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Journal of Ethnopharmacology 87 (2003) 231236

Screening and comparison of antioxidant activity of solvent extracts of herbal medicines used in Korea
Dae Gill Kang a , Chi keun Yun b , Ho Sub Lee a,
a

Department of Herbal Resources, Professional Graduate School of Oriental Medicine and Medicinal Resources Research Center (MRRC), Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea b Department of Health Administration, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea Received 20 March 2002; received in revised form 7 April 2003; accepted 17 April 2003

Abstract The hexane, ethylacetate, n-butanol, and water extracts of 10 Korean herbal medicines were screened and compared for their antioxidant activities in a range of lipid peroxidation system using rat brain homogenates, antihemolysis assay of red blood cells, and other in vitro assays to determine their ability to scavenge superoxide and hydroxyl radicals. All of the 10 Korean herbal medicines have potent antioxidant activities. Among the four solvent extracts, the antioxidant activities of more-polar solvent extracts (BuOH and water extracts) were relatively higher than that of non-polar solvent extracts (hexane and EtOAC extracts). These results will be useful to further analyze those herbal medicines that contain the most antioxidant activity in order to identify the active principles. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Antioxidant; Korean herbal medicines; Solvent extracts; Screening

1. Introduction Reactive oxygen species (ROS), such as hydroxyl radical, hydrogen peroxide, and superoxide anions, are produced as byproducts in aerobic organisms, and have been implicated in the pathology of a vast variety of human diseases including cancer, atherosclerosis, diabetic mellitus, hypertension, AIDS, and aging (Halliwell and Gutteridge, 1984; Wallace, 1999; Lee et al., 2000). Therefore, antioxidant activity is an important in-view of the free radical theory of aging and associated diseases. It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. A great number of plant origin substances have been suggested to appear as antioxidants. Flavonoids and other phenolic compounds of plant origin have been reported as scavenger ROS, thus they are viewed as promising therapeutic drugs for free radical pathologies (Parshad et al., 1998; Lee et al., 2000). The antioxidant activity of plant origin is dependent on the type and polarity of the extracting solvent as well as on the test system and the substrate to be protected by the antioxidant (Heinonen et al., 1998; Moure et al., 2000; Kang

and Lee, 2001). Solvent extraction is frequently used for isolation of the antioxidants and both extraction yield and antioxidant activity of the extracts are strongly dependent on the solvent, due to the different antioxidant potentials of compounds with different polarity. For these reasons, comparative studies for selecting the optimal solvent providing maximum antioxidant activity are required for each substrate. Although the use of different polarity substances can provide more exhaustive information on the properties of the extracts, the literature contains few reports of the polarity-based solvent extraction of medicinal plants. The present study was undertaken to perform the screening of antioxidant properties of 10 traditional medicines grown in Korea, and we selected hexane, ethylacetate, n-BuOH, and water as extract solvents which permits comparison of the antioxidant properties among the polaritybased solvent extracts of medicinal plants.

2. Materials and methods 2.1. Chemicals Hexane, ethylacetate (EtOAC), and n-butanol (n-BuOH) were purchased from Merck (Darmstate, Germany). Phenazine methosulfate (PMS)- -nicotinamide adenine

Corresponding

author. Tel.: +82-63-850-6841; fax: +82-63-850-7324. E-mail address: host@wonkwang.ac.kr (H.S. Lee).

0378-8741/03/$ see front matter 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00142-9

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D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231236

Table 1 Latin names, herbarium voucher specimen numbers, plant parts, and uses in Korea Herbal medicines Inula helenium L. Astragalus membranaceus BUNGE Atratylodes koreana NAKAI Gardenia jasminoides for. Grandiora MAKINO Magnolia liliora DESR. Scutellaria baicalensis GEORGI Siegesbeckia orientalis L. Sinomenium acutum REHDER et WILS. Sorbus amurensis KOEHNE Xanthium strumarium L. Voucher specimen numbers DH-43 DH-14 DH-07 DH-08 DH-34 DH-05 DH-57 DH-15 DH-41 DH-58 Plant parts Root Root Root Fruit Flower Root Whole Root Bark Fruit Uses in Korea Antibacterial, disinsection Cardiotonic, diuretic Sedative, hypoglycemic Sedative, cholagogue Antihypertensive Antipyretic, antiallergy Antihypertensive Analgesia, antiinammation Expectorant Analgesia

dinucleotide (reduced form, NADH), nitroblue tetrazolium (NBT), l-ascorbic acid, cytochrome c, dl-dithiothreitol, thiobarbituric acid (TBA), Butylated hydroxyanisole (BHA), and 2,2 -azo-bis-(2-amidinopropane)dihydrochloride (AAPH) were purchased from Sigma (St. Louise, MO, USA). All other unstated chemicals and reagents were of analytical grade. 2.2. Plant material

USA). l-Ascorbic acid was used as a positive control. The superoxide radical scavenging ratio (%) was calculated using the following formula: Superoxide radical scavenging ratio (%) = A A1 100 A

where A is the absorbance of positive control, and A1 is the absorbance of the test samples. 2.5. Scavenging activities of hydroxyl radicals

All herbal medicines were purchased from a herbal market in Iksan, Jeonbuk Province, South Korea. Dr. Kyu Kwan Jang at the Botanical Garden of Wokwang University identied plant materials. Herbarium voucher specimens were prepared and deposited in the herbarium of the Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, South Korea (Table 1). 2.3. Extraction For the partitioning by solvent, the Korean herbal medicines (100 g) were air-dried at room temperature and reduced to ne powder by milling. The resulting powder was subjected to extraction with 200 ml of methanol, three times, 24 h each. The methanol extract was evaporated and resuspended in H2 O, and sequentially partitioned with n-hexane, EtOAC, and BuOH. 2.4. Scavenging activities of superoxide radicals Scavenging activity of superoxide radicals was determined by the Liu and Ng method (2000), which was slightly modied by Kang and Lee (2001). Superoxide radicals were generated in a PMS- -nicotinamide adenine dinucleotide (reduced form, NADH) system by oxidation of NADH and were assayed by the reduction of NBT in the microplate. The superoxide radicals were generated in 200 l of 16 mM TrisHCl buffer (pH 8.0), which contained 78 M NADH, 50 M NBT, 10 M PMS and samples (10 l) to be tested at different concentrations. The color reaction between superoxide radicals and NBT was detected at A560 nm using a microplate reader (Molecular Devices, Synnyvate, CA,

Scavenging activity of hydroxyl radicals was determined by the Liu and Ng method (2000), which was slightly modied by Kang and Lee (2001). The hydroxyl radicals were generated in a l-ascorbic acidCuSO4 system by reduction of Cu2+ and were assayed by the oxidation of cytochrome c in the 96-well microplate. The hydroxyl radicals were generated in 200 l of 10 mM sodium phosphate buffer (pH 7.4), containing 100 M l-ascorbic acid, 100 M CuSO4 , 12 M cytochrome c and the samples to be tested at different concentrations. The reduced cytochrome c was produced by addition of excess dl-dithiothreitol and followed by Sephadex G-15 chromatography (bed volume, 10 ml) to remove excess dl-dithiothreitiol. The change in absorbance caused by the oxidation of cytochrome c was measured at 550 nm using a microplate reader (Molecular Devices). Thiourea was used as a positive control. The scavenging activity of hydroxyl radical by 500 g/ml of thiourea was taken as 100%. The scavenging activity of hydroxyl radical was calculated using the following formula: Hydroxyl radical scavenging activity (%) = A A0 100 AT A 0

where A is the absorbance of samples, and AT and A0 are the absorbance of the thiourea and the control, respectively. 2.6. Inhibitory effects on erythrocyte hemolysis Whole blood was obtained carefully by cannulation of femoral artery in SpragueDawley rats and collected in heparinized tubes. Erythrocytes were isolated from plasma by centrifugation (1000 g for 20 min) and washed three times

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with 10 volumes of saline solution. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system (Niki et al., 1988). A 10% suspension of erythrocytes in 10 mM of phosphate-buffered saline (PBS, pH 7.4) was added to the same volume of 200 mM AAPH in PBS solution containing samples to be tested at different concentrations. The reaction mixture was shaken gently while being incubated at 37 C for 2 h. The reaction mixture was then removed by centrifugation (1000 g for 20 min), diluted with eight volumes of PBS and centrifuged at 1000 g for 20 min. The absorbance (A) of supernatant was read at 540 nm using spectrophotometer (Milton Roy, Rochester, NY, USA). Similarly, the reaction mixture was treated with eight volumes of distilled water to achieve complete hemolysis, and the absorbance (B) of the supernatant obtained after centrifugation was measured at 540 nm. The inhibition of hemolysis (%) was calculated by the equation (1 A/B) 100. l-Ascorbic acid was used as a positive control. 2.7. Inhibitory effects on lipid peroxide (LPO) production in brain homogenates For the in vitro studies, the brains of normal Sprague Dawley rats were isolated and homogenized with Polytron homogenizer (Switzerland) in ice-cold TrisHCl buffer (20 mM, pH 7.4) to produce a 10% (w/v) homogenate. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant (0.5 ml) was incubated with the test samples in the presence of 10 M FeSO4 and 0.1 mM ascorbic acid at 37 C for 1 h. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (TCA, 28%, w/v) and 0.75 ml thiobarbituric acid (TBA, 1%, w/v) in succession, and the solution was then heated at 100 C for 15 min. After centrifugation to remove precipitated protein, the color of the malondialdehyde (MDA)-TBA complex was detected at OD 532 nm using spectrophotometer (Milton Roy). BHA was used as a positive control. The inhibition ratio (%) was calculated using the following formula: Inhibition ratio (%) = A A1 100 A

3.1. Scavenging effects of solvent extract on O2 radical The four solvent extracts of 10 herbal medicines were screened for their superoxide-scavenging activity in the PMS/NADHNBT system, and the results are shown in Table 2. In the PMS/NADHNBT system, superoxide anion derived from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The decrease of absorbance at 560 nm with antioxidants thus indicates the consumption of superoxide anion in the reaction mixture. There was a difference in the overall scavenging ability among the extract solvents from the 40 extracts and even among the same species. Seven of the extracts, at 200 g/ml assay, displayed scavenging activities that were greater than 50%, while seven extracts exhibited a nearly zero-scavenging activity of the superoxide radical. Among them, the water extract of Sinomenium acutum showed the highest scavenging activity of superoxide radical in this system. 3.2. Scavenging effects of solvent extract on OH radical Table 3 shows the scavenging effect of solvent extracts of 10 Korean herbal drugs on hydroxyl radicals generated by l-ascorbic acid/CuSO4 cytochrome c system. The hydroxyl radicals were generated in a l-ascorbic acid/CuSO4 system by reduction of Cu2+ and were assayed by the oxidation of cytochrome c. High-scavenging activity (50% at 200 g/ml assay) was found in the BuOH extracts of Astragalus membranaceus, Siegesbeckia orientalis, and Sorbus amurensis, and EtOAC extracts of Scutellaria baicalensis and Sinomenium acutum, and hexane extracts of Sorbus amurensis. 3.3. Inhibitory effects on erythrocyte hemolysis The azo compound generates few radicals by its unimolecular thermal decomposition. The rate of generation of peroxyl radicals can be easily controlled and measured by adjusting the concentration of AAPH (Miki et al., 1987). Therefore, hemolysis induced by AAPH must provide suitable means for studying the oxidative erythrocyte membrane damage by peroxyl radical attack from the outside of the membrane (Ng et al., 2000). Of the extracts studied, BuOH extracts of Astragalus membranaceus, Atratylodes koreana, Magnolia liliora, Xanthium strumarium, and Scutellaria baicalensis, and water extracts of Gardenia jasminoides and Sinomenium acutum, and EtOAC extracts of Scutellaria baicalensis, tested at 100 and 200 ug/ml, markedly inhibited erythrocyte hemolysis in this system (Table 4). 3.4. Inhibitory effects on LPO production in brain homogenates Quantication of MDA, one of the products of lipid peroxidation, with TBA is the most common assay used for determination of the rate extent of lipid peroxidation.

where A is the absorbance of control, and A1 is the absorbance of the test samples.

3. Results and discussion The methanol extracts of 10 Korean herbal drugs were divided into four fractions with different polarities by partitioning it in various solvents such as hexane, EtOAC, n-BuOH, and H2 O. Then these solvent extracts were tested for their antioxidant activity in a range of lipid peroxidation systems using rat brain homogenates, red blood cells and other in vitro assay to determine their ability to scavenge ROS.

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Table 2 Effect of solvent extracts of Korean herbal medicines on superoxide radicals generated by PMS/NADH system Samples Concentration ( g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 7.5 4.2 18.2 3.7 nz nz nz nz 17.1 7.0 45.1 1.4 nz nz nz 16.7 1.0 9.4 2.7 16.7 2.4 6.1 1.2 8.2 2.1 nz nz nz nz EtOAC 5.76 2.3 12.4 2.8 nz nz nz nz 18.8 3.5 47.9 3.2 5.8 1.1 12.4 2.2 65.9 1.4 83.8 1.3 nz 20.0 6.3 13.9 2.9 16.0 0.1 nz 5.5 7.8 14.7 3.5 31.7 1.3 n-BuOH 21.7 1.8 31.6 9.1 20.2 5.8 40.1 6.5 38.2 1.3 56.5 3.6 29.8 3.5 44.4 5.6 27.9 1.2 37.9 3.4 56.8 1.7 75.6 1.7 30.7 5.6 49.6 2.7 nz 14.3 3.1 60.1 1.6 78.1 3.5 31.9 6.5 61.7 2.6 H2 O 22.3 0.9 23.2 8.3 29.0 8.0 33.2 4.3 6.5 7.0 13.5 3.2 54.6 0.4 68.8 1.2 19.8 3.6 52.5 1.5 26.3 3.8 30.1 1.1 53.5 5.5 67.6 0.6 90.2 0.5 91.7 0.1 34.6 1.1 41.1 4.1 14.6 4.0 49.1 4.1

Results show mean S.E. (n = 3) of the inhibition of superoxide radical (%); nz: nearly zero.

Table 3 Effect of solvent extracts of Korean herbal medicines on hydroxyl radicals generated by l-ascorbic acid/Cu2+ system Samples Concentration ( g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 12.4 1.9 16.7 3.7 11.4 2.7 15.7 3.7 15.8 5.0 27.6 6.1 nz nz 12.9 1.5 29.3 2.9 25.8 2.5 38.7 1.9 9.2 0.9 19.1 1.6 5.5 2.3 13.6 1.6 32.8 0.8 51.9 8.8 13.4 2.9 14.9 2.4 EtOAC 31.5 2.6 45.8 2.5 23.5 2.2 28.8 2.6 13.7 7.0 24.3 5.3 32.1 4.6 46.7 0.9 10.0 0.8 28.8 1.0 32.5 4.8 63.4 3.7 25.3 3.6 28.1 2.2 34.7 3.8 85.5 3.7 10.7 1.1 15.0 1.5 24.5 6.0 28.2 1.5 n-BuOH 6.7 0.4 17.1 0.3 40.6 7.7 49.8 4.6 19.1 2.1 31.4 5.9 16.7 7.0 40.2 4.1 12.8 1.9 30.9 0.9 34.0 2.8 44.8 1.0 67.1 0.3 84.5 4.4 27.0 4.4 33.6 0.5 28.1 2.0 48.9 3.1 11.1 2.8 30.2 0.5 H2 O 5.7 1.9 10.3 0.5 20.7 3.3 31.5 7.3 6.2 1.0 21.1 2.2 24.8 1.4 39.1 1.0 5.23 1.2 9.96 1.6 11.0 1.2 10.5 2.8 26.5 9.7 41.4 2.0 34.6 1.7 41.8 3.7 7.1 3.4 16.3 1.7 5.5 1.4 10.2 1.4

Results show mean S.E. (n = 3) of the inhibition of hydroxyl radical (%); nz: nearly zero.

D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231236 Table 4 Effect of solvent extracts of Korean herbal medicines on the inhibition of hemolysis by AAPH system Samples Concentration ( g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 18.1 3.5 28.2 0.7 6.5 4.8 24.4 2.8 8.7 6.8 12.0 0.1 23.8 3.4 24.5 2.0 12.2 2.0 16.1 0.6 9.8 0.2 12.2 0.8 21.7 1.1 21.6 0.9 17.0 2.9 25.2 3.5 11.4 1.5 16.4 2.7 8.6 5.6 12.7 2.3 EtOAC 20.9 2.3 40.8 2.2 8.4 1.9 14.6 0.3 12.2 2.5 13.2 0.2 37.6 3.3 65.9 7.4 13.4 1.7 25.2 0.4 96.9 0.9 97.6 1.6 16.1 2.8 22.1 1.0 32.2 3.8 43.9 2.7 13.5 3.3 20.9 1.5 43.2 0.5 58.3 1.1 n-BuOH 58.8 2.0 73.6 0.2 94.9 1.0 94.5 0.4 96.4 1.1 97.8 0.1 56.3 0.8 79.3 1.6 97.2 1.0 98.7 0.2 87.9 1.4 97.1 0.3 56.3 3.9 76.3 0.2 11.4 3.6 30.2 5.2 64.6 2.0 78.9 2.1 94.8 0.4 95.3 0.1 H2 O

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12.3 3.7 21.6 2.9 38.8 2.5 48.6 2.3 7.2 5.9 14.3 4.9 92.8 0.2 96.7 0.4 33.3 2.5 55.2 1.3 26.9 2.2 54.5 0.7 41.1 4.6 75.2 2.1 93.2 1.3 98.5 0.2 23.0 0.2 28.9 1.3 43.0 1.4 69.4 1.2

Results show mean S.E. (n = 3) of the inhibition of erythrocyte hemolysis (%).

Our experiment proved that incubation of the rat brain homogenate with Fe2+ /ascorbate at pH 7.4 causes rapid peroxidation, detectable by the TBA method. Table 5 shows the activities of the representative 10 extracts, which showed high-antihemolysis activity, against lipid peroxidation of the rat brain homogenate. All the extracts markedly inhibited LPO production in the brain homogenates (Table 5). Medicinal herbs are known to contain a variety of antioxidants. Numerous substances have been suggested to appear as antioxidants. The most detailed investigations so far were concerned with reactions involving phenolic compounds, ranging from polymer chemistry to biochemistry
Table 5 Inhibitory effect of solvent extracts of Korean herbal medicines on the lipid peroxide production in brain homogenates Plants Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium Concentration ( g/ml) 200 200 200 200 200 200 200 200 200 200 Solvents BuOH BuOH BuOH H2 O BuOH EtOAC BuOH H2 O BuOH BuOH Inhibition (%) 88.8 90.8 90.9 83.8 93.7 96.2 90.5 91.2 90.7 88.8 0.2 0.1 1.5 0.8 0.1 0.0 1.1 0.2 0.7 1.4

Results show mean S.E. (n = 3) of the inhibition of production of LPO.

and food chemistry (Bravo, 1998). It has been revealed that various phenolic antioxidants, such as avonoids, tannins, coumarins, xanthones and more recently procyanidins scavenge radicals dose-dependently, thus they are viewed as promising therapeutic drugs for free radical pathologies (Lee et al., 2000). Flavonoids are 15-carbon aromatic pigments found in green plants and include chalcones, avonones, avones, biavonoids, dihydroavonols, anthrocyanidins, and avonols (VanderJagt et al., 2002). More than 4000 naturally occurring avonoids have been described (Hollman and Katan, 1998). The present study suggests that more-polar components present in herbal medicinal extracts contributed towards the increased ROS-scavenging activity. Although there are no direct evidences in this study, the antioxidant activities shown by BuOH extracts and/or water extracts of herbal medicines could be related to the presence of phenolic compounds such as tannins and avonoids because they contain an aromatic hydroxyl moiety (Yesilada et al., 2000; Ramezani et al., 2001). As well known, polyphenols are very important constituents because of their scavenging ability with ROS and chelating ability with divalent cations due to their hydroxyl groups (de las Heras et al., 1998; Hatano et al., 1989; Lopes et al., 1999). Although the active principles responsible for the antioxidant activity of the tested extracts have not yet been isolated in this study, it will be useful to further analyze those herbal medicines that contain the most antioxidant activity

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in order to identify the active principles. Then it would lead to a better understanding of the kinds of antioxidants used in Korea as herbal medicines.

Acknowledgements This study was supported by the Brain Korea 21 Project and grant of the Oriental Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea (HMP-00-CO03-0003).

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