Application of saposhnicovia divaricata essential oil in preparation of product for treating and preventing acne

Abstract

The invention discloses an application of radix saposhnikoviae essential oil in preparation of products for treating and preventing acnes, and the essential oil extracted from Chinese herbal medicine radix saposhnikoviae can prevent and treat acnes through anti-inflammation and anti-oxidation. A mouse ear acne model induced by propionibacterium acnes verifies that the saposhnicovia divaricata essential oil has a relieving effect on red and swollen ears of acne-like mice, inflammatory factor aggregation, release of active oxygen in cells and the like. The saposhnicovia divaricata essential oil shows an inhibition effect on NLRP3 inflammasome activation on a mouse ear skin acne model induced by propionibacterium acnes, and the expression of IL-1beta, IL-6 and TNF-alpha inflammatory factors in mouse tissues is remarkably reduced. The composition has remarkable activity in the aspects of diminishing inflammation, resisting oxidation and removing acnes, and has a better prospect in preparation of products for treating and preventing inflammatory acnes.

Description

Technical Field

[0001] The invention relates to application of fangfeng essential oil in preparing products for treating and preventing acne. Background Art

[0002] Acne is a common chronic inflammatory skin disease caused by abnormal secretion of sebaceous glands, which manifests as lesions such as pustules, eczema, papules and cysts, usually on the face and chest. Since 80% to 90% of adolescents suffer from moderate to severe acne vulgaris, which can persist into adulthood, acne vulgaris has become a common skin condition in adolescents and young people. Although acne is generally considered a cosmetic problem, the disease can cause permanent scarring and even disfigurement, and is also associated with various psychological problems, such as depression, anxiety, frustration and social isolation. Propionibacterium acnes is a pathogen that is extremely closely related to it. Relevant literature reports that infection with Propionibacterium acnes can cause the production of ROS and activate a series of inflammatory responses, activating the NLRP 3 inflammasome and increasing the expression of inflammatory factors such as interleukin (IL)-1β. From inflammatory herpes and pustules to post-inflammatory erythema and post-inflammatory pigmentation, the inflammatory response runs through the entire process of acne pathogenesis. This is the main pathological feature of acne and is also a problem that patients are particularly worried about. At present, hormones and retinoic acid drugs are commonly used in clinical treatment of acne. The therapeutic effect of these drugs is relatively ideal, but long-term use is prone to produce some toxic side effects on the human body; after antibacterial drugs kill pathogens, the residual bacteria and antigenic proteins will still stimulate facial inflammation. Steroidal anti-inflammatory drugs have good anti-inflammatory activity but are prone to pigmentation and cannot be accepted by patients. The essential oil extracted from the Chinese herbal medicine Fangfeng plant has significant anti-inflammatory effects. Because of its characteristics of small toxic side effects, high safety, good permeability, low-priced and easy-to-obtain raw materials, it will have a huge market space.

[0003] Microbial infection, endogenous danger signals and environmental stimulation are all associated with the NLRP3 inflammasome, a complex composed of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck protein (ASC) and pro-caspase-1. Activation of the NLRP3 inflammasome is manifested by increased expression of Caspase1 and IL-1β.

[0004] Saposhnikovia divaricata is a perennial herb widely distributed in eastern Siberia and northern Asia. Its dried root is a traditional Chinese medicine for treating immune and respiratory diseases. The essential oil extracted from the root of Saposhnikovia divaricata by steam distillation has been reported to inhibit the production of IL-1β inflammatory factor. However, whether it regulates the process of IL-1β production mediated by NLRP 3 inflammasome has not been revealed. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide an application of fangfeng essential oil in the preparation of products for treating and preventing acne. The fangfeng essential oil has significant antioxidant and skin inflammation improving activity and thus improves acne, and can be used to prepare functional cosmetics such as acne removal and repair, traditional Chinese medicine gel and other applications.

[0006] The technical solution to achieve the above purpose is: application of fangfeng essential oil in preparing products for treating and preventing acne.

[0007] The application of the above-mentioned fangfeng essential oil, wherein the essential oil is obtained by crushing the Chinese herbal medicine fangfeng and then extracting it through a steam distillation method.

[0008] The application of the above-mentioned Saposhnikovia divaricata essential oil, wherein the essential oil inhibits the generation of ROS caused by Propionibacterium acnes in vivo and has antioxidant activity.

[0009] The application of the above-mentioned fangfeng essential oil, wherein the essential oil inhibits the expression of IL-1β, IL-6 and TNF-α inflammatory factors in the body, and prevents the activation of NLRP3 inflammasome caused by Propionibacterium acnes, has anti-inflammatory activity, and inhibits the activation of inflammasome.

[0010] The above-mentioned application of the fangfeng essential oil, wherein the inhibition of inflammasome activation is to inhibit the cleavage of Caspase-1 protein in inflammasome-induced mice, inhibit the maturation of IL-1β in inflammasome-induced mice, inhibit the secretion of IL-1β in inflammasome-induced mice, and inhibit the formation of GSDMD-NT in inflammasome-induced mice.

[0011] The above-mentioned application of the Saposhnikovia divaricata essential oil, wherein the products include medicines and daily chemical products.

[0012] The application of the above-mentioned fangfeng essential oil, wherein the weight content of the essential oil in the product is 0.1%-1%.

[0013] The above-mentioned application of the fangfeng essential oil, wherein the dosage form of the medicine is at least one of capsules, pills, injections, aerosol powders, films, patches, emulsions, ointments, liniments, creams and lotions.

[0014] The application of the above-mentioned windproof essential oil, wherein the daily chemical product is at least one of shampoo, shampoo cream, shower gel, body lotion, essential oil soap, cooling oil, wind oil essence, toner, floral water, facial cream, facial mask and facial essence.

[0015] The invention discloses an application of the fangfeng essential oil in preparing a product for treating and preventing acne. The fangfeng essential oil has significant anti-oxidation and skin inflammation improving activities, thereby improving acne. After preparing an ultrasonically broken Propionibacterium acnes induced mouse acne model, hydrogels of fangfeng essential oil extracts in different proportions are given, and by measuring the degree of mouse skin swelling and the expression of macrophages and inflammatory factors in the skin, it is confirmed that the fangfeng essential oil has the effect of improving the mouse acne model induced by Propionibacterium acnes, indicating that the fangfeng essential oil can be used for preventing and treating inflammatory acne, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the total ion chromatogram of GC-MS analysis of fangfeng essential oil;

[0017] Figure 2 Toxicity of Saposhnikovia essential oil on J774A.1 macrophages;

[0018] Figure 3 To study the effect of fangfeng essential oil on the expression of inflammatory factors in J774A.1 cells induced by Propionibacterium acnes;

[0019] Figure 4 To study the effect of fangfeng essential oil on ear redness and swelling in acne mice induced by Propionibacterium acnes;

[0020] Figure 5 To study the effect of fangfeng essential oil on ear thickness in mice with acne induced by Propionibacterium acnes.

[0021] Figure 6 This is a comparison of the effect of Fangfeng essential oil on the ear skin of acne mice induced by Propionibacterium acnes.

[0022] Figure 7 This is a comparison of the DHE staining of the ear skin of acne mice induced by Propionibacterium acnes using saposhnikovia essential oil;

[0023] Figure 8 To study the effect of fangfeng essential oil on macrophages in the ear skin of acne mice induced by Propionibacterium acnes;

[0024] Fig. 9 The effect of Fangfeng essential oil on the expression level of inflammatory factors in the ear skin tissue fluid of acne mice induced by Propionibacterium acnes (A is TNF-α inflammatory factor, B is IL-1β inflammatory factor, C is IL-6 inflammatory factor);

[0025] Fig.10 Western Blotting analysis of proteins in the ear skin of acne mice induced by Propionibacterium acnes was performed for the essential oil of Saposhnikovia divaricata. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods thereof are described in detail below in conjunction with the accompanying drawings:

[0027] Embodiment 1: identification of chemical components of Radix Saposhnikoviae essential oil

[0028] (1) Preparation of test samples

[0029] Take 700±5 mg of Saposhnikovia essential oil in a 20 mL headspace bottle, and then add 10 μL of 2-octanol (10 mg / L stock indH2O) as the internal standard.

[0030] (2) GC-MS conditions

[0031] In the SPME cycle of the PAL track system, the extraction temperature was 60 °C, the preheating time was 15 min, the extraction time was 30 min, and the desorption time was 4 min. This experiment used an Agilent 7890 gas chromatograph system and a 5977B mass spectrometer and a DB-Wax (30 m × 250 μm × 0.25 μm) chromatographic column. Helium was used as the carrier gas, and the septum purge flow rate was 3 mL min -1 The gas flow rate through the column was 1 mL min -1 The initial temperature was maintained at 40 °C for 4 min, then at 5 °C min -1 The rate was increased to 245°C and maintained for 5 min. The front inlet temperature, transfer line temperature, ion source temperature and quadrupole temperature were 250°C, 250°C, 230°C and 150°C, respectively. The ionization voltage in electron impact mode was -70 eV. Mass spectral data were obtained in scanning mode with a mass range of 20-400 and a solvent delay range of 0 min. The GC-MS total ion current of Fangfeng essential oil is shown in Figure 2. Figure 1 The identification results of the main chemical components of Fangfeng essential oil are shown in Table 1.

[0032] Table 1, chemical composition of windproof essential oil:

[0033]

[0034]

[0035] Example 2: Evaluation of the toxicity of Saposhnikovia essential oil to J774A.1 macrophages

[0036] See also Figure 2 J774A.1 cells were cultured at a rate of 5 × 10 3After the cells / well were inoculated in a 96-well plate for 16 hours, 100 μg / mL of the windproof essential oil emulsion was diluted twice to a final concentration of 3.125 μg / mL for a total of 6 concentrations. Three replicate wells were set for each concentration. DMEM medium containing 8% fetal bovine serum was used as a control and incubated in a 5% CO2 incubator for 24 hours. 20 μL of MTT solution was added to each well of the plate, and the culture plate was incubated in the incubator for 4 hours, the supernatant was discarded, 200 μl of DMSO was added and shaken for 5 minutes, and the absorbance at 450 nm was measured using an enzyme reader (BioTek, 800TS).

[0037] The results are as follows Figure 2 As shown in the data, concentrations of 50 μg / mL and below had no significant effect on the viability of J774A.1 cells, indicating that fangfeng essential oil is safe within this concentration range.

[0038] Example 3 Effect of Fangfeng essential oil on the expression level of IL-1β inflammatory factor in J774A.1 cells induced by Propionibacterium acnes

[0039] (1) Sample preparation: J774A.1 cells were cultured at a rate of 1×10 6 Cells / well were inoculated in 6-well plates. On the second day, 200ng / ml LPS was added to each well and incubated for 3h. The supernatant was discarded and 30μg / mL, 10μg / mL, and 3μg / mL of windproof essential oil emulsion were added and incubated for 30min. Then, Propionibacterium acnes with an MOI of 100 was added and incubated overnight. The supernatant was taken as a sample. It can be used immediately or stored at -80℃ for later use.

[0040] (2) ELISA: Follow the instructions for use, place the ELISA plate in an ELISA reader, use 630 nm as the calibration wavelength, read the absorbance at 450 nm, and calculate the IL-1β concentration in the sample based on the standard curve.

[0041] The results are as follows Figure 3 As shown, fangfeng essential oil can dose-dependently reduce the IL-1β expression level of J774A.1 cells caused by Propionibacterium acnes.

[0042] Example 4: Effect of Fangfeng essential oil on redness and swelling induced by Propionibacterium acnes in ICR mice

[0043] The experiment was conducted using 6-week-old ICR male mice in an SPF-grade breeding environment. Modeling method: (1) 5-week-old ICR male mice weighing about 25 g were purchased and stably raised for one week. After that, 20 μL of 1×10 9CFU / ml of Propionibacterium acnes. The experiment set up 6 treatment methods, each with 6 biological replicates, for a total of 36 mice. The drug was applied immediately after modeling, and then administered again after 12 hours. The mice were killed after 24 hours. The treatment methods are as follows:

[0044] Treatment 1 (blank group), mice were not treated with any treatment;

[0045] Treatment 2 (modeling group), Propionibacterium acnes was injected into the ear to create the model, which was the acne model group;

[0046] Treatment 3 (andrographolide group), the ear was given 0.5% andrographolide topical application since the injection of Propionibacterium acnes modeling, which was the positive drug group;

[0047] Treatment 4 (low-dose administration group), the ears were given topical application of 0.25% fangfeng essential oil since the injection of Propionibacterium acnes modeling, i.e. the low-dose administration group;

[0048] Treatment 5 (medium-dose administration group), the ears were given topical application of 0.5% fangfeng essential oil since the injection of Propionibacterium acnes modeling, which is the medium-dose administration group;

[0049] Treatment 6 (high-dose administration group), the ears were given topical application of 1% fangfeng essential oil since the injection of Propionibacterium acnes modeling, that is, the high-dose administration group.

[0050] The other daily management of mice in the six treatments remained the same. Figure 4 , Figure 5 and Figure 6 After the experiment, the mice were sacrificed to collect tissue samples, and the redness and swelling of the ears and pathological HE staining were analyzed. Figure 4 As shown in the figure, compared with the control group, the Propionibacterium acnes model mice showed ear redness and swelling. Compared with the model mice, the high, medium and low dose groups of Saposhnikovia divaricata essential oil all improved ear redness and swelling, and the high dose group had a more significant relief effect. The positive control group andrographolide had a very significant effect. HE staining analysis of the mouse ears was performed as shown in the figure. Figure 6 As shown in the figure, compared with the control group, the diameter of the sebaceous glands of the Propionibacterium acnes model mice was significantly enlarged, and the inflammatory cell infiltration was severe. Compared with the modeling group, the high, medium and low doses of Saposhnikovia essential oil treatment groups can improve the enlargement of the sebaceous gland diameter and inflammatory cell infiltration in the ears of mice, among which the effect of Saposhnikovia essential oil in the high dose treatment group was more significant. Under a 50x microscope, the ear thickness of each slice modeling position was measured. The ear thickness of six mice in each group was measured, and the average value was taken for each mouse after 3 measurements. The statistical results are shown in the figure. Figure 5As shown in the figure, compared with the control group, the modeling group had abscesses and increased ear thickness. Compared with the modeling group, the high, medium and low doses of saposhnikovia essential oil treatment groups can improve the abscesses produced by Propionibacterium acnes and reduce the ear thickness. The positive drug andrographolide has a significant effect and the ear thickness is close to that of the control group. Therefore, saposhnikovia essential oil has a certain effect on alleviating the redness, swelling and abscesses of the mouse ears induced by Propionibacterium acnes, and may become a safe and low-toxic new drug for acne disease.

[0051] Example 5: Effect of Fangfeng essential oil on reactive oxygen species in the ear skin of acne mice induced by Propionibacterium acnes

[0052] Related studies have reported that a large amount of reactive oxygen species are produced in acne models. In order to verify whether the windproof essential oil exerts its efficacy through antioxidant activity, DHE (Dihydroethidium) staining was performed on the ear tissue of mice as follows:

[0053] (1) Quenching tissue autofluorescence: rewarm the frozen sections at room temperature and control the moisture. Draw a circle around the tissue with a tissue pen, add autofluorescence quencher for 5 minutes, and rinse with running water for 10 minutes.

[0054] (2) Staining: Add ROS dye solution into the circle and incubate at 37° in a light-proof incubator for 30 min.

[0055] (3) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 min each time. Add DAPI staining solution and incubate at room temperature for 10 min in the dark.

[0056] (4) Sealing: Place the slides in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 min each time. Seal the slides with an anti-fluorescence quenching mounting medium.

[0057] (5) Image acquisition: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555nm; CY3 excitation wavelength 510-560nm, emission wavelength 590nm; CY5 excitation wavelength 608-648nm, emission wavelength 672-712nm.

[0058] The results are as follows Figure 7 As shown, the cell nucleus of the DAPI channel is blue, and the positive CY3 channel is red. In the modeling group, Propionibacterium acnes caused a large amount of reactive oxygen species to be produced in the mouse tissues, and the red light was strong. The drug-treated group was significantly improved and showed a dose-dependent manner.

[0059] Example 6, Effect of Fangfeng Essential Oil on Macrophages in the Ear Skin of Acne Mice Induced by Propionibacterium acnes

[0060] In order to explore the relationship between acne model and inflammation, F4 / 80 immunohistochemistry experiment was performed on mouse tissues.

[0061] The experimental steps are as follows:

[0062] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water.

[0063] (2) Antigen repair: See the table above for repair. During this process, prevent the buffer from evaporating excessively and do not dry the slide. After natural cooling, place the slide in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue)

[0064] (3) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time.

[0065] (4) Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue and block at room temperature for 30 minutes.

[0066] (5) Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain ratio with PBS on the slices, and incubate the slices flat in a humidified chamber at 4°C overnight.

[0067] (6) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP-labeled) of the same species as the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0068] (7) DAB color development: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle. The color development time is controlled under a microscope. The positive color is brown-yellow. Rinse the slices with tap water to stop the color development.

[0069] (8) Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water.

[0070] (9) Dehydration and sealing: Place the slices in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, let them dry slightly, and seal them with sealing medium.

[0071] (10) Microscopic examination: Place the sample under a white light microscope to interpret the results.

[0072] The results are as follows Figure 8 As shown, hematoxylin stains the nucleus in blue, and DAB shows positive expression in brown-yellow. Compared with the blank group, the number of macrophages in the modeling group increased, and a large number of macrophages gathered at the modeling site. Compared with the modeling group, the group given fangfeng essential oil and andrographolide significantly improved this condition.

[0073] Example 7, Effect of Fangfeng essential oil on the expression level of inflammatory factors in ear skin tissue fluid of acne mice induced by Propionibacterium acnes

[0074] Sample preparation: After the ear tissue of the sacrificed mouse was ground with liquid nitrogen, PBS containing protease inhibitors was added, and ultrasonic treatment was performed at a power of 8% for a total time of 2 minutes. After the ultrasonic treatment, the tissue was centrifuged in a 4°C precooled centrifuge (8000 rpm, 10 minutes). The supernatant was taken as the sample and placed in a -80°C refrigerator for later use.

[0075] ELISA: Follow the instructions and place the ELISA plate in an ELISA reader. Calculate the IL-1β concentration in the sample according to the standard curve and place the ELISA plate in an ELISA reader at the corresponding detection wavelength (including IL-1β, IL-6 and TNF-α) and calibration wavelength.

[0076] The results are as follows Fig. 9 As shown in the results, Propionibacterium acnes can induce the production of IL-1β, IL-6 and TNF-α inflammatory factors in mice. Fangfeng essential oil can reduce the expression level of inflammatory factors in mice caused by Propionibacterium acnes in a dose-dependent manner, and andrographolide has a more significant effect in reducing inflammatory factors.

[0077] Example 8, Western Blotting Analysis of Fangfeng Essential Oil on Proteins in the Ear Skin of Acne Mice Induced by Propionibacterium acnes

[0078] The ear tissue of the sacrificed mouse was cryogenically ground with liquid nitrogen, and then lysed in an ice-water mixture with RIPA lysis buffer containing protease inhibitors for 30 minutes, and then ultrasonically treated at a power of 8% for a total time of 2 minutes. After the ultrasonic treatment, the tissue was centrifuged in a 4°C precooled centrifuge (8000 rpm, 10 minutes), and the supernatant was taken for BCA quantification. The expression of NLRP3 inflammasome-related proteins in mouse ear tissue was measured by Western blot.

[0079] The results are as follows Fig.10As shown in the figure, compared with the normal group, pro-IL-1β, pro-Caspase-1, and GSDMD-FL in the modeling group were cleaved to generate IL-1β, Caspase-1, and GSDMD-NT, indicating that the acne model activated the NLRP3 inflammasome. After modeling, the mice were given fangfeng essential oil drug treatment, and it was found that IL-1β, Caspase-1, and GSDMD-NT proteins were downregulated, indicating that fangfeng essential oil can inhibit the activation of NLRP3 inflammasome.

[0080] In summary, the application of the windproof essential oil of the present invention in the preparation of products for the treatment and prevention of acne, the windproof essential oil can inhibit the release of IL-1β inflammatory factors of J774A.1 cells induced by Propionibacterium acnes. The mouse ear acne model induced by Propionibacterium acnes verified the alleviating effect of the windproof essential oil on the redness and swelling of the acne-like mouse ears, the accumulation of inflammatory factors, the release of intracellular reactive oxygen species, etc. Therefore, it has certain application prospects in the preparation of products for the treatment and prevention of acne.

[0081] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. As long as they are within the spirit of the present invention, any changes or modifications to the above embodiments will fall within the scope of the claims of the present invention.

Claims

1. Application of siler essential oil in preparing products for treating and preventing acne.

2. The application of the windproof essential oil according to claim 1, characterized in that, The essential oil is obtained by crushing the Chinese herbal medicine Saposhnikovia divaricata and extracting it through a steam distillation method.

3. The application of the windproof essential oil according to claim 1, characterized in that, The essential oil inhibits the generation of ROS caused by Propionibacterium acnes in vivo and has antioxidant activity.

4. The use of the windproof essential oil according to claim 1, characterized in that: The essential oil inhibits the expression of IL-1β, IL-6 and TNF-α inflammatory factors in vivo, and prevents the activation of NLRP3 inflammasome caused by Propionibacterium acnes, has anti-inflammatory activity, and inhibits the activation of inflammasome.

5. The use of the windproof essential oil according to claim 4, characterized in that: The inhibition of inflammasome activation includes inhibiting the shearing of Caspase-1 protein in inflammasome-induced mice, inhibiting the maturation of IL-1β in inflammasome-induced mice, inhibiting the secretion of IL-1β in inflammasome-induced mice, and inhibiting the formation of GSDMD-NT in inflammasome-induced mice.

6. The use of the windproof essential oil according to claim 1, characterized in that: The products include medicines and daily chemical products.

7. The use of the siler essential oil according to claim 1 or 6, characterized in that: The weight content of the essential oil in the product is 0.1%-1%.

8. The use of the windproof essential oil according to claim 7, characterized in that: The dosage form of the drug is at least one of capsules, pills, injections, aerosol powders, films, patches, emulsions, ointments, liniments, creams and lotions.

9. The use of the windproof essential oil according to claim 7, characterized in that: The daily chemical product is at least one of shampoo, shampoo cream, shower gel, body lotion, essential oil soap, cooling oil, wind oil essence, toner, floral water, face cream, facial mask and facial essence.

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