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Article history:
Received 26 September 2013
Received in revised form 14 January 2014
Accepted 21 January 2014
Available online 30 January 2014
Keywords:
Chemical proling
Cinnamoyl amides
Furofuran lignans
UPLC-DAD-ESI-QTOFMS/MS
Zanthoxylum armatum
a b s t r a c t
A rapid and simple ultra performance liquid chromatography-diode array detection (UPLC-DAD) method
has been developed for the simultaneous quantication of four biologically important furofuran lignans,
asarinin, sesamin, fargesin and kobusin, and an amide, armatamide in Zanthoxylum armatum within
7 min. The separation was carried out on a BEH C18 column (2.1 mm 100 mm, 1.7 m particle size)
with 0.05% formic acid aqueous solution and acetonitrile as mobile phase under gradient conditions at
25 C. The method was validated and found to be linear (R2 0.9997), precise in terms of peak areas
(intra-day RSDs 0.62% and inter-day RSDs 2.95%) and accurate (95.6104.0%). The developed method
was applied to the quality assessment of different parts (leaves, bark and seeds) of Z. armatum including
locational variation of leaves samples. Signicant variation in the amount of amides and furofuran lignans
was observed. Tandem electrospray ionizationmass spectrometry (UPLC-DAD-ESIMS/MS) of samples
led to the identication of sixteen compounds in the category of amides and furofuran lignans.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Zanthoxylum armatum DC [syn. Zanthoxylum alatum Roxb], family Rutaceae, is commonly known as Indian prickly ash, Nepal
pepper or toothache tree. It is found throughout China, Taiwan,
Nepal, Malaysia, Pakistan and Japan at altitudes of 13001500 m.
In India, it is mainly distributed in tropical and sub-tropical parts
and extensively used in the Indian system of medicines as a carminative, stomachic and anthelmintic. The bark is pungent and sticks
prepared from it are used for preventing toothache. The fruits
and seeds are employed as an aromatic tonic in fever, dyspepsia and expelling roundworms [1]. Its seeds and leaves are also
known to possess antiinammatory, insecticidal, anti-fungal and
anti-microbial activities [2]. Several types of secondary metabolites including alkaloids, amides, lignans, avonoids, coumarins,
terpenoids, etc. have been isolated from various parts of this plant
[2].
The pharmacological activities of Z. armatum are mainly
attributed to the presence of furofuran lignans and amides. Guo
et al. have reported the antinociceptive and antiinammatory
activities of ethyl acetate fraction of ethanol extract and identied eight furofuran lignans as the major constituents of that
fraction using HPLCMS studies [3]. Furofuran lignans are one of
24
i.e. n-hexane (12.5 g), chloroform (34.3 g), ethyl acetate (12.1 g),
n-butanol (32.4 g) and aqueous fraction (38.1 g). Chloroform fraction (25.0 g) was subjected to column chromatography over
silica-gel (60120 mesh) and eluted with 10%, 20%, 30%, 50%, 75%
and 100% ethyl acetate in n-hexane. Repeated column chromatography of combined fractions eluted in 20% ethyl acetate in n-hexane
led to the isolation of asarinin (16, 234 mg) and sesamin (15,
460 mg). Column chromatography of fractions (30% ethyl acetate
in n-hexane) resulted in the isolation of fargesin (14, 547 mg) and
kobusin (13, 650 mg). Repeated column chromatography of fractions obtained in 50% ethyl acetate/n-hexane led to the isolation of
armatamide (7, 480 mg).
2.4. Sample preparation
Leaves, bark and seeds of Z. armatum were ground into ne
powder. The powdered sample (100 mg) was extracted twice with
methanol:ethyl acetate (1:1, v/v, 10 mL) in an ultrasonic water
bath at 50 C for 15 min. The resultant solution was ltered and
the solvent was removed under reduced pressure. The extract thus
obtained was dissolved in 8 mL of LC grade acetonitrile and ltered.
1 L of the sample was injected into UPLC system for analysis.
2.5. Standard solutions
Accurately weighed ve compounds 7, 1316 were dissolved
in acetonitrile to prepare stock solutions. The concentrations of
stock solutions were 250 g/mL for 7 and 500 g/mL for 1316
each. 1 mL of each stock solution was placed in a 5 mL volumetric
ask to make standard mixture at the concentration of 50 g/mL
for 7 and 100 g/mL for 1316 each. The solution was then diluted
stepwise with acetonitrile to give seven different concentrations in
the range of 0.850 g/mL for 7 and 1.6100 g/mL for 1316 each,
for construction of calibration curves.
2.6. UPLC conditions
The UPLC system consisted of ACQUITY Ultra High Performance
LC system (Waters, Milford, MA, USA) and software MassLynx v4.1.
Table 1
Regression equations, linear ranges, LOD and LOQ of ve investigated compounds and recovery study on ZAB-1 (n = 3).
Regression equationa n = 3
R2
Linear range
(g/mL)
LODb (g/mL)
LOQc (g/mL)
Recovery
Original (g)
Spiked (g)
Detected
(g)
Average
recovery
(%)e
RSD (%)
Armatamide
(7)
0.9998
0.850
0.05
0.16
0.16
2.58
151.6
85.6
153.5
195.4
240.2
306.4
345.7
103.5
100.8
99.3
2.2
1.9
2.0
Kobusin
(13)
0.9998
1.6100
0.19
0.65
0.46
2.24
241.9
116.2
205.6
350.6
354.0
440.2
583.4
96.4
96.4
97.4
3.5
2.5
1.7
Fargesin
(14)
0.9997
1.6100
0.19
0.65
0.44
2.95
207.6
100.8
180.7
250.2
312.5
384.9
463.1
104.0
98.1
102.1
4.1
2.8
1.0
Sesamin
(15)
0.9998
1.6100
0.10
0.33
0.52
2.60
222.9
102.8
180.6
280.5
328.2
398.0
501.3
102.4
96.9
99.2
2.3
1.6
3.1
Asarinin
(16)
0.9998
1.6100
0.19
0.65
0.62
2.91
82.3
50.4
88.3
150.5
130.5
171.2
235.7
95.6
100.6
101.9
2.5
1.9
1.6
a
b
c
d
e
The regression equations were presented as y = mx + c. y and x were dened as peak area and concentration of compound, respectively.
LOD, limit of detection, S/N = 3.
LOQ, limit of quantication, S/N = 10.
Intra and inter-day precision was determined on the basis of peak area. RSD (%) = (SD/mean) 100.
Average recovery (%) = (detected amount original amount)/spiked amount 100.
Analyte
25
26
Fig. 2. ESIMS/MS spectra and mass fragmentation of (a) armatamide 7, (b) hydroxy--sanshool 9 and (c) kobusin 13.
27
Fig. 3. UPLC-DAD chromatograms (left) and TIC (right) of (a) standard mixture, (b) bark sample ZAB-1, (c) leaves sample ZAL-4 and (d) seeds sample ZAS-1 used for quantitative
and qualitative analysis.
28
Table 2
Retention time, UV spectral data, mass fragmentation of compounds identied in Z. armatum by UPLC-DAD-ESIMS/MS.
Peak no.
tR (min)
[M+H]+
Error (ppm)
MS2
Identication
Sample
1
2
3
2.16
2.49
2.64
372.1834
356.1475
342.1722
6.17
6.45
4.96
ZAB-1
ZAB-1
ZAB-1
2.81
233, 278
387.1831
5.94
Rubemamin [1820]
Zanthosin [1820]
N-(4 -methoxy-phenethyl)3,4-dimethoxy-cinnamamide
[18]
Eudesmin [17]
2.96
231, 277
417.1904
2.15
6
7
8
9
10
11
12
13
2.98
3.04
3.06
3.08
3.22
3.44
3.45
3.61
264.1933
326.1378
357.1349
264.1926
264.1940
197.0829
340.1164
371.1475
11.70
-4.29
3.08
14.3
9.08
7.61
6.17
5.38
Isomer of hydroxy-sanshool
Armatamide [13,18]
Horseldin [3,17]
Hydroxy--sanshool
Isomer of hydroxy-sanshool
Xanthoxylin
Dioxamin [1820]
Kobusin [3,17]
ZAS-1
ZAB-1
ZAL-1 to ZAL-5
ZAS-1
ZAS-1
ZAL-4
ZAB-1
ZAB-1, ZAL-1 to ZAL-5
14
4.17
233, 283
371.1479
4.31
Fargesin [3,17]
15
4.67
237, 285
355.1159
6.47
Sesamin [3,17]
16
5.32
238, 285
355.1160
6.19
Asarinin [3,17]
Table 3
Comparison of the amount of investigated compounds in different plant parts (bark, leaves and seeds) and leaves samples from different locations (n = 3).
Sample
ZAB-1
ZAS-1
ZAL-1
ZAL-2
ZAL-3
ZAL-4
ZAL-5
1516 29
2419
761
752
1328
1123
1106
2076
1938
2404
2531
2773
2456
2229
481
619
466
477
550
34
8
8
21
16
22
24
38
41
25
36
37
41
11
18
7
10
19
541
703
593
376
750
29
13
21
6
4
14
3721
4478
4918
4749
4862
part], 147, 139 and 107 (Fig. 2 and Table 2). The structure of this
compound was conrmed as hydroxyl--sanshool by comparison
with isolated reference compound. Two other compounds 6 and
10 having similar UV max and mass spectra were observed and
identied as isomers of hydroxy-sanshool.
3.5. Quantitative analysis of different medicinal parts and
locational variation amongst leaves samples
The developed method was applied for the simultaneous quantication of four furofuran lignans and an amide in different plant
parts, viz. bark, leaves and seeds collected from the same shrub.
Typical chromatograms for standard and samples of different plant
parts are shown in Fig. 3. Contents of these compounds are summarized in Table 3. While all the compounds (7, 1316) were detected
in highest amount in bark, none of these was present in seed
sample. In view of this observation, and previous reports on the
antiinammatory and analgesic activities of the investigated lignans [6], the use of Z. armatum bark in Indian traditional medicinal
system for the treatment of severe toothache may be warranted.
In seed sample, only sanshool compounds were detected as major
constituents, which are previously reported to have antipyretic and
analgesic activities [10], indicating that sanshool compounds may
be responsible for biological activities of Z. armatum seeds. Compound 7 was not detected in any of the leaves samples. Among
four lignans detected in leaves, compound 14 was signicantly
higher than other ones, whereas in bark sample compound 13 was
detected in highest amount. The total content of all investigated
compounds in bark was more than twice as compared to leaves.
The comparison of different leaves samples showed the absence of
compound 7 in all leaves samples. Amongst the investigated compounds, compound 14 was found in highest concentration in all
the samples (1938.332773.18 g/g). The results revealed that the
concentration of compound 13 was highest in sample from Sarahan
region (ZAL-3, 1328.66 g/g) and that of compound 14 was highest in Shahpur sample (ZAL-4, 2773.18 g/g), whereas content of
compounds 15 and 16 was moderate in all the samples. Regarding
previous reports on furofuran lignans, Schwertner and Rios developed an HPLC method to study the contents of asarinin and sesamin
in sesame oil samples [21]. The concentrations of sesamin and
asarinin in Orchids and Sigma sesame oil were 0.4% and 0%, and
0.19% and 0.09%, respectively. Fang et al. reported on quantication
of fargesin and kobusin in Magnolia spp. and the contents of these
compounds in various samples were found to be 0.00160.7546%
and 0.00230.1771%, respectively [22]. However, this is the rst
report on the quantitative analysis of these compounds in Z. armatum.
4. Conclusions
A rapid, sensitive and reliable UPLC-DAD/ESIMS/MS method
has been developed for the quality assessment of Z. armatum. The
developed method showed good linearity, precision and accuracy
for the quantication of four lignans and an amide in Z. armatum.
The contents of these compounds varied remarkably in leaves of
Z. armatum collected from different regions. The current method
involved lower ow rate and much shorter analysis time, and
helped in the identication of sixteen compounds in various Z.
armatum samples. The study showed that sanshool amides might
29
be responsible for biological activities of seeds, and furofuran lignans and cinnamoyl amides might be responsible for biological
activities of bark, whereas the biological activities of leaves may
be attributed to furofuran lignans. This study might provide a comprehensive method for the qualitative and quantitative analysis of
Z. armatum.
Acknowledgements
Authors are grateful to CSIR, India for nancial assistance
(NaPAHA, CSC-0130). Mr. V.K. is also thankful to UGC, India for
granting senior research fellowship.
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