Toxicol. Res.

Val. 25, No. 4, pp. 243-251 (2009) . , . Official Journal of

~ Korean Society of Toxicology
Available online at http://www.toxmut.or.kr

Evaluation of Antioxidative Activity of Agrimonia pi/osa-Ledeb Leaves

on Non-lipid Oxidative Damage
Dae-Sik Hah \ Chung-Hui Kim 2 , Euikyung Kim 3 and Jong-Shu Kim 3
IGyeongnam Livestock Promotion Institute Middle-branch, Changwon 541-703
20epartment of Animal Science and Biotechnology, Jinju National University, Jinju 660-758
3College of Veterinary Medicine, Gyeongsang National University (Institute of Animal Science), Jinju 660-701, Korea

(Received June 16, 2009; Revised July 30, 2009; Accepted August 5, 2009)

Present study was conducted to evaluate the antioxidative activity of the Agrimonia pilosa-Ledeb
leaves on non-lipid oxidative damage. The antioxidative activity of methanolic (MeOH) extract of the
Agrimonia pilosa-Ledeb leaves on non-lipid oxidation, inciuding liposome oxidation, deoxyribose oxi-
dation, protein oxidation, chelating activity against metal ions, scavenging activity against hydrogen
peroxide, scavenging activity against hydroxyl radical and 2'-deoxyguanosine (2'-dG) oxidation were
investigated. The MeOH extract of the Agrimonia pilosa-Ledeb leaves exhibited high antioxidative
activity in the liposome model system. Deoxyribose peroxidation was inhibited by the MeOH extract
of the Agrimonia pilosa-Ledeb leaves and MeOH extract of the Agrimonia pilosa-Ledeb leaves pro-
vided remarkable protection against damage to deoxyribose. Protective effect of MeOH extracts of
the Agrimonia pilosa-Ledeb leaves on protein damage was observed at 600 ~lg level (82.05%). The
MeOH extracts of the Agrimonia pilosa-Ledeb leaves at 300 ~g revealed metal binding ability
(32.64%) for hydrogen peroxide. Furthermore, the oxidation of 2'-deoxyguanosine (2'-dG) to 8-
hydroxy-2-deoxyguanosine (8-0H-2'dG) was inhibited by MeOH extracts of the Agrimonia pilosa-
Ledeb leaves and scavenging activity for hydroxyl radical exhibited a remarkable effecL From the
results in the present study on biological model systems, we concluded that MeOH extract of the
Agrimonia pilosa-Ledeb leaves was effective in the protection of non-lipids against various oxidative
model systems.
Key words: Antioxidative activity, Agrimonia pilosa leaves, 2'-Deoxyguanosine (2'-dG), Non-lipid oxi-
dative model systems

INTRODUCTION exogenous source of ROS are also produced by expo-

sure to various environmental factors, for instance, UV
Free radicals such as reactive oxygen speeies (ROS) light, ionizing radiations or tobacco (Hanato, 1995;
are formed during a variety biological and cellular func- Lander, 1997; Freidovich, 1999; Salles et al. , 1999;
tions in living organisms. All cells having aerobic respi- MeCord, 2000; Fang et al., 2002). Antioxidant defenses
ration, using oxygen as a nutrient, are continuously have evolved to protect biological systems against reac-
exposed to a number of exogenous and endogenous tive oxygen speeies, and a sophisticated, co-operative
ROS or radicals that cause lipid, protein and DNA dam- array of antioxidant defense mechanisms is found in
age (Salles et al., 1999; Droge, 2002). The endoge- biological systems. Antioxidant defenses system pre-
nous source of ROS are formed from the mechanism of vent generation and counteract the damaging effects of
oxidative phosphorylation in which oxygen is reduced to reactive speeies produced within the organism from
water through a four step addition of electrons. The molecular oxygen (Kimie et al., 1988; Masaki et al.,
1995; Nobuyki et al., 1996; Benzie, 2000). But an imbal-
Correspondence to: Jong-Shu Kim, College of Veterinary ance between the production of various reactive spe-
Medicine, Gyeongsng National University (Institute of Animal
Science), Jinju 660-701, Korea eies and the ability of the organisms natural protective
E-mail: [email protected] mechanism to cope with these reactive compounds was
Abbreviations: MeOH, methanol; 2'dG, 2'-deoxyguanosine; 8- occurred, this condition was called oxidative stress. Oxi-
OH-2'dG, 8-hydroxy-2-deoxyguanosine. dative stress causes a net stress on normal body func-

243
244 0.-8. Hah et al.

tions and may result in many specific diseases. It also

appears to contribute to the general decline in optimum
body functions commonly known as ageing (Sohai and
Weindruch, 1996; Wen et a/., 1999; Yun et al., 2002;
Sala et a/., 2003). Oxidative stress meditated many
chronic fatigue disease such as Alzheimer's disease,
autoimmune, diabetes, and cancer et a/. (Floyd, 1990; OH
q 1) qlU'J"t. (2)
Halliwell, 1991; Anne and Andrew, 2000; Selvendiran et
a/., 2003; Valko et a/., 2007). From ancient, traditional
medicinal plants have been known to posse antioxida-
tive activity (Pratt and Watts., 1964; Fukuzawa and
Takaishi, 1990; Inatain et a/., 1996). For several years,
there are current interested in the development of thera-
peutic and chemopreventive antioxidative agents which
have non-cytotoxicity. Many researchers suggest that
antioxidants, in particular plants diet-derived antioxi-
dants, might have health benefits as prophylactic agents
(Aruoma et a/., 1999).
The Agrimonia pilosa-Ledeb. Rosaceae is traditional
Fig. 1. Chemical structure of Agrimonia pilosa leaves.
medicinal plant, which have already been known to
have diverse properties such as antioxidative activity
including antibacterial, anti-inflammatory, antipyretic, Research Institute of Traditional Medicine Plants of
haemostatic, vasoconstrictor, analgesic and anti-tumor Gyeongnam (Hamyang, Gyeongnam, Korea) and was
(Sala et al., 2003). identified by the head of Research Institute. Voucher
Although, the protective or remedy mechanism of specimens were deposited in the Research Institute
Agrimonia pilosa-Ledeb have not been fully elucidated, and Its chemical structure shown in Fig. 1.
It has been commonly used in Korea and other Asian Agrimonia pi/osa leaves were air dried under shade
countries such as China, India , Japan as a natural fla- and cut into small pieces and stored at 4°C until use.
voring agent for prevent, or treatment of various Agrimonia pilosa leaves 300 9 were extracted with
chronic-fatigue syndrome diseases (Su et al., 1984; 80% 900 ml methanol in a shaking incubator at 80°C
Isao et a/., 1988; Pei et a/., 1990; Cha et a/., 1997; Sala for 12 hr. The residue was re-extracted under the same
et al., 2003). Therefore, considerable attention has been condition 3 times. The extracts obtained were com-
focused on quenching free radical oxidation from the bined and filtered. The combined methanol specimens
natural medicinal plants and synthetic phenolic antioxi- were evaporated to dryness using a vacuum rotary
dants such as butylated hydroxytoluene (BHT), buty- evaporator and weighted (98.88 9 dry base) to deter-
lated hydroxyanisole (BHA) are commonly added to mine the yield of soluble constituents. The extract
foods to inhibit free radical damage to lipid; however, obtained was subject to evaluate the antioxidative activ-
BHA, BHT are suspected as possible carcinogens ity on non-lipid oxidative damage.
(Branen, 1975; Duh et a/., 1999). Therefore, It seems to
be need to find out the nature anti-oxidants without side Determination of the antioxidative activity on lipo-
effect from natural medicinal plants. Also, where or not some peroxidation. 580 mg Lecithin (ICN, USA) con-
occurred oxidative stress on non-lipid and/or be able to taining 58 ml, 10 mM phosphate buffer (pH 7.4) was
protect oxidative damage against free radicals using ultrasonicated in ultrasonic cleaner (Branson B-221
Agrimonia pilosa-Ledeb are not under clean. Thus, the Smithkline Company, USA) for 2 hours. The sonicated
objectives of present study were conducted to evaluate solution (10 mg lecithin/mi), Fecl 3 , ascorbic acid and
the antioxidative activity of Agrimonia pilosa-Ledeb MeOH extract (50-1200 fl9) were mixed to produced a
leaves methanolic extract on non-lipid oxidative damage. final concentration of 3.12 mg lecithin/mi, 125 flM FeCI3 ,
and 125 flM ascorbic acid. The mixture was incubated
MATERIALS AND METHODS for 1 hr at 37°C by the thiobarbituric acid (TBA) method
(Tamura and Shibamoto, 1991). The absorbance of the
Plants materials and extracts preparation. The mixture was read at 532 nm against a blank, which con-
Agrimonia pilosa leaves were obtained from the tained all reagents except lecithin. The extinction coeffi-
 Evaluation of Antioxidative Activity of Agrimonia pilosa-Ledeb Leaves on Non-lipid Oxidative Damage 245

cient of TBA-malonaldehyde product of 1.56 x 105 M"1 bance of hydrogen peroxide at 230 nm was deter-
cm- 1 was used to convert absorbance values into con- mined 10 min later in a spectrophotometer against a
centrations of secondary reaction products. blank solution containing MeOH extract in PBS without
hydrogen peroxjde. Hydrogen peroxide concentration
Determination of the antioxidative activity on pro- was determined spectrophotometrically at 230 nm using
tein carbonyl. The reaction mixture (1.2 ml), contain- a molar extraction coefficient for hydrogen peroxide of
ing MeOH mixture (50-1200 fl9), phosphate buffer (20 81 M-1 cm- 1 .
mM, pH 7.4), bovine serum albumin (20 mg/mi), FeCI3
(100 flM), H20 2 (2.0 mM), ascorbic acid (200 flM), was Assay of the antioxidative activity on hydroxyl
incubated for 1 hr at 37°C, and 1 ml 20 mM in 2 M HCI radicals. Scavenging effect of MeOH extract on
was added to the reaction mixture and centrifuged at hydroxyl radicals was assayed by the Shi et a/. (1991)
3000 9 for 10min. The protein was washed three times method. Briefly, The reaction solution containing 3 ml
with 2 ml ethanol-ethyl acetate (1: 1) and dissolved in sodium phosphate buffer (0.15 mM, KH 2 P04-K2 HP04 ,
2 ml 6 M guanidine-HCI (pH 6.5). The absorbance of pH 7.4) was initiated by the addition of 100 flM vitamin
the sam pie was read at 370 nm (Lenz et a/., 1989). All C, 100 flM CuS04 , 12 flM cytochrosome C. After initia-
analyses were run in three replicates and averaged. tion, the MeOH extract (50-1200 fl9) was added to
reaction mixture and incubated at 25°C for 90 min. The
Measurement of the effect on oxidation of deox- absorbance of the color changed in cytochrome C was
yribose. The reaction mixture (3.5 ml), wh ich contained read at 550 nm. Thiourea served as the positive con-
MeOH mixture (50-1200 fl9), deoxyribose (6 mM), H2 0 2 trol, Inhibition rate (%) of hydroxyl radical was calcu-
(3 mM), KH 2 P04-K2 HP04 buffer (20 mM, pH 7.4), FeCI3 lated as following formula.
(400 flM), ethyenediaminetetraacetic acid (EOTA; 400
flM) and ascorbic acid (400 flM) was incubated at 37°C Inhibition rate (%) = T - T2 x 100
T - T1
for 1 hr. The extent of deoxyribose degradation was
tested by the TBA method. 1 ml 1% TBA and 1 ml T: The transmittance of OH radical generation system,
2.8% trichloroacetic acid (TCA) were added to the mix- T1: The transmittance of control system
ture, which was then heated in a water bath at 90°C for T2: The transmittance of test sam pie system
20 min. The absorbance of the mixture was read spec-
trophotometerically at 532 nm (HaIliweIl et a/., 1987). All Assay of the antioxidative effect on 2'-deoxyua-
analyses were run in three replicates and averaged. nosine oxidation. This assay was determined accord-
ing to the Kasai and Nishimura method (1984). The
Measurement of chelating activity on metal ions. reaction mixture (1.4 ml), containing MeOH mixture (50-
The chelating activity of MeOH extract on Fe2+ was 1200 fl9), 2'-dG (0.5 mM), KH 2 P04-K2 HP04 buffer (0.1
measured according to the Carter method (1971). The M, pH 7.4), was initiated by the Fenton reaction model
MeOH extract (0.2 ml, 50-1200 fl9), was incubated with system, which contained H20 2 (50 mM), FeCI 3 6H 2 0 2
0.05 ml Fecl 2 4H 20 (2.0 mM). the reaction mixture was (1.3 nM), EOTA (6.5 mM) and ascorbic acid (15 mM).
initiated by the addition of 0.2 ml ferrozine (5.0 mM), All the mixture was incubated at 37°C for 30 min, and
and finally quantified to 0.8 ml with methanol. After the incubation was terminated by placing the mixture in an
mixture had reached equilibrium (10 min), the absor- ice-bath, and then filtering through a 0.45 flm filter
bance at 562 nm was read. EOTA served as the posi- before use. The filtrate was analyzed by HPLC (Water,
tive control, and an untreated sampie served as the USA), using the Water E2241 F (103 mm x 1 mm, 0.5
negative control. All analyses were run in three repli- flm) and UV detector (measured at 254 nm). The col-
cates and averaged. umn was equilibrated with 50 mM KH 2 P04 (pH 4.61)-
methanol (93.5:6.5, v/v) at a flow rate of 0.5 ml/min. 2'-
Determination of the antioxidative activity on dG and 8-0H-2'dG were identified by comparison of
hydrogen peroxides. Antioxidative activity of MeOH their retention times with those of known standards and
extract to scavenge hydrogen peroxides was measured determined by peak areas from the chromatograms. All
according to the Ruch et a/. (1989). The MeOH extract analyses were run in three replicates and averaged.
(50-1200 fl9) was added to hydrogen peroxide solution
(0.6 ml, 2 mM) preparing in phosphate buffer saline Statistical analysis. The results are expressed as
(PBS, pH 7.4, at 20°C). MeOH extract was decolorized mean ± standard deviation (S.O). Oifferences between
with cartridge (Sep-pack C18, Waters USA). Absor- groups were assessed by one-way analysis of variance
246 0.-8. Hah et al.

using the SAS software package for Windows. If in a

one-way analysis of variance test a significant F-value
of p < 0.05 was obtained, a Ounnett's multiple compari-
son test between the treated and control group was
..
]60
100

80
:;::: I ; --+-- A. P . l.
---- BHA
-
-+- TCP
BHT

conducted. Oifferences between two groups were statis- ""<10

tically evaluated by at-test.
'"~
20

RESULTS o 100 200 :m 400 500 600 700 800 000 100011001200
Amount 01 extract(pg/m I)
Determination of the antioxidative activity on lipo-
some oxidation. Phospholipids wh ich named as Fig. 2. Antioxidative effect of methanolic extract at different
derivatives of phosphatidic acid such as phosphatidyl- concentrations of the Agrimonia Pilosa leaf on protein oxida-
tive damage. AP.L.: Agrimonia Pilosa leaf, BHT: butylated
choline (lecithin) are the main components of cell mem-
hydroxytoluene, BHA: butylated hydroxyanisole, TCP: toco-
brane. To evaluate antioxidative activity of MeOH pherol.
extract in a liposome model system, The lecithin, pre-
pare as a liposome, was used. Antioxidative effect of
MeOH extract against the inhibitory activity of lipid per- and ascorbic acid was showed in Fig. 2. At 100-600 flg
oxidation in cell membranes were assessed by the amounts of MeOH extract used, the inhibition of protein
amount of malondialdehyde (MOA) produced. MeOH oxidation increased with increasing concentration of
extracts in the range of 100-600 flg showed 16.28- MeOH extract. As results to compare the MeOH extract
30.12% inhibition of peroxidation. The incidence of with BHA, BHT, and TCP currently used as commercial
observed inhibition was dependent on amount up to antioxidants, BHA, BHT, and TCP, which at 100-
600 fl9, but inhibition was decreased (29.14%) at 1200 fl9 showed 92.92-94.25%, 92.92-94.75%, and
1200 fl9. As results to compare the MeOH extract with 83.36-92.35% inhibition of protein carbonyl, respec-
butylated hydroxyanisol (BHA), butylated hydroxytolu- tively. Although, inhibition of protein carbonyl of MeOH
ene (BHT), and tocopherol (TCP) currently used as extract was exhibited higher values (71.7-82.05%), its
commercial antioxidants, BHA, BHT, and TCP, wh ich at activity was lower than those of BHA, BHT, and TCP.
600 flg showed 17.85%, 52.68% and 11.27% inhibition But no significant difference (p< 0.05) in the values of
of peroxidation, respectively. Obviously, BHT at 600 flg protein carbonyl formation was found between the sam-
showed a higher antioxidative activity than did 600 flg pie and comparative groups.
MeOH extract, However, BHA and TCP were lower
than that of MeOH extract (Fig. 1). Measurement of the effect on oxidation of
deoxyribose. The antioxidative activity of MeOH
Determination of the antioxidative activity on pro- extract on deoxyribose damage induced by Fe3+/ H20 2 ,
tein carbonyl. The effect of MeOH extract on protein measured by the thiobarbituric acid method, was not
carbonyl formation in albumin, induced by FeCI3 , H20 2 , amount-dependent. The extracts at the range of 100-
1200 fl9 showed 63.32,63.16,67.19, and 66.37% inhi-
60
--+-- A. P. L
50 ---- BHA
-+-- A. P. L
--'- BHT
- BHA
-+- TCP - - BHT
-+- TO'

10 20
10
o '--~~~~~~~~~~~~~-
100 200 :m 400 500 600 700 800 000 1000 11 00 1200
o 100 200 :n:J 400 500 600 700 600 000 1000 1100 1200
Amount 01 extract(pg/m Il Amount 01 extract(J.IO/m I)

Fig. 1. Antioxidative effect of methanolic extract at different Fig. 3. Antioxidative effect of methanolic extract at different
concentrations of the Agrimonia Pilosa leaves on liposome concentrations of the Agrimonia Pilosa leaf on deoxyribose
model system. AP.L.: Agrimonia Pilosa leaf, BHT: butylated oxidative damage. AP.L.: Agrimonia Pilosa leaf, BHT: buty-
hydroxytoluene, BHA: butylated hydroxyanisole, TCP: toco- lated hydroxytoluene, BHA: butylated hydroxyanisole, TCP:
pherol. tocopherol.
 Evaluation of Antioxidative Activity of Agrimonia pilosa-Ledeb Leaves on Non-lipid Oxidative Damage 247

45 1200 Ilg) on hydrogen peroxide was 5.45%, 24.39%,

35 --+- A P . L. 23.71%, and 24.23%, respectively. At all amounts of
MeOH used, the inhibition of hydrogen peroxides was
....... EDTA
more lower than those of BHA, BHT, and TCP. Espe-
cially, in the presence of 100 Ilg MeOH extract, almost
incomplete suppression of hydrogen peroxides occurred,
Whereas protection of hydrogen peroxides was 24.23%
when the concentration of MeOH extract was at 1200 Ilg,
-5 100 200 300 400 500 600 700 800 00) 100011001200
Am ount 01 ex\ra ctCotlm l)
indicating that the greater concentration (1200 Ilg) of
MeOH extract, the less protection on hydrogen perox-
Fig. 4. Chelating effect of methanolic extract at different ides (Fig. 5).
concentrations of the Agrimonia Pilosa leaf on ferrous ion.
A.P.L.: Agrimonia Pilosa leaf, EDTA: Ethylenediaminetet- Assay of the antioxidative activity on hydroxyl
raacetic acid.
radical. The scavenging effect of different amount of
MeOH extract (50, 100, 300, 600, and 1200 Ilg) on
bition of deoxyribose damage. Obviously, BHA and BHT hydroxyl radical was 75%, 80%, 86%, 90%, and 92%,
at 100-1200 Ilg showed a higher inhibition of deoxyribose respectively. MeOH extract markedly scavenged the
damage than did MeOH extract, but TCP was lower hydroxyl radical , and Its scavenging effect increased
than that of MeOH extract except at 1200 Ilg (Fig. 3). with increasing amounts of MeOH extract as dose-
dependent manner. Its scavenging effect was higher
Measurement of chelating activity on metal ions. than BHA, TCP. But BHT was similar to the observa-
The chelating effect of MeOH extract on ferrous ions is tion of MeOH extract (Fig. 6).
plotted in Fig. 4. As expected, the chelating effect
increased with increasing amount of MeOH extract. Assay of the antioxidative effects on 2'-deoxyua-
MeOH extract at 300 Ilg has the highest effect on Fe2+ nosine oxidation. The protective effect of MeOH
binding (32.64%), whereas at 1200 Ilg MeOH extract extract on 8-0H-2'-dG formation exhibited 55-59.7% at
showed a 32.35% chelating effect and 100 Ilg revealed range of 100-1200 Ilg. The incidence of 8-0H-2'-dG
the most lower chelating effect (16.63%). Although this formation was decreased with increasing amounts of
amount (100 Ilg) has more lower value than the other MeOH extract. When compared with the BHA, BHT,
amounts, it is relatively higher when compared with that and TCP, MeOH extract posses less an inhibitory effect
of EDTA (2%). It may be significant because it mini- on the oxidation of 2'-dG (Fig. 7). The result of Fig. 7
mizes the concentration of metal in the Fenton reaction. was supported by Fig. 8 as showing with decreasing 8-
OH-2'-dG formation than 2'-dG formation. The forma-
Determination of the antioxidative activity on tion of 8-0H-2'-dG in the presence of Fe3+/EDTAlH2 0 2
hydrogen peroxides. The scavenging effect of differ- was analyzed by HPLC (water, USA). The retention
ent concentration of MeOH extract (100, 300, 600, and time for 2'-dG and 8-0H-2'-dG were 4.517 and 5.358

100
60 - -BHA
- -A, p, L, ;<
-::;00 --- BHT
50

;
o
~40
---BHA
- -BHT
.e ....... TCP

~;
!i
~ 60 - -A. p, L
Cl)
- - TCP c

.
:::: 30 .~ 40

1°0 !/
~~~~~~~~~~--~~~
>
Jl 20

o ~--~----~----~----~--~
+50 OH + 100 OH +300 OH +600 OH + 1200
o 100 200 300 400 500 600 700 800 00) 1000 1100 1200 OH
Amount 01 exbacgt(I'Q/m Il OH buffer and Sam pie Concenlration(/lOlm l)

Fig. 5. Scavening effect of methanolic extract at different Fig. 6. Scavenging effect of methanolic extracts of Agrimonia
concentrations of the Agrimonia Pilosa leaf on hydrogen Pilosa leaf on hydroxyl radical. A.P.L.: Agrimonia Pilosa leaf,
peroxide. APL: Agrimonia polisa leaves, BHA: Butylated BHT: butylated hydroxytoluene, BHA: butylated hydroxyanisole,
hydroxyanisol. BHT: Butylated hydroxytoluene, TCP: Toco- TCP: tocopherol. OH: Hydroxyl radical generation system
pherol. (100 I-lM Vitamin C, 100 I-lM CUS04 , 121-lM Cytochrome C).
248 0.-8. Hah et al.

:
90
--A, P . L
and ONA are the components of the cell that are most
75 sensitive to oxidative damage induced by ROS. (Hatano,

~~
~ BHA

~60 --'- BHT 1995; Masaki et al., 1995; Benzie, 2000; Karioti et al.,
8 ---- TC?
2004). The assessment of oxidative stress status was
.: : 45
.J>
approached through the measurement the degree of
-230
oxidative damage on lipids, proteins, and ONA (Ayres,
15
1949; Ozcelik et al., 2003; Sioane and William, 1997;
0 Kano et al. , 2005). Although, antioxidative effect of
o 100 200 300 400 500 600 700 600 900 1000 11 00 1200
Amounl 01 Extract(pu!ml)
MeOH extract during linoleic acid oxidation, determined
by the OPPH and thiocyanate methods, was greater
Fig. 7. Inhibitory effect of methanolic extracts of Agrimonia than BHA and TOC in a previous our study and
Pilosa leat on 2'-deoxyguanosine (2'-dG) oxidative damage. because antioxidatine effect of control methanol solvent
A.P.L.: Agrimonia Pilosa leaf, BHT: butylated hydroxytolu-
showed previous our study, its effect did not show in
ene. BHA: butylated hydroxyanisole, TCP: tocopherol.
this study (Hah et al., 2005, 2007), the antioxidative
activity of MeOH extract during lecithine liposome oxida-
tion, determined by the TBA method in present study,
O,OlöOO was inferior to commercial BHA, and BHT. This result
are consistent with previous studies showing that anti-
oxidative activity of natural antioxidants was showed a
wide range of activities and has different activity
depending on substrates used and various system
0 .0100(
(Imaida et al. , 1983; Frankel et al., 1997; Ouh et al.,
1999). Lipid peroxidation was measured by indirectly
measure the MAO formed after lipid oxidation and MOA
has been recognized as a good biomarker for lipid oxi-
0 .0050( dation. MOA is associated with lipid oxidation and very

J
B reactive. Moreover, it can act as a catalyst in the forma-
tion of N-nitrosamines in foods containing secondary
amines and nitrite (Eriksson, 1987). We selected meth-
0 .0000(
~ f8
!'i.
~
si
!ll
-
anol as extract solvent according to our previous study
(Hah et al., 2005, 2007). From our results Fig. 1, MeOH
extract showed significantly (p < 0.05) lower MOA when
compared with BHA, TCP. This data implies that MeOH
Fig. 8. HPLC chromatograms of 2'-deoxyguanosine oxida- extract may protect against damage to cell membrane
tion. A: 2'-deoxyguanosine, B; 8-hydroxy-2'deoxyguanosine. since they reduce the level of lipid peroxidation.
As showing the Fig. 2, The inhibition of protein oxida-
tion increased with increasing amounts up to 600 I-1g
min, respectively (Fig. 8). (82.05%) and then decreased with increasing amounts
at 1200l-1g (80.7%). It seems to be that the protection
DISCUSSION of protein carbonyl formation was not complete. This
finding is consistent with observation of Antosiewicz et
In general terms, an antioxidant is anything which can al. (1995) who showed that inhibition effect of indolinic
prevent or inhibit oxidation (HaIliweIl, 1991). This can be acid and quinolinic aminooxylson on protein of mice
achieved by preventing the generation of ROS, or by microsome and lipid oxidation. But disagree with pub-
inactivating ROS (Benzie, 2000; Eklund et al. , 2005). lished Mung bean Hull methanolic extract antioxidative
The membrane phospholipid is susceptible to free radi- activity (Ouh, 1999). This difference was estimated by
cal attack and leads to lipid peroxidation, membrane presuming that the components of sam pie used was
disruption, and changes the structure and function of not same sam pie used in this study.
key cellular constituents, resulting in mutation, cell dam- Halliwell et al. (1987) and Aruoma (1991) report that
age and death (Chen and Stevens, 1991; Strain and FeCI 3-EOTA and H20 2 mixed solution is incubated with
Benzie, 1999; Halliwell and Gutteridge, 1987). Numer- deoxyribose in phosphate buffer (pH 7.4), the hydroxyl
ous studies suggested that unsaturated lipid, proteins, radical formed attach to the deoxyribose and result seri-
 Evaluation of Antioxidative Activity of Agrimonia pilosa-Ledeb Leaves on Non-lipid Oxidative Damage 249

ous of reactions that result in the formation of MOA. may be concerned with its higher scavenging of
Consequently, the ability to diminish the amount of color hydroxyl radical.
formation has been adapted as one measurement of The formation of 8-0H-2'-dG was induced by various
antioxidative properties. When any hydroxyl radical environmental factors, such as ionizing radiation, UV
scavenger added to the this reaction mixture, It would light or Fenton reaction, indicating that the formation of
compete with deoxyribose for hydroxyl radical and its 8-0H-2'-dG occurred through the reaction of ONA with
concentration is higher than that of deoxylribose, hydroxyl hydroxyl radicals or singlet oxygen (Ames and Gold,
radical was separated from deoxyribose to scavenger, 1991; Floyd, 1990). Kasai et a/. (1986) mentioned that
deoxyribose was protected from hydroxyl radical (Zhao generation of oxygen radicals in vivio is thought to be
and Jung, 1995). This finding was found in present relevant to carcinogenesis, and 8-0H-2'-dG formation in
study, therefore, MeOH extract may be related to the ONA may be related to tumor-genesis. As shown in Fig.
protecting deoxyribose damage against oxidative stress. 7. Although, the inhibitory effect on 8-0H-2'-dG oxida-
If some amino acid residues are oxidized, it convert to tion damage was seems to be different from 55-59.7%
carbonyl derivatives; consequently, it could be used as depending amounts, We suggested that MeOH extract
one measure of protein damage (Stadman and Shibam- was expected for strong antioxidant to protect free radi-
oto, 1992; Neuzil et a/., 1993). cal inducing ONA damage.
lron and copper irons are powerful promoters of free In conciusion, the present results on biological model
radical damage, causing formation of hydroxyl radicals system showed that Agrimonia pilosa leaves was effec-
and accelerating lipid peroxidation (Gutteridge and tive in the protection of lipids and non-lipids against vari-
Stocks, 1981). Antioxidant may have a direct antioxida- ous oxidative stress model system. This might be
tive effect by scavenging free radicals as weil as bind- serving as a commercial, useful antioxidant in food pro-
ing metal ions to reduced their absorption. The cessing and might be a potential adjutant antioxidant in
molecular that can inhibit deoxyribose degradation are pharmaceutical preparations or in organ preservation
those that can chelate the iron ions from the deoxyri- fluids where oxidative stability is desired. One of the
bose and render them inactive or poorly active in a major factors determining the antioxidative activity of
Fenton reaction (Smith et a/., 1992). As showed in Fig. Agrimonia pilosa leaves against ROS induced cell dam-
4. the chelating effect of MeOH extract on metal ions age is may be due to its components, quercetin, quer-
may relate to their inhibition of deoxyribose degradation. citrin, rutin. However, It is difficult to identify the specific
Superoxide can form hydrogen peroxide and this can, contribution of each single component about these
in turn, form the highly reactive hydroxyl radical. effects. Which components critical role are under fur-
Removal of superoxide, therefore, is a key antioxidant ther investigation.
defense mechanism (Fridovich, 1974). Hydrogen perox-
ide is one of the active oxygen specie capable of injur- REFERENCES
ing ONA (Imlay and Linn, 1988). Indeed, H20 2 has
been implicated in the induction of the nuciear transcrip- Ames, B.N. and Gold, L.S. (1991). Endogenous mutagens
tion factor NFkB in Jurket cells (Ginn and Whisler, and cause of aging and cancer. Mut. Res., 250, 3-16.
1998; Schreck et a/., 1991). High concentration of H2 0 2 Anne, C.T. and Andrew, R.C. (2000). Recovery of human
induced necrosis to cell while low concentration induced Iymphocytes from oxidative DNA damage; the apparent
apoptosis (Lennon et al., 1991). Hydrogen peroxide enhancement of DNA repair by cartenoids is probably
also induces ONA damage through Fenton reaction in simply an antioxidant effect. Eur. J. Nutr., 39, 80-85.
Antosiewicz, J., Popinigis, J., Wozniak, M., Damiani, E., Car-
vivio and in vitra (Aruoma et a/., 1999; Imlay and Linn,
loni, P. and Greci, L. (1995). Effects of indolinic and quin-
1988). Hence, the ability of MeOH extract to scavenge olinic aminoxylson protein and lipid peroxidation of rat
hydrogen peroxide may contribute to the inhibitory effect liver microsoned. Free Rad. Bio. Med., 18, 913-917.
on oxidative system. Aruoma, 0.1. (1991). Pro-oxidant properities: An important
The scavenge effect of MeOH extract on hydroxyl consideration for food additive and/or nutrient compo-
radical was increased with increasing amount. The nents.: In free radical and food additives (0.1. Aruoma
inhibitory effect of antioxidants on the oxidative dam- and B. Hailliwell, Eds.). Taylor & Francis, London, pp 173-
194.
age of non-lipids was due to the scavenging of hydroxyl
Aruoma, 0.1., Spencer, J.P. and Mahmood, E. (1999). Protec-
radical, it is essential to evaluate whether antioxidants is tion against oxidative damage and cell death by the natu-
able to scavenge hydroxyl radicalor not. The results of ral antioxidant Ergothineine. Food and Chern. Toxicol., 37,
Fig. 6. indicated that the marked inhibitory effect of 1043-1053.
MeOH extract on the oxidative damage of non-lipids Ayres, GH. (1949). Evaluation of accuracy in photometrie
250 D.-S. Hah et al.

analysis. Anal. Chem., 21,652-657. non-lipid oxidative damage. Korea J Vet. Res., 47, 25-32.
Benzie, I. F. F. (2000). Evolution of antioxidant defence mecha- Halliwell, B. (1991). Drug antioxidant effects. Drugs, 42, 569-
nism. Eur. J Nutr., 39, 53-21. 605.
Branen, A.L. (1975). Toxicology and biochemistry of buty- Halliwell, B., Gutteridge, J.M.C. and Aruoma, 0.1. (1987). The
lated hydroxy anisoie and butylated hydroxytoluene. deoxyribose method: a simple "teste-tube" assay for
JA.o.C.S., 52. 59-63. determination of rate constants for reaction of hydroxyl
Ca rte r, P. (1971). Spectrophotometric determination of serum radicals. Ana/. Bioehem., 165,215-219.
iron at the submicrogram level with a new reagent (fer- Hatano, 1. (1995). Constituents of natural medicines with
rozine). Ana/y. Biochem., 40, 450-458. scavenging effects on active oxygen species-Tannins and
Cha, B.C., Park, H.J., Lee, E., Choi, MY and Rhim, TJ. related polyphenols. Natu. Medi., 49, 357-363.
(1996). Comparison of antioxidant activity and composi- Imaida, K., Fukushima, S., Hivai, 1., Ohtani, M., Nakanish, K.
tion in G/ycine max. and G/ycine soja Siebo/d et Zucc. and Ito, N. (1983). Promoting activitied of butylated
Kor. J Pharmacogn., 27, 190-195. hydroxyanisol and butylated hydroxytpluene on 2-stage
Cha, B.C., Lee, SK, Lee, H.W., Lee, U., Choi, MY, Rhim, urinary bladder carcinogenesis and inhibition of r-glutamyl
TJ. and Park, H.J. (1997). Antioxidative effects of medici- transpeptase-positive foci development in the liver of rats.
nal plants in Korea. Kor. J Pharmacogn., 28, 15-20. Carcino., 4, 895-899.
Chen, G and Stevens, J.L. (1991). Inhibition of iodoaceta- Imlay, JA and Linn, S. (1988). DNA damage and oxygen
mide and t-butyl hydroperoxide toxicity in LLC-PKI cells radical toxicity. Science, 240, 1302-1309.
by antioxidants: a role for lipid peroxidation in alkylation Inatani, R, Nakatani, N. and Fuwa, H. (1996). Antioxidative
induced cytotoxicity. Arch. Biochem. Biophy., 284, 422- effect the constituent of rosemary and their derivatives.
430. Agric. Biol. Chem., 47, 521-525.
Droge, W. (2002). Free radicals in the physiological control of Isao, K., Naosuke, B., Yumiko, O. and Nobusuke, K. (1988).
cell function. Physi%gica/ Reviews, 82, 47-95. Triterpenoids from Agrimonia pilosa. Phytochemistry, 27,
Duh, P.D., Du, PC. and Yen, GC. (1999). Action of methan- 297-299.
olic extract of Mung bean hulls as inhibitors of lipid peroxi- Kano, M., Takayanagi, 1., Harada, K., Makino, K. and Ish-
dation and non-lipid oxidative damage. Food Chem. ikawa, F. (2005). Antioxidative activity of anthocyan ins
Toxico/., 37, 1055-1061. from purple sweet potato /pomoera batatas cultivar Aya-
Eklund, P.C., Langvik, O.K., Warna, J.P, Salmi, 1.0., Willfor, murasaki. Biosei. Biotechno. and Biochemi., 69, 979-988.
S.M. and Sjoholm, RE. (2005). Chemical studies on anti- Karioti, D., Hadjipavlou, L., Mensah, M.L., Fleischer, TC. and
oxidant mechanisms and free radical scavenging proper- Skaltsa, H. (2004). Composition and antioxidant activity of
ti es of lignans. Orga. Bimo/ec. Chem., 21, 3336-3347. the essential oils of Xylopia aethiopica (Dun) A. Rich.
Eriksson, C.E. (1987). Oxidation of lipids in food systems: In (Annonaceae) leaves, stem bark, root bark, and fresh and
Autooxidation of unsaturated lipids (H.W.S. Chan, Ed.). dried fruits, growing in Ghana. J Agri. Food Chemi., 52,
Academic Press, London, pp. 207-231. 8094-8098.
Fang, Y.Z., Yang, S. and Wu, G (2002). Free radical, antioxi- Kasai, H. and Nibishimura, S. (19894). Hydroxylation of deox-
dants and nutrition. Nutr., 18, 872-879. yguanisine at the C-8 position by ascorbic acid and other
Floyd, RA (1990). Role of oxygen free radicals in carcino- reducing agents. Nuc/eie Acids., 12, 2137-2145.
genesis and brain ischemia. FA SEB , 4, 2587-2597. Kimie, 1., Tachio, A., Masaki, S., Yoshisa, K., Ryokei, K. and
Frankei, E.N., Huang, S.w. and Aeschbach, R (1997). Anti- Toshiro, M. (1988). Antioxidative effect of protoporphyrin
oxidant ativity of green teas in different lipid systems. J on lipid rat liver subcellular fractions. Chern. Pharrn. Bull.,
Ame. Oil Chern. Soc., 74, 1309-1315. 36,1104-1109.
Freidovich, I. (1999). Fundamental aspects of reactive oxy- Lander, H.M. (1997). An essential role for free radicals and
gen species, or what's the matter with oxygen? Ann. NY derived species in signal transduction. FASEB J, 11, 118-
Acad. Sei., 893, 13-15. 126.
Fridovich, I. (1974). Superoxide and evolution. Horizons Bio- Lennon, Sv., Marin, S.J. and Cotter, TG (1991). Dose-
ehern. Biophys., 1, 1-37. dependent induction of apoptosis in human tumor cell
Fukuzawa, K. and Takaishi, Y. (1990). Antioxidants. J Act. lines by widely diverging stimuli. Cell Pro/ite., 24, 203-14.
Oxyg. Free Rad., 1, 55-70. Lenz, A.G, Costable, U., Shaltei, S. and Cevine, RL. (1989).
Ginn, PM.E. and Hisler, RL. (1998). Redxo signals and Determinationof carboyl groups inoxidatively modified pro-
NFkB activation in T Gell. Free Rad. Bio. Med., 25, 346- teins by reduction withtritiated sodium borohydride. Ana/y.
361. Bioehem., 177,419-425.
Gutteridge, J.M.C. and Stocks, J. (1981). Caeruloplasmin : Masaki, H., Sakaki, S., Atsumi, 1. and Sakurai, H. (1995).
physiological and pathological perspectives. Crit. Rev. Active-oxygen scavenging activity of plant extracts. Bio/.
Clin. Labo. Sei., 14, 257-329. Pharrn. Bull., 18, 162-166.
Hah, D.S., Kim, C.H., Kim, E.G and Kim, J.S. (2005). Antioxi- MeCord, J.M. (2000). The evolution of free radicals and oxi-
dative effects of traditional medicinal plants on lipid peroxi- dative stress. Am. J Med., 108, 652-662.
dation. Korea J Veto Res., 45, 341-350. Neuzil, J., Gebicki, J.M. and Stocker. R (1993). Radical-
Hah, D.S., Kim, C.H., Kim, E.G, Kang, J.B. and Kim, J.S. induced chain oxidation of proteins and its inhibition by
(2007). Antioxidative effects of Houttuynia cordata root on chain breaking antioxidants. Bioehern. J, 293, 601-606.
 Evaluation of Antioxidative Activity of Agrimonia pilosa-Ledeb Leaves on Non-lipid Oxidative Damage 251

Nobuyki, S., Shunji, U., Yoshinori, F. and Masayasu, S. Food Chem. Toxieol., 29, 1-6.
(1996). Protective effect of vitamin E on chromium (VI)- Sioane, H.8. and William, S.G (1977). Spectrophotometric
induced cytotoxicity and lipid peroxidation in primary cul- accuracy, linearity and adherence to Beer's law. Appl.
tures of rat hepatocytes. Arch. Toxieol., 71, 20-24. Speetra., 31, 25-30.
Ozcelik, B., Lee, J.H. and Min, D.B. (2003). Effects of light, Smith, C., Halliwell, B. and Aruoma, 0.1. (1992). Protecting
oxygen, and pH on the absorbance of 2,2-diphenyl-1-pic- byalbumin against the pro-oxidant action of phnolic dietary
rylhydrazyl. J. Food Sei., 68, 487-490. components. Food Chem. Toxieo., 30, 483-489.
Park, J.H., Kang, K.C., Baek, S.8., Lee, Y.H. and Rhee, K.S. Sohal, R.S. and Weindruch, R. (1996). Oxidative stress,
(1991). Separation of antioxidants compounds from edi- caloric restriction, and agibg. Sei., 128, 372-379.
ble marin algae. Korean J. Food Sei. Technoi., 23, 256- Stadtman, H. and Shibamoto, T (1991). Antioxidative activity
261. measurement in lipid peroxidation system with malonalde-
Pei, Y.H., Li, X. and Zhu, TR. (1989). Studies on the chemi- hyde and 4-hydroxy nonenal. J. Am. Oil Chem. Soe., 68,
cal constitutes from the root-sprouts of Agrimonia pilosa 941-943.
Ledeb. Yao. Hsueh Hsueh Pao., 25, 267-270. Strain, J.J. and Benzie, I.F.F. (1999). Free radicals in biology
PraU, D.E. and Watts, B.W. (1964). The antioxidative activity and medicine. Clarendon Press, Oxford. Antioxidants: diet
of vegetable extracts. I. Flavone aglyeones. J. Food Sei., and antioxidant defence. In: The Encyclopedia of human
29, 17-24. nutrition (M. Sadler, J.J. Strain and B. Cabellero, Eds.).
Ruch, K.J., Cheng, S.J. and Klauing, J.E. (1989). Prevention Academic Press, London, pp. 95-106.
of cytotoxity and inhibition of intracellular communication Su, G, Su, S. and Zhu, T (1984). Studies on bacteriostatic
by antioxidant catechin isolated from Chinese green tea. components from Agrimonia pilosa Ledeb. Shenyang
Careinogenesis., 10, 1003-1008. Yaoxueyuan Xuebao., 1,44-50.
Sala, A, Recio, M.C., Schinella, GR., Manez, S., Giner, R.M., Tamura, H. and Shibamoto, T (1991). Antioxidative activity
Cerda, N.M. and Rios, J.L. (2003). Assessment of the measurement in lipi peroxidation system with malonalde-
anti-inflammatory activity and free radical scavenger activ- hyde and 4-hydroxy nonenal. J. Am. Oil Chem. Soe., 68,
ity of tiliroside. Eur. J. Pharm., 461, 53-61. 941-943.
Salles, B., Sattler, U., Bozzato, C. and Calsou. E. (1999). Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M.
Repair of oxidative DNA damage in vitra: A tool for and Telser, J. (2007). Free radicals and antioxidants in
screening antioxidative compounds. Food Chem. Toxieo., normal physiological functions and human disease, Inter.
37,1009-1014. J. Bioehem. Cell Bio., 39, 44-84.
Schreck, R., Rieber, P. and Baeuerle, PA (1991). Reactive Wen, H.C., Beth, AV. and Xin, GL. (1999). High level dietary
oxygen intermediate as apparently widely used messen- vitamin E do not replace cellular glutathione peroxidase in
gers in the activation of the NFkB transcription factor and protecting mice from acute oxidative stress. Bioehem.
HIV-1. EMBO J., 10, 2247-2258. Mole. Action Nutr., 98, 1951-1957.
Selvendiran, K.J., Singh, pv., Kirshnan, K.B. and Sakthiseka- Yun, Z.F., Sheng, Y. and Guoyao, W. (2002). Free radicals,
ran, D. (2003). Cytoprotective effect of piperine against antioxidants, and Nutrition. Nutrition, 18, 872-879.
beno[a]pyrene induced lung cancer with reference to lipid Zhao, M.J. and Jung, L. (1995). Kinetis of the competive deg-
peroxidation and antioxidant system in Swiss albino mice. radation of deoxyribose and other molecules by hydroxyl
Fitoterapia, 74, 109-115. radicals produced by the Fenton reaction in the presence
Shi, X., Dalal, N.S. and Jain, AC. (1991). Antioxidant behav- of ascorbic acid. Free Rad. Res., 23, 229-243.
iour of caffeine: efficient scavenging of hydroxyl radicals.