Anti-aging composition for vitalization of retinoic acid receptor that contains mixed extract of Chinese matrimony vine and pine needles as effective component

Abstract

Provided are an anti-aging composition that contains a mixed extract of Chinese matrimony vine and pine needles as an effective component and a method of inhibiting aging of skin. The composition is for an external application to the skin, wherein a mixed extract of Chinese matrimony vine and pine needles, which are the natural ingredients, is contained such that excellent anti-aging or wrinkle improvement effects to the skin can be obtained through the vitalization of a retinoic acid derivative.

Claims

1. A method of inhibiting the appearance of aging of the skin of a subject in need thereof, comprising applying an effective amount of a cosmetic composition containing a mixture of a Lycium chinense fruit extract and a pine needle extract to the skin of the subject, wherein the Lycium chinense fruit extract is obtained by extracting Lycium chinense fruit with aqueous ethanol to produce a mixture; filtering the mixture; and re-extracting the filtered mixture with ethyl acetate; and wherein the pine needle extract is obtained by extracting pine needles with aqueous ethanol to produce a mixture; filtering the mixture; and re-extracting the filtered mixture with ethyl acetate.

2. The method of claim 1, wherein the content of the Lycium chinense extract is 40-60 wt% based on the total weight of the mixture.

3. The method of claim 1, wherein the content of the pine needle extract is 40-60 wt% based on the total weight of the mixture.

4. The method of claim 1, wherein the weight ratio of Lycium chinense extract to pine needle extract in the mixture is 1:1.

5. The method of claim 1, wherein said applying reduces wrinkles of the skin.

6. The method of claim 1, wherein the composition is in a formulation selected from the group consisting of skin lotion, milk lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nourishing essence, cream, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cream, and facial mask formulations.

7. A method of inhibiting the appearance of aging of the skin of a subject in need thereof, comprising applying an effective amount of a pharmaceutical composition containing a mixture of a Lycium chinense fruit extract and a pine needle extract to the skin of the subject, wherein the Lycium chinense fruit extract is obtained by extracting Lycium chinense fruit with aqueous ethanol to produce a mixture; filtering the mixture; and re-extracting the filtered mixture with ethyl acetate; and wherein the pine needle extract is obtained by extracting pine needles with aqueous ethanol to produce a mixture; filtering the mixture; and re-extracting the filtered mixture with ethyl acetate.

8. The method of claim 7, wherein the weight ratio of Lycium chinense extract to pine needle extract in the mixture is 1:1.

9. The method of claim 7, wherein said applying reduces wrinkles of the skin.

10. The method of claim 7, wherein the composition is in a formulation selected from the group consisting of suspension, ointment, lotion, and spray formulations.

Description

EXAMPLE 1

Preparation of Lycium chinense Fruit Extract

(1) Lycium chinense fruits were washed with purified water, washed and finely powdered. 200 g of the Lycium chinense fruit powder was added to 1 liter of an aqueous solution of 70% ethanol and extracted by heating for 12 hours in an extractor equipped with a cooling condenser. The extract was filtered through 300-mesh filter cloth, and the filtrate was allowed to stand at 4-15 C. for 7 days, and then filtered through Whatman No. 2 filter paper.

(2) The filtered extract was placed in a 3-liter separatory funnel, and 1 liter of ethyl acetate was added thereto. Then, the solution was mixed well by shaking. After the solution was completely separated into two layers, the upper layer (ethyl acetate layer) was collected. The lower layer (aqueous layer) was extracted twice more using a separatory funnel.

(3) The separated upper layers were combined with each other and concentrated under reduced pressure at 50 C. using a distillation apparatus equipped with a cooling condenser, and the concentrate was dried, thereby obtaining 38.5 g (dry weight) of a Lycium chinense fruit extract.

EXAMPLE 2

Preparation of Pine Needle Extract

(4) Pine needles were washed with purified water, washed and finely powdered. 200 g of the pine needle powder was added to 1 liter of an aqueous solution of 70% ethanol and extracted by heating for 12 hours in an extractor equipped with a cooling condenser. The extract was filtered through 300-mesh filter cloth, and the filtrate was allowed to stand at 4-15 C. for 7 days, and then filtered through Whatman No. 2 filter paper.

(5) The filtered extract was placed in a 3-liter separatory funnel, and 1 liter of ethyl acetate was added thereto. Then, the solution was mixed well by shaking. After the solution was completely separated into two layers, the upper layer (ethyl acetate layer) was collected. The lower layer (aqueous layer) was extracted twice more using a separatory funnel.

(6) The separated upper layers were combined with each other and concentrated under reduced pressure at 50 C. using a distillation apparatus equipped with a cooling condenser, and the concentrate was dried, thereby obtaining 24.9 g (dry weight) of a pine needle extract.

TEST EXAMPLE 1

Wrinkle-reducing Effect

(7) Using the Lycium chinense fruit extract obtained in Example 1, the pine tree extract obtained in Example 2, and a 1:1 (w/w) mixture (Example 3) of a Lycium chinense fruit extract obtained by extracting 100 g of Lycium chinense fruits according to the method of Example 1 and a pine needle extract obtained by extracting 100 g of pine needles according to the method of Example 2, their effects on the inhibition of collagenase expression (collagenase production) were measured in the following manner. As a positive control, retinoic acid known to inhibit collagenase expression was used.

(8) First, human fibroblasts were added to a 96-well microtiter plate containing DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal bovine serum at a density of 5,000 cells/well and were cultured to a confluence of 90%. Then, the cells were cultured in serum-free DMEM medium for 24 hours and treated with each of 100 g/ml of the extract of Example 1, 100 g/ml of the extract of Example 2, a mixed extract (Example 3) obtained by mixing 100 g/ml of the extract of Example 1 with 100 g/ml of the extract of Example 2, and 1 M of retinoic acid, which were test materials dissolved in serum-free DMEM medium, for 24 hours. Then, the cell cultures were harvested.

(9) Next, the degree of collagenase production in each of the harvested cell cultures was measured using a collagenase measurement device (Amersham Phamasia, USA). Specifically, each of the harvested cell cultures was placed added to a 96-well plate having primary collagenase antibody applied uniformly thereto and were subjected to an antigen-antibody reaction in an incubator for 3 hours. After 3 hours, chromophore-conjugated secondary collagen antibody was added to the 96-well plate and allowed to react for 15 minutes. After minutes, a substance that induces color development was added to the 96-well plate, and color development was induced at room temperature for 15 minutes. When 1 M sulfuric acid was added to the 96-well plate to stop the color development reaction, the reaction solution had a yellow color, and the intensity of the yellow color varied depending on the degree of progression of the reaction.

(10) The absorbance of the 96-well plate having a yellow color was measured at 405 nm using an absorbance spectrometer, and the expression of collagenase was calculated using the following Equation 1. The results of the calculation are shown in Table 1 below. Herein, the absorbance of an untreated group was used as a control group. That is, the expression of collagenase in the control group was set at 100, and the expression of collagenase in each test group was calculated relative to the control group.
Collagenase expression (%)=A/B100Equation 1
wherein A is the absorbance of the cell group treated with each test material, and B is the absorbance of the control group.

(11) TABLE-US-00001 TABLE 1 Test material Collagenase expression (%) Control 100 Retinoic acid (positive control) 51.3 Example 1 81.3 Example 2 75.7 Example 3 52.9

(12) As can be seen in Table 1 above, the collagenase expression inhibitory activity of the Lycium chinense extract/pine needle extract mixture of Example 3 was 2.1 times higher than those of the Lycium chinense extract of Example 1 and the pine needle extract of Example 2. This suggests that the use of the mixture of the Lycium chinense extract and the pine needle extract effectively inhibits collagenase expression compared to the use of the Lycium chinense extract or the pine needle extract alone.

TEST EXAMPLE 2

Effects on Retinoic Acid Receptor / (RAR-/) Transcriptional Activities

(13) Using the Lycium chinense extract obtained in Example 1, the pine needle extract obtained in Example 2, and the Lycium chinense extract/pine needle extract mixture (Example 3) obtained by mixing the extract of Example 1 with the extract of Example 2, their effects on retinoic acid receptor / (RAR-/) transcriptional activities were measured in the following manner. As a positive control, retinoic acid known to activate retinoic acid receptor was used.

(14) First, monkey kidney epithelial CV-1 cells were seeded into a 24-well plate at a concentration of 410.sup.4 cells/well and cultured for 12-16 hours. Before transfection, the plate was washed with phosphate buffered saline (PBS), and then the medium was cultured. Meanwhile, a reporter plasmid, a receptor plasmid, a -galactosidase expression vector and the carrier DNA pBluscript (pBS) were combined with each other to a total weight of 0.5 g, and then added to each well containing the CV-1 cells. Then, the cells were treated with each of 100 g/ml of the extract of Example 1, 100 g/ml of the extract of Example 2, the mixed extract (Example 3) obtained by mixing 100 g/ml of the extract of Example 1 with 100 g/ml of the extract of Example 2, and 1 M of retinoic acid, for 20 hours, and 0.25 M Tris-HCl (pH 8.0) was added thereto to prepare cell lysates. Then, each of the cell lysates was transferred to a cell culture plate (Costar) for a 96-well luminometer, and luciferase activity for each cell lysate was measured using Luciferase Assay System kit (Promega Corporation) according to the manufacturer's instruction.

(15) In order to measure -galactosidase activity, 20 l of each of the cell lysates was transferred to a 96-well analysis plate (Falcon, Cat. No. 353911), and 100 l of ONPG (onitrophenyl -galacti-pyranoside) solution was added to each well, after which the plate was allowed to stand in an incubator at 37 C. for 2 hours.

(16) Next, 50 l of 1 M sodium carbonate (Na.sub.2CO.sub.3) solution was added to each well, and the absorbance of each well at 415 nm was measured. The efficiency of transformation in each cell lysate was corrected with the -galactosidase activity measured as described above, and the relative activity of luciferase to the -galactosidase activity was calculated and compared between Examples. The results are shown in Table 2 below.

(17) TABLE-US-00002 TABLE 2 Relative activity Relative activity (%) of luciferase (%) of luciferase Test material (RAR-) (RAR-) Control 100 100 Retinoic acid 343 385 (positive control) Example 1 187 195 Example 2 209 179 Example 3 357 315

(18) As can be seen in Table 2 above, the Lycium chinense extract/pine needle extract mixture of Example 3 showed retinoic acid receptor / (RAR-/) transcriptional activities that are 2.6 times and 2.45 times higher than those of each of the Lycium chinense extract of Example 1 and the pine needle extract of Example 2.

(19) The composition according to the present invention can be prepared in the following formulation examples, but is not limited thereto.

FORMULATION EXAMPLE 1

Milk Lotion

(20) According to the composition shown in Table 3 below, milk lotion can be prepared by a conventional method.

(21) TABLE-US-00003 TABLE 3 Components Content (wt %) Glycerin 8.0 Butylene glycol 4.0 Hyaluronic acid extract 5.0 Beta-glucan 7.0 Carbomer 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Caprylic/capric triglyceride 8.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Preservative q.s. Fragrance q.s. Pigment q.s. Triethanoamine 0.1 Purified water Balance

FORMULATION EXAMPLE 2

Nourishing Lotion

(22) According to the composition shown in Table 4 below, nourishing lotion can be prepared by a conventional method.

(23) TABLE-US-00004 TABLE 4 Components Content (wt %) Purified water Balance Glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 5.0 Beta-glucan 7.0 Carbomer 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Caprylic/capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.5 Preservative q.s. Fragrance q.s. Pigment q.s. Triethanolamine 0.1

FORMULATION EXAMPLE 3

Nourishing Cream

(24) According to the composition shown in Table 5 below, nourishing cream can be prepared by a conventional method.

(25) TABLE-US-00005 TABLE 5 Components Content (wt %) Glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta-glucan 7.0 Carbomer 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Caprylic/capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Preservative q.s. Fragrance q.s. Pigment q.s. Triethanolamine 0.1 Purified water Balance

FORMULATION EXAMPLE 4

Massage Cream

(26) According to the composition shown in Table 6 below, massage cream can be prepared by a conventional method.

(27) TABLE-US-00006 TABLE 6 Components Content (wt %) Glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta-glucan 7.0 Carbomer 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Caprylic/capric triglyceride 3.0 Beeswax 4.0 Cetearyl glucoside 1.5 Sorbitan sesquioleate 0.9 Vaseline 3.0 Preservative q.s. Fragrance q.s. Pigment q.s. Paraffin 1.5 Purified water Balance

FORMULATION EXAMPLE 5

Facial Pack

(28) According to the composition shown in Table 7 below, a facial pack formulation can be prepared by a conventional method.

(29) TABLE-US-00007 TABLE 7 Components Content (wt %) Glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta-glucan 7.0 Allantoin 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Nonyl phenylether 0.4 Polysorbate 60 1.2 Preservative q.s. Fragrance q.s. Pigment q.s. Ethanol 6.0 Purified water Balance.

FORMULATION EXAMPLE 6

Ointment

(30) According to the composition shown in Table 8 below, an ointment can be prepared by a conventional method.

(31) TABLE-US-00008 TABLE 8 Components Content (wt %) Glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta-glucan 7.0 Carbomer 0.1 Mixture of Lycium chinense extract and 1.0 pine needle extract Caprylic/capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Preservative q.s. Fragrance q.s. Pigment q.s. Beeswax 4.0 Purified water Balance

INDUSTRIAL APPLICABILITY

(32) The composition according to the present invention contains a mixture of Lycium chinense fruit extract and pine needle extract, which activates retinoic acid receptor and inhibits the expression of collagenase to thereby exhibit wrinkle-reducing effects and anti-aging effects. In addition, the composition containing the mixture of the extracts has less side effects and is safe, and thus can be used in various applications, including cosmetic or drug products.