Letter - spectral assignment Received: 20 February 2014
Revised: 21 July 2014
Accepted: 1 August 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4134
A new secoiridoid glycosidic lignan ester from the leaves of Olea ferruginea Muhammad Ali Hashmi,a Afsar Khan,a* Umar Farooq,a* Mubeen Rani,b Viqar Uddin Ahmadb and Abdur Rahman Khana
Introduction Olea ferruginea, an indigenous medicinal plant belonging to family Oleaceae, is locally known as Kahoo or Khoona in Pakistan.[1] Folk practitioners use its leaves and bark to treat various ailments. Different parts of this plant such as leaves, bark, and gum possess various activities. Its stem bark is used by local people for fever.[2] The oil extracted from the fruits of O. ferruginea is used as massage in the treatment of rheumatism and dislocation of bones.[3] The fruit, leaves, and bark of the plant are used by the local people of Pakistan as astringent, antiperiodic, uretic, antiseptic, rubefacient,[4] and also to cure toothache, hoarseness of voice, diabetes, and asthma. It is also used as antimalarial, anthelmintic, antileprosy, and in wound dressing.[5] Ripe fruits of O. ferruginea are a very good source of antioxidants.[6] The n-hexane and n-butanol extracts of the leaves of O. ferruginea have shown very good antibacterial and antifungal activities.[7] The genus Olea is a rich source of secoiridoids, secoiridoid glycosides, flavonoids, lignans, terpenoids, and other phenolic compounds with very good activities.[8–11] In the course of our continuing search for secondary metabolites of biological significance from indigenous medicinal plants, we investigated the ethyl acetate soluble fraction of the leaves of O. ferruginea. In the present paper, we report the isolation and structural elucidation of new secoiridoid glycosidic lignan ester (1) (Fig. 1) isolated from this plant, using different spectroscopic methods. 1D and 2D NMR techniques, including 1H–1H COSY, HSQC, HMBC, and NOESY, were utilized in the structure elucidation and complete assignments of 1H and 13C NMR spectra.
Results and Discussion Compound 1 was obtained as a colorless gum by purification of ethyl acetate soluble extract from the methanol extract of the leaves of O. ferruginea. Structure of the compound was elucidated essentially by 1H and 13C NMR spectroscopy including 2D NMR spectroscopy and fast atom bombardment (FAB) spectrometry. Compound 1 was assigned the molecular formula C47H62O24 as determined from its quasi-molecular ion peak at m/z 1011 [M + H]+ in the FAB-MS in positive mode. FAB-MS (+ve) after peak matching confirmed the molecular ion by showing a [M + H]+ peak at m/z 1011.3675. A fragment ion at m/z 996 appeared because of a loss of methyl group, i.e. [M + H 15]+. The peak at m/z 239 confirmed the presence of a secoiridane moiety in the structure. The infrared (IR) spectrum showed the absorption maxima for hydroxyl groups at 3397, C–H stretch at 3011, ester carbonyls at 1733,
Magn. Reson. Chem. (2014)
aromatic C = C at 1515, and for ether linkages at 1266, 1231, 1075, and 1042 cm1. The ultraviolet (UV) absorption maxima of 1 at 273 and 248 nm revealed the presence of substituted ester groups and an α,β-unsaturated carbonyl functionality. 1H and 13C NMR spectral data of compound 1 indicated a 7′,9″-monoepoxylignan structure substituted with a sugar molecule and a secoiridoid glycoside moiety (Table 1). To the best of our knowledge, the compound has never been previously reported in the literature and the structure of the lignan moiety is also different from previously reported lignans from different plants.[12,13] In compound 1, the lignan moiety contains a hydroxyl group at C-7″ and an acetoxy group at C-8″; the simultaneous presence of these groups in lignans is not reported previously in the literature. The basic structure of the furan ring system was confirmed by HMBC correlations (Fig. 2). The C-9″ methylene protons (δ 4.25 and 4.31) and the C-7″ methine proton (δ 5.02) showed HMBC interactions with C-8″ (δ 98.8). Another important assignment was the position of an acetyl group that could be placed at C-7″ and C-8″ equally well, but it was placed at C-8″. If it were placed at C-7″, then H-7″ must show HMBC correlation with the C-11″ carbonyl carbon, but no such correlation was observed in the HMBC spectrum that supported its position to be at C-8″ that is a quaternary carbon. The aromatic region of 1H NMR spectrum showed the presence of two ABX systems with 1, 3, and 4 substituted positions of the aromatic rings. The assignments of the aromatic protons and the methoxy groups to the two aromatic systems were made on the basis of HMBC and 1H–1H COSY spectra (Fig. 2). The chemical shift of the first aromatic set of protons was observed at δ 7.00 (d, J = 1.2 Hz), 6.85 (dd, J = 8.4, 1.2 Hz), and 6.78 (d, J = 8.4 Hz) showing an ABX system. Similarly, the second aromatic set of protons was observed at δ 6.99 (d, J = 1.8 Hz), 6.93 (dd, J = 8.4, 1.8 Hz), and 7.12 (d, J = 8.4 Hz). The aromatic moieties were connected to the tetrahydrofuran ring at C-7′ and C-7″ on the basis of HMBC correlations. Thus, the methine proton at C-7″ (δ 5.02, s) showed HMBC correlations to the aromatic carbons C-1″ (δ 132.9), C-2″ (δ 114.6), and C-6″ (δ 122.6) while the methine proton at C-7′ (δ 4.78) showed HMBC correlations to the aromatic carbons
* Correspondence to: Afsar Khan and Umar Farooq, Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan. E-mail: [email protected]; [email protected] a Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan b H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
Copyright © 2014 John Wiley & Sons, Ltd.
M. A. Hashmi et al.
Figure 1. Compound 1 isolated from the EtOAc extract of O. ferruginea leaves.
C-1′ (δ 132.8), C-2′ (δ 111.2), and C-6′ (δ 120.3) of the other ring. The relative configuration of the lignan unit was proposed to be 7′ S,8′ S,8″S with the help of coupling constant data and NOESY spectra and on the basis of proposition of Birch and Smith for assigning the stereochemistry of lignans.[14] The presence of secoiridane moiety in the molecule was confirmed by its characteristic signals of carbons at δ 95.1 for C-1, 109 and 155 for doubly bonded C-3 and C-4, and 124.8 and 13.8 for Table 1.
1
C-8 and C-10, respectively. Besides, the two carboxyl groups for both the ester moieties present on secoiridane unit were also indicated by 13C broadband spectrum. The relative stereochemistry at positions 1 and 5 of secoiridane moiety was also established on the basis of NOESY spectra and comparison with the literature data.[15] The 1H NMR data also revealed the presence of two glucose moieties in the molecule whose anomeric configurations were assigned as β on the basis of large coupling constants of the anomeric proton signals. The corresponding anomeric carbon signals of the sugar moieties were observed at δ 100.9 and 102.6, respectively. The connectivity of one of the glucose unit at C-1 of secoiridane ring was confirmed by HMBC spectra where the secoiridane proton H-1 (δ 5.90) showed strong correlations with the anomeric carbon of the glucose unit (δ 100.9) while the anomeric proton of the glucose unit H-1′″ (δ 4.79) showed inverse HMBC interactions with C-1 (δ 95.1) of the secoiridane moiety. The connectivity of the lignan moiety with secoiridane via this glucose unit and to the other sugar unit was also confirmed by HMBC spectra. The H-6″′ (δ 3.87 and 3.64–3.69) of the first sugar showed HMBC interactions with C-9′ (δ 71.3) of the lignan moiety while the H-9′ (δ 3.76 and 4.45) of the lignan moiety showed inverse HMBC interactions with C-6″′ (δ 62.7) of the sugar; thus confirming
13
H (600 MHz) and C (150 MHz) NMR data for compound 1 in CD3OD (δ in ppm) δC
δH (J in Hz)
Secoiridane 1 2 3 4 5 6
95.1 — 155.1 109.4 31.8 41.2
7 8 9
173.5 124.8 130.3
5.90, br. s — 7.51, s — 3.98 (dd, 9.0, 4.5) 2.73 (dd, 14.1, 4.5) 2.44 (dd, 14.1, 9.0) — 6.10 (q, 7.2) —
10 11 12 13
13.5 168.6 51.8 52.1
1.72 (d, 7.2) — 3.70, s 3.62, s
Position
Lignan 1′ 2′ 3′ 4′ 5′
132.8 111.2 148.0 149.1 116.1
— 7.00 (d, 1.2) — — 6.78 (d, 8.4)
6′ 7′ 8′ 9′
120.3 86.7 60.1 71.3
10′ 11′
56.8 56.8
6.85 (dd, 8.4, 1.2) 4.78 (d, 5.4) 3.41–3.38, m 4.45 (dd, 9.0, 8.4) 3.76 (dd, 9.0, 5.0) 3.85, s 3.85, s
Position
δC
δH (J in Hz)
Lignan 1″ 2″ 3″ 4″ 5″ 6″
132.9 114.6 150.3 147.5 117.3 122.6
— 6.99 (d, 1.8) — — 7.12 (d, 8.4) 6.93 (dd, 8.4, 1.8)
7″ 8″ 9″
88.5 98.8 75.7
10″ 11″ 12″
56.8 171.1 20.9
Glc′′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′
100.9 74.9 78.4 71.4 77.9 62.7
4.79 (d, 7.8) 3.50–3.43, m 3.31–3.28 3.31–3.28 3.50–3.43, m 3.87 (br. d, 7.8) 3.69–3.64, m
Glc′′′′ 1′′′′ 2′′′′ 3′′′′
102.6 74.7 78.2
4.90 (d, 7.8) 3.31–3.28 3.41–3.38, m
4′′′′ 5′′′′ 6′′′′
71.3 77.8 62.5
3.41–3.38, m 3.50–3.43, m 3.69–3.64, m 3.69–3.64, m
5.02, s — 4.25 (d, 10.8) 4.31 (d, 10.8) 3.85, s — 1.66, s
Glc, β-D-Glucopyranosyl
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Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2014)
A new secoiridoid glycosidic lignan ester from Olea ferruginea
Figure 2. Key COSY, HMBC, and NOESY interactions of compound 1.
the point of attachment of the sugar unit at position C-9′ of the lignan unit. The other sugar was confirmed to be present at C-4″ of aromatic ring of the lignan by the HMBC correlations of its anomeric proton (δ 4.90) with the C-4″ (δ 147.5) of the aromatic ring. Thus, the final structure of compound 1 was found to be methyl (rel. 1R,5S,Z)-1-(((O-β-D-glycopyranosyl)-6′′′-O-9′-(rel. 7′S,8′S,8″S)-[(8″acetoxy-7′-(3′,4′-dimethoxyphenyl)-7″-hydroxy, (3″-methoxy-4″-O-βD-glycopyranosylphenyl)-8′-8″,7′-O-9″-lignan]-1,5-dihydropyran6-ethylidene-5-methylacetate-4-methylcarboxylate. The compound was trivially named as oleferrugine A and is unprecedented. The salient features of compound 1 are that no such lignans have been previously reported in the literature containing a hydroxyl group at C-7″ and an acetoxy group at C-8″ simultaneously. Furthermore, this is also the first report of a molecule containing the lignan and secoiridane moieties in the same molecule.
Materials and Methods General IR spectra were recorded on JASCO 302-A infrared spectrophotometer using KBr disc. UV spectra were obtained on a Shimadzu UV 240 spectrophotometer. FAB-MS spectra were recorded on JMSHX-110 spectrometer, with data system. The extracts/compound were dried on rotary vacuum evaporator (Heidolph Laborota 4000 Efficient). Column chromatography was performed on silica gel (230–400 mesh, E-Merck) and Sephadex LH-20 (Sigma-Aldrich). The high performance liquid chromatography was performed with LC-908 W Recycling Preparative HPLC (Japan Analytical Industries (JAI) Co. Ltd.) that was equipped with Hibar LiChrosorb RP-18 column (7 μm, 250 × 25 mm; Merck) using a JAI RI-5 refractive index detector and a JAI UV-310 detector (254 nm), simultaneously. The flow rate was kept constant at 3 ml/min. Pre-coated thin-layer chromatography plates with silica gel F254 (Merck) were used to observe the purity of the isolated compound by spraying with 10% H2SO4–ammonium cerium(IV) sulfate dihydrate, followed by heating. Plant material Leaves of O. ferruginea were collected from the mountains of Abbottabad, Pakistan, and the plant was identified by a taxonomist, Dr. Gul Jan at the Department of Botany, Hazara University, Mansehra, Pakistan. A voucher specimen (no. 3544) has been submitted in the herbarium of the same department.
Magn. Reson. Chem. (2014)
Extraction and isolation Plant material was air and shade dried and ground to a fine powder. The powdered material (16 Kg) was soaked in MeOH, and the filtrate was condensed under reduced pressure to obtain a brownish gum (1285 g). This procedure was repeated three times. The gum was then suspended in distilled water and sequentially partitioned between n-hexane (250 g), CHCl3 (37.5 g), EtOAc (162 g), and n-butanol (280 g). The resulting extracts were dried on a rotary evaporator and stored under refrigeration conditions. The EtOAc extract (162 g) was subjected to silica gel CC with a gradient of CHCl3/MeOH (100 : 0–0 : 100) to give 14 major fractions (A1–A14). The fraction A5 (30 g) was further chromatographed on a silica gel column, eluted with CHCl3/MeOH (95 : 5–0 : 100) to produce nine subfractions (A5-1–A5-9). Subfraction A5-9 was subjected to repeated column chromatography (silica gel CC, CHCl3/MeOH, 100 : 0–0 : 100; Sephadex LH-20, CHCl3/MeOH, 1 : 1) and resulted into seven subfractions (Q1–Q7). Subfraction Q7 was then subjected to recycling preparative HPLC to yield compound 1 (6 mg) with a retention time (tR) of 30 min upon elution with CH3CN/H2O (1 : 1) with a flow rate of 3 ml/min (Fig. 1).
NMR data The NMR spectra were recorded at 298 K using a Bruker Avance AV 600 spectrometer using 5-mm PATXI 1H/13C probe operating at 600 MHz for 1H and 150 MHz for 13C. 1D and 2D NMR experiments (COSY, HSQC, NOESY, and HMBC) were performed using standard Bruker pulse programs (XWinNMR). Samples were dissolved in CD3OD and chemical shift values were reported in δ (ppm) with reference to the residual non-deuterated solvent peaks (δH 3.31 and δC 49.0 for CD3OD) and coupling constants (J) were measured in Hz. 1 H NMR spectrum was acquired with relaxation delay (D1) of 1 s, spectrometer frequency (SF) 600.03 MHz, acquisition time (AQ) 1.704 s, number of scans (NS) 32, digital resolution (DR) 0.58, and temperature (TE) 302.6 K. 13 C NMR spectrum was recorded with D1 of 1.5 s, SF 150.895 MHz, AQ 0.911 s, NS 11 585, DR 1.09, and TE 306.7 K. 1 H–1H COSY spectrum: SF01 600.032 MHz, NS 8, AQ 0.190 s, TE 302.3 K. NOE spectroscopy using Bruker library pulse sequence ‘noesygpph’: D1 2 s, SF 600.032 MHz, NS 16, TE 302.5 K, SWH 5387.931 Hz.
Copyright © 2014 John Wiley & Sons, Ltd.
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M. A. Hashmi et al.
MAH acknowledges the funding from Higher Education Commission of Pakistan for his PhD studies.
[2] S. W. Khan, S. Khatoon. Pakistan J. Bot. 2007, 39, 699–710. [3] W. Murad, A. Ahmad, S. A. Gilani, M. A. Khan. J. Med. Plants Res. 2011, 5, 1072–1086. [4] A. Jabeen, M. A. Khan, M. Ahmad, M. Zafar, F. Ahmad. Afr. J. Biotechnol. 2009, 8, 763–784. [5] A. Geyid, D. Abebe, A. Debella, Z. Makonnen, F. Aberra, F. Teka, T. Kebede, K. Urga, K. Yersaw, T. Biza, B. H. Mariam, M. Guta. J. Ethnopharmacol. 2005, 97, 421–427. [6] R. K. Sharma, N. Sharma, S. S. Samant, S. K. Nandi, L. M. S. Palni. Int. J. Biosci. Biochem. Bioinforma. 2013, 3, 154–156. [7] A. Amin, M. A. Khan, S. Shah, M. Ahmad, M. Zafar, A. Hameed. Pak. J. Pharm. Sci. 2013, 26, 251–254. [8] A. Cárdeno, M. Sánchez-Hidalgo, M. A. Rosillo, C. A. de la Lastra. Nutr. Cancer 2013, 65, 147–156. [9] E. E. Rufino-Palomares, F. J. Reyes-Zurita, L. García-Salguero, K. Mokhtari, P. P. Medina, J. A. Lupiáñez, J. Peragón. J. Proteomics 2013, 83, 15–25. [10] Y. Allouche, F. Warleta, M. Campos, C. Sanchez-Quesada, M. Uceda, G. Beltran, J. Juan Gaforio. J. Agric. Food Chem. 2011, 59, 121–130. [11] S. Christophoridou, P. Dais, L. H. Tseng, M. Spraul. J. Agric. Food Chem. 2005, 53, 4667–4679. [12] U. Sriphana, Y. Thongsri, P. Ardwichai, K. Poopasit, C. Prariyachatigul, S. Simasathiansophon, C. Yenjai. Fitoterapia 2013, 91, 39–43. [13] B. Schumacher, S. Scholle, J. Hölzl, N. Khudeir, S. Hess, C. E. Müller. J. Nat. Prod. 2002, 65, 1479–1485. [14] A. J. Birch, M. Smith. J. Chem. Soc. 1964, 2705–2708. [15] P. Gariboldi, G. Jommi, L. Verotta. Phytochemistry 1986, 25, 865–869.
References
Supporting Information
Heteronuclear single-quantum correlation using Bruker library pulse sequence ‘hsqcetgpsi’: D1 2 s, SF 600.032 MHz, NS 32, TE 302.9 K, SWH 5387.931 Hz. Heteronuclear multiple-bond correlation using Bruker library pulse sequence ‘hmbcgplpndqf’: D1 2 s, SF 600.032 MHz, NS 64, TE 301.3 K, SWH 5387.931 Hz. Spectroscopic data of compound 1 Colorless gum; ½α28 D : 0.648; UV (MeOH) λmax (log ε): 248 (4.82), 273 (4.69); IR (KBr) νmax 3397 (OH stretch), 3011 (aromatic C–H stretch), 1733 (C = O ester), 1515 (aromatic C = C), 1266 (ether linkage), 1231 (ether linkage), 1075 (ether linkage), 1042 (ether linkage) cm1; 1 H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data, refer to Table 1; HR-FAB-MS (by peak matching) m/z 1011.3675 [M + H]+ (calculated for C47H62O24 + H, 1011.3709). FAB-MS (+ve mode) m/z 1011 [M + H]+, 996, 239. Acknowledgement
[1] M. A. Ginai, Bureau of Agriculture Information, Publication Division. Department of Agriculture, Government of West Pakistan, Lahore, 1968, 450.
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Additional supporting information may be found in the online version of this article at the publisher’s website.
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2014)
Revised: 21 July 2014
Accepted: 1 August 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4134
A new secoiridoid glycosidic lignan ester from the leaves of Olea ferruginea Muhammad Ali Hashmi,a Afsar Khan,a* Umar Farooq,a* Mubeen Rani,b Viqar Uddin Ahmadb and Abdur Rahman Khana
Introduction Olea ferruginea, an indigenous medicinal plant belonging to family Oleaceae, is locally known as Kahoo or Khoona in Pakistan.[1] Folk practitioners use its leaves and bark to treat various ailments. Different parts of this plant such as leaves, bark, and gum possess various activities. Its stem bark is used by local people for fever.[2] The oil extracted from the fruits of O. ferruginea is used as massage in the treatment of rheumatism and dislocation of bones.[3] The fruit, leaves, and bark of the plant are used by the local people of Pakistan as astringent, antiperiodic, uretic, antiseptic, rubefacient,[4] and also to cure toothache, hoarseness of voice, diabetes, and asthma. It is also used as antimalarial, anthelmintic, antileprosy, and in wound dressing.[5] Ripe fruits of O. ferruginea are a very good source of antioxidants.[6] The n-hexane and n-butanol extracts of the leaves of O. ferruginea have shown very good antibacterial and antifungal activities.[7] The genus Olea is a rich source of secoiridoids, secoiridoid glycosides, flavonoids, lignans, terpenoids, and other phenolic compounds with very good activities.[8–11] In the course of our continuing search for secondary metabolites of biological significance from indigenous medicinal plants, we investigated the ethyl acetate soluble fraction of the leaves of O. ferruginea. In the present paper, we report the isolation and structural elucidation of new secoiridoid glycosidic lignan ester (1) (Fig. 1) isolated from this plant, using different spectroscopic methods. 1D and 2D NMR techniques, including 1H–1H COSY, HSQC, HMBC, and NOESY, were utilized in the structure elucidation and complete assignments of 1H and 13C NMR spectra.
Results and Discussion Compound 1 was obtained as a colorless gum by purification of ethyl acetate soluble extract from the methanol extract of the leaves of O. ferruginea. Structure of the compound was elucidated essentially by 1H and 13C NMR spectroscopy including 2D NMR spectroscopy and fast atom bombardment (FAB) spectrometry. Compound 1 was assigned the molecular formula C47H62O24 as determined from its quasi-molecular ion peak at m/z 1011 [M + H]+ in the FAB-MS in positive mode. FAB-MS (+ve) after peak matching confirmed the molecular ion by showing a [M + H]+ peak at m/z 1011.3675. A fragment ion at m/z 996 appeared because of a loss of methyl group, i.e. [M + H 15]+. The peak at m/z 239 confirmed the presence of a secoiridane moiety in the structure. The infrared (IR) spectrum showed the absorption maxima for hydroxyl groups at 3397, C–H stretch at 3011, ester carbonyls at 1733,
Magn. Reson. Chem. (2014)
aromatic C = C at 1515, and for ether linkages at 1266, 1231, 1075, and 1042 cm1. The ultraviolet (UV) absorption maxima of 1 at 273 and 248 nm revealed the presence of substituted ester groups and an α,β-unsaturated carbonyl functionality. 1H and 13C NMR spectral data of compound 1 indicated a 7′,9″-monoepoxylignan structure substituted with a sugar molecule and a secoiridoid glycoside moiety (Table 1). To the best of our knowledge, the compound has never been previously reported in the literature and the structure of the lignan moiety is also different from previously reported lignans from different plants.[12,13] In compound 1, the lignan moiety contains a hydroxyl group at C-7″ and an acetoxy group at C-8″; the simultaneous presence of these groups in lignans is not reported previously in the literature. The basic structure of the furan ring system was confirmed by HMBC correlations (Fig. 2). The C-9″ methylene protons (δ 4.25 and 4.31) and the C-7″ methine proton (δ 5.02) showed HMBC interactions with C-8″ (δ 98.8). Another important assignment was the position of an acetyl group that could be placed at C-7″ and C-8″ equally well, but it was placed at C-8″. If it were placed at C-7″, then H-7″ must show HMBC correlation with the C-11″ carbonyl carbon, but no such correlation was observed in the HMBC spectrum that supported its position to be at C-8″ that is a quaternary carbon. The aromatic region of 1H NMR spectrum showed the presence of two ABX systems with 1, 3, and 4 substituted positions of the aromatic rings. The assignments of the aromatic protons and the methoxy groups to the two aromatic systems were made on the basis of HMBC and 1H–1H COSY spectra (Fig. 2). The chemical shift of the first aromatic set of protons was observed at δ 7.00 (d, J = 1.2 Hz), 6.85 (dd, J = 8.4, 1.2 Hz), and 6.78 (d, J = 8.4 Hz) showing an ABX system. Similarly, the second aromatic set of protons was observed at δ 6.99 (d, J = 1.8 Hz), 6.93 (dd, J = 8.4, 1.8 Hz), and 7.12 (d, J = 8.4 Hz). The aromatic moieties were connected to the tetrahydrofuran ring at C-7′ and C-7″ on the basis of HMBC correlations. Thus, the methine proton at C-7″ (δ 5.02, s) showed HMBC correlations to the aromatic carbons C-1″ (δ 132.9), C-2″ (δ 114.6), and C-6″ (δ 122.6) while the methine proton at C-7′ (δ 4.78) showed HMBC correlations to the aromatic carbons
* Correspondence to: Afsar Khan and Umar Farooq, Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan. E-mail: [email protected]; [email protected] a Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan b H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
Copyright © 2014 John Wiley & Sons, Ltd.
M. A. Hashmi et al.
Figure 1. Compound 1 isolated from the EtOAc extract of O. ferruginea leaves.
C-1′ (δ 132.8), C-2′ (δ 111.2), and C-6′ (δ 120.3) of the other ring. The relative configuration of the lignan unit was proposed to be 7′ S,8′ S,8″S with the help of coupling constant data and NOESY spectra and on the basis of proposition of Birch and Smith for assigning the stereochemistry of lignans.[14] The presence of secoiridane moiety in the molecule was confirmed by its characteristic signals of carbons at δ 95.1 for C-1, 109 and 155 for doubly bonded C-3 and C-4, and 124.8 and 13.8 for Table 1.
1
C-8 and C-10, respectively. Besides, the two carboxyl groups for both the ester moieties present on secoiridane unit were also indicated by 13C broadband spectrum. The relative stereochemistry at positions 1 and 5 of secoiridane moiety was also established on the basis of NOESY spectra and comparison with the literature data.[15] The 1H NMR data also revealed the presence of two glucose moieties in the molecule whose anomeric configurations were assigned as β on the basis of large coupling constants of the anomeric proton signals. The corresponding anomeric carbon signals of the sugar moieties were observed at δ 100.9 and 102.6, respectively. The connectivity of one of the glucose unit at C-1 of secoiridane ring was confirmed by HMBC spectra where the secoiridane proton H-1 (δ 5.90) showed strong correlations with the anomeric carbon of the glucose unit (δ 100.9) while the anomeric proton of the glucose unit H-1′″ (δ 4.79) showed inverse HMBC interactions with C-1 (δ 95.1) of the secoiridane moiety. The connectivity of the lignan moiety with secoiridane via this glucose unit and to the other sugar unit was also confirmed by HMBC spectra. The H-6″′ (δ 3.87 and 3.64–3.69) of the first sugar showed HMBC interactions with C-9′ (δ 71.3) of the lignan moiety while the H-9′ (δ 3.76 and 4.45) of the lignan moiety showed inverse HMBC interactions with C-6″′ (δ 62.7) of the sugar; thus confirming
13
H (600 MHz) and C (150 MHz) NMR data for compound 1 in CD3OD (δ in ppm) δC
δH (J in Hz)
Secoiridane 1 2 3 4 5 6
95.1 — 155.1 109.4 31.8 41.2
7 8 9
173.5 124.8 130.3
5.90, br. s — 7.51, s — 3.98 (dd, 9.0, 4.5) 2.73 (dd, 14.1, 4.5) 2.44 (dd, 14.1, 9.0) — 6.10 (q, 7.2) —
10 11 12 13
13.5 168.6 51.8 52.1
1.72 (d, 7.2) — 3.70, s 3.62, s
Position
Lignan 1′ 2′ 3′ 4′ 5′
132.8 111.2 148.0 149.1 116.1
— 7.00 (d, 1.2) — — 6.78 (d, 8.4)
6′ 7′ 8′ 9′
120.3 86.7 60.1 71.3
10′ 11′
56.8 56.8
6.85 (dd, 8.4, 1.2) 4.78 (d, 5.4) 3.41–3.38, m 4.45 (dd, 9.0, 8.4) 3.76 (dd, 9.0, 5.0) 3.85, s 3.85, s
Position
δC
δH (J in Hz)
Lignan 1″ 2″ 3″ 4″ 5″ 6″
132.9 114.6 150.3 147.5 117.3 122.6
— 6.99 (d, 1.8) — — 7.12 (d, 8.4) 6.93 (dd, 8.4, 1.8)
7″ 8″ 9″
88.5 98.8 75.7
10″ 11″ 12″
56.8 171.1 20.9
Glc′′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′
100.9 74.9 78.4 71.4 77.9 62.7
4.79 (d, 7.8) 3.50–3.43, m 3.31–3.28 3.31–3.28 3.50–3.43, m 3.87 (br. d, 7.8) 3.69–3.64, m
Glc′′′′ 1′′′′ 2′′′′ 3′′′′
102.6 74.7 78.2
4.90 (d, 7.8) 3.31–3.28 3.41–3.38, m
4′′′′ 5′′′′ 6′′′′
71.3 77.8 62.5
3.41–3.38, m 3.50–3.43, m 3.69–3.64, m 3.69–3.64, m
5.02, s — 4.25 (d, 10.8) 4.31 (d, 10.8) 3.85, s — 1.66, s
Glc, β-D-Glucopyranosyl
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Magn. Reson. Chem. (2014)
A new secoiridoid glycosidic lignan ester from Olea ferruginea
Figure 2. Key COSY, HMBC, and NOESY interactions of compound 1.
the point of attachment of the sugar unit at position C-9′ of the lignan unit. The other sugar was confirmed to be present at C-4″ of aromatic ring of the lignan by the HMBC correlations of its anomeric proton (δ 4.90) with the C-4″ (δ 147.5) of the aromatic ring. Thus, the final structure of compound 1 was found to be methyl (rel. 1R,5S,Z)-1-(((O-β-D-glycopyranosyl)-6′′′-O-9′-(rel. 7′S,8′S,8″S)-[(8″acetoxy-7′-(3′,4′-dimethoxyphenyl)-7″-hydroxy, (3″-methoxy-4″-O-βD-glycopyranosylphenyl)-8′-8″,7′-O-9″-lignan]-1,5-dihydropyran6-ethylidene-5-methylacetate-4-methylcarboxylate. The compound was trivially named as oleferrugine A and is unprecedented. The salient features of compound 1 are that no such lignans have been previously reported in the literature containing a hydroxyl group at C-7″ and an acetoxy group at C-8″ simultaneously. Furthermore, this is also the first report of a molecule containing the lignan and secoiridane moieties in the same molecule.
Materials and Methods General IR spectra were recorded on JASCO 302-A infrared spectrophotometer using KBr disc. UV spectra were obtained on a Shimadzu UV 240 spectrophotometer. FAB-MS spectra were recorded on JMSHX-110 spectrometer, with data system. The extracts/compound were dried on rotary vacuum evaporator (Heidolph Laborota 4000 Efficient). Column chromatography was performed on silica gel (230–400 mesh, E-Merck) and Sephadex LH-20 (Sigma-Aldrich). The high performance liquid chromatography was performed with LC-908 W Recycling Preparative HPLC (Japan Analytical Industries (JAI) Co. Ltd.) that was equipped with Hibar LiChrosorb RP-18 column (7 μm, 250 × 25 mm; Merck) using a JAI RI-5 refractive index detector and a JAI UV-310 detector (254 nm), simultaneously. The flow rate was kept constant at 3 ml/min. Pre-coated thin-layer chromatography plates with silica gel F254 (Merck) were used to observe the purity of the isolated compound by spraying with 10% H2SO4–ammonium cerium(IV) sulfate dihydrate, followed by heating. Plant material Leaves of O. ferruginea were collected from the mountains of Abbottabad, Pakistan, and the plant was identified by a taxonomist, Dr. Gul Jan at the Department of Botany, Hazara University, Mansehra, Pakistan. A voucher specimen (no. 3544) has been submitted in the herbarium of the same department.
Magn. Reson. Chem. (2014)
Extraction and isolation Plant material was air and shade dried and ground to a fine powder. The powdered material (16 Kg) was soaked in MeOH, and the filtrate was condensed under reduced pressure to obtain a brownish gum (1285 g). This procedure was repeated three times. The gum was then suspended in distilled water and sequentially partitioned between n-hexane (250 g), CHCl3 (37.5 g), EtOAc (162 g), and n-butanol (280 g). The resulting extracts were dried on a rotary evaporator and stored under refrigeration conditions. The EtOAc extract (162 g) was subjected to silica gel CC with a gradient of CHCl3/MeOH (100 : 0–0 : 100) to give 14 major fractions (A1–A14). The fraction A5 (30 g) was further chromatographed on a silica gel column, eluted with CHCl3/MeOH (95 : 5–0 : 100) to produce nine subfractions (A5-1–A5-9). Subfraction A5-9 was subjected to repeated column chromatography (silica gel CC, CHCl3/MeOH, 100 : 0–0 : 100; Sephadex LH-20, CHCl3/MeOH, 1 : 1) and resulted into seven subfractions (Q1–Q7). Subfraction Q7 was then subjected to recycling preparative HPLC to yield compound 1 (6 mg) with a retention time (tR) of 30 min upon elution with CH3CN/H2O (1 : 1) with a flow rate of 3 ml/min (Fig. 1).
NMR data The NMR spectra were recorded at 298 K using a Bruker Avance AV 600 spectrometer using 5-mm PATXI 1H/13C probe operating at 600 MHz for 1H and 150 MHz for 13C. 1D and 2D NMR experiments (COSY, HSQC, NOESY, and HMBC) were performed using standard Bruker pulse programs (XWinNMR). Samples were dissolved in CD3OD and chemical shift values were reported in δ (ppm) with reference to the residual non-deuterated solvent peaks (δH 3.31 and δC 49.0 for CD3OD) and coupling constants (J) were measured in Hz. 1 H NMR spectrum was acquired with relaxation delay (D1) of 1 s, spectrometer frequency (SF) 600.03 MHz, acquisition time (AQ) 1.704 s, number of scans (NS) 32, digital resolution (DR) 0.58, and temperature (TE) 302.6 K. 13 C NMR spectrum was recorded with D1 of 1.5 s, SF 150.895 MHz, AQ 0.911 s, NS 11 585, DR 1.09, and TE 306.7 K. 1 H–1H COSY spectrum: SF01 600.032 MHz, NS 8, AQ 0.190 s, TE 302.3 K. NOE spectroscopy using Bruker library pulse sequence ‘noesygpph’: D1 2 s, SF 600.032 MHz, NS 16, TE 302.5 K, SWH 5387.931 Hz.
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M. A. Hashmi et al.
MAH acknowledges the funding from Higher Education Commission of Pakistan for his PhD studies.
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References
Supporting Information
Heteronuclear single-quantum correlation using Bruker library pulse sequence ‘hsqcetgpsi’: D1 2 s, SF 600.032 MHz, NS 32, TE 302.9 K, SWH 5387.931 Hz. Heteronuclear multiple-bond correlation using Bruker library pulse sequence ‘hmbcgplpndqf’: D1 2 s, SF 600.032 MHz, NS 64, TE 301.3 K, SWH 5387.931 Hz. Spectroscopic data of compound 1 Colorless gum; ½α28 D : 0.648; UV (MeOH) λmax (log ε): 248 (4.82), 273 (4.69); IR (KBr) νmax 3397 (OH stretch), 3011 (aromatic C–H stretch), 1733 (C = O ester), 1515 (aromatic C = C), 1266 (ether linkage), 1231 (ether linkage), 1075 (ether linkage), 1042 (ether linkage) cm1; 1 H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data, refer to Table 1; HR-FAB-MS (by peak matching) m/z 1011.3675 [M + H]+ (calculated for C47H62O24 + H, 1011.3709). FAB-MS (+ve mode) m/z 1011 [M + H]+, 996, 239. Acknowledgement
[1] M. A. Ginai, Bureau of Agriculture Information, Publication Division. Department of Agriculture, Government of West Pakistan, Lahore, 1968, 450.
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Additional supporting information may be found in the online version of this article at the publisher’s website.
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Magn. Reson. Chem. (2014)
