Preparation method of Huangshan pine needle polyphenols and acne-removing and anti-inflammatory essence

Abstract

The present invention discloses a preparation method of Huangshan pine needle polyphenols and an acne-removing and anti-inflammatory essence. The Huangshan pine needle polyphenols are prepared by a microbial solid fermentation method, which significantly promotes the release of soluble polyphenols in Huangshan pine needles. Compared with the unfermented group, the pine needle polyphenol content is as high as 146.32±2.53 mg / g, which is increased by 1.89 times. The pine needle polyphenol component is prepared by ultrasonic-assisted deep eutectic solvent extraction, membrane filtration and separation and purification technology, which has the advantages of short extraction time, low extraction temperature, and environmental friendliness. It maintains the integrity of the pine needle polyphenol structure to the greatest extent and significantly improves the antibacterial activity of the polyphenols. The acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols has a soft formula and strong safety. It has significant dual effects of moisturizing and removing acne, can effectively inhibit skin inflammation, promote the repair of acne skin, maintain the healthy state of the skin, meet people's needs, and can be widely used in the field of skin care products.

Description

Technical Field

[0001] The invention relates to the field of skin care product preparation, in particular to a preparation method of Huangshan pine needle polyphenol and an acne-removing and anti-inflammatory essence. Background Art

[0002] Acne is a common chronic inflammatory disease of the hair follicles and sebaceous glands, with the main symptoms being comedones, papules, pustules, nodules, and cysts. Acne is more common in areas with high sebum secretion, such as the face and back, and is prone to post-inflammatory pigmentation. Facial acne, in particular, can easily affect a person's appearance, and in severe cases, it can even affect a person's mental health. Currently, it is generally believed that the occurrence of acne is mainly related to five factors: (1) increased sebum secretion and changes in composition; (2) abnormal keratinization of the hair follicle sebaceous gland ducts; (3) increased proliferation of Propionibacterium acnes; (4) inflammatory response of the skin; and (5) changes in the skin's microecological environment. There is a lack of research on the safety and efficacy evaluation of acne-removing skin care products on the market. However, consumer demand for acne-removing skin care products is constantly expanding. Therefore, research on the acne-removing and anti-inflammatory effects of natural plant extracts has become a hot topic in the cosmetics industry.

[0003] my country boasts abundant pine resources, encompassing over 120 species across 10 genera. Currently, pine tree utilization primarily focuses on timber and resin extraction, while pine needles are often used as fuel or waste. Pine needles are the leaves of the genus Pinus in the Pinaceae family. The Compendium of Materia Medica states: "Pine needles are bitter, warm, and non-toxic. Long-term consumption can prevent aging, lighten the body, and invigorate the Qi. They are primarily used to treat rheumatism, promote hair growth, soothe the five internal organs, maintain balance, and prevent hunger and prolong life." Modern pharmacological research has shown that pine needles are rich in nutrients and bioactive components, including proteins, fats, minerals, volatile oils, and flavonoids. These ingredients possess analgesic, anti-inflammatory, antipruritic, sedative, antitussive, expectorant, antiasthmatic, lipid-regulating, antioxidant, anti-aging, anti-mutagenic, and anti-tumor properties. Therefore, pine needles have applications in cosmetics, food, and medicine, making them a renewable resource awaiting development and utilization. At present, the species that have been studied more are mainly Masson pine, Black pine, Elliot pine, Korean pine, Yunnan pine, etc., but there has been no relevant research report on Huangshan pine needles.

[0004] Huangshan pine is a species endemic to China, formed under the unique topography and climatic conditions of Mount Huangshan in Anhui Province. Its needles are thicker and shorter than those of Pinus elliottii and Pinus massoniana, and its resin ducts differ from those of Pinus tabulaeformis. It grows on peaks, steep slopes, ridges, and bare rock at altitudes above 800 meters. Due to the uniqueness of Huangshan pine, the extraction of its polyphenols using existing methods results in low yield, low purity, and weak activity. Therefore, improvements to the pine needle processing methods are needed. Furthermore, there are currently no reports on the application of Huangshan pine needles. To increase the utilization value of Huangshan pine needles, it is necessary to develop an anti-acne and anti-inflammatory serum containing Huangshan pine needle polyphenols to enhance their effectiveness. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for preparing Huangshan pine needle polyphenols. The method promotes the release of soluble polyphenols in Huangshan pine needles through microbial solid-state fermentation, adopts ultrasound-assisted deep eutectic solvent extraction technology to prepare pine needle polyphenol components, utilizes ultrafiltration membrane interception technology to remove biomacromolecules such as polysaccharides and proteins therein, and combines macroporous adsorption resin for enrichment and purification to obtain Huangshan pine needle polyphenols with high purity and strong activity.

[0006] Another object of the present invention is to provide an acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols to improve the acne-removing and anti-inflammatory effects.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing Huangshan pine needle polyphenols, comprising the following steps:

[0008] (1) Raw material pretreatment: Pick fresh Huangshan pine needles, wash and dry them, cut them into 1-2 cm pieces, and sterilize them with high pressure wet heat;

[0009] (2) Microbial solid fermentation: 0.1% to 10% microbial suspension is inoculated into the sterilized Huangshan pine needles, and the fermentation culture time is 3 to 14 days; the strain of the microbial suspension is at least one of the following: Yunzhi ( Coriolus vericolor ) 、Agaricus bisporus( Agaricus bisporus )、Ganoderma lucidum( Ganoderma lucidum ) 、Fomyces oxysporum ( Fomes fomentarius ) 、Poria ( Grifola umbellate ) ,mushroom( Lentinula edodes ) 、Fomyces schrenkiana ( Phellinus linteus )、Agaricus blazei( Agaricus blazeimurii ) 、Pleurotus eryngii( Pleurotus eryngii ), Schizophyllum ( Schizphyllum commune ), the concentration of microbial suspension is 1×10 6 ~1×10 8 CFU / mL, and the fermentation culture temperature was 28℃~45℃.

[0010] (3) Drying and pulverization: The solid fermented Huangshan pine needles were freeze-dried and transferred to a wall-breaking machine for air flow pulverization. The temperature inside the machine was adjusted to -10℃~20℃, and a high-speed air flow was introduced. The air flow pulverization pressure was 0.5~2.5 MPa, and the pulverization time was 2~5 min to obtain the Huangshan pine needle fermentation product powder.

[0011] (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:1 to 5:1 to 5:1 to 6. After mixing, place in a water bath shaker at 45°C to 75°C and 300-1200 r / min to react for 0.5-2 h to form a clear and transparent liquid. If no precipitation occurs after standing overnight, the deep eutectic solvent is successfully prepared and sealed for later use.

[0012] (5) Ultrasonic-assisted deep eutectic solvent extraction: A deep eutectic solvent was added to the powder of the fermentation product of Huangshan pine needles. The mass ratio of the powder to the deep eutectic solvent was 1:2 to 10. The powder was completely immersed in the deep eutectic solvent. The powder was then treated with ultrasound at a power of 100 to 400 W. The treatment temperature was set at 35°C to 65°C and the treatment time was set at 20 to 120 min. The mixture was centrifuged at a speed of 2500 to 10000 rpm for 10 to 30 min. The supernatant was collected and concentrated by rotary evaporation to obtain the Huangshan pine needle fermentation extract.

[0013] (6) Membrane filtration: The Huangshan pine needle fermentation extract prepared in (5) was filtered through a 3 kD~10 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain the Huangshan pine needle polyphenol filtrate.

[0014] (7) Separation and purification: The polyphenol filtrate prepared in (6) is added to a non-polar or weakly polar macroporous resin (the macroporous resin is at least one of the following: NKA-II, XDA-7, D101, ADS-17), and dynamic elution is performed according to an elution solvent of 5% to 85% ethanol solution and an elution rate of 1.2 to 3.5 BV / h. The eluted solution is concentrated and then vacuum freeze-dried at -45°C to -55°C for 4 to 9 hours to obtain Huangshan pine needle polyphenols.

[0015] The present invention also provides an acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols. The acne-removing and anti-inflammatory essence has the following components by weight percentage: Huangshan pine needle polyphenols 0.05% to 1.5%, Chimonanthus chinensis leaf extract 0.02% to 0.2%, Astragalus membranaceus root extract 0.02% to 0.2%, Saposhnikovia root extract 0.02% to 0.2%, Calendula officinalis flower extract 0.02% to 0.2%, Albizia julibrissin flower extract 0.02% to 0.2%, Gastrodia elata root extract 0.02% to 0.2%, Phellodendron amurense bark extract 0.02% to 0.2%, Salix alba bark extract 0.02% to 0.2%, Scutellaria baicalensis root extract 0.02% to 0.2%, Houttuynia cordata extract 0.03% to 0.3%, sodium hyaluronate 0.05% to 0.2%, allantoin 0.05% to 0.2%, acrylates / C10-30 Alkyl acrylate crosspolymer 0.1% to 0.3%, butylene glycol 2% to 7%, arginine 0.1% to 0.3%, gum arabic salicylic acid 0.1% to 0.3%, quaternium-73 0.001% to 0.01%, PEG-40 hydrogenated castor oil 0.02% to 0.1%, preservatives 3.0% to 6.5% and the remainder water, wherein the water is deionized water, distilled water or purified water.

[0016] The preservatives include 0.1% to 0.6% p-hydroxyacetophenone, 0.1% to 0.5% 1,2-hexanediol, and 1.5% to 6% butylene glycol.

[0017] The preparation process of the acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols comprises the following steps:

[0018] (1) Sodium hyaluronate, allantoin, and acrylic acid (esters) / C10-30 alkyl acrylate cross-polymer were added to an emulsifier in the proportions of the above-mentioned acne-removing and anti-inflammatory essence. Water was added and the mixture was heated and stirred at 80°C to 95°C to dissolve the mixture and form an aqueous phase. After cooling to 45°C to 65°C, arginine was slowly added to the aqueous phase. The mixture was sheared at high speed using a 5000-12000 rpm high-speed shear homogenizer and treated with 100-400 W ultrasonic waves at a temperature of 35°C to 55°C for 5-10 minutes. The mixture was then cooled in an ice water bath to obtain Liquid A.

[0019] (2) Take an appropriate amount of butylene glycol according to the proportion of the components of the above-mentioned acne-removing and anti-inflammatory essence, add quaternary ammonium salt-73, and stir with a blender until completely dissolved at a speed of 2000~5000 rpm for 5~10 minutes to obtain liquid B.

[0020] (3) Take hydroxyacetophenone, 1,2-hexanediol and butanediol according to the proportion of the components of the above-mentioned acne-removing and anti-inflammatory essence, heat and stir at 45℃~50℃ to dissolve, and obtain liquid C.

[0021] (4) According to the proportion of each component of the above-mentioned acne-removing and anti-inflammatory essence, slowly add liquid B cooled to room temperature to the liquid A in the emulsifier, and stir with a blender until completely mixed; then add Huangshan pine needle polyphenols, willow-leaf osmanthus leaf extract, membranaceus root extract, windproof root extract, calendula extract, albizzia julibrissin flower extract, Gastrodia elata root extract, Phellodendron amurense bark extract, white willow 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 5000-12000 rpm, intermittently three times, each time for 10-30 seconds; heat the external circulation water temperature to 55℃-75℃ and sterilize for 15-35 minutes; finally add liquid C and stir evenly.

[0022] (5) Adjust the pH of the material within the range of 5.5 to 6.5, adjust the viscosity within the range of 500 to 2500 mPa·s, and vacuum degas at 30°C to 37°C to form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne and anti-inflammatory essence.

[0023] Beneficial effects of the present invention: The present invention uses a microbial solid fermentation process to treat pine needles during the preparation of Huangshan pine needle polyphenols, which can significantly promote the release of soluble polyphenols in Huangshan pine needles. Compared with the unfermented group, the pine needle polyphenol content is as high as 146.32±2.53 mg / g, which is 1.89 times higher. It not only saves production costs, but also greatly simplifies the production steps, so that the fermentation technology can achieve mass production and industrial production. At the same time, the ultrasonic-assisted deep eutectic solvent extraction technology is used to prepare pine needle polyphenol components, which greatly improves the polyphenol extraction rate, has the advantages of short extraction time, low extraction temperature, and environmental friendliness, and maintains the integrity of the pine needle polyphenol structure to the greatest extent, significantly improving the antibacterial activity of the polyphenols. The ultrafiltration membrane interception technology is then used to remove biomacromolecules such as polysaccharides and proteins, and the macroporous adsorption resin is used for enrichment and purification, which has the advantages of high recovery rate, good separation effect, and high preparation purity.

[0024] The acne-removing and anti-inflammatory essence prepared by the present invention has excellent acne-removing and anti-inflammatory effects and stable performance. The functional ingredients of the present invention are all derived from natural plants, and through the systematic compatibility between the functional components, a synergistic effect is achieved. Clinical use has shown that the Huangshan pine needle acne-removing and anti-inflammatory essence of the present invention has a gentle formula, strong safety, and significant dual effects of moisturizing and removing acne. It can effectively inhibit skin inflammation and promote the repair of acne skin. The acne / papule count of patients has been significantly reduced by 46%, maintaining the healthy state of the skin and meeting people's needs.

[0025] The present invention will be described in more detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The enzyme activity changes of the microorganisms fermenting Huangshan pine needles in the present invention.

[0027] Figure 2 This is the changing trend of the polyphenols from Huangshan pine needles fermented by microorganisms in the present invention.

[0028] Figure 3 This is the Visia-CR-red zone diagram of some volunteers in Group 1 of the present invention. DETAILED DESCRIPTION

[0029] Example 1: A method for preparing Huangshan pine needle polyphenols, comprising the following steps:

[0030] (1) Raw material pretreatment: Pick fresh Huangshan pine needles, wash and dry them, cut them into 1-2 cm pieces, and sterilize them with high pressure moist heat.

[0031] (2) Microbial solid fermentation: Inoculate 8% of Coriolus vericolor bacterial suspension into (1) at a concentration of 1×10 8 CFU / mL, the fermentation temperature was 28℃, and the fermentation time was 14 days.

[0032] (3) Drying and pulverization: The solid fermented Huangshan pine needles were freeze-dried and transferred to a wall-breaking machine for air flow pulverization. The temperature inside the machine was adjusted to 4°C, and a high-speed air flow was introduced. The air flow pulverization pressure was 2.0 MPa, and the pulverization time was 3 min to obtain the Huangshan pine needle fermentation product powder.

[0033] (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:2:2:2. Place the mixture in a water bath shaker at 55°C and 1000 rpm for 0.5 h to form a clear, transparent liquid. If no precipitation occurs after standing overnight, the deep eutectic solvent is successfully prepared and sealed for later use.

[0034] (5) Ultrasonic-assisted deep eutectic solvent extraction: A deep eutectic solvent was added to the powder of the fermentation product of Huangshan pine needles at a mass ratio of 1:10. During the treatment, the powder of the fermentation product of Huangshan pine needles was completely immersed in the deep eutectic solvent. Then, ultrasonic treatment was performed at a power of 300 W, the temperature was set to 35°C, and the time was 20 min. Finally, centrifugation was performed at a speed of 10,000 rpm for 15 min. The supernatant was collected and concentrated by rotary evaporation to obtain the Huangshan pine needle fermentation extract.

[0035] (6) Membrane filtration: The Huangshan pine needle fermentation extract prepared in (5) was filtered through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain a polyphenol filtrate.

[0036] (7) Separation and purification: The polyphenol filtrate prepared in (6) was added to a non-polar or weakly polar macroporous resin, wherein the macroporous resin was ADS-17, and dynamic elution was performed according to an elution solvent of 35% ethanol solution and an elution rate of 2 BV / h. The eluted solution was concentrated and then vacuum freeze-dried at -45°C for 8 h to obtain Huangshan pine needle polyphenols.

[0037] Example 2: A method for preparing Huangshan pine needle polyphenols, comprising the following steps:

[0038] (1) Raw material pretreatment: Pick fresh Huangshan pine needles, wash and dry them, cut them into 1-2 cm pieces, and sterilize them with high pressure moist heat.

[0039] (2) Microbial solid fermentation: Inoculate 8% of Ganoderma lucidum ( Ganoderma lucidum ) Bacterial suspension, the concentration of bacterial suspension is 1×10 8 CFU / mL, the fermentation temperature was 28℃, and the fermentation time was 14 days.

[0040] (3) Drying and pulverization: The solid fermented Huangshan pine needles were freeze-dried and transferred to a wall-breaking machine for air flow pulverization. The temperature inside the machine was adjusted to 4°C, and a high-speed air flow was introduced. The air flow pulverization pressure was 2.0 MPa, and the pulverization time was 3 min to obtain the Huangshan pine needle fermentation product powder.

[0041] (4) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:2:2:2. Place the mixture in a water bath shaker at 55°C and 1000 rpm for 0.5 h to form a clear, transparent liquid. If no precipitation occurs after standing overnight, the deep eutectic solvent is successfully prepared and sealed for later use.

[0042] (5) Ultrasonic-assisted deep eutectic solvent extraction: A deep eutectic solvent was added to the powder of the fermented product of Huangshan pine needles at a mass ratio of 1:10. During the treatment, the powder of the Chinese medicine composition was ensured to be completely immersed in the deep eutectic solvent. Ultrasonic treatment was performed at a power of 300 W, the temperature was set to 35°C, and the time was 20 min. The mixture was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected and concentrated by rotary evaporation to obtain the Huangshan pine needle fermented extract.

[0043] (6) Membrane filtration: The Huangshan pine needle fermentation extract prepared in (5) was filtered through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain a polyphenol filtrate.

[0044] (7) Separation and purification: The polyphenol filtrate prepared in (6) was added to a non-polar or weakly polar macroporous resin, wherein the macroporous resin was ADS-17, and dynamic elution was performed according to an elution solvent of 35% ethanol solution and an elution rate of 2 BV / h. The eluted solution was concentrated and then vacuum freeze-dried at -45°C for 8 h to obtain Huangshan pine needle polyphenols.

[0045] Comparative Example 1: A method for preparing Huangshan pine needle polyphenols, comprising the following steps:

[0046] (1) Raw material pretreatment: Wash and dry the Huangshan pine needles and cut them into 1-2 cm pieces.

[0047] (2) Drying and crushing: The dried Huangshan pine needles were transferred to a wall-breaking machine for air flow crushing. The temperature inside the machine was adjusted to 4°C, and a high-speed air flow was introduced. The air flow crushing pressure was 2.0 MPa, and the crushing time was 3 min to obtain Huangshan pine needle powder.

[0048] (3) Preparation of deep eutectic solvent: Mix choline chloride, glycerol, lactic acid, and water in a molar ratio of 1:2:2:2. Place the mixture in a water bath shaker at 55°C and 1000 rpm for 0.5 h to form a clear, transparent liquid. If no precipitation occurs after standing overnight, the deep eutectic solvent is successfully prepared and sealed for later use.

[0049] (4) Ultrasonic-assisted deep eutectic solvent extraction: A deep eutectic solvent was added to the Huangshan pine needle powder at a mass ratio of 1:10. During the treatment, the powder of the Chinese medicine composition was ensured to be completely immersed in the deep eutectic solvent. Ultrasonic treatment was performed at a power of 300 W, the temperature was set to 35°C, and the time was 20 min. The mixture was centrifuged at 10,000 rpm for 15 min. The supernatant was collected and concentrated by rotary evaporation to obtain the Huangshan pine needle extract.

[0050] (5) Membrane filtration: The Huangshan pine needle extract prepared in (4) was filtered through a 5 kD ultrafiltration membrane to retain proteins and polysaccharides to obtain a polyphenol filtrate.

[0051] (6) Separation and purification: The polyphenol filtrate prepared in (5) was added to a non-polar or weakly polar macroporous resin (the macroporous resin was ADS-17), and dynamic elution was performed using a 35% ethanol solution as the elution solvent at an elution rate of 2 BV / h. The eluted solution was concentrated and vacuum freeze-dried at -45°C for 8 h to obtain Huangshan pine needle polyphenols.

[0052] 1. Solid-state fermentation of Huangshan pine needles and determination of the diameter of the fungal growth

[0053] The growth diameter of each fungus during the solid fermentation of Huangshan pine needles was measured at 3, 5, 7, 10, and 14 days. Unfermented Huangshan pine needles were used for comparison with Example 1, which was inoculated with an equal volume of sterile distilled water.

[0054] Table 1 shows the culture conditions of different medicinal (edible) fungi

[0055]

[0056] Table 2 shows the growth changes of various fungi after solid-state fermentation of Huangshan pine needles with Coriolus versicolor and Ganoderma lucidum. In the early growth phase (days 0-10), the mycelia of Examples 1 and 2 grew rapidly. As fermentation progressed, the mycelia expanded in a divergent pattern, gradually increasing in number and forming mycelial bundles. By day 10, the surface of the Huangshan pine needles was completely covered with hyphae, showing a pure white color. The diameters of the Coriolus versicolor and Ganoderma lucidum mycelia reached 30±0.0 and 29±1.1 cm, respectively, demonstrating that both Coriolus versicolor and Ganoderma lucidum can fully utilize Huangshan pine needles for solid-state fermentation.

[0057] Table 2 shows the changes in the diameter of the fungus growth of Huangshan pine needles fermented by microorganisms (cm)

[0058]

[0059] 2. Determination of enzyme activity before and after solid-state fermentation of Huangshan pine needles

[0060] 2.5 g of solid-state fermentation samples of Pinus tsutchuenensis needles from 0, 3, 5, 7, 10, and 14 days of age were added to 50 mL of 0.1 mol / L pH 6.0 citric acid buffer and stirred thoroughly for 30 minutes. The mixture was centrifuged at 4°C and 10,000 rpm for 20 minutes, and the supernatant was collected as the crude enzyme solution. Amylase activity was determined using the DNS reagent method, where a unit of amylase activity is defined as the amount of enzyme required to hydrolyze 1 µg of glucose per minute at 37°C. Protease activity was determined using the Folin reagent method, where a unit of protease activity is defined as the amount of enzyme required to hydrolyze 1 µg of tyrosine per minute at 40°C. Cellulase activity was determined using the CMC saccharification capacity method, where a unit of cellulase activity is defined as the amount of enzyme required to hydrolyze 1 µg of glucose per minute at 37°C.

[0061] Versicolor and Ganoderma lucidum were used to solid-state ferment the pine needles of Huangshan Mountain. The amylase activity, protease activity and cellulase activity in the pine needles of Huangshan Mountain were compared before and after fermentation. Figure 1 The amylase activity in Example 1 was relatively low, only 3.00 units / g. After fermentation with Yunzhi and Ganoderma lucidum, the cellulase activity of Example 1 and Example 2 was significantly increased ( P<0.05), were 338.40±8.25, 56.41±9.91 unit / g respectively. Compared with Comparative Example 1, the protease activity and cellulase activity in Huangshan pine needles after fermentation in Example 1 and Example 2 were significantly improved ( P <0.05), wherein the protease activity and cellulase activity in the fermented Huangshan pine needles of Example 1 were higher than those of Example 2. The results showed that the two medicinal (edible) fungi, Versicolor and Ganoderma lucidum, could produce highly active amylase, protease, and cellulase during the fermentation of Huangshan pine needles, which was beneficial for increasing the content of bioactive substances in the fermented Huangshan pine needles and improving the functional activity of the Huangshan pine needles.

[0062] 3. Determination of Polyphenol Content in Solid-State Fermentation of Pinus truncatula

[0063] Using the Folin-Ciocalteu method, 0.000, 0.025, 0.050, 0.100, 0.150, and 0.200 mL of tannic acid (1 mg / mL) were prepared and the solution was diluted to 1 mL to prepare the test solution. 0.05 mL of each test solution was mixed with 0.05 mL of Folin-Ciocalteu reagent (0.25 mol / L) for 3 minutes. After that, 1 mL of 0.7 mol / L sodium carbonate solution was added. The solution was allowed to stand at 30°C for 1 hour for color development. Accurately aspirated 0.2 mL of the solution was then loaded onto a microplate reader and the absorbance was measured at 750 nm. The Folin-Ciocalteu reagent was replaced with deionized water as a blank control. A standard curve was constructed between mass concentration and absorbance. The polyphenol content of the microbial fermentation and non-fermentation groups of Huangshan pine needles was determined using the same method. The results are expressed as milligrams of equivalent tannic acid per gram of extract (mg TA / g).

[0064] Figure 2 The results show that the polyphenol content of Huangshan pine needles in Example 1 and Example 2 increased significantly with the extension of fermentation time during the fermentation period of 0 to 10 days. On the 10th day of fermentation, the polyphenol content of Huangshan pine needles in Example 1 reached the highest, which was 146.32±2.53 mg / g. When fermented with the microorganism Coriolus versicolor, the polyphenol content of Huangshan pine needles in Example 1 increased by 1.89 times compared with that in Comparative Example 1. The results confirmed that the microbial solid-state fermentation method of the present invention can significantly promote the release of Huangshan pine needle polyphenols, which may be related to the fact that the two medicinal (edible) fungi, Coriolus versicolor and Ganoderma lucidum, can produce highly active amylase, protease and cellulase during the fermentation of Huangshan pine needles.

[0065] 4. Antibacterial effect of Huangshan pine needle polyphenols.

[0066] 4.1 Activation of bacterial strains and preparation of bacterial suspension: Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, and Pseudomonas aeruginosa were inoculated onto the slant of the test tube culture medium for activation and cultured in a 37°C constant temperature incubator for 24 hours. After the colonies grew, the activated bacterial strains were picked out on the sterile operating table and inoculated into sterile saline. The concentration of each bacterial suspension was prepared to be approximately 10 6 ~10 7 CFU / mL.

[0067] 4.2 Determination of antibacterial activity by zone of inhibition assay: Pour the molten and sterilized culture medium into a sterile culture dish in a sterile operating chamber. After cooling and solidification, add 100 μL of the bacterial suspension and spread evenly with a spreading rod. Use a pipette to add 10 μL of sample to the center of a sterile 6 mm diameter filter paper. Use sterile distilled water as a blank control and propylparaben (PP) as a positive control. Incubate the plate in an inverted manner at 37°C for 24 h. Measure the diameter of the zone of inhibition. Repeat three times and calculate the average value.

[0068] 4.3 Table 3 shows the antibacterial effect results of the Huangshan pine needle 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 which 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 propyl paraben. The research results revealed for the first time that Huangshan pine needle polyphenols have a significant inhibitory effect on the four tested Gram-positive and Gram-negative bacteria, indicating that the present invention enhances the antibacterial efficacy of Huangshan pine needle polyphenols through the solid fermentation method of Yunzhi and Ganoderma lucidum, laying a scientific basis for the development of functional cosmetics of Huangshan pine needle polyphenols.

[0069] Table 3 shows the diameters of the inhibition zones of Example 1, Example 2 and Comparative Example 1 against four kinds of bacteria

[0070]

[0071] Example 3: An acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols, the acne-removing and anti-inflammatory essence comprising the following components by weight percentage: 1% Huangshan pine needle polyphenols, 0.1% Chimonanthus willowii leaf extract, 0.1% Astragalus membranaceus root extract, 0.1% Saposhnikovia root extract, 0.1% Calendula officinalis flower extract, 0.05% Albizia julibrissin flower extract, 0.05% Gastrodia elata root extract, 0.05% Phellodendron amurense bark extract, 0.1% Salix alba bark extract, 0.05% Scutellaria baicalensis root extract, 0.1% Houttuynia cordata extract, 0.1% sodium hyaluronate, 0.15% allantoin, 0.2% acrylates / C10-30 alkyl acrylate crosspolymer, 7% butylene glycol, 0.15% arginine, 0.2% gum arabic salicylic acid, 0.007% quaternary ammonium salt-73, 0.1% PEG-40 hydrogenated castor oil, 6% preservative, and the remainder being deionized water, distilled water, or purified water. Preservatives include 0.4% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, and 4.5% butylene glycol.

[0072] The preparation process of the acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols comprises the following steps:

[0073] (1) Sodium hyaluronate, allantoin, and acrylic acid (esters) / C10-30 alkyl acrylate cross-polymer were added to an emulsifier in the proportions of the components in the acne-removing and anti-inflammatory essence. Water was added and the mixture was heated and stirred at 80°C to dissolve the mixture and form an aqueous phase. After cooling to 45°C, arginine was slowly added to the aqueous phase. The mixture was sheared at high speed using a 10,000 rpm high-speed shear homogenizer and treated with 300 W ultrasonic waves at 35°C for 10 minutes. The mixture was cooled in an ice water bath to obtain Liquid A.

[0074] (2) Take an appropriate amount of butylene glycol according to the proportion of each component in the acne-removing and anti-inflammatory essence, add quaternary ammonium salt-73, and stir with a blender until it is completely dissolved at a speed of 3000 rpm for 10 minutes to obtain liquid B.

[0075] (3) Take hydroxyacetophenone, 1,2-hexanediol and butanediol according to the proportion of each component in the acne-removing and anti-inflammatory essence, heat and stir at 45°C to dissolve, and obtain liquid C.

[0076] (4) According to the proportion of each component in the acne-removing and anti-inflammatory essence, slowly add liquid B cooled to room temperature to the liquid A in the emulsifier, and stir with a blender until completely mixed; then add Huangshan pine needle polyphenols, willow-leaf osmanthus leaf extract, membranaceus root extract, windproof root extract, calendula extract, albizzia julibrissin flower extract, Gastrodia elata root extract, Phellodendron amurense bark extract, white willow 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℃ and sterilize for 25 min; finally add liquid C and stir evenly.

[0077] The preparation methods of the Chimonanthus willowii leaf extract, Astragalus membranaceus root extract, Saposhnikovia root extract, Calendula officinalis flower extract, Albizia julibrissin flower extract, Gastrodia elata root extract, Phellodendron amurense bark extract, Salix alba bark extract, Scutellaria baicalensis root extract, and Houttuynia cordata extract are as follows: each raw material is crushed into powder, extracted with 70% wt% ethanol, centrifuged and filtered after extraction, the filtrate is concentrated to remove ethanol, and vacuum dried to a powdery state to obtain each extract.

[0078] (5) Adjust the pH of the material to within 6.0, adjust the viscosity to 2000 mPa·s, and perform vacuum degassing at 30°C to form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne essence.

[0079] Comparative Example 2: An acne-removing and anti-inflammatory essence, the acne-removing and anti-inflammatory essence comprising the following components by weight percentage: 0.1% calycanthus chinensis leaf extract, 0.1% astragalus membranaceus root extract, 0.1% siler root extract, 0.1% calendula flower extract, 0.05% julibrissin flower extract, 0.05% gastrodia elata root extract, 0.05% phellodendron amurense bark extract, 0.1% willow bark extract, 0.05% scutellaria baicalensis root extract, 0.1% houttuynia cordata extract, 0.1% sodium hyaluronate, 0.15% allantoin, 0.2% acrylates / C10-30 alkyl acrylate crosspolymer, 7% butylene glycol, 0.15% arginine, 0.2% gum arabic salicylic acid, 0.007% quaternary ammonium salt-73, 0.1% PEG-40 hydrogenated castor oil, 6% preservative, and the remainder being deionized water, distilled water, or purified water. Preservatives include 0.4% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, and 4.5% butylene glycol.

[0080] The preparation process of the acne-removing and anti-inflammatory essence comprises the following steps:

[0081] (1) Sodium hyaluronate, allantoin, and acrylic acid (esters) / C10-30 alkyl acrylate cross-polymer were added to an emulsifier in the proportions of the components in the acne-removing and anti-inflammatory essence. Water was added and the mixture was heated and stirred at 80°C to dissolve the mixture and form an aqueous phase. After cooling to 45°C, arginine was slowly added to the aqueous phase. The mixture was sheared at high speed using a 10,000 rpm high-speed shear homogenizer and treated with 300 W ultrasonic waves at 35°C for 10 minutes. The mixture was cooled in an ice water bath to obtain Liquid A.

[0082] (2) Take an appropriate amount of butylene glycol according to the proportion of each component in the acne-removing and anti-inflammatory essence, add quaternary ammonium salt-73, and stir with a blender until it is completely dissolved at a speed of 3000 rpm for 10 minutes to obtain liquid B.

[0083] (3) Take hydroxyacetophenone, 1,2-hexanediol and butanediol according to the proportion of each component in the acne-removing and anti-inflammatory essence, heat and stir at 45°C to dissolve, and obtain liquid C.

[0084] (4) According to the proportion of each component in the acne-removing and anti-inflammatory essence, slowly add liquid B cooled to room temperature to the liquid A in the emulsifier, and stir with a blender until completely mixed; then add willow-leaf osmanthus leaf extract, astragalus root extract, siler root extract, calendula extract, albizzia julibrissin flower extract, gastrodia root extract, phellodendron bark extract, willow bark extract, scutellaria 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 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.

[0085] (5) Adjust the pH of the material to within 6.0, adjust the viscosity to 2000 mPa·s, and perform vacuum degassing at 30°C to form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne essence.

[0086] Comparative Example 3: A commercially available general acne-removing essence product, the ingredients of which include water, Pichia pastoris fermentation lysate filtrate, butylene glycol, dipropylene glycol, 1,3-propylene glycol, betaine, PEG / PPG-14 / 7 dimethyl ether, glycerin, 1,2-hexanediol, Pueraria lobata root extract, ammonium acryloyldimethyltaurate / VP copolymer, etc.

[0087] 5. Efficacy evaluation

[0088] Ninety volunteers (aged 18-40, with mild to moderate acne, according to the modified international classification system, with a certain number of comedones and papules, but not nodules or cysts that are difficult to improve with clinical preparations) were randomly divided into three groups. Group 1 used the product of Example 3, Group 2 used the product of Comparative Example 2, and Group 3 used the product of Comparative Example 3. The testers used the product daily, morning and evening: first wet the face with clean water, then apply an appropriate amount of the product to the face and gently massage until absorbed. The product was used for 28 days.

[0089] Scoring criteria: 1. "Agree" = 5 points; "Somewhat agree" = 4 points; "Fair" = 3 points; "Somewhat disagree" = 2 points; "Disagree" = 1 point. 2. The percentage of subjects who scored 4 points or higher is the percentage of subject satisfaction.

[0090] Table 4 is the statistical table of the self-evaluation results of the first group of subjects (28 days after using the product) (n=30)

[0091]

[0092] Table 5 is the statistical table of the self-evaluation results of the second group of subjects (28 days after using the product) (n=30)

[0093]

[0094] Table 6 is the statistical table of the self-evaluation results of the third group of subjects (28 days after using the product) (n=30)

[0095]

[0096] 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 polyphenols has more obvious moisturizing and acne-removing effects, can effectively inhibit skin inflammation, promote the repair of acne skin, and maintain the health of the skin. Figure 3 , where the number of acne / pimples in patients decreased significantly by 46%. 28 days after using the product in Example 3, 100% of the subjects felt that the pain / itching associated with acne was improved, the number of acne / pimples on the skin was reduced, and the overall redness and number of acne spots on the skin were effectively improved, with a subject satisfaction rate of 100%. As shown in Table 6, although Comparative Example 3 has a certain acne-removing effect, compared with the Huangshan pine needle polyphenol anti-acne and anti-inflammatory essence developed by the present invention, the proportion of subjects who felt that the overall acne condition of the skin improved was only 33.33%, and the proportion of skin adverse reactions was as high as 10%.

[0097] The present invention has been described above with reference to the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described method. Any non-substantial improvements made using the method concepts and technical solutions of the present invention, or any direct application of the above-described concepts and technical solutions to other situations without modification, fall within the scope of protection of the present invention.

Claims

1. A method for preparing Huangshan pine needle polyphenols, comprising the following steps: (1) Raw material pretreatment: Pick fresh Huangshan pine needles, wash and dry them, and then sterilize them; (2) Microbial solid fermentation: 0.1% to 10% of a microbial suspension is inoculated into sterilized Huangshan pine needles for 3 to 14 days. The strain of the microbial suspension is Coriolus versicolor or Ganoderma lucidum. (3) Drying and pulverizing: freeze-drying the solid fermented Huangshan pine needles and then pulverizing them to obtain Huangshan pine needle fermentation product powder; (4) Preparation of deep eutectic solvent: choline chloride, glycerol, lactic acid and water were mixed in a molar ratio of 1:1~5:1~5:1~6; after mixing, the mixture was placed in a water bath shaker at 45℃~75℃ and 300~1200 r / min to react for 0.5~2 h, and then allowed to stand to obtain a deep eutectic solvent, which was then sealed for later use; (5) Ultrasonic-assisted deep eutectic solvent extraction: A deep eutectic solvent was added to the powder of the fermentation product of Huangshan pine needles. The mass ratio of the powder of the fermentation product of Huangshan pine needles to the deep eutectic solvent was 1:2 to 10, so that the powder of the fermentation product of Huangshan pine needles was completely immersed in the deep eutectic solvent. The powder was treated with an ultrasonic wave with a power of 100 to 400 W. The treatment temperature was set to 35°C to 65°C and the treatment time was set to 20 to 120 min. After centrifugation at a speed of 2500 to 10000 rpm for 10 to 30 min, the supernatant was collected and concentrated by rotary evaporation to obtain the Huangshan pine needle fermentation extract. (6) Membrane filtration: The fermented extract of pine needles prepared in (5) was filtered through a 3 kD to 10 kD ultrafiltration membrane to retain proteins and polysaccharides, thereby obtaining the pine needle polyphenol filtrate; (7) Separation and purification: The polyphenol filtrate prepared in (6) is added to a non-polar or weakly polar macroporous resin and dynamically eluted using a 5% to 85% ethanol solution as the elution solvent and an elution rate of 1.2 to 3.5 BV / h. The eluted solution is concentrated and then freeze-dried in a vacuum at -45°C to -55°C for 4 to 9 h to obtain Huangshan pine needle polyphenols.

2. The method for preparing Huangshan pine needle polyphenols according to claim 1, wherein: The concentration of the microbial suspension is 1×10 6 ~1×10 8 CFU / mL, and the fermentation culture temperature was 28℃~45℃.

3. The method for preparing Huangshan pine needle polyphenols according to claim 1, wherein: The pulverization in step (3) is as follows: freeze-drying the solid fermented Huangshan pine needles and transferring them to a wall-breaking machine for air flow pulverization, adjusting the temperature inside the machine to -10°C~20°C, introducing a high-speed air flow, and the air flow pulverization pressure is 0.5~2.5 MPa, and the pulverization time is 2~5 min.

4. The method for preparing Huangshan pine needle polyphenols according to claim 1, wherein: The macroporous resin in step (7) is a mixture of one or more of NKA-II, XDA-7, D101, and ADS-17.

5. The method for preparing Huangshan pine needle polyphenols according to claim 1, wherein: In the step (1), the Huangshan pine needles are washed, dried, cut into small segments of 1 to 2 cm, and then sterilized with high pressure wet heat.

6. An acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols, characterized in that: The anti-acne and anti-inflammatory essence comprises the following components by weight percentage: 0.05% to 1.5% of Huangshan pine needle polyphenols, 0.02% to 0.2% of Chimonanthus willowii leaf extract, 0.02% to 0.2% of Astragalus membranaceus root extract, 0.02% to 0.2% of Saposhnikovia root extract, 0.02% to 0.2% of Calendula officinalis flower extract, 0.02% to 0.2% of Albizia julibrissin flower extract, 0.02% to 0.2% of Gastrodia elata root extract, 0.02% to 0.2% of Phellodendron amurense bark extract, 0.02% to 0.2% of Salix alba bark extract, 0.02% to 0.2% of Scutellaria baicalensis root extract, 0.03% to 0.3% of Houttuynia cordata extract, 0.05% to 0.2% of sodium hyaluronate, 0.05% to 0.2% of allantoin, 0.1% to 0.3% of acrylates / C10-30 alkyl acrylate crosspolymer, 2% to 1% of butylene glycol. 7%, arginine 0.1%~0.3%, gum arabic salicylic acid 0.1%~0.3%, quaternium-73 0.001%~0.01%, PEG-40 hydrogenated castor oil 0.02%~0.1%, preservatives 3.0%~6.5% and the remaining amount of water.

7. The acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols according to claim 6, characterized in that: The preservatives include 0.1% to 0.6% p-hydroxyacetophenone, 0.1% to 0.5% 1,2-hexanediol, and 1.5% to 6% butanediol; and the water is deionized water, distilled water, or purified water.

8. The process for preparing the acne-removing and anti-inflammatory essence containing Huangshan pine needle polyphenols according to claim 6 or 7, comprising the following steps: (1) Sodium hyaluronate, allantoin, and acrylic acid (esters) / C10-30 alkyl acrylate cross-linked polymer are sequentially added to an emulsifier, and water is added. The mixture is heated and stirred at 80°C to 95°C to dissolve the mixture and form an aqueous phase. After cooling to 45°C to 65°C, arginine is slowly added to the aqueous phase, and the mixture is sheared at high speed using a 5000-12000 rpm high-speed shear homogenizer and treated with 100-400W ultrasonic waves. The treatment temperature is 35°C to 55°C, and the treatment time is 5-10 min. The mixture is then cooled in an ice water bath to obtain liquid A. (2) Take an appropriate amount of butanediol, add quaternary ammonium salt-73, and stir with a stirrer until completely dissolved at a stirring speed of 2000-5000 rpm for 5-10 minutes to obtain solution B; (3) Heat hydroxyacetophenone, 1,2-hexanediol, and butanediol at 45°C to 50°C and stir to dissolve to obtain Solution C; (4) Slowly add liquid B cooled to room temperature to liquid A in the emulsifier, and stir with a blender until completely mixed; then add Huangshan pine needle polyphenols, willow leaf osmanthus leaf extract, membranaceus root extract, windproof root extract, calendula extract, Albizia julibrissin flower extract, Gastrodia elata root extract, Phellodendron chinense bark extract, white willow 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 5000-12000 rpm, intermittently three times, each time for 10-30 seconds; then heat the external circulating water temperature of the emulsifier to 55℃-75℃ and sterilize for 15-35 minutes; finally, add liquid C and stir evenly; (5) Adjust the pH of the material within the range of 5.5 to 6.5, adjust the viscosity within the range of 500 to 2500 mPa·s, and vacuum degas at 30°C to 37°C to form a light yellow, transparent, uniform liquid with a characteristic odor, which is the anti-acne and anti-inflammatory essence.

Images