Chinese Medicine, 2013, 4, 148-156
Published Online December 2013 (http://www.scirp.org/journal/cm)
http://dx.doi.org/10.4236/cm.2013.44018
Open Access CM
Antioxidant Activity of 50 Traditional Chinese Medicinal
Materials Varies with Total Phenolics
Zhengyou He1,2*, Minbo Lan1, Dongying Lu1, Hongli Zhao1, Huihui Yuan1
1Research Center of Analysis & Test and Institute of Advanced Materials, East China University of Science & Technology,
Shanghai, China
2Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, China
Received September 15, 2013; revised November 14, 2013; accepted November 29, 2013
Copyright © 2013 Zhengyou He et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
This study was designed to determine the total phenolic content of 50 herbs and to examine their antioxidant potential.
In the sample preparation, 60% ethanol was chosen as the extraction solvent for the subsequent experiments. Fo-
lin-Cicolteau phenol reagent and a colorimetric method were used to determine the total phenolic content of the selected
herbs. The result showed that total phenolic content of those herbs ranged from 2 to 185 mg/g. In antioxidant assay, the
ferric reducing/antioxidant power (FRAP) values ranged from 2 to 134 mg GAE/g; the IC50 values of DPPH•, •OH and
scavenging were in the range of 0.06 - 5.50 mg/mL, 0.017 - 0.636 mg/mL and 0.050 - 0.681 mg/mL respectively.
Flos caryophylli was the exceptant in the scavenging assay because there was no linear relation between the con-
centration and the scavenging percentage. Compared to gallic acid, ascorbic acid and butylated hydroxytoluene (BHT)
in antioxidant assay as positive control, the most potential antioxidant herbs were Cacumen platycladi, Radix et Rhi-
zoma rhei, Rhizoma rhodiolae crenulatae, and Rhizoma sanguisorbae with considerable content of phenolics. Espe-
cially, a positive and significant correlation was found between the total phenolic content and FRAP value or DPPH•
scavenging percentage.
•
2
O
•
2
O
Keywords: Traditional Chinese Medicinal Material; Total Phenolics; Antioxidant Activity;
Ferric Reducing/Antioxidant Power; Free Radical Scavenging Activity
1. Introduction
Roles of the reactive oxygen species (ROS) and reactive
nitrogen species (RNS) are increasingly recognized in
physiological processes, pathogenesis of many diseases,
and molecular mechanisms in many drug-therapies [1].
ROS are generated by all aerobic organisms and their
production seems to be essential for signal-transduction
pathways that regulate multiple physiological processes.
Excessive amount of ROS, however, can initiate toxic
and lethal chain reactions, which disable the biological
structures that are required for cellular integrity and sur-
vival. Recently, there is a growing interest in substances
exhibiting antioxidant properties that are supplied to hu-
man and animal organisms as food components or as
specific redox-therapy drugs [1]. Substantive experi-
ments have already testified that many phytochemicals
and extracts from plants possess antioxidant effects.
Many synthetic antioxidants, such as butylated hy-
droxyanisole (BHA), butylated hydroxytoluene (BHT)
and tert-butylhydro-quinone (TBHQ), are widely used in
food and pharmaceutical industries against oxidative
damage. However, animal tests have demonstrated that
those synthesized compounds would accumulate in rats
and result in liver-damage and carcinogenesis [2]. Inter-
estingly, some important antioxidants, including ascorbic
acid and the tocopherols, cannot be synthesized by hu-
mans and must be taken in diet [3]. It has long been rec-
ognized that some naturally occurring substances in
plants process antioxidant activity. Therefore, the devel-
opment and utilization of more effective and non-toxic
antioxidants from natural products are desired, not only
for the food and drug storage, but also for the nutritional
and clinical applications.
It is well known that the traditional Chinese herbs have
been used in food and medicine over two thousand years.
There are more than 11,000 officinal plants, 1500 offici-
*Corresponding author.

Z. Y. HE ET AL. 149
nal animals and 80 officinal minerals used as the tradi-
tional Chinese medicine [4]. For the reason of biodiver-
sity, the chemical composition and bioactivity of the me-
dicinal materials are also varied. Epidemiological studies
have shown that many natural antioxidant compounds
possess anti-inflammatory, antiatherosclerotic, antitumor,
antimutagenic, anticarcinogenic, antibacterial, or antivi-
ral activities to a greater or lesser extent [5,6]. Appar-
ently, the Chinese medicinal plants may contain a wide
variety of chemical composition, including phenolic
compounds (e.g. phenolic acids, flavonoids, quinones,
coumarins, lignans, stilbenes, tannins), nitrogen com-
pounds (alkaloids, amines, betalains), vitamins, terpe-
noids (including carotenoids), with potential antioxidant
activities [7]. In free radical biology, the balance between
antioxidation and oxidation is believed to be a critical
concept to maintain a healthy biological system, which is
similar to the concept of the balance between “Yin” and
“Yang” in the Traditional Chinese Medicine (TCM). The
effective compositions in the yin-tonic herbs were mainly
flavonoids with strong antioxidant activities six times
higher than that of the yang-tonic herbs [8]. Contrarily,
Szeto and Benzie indicated that the yin nature of herbs
may not be necessarily associated with superior antioxi-
dative effect to yang-tonic herbs, at least in terms of
DNA protection against oxidant challenge [9]. The syn-
ergetic antioxidant effects of the traditional Chinese
herbs should be considered in the view of systems biol-
ogy [10], but the literature partially revealed the inner
correlation between the antioxidant capacity and the tra-
ditional usages. Consequently, it is necessary to evaluate
the antioxidant activity of traditional Chinese herbs sys-
tematically using different types of free radical.
50 Traditional Chinese herbs, grown and processed
under the standard operating procedures, were selected
and prepared for the initial investigation. According to
the classification of their traditional usages [4], 18 of tho-
se herbs, including 11 stanchers, are used as the haematic.
The next is the heat-clearing drug, and 10 medicinal ma-
terials are ranged to this class. The third is the tonic, in-
cluding one yin-tonic, eight yang-tonics, and four weak-
tonics. Other medicinal materials are sorted into diapho-
retic, damp-resolving, cathartic, and antitussive respec-
tively. The main objectives of this paper were a) to de-
termine the content of total phenolics in above medicinal
materials; b) to evaluate their in vitro antioxidant activity
of ferric reducing and antioxidant power (FRAP), and
free radicals (DPPH•, •OH and ) scavenging capaci-
ties.
•
2
O
2. Materials and Methods
2.1. Materials from the Traditional Chinese
Medicine
50 Chinese medicinal materials were purchased from a
local pharmacy (Jiamei medicine chain Co., Ltd., Shang-
hai, China). The planting, harvesting, drying, processing,
and storage of the medicinal materials were conducted
according to strict traditional procedures, namely the
standard operating procedures implemented in China.
Names of those Chinese medicinal materials are listed in
Table 1, all of them have been identified according to the
literature [4]. All the voucher specimens have been de-
posited at the Specimen-room of the Research Center of
Analysis & Test, East China University of Science and
Technology, Shanghai, China.
2.2. Preparation of Extracts
Dried and pulverized sample (1 g) was extracted using 20
ml of 60% (v/v) ethanol. It was mixed continuously with
magnetic stirrer under refluxing at 60˚C for 1 h. Then,
the extracts were filtered over Xinhua filter paper. The
residue was re-extracted under the same conditions. The
obtained extracts were conflated and concentrated in
vacuo under 40˚C using a rotary evaporator (ZX98-1
Rotavapor, Shanghai Organic Chemistry Institute,
Shanghai, China) to yield dry extracts, which were stored
at 4˚C for further analysis.
2.3. Total Phenolic Contents (TPCs) Analysis
The TPCs of those extracts were analyzed using Folin-
Ciocalteu’s phenol reagent [11]. The extracts were dis-
solved in 60% (v/v) ethanol at the concentrations to fit
the TPC analysis. The solutions (0.5 mL) of different
concentrations were put in a 10 mL volumetric flask, 4.5
mL of distilled water and 1.0 mL of Folin-Ciocalteu re-
agent were added, and the flask was shaken thoroughly.
After 3 min, 4 mL of 2% Na2CO3 was added, and the
mixture was allowed to stand for 2 h with intermittent
shaking. The absorbance was measured at 770 nm (UV-
2102, Unico Instruments Co., Ltd., Shanghai, China).
Experiments were carried out in triplicate. The results
were expressed as gallic acid equivalent per gram raw
material (mg GAE/g). The same procedure was repeated
Table 1. Extraction efficiencies of various dilutions of etha-
nol in water on Folium artemistae argyi, Rhizoma rhodiolae
crenulatae, and Cortex eucommiae.
TPC a (mg GAE/g dried sample)
Species 95% ethanol60% ethanol 30% ethanol 10% ethanol
Folium
artemistae
argyi
18.49 ± 0.4634.72 ± 0.72 28.33 ± 0.70 24.13 ± 0.56
Rhizoma
rhodiolae
crenulatae 93.61 ± 1.72184.56 ± 3.78 169.02 ± 3.95 151.70 ± 1.87
Cortex
eucommiae 22.07 ± 0.6140.04 ± 0.80 34.15 ± 0.66 31.28 ± 0.59
aResults are means ± SD (n = 3).
Open Access CM

Z. Y. HE ET AL. 150
for all of the standard gallic acid solutions (0 - 10,000
μg/mL), and the standard curve was determined using the
equation:


Absorbance0.0011gallic acidμg0.0027.
2.4. Antioxidant Screening
2.4.1. Ferric Reducing and Antioxidant Power
(FRAP) Assay
The total antioxidant potential of those herbs was deter-
mined using ferric reducing and antioxidant power
(FRAP) assay [12]. FRAP reagent was freshly prepared
and mixed in the proportion of 10:1:1 (v:v:v) for A:B:C
solutions, where A = 300 mmol/L sodium acetate tri-
hydrate in glacial acetic acid buffer (pH = 3.6); B = 10
mmol/L TPTZ in 40 mmol/L HCl; and C = 20 mmol/L
FeCl3. Gallic acid was used for a standard curve with all
solutions, including samples dissolved in 60% ethanol.
The assay was carried out at 37˚C (pH = 3.6) using 0.4
mL sample or standard solution plus 4.0 mL FRAP re-
agent shown above. After 10 min incubation at room
temperature, the absorbance was read at 593 nm. Results
were expressed in mg gallic acid equivalent per gram
dried herb weight (mg GAE/g). Experiments were car-
ried out in triplicate.
2.4.2. DPPH Radical Scavenging Activity Assay
This spectrophotometric assay used the stable DPPH
radical as the reagent to determine the DPPH• scaveng-
ing activity [13]. The extracts and standards were dis-
solved in 60% (v/v) ethanol at the concentrations to fit
the DPPH assay. Ethanolic extracts or standards of 0.1
mL at various concentrations was added to 4.0 mL 0.004%
DPPH• methanol solution in a 10 mL test tube respec-
tively. After 30 min incubation at room temperature, the
absorbance was read against a contrast only containing
all solvents at 517 nm. Inhibition of the free radical of
DPPH in percent (I%) was calculated as follow:

blank sample blank
Inhibition% AAA100%

 
 
where Ablank is the absorbance of the control reaction
(containing all of the reagents except the test compound)
and Asample is the absorbance of the test samples. Exact
concentration providing 50% inhibition (IC50) was cal-
culated from the graph plotted from the regression analy-
sis as inhibition percentage against concentration of the
medicinal materials. Gallic acid, ascorbic acid, and BHT
were measured at the same procedure. Tests were carried
out in triplicate. Results were expressed in milligram
medicinal materials per milliliter (mg/mL).
2.4.3. •OH Scavenging Activity Assay
The scavenging ability of different extracts on hydroxide
radical was measured in the CuSO4-Phen-Vc-H2O2 che-
miluminescence (CL) system. The CL of hydroxyl radi-
cal formation was monitored under the described method
[14] using a BPCL Ultra-weak luminescence analyzer
(Institute of Biophysics, Academia Sinica, China). The
extracts were dispersed in 1% Tween 20 and standards
were dissolved in redistilled water, those solutions were
diluted to fit the •OH scavenging assay. The volume of
the reaction was composed of 50 μL of the sample solu-
tion, 50 μL of 1.0 mmol/L CuSO4 solution, 50 μL of 1
mmol/L 1,10-phenanthroline solution, 700 μL of 0.05
mol/L borate buffer (pH 9.0), 100 μL of 1 mmol/L ascor-
bic acid solution, and 50 μL of 1% H2O2 solution. The
reaction was initiated immediately after the injection of
H2O2 solution, and kinetic curves were obtained at 2 s
intervals over a period of 400 s. Varying degrees of sud-
den drops of CL counts observed represent the different
degrees of •OH scavenging abilities. As the inhibiting
percentage of CL counts had been calculated, compari-
son of the correlativity between the •OH scavenging ef-
ficacy and the concentration of each sample is possible.
The integrated area of the curve expressed the relative
luminescent intensity. The scavenging activity was rep-
resented by the following formula:
 

control 0sample 0
control 0
CLCL CLCL
Inhibition% CL CL
100%


 




where CLcontrol is the relative luminescent intensity of the
control group, CL0 is the relative luminescent intensity of
the background group, and CLsample is the relative lumi-
nescent intensity of the experimental group. Exact con-
centration providing 50% inhibition (IC50) was calculated
from the graph plotted as scavenging percentage against
concentration of medicinal materials. Gallic acid was
also measured at the same procedure. Tests were carried
out in triplicate. IC50 values were expressed in milligram
medicinal materials per milliliter (mg/mL).
2.4.4. Scavenging Activity Assay
•
2
O
-
•
2
O
The

scavenging activity of the selected herbs was
determined by the nitrite reduction method [15]. The
tested solutions were prepared in the •OH scavenging
assay and diluted to fit the scavenging assay. The
reaction mixture contained 0.6 mL 1 mmol/L hypoxan-
thine, 0.3 mL 220 μmol/L hydroxylammonium-chloride,
1mL buffer solution (pH 8.2, the solution containing 15.6
mmol/L Na2B4O7 and 20.8 mmol/L KH2PO4), and 40 μL
0.7 U/mL xanthine oxidase. The diluted solution of 1.0
mL was added to the reaction mixture and incubated for
30 min at 37˚C. Then 2.0 mL 1.73 mmol/L sulfanilic acid,
which was dissolved in 1.36 mmol/L acetic acid, and
2.0mL of 19.29 μmol/L N-1-naphthylethylenediamine
were injected to the solution and shook. After standing at
room temperature in the dark for 20 min, the absorbance
•
2
O
Open Access CM

Z. Y. HE ET AL. 151
was measured at 550 nm. A control solution was meas-
ured, in which sample was replaced by redistilled water.
The scavenging rate was obtained according to the for-
mula:
Scavenging rate(%)100%
cs
cb
AA
AA



where Ac is the absorbance of the control solution, As is
the absorbance of the test sample, and Ab represents the
absorbance of the blank, in which xanthine oxidase was
replaced by the buffer. Exact concentration providing 50
% inhibition (IC50) was calculated from the graph plotted
from the regression analysis as inhibition percentage
against the concentration. The results were expressed in
milligram raw materials per milliliter (mg/mL). Gallic
acid and ascorbic acid were measured at the same pro-
cedure. Experiments were carried out in triplicate.
2.5. Data Analysis
Data were processed using origin 6.1 software (Microcal
Software, Inc., Northampton, MA, USA). The regression
equations and correlation coefficients were fitted by the
least-squares method. All experiments were repeated at
least three times. The results were expressed as means ±
SD. Standard differences were considered significant at
P < 0.05.
3. Results and Discussions
3.1. Selection of Extraction Solvents
In order to select the best solvent for extraction of those
medicinal materials, four different percentages of ethanol
(10%, 3 %, 60% and 95% v/v) were used in the extrac-
tion of Folium artemistae argyi, Rhizoma rhodiolae
crenulatae, and Cortex eucommiae respectively. The
extraction solvent of 60% ethanol, indicated by the TPC
values (Table 2), was found to give the highest extrac-
tion efficiency for the selected three herbs, while 95%
ethanol had the lowest extraction efficiency. Conse-
quently, 60% ethanol was chosen as the extraction sol-
vent for the subsequent antioxidant assays. The average
extraction efficiency of 60% ethanol was determined by
multiple extraction experiments and was found in range
from 95% to 97% after the first and the second extraction
depending on the selected medicinal materials (Table 3).
Therefore, the selected medicinal materials were ex-
tracted twice using 60% ethanol under refluxing for fur-
ther investigations respectively.
3.2. Total Phenolic Contents of 50 Medicinal
Materials
There was a wide range of the total phenolic contents
among the selected medicinal materials. As shown in Ta-
ble 1, the TPC values, determined by the Folin-Ciocal-
teau method, varied from 2 to 185 mg GAE/g (average
39.9 mg GAE/g) depending on the biological origin of
the plant. It is well known that plant polyphenols are
widely distributed in the plant kingdom and sometimes in
surprisingly high concentrations [16,17]. According to
the results, there are 7 medicinal materials with the low-
est total phenolics concentration (<10 mg GAE/g), in-
cluding Semen nelumbinis < Rhizoma atractylodis mac-
rocephalae < Flos magnolia officinalis < Herba portu-
lacae < Semen ginkgo < Folium mori < Radix dipsaci.
Five herbs had total phenolics concentrations > 90 mg
GAE/g: Rhizoma rhodiolae crenulatae > Herba cirsii
japonici > Rhizoma sanguisorbae > Radix rubiae >
Radix et rhizoma rhei. The highest total phenolics con-
tent (> 150 mg GAE/g) was found in Rhizoma rhodiolae
crenulatae, the roots collected from Rhodiola crenulata
(Hook. f. et. Thoms.) H. Ohba. According to the litera-
ture [11], various phenolic compounds have different
responses in TPC assay. The molar response of this
method is roughly proportional to the number of phenolic
hydroxyl groups in a given substrate, whereas the reduc-
ing capacity is enhanced when two phenolic hydroxyl
groups are oriented ortho or para [18]. Since these struc-
tural features of phenolic compounds are also responsible
for antioxidant activity, measurements of phenols in food
or medicinal materials may be related to their antioxidant
properties.
3.3. Antioxidant Capacity
3.3.1. FRAP of 50 Medicinal Materials
As shown in Table 1, the total antioxidant capacities
(FRAP) are different from each other between the se-
lected 50 medicinal materials. The FRAP values varied
from 2 to 134 (the mean was calculated as 25.6) mg
GAE/g of the dried material weight. In FRAP assay, the
Table 2. Extraction efficiencies of Folium artemistae argyi,
Rhizoma rhodiolae crenulatae, and Cortex eucommiae.
Species Extraction
Average TPCa
(mg GAE/g dried
sample)
Average
extraction
efficiencies (%)
1st 30.09 ± 0.67 85.38 ± 1.83
2nd 3.61 ± 0.16 10.24 ± 0.46
Folium
artemistae
argyi 3rd 1.54 ± 0.09 4.37 ± 0.28
1st 139.24 ± 3.45 74.83 ± 1.76
2nd 33.04 ± 1.52 17.76 ± 0.83
Rhizoma
rhodiolae
crenulatae 3rd 13.80 ± 0.94 7.42 ± 0.45
1st 35.34 ± 0.87 87.82 ± 2.03
2nd 3.61 ± 0.18 8.97 ± 0.44
Cortex
eucommiae
3rd 1.29 ± 0.08 3.20 ± 0.21
aThe average of TPC and extraction efficiencies were based on triplicates
from a single batch; the results are means ± SD (n = 3).
Open Access CM

Z. Y. HE ET AL.
Open Access CM
152
Table 3. The total phenolic content (TPC) values and the in vitro antioxidant activities of fifty traditional Chinese medicinesa.
Plant materials
(medicinal name)
Total phenolic
contentsb
(mg GAE/g)
FRAPb
(mg GAE/g)
IC50 of DPPH
scavenging
activityc (mg/mL)
IC50 of OH
scavenging
activityc (mg/mL)
IC50 of O2-
scavenging
activityc (mg/mL)
Cacumen platycladi 74.59 ± 1.49 47.99 ± 0.96 0.140 ± 0.004 0.045 ± 0.001 0.076 ± 0.001
Cortex eucommiae 40.04 ± 0.80 26.59 ± 0.55 0.262 ± 0.005 0.270 ± 0.004 0.220 ± 0.006
Cortex magnoliae officinalis 24.34 ± 0.44 16.59 ± 0.31 0.504 ± 0.010 0.057 ± 0.001 0.069 ± 0.002
Cortex moutan 80.09 ± 1.50 53.48 ± 1.00 0.127 ± 0.002 0.089 ± 0.001 0.093 ± 0.003
Flos caryophylli 53.99 ± 1.10 36.64 ± 0.78 0.198 ± 0.006 0.021 ± 0.001 ndd
Flos chrysanthemi 11.08 ± 0.20 8.43 ± 0.21 1.037 ± 0.019 0.099 ± 0.001 0.083 ± 0.001
Flos chrysanthemi indici 10.76 ± 0.22 7.15 ± 0.14 0.994 ± 0.022 0.208 ± 0.006 0.143 ± 0.005
Flos magnolia officinalis 5.44 ± 0.09 3.68 ± 0.08 2.066 ± 0.046 0.047 ± 0.001 0.050 ± 0.002
Folium artemistae argyi 34.72 ± 0.72 21.71 ± 0.41 0.297 ± 0.008 0.132 ± 0.004 0.094 ± 0.002
Folium eucommiae ulmoides 23.54 ± 0.41 11.25 ± 0.23 0.505 ± 0.010 0.282 ± 0.005 0.108 ± 0.002
Folium ginkgo 36.64 ± 0.73 21.32 ± 0.46 0.281 ± 0.008 0.087 ± 0.002 0.084 ± 0.001
Folium mori 8.94 ± 0.20 6.30 ± 0.11 1.213 ± 0.025 0.227 ± 0.003 0.127 ± 0.002
Folium nelumbinis 14.17 ± 0.25 9.30 ± 0.20 0.725 ± 0.014 0.088 ± 0.001 0.064 ± 0.001
Folium phyllostachydis henonis 40.32 ± 0.77 25.84 ± 0.54 0.253 ± 0.006 0.450 ± 0.012 0.477 ± 0.010
Fructus arctii 17.07 ± 0.30 9.19 ± 0.15 0.704 ± 0.017 0.234 ± 0.005 0.158 ± 0.003
Fructus crataegi 44.97 ± 0.82 26.95 ± 0.59 0.232 ± 0.006 0.098 ± 0.002 0.093 ± 0.001
Fructus lycii 27.16 ± 0.54 17.98 ± 0.37 0.411 ± 0.010 0.089 ± 0.002 0.152 ± 0.003
Fructus psoraleae 36.86 ± 0.71 23.84 ± 0.52 0.297 ± 0.003 0.135 ± 0.003 0.364 ± 0.012
Herba asari 18.65 ± 0.32 10.57 ± 0.23 0.610 ± 0.014 0.533 ± 0.011 0.431 ± 0.008
Herba cirsii 82.06 ± 1.51 51.90 ± 1.02 0.128 ± 0.004 0.094 ± 0.002 0.101 ± 0.002
Herba cirsii japonici 147.64 ± 2.99 90.36 ± 1.83 0.085 ± 0.002 0.102 ± 0.002 0.076 ± 0.001
Herba epimedii 28.38 ± 0.57 14.24 ± 0.28 0.365 ± 0.007 0.296 ± 0.007 0.075 ± 0.001
Herba erodii 10.06 ± 0.20 5.64 ± 0.12 1.085 ± 0.026 0.131 ± 0.003 0.086 ± 0.002
Herba moslae 16.03 ± 0.30 11.02 ± 0.21 0.645 ± 0.015 0.636 ± 0.012 0.478 ± 0.010
Herba portulacae 6.06 ± 0.11 6.94 ± 0.15 1.686 ± 0.034 0.150 ± 0.005 0.142 ± 0.004
Herba senecionis scandentis 17.27 ± 0.38 13.09 ± 0.27 0.607 ± 0.011 0.094 ± 0.002 0.100 ± 0.002
Radix angelicae sinensis 19.92 ± 0.42 13.50 ± 0.28 0.519 ± 0.009 0.148 ± 0.004 0.076 ± 0.001
Radix astragali 27.75 ± 0.56 19.11 ± 0.36 0.380 ± 0.007 0.216 ± 0.002 0.108 ± 0.002
Radix dipsaci 9.50 ± 0.20 6.76 ± 0.15 1.168 ± 0.022 0.102 ± 0.003 0.058 ± 0.001
Radix et rhizoma rhei 90.21 ± 1.90 57.83 ± 1.21 0.126 ± 0.002 0.085 ± 0.001 0.078 ± 0.002
Radix glycyrrhizae 26.71 ± 0.55 16.67 ± 0.32 0.379 ± 0.007 0.092 ± 0.001 0.060 ± 0.001
Radix notoginseng 27.81 ± 0.59 12.84 ± 0.26 0.362 ± 0.006 0.174 ± 0.003 0.111 ± 0.002
Radix paeoniae alba 31.31 ± 0.63 21.20 ± 0.47 0.338 ± 0.008 0.152 ± 0.005 0.106 ± 0.003
Radix paeoniae rubra 46.21 ± 0.92 30.30 ± 0.69 0.232 ± 0.005 0.101 ± 0.002 0.089 ± 0.002
Radix pulsatillae 16.51 ± 0.34 11.72 ± 0.24 0.685 ± 0.014 0.187 ± 0.003 0.225 ± 0.005
Radix rubiae 93.56 ± 1.87 56.45 ± 1.12 0.109 ± 0.002 0.099 ± 0.002 0.222 ± 0.006
Radix scutellartae 80.24 ± 1.62 52.14 ± 1.01 0.146 ± 0.005 0.056 ± 0.001 0.093 ± 0.002
Ramulus uncariae cum uncis 31.53 ± 0.65 20.40 ± 0.30 0.340 ± 0.009 0.091 ± 0.002 0.153 ± 0.002
Rhizoma atractylodis macrocephalae2.88 ± 0.06 4.73 ± 0.05 4.175 ± 0.081 0.074 ± 0.002 0.086 ± 0.002
Rhizoma belamcandae 23.89 ± 0.53 14.71 ± 0.31 0.488 ± 0.013 0.073 ± 0.001 0.163 ± 0.004
Rhizoma chuanxiong 27.62 ± 0.56 16.97 ± 0.33 0.406 ± 0.007 0.244 ± 0.008 0.249 ± 0.007
Rhizoma cimicifugae 22.27 ± 0.45 12.67 ± 0.25 0.511 ± 0.018 0.313 ± 0.010 0.412 ± 0.008
Rhizoma polygont cuspidati 60.96 ± 1.25 41.38 ± 0.53 0.190 ± 0.006 0.097 ± 0.003 0.064 ± 0.002
Rhizoma rhodiolae crenulatae 184.56 ± 3.78 133.98 ± 2.73 0.062 ± 0.002 0.017 ± 0.001 0.109 ± 0.002
Rhizoma sanguisorbae 128.93 ± 2.56 72.38 ± 1.55 0.084 ± 0.002 0.035 ± 0.001 0.059 ± 0.001
Semen euryales 48.42 ± 0.99 30.98 ± 0.66 0.213 ± 0.003 0.225 ± 0.007 0.153 ± 0.005

Z. Y. HE ET AL. 153
Continued
Semen ginkgo 7.81 ± 0.20 4.96 ± 0.07 1.569 ± 0.031 0.217 ± 0.003 0.681 ± 0.009
Semen nelumbinis 2.20 ± 0.05 2.34 ± 0.05 5.477 ± 0.111 0.303 ± 0.009 0.125 ± 0.003
Spica prunellae 13.86 ± 0.24 9.17 ± 0.16 0.828 ± 0.018 0.216 ± 0.006 0.516 ± 0.015
Thallus eckloniae 59.97 ± 1.22 38.75 ± 0.79 0.175 ± 0.005 0.175 ± 0.004 0.151 ± 0.006
Gallic acid -f - 0.0153 ± 0.0004 0.0123 ± 0.0004 0.101 ± 0.003
Ascorbic acid - 510 ± 7 0.0042 ± 0.0001 - 0.0248 ± 0.0006
BHTe - - 0.0191 ± 0.0003 - -
aResults were means ± SD (n = 3); bTotal phenolic contents were expressed in gallic acid equivalent of the dried medicinal materials; cIC50 was defined as the
concentration sufficient to obtain 50% scavenging activity; dThe linear relation could not be constructed; eBHT represents butylated hydroxytoluene; fNot de-
tected.
antioxidant activity was based on the ability of the anti-
oxidant components in the samples to reduce Fe3+ to Fe2+
in a redox-linked colourimetric reaction that involves
single electron transfer [12]. According to their reducing
ability/antioxidant power (FRAP) values, 50 medicinal
plants can be divided into five groups: a) very low FRAP
(<5 mg GAE/g), n = 4; b) low FRAP (5 - 30 mg GAE/g),
n = 32; c) good FRAP (30 - 50 mg GAE/g), n = 6; d)
high FRAP (50 - 100 mg GAE/g), n = 7; and e) very high
FRAP (>100 mg GAE/g) n = 1. On the basis of the
FRAP values of the selected chemicals, the ratio of the
slope of the linear curve of ascorbic acid to that of
FeSO4•7H2O was 1.96, and the ratio of gallic acid to
FeSO4•7H2O was 4.02. Gallic acid, bearing a pyrogallol
moiety, exhibited more potent activity than ascorbic acid
(gallic acid vs asorbic acid = 2.05:1). The significant
linear correlation (coefficient “r” = 0.9918, and two-
tailed “P”-value < 0.0001) was confirmed between TPC
values and their related FRAP values of the selected me-
dicinal materials (Figure 1).
There are many methods to determine the total anti-
oxidant capacity [19]. These in vitro and in vivo methods
differ in terms of their assay principles and experimental
conditions. Consequently, antioxidant components may
individually have varying contributions to the total anti-
oxidant capability in different methods. Because FRAP
assay is quick and simple to perform, and the reaction is
reproducible and linearly related to the molar concentra-
tion of the antioxidant(s) [20], the FRAP assay was ap-
plied in the determination of the total antioxidant capac-
ity of those herbs. This method was initially developed to
assay plasma antioxidant capacity, and popularly used to
measure the antioxidant capacity from a wide range of
biological samples in recent years, including teas, vege-
tables, fruits, wines, plants, and animal tissues [21,22]. In
sharp contrast to the medicinal plants with high or very
high FRAP values, the positive properties of the medici-
nal plants with very low FRAP are unlikely related to
their antioxidant capacity. As a result, eight medicinal
materials, namely Rhizoma rhodiolae crenulatae, Herba
cirsii japonici, Rhizoma sanguisorbae, Radix et rhizoma
rhei, Radix rubiae, Cortex moutan, Radix scutellartae,
and Herba cirsii, have the highest FRAP values among
those selected herbs.
3.3.2. DPPH Radical Scavenging Activity
DPPH assay was applied to test the ability of the anti-
oxidative compounds as well as different plant extracts
functioning as proton radical scavengers or hydrogen
donors [23]. IC50 values of DPPH radicals scavenging
activity were in the range of 0.06 - 5.50 mg/mL accord-
ing to the results listed in Table 1. A negative correlation
was found between the TPCs and IC50 values, indicating
that the materials or the extracts at high TPC levels
would have low IC50 values but strong potency to scav-
enge DPPH radicals. Among 50 selected materials, 12
medicinal materials with lowest IC50 values (<0.2 mg/mL)
were Rhizoma rhodiolae crenulatae < Rhizoma san-
guisorbae < Herba cirsii japonici < Radix rubiae <
Radix et rhizoma rhei < Cortex moutan < Herba cirsii <
Cacumen platycladi < Radix scutellartae < Thallus eck-
loniae < Rhizoma polygont cuspidati < Flos caryophylli.
Gallic acid and BHT, determined with the IC50 values of
15.3 μg/mL and 19.6 μg/mL respectively, were used as
the positive control in DPPH assay. The correlation was
investigated between the concentration and the antioxi-
dant capacity at different concentrations of individual
medicinal materials. The results indicated that the se-
lected herbs have liner relation between the DPPH radi-
cals scavenging percentages and the TPC concentrations.
It could be concluded that the DPPH radicals scavenging
nature of those materials might depend on their total
phenolics tentatively. As a result, a parabola regressive
model could be built from those data. In other words, the
reciprocals of the TPCs values were linear to the IC50
values (coefficient “r” = 0.9985, and two-tailed
“P”-value <0.0001) (Figure 2).
3.3.3. •OH and Scavenging Activities
•
2
O
-
To evaluate the ROS scavenging properties of those me-
dicinal materials, we have used two different reactive
oxygen species (ROS): the hydroxyl radical and super-
oxide anion radical. •OH was produced and monitored by
the CuSO4-Phen-Vc-H2O2 chemiluminescence system,
Open Access CM

Z. Y. HE ET AL. 154
050100 150 200
0
20
40
60
80
100
120
140
FRAP (mg GAE/g)
Total Phenolic Contents (mg GAE/g)
Figure 1. Linear correlation between the amount of total
phenolics and the total antioxidant capacity (FRAP), y =
0.6509x − 0.3783. Correlation coefficient “r” = 0.9918. The
two-tailed P value is <0.0001, considered extremely signifi-
cant.
0.00.10.20.30.4
0
1
2
3
4
5
6
0.5
IC50 of DPPH rad ical ( m g / mL )
1/(Total Phenolic Contents) (mg GAE/g)-1
Figure 2. Linear correlation between the reciprocals of the
total phenolic content and IC50 values of DPPH scavenging
activity, y = 11.9209x − 0.0406. Correlation coefficient “r” =
0.9985. The two-tailed P value is < 0.0001, considered ex-
tremely significant.
whereas was generated by the hypoxanthinexan-
thine oxidase system and detected by UV-Vis spectro-
photometry. The results of the ROS scavenging capaci-
ties, in the form of IC50 values of those herbs, were pre-
sented in Table 1. The IC50 values of •OH and •
2
O
•
2
O

v
ied in the ranges of 0.017 - 0.636 mg/mL and 0.050 -
0.681 mg/mL respectively. In the superoxide anion radi-
cal assay, only Flos caryophylli did not exhibit correla-
tion between the free radicals scavenging percentage and
the concentration. Comparison of the ROS scavenging
characteristics of those medicinal materials, Herba
moslae has the highest potency to scavenge the hydroxyl
radicals, whereas Semen ginkgo is the highest in the su-
peroxide anion radical assay. According to the results,
there was a weak linear relation between IC50 values of
the hydroxyl radical and the superoxide anion radical
scavenging activities (coefficient “r” = 0.6442, and
two-tailed “P”-value <0.0001). The individual extracts,
which could scavenge the hydroxyl radicals, can not
necessarily eliminate the superoxide anion radicals. As a
result, traditional Chinese herbs have specific ROS
scavenging properties respectively, which can be applied
in the explanation of the rules of compatibility of medi-
cines in the traditional Chinese medicine.
In the ROS scavenging experiments, there was no lin-
ear response between the total phenolic contents and the
free radicals scavenging activities, other factors should
be considered in the evaluation of the ROS scavenging
capacities. There were several methods to screen the
ROS scavengers, different methods could give varied
results for the unstable characteristics of the ROS in
chemical or biochemical systems. The total phenolics
content in the extracts could be correlated linearly with
the oxygen depletion, but not with the ROS scavenging
effect by different methods using ESR spin trapping and
electrochemical measurement [24]. Other scientists also
have not found linear response between the total pheno-
lics and the ROS scavenging capacities [8]. The differ-
ence between the sterical structures of antioxidants or the
free radicals played a more important role in the abilities
to scavenge different types of free radicals [25], which
could be applied in the explanation of the antioxidant
variations between the DPPH•, •OH, and •
2
O

scaveng-
ing activities. In DPPH assay, the 60 % ethanol extracts
showed higher scavenging activity than the 95 % ethanol
extracts. The similar conclusion could be drawn from the
results of the extracts by different polar solvents in •OH
and •
2
O

assays. It suggests that more-polar components
presented in extracts have contributed towards the in-
creased ROS scavenging activities. Although there was
no direct evidence in this study, the antioxidant activities
of 60% ethanol extracts could be related to the presence
of phenolic compounds, peptides, saccharides, and other
polar compounds because they contain hydroxyl moiety
[26,27].
3.3.4. Comparison of Antioxidant Activities of 50
Medicinal Materials
Influenced by several biofactors, such as the ROS and
other free radicals occurrence, the redox status in human
body, and the bioavailability of the phytochemicals, the
traditional Chinese herbs would act as more complicated
roles in the life processes than the chemical or bio-
chemical systems in vitro. According to the classification
of their medicinal usages in the traditional Chinese
medicine, 18 of those herbs, including 11 stanchers, were
traditionally used as the haematic. Those haematic drugs,
especially the stanchers, have highest TPC values and
ar-
Open Access CM

Z. Y. HE ET AL. 155
strongest antioxidant capacities in comparison with other
herbs. The next is the heat-clearing drugs, totally 10
herbs are ranged to this class. According to the results,
those heat-clearing drugs owned higher total phenolic
contents and moderate antioxidant activities. The other
medicinal materials, traditionally defined as the tonic, the
diaphoretic and damp-resolving, have quite low TPC
values and low antioxidant activities. On the other hand,
the diseases are usually treated by complex prescriptions
using the drug matching principles in traditional Chinese
medicine. So, it would be of great importance to investi-
gate the antioxidant characteristics of the traditional
Chinese medicinal materials using the different antioxi-
dant screening systems.
The total antioxidant (FRAP) and DPPH•, •OH and
scavenging activities have different mechanisms in
the antioxidant effects, so the herbs with the highest ca-
pacities were chose as the potential antioxidants. Four
traditional Chinese herbs, namely Cacumen platycladi,
Radix et rhizoma rhei, Rhizoma rhodiolae crenu lata e and
Rhizoma sanguisorbae, have antioxidant potency in
comparison with some well known natural and synthetic
antioxidants. Contrarily, Folium mori, Fructus arctii,
Semen ginkgo, Semen nelumbinis and Spica prunellae
were the weak antioxidants correspondingly. It has been
revealed that various phenolic antioxidants, such as fla-
vonoids, tannins, coumarins, xanthones, and procyanid-
ins, can scavenge free radicals dose-dependently [28],
thus they are viewed as promising therapeutic drugs for
the free radical related disorders or illnesses. There are
more than 4000 naturally occurring flavonoids described
in the literature [29], including chalcones, flavonones,
flavones, biflavonoids, dihydroflavonols, anthrocyanid-
ins, and flavonols. Other polar natural products, such as
proteins, saccharides, etc., also have the antioxidant ca-
pacities, but as a rule, phenolic compounds were applied
in the evaluation of the correlation between the results of
the antioxidant capacities and the botanic materials
[26,27]. Therefore, the antioxidant activities of plant
original medicinal materials are dependent on the
chemical type of antioxidant compounds, the polarity of
the extracting solvent, and the test systems or the sub-
strates to be protected.
•
2
O
Interestingly, many complex prescriptions can be as-
sembled from the selected 50 herbs according to the tra-
ditional Chinese medicine. Among those prescriptions,
the herbs from at least two types are discriminated as
monarch, minister, assistant and guide by the roles of
their actions in the diseases treatment. The composition
of abundant substances in the complex prescription will
provide more complicated and synergistic antioxidant
effects in the human body than the individual herbs.
4. Conclusion
In conclusion, our results further support the point of
view that some medicinal materials are promising
sources of natural antioxidants. Among 50 selected tradi-
tional Chinese herbs, the total phenolic content and the
antioxidant capacity differed significantly. There were
significant linear correlations between the total phenolic
concentration and the values of FRAP or DPPH radicals
scavenging percentage. We also have found that three
stanchers, namely Cacumen platycladi, Rhizoma Rho-
diolae crenulatae, Rhizoma sanguisorbae, and one ca-
thartic, that is Radix et rhizoma rhei, have significant
ferric reducing power and free radicals scavenging ac-
tivities. Those traditional Chinese medicines have been
certified with low profile of side effects and toxicities for
thousands of years. Several herbs, popularly used in the
traditional Chinese medicine, have already been on
schedule to be investigated for their phytochemistry and
their medicinal applications.
5. Acknowledgements
We gratefully acknowledge Shanghai Nanotechnology
Promotion Center (Grant No. 0552nm018) for the finan-
cial support.
REFERENCES
[1] B. Halliwell and M. C. G. John, “Free Radicals in Biol-
ogy and Medicine,” 2 Edition, Oxford University Press,
Oxford, 1989, pp. 299-508.
[2] N. Ito, S. Fukushima and H. Tsuda, “Carcinogenicity and
Modification of the Carcinogenic Response by BHA,
BHT, and Other Antioxidants,” Critical Reviews in Toxi-
cology, Vol. 15, No. 2, 1985, pp. 109-150.
http://dx.doi.org/10.3109/10408448509029322
[3] R. G. Cutler, “Antioxidants and Aging,” The American
Journal of Clinical Nutrition, Vol. 53, No. 1, 1991, pp.
373S-379S.
[4] X. M. Hu, “Chinese Materia Medica (in Chinese),” Vol. 1,
Shanghai Science and Technology Press, Shanghai, 1999,
pp. 1-252.
[5] Y. Cai, et al., “Antioxidant Activity and Phenolic Com-
pounds of 112 Traditional Chinese Medicinal Plants As-
sociated with Anticancer,” Life Sciences, Vol. 74, No. 17,
2004, pp. 2157-2184.
http://dx.doi.org/10.1016/j.lfs.2003.09.047
[6] P. Scartezzini and E. Speroni, “Review on Some Plants of
Indian Traditional Medicine with Antioxidant Activity,”
Journal of Ethnopharmacology, Vol. 71, No. 1-2, 2000,
pp. 23-43.
http://dx.doi.org/10.1016/S0378-8741(00)00213-0
[7] Y. Z. Cai, et al., “Structure-Radical Scavenging Activity
Relationships of Phenolic Compounds from Traditional
Chinese Medicinal Plants,” Life Sciences, Vol. 78, No. 25,
2006, pp. 2872-2888.
http://dx.doi.org/10.1016/j.lfs.2005.11.004
[8] B. Ou, et al., “When the East Meets West: The Relation-
ship between Yin-Yang and Antioxidation-Oxidation,”
Open Access CM

Z. Y. HE ET AL.
Open Access CM
156
The FASEB Journal: Official Publication of the Federa-
tion of American Societies for Experimental Biology, Vol.
17, No. 2, 2003, pp. 127-129.
[9] Y. T. Szeto and I. F. Benzie, “Is the Yin-Yang Nature of
Chinese Herbal Medicine Equivalent to Antioxidation-
Oxidation?” Journal of Ethnopharmacology, Vol. 108,
No. 3, 2006, pp. 361-366.
http://dx.doi.org/10.1016/j.jep.2006.05.033
[10] D. Wormuth, et al., “Redox Regulation and Antioxidative
Defence in Arabidopsis Leaves Viewed from a Systems
Biology Perspective,” Journal of Biotechnology, Vol. 129,
No. 2, 2007, pp. 229-248.
http://dx.doi.org/10.1016/j.jbiotec.2006.12.006
[11] V. L. Singleton and J. A. Rossi, “Colorimetry of Total
Phenolics with Phosphomolybdic-Phosphotungstic Acid
Reagents,” American Journal of Enology and Viticulture,
Vol. 16, No. 3, 1965, pp. 144-158.
[12] I. F. F. Benzie and J. J. Strain, “The Ferric Reducing
Ability of Plasma (FRAP) as a Measure of ‘Antioxidant
Power’: The FRAP Assay,” Analytical Biochemistry, Vol.
239, No. 1, 1996, pp. 70-76.
http://dx.doi.org/10.1006/abio.1996.0292
[13] T. Katsube, et al., “Screening for Antioxidant Activity in
Edible Plant Products: Comparison of Low-Density Lipo-
protein Oxidation Assay, DPPH Radical Scavenging As-
say, and Folin-Ciocalteu Assay,” Journal of Agricultural
and Food Chemistry, Vol. 52, No. 8, 2004, pp. 2391-6.
http://dx.doi.org/10.1021/jf035372g
[14] C. H. Tsai, et al., “Rapid and Specific Detection of Hy-
droxyl Radical Using an Ultraweak Chemiluminescence
Analyzer and a Low-Level Chemiluminescence Emitter:
Application to Hydroxyl Radical-Scavenging Ability of
Aqueous Extracts of Food Constituents,” Journal of Ag-
ricultural and Food Chemistry, Vol. 49, No. 5, 2001, pp.
2137-2141. http://dx.doi.org/10.1021/jf001071k
[15] Y. Oyangui, “Reealuative of Assay Methods and Estab-
lishment of Kit for Superoxide Dismutase Activity,” Ana-
lytical Biochemistry, Vol. 142, No. 2, 1984, pp. 290-296.
http://dx.doi.org/10.1016/0003-2697, No. 84)90467-6
[16] J. B. Harborne and C. A. Williams, “Anthocyanins and
Other Flavonoids,” Natural Product Reports, Vol. 18, No.
3, 2001, pp. 310-333. http://dx.doi.org/10.1039/b006257j
[17] J. B. Harborne and C. A. Williams, “Advances in Flavon-
oid Research Since 1992,” Phytochemistry, Vol. 55, No. 6,
2000, pp. 481-504.
http://dx.doi.org/10.1016/S0031-9422(00)00235-1
[18] E. N. Frankel, A. L. Waterhouse and P. L. Teissedre,
“Principal Phytochemicals in Selected California Wines
and Their Antioxidant Activity in Inhibiting Oxidation of
Human Low-Density Lipoproteins,” Journal of Agricul-
tural and Food Chemistry, Vol. 43, No. 4, 1995, pp. 890-
894. http://dx.doi.org/10.1021/jf00052a008
[19] G. Bartosz, “Total Antioxidant Capacity,” In: H. E.
Spiegel, Ed., Advances in Clinical Chemistry, Vol. 37,
2003, Academic Press, New York, pp. 219-292.
[20] I. F. F. Benzie, W. Y. Chung and J. J. Strain, “Antioxi-
dant (Reducing) Efficiency of Ascorbate in Plasma Is Not
Affected by Concentration,” The Journal of Nutritional
Biochemistry, Vol. 10, No. 3, 1999, pp. 146-150.
http://dx.doi.org/10.1016/S0955-2863(98)00084-9
[21] D. Modun, et al., “The Increase in Human Plasma Anti-
oxidant Capacity after Red Wine Consumption Is Due to
Both Plasma Urate and Wine Polyphenols,” Atheroscle-
rosis, 2007.
[22] V. Katalinic, et al., “Screening of 70 Medicinal Plant
Extracts for Antioxidant Capacity and Total Phenols,”
Food Chemistry, Vol. 94, No. 4, 2006, pp. 550-557.
http://dx.doi.org/10.1016/j.foodchem.2004.12.004
[23] T. Nomura, et al., “Proton-Donative Antioxidant Activity
of Fucoxanthin with 1,1-Diphenyl-2-picrylhydrazyl
(DPPH),” Biochemistry and Molecular Biology Interna-
tional, Vol. 42, No. 2, 1997, pp. 361-370.
[24] H. L. Madsen, et al., “Screening of Antioxidantive Activ-
ity of Spices. A Comparison between Assays Based on
ESR Spin Trapping and Electrochemical Measurement of
Oxygen Consumption,” Food Chemistry, Vol. 57, No. 2,
1996, pp. 331-337.
http://dx.doi.org/10.1016/0308-8146(95)00248-0
[25] Q. Guo, et al., “ESR and Cell Culture Studies on Free
Radical-Scavenging and Antioxidant Activities of Isofla-
vonoids,” Toxicology, Vol. 179, No. 1-2, 2002, pp. 171-
180. http://dx.doi.org/10.1016/S0300-483X(02)00241-X
[26] L. E. Netto, et al., “Reactive Cysteine in Proteins: Protein
Folding, Antioxidant Defense, Redox Signaling and More.
Comparative Biochemistry and Physiology,” Toxicology
& Pharmacology: CBP, Vol. 146, No. 1-2, 2007, pp. 180-
193.
[27] A. Kardosová and E. Machová, “Antioxidant Activity of
medicinal Plant Polysaccharides,” Fitoterapia, Vol. 77,
No. 5, 2006, pp. 367-373.
http://dx.doi.org/10.1016/j.fitote.2006.05.001
[28] Y. C. Zhou and R. L. Zheng, “Phenolic Compounds and
an Analog as Superoxide Anion Scavengers and Antioxi-
dants,” Biochemical Pharmacology, Vol. 42, No. 6, 1991,
pp. 1177-1179.
http://dx.doi.org/10.1016/0006-2952(91)90251-Y
[29] E. Grotewold, “The Science of Flavonoids,” Springer
Science & Business Media, Inc., New York, 2006, pp.
1-47.
Abbreviations
FRAP: ferric reducing/antioxidant power
GAE: gallic acid equivalent
IC50: exact concentration providing 50% inhibition
BHT: butylated hydroxytoluene
DPPH•: the free radical of di(phenyl)-(2,4,6-trinitro-
phenyl)iminoazanium
ROS: reactive oxygen species
RNS: reactive nitrogen species
TPC: total phenolic content
CL: chemiluminescence