Perilla essential oil, and preparation method and application thereof

Abstract

The present application provides a perilla essential oil and a preparation method and application thereof. The perilla essential oil contains the following components in terms of relative percentage: perilla aldehyde 46-50%, limonene 30-35%, trans-caryophyllene 8-12%, linalool 3-5%, (3Z,6E)-alpha-farnesene 2-5%, and trans-4-(1-methylethenyl)-cyclohexanemethanol 1-4%. The comprehensive performance of the perilla essential oil is significantly better than that of crude perilla essential oil or refined perilla essential oil containing other components, and the perilla essential oil has excellent effects in promoting human epidermal cell migration, promoting tight junction protein expression, promoting LOR expression, and maintaining the homeostasis of skin secreted ceramide, cholesterol and free fatty acid. Compared with previous products for skin problems related thereto, the perilla essential oil in the present application is natural and safe, and the cost is controllable, which can essentially solve the skin problems.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a perilla essential oil, its preparation method, and its application. Background Technology

[0002] Perilla (Perilla frutescens) is an annual herb belonging to the genus Perilla in the family Lamiaceae, and is a traditional medicinal plant. Perilla leaves contain abundant volatile components, and perilla essential oil can be obtained through methods such as steam distillation. Current research indicates that perilla essential oil contains various terpenoids such as perillaldehyde and limonene, possessing diverse biological activities including antioxidant and antibacterial properties, and shows broad application prospects in cosmetics, aromatherapy, and medicine.

[0003] The chemical composition of plant essential oils directly affects their bioactivity and application value. However, the composition of crude perilla essential oil obtained by traditional steam distillation is affected by various factors such as the origin of the raw material, the harvest season, and the distillation conditions, resulting in poor batch-to-batch stability. Furthermore, it contains some low-boiling-point impurities or irritating components, limiting its application in high-quality products. How to purify and refine perilla essential oil to obtain essential oil preparations with stable components and enhanced activity has become a pressing technical problem in this field. In addition, existing research on the application of perilla essential oil mainly focuses on its crude extract; the relationship between the composition and bioactivity of refined perilla essential oil preparations lacks in-depth exploration. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a perilla essential oil, its preparation method, and its applications. The preparation method of the present invention yields a perilla essential oil with a stable composition and significantly enhanced bioactivity.

[0005] To achieve the above objectives, the present invention provides a perilla essential oil, which, by relative percentage, comprises the following components: perillaldehyde 46-50%, limonene 30-35%, trans-caryophyllene 8-12%, linalool 3-5%, (3Z,6E)-α-farnesene 2-5%, and trans-4-(1-methylvinyl)-cyclohexamethanol 1-4%.

[0006] According to a specific embodiment of the present invention, the present invention uses GC-MS (gas chromatography-mass spectrometry) analysis to obtain a total ion chromatogram (TIC) under set chromatographic and mass spectrometric conditions. Each chromatographic peak corresponds to a volatile component, and the peak area of ​​each component is obtained by integration. The mass spectrum corresponding to each chromatographic peak is searched and compared with the NIST14 database to confirm the chemical name of each component.

[0007] According to a specific embodiment of the present invention, the peak area normalization method is used to obtain the relative percentage content of a certain component in perilla essential oil; the calculation formula is: relative percentage content of a certain component = (peak area of ​​the component / sum of peak areas of all components) × 100%.

[0008] In the above preparation method, preferably, the perilla essential oil contains the following components in relative percentage: perillaldehyde 46-47%, limonene 30-33%, trans-caryophyllene 8-10%, linalool 3-4%, (3Z,6E)-α-farnesene 2-3%, and trans-4-(1-methylvinyl)-cyclohexamethanol 1.5-3%.

[0009] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46-46.5%, limonene 31-32%, trans-caryophyllene 8.5-9.5%, linalool 3.2-3.8%, (3Z,6E)-α-farnesene 2.2-2.5%, and trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.5-2%.

[0010] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46-50%, limonene 30-35%, trans-caryophyllene 8-12%, linalool 3-5%, (3Z,6E)-α-farnesene 2-5%, trans-4-(1-methylvinyl)-cyclohexamethanol 1-4%, α-pinene 0.5-2%, β-pinene 0.5-2%, and α-ruthene 0.5-2%.

[0011] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46-47%, limonene 30-33%, trans-caryophyllene 8-10%, linalool 3-4%, (3Z,6E)-α-farnesene 2-3%, trans-4-(1-methylvinyl)-cyclohexamethanol 1.5-3%, α-pinene 0.5-2%, β-pinene 0.5-2%, and α-ruthene 0.5-2%.

[0012] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46-46.5%, limonene 31-32%, trans-caryophyllene 8.5-9.5%, linalool 3.2-3.8%, (3Z,6E)-α-farnesene 2.2-2.5%, trans-4-(1-methylvinyl)-cyclohexamethanol 1.5-2%, α-pinene 0.5-1%, β-pinene 0.5-1%, and α-ruthene 0.5-1%.

[0013] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46.17%, limonene 31.68%, trans-caryophyllene 9.03%, linalool 3.63%, (3Z,6E)-α-farnesene 2.38%, and trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.787%.

[0014] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: perillaldehyde 46.17%, limonene 31.68%, trans-caryophyllene 9.03%, linalool 3.63%, (3Z,6E)-α-farnesene 2.38%, trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.787%, α-pinene 0.8598%, β-pinene 0.8742%, and α-ruthene 0.7308%.

[0015] In some specific embodiments, preferably, the perilla essential oil comprises, by relative percentage, the following components: α-pinene 0.5-1%, 3-thujone 0.01-0.03%, β-pinene 0.5-1%, sapinene 0.1-0.3%, β-myrcene 0.1-1%, limonene 30-33%, β-phellandrene 0.05-0.1%, eucalyptol 0.1-0.2%, γ-terpinene 0.01-0.03%, terpinene 0.1-0.3%, 6-methyl-5-hepten-2-one 0.01-0.03%, and 1-octen-3-ol 0.01-0.1%. Linalool 3-4%, β-elemene 0.02-0.05%, trans-caryophyllene 8-10%, 4-terpinenol 0.02-0.06%, α-rutane 0.5-1%, cis-citral 0.05-0.3%, α-terpineol 0.1-0.5%, dextrorotatory geraniol 0.05-0.3%, (3Z,6E)-α-farnesene 2-3%, perillaldehyde 46-47%, nerol 0.05-0.3%, trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.5-3%, caryophyllein 0.05-0.2%, perillol 0.1-0.3%.

[0016] In some specific embodiments, preferably, the perilla essential oil comprises the following components in relative percentage: α-pinene 0.8598%, 3-thujone 0.024%, β-pinene 0.8742%, sapinene 0.217%, β-myrcene 0.467%, limonene 31.6766%, β-phellandrene 0.0793%, eucalyptol 0.1708%, γ-terpinene 0.0233%, terpinene 0.14%, 6-methyl-5-hepten-2-one 0.0217%, 1-octen-3-ol 0.0876%, and linalool 3%. 6329%, β-elemene 0.0349%, trans-caryophyllene 9.0303%, 4-terpinenol 0.0466%, α-rutane 0.7308%, cis-citral 0.1265%, α-terpineol 0.2257%, dextrorotatory geraniol 0.1164%, (3Z,6E)-α-farnesene 2.377%, perillaldehyde 46.1743%, nerol 0.1086%, trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.7848%, caryophyllein 0.0863%, perillol 0.2109%.

[0017] The perilla essential oil of the present invention can be used directly after dilution or added to other formulas to exert its effects.

[0018] On the other hand, the present invention also provides a method for preparing perilla essential oil, comprising the following steps: (1) Perilla leaves were steam distilled to obtain crude perilla essential oil; (2) Molecular distillation is performed on the crude perilla essential oil to obtain a light component, namely refined perilla essential oil; The extraction conditions for molecular distillation include: vacuum pressure of 90-100 mbar, heating temperature of 85-95℃, injection flow rate of 20-25 drops / min, rotation speed of 120-140 rpm, and condensation temperature of 4-10℃.

[0019] According to a specific embodiment of the present invention, preferably, the separation conditions of the molecular distillation include: vacuum pressure 98-100 mbar, heating temperature 89-90℃, injection flow rate 23-24 drops / min, rotation speed 130-135 rpm, and condensation temperature 5-8℃.

[0020] According to a specific embodiment of the present invention, preferably, the molecular distillation is performed using a molecular distillation apparatus, such as the KDL2 short-path thin-film distillation apparatus (UIC GmbH, Germany).

[0021] According to a specific embodiment of the present invention, preferably, the steam distillation process includes: mixing perilla leaves with water, distilling, condensing and refluxing, and collecting crude perilla essential oil.

[0022] According to a specific embodiment of the present invention, preferably, during the steam distillation process, the mass ratio of the perilla leaves to water is 1:2-5.

[0023] According to a specific embodiment of the present invention, preferably, the steam distillation temperature is 180-220°C.

[0024] According to a specific embodiment of the present invention, preferably, the crude perilla essential oil comprises the following components in relative percentage: perillaldehyde 44-46%, limonene 28-29%, trans-caryophyllene 10-12%, (3Z,6E)-α-farnesene 4-5%, linalool 2-4%, and trans-4-(1-methylvinyl)-cyclohexamethanol 1.5-3%.

[0025] In some specific embodiments, preferably, the crude perilla essential oil comprises the following components in relative percentage: perillaldehyde 44-46%, limonene 28-29%, trans-caryophyllene 10-12%, (3Z,6E)-α-farnesene 4-5%, linalool 2-4%, trans-4-(1-methylvinyl)-cyclohexamethanol 1.5-3%, α-pinene 0.5-2%, β-pinene 0.5-2%, and α-ruthene 0.5-2%.

[0026] In some specific embodiments, preferably, the crude perilla essential oil comprises the following components in relative percentage: perillaldehyde 45.89%, limonene 28.42%, trans-caryophyllene 10.32%, (3Z,6E)-α-farnesene 4.51%, linalool 2.99%, and trans-4-(1-methylvinyl)-cyclohexyl alcohol 2.01%.

[0027] In some specific embodiments, preferably, the crude perilla essential oil comprises, in relative percentages, the following components: perillaldehyde 45.89%, limonene 28.42%, trans-caryophyllene 10.32%, (3Z,6E)-α-farnesene 4.51%, linalool 2.99%, trans-4-(1-methylvinyl)-cyclohexylethanol 2.01%, α-pinene 0.7607%, β-pinene 0.7588%, and α-ruthene 0.9787%.

[0028] In some specific embodiments, preferably, the crude perilla essential oil comprises, by relative percentage, the following components: α-pinene 0.7607%, 3-thujone 0.0204%, β-pinene 0.7588%, sapinene 0.1868%, β-myrcene 0.3987%, limonene 28.4202%, β-phellandrene 0.0692%, eucalyptol 0.1438%, γ-terpinene 0.0216%, terpinene 0.1205%, 1-octen-3-ol 0.0739%, elemene isomer 0.0272%, linalool 2.9863%, β-elemene 0.0513%, and trans-caryophyllene 10%. 0.3245%, 4-terpinenol 0.0399%, α-terpinene 0.9787%, trans-β-farnesene 0.024%, cis-citral 0.1106%, α-terpineol 0.1987%, dextrorotatory geraniol 0.185%, (3Z,6E)-α-farnesene 4.5098%, α-farnesene 0.1036%, perillaldehyde 45.8884%, nerol 0.1022%, trans-4-(1-methylvinyl)-cyclohexyl alcohol 2.0114%, caryophyllein 0.3269%, perillol 0.2793%, nerol 0.0985%, eucalyptol 0.0637%.

[0029] On the other hand, the present invention also provides a perilla essential oil preparation, wherein the perilla essential oil preparation comprises the above-mentioned perilla essential oil. And physiologically or pharmaceutically acceptable excipients; The concentration of perilla essential oil in the perilla essential oil preparation is 5 μg / mL-100 μg / mL.

[0030] According to a specific embodiment of the present invention, preferably, the concentration of perilla essential oil in the perilla essential oil preparation is 5 μg / mL-50 μg / mL; more preferably, it is 5 μg / mL-10 μg / mL, 20 μg / mL-30 μg / mL, or 40 μg / mL-50 μg / mL; and even more preferably, the concentration of perilla essential oil in the perilla essential oil preparation is 5 μg / mL, 25 μg / mL, or 50 μg / mL.

[0031] On the other hand, the present invention also provides the use of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of products for promoting human epidermal cell migration.

[0032] The perilla essential oil of the present invention has the ability to promote cell migration, promote skin wound closure, promote epidermal reconstruction, and prevent the formation of persistent skin wounds.

[0033] On the other hand, the present invention also provides the use of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of products for promoting tight junction protein expression.

[0034] According to a specific embodiment of the present invention, preferably, the tight junction protein is Claudin-1. Claudin-1 is a core transmembrane protein of the tight junctions in the granular layer of the epidermis, and its core function is to regulate substance permeability and participate in immune homeostasis. The perilla essential oil of the present invention can promote the expression of the skin tight junction protein Claudin-1 and maintain the stability of skin function.

[0035] On the other hand, the present invention also provides the application of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of products for promoting the expression of loricrin (LOR) in the skin. Loricrin (LOR) is a core structural protein of the keratinized capsule (CE) of the skin, accounting for approximately 60%-80% of the total CE. It can maintain the mechanical strength, water retention capacity, and resistance to external aggressors of the skin. The perilla essential oil of the present invention can significantly promote the production of loricrin, improve the skin's water retention capacity, and maintain the stability of skin function.

[0036] On the other hand, the present invention also provides the use of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of products for maintaining the homeostasis of skin ceramides, cholesterol and free fatty acids.

[0037] Ceramides are a core component of the intercellular lipids in the stratum corneum of the skin (accounting for approximately 50%), and are key lipid molecules for maintaining normal skin function and water retention. Insufficient ceramide synthesis or abnormal ceramide structure can lead to disordered intercellular lipid structure, increased TEWL (transient endothelial-weighted oil), dry and sensitive skin, and easily induce related diseases such as atopic dermatitis, eczema, and asteatotic eczema. The perilla essential oil of this invention can significantly promote the production of ceramides, avoiding the risk of related diseases induced by reduced ceramide expression levels.

[0038] Cholesterol is a crucial component of the intercellular lipids in the stratum corneum of the skin (accounting for approximately 25%), and together with ceramides and free fatty acids, it constitutes the main lipid components of the skin. Insufficient cholesterol levels can lead to disruption of the intercellular lipid layer structure and abnormal fluidity, resulting in decreased barrier density and increased permeability. This leads to increased TEWL (transient endothelial wound healing), dry and sensitive skin, and a predisposition to asteatotic eczema, atopic dermatitis, and other skin conditions. Furthermore, it can further reduce the barrier function efficiency of ceramides. After applying the perilla essential oil of this invention, an increase in cholesterol levels can be detected, thus preventing the induction of related diseases caused by decreased cholesterol expression levels.

[0039] The effects of free fatty acids depend on a balanced ratio; excessive amounts can disrupt the golden ratio of intercellular lipids, inducing skin sensitivity, inflammation, and triggering / worsening acne. Stimulation with the perilla essential oil of this invention can significantly reduce the content of free fatty acids, thereby lowering the risk of skin sensitivity, psoriasis, and acne.

[0040] On the other hand, the present invention also provides an application of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of a product that is used for any one or more of the following purposes: (1) Used to promote the migration of human epidermal cells; (2) Used to promote the expression of tight junction proteins; (3) Used to promote the expression of LOR protein in the skin; (4) Used to maintain the homeostasis of skin secretion of ceramides, cholesterol and free fatty acids.

[0041] The present invention also provides the use of the above-mentioned perilla essential oil or perilla essential oil preparation in the preparation of a product for treating rosacea.

[0042] The perilla essential oil of the present invention can promote human epidermal cell migration, tight junction protein expression, lobe protein (LOR) expression, and maintain the homeostasis of ceramides, cholesterol and free fatty acids in the skin, so it can be further used to prepare products for the treatment of rosacea.

[0043] According to a specific embodiment of the present invention, preferably, the product is a pharmaceutical or skin care product.

[0044] According to a specific embodiment of the present invention, preferably, the perilla essential oil can be used as a topical skin preparation within the experimental concentration range.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a perilla essential oil, its preparation method, and its applications. The perilla essential oil obtained through refining comprises perillaldehyde, limonene, trans-caryophyllene, linalool, (3Z,6E)-α-farnesene, and trans-4-(1-methylvinyl)-cyclohexylethanol within a specific percentage range. Its comprehensive efficacy is significantly superior to crude perilla essential oil or refined perilla essential oil containing other components. It exhibits excellent effects in promoting human epidermal cell migration, promoting tight junction protein expression, promoting lobe protein (LOR) expression, and maintaining the homeostasis of ceramide, cholesterol, and free fatty acid secretion in the skin. Compared with previous products targeting skin problems, the perilla essential oil in this invention is naturally derived, safe, and has controllable acquisition costs. It does not have skin absorption issues and can fundamentally solve skin damage problems. Attached Figure Description

[0046] Figure 1 This is a TIC scan of the components of crude perilla essential oil extracted in Example 1.

[0047] Figure 2 TIC scan of the refined perilla essential oil components in Example 1.

[0048] Figure 3The results show the effects of essential oil preparations on the migration ability of human epidermal cells.

[0049] Figure 4 The results show the effects of essential oil preparations on intercellular lipid ceramides.

[0050] Figure 5 The results are experimental findings on the effects of essential oil preparations on intercellular lipid cholesterol.

[0051] Figure 6 These are experimental results regarding the effects of essential oil preparations on free fatty acids in intercellular lipids.

[0052] Figure 7 The results show the effects of essential oil preparations on the keratinized membrane of cells.

[0053] Figure 8 The results of immunohistofluorescence assay for the expression of tight junction protein Claudin-1.

[0054] Figure 9 TIC scan of perilla essential oil components in Comparative Example 1.

[0055] Figure 10 The results show the effects of perilla essential oil treatment on ceramides in Example 1 and Comparative Example 1.

[0056] Figure 11 The results show the effects of perilla essential oil treatment on cholesterol change rate in Example 1 and Comparative Example 1.

[0057] Figure 12 The results show the effects of perilla essential oil treatment on the rate of change of free fatty acids in Example 1 and Comparative Example 1. Detailed Implementation

[0058] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0059] It should be noted that, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0060] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0061] It should be understood that the terms “comprising,” “including,” and / or “containing” as used herein specify the presence of the stated features, integers, steps, components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0062] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0063] In this invention, the molecular distillation instrument can be a short-path thin-film distillation instrument KDL2, which mainly consists of: a feeding system, a discharging system, a distillation unit, a built-in condenser, an external condenser, a cold trap, a vacuum system, a heating / condensing system, an equipment support, and spare parts.

[0064] Example 1

[0065] This embodiment provides a method for preparing perilla essential oil and the component detection of perilla essential oil.

[0066] 1. Preparation method of perilla essential oil

[0067] (1) Add 1000g of fresh perilla leaves to a 5000mL distillation flask, then add 3000mL of water. Turn on the reflux condenser and place the flask in an electric kettle for heating. Set the heating temperature to 200℃ until no oil layer precipitates in the separatory funnel. Turn off the power to stop heating. The extraction time is about 4 hours. After the separatory funnel cools down, rotate the stopcock to release the lower layer of water and collect the upper layer of liquid. Centrifuge the liquid and take the upper layer as the crude perilla essential oil. Collect the crude perilla essential oil in a glass essential oil bottle and seal it. After weighing, store it in a refrigerator at 2-8℃ for later use.

[0068] (2) The crude perilla essential oil obtained in step (1) above is subjected to molecular distillation separation using a molecular distillation instrument (short-path thin film distillation instrument KDL2). The separation conditions are: vacuum pressure 98-100 mbar, heating temperature 89-90℃, injection flow rate 23-24 drops / min, rotation speed 130-135 rpm, and condensation temperature 5-8℃ to obtain the light component, i.e., the refined perilla essential oil.

[0069] 2. Extraction rate determination: The extraction experiment of crude perilla essential oil was repeated three times in parallel to calculate the average yield of crude perilla leaf essential oil obtained by steam distillation. The calculation formula is: Essential oil extraction rate = (oil yield / amount of perilla leaves weighed) × 100%.

[0070] The extraction rate of crude perilla essential oil in this embodiment was determined to be 2±0.05%.

[0071] 3. Essential oil component analysis

[0072] The components of crude perilla essential oil and perilla essential oil were analyzed using GC-MS technology. The experimental parameters are as follows: (1) Chromatographic conditions: DB-WAX (30m×250μm×0.25μm); carrier gas: 99.999% helium; injection 1μL; heating parameter settings: initial temperature 60℃, hold for 2min; then heat to 80℃, heating rate 4℃ / min, hold for 5min, then heat to 180℃ at 2℃ / min, hold for 5min; finally heat to 200℃ at 10℃ / min, hold for 2min.

[0073] (2) Mass spectrometry conditions: an (EI) ion source was used; the ion source temperature was 230℃; the interface temperature was 280℃; the electron energy was 70eV; the multiplication voltage was 1300V; and the mass scan range was 30~350 m / z.

[0074] The relative percentage content of each component was obtained by searching the Nest14 database and then using the peak area normalization method.

[0075] The test results are as follows: In this embodiment, 30 components were detected and identified in the crude perilla essential oil. The six components with the highest content were: perillaldehyde (45.89%), limonene (28.42%), trans-caryophyllene (10.32%), (3Z,6E)-α-farnesene (4.51%), linalool (2.99%), and trans-4-(1-methylvinyl)-cyclohexylethanol (2.01%). The TIC scan chromatogram of the crude perilla essential oil components is shown below. Figure 1 As shown.

[0076] In this embodiment, 26 components were detected and identified in the refined perilla essential oil. The six components with the highest content were: perillaldehyde (46.17%), limonene (31.68%), trans-caryophyllene (9.03%), linalool (3.63%), (3Z,6E)-α-farnesene (2.38%), and trans-4-(1-methylvinyl)-cyclohexylethanol (1.787%). The TIC scan chromatogram of the refined perilla essential oil components is shown below. Figure 2 As shown.

[0077] The comparison results of the specific components and contents of crude perilla essential oil and refined perilla essential oil are shown in Table 1.

[0078] Table 1. Comparison of component composition between crude and refined perilla essential oil.

[0079] Example 2: Effects of essential oil preparations on the migration ability of skin epidermal cells (HaCat)

[0080] Test methods

[0081] Marking: First, draw the first straight line parallel to the long side of the plate on the back of the 6-hole plate, with the hole dot as the line. Then, draw two parallel lines 1 cm above and below the first line.

[0082] 1. Cell seeding: Take the digested single-cell suspension and seed it at a ratio of 5 × 10⁻⁶ cells / mL. 5 Inoculate 2 mL per well into 6-well plates at a density of 1 / mL (top side), and incubate evenly.

[0083] 2. Cell adhesion and fusion: After 24 hours, culture until the cell fusion rate reaches 90%-100%.

[0084] 3. Scratch: Using a sterile 1mL pipette tip, gently and straighten the pipette along the direction of the scribing line on the back of the 6-well plate to form a uniform scratch; one scratch per well, ensuring that the scratch is straight and of consistent width.

[0085] 4. Wash away suspended cells: Gently rinse each well 2-3 times with PBS to remove the suspended cells that have been swept off, preventing them from adhering to the wall and affecting the experimental results. Set up groups as follows: crude perilla essential oil extracted in Example 1 (A), perilla essential oil extracted in Example 1 (B), perilla aldehyde monomer group (D), and CK control group. Then, add complete culture medium containing 2.5 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL of essential oil preparation to each well in the essential oil group. Add the corresponding concentration of perilla aldehyde monomer group to the crude perilla essential oil by calculation. The CK control group is the essential oil group with corresponding cosolvent concentrations of 2.5 μg / mL (concentration 1), 5 μg / mL (concentration 2), 25 μg / mL (concentration 3), and 50 μg / mL (concentration 4).

[0086] 5. Photograph record: Immediately under a microscope, using the scratch line on the back as a reference, take a photo of the scratch at 0h; then put the plate back into the incubator and take photos again at the same location at 24h, 48h, etc.

[0087] 6. Data Analysis: Open the photo with ImageJ software and measure the width of the scratches at different time points.

[0088] Calculate the scratch healing rate: Healing rate = [(0h scratch width - scratch width at a certain time point) / 0h scratch width] × 100%.

[0089] Test results are available Figure 3 (Different lowercase letters indicate significant differences between different treatments at the same concentration and time). It can be seen that the essential oils prepared by this invention all promote scratch healing, i.e., they have the ability to promote cell migration. Furthermore, the refined perilla essential oil, at a concentration of 2.5 μg / mL, achieved a healing rate as high as 93.9% after 48 hours of treatment. Simultaneously, the crude perilla essential oil also exhibited a high scratch healing rate; at a concentration of 25 μg / mL, the healing rate reached 79.6% after 48 hours of treatment. However, after refining using the process of this invention, the ability of perilla essential oil to promote cell migration was enhanced by nearly 15% compared to the crude perilla essential oil, a significant improvement. This experiment further verifies that the cell migration effect of the essential oil preparation is not primarily driven by its main component, perillaldehyde, but rather influenced by the overall formulation of the essential oil preparation.

[0090] Example 3: Effects of essential oil preparations on cellular secretion of ceramides, cholesterol, and free fatty acids

[0091] Test method: Reagents: Human ceramide ELISA kit (batch number: 20260107-N917780C), total cholesterol (TC) test kit / micro method (batch number: 20260107-N138878D), free fatty acid (FFA) test kit (batch number: 20260107-N350135D) were all purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., PEG40, and 0.1% sodium dodecyl sulfate (SLS).

[0092] The concentration of HaCaT cell suspension was prepared to be 3.0 × 10⁻⁶. 5 Inoculate 3 mL of the solution per mL into each culture dish after shaking well. After 24 hours, remove the supernatant from each well and add 3 mL of the corresponding essential oil preparation according to the group. The experimental groups are shown in Table 2.

[0093] Table 2 Experimental Groups

[0094] The supernatant was collected at 0, 3, and 24 hours, and the contents of ceramide, cholesterol, and free fatty acids in the supernatant were detected using a kit.

[0095] Correction value for each supernatant content = actual detection volume over 3 hours or 24 hours / initial detection volume over 0 hours

[0096] Result: As Figure 4(Different letters indicate significant differences between treatments under the same treatment duration.) As shown, the ceramide content decreased significantly after modeling. Treatment with the essential oil formulation of this invention revealed that the formulation promoted ceramide production. Although the crude extract was more effective for a short treatment time, with prolonged treatment time, the refined perilla essential oil at high and medium concentrations significantly outperformed the crude extract, and was also superior to perilla aldehyde alone.

[0097] like Figure 5 (Different letters indicate significant differences between treatments under the same treatment duration.) As shown, after modeling, cholesterol levels decreased. After stimulation with essential oil preparations, cholesterol levels increased, with the low-concentration refined perilla essential oil group showing the best performance, further demonstrating the superior performance of perilla essential oil prepared by the process of this invention.

[0098] like Figure 6 (Different letters indicate significant differences between treatments under the same treatment duration.) As shown, the content of free fat increased significantly after modeling, and decreased significantly after stimulation with essential oil preparations. The best performance was achieved with medium-concentration perilla essential oil, which reduced the content by approximately 45% in a short time, while the long-term reduction efficiency reached over 57%. Refined perilla essential oil was superior to crude perilla essential oil. Perilla aldehyde monomers, at a certain concentration, actually exacerbated the expression of fatty acids, further demonstrating the necessity of the perilla essential oil of this invention.

[0099] Example 4: Effect of essential oil preparations on the keratinization capsule of keratinocytes

[0100] Experimental method: Same as in Example 3. After treatment with essential oil preparation for 24 hours, the supernatant was collected, the cells were washed once with PBS, then digested with trypsin, centrifuged at 1000×g for 5 minutes, and the cells were collected. The collected cells were washed three times with cold PBS. 1 mL of PBS was added to the cells collected from each group for resuspension, and the cells were lysed by repeated freeze-thaw cycles. The cell suspension was centrifuged at 1500×g for 10 minutes, and the supernatant was collected for analysis. The content of keratinized capsid protein in the cell lysate was detected.

[0101] Results: After modeling, the content of pectin decreased, showing a significant difference compared to the control group (CK), indicating the effectiveness of the modeling. Treatment with essential oil preparations significantly increased the pectin content, with a maximum increase of approximately 99%. The high-concentration perilla essential oil showed the best effect, exceeding the efficacy of the high-concentration crude perilla essential oil group by nearly 10%. Furthermore, the experiment further verified that the effect of perilla essential oil in promoting pectin expression is not solely due to perillaldehyde, but rather a synergistic effect of all components. See details. Figure 7 (“ "This indicates that there is a significant difference between the model group and other groups, while there is no significant difference between the high-concentration perillaldehyde group and the model group."

[0102] Example 5: Effects of essential oil preparations on tight junction proteins

[0103] Test method: 1. Preparation of experimental reagents: (1) Preparation of IL-33 diluent For example, 1 mL can be prepared by adding 980 μL of serum-free culture medium (sterile) and 20 μL of heat-inactivated FBS (sterile). This will produce a concentration of 2% (v / v).

[0104] (2) Preparation of PBST

[0105] For example, 10 mL: 9.995 mL of 1× PBS (sterile) + 5 μL of Tween-20 stock solution (sterile) will prepare a final concentration of 0.05% (v / v).

[0106] (3) Preparation of blocking solution and primary and secondary antibody diluents

[0107] 9.495 mL of 1× PBS (sterile) + 5 μL of Tween-20 stock solution (sterile) + 0.5 mL of normal goat serum (sterile) resulted in a final goat serum concentration of 5% (v / v).

[0108] 2. When the experimental cells (HaCat) reach approximately 80% confluence, digest the cells with trypsin and prepare a cell suspension with a concentration of 2.0 × 10⁻⁶. 5 per mL.

[0109] (1) Resuspend the cell pellet, place the corresponding cell smears in a 24-well plate, add a small amount of culture medium to make them adhere tightly, and seed 400 μL of each cell smear evenly in a 24-well plate; (2) After the cells adhered and grew for 24 hours, the 24-well plate was washed with PBS buffer three times to remove the serum; (3) After 24 hours, except for the CK group, the MD (model group) group and the essential oil group were stimulated with 400 μL of 100 ng / mL IL-33, and the CK group was stimulated with an equal amount of complete culture medium. (4) After 6 hours of intervention, the liquid of each group was aspirated, each well was washed 3 times with PBS buffer, 400 μL of each essential oil of the corresponding diluted concentration (5 μg / mL, 25 μg / mL and 50 μg / mL respectively) was added to the essential oil group, an equal amount of IL-33 dilution was added to the model group, and an equal amount of complete culture medium was added to the CK group. (5) After 12 hours of intervention, the liquid of each group was aspirated, and the cell crawling slides were gently rinsed twice with pre-cooled PBS for 1 min each time to remove residual culture medium. 300 μL of 4% paraformaldehyde (PFA) was added to cover the crawling slides and fixed at room temperature for 30 min. (6) After fixation, wash each well three times with PBS buffer for 5 minutes each time to completely remove the fixative and prevent residual PFA from affecting subsequent experiments. Add 300 mL of 0.3% Triton X-100 to each well and permeate at room temperature for 20 minutes. (7) After the permeabilization is completed, wash each well three times with PBS buffer for 5 minutes each time to remove the residual permeabilization solution. Gently blot the PBS off the edge of the slide with clean absorbent paper. Add 50 μL of 5% normal goat serum working solution to each well and block at 37°C for 30 minutes. (8) After the blocking is completed, the blocking solution is aspirated (no rinsing is required, just aspirate and discard), and the corresponding pre-cooled antibody Claudin-1 concentration of 1:200 is added to each well; (9) On the second day, take out the slides and incubate them at 37°C for 1 hour. Wash each well three times with PBST buffer for 5 minutes each time. Gently shake the culture dish to ensure that the unbound primary antibody is completely washed away. Use clean absorbent paper to gently dry the PBST on the edge of the slide. (10) Add 50 μL of 488-labeled fluorescent secondary antibody to each well at a concentration of 1:800. Incubate at 37°C for 1 hour. Wash each well three times with PBS buffer. Rinse with PBS for the last time to remove residual Tween-20 and reduce background fluorescence. Mount the slide with DAPI-containing mounting medium. (11) A laser confocal microscope was used: excitation wavelength 490-495 nm, emission wavelength 515-520 nm; (12) Data analysis: Open the fluorescence image with ImageJ software and measure its gray value.

[0110] The grayscale values ​​of the tight junction protein Claudin-1 expression are shown in Table 3. The results of immunofluorescence assay for Claudin-1 expression are as follows: Figure 8 As shown, where, Figure 8 The first row, from left to right, shows the effects of crudely extracted perilla essential oil from low to high concentrations and the control group; the second row, from left to right, shows the effects of refined perilla essential oil from low to high concentrations and the model group.

[0111] From Table 3 and Figure 8It can be seen that the expression of Claudin-1 was significantly reduced after modeling. Both crude and refined perilla essential oils promoted the expression of the protein, with significant differences. The refined perilla essential oil showed the best expression-promoting effect, which was significantly better than that of the crude perilla essential oil. Compared with the best effect of the two, the efficacy was improved by nearly 8% after refined extraction.

[0112] Table 3. Gray-scale values ​​of Claudin-1 expression, a tight junction protein.

[0113] The above results show that the perilla essential oil extracted by the extraction process of the present invention has the effects of promoting cell migration, improving the secretion of lipids in damaged intercellular cells, promoting the expression of keratinocyte capsule proteins, and promoting the expression of tight junction proteins at appropriate concentrations.

[0114] Comparative Example 1

[0115] This comparative example provides a perilla essential oil, its preparation method, and the component detection of perilla essential oil.

[0116] The preparation method of perilla essential oil is as follows: (1) Add 1000g of fresh perilla leaves to a 5000mL distillation flask, then add 3000mL of water. Turn on the reflux condenser and place the flask in an electric kettle for heating. Set the heating temperature to 200℃ until no oil layer precipitates in the separatory funnel. Turn off the power to stop heating. The extraction time is about 4 hours. After the separatory funnel cools down, rotate the stopcock to release the lower layer of water and collect the upper layer of liquid. Centrifuge the liquid and take the upper layer as the crude perilla essential oil. Collect the crude perilla essential oil in a glass essential oil bottle and seal it. After weighing, store it in a refrigerator at 2-8℃ for later use.

[0117] (2) The crude perilla essential oil obtained in step (1) above is separated by molecular distillation using a molecular distillation instrument (short-path thin film distillation instrument KDL2). The separation conditions are: vacuum pressure 98-100 mbar, heating temperature 89-90℃, injection flow rate 23-24 drops / min, rotation speed 130-135 rpm, condensation temperature 5-8℃, and the distillate (heavy component) is obtained, which is perilla essential oil.

[0118] The detection method is the same as in Example 1.

[0119] In this comparative example, 27 components were detected in perilla essential oil. The components with the highest content were perillaldehyde (49.2117%), trans-caryophyllene (17.0516%), (3Z,6E)-α-farnesene (15.7155%), trans-4-(1-methylvinyl)-cyclohexylethanol (3.8386%), and α-rutoxene (2.4595%). The composition of other components is detailed in Table 4. The TIC scan chromatogram of perilla essential oil components is shown below. Figure 9 As shown. The data in Table 4 was derived from Data Analysis. Figure 9 Generated by Qualitative Analysis software.

[0120] Table 4

[0121] The perilla essential oil obtained in Comparative Example 1 was applied to skin epidermal cells (HaCat) to investigate its effects on intercellular lipids, ceramides, cholesterol, and free fatty acids. The experimental method was the same as in Example 3. Three concentration groups (B1, B2, B3) of the perilla essential oil obtained in Example 1, three concentration groups (C1, C2, C3) of the perilla essential oil obtained in Comparative Example 1, a model group (ZM), and a CK group were established. Changes in intercellular lipids in each group were detected after 24 hours of treatment. The rate of change in intercellular lipids was: Change rate = [(Detection value after 24 hours of treatment - Initial detection value) / Initial detection value] × 100% Results: After 24 hours, ceramide levels decreased in the CK group (normal group), and decreased rapidly in the ZM group after modeling, showing a significant difference from the CK group (p<0.05). Ceramide levels recovered somewhat after treatment with different essential oil preparations, with the high-concentration group (group B) showing the best stabilization and recovery effect, showing a significant difference from the modeling group (p<0.05). Although group C, after refining, still contained characteristic components of perilla essential oil, its effect was significantly less than group B, and it may even lead to excessive ceramide secretion. (See details...) Figure 10 (Different lowercase letters indicate significant differences between treatments, p<0.05).

[0122] Changes in cholesterol levels were monitored. After 24 hours, the cholesterol content in the model group was significantly increased compared to the normal group. Treatment of cells with perilla essential oil from Example 1 and Comparative Example 1 significantly reduced cholesterol levels at medium concentrations, showing a significant difference compared to the ZM group (p<0.05). At the optimal concentration, there was no difference in effect between the two (p>0.05). See details... Figure 11 (Different lowercase letters indicate significant differences between treatments, p<0.05).

[0123] The changes in free fatty acids were examined. After modeling, compared with the normal control group (CK), the content of free fatty acids was significantly increased (p<0.05). After treatment with perilla essential oil from Example 1 and Comparative Example 1, at medium concentrations, the perilla essential oil from Example 1 showed the best effect, significantly reducing the content of free fatty acids, and its effect was far superior to the best high-concentration group of Comparative Example 1 (p<0.05). Comparing the two optimal concentration groups of groups B and C, it was found that the efficacy of perilla essential oil from Example 1 was improved by 76.55%. (See details...) Figure 12 (Different lowercase letters indicate significant differences between treatments, p<0.05).

[0124] The above results indicate that the perilla essential oil of Example 1 has a significantly better effect on stabilizing cell lipids than the perilla essential oil of Comparative Example 1.

[0125] In summary, the present invention provides a perilla essential oil and its preparation method, wherein the perilla essential oil comprises perillaldehyde, limonene, trans-caryophyllene, linalool, (3Z,6E)-α-farnesene, trans-4-(1-methylvinyl)-cyclohexamethanol and other components in a specific range of percentages.

[0126] The invention further verified its multifaceted efficacy through experiments. The results showed that the perilla essential oil in the invention can promote skin cell migration, control the expression of keratinocyte capsule and intercellular lipid composition, and at the same time promote the expression of tight junction proteins.

[0127] Compared to crude perilla essential oil and perilla essential oil with varying component contents, the perilla essential oil of this invention exhibits significantly enhanced efficacy. Specifically: 1. The perilla essential oil of the present invention has the ability to promote cell migration. After an action time of 48 hours at a concentration of 2.5 μg / mL, the healing rate is as high as 93.9%, which is nearly 15% higher than that of crude perilla essential oil. 2. The perilla essential oil of the present invention has the effect of maintaining the homeostasis of intercellular lipids, and its efficacy is significantly better than that of crude perilla essential oil and refined perilla essential oil with different component contents. 3. The perilla essential oil of the present invention can significantly increase the content of pectin, with a maximum increase of about 99%, and its efficacy exceeds that of the high-concentration group of crude perilla essential oil by nearly 10%; 4. The perilla essential oil of the present invention can promote the expression of Claudin-1 protein in tight junctions, and its efficacy is improved by nearly 8% compared with crude perilla essential oil.

[0128] The above embodiments illustrate and describe the main features and advantages of the present invention in detail. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. A perilla essential oil, wherein, The perilla essential oil contains the following components in relative percentage: perillaldehyde 46-50%, limonene 30-35%, trans-caryophyllene 8-12%, linalool 3-5%, (3Z,6E)-α-farnesene 2-5%, and trans-4-(1-methylvinyl)-cyclohexamethanol 1-4%.

2. The perilla essential oil according to claim 1, wherein, The perilla essential oil contains the following components in relative percentage: perillaldehyde 46-47%, limonene 30-33%, trans-caryophyllene 8-10%, linalool 3-4%, (3Z,6E)-α-farnesene 2-3%, and trans-4-(1-methylvinyl)-cyclohexanol 1.5-3%.

3. The perilla essential oil according to claim 1, wherein, The perilla essential oil comprises, by relative percentage, the following components: perillaldehyde 46-50%, limonene 30-35%, trans-caryophyllene 8-12%, linalool 3-5%, (3Z,6E)-α-farnesene 2-5%, trans-4-(1-methylvinyl)-cyclohexamethanol 1-4%, α-pinene 0.5-2%, β-pinene 0.5-2%, and α-thiazolinone 0.5-2%.

4. The perilla essential oil according to claim 1, wherein, The perilla essential oil, by relative percentage, comprises the following components: α-pinene 0.5-1%, 3-thujone 0.01-0.03%, β-pinene 0.5-1%, sapinene 0.1-0.3%, β-myrcene 0.1-1%, limonene 30-33%, β-phellandrene 0.05-0.1%, eucalyptol 0.1-0.2%, γ-terpinene 0.01-0.03%, terpinene 0.1-0.3%, 6-methyl-5-hepten-2-one 0.01-0.03%, 1-octen-3-ol 0.01-0.1%, and linalool 3-4%. β-elemene 0.02-0.05%, trans-caryophyllene 8-10%, 4-terpinenol 0.02-0.06%, α-rutane 0.5-1%, cis-citral 0.05-0.3%, α-terpineol 0.1-0.5%, dextrorotatory geraniol 0.05-0.3%, (3Z,6E)-α-farnesene 2-3%, perillaldehyde 46-47%, nerol 0.05-0.3%, trans-4-(1-methylvinyl)-cyclohexyl alcohol 1.5-3%, caryophyllein 0.05-0.2%, perillol 0.1-0.3%.

5. A method for preparing perilla essential oil according to any one of claims 1-4, wherein, The preparation method includes the following steps: (1) Perilla leaves were steam distilled to obtain crude perilla essential oil; (2) Molecular distillation is performed on the crude perilla essential oil to obtain a light component, namely refined perilla essential oil; The extraction conditions for molecular distillation include: vacuum pressure of 90-100 mbar, heating temperature of 85-95℃, injection flow rate of 20-25 drops / min, rotation speed of 120-140 rpm, and condensation temperature of 4-10℃.

6. The preparation method according to claim 5, wherein, The separation conditions for molecular distillation include: vacuum pressure of 98-100 mbar, heating temperature of 89-90℃, injection flow rate of 23-24 drops / min, rotation speed of 130-135 rpm, and condensation temperature of 5-8℃.

7. The preparation method according to claim 5, wherein, During steam distillation, the mass ratio of perilla leaves to water is 1:2-5.

8. The preparation method according to claim 5, wherein, The steam distillation temperature is 180-220℃.

9. The preparation method according to claim 5, wherein, The crude perilla essential oil contains the following components in relative percentage: perillaldehyde 44-46%, limonene 28-29%, trans-caryophyllene 10-12%, (3Z,6E)-α-farnesene 4-5%, linalool 2-4%, and trans-4-(1-methylvinyl)-cyclohexanol 1.5-3%.

10. A perilla essential oil preparation, wherein the perilla essential oil preparation comprises the perilla essential oil according to any one of claims 1-4. And physiologically or pharmaceutically acceptable excipients; in, The concentration of perilla essential oil in the perilla essential oil preparation is 5 μg / mL-100 μg / mL.

11. The use of the perilla essential oil according to any one of claims 1-4 or the perilla essential oil preparation according to claim 10 in the preparation of a product used for any one or more of the following purposes: (1) Used to promote the migration of human epidermal cells; (2) Used to promote the expression of tight junction proteins; (3) Used to promote the expression of LOR protein in the skin; (4) Maintain the homeostasis of skin secretion of ceramides, cholesterol and free fatty acids.

12. The use of the perilla essential oil according to any one of claims 1-4 or the perilla essential oil preparation according to claim 10 in the preparation of a product for treating rosacea.

Images