pg_0001

Botanical Studies (2008) 49: 311-322.

There two authors contributed equally to this work.

Corresponding author: E-mail: yschang@mail.cmu.edu.tw;

Tel: +886-4-22030380; Fax: +886-4-22083362.

INTRODUCTION

It is commonly accepted that under situations of oxida-

tive stress, reactive oxygen species, such as superoxide

·-

), hydroxyl (OH

·-

), and peroxyl (

OOH, ROO

) radicals,

were generated. These reactive oxygen species play an im-

portant role in degenerative or pathological processes, such

as aging (Burns et al., 2001), cancer, coronary heart dis-

ease, Alzheimer’s disease (Ames, 1983; Gey, 1990; Smith

et al., 1996; Diaz et al., 1997), neurodegenerative disor-

ders, atherosclerosis, diabetes, and inflammation (Aruoma,

1998; Chen et al., 2006). The use of traditional medicine

is comprehensive, and plants were still a large source of

natural antioxidants which might serve as leads for the

development of novel drugs. Several anti-inflammatory,

digestive, anti-necrotic, neuroprotective, and hepatopro-

Antioxidant and antiproliferative activities of the four

Hydrocotyle species from Taiwan

Shyh-Shyun HUANG

1,5

, Guan-Jhong HUANG

1,5

, Yu-Ling HO

, Yaw-Huei LIN

, Hsin-Jung HUNG

Tien-Ning CHANG

, Man-Jau CHANG

, Jial-Jhen CHEN

, and Yuan-Shiun CHANG

Institute of Chinese Pharmaceutical Sciences, College of Pharmacy, China Medical University, Taichung 404, Taiwan,

ROC

Department of Nursing, Hung Kuang University, Sha Lu, Taichung 433, Taiwan, ROC

Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, ROC

Department of Applied Cosmetics Science, Ching Kuo Institute of Management and Health, Keelung 203, Taiwan, ROC

(Received February 5, 2008; Accepted May 22, 2008)

ABSTRACT.

The aim of this study was to examine the possible antioxidant and antiproliferative activi-

ties of the ethanol and water extracts of four Hydrocotyle species from Taiwan. ABTS radical monocation

scavenging, FRAP method, DPPH radical scavenging, reducing power method, total polyphenol content, total

flavonoid content, total flavonol content, and inhibition of cancer cell proliferation methods were employed.

The results showed that the water extracts of all the samples had higher antioxidant and antiproliferative ac-

tivities than the ethanol extracts. All tested extracts were weaker than the positive controls (BHT and GSH)

in the antioxidant activity. We also found that the water extracts of all the samples had higher content of

polyphenol compounds, but lower content of flavonoid compounds than the ethanol extracts. In ABTS radical

scavenging assay, the TEAC (trolox equivalent antioxidant capacity) values of the water extracts samples were

in descending order: H. nepalensis (HN) > H. setulosa (HSe) > H. batrachium (HB) > H. sibthorpioides (HSi).

The correlation coefficient (R

) values of TEAC and total polyphenol content showed a higher correlation

(water extracts, R

=0.934; ethanol extracts, R

=0.904). The R

values of TEAC and total flavonoid content

for the water and ethanol extracts were 0.995 and 0.785 respectively. The R

values of TEAC and FRAP also

showed a higher correlation (water extracts, R

=0.984; ethanol extracts, R

=0.971). In HPLC analysis, the

chromatograms of the water and ethanol extracts of HN with the highest antioxidant activity were established.

Rutin might be an important bioactive compound in HN extracts. The antiproliferative activities of the four

Hydrocotyle species were studied in vitro using human hepatoma Hep3B cells, and the results were consistent

with their antioxidant capacities. The water extract of HN had the highest antiproliferative activity with an IC

of 435.88 ± 8.64 μg/mL. The ethanol extracts of HB and HSi had the lowest antiproliferative activities (IC

2000 μg/mL) under the experimental conditions. We suggested that HN might be served as a good source of

natural antioxidant among the Hydrocotyle genus.

Keywords: Antioxidant; Antiproliferative; Free radical; Flavonoid; Hep3B; Hydrocotyle; Polyphenol.

Abbreviations: TEAC, trolox equivalent antioxidant capacity; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyl-tetrazolium bromide; GSH, glutathione reduced form; BHT, butylate hydroxyltoluene; DPPH, 1,

1-diphenyl-2-picrylhydrazyl; FRAP, ferric reducing antioxidant power; TCA, trichloroacetic acid; FBS, fetal

bovine serum; DMEM, Dulbecco’s modified Eagle medium; EDTA, ethylenediamine tetraacetic acid; IC

concentration with 50% inhibition; ABS, absorbance; HPLC, high performance liquid chromatography.

BIOChemISTRy

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312

Botanical Studies, Vol. 49, 2008

tective drugs recently have been shown to have an antioxi-

dant and/or radical scavenging mechanism as part of their

activity (Perry et al., 1999; Lin and Huang, 2002; Repetto

and Llesuy, 2002). In searching for sources of natural anti-

oxidants and compounds with radical scavenging activity

during the last few years, some had been found, such as

echinacoside in Echinaceae root (Hu and Kitts, 2000), an-

thocyanin (Espin et al., 2000), phenolic compounds (Rice-

Evans et al., 1997), and the extracts of water spinach and

sweet potato organs (Huang et al., 2004; Huang et al.,

2005).

The Hydrocotyle species (Apiaceae family) were the

important original plants of Pai-Tsao-Tsa, which was a

kind of popular folk drink inherited from ancient time

in Taiwan. Pai-Tsao-Tsa made with either traditional

Chinese medicine or Taiwan folk medicine has efficacy

and beneficial effects to our health (Chiu, 2004). The

whole plants of the Hydrocotyle species are often used in

Taiwan folk medicine for treating common cold, tonsillitis,

cephalitis, enteritis, dysentery, zoster, eczema, period

pain, hepatitis and jaundice (Chang et al., 2003). Only

few studies have confirmed the pharmacological activity

of members of the genus Hydrocotyle. For example,

H. sibthorpioides (HSi) could inhibit the growth of

transplanted tumors in mice, such as hepatic carcinoma

(Hep), sarcoma (S

180

) and uterine cervical carcinoma

). Both H. leucocephala Cham. & Schlecht. and HSi

have immunomodulatory effects (Ramos et al., 2006; Yu

et al., 2007). However, no studies to date had been able

to demonstrate the antioxidant effect of the Hydrocotyle

species. The antitumor effect was also not fully

understood, and the pharmacological data for these herbs

were incomplete.

The objectives of this work were to investigate the an-

tioxidant and antiproliferative property of crude extracts

from H. batrachium Hance (HB), H. nepalensis Hook

(HN), H. setulosa Hayata (HSe), and H. sibthorpioides

Lam. (HSi) in comparison with chemical compounds such

as BHT, GSH or rutin and the level of inhibition of the

growth of cancer cells in series of in vitro tests.

mATeRIALS AND meThODS

materials

BHT, GSH, potassium peroxodisulfate (K

DPPH, Tris (hydroxylmethyl) aminomethane, tryp-

sin, potassium ferricyanide (K

Fe(CN)

), TCA, ferric

chloride (FeCl

), (+)-catechin, MTT, aluminum chloride

hexahydrate (AlCl

·6H

O), rutin, 2, 2’-azinobis-(3-

ethylbenzothiazoline)-6-sulphonic acid (ABTS), sodium

bicarbonate (Na

), sodium phosphate dibasic

(Na

HPO

), sodium phosphate monobasic (NaH

)

and other chemicals were purchased from Sigma Chemi-

cal Co. (St. Louis, MO, USA). Folin-Ciocalteu solution

and 95% ethanol were purchased from Merck Co. (Santa

Ana, CA, USA). FBS was purchased from Gibco BRL Co.

(Gaithersburg, MD, USA). DMEM was purchased from

Invitrogen Corp. (Carlsbad, CA, USA). Plant materials

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 Recourses, China Medi-

cal University, Taichung, Taiwan.

Preparation of the ethanol extracts of plant

materials

Dried whole herb (100 g each) was macerated with

1000 mL 95% ethanol for 24 h at room temperature. Fil-

tration and collection of the extract were done three times.

Then the ethanol extract (3000 mL) was evaporated to 10

mL and dried in vacuum at 40°C. The dried extract was

weighted and dissolved in 95% ethanol (stock 4 mg/mL)

and stored in -20°C for further use.

Preparation of the water extracts of plant

materials

Dried whole herb (100 g each) was boiled with 1000

mL distilled water for 1 h. Filtration and collection of the

extracts were done three times. Resulting decoction (about

1000 mL) was evaporated to 10 mL and dried in vacuum

at 50°C. The dried extract was weighted and dissolved in

distilled water (stock 4 mg/mL) and stored in -20°C for

further use. For each sample, yields were calculated in

percentages on the basis of dry weight of the whole herb

used (100 g) and the quantity of dry mass obtained after

the extraction.

Determination of antioxidant activity by ABTS

·+

scavenging ability

The ABTS

·+

scavenging ability was determined accord-

ing to the method of Re et al. (1999). Aqueous solution of

ABTS (7 mM) was oxidized with potassium peroxodisul-

fate (2.45 mM) for 16 hours in the dark at room tempera-

ture. The ABTS

·+

solution was diluted with 95% ethanol

to an absorbance of 0.75 ± 0.05 at 734 nm (Beckman UV-

Vis spectrophotometer, Model DU640B). An aliquot (20

μL) of each sample (125 μg/mL) was mixed with 180 μL

ABTS

·+

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, 15.625,

31.25, 62.5, 125, 250, 500 μM concentration. The TEAC

was expressed as millimolar concentration of trolox solu-

tion with the antioxidant equivalent to a 1000 ppm solu-

tion of the sample under investigations.

Ferric reducing antioxidant power assay

The ferric reducing antioxidant power (FRAP)

assay of the crude extracts was determined according

to the method of Benzie and Strain (1996). This assay

measured the change in the absorbance at 593 nm owing

to the formation of a blue colored Fe

-tripyridyltriazine

compound from colorless oxidized Fe

form by the action

of electron donating antioxidants. To prepare the FRAP

reagent, a mixture of 0.1 M acetate buffer (pH 3.6), 10

pg_0003

HUANG et al. — Antioxidant and antiproliferative activities of the four

Hydrocotyle

species

313

mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and 20 mM

ferric chloride (10:1:1, v/v/v) was made. An aliquot (10

μL) of each sample (125 μg/mL concentration) was mixed

with 300 μL FRAP reagent and the absorbance was read

at 593 nm after 15 min. A standard curve was constructed

for FeSO

·7H

O at 0, 31.25, 62.5, 125, 250, 500, 1000

μg/mL concentration. In the FRAP assay, the antioxidant

efficiency of the sample was calculated with reference

to the reaction signal given by an Fe

solution of known

concentration, which represented a one-electron exchange

reaction. The results were corrected for dilution and

expressed in μmol Fe

/mg.

Determination of antioxidant activity by DPPh

radical scavenging ability

The effect of crude extracts and positive controls (GSH,

BHT and rutin) on the DPPH radical was estimated ac-

cording to the method of Yamaguchi et al. (1998). An

aliquot (20 μL) of crude extracts at various concentrations

was mixed with 100 mM Tris-HCl buffer (80 μL, pH 7.4)

and then with 100 μL of the DPPH in ethanol to a final

concentration of 250 μM. The mixture was shaken vigor-

ously and left to stand at room temperature for 20 min in

the dark. The absorbance at 517 nm of the reaction solu-

tion was measured spectrophotometrically. The percentage

of DPPH decolorization of the samples were calculated ac-

cording to the equation: % decolorization = [1- (ABS

sample

/ABS

control

) ] ×100. IC

value was the effective concen-

tration at which DPPH radicals were scavenged by 50%

and was obtained by interpolation from linear regression

analysis. A lower IC

value indicated a greater antioxidant

activity.

measurement of reducing power

The reducing power of the crude extracts and positive

controls (GSH and BHT) was determined according to the

method of Yen and Chen (1995). The samples (0, 31.25,

62.5, 125, 250, 500, and 1000 μg/mL) were mixed with an

equal volume of 0.2 M phosphate buffer, pH 6.6, and 1%

potassium ferricyanide. The mixture was incubated at 50

°C for 20 min, after which an equal volume of 1% TCA

was added to the mixture, which was then centrifuged at

5,000 g for 10 min. The upper layer of the solution was

mixed with distilled water and 0.1% FeCl

with a ratio

of 1:1:2, and the absorbance at 700 nm was measured.

Increased absorbance of the reaction mixture indicated an

increase in reducing power.

Determination of total polyphenol content

The total polyphenol content of the crude extracts was

determined according to the method of Ragazzi and Ve-

ronese (1973). 20 μL of each extract (125 μg/mL) was

added to 200 μL distilled water and 40 μL of Folin-Cio -

calteu reagent. The mixture was allowed to stand at room

temperature 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 polyphe-

nol content was calibrated using the linear equation based

on the calibration curve. The total polyphenol content was

expressed as mg (+)-catechin equivalent/g dry weight. The

dry weight indicated was the sample dry weight.

Determination of total flavonoid content

The total flavonoid content of the crude extracts was

determined according to the method of Lamaison and

Carnet (1990). Aliquots of 1.5 mL of extracts were added

to equal volumes of a solution of 2% AlCl

·6H

O (2 g in

100 mL methanol). The mixture was vigorously shaken,

and the absorbance at 430 nm was read after 10 min of in-

cubation. Rutin was used as a standard for the calibration

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 equivalent/g

dry weight. The dry weight indicated was the sample dry

weight.

Determination of total flavonol content

The total flavonol content of the crude extracts was de-

termined according to the method of Arnous et al. (2001).

Aliquots of 200 μL of extracts were added to 1 mL of 0.1%

p-dimethylaminocinnamaldehyde (DMACA) in methanol/

HCl (3:1, v/v). The mixture was vigorously shaken, and

the absorbance at 640 nm was read after 10 min of incuba-

tion. (+)-Catechin was used as a standard for the calibra-

tion curve. The total flavonol content was calibrated using

the linear equation based on the calibration curve. The

total flavonol content was expressed as mg (+)-catechin

equivalent/g dry weight. The dry weight indicated was the

sample dry weight.

Analysis of rutin, quercetin, hNW, and hNe by

hPLC

A moderate amount of the ethanol and water extracts

from H. nepalensis Hook. (HN) were weighed and dis-

solved in ethanol and water, respectively. At first, the

solutions were filtered through 0.45 μm PVDF filters.

The HPLC (Waters 2695 separations module; detector:

Waters 996 photodiode array detector) analysis was car-

ried out under the following conditions; the Waters XTerra

RP18 column (5 μm, 4.6 × 250 mm) was used with 0.05%

phosphate buffer as mobile phase A, and acetonitrile was

used as mobile phase B; the gradient elution was run with

30% of solution B at 0 min, 35% of solution B at 30 min

at a flow rate of 0.8 mL/min; the injection volume was

10 μL, and a wavelength of 254 nm was used for detec-

tion. Pure compounds, including rutin and quercetin, were

also analyzed by HPLC with the same conditions, and the

retention time was used to identify the flavonoids in the

samples.

Culture and harvest of human hepatoma cell

line

Hep3B cells were cultured with DMEM with 10% FBS

pg_0004

314

Botanical Studies, Vol. 49, 2008

in a T75 flask at 37°C, 5 % CO

, and 90% relative humid-

ity. To harvest cells, Hep3B cells were washed with PBS

buffer and treated with 4 mL of trypsin-EDTA for 3 min.

The reaction was stopped by adding 8 mL of DMEM with

10% FBS. The mixture was then transferred into a tube

and centrifuged at 200 g at room temperature for 5 min.

After removing the supernatant, cell pellet was resuspend-

ed in 4 mL of DMEM with 10% FBS.

mTT assay for cell proliferation

The colorimetric assay for cellular growth and survival

was based on Hansen et al. (1989). Suspensions of hu-

man Hep3B cells (2 × 10

cells/mL) were cultured with or

without the test samples (at various concentrations in 10

μL of suspension) in a 96-well microplate (90 μL suspen-

sion/well). After 72 h, 10 μL of MTT solution was added

to each well, and the cells were incubated at 37°C for 4

h. Then, 100 μL of lysis buffer were added to each well,

and the cells were again incubated at 37°C for 1 hour to

dissolve the dark blue crystals. Each well was completely

pipetted, and then the absorption at 570 nm of formazan

product was measured using a microplate reader. Each

sample was repeated with the above procedures in order

to determine the cell proliferation. The decolorization was

plotted against the concentration of the sample extract, and

the IC

, which was the amount of the sample necessary to

decrease 50% of the absorbance of MTT, was calculated.

Statistical analysis

Experimental results were presented as the mean ± stan-

dard deviation (SD) of three parallel measurements. The

statistical analyses were performed by one-way ANOVA,

followed by Dunnett’s t test. The difference was consid-

ered to be statistically significant when the p value was

less than 0.05.

ReSULTS AND DISCUSSION

extraction yields

The yields in the water and ethanol extracts of the four

Hydrocotyle species were given in Table 1. The percent-

ages of the ethanol extract (code as E) yields in descending

order were as follows: HSiE (20.26%) > HBE (13.14%) >

HNE (12.95%) > HSeE (11.16%). On the other hand, the

water extract (code as W) yields in decreasing order were

as follows: HSiW (28.55%) > HBW (26.37%) > HSeW

(22.68%) > HNW (11.55%).

Antioxidant activity estimated by ABTS assay

ABTS assay are often used in evaluating total

antioxidant power of single compounds and complex

mixtures of various plants (Katalinic et al., 2006; Chang et

al., 2007a, b). In this assay, ABTS radical monocation was

generated directly in stable form from potassium perox-

odisulfate. Generations of radical before the antioxidants

were added to prevent interference of compounds, which

affected radical formation. This modification made

the assay less susceptible to artifacts and prevented

overestimation of antioxidant power (Sanchez-Moreno,

2002). The antioxidant sample was added to the reaction

medium when the stable absorbance was obtained,

and the antioxidant activity was measured in terms of

decolorization.

ABTS assay was expressed as TEAC value. Higher

TEAC value represented that the sample had a stronger

antioxidant activity. TEAC values determined from the

calibration curve for the four Hydrocotyle species were

shown in Figure 1A. Antioxidant activities of the water

and ethanol extracts of the four Hydrocotyle species

were in the following decreasing order: HNW (0.48 ±

0.03 mM) > HSeW (0.40 ± 0.02 mM) > HBW (0.36 ±

0.01 mM) > HSiW (0.23 ± 0.01 mM) > HSeE (O.22 ±

0.00 mM) > HNE (0.17 ± 0.00 mM) > HBE (0.16 ± 0.00

mM) > HSiE (0.08 ± 0.00 mM). Thus, it was observed that

the water extracts of all the samples had higher antioxi-

dant potencies than the ethanol extracts, and HNW had the

highest activity.

Ferric reducing antioxidant power assay

As shown in Figure 1B, there was a significant

difference in total antioxidant power (FRAP) between the

samples. The FRAP values varied from 1.55 to 4.02 μmol

/mg in the water extracts, and 0.51 to 1.29 μmol Fe

mg in the ethanol extracts. The FRAP values of the eight

samples decreased in the following order: HNW (4.02 ±

0.03 μmol Fe

/mg) > HSeW (3.43 ± 0.02 μmol Fe

/mg)

> HBW (2.70 ± 0.05 μmol Fe

/mg) > HSiW (1.55 ± 0.01

μmol Fe

/mg) > HSeE (1.29 ± 0.07 μmol Fe

/mg) > HNE

(1.06 ± 0.04 μmol Fe

/mg) > HBE (0.87 ± 0.03 μmol

/mg) > HSiE (0.51 ± 0.00 μmol Fe

/mg). Thus, it was

Table 1. The yields in the water and ethanol extracts of four Hydrocotyle species.

Species

Water extract yield (% w/w)

Ethanol extract yield (% w/w)

H. batrachium (HB)

26.37%

13.14%

H. nepalensis (HN)

11.55%

12.95%

H. setulosa (HSe)

22.68%

11.16%

H. sibthorpioides (HSi)

28.55%

20.26%

Dried weight basis.

pg_0005

HUANG et al. — Antioxidant and antiproliferative activities of the four

Hydrocotyle

species

315

also observed that the water extracts of all the samples had

higher antioxidant potencies than the ethanol extracts, and

HNW had the highest activity.

Both FRAP and TEAC assay were used to estimate

the total antioxidant power because they were quick and

simple to perform, and the reaction was reproducible

and linearly related to the molar concentration of the

antioxidants (Benzie et al., 1999). FRAP assay was

initially developed to assay the plasma antioxidant

capacity, but could also be used to measure the antioxidant

capacity of a wide rage of biological samples, pure

compounds, fruits, wines, and animal tissues (Katalinic et

al., 2006).

The correlation coefficients (R

) of FRAP and TEAC

assay of the water and ethanol extracts of the four

Hydrocotyle species were shown in Figure 2A and 2B.

Relationship between FRAP and TEAC assay of the water

extracts was shown in Figure 2A (R

=0.984, p < 0.001),

and that of the ethanol extracts was shown in Figure 2B

=0.971, p < 0.001). The R

values of FRAP and TEAC

assay showed higher correlation. The higher the FRAP

activity, the higher the TEAC activity of the samples.

Scavenging activity against 1,1-diphenyl-2-

picrylhydrazyl radical

The relatively stable organic radical DPPH was widely

used in the model system to investigate the scavenging

activities of several natural compounds, such as phenolics

and anthocyanins, or crude mixtures, such as the ethanol

or water extract of plants. DPPH radical was scavenged

by antioxidants through the donation of a proton forming

the reduced DPPH. The color changed from purple to yel-

low after the reduction, which could be quantified by its

decrease of the absorbance at wavelength 517 nm. Radical

scavenging activity increased with increasing percent-

age of the free radical inhibition. Table 2 showed the IC

values for the radical-scavenging activity of the different

extract fractions of the four Hydrocotyle species, GSH,

BHT, and rutin using the DPPH colorimetric method. It

was found that HNW had the lowest IC

value (84.20 ±

1.84 μg/mL), followed by HSeW (117.01 ± 1.73 μg/mL),

HBW (178.99 ± 1.87 μg/mL), HSeE (197.87 ± 2.65 μg/

mL), HNE (314.51 ± 1.04 μg/mL), HSiW (375.96 ± 2.37

μg/mL), HBE (403.31 ± 0.64 μg/mL), and HSiE (919.47

± 1.31 μg/mL). The eight extract fractions showed signifi-

Figure 1. TEAC (A) and FRAP (B) values of the water and ethanol extracts of the four Hydrocotyle species. HB: H. batrachium; HN:

H. nepalensis; HSe: H. setulosa; HSi: H. sibthorpioides. Each value represented mean ± S.D. of three parallel measurements (P < 0.05).

Figure 2. Correlation coefficients (R

) of TEAC and FRAP in the water (A) and ethanol (B) extracts of the four Hydrocotyle species.

pg_0006

316

Botanical Studies, Vol. 49, 2008

cant differences (p<0.05) in radical-scavenging activity.

Observed from the above results, the most active sample

was HNW, however, its capacity was still lower than the

three positive controls in DPPH assay.

measurement of Reducing Power

We investigated the reducing capacity of the four

Hydrocotyle species extracts by measuring the Fe

-Fe

conversion. The reducing capacity of a compound may be

served as a significant indicator of its potential antioxi-

dant activity (Meir et al., 1995). The antioxidant activities

of putative antioxidants have been attributed to various

mechanisms, such as prevention of chain initiation, bind-

ing of transition metal ion catalysts, decomposition of

peroxides, prevention of continued proton abstraction, and

radical scavenging (Diplock, 1997). The reducing power

of the different extract fractions from the four Hydrocotyle

species was shown in Figure 3. Both reduced GSH and

BHT were used as the positive controls. The different ex-

tract fractions from the four Hydrocotyle species exhibited

a dose-dependent reducing power activity within concen-

tration range of 0, 31.25, 62.5, 125, 250, 500, and 1000

μg/mL. HNW had the highest reducing power, followed by

HBW, HSeW, HSeE, HNE.HBE, HSiW, and HSiE. The

water extracts of the four Hydrocotyle species had higher

reducing power than the ethanol extracts. The different ex-

tract fractions showed significant differences (p<0.05) in

reducing power.

Total polyphenol, flavonoid, and flavonol

content

The total polyphenol, flavonoid, and flavonol content of

the four Hydrocotyle species were shown in Table 3. The

total polyphenol content was expressed as μg of (+)-cat-

echin equivalent per milligram of dry weight. The total

polyphenol content of the extracts of the four Hydrocotyle

species ranged from 34.17 to 115.77 μg CE/mg, and

decreased in the following order: HNW > HSeW > HSeE

> HBW > HNE > HBE > HSiW > HSiE. The water ex-

tracts of the four Hydrocotyle species had higher polyphe-

nolic content than the ethanol extracts.

The total flavonoid content was expressed as μg of rutin

equivalent per milligram of dry weight. The total flavonoid

content of the extracts of the four Hydrocotyle species

ranged from 15.68 to 86.67 μg RE/mg, and decreased

as the following order: HSeE > HNE > HBE > HSiE >

HNW> HSeW > HBW > HSiW. The ethanol extracts of

the four Hydrocotyle species had higher flavonoid content

than the water extracts.

The total flavonol content was expressed as μg of

(+)-catechin equivalent per milligram of dry weight.

The total flavonol content of the extracts of the four

Hydrocotyle species ranged from 4.10 to 21.09 μg CE/mg,

and decreased as the following order: HSeE > HSiE >

HNW > HBW > HNE > HSeW > HBE > HSiW. The

ethanol extracts of HSe and HSi had higher flavonol

content than the water extracts. The ethanol extracts of HB

and HN had lower flavonol content than the water extracts.

Figu re 3. Antioxidant activities of the water and the ethanol

extracts (0, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL) of the

four Hydrocotyle species, as measured by the reducing power

method. BHT and GSH were used as the positive controls. Each

value represented mean ± S.D. of three parallel measurements

(P<0.05).

Table 2. IC

values of the water and ethanol extracts of four Hydrocotyle species in DPPH radical scavenging activity

Species and positive controls

Water extract IC

(μg/mL)

Ethanol extract IC

(μg/mL)

H. batrachium (HB)

178.99 ± 1.87

403.31 ± 0.64

H. nepalensis (HN)

84.20 ± 1.84

314.51 ± 1.04

H. setulosa (HSe)

117.01 ± 1.73

197.87 ± 2.65

H. sibthorpioides (HSi)

375.96 ± 2.37

919.47 ± 1.31

GSH

49.63 ± 0.25

N.D.

BHT

N.D.

41.06 ± 0.76

Rutin

N.D.

15.96 ± 0.55

Values represented mean ± S.D. of three parallel measurements (P<0.05).

N.D.: Not detected.

pg_0007

HUANG et al. — Antioxidant and antiproliferative activities of the four

Hydrocotyle

species

317

Both flavonoid and flavonol belong to polyphenolic

compounds. Polyphenolic compounds have an impor-

tant role in stabilizing lipid oxidation and are associated

with antioxidant activity (Yen et al., 1993). The phenolic

compounds may contribute directly to antioxidative ac-

tion (Duh et al., 1999). It is suggested that polyphenolic

compounds have inhibitory effects on mutagenesis and

carcinogenesis in humans when up to 1.0 g is daily in-

gested from a diet rich in fruits and vegetables (Tanaka

et al., 1998). The antioxidative activities observed could

be ascribed both to the different mechanisms exerted by

different phenolic compounds and to the synergistic ef-

fects of different compounds. The antioxidant assay used

in this study measured the oxidation products at the early

and final stages of oxidation. The antioxidants had differ-

ent functional properties, such as reactive oxygen species

scavenging, e.g. quercetin, rutin, and catechin (Hatano et

al., 1989; Liu et al., 2008); inhibition of the generation of

free radicals and chain-breaking activity, e.g. p-coumaric

acids (Laranjinha et al., 1995) and metal chelation (Van-

Acker et al., 1998). These compounds were normally

phenolic compounds, which were effective proton donors,

including tocopherols, flavonoids, and other organic acids.

However, the components which were responsible for the

antioxidative activity of the four Hydrocotyle species were

currently unclear. Therefore, further work must be per-

formed to isolate and identify these components.

Relationship between total antioxidant power

and total polyphenol, flavonoid, and flavonol

content

The correlation coefficients (R

) of total antioxidant

power (TEAC) and total polyphenol, TEAC and total

flavonoid, and TEAC and total flavonol of the water and

Table 3. The total polyphenol, flavonoid, and flavonol content in the water and ethanol extracts of four Hydrocotyle species

Species

Total polyphenols

(

g CE/mg)

Total flavonoids

(

g RE/mg)

Total flavonols

(

g CE/mg)

Water extract

H. batrachium (HB)

80.83 ± 0.55

24.81 ± 2.82

16.38 ± 0.07

H. nepalensis (HN)

115.77 ± 1.61

31.48 ± 0.74

18.00 ± 0.37

H. setulosa (HSe)

110.27 ± 0.86

27.05 ± 0.61

11.45 ± 0.35

H. sibthorpioides (HSi)

34.63 ± 0.37

15.68 ± 0.34

4.10 ± 0.14

Ethanol extract

H. batrachium (HB)

53.93 ± 0.79

47.32 ± 0.56

9.43 ± 0.17

H. nepalensis (HN)

74.53 ± 1.73

66.07 ± 2.65

12.35 ± 0.01

H. setulosa (HSe)

84.47 ± 6.14

86.67 ± 1.17

21.09 ± 0.35

H. sibthorpioides (HSi)

34.17 ± 0.73

41.53 ± 3.71

18.26 ± 0.40

Values represented mean ± S.D. of three parallel measurements.

Data expressed in

g (+)-catechin equivalent / mg dry weight (

g CE/mg).

Data expressed in

g rutin equivalent / mg dry weight (

g RE/mg).

Figu re 4. Correlation coefficients (R

) of TEAC and total

polyphenol content in the water (A) and the ethanol (B) extracts

of the four Hydrocotyle species.

pg_0008

318

Botanical Studies, Vol. 49, 2008

ethanol extracts were shown in Figures 4, 5 and 6. The

values of TEAC and total polyphenol content of the

water (Figure 4A) and the ethanol (Figure 4B) extracts

were 0.934 and 0.904, respectively. Similarly, R

values of

TEAC and total flavonoid content of the water (Figure 5A)

and the ethanol (Figure 5B) extracts were 0.995 and 0.785

respectively. R

values of TEAC and total flavonol content

of the water (Figure 6A) and the ethanol (Figure 6B) ex-

tracts were 0.766 and 0.008 respectively. Among the above

3 statistics, we could see that there were higher correlation

between the TEAC and total polyphenol / total flavonoid.

The higher the TEAC activity, the higher the total poly-

phenol and flavonoid content of the samples.

Compositional analysis of rutin, quercetin,

hNW, and hNe by hPLC

The water and ethanol extracts of H. nepalensis (HN)

were found to have higher antioxidant activities and

polyphenolic compounds than most of other samples.

HNW and HNE were analyzed by HPLC and their chro-

matograms were shown in Figure 7. Both HNW and HNE

had rutin, but quercetin was not found in the samples. Ru-

tin, a glycoside comprised of the flavonol quercetin and the

disaccharide rutinose, widely distributes in plant kingdom,

and shows remarkable antioxidant, anti-inflammatory and

anticancer activities. It also has relaxing effects on smooth

muscles (Liu et al., 2008). In DPPH assay, we found that

rutin had much lower IC

value (15.96 ± 0.55 μg/mL)

than all the samples. Rutin might be an important compo-

nent in the antioxidant activity of the HN extracts.

Previous chemical studies of the members of the

genus Hydrocotyle had resulted in isolation of trans-

β-farnesene, α-terpinenes, and thymol methyl ether

from H. sibthorpioides Lam. and H. maritime Honda

(Asakawa et al., 1982), quercetin-3-O-galactoside from

H. umbellata L. (Adams et al., 1998), monogalactosyl

monoacylglycerol from H. ramiflora Maxim. (Kwon et

al., 1998), oleane and ursane type glycosides from H .

ranunculoides Blume (Della Greca et al., 1994a, b) and H.

sibthorpioides (Matsushita et al., 2004), diacetylene from

H. leucocephala Cham. & Schlecht. (Ramos et al., 2006).

However, there were no chemical studies on H. batrachi-

um Hance, H. nepalensis Hook. and H. setulosa Hayata.

There were few reports on the flavonoid compounds of

the genus Hydrocotyle. Therefore, it is worthy to study the

phytochemicals of the genus Hydrocotyle.

Figu re 5. Correlation coefficients (R

) of TEAC and total

flavonoid content in the water (A) and the ethanol (B) extracts of

the four Hydrocotyle species.

Fi gu re 6. Correla tion coefficie nt s (R

) of TE AC a nd tota l

flavonol content in the water (A) and the ethanol (B) extracts of

the four Hydrocotyle species.

pg_0009

HUANG et al. — Antioxidant and antiproliferative activities of the four

Hydrocotyle

species

319

measurement of cell proliferation

Antiproliferative activities of the different extract frac-

tions from the four Hydrocotyle species on the growth

of the human hepatoma 3B cell line in vitro were sum-

marized in Figure 8 and Table 4. The cell proliferation

was analyzed 72 h after 3B cells had been cultured with

an extract fraction of 0, 62.5, 125, 250, 500, 1000, 2000

μg/mL in the final concentration using the MTT assay. 3B

cell proliferation was inhibited in a dose-dependent man-

ner after exposure to the different extract fractions. The

antiproliferative activities of each fraction were expressed

as the median IC

. A lower IC

value indicating a higher

antiproliferative activity. The water extracts of the four

Figure 8. Pe rce nt inhi bit ion of 3B c el l pro lif era tio n by

diffe rent e xtracts from the four Hydrocotyle species . Each

value represented mean ± S.D. of three parallel measurements

(P<0.05).

Figure 7. HPLC of HNW and HNE extracts. (A) standards, (B)

HNW, and (C) HNE.

Hydrocotyle species had a higher antiproliferative activity

than the ethanol extracts. HNW had the highest antipro-

liferative activity with the lowest EC

of 435.88 ± 8.64

μg/mL, followed by HSeW (560.06 ± 1.90 μg/mL), HSiW

(952.34 ± 3.27 μg/mL), HBW (1216.99 ± 1.79 μg/mL),

HSeE (1249.45 ± 3.24 μg/mL), HNE (1301.59 ± 3.38

μg/mL). Both HBE and HSiE had the highest IC

values

(> 2000 μg/mL), and it was shown that the two samples

had the lowest antiproliferative activities under the experi-

mental conditions. Significant differences (p<0.05) in anti-

proliferative activity appeared among the different extract

fractions.

The antioxidant activities of the different extract frac-

tions were directly correlated to the total amount of

polyphenols and flavonoids found in each fraction. Their

antiproliferative activities were consistent with their an-

tioxidant activities. This experiment suggested that the

antiproliferative activities of the four Hydrocotyle species

might be also correlated to the total amount of polyphenols

and flavonoids found in each fraction.

In conclusion, the results from in vitro experiments,

including ABTS radical monocation scavenging (Fig-

ure 1A), FRAP method (Figure 1B), DPPH radical

scavenging (Table 2), reducing power method (Figure

3), total polyphenol content, total flavonoid content and

total flavonol content (Table 3), HPLC assay (Figure 7),

and inhibition of cancer cell proliferation (Figure 8 and

Table 4), demonstrated that the phytochemicals in the four

Hydrocotyle species might have a significant effect on

antioxidant and anticancer activities, which were directly

related to the total amount of polyphenols and flavonoids

found in the four Hydrocotyle species extracts. The ad-

ditive roles of phytochemicals might contribute signifi-

cantly to the potent antioxidant activity and the ability to

inhibit tumor cell proliferation in vitro. Hence, the four

Hydrocotyle species could be used as an easy accessible

source of natural antioxidants in pharmaceutical and medi-

cal industries. For this reason, further work should be

performed to isolate and identify the antioxidative or anti-

proliferative components of the four Hydrocotyle species.

Table 4. IC

values of the water and ethanol extracts of four

Hydrocotyle species in inhibiting Hep3B cell proliferation

Species

Water extract IC

(μg/mL)

Ethanol extract IC

(μg/mL)

H. batrachium (HB) 1216.99 ± 1.79 > 2000

H. nepalensis (HN) 435.88 ± 8.64 1301.59 ± 3.38

H. setulosa (HSe)

560.06 ± 1.90 1249.45 ± 3.24

H. sibthorpioides (HSi) 952.34 ± 3.27

> 2000

Values represented mean ± S.D. of three parallel measure-

ments (P<0.05).

pg_0010

320

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