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The antioxidant activity (AA), total phenolic content (TPC) and total flavonoids content (TFC) in Dong quai (DQ, Angelica sinensis) raw materials and dietary supplements (DS) containing this plant were determined using the CUPRAC, FRAP and fluorescence methods. The antioxidant activity for DQ aqueous extracts revealed by CUPRAC was (1330.45 ± 1.30) μmol Trolox equivalent (TE) per 100 g of dry mass (DM), whereas the antioxidant activity as determined by FRAP was (1813.9 ± 2.0) μmol of TE per 100 g of DM. Lower values were noted for the fluorescence method than for CUPRAC and FRAP (ranging from (35.96 ± 0.3) to (304.6 ± 1.4) μmol of TE per 100 g of DM). The highest TPC values were determined for an aqueous extract of DQ ((3330.3 ± 2.3) μmol of TE per 100 g of DM), while TFC for ethanolic extracts of DQ was ((146.50 ± 0.5) mg of quercetin equivalent (QE) per 100 g of DM). Cinnamic acid, isomers of benzoic acid and derivatives of quercetin were analysed by HPLC-PDA. The ferulic acid concentration in an ethanolic extract of DQ was (21.83 ± 0.07) mg per 100 g of DM. Of the flavonols detected, rutin exhibited the highest concentration in ethanolic extract of DQ ((3.32 ± 0.13) mg of QE per 100 g of DM). Other phytochemicals (alkaloids, saponins, flavonoids, anthraquinones, tannins, steroids, etc.) were identified by phytoscreening colour reaction. The results were analysed by principal component analysis (PCA), cluster analysis and one-way ANOVA tests.
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Chemical Papers 68 (4) 493–503 (2014)
DOI: 10.2478/s11696-013-0485-7
ORIGINAL PAPER
Evaluation of antioxidants in Dong quai ( )
and its dietary supplements
Anna Filipiak-Szok*, Marzanna Kurzawa, Edward Szlyk
Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7 St., 87-100 Toru´n, Poland
Received 16 April 2013; Revised 11 July 2013; Accepted 12 July 2013
The antioxidant activity (AA), total phenolic content (TPC) and total flavonoids content (TFC)
in Dong quai (DQ, Angelica sinensis) raw materials and dietary supplements (DS) containing this
plant were determined using the CUPRAC, FRAP and fluorescence methods. The antioxidant
activity for DQ aqueous extracts revealed by CUPRAC was (1330.45 ±1.30) µmol Trolox equivalent
(TE) per 100 g of dry mass (DM), whereas the antioxidant activity as determined by FRAP was
(1813.9 ±2.0) µmol of TE per 100 g of DM. Lower values were noted for the fluorescence method
than for CUPRAC and FRAP (ranging from (35.96 ±0.3) to (304.6 ±1.4) µmol of TE per 100 g
of DM). The highest TPC values were determined for an aqueous extract of DQ ((3330.3 ±2.3)
µmol of TE per 100 g of DM), while TFC for ethanolic extracts of DQ was ((146.50 ±0.5) mg of
quercetin equivalent (QE) per 100 g of DM). Cinnamic acid, isomers of benzoic acid and derivatives
of quercetin were analysed by HPLC-PDA. The ferulic acid concentration in an ethanolic extract
of DQ was (21.83 ±0.07) mg per 100 g of DM. Of the flavonols detected, rutin exhibited the
highest concentration in ethanolic extract of DQ ((3.32 ±0.13) mg of QE per 100 g of DM).
Other phytochemicals (alkaloids, saponins, flavonoids, anthraquinones, tannins, steroids, etc.) were
identified by phytoscreening colour reaction. The results were analysed by principal component
analysis (PCA), cluster analysis and one-way ANOVA tests.
c
2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Dong quai, dietary supplements, phenolic acids, flavonols, antioxidant activity
Introduction
Angelica sinensis (Dong quai, Chinese angelica,
Danggui, Tan kue bai zhi, Tang kuei) is a herb in-
digenous to China. Dong quai (DQ) root is used as a
spice, tonic or medicine in China, Korea and Japan
(Mei et al., 1991; Pharmacopoeia Commission, 1998,
2000). Furthermore, this herb is in common use in
dietary supplements or as an ingredient of cosmetics
available in China, USA and Europe (Upton, 2003;
Lu et al., 2005). Its medicinal value has been demon-
strated in numerous clinical trials, pre-clinical studies
and traditional practices (Mei et al., 1991; Ross, 2001;
Sun et al., 2005; Circosta et al., 2006; Wojcikowski et
al., 2009; Shou & Liu, 2011). Analytical studies of ma-
trices containing Dong quai revealed the presence of
over 70 compounds; however, the main pharmacolog-
ically active components were detected in the essen-
tial oils obtained from this plant (Ross, 2001; Huang
et al., 2004; Song et al., 2004; Sun et al., 2005). The
HPLC-PDA results confirmed the presence of polysac-
charides, ferulic acid, ligustilide and phthalides (e.g.,
butylidenephthalide) (Zhao et al., 2003; Lao et al.,
2004; Li et al., 2006; Wang et al., 2007). Also, the
HPLC (Li et al., 2006; Wang et al., 2007) and GC
(Zhao et al., 2003; Lao et al., 2004) results on the
determination of chemical components in DQ are lim-
ited, due to the lack of standards.
To the best of our knowledge, the amount of data
on the antioxidant activity of Dong quai is limited.
The reported value of the DQ DPPH free radical-
scavenging activity was as low as (3.6 ±0.1) gmL
1
(Ho et al., 2009). Huang et al. (2008) claimed that the
antioxidant activity could be estimated by scavenging
DPPH, inhibiting lipid peroxidation and NO produc-
tion. The latter parameters correlate positively and
*Corresponding author, e-mail: ania f@doktorant.umk.pl
494 A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014)
linearly with the ferulic acid and total phenolic acids
contents in DQ extracts.
Hence, the present work focused on the determina-
tion of bioactive constituents and evaluation of antiox-
idant activity (AA) of DQ by CUPRAC (cupric ion re-
ducing antioxidant activity), FRAP (ferric ion reduc-
ing antioxidant parameter) and fluorescence methods.
It should be noted that the fluorescence (FL) as-
say is rarely used for the determination of antioxidant
activity. The fluorescence of luminol implies the ox-
idation of luminol in a basic medium, generating an
energy-rich intermediate with subsequent emission by
aminophtalic acid. The reaction is catalysed by Cu(II),
and requires the presence of a potent oxidising agent
such as H2O2or ascorbic acid, in order to determine
the oxidant and antioxidant compounds that interact
with them.
In this work, the modified fluorescence method for
the determination of antioxidant activity was opti-
mised and applied to analyses of plant and dietary
supplements extracts. The optimal concentrations of
reagents (luminol, H2O2) were determined, as well as
the optimal reaction conditions (wavelength, pH, tem-
perature, sample volume). All measurements were per-
formed at the wavelength of the emission maximum of
the oxidised form of luminol (413 nm).
In addition, the HPLC determination of deriva-
tives of quercetin, benzoic and cinnamic acids was
performed. The total amounts of flavonols and phe-
nolic acids determined by HPLC were compared with
the total polyphenols content (TPC) using Folin–
Ciocalteu’s method and with total flavonoids content
(TFC). Tests for the identification of phytochemi-
cal components (alkaloids, saponins, flavonoids, an-
thraquinones, tannins, steroids, etc.) were also per-
formed. The matrices studied were dietary supple-
ments (DS) containing DQ (denoted as Vm, VF, DqS,
DqM) available on the Polish market, in which the
antioxidant activity was determined. Using modified
spectrophotometric and chromatographic methods,
the phytochemical components (especially flavonols
and phenolic acids) present in Angelica sinensis were
determined.
Experimental
Dong quai raw material was purchased from Stan-
dard (Lublin, Poland), while dietary supplements con-
taining Dong quai in the form of pills (denoted as Vm,
VF, DqS, DqM) were bought in a local pharmacy. Ac-
cording to the data on the labels, the dietary supple-
ments denoted as DqS and DqM contained only DQ,
while Vm and VF were mixtures of DQ, vitamins and
minerals.
Methanol, isopropanol, tetrahydrofuran, quercetin
derivatives (quercetin (Q), rutin (R), hyperoside (H),
kaempferol (K), rhamnetin (Rh), quercitrin (Qc),
myricetin (M)), caffeic acid (CA), sinapic acid (SA), p-
coumaric acid (pCA), chlorogenic acid (ChA), Folin–
Ciocalteu reagent (FC reagent, 2 M), neocuproine
(2,9-dimethyl-1,10-phenantroline), luminol (5-amino-
2,3-dihydro-1,4-phthalazinedione), 2,4,6-tri-2-pyridyl-
s-triazin (TPTZ, 99 mass %) and iron(III) chloride
hexahydrate, 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (Trolox (T), 97 mass %), were sup-
plied by Sigma–Aldrich (Germany). Methanol (99.8
mass %), ethanol (99.9 mass %), glacial acetic acid,
hydrochloric acid (35–38 mass %), sodium hydroxide,
sodium acetate, sulphuric acid, ammonia (24 mass %
solution), sodium hydrogen carbonate and sodium car-
bonate were purchased from Chempur (Poland). Gal-
lic acid (GA), p-hydroxybenzoic acids (pBA), cop-
per chloride, copper sulphate, ammonium acetate, hy-
drogen peroxide and chloroform were purchased from
POCh (Poland) and ferulic acid (FA) from Fluka
(USA). Methanol, isopropanol, tetrahydrofuran, phe-
nolic acids and flavonols were of HPLC grade, while
other chemicals were of analytical grade. Deionised
water (0.3 S) was used for the preparation of solu-
tions.
The samples (1.00 ±0.01) g of DQ and dietary
supplements (Vm, VF, DqS, DqM) were first ground
and oven-dried at 105
C, then extracted with three
portions of ethanol or water (20 mL each) in an ultra-
sonic water-bath (at 45
C for 30 min) and centrifuged
(4500 min1, 15 min). The DQ samples and dietary
supplements were ground to a grain size of 0.42 mm.
The aqueous extracts were denoted as: DQ-W, Vm-
W, VF-W, DqS-W, DqM-W, while ethanolic: DQ-E,
Vm-E, VF-E, DqS-E, DqM-E.
Phytochemical screening
Aqueous and ethanolic extracts of DQ and di-
etary supplements were used for identifying the phyto-
constituents as previously described (Cisowski, 1995;
Senthamarai et al., 2012). The extracts were de-
colourised with a solution of lead acetate (0.01 M),
alkalised with NaOH (0.1 M) and used for test-
ing.
Flavonoids were tested using the Shinoda test. To
a purified extract of the raw material (methanol), a
small amount of magnesium was added, followed by
the drop-wise addition of concentrated hydrochloric
acid; the resultant colour indicates: orange: flavones;
raspberry-red: flavonols; violet: flavanones; absence of
colour: chalcones and aurones.
The alkaloids were tested by Mayer’s reagent,
which yielded white or yellowish precipitates of a gen-
eral formula [(HnAlkaloid)+
n]2(HgI2
4)n. The second
test was performed by the Sonnenschein reagent (12
MoO3:H
3PO4:H
2O, mass ratio), which is sensi-
tive to alkaloids, by forming light-yellow or brownish-
yellow precipitates that eventually turn blue or green.
Anthraquinones were detected by the Bornträger
reaction, in which an acidified extract (by 1 M HCl)
A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014) 495
was mixed with diethyl ether and the organic layer
treated with an ammonia water solution, resulting in
the red-stained solution.
Tannins were found in the reaction of a few drops
of 0.1 % ferric chloride with aqueous extracts, which in
the presence of tannins turned into a brownish-green
or a blue- black solution.
Phlobatannins: boiled aqueous extracts were tested
by their reaction with 1 mass % HCl, which in the
presence of phlobatannins yield red precipitates. Pre-
cipitates with tannins were analysed using the tannins
colour reaction (referred to above).
Saponins were identified by the foam test, in which
aqueous extracts were shaken vigorously until a sta-
ble foam was noted; this was mixed with 5 drops of
olive oil and shaken. Saponins were identified by the
formation of an emulsion.
The presence of terpenoids was detected by the
reaction of aqueous extracts with chloroform followed
by the addition of concentrated H2SO4(3 mL) to form
a layer which turns reddish-brown in the event of a
positive test.
Phytosterols were detected when extracts were
mixed with a few drops of acetic acid (3 mass %).
Subsequently, acetic anhydride was added followed by
a few drops of concentrated sulphuric acid. A blue-
green colour indicated the presence of phytosterol.
Apparatus
UV-VIS spectra were recorded on a UV-VIS
HELIOS-αspectrophotometer (Unicam, Cambridge,
UK) in 1 cm quartz cell.
The HPLC (Shimadzu, Kyoto, Japan) system was
equipped with an auto-sampler (SIL-20AC HT) and
photodiode multi-wavelength detector (SPD-M20A).
The chromatographic data were recorded and pro-
cessed using the LC solution version 1.23 SP.
Fluorimetric measurements were performed on a
Hitachi F-7000 fluorescence spectrophotometer (Japan)
using a xenon lamp source for excitation in the 1 cm
quartz cell. Excitation and emission slit widths were
5 nm for the wavelength response of the system. In-
dividual and three-dimensional spectra were recorded
in the range of 200–900 nm. The spectra were evalu-
ated using the PC software package supplied with the
spectrophotometer (FL-Solution 2.1 for F-7000).
Determination of antioxidant activity
The cupric ion reducing antioxidant activity
(CUPRAC) method was applied to the antioxidant ac-
tivity determination of DQ and dietary supplements
(Vm,VF,DqS,DqM).Theprocedureisbasedon
the reduction of Cu(II) (0.01 M CuCl2)toCu(I)in
the presence of 0.01 M neocuproine (2,9-dimethyl-
1,10-phenanthroline) by antioxidants in the sample
(Apak et al., 2008). In the next stage, the Cu(I)
complex was incubated at 22
C for 30 min and ab-
sorbance measured at λmax = 455 nm against a solu-
tion blank. The calibration curves were prepared on
the basis of the least-squares method using five repeat
determinations of 15.0–60.0 M working methanolic
solutions of Trolox (T), resulting in the equation:
y= (0.0124 ±0.0003)x+ (0.0920 ±0.0128) (R2=
0.9989, RSDslope = 1.92 %, DL = 3.75 MandQL
=9.39 M). The limit of detection was calculated as
DL = (3.3Sx/y )/a, with the limit of quantifica-
tion as QL = (10Sx/y)/a, where Sx/y is the stan-
dard deviation of y-residuals. The mean recovery was
(98.10 ±0.32) % and the molar absorption coefficient
was 1.49 ×104dm 3mol1cm1. Determinations
of AA for DQ were performed as for the calibration
curve. The results were expressed as micromoles of
Trolox equivalent (TE) per 100 g of dry mass (DM)
of the DQ samples.
Ferric ion reducing antioxidant parameter – FRAP
method: freshly prepared FRAP reagent: 2.5 mL of
TPTZ solution (10 mM) in 2.5 mL FeCl3(20 mM)
and 25 mL acetate buffer (0.3 M, pH 3.6) was heated
to 37
C and incubated for 15 min. DQ and dietary
supplements extracts (0.2–0.5 mL) were mixed with
2.0 mL of FRAP reagent, made up to the mark
in a 10 mL volumetric flask and kept at ambient
temperature. for 20 min; the absorbance was mea-
sured at 593 nm against blank. Five calibration curves
were prepared using working solutions of Trolox (2.0–
20.0 M) in methanol resulting in the linear equation:
y= (0.0425 ±0.0015)x+ (0.0137 ±0.0148) (R2=
0.9991, RSDslope = 2.25 %, DL = 0.73 MandQL=
1.76 M). FRAP values were expressed as mol of TE
per 100 g of DM. The calculated mean recovery was
(97.81 ±0.14) %, while the molar absorption coeffi-
cient was 4.52 ×104dm3mol1cm1.
Fluorescence method
AA of DQ samples was determined by the modi-
fied fluorescence method (Novas et al., 2004; Pogačnik
& Ulrih, 2012). In this procedure, the Trolox solution
(2.0–6.0 mL, 0.001 M) in a 10 mL volumetric flask
into which luminol (1 mL, 0.003 M), CuSO4(1 mL,
0.005 M) and 1 mL H2O2were added, was made up
to volume with redistilled water, incubated at 22
C
for 60 min and the emission was measured at λ=
412 nm (excitation λ= 351 nm). These parameters
(time and emission wavelength) were set experimen-
tally (Fig. 1). The calibration curve was calculated by
plotting emission intensity as a function of the Trolox
content ( mol T per 100 g of DM). The least-square
calibration line equation (the average of five measure-
ments of calibration curves): y= (–48.36 ±2.08)x+
(376.8 ±10.08), (R2= 0.9986, RSDslope =1.05%,
DL = 0.30 mol, QL = 1.39 mol). The tested recov-
ery was (102.02 ±0.23) %. The antioxidant activity of
the DQ and dietary supplements was determined us-
496 A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014)
Fig. 1. 3D luminescence spectrum of combination of Trolox, luminol and H2O2.
ing the same method as for the standard curve (Trolox
test).
Determination of total phenolic compounds
(TPC)
The Folin–Ciocalteu reagent and saturated sodium
carbonate solution (0.15 g mL1) were added to the
standard solution or to DQ and dietary supplement
extracts, mixed, filtered, incubated at 22
C for 1 hour
and the absorbance measured (λ= 725 nm). The
range of Trolox concentration was 25.0–150.00 M
and the calibration curves equation (based on five
repeat measurements of calibration curves): y=
(0.0055 ±0.0004)x+ (0.0040 ±0.0013), (R2= 0.9992,
RSDslope = 1.69 %, DL = 4.60 M, QL = 11.20 M).
The results were expressed as mol of TE per 100 g of
DM of DQ samples. The accuracy of the method was
determined by adding appropriate amounts of stan-
dard solutions and calculating the mean recoveries
(98.64 ±0.35) %. The molar absorption coefficient
was 6.14 ×103dm3mol1cm1.
Determination of total flavonoids content
(TFC)
The total flavonoids content (TFC) was deter-
mined using the modified spectrophotometric method
described in Polskie Towarzystwo Farmaceutyczne
(2002). Quercetin (Q) was used as a calibration stan-
dard. The range of quercetin concentration was 1.00–
15.00 mg L1, and the calibration curves equation y=
(0.0651 ±0.0011)x– (0.0088 ±0.0097), (R2= 0.9997,
RSDslope = 1.09 %, DL = 0.31 mg L1, QL = 0.77
mg L1) for five repeat measurements of the calibra-
tion curves. In the procedure, to a standard solution
of DQ and dietary supplements extracts in a 10 mL
volumetric flask, 1.20 mL of aluminium chloride (20
gL
1) and diluted with the mixture of acetic acid
(1.02 kg L1) and methanol (1 : 19 vol.) were added.
The test solution and samples were incubated at 23
C
for 30 min in the dark and the absorbance measured
at 428 nm against a blank solution. The molar absorp-
tion coefficient was 2.14 ×104dm3mol1cm1.
Determination of phenolic acids and flavonols
by HPLC-PDA
A modified RP-HPLC-PDA (Filipiak-Szok et al.,
2012a, 2012b) with gradient elution was used for
the identification and determination of phenolic acids
(gallic (GA), caffeic (CA), chlorogenic (ChA), fer-
ulic (FA), sinapic (SA), p-hydroxybenzoic (pBA), p-
coumaric (pCA) acid) and flavonols (quercetin (Q),
rutin(R),hyperoside(H),kaempferol(K),rhamnetin
(Rh), quercitrin (Qc), myricetine (M)) in the DQ sam-
ples. The column thermostat (Discovery C-18, Su-
pelco, 150 mm ×4.6 mm, 5 m particle size) was
setat25
C for flavonols and 30
C for phenolic acids.
Peaks were identified by retention time and by spiking
A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014) 497
Table 1 . Calibration curves and parameters of phenolic acids determination
Linear model Concentration
Phenolic acid y=ax +bR2range DL QL
a±Sab±Sbmg L1
GA 16480 ±112 3876 ±803 0.9987 1.00–100 0.36 1.09
ChA 23215 ±212 123562 ±71213 0.9992 10.00–200 2.98 9.04
CA 45365 ±354 34848 ±3541 0.9994 10.00–200 3.55 9.87
FA 52474 ±205 12115 ±1957 0.9997 15.00-200 4.63 14.04
SA 45861 ±764 72550 ±18420 0.9990 10.00–100 3.30 9.54
pCA 28732 ±285 16172 ±2219 0.9982 25.00–200 7.39 22.40
hBA 5123 ±102 17632 ±4297 0.9989 20.00–200 6.66 19.43
x– concentration; y–peakarea;a–slope;b– intercept, ±Sa,±Sb– standard deviation of slope and intercept; R2– determination
coefficient, limit of detection DL = (3.3Sx/y)/a; limit of quantification QL = (10Sx/y )/a; Sx/y – standard deviation of y-residuals.
Table 2 . Calibration curves and parameters of flavonols determination
Linear model Concentration
Flavonols y=ax +bR2range DL QL
a±Sab±Sbmg L1
R 21354 ±218 4232 ±621 0.9990 6.00–100 1.56 5.02
H 157812 ±1331 –31218 ±6203 0.9991 2.00–50 0.25 0.81
Qc 42012 ±565 –76560 ±1171 0.9989 10.0–100 1.94 5.62
M 18197 ±389 –12134 ±2243 0.9990 2.00–100 0.58 1.75
Q 30023 ±412 4989 ±854 0.9991 6.00–100 1.43 4.78
K 38724 ±3215 –16247 ±2193 0.9996 2.00–100 0.41 1.34
Rh 21354 ±503 –3278 ±302 0.9987 5.00–100 0.85 2.89
x– concentration; y–peakarea;a–slope;b– intercept, ±Sa,±Sb– standard deviation of slope and intercept; R2– determination
coefficient, limit of detection DL = (3.3Sx/y)/a; limit of quantification QL = (10Sx/y )/a; Sx/y – standard deviation of y-residuals.
extracts with standards of phenolic acids or quercetin
derivatives.
Phenolic acids were detected using 2 mass % acetic
acid (A) and methanol (B) at a total flow-rate of
1mLmin
1and the gradient programme: 0 min (0 %
B), 0.02–11 min (to 25 % B), 11–15 min (to 28.75 %
B) 15–25 min (to 36 % B), 25–35 min (to 45 % B), 35–
38 min (to 65 % B), 38–41 min (65 % B), 41–44 min
(to 0 % B) and, finally, 0 % B. The injection volume
was 10 L. GA, pBA were detected at 254 nm, while
CA, ChA, FA, SA and pCA were detected at 325 nm.
Flavonols were separated applying mobile phase A:
water–isopropanol (95 : 5 vol.) mixture and phase B
water–isopropanol–THF (50 : 40 : 10 vol.). The non-
linear gradient programme: 0–2 min (to 30 % B), 2–11
min (to 45 % B), 11–13 min (45 % B), 15–25 min (to
55 % B), 25–27 min (55 % B), 27–32 min (to 60 %
B), 32–35 min (to 65 % B), 35–40 min (to 70 % B),
40–44 min (to 0 % B) and, finally, 0 % B at a constant
flow-rate of 1 mL min1and detection at 360 nm was
used. The injection volume was 20 L.
Calibration curves
The phenolic acids analysed were dissolved in a
methanol–water mixture (8 : 2 vol.), whereas the
flavonols were dissolved in ethanol. The calibration
curves were obtained by the analysis of standard so-
lutions at 10 concentration levels each measured five
times (Tables 1 and 2).
Statistical analysis
The results of antioxidant activity, total content of
phenolic compounds (TPC) and flavonoids (TFC) in
the Dong quai and dietary supplements extracts (Vm,
VF, DqS, DqM) are listed in Table 3. The Pearson cor-
relation test was used to determine the correlations
between variables, the antioxidant activity (AA) re-
sults and TPC, TFC in the different plant samples.
The mean differences were considered significant at
the p<0.05 level. One-way ANOVA, followed by the
Duncan test, were performed to analyse the significant
differences between CUPRAC and FRAP data (p<
0.05).
Principal component analysis (PCA) and cluster
analysis were performed for the results of antioxidant
activity, TPC and TFC in the DQ samples using Sta-
tistica (Windows software package, version 8.0). The
PCA score plot was used to determine whether AP at
samples from various sources could be grouped into
different classes.
498 A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014)
Table 3 . Antioxidant activity, TPC and TFC determination in Dong quai and dietary supplements
Antioxidant activity, mol
of TE per 100 g of DM ±SD (n=3) TPC, mol TFC, mol
Samples of TE per 100 g of QE per 100 g
CUPRAC FRAP Fluorescence of DM ±SD (n=3) ofDM±SD (n=3)
DQ W 1813.9 ±2.0h,x 1330.5 ±1.3h,y 233.8 ±0.9i3330.3 ±2.3i133.3 ±0.4f
E 1171.3 ±2.1g,x 924.4 ±0.8g,y 304.6 ±1.4j2445.3 ±2.2h146.5 ±0.5g
Vm W 733.40 ±2.8d,x 295.7 ±0.5e,y 36.0 ±0.3a1078.7 ±1.0e37.0 ±0.2b
E 542.20 ±0.9c,x 215.7 ±0.7d,y 136.8 ±1.1f783.3 ±1.0d40.1 ±0.4c
VF W 515.20 ±0.4c,x 207.8 ±1.0c,d,y 45.4 ±0.5b607.5 ±1.2b25.8 ±0.2a
E 375.90 ±0.9a,x 151.8 ±0.7a,y 64.7 ±0.4c429.8 ±1.3a37.1 ±0.2b
DqS W 822.30 ±2.7f,x 327.6 ±0.2f,y 82.3 ±0.6d1466.2 ±1.8g107.8 ±0.3d
E 472.90 ±2.0b,x 167.9 ±0.5b,y 183.5 ±0.3h727.3 ±0.4c121.2 ±0.2e
DqM W 785.87 ±1.2e,x 313.6 ±1.2e,y 98.6 ±0.7e1104.5 ±0.6f107.5 ±0.2d
E 490.50 ±0.8b,x 201.2 ±0.7c,y 175.2 ±0.8g745.8 ±0.8c120.8 ±0.1e
W – water; E – ethanol; SD – standard deviation.
Results and discussion
Analysis of phytochemicals
Flavonoids, alkaloids, anthraquinones, tannins, sa-
ponins, phlobatannins, terpenoids and phytosterols
were detected in the ethanolic extracts, whereas
flavonoids, tannins, phlobatannins and phytosterols
were detected in the aqueous extracts.
Preliminary phytochemical tests are useful for the
detection of the pharmacologically active main com-
ponents in plants. Natural products such as polyphe-
nol and steroids have received considerable attention
in recent years due to their diverse pharmacological
properties. Hence, phytochemical screening of medic-
inal plants is important for the identification of new
sources of therapeutically and industrially important
compounds.
Antioxidant activity
The results of antioxidant activities (AA), total
phenolic content (TPC) and total flavonoids content
(TFC) are listed in Table 3.
The highest value of AA observed for DQ-W was
((1813.9 ±2.0) molofTEper100gofDMby
CUPRAC; by FRAP it was (1330.5 ±1.3) mol of
TE per 100 g of DM). The results indicated that the
CUPRAC value were higher than FRAP. In every
case, the aqueous extracts demonstrated higher values
than the ethanolic samples (e.g., (1813.9 ±2.0) mol
TE per 100 g of DM and (1171.3 ±2.1) mol TE
per 100 g of DM, for DQ-W and DQ-E, respectively).
It is also worth noting that the dietary supplements
with DQ exhibited smaller values of AA than the DQ
extracts (e.g., by CUPRAC (822.3 ±2.7) mol TE
per 100 g of DM for DqS-W and (515.2 ±0.4) mol
TE per 100 g of DM for VF-W).
On the other hand, higher values of antioxidant
activity for the ethanolic extracts than for the aqueous
extracts (e.g., (304.6 ±1.4) mol TE per 100 g of
DM and (233.8 ±0.9) mol TE per 100 g of DM for
DQ-E and DQ-W, respectively) were found by using
the fluorescence method. It is worth noting that the
fluorescence values were smaller than those obtained
by using the CUPRAC and FRAP methods.
The superscripts in Table 3 are in accordance with
those received by one-way ANOVA followed by the
Duncan test and represent the differences between the
results obtained. In Table 3, the results are compared
in columns (superscripts x,y) and in rows (super-
scripts aj). The CUPRAC and FRAP results were
compared in columns, the other results in rows. It
is apparent that the antioxidant activity in the sam-
ples studied differs markedly (Duncan test, one-way
ANOVA, Table 3). First, the aqueous extracts differ
from the ethanolic extracts. In addition, the CUPRAC
and FRAP values also vary within the same samples.
This variability can be explained by the influence of
genetics, agronomic, and environmental factors, differ-
ent geographical region, different growing conditions,
extraction procedure, storage and purchasing sources,
all of which would affect the type and concentration
of antioxidants.
The DPPH free radical-scavenging activities and
lipid peroxidation inhibitory activities of DQ extracts
were reported by Huang et al. (2008). The authors
showed that DQ-E extracted for 15 min exhibited a
higher inhibitory activity towards lipid peroxidation
((76.8 ±2.3) % control) than DQ-E which underwent
extraction for a longer time. The results of antioxidant
evaluations suggest that a short extraction time (15
min) is sufficient to obtain a product with a favourable
antioxidant effect.
A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014) 499
Ho et al. (2009) revealed by using the DPPH test
that the isolated compound coniferyl ferulate pre-
sented potent DPPH free radical-scavenging activity,
with an EC50 value of (3.6 ±0.1) gmL
1.The
same test with Trolox recorded EC50 =(2.6±0.2)
gmL
1, while for ferulic acid the result was EC50
=(7.9±0.3) gmL
1. The antioxidant activi-
ties of the isolated compounds decreased as follows:
coniferyl ferulate >ferulic acid >Z-ligustilide >
11-angeloylsenkyunolide F. The phenolic compounds,
coniferyl ferulate and ferulic acid, exhibited higher
radical-scavenging activity than the phthalides, Z-
ligustilide and 11-angeloylsenkyunolide F.
Total phenolic and total flavonoids contents
The results of the TPC and TFC analysis are listed
in Table 3. Because AlCl3is not selective for quercetin
derivatives, the values obtained were related to the
various types of flavonoids in the sample and listed as
total flavonoids content (Table 3). The TPC method
revealed higher values for the aqueous extracts than
for the ethanolic extracts, in contrast to TFC. The
TPC and TFC values for the W and E samples differ
significantly according to one-way ANOVA followed
by the Duncan test, (Table 3, superscript x,yand
ajas explained above). In each case, the TPC val-
ues were higher than those obtained by the CUPRAC,
FRAP and fluorescence methods. Higher phenols and
flavonoids contents were noted for DQ than for the di-
etary supplements. Moreover, the Vm and VF samples
revealed smaller amounts of TPC and TFC than DqS
and DqM; this may be due to the fact that DqS and
DqM contain only DQ, while Vm and VF are com-
plex matrices of different plants, vitamins and min-
erals, thus causing interference with the compounds
analysed.
Phenolic acids and flavonols content in Dong
quai extract and dietary supplements deter-
mined by HPLC-PDA
Table 4 shows the results of the determination
of the quercetin, benzoic and cinnamic acids deriva-
tives. Typical chromatograms for the separation of
phenolic acids and flavonols standards and for DQ are
presented in Figs. 2 and 3. The peaks on the chro-
matograms were identified by the retention times us-
ing the reference standards under the same chromato-
graphic conditions or by spiking the extracts with the
reference standards. Due to the complexity of the nat-
ural samples extracts, the lack of standards and the
detector applied, only selected peaks were identified.
The contents of phenolic acids and flavonols
thereby obtained were statistically analysed by one-
way ANOVA followed by the Duncan test (Table 4).
The results were compared against each other in lines
(e.g., DQ-E with DG-W) and significant differences
Table 4. Content of phenolic acids and flavonols in DQ and dietary supplement extracts
Content of phenolic acids, mg per 100 g of DM ±SD (n= 3) Content of flavonols, mg per 100 g of DM ±SD (n=3)
Samples
GA ChA CA FA SA pCA hBA R H Qc M Q K Rh
DQ W2.25±0.02eND 1.24 ±0.02c19.21 ±0.04iND 1.65 ±0.01f0.65 ±0.09f3.29 ±0.02e0.80 ±0.02cND ND 1.65 ±0.03fND ND
E2.22±0.01e0.21 ±0.01a1.15 ±0.02b21.83 ±0.07jND 1.67 ±0.01f0.53 ±0.01e3.32 ±0.01e0.90 ±0.01e0.56 ±0.01eND 2.15 ±0.03g0.34 ±0.00dND
Vm W1.20
b±0.00 ND 1.10 ±0.01a2.49 ±0.01cND 1.30 ±0.00bND 1.46 ±0.01aND 0.72 ±0.01fND 0.89 ±0.01c0.14 ±0.00aND
E1.36±0.01cND 1.20 ±0.00c3.42 ±0.01dND 1.17 ±0.01aND 1.45 ±0.02aND 0.69 ±0.02fND 0.80 ±0.01b0.13 ±0.00aND
VF W1.19±0.01aND 1.24 ±0.00c1.27 ±0.02aND 1.40 ±0.00c0.13 ±0.01a1.52 ±0.01b0.32 ±0.01a0.12 ±0.01b0.50 ±0.00b0.55 ±0.01aND ND
E1.15±0.01a0.30 ±0.00b1.40 ±0.00f2.25 ±0.00bND 1.60 ±0.01e0.10 ±0.01a1.58 ±0.02b0.33 ±0.01a0.14 ±0.01b0.63 ±0.01c0.60 ±0.01aND ND
DqS W 2.47 ±0.1fND 1.25 ±0.02c10.99 ±0.02eND ND 0.27 ±0.01c2.70 ±0.02c0.72 ±0.01b0.14 ±0.01bND 1.55 ±0.02e0.22 ±0.00bND
E2.40±0.1fND 1.46 ±0.02f13.66 ±0.04fND ND 0.20 ±0.01b2.80 ±0.01d0.85 ±0.01d0.06 ±0.01aND 2.30 ±0.01h0.22 ±0.00bND
DqM W1.63±0.01dND 1.30 ±0.01e14.71 ±0.02gND 1.13 ±0.01a0.40 ±0.01d2.75 ±0.02c0.88 ±0.02e0.40 ±0.01c0.42 ±0.01a1.48 ±0.01d0.40 ±0.00eND
E1.42±0.01c0.28 ±0.01b1.28 ±0.01d18.64 ±0.02hND 1.27 ±0.01b0.52 ±0.02e2.84 ±0.02d0.95 ±0.02f0.48 ±0.01d0.53 ±0.01b2.45 ±0.01i0.30 ±0.00cND
W – water; E – ethanol; SD – standard deviation; ND – not detected.
500 A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014)
Fig. 2. HPLC chromatogram of phenolic acids for standard (1
–GA,2–ChA,3–CA,4–FA,5–SA,6–hBA,7–
pCA and DQ-W extract.
Fig. 3. HPLC chromatogram at 360 nm for flavonol standards
(1’–R,2’–H,3’–Qc,4’–M,5’–Q,6’–K,7’–Rh)
and DQ-E extract.
were allocated a different superscript (ah)andsimi-
larities by the same letter in superscript. It is notewor-
thy that, in many cases, the phytochemicals content
in the samples in the study exhibited significant differ-
ences. However, for some phenolic acids and flavonols
(e.g., GA in DQ-W and DQ-E, pCA in DQ-E and W,
R in Vm-W and E, etc.), the results obtained for aque-
ous extracts did not differ from those for the ethanolic
extracts. Significant differences were observed for the
other samples.
The predominant phenolic acids in DQ-E are: fer-
ulic ((21.83 ±0.07) mg per 100 g of DM), gallic
((2.25 ±0.01) mg per 100 g of DM) and p-coumaric
((1.67 ±0.01) mg per 100 g of DM), while the high-
est concentration of flavonols was obtained for rutin
((3.32 ±0.01) mg per 100 g of DM). In the DQ ex-
tracts, other phytocompounds were determined in mi-
nor amounts. In the samples examined, sinapic acid
and rhamnetin were not detected, while chlorogenic
and p-hydroxybenzoic acids, myricetine, kaempferol,
hyperoside and quercitrin were only observed in some
extracts. There were lower concentrations of flavonols
and phenolic acids in the dietary supplements than
in the DQ extracts. In addition, concentrations of
the latter compounds in the DqS and DqM samples
were higher than in VF and Vm. On the other hand,
phenolic compounds are known to exhibit a positive
impact on human health, hence their high concen-
tration in dietary supplements is desirable (Cook &
Samman, 1996; Clifford, 2004; Lafay & Gil-Izquierdo,
2008; Szajdek & Borowska, 2008; Weng & Yen, 2012).
To the best of our knowledge, the literature in-
cludes few data on the content of flavonols and pheno-
lic acids in Dong quai. Huang et al. (2008) determined
ferulic, p-coumaric, vanillic, caffeic, trans-cinnamic,
nicotinic, protocatechuic and phthalic acids by HPLC.
Ferulic acid predominated (for aqueous extracts, it
ranged from 15.1 to 18.9 mg mL1and for ethano-
lic extracts from 13.9 to 17.1 mg mL1, whereas other
phenolic acids were below 2.46 mg mL1). Hence, the
concentration of ferulic acid in the present research is
similar to the results presented by Huang et al. (2008).
Lu et al. (2005) analysed free ferulic acid and total fer-
ulic acids in DQ by RP-HPLC. The concentration of
ferulic acid obtained in DQ varies within the range of
0.21–1.43 mg g1(Zhao et al., 2003; Lu et al., 2005).
Some authors have used ferulic acid concentration as
one of the marker compounds for the assessment of
DQ and quality of its products (Zhao et al., 2003; Lu
et al., 2005). On the other hand, Li et al. (2006) show
that the ferulic acid content ranged between 783.8 and
569 calculated as the peak area, hence these results are
not comparable with those presented in this study.
Correlations between total content of pheno-
lics, flavonoids and antioxidant activity in An-
gelica sinensis extracts
Regression analysis was performed for correlations
between TFC, TPC, CUPRAC, FRAP and the fluo-
rescence of DQ extracts and dietary supplements; the
results are listed in Table 5. A good linear relation-
ship was found between CUPRAC and FRAP, (r=
0.97, p<0.05 level). TPC also correlated positively
with CUPRAC and FRAP value (r= 0.99 and 0.98,
respectively, p<0.05 level). Hence, the total pheno-
lic content parameter represents a suitable measure
of the antioxidant activity for the samples analysed.
Only for fluorescence and the other AA method was
a lower correlation (below r= 0.69, p<0.05 level)
observed. The lowest linear correlation was noted for
fluorescence–CUPRAC (r= 0.57, p<0.05 level). For
fluorescence and TFC, a correlation of r=0.80was
obtained; this can be related to the ethanolic extracts
having higher values than the aqueous extracts.
Cluster and principal components analysis
Principal component analysis (PCA) was applied
to observe any possible clusters within the DQ sam-
ples analysed (Fig. 3). A set of nine orthogonal vari-
A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014) 501
Table 5 . Correlation coefficients between antioxidant activity determined by three analytical methods (CUPRAC, FRAP and
fluorescence), total phenolic content (TPC) and total flavonoids content (TFC) in all samples studied
TFC TPC CUPRAC FRAP Fluorescence
TFC – 0.63 0.76 0.57 0.80
TPC 0.63 – 0.99 0.98 0.67
CUPRAC 0.76 0.99 0.97 0.57
FRAP 0.57 0.98 0.97 0.69
Fluorescence 0.80 0.69 0.57 0.69
Significant at p<0.05 level.
Table 6 . Principal Component Analysis (PCA): percentage of
variance for each component (initial eigenvalues) and
correlation coefficients (component matrix) of each
variable with component 1 (PC1) and 2 (PC2)
Initial eigenvalues
Component
Total Variance/% Cumulative variance/%
PC1 8.52 50.09 50.09
PC2 3.05 17.93 68.02
PC3 2.31 13.61 81.64
PC4 1.95 11.47 93.118
PC5 0.63 3.70 96.82
PC6 0.26 1.55 98.37
PC7 0.17 1.02 99.40
PC8 0.08 0.47 99.87
PC9 0.02 0.13 100.00
Fig. 4. Score plot of first two principal components (PC1 and
PC2) for classification of DQ in various extracts of
plants and from dietary supplements.
ables (PCs) was generated by PCA. The first two
principal components represented 68.02 % (PC1 =
50.09 %, PC2 = 17.93 %), while the first four rep-
resented 93.10 % (PC3 = 13.61, PC4 = 11.47) of the
total variation. The first principal component (PC1)
revealed the highest eigenvalue (8.52) and accounted
for 50.09 % of the variability in the data set (Table 6).
The scores of the first two principal components
Fig. 5. Cluster analysis of DQ and dietary supplements.
(PC1 and PC2) for the DQ extracts studied in var-
ious extracts of plants, different dietary supplements
are presented in Fig. 4. It is apparent that the dietary
supplements formed five distinct groups (Vm-E and
Vm-W, Vf-E and Vf-W, DqM-E and DqS-E, DqM-W
and DqS-W), and DQ (DQ-E and DQ-W). The PCA
graph revealed that the dietary supplements DqM-E,
DqS-E, DqM-W and DqS-W with high antioxidant ac-
tivities, total content of phenolics and flavonoids, were
located to the left in the score plot (in the top-left
quadrant), whereas the dietary supplement samples
with low CUPRAC, FRAP, luminescence, TPC and
TFC values were situated on the right in the diagram.
Fig. 5 presents the cluster analysis. The dietary
supplements revealed some similar properties. First
of all, the ethanolic extracts of DqM and DqS were
the closest, whereas DQ-E and DQ-W were the most
different. Moreover, the branch of DQ differed from
dietary supplements (Vf, Vm, DqM, DqS). Ethanolic
extracts of DqM, DqS and Vm formed one branch. The
same situation can be observed for aqueous extracts
of these latter dietary supplements.
Conclusions
The results presented in this study indicate that
the proposed simple method using the characteris-
tic colour reactions can be successfully applied to the
502 A. Filipiak-Szok et al./Chemical Papers 68 (4) 493–503 (2014)
determination of phytochemical constituents in Dong
quai raw material and dietary supplements and can
have applications in the food and pharmaceutical in-
dustries.
Moreover, the CUPRAC, FRAP and fluorescence
methods can be used for the description of antioxi-
dant activity in DQ and some dietary supplements
extracts. The proposed methods are relatively simple,
precise and convenient for the determination of AA in
the various matrices studied. The DQ extracts studied
are rich in antioxidants and can be a potential source
of natural antioxidants and nutrients. Moreover, the
HPLC-PDA method can be used for the determina-
tion of phenolic acids and flavonols. The present study
focuses not only on methods for the evaluation of an-
tioxidants (phenolic acids and flavonols determined
by the HPLC-PDA method) and antioxidant activ-
ity but also on a comparison of AA levels in different
sources of DQ. Information on the various properties
and chemical composition of DQ presented here can
be of use in the production of dietary supplements.
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... Angelica tsinlingensis is endemic in Shaanxi province (China), and is used as Angelica sinensis, a traditional Chinese herb used in female health related diseases, in folk (Li et al. 1993;Mazaro-Costa et al. 2010). In addition to its medicinal uses, A. sinensis is also used as a health food, a cosmetic and a dietary supplement in Asia, Europe and America (Filipiak-Szok et al. 2014). Owing to important medicinal value, wild A. sinensis has been over-exploited and become quite rare in China during over 2000 years (Zhang et al. 2012). ...
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Angelica tsinlingensis is endemic in Shaanxi province (China), and is used as Angelica sinensis in folk. Owing to important medicinal value, wild A. sinensis has been over-exploited and become quite rare in China during over 2000 years. Angelica sensu lato (s.l.; Apiaceae subfamily Apioideae) is a taxonomically complex and controversial group, and A. tsinlingensis is clearly different from members of the Angelica s.s. clade with the morphological and molecular results. The complete chloroplast DNA sequence of A. tsinlingensis (GenBank accession number: MF924726) was determined in our study. The size of chloroplast genome of A. tsinlingensis is 147,104 bp, including a large single-copy (LSC) region of 93376 bp and a small single-copy (SSC) region of 17,574 bp separated by a pair of identical inverted repeat regions (IRs) of 18,077 bp each. A total of 125 genes were successfully annotated containing 83 protein-coding genes, 34 tRNA genes and 8 rRNA genes. GC content of IRs region is the highest (44.5%). The result of Phylogenomic analysis supports the difference of A. tsinlingensis from Angelica s.s. clade. © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
... In this article, angelica has nourish blood, invigorate the circulation, stop pain, moisten the intestinal function [14]. Astragalus has to fill the function of the spleen and kidney, strengthens the body protection [15][16][17][18][19][20]. ...
... FRAP method (Ferric ion reducing antioxidant parameter) was performed according to Filipiak-Szok et al. procedure (Filipiak-Szok et al. 2014). Five calibration curves were prepared using working solutions of Trolox (TE) (2.00-25.00 ...
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