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Phytochemistry Letters 1 (2008) 85–88 www.elsevier.com/locate/phytol
Sesquiterpene lactones from Algerian Artemisia herba-alba Messai Laid a,*, Mohamed-Elamir F. Hegazy b, Ahmed A. Ahmed c, Kalla Ali a, Djaballah Belkacemi d, Shinji Ohta e b
a Department of Chemistry, Faculty of Science, Tebessa University, Algeria Chemistry of Medicinal Plant Department, National Research Center, Dokki, Cairo, Egypt c Department of Chemistry, Faculty of Science, El-Minia University, Egypt d Department of Chemistry, Faculty of Science, Oum El-Bouaghi University, Algeria e Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan
Received 6 February 2008; received in revised form 4 April 2008; accepted 7 April 2008 Available online 2 May 2008
Abstract The phytochemical investigation of the methylene chloride/methanol extract of the aerial parts of Artemisia herba-alba afforded two new natural sesquiterpene lactones 1b,9b-diacetoxyeudesm-3-en-5a,6b,11bH-12,6-olide (1) and 1b,9b-diacetoxyeudesm-4-en-6b,11bH-12,6-olide (2). The structures of the compounds were determined by comprehensive NMR studies, including DEPT, COSY, NOESY, HMQC, HMBC and HRMS. # 2008 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Keywords: Artemisia herba-alba; Sesquiterpene lactones
1. Introduction Plants of the genus Artemisia (family Asteraceae, tribe Anthemideae) have been used in folk medicine by many cultures since ancient times. Herbal tea from these species has been used as analgesic, antibacterial, antispasmodic, and hemostatic agents. Historically, Artemisia has been a productive genus in the search for new biologically active compounds. Phytochemical investigations have proven that this genus is rich in sesquiterpenes and monoterpenes (Ahmed et al., 1990; Tang et al., 2000; Tan et al., 1998; Jiangsu, 1977). The guaianolide structural type is the main sesquiterpene class of this genus, compounds with this fused 5,7,5-ring system have been reported to possess cytotoxic, root-growth stimulatory, germination inhibitory, and immunomodulatory activity (Ignatkov et al., 1990; El-Thaher et al., 2001; Davis et al., 1999; Singh et al., 1988). Artemisinin, an endoperoxide sesquiterpene lactone, is a constituent of the annual herb Artemisia annua (Bouwmeester et al., 1999) and this metabolite and its derivatives are valuable antimalarial drugs (Frederich et al., 2002; Wilair-
* Corresponding author. E-mail address: [email protected]
atana and Looareesuwan, 2002). The importance of this genus has led us to examine Artemisia herba-alba, a native perennial of Algeria. Organic solvent extraction of the aerial parts of A. herbaalba followed by fractionation on silica gel, Sephadex LH-20 and preparative TLC has yielded two new sesquiterpene lactones 1 and 2. 2. Results and discussion In continuation of our research on the chemical constituents of Algerian medicinal plants, the re-investigation of the aerial parts of A. herba-alba afforded two new sesquiterpene lactones 1 and 2. Compound 1 was isolated as a yellow oil, ½a25 D ¼ þ3:26 (c = 9.28, CHCl3). Complete structural information was obtained from the 1H NMR, 13C NMR, DEPT, 1H–1H COSY, 1 H–13C COSY, HMBC, NOESY and mass spectra. The EIMS did not exhibit the molecular ion peak; however, a medium strong peak at m/z 290 indicative of loss of acetic acid and a significant peak at m/z 230 indicating loss of another molecule of acetic acid, were observed. The exact mass determination of the 230 ion established the elemental composition of C15H18O2, calcd. 230.1307; exp. 230.1283 confirming the molecular formula of 1 as C19H26O6.
1874-3900/$ – see front matter # 2008 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2008.04.002
M. Laid et al. / Phytochemistry Letters 1 (2008) 85–88
The 1H NMR spectrum indicated the presence of an olefinic proton at dH 5.36 (brs, H-3), an olefinic methyl at dH 1.74 (s, H15), and a tertiary methyl at dH 1.08 (s, H-14). In addition, it revealed the presence of two singlet signals at dH 1.97 and 2.04 for the two methyls of the acetyl groups located at C-1 and C-9, respectively, and a double doublet signal at dH 5.02 (1H, dd, J = 8.0, 5.5 Hz, H-1), showed in the 1H–1H COSY spectrum correlations with two signals at dH 1.84 (1H, m, H-2b) and 2.44 (1H, m, H-2a). A secondary methyl group appeared at dH 1.12 (d, J = 7 Hz), and correlated with a methyl carbon signal at dC 12.5 in the HMQC spectrum (C-13). This correlated with a multiplet signal at dH 2.24 (1H, m) in 1H–1H COSY spectrum (H-11) suggesting an a-methyl-g-lactone. In the 1H–1H COSY spectra, clear correlations were observed between the signal at dH 3.84 (1H, dd, J = 9.5, 8.5 Hz, H-6) with the signals at dH 2.42 (1H, d, J = 9.5, H-5) and 1.65 (1H, m, H-7), suggesting the presence of a C5H–C6H(O)–C7H moiety. Besides, the signals at dH 1.56 (1H, m, H-8b) and 2.06 (1H, m, H-8a) correlated with two signals at dH 1.65 (1H, m, H-7) and 4.98 (1H, dd, J = 8.5, 4.0, H-9), indicating the presence of a C7(H)–C8H2–C9H(O) moiety. Accordingly, compound 1 contained a C5H–C6H(O)–C7H–C8H2–C9H(O) moiety. The 13C NMR spectrum exhibited 19 carbon signals which were classified by a DEPT experiment as follows: three
quaternary carbons at dc 178.7, 169.8 and 170.1 for C-12 and two carbonyls of the two acetyl groups; five methyl carbons at dc 12.5, 8.8, 23.3, 21.6 and 21.4 for C-13, C-14, C-15 and two methyls of the two acetyl groups and three oxygenated carbons at dc 77.3, 79.6 and 76.3 for C-1, C-6 and C-9. The remaining proton and carbon signals were listed in Table 1. Moreover, all proton and carbon signals were determined by 1H–1H COSY, HMQC and HMBC. The connectivity of the partial moieties and the position of the acyl groups and the lactonization were established by the HMBC spectrum of 1 (Table 1). The relative stereochemistry of 1 was established from the coupling constants and NOESY experiments. The relative configuration and stereochemistry at C-5, C-6 and C-7 were derived from the coupling constants (J5,6 = 9.5 and J6,7 = 8.5 Hz), which agree with the trans-diaxial disposition of the protons at C-5 (a), C-6 (b) and C-7 (a). The NOESY spectrum indicated clear effects between H-5 (dH 2.42) with H-1 (dH 5.02), indicating the a-orientation of H-1. Additionally, correlations between H-6 (dH 3.84) with CH3-14 (dH 1.08), and H-11 (dH 2.24), indicated the b-orientation of H-11 and CH3-14. Compound 1 was therefore, identified as 1b,9b-diacetoxyeudesm-3-en-5a,6b,11bH-12,6-olide and was a new natural compound and is reported here for the first time from a natural source (Sokoloff and Sfgal, 1977). Compound 2 was isolated as an yellow oil, ½a25 D ¼ þ4:16 (c = 0.048, CHCl3). The EIMS did not exhibit the molecular ion peak; however, three strong peaks at m/z 290, 248 and 230, resulting from loss of acetic acid, ketene and water, respectively, were observed, indicating the presence of two acetoxy groups. The high-resolution mass spectrum exhibited an ion peak [M– CH3COOH]+ at m/z 290.1528 (calcd. 290.1518), suggesting a molecular formula of C19H26O6. The 1H NMR and 13C NMR spectral data of 2 established the presence of a eudesmanolide-
Table 1 1 H and 13C NMR data for 1 and 2 Proton
dH 1 a
H-1 H-2a H-2b H-3a H-3b H-5 H-6b H-7a H-8a H-8b H-9a
5.02 2.44 1.84 5.36
dd (8.0, 5.5) m m brs
2.42 3.84 1.65 2.06 1.56 4.98
d (9.5) dd (9.5, 8.5) m m m dd (8.5, 4.0)
H-13a H-14b H-15 OAc-C1 OAc-C9
1.12 1.08 1.74 1.97 2.04
d (7.0) s s s s
5.16 dd (7.7, 3.3) 1. 64 m 1.63 m 1.65 m 1.86 m
4.09 1.18 1.82 1.10 4.82
br d (10.6) dddd (12.1, 11.5, 10.6, 2.2) ddd (11.5, 4.8, 2.2) ddd (11.5, 11.5, 10.6) dd (10.6, 4.8)
1.50 dq (12.1, 7.0) C-7, C-11, C-12 C-1, C-5, C-9, C-10 C-3, C-4, C-5 CO of acetate CO of acetate
0.94 1.13 1.90 1.78 1.76
d (7.0) s brs s s
*Overlapped. a Measured in CDCl3 (1H, 600 MHz; 13C, 150 MHz; TMS as an internal standard). b Measured in C6D6 (1H, 600 MHz; 13C, 150 MHz; TMS as an internal standard).
C-7, C-11, C-12 C-1, C-5, C-9, C-10 C-3, C-4, C-5 CO of acetate CO of acetate
C-1 C-2 C-3 C-4
77.3 29.4 121.4 132.1
74.5 24.5 30.7 129.1
C-5 C-6 C-7 C-8
49.5 79.6 49.5 30.0
128.5 80.8 49.2 29.9
C-9 C-10 C-11 C-12 C-13 C-14 C-15 OAc-C1 OAc-C9
76.3 42.7 40.6 178.7 12.5 23.3 8.8 169.8, 21.6 170.1, 21.4
76.4 45.0 40.8 176.8 12.6 16.0 20.4 169.4*, 21.0 169.4*, 21.1
M. Laid et al. / Phytochemistry Letters 1 (2008) 85–88
Fig. 1. Key NOEs for 2.
type sesquiterpene with two acetoxy groups at dH = 1.78 and 1.76 (s, both 3H) in the 1H NMR spectrum and dC = 21.0 (q), 21.1 (q) and two carbonyls at dC = 169.4 (s) in the 13C NMR spectrum. Moreover, the 1H NMR spectrum revealed the presence of the following signals: two singlet signals at dH 1.13 and 1.90 for H-14 and H-15, respectively, two double doublets at dH 5.16 (J = 7.7, 3.3 Hz) and 4.82 (10.6, 4.8 Hz) for H-1 and H-9, respectively. Furthermore, a broad doublet at dH 4.09 (J = 10.6 Hz) for H-6, showed a correlation with a signal at dH 1.18 (dddd, J = 12.1, 11.5, 10.6, 2.2 Hz, H-7) in the 1H–1H COSY spectrum. The 1H NMR spectrum showed a clear doublet at dH 0.94 (J = 7.0 Hz) which correlated with a methyl carbon at dC = 12.6 (C-13) in the HMQC spectrum. The latter protons correlated with a signal which integrated for one proton at dH 1.50 (1H, dq, J = 12.1, 7.0 Hz, H-11) in the in 1H–1H COSY spectrum, indicating the presence of an a-methyl-g-lactone. The 13C NMR data (Table 1) revealed the presence of 19 carbon atoms and their multiplicities by DEPT analysis confirmed the number of hydrogen atoms of the formula given above. The carbon atoms were assigned as five methyl carbons at dC 12.6 (C-13), 16.0 (C-14), 20.4 (C-15) and 21.0, 21.1 for the two methyls of the acetyl groups; three methylene carbons at dC 30.7 (C-3), 29.9 (C-8) and 24.5 (C-2); five methine carbons at dC 74.5 (C-1), 80.8 (C-6), 49.20 (C-7), 76.4 (C-9) and 40.8 (C-11) and three quaternary carbons at dC 45.0 (C-10), 129.1 (C-4) and 128.5 (C-5). Moreover, all proton and carbon signals were determined by 1H–1H COSY, HMQC and HMBC spectra. Confirmation of the structure of compound 2 was obtained by the results of the 2D long-range heteronuclear correlation (HMBC) analysis (Table 1). The stereochemistry of 2 was deduced from the chemical shifts and the values of the coupling constants and confirmed by the NOESY spectrum with inspection of Dreiding models. The NOESY spectrum indicated effects between H-6 (dH 4.09) with H-14 (dH 1.13) and H-11 (dH 1.50), indicating the b-orientation of H-11 and H-14. Furthermore, clear NOEs were observed between H-7 ((dH 1.18) and H-9 (dH 4.82), which showed an NOE with H-1 (dH 5.16), indicating the a-orientation of H-1 and H-9 (Fig. 1).
Although, the NMR spectral data of 2 was very close with those of the previously synthesized compound (Sokoloff and Sfgal, 1977), compound 2 showed opposite optical rotation sign ½a25 D ¼ þ4:16 (c = 0.048, CHCl3), while the synthetic compound showed an ½a25 D of 33 (c = 0.03, CHCl3). Compound 2 was therefore the enantiomer of the reported synthetic compound and was identified as 1b,9b-diacetoxyeudesm-4en-6b,11bH-12,6-olide, a new natural compound. 3. Experimental 3.1. General 1
H NMR (600 MHz, CDCl3), 13C NMR (150 MHz, CDCl3) and the 2D spectra were recorded on a JEOL (ECA) 600 MHz, with TMS as an internal standard. The 1H assignments were achieved by 1H–1H correlation spectroscopy (COSY). The 13C assignments were achieved by HMQC and HMBC. EIMS was recorded on a JEOL SX102A mass spectrometer. 3.2. Plant material The aerial parts of A. herba-alba were collected by the authors during the flowering stage, September 2005, in Tebessa (East of Algeria). A voucher specimen of the collection (A 128) was identified by Prof. Dr. Mohei Kamel and was deposited at the Department of Botany, El-Minia University, Egypt. 3.3. Extraction and isolation Air-dried plant material (1 kg) was ground and extracted with CH2Cl2–MeOH (1:1) at room temperature. The extract was concentrated in vacuo to obtain a residue of 60 g. The residue was prefractionated by CC (6 cm 120 cm) on silica gel eluting with n-hexane (2 L) followed by a gradient of n-hexane–CH2Cl2 up to 100% CH2Cl2 and CH2Cl2–MeOH up to 15% MeOH (2 L each of the solvent mixture). The n-hexane–CH2Cl2 (1:1) fraction was subjected to silica gel column chromatography (2 cm 60 cm),
M. Laid et al. / Phytochemistry Letters 1 (2008) 85–88
eluted with n-hexane–CH2Cl2, with increasing polarity up to 100% CH2Cl2 to give pure compounds 1 (25 mg) and 2 (8 mg). 3.4. 1b,9b-Diacetoxyeudesm-3-en-5a,6b,11bH-12,6olide(1) 1 13 C Yellow oil: ½a25 D ¼ þ3:26 (c = 0.092, CHCl3). H and NMR data (CDCl3) Table 1. EIMS m/z (%) = 290 [M– (AcOH)]+, 230 [M–2(AcOH)]+ (Calcd. for C15H18O2, 230.1307; exp. 230. 1283).
3.5. 1b,9b-Diacetoxyeudesm-4-en-6b,11bH-12,6-olide(2) 1 13 Yellow oil: ½a25 C D ¼ þ4:16 (c = 0.048 CHCl3). H and NMR data (C6D6) Table 1. EIMS m/z (%) = 290 [M–(AcOH)]+, 248 [M–CH2CO]+, 230 [M–H2O]+, 290.1528 (calcd. for C17H22O4; exp. 290.1518).
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