阅读说明：本技术 一种促进皮肤细胞新生的青蒿精油、白茶精油及它们的组合物 (Artemisia annua essential oil and white tea essential oil for promoting skin cell regeneration and their composition ) 是由 汤凯 于 2021-09-30 设计创作，主要内容包括：本发明涉及一种促进皮肤细胞新生的青蒿精油、白茶精油及它们的组合物。所述青蒿精油采用以下方法制备：采集新鲜青蒿嫩苗,取叶子,干燥,切成碎段,先后使用碱性蛋白酶和纤维素酶酶解,再离心、萃取。所述白茶精油采用以下方法制备：采集新鲜白茶嫩芽,于背光的室内在温度30-35℃,湿度50％-60％的条件下萎凋至减重率为40％-60％,干燥,粉碎,先后使用果胶酶和纤维素酶酶解,再离心、萃取。本发明证实所述青蒿精油、白茶精油和其组合物对于对HaCat细胞迁移、人皮肤成纤维细胞和表皮干细胞的增殖以及人皮肤成纤维细胞合成I型胶原蛋白的能力有显著的促进作用,在抗皮肤衰老方面具有应用潜力。(The invention relates to sweet wormwood essential oil and white tea essential oil for promoting skin cell regeneration and a composition thereof. The sweet wormwood essential oil is prepared by the following method: collecting fresh herba Artemisiae Annuae tender seedling, collecting leaves, drying, cutting into pieces, sequentially performing enzymolysis with alkaline protease and cellulase, centrifuging, and extracting. The white tea essential oil is prepared by the following method: collecting fresh white tea tender shoots, withering the fresh white tea shoots in a backlight room at the temperature of 30-35 ℃ and the humidity of 50-60% until the weight loss rate is 40-60%, drying, crushing, sequentially carrying out enzymolysis by using pectinase and cellulase, centrifuging and extracting. The invention proves that the sweet wormwood essential oil, the white tea essential oil and the composition thereof have remarkable promoting effects on HaCat cell migration, proliferation of human skin fibroblasts and epidermal stem cells and the capability of the human skin fibroblasts for synthesizing type I collagen, and have application potential in the aspect of resisting skin aging.)

1. The application of white tea essential oil in preparing cosmetics or medicines for resisting skin aging or promoting skin cell regeneration is characterized in that the white tea essential oil is prepared by the following method: collecting tender shoots of fresh white tea, regularly spreading in a backlight room until the thickness is 3-5cm, controlling the temperature at 30-35 ℃, controlling the humidity at 50-60%, withering until the weight loss rate is 50-60%, and then placing in a dryer for drying at the temperature of 100 ℃ and 120 ℃ for 20-40 min; crushing dried white tea leaves, adding 14-18 times of distilled water, adjusting the pH value to 3.8-4.2, adding 40-60U/g of pectinase, reacting for 3.5-4.5h at 45-55 ℃ in a water bath, adjusting the pH value to 4.5-5.0, adding 40-60U/g of cellulase, and reacting for 1.5-2.5h at 45-55 ℃ in the water bath; and after the enzymolysis is finished, centrifuging, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and removing the diethyl ether.

2. The use according to claim 1, wherein the white tea is Yunnan big-leaf white tea.

3. The use according to claim 1, wherein the dried leaves of white tea are ground to a 10-20 mesh coarse powder.

4. The use of claim 1, wherein the centrifugation parameters are 2000-5000r/min, and the centrifugation time is 10-30 min; the method for removing the diethyl ether is to volatilize the diethyl ether in a constant-temperature water bath at the temperature of 45-55 ℃.

5. Use according to claim 1, wherein the skin cells are epidermal stem cells, dermal fibroblasts or epidermal cells.

6. The application of the sweet wormwood essential oil in preparing cosmetics or medicines for resisting skin aging or promoting skin cell regeneration is characterized in that the sweet wormwood essential oil is prepared by the following method: cleaning sweet wormwood leaves, drying, cutting into segments, adding 4-6 times of distilled water, adjusting pH to 9.5-10.5, adding alkaline protease with the amount of 80-120U/g, reacting at 45-55 deg.C in a water bath for 2.5-3.5h, adjusting pH to 4.5-5.0, adding cellulase with the amount of 10-30U/g, and reacting at 45-55 deg.C in a water bath for 2.5-3.5 h; and after the enzymolysis is finished, centrifuging, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and removing the diethyl ether.

7. The use as claimed in claim 6, wherein the leaves of Artemisia annua are leaves of Artemisia annua seedlings and the origin of Artemisia annua is Henan.

8. The use according to claim 6, wherein the drying is carried out at a temperature below 60 ℃ for 2-5 hours and the size of the fragments is 4-6 mm.

9. The use of claim 6, wherein the centrifugation parameters are 2000-5000r/min, and the centrifugation time is 10-30 min; the method for removing the diethyl ether is to volatilize the diethyl ether in a constant-temperature water bath at the temperature of 45-55 ℃.

10. Use according to claim 6, wherein the skin cells are epidermal stem cells, dermal fibroblasts or epidermal cells.

11. Use of a plant essential oil composition comprising the white tea essential oil according to any one of claims 1 to 5 and the artemisia apiacea essential oil according to any one of claims 6 to 10 for preparing cosmetics or medicines for resisting skin aging or promoting skin cell regeneration.

12. The use as claimed in claim 11, wherein the ratio of the sweet wormwood essential oil and the white tea essential oil is 1 (0.01-20).

Technical Field

The invention relates to essential oil extraction of plants, in particular to sweet wormwood essential oil and white tea essential oil which are used for promoting skin cell regeneration, a composition of the sweet wormwood essential oil and the white tea essential oil, a preparation method of the composition and application of the composition.

Background

With the increase of living matter level, people have an increasing demand for skin anti-aging. Skin aging is a result of the combined action of various factors, and can be classified into two types according to the factors causing skin aging: one is endogenous aging, also called natural aging, which is mainly affected by genetic factors and is a process of natural progressive aging; the other is exogenous aging caused by external environmental factors such as ultraviolet radiation, smoking, attraction, chemicals, etc., wherein over 80% of facial skin aging is caused by ultraviolet radiation and is also called photoaging, and the skin aging is characterized by rough texture, dark complexion, pigmentation and loss of skin elasticity.

Human skin is divided into three layers: epidermis, dermis, subcutaneous tissue. The epidermis is the outermost layer of the skin and serves to prevent the invasion of harmful substances and bacteria. The dermis is located below the epidermis and is composed of cells and extracellular matrix, and fibroblasts are the main cells of the dermis and maintain the normal structure and physiological functions of the skin through normal proliferation and differentiation. The dermis layer is an important part directly related to skin aging. The subcutaneous tissue is located under the dermis, and the main function is to buffer the impact of external force.

The essential oil is prepared from leaves, flowers, seeds, fruits, roots, barks, resins, and hearts of plants by steam distillation, organic solvent extraction, and CO₂The extract is extracted by supercritical extraction, cold pressing, ultrasonic extraction, enzyme extraction, microwave extraction, crystallization, etc., and has high concentration of aromatic and volatile substances. Is widely applied in the industries of medicine, food health care and cosmetics. As a natural product, the product has the advantages of less adverse reaction, health and safety when being used as a medicine or a cosmetic.

The herba Artemisiae Annuae essential oil is volatile oil extracted from herba Artemisiae Annuae (Artemisia annua L.). Modern researches have proved that the volatile oil of sweet wormwood has the activities of antibiosis, anticancer, antivirus, anti-inflammatory, mosquito repellent, insect killing, antioxidation and the like. The main uses are acne treatment, influenza virus inhibition, inflammation diminishing, ulcer resistance, local stimulation and heart strengthening, fever relieving, cough relieving, phlegm eliminating, asthma relieving and pain easing, and can be used as a health food and can also be used for mosquito and insect repelling.

The white tea is a special treasure in Chinese tea, belongs to micro-fermented tea, and is prepared by withering leaves and buds of white tea tree in tea (Camellia sinensis (L.) O.Ktze.), baking (or drying in the shade), picking out, and annealing through a special process. The white tea essential oil is volatile oil extracted from tea leaves and buds of white tea. At present, the research on the efficacy of white tea essential oil is less.

Journal literature (Yifang, a beautiful gem, Zhang Bei, etc. Artemisinin has inhibitory effect on human skin keloid fibroblasts and the initial investigation of relevant mechanisms [ J ] Nanjing university of medical science (Nature science edition), 2020(8) ]) discloses that Artemisinin has inhibitory effect on human skin keloid fibroblasts. Journal literature (dawn Lin, Deng Ling, Lihao, et al, research on anti-skin scar of artemisinin and artesunate [ J ]. Zhonghua dermatology journal, 2009,42(006): 421) and 424.) discloses that an external ointment prepared from artemisinin and artesunate can be used for treating a rabbit ear ventral hyperplastic scar model, and the result shows that the artemisinin and artesunate ointment can effectively inhibit experimental skin scar of animals. Patent document CN103550407A discloses a natural skin repair cream, which contains the following substances in parts by weight: 8-17 parts of broccoli juice, 3-13 parts of watermelon juice, 2-11 parts of bird's nest powder, 6-17 parts of green tea powder, 6-15 parts of red bean powder, 4-12 parts of soybean powder, 1-6 parts of borneol, 3-9 parts of salt, 2-9 parts of rapeseed oil and 23-39 parts of distilled water. The natural skin repair cream has the effects of effectively improving the activity of skin cells, enabling the cells to become plump, delaying skin aging, enhancing skin tension, eliminating small wrinkles and the like, preventing wrinkles from generating, avoiding the skin from becoming rough, remarkably enhancing the anti-injury capacity of the skin, also being beneficial to repairing damaged skin, regulating skin metabolism and maintaining the normal functions of skin cell tissues.

However, the research on the promotion of skin regeneration by the sweet wormwood essential oil and the green tea essential oil is not found at present.

Disclosure of Invention

The invention aims to provide sweet wormwood essential oil and white tea essential oil which are used for promoting skin cell regeneration, and a composition, a preparation method and application thereof, aiming at the defects in the prior art.

In a first aspect, the present invention provides a use of white tea essential oil in cosmetics or medicines for resisting skin aging or promoting skin cell regeneration, the white tea essential oil being prepared by the following method: collecting tender shoots of fresh white tea, regularly spreading in a backlight room until the thickness is 3-5cm, controlling the temperature at 30-35 ℃, controlling the humidity at 50-60%, withering until the weight loss rate is 50-60%, and then placing in a dryer for drying at the temperature of 100 ℃ and 120 ℃ for 20-40 min; crushing dried white tea leaves, adding 14-18 times of distilled water, adjusting the pH value to 3.8-4.2, adding 40-60U/g of pectinase, reacting for 3.5-4.5h at 45-55 ℃ in a water bath, adjusting the pH value to 4.5-5.0, adding 40-60U/g of cellulase, and reacting for 1.5-2.5h at 45-55 ℃ in the water bath; and after the enzymolysis is finished, centrifuging, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and removing the diethyl ether.

Preferably, the white tea is Yunnan big-leaf white tea.

Preferably, the dried white tea leaves are crushed into 10-20 meshes of coarse powder.

Preferably, the centrifugation parameters are 2000-5000r/min of rotation speed and 10-30min of centrifugation time; the method for removing the diethyl ether is to volatilize the diethyl ether in a constant-temperature water bath at the temperature of 45-55 ℃.

Preferably, the skin cell is an epidermal stem cell, a skin fibroblast or an epidermal cell.

In a second aspect, the invention provides an application of sweet wormwood essential oil in preparing cosmetics or medicines for resisting skin aging or promoting skin cell regeneration, wherein the sweet wormwood essential oil is prepared by the following method: cleaning sweet wormwood leaves, drying, cutting into segments, adding 4-6 times of distilled water, adjusting pH to 9.5-10.5, adding alkaline protease with the amount of 80-120U/g, reacting at 45-55 deg.C in a water bath for 2.5-3.5h, adjusting pH to 4.5-5.0, adding cellulase with the amount of 10-30U/g, and reacting at 45-55 deg.C in a water bath for 2.5-3.5 h; and after the enzymolysis is finished, centrifuging, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and removing the diethyl ether.

Preferably, the southernwood leaves are leaves of southernwood seedlings, and the production place of the southernwood is Henan.

Preferably, the drying mode is drying at 60 ℃ or below for 2-5 hours, and the size of the fragments is 4-6 mm.

Preferably, the centrifugation parameters are 2000-5000r/min of rotation speed and 10-30min of centrifugation time; the method for removing the diethyl ether is to volatilize the diethyl ether in a constant-temperature water bath at the temperature of 45-55 ℃.

Preferably, the skin cell is an epidermal stem cell, a skin fibroblast or an epidermal cell.

In a third aspect, the invention provides application of a plant essential oil composition in preparing cosmetics or medicines for resisting skin aging or promoting skin cell regeneration, wherein the plant essential oil composition contains the artemisia apiacea essential oil and the white tea essential oil.

Preferably, the ratio of the sweet wormwood essential oil to the white tea essential oil is 1 (0.01-20).

The sweet wormwood essential oil is extracted from Artemisia annua L of Compositae. The white tea essential oil is extracted from Camellia sinensis (L.) Siemens of Theaceae.

The invention has the advantages that:

1. the sweet wormwood essential oil and the white tea essential oil are prepared by the method. The influence of two essential oils and essential oil compositions on the proliferation, migration or collagen synthesis of HaCat cells, human skin fibroblasts and epidermal stem cells is examined, and the effects on the migration of HaCat cells, the proliferation of human skin fibroblasts and epidermal stem cells and the capability of the human skin fibroblasts for synthesizing type I collagen are obviously promoted.

2. The invention optimizes the preparation method of the essential oil and improves the activity of the essential oil.

The research shows that stem cells play a very important role in maintaining the body homeostasis and ensuring the repair and regeneration of tissues and organs, the stem cells play an important role in the skin anti-aging process, the migration and metabolism speed of epidermal cells determine the state of the skin, and the content of collagen in the skin is an important influence factor for determining the skin elasticity and maintaining the young state, so the invention suggests that the artemisia apiacea essential oil, the white tea essential oil and the composition thereof have good application prospects in the aspect of resisting skin aging.

Drawings

FIG. 1: CCK8 measures the effect of different concentrations of essential oils on HaCat cell production. A blank group; b250 mu g/mL sweet wormwood essential oil; c250 mug/mL white tea essential oil; d250 mug/mL of formula essential oil.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1 Artemisia apiacea essential oil of the invention and preparation thereof

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding 100U/g of alkaline protease, reacting at 50 ℃ in a water bath for 3h, adjusting pH to 4.8, adding 20U/g of cellulase, and reacting at 50 ℃ in the water bath for 3 h; after the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 2 Artemisia apiacea essential oil of the invention and preparation thereof

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding 80U/g of alkaline protease, reacting at 50 ℃ in a water bath for 3h, adjusting pH to 4.8, adding 20U/g of cellulase, and reacting at 50 ℃ in the water bath for 3 h; after the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 3 Artemisia annua essential oil of the invention and preparation thereof

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding alkaline protease with the dosage of 120U/g, reacting in a water bath kettle at 50 ℃ for 3h, adjusting pH to 4.8, adding cellulase with the dosage of 20U/g, and reacting in the water bath kettle at 50 ℃ for 3 h; after the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 4 Artemisia apiacea essential oil of the invention and preparation thereof (IV)

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 2 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 4 times of distilled water, adjusting pH to 10, adding 80U/g of alkaline protease, reacting at 45 ℃ in a water bath for 2.5h, adjusting pH to 4.8, adding 10U/g of cellulase, and reacting at 45 ℃ in the water bath for 3.5 h; after the enzymolysis is finished, centrifuging for 10min at 5000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 45 ℃.

Example 5 Artemisia apiacea essential oil of the invention and preparation thereof (V)

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 2 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 6 times of distilled water, adjusting pH to 10, adding alkaline protease with the dosage of 120U/g, reacting at 35 ℃ in a water bath for 1.5h, adjusting pH to 4.8, adding cellulase with the dosage of 30U/g, and reacting at 55 ℃ in the water bath for 2.5 h; after the enzymolysis is finished, centrifuging for 30min at 2000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 55 ℃.

Example 6 Artemisia annua essential oil of the invention and preparation thereof (VI)

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 2 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 6 times of distilled water, adjusting pH to 10, adding 80U/g of alkaline protease, reacting at 45 ℃ in a water bath for 2.5h, adjusting pH to 4.8, adding 10U/g of cellulase, and reacting at 45 ℃ in the water bath for 3.5 h; after the enzymolysis is finished, centrifuging for 30min at 2000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 55 ℃.

Example 7 Artemisia apiacea essential oil of the invention and preparation thereof (seven)

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding 100U/g of alkaline protease, reacting at 40 ℃ in a water bath for 2h, adjusting pH to 4.8, adding 30U/g of cellulase, and reacting at 55 ℃ in the water bath for 2.5 h; after the enzymolysis is finished, centrifuging for 30min at 2000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 55 ℃.

Example 8 white tea essential oil of the invention and its preparation

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 35 ℃, the humidity at 55 to 60 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 110 ℃ for 30min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 16 times of distilled water, adjusting pH to 4.0, adding 50U/g pectase, reacting at 50 deg.C in water bath for 4 hr, adjusting pH to 4.8, adding 50U/g cellulase, and reacting at 50 deg.C in water bath for 2 hr. After the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 9 white tea essential oil of the invention and its preparation (II)

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 35 ℃, the humidity at 55 to 60 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 110 ℃ for 30min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 16 times of distilled water, adjusting pH to 4.0, adding 60U/g of pectinase, reacting at 50 deg.C for 4h, adjusting pH to 4.8, adding 50U/g of cellulase, and reacting at 50 deg.C for 2 h. After the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 10 white tea essential oil of the invention and its preparation (III)

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 30 ℃, controlling the humidity at 50 to 55 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 120 ℃ for 20min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 14 times of distilled water, adjusting pH to 4.0, adding 60U/g of pectinase, reacting at 55 deg.C for 3.5h, adjusting pH to 4.8, adding 40U/g of cellulase, and reacting at 55 deg.C for 2.5 h. After the enzymolysis is finished, centrifuging for 30min at 2000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 45 ℃.

Example 11 white tea essential oil of the invention and its preparation (IV)

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 32 ℃, controlling the humidity at 50 to 55 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 110 ℃ for 40min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 18 times of distilled water, adjusting pH to 4.0, adding 40U/g of pectinase, reacting at 45 deg.C for 4.5h in a water bath, adjusting pH to 4.8, adding 60U/g of cellulase, and reacting at 45 deg.C for 1.5h in a water bath. After the enzymolysis is finished, centrifuging for 10min at 5000r/min, collecting a liquid phase, extracting with diethyl ether, collecting an upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing the diethyl ether in a constant-temperature water bath at 55 ℃.

Comparative example 1 Artemisia apiacea essential oil control I and preparation thereof

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding 140U/g of alkaline protease, reacting at 50 ℃ in a water bath for 3h, adjusting pH to 4.8, adding 20U/g of cellulase, and reacting at 50 ℃ in the water bath for 3 h; after the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Comparative example 2 Artemisia apiacea essential oil control two and preparation thereof

Collecting fresh herba Artemisiae Annuae young seedling of 5 months in Henan, removing stem and useless parts, cleaning leaves, filtering to remove water, and drying in an electrothermal constant temperature drying oven at temperature below 60 deg.C for 3 hr; cutting dried sweet wormwood into 4-6mm fragments by a cutting machine, adding 5 times of distilled water, adjusting pH to 10, adding alkaline protease with the dosage of 160U/g, reacting in a water bath kettle at 50 ℃ for 3h, adjusting pH to 4.8, adding cellulase with the dosage of 20U/g, and reacting in the water bath kettle at 50 ℃ for 3 h; after the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Comparative example 3 white tea essential oil control one and preparation thereof

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 35 ℃, the humidity at 55 to 60 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 110 ℃ for 30min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 16 times of distilled water, adjusting pH to 4.0, adding 70U/g of pectinase, reacting at 50 deg.C for 4h, adjusting pH to 4.8, adding 50U/g of cellulase, and reacting at 50 deg.C for 2 h. After the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Comparative example 4 white tea essential oil control two and preparation thereof

Taking large-leaf white tea in Puer region of Yunnan as a material, picking tender leaves of one heart and two hearts with the same size by hand at 6 to 9 o' clock in the morning, regularly spreading the tender leaves in a backlight room until the thickness is 3 to 5cm, controlling the temperature at 35 ℃, the humidity at 55 to 60 percent, withering until the weight loss rate is 50 to 55 percent, then placing the tender leaves into a dryer for drying at 110 ℃ for 30min, and crushing the white tea leaves and sieving the crushed leaves with a 10-mesh sieve to obtain coarse powder. Adding 16 times of distilled water, adjusting pH to 4.0, adding 80U/g of pectinase, reacting at 50 deg.C for 4h, adjusting pH to 4.8, adding 50U/g of cellulase, and reacting at 50 deg.C for 2 h. After the enzymolysis is finished, centrifuging at 3000r/min for 20min, collecting liquid phase, extracting with diethyl ether, collecting upper layer, dehydrating with anhydrous sodium sulfate, and volatilizing diethyl ether in 50 deg.C constant temperature water bath.

Example 12 Effect of Artemisia Annua essential oil, white tea essential oil and formulation essential oil on HaCat cell scratch

1 materials of the experiment

1.1 Experimental cells and animals

Hacat cells.

1.2 reagents and reagents

Examples 1-3, Artemisia apiacea essential oil of comparative examples 1-2, examples 8-9, white tea essential oil of comparative examples 3-4, composition of Artemisia apiacea essential oil and white tea essential oil of example 8 (the mass ratio of Artemisia apiacea essential oil to white tea essential oil is 4:1), DMEM high-sugar medium, pancreatin, fetal bovine serum, PBS buffer solution, CCK8 detection kit.

1.3 instruments and devices

Biological safety cabinet, cell constant temperature incubator, inverted microscope, full-automatic enzyme labeling instrument, centrifuge, water bath, 80 degree refrigerator, 20 degree refrigerator, 4 degree refrigerator, 96 pore plate, 6 pore plate, disposable straw, 1000ul, 200ul, 10ul and other different specifications of gun and gun head, 50ml, 15ml and 2ml centrifuge tube, cell plug-in.

2 method of experiment

2.1 Hacat cell culture, passage

Hacat cell culture is carried out for 2 hours, then fresh culture medium is timely replaced, and culture is continued. When the cells grow to the logarithmic growth phase, the culture medium is discarded, the cells are washed for 3 times by PBS, 1ml of pancreatin is added, the cells are placed in an incubator for 5min and then observed under an inverted microscope, if the cell gaps become larger, the cells become round, 2ml of culture medium is added to stop digestion, and the cell suspension is sucked into a 15ml centrifuge tube. Centrifuging at 1000rpm at room temperature for 5min, removing supernatant, adding 3ml complete culture medium, pumping, mixing, dividing into 10cm cell culture dishes, adding 10ml DMEM culture medium into each dish, and culturing at 37 deg.C with 5% CO₂And (5) continuously culturing in an incubator with saturated humidity.

2.2 CCK8 method for detecting the effect of single-component essential oil and component essential oil on the survival activity of keratinocytes

Hacat cells were cultured at 5X 10⁴Inoculating 100 μ l/well of each cell in 96-well plate, culturing in incubator, adding essential oil of different concentrations and single essential oil, and incubating for 24 hr. CCK8 was diluted with the culture medium at a ratio of 1:9, and the liquid in the 96-well plate was aspirated off, and 100. mu.l of the diluted CCK8 solution was added to each well. Incubate in incubatorAfter 30min, the wells were removed and the absorbance of each well was measured at 450nm using a microplate reader.

2.3 testing the Effect of Single component essential oils and component essential oils on keratinocyte migration

The cell scratch test is a commonly used in-vitro test method for researching cell migration, simulates the process of cell migration in vivo to a certain extent, and comprises the following specific operation steps: (1) placing a cell plug-in each hole of the 24-hole plate; (2) hacat cells were cultured at 5X 10⁵Inoculating each cell/ml in a cell insert, removing the culture medium when the cells grow to 80-90% fusion, and adding medicine for incubation (prepared by culture medium containing 1% serum); (3) observing and photographing under an inverted microscope at different time points, and calculating the healing rate of the scratch damage area by using software Image J; (4) mobility ═ original scratch area-residual area/original scratch area × 100%; relative mobility (dosing-blank mobility)/blank mobility.

3 results of the experiment

3.1 CCK8 results

The results show that the single-component essential oil and the formula essential oil with the concentrations of 1000 mug/mL, 500 mug/mL and 250 mug/mL have no significant influence on the growth of HaCat cells.

TABLE 1 CCK8 examination of the effect of different concentrations of essential oils on HaCat cell growth

3.2 cell scratch test results

The result shows that the relative mobility is 85.63% when the concentration of the formula essential oil is 125 mug/mL. The results of the single essential oil show that: the mobility of the sweet wormwood herb essential oil group in 125 mu g/mL of the examples 1 to 3 is more than 45 percent, and the mobility of the white tea essential oil group in 125 mu g/mL of the examples 8 to 9 is more than 43 percent. Compared with the sweet wormwood herb essential oil group in examples 1-3 and the white tea essential oil group in examples 8-9, the mobility of the formula essential oil group is significantly different (P is less than 0.01).

The mobility of the artemisia apiacea essential oil group in the comparative example 1 and the comparative example 2 is smaller than that in the examples 1-3, and the difference has statistical significance (P is less than 0.01). The mobility of the white tea essential oil groups of comparative example 3 and comparative example 4 was smaller than that of the white tea essential oil groups of examples 8-9, and the difference was statistically significant (P < 0.01).

Table 2 effect of different essential oils on HaCat cell migration: (n＝6)

Note: examples 1-3 essential oil of artemisia apiacea vs. comparative example 1 essential oil of artemisia apiacea P < 0.01; examples 1-3 essential oil of Artemisia annua vs. comparative example 2 essential oil of Artemisia annua, # P < 0.01; examples 8-9 white tea essential oil vs. comparative example 3 white tea essential oil, Δ P < 0.01; white tea essential oil vs. comparative example 4, P is less than 0.01; (v, the formula essential oil group, & & P & is less than 0.01).

Example 13 Effect of Artemisia Annua essential oil, white tea essential oil and formula essential oil on human skin fibroblast proliferation and type I collagen Synthesis

1 method of experiment

1.1 pharmaceutical formulation

Single essential oil: the sweet wormwood essential oil and the white tea essential oil are respectively dissolved in DMSO and diluted by complete culture medium to the using concentration of 250 mu g/mL.

The formula of the essential oil is as follows: the sweet wormwood essential oil and the white tea essential oil contain 400 mu L of 250 mu g/mL sweet wormwood essential oil and 100 mu L of 250 mu g/mL white tea essential oil per 1mL, and the rest is supplemented to the using concentration of 125 mu g/mL by using a complete culture medium.

1.2 CCK8 method for detecting effect of essential oil on human skin fibroblast proliferation

In order to examine the influence of the essential oil on the proliferation of mouse mononuclear macrophage RAW 264.7 cells, human skin fibroblasts (ESF-1, present in the dermatosis hospital of Shanghai) in the logarithmic growth phase are taken and prepared into cell suspension by DMEM complete culture solution,then 4 × 10⁴The cells were plated at a density of 200. mu.l/well in 96-well plates and incubated for 24 h. The dilution concentration of the essential oil was 250. mu.g/ml, 6 replicates per group. The experiment is provided with a blank control group and a positive control group, the blank control group is added with cell culture solution with corresponding volume, and the positive control group is added with 0.001 mu mol.L^-1Estradiol (Sigma). Discard the culture medium on the 96-well plate, add 100 μ l of 10% CCK8 serum-free DMEM solution at 37 deg.C and 5% CO₂Incubating in cell culture box for 30min, and measuring OD with enzyme-labeling instrument₄₉₀And (4) calculating the cell survival rate. Cell proliferation rate (%) - (oil essential oil OD value-blank OD value)/blank OD value × 100%.

1.3 detection of Effect of essential oils on human skin fibroblast type I collagen Synthesis

The concentration of 250. mu.g/mL of essential oil was selected for the determination of type I collagen synthesis by essential oil on ESF-1 cells. Collecting ESF-1 cells in logarithmic growth phase, preparing into cell suspension with DMEM complete culture solution, and culturing at 4 × 10⁴The cells were plated at a density of 200. mu.l/well in 96-well plates and incubated for 36 h. The dilution concentration of the essential oil was 250. mu.g/ml, 6 replicates per group. The experiment is provided with a blank control group and a positive control group, the blank control group is added with cell culture solution with corresponding volume, and the positive control group is added with 0.001 mu mol.L^-1Estradiol. After the culture is finished, collecting cell culture supernatant, and determining the content of the type I collagen according to the specification of an enzyme-linked immunosorbent assay (ELISA) assay kit for the type I collagen.

1.4 statistical treatment

Statistical analysis is carried out on the data by adopting SPSS 20.0 software, the data are expressed by mean +/-standard deviation, the One-way ANOVA method is adopted for comparing the mean values of a plurality of groups, the LSD method is selected when the variance is uniform in two-two comparison among the groups, and the Dunnett's T3 method is selected when the variance is not uniform. The difference is statistically significant when P is less than 0.05.

3 results of the experiment

3.1 Effect on human skin fibroblast proliferation

The cell proliferation rate of each group is calculated, and compared with a blank group, the positive control group, the artemisia apiacea essential oil group in examples 1-3, the white tea essential oil group in examples 8-9 and the formula essential oil group can obviously promote the ESF-1 cell proliferation, the difference between the cell proliferation rate and the blank group has statistical significance (P is less than 0.05 or P is less than 0.01), while the cell proliferation rate of the white tea essential oil group in comparative example 4 is less than that of the blank group and has statistical significance (P is less than 0.05), which shows that the white tea essential oil group has certain inhibition effect on the ESF-1 cell proliferation. Comparing the cell proliferation rates of the essential oil components of the southernwood of the examples 1-3 and the essential oil components of the southernwood of the comparative example 1, the results show that the cell proliferation rate of the essential oil components of the southernwood of the examples 1-3 is obviously higher than that of the essential oil components of the southernwood of the comparative example 1 (P < 0.05 or P < 0.01). Comparing the cell proliferation rates of the artemisia apiacea essential oil group in the examples 1-3 and the artemisia apiacea essential oil group in the comparative example 2, the result shows that the cell proliferation rate of the artemisia apiacea essential oil group in the examples 1-3 is obviously higher than that of the artemisia apiacea essential oil group in the comparative example 2 (P is less than 0.05 or P is less than 0.01). Comparing the cell proliferation rates of the white tea essence oil group of examples 8 to 9 with that of the white tea essence oil group of comparative example 3, the results showed that the cell proliferation rate of the white tea essence oil group of examples 8 to 9 was significantly higher than that of the white tea essence oil group of comparative example 3 (P < 0.05 or P < 0.01). Comparing the cell proliferation rates of the white tea essence oil group of examples 8 to 9 with that of the white tea essence oil group of comparative example 4, the results showed that the cell proliferation rate of the white tea essence oil group of examples 8 to 9 was significantly higher than that of the white tea essence oil group of comparative example 4 (P < 0.01). Compared with the positive control group, the cell proliferation rate of the formula essential oil group is obviously higher than that of the positive control group (P is less than 0.05).

TABLE 3 Effect of different essential oils on ESF-1 cell proliferation: (n＝6)

Note: p < 0.05, P < 0.01 compared to blank; examples 1-3 essential oil of Artemisia annua vs. comparative example 1 essential oil of Artemisia annua, # P < 0.05, # P < 0.01; examples 1-3 essential oil of Artemisia annua vs. comparative example 2 essential oil of Artemisia annua, delta P < 0.05, delta P < 0.01; white tea essential oil of comparative example 3, P is less than 0.05, P is less than 0.01; examples 8-9 white tea essential oil set vs. comparative example 4 white tea essential oil set, & & P < 0.01; single-component essential oil group vs. component essential oil group, & & P & lt 0.01; compared with the positive control group, the P is less than 0.05, and the P is less than 0.01.

3.2 Effect on human skin fibroblast type I collagen Synthesis

The content of the type I collagen in the supernatant of the human skin fibroblasts is detected, and the result shows that compared with the blank group, the content of the type I collagen in each drug treatment group except the artemisia apiacea essential oil group in the comparative example 2 and the white tea essential oil group in the comparative example 3 is lower than that in the blank group, and the content of the type I collagen in each group is improved, so that the effect of promoting the human skin fibroblasts to synthesize the type I collagen is shown, wherein the positive control group, the artemisia apiacea essential oil groups in the examples 1 to 3, the white tea essential oil groups in the examples 8 to 9 and the formula essential oil group are obviously higher than that in the blank group, and the difference is statistically different (P is less than 0.05 or P is less than 0.01). Comparing each essential oil treatment group with a positive control group, and investigating the capability of the essential oil for promoting human skin fibroblasts to synthesize type I collagen and the height of estrogen, the results show that the content of the type I collagen in the artemisia apiacea essential oil group, the white tea essential oil group and the formula essential oil group in the example 1 is obviously higher than that in the positive control group (P is less than 0.05 or P is less than 0.01), and the capability of promoting the synthesis of the type I collagen is better than that of the estrogen. Comparing the collagen I contents of the essential oil groups of the southernwood of the examples 1-3 with that of the essential oil group of the southernwood of the comparative example 1, the collagen I contents of the essential oil groups of the southernwood of the examples 1-3 are all found to be significantly higher than that of the essential oil group of the comparative example 1 (P < 0.05 or P < 0.01), comparing the collagen I contents of the essential oil groups of the southernwood of the examples 1-3 with that of the essential oil group of the comparative example 2, the collagen I contents of the essential oil groups of the southernwood of the examples 1-3 are all found to be significantly higher than that of the essential oil group of the comparative example 2 (P < 0.01), and comparing the essential oil groups of the white tea of the examples 8-9 with that of the essential oil group of the comparative example 3 with that of the comparative example 3, the collagen I contents are significantly higher than that of the essential oil group of the white tea of the comparative example 3 (P < 0.01). The results of comparing the synthesis of the white tea essential oil group of examples 8 to 9 with the white tea essential oil group I type collagen of the comparative example 4 show that the content of the white tea essential oil group I type collagen of the examples 8 to 9 is significantly higher than that of the white tea essential oil group of the comparative example 4 (P < 0.05 or P < 0.01). Compared with the I-type collagen content of other single essential oil groups, the I-type collagen content of the formula essential oil group is obviously higher than that of each single essential oil administration group, and the difference has statistical significance (P is less than 0.05 or P is less than 0.01).

TABLE 4 Effect of different essential oils on human skin fibroblast type I collagen Synthesis: (n＝6)

Note: p < 0.05, P < 0.01 compared to blank; compared with the positive control group, # P < 0.05, # P < 0.01; examples 1-3 essential oil of Artemisia annua vs. comparative example 1 essential oil of Artemisia annua, delta P < 0.05, delta P < 0.01; examples 1-3. the essential oil group vs. comparative example 2. the essential oil group a P is less than 0.05, a P is less than 0.01; examples 8-9 white tea essential oil set vs. comparative example 3 white tea essential oil set, & & P < 0.01; examples 8-9 white tea essential oil vs. comparative example 4 white tea essential oil, P < 0.05 and P < 0.01. Single essential oil group vs. formula essential oil group, @ P < 0.05 and @ P < 0.01.

Example 14 Effect of Artemisia Annua essential oil, white tea essential oil and formulation essential oil on epidermal stem cell proliferation

1 method of experiment

1.1 pharmaceutical formulation

Single essential oil: the sweet wormwood essential oil and the white tea essential oil are respectively dissolved by DMSO and diluted to 250 mu g/mL by complete culture medium.

The formula of the essential oil is as follows: the sweet wormwood essential oil and the white tea essential oil contain 400 mu L of 250 mu g/mL sweet wormwood essential oil and 100 mu L of 250 mu g/mL white tea essential oil per 1mL, and the rest is supplemented with a complete culture medium.

1.2 isolation and culture of epidermal Stem cells

Taking C57BL/6 suckling mouse, dislocating cervical vertebra, killing, peeling to obtain skin sheet, separating epidermis by trypsin and EDTA combined digestion method, preparing single cell suspension by using complete culture medium of epidermal stem cells, inoculating in cell culture bottle coated with matrigel, and culturing at 37 deg.C with 5% CO by volume fraction₂Culturing under the condition. Changing the solution 1 time every 2-3 days. Passages were performed when cells grew to 70% -80% confluence. Detection by immunofluorescenceExpression of integrin α 6 and CD71 was identified as epidermal stem cells.

1.3 CCK8 method for detecting influence of essential oil on proliferation of epidermal stem cells

Preparing cell suspension from epidermal stem cells of mouse transferred to 3 rd generation, adding complete culture medium of epidermal stem cells, and culturing at 4 × 10⁴The cells were inoculated at a density of 200. mu.l per well in 96-well plates pre-plated with matrigel and incubated for 24 h. The single-component essential oil dilution concentration is 250 mug/ml, the formula essential oil concentration is 125 mug/ml, and each group has 6 compound holes. A blank control group is set in the experiment, and the blank control group is added with the complete culture solution of the epidermal stem cells with corresponding volume. CCK8 was diluted with the culture medium at a ratio of 1:9, and the liquid in the 96-well plate was aspirated off, and 100. mu.l of the diluted CCK8 solution was added to each well. And (3) incubating in the incubator for 30min, taking out, and detecting the absorbance value of each hole at 570nm by using a microplate reader. And (5) calculating the cell proliferation rate. Cell proliferation rate (%) - (oil essential oil OD value-blank OD value)/blank OD value × 100%.

3 results of the experiment

The influence of the single-component essential oil and the formula essential oil on the proliferation of the epidermal stem cells is detected, and the results show that the cell proliferation rates of the single-component essential oil group and the formula essential oil group are obviously increased compared with the blank group, and the difference is statistically significant (P is less than 0.05 or P is less than 0.01). Comparing the capacity of single essential oil and the formula essential oil for promoting the proliferation of the epidermal stem cells of the mice, the cell proliferation rate of the formula essential oil group is found to be remarkably greater than that of each single essential oil group, and the difference has statistical significance (P is less than 0.05). The above results indicate that the treatment of the artemisia apiacea essential oil, the white tea essential oil and the formula essential oil group helps to activate and maintain the proliferative activity of the epidermal stem cells.

TABLE 5 Effect of different essential oils on ESF-1 cell proliferation: (n＝6)

Note: p < 0.05, P < 0.01 compared to blank; single essential oil group vs. group essential oil group, # P < 0.05.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.