Preparation method and application of oldenlandia diffusa extracting solution

Abstract

The invention discloses a hedyotis diffusa extracting solution, which contains 2500-4000 ppm of asperuloside, 4500-6500 ppm of deacetylated asperuloside methyl ester + paederia scandens-glycoside methyl ester + asperuloside, less than 100 ppm of total flavone, no characteristic chromogenic reaction in anthraquinone identification, less than 100 ppm of asperuloside acid, less than 100 ppm of deacetylated asperuloside acid, less than 100 ppm of geniposidic acid, and 6.0-7.0 of pH (potential of hydrogen). The method comprises the following steps: carrying out vacuum ultrasonic treatment to obtain an extracting solution, carrying out centrifugal separation and vacuum concentration, adjusting the pH value to 2.5-3.0 by using an acid, aging, carrying out membrane filtration, and carrying out ion exchange column chromatography refining to obtain a permeate A and an oldenlandia diffusa alkaloid eluent J1; and filtering the permeate A with a membrane, carrying out molecular imprinting polymerization adsorption separation, carrying out vacuum concentration, carrying out ultrafiltration, adjusting the pH value to 6-7 with J1, and carrying out heat preservation and sterilization to obtain the oldenlandia diffusa extract.

Description

Technical Field

[0001] The invention relates to a bioactive raw material in the field of daily chemicals, in particular to an extract of Herba Ophiopogonis. Background Art

[0002] Hedyotis diffusa is a common Chinese herbal medicine, which is the whole herb of Hedyotis diffusa Willd., also known as Oldenlandia diffusa Roxb., a plant of the genus Hedyotis in the Rubiaceae family. It was first recorded in the "Guangxi Chinese Materia Medica" and has the effects of clearing away heat, detoxifying, promoting dampness and relieving jaundice. It is mainly used to treat carbuncle, ulcer, snake bite, edema, damp-heat jaundice, etc., and has a wide range of application history and value. The main components of Hedyotis diffusa include iridoids, flavonoids and anthraquinone compounds, followed by organic acids, sterol compounds, etc. The common iridoid compounds in Hedyotis diffusa mainly include cephaloside methyl ester, deacetylcephaloside methyl ester, cephaloside acid, cephaloside glycoside and cephaloside glycoside. Some of the ingredients here have application defects when used in the daily chemical field. For example, flavonoids are easy to change color and change color with changes in pH value, resulting in product instability; anthraquinone ingredients have safety risks and may cause allergies, and are also prone to cause discoloration of product formulas; some ingredients in organic acids are irritating to a certain extent; coumarins are prone to phototoxicity and photosensitivity when used externally; relatively safe and effective are the rich varieties of cyclopentane saponins contained in it, but although this type of ingredient has strong efficacy, there are also some problems. For example, cyclopentane saponins are not very stable in solution and are easily degraded. Some cyclopentane saponins also have carboxylic acid groups and are mildly irritating. It is difficult to obtain a stable, safe and effective Houttuynia cordata extract, but it has great application value. Summary of the invention

[0003] The present invention provides an extract of Hedyotis diffusa to overcome the defects of the prior art that the content of iridoid compounds such as hedyotis diffusa is high, the irritation is strong, and the extract is not stable in the solution.

[0004] Another object of the present invention is to provide a method for preparing the Hedyotis diffusa extract.

[0005] Another object of the present invention is to provide the use of the Hedyotis diffusa extract in the preparation of soothing products and anti-inflammatory and antioxidant daily chemical products.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: A Hedyotis diffusa extract contains 2500-4000 ppm of calendula glycoside, 4500-6500 ppm of deacetylated calendula methyl ester + scutellaria glycoside methyl ester + calendula glycoside, total flavonoids content less than 100 ppm, no characteristic color reaction for anthraquinone identification, calendula glycoside acid less than 100 ppm, deacetylated calendula glycoside acid less than 100 ppm, geniposide acid less than 100 ppm, and pH value of 6.0-7.0.

[0007] The present invention purifies and separates several specific iridoid glycosides in Hedyotis diffusa, quantifies the contents of these components, and limits the limits of other components with great potential risks in stability and safety, and uses the components with this specific content to form Hedyotis diffusa extract as a product, and provides a corresponding preparation method. Compared with conventional Hedyotis diffusa extract, the Hedyotis diffusa extract has better stability, safety and efficacy when used in the field of daily chemicals. The specific process of the present invention can reduce the decomposition of effective ingredients, increase the content of the main ingredient, calendula glycoside, and control the content of impurities, providing a safe, mild, low-irritation, extremely stable raw material for use in the field of daily chemicals with soothing, anti-inflammatory, repair and other effects.

[0008] The invention also provides a method for preparing an oldenlandia diffusa extract, which comprises obtaining an extract by vacuum ultrasound, centrifugal separation, and vacuum concentration, adjusting the pH value to 2.5-3.0 with acid, aging, membrane filtration, and then refining by ion exchange resin column chromatography to obtain a permeate A and an oldenlandia diffusa alkaloid eluate J1; the permeate A is filtered through a membrane, and then separated by molecular imprinting polymerization adsorption, the separated eluate is vacuum concentrated and ultrafiltered, and finally the oldenlandia diffusa alkaloid eluate J1 is adjusted to pH 6-7, and heat-insulated and sterilized to obtain the oldenlandia diffusa extract.

[0009] The specific steps of the preparation method of the Hedyotis diffusa extract are as follows: The first step is ultrasonic extraction: crushing Hedyotis diffusa powder with ethanol aqueous solution as extraction solvent, vacuum ultrasonic reflux extraction to obtain extract, and centrifugal separation; The second step is concentration and aging: the supernatant is concentrated under reduced pressure, adjusted to pH 2.5-3 by acid, aged for 48-72 hours, and filtered through a membrane until the filtrate is clear; The third step is column chromatography purification: the filtrate is first passed through a cation exchange resin column, and the permeate is then passed through an anion exchange resin column to obtain a permeate A; the cation exchange resin column is rinsed with pure water, and then eluted with a 40-50% ethanol solution containing 8-10% citric acid, and the elution end point is that the pH of the eluate mixture is 5-5.5 to obtain an eluate, which is concentrated under reduced pressure to a solid content of 5-8%, and a solvent is added to adjust the concentration, and a base is added to adjust the pH to 7.5-8.0 to obtain a Herba Ophiopogonis alkaloids eluate J1; Step 4, molecular imprinting polymer adsorption separation: the permeate A is concentrated by membrane and separated by molecular imprinting polymer adsorption, and the eluate is concentrated under reduced pressure and then solvent is added to adjust the concentration to obtain eluate B; Step 5, ultrafiltration: the eluent B is subjected to ultrafiltration again, and the concentration of the permeate is adjusted with the Hedyotis diffusa alkaloid eluent J1 to prepare the Hedyotis diffusa extract.

[0010] In the third step, the loading ratio of the cation exchange resin column is filtrate volume: resin volume = 3-5:1; the loading ratio of the anion exchange resin column is filtrate volume: resin volume = 2-4:1.

[0011] During the molecular imprinting polymer adsorption separation in the fourth step, the loading ratio is filtrate volume: molecular imprinting polymer mass = 5-7 mL / g.

[0012] In the fourth step, the permeate A is concentrated to 300-400% of the crude drug amount by a membrane, the membrane cut-off molecular weight is 100-150 Daltons, and the pressure is 10-15 bar.

[0013] The solvent is a 45-55% polyol solution, and the polyol is any one of butanediol, dipropylene glycol and pentanediol.

[0014] In the fourth step, the molecular imprinted polymer is separated by adsorption, eluted with ethanol 3 to 4 times the mass of the molecular imprinted polymer, the eluent is decompressed at 60 to 65° C., the vacuum degree is ≥ 0.07, and concentrated to a content of 4500 to 9000 ppm of phytosylvatica glycoside.

[0015] The present invention also provides an application of an oldenlandia diffusa extract in the preparation of soothing products and anti-inflammatory and antioxidant products.

[0016] It is mainly used in products that reduce skin redness caused by inflammation by inhibiting inflammatory mediators such as P65, iNOS, and histamine; and products that reduce skin dullness and yellowing caused by inflammatory free radicals by scavenging free radicals; products that promote the skin's own soothing ability by increasing the expression of NRF2, NQO1, and HO-1, further strengthening the soothing process, reducing skin dullness, yellowing, and redness caused by skin inflammation, and making the skin more even-toned.

[0017] Compared with the prior art, the present invention has the following beneficial effects: By extracting and refining non-carboxylic acid cyclopentane glycosides and controlling the limit of specific harmful components, a specific Hedyotis diffusa extract is obtained, which has higher stability, safety and efficacy.

[0018] The obtained Hedyotis diffusa extract has excellent soothing effect compared with similar soothing products. It can relieve oxidative stress response through multiple pathways and has a multi-target synergistic soothing effect. It reduces skin redness caused by the inflammatory process by inhibiting inflammatory mediators such as P65, iNOS, and histamine; reduces skin dullness and yellowing caused by inflammatory free radicals by scavenging free radicals; promotes the skin's own soothing ability by increasing the expression of NRF2, NQO1, and HO-1, further strengthens the soothing process, reduces skin dullness, yellowing, and redness caused by skin inflammation, and makes the skin more even. Through the ultrasonic reflux integrated extraction technology, the extraction efficiency is improved, the degradation of active ingredients is reduced, and the total content of target active ingredients is increased; through acidic aging, the sarcosyl acid is converted into sarcosyl glycoside, which not only reduces the amount of acidic components and irritation, but also increases the content of target active ingredients, and is also beneficial to the removal of flavonoids and anthraquinone components; through the three processes of ion exchange, molecular imprinting separation, and ultrafiltration, the specific target components are obtained and the target impurities are removed; the alkaloid components in the Hedyotis diffusa alkaloid eluate are used to stabilize and control the product pH, thereby enhancing the long-term stability of the target components. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the liquid chromatogram of the standard substance of cypermethrin.

[0020] Figure 2 This is the liquid chromatogram of the standard substance of scutellaria baicalensis.

[0021] Figure 3 This is the liquid chromatogram of the standard sample of cypermethrin at 55°C for 3 days.

[0022] Figure 4 This is the liquid chromatogram obtained after the standard sample of scutellaria baicalensis was placed at 55°C for 3 days, then acidified and stirred at 60°C for 2 hours.

[0023] Figure 5 This is a WB test result diagram of an embodiment of the present invention.

[0024] Figure 6 It is the inhibition rate curve diagram of DPPH of Example 1 of the present invention.

[0025] Figure 7 It is the inhibition rate curve diagram of DPPH of Example 3 of the present invention.

[0026] Figure 8 It is the inhibition rate curve diagram of DPPH of Comparative Example 4 of the present invention.

[0027] Fig. 9 It is the inhibition rate curve diagram of DPPH of Comparative Example 5 of the present invention.

[0028] Fig.10 It is the inhibition rate curve diagram of DPPH of Comparative Example 6 of the present invention.

[0029] Fig.11 It is the liquid phase diagram of Examples 1 to 3 of the present invention.

[0030] Fig.12 It is the liquid phase diagram of comparative examples 4 to 6 of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific implementation methods.

[0032] Unless otherwise specified, the percentage concentrations mentioned in the present invention are all mass percentage concentrations.

[0033] The composition characteristics of the extract of Hedyotis diffusa contain: calendula glycoside content 2500-4000 ppm; deacetylcalendula methyl ester + gentiana glycoside methyl ester + calendula glycoside content 4500-6500 ppm; total flavonoids content <100 ppm; no characteristic color reaction for anthraquinone identification; calendula glycoside <100 ppm; deacetylcalendula glycoside <100 ppm; geniposide <100 ppm; pH=6.0-7.0.

[0034] Process steps: 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure ultrasonic reflux for 1 to 1.5 hours, using 40 to 50% ethanol as the extraction solvent, and the solvent reflux temperature is 30 to 40°C. Refer to ZL 2024 1 0591953.3: Ultrasonic extraction equipment and ultrasonic extraction method for medicinal materials to extract until the mass of the concentrated liquid is 0.8 to 1 times the mass of the medicinal material, and obtain the extract; 3. Centrifuge the extract until it is clear at a speed of 15,000 to 18,000 rpm; 4. The supernatant is concentrated under reduced pressure, with a vacuum degree of ≥0.07 and a temperature of 65-75°C, to a concentration of 150-200% of the crude drug content; 5. Adjust pH to 2.5-3.0 with citric acid and age at 0-4℃ for 48-72h; 6. Filter through 0.45μm membrane until clear; 7. Column chromatography purification: The filtrate of step 6 is first passed through a cation exchange resin column, and the loading ratio is filtrate volume: resin volume = 3-5:1; the permeate is then passed through an anion exchange resin column, and the loading ratio is filtrate volume: resin volume = 2-4:1, to obtain permeate A; the cation exchange resin column is rinsed with 2 times column volume of pure water, and then eluted with 40-50% ethanol solution containing 8-10% citric acid, and the elution end point is that the pH of the eluate mixture is 5-5.5, to obtain the eluate, which is concentrated to a solid content of 5-8% according to the step 4 method, and polyols are added to mix until the polyol content accounts for 45-55%, and sodium hydroxide solution is added to adjust the pH to 7.5-8.0 to obtain snake grass alkaloid eluate J1; the cation exchange resin model is: HD-2, HZD-2; the anion exchange resin model is: D201, D293; Shanghai Huazhen Technology Co., Ltd.; 8. The permeate A is concentrated to 300-400% of the crude drug amount by a membrane, the membrane molecular weight cut-off is 100-150 Daltons, and the pressure is 10-15 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 5-7 mL / g, eluted with 95% ethanol 3-4 times the mass of the molecularly imprinted polymer, the eluate is concentrated at 60-65°C under reduced pressure (vacuum degree ≥ 0.07) to a content of 4500-9000 ppm of phytosan; deacetylated phytosan methyl ester + methyl ester of scutellaria baicalensis + phytosan content of 8000-15000 ppm, and polyol is added and mixed until the polyol content accounts for 45-55%; the molecularly imprinted material is the molecularly imprinted polymer obtained in Example 2 of patent CN2024114181839; 10. Ultrafiltration, membrane cutoff molecular weight is 1000 Dalton, operating pressure is 8-10 bar, when ultrafiltration is performed to the point where the retentate is 30% of the mother liquor, add the solvent composed of the mother liquor, i.e., 45-55% polyol solution, and continue ultrafiltration until the permeate content satisfies the content of calomelanoside (C1) of 2500-4000ppm; the content of deacetylcalomelanoside methyl ester + galomelanoside methyl ester + calomelanoside (C2) of 4500-6500ppm; the content of deacetylcalomelanoside methyl ester + galomelanoside methyl ester + calomelanoside + calomelanoside acid + deacetylcalomelanoside + geniposide acid is recorded as C3; 11. Use J1 to adjust the pH to 6-7, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane to obtain Hedyotis diffusa extract.

[0035] The polyol is any one of butanediol solution, dipropylene glycol and pentanediol.

[0036] Note: Amount of crude drug = the mass of raw materials fed into the process ÷ the mass of the material obtained in this step × 100%.

[0037] Example 1 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure ultrasonic reflux for 1.5 hours, using 40% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Method for Ultrasonic Extraction of Medicinal Materials], and extract until the mass of the concentrated liquid is 1 times the mass of the medicinal material to obtain the extract; 3. Centrifuge until clear at 15,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 65°C, and concentrate to 200% of the crude drug content; 5. Adjust pH to 2.5 with citric acid and age at 0-4℃ for 72h; 6. Filter through 0.45μm membrane until clear; 7. Column chromatography purification: The filtrate of step 6 is first passed through a cation exchange resin column HD-2, with a loading ratio of filtrate volume: resin volume = 3:1; the permeate is then passed through an anion exchange resin column D201, with a loading ratio of filtrate volume: resin volume = 2:1, to obtain permeate A; the cation exchange resin column is rinsed with 2 column volumes of pure water, and then eluted with a 50% ethanol solution containing 10% citric acid, with the elution end point being the pH of the eluate mixture of 5.5, to obtain the eluate, which is concentrated to a solid content of 6.5% according to the method of step 4, and butanediol is added and mixed until the butanediol content accounts for 50%, and 10% sodium hydroxide solution is added to adjust the pH to 8.0, to obtain the snake grass alkaloid eluate J1; 8. The permeate A is concentrated to 300% of the crude drug amount using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 10 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 5 mL / g, eluted with 95% ethanol 3 times the mass of molecularly imprinted polymer, the eluate was concentrated at 60℃ under reduced pressure (vacuum degree ≥ 0.07) to a content of 8834.1 ppm of phytophthora glycoside; the content of deacetylated phytophthora methyl ester + methyl ester of scutellaria serrata + phytophthora glycoside was 13546.2 ppm, butanediol was added and mixed until the content of butanediol accounted for 50%; 10. Ultrafiltration, membrane cut-off molecular weight is 1000 Dalton, 10 bar operating pressure, when ultrafiltration to the retentate liquid is 30% of the mother liquor, add the solvent composed of the mother liquor, i.e. 50% butanediol solution, and continue ultrafiltration to the permeate content: C1=3969.8 ppm; C2=6091.2 ppm; C3=6275.0 ppm; 11. Use J1 to adjust the pH to 6.5, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane, and obtain Hedyotis diffusa extract.

[0038] Example 2 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure and ultrasonic reflux for 1 hour, using 50% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Equipment for ultrasonic extraction of medicinal materials and method for ultrasonic extraction of medicinal materials], and extract the concentrated solution to a mass of 0.8 times the mass of the medicinal material to obtain the extract; 3. Centrifuge until clear at a speed of 18,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 75°C, and concentrate to 150% of the crude drug amount; 5. Adjust pH to 3 with citric acid and age at 0-4℃ for 48h; 6. Filter through 0.45 μm membrane until clear; 7. Column chromatography purification: The filtrate from step 6 is first passed through a cation exchange resin column HZD-2, with a loading ratio of filtrate volume: resin volume = 5:1; the permeate is then passed through an anion exchange resin column D293, with a loading ratio of filtrate volume: resin volume = 4:1, to obtain a permeate A; the cation exchange resin column is rinsed with 2 times the column volume of pure water, and then eluted with a 40% ethanol solution containing 8% citric acid. The elution end point is that the pH of the eluate mixture is 5.0, to obtain an eluate, which is concentrated to a solid content of 5% according to the method in step 4, and dipropylene glycol is added to mix until the dipropylene glycol content is 45%, and 1% sodium hydroxide solution is added to adjust the pH to 7.5 to obtain a snake grass alkaloid eluate J1.

[0039] 8. The permeate A is concentrated to 400% of the crude drug amount using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 15 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 7 mL / g, eluted with 95% ethanol 4 times the mass of molecularly imprinted polymer, the eluate was concentrated at 65℃ under reduced pressure (vacuum degree ≥ 0.07) to a content of 8275.2 ppm of phytosan; the content of deacetylated phytosan methyl ester + methyl ester of scutellaria serrata + phytosan methyl ester was 11401.8 ppm, and dipropylene glycol was added and mixed until the content of dipropylene glycol accounted for 45%; 10.Ultrafiltration, membrane cut-off molecular weight is 1000 Dalton, 8 bar operating pressure, when ultrafiltration to the retentate of 30% of the mother liquor, add the mother liquor and the same composition of the solvent, i.e. 45% dipropylene glycol solution, continue ultrafiltration to the permeate content: C1 = 4153.7 ppm; C2 = 5661.6 ppm; C3 = 5680.8 ppm; 11. Use J1 to adjust the pH to 7, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane, and obtain Hedyotis diffusa extract.

[0040] Example 3 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure and ultrasonic reflux for 75 minutes, using 45% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Equipment for ultrasonic extraction of medicinal materials and method for ultrasonic extraction of medicinal materials], and extract until the mass of the concentrated liquid is 0.9 times the mass of the medicinal material, and obtain the extract; 3. Centrifuge until clear at a speed of 17,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 70°C, and concentrate to 180% of the crude drug amount; 5. Adjust pH to 2.7 with citric acid and age at 0-4℃ for 60h; 6. Filter through 0.45 μm membrane until clear; 7. Column chromatography purification: The filtrate of step 6 is first passed through a cation exchange resin column HD-2, with a loading ratio of filtrate volume: resin volume = 4:1; the permeate is then passed through an anion exchange resin column D201, with a loading ratio of filtrate volume: resin volume = 3:1, to obtain a permeate A; the cation exchange resin column is rinsed with 2 column volumes of pure water, and then eluted with a 45% ethanol solution containing 8.6% citric acid, and the elution end point is that the pH of the eluate mixture is 5.2, to obtain an eluate, which is concentrated to 8% solid content according to the method of step 4, and pentanediol is added and mixed until the pentanediol content accounts for 55%, and 5% sodium hydroxide solution is added to adjust the pH to 7.8 to obtain a snake grass alkaloid eluate J1; 8. The permeate A is concentrated to 350% of the crude drug amount using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 12 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 6 mL / g, eluted with 95% ethanol 3.5 times the mass of molecularly imprinted polymer, the eluate is concentrated at 65℃ under reduced pressure (vacuum degree ≥ 0.07) to a content of 8903.5 ppm of phytophthora glycoside; the content of deacetylated phytophthora methyl ester + methyl ester of scutellaria serrata + phytophthora glycoside is 12598.3 ppm, and pentanediol is added and mixed until the content of pentanediol accounts for 55%; 10. Ultrafiltration, membrane cutoff molecular weight is 1000 Dalton, 9 bar operating pressure, when ultrafiltration to the retentate liquid is 30% of the mother liquor, add the solvent composed of the mother liquor, i.e. 55% pentanediol solution, and continue ultrafiltration to the permeate content: C1=3688.5 ppm; C2=5085.5 ppm; C3=5251.0 ppm; 11. Use J1 to adjust the pH to 6, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane to obtain Hedyotis diffusa extract.

[0041] Comparative Example 1 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure ultrasonic reflux for 1.5 hours, using 40% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Method for Ultrasonic Extraction of Medicinal Materials], and extract until the mass of the concentrated liquid is 1 times the mass of the medicinal material to obtain the extract; 3. Centrifuge until clear at 15,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 65°C, and concentrate to 200% of the crude drug content; 5. Adjust pH to 5 with citric acid and age at 0-4℃ for 72h; 6. Filter through a 0.45 μm membrane until clear.

[0042] Comparative Example 2 Steps 1 to 10 are the same as in Example 1. 11. Use 10% sodium hydroxide solution to adjust the pH to 6.5, sterilize at 90-95℃ for 30 minutes, cool to <45℃, filter through 0.22μm filter membrane, and obtain Hedyotis diffusa extract. Comparative Example 3 Steps 1 to 10 are the same as in Example 1. 11. Sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane to obtain Hedyotis diffusa extract (measured pH=4.9).

[0043] Comparative Example 4 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure ultrasonic reflux for 45 minutes, using 55% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Equipment for ultrasonic extraction of medicinal materials and methods for ultrasonic extraction of medicinal materials], and extract the concentrated solution to a mass of 0.7 times the mass of the medicinal material to obtain the extract; 3. Centrifuge until clear at 14,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 60°C, and concentrate to 125% of the crude drug content; 5. Adjust pH to 2.0 with citric acid and age at 0-4℃ for 36h; 6. Filter through 0.45μm membrane until clear; 7. Column chromatography purification: The filtrate of step 6 is first passed through a cation exchange resin column HD-2, with a loading ratio of filtrate volume: resin volume = 6:1; the permeate is then passed through an anion exchange resin column D201, with a loading ratio of filtrate volume: resin volume = 5:1, to obtain permeate A; the cation exchange resin column is rinsed with 2 column volumes of pure water, and then eluted with a 55% ethanol solution containing 7% citric acid, with the elution end point being the pH of the eluate mixture being 4.5, to obtain the eluate, which is concentrated to a solid content of 4.5% according to the method of step 4, and butanediol is added and mixed until the butanediol content is 40%, and 10% sodium hydroxide solution is added to adjust the pH to 7.0, to obtain the snake grass alkaloid eluate J1; 8. The permeate A is concentrated to 250% of the crude drug amount using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 8 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 4 mL / g, eluted with 95% ethanol 2.5 times the mass of molecularly imprinted polymer, the eluate was concentrated at 55℃ under reduced pressure (vacuum degree ≥ 0.07) to a content of 7128.4 ppm of phytosan; the content of deacetylated phytosan methyl ester + methyl ester of scutellaria serrata + phytosan methyl ester was 10622.7 ppm, butanediol was added and mixed until the content of butanediol accounted for 40%; 10. Ultrafiltration, membrane cutoff molecular weight is 1000 Dalton, 7 bar operating pressure, when ultrafiltration to the retentate liquid is 30% of the mother liquor, add the solvent composed of the mother liquor, i.e. 40% butanediol solution, and continue ultrafiltration to the permeate content: C1=3504.0 ppm; C2=5159.4 ppm; C3=6311.9 ppm.

[0044] 11. Use J1 to adjust the pH to 5.5, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane to obtain Hedyotis diffusa extract.

[0045] Comparative Example 5 1. Crush the medicinal materials into 5-10 mesh; 2. Extract under reduced pressure ultrasonic reflux for 2 hours, using 30% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Method for Ultrasonic Extraction of Medicinal Materials], and extract until the mass of the concentrated liquid is 1.2 times the mass of the medicinal material; 3. Centrifuge until clear at 14,000 rpm; 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 80°C, and concentrate to 230% of the crude drug amount; 5. Adjust pH to 4.0 with citric acid and age at 0-4℃ for 84h; 6. Filter through 0.45μm membrane until clear; 7. Column chromatography purification: The filtrate from step 6 is first passed through a cation exchange resin column HD-2, with a loading ratio of filtrate volume: resin volume = 2.5:1; the permeate is then passed through an anion exchange resin column D201, with a loading ratio of filtrate volume: resin volume = 1.5:1, to obtain a permeate A; the cation exchange resin column is rinsed with 2 times the column volume of pure water, and then eluted with a 30% ethanol solution containing 12% citric acid. The elution end point is that the pH of the eluate mixture is 6.0, to obtain an eluate, which is concentrated to a solid content of 8.5% according to the method in step 4, and butanediol is added and mixed until the butanediol content accounts for 60%, and 10% sodium hydroxide solution is added to adjust the pH to 8.5 to obtain a snake grass alkaloid eluate J1.

[0046] 8. The permeate A is concentrated to 450% of the crude drug amount using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 17 bar; 9. Molecularly imprinted polymer adsorption separation, the loading ratio is filtrate volume: molecular imprinted polymer mass = 8 mL / g, eluted with 95% ethanol 4.5 times the mass of molecularly imprinted polymer, the eluate was concentrated at 70℃ under reduced pressure (vacuum degree ≥ 0.07) to a content of 8944.1 ppm of phytophthora glycoside; the content of deacetylated phytophthora methyl ester + methyl ester of scutellaria serrata + phytophthora glycoside was 13743.8 ppm, butanediol was added and mixed until the content of butanediol accounted for 60%; 10. Ultrafiltration, membrane molecular weight cutoff is 1000 Dalton, 12 bar operating pressure, when ultrafiltration to the retentate of 30% of the mother liquor, add the mother liquor and the same composition of the solvent, i.e. 60% butanediol solution, continue ultrafiltration to the permeate content: C1 = 3232.4 ppm; C2 = 4715.0 ppm; C3 = 6291.0 ppm; 11. Use J1 to adjust the pH to 7.5, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm filter membrane to obtain Hedyotis diffusa extract.

[0047] Comparative Example 6 (conventional process) 1. Crush the medicinal materials into 5-10 mesh; 2. Add 60% ethanol (W / W) 8 times the weight of the medicinal material and reflux for 3 hours at a reflux temperature of 60-70°C. Repeat once, combine the extracts, and filter through a 0.45 μm membrane until clear. 3. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 65°C, until there is no alcohol taste; 4. Add pure water to dilute to the mass of medicinal material: sample volume = 0.1g / mL sample solution; 5. Aging at 0-4℃ for 72h; 6. Filter through 0.45μm membrane until clear; 7. The sample solution was loaded onto the D101 resin column at a flow rate of 2 BV / h. The sample volume was equivalent to the mass of the medicinal material: the volume of the D101 resin = 1:1. The resin column was rinsed with 3 BV of pure water, and then eluted with 3 BV of 40% ethanol. The flow rate was 2 BV / h. The eluates were combined and concentrated, and butanediol was added to make a 50% butanediol solution. The endpoint detection content was: C1 = 2666.8 ppm; C2 = 5088.6 ppm; C3 = 11287.6 ppm. After filtering through a 0.22 μm filter membrane, the Hedyotis diffusa extract was obtained. Comparative Example 7 Steps 1 to 6 are the same as in Example 1. 7. Column chromatography purification: The filtrate from step 6 is first passed through an anion exchange resin column D201, with the loading ratio being filtrate volume: resin volume = 2:1; the permeate is then passed through a cation exchange resin column HD-2, with the loading ratio being filtrate volume: resin volume = 3:1 to obtain permeate A.

[0048] Table 1: Intermediate step content test (all converted to 200% crude drug amount)

[0049] From the data comparison between the embodiment and the comparative example 4 step 4, it can be seen that when the methods are consistent and the crude drug concentration is the same, the content of calendula glycoside and calendula glycoside acid is lower than that of the embodiment due to the low extraction efficiency caused by different process conditions; from the data comparison between the embodiment and the comparative example 6 step 4, it can be seen that when the methods are inconsistent and the crude drug concentration is the same, the content of calendula glycoside and calendula glycoside acid in the comparative example 6 is lower than that of the embodiment. It is speculated that the reason is caused by the low extraction efficiency and the thermal degradation of some components, because the ultrasonic reflux extraction process temperature adopted in the embodiment is lower than the reflux extraction temperature adopted in the conventional process of the comparative example 6, and the heating time is short, and the cyclopentadiene ether terpenoid components such as calendula glycoside have many unsaturated bonds, so they are easily oxidized by heat; Comparing the data of comparative example 7 and step 7 of example 1, it can be seen that the yield of calendula glycoside is different due to the different order of ion exchange columns. This may be related to the five-membered ring lactone structure of calendula glycoside. Alkaline conditions may cause the lactone to open and degrade. Comparative example 7 first passes through the anion exchange column to make the permeate alkaline. This period of time may cause the ring-opening degradation of cyclopentadione lactone components such as calendula glycoside. Therefore, the order of the ion exchange columns in the example cannot be changed.

[0050] Comparison of the data of comparative example 1 and step 6 of the example shows that the sarcosyl acid in the example is largely converted into sarcosyl glycoside, increasing the content of the effective ingredient. This result is also speculated to reduce irritation due to the reduction of the acid content. A degradation experiment of sarcosyl glycoside standard also proves this point; and the comparison of comparative example 6 and the example shows that the conventional extraction scheme does not have the effect of converting sarcosyl acid.

[0051] Degradation experiment of calendula glycoside: The present invention takes the standard product of calendula glycoside and places it at 55°C for 3 days under the condition of pH=5, detects the change of the liquid phase spectrum, and finds that calendula glycoside acid is decomposed, and adds acid to adjust the pH to 2.6 and stirs and heats at 60°C for 2h. After repeated measurement, the area of ​​calendula glycoside acid decreases by 16.5% [586.1→489.6], and the area of ​​calendula glycoside increases by 6.1% [9818.2→10420.6]. See the attached Figures 1 to 4 .

[0052] Table 2 Comparison of the content of calendula glycosides after 0 days and 48℃ for 30 days

[0053] Table 2 shows the results of the test on the content change of calendula glycoside in Comparative Examples 2, 3 and Example 1 after being placed at 48°C for 30 days. From the comparison of the data in Table 2, it can be seen that if the pH is not controlled or adjusted with sodium hydroxide solution, the content will decrease during the sterilization process, and the decrease will be even greater if the pH is not adjusted. From the 30-day high temperature stability, it can be seen that adjusting the pH of the Hedyotis diffusa extract with sodium hydroxide solution and not adjusting the pH will cause a significant decrease in the content of calendula glycoside, and the decrease is similar.

[0054] Table 3 Comparison of physical, chemical, safety and stability data between the examples and comparative examples

[0055] From the comparison between the comparative examples and the examples in Table 3, it can be seen that under the conditions of the examples, the content of the main ingredient sargentioside decreased significantly, and the contents of impurity components such as anthraquinone, iridoid acid, and total flavonoids were significantly reduced, resulting in a significant improvement in safety (chicken embryo test), and the original sample and the sample accelerated at high temperature for 2 months were both non-irritating; while the sargentioside content of the comparative example decreased greatly, and the impurity content was very high. The irritation of the original sample and the sample accelerated at high temperature for 2 months was greater than that of the examples, among which the conventional process of comparative example 6 had poor refining and impurity removal effect, and there was no effective measure to inhibit the degradation of the active ingredient, so the effect was the worst; the sargentioside content of comparative examples 4 and 5 was significantly lower than that of comparative example 6, and it is speculated that the main reason is that the molecular imprinting polymer specific separation in step 9 of comparative examples 4 and 5 played an important role, and it is speculated that more sargentioside was separated and removed in this step, while the final content of sargentioside in comparative example 6 without this step was relatively higher.

[0056] Note: The geniposide, deacetylgeniposide and geniposide listed in Table 3 of Examples 4 and Comparative Examples 4-5 are the detection data of step 10.

[0057] 1. Anthraquinone color reaction: Take 5 mL of sample, add 1 mL of 0.5% MgAc2 alcohol solution, mix well, adjust pH to 10 with 5% sodium hydroxide solution, and an orange to red precipitate will appear.

[0058] 2. Liquid phase detection method for phytosan, deacetylphytosan methyl ester, gentiana glycoside methyl ester, geniposide, phytosan glycoside, deacetylphytosan methyl ester: Chromatographic column: Inertsil ODS-HL 5UM4.6MM*250MM; flow rate: 0.8 mL / min; detection wavelength: 238nm; column temperature: 40℃; injection volume: 10 μL Mobile phase: 0.1% phosphoric acid as phase A and acetonitrile as phase B.

[0059] Table 4 Liquid chromatography peak timetable

[0060] Approximate peak time: calendula glycoside (tR = 29.169min), deacetylcalendula methyl ester (tR = 15.922min), deacetylcalendula acid (tR = 5.604min), gentiana glycoside methyl ester (tR = 18.877min), geniposide (tR = 14.672min), calendula glycoside (tR = 24.016min), liquid phase spectrum see attached Figures 11-12 ; Degradation rate of sarmentosin: The sample was placed at 48°C for 60 days. The sarmentosin content of the sample at 0 day and 60 day (48°C) was recorded as A1 and A2 respectively. Then the degradation rate of calendula glycoside = (A1-A2) ÷ A1 × 100%; 4. Total flavonoids detection method: using rutin as the standard, sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method (ultraviolet spectrophotometry); 5. Chicken embryo test: SNT2329-2009-Cosmetic eye irritation-Corrosive chicken embryo chorioallantoic membrane test, IS < 1 means no irritation; 1 ≤ IS < 5 means mild irritation; 5 ≤ IS < 9 means moderate irritation. Test the irritation of the sample at 0 days and 48°C for 60 days.

[0061] Table 5 Comparison of soothing, anti-inflammatory and antioxidant efficacy data

[0062] Statistical analysis was performed using the t-test method. Compared with the model group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **. Nrf2 belongs to the CNC subfamily of basic leucine zipper (bZIP) transcription factors. Under normal physiological conditions, Nrf2 maintains a low activity state in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (Keap1). When the body is exposed to electrophilic substances, reactive oxygen species (ROS) or other active substances, Nrf2 is phosphorylated and dissociates from Keap1; Nrf2 enters the nucleus and forms a heterodimer with small musculoaponeurotic fibrosarcoma proteins (sMAF), and then binds to the antioxidant response elements (ARE) sequence, inducing the expression of downstream antioxidant and anti-inflammatory proteins and related genes such as HO-1 (heme oxygenase-1) and phosphoamidite adenine dinucleotide quinone oxidoreductase-1 (NADpH quinone oxidoreductase 1, NQO1).

[0063] NF-κB is involved in a variety of biological processes and is a key inflammatory pathway in the body. After stimulation, it is transferred to the cell nucleus, promoting the release of inflammatory genes, and then promoting the production of TNF-α, IL-6, and IL-1β. NF-κB P65 is a member of the NF-κB family. Studies have shown that activation of the Nrf2 signaling pathway can inhibit the activity of NF-κB and reduce inflammatory responses. ROS can promote inflammatory responses and activate MAPK and NF-κB signaling pathways. iNOS and NO are two important signal transduction molecules in the NF-κB signaling pathway. The promoter sequence of the iNOS gene contains a κB site (GGGACTTTCC), which specifically binds to NF-κB, thereby initiating gene transcription and producing a large amount of NO. NO is released and acts on smooth muscle cells, capillary dilation leads to skin irritation, heat sensation, skin redness and other discomfort.

[0064] When the skin is stimulated, keratinocytes release inflammatory factors, which are transmitted to Langerhans cells, activating T cells and B cells, producing IgE and combining with mast cells to produce histamine. Histamine is a medium released by mast cells during allergic reactions, which can promote the synthesis and secretion of proinflammatory cytokines and chemokines such as IL-1α, IL-1β, IL-6, IL-8 or RANTES by various cells and tissues, causing itching, capillary dilation and increased permeability, smooth muscle spasm, and increased secretory activity. Clinically, it can manifest as urticaria-like manifestations such as redness, swelling, itching, and blistering. Histamine also stimulates nerve endings in the skin, directly causing itching and releasing substance P (SP), which promotes mast cell degranulation and releases histamine, further causing itching. At the same time, scratching caused by itching can lead to the release of inflammatory mediators, aggravating the degree of itching.

[0065] There is a complex relationship between oxidative stress and inflammation. Oxidative stress is an important factor leading to inflammation, and inflammation also has the manifestation of oxidative stress. Therefore, it is feasible to find models and raw materials with dual antioxidant and anti-inflammatory effects.

[0066] As can be seen from Table 5, the embodiment has a significant inhibitory effect on free radicals, that is, it has a significant antioxidant effect; it has a significant inhibitory effect on the itching caused by histamine and the expression of P65 (NF-κB) and iNOS, and has an anti-inflammatory effect; and it can significantly promote the expression of NRF2, NQO1, and HO-1 proteins, and has an anti-oxidative stress effect. The embodiment has positive effects on the three pathways of anti-oxidation, anti-inflammatory, and anti-oxidative stress at the same time, which is its characteristic as a soothing raw material. However, due to the difference in process conditions, the comparative example is obviously worse than the embodiment in the effect of each target. It is inferred that the effect of specific process conditions on the ingredients and efficacy, especially the improper treatment of impurity components, will cause the efficacy to decrease: Compared with Example 1, the total amount of iridoid glycosides C3 in Comparative Examples 4 and 5 is similar, but the difference is that the proportion of non-carboxylic acid iridoid glycosides C2 (i.e. C2 / C3) in Comparative Examples 4 and 5 is significantly lower than that in Example 1; while the C3 content of Comparative Example 6 is much higher than that in Example 1, but the proportion of C2 is very low. Although the antioxidant capacity is acceptable, the efficacy of anti-inflammatory and anti-oxidative stress is seriously reduced.

[0067] Detection Methods Method 1: Histamine-induced pruritus model in mice Test samples (diluted with saline to 0.2% for use), positive control (compound dexamethasone acetate cream, batch number: 2207004X, Guangdong China Resources Shunfeng Pharmaceutical Co., Ltd.), deionized water, saline, Shanghai Chuangsai Technology Co., Ltd., histamine phosphate, batch number: C28GS150074, Shanghai Yuanye Biotechnology Co., Ltd. Kunming mice, 18-22 g, were provided by Shanghai Jiesijie Experimental Animal Co., Ltd.

[0068] The effect of subcutaneous injection of histamine phosphate on the antipruritic effect of mice was studied. The mice were randomly divided into half male and half female, with 10 mice in each group. ① Model control group, ② Positive control group, and ③ Sample group. After adaptive feeding, the hair on the back of the neck of the mice was cut with scissors one day before the experiment, and then the hair was removed with a depilatory cream to expose the skin. The depilatory area was about 2cm*2cm. The mice with no skin damage were taken for smearing and administration. Each drug was administered once a day for 2 consecutive days. Itching: 1 hour after the last administration, each mouse in each group was subcutaneously injected with 0.2 mL (10 mL / kg) of 0.05% histamine phosphate saline injection. The itch reaction index was observed by scratching the head with the front paws, scratching the trunk with the hind paws, and biting all parts of the body with the mouth. The number of itching times of mice in each group within 30 minutes was observed and recorded, and the itching inhibition rate was calculated. The strength of the antipruritic effect of the test group was judged according to the itching inhibition rate.

[0069] Inhibition rate = ×100% The method of administration is shown in Table 6.

[0070] Table 6 Drug administration and dosage of each group

[0071] Method 2: UVB-induced mouse skin inflammation model 2.1 Experimental equipment and reagents used for model making Kangfuxin solution (positive control) (Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., batch number: 231104) Ninety KM mice weighing 18-20 g were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0072] Test sample: diluted to 2% with saline.

[0073] Physiological saline (0.9% NaCl, sterile) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., product number: ST341-500mL Ultraviolet light therapy device: Model: SS01B, manufacturer: Sigma.

[0074] 2.2 Reagents used for index detection The reagents used in Western Blot experiments are shown in Table 7 below. Table 7 Main experimental reagents for Western Blot experiment

[0075] 2.3 Model preparation and drug administration regimen Mice were randomly divided into groups (blank, model, positive control, test group), with 10 mice in each group. The back of the mice was depilated, with an area of ​​4cm×2cm. Gauze was spread flat on the exposed skin of the mice 30 minutes before irradiation with the ultraviolet light therapy device. The drugs were administered at 9 o'clock every morning. No drugs were applied externally in the blank group, 1 mL of normal saline was evenly applied externally in the model group, and 1 mL of drugs were evenly applied externally in the positive control and sample groups for 10 minutes.

[0076] The blank group was not given UVB intervention. The other groups were given medication once a day before irradiation for 7 consecutive days. The irradiation distance was determined to be 20 cm from the mouse back, and the UVB irradiation intensity was 13 mW / cm2 for one week. The irradiation time on the first and last day was 10 min, and the remaining 5 days were 15 min, with a total irradiation dose of 74.1 J / cm2.

[0077] A large amount of pigmentation, scaling, erythema, and wrinkles appeared in the irradiated area on the back of the mice, and even ulceration, scabs, and leather-like touch appeared locally, indicating that the model was successful. After the experiment, on the 8th day, the mice were killed, and three mice were randomly sampled from each group. The skin was collected for Wb to detect the expression levels of P65, NRF2, NQO1, INOS, and HO-1 proteins. First, the total protein of the cells was extracted with lysis buffer; total protein of the tissue was extracted; protein denaturation; SDS-PAGE electrophoresis; transfer; immunoreaction; chemiluminescence; WB results and analysis indicators gray value / internal reference gray value. GraphHPad prism8 software was used for analysis, and the data of each group were expressed as mean ± standard deviation ( ) indicates that the inter-group comparison was performed using the student t-test. Compared with the model group, the difference was considered statistically significant when P < 0.05.

[0078] Table 8 Modeling and drug administration scheme of light-damaged mice

[0079] Method 3: Inhibition of DPPH activity by samples 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) reagent was purchased from MacLean Reagent. Vitamin C was purchased from Yuanye Biotechnology Co., Ltd. The sample stock solution was diluted with deionized water for use; Experimental instruments: electronic balance, model: FA2004, manufacturer: Shanghai Sunyu Hengping Scientific Instrument Co., Ltd.; ultrasonic cleaning machine, model: JP-030S, manufacturer: Shenzhen Jiemeng Cleaning Equipment Co., Ltd.; electric constant temperature incubator, model: 303-2B, manufacturer: Shaoxing Shangyu District Huyue Instrument Equipment Factory; ultraviolet spectrophotometer, model: UV-754N, manufacturer: Opler Instrument Co., Ltd.

[0080] Add each reagent according to the reaction system in Table 9, shake well, and let stand for 30 min. Read the absorbance at 517 nm on a UV spectrophotometer, measure three times in parallel, and calculate the inhibition rate according to the scavenging rate formula. The samples were diluted at different times to calculate the DPPH free radical scavenging rate, and the different scavenging rates obtained at different concentrations were used to calculate the IC50 using grapHpad prism8 software.

[0081] Table 9 Sampling method for each group of samples

[0082] DPPH free radical scavenging rate (SR) = (1-(Ai-Aj) / Ac) × 100% Wherein, Ai: reaction system with the sample to be tested and DPPH solution added; Aj: control with the sample to be tested and anhydrous ethanol added; Ac: blank control with the DPPH solution and deionized water added.

[0083] Several product formulations prepared using the products of the present invention are provided below.

[0084] Table 10 Cream formula

[0085] Table 11 Toner formula

[0086] Table 12 Essence formula

[0087] Use this serum for testing: Comparison: Before and after comparison Subjects: A total of 33 subjects, all female, aged 29 to 58 years old.

[0088] How to use the product: After cleansing, apply an appropriate amount of the product evenly on the face until it is fully absorbed. Use once in the morning and evening for 28 consecutive days, test the face on D0 / D28, and conduct subjective and semi-subjective evaluations. The semi-subjective evaluation uses a lactic acid stinging test, and finally an instrument is used for objective quantitative evaluation.

[0089] Subjective evaluation: professional dermatologists asked the subjects whether they experienced dryness, oiliness, scaling, redness, stinging and other symptoms during the use of this product, and observed and recorded whether there were rashes, redness, swelling and scaling on the face, and scored them. No subjects had adverse reactions during the trial. Using the 7-level scoring method, 100% of the skin feel, effect and mildness of the product agreed on D28 that "the product is mild and non-irritating" and "the skin does not feel tight after use"; 96.97% agreed on D28 that "improved skin tolerance".

[0090] Semi-subjective evaluation: 50 µL of 10% lactic acid solution was placed on filter paper (0.8*0.8 cm) at room temperature and placed on the nasolabial groove and any cheek. The subjects' sensitivity was rated at 4 levels at 0.5, 2.5 and 5 minutes, and then the 3 scores were added together. The smaller the score value, the lower the degree of lactic acid stimulation.

[0091] Table 13 Results of lactic acid sting test (n=33)

[0092] The results showed that compared with the baseline value D0 before use, the mean value of the lactic acid sting test showed a downward trend after using the product, and decreased by 28.76% after 28 days of use D28, which was extremely significantly different from the baseline value p<0.01.

[0093] Objective evaluation: Table 14 Instrument measurement parameters and related equipment:

[0094] TEWL value change rate = (TEWL value after using the product - TEWL value before using the product) / TEWL value before using the product * 100% Table 15 Descriptive statistics of skin TEWL mean values ​​(g / (h·m2), n=33)

[0095] Table 16 Analysis of the difference in the average values ​​of skin TEWL (g / (h·m2), , n=33)

[0096] Compared with the baseline value D0 before use, the average skin TEWL showed a downward trend after use, and decreased by 31.40% after 28 days of use D28, which was significantly different from the baseline value p<0.01. The average skin TEWL value after using the product was lower than the baseline value.

[0097] In summary, after 28 days of using 1% of the essence prepared by the product of the present invention, the effect of lactic acid on the stinging reaction of subjects with sensitive skin can be improved, the water loss rate of facial skin can be reduced, and the repair effect can be achieved. In the subjective evaluation, more than 90% of the subjects gave positive comments on the skin feel and soothing effect of the product, and 100% of the subjects gave positive comments on the mildness of the product. This shows that the product of the present invention is mild and non-irritating, has a repair effect, and is suitable for sensitive skin.

[0098] The above is only an implementation method of the invention, and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the invention, which still fall within the scope of patent protection.

Claims

1. A Hedyotis diffusa extract, characterized in that: The content of calendula glycoside is 2500-4000 ppm, the content of deacetylcalendula methyl ester + gentiana glycoside methyl ester + calendula glycoside is 4500-6500 ppm, the total flavonoids content is <100 ppm, there is no characteristic color reaction for anthraquinone identification, calendula glycoside acid <100 ppm, deacetylcalendula glycoside acid <100 ppm, geniposide acid <100 ppm, pH=6.0-7.

0.

2. The method for preparing the Hedyotis diffusa extract according to claim 1, characterized in that: The extract is obtained by vacuum ultrasound, centrifugal separation and vacuum concentration, acid adjustment to pH 2.5-3.0 and then aging, membrane filtration and then purification by ion exchange resin column chromatography to obtain permeate A and Hedyotis diffusa alkaloid eluate J1; the permeate A is filtered through a membrane, and then separated by molecular imprinting polymerization adsorption, the separated eluate is vacuum concentrated and then ultrafiltered, and finally the Hedyotis diffusa alkaloid eluate J1 is adjusted to pH 6-7 and heat-insulated and sterilized to obtain the Hedyotis diffusa extract.

3. The method according to claim 2, characterized in that The specific steps are: The first step is ultrasonic extraction: crushing Hedyotis diffusa powder with ethanol aqueous solution as extraction solvent, vacuum ultrasonic reflux extraction to obtain extract, and centrifugal separation; The second step is concentration and aging: the supernatant is concentrated under reduced pressure, adjusted to pH 2.5-3 by acid, aged for 48-72 hours, and filtered through a membrane until the filtrate is clear; The third step is column chromatography purification: the filtrate is first passed through a cation exchange resin column, and the permeate is then passed through an anion exchange resin column to obtain a permeate A; the cation exchange resin column is rinsed with pure water, and then eluted with a 40-50% ethanol solution containing 8-10% citric acid, and the elution end point is that the pH of the eluate mixture is 5-5.5 to obtain an eluate, which is concentrated under reduced pressure to a solid content of 5-8%, and a solvent is added to adjust the concentration, and a base is added to adjust the pH to 7.5-8.0 to obtain a Herba Ophiopogonis alkaloids eluate J1; Step 4, molecular imprinting polymer adsorption separation: the permeate A is concentrated by membrane and separated by molecular imprinting polymer adsorption, and the eluate is concentrated under reduced pressure and then solvent is added to adjust the concentration to obtain eluate B; Step 5, ultrafiltration: the eluent B is subjected to ultrafiltration again, and the concentration of the permeate is adjusted with the Hedyotis diffusa alkaloid eluent J1 to prepare the Hedyotis diffusa extract.

4. The method according to claim 3, characterized in that In the third step, the loading ratio of the cation exchange resin column is filtrate volume: resin volume = 3 to 5:1; the loading ratio of the anion exchange resin column is filtrate volume: resin volume = 2 to 4:

1.

5. The method according to claim 3, characterized in that In the fourth step, when the molecular imprinting polymer is adsorbed and separated, the loading ratio is filtrate volume: molecular imprinting polymer mass = 5-7 mL / g.

6. The method according to claim 3, characterized in that In the fourth step, the permeate A is concentrated to 300-400% of the crude drug amount by a membrane, the membrane cut-off molecular weight is 100-150 Daltons, and the pressure is 10-15 bar.

7. The method according to claim 3, characterized in that The solvent is a 45-55% polyol solution, and the polyol is any one of butanediol, dipropylene glycol and 1,3-propylene glycol.

8. The method according to claim 3, characterized in that In the fourth step, the molecular imprinting polymer is separated by adsorption, eluted with ethanol 3 to 4 times the mass of the molecular imprinting polymer, the eluent is decompressed at 60 to 65° C., the vacuum degree is ≥ 0.07, and concentrated to a content of 4500 to 9000 ppm of phytosylvatica glycoside.

9. Use of the Hedyotis diffusa extract according to claim 1 in the preparation of soothing products and anti-inflammatory and antioxidant daily chemical products.

10. The use according to claim 9, characterized in that It is mainly used in products that reduce skin redness caused by inflammation by inhibiting inflammatory mediators such as P65, iNOS, and histamine; and products that reduce skin dullness and yellowing caused by inflammatory free radicals by scavenging free radicals; products that promote the skin's own soothing ability by increasing the expression of NRF2, NQO1, and HO-1, further strengthening the soothing process, reducing skin dullness, yellowing, and redness caused by skin inflammation, and making the skin more even-toned.

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