Antioxidant Activities and Cytoprotective Effects of Lonicera japonica Thunb. Extract and Fraction against Oxidative Stress

인동덩굴 추출물과 분획물의 항산화 활성 및 산화적 스트레스에 대한 세포 보호 효과

이예슬, 윤믿음, 이윤주, 박영민, 이상래, 박수남*

서울과학기술대학교정밀화학과

,

Received: November 29, 2017 / Revised: January 24, 2018 / Accepted: March 8, 2018

서 론

피부노화는주요발생원인에따라내인성노화와외인 성노화로구분할수있다

^.

^.

^,

^.

^.

관찰된광노화에의한피부변화에는두꺼워진표피

^,

^.

^[1

^3].

피부의광노화에대한가설중자유라디칼

(free radical)

^,

(reactive oxygen species, ROS)

^·

^[4].

^{, ROS}

이다

^{. ROS}

라디칼

^{(· OH),}

^(O

),

^(ROO·)

⁽

^O

),

^(H

O

),

^(ROOH)

^[5].

Antioxidant Activities and Cytoprotective Effects of Lonicera japonica Thunb. Extract and Fraction against Oxidative Stress Ye Seul Lee, Mid Eum Yun, Yun Ju Lee, Young Min Park, Sang Lae Lee, and Soo Nam Park*

Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea

In this study, the antioxidant activities and cytoprotective effects against oxidative stress of Lonicera japonica Thunb. 50% ethanol extract and ethyl acetate fraction were investigated. Using the 1,1-diphenyl- 2-picrylhydrazyl assay, the free radical scavenging activity (FSC50) of L. japonica Thunb. 50% ethanol extract and ethyl acetate fraction was determined as 152.00 and 77.25 μg/ml, respectively. To measure the reactive oxygen species (ROS) scavenging activity, the total antioxidant capacity (OSC50) was deter- mined by using a luminol-dependent chemiluminescence assay. The antioxidant activity of the ethyl ace- tate fraction (0.33 μg/ml) was approximately four times stronger than that of the 50% ethanol extract (1.12 μg/ml). The protective effect against

O

-induced cellular damage of human erythrocytes ( τ

) was 46.0 min at 10 μg/ml of the 50% ethanol extract and 52.3 min at 1 μg/ml of the ethyl acetate fraction. We also investigated the cytoprotective effects against oxidative stress induced by H

O

and the intracellular ROS scavenging activity in response to UVB irradiation and found that the extract and fraction protected human skin cells from damage and reduced ROS. These results confirmed that L. japonica Thunb. was a valuable plant-derived natural antioxidant with potential for development as an antioxidative functional ingredient.

Keywords: Lonicera japonica Thunb., reactive oxygen species, antioxidative activity, cytoprotective effect, oxidative stress

*Corresponding author

Tel: +82-2-970-6451, Fax: +82-2-972-9585 E-mail: snpark@seoultech.ac.kr

© 2018, The Korean Society for Microbiology and Biotechnology

앞서언급한활성산소의공격으로부터신체를보호하기위 해우리몸은다양한항산화네트워크를구축하고있다

^.

superoxide dismutase (SOD)

^C,

^E

^.

^,

^ROS

워크가무너지게되고피부노화가촉진된다

^{[7, 8].}

노화로부터피부를보호하고항산화네트워크를유지하기 위해항산화물질을보충해줘야한다

^.

^,

^[9].

본연구에서사용한인동덩굴

(Lonicera japonica Thunb.)

^,

^“

^”

^.

,

,

,

,

^.

^[10],

^[11],

^[12]

^.

^[13],

^ROS

^.

^,

Fe

-EDTA/H

O

^O

^.

부각질형성세포인

^HaCaT

선으로유도된산화적스트레스에대한인동덩굴추출물및 분획물에대한세포보호효과를조사하였다

^.

본연구는피부광노화에서중요한항산화평가법을이용 하여식물유래천연항산화제로서인동덩굴의각종

^ROS

^.

재료 및 방법

기기 및 시약

UV-visible spectrophotometer

Cary 50 (Varian, Australia),

6-channel LB9505 LT (Berthold, Germany)

^{. HPLC}

Shim-pack VP-ODS C18 column (250 mm × 4.6 mm, 5

m) (Shimadzu, Japan)

. Free radical

^1,1-

diphenyl-2-picrylhydrazyl (DPPH), EDTA, luminol, Folin- ciocalteu’s phenol reagent

Sigma chemical Co. (USA)

^.

^FeCl

· 6H

O

Junsei Chemical Co.

(Japan)

^{, H}

O

Dae Jung Chemical & Metals (Korea)

^.

^(EtOH),

^(MeOH),

(EtOAc), n-

^.

^(+)-

-tocopherol (1,000 IU vitamin E/g), L-ascorbic acid, apigenin, caffeic acid, coumaric acid, luteolin

Sigma Chemical Co.

(USA)

^.

²⁰¹⁷

4

^.

인동덩굴의 추출/분획 및 수율

실험에사용한인동덩굴은

^{Fig. 1}

^.

^{400 g}

^50%

^{8 L}

^{24 h}

^.

^.

^50%

³

^.

^·

얻었다

^.

^{(400 g)}

였다

^.

^50%

^4.85%,

^0.41%

^.

인동덩굴 추출물의 항산화 효과 측정

DPPH

DPPH

^,

Fig. 1. Preparation procedure of extract and fractions of

dried L. japonica Thunb.

안정한라디칼인

^DPPH

^.

0.2 mM DPPH

^{1 ml}

^{1 ml}

고여러농도의인동덩굴추출물

^{1 ml}

음 실온에서

^{10 min}

spectrophotometer

517 nm

^.

넣지않은경우를대조군

^(control)

을실험군

(experiment)

^.

은

^DPPH

^50%

(free radical scavenging activity, FSC

,

^g/ml)

^,

^(DPPH)

^(%)

^.

Radical Scavenging (%) =

Luminol

-EDTA/H

O

^Fe

^-EDTA/H

O

ROS (O

, · OH

^H

O

)

^,

^.

^ROS

^,

^.

^ROS

^ROS

^.

에증류수

^{1.78 ml}

넣었다

^.

2.5 mM EDTA 40

^l

^{5 mM FeCl}

· 6H

O 10

^l

35 mM luminol 80

^l

^.

cell holder

^{5 min}

^{150 mM H}

O

40

^l

^{25 min}

^.

^(control)

^,

^(blank)

^H

O

^FeCl

· 6H

O

^.

6-channel LB9505 LT

.

^(%)

^,

(counts per minute, cpm)

^50%

^(reactive oxygen species scavenging activity, OSC

)

^.

ROS Scavenging (%) =

광용혈(Photohemolysis)법을 이용한 세포 보호 효과 측정 적혈구 현탁액 제조. 적혈구는건강한성인남녀로부터얻 었다

^.

^heparin

^{, 1,518}

×g

^{5 min}

고

^,

0.9% saline phosphate buffer (pH 7.4, Na

HPO

· 12H

O 9.6 mM, NaH

PO

· 2H

O 1.6 mM)

^.

³

^,

⁴

^{12 h}

^.

[6].

^{700 nm}

^O.D.

0.6

^{1.5 × 10}

^cells/ml

^.

인동덩굴 추출물 및 분획물의 광용혈 억제 효과. 적혈구현 탁액

^{3.5 ml}

^{(No. 9820)}

^,

^.

⁵⁰

^l

^{30 min}

pre-incubation

^,

rose-bengal (13

^{M) 0.5 ml}

^(Whatman laboratory sealing film, UK)

^{15 min}

^.

50 cm × 20 cm × 25 cm

^{20 W}

^,

^{5 cm}

후

^{15 min}

^.

(post-incubation)

^{15 min}

^{700 nm}

(transmittance, %)

^.

^.

²⁰

^.

post-incubation

^50%

^.

HaCaT 세포 보호 효과 측정

HaCaT

^HaCaT

^CLS Cell Line Service GmbH (Eppelheim, Germany)

^.

10% fetal bovine serum (FBS)

^1%

penicillin/streptomycin (P/S)

³⁷

^{, 5% CO}

^.

인동덩굴 추출물 및 분획물의 HaCaT 세포 독성 평가. 인동덩

굴

^50%

성세포인

^HaCaT

위해

3-(4,5-dimethyl-2-thiazolyl)-2, 5-di-phenyltetrazolium bromide (MTT, Sigma, USA) assay

. 96-well plate

1 × 10

cells/well

⁶⁰

^80%

^.

배지에인동덩굴

^50%

획물을농도별로희석하여

^{24 h}

^,

두제거하고

0.5 mg/ml MTT

^well

^{2 h} 1 A _experiment – A sample blank

A _control ---

⎝ – ⎠

⎛ ⎞ 100 ×

Cpm _control – Cpm _experiment

( )

Cpm _control – Cpm _blank

( )

---

⎩ ⎭

⎨ ⎬

⎧ ⎫ × 100

동안

³⁷

^.

^formazan

^dimethyl sulfoxide (DMSO)

ELISA reader (Tecan, Austria)

이용하여

^{570 nm}

^.

과산화수소로 유도된 세포 손상에 대한 세포 보호 효과.

HaCaT

^{1 × 10}

cells/well

96-well plate

^{24 h}

³⁷

^{, 5% CO}

^incubator

. 24 h incubation

^,

지에인동덩굴

^50%

물을농도별로희석하여

^{24 h}

^{. PBS 100}

^l

¹

^,

^{2 mM}

^{(in PBS)}

^{30 min}

^.

^{, PBS 100}

^l

²

^.

^,

³⁷

^{, 5% CO}

incubator

^{24 h}

. 24 h incubation

^{, MTT}

assay

손상에대한인동덩굴

^50%

보호효과를확인하였다

^.

세포 내 ROS 소거활성 평가. 세포내

^ROS

2’,7’-dichlorodihydrofluorescein diacetate H

DCF-DA)

사용하여

fluorescence

^.

HaCaT

96-well plate

^{1 × 10}

cells/well

분주하여

^{24 h}

^,

리하여

^{24 h}

. Dulbecco’s phosphate-buffered saline (DPBS)

²

^{. 20}

^M H

DCF-DA

^{30 min}

³⁷

^{, CL-1000} ultraviolet crosslinker

^DPBS

²⁰⁰ mJ/cm

UVB

^ROS

^, fluorescence ELISA reader (excitation, 490 nm emission,

530 nm)

^.

TLC 및 HPLC를 이용한 인동덩굴 추출물의 성분 분석

^100%

, syringe filter (Milliopore 0.45

^m)

^TLC (normal phase)

HPLC (C18)

^. TLC

^{Table 1}

^.

^,

^R

NP-PEG (natural products-polyethylene glycol, 2- aminoethyl diphenylborinate)

^{. HPLC}

2% acetic acid

0.5% acetic acid

50% acetonitrile

^{, HPLC}

Table 2

^.

총 페놀성 화합물 및 총 플라보노이드 함량 측정

인동덩굴추출물의총페놀성화합물함량측정은

^Alves

등의방법을이용하였다

^[23].

⁸⁰

^l

Folin-Ciocalteu

^{(50% v/v)}

²⁰

^l

^{, 25}

^{5 min}

^.

^Na

CO

(2% w/v) 200

^l

Table 1. TLC mobile phase for separation of EtOAc fraction of

L. japonica Thunb.

Eluent System EtOAc

Fraction Hexane : Ethyl Acetate : Acetic Acid = 21 : 14 : 5 (v/v)

Table 2. HPLC condition for the separation of EtOAc fraction of L. japonica Thunb.

Condition of HPLC Analysis

Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm)

Detector UVD 170s DIONEX

Detection Wavelength 254 −400 nm

Flow Rate 1.0 ml/min

Injection Volume 20

^l

Mobile phase conditions

for HPLC gradient-elution

Program Order

Time (min)

2% AA

in Water (%)

0.5% AA

in 50% ACN

(%)

1 0 100 0

2 10 100 0

3 150 50 50

4 200 0 70

5 210 0 70

6 215 100 0

7 220 100 0

1)

AA: Acetic acid,

ACN: Acetonitrile.

가하여 혼합하고

²⁵

^{30 min}

^ELISA reader (Tecan, Austraia)

^{760 nm}

. Caffeic acid

^,

^.

인동덩굴추출물의총플라보노이드함량은

^Zhishen

^[24].

³⁰

^l

¹²⁰

^l

^{, NaNO}

(25% w/v) 9

^l

²⁵

^{5 min}

^.

^AlCl

(10% w/v) 9

^l

^, 1 min

NaOH (1 M) 60

^l

⁷²

^l

510 nm

^.

^luteolin

^,

^.

통계처리

모든실험은

³

^mean

^SD

^.

GraphPad Prism 5.0 (USA)

, Student’s t-test

^one- way ANOVA

p < 0.05

p < 0.01

^.

결과 및 고찰

인동덩굴 추출물의 항산화 활성

DPPH

^.

(free radical scavenging concentration, FSC

)

^[6].

본실험에서는비교적안정한라디칼인

^DPPH

여인동덩굴의

^50%

^.

^(+)-

-tocopherol

^,

^50%

^FSC

^50%

에탄올추출물에서

^152.00

^g/ml,

^77.25

^g/ml

^{(Fig. 2).}

^(+)-

-tocopherol (FSC

, 8.98

^g/ml)

^,

획물이

^50%

²

높은자유라디칼소거능을나타냄을확인하였다

^.

Luminol

-EDTA/H

O

^ROS

^Fe

^-EDTA/H

O

^Fenton

^.

^Fenton

^H

O

, O

, ·OH

^.

^(ROS scavenging concentration, OSC

)

^luminol

⁴²⁰

^{450 nm}

^[4].

활성산소가

^50%

^OSC

^50%

에탄올추출물에서

^1.12

^g/ml,

^0.33

^g/ml

^{(Fig. 3).}

L-ascorbic acid (4.95

^g/ml)

^.

^,

^50%

⁴

, L-ascorbic

acid

였다

. L-Ascorbic acid

^50%

Fig. 2. Free radical scavenging activities of 50% EtOH extract and EtOAc fraction of L. japonica Thunb., and (+)-α-tocoph- erol. Data are presented as the mean ± SD.

p < 0.05 compared with (+)- α-tocopherol.

Fig. 3. ROS scavenging activities of 50% EtOH extract and

EtOAc fraction of L. japonica Thunb., and L-ascorbic acid in

Fe

-EDTA/H

O

system by luminol-dependent chemilumi-

nescence assay. Data are presented as the mean ± SD.

p < 0.05

and

p < 0.01 compared with L-ascorbic acid.

출물과에틸아세테이트분획물은모두통계적으로유의한 차이를나타내었다

^{. Fig. 2}

^.

1

O

porphyrin

riboflavin

^O

^ROS

^.

^O

^.

^[14].

증감제인

rose-bengal

의피부광노화에서의항산화능및세보보호효과를평가 하였다

^.

^,

^Fenton

^ROS

^.

^50%

⁽

, min)

^.

^50%

^{1, 5, 10}

²⁵

^g/ml

24.8, 36.2, 46.0, 31.8 min

0.1, 0.5, 1, 5

¹⁰

^g/ml

26.2, 39.0, 52.3, 47.1, 16.9 min

(Table 3).

^50%

²⁵

^g/

ml

^.

^50%

¹⁰

^g/ml

^(+)-

-tocopherol (37.5 min)

^.

^, 5

^g/ml

10

^g/ml

였다

^.

¹

^g/ml

^,

^(+)-

-tocopherol (31.1 min)

^.

^{, 1}

^g/ml

^50%

2

^{(Fig. 4).}

고농도에서의인동덩굴추출물및분획물의세포보호효 과감소를통해

^,

^.

¹⁰

^g/ml

물에서대조군

^{(30.8 min)}

로미루어보아이물질이에틸아세테이트분획에더많이함 유되어있을것으로예상된다

^.

HaCaT

인동덩굴 추출물 및 분획물의 HaCaT 세포 독성 평가.

^MTT

Table 3. Cellular protective effects of 50% EtOH Extract and EtOAc fraction of L. japonica Thunb., and (+)-α-Tocopherol on the rose-bengal sensitized photohemolysis of human erythrocytes.

τ

(Half time of hemolysis)

Concentration ( μg/ml) Control 0.1 0.5 1 5 10 25

50% EtOH extract 28.5

(±0.8) - - 24.8

(±0.5)

36.2 (±0.9)

46.0 (±0.3)

31.8 (±1.4)

EtOAc fraction 30.8

(±1.2)

26.2 (±0.4)

39.0 (±0.6)

52.3 (±0.5)

47.1 (±1.8)

16.9

(±0.4) -

(+)- α-tocopherol 30.3

(±1.3) - - 31.1

(±1.6)

36.6 (±1.5)

37.5 (±1.3)

38.7 (±1.6) Fig. 4. Cellular protective effects of 50% EtOH extract and EtOAc fraction of L. japonica Thunb., and (+)-α-tocopherol at 1 μg/ml and 5 μg/ml on rose-bengal sensitized photohemo- lysis of human erythrocytes. Data are presented as the mean

± SD.

p < 0.05 and

p < 0.01 compared with (+)- α-tocopherol by

one-way ANOVA.

p < 0.05 1 μg/ml versus 5 μg/ml of 50% EtOH

extract by Student’s t-test.

assay

^50%

이트분획물의각질형성세포인

^HaCaT

을확인함으로써앞으로의실험에사용될시료의농도범위 를결정하였다

^{. 0.4}

^400.0

^g/ml

^50%

^HaCaT

^{24 h}

^.

^,

^50%

²⁵

^g/ml,

^12.5

^g/ml

성을나타내지않았다

^{(Fig. 5).}

인동덩굴

^50%

최고농도를

^12.5

^g/ml

^.

과산화수소로 유도된 세포 손상에 대한 세포 보호 효과. 과산 화수소는세포내에서

^O

^SOD

^.

^ROS

^[19].

^50%

에탄올추출물과에틸아세테이트분획물이과산화수소로유

도된

^HaCaT

확인하였다

^.

실험결과

^,

^65%

(Fig. 6A). 0.4

Fig. 5. Effect of L. japonica Thunb. 50% EtOH extract and EtOAc fraction treatment on HaCaT cell viability. HaCaT cells were treated with various concentrations of samples for 24 h and cell viability was determined using the MTT assay. Data are presented as the mean ± SD.

p < 0.05,

p < 0.05 compared with untreated control by one-way ANOVA.

Fig. 6. (A) Cell protective effects of 50% EtOH extract and EtOAc fraction of L. japonica Thunb. on H

O

-induced HaCaT cell.

HaCaT cells were treated with different concentration of sample for 24 h after being exposed to oxidative stress. (B) Rate of increase in

proliferation of cell protective effects. Data are presented as the mean ± SD.

p < 0.05 compared with H

O

treated control in 50% EtOH

extract dose-treated groups by one-way ANOVA,

p < 0.05 compared with H

O

treated control in EtOAc fraction dose-treated groups

by one-way ANOVA.