A cyclic peptide compound with anti-inflammatory effect, its preparation method and application

Abstract

The present invention relates to a cyclic peptide compound with anti-inflammatory effects, and its preparation method and application. The preparation method includes the following steps: Select dichloro resin as the loading resin, adopt the Fmoc solid-phase synthesis strategy, repeat condensation, remove the Fmoc protecting group, wash and dry to obtain Met-Leu-Pro-Ile-Pro-Gln(Trt)-Thr(tBu)-Gly-resin with exposed α-amino group. Cyclize the crude polypeptide solid-phase to obtain the reaction mixture; add hydrochloric acid, stir, and obtain a yellow oil after drying under reduced pressure; add cleavage reagent, filter, purify, and freeze-dry to obtain the cyclic peptide compound with anti-inflammatory effects. The present invention uses a method with simple operation, high extraction yield, and high product purity to prepare the cyclic peptide compound contained in the fruit of Annona cherimola Mill. and apply it to cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and more specifically, relates to a cyclic peptide compound with anti-inflammatory effects, its preparation method and application. Background Art

[0002] Climate, environmental factors and other irritating factors have a significant impact on the appearance of sensitive skin and skin inflammation. The above-mentioned other irritating factors include, for example, pollen, dust mites, pet dander or certain chemicals. In order to reduce the impact of the foregoing factors on skin inflammation and allergic reactions, most of the existing related studies focus on adding anti-inflammatory raw materials to cosmetics. For example, Chinese Patent Publication CN112912063A provides an extract of Eucommia ulmoides bark, which can inhibit the expression of PGE2 synthase protein increased by heat treatment and has anti-inflammatory effects; Chinese Patent CN115089521B provides a soothing and anti-inflammatory composition including sodium carboxymethyl β-glucan, purslane extract, asiatic pennywort extract, sea fennel extract and panthenol, which can relieve skin sensitivity from multiple dimensions; Chinese Patent Publication CN117919143A provides a soothing and anti-inflammatory composition, which includes rosemary leaf extract, asiatic pennywort extract, glabrous licorice root extract and sophora flavescens root extract, and has effects such as soothing inflammation, repairing the skin, and relieving redness. However, some plant extracts may affect the odor, color, skin feel, etc. of the product.

[0003] Cherimoya (Annona cherimola), a plant belonging to the genus Annona of the Annonaceae family in the order Magnoliales, the cyclic peptide compound contained in its fruit, cherimoya (Annona cherimola Miller), can down-regulate histamine, TNF-α and substance P, and reduce the expression of IL-31 receptor, thereby reducing skin sensitivity and alleviating skin inflammatory itching symptoms. However, due to the extremely low content of cyclic peptide compounds in cherimoya fruit and the high difficulty of extraction and separation, it is very difficult and extremely inefficient to extract the cyclic peptide compounds from cherimoya fruit by traditional separation processes.

[0004] Based on this, there is an urgent need to propose a cyclic peptide compound with anti-inflammatory effects, its preparation method and application, so as to solve the technical problems of the extremely low content of cyclic peptide compounds contained in cherimoya fruit and the high difficulty of extraction and separation by chemical synthesis. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the main object of the present invention is to provide a cyclic peptide compound with anti-inflammatory effects, its preparation method and application, and solve the technical problems that it is extremely difficult to extract and separate cyclic peptide compounds from Annona cherimola fruits by traditional separation processes due to the extremely low content of cyclic peptide compounds in Annona cherimola fruits and the high difficulty of extraction and separation, with extremely low efficiency, through chemical synthesis.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a cyclic peptide compound with anti-inflammatory effects, and the cyclic peptide compound has the structure shown in formula (Ⅰ):

[0008]

[0009] In the second aspect, the present invention provides a preparation method of a cyclic peptide compound with anti-inflammatory effects, and the preparation method includes the following steps:

[0010] Step S1: Select dichlororesin as the loading resin, provide an activated Fmoc-Gly-OH solution, adopt the Fmoc solid-phase synthesis strategy to condense and prepare Fmoc-Gly-resin, and remove the Fmoc protecting group to obtain Gly-resin with exposed α-amino group;

[0011] Step S2: Sequentially condense and remove the Fmoc protecting group of the Gly-resin with the activated Fmoc-Thr(tBu)-OH solution, Fmoc-Gln(Trt)-OH solution, Fmoc-Pro-OH solution, Fmoc-Ile-OH solution, Fmoc-Pro-OH solution, Fmoc-Leu-OH solution, and Fmoc-Met-OH solution according to the Fmoc solid-phase synthesis strategy. After all the condensation and removal of the Fmoc protecting group operations are completed, wash with DMF and methanol in sequence, and after washing, perform drying treatment to obtain Met-Leu-Pro-Ile-Pro-Gln(Trt)-Thr(tBu)-Gly-resin with exposed α-amino group;

[0012] Step S3: Stir and swell the Met-Leu-Pro-Ile-Pro-Gln(Trt)-Thr(tBu)-Gly-resin with exposed α-amino group in a DCM solution of TFA, filter and collect the solvent to obtain a crude polypeptide product;

[0013] Step S4: Add the coupling system formed by DIPEA, TBTU, and HOBT to the crude polypeptide product, stir and react until the crude polypeptide product completes solid-phase cyclization to obtain a post-reaction mixture;

[0014] Step S5: Add hydrochloric acid to the post-reaction mixture to adjust the pH value to 6 to 7, stir and collect the organic phase, and obtain a yellow oil after drying under reduced pressure;

[0015] Step S6: Slowly add the yellow oil to a mixed solution formed by a TFA / EDT / H2O / TIPS cleavage reagent pre-cooled to 0 °C, and then slowly add the mixed solution to an MTBE solution to precipitate a white solid, which is filtered, purified by reverse-phase HPLC, and freeze-dried to obtain the cyclic peptide compound with anti-inflammatory effect.

[0016] Preferably, in the step S1, the activated Fmoc-Gly-OH solution is obtained by activating Fmoc-Gly-OH with an activator, and the activator is DIPEA (N,N-diisopropylethylamine).

[0017] It should be noted that Fmoc-Gly-OH needs to be activated by DIPEA to react with the chloromethyl functional group on the dichlororesin.

[0018] Preferably, in the step S2, the sequential washing with DMF and methanol includes: washing the resin with DMF 5 times, and then washing the resin with methanol 3 times; the drying treatment includes vacuum drying. Among them, washing the resin with DMF (dimethylformamide) 5 times can remove the reactants, by-products and excess Fmoc reagents that may remain on the resin surface and in the pores; since methanol is a strongly polar solvent, washing the resin with methanol 3 times can fully remove the residual DMF and other polar impurities on the loaded resin.

[0019] Preferably, in the step S3, the swelling time of stirring is 10 min, the volume percentage concentration of TFA in the DCM solution containing TFA is 1%, and the number of times of repeating the above-mentioned operations of stirring and swelling, filtering and collecting the solvent is 3 times.

[0020] Preferably, in the step S4, the stirring reaction is carried out under ice bath conditions, and the stirring reaction time is 4 h.

[0021] It should be noted that in the step S4, in the coupling system formed by DIPEA (N,N-diisopropylethylamine), TBTU (2,4,6-tribromophenyl carbonate), and HOBT (1-hydroxybenzotriazole): DIPEA is an organic base used to deprotonate the carboxylic acid component to generate carboxylate salts, thereby promoting the coupling reaction between carboxylic acids and isocyanates or activated carboxylic acids; TBTU is a peptide coupling reagent that can react with amines to generate reactive intermediates, which can react with another carboxylic acid to form peptide bonds; HOBT is an additive that can react with carboxylic acids and amines to generate active esters, promoting the coupling reaction. Additionally, the reaction system is placed in an ice bath to control the reaction temperature, which helps to slow down the reaction rate, reduce side reactions, and improve reaction selectivity.

[0022] It should be noted that in step S4, the completion of solid-phase cyclization of the crude polypeptide can be monitored by LCMS (Liquid Chromatography-Mass Spectrometry).

[0023] Preferably, in the step S5, the concentration of the hydrochloric acid is 1N HCl / H2O, and the stirring duration is 0.5 h. The 1N HCl / H2O indicates that the hydrochloric acid solution has a concentration of 1 equivalent, which is mainly used to remove some basic substances such as DIPEA; it can also quench the reaction and stop it from proceeding.

[0024] Preferably, in the step S6, the yellow oil is slowly added to a mixed solution formed by a TFA / EDT / H2O / TIPS cleavage reagent pre-cooled to 0 °C, and stirred for 2 h.

[0025] It should be noted that the yellow oil is slowly added to a mixed solution formed by a TFA / EDT / H2O / TIPS (triisopropylsilane) cleavage reagent pre-cooled to 0 °C, and then the mixed solution is slowly added to a pre-cooled MTBE (methyl tert-butyl ether) solution, which can control the reaction rate and reduce side reactions.

[0026] In a third aspect, the present invention provides an application of a cyclic peptide compound with anti-inflammatory effects in cosmetics.

[0027] Preferably, the mass percentage value range of the cyclic peptide compound in the cosmetics is 0.008% - 0.016%.

[0028] Preferably, the type of the cosmetics is selected from any one of lotions, creams, gels, aqueous solutions, and facial masks.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] (1) The preparation method of a cyclic peptide compound with anti-inflammatory effect provided by the present invention is simple in operation, high in extraction yield and high in product purity, realizing the industrial batch synthesis of the same cyclic peptide compound as that contained in Annona cherimola Mill. fruit, and avoiding the problem that it is difficult to utilize directly due to the extremely low content of the cyclic peptide compound in traditional Annona cherimola Mill. fruit and the high difficulty in direct extraction and separation.

[0031] (2) The cyclic peptide compound prepared by the preparation method of a cyclic peptide compound with anti-inflammatory effect provided by the present invention not only has good skin permeability, but also has anti-inflammatory activity, can be applied to the cosmetic field, and has great application prospects and commercial value. Description of the Drawings

[0032] Figure 1 Shows the synthesis route of the cyclic peptide compound with anti-inflammatory activity in an embodiment of the present application;

[0033] Figures 2a - 2b Shows the two-dimensional nuclear magnetic resonance spectrum characterization of the cyclic peptide compound with anti-inflammatory activity in an embodiment of the present application;

[0034] Figures 3a - 3b Shows the anti-inflammatory experiment of the cyclic peptide compound on zebrafish;

[0035] Figure 4 Shows the cell anti-inflammatory test experiment of the cyclic peptide compound. Detailed Embodiments

[0036] The technical solution of the present invention will be further described below in conjunction with the drawings and through specific embodiments. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the patent protection of the present invention. The scope of protection of the present invention is subject to the claims.

[0037] Example 1

[0038] Refer to Figure 1 , the preparation method of a cyclic peptide compound with anti-inflammatory effect in this embodiment, the preparation method includes the following steps:

[0039] Step S1: Take 1 mol of dichloro resin (loading capacity: 0.56 mol / kg) as the supported resin. Stir the dichloro resin in 6.5 L of DCM (dichloromethane) for 30 min, add 774 g (6 mol) of DIPEA (diisopropylethylamine) and 891.2 g (3 mol) of Fmoc-Gly-OH for condensation reaction, and stir at room temperature for 2.5 h; add 650 ml of MeOH (methanol) and stir for 0.5 h; wash the supported resin 3 times with 6.5 L of DMF (N,N-dimethylformamide); add 6.5 L of DMF containing 20% piperidine to the supported resin, react at room temperature for 20 min, and wash with 6.5 L of DMF 5 times to remove the Fmoc protecting group, obtaining Gly-resin with exposed α-amino group;

[0040] Step S2: Mix 658.8 g (5.4 mol) of DIC (1,3-diisopropylcarbodiimide), 729.1 g (5.4 mol) of HOBT (1-hydroxybenzotriazole), and 1.2 kg (3 mol) of Fmoc-Thr(tBu)-OH in 6.5 L of DMF, and add the mixture to the supported resin for condensation reaction. Stir at 0 °C to room temperature for about 2 h, then add 20% piperidine and stir at room temperature for 20 min. Wash the supported resin 5 times with 6.5 L of DMF to remove the Fmoc protecting group, obtaining Thr(tBu)-Gly-resin with exposed α-amino group;

[0041] Repeat the above condensation and Fmoc protecting group removal steps for the following amino acids in sequence. The following amino acids include: Fmoc-Gln(Trt)-OH (1.83 kg, 3 mol), Fmoc-Pro-OH (1.01 kg, 3 mol), Fmoc-Ile-OH (1.06 kg, 3 mol), Fmoc-Pro-OH (1.01 kg, 3 mol), Fmoc-Leu-OH (1.06 kg, 3 mol), and Fmoc-Met-OH (1.11 kg, 3 mol); after the coupling of the entire sequence is completed, wash the supported resin 5 times with 6.5 L of DMF, then wash 3 times with 10 L of MeOH, and dry the resin by vacuum drying method to obtain 1.8 kg of yellow solid supported resin;

[0042] Step S3: Swell 1.8 kg of yellow solid supported resin in 10 L of 1% TFA / DCM, stir for 10 min, filter and collect the solvent, and repeat the above steps 3 times with 10 L of 1% TFA / DCM to obtain the crude polypeptide;

[0043] Step S4: DIPEA (387.2 g, 3 mol), TBTU (641.0 g, 2 mol) and HOBT (270.0 g, 2 mol) were added to the above 1000 L DCM solution under ice bath conditions and stirred for 4 h until the solid phase cyclization of the crude product was completed to obtain a reaction mixture;

[0044] Step S5: adding 1N, 50 L hydrochloric acid to the reaction mixture until the pH value reaches the range of 6-7, stirring for 0.5 h, collecting the organic phase, and drying under reduced pressure to obtain 0.98 kg of yellow oil;

[0045] Step S6: The 0.98 kg yellow oil was slowly added to a TFA / EDT / H2O / TIPS cutting reagent at 0°C to form a mixed solution, and stirred for 2 hours. The volume percentage concentration of TFA in the TFA / EDT / H2O / TIPS system was 92.5%. The mixed solution was then slowly added to a pre-cooled MTBE (methyl tert-butyl ether) solution (70 L) until a white solid was completely precipitated. 480 g of a crude white solid was obtained by filtration, which was purified by reverse-phase HPLC and lyophilized to obtain the cyclic peptide compound with anti-inflammatory activity.

[0046] Table 1. Cyclic peptide A and cyclic peptide compounds from Annona chebula 13 C and 1 H NMR data

[0047]

[0048]

[0049]

[0050] Table 1 13 C and 1 H NMR data are from the literature: Tetrahedron 60 (2004) 405–414; 13 C and 1 The H NMR data is the result of the two-dimensional NMR spectrum analysis of the sample (cyclic peptide compound), the solvent is deuterated DMSO, and the NMR spectrum is as follows Figures 2a - 2b As shown in Table 1, the structure of the cyclic peptide compound prepared by the preparation method of the cyclic peptide compound described in Example 1 is the same as the structure of the cyclic peptide A of the Annona chebula recorded in the existing literature, indicating that the present application has achieved the industrial batch synthesis of the cyclic peptide compound identical to the cyclic peptide compound contained in the Annona chebula fruit, avoiding the problem that the cyclic peptide compound in the traditional Annona chebula fruit is difficult to be utilized due to the extremely low content of the cyclic peptide compound contained in the fruit and the high difficulty in direct extraction and separation.

[0051] Test Example 1

[0052] Anti-inflammatory Experiment on Zebrafish

[0053] Select zebrafish Tg(mpx:dsRED) with neutrophils labeled by red fluorescence. The breeding method of zebrafish is carried out according to 《The zebrafish book》. Keep the breeding water temperature at about 28.5°C, with 14 hours of light and 10 hours of darkness every day, and feed once in the morning and once in the evening. One female fish and two male fish were placed in the spawning bar the night before collecting embryos, separated by a partition, and placed in a dark environment. The next morning, after the light was turned on, the partition was removed, and the embryos were collected after spawning was completed. The embryos were placed in egg water and cultured in a light incubator at 28.5°C.

[0054] On the day of collecting embryos, at about 7 - 8 hpf, the embryos were thoroughly changed and washed, and the embryos in poor condition were removed, fresh egg water was replaced, and they were continued to be cultured in an incubator at 28.5°C until 3 dpf (during the cultivation period, the poor embryos were picked out in time, and fresh egg water was replaced every day). The 3-dpf zebrafish were randomly grouped, with 15 in each group: a. Normal control group: containing zebrafish and standard dilution water. b. Model control group: containing zebrafish and 1 μM copper sulfate solution. c. Positive control group: containing zebrafish, 1 μM copper sulfate solution and positive drug (in this experiment, 5 μg / mL dexamethasone was used as the positive drug). d. Test group of test substance: containing zebrafish, 1 μM copper sulfate solution and test substance (100 μg / mL cyclic peptide compound).

[0055] According to the experimental requirements, a sufficient number of zebrafish at 72 hpf with consistent developmental status were pre-screened, and the zebrafish were randomly assigned to a six-well plate, 15 in each well. Without harming the embryos, the standard dilution water in the six-well plate was removed, and 3 mL of the test substance solution was quickly added to each well. After thorough mixing, the culture plate panel was covered and wrapped with aluminum foil, and incubated in the dark in a biochemical incubator at 28°C for 2 hours. After the incubation, zebrafish were randomly selected from the zebrafish with normal phenotypes and behaviors, fixed with 3% methyl cellulose, observed and photographed under a stereoscopic fluorescence microscope. The photographing results of all zebrafish must be completed under the same instrument and environmental conditions, and the body positions of the zebrafish should be kept consistent.

[0056] Table 2. Experimental Results of Zebrafish Anti-inflammatory Model

[0057] Group Concentration Anti - inflammatory effect (%) Significant difference Dexamethasone 5μg / mL 56.18 *** Cyclic peptide compound 100μg / mL 99.82 ***

[0058] Zebrafish is a newly emerging experimental animal model that can be used for high-throughput screening. The positions of neuromasts in zebrafish are relatively fixed and are generally distributed along the lateral line corresponding to different myomeres. When inflammation is induced by copper sulfate, neutrophils that are normally located in the tail hematopoietic tissue and posterior blood islands of zebrafish will migrate. A large number of neutrophils will accumulate at the damaged lateral line neuromast positions of zebrafish. As Figures 3a - 3b shown, after treatment with the cyclic peptide compound, the migration of neutrophils to the lateral line neuromast cell region can be inhibited, indicating that the cyclic peptide compound is an anti-inflammatory active substance.

[0059] Test Example 2

[0060] Cell anti-inflammatory test

[0061] HaCaT cells were seeded at 5×10 5 cells / well in a six-well plate and cultured under the conditions of 37°C and 5% CO2. After the cells adhered, they were washed with PBS, and then 0, 4, 8, 16 μg / mL of the cyclic peptide compound were added respectively. After UVB (13 m / cm 2 , 4 min) irradiation, 200 μL of lysis buffer was added to each well, and gently shaken evenly until the lysis buffer covered the bottom of the well. Lysed on ice for 20 minutes, shaking several times during this period. The cells were scraped off with a scraper, collected in a 1.5 mL EP tube, centrifuged at 4°C and 12,000 rpm for 30 minutes, and the supernatant was stored at -80°C for further testing. The contents of IL-1β and IL-6 were detected by the method of an Elisa enzyme-linked immunosorbent assay kit.

[0062] The results are as Figure 4 shown. After UVB irradiation, the contents of IL-1β and IL-6 in HaCaT cells can be induced to increase. And the cyclic peptide compounds with concentrations of 4, 8, 16 μg / mL can significantly reduce the content of IL-1β in the model group, and the cyclic peptide compounds with concentrations of 8, 16 μg / mL can significantly reduce the content of IL-6 in the model group, indicating that the cyclic peptide compound has a certain anti-inflammatory effect.

[0063] Test Example 3

[0064] Skin permeability test

[0065] The tape stripping method was used to collect and test the content of the cyclic peptide compound in the superficial epidermis of the skin. A 3.8 cm 2 -sized intact and flat skin on the inner side of the forearm of healthy subjects was selected as the test area and marked. 20 μL of a 50 mg / mL cyclic peptide compound solution was applied to this area and absorbed with a clean finger cot. 2 h after drug administration and absorption, the surface residue was rinsed with mild running water and gently dried. 10 layers of tape were continuously pasted on the drug administration area, and the average pressure applied to the tape was 225 g / cm 2, Collect the tape of each layer in a centrifuge tube. Methanol is used as the extraction solution. The LC-MS method is used to test the residence amount of the cyclic peptide compound in each layer of the human superficial epidermis, and the residence amount in the epidermis per unit area is calculated. As shown in Table 3, the experimental results show that at 2 h, the cyclic peptide compound can penetrate into the skin epidermis and has a certain residence effect.

[0066] Table 3. Skin permeability results of cyclic peptide compounds

[0067] Subject number <![CDATA[Residual amount in the epidermis per unit area (ng / cm 2 )]]> 1 759.62 2 1507.89 3 681.71

[0068] Application Example 1

[0069] Application of cyclic peptide compounds in creams

[0070] Table 4. A cream formula containing cyclic peptide compounds

[0071]

[0072] Preparation method: Prepare according to the formula ratio shown in Table 4, and make up to 100% with deionized water. The preparation amount of Formula 1 is 200 g. Premix the raw materials in Phase B of the formula and add them to the water phase pot, stir at low speed for 5 - 10 minutes until completely uniform, and keep warm at 75 - 85 °C for 20 minutes; dissolve the raw materials in Phase A completely in the oil phase pot at 80 - 85 °C and keep warm for standby. Transfer the raw materials in Phase B to the emulsifying pot, heat up to 75 - 85 °C, stir at medium speed and homogenize for 10 minutes until completely dispersed and uniform. While the emulsifying pot is stirring, evacuate the air, slowly pump in Phase A, stir at medium - high speed for 5 - 10 minutes, homogenize for 3 - 8 minutes, and then continue to stir and cool to 75 °C and add the raw materials in Phase C. When continuing to cool below 45 °C, add the raw materials in Phase D in sequence. After passing the physical and chemical index detection, filter and discharge.

[0073] Application Example 2

[0074] Application of cyclic peptide compounds in lotions

[0075] Table 5. A lotion formula containing cyclic peptide compounds

[0076]

[0077] Preparation method: Formula 2 is prepared according to the formula ratio shown in Table 5, and the specific preparation process is the same as that of Formula 1.

[0078] Application Example 3

[0079] Application of cyclic peptide compounds in facial masks

[0080] Table 6. A facial mask formula containing cyclic peptide compounds

[0081]

[0082]

[0083] Preparation method: Prepare according to the formulation ratio shown in Table 6, and make up to 100% with deionized water. The preparation amount of Formulation 3 is 200 g. The preparation process is as follows: Add the raw materials of Phase A in the formulation to the water phase kettle and fully hydrate until complete. Premix the raw materials of Phase B and then add them to the water phase kettle. Heat up to 75 - 85 °C and stir until completely dissolved; cool down and stir. Add Phase C at 75 °C and stir until complete. Add the raw materials of Phase D at 55 °C and stir until complete. Add the raw materials of Phase E successively below 45 °C and stir evenly. After passing the physical and chemical index tests, filter and discharge.

[0084] Stability investigation

[0085] Perform stability investigation tests on the formulations of Application Examples 1 - 3. Respectively test for 30 days, 60 days, and 90 days at -15 °C, 25 °C, 40 °C, 49 °C, and under light conditions to test the stability of the cyclic peptide compounds of the present invention. The stability investigation results are shown in Table 7.

[0086] Table 7. Stability investigation results of several formulations containing cyclic peptide compounds

[0087]

[0088]

[0089] Thus, it can be seen that the stabilities of the products obtained by applying the formulations corresponding to Application Examples 1 - 3 to creams, lotions, and facial masks all meet the standards.

[0090] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Application of a cyclic peptide compound with anti-inflammatory effect in cosmetics, characterized in that, The cyclic peptide compound has the structure shown in formula (I): Formula (I); wherein, The mass percentage value range of the cyclic peptide compound in the cosmetic is 0.008% - 0.016%; The cyclic peptide compound can penetrate into the skin epidermis layer and stay therein.

2. Use of the cyclic peptide compound with anti-inflammatory effect according to claim 1 in cosmetics, characterized in that, The type of the cosmetic is selected from any one of lotion, cream, gel, aqueous solution, and facial mask.

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