Galangin protects hydrogen peroxide induced oxidative stress via the scavenging of reactive oxygen species

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eJ띤rnat01Medicineand Life SCience

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GaJangin protects hydrogen peroxide induced oxidative stress via the scavenging of - - reactlve oxygen speCles

Mei .Jing Piao

，

Kyoung Ah Kang

，

and .Jin Won Hyun Departmenl018iochemistry，Jeju NationalUniversitySCh。이 이 Medicine.Jeju，Korea

Abstract

Flavonoids are a class 01 secondary metaboliles abundanlly found in fruits and vegetables. In addition，flavonoids have been reported as petenl antioxidants wilh beneficial effecfs againsl oxidative stress retaled diseases such as cancer，aging，and diabeles. Galangin (3，5，7-trihydroxyfravone)，a member of lhe Ilavonol class 01 flavonoid，is present in high concenfrations in medicinal plants (8.9. A/pinia officinarum) and propolis，a natural beehive product. The preSenl'_lstudy was car끼ed oul t

。

invesligate the prolective effecls of galangin against hydrogen peroxide (H202Hnduced oxidalive stress. Galanginwas found t

。

quench the l，l-diphenyl-2-picrylhydrazyl (OPPH)radical and inlracellular reaclive oxygen species generated by H202lrealment in cells，which is detecled by a spectr，。ω。rometer after staining of 2’，7' -dichlα 。dihydrofluorescein diacetale (OCF-OA). These results suggesl lhat galangin protecled V79-4 lung fibroblasl cells against H202-induced oxidalive stress via Ihe scavenging of reaclive oxygen species (ROS)，(J Med Ule SCi 2009;6:362-364)

Key Words : Galangin. Reactive oxygen species

Introduction

Galangin (3，5，7-σihydroxyf1avone) (F'ig， 1)‘a member of flavonol c1assof flavonoid. is present in hi영1 eoncenσ'ations in honey and AJpÍniB o[fiαn 외llm. a plant which has been

used as spice and as a herbal medieine for a v없iety of

ailments in Asia for eenturies. From the ethanol exσ'aet of

A. o[fiCÍnBrum root. galangin m어{es up approximately 10% of the extraetl) ，Galangin is also present in high

eoneen1rations in propoJis. whieh is a naωral eomposite b외sam produeed by honeybees from ilie 밍m of vaIious plants. with the following eomponents: galangin (9%) ehrysin (4%). and quereetin (2%)2)，Galangin possesses

eertain biologieal aetivities. inclu이ng mutagenic3).

anti-c1astogenico1l.anti-oxidative and radical scavengin양 6). and

metabo1ieenzyme modulating aetivities7l

Reaetive oxygen species，including the superoxide anion‘

hydroxyl' radieal. single oxygen. and hydrogen peroxide. 밍견

oxygen eontaining moleeules with unpaired eleetrons 01 ahstraet eleetrons from other moleeules π1ese reaetive oxygen speeies ean lead to functional damage in Iipid proteins and DNA. whieh ean eventually result in eell

Addressfor eorrespandence: Jin WonHyun

Departmen101Biochemistry，‘Jeju NationalUniversitySCh。이01

Medieine，66 Jejudaehakno.690-756. Jeju，Korea 5에1ail: jinwonh@jejunu.ac，kr

362

death8'，Furtheπnore，the oxidative sσess indueed by the overproduetion of ROS plays an important role in various lung pa1hologies，including bronehial asthma‘양stie fibrosis

pulmonary sareoidosis，and lung eaneer9. 10.l}) and lung fibroblast is very sensitive to oxidative slress12)

ηlis study focused on evaluating 1he protective eITeet of galangin on fùÜ2-indueed oxidative stress in V79-4 lung fibroblast eells

Materials and methods

1. Reagents

Ga1angin (Fig. I) was obtained from Professor Sam Sik Kang of SeoulNational University. Republie of Korea Galangin was freshly dissolved in dimethyl sulfoxide (DMSO)，yielding a final concenσation，which did not exeeed 0.1% η1e I.1-diphenyi-2-picrylhydl'azyi (DPPH) I'adical와1d

2’.7’-diehlorodihydrofluoreseein diaeetate (DCF-DA) were purchased from Sigma Chemicat Company (St. Louis，MO) AIl other ehemieals and reagents 야ed were of an~써ticat

grade

2，Cell culture

Previous reports have shown that the lung is an organ whieh is sensitive to oxidative stress12.13l，To study 1he

Galangin protects hydrogen peroxide induced oxidative stress via the scaνenging of reactive oxygen species

hamster lung fibroblasls (V79-4 cellsl 만1e V79-4 cells were obtained from the American Type Culture Collection and maintained at 37 "C in an incubator at a humidified

atmosphere of 5% C02 and cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-ìnactivated fetal calf serum ，streptomycin (10C μg/mll and penicillin (100 unils/m!)

5. Statistical analysis

All measurements were made ìn triplicate 밍1d' all values

were expressed as the means :t standard error. The results were subjected to an analysis of γariance (ANOVA) using

야1e Tukey test to analyze the difference. P

<

0.05 was considered significant1y

3. DPPH radical scavenging activity Results and Discussion Galangin at concentrations of 0.1，1 and 10 μg/ml were

added to a 1 X 10→ M solution of DPPH in methanol ，and the reaction mixture was shaken vigorously.' After 30 min， the amount of DPPH remaining was determined at 520 nm14l. The DPPH radical-scavengin

냥

activity (%) was calculated as 100 x [{optical density _of DPPH radical treatment) ← (。pdcal dens@ of galangm

y

며

DPPH radical

treatment)]/(optical density of DPPH radical 따a디nent)

The radical-scavenging effects of galan밍n on the DPPH

radical and intracellular ROS were measured. The DPPH radical-scavenging activity of galangin showed dose dependent matIDer，3.3% at 0.1 μg/m!. 7% at 1 앵1m!. and

35% at 10 μg/ ml. The DPPH radical-scavenging activity of Figure 2. Effect of galangin on 야1e scavenging of DPPH radical. Measurements were made in triplicate and values are expressed as means :t standard error. *Significan찌y different from control (P

<

0.05)

4. Intracellular ROS measurement

Figure 1. Chemical structure of g"alangin

‘

•

、

NAC NAC 10

.

10 」↑y 0.1 0.1 o o 0 0 100

‘。

i 깅

흥

60 얄 @ 동 60 g Q m

입

40 。: 흐 르 20 m 엉 E

Fi밍Jre 3. Effect of galangin on the scavenging intracellular

ROS 꺼1e intracellular 'ROS generated were detected with spectrof1uorometer after DCF--:-DAtreatment. Measurements were made in triplicate and values are expressed as me입1S:t standard error. *Significantly different from

control (P

<

0.05) 100 용 강@

z

를) 를’@ g

현

40 잉

훌

m

o

。

H

。

。

OH

HO

To detect intracellular ROS， the DCF-DA !llethod was used. DCFcDA때ffuses into cells，where it is hyclrolysed by ìntracellular，、esterase to _polar 2'，7→dichlorodihyclrof1uorescein

This non-f1uorescent f1uorescein analogue is tmpped in cells and can be oxidised to the highly fluorescent 2' ，7' dichlorof1uorescein by intracellular oxidants151. The V79-4 cells were seeded in a 96-wel1 plate at 2 x 10'‘cells/wel1 Sixteen hours ，after plating ， the cells were treated with

gala.r멍in at concenσations of 0.1，1 and，10 μg/ml. After 30

min，1 mM lb02 was added to the plate. The cells were incubated for an additional 30 min at，37 "C. After the addition of 25 μM DCF-DA solution for 10 min，the f1uorescence of 2'，7' -dichlorof1uorescein was detected using a Perkin-Ehner 18-5B spectrof1uorometer. The intracellular ROS scavenging activity (%) was calculated as 100 x [(optical density of ffi02 treatment) - (optical density of galangin wi야1 H202 treatment)] / (optical -density of l--bo

，

σ않tment)

MeiJing Piao，KyoungAh Kang，Jin Won Hyun

N-acetylcystein (NAC)，a major antioxidant used as a positìve control ，showed 58% at 2 mM (Fig. 2). The intracellular ROS scaven밍ng activity of galangin showed

dose dependent manner，6% at 0.1 μg/rrù，13% at 1 μg/rrù， and 28% at 10 μg/nι The intracellular ROS scavenging activity of NAC was 77% at 2 mM (Fig. 3). These results indicate a reduction of ROS by galangin σeatment ，잉ld

suggest that galangin possesses antioxidant properties Flavonoids are polyphenolic compOllllds present ubiquitously in fruits，vegetables ，and beverages such as tea and red wine16 η) η1ey have been shown to possess a variety of

biolo밍C꾀activities at non-to잉c concentrations in organisms

Polyphenols have an ideal and intrinsic struct 미re for

capturing free radica1s and electron delocalization，causing higher antioxidant activitylB). The antioxidant activity of phenolic componnds is mainly due to their redox properties， which allow them to act as reducing agents ，hydrogen donors ，and singlet-oxygen quenchers19l. Gaiangin ，a member of the flavonol class of flavonoid，is present in high concentrations in medicinal plants (e.g. A1pinia. oflïcina.rum) and propolis，a natural beehive productU. Results from V79 4 cells studies iI띠icate 암1at g외angin with anti-oxidative and free radica1 scaven밍ng activities 만1e antioxidant effect of galangin is attributed to this polyphenolic structure

L

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