Andrographolide with soothing function and preparation method and application thereof

Abstract

This invention proposes a soothing andrographolide, its preparation method, and its applications, belonging to the field of cosmetic technology. The method involves sterilizing and inducing culture of *Andrographis paniculata* leaves, followed by crushing, enzymatic hydrolysis, and extraction with a deep eutectic solvent to obtain the extract. This extract is then added to an adsorbent, stirred for adsorption, filtered, desorbed, and reacted with salicylic acid to obtain andrographolide salicylate ester. Alcoholic esters are then prepared, thus yielding the soothing andrographolide. The soothing andrographolide prepared by this invention significantly improves the product's water solubility and bioavailability, is safe and non-irritating to the skin, and has anti-redness, anti-allergic, anti-inflammatory, and soothing effects. The addition of alcohol improves the flexibility and extensibility of the structure, resulting in better skin permeability than liposomes and stronger transdermal penetration, thereby expanding its application scope.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to andrographolide with soothing properties, its preparation method, and its application. Background Technology

[0002] Andrographolide is a diterpenoid lactone compound extracted from the plant *Andrographis paniculata*, belonging to the Acanthaceae family. It possesses significant pharmacological effects, including antibacterial, anti-inflammatory, antitumor, antiviral, and immunomodulatory properties. It has been widely used in traditional medicine to treat various infectious and inflammatory diseases.

[0003] The preparation of andrographolide typically involves soaking andrographis paniculata leaves in 95% ethanol, followed by decolorization with activated carbon, ethanol recovery by distillation, and obtaining crude crystals from the concentrate. The crude crystals then undergo dissolution, decolorization, filtration, recrystallization, and washing purification to finally obtain the pale yellow andrographolide product. The overall yield of this process is approximately 1-1.5%. Alternatively, solvent extraction via pressing can be used; however, this method has the disadvantage of difficulty in completely removing organic solvents, and the resulting andrographolide is poorly soluble in aqueous solutions, resulting in low bioavailability, thus significantly limiting its application.

[0004] Andrographolide has a wide range of clinical applications, including the treatment of bacillary dysentery, leptospirosis, meningitis, and pneumonia. It is also used to enhance adrenal cortex function and treat upper respiratory tract infections. Andrographolide derivatives, such as andrographolide acetonide, andrographolide styrax, have been developed to improve its water solubility and bioavailability; these derivatives also have wide clinical applications.

[0005] However, the use of andrographolide may also be accompanied by some adverse reactions, such as vomiting, stomach discomfort, and loss of appetite when taken orally. In some cases, injectable andrographolide preparations may cause drug rash, upper abdominal pain, and anaphylactic shock. Nevertheless, the toxic side effects of andrographolide are relatively minor, with a median lethal dose (LD50) of [missing information]. 50 The concentration in mice was 13.19 g / kg. Summary of the Invention

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing andrographolide with soothing function. The method involves disinfecting and inducing culture of andrographis paniculata leaves, crushing and enzymatically hydrolyzing them, and then extracting them with a deep eutectic solvent to obtain an extract. The extract is then added to an adsorbent, stirred for adsorption, filtered, desorbed, and reacted with salicylic acid to obtain andrographolide salicylate ester. The alcohol body is then prepared to obtain andrographolide with soothing function.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Wash fresh Andrographis paniculata leaves with water, disinfect them in sequence with alcohol, sodium hypochlorite solution and mercuric chloride solution, take them out and wash them clean, and carry out callus culture in culture medium.

[0010] S2. Add the composition to the culture medium, and then carry out induction culture under warm and red light conditions to obtain induced callus tissue;

[0011] S3. The induced callus tissue is added to water, broken down, hydrolyzed with a compound enzyme, and the enzyme is inactivated to obtain the enzymatic hydrolysis composition.

[0012] S4. Add the enzymatic hydrolysis composition to an aqueous solution containing a deep eutectic solvent, extract with ultrasound, filter, add sodium phosphate to the filtrate to precipitate, dry the filtrate, add acetone, add sodium phosphate to precipitate, filter, collect the liquid, dry, and obtain the extract;

[0013] S5. The extract was added to the adsorbent, stirred and adsorbed, filtered, the solid was added to acetone for desorption, filtered, the solid was dried and recycled, the solvent was removed from the liquid under reduced pressure, the product was added to toluene, salicylic acid and catalyst were added, the reaction was heated and stirred, filtered, washed and dried to obtain andrographolide salicylate.

[0014] S6. Andrographolide salicylate, phospholipids, and cholesterol were dissolved in ethanol, and the ethanol was removed by rotary evaporation under reduced pressure to obtain a transparent film. An ethanol solution was added, and the film was sonicated and filtered through a microporous membrane to obtain andrographolide with soothing function.

[0015] 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, and the culture medium is MS or 1 / 2 MS medium with 1-3mg / L 6-BA added.

[0016] As a further improvement of the present invention, the temperature for heat preservation in step S2 is 30-35°C, the 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%.

[0017] As a further improvement of the present invention, the mass ratio of the induced callus tissue to water in step S3 is 1:5-7, the amount of the compound enzyme added is 2-3 wt% of the total mass of the system, the compound enzyme is cellulase and pectinase in a mass ratio of 10:4-7, the enzymatic hydrolysis temperature is 35-45℃, and the time is 2-4h.

[0018] As a further improvement of the present invention, the aqueous solution containing the eutectic solvent in step S4 has a water content of 40-60 wt%, the eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 2-4:1, the ultrasonic extraction power is 1500-2000W, the time is 1-3h, and the mass ratio of the product to sodium phosphate is 10:1-2.

[0019] As a further improvement of the present invention, the mass ratio of the extract to the adsorbent in step S5 is 2-3:10, the mass ratio of the product, salicylic acid and 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℃, and the time is 5-7h.

[0020] Preferably, the adsorbent is a MOF.

[0021] As a further improvement of the present invention, the mass ratio of andrographolide salicylate, phospholipid 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.

[0022] This invention further protects a soothing andrographolide prepared by the above-described preparation method.

[0023] This invention further protects the application of the above-mentioned andrographolide with soothing function in the preparation of cosmetics.

[0024] The present invention has the following beneficial effects:

[0025] This invention utilizes callus culture technology to obtain Andrographis paniculata callus, resulting in stable andographolide extraction rates. The process is green, free of pesticide residues and other hazardous substances. Furthermore, the biomass production of andrographolide can be increased by regulating the culture environment to induce callus formation, thereby improving yield. Temperatures of 30-35℃ are beneficial for increasing andrographolide content, and red light treatment further enhances this effect. Increasing sulfur application (thiourea) promotes the synthesis and accumulation of andrographolide in Andrographis paniculata. Organic nitrogen sources (urea, thiourea) can upregulate... HMGR , DXS , GGPS and ApCPSThe expression level is increased, thereby increasing the content of andrographolide. Under high phosphorus (sodium phosphate) conditions, the synthesis content of andrographolide is significantly increased.

[0026] This invention induces callus tissue to undergo enzymatic hydrolysis with a complex enzyme, resulting in the rupture of most cells and promoting the release of a high amount of andrographolide, thereby greatly improving the yield. In deep eutectic solvent extraction, it has low volatility and high solubility, which is a significant advantage in the extraction of andrographolide compounds, thus greatly improving the extraction rate of andrographolide.

[0027] The MOF mixture of this invention has a good specific adsorption effect on andrographolide, and has an excellent separation effect, thus obtaining andrographolide with high purity, which facilitates subsequent reactions. It also has a large specific surface area, large loading capacity, and is easy to desorb and reuse, thereby reducing the cost of separation and purification.

[0028] Salicylic acid is one of the earliest nonsteroidal anti-inflammatory drugs (NSAIDs), but due to its strong irritant properties, it is often modified into a prodrug in clinical practice to obtain corresponding derivatives before use. The andrographolide salicylate ester prepared in this invention not only possesses the good anti-inflammatory effects of salicylic acid, but also exhibits significantly reduced skin irritation and a soothing effect under the action of andrographolide. The resulting ester derivative has low skin irritation and possesses anti-redness, anti-allergic, and anti-inflammatory effects. Compared to directly adding a mixture of the two, the coupled ester derivative significantly reduces the adverse effects of salicylic acid on the skin, while simultaneously improving the water solubility and bioavailability of andrographolide, thereby enhancing efficacy, reducing dosage, lowering costs, and improving transdermal absorption.

[0029] In this invention, the liposome is composed of phospholipids, polyols, and water, effectively improving the skin penetration efficiency of the drug. The addition of alcohol enhances the flexibility and extensibility of the structure, resulting in superior skin permeability compared to liposomes and stronger transdermal penetration. Encapsulating andrographolide salicylate in the liposome effectively improves the transdermal penetration of the active ingredient, enabling skin retention and enhancing water solubility, thus significantly improving the product's bioavailability and expanding its application scope.

[0030] The andrographolide prepared by this invention has greatly improved the water solubility and bioavailability of the product. It is safe and non-irritating to the skin and has the effects of reducing redness, anti-allergy, anti-inflammation and soothing. The addition of alcohol improves the flexibility and extensibility of the structure, and the skin permeability is better than that of liposomes, with stronger transdermal properties, thereby expanding its application scope. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Cellulase (10,000 U / g) and pectinase (10,000 U / g) were both purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0033] Preparation Example 1: Preparation of MOFs

[0034] The method is as follows: Weigh 1.67g of terephthalic acid, 4g of chromium nitrate, 2g of cobalt chloride, 1g of zinc nitrate, 0.5g of HF, and 50mL of water into a stainless steel high-pressure reactor lined with polytetrafluoroethylene. React at a constant temperature of 205℃ for 10h. After cooling, take out the product, wash it with dichloromethane, and dry it to obtain metal-organic frameworks (MOFs).

[0035] Test Example 1: Static adsorption of andrographolide:

[0036] Dissolve an appropriate amount of andrographolide standard in 60% methanol-water solution to prepare a 5 mg / mL sample solution. Take 5 mL of the sample solution and add MOFs (andrographolide:MOFs mass ratio of 2:10, 2.5:10, and 3:10). Stir at room temperature for 4 hours. Centrifuge.

[0037] Collect the supernatant. Analyze the concentrations of the original sample solution and the supernatant after adsorption using HPLC, and calculate the mass adsorption rate (%).

[0038] Mass adsorption rate (%) = [(Initial concentration - Residual concentration after adsorption) × Solution volume] / Mass of MOFs × 100%

[0039] The results are shown in Table 1.

[0040] Table 1

[0041]

[0042] As shown in the table above, the MOFs of this invention have a good adsorption effect on andrographolide.

[0043] Example 1

[0044] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0045] S1. Wash fresh Andrographis paniculata leaves with water, then disinfect them sequentially with 75% alcohol for 10 seconds, 2wt% sodium hypochlorite solution for 20 minutes, and 1wt% mercuric chloride solution for 10 minutes. Remove and wash them, then culture the callus tissue in MS medium containing 1 mg / L 6-BA (cytokinin) for 1 day.

[0046] S2. Add 3 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:7:10, to the culture medium. Then, under red light conditions with an intensity of 7000 Lux at 30°C for 12 hours and darkness for the rest of the time, induce the culture for 5 days to obtain induced callus tissue.

[0047] S3. Add the induced callus tissue to 5 times its volume of water, mince it, add a compound enzyme, the amount of the compound enzyme added is 2wt% of the total mass of the system, enzymatically hydrolyze at 35℃ for 2h, inactivate the enzyme, and obtain the enzymatic hydrolysis composition.

[0048] The composite enzyme is cellulase and pectinase in a mass ratio of 10:4.

[0049] S4. Add 10g of the enzymatic hydrolysis composition to 200mL of an aqueous solution (water content 40wt%) containing a deep eutectic solvent, extract with ultrasound at 1500W for 1h, filter, dry the filtrate, add 1g of the product to 50mL of water, add 1g of sodium phosphate to precipitate, filter, collect the liquid, dry, and obtain the extract.

[0050] The deep eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 2:1;

[0051] S5. Add 2g of the extract to 10g of the metal-organic framework (MOF) prepared in Preparation Example 1, add 100mL of water, stir and adsorb for 10min, filter, add the solid to acetone for desorption, filter, dry the solid and recycle, remove the solvent under reduced pressure in the liquid, add 3g of the product to 200mL of toluene, add 1.3g of salicylic acid and 0.2g of concentrated sulfuric acid, heat to 150℃, stir and react for 5h, filter, wash, dry to obtain andrographolide salicylate;

[0052] S6. Dissolve 5 mg of andrographolide salicylate, 12 mg of phospholipid, and 6 mg of cholesterol in 20 mL of ethanol. Remove the ethanol by rotary evaporation under reduced pressure to obtain a transparent film. Add 20 mL of 50 wt% ethanol solution, sonicate at 1000 W for 10 min, and filter with a 0.22 μm microporous membrane to obtain andrographolide with soothing function.

[0053] Example 2

[0054] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0055] S1. Wash fresh Andrographis paniculata leaves with water, then disinfect them sequentially with 75% alcohol for 20 seconds, 4wt% sodium hypochlorite solution for 30 minutes, and 2wt% mercuric chloride solution for 15 minutes. Remove and wash them, then culture the callus tissue in 1 / 2 MS medium containing 3 mg / L 6-BA for 2 days.

[0056] S2. Add 5 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 2:10:15, to the culture medium. Then, under red light conditions with an intensity of 8000 Lux at 35°C for 15 h and darkness for the rest of the time, induce the culture for 7 days to obtain induced callus tissue.

[0057] S3. Add the induced callus tissue to 7 times its volume of water, mince it, add a compound enzyme, the amount of the compound enzyme added is 3 wt% of the total mass of the system, enzymatically hydrolyze at 45℃ for 4 h, inactivate the enzyme, and obtain the enzymatic hydrolysis composition.

[0058] The composite enzyme is cellulase and pectinase in a mass ratio of 10:7.

[0059] S4. Add 10g of the enzymatic hydrolysis composition to 200mL of an aqueous solution (water content is 60wt%) containing a deep eutectic solvent, extract with ultrasound at 2000W for 3h, filter, dry the filtrate, add 1g of the product to 50mL of water, add 2g of sodium phosphate to precipitate, filter, collect the liquid, dry, and obtain the extract.

[0060] The deep eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 4:1;

[0061] S5. Add 3g of extract to 10g of metal-organic frameworks (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 and recycle, remove the solvent under reduced pressure in the liquid, add 4g of product to 200mL of toluene, add 1.4g of salicylic acid and 0.4g of concentrated sulfuric acid, heat to 160℃, stir and react for 7h, filter, wash, dry to obtain andrographolide salicylate;

[0062] S6. Dissolve 8 mg of andrographolide salicylate, 17 mg of phospholipid, and 8 mg of cholesterol in 20 mL of ethanol. Remove the ethanol by rotary evaporation under reduced pressure to obtain a transparent film. Add 20 mL of 70 wt% ethanol solution, sonicate at 1200 W for 20 min, and filter with a 0.22 μm microporous membrane to obtain andrographolide with soothing function.

[0063] Example 3

[0064] This embodiment provides a method for preparing andrographolide with soothing function, comprising the following steps:

[0065] S1. Wash fresh Andrographis paniculata leaves with water, then disinfect them sequentially with 75% alcohol for 15 seconds, 3wt% sodium hypochlorite solution for 25 minutes, and 1.5wt% mercuric chloride solution for 12 minutes. Remove and wash them, then culture the callus tissue in MS medium containing 2 mg / L 6-BA for 1.5 days.

[0066] S2. Add 4 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, to the culture medium. Then, under red light conditions with an intensity of 7500 Lux at 32°C for 13 hours and darkness for the rest of the time, induce the culture for 6 days to obtain induced callus tissue.

[0067] S3. Add the induced callus tissue to 6 times its volume of water, mince it, add a compound enzyme, the amount of the compound enzyme added is 2.5 wt% of the total mass of the system, enzymatically hydrolyze at 40℃ for 3 h, inactivate the enzyme, and obtain the enzymatic hydrolysis composition.

[0068] The composite enzyme is cellulase and pectinase in a mass ratio of 10:6.

[0069] S4. Add 10g of the enzymatic hydrolysis composition to 200mL of an aqueous solution (50wt% water content) containing a deep eutectic solvent, extract with ultrasound at 1700W for 2h, filter, dry the filtrate, add 1g of the product to 50mL of water, add 1.5g of sodium phosphate to precipitate, filter, collect the liquid, dry, and obtain the extract.

[0070] The deep eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 3:1;

[0071] S5. Add 2.5g of the extract to 10g of the metal-organic framework (MOF) prepared in Preparation Example 1, add 100mL of water, stir and adsorb for 15min, filter, add the solid to acetone for desorption, filter, dry the solid and recycle, remove the solvent under reduced pressure in the liquid, 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℃, stir and react for 6h, filter, wash, dry to obtain andrographolide salicylate;

[0072] S6. Dissolve 7 mg of andrographolide salicylate, 15 mg of phospholipid, and 7 mg of cholesterol in 20 mL of ethanol. Remove the ethanol by rotary evaporation under reduced pressure to obtain a transparent film. Add 20 mL of 60 wt% ethanol solution, sonicate at 1100 W for 15 min, and filter with a 0.22 μm microporous membrane to obtain andrographolide with soothing function.

[0073] Comparative Example 1

[0074] The difference from Example 3 is that no composition was added in step S2.

[0075] Specifically as follows:

[0076] S2. The culture system was induced and cultured for 6 days under red light conditions of 7500 Lux at 32℃ for 13 hours and in darkness for the rest of the time to obtain induced callus tissue.

[0077] Comparative Example 2

[0078] The difference from Example 3 is that the temperature in step S2 is set to 25°C.

[0079] Specifically as follows:

[0080] S2. Add 4 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, to the culture medium. Then, under red light conditions with an intensity of 7500 Lux at 25°C for 13 hours and in darkness for the remaining time, induce callus tissue for 6 days to obtain the induced callus tissue.

[0081] Comparative Example 3

[0082] The difference from Example 3 is that white light is used instead of red light in step S2.

[0083] Specifically as follows:

[0084] S2. Add 4 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, to the culture medium. Then, under white light conditions of 7500 Lux at 325°C for 13 hours and in darkness for the remaining time, induce callus tissue for 6 days to obtain the induced callus tissue.

[0085] Comparative Example 4

[0086] The difference from Example 3 is that 50wt% ethanol is used instead of the deep eutectic solvent in step S4.

[0087] Specifically as follows:

[0088] S4. Add 10g of the enzymatic hydrolysis composition to 200mL of 50wt% ethanol, extract with ultrasound at 1700W for 2h, filter, dry the filtrate, add 1g of the product to 50mL of water to obtain the extract.

[0089] Comparative Example 5

[0090] The difference from Example 3 is that the metal-organic frameworks (MOFs) prepared in Preparation Example 1 were not used for separation and purification in step S5.

[0091] Specifically as follows:

[0092] S5. Add the 3.5 extract 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 for 6 h, filter, wash, and dry to obtain andrographolide salicylate.

[0093] Comparative Example 6

[0094] 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).

[0095] Specifically as follows:

[0096] S6. Dissolve 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 phospholipid, and 7 mg of cholesterol in 20 mL of ethanol. Remove the ethanol by rotary evaporation under reduced pressure to obtain a transparent film. Add 20 mL of 60 wt% ethanol solution, sonicate at 1100 W for 15 min, and filter with a 0.22 μm microporous membrane to obtain andrographolide with soothing function.

[0097] Comparative Example 7

[0098] The difference from Example 3 is that step S6 was not performed.

[0099] Specifically as follows:

[0100] S1. Wash fresh Andrographis paniculata leaves with water, then disinfect them sequentially with 75% alcohol for 15 seconds, 3wt% sodium hypochlorite solution for 25 minutes, and 1.5wt% mercuric chloride solution for 12 minutes. Remove and wash them, then culture the callus tissue in MS medium containing 2 mg / L 6-BA for 1.5 days.

[0101] S2. Add 4 wt% of the composition, which includes urea, sodium hydrogen phosphate and thiourea in a mass ratio of 1:8:12, to the culture medium. Then, under red light conditions with an intensity of 7500 Lux at 32°C for 13 hours and darkness for the rest of the time, induce the culture for 6 days to obtain induced callus tissue.

[0102] S3. Add the induced callus tissue to 6 times its volume of water, mince it, add a compound enzyme, the amount of the compound enzyme added is 2.5 wt% of the total mass of the system, enzymatically hydrolyze at 40℃ for 3 h, inactivate the enzyme, and obtain the enzymatic hydrolysis composition.

[0103] The composite enzyme is cellulase and pectinase in a mass ratio of 10:6.

[0104] S4. Add 10g of the enzymatic hydrolysis composition to 200mL of an aqueous solution (50wt% water content) containing a deep eutectic solvent, extract with ultrasound at 1700W for 2h, filter, dry the filtrate, add 1g of the product to 50mL of water, add 1.5g of sodium phosphate to precipitate, filter, collect the liquid, dry, and obtain the extract.

[0105] The deep eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 3:1;

[0106] S5. Add 2.5g of the extract to 10g of the metal-organic framework (MOF) prepared in Preparation Example 1, add 100mL of water, stir and adsorb for 15min, filter, add the solid to acetone for desorption, filter, dry the solid and recycle, remove the solvent under reduced pressure, 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℃, stir and react for 6h, filter, wash, dry to obtain andrographolide salicylate, which is andrographolide with soothing function.

[0107] Test Example 2

[0108] Test subject: Andrographolide with soothing function prepared in Examples 1-3 and Comparative Examples 1-7 of this invention.

[0109] Test method: as shown in Table 2 below.

[0110] Table 2

[0111]

[0112] According to the test protocol in Table 2, the model was transferred to a 6-well plate (with 0.9 mL of EpiGrowth medium added beforehand), and the test group number was labeled on the 6-well plate. 12.5 μL of 0.2% SLS solution and 12.5 μL of the corresponding concentration of working solution were added to the surface of the sample group, distributing the sample on the model surface. The plate was then incubated in a CO2 incubator (37℃, 5% CO2) for 24 h. After incubation, the residual test material on the model surface was washed with sterile PBS solution, and any remaining material inside and outside the model was wiped away with sterile cotton swabs. Detection and analysis were performed according to the instructions of the IL-1α ELISA kit, TNF-α ELISA kit, and PGE2 ELISA kit.

[0113] The results are shown in Table 3.

[0114] Table 3

[0115]

[0116] As shown in the table above, the andrographolide with soothing function prepared in Examples 1-3 of this invention can significantly reduce the content of IL-1α, TNF-α and PGE2.

[0117] Test Example 2: Human Skin Patch Test and Allergy Test

[0118] Test subject: Andrographolide with soothing function prepared in Examples 1-3 and Comparative Examples 1-7 of this invention.

[0119] Experimental Methods: Subjects aged 24-54 years were randomly divided into 10 groups of 6 subjects each. A 5cm x 5cm area was marked on the flexor surface of the right forearm of each subject and medically disinfected. The marked area was then treated by introducing nutrient solution through a 36-needle microneedle for 30 seconds, followed by wiping away any remaining liquid with a saline-soaked cotton ball to keep the marked area dry. Test samples were placed into the wells of a 1-10 spectrophotometer, with a volume of 10-20 μL. Bathing was prohibited during the test. A negative reaction was considered normal. The test samples were applied to the subject's face, neck, and behind the ear. Skin condition was observed after 10 minutes.

[0120] The results are shown in Table 4.

[0121] Table 4

[0122]

[0123] As shown in the table above, the andrographolide patches with soothing function prepared in Examples 1-3 and Comparative Examples 1-7 of this invention showed negative reactions, indicating they are safe to use and that no allergic reactions were observed.

[0124] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing andrographolide with soothing properties, characterized in that, Includes the following steps: S1. Wash fresh Andrographis paniculata leaves with water, disinfect them in sequence with alcohol, sodium hypochlorite solution and mercuric chloride solution, take them out and wash them clean, and carry out callus culture in culture medium. S2. Add the composition to the culture medium, and then induce callus formation under incubation and red light conditions to obtain induced callus tissue; the incubation temperature is 30-35℃, the red light intensity is 7000-8000 Lux, the illumination time is 12-15h, and the content of the composition in the culture medium is 3-5wt%; the composition includes urea, sodium hydrogen phosphate, and thiourea in a mass ratio of 1-2:7-10:10-15; S3. The induced callus tissue is added to water, broken down, hydrolyzed with a compound enzyme, and the enzyme is inactivated to obtain the enzymatic hydrolysis composition. S4. The enzymatic hydrolysis composition is added to an aqueous solution containing a deep eutectic solvent, ultrasonically extracted, filtered, the filtrate dried, the product added to water, sodium phosphate precipitate added, filtered, the liquid collected, dried, and the extract obtained; the aqueous solution containing the deep eutectic solvent has a water content of 40-60 wt%, and the deep eutectic solvent is a mixture of glycerol and choline chloride prepared in a molar ratio of 2-4:1; S5. The extract is added to the adsorbent, stirred for adsorption, filtered, the solid is added to acetone for desorption, filtered, the solid is dried and recycled, the solvent is removed from the liquid under reduced pressure, the product is added to toluene, salicylic acid and catalyst are added, heated and stirred for reaction, filtered, washed, and dried to obtain andrographolide salicylate; the adsorbent is a metal-organic framework (MOF). S6. Andrographolide salicylate, phospholipids, and cholesterol were dissolved in ethanol, and the ethanol was removed by rotary evaporation under reduced pressure to obtain a transparent film. An ethanol solution was added, and the film was sonicated and filtered through a microporous membrane to obtain andrographolide with soothing function.

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

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

4. The preparation method according to claim 1, characterized in that, The ultrasonic extraction in step S4 uses a power of 1500-2000W and a time of 1-3 hours.

5. The preparation method according to claim 1, 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 catalyst is 3-4:1.3-1.4:0.2-0.4, the catalyst is concentrated sulfuric acid, and the heating and stirring reaction is carried out at a temperature of 150-160℃ for 5-7 hours.

6. The preparation method according to claim 1, 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 50-70 wt%, the power of the ultrasound is 1000-1200 W, the time is 10-20 min, and the pore size of the microporous filter membrane is 0.22 μm.

7. Andrographolide with soothing function prepared by the preparation method according to any one of claims 1-6.

8. The use of andrographolide with soothing function as described in claim 7 in the preparation of cosmetics.

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