28

Thai J. Pharm. Sci. 30 (2006) 28-35

Antioxidant activity, phenolic compound contents and antimutagenic

activity of some water extract of herbs
Kalyarat Kruawan and Kaew Kangsadalampai*
Division of Food and Nutrition Toxicology, Institute of Nutrition, Mahidol University, Salaya,
Nakhon Pathom 73170, Thailand
*Corresponding author. Tel: (662) 8002380 ext. 399, Fax: (662) 4419344, E-mail address: nukks@mahidol.ac.th

Abstract:
Ten water extracts from herbs were prepared by boiling in hot water for 10 min. The herbal extracts
were studied for their antioxidant activity and phenolic compound contents. In this study, the extracts of
Carthamus tinctorius L., Hibiscus sabdariffa L., Chrysanthemum indicum L., Aegle marmelos L. and Jubliang
exhibited strong scavenging activity against DPPH radicals (> 90%), and their ferric reducing antioxidant
power (FRAP) value ranged from 1140.5 to 2295.5 mol/g. The content of phenolic compound in the
extracts was determined using the Folin-Ciocalteu reagent and calculated as gallic acid equivalents (GAE).
The herbal extracts that gave high phenolic compound contents (GAE > 130 mg/g) were Jubliang,
A. marmelos L., C. indicum L., H. sabdariffa L., C. tinctorius L. They are related with ferric ion reduction
(FRAP value) and the percentages of scavenging activity from DPPH assay. Afterwards, the seven herbal
extracts (i.e. A. marmelos L., Andrographis paniculata Nees, C. indicum L., Cymbopogon citratus Stapf.,
H. sabdariffa L., Jubliang and Zingiber officinale Rosc.) of different phenolic compound contents and
antioxidant activity were selected for antimutagenicity assay using Ames test. At the concentrations of
8.13 - 9.26 mg/plate, the antimutagenicity of Jubliang and A. paniculata Nees was strong (> 60% inhibition)
while that of A. marmelos L. and C. indicum L. was moderate (40 - 60% inhibition). The present study has
revealed that phenolic contents of herbal drinks, except that of A. paniculata Nees, were correlated
with antimutagenic activity. Thus, herbal extracts exhibited good sources of water soluble antioxidants,
phenolic compounds and antimutagens.
Key words: Antimutagenic activity, Antioxidant activity, Herbal drinks, Phenolic compounds

K. Kruawan and K. Kangsadalampai

Introduction
Free radicals and other reactive oxygen species
are derived either from normal essential metabolic
processes in the human body or from external sources
such as exposure to radiation, ozone, cigarette
smoking, air pollutants and industrial chemicals [1]. The
free radicals are major cause of human cancer and
other diseases. That the risk of diseases can be
reduced by increased consumption of antioxidants
which are abundant in food [2]. Herbal drinks are
becoming popular especially among health-conscious
consumers since these beverages are prepared
from natural ingredients. Indigenous people have been
using herbs, such as Hibiscus sabdariffa L., Chrysanthemum indicum L., Aegle marmelos L. for a long time as
panacea drinks. Many investigators reported that
some herbal drinks contain many compounds such as
polyphenols, flavonoids, isoflavones, glucosinolates.
Polyphenols belong to the category of natural antioxidants [3]. A wide variety of phenolic substances derived
from edible plants have been reported to retain
marked antioxidant and anti-inflammatory activities,
which contribute to their chemopreventive potential [4,5].
Tseng et al. [6] reported that Thai herbs inhibited the
mutagenesis induced by various chemicals in animal
experiment. Hence, it is of importance to investigate
the phenolic contents, the antioxidant activity and
antimutagenicity of some water extract of herbs often
consumed by Thai people, and to determine the
relationships between antioxidant activity, phenolic
contents and the antimutagenic activity.

Materials and Methods

Plant material
Ten commercial grinded herbs: fruits of Aegle
marmelos L. and Garcinia atroviridis Griff. ex T.
Anderson., dried leaves of Andrographis paniculata Nees
and Schefflera leucantha R. Vig. (Hanumaanprasankhai),
dried flowers of Carthamus tinctorius L., Chrysanthemum
indicum L. and Hibiscus sabdariffa L., stems of
Cymbopogon citratus Stapf., rhizome of Zingiber officinale
Rosc. and Jubliang (a mixture of eight herbs namely,

29
Bombax ceiba L., Chrysanthemum morifolium, Imperata
cylindrical (L.) P. Beauv., Lophatherum gracile Brogn.,
Nelumbo nucifera Gaertn, Oroxylum indicum L., Pragmites
communis Trin and Prunella vulgaris) were selected
for the study. These were purchased from a health
market in Bangkok.
Preparation of water extract of herbs
Each sample of 1 g was extracted with 150 ml of
boiling water on a hot plate for 10 min. The extract was
then filtered through a cotton mesh and filter paper
Whatman no. 1. The filtrate was used directly for
antioxidant assay without storage.
Antioxidant activity assay
Free radical scavenging activity (DPPH assay) :
The antioxidant activity of the water extract from
each herb was tested using a stable radical 2,
2-diphenyl-1-picrylhydrazyl (DPPH) as described by
Fukumoto and Mazza [7] with some modifications. The
extract was allowed to react with DPPH in order to
evaluate the free radical scavenging activity. The activity
was monitored by a decrease in an absorbance at
520 nm. An aliquot of 22 l of the extract or blank
reagent (dimethyl sulfoxide, DMSO) or standard Trolox
(0.04-1.28 mM in 80% methanol) was added to 200
l of DPPH in 80% methanol (150 M) in a 96-well
flat-bottom microplate (Bibby Sterilin Ltd, UK). After
incubation at 37 C for 30 min, the absorbance of the
solution was read in a microplate reader (Sunrise,
Tecan Co., Austria) using a 520 mm filter. The radical
scavenging activity was calculated as a percentage
of DPPH scavenging activity using the equation: %
scavenging activity = 100 [1- (AE/AD)], where AE
is the absorbance of the DPPH solution with an extract
added, and AD is the absorbance of the DPPH solution
with nothing added [8].
Ferric reducing antioxidant power (FRAP)
assay :
Each of 20 l of extract or standard (ferrous
sulfate) or appropriate blank reagent (DMSO) was
added to each well in a 96-well microtiter plate and was

30
run in triplicate. FRAP reagent (150 l), freshly prepared
and warmed at 37 C according to the procedure
described by Griffin and Bhagooli [9], was added to
each well. The change in absorbance at 600 nm from
the initial blank was read after 8 min using a microplate
reader and compared to that of a standard solution.
The FRAP values of the extracts were determined
from a calibration curve of ferrous sulfate solutions at
concentrations of 62.5, 125, 250, 500 and 1000
M, expressed as M of ferric iron reduced per gram
of dried sample. The antioxidant activity was measured
by its ability to reduce the Fe3+/ferricyanide complex by
forming ferrous products. Fe2+ can be monitored by
measuring the formation of Perls Prussian blue at 600
nm. Increased absorbance at 600 nm indicates a
stronger reducing power [10].
Determination of total phenolic contents
The total phenolic contents of water extracts were
determined according to the method described
by Amarowicz et al. [8], Swain and Hillis [11], Naczk and
Shahidi [12] and modified the procedures of measurement
by using a microplate reader. Briefly, 10 l of
each extract was transferred into a 96-well microplate
containing 160 l of distilled water. After mixing the
contents, 10 l of Folin-Ciocalteu reagent and 20 l of
a saturated sodium carbonate solution were added.
The plate was mixed well and the absorbance of blue
mixtures was recorded at 750 nm with microplate reader
after 30 min incubation. The readings of sample and
reagent blanks were subtracted from the reading of
reagent with extract. The total phenolic contents were
calculated as a gallic acid equivalent (GAE) from a
calibration curve of gallic acid standard solutions
(ranging from 25 to 800 mg/ml), and expressed as mg
of gallic acid per gram of dry sample. All measurements
were done in triplicate.
Antimutagenicity assay
Seven herbal extracts (Aegle marmelos L.,
Andrographis paniculata Nees, Chrysanthemum
indicum L., Cymbopogon citratus Stapf., Hibiscus
sabdariffa L., Jubliang and Zingiber officinale Rosc.)

Thai J. Pharm. Sci. 30 (2006) 28-35

were concentrated and used for evaluating the antimutagenic activity. The pre-incubation method of Ames
test described by Yahagi et al. [13] was used to determine
the antimutagenicity. One hundred microliters of the
Salmonella typhimurium strain TA100 were added to
the test tube containing 500 l of 0.5 M trisodium
phosphate-potassium chloride buffer and 100 l of the
selected concentrations of each extract. One hundred
microliters of a direct mutagen, nitrite-treated aminopyrene
that was prepared by the method described by
Kangsadalampai et al. [14], was added to the test tube.
The mixture was incubated at 37 C for 20 min in a
shaking water bath. Then, 2 ml of top agar with 0.5 mM
L-histidine and 0.5 mM D-biotin was added to the
mixture. The sample tube was mixed and quickly
poured onto an agar plate. The plate was inverted
and incubated at 37 C for 48 h. Colonies of the
His+ revertant were counted. The inhibitory effect of
each herbal extracts on mutagenicity of the standard
direct mutagen was determined as percentage of
inhibition, which is calculated as follows:
Percentage of inhibition = (A-B)/(A-C) 100
Where A is the number of revertants per plate
induced by the mutagen; B is the number of revertants
per plate induced by the mutagen in the presence
of the extracts; and C is the number of spontaneous
revertants per plate. The inhibition of herbal extracts
was considered as being strong, moderate or weak
when the value is higher than 60%, 40-60% or
20-40%, respectively, and negligible effect when the
value is less than 20% [15].

Results
The antioxidant activity and total phenolic
contents vary considerably among herbs. The
antioxidant activity of herbal extracts was investigated
using DPPH and FRAP assays. The reduction of DPPH
by antioxidants in the herbal extracts expressed as
the percentage of radical scavenging activity was
ranked (from highest to lowest) as follows: Carthamus
tinctorius L., Jubliang, Aegle marmelos L., Chrysanthemum
indicum L., Hibiscus sabdariffa L., Zingiber officinale

K. Kruawan and K. Kangsadalampai

Rosc., Cymbopogon citratus Stapf., Schefflera
leucantha R.Vig., Andrographis paniculata Nees,
Garcinia atroviridis Griff. ex T. Anderson. (Table 1). For
the reducing (FRAP value) power, the extracts
obtained from Jubliang, Hibiscus sabdariffa L.,
Chrysanthemum indicum L., Aegle marmelos L.,
Carthamus tinctorius L., Zingiber officinale Rosc.,
Cymbopogon citratus Stapf., Schefflera leucantha
R. Vig., Andrographis paniculata Nees and Garcinia
atroviridis Griff. ex T. Anderson had the FRAP values
of 2295.5 26.22, 2220 39.18, 1626 153.54,
1444.43 61.11, 1140.5 5.05, 1030.50 11.49,
794.31 18.37, 523.36 9.09, 486.92 7.14 and
376.93 15.79 mol/g, respectively.
Total phenolic contents were determined using
the Folin-Ciocalteu reagent and expressed as gallic
acid equivalents (GAE) per gram. The total phenolic
contents, calculated using the standard curve of gallic
acid (R2 = 0.9988), were varied from 36.76 to 283 mg
of GAE per gram. The total phenolic contents among
different herbal extracts were as follows: Jubliang >
Aegle marmelos L. > Chrysanthemum indicum L. >
Hibiscus sabdariffa L. > Carthamus tinctorius L. >
Schefflera leucantha R. Vig. > Cymbopogon citratus Stapf.
> Zingiber officinale Rosc. > Andrographis paniculata
Nees > Garcinia atroviridis Griff. ex T. Anderson
(Table 1). Overall, the highest and the lowest phenolic
content were found in Jubliang and Garcinia atroviridis
Griff. ex T. Anderson., respectively. Similar observation
was found for their antioxidant activity.
Almost all of the extracts showed that the total
phenolic contents were related to the ferric ion
reduction (FRAP value) and the scavenging activity
(DPPH assay). The herbal extracts with high antioxidant
activity exhibited relatively high total phenolic contents.
From the data, the herbal extracts can be classified
into three antioxidant activity groups, as shown in
Table 2.
Seven extracts from Aegle marmelos L.,
Andrographis paniculata Nees, Chrysanthemum indicum
L., Cymbopogon citratus Stapf., Hibiscus sabdariffa
L., Jubliang and Zingiber officinale Rosc. containing
different phenolic contents or antioxidant activity

31
were used for antimutagenic assay. The results
showed that almost all of the extracts were not
mutagenic on S. typhimurium strain TA100 (data not
shown). The effect of herbal extracts on the mutagenic
activity of nitrite treated 1-aminopyrene in the absence
of metabolic activation is shown in Figure 1. The results
revealed that the mutagenicity of nitrite treated
1-aminopyrene was inhibited to a greater or lesser
extent by an addition of herbal extracts on S. typhimurium
strain TA100. At the high concentrations of the extracts
(8.13-9.26 mg/plate), Jubliang and Andrographis
paniculata Nees showed strong antimutagenicity (> 60%
inhibition) while Aegle marmelos L. showed moderate
activity (40-60% inhibition). At low and moderate
concentrations, almost all of the extracts had weak
antimutagenicity (20-40% inhibition) except for the
extract of Hibiscus sabdariffa L. (at moderate
concentration) showing moderate activity. Interestingly,
the lowest concentration of the extract from Hibiscus
sabdariffa L. showed nearly moderate antimutagenicity
(39% inhibition). Although, Andrographis paniculata
Nees had low total phenolic contents and antioxidant
properties, it showed high antimutagenicity. From the
data, the phenolic contents did not always correspond
with the antimutagenicity activity.

Discussion
In this study, ten herbs were extracted with hot
water, suitable for the evaluation of antioxidant activity
and total phenolic contents of actual condition of herbal
drinks. Jubliang, Aegle marmelos L., Chrysanthemum
indicum L., Hibiscus sabdariffa L. and Carthamus
tinctorius L. which contained high phenolic compounds
exhibited high antioxidant activity when determined
by DPPH and FRAP assays. It, thus, confirms that
phenolic compounds have an important role in
antioxidant activities [16]. A good correspond between
antioxidant activity and phenolic compounds was
found in Bulgarian medicinal plants [17], Chinese
medicinal plants [18], some fruits, vegetables and grain
products [19]. The phenolic hydroxyl groups present in
plant antioxidants have redox properties [20,21] allowing
them to act as a reducing agent and a hydrogen
donator in the two assays. Thus, phenolic compounds

32

Thai J. Pharm. Sci. 30 (2006) 28-35

Table 1. Antioxidant activity and total phenolic contents of tested herbal extracts.
Total phenolic
contents
(mg of GAE/g)
36.76 0.49
38.65 4.26
59.86 9.02
83.83 18.53
129.86 1.95
139.98 18.02
210.72 14.62
214.34 17.09
267.22 17.79
283 14.23

Herbal extracts
Garcinia atroviridis Griff. ex T.Anderson. ()
Andrographis paniculata Nees ()
Zingiber officinale Rosc. ()
Cymbopogon citratus Stapf. ()
Schefflera leucantha R.Vig. ()
Carthamus tinctorius L. ()
Hibiscus sabdariffa L. ()
Chrysanthemum indicum L. ()
Aegle marmelos L. ()
Jubliang ()

Antioxidant activity
FRAP value
DPPH
(mol/g)
(% Scavenging effect)
376.93 15.79
46.52
486.92 7.14
58.02
1030.5 11.49
84.88
794.31 18.37
81.69
523.36 9.09
64.45
1140.5 5.05
96.65
2220 39.18
93.12
1626 153.54
93.33
1444.43 61.11
94.07
2295.5 26.22
95.59

Values expressed are means S.D. of three replicate experiments

Table 2. Classification of antioxidant activity of water extracts from herbs.

Herbal extracts

Phenolic contents FRAP value

(mg of GAE/g)
(mol/g)

DPPH
Antioxidant activity
(% Scavenging effect)

Jubliang
Aegle marmelos L.
Chrysanthemum indicum L.
Hibiscus sabdariffa L.
Carthamus tinctorius L.

>130

>1000

>90%

High

Schefflera leucantha R.Vig.

Cymbopogon citratus Stapf.
Zingiber officinale Rosc.

50-130

500-1000

60-90%

Moderate

<50

<500

<60%

Low

Andrographis paniculata Nees

Garcinia atroviridis Griff. Ex T.Anderson.

K. Kruawan and K. Kangsadalampai

could be the major antioxidant in these herbal
drinks. An exception was found on Schefflera leucantha
R. Vig. which had high phenolic contents but it showed
moderate antioxidant activity. It implied that the
phenolic contents might not correspond to the
antioxidant activity. The finding was in agreement
with the study of Kahkonen et al. [22], who found no
correspond between antioxidant activity and phenolic
contents on some plant extracts. Ivanova [17] proposed
that not all phenolic compounds possessed radical
quenching activity. The method of extracting the
herbs with hot water might give the different results
in antioxidant activity from other published reports.
The difference would be due to organic solvents
used in isolation that extracted only some selective
components. Tiwari et al. [23] suggested that individual
components may lose their synergistic characteristics
probably present in their natural mixture form.
Among the herbal drinks Jubliang showed the
highest FRAP value, highest phenolic contents and
highest antimutagenic activity. Jubliang is the only
herbal drink that consists of eight herbs. Some of the
components of Jubliang, namely Pragmites communis [24]
and Lophatherum gracile [25] had been reported to have
high antioxidant activity in DPPH assay. Possible
synergistic interactions among herbal components that
had high antioxidant and antimutagenic activity in Jubliang
are warrant for further study.
The antioxidant properties of herbs may be
attributed to the plant pigments that are the main
components of each herbal extract. The red pigment
presented in the flowers of Hibiscus species are
anthocyanins (cyanidin-3-glucoside and delphinidin-3glucoside) which may act as an antioxidant [26, 27].
Stich and Rosin [28] proposed that phenolics had
inhibitory effects on genotoxicity of several mutagens.
It was found that such compounds were associated
with low incidence of various cancers [29-31]. The present
study has revealed that phenolic contents of herbal
extracts were correlated with antimutagenic activity.
An exception was found on the extract from leaves
of Andrographis paniculata Nees. It had strong

33
antimutagenic effect but had less phenolic contents. It
is proposed that the antimutagenic effect was due to
the high chlorophyll content extracted with hot water.
Several researchers [32-34] found that chlorophylls had
antimutagenic activity in in vitro against aromatic nitro
compounds, which is in agreement with the present
results. Some investigators found that chlorophylls
exhibited antimutagenic activity (in Ames test) by
complex formation [35]. The purified chlorophyll protects
against the adduct formation of DNA in isolated hepatocytes by interfering with carcinogen activation [36].
Although the extract from Cymbopogon citratus
Stapf. has moderate antioxidant activity, it showed low
antimutagenic activity. Although Vinitketkumnuen et al. [37]
found that mutagenicity of aflatoxin B1, Trp-P-1,
Trp-P-2, Glu-P-1, Glu-P-2, IQ, MNNG and AF-2 was
inhibited by the extract of lemon grass in a dose
dependent manner, but no effect was found on the
mutagenic activity of benzo[a]pyrene in Salmonella
typhimurium strains TA98 and TA100. Benzo[a]pyrene is
a polycyclic aromatic hydrocarbon which has a
structure related to nitropyrene, a standard mutagen in
this experiment.
The non-enzymatic formation of direct mutagens
such as products from treating 1-aminopyrene with
nitrite in acidic solution (pH 3.0-3.4) is physiologically
important, especially with regard to the etiology of
gastric cancer. The reduction of His+ revertant of
TA100 induced by nitrite-treated aminopyrene suggests
that the herbal extracts may contain some compounds
that interfere with the mutagenic metabolism of ultimate
mutagen/carcinogen, or form a complex of phenolic
compounds with 1-nitropyrene, or scavenge the
electrophilic metabolites [38].

Conclusion
The study indicates the presence of antioxidants,
phenolic compounds and antimutagens in hot water extract
of herbs consumed in Thailand. The high antioxidant
and antimutagenic activity found in some herbal
extracts may help to protect against free radical and
reduce mutagen formed in the pH of stomach digestion.

34

Thai J. Pharm. Sci. 30 (2006) 28-35

100
Aegle marmelos
Andrographis paniculata
Chrysanthemum indicum
Cymbopogon citratus
Hibiscus sabdariffa
Jub-liang
Zingiber officinale

90

Percentage of inhibition

80
70
60

Strong

50

Moderate

40
30

Weak

20
10

No effect

lowest conc.

low conc.

moderate conc.

8.43

8.21

3.7

1.66

1.63

0.37

0.33

0.15

0.06

0.01

high conc.

Amount of extracts (mg/plate)

Figure 1. Effect of herbal extracts on the mutagenic inhibition of nitrite treated 1-aminopyrene to Salmonella typhimurium strain TA100.

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