Platycladi Cacumen Extract, Preparation Method Thereof and Application

Abstract

The application discloses a cypress leaf extract, a preparation method and application thereof, and belongs to the field of cosmetics. The cypress leaf is crushed, and then soaked in deionized water at low temperature. Then, flocculation is performed by using 100 kd chitosan, molecular interception is performed, macroporous resin adsorption is performed, and elution is performed by using ethanol with a certain concentration. The obtained cypress leaf extract can improve the activity of human keratinocytes, inhibit the enzyme activity of human keratinocytes KLK5, and improve the ATP content in hair papilla cells and / or human keratinocytes. The cypress leaf extract can be used for preparing cosmetics, skin care products or external preparations with the effects of improving the activity of human keratinocytes and / or inhibiting the enzyme activity of human keratinocytes KLK5 and / or promoting the ATP synthesis in hair papilla cells and / or human keratinocytes.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to an extract of Platycladus orientalis leaves, its preparation method, and its application. Background Technology

[0002] Kallikrein-5 (KLK5) is one of the major serine proteases in the epidermis, playing a crucial role in the dynamic balance of stratum corneum shedding and skin barrier function. Under normal physiological conditions, KLK5 regulates the normal shedding of keratinocytes (the shedding process) by precisely cleaving junction proteins between keratinocytes (such as desmosome core protein and keratinocyte granules), thereby maintaining the thickness of the stratum corneum and the integrity of the skin barrier.

[0003] However, abnormally upregulated KLK5 activity can lead to a series of skin dysfunctions. Overactivated KLK5 degrades intercellular junction proteins uncontrollably, causing excessive exfoliation of the stratum corneum and directly disrupting the physical integrity of the epidermal barrier. Simultaneously, KLK5 can hydrolyze and activate the antimicrobial peptide LL-37, thereby activating the protease-activated receptor-2 (PAR2) and driving the release of various pro-inflammatory cytokines, inducing or exacerbating skin inflammation. This plays a significant role in the development of skin diseases such as rosacea, atopic dermatitis, and Netherton's syndrome. More importantly, studies have revealed a close correlation between abnormal KLK family activity and hair abnormalities such as hair shaft defects and follicular atrophy. Therefore, effectively inhibiting excessive KLK5 activity in keratinocytes not only helps rebuild the skin barrier and reduce secondary inflammation caused by barrier damage, but may also indirectly provide good growth support for dermal papilla cells by maintaining the stability of the microenvironment around the hair follicle, thus improving skin aging and preventing hair loss.

[0004] Besides KLK5-mediated barrier damage and inflammatory responses, cellular energy metabolism imbalance is also a significant factor leading to skin aging and hair follicle atrophy. Adenosine triphosphate (ATP) is the most important energy currency within cells. When ATP synthesis decreases due to oxidative stress, ultraviolet radiation, or metabolic abnormalities, it triggers a series of functional disorders. In fibroblasts, decreased ATP levels can lead to reduced extracellular matrix synthesis, accelerating wrinkle formation; in keratinocytes, ATP deficiency weakens the epidermal barrier repair capacity; and in dermal papilla cells, reduced ATP levels inhibit the secretion of hair follicle growth-related factors, causing hair follicles to prematurely enter the regression phase, resulting in follicle atrophy, thinning hair, and even hair loss. Therefore, maintaining or increasing ATP levels in these cells is also a crucial strategy for protecting skin and hair follicle health.

[0005] The leaves of Platycladus orientalis (L.) Franco, a plant in the Cupressaceae family, are the dried branches and leaves. They are a traditional Chinese medicinal material and are included in all editions of the Chinese Pharmacopoeia. Platycladus orientalis leaves are cold in nature and bitter in taste, entering the lung, liver, and spleen meridians. They have the effects of cooling the blood and stopping bleeding, resolving phlegm and relieving cough, and promoting hair growth and darkening hair. Modern pharmacological studies have shown that Platycladus orientalis leaves contain various active ingredients such as flavonoids, terpenes, volatile oils, and tannins, possessing various biological activities including anti-inflammatory, antioxidant, antibacterial, procoagulant, and neuroprotective effects. In the skin and hair care field, Platycladus orientalis leaf extract has been widely used in hair loss prevention and hair growth products. Studies have reported that it can promote hair growth through mechanisms such as activating the Wnt / β-catenin signaling pathway, promoting dermal papilla cell proliferation, prolonging the hair follicle growth phase, and inhibiting 5α-reductase activity.

[0006] Chinese Patent Publication No. CN120983309A discloses a composition for promoting dermal papilla cell metabolism, its preparation method, and its application. The composition comprises the following components: Platycladus orientalis leaf extract, trehalose, betaine, acetyl tetrapeptide-3, and myristoyl pentapeptide-4, which significantly enhances the mitochondrial membrane potential of dermal papilla cells. However, enhancement of mitochondrial membrane potential does not necessarily lead to an increase in intracellular ATP levels, and excessively high membrane potential may induce oxidative stress damage.

[0007] However, existing research mainly focuses on the regulatory effects of Platycladus orientalis leaves on cell proliferation or signaling pathways, and there are no systematic reports on the role of Platycladus orientalis leaf extract in protecting the skin barrier, reducing inflammation, and preventing hair loss by inhibiting KLK5 enzyme activity. Therefore, discovering natural extracts that can inhibit KLK5 enzyme activity while also promoting skin cell energy metabolism is of great significance for developing novel cosmetics or drugs for dandruff control, anti-skin aging, skin barrier repair, hair loss prevention, and anti-inflammation. Summary of the Invention

[0008] The purpose of this invention is to provide an extract of Platycladus orientalis leaves, its preparation method and application. The obtained Platycladus orientalis leaf extract can improve the vitality of human keratinocytes, inhibit the activity of KLK5 enzyme in human keratinocytes, and increase the ATP content in dermal papilla cells and / or human keratinocytes.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: A method for preparing an extract of Platycladus orientalis leaves includes the following steps: pulverizing Platycladus orientalis leaves to 80-120 mesh, adding 14-16 times the weight of the Platycladus orientalis leaves in deionized water, soaking at room temperature for more than 2 hours, heating to 40-60℃, ultrasonically extracting for 45-75 minutes, performing solid-liquid separation, and obtaining filtrate 1. The obtained filtrate 1 is concentrated and adjusted to 5-6 times the weight of the arborvitae leaves using a reverse osmosis membrane to obtain solution 1. Chitosan solution of 12-14% of the liquid volume of solution 1 is added, and the mixture is stirred and flocculated for 25-30 minutes. The mixture is then placed in an environment below 4°C and allowed to settle for more than 12 hours. After filtration until clear, filtrate 2 is obtained. The molecular weight of the chitosan is above 100 kDa. The obtained filtrate 2 was filtered through a 3-8 kd ultrafiltration membrane. The filtrate 2 was concentrated under reduced pressure at a temperature below 60°C and the volume was adjusted to 1.8-2.2 times the weight of the arborvitae leaves. Then it was placed in an environment below 4°C for sedimentation for more than 12 hours and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at a temperature below 60°C for the second time and the volume was adjusted to 0.4 to 0.6 times the weight of the arborvitae leaves to obtain solution 2. Ethanol with a concentration of 90° or higher was added to solution 2 to adjust the alcohol content of solution 2 to 60 to 70°. Then, it was placed in an environment below 4°C for sedimentation for more than 12 hours and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure at a temperature below 60°C for the third time to a volume of 1.8 to 2.2 times the weight of the arborvitae leaves. It was then adsorbed by macroporous resin and washed with water to remove impurities. Subsequently, elution was performed using 3 BV 10~20% ethanol solution at a flow rate controlled at 1 BV / h, with the temperature ≤50℃ throughout the process. The target eluent was collected, filtered until clear, and then concentrated under reduced pressure for the fourth time at below 60℃ and dried to obtain the Platycladus orientalis leaf extract.

[0010] Preferably, the concentration of the chitosan solution is 1%.

[0011] Preferably, the vacuum degree of the reduced pressure concentration is -0.08 to -0.09 MPa.

[0012] Preferably, the drying process is freeze drying or vacuum drying, and the temperature throughout the process does not exceed 60°C.

[0013] Preferably, the macroporous resin is one of the following: AB~8, HPD100, or HP20 type macroporous resin.

[0014] Preferably, during the adsorption of the macroporous resin, the flow rate is controlled at 1 BV / h, and the weight ratio of the macroporous resin to the arborvitae leaf is 1:0.5.

[0015] Preferably, the water washing and impurity removal process is as follows: the resin column is rinsed with 2~3 BV of pure water at a flow rate of 2 BV / h.

[0016] The Platycladus orientalis leaf extract obtained by the preparation method of the present invention can improve the vitality of human keratinocytes, inhibit the activity of KLK5 enzyme in human keratinocytes, and increase the ATP content in dermal papilla cells and / or human keratinocytes.

[0017] The application of the Platycladus orientalis leaf extract described in this invention in the preparation of cosmetics, skin care products or topical formulations that have the effects of viability of human keratinocytes and / or inhibition of KLK5 enzyme activity in human keratinocytes and / or promotion of ATP synthesis in dermal papilla cells and / or human keratinocytes.

[0018] The present invention relates to the use of the Platycladus orientalis leaf extract in the preparation of medicaments or cosmetics for the treatment or prevention of skin diseases associated with abnormal upregulation of KLK5 activity.

[0019] The present invention relates to the use of the Platycladus orientalis leaf extract in the preparation of medicaments or cosmetics for the treatment or prevention of skin diseases related to ATP content in dermal papilla cells and / or human keratinocytes.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: In the extraction process of Platycladus orientalis leaf extract of this invention, a crude extract of Platycladus orientalis leaf is prepared by low-temperature deionized water soaking, ultrasonic extraction, chitosan flocculation and low-temperature precipitation, molecular retention and alcohol precipitation concentration, etc., and then by a process route of adsorption and elution separation of specific polar extracts through macroporous resin, thus enriching Platycladus orientalis leaf extract with specific active fractions. Compared with Platycladus orientalis leaf extract and crude extract of common processes and other eluted extracts, this fraction of extract can significantly improve the viability of human keratinocytes, inhibit the enzyme activity of kallikrein 5 (KLK5) in keratinocytes, and increase the ATP content in human keratinocytes and hair papilla cells with abnormal energy metabolism caused by ultraviolet radiation.

[0021] The Platycladus orientalis leaf extract described in this invention can be used to prepare drugs, cosmetics or health products that inhibit KLK5 activity, and then used to prevent or treat skin diseases, skin damage, skin aging, hair follicle atrophy and hair loss problems related to abnormal upregulation of KLK5 activity. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments.

[0023] The weight of the arborvitae leaves used in the embodiments and comparative examples of this invention is 100g.

[0024] Example 1 A method for preparing a cypress leaf extract, comprising the following steps: 100g of cypress leaves are pulverized to 80 mesh, 1400g of deionized water is added, and the mixture is soaked at room temperature for 2 hours. Then, the mixture is heated to 40°C, ultrasonically extracted for 45 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated to a volume of 500g using a reverse osmosis membrane, and 65g of a 1% chitosan solution was added. The mixture was stirred and flocculated for 30 minutes, then allowed to settle at 4°C for 12 hours. The solution was then filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 3kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 60°C and the volume was adjusted to 200g. It was then placed in an environment below 4°C for 12h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at a temperature below 60°C for the second time and the volume was adjusted to 50g. Ethanol of 90° or higher was added to the liquid to adjust the alcohol content of the clear liquid to 60°. Then, it was placed in an environment below 4°C for 12 hours to settle. After filtration until clear, filtrate 4 was obtained. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 60℃ to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was washed with 2BV of pure water at a flow rate of 2BV / h to remove impurities. Elution was carried out with 3BV of 12% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50℃ throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 60℃ and dried to obtain 18.2g of Platycladus orientalis leaf extract.

[0025] Example 2 A method for preparing a cypress leaf extract, comprising the following steps: 100g of cypress leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g by reverse osmosis membrane, and 60g of 1% chitosan solution was added. The mixture was stirred and flocculated for 25min, and then allowed to settle at 2℃ for 18h. After that, it was filtered until clear to obtain filtrate 2. The molecular weight of the chitosan was above 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 180g. It was then placed in an environment below 2°C for 18h to settle. After filtration until clear, filtrate 3 was obtained. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C to settle for 18h and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 18.7g of Platycladus orientalis leaf extract.

[0026] Example 3 A method for preparing a Platycladus orientalis leaf extract includes the following steps: 100g of Platycladus orientalis leaves are pulverized to 120 mesh, 1600g of deionized water is added, and the mixture is soaked at room temperature for 4 hours. Then, the mixture is heated to 60°C, ultrasonically extracted for 75 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 600g by reverse osmosis membrane, and 65g of 1% chitosan solution was added. The mixture was stirred and flocculated for 35min, and then allowed to settle at 3℃ for 24h. After that, it was filtered until clear to obtain filtrate 2. The molecular weight of the chitosan was above 100kd. The obtained filtrate 2 was filtered through an 8kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at below 50°C and the volume was adjusted to 220g. It was then placed in an environment at below 3°C for 24h to settle. After filtration until clear, filtrate 3 was obtained. The obtained filtrate 3 was concentrated under reduced pressure at a temperature below 50°C to a final volume of 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. The liquid was then allowed to settle at a temperature below 2°C for 18 hours and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 18.7g of Platycladus orientalis leaf extract.

[0027] Example 4 A method for preparing a cypress leaf extract, comprising the following steps: 100g of cypress leaves are pulverized to 100 mesh, 1480g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 68g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 32min. It was then allowed to settle at 2℃ for 18h and subsequently filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 210g. It was then placed in an environment below 4°C for 12h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 4°C for 12h to settle. After filtration until clear, filtrate 4 was obtained. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was washed with 2BV of pure water at a flow rate of 2BV / h to remove impurities. Elution was carried out with 3BV of 12% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 60°C and dried to obtain 18.4g of Platycladus orientalis leaf extract.

[0028] Example 5 A method for preparing a Platycladus orientalis leaf extract, comprising the following steps: 100g of Platycladus orientalis leaves are pulverized to 80 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 60°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g by reverse osmosis membrane, and 70g of 1% chitosan solution was added. The mixture was stirred and flocculated for 30min, and then allowed to settle at 2℃ for 18h. After that, it was filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of more than 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C to settle for 18h and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 18.6g of Platycladus orientalis leaf extract.

[0029] Example 6 A method for preparing a Platycladus orientalis leaf extract includes the following steps: 100g of Platycladus orientalis leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 65g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h and subsequently filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C to settle for 18h and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of HPD100 macroporous resin at a flow rate of 1BV / h. The resin column was washed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 18.8g of Platycladus orientalis leaf extract.

[0030] Comparative Example 1 A method for preparing a cypress leaf extract includes the following steps: 100g of cypress leaves are pulverized to 200 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 1 hour. Then, the mixture is heated to 70°C, ultrasonically extracted for 20 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 400g using a reverse osmosis membrane. 65g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h and subsequently filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 10kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at below 55℃ and the volume was adjusted to 200g. Then it was placed in an environment below 2℃ for 18h to settle. After filtration until clear, filtrate 3 was obtained. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 50°. Then it was placed in an environment below 2°C for 18h to settle. After filtration until clear, filtrate 4 was obtained. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 16.2g of Platycladus orientalis leaf extract.

[0031] The difference between Comparative Example 1 and the embodiment of the present invention is that the particle size of the pulverized arborvitae leaves is larger, the soaking time at room temperature is too short, the ultrasonic extraction temperature is higher, the ultrasonic extraction time is shorter, the volume after concentration by reverse osmosis membrane is smaller, the amount of ultrafiltration membrane used has a lower rejection ratio, the alcohol content of alcohol precipitation is lower, and the yield of the obtained arborvitae leaf extract is lower.

[0032] Comparative Example 2 A method for preparing a Chinese arborvitae leaf extract includes the following steps: 100g of Chinese arborvitae leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 55g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 60min. It was then allowed to settle at 8℃ for 6h, and finally filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 8°C for 6 hours to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at a temperature below 70°C to a final volume of 40g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. The liquid was then allowed to settle at a temperature below 8°C for 6 hours and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 70°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 19.2g of Platycladus orientalis leaf extract.

[0033] The difference between Comparative Example 2 and the embodiment of the present invention is that the amount of flocculant added is less, the flocculation stirring time is too long, the flocculation temperature is higher, the flocculation settling time is shorter, the settling temperature is higher, and the settling time is shorter.

[0034] Comparative Example 3 A method for preparing a Chinese arborvitae leaf extract includes the following steps: 100g of Chinese arborvitae leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g by reverse osmosis membrane, and 71.5g of 1% chitosan solution was added. The mixture was stirred and flocculated for 30min, and then allowed to settle at 2℃ for 18h. After that, it was filtered until clear to obtain filtrate 2. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 60g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C for 18h to settle. After filtration until clear, filtrate 4 was obtained. The resulting filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 18.7g of Platycladus orientalis leaf extract.

[0035] The difference between Comparative Example 3 and the embodiments of the present invention is that the flocculant does not retain molecules.

[0036] Comparative Example 4 A method for preparing a cypress leaf extract, comprising the following steps: 100g of cypress leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 71.5g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h, and finally filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 55g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C to settle for 18h and filtered until clear to obtain filtrate 4. The obtained filtrate 4 was concentrated under reduced pressure for the third time at below 55°C to a final volume of 200g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 3BV / h. The resin column was washed with 3BV of pure water at a flow rate of 5BV / h to remove impurities. Elution was carried out with 3BV of 30% ethanol solution at a flow rate of 5BV / h, with the temperature maintained at 65~75°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure for the fourth time at 55°C and dried to obtain 16.2g of Platycladus orientalis leaf extract.

[0037] The difference between Comparative Example 4 and the embodiments of the present invention is that the resin adsorption rate, water washing column volume, water washing rate, alcohol concentration during alcohol washing, alcohol washing rate, and elution temperature are all higher.

[0038] Comparative Example 5 A method for preparing a Chinese arborvitae leaf extract, comprising the following steps: 100g of Chinese arborvitae leaves are pulverized to 100 mesh, 1500g of 55% ethanol aqueous solution is added, the mixture is soaked at room temperature for 3 hours, then heated to 50℃, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 71.5g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h, and finally filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 52g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C for 18h to settle. After filtration until clear, filtrate 4 was obtained. Then it was concentrated under reduced pressure at 55°C for the third time and dried to obtain 21.3g of Platycladus orientalis leaf extract.

[0039] The difference between Comparative Example 5 and the embodiments of the present invention is that the room temperature soaking used is not deionized water, but a 55% volume fraction ethanol aqueous solution, and it does not undergo macroporous resin adsorption, water washing, or alcohol washing.

[0040] Comparative Example 6 A method for preparing a Chinese arborvitae leaf extract, comprising the following steps: 100g of Chinese arborvitae leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 71.5g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h, and finally filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The resulting filtrate 3 was concentrated under reduced pressure at below 55°C to a final volume of 50g, and then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. The resin column was rinsed with 2BV of pure water at a flow rate of 2.5BV / h to remove impurities. Elution was carried out with 3BV of 15% ethanol solution at a flow rate of 1BV / h, with the temperature ≤50°C throughout the elution process. The target eluent was collected. The eluent was filtered until clear, and then concentrated under reduced pressure at 55°C for the fourth time and dried to obtain 18.7g of Platycladus orientalis leaf extract.

[0041] The difference between Comparative Example 6 and the embodiments of the present invention is that alcohol precipitation was not performed.

[0042] Supplementary Comparison A method for preparing an extract of Platycladus orientalis leaves includes the following steps: 100g of Platycladus orientalis leaves are pulverized to 100 mesh, 1500g of deionized water is added, and the mixture is soaked at room temperature for 3 hours. Then, the mixture is heated to 50°C, ultrasonically extracted for 60 minutes, filtered, and filtrate 1 is obtained. The obtained filtrate 1 was concentrated and brought to a volume of 550g using a reverse osmosis membrane. 65g of a 1% chitosan solution was added, and the mixture was stirred and flocculated for 30min. It was then allowed to settle at 2℃ for 18h and subsequently filtered until clear to obtain filtrate 2. The chitosan had a molecular weight of over 100kd. The obtained filtrate 2 was filtered through a 5kd ultrafiltration membrane. Filtrate 2 was concentrated under reduced pressure at a temperature below 55°C and the volume was adjusted to 200g. It was then placed in an environment below 2°C for 18h to settle and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at 55°C for the second time and the volume was adjusted to 50g. 95° ethanol was added to the liquid to adjust the alcohol content of the clear liquid to 65°. Then it was placed in an environment below 2°C to settle for 18h and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure at 55°C for the third time and brought to a final volume of 200g. It was then adsorbed onto 200g of AB-8 macroporous resin at a flow rate of 1BV / h. Then proceed with the following steps in sequence: The resin column was rinsed with 1 BV of pure water at a flow rate of 1 BV / h and the temperature was ≤50℃ throughout the elution process. The target eluent A was collected. Elute with 1 BV 10% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent B; Elute with 1 BV 20% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent C; Elute with 1 BV 30% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent D; Elute with 1 BV 50% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent E; Elute with 1 BV 70% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent F; Elute with 1 BV 90% ethanol solution at a flow rate of 1 BV / h and a temperature ≤50℃ throughout the elution process, and collect the target eluent G; The eluents AG were filtered until clear, and then concentrated and dried under reduced pressure at 55°C to obtain Platycladus orientalis leaf extract AG.

[0043] I. Determination of the ethanol concentration used for ethanol elution 1. Effects of the obtained Platycladus orientalis leaf extract AG on Hacat cell viability 1.1 Experimental Methods All the elution fractions of the supplementary comparative example were taken, and the extracts of Platycladus orientalis leaves were dissolved in deionized water under sterile conditions and quantified to 100g to obtain each test sample with a concentration of 100%. Before the test, the test samples were diluted to the test concentration with DMEM medium.

[0044] Hacat cell suspension in the logarithmic growth phase and in good condition was seeded into 96-well plates in DMEM medium, with 8 × 10⁶ cells per well. 3 After culturing for 24 h, samples of different concentrations were added and cultured for another 24 h. The cells were then washed twice with PBS, and 100 μL of CCK-8 working solution (CCK-8 reagent: DMEM medium = 1:10) was added to each well. The cells were incubated at 37 °C and 5% CO2 for 2 h, and the absorbance was measured at 450 nm using an ELISA reader.

[0045] Relative cell viability (%) = (OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group)%.

[0046] Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0047] 1.2 Experimental Results The test results are shown in Table 1.

[0048] Table 1 Note:* p <0.05,** p <0.01 vs blank control The test results show that the Platycladus orientalis leaf extract G obtained from the 90° ethanol elution phase is cytotoxic at a concentration of 3000 ppm, while the Platycladus orientalis leaf extract AF obtained from other elution phases has no significant effect on the cell viability of Hacat cells within the tested concentration range.

[0049] 2. Effects of the obtained Platycladus orientalis leaf extract AG on KLK5 enzyme activity in Hacat cells 2.1 Experimental Methods All the elution fractions of the supplementary comparative example were taken, and the extracts of Platycladus orientalis leaves were dissolved in deionized water under sterile conditions and quantified to 100g to obtain each test sample with a concentration of 100%. Before the test, the test samples were diluted to the test concentration with DMEM medium.

[0050] Human HaCaT cells (HaCaT) in the logarithmic growth phase and in good growth were seeded into 6-well plates containing 10% fetal bovine serum and 1% DMEM medium containing penicillin and antibiotics, with 3 × 10⁶ cells per well. 5 After culturing for 24 hours, 1,25-dihydroxyvitamin D3 (100 nM) and different concentrations of test samples were added and incubated for another 24 hours. The culture supernatant and cell lysate were collected and incubated with 100 μM Boc-VPR-AMC fluorescent peptide substrate at room temperature for 5 min. After incubation, the fluorescence intensity was measured at Ex 380 nm / Em 460 nm.

[0051] Inhibition rate (%) = (1 - KLK5 enzyme activity in sample group / KLK5 enzyme activity in negative control group) * 100% Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0052] 2.2 Experimental Results The experimental results are shown in Table 2.

[0053] Table 2 The test results show that, under the same preparation method, the effects of Platycladus orientalis leaf extracts obtained from different elution stages on KLK5 enzyme activity differed. Compared with the negative control group, Platycladus orientalis leaf extract B obtained from the 0-10° ethanol elution stage inhibited KLK5 enzyme activity at high concentrations, while Platycladus orientalis leaf extract C obtained from the 10-20° ethanol elution stage inhibited KLK5 enzyme activity within the tested concentration range. However, Platycladus orientalis leaf extract A obtained from the pure water elution stage and Platycladus orientalis leaf extract DG obtained from the 20-90° ethanol elution stage had no significant effect on KLK5 enzyme activity. Inhibition rate calculations revealed that as the ethanol concentration in the eluent increased, the obtained Platycladus orientalis leaf extracts actually promoted KLK5 enzyme activity, and this promotion increased with increasing ethanol concentration.

[0054] II. Effects of different Platycladus orientalis leaf extracts on Hacat cell viability 1. Experimental Methods Take 1g of the Platycladus orientalis leaf extract powder obtained in Examples 1-6 and Comparative Examples 1-6, dissolve it in deionized water under sterile conditions, and dilute the concentration to 100% (m / m) of the crude drug weight to obtain the sample to be tested. Take a suspension of well-grown Hacat cells in the logarithmic growth phase and seed them in a 96-well plate of DMEM medium, with 8 × 10⁶ cells per well. 3 After culturing for 24 h, samples of different concentrations were added and cultured for another 24 h. The cells were then washed twice with PBS, and 100 μL of CCK-8 working solution (CCK-8 reagent: DMEM medium = 1:10) was added to each well. The cells were incubated at 37℃ and 5% CO2 for 2 h, and the absorbance was measured at 450 nm using an ELISA reader.

[0055] Relative cell viability (%) = (OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group) % Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0056] 2. Experimental Results The experimental results are shown in Tables 3 and 4.

[0057] Table 3 Note:* p <0.05,** p <0.01 vs blank control Table 4 Note:*p <0.05,** p <0.01 vs blank control As shown in Tables 3 and 4, the Platycladus orientalis leaf extracts described in Examples 1-6 of this invention significantly enhanced HACAT cell viability at a concentration of 750 ppm. In contrast, the Platycladus orientalis leaf extracts described in Comparative Examples 1, 2, 6, and 13 exhibited an inhibitory effect on HACAT cell viability. While the Platycladus orientalis leaf extracts described in Comparative Examples 3, 4, and 5 also showed an effect of enhancing HACAT cell viability, the rate of enhancement was significantly lower than that of this invention.

[0058] Examples 1, 2, 4, and 6 significantly enhanced HACAT cell viability at 750 ppm, with Examples 2 and 6 showing a significant enhancement at 375 ppm. Examples 1-6 did not exhibit cytotoxicity within the tested concentration range. Comparative Examples 1-6, however, did not significantly enhance HACAT cell viability within the tested concentration range, with Comparative Example 5 exhibiting cytotoxicity at 3000 ppm.

[0059] The experimental results show that the Platycladus orientalis leaf extract obtained under this preparation process can enhance the viability of Hacat cells, while the Platycladus orientalis leaf extract obtained without this preparation process did not show the effect of enhancing Hacat cell viability and may have certain cytotoxicity.

[0060] III. Effects of different Platycladus orientalis leaf extracts on KLK5 enzyme activity in Hacat cells 1. Experimental Methods Take 1g of the Platycladus orientalis leaf extract powder obtained in Examples 1-6 and Comparative Examples 1-6, dissolve it in deionized water under sterile conditions, and dilute the concentration to 100% (m / m) of the crude drug to obtain the sample to be tested. Take a suspension of well-grown human HaCaT cells in the logarithmic growth phase and seed them in 6-well plates containing 10% fetal bovine serum and 1% DMEM medium containing double antibiotics, with 3 × 10 cells per well. 5 After culturing for 24 hours, 1,25-dihydroxyvitamin D3 (100 nM) and different concentrations of test samples were added and incubated for another 24 hours. The culture supernatant and cell lysate were collected and incubated with 100 μM Boc-VPR-AMC fluorescent peptide substrate at room temperature for 5 min. After incubation, the fluorescence intensity was measured at Ex 380 nm / Em 460 nm.

[0061] Inhibition rate (%) = (1 - KLK5 enzyme activity in sample group / KLK5 enzyme activity in negative control group) * 100%.

[0062] Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0063] 2. Experimental Results The experimental results are shown in Table 5.

[0064] Table 5 Note: ##p<0.01 vs blank control, *p<0.05, **p<0.01 vs negative control The experimental results show that, compared with the negative control, the Platycladus orientalis leaf extract obtained by the embodiments of the present invention showed a significant inhibitory effect on the KLK5 enzyme activity of Hacat cells. Under the condition of 500 ppm of Platycladus orientalis leaf extract, the inhibition rate of KLK5 enzyme activity of Hacat cells reached more than 43%.

[0065] Examples 1 and 2 showed inhibitory effects on KLK5 enzyme activity in Hacat cells within the tested concentration range. Examples 3, 4, 5, and 6 showed inhibitory effects on KLK5 enzyme activity in Hacat cells at medium to high concentrations. Comparative Examples 2, 3, and 6 also showed inhibitory effects on KLK5 enzyme activity in Hacat cells at medium to high concentrations, but the inhibition rate was lower than that of Examples 1-6. Comparative Example 4 also showed inhibitory effects on KLK5 enzyme activity in Hacat cells at high concentrations, but the inhibition rate was lower than that of Examples 1-6. Comparative Examples 1 and 5 did not show any effect on KLK5 enzyme activity in Hacat cells.

[0066] The inhibitory effect of the arborvitae leaf extract obtained in the comparative proportion on the KLK5 enzyme activity of hacat cells was significantly lower than that in the embodiments of the present invention, and it may even have shown the effect of promoting the KLK5 enzyme activity of hacat cells.

[0067] IV. Effects of different Platycladus orientalis leaf extracts on Hacat cell ATP levels 1. Experimental Methods Take 1g of the Platycladus orientalis leaf extract powder obtained in Examples 1-6 and Comparative Examples 1-6, dissolve it in deionized water under sterile conditions, and dilute the concentration to 100% (m / m) of the crude drug to obtain the sample to be tested. Take a suspension of human keratinocytes (HaCaT) cells in the logarithmic growth phase and inoculate them into 6-well plates containing 10% fetal bovine serum and 1% DMEM medium containing double antibiotics, with 1×10 cells per well. 5After culturing for 24 hours, different concentrations of test samples were added and reacted for another 24 hours. Then, PBS was replaced, and except for the blank group, the samples were irradiated with ultraviolet (UVB) at 400 mJ / cm². 2 After administration, the PBS was replaced with culture medium and the cells were cultured for another 24 hours. Cells were lysed using lysis buffer and centrifuged at 4°C, 10,000 rpm for 5 minutes. The supernatant was collected as the test sample. An ATP assay kit was used for detection, and the OD value was measured using a chemiluminescence analyzer to calculate the relative ATP promotion rate of the sample.

[0068] Increase rate (%) = (ATP concentration in sample group / ATP concentration in negative control group - 1) * 100%.

[0069] Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0070] 2. Experimental Results Table 6 shows the effect of the extract of Platycladus orientalis leaf on the ATP level of Hacat cells in the embodiments and comparative examples of the present invention, and Table 7 shows the rate of increase in ATP level of Hacat cells by the extract of Platycladus orientalis leaf in the embodiments and comparative examples of the present invention.

[0071] Table 6 Note: ##p<0.01 vs blank control, *p<0.05, **p<0.01 vs negative control Table 7 The experimental results show that, compared with the negative control, the Platycladus orientalis leaf extract obtained in the embodiments of the present invention all showed an effect of increasing the intracellular ATP level of Hacat cells at a concentration of 500 ppm, with an increase rate of more than 15%. In contrast, the increase rate of Platycladus orientalis leaf extract at a concentration of 500 ppm on the intracellular ATP level of Hacat cells was ≤8%, and Comparative Examples 1, 3, 5, and 6 did not show any effect on the intracellular ATP level of Hacat cells.

[0072] V. Effects of different Platycladus orientalis leaf extracts on ATP levels in dermal papilla cells 1. Experimental Methods Take 1g of the Platycladus orientalis leaf extract powder obtained in Examples 1-6 and Comparative Examples 1-6, dissolve it in deionized water under sterile conditions, and dilute the concentration to 100% (m / m) of the crude drug to obtain the sample to be tested. Take a suspension of human dermal papilla cells (HDPC) in the logarithmic growth phase and seed them in 6-well plates containing 10% fetal bovine serum and 1% DMEM medium containing double antibiotics, with 1×10 cells per well. 5 After culturing for 24 hours, different concentrations of test samples were added and reacted for another 24 hours. Then, PBS was replaced, and except for the blank group, the samples were irradiated with ultraviolet (UVB) at 400 mJ / cm². 2 After administration, the PBS was replaced with culture medium and the cells were cultured for another 24 hours. Cells were lysed using lysis buffer and centrifuged at 4°C, 10,000 rpm for 5 minutes. The supernatant was collected as the test sample. An ATP assay kit was used for detection, and the OD value was measured using a chemiluminescence analyzer to calculate the relative ATP promotion rate of the sample.

[0073] Increase rate (%) = (ATP concentration in sample group / ATP concentration in negative control group - 1) * 100%.

[0074] Statistical analysis: All experimental data are expressed as mean ± standard deviation (SD). Mean ± s) is expressed as mean ± s. Differences between groups were analyzed using one-way ANOVA, and pairwise comparisons were performed using Dunnett's multiple comparison test, with a significance level of α = 0.05.

[0075] 2. Experimental Results Table 8 shows the effect of the extract of Platycladus orientalis leaf on the ATP level of dermal papilla cells in the embodiments and comparative examples of the present invention, and Table 9 shows the rate of increase in ATP level of dermal papilla cells by the extract of Platycladus orientalis leaf on the embodiments and comparative examples of the present invention.

[0076] Table 8 Note: ##p<0.01 vs blank control, *p<0.05, **p<0.01 vs negative control Table 9 The experimental results show that, compared with the negative control, the Platycladus orientalis leaf extract obtained in this embodiment of the invention exhibited an effect of increasing the intracellular ATP level of dermal papilla cells at both low and high concentrations. At a concentration of 20 ppm, the increase in intracellular ATP levels was ≥10%; at 100 ppm, it was ≥25%; and at 500 ppm, it was ≥36%. In contrast, the Platycladus orientalis leaf extract obtained in the comparative example showed a weaker effect on increasing intracellular ATP levels in dermal papilla cells, with an increase of ≤8% at 20 ppm, ≤14% at 100 ppm, and ≤24% at 500 ppm.

[0077] Examples 1, 2, 4, and 5 all showed an effect of increasing the intracellular ATP level of dermal papilla cells within the tested concentration range. Examples 3 and 6 showed an effect of increasing the intracellular ATP level of dermal papilla cells at medium to high concentrations. Comparative Examples 3 and 6 also showed an effect of increasing the intracellular ATP level of dermal papilla cells at medium to high concentrations, but the rate of increase was lower than that of Examples 1-6. Comparative Example 2 showed an effect of increasing the intracellular ATP level of dermal papilla cells at high concentrations, but the rate of increase was lower than that of Examples 1-6. Comparative Examples 1, 4, and 5 did not show any effect on the intracellular ATP level of dermal papilla cells.

Claims

1. A method for preparing a extract of biota orientalis, characterized in that, Includes the following steps: The leaves of Platycladus orientalis were crushed to 80-120 mesh, and 14-16 times the weight of the leaves of Platycladus orientalis were added to deionized water. The mixture was soaked at room temperature for more than 2 hours, heated to 40-60℃, and ultrasonically extracted for 45-75 minutes. Solid-liquid separation was then performed to obtain filtrate 1. The obtained filtrate 1 is concentrated and adjusted to 5-6 times the weight of the arborvitae leaves using a reverse osmosis membrane to obtain solution 1. Chitosan solution of 12-14% of the liquid volume of solution 1 is added, and the mixture is stirred and flocculated for 25-30 minutes. The mixture is then placed in an environment below 4°C and allowed to settle for more than 12 hours. After filtration until clear, filtrate 2 is obtained. The molecular weight of the chitosan is above 100 kDa. The obtained filtrate 2 was filtered through a 3-8 kd ultrafiltration membrane. The filtrate 2 was concentrated under reduced pressure at a temperature below 60°C and the volume was adjusted to 1.8-2.2 times the weight of the arborvitae leaves. Then it was placed in an environment below 4°C for sedimentation for more than 12 hours and filtered until clear to obtain filtrate 3. The obtained filtrate 3 was concentrated under reduced pressure at a temperature below 60°C for the second time and the volume was adjusted to 0.4 to 0.6 times the weight of the arborvitae leaves to obtain solution 2. Ethanol with a concentration of 90° or higher was added to solution 2 to adjust the alcohol content of solution 2 to 60 to 70°. Then, it was placed in an environment below 4°C for sedimentation for more than 12 hours and filtered until clear to obtain filtrate 4. The resulting filtrate 4 was concentrated under reduced pressure at a temperature below 60°C for the third time to a volume of 1.8 to 2.2 times the weight of the arborvitae leaves. It was then adsorbed by macroporous resin and washed with water to remove impurities. Subsequently, elution was performed using 3 BV 10~20% ethanol solution at a flow rate controlled at 1 BV / h, with the temperature ≤50℃ throughout the process. The target eluent was collected, filtered until clear, and then concentrated under reduced pressure for the fourth time at below 60℃ and dried to obtain the Platycladus orientalis leaf extract.

2. The preparation method according to claim 1, characterized in that, The chitosan solution concentration is 1%, and the vacuum degree of the reduced pressure concentration is -0.08 to -0.09 MPa.

3. The preparation method according to claim 1, characterized in that, The drying process is either freeze-drying or vacuum drying, with the temperature not exceeding 60°C throughout.

4. The preparation method according to claim 1, characterized in that, The macroporous resin is one of the following: AB~8, HPD100, or HP20 type macroporous resin.

5. The preparation method according to claim 1, characterized in that, During the adsorption of the macroporous resin, the flow rate is controlled at 1 BV / h, and the weight ratio of the macroporous resin to the arborvitae leaf is 1:0.

5.

6. The preparation method according to claim 1, characterized in that, The water washing and impurity removal process is as follows: the resin column is rinsed with 2~3 BV of pure water at a flow rate of 2 BV / h.

7. The Platycladus orientalis leaf extract obtained by the preparation method according to any one of claims 1 to 6, characterized in that, It can enhance the vitality of human keratinocytes, inhibit the activity of KLK5 enzyme in human keratinocytes, and increase the ATP content in dermal papilla cells and / or human keratinocytes.

8. The use of the Platycladus orientalis leaf extract obtained by the preparation method according to any one of claims 1 to 6 in the preparation of cosmetics, skin care products or topical formulations that have the effects of human keratinocyte viability and / or inhibiting human keratinocyte KLK5 enzyme activity and / or promoting ATP synthesis in dermal papilla cells and / or human keratinocytes.

9. The use of the Platycladus orientalis leaf extract obtained by the preparation method according to any one of claims 1 to 6 in the preparation of medicaments or cosmetics for treating or preventing skin diseases associated with abnormal upregulation of KLK5 activity.

10. The use of the Platycladus orientalis leaf extract obtained by the preparation method according to any one of claims 1 to 6 in the preparation of medicaments or cosmetics for treating or preventing skin diseases related to ATP content in dermal papilla cells and / or human keratinocytes.

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