Evaluation of Antioxidative Activity of Agrimonia pilosa-Ledeb Leaves on Non-lipid Oxidative Damage

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, including liposome oxidation, deoxyribose oxidation, 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 provided remarkable protection against damage to deoxyribose. Protective effect of MeOH extracts of the Agrimonia pilosa-Ledeb leaves on protein damage was observed at 600 µg level (82.05%). The MeOH extracts of the Agrimonia pilosa-Ledeb leaves at 300 µg revealed metal binding ability (32.64%) for hydrogen peroxide. Furthermore, the oxidation of 2’-deoxyguanosine (2’-dG) to 8-hydroxy-2-deoxyguanosine (8-OH-2’dG) was inhibited by MeOH extracts of the Agrimonia pilosa-Ledeb leaves and scavenging activity for hydroxyl radical exhibited a remarkable effect. 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.


INTRODUCTION
Free radicals such as reactive oxygen speeies (ROS) are formed during a variety biological and cellular functions in living organisms. All cells having aerobic respiration, using oxygen as a nutrient, are continuously exposed to a number of exogenous and endogenous ROS or radicals that cause lipid, protein and DNA damage (Salles et al., 1999;Droge, 2002). The endogenous source of ROS are formed from the mechanism of oxidative phosphorylation in which oxygen is reduced to water through a four step addition of electrons. The exogenous source of ROS are also produced by exposure to various environmental factors, for instance, UV light, ionizing radiations or tobacco (Hanato, 1995;Lander, 1997;Freidovich, 1999;Salles et al. , 1999;MeCord, 2000;Fang et al., 2002). Antioxidant defenses have evolved to protect biological systems against reactive oxygen speeies, and a sophisticated, co-operative array of antioxidant defense mechanisms is found in biological systems. Antioxidant defenses system prevent generation and counteract the damaging effects of reactive speeies produced within the organism from molecular oxygen (Kimie et al., 1988;Masaki et al., 1995;Nobuyki et al., 1996;Benzie, 2000). But an imbalance between the production of various reactive speeies and the ability of the organisms natural protective mechanism to cope with these reactive compounds was occurred, this condition was called oxidative stress. Oxidative stress causes a net stress on normal body func-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;Halliwell, 1991;Anne and Andrew, 2000;Selvendiran et a/., 2003;Valko et a/., 2007). From ancient, traditional medicinal plants have been known to posse antioxidative activity (Pratt and Watts., 1964;Fukuzawa and Takaishi, 1990;Inatain et a/., 1996). For several years, there are current interested in the development of therapeutic and chemopreventive antioxidative agents which have non-cytotoxicity. Many researchers suggest that antioxidants, in particular plants diet-derived antioxidants, might have health benefits as prophylactic agents (Aruoma et a/., 1999).
The Agrimonia pilosa-Ledeb. Rosaceae is traditional medicinal plant, which have already been known to have diverse properties such as antioxidative activity including antibacterial, anti-inflammatory, antipyretic, haemostatic, vasoconstrictor, analgesic and anti-tumor (Sala et al., 2003).
Although, the protective or remedy mechanism of Agrimonia pilosa-Ledeb have not been fully elucidated, It has been commonly used in Korea and other Asian countries such as China, India , Japan as a natural flavoring agent for prevent, or treatment of various chronic-fatigue syndrome diseases (Su et al., 1984;Isao et a/., 1988;Pei et a/., 1990;Cha et a/., 1997;Sala et al., 2003). Therefore, considerable attention has been focused on quenching free radical oxidation from the natural medicinal plants and synthetic phenolic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) are commonly added to foods to inhibit free radical damage to lipid; however, BHA, BHT are suspected as possible carcinogens (Branen, 1975;Duh et a/., 1999). Therefore, It seems to be need to find out the nature anti-oxidants without side effect from natural medicinal plants. Also, where or not occurred oxidative stress on non-lipid and/or be able to protect oxidative damage against free radicals using Agrimonia pilosa-Ledeb are not under clean. Thus, the objectives of present study were conducted to evaluate the antioxidative activity of Agrimonia pilosa-Ledeb leaves methanolic extract on non-lipid oxidative damage.

Plants materials and extracts preparation. The
Agrimonia pilosa leaves were obtained from the OH q 1) qlU'J"t. Research Institute of Traditional Medicine Plants of Gyeongnam (Hamyang, Gyeongnam, Korea) and was identified by the head of Research Institute. Voucher specimens were deposited in the Research Institute and Its chemical structure shown in Fig. 1.
Agrimonia pi/osa leaves were air dried under shade and cut into small pieces and stored at 4°C until use.
Agrimonia pilosa leaves 300 9 were extracted with 80% 900 ml methanol in a shaking incubator at 80°C for 12 hr. The residue was re-extracted under the same condition 3 times. The extracts obtained were combined and filtered. The combined methanol specimens were evaporated to dryness using a vacuum rotary evaporator and weighted (98.88 9 dry base) to determine the yield of soluble constituents. The extract obtained was subject to evaluate the antioxidative activity on non-lipid oxidative damage.
Determination of the antioxidative activity on liposome peroxidation. 580 mg Lecithin (ICN, USA) containing 58 ml, 10 mM phosphate buffer (pH 7.4) was ultrasonicated in ultrasonic cleaner (Branson B-221 Smithkline Company, USA) for 2 hours. The sonicated solution (10 mg lecithin/mi), Fecl 3 , ascorbic acid and MeOH extract (50-1200 fl9) were mixed to produced a final concentration of 3.12 mg lecithin/mi, 125 flM FeCI3, and 125 flM ascorbic acid. The mixture was incubated for 1 hr at 37°C by the thiobarbituric acid (TBA) method (Tamura and Shibamoto, 1991). The absorbance of the mixture was read at 532 nm against a blank, which contained all reagents except lecithin. The extinction coeffi-cient of TBA-malonaldehyde product of 1.56 x 10 5 M"1 cm-1 was used to convert absorbance values into concentrations of secondary reaction products.
Measurement of chelating activity on metal ions. The chelating activity of MeOH extract on Fe 2 + was measured according to the Carter method (1971). The MeOH extract (0.2 ml, 50-1200 fl9), was incubated with 0.05 ml Fecl2 4H20 (2.0 mM). the reaction mixture was initiated by the addition of 0.2 ml ferrozine (5.0 mM), and finally quantified to 0.8 ml with methanol. After the mixture had reached equilibrium (10 min), the absorbance at 562 nm was read. EOTA served as the positive control, and an untreated sampie served as the negative control. All analyses were run in three replicates and averaged.
Determination of the antioxidative activity on hydrogen peroxides. Antioxidative activity of MeOH extract to scavenge hydrogen peroxides was measured according to the Ruch et a/. (1989). The MeOH extract (50-1200 fl9) was added to hydrogen peroxide solution (0.6 ml, 2 mM) preparing in phosphate buffer saline (PBS, pH 7.4, at 20°C). MeOH extract was decolorized with cartridge (Sep-pack C18, Waters USA). Absor-bance of hydrogen peroxide at 230 nm was determined 10 min later in a spectrophotometer against a blank solution containing MeOH extract in PBS without hydrogen peroxjde. Hydrogen peroxide concentration was determined spectrophotometrically at 230 nm using a molar extraction coefficient for hydrogen peroxide of 81 M-1 cm-1 .
Assay of the antioxidative activity on hydroxyl radicals. Scavenging effect of MeOH extract on hydroxyl radicals was assayed by the Shi et a/. (1991) method. Briefly, The reaction solution containing 3 ml sodium phosphate buffer (0.15 mM, KH 2 P0 4 -K 2 HP0 4 , pH 7.4) was initiated by the addition of 100 flM vitamin C, 100 flM CuS04, 12 flM cytochrosome C. After initiation, the MeOH extract (50-1200 fl9) was added to reaction mixture and incubated at 25°C for 90 min. The absorbance of the color changed in cytochrome C was read at 550 nm. Thiourea served as the positive control, Inhibition rate (%) of hydroxyl radical was calculated as following formula.

Inhibition rate (%) = T -T2 x 100
T -T1 T: The transmittance of OH radical generation system, T1: The transmittance of control system T2: The transmittance of test sam pie system Assay of the antioxidative effect on 2'-deoxyuanosine oxidation. This assay was determined according to the Kasai and Nishimura method (1984). The reaction mixture (1.4 ml), containing MeOH mixture (50-1200 fl9), 2'-dG (0.5 mM), KH 2 P0 4 -K 2 HP0 4 buffer (0.1 M, pH 7.4), was initiated by the Fenton reaction model system, which contained H20 2 (50 mM), FeCI3 6H20 2 (1.3 nM), EOTA (6.5 mM) and ascorbic acid (15 mM). All the mixture was incubated at 37°C for 30 min, and incubation was terminated by placing the mixture in an ice-bath, and then filtering through a 0.45 flm filter before use. The filtrate was analyzed by HPLC (Water, USA), using the Water E2241 F (103 mm x 1 mm, 0.5 flm) and UV detector (measured at 254 nm). The column was equilibrated with 50 mM KH2P04 (pH 4.61)methanol (93.5:6.5, v/v) at a flow rate of 0.5 ml/min. 2'-dG and 8-0H-2'dG were identified by comparison of their retention times with those of known standards and determined by peak areas from the chromatograms. All analyses were run in three replicates and averaged.
Statistical analysis. The results are expressed as mean ± standard deviation (S.O). Oifferences between groups were assessed by one-way analysis of variance 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 comparison test between the treated and control group was conducted. Oifferences between two groups were statistically evaluated by at-test.

RESULTS
Determination of the antioxidative activity on liposome oxidation. Phospholipids wh ich named as derivatives of phosphatidic acid such as phosphatidylcholine (lecithin) are the main components of cell membrane. To evaluate antioxidative activity of MeOH extract in a liposome model system, The lecithin, prepare as a liposome, was used. Antioxidative effect of MeOH extract against the inhibitory activity of lipid peroxidation in cell membranes were assessed by the amount of malondialdehyde (MOA) produced. MeOH extracts in the range of 100-600 flg showed 16.28-30.12% inhibition of peroxidation. The incidence of observed inhibition was dependent on amount up to 600 fl9, but inhibition was decreased (29.14%) at 1200 fl9. As results to compare the MeOH extract with butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), and tocopherol (TCP) currently used as commercial antioxidants, BHA, BHT, and TCP, wh ich at 600 flg showed 17.85%, 52.68% and 11.27% inhibition of peroxidation, respectively. Obviously, BHT at 600 flg showed a higher antioxidative activity than did 600 flg MeOH extract, However, BHA and TCP were lower than that of MeOH extract (Fig. 1).
Determination of the antioxidative activity on protein carbonyl. The effect of MeOH extract on protein carbonyl formation in albumin, induced by FeCI3, H20 2,   bition of deoxyribose damage. Obviously, BHA and BHT at 100-1200 Ilg showed a higher inhibition of deoxyribose damage than did MeOH extract, but TCP was lower than that of MeOH extract except at 1200 Ilg (Fig. 3). Fig. 4. As expected, the chelating effect increased with increasing amount of MeOH extract. MeOH extract at 300 Ilg has the highest effect on Fe2+ binding (32.64%), whereas at 1200 Ilg MeOH extract showed a 32.35% chelating effect and 100 Ilg revealed the most lower chelating effect (16.63%). Although this amount (100 Ilg) has more lower value than the other amounts, it is relatively higher when compared with that of EDTA (2%). It may be significant because it minimizes the concentration of metal in the Fenton reaction. 1200 Ilg) on hydrogen peroxide was 5.45%, 24.39%, 23.71%, and 24.23%, respectively. At all amounts of MeOH used, the inhibition of hydrogen peroxides was more lower than those of BHA, BHT, and TCP. Especially, 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, indicating that the greater concentration (1200 Ilg) of MeOH extract, the less protection on hydrogen peroxides (Fig. 5).

Measurement of chelating activity on metal ions. The chelating effect of MeOH extract on ferrous ions is plotted in
Assay of the antioxidative activity on hydroxyl radical. The scavenging effect of different amount of MeOH extract (50, 100, 300, 600, and 1200 Ilg) on hydroxyl radical was 75%, 80%, 86%, 90%, and 92%, respectively. MeOH extract markedly scavenged the hydroxyl radical , and Its scavenging effect increased with increasing amounts of MeOH extract as dosedependent manner. Its scavenging effect was higher than BHA, TCP. But BHT was similar to the observation of MeOH extract (Fig. 6).

DISCUSSION
In general terms, an antioxidant is anything which can prevent or inhibit oxidation (HaIliweIl, 1991). This can be achieved by preventing the generation of ROS, or by inactivating ROS (Benzie, 2000;Eklund et al. , 2005). The membrane phospholipid is susceptible to free radical attack and leads to lipid peroxidation, membrane disruption, and changes the structure and function of key cellular constituents, resulting in mutation, cell damage and death (Chen and Stevens, 1991;Strain and Benzie, 1999;Halliwell and Gutteridge, 1987). Numerous studies suggested that unsaturated lipid, proteins, and ONA are the components of the cell that are most sensitive to oxidative damage induced by ROS. (Hatano, 1995;Masaki et al., 1995;Benzie, 2000;Karioti et al., 2004). The assessment of oxidative stress status was approached through the measurement the degree of oxidative damage on lipids, proteins, and ONA (Ayres, 1949;Ozcelik et al., 2003;Sioane and William, 1997;Kano et al. , 2005). Although, antioxidative effect of MeOH extract during linoleic acid oxidation, determined by the OPPH and thiocyanate methods, was greater than BHA and TOC in a previous our study and because antioxidatine effect of control methanol solvent showed previous our study, its effect did not show in this study (Hah et al., 2005(Hah et al., , 2007, the antioxidative activity of MeOH extract during lecithine liposome oxidation, determined by the TBA method in present study, was inferior to commercial BHA, and BHT. This result are consistent with previous studies showing that antioxidative activity of natural antioxidants was showed a wide range of activities and has different activity depending on substrates used and various system (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 oxidation. MOA is associated with lipid oxidation and very reactive. Moreover, it can act as a catalyst in the formation of N-nitrosamines in foods containing secondary amines and nitrite (Eriksson, 1987). We selected methanol as extract solvent according to our previous study (Hah et al., 2005(Hah et al., , 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 extract may protect against damage to cell membrane since they reduce the level of lipid peroxidation.
As showing the Fig. 2, The inhibition of protein oxidation increased with increasing amounts up to 600 I-1g (82.05%) and then decreased with increasing amounts at 1200l-1g (80.7%). It seems to be that the protection of protein carbonyl formation was not complete. This finding is consistent with observation of Antosiewicz et al. (1995) who showed that inhibition effect of indolinic acid and quinolinic aminooxylson on protein of mice microsome and lipid oxidation. But disagree with published Mung bean Hull methanolic extract antioxidative activity (Ouh, 1999). This difference was estimated by presuming that the components of sam pie used was not same sam pie used in this study. Halliwell et al. (1987) and Aruoma (1991) report that FeCI 3 -EOTA and H 2 0 2 mixed solution is incubated with deoxyribose in phosphate buffer (pH 7.4), the hydroxyl radical formed attach to the deoxyribose and result seri-ous of reactions that result in the formation of MOA. Consequently, the ability to diminish the amount of color formation has been adapted as one measurement of antioxidative properties. When any hydroxyl radical scavenger added to the this reaction mixture, It would compete with deoxyribose for hydroxyl radical and its concentration is higher than that of deoxylribose, hydroxyl radical was separated from deoxyribose to scavenger, deoxyribose was protected from hydroxyl radical (Zhao and Jung, 1995). This finding was found in present study, therefore, MeOH extract may be related to the protecting deoxyribose damage against oxidative stress. If some amino acid residues are oxidized, it convert to carbonyl derivatives; consequently, it could be used as one measure of protein damage (Stadman and Shibamoto, 1992;Neuzil et a/., 1993). lron and copper irons are powerful promoters of free radical damage, causing formation of hydroxyl radicals and accelerating lipid peroxidation (Gutteridge and Stocks, 1981). Antioxidant may have a direct antioxidative effect by scavenging free radicals as weil as binding metal ions to reduced their absorption. The molecular that can inhibit deoxyribose degradation are those that can chelate the iron ions from the deoxyribose and render them inactive or poorly active in a Fenton reaction (Smith et a/., 1992). As showed in Fig.  4. the chelating effect of MeOH extract on metal ions may relate to their inhibition of deoxyribose degradation.
Superoxide can form hydrogen peroxide and this can, in turn, form the highly reactive hydroxyl radical. Removal of superoxide, therefore, is a key antioxidant defense mechanism (Fridovich, 1974). Hydrogen peroxide is one of the active oxygen specie capable of injuring ONA (Imlay and Linn, 1988). Indeed, H20 2 has been implicated in the induction of the nuciear transcription factor NFkB in Jurket cells (Ginn and Whisler, 1998;Schreck et a/., 1991). High concentration of H20 2 induced necrosis to cell while low concentration induced apoptosis (Lennon et al., 1991). Hydrogen peroxide also induces ONA damage through Fenton reaction in vivio and in vitra (Aruoma et a/., 1999;Imlay and Linn, 1988). Hence, the ability of MeOH extract to scavenge hydrogen peroxide may contribute to the inhibitory effect on oxidative system. The scavenge effect of MeOH extract on hydroxyl radical was increased with increasing amount. The inhibitory effect of antioxidants on the oxidative damage of non-lipids was due to the scavenging of hydroxyl radical, it is essential to evaluate whether antioxidants is able to scavenge hydroxyl radicalor not. The results of Fig. 6. indicated that the marked inhibitory effect of MeOH extract on the oxidative damage of non-lipids may be concerned with its higher scavenging of hydroxyl radical.
The formation of 8-0H-2'-dG was induced by various environmental factors, such as ionizing radiation, UV light or Fenton reaction, indicating that the formation of 8-0H-2'-dG occurred through the reaction of ONA with hydroxyl radicals or singlet oxygen (Ames and Gold, 1991;Floyd, 1990). Kasai et a/. (1986) mentioned that generation of oxygen radicals in vivio is thought to be relevant to carcinogenesis, and 8-0H-2'-dG formation in ONA may be related to tumor-genesis. As shown in Fig.  7. Although, the inhibitory effect on 8-0H-2'-dG oxidation damage was seems to be different from 55-59.7% depending amounts, We suggested that MeOH extract was expected for strong antioxidant to protect free radical inducing ONA damage.
In conciusion, the present results on biological model system showed that Agrimonia pilosa leaves was effective in the protection of lipids and non-lipids against various oxidative stress model system. This might be serving as a commercial, useful antioxidant in food processing and might be a potential adjutant antioxidant in pharmaceutical preparations or in organ preservation fluids where oxidative stability is desired. One of the major factors determining the antioxidative activity of Agrimonia pilosa leaves against ROS induced cell damage is may be due to its components, quercetin, quercitrin, rutin. However, It is difficult to identify the specific contribution of each single component about these effects. Which components critical role are under further investigation.