WO2008147022A1

WO2008147022A1 - Anti-wrinkle composition for external applications to the skin containing biflavonoid derivatives

Info

Publication number: WO2008147022A1
Application number: PCT/KR2008/001038
Authority: WO
Inventors: Chanwoo Lee; Eunrhan Woo; Hansung Kim; Song Yi Lee; Hyunjung Choi; Ji Yeong Kim; Jinwong Kim; Duckhee Kim; Han Kon Kim; Ihseop Chang
Original assignee: Amorepacific Corporation; Industry-Academic Cooperation Foundation, Chosun University
Priority date: 2007-06-01
Filing date: 2008-02-22
Publication date: 2008-12-04
Also published as: KR20080105730A; KR100902173B1; CN101730520A

Abstract

The present invention relates to a cosmetic composition for improving skin wrinkles, which contains biflavonoid derivatives as an active ingredient. The cosmetic composition containing the biflavonoid derivatives of the present invention not only is safe for application on the skin without side-effects but also improves wrinkles caused by UV through procollagen production and MMP-I inhibition.

Description

[DESCRIPTION]

[invention Title]

ANTI-WRINKLE COMPOSITION FOR EXTERNAL APPLICATIONS TO THE SKIN CONTAINING BIFLAVONOID DERIVATIVES

[Technical Field]

The present invention relates to a cosmetic composition containing biflavonoid derivatives as an active ingredient for improving skin wrinkles caused by aging.

[Background Art]

The present invention relates to an anti-wrinkle cosmetic composition containing biflavonoid derivatives as an active ingredient, which is safe for the application on the skin without side-effects, induces procollagen production, inhibits MMP-I, and has the effect of improving wrinkles generated by UV.

Every living bodies experience aging as they get old and the skin is not an exception. The effort to postpone aging has been constantly made and questions on 'what is aging' and 'what causes aging' have been continuously asked.

Skin aging is largely divided into two groups according to the reason; one is intrinsic aging caused by the aging of the structureand physiological functions of the skin and the other is extrinsic aging caused by accumulated stress given from outside such as sunrays, etc. In particular, UV among the sunrays is well-known as a reason of aging. When the skin is exposed on UV for a long time, the horny layer of the skin becomes thicker and the major structural constituents of the skin, collagen and elastin, are denatured, resulting in wrinkles by lack of elasticity.

Skin aging accompanies various functional, structural changes. As for structural changes on skin by aging, the skin composing components, epidermis, dermis and subcutaneous tissue become thinner. In addition, ECM (extracellular matrix) of dermis which is responsible for elasticity and tension of skin is denatured. ECM is composed of two main components. One is elastic fiber taking 2-4% of ECM and the other is collagen taking 70-80% of ECM. According to aging, the skin greatly loses its elasticity, which is resulted from the decrease of collagen and elastin. The collagen and elastin are regulated by various factors. For example, collagen and elastin are decomposed by the expression of matrix metallo protease such as collagenase and elastase, so that the collagen content in skin is reduced. Once collagen and elastin are reduced in dermis, skin becomes rough and loses elasticity, one of the symptoms of aging, and gets more wrinkles. So, studies have been undergoing to prevent the decrease of collagen and elastin, the cause of elasticity loss.

The most effective method to prevent skin aging so far is to use retinol and retinoic acid. The improvement effect of retinol and retinoic acid on wrinkles and elasticity has been well-informed through previous reports (Dermatology therapy, 1998, 16, 357~364) . However, although the retinoid has positive effects such as wrinkle or elasticity improvement, it has problems of irritating skin even with only a small amount and being so unstable that it is easily oxidized and denatured when being exposed on the air, -and it causes the limitation to use. Therefore, studies have been going on to make the retinoid more stable, but skin irriation, the stability problems, remains unsolved. As an alternative, a method inhibiting the activity of enzymes hydrolyzing collagen and elastin such as collagenase, elastase and matrix metallo protease has been proposed and thus inhibitors capable of inhibiting the activity of such enzymes have been actively studied. Particularly, natural plant materials are safe and have many useful components, so that they can be a good target for the screening of an anti-wrinkle agent. In fact, it has been long tried to find out a natural plant material which is safe in human skin and has anti-wrinkle effect.

It has been disclosed recently that the increase of the activities of collagenase decomposing collagen and matrix metallo protease is one of the reasons of wrinkle generation. Therefore, these enzymes are drawing our attention as targets for anti-wrinkle activity.

[Disclosure]

[Technical Problem]

The present inventors studied to find out a novel anti- aging material that is safe in human skin and has excellent anti-wrinkle effect among natural plant materials. As a result, the present inventors found out that the natural plant originated biflavonoid derivatives not only inhibited the activity of collagenase but also exhibited improvement effect on wrinkles caused by UV and at the same time excellent safety on human skin. The present inventors further observed that the biflavonoid derivatives had excellent preventive and therapeutic effect on skin aging. So, the present inventors completed this invention by confirming that a cosmetic composition for improving skin wrinkles of the invention which contained biflavonoid derivatives by 0.001-10 weight% more preferably 0.01-5 weight! for the total weight of the composition had excellent wrinkle improvement effect.

Therefore, it is an object of the present invention to provide a cosmetic composition for improvement and prevention of wrinkles that contains biflavonoid derivatives as an active ingredient so as to improve wrinkles without side effects.

[Technical Solution)

To achieve the above object, the present invention is completed by the following steps: preparing an extract containing biflavonoid derivatives from natural plants by using water or an organic solvent; separating the biflavonoid derivatives by separation/purification process; measuring procollagen productivity and inhibition of collagenase decomposition; and confirming suppressing effect of the biflavonoid derivatives on wrinkles caused by UV.

In the structure of the biflavonoid derivatives, two flavonoid molecules form a dimer by C-C or C-O-C bond. The biflavonoid derivatives can be extracted from medicinal herbs or plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica, Lonicera japonica, etc, but not always limited thereto.

The biflavonoid derivatives can be included by 0.001-10 weight% for the total weight of the cosmetic composition for prevention and improvement of wrinkles and more preferably included by 0.01-5 weight%.

Hereinafter, biflavonoid which is the active ingredient of the composition for prevention and improvement of wrinkles is described in detail. The b^'iflavonoid derivatives, the active ingredient of the cosmetic composition for improving wrinkles, are the compounds represented by the following formulas 1 - 7.

Sumaflavone

2' , 8"-biapigenin

Taiwaniflavone

Amentoflavone

Robustaflavone

<Formula 6> Ginkgetin

Ochnaflavone

The biflavonoid derivatives represented by formulas 1 - 7 can be obtained from natural medicinal herbs or plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica, by the conventional extraction and separation method. To extract a target material, stems, roots and leaves were dehydrated properly, followed by macerated or dehydrated only. The target material was purified by the conventional purification method known to those in the art.

The synthetic compound or its derivatives corresponding to the above plant originated biflavonoid derivatives represented by formulas 1 - 7 .can be purchased on the market or can be synthesized by the conventional method well-known to those in the art. Specifically, the compound can be extracted from medicinal herbs or plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica, or synthesized by the conventional chemical synthesis.

As other preferable compounds of the present invention, panduratin derivatives represented by formulas 1 - 7 can be separated and purified from the extract extracted from Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica by using an organic solvent or from oil obtained from the above by compressing.

Biflavonoid derivatives can be extracted from such plants as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica, but not always limited thereto.

Biflavonoid is a compound that is naturally generated in vascular plants and has the morphology of dimer formed by C-C or C-O-C bond. It is believed that the reason why the biflavonoid is in dimer form in plants is that when a stimulus is coming in from outside, a plant activates its in vivo protection system to protect itself, in which single molecular polyphenol structure is developed into biflavonoid. The structure of biflavonoid is divided largely into two groups according to the bond types; one is amentoflavone having C-C bond and the other is ochnaflavone having C-O-C bond. Biological functions and effects of biflavonoid have also been reported regarding antitumor (J Nat Prod, 1999, 1668-71.)/ antimicrobial (Phytochem, 2005, 1922-26) , antiparasitic, Neuroprotective (Bioorg and Medichem.Lett, 2005, 3588-91), antioxidant (Phytochem, 2005, 2238-47), antifungal (Arch Pharm Res, 2006, 746-751) and Antiinflamination (Pharma Res, 2005, 539-546., Arch Biochem and Biophy, 2006, 136-146) activities. However, there has been no report on anti-wrinkle effect of biflavonoid derivatives, yet.

The biflavonoid derivatives of the present invention can be separated and purified from the organic solvent extract or oil prepared from medicinal herbs or plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica. The extraction solvent can be water, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether or a mixture thereof. The separation and purification of biflavonoid derivatives from the natural plant extract can be performed by column chromatography filled with synthetic resins such as silica gel or active alumina and High performance liquid chromatography (HPLC) respectively or together. However, the methods for extraction, separation and purification of the active ingredient are not always limited thereto.

The preferable content of the biflavonoid derivatives of the present invention in a cosmetic composition for improving wrinkles is 0.001-10 weight% for the total weight of the composition. If the content is less than 0.001 weight%, anti- wrinkle effect will not be obtained. In the meantime, if the content is more than 10 weight%, safety or formulation will matter. To enhance the anti-wrinkle effect and convenience in formulation, the biflavonoid derivatives are preferably contained by 0.01-5 weight% for the total weight of the composition. However, the content of the biflavonoid derivatives is not always limited thereto. The cosmetic composition for improving wrinkles of the present invention can additionally include other anti-wrinkle elements that can raise the anti-wrinkle effect of the composition as long as they do not damage the original targeted anti-wrinkle effect. The formulation forms of the cosmetic composition for improving wrinkles of the present invention are not limited, and can be nutritional toner, softener, nutritional cream, massage cream, pack, gel, astringent, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, powder, body lotion, body oil and body essence, etc.

Method for extracting biflavonoid derivatives

As an example of methods for extracting the biflavonoid derivatives of the present invention, the extraction method of sumaflavone is described in detail hereinafter.

The biflavonoid derivatives can be isolated from natural plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica, but herein Selaginella tamariscina was selected as an example.

Dried Selaginella tamariscina was pulverized and mixed with alcohol, followed by elution. Then, the solvent was eliminated and the crude extract was concentrated. The concentrated crude extract was mixed with ethyl acetate, and then ethyl acetate soluble component was extracted. Preferably, this extraction process was repeated at least three times. The extracted ethyl acetate soluble component contains biflavonoid derivatives, which already exhibited collagenase inhibitory activity despite it was not a single component. Then, ethyl acetate was eliminated to concentrate ethyl acetate soluble component. According to the difference of polarity, each component was separated. For example, a solvent was prepared by mixing chloroform, methanol and water at the ratio of 10 : 1 : 0.5 (v/v) . The component was mixed with this solvent and 10 fractions were separated thereby. The fractions proceeded to column chromatography to separate pure single anti-wrinkle component.

The anti-wrinkle component obtained from the above extraction and separation was proved to be amentoflavone having the structure of formula 1.

The methods for extracting biflavonoid derivatives, amentoflavone, 2' , 8"-biapigenin, taiwaniflavone, robustaflavone, ginkgetin and ochnaflavone, from Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana, Nandina domestica and Lonicera japonica are similar to the above method.

[Description of Drawings] The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which: Fig. 1 is a set of photographs illustrating the improvement of wrinkles caused by ultraviolet (UV) by biflavonoid derivatives.

[Best Mode]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Extraction of sumaflavone 1.8 kg of dried Selaginella tamariscina was cut into small pieces, followed by extraction with methanol at 80^°C for 4 hours, which was repeated three times. The obtained methanol extract was concentrated under the reduced pressure. 78.7 g of the concentrate was dissolved in water, to which methylene chloride was added as an extraction solvent, followed by extraction over three times. The methylene chloride soluble fraction (21.7g) was obtained and the remnants proceeded to extraction again using ethyl acetate or butanol as an extraction solvent over three times. As a result, the ethyl acetate soluble fraction (β.8g), the butanol soluble fraction (7.8g) and the remaining water fraction (16.2 g) were obtained. β.8g of the ethyl acetate soluble fraction proceeded to silica gel column chromatography. A mixed solvent comprising chloroform, methanol and water at the ratio of 10:1:0.1 (v/v) was used as a column elution buffer. Column chromatography was performed with raising methanol content slowly to increase polarity of the mixed solvent, resulting in 6 fractions (El- E6) . Among these 6 fractions, the third fraction (E3) proceeded to silica gel column chromatography (chloroformimethanol : water=8 : 1 : 0.1) , RP-18 column chromatography (methanol : water=2 : 1, Merck, Lichroprep RP-18, 40-63 μm, L610400138), and sephadex LH-20 column chromatography (methanol :water=4 : 1, Lipophilic Sephadex LH 20, 25-100 μm, Sigma, Lot 81klO92) stepwise to give compound 1 (sumaflavone) .

Amentoflavone, 2' , 8"-biapigenin, taiwaniflavone, robustaflavone, ginkgetin and ochnaflavone were also prepared by the same manner as described above.

Example 2 : Determination of the structure of panduratin A compound

To determine the structure of the single material separated in Example 1, ¹H-NMR spectrum and ¹³C-NMR spectrum were measured at 500 MHz and 125MHz (solvent : DMSO) respectively. The results were as follows.

The results of ¹H-NMR and ¹³C-NMR were compared with the conventional study results to identify the structure and the references are shown below.

Lin, L. -C, Kuo, Y. -C, and Chou, C-J. (2000) Cytotoxic biflavonoids from Selaginella delicatula. J. Nat. Prod., 63, 627-630.

Markham, K. R., Sheppard, C, and Geiger, H. (1987) ¹³C NMR studies of some naturally occurring amentoflavone and hinokiflavone biflavonoids. Phytochemistry, 26, 3335-3337.

Chen, S. -C, Liu, H. -K., and Kuo, Y. -H. (2004) Two new compounds from the leaves of Calocedrus microlepic var. formosana. Chem. Pharm. Bull., 52, 762-763. Chari, V. M., Ilyas, M., Wagner, H., Neszmelyi, A., Chen, F. -C, Chen,

L. -K., Lin, Y. -C, and Lin, Y. -M. (1977) ¹³C-NMR spectroscopy of biflavonoids. Phytochemistry, 16, 1273-1278.

The structures of 2' , 8"-biapigenin, taiwaniflavone, robustaflavone, amentoflavone , ginkgetin and ochnaflavone were also determined by the same manner as described above and the results are shown below.

Compound 1 (sumaflavone)

¹H-NMR (CD₃OD, 500 MHz) δ 6.25 (IH, s, H-3) , 6.12 (2H, s, H-6, H-8), 8.23 (IH, s, H-2 ' ) , 7.08 (IH, d, J=8.5 Hz, H-5'), 7.86 (IH, d, J=8.5 Hz, H-6¹) , 6.58 (IH, s, H-3") , 7.63(2H, d, J=8.0 Hz, H-2^{1 1 1} , H-6' " ) , 6.60 (2H, d, J=8.0 Hz, H-3^{I I ?}, H- 5' ") .

¹³C-NMR (CD₃OD, 125MHz) 5162.34 (C-2) , 103.53(03) , 183.70(04) , 163.09(05) , 100.94 (06) , 163.09(07) , 95.74 (08) , 159.56(09) , 104.75(C-IO) , 121.75(C-I' ) , 132.90(02') ,

124.76(03') , 166.54 (C-4¹) , 120.71(05' ) , 127.81(06') ,

162.76(02") , 103.17 (03") , 183.97 (C-4" ) , 163.73(05", C-6", C-7") , 108.65(08") , 156.73(09") , 104.10 (C-IO") , 123.28 (C- I" ¹ ) , 129.44 (02' " ) , 116.96(03' ") , 165.58 (04' " ) ,

116.96(05" ' ) , 129.44 (06' " ) .

Compound 2 (2' , 8"-biapigenin)

¹H-NMR (CD₃OD, 500 MHz) δ 6.51 (IH, S, H-3) , 6.13 (IH, s,

H-6) , 6.39 (IH, s, H-8) ,7.92 (IH, s, H-3¹) , 7.04 (IH, d, J=7. O Hz, H-5¹ ) , 7.76 (IH, d, J=7.0 Hz, H-6¹) , 6.51 (IH, s, H-3") ,

6.32(1H, s, H-6") , 7.44 (2H, d, J=8.0 Hz, H-2" ¹, H-6" ¹),

6.68 (2H, d, J=8.0 Hz, H-3' " , H-5" ') .

¹³C-NMR (CD₃OD, 125 MHz) δ 166.22 (C-2) , 103.43(03) , 184.81(04) , 163.24 (05) , 100.32(06) , 166.02(07) , 95.30(08) , 159.38 (09) , 105.49^* (C-IO) , 121.60(C-I¹) , 121.60 (C-2 ' ) ,

132.91(03') , 161.00(04") , 117.42(05') , 129.05(06'),

162.57 (C-2") , 104.07 (03") , 184.23 (C-4" ) , 163.59(05") ,

100.11(06") , 163.59(07") , 105.32(08") , 156.53(09") ,

105.39^*(O10") , 123.29(C-I' " ) , 129.41(02" ' ) , 116.94 (03' " ) , 162.57 (04" ') , 116.94 (C-S^{1 1 1} ) , 129.41(0-6' " ) . Compound 3 (taiwanif lavone)

¹H-NMR (CD₃OD, 500 MHz) δ 6.53 (IH, s, H-3) , 6.08 (IH, s,

H-6) , 7.64 (2H, d, J=8.5 Hz, H-2 ' , H-6') , 6.57-6.59 (3H, s, H- 3', H-5¹, H-3") , 6.53 (IH, s, H-6") , 6.08 (IH, s, H-8") ,

8.26(1H, br d, J=2.0 Hz, H-2'") , 7.08 (IH, d, J=8.5 Hz, H-

5' " ) , 7.86 (IH, br dd, J=8.5, 2.0 Hz, H-6' " ) .

¹³C-NMR (CD₃OD, 125MHz) 5166.05^a (C-2) , 109.07 (C-3) , 183.89^b(C-4) , 165.63^a(C-5) , 102.93(C-6) , 163.71^a (C-7) , 96.72(C- 8) , 159.84^c(C-9) , 103.84(C-IO) , 122.84 (C-I' ) , 129.44 (C-2 ' ) , 117.22 (C-3¹ ) , 163.47^a(C-4' ) , 117.22 (C-5 ' ) , 129.44 (C-6 ' ) ,

165.63^a(C-2") , 103.23(C-3") , 189.29^b (C-4" ) , 162.83^a (C-5" ) , 102.93 (C-6") , 162.23^a(C-7") , 96.72 (C-8") , 159.80^c (C-9" ) ,

103.65(C-IO") , 121.87 (C-I" ' ) , 132.76 (C-2 ' " ) , 125.14 (C-3 ' " ) , 156.71 (C-4' ") , 121.02 (05' " ) , 127.50(06" ') . Compound 4 (ament of lavone)

¹H-NMR (Pyridine-d₅, 500 MHz) 57.03 (IH, s, H-3) , 6.84 (IH, s, H-6) , 6.79 (IH, d, J=2.0 Hz, H-8) , 8.54 (IH, d, J=2.0 Hz, H-2') , 7.40 (IH, d, J=8.0 Hz, H-5' ) , 7.87 (IH, d, J=7.0 Hz, H- 6' ) , 6.92 (IH, s, H-3") , 6.71(1H, d, J=2.0 Hz, H-6") , 7.89 (2H, d, J=8.5 Hz, H-2' ", H-6' " ) , 7.14 (2H, d, J=8.5 Hz, H- 3' ' ' , H-5' " ) .

¹³C-NMR (Pyridine-d5, 125 MHz) δ 165.01 (C-2) , 104.44 (C-3) , 183.41(04) , 163.43(05) , 100.34 (06) , 164.95(07) , 95.19(08) , 158.85(C-9), 105.38(C-IO), 122.08(C-I'), 128.66 (C-2 ' ) , 122.83(03'), 161.66(C-4') , 117.60 (C-5 ' ) , 132.92 (C-6 ' ) , 166.15(02"), 103.92(C-3"), 183.06 (C-4" ) , 162.77 (C-5" ) , 100.14 (C-6") , 164.06(C-7"), 105.61 (C-8") , 156.19 (C-9") , 105.27(C-IO"), 122.76(C-I¹¹¹), 129.16 (C-2 ' ' ' ) , 117.18 (C-3 ' ' ' ) , 162.98 (C-4' "), 117.18 (C-5¹ ''), 129.16 (C-6 ^• ' ' ) . Compound 5 (robustaflavone)

¹H-NMR (DMSO-D₆, 500 MHz) 56.81 (IH, s, H-3) , 6.48 (2H, br d, J=2.0 Hz, H-6, H-8), 7.82 (IH, br s, H-2 ^• ) , 7.02 (IH, d, J=8.5 Hz, H-5¹), 7.90 (IH, dd, J=8.5, 2.5 Hz, H-6¹), 6.77 (IH, s, H-3"), 6.6O(1H, s, H-8"), 7.96 (2H, d, J=8.5 Hz, H-2^{1 11}, H-

6^{11 1}), 6.95 (2H, d, J=8.5 Hz, H-3¹¹¹, H-5¹¹¹).

¹³C-NMR (DMSO-D₆, 125 MHz) δ 164. ll^a (C-2) , 102.81^b (C-3) , 181.82^c(C-4) , 159.09(05), 98.82(06), 163.54(07), 94.04^d(C-8), 157.37(09), 103.71(C-IO), 121.28(C-I¹), 127.41(02'),

120.54 (C-3¹ ), 161.47^e(C-4') , 116.42 (C-5 ' ) , 130.92(06'), 164.01^a(C-2") , 102.75^b(C-3") , 181.73^C (C-4") , 161.17^e (C-5" ) , 103.29(06"), 163.54(07"), 93.73^d (C-8" ) , 156.42 (C-9" ) ,

103.71(C-IO"), 121.21(C-I^{1 1} ') , 128.51(02" '), 116.02(03' "), 161.47^e(C-4^! ' ' ) , 116.02(05" '), 128.51(06" ') . Compound 6 ( ginkgetin)

¹H-NMR (DMSO-D₆, 500 MHz) δ 6.88 (IH, s, H-3), 6.36 (IH, d, J=2.2 Hz, H-6), 6.79 (IH, s, H-8), 8.09 (IH, d, J=2.1 Hz, H- 2¹), 7.36 (IH, d, J=8.6 Hz, H-5¹), 8.22 (IH, d, J=8.7 Hz, H- 6' ) , 6.99 (IH, s, H-3") , 6.38(1H, s, H-6") , 7.49 (2H, d, J=8.8 Hz, H-2' ^{1 1}, H-6' " ) , 6-78 (2H, d, J=8.8 Hz, H-3' ^{! I}, H- 5¹ ") - 3.79(s, 3H, OCH3) , 3.88(s, 3H, OCH3)

¹³C-NMR (DMSO-D₆, 125 MHz) 5163.01 (C-2) , 103.7 (C-3) , 181.9MC-4) , 161.09(C-5) , 98.74 (C-6) , 165.22 (C-7) , 92.69(C-8) ,

157.42 (C-9) , 104.43(C-IO) , 122.38 (C-I¹ ) , 128.24 (C-2 ' ) ,

121.83(C-3') , 160.66(C-4' ) , 117.60 (C-5 ' ) , 130.92 (C-6 ' ) ,

163.15(C-2") , 102.92(C-3") , 181.06 (C-4") , 160.07 (C-5" ) ,

98.84 (C-6") , 162.06(07") , 103.61 (C-8" ) , 154.19(09") , 103.27 (C-IO") , 121.76(C-I^{1 1 1}) , 127.86(02" ' ) , 115.38 (C-3 ' " ) ,

161.28 (C-4¹ " ) , 115.59(C-S¹ ") , 127.46 (C-6 • " ) . 55.76(OCH3) ,

56.37 (OCH3)

Compound 7 (ochnaf lavone) 1H-NMR (DMSO-D₆, 500 MHz) 56.41 (IH, s, H-3) , 6.17 (IH, s,

H-6) , 6.37 (IH, s, H-8), 7.82 (IH, s, H-2 ' ) , 7.01 (IH, d, J=7.2 Hz, H-5¹ ) , 7.56 (IH, d, J=I .2 Hz, H-6' ) , 6.52 (IH, s, H- 3") , 6.35(1H, s, H-6") , 6.75(1H, s, H-8") , 7.48 (2H, d, J=8.2 Hz, H-2' ", H-6" ') , 6.72 (2H, d, J=8.2 Hz, H-3" ', H-5' " ) . 1³C-NMR (DMSO-D₆, 125 MHz) 5164.02 (C-2) , 103.60 (C-3) ,

182.21(04) , 161.74(05) , 98.32(06) , 165.02(07), 94.30(08), 157.78 (09) , 103.59* (C-IO) , 122.33(C-I' ) , 121.22 (C-2 ' ) ,

141.5KC-3¹) , 153.56(04' ) , 117.92 (C-5 ' ) , 125.35 (C-6¹ ) ,

162.62 (C-2") , 104.15(03") , 181.23 (C-4") , 161.59 (C-5") , 99.1MC-6"), 164.59(C-7"), 94.32 (C-8") , 157.53(09"), 103.39^*(C-10"), 124.29(C-I'"), 127.91(02'"), 116.64(C-S^{1 1}'), 160.57 (C-4¹ ''), 116.74 (C-5¹ "), 128.41 (C-6 " ' ) .

Experimental Example 1: Procollagen biosynthesis

Fibroblasts separated from human skin were inoculated in a 24-well plate by 10⁶ cells/well, followed by culture until they grew 90%. Then, the cells were cultured in a serum-free medium for 24 hours. 4-substituted benzoic acid derivative compound having 3, 4-methylenedioxy or 3, 4-ethylenedioxybenzene moiety, obtained in Examples 1-2, was dissolved in the serum- free medium was treated thereto at the concentration of 10^~4, followed by culture in a CO₂ incubator for 24 hours. Supernatant was separated and procollagen level was measured using procollagen type 1 ELISA kit. The results are shown in Table 1 and the biosynthesis capacity was presented by the ratio to non-treated group (considered as 100%) . [Table 1]

As shown in Table 1, the biflavonoid derivatives were proved to increase collagen biosynthesis.

Experimental Example 2: Collaqenase (MMP-I) inhibitory activity

Human fibroblasts were inoculated in a 96-well microtiter plate containing DMEM (Dulbecco's Modified Eagle's Media) supplemented with 2.5% FBS at the concentration of 5,000 cells/well, followed by culture till the cells grew 70-80%. The sample compound was treated thereto at the concentration of 10^"4 for 24 hours and then the cell culture solution was collected. Collagenase production in the cell culture solution was measured by using a commercial collagenase measuring kit (Catalog #: RPN 2610, Amersham Pharmacia, USA). First, the collected cell culture solution was loaded in a 96-well plate evenly distributed with the first collagenase antibody, followed by antigen-antibody reaction in a thermostat for 3 hours. Three hours later, the chromophore-conjugated second collagen antibody was added to the 96-well plate, followed by reaction for 15 minutes. 15 minutes later, coloring material was added thereto to induce color development for 15 minutes at room temperature and then the reaction was terminated by adding IM sulfuric acid. The reaction solution was yellow. According to the degree of the reaction, the degree of yellow color varied, optical density^s (OD405) of the yellow 96-well plate was measured by using a spectrophotometer and collagenase production was calculated by mathematical formula 1. At this time, the OD of the cell culture solution not- treated with the sample compound was considered as control.

[Mathematical Formula 1]

Collagenase Expression Level (%) = (OD of the group treated with the sample compound/0D of control) XlOO

[Table 2]

As shown in Table 2, the biflavonoid derivatives were proved to have inhibitory effect on matrix metallo protease (MMP-I) .

Experimental Example 3: Improvement effect on wrinkles caused by UV

To investigate anti-aging effect of the anti-wrinkle material of the present invention on wrinkles caused by UV, the biflavonoid derivatives were dissolved in a vehicle (propylene glycol :ethanol: water = 5:3:2 (v/v) ) at the concentration of 1%. 6-week old female SKH-I hairless mice were divided into 4 experimental groups and 1 control group

(10 mice per group) . UV was irradiated to the mice from 70 cm distance three times a week for 10 weeks (total 30 times) to cause wrinkles. The sample material and the positive control material were transdermally administered by 100 μL, twice a day (morning/afternoon) , 5 days a week (Monday - Friday) for

10 weeks. This administration started simultaneously with the wrinkle inducing irradiation. Skin replica was taken to evaluate anti-wrinkle effect.

As the experimental materials, 3 fractions and biflavonoid fraction (BFF) that exhibited particularly high procollagen biosynthesis capacity and collagenase expression inhibiting activity were selected and the results are shown in Fig. 1.

As shown in Fig. 1, when biflavonoid derivatives were administered, anti-wrinkle effect was significant, compared with when only a vehicle was administered. The formulation forms of a cosmetic composition for improving wrinkles containing biflavonoid derivatives (sumaflavone, 2' , 8"-biapigenin, taiwaniflavone, amentoflavone, robustaflavone, ginkgetin and ochnaflavone) can be selected at random from the group consisting of nutritional cream, nutritional lotion, massage cream, nutritional essence, and softener, etc.

Manufacturing Example 1: Nutritional toner (milk lotion) Nutritional toner containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

Manufacturing Example 2: Softener (skin lotion)

Softener containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

Manufacturing Example 3: Nutritional cream

Nutritional cream containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

Manufacturing Example 4: Massage cream

Massage cream containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

Manufacturing Example 5: Pack

Pack containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

Manufacturing Example 6: Gel

Gel containing biflavonoid derivatives was prepared by the conventional method according to the composition shown in the below table.

[industrial Applicability] As explained hereinbefore, the biflavonoid derivatives of the present invention interact with biflavonoid receptor to increase procollagen biosynthesis, so that they can have the effect of inhibiting wrinkle generation caused by lack of skin elasticity resulted from the denaturation of collagen, one of the important skin components, and of inhibiting the expression of collagenase, the enzyme decomposing collagen, and thereby increases improvement of wrinkles caused by UV. Therefore, the cosmetic composition containing the biflavonoid derivatives as an active ingredient of the present invention can be effectively used as a cosmetic composition or an agent for improvement and prevention of wrinkles without side effects .

Claims

[CLAIMS]

[Claim l]

A cosmetic composition for improving skin wrinkles containing biflavonoid derivatives as an active ingredient.

[Claim 2]

The cosmetic composition for improving skin wrinkles according to claim 1, wherein the biflavonoid derivatives are extracted from natural medicinal herbs or plants such as Selaginella tamariscina, Ginkgo biloba, Cephalotaxus koreana,

Nandina domestica and Lonicera japonica.

[Claim 3]

The cosmetic composition for improving skin wrinkles according to claim 2, wherein the biflavonoid derivatives are obtained from ethyl acetate after adding ethyl acetate and water to methanol extract of the medicinal herbs.

[Claim 4] The cosmetic composition for improving skin wrinkles according to claim 3, wherein the biflavonoid derivatives are represented by formulas 1-7. <Formula 1>

[Claim 5] The cosmetic composition for improving skin wrinkles according to claim 4, wherein the biflavonoid derivatives increase procollagen biosynthesis.

[claim 6] The cosmetic composition for improving skin wrinkles according to claim 4, wherein the bioflavonoid derivatives inhibit the expression of collagenase.

[Claim 7] The cosmetic composition for improving skin wrinkles according to claim 4, wherein the biflavonoid derivatives improve wrinkles caused by UV.

[Claim 8]

The cosmetic composition for improving skin wrinkles according to claim 4, wherein the content of the biflavonoid derivatives is 0.001 - 10 weight% for the total weight of the composition.

[Claim 9]

The cosmetic composition for improving skin wrinkles according to claim 4, wherein the content of the biflavonoid derivatives is 0.01 - 5 weight% for the total weight of the composition.

[Claim 10]

The cosmetic composition for improving skin wrinkles according to one of claims 1 - 3, wherein the cosmetic composition can be formulated as softener, astringent, nutritional toner, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, gel and body essence.