MS

Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 23–29

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Short communication

Quantitative and structural analysis of amides and lignans in Zanthoxylum armatum by UPLC-DAD-ESI-QTOF–MS/MS夽 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 profiling Cinnamoyl amides Furofuran lignans UPLC-DAD-ESI-QTOF–MS/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 quantification 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 (95.6–104.0%). 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. Significant variation in the amount of amides and furofuran lignans was observed. Tandem electrospray ionization–mass spectrometry (UPLC-DAD-ESI–MS/MS) of samples led to the identification 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 1300–1500 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 antiinflammatory, insecticidal, anti-fungal and anti-microbial activities [2]. Several types of secondary metabolites including alkaloids, amides, lignans, flavonoids, 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 antiinflammatory activities of ethyl acetate fraction of ethanol extract and identified eight furofuran lignans as the major constituents of that fraction using HPLC–MS studies [3]. Furofuran lignans are one of

夽 IHBT Communication No. 3450. ∗ Corresponding author. Tel.: +91 1894 230426; fax: +91 1894 230433. E-mail addresses: [email protected], [email protected] (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, antiinflammatory, 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 antiinflammatory, 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 HPLC–DAD 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 quantification 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 identification 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) 23–29

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

knowledge, this is the first report on the simultaneous qualitative and quantitative analysis of amides and lignans in Z. armatum using UPLC-DAD/ESI–MS/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 identified by comparison of their spectral data (MS, 1D NMR and 2D NMR) with that reported in literatures [13–16]. 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, 13–16. 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 (60–120 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 fine 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 filtered and the solvent was removed under reduced pressure. The extract thus obtained was dissolved in 8 mL of LC grade acetonitrile and filtered. 1 ␮L of the sample was injected into UPLC system for analysis. 2.5. Standard solutions Accurately weighed five compounds 7, 13–16 were dissolved in acetonitrile to prepare stock solutions. The concentrations of stock solutions were 250 ␮g/mL for 7 and 500 ␮g/mL for 13–16 each. 1 mL of each stock solution was placed in a 5 mL volumetric flask to make standard mixture at the concentration of 50 ␮g/mL for 7 and 100 ␮g/mL for 13–16 each. The solution was then diluted stepwise with acetonitrile to give seven different concentrations in the range of 0.8–50 ␮g/mL for 7 and 1.6–100 ␮g/mL for 13–16 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 five 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

0.8–50

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

1.6–100

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

1.6–100

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

1.6–100

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

1.6–100

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 defined as peak area and concentration of compound, respectively. LOD, limit of detection, S/N = 3. LOQ, limit of quantification, 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) 23–29

Analyte

25

26

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 23–29

Fig. 2. ESI–MS/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: 0.0–0.8 min, 60%; 1.5–3.2 min, 50%; 3.5–4.5 min, 45%; 5.0–6.0 min, 60% of A. The flow 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 × 10−3 mbar and ion energy 23.0 eV.

2.7. ESI–MS/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 flow rates were checked. The best separation was achieved with mobile phase water (0.05% formic acid):acetonitrile and column temperature 25 ◦ C at a flow 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) 23–29

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 efficient 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 find 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 (96.4–100.8%) 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 (74.3–81.7%) under same conditions. These conditions were applied for the quantification of analytes in various samples.

28

V. Kumar et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 23–29

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

tR (min)

UV, max (nm)

[M+H]+

Error (ppm)

MS2

Identification

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

4

2.81

233, 278

387.1831

5.94

Rubemamin [18–20] Zanthosin [18–20] N-(4 -methoxy-phenethyl)3,4-dimethoxy-cinnamamide [18] Eudesmin [17]

5

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] Horsfieldin [3,17] Hydroxy-␣-sanshool Isomer of hydroxy-sanshool Xanthoxylin Dioxamin [18–20] 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+H–H2 O], 351 [M+H–2H2 O], 201, 151 399 [M+H–H2 O], 381 [M+H–2H2 O], 329, 151 246, 223, 129, 93 175, 145, 135, 105 339 [M+H–H2 O], 321 [M+H–2H2 O] 246, 175, 147, 139, 107 246, 175, 139, 107 179, 119 175, 165, 145, 135 353 [M+H–H2 O], 335 [M+H–2H2 O], 203, 135 353 [M+H–H2 O], 335 [M+H–2H2 O], 151, 135 337 [M+H–H2 O], 319 [M+H–2H2 O], 135 337 [M+H–H2 O], 319 [M+H–2H2 O], 135

3.3. UPLC method validation The linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy for the quantification of analytes were validated. The results are summarized in Table 1. All calibration curves showed good linearity with high correlation coefficient (R2 ≥ 0.9997) over the tested range. The LOD and LOQ values for the analytes were between 0.048–0.195 and 0.16–0.65 ␮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 95.6–104.0% with RSD 1.0–4.1%, showing that the developed method was reproducible with all the values within acceptable range (Table 1). 3.4. Qualitative analysis using UPLC-DAD-ESI–MS/MS The identification 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 five lignans (1, 2, 5, 7, 9, 12–16) 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 identified by comparison of their UV and MS/MS data with earlier reported MS/MS fragmentation pattern. The chemical structures of the identified 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+H–amine part], 145 [M+H–amine part–OCH2 ], 135 [M+H–C10 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 confirmed by comparison with isolated reference compounds. Compounds 1, 2 and 12 are first 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+H–H2 O], [M+H–2H2 O], 203 and 135 (Fig. 2 and Table 2). Other standard lignans 14–16 showed similar UV and mass spectra. Other identified compounds of this type were 4, 5 and 8, which were characterized as eudesmin, magnolin and horsfieldin, respectively. The sample ZAL-4 showed one extra peak (compound 11) having UV–vis max at 280 nm and molecular ion at m/z 197 [M+H]+ . The structure of this compound was confirmed 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+H–H2 O], 175 [M+H–amine

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) 23–29

part], 147, 139 and 107 (Fig. 2 and Table 2). The structure of this compound was confirmed 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 identified 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 quantification 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, 13–16) 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 antiinflammatory 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 significantly 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 (1938.33–2773.18 ␮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 quantification of fargesin and kobusin in Magnolia spp. and the contents of these compounds in various samples were found to be 0.0016–0.7546% and 0.0023–0.1771%, respectively [22]. However, this is the first report on the quantitative analysis of these compounds in Z. armatum. 4. Conclusions A rapid, sensitive and reliable UPLC-DAD/ESI–MS/MS method has been developed for the quality assessment of Z. armatum. The developed method showed good linearity, precision and accuracy for the quantification 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 flow rate and much shorter analysis time, and helped in the identification 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 financial assistance (NaPAHA, CSC-0130). Mr. V.K. is also thankful to UGC, India for granting senior research fellowship. References [1] The Wealth of India: Raw Materials; PID, Council of Scientific and Industrial Research (CSIR), New Delhi 2, 1976, pp. 18. [2] T.P. Singh, O.M. Singh, Phytochemical and pharmacological profile of Zanthoxylum armatum DC. – an overview, Ind. J. Nat. Prod. Res. 2 (2011) 275–285. [3] T. Guo, Y.-X. Deng, H. Xie, C.-Y. Yao, C.-C. Cai, S.-L. Pan, Y.-L. Wang, Antinociceptive and antiinflammatory activities of ethyl acetate fraction from Zanthoxylum armatum in mice, Fitoterapia 82 (2011) 347–351. [4] H.L. Teles, J.P. Hemerly, P.M. Pauletti, J.R.C. Pandolfi, A.R. Araujo, S.R. Valentini, H.C.M. Young, V.D.S. Bolzani, H.S. Dulce, Cytotoxic lignans from the stems of Styrax camporum (Styracaceae), Nat. Prod. Res. 19 (2005) 319–323. [5] B.-S. Min, M.-K. Na, S.-R. Oh, K.-S. Ahn, G.-S. Jeong, G. Li, S.-K. Lee, H. Joung, H.-K. Lee, New furofuran and butyrolactone lignans with antioxidant activity from the stem bark of Styrax japonica, J. Nat. Prod. 67 (2004) 1980–1984. [6] W.D. MacRae, G.H.N. Towers, Biological activities of lignans, Phytochemistry 23 (1984) 1207–1220. [7] P. Budowski, Recent research on sesamin, sesamolin, and related compounds, J. Am. Oil Chem. Soc. 41 (1964) 280–285. [8] Y.C. Wu, G.Y. Chang, F.N. Ko, C.M. Teng, Bioactive constituents from the stems of Annona montana, Planta Med. 61 (1995) 146–149. [9] S.A. Ross, G.N.N. Sultana, C.L. Burandt, M.A. Elsohly, J.P.J. Marais, D. Ferreira, Syncarpamide, a new antiplasmodial (+)-norepinephrine derivative from Zanthoxylum syncarpum, J. Nat. Prod. 67 (2004) 88–90. [10] D.M. Bautista, Y.M. Sigal, A.D. Milstein, J.L. Garrison, J.A. Zorn, P.R. Tsuruda, R.A. Nicoll, D. Julius, Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels, Nat. Neurosci. 11 (2008) 772–779. [11] E. Sugai, Y. Morimitsu, K. Kubota, Quantitative analysis of sanshool compounds in Japanese pepper (Xanthoxylum piperitum DC.) and their pungent characteristics, Biosci. Biotechnol. Biochem. 69 (2005) 1958–1962. [12] S. Wang, J. Xie, W. Yang, B. Sun, Preparative separation and purification of alkylamides from Zanthoxylum bungeanum maxim by high-speed counter-current chromatography, J. Liq. Chromatogr. Relat. Technol. 34 (2011) 2640–2652. [13] N.K. Kalia, B. Singh, R.P. Sood, A new amide from Zanthoxylum armatum, J. Nat. Prod. 62 (1999) 311–312. [14] T. Iida, M. Nakano, K. Ito, Hydroperoxysesquiterpene and lignan constituents of Magnolia kobus, Phytochemistry 21 (1982) 673–675. [15] R.C.D. Brown, C.J.R. Bataille, G. Bruton, J.D. Hinks, N.A. Swain, C H insertion approach to the synthesis of endo,exo-furofuranones: Synthesis of (±)-asarinin, (±)-epimagnolin A, and (±)-fargesin, J. Org. Chem. 66 (2001) 6719–6728. [16] H. Kakisawa, Y.P. Chen, H.Y. Hsu, Lignans in flower buds of Magnolia fargesii, Phytochemistry 11 (1972) 2289–2293. [17] T.J. Schmidt, S. Hemmati, E. Fuss, A.W. Alfermann, A combined HPLC–UV and HPLC–MS method for the identification of lignans and its application to the lignans of Linum usitatissimum L. and L. bienne Mill., Phytochem. Anal. 17 (2006) 299–311. [18] C. Guo, K. Jiang, L. Yue, Z. Xia, X. Wang, Y. Pan, Intriguing roles of reactive intermediates in dissociation chemistry of N-phenylcinnamides, Org. Biomol. Chem. 10 (2012) 7070–7077. [19] S.K. Adesina, Three new amides from Zanthoxylum rubescens, Planta Med. 55 (1989) 324–326. [20] S.K. Adesina, J. Reisch, Amides from Zanthoxylum rubescens, Phytochemistry 28 (1989) 839–842. [21] H.A. Schwertner, D.C. Rios, Analysis of sesamin, asarinin, and sesamolin by HPLC with photodiode and fluorescent detection and by GC/MS: application to sesame oil and serum samples, J. Am. Oil Chem. Soc. 89 (2012) 1943–1950. [22] Z. Fang, C.M. Shen, D.C. Moon, K.H. Son, J.K. Son, M.H. Woo, Quantitative and pattern recognition analyses for the quality evaluation of Magnoliae flos by HPLC, Bull. Korean Chem. Soc. 31 (2010) 3371–3381.