Three new acyltyramines from Anisodus luridus Link et Otto (Solanaceae)

Abstract Three new acyltyramines, N-[2-(4-hydroxyphenyl)ethyl]hentriacontanamide (1), N-[2-(4-hydroxyphenyl)ethyl]nonacosanamide (2) and N-[2-(4-hydroxyphenyl)ethyl]heneicosanamide (3) have been isolated from n-hexane extract of leaves of Anisodus luridus (Solanaceae). Successive extraction of defatted leaves of A. luridus with methanol afforded a residue on removal of solvent under reduced pressure. Residue was partitioned by means of chloroform and n-butanol. Chromatographic resolution of n-BuOH extract afforded six known compounds, apigenin (4), luteolin (5), quercetin (6), quercetin 3-O-α-l-rhamnoside (7), kaempferol 3-O-α-rhamnoside (8) and quercetin 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (9). The structures of the isolated compounds were assigned with the help of spectroscopic techniques. This is the first report of isolation of these compounds from this plant.


Introduction
More than 75% of the Nepalese still depend on herbal plants as a local source of medicines for their primary health care (Devkota & Dutta 2001). Moreover, there are more than 90% ailments which are cured by medicinal and aromatic plants by local healers (Baidyas). However, some cases of ailments cannot be cured because of incomplete or lack of scientific knowledge and diagnosis. Anisodus luridus Link et Otto is one of the medicinal plants which belongs to Solanaceae family. It is mostly found in cultivated area around village regions and open forests (2300-4000 m) of Nepal. It is distributed from Northern Sino-Himalayan region of China, Tibet, Nepal, and India. The roots and seeds are used for alleviating pain and spasms, used as bio-pesticides in industries and traditional Chinese medicines (Gewali 2008). The plant is also dried and stored as winter fodder (Sherpa 2001). In Manang district of Nepal, plant is put on fire and the smoke is inhaled to treat wounds inside the nose. Crushed dried flowers are mixed with tobacco and smoked to treat gingivitis and toothache (Shrestha et al. 1995). Roots and seeds are also commonly used for plague (Quattrocchi 2012). Seeds are also used as anthelmintic, in sinusitis and colic pain (Bhattarai 2003). The plant is used in stomach ache, cholecystalgia, acute gastroenteritis and chronic gastroenteritis as a Chinese traditional medicine (Zhou et al. 2011). Roots are rich in alkaloids, whereas phytochemistry of leaves is still not explored. Only few alkaloids such as cuskohygrine (Rabinovich & Konovalova 1946), hyoscyamine (Gritsaeva & Prozorovskii 1956), scopolamine (Jovankovics 1966;Qin et al. 2014), 3-tropanol (Zhou et al. 2011) and apohyoscyamine (Verma et al. 2015) are reported from the roots of this plant. This motivated us to explore the phytochemical study of leaves of A. luridus.
HR-eSI-MS analysis of 1 exhibited a quasimolecular ion [M + Na] + peak at m/z 608.5372 (Calcd 608.5377 for C 39 H 71 NO 2 Na + ) which is consistent with molecular formula C 39 H 71 NO 2 . FT-IR spectrum exhibited distinguishable absorption bands for hydroxyl (br, 3450 cm −1 ), secondary amine (3306 cm −1 ), amide carbonyl (1641 cm −1 ) and aromatic (1555 cm −1 ) functionalities. The 1 H NMR (500 MHz, CDCl 3 ) spectrum exhibited two proton doublets at δ H 7.05, 2H (J = 8.0 Hz) and at δ H 6.78, 2H (J = 8.0 Hz) which were attributed to altogether four ortho-coupled hydrogens of an aromatic ring as an a 2 B 2 spin system. Two broad singlets at δ H 5.37 and 4.70 were considered to be originated due to phenolic hydrogen and amide hydrogen, respectively. a quintet at δ H 3.47 was probably originated due to methylene bound to an amide group (ar-CH 2 -CH 2 -NH-CO-). The spectrum also showed two triplets: one centred at δ H 2.74 probably due to benzylic methylene group (ar-CH 2 -CH 2 -NH-) and other for terminal methyl protons at δ H 0.88 (3H) indicating CH 3 -CH 2 -moiety in the molecule. a broad singlet for the several methylene protons was observed at δ H 1.25 (56H) indicating the presence of long alkyl chain in the molecule. 13 C NMR (125 MHz, CDCl 3 -CD 3 OD) spectrum showed the presence of phenolic carbon (δ C 155.3), benzylic carbon (δ C 36.6), methylene bound to amide function (δ C 40.7), amide carbonyl carbon (δ C 173.9), methylene carbon bound to amide carbonyl (δ C 34.6), several carbons of methylenes (δ C 22.6-25.7) and terminal methyl carbon (δ C 13.9) in the molecule. The 1 H and 13 C NMR data of 1 have also been compared with other acyltyramine derivatives reported earlier (achenbach et al. 1994;Xu et al. 2012). Careful analysis of these spectral data clearly suggested 1 to be an acyltyramine, N-hentriacontanoyltyramine.
Similarly, HR-eSI-MS showed quasimolecular ion [M + Na] + peaks at m/z 580.5058 (Calcd 580.5064 for C 37 H 67 NO 2 Na + ) and 468.3826 (Calcd 468.3812 for C 29 H 51 NO 2 Na + ) suggesting the molecular formula C 37 H 67 NO 2 and C 29 H 51 NO 2 , respectively, for 2 and 3 which were found to be congeners of 1 and differ only in methylenes present in the side acyl chain. Consequently, compounds 2 and 3 were characterised as N-nonacosanoyltyramine and N-heneicosanoyltyramine, respectively.

General experimental procedures
HR-eSI-MS and FaB-MS were performed on Jeol JMS-700 mass spectrometer, in m/z. Melting points were taken using a Yazawa hot stage microscope and are uncorrected. Optical rotations were measured on JaSCO DIP-360 polarimeter (cell length 5 cm). uV spectra were recorded on Shimadzu uV-1600PC spectrophotometer or JaSCO uV/visible spectrophotometer (model No. 7800). FT-IR spectra were recorded using either VaRIaN-640 or JaSCO FT-IR 5300 spectrometer. NMR spectra were measured using either Bruker DRX 500 MHz ( 1 H)/125 MHz ( 13 C) or Bruker DRX 300 MHz ( 1 H)/75 MHz ( 13 C) spectrometer; in CDCl 3 or CDCl 3 -CD 3 OD; δ (H) in ppm, rel. to Me 4 Si, J in Hz; δ (C) in ppm referenced to the solvent CDCl 3 or CD 3 OD.
Column chromatography was carried out using silica gel 70-325 mesh (Centron Research Laboratories, India) and 60-120 mesh (Merck, India). Thin-layer chromatography plates were prepared with silica gel G (Merck, India). Pre-coated TLC plates were also used (Merck Kgaa, 64,271 Darmstadt, Germany). The spots were visualised in iodine vapour followed by Lieberman Burchard reagent spray and heating the plates at 110 °C for 5-10 min. all the compounds were routinely dried over P 2 O 5 for 24 h in vacuo and were tested for purity by HPLC. anhydrous sodium sulphate was routinely used for drying the organic solvents.

Plant material
The leaves of A. luridus Link et Otto were collected from the district Mustang, Nepal, in October 2011. Plant specimen was identified by Prof S.D. Dubey, Department of Dravyaguna, Banaras Hindu university, Varanasi, India. a voucher specimen (No. LKLD/aLL/01) is preserved in the department.

Extraction and isolation
The fresh leaves of A. luridus (4.7 kg) were dried and powdered coarsely. The powdered leaves (3.5 kg) were extracted repeatedly with n-hexane (6 L) with the help of Soxhlet extractor (36 h). The n-hexane extract (186 g) was obtained on removal of solvent by distillation. n-Hexane extract was subjected to column chromatography over silica gel (70-325 mesh). The column was eluted (flow rate 5 mL/min) with different solvents in order of increasing polarity, viz. n-hexane (250 mL each, 3 L), benzene (250 mL each, 3.5 L), chloroform (100 mL each, 0.5 L; 300 mL each, 6 L), ethyl acetate (50 mL each, 2 L) and methanol (50 mL each, 2 L). n-Hexane, benzene and methanol fractions were discarded. Different fractions eluted from column chromatography were collected and monitored by TLC. Fractions 32-37 were pooled after TLC and yielded a yellowish white compound 1 (7.9 mg). Fractions 39-44 showed single-spot chromatogram on TLC plate. These fractions were pooled and yielded yellowish white compound 2 (8.2 mg). Fractions 45-49 were also pooled after TLC study and furnished yellowish white compound 3 (7.7 mg). Defatted leaf material of A. luridus (3.31 kg) was extracted repeatedly with methanol (6 L, 36 h) in a Soxhlet extractor. Removal of solvent by distillation afforded methanolic extract which was concentrated under reduced pressure and afforded a residue (400 g) which was stirred with water (1.2 L) for 12 h and partitioned with CHCl 3 (0.5 L × 3) and yielded CHCl 3 and aqueous layer. aqueous layer was, further, partitioned with n-BuOH (0.5 L × 3) and afforded n-BuOH soluble fraction. n-BuOH was removed under reduced pressure and yielded n-BuOH extract (