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Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica ab
c
cd
Sabrin R.M. Ibrahim , Mostafa A. Fouad , Ahmed Abdel-Lateff , e
Tatsufumi Okino & Gamal A. Mohamed
df
a
Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia b
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt c
Department of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt d
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia e
Faculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan f
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt Published online: 12 Aug 2014.
To cite this article: Sabrin R.M. Ibrahim, Mostafa A. Fouad, Ahmed Abdel-Lateff, Tatsufumi Okino & Gamal A. Mohamed (2014): Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947489 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947489
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947489
Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica
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Sabrin R.M. Ibrahimab*, Mostafa A. Fouadc, Ahmed Abdel-Lateffcd, Tatsufumi Okinoe and Gamal A. Mohameddf a Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia; bDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; cDepartment of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt; dDepartment of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia; eFaculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan; fDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
(Received 23 May 2014; final version received 19 July 2014) Extensive chromatographic investigation of the ethanolic extract of Alnus japonica Steud stem bark led to the isolation of a new diarylheptanoid named alnuheptanoid A [(5S)-7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-5-methoxyheptan-3-one] (8), together with seven known diarylheptanoid derivatives: platyphyllenone (5), (5S)1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one (6), 1-(3,4-dihydroxyphenyl)-7-(4hydroxyphenyl)-4-hepten-3-one (7), hirsutenone (9), (5R)-O-methylhirsutanonol (10), hirsutanonol (11) and oregonin (13), three triterpenes: a-amyrin (1), betulinaldehyde (3) and betulinic acid (4), and two sterols: b-sitosterol (2) and daucosterol (12). Compound 6 was isolated for the first time from natural source. The structures of the isolated compounds were determined on the basis of spectroscopic measurements (UV, IR, HR-ESI-MS, 1D and 2D NMR). Keywords: Alnus japonica; Betulaceae; alnuheptanoid A; diarylheptanoids; triterpenoids; sterols
1. Introduction The genus Alnus (family Betulaceae) comprises 30 species of alder trees which ranges from large size to shrubs. The species of Alnus were used in oriental traditional medicine as remedies for fever, haemorrhage, burn injuries, diarrhoea and alcoholism. This genus is considered as a potential source of different triterpenes, tannins, flavonoids and diarylheptanoids (Karchesy et al. 1974; Nomura et al. 1981; Aoki et al. 1990; Lee et al. 1992, 1999; Tori et al. 1995; Kuroyanagi et al. 2005). Alnus japonica Steud is a deciduous tree, which is indigenous in the damp parts of the fields and forests of Japan. Hannoki (Japanese name) is a common tree in low mountainous areas of Japan (Nomura et al. 1981; Lee et al. 1992; Wada et al. 1998; Kuroyanagi et al. 2005). Diarylheptanoid is a class of compounds, which bears 1,7-diphenylheptane skeleton. They were isolated from different sources such as Zingiber, Curcuma, Alpinia, Alnus and Myrica. About 400 diarylheptanoids were published and recognised as potential therapeutic agents with different biological activities: anti-inflammatory, antioxidant, antitumour, oestrogenic, leishmanicidal, hepatoprotective and neuro-protective (Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Shin et al. 2002; Agarwal et al. 2003; Kang et al. 2004; Jin et al. 2007; Park et al. 2010; Lv & She 2012). Many diarylheptanoid derivatives isolated from A. japonica
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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exhibited interesting biological properties including hepatoprotective, antioxidant (Lee et al. 2003, 2005; Kim et al. 2004), antitumour (Joo et al. 2002; Choi et al. 2008), antiviral against avian influenza virus (Tung et al. 2010) and protease inhibitor in severe acute respiratory syndrome coronavirus (Park et al. 2012). Moreover, hirsutenone isolated from the bark of A. japonica acts by mimicking calcineurin inhibitor. So, it may be a new topical drug candidate for treatment of atopic dermatitis (Joo et al. 2009). We report herein the isolation and identification of the metabolites from A. japonica stem bark (Figure 1). The different chromatographic techniques and final purification using HPLC led to isolation of 13 compounds including three triterpenes (1, 3 and 4), two sterols (2 and 12) and eight diarylheptanoids (6– 11 and 13) one of them is new, alnuheptanoid A (8), and another one (6) isolated for the first time from natural source. Their structures were elucidated using different spectral methods (UV, IR, HR-ESI-MS, 1D and 2D NMR). 2. Results and discussion Compound 6 was isolated as a brown residue. It had a molecular formula C20H24O4 based on HR-ESI-MS pseudo-molecular ion peak at m/z ¼ 327.1677 [M –H]þ, requiring nine degrees of unsaturation: eight for two phenyl moieties and one for ketone carbonyl group. The presence of ketone carbonyl was confirmed by the IR absorption band at 1712 cm21 and 13C NMR signal at dC 210.7 (C-3). Also, the IR spectrum showed characteristic absorption bands due to a hydroxyl group (3395 cm21) and phenyl (1608 and 1521 cm21) moieties. Compound 6 had UV absorption maxima at 280 and 222 nm characteristic for the presence of aromatic moiety (Itokawa et al. 1985; Uehara et al. 1985). The 1H and 13C NMR spectra showed signals at dH 6.96 (2H, dd, J ¼ 8.2 and 2.2 Hz, H-20 , 60 )/dC 129.9 (C-20 , 60 ), 6.98 (2H, dd, J ¼ 8.2, 2.2 Hz, H-200 , 600 )/130.0 (C-200 , 600 ), 6.66 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 )/115.8 (C-30 , 50 ), 6.69 (2H, dd, J ¼ 8.2, 2.2 Hz, H-300 , 500 )/115.9 (C-300 , 500 ), 132.7 (C-10 ), 133.6 (C-100 ), 156.4 (C-40 ) and 156.5 (C-400 ) characteristic for the presence of two 4-oxyphenyl moieties (Ohta et al. 1984; Wada et al 1998;
R
H R 2 H 12 Glu
HO 1
RO
O
R1
R2
HO
1` 1
R2 3` A
HO 5 7 9
1
HO 3`
1`
HO 4`
6`
R1 H H OH
R2 H OH OH
O
OCH3
3
5
10
OH
HO 4`
6 CH3 8 CH3 11 H 13 Xyl 3``OH
6``
4`` OH
Figure 1. Structures of the isolated compounds (1 – 13).
OR1
3
5
7 1``
3``R3
B
6` R1
7 1``
O
R 3 CHO 4 COOH
6`` R2
R3
H H OH OH
H OH OH OH
4`` OH
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Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Park et al. 2010) (Supplementary Figures S1 and S2). They were established by the observed 1H – 1H COSY cross peaks of H-20 , H-60 /H-30 , H-50 and H-200 , H-600 /H-300 , H-500 . They were further confirmed by the HMBC correlations of H-20 and H-60 to C-10 , C-30 , C-40 and C-50 , H-30 and H-50 with C-10 , C-20 , C-40 and C-60 , H-200 and H-600 to C-100 and C-400 , and H-300 and H-500 with C-100 , C-200 , C-400 and C-600 (Supplementary Figure S4). Moreover, the 1H – 1H COSY showed two coupled methylene groups at dH 2.79 (2H, t, J ¼ 6.4 Hz, H-1) and 2.72 (2H, t, J ¼ 6.4 Hz, H-2), which correlated with the carbon signals resonating at dC 30.4 (C-1) and 46.2 (C-2), respectively, to give ZCH2ZCH2Z moiety. In the 1H and 13C NMR spectra, signals for three methylene groups at dH 2.66 (1H, m, H-4A) and 2.46 (1H, m, H-4B)/dC 48.0 (C-4), 1.71 (1H, m, H-6A) and 1.66 (1H, m, H-6B)/37.0 (C-6) and 2.52 (2H, m, H-7)/31.3 (C-7) and an oxymethine at dH 3.62 (1H, pintet, 6.6 Hz, H-5)/77.7 (C-5) were observed. The correlations observed in the 1H – 1H COSY displayed the connectivity from H-4 to H-7 to give ZCH2ZCHOHZCH2ZCH2Z moiety. The 3J HMBC correlations of H-1 and H-5 to C-3, H-2 and H-6 to C-4, and H-7 to C-5 confirmed the presence of 5-hydroxy heptan-3-one moiety (Ohta et al. 1984; Wada et al 1998; Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Park et al. 2010; El-Halawany & Hattori 2012). Signals for methoxy group at dH 3.25/dC 56.9 were observed. The location of methoxy group was established by its HMBC cross peak to C-5 of the 5-hydroxy heptan-3-one moiety. The linkages of the two 4-oxyphenyl moieties on the alkyl group at C-1 and C-7 were confirmed by HMBC correlations from H-1 to C-10 , C-20 and C-60 , H20 and H-60 to C-10 , H-7 to C-100 , C-200 and C-600 , and H-200 and H-600 to C-7. The absolute stereochemistry at position C-5 was suggested to be S-form on the basis of positive sign optical rotation of 6 compared to negative sign optical rotations in the R-form of a series of analogous compounds (Giang et al. 2006; Park et al. 2010). This was further secured by the comparison 1 H NMR chemical shift of H-5 (dH 3.62) in 6 with those of related compounds in the literature [R-form (H-5 at dH 3.99– 4.10) and S-form (H-5 at dH 3.62)] (Itokawa et al. 1985; Li et al. 2003; Kuroyanagi et al. 2005; Park et al. 2010). On the basis of the NMR spectral data, 6 was identified as (5S)-1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one. Compound 6 was available from a commercial source and this is the first report for its isolation and structural characterisation from a natural source. Compound 8 was isolated as a brown residue. Its molecular formula C20H24O5 was supported by the presence of a quasi-molecular ion peak at m/z ¼ 343.1625 [M 2 H]þ in HRESI-MS. Compound 8 is 16 mass units more than 6, indicating the presence of an additional hydroxyl group in 8. It had UV absorption maxima at 284 and 225 nm characteristic for the presence of aromatic moiety (Itokawa et al. 1985; Uehara et al. 1985). The IR spectrum of 8 showed absorption bands for a hydroxyl at 3394 cm21, ketone carbonyl at 1710 cm21 and an aromatic ring at 1606 and 1524 cm21. The 13C and DEPT NMR spectra showed resonances for 20 carbons including 1 methoxy, 5 methylenes, 8 methines and 6 quaternary carbons; one of them for ketone carbonyl at dC 211.5 (C-3) and three for oxygen-bonded aromatic carbons at dC 156.4 (C-40 ), 146.2 (C-300 ) and 144.5 (C-400 ). The 1H and 13C NMR spectra of 8 were quite similar to those of 6, but the signals associated with one of the 4-oxyphenyl moieties were absent. Instead, new signals for 1,3,4-tri-substituted phenyl moiety at dH 6.65 (d, J ¼ 2.2 Hz, H-200 )/dC 116.4 (C-200 ), 6.73 (d, J ¼ 8.2 Hz, H-500 )/116.5 and 6.64 (dd, J ¼ 8.2, 2.2 Hz, H-600 )/120.6, 134.0 (C-100 ), 146.2 (C-300 ) and 144.5 (C-400 ) were observed. The 1H and 13C NMR spectra revealed the presence of 5-methoxy heptan-3-one moiety in 8 (Supplementary Figures S5 and S6). The resonances at dH 7.05 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 )/dC 130.3, 6.71 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 )/116.2 (C-30 , 50 ), 133.2 (C-10 ), and 156.4 (C-40 ) in 1H and 13C NMR spectra indicated the presence of a 1,4-di-substituted phenyl moiety. The S configuration at position 5 was assigned based on positive optical rotation value of 8 (Giang et al. 2006; Park et al. 2010), as well as the comparison of the 1H shifts of H-5 with those reported in related diarylheptanoids (Itokawa et al. 1985; Li et al. 2003; Kuroyanagi et al. 2005; Park et al. 2010). On the basis of the above pieces of
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evidence and by comparison of the spectral data with the literature, the structure of 8 was elucidated as (5S)-7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-5-methoxyheptane-3-one (8) and named alnuheptanoid A. The known compounds were identified through the analysis of their spectroscopic data (Supplementary material) and comparison of these data with the literature as a-amyrin (1) (Fingolo et al. 2013), b-sitosterol (2) (Sayed et al. 2007), betulinaldehyde (3) (Mahato & Kundu 1994), betulinic acid (4) (Mahato & Kundu 1994), platyphyllenone (5) (Fuchino et al. 1996), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-4-hepten-3-one (7) (Ohta et al. 1984; Chen et al. 1998), hirsutenone (9) (Ohta et al. 1984), (5R)-O-methylhirsutanonol (10) (Kuroyanagi et al. 2005), hirsutanonol (11) (Ohta et al. 1984), daucosterol (12) (Sayed et al. 2007) and oregonin (13) (Lee, Kim, et al. 2000; Lee, Park, et al. 2000). 3. Experimental 3.1. General experimental procedures Melting points were carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The UV spectra were determined using Perkin Elmer double beam spectrophotometer Model 550S (Perkin-Elmer, Waltham, MA, USA), attached to a Hitachi recorder Model 561, using 1 cm quartz cell. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were recorded on a Perkin-Elmer Model 341 LC Polarimeter (Perkin-Elmer). Electron impact mass spectra were recorded on a Finnigan MAT TSQ 7000 mass spectrometer (Thermo Finnigan, Bremen, Germany). ESI-MS were obtained with an LCQ DECA mass spectrometer (Thermo Finnigan) coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. HR-ESI-MS were recorded on an LTQ Orbitrap (Thermo Finnigan). 1D (1H and 13C) and 2D (COSY, HSQC and HMBC) NMR spectra were recorded on Bruker ARX 400 NMR spectrometers (Bruker BioSpin, Billerica, MA, USA). Semi-preparative HPLC (Merck, Darmstadt, Germany) coupled with UV detector L7400 (UV detection at 280 nm) was used. Separation column (250 £ 8 mm, i.d.) pre-packed with Eurosphere 100-C18 (Knauer, Berlin, Germany). Separation was achieved by applying a linear gradient from 80% H2O (pH 2.0) and 20% MeOH to 100% MeOH over 45 min. Solvents were distilled prior spectroscopic measurements. Column chromatographic separations were performed on silica gel 60 (0.04 – 0.063 mm, Merck). TLC analysis was performed on pre-coated TLC plates with silica gel 60 F254 (0.2 mm, Merck). The compounds were detected by UV absorption at lmax 255 and 366 nm followed by spraying with p-anisaldehyde –H2SO4 reagent and heating at 1108C for 1 –2 min. 3.2. Plant material A. japonica stem bark was collected in March 2005 from the authorised trees growing in the Botanical Garden, Hieinrich-Heine University, Du¨sseldorf, Germany. The plant was taxonomically identified by Prof. Dr Peter Westhoff (Professor of Development and Molecular Biology of Plants, Hieinrich-Heine University, Du¨sseldorf, Germany). A voucher specimen has been deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt (Registration code AJB-2005). 3.3. Extraction and isolation The dried powdered stem bark of A. japonica (200 g) was exhaustively extracted with 70% EtOH (4 £ 2 L). The combined extracts were concentrated under reduced pressure to afford a dark brown residue (12 g). The EtOH extract was subjected to vacuum liquid chromatography
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(VLC) using 50% n-hexane –CHCl3 (4 £ 500 mL) and EtOAc (4 £ 500 mL), which were separately concentrated to give 2.6 and 4.1 g, respectively. The 50% n-hexane –CHCl3 fraction (2.6 g) was subjected to repeated silica gel chromatographic columns (120 g £ 50 cm £ 3 cm) using n-hexane –EtOAc in order of increasing polarity to afford compounds 1 (5.9 mg, white fine needles), 2 (15 mg, white fine needles), 3 (8 mg, white crystals) and 4 (6.5 mg, white crystals). The EtOAc fraction (4.1 g) was subjected to VLC using CHCl3 –MeOH gradient elution to give six sub-fractions (A to F). Sub-fraction B (340 mg) was chromatographed on silica gel column (60 g £ 50 cm £ 2 cm) and eluted with CHCl3, followed by CHCl3 –MeOH gradient to obtain compound 5 (5.2 mg, brown residue). Silica gel column chromatography (80 g £ 50 cm £ 3 cm) of sub-fraction C (911 mg) using CHCl3 – MeOH gradient as an eluent gave impure compounds 6, 7 and 8. They were further purified by semi-preparative HPLC to yield 6 (4.5 mg, brown residue), 7 (3.7 mg, brown residue) and 8 (2.6 mg, brown residue). Subfraction D (760) was treated similarly as sub-fraction C to obtain semi-pure compounds 9, 10 and 11, which were further purified by semi-preparative HPLC to give 9 (2.9 mg, brown residue), 10 (2.3 mg, brown residue) and 11 (4.6 mg, brown residue). Sub-fraction E (1.2 g) was chromatographed on silica gel column (120 g £ 50 cm £ 3 cm) and eluted with CHCl3, followed by CHCl3 – MeOH gradient to obtain compounds 12 (14 mg, brown residue) and 13 (11 mg, white powder). 3.4. Spectral data (5S)-1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one (6): brown residue (4.5 mg); ½a22 D þ 21.6 (c ¼ 1.0, MeOH); UV (MeOH) lmax (log 1): 280 (3.96), 222 (4.35) nm; IR (KBr) gmax: 3395, 2986, 1712, 1608, 1521, 1418, 1145, 1050, 980 cm21; 1H NMR (CD3OD, 400 MHz): dH 2.79 (2H, t, J ¼ 6.4 Hz, H-1), 2.72 (2H, t, J ¼ 6.4 Hz, H-2), 2.66 (1H, m, H-4A), 2.46 (1H, m, H-4B), 3.62 (1H, pintet, J ¼ 6.6 Hz, H-5), 1.71 (1H, m, H-6A), 1.66 (1H, m, H-6B), 2.52 (2H, m, H-7), 6.96 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 ), 6.66 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 ), 6.98 (2H, dd, J ¼ 8.2, 2.2 Hz, H-200 , 600 ), 6.69 (2H, dd, J ¼ 8.2, 2.2 Hz, H-300 , 500 ), 3.25 (3H, s, 5-OCH3); 13C NMR (CD3OD, 100 MHz): dC 30.4 (t, C-1), 46.2 (t, C-2), 210.7 (s, C-3), 48.0 (t, C-4), 77.7 (d, C-5), 37.0 (t, C-6), 31.3 (t, C-7), 132.7 (s, C-10 ), 129.9 (d, C-20 , 60 ), 115.8 (d, C-30 , 50 ), 156.4 (s, C-40 ), 133.6 (s, C-100 ), 130.0 (d, C-200 , 600 ), 115.9 (d, C-300 , 500 ), 156.5 (s, C-400 ), 56.9 (q, 5-OCH3); HR-ESI-MS m/z 327.1677 (calcd for C20H23O4 [M 2 H]þ, 327.1675). Alnuheptanoid A (8): brown residue (2.6 mg); ½a22 D þ 19.1 (c ¼ 1.0, MeOH); UV (MeOH) lmax (log 1): 284 (4.11), 225 (4.52) nm; IR (KBr) gmax: 3394, 2980, 1710, 1606, 1524, 1285, 1127, 1052, 952 cm21; 1H NMR (CD3OD, 400 MHz): dH 2.80 (2H, t, J ¼ 6.4 Hz, H-1), 2.75 (2H, t, J ¼ 6.4 Hz, H-2), 2.68 (1H, m, H-4A), 2.52 (1H, m, H-4B), 3.65 (1H, pintet, J ¼ 6.5 Hz, H-5), 1.77 (1H, m, H-6A), 1.70 (1H, m, H-6B), 2.55 (2H, m, H-7), 7.05 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 ), 6.71 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 ), 6.65 (1H, d, J ¼ 2.2 Hz, H- 200 ), 6.73 (1H, d, J ¼ 8.2 Hz, H-500 ), 6.64 (1H, dd, J ¼ 8.2, 2.2 Hz, H-600 ), 3.30 (3H, s, 5-OCH3); 13C NMR (CD3OD, 100 MHz): dC 29.8 (t, C-1), 45.3 (t, C-2), 211.5 (s, C-3), 47.3 (t, C-4), 77.9 (d, C-5), 36.2 (t, C-6), 30.7 (t, C-7), 133.2 (s, C-10 ), 130.3 (d, C-20 , 60 ), 116.2 (d, C-30 , 50 ), 156.4 (s, C-40 ), 134.0 (s, C-100 ), 116.4 (d, C-200 ), 146.2 (s, C-300 ), 144.5 (s, C-400 ), 116.5 (d, C-500 ), 120.6 (d, C-600 ), 57.1 (q, 5-OCH3); HR-ESI-MS m/z 343.1625 (calcd for C20H23O5 [M 2 H]þ, 343.1623). 4. Conclusions A new diarylheptanoid, alnuheptanoid A (8), and 12 known compounds, one of them isolated for the first time from natural source (6), were isolated from the stem bark of A. japonica. Their structures were elucidated using different spectroscopic methods including UV, IR, HR-ESIMS, 1D (1H and 13C) and 2D (1H – 1H COSY, HSQC and HMBC) NMR.
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Supplementary material Experimental details relating to this paper are available online.
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica ab
c
cd
Sabrin R.M. Ibrahim , Mostafa A. Fouad , Ahmed Abdel-Lateff , e
Tatsufumi Okino & Gamal A. Mohamed
df
a
Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia b
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt c
Department of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt d
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia e
Faculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan f
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt Published online: 12 Aug 2014.
To cite this article: Sabrin R.M. Ibrahim, Mostafa A. Fouad, Ahmed Abdel-Lateff, Tatsufumi Okino & Gamal A. Mohamed (2014): Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947489 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947489
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947489
Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica
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Sabrin R.M. Ibrahimab*, Mostafa A. Fouadc, Ahmed Abdel-Lateffcd, Tatsufumi Okinoe and Gamal A. Mohameddf a Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia; bDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; cDepartment of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt; dDepartment of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia; eFaculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan; fDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
(Received 23 May 2014; final version received 19 July 2014) Extensive chromatographic investigation of the ethanolic extract of Alnus japonica Steud stem bark led to the isolation of a new diarylheptanoid named alnuheptanoid A [(5S)-7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-5-methoxyheptan-3-one] (8), together with seven known diarylheptanoid derivatives: platyphyllenone (5), (5S)1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one (6), 1-(3,4-dihydroxyphenyl)-7-(4hydroxyphenyl)-4-hepten-3-one (7), hirsutenone (9), (5R)-O-methylhirsutanonol (10), hirsutanonol (11) and oregonin (13), three triterpenes: a-amyrin (1), betulinaldehyde (3) and betulinic acid (4), and two sterols: b-sitosterol (2) and daucosterol (12). Compound 6 was isolated for the first time from natural source. The structures of the isolated compounds were determined on the basis of spectroscopic measurements (UV, IR, HR-ESI-MS, 1D and 2D NMR). Keywords: Alnus japonica; Betulaceae; alnuheptanoid A; diarylheptanoids; triterpenoids; sterols
1. Introduction The genus Alnus (family Betulaceae) comprises 30 species of alder trees which ranges from large size to shrubs. The species of Alnus were used in oriental traditional medicine as remedies for fever, haemorrhage, burn injuries, diarrhoea and alcoholism. This genus is considered as a potential source of different triterpenes, tannins, flavonoids and diarylheptanoids (Karchesy et al. 1974; Nomura et al. 1981; Aoki et al. 1990; Lee et al. 1992, 1999; Tori et al. 1995; Kuroyanagi et al. 2005). Alnus japonica Steud is a deciduous tree, which is indigenous in the damp parts of the fields and forests of Japan. Hannoki (Japanese name) is a common tree in low mountainous areas of Japan (Nomura et al. 1981; Lee et al. 1992; Wada et al. 1998; Kuroyanagi et al. 2005). Diarylheptanoid is a class of compounds, which bears 1,7-diphenylheptane skeleton. They were isolated from different sources such as Zingiber, Curcuma, Alpinia, Alnus and Myrica. About 400 diarylheptanoids were published and recognised as potential therapeutic agents with different biological activities: anti-inflammatory, antioxidant, antitumour, oestrogenic, leishmanicidal, hepatoprotective and neuro-protective (Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Shin et al. 2002; Agarwal et al. 2003; Kang et al. 2004; Jin et al. 2007; Park et al. 2010; Lv & She 2012). Many diarylheptanoid derivatives isolated from A. japonica
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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exhibited interesting biological properties including hepatoprotective, antioxidant (Lee et al. 2003, 2005; Kim et al. 2004), antitumour (Joo et al. 2002; Choi et al. 2008), antiviral against avian influenza virus (Tung et al. 2010) and protease inhibitor in severe acute respiratory syndrome coronavirus (Park et al. 2012). Moreover, hirsutenone isolated from the bark of A. japonica acts by mimicking calcineurin inhibitor. So, it may be a new topical drug candidate for treatment of atopic dermatitis (Joo et al. 2009). We report herein the isolation and identification of the metabolites from A. japonica stem bark (Figure 1). The different chromatographic techniques and final purification using HPLC led to isolation of 13 compounds including three triterpenes (1, 3 and 4), two sterols (2 and 12) and eight diarylheptanoids (6– 11 and 13) one of them is new, alnuheptanoid A (8), and another one (6) isolated for the first time from natural source. Their structures were elucidated using different spectral methods (UV, IR, HR-ESI-MS, 1D and 2D NMR). 2. Results and discussion Compound 6 was isolated as a brown residue. It had a molecular formula C20H24O4 based on HR-ESI-MS pseudo-molecular ion peak at m/z ¼ 327.1677 [M –H]þ, requiring nine degrees of unsaturation: eight for two phenyl moieties and one for ketone carbonyl group. The presence of ketone carbonyl was confirmed by the IR absorption band at 1712 cm21 and 13C NMR signal at dC 210.7 (C-3). Also, the IR spectrum showed characteristic absorption bands due to a hydroxyl group (3395 cm21) and phenyl (1608 and 1521 cm21) moieties. Compound 6 had UV absorption maxima at 280 and 222 nm characteristic for the presence of aromatic moiety (Itokawa et al. 1985; Uehara et al. 1985). The 1H and 13C NMR spectra showed signals at dH 6.96 (2H, dd, J ¼ 8.2 and 2.2 Hz, H-20 , 60 )/dC 129.9 (C-20 , 60 ), 6.98 (2H, dd, J ¼ 8.2, 2.2 Hz, H-200 , 600 )/130.0 (C-200 , 600 ), 6.66 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 )/115.8 (C-30 , 50 ), 6.69 (2H, dd, J ¼ 8.2, 2.2 Hz, H-300 , 500 )/115.9 (C-300 , 500 ), 132.7 (C-10 ), 133.6 (C-100 ), 156.4 (C-40 ) and 156.5 (C-400 ) characteristic for the presence of two 4-oxyphenyl moieties (Ohta et al. 1984; Wada et al 1998;
R
H R 2 H 12 Glu
HO 1
RO
O
R1
R2
HO
1` 1
R2 3` A
HO 5 7 9
1
HO 3`
1`
HO 4`
6`
R1 H H OH
R2 H OH OH
O
OCH3
3
5
10
OH
HO 4`
6 CH3 8 CH3 11 H 13 Xyl 3``OH
6``
4`` OH
Figure 1. Structures of the isolated compounds (1 – 13).
OR1
3
5
7 1``
3``R3
B
6` R1
7 1``
O
R 3 CHO 4 COOH
6`` R2
R3
H H OH OH
H OH OH OH
4`` OH
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Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Park et al. 2010) (Supplementary Figures S1 and S2). They were established by the observed 1H – 1H COSY cross peaks of H-20 , H-60 /H-30 , H-50 and H-200 , H-600 /H-300 , H-500 . They were further confirmed by the HMBC correlations of H-20 and H-60 to C-10 , C-30 , C-40 and C-50 , H-30 and H-50 with C-10 , C-20 , C-40 and C-60 , H-200 and H-600 to C-100 and C-400 , and H-300 and H-500 with C-100 , C-200 , C-400 and C-600 (Supplementary Figure S4). Moreover, the 1H – 1H COSY showed two coupled methylene groups at dH 2.79 (2H, t, J ¼ 6.4 Hz, H-1) and 2.72 (2H, t, J ¼ 6.4 Hz, H-2), which correlated with the carbon signals resonating at dC 30.4 (C-1) and 46.2 (C-2), respectively, to give ZCH2ZCH2Z moiety. In the 1H and 13C NMR spectra, signals for three methylene groups at dH 2.66 (1H, m, H-4A) and 2.46 (1H, m, H-4B)/dC 48.0 (C-4), 1.71 (1H, m, H-6A) and 1.66 (1H, m, H-6B)/37.0 (C-6) and 2.52 (2H, m, H-7)/31.3 (C-7) and an oxymethine at dH 3.62 (1H, pintet, 6.6 Hz, H-5)/77.7 (C-5) were observed. The correlations observed in the 1H – 1H COSY displayed the connectivity from H-4 to H-7 to give ZCH2ZCHOHZCH2ZCH2Z moiety. The 3J HMBC correlations of H-1 and H-5 to C-3, H-2 and H-6 to C-4, and H-7 to C-5 confirmed the presence of 5-hydroxy heptan-3-one moiety (Ohta et al. 1984; Wada et al 1998; Lee, Kim, et al. 2000; Lee, Park, et al. 2000; Park et al. 2010; El-Halawany & Hattori 2012). Signals for methoxy group at dH 3.25/dC 56.9 were observed. The location of methoxy group was established by its HMBC cross peak to C-5 of the 5-hydroxy heptan-3-one moiety. The linkages of the two 4-oxyphenyl moieties on the alkyl group at C-1 and C-7 were confirmed by HMBC correlations from H-1 to C-10 , C-20 and C-60 , H20 and H-60 to C-10 , H-7 to C-100 , C-200 and C-600 , and H-200 and H-600 to C-7. The absolute stereochemistry at position C-5 was suggested to be S-form on the basis of positive sign optical rotation of 6 compared to negative sign optical rotations in the R-form of a series of analogous compounds (Giang et al. 2006; Park et al. 2010). This was further secured by the comparison 1 H NMR chemical shift of H-5 (dH 3.62) in 6 with those of related compounds in the literature [R-form (H-5 at dH 3.99– 4.10) and S-form (H-5 at dH 3.62)] (Itokawa et al. 1985; Li et al. 2003; Kuroyanagi et al. 2005; Park et al. 2010). On the basis of the NMR spectral data, 6 was identified as (5S)-1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one. Compound 6 was available from a commercial source and this is the first report for its isolation and structural characterisation from a natural source. Compound 8 was isolated as a brown residue. Its molecular formula C20H24O5 was supported by the presence of a quasi-molecular ion peak at m/z ¼ 343.1625 [M 2 H]þ in HRESI-MS. Compound 8 is 16 mass units more than 6, indicating the presence of an additional hydroxyl group in 8. It had UV absorption maxima at 284 and 225 nm characteristic for the presence of aromatic moiety (Itokawa et al. 1985; Uehara et al. 1985). The IR spectrum of 8 showed absorption bands for a hydroxyl at 3394 cm21, ketone carbonyl at 1710 cm21 and an aromatic ring at 1606 and 1524 cm21. The 13C and DEPT NMR spectra showed resonances for 20 carbons including 1 methoxy, 5 methylenes, 8 methines and 6 quaternary carbons; one of them for ketone carbonyl at dC 211.5 (C-3) and three for oxygen-bonded aromatic carbons at dC 156.4 (C-40 ), 146.2 (C-300 ) and 144.5 (C-400 ). The 1H and 13C NMR spectra of 8 were quite similar to those of 6, but the signals associated with one of the 4-oxyphenyl moieties were absent. Instead, new signals for 1,3,4-tri-substituted phenyl moiety at dH 6.65 (d, J ¼ 2.2 Hz, H-200 )/dC 116.4 (C-200 ), 6.73 (d, J ¼ 8.2 Hz, H-500 )/116.5 and 6.64 (dd, J ¼ 8.2, 2.2 Hz, H-600 )/120.6, 134.0 (C-100 ), 146.2 (C-300 ) and 144.5 (C-400 ) were observed. The 1H and 13C NMR spectra revealed the presence of 5-methoxy heptan-3-one moiety in 8 (Supplementary Figures S5 and S6). The resonances at dH 7.05 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 )/dC 130.3, 6.71 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 )/116.2 (C-30 , 50 ), 133.2 (C-10 ), and 156.4 (C-40 ) in 1H and 13C NMR spectra indicated the presence of a 1,4-di-substituted phenyl moiety. The S configuration at position 5 was assigned based on positive optical rotation value of 8 (Giang et al. 2006; Park et al. 2010), as well as the comparison of the 1H shifts of H-5 with those reported in related diarylheptanoids (Itokawa et al. 1985; Li et al. 2003; Kuroyanagi et al. 2005; Park et al. 2010). On the basis of the above pieces of
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evidence and by comparison of the spectral data with the literature, the structure of 8 was elucidated as (5S)-7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-5-methoxyheptane-3-one (8) and named alnuheptanoid A. The known compounds were identified through the analysis of their spectroscopic data (Supplementary material) and comparison of these data with the literature as a-amyrin (1) (Fingolo et al. 2013), b-sitosterol (2) (Sayed et al. 2007), betulinaldehyde (3) (Mahato & Kundu 1994), betulinic acid (4) (Mahato & Kundu 1994), platyphyllenone (5) (Fuchino et al. 1996), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-4-hepten-3-one (7) (Ohta et al. 1984; Chen et al. 1998), hirsutenone (9) (Ohta et al. 1984), (5R)-O-methylhirsutanonol (10) (Kuroyanagi et al. 2005), hirsutanonol (11) (Ohta et al. 1984), daucosterol (12) (Sayed et al. 2007) and oregonin (13) (Lee, Kim, et al. 2000; Lee, Park, et al. 2000). 3. Experimental 3.1. General experimental procedures Melting points were carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The UV spectra were determined using Perkin Elmer double beam spectrophotometer Model 550S (Perkin-Elmer, Waltham, MA, USA), attached to a Hitachi recorder Model 561, using 1 cm quartz cell. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were recorded on a Perkin-Elmer Model 341 LC Polarimeter (Perkin-Elmer). Electron impact mass spectra were recorded on a Finnigan MAT TSQ 7000 mass spectrometer (Thermo Finnigan, Bremen, Germany). ESI-MS were obtained with an LCQ DECA mass spectrometer (Thermo Finnigan) coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. HR-ESI-MS were recorded on an LTQ Orbitrap (Thermo Finnigan). 1D (1H and 13C) and 2D (COSY, HSQC and HMBC) NMR spectra were recorded on Bruker ARX 400 NMR spectrometers (Bruker BioSpin, Billerica, MA, USA). Semi-preparative HPLC (Merck, Darmstadt, Germany) coupled with UV detector L7400 (UV detection at 280 nm) was used. Separation column (250 £ 8 mm, i.d.) pre-packed with Eurosphere 100-C18 (Knauer, Berlin, Germany). Separation was achieved by applying a linear gradient from 80% H2O (pH 2.0) and 20% MeOH to 100% MeOH over 45 min. Solvents were distilled prior spectroscopic measurements. Column chromatographic separations were performed on silica gel 60 (0.04 – 0.063 mm, Merck). TLC analysis was performed on pre-coated TLC plates with silica gel 60 F254 (0.2 mm, Merck). The compounds were detected by UV absorption at lmax 255 and 366 nm followed by spraying with p-anisaldehyde –H2SO4 reagent and heating at 1108C for 1 –2 min. 3.2. Plant material A. japonica stem bark was collected in March 2005 from the authorised trees growing in the Botanical Garden, Hieinrich-Heine University, Du¨sseldorf, Germany. The plant was taxonomically identified by Prof. Dr Peter Westhoff (Professor of Development and Molecular Biology of Plants, Hieinrich-Heine University, Du¨sseldorf, Germany). A voucher specimen has been deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt (Registration code AJB-2005). 3.3. Extraction and isolation The dried powdered stem bark of A. japonica (200 g) was exhaustively extracted with 70% EtOH (4 £ 2 L). The combined extracts were concentrated under reduced pressure to afford a dark brown residue (12 g). The EtOH extract was subjected to vacuum liquid chromatography
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(VLC) using 50% n-hexane –CHCl3 (4 £ 500 mL) and EtOAc (4 £ 500 mL), which were separately concentrated to give 2.6 and 4.1 g, respectively. The 50% n-hexane –CHCl3 fraction (2.6 g) was subjected to repeated silica gel chromatographic columns (120 g £ 50 cm £ 3 cm) using n-hexane –EtOAc in order of increasing polarity to afford compounds 1 (5.9 mg, white fine needles), 2 (15 mg, white fine needles), 3 (8 mg, white crystals) and 4 (6.5 mg, white crystals). The EtOAc fraction (4.1 g) was subjected to VLC using CHCl3 –MeOH gradient elution to give six sub-fractions (A to F). Sub-fraction B (340 mg) was chromatographed on silica gel column (60 g £ 50 cm £ 2 cm) and eluted with CHCl3, followed by CHCl3 –MeOH gradient to obtain compound 5 (5.2 mg, brown residue). Silica gel column chromatography (80 g £ 50 cm £ 3 cm) of sub-fraction C (911 mg) using CHCl3 – MeOH gradient as an eluent gave impure compounds 6, 7 and 8. They were further purified by semi-preparative HPLC to yield 6 (4.5 mg, brown residue), 7 (3.7 mg, brown residue) and 8 (2.6 mg, brown residue). Subfraction D (760) was treated similarly as sub-fraction C to obtain semi-pure compounds 9, 10 and 11, which were further purified by semi-preparative HPLC to give 9 (2.9 mg, brown residue), 10 (2.3 mg, brown residue) and 11 (4.6 mg, brown residue). Sub-fraction E (1.2 g) was chromatographed on silica gel column (120 g £ 50 cm £ 3 cm) and eluted with CHCl3, followed by CHCl3 – MeOH gradient to obtain compounds 12 (14 mg, brown residue) and 13 (11 mg, white powder). 3.4. Spectral data (5S)-1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one (6): brown residue (4.5 mg); ½a22 D þ 21.6 (c ¼ 1.0, MeOH); UV (MeOH) lmax (log 1): 280 (3.96), 222 (4.35) nm; IR (KBr) gmax: 3395, 2986, 1712, 1608, 1521, 1418, 1145, 1050, 980 cm21; 1H NMR (CD3OD, 400 MHz): dH 2.79 (2H, t, J ¼ 6.4 Hz, H-1), 2.72 (2H, t, J ¼ 6.4 Hz, H-2), 2.66 (1H, m, H-4A), 2.46 (1H, m, H-4B), 3.62 (1H, pintet, J ¼ 6.6 Hz, H-5), 1.71 (1H, m, H-6A), 1.66 (1H, m, H-6B), 2.52 (2H, m, H-7), 6.96 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 ), 6.66 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 ), 6.98 (2H, dd, J ¼ 8.2, 2.2 Hz, H-200 , 600 ), 6.69 (2H, dd, J ¼ 8.2, 2.2 Hz, H-300 , 500 ), 3.25 (3H, s, 5-OCH3); 13C NMR (CD3OD, 100 MHz): dC 30.4 (t, C-1), 46.2 (t, C-2), 210.7 (s, C-3), 48.0 (t, C-4), 77.7 (d, C-5), 37.0 (t, C-6), 31.3 (t, C-7), 132.7 (s, C-10 ), 129.9 (d, C-20 , 60 ), 115.8 (d, C-30 , 50 ), 156.4 (s, C-40 ), 133.6 (s, C-100 ), 130.0 (d, C-200 , 600 ), 115.9 (d, C-300 , 500 ), 156.5 (s, C-400 ), 56.9 (q, 5-OCH3); HR-ESI-MS m/z 327.1677 (calcd for C20H23O4 [M 2 H]þ, 327.1675). Alnuheptanoid A (8): brown residue (2.6 mg); ½a22 D þ 19.1 (c ¼ 1.0, MeOH); UV (MeOH) lmax (log 1): 284 (4.11), 225 (4.52) nm; IR (KBr) gmax: 3394, 2980, 1710, 1606, 1524, 1285, 1127, 1052, 952 cm21; 1H NMR (CD3OD, 400 MHz): dH 2.80 (2H, t, J ¼ 6.4 Hz, H-1), 2.75 (2H, t, J ¼ 6.4 Hz, H-2), 2.68 (1H, m, H-4A), 2.52 (1H, m, H-4B), 3.65 (1H, pintet, J ¼ 6.5 Hz, H-5), 1.77 (1H, m, H-6A), 1.70 (1H, m, H-6B), 2.55 (2H, m, H-7), 7.05 (2H, dd, J ¼ 8.2, 2.2 Hz, H-20 , 60 ), 6.71 (2H, dd, J ¼ 8.2, 2.2 Hz, H-30 , 50 ), 6.65 (1H, d, J ¼ 2.2 Hz, H- 200 ), 6.73 (1H, d, J ¼ 8.2 Hz, H-500 ), 6.64 (1H, dd, J ¼ 8.2, 2.2 Hz, H-600 ), 3.30 (3H, s, 5-OCH3); 13C NMR (CD3OD, 100 MHz): dC 29.8 (t, C-1), 45.3 (t, C-2), 211.5 (s, C-3), 47.3 (t, C-4), 77.9 (d, C-5), 36.2 (t, C-6), 30.7 (t, C-7), 133.2 (s, C-10 ), 130.3 (d, C-20 , 60 ), 116.2 (d, C-30 , 50 ), 156.4 (s, C-40 ), 134.0 (s, C-100 ), 116.4 (d, C-200 ), 146.2 (s, C-300 ), 144.5 (s, C-400 ), 116.5 (d, C-500 ), 120.6 (d, C-600 ), 57.1 (q, 5-OCH3); HR-ESI-MS m/z 343.1625 (calcd for C20H23O5 [M 2 H]þ, 343.1623). 4. Conclusions A new diarylheptanoid, alnuheptanoid A (8), and 12 known compounds, one of them isolated for the first time from natural source (6), were isolated from the stem bark of A. japonica. Their structures were elucidated using different spectroscopic methods including UV, IR, HR-ESIMS, 1D (1H and 13C) and 2D (1H – 1H COSY, HSQC and HMBC) NMR.
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Supplementary material Experimental details relating to this paper are available online.
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