ANTI-ALLERGIC COSMETIC COMPOSITION

Abstract

The present invention relates to the use of a combination of D-panthenol, allantoin and oat kernel extract in protecting mast cells thereby anti-allergy. The present invention also provides an anti-allergic cosmetic composition having the function of protecting mast cells, comprising a combination of D-panthenol, allantoin and oat kernel extract, and ingredients commonly used in cosmetics; and an anti-allergic cosmetic method, comprising applying the cosmetic composition to the skin.

Claims

1. Use of the combination of D-panthenol, allantoin and oat kernel extract in protecting mast cells thereby anti-allergy.

2. An anti-allergic cosmetic composition comprising a combination of D-panthenol, allantoin and oat kernel extract, and ingredients commonly used in cosmetics.

3. The cosmetic composition according to claim 2, wherein the amount of D-panthenol is about 0.02-3%, the amount of allantoin is about 0.01-1.5%, and the amount of oat kernel extract is about 0.2-5%, based on the total weight of the cosmetic composition.

4. The cosmetic composition according to claim 3, wherein the amount of D-panthenol is about 0.05-2%, the amount of allantoin is about 0.03-1%, and the amount of oat kernel extract is about 0.5-4%.

5. An anti-allergic cosmetic method, comprising applying a cosmetic composition to the skin, wherein the cosmetic composition comprises a combination of D-panthenol, allantoin and oat kernel extract, and ingredients commonly used in cosmetic compositions.

6. The cosmetic method according to claim 5, wherein the amount of D-panthenol is about 0.02-3%, the amount of allantoin is about 0.01-1.5%, and the amount of oat kernel extract is about 0.2-5%, based on the total weight of the cosmetic composition.

7. The cosmetic method according to claim 6, wherein the amount of D-panthenol is about 0.05-2%, the amount of allantoin is about 0.03-1%, and the amount of oat kernel extract is about 0.5-4%.

Description

EXAMPLES

[0031] The present invention is further described in detail with reference to examples below. It should be understood that the examples described herein merely illustrate the technical solution of the present invention and are not intended to limit the scope of the present invention. All similar substitutions and modifications are obvious to those skilled in the art, and considered to be included in the scope of the present invention.

Example 1: Preparation of Anti-Allergic Spray A

[0032] In this example, an anti-allergic spray A having the following ingredients was prepared.

TABLE-US-00001 Spray A Ingredients (wt. %) Water 96.6 Panthenol 0.5 Allantoin 0.2 Oat kernel extract 1.3 Glycerin 0.8 Betaine 0.6

[0033] The preparation procedure of spray A was as follows:

[0034] (1) heating water to 80° C., adding allantoin, glycerin, panthenol and betaine in turn, mixing, stirring and dissolving uniformly;

[0035] (2) cooling the mixture to 40° C., adding oat kernel extract, and discharging.

[0036] The resulting spray A was a pale yellow clear liquid.

Example 2 (Comparative Example): Preparation of Control Sprays B, C, D and E

[0037] In this example, control sprays B, C, D, and E having the following ingredients were prepared using a procedure similar to the above, and all of the control sprays did not contain a combination of panthenol, allantoin and oat kernel extract. The resulting spray B was a colorless clear liquid, and the sprays C, D, and E were pale yellow clear liquids.

TABLE-US-00002 Ingredients (wt. %) Spray B Spray C Spray D Spray E Water 96.6  96.6  96.6  96.6  Panthenol 1.6 — — 0.4 Allantoin 0.4 — 0.4 — Oat kernel extract — 2.0 1.6 1.6 Glycerin 0.8 0.8 0.8 0.8 Betaine 0.6 0.6 0.6 0.6

Example 3: Effect of Sprays A-E on Allergic Reaction of RBL-2H3 Cells

[0038] The effect of the prepared sprays A-E on allergic reaction of cells was tested according to following methods.

[0039] Experimental principle: Compound 48/80 is a tool drug to induce mast cell degranulation, the mechanism is that it acts on mast cell membrane and induces an increase of intracellular calcium ions to change the amount of second messengers cAMP and cGMP, resulting in mast cell degranulation and histamine release.

[0040] Experimental materials: RBL-2H3 cell line was provided by the animal center of Zhejiang Academy of Medical Sciences; the test samples included 5 sprays A-E prepared above; the reagents included fetal bovine serum, RPM1640 medium, Tyrode's salt (mainly composed of sodium chloride, phosphate, calcium chloride, glucose, etc.), Compound 48/80 and histamine Elisa kit.

[0041] Experimental procedures:

TABLE-US-00003 Preparation of 1.0 mg of 48/80 powder was weighed and diluted with 1 mL of Compound Tyrode's solution, and 50 uL of the dilution was diluted to 48/80 solution 1000 uL, i.e. 48/80 solution at a concentration of 50 ug/mL, and the solution should be used right after it was ready. Preparation of 9.6 g of Tyrode's salt was diluted with 80 mL of sterilized Tyrode's deionized water, constant-volumed to 100 mL to prepare 10 solution times concentrate of Tyrode's solution, and autoclaved. Pre-treatment Sterilization and pH determination were carried out ahead, of water 900 uL of water sample was mixed with 1000 uL of 10 times sample Tyrode's solution, ready for use. Cell treatment RBL-2H3 cells in logarithmic growth phase were spreaded to the bottom of a culture flask and digested with a digestive juice containing 0.25% trypsin and 0.03% EDTA, centrifuged at 1200 rpm for 5 mins; resuspended with 1 time Tyrode's solution and counted, and diluted to 1 × 10.sup.4/mL; centrifuged again at 1200 rpm for 5 mins to remove the supernatant. Water sample Model group: 700 uL of cell suspension was centrifuged and grouping and resuspended with Tyrode's solution, stood at 37° C. for 30 experiments mins; sample group: 700 uL of cell suspension was centrifuged and resuspended with sample solution, stood at 37° C. for 30 mins; the sample was mixed uniformly and dispensed into 3 tubes, 200 uL per tube; the model group and the 5 spray sample groups: 5 uL of 48/80 solution was added to the cell suspension at a final concentration of 10 ug/mL, and stood at 37° C. for 20 mins. Histamine The above groups of samples were cooled in an ice box for 10 determination mins to terminate the reaction. The samples after termination of the reaction were centrifuged (4° C., 1500 rpm, 30 mins) to precipitate the cells at the bottom of EP tube, and the supernatants were transferred to new centrifuge tubes. The precipitated cells were resuspended with 250 μL of 1 time Tyrode's solution, heated in a 90° C. water bath, taken out after 5 mins and immediately placed in an ice box, after the samples were completely cooled, they were taken out and heated again in a 90° C. water bath, and frozen-thawed repeatedly several times to break cells. The supernatants and the cell solutions were diluted 10 times, and the stock solutions were stored in a refrigerator at −20° C. The diluted samples were determined for histamine using an imported histamine kit and calculated. The histamine release was calculated using the following formula: histamine release (%) = extracellular histamine concentration/(extracellular histamine concentration + intracellular histamine concentration) × 100%

[0042] Experimental results: The experimental results are summarized in Table 1 below.

TABLE-US-00004 TABLE 1 Test results of histamine release in cell model group Release % P value (vs. model) P value (vs. spray A) Model 66.4 ± 8.7 — 0.000 group Spray A 36.6 ± 9.2 0.000 — Spray B 50.0 ± 6.5 0.001 0.010 Spray C 45.5 ± 6.0 0.001 0.005 Spray D 47.4 ± 7.8 0.000 0.025 Spray E 49.6 ± 8.1 0.000 0.039

[0043] The results of the above cell experiments showed that spray A in the scope of the present invention significantly reduced histamine release as compared to control sprays B-E, indicating that the combination of panthenol, allantoin and oat kernel extract had synergistic effect on inhibition of histamine release.

Example 4: Protective Effect of Sprays A-E on Mast Cells of Zebrafish Larvae

[0044] The protective effect of sprays A-E prepared above on mast cells of zebrafish larvae was measured according to following methods.

[0045] Experimental animals: Zebrafish 5dpf (days post fertilization)

[0046] Experimental reagents: Substance P (SP, molecular formula C63H98N18O13S) is a neuropeptide, belonging to the tachykinin neuropeptide-family. SP can induce mast cell degranulation, cause rapid release of histamine, tryptase and other mediators, and directly mediate a series of immunoinflammatory reactions caused by mast cells.

[0047] Index of evaluation: Tryptase release

[0048] Experimental samples: Sprays A-E prepared above, and 60 ug/ml ketotifen used as a positive control.

[0049] Experimental methods: Wild-type zebrafish embryos were collected and cultured in a 28.5° C. incubator with E3 buffer (for larvae culture, containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl.sub.2), 0.33 mM MgSO.sub.4, supplemented with water to the final volume) to 5 dpf (days post fertilization), the buffer was changed every day, and the zebrafish 5 dpf was randomly transferred to 48-well cell culture plates in groups of 10 tails per well, 4 duplicate wells per group, grouped as follows:

[0050] Model group Msp: pure water+15 μg/ml SP

[0051] Positive group Tsp: 60 μg/ml ketotifen+15 μg/ml SP

[0052] Sample group to be tested X.sub.SP: spray sample to be tested+15 μg/ml SP wherein, positive drug ketotifen was formulated into a mother liquor with DMSO and stored at −20° C., and it was formulated into 60 μg/ml fluid with pure water when using in the experiment.

[0053] Corresponding to the above degranulation groups with SP, a negative control group without SP was established, 4 duplicate wells per group; corresponding to the SP-induced degranulation group and the negative control group without SP, a background control group (without zebrafish larvae) was established, 2 duplicate wells per group, and the E3 buffer involved in each group of wells was absorbed, and 250 μl of a solution corresponding to each group was added, and reacted in darkness in 28.5° C. incubator for 60 mins; after 60 mins, 200 μl of the supernatant of each group was placed in a 96-well cell culture plate, and added with enzyme reaction substrate BAPNA (trypsin substrate) respectively to reach a concentration of 400 μg/ml, and the 96-well plate was covered in darkness and placed in a 28.5° C. incubator, and reacted for 2 hours. The full plate was measured for light absorption value at 405 nm after 2 hours. The value reflects tryptase release in the mast cells of zebrafish. The protection rate of mast cells to be tested was calculated based on the following formula:

protection rate of mast cells of sample to be tested=[(M.sub.sp−M.sub.sp.bg)−(RO−RO.sub.bg)]−[(X.sub.sp−X.sub.sp.bg)−(X−X.sub.bg)]/(M.sub.sp−M.sub.sp.bg)−(RO−RO.sub.bg)×100%

[0054] wherein:

[0055] M.sub.sp−M.sub.sp.bg represents the absorbance of the SP-induced degranulation group (i.e. model group) minus the absorbance of its corresponding background control group (without zebrafish larvae);

[0056] RO−RO.sub.bg represents the absorbance of the ultra-clean water group without SP (i.e. the negative control group of the model group) minus the absorbance of its corresponding background control group;

[0057] X.sub.sp−X.sub.sp.bg represents the absorbance of the sample group of SP-induced degranulation minus the absorbance of its corresponding background control group;

[0058] X−X.sub.bg represents the absorbance of the sample group without SP minus the absorbance of its corresponding background control group.

[0059] The experimental results were summarized in Table 2 below.

TABLE-US-00005 TABLE 2 Experimental results of Zebrafish larvae mast cell protection model effects Mast cell protection Group Xsp-Xsp•bg X-Xbg rate Model group 0.061 0.006 — Positive group 0.006** 0.003 95.45% Spray A 0.002** 0.001 97.73% Spray B 0.220** 0.013 0 Spray C 0.028** 0.018 82.27% Spray D 0.016** 0.009 88.18% Spray E 0.028** 0.022 88.64% Note: * indicates p < 0.05, **indicates p < 0.01 (as compared to the model group); if the mast cell protection rate is greater than 100%, it is regarded as 100%, and if the mast cell protection rate is less than 0, it is regarded as 0.

[0060] The results in Table 2 showed that spray A in the scope of the present invention had a significantly improved mast cell protection effect as compared to the model group and the control sprays B-E, indicating that the combination of oat kernel extract, panthenol and allantoin had synergistic effect in protecting mast cell degranulation.

Example 5: Anti-Inflammatory Effect of Sprays A-E in Mouse Models

[0061] The anti-inflammatory effect of sprays A-E in mouse models were measured according to following methods.

[0062] Experiments on Irritant Contact Dermatitis

[0063] Irritant contact dermatitis (ICD) is caused by the strong irritation (such as strong acid and alkali) or toxicity of contactant itself. Its histopathological manifestations include vasodilation, plasma exudation, and infiltration of leukocytes in blood into local tissues to cause an inflammatory reaction. It is generally believed that ICD is a non-immune inflammatory reaction. In this experimental model, chemical reagents were used to stimulate abdominal nude skin of mice (10% sodium dodecyl sulfate, SDS), causing damage of skin barrier and aggravating local inflammatory reaction of the skin. The inflammatory factor index was used to determine whether the model was established for later anti-inflammatory efficacy evaluation experiments.

[0064] Experimental animals: ICR mice, 20-25 g, male.

[0065] Experimental materials: 10% sodium dodecyl sulfate solution (SDS), 1% hydrocortisone ointment, depilatory paste.

[0066] Experimental methods: ICR mice were randomly divided into groups according to body weight, 12 mice in each group, including normal group, model group, positive group, and spray test groups. At 1 day before modeling, the mice in each group were treated with depilatory paste to remove about 2*2 cm area of the hair in the abdomen of the mice. The model group, the positive group, and the test groups: on the day of modeling, 50 μL of a 10% sodium dodecyl sulfate solution was pipetted and evenly applied to the nude skin of the mice for 5 days. After modeling: the positive group was treated with 1% hydrocortisone ointment 3 times a day continuously for 2 days; the model group was not treated. Each test group solution was administered in the same manner as the positive group. The mice in the normal group were treated by the same method to remove the abdominal hair, fed for 7 days and compared. On the 7th day of the experiment, the skin condition of each group of experimental mice was observed, and the serum of mice was collected for determining the amount of inflammatory factor IL-1a.

[0067] Index of evaluation: Determination of the amount of inflammatory factor IL-1a in serum

[0068] Index explanation: Acute barrier dysfunction can activate MyD pathway and increase the synthesis of inflammatory factor IL-1a downstream of MyD pathway, which is repeatedly observed in the inflammatory state of the skin. In this experiment, the feasibility of irritant dermatitis caused by 10% SDS was observed by comparing model group, positive group and normal group, and tested by the treatment with a positive drug hydrocortisone.

[0069] Experiments on allergic contact dermatitis Cosmetic allergy is commonly referred to as cosmetic allergic contact dermatitis, which is as high as 30.0-46.2% of cosmetic dermatitis. Cosmetic allergic contact dermatitis is often more severe than irritant dermatitis, and cross-allergic reactions occur, and treatment is relatively difficult. Therefore, 2,4-dinitrofluorobenzene (DNFB) was used to sensitize and stimulate mice to establish allergic contact dermatitis model (ACD), and 1% hydrocortisone ointment was administered as a positive control, and efficacy evaluation experiments were carried out in the test groups.

[0070] Experimental animals: ICR mice, 20-25 g, male.

[0071] Experimental materials: DNFB acetone/olive oil (4:1) solution, 1% hydrocortisone ointment, depilatory paste.

[0072] Experimental methods: ICR mice were randomly divided into groups according to body weight, 12 mice in each group, including normal group, model group, positive group, and spray test groups. At 1 day before modeling, the mice in each group were treated with depilatory paste to remove about 2*2 cm area of the hair in the abdomen of the mice. On the day of modeling, the mice in the model group, the positive group and the test groups were sensitized by applying 100 μL of a 5% DNFB acetone/olive oil solution evenly to the nude skin of the mice continuously for 2 days. On the 5th day, the mice were stimulated by applying 30 μL/ear of 1% DNFB acetone/olive oil solution evenly to the inner and outer auricular surfaces of both ears in the mice to establish an allergic contact dermatitis model. The mice in the normal group were treated with acetone/olive oil (4:1) solution in the abdomen and ears.

[0073] After 16 hours of stimulation, the drug was administered. For the test groups, 30 μL/ear of the sample was pipetted and evenly applied to the inner and outer auricular surfaces with cotton sticks, three times a day, morning, noon and night, continuously for 2 days. The model group was sprayed with distilled water, and the positive group was sprayed with 1% hydrocortisone ointment. Whole blood was collected 4 h later after the last administration, and the mice were executed.

[0074] Index of Evaluation

[0075] Ear thickness index: After execution, the thickness of mice's ears was measured, and the average thickness of both ears was obtained.

[0076] Ear weight index: After execution, each mouse's ears were collected by a perforator (Deli, 8 mm diameter), and the total weight was weighed.

[0077] Inflammatory index determination: The serum of mice was collected, and the amount of inflammatory factor IFN-γ in the ear tissues of the mice was detected by ELISA kit.

[0078] Index explanation: Th1/Th2 imbalance is an important link or initiating factor in the pathogenesis of allergic diseases. Normally, Th1 cells and Th2 cells are in dynamic equilibrium, and inhibit each other by secreting their own cytokines (Th1 cytokines such as IFN-r, Th2 cytokines such as IL-4). Th1 cells produce IFN-Y, TNF-α, IL-2 and the like, causing contact allergy; Th2 cells produce IL-4, IL-5, IL-10, IL-13 and the like, inhibiting contact hypersensitivity.

[0079] The experimental results were summarized in Table 3 below.

TABLE-US-00006 TABLE 3 Anti-inflammatory effect test in mouse models Ear thickness Ear weight INF-γ IL-1a (mm) (mg) Th1/Th2 (pg/ml) (pg/ml) Mean P Mean P Mean P Mean P Mean P value value value value value value value value value value Model group 0.39 — 22.08 — 2.53 — 99.2 — 30.54 — Normal group 0.18 0.00 8.88 0.00 1.21 0.00 72.2 0.02 20.85 0.00 Positive group 0.27 0.00 14.17 0.00 1.36 0.01 82.6 0.01 19.17 0.00 Spray A 0.27 0.00 13.71 0.00 1.07 0.00 69.3 0.00 19.40 0.00 Spray B 0.32 0.02 19.33 0.01 1.80 0.05 82.3 0.01 24.36 0.03 Spray C 0.30 0.00 17.67 0.01 1.58 0.03 80.2 0.03 24.46 0.04 Spray D 0.30 0.00 17.83 0.01 1.59 0.04 81.3 0.05 24.49 0.05 Spray E 0.31 0.00 19.29 0.02 1.59 0.04 80.6 0.04 24.37 0.04

[0080] The above results showed that the anti-inflammatory effect of spray A in the scope of the invention was significantly better than that of sprays B-E in all indexes, indicating that the combination of oat kernel extract, panthenol and allantoin had synergistic effect on the relief of contact dermatitis in mice.

Example 6: Preparation of Anti-Allergic Toner

[0081] This example provided an anti-allergic toner composition comprising the following ingredients:

TABLE-US-00007 Ingredients Amount (wt %) Birch juice 75.25 Panthenol 1.2 Allantoin 0.3 Oat kernel extract 3 Glycerin 5 Betaine 3 Glycereth-26 0.5 phytosteryl/octyldodecyl lauroyl glutamate 1 Butanediol 5 Pentanediol 2 Glyceryl tri(2-ethylhexanoate) 2 PEG-60 hydrogenated castor oil 1 Citric acid 0.1 Arginine 0.1 Methyl hydroxybenzoate 0.15 Phenoxyethanol 0.4

[0082] The toner composition was prepared as follows:

[0083] 1. The birch juice was heated to 80° C., and citric acid, panthenol, allantoin, 3 parts of glycerin, methyl hydroxybenzoate, butanediol, pentanediol and betaine were added in turn, mixed, stirred and dissolved uniformly;

[0084] 2. Phytosteryl/octyldodecyl lauroyl glutamate, glyceryl tri(2-ethylhexanoate), 2 parts of glycerin, glycereth-26 and PEG-60 hydrogenated castor oil were heated to 80° C., stirred uniformly, and then added to the mixture obtained in step 1;

[0085] 3. the resultant was cooled to 40° C., added with oat kernel extract and phenoxyethanol in turn, the pH was regulated with arginine, and the product was discharged.

[0086] The toner exhibited excellent anti-allergic, soothing and other effects in skin care.

Example 7: Preparation of Anti-Allergic Lotion

[0087] This example provided an anti-allergic lotion composition comprising the following ingredients:

TABLE-US-00008 Ingredients Amount (wt %) Water 78.1 Panthenol 1.5 Allantoin 0.5 Oat kernel extract 3 Glyerin 5 Hydrolyzed sodium hyaluronate 1 Dipropylene glycol 3 Caprylic/capric triglyceride 3 Shea butter 1 Carbomer 0.15 Xanthan gum 0.1 Cetyl alcohol 1 Glyceryl stearate/PEG-100 1 stearate Glyceryl tri(2-ethylhexanoate) 1 Phenoxyethanol 0.4 Methyl hydroxybenzoate 0.1 Arginine 0.15

[0088] The above anti-allergic lotion composition was prepared as follows:

[0089] 1. Aqueous phase: water, glycerin, panthenol, allantoin, hydrolyzed sodium hyaluronate, dipropylene glycol, xanthan gum, carbomer, and methyl hydroxybenzoate were mixed and heated to 80° C., stirred and dissolved uniformly;

[0090] 2. Oil phase: cetyl alcohol, glyceryl stearate/PEG-100 stearate, caprylic/capric triglyceride, glyceryl tri(2-ethylhexanoate), and shea butter. The raw materials were heated to 80° C., stirred and dissolved uniformly;

[0091] 3. The aqueous phase and the oil phase were mixed and homoemulsified for 5 mins, upon the completion of the emulsification, arginine dissolved in water was added, stirred for 15 mins, cooled to 40° C., and added with oat kernel extract and phenoxyethanol in turn.

[0092] The lotion exhibited excellent anti-allergy, soothing and other effects in skin care.

Example 8: Preparation of Anti-Allergic Facial Cream

[0093] This example provided an anti-allergic facial cream composition comprising the following ingredients:

TABLE-US-00009 Ingredients Amount (wt %) Water 75.15 Panthenol 1 Allantoin 0.6 Oat kernel extract 2 Glyerin 6 Hydrolyzed sodium hyaluronate 0.5 Glycereth-26 2 Xanthan gum 0.1 Glyeryl caprylate/caprate 2 Shea butter 1 Cetyl alcohol 1 Glyceryl stearate/PEG-100 stearate 1.5 SIMUGEL EG 1 Pentaerythritol tetra(2-ethyl hexanoate) 3 Polydimethylsiloxane 2 Methyl hydroxybenzoate 0.2 Propyl hydroxybenzoate 0.1 Carbomer 0.1 Phenoxyethanol 0.4 Caprylyl glycol 0.05 arginine 0.3

[0094] The above anti-allergic facial cream composition was prepared as follows:

[0095] 1. Aqueous phase: water, glycerin, panthenol, allantoin, hydrolyzed sodium hyaluronate, glycereth-26, xanthan gum, carbomer, and methyl hydroxybenzoate;

[0096] 2. Oil phase: cetyl alcohol, caprylate/caprate, pentaerythritol tetra(2-ethyl hexanoate), glyceryl stearate/PEG-100 stearate, shea butter, polydimethylsiloxane, propyl hydroxybenzoate. The raw materials were heated to 80° C., stirred and dissolved uniformly;

[0097] 3. The aqueous phase and the oil phase were mixed and homoemulsified for 5 mins, upon the completion of the emulsification, arginine dissolved in water was added, stirred for 15 mins, cooled to 40° C., and added with oat kernel extract, phenoxyethanol and caprylyl glycol in turn.

[0098] The facial cream exhibited excellent anti-allergy, soothing and other effects in skin care.

[0099] The technical solutions of the above examples were preferred embodiments of the present invention. In addition, the soothing and anti-sensitive composition of the present invention can be applied to various cosmetics such as essence, BB cream, sunscreen and so on. Some improvements and changes can be made without departing from the principles of the present invention, and such improvements and changes should also be considered in the protection scope of the present invention.