Application of litsea cubeba oil or citral serving as active ingredient of litsea cubeba oil

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of litsea cubeba oil or citral serving as an active ingredient of the litsea cubeba oil in preparation of a product for preventing and treating photoaging. The invention further discloses a preparation for preventing and treating light aging. The preparation comprises litsea cubeba oil or citral serving as an active ingredient of the litsea cubeba oil. The invention overcomes the side effects of high price, skin irritation risk and the like of the existing photoaging treatment means (instruments / chemical drugs), develops an alternative scheme based on natural components, and provides a new application of natural and low-toxicity litsea cubeba oil or citral as an active component thereof in preventing and treating UVA-induced photoaging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an application of Litsea Cubeba Oil or its active ingredient citral. BACKGROUND

[0002] Photoaging is the premature aging of human skin caused by continuous exposure to ultraviolet radiation (UVR). In fact, 80% of the signs of skin aging are caused by UVR exposure. Photoaging leads to major changes in the skin, including irregular pigmentation, telangiectasia, roughness, deep wrinkles, dryness, wrinkles, elastosis, and precancerous lesions. Although many methods for treating photoaging have been proposed, such as various acoustic, light, and electrical-based devices (laser, radiofrequency device, and high-intensity focused ultrasound) and topical drugs such as tretinoin. However, the use of these devices requires medical qualifications, is expensive, and sometimes accompanied by patient discomfort, and even the risk of skin pigmentation and burns in the treated area. Tretinoin is widely considered the gold standard for preventing and treating photoaging, but it carries the risk of varying degrees of irritation, such as skin dryness, peeling, and skin redness.

[0003] According to the wavelength, ultraviolet radiation is divided into three types: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm). UVC radiation is absorbed by the ozone layer, so the radiation cannot reach the ground; UVB has weak penetration ability of the skin, mainly causing damage to the epidermis and superficial dermis; UVA has strong penetration ability and can penetrate through the stratum corneum, epidermis, and even subcutaneous tissue into the skin, which is the main cause of photoaging.

[0004] Therefore, there is a need for a new technical means that can overcome the many inconveniences and side effects of the use of instruments or chemical drugs in existing photoaging treatment methods. SUMMARY

[0005] The purpose of the present application is to provide an application of Litsea Cubeba Oil or its active ingredient citral to solve the problem of the many inconveniences and side effects of the use of instruments or chemical drugs in existing photoaging treatment methods proposed in the background.

[0006] To achieve the above-mentioned purpose, the present application provides an application of Litsea Cubeba Oil or its active ingredient citral, which is applied in the preparation of a product for preventing and treating photoaging.

[0007] In a specific embodiment, the photoaging is UVA-induced or UVA+UVB-induced photoaging.

[0008] The present application also provides a preparation for preventing and treating photoaging, which comprises Litsea Cubeba Oil or its active ingredient citral.

[0009] In a specific embodiment, when the preparation contains Litsea cubeba oil, the concentration of Litsea cubeba oil is less than or equal to 100 μg / mL; and when the preparation contains citral, the concentration of citral is less than or equal to 100 μM.

[0010] In a specific embodiment, the preparation is an external cream.

[0011] In a specific embodiment, the external cream does not contain tretinoin.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] The present application overcomes the many inconveniences and side effects of the existing photoaging treatment means (devices / chemical drugs), develops an alternative solution based on natural ingredients, and provides a new use of natural and low-toxic Litsea cubeba oil or its active ingredient citral in preventing and treating UVA-induced photoaging.

[0014] The present application uses Litsea cubeba oil extracted from fresh Litsea cubeba fruits, which has the characteristics of being natural and low irritating; and Litsea cubeba is a evergreen tree or shrub, which is originally from the subtropical and tropical regions of Asia, has geographical advantages for planting, can be planted on a large scale, can effectively reduce the production cost of Litsea cubeba oil, and further reduce the cost of related preparations.

[0015] The cream prepared from Litsea cubeba oil and its main component citral has no obvious irritation to the skin, and can significantly improve the epidermal thickening, skin erythema and deep wrinkles, abnormal collagen metabolism and aging indicators, and the increase of inflammatory factors caused by photoaging.

[0016] In addition to the purposes, features and advantages described above, the present application has other purposes, features and advantages. The present application is further described in detail below. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, illustrate the preferred embodiments of the application and assist in

[0018] Figure 1 is a 24-hour effect diagram of Litsea cubeba oil on the cell viability of human fibroblasts;

[0019] Figure 2 is a 48-hour effect diagram of Litsea cubeba oil on the cell viability of human fibroblasts;

[0020] Figure 3 is a 24-hour effect diagram of citral on the cell viability of human fibroblasts;

[0021] Figure 4 Figure 5 is a graph showing the 48-hour effect of citral on the cell viability of human fibroblasts;

[0022] Figure 5 Figure 6 is a graph showing the effect of citral and Litsea cubeba oil on the ROS level of photoaged fibroblasts;

[0023] Figure 6 Figure 7 is a graph showing the effect of citral and Litsea cubeba oil on the mRNA level of MMP1 of photoaged fibroblasts;

[0024] Figure 7 Figure 8 is a graph showing the effect of citral and Litsea cubeba oil on the mRNA level of p21 of photoaged fibroblasts;

[0025] Figure 8 Figure 9 is a graph showing the effect of citral and Litsea cubeba oil on the mRNA level of p53 of photoaged fibroblasts;

[0026] Figure 9 Figure 10 is a graph showing the effect of citral and Litsea cubeba oil on the mRNA level of IL-1β of photoaged fibroblasts;

[0027] Figure 10 Figure 11 is a graph showing the effect of citral and Litsea cubeba oil on the mRNA level of TNF-α of photoaged fibroblasts;

[0028] Figure 11 Figure 12 is a graph showing the effect of citral and Litsea cubeba oil on the protein level of collagen metabolism-related factors and aging markers of photoaged fibroblasts;

[0029] Figure 12 Figure 13 is a graph showing the effect of citral and Litsea cubeba oil on the skin of photoaged model mice;

[0030] Figure 13 Figure 14 is a graph showing the HE staining of the effect of citral and Litsea cubeba oil on the skin of photoaged model mice;

[0031] Figure 14 Figure 15 is a graph showing the relative expression amount of collagen metabolism-related factor COL1A1 of the skin of photoaged model mice treated with citral and Litsea cubeba oil;

[0032] Figure 15 Figure 16 is a graph showing the relative expression amount of p21 of the skin of photoaged model mice treated with citral and Litsea cubeba oil;

[0033] Figure 16 Figure 17 is a graph showing the relative expression amount of p53 of the skin of photoaged model mice treated with citral and Litsea cubeba oil;

[0034] Figure 17 Figure 18 is a graph showing the relative expression amount of IL-1β of the skin of photoaged model mice treated with citral and Litsea cubeba oil;

[0035] Figure 18 Figure 3 is a comparison chart of relative expression of IL-6 in the skin of a photoaging model mouse treated with limonene and Litsea cubeba oil;

[0036] Figure 19 Figure 4 is a comparison chart of relative expression of TNF-α in the skin of a photoaging model mouse treated with limonene and Litsea cubeba oil. DETAILED DESCRIPTION

[0037] The following detailed description of the embodiments of the present application is provided for the purpose of explanation only and is not intended to limit the present application.

[0038] The present application provides an application of Litsea cubeba oil or its active ingredient limonene in the preparation of a product for preventing and treating photoaging.

[0039] The photoaging is UVA-induced or UVA+UVB-induced photoaging.

[0040] The present application also provides a preparation for preventing and treating photoaging, which comprises Litsea cubeba oil or its active ingredient limonene.

[0041] When the preparation comprises Litsea cubeba oil, the concentration of Litsea cubeba oil is less than or equal to 100 μg / mL; when the preparation comprises limonene, the concentration of limonene is less than or equal to 100 μM.

[0042] The preparation is an external cream.

[0043] The external cream does not contain tretinoin.

[0044] The external cream further comprises jojoba oil (5-8%), cetyl stearyl alcohol (2-4%), vitamin E (0.5%), phenoxyethanol (0.5%), propylene glycol (3-5%), glycerol (4-6%), Tween-80 (1-3%), xanthan gum (0.1-0.3%), and deionized water, wherein the above proportions are mass fractions. Preferably, the external cream comprises 5% jojoba oil, 2% cetyl stearyl alcohol, 0.5% vitamin E, 0.5% phenoxyethanol, 3% propylene glycol, 5% glycerol, 2% Tween-80, 0.2% xanthan gum, and deionized water.

[0045] The specific preparation process of the external cream is as follows:

[0046] S1, oil phase preparation: mix Litsea cubeba oil or limonene, jojoba oil, cetyl stearyl alcohol, vitamin E, phenoxyethanol, and propylene glycol, heat to melt, and stir uniformly; preferably, the mass fraction of Litsea cubeba oil is 0.5-1% or the mass fraction of limonene is 0.5-1%. More preferably, the mass fraction of Litsea cubeba oil is 0.5%.

[0047] S2, water phase preparation: deionized water is heated, glycerol, Tween-80, xanthan gum is dissolved;

[0048] S3, emulsification: the oil phase is slowly added to the water phase, heated and stirred, the pH is adjusted with citric acid or triethanolamine, and the temperature is reduced to below 45℃;

[0049] S4, the external cream is divided into subpackaging boxes to avoid light and high temperature.

[0050] The melting temperature of the oil phase preparation of the external cream is 75℃; the deionized water for water phase preparation is heated to 75℃; the heating and stirring time for emulsification is 5 minutes, and the pH value is adjusted to 5.5-6.0 with citric acid or triethanolamine.

[0051] Example 1

[0052] Verification experiment:

[0053] 1. Human fibroblast separation

[0054] (1) Take the normal prepuce tissue included in the outpatient operating room, remove the subcutaneous tissue of the tissue block as much as possible with a sterile ophthalmic scissors, and only leave the true epidermis. The prepuce is cut to a diameter of 4-5 cm with a sterile ophthalmic scissors, and then 4 mg / mL dispase II digestion solution is added and incubated at 4℃ in the dark overnight;

[0055] (2) The next day, after separating the epidermis and dermis, the dermis is cut into pieces with scissors, and 2 mg / mL collagenase solution is added and shaken at 37℃ for 1-2 h;

[0056] (3) The tissue mixture is sucked and filtered into a cell screen;

[0057] (4) Collect the filtered liquid, set the relative centrifugal force to 300g, and centrifuge at room temperature for 5 min, and evenly seed the cells into a T25 culture bottle.

[0058] 2. Human skin fibroblast culture

[0059] (1) Human skin primary fibroblasts are cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin-penicillin, and placed in a 37℃, 5% CO2 saturated humidity incubator;

[0060] (2) When the confluence is 80%, discard the culture solution and wash the cells with PBS;

[0061] (3) The cells are digested with trypsin (0.25%, containing 0.02% EDTA) for 3 min;

[0062] (4) DMEM complete medium is used to terminate digestion and collect cells, and the relative centrifugal force is set to 300g for centrifugation for 5 min;

[0063] (5) Remove the culture medium, resuspend the cell pellet in DMEM complete medium, passage at a ratio of 1:3, and culture in a cell culture incubator (37℃, 5% CO2).

[0064] (6) Select cells in good growth condition from the 3rd to 5th generation for experiments.

[0065] 3. CCK8 experiment.

[0066] (1) Human fibroblasts in the logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 3 / wells are evenly inoculated in a 96-well plate;

[0067] (2) Set up treatment groups of 50μM, 100μM, 150μM, and 200μM citral, and treatment groups of 25μg / mL, 50μg / mL, 75μg / mL, and 100μg / mL Litsea cubeba oil. The control group was given DMEM complete culture medium.

[0068] (3) 24h and 48h after administration, 10μL of CCK8 reagent was added under light-protected conditions, and the mixture was incubated at 37℃ for 1h under light-protected conditions. The OD value was then detected at 450nm.

[0069] 4. Measurement of cellular ROS levels.

[0070] (1) Human skin fibroblast cells (1×10 5 ( / well) inoculated into a 24-well plate;

[0071] (2) Set up a blank control group, a UVA modeling group, a 50μM and 100μM citral treatment group, and a 50μg / mL and 100μg / mL Litsea cubeba oil treatment group. The drugs were administered after UVA modeling.

[0072] (3) Dilute the stock solution of the fluorescent probe DCFH-DA (10mM) with serum-free DMEM medium to make its final concentration 10μM.

[0073] (4) Remove the old culture medium, add 1 mL of diluted DCFH-DA solution to each well, and incubate at 37°C for 30 min;

[0074] (5) Wash the cells three times with serum-free culture medium to fully remove the DCFH-DA probes that failed to penetrate and remained outside the cells;

[0075] (5) Observe cells using a fluorescence inverted microscope.

[0076] 5. Quantitative polymerase chain reaction (RT-qPCR).

[0077] (1) Extracting RNA from cells or tissues using Trizol:

[0078] Add pre-cooled Trizol into the six-hole plate, 1 mL per hole, and blow with a pipette;

[0079] Move the sample into a 1.5 mL EP tube, add 0.2 mL chloroform, shake for 15 s, and stand at room temperature for 5 min;

[0080] Centrifuge at 12000 rpm / min, 4°C, for 15 min;

[0081] Aspirate the supernatant water phase in the EP tube into a new EP tube;

[0082] Add an equal volume of isopropanol into the supernatant and mix well, and stand at room temperature for 10 min;

[0083] Centrifuge at 12000 rpm / min, 4°C, for 15 min;

[0084] Remove the supernatant in the EP tube, and add 900 μL of pre-cooled 75% ethanol and mix well;

[0085] Centrifuge at 12000 rpm / min, 4°C, for 5 min;

[0086] Aspirate the supernatant, invert the EP tube, and after the 75% ethanol evaporates, add 10-30 μL of enzyme-free water, gently blow and mix well to dissolve the RNA precipitate.

[0087] (2) According to the Takara reverse transcription kit instructions, add reagents to convert RNA to cDNA;

[0088] (3) Add 0.3 μL of forward primer and reverse primer, 2.4 μL of ddH2O, 2 μL of sample, and 5 μL of fluorescence quantitative reagent. Measure the mRNA expression according to the threshold cycle value based on the standard curve, and normalize according to GADPH. The experiment is repeated at least 3 times.

[0089] 6. Western blot detection of protein expression level.

[0090] (1) Extract cell or tissue protein:

[0091] After centrifugation of cell digestion, collect the cell precipitate, add protein lysis solution, and lyse on ice for 30 min; ultrasonic on ice for 3-5 min; centrifuge at 12000 rpm / min, 4°C, for 10 min, aspirate the supernatant into a new EP tube; use Thermo Fisher BCA protein concentration determination kit to determine the total protein sample concentration; according to the measured total protein concentration, calculate the required total protein, 5xloading Buffer and 1xloading Buffer volume of each sample; denature in a 100°C metal bath for 5 minutes;

[0092] (2) Fill the electrophoresis tank with electrophoresis buffer, put the prepared gel into the electrophoresis buffer, cover the gel with the electrophoresis buffer, pull out the comb, and then add the sample and protein Marker into the hole in turn;

[0093] After the electrophoresis is completed, take out the gel, cut the gel according to the size of the band, cut the PVDF membrane according to the size of the gel, and soak in methanol for 30 s to activate;

[0094] (3) Open the membrane transfer clamp, put the membrane transfer device in turn and fix it. Put the clamp into the membrane transfer tank, fill it with pre-cooled membrane transfer solution, and then transfer the membrane;

[0095] (4) After the membrane transfer is completed, take out the membrane, and seal the protein with a quick sealing solution for 15 min;

[0096] (5) Dilute the primary antibody with TBST according to the recommended proportion in the instruction, and incubate at 4°C overnight;

[0097] (6) After washing the membrane with TBST, dilute the secondary antibody according to the recommended proportion in the instruction, and shake on a shaker at room temperature for 1 h, and then develop.

[0098] 7. Preparation of external cream.

[0099] (1) Preparation of oil phase: Mix Litsea cubeba oil / citral, jojoba oil, cetylstearyl alcohol, vitamin E, phenoxyethanol, and propylene glycol, heat to 75°C to melt, and stir uniformly.

[0100] (2) Preparation of water phase: Heat deionized water to 75°C, and dissolve glycerol, Tween-80, and xanthan gum.

[0101] (3) Emulsification: Slowly add the oil phase to the water phase, heat and stir for 5 min, adjust the pH to 5.5-6.0 with citric acid or triethanolamine, and cool to below 45°C.

[0102] (4) Distribute the cream to the distribution box to avoid light and high temperature.

[0103] 8. Animal modeling.

[0104] 8-week-old BALB / c female mice (16-20 g) were raised in cages with clean and sterile bedding, controlled temperature (23±2°C), humidity (50±5%), and light (12 h light / dark cycle). The adaptive feeding was performed for 1 week before the experiment. The groups were blank group, model group, solvent control group, 0.5% citral group, 1% citral group, 0.5% Litsea cubeba oil group, and 1% Litsea cubeba oil group. The mice were all depilated on the back, placed on a platform 10 cm away from the lamp, and irradiated with UVB+UVA at 100 mJ / cm 2 for 5 times a week for 6 weeks of modeling. All animal experiments were approved by the Animal Investigation Ethics Committee of Xiangya Second Hospital of Central South University.

[0105] 9. Hematoxylin-eosin staining.

[0106] After the mice were sacrificed, the mouse skin tissue was immersed in 4% paraformaldehyde fixative and fixed for 24 hours. After fixation, the tissue was dehydrated, embedded and sectioned, and then hematoxylin and eosin (HE) staining was performed.

[0107] Results of the experiment:

[0108] 1. Cytotoxicity of citral and Litsea cubeba oil on human normal fibroblasts.

[0109] Safety is the primary prerequisite for any drug, therefore, we first used CCK-8 assay to determine the cytotoxicity of citral and Litsea cubeba oil. The results showed that after 24h and 48h of drug treatment, citral at a concentration of 100 μM and below and Litsea cubeba oil at 100 μg / mL and below had no obvious toxicity to human skin fibroblasts, and the cell viability was maintained at more than 80% ( Figures 1-4 ).

[0110] 2. Effect of citral and Litsea cubeba oil on ROS levels in photoaged fibroblasts.

[0111] After UVA irradiation, the ROS level in human fibroblasts increased significantly ( Figure 5 ), however, after intervention with different concentrations of citral and Litsea cubeba oil, the ROS level of human fibroblasts cells decreased significantly.

[0112] 3. Effect of citral and Litsea cubeba oil on the mRNA levels of collagen metabolism-related factors, aging markers, and inflammatory factors in photoaged fibroblasts.

[0113] MMP-1 is a matrix metalloproteinase, an important enzyme that degrades extracellular matrix components, mainly hydrolyzing types I, II, and III collagen. Therefore, inhibiting its activity can reduce the degradation of extracellular matrix. p21, p53 are classic markers of cell aging, which can induce cells to enter cycle arrest. IL-1β, TNF-α are pro-inflammatory cytokines, which can induce a variety of pro-inflammatory mediators and play an important role in inflammatory response. After UVA photoaging modeling, MMP-1, p21, p53, L-1β, TNF-α were all increased, while after treatment with citral and Litsea cubeba oil, the mRNA relative expression of the above indicators was significantly reduced ( Figures 6-10 ).

[0114] 4. Effect of citral and Litsea cubeba oil on the protein levels of collagen metabolism-related factors and aging markers in photoaged fibroblasts.

[0115] After UVA light aging modeling, the collagen metabolism related factors MMP-1, the aging markers p21, p53, p16 were all increased, and COL1A1 was decreased, while after treatment with 100 μM citral and 50 μg / mL Litsea cubeba oil, 100 μg / mL Litsea cubeba oil, the above index protein expression was significantly improved. Figure 11 )。

[0116] 5, the influence of citral and Litsea cubeba oil on the skin of the light aging model mice.

[0117] From a macro perspective, the skin of the blank group mice remained in the best state without any wrinkles or erythema. The model group appeared erythema and wide wrinkles, accompanied by pigmentation. The solvent group still had a large area of erythema caused by sunburn, but the wrinkles were not obvious. The 0.5% citral group, 1% citral group, 0.5% Litsea cubeba oil group, and 1% Litsea cubeba oil group only had a small amount of erythema, no wrinkles, pigmentation, and other skin photoaging symptoms, and the overall appearance of the skin was normal. Figure 12 ) HE staining results showed that after the mouse back skin was exposed to UVA+UVB, the epidermis was significantly thickened, the collagen fibers in the dermis were tangled and degraded, and there was inflammatory cell infiltration. After treatment with different concentrations of citral and Litsea cubeba oil, the epidermal thickening of the mice was significantly reduced, the collagen fibers in the dermis were arranged in order, and there was almost no obvious inflammatory cell infiltration. Figure 13

[0118] 6, the influence of citral and Litsea cubeba oil on the mRNA levels of collagen metabolism related factors, aging markers, and inflammatory factors in the skin of light aging model mice.

[0119] After UVA+UVB modeling, p21, p53, IL-1β, IL-6, TNF-α were significantly increased, and COL1A1 was decreased, which was consistent with the phenotype changes of photoaging. After treatment with citral and Litsea cubeba oil, the mRNA relative expression of IL-1β, IL-6, TNF-α and other inflammatory indicators was significantly down-regulated, which proved the strong anti-inflammatory effect of citral and Litsea cubeba oil, and the classic aging marker p21 was also down-regulated, and had statistical significance. Figures 14-19

[0120] The above is a further detailed description of the present application in combination with specific preferred embodiments, which cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the concept of the present application, a number of simple deductions and substitutions can be made, which should be considered as belonging to the protection scope of the present application.​​

Claims

1. An application of Litsea cubeba oil or its active ingredient citral, characterized in that, It is used in the preparation of products for preventing and treating photoaging.

2. An application according to claim 1, characterized in that, The photoaging is UVA-induced or UVA+UVB-induced photoaging.

3. A formulation for preventing and treating photoaging, characterized in that, The preparation contains Litsea cubeba oil or its active ingredient citral.

4. The formulation for preventing photoaging according to claim 3, characterized in that, When the formulation contains Litsea cubeba oil, the concentration of Litsea cubeba oil is less than or equal to 100 μg / mL; when the formulation contains citral, the concentration of citral is less than or equal to 100 μM.

5. The formulation for preventing photoaging according to claim 3, characterized in that, The preparation is a topical cream.

6. The formulation for preventing photoaging according to claim 4, characterized in that, The topical cream does not contain retinoic acid.

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