potent antioxidant radical-scavenging activity chenpi

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J UOEH31 (4):311-324(2009) 31!

[Original]

Potent AntioxidantCompensatory and

Citric Acid

and Radical-Scavenging Activity of Chenpi -

Cooperative Actions of Ascorbic Acid and

KiyokaHIGAsHt-OKAIi, Akiko IsHIKAwAi, Sayuri YAsuMoToi and Yasuji OKAI2

EDepartment of Food and Nutritional Environment, College qf Human Lijle Environmen4 Kiqio Gakuin

Uitiversiry, Mbriyama-KLt, Nagoya 463-8521, Jopan2Section

Qf Food and AItttrition, Deparnnent qf' Human Life Science, Osaka Kun-Ei IVbmen's College,

Sets City, Osaka, 566-850J, .lopan

Abstract:Dried peels of Satsuma mandarin (Citr"s unshiu Marcov. ) have been used as

traditional Chinese and Japanese medicine, which are called `Chenpi'.

inour present study, cold and hot water extracts of Chenpi exhibited a strong in-

hibitory actiyity against linoleic acid peroxidation and 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging activity compared with o-methanol ex-

tract. When these extracts were separated into ethanol-soluble(ES) and etha-

nol-precipitable fractions, the dominant antioxidant and radical-scavenging

activities were detected in theES firactions, which suggests that these antioxi-dant and radical-scavenging activi'ties are responsible fbr water-soluble and

low-molecular-weight substances. As possible active principles for antioxi-

dant activities in the water extracts, the contents of ascorbic acid and citric

acid in these extracts were measured, and the antioxidant and radical-scay-

enging activities of these substances were assayed at various concentrationg.

The experimental results indicate that the antioxidant activity against lipid

peroxidation in the water extracts is dominantiy associated with citric acid,

and the DPPH radical-scayenging activity of the water extracts is majorly re-

sponsible for ascorbic acid, suggsting a compensatory action of ascorbic acid

and citric acid in expression of the antioxidant and radical-scavenging activi-

ties of Chenpi.

Kby wotzty:Chenpi, antioxidant arid radical-scavenging activities, ascorbic acid,

acid.

citric

(Received 22 June 2009, accepted14 October 2009)

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312 K HIGASHI-OKAI et al

Introduction

'Free

radi'cal-mediated damage to body cells is associated with the causation and progres-

sion of chronic diseases such as atherosclerosis, diabetes and cancer [1,2]. On the other

hand, various vegetables and fruits have been considered as preventive factors against these

chronic djseases, and antioxidant components in these fbods are important because they have

antioxidant and radical-scayenging activities against the free radicai attack on proteins, nu-

cleic acids and lipids in body cells [3,4]. Among these edible plants, citms fruits have been traditional]y used as healthy fbods.

Pliny, an acient Roman naturalist, first used the word `Citms'

which labeled the fruit a medi-

cine. Among citrus fruits, the dried peels of citms fruits, called Chenpi, hewe been tradition-

ally used as a medicine fbr improvement of bronchial and ashmatic conditions or cardiac and

blood circulation in China and Japan [5]. However, the details of the antioxidant activity of citrus peels has not been ana].yzed except

in several studies. For example, the methanol extracts of citrus peels including several phe-nolic substances showed relatively weaker antioxidant activity than the methanol extracts of

citrus seeds [6]. A flavanone glycoside, hesperdin, is contained abundantly in citrus peels[7] and is considered as one of･main antioxidant substances in Chenpi. Furthermore, other

researchers studied the contents of flavonoids and phenolic compounds and antioxidant activ-

ity in hot water extracts of citrus peels [8,9]. We then compared the effects of the extracts

treated with various solvents which were prepared from fresh and dried peels of Satsuma

mandEuin (Citrus unshiu Marcov. ) on lipid peroxidation, and found that the hot water extract

of dried peels showed the strongest antioxidant activity among various extracts [10]. In this

study, the content of ascorbic acid in the hot water extract of Chenpi could not explain its re-

marlcal)le antioxidant activity, which suggested the existence of other unknown factors re-

sponsible fbr the strong antioxidant activity.

In our present study, cold- and hot-water extracts of Chenpi showed very strong antioxi-

dant and radical-scavengjng acivities, and we elucidated the compensatory and cooperative

actions of ascorbic acid and citritic acid as acti've principles in these extracts.



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Antiexidant and Radical-Scavenging Activity of Chenpi 3B

Materials and Methods

Chemical reagents

Potassium iodide, aluminium chloride, soluble starch, 2,6-dichlorophenol-indophenol

(DCIP), ethylenediamine tetraacetic acid (EDTA), metaphosphoric acid and 1,1-diphenyl-2-

picrylhydrazyl (DPPH) were purchased from Sigma-A]drich Japan Co. (Tokyo, Japan). o-

phenanthroline and linoleic acid were purchased from Kanto Chemical Co. (Tokyo, Japan).

Ascerbic acid and citric acid were puchased from Wako Pure Chemical Co. (Osaka, Japan).

Potassium iodide solution was prepared by 2 g of KI in 100 ml of ethanol, and aluminium

chloride solution was prepared from 2 g of AIC13 and 20 pg of o-phenanthroline jn 100 ml of

ethanol. A starch solution contained 1 g of soluble starch and 20 g of NaCl in 10 ml of dis-

tilled water. Other chemical reagents used in the present study were purchased from Sigma-

Aldorich Japan, except fbr specific reagents which are described in the text.

Preparation ofthe extracts from thepeels ofSatsuma nzandarin (Cheupi) The peels were separated from the fresh Satsuma mandarin (Citrus "nshiu Marcov. ) sup-

plied from the Shizuoka branch of the Japanese Agriculture AssQeiatiQn (Shizuoka, Japan).

The fresh pee]s were dried in a silica gel desicator at 4℃ , and the weight of dried peels ba-

came approximately 25% of the total weight of the fresh peels. The dried peels were

minced extensively by an electric mi11, combined, mixed with 10 volumes of cold water (4℃),hot water (90℃) or methanol and stood for 3 days at 4℃. The extract was centrifuged at

1500 × g for 15 min and the supernatant was used as the test sample

Separation qf ethanol-soluhle (EIS) and ethanolTprecipitabie (EP) .fractions from Cheupi ex-tracts

After each extract (10 ml) of Chenpi was combined and mixed with three volumes of cold

ethanol in the presence of 30 mM NaCl, it was kept in a deep freezer fbr 3 hr at -40 ℃. [[he

mixture was centrifuged at 1500 × g for 15 min and the supematant was recovered and con-

centrated with a rotary evaporator at 4℃ as ES fraction (10 ml). The precipitate was dis-

solved in 10 ml of cold distilled water or methanol as EP fraction. The ES and EP fractions

were stored in a deep freezer at -40 ℃.

Measurement ofascorbic acid content

Ascorbic acid content was measured by a modification of the previous method [11]. One

ml of the test samples incguding ascorbic acid was mixed with 1 ml of ice-cold 10% meta-

phosphQric acid and 2 mM EDTA for deproteinization and stabilization of ascorbic acid.

After 600 pl of 50 mM citratelacetate buffer (pH 3.5) was added to the mixture, 300 pl of

DCIP solution (O.1 mglml) was added and mixed. One min later, the absorbance of the mix-

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314 K HIGAsm-OKAI et at

ture was measued at 520 nm. Standard ascorbic acids were dissolved in 5% metaphosphoric

acid and the standard curve of ascorbic acid was made by using the assay method described

above.

Measurement ofcitric acid content

The citric acid content was measured by a modfication of the previous method [12]. O.1

M NaOH solution in water (WfV) was exactly prepared by the quantitation with O.05 M ox-

alic acid. 20 pl of 1% phenolphthalein solution in ethanol (WtrV) was added and mixed in

10 ml Qf Chenpi extract and then 10 ptl of NaOH solution was added and mixed repeatedly

until the pinkish.color of the Chenpi extract disappeared. The citric acid content in the

Chenpi extract (mgltnl) was calculated by the following equation : O.64 × (VflOOe) 1 (10.02+ VllOOO) ( V : Volume of O.1 M NaOH solution (pl))

Assay for the antioxidunt activity against lipid peroxidation

Hydroperoxide generation from oxidized linoleic acid was assayed by the modified method

of Asakawa and Matsushita [13]. One ml of each extract or test solution was mixed with 1

ml of linoleic acid solution and 2 ml of 50 mM phosphate buffer (pH 7,5) in a glass test tube

and stood for two weeks at 33℃. As a negative control experiment, l ml of each solvent

was used in place of 1 ml of the extract or test solution. The test solution was prepared, in-

cluding oxidized linoleic acid solution (200 pl), 250 pt of KI solution, 250 pl of aluminium

chloride solution and1 ml of hexane were added to a test tube, mixed and incubated for 5

min at 37℃ under dark condition. Starch solution (O.25 ml) and 7.5 ml of 10 mM HCI were

added, mixed and shaken vigorously. The solution was centrifuged at 1,500 × g for 5 min,

and the absorbance of the lower layer was measured at 560 nm using a Shimadzu UN-1600

spectrophotometer.

Assay for radical-scavenging activity

The radical-scavenging activity was measured using 1,1-diphenyl-2-picrylhydrazyl

(DPPH) a ¢ cording to the modified method of Brand-Williams [14]. An aliquat of water or

methanol (200 p]) containing different test substances was mixed with 3,8 ml of O.1 mM

DPPH in methanol prepared daily. The extract or test solution was diluted successiyely with

water or methanol, and a control experiment was perfbrmed with water or methanol alone and

compared with that of the test solutions. The decrease of absorbance at 520 nm was meas-

ured at 1 5 min intervals fbr 3 hrs using a Shimadzu UV-265 spectrophotometer.

Statisticat analysis

The experimental result was expressed as the mean and standard error of triplicate assays.

Relative activity(%) was expressed by the fbllowing equation : (the mean value of sample-

treated experimentl the mean value of the control experirnent) × 1OO%. The statistical com-




Antioxidant and Radical-Scavenging Activi'Ly of Chenpi 31S

parison between

Student' s t test.thePcontrol

2vid sarnple-treated experimental groups was canied out using

value less than O.05 was considered to be significantly different.

Results

Antioxiclant activity of Cherrpi extracts against lipid peroxidation

[lhe dned peels of Satsuma mandarin (Chenpi) were milled, and their extracts were pre-

pared by cold water, hot water and methanel, and tlie effects of these extracts on the hydro-

peroxide generation from oxidized linoleic acid were compared. As shown in Fig. 1, strong

antioxidant activities were observed in cold and hot water extracts, which showed 72 and

77.1% inhibition against hydroperoxide generation, respectively (P<O.OOI), The methanol

extract exhibited a relatively weak antioxidant activity (30.196 mhibition, P<O.05). Then,

these extracts were treated with ethanol and separated into ethanol-soluble (ES) and ethano]-

precipitable (EP) fractions and the antioxidant activities of both fractions were assayed, As

indicated in Fig. 2, the ES fractions of cold and hot water extracts showed relatively strong

activities (51,4 and 58.I% inhibition, P<O.OOI) compared with that of methanol extract (20%inhibition, .P<O.025). However, the EP fractions of all extracts did not exhibit any signifi-

cant antioxidant activities. This result indicates that the major antioxidant activtties against

lipid peroxidation in Chenpi are associated with water- and ethanol-soluble low-molecular-

weight substances.

As possible active substances responsible fbr the antioxidant activity against lipid peroxi-dation jn the water extracts of Chenpi, we assumed ascorbic acid and citric acid, acording to

previous studies [3,4]. Then we measured the contents of these substances in cold- and hot-

ge loe-l;l-z-

g-op'･R 509&9vx=

o

Fig. 1.

****

*

Control CoidWater HotWater Methanol

Antioxidant etTect of Chenpi extracts on hydrQperoxide genration in

lipid peroxidation. The column and bar represent the mean and

SD of triplicate assays. The statisticai comparison between the

contrel and sample-treated experiment was carried out using Stu-dent'st test. P values of the extract-treated experiments are

shown as follows. SD : standard deviation, *: P <O.05, **: P <

O.OOI,



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gE loo1-egz･8.

soE"m.8.=o

Fig. 2.

ER 10o1･2geo.Po-rs

50g:.fi

*

li'c***

Centrol ColdWaterHotWater Methanol ColdWaterHotWater Methaiiol

･ES fraction EP fraction

Antioxidant effect of ES and EP fractions of Chenpi extracts on hy-

droperoxide genration in Hpid peroxidatien. The column and barrepresent the mean and SD of triplicate assays. The statistical

cemparison between the control and sarnple-treated experiment was

carried out using Student'st test. P values of the extract-treated

experiments are shown as fbllews. ES : ethanol-solubje, EP :

ethanol-precipitable, SD : standard deviation, *: P<O.025, **: P<

O,OOI.

o Coptrol 50 100 250 (ggim1) Ascorbic acid

Fig. 3. Antioxidant effect of ascorbic acid on hydroperoxide genration in

lipid peroxidation. The column and bar represent the mean and

SD of triplicate assays. The statistical comparison between the

control and sample-treated experiment was carried out using Stu-

dent'st test. P values of the extract-treated experiments are

shown as follows. SD : standard deviation, *: P <O.05.

water extracts. The ascorbic acid contents in cold- and hot-water extracts were estimated to

be 250 and 220 pgim1, respectively. As shown in Fig. 3, the ascorbic acid concentrations of

50-250 pglml caused apparent weak antioxidant effects without a dose-dependency (12.7-19.996 inhibition, P<O.05). [I;his result indicates that the antioxidant activity of ascorbic

acid itself is too insufficient to explain the strong antioxidant activity of water extracts of

Chenpi, as shown in Fig. 1,




Antiexidmit and Radical-Scavenging Activity of Chenpi 317

ge 100u･8gg8'ft

509gEVh:

Fig

o Control 250 500 10oo ("gfml) Citricacid

. 4. Antioxidant effect ofcitric acid on hydroperoxide genration in lipid

peroxidation. The columm and bar represent the mean and SD of

triplicate assays. The statistical comparison between the control

and sample-treated experirnent was canied out using Student' s t test,

P values of the extract-treated experiments are shown as follows,

SD:standard deviation, *: P <O,05, **: P <O.OOI.

ge 100Uvo･g:puAE

sogts

****

*

o Control Cold Water Hot Water Methanol

Fig. 5. Scavenging activity of Chenpi extracs against DPPH radical. [[1ie

colurrm and bar represent the mean and SD of triplicate assays.

The statistical comparison between the control and sample-treuted

experirnent was carried out using Student' s t test. P values of the

extract-treated experirnents are shown as fbllows. DPPH : 1, 1-

diphenyl-2-picrylhydrazyl, SD : standard deviation, *: P <O.OOI, *

*: P <O.OOOI.

Next, the ciuic acid contents in the cold and hot water extracts were estimated to be 1.0 and

1.3 mgim1, respectively, As shown in Fig. 4, when different concentrations of citric acid

(250- 1000 pg/ml) were added to the lipid peroxidation system, they showed dose-dependent

inhibitory activities. Especially, the high eoncentration of citric acid (l mglml) equivalent

to that of cold water extract showed about 80% inhibition against lipid peroxidation (P <O,OOI), which was roughly equal to the activities in the cold and hot water extracts of Chenpi.



The UOEHAssociation of HealthSciences

318 K HIGAsm-OKAI el at

These results suggest that the dominant antioxidant activity against lipid peroxidation in the

water extracts of Chenpi is associated with citric acid, and ascorbic acid plays a minor role in

expressing antioxidant activity in water extracts.

DPPH radicat-scavenging activity qf Cheupi extracts

Next, we analyzed radical-scavenging activities of cold and hot water and methanol ex-

tracts of Chenpi. As shown in Fig. 5, the cold and hot water extracts showed remarkable

radical-scavenging activities (98.3 and 95.3% inhibition, P<O.OOOI), and methanol extract

also expressed a relatively strong activity (about 7096 inhibition, P<O.OOI). These extracts

were then sepai:ated into ES and EP fractions by ethanol treatment. As indicated in Fig. 6,

the dominant radical-sacvenging activities of these extracts were observed in the ES fractions

of ali the extracts, and the ES fractions of the water extracts exhibited much stronger activity

than that of the methanol extract. Howeyer, the radical-scavenging activities in the EP frac-

tions of all the extracts were relatively low (Fig. 6). This result suggests that the major radi-

cal-scavenging activities in Chenpi are associated with water- and ethanol-soluble and low-

molecular-weight substances. We also assumed ascorbic acid and citric acid as possible ac-

tive principles responsible for DPPH radicai-scavenging avtivities in the water extracts. As

shown in Fig. 7, ascorbic acid showed strong radical-scavenging activities at IOO, 250 and

500 pgifml in a dose-dependent fashion (82.2, 95.3 and 98.3% inhibition). Especially, the

concentration equivalent to that in the cold water extract (250 pglml) showed a remarkable

radical-scavenging activity. However, citric acid exhibited relatively weak radical-scaveng-

ing acti.vities at 100-1000 pglml <17.8-22% inhibition) and no dose-dqpendency was ob-

served (Fig. 8). These results suggest that DPPH radical-scavenging activity in the water

g loo1.e#ett,y

50g-=mbQ

o

Fig. 6.

fi **

**

****

Control ColdWaterHetWater Methanol Co]dWaterHotWater Methanol

ES fraction EP fraetion

Scavenging uctivity of ES and EP fractions of Chenpi extracs

against DPPH radical. The column and bar represent the mean and

SD of triplicate assays, The statis'tical comparison between thecontrol and sample-treated experiment was carried out using Stu-dent'st test. P values of the extract-treated experiments are

shown as follows. ES : ethunol-soLuble, EP : ethanot-precipitable,

DPPH : 1, 1-diphenyl-2-picrylhydrazyl, *: P <O,05, **: P <O.OOI.




Antioxidant and Radical-Scavenging Activity of Chenpi 319

ge 100vv

o.g

esE

£

gE so

za

Fig. 7.

*

**

*** ***

o Control 50 100 250 500 (,org!ml) Ascorbic acid

Scavenging activity of ascorbic acid against DPPH radi'cal, The column

and bar represent the mean and SD of triplicate assays. The statistical

comparisop between the control and sample-treated experiment was carried

out usi"g Student's t test. P values of the extract-treated expeTiments are

shown as follows. DPPH : 1, 1-diphenyl-2-picrylhydrazyl, SD : standard

deviation, *: P <O.Ol, **: P <O.OOI, ***: P <O.OOOI.

3t ioo8,:EpuAE

soE8

Fig. 8.

o (-) loo 2so soo looo(xig/ml)

Citric acid

Scavenging activity of citric acid against DPPH radical. The column and

bar represent the mean and SD of triplicate assays. The statistical com-

parison between the control and sarnple-treated experiment was carried out

using Student'st test. P values of the extract-treated experirnents are

shown as fbllows, DPPH : 1, 1-di'phenyl-2-picrylhydrazyl, SD : standard

deviation, *: P <O.05.

* * x tst

extracts of Chenpi is majorly responsible fbr ascorbi'c acid and a minor radical-scavenging ac-

tivity is derived from citric acid,

Discussion

A partof our presentstudy confirms our previous result that the waterextractsof dried





peels of Satsuma mandarin (Chenpi) show much stronger antioxidant activities than the

methanol extract against lipid peroxidation [10]. We then focussed on the antioxidant and

radical-scavenging activities of cold and hot water extracts of Chenpi and fUrther analyzed

the active principles in the extracts responsible fbr these activities. [Ihe dominant activities

of these extracts were observed in ES fractions prepared by ethanol treatment (Fig. 2 and 6),

which indicates that the antioxidant and radical-scavenging activities of Chenpi extracts are

associated with water-soluble and low-molecular-weight substances. We assumed ascorbic

acid and citric acid as possible principtes responsible fbr the antioxidant and radical-scaveng-

ing activities of Chenpi extracts. Interesting]y, both substances showed qualitatively differ-

ent antioxidant and radical-scavenging activities.

Citric acid sbowed the strong antioxidant activity against lipid peroxidation (Fig, 4), but

exhibited very weak radical-scavenging activity against DPPH (Fig. 7). As a possible

mechanism for the antioxidant activity of citric acid against lipid peroxidation, we considered

a chelating abjlity of ciuic acid for transition metal ions such as ferrous and copper ions.

Namely, lipid peroxidation is acceralated by metal ion-dependent reactien, which promoteshydroperoxide generation [15]. Although the radical-scavenging activity of citric acid itself

is not so strong (Fig. 7), its chelating activity might cause the strong suppression against hy-

droperoxide generation in lipid peroxidation (Fig, 4).

In contrast, ascorbic acid exhibited a strong scavenging activity against DPPH radical (Fig.6), but showed very weak Emtioxidant activities against lipid peroxidation (Fig. 3). This re-

sult can be explained by previous studies by other researchers. The.reductive form of ascor-

bic acid has a strong radical-scavenging activity, but reacts with a trace amount of ferrous or

copper ion and ¢ onverts to its oxidized fOrm. During these reactions, highly reactive radj-

cals such as superoxide anion, hydrogen peroxide and hydroxyl radical are generated [16,17].Possibly, this property of ascrobic acid induces a prooxidant effect on lipid peroxidation.On the other hand, a chelating activity of citric acid rnight cause a protective effect against

lipid peroxidation induced by meta1 ion and ascorbic acid. To confirm the above mentioned

notion, we canied out the fbl]owing experiment. As shown in Fig. 9, the combined supple-

ment with 200 pgim1 ascrobic acid and 2 pM FeC13 showed a prooxidarit effect on lipid per-oxidation compared with the negative control experiment. Then the addition of citric acid

(250-1000 pg/ml) caused a dose-dependent inhibition against lipid peroxidation induced by

ferrous ion and ascorbic acid. This result indicates a posisibility that the strong antioxidant

activities of the cold- and hot-water extracts of Chenpi might be responsible fbr the coopera-

tive action of ascorbic acid and citric acid /in the extracts.

Furthermore, in our present study, the methanol extract of Chenpi also showed significant

antioxidant and radical-scavenging activities in spite of its relatively weak activities com-

pared with the water extracts (Fig. 1 and 5 ). As possible antioxidants in the methanol ex-

tract of Chenpi, the fiavonoid group including flavones, flavanones and phenolic acids can be

considered according to previous studies [6-9].




Antioxidant and Radical-Scavenging Activity of Chenpi 321

***

ge-ll･g-

loogll).a2

50&e8

ts:

Fig. 9.

T

o Control Ascorbicacid 250 500 1000("g!ml) + FeCli

Citric acid

Effect of citric acid against lipid peroxidation induced by ascorbic acid and

ferrous ion. [[he column and bar represent the mean and SD o/f triplicate

assays. As a positive control expertment, 200 "gim1 ascrobic acid and 2

pM FeCli were added to the lipid oxidation system. The statistical com-

parison between the negative and positive control experiments was carried

out using Student'st test. (*: P<O,Ol). Furthermore, different concen-

trations (250, 500 and 1OOO ptgim1) of citric acid were applied to the lipid

oxidation system induced by ascorbic acid and ferrous ion. The statistical

comparison between the positive control and citric acid-treated experiments

wus canied out using Student' s t test. P values are shown as fo11ows,

**: P<O.OOI, ***: P<O.OO05,

Although a detailed pharmacolagical analysis of flavonoids in Chenpi has not been per-formed, some flavonoids might be responsible fbr i'ts chemopreventive efiiect on chronic dis-

eases. For example, citrus-derived flavonoids such as tangeretin and nobiletin caused hy-

polipidemic effects on diet-induced hypercholesterolemia in hamsters [1 8], which may be as-

sociated with their antioxidant or radical-scavenging activities.

As another interesting problem, the consumption of cirms peels, but not the consumption

of citrus juices, is associated with a reduced risk of human skin cancer [19]. Generally, the

antioxidant substances in citrus peel extracts are more abundant than those in citms juices.For example, ascorbic acid, flavonoids and phenolic acids in citruis peels showed higher con-

centrations than those of the citrus juices [7, 20-21].

Possibly, abundant amounts of ascorbic acid, citric acid, fiavonoids and other antioxidant

substances in citrus peels seem to show additive or synergestic effects on expressing the anti-

oxidant or radical-scavenging activities, possibly associated with the preventive effects an

various chronic diseases such as cardiovascular diseases and cancer.





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Antioxidant and Radical-Scuvenging Activity of Chenpi 323

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Agric 57 : 417-426



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324 KHIGASHI −OKAI 　 et 　at

陳皮の抗酸化・ラジカル消去活性におけるアスコルビン酸とクエン酸の補完・協同

作用について

岡井（東）　紀代香1 ・

石河　暁子1 ・安友　小百合

’ ・岡井　康二

2

1金城学院大学・生活環境学部・食環境栄養学科

2大阪薫英女子短期大学・生活科学科・食物栄養専攻

要旨：　中国や口本では古来より乾燥した温州みかんの皮を「陳皮」と称して循環器系疾患や

　　呼吸器系疾患の予防や改善の目的で漢方薬・生薬・薬膳料理などに利用してきた．

　　本研究ではこれらの陳皮の薬理効果の作用メカニズムのひとつとして抗酸化・ラジ

　　　カル消去活性に注目して分析した結果，陳皮の冷水・熱水抽出液にきわめて強い抗

　　酸化・ラジカル消去活性が認められた．そこで水抽出液をエタノー

ル可溶性画分と

　　沈澱画分に分けたところ，主要な活性はエタノール可溶性画分に認められた．そこ

　　　でその活性本体に対応する水溶性・低分子性物質としてアスコルビン酸とクエン酸

　　　を仮定してそれらの陳皮水抽出液中の濃度を測定し，その濃度に対応する両者の抗

　　酸化・ラジカル消去活性を分析したところアスコルビン酸は強い 1 ，1 −diphenyl−2 −

　　 picrylhydrazyl（DPPH ）ラジカル消去活性を示したが，リノール酸の過酸化脂質の生成

　　　に対する作用は弱かった．ところが，クエン酸は逆に DPPH ラジカル消去活性は弱

　　　かったが，リノール酸の過酸化脂質の生成に対して強い抑制作用を示した．さらに

　　　クエン酸は微量金属とアスコルビン酸によるリノー

ル酸の過酸化脂質の生成の促進

　　　を消去・抑制することを見出した．以上の実験結果により陳皮の水抽出液の示す強

　　　い抗酸化・ラジカル消去活性はアスコルビン酸とクエン酸がお互いにそれぞれの作

　　用を補完するとともに一

方の物質の酸化促進作用を他の物質が消去・抑制するため

　　　であることが示唆された．

キー

ワード：　陳皮，抗酸化，ラジカル消去，アスコルビン酸，クエン酸．

JUOEH （産業医大誌）31 （4）：3U − 324 （2009）

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potent antioxidant radical-scavenging activity chenpi

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