Lipoxygenase inhibitory sphingolipids from Launaea ...

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Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis a

a

a

Naheed Riaz , Shehla Parveen , Muhammad Saleem , b

b

c

Muhammad Shaiq Ali , Abdul Malik , Muhammad Ashraf , c

Iftikhar Afzal & Abdul Jabbar

a

a

Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100, Bahawalpur, Pakistan b

HEJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi, 75270, Pakistan c

Department of Biochemistry and Biotechnology, Baghdadul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan Available online: 15 May 2012

To cite this article: Naheed Riaz, Shehla Parveen, Muhammad Saleem, Muhammad Shaiq Ali, Abdul Malik, Muhammad Ashraf, Iftikhar Afzal & Abdul Jabbar (2012): Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis , Journal of Asian Natural Products Research, 14:6, 545-554 To link to this article: http://dx.doi.org/10.1080/10286020.2012.680440

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Journal of Asian Natural Products Research Vol. 14, No. 6, June 2012, 545–554

Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis Naheed Riaza, Shehla Parveena, Muhammad Saleema, Muhammad Shaiq Alib, Abdul Malikb, Muhammad Ashrafc, Iftikhar Afzalc and Abdul Jabbara* a


Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan; bHEJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan; c Department of Biochemistry and Biotechnology, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan (Received 26 October 2011; final version received 26 March 2012)

This paper is dedicated to Dr Muhammad Arshad (late), Ex-Director CIDS, The Islamia University of Bahawalpur, whose death (March 31, 2011) is a big loss for the Institute as well as for the University

Four new sphingolipids: nudicaulin A [(2S,3S,4R,14E)-2-{[octadecanoyl]amino} tetraeicos-14-ene-1,3,4-triol; 1], nudicaulin B [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 2], nudicaulin C [(2S,3S,4R, 14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol-1-O-b-D glucopyranoside; 3], and nudicaulin D [(2S,3S,4R)-2-{[(2R,3S,12E)-2,3-dihydroxyeicos-12-enoyl]amino}octadecane-1,3,4-triol; 4] together with 1-hexatriacontanol, b-sitosterol, octadecyl 4-hydroxycinnamate, elaidic acid, cholesta-5,22-diene-3,7-diol, oleanolic acid, apigenin, and b-sitosterol 3-O-b-D -glucopyranoside were isolated from the methanolic extract of the whole plant of Launaea nudicaulis. Their structures were elucidated using 1H and 13C NMR spectra and 2D NMR analyses (HMQC, HMBC, and COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HRFAB-MS) experiments and comparison with literature data of related compounds. Compounds 1 – 4 displayed moderate inhibitory potential against enzyme lipoxygenase in concentration-dependent manner with IC50 value ranges 103– 193 mM. Keywords: Launaea nudicaulis; sphingolipids; secondary metabolite; lipoxygenase inhibition

1.

Introduction

The genus Launaea of Compositae family is known for antitumor, insecticidal, and cytotoxic activities and used in folk medicine against stomach ache and skin diseases [1– 3]. In Pakistan, this genus is represented by 20 species mostly with galactagogue, soporific, diuretic, and aperient properties [4]. Some species are important for the treatment of infected wounds and fever [5,6]. Literature survey reveals the presence of

sesquiterpene lactones, triterpenes, steroids, flavonoids, and coumarins from Launaea species [7–12]. Launaea nudicaulis is an important member of this genus, wildly growing in Cholistan Desert and is known to be effective against itches, ulcers, cuts, swellings, bilious fever, eczema eruptions, cancer, microbial pathogens, insects, and rheumatism [13,14]. The leaves of L. nudicaulis are used as antipyretic in children, whereas its latex is used to relieve

*Corresponding author. Email: [email protected]; [email protected] ISSN 1028-6020 print/ISSN 1477-2213 online q 2012 Taylor & Francis http://dx.doi.org/10.1080/10286020.2012.680440 http://www.tandfonline.com

546

N. Riaz et al. secondary amide (1650 cm21), and olefinic (1625 cm21) groups. The molecular formula C42H83NO4 was deduced by HR-EI-MS showing molecular ion peak at m/z 665.6330 [M]þ. It indicated that the molecule has two double bond equivalences. The 1H NMR spectrum of 1 (Table 1) displayed signal for a secondary amide at d 6.75 (1H, d, J ¼ 8.0 Hz); an oxygenated methylene at d 3.94 (1H, dd, J ¼ 11.5, 3.5 Hz) and 3.57 (1H, dd, J ¼ 11.5, 5.5 Hz); two oxymethines at d 3.47 (1H, dt, J ¼ 5.3, 4.1 Hz) and 3.43 (1H, dd, J ¼ 4.5, 4.1 Hz). A signal at d 3.97 (1H, m) was ascribed for the methine proton vicinal to the nitrogen atom of the amide group. The 1H NMR spectrum also displayed signals for a double bond and two primary methyls. The 13C NMR spectrum of 1 (Table 1) disclosed the signal for an amide carbonyl (d 174.4), a methine carbon (d 51.6), one double bond (d 130.8, 129.0), oxygenated methines and a methylene (d 75.7, 72.4, 60.9), and aliphatic chain (d 32.6–29.2). These data fully supported the 1 H NMR data and declared 1 to be a sphingolipid [15,16]. A methylene triplet at d 2.10 (J ¼ 8.0 Hz) indicated the absence of hydroxyl group at C-20 [15]. The entire


constipation [13]. The diverse biological activities of L. nudicaulis prompted us to carry out phytochemical studies on this plant. Herein, we report the isolation, structure elucidation, and biological studies of four new sphingolipids (1 –4) together with known metabolites: 1-hexatriacontanol, bsitosterol, octadecyl 4-hydroxycinnamate, elaidic acid cholesta-5,22-diene-3,7-diol, oleanolic acid, apigenin, and b-sitosterol 3Ob-D -glucopyranoside from the n-hexanesoluble fraction of methanolic extract of the whole plant material. 2. Results and discussion The methanolic extract of L. nudicaulis was divided into n-hexane, ethyl acetate, and n-butanol fractions. The n-hexane soluble fraction on column chromatography (CC) yielded four new sphingolipids (1 – 4), together with above-mentioned known compounds. Compound 1 was isolated as a colorless amorphous powder. The UV spectrum of 1 indicated the absence of UV-active chromophores, while infrared (IR) spectrum showed the presence of hydroxyl (3640 cm21), Table 1. 1H and 100 MHz). Position 1 2 3 4 5 6-12,17-23, 40 -170 13,16 14,15 24,180 NH 10 20 30

13

C NMR spectral data, HMBC, and COSY correlations of 1 (CDCl3; 400,

dH (J in Hz)

dC

HMBC (H ! C)

COSY (H ! H)

3.94 (1H, dd, 11.5, 3.5) 3.57 (1H, dd, 11.5, 5.5) 3.97 (1H, m,) 3.43 (1H, dd, 4.5, 4.1) 3.47 (1H, dt, 5.3, 4.1) 1.56, 1.32 (1H each, m) 1.15 – 1.30 (56H, br s)

60.9

2,3

H-1/H-2

51.6 75.7 72.4 32.6 29.2– 31.5

1,3,4,10 1,2,4,5 2,3,5,6 3,4,6 5,13,24,180

1.86 – 1.93 (4H, m) 5.31 (2H, dt, 16.5, 5.2) 0.79 (6H, t, 7.0) 6.75 (1H, d, 8.0) – 2.10 (2H, t, 8.0) 1.51 (2H, m)

32.4 129.0, 130.8 13.9 – 174.4 36.4 25.7

H-2/H-1,3,N-H H-3/H-2,4 H-4/H-3,5 H-5/H-4,6 H-6,12/H-5,13; H-17,23/H-16,24; H-40 ,170 /H-30 ,180 H-13,16/H-12,14,15,17 H-14,15/H-13,16 H-24,180 /H-23,170 N-H/H-2 – H-20 /H-30 H-30 /H-40

14,15 13,16 23,170 1,2,3,10 – 10 ,30 20 ,40


Journal of Asian Natural Products Research sequence of the skeleton and the substitutions were fixed by 1H– 1H COSY and long-range HMBC correlations (Table 1). The length of the fatty acid chain was determined by characteristic fragments at m/z 268, 369 and the amine chain containing a double bond at m/z 427 and 341 (Figure 1). Methanolysis [18] of 1 with methanolic HCl provided the aliphatic amine and the methyl ester of fatty acid, which after acetylation [17] were analyzed by GC-MS and were identified as methyl octadecanoate (m/z 298) and 2-acetamino-1,3,4-triacetoxytetraeicosene (m/z 567). The position of double bond was fixed between C-14 and C-15 by permanganate/periodate oxidative cleavage [18] of 2-acetamino-1,3,4-triacetoxytetraecosene, yielding a mixture of carboxylic acids which on methylation and GC-MS analysis provided peaks for 2-acetamino1,3,4-triacetoxytetradecanoic acid (m/z 473) and decanoic acid (m/z 186). The stereochemistry at the three stereogenic centers was determined by optical rotations of 1 ([a ]D ¼ þ16.9) and its methylated amine ([a ]D ¼ þ11.2) which were found similar with those having (2S,3S,4R)-configurations [19–22]. Based on these evidences, 1 could be assigned as (2S,3S,4R,14E)-2-{[octadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol and named nudicaulin A. Compound 2 was obtained as a white amorphous powder. The IR spectrum of 2 was similar to that of 1. The HR-EI-MS

547

showed the molecular ion peak at m/z 681.6280 [M]þ corresponding to the molecular formula C42H83NO5 indicating that 2 could be an oxidative derivative of 1. The 1H and 13C NMR spectra of 2 (Table 2) were almost similar to those of 1 with a minor difference of an additional signal for oxymethine at d 3.94 (1H, dd, J ¼ 6.4, 3.6 Hz) correlated with the carbon at d 71.9. The signal of CH2-20 observed in 1 was missing in the NMR spectra of 2 further supported that C-20 is hydroxylated [16,19 – 22]. The positions of all the substituents were assigned with the help of 1H – 1H COSY and HMBC spectra (Table 2) and mass fragmentation patterns (Figure 2). The length of the fatty acid and the amine chains, and the position of double bond were tentatively fixed by the combination of mass fragmentation pattern (Figure 2) and methanolysis (see experimental) as done for 1. The configuration at the stereogenic centers was deduced by optical rotations of 2 ([a ]D ¼ þ 39.7) and its methanolysis products ([a ]D ¼ 2 7.1 and þ 19.1), which were comparable with those of sphingolipids with a 2S,3S,4R,20 R configurations [20 –22]. Based on these evidences, nudicaulin B (2) could be assigned the structure (2S,3S,4R,14E)-2{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol. Compound 3 with a molecular formula C49H94NO10, isolated as a colorless gummy

441 268

O 282

HN

18'

1'

CH3

2'

OH 2

3

CH3

4 15

1

498

OH 384

24

14

HO

386

552

356 339

Figure 1. Structure and mass fragmentation pattern of 1.

23

548

N. Riaz et al.

Table 2. 1H and 100 MHz).

13


dH (J in Hz)

Position 1

3.71 (1H, dd, 11.6, 4.0) 3.63 (1H, dd, 11.6, 4.8) 3.99 (1H, m) 3.45 (1H, dd, 4.2, 3.9) 3.41 (1H, dt, 6.3, 4.2) 1.59 (2H, m) 1.15 – 1.29 (36H, br s)


2 3 4 5 6 – 12, 17 – 23, 40 -170 13,16 14,15 24,180 NH 10 20 30

1.90 (4H, 5.29 (2H, 0.77 (6H, 7.42 (1H, – 3.94 (1H, 1.52 (2H,

m) dt, 15.3, 6.8) t, 6.4) d, 8.8) dd, 6.4, 3.6) m)

HMBC (H ! C)

dC

COSY (H ! H)

61.0

2,3

H-1/H-2

51.5 75.5 72.1 32.8 28.5 – 31.3

1,3,4,10 1,2,4,5 2,3,5,6 3,4,6 5,13,24,180

32.5 130.8, 129.7 13.9 – 175.7 71.9 32.3

14,15 13,16 23,170 1,2,3,10 ,20 – 10 ,30 ,40 10 ,20 ,40

H-2/H-1,3 H-3/H-2,4 H-4/H-3,5 H-5/H-4,6 H-6,12/H-5,13; H-17,23/ H-16,24; H-40 ,170 / H-30 ,180 H-13,16/H-12,15,17 H-14,15/H-13,16 H-24,180 /H-23,170 N-H/H-2 – H-20 /H-30 H-30 /H-20 ,40

solid, was found to be a spingolipid glycoside based on its NMR data when compared with those of 1 and 2. In addition to the usual signals for a sphingolipid skeleton, the 1H NMR spectrum of 3 (Table 3) displayed signals for a sugar moiety at d 4.15 (1H, d, J ¼ 7.6 Hz, H-100 ), 3.40 (1H, m, H-200 ), 3.28 (1H, t, J ¼ 7.4 Hz, H-300 ), 3.23 (1H, t, J ¼ 7.4 Hz, H-400 ), 3.16 (1H, m, H500 ), 3.79 (1H, dd, J ¼ 10.8, 4.9 Hz, H-600 ), and 3.61 (1H, dd, J ¼ 10.8, 2.9 Hz, H-600 ). The 13C NMR spectrum of 3 (Table 3) also supported the above data for a

glycosphingolid [21,22]. A downfield shift (d 68.5) of C-1 was due to the sugar unit attached to it, which was further confirmed by HMBC correlation of anomeric methine at d 4.15 with C-1 at d 68.5 (Table 3). In addition to the presence of glucose moiety, mass spectrometry (Figure 3), methanolysis, and oxidative cleavage revealed that compound 3 has C17 fatty acid chain and C26 amine chain with a double bond at C-14,15 [20,23]. The stereochemistry at various stereogenic centers was found similar to those for 2. Based on these evidences,

456 283

O OH HN

398

18'

1'

CH3

2'

OH 2

3

CH3

4 15

1 383

24

14

HO

OH

113

279 167 339


23

Journal of Asian Natural Products Research Table 3. 1H and 100 MHz).

13


dH (J in Hz)

Position 1


2 3 4 5 6 – 12, 17 – 25,40 -160 13,16 14,15 26,170 NH 10 20 30 100 200 300 400 500 600

549

3.92 (1H, dd, 11.3, 4.8) 3.71 (1H, dd, 11.3, 3.4) 4.09 (1H, m) 3.12 (1H, dd, 4.8, 3.8) 3.43 (1H, dt, 5.8, 4.8) 1.52 (2H, m) 1.12 – 1.28 (58H, br s) 1.86 (4H, 5.26 (2H, 0.77 (3H, 7.48 (1H, – 3.88 (1H, 1.64 (2H, 4.15 (1H, 3.40 (1H, 3.28 (1H, 3.23 (1H, 3.16 (1H, 3.79 (1H, 3.61 (1H,

HMBC (H ! C)

dC

m) dt, 17.3, 5.4) t, 6.4) d, 8.8) t, 6.9) m) d, 7.6) m) t, 7.4) t, 7.4) m) dd, 10. 8, 4.9) dd, 10. 8, 2.9)

COSY (H ! H)

68.5

2,3,100

H-1/H-2

51.5 76.1 74.2 32.1 28.1 –30.9

1,3,4,10 1,2,4,5 2,3,5,6 3,4,6 5,13,26,170

32.4 130.8, 129.7 13.9 – 175.7 71.9 34.2 102.8 73.1 76.1 69.7 76.5 61.2

14,15 13,16 25,160 1,2,3,10 – 10 ,30 10 ,20 ,40 1,200 ,300 100 ,300 ,400 100 ,200 ,400 ,500 300 ,500 300 ,600 500 ,400

H-2/H-1,3 H-3/H-2,4 H-4/H-3,5 H-5/H-4,6 H-6,12/H-5,13; H-17,25/H-16,26; H-40 ,160 /H-30 ,170 H-13,16/H-12,14,15,17 H-14,15/H-13,16 H-26,170 /H-25,160 N-H/H-2 – H-20 /H-30 H-30 /H-20 ,40 H-100 /H-200 H-200 /H-100 ,300 H-300 /H-200 ,400 H-400 /H-300 ,500 H-500 /H-400 ,600 H-600 /H-500

nudicaulin C (3) could be assigned the structure (2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene1,3,4-triol-1-O-b-D -glucopyranoside. The sphingolipid 4 was obtained as colorless shiny powder with molecular formula C38H75NO6. The 1H NMR spectrum of 4 (Table 4) was similar to that of 2 with an additional oxymethine signal at d 3.62 (1H, dt, J ¼ 6.2, 4.3 Hz, H-30 ),

corresponding to the carbon at d 72.8. This information indicated that 4 could be an oxidative derivative of 2 with slight variation in lengths of both the chains (Figure 4). All assignments were accomplished by careful analysis of mass fragmentation and 1D and 2D NMR data (Table 4). The optical rotations of 4 ([a ]D ¼ þ29.9) and the subsequent methanolysis products ([a ]D ¼ þ9.6 and þ16.9)

269

O OH 1'

HN

OH 4''

HO HO

6''

CH3

OH O OH1''

26

14

O

5'' 2'' 3''

17'

2'

2

3

CH3

4

1

25

15

OH 394

307

662

367

694


141

550

N. Riaz et al.

Table 4. 1H and 100 MHz).

13


dH (J in Hz)

Position 1

3.60 (1H,dd, 11.6, 4.8) 3.54 (1H, dd, 11.6, 3.2) 3.94 (1H, m) 3.35 (1H, dd, 4.8, 4.1) 3.31 (1H, dt, 5.1, 4.8) 1.52 (2H, m) 1.06 (44H, br s)


2 3 4 5 6 – 17, 50 – 100 ,150 —190 18,200 NH 10 20 30 40 120 ,130 110 ,140

0.68 (6H, 7.42 (1H, – 3.82 (1H, 3.62 (1H, 1.58 (2H, 5.21 (2H, 1.81 (4H,

HMBC (H ! C)

dC

t, 6.4) d, 8.8) d, 4.3) dt, 6.2, 4.3) m) dt, 16.5, 5.0) m)

COSY (H ! H)

60.8

2,3

H-1/H-2

51.7 75.2 72.0 32.9 29.4-22.4

1,3,4,10 1,2,4,5 2,3,5,6 3,4,6 5,17,18, 200 ,140

13.7 – 174.8 74.3 72.8 31.6 130.5, 129.6 32.3

17,190 1,2,3,4,10 – 10 ,30 ,40 , 10 ,20 ,40 , 20 ,30 ,50 , 110 ,140 , 120 ,130

H-2/H-1,3 H-3/H-2,4 H-4/H-3,5 H-5/H-4,6 H-6,17/H-5,18; H-50 ,100 /H-40 , H-110 ; H-150 ,190 / H-140 ,200 H-18,200 /H-17,190 N-H/H-2 – H-20 /H-30 H-30 /H-20 ,40 H-40 /H-30 ,50 H-120 ,130 /H-110 ,140 H-110 ,140 /H-120 ,130

325 237

OH

O

139

OH

340 1'

HN

2'

85

13'

20'

3'

CH3

12' 267

OH

18

HO 1

2

3

CH3

4

OH 227


were comparable with reported ceramides having same configurations [15–17]. Based on these evidences, nudicaulin D (4) was assigned the structure (2S,3S,4R,)-2{[(2R,3S,12E)-2,3-dihydroxyeicos-12enoyl]amino}octadecane-1,3,4-triol. Lipoxygenases (EC 1.13.11.12) are potential target for the rational drug design and discovery of mechanism-based inhibitors for the treatment of a variety of disorders such as bronchial asthma, inflammation, cancer, and autoimmune diseases. Compounds 1 – 4 were evaluated for lipoxygenase inhibitory potential using baicalein (Aldrich Chem. Co., Seelze,

Germany) as positive control in the assay. The results (Table 5) showed that compounds 1 – 4 were moderate inhibitors of enzyme lipoxygenase. Table 5. In vitro quantitative inhibition of lipoxygenase by compounds 1 – 4. Sr. no. 1 2 3 4 5 a b

Compounds

IC50 ^ SEM (mM)a

1 2 3 4 Baicaleinb

193 ^ 1.11 105 ^ 1.34 103 ^ 1.74 163 ^ 1.25 22.4 ^ 1.3

Standard error of the mean of five assays. Standard inhibitor of the lipoxygenase enzyme.

Journal of Asian Natural Products Research


3. Experimental 3.1 General experimental procedures Melting points were determined by a Buchi 434 melting point apparatus (Flavil, Switzerland). IR spectra were recorded on Shimadzu 460 spectrometer (Duisburg, Germany). 1H (400, 500 MHz), 13C NMR (100, 125 MHz), and 2D NMR (HMQC, HMBC, and COSY; 400, 500 MHz) spectra were recorded on Bruker spectrometer (Zurich, Switzerland). The chemical shift values (d) are reported in ppm and the coupling constant (J) is in Hz. EI-MS, HREI-MS, FAB-MS, and HR-FAB-MS were recorded on Finnigan (Varian MAT, Waldbronn, Germany) JMS H £ 110 with a data system and JMSA 500 mass spectrometers, respectively. The gas chromatography (GC) was performed on a Shimadzu gas chromatograph (GC-9A; Noisiel, France) (3% OV-1 silanized chromosorb W, column temperature 1808C, injection port and detector temperature 275–3008C, flow rate 35 ml/min, and flame-ionization detector). Chromatographic separations were carried out using aluminum sheets pre-coated with silica gel 60 F254 (20 £ 20 cm, 0.2 mm thick; E. Merck; Darmstadt, Germany) for thin layer chromatography (TLC) and silica gel (230–400 mesh, Darmstadt, Germany) for CC. TLC plates were visualized under UV at 254 and 366 nm and by spraying with ceric sulphate reagent solution (by heating). 3.2 Plant material The whole plant material of L. nudicaulis was collected from Cholistan Desert (Punjab), Pakistan in April 2008. It was identified by Dr Muhammad Arshad, Plant Taxonomist, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur, where a voucher specimen is deposited (0022-LN/CIDS/08). 3.3 Extraction and isolation The whole plant of L. nudicaulis (26 kg) was shade dried, ground, and extracted with

551

methanol. The methanolic extract was evaporated under vacuum to a dark greenish mass (1.2 kg) which was suspended in water and extracted with n-hexane and ethyl acetate. The n-hexane soluble fraction (150 g) was subjected to CC over silica gel eluting with n-hexane, n-hexane–dichloromethane, dichloromethane, dichloromethane – methanol, and methanol in increasing order of polarity. The subfractions (fA –fI) obtained from the main column subjected to further CC using isocratic 15% n-hexane–dichloromethane yielded 1-hexatriacontanol and b-sitosterol; 20% n-hexane in dichloromethane, octadecyl 4-hydroxycinnamate and elaidic acid; 30% n-hexane–dichloromethane, cholesta5,22-diene-3,7-diol and oleanolic acid; 70% n-hexane–dichloromethane, apigenin; 1% methanol in dichloromethane, nudicaulin A (1); 3% methanol in dichloromethane, bsitosterol 3-O-b-D -glucopyranoside; 5% methanol in dichloromethane, nudicaulin B (2); 7% methanol in dichloromethane, nudicaulin D (4); and 10% methanol in dichloromethane, nudicaulin C (3), respectively. 3.3.1

Nudicaulin A (1)

Colorless amorphous powder (49 mg). mp 106 –1088C; ½a24 D þ 16.9 (c 0.2, MeOH); IR y max (KBr) cm21: 3640, 2910, 1650, 1625; 1H and 13C NMR spectral data, see Table 1; HR-EI-MS: m/z 665.6330 [M]þ (calcd for C42H83NO4, 665.6322). 3.3.2

Nudicaulin B (2)

White amorphous powder (35 mg). mp 114 – 1168C; ½a24 D þ 39.7 (c 0.15 g/ml, MeOH); IR y max (KBr) cm21: 3334, 3215, 2919, 1622, 1602; 1H and 13C NMR spectral data, see Table 2; HR-EI-MS: m/z 681.6280 [Mþ (calcd for C42H83NO5, 681.6271). 3.3.3 Nudicaulin C (3) Colorless gummy solid (44 mg). ½a24 D þ 32.1 (c 0.12 g/ml, MeOH); IR y max (KBr)

552

N. Riaz et al.

cm21: 3346, 3242, 2910, 1639, 1616; 1 H and 13C NMR spectral data, see Table 3; HR-FAB-MS (2 ve mode) m/z 856.6884 [M – H]2 (calcd for C49H94NO10, 856. 6877).


3.3.4

Nudicaulin D (4)

Colorless shiny powder (57 mg). mp 139– 1418C; ½a24 D þ 29.9 (c 0.22, MeOH); IR y max (KBr) cm21: 3440, 3296, 2914, 1663, 1616; 1H and 13C NMR spectral data, see Table 4; HR-EI-MS: m/z 641.5600 [M]þ (calcd for C38H75NO6, 641.5594).

3.4

3.4.1

3.4.3

Methyl ester derived from 3

2 11.2 (c 0.011); 1H NMR (CDCl3, 400 MHz): d 4.15 (1H, t, J ¼ 6.6 Hz, H-20 ), 3.56 (3H, s, MeO), 1.99 (3H, s, MeCO), 1.18 –1.29 (26H, br s, CH2-40 -160 ), 0.86 (3H, t, J ¼ 6.9 Hz, CH3-170 ); GC-MS: m/z 342. ½a24 D

Methanolysis

Compounds 1 – 4 (12 mg each) were refluxed separately with 6 ml of 1N HCl and 25 ml of MeOH for 15 h. The reaction mixture was then extracted with n-hexane to obtain the corresponding fatty acid methyl esters, which were analyzed by GCMS after acetylation with aceticanhydride –pyridine. The aqueous layer of 1, 2, and 4 was evaporated, and the residue was acetylated. Purification over Sephadex LH-20 and elution with CH2Cl2/MeOH (1:1) gave the corresponding acetylated sphingosines, which were analyzed by GCMS. The aqueous layer of 3 was evaporated to dryness, and the residue was separated by silica gel CC as sphingosine base and methylated sugar. The base was acetylated and analyzed by GC-MS. The sugar was identified as methyl b-D -glucopyranoside based on the sign of optical rotation [a ]D þ 76.2 (c 0.1, MeOH) and Co-TLC profile (Rf 0.45 (EtOAc/MeOH/H 2O; 5:2:0.5)).

1

3.4.2 Methyl ester derived from 2 1 ½a24 D 2 7.1 (c 0.01); H NMR (CDCl3, 400 MHz): d 4.12 (1H, t, J ¼ 6.9 Hz, H-20 ), 3.53 (3H, s, MeO), 1.98 (3H, s, MeCO), 1.17 –1.27 (28H, br s, CH2-40 -170 ), 0.83 (3H, t, J ¼ 6.7 Hz, CH3-180 ); GC-MS: m/z 356.


H NMR (CDCl3, 400 MHz): d 3.51 (s, MeO), 2.32 (2H, t, J ¼ 6.5 Hz, H-20 ), 1.51 (2H, m, H-30 ), 1.19 –1.28 (28H, br s, CH240 -170 ), 0.84 (3H, t, J ¼ 6.8 Hz, CH3-180 ); GC-MS: m/z 298.

3.4.4


þ 9.6 (c 0.012); 1H NMR (CDCl3, 400 MHz): d 5.28 (2H, dt, J ¼ 16.0, 5.6 Hz, H-12,13), 4.19 (1H, d, J ¼ 4.3 Hz, H-20 ), 4.11 (1H, dt, J ¼ 6.1, 4.3 Hz, H-30 ), 3.50 (3H, s, MeO), 2.00 (6H, s, 2 £ MeCO), 1.15 –1.25 (22H, br s, CH2-50 -100 ,150 -190 ), 0.90 (3H, t, J ¼ 6.5 Hz, CH3-200 ); GC-MS: m/z 440. ½a24 D

3.4.5

Acetylsphingamine derived from 1

1 ½a24 D þ 11.2 (c 0.01); H NMR (CDCl3, 400 MHz): d 8.06 (1H, d, J ¼ 7.6 Hz, NH), 5.31 (2H, dt, J ¼ 15.1, 5.2 Hz, H-14,15), 4.56 (1H, dd, J ¼ 5.1, 4.3 Hz, H-4), 4.50 (1H, m, H-2), 4.42 (1H, dd, J ¼ 10.3, 5.6 Hz, H-1), 4.33 (1H, dd, J ¼ 10.3, 3.3 Hz, H-1), 4.17 (1H, dd, J ¼ 5.1, 3.2 Hz, H-3), 2.00 (6H, 2 £ MeCO), 1.98 (6H, 2 £ MeCO), 1.13 –1.21 (26H, br s, CH2-7-12,17-23), 0.89 (t, J ¼ 6.5 Hz, CH324); GC-MS: m/z 567.

3.4.6


þ 19.1 (c 0.015); 1H NMR (CDCl3, 400 MHz): d 7.96 (1H, d, J ¼ 7.9 Hz, NH), 5.33 (2H, dt, J ¼ 15.5, 5.5 Hz, H-14,15), 4.66 (1H, dd, J ¼ 5.0, 4.1 Hz, H-4), 4.55 (1H, m, H-2), 4.44 (1H, dd, J ¼ 10.2, ½a24 D

Journal of Asian Natural Products Research 5.4 Hz, H-1), 4.33 (1H, dd, J ¼ 10.2, 3.1 Hz, H-1), 4.19 (1H, dd, J ¼ 5.0, 3.1 Hz, H-3), 2.00 (12H, 4 £ MeCO), 1.15–1.25 (26H, br s, CH2-7-12,17-23), 0.85 (t, J ¼ 6.6 Hz, CH3-24); GC-MS: m/z 567.


3.4.7


1 ½a24 D þ 19.1 (c 0.013); H NMR (CDCl3, 400 MHz): d 7.90 (1H, d, J ¼ 8.2 Hz, NH), 5.29 (2H, dt, J ¼ 16.1, 5.8 Hz, H-14,15), 4.60 (1H, dd, J ¼ 5.1, 4.0 Hz, H-4), 4.52 (1H, m, H-2), 4.40 (1H, dd, J ¼ 10.6, 5.3 Hz, H-1), 4.30 (1H, dd, J ¼ 10.6, 3.0 Hz, H-1), 4.16 (1H, dd, J ¼ 5.1, 3.2 Hz, H-3), 2.01 (12H, 4 £ MeCO), 1.16–1.26 (30H, br s, CH2-7-12,17-25), 0.87 (t, J ¼ 6.2 Hz, CH3-26); GC-MS: m/z 595.

3.4.8


þ 16.9 (c 0.012); 1H NMR (CDCl3, 400 MHz): d 8.16 (1H, d, J ¼ 7.9 Hz, NH), 4.56 (1H, dd, J ¼ 5.2, 3.9 Hz, H-4), 4.50 (1H, m, H-2), 4.39 (1H, dd, J ¼ 11.0, 5.1 Hz, H-1), 4.30 (1H, dd, J ¼ 11.0, 3.0 Hz, H-1), 4.15 (1H, dd, J ¼ 5.2, 3.0 Hz, H-3), 2.00 (12H, 4 £ MeCO), 1.19–1.30 (24H, br s, CH2-6-17), 0.86 (t, J ¼ 6.5 Hz, CH3-18); GC-MS: m/z 485.

½a24 D

3.5 Oxidative cleavage of the double bond in 1 – 4 Separately, to the solution of acetylsphingamines of compounds 1 –3 and the methyl ester derived from 4 (4 mg each) in acetone, 1 ml of 0.04 M solution of K2CO3, 6 ml of an aqueous solution of 0.025 M KMnO4, and 0.09 M NaIO4 were added in 100 ml round-bottomed flask. The reaction was allowed to proceed at 378C for 18 h. After acidification with 5 N H2SO4, the solution was decolorized with a 1 M solution of oxalic acid and extracted with Et2O (3 – 10 ml). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. The resulting carboxylic acids were methylated with ethereal

553

solution of diazomethane and analyzed by GC-MS. 3.6 Lipoxygenase assay The lipoxygenase activity was assayed according to the method of Tappel et al. [24] with slight modifications. A total volume of 200 ml assay mixture contained 160 ml sodium phosphate buffer (100 mM, pH 8.0), 10 ml of test compound, and 20 ml purified lipoxygenase (Sigma Chemicals, Seelze, Germany). The contents were preincubated for 10 min at 258C. The reaction was initiated by addition of 10 ml linoleic acid (substrate solution). The change in absorbance was observed after 6 min at 234 nm. All reactions were performed in triplicates in 96-well microplate reader (Synergy HT, Biotek, Winooski, Vermont, USA). The positive and negative controls were included in the assay. The percentage inhibition (%) was calculated by formula: Inhibition ð%Þ ¼ ðcontrol 2 testÞ £ 100: Control is the total enzyme activity without inhibitor and test is the activity of the test compound. IC50 values (concentration at which there is 50% enzyme inhibition) of selected samples were calculated using EZFit Enzyme kinetics software (Perrella Scientific Inc. Amherest, MA, USA).

Acknowledgments The authors are thankful to Higher Education Commission (HEC) Pakistan and Alaxander von Humboldt (AvH) Foundation, Germany for financial support. We are also obliged to Third World Academy of Science (TWAS), Italy, for providing some of the basic laboratory facilities in Chemistry Department of IUB.

References [1] P.P.J. Herman, E. Retief, M. Koekemoer, and W.G. Welman, Strelitzia 10, 101 (2000). [2] K.R. Prabhu and V. Venkateswarlu, J. Indian Chem. Soc. 46, 176 (1969).


554

N. Riaz et al.

[3] S. Rashid, M. Ashraf, S. Bibi, and R. Anjum, Pak. J. Biol. Sci. 3, 808 (2000). [4] A. Krishnamurthi, The Wealth of India (Council of Scientific and Industrial Research (CSIR), New Delhi, 1969), Vol. 1, p. 68. [5] E. Nasir and S.I. Ali, Flora of West Pakistan (Fakhri Printing Press, Karachi, 1972), Vol. 712. [6] S.R. Baquar, Medicinal and Poisonous Plants of Pakistan (Printas Printing Press, Karachi, 1989), Vol. 31&258. [7] M.M. Gupta, R.K. Verma, and S.C. Singh, Fitoterapia 60, 476 (1989). [8] R.N. Yadava and N. Chakravarti, Indian J. Chem. Section B 48B, 83 (2009). [9] R.M.A. Mansour, A.A. Ahmed, and N.A.M. Saleh, Phytochemistry 22, 2630 (1983). [10] F. Moussaoui, A. Zellagui, N. Segueni, A. Touil, and S. Rhouati, Rec. Nat. Prod. 4, 91 (2010). [11] D. Ali, S.M.S. Hussain, A. Malik, and Z. Ahmed, J. Chem. Soc. Pak. 25, 341 (2003). [12] A.A. El-Bassuony and A.M. Kabbash, Pharm. Mag. 4, 249 (2008). [13] M.M. Bhandari, Flora of Indian Desert (Mps Repros, Jodhpur, 1988), p. 182. [14] S. Rashid, M. Ashraf, S. Bibi, and R. Anjum, Pak. J. Biol. Sci. 3, 630 (2000).

[15] H.C. Kwon, K.C. Lee, O.R. Cho, Y. Jung, S.Y. Cho, S.Y. Kim, and K.R. Lee, J. Nat. Prod. 66, 466 (2003). [16] V.U. Ahmad, M. Zubair, M.A. Abbasi, F. Kousar, M.A. Rashid, N. Rasool, and R.B. Tareen, Nat. Prod. Res. 20, 69 (2006). [17] P. Muralidhar, M.M. Kumar, N. Krishna, C.B. Rao, and D.V. Rao, Chem. Pharm. Bull. 53, 168 (2005). [18] E. Ahmed, A. Malik, N. Afza, N. Riaz, I. Anis, A. Sharif, S. Farheen, M.A. Lodhi, and M.I. Choudhary, Nat. Prod. Res. 21, 69 (2007). [19] H.S. Garg and S. Agrawal, J. Nat. Prod. 58, 442 (1995). [20] P. Muralidhar, N. Krishna, M.M. Kumar, C.B. Rao, and D.V. Rao, Chem. Pharm. Bull. 51, 1193 (2003). [21] T. Natori, M. Morita, K. Akimoto, and Y. Koezuka, Tetrahedron 50, 2771 (1994). [22] N. Riaz, S.A. Nawaz, N. Mukhtar, A. Malik, N. Afza, S. Ali, S. Ullah, P. Muhammad, and M.I. Choudhary, Chem. Biodiver. 4, 72 (2004). [23] W. Jin, K.L. Rinehart, and E.A. JaresErijman, J. Org. Chem. 59, 144 (1994). [24] A.L. Tappel, Arch. Biochem. Biophy. 44, 378 (1953).