Constituents of Nepeta crassifolia (Lamiaceae)

Turk J Chem 31 (2007) , 463 – 470. c TÜBİTAK
Constituents of Nepeta crassifolia (Lamiaceae) Syed Amir IBRAHIM and Muhammad Shaiq ALI∗ H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, 75270, Karachi-PAKISTAN e-mail: Received 18.07.2006 A long chain ketone (crassifone), a pentacyclic triterpenoid coupled with fatty acid moiety (crassifoate), and an acyclic diterpenoid (crassifol) have been isolated from the ethanol soluble part of Nepeta crassifolia (Lamiaceae) collected from Kangavar, Iran. Structures of all the metabolites were elucidated with the aid of spectroscopic techniques, including 2D NMR experiments. Key Words: Nepeta crassifolia, Lamiaceae, spectroscopic characterization, crassifone, crassifoate, crassifol. Introduction Nepeta is a multiregional genus of the family Lamiaceae (Labiatae), comprising about 250 species distributed mainly in southwest and central Asia, Europe, North Africa, and North America.1,2 Several species of the genus Nepeta are rich in interesting biological activities and, for this reason only, many members of Nepeta have been investigated for bioactive constituents.3 Among the various medicinal properties, Nepeta species are famous for treating cardiovascular complaints, such as angina pectoris, cardiac thrombosis, tachycardia, and weakness of the heart.4−6 Several Iranian Nepeta species have been of great interest for use in Iranian folk and traditional medicines, and are used in the treatment of various diseases,7 including N. hindostana for sore throat8 and its decoction for fever and pain, including ear and tooth aches.9 N. glomerulosa is used to treat digestive troubles, pneumonia, and itching.10 Most Nepeta plants are rich in essential oils and, among their constituents, triterpenes are the most common.11−14 The present work describes the isolation and characterization of 3 new constituents: a long chain ketone (1, crassifone), a fatty acid coupled pentacyclic triterpene (2, crassifoate), and an acyclic diterpene (3, crassifol). The structures of 1-3 were elucidated by spectroscopic techniques, including modern NMR experiments. ∗ Corresponding author 463 Constituents of Nepeta crassifolia (Lamiaceae), S. A. IBRAHIM, M. S. ALI Experimental General techniques Melting points were determined on a Gallenkamp apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 digital polarimeter in CHCl3 using a 10-cm cell tube. IR spectra were recorded on a Jasco-320-A spectrometer. Mass spectra (EI, FD, and HR-EI) were measured in an electron impact mode on a Finnigan MAT 12 or MAT 312 spectrometer, and ions are given in m/z (%). 1 H- and 13 C-NMR spectra were recorded in CDCl3 with a Bruker AM-400 spectrometer at 400 MHz and at 100 MHz. Chemical shifts are given relative to TMS as an internal standard. TLC was performed with precoated silica gel G-25-UV254 plates and detection was made by spraying with ceric sulphate in 10% H2 SO4 . Silica gel (E. Merck, 230-400 mesh) was used for column chromatography. Plant material Nepeta crassifolia (all parts) was collected from Kangavar, Iran, in August 2003 and identified by Prof. Sanei Chariat Pannahi at Karaj Agriculture College, University of Tehran, where voucher specimens of the collected plant material are deposited in the herbarium. Extraction and isolation Shade-dried Nepeta crassifolia (2 kg, whole plant) was soaked in ethanol (5 L) for 10 days. The ethanol was evaporated under reduced pressure to avoid thermal decomposition. The crude ethanol soluble part thus obtained (71 g) was subjected to vacuum-liquid chromatography (VLC) using flesh silica (Merck, mesh size: 230-400 µm). Elution was carried out using 20%, 40%, 60%, and 80% ethyl acetate in hexane, and finally pure ethyl-acetate as the mobile phase. The fraction obtained with 20% ethyl acetate in hexane was re-chromatographed on a silica gel column using hexane, hexane-chloroform, and finally pure chloroform as the mobile phase, yielding compounds 1- 3. Fraction eluted with 20% chloroform in hexane yielded 1 as a white solid (15 mg). Crassifone (1) mp: 164 ◦ C; IR (CHCl3 ): 1725 (ketone C=O) cm−1 ; EIMS: m/z 492 [M]+ , 477 [M-CH3 ]+ , 449 [M- COCH3 ]+ ; HR-EIMS: m/z 492.5273 (C34 H68 O requires m/z 492.5269), 477. 5039 (C33 H65 O requires m/z 477.5035), 449.5090 (C32 H65 requires m/z 449.5086); 1 H-NMR (CDCl3 , 400 MHz): δ 2.39 (2H, t, J = 7.4 Hz, H-3), 2.11 (3H, s, H-1), 1.54 (2H, m, H-4), 1.24 (br. s, chain), and 0.86 (3H, t, J = 6.9 Hz, H-34); 13 C-NMR: (CDCl3 , 100 MHz): δ 29.8 (C-1), 209.5 (C-2), 43.8 (C-3), 23.9 (C-4), 29.2 (C-31), 31.9 (C-32), 22.7 (C-33), 14.1 (C-34), and 29.3-29.7(chain); HMBC: See Figure 1. Fraction eluted with 25% chloroform in hexane yielded 2 as a white solid (10 mg). Crassifoate (2) ◦ mp: 112 ◦ C; [α]28 D = –76 (chloroform, c 0.665); IR (CHCl3 ): 1735 (ester C=O), 1640 (C=C), 1190 (C–O) cm−1 ; EIMS: m/z 706 [M]+ , 423 [M-stearyl]+ , 405 [M-stearyl + H2 O]+ , 299 [rDA], and 283 [stearyl]+ ; HR-EIMS: m/z 706.6267 (C48 H82 O3 requires m/z 706.6263), 423.3631 (C30 H47 O requires m/z 464 Constituents of Nepeta crassifolia (Lamiaceae), S. A. IBRAHIM, M. S. ALI 423.3626), 405.3526 (C30 H45 requires m/z 405.3521), 299.2379 (C21 H31 O requires m/z 299.2374), and 283.2640 (C18 H35 O2 requires m/z 283.2636); 1 H-NMR (CDCl3 , 400 MHz): δ 5.52 (1H, dd, J = 8.0, 3.2 Hz, H-15), 4.50 (1H, dd, J = 10.1, 6.2 Hz, H-3α), 3.09 (1H, dd, J = 5.0, 4.6 Hz, H-11β), 2.78 (1H, d, J = 4.6 Hz, H-12β), 2.28 (2H, t, J = 7.4 Hz, H-2 ), 1.60 (m, H-3 ), 1.23 (chain, 28H), 1.08, 1.06, 0.98, 0.94, 0.88 (3H each, s, 5 x CH3 ), 0.87 (3H, t, J = 7.4 Hz, H-18 ), 0.86, 0.84, and 0.80 (3H each, s, 3 x CH3 ); 13 C-NMR: See Table 1; HMBC: See Figure 2. Table 1. 13 C-NMR Spectral Data of Crassifoate (2) and 11α, 12α-Oxidotaraxerol (2a). Carbon (2) (2a) Carbon (2) (2a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 38.0 23.3 80.3 37.7 54.7 18.8 33.2 38.9 51.9 37.6 53.5 58.2 36.6 157.1 118.9 35.3 35.4 48.1 38.2 26.9 79.0 38.7 54.6 18.9 33.2 38.9 52.0 37.5 53.6 58.3 36.6 157.1 118.9 35.2 35.4 48.1 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 17 18 Chain 40.3 28.7 36.6 38.2 27.9 17.0 16.6 27.0 30.2 29.9 33.7 19.5 173.5 34.8 25.2 22.7 14.1 29.2-29.7 40.3 28.7 36.6 38.2 27.9 17.0 15.4 27.1 30.2 29.9 33.7 19.6 – – – – – – In CDCl3 at 100 MHz. Fraction eluted with 30% chloroform in hexane yielded 3 as a mobile oil (0.7 mg). Crassifol (3) ◦ [α]28 (chloroform, c 0.778); IR (CHCl3 ): 3425 (OH), 1720 (ketone C=O) cm−1 ; EIMS: m/z D = +72.3 312 [M]+, 294 [M-H2 O]+ , 269 [M-COCH3 ]+ , and 251 [M-COCH3 + H2 O]+ ; HR-EIMS: m/z 312.3032 (C20 H40 O2 requires m/z 312.3028), 294.2926 (C20 H38 O requires m/z 294.2922), 269.2849 (C18 H37 O requires m/z 269.2844), and 251.2743 (C18 H35 requires m/z 251.2738); 1 H-NMR (CDCl3 , 400 MHz): δ 2.38 (2H, t, J = 7.5 Hz, H-4), 2.11 (3H, s, H-1), 1.51 (3H, s, H-3a), 0.86 (6H, d, J = 6.6 Hz, H-15a & 16), 0.85 ( (...truncated)