Volatile Constituents and Antimicrobial Activity of Naio ( Myoporum sandwicense A. Gray), a Native Hawaiian Tree

: Myoporum sandwicense A. Gray (naio) is one of the characteristic trees of Hawaiian montane– subalpine mesic forests. In this study, lab-distilled oils of M. sandwicense leaves, wood, and twigs growing on the island of Hawaii, as well as industrially produced wood oils, were characterized by gas chromatography–mass spectrometry (GC-MS). The lab-distilled oils were screened for antimicrobial activity. M. sandwicense leaf essential oil was rich in β -caryophyllene (15.1%), α -humulene (12.8%), germacrene D (7.9%), bicyclogermacrene (12.5%), brigalow ketol (9.6%), and myoporone (16.8%), while the wood essential oils were dominated by α -bisabolol and trans - α -bisabolol oxide B. The sapwood oil was dominated by palmitic acid (35.5%), linoleic acid (19.7%), oleic acid (31.9%), and stearic acid (5.7%), whereas the oil from twigs was rich in tricosane (77.3%) and pentacosane (13.1%). M. sandwicense essential oils were screened for antimicrobial activity against a panel of potentially pathogenic bacteria and fungi. The leaf essential oil of M. sandwicense showed excellent antibacterial activity against S. pyogenes and antifungal activity against A. fumigatus . The wood essential oil showed notable activity against S. pyogenes , A. fumigatus, A. niger, and M. gypseum . The twig oil was remarkably active against mold species. This work is the ﬁrst report we are aware of on the composition and antimicrobial properties of naio essential oils.


Introduction
Myoporum sandwicense A. Gray (Scrophulariaceae), commonly known as "Naio", "false sandalwood", or "bastard sandalwood", is a notoriously polymorphic evergreen shrub or tree that can grow as a creeping, prostrate shrub or it may occur as a tree around 15 m tall [1]. Furthermore, there are several varieties of M. sandwicense (e.g., M. sandwicense var.  [1]. The fruits of M. sandwicense are important food sources for the Hawaiian thrush (omao, Phaeornis obscurus obscurus) [3] and were apparently the exclusive food source of the now-extinct Kona grosbeak (Chloridops kona) [4]. Myoporum sandwicense populations have been adversely affected by habitat damage by feral ungulates [5] and by an introduced insect. Klambothrips myopori Mound and Morris (Phlaeothripidae) is a potential threat to the tree form of naio [6].
We have been interested in examining the essential oils of tropical plants for potential cultivation for the fragrance industry [7,8]. The purpose of this work was to obtain the

Gas Chromatography-Mass Spectrometry
M. sandwicense essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software v. 4.20 (Shimadzu Scientific Instruments, Columbia, MD, USA). The GC column was a ZB-5 ms fused silica capillary column (Phenomenex, Torrance, CA, USA) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium, with a column head pressure of 552 kPa and a flow rate of 1.37 mL/min. The injector temperature was 260 • C and the ion source temperature was 260 • C. The GC oven temperature was programmed for 50 • C initial temperature; temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared, and 0.1 µL was injected with a splitting mode (30:1). Identification of the oil components was based on their retention indices, determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the databases [9][10][11][12].

Antimicrobial
All bacteria were cultured on tryptic soy agar (Sigma-Aldrich, St. Louis, MO, USA) except for H. pylori and Streptococcus pneumoniae, which were grown on tryptic soy agar supplemented with 7% (v/v) defibrillated whole sheep blood (Cleveland Scientific, Ohio, USA) under micro-aerophilic conditions for 3 days [14]. All fungi were cultured on yeast malt agar (Sigma-Aldrich, St. Louis, MO, USA). For bacteria, a 50-µL volume of 1% (w/v) solution of the samples in DMSO was diluted in 50 µL of cation-adjusted Mueller-Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA). The sample solutions were then serially diluted (1:1) in fresh CAMHB to obtain concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 20 µg/mL. The microbes were harvested from a fresh culture and added to each well at a concentration of approximately 1.5 × 10 8 colony-forming unit (CFU)/mL for bacteria, and 7.5 × 10 7 CFU/mL for fungi. The 96-well microdilution plates for bacteria were incubated at 37 • C, and the fungi were incubated at 35 • C for 24 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration with no turbidity. Gentamicin (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive antibiotic control, and DMSO was used as the negative control (50 µL DMSO diluted in 50 µL broth medium, and then serially diluted, as above). For fungi, the above-mentioned method was implemented using yeast nitrogen base growth medium (Sigma-Aldrich, St. Louis, MO, USA) and Amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) as a positive antifungal control.

Essential Oil Composition
Steam distillation of the leaves of M. sandwicense produced a yield of 0.11%. Analysis of the leaf essential oil by GC-MS showed the oil to be rich in the sesquiterpene hydrocarbons β-caryophyllene (15.1%), α-humulene (12.8%), germacrene D (7.9%), bicyclogermacrene (12.5%), and the furanoterpenoids brigalow ketol (9.6%) and myoporone (16.8%) ( Table 1). Two samples of M. sandwicense wood were steam-distilled in a lab setting and analyzed by GC-MS ( Table 2). The average oil yield was 0.34%. The oil was yellow to golden in color and had a woody, sweet, slightly spicy, and sandalwood-like aroma. Twentythree industrially produced M. sandwicense wood oils (N1-N23) were analyzed by GC-MS (Table 3). Both lab-distilled and industrially distilled M. sandwicense wood essential oils were dominated by α-bisabolol and trans-α-bisabolol oxide B. Based on M. sandwicense essential oil compositions, a hierarchical cluster analysis of the oils from this work was carried out. The dissimilarity index was very small, indicating no significant differences in the essential oil compositions of the tested samples ( Figure 1).  Steam distillation of a sample of sapwood and a sample of twigs did not produce a separable essential oil. The aqueous distillate (hydrosol) was extracted with dichloromethane, however, to provide vanishingly small quantities of volatiles that could be analyzed by GC-MS (Table 4). The sapwood essential oil was dominated by fatty acids, palmitic acid (35.5%), linoleic acid (19.7%), oleic acid (31.9%, and stearic acid (5.7%), and derivatives of fatty acids. The essential oil from the twigs was rich in n-alkanes, tricosane (77.3%), and pentacosane (13.1%), probably reflecting a waxy coating on the twigs. 1447  -------1640  1638  Gossonorol  ------------------tr  ----1647 1646  ----------tr  tr  tr  tr  tr  tr  -------1820  1824  Avocadynofuran  tr  tr  ---tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  tr  1959 1958  There have been several previous investigations on essential oils of Myoporum species reported in the literature. Furanosesquiterpenoids have generally been the dominant constituents (see Table 5). Myoporone (16.0%), bicyclogermacrene (10.5%), germacrene D (9.0%), 10,11-dehydroisomyodesmone (5.2%), 10,11-dehydromyodesmone (4.5%) [20] M. tetrandrum (Labill.) Domin Leaf Dehydrongaione (78%), ngaione (7%), myoporone (11%) [17] 3.2. Antimicrobial Activity M. sandwicense essential oils were screened for antimicrobial activity against a panel of potentially pathogenic bacteria and fungi ( Table 6). The leaf essential oil of M. sandwicense showed excellent antibacterial activity against S. pyogenes (MIC = 78 µg/mL) and antifungal activity against A. fumigatus (MIC = 39 µg/mL). In fact, these two organisms were the most susceptible in our panel. The major components in the leaf essential oil: β-caryophyllene, α-humulene, bicyclogermacrene, and myoporone, may be responsible for the antimicrobial activity. Both β-caryophyllene and α-humulene have shown antimicrobial activity [21]. In earlier investigation, the essential oil of Centella asiatica, also rich in β-caryophyllene, α-humulene, and bicyclogermacrene, showed antibacterial activity [22]. Myoporone has shown antibacterial activity [20,23]. The wood essential oil of M. sandwicense, which was dominated by α-bisabolol and α-bisabolol oxide B, also showed notable activity against S. pyogenes and A. fumigatus (MIC = 78 µg/mL for each) as well as A. niger and M. gypseum (MIC = 39 µg/mL for each). α-Bisabolol has shown marginal antibacterial activity [24], but good antifungal activity [25,26]. In addition, α-bisabolol has been shown to potentiate the antibacterial activities of several antibiotics [27,28]. The antifungal activity of the essential oil from the twigs against the mold species was surprising. The twig essential oil was composed of 95.4% n-alkanes. Yin and co-workers have examined the antifungal activity of cuticular wax from Asian pear fruit (Pyrus bretchneideri) and concluded that long-chain alkanes have antifungal activity [29]. The flower SFE-CO2 extract of black elderberry, rich in ethyl palmitate, n-pentacosane, n-tricosane, and n-heneicosane, showed antifungal activity [30]. In contrast, the alkane-rich wood essential oil of agarwood (Aquilaria sinensis) showed only marginal antifungal activity [31]. Similarly, n-alkane-rich fractions from a hexane extract of Cupressus lusitanica leaves (>90% alkanes) showed no antifungal activity against a panel of dermatophytes [32]. It is likely that synergistic interactions with the minor components account for the observed antifungal activity of the twig essential oil. The non-polar fraction of surface wax from Asian pear fruits, dominated by long-chain n-alkanes showed inhibition of germination of Alternaria alternata [29]. Likewise, sorghum leaf wax, rich in long-chain alkanes, suppressed the growth of A. alternata [33]. Several long-chain n-alkanes have been screened for inhibition of mycelia and conidial germination of A. alternata and were found to be inactive [34]. Since alkanes do not have functional groups, they are unlikely to interact with biological targets involved with fungal metabolic pathways (e.g., glyoxylate cycle, pyrimidine biosynthesis, cytochrome P450 enzymes, iron metabolism, heme biosynthesis, and acetate metabolism), signal transduction pathways (e.g., MAP kinase, PDK1, and calcium signaling), or gene expression [35]. They are, however, non-polar compounds and would be expected to interact with fungal membranes and disturb membrane integrity, affecting membrane permeability. Thus, n-alkanes may act synergistically with other components in the essential oil, allowing these components to diffuse into the fungal cells.

Conclusions
This work is the first report on the chemical composition and antimicrobial properties of Myoporum sandwicense A. Gray (naio) essential oils. The leaf essential oil was made of β-caryophyllene, α-humulene, germacrene D, bicyclogermacrene, brigalow ketol, and myoporone, while the wood essential oil was dominated by α-bisabolol and trans-α-bisabolol oxide B. Palmitic acid, linoleic acid, oleic acid, and stearic acid were the major components of the sapwood oil, whereas the oil from twigs was rich in tricosane and pentacosane. The leaf essential oil of M. sandwicense showed excellent antibacterial activity against Streptococcus pyogenes and antifungal activity against A. fumigatus. The wood essential oil showed notable activity against S. pyogenes, A. fumigatus, A. niger, and M. gypseum. The twig oil was remarkably active against mold species.