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New acylated flavone and cyanogenic glycosides from Linum grandiflorumMagdy M. D. Mohammed abc; Lars P. Christensen a; Nabaweya A. Ibrahim c; Nagwa E. Awad c; Ibrahim F.Zeid d; Erik B. Pedersen b

a Department of Food Science, Danish Institute of Agricultural Sciences, Research Center Aarslev, Aarslev,Denmark b Nucleic Acid Center, Institute of Physics and Chemistry, University of Southern Denmark, OdenseM, Denmark c Pharmacognosy Department, National Research Center, Cairo, Egypt d Faculty of Science,Chemistry Department, El-Menoufia University, El-Menoufia, Egypt

Online Publication Date: 01 January 2009

To cite this Article Mohammed, Magdy M. D., Christensen, Lars P., Ibrahim, Nabaweya A., Awad, Nagwa E., Zeid, Ibrahim F. andPedersen, Erik B.(2009)'New acylated flavone and cyanogenic glycosides from Linum grandiflorum',Natural ProductResearch,23:5,489 — 497

To link to this Article: DOI: 10.1080/14786410802364168

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Natural Product ResearchVol. 23, No. 5, 20 March 2009, 489–497

New acylated flavone and cyanogenic glycosides from Linum grandiflorum

Magdy M.D. Mohammedabc*, Lars P. Christensena, Nabaweya A. Ibrahimc,Nagwa E. Awadc, Ibrahim F. Zeidd and Erik B. Pedersenb

aDepartment of Food Science, Danish Institute of Agricultural Sciences, Research Center Aarslev,Aarslev, Denmark; bNucleic Acid Center, Institute of Physics and Chemistry, University ofSouthern Denmark, Odense M, Denmark; cPharmacognosy Department, National Research Center,Cairo, Egypt; dFaculty of Science, Chemistry Department, El-Menoufia University,El-Menoufia, Egypt

(Received 8 March 2008; final version received 8 August 2008)

The first investigation of Linum grandiflorum resulted in the isolation of one newacylated flavone O-diglycoside known as luteolin 7-O-a-D-(60 00-E-feruloyl)gluco-pyranosyl (1! 2)-�-D-glucopyranoside, and one new cyanogenic glycosideknown as 2-[(30-isopropoxy-O-�-D-glucopyranosyl)oxy]-2-methylbutanenitrile,together with four known flavonoid glycosides, three known cyanogenicglycosides and one alkyl glycoside. The new compounds were structurallyelucidated via the extensive 1D, 2D NMR and DIFNOE together with ESI-TOF-CID-MS/MS and HR-MALDI/MS.

Keywords: Linum grandiflorum; linaceae; acylated flavonoids; cyanogenic glyco-side; ESI-TOF-CID-MS/MS of flavonoids

1. Introduction

Linum grandiflorum (Linum; Linaceae) is native to North Africa and Southern Europe,and it can be cultivated in moderate climates (Duke, 2002; Hortus, 1976). Seed oil ofL. grandiflorum is used to improve fertility, is cyanogenetic, laxative, analgesic, emollient,expectorant and resolving (Bown, 1995), and is used as a treatment for cancer (Phillips &Foy, 1991). A literature survey of this species shows the following: anthocyanidintriglycoside was isolated from the flowers (Kenjiro, Norio, Koji, Atsushi, & Toshio, 1995)and there has been an evaluation of the fatty acid composition of the plant seeds (Plesser,1966; Yermanos, 1966; Yermanos, Bcard, Gill, & Anderson, 1966). So we started aphytochemical investigation of the aerial parts (leaves and seeds), which resulted in theisolation of two new compounds (1 and 6), together with eight known compounds, and thenew metabolites were structurally elucidated by both physical and chemical methods andon the bases of 1D, 2D NMR, DIFNOE spectra, ESI/MS and MALDI/MS.

2. Results and discussion

The MeOH soluble portion of the methanolic extract was subjected to fractionation withpreparative reversed-phase HPLC as described in the experimental section, and this

*Corresponding author. Email: melhenawy111@gmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

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DOI: 10.1080/14786410802364168

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resulted in the isolation of 10 compounds: flavone glycosides (1–5), which all gave a

positive Shinoda and Molish–Udransky reactions, four cyanogenic glycosides (6–9) and

one alkyl glycoside (10).Compounds 2–5, 7–9 and 10, as shown in Figure 1, were identified as vicenin 1, vicenin

2, vicenin 3 (Seikel, Chow, & Feldman, 1966), luteolin 7-O-glucoside (glucoluteolin)

(Harborne, 1994), linamarin, lotaustralin, neolinustatin (Cecil, David, Roger, Miller, &

Oscar, 1980) and butan-2-O-�-D-glucopyranoside (Cable & Nocke, 1975), by comparison

of their spectroscopic data with those in the literature.Compound 1 was obtained as a yellow amorphous solid, and its molecular

formula was determined to be C37H38O19 by HRESIMS. The UV spectrum of 1 was

similar to that of luteolin 7-O-glycoside; shift reagents confirmed that the 7-hydroxy group

O

O

OH

OH

R3O

R2

R1

R4

Compound R1 R2 R3 R4

Vicenin 1 Xylosyl Glucosyl H H

Vicenin 2 Glucosyl Glucosyl H H

Vicenin 3 Glucosyl Xylosyl H H

Glucoluteolin H H Glucosyl OH

C

CH3

O

HOOH

HO

O

R1O

R2R3

Compound R1 R2 R3

Linamarin H CH3 CN

Lotaustralin H CH2CH3 CN

Neolinustatin O-β-D-glucopyranosyl CH2CH3 CN

Butan-2-O-β-D-glucopyranoside

O-β-D-glucopyranosyl CH2CH3 H

Figure 1. The isolated known compounds.

490 M.M.D. Mohammed et al.

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was not free. The 1HNMR spectrum in DMSO-d6 (Table 1) showed a singlet signal

(�H6.67) characteristic for the flavone type, a hydrogen-bonded hydroxyl proton

(�H12.95), two anomeric glucose protons at (�H 5.24) and (�H 5.18) representing �- anda-glucopyranoside, which were confirmed from the 3JH-1,H-2 coupling constant (7.3 and

3.7Hz), respectively (Day & Harborne, 1989), as determined from acid hydrolysis of 1.

H2SO4 released a-, �-glucose and luteolin, which were identified by PPC and TLC

comparison with authentic samples; alkaline hydrolysis of 1 gave ferulic acid, which was

identified by comparing with an authentic sample (Mabry, Markham, & Thomas, 1970).

The attachment positions of the two glucose moieties were established, as shown in

Figure 2, by the negative nuclear Overhauser enhancement (DIFNOE) difference

spectroscopy (David & Michael, 1989, 2000; Kondo, Kawai, Tamura, & Gota, 1987).

Table 1. 1H NMR datab for compounds 1 and 6.

� 1H (mult, J¼Hz)

Position 1a 6a

3 6.66 (1H, s) 1.46 (2H, q, 7.6)4 0.86 (3H, t, 7.6)6 6.46 (1H, d, 2.1)8 6.74 (1H, d, 2.1)20 7.41 (1H, brd, 8.5)50 6.95 (1H, d, 8.4)60 7.41 (1H, brd, 8.5)

Glc I100 5.24 (1H, d, 7.3) 4.29 (1H, d, 7.7)200 3.58c (1H, brd, 8.0) 3.06 (1H, dd, 7.7)c

300 3.00–3.55 (3H, overlapped)d 4.74 (1H, t, 9.2)c

400 3.00–3.55 (3H, overlapped)d 3.24 (1H, brd, 8.1)c

500 3.00–3.55 (3H, overlapped)d 3.48 (1H, m)c

600 (3.78–3.90)c (2H, m)d 3.64 (2H, m)c

Glc II1000 5.18 (1H, d, 3.7)2000 3.26c (1H, brd, 9.0)3000–50 00 3.00–3.55 (3H, overlapped)d

6000 (4.15–4.27)c (2H, m)d

Feruloyl at C-60 00

20000 7.12 (1H, d, 1.5)50000 6.70 (1H, d, 8.1)60000 6.89 (1H, brd, 8.1)a- 6.37 (1H, d, 15.8)�- 7.45 (1H, d, 15.8)OMe 3.73 (3H, s)CH3 at C-2 1.27 (3H, s)CH¼(2CH3) 3.67 (1H, sep, 6.2)c

CH¼(2CH3) 1.14 (6H, d, 6.2)

Notes: aJ-values given in Hz in parentheses.bIn DMSO-d6.cAssignments were confirmed by 1H�1H COSY.dMultiplicity was not determined due to overlapping and/or broadening of thesignals.

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Irradiation at H-1 (�H 5.24) of Glc I caused a strong negative nuclear Overhauserenhancements (NOEs) at (�H 5.18, �H6.47 and �H 6.74) corresponding to H-1 of Glc II,H-6 and H-8, respectively, confirmed the attachment of Glc I to be at C-7 hydroxyl ofluteolin, and irradiation at H-1 (�H5.18) of Glc II caused a strong negative NOE at �H 5.24H-1 of Glc I indicated that the interglycosidic linkage to be a-D-glucopyranosyl (1! 2)-�-D-glucopyranoside. 2D COSY spectra showed a cross-peak of two doublet signals at(�H6.37) and (�H 7.45) with J value 15.80Hz, corresponding to the AM system, the down-field shift of 6-CH2OH of the Glc II at (�H 4.15–4.27) assigned from 1H–1H COSY, and theirradiation of the a-H of the E-feruloyl at (�H 6.37) caused a weak negative NOE at�H 4.15–4.27 which confirmed the attachment of E-feruloyl to be at the C-6000 hydroxyl ofGlc II. This proposed structure was confirmed by analysis with nano-ESI-CID-MS/MS,and it has been investigated that the interglycosidic linkage type (1! 2) or (1! 6) can beconcluded from the relative abundances of the Y0 and Y1 ions obtained from theprotonated molecule (Es-Safi, Krhoas, Einhorn, & Ducrot, 2005; Ma, Vedernikova,Van Den Heuvel, & Claeys, 2000), and the relative abundance of the Y* ion providesinformation on the nature of the aglycone and on the linkage position of the disaccharide(Cuyckens, Rozenberg, Hoffmann, & Claeys, 2001; Ma, Cuyckens, Van Den Heuvel, &Claeys, 2001). Characterisation of the aglycone part and the interglycosidic linkage ofcompound 1 was done by CID-MS/MS spectra for the [MþH]þ. Figure 3 shows MIspectra of 1 with [MþH]þ ion at m/z 787, protonated aglycone Yþ0 ion at m/z 287,

Glc I

Glc II

1″ O

1′′′

2″″

O

O

OH

OH

OH

H

H

A C

B

O H

HOHO

HO

O

HOHO

HO

H

O

C

O

O

H3CO

HO

Hb

Ha

H

H

C

CH3

CN

O

HO OHO

O

CH

HO

CH2H

H3C CH3

CH3

H

1

2

3 43′

Figure 2. Nuclear Overhauser enhancements are indicated by arrows and 1H�1H COSY is indicatedby bold bonds for 1 and 6.

492 M.M.D. Mohammed et al.

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fragments at m/z 177 and 339 of feruloylþ and feruloylhexoseþ, respectively (Cuyckenset al., 2003), and the weak abundance of the Y* ion at m/z 625 confirmed the flavone type,where the high relative abundance of Y0 at m/z 287 compared with Y1 at m/z 449 (Y04Y1)confirmed the interglycosidic linkage to be (1! 2) agreed with DIFNOE spectra, and itwas confirmed from different NOE the exact location of the E-feruloyl was at the C-6000

hydroxyl of Glc II, from the CID-MS/MS spectra; the presence of molecular ion 0,4X1 atm/z 321 confirmed the attachment of E-feruloyl at the C-6000 hydroxyl of Glc II (Bylka,Franski, & Stobiecki, 2002). Hence, compound 1 was confirmed to be luteolin 7-O-a-D-(6000-E-feruloyl)glucopyranosyl (1! 2)-�-D-glucopyranoside.

Compound 6 was isolated as a yellow amorphous solid, and its molecular formula wasdetermined to be C14H25NO6 by HRMALDIMS. The IR spectral absorption showed thepresence of hydroxyl (3499 cm�1), C–H stretching (2859–2928 cm�1), nitrile (2252 cm�1),and ether stretching (1011–1094 cm�1) in the molecule. The UV spectrum showedabsorption band at 274 and 320 nm (sh). The 1HNMR in DMSO-d6 (Table 1) showed thespectrum looks like that of compound 8, with extra three signals: doublet (�H1.14 and�C 22.43), multiplet (�H3.67 and �C 73.80), and triplet (�H4.74 and �C 81.98). Analysis of1HNMR and 1H�1H COSY spectra showed a cross-peak correlate triplet methyl signal(�H0.86) with quartet methylene signal (�H 1.46), a cross-peak correlate doublet signalat �H1.14 (6H, –CH¼(2CH3)) with the methene group at �H 3.67 (1H, –CH¼(2Me))corresponding to the isopropyl group, the downfield shift of the methene group (�H3.67)suggested the form of the isopropoxy group, which indicated an attachment to the30-hydroxy of glucose because of the downfield shift of H-30 (�H 4.74), C-30 (�C 81.98); thiswas confirmed by the irradiation of the methene proton (�H 3.67), which caused a strongNOE enhancement at (�H4.74) and (�H1.14) corresponding to H-30 and the doublet signalof the two methyls, respectively. For 13CNMR, see Section 3.

3. Experimental section

3.1. General experimental procedures

UV spectra: Shimadzu MPS-2000. NMR: 300MHz (Varian VXR-Unite) (1H, 1H�1HCOSY and NOE difference) in DMSO-d6. 2D spectra were obtained using a pulse

Figure 3. Collision induced dissociation product ion spectra obtained for [MþH]þ ions and ionnomenclature for 1.

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sequence supplied from Varian. Chemical shifts were given in values (ppm) relative to

trimethylsilane (TMS) as an internal reference. High-resolution ESI/MS: was obtained

using nano-electrospray tandem (MS/MS) mass spectrometry on a hybrid quadrupole

time-of-flight (Q-TOF) MS instrument equipped with Protana’s nano-ESI source for HR

ESI-MS and nano-spray needles from Proxeon (Applied Biosystems/MDS Sciex)

(QSTAR, prototype, PE-Sciex, Canada). Tandem (MS/MS) spectra were interpreted

using the programs BioMultiView (PE Sciex, Canada) and GPMAW (Lighthouse Data,

Denmark). For accurate mass measurements, the instrument was calibrated using a

10mM solution of NaI in isopropanol/water. The instrument’s mass scale was calibrated

for each determined ion mass using the cluster ions Nanþ1Iþn closest to the sought mass.

Collision induced dissociation (CID) spectra were obtained using N2 in the collision cell

and collision energies between 30–40 eV (Elab). Analyses were first conducted using ESI/

MS in positive mode to obtain ionised molecular species. Then tandem MS/MS spectra

were obtained by CID of the [MþH]þ ion (Nielsen, Freese, Cornett, & Dragsted, 2000;

Hakkinen & Auriola, 1998). Protonation is believed to occur preferentially in the aglycone

part of the molecule, in particular at the carbonyl oxygen atom. Charge delocalisation in

the C-ring can lead to a protonated molecular species, which are highly stabilised by

resonance. Subsequent charge-remote rearrangements take place, resulting in the Y0 and

Y1 ions, most likely involving hydrogen rearrangement from hydroxyl groups, which can

sterically approach the glycosidic bonds (Ma et al., 2000). The product ion spectra were

obtained in the continuous mode of acquisition of the quadruple analyser. Sequence ion

notations have been used, e.g. the Y1 and Y0 corresponding to the [MþH� (176uþ 162)]þ

and [MþH� (176uþ 162þ 162)]þ ions, respectively (Domon & Costello, 1988). An

irregular ion [MþH� 162]þ corresponding to the loss of an internal dehydrated glucose

residue from the precursor ions is denoted as a Y* ion (Ma et al., 2000). High-resolution

MALDIMS was recorded on an IonSpec Fourier Transform Ion Cyclotron resonance

mass spectrometer. HPLC consists of L-6200 Intelligent Pump (Merck-HITACHI)

connected with UV-VIS Detector SPD-10AV (SHIMADZU). HPLC solvents used for all

analyses were of grade M (Sigma-Aldrich Chemie, UK) and ultra-pure water; the

petroleum ether, chloroform and methanol for plant extraction were of AR grade; Kiesel

gel 60 F254 (Merck) was used for analytical TLC.

3.2. Plant material

The aerial parts (leaves and seeds) of L. grandiflorum were collected in March 2006 from

El-Orman Garden, Giza Governorate, Egypt. The plant samples were kindly identified by

Miss Tressa Labib, Head of Specialists at the Garden. A voucher specimen of the plant is

kept at the Herbarium of the National Research Center.

3.3. Extraction and isolation

The air-dried aerial parts (leaves (2.4 kg) and seeds (184.46 gm)) of L. grandiflorum were

extracted with hot MeOH which was defatted with petroleum ether and then the residue

was dissolved in H2O, followed by fractionation with CHCl3. Then the H2O soluble

fraction was concentrated till dryness and extracted with MeOH, which was examined with

TLC (silica gel 60 F254, Fluka) for flavonoids with different solvent systems; the spots were

494 M.M.D. Mohammed et al.

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visualised by the dipping solutions (Jork, Funk, Fischer, & Wimmer, 1990, 1994; Marston

& Hostettmann, 2006). The MeOH fraction (15.5 gm) was dissolved in MeOH–H2O (1 : 1)

and subjected to fractionation (8mL injected into 10mL loop, after prefiltration with

nylon filter 0.45mm) using preparative ODS-HPLC (20’� 250mm, Develosil ODS-HG-5,

Nomura Chemicals) at room temperature. Solvent A¼ 1% HCOOH and Solvent

B¼ 100% MeOH were used in the elution profile 90% A, 50% A, and finally with

100% B, monitoring at 340 nm, with flow rate 3mLmin�1. This accumulated three main

fractions (I, II and III). After TLC examinations, fraction II (50% A) was chosen to be

refractionated using preparative ODS-HPLC at room temperature, linearly with 60% A,

monitoring at 280 nm, with 0.8mLmin�1 flow rate, and led to the isolation of 1 (5mg),

2 (12mg), 3 (8mg), 4 (15mg), 5 (10mg), 6 (10mg), 7 (8mg), 8 (12mg), 9 (6mg) and

10 (11mg).

3.3.1. Luteolin 7-O-a-D-(6000-E-feruloyl)glucopyranosyl (1!2)-�-D-glucopyranoside (1)

Pale yellow amorphous; UV �max (MeOH): 252, 269, 300(sh), 337; (NaOMe): 268, 390;

(AlCl3): 275, 300(sh), 325, 360(sh), 423; (AlCl3/HCl): 277, 298(sh), 325, 385; (NaOAc):

265(sh), 269(sh), 335, 408; (NaOAc/H3BO3): 258, 296(sh), 335, 376; 1HNMR

(DMSO-d6, 300MHz) � 5.24 (1H, d, J¼ 7.32Hz, H-100 of Glc I), 3.58 (1H, brd,

J¼ 8.0Hz, H-200), 3.78–3.90 (2H, m, H-600), 5.18 (1H, d, J¼ 3.66Hz, H-1000 of Glc II),

3.26 (1H, brd, J¼ 9.0Hz, H-2000), 4.15–4.27 (2H, m, H-6000), 3.00–3.55 (6H, overlapped,

H-300, 400, 500, 3000, 4000 and 5000) 6.67 (1H, s, H-3), 6.46 (1H, d, J¼ 2.1Hz, H-6), 6.74

(1H, d, J¼ 2.1Hz, H-8), 7.41 (2H, brd, J¼ 8.5Hz, H-20, 60), 6.95 (1H, d, J¼ 8.4Hz,

H-50), 3.73 (3H, s, feruloyl-OMe), 7.12 (1H, d, J¼ 1.5Hz, H-20000), 6.70 (1H, d,

J¼ 8.1 Hz, H-50000), 6.89 (1H, brd, J¼ 8.1 Hz, H-60000), 6.37 (1H, d, J¼ 15.80Hz, H-a),7.45 (1H, d, J¼ 15.8Hz, H-�); DIFNOE and COSY spectra: (Figure 2); HRESIMS

(Positive mode) m/z 809.1917 [MþNa]þ (Calcd for C37H38O19Na, 809.1905) (see

Figure 3 for the fragmentations).

3.3.2. 2-[(30-isopropoxy-O-�-D-glucopyranosyl)oxy]-2-methylbutanenitrile (6)

Yellow amorphous; IR �max 3499, 2859, 2928, 2252, 1094, 1011 cm�1; UV �max (MeOH):

274, 320 (sh); 1HNMR (DMSO-d6, 300MHz) � 0.86 (3H, t, J¼ 7.61Hz, CH3-4), 1.14 (6H,

d, J¼ 6.23Hz, –CH¼(2CH3)), 1.27 (3H, s, CH3), 1.46 (2H, q, J¼ 7.61Hz, CH2-3), 3.06

(1H, dd, J¼ 7.69Hz, H-20), 3.24 (1H, brd, J¼ 8.05Hz, H-40), 3.48 (1H, m, H-50), 3.64 (2H,

m, H-60), 3.67 (1H, sep, J¼ 6.23 Hz, –CH¼(2Me)), 4.29 (1H, d, J¼ 7.69Hz, H-10), 4.74

(1H, t, J¼ 9.16Hz, H-30); 13CNMR (DMSO-d6, 125MHz) �C 121.5 (C�N), 74.5 (C-2),

32.9 (C-3), 8.6 (C-4), 104.5 (C-10), 74.6 (C-20), 81.9 (C-30), 71.9 (C-40), 78.1 (C-50), 62.6

(C-60), 24.6 (CH3-2), 73.8 (–CH¼(2CH3)), 22.4 (–CH¼(2CH3)); DIFNOE and COSY

spectra: (Figure 2); HRMALDIMS (Positive mode) m/z 326.1579 [MþNa]þ, (Calcd

for C14H25NO9Na, 326.1575) m/z 303 [MþH]þ, m/z 284 [MþNa� (:C(CH3)2)]þ, m/z 611

[2MþNa�H2O]þ.

Acknowledgements

The authors are grateful to the Danish Institute of Agricultural Sciences, Research Center Aarslev,Denmark for financial support, and to the University of Southern Denmark, Odense, Denmark, forproviding the facilities for instrumental analysis.

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