Compound essential oil for repairing skin barrier and pharmaceutical composition containing compound essential oil

Abstract

The invention provides compound essential oil for repairing skin barrier, which is prepared from the following components: sandalwood essential oil, permanent flower essential oil, black red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and fructus forsythiae essential oil in a volume ratio of 2: 0.7: 1.8: 1.7: 3.7: 2: 2. The compound essential oil disclosed by the invention has an excellent effect of repairing a skin barrier. The invention also provides a pharmaceutical composition containing the compound essential oil.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a compound essential oil for repairing skin barrier and a pharmaceutical composition containing the compound essential oil. Background Art

[0002] Defective epidermal barrier function is the main feature of the most common inflammatory skin diseases. When the epidermal barrier is damaged, the amount of exogenous allergens and microorganisms that pass through the epidermis will increase, resulting in immune activation and subsequent inflammatory response. Therefore, barrier repair therapy is an important strategy for the treatment of chronic inflammatory skin diseases.

[0003] In clinical practice, the external treatment of barrier repair therapy mainly includes moisturizing with topical moisturizers and sealants, and anti-inflammatory, anti-proliferative and immunosuppressive drugs represented by topical glucocorticoids and calcineurin inhibitors. In recent years, plant-derived barrier repair topical medications have become increasingly popular with patients and the market. Plant-derived topical medications are usually extracted from natural plants. Compared with synthetic drugs, they have the advantages of safety, fewer side effects, milder on the skin, rarely causing irritation or allergic reactions, and being environmentally friendly and less polluting. At the same time, plant-derived topical medications are usually active ingredients extracted from plants, which have multiple biological activities such as anti-inflammatory, antibacterial, antioxidant, and moisturizing, and can promote wound healing, skin repair, and barrier function recovery. The combined use of different plant active ingredients often has a synergistic effect.

[0004] Essential oils are extracted from leaves, flowers, seeds, fruits, roots, bark, resins, wood cores and other parts of plants by steam distillation, cold pressing, liposorption, supercritical carbon dioxide extraction and other methods. Due to its natural origin, it is widely regarded as closer to nature than synthetic drugs and a healthier and more sustainable choice. Essential oils are complex mixtures of various chemical components, such as volatile oils, phenols, alcohols, esters, etc., which give essential oils multiple functions such as antibacterial, anti-inflammatory, antioxidant, analgesic, etc., and work together to produce an overall effect, making them widely used in skin care and other external applications.

[0005] Due to the existence of the epidermal barrier, how to enhance the permeability of drugs in the skin has also become an important research topic. Commonly used chemical penetration enhancers often cause skin irritation (local inflammation). The use of special carrier systems such as nanoparticles, electric field forces, lasers, ultrasound and microneedles provides more research space for transdermal drug delivery, but there are not many products that have been converted into clinical applications. Compounds with a molecular weight of less than 500 Daltons usually have good skin permeability, and most essential oils have a molecular weight of less than 250 Daltons, which can more easily penetrate the skin surface and penetrate into different layers of the skin, thereby achieving better absorption. Essential oil molecules are usually fat-soluble, which can better be compatible with the lipid layer of the skin and more easily penetrate into the deep layers of the skin to exert their effects. In addition, the essential oil ingredients themselves have the property of permeation enhancement, which can improve the permeability of other substances, promote the absorption of other active ingredients, and enhance their effects in the skin.

[0006] Single essential oils contain limited medicinal material basis, and their single efficacy, limited therapeutic range, odor stimulation and other factors have greatly restricted their application. Compound essential oils are compound essential oils made by mixing various essential oil components with certain efficacy or clear medicinal material basis in a certain proportion. They are derived from the classification and combination of single essential oils with certain efficacy. After laboratory safety, pharmacology and toxicology research and clinical efficacy verification, they can be classified as component Chinese medicine. Component Chinese medicine is a way to innovate Chinese medicine research. It is guided by traditional Chinese medicine theory, follows the theory and principles of prescription compatibility, and combines modern drug development methods and technologies to form Chinese medicine composed of effective components. The characteristics of component Chinese medicine are that the medicinal substances and action mechanisms are relatively clear. They can play a role from different angles and multiple aspects in the treatment of clinical diseases or symptoms, improve the efficacy and reduce adverse reactions, and increase the treatment range. In addition, for essential oils with bad smell but strong functionality, the smell can be reconciled through formulas to make them feel better. In short, compound essential oils can improve the synergistic pharmacological effects of single essential oils, and their application fields are constantly expanding and deepening. Summary of the invention

[0007] The object of the present invention is to provide a compound essential oil for repairing the skin barrier, wherein the compound essential oil achieves the effect of repairing the skin barrier by moisturizing, increasing ceramide expression and promoting cell migration.

[0008] The compound essential oil of the invention consists of the following components: sandalwood essential oil, helichrysum essential oil, dark red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and forsythia essential oil.

[0009] Preferably, in the compound essential oil of the present invention, the volume ratio of sandalwood essential oil, helichrysum essential oil, dark red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and forsythia essential oil is 2:0.7:1.8:1.7:3.7:2:2.

[0010] The present invention also provides a pharmaceutical composition for repairing skin barrier, comprising the above compound essential oil and physiologically or pharmaceutically acceptable auxiliary materials.

[0011] Preferably, the content of the compound essential oil in the pharmaceutical composition of the present invention is: 10 μg / mL to 500 μg / mL.

[0012] More preferably, the content of the compound essential oil in the pharmaceutical composition of the present invention is 50 μg / mL to 100 μg / mL.

[0013] The auxiliary materials can be selected from vaseline, solid paraffin, liquid paraffin, ozokerite, microcrystalline wax, saturated fatty acid esters, unsaturated fatty acid esters, antioxidants, dimethyl silicone oil, lanolin, cholesterol, lanosterol and other steroids, acetyl steroids or polybasic esters (such as span), methylcellulose, sodium carboxymethylcellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, gac, pectin, alginic acid, xanthan gum, guar gum, agar, tea polyphenols (TP), tocopherol, etc.

[0014] The present invention also provides a cosmetic composition, which comprises the compound essential oil and a cosmetic base.

[0015] The present invention also provides use of the compound essential oil in moisturizing and repairing skin barrier.

[0016] The prescription basis of the compound essential oil of the present invention is:

[0017] Sandalwood essential oil: The main ingredients are santalol and santalene; the main ingredients' functions: balancing endocrine glands, balancing immune function, skin care, and inhibiting tyrosinase.

[0018] Helichrysum essential oil: The main ingredients are diketone and neryl acetate; the main ingredients' effects: removing blood stasis, promoting cell regeneration, and anti-spasmodic.

[0019] Dark red rose essential oil: the main ingredients are geraniol, citronellol, phenylethyl alcohol, and rose wax; the main ingredients have the functions of: detoxifying the liver, strengthening the nervous and reproductive systems, cooling blood and dissipating heat, and anti-oxidation.

[0020] Jasmine essential oil: The main ingredients are methyl anthranilate, indole, benzyl acetate and jasmine ketone; the main ingredients' functions: improve physical fitness, regulate yin and yang, strengthen the uterus, aid childbirth, anti-aging and firming.

[0021] White tea essential oil: The main ingredients are linalool, geraniol, and alkyd; the main ingredients function: inhibit the activity of elastase, reduce the decomposition of elastic fibers, and are used for anti-aging; antioxidant and free radical scavenging.

[0022] Honeysuckle essential oil: The main components are linalool, geraniol, benzyl acetate, etc. The main components have the effects of anti-pathogenic microorganisms, anti-inflammatory, immunomodulatory, hypolipidemic, and central nervous system excitation.

[0023] Forsythia essential oil: The main components are monoterpenes and monoterpenoids; the main components have multiple biological activities such as antibacterial and anti-inflammatory, antioxidant, and effective scavenging of organic free radicals.

[0024] The prescription of the compound essential oil of the present invention (represented by numbering 2T herein) is based on the following:

[0025] White tea tastes sweet and bitter, is cold in nature, enters the lung meridian, governs the skin and fur, has the effects of clearing away heat and promoting body fluid, detoxifying and relieving itching, resisting oxidation, and delaying aging, and is the main medicine;

[0026] Dark red rose is warm in nature, sweet and slightly bitter in taste, and belongs to the liver meridian. It has the effects of regulating qi and relieving depression, promoting blood circulation and removing blood stasis, and reducing swelling and relieving pain. Helichrysum is pungent and bitter in taste, and belongs to the lung and spleen meridians. It can promote blood circulation and remove blood stasis, resist bacteria and reduce inflammation, help blood circulation, and promote repair. The two together play the role of promoting blood circulation, reducing inflammation, reducing swelling and relieving pain, and are combined as ministerial drugs.

[0027] Jasmine tastes spicy, slightly sweet, warm, and enters the spleen, stomach, and liver meridians. It can clear away internal heat, treat sores, harmonize the middle and lower qi, and calm the liver and regulate qi. Honeysuckle is the dried buds or flowers with just opened flowers of the honeysuckle plant of the Caprifoliaceae family. Honeysuckle has a fragrant smell, a light texture, and a dispersing nature. It is sweet and cold and enters the lungs and stomach. It has the function of clearing away superficial heat and various heats in the upper part of the body, and has the function of penetrating the blood and transforming qi. It is an important medicine for treating all internal and external abscesses caused by heat toxins. Forsythia suspensa is the dried buds of the forsythia suspensa plant of the Oleaceae family. The dry fruit is bitter and slightly cold in nature, and it enters the lung, heart, and small intestine meridians. It can disperse the wind-heat in the upper part of the body, penetrate the surface evil, and clear the heart fire and resolve the sore poison; honeysuckle essential oil, forsythia essential oil, and jasmine essential oil are used together as adjuvants, which can assist the ministerial medicine in clearing heat and detoxifying, and can also consolidate and protect the qi, dispel the surface evil, and promote the repair; sandalwood is pungent and warm in nature, and it enters the spleen, stomach, heart, and lung meridians. It has the effects of aromatic turbidity, pungent dispersing and warming, and is the adjuvant. All the medicines are used together to play the role of clearing heat and eliminating evil, regulating qi and reducing swelling, and promoting blood circulation and consolidating the surface.

[0028] Compared with each single essential oil and other compound essential oils, the compound essential oil 2T of the present invention has a larger safe concentration range for acting on cells, and is particularly effective in inhibiting hyaluronidase activity, promoting hydration gene expression, promoting cell proliferation, migration, and increasing the content of ceramide in the skin of mice with mechanical skin damage. In addition, under the premise of achieving the same effect, the dosage of each single essential oil in the compound essential oil 2T of the present invention is small, especially when the compound essential oil is put into use in mass production, the compound essential oil 2T of the present invention has the advantage of low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Effects of essential oils on the vitality of skin keratinocytes;

[0030] Figure 2 : Effects of essential oils on the vitality of skin keratinocytes;

[0031] Figure 3 : Effects of essential oils on the migration of skin keratinocytes;

[0032] Figure 4 : Linear relationship between hyaluronidase concentration and OD value;

[0033] Figure 5 :Effects of essential oils on ceramide content in skin of mice with mechanical barrier damage; Compared with the blank group, ***P<0.001; Compared with the model group, ###P<0.001, ####P<0.0001;

[0034] Figure 6 : GC-MS test results of sandalwood essential oil;

[0035] Figure 7 :The results of gas chromatography-mass spectrometry (GC-MS) of helichrysum essential oil;

[0036] Figure 8 : The results of gas chromatography-mass spectrometry (GC-MS) of black red rose essential oil;

[0037] Fig. 9 : GC-MS test results of jasmine essential oil;

[0038] Fig.10 : GC-MS test results of white tea essential oil;

[0039] Fig.11 : GC-MS test results of honeysuckle essential oil;

[0040] Fig.12 : Detection results of Forsythia suspensa essential oil by gas chromatography-mass spectrometry. DETAILED DESCRIPTION

[0041] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0042] The essential oils used in the following Examples 2-6 were purchased from Mujiuye (Shanghai) Technology Co., Ltd. or extracted by the method of Example 1.

[0043] Sandalwood essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO00381012305, enterprise standard: relative density (20℃): 0.893~0.951; refractive index (20℃): 1.470~1.478; the cosmetic raw material safety information reporting code for sandalwood essential oil is 006568-02571-5954.

[0044] Helichrysum essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO09211012302, enterprise standard: relative density (20℃): 0.889~0.950; refractive index (20℃): 1.450~1.480; the cosmetic raw material safety information reporting code for Helichrysum essential oil is 004210-02571-3660.

[0045] Dark red rose essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: SEO0411201202307, enterprise standard: relative density (20℃): 0.911-0.963; refractive index (20℃): 1.485-1.552; the cosmetic raw material safety information reporting code for dark red essential oil is 004636-02571-8941.

[0046] Jasmine essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO0861201303, enterprise standard: relative density (20℃): 0.925~0.998; refractive index (20℃): 1.481~1.525; the cosmetic raw material safety information reporting code of jasmine essential oil is 004723-02571-4822;

[0047] White tea essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: XYJY0037, enterprise standard: relative density (20℃): 0.875~0.950; refractive index (20℃): 1.470~1.495. The cosmetic raw material safety information reporting code for white tea essential oil is 001605-02571-4211;

[0048] Honeysuckle essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: XYJY0070, enterprise standard: relative density (20℃): 0.922-0.965; refractive index (20℃): 1.470-1.520. The cosmetic raw material safety information reporting code for honeysuckle essential oil is 005525-02571-6901;

[0049] Forsythia essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: JY0109, enterprise standard: relative density (20℃), 0.859-0.920; refractive index (20℃), 1.467-1.479. The cosmetic raw material safety information reporting code for Forsythia essential oil is 004290-02571-1083.

[0050] [Example 1] Preparation of unilateral essential oil

[0051] 1.1 Preparation of sandalwood essential oil

[0052] Sandalwood essential oil is a volatile oil obtained by steam distillation using sandalwood (Santalum album L.) as a raw material. The sandalwood essential oil is obtained after processes such as raw material harvesting, selecting qualified woody parts, water distillation, cooling the distillation product, oil-water separation, filtration, alcoholization, mixing, packaging, testing, and warehousing.

[0053] 1.2 Preparation of Helichrysum essential oil

[0054] Helichrysum essential oil is a volatile oil obtained by steam distillation using Helichrysum italicum (Roth) G.Don as a raw material, and the Helichrysum essential oil is obtained through processes such as raw material collection, selection of qualified flower parts, distillation, cooling of distillation products, oil-water separation, filtration, canning, alcoholization, mixing, subpackaging, testing, and storage.

[0055] 1.3 Preparation of dark red rose essential oil

[0056] The black red rose essential oil is obtained by using black red rose (Rosa chinese Jacq Crimson Glory) as a raw material, extracting crude oil from the flower part of the plant through solvent, and then extracting volatile oil through molecular distillation. The black red rose essential oil is obtained after raw material collection, selecting qualified flower parts, extracting crude oil through solvent, extracting volatile oil through molecular distillation, cooling the product, oil-water separation, filtering, canning, alcoholization, mixing, packaging, testing, warehousing and other processes;

[0057] 1.4 Preparation of jasmine essential oil

[0058] Jasmine essential oil is a volatile oil extracted from jasmine (Jasminum sambac (L.) Aiton) as a raw material by solvent extraction, and the jasmine essential oil is obtained after the processes of raw material harvesting, selecting qualified flower and leaf parts, extraction, cooling volatile oil, oil-water separation, filtration, canning, alcoholization, mixing, packaging, testing, warehousing, etc.;

[0059] 1.5 Preparation of white tea essential oil

[0060] The large-leaf white tea (Camellia sinensis (L.) O.Ktze) from Pu'er, Yunnan was used as the material.

[0061] At 6 to 9 in the morning, hand-pick one-heart and two-heart young leaves of equal size, and spread each young bud neatly. Dry them in the sun as if there is sunlight during the local daytime, and dry them in the shade indoors at night for 3 consecutive days, and then put them into the dryer to dry at 110℃. Then crush the white tea leaves through a 10-20 mesh sieve to make coarse powder, put the weighed tea coarse powder into a sandbag, and put it into the extractor, and seal the top cover. Turn on the power of the thermostatic bath. Open the valve of the carbon dioxide cylinder, so that the gas passes through the dryer along the pipeline, cools the condenser (immersed in an ice water bath), and the plunger pump, and enters the booster buffer bottle. When the pressure of the booster buffer bottle greatly exceeds the experimental pressure, open valve V1 to boost the extractor to the experimental pressure, close valve V1, let the tea pre-soak for 10 minutes, and extract with supercritical CO2 fluid. The CO2 fluid flow rate is 1L / min, the extraction temperature is 50℃, the extraction pressure is 25MPa, the separation temperature is 50℃, the separation pressure is 4.5-5.5MPa, and the extraction time is 1.5-2.5h to obtain white tea essential oil.

[0062] 1.6 Preparation of honeysuckle essential oil

[0063] Honeysuckle essential oil is a volatile oil obtained by the following supercritical CO2 extraction method.

[0064] First, crush the dried honeysuckle produced in Shandong according to 10-20 mesh, put the weighed medicinal powder into a sandbag, put it into the extractor, and seal the top cover. Turn on the power of the thermostatic bath. Open the valve of the carbon dioxide cylinder, let the gas pass through the dryer along the pipeline, cool the condenser (immersed in an ice water bath), and the plunger pump to enter the booster buffer bottle. After the pressure of the booster buffer bottle greatly exceeds the experimental pressure, open the valve to boost the extractor to the experimental pressure, close the valve, let the dried honeysuckle flowers pre-soak for 10 minutes, and extract with supercritical CO2 fluid. The CO2 fluid flow rate is 1L / min, the extraction temperature is 50℃, the extraction pressure is 25MPa, the separation temperature is 50℃, the separation pressure is 4.5-5.5MPa, and the extraction time is 2-2.5h to obtain honeysuckle essential oil paste. Using the honeysuckle essential oil paste as the raw material, supercritical CO2 extraction is performed again, the temperature remains unchanged, the pressure is changed to 40-45MPa, and the extraction time is 2-3h again. Use 95.0% ethanol to extract the honeysuckle essential oil paste and ethanol in a ratio of 1:9, filter to remove part of the wax, let the filtrate stand overnight, filter again the next day, and vacuum the filtrate at 50°C and a vacuum degree of -0.015MPa to extract the ethanol to obtain the honeysuckle essential oil.

[0065] 1.7 Preparation of Forsythia suspensa essential oil

[0066] Forsythia essential oil is a volatile oil obtained by the following steam distillation method.

[0067] The material is the fruit of Forsythia suspensa produced in Shanxi.

[0068] Wash the shell of Forsythia suspensa, drain the water, and place it in an electric constant temperature drying oven for drying. The temperature is controlled to be maintained at 60 degrees Celsius. Grind it into a 60-mesh sieve, wrap it with gauze, place it in the inner distillation tank, add an equal amount of distilled water, pour it into the outer distillation tank, and place the distillation tank on the water tank. Seal the outer distillation tank and distill it after soaking for 1.5 hours. The extraction temperature is 90℃, the condenser temperature is 15-25℃, and the distillation time is 1.5 hours. The essential oil pure dew mixture is obtained, which is separated by a separator to obtain Forsythia suspensa essential oil.

[0069] [Example 2] Preparation of compound essential oil

[0070] In this article, sandalwood essential oil, helichrysum essential oil, dark red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and forsythia essential oil are represented by codes 2A, 2B, 2C, 2D, 2E, 2X-1 and 2X-2 respectively.

[0071] 2A, 2B, 2C, 2D, 2E, 2X-1 and 2X-2 obtained in Examples 1.1 to 1.7 or purchased from Mu Jiuye (Shanghai) Technology Co., Ltd. are uniformly mixed together to obtain the compound essential oil 2T of the present invention. The compound essential oil 2T of the present invention is sandalwood essential oil + helichrysum essential oil + black red rose essential oil + jasmine essential oil + white tea essential oil + honeysuckle essential oil + forsythia essential oil, and the volume ratio is 2: 0.7: 1.8: 1.7: 3.7: 2: 2. That is, in 100ml of the compound essential oil 2T, the content of sandalwood essential oil is 14.38ml, the content of helichrysum essential oil is 5.03ml, the content of black red rose essential oil is 12.94ml, the content of jasmine essential oil is 12.23ml, the content of white tea essential oil is 26.61ml, the content of honeysuckle essential oil is 14.38ml, and the content of forsythia essential oil is 14.38ml.

[0072] 2A, 2B, 2C, 2D and 2E obtained in Examples 1.1 to 1.5 or purchased from Mu Jiuye (Shanghai) Technology Co., Ltd. were uniformly mixed together to obtain the control group compound essential oil 2F. Compound essential oil 2F is sandalwood essential oil + helichrysum essential oil + black red rose essential oil + jasmine essential oil + white tea essential oil, with a volume ratio of 2: 0.7: 1.8: 1.7: 3.7. That is, the content of sandalwood essential oil in 100ml of compound essential oil 2F is 20.2ml, the content of helichrysum essential oil is 7.07ml, the content of black red rose essential oil is 18.18ml, the content of jasmine essential oil is 17.17ml, and the content of white tea essential oil is 37.37ml.

[0073] [Example 3] Study on the moisturizing effect of essential oils on skin

[0074] 1. Experimental Materials

[0075] 3.1.1 Experimental animals

[0076] BALB / c mice, male, weighing 20-23 g, were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., license number: SCXK (Shanghai) 2018-0004

[0077] 3.1.2 Test drugs and reagents

[0078] Hyaluronidase (500U / mg), hyaluronic acid (HA), single essential oil, compound essential oil 2F, compound essential oil 2T, DNS staining solution, olive oil, DMSO, RT-PCR kit, primers.

[0079] 3.1.3 Instruments and equipment

[0080] ELISA analyzer, real-time fluorescence quantitative PCR instrument.

[0081] 2. Experimental Methods

[0082] 3.2.1 Study on the inhibition of hyaluronidase in vitro

[0083] ① DNS method to draw the enzyme activity standard curve of hyaluronidase and substrate HA reaction

[0084] Use deionized water (ddH2O) to prepare hyaluronidase to 2000U / ml. Prepare the reaction system according to Table 1, shake and mix, react at 37℃ for 30min, and boil in water for 5min. Take 200μl of the reaction solution, add 400μl DNS solution, shake and mix, boil in water for 5min, and read the OD value at 540nm with a microplate reader after cooling. Each sample has 3 replicates. Draw a standard curve based on the corresponding relationship between the OD value at A540 and the hyaluronidase concentration.

[0085] Table 1 Hyaluronidase standard solution and substrate HA reaction system

[0086]

[0087]

[0088] ② Preparation of reaction system for essential oil samples to be tested

[0089] The compound essential oil and the single essential oil were diluted with DMSO to form 200 mg / ml essential oil solutions, respectively, to prepare essential oil reaction system 1: 300 μl of 2000 U / ml hyaluronidase solution + 180 μl of ddH2O + 500 μl of 0.5% hyaluronic acid + 20 μl of essential oil sample, oscillated and mixed, reacted at 37°C for 30 min, and boiled in water for 5 min. The concentration of essential oil in this reaction system was 4 mg / ml; essential oil reaction system 2: 300 μl of 2000 U / ml hyaluronidase solution + 150 μl of ddH2O + 500 μl of 0.5% hyaluronic acid + 50 μl of essential oil sample, oscillated and mixed, reacted at 37°C for 30 min, and boiled in water for 5 min. The concentration of essential oil in this reaction system was 10 mg / ml.

[0090] ③ DNS method to determine the effect of essential oil samples on hyaluronidase activity

[0091] Take 200 μl of the prepared essential oil reaction system, add 400 μl of DNS solution, shake and mix, boil in water bath for 5 min, and read the OD value at 540 nm with a microplate reader after cooling. Each sample has 3 replicates. Calculate the hyaluronidase activity of the essential oil group according to the standard curve of hyaluronidase.

[0092] 3.2.2 Detection of mouse skin hydration-related gene expression (AQP3, FLG, LOR)

[0093] BALB / c mice were randomly divided into a blank group (olive oil), a positive group (1% hyaluronic acid), and an essential oil group (each essential oil was diluted to 10% with olive oil), with 3 mice in each group. Back hair removal was performed one day before the experiment, and the hair removal area was 2cm*2cm. The blank group was smeared with olive oil, the positive group was smeared with 1% hyaluronic acid solution, and each essential oil group was given 10% of different single essential oils and compound essential oils. Each mouse was given 100μl of solution once a day. After 7 consecutive days of application, the mice were anesthetized and about 100mg of skin was taken for detection of hydration-related genes AQP3, FLG, and LOR.

[0094] Total RNA from the back skin of mice was extracted and reverse transcribed according to the kit instructions. According to the gene sequence, the synthetic primers were designed as follows. AQP3: upstream primer 5'-CTCACCATCAAC TTGGCTTTTG-3', downstream primer 5'-CATCGTAGTACAGCCCAAAAAC-3'; FLG: upstream primer 5'-AGACTGGGAGGCAAGCTACA-3', downstream primer 5'-GAGATGGTTTG GAGTGGGATT-3'; LOR: upstream primer 5'-TTACTCCTCTCAGCAGACCAGTCAG-3', downstream primer 5'-CCTCCACAGCTACCACCTCCTC-3'. Real-time quantitative PCR reaction was performed according to the conditions of 95℃30s, 95℃5s, 60℃30s, 95℃15s, 60℃1min, 95℃15s, for a total of 45 cycles. Each index was repeated 3 times, and the average Ct value was taken. GAPDH was used as the internal reference gene, and the relative quantification method was used. The relative expression content of the target gene = 2 -△△Ct .

[0095] 3. Experimental Results

[0096] 3.3.1 In vitro hyaluronidase inhibition

[0097] Hyaluronic acid (HA) is a polysaccharide substance and the main component of the interstitial and extracellular matrix of skin cells. Its unique ability to bind and retain water molecules in the dermis and epidermis determines the hydration of the skin. HA is extremely hydrophilic and can absorb a large number of water molecules to form a layer of moisture film, effectively locking the moisture of the skin and having good moisturizing properties. Hyaluronic acid synthase synthesizes HA, and hyaluronidase degrades HA. The greater the inhibition rate of hyaluronidase, the weaker the degradation effect on HA, and the higher the HA content, the more obvious the moisturizing effect on the skin.

[0098] Draw a standard curve based on the corresponding relationship between hyaluronidase concentration and OD value, see Figure 4 The hyaluronidase solution has a good linear relationship with the OD value in the range of 400U / ml-1000U / ml, and the linear equation is: y=0.0008x-0.1418, R 2 =0.995. The hyaluronidase concentration in the essential oil reaction system was calculated according to the linear regression equation of the standard curve, and the effect of essential oil on enzyme activity was evaluated.

[0099] The results are shown in Table 2.

[0100] Table 2 Essential oil in vitro hyaluronidase inhibition experiment

[0101]

[0102] 0* means no inhibitory effect on hyaluronidase.

[0103] According to the results of Table 2, the compound essential oil 2F has no effect on the hyaluronidase activity at the concentrations of 4 mg / mL and 10 mg / mL; while the inhibition rates of the compound essential oil 2T of the present invention on the hyaluronidase activity at the concentrations of 4 mg / mL and 10 mg / mL are 12.22% and 9.28%, respectively. The above results show that the compound essential oil 2T can reduce the degradation of hyaluronic acid by inhibiting the activity of hyaluronic acid, ensure the content of hyaluronic acid, and indicate that it has a certain moisturizing effect.

[0104] 3.3.2 Effects of essential oils on the expression of genes related to skin hydration in mice

[0105] Hydration-related genes such as AQP3 (Aquaporin-3) are the most abundant subtypes of the aquaporin family in human skin, which can enhance the hydration of the stratum corneum. FLG (Filaggrin) can bind to the intermediate filaments of keratin to provide a skeleton for epidermal cells. LOR (loricrin) is mainly located between the granular layer and the stratum corneum, constituting 70% of the total amount of epidermal keratin. FLG and LOR are the main proteins that maintain the skin barrier function. The loss of both will lead to skin barrier damage, skin hydration dysfunction or water loss in cells.

[0106] The mouse skin was treated with essential oils once a day for 7 consecutive days. RT-PCR was used to detect the expression of hydration-related genes (AQP3, FLG, LOR) in skin tissue. The results are shown in Table 3.

[0107] Table 3 Effects of essential oils on the expression of genes related to skin hydration (Mean±SD, n=3)

[0108]

[0109] Note: Compared with the blank group: *P<0.05, **P<0.01, ***P<0.001; compared with the positive group: #P<0.05, ##P<0.01, ###P<0.001.

[0110] Compared with the blank group, the control group compound essential oil 2F and the compound essential oil 2T of the present invention can significantly promote the expression of FLG and LOR genes (P<0.001), and the compound essential oil 2T of the present invention has a more significant effect on promoting the expression of hydration genes FLG and LOR relative to the positive control group and compound essential oil 2F: 35.77±3.55 vs 20.96±4.14 (the expression amount of the essential oil group is 29 times vs 6 times of the blank group), 29.25±0.42 vs 4.56±0.52 (the expression amount of the essential oil group is 35.7 times vs 17 times of the blank group). In addition, the compound essential oil 2T of the present invention can also promote the expression of AQP3 gene (the expression amount of the essential oil group is 2.85 times of the blank group, P<0.001). From the above data, it can be seen that the compound essential oil 2T of the present invention has an outstanding effect on promoting the expression of hydration-related genes.

[0111] [Example 4] Study on the effect of essential oils on skin barrier repair

[0112] 1. Experimental Materials

[0113] 4.1.1 Experimental cells

[0114] Human immortalized keratinocyte cell line HaCaT cells

[0115] 4.1.2 Experimental animals

[0116] BALB / c mice, male, weighing 20-23 g, were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., license number: SCXK (Shanghai) 2018-0004

[0117] 4.1.3 Test drugs and reagents

[0118] Compound essential oil 2F, compound essential oil 2T, each single essential oil, olive oil, ceramide kit, DMSO, DMEM high glucose medium, trypsin, fetal bovine serum, PBS buffer, CCK8 detection kit 4.1.4 Instruments and equipment

[0119] ELISA analyzer, CO2 cell culture incubator, Zeiss microscope

[0120] 2. Experimental Methods

[0121] 4.2.1 Essential oil preparation

[0122] 4.2.1.1 Preparation of single essential oils in cell experiments: Take 1 ml of each single essential oil obtained in Example 1 and 1 ml of DMSO and vortex for 2-3 min to mix evenly. Take 200 mg of the above essential oil solution and add it to DMEM culture medium to a volume of 10 mL, and vortex to mix evenly to obtain each essential oil stock solution (20 mg / ml). The culture medium containing essential oils in cell experiments was prepared by diluting the above essential oil stock solution (20 mg / ml) in proportion.

[0123] 4.2.1.2 Preparation of compound essential oil in cell experiment: Take 1 ml of the compound essential oil obtained in Example 2 and 1 ml of DMSO and vortex for 2-3 min to mix evenly, take 200 mg of the above essential oil solution and add DMEM culture medium to a volume of 10 mL, vortex and mix to obtain each essential oil stock solution (20 mg / mL). The composition of 1 ml of compound essential oil 2F is 0.20 ml of sandalwood essential oil, 0.71 ml of helichrysum essential oil, 0.18 ml of black red rose essential oil, 0.17 ml of jasmine essential oil and 0.37 ml of white tea essential oil; the composition of 1 ml of compound essential oil 2T is 0.14 ml of sandalwood essential oil, 0.05 ml of helichrysum essential oil, 0.13 ml of black red rose essential oil, 0.12 ml of jasmine essential oil, 0.27 ml of white tea essential oil, 0.14 ml of honeysuckle essential oil and 0.14 ml of forsythia essential oil. The culture medium containing essential oils in the cell experiments was prepared by diluting the above essential oil stock solution (20 mg / ml) in proportion.

[0124] 4.2.1.3 Preparation of compound essential oil for animal experiments: Take 1 ml of the compound essential oil obtained in Example 2 and 1 ml of olive oil, vortex and mix them evenly for 2 to 3 minutes to obtain 50% concentration essential oil; take 1 ml of the compound essential oil obtained in Example 2 and 3 ml of olive oil, vortex and mix them evenly for 2 to 3 minutes to obtain 25% concentration essential oil.

[0125] 4.2.2 HaCaT cell culture

[0126] HaCaT cell line was cultured in a 37°C cell culture incubator using DMEM medium containing 10% FBS, and the medium was replaced every 24 hours. The cells were passaged to 80% and maintained in logarithmic growth. After the cells were passaged to the third generation and in a stable state, they were used for experiments.

[0127] 4.2.3 Detection of the effect of essential oils on keratinocyte proliferation

[0128] HaCaT cells were cultured at 2×10 4 / well was inoculated in a 96-well plate and cultured in a cell culture incubator. When the cell density was about 80%, different concentrations of essential oils were added and cultured for 24 hours (the concentration gradient was 25, 50, 100, 250, 500 μg / ml), and 3 replicate wells were set up in each group. After that, the culture medium in the 96-well plate was removed, and 100 μL of culture medium containing 10% CCK8 test solution was added to each well. After incubation in the incubator for 1 hour, it was taken out. After continuing to culture in the incubator for 30 minutes, it was taken out, and the absorbance value of each well was detected at 450nm with an enzyme marker to calculate the cell survival rate. Cell survival rate (%) = (OD value of essential oil group-OD value of PBS well) / (OD value of blank control group-OD value of PBS well) × 100%.

[0129] 4.2.4 Detection of the effect of essential oils on cell scratch healing

[0130] HaCaT cells were cultured at 4 × 10 4 / well were inoculated in the cell insert, cultured in a cell culture incubator, and when the cell density was about 80%, the cell insert and the liquid in the 6-well plate were removed, and 2 mL of samples of different concentrations were added, and co-cultured for 24 h (concentration gradient was 25 and 50 μg / ml), and photos were taken at 0 h and 24 h, and the cell scratch area and corresponding migration rate were calculated using ImageJ. Migration rate = (0 h scratch area - 24 h scratch area) / 0 h scratch area.

[0131] 4.2.5 Detection of the skin repair effect of essential oils on mice with skin mechanical barrier disorders

[0132] The skin mechanical barrier damage model was established by the tape method. The specific method is as follows: the medical tape was attached to the shaved mouse back skin and then quickly torn off. Each mouse was stimulated twice a day, and the "tape sticking-tearing" process was repeated 5 times each time. The interval between the two modeling stimulations was 8 hours, and the modeling process lasted for 4 days. The modeling was successful when the mouse skin was red but without bleeding. After that, the mice in each group were given different concentrations of compound essential oils applied to the damaged parts for 4 consecutive days. The intervention group was given essential oils of various concentrations, and the model group was given olive oil.

[0133] 4.2.6 Ceramide content detection

[0134] After the last administration, the mice were anesthetized and the damaged skin tissue was taken out to prepare homogenate, and the content of ceramide in the mouse skin tissue was detected according to the instructions of the kit.

[0135] 3. Experimental Results

[0136] 4.3.1 Effect of essential oils on HaCaT cell survival

[0137] The CCK8 method was used to detect the effects of different concentrations of single essential oils, the control group compound essential oil 2F and the compound essential oil 2T of the present invention on the cell survival rate of skin keratinocytes HaCaT for 24 hours. Figure 1 and Figure 2 .from Figure 1 It can be seen that the compound essential oil 2T of the present invention promotes cell proliferation in a dose-dependent manner within the concentration range of 25-100 μg / ml, and the control group compound essential oil 2F can promote cell proliferation to varying degrees at concentrations of 25 and 50 μg / ml; at 100 μg / ml, it produces cytotoxicity to cells and significantly inhibits cell survival rate. In comparison, the safe concentration range of the compound essential oil 2T of the present invention (0-100 μg / ml) is larger than the safe concentration range of the compound essential oil 2F (0-50 μg / ml).

[0138] 4.3.2 Effects of essential oils on keratinocyte migration

[0139] The cell scratch test is a simple and quick method to measure cell migration and repair ability, similar to the in vitro wound healing model. Detecting cell migration rate can reflect the barrier repair ability of cells. The greater the migration rate, the stronger the barrier repair ability. The cell scratch test was used to detect the effect of compound essential oil on HaCaT cell migration. The results are as follows Figure 3 And as shown in Table 4.

[0140] Table 4-1 Effects of essential oils on the migration rate of skin keratinocytes

[0141]

[0142]

[0143] Table 4-2 Effects of essential oils on the migration rate of skin keratinocytes

[0144]

[0145] 24h after scratching, the cell migration rate of the cells treated with 25μg / ml and 50μg / ml of the compound essential oil 2T of the present invention was significantly increased compared with the blank group (42.02% vs 13.17%; 32.10% vs 13.17%), while the cell migration rate of the control group compound essential oil 2F at the same concentration was significantly weaker than that of the blank group (25μg / ml, 29.98% vs 13.17%; 50μg / ml, 17.64% vs 13.17%). The ability of compound essential oil 2T to promote cell migration is significantly better than that of compound essential oil 2F (42.02% vs 29.98%; 32.10% vs 17.64%), indicating that the ability of compound essential oil 2T of the present invention to promote scratch repair is stronger than that of compound essential oil 2F.

[0146] 4.3.3 Effects of essential oils on ceramide content in the skin of mice with skin mechanical barrier disorder

[0147] The stratum corneum plays an important role in the skin barrier function. The lipids between cells make the keratinocytes tightly bound together like shingles, which can prevent water loss and the invasion of external pathogens. Ceramide accounts for about 50% of the intercellular lipids and has the effects of moisturizing, anti-inflammatory, and promoting the repair of skin barrier function. If ceramide is removed from the epidermis, the barrier function is almost lost. Ceramide is one of the key substances that reflect the skin barrier function. The more ceramide content, the better the skin barrier function; the less ceramide, the worse the skin barrier function.

[0148] The skin of each group was taken to prepare homogenate, and the ceramide content in the skin homogenate was detected by ELISA. Figure 5And as shown in Table 5.

[0149] Table 5 Effects of essential oils on ceramide content in mice (Mean±SD, n=6)

[0150]

[0151] Note: Compared with the blank group, ***P<0.001; compared with the model group, ###P<0.001, ####P<0.0001

[0152] Compared with the blank group mice, the ceramide content in the skin of the model group mice decreased significantly (P<0.001); compared with the model group, the ceramide content of the 25% control group compound essential oil 2F (0.24648±0.04098vs 0.1433±0.0228), 25% compound essential oil 2T of the present invention (0.27556±0.05696vs 0.1433±0.0228) and 50% compound essential oil 2T of the present invention (0.30311±0.03972vs 0.1433±0.0228) groups increased significantly (P<0.001, P<0.0001); compared with the model group, the ceramide content of the 50% 2F group had an increasing trend but no statistical difference. The above results show that the compound essential oil 2T of the present invention can significantly increase the ceramide content of the skin, and its effect is significantly better than 2F.

[0153] [Example 5] 7 kinds of single essential oils of the present invention were detected by gas chromatography-mass spectrometry

[0154] 5.1 Preparation of test samples

[0155] Pipette 0.5 mL of the essential oil to be tested into a 1.5-mL centrifuge tube and centrifuge for 1 min; take 50 μL of the supernatant, weigh it, add ethyl acetate to prepare an essential oil solution with a concentration of 100 mg / mL; add anhydrous sodium sulfate powder at a mass ratio of 1:10, mix well, let stand, and centrifuge; take 10 μL of the supernatant, dilute it 100 times with ethyl acetate to obtain a solution to be tested with a concentration of 1 mg / mL; centrifuge the solution to be tested for 1 min, take the supernatant, and detect it by gas chromatography-mass spectrometry.

[0156] 5.2 Detection Method

[0157] Chromatographic column: Agilent HP-5MS UI (30m×0.25mm×0.25μm)

[0158] Carrier gas: high purity helium

[0159] Flow rate: 1.0mL / min

[0160] Inlet temperature: 250℃

[0161] Split ratio: 10:1

[0162] Injection volume: 1 μL

[0163] Heating program: 50℃—180℃ (3℃ / min)—300℃ (20℃ / min, hold for 5min)

[0164] Ion source: EI+

[0165] Ion source temperature: 230°C

[0166] Scan mode: full ion scan

[0167] Scan range: m / z 40 -600

[0168] Solvent delay: 4min

[0169] 5.3 Test results

[0170] Test results such as Figures 6 to 12 The raw data of the GC-MS detection were analyzed by Agilent MassHunterQualitative Analysis (VB.07.00) software to obtain the peak information of each component (retention time, integrated peak area, total peak area percentage, and peak height).

Claims

1. A compound essential oil for repairing skin barrier, characterized in that: The compound essential oil comprises the following ingredients: sandalwood essential oil, helichrysum essential oil, dark red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and forsythia essential oil.

2. The compound essential oil according to claim 1, characterized in that The volume ratio of sandalwood essential oil, helichrysum essential oil, dark red rose essential oil, jasmine essential oil, white tea essential oil, honeysuckle essential oil and forsythia essential oil in the compound essential oil is 2:0.7:1.8:1.7:3.7:2:

2.

3. The compound essential oil according to claim 1, characterized in that The compound essential oil has the effects of inhibiting hyaluronidase activity, promoting hydration gene expression, promoting cell proliferation and migration, and increasing the ceramide content in the skin.

4. A pharmaceutical composition for repairing skin barrier, characterized in that: The pharmaceutical composition comprises the compound essential oil according to any one of claims 1 to 3 and physiologically or pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The content of the compound essential oil in the pharmaceutical composition is 10 μg / mL to 500 μg / mL.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is an ointment, an emulsion or a cream.

7. Use of the compound essential oil according to any one of claims 1 to 3 in the preparation of a medicament for repairing the skin barrier.

8. A cosmetic composition, characterized in that The cosmetic composition comprises the compound essential oil according to any one of claims 1 to 3 and a cosmetic base.

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