ANTI-WRINKLE COMPOSITION, ANTI-WRINKLE MICRONEEDLE PATCH AND PREPARATION METHOD THEREOF

Abstract

Provided are an anti-wrinkle composition, an anti-wrinkle microneedle patch and a preparation method thereof. The anti-wrinkle microneedle patch comprises a base and multiple needles, the base has an upper surface, and the needles are formed on the upper surface; wherein, a material of each needle comprises a low-molecular hyaluronic acid, a modified hyaluronic acid and a cross-linked hyaluronic acid. A weight ratio of the modified hyaluronic acid to the low-molecular hyaluronic acid is 0.1 to 4, and a weight ratio of the modified hyaluronic acid to the cross-linked hyaluronic acid is less than 30. Accordingly, the anti-wrinkle microneedle patch has effects of moisturizing the skin, elevating the thickness and the strength of the skin, and smoothing and reducing the wrinkles.

Claims

1. An anti-wrinkle composition, comprising a low-molecular hyaluronic acid, a modified hyaluronic acid and a cross-linked hyaluronic acid, and a weight ratio of the modified hyaluronic acid relative to the low-molecular hyaluronic acid being more than or equal to 0.1 and less than or equal to 4, and a weight ratio of the modified hyaluronic acid relative to the cross-linked hyaluronic acid being less than 30.

2. The anti-wrinkle composition as claimed in claim 1, wherein an average molecular weight of the low-molecular hyaluronic acid is more than or equal to 1 kDa and less than or equal to 10 kDa.

3. The anti-wrinkle composition as claimed in claim 1, wherein the modified hyaluronic acid is hyaluronic acid modified with histidine.

4. The anti-wrinkle composition as claimed in claim 1, wherein the cross-linked hyaluronic acid is prepared by subjecting hyaluronic acid to a cross-linking agent for cross-linking reaction.

5. The anti-wrinkle composition as claimed in claim 1, wherein the anti-wrinkle composition further comprises p-hydroxyacetophenone, pentylene glycol, hexanediol, caprylyl glycol, phenoxyethanol, benzyl alcohol, salicylic acid, benzoic acid, sorbic acid, potassium sorbate, propionic acid, dehydroacetic acid, chlorphenesin, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, butyl benzoate, dimethyl oxazolidine, bromochlorophene, dichlorobenzyl alcohol, ethyl lauroyl arginate HCl, 7-ethylbicyclooxazolidine, formic acid, glutaral, hexamidine, sodium sulfite, iodopropynyl butylcarbamate, methylisothiazolinone, butylparaben, methylparaben, phenoxyisopropanol, phenylmercuric acetate, triclocarban, undecylenic acid, zinc pyrithione, 5-bromo-5-nitro-1,3-dioxane, benzylhemiformal, 1,3-dimethylol-5,5-dimethylhydantoin, diazolidinyl urea, imidazolidinyl urea, methenamine, quaternium-15, sodium hydroxymethylglycinate, Leuconostoc/Radish Root Ferment Filtrate, Lactobacillus ferment, Populus tremuloides Bark Extract, Black Currant Fruit Extract or any combination thereof.

6. The anti-wrinkle composition as claimed in claim 2, wherein the anti-wrinkle composition further comprises p-hydroxyacetophenone, pentylene glycol, hexanediol, caprylyl glycol, phenoxyethanol, benzyl alcohol, salicylic acid, benzoic acid, sorbic acid, potassium sorbate, propionic acid, dehydroacetic acid, chlorphenesin, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, butyl benzoate, dimethyl oxazolidine, bromochlorophene, dichlorobenzyl alcohol, ethyl lauroyl arginate HCl, 7-ethylbicyclooxazolidine, formic acid, glutaral, hexamidine, sodium sulfite, iodopropynyl butylcarbamate, methylisothiazolinone, butylparaben, methylparaben, phenoxyisopropanol, phenylmercuric acetate, triclocarban, undecylenic acid, zinc pyrithione, 5-bromo-5-nitro-1,3-dioxane, benzylhemiformal, 1,3-dimethylol-5,5-dimethylhydantoin, diazolidinyl urea, imidazolidinyl urea, methenamine, quaternium-15, sodium hydroxymethylglycinate, Leuconostoc/Radish Root Ferment Filtrate, Lactobacillus ferment, Populus tremuloides Bark Extract, Black Currant Fruit Extract or any combination thereof.

7. The anti-wrinkle composition as claimed in claim 3, wherein the anti-wrinkle composition further comprises p-hydroxyacetophenone, pentylene glycol, hexanediol, caprylyl glycol, phenoxyethanol, benzyl alcohol, salicylic acid, benzoic acid, sorbic acid, potassium sorbate, propionic acid, dehydroacetic acid, chlorphenesin, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, butyl benzoate, dimethyl oxazolidine, bromochlorophene, dichlorobenzyl alcohol, ethyl lauroyl arginate HCl, 7-ethylbicyclooxazolidine, formic acid, glutaral, hexamidine, sodium sulfite, iodopropynyl butylcarbamate, methylisothiazolinone, butylparaben, methylparaben, phenoxyisopropanol, phenylmercuric acetate, triclocarban, undecylenic acid, zinc pyrithione, 5-bromo-5-nitro-1,3-dioxane, benzylhemiformal, 1,3-dimethylol-5,5-dimethylation, diazolidinyl urea, imidazolidinyl urea, methenamine, quaternium-15, sodium hydroxymethylglycinate, Leuconostoc/Radish Root Ferment Filtrate, Lactobacillus ferment, Populus tremuloides Bark Extract, Black Currant Fruit Extract or any combination thereof.

8. The anti-wrinkle composition as claimed in claim 4, wherein the anti-wrinkle composition further comprises p-hydroxyacetophenone, pentylene glycol, hexanediol, caprylyl glycol, phenoxyethanol, benzyl alcohol, salicylic acid, benzoic acid, sorbic acid, potassium sorbate, propionic acid, dehydroacetic acid, chlorphenesin, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, butyl benzoate, dimethyl oxazolidine, bromochlorophene, dichlorobenzyl alcohol, ethyl lauroyl arginate HCl, 7-ethylbicyclooxazolidine, formic acid, glutaral, hexamidine, sodium sulfite, iodopropynyl butylcarbamate, methylisothiazolinone, butylparaben, methylparaben, phenoxyisopropanol, phenylmercuric acetate, triclocarban, undecylenic acid, zinc pyrithione, 5-bromo-5-nitro-1,3-dioxane, benzylhemiformal, 1,3-dimethylol-5,5-dimethylhydantoin, diazolidinyl urea, imidazolidinyl urea, methenamine, quaternium-15, sodium hydroxymethylglycinate, Leuconostoc/Radish Root Ferment Filtrate, Lactobacillus ferment, Populus tremuloides Bark Extract, Black Currant Fruit Extract or any combination thereof.

9. An anti-wrinkle microneedle patch, comprising a base and multiple needles; the base having an upper surface and the multiple needles formed on the upper surface; wherein, a material of each needle comprises the anti-wrinkle composition as claimed in claim 1.

10. A preparation method of the anti-wrinkle microneedle patch as claimed in claim 9, comprising the following steps: step (a): providing a master mold having a datum plane and multiple holes, and the multiple holes formed by recessing from the datum plane; step (b): filling the multiple holes with a needle solution to make a surface of the needle solution level with the datum plane; wherein the needle solution comprises a low-molecular hyaluronic acid, a modified hyaluronic acid and a cross-linked hyaluronic acid, and a weight ratio of the modified hyaluronic acid relative to the low-molecular hyaluronic acid is more than or equal to 0.1 and less than or equal to 4, and a weight ratio of the modified hyaluronic acid relative to the cross-linked hyaluronic acid is less than 30; step (c): drying the needle solution to form multiple needles, wherein a surface of each needle is lower than the datum plane; step (d): filling the multiple holes with a base solution, and the base solution covering the surface of the needles and the datum plane; step (e): drying the base solution to form a base, such that the base is adhered to the multiple needles; and step (f): demolding the needles and the base that are adhered to each other from the master mold to obtain the anti-wrinkle microneedle patch.

11. The preparation method as claimed in claim 10, wherein a sum of a weight of the low-molecular hyaluronic acid, a weight of the modified hyaluronic acid, and a weight of the cross-linked hyaluronic acid in 1000 g of the needle solution is more than or equal to 20 g and less than or equal to 70 g.

12. The preparation method as claimed in claim 10, wherein a viscosity of the needle solution is more than or equal to 2 cP and less than or equal to 50 cP.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Several preparation methods of embodiments are exemplified below to illustrate the implementation of the present invention. One person skilled in the art can easily realize the advantages and effects of the present invention in accordance with the contents disclosed in the specification. Various modifications and variations could be made in order to practice or apply the present invention without departing from the spirit and scope of the invention.

[0054] Description of Reagents

[0055] 1. Low-molecular HA: purchased from Kewpie corporation; the average molecular weight is about 2 kDa; after dissolving in water with 1 wt %, the pH value is 5.0 to 7.0, and the viscosity is less than 2 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0056] 2. Histidine-modified HA: purchased from Industrial Technology Research Institute; after dissolving in water with 1 wt %, the pH value is 5.0 to 7.0, and the viscosity is about 4 cP to 5 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0057] 3. Cross-linked HA aqueous solution 1: purchased from Industrial Technology Research Institute; BDDE is used as the cross-linking agent; the solid content is about 4.5 wt %; the pH value is 5.0 to 7.5; and the viscosity is about 5000 cP to 8000 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0058] 4. Cross-linked HA aqueous solution 2: purchased from Bloomage Biotechnology Corporation Limited; BDDE is used as the cross-linking agent; the solid content is about 5 wt %; the pH value is 5.0 to 7.5; and the viscosity is more than or equal to 250000 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0059] 5. Hyaluronic acid: purchased from Kewpie corporation; the average molecular weight is less than or equal to 10 kDa; after dissolving in water with 0.1 wt %, the pH value is 2.0 to 4.0, and the viscosity is less than 1.5 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0060] 6. Carboxymethyl cellulose (CMC): purchased from Shin-Etsu Chemical Co., Ltd.; the average molecular weight is about 90 kDa; after dissolving in water with 4 wt %, the pH value is 6.5 to 8.5, and the viscosity is more than or equal to 50 cP and less than or equal to 200 cP when measured at a shear rate of 1 s.sup.−1 at 25° C.

[0061] 7. p-Hydroxyacetophenone: purchased from Symrise corporation.

[0062] 8. Pentylene glycol: purchased from ACTIVON Corporation.

[0063] 9. Polysorbate 20 (Tween-20): purchased from CRODA Corporation.

[0064] 10. First HPMC: purchased from Shin-Etsu Chemical Co., Ltd.; product name: 60SH-4000; after dissolving in water with 2 wt %, the viscosity is about 3000 cP to 5000 cP when measured at a shear rate of 1 s.sup.−1 at 20° C.

[0065] 11. Second HPMC: purchased from Shin-Etsu Chemical Co., Ltd.; product name: PHARMACOAT 645; after dissolving in water with 2 wt %, the viscosity is about 3.6 cP to 5.1 cP when measured at a shear rate of 1 s.sup.−1 at 20° C.

[0066] 12. Polyvinylpyrrolidone/vinyl acetate (PVP/VA): purchased from BASF Corporation.

[0067] 13. Lactobacillus ferment: purchased from ACTIVE MICRO Corporation; product name: Leucidal® SF MAX.

[0068] 14. Organic silicon defoamer: purchased from Dow Corning Corporation; Product Name: DOWSIL™ 62 Additive.

Examples 1 to 8: Anti-Wrinkle Microneedle Patch

[0069] First, a master mold with a datum plane and multiple holes was adopted. The multiple holes were formed by recessing from the datum plane, and arranged in array on the master mold. The material of the master mold was polydimethylsiloxane (PDMS), the density of the multiple holes was 75.08 holes/cm.sup.2, the multiple holes were arranged in a square array with a side length of 1 cm, and each hole was in the shape of a pyramid. The depth of each hole, i.e. the vertical distance between the tip of each hole and the datum plan, was about 270 μm to 330 μm, and the maximum width of each hole, i.e. the maximum inner diameter of the cross-section of each hole that aligned to the datum plane, was about 130 μm to 170 μm.

[0070] Next, according to the compositions listed in the following Table 1, suitable amounts of the low-molecular HA, the histidine-modified HA, the cross-linked HA aqueous solution 1, p-hydroxyacetophenone and pentylene glycol were added into water and mixed uniformly to obtain a needle solution. Afterward, about 5 g to 7 g of the needle solution was dropped on the master mold and covered the multiple holes thereof by a dispenser. Then, the master mold with the needle solution was placed into a vacuum oven for evacuation to reduce the pressure to −700 mmHg to −760 mmHg, such that the needle solution flowed downward into the multiple holes from the datum plane, and covered the datum plane and all holes. Next, the needle solution above the datum plane was totally removed to make the surface of the needle solution level with the datum plane, and the master mold with the needle solution was placed into an environment at 30° C. and with the RH of 10% to 30% for 1 hr to dry the needle solution into multiple needles. The surface of the needles was all lower than the datum plane, and a master mold with multiple needles was obtained. The viscosity of the needle solutions of Examples 1 to 8 was measured by a viscometer (model: MCR302; purchased from Anton Paar) at a shear rate of 1 s.sup.−1 at 25° C., and the results were listed in the following Table 1. Besides, the sum of the weight of the low-molecular HA, the weight of the histidine-modified HA and the weight of the cross-linked HA relative to the total weight of 1000 g of the needle solution, abbreviated as “total amount of three HA in needle solution”, of Examples 1 to 8 were also listed in the following Table 1, and the unit was g/kg. Said weight of the cross-linked HA indicated the weight of solid, which could be calculated by multiplying the weight of the cross-linked HA aqueous solution 1 to its solid content (4.5 wt %). For instance, in Example 1, the needle solution contained 6.72 wt % of the cross-linked HA aqueous solution 1, which indicated that 1000 g of the needle solution contained 67.2 g of the cross-linked HA aqueous solution 1. Then, the weight of the cross-linked HA aqueous solution 1 was multiplied to its solid content of 4.5 wt % to obtain the weight of the cross-linked HA, i.e., 3.024 g. Therefore, in Example 1, the sum of the weight of the low-molecular HA, the weight of the histidine-modified HA and the weight of the cross-linked HA relative to the total weight of 1000 g of the needle solution was the sum of 1000 g×4.03 wt %, 1000 g×1.34 wt % and 3.024 g, and was equal to 56.72 g. Besides, the weight ratio of the histidine-modified HA relative to the low-molecular HA and the weight ratio of the histidine-modified HA relative to the cross-linked HA were also listed in the following Table 1. It should be understood that the weight of the cross-linked HA was obtained by multiplying the weight of the cross-linked HA aqueous solution 1 to its solid content (4.5 wt %), and the weight ratio of the histidine-modified HA relative to the cross-linked HA can be calculated by the ratio of the weight of the histidine-modified HA relative to the weight of the cross-linked HA. For instance, in Example 1, the needle solution contained 6.72 wt % of the cross-linked HA aqueous solution 1, which indicated that the needle solution contained 0.3024 wt % of the cross-linked HA. Then, the cross-linked HA was further compared to the histidine-modified HA (1.34 wt %) to obtain the weight ratio of the histidine-modified HA relative to the cross-linked HA was about 4.43.

[0071] Next, suitable amounts of first HPMC, second HPMC, PVP/VA, p-hydroxyacetophenone, pentylene glycol, Tween-20, Lactobacillus ferment and organic silicon defoamer were added into water and mixed uniformly to obtain a base solution; wherein, based on the total weight of the base solution, the content of the first HPMC was about 0.96 wt %, the content of the second HPMC was about 0.48 wt %, the content of PVP/VA was about 1.92 wt %, the content of Tween-20 was about 0.02 v/w %, the content of the Lactobacillus ferment was about 0.8 wt %, the content of p-hydroxyacetophenone was about 0.25 wt %, the content of pentylene glycol was about 0.16 v/w %, and the content of the organic silicon defoamer was about 0.02 wt %. Afterward, about 7 g to 8.5 g of the base solution was dropped on the master mold with multiple needles by a dispenser, and covered the multiple holes thereof. Then, the master mold with the base solution was placed into the vacuum oven for evacuation to reduce the pressure to −700 mmHg to −760 mmHg, such that the base solution flowed downward into the multiple holes from the datum plane, and covered the datum plane and all needles in the holes. Next, the master mold with the base solution was placed into an environment at 23° C. and with the RH of 30% to 50% for 24 hr, and subsequently placed into an environment at 23° C. and with the RH of 55% to 75% for 60 hr to dry the base solution into the base. The base was adhered to the needles and the datum plane of the master mold, and a master mold with multiple needles and the base was obtained.

[0072] Finally, the final product was demolded from the master mold with multiple needles and the base to obtain the anti-wrinkle microneedle patches of Examples 1 to 8.

Comparative Examples 1 to 3: Anti-Wrinkle Microneedle Patch

[0073] The methods of preparing the anti-wrinkle microneedle patches of Comparative Examples 1 to 3 were similar to those of Examples. The main difference was that the needle solutions were prepared according to the compositions of Comparative Examples 1 to 3 listed in the following Table 1, and the rest of the preparation was in accordance with the descriptions in Examples to obtain the anti-wrinkle microneedle patches of Comparative Examples 1 to 3. The viscosity of the needle solutions of Comparative Examples 1 to 3 was measured by the viscometer at a shear rate of 1 s.sup.−1 at 25° C., and the results were listed in the following Table 1. Besides, the results of total amount of three HA in needle solution of Comparative Examples 1 to 3 were also listed in the following Table 1. Also, the weight ratio of the histidine-modified HA relative to the low-molecular HA and the weight ratio of the histidine-modified HA relative to the cross-linked HA were also listed in the following Table 1.

Comparative Examples 4 to 7: Anti-Wrinkle Microneedle Patch

[0074] The methods of preparing the anti-wrinkle microneedle patches of Comparative Examples 4 to 7 were similar to those of Examples. The main difference was that the needle solutions were prepared according to the compositions of Comparative Examples 4 to 7 listed in the following Table 2, and the rest of the preparation was in accordance with the descriptions in Examples to obtain the anti-wrinkle microneedle patches of Comparative Examples 4 to 7. The solid content of the needle solutions and the viscosity of the needle solutions measured by the viscometer at a shear rate of 1 s.sup.−1 at 25° C. of Comparative Examples 4 to 7 were also listed in the following Table 2.

TABLE-US-00001 TABLE 1 The composition of the needle solution, the weight ratio of the histidine-modified HA relative to the low-molecular HA, the weight ratio of the histidine-modified HA relative to the cross-linked HA, the total amount of three HA in needle solution, and viscosity of Examples 1 to 8 (E1 to E8) and Comparative Examples 1 to 3 (CE1 to CE3). Weight Weight ratio of the ratio of the histidine- histidine- The composition of the needle solution modified modified Total Cross- HA HA amount of Low- Histidine- linked HA relative to relative to three HA molecular modified aqueous p- Pentylene the low- the cross- in needle HA HA solution Hydroxyacetophenone glycol molecular linked solution Viscosity No. (wt %) (wt %) 1 (wt %) (wt %) (v/w %) HA HA (g/kg) (cP) E1 4.03 1.34 6.72 0.4 0.11 0.33 4.43 56.72 10.18 E2 4.03 1.35 1.34 0.4 0.11 0.33 22.39 54.40 6.3 E3 1.34 1.35 6.72 0.4 0.11 1.01 4.46 29.92 9.98 E4 1.34 1.35 4.03 0.4 0.11 1.01 7.44 28.71 7.94 E5 2.83 1.42 5.67 0.08 0.09 0.50 5.57 45.05 12.56 E6 1.42 2.83 5.67 0.08 0.09 1.99 11.09 45.05 26.64 E7 2.32 0.46 4.1 0.24 0.46 0.20 2.49 29.65 5.5 E8 4.1 0.46 2.32 0.24 0.46 0.11 4.41 46.64 4.48 CE1 5.67 2.83 1.42 0.08 0.09 0.50 44.29 85.64 21.24 CE2 2.83 5.67 1.42 0.08 0.09 2.00 88.73 85.64 86.18 CE3 1.42 5.67 2.83 0.08 0.09 3.99 44.52 72.17 92.56

TABLE-US-00002 TABLE 2 The composition, solid content and viscosity of the needle solutions of Comparative Examples 4 to 7 (CE4 to CE7). The composition of the needle solution Cross-linked HA aqueous Tween- Solid Viscos- HA solution CMC 20 content ity No. (wt %) 2 (wt %) (wt %) (v/w %) (wt %) (cP) CE4 1 0.135 — — 9.94 13.62 CE5 1 0.067 — — 9.97 8.76 CE6 1 0.026 — — 11.05 9.46 CE7 3 0.15 1 0.000018 3.85 24.08

Test Example 1: Needle Length Evaluation

[0075] The anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7 were adopted for this test example. Specifically, the vertical distance from the base where the needle was formed to the needle tip was directly measured by observing with microscope. 5 needles of each group were randomly picked for the measurement, and the results were averaged to obtain the needle length of the anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7, which were listed in the following Table 3.

Test Example 2: Mechanical Strength Evaluation

[0076] The anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7 were adopted for this test example. Specifically, the anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 and 7 were placed in an universal testing machine (model: 3343, purchased from INSTRON), and then the mechanical strength of the needle of each microneedle patch was measured. In this test example, a compressive test with the displacement set to 10 millimeters (mm) and the speed set to 66 mm/min was conducted, and 500 compressive stress values were received per second at the same time. The mechanical strength of the needles of the microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7 were listed in the following Table 3.

[0077] Table 3: The needle length and the mechanical strength of the needle of the anti-wrinkle microneedle patches of Examples 1 to 8 (E1 to E8) and Comparative Examples 1 to 7 (CE1 to CE 7).

TABLE-US-00003 Mechanical Needle strength length of the needle No. (μm) (N/needle) E1 297.24 0.205 E2 299.44 0.206 E3 290.56 0.190 E4 299.62 0.170 E5 299.34 0.161 E6 302.44 0.144 E7 303.87 0.163 E8 300.75 0.169 CE1 302.38 0.155 CE2 299.38 0.161 CE3 297.64 0.158 CE4 301.48 0.115 CE5 301.48 0.133 CE6 300.78 0.121 CE7 303.24 0.164

[0078] According to the results in Table 3, the needle length of the anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7 were all about 300 μm, which indicated that the needle length of the microneedle patch prepared by the method of the present invention had consistency, and had the advantage of stable quality. Further referring to the results of the mechanical strength of the needle, the mechanical strength of the needle of the anti-wrinkle microneedle patches of Examples 1 to 8 were all higher than 0.058 N/needle, and thus could pierce the stratum corneum without breakage. Therefore, the anti-wrinkle microneedle patches of the present invention could pierce the stratum corneum without bending or breakage, which ensured needle penetration under the skin and exerted the desired effects.

Test Example 3: Dissolution Rate Evaluation

[0079] Samples having the same composition with the needle of the anti-wrinkle microneedle patches of Examples 1 to 8 and Comparative Examples 1 to 7 were prepared according the aforementioned preparation method for the dissolution rate evaluation. Next, the samples of Examples 1 to 8 and Comparative Examples 1 to 7 were cut to the same size and weighed, such that those samples had the same weight. Then, after the samples were soaked in water for 5 minutes, the samples were taken out and weighed again. The weight difference between the sample before soaking and the sample after soaking relative to the weight of the sample before soaking in percentage was set as dissolution rate in water for 5 minutes. Here, the higher dissolution rate in water for 5 minutes indicated HA was dissolved and released more rapidly. The dissolution rate in water for 5 minutes of Examples 1 to 8 and Comparative Examples 1 to 7 were listed in the following Table 4.

TABLE-US-00004 TABLE 4 The dissolution rate in water for 5 minutes of Examples 1 to 8 and Comparative Examples 1 to 7. Dissolution rate in water No. for 5 minutes (%) E1 69.3 E2 74.03 E3 76.64 E4 80.66 E5 85.87 E6 78.41 E7 80.98 E8 70.98 CE1 24.35 CE2 17.13 CE3 16.58 CE4 53.85 CE5 56.25 CE6 52.08 CE7 37.26

[0080] According to the results in Table 4, the dissolution rates in water for 5 minutes of Examples 1 to 8 were all higher than 60%; wherein, the dissolution rates in water for 5 minutes of Examples 2 and 8 were higher than 70%, the dissolution rates in water for 5 minutes of Examples 3 and 6 were higher than 75%, and the dissolution rates in water for 5 minutes of Examples 4, 5 and 7 were higher than 80%. In contrast, the dissolution rates in water for 5 minutes of Comparative Examples 1 to 7 were all lower than 60%, and the difference of dissolution rate between Comparative Example 5 (56.25%, highest in Comparative Examples) and Example 1 (69.3%, lowest in Examples) was still up to 20%. Accordingly, as the anti-wrinkle microneedle patch of the present invention adopted the needle having specific composition, HA actually could rapidly dissolve and be released, thereby exerting the effects of skin moisturizing and increasing skin thickness and strength, and achieving the purpose of smoothing and reducing wrinkles.

Test Example 4: Anti-Wrinkle Evaluation

[0081] The anti-wrinkle microneedle patch of Example 7 was adopted for this test example. Specifically, 20 subjects (6 males; 14 females; age: 31 to 57) having wrinkles at periorbital were recruited. After the subjects were applied with the anti-wrinkle microneedle patch of Example 7, the tests for skin irritation, skin moisturizing, skin thickness, skin strength and wrinkle number were conducted for completely evaluating the effect of anti-wrinkle.

[0082] (1) Skin Irritation Test

[0083] In this test, the anti-wrinkle microneedle patch of Example 7 was cut into a size of 2.5 cm.sup.2 as a test group, and then was flat pasted on an inner side of a lower arm of the subject. Besides, a 0.9 wt % of sodium chloride solution (purchased from TUNGHSIN BIOSCIENCE CO., LTD.) was set as a control group, and then 0.2 ml of the sodium chloride solution was adsorbed by a sterile gauze of a size of 2.5 cm.sup.2. The sterile gauze was also flat pasted on the inner side of the lower arm of the subject and fixed with a waterproof and breathable tape. The evaluation of skin irritation was achieved by observing whether the symptoms of skin irritation appeared on the area applied with the test group after the subject was continuously applied with the test group and the control group for 24 hours. According to the score standard of skin irritation denoted in ISO 10993-10:2010, score 0 indicated no irritated reaction; score 1 indicated weak positive reaction (mild erythema and/or dryness of the skin); score 2 indicated moderate positive reaction (obvious erythema and/or dryness of the skin, and may spread beyond the applied area); and score 3 indicated strong reaction (intense and diffuse erythema with edema and/or eschar formation). Then, the score of skin irritation provided by each subject was added and then divided by the number of the subject to obtain an index of skin irritation of the test group. The index of skin irritation lower than 5 was considered as safe level; the index of skin irritation between 5 and 15 was considered as acceptable level; and the index of skin irritation higher than 15 was considered as mild irritation level.

[0084] After the foresaid skin irritation test for 24 hr, the scores of skin irritation of the anti-wrinkle microneedle patch of Example 7 acquired from the 20 subjects were all 0, i.e., no irritated reaction. Accordingly, the index of skin irritation of the anti-wrinkle microneedle patch of Example 7 was also 0, which represented the safe level without skin irritation.

[0085] (2) Skin Moisturizing Test

[0086] In this test example, the subjects who completed the foresaid skin irritation test were further tested for the effect of skin moisturizing of the anti-wrinkle microneedle patch of Example 7. Specifically, the anti-wrinkle microneedle patch of Example 7 was set as a test group and pasted from the area below the right eye to corner of the right eye; and the needle solution of Example 7 was set as a control group and directly applied from the area below the left eye to corner of the left eye. The applying frequency of each group was 2 times per week for 4 weeks, and the period of applying those group each time was about 6 hr. At the times when those groups were not applied yet, i.e., the 0 week, those groups were continuously applied for 2 weeks, and those groups were continuously applied for 4 weeks, the water content and the ability of moisturizing at the superficial skin of periorbital were measured by Corneometer® (purchased from Courage+Khazaka electronic GmbH) based on bioelectricity. After applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods, the results of water content at the skin of periorbital were measured and listed in the following Table 5; wherein, the higher value indicated better ability of moisturizing, and the unit was represented by A.C.U (arbitrary capacitance units). In Table 5, the “difference relative to 0 week” indicated the water content of the specific week subtracted the water content of 0 week, and then divided by the water content of 0 week and presented in percentage, which represented the elevation degree of the water content.

TABLE-US-00005 TABLE 5 The measured results of the water content at the skin of periorbital after applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods. Anti-wrinkle microneedle Needle solution of patch of Example 7 Example 7 Difference Difference Water relative Water relative Time content to 0 week content to 0 week periods (A.U.C) (%) (A.U.C) (%) 0 week  53.8 — 51.8 — 2 weeks 55.4 3.3 52.6 1.8 4 weeks 56.8 5.8 52.6 1.8

[0087] According to the results of Table 5, during the 4 weeks of applying the anti-wrinkle microneedle patch of Example 7, the measured water content at the skin of periorbital was gradually elevated; wherein, the measured water content of 2 weeks elevated by 3.3% compared to 0 week, and the measured water content of 4 weeks further elevated by 5.8% compared to 0 week. As for the results of the needle solution of Example 7, although the measured water content of 2 weeks elevated by 1.8% compared to 0 week, the measured water content of 4 weeks did not elevate compared to 0 week, and regardless of 2 weeks or 4 weeks, the effect of elevating the water content at the skin of periorbital was obviously worse than the anti-wrinkle microneedle patch of Example 7. Accordingly, further making the needle solution of Example 7 into the microneedle patch actually could provide better effect of elevating the water content at the skin of periorbital and had better ability of moisturizing.

[0088] (3) Skin Thickness and Strength Test

[0089] This test and the foresaid (2) skin moisturizing test were conducted at the same time. At the times when the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 were not applied to the subject yet, i.e., the 0 week, those groups were continuously applied for 2 weeks, and those groups were continuously applied for 4 weeks, the structural change at the dermis of periorbital was evaluated by DermaLab® (purchased from Cortex Technology) based on the recorded ultrasound images of the skin below the eyes and at corner of the eyes. Said DermaLab® was a probe for ultrasound image of the dermis, and in the ultrasonic image, color was used to represent the intensity of ultrasonic echo; wherein, deep color indicated low intensity and white/yellow indicated high intensity. In general, the epidermis had high intensity (white and yellow); the dermis has a combination of different color such as green, red and yellow (color region comprising the structures of collagen and elastin); and the fat, muscle and vessel beneath the dermis had low intensity (deep green and black). After applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods, the measured results of thickness of the skin below the eyes and at corner of the eyes were listed in the following Table 6, and the higher value indicated the thicker thickness, and the unit was represented by μm; the measured results of strength of the skin below the eyes and at corner of the eyes were listed in the following Table 7, and the higher value indicated the higher strength, and the unit was represented by intensity score. In Table 6 and Table 7, the “difference relative to 0 week” indicated the result of the specific week subtracted the result of 0 week, and then divided by the result of 0 week and presented in percentage, which represented the elevation degree of the result.

TABLE-US-00006 TABLE 6 The results of measuring the thickness of the skin below the eyes and at corner of the eyes after applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods. Anti-wrinkle microneedle Needle solution of patch of Example 7 Example 7 Difference Difference relative relative Time Thickness to 0 week Thickness to 0 week Position periods (μm) (%) (μm) (%) Area 0 week  888.8 — 931.1 — below the 2 weeks 1036.0 18.7 979.0  7.0 eyes 4 weeks 1121.4 33.1 1123.3 22.4 Corner of 0 week  989.4 — 892.8 — the eyes 2 weeks 1023.3 11.9 923.1  5.0 4 weeks 1178.1 30.0 1033.8 17.1

[0090] According to the results of Table 6, during the 4 weeks of applying the anti-wrinkle microneedle patch of Example 7, both the thickness of the skin below the eyes and at corner of the eyes had a clear upward trend; wherein, for the thickness of the skin below the eyes, the result of 2 weeks elevated by 18.7% compared to 0 week, and the result of 4 weeks further elevated by 33.1%; and for the thickness of the skin at corner of the eyes, the result of 2 weeks elevated by 11.9% compared to 0 week, and the result of 4 weeks further elevated by 30.0% compared to 0 week. As for the results of the needle solution of Example 7, for the thickness of the skin below the eyes, the results of 2 weeks and 4 weeks respectively elevated by 7.0% and 22.4% compared to 0 week; and for the thickness of the skin at corner of the eyes, the results of 2 weeks and 4 weeks respectively elevated by 5.0% and 17.1% compared to 0 week. Accordingly, the effect of the needle solution of Example 7 for elevating the thickness of the skin below the eyes and at corner of the eyes was obviously worse than the anti-wrinkle microneedle patch of Example 7. That is, further making the needle solution of Example 7 into the microneedle patch actually could provide better effect of elevating the thickness of the skin of periorbital.

TABLE-US-00007 TABLE 7 The results of measuring the strength of the skin below the eyes and at corner of the eyes after applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods. Anti-wrinkle microneedle Needle solution of patch of Example 7 Example 7 Difference Difference Strength relative Strength relative Time (intensity to 0 week (intensity to 0 week Position periods score) (%) score) (%) Area 0 week  27.5 — 29.0 — below the 2 weeks 30.6 15.5 28.7 13.0 eyes 4 weeks 34.8 34.8 28.3 12.3 Corner of 0 week  26.1 — 25.2 — the eyes 2 weeks 28.3 10.3 26.5 10.2 4 weeks 30.0 18.0 27.3 14.8

[0091] According to the results of Table 7, during the 4 weeks of applying the anti-wrinkle microneedle patch of Example 7, both the strength of the skin below the eyes and at corner of the eyes had a clear upward trend; wherein, for the strength of the skin below the eyes, the result of 2 weeks elevated by 15.5% compared to 0 week, and the result of 4 weeks further elevated by 34.8%; and for the strength of the skin at corner of the eyes, the result of 2 weeks elevated by 10.3% compared to 0 week, and the result of 4 weeks further elevated by 18.0% compared to 0 week. As for the results of the needle solution of Example 7, for the strength of the skin below the eyes, the results of 2 weeks and 4 weeks respectively elevated by 13.0% and 12.3% compared to 0 week; and for the strength of the skin at corner of the eyes, the results of 2 weeks and 4 weeks respectively elevated by 10.2% and 14.8% compared to 0 week. Accordingly, the effect of the needle solution of Example 7 for elevating the strength of the skin below the eyes and at corner of the eyes was obviously worse than the anti-wrinkle microneedle patch of Example 7. That is, further making the needle solution of Example 7 into the microneedle patch actually could provide better effect of elevating the strength of the skin of periorbital.

[0092] (4) Wrinkle Reduction Test

[0093] This test and the foresaid (2) skin moisturizing test were conducted at the same time. At the times when the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 were not applied to the subject yet, i.e., the 0 week, those groups were continuously applied for 2 weeks, and those groups were continuously applied for 4 weeks, the number of wrinkle at corner of the eyes was analyzed by VISIA® Evolution ver. 7.0.1 (purchased from Canfield Scientific, Inc.) based on the high-resolution (12 million pixels) image of the skin at corner of the eyes. After applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods, the numbers of wrinkle at corner of the eyes were listed in the following Table 8, and the lower value indicated the less number of wrinkle, and the unit was represented by count. In Table 8, the “difference relative to 0 week” indicated the number of wrinkle of the specific week subtracted the number of wrinkle of 0 week, and then divided by the number of wrinkle of 0 week and presented in percentage, which represented the reduction degree of the wrinkles.

TABLE-US-00008 TABLE 8 The results of the number of wrinkle at corner of the eyes after applied with the anti-wrinkle microneedle patch of Example 7 and the needle solution of Example 7 for different time periods. Anti-wrinkle microneedle Needle solution of patch of Example 7 Example 7 Difference Difference Number of relative Number of relative Time wrinkle to 0 week wrinkle to 0 week periods (count) (%) (count) (%) 0 week  98.5 — 97.2 — 2 weeks 95.0 −1.4 95.2 −1.0 4 weeks 92.2 −4.4 94.0 −2.0

[0094] According to the results of Table 8, during the 4 weeks of applying the anti-wrinkle microneedle patch of Example 7, the measured number of wrinkle at corner of the eyes was gradually decreased; wherein, the measured number of wrinkle of 2 weeks decreased by 1.4% compared to 0 week, and the measured number of wrinkle of 4 weeks further decreased by 4.4% compared to 0 week. As for the results of the needle solution of Example 7, the numbers of wrinkle of 2 weeks and 4 weeks respectively decreased by 1.0% and 2.0% compared to 0 week, which clearly indicated that regardless of being applied with the needle solution of Example 7 for 2 weeks or 4 weeks, the effect of reducing the number of wrinkle at corner of the eyes was obviously worse than the anti-wrinkle microneedle patch of Example 7. Accordingly, further making the needle solution of Example 7 into the microneedle patch actually could provide better effect of smoothing wrinkles and had better ability of wrinkle reduction.

Test Example 5: Comparison Between Anti-Wrinkle Microneedle Patch and Known Anti-Wrinkle Materials

[0095] This test example adopted the effects of elevating the skin moisturizing of periorbital, elevating the thickness and strength of the skin below the eyes and reducing the number of wrinkle at corner of eyes of the anti-wrinkle microneedle patch of Example 7 denoted in the Test Example 4, and further compared to a commercial anti-wrinkle product: La Mer® cream (hereinafter referred to as control group 1) and a known anti-wrinkle substance: retinyl retinoate (hereinafter referred to as control group 2). Specifically, the control group 1 was applied once per day for 4 weeks, so as to show the influences on the skin moisturizing and the number of wrinkles compared to 0 week; and the control group 2 was applied twice per day for 12 weeks to show the influences on the thickness and strength compared to 0 week. For comparing the differences in effect among the different groups, the measured results of the skin moisturizing, the thickness and strength of the skin and the number of wrinkle of the anti-wrinkle microneedle patch of Example 7, the control group 1 and the control group 2 were listed in the following Table 9.

TABLE-US-00009 TABLE 9 The test conditions and the measured results of the skin moisturizing, the thickness and strength of the skin and the number of wrinkle of the anti-wrinkle microneedle patch of Example 7 (E7), the control group 1 (C1) and the control group 2 (C2). Elevation Elevation Reduction Elevation of the of the of the of the skin thickness strength number of Test conditions moisturizing of the skin of the skin wrinkles Applying Time (relative (relative (relative (relative No. frequency period to 0 week) to 0 week) to 0 week) to 0 week) E7 twice/week 4 weeks 5.8% 33.1% 34.8%  4.4% C1 once/day 4 weeks .sup. 6% — — .sup. 3% C2 Twice/day 12 weeks  —  9.3% 25.88% —

[0096] According to the results of Table 9, in comparison with the control group 1, the anti-wrinkle microneedle patch of Example 7 had comparable effect of elevating the skin moisturizing, and had better effect of reducing the number of wrinkles simultaneously. Especially, in the situation that the applying frequency of the anti-wrinkle microneedle patch of Example 7 was obviously lower than that of the control group 1, the anti-wrinkle microneedle patch of Example 7 should substantially have superior effects of elevating the skin moisturizing and reducing the number of wrinkles compared to the control group 1. Further compared to the control group 2, the anti-wrinkle microneedle patch of Example 7 had better effect of elevating the thickness and strength of the skin simultaneously. Besides, the applying frequency of the anti-wrinkle microneedle patch of Example 7 was obviously lower than that of the control group 2. That is to say, the anti-wrinkle microneedle patch of Example 7 should also substantially have superior effects of elevating the thickness and strength of the skin compared to the control group 2. Accordingly, in comparison with the known anti-wrinkle materials, the anti-wrinkle microneedle patch of the present invention actually had better effects of elevating the skin moisturizing, elevating the thickness and strength of the skin and reducing the number of wrinkles.

[0097] In conclusion, by controlling the composition and the ratio of each ingredient of the anti-wrinkle composition of the present invention, and further taking the anti-wrinkle composition as the material of the needles of the microneedle patch, the prepared anti-wrinkle microneedle patch not only has good mechanical strength contributing to applying the microneedle patch, but also makes HA rapidly dissolve and released, so as to exert the effects of skin moisturizing and increasing skin thickness and strength, and achieve the purpose of smoothing and reducing wrinkles, thereby having great commercial value.

[0098] Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.