Composition comprising natural complex extract as active ingredient

Abstract

The present invention relates to a composition comprising a natural complex extract. It is confirmed that the composition has synergistic effects on antioxidant activity, immune enhancement, whitening, anti-wrinkle and collagenase inhibition with low cytotoxicity, and therefore, the composition is suitable for uses of antioxidation, immune, whitening and skin anti-wrinkle.

Claims

1. A composition comprising an extract of goji berry, fig and Korean mint, wherein the extract is effective for immune enhancement, for skin whitening, or a combination thereof, and wherein the extract is prepared by extracting a mixture of 5-20 parts by weight of goji berry, 5-20 parts by weight of fig, and 60-90 parts by weight of Korean mint with hot water.

2. The composition according to claim 1, wherein the extract is contained in an amount of 0.01-100 parts by weight based on 100 parts by weight of the composition.

3. A composition comprising an extract of goji berry, fig and persimmon leaf, wherein the extract is effective for skin whitening, wherein the extract is prepared by extracting a mixture of 5-20 parts by weight of goji berry, 5-20 parts by weight of fig, and 60-90parts by weight persimmon leaf with hot water.

4. The composition according to claim 3, wherein the extract is contained in an amount of 0.01-100 parts by weight based on 100 parts by weight of the composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the antioxidative effect of the natural complex extract.

(2) FIG. 2 is a graph showing the cell viability of the natural complex extract.

(3) FIG. 3 is a graph showing the effect of the natural complex extract on the TNF- production rate of macrophages.

(4) FIG. 4 is a graph showing the effect of the natural complex extract on the melanin production.

(5) FIG. 5 is the result of electrophoresis showing the effect of the natural complex extract on the inhibition of keratinocyte-derived MMP-1 gene expression.

BEST MODE FOR CARRYING OUT THE INVENTION

(6) Various changes in form and details may be made to the presently disclosed embodiment and thus should not be construed as being limited to the aspects set forth herein. The presently disclosed embodiment is not limited to the aspects described in the present description, and thus it should be understood that the presently disclosed embodiment does not include every kind of variation example or alternative equivalent included in the spirit and scope of the presently disclosed embodiment. Also, while describing the aspects, detailed descriptions about related well-known functions or configurations that may diminish the clarity of the points of the aspects of the presently disclosed embodiment will be omitted.

PREPARATIVE EXAMPLE 1

Preparative Example 1-1

(7) Each of goji berry 50 g, fig 50 g, Korean mint 50 g and persimmon leaf 50 g was mixed with 10 times of distilled water, and then heated and extracted at 95 C. for 4 hours by using a heating mantle equipped with a reflux cooling system. The extract was filtered through a filter paper (Whatman No. 2), concentrated at reduced pressure and at a temperature of 50 C. to 60 C., pre-frozen at a temperature of 40 C., and then dried at 40 C. for 48 hours by using a freeze dryer to the water content of 3% to 5%, to obtain each extract.

Preparative Example 1-2

(8) The goji berry extract, the fig extract and Korean mint extract of Preparative Example 1-1 were mixed at weight ratio of 1:1:1 and 2:1:2, respectively.

Preparative Example 1-3

(9) The goji berry extract, the fig extract and the persimmon leaf extract of Preparative Example 1-1 were mixed at weight ratio of 1:1:1 and 2:1:2, respectively.

PREPARATIVE EXAMPLE 2

Preparative Example 2-1

(10) Goji berry 14.93 g, fig 9.29 g and Korean mint 75.78 g were mixed, 10 times of distilled water was added thereto, and then heated and extracted at 95 C. for 4 hours by using a heating mantle equipped with a reflux cooling system. The extract was filtered, concentrated and then spray dried to obtain an extract.

Preparative Example 2-2

(11) Goji berry 17.5 g, fig 10.8 g and persimmon leaf 71.7 g were mixed, 10 times of distilled water was added thereto, and then heated and extracted at 95 C. for 4 hours by using a heating mantle equipped with a reflux cooling system. The extract was filtered, concentrated and then spray dried to obtain an extract.

TEST EXAMPLE 1

Measurement of Antioxidant Activity

(12) Antioxidative effect was examined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Each sample was added to the DPPH (250 M) solution (Positive control: Vitamin C, 50 g/mL), and a reaction was induced at 37 C. for 30 min. The antioxidation effect was calculated from OD values obtained by measuring absorbance at 517 nm after the reaction was completed, and the results were shown in FIG. 1.

(13) As shown in FIG. 1, from the results of measuring the antioxidative effect at the test group wherein each of the goji berry, the fig and Korean mint was extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2, respectively; and the test group wherein the goji berry, the fig and Korean mint of 14.93, 9.29 and 75.78 parts by weight, respectively, were mixed, and then extracted and spray-dried, it was confirmed that the antioxidative effect of the test group of extracting after mixing showed the highest antioxidative effect. From the results of measuring the antioxidative effect at the test group wherein each of the goji berry, the fig and the persimmon leaf was extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2, respectively; and the test group wherein the goji berry, the fig and the persimmon leaf of 17.5, 10.8 and 71.7 parts by weight, respectively, were mixed, and then extracted and spray-dried, it was confirmed that the antioxidative effect of the test group of extracting after mixing showed the highest antioxidative effect. From FIG. 1, it was confirmed that the method of mixing the goji berry, the fig and Korean mint and then extracting the mixture showed more remarkable synergistic effect on the antioxidative efficacy than the method of extracting each of the goji berry, the fig and Korean mint separately, and then mixing the extracts.

TEST EXAMPLE 2

Measurement of Cell Viability

(14) 2-1. Cell Culture

(15) Mouse macrophage cell line, RAW264.7 cells were cultured at the density of 70% to 80% in a 100 mm cell culture dish with RPMI 1640 medium containing penicillin (100 IU/ml), streptomycin (100 g/ml) and 10% FBS.

(16) 2-2. Measurement of Cell Viability

(17) The cell viability was analyzed by using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide) assay. The concentration of the cultured mouse macrophage cell line, RAW264.7 was controlled by using RPMI 1640 medium to 110.sup.6 cell/ml. The cells were inoculated in a 96 well plate, and pre-cultured for 18 hours at a condition of 5% CO.sub.2 and 37 C. Then the medium was removed and medium containing the extracts at a concentration of 100, 200 and 400 g/mL, respectively, was added thereto, and the cells were cultured. After 24 hours, MTT solution (stock concentration: 5 mg/ml) 10 l was added thereto and an additional reaction was induced for 3 hours. For completing the reaction and dissolving formazan crystal, DMSO 100 l was additionally added to each well. As the cell viability, the amount that the MTT was reduced to the formazan was calculated from OD values obtained by measuring absorbance at 570 nm, and the results were shown in FIG. 2.

(18) As shown in FIG. 2, it was confirmed that the cell viabilities of the group wherein each of the goji berry, the fig and Korean mint was separately extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2, respectively; and the group wherein the goji berry, the fig and Korean mint of 14.93, 9.29 and 75.78 parts by weight, respectively, were mixed, and then extracted and spray-dried are within the range from 80% to 120%. It was confirmed that the cell viabilities of the group wherein each of the goji berry, the fig and the persimmon leaf was extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2, respectively; and the group wherein the goji berry, the fig and the persimmon leaf of 17.5, 10.8 and 71.7 parts by weight, respectively, were mixed, and then extracted and spray-dried are within the range from 80% to 120%. From the results, it was confirmed that the extracts did not affect to the cell viability at the concentration range tested above.

TEST EXAMPLE 3

Immune Enhancement Effect According to Extract ConcentrationMeasurement of Macrophage-derived TNF- Production

(19) The concentration of the cultured mouse macrophage cell line, RAW264.7 was controlled by using RPMI 1640 medium to 110.sup.6 cell/ml. The cells were inoculated in a 96 well plate, and pre-cultured for 18 hours at a condition of 5% CO.sub.2 and 37 C. Then the medium was removed and medium containing positive control (LPS, 100 ng/mL) and the extracts at a concentration of 100, 200 and 400 g/mL, respectively, was added thereto, and the cells were cultured. After 24 hours, the supernatant 100 l was transferred to another 96 well plate, the TNF- concentration was measured by using an enzyme-linked immunosorbent assay (ELISA) kit of R&D systems according to instructions of the manufacturer. The results were shown in FIG. 3.

(20) As shown in FIG. 3, from the results of measuring the TNF-a production at the test group wherein each of the goji berry, the fig and Korean mint was separately extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2; and the test group wherein the goji berry, the fig and Korean mint of 14.93, 9.29 and 75.78 parts by weight, respectively, were mixed, and then extracted and spray-dried, it was confirmed that the TNF- production of the test group of extracting after mixing showed the highest antioxidative effect. From the results of measuring the TNF- production at the group wherein each of the goji berry, the fig and the persimmon leaf was separately extracted with hot water and then they were mixed at the ratios of 1:1:1 and 2:1:2; and the group wherein the goji berry, the fig and the persimmon leaf of 17.5, 10.8 and 71.7 parts by weight, respectively, were mixed, and then extracted and spray-dried, it was confirmed that the TNF- production of the test group of extracting after mixing showed the highest antioxidative effect. From the results, it was confirmed that the method of mixing the goji berry, the fig, Korean mint and the persimmon leaf and then extracting the mixture showed more remarkable synergistic effect on the immune enhancement efficacy than the method of extracting each of the goji berry, the fig, Korean mint and the persimmon leaf separately, and then mixing the extracts.

TEST EXAMPLE 4

Measurement of Melanin Production

(21) 4-1. Cell Culture

(22) Mouse melanoma cell line, B16-F10 cells were cultured the density of 70% to 80% in a 100 mm cell culture dish with DMEM medium containing penicillin (100 IU/ml), streptomycin (100 g/ml) and 10% FBS.

(23) 4-2. Measurement of Melanin Concentration

(24) The concentration of the cultured mouse melanoma cell line, B16-F10 cell was controlled by using DMEM medium containing penicillin (100 IU/ml), streptomycin (100 g/ml) and 10% FBS to 110.sup.5 cell/ml. The cells were inoculated in a 12 well plate, and pre-cultured for 18 hours at a condition of 5% CO.sub.2 and 37 C. Then the medium was removed, and medium containing the extracts at a concentration of 1000 g/mL, respectively, and alpha-Melanocyte-stimulating hormone (-MSH) were added thereto at the same time. The cells were cultured for 24 hours, the medium was removed, and then 1N NaOH was added thereto for dissolving melanin. The melanin content was calculated from OD values obtained by measuring absorbance at 405 nm, and the results were shown in FIG. 4.

(25) As shown in FIG. 4, from the results of measuring the melanin production at the group extracting each of the goji berry, the fig and Korean mint with hot water and mixing the three extracts at a ratio of 1:1:1 and 2:1:2, respectively; and the group mixing the goji berry, the fig and Korean mint of 14.93, 9.29 and 75.78 parts by weight, respectively, and then extracting and spray drying the mixture, it was confirmed that the melanin production was reduced. From the results of measuring the melanin production at the group extracting each of the goji berry, the fig and the persimmon leaf with hot water and mixing the three extracts at a ratio of 1:1:1 and 2:1:2, respectively; and the group mixing the goji berry, the fig and the persimmon leaf of 17.5, 10.8 and 71.7 parts by weight, respectively, and then extracting and spray drying the mixture, it was confirmed that the melanin production was reduced.

TEST EXAMPLE 5

Measurement of Keratinocyte-derived MMP-1 Gene Expression

(26) 5-1. Cell Culture

(27) Human keratinocyte cell line, HaCaT cells were cultured in a 100 mm cell culture dish with DMEM medium containing penicillin (100 IU/ml), streptomycin (100 g/ml) and 10% FBS to the density of 70% to 80%.

(28) 5-2. Measurement of MMP-1 Gene Expression

(29) In order to examine the degree of MMP-1 expression at the transcription level, each sample was treated to the cells for a predetermined time, UVB was irradiated thereto. Then total RNA was extracted by using Trizol reagent. cDNA was manufactured from the extracted total RNA by using a First strand cDNA synthesis kit (Thermo scientific), and the same amount of cDNA was amplified by PCR. At this time, the sense and antisense primer sequences of the used target protein were manufactured by referring the existing documents, GAPDH was used as a control gene. PCR amplification was performed by using a Hipi PCR kit (Elpis biotech) 20 l containing dNTP 250 M, Tris-HCL (pH 8.3) 10 mM, KCl 50 mM, NgCl.sub.2 1.5 mM, with each test group cDNA, MMP-1 primers and control GAPDH primers. PCR was performed 30 cycles at the condition of denaturing at 95 C. for 45 sec, annealing at 60 C. for 45 sec and extension at 72 C. for 1 min. The DNA amplified by PCR was subjected to electrophoresis on a 1.5 agarose gel. The intensity of the fractionated DNA band was measured, and the results were shown in FIG. 5. MMP-1 forward 5-ATT CTA CTG ATA TCG GGG CTT TGA-3, MMP-1 reverse 5-ATG TCC TTG GGG TAT CCG TGT AG-3 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5-CCA GCC GAG CCA CAT CGC TC-3, GAPDH reverse 5-TGA CCT TGG CCA GGG GTG CT-3

(30) As shown in FIG. 5, from the results of measuring the MMP-1 gene expression at the group extracting each of the goji berry, the fig and Korean mint with hot water and then mixing the three extracts at a ratio of 1:1:1 and 2:1:2; and the group mixing the goji berry, the fig and Korean mint of 14.93, 9.29 and 75.78 parts by weight, respectively, and then extracting and spray drying the mixture, it was confirmed that the gene expression was reduced. From the results of measuring the MMP-1 gene expression at the group extracting each of the goji berry, the fig and the persimmon leaf with hot water and then mixing the three extracts at a ratio of 1:1:1 and 2:1:2; and the group mixing the goji berry, the fig and the persimmon leaf of 17.5, 10.8 and 71.7 parts by weight, respectively, and then extracting and spray drying the mixture, it was confirmed that the gene expression was reduced.

(31) As can be seen form the result of Test Examples, the composition comprising a natural complex extract according to the present invention exhibits effects on antioxidation, immune enhancement, whitening, collagenase inhibition and anti-wrinkle, and the effects result from the synergistic action among the extract of goji berry, fig and Korean mint or the extract of goji berry, fig and persimmon leaf.

(32) Although exemplary embodiments of the present disclosure have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure. Therefore, exemplary embodiments of the present disclosure have not been described for limiting purposes. Accordingly, the scope of the disclosure is not to be limited by the above embodiments but by the claims and the equivalents thereof.