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Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

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Journal of Pharmaceutical and Biomedical Analysis


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Short communication

Quantitative and structural analysis of amides and lignans in


Zanthoxylum armatum by UPLC-DAD-ESI-QTOFMS/MS
Vishal Kumar, Shiv Kumar, Bikram Singh, Neeraj Kumar
Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India

a r t i c l e

i n f o

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

IHBT Communication No. 3450.


Corresponding author. Tel.: +91 1894 230426; fax: +91 1894 230433.
E-mail addresses: neeraj@ihbt.res.in, neerajnpp@rediffmail.com (N. Kumar).
0731-7085/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2014.01.028

the largest groups of lignans that are of special interest owing


to their powerful antitumor, antiinammatory, antioxidant and
insecticidal properties, along with phosphodiesterase inhibition
and hypocholesterolemic activities in humans [4,5]. Many in vitro
and animal studies involving two important furofuran lignans,
sesamin and asarinin dietary supplements have been performed
over the past decade [6,7]. Cinnamoyl and long chain isobutylamides isolated from various Zanthoxylum species and other plants
have shown a wide spectrum of biological activities such as antiinammatory, antiplasmodial, antiviral, antibacterial, antiplatelet
aggregation, eukotriene biosynthesis in human polymorphonuclear leukocytes and anticancer activities [8,9]. An isobutylamide,
hydroxy--sanshool, widely present in Zanthoxylum genus is a
well-known analgesic and act as agonist at the pain-integrating
cation channels TRPV1 and TRPA1 [10].
A few analytical methods based on HPLCDAD have been
reported for the analysis of isobutylamides in Zanthoxylum genus
[11,12]. However, no report is available on the quantitative or
qualitative analysis of furofuran lignans and amides in Zanthoxylum. In this context, a UPLC-DAD method has been developed
and validated for the simultaneous quantication of four furofuran lignans, i.e. kobusin 13, fargesin 14, sesamin 15 and asarinin
16, and an amide, armatamide 7 in Z. armatum. The method
was successfully employed to determine the locational variation
of these compounds in the leaves samples collected from various regions of Himachal Pradesh, India. Further, the method was
also applied for the identication of sixteen biologically important amides and furofuran lignans in this plant. To the best of our

24

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

Fig. 1. Chemical structures of identied compounds in Z. armatum.

knowledge, this is the rst report on the simultaneous qualitative


and quantitative analysis of amides and lignans in Z. armatum using
UPLC-DAD/ESIMS/MS.
2. Experimental
2.1. Chemicals and reagents
Standards of armatamide 7, kobusin 13, fargesin 14, sesamin 15
and asarinin 16 were isolated from Z. armatum and identied by
comparison of their spectral data (MS, 1D NMR and 2D NMR) with
that reported in literatures [1316]. Their structures are displayed
in Fig. 1. The purities were above 98% as determined by LC analysis.
All LC grade solvents were purchased from J.T. Baker (Mallinckrodt
Baker Inc., St. Louis, MO, USA). Formic acid was purchased from S.D.
Fine Chemicals Ltd. (Mumbai, India).
2.2. Plant materials
The leaves samples of Z. armatum were collected from different regions of Himachal Pradesh, India in June 2011 and labelled as
ZAL-1 (Palampur, 1200 m), ZAL-2 (Sarkaghat, 910 m), ZAL-3 (Sarahan, 1550 m), ZAL-4 (Shahpur, 680 m) and ZAL-5 (Kullu, 1220 m).
The bark and seeds of Z. armatum were collected from Palampur, India and labelled as ZAB-1 and ZAS-1. The plant materials
were authenticated by Dr. Brij Lal, CSIR-Institute of Himalayan
Bioresource Technology, Palampur, India and a voucher specimen
was deposited in the herbarium (voucher no. PLP 16528) of CSIRInstitute of Himalayan Bioresource Technology, Palampur, India.
2.3. Extraction and isolation of standard compounds
Bark sample was used for the isolation of compounds 7, 1316.
Air dried powder of bark (1.0 kg) of Z. armatum was extracted with
methanol (3 4 L) in a percolator at room temperature. Combined
percolations were dried under reduced pressure to yield 138.2 g of
methanol extract. The crude extract thus obtained was suspended
in water and sequentially fractionated with n-hexane, chloroform, ethyl acetate and n-butanol to get corresponding fractions,

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)

Intra-day RSD Inter-day


RSD (%)d
(%)d n = 6
n=3

Recovery

Original (g)

Spiked (g)

Detected
(g)

Average
recovery
(%)e

RSD (%)

Armatamide
(7)

y = (149.26 1.26)x 11.26 0.37

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)

y = (73.57 1.06)x 16.59 0.24

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)

y = (62.31 0.94)x 14.99 0.41

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)

y = (67.48 0.82)x 7.69 0.21

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)

y = (66.57 0.75)x 13.17 0.18

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.

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

Analyte

25

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V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

Fig. 2. ESIMS/MS spectra and mass fragmentation of (a) armatamide 7, (b) hydroxy--sanshool 9 and (c) kobusin 13.

Chromatographic separations of analytes were carried out on BEH


C18 column (2.1 mm 100 mm, 1.7 m particle size) from Waters
at 25 C. The mobile phase consisted of water (0.05% formic acid)
as solvent A and acetonitrile as solvent B, with a linear gradient
programme as: 0.00.8 min, 60%; 1.53.2 min, 50%; 3.54.5 min,
45%; 5.06.0 min, 60% of A. The ow rate was 0.3 mL/min and the
injection volume was 1 L. Detector wavelength was set at 280 nm.

200 C, desolvation gas 500 L/h, argon collision gas pressure


3.2 103 mbar and ion energy 23.0 eV.

2.7. ESIMS/MS conditions

For the optimization of chromatographic conditions, different


mobile phases such as acetonitrile:water, methanol:water and
water (0.05% formic acid):acetonitrile, column temperatures 25, 30,
35, and 40 C and ow rates were checked. The best separation was
achieved with mobile phase water (0.05% formic acid):acetonitrile
and column temperature 25 C at a ow rate of 0.3 mL/min. These
optimized conditions were further developed for resolution, baseline and analysis time.

MS/MS was performed on a Q-TOF mass spectrometer equipped


with an ESI source (Micromass, Manchester, UK) and software
Masslynx v4.1. The parameters of the mass spectrometer under
the ESI mode were as follows: capillary voltage 3.1 kV, cone
voltage 22 V, source block temperature 80 C, RF lens1 45, aperture 0.5, RF lens2 0.6, cone gas 50 L/h, desolvation temperature

3. Results and discussion


3.1. Optimization of chromatographic conditions

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

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.

3.2. Optimization of extraction method


Ultrasound assisted extraction is a rapid and simple extraction technique which consumes less fossil energy and provides
high yields. Hence, ultrasonication was selected as the technique
of choice for the extraction of active constituents of Z. armatum.
The bark sample, ZAB-1 was used to optimize the best extraction
method. To achieve efcient extraction of active components, various factors such as extraction solvent (methanol, ethanol, ethyl
acetate, methanol/water (1:3, 1:1 and 3:1, v/v) and methanol/ethyl

acetate (1:1, v/v)), ultrasonication time (15, 30, 45 and 60 min)


and temperature (25, 50 and 60 C) were investigated. In order
to nd best extraction method in terms of recovery of analytes,
spiked samples were also analysed under all the test conditions.
The highest extraction and recovery of the analytes (96.4100.8%)
was achieved with methanol/ethyl acetate (1:1, v/v) with ultrasonication at 50 C for 15 min, whereas methanol/water (1:3) provided
lowest recovery of analytes (74.381.7%) under same conditions.
These conditions were applied for the quantication of analytes in
various samples.

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V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

Table 2
Retention time, UV spectral data, mass fragmentation of compounds identied in Z. armatum by UPLC-DAD-ESIMS/MS.
Peak no.

tR (min)

UV, max (nm)

[M+H]+

Error (ppm)

MS2

Identication

Sample

1
2
3

2.16
2.49
2.64

231, 285, 316


234, 281, 320
236, 291, 315

372.1834
356.1475
342.1722

6.17
6.45
4.96

191, 165, 129


175, 165, 145
191, 163, 135

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

Magnolin or epimagnolin [17]

ZAB-1, ZAL-1 to ZAL-5

6
7
8
9
10
11
12
13

2.98
3.04
3.06
3.08
3.22
3.44
3.45
3.61

262, 272, 282


218, 282, 320
237, 281
260, 270, 280
257, 267, 277
286
229, 283, 323
233, 283

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]

ZAB-1, ZAL-1 to ZAL-5

15

4.67

237, 285

355.1159

6.47

Sesamin [3,17]

ZAB-1, ZAL-1 to ZAL-5

16

5.32

238, 285

355.1160

6.19

Asarinin [3,17]

ZAB-1, ZAL-1 to ZAL-5

369 [M+HH2 O], 351 [M+H2H2 O],


201, 151
399 [M+HH2 O], 381 [M+H2H2 O],
329, 151
246, 223, 129, 93
175, 145, 135, 105
339 [M+HH2 O], 321 [M+H2H2 O]
246, 175, 147, 139, 107
246, 175, 139, 107
179, 119
175, 165, 145, 135
353 [M+HH2 O], 335 [M+H2H2 O],
203, 135
353 [M+HH2 O], 335 [M+H2H2 O],
151, 135
337 [M+HH2 O], 319 [M+H2H2 O],
135
337 [M+HH2 O], 319 [M+H2H2 O],
135

3.3. UPLC method validation


The linearity, limit of detection (LOD), limit of quantication
(LOQ), precision and accuracy for the quantication of analytes
were validated. The results are summarized in Table 1. All calibration curves showed good linearity with high correlation coefcient
(R2 0.9997) over the tested range. The LOD and LOQ values for the
analytes were between 0.0480.195 and 0.160.65 g/mL, respectively. The analysis of intra- and inter-day precision was conducted
by six repetitive injections on the same day and consecutive three
days. The RSD values for intra-day precision was 0.62% and that
for inter-day precision was 2.95%. Mean recovery results were
in the range of 95.6104.0% with RSD 1.04.1%, showing that the
developed method was reproducible with all the values within
acceptable range (Table 1).
3.4. Qualitative analysis using UPLC-DAD-ESIMS/MS
The identication and characterization of compounds was done
by comparison of their retention times, UV spectrum and MS/MS
data with isolated reference compounds and information available
in literature. Three cinnamoyl amides, one long chain isobutylamide, one acetophenone derivative and ve lignans (1, 2, 5, 7,
9, 1216) were conclusively characterized by comparison of their
retention times, UV and MS/MS fragmentation pattern with isolated reference compounds. Two cinnamoyl amides, two long chain
amides and two lignans were tentatively identied by comparison
of their UV and MS/MS data with earlier reported MS/MS fragmentation pattern. The chemical structures of the identied compounds
are shown in Fig. 1.

ZAB-1, ZAL-1 to ZAL-5

In cinnamoyl amides, fragment by loss of amine part was


observed as base peak. The mass spectrum of standard cinnamoyl
amide, armatamide 7, showed a molecular ion peak at m/z 326
[M+H]+ and fragment ions at m/z 175 [M+Hamine part], 145
[M+Hamine partOCH2 ], 135 [M+HC10 H8 NO3 ] and 105 (Fig. 2
and Table 2). Similar pattern of UV and mass spectra was observed
for compounds 1, 2, 3 and 12 and were characterized as rubemamin,
zanthosin, N-(4 -methoxyphenethyl)-3,4-dimethoxycinnamamide
and dioxamin, respectively (Table 2). The structures of compounds 1 and 2 were further conrmed by comparison with
isolated reference compounds. Compounds 1, 2 and 12 are rst
time reported from Z. armatum and compound 3 is a new
compound.
Mass fragmentation of furofuran lignans, showed the successive loss of two water molecules as previously reported [17]. Mass
spectrum of Kobusin 13 showed the molecular ion peak at m/z 371
[M+H]+ and characteristic fragment ions at m/z 353 [M+HH2 O],
[M+H2H2 O], 203 and 135 (Fig. 2 and Table 2). Other standard lignans 1416 showed similar UV and mass spectra. Other identied
compounds of this type were 4, 5 and 8, which were characterized
as eudesmin, magnolin and horseldin, respectively. The sample
ZAL-4 showed one extra peak (compound 11) having UVvis max
at 280 nm and molecular ion at m/z 197 [M+H]+ . The structure of
this compound was conrmed as xanthoxylin by comparison with
isolated reference compound.
Seed sample (ZAS-1) showed the presence of long chain
isobutylamides instead of cinnamoyl amides or lignans and three
isobutylamides were detected (6, 9 and 10). In the mass spectrum
of compound 9, the molecular ion peak was observed at m/z 264
[M+H]+ and fragment ions at m/z 246 [M+HH2 O], 175 [M+Hamine

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

Armatamide (7) (g/g)

Kobusin (13) (g/g)

Fargesin (14) (g/g)

Sesamin (15) (g/g)

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

Asarinin (16) (g/g)


823

541
703
593
376
750

29
13
21
6
4
14

Total lignans (g/g)


7547

3721
4478
4918
4749
4862

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 2329

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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