Article pubs.acs.org/jnp
HPLC-NMR and HPLC-MS Profiling and Bioassay-Guided Identification of Secondary Metabolites from the Australian Plant Haemodorum spicatum Robert Brkljača and Sylvia Urban* School of Applied Sciences (Discipline of Chemistry), Health Innovations Research Institute (HIRi), RMIT University, GPO Box 2476 V Melbourne, Victoria 3001, Australia S Supporting Information *
ABSTRACT: Phytochemical dereplication was undertaken on the bioactive crude CH2Cl2 extract of the bulbs of the Australian plant Haemodorum spicatum employing HPLCNMR and HPLC-MS methodologies. Subsequent bioassayguided isolation resulted in the identification of two new phenylphenalenones [haemoxiphidone (8) and haemodoronol (17)] and two new chromenes [haemodordione (13) and haemodordiol (16)], together with seven previously described compounds. Antimicrobial testing showed that the compounds displayed selective antibacterial activity. Most noteworthy were the activities displayed by several of the compounds against multi-drug-resistant Pseudomonas aeruginosa.
T
he Haemodorum genus (Haemodoraceae family) consists of 20 different species.1 Various studies have reported that the Haemodoraceae family contains chemotaxonomic markers made up of three distinct secondary metabolite structural classes including the phenylphenalenones, the oxabenzochrysenones, and the chromenes.2−4 Altogether, 30 phenylphenalenones have been reported from the family Haemodoraceae, including four from the Haemodorum genus, of which 15 possess a 9-substituted aromatic moiety, while the others display a 4-, 6-, or 7-substituted aromatic ring.2−24 Phenylphenalenones containing sugar moieties appear to favor the 7substituted aromatic position. Only six oxabenzochrysenones have been reported from the family Haemodoraceae, half of which occur in the Haemodorum genus, and, unlike the phenylphenalenones, the oxabenzochrysenones contain no terminal aromatic ring.2,3,7,11,15,19,25−29 The final structural class associated with the family Haemodoraceae is the chromenes, which can contain either one or two carbonyl functional groups in the lower right ring system. Twenty-three chromenes have been reported from the family Haemodoraceae, of which five occur within the genus Haemodorum.2,3,5−11,18,19,26,30,31 All but one of the chromenes have a 7-substituted aromatic ring. Biosynthetically, the phenylphenalenones are derived from a diarylheptanoid intermediate formed from phenylalanine and tyrosine.14,32−36 The chromenes are produced by the same diarylheptanoid intermediate pathway, but this most likely undergoes oxidative rearrangement and decarboxylation to incorporate the additional oxygen into the ring system.37 No indepth studies have been conducted to conclude the biosynthetic pathway of the oxabenzochrysenones. However, Published XXXX by the American Chemical Society
two reports have suggested that oxidative cyclization may be involved in converting phenylphenalenones to oxabenzochrysenones.27,28 As part of continuing efforts to study the chemical diversity and ethnopharmacology of Australian plants, particularly of the family Haemodoraceae, the chemistry of Haemodorum spicatum R. Br. was investigated. Of significance is the fact that the bulbs of H. spicatum are reported to have been used as a traditional medicine by the Australian Aborigines to treat dysentery.38 The present investigation on the bulbs of H. spicatum has yielded two new [(haemoxiphidone (8) and haemodoronol (17)] and two previously reported phenylphenalenones (2 and 15), four known oxabenzochrysenones (1, 5, 11, and 12), and two new [haemodordione (13) and haemodordiol (16)] chromenes as well as a reported (3) chromene. For three (2, 5, and 11) of the known compounds identified, this represents their first occurrence as natural products. The complete 2D NMR spectroscopic characterization of 2, 5, and 11 is also reported herein for the first time. All isolated compounds were evaluated for their selective antibacterial activity.
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RESULTS AND DISCUSSION The bulbs and stems of H. spicatum were extracted with 3:1 MeOH/CH2Cl2, evaporated under reduced pressure, and then sequentially and exhaustively solvent partitioned into CH2Cl2and MeOH-soluble fractions, respectively. Antimicrobial testing was carried out on both the CH2Cl2- and MeOH-soluble crude Received: November 12, 2014
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DOI: 10.1021/np500905g J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 1. Stop-flow Wet1D proton NMR spectra (500 MHz, 50% CH3CN/D2O, suppression of HDO and CH3CN at δH 4.64 and 2.38 ppm, respectively) of peaks A−I resulting from analysis of the CH2Cl2 crude extract of H. spicatum.
NMR system utilizes only D2O and CH3CN as mobile-phase solvents. MeOH as a solvent for elution is found to stick irreversibly to the HPLC tubing and flow cell and therefore is not recommended for HPLC-NMR use. In addition, samples must be dissolved in either D2O or HPLC-NMR grade CH3CN or a combination of the two solvents for similar reasons. The CH2Cl2 crude extract of the bulbs of H. spicatum displayed poor solubility in acetonitrile. In terms of HPLC-NMR chemical profiling this meant that only WET1D (stop-flow proton NMR) and gCOSY NMR spectra could be obtained for the compounds present, given the small amounts that could be loaded onto the HPLC column. These two NMR experiments alone are not sufficient for an unequivocal structure identification; however by adopting the use of chemical databases such as the Dictionary of Natural Products (www. dnp.chemnetbase.com) and SciFinder (https://scifinder.cas. org), dereplication may assist in concluding the structure classes present and often the most likely identity for some of the compounds detected. During HPLC-MS analysis it was noted that some of the compounds (11 and 12) displayed an inability to ionize or would only ionize on the low-resolution
extracts of the bulbs and the aerial parts of the plant. It was found that the CH2Cl2-soluble extract of the bulbs displayed significant antibacterial activity. On the basis of the observed activity along with the known ethnopharmacology associated with the crude extract derived from the bulbs, the CH2Cl2 crude extract was prioritized for further chemical investigation. Dereplication of the secondary metabolites present in the bioactive CH2Cl2 crude extract was achieved using HPLCNMR and HPLC-MS in combination with the use of selected databases. Nine peaks (peaks A−I in Figure 1) were detected by HPLC-NMR and HPLC-MS. Analysis of the HPLC-NMR data showed the presence of characteristic proton NMR signals that suggested the presence of aromatic and methoxy moieties, and further examination of the UV profiles obtained (as detected by PDA) confirmed the presence of three distinct chemical classes. These were the phenylphenalenones (λ >400 nm),18 oxabenzochrysenones (λ >500 nm),27 and chromenes (λ
