Andrographolide with soothing function as well as preparation method and application of andrographolide

Abstract

The invention provides andrographolide with a soothing function as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The preparation method comprises the following steps: performing disinfection, induction culture, crushing and enzymolysis on andrographis paniculata leaves, then performing deep eutectic solvent extraction to obtain an extract, adding the extract into an adsorbent, performing stirring adsorption, filtration and desorption, and reacting with salicylic acid to prepare andrographolide salicylate and ethosome, thereby obtaining the andrographolide with the soothing function. According to the andrographolide with the soothing function, the water solubility and bioavailability of a product are greatly improved, the andrographolide is safe and non-irritating to skin and has the effects of fading redness, resisting allergy, diminishing inflammation and soothing, the flexibility and ductility of the structure are improved due to the addition of alcohol, the skin permeability is superior to that of lipidosome, and the transdermal property is higher, so that the application range of the andrographolide is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to andrographolide with soothing function, a preparation method and application thereof. Background Art

[0002] Andrographolide is a diterpene lactone compound extracted from the Acanthaceae plant Andrographis paniculata, which has significant pharmacological effects, including antibacterial, anti-inflammatory, anti-tumor, antiviral, and immunomodulatory effects. It is widely used in traditional medicine to treat various infections and inflammatory diseases.

[0003] The preparation method of andrographolide usually includes soaking andrographolide leaves with 95% ethanol, followed by decolorization with activated carbon, distillation to recover ethanol, and obtaining a crude crystal product from the concentrated solution. The crude crystal product is then subjected to steps such as dissolution, decolorization, filtration, recrystallization, washing and refining to finally obtain a light yellow andrographolide product. The total yield of this process is approximately between 1-1.5%. Alternatively, a squeeze solvent extraction method is used as the main method, but the disadvantage of this method is that the organic solvent is difficult to completely remove, and the obtained andrographolide is not well soluble in an aqueous solution system, and has low bioavailability, which greatly limits its application.

[0004] The clinical application of andrographolide is very extensive, including the treatment of bacillary dysentery, leptospirosis, meningitis, pneumonia, etc. In addition, it is also used to enhance adrenal cortex function and treat upper respiratory tract infections. Andrographolide derivatives, such as Chuanhuning, Yanhuning and Lianbizhi, have been developed to improve their water solubility and bioavailability, and these derivatives are also widely used in clinical practice.

[0005] However, the use of andrographolide may also be accompanied by some adverse reactions, such as oral administration may cause vomiting, gastric discomfort and loss of appetite. In some cases, injection of andrographolide preparations may cause drug rash, upper abdominal pain and anaphylactic shock. Nevertheless, the toxicity and side effects of andrographolide are relatively small, and its median lethal dose (LD50) is 1. 50 ) is 13.19 g / kg in mice. Summary of the invention

[0006] The purpose of the present invention is to propose andrographolide with soothing function and its preparation method and application. Not only is the preparation method simple and the raw material source is wide, the prepared aerosol can be directly delivered to the lungs through spray administration, has the unique performance of magnetic targeted drug delivery, releases drugs in a targeted and fixed point, improves the efficacy, effectively promotes tumor cell apoptosis, and plays an anti-lung cancer and anti-pneumonia role.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a method for preparing andrographolide with a soothing function. The method comprises the following steps: disinfecting andrographolide leaves, inducing culture, crushing, enzymolysis and then extracting with a deep eutectic solvent to obtain an extract, adding the extract to an adsorbent, stirring for adsorption, filtering, desorbing, reacting with salicylic acid to obtain andrographolide salicylate, preparing an alcohol body, and thus obtaining andrographolide with a soothing function.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with alcohol, sodium hypochlorite solution, and mercuric chloride solution, remove them, wash them, and culture callus in a culture medium;

[0011] S2. The composition is added to the culture medium, and then cultured under heat preservation and red light conditions to obtain induced callus;

[0012] S3. The induced callus is added to water, crushed, added with a complex enzyme for enzymatic hydrolysis, and the enzyme is inactivated to obtain an enzymatic composition;

[0013] S4. adding the enzymatic hydrolysis composition to an aqueous solution containing a deep eutectic solvent, extracting with ultrasound, filtering, adding sodium phosphate to the filtrate for precipitation, drying the filtrate, adding to acetone, adding sodium phosphate for precipitation, filtering, collecting the liquid, and drying to obtain an extract;

[0014] S5. adding the extract to an adsorbent, stirring for adsorption, filtering, adding the solid to acetone for desorption, filtering, drying the solid, recycling, removing the solvent from the liquid under reduced pressure, adding the product to toluene, adding salicylic acid and a catalyst, heating and stirring for reaction, filtering, washing, and drying to obtain andrographolide salicylate;

[0015] S6. Add andrographolide salicylate, phospholipids and cholesterol into ethanol and dissolve them. Remove the ethanol by vacuum evaporation to obtain a transparent film. Add ethanol solution, perform ultrasound, and filter with a microporous membrane to obtain andrographolide with soothing function.

[0016] As a further improvement of the present invention, the alcohol in step S1 is 75% alcohol, the disinfection time is 10-20s, the concentration of the sodium hypochlorite solution is 2-4wt%, the disinfection time is 20-30min, the concentration of the mercuric chloride solution is 1-2wt%, the disinfection time is 10-15min, the culture medium is MS or 1 / 2MS culture medium, and 1-3mg / L 6-BA is added.

[0017] As a further improvement of the present invention, the insulation temperature in step S2 is 30-35°C, the illumination intensity of the red light is 7000-8000Lux, the illumination time is 12-15h, the composition includes urea, sodium hydrogen phosphate, and thiourea in a mass ratio of 1-2:7-10:10-15, and the content of the composition in the culture medium is 3-5wt%.

[0018] As a further improvement of the present invention, the mass ratio of the induced callus tissue and water in step S3 is 1:5-7, the added amount of the complex enzyme is 2-3wt% of the total mass of the system, the complex enzyme is cellulase and pectinase, the mass ratio is 10:4-7, the temperature of the enzymatic hydrolysis is 35-45°C, and the time is 2-4h.

[0019] As a further improvement of the present invention, the water content of the aqueous solution containing the deep eutectic solvent in step S4 is 40-60wt%, the deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 2-4:1, the power of the ultrasonic extraction is 1500-2000W, the time is 1-3h, and the mass ratio of the product to sodium phosphate is 10:1-2.

[0020] As a further improvement of the present invention, the mass ratio of the extract and the adsorbent in step S5 is 2-3:10, the mass ratio of the product, salicylic acid and the catalyst is 3-4:1.3-1.4:0.2-0.4, the catalyst is concentrated sulfuric acid, the temperature of the heating and stirring reaction is 150-160°C, and the time is 5-7h.

[0021] Preferably, the adsorbent is MOFs.

[0022] As a further improvement of the present invention, the mass ratio of andrographolide salicylate, phospholipids and cholesterol in step S6 is 5-8:12-17:6-8, the concentration of the ethanol solution is 50-70wt%, the power of the ultrasound is 1000-1200W, the time is 10-20min, and the pore size of the microporous filter membrane is 0.22μm.

[0023] The present invention further protects andrographolide with soothing function obtained by the above preparation method.

[0024] The present invention further protects the use of the andrographolide with soothing function in the preparation of cosmetics.

[0025] The present invention has the following beneficial effects:

[0026] The present invention uses callus culture technology to obtain Andrographolide callus, which has a stable lactone extraction rate, is green, does not contain risk substances such as pesticide residues, and can regulate the cultivation environment to induce callus to increase the bioproduction of andrographolide, thereby improving the yield. The temperature condition of 30-35°C is conducive to increasing the content of andrographolide, and red light treatment increases the content of andrographolide. Increasing the amount of sulfur applied (thiourea) can promote the synthesis and accumulation of andrographolide in Andrographolide. Organic nitrogen sources (urea, thiourea) can increase the expression of HMGR, DXS, GGPS and ApCPS, thereby increasing the content of andrographolide. Under high phosphorus (sodium phosphate) conditions, the synthesis content of andrographolide is significantly improved.

[0027] After the induced callus tissue of the present invention is hydrolyzed by the composite enzyme, most of the cells are broken, thereby promoting the release of highly generated andrographolide, thereby greatly improving the yield. In the extraction of deep eutectic solvents, it has lower volatility and higher solubility, which has great advantages in extracting andrographolide compounds, thereby greatly improving the extraction rate of andrographolide.

[0028] The MOFs mixture of the present invention has a good specific adsorption effect on andrographolide and has an excellent separation effect, thereby obtaining andrographolide with higher purity, which is convenient for subsequent reactions, and has a large specific surface area and a large loading capacity, and is easy to desorb and reuse, thereby reducing the cost of separation and purification.

[0029] Salicylic acid is one of the earliest non-steroidal anti-inflammatory drugs, but due to its strong irritation, salicylic acid is often used clinically after being modified with prodrugs to obtain corresponding derivatives. The andrographolide salicylate prepared by the present invention not only has the good anti-inflammatory effect of salicylic acid, but also greatly reduces the skin irritation under the action of andrographolide, and has the effect of soothing the skin. The prepared ester derivative has low skin irritation and has the effects of redness reduction, anti-sensitization and anti-inflammatory. Compared with directly adding a mixture of the two, the coupled ester derivative greatly reduces the adverse effects of salicylic acid on the skin, and at the same time, improves the water solubility and bioavailability of andrographolide, thereby improving the efficacy, reducing the usage, reducing the cost, and at the same time, also improving the effect of transdermal absorption.

[0030] The ethosomes in the present invention are composed of phospholipids, polyols and water, which effectively improve the skin penetration efficiency of the drug. The addition of alcohol improves the flexibility and ductility of the structure, and the skin penetration performance is better than that of liposomes, and the transdermal property is stronger. After the andrographolide salicylate is wrapped by the ethosomes, the transdermal penetration ability of the active ingredient can be effectively improved, the active ingredient can be retained in the skin, and the water solubility is improved, which greatly improves the bioavailability of the product, thereby expanding its application area.

[0031] The andrographolide with soothing function prepared by the present invention greatly improves the water solubility and bioavailability of the product, is safe and non-irritating to the skin, has the effects of reducing redness, anti-allergy, anti-inflammatory and soothing, and the addition of alcohol improves the flexibility and ductility of the structure, has better skin permeability than liposomes, and has stronger transdermal properties, thereby expanding its application range. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Cellulase, 10,000 U / g, and pectinase, 10,000 U / g, were purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0034] Preparation Example 1 Preparation of MOFs

[0035] The method is as follows: weigh 1.67 g of terephthalic acid, 4 g of chromium nitrate, 2 g of cobalt chloride, 1 g of zinc nitrate, 0.5 g of HF, and 50 mL of water into a stainless steel high-pressure reactor lined with polytetrafluoroethylene, react at a constant temperature of 205°C for 10 hours, take out the product after cooling, wash it with dichloromethane, and dry it to obtain metal organic frameworks MOFs.

[0036] Test Example 1: Static adsorption of andrographolide:

[0037] Take an appropriate amount of andrographolide standard and dissolve it in 60% methanol-water solution to prepare a sample solution with a concentration of 5 mg / mL. Take 5 mL of sample solution in a vial and add MOFs (the mass ratio of andrographolide:MOFs is 2:10, 2.5:10, 3:10). Stir at room temperature for 4 hours. Centrifuge,

[0038] The supernatant was collected and the concentrations of the original sample solution and the supernatant after adsorption were detected by HPLC, and the mass adsorption rate (%) was calculated.

[0039] Mass adsorption rate (%) = [(initial concentration - residual concentration after adsorption) × solution volume] / mass of MOFs × 100%

[0040] The results are shown in Table 1.

[0041] Table 1

[0042] Group Mass adsorption rate (%) The mass ratio of andrographolide:MOFs is 2:10 92.5 The mass ratio of andrographolide:MOFs is 2.5:10 94.1 The mass ratio of andrographolide:MOFs is 3:10 95.7

[0043] It can be seen from the above table that the MOFs of the present invention have a good adsorption effect on andrographolide.

[0044] Example 1

[0045] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0046] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with 75% alcohol for 10 seconds, 2wt% sodium hypochlorite solution for 20 minutes, and 1wt% mercuric chloride solution for 10 minutes, take them out and wash them, and culture callus in MS medium containing 1 mg / L 6-BA (cytokinin) for 1 day;

[0047] S2. Add 3 wt % of a composition to the culture medium, wherein the composition comprises urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:7:10, and then culture the culture medium at a temperature of 30°C and a red light intensity of 7000 Lux for 12 h, and the rest of the time in darkness for 5 days to obtain induced callus;

[0048] S3. The induced callus was added to 5 times the volume of water, minced, and a composite enzyme was added, wherein the amount of the composite enzyme added was 2wt% of the total mass of the system, and the enzymatic hydrolysis was carried out at 35°C for 2h, and the enzyme was inactivated to obtain an enzymatic composition;

[0049] The complex enzyme is cellulase and pectinase, with a mass ratio of 10:4;

[0050] S4. 10 g of the enzymatic composition was added to 200 mL of an aqueous solution containing a deep eutectic solvent (water content of 40 wt%), ultrasonically extracted at 1500 W for 1 h, filtered, the filtrate was dried, 1 g of the product was added to 50 mL of water, 1 g of sodium phosphate was added for precipitation, filtered, the liquid was collected, and dried to obtain an extract;

[0051] The deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 2:1;

[0052] S5. Add 2 g of the extract to 10 g of the metal organic framework MOFs prepared in Preparation Example 1, add to 100 mL of water, stir and adsorb for 10 min, filter, add the solid to acetone for desorption, filter, dry the solid, recycle, remove the solvent from the liquid under reduced pressure, add 3 g of the product to 200 mL of toluene, add 1.3 g of salicylic acid and 0.2 g of concentrated sulfuric acid, heat to 150° C., stir and react for 5 h, filter, wash, and dry to obtain andrographolide salicylate;

[0053] S6. Add 5 mg of andrographolide salicylate, 12 mg of phospholipids, and 6 mg of cholesterol into 20 mL of ethanol and dissolve them. Remove the ethanol by vacuum evaporation to obtain a transparent film. Add 20 mL of 50 wt% ethanol solution, ultrasonicate at 1000 W for 10 min, and filter with a microporous filter membrane with a pore size of 0.22 μm to obtain andrographolide with soothing function.

[0054] Example 2

[0055] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0056] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with 75% alcohol for 20 seconds, 4wt% sodium hypochlorite solution for 30 minutes, and 2wt% mercuric chloride solution for 15 minutes, take them out and wash them, and culture callus in 1 / 2MS medium containing 3mg / L 6-BA for 2 days;

[0057] S2. Add 5 wt % of the composition to the culture medium, wherein the composition includes urea, sodium hydrogen phosphate, and thiourea in a mass ratio of 2:10:15, and then culture for 7 days at a temperature of 35 ° C and a red light intensity of 8000 Lux for 15 h, and the rest of the time in the dark to obtain induced callus;

[0058] S3. The induced callus was added to 7 times the volume of water, minced, and a composite enzyme was added, wherein the amount of the composite enzyme added was 3wt% of the total mass of the system, and the enzymatic hydrolysis was performed at 45°C for 4h, and the enzyme was inactivated to obtain an enzymatic composition;

[0059] The complex enzyme is cellulase and pectinase, with a mass ratio of 10:7;

[0060] S4. 10 g of the enzymatic composition was added to 200 mL of an aqueous solution containing a deep eutectic solvent (water content of 60 wt%), and ultrasonic extraction was performed at 2000 W for 3 h, filtered, and the filtrate was dried. 1 g of the product was added to 50 mL of water, 2 g of sodium phosphate was added for precipitation, filtered, the liquid was collected, and dried to obtain an extract;

[0061] The deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 4:1;

[0062] S5. Add 3g of the extract to 10g of the metal organic framework MOFs prepared in Preparation Example 1, add to 100mL of water, stir and adsorb for 20min, filter, add the solid to acetone for desorption, filter, dry the solid, recycle, remove the solvent from the liquid under reduced pressure, add 4g of the product to 200mL of toluene, add 1.4g of salicylic acid and 0.4g of concentrated sulfuric acid, heat to 160°C, stir and react for 7h, filter, wash, and dry to obtain andrographolide salicylate;

[0063] S6. Add 8 mg of andrographolide salicylate, 17 mg of phospholipids, and 8 mg of cholesterol into 20 mL of ethanol and dissolve them. Remove the ethanol by vacuum evaporation to obtain a transparent film. Add 20 mL of 70 wt% ethanol solution, ultrasonicate at 1200 W for 20 min, and filter with a microporous filter membrane with a pore size of 0.22 μm to obtain andrographolide with soothing function.

[0064] Example 3

[0065] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0066] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with 75% alcohol for 15 seconds, 3wt% sodium hypochlorite solution for 25 minutes, and 1.5wt% mercuric chloride solution for 12 minutes, take them out and wash them, and culture callus in MS medium containing 2 mg / L 6-BA for 1.5 days;

[0067] S2. Add 4 wt % of a composition to the culture medium, wherein the composition comprises urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, and then culture the culture medium at a temperature of 32 ° C. and a red light intensity of 7500 Lux for 13 h, and the rest of the time in the dark for 6 days to obtain induced callus;

[0068] S3. The induced callus was added to 6 volumes of water, minced, and a composite enzyme was added, wherein the amount of the composite enzyme added was 2.5wt% of the total mass of the system, and the enzymatic hydrolysis was performed at 40°C for 3h, and the enzyme was inactivated to obtain an enzymatic composition;

[0069] The complex enzyme is cellulase and pectinase, with a mass ratio of 10:6;

[0070] S4. 10 g of the enzymatic composition was added to 200 mL of an aqueous solution containing a deep eutectic solvent (water content of 50 wt%), ultrasonically extracted at 1700 W for 2 h, filtered, and the filtrate was dried. 1 g of the product was added to 50 mL of water, 1.5 g of sodium phosphate was added for precipitation, filtered, the liquid was collected, and dried to obtain an extract;

[0071] The deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 3:1;

[0072] S5. Add 2.5 g of the extract to 10 g of the metal organic framework MOFs prepared in Preparation Example 1, add to 100 mL of water, stir and adsorb for 15 min, filter, add the solid to acetone for desorption, filter, dry the solid, recycle, remove the solvent from the liquid under reduced pressure, add 3.5 g of the product to 200 mL of toluene, add 1.35 g of salicylic acid and 0.3 g of concentrated sulfuric acid, heat to 155 ° C, stir and react for 6 h, filter, wash, and dry to obtain andrographolide salicylate;

[0073] S6. Add 7 mg of andrographolide salicylate, 15 mg of phospholipids, and 7 mg of cholesterol into 20 mL of ethanol and dissolve them. Remove the ethanol by vacuum evaporation to obtain a transparent film. Add 20 mL of 60 wt% ethanol solution, ultrasonicate at 1100 W for 15 min, and filter with a microporous filter membrane with a pore size of 0.22 μm to obtain andrographolide with soothing function.

[0074] Comparative Example 1

[0075] Compared with Example 3, the difference is that no composition is added in step S2.

[0076] The details are as follows:

[0077] S2. The culture system was placed at 32°C, with a red light intensity of 7500 Lux, the illumination time was 13 h, and the rest of the time was in darkness. The induction culture was carried out for 6 days to obtain induced callus tissue.

[0078] Comparative Example 2

[0079] Compared with Example 3, the difference is that the temperature in step S2 is set to 25°C.

[0080] The details are as follows:

[0081] S2. Add 4 wt % of a composition comprising urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12 to the culture medium, and then culture the culture medium under a temperature of 25°C and a red light condition of 7500 Lux for 13 h, with the rest of the time being in the dark, for 6 days to obtain induced callus.

[0082] Comparative Example 3

[0083] Compared with embodiment 3, the difference is that white light is used instead of red light in step S2.

[0084] The details are as follows:

[0085] S2. Add 4 wt % of a composition comprising urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12 to the culture medium, and then culture the culture medium at a temperature of 325°C and under white light conditions with an intensity of 7500 Lux for 13 h, and the rest of the time in darkness for 6 days to obtain induced callus.

[0086] Comparative Example 4

[0087] Compared with Example 3, the difference is that 50 wt % ethanol is used instead of the deep eutectic solvent in step S4.

[0088] The details are as follows:

[0089] S4. 10 g of the enzymatic hydrolysis composition was added to 200 mL of 50 wt% ethanol, and ultrasonic extraction was performed at 1700 W for 2 h. The mixture was filtered, the filtrate was dried, and 1 g of the product was added to 50 mL of water to obtain an extract.

[0090] Comparative Example 5

[0091] Compared with Example 3, the difference is that the metal organic framework MOFs prepared in Preparation Example 1 are not used for separation and purification in step S5.

[0092] The details are as follows:

[0093] S5. Add the extract from 3.5 into 200 mL of toluene, add 1.35 g of salicylic acid and 0.3 g of concentrated sulfuric acid, heat to 155°C, stir to react for 6 hours, filter, wash, and dry to obtain andrographolide salicylate.

[0094] Comparative Example 6

[0095] Compared with Example 3, the difference is that in step S6, andrographolide salicylate is replaced by a mixture of andrographolide and salicylate (the mass ratio of andrographolide to salicylate is 3.5:1.35).

[0096] The details are as follows:

[0097] S6. Add 7 mg of a mixture (a mixture of andrographolide and salicylate, wherein the mass ratio of andrographolide to salicylate is 3.5:1.35), 15 mg of phospholipids, and 7 mg of cholesterol into 20 mL of ethanol and dissolve them. Remove the ethanol by rotary evaporation under reduced pressure to obtain a transparent film. Add 20 mL of a 60 wt% ethanol solution, ultrasonically control the mixture at 1100 W for 15 min, and filter the mixture through a microporous filter membrane with a pore size of 0.22 μm to obtain andrographolide with a soothing function.

[0098] Comparative Example 7

[0099] Compared with the embodiment 3, the difference is that step S6 is not performed.

[0100] The details are as follows:

[0101] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with 75% alcohol for 15 seconds, 3wt% sodium hypochlorite solution for 25 minutes, and 1.5wt% mercuric chloride solution for 12 minutes, take them out and wash them, and culture callus in MS medium containing 2 mg / L 6-BA for 1.5 days;

[0102] S2. Add 4 wt % of a composition to the culture medium, wherein the composition comprises urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, and then culture the culture medium at a temperature of 32 ° C. and a red light intensity of 7500 Lux for 13 h, and the rest of the time in the dark for 6 days to obtain induced callus;

[0103] S3. The induced callus was added to 6 volumes of water, minced, and a composite enzyme was added, wherein the amount of the composite enzyme added was 2.5wt% of the total mass of the system, and the enzymatic hydrolysis was performed at 40°C for 3h, and the enzyme was inactivated to obtain an enzymatic composition;

[0104] The complex enzyme is cellulase and pectinase, with a mass ratio of 10:6;

[0105] S4. 10 g of the enzymatic composition was added to 200 mL of an aqueous solution containing a deep eutectic solvent (water content of 50 wt%), ultrasonically extracted at 1700 W for 2 h, filtered, and the filtrate was dried. 1 g of the product was added to 50 mL of water, 1.5 g of sodium phosphate was added for precipitation, filtered, the liquid was collected, and dried to obtain an extract;

[0106] The deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 3:1;

[0107] S5. Add 2.5g of the extract to 10g of the metal organic framework MOFs obtained in Preparation Example 1, add to 100mL of water, stir and adsorb for 15min, filter, add the solid to acetone for desorption, filter, dry the solid, recycle, reduce the pressure on the liquid to remove the solvent, add 3.5g of the product to 200mL of toluene, add 1.35g of salicylic acid and 0.3g of concentrated sulfuric acid, heat to 155°C, stir and react for 6h, filter, wash, and dry to obtain andrographolide salicylate, which is andrographolide with soothing function.

[0108] Test Example 2

[0109] Test object: andrographolide with soothing function prepared in Examples 1-3 and Comparative Examples 1-7 of the present invention.

[0110] Test method: as shown in Table 2.

[0111] Table 2

[0112]

[0113]

[0114] According to the test scheme in Table 2, the model was transferred to a 6-well plate (0.9 mL EpiGrowth culture medium was added in advance), and the test group number was marked on the 6-well plate. 12.5 μL of 0.2% SLS solution and 12.5 μL of sample working solution of the corresponding mass concentration were added to the surface of the sample group, and the samples were distributed on the surface of the model and placed in a CO2 incubator (37 ° C, 5% CO2) for 24 hours. After the incubation, the residual test substance on the surface of the model was washed with sterile PBS solution, and the residual body inside and outside the model was wiped off with a sterile cotton swab, and the detection and analysis were performed according to the operating instructions of the IL-1α ELISA kit, TNF-α ELISA kit, and PGE2 ELISA kit.

[0115] The results are shown in Table 3.

[0116] Table 3

[0117] Group IL-1α content (pg / mL) TNF-α content (pg / mL) PGE2 content (pg / mL) Blank control 6.22 16.29 25.18 Negative control 152.61 57.66 129.45 Positive Control 126.82 18.49 36.63 Example 1 62.27 21.10 42.56 Example 2 60.92 20.89 42.83 Example 3 59.57 20.15 40.72 Comparative Example 1 72.51 26.98 65.26 Comparative Example 2 75.19 28.63 67.81 Comparative Example 3 70.83 27.09 66.09 Comparative Example 4 79.56 31.25 72.25 Comparative Example 5 94.26 40.66 89.92 Comparative Example 6 76.08 29.47 69.42 Comparative Example 7 102.25 44.56 92.58

[0118] It can be seen from the above table that the andrographolide with soothing function prepared in Examples 1-3 of the present invention can significantly reduce the contents of IL-1α, TNF-α and PGE2.

[0119] Test Example 2 Human skin patch test and allergy test

[0120] Test object: andrographolide with soothing function prepared in Examples 1-3 and Comparative Examples 1-7 of the present invention.

[0121] Experimental method: Subjects aged 24-54 were selected and randomly divided into 10 groups, with 6 people in each group. Among them, the subject's right forearm flexor side was marked with a 5cm×5cm area and medical disinfection was used to create an injury in the marked area. The method was: 36-needle microneedle introduced nutrient solution for 30s, and a cotton ball soaked in physiological saline was used to gently wipe the residual liquid to keep the marked area dry; the test samples were placed in the 1-10 spot tester holes, respectively, with a dosage of 10-20μL. Bathing was prohibited during the test, and the normal reaction was negative. The test samples were applied to the face, neck and back of the ear of the subject, and the skin condition of the subject was observed after 10 minutes.

[0122] The results are shown in Table 4.

[0123] Table 4

[0124] Group Patch reaction Facial skin Neck skin Skin behind the ear Example 1 Negative No allergy No allergy No allergy Example 2 Negative No allergy No allergy No allergy Example 3 Negative No allergy No allergy No allergy Comparative Example 1 Negative No allergy No allergy No allergy Comparative Example 2 Negative No allergy No allergy No allergy Comparative Example 3 Negative No allergy No allergy No allergy Comparative Example 4 Negative No allergy No allergy No allergy Comparative Example 5 Negative No allergy No allergy No allergy Comparative Example 6 Negative No allergy No allergy No allergy Comparative Example 7 Negative No allergy No allergy No allergy

[0125] As can be seen from the above table, the patch reactions of andrographolide with soothing function obtained in Examples 1-3 of the present invention and Comparative Examples 1-7 are negative reactions, and are safe to use without allergic reactions.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing andrographolide with soothing function, characterized in that: The leaves of Andrographis paniculata are disinfected, induced for culture, crushed, enzymatically hydrolyzed and then extracted with a deep eutectic solvent to obtain an extract, which is added to an adsorbent, stirred for adsorption, filtered, desorbed, and reacted with salicylic acid to obtain andrographolide salicylate, to prepare an alcohol body, and thus to obtain andrographolide with a soothing function.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Wash fresh Andrographis paniculata leaves with water, disinfect them with alcohol, sodium hypochlorite solution, and mercuric chloride solution, remove them, wash them, and culture callus in a culture medium; S2. The composition is added to the culture medium, and then cultured under heat preservation and red light conditions to obtain induced callus; S3. The induced callus is added to water, crushed, added with a complex enzyme for enzymatic hydrolysis, and the enzyme is inactivated to obtain an enzymatic composition; S4. adding the enzymatic hydrolysis composition to an aqueous solution containing a deep eutectic solvent, extracting with ultrasound, filtering, adding sodium phosphate to the filtrate for precipitation, drying the filtrate, adding to acetone, adding sodium phosphate for precipitation, filtering, collecting the liquid, and drying to obtain an extract; S5. adding the extract to an adsorbent, stirring for adsorption, filtering, adding the solid to acetone for desorption, filtering, drying the solid, recycling, removing the solvent from the liquid under reduced pressure, adding the product to toluene, adding salicylic acid and a catalyst, heating and stirring for reaction, filtering, washing, and drying to obtain andrographolide salicylate; S6. Add andrographolide salicylate, phospholipids and cholesterol into ethanol and dissolve them. Remove the ethanol by vacuum evaporation to obtain a transparent film. Add ethanol solution, perform ultrasound, and filter with a microporous membrane to obtain andrographolide with soothing function.

3. The preparation method according to claim 2, characterized in that: In step S1, the alcohol is 75% alcohol, the disinfection time is 10-20s, the concentration of the sodium hypochlorite solution is 2-4wt%, the disinfection time is 20-30min, the concentration of the mercuric chloride solution is 1-2wt%, the disinfection time is 10-15min, the culture medium is MS or 1 / 2MS culture medium, and 1-3mg / L 6-BA is added.

4. The preparation method according to claim 2, characterized in that: The insulation temperature in step S2 is 30-35° C., the illumination intensity of the red light is 7000-8000 Lux, the illumination time is 12-15 h, the composition includes urea, sodium hydrogen phosphate, and thiourea in a mass ratio of 1-2:7-10:10-15, and the content of the composition in the culture medium is 3-5 wt %.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass ratio of the induced callus tissue to water is 1:5-7, the added amount of the complex enzyme is 2-3wt% of the total mass of the system, the complex enzyme is cellulase and pectinase, the mass ratio is 10:4-7, the enzymolysis temperature is 35-45°C, and the time is 2-4h.

6. The preparation method according to claim 2, characterized in that: The water content of the aqueous solution containing the deep eutectic solvent in step S4 is 40-60wt%, the deep eutectic solvent is a mixture of glycerol and choline chloride in a molar ratio of 2-4:1, the power of the ultrasonic extraction is 1500-2000W, the time is 1-3h, and the mass ratio of the product to sodium phosphate is 10:1-2.

7. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of the extract to the adsorbent is 2-3:10, the mass ratio of the product, salicylic acid and the catalyst is 3-4:1.3-1.4:0.2-0.4, the catalyst is concentrated sulfuric acid, the temperature of the heating and stirring reaction is 150-160°C, and the time is 5-7h.

8. The preparation method according to claim 2, characterized in that: In step S6, the mass ratio of andrographolide salicylate, phospholipid and cholesterol is 5-8:12-17:6-8, the concentration of the ethanol solution is 50-70wt%, the power of the ultrasound is 1000-1200W, the time is 10-20min, and the pore size of the microporous filter membrane is 0.22μm.

9. Andrographolide with soothing function obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the andrographolide with soothing function as claimed in claim 9 in the preparation of cosmetics.

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