Naucline, a New Indole Alkaloid from the Bark of Nauclea officinalis †

A new indole alkaloid, naucline (1) together with four known alkaloids, angustine (2), angustidine (3), nauclefine (4) and naucletine (5), were isolated from the bark of Nauclea officinalis. The structures of all isolated compounds were elucidated with various spectroscopic methods such as 1D- and 2D- NMR, IR, UV and LCMS-IT-TOF. In addition to that of alkaloid 1, the complete 13C-NMR data of naucletine (5) were also reported. Naucline (1) showed a moderate vasorelaxant activity (90% relaxation at 1 × 10−5 M) whereas, angustine (2), nauclefine (4), and naucletine (5) showed potent vasorelaxant activity (more than 90% relaxation at 1 × 10−5 M) on an isolated rat aorta.


Introduction
The Rubiaceae family is known as Madder or Bedstraw, comprising 650 genera and 10,500 species worldwide [1]. Most of them are distributed primarily in the tropical regions and are mainly woody trees and shrubs [2]. A number of monoterpenoid indole alkaloids have been isolated from the Nauclea genus [3]. Some of these alkaloids were reported to exhibit certain biological activities such as anticonvulsant, antiproliferative, antimalarial, antimicrobial and antiparasitic properties [4][5][6][7][8]. Nauclea officinalis, a traditional Chinese medicine, is reported to contain alkaloids and terpenoids as major components [9,10].

Results and Discussion
Naucline (1) was isolated as a brownish amorphous solid. The LCMS-IT-TOF spectrum revealed a pseudomolecular ion peak [M+H] + at m/z 319.1450, corresponding to the molecular formula of C 20 H 18 N 2 O 2 . In the IR spectrum, an absorption band due to a conjugated carbonyl stretching vibration was observed at 1638 cm −1 .
In the 1 H-NMR spectrum, the presence of four aromatic protons, a broad peak of -NH-and one -CH 2 -CH 2 -N-group were observed, suggesting a β-carboline skeleton [10]. Two of the four aromatic protons in ring A appeared as doublets at δ H 7.48 and 7.33, and the other as two doublet of doublets (dd) at δ H 7.10 and 7.21 were attributed to H-9, H-12, H-10, and H-11, respectively. H-14 of ring D was revealed as a singlet at δ H 6.32 indicating that a double bond could be formed between C-15 and C-16. In addition, two upfield signals of H-19 (δ H 5.76, q, J = 6.6 Hz) and methyl protons (δ H 1.46, d, J = 6.6 Hz) characteristic of trisubstituted olefin group were observed [10]. The 13 C-NMR and DEPT spectra of naucline (1) indicated a total of 20 carbon signals; one methyl, one carbonyl, four methylenes, six methines and eight quaternary carbons. The presence of a carbonyl carbon was observed at δ C 163.1. The signals at δ C 58.9 and 66.6 could be assigned as the resonances of two oxymethylenes, C-17 and C-21, respectively.

General Procedures
Spectra were recorded on the following instruments: UV, Shimadzu UV-250 UV-visible spectrophotometer; IR, Perkin Elmer 1600; NMR, JEOL ECA 400 MHz; LCMS-IT-TOF, Shimadzu. All solvents, except those used for bulk extraction are AR grade. Silica gel 60 F 254 for thin layer chromatography (TLC) was used for column chromatography. Glass and aluminum supported silica gel 60 F 254 plates were used for TLC. TLC spots were visualized under UV light (254 and 365 nm) followed by spraying with Dragendorff's reagent for alkaloid detection.

Plant Material
The bark of Nauclea officinalis was collected at Hutan Simpan Madek, Keluang, Johor, Malaysia by the phytochemical group of the Department of Chemistry, Faculty of Science, University of Malaya. The voucher specimen (KL 5655) of this plant has been deposited at the Herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia.

Extraction and Isolation
Dried, grounded bark of the plant (2.0 kg) was first defatted with hexane (17 litres) for one night. The dried materials then were extracted using CH 2 Cl 2 (17 litres) twice for a 3-day period. The supernatant obtained was concentrated using rotary evaporator under reduced pressure to a volume of 500 mL and examined for its alkaloid content (using TLC and confirmed by spraying with Dragendorff's reagent). The extract was finally concentrated to give crude alkaloids (11.0 g). The crude alkaloid (8.0 g) was subjected to column chromatography over silica gel using dichloromethane and methanol solvent (100:0

Vasodilation Assay
A male Wistar rat weighing 260 g was sacrificed by bleeding from carotid arteries under anesthetization. A section of the thoracic aorta between the aortic arch and the diaphragm was removed and placed in oxygenated, modified Krebs-Henseleit solution (KHS: 118.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO 3 , 1.8 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 1.2 mM MgSO 4 , and 11.0 mM glucose). The aorta was cleaned of loosely adhering fat and connective tissue and cut into ring preparations 3 mm in length. The tissue was placed in a well-oxygenated (95% O 2 , 5% CO 2 ) bath of 5 mL KHS solution at 37 °C with one end connected to a tissue holder and the other to a force-displacement transducer (Nihon Kohden, TB-611T). The tissue was equilibrated for 60 min under a resting tension of 1.0 g. During this time the KHS in the tissue bath was replaced every 20 min.