ARTICLE

[Link]/JAFC

Antioxidant and Anti-inflammatory Activities of Selected Medicinal

Plants Containing Phenolic and Flavonoid Compounds
Lin Zhang,† Anjaneya S. Ravipati,† Sundar Rao Koyyalamudi,*,† Sang Chul Jeong,† Narsimha Reddy,†
Paul T. Smith,† John Bartlett,† Kirubakaran Shanmugam,§,‡ Gerald M€unch,‡ and Ming Jie Wu#
†
School of Natural Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia
§
School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland 4811, Australia
‡
School of Medicine and #School of Biomedical and Health Sciences, University of Western Sydney, Sydney, Australia

ABSTRACT: The antioxidant, anti-inflammatory, and cytotoxic activities of water and ethanol extracts of 14 Chinese medicinal
plants were investigated and also their total phenolics and flavonoid contents measured. The antioxidant activity was evaluated in a
biological assay using Saccharomyces cerevisiae, whereas the radical scavenging activity was measured using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) method. Total phenolics and flavonoid contents were estimated by Folin
Ciocalteu and aluminum
chloride methods, respectively. The anti-inflammatory activities of the plant extracts were determined by measuring the inhibition of
production of nitric oxide (NO) and TNF-α in LPS and IFN-γ activated RAW 264.7 macrophages. Their cytotoxic activities against
macrophages were determined by Alamar Blue assay. Four plants, namely, Scutellaria baicalensis, Taxillus chinensis, Rheum officinale,
and Sophora japonica, showed significant antioxidant activity in both yeast model and also free radical scavenging methods. The
ethanol extract of S. japonica showed highest levels of phenolics and flavonoids (91.33 GAE mg/g and 151.86 QE mg/g,
respectively). A positive linear correlation between antioxidant activity and the total phenolics and flavonoid contents indicates that
these compounds are likely to be the main antioxidants contributing to the observed activities. Five plant extracts (S. baicalensis,
T. chinensis, S. japonica, Mahonia fortunei, and Sophora flavescens) exhibited significant anti-inflammatory activity by in vitro
inhibition of the production of NO and TNF-α with low IC50 values. These findings suggest that some of the medicinal herbs
studied in this paper are good sources of antioxidants.
KEYWORDS: medicinal plants, antioxidant activity, anti-inflammatory, phenolics, flavonoids

’ INTRODUCTION tissue damage.7,8 It is also important to note that NO reacts with

Free radicals and reactive oxygen species (ROS) are constantly free radicals, such as superoxides, to produce highly damaging
produced as byproducts in the human body during cell metabo- peroxynitrates, which can oxidize low-density lipoproteins that
lism. These harmful byproducts, if not eliminated immediately, can lead to irreversible damage to cell membranes.7,8 Hence, inhibi-
cause oxidative damage to functional macromolecules such as tion of production of such pro-inflammatory molecules (NO and
DNA, proteins, and lipids.1,2 This increases the chance of oc- TNF-α) is expected to have therapeutic value against inflamma-
currence of age-related disorders, cancer, atherosclerosis, neu- tory diseases.
rodegenerative diseases, and inflammation.2,3 Consumption of The free radical scavenging activity (antioxidant activity) has
antioxidants, through diet and supplements, is expected to remove been evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
ROS from the living system and provide health benefits. Several method, which is regarded as an easy, reliable, sensitive, and rapid
studies demonstrated that medicinal plants are a rich source of method as compared with other antiradical methods.9 A review
antioxidant compounds such as phenolics, flavonoids, quinones, by Moon and Shibamoto also reported that 90% of antioxidant
vitamins, coumarins, and alkaloids, which can decrease the in- activity measurements are conducted by this method. In vitro
cidence of oxidative stress and associated diseases.3 Recent antioxidant activities of the plants were evaluated on the basis of
studies demonstrated that plant phenolic compounds and flavo- their ability to inhibit H2O2 induced oxidative stress in yeast cells
noids act as reducing agents (either by donating hydrogen atom (Saccharomyces cervisiae). This method has been employed for
and/or by quenching singlet oxygen), which explains their antiox- in vivo antioxidant studies as yeast is the ideal model organism for
idant activities.4 monitoring the biological mechanism.10,11 The anti-inflamma-
The literature strongly suggests that plant polyphenols (especially tory properties of selected plants were evaluated on the basis of
phenolics and flavoinoids) inhibit the inflammation process by inhibitory ability against the production of NO and TNF-α using
regulating the production of pro-inflammatory molecules.5 the Griess reagent method and sandwich ELISA, respectively.
Pro-inflammatory molecules such as cytokine (TNF-α), leuko-
cyte adhesion, and nitric oxide (NO) produced during inflam- Received: August 7, 2011
matory reactions have been shown to play a crucial role in Revised: October 20, 2011
immune-inflammatory response.6,7 Excess production of NO Accepted: October 24, 2011
and TNF-α is known to cause host cell death and inflammatory Published: October 24, 2011

r 2011 American Chemical Society 12361 [Link]/10.1021/jf203146e | J. Agric. Food Chem. 2011, 59, 12361–12367
Journal of Agricultural and Food Chemistry ARTICLE

Table 1. Chinese Medicinal Herbs Used in This Study

plant plant name family name Chinese name herb parts used medicinal use ref

1 Sarcandra glabre (Thunb.) Nakai Chloranthaceae Zhong jie feng whole plant antitumor and antibacterial 27
2 Codonopsis pilosula (Franch.) Nannf. Campanulaceae Dang shen root immunomodulatory activity 28
3 Curcuma aromatica Salisb Zingiberaceae Yu jin root anti-inflammatory, antioxidant, and 29
anticarcinogenic
4 Scutellaria baicalensis Georgi. Labiatae Huang qin root anti-inflammatory and antitumor 30
5 Taxillus chinensis (DC) Danser Loranthaceae Sang ji sheng stem and branch obesity 31
6 Eucommia ulmoides Oliver Eucomiaceae Engler Du zhong bark anticancer and antifungal 32
7 Atractylodes macrocephala Koidz. Compositae Bai zhu rhizome anti-inflammatory activity 33
8 Rheum officinale Bail Polygonaceae Da huang rhizome digestive system diseases, treatment of 22
various hemorrhages, and trauma
9 Sophora japonica (L.) Schott. Fabaceae Huai hua flower anti-inflammatory activity 34
10 Polygonum cuspidatum Sieb. et Zucc Polygonacae Hu Zhang root and rhizome antioxidant activty and anti-inflammatory 35
activity
11 Saposhnikovia divaricata Apiaceae Fang feng root anti-inflammatory 36
(Turcz.) Schischk (alt. Umbelliferae)
12 Mahonia fortunei (Lindl.) Fedde Berberidaceae Shi da gong lao leaf antibacterial and antifungal 37
13 Sophora flavescens Ait. Fabaceae Ku shen root antimicrobial 38
14 Pinellia ternate (Thunb.) Breit. Araceae Ban xia rhizome antimicrobial and anti-inflammatory 39

The toxicity effects of the plant extracts were evaluated using the Ethanol Extracts. Ground samples (3 gms) were extracted with
Alamar Blue assay. 45 mL of 95% ethanol on a water bath at 70 °C for 6 h. The extracted
The approaches for selecting plants to be tested for their activity samples were centrifuged, and the supernatant was transferred into
vary from random selection to more guided selection strategies a 50 mL volumetric flask and its volume and adjusted to 50 mL with
such as the ethnopharmacological approach12,13 and the experi- 95% ethanol. The samples were stored at
4 °C until analysis. All water
ence derived from traditional practice.14,15 For the study reported and ethanol extracts were filtered before analysis.
here, 14 medicinal plants (Table 1) have been carefully selected on Determination of Total Phenolic Compounds. The total
the basis of ethnopharmacological importance13 and traditional phenolics content was determined by F-C colorimetric method.16
practice.12,14 The main aim of the current study is to discover the Briefly, 50 μL of sample and 50 μL of F-C reagent were pipetted into
plants that are good sources of natural antioxidants. Such plants an eppendorf tube. The contents were vortexed for 10 s and then left at
will be useful for isolating newer antioxidants. room temperature for 2 min. After 2 min, 500 μL of 5% (w/v) sodium
carbonate solution was added to stop the reaction, and then 400 μL of
distilled water was added to make up to 1 mL. The vortexed reaction
’ MATERIALS AND METHODS
mixture was heated in a water bath at 45 °C for 30 min and then cooled
Plant Materials. The dried plant materials were obtained from rapidly in an ice bath. Absorbance was measured at 760 nm. Gallic acid
Beijing Tong Ren Tang Chinese Herbal Medicine shop, Sydney, concentrations ranging from 0 to 300 μg/mL were prepared, and
Australia. A voucher specimen of each plant has been deposited in the the calibration curve was obtained using a linear fit (r2 = 0.9961). The
laboratory. The scientific names and family names are given in Table 1. samples were analyzed in duplicate.
The plant materials were ground to a fine powder in a grinder before Determination of Total Flavonoids. Total flavonoids were
extraction. estimated according to the aluminum chloride method.17 Briefly,
Chemicals and Reagents. Gallic acid, quercetin, DPPH, dimethyl 0.5 mL of each sample and 300 μL of NaNO2 (1:20 w/v) were pipetted
sulfoxide (DMSO), sodium carbonate, aluminum chloride (AlCl3), into a test tube. The contents were vortexed for 10 s and left at room
sodium nitrate (NaNO2), sodium hydroxide (NaOH), hydrogen per- temperature for 5 min. Into the mixture were then added 300 μL of
oxide (H2O2), Folin
Ciocalteu (F-C) reagent, ascorbic acid, 95% AlCl3 (1:10 w/v), 2 mL of 1 M NaOH, and 1.9 mL of distilled water.
ethanol, bovine serum albumin (BSA), lipopolysaccharide (LPS: Escher- After 10 s of vortexing, the absorbance for each sample was measured at
ichia coli serotype 0127:B8), N-(1,1-naphthyl)ethylenediamine dihy- 510 nm. Quercetin concentrations ranging from 0 to 1200 μg/mL were
drochloride, penicillin G sodium benzyl, resazurin sodium 10%, prepared, and the standard calibration curve was obtained using a linear
streptomycin, sulfanilamide, tetramethylbenzidine (TMB), and Trypan fit (r2 = 0.9980). The samples were analyzed in duplicate.
Blue were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd. Free Radical DPPH Scavenging Assay. The DPPH radical
(Australia). Antibiotics, Dulbecco’s modified eagle’s medium (DMEM), scavenging assay was carried out by using the Blois method.18 Each plant
fetal bovine serum (FBS), and glutamine were purchased from GIBCO. extract (50 μL) (water or ethanol) was added to a 150 μL of 62.5 μM
Interferon-γ (murine) and tumor necrosis factor-α (TNF-α) enzyme-linked DPPH. After 30 min of incubation, the absorbance of the reaction
immunosorbent assay (ELISA) kits were purchased from Peprotech. mixtures was measured at 492 nm using a microplate reader (Multiskan
Preparation of the Water Extracts. Approximately 3 g of each EX, Thermo Electron, USA). Ascorbate (vitamin C), an antioxidant, was
ground plant material was autoclaved with 45 mL of deionized water used as a positive control. A standard curve was included for each plate
(DIW) at 121 °C for 1 h. The extracted samples were centrifuged at with a series of ascorbate concentrations (0, 10, 20, 40, 60, 80, 100, 200,
10447g for 20 min), the supernatant was transferred into a 50 mL 400, and 1000 μM). The free radical reduction capacity for each herbal
volumetric flask, and the volume was adjusted to 50 mL. The samples extract was calculated as the ascorbate equivalent against the ascorbate
were stored at
20 °C until analysis. standard curve (r2 = 0.9924).

12362 [Link]/10.1021/jf203146e |J. Agric. Food Chem. 2011, 59, 12361–12367

Journal of Agricultural and Food Chemistry ARTICLE

Assay for Screening Scavenging Activity in a 96-Well TNF-α Determination by ELISA. Sandwich ELISA was used
Microplate by Using S. cerevisiae. The antioxidant capacities of according to the manufacturer’s manual (Peprotech) to determine
the herbal extracts were also measured using a S. cerevisiae-based high TNF-α concentration. Capture antibody was used at a concentration of
throughput assay. S. cerevisiae BY4743 was cultured overnight in a 50 mL 0.5 μg/mL in PBS (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM
volume by inoculation of a single colony. The culture was then diluted to NaCl; pH 7.4). Serial dilutions of TNF-α standard from 0 to 1000 pg/mL
an optical density at 600 nm (OD600) of 0.2 in media, and 180 μL of each in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal
strain was added into a well in a 96-well microtiter plate, where 10 μL per standard. TNF-α was detected with a biotinylated second antibody and an
well of each herbal extract was also added in duplicate. Ten microliters of avidin peroxidase conjugate with TMB as detection reagent. The color
H2O2 was added to a final concentration of 4 mM. The initial OD600 development was monitored at 655 nm, with readings taken after every
reading was taken using a microplate reader (Multiskan EX, Thermo 5 min. After 25 min, the reaction was stopped using 0.5 M sulfuric acid,
Electron, USA), and the plates were then incubated in a 30 °C incubator and the absorbance was measured at 450 nm.
with shaking at 750 rpm. Yeast growth was monitored by reading OD600 Data Presentation and Analysis. As the experiments were done
at the end of 20 h. Ascorbic acid was used as a positive control.19 The net in duplicate, the results were expressed as the mean ( standard
growth of H2O2-induced yeast cells after the treatment of selected plant deviation. In addition, linear relationships and significance tests of these
extracts was measured using the equation data sets were also conducted. GraphPad Prism 5.01 was used for growth
  curve analysis in dose-dependent experiments and to determine the IC50
Psample
Pcontrol values for NO and TNF-α inhibition.
Pyeastgrowth ¼  100
Pcontrol

where Pyeast growth = net growth of H2O2-induced yeast cells after ’ RESULTS AND DISCUSSION
treatment with plant extracts, Psample = observed optical density of yeast
Total Phenolics and Flavonoids Contents in the Selected
cells with the treatment of plant extracts, and Pcontrol = observed optical
Plants. The total phenolics and flavonoid contents of the selected
density of yeast cells with the treatment of negative control (H2O2).
14 herbs were measured using F-C reagent and aluminum chloride
Maintenance, Preparation, and Activation of RAW 264.7
Macrophages. RAW 264.7 macrophages were grown in 175 cm2
methods, respectively. These results obtained for water and
flasks on DMEM containing 5% FBS that was supplemented with
ethanol extracts of the plants are presented in Table 2. As
antibiotics (1%) and glutamine (1%). The cell line was maintained in can be seen from the table, significant phenolics content was
5% CO2 at 37 °C, with media being replaced every 3
4 days. Once cells observed in water extracts of S. baicalensis (60.52 GAE mg/g) and
had grown to confluence in the culture flask, they were removed using a R. officinale (33.18 GAE mg/g). Moderate levels of phenolics
rubber policeman, as opposed to using trypsin, which can remove content were found in water extracts of T. chinensis, S. japonicas,
membrane-bound receptors such as RAGE.20 Cell suspension was and M. fortunei (21.46, 27.84, and 28.6 GAE mg/g, respectively).
concentrated by centrifugation for 3 min at 900 rpm and resuspension The highest phenolics content was observed in the ethanol extracts
in a small volume of fresh DMEM (with 1% antibiotics and 5% FBS). of S. japonica (91.33 GAE mg/g) followed by P. cuspidatum
Cell densities were estimated using a Neubauer counting chamber. Cell (49.07 GAE mg/g), S. baicalensis (46.85 GAE mg/g), R. officinale
concentration is adjusted with DMEM (with 1% antibiotics and 5% (42.77 GAE mg/g), and T. chinensis (26.54 GAE mg/g).
FBS) to obtain 75000 cells/well when 100 μL cell suspension was The mean values of total flavonoids content varied from 0.73
dispensed into the 60 inner wells of 96-well plates. Sterile distilled water to 78.52 QE mg/g, whereas in ethanol extracts it ranged from
was added to the outer row of wells and incubated at 37 °C and 5% CO2 1.05 to 151.86 QE mg/g. Among all of the water extracts, the
for 12 h. From each well conditioned medium was replaced with fresh highest flavonoid contents were found in T. chinensis (78.52 QE
serum-free medium. For assays with extracts, a 50 μL volume of the mg/g), S. japonica (66.76 QE mg/g), M. fortunei (59.13 QE mg/
dilutions (in water) was added an hour prior to addition of activator. Due g), and R. officinale (54.57 QE mg/g). In addition to these plants,
to the often inconsistent nature of LPS at activating cells, a combination moderate levels of total flavonoids content were also found in
of 25 μg/mL LPS and 10 U/mL IFN-γ, both in DMEM, was used for Sarcandra glabre, S. baicalensis, and P. cuspidatum. The ethanol
activation. Usually a maximum dose of the extracts used was 2.5 mg/mL, extracts of S. japonica (151.86 QE mg/g), P. cuspidatum (84.73
and a minimum of 6 doses were made by serial dilution. Then the cells QE mg/g), R. officinale (98.51 QE mg/g), T. chinensis (64.64 QE
were incubated for 24 h at 37 °C and 5% CO2. Cells with media alone mg/g), and S. baicalensis (58.46 QE mg/g) showed significantly
were used as negative control and activated cells as positive control. high flavonoid contents. As can be seen from Table 2, the
Determination of Nitric Oxide Production by Griess Assay. phenolics/flavonoid contents varied among all selected plants
Nitric oxide is determined by Griess reagent quantification of nitrite, one
and in different extracts.
of its stable reaction products. Griess reagent is freshly made up of equal
Antioxidant Activities of Selected Plant Extracts. The
volumes of 1% sulfanilamide and 0.1% naphthylethylenediamine in
5% HCl. In the presence of nitrite this reagent forms a violet color. From
antioxidant activity of the selected medicinal herbs was evaluated
each well 70 μL of supernatant was transferred to a fresh 96-well plate and by using DPPH free radical scavenging method and also yeast
mixed with 70 μL of Griess reagent, and the color produced was measured (S. cervicea) model.
at 540 nm. The remaining supernatant that was removed from each well The results of free radical scavenging capacity of the selected
was used for TNF-α assay using a commercial sandwich ELISA. herbs are presented in Table 2. The scavenging capacity of water
Determination of Cell Viability by Alamar Blue Assay. The extracts ranged from 19.9 μM to 202.4 μM ascorbate equiv/g
Alamar Blue assay is a colorimetric assay involving the cellular reduction while for ethanol extracts the range is from 8.14 to 111.36 μM
of resazurin to resorufin. One hundred microliters of Alamar Blue ascorbate equiv/g. High scavenging capacity was shown by water
solution (10% Alamar Blue (Resazurin) in DMEM medium) was added extracts of S. baicalensis, R. officinale, S. japonica, and Polygonum
to each well and incubated at 37 °C for 1
2 h. Fluorescence was cuspidatum (Table 2). Among all the ethanol extracts S. japonica,
measured (excitation at 545 nm and emission at 595 nm) and expressed S. baicalensis, R. officinale, and P. cuspidatum exhibited good
as a percentage of that in control cells after background fluorescence was scavenging activity (Table 2). S. japonica and S. baicalensis showed
subtracted. significant scavenging activity in both water and ethanol extracts.
12363 [Link]/10.1021/jf203146e |J. Agric. Food Chem. 2011, 59, 12361–12367
Journal of Agricultural and Food Chemistry ARTICLE

Table 2. Antioxidant Activities of Selected Chinese Medicinal Herbs along with Their Total Phenolics and Flavonoid Contents

DPPH scavenging activitya (ascorbate equivalent μM) % yeast cell growthb phenolics contentc (GAE mg/g) flavonoid contentc (QE mg/g)

plant water extracts ethanol extracts water extracts water extracts ethanol extracts water extracts ethanol extracts

1 126.9 ( 9.19 68.5 ( 0.00 7.15 11.4 ( 2.73 6.64 ( 0.78 32.37 ( 3.91 24.78 ( 5.87
2 40.9 ( 2.12 8.14 ( 0.51 8.45 5.54 ( 1.36 3.78 ( 3.79 4.09 ( 0.98 12.91 ( 0.98
3 42.9 ( 3.54 30.29 ( 2.53 3.23 2.04 ( 0.27 0.82 ( 0.65 0.73 ( 0.00 2.46 ( 0.00
4 201.9 ( 0.71 107.43 ( 1.52 11.76 60.52 ( 0.96 46.85 ( 3.92 45.47 ( 5.87 58.46 ( 4.89
5 152.4 ( 0.00 91.36 ( 0.00 15.85 21.46 ( 0.41 26.54 ( 0.65 78.52 ( 0.00 64.64 ( 0.00
6 121.9 ( 0.71 57.07 ( 4.04 14.81 5.28 ( 0.00 6.84 ( 0.00 6.32 ( 0.98 29.03 ( 0.00
7 35.9 ( 4.95 49.21 ( 1.01 16.03 2.56 ( 2.46 2.05 ( 4.28 4.39 ( 1.96 10.99 ( 2.93
8 202.4 ( 0 98.14 ( 1.52 13.07 33.18 ( 2.59 42.77 ( 1.31 54.57 ( 0.98 98.51 ( 0.00
9 196.9 ( 0.71 111.36 ( 1.01 15.42 27.84 ( 3.28 91.33 ( 0.00 66.76 ( 1.96 151.86 ( 0.00
10 189.4 ( 4.24 91.71 ( 0.51 3.41 17.14 ( 3.28 49.07 ( 4.05 47.61 ( 0.00 84.73 ( 0.00
11 53.9 ( 0.71 20.29 ( 2.53 18.72 7.07 ( 0.27 6.03 ( 0.44 3.39 ( 1.96 40.59 ( 2.93
12 171.9 ( 3.54 84.57 ( 0.51 5.03 28.6 ( 4.37 9.82 ( 0.3 59.13 ( 0.98 40.32 ( 2.93
13 19.9 ( 2.12 35.64 ( 1.01 18.72 7.35 ( 0.96 10.05 ( 0.44 2.18 ( 0.00 9.01 ( 2.93
14 25.4 ( 2.83 32.79 ( 1.01 5.84 1.26 ( 0.14 1.09 ( 1.57 1.33 ( 0.98 1.05 ( 2.93
a
DPPH free radical scavenging activity was measured in terms of equivalent of ascorbate (μM). b Yeast oxidative stress was measure on the basis of
survival of yeast cells (yeast growth) after treatment with H2O2. c Total phenolics and flavonoid contents were expressed in gallic acid equivalent (GAE
mg/g) and quercetin equivalent (QE equiv/mg), respectively.

Table 3. Anti-inflammatory Activities of the Selected Plants

IC50 for inhibition of NO production IC50 for inhibition of TNF-α production
plant (mg/mL) cell viabilitya (% of cell survival) (mg/mL) cell viabilitya (% of cell survival)

1 0.11 ( 0.02 91 ( 8.8 0.4 ( 0.15 64.2 ( 1.6

2 0.62 ( 0.09 90.3 ( 10.7 >2.5 NAb
3 0.46 ( 0.12 94.7 ( 0.5 >2.5 NA
4 0.04 ( 0 91.8 ( 4.6 0.18 ( 0.05 73.6 ( 0.6
5 0.05 ( 0 87 ( 0 0.14 ( 0.05 63.45 ( 7.7
6 0.07 ( 0 95.7 ( 0 0.8 ( 0.2 86.7 ( 0.5
7 1.09 ( 0.2 104 ( 5.7 >2.5 NA
8 0.15 ( 0.08 99.5 ( 2.2 0.58 ( 0.24 65.7 ( 3.6
9 0.06 ( 0.02 91 ( 2.5 0.18 ( 0.08 78.7 ( 0.5
10 0.12 ( 0.04 95.4 ( 2.6 0.55 ( 0.12 56.9 ( 5.7
11 0.42 ( 0.36 104.7 ( 7.6 >2.5 NA
12 0.09 ( 0 95 ( 0 0.17 ( 0 93.6 ( 1.1
13 0.13 ( 0.08 89.6 ( 6.7 0.08 94.6 ( 6.7
14 1.05 ( 0.05 108.5 ( 9.2 >2.5 NA
a
Cell viability was measured at appropriate IC50 values corresponding to the inhibition of NO and TNF-α. b NA, not analyzed.

Antioxidant activities of water extracts of the selected plants down-regulated NO production with IC50 values of <0.1 mg/mL
measured by yeast model are presented in Table 2. These results without significantly affecting cell viability (>85%). As can be
revealed that the extracts of S. flavescens (18.72%), Saposhnikovia seen from Table 3, the inhibition efficiency of plant extracts with
divaricata (18.72%), Atracylodes macrocephala (16.03%), S. respect to NO production was superior when compared to that of
japonica (15.42%), T. chinensis (15.85%), Eucommia ulmoides TNF-α production. Among all of the plants, M. fortunei (IC50 =
(14.81%), R. officinale (13.07%) and S. baicalensis (11.76%) have 0.17 mg/mL), S. baicalensis (IC50 = 0.18 mg/mL), T. chinensis
high activity. It is interesting to note that the plants Scutellaria (IC50 = 0.14 mg/mL), S. japonica (IC50 = 0.18 mg/mL), and
baicalensis, Taxillus chinensis, and Sophora japonica showed sig- S. flavescens (IC50 = 0.08 mg/mL) have shown significant
nificant activity in both the DPPH method and yeast model. inhibition of TNF-α production with good cell viabilities.
Anti-inflammatory Activity of Plant Extracts. The anti- It is important to note that the water extracts of three plants,
inflammatory activities of plants as measured using the RAW namely, S. baicalensis, T. chinensis, and P. cuspidatum, have
264.7 macrophage model are presented in Table 3. significant anti-inflammatory activity in terms of inhibiting
As can be seen from these results (Table 3), the extracts of S. the production of both NO and TNF-α with significant cell
baicalensis, T. chinensis, , E. ulmoides, S. japonica, and M. fortunei have viability (Table 3).
12364 [Link]/10.1021/jf203146e |J. Agric. Food Chem. 2011, 59, 12361–12367
Journal of Agricultural and Food Chemistry ARTICLE

Figure 1. Correlation between DPPH free radical scavenging activity and the total phenolics content in (A) water extracts and (C) ethanol extracts and
between DPPH free radical scavenging activity and total flavonoid content in (B) water extracts (D) and ethanol extracts.

Correlation of Antioxidant Activities of Selected Plants in is a fair degree of correlation between the anti-inflammatory
Terms of Their Total Phenolics and Flavonoid Contents. To activities and total phenolics and flavonoid contents. For instance,
understand the antioxidant activities of the selected medicinal Scutellaria baicalensis, Taxillus chinensis, and Sophora japonica
herbs in terms of their antioxidant content (total phenolics and inhibited the production of NO and TNF-α (low IC50 values)
flavonoids), correlation plots were developed (Figure 1). In water and also have significant levels of total phenolics and flavonoid
extracts the DPPH scavenging activity showed significant correla- contents. Other plants such as Codonopsis pilosula, Curcuma
tion with total phenolics content (Figure 1A, 0.647, p < 0.05) and aromatic, Atractylodes macrocephala, Saposhnikovia divaricata
also with the total flvavoid content (Figure 1C, 0.7673, p < 0.05). and Pinellia ternate, were found to contain low levels of
The correlation of antioxidant properties to their total phenolics phenolics/flavonoid contents and also lower antioxidant
(Figure 1B, 0.6248, p < 0.05) and flavonoid (Figure 1D, 0.6256 activity (Table 2).
p < 0.05) contents was also significant in ethanol extracts. These results are in agreement with the previous studies that
The results presented above are in agreement with the fact that phenolics and flavonoids influence anti-inflammatory activities.25
the total phenolics and flavonoid contents are major contributors Recently, several compounds that have been isolated from
to the antioxidant activity of herbal medicine, which is in accor- medicinal herbs showed strong anti-inflammatory activities.
dance with the literature.21
24 The herbs S. baicalensis, T. chinensis, For instance, phenolic compounds, namely, baicalein, oroxylin
R. officinale, S. japonica, and P. cuspidatum studied here showed A, and wogonin, isolated from S. baicalensis significantly inhibited
high DPPH scavenging activity and also have high total phenolics the production of NO.26 The literature also supports that
and flavonoid contents (Table 2). It is also interesting to note that isoflavone glycosides isolated from S. japonica show significant
several flavonoid and flavonoid glycosides have been isolated from inhibition of chemical modulators formed during inflammatory
S. japonicas.23 Results reported in the literature24 confirm that response.
P. cuspidatum possesses high phenolics and flavonoid contents. On the other hand, the anti-inflammatory activities of some of
Although the correlations of antioxidant activity with total the plants are not consistent with the total phenolics and flavonoid
phenolics and flavonoids presented in this paper are significant contents. For instance, S. flavescens is found to have significant anti-
(Figure 1), some of the herbs significantly deviated from the inflammatory activities (inhibition of production of both NO and
linear correlation. For instance, Eucommia ulmoides and Sarcan- TNF-α), but this plant contains significantly low levels of phe-
dra glabre have been found to have high antioxidant activity but nolics and flavonoids. Hence, it is concluded that chemical
low levels of phenolics/flavonoid contents (Table 2). It is constituents other than phenolics and flavonoids may also be
therefore reasonable to conclude that in addition to phenolics responsible for the anti-inflammatory properties of such plants.
and flavonoids there are other antioxidants that may be present in From the results presented above, it is clear that some of the
medicinal herbs and contribute to their antioxidant activities. plants studied (S. baicalensis, T. chinensis, S. japonica, and M.
Correlation of Anti-inflammatory Activities of Selected fortunei) are good sources of antioxidant/anti-inflammatory
Plants in Terms of Their Total Phenolics and Flavonoid compounds. Currently, the isolation of bioactive compounds
Contents. It is interesting to note from the results that there from T. chinensis and other plants is underway in our laboratory.
12365 [Link]/10.1021/jf203146e |J. Agric. Food Chem. 2011, 59, 12361–12367
Journal of Agricultural and Food Chemistry ARTICLE

Conclusion. In this study, total phenolics and flavonoid (15) Huang, Y. C.; Guh, J. H.; Teng, C. M. Denbinobin-mediated
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logical activities of the target compounds of the lead plants. based on oxidant induced growth arrest in Saccharomyces cerevisiae.
FEMS Yeast Res. 2011, 11, 379–387.
(20) Shanmugam, K.; Holmquist, L.; Steele, M.; Stuchbury, G.;
’ AUTHOR INFORMATION Berbaum, K.; Schulz, O.; Benavente-García, O.; Castillo, J.; Burnell, J.;
Corresponding Author Garcia Rivas, V.; Dobson, G.; M€unch, G. Plant-derived polyphenols
*Phone: +61-2-96859987. Fax: +61-2-96859915. E-mail: attenuate lipopolysaccharide-induced nitric oxide and tumour necrosis
factor production in murine microglia and macropgages. Mol. Nutr. Food
[Link]@[Link]. Res. 2008, 52, 427–438.
(21) Kim, D. O.; Jeong, S. W.; Lee, C. Y. Antioxidant capacity of
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