Application of relieving and moisturizing effects of cineole in facial dermatitis rehabilitation

Abstract

The invention belongs to the technical field of application of cineole, discloses application of relieving and moisturizing effects of cineole in facial dermatitis rehabilitation, and comprises application of cineole in preparation of a medicine for facial dermatitis rehabilitation. The Cin can play an antioxidant role by regulating expression of gene transcription and protein levels of Bax, Bcl-2 and inflammatory factors IL-1beta and regulating ROS, SOD and MDA, and plays a role in protecting H2O2-induced HaCaT cells and menadione-induced oxidative stress of zebrafish, so that the cineole is proved to have a protective role in epidermal keratinocytes under oxidative stress, and the cineole can be used for preparing a medicine for treating the oxidative stress of the zebrafish. Treating zebra fish with sodium chloride, observing the influence of the tail area of the zebra fish, and evaluating the moisturizing effect of Cin; by inhibiting expression of TRPV1 and TRPV4, opening of ion channels is inhibited, nerve impulse conduction is inhibited to play a soothing role, and dermatitis or skin discomfort caused by stimulation of ultraviolet rays, polluted air, drugs and the like on skin can be treated or relieved.

Description

Technical Field

[0001] The invention relates to the technical field of application of eucalyptol, and in particular to application of eucalyptol's soothing and moisturizing effects in the rehabilitation of facial dermatitis. Background Art

[0002] The skin is the largest organ in the human body. Various internal and external stimuli can cause oxidative stress in the skin and stimulate the production of reactive oxygen species (ROS). Excessive ROS will produce relatively stable oxidants in the body, including 4-hydroxy-2-nonenal and malondialdehyde (MDA). These oxidants can change the structure of proteins, induce cell apoptosis, and regulate the release of inflammatory cytokines, thereby exacerbating skin aging and inflammation. In addition, the occurrence and development of various skin diseases are closely related to oxidative stress and inflammatory response. Previous studies on atopic dermatitis, psoriasis, alopecia areata, lichen planus, pemphigus, skin malignancies and other diseases have found an increase in ROS-related markers and inflammatory cytokines. Therefore, studying the clinical application of new antioxidants is of great significance for the treatment of various skin diseases related to oxidative stress.

[0003] Cineole (1,8-cineole, Cin), also known as eucalyptol, is a monoterpene oil found in many plants (mainly eucalyptus species) and is the main component of eucalyptus oil. When it was first purified into essential oils in the 1960s, it was mostly used in herbicides because of its herbicidal and insecticidal activity. With the in-depth study of Cin, it has been found that Cin also has antibacterial and analgesic pharmacological effects, so it is gradually used to treat cardiovascular diseases, respiratory diseases, digestive diseases, Alzheimer's disease and other diseases. Studies have shown that Cin has great potential in antioxidants in the field of dermatology. There are no reports on the application of eucalyptol in the rehabilitation of facial dermatitis, and there is no verification method for whether eucalyptol can act on the rehabilitation of facial dermatitis.

[0004] Therefore, it is necessary to study the application of eucalyptus in the preparation of drugs for the recovery of facial dermatitis to solve the above problems. Summary of the invention

[0005] The present invention aims to provide an application of the soothing and moisturizing effect of eucalyptol in the rehabilitation of facial dermatitis, so as to solve the deficiencies in the above-mentioned prior art.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] Application of eucalyptol in preparing medicine for recovery of facial dermatitis.

[0008] Furthermore, the concentration of eucalyptol is 0.00625%.

[0009] Furthermore, the causes of dermatitis include skin being subjected to various physical and chemical stimuli inside and outside the body, including ultraviolet rays, polluted air, drugs and cosmetics.

[0010] Furthermore, the cineole is prepared by freezing and separating eucalyptus oil after distillation.

[0011] The beneficial effects of the technical solution are:

[0012] The Cin of the present invention can play an antioxidant role by regulating the expression of Bax, Bcl-2 and inflammatory factor IL-1β gene transcription and protein level, as well as regulating ROS, SOD and MDA, and has an anti-oxidative effect on H 2 O 2 The results showed that eucalyptus buds had a protective effect on oxidative stress in induced HaCaT cells and zebrafish induced by menadione, proving that eucalyptus buds had a protective effect on epidermal keratinocytes under oxidative stress. The moisturizing effect of Cin was evaluated by treating zebrafish with sodium chloride and observing the effect on their tail area. It inhibited the expression of TRPV1 and TRPV4, thereby inhibiting the opening of ion channels and inhibiting the conduction of nerve impulses. It can treat or relieve dermatitis or skin discomfort caused by skin stimulation such as ultraviolet rays, polluted air, drugs and cosmetics. Specifically, through in vivo experiments, after pre-treating HaCaT cells with Cin, H 2 O 2 Stimulate cells to cause oxidative stress, and then observe the proliferation activity of HaCaT cells, the expression of apoptotic proteins, the levels of ROS-related markers and inflammatory indicators; in the in vivo experiment, after incubating zebrafish with Cin and menadione at the same time, the fluorescence intensity of the zebrafish yolk sac and the levels of SOD and MDA in the body were detected to explore the antioxidant effect of Cin in zebrafish. Both in vivo and in vitro experiments have proved that eucalyptol has an antioxidant effect, and 0.00625% Cin has the best effect. At the same time, 0.00625% Cin can reverse the decrease in SOD and the increase in MDA in zebrafish caused by menadione stimulation, that is, Cin can alleviate oxidative stress damage in zebrafish. Whether in vivo or in vitro, Cin has a certain antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is an analytical curve diagram of the free radical scavenging ability of eucalyptol detected by the DPPH method and the PTIO method in Example 1 of the present invention.

[0014] Figure 2 In Example 2 of the present invention, different concentrations of Cin / H 2 O 2The proliferation activity of HaCaT cells treated at different times, in which A is a histogram of relative proliferation activity of HaCaT cells incubated with different concentrations of Cin for 24 hours; B is a histogram of relative proliferation activity of HaCaT cells treated with different concentrations of Cin for 24 hours; 2 O 2 Histogram of relative proliferation activity of HaCaT cells after incubation for 24 h; C is 0.5 mM H 2 O 2 Histogram of relative proliferation activity of HaCaT cells after incubation for different time periods;

[0015] Figure 3 In Example 3 of the present invention, 0.5 mM H 2 O 2 Comparison histogram of intracellular reactive oxygen species levels in HaCaT cells after 24 h of treatment compared with cells in the blank group;

[0016] Figure 4 In Example 4 of the present invention, HaCaT cells were pretreated with Cin at different concentrations (0, 0.001%, 0.01%) for 24 h and then 0.5 mM H 2 O 2 Electrophoresis and protein relative expression histograms after stimulating cells for 24 hours; in the figure, A is the electrophoresis of Bax and Bcl-2 proteins; B is the histogram of Bax protein relative expression; C is the histogram of Bcl-2 protein relative expression; D is the histogram of Bcl-2 / Bax ratio;

[0017] Figure 5 The figure is a histogram of the relative expression of Bax and Bcl-2 at the mRNA level in Example 5 of the present invention; in the figure, A is a histogram of the relative expression of Bax at the mRNA level; B is a histogram of the relative expression of Bcl-2 at the mRNA level; C is a histogram of the Bcl-2 / Bax ratio;

[0018] Figure 6 The figure is a histogram of the relative expression of IL-1β and IL-6 at the mRNA level in Example 5 of the present invention; in the figure, A is a histogram of the relative expression of IL-1β at the mRNA level; B is a histogram of the relative expression of IL-6 at the mRNA level;

[0019] Figure 7 The figure is a histogram of the relative expression of Nrf-2 and HO-1 at the mRNA level in Example 5 of the present invention; in the figure, A is a histogram of the relative expression of Nrf-2 at the mRNA level; B is a histogram of the relative expression of HO-1 at the mRNA level;

[0020] Figure 8The figure is a histogram of the expression of SOD and MDA in the cell supernatant in Example 6 of the present invention; in the figure, A is a histogram of the expression of SOD in the cell supernatant; B is a histogram of the expression of MDA in the cell supernatant;

[0021] Fig. 9 Schematic diagram of the fluorescence intensity of zebrafish yolk sac after co-incubation of zebrafish with different concentrations (0.00625% and 0.0125%) of Cin or 0.0125% glutathione and menadione for 22 hours in Example 7 of the present invention, in which A: normal control group, B: solvent control group, C: model control group; D: 0.0125% glutathione positive control group; E: 0.00625% Cin group; F: 0.0125% Cin group;

[0022] Fig.10 The relative quantitative histogram of zebrafish yolk sac fluorescence after incubating zebrafish with different concentrations (0.00625% and 0.0125%) of Cin or 0.0125% glutathione and 1.25 μM menadione for 22 hours in Example 7 of the present invention;

[0023] Fig.11 The figure is a histogram showing the effect of different concentrations of eucalyptol on SOD in zebrafish in Example 7 of the present invention, in which zebrafish were co-incubated with different concentrations (0.00625% and 0.0125%) of Cin or 0.0125% glutathione and 1.25 μM menadione for 22 hours; in the figure, A shows the effect of different concentrations of eucalyptol on SOD in zebrafish; B shows the effect of different concentrations of eucalyptol on MDA in zebrafish;

[0024] Fig.12 This is a typical moisturizing diagram of 0.05% cosmetic grade sodium hyaluronate and 0.00625% eucalyptol (eucalyptol) in Example 8 of the present invention;

[0025] Fig.13 This is a bar graph of the relative expression level of the TRPV1 gene in Example 8 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0027] Example 1

[0028] Study on the antioxidant activity of Cin.

[0029] (1) 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging ability

[0030] Take 1 mg of DPPH solid and dissolve it in 24 ml of anhydrous ethanol. After sufficient shaking and mixing, store it in the dark. Take 5 ml of the DPPH solution prepared in the above steps and add an appropriate amount of anhydrous ethanol so that the absorbance measured at a wavelength of 517 nm is between 0.6-1.0. Add 100 ul of the prepared DPPH-anhydrous ethanol solution and 100 ul of Cin solution of different volume concentrations to a 96-well plate in sequence. Make three replicate wells for each concentration, mix well, react at room temperature in the dark for 2 hours, and then measure the absorbance A at a wavelength of 517 nm; use distilled water instead of the sample as a blank control group, mix it with 100 ul of DPPH-anhydrous ethanol solution, measure the absorbance A0, and calculate the DPPH free radical scavenging rate I:

[0031]

[0032] (2) Free radical scavenging ability of 3-oxo-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxide (PTIO)

[0033] Take 3mg of PTIO solid powder and dissolve it in 20ml of distilled water. After sufficient shaking and mixing, obtain PTIO test solution. Take 5ml of the PTIO test solution prepared in the above steps and add an appropriate amount of distilled water so that the absorbance measured at a wavelength of 557nm is between 0.6-1.0. Add 100ul of the prepared PTIO test solution and 100ul of Cin solution of different volume concentrations to a 96-well plate in sequence. Make three replicate wells for each concentration, mix well, react at room temperature in the dark for 2h, and then measure the absorbance E at a wavelength of 557nm; use distilled water instead of the sample as a blank control group, mix with 100ul of PTIO test solution, measure the absorbance value E0, and calculate the PTIO free radical scavenging rate K:

[0034]

[0035] From this we can draw the following conclusions: Figure 1 As shown in the results, within the experimental concentration range, with the increase of eucalyptol concentration, the DPPH free radical scavenging rate gradually increased, showing a positive correlation (r = 1.00), and the IC50 of eucalyptol scavenging DPPH free radicals was 48.646%. Similarly, eucalyptol also has a good scavenging effect on PTIO free radicals. With the increase of eucalyptol concentration, the PTIO free radical scavenging rate is positively correlated with the volume concentration (r = 1.00), and the IC50 of eucalyptol scavenging PTIO free radicals is 50.615%.

[0036] Example 2

[0037] CCK8 method was used to detect different concentrations of Cin and H 2 O 2 Effects on the proliferation activity of HaCaT cells.

[0038] The 3rd-4th generation HaCaT cells in good growth state and in the logarithmic growth phase were selected and plated in 96-well plates with 8×10^3 HaCaT cells per well. After gently shaking by the cross method, the following groups were randomly divided into the following groups: blank control group (containing only culture medium), normal control group (containing only HaCaT cells and equal amount of culture medium), experimental group ((cells + different concentrations of Cin solution (0.001%, 0.01%, 0.1% (v / v)) / (cells + different concentrations of H 2 O 2 The 96-well plate with cells was placed at 37°C and 5% CO 2 The cells were placed in an incubator overnight. On the second day, after the HaCaT cells were observed to be attached to the wall and growing under a microscope, the old culture medium was removed and 100 μl of culture medium with different concentrations of Cin solution or ordinary culture medium was added to each well according to the grouping. After gently shaking, the cells were placed back in an incubator at 37°C and 5% CO. 2 Incubate in the incubator for 6h / 18h / 24h according to the experimental design. After the incubation time, remove the old culture medium, wash once with PSB, add 110μl culture medium containing 10μl CCK-8 to each well and continue to incubate in the incubator in the dark for 2h, then use a microplate reader to detect the absorbance at 450nm. The relative cell proliferation rate is calculated as follows: relative cell proliferation rate = (absorbance value of each experimental group - absorbance value of the blank group) / (absorbance value of the control group - absorbance value of the blank group) × 100%, and perform statistical analysis on the data.

[0039] In summary, the CCK8 method was used to detect different concentrations (0, 0.001%, 0.01% and 0.1%) of Cin to treat HaCaT cells for 24 hours. The results showed that after treating HaCaT cells with different concentrations of Cin, the cell growth rate of the 0.1% Cin 24-hour treatment group was significantly different from that of the blank group (P<0.05). However, there was no significant difference in the cell growth rate of the 0.001% and 0.01% Cin groups compared with the blank group (P>0.05). Therefore, 0.01% Cin was selected as the optimal experimental concentration for subsequent experiments, such as Figure 2 As shown in A (compared with the control group, ns: P>0.05; *: P<0.05; ###: P<0.0001).

[0040] CCK8 assay was used to detect H 2 O 2 HaCaT cells were treated for 24 h. 2 O 2After treatment of HaCaT cells with 0.5 mM, 1.0 mM and 2.0 mM H 2 O 2 Compared with the blank group, the cell growth rate of the two groups was significantly different (P<0.05), but the 1.0nM and 2.0mM H 2 O 2 After treatment of HaCaT cells, the cell activity decreased significantly, affecting the subsequent extraction of mRNA and protein detection. Therefore, 0.5 mM H was selected to induce oxidative stress in HaCaT cells without causing excessive cell death, allowing for the next step of intervention and treatment. 2 O 2 The concentrations were used in subsequent experiments, such as Figure 2 As shown in B (compared with the control group, ns: P>0.05; *: P<0.05; ###: P<0.0001).

[0041] The CCK8 assay was used to detect 0.5 mM H 2 O 2 HaCaT cells were treated for 0, 6, 18 and 24 hours. The results showed that 0.5mMH 2 O 2 After treating HaCaT cells for different time periods, the cell growth rates of the 24h treatment group were significantly different from those of the blank group (P<0.05), while the cell growth rates of the 6h and 18h ​​treatment groups were not significantly different from those of the blank group (P>0.05). 2 O 2 HaCaT cells were treated for 24 h as the treatment condition for inducing oxidative stress in HaCaT cells for subsequent experiments, see Figure 2 C (compared with the control group, ns: P>0.05; *: P<0.05; ###: P<0.0001).

[0042] Example 3

[0043] Human immortalized keratinocytes (HaCaT cells) used in the in vitro experiments were purchased from the Cell Bank of the Chinese Academy of Sciences (cell line number: SCSP-5091). The steps for establishing the HaCaT cell oxidative stress model are as follows:

[0044] S1. Preparation of HaCaT cells:

[0045] (1) Select HaCaT cells in good growth state and in the logarithmic growth phase and plate 96-well plates at a density of 8 × 103 HaCaT cells per well;

[0046] (2) Cross method: Gently shake and place at 37°C and 5% CO 2Incubate overnight in an incubator to allow the cells to adhere;

[0047] (3) After the cells were completely attached, the cells were treated according to the following groups: ① Blank group: incubated in complete medium for 24 h and then replaced with new complete medium for another 24 h; ② 2 O 2 Stimulation group: incubate in complete medium for 24 h and then add 0.5 mM H 2 O 2 The solution was incubated for 24 h; ③0.01% Cin group: HaCaT cells were pretreated with 0.01% Cin for 24 h, and then 0.5 mM H 2 O 2 The solution was incubated for 24 h; 5 replicate wells were set up for each group.

[0048] S2. Detect the level of reactive oxygen species (ROS) in cells:

[0049] (1) After the HaCaT cells in the 96-well plate were grouped and treated as in S1, the old culture medium in each well was removed with a pipette and the cells were washed once with phosphate buffered saline (PBS);

[0050] (2) Add 100 μl of DCFH-DA solution diluted 1:1000 in basal medium to each well, mix gently, and place at 37°C and 5% CO 2 Incubate in an incubator away from light for 20 min;

[0051] (3) Remove the DCFH-DA solution and wash the cells three times with PBS buffer to fully remove the DCFH-DA that has not entered the cells;

[0052] (4) Use a fluorescence microplate reader to detect the DCF value at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and perform statistical analysis on the data.

[0053] In summary, the fluorescent probe DCFH-DA method was used to detect H 2 O 2 The level of reactive oxygen species in HaCaT cells after 24 h of treatment. The results showed that 0.5 mM H 2 O 2 Compared with the blank control group, the intracellular reactive oxygen species level of HaCaT cells treated with the 24-hour irradiation was significantly different (P<0.01). Figure 3 (Compared with the control group, **: P < 0.01). 0.5 mM H 2 O 2 After 24h of stimulation, the cell activity was different from that of the blank group, and ROS was significantly increased, so it was considered that the oxidative stress model was successfully established.

[0054] Example 4

[0055] Western Blot was used to detect the effect of Cin pretreatment on H 2 O 2 Effect of Bax and Bcl-2 protein expression on cell apoptosis caused by stimulation.

[0056] Total protein extraction:

[0057] (1) Take HaCaT cells in good growth condition and plate them in 6-well plates at 3×10^5 cells per well. Incubate at 37°C and 5% CO 2 (1) Incubate the cells overnight in an incubator to allow the cells to adhere; (2) After the cells are completely attached, group the cells according to step S1 of Example 2; (3) Remove the old culture medium with a pipette on ice, add 1 ml of pre-cooled PBS to wash twice, and discard the PBS; (4) Add 80 ul of cell lysis buffer RIPA containing protease inhibitors (PMSF) to each well, and place the 6-well plate on an ice shaker for 30 minutes; (5) Scrape the cells with the thick end of the pipette to allow the cells to fall off completely, and transfer the cell solutions of each group into a pre-cooled new EP tube; (6) Centrifuge at 12000 rpm / min, 4°C for 15 minutes, and transfer the supernatant into a new EP tube.

[0058] BCA protein quantification:

[0059] (1) Prepare BCA reagent (liquid A: liquid B = 50:1) for use; (2) Use a 96-well plate for protein quantitative loading, dilute the BCA standard into 8 concentration gradients with PBS, add 20 μl of different concentrations of standard and 200 μl of BCA reagent to the standard well, add 20 μl of 10-fold diluted sample and 200 μl of BCA reagent to the sample well, mix gently, place the 96-well plate in a 37°C incubator for 30 min, and then measure the absorbance of the sample at 562 nm; (3) Draw a standard curve, calculate the protein concentration before sample dilution, then configure the system according to the required concentration and load the samples one by one; (4) Preheat the water bath to 100°C, heat for 20 min to fully denature the protein, then divide the sample into smaller pieces and store them in a -20°C refrigerator.

[0060] SDS-PAGE protein electrophoresis

[0061] Glue making: (1) Fix two thick and thin glass plates on the glue making rack and keep the bottom of the plates flush; (2) According to the instructions for quick glue making, take equal volumes of Resolver A and Resolver B, Stacker A and Stacker B and mix them for later use; (3) Add an appropriate amount of APS to the lower glue mixed solution of step (2), mix it thoroughly immediately, and then inject it into the glue making glass plate so that the liquid level is about 1.5 cm from the upper edge of the short glass plate; (4) Add an appropriate amount of APS to the upper glue mixed solution of step (2), mix it thoroughly immediately, and without waiting for the lower glue to solidify, you can gently inject the upper glue solution into the glue making glass plate along the long glass plate and gently insert the comb teeth. (5) Let it stand at room temperature for 15-20 minutes. After the glue is completely solidified, it can be used for electrophoresis.

[0062] Loading and running the gel: (1) Take the sample out of the refrigerator and thaw it; (2) After the gel solidifies, take out the glass plate and fix it, place it in the electrophoresis tank, pour in the electrophoresis solution, and then pull out the comb vertically with both hands. Be careful not to damage the sample wells when pulling it out; (3) Shake the sample well, and add the marker and the sample to be tested into the wells respectively; (4) Cover the electrophoresis tank, turn on the power, and run at 160V for 60 minutes; (5) When the band reaches the bottom of the gel, turn off the power, stop electrophoresis, and take out the glass plate;

[0063] Transfer: (1) First, cut the PVDF membrane and filter paper of corresponding size according to the size of the gel, mark the PVDF membrane, and place it in a box filled with methanol for activation; (2) Gently pry up the glass plate, cut the gel according to the molecular weight of the target protein and the marker, and gently move the gel to a tray filled with transfer solution; (3) Open the transfer clamp in the transfer solution, with the black side facing the bottom, and place the sponge, filter paper, gel, PVDF membrane, filter paper and sponge in order, and then clamp the transfer clamp. During operation, be careful to avoid bubbles between the gel and the membrane; (4) Place the transfer clamp in the electrophoresis tank filled with transfer solution, place the electrophoresis tank on ice, cover it, connect the power supply, and transfer at a constant current of 330mA for 120 minutes.

[0064] Blocking, primary and secondary antibody incubation, development and result analysis: (1) After the transfer, remove the PVDF membrane from the electrophoresis tank and place it in a box containing 5% skim milk prepared with TBST solution. Block it on a shaker at room temperature for 1.5-2 hours; (2) Wash the membrane once with TBST solution; (3) Pick up the PVDF membrane with plastic tweezers, place it on plastic wrap, cover it, and use a blade to cut the membrane fragment where the target band is located; (4) Primary antibody incubation: Incubate overnight at 4°C (where the dilution ratio of β-actin is 1:5 000, Bax is 1:1000, Bcl-2 is 1:1000); (5) Washing: Wash with TBST on a shaker for 3 times, 10 min each time; (6) Secondary antibody incubation: Incubate at room temperature for 1 h (the dilution ratio of goat anti-rabbit and goat anti-mouse is 1:10000); (7) Washing: Wash with TBST on a shaker for 3 times, 10 min each time; (8) Developing: Take equal volumes of A and B solutions to prepare ECL chemiluminescent solution, evenly cover the entire membrane with the luminescent solution and place it in the developer for processing; (9) Use Image J software to process and analyze the results. Figure 4 A shows the electrophoresis diagram of Bax and Bcl-2 proteins.

[0065] In summary, Western Blot was used to detect the effect of different concentrations (0, 0.001% and 0.01%) of Cin pretreatment on HaCaT cells for 24 h and then 0.5 mM H 2 O 2 The effect of 24h stimulation on the expression of Bax and Bcl-2 proteins. The results showed that compared with the blank group, H 2 O 2 The expression of Bax in the stimulation group increased significantly (P<0.01), suggesting that H 2 O 2 The oxidative stress caused by H 2 O 2 Compared with the stimulation group, with the increase of Cin concentration, the expression of Bax protein decreased more obviously, and the result of 0.01% Cin group was statistically significant (P<0.0001), indicating that Cin has an important effect on H 2 O 2 The induced pro-apoptotic effect has an inhibitory effect and a protective effect on HaCaT cells, such as Figure 4 As shown in B (compared with the control group, ns: P>0.05; **: P<0.01; ###: P<0.0001).

[0066] Compared with the blank group, H 2 O 2 The expression of Bcl-2 protein in the stimulation group was decreased, but there was no statistical difference (P>0.05). 2 O 2Compared with the stimulation group, with the increase of Cin concentration, the protein expression of Bcl-2 gradually increased, but compared with H 2 O 2 There was no statistical difference in the results between the stimulation groups (P>0.05). Figure 4 As shown in C (compared with the control group, ns: P>0.05; **: P<0.01; ###: P<0.0001);

[0067] Compared with the blank group, H 2 O 2 Although the Bcl-2 / Bax ratio in the stimulation group decreased, the results were not statistically significant (P>0.05), suggesting that oxidative stress promoted cell apoptosis to a certain extent. 2 O 2 Compared with the stimulation group, the Bcl-2 / Bax ratio of the two Cin groups increased, but there was no statistical difference (P>0.05), suggesting that Cin plays a protective role to a certain extent and inhibits cell apoptosis. Figure 4 D (compared with the control group, ns: P>0.05; **: P<0.01; ###: P<0.0001).

[0068] Example 5

[0069] The qPCR method was used to detect the effects of Cin pretreatment on apoptotic proteins (Bax and Bcl-2), inflammatory factors (IL-1β and IL-6) and Nrf-2 pathway indicators (Nrf-2 and HO-1) in HaCaT cells.

[0070] S1. Kit for RNA extraction:

[0071] (1) Plate a 6-well plate according to the requirements for total protein extraction (1) and place it in a 37°C, 5% CO 2 Incubate in an incubator overnight to allow cells to adhere;

[0072] (2) After the cells are completely attached, the cells are grouped and treated according to S1(3) in Example 3;

[0073] (3) Remove the old culture medium with a pipette, add 1 ml of PBS to wash the cells once, and discard the PBS;

[0074] (4) Add 500 μl of FreeZol Reagent and pipette the cells several times;

[0075] (5) Collect cells from each group into different RNase-free EP tubes, vortex to fully lyse the cells, and let stand at room temperature for 5 min;

[0076] (6) Add 2 / 5 volume of Dilution Buffer to the above lysate (add 200 μl Dilution Buffer for every 500 μl FreeZol Reagen), cover the centrifuge tube tightly, vortex thoroughly until the solution is uniform and milky, and let it stand at room temperature for 5 min;

[0077] (7) Centrifuge at 11200 rpm for 15 min at room temperature and discard the supernatant;

[0078] (8) Carefully remove the centrifuge tube and transfer the supernatant solution (about 550 μl) to a new enzyme-free EP tube;

[0079] (9) Add an equal volume of isopropanol to the above EP tube, mix thoroughly by inversion, and let stand at room temperature for 10 min;

[0080] (10) Centrifuge at 11200 rpm for 10 min at room temperature. A white gelatinous precipitate can be seen on the sides and bottom of the tube. Carefully discard the supernatant and keep the precipitate.

[0081] (11) Add 1 ml of RNase-free ddH 2 Add 75% ethanol prepared in 0.5% ethanol, tap the bottom of the tube gently, and turn it upside down several times to suspend the precipitate;

[0082] (12) Centrifuge at 9100 rpm for 3 min at room temperature, discard the supernatant and keep the precipitate;

[0083] (13) Repeat steps 11 and 12 above and discard the supernatant;

[0084] (14) Allow to dry at room temperature and add 20-100 μl RNase-free ddH 2 O to dissolve the precipitate, and vortex for 3 minutes to fully dissolve the RNA precipitate;

[0085] (15) Perform a purity test on the RNA, label it, and then store the RNA sample in a -80°C refrigerator or directly perform reverse transcription.

[0086] S2, Reverse transcription RT-PCR:

[0087] Remove genomic DNA; prepare reverse transcription reaction system; the product obtained by reverse transcription reaction in PCR instrument can be used immediately for qPCR reaction, or can be packaged and frozen in -80℃ refrigerator.

[0088] S3, qPCR:

[0089] For primer synthesis, prepare a mixed solution in a qPCR tube. After the system is configured, use a centrifuge to instantly centrifuge the liquid in the tube; perform qPCR reaction in a qPCR instrument under the following conditions.

[0090] In summary, RT qPCR detected the expression of Cin in HaCaT cells after 24 h of pretreatment with 0.5 mM H 2 O 2 The effect of 24h stimulation on the expression of Bax and Bcl-2 mRNA levels. The results showed that compared with the blank group, H 2 O 2 The expression of Bax in the stimulation group increased, and the difference was statistically significant (P<0.05). 2 O 2 Compared with the stimulation group, the 0.01% Cin group was 2 O 2 The expression of Bax in the stimulation group decreased, and the results were statistically significant (P<0.05), suggesting that Cin can affect H 2 O 2 Induced transcription of the Bax gene, see Figure 5 As shown in A (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01).

[0091] Compared with the blank group, H 2 O 2 The mRNA expression of Bcl-2 in the stimulation group increased, and the result was statistically significant (P<0.01); 2 O 2 Compared with the stimulation group, the expression of Bcl-2 in the 0.01% Cin group decreased, but the results were not statistically significant (P<0.05). Figure 5 As shown in B (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01).

[0092] Compared with the blank group, H 2 O 2 The Bcl-2 / Bax ratio in the stimulation group increased significantly, and the result was statistically significant (P<0.01); 2 O 2 Compared with the stimulation group, the 0.01% Cin group was lower, but the results were not statistically different (P>0.05). Figure 5 As shown in C (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01).

[0093] RT qPCR detection of different concentrations (0, 0.001% and 0.01%) of Cin pretreatment of HaCaT cells for 24 h and then adding 0.5 mM H 2 O 2 Effect of 24h stimulation on the expression of IL-1β and IL-6 mRNA levels.

[0094] The results showed that compared with the blank group, H 2 O 2 The mRNA expression of IL-1β in the stimulation group increased, and the result was statistically significant (P<0.01); 2 O 2 Compared with the stimulation group, the expression of IL-1β in the 0.01% Cin group was downregulated, and the results were statistically significant (P<0.05), suggesting that Cin can alleviate H 2 O 2 The inflammatory response induced by the inflammatory reaction has a protective effect on HaCaT cells, such as Figure 6 As shown in A (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01; ###: P<0.0001);

[0095] Compared with the blank group, H 2 O 2 The expression of IL-6 in the stimulation group increased significantly, and the difference was statistically significant (P<0.0001); 2 O 2 Compared with the stimulation group, the expression of IL-6 in the 0.01% Cin group had no statistical difference (P>0.05). Figure 6 As shown in B (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01; ###: P<0.0001).

[0096] RT qPCR detection of HaCaT cells pretreated with Cin for 24 h and then added with 0.5 mM H 2 O 2 The effect of 24h stimulation on the mRNA expression of some indicators in the Nrf-2 signaling pathway. The results showed that compared with the blank group, H 2 O 2 The expression of Nrf-2 in the stimulation group increased significantly, and the difference was statistically significant (P<0.001); 2 O 2 Compared with the stimulation group, the expression of Nrf-2 in the 0.01% Cin group decreased, but the results were not statistically significant (P>0.05). Figure 7 As shown in A (compared with the control group, ns: P>0.05; **: P<0.01; ***: P<0.001);

[0097] Compared with the blank group, H 2 O 2 The expression of HO-1 in the stimulation group increased, and the difference was statistically significant (P<0.01). 2 O 2Compared with the stimulation group, the expression of HO-1 in the 0.01% Cin group decreased, and the result was statistically significant (P<0.01), suggesting that Cin may protect HaCaT cells by regulating the HO-1 factor of the Nrf-2 pathway. Figure 7 As shown in B (compared with the control group, ns: P>0.05; **: P<0.01; ***: P<0.001).

[0098] Example 6

[0099] ELISA kits were used to detect the effects of Cin pretreatment on the expressions of SOD and MDA in HaCaT cells.

[0100] (1) Spread a 6-well plate according to the requirements of total protein extraction (1), and then place the 6-well plate at 37°C and 5% CO 2 Incubate in an incubator overnight to allow cells to adhere;

[0101] (2) After the cells are completely attached to the wall, the cells are grouped and treated according to S1(4) in Example 3;

[0102] (3) Place all reagents at room temperature for at least 30 minutes in advance and prepare the reagents according to the method in the kit;

[0103] (4) The cell culture supernatant of each group was collected and centrifuged at 3000 × g for 20 min at 4°C. The supernatant was used for subsequent experiments.

[0104] (5) Add 50 μl of standard or sample to be tested to each well and gently shake to mix;

[0105] (6) Add 100 μl of enzyme-labeled reagent to each well, except for the blank well;

[0106] (7) Seal the plate with a sealing film and incubate at 37°C for 60 min;

[0107] (8) Carefully remove the sealing film covering the surface, discard the liquid, spin dry, fill each well with 20-fold diluted washing solution, let stand for 30 seconds and then discard, repeat this step 5 times, and pat dry;

[0108] (9) Add 50 μl of chromogen A and 50 μl of chromogen B to each well, shake gently to mix, and develop at 37°C in the dark for 15 min.

[0109] (10) Add 50 μl of stop solution to each well in the same order to terminate the reaction;

[0110] (11) Use the blank well as the zero value and measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader within 15 min after terminating the reaction.

[0111] In summary, ELISA was used to detect the expression of Cin in HaCaT cells after 24 h of pretreatment with 0.5 mM H 2 O 2 After stimulating the cells for 24 hours, the results showed that compared with the blank group, H 2 O 2 The expression of SOD in the stimulation group decreased, and the difference was statistically significant (P<0.01). 2 O 2 Compared with the stimulation group, the SOD expression in the 0.01% Cin group was increased, and the results were statistically significant (P<0.05), indicating that Cin has an antioxidant effect to a certain extent. 2 O 2 Stimulated HaCaT cells have protective effects, such as Figure 8 As shown in A (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01);

[0112] Compared with the blank group, H 2 O 2 The expression of MDA in the stimulation group increased, and the difference was statistically significant (P<0.01); 2 O 2 Compared with the stimulation group, the MDA expression in the 0.01% Cin group was downregulated, but the results were not statistically significant (P>0.05). Figure 8 As shown in B (compared with the control group, ns: P>0.05; *: P<0.05; **: P<0.01).

[0113] Example 7

[0114] The zebrafish used in the in vivo experiment were melanin allele mutant zebrafish (Albino), 2 days post fertilization (2dpf), and were obtained from Shanghai Huante Zhiyu Youjian Biotechnology Co., Ltd.

[0115] Zebrafish in vivo model to explore the antioxidant effect of Cin

[0116] S1. Determine the maximum detection concentration (MTC) of Cin in zebrafish

[0117] (1) Randomly select zebrafish 2 days post fertilization (2dpf) in a 6-well plate, 15 per well. (2) Water-soluble samples were administered at concentrations of 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05% and 0.1%. A normal control group, solvent group and model group were also set up, with a volume of 3 mL per well. (3) Except for the normal control group, the other experimental groups were administered 1.25μM menadione in water to establish an oxidative stress model for zebrafish. (4) Incubate at 28℃ in the dark for 22h. (5) Determine the MTC of the antioxidant effect of the sample based on the death and toxicity of zebrafish observed under a microscope.

[0118] S2. Effects of different concentrations of Cin on ROS in zebrafish

[0119] (1) 2 dpf zebrafish were randomly selected and placed in a 6-well plate, with 15 zebrafish per well.

[0120] (2) Cin with concentrations of 0.003125%, 0.00625% and 0.0125% were prepared respectively. A normal control group, a solvent control group (DMSO + menadione), a model control group (menadione) and a positive control L-reduced glutathione 0.0125% were set up at the same time. The capacity of each well was 3 mL.

[0121] (3) Except for the normal control group, the other experimental groups were given 1.25 μM menadione in water to establish the zebrafish oxidative stress model.

[0122] (4) Incubate at 28°C in the dark for 22 h.

[0123] (5) The zebrafish were stained with a specific ROS fluorescent reagent. After 1 hour of treatment, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The data were analyzed and collected using advanced image processing software. The fluorescence intensity (S) of the zebrafish yolk sac was analyzed and the antioxidant effect of the sample was calculated and determined according to the following formula:

[0124] (6) Five biological replicates.

[0125] S3. ELISA kit to detect the effect of Cin pretreatment on SOD / MDA in zebrafish tissues:

[0126] (1) 2 dpf zebrafish were randomly selected and placed in a 6-well plate, with 30 zebrafish per well.

[0127] (2) Water-soluble administration samples with concentrations of 0.003125%, 0.00625% and 0.0125%. A normal control group, a solvent control group, a model control group and a positive control L-reduced glutathione 0.0125% were set up. The volume of each well was 3 mL, and three biological replicates were performed.

[0128] (3) Except for the normal control group, the other experimental groups were given 1.25 μM menadione in water to establish the zebrafish oxidative stress model.

[0129] (4) Incubate at 28°C in the dark for 22 h.

[0130] (5) After the experiment, the zebrafish samples from each group were collected, ground and homogenized, and the supernatant was obtained by centrifugation. The SOD activity / MDA content detection kit was used to collect data using an ELISA instrument to analyze the SOD activity (A) / MDA content (C) in the zebrafish. The following formula was used to calculate and determine whether the sample had an antioxidant effect:

[0131]

[0132] In summary, the state of zebrafish 2 days after fertilization (2dpf) incubated with different concentrations of Cin was observed under a microscope for 22 hours. The results showed that the state of zebrafish incubated with 0.003125%, 0.00625% and 0.0125% Cin was similar to that of the model control group, the state of zebrafish incubated with 0.025% and 0.05% Cin was poor under the microscope, and all zebrafish incubated with 0.1% Cin died. Therefore, the MTC of the antioxidant effect of eucalyptol is 0.0125%, so Cin concentrations of 0.00625% and 0.0125% were selected for subsequent experiments, see Table 1.

[0133] Table 1 Results of the experimental study on the concentration of Cin for antioxidant efficacy (n=15)

[0134]

[0135] By comparing the fluorescence intensity of zebrafish yolk sac after incubation of zebrafish with different concentrations (0.00625% and 0.0125%) of Cin or 0.0125% glutathione and menadione for 22 hours, we found Fig. 9 As shown. The results showed that the fluorescence intensity of the zebrafish yolk sac in the model group was significantly higher than that in the normal control group, indicating that compared with the normal control group, the ROS in the model group increased significantly, and the oxidative stress model was successfully established; compared with the model group, the fluorescence intensity of the zebrafish yolk sac in the 0.00625% Cin and 0.0125% glutathione groups was significantly weakened, and the results were statistically significant (P < 0.001), while there was no statistical difference between the 0.0125% Cin group and the solvent control group (P> 0.05), indicating that Cin at a certain concentration can reduce the increase in ROS caused by menadione stimulation and has a protective effect on zebrafish, see Fig.10 As shown (ns: P>0.05; ***: P<0.001).

[0136] The ELISA method was used to detect the SOD / MDA levels in zebrafish after they were incubated with different concentrations (0.00625% and 0.0125%) of Cin or 0.0125% glutathione and menadione for 22 hours.

[0137] The results showed that compared with the normal control group, the SOD expression in the model group decreased significantly, and the result was statistically significant (P<0.05), indicating that menadione induced oxidative stress in zebrafish; compared with the model group, the SOD activity in zebrafish in the 0.00625% Cin and 0.0125% glutathione groups increased significantly, and the difference was statistically significant (P<0.05), while there was no statistical difference between the 0.0125% Cin group and the model group (P>0.05); compared with the glutathione group, there was no difference in SOD expression in the 0.00625% Cin group (P>0.05), while there was a difference in 0.0125% Cin group, and the result was statistically significant (P<0.05), indicating that 0.00625% Cin can reverse the decrease in SOD activity induced by menadione and protect zebrafish, and its effect is similar to that of 0.0125% glutathione, such as Fig.11 As shown in A (ns: P>0.05; *: P<0.05; **: P<0.01).

[0138] Compared with the normal control group, the MDA content in the model group was significantly increased, and the result was statistically significant (P<0.01); compared with the model group, the MDA content in zebrafish in the 0.00625% and 0.0125% Cin groups and the 0.0125% glutathione group was decreased, and the difference was statistically significant (P<0.01); compared with the glutathione group, there was no difference in the MDA expression in the 0.00625% and 0.0125% Cin groups (P>0.05), indicating that Cin at a concentration of 0.0125% and below can reverse the increase in MDA content induced by menadione and play a protective role in zebrafish, and its effect is similar to that of 0.0125% glutathione, see Fig.11 As shown in B (ns: P>0.05; *: P<0.05; **: P<0.01).

[0139] In summary, the present invention firstly determines the radical antioxidant activity of Cin by DPPH and PTIO free radical scavenging experiments, then screens out the Cin concentration that has no effect on the proliferation activity of HaCaT cells by CCK8 method in in vitro experiments, and simultaneously detects ROS by fluorescent probe method to determine the H that stimulates HaCaT cells to produce oxidative stress. 2 O 2 The concentration and time were finally determined to be 0.01% Cin pretreatment for 24 h and then 0.5 mM H 2 O 2HaCaT cells were stimulated, and the expressions of Bax, bcl-2, IL-1β, IL-6, NRF-2 and HO-1 at the mRNA or protein level were measured, and the secretion of SOD and MDA in the cell supernatant was detected; in the in vivo experiment, the ROS, SOD and MDA contents produced by zebrafish incubated with menadione at different concentrations of Cin were detected, and the following conclusions were drawn: Cin itself has the ability to scavenge DPPH and PTIO free radicals; 0.5mM H 2 O 2 Treatment for 24 h successfully induced the oxidative stress model of HaCaT cells; Cin could play an antioxidant role by regulating the gene transcription and protein expression of Bax, Bcl-2 and inflammatory factor IL-1β, as well as regulating ROS, SOD and MDA, and had an anti-oxidative effect on H 2 O 2 The results showed that eucalyptol has a protective effect on oxidative stress in induced HaCaT cells and zebrafish induced by menadione, which proves that eucalyptol has a protective effect on epidermal keratinocytes under oxidative stress, and can treat or alleviate dermatitis or skin discomfort caused by ultraviolet rays, polluted air, drugs and cosmetics. Among them, the Nrf-2 pathway can participate in the antioxidant mechanism of eucalyptol.

[0140] Example 8

[0141] The moisturizing effect of eucalyptol was tested. The test method and process are shown in Table 2:

[0142] Table 2 Detection method and process of moisturizing effect of eucalyptol

[0143]

[0144] The test results are shown in Table 3 below:

[0145] Table 3 Eucalyptol moisturizing effect test results

[0146]

[0147] From the above results and Fig.12 (The red dotted line is the quantitative area) The following conclusions can be drawn:

[0148] The tail area of ​​the sample eucalyptol (cineole) group was significantly increased compared with the model control group, revealing that the sample eucalyptol (cineole) has a moisturizing effect.

[0149] Eucalyptol was tested for its soothing effect. The test method and process are shown in Table 4:

[0150] Table 4 Eucalyptol soothing effect detection method and process

[0151]

[0152]

[0153] The test results are shown in Table 5 below:

[0154] Table 5 Eucalyptol soothing effect test results

[0155]

[0156] From the above results and Fig.13 (Compared with the model control group, *p<0.05, ***p<0.001) The following conclusions can be drawn:

[0157] The relative expression level of TRPV1 gene in the sample eucalyptol (cineole) group was significantly downregulated compared with the model control group, revealing that the sample eucalyptol (cineole) has a soothing effect.

[0158] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of eucalyptol in the preparation of medicine for the recovery of facial dermatitis.

2. The use according to claim 1, characterized in that: The concentration of eucalyptol is 0.00625%.

3. The use according to claim 1, characterized in that: The causes of dermatitis include the skin being subjected to various physical and chemical stimuli from both inside and outside the body, including ultraviolet rays, polluted air, drugs and cosmetics.

4. The use according to any one of claims 1 to 3, characterized in that: The cineole is prepared by freezing and separating eucalyptus oil after distillation.

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