Cosmetic compositions to prevent and/or ameliorate skin aging and methods of applications

Abstract

A cosmetic composition to prevent and/or ameliorate skin aging and applications thereof are provided in the present disclosure and the cosmetic composition comprises cyclic phosphatidic acid and citrus flavonoids which is hesperidin and/or hesperetin wherein the preventing and/or ameliorating skin aging include, without limitation, promoting moisture retention of stratum corneum, promoting skin elasticity, increasing presence or elasticity of collagens and elastins in fibroblasts, increasing transfers of fibroblasts, promoting anti-oxidation, increasing generation of hyaluronic acid, alleviating wrinkles and skin textures, moderating melanin, reducing skin oil and preventing transepidermal water losses.

Claims

1. A cosmetic composition for preventing and/or ameliorating skin aging, comprising (a) cyclic phosphatidic acid, (b) propylene or butylene glycol, (c) glycerin and (d) citrus flavonoid; wherein the citrus flavonoid is hesperidin and/or hesperetin; wherein the cyclic phosphatidic acid is represented by a structural formula (I): ##STR00004## where R is linoleic acid, palmitic acid or oleic acid; M is sodium (Na) or hydrogen (H); and the molecular weight of the cyclic phosphatidic acid ranges from 300 to 500.

2. The cosmetic composition as claimed in claim 1, wherein the cyclic phosphatidic acid is sodium cyclic lysophosphatidic acid (NcPA).

3. The cosmetic composition as claimed in claim 1, wherein the cyclic phosphatidic acid features the weight percentage between 0.01% and 0.5%.

4. The cosmetic composition as claimed in claim 1, wherein the citrus flavonoid features the weight percentage between 0.01% and 0.5%.

5. The cosmetic composition as claimed in claim 1, wherein the cosmetic composition is in the form of an ointment, a lotion, a cream, a gel, liquid drops, a spray, a solution, a facial mask or an agent acceptable in pharmacy or cosmetology.

6. The cosmetic composition as claimed in claim 1, wherein the cosmetic composition further comprise moisturizing agents, surfactants, UV absorbents, fragrances, anti-oxidants, preservatives, body pigments, color pigments, pH adjusting agents, solvents or any other ingredients for general cosmetics or topical dermatologic drug compositions.

7. A method for preventing and/or ameliorating skin aging, in which a cosmetic composition is applied on skin to be cured transdermally or on local skin topically at room temperature, wherein the cosmetic composition comprising the composition of claim 1.

8. The method as claimed in claim 7, wherein the skin includes normal skin and aging skin.

9. The method as claimed in claim 7, wherein the skin aging is selected from a combination of wrinkles, fine lines, skin darkening, dehydration, absence of pigments and absence of skin elasticity.

10. The method as claimed in claim 7, wherein the preventing and/or ameliorating skin aging means promoting moisture retention of stratum corneum, promoting skin elasticity, increasing presence or elasticity of collagens and elastin in fibroblasts, increasing transfers of fibroblasts, promoting anti-oxidation, increasing generation of hyaluronic acid, alleviating wrinkles and skin textures, moderating melanin, reducing skin oil and preventing transepidermal water losses.

11. A method used for preventing and/or ameliorating skin aging, comprising topically or transdermally administering a composition of claim 1.

12. The cosmetic composition as claimed in claim 1, wherein the cyclic phosphatidic acid is a non-liposomal cyclic phosphatidic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 demonstrates the reference standard of the hyaluronic acid score for Score 3, Score 5 and Score 7 in Embodiment 11;

(3) FIG. 2 demonstrates hyaluronic acid staining for a subject's skin specimen changing from the baseline to that on Week 12 in Embodiment 11;

(4) FIG. 3 demonstrates the reference standard of the Type III collagen score for Score 3, Score 5 and Score 7 in Embodiment 12;

(5) FIG. 4 demonstrates Type III collagen staining for a subject's skin specimen changing from the baseline to that on Week 12 in Embodiment 12;

(6) FIG. 5 demonstrates the reference standard of the elastic fiber score for Score 3, Score 5 and Score 7 in Embodiment 13;

(7) FIG. 6 demonstrates elastic fiber staining for a subject's skin specimen changing from the baseline to that on Week 12 in Embodiment 13;

(8) FIG. 7 demonstrates test results by the VISIA Skin Analysis System for a subject's wrinkles on the right face changing from the baseline to that on Week 12 in Embodiment 14;

(9) FIG. 8 demonstrates test results by the VISIA Skin Analysis System for a subject's wrinkles on the left face changing from the baseline to that on Week 12 in Embodiment 14;

(10) FIG. 9 demonstrates test results by the VISIA Skin Analysis System for a subject's skin textures on the right face changing from the baseline to that on Week 12 in Embodiment 15; and

(11) FIG. 10 demonstrates test results by the VISIA Skin Analysis System for a subject's skin textures on the left face changing from the baseline to that on Week 12 in Embodiment 15.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(12) Unless otherwise stated, all technical and scientific terms in the present application, including the specification and claims, have definitions known to the persons who are skilled and have general knowledge in the art.

(13) A cosmetic composition for preventing and/or ameliorating skin aging for skin care in the present disclosure is characteristic of a dosage form, which is adjusted flexibly and not specified, and particularly a formulation topically applied on skin.

(14) As a topical product for skin, the cosmetic composition in the present disclosure could be made as a cosmetic, a medicine or a topical drug. Furthermore, a cosmetic composition features the dosage form including, without limitation, a solution, a lotion, a cream, a cleanser, a gel, an ointment or a paste.

(15) In addition to primary ingredients, the cosmetic composition for the topical application in the present disclosure may comprise moisturizing agents, surfactants, UV absorbents, fragrances, anti-oxidants, preservatives, body pigments, color pigments, pH adjusting agents, solvents or any other acceptable ingredients for general cosmetics or topical dermatologic drug compositions, all of which have no negative effect on the scope of the present application.

(16) In the cosmetic composition, an agent acceptable in pharmacy or cosmetology may comprise one or more reagents selected from a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, a surfactant and other similar agents or agents applicable to the cosmetic composition.

(17) In the present disclosure, the cosmetic composition for preventing and/or ameliorating skin aging comprises cyclic phosphatidic acid and citrus flavonoid wherein the citrus flavonoid is hesperidin and/or hesperetin.

(18) The cyclic phosphatidic acid is represented by the structural formula (I):

(19) ##STR00002##
where R is linoleic acid, palmitic acid or oleic acid, M is sodium (Na) or hydrogen (H), and the molecular weight of sodium cyclic phosphoric ranges from 300 to 500.

(20) In embodiments of the present disclosure, the cyclic phosphatidic acid is sodium cyclic lysophosphatidic acid (NcPA).

(21) In the present disclosure, hesperidin is a natural glucoside existing in citrus and can be transferred to hesperetin after acidification based on a chemical reaction as follows:

(22) ##STR00003##

(23) The cosmetic composition for preventing and/or ameliorating skin aging is demonstrated in embodiments hereinafter that do not limit the scope of the cosmetic composition. The medicines or biological materials used hereinafter are available in the market and indicated in the following embodiments.

Embodiment 1: Ingredients and the Manufacturing Process of a Lotion with Cyclic Phosphatidic Acid and Hesperidin

(24) Ingredients and weight percentages of ingredients in a lotion with cyclic phosphatidic acid and hesperidin are shown in Table 1.

(25) TABLE-US-00001 Ingredient Weight Percentage Water  >80% Propylene Glycol 1-10% Glycerin 1-10% Bis-Ethoxydiglycol Cyclohexane 1,4-Dicarboxylate 1-10% Sorbitol   <1% Betaine   <1% PEG-60 Hydrogenated Castor Oil   <1% Carbomer   <1% Triethylhexanol   <1% Phenyl Trimethicone   <1% Potassium Hydroxide   <1% Polyglyceryl-10 Myristate   <1% Hesperidin   <1% Sodium Cyclic Lysophosphatidic Acid (NcPA)   <1% Phenoxyethanol   <1% Methylparaben   <1%

(26) In Table 1, the preferred contents of hesperidin and cyclic phosphatidic acid are 0.01-0.5 wt % and 0.01-0.5 wt %, respectively.

(27) The manufacturing process of the lotion is shown as follows:

(28) Oil phase: Bis-ethoxydiglycol cyclohexane 1,4-dicarboxylate, PEG-60 hydrogenated castor oil, triethylhexanol, phenyl trimethicone, polyglyceryl-10 myristate, phenoxyethanol and methylparaben are added into Container A in which temperature is heated to 70° C. and the mixture is stirred well.

(29) Aqueous phase: Carbomer is slowly added into Container B with water inside, stirred and dissolved in water. Propylene Glycol, glycerin and sorbitol are sequentially added into Container B in which mixtures are stirred well and potassium hydroxide is further added for better consistency. Container B is heated to 70° C. at which mixtures are stirred continuously and phenoxyethanol as well as methylparaben are further added for stirring well.

(30) Oil-water mixture: The mixture in Container A is added into Container B in which a new mixture is stirred well at 70° C. and then temperature is lowered to 50° C.

(31) Lotion: Hesperidin and sodium cyclic lysophosphatidic acid (NcPA) are added into Container B in turn; the mixture in Container B is stirred well for production of the lotion.

Embodiment 2: Ingredients and the Manufacturing Process of a Lotion with Cyclic Phosphatidic Acid and Hesperetin

(32) Ingredients and weight percentages of ingredients in a lotion with cyclic phosphatidic acid and hesperetin are shown in Table 2.

(33) TABLE-US-00002 TABLE 2 Weight Ingredient Percentage Water >80%  Propylene Glycol 1-10%   Glycerin 1-10%   Bis-Ethoxydiglycol Cyclohexane 1-10%   1,4-Dicarboxylate Sorbitol <1% Betaine <1% PEG-60 Hydrogenated Castor Oil <1% Carbomer <1% Triethylhexanol <1% Phenyl Trimethicone <1% Potassium Hydroxide <1% Polyglyceryl-10 Myristate <1% Hesperetin <1% Sodium Cyclic Lysophosphatidic <1% Acid (NcPA) Phenoxyethanol <1% Methylparaben <1%

(34) In Table 2, the preferred contents of hesperetin and cyclic phosphatidic acid are 0.01-0.5 wt % and 0.01-0.5 wt %, respectively.

(35) The manufacturing process of the lotion is shown as follows:

(36) Oil phase: Bis-ethoxydiglycol cyclohexane 1,4-dicarboxylate, PEG-60 hydrogenated castor oil, triethylhexanol, phenyl trimethicone, polyglyceryl-10 myristate, phenoxyethanol and methylparaben are added into Container A in which temperature is heated to 70° C. and the mixture is stirred well.

(37) Aqueous phase: Carbomer is slowly added into Container B with water inside, stirred and dissolved in water. Propylene glycol, glycerin and sorbitol are sequentially added into Container B in which the mixture is stirred well and potassium hydroxide is further added for better consistency. Container B is heated to 70° C. at which the mixture is stirred continuously and phenoxyethanol as well as methylparaben are further added for stirring well.

(38) Oil-water mixture: The mixture in Container A is added into Container B in which a new mixture is stirred well at 70° C. and then temperature is lowered to 50° C.

(39) Lotion: Hesperidin and sodium cyclic lysophosphatidic acid (NcPA) are added into Container B in turn; the mixture in Container B is stirred well for production of the lotion.

Embodiment 3: Ingredients and the Manufacturing Process of an Essence with Cyclic Phosphatidic Acid and Hesperidin

(40) Ingredients and weight percentages of ingredients in an essence with cyclic phosphatidic acid and hesperidin are shown in Table 3.

(41) TABLE-US-00003 TABLE 3 Weight Ingredient Percentage Water >75%  Butylene Glycol 5-15%   Glycerin 5-15%   Hydrogenated Lecithin <1% Bis-Ethoxydiglycol Cyclohexane <1% 1,4-Dicarboxylate Phenoxyethanol <1% Carbomer <1% Cholesterol <1% Methylparaben <1% Hesperidin <1% Sodium Cyclic Lysophosphatidic <1% Acid (NcPA) Sodium Hydroxide <1% Xanthan Gum <1% Dipotassium Glycyrrhizate <1% Hydrogenated Lysolecithin <1%

(42) In Table 3, the preferred contents of hesperidin and cyclic phosphatidic acid are 0.01-0.5 wt % and 0.01-0.5 wt %, respectively.

(43) The manufacturing process of the essence is shown as follows:

(44) Oil phase: Glycerin, hydrogenated lecithin, bis-ethoxydiglycol cyclohexane 1,4-dicarboxylate, phenoxyethanol and hydrogenated lysolecithin are added into Container A at room temperature at which the mixture is stirred well; cholesterol, methylparaben and hesperidin are added into Container A in turn and dissolved in the mixture therein.

(45) Aqueous phase: Carbomer is slowly added into Container B with water inside, stirred and dissolved in water.

(46) Essence: The mixture in Container A is added into Container B in which a new mixture is stirred well; sodium hydroxide is slowly added into Container B for a pH value adjustment.

Embodiment 4: Ingredients and the Manufacturing Process of an Essence with Cyclic Phosphatidic Acid and Hesperetin

(47) Ingredients and weight percentages of ingredients in an essence with cyclic phosphatidic acid and hesperetin are shown in Table 4.

(48) TABLE-US-00004 TABLE 4 Weight Ingredient Percentage Water >75%  Butylene Glycol 5-15%   Glycerin 5-15%   Hydrogenated Lecithin <1% Bis-Ethoxydiglycol Cyclohexane <1% 1,4-Dicarboxylate Phenoxyethanol <1% Carbomer <1% Cholesterol <1% Methylparaben <1% Hesperetin <1% Sodium Cyclic Lysophosphatidic <1% Acid (NcPA) Sodium Hydroxide <1% Xanthan Gum <1% Dipotassium Glycyrrhizate <1% Hydrogenated Lysolecithin <1%

(49) In Table 4, the preferred contents of hesperetin and cyclic phosphatidic acid are 0.01-0.5 wt % and 0.01-0.5 wt %, respectively.

(50) The manufacturing process of the essence is shown as follows:

(51) Oil phase: Glycerin, hydrogenated lecithin, bis-ethoxydiglycol cyclohexane 1,4-dicarboxylate, phenoxyethanol and hydrogenated lysolecithin are added into Container A at room temperature at which the mixture is stirred well; cholesterol, methylparaben and hesperidin are further added into Container A.

(52) Aqueous phase: Carbomer is slowly added into Container B with water inside, stirred and dissolved in water.

(53) Essence: The mixture in Container A is added into Container B in which a new mixture is stirred well; sodium hydroxide is slowly added into Container B for a pH value adjustment.

Embodiment 5: In-Vitro Skin Permeability Test Based on Formula Product with Hesperidin or Hesperetin

(54) A series of cosmetics prepared with hesperidin or hesperetin, for example, lotions and essences, are tested by in-vitro skin permeation to investigate the percutaneous absorptions of cosmetics.

(55) Conditions for the in-vitro skin permeation test:

(56) Skin Specimen: Human cadaver skin;

(57) Apparatus for the skin permeation test: Horizontal skin permeation device;

(58) Area of skin effectively permeated: 0.64 cm.sup.2;

(59) Acceptor vehicle: 3.5 mL water solutions with 10% PEG 400 dissolved in water;

(60) Temperature: 32° C.;

(61) Test time: 24 hours;

(62) Number of trials: Each specimen tested three times.

(63) The steps of the skin permeation test are shown as follows: 1. Human cadaver skin is placed on a horizontal skin permeation device in which 10% PEG 400 solution is used as the receptor vehicle, at 32° C.; a formula product is applied on the skin surface in triplicate. 2. Study period: The test is terminated after 24 hours; specimens are collected from the receptor vehicle. 3. Processing of residues on outer layer skin: After termination of the test, skin on which a formula product is applied on is removed from the horizontal skin permeation device and laid on a slab. A piece of Scotch tape (size: 1×3 cm.sup.2) is placed on the skin surface and a gentle pressure is applied to the tape to strip the surface skin layer. Note: The practice is repeated for a total of five times to remove any possible cosmetic residue left on skin surface. The residues on the tape are not analyzed. 4. Specimens on epidermis: After the process in step 3, the same practice as described in Step 3 is repeated 15 times each with a piece of Scotch tape (size: 1×3 cm.sup.2). Note: Each piece of tape after skin stripping is collected together in a 20 mL scintillation vial for extraction of cosmetic ingredients using methanol as a solvent. 5. Specimens on dermis: Skin after the process in step 4 is cut into pieces with a scissors. The pieces are collected into a 20 mL scintillation vial. Methanol is added into the vial to extract the cosmetic ingredients by using a homogenizer. 6. Specimens processed from step 4 to step 5 are analyzed for the content of hesperidin or hesperetin using HPLC.

(64) Conditions of HPLC:

(65) Equipment: Waters, Photodiode Array Detector equipped with an autosampler

(66) Column: C18, 5 μm; 4.6×250 mm

(67) Temperature: 45±5° C.

(68) Mobile phase: 0.2% acetic acid (pH=3):methanol=70:30 by volume

(69) Flow velocity: 1.0 mL/min

(70) Test results for formula products with hesperidin are shown in Table 5:

(71) TABLE-US-00005 TABLE 5 Content of hesperidin Formula Lot Epidermis Dermis Skin permeation of product Number (μg/g) (μg/g) hesperidin (μg/cm.sup.2/24 hrs) Hydrating P28736-02 1,462 8 0.16 lotion Hydrating P29422-01 0 0 0 night cream

(72) A hydrating lotion and a hydrating night cream, each of which is prepared with hesperidin mixed, are produced for studying skin permeation of hesperidin according to the content of hesperidin on each skin specimen processed in previous steps.

(73) As shown in Table 5, the test results are summarized as follows: (a) hydrating lotion: the contents of hesperidin in a hydrating lotion on 1 g epidermis and 1 g dermis are 1.4 mg and 8 μg, respectively; skin permeation of hesperidin permeating through skin and assayed in the receptor vehicle is 0.16 μg/cm.sup.2/24 hrs; (b) hydrating night cream: no hesperidin is detected in the receptor vehicle meaning no hesperidin in a hydrating night cream deposits on epidermis or dermis.

(74) Test results for formula products with hesperetin are shown in Table 6:

(75) TABLE-US-00006 TABLE 6 Content of hesperetin Formula Lot Epidermis Dermis Skin permeation of product Number (μg/g) (μg/g) hesperetin (μg/cm.sup.2/24 hrs) Anti- P29433-01 14 14 1.73 aging lotion Anti- P29429-01 26 131 2.35 aging essence Anti- P29431-01 1 1 0 aging cream

(76) An anti-aging lotion, an anti-aging essence and an anti-aging cream, each of which is prepared with hesperetin mixed, are produced for studying skin permeation of hesperetin according to the content of hesperetin on each skin specimen processed in previous steps.

(77) As shown in Table 6, the test results are summarized as follows: (a) anti-aging lotion: the contents of hesperetin in an anti-aging lotion on 1 g epidermis and 1 g dermis are 14 μg and 14 μg, respectively; skin permeation of hesperetin permeating through skin and assayed in the receptor vehicle is 1.73 μg/cm.sup.2/24 hrs; (b) anti-aging essence: the contents of hesperetin in an anti-aging essence on 1 g epidermis and 1 g dermis are 26 μg and 131 μg, respectively; skin permeation of hesperetin permeating through skin and assayed in the receptor vehicle is 2.35 μg/cm.sup.2/24 hrs; (c) anti-aging cream: the contents of hesperetin in an anti-aging cream on 1 g epidermis and dermis are 1 μg and 1 μg, respectively; no hesperetin is detected in the receptor vehicle meaning no hesperetin in an anti-aging cream deposits on epidermis or dermis.

(78) It can be seen from test results that hesperidin or hesperetin mixed in a cosmetic is transmitted to epidermis and dermis. For example, 0.1 mg-5 mg hesperidin and 1 μg-50 μg hesperidin are absorbed by 1 g epidermis and 1 g dermis, respectively; 0.1 μg-50 μg hesperetin and 0.1 μg-10 μg hesperetin are absorbed by 1 g epidermis and 1 g dermis, respectively. Among these cosmetics, the anti-aging essence, lot number P29429-01, with ingredients of hesperetin (0.01-0.5 wt %) and NcPA (0.01-0.5 wt %) presents better skin permeation and is selected as a test object of a clinic trial for the effect of preventing skin aging.

Embodiment 6: Human Clinic Trial Based on the Anti-Aging Essence with Cyclic Phosphatidic Acid and Hesperetin

(79) In this embodiment, the effect of a cosmetic composition with cyclic phosphatidic acid and hesperetin on prevention and/or alleviation of skin aging is tested in a human clinic trial through a formula product, lot number P29429-01, for its effect and safety.

(80) The formula product is the anti-aging essence, lot number P29429-01, in which hesperetin (0.01-0.5 wt %) and NcPA (0.01-0.5 wt %) are mixed.

(81) Clinic trial Protocol: 35 subjects aged from 40 to 65 years old with facial skin aging were treated with the formula P29429-01 for 12 weeks, and the effects on skin status were tested to evaluate the efficacy. 12 of the subjects were additionally received an adjunct study of skin tissue sections. The dosage of the formula P29429-01 is about a finger pulp (approximately 0.5 c.c.) and was applied once in the morning and at night.

Embodiment 7: Promotion of the Water Content in Stratum Corneum and Skin Elasticity

(82) In Embodiment 7, any changes in the water content of facial skin (the skin elasticity) in Week 4 and Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(83) The statistics for the water content of subjects' facial skin are shown in Table 7.

(84) TABLE-US-00007 TABLE 7 Time N Mean S.D. Min. Max. Baseline 35 61.9 15.19 24.5 84.2 Week 4 35 67.0 13.72 38.7 97.6 Week12 35  67.6* 12.17 38.5 88.2 Asterisk (*) means a significant difference between the statistic in Week 4 (or Week 12) and the baseline (* p < 0.05; ** p < 0.01; *** p < 0.001)

(85) As shown in Table 7 for the comparison between the test data for the water content of subjects' skin and the baseline, the promotion in Week 12 indicates the statistically significant effect (the mean in Week 12, 67.60 (±12.17), relative to the baseline, 61.86 (±15.19); p=0.0148), that is, the water content of stratum corneum increases significantly. Moreover, the water content of subjects' stratum corneum in Week 4 also increases (mean=67.00 (±13.72)) and moisture retention of subjects' stratum corneum in Week 4 increase by 10% stably.

(86) The statistics for skin elasticity of subjects' facial skin are shown in Table 8.

(87) TABLE-US-00008 TABLE 8 Time N Mean S.D. Min. Max. Baseline 35 0.66 0.08 0.467 0.857 Week 4 35 0.71 0.07 0.58 0.879 Week12 35   0.73*** 0.1 0.543 1 Asterisk (*) means a significant difference between the statistic in Week 4 (or Week 12) and the baseline (* p < 0.05; ** p < 0.01; *** p < 0.001)

(88) As shown in Table 8 for the comparison between the test data for skin elasticity of subjects' skin and the baseline, the promotion in Week 12 indicates the statistically significant effect (the mean in Week 12, 0.73 (±0.1), relative to the baseline, 0.66 (±0.08); p<0.0001), that is, skin elasticity increases by 10% in Week 12. Accordingly, the cosmetic composition in the present disclosure proves effectiveness in the better water content of skin and skin elasticity.

Embodiment 8: Improvement of Melanin

(89) In Embodiment 8, any changes in melanin on subjects' faces in Week 4 and Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(90) The statistics for melanin on subjects' facial skin are shown in Table 9.

(91) TABLE-US-00009 TABLE 9 Time N Mean S.D. Min. Max. Baseline 35 185.03 31.23 136 258 Week 4 35 182.74 29.0 129 258 Week12 35 190.29 38.66 125 290

(92) As shown in Table 9 for the comparison between the test data for melanin on subjects' facial skin and the baseline, melanin on subjects' facial skin in Week 12 is improved by 3% (from the baseline (185.03 (±31.23)) to the mean in Week 12 (190.29 (±38.66))). Accordingly, the cosmetic composition in the present disclosure proves effectiveness in improvement of melanin.

Embodiment 9: Alleviation of Skin Oil

(93) In Embodiment 9, any changes in skin oil on subjects' faces in Week 4 and Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(94) The statistics for skin oil on subjects' facial skin are shown in Table 10.

(95) TABLE-US-00010 TABLE 10 Time N Mean S.D. Min. Max. Baseline 35 47.29 35.42 5 133 Week 4 35 44.51 32.28 3 143 Week12 35 41.8 32.41 3 154

(96) As shown in Table 10 for the comparison between the test data for skin oil on subjects' facial skin and the baseline, skin oil on subjects' facial skin in Week 12 decreases by 11% (from the baseline (47.29 (±35.42)) to the mean in Week 12 (41.8 (±32.41)). Accordingly, the cosmetic composition in the present disclosure proves effectiveness in alleviating skin oil.

Embodiment 10: Alleviation of Transepidermal Water Losses

(97) In Embodiment 10, any changes in transepidermal water losses (TEWL) from subjects' skin in Week 4 and Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(98) TABLE-US-00011 TABLE 11 Time N Mean S.D. Min. Max. Baseline 35 13.55 17.45 −29.7 99.9 Week 4 35 10.99 5.69 4.0 28.6 Week12 35 11.18 5.87 2.7 31.8

(99) As shown in Table 11 for the comparison between the test data for transepidermal water losses of subjects' facial skin and the baseline, transepidermal water losses from subjects' facial skin in Week 12 are alleviated by 18% (from the baseline (13.55 (±17.45)) to the mean in Week 12 (11.18 (±5.87)). Accordingly, the cosmetic composition in the present disclosure proves effectiveness in alleviating transepidermal water losses.

Embodiment 11: Increase of the Hyaluronic Acid Score Based on Live Skin Slices

(100) In Embodiment 11, any changes in the hyaluronic acid score based on live skin slices of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(101) The hyaluronic acid score is created according to test results of hyaluronic acid staining with which target cells stained by colloidal iron stains present Prussian blue that is considered as collections of hyaluronic acids, particularly more hyaluronic acids displayed by a thicker and more intensive blue color (equivalent to a higher hyaluronic acid score). FIG. 1 demonstrates the reference standard of hyaluronic acid score, for example, Score 3, Score 5 and Score 7. In Embodiment 11, the statistics for changes in the hyaluronic acid score collected from subjects in Week 12 relative to the baseline are shown in Table 12.

(102) TABLE-US-00012 TABLE 12 Hyaluronic acid score Mean hyaluronic acid 2 3 4 5 6 7 score Baseline 1 4 2 1 4 0 4.25 (Case count) Week 12 0 0 2 4 3 3 5.58 (Case count)

(103) As shown in Table 12, the mean hyaluronic acid score for live skin slices changes from 4.25 (baseline) to 5.58 (Week 12) that means the hyaluronic acid score increases by 31% after 12 weeks during which subjects applied the cosmetic product on their skin. Moreover, the significant difference for case counts between the baseline and Week 12 is observed through the Kruskal-Wallis Test (p-value=0.033572).

(104) For example, the test result of hyaluronic acid staining for one subject in FIG. 2 indicates the hyaluronic acid score increases from 3 (baseline) to 7 (Week 12) and the blue color is thicker and more intensive. Accordingly, the cosmetic composition in the present disclosure proves effectiveness in increasing the hyaluronic acid score.

Embodiment 12: Increase of the Collagen Score Based on Live Skin Slices

(105) In Embodiment 12, any changes in the collagen score based on live skin slices of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(106) The collagen score is created according to test results of collagen staining with which stained target cells present a deep and intensive pigment that is granted a higher collagen score and considered as collections of more collagens. In Embodiment 12, two types of collagens, Type I collagen and Type III collagen, are checked and any changes in the collagen score in Week 12 relative to the baseline are compared. The statistics collected from subjects for Type I collagen are shown in Table 13.

(107) TABLE-US-00013 TABLE 13 Type I Collagen Score Mean Type I Collagen 3 4 5 6 7 Score Baseline 1 2 8 1 0 4.75 (Case count) Week 12 0 1 6 4 1 5.42 (Case count)

(108) As shown in Table 13, the mean Type I collagen score for live skin slices changes from 4.75 (baseline) to 5.42 (Week 12) that means the Type I collagen score increases by 15% after 12 weeks during which subjects applied the cosmetic product on their skin. Moreover, the significant difference for case counts between the baseline and Week 12 is observed through the Kruskal-Wallis Test (p-value=0.0313).

(109) FIG. 3 demonstrates the reference standard of the Type III collagen score, for example, Score 3, Score 5 and Score 7. The statistics collected from subjects for Type III collagen are shown in Table 14.

(110) TABLE-US-00014 TABLE 14 Type III Collagen Score Mean Type III 4 5 6 7 Collagen Score Baseline 5 5 2 0 4.75 (Case count) Week 12 1 7 2 2 5.42 (Case count)

(111) As shown in Table 14, the mean Type III collagen score for live skin slices changes from 4.75 (baseline) to 5.42 (Week 12) that means the Type III collagen score increases by 15% after 12 weeks during which subjects applied the cosmetic product on their skin. Moreover, the significant difference for case counts between the baseline and Week 12 is observed through the Kruskal-Wallis Test (p-value=0.0156).

(112) For example, the test result of Type III collagen staining for one subject in FIG. 4 indicates the collagen score increases from 5 (baseline) to 7 (Week 12). Accordingly, the cosmetic composition in the present disclosure proves effectiveness in increasing the collagen score.

Embodiment 13: Increase of the Elastic Fiber Score Based on Live Skin Slices

(113) In Embodiment 13, any changes in the elastic fiber score based on live skin slices of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(114) The staining status of elastic fiber debris attributed to skin photoaging is evaluated by the ranking score that represents different levels of elastic fiber debris. Moreover, elastic fibers vertically arranged between dermis and epidermis are the symbol of “younger skin” but rare in aging skin. FIG. 5 demonstrates the reference standard of the elastic fiber score, for example, Score 3, Score 5 and Score 7. In Embodiment 13, any changes in the elastic fiber score based on live skin slices of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are compared and statistics collected from subjects are shown in Table 15.

(115) TABLE-US-00015 TABLE 15 Elastic fiber score Mean elastic fiber 3.5 3.8 4 4.3 4.5 4.8 5 score Baseline 3 2 1 2 1 2 1 4.15 (Case count) Week 12 0 0 2 1 2 3 4 4.65 (Case count)

(116) As shown in Table 15, the mean elastic fiber score for live skin slices changes from 4.15 (baseline) to 4.65 (Week 12) that means the elastic fiber score increases by 12% after 12 weeks during which subjects applied the cosmetic product on their skin. Moreover, the significant difference for case counts between the baseline and Week 12 is observed through the Kruskal-Wallis Test (p-value=0.0029).

(117) For example, the test result of elastic fiber staining for one subject in FIG. 6 indicates the elastic fiber score increases from 3 (baseline) to 7 (Week 12). Accordingly, the cosmetic composition in the present disclosure proves effectiveness in increasing the elastic fiber score.

Embodiment 14: Evaluation of Wrinkles by the VISIA Skin Analysis System

(118) In Embodiment 14, any changes in facial wrinkles of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated and taken as the major indicator of the curative effect.

(119) For example, alleviations of a subject's wrinkles on left and right faces are evaluated by the VISIA Skin Analysis System.

(120) As shown in FIG. 7, the percentile of the mean for the subject's wrinkles on the right face changes from 24% (baseline) to 43% (Week 12) that means wrinkles on the right face are improved by 79%. The test results are summarized in Table 16:

(121) TABLE-US-00016 TABLE 16 Feature count Score Percentile Baseline 45 10.645 24% Week 12 22 7.464 43%

(122) As shown in FIG. 8, the percentile of the mean for the subject's wrinkles on the left face changes from 13% (baseline) to 49% (Week 12) that means wrinkles on the left face are improved by 277%. The test results as shown in Table 17:

(123) TABLE-US-00017 TABLE 17 Feature count Score Percentile Baseline 45 13.919 13% Week 12 30 6.742 49%

(124) Accordingly, the cosmetic composition in the present disclosure proves effectiveness in smoothing away wrinkles.

Embodiment 15: Evaluation of Skin Textures by the VISIA Skin Analysis System

(125) In Embodiment 15, any changes in skin textures of subjects in Week 12 relative to the baseline (data collected one week before the human clinic trial) are evaluated by the VISIA Skin Analysis System and taken as the major indicator of the curative effect.

(126) For example, alleviations of skin textures on a subject's left and right faces are evaluated by the VISIA Skin Analysis System.

(127) As shown in FIG. 9, the percentile of the mean for skin textures on the subject's right face changes from 81% (baseline) to 94% (Week 12) that means skin textures on the subject's right face are improved by 16%. The test results are summarized in Table 18:

(128) TABLE-US-00018 TABLE 18 Feature count Score Percentile Baseline 494 1.948 81% Week 12 257 0.845 94%

(129) As shown in FIG. 10, the percentile of the mean for skin textures on the subject's left face changes from 70% (baseline) to 89% (Week 12) that means skin textures on the subject's left face are improved by 27%. The test results are summarized in Table 19:

(130) TABLE-US-00019 TABLE 19 Feature count Score Percentile Baseline 934 3.433 70% Week 12 433 1.464 89%

(131) Accordingly, the cosmetic composition in the present disclosure proves effective in smoothing away skin textures.

(132) The present disclosure offers a method to prevent and/or ameliorate skin aging, in which the cosmetic composition with cyclic phosphatidic acid and citrus flavonoid is applied on skin to be cured transdermally or on local skin topically at room temperature, wherein: the citrus flavonoid is hesperidin and/or hesperetin; the skin includes normal skin and ageing skin; the skin aging is selected from a combination of wrinkles, fine lines, skin darkening, dehydration, absence of pigments and absence of skin elasticity.

(133) In the present disclosure, the steps for the method for preventing and/or ameliorating skin aging include, without limitation, promoting moisture retention of stratum corneum, promoting skin elasticity, increasing presence or elasticity of collagens and elastins in fibroblasts, increasing transfers of fibroblasts, promoting anti-oxidation, increasing generation of hyaluronic acid, alleviating wrinkles and skin textures, moderating melanin, reducing skin oil and preventing transepidermal water losses.

(134) In summary, the cosmetic composition applied on skin contributes to significantly increasing moisture retention of skin, moderating transepidermal water losses, sustaining the water content of skin, preventing skin from fast aging due to dehydration, decreasing skin oil inside skin, alleviating melanin and making skin delicate and compact. In addition, the cosmetic composition, which is favorable to syntheses of hyaluronic acid and collagen/elastic fiber fragments for revived elastic fibers/skin elasticity, further proves effectiveness in moderating wrinkles and skin textures in the clinic trial and consists of natural ingredients without side effects for practical effects and safety.

(135) The descriptions presented in embodiments of the specification are only technical philosophy and characteristics of the present application that can be understood and practiced by a person skilled in the art; the embodiments which should not be taken as examples to limit claims thereinafter may be modified or changed by the person based on the disclosed embodiments in the present disclosure without departing from the spirit of claims.

(136) A cosmetic composition to prevent and/or ameliorate skin aging and applications thereof in the present application for multiple effects meets novelty and non-obviousness for patentability.