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Further Secondary Metabolites from Seriphidium stenocephalum.

Byline: Nusrat Shafiq, Liaqat Ali, Naheed Riaz, Tasneem Khatoon, Muhammad Imran Touseef, Abdul Jabbar, Rasool Bakhsh Tareen and Muhammad Saleem

Summary:

In continuation of our search for bioactive secondary metabolites from Seriphidium stenocephalum, chromatographic analysis of some ignored fractions from previous work on ethyl acetate part of the methanolic extract yielded five new sphingolipids; seriphidalin A [(2S,3S,4R,12E)-2-{[heptdecanoyl]amino}unieicos-12-ene-1,3,4-triol; 1], seriphidalin B [(2S,3S,4R,12E)-2-{[(2R)-2-hydroxydodidecanoyl]amino}trididec-12-ene-1,3,4-triol; 2], seriphidalin C [(2S,3S,4R,9E)-2-{[(2R)-2-hydroxydodidecanoyl]amino}didec-9-ene-1,3,4-triol; 3], seriphidalin D [(2S,3S,4R)-2-{[(2R,5E)-2-hydroxyheptadec-5-enoyl]amino}pentadecane-1,3,4-triol- 1-O-AY-D-galactopyranoside; 4] and seriphidalin E [(2S,3S,4R,7E,12E)-2-{[(2R)-2- hydroxyheptanoyl]amino}heptadeca-7,12-diene-1,3,4-triol-1-O-AY-D-glucopyranoside; 5] together with five known compounds (6-10). The structures of the isolated compounds were determined by 1D and 2D NMR and high resolution mass spectrometry.

Keywords: Seriphidium stenocephalum; Methanolic extract; Sphingolipids; Characterization

Introduction

The genus Seriphidium consists of about 400 species, mostly are herbs and hardy shrubs growing in dry or semi-dry habitats [1]. The strong aroma and bitter taste of most of the Seriphidium species is due to the presence of sesquiterpene lactones and other terpenoids, which exist as an adaptation to discourage herbivores. Some Seriphidium relatives are also used as food plants [2]. In Pakistan, Seriphidium stenocephalum is known as one of the medicinally important plant, growing along gravelly dry riverbanks with coarse sand-mixed clay soils in Punjab and Baluchistan [3]. Several species of Seriphidium have been reported to produce antioxidant and anticancer compounds such as flavonoids, flavonoid glycosides, and terpenoids [4].

Previously, we isolated seven phenolics [5] from ethyl acetate soluble part of the methanolic extract of Seriphidium stenocephalum. This paper describes the isolation and characterization of further metabolites from some ignored sub-fraction of the ethyl acetate soluble part of the same extract. As a result of chromatographic purification of the said fraction five new sphingolipids: seriphidalin A- E (1-5) together with five known compounds: 2,3-dihydroxypropyl hexacosanoate (6), 2,3- dihydroxypropyl triacontanoate (7) [6], tricosyl-p- (E)-coumarate (8) [7], taraxerol acetate (9) [8] and taraxerol (10) [9] (Fig. 1) were isolated and characterized due to spectroscopic analysis.

Experimental

General Experimental Procedures

Purification was carried out on aluminium sheets pre-coated with silica gel 60 F254 (20A-20 cm, 0.2 mm thick, E. Merck; Darmstadt, Germany) and silica gel (230-400 mesh) column chromatography. TLC plates were visualized under UV-light (254 and 366 nm) and by spraying with ceric sulphate solution followed by heating. The IR spectra were scanned on IR spectrophotometer Shimadzu 460 (Duisburg, Germany). 1H and 2D-NMR (HSQC, COSY, HMBC) spectra were recorded on Bruker (Zurich, Switzerland) 300, 400 and 500 MHz, whereas, 13C-NMR spectra were measured on the same machine operating at 125, 100 and 75 MHz. The chemical shift values () are reported in ppm and the coupling constants (J) in Hz. EIMS, HR-EI- MS, FAB-MS and HR-FAB-MS were performed on Finnigan (Varian MAT, Waldbronn, Germany) JMS-HA-110 with a data system and JMS-A 500 mass spectrometers, respectively. Optical rotation was measured on a Jasco DIP-360 digital polarimeter.

Collection and Identification of the Plant Material

Seriphidium stenocephalum was collected from District Ziarat, Baluchistan in September 2010 and was identified by Prof. Dr. Rasool Bakhsh Tareen, Plant Taxonomist, Department of Botany, University of Baluchistan, Quetta, where the voucher specimen (RBT-SS-09) has been deposited in the herbarium.

Extraction and Isolation

The collected plant material was dried under shade for two weeks, ground to a semi- powder and was extracted in methanol for one week twice. The methanolic extract was concentrated under vacuum to a dark brown gummy mass (150 g) and was suspended in distilled water (1 L). The suspension was then fractionated into n-hexane and ethyl acetate (EtOAc) fractions. The EtOAc fraction was chromatographed on silica gel column eluting with a gradient of n-hexane, n-hexane:EtOAc, EtOAc, EtOAc:MeOH and MeOH to get nine fractions (SS-1 to SS-9). The sub-fraction SS-3 was chromatographed on silica gel column eluting with a gradient of n-hexane: EtOAc and five sub- fractions SS-3b1-SS-3b5 were obtained. The sub- fraction SS-3b3 was further subjected to silica gel column chromatography eluting with gradient of n- hexane:EtOAc to get three sub-fractions SS-3b3a to SS-3b3c.

Out of these, SS-3b3b sub-fraction was further purified on silica gel column by using n- hexane:EtOAc (7:3) and n-hexane:EtOAc (5:5), that yielded compounds 1 (49 mg), and 3 (44 mg), respectively. The sub-fraction SS-3b3c was further purified on silica gel column chromatography by using n-hexane:EtOAc (7:3) to get compound 2 (35 mg). The main fraction SS-8 was again chromatographed on silica gel column eluting with n-hexane:EtOAc that yielded 4 sub-fractions SS-8a- SS-8d. The sub-fraction SS-8a was further purified on silica gel column eluting with n-hexane:EtOAc (6:4) to get 4 (57 mg). The sub-fraction SS-8d was passed through silica gel column with n- hexane:EtOAc (5:5) to get 5 (47 mg) and 7 (34 mg). The SS-6 from the main column was also subjected to repeated silica gel column chromatography eluting with a gradient of n-hexane:EtOAc, that yielded a semi-pure fraction, which was washed with MeOH to get 8 (38.5 mg).

The sub-fraction SS-3b1 on chromatographic purification with an isocratic of n-hexane:EtOAc (5:5) gave compound 6 (19.5 mg). The fraction SS-1 from the main column was again passed through a silica gel column eluting with a gradient of by n-hexane:EtOAc to get three sub-fractions SS-1a-SS-1b. The sub-fraction SS-1a was finally purified over silica gel column using an isocratic of n-hexane:EtOAc (7:3) to get compound 9 (32 mg) and with n-hexane:EtOAc (8:2) to get compound 10 (45 mg).

Seriphidalin A (1)

Colorless amorphous powder (49 mg); [a]D24 = + 19.3 (c 0.001, MeOH); IR max (KBr): 3450, 3332, 1654, 1625 cm-1; 1H and 13C NMR data, see Table-1; EIMS: m/z 749, 539, 524, 510, 481, 456, 437, 293, 268, 239, 211; HR-EI-MS: m/z 749.7238 (calcd. for C48H95NO4, 749.7261).

Seriphidalin B (2)

White amorphous powder (35 mg); [a]D24 = + 37.6 (c 0.0015, MeOH); IR max (KBr): 3365, 3210, 1621, 1601 cm-1; 1H and 13C-NMR data, see Table-2; EI-MS m/z: 709, 398, 355, 354, 339, 311, 281, 153, 99; HR-EI-MS: m/z 709.6512 (calcd. for C44H87NO5, 709.6584).

Seriphidalin C (3)

Colorless gummy solid (44 mg); [a]D24 = + 29.1 (c 0.0012, MeOH); IR max (KBr): 3343, 3234, 2911, 1637, 1614 cm-1; 1H and 13C-NMR data, see Table-3; EI-MS: m/z 681, 354, 342, 339, 327, 311, 281, 181, 127; HR-EI-MS: m/z 681.6285 (681.6271 calcd. for C42H83NO5).

Seriphidalin D (4)

Colorless shiny powder (57 mg); [a]D24 = + 31.6 (c 0.0013, MeOH); IR max (KBr): 3410, 3266, 2919, 1653, 1615 cm -1; 1H and 13C-NMR data, see Table-4; HR-FAB-MS: m/z 702.5127 (calcd. for C38H72NO10, 702.5156 corresponding to the formula C38H73NO10).

Seriphidalin E (5)

Colorless shiny powder (47 mg); [a]D24 = + 41.5 (c 0.0022, MeOH); IR max (KBr): 3409, 3257, 2924, 1653, 1610 cm -1; 1H and 13C-NMR data, see Table-5; HR-FAB-MS: m/z 588.3760 (calcd. for C30H54NO10, 588.3748 corresponding to the formula C30H55NO10).

Table-1: 1H- and 13C-NMR data and HMBC and COSY correlations of 1 (CDCl3, 500 and 125MHz).

###Position###H (J in Hz)###C###HMBC (HC)###COSY (HH)

###3.75 (1H, dd, 11.6, 3.6)

###1###61.3###2,3###H-1/H-2

###3.66 (1H, dd, 11.6, 5.2)

###2###4.02 (1H, m)###51.1###1,3,4,1###H-2/H-1,3,NH

###3###3.46 (1H, dd, 8.0, 3.6)###75.7###1,2,4,5###H-3/H-2,4

###4###3.98 (1H, dd, 8.4, 3.6)###72.0###2,3,5,6###H-4/H-3,5

###5###1.90 (2H, m)###32.5###3,4,6###H-5/H-4,6

###6-10###1.90 (10H, m)###29.6###4,5,11###H-5-10/H-5,11

###11,14###1.96 (4H, m)###34.3, 32.4###10,12,13###H-11,14/H-10,12,15

###12,13###5.33 (2H, m, W1/2, 19.5)###128.7, 131.8###10,11,14,15###H-12,13/H-11,14

###15-21###1.18 (14H, m)###29.6-29.3###13,12###H-15-21/H-14,22

###22-28###1.16 (14H, m)###29.6-29.3###21,29,30###H-22-28/H-21,29

###29,30###1.23 (4H, m)###31.8, 22.0###28,31###H-29,30/H-28,31

###31###0.82 (3H, t, 7.1)###14.1###29,30###H-31/H-30

###NH###7.41 (1H, d, 8.0)###-###1,2,3,1,2###N-H/H-2

###1###-###173.0###-###-

###2###2.11 (2H, t, 7.8)###36.2###1,3###H-2/H-3

###3-16###1.18 (28H, br s)###22.6, 29.3, 29.5, 31.8###1,2,4###H-3-16/H-2,17

###17###0.84 (3H, t, 6.8)###14.1###16###H-17/H-16

Table-2: 1H- and 13C-NMR data and HMBC and COSY correlations of 2 (CDCl3, 500 and 125MHz).

###Position###H (J in Hz)###C###HMBC (HC)###COSY (HH)

###1###3.66 (1H, dd, 13.5, 7.0)###61.0###2,3###H-1/H-2

###3.72 (1H, dd, 11.5, 4.0)

###2###4.01 (1H, dd, 8.0, 4.0)###51.5###1,3,4,1###H-2/H-1,3,NH

###3###3.44 (1H, dd, 3.5, 1.5)###75.6###1,2,4,5###H-3/H-2,4

###4###3.46 (1H, m)###72.0###2,3,5,6###H-4/H-3,5

###5###1.87 (2H, m)###32.5###3,4,6###H-5/H-4,6

###6-10###1.71 (10H, m)###29.5, 29.0###4,5,11,12###H-6-10/H-5,11

###11,14###1.99 (4H, m)###34.2, 32.5###10,12,15###H-11,14/H-10,12,15

###12,13###5.32 (2H, m, W1/2, 20.2)###129.6, 130.6###10,11,14,15###H-12,13/H-11,14

###15-21###1.69 (14H, br s)###29.7-29.3###11,12,22###H-15-21/H-14,22

###22###0.88 (3H, t, 7.3)###14.0###21###H-22/H-21

###NH###7.42 (1H, d, 8.0)###-###1,2,3,1,2###N-H/H-2

###1###-###175.6###-###-

###2###3.95 (1H, dd, 8.5, 3.0)###71.9###1,3,4###H-2/H-3

###3###1.70 (2H, m)###34.0###1,2,4,5###H-3/H-2,4

###4###1.52 (2H, m)###25.7###2,3,5###H-4/H-3,5

###5-21###1.19 (34H, br s)###22.5, 29.0, 31.7###3,4,22###H-5-21/H-4,22'

###22###0.91 (3H, t, 7.3)###13.9###21###H-22/H-21

Table-3: 1H- and 13C-NMR data, HMBC and COSY correlation of 3 (CDCl3, 400, 100 MHz).

###Position###H (J in Hz)###C###HMBC (HC)###COSY (HH)

###1###3.58 (1H, dd, 3.0, 6.6)###60.8###2,3###H-1/H-2

###3.66 (1H, dd, 8.4, 4.2)

###2###3.94 (1H, m)###51.3###1,2,4,1###H-2/H-1,3,N-H

###3###3.35 (1H, m)###75.3###1,2,4,5###H-3/H-2,4

###4###3.37 (1H, m)###71.9###2,3,5###H-4/H-3,5

###5-7###1.24 (6H, m)###29.0, 29.5, 32.3###3,4,8###H-5-7/H-4,8

8,11-19,3-21###1.85 (58H, m)###22.3-32.3###7,9,1,2,22###H-8,11-19,3-21/H-7,9,20,2,22

###9,10###5.24 (2H, m)###129.6, 130.5###7,8,11###H-9,10/H-8,11

###20,22###0.85 (6H, m)###13.7###19,21###H-20,22/H-19,21

###N-H###7.37 (1H, d, 7.8)###-###1,1,2###N-H/H-2

###1###-###175.5###-###-

###2###3.89 (1H, t, 7.5)###71.7###1,3,4###H-2/H-3

###3###1.62 (2H, m)###34.1###1,2,4###H-3/H-2,4

Table-4: 1H- and 13C-NMR spectral data, HMBC and COSY correlations of 4 (CDCl3; 400, 100 MHz).

###Position###H (J in Hz)###C###HMBC (HC)###COSY (HH)

###1###3.78 (1H, dd, 11.2, 5.0)###68.4###2,3,1''###H-1/H-2

###3.73 (1H, dd, 11.2, 3.2)

###2###4.15 (1H, m)###50.5###1,3,4,1'###H-2/H-1,3

###3###3.49 (1H, dd, 4.6, 4.0)###74.5###1,2,4,5###H-3/H-2,4

###4###3.42 (1H, dt, 5.8, 4.6)###71.2###2,3,5,6###H-4/H-3,5

###5###1.73 (2H, m)###34.3###3,4,6###H-5/H-4,6

###6-13,9-15###1.13-1.30 (26H, br s)###28.2-31.0###-###-

###15###0.82 (3H, t, 7.0)###14.0###13,14###H-15/H-14

###14,16###1.16 (4H, m)###22.6###14,15###H-13,16/H-12

###NH###7.47 (1H, d, 8.8)###-###1,2,3,1###N-H/H-2

###1###-###173.9###-###-

###2###3.94 (1H, dd, 8.8, 4.2)###72.0###1,3###H-2'/H-3

###3###1.73 (2H, m)###34.3###1,2,4###H-3/H-2,4

###4###1.18 (2H, m)###32.5###2,3,5###H-4'/H-3',5'

###5,6###5.32 (2H, dt, 17.3, 5.4)###130.7, 129.7###4,7###H-5,6/H-4,7

###7###1.89 (2H, m)###32.5###4,5###H-7/H-6,8

###8###1.18 (2H, m)###25.8###-###-

###16###1.20 (2H, m)###22.6###-###H-16/H-17,15

###17###0.83 (3H, t, 6.9)###14.0###-###H-17/H-16

###1###4.21 (1H, d, 7.6)###103.1###1,2,3###H-1/H-2

###2###3.14 (1H, m)###76.1###1,3,4###H-2/H-1,3

###3###3.31 (1H, dd, 7.4, 2.0)###76.8###1,2,4,5###H-3/H-2,4

###4###3.16 (1H, d, 2.0)###73.0###3,5###H-4/H-3,5

###5###3.18 (1H, m)###75.0###3,6###H-5/H-4,6

###6###3.76 (1H, dd, 10.8, 4.9)###61.0###5,4###H-6/H-5

###3.62 (1H, dd, 10.8, 2.9)

Table-5: 1H- and 13C-NMR spectral data, HMBC and COSY correlations of 5 (CD3OD; 400, 100 MHz).

###Position###H, J in Hz###C###HMBC (HC)###COSY (HH)

###1###3.79 (1H, dd, 11.5, 5.0)###69.9###2,3,1###H-1/H-2

###4.06 (1H, dd, 11.5, 4.2)

###2###4.25 (1H, m)###51.6###1,3,4,1###H-2/H-1,3

###3###3.60 (1H, m)###75.5###1,2,4,5###H-3/H-2,4

###4###3.51 (1H, m)###72.8###2,3,5,6###H-4/H-3,5

###5###1.41, 1.38 (2H, m)###32.7###3,4,6,7###H-5/H-4,6

###6###1.58 (2H, m)###33.0###4,5,7,8###H-6/H-5,7

###7,8###5.41 (2H, br m, W1/2 20.1)###130.9, 131.4###5,6,9,10###H-7,8/H-6,9

###9,14###1.97 (4H, m)###33.7, 33.9,###7,8,13,15###H-9,14/H-8,13,15

###10###1.04 (2H, m)###28.2###8,9,1211###H-10/H-9,11

###11###1.33 (2H, m)###32.7###9,10,12###H-11/H-10,12

###12,13###5.31 (2H, br m, W1/2 20.1)###130.7, 131.5###10,11,14,15###H-12,13/H-11,14

###15,16,5,6###1.28 (8H, m)###23.7-30.8###13,14,17,3',4,7'###H-15,16,5,6/H-13,17,4,7

###17,7###0.90 (6H, m)###14.4###15,16,5,6###H-17,7/H-16,6

###NH###7.88 (1H, d, 7.9)###-###1,2,3,1,2###N-H/H-2

###1###-###177.1###-###-

###2###4.02(1H, t, 7.3)###72.9###1,3,4###H-2/H-3

###3###1.72 (1H, m)###35.7###1,4,5###H-3/H-2,4

###1###4.28 (1H, d, 8.0)###104.6###1,3###H-1/H-2

###2###3.16 (1H, m)###75.0###1,3###H-2/H-1,3

###3###3.31 (1H, m)###77.8###2,5###H-3/H-2,4

###4###3.25 (1H, m)###72.8###1,2,5###H-4/H-3,5

###5###3.26 (1H, m)###78.0###3,4,6###H-5/H-4,6

###6###3.67 (1H, m)###62.6###4,5###H-6/H-5

###3.87 (1H, m)

Methanolysis

Compounds 1-5 (12 mg each) were refluxed separately with 6 ml of 1N HCl and 25 mL of MeOH for 15 hrs. The reaction mixture was then extracted with n-hexane to obtain the corresponding fatty acid methyl esters, which were analyzed by GC-MS after acetylation with acetic anhydride-pyridine. The aqueous layer of 1-3 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 was analyzed by GC-MS. The aqueous layer of 4,5 was evaporated to dryness, and the residue was separated by silica gel column chromatography as sphingosine base and methylated sugar. The base was acetylated and analyzed by GC-MS. The sugar was identified as methyl AY-D-galactopyranoside in case of 4 and AY-D- glucopyranoside for 5 based on the sign of optical rotation ([]D + 80.2; + 74.3) and Co-TLC profile (Rf 0.45 (EtOAc/MeOH/H2O; 5:2:0.5).

Methyl Ester Derived From 1 1H-NMR (CDCl3, 400 MHz): 3.53 (s, MeO), 2.33 (2H, t, J = 6.5 Hz, H-2'), 1.52 (2H, m, H- 3'), 1.22-1.30 (28H, br s, CH2-3'-16'), 0.87 (3H, t, J = 6.7, CH3-17'); GC-MS: m/z 283.

Methyl Ester Derived From 2 [a]D24 = + 9.1 (c 0.002); 1H-NMR (CDCl3, 400 MHz): 4.12 (1H, t, J = 6.7 Hz, H-2'), 3.50 (3H, s, MeO), 1.98 (3H, s, MeCO), 1.18-1.31 (36H, br s, CH2-4'-21'), 0.85 (3H, t, J = 6.7 Hz, CH3-22'); GC-MS: m/z 411.

Methyl Ester Derived From 3 [a]D24 = + 10.1 (c 0.001); 1H-NMR (CDCl3, 400 MHz): 4.14 (1H, t, J = 6.6 Hz, H-2'), 3.53 (3H, s, MeO), 1.99 (3H, s, MeCO), 1.18-1.28 (36H, br s, CH2-4'-21'), 0.87 (3H, t, J = 6.9 Hz, CH3-22'); GC-MS: m/z 411.

Methyl Ester Derived From 4 [a]D24 = + 11.5 (c 0.0012); 1H-NMR (CDCl3, 400 MHz): 5.28 (2H, dt, J = 15.9, 5.4 Hz, H-5',6'), 4.14 (1H, d, J = 6.3 Hz, H-2'), 3.52 (3H, s, MeO), 2.00 (3H, s, MeCO), 1.18-1.28 (18H, br s, CH2-8'- 16'), 0.92 (3H, t, J = 6.6 Hz, CH3-17'); GC-MS: m/z 341.

Methyl Ester Derived From 5 [a]D24 = + 13.1 (c 0.0013); 1H-NMR (CDCl3, 400 MHz): 4.12 (1H, d, J = 6.4 Hz, H-2'), 3.52 3H, s, MeO), 1.98 (3H, s, MeCO), 1.21-1.26 (6H, br s, CH2-4'-6'), 0.88 (3H, t, J = 6.7 Hz, CH3-7'); GC- MS: m/z 203.

Acetylsphingamine Derived From 1 [a]D24 = + 21.1 (c 0.0013); 1H-NMR (CDCl3, 400 MHz): 7.85 (1H, d, J = 7.6 Hz, NH), 5.28 (2H, dt, J = 15.6, 5.0 Hz, H-12,13), 4.57 (1H, td, J = 6.1, 5.6 Hz, H-4), 4.51 (1H, m, H-2), 4.41 (1H, dd, J = 10.8, 5.1 Hz, H-1), 4.28 (1H, dd, J = 10.8, 3.0 Hz, H-1), 4.16 (1H, dd, J = 5.0, 3.2 Hz, H-3), 2.02 (6H, 2A-MeCO), 1.98 (6H, 2A-MeCO), 1.18-1.29 (42H, br s, CH2-5-9,15-30), 0.88 (3H, t, J = 6.6 Hz, CH3-31); GC-MS: m/z 666.

Acetylsphingamine Derived From 2 [a]D24 = + 29.1 (c 0.0015); 1H-NMR (CDCl3, 400 MHz): 7.98 (1H, d, J = 7.8 Hz, NH), 5.32 (2H, dt, J = 15.2, 5.3 Hz, H-12,13), 4.66 (1H, td, J = 6.2, 4.1 Hz, H-4), 4.53 (1H, m, H-2), 4.44 (1H, dd, J = 10.5, 5.0 Hz, H-1), 4.31 (1H, dd, J = 10.5, 2.8 Hz, H-1), 4.17 (1H, dd, J = 5.0, 4.1 Hz, H-3), 1.98 (12H, 4A-MeCO), 1.16-1.27 (24H, br s, CH2-6-10,15-21), 0.87 (3H, t, J = 6.7 Hz, CH3-22); GC-MS: m/z 538.

Acetylsphingamine Derived From 3 [a]D24 = + 23.1 (c 0.0013); 1H-NMR (CDCl3, 400 MHz): 8.06 (1H, d, J = 8.0 Hz, NH), 5.31 (2H, dt, J = 16.0, 5.6 Hz, H-9,10), 4.61 (1H, td, J = 6.5, 4.0 Hz, H-4), 4.51 (1H, m, H-2), 4.42 (1H, dd, J = 10.8, 5.0 Hz , H-1), 4.31 (1H, dd, J = 10.8, 2.5 Hz, H-1), 4.17 (1H, dd, J = 4.0, 3.2 Hz, H-3), 2.01 (12H, 4A-MeCO), 1.17-1.28 (20H, br s, CH2-6,7,12-19), 0.87 (3H, t, J = 6.5 Hz, CH3-20); GC-MS: m/z 527.

Acetylsphingamine Derived From 4 [a]D24 = + 17.3 (c 0.0012); 1H-NMR (CDCl3, 400 MHz): 8.11 (1H, d, J = 7.8 Hz, NH), 4.56 (1H, td, J = 6.2, 4.0 Hz, H-4), 4.51 (1H, m, H-2), 4.48 (1H, dd, J = 11.0, 5.0 Hz, H-1), 4.31 (1H, dd, J = 11.0, 2.5 Hz, H-1), 4.14 (1H, dd, J = 4.0, 3.0 Hz, H-3), 2.02 (12H, 4A-MeCO), 1.19-1.27 (18H, br s, CH2-6-14), 0.88 (3H, t, J = 6.6 Hz, CH3-15); GC-MS: m/z 443.

Acetylsphingamine Derived From 5 [a]D24 = + 14.3 (c 0.0011); 1H-NMR (CDCl3, 400 MHz): 8.13 (1H, d, J = 7.8 Hz, NH), 4.51 (1H, td, J = 6.2, 4.1 Hz, H-4), 4.52 (1H, m, H-2), 4.47 (1H, dd, J = 10.8, 5.0 Hz, H-1), 4.31 (1H, dd, J = 10.8, 3.0 Hz, H-1), 4.17 (1H, dd, J = 4.1, 3.4 Hz H-3), 1.98 (12H, 4A-MeCO), 1.21-1.27 (6H, br s, CH2-10,15,16), 0.85 (3H, t, J = 6.5 Hz, CH3-17); GC- MS: m/z 468.

Results and Discussion

Compound 1 was isolated as white amorphous solid, which displayed molecular ion peak in HR-EI-MS at m/z 749.7238 for a molecular formula C48H95NO4. The IR spectrum of 1 revealed hydroxyl group (3450 cm-1), secondary amine (3332 cm-1), amide function (1654 cm-1) and olefinic system (1625 cm-1). An amide hydrogen nucleus resonated in the 1H-NMR of 1 (Table-1) at 7.41 (1H, d, J = 8.0 Hz), which was correlated in COSY spectrum with a proton at 4.02 (1H, m), which in turn was correlated with a carbon at 51.1 in HSQC spectrum and was thus attested for azomethine. Two oxymethines displayed their positions in the 1H-NMR spectrum at 3.98 (1H, dd, J = 8.4, 3.6 Hz) and 3.46 (1H, dd, J = 8.0, 3.6 Hz), besides, two resonances due to an oxymethylene at 3.75 (1H, dd, J = 11.6, 3.6 Hz) and 3.66 (1H, dd, J = 11.6, 5.2 Hz) and one due to an olefinic system at 5.33 (2H, br m, W1/2 = 19.5 Hz) were also observed.

The same spectrum further displayed the resonance of an aliphatic methylene at 2.11 (2H, t, J = 7.8 Hz), which showed cross peak in COSY spectrum with a broad singlet of several hydrogen at 1.18-1.96, characteristic of an aliphatic chain. The signal of triplet methyl at 0.82 (6H, J = 7.1 Hz) substantiated the above evidence. Therefore, the above-discussed data could be attested for a sphingolipid [10,11]. The 13C-NMR spectrum of 1 (Table-1) supported the sphingolipid skeleton of 1, as the carbon nucleus resonating at 173.0 was attributed to the amide carbonyl, with other carbon resonances were observed at 131.8 (CH), 128.7 (CH), 75.7 (CH), 72.0 (CH) and 61.3 (CH2), 22.7- 32.4 (aliphatic chain) and 14.1 (CH3). COSY and HMBC analyses (Table-1) of 1 helped to fix various substitutions and overall molecular structure. The number of carbon atoms in fatty acid chain was determined due to the characteristic mass fragments at m/z 268, 253 and 211.

The fragment ions at m/z 210 and 539 (Fig. 2) were attributed to the McLafferty fragmentation process. Similarly, the length of the amine chain containing a double bond could be determined due to fragment ions at m/z 496 and 481 (Fig. 2), whereas, the fragment ions at m/z 293 and 239 were attributed to the allelic cleavage that revealed the location of olefinic system. The geometry of this olefin was found to be E in nature due to the C NMR shifts of C-11 ( 34.3) and C-14 ( 32.4), and J value of the signal at half height (W1/2 = 19.5 Hz) [12]. On methanolysis [13], compound 1 gave the aliphatic amine and the methyl ester of fatty acid, which were acetylated and were identified through GC-MS as 2-acetamino-1,3,4- triacetoxyunieicosene (m/z 666) and methyl heptadecanoate (m/z 283), respectively. The stereochemistry at various centers could be determined as 2S,3S,4R, due to the optical rotations of 1 ([a]D = + 19.3) and its methylated amine ([a]D = + 21.1) [14,15].

Based on the above disc ussion and the evidences, 1 was identified as (2S,3S,4R,12E)-2- {[heptdecanoyl]amino}unieicos-12-ene-1,3,4-triol and names as seriphidalin A. The IR spectrum of compound 2 was identical to that of 1, whereas, its HR-EI-MS (m/z 709.6512) depicted the molecular formula as C44H87NO5. The NMR data of 2 (Table-2) was also nearly similar to that of 1 with fewer differences. The 1H-NMR spectrum of 2 showed the absence of a triplet methylene ( 2.10) as was observed in 1, instead it displayed an additional oxymethine proton at 3.95 (1H, dd, J = 8.5, 3.0 Hz), which was correlated with a carbon in C-NMR spectrum at 71.9 (Table-2). This observation clearly indicated that 2 must be C-2 oxidized product [16] of one of the analogue of compound 1. As a whole the molecule could be assembled and confirmed through 1H-1H COSY and HMBC correlations (Table-2).

The number of carbon atoms in fatty acid and the amine chains, and position of the double bond were elucidated due to the combination of mass spectrometry (Fig. 2) and methanolysis, whereas, the stereochemistry as 2S,3S,4R,2R could be established due to optical rotation values of 2 ([a]24D = + 37.6), its methanolysis products ([a] D = 9.1 and + 29.1), and in comparison with the data of known similar sphingolipids [11,15].

The above data finally led to the structure of seriphidalin B (2) as (2S,3S,4R,12E)-2-{[(2R)-2- hydroxydodidecanoyl]amino}trididec-12-ene-1,3,4- triol. Compound 3 with molecular formula C42H83NO5 was also found to be a sphingolipid as it displayed similar spectral features to that of 1 and 2. The main difference lies in the number of carbon atoms in fatty acid (22) and amine (20) chains, which could be determined through EI-MS (Fig. 2).

It also revealed the position of double bond between carbon 9 and 10 in the base chain. This deduction was substantiated through the similar chemical degradation/modification as were done for 1 and 2, which yielded acid and base chains as methyl-2- acetoxydodidecanoate (m/z 411) and 2-acetamino- 1,3,4-triacetoxydodecene (m/z 527). The stereochemistry could also be established due to the optical rotation value and found similar to that of 2. The combination of the whole spectroscopic data and comparison with the previously discussed data finally led to the structure of 3 as (2S,3S,4R,9E)-2-{[(2R)-2- hydroxydodidecanoyl]amino}didec-9-ene-1,3,4-triol and named as seriphidalin C.

FAB-MS in negative mode of compound 4 exhibited pseudo-molecular ion at m/z 702.5127 corresponding to the molecular formula C38H73NO10 with three DBE. The NMR data (Table-4) of 4 and its comparison with those of the data of compounds 1-3 revealed a glycosphingolipid nature of 4. The 1H- NMR signals for a sphingolipid part were observed at their usual position, whereas, the glycon moiety was depicted due to the resonance of anomeric proton at 4.21 (1H, d, J = 7.6 Hz, H-1''), with other sugar protons displayed their positions at 3.14 (1H, m, H-2''), 3.31 (1H, dd, J = 7.4, 2.0 Hz, H-3''), 3.16 (1H, d, J = 2.0 Hz, H-4''), 3.18 (1H, m, H-5''), 3.76 (1H, dd, J = 10.8, 4.9 Hz, H-6''), 3.62 (1H, dd, J = 10.8, 2.9 Hz, H-6''). The amount of coupling constant (J = 7.6 Hz) of anomeric hydrogen could be attributed to a AY-hexose, whereas, the smaller coupling constant of H-4 (J = 2.0) indicated the hexose could be galactose, which was substantiated through 13C NMR shifts of the sugar unit.

The downfield shift ( 68.4) of C-1 in 13C-NMR spectrum (Table-4) revealed the glycosylation at C-1, which could further be confirmed through HMBC analysis, in which the anomeric proton ( 4.21) exhibited correlation with C-1 ( 68.4). The combination of the whole data and comparison with the reported values of similar compounds, compound 4 was found to be a glycosphingolipid [11,15]. Number of carbon atoms in acid (C17) and base (C15) chains was determined through mass fragmentation pattern (Fig. 2) and methanolysis, whereas, the position of C=C could be determined at C-5,6 in fatty acid chain. The stereochemistry at the stereogenic centers was determined by the same method as done for 2,3 and was found similar. Based on these evidences, seriphidalin D (4) was assigned the structure as (2S,3S,4R)-2-{[(2R,5E)-2-hydroxyheptadec-5- enoyl]amino} pentadecane-1,3,4-triol-1-O-AY-D- galactopyranoside.

Compound 5 showed a pseudo-molecular ion peak in the (-ve)-HR-FAB-MS at m/z 588.3760 corresponding to the molecular formula C30H55NO10 with four DBE. The 1H- and 13C-NMR spectra (Table-5) of 5 were nearly identical to that for 4 as it afforded the usual signals for glycosphingolipid skeleton that accommodated two DBE. The remaining two DBE could be attributed to two E- double bonds at 5.41 (2H, br m, W1/2 = 20.1 Hz) and 5.31 (2H, br m, W1/2 = 20.1 Hz) in the 1H-NMR spectrum. The length of both fatty acid and amine chain (containing two double bonds) were fixed through COSY spectrum (Table-5) and mass fragmentation pattern (Fig. 2) and methanolysis as was done for 1-4. The E-geometry of both the double bonds could be substantiated due to the carbon shifts of C-6 ( 33.0), C-9 ( 33.9), C-11 ( 32.7) and C-14 ( 33.7).

The presence of glucose in 5 was confirmed because of the resonances at H [4.28 (1H, d, J = 8.0 Hz, H-1), 3.16 (1H, m, H-2), 3.31 (1H, m, H-3), 3.25 (1H, m, H-4), 3.26 (1H, m, H- 5), 3.67 (1H, m, H-6), 3.87 (1H, m, H-6)] and C (104.6, 78.0, 77.8, 75.0, 72.8, 62.6). The downfield shift ( 69.9) of C-1 depicted the sugar linkage at this carbon, which was further confirmed through HMBC spectrum (Table-5); in which oxymethylene ( 4.06 and 3.79) exhibited HMBC correlations with anomeric carbon at 104.6 and the anomeric proton ( 4.28) correlated with C-1 ( 69.9). Further the methanolysis of 5 produced a fatty acid methyl ester, an aliphatic chain base and a mixture of - and AY- anomer of methyl glucoside. GC-MS analysis identified the fatty acid, whereas, the optical rotation of methyl glucoside ([a]D = + 74.3) was comparable to the value [a]D = + 77.3 of D-glucose [17,18]. The stereochemistry on the chiral centers was confirmed through optical rotations and found similar to that for 1-4.

Based on the above data, 5 was identified as (2S,3S,4R,7E,12E)-2-{[(2R)-2- hydroxyheptanoyl]amino}heptadeca-7,12-diene- 1,3,4-triol-1-O-AY-D-glucopyranoside and named as seriphidalin E.

Acknowledgements

The authors are thankful to Higher Education Commission (HEC) Pakistan and Alexander von Humboldt (AvH) Foundation, Germany for providing equipment facilities.

References

1. L. E. Watson, Molecular Phylogeny of Subtribe Artemisiinae (Asteraceae), including Artemisia and its Allied and Segregate Genera, Biomed. Cent. Evolut. Biol., 2, 17 (2002).

2. L. M. Shultz, Flora of North America, 199 (2006).

3. Flora of Pakistan, 207, 156 (2002) http://www.efloras.org/florataxon.aspxflora_id=5andta xon_id=242101188

4. Y. R. Deng, A. X. Song and H. Q. Wang, Chemical Components of Seriphidium Santolium, Poljak, J. Chin. Chem. Soc., 51, 629 (2004).

5. N. Shafiq, N. Riaz, S. Ahmed, M. Ashraf, S. A. Ejaz, I. Ahmed, M. Saleem, M. I. Touseef, R. B. Tareen, A. Jabbar, Bioactive Phenoics from Seriphidium Stenocephalum, J. Asian Nat. Prod. Res., 15, 286 (2013).

6. J. P. Singh, A. K. Singh, A. Singh and R. Ranjan, Chemical Constituents of Artabotrys odoratissimus (seeds), Ras. J. Chem., 2, 156 (2009).

7. F. Bohlmann, C. Zdero, R. M. Kings and H. Robinsons, Neue Cadinen-Derivate Und Andere Inhaltsstoffe Aus Chromolaena-Arten, Phytochem., 18, 1177 (1979).

8. W. H. Hui and M. M. Li, Lupene Triterpenoids from Glochidion eriocarpum, Phytochem., 15, 561 (1976).

9. V. U. Ahmad and A. Rehman, Handbook of Natural product data, Elsevier, Vol. II. p. 516 (1994).

10. H. C. Kwon, K. C. Lee, O. R. Cho, Y. Jung, S. Y. Cho, S. Y. Kim and K. R. Lee, Sphingolipids from Bombycis Corpus 101A and their Neurotrophic Effects, J. Nat. Prod., 66, 466 (2003).

11. N. Riaz, S. A. Nawaz, N. Mukhtar, A. Malik, N. Afza, S. Ali, S. Ullah, P. Muhammad and M. I. Choudhary, Isolation and Enzyme-Inhibition Studies of the Chemical Constituents from Ajuga bracteosa, J. Chem. and Biodiv., 4, 72 (2007).

12. M. S. Ali, W. Ahmed, M. Saleem and M. A. Ali, Longifoside-A and -B: Two New Cerebrosides from Mentha longifolia (Lamiaceae), Nat. Prod. Res., 20, 715 (2006).

13. P. Muralidhar, M. M. Kumar, N. Krishna, C. B. Rao and D. V. Rao, New Sphingolipids and a Sterol from a Lobophytum Species of the Indian Ocean, Chem. Pharm. Bull., 53, 168 (2005).

14. H. S. Garg and S. Agrawal, A Novel Sphingosine Derivative from the Sponge Spirastrella inconstans, J. Nat. Prod., 58, 442 (1995).

15. T. Natori, M. Morita, K. Akimoto and Y. Koezuka, Agelasphins, Novel Antitumor and Immunostimulatory Cerebrosides from the Marine Sponge Agelas mauritianus, Tetrahedron, 50, 2771 (1994).

16. R. F. Wang, R. N. Liu, T. Zhang and T. Wu, A New Natural Ceramide from Trollius chinensis Bunge, Molecules, 15, 7467 (2010).

17. J. M. Gaoa, L. Hua, Z. J. Donga and J. K. Liua, New Glycosphingolipid from Basidiomycete polyporus ellisii, Lipids, 36, 521 (2001).

18. W. Jin, K. L. Rinehart and E. R. Jares-Erijman, Ophidiacerebrosides: Cytotoxic Glycosphingolipids Containing a Novel Sphingosine from a Sea Star, J. Org. Chem., 59, 144 (1994).
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