Botanical Studies (2007) 48: 397-406.
4
These two authors contributed equally to this work.
*
Corresponding author: E-mail: hstsay@cyut.edu.tw; Te l:
+886-4-2330-4920; Fax: + 886-4-2330 4921.
INTRODUCTION
In the Chinese traditional medicine system, "Gusuibu"
has long been used in the treatment of bone injuries.
It has been proved very effective for the treatment of
inflammation, hyperlipemia, and arteriosclerosis (Editorial
Board of Zhong Hua Ben Cao [China Herbal], State
Administration of Traditional Chinese Medicine, 1999).
Also, several recent studies have claimed the medicine has
therapeutic effects on bone healing (Ma et al., 1996; Sun
et al., 2004; Jeong et al., 2005). However, in the Chinese
Material Medica, different herbs from different areas with
similar common names have been mentioned for treatment
of the same disease. This is evident from field records,
visits to local traditional doctors, review of specimens
in herbaria, and the available literature. Rhizomes o f
Drynaria fortunei (Kze.) J. Sm., Pseudodrynaria coronans
(Wall. ex Mett.) Ching (both from Polypodiaceae), Daval-
lia divaricata Bl., Davallia mariesii Moore ex Bak, Da-
vallia solida (Forst.) Sw., and Humata griffithiana (Hk.) C.
Chr. (from Davalliaceae) are used as or called "Gusuibu"
or "Shibu" in Taiwan. These have been claimed to cure
physique ache, inflammation, cancer, ageing, blood stasis,
and bone injuries. In a publication by the Pharmacopoeia
Commission of the People¡¦s Republic of China, only
Drynaria fortunei has been reported to be a source
of "Gusuibu" (ChPC, 2005). However, no systematic
investigation has been carried out so far to evaluate the
comparative values of these different sources.
Over the past few years, investigations for phenolic
compounds in medicinal herbs have gained importance
due to their high antioxidative activity (Zhu et al., 2004).
A large number of reports have demonstrated that these
compounds are of great value in preventing the onset and/
or progression of many human diseases (Parshad et al.,
1998; Lee et al., 2000). Polyphenols have many favorable
effects on human health, like inhibiting the oxidization
of low-density proteins (Frankel et al., 1993), thereby
decreasing the risk of heart disease (Williams and Elliot,
Antioxidant activities and polyphenol contents of six folk
medicinal ferns used as "Gusuibu"
Hung-Chi CHANG
1,4
, Guan-Jhong HUANG
1,4
, Dinesh Chandra AGRAWAL
2
, Chao-Lin KUO
1, 3
,
Chi-Rei WU
1
,
and Hsin-Sheng TSAY
1, 2,
*
1
Institute of Chinese Pharmaceutical Science, China Medical University, Taichung 40402, Taiwan
2
Graduate Institute of Biotechnology, Chaoyang University of Technology, Wufong, Taichung 40413, Taiwan
3
School of Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan
(Received November 29, 2006; Accepted April 17, 2007)
ABSTRACT.
In the traditional Chinese system of medicine, the folk remedy "Gusuibu," renowned for its
therapeutic effects on bone is sourced from six different ferns. However, no scientific investigation has been
carried out so far, to evaluate the comparative values of these sources. In the present report, ethanol and
aqueous extracts of these six sources were characterized for their antioxidant, scavenging activities, reducing
power, total polyphenols, flavonols, flavonoids, condensed tannins, and proanthocyanidin contents. Results
showed wide variation among the six sources. Most samples in aqueous extracts had higher antioxidant
potencies and polyphenol contents than the ethanol extracts, indicating that the aqueous preparation of
"Gusuibu" is more potent than the ethanol one. EC
50
values of reducing capacities, and scavenging activities
against DPPH radicals showed significant variation among the six sources, within ethanol or aqueous extracts
and between the two solvents. The maximum (1.27) Trolox Equivalent Antioxidant Capacity (TEAC) was
recorded in aqueous extract of fern Davallia mariesii. The correlation coefficient (R
2
) values of TEAC and
total polyphenol contents showed a higher correlation (aqueous extract, R
2
= 0.971; ethanol extract, R
2
= 0.981).
Keywords: Antioxidant activity; Gusuibu; Medicinal fern; Polyphenols contents; Radical scavenging activity;
Reducing capacity.
Abbreviations: RSC, radical scavenging capacity; DPPH, 1,1-diphenylpicrylhydrazyl free radical; TEAC,
Trolox equivalent antioxidant capacity.
BIOChemISTRy
pg_0002
398
Botanical Studies, Vol. 48, 2007
1997). These compounds have anti-inflammatory and
anti-carcinogenic properties (Carrol et al., 1999; Maeda-
Yamamoto et al., 1999). Also, flavonoids and many other
phenolic compounds of plant origin have been reported as
scavenger reactive oxygen species (ROS), and are viewed
as promising therapeutic drugs for free radical pathologies
(Parshad et al., 1998; Lee et al., 2000). Thus, measurement
of the polyphenols and antioxidant activity in herbs have
become important tools to understand the relative values
of plant species, especially from a health point of view.
The main objective of the present study was to
characterize the antioxidative potencies, scavenging
activities against DPPH radical, reducing power, and
estimation of polyphenol contents in six sources of
"Gusuibu."
mATeRIALS AND meThODS
Plant materials
Plant materials of six sources of "Gusuibu" were
collected from the counties of Hsinchu, Taichung,
Nantou, and Taitung in Taiwan. These were identified
and authenticated by Professor Chung-Chuan Chen of
the Institute of Chinese Pharmaceutical Science, China
Medical University, Taichung, Taiwan. Particulars of these
sources including species, families, and their uses have
been listed in Table 1.
extractions
Ethanol extracts: Dried rhizome (100 g each) was
macerated with 1,000 ml ethanol for 24 h at room
temperature. Filtration and collection of the extract was
done thrice. Then, the ethanol with crude extract (3,000
ml) was evaporated to 10 ml and dried in vacuo at 40¢XC.
The dry extract was weighted and dissolved in ethanol
(stock 5mg/ml) and stored in -20¢XC until further use.
Aqueous extracts: Dried rhizome (100 g each) was
used for decoction with 1,000 ml distilled water for 1 h.
Filtration and collection of extract was done thrice. The
resulting decoction (about 1,000 ml) was evaporated to
10 ml and dried in vacuo at 50¢XC. Each dry extract was
weighted, dissolved in distilled water (stock 5 mg/ml) and
stored in -20¢XC until further use.
For each sample, yields were calculated in percentages
on the basis of dry weight of rhizome used (100 g) and
quantity of dry mass obtained after extraction.
Antioxidant activity by ABTS assay
The assay was carried out as described earlier (Re
et al., 1999). An aqueous solution of 2, 2-Azinobis
[3-ethylbenzothiazoline-6-sulfonate] (ABTS) (7 mM) was
oxidized using potassium peroxodisulfate (2.45 mM) for
16 h in dark. The ABTS
+
solution was diluted with ethanol
to an absorbance of 0.75 ¡Ó 0.05 at 734 nm (Beckman
UV-Vis spectrophotometer, Model DU640B). A standard
calibration curve was constructed for Trolox at 0, 0.1, 0.2,
0.5, 1.0, 2.0 mM concentration. An aliquot (10 £gl) of each
sample (100
£g
g ml
-1
concentration) was mixed with 1.0 ml
of ABTS
+
radical cation solution in cuvette and absorbance
was read at 734 nm after 1 min. Antioxidant properties of
"Gusuibu" extracts were expressed as Trolox Equivalent
Antioxidant Capacity (TEAC), calculated from at least
three different concentrations of extract tested in the assay
giving a linear response.
Table 1. Particulars of six sources of medicinal fern used as "Gusuibu" and their comparative yields in ethanol and aqueous
extracts.
Species
Common name Ethanol extract Aqueous extract Medical use/ disease treated
Code Yield (%)
a
Code Yield (%)
Drynaria fortunei
(Polypodiaceae)
Gusuibu DFE 11.2 DFW 12.8 Inflammation, hyperlipemia and arteriosclerosis
(ChPC, 2005); Cancer (Cai et al., 2004).
Pseudodrynaria
coronans
(Polypodiaceae)
Gusuibu PCE 15.0 PCW 18.6 Bone injuries, tinnitus and lumbago (Editorial
Board of China Herbal, State Administration of
Traditional Chinese Medicine, 1999).
Davallia divaricata
(Davalliaceae)
Dayegusuibu DDE 14.6 DDW 20.4 Bone injuries, tinnitus and lumbago (Editorial
Board of China Herbal, State Administration
of Traditional Chinese Medicine, 1999); Joint
pain.(Hwang et al., 1989)
Davallia mariesii
(Davalliaceae)
Haizhougusuibu DME 8.5 DMW 29.0 Common cold, neuralgia, stomach cancer,
lumbago, rheumatalgia, odontalgia and tinnitus
(Cui et al., 1990)
Davallia solida
(Davalliaceae)
Koyegusuibu DSE 12.7 DSW 14.5 Physique ache, inflammation, cancer, and bone
injuries.
Humata griffithiana
(Davalliaceae)
Begaigusuibu HGE 6.7 HGW 16.1 Physique ache, inflammation, cancer, and bone
injuries.
a
Dry weight basis.
pg_0003
CHANG et al. ¡X Antioxidant activities of six medicinal ferns used as Guisuibu
399
Antioxidant activity by Dot-Blot and DPPh
staining
An aliquot (3 £gl) of each sample was carefully loaded
on a 20 cm ¡Ñ 20 cm TLC layer (silica gel 60 F
254
; Merck)
and allowed to dry (3 min). Drops of each sample were
loaded in order of decreasing concentration along the row.
The staining of the silica plate was based on the procedure
of Soler-Rivas et al. (2000). The sheet bearing the dry
spots was placed upside down for 10 s in a 0.4 mM DPPH
solution. The excess solution was then removed with a
tissue paper, and the layer was dried with a hair-dryer
blowing cold air. A stained silica layer revealed a purple
background with white spots at the location where radical
scavenger capacity was present. The intensity of the white
color depends upon the amount and nature of radical
scavenger present in the sample. The GSH was used for
the positive control in the aqueous extract, and the BHT
was used for the positive control in ethanolic extracts.
Determination of "Reducing power"
The reducing power of the extracted samples (dissolved
in ethanol or distilled water), glutathione (GSH, dissolved
in distilled water) or butylated hydroxytoluene (BHT,
dissolved in ethanol) were determined according to
the method of Jayaprakasha et al. (2002). Different
concentrations (12.5, 25, 50, 100, 200 and 400 £gg ml
-1
)
of each extract, GSH or BHT 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
¢XC for 20 min, to reduce ferricyanide into ferrocyanide.
Thereafter, an equal volume of 1% trichloroacetic acid
was added to the mixture and centrifuged at 6,000 rpm for
10 min. The upper layer of the solution was collected and
mixed with distilled water and 0.1% ferric chloride at a
ratio of 1:1:0.2. Absorbance was measured to determine
the amount of ferric ferrocyanide (Prussian Blue)
formed at 700 nm against a blank in a DU 640
.
UV/Vis
spectrophotometer (Beckmann). The GSH was used for
the positive control in the aqueous extract, and the BHT
was used for the positive control in ethanolic extracts. The
reducing power tests were run in triplicate. Increase in
absorbance of the reaction indicated the reducing power of
the samples. EC
50
value (£gg extract ml
-1
) was the effective
concentration at which the absorbance was 0.5 for
reducing capacity and was obtained by interpolation from
linear regression analysis.
Scavenging activity against DPPh radical
The effect of each extract sample on the DPPH radical
was estimated according to the method reported by Blois
(1958) with minor modification. Stock solution (500.0
mg ml
-1
) of each sample was diluted to the concentrations
of 400, 200, 100, 50, 25 and 12.5 mg ml
-1
, in ethanol or
distilled water. An aliquot of each sample (20 £gl) was
mixed with 100 mM Tris-HCl buffer (80 £gl, pH 7.4),
and then 100 £gl of the DPPH in ethanol with a final
concentration of 200 £gM was added. The mixture was
shaken vigorously and left to stand at room temperature for
20 min in dark. The absorbance was measured at 517 nm
against a blank in a DU 640
.
UV/Vis spectrophotometer
(Beckmann). The percentage of DPPH discoloration of
the sample was calculated according to the equation: %
discoloration = (1-Abs sample /Abs control) ¡Ñ 100. EC
50
value (£gg extract/ml) is the effective concentration at
which DPPH radicals were scavenged by 50% and was
obtained by interpolation from linear regression analysis.
The GSH was used for the positive control in the aqueous
extract, and the BHT was used for the positive control in
ethanolic extracts.
Total polyphenols content (TPC)
Total phenolic compounds were estimated using the
Folin-Ciocalteu method (Ragazzi and Veronese, 1973).
Twenty £gl of each extract (100 £gg ml
-1
) was added to 200
£gl distilled water and 40 £gl of Folin-Ciocalteu phenol
reagent (Merck-Schuchardt, Hohenbrun, Germany).
The mixture was allowed to stand at room temperature
for 5 min, and then 40 £gl of 20% sodium carbonate was
added to the mixture. The resulting blue complex was
then measured at 680 nm. The TPC was expressed as £gg
catechin equivalent/mg dry weight by reference to the
(+)-catechin standard calibration curve.
Total flavonoid content
The AlCl
3
method (Lamaison and Carnet, 1990) was
used for estimation of the total flavonoids content of the
extracted samples. An aliquot of 100 £gl of each extract
(100 £gg ml
-1
) was added individually to equal volumes
of solution of 2% AlCl
3
¡P6H
2
O (2 g in 100 ml methanol).
The mixture was vigorously shaken, and after 10 min of
incubation, absorbance was taken at 430 nm. Flavonoids
contents were calculated from the calibration curve of rutin
standard solutions, and expressed as
£g
g rutin equivalent/
mg dry weight.
Total flavanol content
The total flavanol content was estimated using the p-di
methylaminocinnamaldehyde (DMACA) method (Arnous
et al., 2001). This method has a great advantage over the
widely used vanillin method since there is no interference
by anthocyanins. Furthermore, it provides higher
sensitivity and specificity (Li
et al., 1996). Forty £gl of
each extract (100 £gg/ml) was added to 200 £gl of DMACA
solution [0.1% in methanol/HCl (3:1, v/v)]. The mixture
was vortexed and allowed to react at room temperature for
10 min. The absorbance at 640 nm was then read against
a blank prepared without DMACA. The concentration
of total flavanols was estimated from a calibration curve.
Results are expressed as £gg
catechin equivalent/mg dry
weight.
Condensed tannin content
Condensed tannin content was estimated using the
vanillin assay method (Julkunen-Titto, 1985). Twenty
pg_0004
400
Botanical Studies, Vol. 48, 2007
five £gl of extract (100 £gg ml
-1
) was added to 750 £gl of
vanillin/methanol solution (4%, w/v) and vortexed. Then,
concentrated HCl (375 £gl) was added and allowed to react
at room temperature for 20 min. The absorbance at 550 nm
was then read against a blank. Concentration of tannins
was calculated as £gg catechin equivalent/mg dry weight
from a calibration curve.
Proanthocyanidin content
The proanthocyanidin content was estimated using
HCl/butanol assay method (Porter et al., 1986). An aliquot
(0.2 ml) of each extract (stock 100 £gg/ml) was added to
0.2 ml solution of 0.04 M FeSO
4
¡P7H
2
O (in 2 M HCl) in a
1.5 ml centrifuge tube, followed by 0.8 ml of butanol. The
tube was incubated for 30 min at 95¢XC. The absorbance
of the red colouration was read at 550 nm. Results were
expressed in mg cyanidin chloride g
-1
fresh weight.
Statistical analysis
All analyses were carried out in triplicate. Data are
expressed as mean ¡Ó standard deviation (SD). Differences
were estimated by one-way analysis of variance (ANOVA)
followed by least significance difference (LSD) test.
Probability values of less than 0.05 were considered
statistically significant. All statistical analyses were
performed using SAS statistical software package (SAS
R
Inc., 2001).
ReSULTS
extraction yields
Ethanol extract yields of six sources of "Gusuibu" are
given in Table 1. Species-wise percentages of ethanol
extract yields in decreasing order were as follows:
Pseudodrynaria coronans (PCE) (15.0), Davallia
divaricata (DDE) (14.6), Davallia solida (DSE) (12.7),
Drynaria fortunei (DFE) (11.2), Davallia mariesii (DME)
(8.5), and Humata griffithiana (HGE) (6.7). The aqueous
extract yields in decreasing order were as follows:
Davallia mariesii (DMW) (29.0), Davallia divaricata
(DDW) (20.4), Pseudodrynaria coronans (PCW) (18.6)
Humata griffithiana (HGW) (16.1), Davallia solida
(DSW) (14.5), Drynaria fortunei (DFW) (12.8).
Antioxidant activities estimated by ABTS assay
method
TEAC values determined from the calibration curve
for six sources of "Gusuibu" are shown in Figure 1.
Antioxidant activities of both aqueous and ethanol extracts
of the six sources were in the following decreasing order:
DMW (1.27 mM) > DDW (0.96 mM) > DSW (O.91 mM)
> PCW (0.77 mM) > DSE (O.32 mM) > DFW (0.26 mM)
> HGE (0.18 mM) > DME and DDE (0.17 mM) > PCE
(0.11 mM) > DFE (0.07 mM). Thus, it was observed that
most samples in aqueous extracts had higher antioxidant
potencies than ethanol extracts.
Antioxidant activities estimated by "dot-blot"
method
In order to visualize semi-quantitatively and for rapid
screening, each diluted sample was applied as a dot on a
TLC plate which was then stained with DPPH solution
(Figure 2). White spots of strong intensity appeared
quickly up to the dilution of 25 £gg ml
-1
for DSE (the final
amount in the spot: 0.075 £gg dry matter), 50-100 £gg/ml for
PCE, PCW, DDE, DDW, DSW and HGE (final amount:
0.15-0.3 £gg dry matter); and the lowest intensities of DFE,
DFW and HGW. For antioxidant analysis, fast-reacting
and strong intensity white spots appeared up to dilutions of
50-100 £gg ml
-1
for BHT and GSH (final amount: 0.15-0.3
£gg dry matter). Appropriate dilutions react positively
with DPPH, depending upon their free radical scavenging
capacity (RSC) and nature (Chang
et al., 2002) and result
in dots of a certain diameter and color intensity, which
indicate radical scavenging capacity. Darker dots indicate
higher RSC values. According to the color intensities,
Figure 1. Trolox equivalent antioxidant capacity (TEAC) values
of aqueous and ethanol extracts of six s ources of Gus uibu.
DF: D. fortunei; PC: P. coronans; DD: D. divaricata; DM: D.
mariesii; DS: D. solida; HG: H. griffithiana. In text, all ethanol
extracts have been designated as E and aqueous extracts as W
after the species code.
Figu re 2. Dot blot assay of free radical scavenging capacity
(RSC) on a silica sheet stained with a DPPH solution in
methanol. Each 3 £gl of plant extract [400, 200, 100, 50, and 25
£gg ml
-1
) was applied from top to down. Dots from left to right
are: D. fortunei (E
1
, W
1
), P. coronans (E
2
, W
2
), D. divaricata (E
3
,
W
3
), D. mariesii (E
4
, W
4
), D. solida (E
5
, W
5
) and H. griffithiana
(E
6
, W
6
). Antioxidants; butylated hydroxytoluene (BHT) (S
1
),
glutathione (GSH) (S
2
) were used as positive controls. (E
1
-E
6
:
Ethanol extracts; W
1
-W
6
: Aqueous extracts).
pg_0005
CHANG et al. ¡X Antioxidant activities of six medicinal ferns used as Guisuibu
401
RSC values of ethanol extracts in decreasing order were
as follows: DSE (E
5
) > DD E (E
3
) > HG E (E
6
) > D ME
(E
4
) > PCE (E
2
) > DFE (E
1
) while RSC values of aqueous
extracts in the decreasing order were as follows: DMW
(W
4
) > DSW (W
5
) > DDW (W
3
) > PCW (W
2
) > DFW (W
1
)
> HGW (W
6
). Between the two standards, BHT (S
1
) had
higher RSC compared to glutathione (S
2
) (Figure 2).
eC
50
of scavenging activity against DPPh
radical
Each sample extract exhibited the scavenging activity
and was found to be dose-dependent (data not shown). The
EC
50
values calculated at 20 min incubation are given in
Table 2. It was found that ethanol extract of fern species
Davallia solida (DSE) had the highest radical-scavenging
activity (expressed as
£g
g extract/ml) (26.89), followed by
DDW (52.81), DDE (87.95), HGE (93.50), PCW (98.23),
DSW (166.72), DMW (173.03) and PCE (190.81). Lower
radical-scavenging activities (>400) were observed in
case of DFE, DFW and HGW. In this assay condition the
scavenging activity of GSH (89.64) against DPPH radicals
is not as effective as that of BHT (42.28).
eC
50
of reducing power
Extracts of all six sources of "Gusuibu" exhibited the
reducing powers and were concentration dependent (data
not shown). The reducing powers (expressed as £gg extract/
ml) of the samples studied have been given in Table 2.
Lower EC
50
values means higher reducing capacity. It was
found that the aqueous extract of fern species Davallia
mariesii (DMW) had a higher degree of reducing power
(26.58), followed by DSW (32.81), PCW (38.96), DDW
(43.36), DSE (185.10), DFW (193,55), HGE (317.8), DDE
(349.46) and DME (364.55). DFE, PCE and HGW showed
lower reducing power (>400).
Total polyphenol content
The total polyphenols, flavonoids, flavonols,
condensed tannin, and proanthocyanidin in ethanol and
aqueous extracts of six sources of "Gusuibu" are given
in Table 3. Total polyphenol contents (expressed as £gg
catechin equivalent/mg dry weight) varied significantly
among the ethanol or aqueous extracts of six sources of
"Gusuibu." The quantities of total polyphenols ranged
from a minimum 53.44 £gg in ethanol extract of Drynaria
fortunei (DFE) to the maximum 1635.16 £gg in aqueous
extract of Davallia mariesii (DMW) (Table 3). Depending
upon species (source of Gusuibu), the quantities of total
polyphenols in aqueous extracts were 5-12 times higher
compared to ethanol extracts. The only exception was fern
species Humata griffithiana (HG), where the margin of
difference in total polyphenols in both the solvents was
narrow.
Total flavonoid contents (expressed as £gg rutin
equivalent/mg dry weight) ranged from 0.0 to 122.44 £gg
in ethanol extracts and from 3.91 £gg to 123.98 £gg in the
aqueous extracts of the six sources. The ethanol extracts
of fern species Pseudodrynaria coronans (PC), Davallia
mariesii (DM) and Davallia solida (DS) had no flavonoid
content (Table 3).
Quantities of total flavonols in ethanol and aqueous
extracts of six sources of "Gusuibu" varied significantly
(Table 3). With the exception of fern species Humata
griffithiana (HG), aqueous extracts had 3 to 22 times
higher total flavonol contents compared to ethanol extracts.
However, the ethanol extract of Humata griffithiana
Table 2. EC
50
values of ethanol and aqueous extracts in antioxidant properties of six sources of Gusuibu.
EC
50
value (£gg extract ml
-1
)
a
Sources of Gusuibu
b
Reducing capacity
Scavenging ability on DPPH radicals
Ethanol extract Aqueous extract Ethanol extract Aqueous extract
Drynaria fortunei (DF)
>400
193.55 ¡Ó 1.31 A
>400
>400
Pseudodrynaria coronans (PC)
>400
38.96 ¡Ó 0.14 CD 190.81 ¡Ó 4.15 A 98.23 ¡Ó 14.36 B
Davallia divaricata (DD)
349.46 ¡Ó 2.53 A
c
43.36 ¡Ó 1.53 C
87.95 ¡Ó 0.88 B 52.81 ¡Ó 1.01 C
Davallia mariesii (DM)
364.55 ¡Ó 15.83 A 26.58 ¡Ó 0.26 E 95.87 ¡Ó 8.42 B 173.03 ¡Ó 10.68 A
Davallia solida (DS)
185.10 ¡Ó 15.25 D 32.16 ¡Ó 0.04 DE 26.89 ¡Ó 0.53 D 166.72 ¡Ó 7.24 A
Humata griffithiana (HG)
317.80 ¡Ó 19.90 B
>400
93.50 ¡Ó 8.60 B
>400
Glutathione (GSH)
N.D
d
135.11 ¡Ó 12.25 B
N.D
89.64 ¡Ó 2.88 B
2, 6-Di-tert-butyl-4-methylphenol (BHT) 229.01 ¡Ó 0.60 C
N.D
42.28 ¡Ó 0.87 C
N.D
a
EC
50
value: the effective concentraion at which the absorbance was 0.5 for reducing capacity; 1, 1¡¦-diphenyl 2-picrylhydrazyl
(DPPH) radicals were scavenged by 50%. EC
50
value was obtained by interpolation from linear regression analysis.
b
In text, all ethanol extracts have been designated as E and aqueous extracts as W after the species code.
c
Each value is expressed as mean ¡Ó standard deviation (n=3). Means with different letters within a column are significantly different
(p<0.05).
d
N.D: Not detected.
pg_0006
402
Botanical Studies, Vol. 48, 2007
showed a 2.5-times higher quantity than aqueous extract.
The minimum quantity of total flavonols (0.75 £gg) was
found in Drynaria fortunei (DFE), and the maximum
(295.70 £gg) was in Davallia mariesii (DMW) (Table 3).
In the case of condensed tannins and proanthocyanidin,
trends more or less similar to total flavonols were observed
among the six sources. Aqueous extracts had several
times higher values compared to ethanol extracts with the
exception of Humata griffithiana (Table 3).
Relationship between antioxidant activity and
total polyphenols, and total flavonoid contents
Correlation cofficients (R
2
) of antioxidant capacity
(TEAC) and total polyphenols, and TEAC and total
flavonoids of both aqueous and ethanol extracts are
shown in Figures 3 and 4. R
2
values for TEAC and total
polyphenol contents for aqueous (Figure 3A) and ethanol
(Figure 3 B) extracts were 0.971 and 0.981, respectively.
Similarly, R
2
values for TEAC and total flavonoid contents
Table 3. Phenolic contents in ethanol and aqueous extracts of six sources of Gusuibu.
Sources of Gusuibu
d
Total polyphenols
a
Total flavonoids
b
Total flavonols
a
Condensed tannins
a
Proanthocyanidins
c
Ethanol extract
Drynaria fortunei (DF)
53.44 ¡Ó 2.78 2.3 6¡Ó 1.54 0.75 ¡Ó 0.00 3.17 ¡Ó 5.63 89.17 ¡Ó 0.00
Pseudodrynaria coronans (PC) 108.18 ¡Ó 3.21
0
12.91 ¡Ó 0.21 32.73 ¡Ó 11.26 103.62 ¡Ó 1.28
Davallia divaricata (DD) 134.15 ¡Ó 12.13
0
49.39 ¡Ó 2.24 108.76 ¡Ó 14.07 128.63 ¡Ó 1.11
Davallia mariesii (DM)
140.64 ¡Ó 8.50
0
30.41 ¡Ó 0.86 53.85 ¡Ó 11.26 128.63 ¡Ó 1.11
Davallia solida (DS)
291.86 ¡Ó 7.00 122.44 ¡Ó 1.78 66.14 ¡Ó 2.61 260.82 ¡Ó 22.52 180.88 ¡Ó 2.12
Humata griffithiana (HG) 166.62 ¡Ó 5.79 11.64 ¡Ó 0.00 31.03 ¡Ó 0.86 79.19 ¡Ó 11.26 168.09 ¡Ó 3.85
Aqueous extract
Drynaria fortunei (DF)
326.18 ¡Ó 12.13 16.28 ¡Ó 0.00 16.51 ¡Ó 0.56 108.76 ¡Ó 5.63 132.15 ¡Ó 3.39
Pseudodrynaria coronans (PC) 1120.29 ¡Ó 20.51 41.01 ¡Ó 1.54 145.31 ¡Ó 2.61 636.73 ¡Ó 14.07 341.13 ¡Ó 2.56
Davallia divaricata (DD) 1203.78 ¡Ó 12.55 3.91 ¡Ó 1.54 263.81 ¡Ó 4.85 1029.54 ¡Ó 30.97 258.13 ¡Ó 3.85
Davallia mariesii (DM)
1635.16 ¡Ó 48.39 3.91 ¡Ó 0.00 295.70 ¡Ó 3.33 1422.35 ¡Ó 14.07 467.85 ¡Ó 2.56
Davallia solida (DS)
1400.45 ¡Ó 34.87 123.98 ¡Ó 5.42 246.93 ¡Ó 5.02 1164.70 ¡Ó 25.34 429.32 ¡Ó 3.85
Humata griffithiana (HG) 183.32 ¡Ó 49.88 14.22 ¡Ó 1.78 12.041 ¡Ó 1.07 32.73 ¡Ó 14.07 106.21 ¡Ó 1.28
a
Expressed as £gg catechin equivalent/mg dry weight.
b
Expressed as £gg rutin equivalent/mg dry weight.
c
Expressed as £gg cyanidin chloride equivalent/mg dry weight.
d
In text, all ethanol extracts have been designated as E and aqueous extracts as W after the species code.
Figure 3. Correlation coefficients (R
2
) of TEAC and total polyphenol contents in aqueous (A) and ethanol (B) extracts.
pg_0007
CHANG et al. ¡X Antioxidant activities of six medicinal ferns used as Guisuibu
403
for aqueous (Figure 4A) and ethanol (Figure 4B) extracts
were 0.018 and 0.764, respectively.
DISCUSSION
Antioxidants have been defined as substances that,
when present at low concentrations compared with
oxidizable compounds (e.g. DNA, protein, lipid, or
carbohydrate), delay or prevent oxidative damage due
to the presence of reactive oxygen species (ROS). These
ROS undergo redox reactions with phenolics, resulting
in inhibition of antioxidant activity in a concentration
dependant manner (Halliwell and Gutteridge, 1990). Thus,
measurement of total polyphenols and its constituents
along with antioxidant activity has increasingly been
used in plant samples and has become an important tool
for investigation. The TEAC assay is one of the most
frequently used analytical strategies for antioxidant
activity. The assay shows a good correlation with the
other methods as well, such as the 2, 2¡¦-diphenyl-
l-picrylhydrazyl assay (DPPH), the total radical-
trapping antioxidant parameter assay (TRAP), the
photochemiluminescence assay (PCL), and the ferric
reducing ability of plasma assay (FRAP). In the present
study, it was observed that the greatest antioxidant
activities (measured as TEAC values) more or less had
direct correlation with quantities of total polyphenols both
in aqueous as well as ethanol extracts. Similar findings
have been reported earlier (Zhu et al., 2004) suggesting a
causative relationship between total polyphenols content
and antioxidant activity and indicating that phenolic
compounds present in the extracts of six sources of
"Gusuibu" may be responsible for these antioxidant
properties.
Several methods have been used to measure free radical
scavenging capacities (RSC), regardless of the individual
compounds which contribute to the total capacity of a
plant product in scavenging free radicals. Most methods
are based on the inhibition of the accumulation of oxidized
products, since the generation of free radical species
is inhibited by the addition of antioxidants resulting in
reduction of the end point by scavenging free radicals.
The reliable method to determine RSC involves the
measurement of the disappearance of free radicals, such as
the 2, 2¡¦-azino-bis (3-ethylbenzenthiazoline-6-sulphonic)
acid radical (ABTS
¡E +
), the 2, 2-diphenyl-1-picrylhydrazyl
radical (DPPH
¡E +
), or other colored radicals, with a
spectrophotometer (Miller and Rice-Evans, 1997). The
DPPH radical has been widely used as a model system
to investigate the scavenging activities of several natural
compounds including phenolic compounds, flavonoids,
or crude mixtures such as ethanol or water extracts of
plants. DPPH radical is scavenged by antioxidants through
the donation of a hydrogen atom, forming the reduced
DPPH-H. The color changes from purple to yellow after
reduction, and this can be quantified by its decrease in
absorbance at wavelength 517 nm. In the present study,
all the samples irrespective of solvent showed scavenging
activity in a concentration dependent manner. Stained
silica layer revealed a purple background with white spots
at the location of drops, which showed radical scavenging
capacity. The intensity of the white color depended upon
the amount and nature of radical scavenger present in the
samples. Thus, TLC screening and DPPH staining methods
demonstrated that ethanol and aqueous extracts had
significant variations in free radical scavenging capacities.
Most samples in water extract were found to have
higher antioxidant potencies than ethanol extracts. This
observation is in conformity with an earlier report, which
observed that the antioxidant activities of more-polar
solvent extracts (BuOH and water extracts) exceeded those
of non-polar solvent extracts (hexane and EtOAC extracts).
The much higher antioxidant activity of the water extracts
of "Gusuibu" preparations indicates that the herb may be
most effective when taken with water. In contrast to this
report, alcoholic preparations of the herbal drug Uncaria
tomentosa showed higher antioxidant activity than water
extract (Pietta et al., 1998). Thus it is essential to carry out
a study on different solvents.
Figure 4. Correlation coefficients (R
2
) of TEAC and total flavonoid contents in aqueous (A) and ethanol (B) extracts.
pg_0008
404
Botanical Studies, Vol. 48, 2007
In the present study, all the extracts of six sources
exhibited the reducing power in a concentration dependent
manner. The reducing properties are generally associated
with the presence of reductones (Duh, 1998). It has been
reported that the antioxidant action of reductone was based
on the breaking of the free radical chain by donating a
hydrogen atom (Gordon, 1990). Reductones also react with
certain precursors of peroxide, thus preventing peroxide
formation. It was reported earlier that the antioxidative
properties of extracts from Garcinia subelliptica and
G.arcinia mangostana were due to presence of various
xanthones with phenolic functional groups (Minami et al.,
1996; Mahabusarakam et al., 2000). These compounds
act similarly to reductones by donating the electrons and
reacting with free radicals to convert them to a more stable
product, and by terminating the free radical chain reaction.
Results in the present study demonstrate that aqueous
extracts exert a reducing activity 2-14 times higher than
the ethanol extracts, indicating that some enzymatic
protein molecules may be involved in the ferricyanide
reduction. In fact, several enzymes such as the cytochrome
c reductase (Rafferty and Malech, 1996) or the lactate
dehydrogenase may catalyze the ferricyanide reduction.
It has been reported that the antioxidant activity of
many compounds of botanical origin is proportional to
the phenolic content (Rice-Evans et al., 1997), suggesting
a causative relationship between total phenolic content
and antioxidant activity (Veglioglu
et al., 1998). Cai et al.
(2004) showed a linear correlation between antioxidant
activity and total phenolic content (R
2
values > 0.95) in
the 112 traditional Chinese medicinal plants associated
with anticancer properties. In the present study, higher
correlation coefficients (R2) values for TEAC and total
polyphenol contents for aqueous or ethanol extracts
were observed compared to R
2
values for total flavonoid
contents. The results suggest that the total polyphenol
compounds in extracts of six folk medicinal ferns used
as "Gusuibu" contributed significantly to the antioxidant
capacities.
CONCLUSION
The present investigations provide useful information
on antioxidant properties and polyphenolic contents of
six sources of "Gusuibu." Antioxidant activities of both
ethanol and aqueous extracts among the six sources
varied significantly and had a strong correlation with total
polyphenol contents. The methods employed in the present
study are easily used and provide reproducible results.
Much higher antioxidant activities of the aqueous extracts
have given evident assumption that the water preparation
of "Gusuibu" is more potent than the ethanol one from a
medical point of view.
Acknowledgments. The authors acknowledge the
supply of plant material in the form of the fern Davallia
solida by Mr. Jinn-Fen Chen, Research Assistant, Taitung
District Agricultural Research Station, Taiwan. Help
in statistical analysis of data by Dr. Wen-Huang Peng,
Associate Professor, Institute of Chinese Pharmaceutical
Science, China Medical University, Taichung is gratefully
acknowledged.
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