Cortex fraxini and wormwood composition and application thereof

Abstract

The invention relates to a preparation method of a cortex fraxini and wormwood composition. The preparation method comprises the following steps: mixing sterilized cortex fraxini powder with an aqueous solution containing zymophyte and biological enzyme, fermenting and performing enzymolysis; adding water for extraction, filtering, concentrating under reduced pressure, centrifuging, taking supernate, enabling the supernate to pass through a polyamide resin column, firstly eluting with water, then eluting with 10%-30% ethanol, finally eluting with 70%-90% ethanol, collecting eluent, and concentrating under reduced pressure until the eluent is dry, so as to obtain a cortex fraxini lignan component extract; adding DES with the water content of 10%-30% and cellulase with the water content of 2%-5% into the wormwood powder, extracting, filtering, concentrating filtrate under reduced pressure, centrifuging, taking supernate, passing through a medium pressure to prepare chromatography, eluting with 30%-50% ethanol, collecting eluent, and concentrating under reduced pressure until the eluent is dry, so as to obtain wormwood chlorogenic acid component extract; the cortex fraxini and wormwood composition is a mixture of cortex fraxini lignan component extract and / or wormwood chlorogenic acid component extract. The cortex fraxini and wormwood composition has antibacterial, anti-inflammatory and anti-allergic effects and can be widely applied to prevention and treatment of acne.

Description

Technical Field

[0001] The invention belongs to the technical field of drug extraction and application, and specifically relates to a qinpi wormwood composition and application thereof. Background Art

[0002] Acne vulgaris is a chronic inflammatory skin disease that occurs in adolescence and mainly affects the hair follicle sebaceous gland unit on the face. Epidemiological studies have shown that acne has become the eighth most common disease in the world. Therefore, how to effectively prevent and treat acne has great socioeconomic significance. Currently, the clinical treatment of acne is mainly based on retinoic acid, benzoyl peroxide, antibiotics, azelaic acid chemical drugs, etc., which have a positive effect, but are usually accompanied by adverse reactions, such as skin irritation, dry skin, organ damage and bacterial resistance.

[0003] Traditional Chinese medicine has a long history in treating acne, and has the characteristics of low toxicity and side effects, high effectiveness, mild and non-irritating to the skin, etc., which can better make up for the disadvantage of chemical drugs with large side effects. Based on this, this application was developed. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a Qinpi Artemisia argyi composition, which has antibacterial, anti-inflammatory and anti-allergic effects and can be widely used in the prevention and treatment of acne, in order to provide a new choice for the clinical development of drugs for the treatment of acne.

[0005] The present invention also provides a preparation method and application of the qinpi wormwood composition.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a wormwood composition comprises the following steps:

[0008] 1) Preparation of extracts of lignan components of the cortex of the Chinese qin: The cortex of the Chinese qin is crushed and passed through a sieve to obtain the cortex of the Chinese qin powder, and the lignan components of the cortex of the Chinese qin are extracted by a combined process of microorganisms, enzymes and ultrasound. The sterilized cortex of the Chinese qin powder is mixed with an aqueous solution containing fermentation bacteria and biological enzymes, and then fermented at 25-35°C for 24-72 hours, and then heated to 40-60°C for enzymatic hydrolysis for 3-6 hours; then water is added for ultrasonic extraction at 75-90°C for 0.5-2 hours, filtered, concentrated under reduced pressure, centrifuged, and the supernatant is passed through a polyamide resin column, first eluted with water until the eluent is nearly colorless (the water eluent is discarded), and then eluted with 10%-30% ethanol to remove impurities, and finally eluted with 70%-90% ethanol and the 70%-90% ethanol eluent is collected, and concentrated to dryness under reduced pressure to obtain the extract of lignan components of the cortex of the Chinese qin;

[0009] 2) Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder and use ultrasound-enzyme-assisted low eutectic solvent method to extract chlorogenic acid components of wormwood. Add DES with a water content of 10%-30% and cellulase with a mass of 2%-5% of wormwood powder to the wormwood powder, then ultrasonically extract at 30-50°C for 30-60min, filter, concentrate the filtrate under reduced pressure, centrifuge, take the supernatant and enrich it through medium pressure preparative chromatography, use C18 (octadecylsilane bonded silica gel) filler to pack the column, directly load the sample at a pressure of 0-10MPa, control the sample flow rate at 1-5mL / min, elute with 30-50% ethanol (elution flow rate is controlled at 5-10mL / min), collect the 30-50% ethanol eluate, and concentrate to dryness under reduced pressure to obtain the chlorogenic acid extract of wormwood;

[0010] The qinpi wormwood composition is a mixture of qinpi lignan extracts and / or wormwood chlorogenic acid extracts. The contents of qinpi lignan extracts and wormwood chlorogenic acid extracts can be determined by HPLC external standard method, and the results are expressed as percentage (%).

[0011] Specifically, in step 1), the sterilized Fraxinus fraseri powder can be mixed with an aqueous solution containing 2%-4% fermentation bacteria and 4-6% biological enzyme at a mass ratio of 1:3-6.

[0012] Furthermore, the fermentation bacteria may be Saccharomyces cerevisiae. The biological enzyme may be a mixture of cellulase and pectinase.

[0013] Specifically, in step 1), 8-14 times the weight of water of the Fraxinus Fraxinus powder can be added for ultrasonic extraction.

[0014] Furthermore, in steps 1) and 2), the concentration is stopped when the ratio of the original medicinal material to the concentrated solution is 1g:1.8-3ml. In other words, taking 100g of Fraxinus chinensis as an example, the concentration is stopped when the volume of the concentrated solution is 180-300ml.

[0015] Specifically, in step 2), DES with a water content of 10%-30% can be added at a material-liquid ratio of 1g:10-50mL. The DES can be a betaine-fructose mixture, a betaine-glucose mixture, a betaine-fructose-glycerol mixture, or a betaine-glucose-urea mixture. Further preferably, the DES can be a betaine-fructose mixture with a molar ratio of 1:1-2, a betaine-glucose mixture with a molar ratio of 1:1-2, a betaine-fructose-glycerol mixture with a molar ratio of 1:1-2:1-2, or a betaine-glucose-urea mixture with a molar ratio of 1:1-2:1-2.

[0016] The present invention also provides a herbaceous wormwood composition prepared by the above-mentioned preparation method.

[0017] The present invention also provides the use of the above-mentioned qinpi wormwood composition in the preparation of antibacterial, anti-inflammatory, anti-allergic drugs or acne treatment drugs.

[0018] The cortex qinpi is cold and bitter, and has the effects of clearing away heat and dampness, astringing, and improving eyesight. Modern studies have found that the lignans in cortex qinpi are one of the important active ingredients of cortex qinpi, and have multiple pharmacological activities such as liver protection and anti-oxidation. Artemisia argyi is bitter and warm in nature and has the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain. Modern studies have shown that the chlorogenic acid components in artemisia argyi are one of the important active ingredients of artemisia argyi. At present, the research on the antibacterial and anti-inflammatory active ingredients of cortex qinpi and artemisia argyi is mostly focused on coumarins and volatile oil components, and there has been no relevant research and report on the compatibility of cortex qinpi lignan component extracts and artemisia argyi chlorogenic acid component extracts in the treatment of acne. The present invention conducts in-depth research on the antibacterial, anti-inflammatory, and anti-allergic effects and mechanisms of the compatibility of cortex qinpi lignan component extracts and artemisia argyi chlorogenic acid component extracts in the treatment of acne, in order to provide new options for the clinical development of drugs for the treatment of acne.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The extract of lignan components of Fragrant Cortex and the extract of chlorogenic acid components of Artemisia argyi in the present invention are extracted and refined from Fragrant Cortex and Artemisia argyi, which can not only significantly inhibit Propionibacterium acnes and Staphylococcus aureus, but also have anti-inflammatory, anti-allergic and good acne inhibition effects and have no obvious adverse reactions to the human body.

[0021] 2) The yield of the extract of the qinpi lignans components obtained by the present invention is more than 2.55%, and the content is more than 20.17%; the yield of the extract of the chlorogenic acid components of wormwood is more than 3.05%, and the total content is more than 50.06%, and the extract of the qinpi lignans components and the extract of the chlorogenic acid components of wormwood have synergistic effects in terms of antibacterial, anti-inflammatory and anti-allergic activities, and can be widely used in the prevention and treatment of acne. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HPLC chromatogram of the extract of the lignans components of the cortex quercetin obtained in Example 1; in the figure, 1 is pinoresinol, and 2 is acetoxypinoresinol;

[0023] Figure 2 This is a HPLC chromatogram of the chlorogenic acid component extract of Artemisia argyi prepared in Example 1, in which 1 is chlorogenic acid, 2 is cryptochlorogenic acid, 3 is isochlorogenic acid B, 4 is isochlorogenic acid A, and 5 is isochlorogenic acid C;

[0024] Figure 3This is a comparison chart of 3 tests on 5 subjects before and after using the product. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0026] In the following examples, the raw materials used are all common commercial products that can be directly purchased or can be prepared using conventional techniques in the art. For example, Saccharomycopsis fibuligera was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (strain number BNCC 190191). Cellulase and pectinase were purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the enzyme activity was 100u / mg.

[0027] In the present invention, ethanol refers to the volume concentration, and other concentrations not otherwise specified refer to the mass percentage concentration.

[0028] Room temperature refers to 25±5°C.

[0029] Example 1

[0030] A method for preparing a wormwood composition comprises the following steps:

[0031] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (2% Saccharomyces cerevisiae) and biological enzymes (5% cellulase) in a mass ratio of 1:3, and then fermented at 25°C for 48h, and then heated to 40°C for enzymatic hydrolysis for 3h; then 8 times the weight of water of the Cortex Fraxini powder is added for ultrasonic extraction at 75°C for 0.5h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 10% ethanol to remove impurities, and then eluted with 70% ethanol, the 70% ethanol eluate is collected and concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0032] Preparation of chlorogenic acid extract of mugwort: After grinding mugwort, pass through a 30-mesh sieve, take the mugwort powder, and extract the chlorogenic acid components of mugwort using an ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-fructose, molar ratio 1:1) with a water content of 10% and cellulase accounting for 2% of the mass of mugwort powder at a solid-liquid ratio of 1:10 (g / mL), ultrasonic extraction for 30 minutes, extraction temperature 30°C, filter, concentrate under reduced pressure until the ratio of the original medicinal material mugwort to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 (octadecylsilane bonded silica gel) filler is packed into the column, and the sample is directly loaded under a pressure of 3 MPa, the loading flow rate is controlled at 1 mL / min, 30% ethanol is used for elution, and the elution flow rate is controlled at 5 mL / min. The ethanol eluate is collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0033] Figure 1 The HPLC chromatogram of the extract of the lignans from the cortex quercetin prepared in Example 1 is given; it can be seen from the figure that the main components of the extract are pinoresinol and acetoxypinoresinol.

[0034] Figure 2 The HPLC chromatogram of the chlorogenic acid component extract of Artemisia argyi prepared in Example 1 is given. It can be seen from the figure that the main components of this extract are chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C.

[0035] Example 2

[0036] A method for preparing a wormwood composition comprises the following steps:

[0037] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (3% Saccharomyces cerevisiae) and biological enzymes (5% pectinase) in a mass ratio of 1:4, and then fermented at 28°C for 24h, and then heated to 50°C for enzymatic hydrolysis for 4h; then water 10 times the weight of the Cortex Fraxini powder is added and ultrasonically extracted at 80°C for 1h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 20% ethanol to remove impurities, and then eluted with 80% ethanol, the 80% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0038] Preparation of chlorogenic acid extract of mugwort: After grinding mugwort, pass through a 30-mesh sieve, take the mugwort powder, and extract the chlorogenic acid components of mugwort by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-glucose, molar ratio 1:1) with a water content of 20% and cellulase accounting for 3% of the mass of mugwort powder at a solid-liquid ratio of 1:20 (g / mL), ultrasonic extraction for 40 minutes, extraction temperature 40°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material mugwort to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 4 MPa. The loading flow rate was controlled at 2 mL / min. The elution was performed with 40% ethanol and the elution flow rate was controlled at 6 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0039] Example 3

[0040] A method for preparing a wormwood composition comprises the following steps:

[0041] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The cortex qinpi powder (sterilized at 0.1MPa high pressure at 121°C for 20min) was mixed with an aqueous solution containing fermentation bacteria (4% Saccharomyces cerevisiae) and biological enzymes (5%, cellulase: pectinase = 1:1, mass ratio) in a mass ratio of 1:5, and then fermented at 32°C for 36h, and then heated to 60°C for enzymolysis for 5h; then 12 times the weight of water of the cortex qinpi powder was added for ultrasonic extraction at 85°C for 1.5h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material cortex qinpi to the concentrated solution was 1g:2ml, then the concentration was stopped, centrifuged at 5000rpm for 10min, the supernatant was passed through a polyamide resin column, first eluted with water, the water eluate was discarded, and when the eluate was almost colorless, it was eluted with 30% ethanol to remove impurities, and then eluted with 90% ethanol, the 90% ethanol eluate was collected, and the ethanol eluate was concentrated under reduced pressure to dryness, which is the cortex qinpi lignan component extract.

[0042] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-fructose-glycerol, molar ratio 1:1:1) with a water content of 30% and cellulase accounting for 4% of the weight of wormwood powder at a solid-liquid ratio of 1:30 (g / mL), ultrasonic extraction for 50 minutes, extraction temperature 50°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 5 MPa. The loading flow rate was controlled at 3 mL / min. The elution was performed with 50% ethanol and the elution flow rate was controlled at 7 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0043] Example 4

[0044] A method for preparing a wormwood composition comprises the following steps:

[0045] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (2% Saccharomyces cerevisiae) and biological enzymes (5% cellulase) in a mass ratio of 1:6, and then fermented at 35°C for 72h, and then heated to 40°C for enzymatic hydrolysis for 6h; then 14 times the weight of water of the Cortex Fraxini powder is added for ultrasonic extraction at 90°C for 2h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 10% ethanol to remove impurities, and then eluted with 80% ethanol, the 80% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0046] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-glucose-urea, molar ratio 1:1:1) with a water content of 30% and cellulase accounting for 5% of the weight of wormwood powder at a solid-liquid ratio of 1:40 (g / mL), ultrasonic extraction for 60 minutes, extraction temperature 40°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 6 MPa. The loading flow rate was controlled at 4 mL / min. The elution was performed with 30% ethanol and the elution flow rate was controlled at 8 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0047] Example 5

[0048] A method for preparing a wormwood composition comprises the following steps:

[0049] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (3% Saccharomyces cerevisiae) and biological enzymes (5% pectinase) in a mass ratio of 1:3, and then fermented at 28°C for 36h, and then heated to 50°C for enzymolysis for 3h; then water 10 times the weight of the Cortex Fraxini powder is added, and ultrasonic extraction is carried out at 75°C for 0.5h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 10% ethanol to remove impurities, and then eluted with 90% ethanol, the 90% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0050] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-fructose, molar ratio 1:1) with a water content of 10% and cellulase accounting for 3% of the weight of wormwood powder at a solid-liquid ratio of 1:50 (g / mL), ultrasonic extraction for 30 minutes, extraction temperature 40°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 (octadecylsilane bonded silica gel) filler is packed into the column, and the sample is directly loaded under a pressure of 4 MPa, the loading flow rate is controlled at 5 mL / min, 30% ethanol is used for elution, and the elution flow rate is controlled at 9 mL / min. The ethanol eluate is collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0051] Example 6

[0052] A method for preparing a wormwood composition comprises the following steps:

[0053] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (4% Saccharomyces cerevisiae) and biological enzymes (5% cellulase) in a mass ratio of 1:4, and then fermented at 32°C for 48h, and then heated to 40°C for enzymatic hydrolysis for 4h; then water 12 times the weight of the Cortex Fraxini powder is added and ultrasonically extracted at 80°C for 1.5h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 20% ethanol to remove impurities, and then eluted with 70% ethanol, the 70% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0054] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-fructose-glycerol, molar ratio 1:1:1) with a water content of 20% and cellulase accounting for 2% of the weight of wormwood powder at a solid-liquid ratio of 1:50 (g / mL), ultrasonic extraction for 30 minutes, extraction temperature 50°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 5 MPa. The loading flow rate was controlled at 4 mL / min, and eluted with 40% ethanol. The elution flow rate was controlled at 10 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0055] Example 7

[0056] A method for preparing a wormwood composition comprises the following steps:

[0057] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (2% Saccharomyces cerevisiae) and biological enzymes (5% pectinase) in a mass ratio of 1:5, and then fermented at 25°C for 72h, and then heated to 60°C for enzymatic hydrolysis for 6h; then water 10 times the weight of the Cortex Fraxini powder is added, and ultrasonic extraction is carried out at 90°C for 1h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 20% ethanol to remove impurities, and then eluted with 90% ethanol, the 90% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0058] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-glucose, molar ratio 1:1) with a water content of 10% and cellulase accounting for 5% of the weight of wormwood powder at a solid-liquid ratio of 1:30 (g / mL), ultrasonic extraction for 60 minutes, extraction temperature 30°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 2 MPa. The loading flow rate was controlled at 3 mL / min. The elution was performed with 40% ethanol and the elution flow rate was controlled at 10 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0059] Example 8

[0060] A method for preparing a wormwood composition comprises the following steps:

[0061] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (3% Saccharomyces cerevisiae) and biological enzymes (5%, cellulase: pectinase = 1:1, mass ratio) in a mass ratio of 1:6, and then fermented at 28°C for 48h, and then heated to 50°C for enzymolysis for 5h; then 8 times the weight of water of the Cortex Fraxini powder is added for ultrasonic extraction at 75°C for 2h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 30% ethanol to remove impurities, and then eluted with 70% ethanol, the 70% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0062] Preparation of chlorogenic acid extract of mugwort: wormwood, crush, take wormwood powder, and extract chlorogenic acid components of wormwood by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-fructose, molar ratio 1:1) with a water content of 20% and cellulase accounting for 4% of the weight of wormwood powder at a solid-liquid ratio of 1:40 (g / mL), ultrasonic extraction for 40 minutes, extraction temperature 30°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 (octadecylsilane bonded silica gel) filler is packed into the column, and the sample is directly loaded under a pressure of 3 MPa, the loading flow rate is controlled at 5 mL / min, and eluted with 50% ethanol, the elution flow rate is controlled at 5 mL / min, the ethanol eluate is collected, and the ethanol eluate is concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0063] Example 9

[0064] A method for preparing a wormwood composition comprises the following steps:

[0065] Preparation of extract of lignan components of Fraxinus fraxinus: Fraxinus fraxinus is crushed and passed through a 30-mesh sieve, and Fraxinus fraxinus powder is taken, and the lignan components of Fraxinus fraxinus are extracted by a microbial-enzyme-ultrasonic combined process technology. The sterilized (0.1MPa high pressure sterilization at 121°C for 20min) Cortex Fraxini powder is mixed with an aqueous solution containing fermentation bacteria (3% Saccharomyces cerevisiae) and biological enzymes (5%, cellulase: pectinase = 1:1, mass ratio) in a mass ratio of 1:5, and then fermented at 35°C for 36h, and then heated to 40°C for enzymolysis for 3h; then water 12 times the weight of the Cortex Fraxini powder is added and ultrasonically extracted at 85°C for 1h, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material Cortex Fraxini to the concentrated solution is 1g:2ml, then the concentration is stopped, centrifuged at 5000rpm for 10min, the supernatant is passed through a polyamide resin column, first eluted with water, the water eluate is discarded, and when the eluate is almost colorless, it is eluted with 30% ethanol to remove impurities, and then eluted with 80% ethanol, the 80% ethanol eluate is collected, and the ethanol eluate is concentrated under reduced pressure to dryness, which is the Cortex Fraxini lignan component extract.

[0066] Preparation of wormwood chlorogenic acid extract: wormwood, crush, take wormwood powder, and extract wormwood chlorogenic acid components by ultrasound-enzyme-assisted low eutectic solvent method. Add DES (betaine-glucose-urea, molar ratio 1:1:1) with a water content of 30% and cellulase accounting for 2% of the weight of wormwood powder at a solid-liquid ratio of 1:20 (g / mL), ultrasonic extraction for 50 minutes, extraction temperature 50°C, filter, and concentrate under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and enrich it by medium pressure preparative chromatography, and use C 18 The column was packed with (octadecylsilane bonded silica gel) filler and the sample was directly loaded under a pressure of 4 MPa. The loading flow rate was controlled at 4 mL / min. The elution was performed with 50% ethanol and the elution flow rate was controlled at 7 mL / min. The ethanol eluate was collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0067] Comparative Example 1

[0068] Preparation of extracts of lignan components of cortex qinpi (without using microbial-enzymatic hydrolysis process): cortex qinpi, after crushing, pass through a 30-mesh sieve, mix the sterilized cortex qinpi powder with water at a mass ratio of 1:3, and soak for 48 hours. Then add 8 times the weight of water of cortex qinpi powder, ultrasonically extract at 75°C for 0.5 hours, filter, and concentrate under reduced pressure until the ratio of the original medicinal material cortex qinpi to the concentrated solution is 1g:2ml, stop concentrating, centrifuge at 5000rpm for 10 minutes, take the supernatant and pass it through a polyamide resin column, first elute with water, discard the water eluate, elute with 10% ethanol to remove impurities after the eluate is almost colorless, and then elute with 70% ethanol, collect the 70% ethanol eluate and concentrate the 70% ethanol eluate under reduced pressure to dryness, which is the extract of lignan components of cortex qinpi.

[0069] Preparation of chlorogenic acid extract of mugwort (without enzyme-assisted process): wormwood was crushed and passed through a 30-mesh sieve, water was added at a solid-liquid ratio of 1:10 (g / mL), ultrasonic extraction was performed for 30 minutes, the extraction temperature was 30°C, filtered, and concentrated under reduced pressure until the ratio of the original medicinal material wormwood to the concentrated solution was 1g:2ml, then the concentration was stopped, centrifuged at 5000rpm for 10 minutes, and the supernatant was enriched by medium pressure preparative chromatography using C 18 (octadecylsilane bonded silica gel) filler is packed into the column, and the sample is directly loaded under a pressure of 3 MPa, the loading flow rate is controlled at 1 mL / min, 30% ethanol is used for elution, and the elution flow rate is controlled at 5 mL / min. The ethanol eluate is collected and concentrated to dryness under reduced pressure to obtain the chlorogenic acid component extract of Artemisia argyi.

[0070] The yield and content of the extracts of the lignan components of the fraseris chinensis and the extracts of the chlorogenic acid components of the wormwood prepared in Examples 1 to 9 and Comparative Example 1 were determined. The calculation formula is as follows:

[0071] (1) The calculation formula for the yield of component extract is:

[0072] Fraxinus cordata lignans extract rate % = mass of Fraxinus cordata lignans extract / mass of Fraxinus cordata raw material × 100%,

[0073] Yield of chlorogenic acid component extract from mugwort (%) = mass of chlorogenic acid component extract from mugwort / mass of mugwort raw material × 100%. See Table 1 for the results.

[0074] (2) The calculation formula for the content of each component in the component extract is:

[0075] Content of each component of Fraxinus lignans % = mass of each component of Fraxinus lignans / total mass of Fraxinus lignans extract × 100%,

[0076] Content of each component of chlorogenic acid of mugwort (%) = mass of each component of chlorogenic acid of mugwort / total mass of chlorogenic acid component extract of mugwort × 100%. See Table 2 for the results.

[0077] Table 1 Yield of extracts obtained in different embodiments

[0078]

[0079] Table 2 Content of each component in the extracts obtained in different embodiments

[0080]

[0081] It can be seen from Table 1 and Table 2 that the yield and content of the extract of the lignan component of the qinpi and the extract of the chlorogenic acid component of the wormwood obtained under the preparation conditions of Examples 1-9 are significantly higher than the yield and content of the extract of the lignan component of the qinpi and the extract of the chlorogenic acid component of the wormwood obtained under the preparation conditions of Comparative Example 1. Compared with the example, the preparation process of Comparative Example 1 does not add the spore yeast of the button capsule, the low eutectic solvent and the enzyme, which shows that the spore yeast of the button capsule, the low eutectic solvent and the enzyme have a great influence on the extraction of the extract of the lignan component of the qinpi and the extract of the chlorogenic acid component of the wormwood, and can make the lignan component and the chlorogenic acid component in the qinpi and wormwood obtain a good extraction rate. The yield of the lignan component extract of the qinpi obtained by the present invention is more than 2.55%, and the content is more than 20.17%; the yield of the chlorogenic acid component extract of the wormwood obtained is more than 3.05%, and the total content is more than 50.06%.

[0082] Application test

[0083] Modern medicine believes that the pathogenesis of acne is complex, involving the interaction of multiple factors, mainly including excessive sebum secretion, excessive keratinization of the hair follicle sebaceous gland duct, imbalance of the skin microbiome, and the release of inflammatory mediators. Most acne occurs mainly due to the large-scale reproduction of various microorganisms in the skin, especially Propionibacterium acnes and Staphylococcus aureus in the hair follicles and sebaceous glands of the skin, producing toxic enzymes such as lipase, hyaluronic acid lyase, and sialidase, which decompose sebum to produce free fatty acids and cause tissue damage, stimulate hair follicles to cause inflammation, cause damage and rupture of the hair follicle wall, and the accumulated cortex enters the dermis, thereby causing varying degrees of inflammatory and allergic reactions around the hair follicles, promoting the progression of acne.

[0084] Therefore, the extract of the lignan components of the Chinese pectin prepared in Example 1, the extract of the chlorogenic acid components of Artemisia argyi, and the composite composition of the two in different mass ratios (1:1, 1:2, 1:4, 1:6, 6:1, 4:1, 2:1) were subjected to anti-acne efficacy tests, specifically testing the antibacterial activity of the products against Propionibacterium acnes and Staphylococcus aureus, the anti-inflammatory activity against lipopolysaccharide (LPS)-induced Hacat cells, the anti-allergic activity of hyaluronidase in vitro, the anti-inflammatory test on animal acne models, and the human test on acne patients.

[0085] Antibacterial activity test on Propionibacterium acnes and Staphylococcus aureus

[0086] Propionibacterium acnes is an anaerobic Gram-positive bacterium that mainly exists in the deep area of ​​hair follicles. Propionibacterium acnes promotes its pathogenicity by forming biofilms and producing virulence factors that may stimulate the host immune response or promote the release of bacteria. Staphylococcus aureus belongs to the genus Staphylococcus, is a representative of Gram-positive bacteria, and is a common foodborne pathogenic microorganism. Current studies believe that Propionibacterium acnes and Staphylococcus aureus play an important role in the occurrence and development of acne, so inhibiting the growth of Propionibacterium acnes and Staphylococcus aureus is an effective idea and mechanism for treating acne.

[0087] (1) Resuscitation of strains

[0088] The standard strain of Propionibacterium acnes ATCC6919 and the standard strain of Staphylococcus aureus ATCC6538 were revived on slant planes. After resuscitation, Staphylococcus aureus was inoculated on nutrient agar (LB) solid medium and cultured at 37°C for 24 hours; Propionibacterium acnes was inoculated on reinforced Clostridium difficile (RCM) solid medium and cultured anaerobically at 37°C for 48 hours.

[0089] (2) Preparation of bacterial suspension

[0090] Single colonies were picked from the agar plate, and Staphylococcus aureus was inoculated into 10 mL LB liquid medium and cultured at 37°C for 24 h; Propionibacterium acnes was inoculated into 10 mL RCM liquid medium and cultured anaerobically at 37°C for 24 h, and then diluted with sterile water to a concentration of 1×10 6 CFU / mL bacterial suspension for later use.

[0091] (3) Determination of antibacterial activity

[0092] Referring to the Oxford Cup Microbial Inhibition Test of the Pharmacopoeia, the antibacterial activity test of Propionibacterium acnes and Staphylococcus aureus was conducted. Pipette 0.2mL of bacterial suspension into the corresponding culture dish and spread it evenly with a coating rod (both the culture medium and the coating rod need to be sterilized before use), place the Oxford cup (need to be sterilized before use) on the surface of the culture medium, and make the three Oxford cups at a moderate distance, then add 200μL of the test solution with a concentration of 1mg / mL to the Oxford cup, and then put the culture dish into the incubator and culture it at 37℃ for 24h. After the culture is completed, measure the diameter of the inhibition zone, measure it in parallel 3 times, and take the average value as the experimental result.

[0093] The test results are shown in Table 3. The results indicate that the extracts of qinpi lignans and wormwood chlorogenic acid have certain antibacterial effects on Propionibacterium acnes and Staphylococcus aureus when used alone. At the same time, it can be seen that the compositions of the two mixed in different mass ratios have greatly improved inhibitory effects on Propionibacterium acnes and Staphylococcus aureus. Among them, the group of qinpi lignans extract: wormwood chlorogenic acid extract = 1:4 has the strongest antibacterial effect, indicating that the combination of the two has a synergistic effect in antibacterial properties.

[0094] Table 3 Inhibitory effects of extracts of lignans from Cortex Fraxini, chlorogenic acids from Artemisia selengensis, and their composite compositions

[0095]

[0096]

[0097] (4) Determination of minimum inhibitory concentration (MIC)

[0098] The minimum inhibitory concentration (MIC) of each sample was determined by the half-dilution method. First, the drug to be tested was made into an aqueous solution with an initial mass concentration of 1 mg / mL, and then half-diluted with liquid culture medium in turn, with a total of 8 concentrations, that is, the actual detection mass concentrations of each sample were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 3.91 μg / mL. The above solutions were added to a 96-well plate in order according to the mass concentration, 100 μL per well; then 100 μL of each test bacterial suspension was added. Each bacterial liquid was set up with a negative group (100 μL of bacterial liquid and 100 μL of liquid culture medium) and a blank group (only 100 μL of liquid culture medium). The prepared 96-well plate was cultured at 37°C for 24 hours. The clarity of each well was observed, and the lowest dilution concentration for complete sterile growth was taken as the MIC.

[0099] The test results are shown in Table 4. The results show that the combination of different mass ratios of the extract of lignans from the qinpi and the extract of chlorogenic acid from wormwood has a better inhibitory effect on Propionibacterium acnes and Staphylococcus aureus than the use of each alone. Among them, the extract of lignans from the qinpi and the extract of chlorogenic acid from wormwood (1:4) have the best effect against Propionibacterium acnes and Staphylococcus aureus, and their MIC values ​​are 7.81μg / mL and 3.91μg / mL, respectively. It shows that the combination of the two has a synergistic effect in antibacterial, which is consistent with the results of the inhibition zone.

[0100] Table 4 MIC values ​​of extracts of lignans from Cortex Fraxini, extracts of chlorogenic acids from Artemisia selengensis and their composite compositions

[0101]

[0102]

[0103] (5) Determination of minimum bactericidal concentration (MBC)

[0104] On the basis of the previous MIC test, samples from each well without bacterial growth were spread on the corresponding sterile agar plates, cultured at 37°C for 24 hours, and the results were observed. The lowest drug concentration without bacterial growth on the plate was taken as MBC.

[0105] The test results are shown in Table 5. The results show that the MBC values ​​of the composites of the extracts of lignans from the qinpi peel and chlorogenic acid from the wormwood in different mass ratios against Propionibacterium acnes and Staphylococcus aureus are significantly lower than those of the single component groups, among which the group of extracts of lignans from the qinpi peel and chlorogenic acid from the wormwood (1:4) has the lowest MBC values ​​against Propionibacterium acnes and Staphylococcus aureus, which are 15.63μg / mL and 7.81μg / mL, respectively. This shows that the combination of the two has a synergistic effect in antibacterial effects, which is consistent with the results of the inhibition zone and MIC.

[0106] Table 5 MBC values ​​of extracts of lignans from Cortex Fraxini, chlorogenic acids from Artemisia selengensis and their composite compositions

[0107]

[0108]

[0109] Anti-inflammatory test on Hacat cells induced by lipopolysaccharide (LPS)

[0110] Keratinocytes are one of the main components of the hair follicle sebaceous gland unit and play an important role in the skin inflammatory response. They synthesize a variety of cytokines and participate in the immune cascade of acne lesions. Lipopolysaccharide is a bacterial endotoxin found in the cell wall of Gram-negative bacteria. It has been widely used to induce immune responses of various immune cells in vivo and in vitro. Human immortalized keratinocyte Hacat cells activated by LPS will produce a large number of inflammatory factors (such as interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor (TNF-α), etc.), leading to inflammatory response and tissue damage, causing acne. Prostaglandin E2 (PGE2) can promote the occurrence of inflammation and act as an inflammatory mediator that induces pain in the inflammatory response. Therefore, controlling the inflammatory response in the pathological process will help prevent and treat acne.

[0111] (1) Cell culture

[0112] Human immortalized keratinocytes Hacat were cultured in high-glucose DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in a 37°C, 5% CO2 incubator, and passaged when the cell adherence density reached about 80%. Cells in logarithmic growth phase were selected for the experiment.

[0113] (2) CCK8 method to determine cell viability

[0114] Collect the cell pellet and dilute the cells to a density of 1×10 5 / mL, 100μL of cell suspension was added to 96-well plates. After culturing for 24h, 100μL of DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody) was added to the blank group (no cells were inoculated) and the control group (containing 100uL of cell suspension), and 100uL of culture medium containing samples of different concentrations was added to the drug-added group. The pretreatment was performed for 24h, and each group was repeated for 6 wells. Then 100uL of culture medium containing 10% CCK-8 was added in the form of liquid replacement, incubated at 37℃ for 2h, and the OD value at a wavelength of 450nm was measured with an enzyme marker. With normal cells as the control, the cell survival rate was calculated according to the following formula:

[0115] Cell survival rate (%) = [(OD administration - OD blank) / (OD control - OD blank)] x 100%.

[0116] The results are shown in Table 6. The extracts of lignans from the cortex fraxini, chlorogenic acid extracts from wormwood, and the compositions of the two in different mass ratios had cell activity above 90% at a concentration below 125 μg / mL, with no obvious cytotoxicity; at a concentration of 250 μg / mL, cell viability was affected to varying degrees. Therefore, a sample concentration of 125 μg / mL was selected for the next test.

[0117] Table 6 Effects of extracts of lignans from Fraxinus chinensis, chlorogenic acid from Artemisia argyi and their combination on Hacat cell activity %

[0118]

[0119]

[0120]

[0121] (3) Effects on the release of inflammatory factors such as IL-6, IL-1β, TNF-α, and PGE2

[0122] Collect the cell pellet and dilute the cells to a density of 4.0 × 10 5 / mL, 1000uL of cell suspension was added to a 24-well plate. After culturing for 24 hours, the drug group discarded the old culture medium and added 1000uL of culture medium containing different concentrations of the test sample. The control group and the model group were set up, and each group repeated 3 wells. After the drug acted on the cells for 6 hours, the culture medium in the well was aspirated, and LPS solution with a final concentration of 100μg / mL was added to each well of the drug group and the model group. The control group added an equal amount of DMEM culture medium. After continuing to culture for 24 hours, the culture medium supernatant was collected and centrifuged at 4000r / min for 10 minutes. The levels of IL-6, IL-1β, PGE2, and TNF-α in the cell supernatant of each group were detected according to the instructions of the corresponding ELISA kit.

[0123] The results are shown in Table 7. Compared with the control group, the levels of IL-6, IL-1β, TNF-α and PGE2 in the cell supernatant of the model group were significantly increased (P<0.05), indicating that the inflammatory model had been successfully constructed; compared with the model group, the extracts of fraxinus lignans, the extracts of wormwood chlorogenic acid and their combined combination group could significantly inhibit the release of IL-6, IL-1β, TNF-α and PGE2 in the supernatant of Hacat cells (P<0.05), among which the extracts of fraxinus lignans and wormwood chlorogenic acid (1:4) had the best effect, indicating that the extracts of fraxinus lignans, the extracts of wormwood chlorogenic acid and their combined combination had a significant inhibitory effect on the increased level of inflammatory factors induced by LPS, and the combination of the two had a better inhibitory effect on Hacat cell inflammation caused by LPS, playing a synergistic role, which was consistent with the antibacterial results.

[0124] Table 7 Effects of extracts of lignans from qinpi, extracts of chlorogenic acid from mugwort and their combination on the levels of IL-6, IL-1β, TNF-α and PGE-2 in the supernatant of Hacat cells

[0125]

[0126] Note: For the same column of data, different letters indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05), the same below.

[0127] Hyaluronidase inhibition and anti-allergy test in vitro

[0128] Hyaluronidase is a participant in type I allergic reactions. Studies have shown that hyaluronidase is strongly correlated with inflammation and allergies. The anti-allergic activity of a substance is evaluated by the hyaluronidase inhibition rate. The greater the hyaluronidase inhibition rate, the stronger the anti-allergic activity. Currently, Western medicine has a definite effect on the treatment of acne, but it is very easy to have adverse reactions such as allergies and drug resistance. Therefore, it is of great significance to obtain natural anti-acne components with anti-allergic effects.

[0129] According to the Morgan-Elson method, 0.5 mL of 125 μg / mL sample solution and 0.5 mL of hyaluronidase (500 U / mL) were added and kept at 37°C for 20 min; 0.1 mL of CaCl2 solution (2.5 mol / L) was added and kept at 37°C for 20 min; 0.5 mL of sodium hyaluronate (0.5 mg / mL, acetic acid-sodium acetate buffer was used as solvent) was added and kept at 37°C for 40 min, and then placed at room temperature for 10 min, and 0.5 mL of deionized water, 0.1 mL of NaOH solution (5 mol / L), and 1 mL of acetylacetone solution (3.5 mL of acetylacetone dissolved in 50 mL 1.0 mol / L sodium carbonate solution, mix immediately before use), heat in boiling water for 15 minutes, then immediately put in ice bath for 10 minutes, place at room temperature for 10 minutes, then add 1 mL of P-DAB reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 ml of concentrated hydrochloric acid and 15 ml of anhydrous ethanol), shake thoroughly, add anhydrous ethanol to 8 mL, color at room temperature for 30 minutes, and measure its absorbance at a wavelength of 530 nm. The hyaluronidase inhibition rate is calculated according to the following formula:

[0130] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) x 100%

[0131] During the test:

[0132] A-absorbance value of (hyaluronidase + sample + sodium hyaluronate) sample solution;

[0133] B-(acetate buffer + sample + acetate buffer) absorbance of sample blank;

[0134] C-absorbance value of (hyaluronidase + deionized water + sodium hyaluronate) control solution;

[0135] D-(acetate buffer + deionized water + acetate buffer) absorbance value of the control blank.

[0136] Table 8 Results of the inhibition rate of hyaluronidase by extracts of lignans from qinpi, extracts of chlorogenic acids from mugwort and their composite compositions

[0137]

[0138]

[0139] The results are shown in Table 8. The results in Table 8 show that the extracts of qinpi lignans, the extracts of wormwood chlorogenic acid and their combined combination can effectively inhibit the activity of hyaluronidase, among which the group of extracts of qinpi lignans and wormwood chlorogenic acid (1:4) has the highest inhibitory activity, indicating that the extracts of qinpi lignans, the extracts of wormwood chlorogenic acid and their combined combination have anti-allergic potential, and the inhibitory effect of the combination of the two on hyaluronidase has a synergistic effect, which is consistent with the antibacterial and anti-inflammatory results.

[0140] Anti-inflammatory test in animal acne model

[0141] Acne is an inflammatory skin disease, and its main pathogen is the Gram-positive anaerobic Propionibacterium acnes. At present, the mouse acne model is one of the main acne models for studying the pathogenesis of acne and the efficacy of clinical anti-acne drugs. It is mainly completed by intradermal injection of Propionibacterium acnes suspension into the mouse auricle to cause mouse ear infection, simulating the external manifestations and inflammatory reactions of acne caused by abnormal Propionibacterium acnes population. Therefore, this experiment constructed a local inflammation model of mouse auricle acne by intradermal injection of Propionibacterium acnes, and used a mixture of extracts of lignans from qinpi and chlorogenic acid extracts from wormwood to intervene in it, and evaluated its effect on treating auricle acne.

[0142] (1) Experimental animals

[0143] 50 healthy SPF male ICR mice aged 6-8 weeks, weighing 38-45 g, were used for the experiment after acclimatization in the breeding center for one week. The room temperature was (25±2)°C, the humidity was 50-60%, and the animals were allowed to drink water and eat freely.

[0144] (2) Experimental methods

[0145] ICR mice were randomly divided into 5 groups according to body weight (blank group, model group, and treatment groups of lignans from fraseris and chlorogenic acid from wormwood (1:4, low, medium, and high doses), with 10 mice in each group. Except for the blank group, which was injected with 20 μL of normal saline, each mouse in the other groups was injected with Propionibacterium acnes suspension (5×10 7cfu / μL), once a day for 5 consecutive days. The experimental group began gavage treatment 1 day after modeling, with extracts of lignans from qinpi and chlorogenic acid from wormwood (1:4, the gavage doses for the low, medium and high dose groups were 50 mg / kg, 100 mg / kg and 200 mg / kg, respectively, and the blank group and model group were gavaged with 20 mL / kg of normal saline, once a day for 7 consecutive days. After the last administration, the mice were fasted but not watered for 24 hours. The thickness of the middle part of the left and right ears of each group of mice was measured by an electronic digital micrometer, and the measurements were repeated twice. The average value was taken and the auricle swelling rate was calculated according to formula (1). At the same time, blood was collected from the mouse orbits. After standing at room temperature for 4 hours, the mice were centrifuged at 4°C and 3500 r / min for 15 minutes. The supernatant was taken and the levels of IL-6, IL-1β, PGE2 and TNF-α were determined according to the instructions of the kit.

[0146] Auricular swelling rate / % = (right auricular thickness - left auricular thickness) / left auricular thickness × 100% (1)

[0147] The test results are shown in Table 9. As can be seen from Table 9, compared with the blank group, the ear swelling rate of acne mice in the model group and the levels of IL-6, IL-1β, TNF-α, and PGE2 in the serum were significantly increased, which was statistically significant (P<0.05), indicating that the mouse acne inflammation model has been successfully established; compared with the model group, the low, medium, and high dose test groups of the extract of the lignans components of the qinpi and the extract of the chlorogenic acid components of the wormwood (1:4) can significantly reduce the ear swelling rate of acne mice and the levels of IL-6, IL-1β, TNF-α, and PGE2 in the serum (P<0.05), among which the high dose group has the best effect. It shows that the compatibility of the extract of the lignans components of the qinpi and the extract of the chlorogenic acid components of the wormwood (1:4, mass ratio) has an excellent acne treatment effect.

[0148] Table 9 Effects of lignans from qinpi and chlorogenic acid from mugwort (1:4) on the swelling rate of the auricle and the levels of IL-6, IL-1β, PGE2 and TNF-α in serum of mice

[0149]

[0150] Human testing of the composition on acne patients

[0151] 100 healthy subjects with mild or moderate acne on the face (the definition of mild to moderate acne: the main symptom of mild acne is only acne, and the main symptom of moderate acne is inflammatory papules and pustules) were selected, with an age range of 18 to 35 years old. They were randomly divided into 10 groups, half male and half female, with 10 people in each group, a blank group (basic mud mask product without a pharmaceutical composition), test group 1 (mud mask product prepared in Example 1), test group 2 (mud mask product prepared in Example 2), test group 3 (mud mask product prepared in Example 3), test group 4 (mud mask product prepared in Example 4), test group 5 (mud mask product prepared in Example 5), test group 6 (mud mask product prepared in Example 6), test group 7 (mud mask product prepared in Example 7), test group 8 (mud mask product prepared in Example 8), test group 9 (mud mask product prepared in Example 9). The subjects were treated with the corresponding mud mask products, and used the products once in the morning and evening after cleansing the face at the acne site. The adverse reactions were detected after using the products for 7 and 14 days, and the a value of the red area of ​​the acne site was analyzed with the VISIA multifunctional skin imaging analyzer. The test environment temperature of this experiment was 20℃~25℃, and the relative humidity was 40%~60%.

[0152] The preparation method of the mud film prepared by the pharmaceutical composition of the present invention is as follows: (1) 28g of ethylhexyl palmitate, 20g of kaolin, 1g of talc, and 3g of PEG-100 stearate are mixed and heated to 80-85°C and stirred evenly to obtain phase A; (2) 12g of glycerin, 0.9g of xanthan gum, 0.1g of propylparaben, and 30ml of deionized water are mixed and heated to 80-85°C and stirred evenly to obtain phase B; (3) Phase A is added to phase B, emulsified and homogenized for 5-10min, and kept warm and stirred for 20-25min to obtain phase C; (4) Phase C is cooled to 50-55°C, and 1g of the extract of the lignan components of the cortex fraxini and 4g of the extract of the chlorogenic acid components of artemisia argyi in Examples 1-9 are added in sequence and stirred evenly to obtain phase C.

[0153] Preparation method of the blank group mud film: (1) Mix 28g ethylhexyl palmitate, 20g kaolin, 1g talc, and 3g PEG-100 stearate, heat to 80-85°C and stir evenly to obtain phase A; (2) Mix 12g glycerin, 0.9g xanthan gum, 0.1g propylparaben, and 35ml deionized water, heat to 80-85°C and stir evenly to obtain phase B; (3) Add phase A to phase B, emulsify and homogenize for 5-10min, keep warm and stir for 20-25min to obtain phase C; (4) Cool phase C to room temperature.

[0154] The adverse reaction results are shown in Table 10. The results of Table 10 show that the paste product composed of the extract of lignans from the cortex fraxini and the extract of chlorogenic acid from mugwort had no irritation to the skin of all subjects, no toxic side effects, and no adverse reactions. Figure 3 It can be seen that after the subjects applied the mud mask product prepared by the pharmaceutical composition of the present invention on their faces, the acne symptoms were significantly improved. The red zone a value on the 7th day and the 14th day were significantly different from the baseline value (P<0.05), and the a value improvement rate was significantly improved. This shows that applying the product prepared by the present invention on the face has a good anti-acne effect.

[0155] Table 10 Adverse reactions of subjects in human trials

[0156]

[0157]

[0158] Note: According to the 2022 edition of the "Technical Specifications for Safety of Cosmetics", in the grading standard for adverse skin reactions, Grade 0 represents no reaction; Grade 1 represents a suspicious reaction, with only slight erythema; Grade 2 represents a weak positive reaction (erythema reaction), with erythema, infiltration, edema, and may have papules; Grade 3 represents a strong positive reaction (herpes reaction), with erythema, infiltration, edema, papules, and herpes, and the reaction may exceed the test area; Grade 4 represents an extremely strong positive reaction (confluent herpes reaction), with obvious erythema, severe infiltration, edema, and confluent herpes, and the reaction exceeds the test area.

[0159] Table 11 The red zone a value test results of the subjects for 3 times

[0160]

[0161]

[0162] Note: Different letters indicate significant differences among the three results of each group (p<0.05); Improvement rate (%) = (basic value - test value after using the product for N days) / basic value × 100%.

Claims

1. A method for preparing a composition of Artemisia selengensis, characterized in that: The steps include: 1) Preparation of extracts of lignan components of cortex fraxini: Sterilized cortex fraxini powder is mixed with an aqueous solution containing fermentation bacteria and biological enzymes, and then fermented at 25-35°C for 24-72h, and then heated to 40-60°C for enzymatic hydrolysis for 3-6h; then water is added for ultrasonic extraction at 75-90°C for 0.5-2h, filtered, concentrated under reduced pressure, centrifuged, and the supernatant is passed through a polyamide resin column, first eluted with water until the eluent is nearly colorless, then eluted with 10%-30% ethanol, and finally eluted with 70%-90% ethanol and the 70%-90% ethanol eluent is collected, and concentrated to dryness under reduced pressure to obtain extracts of lignan components of cortex fraxini; 2) Preparation of chlorogenic acid extract of mugwort: DES with a water content of 10%-30% and cellulase with a mass content of 2%-5% of mugwort powder are added to mugwort powder, and then ultrasonically extracted at 30-50°C for 30-60 min, filtered, the filtrate is concentrated under reduced pressure, centrifuged, and the supernatant is enriched by medium-pressure preparative chromatography, packed with C18 filler, directly loaded at a pressure of 0-10MPa, eluted with 30-50% ethanol, and the 30-50% ethanol eluate is collected, and concentrated to dryness under reduced pressure to obtain the chlorogenic acid extract of mugwort; The Fragrant Cortex Artemisiae Argyi composition is a mixture of Fragrant Cortex Artemisiae Argyi lignan component extracts and / or Artemisiae Argyi chlorogenic acid component extracts.

2. The method for preparing the herb composition according to claim 1, characterized in that: The sterilized Fraxinus fraseri powder is mixed with an aqueous solution containing 2%-4% fermentation bacteria and 4-6% biological enzyme in a mass ratio of 1:3-6.

3. The method for preparing the herb composition of claim 2, wherein: The fermentation bacteria is Saccharomyces cerevisiae.

4. The method for preparing the qinpi wormwood composition as claimed in claim 2, characterized in that: The biological enzyme is composed of a mixture of cellulase and pectinase.

5. The method for preparing the qinpi wormwood composition according to claim 1, characterized in that: In step 1), 8-14 times the weight of water is added to the Cortex Fraxini powder for ultrasonic extraction.

6. The method for preparing the qinpi wormwood composition according to claim 1, characterized in that: In steps 1) and 2), the concentration is stopped when the ratio of the original medicinal material to the concentrated solution is 1 g:1.8-3 ml.

7. The method for preparing the qinpi wormwood composition according to claim 1, characterized in that: DES with a water content of 10%-30% is added at a material-liquid ratio of 1g:10-50mL, wherein the DES is a betaine-fructose mixture, a betaine-glucose mixture, a betaine-fructose-glycerol mixture or a betaine-glucose-urea mixture.

8. The method for preparing the qinpi wormwood composition as claimed in claim 7, characterized in that: The DES is a betaine-fructose mixture with a mass ratio of 1:1-2, a betaine-glucose mixture with a mass ratio of 1:1-2, a betaine-fructose-glycerol mixture with a mass ratio of 1:1-2:1-2, or a betaine-glucose-urea mixture with a mass ratio of 1:1-2:1-2.

9. The Qinpi Artemisia composition prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Qinpi Artemisia argyi composition according to claim 9 in the preparation of antibacterial, anti-inflammatory, antiallergic drugs or drugs for treating acne.

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