect the surface and place it on a clean bench. Use a sterile cotton swab to pick up Gram-positive cocci, inoculate them into a plate culture medium, and culture them in a 37°C incubator for 2 days. Add wet wipes solution to the culture dish and cover it with culture medium. After leaving it for 1 hour, observe the survival of bacteria and calculate the bacterial survival rate.

[0097] (4) OD value of drug solution: Take the drug solution of wet wipes for indirect enzyme-linked immunosorbent assay, weigh a certain amount of drug solution and dilute it to 10μg / ml with 0.5mol / L CB solution, add it to the ELISA plate, add three wells, 100μL in each well, and coat overnight at 4°C. Discard the liquid in the wells, add 200μL of 0.05mol / L PBS solution to wash, wash three times, add 200μL of 1% BSA solution to each well, and incubate at 37°C for 2h. Repeat the washing steps, add 100μL of 10μg / ml rabbit anti-mulberry silk protein (primary antibody) to each well, and incubate at 37°C for 1h. Repeat the washing steps, add 100μL of HRP-labeled goat anti-rabbit antibody (secondary antibody) to each well, and incubate at 37°C for 1h. Repeat the washing steps and increase the number of washing times to 5 times, add 50 μL of TMB color developer solution A and 50 μL of solution B, shake evenly, react for 10 minutes in the dark, then add 100 μL of H2SO4 to terminate the reaction, and then place it in a microplate reader to measure the absorbance.

[0098] (5) Longest effective time: Take a piece of non-woven fabric and immerse it in the drug solution until it is fully absorbed. Take out the non-woven fabric and place it at room temperature. After leaving it for 1 hour, take a small amount of liquid from it and add it to the culture dish for culturing bacteria (take a culture dish, disinfect the surface and place it on the clean bench, use a sterile cotton swab to dip Gram-positive cocci, inoculate it into the plate culture medium, and place it in a 37°C incubator for 2 days). Observe the survival of the bacteria (the culture dish is replaced each time) until the drug solution has basically no effect on the survival rate of the bacteria. Record the time.

[0099] The anti-ultraviolet and moisturizing tests were performed on the drug solutions obtained in Examples 1-3 and Comparative Examples 1-6. The results are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] Table 2

[0104]

[0105] As can be seen from the data in Tables 1 and 2, compared with Example 1, Comparative Example 1 cannot effectively moisturize for a long time due to the absence of silk protein, and because it cannot moisturize for a long time, the effective ingredients of the liquid medicine will also fail quickly, and due to the lack of silk protein, the ultraviolet light absorption ability of the liquid medicine in Comparative Example 1 is also very poor, and it cannot effectively resist ultraviolet rays, that is, it does not have the effect of anti-aging. Compared with Example 1, Comparative Example 2 has a similar moisture retention rate and ultraviolet absorption due to the presence of silk protein, but there is no eucalyptus leaf component in Comparative Example 2, and the ability of disinfection and sterilization is very poor, so its effective time of sterilization and disinfection is basically zero. Compared with Example 1, Comparative Example 3 has a poor protein binding ability of the solution due to the lack of methylation, and its anti-aging and moisturizing effects are lower than those of Example 1, and other aspects of the ability include sterilization and disinfection, and effective time are relatively low. Compared with Example 1, Comparative Example 4 has a relatively poor stability of the effective ingredients in the liquid medicine that is not glycosylated, so it will reduce the longest effective time of sterilization, and similarly, the liquid medicine is also lower than Example 1 in other aspects. Compared with Example 1, in Comparative Example 5, the methylation degree is too high, resulting in a subsequent glycosylation degree that is too low, so the effective ingredients of the eucalyptus leaf extract cannot be well preserved, which will greatly shorten the shelf life of the medicinal solution, and the antibacterial effect will also decrease to a certain extent. However, at the same time, due to the high degree of methylation, the protein binding ability is strong, resulting in the anti-aging and moisturizing abilities of the medicinal solution becoming stronger. Compared with Example 1, in Comparative Example 6, the glycosylation degree is too high, resulting in a decrease in the methylation degree, so the protein binding ability of the eucalyptus leaf extract becomes poor, resulting in the anti-aging and moisturizing abilities of the medicinal solution becoming poor. At the same time, due to the high degree of glycosylation, the eucalyptus leaf extract can exist more stably in the solution, the shelf life of the medicinal solution will be greatly extended, and the bactericidal effect will also be slightly improved.

[0106] In summary, the present invention, through the glycosylation and methylation of eucalyptus leaf extract, makes the liquid medicine have anti-aging, whitening, moisturizing, disinfection, bactericidal and other effects that other wet wipes liquid medicines do not have, and methylation makes it more combined with silk fibroin, and the whitening, moisturizing and anti-aging effects are more powerful, and glycosylation makes it more stable, which has been greatly enhanced in terms of storage time.

[0107] The above embodiments of the present invention are merely examples for illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing a novel skin care wet wipes liquid, characterized in that: The following steps are involved: (1) Pre-treating silkworm cocoons to obtain silk fibroin powder for later use; (2) crushing eucalyptus leaves into particles, dissolving them in isododecane at 20-40°C for 2-5 hours, filtering them, and then impregnating them with glycerol, filtering and recovering solid particles; the ratio of eucalyptus leaves to isododecane is 1:40-80 g / mL; the eucalyptus leaves are impregnated with glycerol as follows: first, impregnating them with glycerol at 30-50°C for 6-12 hours, and the ratio of eucalyptus leaves to glycerol is 1:50-100 g / mL; then, impregnating them with glycerol at 30-50°C for 8-16 hours, and the ratio of eucalyptus leaves to glycerol is 1:70-120 g / mL; (3) dialyzing the solid particles with water, and then freeze-drying them to obtain eucalyptus leaf extract; the ratio of the solid particles to water is 1:10-30 g / mL; the dialyzing of the solid particles with water is as follows: dissolving the solid particles with water, pouring them into a dialysis bag with a molecular weight cutoff of 1000-5000, and dialyzing them for 2-3 days, and changing the water every 2-4 hours; (4) Mixing the eucalyptus leaf extract with a 0.02-0.04 mol / mL methylase solution, adding a 0.05-0.08 mol / mL S-adenosylmethionine solution, mixing and reacting at 36-37° C. for 2-3 hours, and then performing an enzyme inactivation treatment to obtain solution I; The mass ratio of the eucalyptus leaf extract to the methylase solution is not greater than 5:6; (5) Mix solution I with 0.04-0.06 mol / mL amylosucrase solution and 0.08-0.12 mol / mL sucrose solution, react at 36-37°C for 2-3 hours, and then inactivate the enzyme to obtain solution II for later use; The mass ratio of the eucalyptus leaf extract to the amylosucrase solution is not greater than 6:7; (6) Silk fibroin powder, solution II, glycerol, and water are mixed in a mass ratio of 7-21:7-10:5-8:67-75 to prepare a skin care wet wipes solution.

2. The method for preparing the novel skin care wet wipes liquid as claimed in claim 1, characterized in that: In step (1), the pretreatment is as follows: removing the outer layer of the silkworm cocoons and crushing them to obtain silkworm fragments; boiling the silkworm fragments in a Na2CO3 solution for 50-70 minutes for degumming, repeating the degumming three times, collecting the degumming product, washing and drying the product, adding the obtained silk to a calcium alcohol solution, boiling until the silk is completely dissolved, cooling to room temperature, vacuum filtering, dialyzing the obtained filtrate, and obtaining a silk fibroin solution after dialysis. After freeze-drying, a silk fibroin powder is obtained for later use.

3. The method for preparing the novel skin care wet wipes liquid as claimed in claim 2, characterized in that: The ratio of the silkworm fragments to the Na2CO3 solution is 1:30-100 g / mL; the concentration of the Na2CO3 solution is 0.1-0.8 wt%.

4. The method for preparing the novel skin care wet wipes liquid as claimed in claim 2 or 3, characterized in that: The ratio of the silk to the calcium alcohol solution is 1:30-60 g / mL; the calcium alcohol solution is prepared by dissolving calcium chloride in a mixed solution of ethanol and water, wherein the molar ratio of calcium chloride, anhydrous ethanol and water is 1:2:

8.

5. The method for preparing the novel skin care wet wipes liquid as claimed in claim 1, characterized in that: In step (3), the freeze-drying temperature is -30 to -15°C and the time is 2 to 3 days.

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