Introduction
Asari Radix et Rhizoma (ARR, Xixin in Chinese) is a well known herbal medicine which
has been mainly used to
treat common cold, influenza, rhinitis, or as a local anesthetic agent, etc [1 ], [2 ]. According to the Chinese Pharmacopoeia [3 ], it is derived botanically from the dry roots and rhizomes of Asarum heterotropoides Fr.
Schmidt var. mandshuricum (Maxim.) Kitag., A sieboldii Miq. var. seoulense Nakai., and
A. sieboldii Miq.. Many pharmacological studies reported the extensive biological activities of
ARR, including antimicrobial, antipyretic, anti-inflammatory, and analgesic properties
[4 ], [5 ], [6 ], [7 ]. Essential oils, lignans, and alkamides are the major components in ARR [8 ], [9 ], [10 ] and should
be responsible for the aforementioned activities [11 ].
Among the three Asarum spp. recorded under the same monograph of ARR in the Chinese Pharmacopoeias,
the raw materials from A. heterotropoides var. mandshuricum and A. sieboldii var.
seoulense are called “Liaoxixin” in China, and both Asarum species are mixed in most herbal
farms in northeastern China. The herbal materials from A. sieboldii have been rarely commercially
available due to its depleted wild populations [12 ], [13 ]. Meanwhile, there are many adulterants of ARR in China, such as the roots and rhizomes
of
A. himalaicum Hook. f. et Thoms. ex. Klotzsch., A. caulescens Maxim., A. maximum
Hemsl., and A. forbesii Maxim. [14 ]. Among them, the roots and rhizomes
of A. himalaicum have been used as a folk medicine and are commercially available in a relatively
large amount in some regions of western China [15 ]. However, there have been
still no scientific studies to characterize the rationality of the traditional practice
about ARR and its
adulterants until now, which is a serious problem with regard to their efficacy, quality
control, and safety
[16 ].
According to the Chinese Pharmacopoeia [3 ], the essential oil and asarinin are
used as marker compounds for the quality control of ARR. Several holistic chemical
profiling methods of ARR
have been reported, such as quantification of 2–3 lignans and 1–2 constituents of
essential oils using
(HPLC)-UV [17 ], [18 ], [19 ]. It is worth noting that ARR contains AAs, some of which can cause AAN. The US FDA
has
prohibited the import of the herbal preparations in which the herbal medicines containing
AAs are formulated
[20 ]. Because ARR contains AAs in extremely small amount, an acceptable
limit for AAs in ARR has been established in the Chinese and Japanese Pharmacopoeias.
In the limit tests by
HPLC method, the content of AA-I in dry materials of ARR must be less than 0.001 %
(10 ppm) according to the
Chinese Pharmacopoeia [3 ], and the sample solution of ARR must show no peak at
the retention time corresponding to AA-I from the standard solution in the Japanese
Pharmacopoeia [21 ]. Although AA-I in ARR was determined by HPLC-UV and LC-APCI-MS/MS methods
[22 ], [23 ], [24 ],
few papers investigated the content of other AA compounds in ARR, such as AA-IV a
and AL-I, in relation to
AAN [25 ], [26 ], [27 ].
Thus, it is necessary to develop a method for simultaneous determination of active
or marker components and
AAs in ARR.
Generally, the use of conventional HPLC methods is difficult and time-consuming for
simultaneous
determination of lignans, essential oils, flavanoids, AAs, and alkamides in ARR because
of their relative
low efficient stationary phases. Recently, the use of UHPLC coupled with QTOF/MS has
been an effective
approach for rapid analysis of multi-components of herbal medicines due to its higher
peak capacity, greater
resolution, increased sensitivity, and rich data on accurate molecular formulae for
structural
identification of unknown compounds [28 ], [29 ].
Since many unknown components may be present in ARR, most of which are new or without
reference standards,
using TOF/MS to identify them is important based on the molecular formula and fragmentation
of the known
components.
In the present study, a multi-component quantification fingerprinting approach based
on UHPLC-QTOF/MS
techniques has been developed, which combined the chemical profiling and semi-quantification
of over 20
components in the roots and rhizomes of A. heterotropoides var. mandshuricum and
A. sieboldii var. seoulense and the roots of A. himalaicum . This is the first report
on the comprehensive evaluation of the chemical profile of the roots and rhizomes
of
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense and the
roots of A. himalaicum by UHPLC-QTOF/MS method.
Results and Discussion
In order to obtain satisfactory extraction efficiency for all the analytes, extractive
methods (ultrasonic
and refluxing), solvents (50 %, 75 %, and 100 % methanol), and time (30 and 60 min)
were assessed based on
single factor experiments. The best extraction efficiency was obtained by refluxing
extraction with 100 %
methanol for 30 min.
The methanolic extracts of A. heterotropoides var. mandshuricum (sample #3) and
A. sieboldii var. seoulense (sample #10) were used for the optimization of UHPLC
conditions; their representative UHPLC-UV and UHPLC-MS chromatograms are presented
in [Fig. 1 ]. Using the optimal gradient elution as described in Material and Methods,
all the specific peaks were satisfactorily separated within 22 min.
Fig. 1 Representative UHPLC-UV (a and b , detected at 287 nm) and extracted ion
chromatograms (c and d , base peak intensity in positive ionization mode) of the extracts of
the roots of A. heterotropoides var. mandshuricum (sample #3, a and c ) and
A. sieboldii var. seoulense (sample #10, b and d ). See [Table 1 ] for the peak numbers, and see Materials and Methods for UHPLC-QTOF/MS
conditions. (Color figure available online only.)
In order to characterize the chemical composition, the methanolic extracts of the
root and rhizome of
A. heterotropoides var. mandshuricum (sample #3) and A. sieboldii var.
seoulense (sample #10) were subjected to UHPLC-QTOF/MS analysis. Twenty-two specific peaks
(labeled peaks 1 –22 , [Fig. 1 ]) in the UHPLC chromatograms were
characterized by UV absorptions obtained with Waters DAD. By co-chromatography and
comparison of the
retention time, UV spectra, and characteristic molecular ions and fragment ions with
the authentic
standards, compounds corresponding to 17 peaks were identified ([Table 1 ]).
Table 1 Marker compounds identified from A. heterotropoides var.
mandshuricum, A. sieboldii var. seoulense , and A. himalaicum by
UHPLC-QTOF/MS methods.
Peaks No.
Compounds
tR
UV
[M + H]+
m/z )
Aglycones or diagnostics fragments (m/z )
Comparison with standards
n. d., not detectable
1
(2R )-naringenin 5,7-di-O -glucoside
1.78
277, 320
597.1819
435, 273
yes
2
(2S )-naringenin 5,7-di-O -glucoside
1.99
277, 320
597.1819
435, 273
yes
3
1-O -p -coumaroyl-xylopyranosyl-glucoside
2.00
232, 315
459.1503
481, 476, 165, 147
yes
4
1-O -feruloyl-xylopyranosyl-glucoside
2.64
241, 330
489.1608
511, 506, 195, 177
yes
5
isomer of (2R )-naringenin 5,7-di-O -glucoside
3.95
288, 323
597.1819
435, 273
no
6
AA-IV a
6.03
242, 330
358.0563
312, 297
yes
7
3,4,5-trimethoxytoluene
6.53
269, 300
183.1021
168, 153
no
8
2-methoxyl-methylenedioxypropiophenone
7.52
269, 332
209.0814
176, 161
yes
9
2,4,6-trimethoxytoluene
7.52
225, 280
183.1021
168, 153
no
10
kakuol
8.25
277, 346
195.0657
147
yes
11
pluviatilol
9.67
232, 285
357.1338
339, 289
yes
12
methyleugenol
10.83
231, 280
179.1072
164
yes
13
AL-I
10.92
239, 259
294.0766
n. d.
yes
14
AA-I
13.49
225, 244
342.0614
n. d.
yes
15
safrole
15.94
235, 286
163.0759
n. d.
yes
16
myristicin
16.67
239, 299
193.0865
192
yes
17
sesamin
16.98
241, 286
355.1182
337, 319, 289
yes
18
N -isobutyl-2E,4E,8Z- decatetraenamide
17.30
260
222.1858
167, 152
no
19
asarinin
18.09
241, 286
355.1182
337, 319
yes
20
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide
20.45
235, 259
248.2014
167, 152
yes
21
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide
20.73
235, 259
248.2014
167, 152
yes
22
isomer of N -isobutyl-dodecatetraenamide
21.48
235, 259
248.2014
167, 152
no
Due to absence of reference compounds, the compounds corresponding to the rest 5 compounds
were tentatively
identified by MS/MS determination along with Waters MassFragment software, UV spectra,
and comparison with
literature data ([Fig. 1 ] and [Table 1 ]). Peak
5 (tR 3.95 min) generated the positive molecular ion at m /z 597.1819 [M +
H]+ , corresponding to the molecular formula C27 H32 O15 , and
two fragment ions at m /z 435.1291 (loss of a hexosyl, 162 mass units) and 273.0763 (loss of
two hexosyls, 324 mass units), which were the same as those of (2R )-naringenin
5,7-di-O -glucoside (peak 1 , tR 1.78 min) in the MS chromatograms. The UV spectra of
peak 5 (288, 323 nm) was also similar to that of (2R )-naringenin 5,7-di-O -glucoside
(277, 320 nm), suggesting that it should be a flavanoid. Thus, the compound corresponding
to peak 5
was tentatively identified as the isomer of (2R )-naringenin 5,7-di-O -glucoside. According to
the UV spectra of peak 5 , the compound corresponding to peak 5 may be (2R )-naringenin
4′,7-di-O -hexoside [30 ].
Peaks 7 (tR 6.53 min) and 9 (tR 7.52 min) generated the [M +
H]+ ions at m /z 183.1021, corresponding to the same molecular formula
C10 H14 O3 . High collision energy resulted in two important fragment ions,
at m /z 168.0779 (loss of a methyl, 15 mass units) and 153.0548 (loss of two methyls, 30
mass
units). These fragment ions are in accordance with the fragmentation pathways of 3,4,5-trimethoxytoluene
and
2,4,6-trimethoxytoluene shown in the phytochemical study on ARR [31 ]. According
to the difference of polarity [32 ], peaks 7 and 9 were
tentatively identified as 3,4,5-trimethoxytoluene and 2,4,6-trimethoxytoluene, respectively.
Peak 22 (tR 21.48 min) showed the similar UV absorption at 235 and 259 nm and generated
the same [M + H]+ ion at m /z 248.2014, corresponding to the molecular formula
C16 H25 NO. High collision energy resulted in diagnostic fragment ion at
m /z 167.1310 (loss of C6 H9 , 81 mass units) and m /z
152.1075 (loss of C7 H12 , 95 mass units). The fragment ion was shown in the
fragmentation pathways of a pair of isomers,
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide in the studies by Yasuda et
al. [10 ] and Luo et al. [33 ]. So the compound
corresponding to peak 22 was tentatively identified as isomer of
N -isobutyl-2,4,8,10-dodecatetraenamide.
Peak 18 (tR 17.30 min) generated the [M + H]+ ions at m /z
222.1858, corresponding to the same molecular formula, C14 H23 NO. High collision energy
resulted in two diagnostic fragment ions, at m /z 167.1310 (loss of C4 H7 ,
55 mass units) and m /z 152.1075 (loss of C5 H10 , 70 mass units). The same
fragment ions were shown in MS data of peak 20 and 21 , suggesting that the compound
corresponding to peak 18 should also be an alkamide. According to the fragmentation pathways in the
study by Quang et al. [8 ], the compound corresponding to peak 18 was
tentatively identified as N -isobutyl-2E ,4E ,8Z -decatetraenamide ([Fig. 2 ]).
Fig. 2 Fragmentation pattern and MS spectra of
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (peak 21, a ) and
N -isobutyl-2E,4E,8Z- decatetraenamide (peak 18, b ).
Good linear calibration curves were obtained with 18 tested reference standards (R > 0.995,
[Table 2 ]). Because of a great difference in the contents of
2-methoxyl-4,5-methylene-dioxypropiophenone (8 ), kakuol (10 ), and methyleugenol (12 )
between the herbal materials, two calibrations were established to serve for the low
and high content
levels. The LODs and LOQs were in the range from 0.03 to 24.9 × 10−3 µg/mL and from 0.09 to
82.9 × 10−3 µg/mL, respectively.
Table 2 Calibrations and detection limits for marker compounds.
Compounds
Calibration curve
r
Linear range
LOD−3 µg/mL)
LOQ−3 µg/mL)
(2R )-naringenin 5,7-di-O -glucoside (1 )
Y = 83.7 x -16.06
1.000
0.320–16.00
2.28
7.59
(2S )-naringenin 5,7-di-O -glucoside (2 )
Y = 99.5 x + 19.24
0.999
0.170–17.00
2.00
6.67
1-O -p -coumaroyl-xylopyranosyl-glucoside (3 )
Y = 55.45 x -2.852
1.000
0.200–10.00
3.19
10.6
1-O -feruloyl-xylopyranosyl-glucoside (4 )
Y = 256.6 x + 42.71
0.998
0.050–4.000
0.37
1.25
AA-IV a (6 )
Y = 186.4 x + 4.856
0.999
0.050–5.000
0.71
2.36
2-methoxyl-methylenedioxypropiophenone (8 )
Y = 2076 x + 667.5
0.999
0.400–20.00
0.03
0.09
Y = 1721 x + 0.4944
0.998
0.004–0.400
kakuol (10 )
Y = 818 x + 94.4
0.998
0.200–10.00
0.03
0.10
Y = 1057 x + 3.834
0.999
0.002–0.200
pluviatilol (11 )
Y = 339.7 x -12.55
0.999
0.250–10.00
0.89
2.96
methyleugenol (12 )
Y = 64.80 x -17.19
0.997
5.000–100.0
0.72
2.41
Y = 54.29 x -1.018
0.998
0.100–5.000
AL-I (13 )
Y = 3065 x -82.52
0.998
0.020–2.000
0.12
0.39
AA-I (14 )
Y = 35.86 x + 30.49
0.998
0.020–2.000
0.03
0.14
safrole (15 )
Y = 71.31 x + 98.2
0.998
1.000–10.00
6.12
20.4
myristicin (16 )
Y = 72.33 x + 7.632
0.999
0.200–10.00
24.9
82.9
sesamin (17 )
Y = 358.6 x -103.8
0.999
1.000–15.00
0.73
2.44
asarinin (19 )
Y = 224.3 x + 637.8
0.998
2.000–50.00
0.44
1.47
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetranamide (20 )
Y = 118.8 x -186.5
0.999
5.000–100.0
0.26
0.88
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetranamide (21 )
Y = 151.3 x + 213.0
0.999
2.500–75.00
0.41
1.37
Isomer of N -isobutyl-dodecatetranamide (22 )
Y = 31.62 x -4.544
0.999
0.630–50.00
0.46
1.53
The precision and recovery tests were done for 7 major marker compounds by the methods
described in
Materials and Methods. This method exhibited good reproducibility with intra- and
inter-day variations
(evaluated with RSD) of less than 4.8 % (Table 2S , Supporting Information). The recovery for these
markers ranged from 99.0 % to 103 %, with RSD ranging from 2.0 % to 5.8 % (Table 2S , Supporting
Information). Thus, the analytical procedure is accurate and sufficiently sensitive
for the simultaneous
quantification of the major compounds in the raw materials of three Asarum species.
Using the UHPLC-QTOF/MS method, chemical profiling and quantification of the components
from the roots and
rhizomes of A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense
and the roots of A. himalaicum were carried out ([Fig. 1 ] and [Table 3 ]). There is a significant difference in the chemical profiling patterns
among the roots and rhizomes of the three Asarum species.
Table 3 Content (mg/g) of 18 compounds in the roots and rhizomes of
A. heterotropoides var. mandshuricum, A. sieboldii var. seoulense and the roots of
A. himalaicum
a .
Sample No.
Flavanoids
AAs
Lignans
Essential oils
Alkamides
1
2
3
4
5
6
13
14b
8
10
11
17
19
12
15
16
18
20
21
22
a: The samples #1–#4 were botanically identified as A. heterotropoides var.
mandshuricum , #5–#10 as A. sieboldii var. seoulense , the samples #26 and #27 as
A. himalaicum . The rest samples (#11–#25, called “Liaoxixin” in China herbal
market) were the raw materials from A. heterotropoides var. mandshuricum and
A. sieboldii var. seoulense . n. d., not detectable (< limit of
detection). tr., trace (> limit of detection but < minimum of the linear range). The
numbers in bold represent the compounds as described in [Fig. 1 ]; b: The contents of AA-I #1–#4 are shown by the unit “ppm”
1
0.82
0.87
0.56
0.23
0.82
0.16
0.07
tr.
0.49
0.11
0.15
0.51
2.17
0.15
0.27
0.04
0.13
3.38
3.14
0.58
2
0.88
0.94
0.51
0.18
0.57
0.13
0.03
tr.
1.22
0.25
0.21
0.66
2.62
0.81
0.25
0.78
0.20
3.02
2.46
0.97
3
0.22
0.29
0.07
tr.
0.23
0.07
0.04
tr.
0.93
0.23
0.14
0.49
1.88
0.49
0.41
0.07
0.20
3.27
3.01
0.51
4
0.19
0.22
0.04
0.07
0.23
0.07
0.04
tr.
0.85
0.24
0.16
0.46
1.73
0.47
0.43
0.06
0.11
2.38
2.79
0.33
5
0.36
0.37
0.31
0.15
0.32
0.10
0.04
tr.
tr.
tr.
0.18
0.63
2.68
0.35
0.23
tr.
0.01
1.79
2.25
0.27
6
0.25
0.24
0.07
0.07
0.22
0.06
0.03
tr.
0.04
tr.
0.23
0.73
3.10
1.80
0.82
tr.
0.02
2.40
3.00
0.26
7
0.16
0.16
0.05
0.05
0.14
0.07
0.03
tr.
tr.
tr.
0.22
0.68
2.81
0.49
0.31
tr.
tr.
1.48
1.81
0.24
8
0.23
0.26
0.11
0.08
0.21
0.07
0.03
tr.
0.04
tr.
0.29
0.73
3.09
0.36
0.29
tr.
tr.
2.18
2.77
0.38
9
0.15
0.16
0.08
0.05
0.17
0.06
0.03
tr.
0.03
tr.
0.17
0.53
2.25
0.52
0.24
tr.
0.01
1.32
1.59
0.22
10
0.32
0.28
0.13
0.09
0.34
0.07
0.04
tr.
0.07
0.01
0.12
0.43
1.76
0.28
0.33
tr.
0.05
1.66
1.95
0.62
11
0.78
0.76
0.35
0.11
0.53
0.11
0.07
tr.
0.81
0.14
0.13
0.39
1.63
0.45
0.27
0.25
0.23
2.79
2.28
1.49
12
0.32
0.30
0.23
0.07
0.37
0.07
0.04
tr.
0.01
tr.
0.16
0.50
1.93
0.63
0.37
tr.
tr.
1.92
1.81
0.33
13
0.15
0.16
0.05
0.02
0.21
0.06
0.03
tr.
tr.
tr.
0.16
0.58
2.34
1.91
0.29
tr.
tr.
1.79
2.26
0.39
14
0.21
0.20
0.06
0.01
0.31
0.07
0.03
tr.
tr.
tr.
0.17
0.58
2.35
3.08
0.45
tr.
0.17
2.80
1.81
0.73
15
0.53
0.54
0.32
0.11
0.29
0.10
0.05
tr.
0.26
0.09
0.11
0.38
1.57
0.65
0.14
0.15
0.72
2.53
2.15
1.04
16
0.26
0.21
0.05
0.03
0.15
0.06
0.03
tr.
0.07
0.04
0.19
0.57
2.39
2.13
0.23
0.04
0.04
1.47
2.02
0.49
17
0.20
0.15
0.07
0.03
0.10
0.05
0.02
tr.
0.09
0.02
0.22
0.62
2.66
2.05
0.36
0.06
1.30
1.77
1.83
0.40
18
0.26
0.27
0.09
0.04
0.18
0.06
0.03
tr.
0.20
0.05
0.15
0.51
2.04
0.95
0.20
0.06
0.17
2.19
2.15
0.76
19
0.42
0.50
0.31
0.13
0.56
0.13
0.04
tr.
0.04
tr.
0.20
0.60
2.48
1.96
0.25
0.01
0.02
1.85
2.40
0.68
20
0.62
0.62
0.19
0.20
0.36
0.13
0.04
tr.
tr.
tr.
0.15
0.49
1.96
2.74
0.26
tr.
0.02
1.52
1.83
0.57
21
0.39
0.43
0.31
0.06
0.71
0.08
0.06
tr.
0.37
0.04
0.08
0.34
1.34
0.07
0.37
tr.
0.17
2.80
1.81
0.73
22
0.11
0.07
0.07
0.01
0.18
0.03
0.05
tr.
0.21
0.02
0.03
0.17
0.37
0.01
0.39
tr.
0.11
1.49
0.80
0.30
23
0.40
0.36
0.24
0.06
0.58
0.08
0.07
tr.
0.50
0.05
0.10
0.35
1.47
0.09
0.33
tr.
0.16
3.07
2.23
1.10
24
0.78
0.88
0.55
0.18
0.94
0.15
0.07
tr.
0.62
0.17
0.15
0.55
2.24
0.53
0.36
0.08
0.43
4.23
3.22
0.80
25
0.79
0.75
0.23
0.15
0.37
0.14
0.04
tr.
0.90
0.20
0.11
0.45
1.86
0.74
0.31
0.16
0.29
4.37
2.73
0.81
26
1.11
1.28
n. d.
n. d.
1.13
0.03
0.06
6
0.03
n. d.
n. d.
tr.
tr.
n. d.
0.26
n. d.
n. d.
tr.
tr.
n. d.
27
1.44
1.67
n. d.
n. d.
1.06
0.04
0.06
8
tr.
n. d.
n. d.
tr.
tr.
n. d.
0.27
n. d.
n. d.
tr.
tr.
n. d.
Min
0.11
0.07
n. d.
n. d.
0.10
0.03
0.02
tr.
tr.
n. d.
n. d.
tr.
tr.
n. d.
0.14
n. d.
n. d.
tr.
tr.
n. d.
Max
1.44
1.67
0.56
0.23
0.94
0.16
0.07
8
1.22
0.25
0.29
0.73
3.10
3.08
0.82
0.78
1.30
4.37
3.22
1.49
Twenty-two components were commonly found in two raw materials of ARR (A. heterotropoides var.
mandshuricum and A. sieboldii var. seoulense ), including 5 lignans, 5 essential
oils, 3 AAs, 4 alkamides, and 5 flavanoids ([Table 3 ]).
Asarinin (19 ) (0.37–3.10 mg/g) and sesamin (17 ) (0.17–0.73 mg/g) were the major lignans in the
samples of two Asarum species, asarinin (19 ) being a marker compound for the quality control
of ARR according to the Chinese Pharmacopoeia. 2-methoxyl-4,5-methylenedioxypropiophenone
(8 ) and
kakuol (10 ) were detected in much greater abundance in the samples of A. heterotropoides var.
mandshuricum [0.49–1.22 mg/g for 2-methoxyl-4,5-methylenedioxypropiophenone (8 ) and
0.11–0.25 mg/g for kakuol (10 )] than in those of A. sieboldii var. seoulense
[trace–0.07 mg/g for 2-methoxyl-4,5-methylenedioxypropiophenone (8 ) and trace–0.01 mg/g for kakuol
(10 )], suggesting that both characteristic lignans might contribute to chemically distinguish
the
raw materials of the two Asarum species. Actually, the commercial samples of ARR mostly consist of
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense due to
their mixed plantation in most herbal farms. According to our results, there is not
a relationship between
the proportion of the mixed cultivation of different subspecies and the lignan contents.
However, the
difference in the lignan contents between Asarum heterotropoides var. mandshuricum and
A. sieboldii var. seoulense is significant. For example, kakuol (10 ) and
2-methoxyl-4,5-methylenedioxypropiophenone (8 ) were detected in much greater abundance in the samples
of A. heterotropoides var. mandshuricum than in those of A. sieboldii var.
seoulense , which may serve for the chemical characterization of both Asarum herbal
materials. Since the pharmacological studies reported the in vitro and in vivo antifungal
activity of both lignans [34 ], they should be the candidates for
standardization of herbal materials of ARR, and the mixed cultivation of the two Asarum plants might
not be desirable for the quality control of raw materials of ARR. Zhang et al. [18 ] reported the determination of three major lignans, asarinin (19 ), sesamin (17 )
and kakuol (10 ), in the roots and rhizomes of ARR by HPLC method, in which the used samples were
not
authenticated due to their commercial origins. The plant materials can be easily authenticated
on the basis
of the significant differences in the morphological characteristics of flowers, as
described in Materials
and Methods.
The chemical profiling of the essential oils of ARR was well studied using the GC-MS
method [9 ]. In the present study, only 3 essential oils, methyleugenol (12 ),
safrole (15 ), and myristicin (16 ) were detected and quantified by the UHPLC-UV or
UHPLC-QTOF/MS methods. Safrole (15 ), possessing liver carcinogenic effects, was quantified by the
UHPLC-UV method because it did not produce mass signal response due to its volatility.
Methyleugenol
(12 ), with central inhibition, antibechic and analgetic activity [35 ], [36 ], is a marker compound for the quality control of essential
oils of the raw materials of ARR and the granules of ARR extract.
The limit test for AAs in ARR has been recorded in the Chinese and Japanese Pharmacopoeias
[3 ], [21 ]. Three AAs in ARR were simultaneously
quantified for the first time. AA-I (14 ) was found in trace amounts (less than 10 ppm) in all the
samples of A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense ,
but with a content of 6–8 ppm in those of A. himalaicum . AA-IV a (6 ) and AL-I (13 ) were
detected in A. heterotropoides var. mandshuricum [0.07–0.16 mg/g for AA-IV a (6 ) and
0.03–0.07 mg/g for AL-I (13 )] and A. sieboldii var. seoulense [0.03–0.04 mg/g for
AA-IV a (6 ) and 0.03–0.04 mg/g for AL-I (13 )]. Many papers reported the quantification of AAs
in ARR. Sun et al. [37 ] reported that there is no or just a trace amount of
AA-I (14 ) in the underground parts (namely roots and rhizomes) of A. heterotropoides var.
mandshuricum and A. sieboldii var. seoulense in a small amount of AA-I (14 )
in their aerial parts. The results from the study of Chen et al. [38 ] showed
that AA-IV a was detected in greater abundance than AA-I (14 ) in the A. heterotropoides var.
mandshuricum roots and rhizomes (26.49–51.73 µg/g). According to our study, AA-IV a (6 ) and
AL-I (13 ) were found in greater amount in A. heterotropoides var. mandshuricum than in
A. sieboldii var. seoulense . Since AA-IV a (6 ) and AL-I (13 ) are the
potential compounds for side effects of AAN, the planting of A. sieboldii var. seoulense
should be preferable for GAP of ARR.
Four alkamides were simultaneously quantified or semi-quantified. Among them,
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide (20 ) and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ) were both major
alkamides found in the samples of A. heterotropoides var. mandshuricum and A. sieboldii
var. seoulense [1.32–4.37 mg/g for
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide (20 ) and 0.80–3.22 mg/g
for N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 )]. Some
pharmacological studies indicated that both alkamides showed antioxidant, immunostimulatory,
and
anti-inflammatory effects [39 ], [40 ]. So they
should not be ignored in quality standardization of ARR.
Moreover, 5 flavonoids, (2R )-naringenin 5,7-di-O -β -D-glucoside (1 ),
(2S )-naringenin 5,7-di-O -β -D-glucoside (2 ),
1-O -p -coumaroyl-xylopyranosyl-glucoside (3 ),
1-O -feruloyl-xylopyranosyl-glucoside (4 ), and the isomer of (2R )-naringenin
5,7-di-O -β -D-glucoside (5 ), were quantified and semi-quantified as minor components
in the samples of A. heterotropoides var. mandshuricum and A. sieboldii var.
seoulense as well the content of all of which were less than 1.0 mg/g.
The root of A. himalaicum is one of the adulterants of ARR in China. There is a significant
difference in the chemical composition between the raw materials of A. heterotropoides var.
mandshuricum and/or A. sieboldii var. seoulense , and those of A. himalaicum .
Twelve components were detected in the roots of A. himalaicum , including 3 flavanoids,
(2R )-naringenin 5,7-di-O -β -D-glucoside (1 ), (2S )-naringenin
5,7-di-O -β -D-glucoside (2 ), and the isomer of (2R )-naringenin
5,7-di-O -β -D-glucoside (5 ), 3 AAs, AA-IV a (6 ), AL-I (13 ), and AA-I
(14 ), 3 lignans, 2-methoxyl-4,5-methylene-dioxypropiophenone (8 ), sesamin (17 ), and
asarinin (19 ), 1 essential oil, safrole (15 ), and 2 alkamides,
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide (20 ) and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ). Among them,
the 3 flavanoids were the major components of A. himalaicum . It is worth noting that AA-I (14 )
was detected in higher content in A. himalaicum than in the samples of two certified Asarum
species in spite of AA-IV a (6 ) and AL-I (13 ) being found in similar amounts in the samples of
three Asarum species. The results suggested that A. himalaicum should not be used as a
medicinal substitute of ARR and should be prohibited in herbal markets in China.
Several studies on HPLC determination of 1–3 marker compounds of ARR have been reported
[17 ], [18 ], [19 ], [37 ], [38 ], in which asarinin (19 ), sesamin
(17 ), kakuol (10 ), methyleugenol (12 ), safrole (15 ), AA-I (14 ), and
AA-IV a (6 ) were involved. The contents of these marker compounds determined by UHPLC-QTOF MS
in the
present study were similar to those shown in the mentioned above literatures. We established
a UHPLC-QTOF/MS
profiling and quantification of 5 lignans, 3 AAs, 3 essential oils, 4 alkamides, and
5 flavanoids in 25 min,
which may serve for the chemical characterization of the raw materials of the three
Asarum plants.
Moreover, the established profiles may be more practical for the standardization of
the roots and rhizomes
of A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense than the
HPLC methods. However, it was difficult to distinguish the mixed commercial samples
of ARR using the present
chemical profiling.
To further visualize the difference between the UHPLC-QTOF/MS profiles obtained from
the samples of
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense ,
unsupervised PCA and supervised OPLS-DA were performed to process data and figure
out important components
for their difference.
The score plot obtained by all observations using 6997 Pareto-scaled variables from
the two species is
displayed in [Fig. 3 A ]. A clear separation can be seen between
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense . 57.9 %
of the variables can be explained by two indices, which were calculated by cross validation.
The results
indicated a significant discrimination between the raw materials of two Asarum species in the
profiles.
Fig. 3 PCA scores plot (A ), OPLS-DA Plot (B ), and S-plot (C ) of four
A. heterotropoides var. mandshuricum and six A. sieboldii var. seoulense
samples. Circles and open triangles in (A ) and (B ) represent A. heterotropoides var.
mandshuricum and A. sieboldii var. seoulense , respectively. The points
a ~g in the S-plot (C ) represent the 7 seven leading markers.
To further find the potential chemical markers for the discrimination between two
Asarum samples, 21
marker components (except safrole (15 ), without MS determination) were screened to perform the
OPLS-DA and generate score plot and S-plot ([Fig. 3 B ] and [C ]). In the present study, R2Y (total explained variation for the X matrix) and
Q2 (the predictability of the model) were 0.9909 and 0.9028, respectively, indicating
excellent prediction
ability of the model. Thus, the established OPLS-DA model had a good capability to
distinguish
A. heterotropoides var. mandshuricum from A. sieboldii var. seoulense . In the
S-plot, each point represents an ion tR -m/z pair. The tR -m/z pair points at the two
ends of “S” represent characteristic markers with the most confidence to each group.
The VIP (variable
importance in the projection) value ensured the significance of potential markers.
Five ions, a
(tR 17.29 min, m/z 222.1856, VIP 1.67), b (tR 8.24 min, m/z 195.0660, VIP 1.63), c
(tR 7.48 min, m/z 209.0812, VIP 1.63), d (tR 6.53 min, m/z 183.1022,
VIP 1.51), and e (tR 20.48 min, m/z 248.2015, VIP 1.36), at the bottom left corner, and
two ions, f (tR 18.08 min, m/z 353.1180, VIP 1.52) and g (tR 16.97 min,
m/z 353.1184, VIP 1.51), at the top right corner of “S”, are the marker compounds of
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense ,
respectively, which contribute most to the difference between the raw materials of
the two Asarum
species.
The 7 leading markers between the raw materials of A. heterotropoides var. mandshuricum and/or
A. sieboldii var. seoulense were structurally identified as
N -isobutyl-(2E ,4E ,8Z )–decatrienamide (18 ) (a), kakuol (10 ) (b),
2-methoxyl-4,5-methylene-dioxypropiophenone (8 ) (c), 3,4,5-trimethoxytoluene (7 ) (d),
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ) (e), asarinin
(19 ) (f), and sesamin (17 ) (g), respectively. The results of multivariate statistical
analysis supported the quantification results in our study.
In conclusion, the present study is the first report on a UHPLC-QTOF/MS method for
the rapid structural
elucidation of 5 lignans, 5 essential oils, 3 AAs, 4 alkamides, and 5 flavanoids from
the roots and rhizomes
of A. heterotropoides var. mandshuricum, A. sieboldii var. seoulense and the roots of
A. himalaicum . Moreover, the method was validated and applied for simultaneous quantification and
semi-quantification of 20 compounds among them with good accuracy and precision. The
difference in the
chemical profiles among the samples of three Asarum species is helpful for the standardization and
quality control of plant materials of ARR. Furthermore, the determined markers are
representative according
to multivariate statistical analysis.
Materials and Methods
Chemicals and reagents
Eighteen reference compounds were used in the present study (Fig. 1S , Supporting Information).
Methyleugenol (12 ), AL-I (13 ), and AA-I (14 ) were purchased from National Institutes
for Food and Drug Control (China), and AA-IV a (6 ), kakuol (10 ), safrole (15 ),
sesamin (17 ) and asarinin (19 ) were from Shanghai Forever Biotech Co., Ltd. Other reference
compounds used were isolated from the extracts of the roots and rhizomes of A. heterotropoides
var. mandshuricum and A. sieboldii var. seoulense in our previous studies [41 ], [42 ]. They included (2R )-naringenin
5,7-di-O -β -D-glucopyranoside (1 ), (2S )-naringenin
5,7-di-O -β -D-glucopyranoside (2 ),
1-O -p -coumaroyl-β -D -xylopyranosyl-(1 → 6)-β -D -glucopyraboside
(3 ),
1-O -feruloyl-β -D -xylopyranosyl-(1 → 6)-β -D -glucopyraboside (4 ),
2-methoxyl-4,5-methylene-dioxypropiophenone (8 ), pluviatilol (11 ), and myristicin
(16 ). The identity of these compounds was confirmed by melting point, UV, IR, 1 H-
and 13 C-NMR, and MS, and their purities evaluated with HPLC-DAD were more than 98 %. Two
standards, N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide (20 ) and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ), were
isolated from Echinacea purpurea in our previous study [43 ], and
their structures were confirmed by melting point, UV, IR, 1 H- and 13 C-NMR, MS and
compared with the literature. The standard of an isomer of N -isobutyl-2,4,8,10-dodecatetraenamide
(22 ) was isolated from the extracts of the roots and rhizomes of A. heterotropoides var.
mandshuricum and A. sieboldii var. seoulense , and its structure was tentatively
identified by the UV spectra, MS/MS determination along with Waters MassFragment software,
and comparison
with literature [10 ]. However, the configurations of C2 ,
C4 , C8 , and C10 could not be determined by MS/MS. The purities of the
three compounds evaluated with HPLC-DAD were more than 98 %. ACN (HPLC-MS grade) and
formic acid
(spectroscopy grade) were purchased from Fisher Scientific UK. Ultrapure water (18.2 MΩ ) was daily
prepared with a Milli-Q water purification system (Millipore). Leucine-enkephalin
was obtained from
Sigma–Aldrich.
Plant materials
Twenty-seven samples (samples #1 ~ #27) were directly obtained from China (Table 1S , Supporting
Information). The plant materials were authenticated by Prof. Weining Wang (Liaoning
Institute for Food
and Drug Control, China). They were identified as A. heterotropoides var. mandshuricum or
A. sieboldii var. seoulense according to the shape of the perianth lobes and leaves
[44 ]. The perianth lobe of A. heterotropoides var.
mandshuricum is erect with acuminate leaf blade apex, while that of A. sieboldii var.
seoulense curls with acute leaf blade apex. We could only authenticate some samples according
to
the phytomorphology of the flowers in the fresh materials, while others from herbal
markets were recorded
as “Liaoxixin” according to their commercial names and the general appearance identification.
The voucher
specimens are kept in the reference library for the medicinal herbs in Shenyang Pharmaceutical
University. For voucher specimens numbers and collection details, see Table 1S Supporting
Information.
Instrumentation and chromatographic conditions
UHPLC analysis was performed on a Waters Acquity HSSC18 column (2.1 × 100 mm, 1.8 µm, Waters)
at 40 °C. The mobile phase consisted of (A) water containing 0.2 % formic acid and
(B) acetonitrile
containing 0.2 % formic acid with gradient elution (linear gradient 9 % B in 2.5 min,
linear gradient 9 %
B to 31 % B between 2.5 and 4 min, linear gradient 31 % B between 4 and 15 min, linear
gradient 31 % B to
40 % B between 15 and 16 min, linear gradient 40 % B to 44 % B between 16 and 17.5 min,
linear gradient
40 % B to 44 % B between 16 and 17.5 min, finally linear gradient 44 % B between 17.5
and 21.5 min).
Re-equilibration duration (linear gradient 44 % to 99 % B between 21.5 min and 22 min,
linear gradient
99 % B between 22 and 23 min, linear gradient 99 % B to 9 % B between 23 and 23.1 min,
finally 9 % B
between 23.1 and 25 min) was 2.5 min between individual runs. The flow rate was kept
at 0.6 mL/min, and
2 µL of standard and sample solution were injected in each run.
Identification of marker compounds by UHPLC-QTOF/MS was performed on Waters QTOF Xevo
G2 equipped with an
ESI source, which gives a resolution of 10 000 (FWHM) and mass accuracy error less
than 5 ppm.
Leucine-enkephalin was used as the lock mass to generate an [M + H]+ ion (m/z 556.2771)
in the LockSpray mode at a concentration of 50 pg/µL at an infusion flow rate of 10 µL/min.
The ESI
source was operated in positive ionization mode with the capillary voltage at 3.0 kV,
and the cone
voltage was set to 25 V. Source and desolvation temperatures were set at 130 and 450 °C,
respectively.
The nebulization gas flows were 800 L/h. All data collected in centroid mode were
acquired using
Masslynx™ NT 4.1 software (Waters Corp.).
Two different MS scanning experiments were used. (1) MS
E
experiment (E
represents collision energy) uses an intelligent approach where parallel alternating
scans are acquired
both at low-collision and high-collision energy to obtain precursor ion information
and full-scan mass
fragment with precursor ion information in a single analytical run, respectively.
The
MS
E
experiment in two scan functions was carried out as follows. Function 1:
m /z 100–1200, 0.2 s scan time, 6 V collision energy; and function 2: m /z
100–1200, 0.2 s scan time, collision energy ramp of 20–30 V. (2) MS/MS experiments
were carried out by
ramping collision energies from 20 and 30 V.
Preparation of standard solutions
Seventeen reference compounds, including (2R )-naringenin
5,7-di-O -β -D-glucopyranoside (1 ), (2S )-naringenin
5,7-di-O -β -D-glucopyranoside (2 ),
1-O -p -coumaroyl-β -D -xylopyranosyl-(1 → 6)-β -D -glucopyraboside
(3 ),
1-O -feruloyl-β -D -xylopyranosyl-(1 → 6)-β -D -glucopyraboside (4 ),
AA-IV a (6 ) 2-methoxyl-4,5-methylene-dioxypropiophenone (8 ), kakuol (10 ),
pluviatilol (11 ), methyleugenol (12 ), AL-I (13 ), AA-I (14 ), safrole
(15 ), myristicin (16 ), sesamin (17 ), asarinin (19 ),
N -isobutyl-2E ,4E ,8Z ,10Z -dodecatetraenamide (20 ), and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ), as well as
one tentatively identified compound, the isomer of N -isobutyl-2,4,8,10-dodecatetraenamide
(22 ), were accurately weighed and dissolved in methanol to give individual stock solutions
at
suitable concentration. Series of working standard solutions were prepared by appropriate
dilution of the
stock solution with methanol in order to prepare calibrators. All solutions were stored
at 4 °C in
refrigerator before analysis.
Sample preparation
Powdered herbal materials (0.5 g, passed through a 500 µm mesh sieve) were extracted
by reflux with 50 mL
of methanol for 30 min. The mixtures were centrifuged at 3000 rpm for 5 min, the supernatants
were
evaporated to dry under vacuum at 35 °C, and the residues were dissolved with methanol,
transformed to a
5 mL of volumetric flask and diluted with methanol to volume. An aliquot of each 2 µL
filtrate filtered
through a 0.22 µm PTFE syringe filter (Whatman, MN, Nal-gene, Advantec) was injected
into the UHPLC
instrument for analysis.
Method validation for quantification
Among 22 identified compounds, 18 compounds were quantified on an UHPLC-UV or UHPLC-QTOF/MS.
Safrole
(16 ) was determined at the detective wavelength of 287 nm by UHPLC-UV method due to no
response
in the MS chromatogram. Twelve compounds (3, 4, 6, 8, 10 –15, 17 , and 19 ) were
quantified using quasi-molecular ion chromatograms (XICs, with a 0.02 Da window),
all peak areas of which
were integrated at the expected retention times under full-scan MS conditions (Waters
QuanLynx™ version
4.0 software). Five compounds (1, 2 , and 20 –22 ) were quantified using
quasi-molecular ion chromatograms, all peak areas of which were integrated at the
expected retention
times under MS/MS conditions at m/z 435.1291 (1 and 2 ) and m/z 167.1310
(20 –22 ). Due to lack of standards to complete the validation procedure, the calibration
curves for peaks 5 and 18 were not established. Their contents were calculated by the
method of semi-quantification with the calibrations of peaks 1 and 22 , respectively,
because peak 5 showed the same aglycone ion at m/z 435.1291 as peak 1 , and peak
22 showed the same fragment ion at m/z 167.1310 as peak 18 . A similar assumption
was made by Liu et al. [45 ].
Calibration curves
Calibration curves (5-point) were obtained using external standard calibrations for
18 analytes injecting
each solution in triplicates and then constructed by plotting the peak area versus
the concentration of
each analyte.
Limit of detection and of quantitation
The stock solutions of 18 reference compounds were diluted to a range from 0.03 to
24.9 × 10−3 µg/mL, and the injection volume was 2 µL. LOD and LOQ were determined at a
signal-to-noise ratio (S/N) of about 3 and 10, respectively.
Precision, accuracy, repeatability, stability, and recovery
The intra- and inter-day precisions were evaluated by analyzing known concentrations
of the 7 analytes,
including
1-O -p -coumaroyl-β -D -xylopyranosyl-(1 → 6)-β -D -glucopyraboside
(3 ), 2-methoxyl-4,5-methylene-dioxypropiophenone (8 ), methyleugenol (12 ), AL-I
(13 ), safrole (16 ), asarinin (19 ), and
N -isobutyl-2E ,4E ,8Z ,10E -dodecatetraenamide (21 ), in six
replicates during a single day and by duplicating the experiments on 3 successive
days. Six different
sample solutions prepared from the same sample were analyzed to confirm the repeatability
of the
developed assay. Stability of sample solutions was analyzed at 0, 2, 4, 8, 12 and
24 h at room
temperature, respectively. Variations were expressed by RSD.
The recovery was used to evaluate the accuracy of the method. A known amount of the
7 standards mixed
solutions were added into a certain amount of the samples of “Liaoxixin” (A. heterotropoides var.
mandshuricum and A. sieboldii var. seoulense ) (0.25 g, sample #17). The mixture
was extracted and analyzed using the method mentioned above. Three replicates were
performed for the
test.
Chemometric data analysis
The UHPLC-MS data of A. heterotropoides var. mandshuricum and A. sieboldii var.
seoulense samples were analyzed by MarkerLynx XS software (Waters). The parameters were set
as
following: retention time range 1.5–22.0 min; mass range m/z 100–600 Da; retention time tolerance
0.1 min; mass tolerance 0.05 Da; width of an average peak at 5 % height and peak-to-peak
baseline noise
were automatically calculated; marker intensity threshold 10.0; noise elimination
level 6.0; isotopic
peaks were excluded for analysis.
Supporting information
The chemical structures of the analysed compounds, collection data of the herbal material
samples used in
the study, as well as intra-day, inter-day precision and recovery of the seven major
marker compounds are
available as Supporting Information.
