Preparation method and application of Hedyotis diffusa extract

Abstract

The invention discloses a Hedyotis diffusa extract, comprising a hedyotis diffusa glycoside content of 2500-4000 ppm, deacetylated hedyotis diffusa methyl ester, scutellariae glycoside methyl ester, and hedyotis diffusa glycoside content of 4500-6500 ppm, a total flavonoid content of less than 100 ppm, no characteristic color reaction for anthraquinone identification, hedyotis diffusa acid content of less than 100 ppm, deacetylated hedyotis diffusa acid content of less than 100 ppm, and geniposide acid content of less than 100 ppm, and a pH value of 6.0-7.0. The extract is obtained by vacuum ultrasound, centrifugation, and vacuum concentration, followed by acid adjustment to a pH of 2.5-3.0, followed by aging, membrane filtration, and purification by ion exchange column chromatography to obtain a permeate A and a Hedyotis diffusa alkaloid eluate J1. The permeate A is filtered through a membrane, separated by molecular imprinting polymerization adsorption, vacuum concentrated, and ultrafiltered. Finally, the pH is adjusted to 6-7 with J1, and the extract is sterilized by heat preservation.

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 (Hedyotis diffusa) is a common Chinese herbal medicine, the whole herb of Hedyotis diffusa Willd., also known as Oldenlandia diffusa Roxb., a plant of the Rubiaceae family. First recorded in the "Guangxi Chinese Materia Medica," it has the effects of clearing heat, detoxifying, promoting dampness, and relieving jaundice. It is primarily used to treat boils, ulcers, snake bites, edema, and damp-heat jaundice, and has a long and extensive history of use. The main components of Hedyotis diffusa include iridoids, flavonoids, and anthraquinones, followed by organic acids and sterols. Common iridoids in Hedyotis diffusa include methyl scutellarin, methyl deacetylated scutellarin, scutellarin acid, scutellarin, and scutellarin. 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, causing 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 cause phototoxicity and photosensitivity when used externally; relatively safe and effective are the rich variety of cyclopentane saponins contained in it. However, 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. How to obtain a stable, safe and effective Houttuynia cordata extract is difficult, 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 in that the content of iridoid compounds such as hedyotis diffusa is high, the irritation is strong, and the extract is not stable in 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:

[0007] A Hedyotis diffusa extract contains 2500-4000 ppm of calendulaside, 4500-6500 ppm of deacetylated calendulaside methyl ester + tropaeoside methyl ester + calendulaside, a total flavonoid content of less than 100 ppm, no characteristic color reaction for anthraquinone identification, calendulaside acid less than 100 ppm, deacetylated calendulaside acid less than 100 ppm, geniposide acid less than 100 ppm, and a pH value of 6.0-7.0.

[0008] The present invention purifies and separates specific iridoid glycosides from Hedyotis diffusa, quantifies their contents, and limits the amounts of other components that pose significant stability and safety risks. This specific amount of components is then used to create a Hedyotis diffusa extract as a product, and a corresponding preparation method is provided. Compared to conventional Hedyotis diffusa extracts, this extract exhibits improved stability, safety, and efficacy when used in daily chemical applications. The specific process of the present invention reduces the decomposition of active ingredients, increases the content of the main component, calendulaside, and controls the content of impurities, providing a safe, mild, low-irritation, and highly stable raw material for daily chemical applications with soothing, anti-inflammatory, and restorative properties.

[0009] The present invention also provides a preparation method of Hedyotis diffusa extract, which comprises obtaining an extract by vacuum ultrasound, centrifugal separation, and vacuum concentration, adjusting the pH to 2.5-3.0 with acid, aging the extract, membrane filtration, and purification by ion exchange resin column chromatography to obtain a permeate A and a Hedyotis diffusa alkaloid eluate J1; filtering the permeate A through a membrane, and then separating the permeate by molecular imprinting polymerization adsorption, concentrating the separated eluate under vacuum, and ultrafiltration; and finally adjusting the pH of the Hedyotis diffusa alkaloid eluate J1 to 6-7, heat-insulating, and sterilizing the extract.

[0010] The specific steps of the preparation method of the Hedyotis diffusa extract are as follows:

[0011] The first step is ultrasonic extraction: crushing Hedyotis diffusa powder with ethanol-water as the extraction solvent, and then extracting under reduced pressure and ultrasonic reflux to obtain an extract, which is then centrifuged;

[0012] The second step is concentration and aging: the supernatant is concentrated under reduced pressure, adjusted to pH 2.5-3 with acid, aged for 48-72 hours, and filtered through a membrane until the filtrate is clear;

[0013] 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 eluted with a 40-50% ethanol solution containing 8-10% citric acid. The elution end point is when 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. The pH is then adjusted to 7.5-8.0 by adding alkali to obtain a Herba Hedychium alkaloids eluate J1;

[0014] Step 4, molecularly imprinted polymer adsorption separation: the permeate A is concentrated by membrane and then separated by molecularly imprinted polymer adsorption. The eluate is concentrated under reduced pressure and then solvent is added to adjust the concentration to obtain eluate B.

[0015] Step 5, ultrafiltration: the eluate B is further ultrafiltered, and the permeate is adjusted in concentration with the Hedyotis diffusa alkaloid eluate J1 to prepare the Hedyotis diffusa extract.

[0016] 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.

[0017] During the adsorption separation of the molecularly imprinted polymer in the fourth step, the loading ratio is filtrate volume: molecularly imprinted polymer mass = 5-7 mL / g.

[0018] In the fourth step, the permeate A is concentrated to 300-400% of the crude drug amount using a membrane, the membrane cut-off molecular weight is 100-150 Daltons, and the pressure is 10-15 bar.

[0019] The solvent is a 45-55% polyol solution, and the polyol is any one of butanediol, dipropylene glycol, and pentanediol.

[0020] In the fourth step, the molecularly imprinted polymer is separated by adsorption and eluted with ethanol 3 to 4 times the mass of the molecularly imprinted polymer. The eluate is decompressed at 60 to 65° C. with a vacuum degree of ≥0.07 and concentrated to a content of 4500 to 9000 ppm of styracoside.

[0021] The present invention also provides an application of an extract of Hedyotis diffusa in the preparation of soothing products and anti-inflammatory and antioxidant products.

[0022] 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; and 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] By extracting and refining non-carboxylic acid iridoid glycosides and controlling and limiting specific harmful components, a specific Hedyotis diffusa extract is obtained, which has higher stability, safety and efficacy.

[0025] The resulting Hedyotis diffusa extract has superior soothing effects compared to similar soothing products. It can alleviate oxidative stress responses 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; it reduces skin dullness and yellowing caused by inflammatory free radicals by scavenging free radicals; and it promotes the skin's own soothing ability by increasing the expression of NRF2, NQO1, and HO-1, further strengthening the soothing process and reducing the dullness, yellowing, and redness caused by the skin's inflammatory process, making the skin more even-toned.

[0026] Through 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 sarcosinolic acid is converted into sarcosinoside, 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 three steps of ion exchange, molecular imprinting separation, and ultrafiltration, the product is refined and impurities are removed to obtain specific target components; the alkaloid components in the Houttuynia cordata 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

[0027] Figure 1 This is the liquid chromatogram of the standard of cyperus glutinosin.

[0028] Figure 2 This is the liquid chromatogram of the standard of scutellaria baicalensis.

[0029] Figure 3 This is the liquid chromatogram of the cyperus glutamic acid standard after being placed at 55℃ for 3 days.

[0030] Figure 4 This is the liquid chromatogram of the standard product of scutellariae glycoside after being placed at 55℃ for 3 days, then adjusted with acid and stirred at 60℃ for 2 hours.

[0031] Figure 5 This is a WB test result diagram of an embodiment of the present invention.

[0032] Figure 6 It is the inhibition rate curve diagram of DPPH of Example 1 of the present invention.

[0033] Figure 7 It is the inhibition rate curve diagram of DPPH of Example 3 of the present invention.

[0034] Figure 8 It is the inhibition rate curve diagram of DPPH of Comparative Example 4 of the present invention.

[0035] Figure 9 It is the inhibition rate curve diagram of DPPH of Comparative Example 5 of the present invention.

[0036] Figure 10 It is a graph showing the inhibition rate of DPPH in Comparative Example 6 of the present invention.

[0037] Figure 11 These are liquid phase diagrams of Examples 1 to 3 of the present invention.

[0038] Figure 12 It is the liquid phase diagram of comparative examples 4 to 6 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments.

[0040] Unless otherwise specified, the percentage concentrations mentioned in the present invention are all mass percentage concentrations.

[0041] The composition characteristics of the Hedyotis diffusa extract include: calendulaside content 2500-4000 ppm; deacetylcalendulaside methyl ester + tropaeoside methyl ester + calendulaside content 4500-6500 ppm; total flavonoids content <100 ppm; no characteristic color reaction for anthraquinone identification; calendulaside acid <100 ppm; deacetylcalendulaside acid <100 ppm; geniposide acid <100 ppm; pH = 6.0-7.0.

[0042] Process steps:

[0043] 1. Crush the medicinal materials into 5-10 mesh;

[0044] 2. Perform vacuum ultrasonic reflux extraction for 1-1.5 hours, using 40-50% ethanol as the extraction solvent at a reflux temperature of 30-40°C. Refer to ZL 2024 1 0591953.3: Ultrasonic Extraction Equipment and Method for Medicinal Materials for Extraction, and perform extraction until the mass of the concentrate is 0.8-1 times the mass of the medicinal material to obtain the extract.

[0045] 3. Centrifuge the extract until it is clear at a speed of 15,000-18,000 rpm;

[0046] 4. The supernatant is concentrated under reduced pressure with a vacuum degree ≥ 0.07 and a temperature of 65-75°C to a concentration of 150-200% of the crude drug content;

[0047] 5. Adjust the pH to 2.5-3.0 with citric acid and age at 0-4°C for 48-72 hours.

[0048] 6. Filter through a 0.45 μm membrane until clear;

[0049] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column at a loading ratio of filtrate volume:resin volume = 3-5:1. The permeate was then passed through an anion exchange resin column at a loading ratio of filtrate volume:resin volume = 2-4:1 to obtain permeate A. The cation exchange resin column was rinsed with 2 column volumes of pure water and then eluted with a 40-50% ethanol solution containing 8-10% citric acid. The elution endpoint was the pH of the eluate mixture at 5-5.5. The eluate was concentrated according to the method in step 4 to a solid content of 5-8%, and then polyol was added and mixed until the polyol content was 45-55%. The pH was adjusted to 7.5-8.0 with sodium hydroxide solution to obtain snake grass alkaloid eluate J1. The cation exchange resin models are HD-2 and HZD-2; the anion exchange resin models are D201 and D293. Shanghai Huazhen Technology Co., Ltd.

[0050] 8. The permeate A is concentrated to 300-400% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 10-15 bar;

[0051] 9. Molecularly imprinted polymer adsorption separation: The loading ratio is 5-7 mL / g of molecularly imprinted polymer per filtrate volume. Elution is performed with 3-4 times the molecularly imprinted polymer mass in 95% ethanol. The eluate is concentrated under reduced pressure (vacuum ≥ 0.07) at 60-65°C to a phytosan content of 4500-9000 ppm. Deacetylated phytosan methyl ester, patulin methyl ester, and phytosan content of 8000-15000 ppm are then added with a polyol and mixed until the polyol content is 45-55%. The molecularly imprinted material is the molecularly imprinted polymer obtained in Example 2 of patent CN2024114181839.

[0052] 10. Ultrafiltration, membrane molecular weight cut-off of 1000 Dalton, operating pressure of 8-10 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., a 45-55% polyol solution, and continue ultrafiltration until the permeate content satisfies the following requirements: a calendulaside content (C1) of 2500-4000 ppm; a deacetylcalendulaside methyl ester + patulaside methyl ester + calendulaside content (C2) of 4500-6500 ppm; and a content of deacetylcalendulaside methyl ester + patulaside methyl ester + calendulaside + calendulaside acid + deacetylcalendulaside + geniposide acid is recorded as C3;

[0053] 11. Adjust the pH to 6-7 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, filter through 0.22μm membrane to obtain Hedyotis diffusa extract.

[0054] The polyol is any one of butanediol solution, dipropylene glycol and pentanediol.

[0055] Note: Amount of crude drug = the mass of the raw materials fed into the process ÷ the mass of the material obtained in this step × 100%.

[0056] Example 1

[0057] 1. Crush the medicinal materials into 5-10 mesh;

[0058] 2. Extract under reduced pressure and ultrasonic reflux for 1.5 hours, using 40% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Methods for Medicinal Materials], until the mass of the concentrate is 1 times that of the medicinal material.

[0059] 3. Centrifuge until clear at 15,000 rpm;

[0060] 4. Concentrate under reduced pressure, with a vacuum degree ≥ 0.07 and a temperature of 65°C, until the crude drug content reaches 200%;

[0061] 5. Adjust pH to 2.5 with citric acid and age at 0-4°C for 72 hours.

[0062] 6. Filter through a 0.45 μm membrane until clear;

[0063] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column HD-2 at a loading ratio of filtrate volume:resin volume = 3:1; the permeate was then passed through an anion exchange resin column D201 at a loading ratio of filtrate volume:resin volume = 2:1 to obtain permeate A; the cation exchange resin column was rinsed with 2 column volumes of pure water and eluted with a 50% ethanol solution containing 10% citric acid. The elution endpoint was the pH of the eluate mixture at 5.5, to obtain the eluate. The eluate was concentrated to a solid content of 6.5% according to the method in step 4, and butanediol was added and mixed until the butanediol content was 50%. The pH was adjusted to 8.0 with 10% sodium hydroxide solution to obtain the snake grass alkaloid eluate J1;

[0064] 8. The permeate A is concentrated to 300% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 10 bar;

[0065] 9. Molecularly imprinted polymer adsorption separation, the sample loading ratio is filtrate volume: molecularly imprinted polymer mass = 5 mL / g, eluted with 95% ethanol 3 times the mass of molecularly imprinted polymer, and the eluate is concentrated under reduced pressure at 60°C (vacuum degree ≥ 0.07) to a content of 8834.1 ppm of scutellarin and 13546.2 ppm of deacetylated scutellarin methyl ester + scutellarin methyl ester + scutellarin. Butanediol is added and mixed until the content of butanediol accounts for 50%;

[0066] 10. Ultrafiltration, membrane molecular weight cut-off 1000 Dalton, operating pressure 10 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., 50% butanediol solution, and continue ultrafiltration until the permeate contents are: C1 = 3969.8 ppm; C2 = 6091.2 ppm; C3 = 6275.0 ppm.

[0067] 11. Adjust the pH to 6.5 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, and filter through a 0.22μm filter to obtain the Hedyotis diffusa extract.

[0068] Example 2

[0069] 1. Crush the medicinal materials into 5-10 mesh;

[0070] 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: Ultrasonic Extraction Equipment and Methods for Medicinal Materials], and extract until the mass of the concentrated solution is 0.8 times the mass of the medicinal material;

[0071] 3. Centrifuge until clear at 18,000 rpm;

[0072] 4. Concentrate under reduced pressure, with a vacuum degree of ≥0.07 and a temperature of 75°C, to a concentration of 150% of the crude drug content;

[0073] 5. Adjust pH to 3 with citric acid and age at 0-4°C for 48 hours;

[0074] 6. Filter through a 0.45 μm membrane until clear;

[0075] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column HZD-2 at a loading ratio of filtrate volume:resin volume = 5:1; the permeate was then passed through an anion exchange resin column D293 at a loading ratio of filtrate volume:resin volume = 4:1 to obtain permeate A; the cation exchange resin column was rinsed with 2 column volumes of pure water and eluted with a 40% ethanol solution containing 8% citric acid. The elution end point was the pH of the eluate mixture of 5.0 to obtain the eluate, which was concentrated to a solid content of 5% according to the method in step 4, and dipropylene glycol was added and mixed until the dipropylene glycol content was 45%. 1% sodium hydroxide solution was added to adjust the pH to 7.5 to obtain snake grass alkaloid eluate J1.

[0076] 8. The permeate A is concentrated to 400% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 15 bar;

[0077] 9. Molecularly imprinted polymer adsorption separation, the sample loading ratio is 7 mL / g of the filtrate volume: molecularly imprinted polymer mass = 7 mL / g, eluted with 95% ethanol 4 times the mass of the molecularly imprinted polymer, and the eluate is concentrated under reduced pressure at 65°C (vacuum degree ≥ 0.07) to a content of 8275.2 ppm of calophylloside and 11401.8 ppm of deacetylated calophylloside methyl ester + calophylloside methyl ester + calophylloside. Dipropylene glycol is added and mixed until the dipropylene glycol content is 45%;

[0078] 10. Ultrafiltration, membrane molecular weight cut-off 1000 Dalton, operating pressure 8 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., 45% dipropylene glycol solution, and continue ultrafiltration until the permeate contents are: C1 = 4153.7 ppm; C2 = 5661.6 ppm; C3 = 5680.8 ppm.

[0079] 11. Adjust the pH to 7 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, and filter through a 0.22μm filter to obtain the Hedyotis diffusa extract.

[0080] Example 3

[0081] 1. Crush the medicinal materials into 5-10 mesh;

[0082] 2. Extract under reduced pressure and ultrasonic reflux for 75 minutes, using 45% ethanol as the extraction solvent (see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Method for Medicinal Materials]). Extract until the mass of the concentrate is 0.9 times that of the medicinal material, and obtain the extract;

[0083] 3. Centrifuge until clear at 17,000 rpm;

[0084] 4. Concentrate under reduced pressure, vacuum degree ≥ 0.07, temperature 70°C, and concentrate to 180% of the crude drug amount;

[0085] 5. Adjust the pH to 2.7 with citric acid and age at 0-4°C for 60 hours;

[0086] 6. Filter through a 0.45 μm membrane until clear;

[0087] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column HD-2 at a loading ratio of filtrate volume:resin volume = 4:1; the permeate was then passed through an anion exchange resin column D201 at a loading ratio of filtrate volume:resin volume = 3:1 to obtain permeate A; the cation exchange resin column was rinsed with 2 column volumes of pure water and eluted with a 45% ethanol solution containing 8.6% citric acid. The elution endpoint was the pH of the eluate mixture of 5.2 to obtain the eluate, which was concentrated to 8% solid content according to the method in step 4, and pentanediol was added and mixed until the pentanediol content was 55%. The pH was adjusted to 7.8 with 5% sodium hydroxide solution to obtain the snake grass alkaloid eluate J1;

[0088] 8. The permeate A is concentrated to 350% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 12 bar;

[0089] 9. Molecularly imprinted polymer adsorption separation, the sample loading ratio is 6 mL / g of the filtrate volume: molecularly imprinted polymer mass = 6 mL / g, eluted with 95% ethanol (3.5 times the mass of the molecularly imprinted polymer), and the eluate is concentrated under reduced pressure at 65°C (vacuum degree ≥ 0.07) to a content of 8903.5 ppm of calophylloside and 12598.3 ppm of deacetylated calophylloside methyl ester + calophylloside methyl ester + calophylloside. Pentylene glycol is added and mixed until the pentylene glycol content is 55%;

[0090] 10. Ultrafiltration, membrane molecular weight cut-off 1000 Dalton, operating pressure 9 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., 55% pentanediol solution, and continue ultrafiltration until the permeate contents are: C1 = 3688.5 ppm; C2 = 5085.5 ppm; C3 = 5251.0 ppm.

[0091] 11. Adjust the pH to 6 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, and filter through a 0.22μm filter to obtain the Hedyotis diffusa extract.

[0092] Comparative Example 1

[0093] 1. Crush the medicinal materials into 5-10 mesh;

[0094] 2. Extract under reduced pressure and ultrasonic reflux for 1.5 hours, using 40% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Methods for Medicinal Materials], until the mass of the concentrate is 1 times that of the medicinal material.

[0095] 3. Centrifuge until clear at 15,000 rpm;

[0096] 4. Concentrate under reduced pressure, with a vacuum degree ≥ 0.07 and a temperature of 65°C, until the crude drug content reaches 200%;

[0097] 5. Adjust pH to 5 with citric acid and age at 0-4°C for 72 hours.

[0098] 6. Filter through a 0.45 μm membrane until clear.

[0099] Comparative Example 2

[0100] Steps 1 to 10 are the same as in Example 1.

[0101] 11. Adjust the pH to 6.5 with 10% sodium hydroxide solution, sterilize at 90-95℃ for 30 minutes, cool to <45℃, and filter through a 0.22μm filter membrane to obtain the Hedyotis diffusa extract.

[0102] Comparative Example 3

[0103] Steps 1 to 10 are the same as in Example 1.

[0104] 11. Sterilize at 90-95°C for 30 min, cool to <45°C, and filter through a 0.22 μm filter to obtain the Hedyotis diffusa extract (measured pH = 4.9).

[0105] Comparative Example 4

[0106] 1. Crush the medicinal materials into 5-10 mesh;

[0107] 2. Extract under reduced pressure and ultrasonic reflux for 45 minutes, using 55% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Methods for Medicinal Materials], and extract until the mass of the concentrated solution is 0.7 times the mass of the medicinal material.

[0108] 3. Centrifuge until clear at 14,000 rpm;

[0109] 4. Concentrate under reduced pressure, with a vacuum degree of ≥0.07 and a temperature of 60°C, to a concentration of 125% of the crude drug content;

[0110] 5. Adjust pH to 2.0 with citric acid and age at 0-4°C for 36 hours;

[0111] 6. Filter through a 0.45 μm membrane until clear;

[0112] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column HD-2 at a loading ratio of filtrate volume:resin volume = 6:1; the permeate was then passed through an anion exchange resin column D201 at a loading ratio of filtrate volume:resin volume = 5:1 to obtain permeate A; the cation exchange resin column was rinsed with 2 column volumes of pure water and eluted with a 55% ethanol solution containing 7% citric acid. The elution endpoint was the pH of the eluate mixture at 4.5, to obtain the eluate, which was concentrated to a solid content of 4.5% according to the method in step 4. Butanediol was added and mixed until the butanediol content reached 40%. The pH was adjusted to 7.0 with 10% sodium hydroxide solution to obtain the snake grass alkaloid eluate J1;

[0113] 8. The permeate A is concentrated to 250% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 8 bar;

[0114] 9. Molecularly imprinted polymer adsorption separation, the sample loading ratio is filtrate volume: molecularly imprinted polymer mass = 4 mL / g, eluted with 95% ethanol 2.5 times the mass of molecularly imprinted polymer, and the eluate is concentrated under reduced pressure at 55°C (vacuum degree ≥ 0.07) to a content of 7128.4 ppm of calophylloside and 10622.7 ppm of deacetylated calophylloside methyl ester + calophylloside methyl ester + calophylloside. Butanediol is added and mixed until the content of butanediol accounts for 40%;

[0115] 10. Ultrafiltration, membrane molecular weight cutoff of 1000 Dalton, operating pressure of 7 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., 40% butanediol solution, and continue ultrafiltration until the permeate contents are: C1 = 3504.0 ppm; C2 = 5159.4 ppm; C3 = 6311.9 ppm.

[0116] 11. Adjust the pH to 5.5 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, and filter through a 0.22μm filter to obtain the Hedyotis diffusa extract.

[0117] Comparative Example 5

[0118] 1. Crush the medicinal materials into 5-10 mesh;

[0119] 2. Extract under reduced pressure and ultrasonic reflux for 2 hours, using 30% ethanol as the extraction solvent. For details, see [ZL 2024 10591953.3: Ultrasonic Extraction Equipment and Methods for Medicinal Materials], and extract until the mass of the concentrated solution is 1.2 times that of the medicinal material.

[0120] 3. Centrifuge until clear at 14,000 rpm;

[0121] 4. Concentrate under reduced pressure, with a vacuum degree of ≥0.07 and a temperature of 80°C, to a concentration of 230% of the crude drug content;

[0122] 5. Adjust pH to 4.0 with citric acid and age at 0-4°C for 84 hours;

[0123] 6. Filter through a 0.45 μm membrane until clear;

[0124] 7. Column chromatography purification: The filtrate from step 6 was first passed through a cation exchange resin column HD-2 at a loading ratio of filtrate volume:resin volume = 2.5:1; the permeate was then passed through an anion exchange resin column D201 at a loading ratio of filtrate volume:resin volume = 1.5:1 to obtain permeate A; the cation exchange resin column was rinsed with 2 column volumes of pure water and eluted with a 30% ethanol solution containing 12% citric acid. The elution endpoint was the pH of the eluate mixture of 6.0, to obtain the eluate, which was concentrated to a solid content of 8.5% according to the method in step 4. Butanediol was added and mixed until the butanediol content was 60%. The pH was adjusted to 8.5 with 10% sodium hydroxide solution to obtain the snake grass alkaloid eluate J1.

[0125] 8. The permeate A is concentrated to 450% of the crude drug content using a membrane with a molecular weight cut-off of 100-150 Daltons and a pressure of 17 bar;

[0126] 9. Molecularly imprinted polymer adsorption separation, the sample loading ratio is filtrate volume: molecularly imprinted polymer mass = 8 mL / g, eluted with 95% ethanol 4.5 times the mass of molecularly imprinted polymer, and the eluate is concentrated under reduced pressure at 70°C (vacuum degree ≥ 0.07) to a content of 8944.1 ppm of calophylloside and 13743.8 ppm of deacetylated calophylloside methyl ester + calophylloside methyl ester + calophylloside. Butanediol is added and mixed until the content of butanediol accounts for 60%;

[0127] 10. Ultrafiltration, membrane molecular weight cut-off 1000 Dalton, operating pressure 12 bar. When the retentate is 30% of the mother liquor, add a solvent composed of the mother liquor, i.e., 60% butanediol solution, and continue ultrafiltration until the permeate contents are: C1 = 3232.4 ppm; C2 = 4715.0 ppm; C3 = 6291.0 ppm.

[0128] 11. Adjust the pH to 7.5 with J1, sterilize at 90-95℃ for 30 min, cool to <45℃, and filter through a 0.22μm filter to obtain the Hedyotis diffusa extract.

[0129] Comparative Example 6 (conventional process)

[0130] 1. Crush the medicinal materials into 5-10 mesh;

[0131] 2. Add 8 times the weight of the medicinal material and reflux extract with 60% ethanol (w / w) for 3 hours at 60-70°C. Repeat once, combine the extracts, and filter through a 0.45 μm membrane until clear.

[0132] 3. Concentrate under reduced pressure, with a vacuum degree of ≥0.07 and a temperature of 65°C, until there is no alcohol smell;

[0133] 4. Add pure water to dilute the sample to the mass of the medicinal material: the sample volume = 0.1g / mL sample solution;

[0134] 5. Aging at 0-4°C for 72 hours;

[0135] 6. Filter through a 0.45 μm membrane until clear;

[0136] 7. Load the sample onto a D101 resin column at a flow rate of 2 BV / h. The sample volume is equivalent to the weight of the medicinal material: the volume of D101 resin = 1:1. Rinse the resin column with 3 BV of pure water and then elute with 3 BV of 40% ethanol at a flow rate of 2 BV / h. Combine the eluates, concentrate them, and add butanediol to make a 50% butanediol solution. The endpoint detection content is: C1 = 2666.8 ppm; C2 = 5088.6 ppm; C3 = 11287.6 ppm. Pass through a 0.22 μm filter membrane to obtain the Hedyotis diffusa extract.

[0137] Comparative Example 7

[0138] Steps 1 to 6 are the same as in Example 1.

[0139] 7. Column chromatography purification: The filtrate from step 6 was first passed through an anion exchange resin column D201 with a loading ratio of filtrate volume:resin volume = 2:1; the permeate was then passed through a cation exchange resin column HD-2 with a loading ratio of filtrate volume:resin volume = 3:1 to obtain permeate A.

[0140] Table 1: Intermediate step content test (all converted to 200% crude drug content)

[0141]

[0142] From the data comparison of step 4 of Example 4 and Comparative Example 4, it can be seen that when the methods are consistent and the crude drug concentration is the same, the content of calendulaside and calendulaside acid is lower than that of Example 1 due to low extraction efficiency caused by different process conditions; from the data comparison of step 4 of Example 6 and Comparative Example 6, it can be seen that when the methods are inconsistent and the crude drug concentration is the same, the content of calendulaside and calendulaside acid in Comparative Example 6 is lower than that of Example 1. It is speculated that the reason is caused by the low extraction efficiency and the thermal degradation of some components. The ultrasonic reflux extraction process temperature used in the embodiment is lower than the reflux extraction temperature used in the conventional process of Comparative Example 6, and the heating time is shorter. Since calendulaside and other iridoid components have many unsaturated bonds, they are easily oxidized by heat.

[0143] Comparing the data of Comparative Example 7 and Example 1 step 7, it can be seen that the yield of calendulaside is different due to the different order of ion exchange columns. This may be related to the five-membered ring lactone structure of calendulaside. 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 cyclopentasiloxane lactone components such as calendulaside. Therefore, the order of the ion exchange columns in the example cannot be changed.

[0144] Comparing the data from Comparative Example 1 with that from Step 6 of Example 1, it can be seen that the Example significantly converts calendula acid into calendulaside, increasing the active ingredient content. This result, presumably due to the reduced acid content, can reduce irritation. This is also confirmed by a degradation experiment with a calendulaside standard. A comparison of Comparative Example 6 with the Example shows that the conventional extraction method does not convert calendula acid.

[0145] Degradation experiment of calendula officinalis:

[0146] The present invention takes a standard sample of calendulaside and places it at 55°C for 3 days under pH=5, detects the change of the liquid phase spectrum, and finds that calendulaside acid is decomposed. Acid is added to adjust the pH to 2.6 and heated at 60°C with stirring for 2 hours. After re-testing, the area of ​​calendulaside acid decreases by 16.5% [586.1→489.6] and the area of ​​calendulaside increases by 6.1% [9818.2→10420.6]. See the attached figure for details. Figures 1 to 4 .

[0147] Table 2 Comparison of the content of scutellariae glycosides after 0 days and 48 ° C for 30 days

[0148]

[0149] Table 2 shows the results of the content change test of calendulaside in Comparative Examples 2, 3 and Example 1 after 0 day and 30 days at 48°C. 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 or not adjusting the pH will cause a significant decrease in the content of calendulaside, and the decrease is similar.

[0150] Table 3 Comparison of various physical, chemical, safety and stability data of Examples and Comparative Examples

[0151]

[0152] Comparison of the comparative examples in Table 3 with the examples shows that under the conditions of the examples, the content of the main ingredient, calendulaside, decreased significantly, and the contents of impurities such as anthraquinone, iridoid acid, and total flavonoids were significantly reduced, resulting in a significant improvement in safety (chicken embryo test). Both the original sample and the sample accelerated at high temperature for 2 months were non-irritating. However, the calendulaside content of the comparative examples decreased significantly, 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 them, the conventional process of comparative example 6 had poor refining and impurity removal effect and no effective measures to inhibit the degradation of the active ingredient, so the effect was the worst. The calendulaside content of comparative examples 4 and 5 was significantly lower than that of comparative example 6. It is speculated that the main reason is that the specific separation of the molecularly imprinted polymer in step 9 of comparative examples 4 and 5 played an important role. It is speculated that more calendulaside was separated and removed in this step, while the final content of calendulaside in comparative example 6 was relatively higher without this step.

[0153] Note: The geniposide, deacetylgeniposide, and geniposide listed in Table 3 for Examples 4 and Comparative Examples 4-5 are the test data of step 10.

[0154] 1. Anthraquinone color reaction: Take 5 mL of sample, add 1 mL of 0.5% MgAc2 alcohol solution, mix well, and adjust the pH to 10 with 5% sodium hydroxide solution. An orange to red precipitate will appear.

[0155] 2. Liquid phase detection method for phytosan, deacetylated phytosan methyl ester, gentianin methyl ester, geniposide, phytosan glycoside, deacetylated phytosan methyl ester:

[0156] Chromatographic column: Inertsil ODS-HL 5UM 4.6MM*250MM; flow rate: 0.8 mL / min; detection wavelength: 238 nm; column temperature: 40°C; injection volume: 10 μL

[0157] Mobile phase: 0.1% phosphoric acid as phase A and acetonitrile as phase B.

[0158] Table 4 Liquid chromatography peak timetable

[0159]

[0160] Approximate peak times: calendulaside (tR = 29.169 min), deacetylcalendulaside methyl ester (tR = 15.922 min), deacetylcalendulaside acid (tR = 5.604 min), gentianin methyl ester (tR = 18.877 min), geniposide (tR = 14.672 min), calendulaside (tR = 24.016 min). For the liquid chromatography, see the attached Figures 11-12 ;

[0161] Degradation rate of sarmentosin: The sample was placed at 48℃ for 60 days. The sarmentosin content of the sample at 0 day and 60 day (48℃) was recorded as A1 and A2 respectively.

[0162] Then the degradation rate of schizonepetaside = (A1-A2) ÷ A1 × 100%;

[0163] 4. Total flavonoids detection method: using rutin as the standard, sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method (UV spectrophotometry);

[0164] 5. Chicken embryo test: SNT2329-2009 - Cosmetic Eye Irritation - Corrosive Chicken Embryo Chorioallantoic Membrane Test. IS < 1 indicates no irritation; 1 ≤ IS < 5 indicates mild irritation; 5 ≤ IS < 9 indicates moderate irritation. Test samples for irritation at 0 days and at 48°C for 60 days.

[0165] Table 5 Comparison of soothing, anti-inflammatory and antioxidant efficacy data

[0166]

[0167] Statistical analysis was performed using the t-test method. Compared with the model group, significance was indicated by *, P-value < 0.05 was indicated by *, and P-value < 0.01 was indicated by **.

[0168] Nrf2 belongs to the CNC subfamily of basic leucine zipper (bZIP) transcription factors. Under normal physiological conditions, Nrf2 remains inactive in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (Keap1). Upon exposure to electrophiles, reactive oxygen species (ROS), or other reactive substances, Nrf2 becomes phosphorylated and dissociates from Keap1. Nrf2 then enters the nucleus, forming a heterodimer with small musculoaponeurotic fibrosarcoma proteins (sMAF). Nrf2 then binds to antioxidant response elements (AREs), inducing the expression of downstream antioxidant and anti-inflammatory proteins and related genes, such as HO-1 (heme oxygenase-1) and NADPH quinone oxidoreductase-1 (NQO1).

[0169] NF-κB is involved in numerous biological processes and is a key inflammatory pathway in the body. Upon stimulation, it translocates to the cell nucleus, promoting the release of inflammatory genes and, in turn, 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 NF-κB activity and alleviate inflammatory responses. Reactive oxygen species (ROS) can promote inflammatory responses and activate both the MAPK and NF-κB signaling pathways. iNOS and nitric oxide (NO) are two key signaling molecules in the NF-κB signaling pathway. The iNOS gene promoter sequence contains a κB site (GGGACTTTCC), which specifically binds to NF-κB, initiating gene transcription and producing large amounts of NO. The released NO acts on smooth muscle cells, dilating capillaries and causing skin irritation, a sensation of warmth, and redness.

[0170] When the skin is irritated, keratinocytes release inflammatory factors that are transmitted to Langerhans cells, activating T and B cells. These factors trigger the production of IgE, which binds to mast cells to produce histamine. Histamine, a mediator released by mast cells during allergic reactions, stimulates 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. This leads to itching, increased capillary dilation and permeability, smooth muscle spasm, and increased secretory activity. Clinically, this can manifest as urticaria-like symptoms such as redness, itching, and blistering. Histamine also stimulates nerve endings in the skin, directly causing itching and the release of substance P (SP). This triggers mast cell degranulation and releases histamine, further causing itching. Furthermore, scratching caused by the itching leads to the release of inflammatory mediators, exacerbating the itching.

[0171] There is a complex relationship between oxidative stress and inflammation. Oxidative stress is a key factor in inflammation, while inflammation also manifests as oxidative stress. Therefore, it is feasible to find models and raw materials with dual antioxidant and anti-inflammatory effects.

[0172] As shown in Table 5, the Examples exhibit significant free radical inhibition, i.e., significant antioxidant activity. They also significantly inhibit histamine-induced pruritus and the expression of P65 (NF-κB) and iNOS, demonstrating anti-inflammatory efficacy. They also significantly promote the expression of NRF2, NQO1, and HO-1 proteins, demonstrating antioxidant stress. The Examples exhibit positive effects across three pathways: antioxidant, anti-inflammatory, and antioxidant stress resistance, demonstrating their unique properties as soothing ingredients. However, due to differences in process conditions, the Comparative Examples exhibit significantly inferior effects on various targets compared to the Examples. This suggests that specific process conditions can influence composition and efficacy, particularly impurity handling, leading to decreased efficacy. Comparative Examples 4 and 5 have similar iridoid glycoside content (C3) to Example 1, but the C2 ratio (C2 / C3) of non-carboxylic acid iridoid glycosides in Examples 4 and 5 is significantly lower than that in Example 1. While the C3 content in Comparative Example 6 is significantly higher than in Example 1, the C2 ratio is significantly lower. While the antioxidant capacity of Comparative Example 6 is acceptable, its anti-inflammatory and antioxidant stress efficacy is significantly reduced.

[0173] Detection method

[0174] Method 1: Histamine-induced pruritus model in mice

[0175] Test samples (diluted to 0.2% with saline), positive control (compound dexamethasone acetate cream, batch number: 2207004X, Guangdong China Resources Shunfeng Pharmaceutical Co., Ltd.), deionized water and saline, Shanghai Chuangsai Technology Co., Ltd., and histamine phosphate, batch number: C28GS150074, Shanghai Yuanye Biotechnology Co., Ltd. Kunming mice, 18-22 g, were provided by Shanghai Jiesijie Laboratory Animal Co., Ltd.

[0176] To investigate the antipruritic effect of subcutaneous histamine phosphate injection in mice, mice were randomly divided into 50% male and 50% female groups, with 10 mice in each group. The following groups were used: ① Model control group, ② Positive control group, and ③ Sample group. After adaptive feeding, the hair on the back of the mice's necks was trimmed with scissors the day before the experiment, and then depilatory cream was used to expose the skin. The depilatory area was approximately 2 cm x 2 cm. Mice with intact skin were then treated with the drug. The drug was administered once daily for two consecutive days. One hour after the last dose, each mouse in each group received a subcutaneous injection of 0.2 mL (10 mL / kg) of 0.05% histamine phosphate in normal saline. Itch response was assessed by scratching the head with the forepaws, scratching the trunk with the hind paws, and biting various parts of the body with the mouth. The number of scratches in each group over a 30-minute period was recorded, and the itch inhibition rate was calculated. The itch inhibition rate was used to determine the strength of the antipruritic effect of the test group.

[0177] Inhibition rate = ×100%

[0178] The method of administration is shown in Table 6.

[0179] Table 6: Administration and dosage of each group

[0180]

[0181] Method 2: UVB-induced mouse skin inflammation model

[0182] 2.1 Experimental equipment and reagents used for model making

[0183] Kangfuxin solution (positive control) (Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., batch number: 231104)

[0184] Ninety KM mice weighing 18-20 g were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0185] Test sample: diluted to 2% with saline.

[0186] Physiological saline (0.9% NaCl, sterile) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., product number: ST341-500mL

[0187] Ultraviolet light therapy device: Model: SS01B, Manufacturer: Sigma.

[0188] 2.2 Reagents used for index detection

[0189] The reagents used in Western Blot experiments are shown in Table 7 below.

[0190] Table 7 Main experimental reagents for Western Blot experiment

[0191]

[0192] 2.3 Model preparation and drug administration regimen

[0193] Mice were randomly divided into 10 groups (blank, model, positive control, and test groups). Hair was removed from the back of the mice, covering an area of ​​4 cm × 2 cm. Thirty minutes before UV light therapy, gauze was placed flat on the exposed skin of the mice. Drug administration was performed at 9:00 AM each day. The blank group received no drug application. The model group was evenly coated with 1 mL of saline solution. The positive control and sample groups were evenly coated with 1 mL of drug for 10 minutes.

[0194] The blank group received no UVB treatment. The remaining groups received medication once daily before irradiation for seven consecutive days. The irradiation distance was 20 cm from the back of the mice, and the UVB intensity was 13 mW / cm². This lasted for one week. The irradiation duration was 10 minutes on the first and last day, and 15 minutes on the remaining five days, for a total irradiation dose of 74.1 J / cm².

[0195] A large amount of pigmentation, scales, 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 end of the experiment, that is, 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. GrapHPad prism8 software was used for analysis, and the data of each group were expressed as mean ± standard deviation ( ) indicates that the difference between the two groups was statistically significant. The difference between the two groups was statistically significant when P < 0.05 compared with the model group.

[0196] Table 8 Light-damaged mouse modeling and drug administration regimen

[0197]

[0198] Method 3: Inhibitory effect of samples on DPPH activity

[0199] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) reagent was purchased from Macklin Reagent. Vitamin C was purchased from Yuanye Biotechnology Co., Ltd. The sample stock solutions were diluted with deionized water. Laboratory instruments included: electronic balance, model FA2004, manufactured by Shanghai Sunny Hengping Scientific Instrument Co., Ltd.; ultrasonic cleaner, model JP-030S, manufactured by Shenzhen Jiemeng Cleaning Equipment Co., Ltd.; electric constant temperature incubator, model 303-2B, manufactured by Huyue Instrument Factory, Shangyu District, Shaoxing; and UV spectrophotometer, model UV-754N, manufactured by Opple Instrument Co., Ltd.

[0200] Add each reagent according to the reaction system in Table 9, shake well, and let stand for 30 minutes. Read the absorbance at 517 nm on a UV spectrophotometer. Measure three times in parallel and calculate the inhibition rate according to the scavenging formula. DPPH free radical scavenging rate was calculated by diluting the sample at different times. The different scavenging rates obtained at different concentrations were used to calculate the IC50 using grapHpad Prism8 software.

[0201] Table 9 Sampling method for each group of samples

[0202]

[0203] DPPH radical scavenging rate (SR) = (1-(Ai-Aj) / Ac) × 100%

[0204] Wherein, Ai: reaction system with the test sample and DPPH solution added; Aj: control with the test sample and anhydrous ethanol added; Ac: blank control with the DPPH solution and deionized water added.

[0205] Several product formulations prepared using the products of the present invention are provided below.

[0206] Table 10 Cream formula

[0207]

[0208] Table 11 Toner formula

[0209]

[0210] Table 12 Essence formula

[0211]

[0212] Tested with this serum:

[0213] Comparison: Before and after comparison

[0214] Participants: A total of 33 subjects, all female, aged 29 to 58 years old.

[0215] How to use the product:

[0216] After cleansing, apply an appropriate amount of product evenly to the face until fully absorbed. Use once in the morning and evening for 28 consecutive days. Test the face on Day 0 and Day 28 for subjective and semi-subjective evaluation. The semi-subjective evaluation uses a lactic acid sting test, and finally, an objective quantitative evaluation is performed using an instrument.

[0217] Subjective evaluations were conducted by professional dermatologists, who asked participants whether they experienced dryness, oiliness, scaling, redness, or stinging while using the product. They also observed and recorded any facial rashes, swelling, or scaling, and rated the results accordingly. No subjects experienced adverse reactions during the trial. Using a 7-point scale, the product's skin feel, effectiveness, and mildness were rated as "gentle and non-irritating" and "skin feels less tight after use" by day 28. Dermatologists also rated the product "improved skin tolerance" at 96.97% by day 28.

[0218] For a semi-subjective evaluation, 50µL of a 10% lactic acid solution was placed on a filter paper (0.8 x 0.8cm) at room temperature. The filter paper was then placed in the nasolabial groove and on either cheek. The subjects' sensitivity was rated on a four-point scale at 0.5, 2.5, and 5 minutes. The three scores were then added together. Lower scores indicate less severe lactic acid irritation.

[0219] Table 13 Lactic acid sting test results (n=33)

[0220]

[0221] 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.

[0222] Objective evaluation:

[0223] Table 14 Instrument measurement parameters and related equipment:

[0224]

[0225] TEWL value change rate = (TEWL value after using the product - TEWL value before using the product) / TEWL value before using the product * 100%

[0226] Table 15 Descriptive statistics of skin TEWL mean values ​​(g / (h·m2), n=33)

[0227]

[0228] Table 16 Analysis of the difference in the average value of skin TEWL (g / (h·m2), , n=33)

[0229]

[0230] 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 a very significant difference compared with the baseline value p<0.01. The average skin TEWL after using the product was lower than the baseline value.

[0231] In summary, after 28 days of using a 1% serum prepared with the product of the present invention, the irritation caused by lactic acid in subjects with sensitive skin was alleviated, the facial skin moisture loss rate was reduced, and a repairing effect was achieved. Furthermore, in subjective evaluations, over 90% of subjects gave positive comments on the product's skin feel and soothing effects, and 100% of subjects gave positive comments on its mildness. This demonstrates that the product of the present invention is mild, non-irritating, and has a repairing effect, making it suitable for sensitive skin.

[0232] The above is only an embodiment of the present 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 present invention and still fall within the scope of patent protection.

Claims

1. A method for preparing an extract of Hedyotis diffusa, characterized in that: The specific steps are: The first step is ultrasonic extraction: crushing Hedyotis diffusa powder with ethanol-water as the extraction solvent, and then extracting under reduced pressure and ultrasonic reflux to obtain an extract, which is then centrifuged; The second step is concentration and aging: the supernatant is concentrated under reduced pressure, adjusted to pH 2.5-3 with 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. The elution end point is when 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. A base is added to adjust the pH to 7.5-8.0 to obtain a Houttuynia cordata alkaloid eluate J1; the cation exchange resin models are: HD-2, HZD-2; the anion exchange resin models are: D201, D293; The fourth step is molecularly imprinted polymer adsorption separation: the permeate A is concentrated by membrane and then adsorbed by molecularly imprinted polymer, and then eluted with 95% ethanol with a mass of 3 to 4 times that of the molecularly imprinted polymer. 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 further ultrafiltered, and the permeate is adjusted in concentration with the Hedyotis diffusa alkaloid eluent J1 to obtain the Hedyotis diffusa extract; The solvent in the third and fourth steps is a 45-55% polyol solution, and the polyol is any one of butanediol, dipropylene glycol, and 1,3-propylene glycol.

2. The method according to claim 1, wherein 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.

3. The method according to claim 1, wherein During the adsorption separation of molecularly imprinted polymer in the fourth step, the loading ratio was filtrate volume: molecularly imprinted polymer mass = 5-7 mL / g.

4. The method according to claim 1, wherein In the fourth step, the permeate A is concentrated to 300-400% of the crude drug amount using a membrane, the membrane cut-off molecular weight is 100-150 Daltons, and the pressure is 10-15 bar.

5. The method according to claim 1, wherein In the fourth step, the molecularly imprinted polymer is used for adsorption separation, and the eluate is decompressed at 60-65° C. with a vacuum degree of ≥0.07 and concentrated to a content of 4500-9000 ppm of styracoside.

6. The Hedyotis diffusa extract prepared by the method of any one of claims 1 to 5, characterized in that: The content of calendulaside is 2500-4000 ppm, the content of deacetylcalendula acid methyl ester, gentiana glycoside methyl ester and calendulaside is 4500-6500 ppm, the content of total flavonoids is less than 100 ppm, there is no characteristic color reaction for anthraquinone identification, the content of calendulaside is less than 100 ppm, the content of deacetylcalendula acid is less than 100 ppm, the content of geniposide is less than 100 ppm, and the pH is 6.0-7.

0.

7. Use of the Hedyotis diffusa extract according to claim 6 in the preparation of soothing products and anti-inflammatory and antioxidant daily chemical products.

Images