Preparation method of Huangshan pine needle polyphenol and acne-removing and inflammation-diminishing essence

Abstract

According to the preparation method of the Huangshan pine needle polyphenol and the acne-removing and inflammation-diminishing essence, the Huangshan pine needle polyphenol is prepared by adopting a microbial solid fermentation method, release of soluble polyphenol substances in Huangshan pine needles is remarkably promoted, and compared with an unfermented group, the polyphenol content of pine needles reaches up to 146.32 + / -2.53 mg / g and is increased by 1.89 times. The pine needle polyphenol component is prepared by adopting ultrasonic-assisted deep eutectic solvent extraction, membrane filtration and separation and purification technologies, the method has the advantages of short extraction time, low extraction temperature, environmental friendliness and the like, the structural integrity of pine needle polyphenol is maintained to the greatest extent, and the antibacterial activity of polyphenol is remarkably improved. The acne-removing and inflammation-diminishing essence containing the Huangshan pine needle polyphenol is soft in formula and high in safety, has remarkable dual effects of moisturizing and acne removing, can effectively inhibit skin inflammation, promote repair of acne skin, maintain the health state of the skin and meet the requirements of people, and can be widely applied to the field of skin care products.

Description

Technical Field

[0001] The present invention relates to the field of skin care product preparation, and particularly to a preparation method of Pinus taiwanensis needles polyphenol and an anti-acne and anti-inflammatory essence. Background Art

[0002] Acne is a common chronic inflammatory disease of hair follicles and sebaceous glands, and its main symptoms are comedones, papules, pustules, nodules, cysts, etc. Acne is prone to occur in parts with strong sebum secretion such as the face and back, and it is easy to leave post-inflammatory hyperpigmentation. In particular, facial acne can easily affect a person's appearance and even seriously affect a person's mental health. At present, it is generally believed that the occurrence of acne is mainly related to five factors: (1) increased sebum secretion and compositional changes; (2) abnormal keratinization of the hair follicle sebaceous gland duct; (3) increased proliferation of Propionibacterium acnes; (4) skin inflammatory response; (5) change of skin microecological environment. There is a lack of research on the safety evaluation and efficacy evaluation of acne-removing skin care products on the market. However, consumers' demand for acne-removing skin care products is constantly expanding. Therefore, the research on the acne-removing and anti-inflammatory effects of natural extract plants has become a hot topic in the cosmetics industry.

[0003] China is rich in pine tree resources, with more than 120 species belonging to 10 genera. At present, the utilization of pine trees mainly focuses on timber and resin tapping, while pine needles are often only used as fuel or waste. Pine needles are the leaves of plants of the genus Pinus in the family Pinaceae. Compendium of Materia Medica records that pine needles have a bitter taste, a warm nature, and are non-toxic. Taking them for a long time can make people ageless, lighten the body and strengthen qi. They are mainly used to treat rheumatic sores, promote hair growth, nourish the five internal organs, maintain the middle energizer, not feel hungry and prolong life. Modern pharmacological research shows that pine needles contain rich nutritional components and various bioactive components such as proteins, fats, minerals, volatile oils, and flavonoids, and have activities such as analgesia, anti-inflammation, antipruritus, sedation, antitussive, expectorant, anti-asthmatic, regulating blood lipid, antioxidant, anti-aging, anti-mutation, and anti-tumor. Thus, pine needles can be applied to cosmetics, food, medicine, etc., and are renewable resources to be developed and utilized. At present, the varieties studied more are mainly Pinus massoniana, Pinus thunbergii, Pinus elliottii, Pinus koraiensis, Pinus yunnanensis, etc., but there has been no relevant research report on Pinus taiwanensis needles so far.

[0004] Pinus taiwanensis is a unique species in China formed under the unique landforms and climatic conditions such as Huangshan Mountain in Anhui. Its pine needles are shorter and thicker than those of Pinus elliottii and Pinus massoniana, and the resin canal traits are different from those of Pinus tabuliformis. It grows in sections such as the peaks, steep slopes, ridges, and bare rocks above an altitude of 800 m. Due to the uniqueness of Pinus taiwanensis, when extracting polyphenols by existing process methods, the yield of polyphenols is low, and the purity is low and the activity is weak. Therefore, it is necessary to improve the treatment method of pine needles. At the same time, there is also no relevant report on the application of Pinus taiwanensis needles. In order to improve the utilization value of Pinus taiwanensis needles, it is necessary to develop an anti-acne and anti-inflammatory essence containing Pinus taiwanensis needles polyphenol to improve the anti-acne and anti-inflammatory effects. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a preparation method of Pinus taiwanensis needles polyphenols. This method promotes the release of soluble polyphenols in Pinus taiwanensis needles through solid-state fermentation by microorganisms, uses ultrasonic-assisted deep eutectic solvent extraction technology to prepare the polyphenol components of pine needles, and utilizes ultrafiltration membrane retention technology to remove biological macromolecules such as polysaccharides and proteins therein. Combined with macroporous adsorption resin enrichment and purification, Pinus taiwanensis needles polyphenols with high purity and strong activity are obtained.

[0006] Another objective of the present invention also lies in providing an anti-acne and anti-inflammatory essence containing Pinus taiwanensis needles polyphenols to improve the anti-acne and anti-inflammatory effects.

[0007] The technical solution adopted by the present invention to solve its technical problems is: a preparation method of Pinus taiwanensis needles polyphenols, including the following steps: (1) Raw material pretreatment: Pick fresh Pinus taiwanensis needles, wash and dry the Pinus taiwanensis needles and cut them into 1 - 2 cm, and carry out high-pressure hydrothermal sterilization treatment; (2) Microbial solid fermentation: Inoculate 0.1% - 10% of the microbial suspension into the sterilized Pinus taiwanensis needles, and the fermentation culture time is 3 - 14 d; the strains of the microbial suspension are at least one of the following: Coriolus versicolor ( Coriolus vericolor ), Agaricus bisporus ( Agaricus bisporus ), Ganoderma lucidum ( Ganoderma lucidum ), Fomes fomentarius ( Fomes fomentarius ), Polyporus umbellatus ( Grifola umbellate ), Lentinula edodes ( Lentinula edodes ), Pycnoporus cinnabarinus ( Phellinus linteus ), Agaricus blazei ( Agaricus blazeimurii ), Pleurotus eryngii ( Pleurotus eryngii ), Schizophyllum commune ( Schizphyllum commune ). The concentration of the microbial suspension is 1×10 6 ~1×10 8 CFU / mL, and the temperature of the fermentation culture is 28°C - 45°C.

[0008] (3) Drying and pulverizing: Freeze-dry the solid-fermented Pinus taiwanensis needles, transfer them to a wall breaker for air flow pulverization, adjust the temperature in the machine to -10°C - 20°C, introduce high-speed air flow, the air flow pulverization pressure is 0.5 - 2.5 MPa, and the pulverization time is 2 - 5 min to obtain the powder of the Pinus taiwanensis needles fermentation product.

[0009] (4)Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:1~5:1~5:1~6. After mixing, place it in a water bath shaker at 45°C~75°C and 300~1200 r / min for reaction for 0.5~2 h to form a clear and transparent liquid. If no precipitate appears after standing overnight, the deep eutectic solvent is successfully prepared and sealed for standby.

[0010] (5)Ultrasound-assisted extraction with deep eutectic solvent: Add deep eutectic solvent to the fermented product powder of Pinus taiwanensis needles. The mass ratio of the fermented product powder of Pinus taiwanensis needles to the deep eutectic solvent is 1:2~10, so that the fermented product powder of Pinus taiwanensis needles is completely immersed in the deep eutectic solvent, and ultrasonic treatment is carried out at a power of 100~400 W. Set: the treatment temperature is 35°C~65°C and the time is 20~120 min. Centrifuge at a rotation speed of 2500~10000 rpm for 10~30 min, collect the supernatant and perform rotary evaporation and concentration to obtain the fermented extract of Pinus taiwanensis needles.

[0011] (6)Membrane filtration: Filter the fermented extract of Pinus taiwanensis needles prepared in (5) through a 3 kD~10 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain the polyphenol filtrate of Pinus taiwanensis needles.

[0012] (7)Separation and purification: Add the polyphenol filtrate prepared in (6) to non-polar or weakly polar macroporous resins (the macroporous resins are at least one of the following: NKA-II type, XDA-7 type, D101 type, ADS-17), and perform dynamic elution according to an elution solvent of 5%~85% ethanol solution and an elution rate of 1.2-3.5 BV / h. The elution solution is concentrated and then vacuum freeze-dried at -45°C~ -55°C for 4~9 h to obtain the polyphenols of Pinus taiwanensis needles.

[0013] The present invention also provides an anti-acne and anti-inflammatory essence containing polyphenols from Pinus taiwanensis needles. The components of the anti-acne and anti-inflammatory essence are as follows by weight percentage: polyphenols from Pinus taiwanensis needles 0.05% - 1.5%, extract of Chimonanthus salicifolius leaves 0.02% - 0.2%, extract of Astragalus membranaceus roots 0.02% - 0.2%, extract of Saposhnikovia divaricata roots 0.02% - 0.2%, extract of Calendula officinalis 0.02% - 0.2%, extract of Albizia julibrissin flowers 0.02% - 0.2%, extract of Gastrodia elata roots 0.02% - 0.2%, extract of Phellodendron amurense bark 0.02% - 0.2%, extract of Salix alba bark 0.02% - 0.2%, extract of Scutellaria baicalensis roots 0.02% - 0.2%, extract of Houttuynia cordata 0.03% - 0.3%, sodium hyaluronate 0.05% - 0.2%, allantoin 0.05% - 0.2%, acrylic (ester) / C10 - 30 alkyl acrylate cross-linked polymer 0.1% - 0.3%, butanediol 2% - 7%, arginine 0.1% - 0.3%, arabic gum salicylic acid 0.1% - 0.3%, quaternium - 73 0.001% - 0.01%, PEG - 40 hydrogenated castor oil 0.02% - 0.1%, preservative 3.0% - 6.5% and the balance of water, and the water is deionized water, distilled water or purified water.

[0014] The preservative includes 0.1% - 0.6% of p - hydroxyacetophenone, 0.1% - 0.5% of 1,2 - hexanediol, and 1.5% - 6% of butanediol.

[0015] The preparation process of the anti - acne and anti - inflammatory essence containing polyphenols from Pinus taiwanensis needles includes the following steps: (1) Add sodium hyaluronate, allantoin, and acrylic (ester) / C10 - 30 alkyl acrylate cross - linked polymer to an emulsifying pot in sequence according to the proportions of the components of the anti - acne and anti - inflammatory essence, add water, heat and stir at 80°C - 95°C until dissolved to form an aqueous phase. After cooling to 45°C - 65°C, slowly add arginine to the aqueous phase, perform high - speed shearing homogenization with a high - speed shearing homogenizer at 5000 - 12000 rpm, and perform ultrasonic treatment with 100 - 400 W at a temperature of 35°C - 55°C for a working time of 5 - 10 min, and then cool down by ice - water bath to obtain liquid A.

[0016] (2) Take an appropriate amount of butanediol according to the proportions of the components of the anti - acne and anti - inflammatory essence, add quaternium - 73, and stir with a stirrer until completely dissolved. The stirring speed is 2000 - 5000 rpm and the time is 5 - 10 min to obtain liquid B.

[0017] (3) Take p - hydroxyacetophenone, 1,2 - hexanediol, and butanediol according to the proportions of the components of the anti - acne and anti - inflammatory essence, heat and stir at 45°C - 50°C until dissolved to obtain liquid C.

[0018] (4) Slowly add the B liquid cooled to room temperature to the A liquid in the emulsifying pot according to the proportions of the components of the above acne-removing and anti-inflammatory essence, and stir with a blender until completely mixed; then add polyphenols from Pinus taiwanensis needles, extracts of Chimonanthus salicifolius leaves, extracts of Astragalus membranaceus roots, extracts of Saposhnikovia divaricata roots, extracts of Calendula officinalis flowers, extracts of Albizia julibrissin flowers, extracts of Gastrodia elata roots, extracts of Phellodendron amurense bark, extracts of Salix alba bark, extracts of Scutellaria baicalensis roots, extracts of Houttuynia cordata, arabic gum salicylic acid, and PEG-40 hydrogenated castor oil, and homogenize at a speed of 5000-12000 rpm, intermittently for three times, with each time being 10-30 s; heat the external circulation water temperature to 55°C-75°C and sterilize for 15-35 min; finally add the C liquid and stir evenly.

[0019] (5) Adjust the pH of the material in the range of 5.5-6.5, adjust the viscosity to 500-2500 mPa·s, and evacuate and defoam under vacuum at 30°C-37°C to finally form a light yellow transparent and uniform liquid with a characteristic odor, which is the acne-removing and anti-inflammatory essence.

[0020] The beneficial effects of the present invention: In the preparation process of polyphenols from Pinus taiwanensis needles in the present invention, the needles are treated by a microbial solid fermentation process, which can significantly promote the release of soluble polyphenols in Pinus taiwanensis needles. Compared with the unfermented group, the content of polyphenols from Pinus taiwanensis needles is as high as 146.32±2.53 mg / g, which is 1.89 times higher. It not only saves production costs but also maximally simplifies the production steps, enabling this fermentation technology to achieve large-scale production and industrial production. At the same time, the ultrasonic-assisted deep eutectic solvent extraction technology is used to prepare the polyphenol components from Pinus taiwanensis needles, greatly improving the polyphenol extraction rate, having the advantages of short extraction time, low extraction temperature, environmental friendliness, etc., and maintaining the integrity of the polyphenol structure of Pinus taiwanensis needles to the greatest extent, significantly improving the antibacterial activity of polyphenols. Then, the ultrafiltration membrane retention technology is used to remove biological macromolecules such as polysaccharides and proteins, and macroporous adsorption resins are used for enrichment and purification, having the advantages of high recovery rate, good separation effect, and high preparation purity.

[0021] The acne-removing and anti-inflammatory essence prepared by the present invention has excellent acne-removing and anti-inflammatory effects and stable performance. The active ingredients of the present invention all come from natural plants, and through the systematic compatibility among the active components, they have a synergistic effect. Clinical use shows that the formula of the acne-removing and anti-inflammatory essence of Pinus taiwanensis needles of the present invention is mild and has strong safety, with significant dual effects of moisturizing and acne-removing. It can effectively inhibit skin inflammation and promote the repair of acne skin. Among them, the acne / papule count of patients decreased extremely significantly by 46%, maintaining the healthy state of the skin and meeting the needs of people.

[0022] The following will describe the present invention in more detail with reference to the drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This shows the change in enzyme activity during the microbial fermentation of Pinus taiwanensis needles in the present invention.

[0024] Figure 2 This shows the change trend of polyphenols during the microbial fermentation of Pinus taiwanensis needles in the present invention.

[0025] Figure 3 This is the Visia-CR red area map of part of the volunteers in Group 1 of the present invention. Detailed implementation manners

[0026] Example 1: A method for preparing polyphenols from Pinus taiwanensis needles, comprising the following steps: (1) Raw material pretreatment: Pick fresh Pinus taiwanensis needles, wash and air-dry the raw material of Pinus taiwanensis needles, cut them into 1 - 2 cm pieces, and perform high-pressure hydrothermal sterilization.

[0027] (2) Microbial solid fermentation: Inoculate 8% of Coriolus vericolor bacterial suspension into (1), the concentration of the bacterial suspension is 1×10 8 CFU / mL, the fermentation temperature is 28°C, and the fermentation time is 14 d.

[0028] (3) Drying and pulverization: Freeze-dry the solid-fermented Pinus taiwanensis needles, transfer them to a wall breaker for airflow pulverization, adjust the temperature inside the machine to 4°C, introduce high-speed airflow, the airflow pulverization pressure is 2.0 MPa, and the pulverization time is 3 min to obtain the powder of the fermented product of Pinus taiwanensis needles.

[0029] (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid and water in a molar ratio of 1:2:2:2. Place it in a water bath shaker at 55°C and 1000 r / min for reaction for 0.5 h to form a clear and transparent liquid. After standing overnight without precipitation, the deep eutectic solvent is successfully prepared and sealed for standby.

[0030] (5) Ultrasonic-assisted extraction with deep eutectic solvent: Add deep eutectic solvent with a mass ratio of 1:10 to the powder of the fermented product of Pinus taiwanensis needles, and ensure that the powder of the fermented product of Pinus taiwanensis needles is completely immersed in the deep eutectic solvent during the treatment process. Then perform ultrasonic treatment at a power of 300W, the temperature is set at 35°C, and the time is 20 min. Finally, centrifuge at a rotation speed of 10000 rpm for 15 min, collect the supernatant for rotary evaporation and concentration to obtain the fermentation extract of Pinus taiwanensis needles.

[0031] (6) Membrane filtration: Filter the fermentation extract of Pinus taiwanensis needles prepared in (5) through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides and obtain a polyphenol filtrate.

[0032] (7) Separation and purification: Add the polyphenol filtrate prepared in (6) to non-polar or weakly polar macroporous resin, and the macroporous resin is ADS-17. Perform dynamic elution according to the elution solvent of 35% ethanol solution and the elution rate of 2 BV / h. The elution solution is concentrated and then vacuum freeze-dried at -45°C for 8 h to obtain Pinus taiwanensis needles polyphenols.

[0033] Example 2: A preparation method of Pinus taiwanensis needles polyphenols, comprising the following steps: (1) Raw material pretreatment: Pick fresh Pinus taiwanensis needles, wash, dry and cut the Pinus taiwanensis needle raw materials into 1-2 cm, and perform high-pressure hydrothermal sterilization.

[0034] (2) Microbial solid fermentation: Inoculate 8% of Ganoderma lucidum Ganoderma lucidum ( ) bacterial suspension into (1), and the concentration of the bacterial suspension is 1×10 8 CFU / mL. The temperature of fermentation culture is 28°C and the time of fermentation culture is 14 d.

[0035] (3) Drying and pulverization: Freeze-dry the Pinus taiwanensis needles after solid fermentation and transfer them to a wall breaker for airflow pulverization. Adjust the temperature in the machine to 4°C, introduce high-speed airflow, the airflow pulverization pressure is 2.0 MPa, and the pulverization time is 3 min to obtain the powder of Pinus taiwanensis needles fermentation product.

[0036] (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid and water in a molar ratio of 1:2:2:2. Place it in a water bath shaker at 55°C and 1000 r / min and react for 0.5 h to form a clear and transparent liquid. After standing overnight without precipitation, the deep eutectic solvent is successfully prepared and sealed for standby.

[0037] (5) Ultrasonic-assisted extraction with deep eutectic solvent: Add deep eutectic solvent with a mass ratio of 1:10 to the powder of Pinus taiwanensis needles fermentation product, and ensure that the powder of traditional Chinese medicine composition is completely immersed in the deep eutectic solvent during the treatment process. Perform ultrasonic treatment at a power of 300 W, set the temperature to 35°C, and the time to 20 min. Centrifuge at a speed of 10000 rpm for 15 min, collect the supernatant and perform rotary evaporation and concentration to obtain the fermentation extract of Pinus taiwanensis needles.

[0038] (6) Membrane filtration: Filter the fermentation extract of Pinus taiwanensis needles prepared in (5) through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides and obtain a polyphenol filtrate.

[0039] (7)Separation and purification: Add the polyphenol filtrate prepared in (6) to non-polar or weakly polar macroporous resin, where the macroporous resin is ADS-17. Perform dynamic elution according to the elution solvent of 35% ethanol solution and an elution rate of 2 BV / h. The eluted solution is concentrated and then vacuum freeze-dried at -45°C for 8 h to obtain Pinus taiwanensis needles polyphenols.

[0040] Comparative Example 1: A method for preparing Pinus taiwanensis needles polyphenols, comprising the following steps: (1)Raw material pretreatment: Wash and air-dry the Pinus taiwanensis needles raw material and cut it into pieces of 1 - 2 cm.

[0041] (2)Drying and pulverization: Transfer the air-dried Pinus taiwanensis needles to a crusher for airflow pulverization. Adjust the temperature inside the machine to 4°C, introduce high-speed airflow, the airflow pulverization pressure is 2.0 MPa, and the pulverization time is 3 min to obtain Pinus taiwanensis needles powder.

[0042] (3)Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:2:2:2. Place it in a water bath shaker at 55°C and 1000 r / min for reaction for 0.5 h to form a clear and transparent liquid. Let it stand overnight without precipitation, then the deep eutectic solvent is successfully prepared and sealed for standby.

[0043] (4)Ultrasound-assisted deep eutectic solvent extraction: Add deep eutectic solvent with a mass ratio of 1:10 to the Pinus taiwanensis needles powder, and ensure that the traditional Chinese medicine composition powder is completely immersed in the deep eutectic solvent during the treatment process. Perform ultrasonic treatment at a power of 300 W, set the temperature to 35°C, and the time to 20 min. Centrifuge at a speed of 10000 rpm for 15 min, collect the supernatant and perform rotary evaporation concentration to obtain Pinus taiwanensis needles extract.

[0044] (5)Membrane filtration: Filter the Pinus taiwanensis needles extract prepared in (4) through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides and obtain polyphenol filtrate.

[0045] (6)Separation and purification: Add the polyphenol filtrate prepared in (5) to non-polar or weakly polar macroporous resin (the macroporous resin is ADS-17), perform dynamic elution according to the elution solvent of 35% ethanol solution and an elution rate of 2 BV / h. The eluted solution is concentrated and vacuum freeze-dried at -45°C for 8 h to obtain Pinus taiwanensis needles polyphenols.

[0046] 1. Solid fermentation of Pinus taiwanensis needles and determination of the growth diameter of the fermented mass Measure the growth diameter of each fermented mass during the solid fermentation of Pinus taiwanensis needles on the 3rd, 5th, 7th, 10th, and 14th days respectively. Set up Comparative Example 1 of unfermented Pinus taiwanensis needles, that is, inoculate an equal volume of sterilized distilled water.

[0047] Table 1 shows the culture conditions of different medicinal (edible) fungi

[0048] After the solid fermentation of Pinus taiwanensis needles with Coriolus versicolor and Ganoderma lucidum, the growth changes of each fungal mass are shown in Table 2. In the early growth stage (0 - 10 d), the mycelial growth rates of Example 1 and Example 2 were fast. As the fermentation progressed, the mycelia spread out radially in all directions, the number of mycelia gradually increased, and mycelial bundles were gradually formed. By the 10th day, the surface of Pinus taiwanensis needles was completely covered by each mycelium, and the color was white. The growth diameters of the Coriolus versicolor and Ganoderma lucidum fungal masses reached 30 ± 0.0 and 29 ± 1.1 cm respectively, indicating that both Coriolus versicolor and Ganoderma lucidum, the two medicinal (edible) fungi, could fully utilize Pinus taiwanensis needles for solid fermentation.

[0049] Table 2 shows the changes in the growth diameter of the fungal mass of Pinus taiwanensis needles fermented by microorganisms (cm)

[0050] 2. Determination of enzyme activities before and after solid fermentation of Pinus taiwanensis needles Respectively take 2.5 g of the solid fermentation samples of Pinus taiwanensis needles at 0, 3, 5, 7, 10, and 14 d, add 50 mL of 0.1 mol / L citric acid buffer solution with pH 6.0, and stir well for 30 min. Centrifuge at 4 °C and 10,000 r / min for 20 min, and take the supernatant, which is the crude enzyme solution. The determination of amylase activity refers to the DNS reagent method, where the unit definition of amylase activity (unit) is the amount of enzyme required to hydrolyze and generate 1 µg of glucose per minute at 37 °C. The determination of protease activity refers to the Folin reagent method, where the unit definition of protease activity (unit) is the amount of enzyme required to hydrolyze and generate 1 µg of tyrosine per minute at 40 °C. The determination of cellulase activity refers to the CMC saccharification method, where the unit definition of cellulase activity (unit) is the amount of enzyme required to hydrolyze and generate 1 µg of glucose per minute at 37 °C.

[0051] Coriolus versicolor and Ganoderma lucidum were selected for solid fermentation of Pinus taiwanensis needles, and the amylase activity, protease activity, and cellulase activity in Pinus taiwanensis needles before and after fermentation were compared. The results are shown in Figure 1 . In Comparative Example 1, the amylase activity was relatively low, only 3.00 unit / g. After fermentation with Coriolus versicolor and Ganoderma lucidum, the cellulase activities of Example 1 and Example 2 increased significantly ( P < 0.05), reaching 338.40 ± 8.25 and 56.41 ± 9.91 unit / g respectively. Compared with Comparative Example 1, the protease activity and cellulase activity in Pinus taiwanensis needles after fermentation in Example 1 and Example 2 increased significantly ( P<0.05), and the protease activity and cellulase activity in the fermented Pinus taiwanensis needles in Example 1 were higher than those in Example 2. The results showed that two medicinal (edible) fungi, Coriolus versicolor and Ganoderma lucidum, could produce highly active amylase, protease and cellulase during the fermentation of Pinus taiwanensis needles, which was beneficial to increasing the content of bioactive substances in the fermented Pinus taiwanensis needles and improving the functional activity of Pinus taiwanensis needles.

[0052] 3. Determination of Polyphenol Content in Solid Fermentation of Pinus taiwanensis Needles Using the Folin-Ciocalteu method, 0.000, 0.025, 0.050, 0.100, 0.150, 0.200 mL of tannic acid (1 mg / mL) was taken, and the solution was made up to 1 mL to prepare the test solution. 0.05 mL of each test solution and 0.05 mL of Folin-Ciocalteu reagent (0.25 mol / L) were taken. After reacting for 3 min, 1 mL of 0.7 mol / L sodium carbonate solution was added, and the mixture was left standing at 30 °C for 1 h for color development. 0.2 mL was accurately pipetted and the absorbance was measured at 750 nm with an enzyme-linked immunosorbent assay reader. At the same time, the Folin-Ciocalteu reagent was replaced with deionized water as the blank control group, and the standard curve of mass concentration - absorbance value was drawn. The polyphenol contents of the microbial fermentation group and non-fermentation group of Pinus taiwanensis needles were measured in the same way, and the results were expressed as the milligrams of tannic acid equivalent per gram of extract, that is, mg TA / g.

[0053] Figure 2 It is the change trend of polyphenols in the microbial fermentation of Pinus taiwanensis needles. The results showed that in the stage of 0 - 10 days of fermentation, the polyphenol contents of Pinus taiwanensis needles in Example 1 and Example 2 both increased significantly with the extension of fermentation time; on the 10th day of fermentation, the polyphenol content of Pinus taiwanensis needles in Example 1 reached the highest, which was 146.32 ± 2.53 mg / g. When fermented with Coriolus versicolor, the polyphenol content of Pinus taiwanensis needles in Example 1 increased by 1.89 times compared with Comparative Example 1. The results confirmed that the microbial solid-state fermentation method of the present invention could significantly promote the release of polyphenols in Pinus taiwanensis needles, which might be related to the fact that two medicinal (edible) fungi, Coriolus versicolor and Ganoderma lucidum, could produce highly active amylase, protease and cellulase during the fermentation of Pinus taiwanensis needles.

[0054] 4. Antibacterial Effect of Polyphenols in Pinus taiwanensis Needles.

[0055] 4.1 Bacterial Strain Activation and Bacterial Suspension Preparation: Escherichia coli, Staphylococcus aureus, Propionibacterium acnes and Pseudomonas aeruginosa were inoculated onto the slant of the test tube medium for activation and cultured in a constant temperature incubator at 37 °C for 24 h. After the colonies grew, the activated bacterial strains were each picked and inoculated into sterile physiological saline in a sterile operating table to prepare the concentration of each bacterial suspension to be about 10 6 ~10 7 CFU / mL.

[0056] 4.2 Determination of antibacterial activity by inhibition zone method: Pour the melted and sterilized culture medium into the sterilized petri dish in the sterile operation bench. After cooling and solidifying, add 100 μL of the bacterial suspension and spread it evenly with a spreading rod. Use a pipette to add 10 μL of the sample to the center of a sterile filter paper with a diameter of 6 mm. Use sterilized distilled water as the blank control (Control), and propylparaben (PP) as the positive control. After culturing upside down at 37 °C for 24 h, measure the diameter of the inhibition zone. The results are repeated 3 times and the average value is taken.

[0057] 4.3 Table 3 shows the antibacterial effect results of the Pinus taiwanensis needles polyphenols prepared by the present invention. Compared with Comparative Example 1, Examples 1 and 2 have significant antibacterial effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Propionibacterium acnes. Among them, the antibacterial effect of Example 1 is more significant. At the same concentration of 1 mg / mL, the antibacterial effect of Example 1 is equivalent to that of the positive control propylparaben. The research results first reveal that Pinus taiwanensis needles polyphenols have obvious inhibitory effects on 4 tested Gram-positive bacteria and Gram-negative bacteria, indicating that the present invention enhances the antibacterial efficacy of Pinus taiwanensis needles polyphenols through the solid fermentation method of Coriolus versicolor and Ganoderma lucidum, laying a scientific basis for the development of functional cosmetics containing Pinus taiwanensis needles polyphenols.

[0058] Table 3 shows the inhibition zone diameters of Examples 1 and 2 and Comparative Example 1 against 4 kinds of bacteria

[0059] Example 3: An anti-acne and anti-inflammatory essence containing Pinus taiwanensis needles polyphenols, the components of the anti-acne and anti-inflammatory essence are as follows by weight percentage: Pinus taiwanensis needles polyphenols 1%, Chimonanthus salicifolius leaf extract 0.1%, Astragalus membranaceus root extract 0.1%, Saposhnikovia divaricata root extract 0.1%, Calendula officinalis extract 0.1%, Albizia julibrissin flower extract 0.05%, Gastrodia elata root extract 0.05%, Phellodendron amurense bark extract 0.05%, Salix alba bark extract 0.1%, Scutellaria baicalensis root extract 0.05%, Houttuynia cordata extract 0.1%, Sodium hyaluronate 0.1%, Allantoin 0.15%, Acrylate / C10-30 alkyl acrylate cross-linked polymer 0.2%, Butylene glycol 7%, Arginine 0.15%, Arabic gum salicylic acid 0.2%, Quaternium-73 0.007%, PEG-40 hydrogenated castor oil 0.1%, preservative 6% and the balance is deionized water, distilled water or purified water. The preservative includes p-hydroxyacetophenone 0.4%, 1,2-hexanediol 0.5%, and butylene glycol 4.5%.

[0060] The preparation process of the anti-acne and anti-inflammatory essence containing Pinus taiwanensis needles polyphenols includes the following steps: (1) Sodium hyaluronate, allantoin, and acrylic acid (esters) / C10-30 alkyl acrylate cross-linked polymer were added to an emulsifier in the proportion of each component in the anti-acne and anti-inflammatory essence, and water was added. The mixture was heated and stirred at 80°C to dissolve and fully dissolve to form an aqueous phase. After cooling to 45°C, arginine was slowly added to the aqueous phase, and sheared at high speed using a 10,000 rpm high-speed shear homogenizer, and treated with 300 W ultrasonic waves at a temperature of 35°C for 10 min. The mixture was cooled in an ice water bath to obtain Liquid A.

[0061] (2) Take an appropriate amount of butylene glycol according to the proportion of each component in the anti-acne and anti-inflammatory essence, add quaternary ammonium salt-73, and use a blender to stir until it is completely dissolved at a speed of 3000 rpm for 10 min to obtain liquid B.

[0062] (3) Take hydroxyacetophenone, 1,2-hexanediol and butanediol according to the proportion of each component in the anti-acne and anti-inflammatory essence, heat and stir at 45°C to dissolve, and obtain Liquid C.

[0063] (4) According to the proportion of each component in the anti-acne and anti-inflammatory essence, slowly add liquid B cooled to room temperature into liquid A in the emulsifier, and stir with a blender until completely mixed; then add Huangshan pine needle polyphenols, willow-leaf osmanthus leaf extract, Astragalus membranaceus root extract, Fangfeng root extract, Calendula officinalis extract, Albizzia julibrissin flower extract, Gastrodia elata root extract, Phellodendron chinense bark extract, Salix alba bark extract, Scutellaria baicalensis root extract, Houttuynia cordata extract, gum arabic salicylic acid and PEG-40 hydrogenated castor oil, and homogenize at a speed of 10,000 rpm, intermittently for three times, each time for 20 s; heat the external circulation water temperature to 65°C and sterilize for 25 min; finally, add liquid C and stir evenly.

[0064] The preparation methods of the Chimonanthus willowii leaf extract, Astragalus membranaceus root extract, Saposhnikovia root extract, Calendula officinalis extract, Albizzia julibrissin flower extract, Gastrodia elata root extract, Phellodendron chinense bark extract, Salix alba bark extract, Scutellaria baicalensis root extract and Houttuynia cordata extract are as follows: each raw material is crushed into powder, 70% wt% ethanol is added for extraction, centrifugation is performed after extraction, the filtrate is concentrated to remove ethanol, and vacuum drying is performed to obtain a powder state to obtain each extract.

[0065] (5) The pH value of the material is adjusted to 6.0, the viscosity is adjusted to 2000 mPa·s, and vacuum degassing is performed at 30°C to finally form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne essence.

[0066] Comparative Example 2: An anti-acne and anti-inflammatory essence. The components of the anti-acne and anti-inflammatory essence are as follows by weight percentage: 0.1% of Chimonanthus salicifolius leaf extract, 0.1% of Astragalus membranaceus root extract, 0.1% of Saposhnikovia divaricata root extract, 0.1% of Calendula officinalis extract, 0.05% of Albizia julibrissin flower extract, 0.05% of Gastrodia elata root extract, 0.05% of Phellodendron amurense bark extract, 0.1% of Salix alba bark extract, 0.05% of Scutellaria baicalensis root extract, 0.1% of Houttuynia cordata extract, 0.1% of sodium hyaluronate, 0.15% of allantoin, 0.2% of acrylate / C10-30 alkyl acrylate cross-linked polymer, 7% of butanediol, 0.15% of arginine, 0.2% of arabic gum salicylic acid, 0.007% of quaternium-73, 0.1% of PEG-40 hydrogenated castor oil, 6% of preservative, and the balance is deionized water, distilled water or purified water. The preservative includes 0.4% of p-hydroxyacetophenone, 0.5% of 1,2-hexanediol, and 4.5% of butanediol.

[0067] The preparation process of the anti-acne and anti-inflammatory essence includes the following steps: (1) Add sodium hyaluronate, allantoin, and acrylate / C10-30 alkyl acrylate cross-linked polymer to the emulsifying pot in sequence according to the proportions of the components in the anti-acne and anti-inflammatory essence, add water, heat and stir to dissolve at 80°C to form an aqueous phase after complete dissolution. After cooling to 45°C, slowly add arginine to the aqueous phase, perform high-speed shearing homogenization with a high-speed shearing homogenizer at 10000 rpm, and perform ultrasonic treatment with 300 W, with the temperature at 35°C and the working time of 10 min, and cool down by taking an ice-water bath to obtain Liquid A.

[0068] (2) Take an appropriate amount of butanediol according to the proportions of the components in the anti-acne and anti-inflammatory essence, add quaternium-73, and stir with a stirrer until completely dissolved. The stirring speed is 3000 rpm and the time is 10 min to obtain Liquid B.

[0069] (3) Take p-hydroxyacetophenone, 1,2-hexanediol, and butanediol according to the proportions of the components in the anti-acne and anti-inflammatory essence, heat and stir to dissolve at 45°C to obtain Liquid C.

[0070] (4) Slowly add the cooled-to-room-temperature Liquid B to Liquid A in the emulsifying pot according to the proportions of the components in the anti-acne and anti-inflammatory essence, and stir with a stirrer until completely mixed; then add Chimonanthus salicifolius leaf extract, Astragalus membranaceus root extract, Saposhnikovia divaricata root extract, Calendula officinalis extract, Albizia julibrissin flower extract, Gastrodia elata root extract, Phellodendron amurense bark extract, Salix alba bark extract, Scutellaria baicalensis root extract, Houttuynia cordata extract, arabic gum salicylic acid, and PEG-40 hydrogenated castor oil, and perform homogenization at a speed of 10000 rpm intermittently three times, with each time being 20 s; heat the external circulation water temperature to 65°C and perform sterilization for 25 min; finally add Liquid C and stir evenly.

[0071] (5) The pH value of the material is adjusted to 6.0, the viscosity is adjusted to 2000 mPa·s, and vacuum degassing is performed at 30°C to finally form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne essence.

[0072] Comparative Example 3: A general acne-removing essence product available on the market, whose ingredients include water, Pichia pastoris fermentation lysate filtrate, butylene glycol, dipropylene glycol, 1,3-propylene glycol, betaine, PEG / PPG-14 / 7 dimethyl ether, glycerol, 1,2-hexanediol, Pueraria lobata root extract, ammonium acryloyldimethyltaurate / VP copolymer, etc.

[0073] 5. Efficacy evaluation 90 volunteers with acne-prone skin (18-40 years old, mild to moderate acne patients, according to the International Modified Classification, with a certain number of acne and papules, not nodules or cysts that are difficult to improve with clinical preparations, etc.) were selected and randomly divided into 3 groups. The first group of volunteers used the product of Example 3, the second group of volunteers used the product of Comparative Example 2, and the third group of volunteers used the product of Comparative Example 3. The specific operation method of the testers is: use it every morning and evening, first wet the facial skin with clean water, after cleansing, apply an appropriate amount of the product on the face, and gently massage until absorbed. The product use cycle is 28 days.

[0074] Scoring criteria: 1. "Agree" is 5 points; "Somewhat agree" is 4 points; "Fair" is 3 points; "Somewhat disagree" is 2 points; "Disagree" is 1 point. 2. The proportion of subjects with a score of 4 or more is the proportion of subject satisfaction.

[0075] Table 4 is a statistical table of the self-evaluation results of the first group of subjects (28 days after using the product) (n=30)

[0076] Table 5 is a statistical table of the self-evaluation results of the second group of subjects (28 days after using the product) (n=30)

[0077] Table 6 is a statistical table of the self-evaluation results of the third group of subjects (28 days after using the product) (n=30)

[0078] As shown in Tables 4 and 5, compared with Comparative Example 2, the acne-removing and anti-inflammatory essence Example 3 prepared by adding Huangshan pine needle polyphenol has more obvious moisturizing and acne-removing effects, can effectively inhibit skin inflammation, promote the repair of acne skin, and maintain the healthy state of the skin. Figure 3, in which the acne / papule count of the patient decreased extremely significantly by 46%. 28 days after using the product of Example 3, 100% of the subjects believed that the pain / itching accompanied by acne was improved, the acne / papules on the skin were reduced, the overall acne redness of the skin and the overall number of acne on the skin were effectively improved, and the proportion of subject satisfaction was 100%. It can be seen from Table 6 that although Comparative Example 3 has a certain acne-removing effect, compared with the Pinus taiwanensis needle polyphenol acne-removing and anti-inflammatory essence developed by the present invention, the proportion of subjects who believed that the overall acne state of the skin was improved was only 33.33%, and the proportion of adverse reactions on the skin was as high as 10%.

[0079] The present invention has been described exemplarily in conjunction with the accompanying drawings. Obviously, the specific implementation of the present invention is not limited by the above methods. As long as various non-substantive improvements are made by adopting the method concept and technical solution of the present invention; or without improvement, the above concept and technical solution of the present invention are directly applied to other occasions, they are all within the protection scope of the present invention.

Claims

1. A preparation method of Pinus taiwanensis needles polyphenols, comprising the following steps: (1) Raw material pretreatment: Pick fresh Pinus taiwanensis needles, wash and dry them, and then perform sterilization treatment; (2) Solid-state microbial fermentation: Inoculate 0.1% - 10% of microbial suspension into the sterilized Pinus taiwanensis needles, and the fermentation culture time is 3 - 14 d; (3) Drying and pulverization: Freeze-dry the solid-state fermented Pinus taiwanensis needles and then pulverize them to obtain Pinus taiwanensis needles fermentation product powder; (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid and water in a molar ratio of 1:1 - 5:1 - 5:1 - 6; after mixing, place it in a water bath shaker at 45°C - 75°C, 300 - 1200 r / min and react for 0.5 - 2 h, then let it stand to obtain the deep eutectic solvent, seal and reserve for use; (5) Ultrasonic-assisted deep eutectic solvent extraction: Add the deep eutectic solvent to the Pinus taiwanensis needles fermentation product powder, and the mass ratio of the Pinus taiwanensis needles fermentation product powder to the deep eutectic solvent is 1:2 - 10, so that the Pinus taiwanensis needles fermentation product powder is completely immersed in the deep eutectic solvent, and perform ultrasonic treatment with a power of 100 - 400 W, set: the treatment temperature is 35°C - 65°C, the time is 20 - 120 min; then centrifuge at a rotation speed of 2500 - 10000 rpm for 10 - 30 min, and collect the supernatant for rotary evaporation and concentration to obtain the Pinus taiwanensis needles fermentation extract; (6) Membrane filtration: Filter the Pinus taiwanensis needles fermentation extract prepared in (5) through a 3 kD - 10 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain the Pinus taiwanensis needles polyphenols filtrate; (7) Separation and purification: Add the polyphenols filtrate prepared in (6) to non-polar or weakly polar macroporous resins, and perform dynamic elution according to an elution solvent of 5% - 85% ethanol solution and an elution rate of 1.2 - 3.5 BV / h. The elution solution is concentrated and then vacuum freeze-dried at -45°C - -55°C for 4 - 9 h to obtain Pinus taiwanensis needles polyphenols.

2. The preparation method of Pinus taiwanensis needle polyphenol according to claim 1, characterized in that: The strains of the microbial bacterial suspension are at least one of the following: Coriolus versicolor ( Coriolus vericolor ), Agaricus bisporus ( Agaricus bisporus ), Ganoderma lucidum ( Ganoderma lucidum ), Fomes fomentarius ( Fomes fomentarius ), Polyporus umbellatus ( Grifola umbellate ), Lentinula edodes ( Lentinula edodes ), Phellinus linteus ( Phellinus linteus ), Agaricus blazei ( Agaricus blazeimurii ), Pleurotus eryngii ( Pleurotus eryngii ), Schizophyllum commune ( Schizphyllum commune ). The concentration of the microbial bacterial suspension is 1×10 6 ~1×10 8 CFU / mL, and the temperature for fermentation culture is 28°C~45°C.

3. The preparation method of Pinus taiwanensis needle polyphenol according to claim 1, characterized in that: The pulverization in step (3) is as follows: Freeze-dry the solid-state fermented Pinus taiwanensis needles and transfer them to a wall breaker for air flow pulverization. Adjust the temperature in the machine to -10°C - 20°C, introduce high-speed air flow, the air flow pulverization pressure is 0.5 - 2.5 MPa, and the pulverization time is 2 - 5 min.

4. The preparation method of Pinus taiwanensis needles polyphenol according to claim 1, characterized in that: The macroporous resin in step (7) is one or a mixture of more of NKA-II type, XDA-7 type, D101 type, and ADS-17.

5. The preparation method of Pinus taiwanensis needles polyphenol according to claim 1, characterized in that: In step (1), the Pinus taiwanensis needles are washed, dried and cut into small pieces of 1-2 cm, and then subjected to high-pressure hydrothermal sterilization treatment.

6. An acne-removing and anti-inflammatory essence containing polyphenols from Pinus taiwanensis needles, characterized in that, The anti-acne and anti-inflammatory essence has the following components by weight percentage: 0.05% - 1.5% of Pinus taiwanensis needle polyphenol, 0.02% - 0.2% of Chimonanthus salicifolius leaf extract, 0.02% - 0.2% of Astragalus membranaceus root extract, 0.02% - 0.2% of Saposhnikovia divaricata root extract, 0.02% - 0.2% of Calendula officinalis extract, 0.02% - 0.2% of Albizia julibrissin flower extract, 0.02% - 0.2% of Gastrodia elata root extract, 0.02% - 0.2% of Phellodendron amurense bark extract, 0.02% - 0.2% of Salix alba bark extract, 0.02% - 0.2% of Scutellaria baicalensis root extract, 0.03% - 0.3% of Houttuynia cordata extract, 0.05% - 0.2% of sodium hyaluronate, 0.05% - 0.2% of allantoin, 0.1% - 0.3% of acrylate / C10-30 alkanol acrylate cross-linked polymer, 2% - 7% of butanediol, 0.1% - 0.3% of arginine, 0.1% - 0.3% of gum arabic salicylic acid, 0.001% - 0.01% of quaternary ammonium salt-73, 0.02% - 0.1% of PEG-40 hydrogenated castor oil, 3.0% - 6.5% of preservative and the balance of water.

7. The anti-acne and anti-inflammatory essence containing Pinus taiwanensis needles polyphenols according to claim 6, characterized in that: The preservative includes 0.1% - 0.6% of p-hydroxyacetophenone, 0.1% - 0.5% of 1,2-hexanediol, 1.5% - 6% of butanediol; the water is deionized water, distilled water or purified water.

8. The preparation process of the anti-acne and anti-inflammatory essence containing Pinus taiwanensis needle polyphenol according to claim 6 or 7, comprising the following steps: (1) Sodium hyaluronate, allantoin, acrylate / C10-30 alkanol acrylate cross-linked polymer are added to an emulsifying pan in sequence, water is added, and the mixture is heated and stirred at 80°C - 95°C until dissolved to form an aqueous phase; after cooling to 45°C - 65°C, arginine is slowly added to the aqueous phase, and high-speed shearing and homogenization are carried out with a high-speed shearing homogenizer at 5000 - 12000 rpm, and ultrasonic treatment is carried out with 100 - 400 W ultrasonic waves, the treatment temperature is 35°C - 55°C, the treatment time is 5 - 10 min, and then ice-water bath cooling is adopted to obtain liquid A; (2) An appropriate amount of butanediol is taken, quaternary ammonium salt-73 is added, and stirring is carried out with a stirrer until completely dissolved, the stirring speed is 2000 - 5000 rpm, and the time is 5 - 10 min to obtain liquid B; (3) p-Hydroxyacetophenone, 1,2-hexanediol, and butanediol are taken, and heated and stirred at 45°C - 50°C until dissolved to obtain liquid C; (4) Slowly add the cooled-to-room-temperature Liquid B to Liquid A in the emulsifying pot, and stir with a stirrer until completely mixed; then add polyphenols from Pinus taiwanensis needles, extracts from Chimonanthus salicifolius leaves, extracts from Astragalus membranaceus roots, extracts from Saposhnikovia divaricata roots, extracts from Calendula officinalis flowers, extracts from Albizia julibrissin flowers, extracts from Gastrodia elata roots, extracts from Phellodendron amurense bark, extracts from Salix alba bark, extracts from Scutellaria baicalensis roots, extracts from Houttuynia cordata, gum arabic salicylic acid, and PEG-40 hydrogenated castor oil, and homogenize at a speed of 5000 - 12000 rpm, intermittently for three times, with each time being 10 - 30 s; then heat the water temperature of the external circulation of the emulsifying pot to 55°C - 75°C and sterilize for 15 - 35 min; finally add Liquid C and stir evenly; (5) Adjust the pH of the material to be within the range of 5.5 - 6.5, adjust the viscosity to 500 - 2500 mPa·s, and perform vacuum defoaming at 30°C - 37°C. Finally, a light yellow transparent and uniform liquid with a characteristic odor is formed, which is the acne-removing and anti-inflammatory essence.

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