Anti-wrinkling Effects of Juniperus rigida Sied

노간주나무(Juniperus rigida Sieb.)의 주름개선 효과

전혜지¹, 이수연¹, 김정환¹, 안봉전², 이진영¹*

1호서대학교한방화장품과학과

2대구한의대학교화장품약리학과

Received: July 26, 2013 / Revised: November 8, 2013 / Accepted: November 28, 2013

서 론

자외선에의해손상받은피부는

^collagen

있는데

^,

matrix metallopro- teinase (MMPs)

^,

^MMPs

는피부광노화발생에중요한역할을한다

^{[6]. MMPs}

부의

keratinocytes, fibroblasts

(extracellular matrix, ECM)

(basement membrane, BM)

으로알려져있다

^[22].

^MMPs

관련된 세포 신호 전달 경로는 주로

stress-activated

mitogen-activated protein (MAP) kinase pathway

[4], MAP kinase pathway

activator protein-1 (AP-1)

몇

^MMPs

^{[5]. MMPs}

심부에아연을가지는금속단백분해효소로생체내에서잠

재성전효소

^(zymogen)

^,

위해서구조적변형이일어나아미노말단부위가절단

^,

2-macroglobulin

TIMPs (tissue inhibitors of metalloproteinase)

[6].

유라디칼을소거하고비정상적으로과발현되는

^MMPs

^.

^,

Anti-wrinkling Effects of Juniperus rigida Sied. Jun, Hye-Ji

, Soo-Yeon Lee

, Jeung-Hoan Kim

, Bong-Jeun An

, and Jin-Young Lee

*.

Department of Herbal Cosmetic Science, Hoseo University, Chungnam 336-795, Korea,

Department of Cosmeceutical Science, Daegu Haany University, Gyeongbuk 712-715, Korea

Human skin is constantly exposed to environmental conditions such as UV rays, polluted air, and chemical products. UV rays, in particular, affect skin in many ways causing wrinkles, fine wrinkles, rough skin, and xeroderma through a skin aging process.

The purpose of this study was to investigate the anti-wrinkling effect of Juniperus rigida Sieb., derived from a common cedar tree found the world over. Measuring the elastase to investigate wrinkling efficacy, it was shown that at a concentration level of 1,000 µg/ml of the two extracts, the water extract exhibited a lower than 10% inhibition activity, while the ethanol extract exhib- ited a 68.5% inhibition activity. Collagenase inhibition activity in the water extract and ethanol extract were 44.9% in the former and 97.2% in the latter extract, which in the case of the ethanol extract, is similar to ascorbic acid (99.6%). Moreover, measur- ing the biosynthesis of collagen by fibroblast, a concentration level of 50 µg/ml of ethanol extract produced 151.52% of biosyn- thetic promotion, proving that the ethanol extract acts as a superb anti-wrinkling agent. The result of an investigation conducted on the influence of the ethanol extract on MMP-1 caused by UVA showed that at a concentration level of 100 µg/ml of the etha- nol extract of J. rigida Sieb a 67.1% inhibition activity was noted. At a concentration level of 50 µg/ml of the ethanol extract of J.

rigida Sieb a 35% and 39% inhibition ratio to MMP-1 protein and mRNA were observed respectively, thereby restraining the appearance of the collagen breakdown enzyme MMP-1 and wrinkle creation by skin photo-aging.

Keywords: Juniperus rigida Sieb., anti-wrinkle, procollagen, MMP-1

*Corresponding author

Tel: +82-41-540-9552, Fax: +82-41-540-9538 E-mail: [email protected]

© 2013, The Korean Society for Microbiology and Biotechnology

하고우수한효능을가진천연물에서추출한천연항산화제 에대한개발이이루어지고있다

^[13].

전세계적으로향나무과의일종인다년생수목인노간주 나무속은노가지나무

, needle juniper

^,

^,

^,

^.

(Juniperus rigida Sieb.)

^,

(J. conferta),

^{15 m}

^{3-5 m}

(J. communis),

기본종에비해잎의길이가

^{4-8 mm}

^{6-7 mm}

로잎보다긴곱향나무

(J. communis var. montana)

(Juniperus rigida Sieb. et Zucc var.)

^[11].

⁴

,

^,

워능히살아남을수있다고한다

^[14].

종자에서채취한기름은이뇨제등의약리적효과가있다고 알려져있어부종

^,

^,

^{, insect}

repellent

^,

매인두송실

(juniper berry)

^3-4

⁶

^[11].

중국 본초도감에 따르면 과실에는 α

-pinine, mycene, limonene

^,

^,

^,

^,

^,

^[8].

본연구에서는피부에안전한소재인천연물에대한연구 가활발하게진행됨에따라한국전역에자생하고있는노 간주나무추출물을이용하여주름개선관련실험을통하여 그효능을검증하고새로운기능성화장품소재로서의가능 성을탐색하고자한다

^.

재료 및 방법

재료 및 시료 추출

본실험에사용된노간주나무는충북괴산에서

²⁰¹²

^.

^70%

¹⁰

²⁴

³

^,

¹⁰

⁸⁰

^C

³

²⁴

³

^.

은여과지

(Whatman No. 2)

^EYELA

evaporator

건조하여−

²⁰

^C

^,

^.

실험에 사용된 시약

주름억제효과 측정에사용된 시약인

porcine pancreas

elastase, N-succinyl-L-ala-ala-ala-p-nitroanilide, collagenase, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg

Sigma Chemical Co. (USA)

^.

^collagen

pro-collagen type I C-peptide EIA kit

Takara bio (Japan)

^, MMP-1

^{MMP-1 kit}

^Amersham Bioscience (USA), tumor necrosis factor-

^(TNF-

⁾

Sigma Chemical Co. (USA)

^.

Elastase 저해활성 측정

Elastase

^Cannell

^[1]

^.

N-succinyl-(L-Ala)

-p-nitroanilide (Sigma, USA)

³⁷

^C

³⁰

^p- nitroanilide

^{445 nm}

^.

^,

⁴⁰

^l

^96-well plate

, 50 mM tris-HCl buffer (pH 8.6)

2.5 U/ml porcine pancreas elastase (Sigma, USA)

40

^l

50 mM tris-HCl buffer (pH 8.6)

N-succinyl-(L-Ala)

-p-nitroanilide (0.5 mg/ml)

⁸⁰

^l

³⁰

p-nitroanilide

^{445 nm}

. Elastase

^.

저해율

^{(%) =}

Collagenase 저해활성 측정

Collagenase

^W

^{nsch E}

^Heindrich HG

^[23]

^.

0.1 M tris- HCl buffer (pH 7.5)

^{4 mM CaCl}

, 0.3 mg/ml

4-phenyl azobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Sigma, USA)

¹²⁵

^l

⁵⁰

^l

^{0.2 mg/ml}

collagenase (Sigma, USA) 75

^l

²⁰

6% citric acid 250

^l

, ethyl acetate 1.5 ml

^{320 nm}

. Collagenase

1 시료첨가군의 흡광도–---무첨가군의 흡광도

⎝ ⎠

⎛ ⎞ 100×

나타내었다

^.

^{(%) =}

Procollagen type-1 생합성 측정

세포를

¹

¹⁰

cells/well

96 well plate

^,

^well

^CO

²⁴

^.

.

collagen

procollagen type-I C peptide (PIP) EIA (Takara Bio, Japan)

propeptide

^.

Matrix metalloproteinase-1 (MMP-1) 저해활성 측정 세포를

¹

¹⁰

cells/well

96 well plate

^,

^well

^CO

²⁴

^.

^MMP-1

^TNF-

10 ng/ml

^.

실험에 사용하였으며

^Gross

^[7]

^MMP-1 biotrack activity assay

^.

세포 배양

본 실험에 이용한 각 세포의 배양은

^{10% FBS}

^1%

penicillin/streptomycin (100 U/ml)

^DMEM

^{, 37}

^{C, 5% CO}

incubator

^.

MTT assay에 의한 세포 생존율 측정

세포생존율측정은

Carmichael

^[2]

^.

(CCD-986sk)

96 well plate

⁵

¹⁰

cells/well

^{0.18 ml}

^,

^{0.02 ml}

³⁷

^{C, 5% CO}

incubator

²⁴

^.

^.

^{5 mg/ml}

^MTT

^{0.02 ml}

⁴

^well

DMSO 0.15 ml

30

ELISA reader

^{540 nm}

^.

^.

세포생존율

^{(%) =}

Western blot을 통한 단백질 발현 측정

주름개선인자인

matrix metalloproteinase-1 (MMP-1)

^CCD-986sk

100 mm tissue culture dish

²⁴

정화시켰다

^.

배지로

^24-48

phosphate buffered saline (PBS)

²

^. Radio-immunoprecipitation assay (RIPA) buffer 10 ml

complete mini 1 tab

¹⁰⁰

^l

⁴

^C 13,200 rpm

²⁰

^.

BCA protein assay kit

20

^l

10% SDS-PAGE

^.

semi dry transfer cell (Hofer, USA)

polyvinylidene fluoride (PVDF) membrane

¹

blocking buffer (5% skim milk in TBST)

^{. 1}

⁴

^C

^,

¹⁰

tris-buffered saline and tween 20 (TBST)

³

²

1:1,000

²

^{. 3}

한후

LAS 4,000

.

Total RNA 분리 및 cDNA 합성

세포를

100 mm culture dish

cell seeding

²⁴

²⁴

^.

trizol lysis buffer

well

^{1 ml}

^lysis

chloroform

200 ml

²⁰

^.

13,200 rpm

²⁰

isopropanol 500 ml

^.

13,200 rpm

²⁰

^,

^75%

EtOH-diethylpyrocarbonate water

^{1 ml}

13,200 rpm

⁵

^{. DEPC}

⁵⁰

^l

96 well plate

^{RNA 5}

^l

¹⁹⁵

^l

260 nm, 280 nm

^{total RNA}

. Oligo (dT) 15 primer (500

^{g/ml) 1}

^l,

^{RNA (2}

^g)

nuclease free water

¹⁰

^l

⁷⁵

^C

⁵

5X reaction buffer, MgCl

, PCR necleotide mix, rnasin inhibitor, reverse transcriptase, ruclease free water

²⁵

^C

⁵

^{, 42}

^C

60

^{, 70}

^C

¹⁵

^cDNA

^.

Reverse transcription-polymerase chain reaction

Matrix metalloproteinase-1 (MMP-1)

^mRNA

polymerase chain reaction (PCR)

^.

primer sequences

^{Table 1}

^. PCR tube

5×green GoTaq Flexi buffer, MgCl

, PCR Nucleotide Mix (10 mM), primer, GoTaq DNA Polymerase, nuclease free water,

^cDNA

⎝ ⎠

⎛ ⎞ 100×

1 시료첨가군의 흡광도–---무첨가군의 흡광도

⎝ ⎠

⎛ ⎞ 100×

PCR

^{. MMP-1}

⁹⁴

^{C 30}

^{, 56}

^{C 60}

^{, 72}

^C 1

(35 cycles)

^{. PCR}

^0.002%

ethidium bromide

1.5% agarose gel

^{100 V}

⁴⁰

^{LAS 4,000}

^.

통계처리

모든실험은

³

^,

SPSS10.0 (Evanston, IL, USA) software

^,

^(analysis of variance ANOVA)

^{= 0.05}

^Turkey’s HSD test

^.

결과 및 고찰

Elastase 저해활성 측정 결과

Elastase

ursolic acid

^,

ursolic acid

질을가지고있기때문에제제화하기어려워일반적으로사

용하기어려운문제점이있다

^[15].

한

^elastase

를

^{Fig. 1}

^.

^10%

이하의낮은저해활성을나타낸반면에노간주나무에탄올 추출물의

^1,000

^g/ml

^68.5%

^.

^Jung

^[9]

^1,000

^g/ml

^,

^,

^elastase

3.73%, 8.73%, 15.59%

^Kwak

^[12]

^,

^,

^,

^,

^elastase

^1,000

^g/ml

54%, 52%, 51%, 45%, 57%

^.

Collagenase 저해활성 측정 결과

Collagen

이 알려져 있는 것이 콜라겐

^{type I}

^matrix

metalloproteinase I (MMP-1, collagenase)

있어

^MMP-1

다

^[3].

collagenase

을사용하여측정한결과

^{Fig. 2}

^.

무열수추출물의저해활성은

^1,000

^g/ml

^44.9%

^,

1,000

^g/ml

^97.2%

ascorbic acid (99.6%)

^.

^{Seo [20]}

^1,000

^g/ml

^55.5%

Table 1. Sequence of the primers used for PCR.

Gene Primer Sequence(5’ → 3’)

MMP-1 Forward Reverse

AGCGTGTGACAGTAAGCTAA GTTTTCCTCAGAAAGAGCAGCAT β-actin Forward

Reverse

ATTGTTGCCATCAATGACCC AGTAGAGGCAGGGATGAT

Fig. 1. Inhibition rate of Juniperus rigida Sieb. extracts on elastase.

JRW : Juniperus rigida Sieb. extracted with water, JRE : Juniperus rigida Sieb. extracted with ethanol, Vit. C : L-ascorbic acid. Results are means ± S.D. of triplicate data (Significant as compared to con- trol. *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2. Inhibition rate of Juniperus rigida Sieb. extracts on col- lagenase.

JRW : Juniperus rigida Sieb. extracted with water, JRE : Juniperus

rigida Sieb. extracted with ethanol, Vit. C : L-ascorbic acid. Results

are means ± S.D. of triplicate data (Significant as compared to con-

trol. *p < 0.05, **p < 0.01, ***p < 0.001).

타내었으며

^{, Lee}

^[17]

^1,000

^g/ml

에서

^37.4%

주나무추출물의

collagenase

수있었다

^.

섬유아세포(CCD-986sk)의 생존율 확인

노간주나무에탄올추출물이세포의생존율에미치는영 향을알아보기위하여

5, 10, 50, 100

^g/ml

하고

^{, 24}

^MTT

관찰하였다

^.

^{, 100}

^g/ml

^58.8%

^,

⁵⁰

^g/ml

^{(Fig. 3).}

섬유아세포(CCD-986sk)에서의 pro-collagen type I 생합성량 확인

Pro-collagen

propeptide

^peptide

, propeptide

pro-collagen

^folding

^collagen

^collagen

^,

^.

propeptide

^,

^collagen

^{[18, 21].}

(CCD-986sk)

collagen

pro-collagen type I C-peptide (PICP) enzyme immunoassay

^collagen

^{Fig. 4}

^.

^collagen

은

⁵⁰

^g/ml

^151.52%

다

^{. Lee [16]}

^,

collagen

¹⁰⁰

^g/ml

27.7%, 37.7%, 41.0%

의효과와

^{Kim [10]}

^collagen

⁵⁰

^g/ml

^121.4%

^.

Fig. 3. Cell viability of Juniperus rigida Sieb. ethanol extracts on fibroblast cell (CCD-986sk).

JRE : Juniperus rigida Sieb. extracted with ethanol.

Results are means ± S.D. of triplicate data (Significant as com- pared to control. *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4. Pro-collagen synthesis of Juniperus rigida Sieb. etha- nol extracts on fibroblast cell (CCD-986sk).

JRE : Juniperus rigida Sieb. extracted with ethanol.

Results are means ± S.D. of triplicate data (Significant as com- pared to control. *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 5. Inhibitory effect of extract from Juniperus rigida Sieb.

The expression of MMP-1 in the UVB irradiated human der- mal fibroblasts.

The cells were treated with various concentration of Juniperus rigida Sieb. for 24 h. Each values represents mean ± SD of three individual experiments (Significant as compared to control.

*p < 0.05, **p < 0.01, ***p < 0.001).

MMP-1 저해활성 kit 측정 결과

본연구에서는

^UVA

^MMP-1

^UVA

²⁴

^MMP-1

^enzyme- linked immunosorbent assay (ELISA)

^.

^{Fig. 5}

¹⁰⁰

^g/ml

^67.1%

^MMP-1

^.

^.

MMP-1 단백질 발현 및 mRNA 발현 억제 효과 확인 본연구에서는

MMP family

^MMP-1

^{. MMP-1}

collagenase 1

^, type I

III collagen

, stromelysin 1

^MMP-3

type IV collagne

zymogen

^{pro MMP-1}

^,

^MMPs

^[5].

되어진

^MMP-1

^mRNA

과

, Fig. 6, 7

^.

MMP-1

^mRNA

⁵⁰

^g/ml

각각

^{35%, 39%}

^.

해노간주나무에탄올추출물은

^collagen

MMP-1

성을보이는것으로판단된다

^{. Park}

^[19]

5, 10, 20

^g/ml

^MMP-1

40%, 50%, 70%

^.

요 약

사람의피부는자외선

^,

^,

^.

^,

^,

^,

^.

^(Juniperus rigida Sieb.)

^.

주름개선효과측정으로

^elastase

노간주나무열수추출물

^1,000

^g/ml

^10%

1,000

^g/ml

^68.5%

, collagenase

^1,000

^g/ml

44.9%, 97.2%

ascorbic acid

(99.6%)

^.

섬유아세포를이용한

^collagen

Fig. 6. MMP-1 protein expression rate of ethanol from Junipe- rus rigida Sieb. extracts on fibroblast cell (CCD-986sk).

After CCD-986sk cells were started in serum free medium for 1 h the cells were treated with 5, 25, 50 µg/ml of ethanol extracted of Juniperus rigida Sieb. for 48 h. Histogram show the densitometric of MMP-1 protein normalized to β-actin. Each values represents mean ± SD of three individual experiments (Significant as com- pared to control. *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 7. MMP-1 mRNA expression rate of ethanol from Junipe- rus rigida Sieb. extracts on fibroblast cell (CCD-986sk).

After CCD-986sk cells were started in serum free medium for 1 h

the cells were treated with 5, 25, 50 µg/ml of ethanol extracted of

Juniperus rigida Sieb. for 48 h. Histogram show the densitometric

of MMP-1 protein normalized to β-actin. Each values represents

mean ± SD of three individual experiments (Significant as com-

pared to control. *p < 0.05, **p < 0.01, ***p < 0.001).

간주나무에탄올추출물

⁵⁰

^g/ml

^151.52%

^{. UVA}

현이증가되는

^MMP-1

는영향을알아본결과노간주나무에탄올추출물은

¹⁰⁰

^g/

ml

^67.1%

^MMP-1

^.

주나무에탄올추출물의

^MMP-1

^mRNA

현은

⁵⁰

^g/ml

^{35%, 39%}

^collagen

효소인

^MMP-1

억제활성을보이는것을확인하였다

^.

Acknowledgments

This study was supported by a grant of the Korea Healthcare technology R & D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. : A103017).

References

1. Cannell RJ, Kellam SJ, Owsianka AM, Walker JM. 1988.

Results of a large scale screen of microalgae for the produc- tion of protease inhibitors. Planta Med. 54: 10-14.

2. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB.

1987. Evaluation of a tetrazolium-based semiautomated colo- rimetric assay: assessment of chemosensitivity testing. Can- cer Res. 47: 936-942.

3. Chung JH, Kang S, Varani J, Lin J, Fisher GJ, Voorhees JJ.

2000. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J. Invest. Dermatol. 115: 117-182.

4. Davis RJ. 1994. MAPKs: new JNK expands the group. Trends Biochem. Sci. 19: 470-473.

5. Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, et al. 1996. Molecular basis of sun-induced premature ageing and retinoid antagonism. Nature 379: 335-339.

6. Fisher GJ, Talwar HS, Lin J, Voorhees JJ. 1999. Molecular mechanisms of photoaging in human skin in vivo and their prevention by all-trans retinoic acid. Photochem. Photobiol.

69: 154-157.

7. Gross J, Lapiere CM. 1962. Collagenolytic activity in amphib- ian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. 54:

1197-1204.

8. Jo JO, Kim SM, Kim KS. 1999. Analysis of asarone, coumarin and thujone in medicinal plants used in brewing a Korean tra- ditional folk Wine. J. Korean Soc. Agric. Chem. Biotechnol.

42: 210-217.

9. Jung JH, Kim KJ. 2009. Experimental studies about the inhibi- tory effect on tyrosinase and elastase activities by various herb medicines. The Journal of Korean Medical Ophthalmol- ogy & Otolaryngology & Dermatology 22: 82-91.

10. Kim YH. 2009. Skin whitening and anti-wrinkle activities of vaccinium uliginosm L. and its action mechanism. Ph. D.

dissertation, Kyung Hee University, Seoul, Korea.

11. Kim TW. 1996. Woody plant of Korea, pp. 49. Kyohak Publish- ing, Seoul.

12. Kwak YJ, Lee DH, Kim NM, Lee JS. 2005. Screening and extraction condition of anti-skin aging elastase inhibitor from medicinal plants. Korean J. Medicinal Crop Sci. 13: 213-216.

13. Lim AK, Jung YJ, Kim KS, Kim YH, Kwak JH, Hong JH, et al.

2010. Skin UVB photo aging effect from extract of fermented Reynoutri elliptica. J. Korean Soc. Food Sci. Nutr. 39: 369- 375.

14. Lee CB. 1990. Dendrology, pp. 110-112. Hyangmoon Publish- ing, Seoul.

15. Lee GW, Lee JY. 2008. The effect on skin deposition and moisturizing of ursolic acid in hydrogel system containing Wood vinegar. J. Korean Pharm. Sci. 38: 87-92.

16. Lee JY. 2007. Anti-wrinkle, whitening constituents of Prunus persica Flos and an application for functional cosmetics. Ph.

D. dissertation, Daegu Haany University, Gyeongbuk, Korea.

17. Lee JY, Choi YH, Lee SM, Lee SY, Jun HJ. 2012. Physiologi- cal activity of Aconitum pseudolaeve var erectum Nakai and its effect on perfume on an electroencephalogram (EEG). J.

Life Sci. 22: 1214-1223.

18. Parfitt AM, Simon LS, Villanueva AR, Krane SM. 1987. Pro- collagen type I carboxy-terminal extension peptide in serum as a marker of collagen biosynthesis in bone. Correlation with iliac bone formation rates and comparison with total alkaline phosphatase. J. Bone Miner Res. 2: 427-436.

19. Park SM, Lee GW, Cho YG. 2008. Effect of Rheum undulatum extract on antioxidant activity and activity of matrix metallopro- teinase-1 in human skin fibroblasts. J. Life Sci. 18: 1700-1704.

20. Seo IA. 2010. Studies on cosmeceutical of Phaseolus radia- tus. M. S. dissertation, Daegu Haany University, Gyeongbuk, Korea.

21. Talwar HS, Griffiths CE, Fisher GJ, Hamilton TA, Voorhees JJ.

1999. Reduced type I and type III procollagens in photodam- aged adult human skin. J. Invest. Dermatol. 105: 285-290.

22. Wang Y, Johnson AR, Ye QZ, Dyer RD. 1999. Catalytic activi- ties and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain. J. Biol. Chem. 274:

33043-33049.

23. Wünsch E, Heidrich HC. 1963. Zur quantitativen bestimmung

der collagenase. Z. Physiol. Chem. 333: 149-151.