in vitro antioxidant properties and total phenolic

INTRODUCTION

It is commonly accepted that reactive oxygen species such as superoxide (O2˙-) hydroxyl (OH˙-) and peroxyl (˙OOH ROO˙) radicals are produced under oxidative stress Reactive oxygen species play important roles in de-generative or pathological processes such as aging (Burns et al 2001) cancer coronary heart disease Alzheimerrsquos disease (Diaz et al 1997) neurodegenerative disorders atherosclerosis diabetes and inflammation (Chen et al 2006) Several anti-inflammatory digestive anti-necrotic neuroprotective and hepatoprotective drugs have recently been shown to have antioxidant andor radical scavenging mechanisms as well (Lin and Huang 2002) Some natural antioxidants and compounds with radical scavenging ac-tivity have been identified over the last few years includ-ing echinacoside in Echinaceae root (Hu and Kitts 2000) anthocyanin (Espin et al 2000) phenolic compounds (Rice-Evans et al 1997) and the extracts of water spin-

Botanical Studies (2012) 53 55-66

Corresponding author E-mail gjhuangmailcmuedutw Tel +886-4-22053366 ext 5508 Fax +886-4-22083362

ach and sweet potato tuberous roots (Huang et al 2004 Huang et al 2005)

Medicinal plant parts are commonly rich in phenolic compounds such as flavonoids phenolic acids stilbenes tannins coumarins lignans and lignins These compounds have multiple biological effects including antioxidant activity (Packer et al 1999) In vitro experiments on an-tioxidant compounds in higher plants show how they pro-tect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species (Ali et al 2008) The role of these compounds as potential antioxidants can be inferred by their similarity to synthetic antioxidants of related structures

The multifarious natural environment of Taiwan harbors abundant plant resources Many of these plants includ-ing those with therapeutic potential face endangerment Therefore we investigated wetland medicinal plants and analyzed their antioxidant activities In the present study we collected 31 medicinal wetland plant species that are widely consumed in Taiwan prepared their water and methanolic extracts and analyzed their antioxidant activi-ties and polyphenol contents

In vitro antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan

Yu-Ling HO1 Shyh-Shyun HUANG1 Jeng-Shyan DENG2 Yaw-Huei LIN3 Yuan-Shiun CHANG4 and Guan-Jhong HUANG4

1Department of Nursing Hung Kuang University Sha Lu Taichung 433 Taiwan2Department of Health and Nutrition Biotechnology Asia University Taichung 413 Taiwan3Institute of Plant and Microbial Biology Academia Sinica Taipei 115 Taiwan4 School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources China Medical University Taichung 404 Taiwan

(Received February 10 2011 Accepted July 20 2011)

ABSTRACT The aim of this study was to examine the possible antioxidant activities of the methanol and water extracts of 31 medicinal wetland plants in Taiwan We assayed for such properties such as TEAC DPPH radical scavenging total polyphenol content total flavonoid and total flavonol contents using the reduc-ing power method Our results showed that Rotala rotundifolia Juncus effusus var decipiens Cyperus iria Salix warburgii Lindernia antipoda Kyllinga brevifolia and Typha orientalis possessed both high antioxidant activities and high total polyphenol contents There was a low correlation between TEAC and total polyphenol content (water extracts R2=014 methanol extracts R2=023) thus eliminating high phenolic content as an im-portant factor in determining the wetland plantsrsquo antioxidant capacities Our results demonstrated that although phytochemicals in the wetland medicinal plants may contribute significantly to their antioxidant activities these antioxidant activities were not directly related to the polyphenol quantity Phytochemicals may play key roles in the potent antioxidant activity of wetland medicinal plants The potential of these easily accessible sources of natural antioxidants should be explored by the pharmaceutical medical and health food industries

Keywords Antioxidant Flavonoid Flavonol Polyphenol Wetland medicinal plant

BIOChemISTRy

56 Botanical Studies Vol 53 2012

mATeRIALS AND meThODS

materialsButylated hydroxytoluene (BHT) Glutathione (GSH)

11-Diphenyl-2-picrylhydrazyl (DPPH) 6-Hydroxy-2 5 7 8-tetramethylchroman-2-carboxylic acid (Trolox) po-tassium peroxodisulfate (K2S2O8) Tris (hydroxylmethyl) aminomethane potassium ferricyanide (K3Fe(CN)6) ferric chloride (FeCl3) catechin 22rsquo-azinobis-(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) and other chemicals were purchased from Sigma Chemical Co (St Louis MO USA) Folin-Ciocalteu solution and 95 ethanol were purchased from Merck Co (Santa Ana CA USA) Thirty-one wetland medicinal plants were collected from Taichung Nantou and Hsinchu counties in Taiwan They were identified and authenticated by Dr Chao-Lin Kuo Associate Professor and Chairman Department of Chinese Medicine Resources China Medical University Taichung Taiwan The medicinal wetland plants studied were all described in the Catalogue of Medicinal Plant Resources in Taiwan published by the Committee on Chinese Medicine and Pharmacy Taiwan Department of Health (Lin 2003)

Plant materials methanol extracts preparation Dried whole herbs (100 g for each sample) were macer-

ated with 1L 95 ethanol for 24 hours at room tempera-ture then filtered and extracted three times The ethanol extract (3 L) was then evaporated to 10 mL and dried in a vacuum at 40degC The dried extract was weighed dissolved in 95 ethanol and stored at -20degC for further use

Plant materials water extracts preparation Dried whole herbs (100 g for each sample) were boiled

with 1L distilled water for 1 hour Filtration and extract collection were performed three times The resulting de-coction was evaporated to 10 mL and dried in a vacuum at 50degC The dried extract was weighed dissolved in distilled water and stored at -20degC for further use For each extract the yield was calculated as a percentage of the dry weight of the whole herbs used (100 g) and the quantity of dry mass obtained after extraction (ww)

TEAC antioxidant activity determination A TEAC assay was conducted based on the method of

Ramos et al (1999) The ABTS aqueous solution (7 mM) was oxidized with potassium peroxodisulfate (245 mM) for 16 hours in the dark at room temperature The ABTSmiddot+ solution was diluted with 95 ethanol to an absorbance of 075 plusmn 005 at 734 nm (Beckman UV-Vis spectrophotom-eter Model DU640B) An aliquot (20 μL) of each sample (125 μgmL) was mixed with 180 μL ABTSmiddot+ solution and the absorbance was read at 734 nm after 1 min Trolox was used as a reference standard A standard curve was constructed for Trolox at 0 15625 3125 625 125 250 500 μM concentrations TEAC value was expressed as millimolar concentration of Trolox solution with the an-

tioxidant equivalent to a 1000 ppm solution of the sample under investigation

DPPh radical scavenging antioxidant activity determination

The effects of crude extracts and positive controls (GSH and BHT) on DPPH radicals were estimated based on the method of Yamaguchi et al (1998) Aliquots (20 μL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 μL pH 74) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark The absorbance of the reaction solution was measured spectro-photometrically at 517 nm The percentage of DPPH de-colorization of the samples was calculated according to the equation decolorization = [1- (ABS sample ABS control)] times100 IC50 value was the effective concentration at which DPPH radicals were scavenged by 50 and was obtained by interpolation from linear regression analysis A lower IC50 value indicated a greater antioxidant activity

Reducing power measurement The reducing power of the crude extracts and posi-

tive controls (GSH and BHT) were determined according to the method of Yen and Chen (1995) The samples (0 3125 625 125 250 500 and 1000 μgmL) were each mixed with an equal volume of 02 M phosphate buffer pH 66 and 1 potassium ferricyanide The mixture was incubated at 50degC for 20 min before an equal volume of 1 TCA was added and then centrifuged at 5000 g for 10 min The upper layer of the solution was mixed with distilled water and 01 FeCl3 with a ratio of 1 1 2 and the absorbance was measured at 700 nm Increased ab-sorbance of the reaction mixture indicated an increase in reducing power

Total polyphenol content determination Total polyphenol contents of the crude extracts were

determined according to the method of Ragazzi and Vero-nese (1973) 20 μL of each extract (125 μgmL) was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu re-agent The mixture was allowed to stand at room tempera-ture for 5 min and then 40 μL of 20 sodium carbonate was added to the mixture The resulting blue complex was then measured at 680 nm Catechin was used as a standard for the calibration curve The polyphenol content was cali-brated using the linear equation based on the calibration curve The total polyphenol content was expressed as mg catechin equivalentg dry weight The dry weight indicated was the sample dry weight

Total flavonoid content determination Total flavonoid contents of the crude extracts were de-

termined according to the method of Lamaison and Car-net (1990) Aliquots of 15 mL extracts were each added to an equal volume of 2 AlCl36H2O (2 g in 100 mL

HO et al mdash Antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan 57

methanol) solution The mixture was vigorously shaken and the absorbance was read after 10 min of incubation at 430 nm Rutin was used as the standard for the cali-bration curve The total flavonoid content was calibrated using the linear equation based on the calibration curve The total flavonoid content was expressed as mg rutin equivalentg dry weight The dry weight indicated was the sample dry weight

Total flavonol content determination The total flavonol contents of the crude extracts were

determined according to the method of Arnous et al (2001) Aliquots of 200 μL extracts were each added to 1 mL of 01 p-dimethylaminocinnamaldehyde (DMACA) in methanolHCl (31 vv) The mixture was vigorously shaken and the absorbance was read after 10 min of in-cubation at 640 nm Catechin was used as the standard for the calibration curve The total flavonol content was calibrated using the linear equation based on the calibra-tion curve The total flavonol content was expressed as mg catechin equivalentg dry weight The dry weight indicated was the sample dry weight

Statistical analysisExperimental results were presented as the mean plusmn

standard deviation (SD) of three parallel measurements Statistical analyses were performed by one-way ANOVA followed by Dunnettrsquos t test The difference was consid-ered to be statistically significant when the p value was less than 005

ReSULTS

extraction yieldsThe water and methanol extract yields of the wetland

medicinal plants are presented in Table 1 The water ex-tract yields ranged from 424 to 7018 and the of methanol extract yields ranged from 089 to 3403 Among the water extracts Pistia stratiotes produced the highest yield (7018) followed by Lindernia antipoda (6333) Polygonum plebeium (4599) Alisma ori-entalis (4333) and Torulinium odoratum (3983 ) The highest yield among methanol extracts was obtained from Lindernia antipoda (3403) followed by Cyperus alternifolius subsp flabelliformis (2676) Avicennia marina (2437) Pistia stratiotes (2158) and Cyperus difformis (1784)

Water extractions of the wetland medicinal plants gen-erally yielded more components than methanol extrac-tions It is worth mentioning that water extraction may allow more hydrogen bonding with phenolic compounds than does methanol

TeAC assay antioxidant activity estimation The Trolox-equivalent antioxidant capacity (TEAC) as-

say is often used to evaluate the total antioxidant power of single compounds and complex mixtures of various plants

(Chang et al 2007a b) In this assay ABTS radical mono-cation was generated directly in stable form from potas-sium peroxodisulfate The radicals were generated before the addition of antioxidants to prevent the interference of compounds which affected radical formation This modi-fication made the assay less susceptible to interruptions and prevented the overestimation of antioxidant power

Table 1 The yield of water and methanol extracts of the wet-land medicinal plants

Scientific nameYield ( ww)a

Water extract

Ethanol extract

Acorus gramineus Soland 1296 837Avicennia marina (Forsk) Vierh -leaf 547 583Avicennia marina (Forsk) Vierh -root 1784 2437Alisma orientalis (Sam) Juzep 4333 1484Alternanthera sessilis (L) R Br 1591 1214Cyperus alternifolius L subsp

flabelliformis (Rottb) Kukenthal 1124 2676

Commelina communis L 1176 1421Cyperus difformis L 2322 1784Cyperus imbricatus Retz 3633 979Cyperus iria L 1107 762Eichhornia crassipes (Mart) Solms 1435 1305Echinochloa crus-galli (L) Beauv 677 639Egeria densa Planch 2077 342Euryale ferox Salisb 1444 143Eriocaulon sexangulare L 1245 253Fimbristylis littoralis Gaud 1052 392Hedyotis corymbosa (L) Lam 2119 1066Hygrophila pogonocalyx Hayata 1284 641Juncus effusus L var decipiens Buchen 1304 1134Kyllinga brevifolia Rottb 1218 445Lindernia antipoda (L) Alston 6333 3403Marsilea minuta L 3703 1127Pilea microphylla (L) Liebm 496 1076Phyla nodiflora (L) Greene 786 918Polygonum plebeium R Br 4599 1731Pistia stratiotes L 7018 2158Rotala rotundifolia (Wallich ex Roxb)

Koehne 424 1231

Spirodela punctata G F W Meyer 1396 1142Salix warburgii O Seem 1463 1544Typha orientalis Presl 197 089Torulinium odoratum (L) S Hooper 3983 1276a On dried weight basis


(Sanchez-Moreno 2002) The tested samples were only added to the reaction medium once stable absorbance was obtained Their antioxidant activities were then measured in terms of decolorization This method is recommended for plant extracts because the maximum wavelength ab-sorption of ABTS at 734 nm eliminates color interference (Awika et al 2003) The results were expressed as μM Troloxmg dry weight of plant material

In the TEAC assay the antioxidant capacities of wet-land medicinal plants ranged from 752 μM to 175341 μM Troloxmg for the water extracts and 569 μM to 207435 μM Troloxmg for the methanol extracts (Table 2) The differences in antioxidant capacities were very large up to 233 and 364 fold respectively Among the water extracts Rotala rotundifolia possessed the highest antioxidant capacity (175341 plusmn 7699 μM Troloxmg)

followed by Juncus effusus var decipiens (97114 plusmn 4968 μM Troloxmg) Cyperus iria (76204 plusmn 3380 μM Troloxmg) Salix warburgii (65757 plusmn 1837 μM Troloxmg) and Kyllinga brevifolia (46267 plusmn 949 μM Troloxmg) The plant with the highest antioxidant capacity among the methanol extracts was Juncus effusus var decipiens (207435 plusmn 11619 μM Troloxmg) followed by Salix warburgii (93145 plusmn 8414 μM Troloxmg) Cyperus iria (76941 plusmn 5357 μM Troloxmg) Typha orientalis (65122 plusmn 1495 μM Troloxmg) and Kyllinga brevifolia (34252 plusmn 1091 μM Troloxmg)

Scavenging activity against 11-diphenyl-2-picrylhydrazyl radicals

The relatively stable organic radical DPPH is widely used in modeling systems to investigate the scavenging

Table 2 The TEAC of the water and methanol extracts of the wetland medicinal plants

Scientific name and positive controlsTEACa

(microM Troloxmg plusmn SD)Water extracted Methanol extracted

GSH 182768 plusmn 7684 Not detectedBHT Not detected 1186941 plusmn 3463Acorus gramineus Soland 15952 plusmn 257 22565 plusmn 945Avicennia marina (Forsk) Vierh -leaf 37617 plusmn 1042 5415 plusmn 211Avicennia marina (Forsk) Vierh -root 38185 plusmn 1307 17700 plusmn 213Alisma orientalis (Sam) Juzep 1233 plusmn 344 1908 plusmn 796Alternanthera sessilis (L) R Br 15638 plusmn 948 14846 plusmn 664Cyperus alternifolius L subsp flabelliformis (Rottb) Kukenthal 4723 plusmn 366 8177 plusmn 410Commelina communis L 11302 plusmn 196 9613 plusmn 769Cyperus difformis L 13200 plusmn 483 1533 plusmn 1620Cyperus imbricatus Retz 11235 plusmn 474 2735 plusmn 481Cyperus iria L 76204 plusmn 3380 76941 plusmn 5357Eichhornia crassipes (Mart) Solms 10213 plusmn 266 14333 plusmn 639Echinochloa crus-galli (L) Beauv 44898 plusmn 641 3956 plusmn 2028Egeria densa Planch 3710 plusmn 217 569 plusmn 758Euryale ferox Salisb 1458 plusmn 111 35073 plusmn 413Eriocaulon sexangulare L 7758 plusmn 357 22265 plusmn 086Fimbristylis littoralis Gaud 31173 plusmn 371 8717 plusmn 502Hedyotis corymbosa (L) Lam 28358 plusmn 445 5673 plusmn 718Hygrophila pogonocalyx Hayata 6940 plusmn 094 4338 plusmn 503Juncus effusus L var decipiens Buchen 97114 plusmn 4968 207435 plusmn 11619Kyllinga brevifolia Rottb 46267 plusmn 949 34252 plusmn 1091Lindernia antipoda (L) Alston 32035 plusmn 280 31123 plusmn 1605Marsilea minuta L 9427 plusmn 482 19625 plusmn 1350Pilea microphylla (L) Liebm 16544 plusmn 038 24817 plusmn 3450Phyla nodiflora (L) Greene 9704 plusmn 153 8621 plusmn 1258Polygonum plebeium R Br 36404 plusmn 107 17544 plusmn 947Pistia stratiotes L 752 plusmn 364 3479 plusmn 263Rotala rotundifolia (Wallich ex Roxb) Koehne 175341 plusmn 7699 15990 plusmn 1552Spirodela punctata G F W Meyer 36025 plusmn 770 10417 plusmn 536Salix warburgii O Seem 65757 plusmn 1837 93145 plusmn 8414Typha orientalis Presl 34413 plusmn 548 65122 plusmn 1495Torulinium odoratum (L) S Hooper 3271 plusmn 071 20367 plusmn 5257aValues represented mean plusmn SD of three parallel measurements (Plt005)


activities of several natural compounds such as pheno-lics and anthocyanins as well as crude mixtures such as methanol or water extracts from plants The DPPH radi-cal is scavenged by antioxidants through the donation of electrons forming the reduced DPPH The color changes from purple to yellow after reduction and the accompa-nying decrease in absorbance can be quantified at wave-length 517 nm Table 3 shows the IC50 values for radical-scavenging activities of GSH BHT and different extract fractions of the wetland medicinal plants using the DPPH colorimetric method

In the DPPH assay conducted on the water extracts Rotala rotundifolia had the lowest IC50 value among the medicinal plants (9489 plusmn 031 μgmL) followed by Salix warburgii (11269 plusmn 028 μgmL) Lindernia antipoda (18914 plusmn 455 μgmL) Cyperus iria (19445 plusmn 032

μgmL) Avicennia marina -leaf (27171 plusmn 128 μgmL) and Polygonum plebeium (30152 plusmn 462 μgmL) The pos-itive control glutathione (GSH) had an IC50 value of 7177 plusmn 209 μgmL

For the methanol extracts Salix warburgii had the low-est IC50 value (5958 plusmn 033 μgmL) followed by Juncus effusus var decipiens (10895 plusmn 447 μgmL) Lindernia antipoda (14461 plusmn 253 μgmL) Cyperus iria (16718 plusmn 064 μgmL) Typha orientalis (20801 plusmn 146 μgmL) and Cyperus imbricatus (24255 plusmn 311 μgmL) The positive control BHT also had a low IC50 value (13956 plusmn 296 μgmL) The above IC50 values showed that Salix warburgii and Juncus effusus var decipiens demonstrated even higher radical scavenging activities than the positive control in the DPPH assay

Table 3 The DPPH radical scavenging activity of the water and methanol extracts of the wetland medicinal plants

Scientific name and positive controlsDPPH radical scavenging activitya

(IC50 microgmL)Water extract Methanol extract

GSH 7177 plusmn 209 Not detectedBHT Not detected 13956 plusmn 296Acorus gramineus Soland 89690 plusmn 760 104551 plusmn 069Avicennia marina (Forsk) Vierh -leaf 27171 plusmn 128 gt2000Avicennia marina (Forsk) Vierh -root 40419 plusmn 118 71399 plusmn 024Alisma orientalis (Sam) Juzep gt2000 gt2000Alternanthera sessilis (L) R Br 84469 plusmn 642 94679 plusmn 839Cyperus alternifolius L subsp flabelliformis (Rottb) Kukenthal gt2000 gt2000Commelina communis L gt2000 gt2000Cyperus difformis L 112567 plusmn 122 48904 plusmn 382Cyperus imbricatus Retz 188264 plusmn 392 24255 plusmn 311Cyperus iria L 19445 plusmn 032 16718 plusmn 064Eichhornia crassipes (Mart) Solms gt2000 gt2000Echinochloa crus-galli (L) Beauv 54823 plusmn 462 gt2000Egeria densa Planch gt2000 gt2000Euryale ferox Salisb gt2000 30735 plusmn 161Eriocaulon sexangulare L gt2000 gt2000Fimbristylis littoralis Gaud 81061 plusmn 658 gt2000Hedyotis corymbosa (L) Lam 66889 plusmn 862 gt2000Hygrophila pogonocalyx Hayata 152006 plusmn 525 gt2000Juncus effusus L var decipiens Buchen 45688 plusmn 388 10895 plusmn 447Kyllinga brevifolia Rottb 37952 plusmn 252 52355 plusmn 0091Lindernia antipoda (L) Alston 18914 plusmn 455 14461 plusmn 253Marsilea minuta L 140048 plusmn 32 61376 plusmn 167Pilea microphylla (L) Liebm gt2000 42314 plusmn 561Phyla nodiflora (L) Greene gt2000 78926 plusmn 584Polygonum plebeium R Br 30152 plusmn 462 gt2000Pistia stratiotes L gt2000 gt2000Rotala rotundifolia (Wallich ex Roxb) Koehne 9489 plusmn 031 72189 plusmn 391Spirodela punctata G F W Meyer 43220 plusmn 463 109473 plusmn 1261Salix warburgii O Seem 11269 plusmn 028 5958 plusmn 033Typha orientalis Presl 53359 plusmn 492 20801 plusmn 146Torulinium odoratum (L) S Hooper gt2000 gt2000aValues represented mean plusmn SD of three parallel measurements (Plt005)


1997) The reducing power of different extract fractions from the wetland medicinal plants are shown in Table 4 Both reduced GSH and BHT were used as the positive controls

For the reducing capacity of the water extracts Salix warburgii had the highest value among the medicinal plants examined (164 plusmn 001 Δ700) followed by Rotala rotun-difolia (161 plusmn 005 Δ700) Lindernia antipoda (160 plusmn 007 Δ700) Cyperus iria (156 plusmn 001 Δ700) Polygonum plebeium (117 plusmn 003 Δ700) and Avicennia marina-leaf

Reducing power measurement We investigated the reducing capacity of wetland me-

dicinal plants by measuring Fe3+-Fe2+ conversion The re-ducing capacity of a compound may serve as an important indicator of its potential antioxidant activity (Meir et al 1995) The antioxidant activities of putative antioxidants have been attributed to various mechanisms such as the prevention of chain initiation transition metal ion catalyst binding peroxides decomposition prevention of contin-ued proton abstraction and radical scavenging (Diplock

Table 4 The reducing power of the water and methanol extracts of the wetland medicinal plants

Scientific name and positive controlsReducing power Δ700a

(Mean plusmn SD)Water extract Methanol extract

GSH 180 plusmn 001 Not detectedBHT Not detested 027 plusmn 002Acorus gramineus Soland 036 plusmn 001 004 plusmn 001Avicennia marina (Forsk) Vierh -leaf 111 plusmn 001 006 plusmn 001Avicennia marina (Forsk) Vierh -root 1010 plusmn 002 036 plusmn 002Alisma orientalis (Sam) Juzep 005 plusmn 001 015 plusmn 002Alternanthera sessilis (L) R Br 046 plusmn 001 019 plusmn 002Cyperus alternifolius L subsp flabelliformis (Rottb) Kukenthal 013 plusmn 001 017 plusmn 001Commelina communis L 023 plusmn 001 040 plusmn 001Cyperus difformis L 039 plusmn 001 051 plusmn 002Cyperus imbricatus Retz 027 plusmn 002 041 plusmn 003Cyperus iria L 156 plusmn 001 058 plusmn 003Eichhornia crassipes (Mart) Solms 017 plusmn 001 026 plusmn 001Echinochloa crus-galli (L) Beauv 063 plusmn 002 003 plusmn 001Egeria densa Planch 002 plusmn 001 004 plusmn 001Euryale ferox Salisb 007 plusmn 001 076 plusmn 012Eriocaulon sexangulare L 009 plusmn 001 032 plusmn 001Fimbristylis littoralis Gaud 040 plusmn 001 001 plusmn 001Hedyotis corymbosa (L) Lam 0552 plusmn 002 006 plusmn 000Hygrophila pogonocalyx Hayata 0310 plusmn 001 009 plusmn 002Juncus effusus L var decipiens Buchen 056 plusmn 001 026 plusmn 005Kyllinga brevifolia Rottb 074 plusmn 001 029 plusmn 008Lindernia antipoda (L) Alston 160 plusmn 007 166 plusmn 001Marsilea minuta L 027 plusmn 001 069 plusmn 002Pilea microphylla (L) Liebm 008 plusmn 003 029 plusmn 002Phyla nodiflora (L) Greene 033 plusmn 002 036 plusmn 003Polygonum plebeium R Br 117 plusmn 003 036 plusmn 002Pistia stratiotes L 002 plusmn 001 004 plusmn 001Rotala rotundifolia (Wallich ex Roxb) Koehne 161 plusmn 005 025 plusmn 001Spirodela punctata G F W Meyer 071 plusmn 005 010 plusmn 004Salix warburgii O Seem 164 plusmn 001 168 plusmn 001Typha orientalis Presl 058 plusmn 001 046 plusmn 002Torulinium odoratum (L) S Hooper 009 plusmn 001 027 plusmn 005aValues represented mean plusmn SD of three parallel measurements (Plt005)


(111 plusmn 001 Δ700) The positive control glutathione (GSH) had a high reducing capacity activity of 180 plusmn 001 Δ700

For the methanol extracts Salix warburgii had the high-est reducing capacity value (168 plusmn 001 Δ700) followed by Lindernia antipoda (166 plusmn 001 Δ700) Euryale ferox (076 plusmn 012 Δ700) Marsilea minuta (069 plusmn 002 Δ700) Cyperus iria (058 plusmn 003 Δ700) and Cyperus difformis (051 plusmn 002 Δ700) The positive control BHT also had quite a high reducing capacity (027 plusmn 002 Δ700) The results showed that the reducing capacities for radical-scavenging of all the tested wetland medicinal plants were even higher than those of the positive controls

Total polyphenol flavonoid and flavonol con-tents of the wetland medicinal plants

The total polyphenol flavonoid and flavonol contents of the water and methanol extracts of wetland medicinal plants are shown in Tables 5 and 6 respectively The total polyphenol content is expressed as μg of catechin equiva-lent per milligram of dry weight For the water extracts the total polyphenol content of the wetland medicinal plants ranged from 1791 μg to 56592 μg CEmg as for the methanol extract the total polyphenol content ranged from 769 μg CEmg to 55150 μg CEmg and the differ-ence of antioxidant capacities was also very large up to 31 and 71 fold Rotala rotundifolia had a total polyphenol content of 56592 plusmn 445 μg CEmg followed by Linder-nia antipoda (38925 plusmn 1912μg CEmg) Cyperus iria (38567 plusmn 562 μg CEmg) Cyperus iria (30289 plusmn 2119 μg CEmg) Typha orientalis (23050 plusmn 141 μg CEmg) and Cyperus difformis (16204 plusmn 1016 μg CEmg) in their water extracts (Table 5) Salix warburgii had a total poly-phenol content of 55150 plusmn 1757 μg CEmg followed by Typha orientalis (49417 plusmn 1013 μg CEmg) Juncus ef-fusus var decipiens (48975 plusmn 5328 μg CEmg) Linder-nia antipoda (35670 plusmn 1732 μg CEmg) Cyperus iria (34525 plusmn 981 μg CEmg) and Kyllinga brevifolia (25125 plusmn 190 μg CEmg) in their methanol extracts (Table 6)

The total flavonoid content was expressed as μg of rutin equivalent per milligram of dry weight The total flavonoid contents in the wetland medicinal plant water extracts ranged from 311 to 5392 μg REmg and the total flavonoid contents in their methanol extracts ranged from 119 to 7217 μg REmg furthermore the difference of antioxidant capacities was also very large up to 17 and 60 fold respectively Typha orientalis had the highest total flavonoid content (5392 plusmn 544 μg REmg) followed by Rotala rotundifolia (4624 plusmn 039 μg REmg) and Cyperus iria (2973 plusmn 051 μg REmg) in their water extracts Erio-caulon sexangulare had the highest total flavonoid content (7455 plusmn 150 μg REmg) followed by Polygonum ple-beium (7217 plusmn 333 μg CEmg) Typha orientalis (7189 plusmn 042 μg REmg) and Salix warburgii (7034 plusmn 243 μg REmg) in their methanol extracts

The total flavonol content was expressed as μg of cat-echin equivalent per milligram of dry weight The total flavonol content of the wetland medicinal plants water

extracts ranged from 005 to 1405 μg CEmg and that of the methanol extracts ranged from 046 to 1436 μg CEmg The difference in antioxidant capacities was also very large from 31 up to up to 281 fold Cyperus iria had the highest flavonol content (1405 plusmn 088 μg CEmg) fol-lowed by Polygonum plebeium (1347 plusmn 122 μg CEmg) and Salix warburgii (554 plusmn 018 μg CEmg) for the water extracts Cyperus imbricatus had the highest flavonol con-tent (1436 plusmn 128 μg CEmg) followed by Cyperus iria (1341 plusmn 087 μg CEmg) and Typha orientalis (704 plusmn 010 μg CEmg) for the methanol extracts

Relationship between total antioxidant activity and total polyphenol content

The correlation coefficients (R2) of total antioxidant activity (TEAC) and total polyphenols of the water and methanol extracts are shown in Figure 1A and 1B The R2 values of TEAC and total polyphenol content of the water (Figure 1A) and methanol (Figure 1B) extracts were 014 and 025 respectively From these statistics we deter-mined a low correlation between the TEAC and total poly-phenol contents Linear regression analysis showed a low correlation between antioxidant activity and total phenolic contents Different wetland medicinal plant species may influence the antioxidant activity as well High phenolic content is only one of the antioxidant capacity indicators

DISCUSSION

The best antioxidant activities among the 31 wetland medicinal plants screened based on TEAC assay DPPH radical scavenging reducing power total polyphenol con-tent total flavonoid content and flavonol content results were Rotala rotundifolia Juncus effusus var decipi-ens Cyperus iria Salix warburgii Lindernia antipoda Kyllinga brevifolia and Typha orientalis The therapeutic properties of Rotala rotundifolia have not been reported in any scientific papers before This plant possesses antiradia-tion anti-inflammatory and antibacterial properties The present study provided valuable preliminary data through a demonstration of its efficient antioxidant capacity Isola-tion and characterization of its individual active compo-nents and in vivo relevance await further comprehensive studies

Juncus effusus var decipiens possesses anti-depressant anti-inflammatory and antibacterial effects To our knowl-edge there were no prior reports on the antioxidant activ-ity of this plant This study rendered valuable preliminary data through demonstration of its high antioxidant capac-ity To study the phenolic constituents from the dry stem of Juncus effusus six phenolic constituents were purified and identified as 7-carboxy-2-hydroxy-1-methyl-5-vinyl-9 10-dihydrophenanthrene 23-isopylidene-1-O-ferulic acid glyceride (2S)-2 3-isopylidene-1-0-p-coumaroyl glycer-ide dehydroeffusal p-hydroxybenzaldehyde and luteolin-53rsquo-dimethyl ether (Li et al 2007) Some of these might be antioxidants


Cyperus iria has not been reported in any scientific pa-pers before This plant possesses rheumatic antidiuretic and anti-inflammatory effects The present study showed first-hand data on the antixodiant capacity of Cyperus iria Isolation and characterization of its individual active com-ponents and in vivo relevance of such activity await further comprehensive studies

Salix warburgii possesses anticoagulant anti-inflam-matory and antibacterial properties To our knowledge

there have been no prior reports on the antioxidant activ-ity of this plant This paper studied the antioxidant effects of Salix warburgii for the first time a bioassay-guided in vitro screen has revealed that a 70 methanol extract of the leaves of Salix matsudana showed considerable inhibitory activity against cyclooxygenases (COX-1 and COX-2) (Li et al 2008) A subsequent phytochemical study led to the isolation of some compounds matsudone A luteolin isoquercitrin 7-methoxyflavone luteolin 7-O-

Table 5 Total polyphenol flavonoid and flavonol content of the water extracts of the wetland medicinal plantsa

Scientific nameWater extracted

Polyphenola b

(microg CEmg)Flavonoida c

(microg REmg)Flavonola b

(microg CEmg)Acorus gramineus Soland 9150 plusmn 125 1381 plusmn 493 206 plusmn 014Avicennia marina (Forsk) Vierh -leaf 10925 plusmn 167 2755 plusmn 024 034 plusmn 002Avicennia marina (Forsk) Vierh -root 1021 plusmn 755 778 plusmn 007 042 plusmn 001Alisma orientalis (Sam) Juzep 3704 plusmn 181 371 plusmn 005 106 plusmn 001Alternanthera sessilis (L) R Br 13767 plusmn 381 2644 plusmn 053 156 plusmn 911Cyperus alternifolius L subsp flabelliformis (Rottb) Kukenthal 7845 plusmn 093 463 plusmn 005 123 plusmn 002Commelina communis L 11379 plusmn 576 1572 plusmn 020 104 plusmn 001Cyperus difformis L 16204 plusmn 1016 1896 plusmn 053 115 plusmn 001Cyperus imbricatus Retz 14087 plusmn 701 1370 plusmn 037 131 plusmn 001Cyperus iria L 38567 plusmn 562 2973 plusmn 051 1405 plusmn 088Eichhornia crassipes (Mart) Solms 9012 plusmn 726 997 plusmn 023 114 plusmn 001Echinochloa crus-galli (L) Beauv 10833 plusmn 926 1488 plusmn 133 293 plusmn 198Egeria densa Planch 2466 plusmn 010 585 plusmn 009 105 plusmn 001Euryale ferox Salisb 2816 plusmn 049 328 plusmn 021 193 plusmn 002Eriocaulon sexangulare L 8862 plusmn 091 957 plusmn 025 125 plusmn 002Fimbristylis littoralis Gaud 8750 plusmn 2002 1416 plusmn 036 313 plusmn 008Hedyotis corymbosa (L) Lam 15750 plusmn 753 1704 plusmn 067 071 plusmn 001Hygrophila pogonocalyx Hayata 8125 plusmn 911 786 plusmn 008 122 plusmn 001Juncus effusus L var decipiens Buchen 3733 plusmn 1037 879 plusmn 288 038 plusmn 001Kyllinga brevifolia Rottb 21592 plusmn 123 954 plusmn 044 169 plusmn 001Lindernia antipoda (L) Alston 38925 plusmn 1912 2268 plusmn 057 223 plusmn 002Marsilea minuta L 10241 plusmn 693 1242 plusmn 061 397 plusmn 015Pilea microphylla (L) Liebm 8167 plusmn 359 1000 plusmn 040 005 plusmn 001Phyla nodiflora (L) Greene 13704 plusmn 413 1676 plusmn 010 161 plusmn 001Polygonum plebeium R Br 6410 plusmn 1048 1722 plusmn 025 1347 plusmn 122Pistia stratiotes L 1791 plusmn 010 333 plusmn 004 085 plusmn 001Rotala rotundifolia (Wallich ex Roxb) Koehne 56592 plusmn 445 4624 plusmn 039 450 plusmn 027Spirodela punctata G F W Meyer 6767 plusmn 1158 2137 plusmn 127 081 plusmn 002Salix warburgii O Seem 30289 plusmn 2119 3420 plusmn 136 554 plusmn 018Typha orientalis Presl 23050 plusmn 141 5392 plusmn 544 289 plusmn 001Torulinium odoratum (L) S Hooper 4391 plusmn 055 311 plusmn 004 239 plusmn 004aValues represented mean plusmn SD of three parallel measurementsbData expressed in microg catechin equivalent mg dry weight (microg CEmg)cData expressed in microg rutin equivalent mg dry weight (microg REmg)


glucoside and 4rsquo7-dihydroxyflavone These isolated compounds were found to possess activities in inhibiting against COX-1 or COX-2

Lindernia antipoda possesses analgesic and anti-inflam-matory effects There have been no prior reports on the antioxidant activity of this plant This study provided valu-able data by demonstrating the high antioxidant capacity of Lindernia antipoda for the first time However isolation and characterization of its active components and their in

vivo relevance await further comprehensive studiesKyllinga brevifolia possesses analgesic and anti-inflam-

matory effects Oral administration of doses up to 30 gkg did not provoke any toxic symptoms The toxicity of this plant was observed to be dose dependent and its intraperi-toneal LD50 was found to be 575 mgkg It is used in tra-ditional medicine to alleviate stress or as a sedative agent (Hellioumln-Ibarrola et al 1999)

Table 6 Total polyphenol flavonoid and flavonol content of the methanol extracts of the wetland medicinal plants a

Scientific nameMethanol extracted

Polyphenola b

(microg CEmg)Flavonoida c (microg REmg)

Flavonola b

(microg CEmg)Acorus gramineus Soland 16058 plusmn 6115 1998 plusmn 3155 138 plusmn 001

Avicennia marina (Forsk) Vierh -leaf 769 plusmn 2450 1834 plusmn 2474 149 plusmn 016

Avicennia marina (Forsk) Vierh -root 5976 plusmn 327 4208 plusmn 122 046 plusmn 001

Alisma orientalis (Sam) Juzep 3745 plusmn 028 625 plusmn 193 094 plusmn 001

Alternanthera sessilis (L) R Br 15283 plusmn 1130 2407 plusmn 1878 379 plusmn 038

Cyperus alternifolius L subsp flabelliformis (Rottb) Kukenthal 10558 plusmn 1319 2051 plusmn 302 384 plusmn 005

Commelina communis L 12916 plusmn 499 2121 plusmn 024 136 plusmn 001

Cyperus difformis L 3901 plusmn 605 1531 plusmn 068 229 plusmn 002

Cyperus imbricatus Retz 11207 plusmn 1079 2611 plusmn 991 1436 plusmn 128

Cyperus iria L 34525 plusmn 981 4688 plusmn 047 1341 plusmn 087

Eichhornia crassipes (Mart) Solms 184 plusmn 1912 2560 plusmn 630 308 plusmn 006

Echinochloa crus-galli (L) Beauv 8008 plusmn 880 2421 plusmn 1061 200 plusmn 038

Egeria densa Planch 8179 plusmn 1852 12511 plusmn 541 295 plusmn 001

Euryale ferox Salisb 21358 plusmn 729 1670 plusmn 231 343 plusmn 001

Eriocaulon sexangulare L 13302 plusmn 512 7455 plusmn 150 615 plusmn 086

Fimbristylis littoralis Gaud 10708 plusmn 412 1916 plusmn 542 200 plusmn 037

Hedyotis corymbosa (L) Lam 10367 plusmn 146 2602 plusmn 649 257 plusmn 016

Hygrophila pogonocalyx Hayata 1805 plusmn 856 119 plusmn 053 254 plusmn 134

Juncus effusus L var decipiens Buchen 48975 plusmn 5328 3016 plusmn 507 160 plusmn 005

Kyllinga brevifolia Rottb 25125 plusmn 190 4186 plusmn 219 385 plusmn 026

Lindernia antipoda (L) Alston 35670 plusmn 1732 3521 plusmn 242 212 plusmn 001

Marsilea minuta L 4781 plusmn 958 1205 plusmn 384 145 plusmn 008

Pilea microphylla (L) Liebm 24408 plusmn 1443 1478 plusmn 338 598 plusmn 078

Phyla nodiflora (L) Greene 2953 plusmn 471 1110 plusmn 186 268 plusmn 010

Polygonum plebeium R Br 4432 plusmn 1138 7217 plusmn 333 282 plusmn 039

Pistia stratiotes L 5179 plusmn 112 2022 plusmn 258 497 plusmn 012

Rotala rotundifolia (Wallich ex Roxb) Koehne 12292 plusmn 367 2814 plusmn 548 243 plusmn 033

Spirodela punctata G F W Meyer 20400 plusmn 439 2084 plusmn 153 377 plusmn 029

Salix warburgii O Seem 55150 plusmn 1757 7034 plusmn 243 662 plusmn 031

Typha orientalis Presl 49417 plusmn 1013 7189 plusmn 042 704 plusmn 010

Torulinium odoratum (L) S Hooper 5406 plusmn 1180 1227 plusmn 227 333 plusmn 156aValues represented mean plusmn SD of three parallel measurementsbData expressed in microg catechin equivalent mg dry weight (microg CEmg)cData expressed in microg rutin equivalent mg dry weight (microg REmg)


Typha orientalis is a commonly used Chinese herbal drug which has been shown to possess blood circulation stimulating hypertension relieving and nerve soothing ef-fects There are no prior reports on the antioxidant activity of this plant

Phenolic compounds such as flavonoids phenolic acid and tannins possess anti-inflammatory anti-carcinogenic anti-atherosclerotic and other properties that may be re-lated to their antioxidant activities (Chung et al 1998 Wong et al 2006) Flavonoids and flavonols are two poly-phenolic compounds that play an important role in stabi-lizing lipid oxidation and are associated with antioxidant activity (Yen et al 1993) Phenolic compounds may con-tribute directly to antioxidative action (Duh et al 1999) Polyphenolic compounds may have an inhibitory effect on mutagenesis and carcinogenesis in humans when as much as 10 g is ingested daily from a diet rich in fruits and veg-etables (Tanaka et al 1998) The antioxidative activities observed can be attributed to both the different mecha-nisms exerted by different phenolic compounds and to the synergistic effects of different compounds The antioxidant

assays used in this study measured the oxidative products at the early and final stages of oxidation Antioxidants have different functional properties for example quer-cetin rutin and catechin can scavenge reactive oxygen species (Liu et al 2008) p-coumaric acids on the other hand inhibit the generation of free radicals and chain-breaking activity (Laranjinha et al 1995) and metal chela-tion (Van-Acker et al 1998) These compounds as well as flavonoids and other organic acids are highly effective electron donors However the components responsible for the antioxidative activities of the wetland medicinal plants are still unclear Further work must be performed to isolate and identify these components

In conclusion the results from these in vitro experi-ments including ABTS radical monocation scavenging (Table 2) DPPH radical scavenging (Table 3) reducing power method (Table 4) total polyphenol content total flavonoid content and total flavonol content (Table 5 and 6) demonstrated that phytochemicals in wetland medicinal plants might have significant effects on antioxidant activi-ties However the quantity of polyphenols and flavonoids found in the wetland medicinal plant extracts were not directly related to their antioxidant activities The additive roles of phytochemicals might contribute significantly to the potent antioxidant activity Hence some wetland me-dicinal plants could be used as an easy accessible source of natural antioxidants in pharmaceutical and medical in-dustries For this reason further work should be performed to isolate and identify the antioxidative components of wetland medicinal plants

Acknowledgements This study was supported by a grant CCMP-97-RD-001 from the Committee on Chinese Medicine and Pharmacy Department of Health Execu-tive Yuan Taiwan And China Medical University (CMU) (CMU99-S-29 CCM-P99-RD-042 and CMU99-COL-10) Taiwan Department of Heath Clinical Trial and Re-search Center of Excellence (DOH100-TD-B-111-004) and the Cancer Research Center of Excellence (DOH100-TD-C-111-005)

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Arnous A DP Makris and P Kefalas 2001 Effect of prin-cipal polyphenolic components in relation to antioxidant characteristics of aged red wines J Agric Food Chem 49 5736-5742

Awika R P Wu JM Cisneros-Zevallos LW Awika XL Rooney RL Wu and L Cisneros-Zevallos 2003 Screen-ing methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products J Agric Food Chem 51 6657-6662

Figure 1 Correlation coefficients (R2) of TEAC and total poly-phenol contents in the water (A) and methanol (B) extracts of the wetland medicinal plants


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Chang HC GJ Huang DC Agrawal CL Kuo CR Wu and HS Tsay 2007b Antioxidant activities and polyphenol contents of six folk medicinal ferns used as ldquoGusuiburdquo Bot Stud 48 397-406

Chang HY YL Ho MJ Sheu YH Lin MC Tseng SH Wu GJ Huang and YS Chang 2007a Antioxidant and free radical scavenging activities of Phellinus merrillii ex-tracts Bot Stud 48 407-417

Chen FA AB Wu P Shieh DH Kuo and CY Hsieh 2006 Evaluation of the antioxidant activity of Ruellia tuberosa Food Chem 94 14-18

Chung KT TY Wong YW Huang and Y Lin 1998 Tannins and human health a review Critical Reviews in Food Sci-ence and Nutrition 38 421-464

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Espin JC C Soler-Rivas HJ Wichers and C Viguera-Garcia 2000 Anthocyanin-based natural colorants a new source of antiradical activity for foodstuff J Agric Food Chem 48 1588-1592

Hellioumln-Ibarrola MC DA Ibarrola Y Montalbetti D Villa-lba O Heinichen and EA Ferro 1999 Acute toxicity and general pharmacological effect on central nervous system of the crude rhizome extract of Kyllinga brevifolia Rottb J Ethnopharmacol 66 271-276

Hu C and DD Kitts 2000 Studies on the antioxidant activity of Echinaceae root extract J Agric Food Chem 48 1466-1472

Huang DJ CD Lin HJ Chen and YH Lin 2004 Antioxi-dant and antiproliferative activities of sweet potato (Ipom-oea batatas [L] Lam lsquoTainong 57rsquo) constituents Bot Bull Acad Sin 45 179-186

Huang DJ HJ Chen CD Lin and YH Lin 2005 Antioxi-dant and antiproliferative activities of water spinach (Ipom-oea aquatica Forsk) constituents Bot Bull Acad Sin 46 99-106

Lamaison JLC and A Carnet 1990 Teneurs en principaux flavonoids des fleurs de Crataegeus monogyna Jacq et de Crataegeus laevigata (Poiret D C) en fonction de la vegeta-tion Pharm Acta Helv 65 315-320

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臺灣濕地藥用植物之抗氧化活性和總多酚含量

何玉鈴1　黃世勳1　鄧正賢2　林耀輝3　張永勳4　黃冠中4

1弘光科技大學護理系2亞洲大學保健營養生技學系3中央研究院植物暨微生物學研究所4中國醫藥大學中國藥學暨中藥資源系

本文章研究目的是評估台灣濕地藥用植物之甲醇和水萃取物之抗氧化活性評估的項目包括

ABTS清除清除 DPPH自由基還原力總多酚含量總類黃酮類含量總黃酮醇類含量結果顯示31種濕地藥用植物中以水豬母乳燈心草碎米莎草水柳泥花草短葉水蜈蚣和香蒲共七種其抗氧化物和多酚類均具不錯之效果和含量且由抗氧化活性和總多酚含量之線性相關係數結果得

知水萃取物相關係數為 014和甲醇萃取物相關係數為 023結果顯示植物中化學物質含量可能有助於顯著的抗氧化活性但這種關係並不一定成正比濕地藥用植物未來在醫藥和保健食品行業中將可

作為一個容易取得的天然抗氧化劑的來源

關鍵詞濕地藥用植物抗氧化劑多酚類黃酮類黃酮醇類


mATeRIALS AND meThODS

materialsButylated hydroxytoluene (BHT) Glutathione (GSH)

11-Diphenyl-2-picrylhydrazyl (DPPH) 6-Hydroxy-2 5 7 8-tetramethylchroman-2-carboxylic acid (Trolox) po-tassium peroxodisulfate (K2S2O8) Tris (hydroxylmethyl) aminomethane potassium ferricyanide (K3Fe(CN)6) ferric chloride (FeCl3) catechin 22rsquo-azinobis-(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) and other chemicals were purchased from Sigma Chemical Co (St Louis MO USA) Folin-Ciocalteu solution and 95 ethanol were purchased from Merck Co (Santa Ana CA USA) Thirty-one wetland medicinal plants were collected from Taichung Nantou and Hsinchu counties in Taiwan They were identified and authenticated by Dr Chao-Lin Kuo Associate Professor and Chairman Department of Chinese Medicine Resources China Medical University Taichung Taiwan The medicinal wetland plants studied were all described in the Catalogue of Medicinal Plant Resources in Taiwan published by the Committee on Chinese Medicine and Pharmacy Taiwan Department of Health (Lin 2003)

Plant materials methanol extracts preparation Dried whole herbs (100 g for each sample) were macer-

ated with 1L 95 ethanol for 24 hours at room tempera-ture then filtered and extracted three times The ethanol extract (3 L) was then evaporated to 10 mL and dried in a vacuum at 40degC The dried extract was weighed dissolved in 95 ethanol and stored at -20degC for further use

Plant materials water extracts preparation Dried whole herbs (100 g for each sample) were boiled

with 1L distilled water for 1 hour Filtration and extract collection were performed three times The resulting de-coction was evaporated to 10 mL and dried in a vacuum at 50degC The dried extract was weighed dissolved in distilled water and stored at -20degC for further use For each extract the yield was calculated as a percentage of the dry weight of the whole herbs used (100 g) and the quantity of dry mass obtained after extraction (ww)

TEAC antioxidant activity determination A TEAC assay was conducted based on the method of

Ramos et al (1999) The ABTS aqueous solution (7 mM) was oxidized with potassium peroxodisulfate (245 mM) for 16 hours in the dark at room temperature The ABTSmiddot+ solution was diluted with 95 ethanol to an absorbance of 075 plusmn 005 at 734 nm (Beckman UV-Vis spectrophotom-eter Model DU640B) An aliquot (20 μL) of each sample (125 μgmL) was mixed with 180 μL ABTSmiddot+ solution and the absorbance was read at 734 nm after 1 min Trolox was used as a reference standard A standard curve was constructed for Trolox at 0 15625 3125 625 125 250 500 μM concentrations TEAC value was expressed as millimolar concentration of Trolox solution with the an-

tioxidant equivalent to a 1000 ppm solution of the sample under investigation

DPPh radical scavenging antioxidant activity determination

The effects of crude extracts and positive controls (GSH and BHT) on DPPH radicals were estimated based on the method of Yamaguchi et al (1998) Aliquots (20 μL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 μL pH 74) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark The absorbance of the reaction solution was measured spectro-photometrically at 517 nm The percentage of DPPH de-colorization of the samples was calculated according to the equation decolorization = [1- (ABS sample ABS control)] times100 IC50 value was the effective concentration at which DPPH radicals were scavenged by 50 and was obtained by interpolation from linear regression analysis A lower IC50 value indicated a greater antioxidant activity

Reducing power measurement The reducing power of the crude extracts and posi-

tive controls (GSH and BHT) were determined according to the method of Yen and Chen (1995) The samples (0 3125 625 125 250 500 and 1000 μgmL) were each mixed with an equal volume of 02 M phosphate buffer pH 66 and 1 potassium ferricyanide The mixture was incubated at 50degC for 20 min before an equal volume of 1 TCA was added and then centrifuged at 5000 g for 10 min The upper layer of the solution was mixed with distilled water and 01 FeCl3 with a ratio of 1 1 2 and the absorbance was measured at 700 nm Increased ab-sorbance of the reaction mixture indicated an increase in reducing power

Total polyphenol content determination Total polyphenol contents of the crude extracts were

determined according to the method of Ragazzi and Vero-nese (1973) 20 μL of each extract (125 μgmL) was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu re-agent The mixture was allowed to stand at room tempera-ture for 5 min and then 40 μL of 20 sodium carbonate was added to the mixture The resulting blue complex was then measured at 680 nm Catechin was used as a standard for the calibration curve The polyphenol content was cali-brated using the linear equation based on the calibration curve The total polyphenol content was expressed as mg catechin equivalentg dry weight The dry weight indicated was the sample dry weight

Total flavonoid content determination Total flavonoid contents of the crude extracts were de-

termined according to the method of Lamaison and Car-net (1990) Aliquots of 15 mL extracts were each added to an equal volume of 2 AlCl36H2O (2 g in 100 mL







ReSULTS









Water extract

Ethanol extract




Koehne 424 1231








































DISCUSSION









Polyphenola b











Polyphenola b


Flavonola b






































LITeRATURe CITeD
























































ReSULTS









Water extract

Ethanol extract




Koehne 424 1231








































DISCUSSION









Polyphenola b











Polyphenola b


Flavonola b






































LITeRATURe CITeD
























































































DISCUSSION









Polyphenola b











Polyphenola b


Flavonola b






































LITeRATURe CITeD
























































Polyphenola b











Polyphenola b


Flavonola b






































LITeRATURe CITeD

























































Polyphenola b


Flavonola b






































LITeRATURe CITeD
























































LITeRATURe CITeD


















































in vitro antioxidant properties and total phenolic

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