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Angelica dahurica (Umbelliferae) is widely used as a herbal ingredient in functional foods and folk medicine. This study was designed to examine the antioxidant, anti-inflammatory and antiproliferative activities of water and ethanol extracts of A. dahurica (AD) root. Antioxidant properties of AD root extracts were studied using methods, including 2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and hydroxide radical-scavenging activity at a dose of 0.12–2.0 mg/mL. The IC50 values for DPPH and ABTS radical-scavenging activity were 0.32 and 0.20 mg/mL, respectively, for water extract and 0.24 and 0.13 mg/mL, respectively, for ethanol extract. Lipid peroxidation was also determined as an indicator of oxidative stress. The extracts also showed strong reducing power, superoxide dismutase activity, catalase activity and DNA damage prevention. AD root extracts inhibited the production of nitric oxide in a dose-dependent manner in lipopolysaccharide-treated RAW264.7 cells. The water and ethanol extracts of AD root also showed significant antiproliferative activity against HT-29 and CMT-93 cell lines. Practical ApplicationsIn this study, the biological potentials of different extracts from Angelica dahurica roots (water and ethanol extracts) were evaluated. The roots could be useful as antioxidants and for treatment of chronic inflammatory pathologies associated with overproduction of nitric oxide. The results suggest that A. dahurica contains excellent antioxidant, anti-inflammatory and antiproliferative properties that can provide opportunities for application of A. dahurica root extracts in areas such as food, pharmacy, alternative medicine and natural therapy.
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Department of Applied Biochemistry, College of Biomedical & Health Science, Konkuk University, Chungju 380-701, Republic of Korea
1Corresponding author. Beong Ou Lim,
College of Biomedical & Health Science,
Department of Applied Biochemistry, Konkuk
University, Chungju-si, Chungbuk-do
380-701, Republic of Korea.
TEL: +82-43-840-3570;
FAX: +82-43-856-3572;
Received for Publication November 18, 2012
Accepted for Publication May 12, 2013
Angelica dahurica (Umbelliferae) is widely used as a herbal ingredient in func-
tional foods and folk medicine. This study was designed to examine the antioxi-
dant, anti-inflammatory and antiproliferative activities of water and ethanol
extracts of A. dahurica (AD) root. Antioxidant properties of AD root extracts were
studied using methods, including 2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-
azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and hydroxide radical-
scavenging activity at a dose of 0.12–2.0 mg/mL. The IC50 values for DPPH and
ABTS radical-scavenging activity were 0.32 and 0.20 mg/mL, respectively, for
water extract and 0.24 and 0.13 mg/mL, respectively, for ethanol extract. Lipid
peroxidation was also determined as an indicator of oxidative stress. The extracts
also showed strong reducing power, superoxide dismutase activity, catalase activity
and DNA damage prevention. AD root extracts inhibited the production of nitric
oxide in a dose-dependent manner in lipopolysaccharide-treated RAW264.7
cells. The water and ethanol extracts of AD root also showed significant
antiproliferative activity against HT-29 and CMT-93 cell lines.
In this study, the biological potentials of different extracts from Angelica dahurica
roots (water and ethanol extracts) were evaluated. The roots could be useful as
antioxidants and for treatment of chronic inflammatory pathologies associated
with overproduction of nitric oxide. The results suggest that A. dahurica contains
excellent antioxidant, anti-inflammatory and antiproliferative properties that can
provide opportunities for application of A. dahurica root extracts in areas such as
food, pharmacy, alternative medicine and natural therapy.
The in vivo oxidation of lipids, DNA, membranes and pro-
teins caused by reactive oxygen species (ROS) is regarded as
the pathogenesis of atherosclerosis, tumors and other dis-
eases (Leopold and Loscalzo 2009). Antioxidants are neces-
sary to help treat these diseases, but the use of synthetic
antioxidants could be unsafe (Wanasundara and Shahidi
1994; Wettasinghe and Shahidi 1999). Therefore, greater
attention has been focused on searching for natural antioxi-
dants from medicinal and dietary plants to prevent oxida-
tive damage. Antioxidants like polyphenolics, among others,
are on the front line of investigation due to not only their
natural origin but also their ability to act as free radical
scavengers (Katalinic et al. 2006; Ferreira et al. 2006).
For centuries, traditional medicine had used plants for
their remedial and defensive abilities. Some of the more
widely used plants have been selected for investigation of
their chemical constituents and biological activities such as
anti-inflammatory, antibacterial and anticancer properties
(Ojewole 2005). The extracts of several medicinal plants
have been reported to exhibit antioxidant capacity (Giao
et al. 2007, 2009) as well as a protective effect against degra-
dation of deoxyribose and DNA (Giao et al. 2008). Phenolic
compounds can prevent oxidative damage via a number
of different mechanisms, such as free radical scavenging,
Journal of Food Biochemistry ISSN 1745-4514
281Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
transition metal chelation and interactions with lipid mem-
branes, proteins and nucleic acids (Dai and Mumper 2010).
The radical-scavenging capacities of antioxidants are usually
accessed by trapping 2,2-azino-bis(3-ethylbenzothiazoline-
6-sulfonate) cationic radical (ABTS+) and 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical (Niki 2010).
Angelica is one of the most important genera of medici-
nal plants used in traditional medicinal systems of the Far
East and certain Western countries (Sarker and Nahar
2004). Angelica dahurica (AD) is abundant in Korea, China
and Japan. The dried root of AD has the folkloric reputa-
tion of being used as an analgesic, antibacterial, anti-
inflammatory and diuretic agent, as well as a remedy for
colds and influenza (Sarker and Nahar 2004). It is also
claimed to be effective for treatment of acne, erythema,
headache, toothache, sinusitis, colds and flu (Wagner
1999).The major active compounds derived from AD are
the coumarin compounds, phellopterin, isoimperatorin,
imperatorin, alloimperatorin, byakangelicin, isooxypeuce-
danin and pimpinellin (Ban et al. 2003). These have been
reported to exhibit pharmacological effects such as remark-
able anticancer and antibacterial effects (Ban et al. 2003),
inhibition of acetylcholinesterase (Kim et al. 2002) and
inhibition of gamma-aminobutyric acid transaminase
(Choi et al. 2005). A total of 20 furocoumarins have been
identified or tentatively characterized from the roots of AD
by a high-performance liquid chromatography–diode array
detection–electrospray ionization tandem mass spectrom-
etry (HPLC/DAD/ESI-MSn) technique (Kang et al. 2008).
Thus, the aim of the present work was to quantify pheno-
lic compounds of AD and to evaluate the antioxidant
potentials of extracts from this plant as well as screen for
anti-inflammatory and antiproliferative activities by using
cell-based assay.
DPPH, ABTS, gallic acid, sodium nitrite, Folin–Ciocalteu
reagent (FC reagent), trichloroacetic acid (TCA), butylated
hydroxytoluene (BHT), ascorbic acid (AA), α-tocopherol,
potassium persulfate, linoleic acid, anhydrous sodium phos-
phate (dibasic), anhydrous sodium phosphate (monobasic),
ferrous chloride, ammonium thiocyanate, ethylenedia-
minetetraacetic acid (EDTA), 5,5-dimethyl pyrroline-1-
oxide (DMPO), pyrogallol and ferrous sulfate (FeSO4)were
purchased from Sigma Chemical Co. (St. Louis, MO). Ferric
chloride and sodium hydroxide were obtained from Wako
Pure Chemical Industries Ltd. (Osaka, Japan). Agarose A
was purchased from Bio Basic Inc. (Ontario, Canada). The
catalase assay kit was purchased from Cayman Chemical
Company (Ann Arbor, MI). Roswell Park Memorial Insti-
tute medium (RPMI 1640) and Dulbecco’s modified Eagle’s
medium (DMEM) were purchased from WElGENE Inc.
(Daegu, Korea). Human colon adenocarcinoma cell line
(HT-29) and mouse rectum carcinoma cell line (CMT-93)
were purchased from American Type Culture Collection
(ATCC, Manassas, VA). The pBR322 DNA and 6X DNA
loading dye were purchased from Fermentas Inc. (Cromwell
Park, Glen Burnie, MD). All the other solvents and chemi-
cals used were of analytical grade.
Preparation of Extracts
Water and ethanol extracts of AD roots were prepared
according to the method of Debnath et al. (2011) with
minor modification. About 100 g ground powder was
extracted with 700 mL of distilled water (DW) at 90C for
3 h (water extraction) and 500 mL of 75% (v/v) ethanol at
room temperature (RT) for 24 h. The extracts were filtered
through Whatman filter paper (GE Healthcare UK Limited,
Buckinghamshire, U.K.) and evaporated with a vacuum
rotary evaporator. The residual root extracts were freeze-
dried, weighed and the yield was calculated. Finally, the
extracts were stored in a refrigerator at 20C until used. The
percent yields (w/w) were calculated (Yield [%] =[total
extracted sample mass/total dry sample mass] ×100).
Determination of Total Phenolic Content
The total phenolic content of the extracts was determined
with the FC method with minor modification (Bonoli et al.
2004). Briefly, 10 mg of each extract was dissolved in 1 mL
of DW and different concentrations of gallic acid (0.0078–
1 mg/mL) were prepared in water. Further, 40 μLofeach
sample, 20 μL of 1 M FC reagent and 60 μLofNa
(20%, w/v) were mixed and kept in the dark at RT for
30 min to complete the reaction. Finally, the absorbance was
measured at 700 nm, with a UV–visible spectrophotometer.
Results were expressed as mg of gallic acid equivalents
(GAE) per 100 g dry mass through the calibration curve
with gallic acid. All samples were analyzed in triplicate.
Determination of Total Flavonoid Content
Total flavonoid content was measured using a colorimetric
assay developed by Dewanto et al. (2002) with little modifi-
cation. The concentrations of the sample and standard (cat-
echins) solutions were 10 and 0.015–1 mg/mL, respectively.
Briefly, 25 μL of each sample or standard reagent was mixed
with 125 μL of DW and 8 μL of 5% sodium nitrate solution
was added. After 5 min of incubation, 15 μL of 10% (w/v)
aluminum chloride solution was mixed with the above
mixture and incubated at RT for 6 min. Further, 50 μLof
NaOH (0.1 M) and 27 μL of DW were added to the mixture
282 Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
and the absorbance was measured at 517 nm. Total flavo-
noid content was determined from the standard calibration
curve. Results were expressed as mg of catechin equivalent
(CE) per 100 g dry mass. All samples were analyzed in
DPPH Radical-Scavenging Activity
DPPH radical-scavenging activities of ethanol and water
extracts were determined by the method of Lee et al. (2009).
In brief, different concentrations (0.12–2.00 mg/mL) of
sample and BHT (as positive control) were prepared in
water and methanol, respectively. Further, 80 μLofeach
sample or standard solution was mixed with 80 μL of DPPH
(0.3 mM in methanol) solution. The mixture was shaken
vigorously and left in darkness for 30 min and the absor-
bance was read at 517 nm. Controls were composed of the
corresponding extraction solvent. DPPH radical-scavenging
activity was calculated using the following equation:
DPPH radical-scavenging activity
Absorbance of sampl
(=−1ee Absorbance of control/)×100
ABTS Radical-Scavenging Activity
Determination of the ABTS+radical-scavenging activity was
performed according to the method of Re et al. (1998) with
some modifications. Initially, ABTS was dissolved in water
to make 7 mM concentration and ABTS+was produced by
the reaction of ABTS stock solution with a potassium
persulfate solution (at 2.45 mM final concentration). The
mixture was kept in the dark at RT for 12–16 h before use.
Freshly prepared ABTS+solution was diluted with 0.01 M
phosphate buffered saline (PBS, pH 7.4) and absorbance
was adjusted to 0.70 ±0.02 at 734 nm. Different concentra-
tions of sample and AA (alone or in combination) were
mixed with 0.7 mL of ABTS+solution. Finally, the absor-
bance was measured at 734 nm against the blank after 5 min
of reaction at RT. The controls were composed of the
extraction solvent. The scavenging activity of ABTS free
radical was calculated as
ABTS radical-scavenging activity
Absorbance of samp
(=−1lle Absorbance of control/)×100
Hydroxyl Radical-Scavenging Activity
Hydroxyl radicals were generated by iron-catalyzed Fenton–
Haber–Weiss reaction and the generated hydroxyl radicals
rapidly reacted with nitrone spin trap DMPO (Rosen and
Rauckman 1984). The resultant DMPO-OH adducts was
detected with an electron spin resonance (ESR) spectrom-
eter (Jeol, Tokyo, Japan). The AD root extract (20 μL) was
mixed with DMPO (0.3 M, 20 μL), FeSO4(10 mM, 20 μL)
and H2O2(10 mM, 20 μL) in a phosphate buffer solution
(pH 7.4), and transferred to a 100-μL quartz capillary tube.
After 2.5 min, the ESR spectrum was recorded using an ESR
spectrometer. Experimental conditions were as follows:
magnetic field, 336.5 ±5 mT; power, 1 mW; amplitude,
1×200; modulation width 0.1 mT, sweep width, 10 mT;
sweep time, 30 s. Hydroxyl radical-scavenging ability was
calculated according to the following equation in which A
and A0were relative peak heights of radical signals with and
without sample, respectively.
Radical-scavenging activity =− ×()/1 100
Reducing Power
The reducing power of the samples was determined accord-
ing to the method of Atmani et al. (2009) with a little modi-
fication. Further, 1.0 mL of each sample or standard
reagent (aa as positive control) at various concentrations
(0.125–2.00 mg/mL) was mixed with 2.5 mL of PBS (0.2 M,
pH 6.6) and 2.5 mL of potassium ferricyanide (10 mg/mL)
solution was added to the mixture. The mixture was incu-
bated at 50C in a water bath for 30 min and mixed with
2.5 mL of TCA (100 mg/mL) and centrifuged at 3,000 rela-
tive centrifugal force (rcf) for 10 min. Then, 0.25 mL of
supernatant was mixed with 0.25 mL of DW and finally
0.5 mL of FeCl3(0.1% [w/v]) was added to the mixture. The
absorbance was measured at 700 nm.
Lipid Peroxidation Inhibition Assay
The inhibition of lipid peroxidation was determined by
ferric thiocyanate method (Ak and Gulcin 2008). Briefly,
2.5 mg of sample (2 mg/mL) was dissolved in 1 mL of 0.1 M
sodium phosphate buffer (pH 7.0) and added to a solution
of 1 mL of 50 mM linoleic acid (dissolved in 99% (v/v)
ethanol). Then, 0.5 mL distilled water was added and the
mixture was incubated in a conical flask with a screw cap at
40 ±1C in a dark room and the degree of oxidation was
evaluated by measuring the ferric thiocyanate values. The
ferric thiocyanate value was measured according to the
method of Mitsuta et al. (1996). The reaction solution
(50 μL) incubated in the linoleic acid model system was
mixed with 2.5 mL of 75% ethanol (v/v) and 50 μL of 30%
ammonium thiocyanate. Further, 50 μLof20mMferrous
chloride dissolved in 3.5% (v/v) HCl was added to the
mixture and incubated for 3 min at RT and the thiocyanate
value was measured by reading the absorbance at 500 nm at
each 24 h. The scavenging activity was calculated using the
following equation:
Inhibition Absorbance of sample at nm
(%) ( /=−1 500
of control at nm500 100).×
283Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
Superoxide Dismutase Like
(SOD-Like)-Scavenging Activity Assay
SOD-like activity was determined as described by Marklund
and Marklund (1974) with a little modification. Briefly,
200 μL of each extract (0.125–2.0 mg/mL) was mixed with
3 mL Tris–HCl buffer (50 mM Tris [hydroxymethyl] amino
– methane contained 10 mM EDTA, pH 8.5). Then, 200 μL
of pyrogallol solution (7.2 mM in water) was added and the
mixture was incubated at 25C for 10 min and the reaction
stopped by addition of 1 mL 1 N HCl. AA was used (0.125–
2 mg/mL) as a positive control. Absorbance of the mixture
was determined at 420 nm. Water was used as control. SOD-
like activity was calculated by using the following equation:
Scavenging activity Absorbance of treated
sample at
(%) ( [=−1
nm Absorbance of sample or standard
at nm Absorbanc
]/ ee of control at nm420 100),×
Catalase Activity Assay
Catalase activity was measured according to manufacturer’s
instructions for the kits (Cayman Chemical, Ann Arbor,
MI). Briefly, 100 μL of 100 mM potassium phosphate buffer
(pH 7) and 30 μL of methanol were mixed with 20 μLof
4.25 M formaldehyde standard (0–75 μM) and 10 mg/mL
of 20 μL sample was added. Reaction was started by adding
20 μL of diluted hydrogen peroxide (0.035 M). After 20 min
of incubation at RT on a shaker, 30 μL of diluted potassium
hydroxide (10 M) was added to terminate the reaction and
30 μL of chromagen (in 0.5 M hydrochloric acid) was added
to each well. Further, 10 μL of catalase potassium periodate
(in 0.5 M potassium hydroxide) was added and the absor-
bance of the sample was monitored for 5 min at 240 nm.
Catalase activity was expressed as nmol/min/mL. Bovine
liver was used as a positive control. Changes in absorbance
were taken to be proportional to the breakdown of H2O2.
Prevention of Oxidative DNA Damage
Oxidative DNA damage preventive activity of AD root
extracts was determined as described by Kitts et al. (2000)
with some minor modifications. In brief, 1 μL of Plasmid
pBR 322 DNA (0.5 μg/μL) was treated with 3 μL of FeSO4
(0.08 mM), 4 μL of 30% H2O2(v/v), 3 μL distilled water
and 2 μL test extracts at different concentrations. The
mixture was incubated at 37C for 1 h followed by addition
of 2 μL of 6X DNA loading dye. The mixture was subjected
to 0.8% (w/v) agarose gel electrophoresis. DNA bands
(supercoiled [SC], linear and open circular) were stained
with ethidium bromide. Gels were scanned on a gel docu-
mentation system (Nextep, Gyeonggi-do, Korea) and bands
were quantified using NEXTEP analysis software.
Nitrite Assay
The lipopolysaccharide-induced NO production by the
macrophages was determined as nitrite concentration in
the culture medium according to the Griess reaction
(Kim et al. 1995). RAW 264.7 cells were seeded onto
96-well plates with 5 ×103cells/well and allowed to
adhere overnight. The medium was removed and replaced
with 200 μL of fresh medium alone or containing various
concentration of extracts. After 1 h of incubation, LPS
stimulation was performed at a concentration of 1 μg/mL
for 24 h. An aliquot (100 μL) of the supernatant was
mixed with an equal volume of Griess reagent (1% sulfa-
nilamide in 5% phosphoric acid and 0.1% naphthy-
lethylenediamine dihydrochloride in water). Absorbance of
the mixture was read at 540 nm with an enzyme-linked
immunosorbent assay (ELISA) plate reader (Bio-Rad,
Hercules, CA). The amount of nitrite present in the
samples was calculated by means of a standard curve gen-
erated using serial dilutions of NaNO2in fresh culture
MTT Assay
Cytotoxicity of AD root was determined as described by
Jeong et al. (2010) with a little modification. Human
HT-29 colon adenocarcinoma cell lines and CMT-93-
mouse adenocarcinoma cell lines were cultured in RPMI
1640 supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and 1% penicillin. Murine RAW264.7 mac-
rophages were cultured in DMEM supplemented with 10%
FBS and 1% penicillin. Cells were incubated at 37C in 5%
CO2. Cells in the culture media (4 ×103HT-29, 4 ×103
CMT-93 and 5 ×103RAW264.7 cells) were incubated in
each well of a 96-well microplate (Costar, Corning Inc.,
Corning, NY) for 16 h. After incubation, the medium was
removed from each well leaving cells on the bottom. New
media (100 μL) containing different concentrations of the
freeze-dried extracts were added to the wells. Further,
20 μL MTT solutions (5 mg/mL) was added to each
96-well culture plate and incubated for 4 h at 37C, and the
medium containing MTT was removed. The formazan
crystals incorporated in the viable cells were solubilized
with 100 μL of dimethyl sulfoxide and the absorbance of
each well was read at 540 nm using the ELISA microplate
reader (Bio-Rad). Cell viability (%) was calculated as
Cell viability sample absorbance with cells sample
(%) ([=−
ssorbance without cells]/[control absorbance with cells
conntrol absorbance without cells]) .×100
284 Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
High-Performance Liquid
Chromatography (HPLC)
HPLC analysis was carried out using a Shimadzu UFLC
system (Shimadzu Corporation, Tokyo, Japan) consisting of
a vacuum degasser, an autosampler and a binary pump
equipped with a reversed-phase C18 analytical column
(4.6 ×250 mm, 5-μm particle size, COSMOSIL 5C18-AR-II
packed column). The mobile phase and gradient program
were used as previously described by Karoui et al. (2012).
Briefly, the samples (20 μL) were eluted through the column
with a gradient mobile phase consisting of A (acetonitrile)
and B (0.2% sulfuric acid) with a flow rate of 0.5 mL/min.
The following multistep linear solvent gradient was
employed: 15%A/85%B 0–12 min, 40%A/60%B 12–14 min,
60%A/40%B 14–18 min, 80%A/20%B 18–20 min, 90%A/
10%B 20–24 min, 100%A 24–28 min. Peaks were moni-
tored at 280 nm. Phenolic compounds were identified by
matching the retention time and their spectral characteristic
against those of prepared standards.
Statistical Analysis
Data were reported as mean values calculated from repli-
cates. All analyses were carried out in triplicate. Significant
differences between groups were determined at P<0.05.
The statistical analysis was carried out by using SPSS 15.0
(SPSS Inc., Chicago, IL), Sigma plot 10.0 (Systat Software,
Inc., Richmond, CA), GraphPad Prism 5 (GraphPad Soft-
ware, Inc., La Jolla, CA) and Microsoft Excel 2007.
Extraction Yield, Total Polyphenol and
Flavonoid Contents
Table 1 reveals the extraction yield, total polyphenol and
flavonoid contents of water, and ethanol extracts of AD
roots. The extraction yields were 12.67 and 10.10% for
water and ethanol extract, respectively. Total phenolic and
flavonoid contents of AD root extracts were expressed in mg
of gallic acid (GAE) and catechin equivalents (CE), respec-
tively. Our results showed that the root extracts contained
significant amount of phenolic compounds (9.03 mg GAE/
100 g and 11.17 mg GAE/100 g of dry mass, for water and
ethanol extract, respectively). The flavonoid contents of the
water and ethanol extracts were 1.78 and 2.09 mg CE/100 g
dry mass, respectively.
DPPH, ABTS and Hydroxyl
Radical-Scavenging Activity
The DPPH, ABTS+and •OH radical-scavenging activity of
AD roots are shown in Table 2. DPPH radical-scavenging
activity was 88.63 and 90.83%, for water and ethanol
extract, respectively, at 2 mg/mL (P<0.05 compared with
the BHT standard). The extracts showed dose-dependent
scavenging activity when reacted with the ABTS+radicals. At
2 mg/mL, the water and ethanol extracts of AD roots were
able to inhibit the formation of ABTS+radicals with a
percentage inhibition of 96.73 and 98.70%, respectively. IC50
Total phenolic (mg
GAE/100 g of dry mass)
Total flavonoid (mg CE/100 g
of dry mass) Yield (%)
Water extract 9.03 ±0.04 1.78 ±0.02 12.67
Ethanol extract 11.17 ±0.07 2.09 ±0.07 10.10
Data are means ±SD of three independent experiments.
CE, catechin equivalent; GAE, gallic acid equivalent.
DPPH radical ABTS radical Hydroxyl radical
Scavenging activity (%)
(2 mg/mL)
†IC50 value
Scavenging activity (%)
(2 mg/mL)
†IC50 value
Scavenging activity (%)
(2 mg/mL)
†IC50 value
Water extract 88.63 ±0.60a0.32 ±0.01a96.73 ±0.422a0.20 ±0.003a60.23 ±0.77a0.87 ±0.004a
Ethanol extract 90.83 ±0.92b0.24 ±0.0006b98.70 ±0.25a0.13 ±0.004a73.10 ±0.13b0.55 ±0.002b
Positive control 96.10 ±0.38
0.10 ±0.002c99.67 ±0.13a
(ascorbic acid)
0.02 ±0.00006a95.60 ±0.10c
(ascorbic acid)
0.008 ±0.001c
Data are means ±SD of three independent experiments.
50 value (mg/mL): the concentration in which 50% inhibited.
Different letters in each column (a–c) denote statistically significant difference compared with the positive control group at P<0.05.
ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate; BHT, butylated hydroxytoluene; DPPH, 2-diphenyl-1-picrylhydrazyl.
285Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
values were 0.20 and 0.13 mg/mL, for water and ethanol
extracts, respectively, which is inversely related to its anti-
oxidant ability.
At AD concentrations of 2.0 mg/mL from the water
extract, the •OH scavenging activity was 60.24%, while the
same AD concentration from the ethanol extract resulted in
73.10% •OH scavenging activity.
Estimation of Reducing Power
The reducing capacity of a compound may serve as a sig-
nificant indicator of its potential antioxidant activity. In
Fig. 1, all the extracts showed some degree of electron-
donating capacity in a linear concentration-dependent
manner within a concentration range of 0.12–2 mg/mL.
The values are represented as ascorbate equivalents. There
were significant differences (P<0.05) between the plant
extracts and standard antioxidant BHT.
Linoleic Acid Oxidation Inhibition
The antioxidant activities of AD roots were determined by
its retarding effects on linoleic acid peroxidation using the
thiocyanate method. The antioxidant activities of the
extracts in the linoleic acid system are shown in Fig. 2. On
the fourth day, water extract, ethanol extract, AA and BHT
showed 85.48, 86.67, 96.07 and 87.39% inhibition, respec-
tively, which gradually decreased thereafter. The differences
in antioxidant activities among all the extracts were signifi-
cant (P<0.05).
SOD-Like and Catalase Activities
Bioactivity of AD rood extracts on SOD-like activity was
measured and compared to AA as references (Table 3).
Ethanol extract showed significantly higher activity
(59.22%) than the water extract (51.68%) at 2 mg/mL. AA
was more effective (99.11% at 2 mg/mL) than both extracts.
Ethanol extract of AD root also showed significantly higher
catalase activity (0.95 nmol/min/mL) than the water extract
(Table 3).
Oxidative DNA Damage Preventive Activity
The protective effects of AD root extracts against free
radical-induced DNA damage (pBR322 plasmid DNA) are
0.0 0.5 1.0 1.5 2.0 2.5
Absorbance (700 nm)
1.2 Water extract
Ethanol extract
BHT standard
Data are Mean ±SD of the Three Independent Experiments. P<0.05,
compare to the positive control group.
Storage time (days)
Absorbance at 500 nm
Water extract
Ethanol extract
Ascorbic acid standard
BHT standard
Data are mean ±SD of the three independent experiments. P<0.05,
compare to the positive control group.
Catalase activity
(nmol/min/mL) at 10 mg/mL
Superoxide dismutase
(SOD-like) activity at 2 mg/mL
Water extract 0.88 ±0.01 51.68 ±0.77
Ethanol extract 0.95 ±0.01 59.22 ±0.38
Positive control 2.70 ±0.12
(bovine liver catalase)
99.11 ±1.38
(ascorbic acid)
Data are means ±SD of three independent experiments.
286 Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
shown in Fig. 3. The SC DNA was completely converted to
the linear form due to hydroxyl radical-induced DNA
damage (line 2). SC DNA was restored in a dose-dependent
manner when DNA was treated with AD root extracts
(P<0.05, compared with control).
Cytotoxicity of AD Root Extracts against
RAW264.7 Cell Lines
Murine RAW264.7 macrophages were used to determine
cytotoxicity and anti-inflammatory effects of AD root
extracts. The results showed AD root extracts had no sig-
nificant effect on the cells up to a concentration of
1,000 μg/mL for 24 and 48 h (Fig. 4).
Measurement of Anti-Inflammatory Activity
by Nitrite Quantification
Normal macrophages produced undetectable levels of
nitrite, but treatment with LPS induced a high release of
NO in the culture medium, which was taken as 100%. NO
production was inhibited by AD extract treatment in a
concentration-dependent manner (Fig. 5). At the highest
concentration (1 mg/mL), the extracts significantly sup-
pressed NO production by approximately 67 and 78.07%,
for water and ethanol extracts, respectively (P<0.05).
Inhibition of Cancer Cell Proliferation
AD root extracts suppressed the proliferation of both
HT-29 cells and CMT-93 cells (Fig. 6). Cell viability
decreased in a dose-dependent manner when treated with
1,000 μg/mL. Ethanol extract inhibited the proliferation of
HT-29 and CMT-93 cells more than water extract, with
(a) Lane 1: untreated control DNA; lane 2: FeSO4+H2O2(DNA
damage control); lane 3: FeSO4+H2O2+DNA in the presence of
water extract of AD roots (0.5 and 1 mg/mL, respectively). (b) Lane 1:
untreated control DNA; lane 2: FeSO4+H2O2(DNA damage control);
lane 3: FeSO4+H2O2+DNA in the presence of ethanol extract of AD
roots (0.5 and 1.0 mg/mL, respectively).
(A) Cell viability after 24 h. (B) cell viability after 48 h. Data are
mean ±SD of the three independent experiments.
Data are mean ±SD of the three independent experiments. * Denotes
significant difference (P<0.05).
287Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
increasing inhibitory activity as the concentrations of
extracts increased (P<0.05). After 24 and 48 h, water
extract showed 29.10 and 18.44% cell viability of HT-29
cells, respectively, at a concentration of 1.0 mg/mL, while at
the same concentration ethanol extract showed 20.72 and
15.67% cell viability, respectively. The water extract (1.0 mg/
mL) showed 42.91 and 29.89% cell viability for CMT-93
cells after 24 and 48 h, respectively. After 24 and 48 h,
ethanol extract showed 21.84 and 16.88% cell viability of
CMT-93 cells, respectively, at the same concentration.
HPLC Analyses of the AD Extracts
Chemical composition analysis of AD root extracts was
summarized in Table 4. Nine components were determined
by HPLC analysis of tested extracts. The nine components
were identified as cyanidin, rutin, catechin, epicatechin,
coumarin, kaempferol, ferulic acid, vanillic acid and AA.
The extraction yields were influenced by several parameters,
including solvent polarity, chemical composition and physi-
cal characteristics of the plant material (Dai and Mumper
2010). Higher extraction yields were achieved with water
compared to water/ethanol and ethanol alone because pro-
teins and carbohydrates are more soluble in water than
ethanol (Zielinski and Kozlowska 2000). Several recent
studies have shown that phenolics in plants constitute the
main antioxidant compounds (Maisuthisakul et al. 2007;
Ksouri et al. 2008). The results of the total phenolic and fla-
vonoid content analyses showed that the ethanol extract
from roots of AD had higher phenolics and flavonoids than
water extract (Table 1). Moreover, HPLC analysis of AD
root extract showed presence of polyphenol compound.
The action of polyphenols is believed to be mainly due to
their redox properties, which play an important role in
adsorbing and neutralizing free radicals, quenching singlet
and triplet oxygen, or decomposing peroxides (Itagaki et al.
2009). Polyphenolic compounds like flavonoids contain
conjugated ring structures and hydroxyl groups that have
the potential to function as antioxidants in vitro or cell free
systems by scavenging superoxide anion, singlet oxygen,
lipid peroxyradicals and stabilizing free radicals involved in
oxidative processes through hydrogenation or complexing
with oxidizing species (Klahorst 2002).
The results showed that treatment with AD root extracts
increased the DPPH radical-scavenging activity in a dose-
dependent manner. Ethanol extract was a potent DPPH
radical scavenger (IC50 =0.24 mg/mL), whereas water
extract was slightly less potent, with IC50 values equal to
0.32 mg/mL. The IC50 of water and ethanol extract of AD
root against ABTS+was 0.20 and 0.13 mg/mL, respectively,
(a) Antiproliferative activity against HT-29 cell line after 24 and 48 h.
(b) Antiproliferative activity against CMT-93 cell line after 24 and 48 h.
Data are mean ±SD of the three independent experiments. P<0.05
compared to control.
Water extract
Ethanol extract
Cyanidin nd 3.46
Rutin 0.04 nd
Catechin 0.28 0.18
Epicatechin 0.22 nd
Coumarin 0.47 1.09
Kaempferol 0.97 1.09
Ferulic acid 0.28 1.81
Vanillic acid nd 2.04
Ascorbic acid 0.99 1.13
nd, not detected.
288 Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
indicating strong antioxidant activity. The results suggest
that AD root extracts display scavenging effect on DPPH
and ABTS+radical generation that could help prevent oxi-
dative damage. Six species of Angelica have been previously
shown to have potent DPPH radical-scavenging activity
(Makchuchit et al. 2010). Wang (2012) reported Angelica
sinensis showed DPPH radical-scavenging activity with IC50
value of 1.67 mg/mL.
Hydroxyl radicals cause injury to surrounding organs and
play a vital role in some clinical disorders. Removal of •OH
is therefore one of the most effective defenses against
disease (Lin et al. 1995). The results indicate that AD has a
significant protective effect against the highly toxic •OH
radical. Multiple mechanisms underlying the reaction
between the hydroxyl radical and phenolic compounds have
been reported by Cheng et al. (2002). In a previous study,
the total crude polysaccharides isolated from the roots of
A. dahurica significantly scavenged •HO radicals (Xu et al.
2011). Our chemical analysis suggests that AD root extracts
showed significant reducing power. The reducing power of
the ethanol extract was superior to that of the aqueous
extract, which is confirmatory of other reports (Yildirim
et al. 2001; Kumar et al. 2008).
As shown in Fig. 2, both extracts significantly reduced
peroxidation in the linoleic acid emulsion system through-
out the incubation period when compared to the control
sample (P<0.05). Phenolic compounds afforded their pro-
tective actions in lipid peroxidation by scavenging the lipid
derived radicals (R•, RO• or ROO•) to stop the chain
reactions in a heterogeneous lipid phase (Cheng et al.
2003). Luteolin and caffeic acid have also been shown to
inhibit lipid peroxidation (Lee et al. 2003). In the pro-
cess of inhibiting linoleic acid peroxidation, the antioxi-
dant components in the extract may have acted synergisti-
cally through the various mechanisms, such as scavenging
free radicals, binding metal catalysts, decomposing
peroxides and breaking reaction chains (Adedapo et al.
SOD, an antioxidative enzyme active against the ROS,
protects cells and tissues by converting the toxic superoxide
radical anion (O2)tohydrogenperoxide(H
2O2) and O2
(Lim et al. 2010). SOD is involved in a variety of diseases,
such as degenerative neurological, heart and artery diseases
(Harman 1956; Balin 1982). Substances with SOD-like
activity protect cells by inhibiting the activity of the super-
oxide radicals (Murakami et al. 2003). Lee et al. (2007)
reported that different extracts of A. dahurica showed SOD-
like activity. In another study, ginsenoside from white
ginseng showed significant SOD activity (Lim et al. 2010).
Catalase is a tetrameric heme-containing enzyme found
in all aerobic organisms and is known to play a key role in
protecting cells against oxidative stress (Chaudiere and
Ferrari-Lliou 1999). In a previous study, Yoo et al. (2008)
reported 17 selected commercial herbs enhanced the activi-
ties of antioxidative enzymes, SOD and CAT.
DNA can be divided into three categories based on its
structure as SC, open circular (OC) and linear – when
exposed to hydroxyl radicals derived from Fenton reaction
(Jung and Surh 2001). Treatment with FeSO4and H2O2in
the absence of any extract led to formation of OC DNA by
the strand scission of SC DNA, whereas the presence of AD
root extracts prevented this strand scission to significant
extent (Fig. 3). Fenton reaction is one of the major sources
of •OH, which is produced near DNA molecules in the pres-
ence of transition metal ions such as iron and copper
(Wiseman and Halliwell 1996). In a previous study, DNA
damage induced by the Fenton reaction was prevented by
hydroxyl radical-scavenging flavonoids (Husain et al. 1987).
Hence, it may be concluded that the strong radical-
scavenging activity and prevention of oxidative DNA
damage by AD root extracts may be attributed to its rich
content of phenols and flavonoids.
NO is a widespread intra- and inter-cellular messenger
and cytotoxin (Schmidt and Walter 1994). The excessive
production of NO contributes to the pathology of inflam-
mation, shock and injury to living tissues (Zaki et al. 2005;
Pacher et al. 2007). In addition, it produces secondary active
substances by reacting with oxygen and ROS, producing
toxic effects (Pechanova and Simko 2007; Kim et al. 2008).
Phenolic compounds and terpenoids have been reported to
be beneficial in the treatment of chronic inflammatory dis-
eases associated with overproduction of NO (Jiang and
Dusting 2003). For this reason, numerous plants rich in
these potent antioxidant compounds have been investigated
as potential inhibitors of NO production in inflammatory
reactions (Choi et al. 2007). Our findings suggested that AD
root extracts significantly inhibited NO release in a dose-
dependent manner. Makchuchit et al. (2010) also demon-
strated that some species of Angelica inhibited NO
Natural extracts have been previously documented
as a potential source of anticarcinogenic compounds
(Shoemaker et al. 2005; Kaefer and Milner 2008). Hence, it
is accepted that the chemopreventive and tumor-inhibitory
effect associated with some dietary antioxidant polyphenols
could be due to their capacity to inhibit ROS or free radicals
(Halliwell 1996).
The extracts suppressed the proliferation of both HT-29
colon cancer cells and CMT-93-mouse adenocarcinoma cell
lines in a dose-dependent manner (Fig. 6). The ethanol
extract of AD root exhibited a higher antiproliferative effect
on HT-29 and CMT-93 cells than the water extract after
24 and 48 h. In a preliminary experiment, MTT assay data
showed that 75% aqueous ethanol extract (used as a nega-
tive control) did not show any significant toxicity (data not
shown). A. dahurica compounds, isoimperatorin, cnidicin,
289Journal of Food Biochemistry 38 (2014) 281–292 © 2013 Wiley Periodicals, Inc.
imperatorin, oxypeucedanin, byakagelicol and oxy-
peucedanin hydrate, have been reported to exhibit potent
cytotoxic activity against five cultured human tumor cell
lines, in vitro (Kim et al. 2007). Isoimperatorin, osthol,
imperatorin, psoralen, pangelin, bergapten and xanthotoxin
isolated from A. dahurica showed dose-dependent cytotox-
icity against four cancer cell lines (Thanh et al. 2004). The
result indicates that the synergistic effect of polypheno-
lic compounds of AD root extracts shows potent
antiproliferative activity.
Antioxidant activity was correlated with the amount of total
phenols present in the respective extracts. Ethanol proved to
be the more effective solvent for extraction of antioxidants
from AD roots, as shown by the antioxidant capacity in all
the assays used. Water and ethanol extracts were potent
free radical scavengers and effectively inhibited lipid
peroxidation, which would be expected to protect against
oxidative damage in living systems in relation to aging and
carcinogenesis. Therefore, it can be suggested that AD root
presents a potential nutraceutical and bioactive material.
However, further detailed studies on AD root and its anti-
oxidant activities in vivo are needed.
The authors declare that there are no conflicts of interest.
This work was supported by Konkuk University, South
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... Similarly, A. glauca oil was noted to increase the DPPH• scavenging capacity in a concentration-dependent manner with an IC 50 value of 32.32 µg/mL, but showed lower activity compared to BHT [41]. Moreover, Pervin et al. (2014) reported that A. dahurica root extracts showed a dosedependent increasing DPPH• and ABTS• + scavenging activities [42]. Roh and Shin (2014) demonstrated that A. koreana root EO and its two main components showed less scavenging activity than butylated hydroxyl anisole (BHA) at 1 mg/mL [43]. ...
... Similarly, A. glauca oil was noted to increase the DPPH• scavenging capacity in a concentration-dependent manner with an IC 50 value of 32.32 µg/mL, but showed lower activity compared to BHT [41]. Moreover, Pervin et al. (2014) reported that A. dahurica root extracts showed a dosedependent increasing DPPH• and ABTS• + scavenging activities [42]. Roh and Shin (2014) demonstrated that A. koreana root EO and its two main components showed less scavenging activity than butylated hydroxyl anisole (BHA) at 1 mg/mL [43]. ...
Full-text available
In this study, methanol extracts (MEs) and essential oil (EO) of Angelica purpurascens (Avé-Lall.) Gill obtained from different parts (root, stem, leaf, and seed) were evaluated in terms of antioxidant activity, total phenolics, compositions of phenolic compound, and essential oil with the methods of 2,2-azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid (ABTS•+), 2,2-diphenyl-1-picrylhydrazil (DPPH•) radical scavenging activities, and ferric reducing/antioxidant power (FRAP), the Folin-Ciocalteu, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gas chromatography-mass spectrometry (GC-MS), respectively. The root extract of A. purpurascens exhibited the highest ABTS•+, DPPH•, and FRAP activities (IC50: 0.05 ± 0.0001 mg/mL, IC50: 0.06 ± 0.002 mg/mL, 821.04 ± 15.96 µM TEAC (Trolox equivalent antioxidant capacity), respectively). Moreover, EO of A. purpurascens root displayed DPPH• scavenging activity (IC50: 2.95 ± 0.084 mg/mL). The root extract had the highest total phenolic content (438.75 ± 16.39 GAE (gallic acid equivalent), µg/mL)). Twenty compounds were identified by LC-MS/MS. The most abundant phenolics were ferulic acid (244.39 ± 15.64 μg/g extract), benzoic acid (138.18 ± 8.84 μg/g extract), oleuropein (78.04 ± 4.99 μg/g extract), and rutin (31.21 ± 2.00 μg/g extract) in seed, stem, root, and leaf extracts, respectively. According to the GC-MS analysis, the major components were determined as α-bisabolol (22.93%), cubebol (14.39%), α-pinene (11.63%), and α-limonene (9.41%) among 29 compounds. Consequently, the MEs and EO of A. purpurascens can be used as a natural antioxidant source.
... The vegetable extracts were particularly more effective than BHT in preventing linoleic acid peroxidation within the first 5 days of the experiment. The results differ from those reported for a herb root water extract, which had lower linoleic acid peroxidation inhibitory activity than BHT (Pervin et al. 2014). On days 6 and 7, the inhibitory power of the vegetable extracts was similar to that of BHT, which suggests slight weakening of the strength of the extracts. ...
... The results are similar to that of the root water extract, which was also shown to exhibit slight losses in inhibitory activity at days 6 and 7 (Pervin et al., 2014). In contrast, the absence of BHT or veg- ...
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The aim of this study was to evaluate the nutritional value and antioxidant properties of aqueous extracts of some Bangladesh vegetables using fruits of ash gourd, bitter gourd, brinjal, okra, ridge gourd, snake gourd, and leaves of Indian spinach, kangkong, and stem amaranth. Proximate composition showed that the dried extracts were composed mainly of crude protein (14.6%–46.7%) and non‐fibre carbohydrates (26.4%–53.5%). With the exception of stem amaranth, all the extracts had >40% DPPH radical scavenging ability at 0.5 mg/ml. In contrast metal chelation was lower, except in Indian spinach with ~46%. The ferric reducing antioxidant power (FRAP) was highest for the kangkong (10.9 mM Fe³⁺ reduced), which is similar to the 9.9 mM for butylated hydroxytoluene (BHT). All the extracts suppressed linoleic acid oxidation better than BHT within the first 5 days of the incubation period. We conclude that the Indian spinach, kangkong, and okra could be considered as the most promising sources of antioxidant compounds. Practical applications Vegetables are commonly consumed as part of a regular diet but the high water and fiber contents usually mean that large quantities are required to provide long‐term health benefits. Therefore, in this work, aqueous extracts of nine Bangladesh vegetables were prepared to provide a more concentrated form of nutrients and bioactive compounds. The extracts had strong nutritional value based on the high contents of crude protein, potassium, iron, and non‐fibre carbohydrates. The high content of polyphenolic compounds in the extracts can also provide health benefits, which was demonstrated through strong free radical scavenging, metal chelation, ferric iron reduction, and inhibition of linoleic acid oxidation. These vegetable extracts have the potential to be used as sources of bioactive compounds to prevent or treat non‐communicable diseases that are associated with high oxidative stress.
... The two extracts had potent reducing power and inhibited superoxide dismutase, catalase and DNA damage. It also inhibited the production of NO in a dose-dependent manner in lipopolysaccharide-treated RAW264.7 cells [110]. ...
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Genus Angelica is one of the widely distributed and well-known genera of family Umbelliferae. It is utilized mainly by Chinese and Korean populations especially in their folk medicine. Angelica comprises a lot of medicinally important phytoconstituents such as coumarins, furanocoumarins, flavonoids, essential oils, verbascosides, polysaccharides, etc. Members of this genus play important roles, namely antioxidant, anti-inflammatory, anti-microbial, anti-diabetic, skin-whitening, cytotoxic, hepatoprotective, and many others. This review draws attention to many species of genus Angelica with much focus on A. dahurica being one of the highly medicinally used species within this genus.
... Another phenolic compound, ferulic acid (292) was isolated from the EtOAc-soluble fraction of A. dahurica root (Kwon et al., 1997). In addition, five flavonoids, including cyanidin (293), rutin (294), catechin (295), epicatechin (296) and kaempferol (297) have been found in the water extract or ethanol extract (Pervin et al., 2014) ...
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Angelica dahurica (A. dahurica) root is a famous edible medicinal herb that has been used in China for thousands of years. To date, more than 300 chemical constituents have been discovered from A. dahurica. Among these ingredients, coumarins and volatile oils are the major active compounds. Moreover, a few other compounds have also been isolated from the root of A. dahurica, such as alkaloids, phenols, sterols, benzofurans, polyacetylenes and polysaccharides. Modern pharmacological studies demonstrated that the root of A. dahurica and its active components displayed various bioactivities such as anti-inflammation, anti-tumor, anti-oxidation, analgesic activity, antiviral and anti-microbial effects, effects on the cardiovascular system, neuroprotective function, hepatoprotective activity, effects on skin diseases and so on. Based on these studies, this review focused on the research publications of A. dahurica and aimed to summarize the advances in the traditional uses, phytochemistry and pharmacology which will provide reference for the further studies and applications of A. dahurica.
... dahurica) is a folk medicinal plant for which the roots have been used over the years as a remedy for cold, fever, headache, nasal congestion, toothache, asthma, stomachache and dysmenorrhea in East Asian countries (Korea, China, Russia, Taiwan, etc.) [1][2][3][4]. Several previous studies have also reported that the roots of A. dahurica exhibit various pharmacological functions, including antibacterial, anti-asthmatic, hypotensive, anti-inflammatory, antioxidation, anti-cancer, and anti-Alzheimer effects [5][6][7][8][9][10][11]. Pharmacological functions of A. dahurica are generally attributed to coumarins (oxypeucedanin, bergapten, imperatorin, cnidilin, isoimperatorin, xanthotoxol, oxypeucedanin hydrate, byakangelicin, etc.), which are major constituents of this herb [12][13][14]. ...
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Ionic liquids (ILs) have sparked much interest as alternative solvents for plant materials as they provide distinctive properties. Therefore, in this study, the capacity of ILs to extract oxypeucedanin hydrate and byakangelicin from the roots of Angelica dahurica (A. dahurica) was investigated. The back-extraction method was examined to recover target components from the IL solution as well. Herein, [Bmim]Tf2N demonstrated outstanding performance for extracting oxypeucedanin hydrate and byakangelicin. Moreover, factors including solvent/solid ratio, extraction temperature and time were investigated and optimized using a statistical approach. Under optimum extraction conditions (solvent/solid ratio 8:1, temperature 60 °C and time 180 min), the yields of oxypeucedanin hydrate and byakangelicin were 98.06% and 99.52%, respectively. In addition, 0.01 N HCl showed the most significant ability to back-extract target components from the [Bmim]Tf2N solution. The total content of both oxypeucedanin hydrate (36.99%) and byakangelicin (45.12%) in the final product exceeded 80%. Based on the data, the proposed approach demonstrated satisfactory extraction ability, recovery and enrichment of target compounds in record time. Therefore, the developed approach is assumed essential to considerably reduce drawbacks encountered during the separation of oxypeucedanin hydrate and byakangelicin from the roots of A. dahurica.
In this study, a simple, accurate and green localization method of coumarins in Radix Angelicae dahuricae was established with fresh Radix Angelicae dahuricae as research material to reveal the distribution and accumulation of coumarins, based on frozen section and fluorescence imaging technology. The best frozen section conditions were established by comparing the effects of different cryoprotectants on the quality of Radix Angelicae dahuricae's frozen sections, according to the loss of coumarins and the complexity of the operation process. The coumarin components in Radix Angelicae dahuricae at different stages were located and quantitatively analyzed, and coumarin components distribution positions and content changes were identified using fluorescence imaging combined with LC-MS technology. The results showed that 30μm slice thickness with 15% glycerin protectant treatment is the best condition for frozen section. Fluorescence imaging showed that coumarins in Radix Angelicae dahuricae were mainly distributed in secretory tissue, the content over different periods showed an “S” curve of growth and coumarins reached their highest content in early September. The distribution and accumulation of coumarins in roots were revealed, which provided a reference for the synthesis and metabolism mechanism of metabolites in medicinal plants and the quality evaluation of traditional Chinese medicine.
Introduction: The main chemical components of Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. are coumarins and volatile oils, and coumarins are regarded as the representative constituents with various pharmacological effects. Objective: Based on matrix-assisted laser desorption/ionization time of flight mass spectrometry imaging (MALDI-TOF-MSI), a method for spatial distribution analysis of coumarins in primary root and lateral root of A. dahurica was established. Also, spatial visualization of coumarins in the roots of A. dahurica was realized. Materials and methods: α-Cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, and 9-aminoacridine were used as matrices. MALDI-TOF-MSI was employed to analyze the standards of imperatorin, oxypeucedanin, and osthole. Based on the higher sensitivity and repeatability of MALDI-TOF-MSI, the CHCA matrix was selected. The matrix was used for MALDI-TOF-MSI in positive mode to analyze the distribution of coumarins in primary root and lateral root of A. dahurica. Results: In total, 37 coumarins were detected in primary root and 36 coumarins were detected in lateral root by MALDI-TOF-MSI. The results showed that the coumarin content in primary root was higher than that in lateral root. Coumarins in primary root of A. dahurica were concentrated in the periderm, cortex, and phloem, whereas coumarins in lateral roots were concentrated in the phloem. Conclusion: The coumarins in primary root and lateral root of A. dahurica were directly analyzed without extraction and isolation, and the spatial distribution of coumarins was comprehensively visualized for the first time by MALDI-TOF-MSI, which provided a basis for distinguishing primary root and lateral root.
Current gyrodactylids treatments using chemicals such as rotenone, piperazine and trichlorfon cause environmental and human health issues, especially their toxicity to the host. Therefore, search for alternative anthelmintic agents is urgent and natural compounds from herbs show promising solution to this problem. In our previous study, we screened 33 herbal medicines and found that Angelica dahurica has a good anthelmintic efficacy. In order to identify the active compounds from Angelica dahurica, bioassay-guided isolation was performed using a Carassius auratus - Gyrodactylus kobayashii model in this study. After extraction, purification and characterization step by step, isoimperatorin was eventually separated as the main active ingredient for eradicating G. kobayashii in goldfish. Compared to the control groups, bathing G. kobayashii-infected goldfish in isoimperatorin significantly reduced infection prevalence and intensity, with EC50 values of 0.63 (24 h) and 0.53 mg/L (48 h), respectively. Acute toxicity assays indicated that the LC50 (96 h) of isoimperatorin against goldfish was 10.16 mg/L, which was 19.17 times higher than EC50 (48 h). In addition, scanning electron microscope (SEM) and RT-qPCR were used to explore the anthelminthic mechanism of isoimperatorin. TEM results showed that after exposure to isoimperatorin, morphological alterations in the tegument of G. kobayashii were observed, which disrupted its homeostasis. Besides, isoimperatorin significantly downregulated the expression of ATPase genes (ATP5A1, ATP5C, ATP5F) and oxidative phosphorylation pathway genes (NDUFS8, NDUFA8, SDH). After treatment with isoimperatorin, the content of ATP in the worms decreased significantly. In summary, isoimperatorin showed promising anti G. kobayashii efficacy via damaging tegument, supplying insufficient energy to the G. kobayashii.
Ethnopharmacological relevance The two major theories utilized for diagnosis and treatment in Traditional Thai Medicine (TTM) are the Four Element Theory and the Herbal Flavor Theory. A TTM “Poh-Pu” Remedy has been effectively utilized in Thailand for cancer therapy for centuries. Aims of study To investigate anti-inflammatory activity and liver cancer cytotoxicity of Poh-Pu remedy. To determine relationships between the TTM Herbal Flavor theory and the Four Element theory and total flavonoid content and biological activities of Poh-Pu Remedy plant extracts. Materials and methods Each plant ingredient was macerated with 95% ethanol. The extracts were investigated for cytotoxic activity against liver cancer using a sulforhodamine B assay, and anti-inflammatory activity was evaluated by inhibition of nitric oxide production. The total flavonoid content was determined by an aluminum chloride colorimetric assay. The relationships between the TTM theories, total flavonoid content, and biological activities were evaluated by correlation and cluster analysis. Results Mammea siamensis exerted potent cytotoxicity against hepatocellular carcinoma (HepG2) cell lines with an IC50 of 3.15 ± 0.16 μg/mL and low cytotoxicity to the non-cancerous cells (HaCat) with an IC50 33.39 ± 0.40 μg/mL (Selective index (SI) = 10.6). Tiliacora triandra was selectively cytotoxic to cholangiocarcinama (KKU-M156) cells with an IC50 of 12.65 ± 0.92 μg/mL (SI = 6.4). Curcuma comosa was the most potent anti-inflammatory inhibitor of nitric oxide production with an IC50 of 2.75 ± 0.34 μg/mL. Campomanesia aromatica exhibited the highest total flavonoid content of 259.7 ± 3.21 mg quercetin equivalent/g. Pungent plants were most prevalent in the TTM remedy. Conclusion Pungent, fragrant, bitter and nauseating plants utilized in TTM cancer remedy were successfully investigated and identified several lead plants and components with cytotoxic and antiinflammatory activity that require further study. The TTM wind element theory appeared to be aligned with cancer-related activity. Biological activity results of taste from herbs related with The TTM Herbal Flavor theory. The extra-oral locations of flavor receptors are a promising target for biological activity of TTM which require further scrutiny and identified several lead plants and components with cytotoxic and antiinflammatory activities that also require further study.
Ethnopharmacological relevance Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name “Bai Zhi” as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-tang, for the common cold and influenza. Aim of the study Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. Materials and methods Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. Results The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (μM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80–90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 μM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; −36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. Conclusion Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).
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Ethanol and water extracts of white and fermented ginseng were prepared and their ginsenoside composition and antioxidant effects were assessed. The main ginsenosides in white ginseng were > Re > , and those in fermented ginseng were > Rd > . Ginsenosides Rd and in fermented ginseng were enriched 11 and 58 times, respectively, over that in white ginseng through fermentation with five Bacillus spp. The greatest levels of 2-deoxyribose and superoxide anion dismutase-like activities were found in 50% ethanol extracts of fermented ginseng. Thus, these data suggest that white ginseng has the greatest free radical scavenging activity and that fermented ginseng has the highest antioxidant activity.
Antioxidative activity of aromatic amino acids and indole compounds for the autoxidation of linoleic acid was found to correlate in some extent with the highest occupied molecular orbital energy which represents the electron donor property of respective molecule. 5-Hydroxytryptophan, one of the best electron donor among the compounds tested, was the most effective antioxidant. However, antioxidative activity of some indole compounds could not be interpreted simply by their highest molecular orbital energies.Neither the chelating action for the possible metal traces nor the accelerated decomposition of hydroperoxide produced during the course of the reaction explained these actions of indoles. Tryptophan, while preventing the autoxidation of linoleic acid, underwent the ring cleavage at the position of between C2 and C3 or hydroxylation at C5 to yield formylkynurenine, kynurenine, 3-hydroxykynurenine, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, etc. Following mechanisms which were compatible with the experimental results were proposed for the antioxidative action of indoles; indole donates an electron from its π-pool to linoleic acid radical or peroxy radical produced during the autoxidation of linoleic acid to form a loose charge transfer complex through a “local” interaction; an electron transfer occurs within the complex, which brings cleavage of indole rings and an inhibition of autoxidation.
This article examined the interactive effect among herbs extracts in combined use, compared with the effect of individual herbal extracts on 1,1-diphenyl picryl hydrazyl (DPPH). The Chinese traditional prescription, four substances decoction (FSD) consisted of 25 g of Angelica sinensis (Umbelliferae, AS), 6 g of Ligusticum chuanxiong Hort. (Umbelliferae, LCH), 10 g of Paeonia loactiflora Pall. (Paeoniaceae, PCP), and 15 g of Radix rehmanniae preparata (Scrophulariaceae, RRP). Each herb was quantitatively mixed according to the FSD prescription ratio. The interaction between the herbal extract in two-two combination way were estimated using a spectrophotometer. The results showed the strongest synergistic effect in LCH and PLP system, with IC50 0.25 mg/ml, and the additive effect was obvious in the AS and LCH combined system. The other combined system showed additive effect or sub-additive effect. This implied that the combined system effect have a close relationship with the single component activity.
Harald H. H. W. Schmidt and Ulrich Walter Medizinische Universitatsklinik Wiirzburg Klinische Biochemie und Pathobiochemie Versbacher Strasse 5 D-97078 Wiirzburg Federal Republic of Germany Nitroglycerine has been used for over a century to treat coronary heart disease, and it has long been suggested that humans synthesize oxides of nitrogen (Mitchell et al., 1916). These observations have recently been brought into focus by the demonstration that endogenous nitric oxide (NO) regulates mammalian blood vessels and other systems (Moncada and Higgs, 1991) such that virtually every mammalian cell is under the influence of NO. The three “classics” of NO-mediated functions-endothelium- dependent relaxation (Furchgott and Zawadzki, 1980) neurotransmission (Garthwaite et al., 1988; Gillespie et al., 1989) and cell-mediated immune response (Nathan and Hibbs, 1991)-have suggested principles for the mode of action of NO and for its functions. General Principles Networks In many systems, NO derives from two or more different cellular sources, forming networks of paracrine communi- cation (Figure 1). For example, we now know that vascular and bronchial NO originates not only from endothelial cells, where it iscalledendotheliumderived relaxing factor (EDRF), but also from adventitial nerves and epithelial cells (Schmidt et al., 1992a; Wilcox et al., 1992), where it mediates endothelium-independent smooth muscle relax- ation. Neurons use NO to regulate transmitter release of adjacent neurons (Meffert et al., 1994) and also to match cerebral blood flow with neuronal activity; similarly, bron- chial epithelial and endothelial cells use NO to match venti- lation and perfusion (Gaston et al., 1994). Macula densa renal tubular epithelial cells release NO to dilate the neigh- boring afferent artery and increase glomerular filtration (Wilcox et al., 1992). NO Toxicity NO is a double-edged sword (Table l), beneficial as a messenger or modulator and for immunologic self-defense, but potentially toxic. In several different scenarios (Figure 2) with factors such as oxidative stress, generation of reactive oxygen intermediates (ROls), and deficient anti- oxidant systems, NO switches from friend to foe. A pre- dominant mechanism by which this occurs is through the diffusion-limited reaction of NO with superoxide to gener- ate peroxynitrite (Beckman et al., 1990) which may modu- late signaling functions of NO (Gaston et al., 1994; Moro et al., 1994) and is directly cytotoxic (Beckman, 1991). e.g., by causing extensive protein tyrosine nitration (Beck- man et al., 1994).
The dependence of the extent of aqueous extraction of antioxidant compounds on particle size and contact time was studied for three important medicinal plants, that are commonly used in infusions: agrimony, sage and savoury. The effect of extraction time was dependent on the plant considered; however, ca. 5 min can be taken as the minimum period required to assure an acceptable degree of extraction of those compounds. As expected, a smaller particle size led to a higher extraction extents; a typical value of 0.2 mm is accordingly recommended. Chlorogenic acid was the dominant phenolic compound extracted from agrimony, whereas caffeic acid dominated in the case of sage or savoury. A mathematical model based on Fick’s law was developed from first principles, and its two parameters were suitably fitted to the experimental data generated – in attempts to predict the evolution of antioxidant capacity extracted during contact time, for each plant and each particle size.
Various solvent extracts of Kappaphycus alvarezii, an edible red seaweed (family Solieriaceae) were screened for total phenol content and antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ion chelating activity, reducing power and antioxidant activity assays in a linoleic acid system with ferrothiocyanate reagent (FTC). The total phenol content of different extracts of K. alvarezii varied from 0.683 ± 0.040% to 2.05 ± 0.038%. The radical-scavenging activity of ethanol extract was, as IC 50 3.03 mg ml À1 , whereas that of the water extract was IC 50 4.76 mg ml À1 . Good chelating activity was recorded for methanol extract (IC 50 3.08 mg ml À1) wherein 67.0 ± 0.924% chelation was obtained using 5.0 mg ml À1 of extract. The reducing power of the samples was in the following order: BHT > methanol > ethanol > ethyl acetate > water > hexane. But, in the linoleic acid system, the ethanol extract proved superior to the synthetic antioxidants butylated hydroxytoluene (BHT). Hence, these extracts could be considered as natural antioxidants and may be useful for curing diseases arising from oxidative deterioration.
The objective of this study was to characterise the antioxidant properties of both water and ethanol extracts from the fruit of Gardenia jasminoides Ellis (GJE). The IC50 values for DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt], hydroxyl and superoxide radical-scavenging activities were 0.14, 0.21, 1.08 and 1.43 mg/ml for the water-based extract, and 0.36, 0.39, 1.56 and 1.99 mg/ml for the ethanol-based extract, respectively. The extracts also showed strong reducing power, nitrite-scavenging activity, inhibition of linoleic acid oxidation, superoxide dismutase-like (SOD-like) activity and catalase activity. However, the water extract had a higher antioxidant activity than the ethanol extract. In addition, the antioxidant activities were highly correlated with the observed phenolic and flavonoid contents. Therefore, our study strongly suggests that extracts derived from the fruit of Gardenia jasminoides could be an excellent source of antioxidants as dietary supplements.