Phytochemistry 97 (2014) 38–45
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Composition and antimicrobial activity of the essential oils from the Phebalium squamulosum species complex (Rutaceae) in New South Wales, Australia Nicholas J. Sadgrove a,⇑, Ian R.H. Telford b, Ben W. Greatrex a, Graham L. Jones a a b
Pharmaceuticals and Nutraceuticals Group (School of Science and Technology), University of New England, Armidale, NSW 2351, Australia Botany, School of Environmental and Rural Science and N.C.W. Beadle Herbarium, University of New England, Armidale, NSW 2351, Australia
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 22 October 2013 Available online 15 November 2013 Keywords: Chemotaxonomy Bioactivity Monoterpenes Sesquiterpenes
a b s t r a c t Essential oils have been hydrodistilled and characterized from 21 populations of taxa currently assigned to the endemic Australian species Phebalium squamulosum (Rutaceae: Boronieae) using GC–MS, NMR and quantified using GC–FID. Essential oils were further examined using principle component analysis to distinguish chemotypes, then screened for antimicrobial activity using broth dilution and TLC-bioautography. Collections of subspecies of P. squamulosum, namely subsp. coriaceum, subsp. gracile, subsp. lineare, subsp. squamulosum, subsp. ozothamnoides and subsp. verrucosum, were made from the wild and one from a cultivated plant of known provenance within New South Wales. Results demonstrated considerable intra- and interspecific essential oil component variation, suggesting the existence of distinct chemotypes and supporting previously observed segregate species based on morphological evidence. Antimicrobial testing revealed moderate to high activity for all essential oils dominated by sesquiterpene alcohols; elemol and eudesmol isomers. Conversely, very low antimicrobial activity was observed from essential oils dominated by monoterpenes. This study constitutes the most exhaustive investigation of essential oils from P. squamulosum subspecies to date and provides the first report of antimicrobial activity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The Australian genus Phebalium contains 28 formally named species (Wilson, 2013) with at least six putative new species (Telford and Bruhl, 2013), mostly distributed in southern and eastern Australia. Phebalium squamulosum is a yellow-flowering shrub or small tree exhibiting considerable polymorphism. This variation has resulted in the naming of nine subspecies (Wilson, 1970), six of which are examined here for essential oil composition. The first examination of essential oils from P. squamulosum was undertaken in 1971, shortly after Wilson (1970) revised the species. A novel sesquiterpene ‘squamulosone’ identified spectroscopically, and by conversion to known compounds, was shown to be the major constituent (Batey et al., 1971). Although no single subspecies was specified in that study, the locality of
⇑ Corresponding author. Address: Human Biology, School of Science and Technology, McClymont Building University of New England, Armidale, NSW 2351, Australia. Tel.: +1 61481130595. E-mail addresses: [email protected] (N.J. Sadgrove), [email protected] (G.L. Jones). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.015
the collection (Narrabeen, Sydney) places it as P. squamulosum subsp. squamulosum. Brophy et al. (2006), in their analyses of essential oils from Queensland Phebalium species, reported no squamulosone present in taxa identified as P. squamulosum subsp. squamulosum and P. squamulosum subsp. gracile from south-eastern Queensland. In New South Wales, Southwell (1970) showed that essential oil from leaves of P. squamulosum subsp. ozothamnoides (named as P. ozothamnoides in that study) collected from the Blue Mountains, contained elemol as the major component; however, this is present in fresh leaf material as the heat labile precursor, hedycaryol; prior to chemical alteration in the course of the steam distillation process. Pala-Paul et al. (2009) investigated two of the putative new species, P. stellatum and P. sylvaticum (Telford and Bruhl, 2013) currently assigned to P. squamulosum subsp. squamulosum. The current study contributes additional data to further the on-going survey of leaf essential oil composition of seven of the subspecies from the P. squamulosum species complex, depicted in Fig. 1. In addition, antimicrobial capacity of essential oils from all subspecies was investigated for the first time against nine microbial species, including some human pathogens.
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Fig. 1. Leaf morphology of the Phebalium squamulosum complex (Wilson, 1970), with molecular structures of the main chemotypically significant essential oil components.
2. Results and discussion 2.1. Character of essential oils P. squamulosum subspecies sampled for essential oil analysis are listed in Table 1. Leaf morphology is depicted in Fig. 1 and collection sites in Fig. 2. Essential oil character from populations currently assigned to P. squamulosum subsp. squamulosum infers the existence of two distinct intraspecific chemotypes (Tables 2 and 4); one corresponding to the major constituent squamulosone (81%), which was hydrodistilled from collections from Gloucester Tops. Squamulosone was first reported from a specimen collected at Narrabeen, Sydney (Batey et al., 1971). It may be worthwhile investigating further specimens to identify the southern boundary of this squamulosone chemotype. The other intraspecific chemotype is characterized by the major constituents; elemol (5–19%), a-, b- and c-eudesmol (Tables 2 and 4). This chemotype was hydrodistilled from material collected in north-eastern New South Wales (Fig. 1), so the expectation was that such specimens would produce similar essential oil character to that reported by Brophy et al. (2006). However, specimens collected by Brophy et al. (2006) produced a lower quantity of
these constituents and additionally produced the monoterpene minor constituents a-pinene and limonene, and the monoterpene major constituents myrcene and a- and b-phellandrene. It is unclear if environment and/or seasonal variation and/or growth stage influenced this variable essential oil character relative to the results obtained by us and presented here. Alternatively, essential oil data presented by Brophy et al. (2006) for P. squamulosum subsp. squamulosum may have been for the specimens collected from Mt Barney National Park (Mowburra Peak), indicating a potential third discrete chemotype. The Mowburra Peak population almost certainly represents a new species, P. sylvaticum ined. (Telford and Bruhl, 2013), previously examined for essential oils by Pala-Paul et al. (2009). The specimen of P. squamulosum subsp. squamulosum containing the ketone squamulosone was collected from soil derived from basalt in the Gloucester Tops area of Barrington Tops National Park (Fig. 2). This edaphic/geological aspect contrasts to the more northerly specimens producing elemol and eudesmol isomers, which were growing on soils derived from granite. In addition to differences in habitat preference, morphological differences between these two chemotypes strongly suggest more than one taxon is involved (Telford and Bruhl, 2013). It may be that the presence
Table 1 Vouchers for this study of collections of taxa currently assigned to Phebalium squamulosum. In the text essential oils are cited in shortened form using the collector code, for example, N.J.Sadgrove 186 becomes NJS186 in the text. Taxon P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum
subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp.
coriaceum 1 coriaceum 2 coriaceum 3 coriaceum 4 gracile 1 gracile 2 gracile 3 lineare lineare ozothamnoides 1 ozothamnoides 2 aff. ozothamnoides 1 aff. ozothamnoides 2 squamulosum 1 squamulosum 2 squamulosum 3 verrucosum 1a verrucosum 1b verrucosum 1c verrucosum 2 verrucosum 3
Locality
Date collected
Collector
Split Rock, Warrumbungle NP, NSW Spirey View track, Warrumbungle NP, NSW Spirey View track, Warrumbungle NP, NSW Mt Naman, Warrumbungle NP, NSW Pilliga Nature Reserve, N of Coonabarabran, NSW Cox’s Gap, Goulburn River NP, NSW Goulburn River, Wollara Rd, Goulburn River NP, NSW Wingen Maid Nature Reserve, NW of Scone, NSW Salisbury Trig, Wingen Maid Nature Reserve NW of Scone Cultivated: ex Tinderry Mtn, E of Michelago, NSW Hassans Walls, Lithgow, Blue Mtns, NSW Mushroom Rock, NE of Guyra, NSW Crown Mtn, Warra NP, NE of Guyra, NSW Gloucester Falls track, Barrington Tops NP, NSW Bluff Rock, S of Tenterfield, NSW Donnybrook State Forest, W of Tenterfield, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Tia Falls, E of Walcha, NSW Nymboida River, N of Dorrigo, NSW
May 2012 May 2012 May 2012 February 2013 May 2012 August 2012 August 2012 May 2013 May 2013 August 2012 August 2012 August 2012 August 2012 July 2012 August 2012 September 2012 March 2012 March 2012 March 2012 July 2012 August 2012
N.J. Sadgrove 186 (NE) N.J. Sadgrove 204 (NE) N.J. Sadgrove 207 (NE) N.J. Sadgrove 328 (NE) N.J. Sadgrove 191 (NE) N.J. Sadgrove 223 (NE) N.J. Sadgrove 251 (NE) N.J. Sadgrove 314 (NE) N.J. Sadgrove 334 (NE) I.R. Telford 13427 & N.J. Sadgrove (NE) N.J. Sadgrove 273 (NE) N.J. Sadgrove 289 (NE) N.J. Sadgrove 300 (NE) N.J. Sadgrove 246 (NE) N.J. Sadgrove 282 (NE) N.J. Sadgrove 303 & I. Telford (NE) N.J. Sadgrove 131 (NE) N.J. Sadgrove 132 (NE) N.J. Sadgrove 133 (NE) N.J. Sadgrove 243 (NE) N.J. Sadgrove 280 (NE)
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Fig. 2. Sampling sites for Phebalium squamulosum: d, subsp. squamulosum; j, subsp. coriaceum; +, subsp. gracile; , subsp. lineare; N, subsp. ozothamnoides; ., subsp. aff. ozothamnoides; ⁄, subsp. verrucosum.
of squamulosone supports this taxonomic observation, apparently being a chemical marker for P. squamulosum subsp. squamulosum in the strict sense. In the current study, squamulosone was also found in P. squamulosum subsp. lineare (Tables 2 and 4), with a similar whole essential oil to P. squamulosum subsp. squamulosum from Barrington Tops National Park, with the exception of overall yield, which was higher. This may have been a consequence of seasonal variation, because leaf samples from P. squamulosum subsp. lineare were collected in mid-summer, as opposed to specimens of P. squamulosum subsp. squamulosum, which were collected in winter. Previously it was unclear if P. squamulosum subsp. lineare was indeed a distinct subspecies, as it is morphologically similar to P. squamulosum subsp. gracile (pers. observation). The presence of squamulosone in populations of P. squamulosum subsp. lineare confirms they are distinct from subsp. gracile which lacks that ketone. P. squamulosum subsp. lineare will be raised to species rank (Telford and Bruhl, 2013). Limonene, a-pinene, elemol and eudesmol isomers are the major components of essential oils sourced from P. squamulosum subsp. gracile (Tables 2 and 5). Essential oils from these specimens produced the greatest component variation (within the discrete
subspecies), compared to the subspecies examined in this study, with the exception of P. squamulosum subsp. squamulosum (Tables 2 and 4). The essential oil components presented by Brophy et al. (2006), concerning essential oils hydrodistilled from P. squamulosum subsp. gracile, adds to this variation. Results presented by Brophy et al. (2006) demonstrated a monoterpene composition of more than 50%, compared to results in the current study, showing a range from 20% to 40% monoterpenes. In addition, Brophy et al. (2006) reported geijerene in this essential oil, which has previously been shown to be a characteristic of essential oils distilled from northern (i.e. Queensland) but not southern (i.e. New South Wales) specimens of other species, such as Geijera parviflora (Brophy et al., 2005). Geijerene was not present in any of our specimens of P. squamulosum subsp. gracile, however one specimen produced an as yet unidentified sesquiterpene (not geijerene, C15H26) with a relative abundance of 13.6% by GC–MS (Tables 2 and 5). Components from essential oils of P. squamulosum subsp. ozothamnoides and aff. ozothamnoides demonstrated no distinct chemovariation, other than a slightly higher quantity of a-pinene and elemol in specimens of aff. ozothamnoides (Tables 2 and 6). It is interesting to note however, that a substantially higher yield was obtained from specimens of aff. ozothamnoides (0.3–0.79%)
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Table 2 Chemical composition of essential oils from Phebalium squamulosum subspecies. Quantification data in relative abundance (%) is presented here as a range. Original data is included as supplementary material. Published arithmetic indices (Pub. AI) are from Adams (2007) or Singh et al. (2003) for dihydrotagetone. Calculated arithmetic indices (AI) were produced by the authors. Data related to the essential oil from P. squamulosum subsp. verrucosum was published earlier by Sadgrove et al. (2013) with further details of the enantiomeric composition of dihydrotagetone. Yield
AI
Pub. AI
P.s.s(A) 0.4
P.s.s(B) 0.02–0.7
P.s.l 0.9–1.7
P.s.g 0.2–0.6
P.s.o 0.03–0.8
P.s.c 0.1–0.2
P.s.v 1.6–3.1
Cyclofenchene a-Pinene a-Fenchene Camphene Sabinene b-Pinene Z-Meta-mentha-2,8-diene b-Myrcene d-2-Carene a-Phellandrene a-Terpinene p-Cymene Limonene b-Phellandrene 1,8-Cineol b-E-ocimene c-Terpinene Dihydrotagetone Terpinolene p-Mentha-2,4(8)-diene Linalool Nonanal Citronellal 2-Bornanol Terpinen-4-ol Cryptone Decanal a-Terpineol Citronellol Bornyl acetate C10H10O2 Myrtenyl acetate d-Elemene a-Copaene b-Elemene Dodecanal E-Caryophyllene b-Gurjurene Aromadendrene Citronellyl propionate a-Humulene Alloaromadendrene c-Muurolene Geranyl proprionate a-Selinene d-Selinene Viridiflorene a-Muurolene c-Patchoulene Bicyclogermacrene Epizonarene Germacrene A Geraniol isobutanoate d-Cadinene Elemol E-Dauca-4(ll),7-diene Palustrol Spathulenol Allohedcaryol Globulol Viridiflorol Guaiol Klusinone C15H24O C15H24O Ledol 5-Epi-7-epi-a-eudesmol 10-Epi-c-eudesmol C15H24O C15H26#
929 930 944 947 970 974 989 989 1000 1004 1015 1022 1028 1027 1028 1045 1057 1058 1088 1088 1100 1103 1230 1164 1177 1186 1206 1190 1230 1283 1284 1323 1339 1372 1390 1405 1414 1442 1445 1441 1448 1457 1476 1474 1481 1486 1491 1496 1498 1500 1503 1499 1514 1519 1546 1561 1569 1572 1593 1593 1593 1596 1597 1598 1601 1604 1609 1615 1619 1623
927 932 945 946 969 974 983 988 1001 1002 1014 1022 1024 1025 1026 1044 1054 1055 1086 1085 1095 1100 1223 1166 1174 1183 1201 1186 1223 1287 – 1324 1335 1374 1389 1408 1417 1431 1439 1444 1452 1458 1478 1476 1489 1492 1496 1500 1502 1500 1501 1508 1514 1522 1548 1556 1567 1577 1589 1590 1592 1600 1604 – – 1602 1607 1622 – –
0 1.5 0.7 0 0 0 0 0 0 3.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.9 0 0 0 1.4 0 0 0.2 0 0 6.8 0 0 0 0 0 2.1 0 0 0 0
0 0.2–3.4 0 0 0 0 0 0.9–6 0 0–2 0.1 0.1–0.2 0 0.2–7.1 0 0–0.2 0 0 0 0 0 0–3.7 0 0 0 0 0 0 0 0.2–1.3 0 0–0.5 0 0–0.3 0 0 0.3–0.8 0 0 0 0–0.2 0 0.3–0.6 0 0–0.1 0 0–4.5 0–0.3 0 0–0.5 0–0.6 0 0 0.3–0.7 5.7–18.7 0–0.4 0 0.7–0.9 0 6.3–33.3 0–0.8 1.5–3.9 0–0.4 0 0–3.9 0 0 0–14.1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–0.5 0 0.6–1.1 0 0–0.4 0 0 0–0.3 0 0 0 0 0 0 0 2.3–2.6 0 0 0 0 0 0 0 0 0 0.6–4.1 0 0 0 0–4.2 0 0 0 0 0.9–1.2 0
0 15.6–23.2 0 0–0.3 0–5.3 0.7–0.9 0 3.6–3.7 0 0–0.2 0–1.1 0–0.4 0 0 0 2.7–13.5 0–1.9 0 0–0.6 0 0 0 0 0 0–2.9 0 0 0–0.2 0 0 0–2.6 0 0 0 0.8–0.9 0 0–0.5 0 0 0 0–0.2 0 0 0 0 0 0 0 0–0.7 0.9–9.7 0 0 0 0 13.9–27 0 0 0–3.9 0 0–1.6 0 0–3.6 0 0 0 0–1.7 2.4–6.9 1–10.3 0 0–13.6
0 0.1–5 0 0 0–0.3 0–0.2 0.2–3.6 0–3.1 0 0.3–0.6 0 0–0.8 0 1.5–3.2 0 0 0 0 0 0 0–0.2 0 0 0 0–0.2 0 0 0 0–0.1 0 0 0 0 0 0–0.2 0 0.2–0.8 0 0 0 0–0.1 0 0–0.2 0 0–0.2 0–0.1 0 0 0 1.5–1.9 0–0.2 0–0.2 0 0–0.3 19.7–29.5 0 0 0–1.3 0–0.6 0.4–7.5 0 0–4.7 0 0 0 0 0–1.4 4.2–5.5 0 0
0 26.5–32.3 0 0 0.5–4.3 0.8–1.4 0 1.3–11.9 0–2.5 3.5–5.1 0–0.3 0.7–2.1 0 22.5–31.1 0 0–1.7 0–0.6 0 0 0.4–0.6 0 0 0–0.5 0–0.4 0–0.2 0–0.3 0–0.6 0 0 0–0.6 0 0 0–1.5 0 0 0–0.4 0–2.1 0 0–2.1 0–0.3 0 0 0 0–0.9 0 0 0 0 0 7.5–16.5 0 0 0–1.2 0 0 0 0 0–7.2 0 0–2 0 0 0 0 0 0–1.5 0 0 0 0
0–3.2 0–1.1 0 0 0 0 0 0 0 0 0 0 0–0.1 0 0–3.1 0 0 90.4–98.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–5.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–0.5 0 0
(continued on next page)
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Table 2 (continued) Yield
AI
Pub. AI
P.s.s(A) 0.4
P.s.s(B) 0.02–0.7
P.s.l 0.9–1.7
P.s.g 0.2–0.6
P.s.o 0.03–0.8
P.s.c 0.1–0.2
P.s.v 1.6–3.1
c-Eudesmol
1626 1631 1637 1639 1644 1647 1654 1658 1662 1684 1725 1785 1787
1630 1629 1638 1640 1649 1652 1656 – 1670 – – – 1792
0 0 0 0 0 0 0 0 0 0 0 81 0
1.5–13.1 0–1.3 0.6–2.6 0 1.4–16.3 1.9–9.8 0 0 2.3–3.1 0 0 0 0.2–6
0 0 0 0 0 0 0 0 0 0 0–0.6 85–88 0
6.6–14.3 0 0–0.5 0 4.8–9.7 0–17.8 0 0–1.2 3.9–4 0–1.2 0 0 0–2.2
2.2–5.5 0–0.1 0–1.7 0–1.9 1.6–2.4 3.2–5.7 0–2.4 0 12.4–21 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0–3.3 0 0 0 0 0 0 0 0
Eremoligenol Epi-a-cadinol Hinesol b-Eudesmol a-Eudesmol Valerianol C15H24 Bulnesol C15H22O C15H24O Squamulosone 8-a-Acetoxyelemol
P.s.s(A) – subsp. squamulosum chemotypes A, (B) – chemotypes B; P.s.l – subsp. lineare; P.s.g – subsp. gracile; P.s.o – subsp. ozothamnoides; P.s.c – subsp. coreaceum; P.s.v – subsp. verrucosum. # Unidentified compound in P. squamulosum subsp. gracile may indicate another chemotype m/z: 41 (9), 59 (48), 81 (31), 91 (27), 108 (33), 121 (17), 135 (9), 149 (100), 164 (34), 189 (8) and 204 (7).
than from subsp. ozothamnoides (0.03–0.1%). These differences corroborate the morphological differences indicating two distinct taxa, although environmental factors may be responsible for yield differences, with seasonal factors less plausible because all specimens were collected in winter. It is also interesting to note that a specimen of P. squamulosum subsp. ozothamnoides, collected much earlier by another researcher from the same location as NJS273, produced an essential oil yield at approximately 1.64% (Southwell, 1970). In this particular instance, it is plausible to speculate seasonal variation in essential oil yield, particularly as yields tend to increase in arid summers (unpublished results). The study undertaken by Southwell (1970) revealed that the presence of elemol in the essential oil following steam distillation of leaf material of subsp. ozothamnoides was a result of the thermal Cope rearrangement of the precursor hedycaryol, which was found in the macerated leaf material following light petroleum extraction. It is suggested that elemol is not a natural product per se, but rather an artifact of the distillation process (Southwell, 1970). At present this study, and those completed elsewhere (Brophy et al., 2006), have revealed elemol to be also present in subsp. gracile, subsp. squamulosum and subsp. aff. ozothamnoides. This result may indicate the presence of the hedycaryol precursor in these subspecies as well. Essential oil from P. squamulosum subsp. coriaceum was predominantly monoterpene in character (Tables 2 and 7). Major components were a-pinene (26–33%), b-pinene (1.3%), b-myrcene (1–4%), a-phellandrene (3–5%), b-phellandrene (22–30%), bicyclogermacrene (7–17%) and spathulenol (2–7%). Of the essential oils characterized by Brophy et al. (2006) the most similar in composition to this is that from P. squamulosum subsp. squamulosum, with the exception of the lower quantity of a-pinene (3.3%) and the presence of elemol in subsp. squamulosum. Thus, subsp. coriaceum is unlike any of the previously reported essential oils from Phebalium, and certainly indicates its taxonomic misplacement in P. squamulosum. Hydrodistillation of P. squamulosum subsp. verrucosum (Tables 2 and 8) produced an essential oil predominantly made up of dihydrotagetone (86–99%), which was first characterized by Jones and Smith (1925) from Tagetes glandulifera, now correctly T. minuta. The essential oil of T. minuta typically contains dihydrotagetone at a maximum concentration of 7% of the entire essential oil fraction, but it can reach as high as 43%, depending on the time of year and part of the plant distilled (Chammorro et al., 2008). Lassak and Southwell (1974) revealed the presence of (+)-dihydrotagetone (2,6-dimethyloct-7-en-4-one) in relatively high enantiomeric purity
and concentration (97.4%) in essential oil from P. glandulosum subsp. macrocalyx (Giles et al., 2008) sourced from near Dubbo, New South Wales, but referred to as subsp. glandulosum in their study. Sadgrove et al. (2013) reported dihydrotagetone from other subspecies of P. glandulosum and in P. squamulosum subsp. verrucosum with results strikingly similar to those previously obtained by Lassak and Southwell (1974). Thus, dihydrotagetone appears to be a chemical marker for P. glandulosum and related taxa. Similar essential oil profiles, together with morphological differences (Telford and Bruhl, 2013) indicate the taxonomic misplacement of subsp. verrucosum, having closer affinities to the P. glandulosum group. 2.2. Intra- and interspecific essential oil chemotypes There were striking differences in the components of the essential oils, with the main interspecific variation between being seen in either the presence of dihydrotagetone at greater than 85% of the total essential oil content, corresponding to subsp. verrucosum, or its complete absence, as seen in other subspecies. The only intraspecific variation (subspecific) was seen within the subsp. squamulosum group, with squamulosone present at either >80% in southern specimens or completely absent in northern specimens. To examine the correlation between taxonomic classification and essential oil composition, principle component analysis (PCA) was used. Calculation of the principle components for the most abundant 19 components found in the essential oils showed that there were 7 principal components that together accounted for 91% of the variance of the data set. Principal component 1 (PC1) accounted for 23% of the variance and principal component 3 (PC3) accounted for 13% of the variance seen between different chemotypes. A plot of PC1 against PC3 showed that clusters were seen which corresponded to the taxonomic classification based on plant morphology. These clusters have been circled in Fig. 3 by the authors. PC1 separated subsp. coriaceum from subsp. verrucosum and subsp. lineare which also clustered separately to subsp. ozothamnoides and aff. ozothamnoides. PC3 partially separated subsp. squamulosum from subsp. ozothamnoides/aff ozothamnoides and further separated subsp. gracile from the remaining species. The two subsp., ozothamnoides and aff. ozothamnoides, clustered together indicating that there is no significant variation in the essential oils from these subspecies other than the yield, which was not a parameter in the analysis. There was overlap seen in the clustering of subsp. squamulosum and ozothamnoides and overlap of verucosum and
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Fig. 3. Scoring plot from principal component analysis, showing the five species, with PC1 plotted against PC3.
lineare. The loading plot for PC1 and PC3, Fig. 4, shows that the main positive contributors to PC1 were b-phellandrene, bicyclogemacrene, a-pinene while dihydrotagetone and squamulosone had large negative coefficients. PC3 was determined by the content of b-eudesmol, E-ocimene and c-eudesmol although elemol and a-pinene also had significant effects. Thus, from these results, chemical characterization presented in this study supports classification of six distinct essential oil chemotypes within the P. squamulosum complex; most of which were demonstrated using principal component analysis and are seen in Fig. 3. The first chemotype is dominated by squamulosone, which is characteristic of two subspecies; subsp. squamulosum and subsp. lineare specimens from within the Hunter valley in central New South Wales (Tables 2 and 4: Figs. 1 and 2). It is interesting that PCA (Fig. 3) clustered both chemotypes of subsp. squamulosum together despite the distinct chemotypic representation of squamulosone at >80% in southern, morphologically distinct specimens. Although this makes the essential oil from southern specimens of subsp. squamulosum more similar to subsp. lineare, PCA was additionally able to differentiate between the two. The second and third essential oil chemotypes are primarily characterized by the major chemical component elemol, which is present in the essential oils from four subspecies; P. squamulosum subsp. squamulosum (northern distribution) (Tables 2 and 4: Figs. 1 and 2), subsp. gracile (Tables 2 and 5) and subsp. ozothamnoides or aff. ozothamnoides (Tables 2 and 6). Essential oils from these four subspecies can be distinguished into two groups; P. squamulosum subsp. squamulosum from the northern distribution is predominantly sesquiterpenoid but with low quantities of bulnesol (2–3%) and subsp. ozothamnoides and aff. ozothamnoides contain bulnesol at a concentration higher than other squamulosum subspecies (12–21%). This higher occurrence of bulnesol in essential oils from subsp. ozothamnoides and aff. ozothamnoides indicates that these two subspecies belong to a separate chemotype. Thus, subsp. squamulosum from the northern distribution belongs to the third chemotype, and subsp. ozothamnoides and aff. ozothamnoides belong to the fourth. Although no phytochemical difference has yet been demonstrated between subsp. ozothamnoides and aff. ozothamnoides, other than overall winter essential oil yield, morphological differences are sufficient to demonstrate these two are different species and warrant species revision (pers. observation). Principal component analysis showed a degree of observable difference between essential oils from subsp. gracile and subsp. squamulosum; however, further analysis, using additional specimens,
may strengthen the cluster separation observed in Fig. 3. Additionally, clustering in Fig. 3 may have been offset by sample NS251, which was collected from a geologically and edaphically different site. This factor may be responsible for the unusual essential oil produced by this specimen, which appears to be an outlier at the moment. Essential oil from subsp. squamulosum contained globulol varying in concentration from 6% to 33%, and subsp. gracile contained monoterpenes a-pinene (15–23%) and limonene (3–14%). Additionally, Brophy et al. (2006) reported geijerene in the essential oil from Queensland specimens of subsp. gracile, which may again indicate a further discrete chemotype. Therefore, using this observation and the clustering observed in Fig. 3, it may be sufficient to put subsp. gracile and subsp. squamulosum (northern distribution) into different chemotypes, with subsp. squamulosum in the third and subsp. gracile on its own, in the fifth chemotype. The sixth essential oil chemotype is characterized by essential oil from subsp. coriaceum (Tables 2 and 7; Fig. 3), which is dominated by monoterpenes such as a- and b-pinene, b-myrcene and a- and b-phellandrene. The seventh chemotype is represented by essential oil from subsp. verrucosum (Tables 2 and 8) which is dominated by dihydrotagetone, which clustered around subsp. lineare in Fig. 3. In a previous study, Sadgrove et al. (2013) demonstrated that P. squamulosum subsp. verrucosum had natural dihydrotagetone enantiotypes, but factors contributing to this distinct variation were not further examined. At present it is unclear if subsp. lineare and subsp. squamulosum (southern specimen) can be differentiated into two separate chemotypes. In Fig. 3 they clustered separately, despite both apparently being the same essential oil with squamulosone at a concentration >80%. Further sampling may clarify this. 2.3. Antimicrobial activity of essential oils This study constitutes the first time essential oils from Phebalium species are examined for antimicrobial activity (Table 3). Nil or very low antimicrobial activity was observed for type three and four essential oils (subsp. coriaceum and subsp. verrucosum, respectively). Relatively high antimicrobial activity was observed for type one and two essential oils (subsp. squamulosum, subsp. gracile and subsp. ozothamnoides and aff. ozothamnoides). This high antimicrobial activity was evident only against select microbial species, and did not manifest against others (Table 3). At present it is not clear why this trend emerged. It does not seem to relate to gram-stain patterns.
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Table 3 Mean inhibitory concentrations of essential oils from subspecies of the squamulosum complex. Additional microbial species examined were Salmonella typhimirium and Escherechia coli but no inhibition was observed up to 4% v/v essential oil concentration. The control was tetracycline in lg/ml or nystatin where C. albicans was used. Essential oils MICs are presented as percentages calculated in volume of oil relative to volume of media.
S. aureus S. epidermidis B. subtilis S. pneumoniae K. aeroenes P. aeruginosa C. albicans
131
246
289
282
P. s. ozo
251
191-92-93
223-24-25
300
204-05-06
Control
> > > – > 4–5 >
0.25–0.5 N/A 0.06–0.5 0.02–0.3 > >4 0.5–1
0.06–0.25 0.5–3 0.1–0.25 0.06–0.25 0.1–0.5 2–3 0.5
0.03–0.5 0.5–1 0.03–0.5 0.05–0.25 0.06–0.25 2–3 0.25–1
0.2–0.5 0.5–2 0.25–0.5 0.06–1 0.25–0.5 2–3 0.5–1
0.13–1 0.5–2 0.06–0.5 0.06–1 0.5–1 4–5 0.5–1
0.13–0.5 0.5–2 0.06–0.13 0.06–1 0.1–0.5 2–3 0.1–1
0.25–0.5 0.5–2 0.13–0.25 0.13–1 0.13–0.25 2–3 0.5–1
0.06–0.25 0.5–3 0.06–0.13 0.13–0.5 0.13–0.25 2–3 0.1–1
>4
0.13–1 0.15–2 0.25–8 0.03–0.5 0.25–2 0.75–16 1.25–7
5.5 >4 >4 >4 0.25–0.5
P. squamulosum subsp. verrucosum (NS131), subsp. gracile (NS251, NS191-92-93 and NS223-24-25), subsp. ozothamnoides (P. s. ozo), subsp. aff. ozothamnoides (NS289 and NS300), subsp. coreaceum (204-05-06) and subsp. squamulosum (NS246 and NS282).
Individual discrete components corresponding with zones of antimicrobial activity were not fully resolved by TLC-bioautography, as sesquiterpenols habitually co-migrate in thin layer chromatography. However, results demonstrated that sesquiterpenols as a class were largely responsible for antimicrobial activity. Sesquiterpene alcohols have often been associated with high antifungal and antibacterial activity, particularly with components a- and b-eudesmol and bicyclogermacrene (Koroch et al., 2007). These were present in the type one and two essential oils producing the greatest bioactivity in this study. It is probable that elemol/hedycaryol is also responsible for enhanced antimicrobial ability. 3. Experimental 3.1. Collection of taxa Taxa surveyed (Table 1) are, for the first time: P. squamulosum subsp. coriaceum and subsp. verrucosum; from two subspecies that have not been hitherto examined in New South Wales populations: subsp. gracile and subsp. squamulosum; from an additional population of P. squamulosum subsp. ozothamnoides, and from populations of a putative new species currently included under subsp. ozothamnoides (subsp. aff. ozothamnoides) (Figs. 1 and 2). Aerial parts from P. squamulosum subspecies were collected from the wild in New South Wales (SL100811) (Fig. 2) or from cultivated plants of known provenance. Voucher specimens were lodged with the N.C.W Beadle Herbarium (NE), University of New England, Armidale, NSW, Australia. 3.2. Essential oil extraction and characterization Approximately 500 g of fresh foliage was removed from the twig and chopped into 0.5 mm fragments and placed into a 5 L round bottomed flask with 2.5 L of deionised distilled water. Leaves were distilled using cohobation for approximately 6 h using a 6 L mantle and the steam/oil mix condensed and collected in a 500 ml funnel. Essential oils were then separated from the hydrosol and stored in the dark at 4 °C until used. Prior to GC–MS analysis the essential oils were dried to remove hydrosol emulsions using anhydrous NaSO4 powder (0.5 g in 10 ml essential oil) for more than 24 h. Essential oils were then dissolved in dichloromethane at a ratio of 1:1000. Identification was performed by comparison of mass spectra with an electronic library database (NIST08) and confirmed by comparing arithmetic indices, calculated in our laboratory relative to linear alkanes from C8–C21 (IUPAC, 1997), with values published elsewhere (Adams, 2007). Discrepancies were resolved using NMR and by comparing mass spectra with spectra published elsewhere (Adams, 2007; NIST, 2011). Analyses were performed using an HP 6890 GC coupled to a HP 5973 mass spectrometer. An autosampler unit (HP 7673–8
positions) was used to perform 1 ll injections. Separation was accomplished with a Phenomenex ZB-5MS column (30 m 0.25 mm i.d., 0.25 lm phase thickness). Operating conditions were as follows: Injector: split ratio 25:1; Temperature: 280 °C; carrier gas: helium, 1.0-ml/min, constant flow; column temperature, 60 °C (no hold), 3 °C per min then @ 280 hold 20 min. MS was acquired at 70 eV using a mass scan range of 40–350 m/z; integration parameters: cut-off at 0.5% peak height. 3.3. Structure elucidation of squamulosone The mass spectrum of squamulosum is included as Fig. 4 in supplementary information. The structure of squamulosone was determined by exactly matching 1H (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) with literature data (Batey et al., 1971; Collins et al., 2001). In methanol [a]25D: 205°, mp 45–46 °C [lit. [a]25D: 202°, mp(1) 50–51 °C, mp(2) 45–46 °C]. 3.4. Quantification and analysis of essential oils Quantification was performed using gas-chromatography coupled with flame ionization detection (GC–FID). The relative ionization coefficient of several authentic reference standards was calculated relative to n-decane. To do this, equal masses of n-decane and authentic reference standard were dissolved in dichloromethane, and then injected into the GC–FID for analysis. n-Decane was then used as an internal standard in whole essential oils for quantification using relative ionization data calculated earlier. To do this, equal masses of essential oil and n-decane were dissolved in dichloromethane, and then injected into the GC–FID for analysis. Identification of constituents was achieved by calculating arithmetic indices relative to linear alkanes from C8–C21 and comparing with data from GC–MS identifications. Analyses were performed using a Varian 3300 gas chromatograph. Separation was accomplished with a Phenomenex ZB-5MS column (30 m 0.25 mm i.d., 0.25 lm phase thickness). Operating conditions were as follows. Injector: split turned on; temperature: 250 °C; carrier gas: helium, 4.5 ml/min, constant flow; column temperature, initially 50 °C, 50–150 °C at 5 °C/min, 150 °C hold for 10 min. Prinicipal component analysis of the data was carried out using the SPSS 20.0 statistical package (Polar Engineering and Consulting, Nikiski, IL, USA) for potential clustering of the samples based on their essential oil composition. 3.5. Antimicrobial activity Working stocks of all species were maintained on nutrient agar (NA) except Candida albicans, which used yeast extract peptone agar (YEPA). All growth media was purchased from Oxoid (Thebarton, South Australia) and prepared as per instructions.
N.J. Sadgrove et al. / Phytochemistry 97 (2014) 38–45
The minimum inhibitory concentration (MIC) of the essential oil was determined using micro-titre plates for a serial twofold broth dilution (CLSI, 2009) with the following modifications. Essential oil emulsions were prepared by vortexing a measured combination of essential oil and the appropriate broth with 0.15% w/w agar (Mann and Markham, 1998). Species were assayed in tryptone soya sloppy broth (TSSB) or yeast extract peptone sloppy broth (YEPSB) for C. albicans, produced using the same method as described above (Mann and Markham, 1998). Broth dilutions were performed using 96-well plates. Inoculation was achieved by collecting colonies from fresh working stocks and dispensing into 0.9% w/v NaCl and diluting to match a 0.5 McFarland BaSO4 Turbidity Standard (McFarland, 1907) using a spectrophotometer at 600 or 530 nm for C. albicans. To achieve a final inoculation concentration of 5 105 the adjusted saline suspension was diluted into 40 volumes of the appropriate medium and 20 ll was used to inoculate 80 ll of assay media bringing the total volume to 100 ll and reducing the essential oil concentration to the appropriate target. Following inoculation the 96-well plates were sealed using parafilm and placed into an incubator at 37 °C for approximately 20 h before dispensing 40 ll of sterile 0.2 mg/ml p-iodonitrotetrazolium dye, waiting for 1–4 h depending upon organism and examining visually for red colour development, which indicated organism growth. Experiments were repeated 4 times, varying starting concentrations to elucidate the range of MICs likely to occur from effects related to well-to-well vapour diffusion. Results are reported as a range. 3.6. TLC-bioautography Essential oils were applied undiluted using 2 ll onto aluminium backed silicon TLC plates (Merck kieselgel 60 F254). The solvent system was toluene: ethyl acetate, 90:10 by volume. The solvent chamber was allowed to equilibrate for 30 min before plates were inserted. Following separation the plates were air dried for 3 h to allow solvent to completely evaporate prior to overlay with the target organism in agar. Duplicate TLC plates were prepared and run in the same chamber; one for staining and the other for bioautographic overlay. The bioautography was performed in sterile conditions with Staphylococcus aureus (ATCC 29213) as the target organism overlay in agar (1% w/w). Nutrient agar was maintained as a liquid after autoclaving by placing into a rocking water bath at 40 °C. The agar was seeded with 100 mg/L p-iodonitrotetrazolium dye then inoculated with the test organism after matching to a 0.5 McFarland BaSO4 turbidity standard in saline. Inoculations used saline at approximately the same temperature as the agar (otherwise the organism was not viable). Seeded agar was set over the TLC plate covering a thickness of not more than 3 mm and remained for 20 h at 38 °C incubation before inspection. TLC separated components of essential oils with antibacterial activity appeared as clear zones against a red background. Acknowledgements The authors would like to thank the New South Wales National Parks and Wildlife Service for permission to collect on reserves
45
under its administration; collections were made under permit SL100811. Additionally, the authors would like to express gratitude for photographs of Phebalium squamulosum specimens and support provided by Professor Jeremy J. Bruhl from the University of New England, Armidale, Australia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 10.015. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, fourth ed. Allured Publishing Corporation (ISBN 978-1-932633-21-4). Batey, I.L., Hellyer, R.O., Pinhey, J.T., 1971. The structure of squamulosone, a new sesquiterpene ketone from Phebalium squamulosum. Aust. J. Chem. 24, 2173– 2177. Brophy, J.J., Goldsack, R.J., Forster, P.I., 2005. The leaf oils of coatesia and Geijera (Rutaceae) from Australia. J. Essent. Oil Res. 17, 169–174. Brophy, J.J., Goldsack, R.J., Forster, P.I., 2006. Leaf essential oils of the Queensland species of Phebalium (Rutaceae: Boronieae). J. Essent. Oil Res. 18, 386–391. Chammorro, E.R., Ballerini, G., Sequeira, A.F., Velasco, G.A., Zalazar, M.F., 2008. Chemical composition of essential oil from Tagetes minuta L. leaves and flowers. J. Argentine Chem. Soc. 96, 80–86. Collins, D.O., Buchanan, G.O., Reynolds, W.F., Reese, P.B., 2001. Biotransformation of squamulosone by Curvularia lunata ATCC 12017. Phytochemistry 57, 377–383. CLSI, 2009. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically. Approved Standard – Eight Edition. M07–A8 29(2), 1–66. Giles, R.L., Drinnan, A.N., Walsh, N.G., 2008. Variation in Phebalium glandulosum subsp. glandulosum (Rutaceae). Aust. Sys. Bot. 21, 271–288. IUPAC, 1997. Compendium of Chemical Terminology, second ed. (the ‘‘Gold Book’’). Blackwell Scientific Publications, Oxford. XML on-line corrected version: http:// goldbook.iupac.org 2006. created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. DOI: 10.1351/goldbook. Jones, T.G.H., Smith, F.B., 1925. CCCXLIX – Olefinis terpene ketones from the volatile oil of flowering Tagetes glandulifera. Part 1. J. Chem. Soc. Trans. 127, 2530–2539. Koroch, A.R., Juliani, H.R., Zygadlo, J.A., 2007. Bioactivity of essential oils and their components. In: Berger, R.G. (Ed.), Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Springer, New York. Lassak, E.V., Southwell, I.A., 1974. Occurrence of some unusual compounds in the leaf oil of Eriostemon obovalis and Phebalium glandulosum subsp. glandulosum. Aust. J. Chem. 27, 2703–2705. Mann, C.M., Markham, J.L., 1998. A new method for determining the minimum inhibitory concentration of essential oils. J. Appl. Microbiol. 84 (4), 538–544. McFarland, J., 1907. Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Am. Med. Assoc. 14, 1176–1178. NIST, 2011. NIST Chemistry WebBook – NIST Standard Reference Database Number 69 Retrieved August, 2012. Avaialble from: . Pala-Paul, J., Copeland, L.M., Brophy, J.J., Goldsack, R.J., 2009. Essential oil composition of two new species of Phebalium (Rutaceae) from north-eastern New South Wales, Australia. Nat. Prod. Commun. 4, 983–986. Sadgrove, N.J., Telford, I.R.H., Greatrex, B.W., Dowell, A., Jones, G.L., 2013. Dihydrotagetone, an unusual fruity ketone, is found in enantiopure and enantioenriched forms in additional Australian taxa of Phebalium (Rutaceae: Boronieae). Nat. Prod. Commun. 8, 737–740. Singh, G., Singh, O.P., de Lampasona, M.P., Catalán, C.A.N., 2003. Studies on essential oils. Part 35: chemical and biocidal investigations on Tagetes erecta leaf volatile oil. Flavour Frag. J. 18, 62–65. Southwell, I.A., 1970. A new occurrence of hedycaryol, the precursor of elemol, in Phebalium ozothamnoides (Rutaceae). Phytochemistry 9, 2243–2245. Telford, I.R.H., Bruhl, J.J., 2013. Ongoing morphological investigation of Phebalium (Rutaceae) species. University of New England, Armidale NSW Australia 2351. Wilson, P.G., 1970. A taxonomic revision of the genera Crowea, Eriostemon and Phebalium (Rutaceae). Nuytsia 1, 5–55. Wilson, P.G., 2013. Phebalium. Flora of Australia, vol. 26, pp. 458-480.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Composition and antimicrobial activity of the essential oils from the Phebalium squamulosum species complex (Rutaceae) in New South Wales, Australia Nicholas J. Sadgrove a,⇑, Ian R.H. Telford b, Ben W. Greatrex a, Graham L. Jones a a b
Pharmaceuticals and Nutraceuticals Group (School of Science and Technology), University of New England, Armidale, NSW 2351, Australia Botany, School of Environmental and Rural Science and N.C.W. Beadle Herbarium, University of New England, Armidale, NSW 2351, Australia
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 22 October 2013 Available online 15 November 2013 Keywords: Chemotaxonomy Bioactivity Monoterpenes Sesquiterpenes
a b s t r a c t Essential oils have been hydrodistilled and characterized from 21 populations of taxa currently assigned to the endemic Australian species Phebalium squamulosum (Rutaceae: Boronieae) using GC–MS, NMR and quantified using GC–FID. Essential oils were further examined using principle component analysis to distinguish chemotypes, then screened for antimicrobial activity using broth dilution and TLC-bioautography. Collections of subspecies of P. squamulosum, namely subsp. coriaceum, subsp. gracile, subsp. lineare, subsp. squamulosum, subsp. ozothamnoides and subsp. verrucosum, were made from the wild and one from a cultivated plant of known provenance within New South Wales. Results demonstrated considerable intra- and interspecific essential oil component variation, suggesting the existence of distinct chemotypes and supporting previously observed segregate species based on morphological evidence. Antimicrobial testing revealed moderate to high activity for all essential oils dominated by sesquiterpene alcohols; elemol and eudesmol isomers. Conversely, very low antimicrobial activity was observed from essential oils dominated by monoterpenes. This study constitutes the most exhaustive investigation of essential oils from P. squamulosum subspecies to date and provides the first report of antimicrobial activity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The Australian genus Phebalium contains 28 formally named species (Wilson, 2013) with at least six putative new species (Telford and Bruhl, 2013), mostly distributed in southern and eastern Australia. Phebalium squamulosum is a yellow-flowering shrub or small tree exhibiting considerable polymorphism. This variation has resulted in the naming of nine subspecies (Wilson, 1970), six of which are examined here for essential oil composition. The first examination of essential oils from P. squamulosum was undertaken in 1971, shortly after Wilson (1970) revised the species. A novel sesquiterpene ‘squamulosone’ identified spectroscopically, and by conversion to known compounds, was shown to be the major constituent (Batey et al., 1971). Although no single subspecies was specified in that study, the locality of
⇑ Corresponding author. Address: Human Biology, School of Science and Technology, McClymont Building University of New England, Armidale, NSW 2351, Australia. Tel.: +1 61481130595. E-mail addresses: [email protected] (N.J. Sadgrove), [email protected] (G.L. Jones). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.015
the collection (Narrabeen, Sydney) places it as P. squamulosum subsp. squamulosum. Brophy et al. (2006), in their analyses of essential oils from Queensland Phebalium species, reported no squamulosone present in taxa identified as P. squamulosum subsp. squamulosum and P. squamulosum subsp. gracile from south-eastern Queensland. In New South Wales, Southwell (1970) showed that essential oil from leaves of P. squamulosum subsp. ozothamnoides (named as P. ozothamnoides in that study) collected from the Blue Mountains, contained elemol as the major component; however, this is present in fresh leaf material as the heat labile precursor, hedycaryol; prior to chemical alteration in the course of the steam distillation process. Pala-Paul et al. (2009) investigated two of the putative new species, P. stellatum and P. sylvaticum (Telford and Bruhl, 2013) currently assigned to P. squamulosum subsp. squamulosum. The current study contributes additional data to further the on-going survey of leaf essential oil composition of seven of the subspecies from the P. squamulosum species complex, depicted in Fig. 1. In addition, antimicrobial capacity of essential oils from all subspecies was investigated for the first time against nine microbial species, including some human pathogens.
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N.J. Sadgrove et al. / Phytochemistry 97 (2014) 38–45
Fig. 1. Leaf morphology of the Phebalium squamulosum complex (Wilson, 1970), with molecular structures of the main chemotypically significant essential oil components.
2. Results and discussion 2.1. Character of essential oils P. squamulosum subspecies sampled for essential oil analysis are listed in Table 1. Leaf morphology is depicted in Fig. 1 and collection sites in Fig. 2. Essential oil character from populations currently assigned to P. squamulosum subsp. squamulosum infers the existence of two distinct intraspecific chemotypes (Tables 2 and 4); one corresponding to the major constituent squamulosone (81%), which was hydrodistilled from collections from Gloucester Tops. Squamulosone was first reported from a specimen collected at Narrabeen, Sydney (Batey et al., 1971). It may be worthwhile investigating further specimens to identify the southern boundary of this squamulosone chemotype. The other intraspecific chemotype is characterized by the major constituents; elemol (5–19%), a-, b- and c-eudesmol (Tables 2 and 4). This chemotype was hydrodistilled from material collected in north-eastern New South Wales (Fig. 1), so the expectation was that such specimens would produce similar essential oil character to that reported by Brophy et al. (2006). However, specimens collected by Brophy et al. (2006) produced a lower quantity of
these constituents and additionally produced the monoterpene minor constituents a-pinene and limonene, and the monoterpene major constituents myrcene and a- and b-phellandrene. It is unclear if environment and/or seasonal variation and/or growth stage influenced this variable essential oil character relative to the results obtained by us and presented here. Alternatively, essential oil data presented by Brophy et al. (2006) for P. squamulosum subsp. squamulosum may have been for the specimens collected from Mt Barney National Park (Mowburra Peak), indicating a potential third discrete chemotype. The Mowburra Peak population almost certainly represents a new species, P. sylvaticum ined. (Telford and Bruhl, 2013), previously examined for essential oils by Pala-Paul et al. (2009). The specimen of P. squamulosum subsp. squamulosum containing the ketone squamulosone was collected from soil derived from basalt in the Gloucester Tops area of Barrington Tops National Park (Fig. 2). This edaphic/geological aspect contrasts to the more northerly specimens producing elemol and eudesmol isomers, which were growing on soils derived from granite. In addition to differences in habitat preference, morphological differences between these two chemotypes strongly suggest more than one taxon is involved (Telford and Bruhl, 2013). It may be that the presence
Table 1 Vouchers for this study of collections of taxa currently assigned to Phebalium squamulosum. In the text essential oils are cited in shortened form using the collector code, for example, N.J.Sadgrove 186 becomes NJS186 in the text. Taxon P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum squamulosum
subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp.
coriaceum 1 coriaceum 2 coriaceum 3 coriaceum 4 gracile 1 gracile 2 gracile 3 lineare lineare ozothamnoides 1 ozothamnoides 2 aff. ozothamnoides 1 aff. ozothamnoides 2 squamulosum 1 squamulosum 2 squamulosum 3 verrucosum 1a verrucosum 1b verrucosum 1c verrucosum 2 verrucosum 3
Locality
Date collected
Collector
Split Rock, Warrumbungle NP, NSW Spirey View track, Warrumbungle NP, NSW Spirey View track, Warrumbungle NP, NSW Mt Naman, Warrumbungle NP, NSW Pilliga Nature Reserve, N of Coonabarabran, NSW Cox’s Gap, Goulburn River NP, NSW Goulburn River, Wollara Rd, Goulburn River NP, NSW Wingen Maid Nature Reserve, NW of Scone, NSW Salisbury Trig, Wingen Maid Nature Reserve NW of Scone Cultivated: ex Tinderry Mtn, E of Michelago, NSW Hassans Walls, Lithgow, Blue Mtns, NSW Mushroom Rock, NE of Guyra, NSW Crown Mtn, Warra NP, NE of Guyra, NSW Gloucester Falls track, Barrington Tops NP, NSW Bluff Rock, S of Tenterfield, NSW Donnybrook State Forest, W of Tenterfield, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Long Point, E of Armidale, NSW Oxley Wild Rivers NP, Tia Falls, E of Walcha, NSW Nymboida River, N of Dorrigo, NSW
May 2012 May 2012 May 2012 February 2013 May 2012 August 2012 August 2012 May 2013 May 2013 August 2012 August 2012 August 2012 August 2012 July 2012 August 2012 September 2012 March 2012 March 2012 March 2012 July 2012 August 2012
N.J. Sadgrove 186 (NE) N.J. Sadgrove 204 (NE) N.J. Sadgrove 207 (NE) N.J. Sadgrove 328 (NE) N.J. Sadgrove 191 (NE) N.J. Sadgrove 223 (NE) N.J. Sadgrove 251 (NE) N.J. Sadgrove 314 (NE) N.J. Sadgrove 334 (NE) I.R. Telford 13427 & N.J. Sadgrove (NE) N.J. Sadgrove 273 (NE) N.J. Sadgrove 289 (NE) N.J. Sadgrove 300 (NE) N.J. Sadgrove 246 (NE) N.J. Sadgrove 282 (NE) N.J. Sadgrove 303 & I. Telford (NE) N.J. Sadgrove 131 (NE) N.J. Sadgrove 132 (NE) N.J. Sadgrove 133 (NE) N.J. Sadgrove 243 (NE) N.J. Sadgrove 280 (NE)
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Fig. 2. Sampling sites for Phebalium squamulosum: d, subsp. squamulosum; j, subsp. coriaceum; +, subsp. gracile; , subsp. lineare; N, subsp. ozothamnoides; ., subsp. aff. ozothamnoides; ⁄, subsp. verrucosum.
of squamulosone supports this taxonomic observation, apparently being a chemical marker for P. squamulosum subsp. squamulosum in the strict sense. In the current study, squamulosone was also found in P. squamulosum subsp. lineare (Tables 2 and 4), with a similar whole essential oil to P. squamulosum subsp. squamulosum from Barrington Tops National Park, with the exception of overall yield, which was higher. This may have been a consequence of seasonal variation, because leaf samples from P. squamulosum subsp. lineare were collected in mid-summer, as opposed to specimens of P. squamulosum subsp. squamulosum, which were collected in winter. Previously it was unclear if P. squamulosum subsp. lineare was indeed a distinct subspecies, as it is morphologically similar to P. squamulosum subsp. gracile (pers. observation). The presence of squamulosone in populations of P. squamulosum subsp. lineare confirms they are distinct from subsp. gracile which lacks that ketone. P. squamulosum subsp. lineare will be raised to species rank (Telford and Bruhl, 2013). Limonene, a-pinene, elemol and eudesmol isomers are the major components of essential oils sourced from P. squamulosum subsp. gracile (Tables 2 and 5). Essential oils from these specimens produced the greatest component variation (within the discrete
subspecies), compared to the subspecies examined in this study, with the exception of P. squamulosum subsp. squamulosum (Tables 2 and 4). The essential oil components presented by Brophy et al. (2006), concerning essential oils hydrodistilled from P. squamulosum subsp. gracile, adds to this variation. Results presented by Brophy et al. (2006) demonstrated a monoterpene composition of more than 50%, compared to results in the current study, showing a range from 20% to 40% monoterpenes. In addition, Brophy et al. (2006) reported geijerene in this essential oil, which has previously been shown to be a characteristic of essential oils distilled from northern (i.e. Queensland) but not southern (i.e. New South Wales) specimens of other species, such as Geijera parviflora (Brophy et al., 2005). Geijerene was not present in any of our specimens of P. squamulosum subsp. gracile, however one specimen produced an as yet unidentified sesquiterpene (not geijerene, C15H26) with a relative abundance of 13.6% by GC–MS (Tables 2 and 5). Components from essential oils of P. squamulosum subsp. ozothamnoides and aff. ozothamnoides demonstrated no distinct chemovariation, other than a slightly higher quantity of a-pinene and elemol in specimens of aff. ozothamnoides (Tables 2 and 6). It is interesting to note however, that a substantially higher yield was obtained from specimens of aff. ozothamnoides (0.3–0.79%)
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N.J. Sadgrove et al. / Phytochemistry 97 (2014) 38–45
Table 2 Chemical composition of essential oils from Phebalium squamulosum subspecies. Quantification data in relative abundance (%) is presented here as a range. Original data is included as supplementary material. Published arithmetic indices (Pub. AI) are from Adams (2007) or Singh et al. (2003) for dihydrotagetone. Calculated arithmetic indices (AI) were produced by the authors. Data related to the essential oil from P. squamulosum subsp. verrucosum was published earlier by Sadgrove et al. (2013) with further details of the enantiomeric composition of dihydrotagetone. Yield
AI
Pub. AI
P.s.s(A) 0.4
P.s.s(B) 0.02–0.7
P.s.l 0.9–1.7
P.s.g 0.2–0.6
P.s.o 0.03–0.8
P.s.c 0.1–0.2
P.s.v 1.6–3.1
Cyclofenchene a-Pinene a-Fenchene Camphene Sabinene b-Pinene Z-Meta-mentha-2,8-diene b-Myrcene d-2-Carene a-Phellandrene a-Terpinene p-Cymene Limonene b-Phellandrene 1,8-Cineol b-E-ocimene c-Terpinene Dihydrotagetone Terpinolene p-Mentha-2,4(8)-diene Linalool Nonanal Citronellal 2-Bornanol Terpinen-4-ol Cryptone Decanal a-Terpineol Citronellol Bornyl acetate C10H10O2 Myrtenyl acetate d-Elemene a-Copaene b-Elemene Dodecanal E-Caryophyllene b-Gurjurene Aromadendrene Citronellyl propionate a-Humulene Alloaromadendrene c-Muurolene Geranyl proprionate a-Selinene d-Selinene Viridiflorene a-Muurolene c-Patchoulene Bicyclogermacrene Epizonarene Germacrene A Geraniol isobutanoate d-Cadinene Elemol E-Dauca-4(ll),7-diene Palustrol Spathulenol Allohedcaryol Globulol Viridiflorol Guaiol Klusinone C15H24O C15H24O Ledol 5-Epi-7-epi-a-eudesmol 10-Epi-c-eudesmol C15H24O C15H26#
929 930 944 947 970 974 989 989 1000 1004 1015 1022 1028 1027 1028 1045 1057 1058 1088 1088 1100 1103 1230 1164 1177 1186 1206 1190 1230 1283 1284 1323 1339 1372 1390 1405 1414 1442 1445 1441 1448 1457 1476 1474 1481 1486 1491 1496 1498 1500 1503 1499 1514 1519 1546 1561 1569 1572 1593 1593 1593 1596 1597 1598 1601 1604 1609 1615 1619 1623
927 932 945 946 969 974 983 988 1001 1002 1014 1022 1024 1025 1026 1044 1054 1055 1086 1085 1095 1100 1223 1166 1174 1183 1201 1186 1223 1287 – 1324 1335 1374 1389 1408 1417 1431 1439 1444 1452 1458 1478 1476 1489 1492 1496 1500 1502 1500 1501 1508 1514 1522 1548 1556 1567 1577 1589 1590 1592 1600 1604 – – 1602 1607 1622 – –
0 1.5 0.7 0 0 0 0 0 0 3.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.9 0 0 0 1.4 0 0 0.2 0 0 6.8 0 0 0 0 0 2.1 0 0 0 0
0 0.2–3.4 0 0 0 0 0 0.9–6 0 0–2 0.1 0.1–0.2 0 0.2–7.1 0 0–0.2 0 0 0 0 0 0–3.7 0 0 0 0 0 0 0 0.2–1.3 0 0–0.5 0 0–0.3 0 0 0.3–0.8 0 0 0 0–0.2 0 0.3–0.6 0 0–0.1 0 0–4.5 0–0.3 0 0–0.5 0–0.6 0 0 0.3–0.7 5.7–18.7 0–0.4 0 0.7–0.9 0 6.3–33.3 0–0.8 1.5–3.9 0–0.4 0 0–3.9 0 0 0–14.1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–0.5 0 0.6–1.1 0 0–0.4 0 0 0–0.3 0 0 0 0 0 0 0 2.3–2.6 0 0 0 0 0 0 0 0 0 0.6–4.1 0 0 0 0–4.2 0 0 0 0 0.9–1.2 0
0 15.6–23.2 0 0–0.3 0–5.3 0.7–0.9 0 3.6–3.7 0 0–0.2 0–1.1 0–0.4 0 0 0 2.7–13.5 0–1.9 0 0–0.6 0 0 0 0 0 0–2.9 0 0 0–0.2 0 0 0–2.6 0 0 0 0.8–0.9 0 0–0.5 0 0 0 0–0.2 0 0 0 0 0 0 0 0–0.7 0.9–9.7 0 0 0 0 13.9–27 0 0 0–3.9 0 0–1.6 0 0–3.6 0 0 0 0–1.7 2.4–6.9 1–10.3 0 0–13.6
0 0.1–5 0 0 0–0.3 0–0.2 0.2–3.6 0–3.1 0 0.3–0.6 0 0–0.8 0 1.5–3.2 0 0 0 0 0 0 0–0.2 0 0 0 0–0.2 0 0 0 0–0.1 0 0 0 0 0 0–0.2 0 0.2–0.8 0 0 0 0–0.1 0 0–0.2 0 0–0.2 0–0.1 0 0 0 1.5–1.9 0–0.2 0–0.2 0 0–0.3 19.7–29.5 0 0 0–1.3 0–0.6 0.4–7.5 0 0–4.7 0 0 0 0 0–1.4 4.2–5.5 0 0
0 26.5–32.3 0 0 0.5–4.3 0.8–1.4 0 1.3–11.9 0–2.5 3.5–5.1 0–0.3 0.7–2.1 0 22.5–31.1 0 0–1.7 0–0.6 0 0 0.4–0.6 0 0 0–0.5 0–0.4 0–0.2 0–0.3 0–0.6 0 0 0–0.6 0 0 0–1.5 0 0 0–0.4 0–2.1 0 0–2.1 0–0.3 0 0 0 0–0.9 0 0 0 0 0 7.5–16.5 0 0 0–1.2 0 0 0 0 0–7.2 0 0–2 0 0 0 0 0 0–1.5 0 0 0 0
0–3.2 0–1.1 0 0 0 0 0 0 0 0 0 0 0–0.1 0 0–3.1 0 0 90.4–98.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–5.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0–0.5 0 0
(continued on next page)
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Table 2 (continued) Yield
AI
Pub. AI
P.s.s(A) 0.4
P.s.s(B) 0.02–0.7
P.s.l 0.9–1.7
P.s.g 0.2–0.6
P.s.o 0.03–0.8
P.s.c 0.1–0.2
P.s.v 1.6–3.1
c-Eudesmol
1626 1631 1637 1639 1644 1647 1654 1658 1662 1684 1725 1785 1787
1630 1629 1638 1640 1649 1652 1656 – 1670 – – – 1792
0 0 0 0 0 0 0 0 0 0 0 81 0
1.5–13.1 0–1.3 0.6–2.6 0 1.4–16.3 1.9–9.8 0 0 2.3–3.1 0 0 0 0.2–6
0 0 0 0 0 0 0 0 0 0 0–0.6 85–88 0
6.6–14.3 0 0–0.5 0 4.8–9.7 0–17.8 0 0–1.2 3.9–4 0–1.2 0 0 0–2.2
2.2–5.5 0–0.1 0–1.7 0–1.9 1.6–2.4 3.2–5.7 0–2.4 0 12.4–21 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0–3.3 0 0 0 0 0 0 0 0
Eremoligenol Epi-a-cadinol Hinesol b-Eudesmol a-Eudesmol Valerianol C15H24 Bulnesol C15H22O C15H24O Squamulosone 8-a-Acetoxyelemol
P.s.s(A) – subsp. squamulosum chemotypes A, (B) – chemotypes B; P.s.l – subsp. lineare; P.s.g – subsp. gracile; P.s.o – subsp. ozothamnoides; P.s.c – subsp. coreaceum; P.s.v – subsp. verrucosum. # Unidentified compound in P. squamulosum subsp. gracile may indicate another chemotype m/z: 41 (9), 59 (48), 81 (31), 91 (27), 108 (33), 121 (17), 135 (9), 149 (100), 164 (34), 189 (8) and 204 (7).
than from subsp. ozothamnoides (0.03–0.1%). These differences corroborate the morphological differences indicating two distinct taxa, although environmental factors may be responsible for yield differences, with seasonal factors less plausible because all specimens were collected in winter. It is also interesting to note that a specimen of P. squamulosum subsp. ozothamnoides, collected much earlier by another researcher from the same location as NJS273, produced an essential oil yield at approximately 1.64% (Southwell, 1970). In this particular instance, it is plausible to speculate seasonal variation in essential oil yield, particularly as yields tend to increase in arid summers (unpublished results). The study undertaken by Southwell (1970) revealed that the presence of elemol in the essential oil following steam distillation of leaf material of subsp. ozothamnoides was a result of the thermal Cope rearrangement of the precursor hedycaryol, which was found in the macerated leaf material following light petroleum extraction. It is suggested that elemol is not a natural product per se, but rather an artifact of the distillation process (Southwell, 1970). At present this study, and those completed elsewhere (Brophy et al., 2006), have revealed elemol to be also present in subsp. gracile, subsp. squamulosum and subsp. aff. ozothamnoides. This result may indicate the presence of the hedycaryol precursor in these subspecies as well. Essential oil from P. squamulosum subsp. coriaceum was predominantly monoterpene in character (Tables 2 and 7). Major components were a-pinene (26–33%), b-pinene (1.3%), b-myrcene (1–4%), a-phellandrene (3–5%), b-phellandrene (22–30%), bicyclogermacrene (7–17%) and spathulenol (2–7%). Of the essential oils characterized by Brophy et al. (2006) the most similar in composition to this is that from P. squamulosum subsp. squamulosum, with the exception of the lower quantity of a-pinene (3.3%) and the presence of elemol in subsp. squamulosum. Thus, subsp. coriaceum is unlike any of the previously reported essential oils from Phebalium, and certainly indicates its taxonomic misplacement in P. squamulosum. Hydrodistillation of P. squamulosum subsp. verrucosum (Tables 2 and 8) produced an essential oil predominantly made up of dihydrotagetone (86–99%), which was first characterized by Jones and Smith (1925) from Tagetes glandulifera, now correctly T. minuta. The essential oil of T. minuta typically contains dihydrotagetone at a maximum concentration of 7% of the entire essential oil fraction, but it can reach as high as 43%, depending on the time of year and part of the plant distilled (Chammorro et al., 2008). Lassak and Southwell (1974) revealed the presence of (+)-dihydrotagetone (2,6-dimethyloct-7-en-4-one) in relatively high enantiomeric purity
and concentration (97.4%) in essential oil from P. glandulosum subsp. macrocalyx (Giles et al., 2008) sourced from near Dubbo, New South Wales, but referred to as subsp. glandulosum in their study. Sadgrove et al. (2013) reported dihydrotagetone from other subspecies of P. glandulosum and in P. squamulosum subsp. verrucosum with results strikingly similar to those previously obtained by Lassak and Southwell (1974). Thus, dihydrotagetone appears to be a chemical marker for P. glandulosum and related taxa. Similar essential oil profiles, together with morphological differences (Telford and Bruhl, 2013) indicate the taxonomic misplacement of subsp. verrucosum, having closer affinities to the P. glandulosum group. 2.2. Intra- and interspecific essential oil chemotypes There were striking differences in the components of the essential oils, with the main interspecific variation between being seen in either the presence of dihydrotagetone at greater than 85% of the total essential oil content, corresponding to subsp. verrucosum, or its complete absence, as seen in other subspecies. The only intraspecific variation (subspecific) was seen within the subsp. squamulosum group, with squamulosone present at either >80% in southern specimens or completely absent in northern specimens. To examine the correlation between taxonomic classification and essential oil composition, principle component analysis (PCA) was used. Calculation of the principle components for the most abundant 19 components found in the essential oils showed that there were 7 principal components that together accounted for 91% of the variance of the data set. Principal component 1 (PC1) accounted for 23% of the variance and principal component 3 (PC3) accounted for 13% of the variance seen between different chemotypes. A plot of PC1 against PC3 showed that clusters were seen which corresponded to the taxonomic classification based on plant morphology. These clusters have been circled in Fig. 3 by the authors. PC1 separated subsp. coriaceum from subsp. verrucosum and subsp. lineare which also clustered separately to subsp. ozothamnoides and aff. ozothamnoides. PC3 partially separated subsp. squamulosum from subsp. ozothamnoides/aff ozothamnoides and further separated subsp. gracile from the remaining species. The two subsp., ozothamnoides and aff. ozothamnoides, clustered together indicating that there is no significant variation in the essential oils from these subspecies other than the yield, which was not a parameter in the analysis. There was overlap seen in the clustering of subsp. squamulosum and ozothamnoides and overlap of verucosum and
N.J. Sadgrove et al. / Phytochemistry 97 (2014) 38–45
43
Fig. 3. Scoring plot from principal component analysis, showing the five species, with PC1 plotted against PC3.
lineare. The loading plot for PC1 and PC3, Fig. 4, shows that the main positive contributors to PC1 were b-phellandrene, bicyclogemacrene, a-pinene while dihydrotagetone and squamulosone had large negative coefficients. PC3 was determined by the content of b-eudesmol, E-ocimene and c-eudesmol although elemol and a-pinene also had significant effects. Thus, from these results, chemical characterization presented in this study supports classification of six distinct essential oil chemotypes within the P. squamulosum complex; most of which were demonstrated using principal component analysis and are seen in Fig. 3. The first chemotype is dominated by squamulosone, which is characteristic of two subspecies; subsp. squamulosum and subsp. lineare specimens from within the Hunter valley in central New South Wales (Tables 2 and 4: Figs. 1 and 2). It is interesting that PCA (Fig. 3) clustered both chemotypes of subsp. squamulosum together despite the distinct chemotypic representation of squamulosone at >80% in southern, morphologically distinct specimens. Although this makes the essential oil from southern specimens of subsp. squamulosum more similar to subsp. lineare, PCA was additionally able to differentiate between the two. The second and third essential oil chemotypes are primarily characterized by the major chemical component elemol, which is present in the essential oils from four subspecies; P. squamulosum subsp. squamulosum (northern distribution) (Tables 2 and 4: Figs. 1 and 2), subsp. gracile (Tables 2 and 5) and subsp. ozothamnoides or aff. ozothamnoides (Tables 2 and 6). Essential oils from these four subspecies can be distinguished into two groups; P. squamulosum subsp. squamulosum from the northern distribution is predominantly sesquiterpenoid but with low quantities of bulnesol (2–3%) and subsp. ozothamnoides and aff. ozothamnoides contain bulnesol at a concentration higher than other squamulosum subspecies (12–21%). This higher occurrence of bulnesol in essential oils from subsp. ozothamnoides and aff. ozothamnoides indicates that these two subspecies belong to a separate chemotype. Thus, subsp. squamulosum from the northern distribution belongs to the third chemotype, and subsp. ozothamnoides and aff. ozothamnoides belong to the fourth. Although no phytochemical difference has yet been demonstrated between subsp. ozothamnoides and aff. ozothamnoides, other than overall winter essential oil yield, morphological differences are sufficient to demonstrate these two are different species and warrant species revision (pers. observation). Principal component analysis showed a degree of observable difference between essential oils from subsp. gracile and subsp. squamulosum; however, further analysis, using additional specimens,
may strengthen the cluster separation observed in Fig. 3. Additionally, clustering in Fig. 3 may have been offset by sample NS251, which was collected from a geologically and edaphically different site. This factor may be responsible for the unusual essential oil produced by this specimen, which appears to be an outlier at the moment. Essential oil from subsp. squamulosum contained globulol varying in concentration from 6% to 33%, and subsp. gracile contained monoterpenes a-pinene (15–23%) and limonene (3–14%). Additionally, Brophy et al. (2006) reported geijerene in the essential oil from Queensland specimens of subsp. gracile, which may again indicate a further discrete chemotype. Therefore, using this observation and the clustering observed in Fig. 3, it may be sufficient to put subsp. gracile and subsp. squamulosum (northern distribution) into different chemotypes, with subsp. squamulosum in the third and subsp. gracile on its own, in the fifth chemotype. The sixth essential oil chemotype is characterized by essential oil from subsp. coriaceum (Tables 2 and 7; Fig. 3), which is dominated by monoterpenes such as a- and b-pinene, b-myrcene and a- and b-phellandrene. The seventh chemotype is represented by essential oil from subsp. verrucosum (Tables 2 and 8) which is dominated by dihydrotagetone, which clustered around subsp. lineare in Fig. 3. In a previous study, Sadgrove et al. (2013) demonstrated that P. squamulosum subsp. verrucosum had natural dihydrotagetone enantiotypes, but factors contributing to this distinct variation were not further examined. At present it is unclear if subsp. lineare and subsp. squamulosum (southern specimen) can be differentiated into two separate chemotypes. In Fig. 3 they clustered separately, despite both apparently being the same essential oil with squamulosone at a concentration >80%. Further sampling may clarify this. 2.3. Antimicrobial activity of essential oils This study constitutes the first time essential oils from Phebalium species are examined for antimicrobial activity (Table 3). Nil or very low antimicrobial activity was observed for type three and four essential oils (subsp. coriaceum and subsp. verrucosum, respectively). Relatively high antimicrobial activity was observed for type one and two essential oils (subsp. squamulosum, subsp. gracile and subsp. ozothamnoides and aff. ozothamnoides). This high antimicrobial activity was evident only against select microbial species, and did not manifest against others (Table 3). At present it is not clear why this trend emerged. It does not seem to relate to gram-stain patterns.
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N.J. Sadgrove et al. / Phytochemistry 97 (2014) 38–45
Table 3 Mean inhibitory concentrations of essential oils from subspecies of the squamulosum complex. Additional microbial species examined were Salmonella typhimirium and Escherechia coli but no inhibition was observed up to 4% v/v essential oil concentration. The control was tetracycline in lg/ml or nystatin where C. albicans was used. Essential oils MICs are presented as percentages calculated in volume of oil relative to volume of media.
S. aureus S. epidermidis B. subtilis S. pneumoniae K. aeroenes P. aeruginosa C. albicans
131
246
289
282
P. s. ozo
251
191-92-93
223-24-25
300
204-05-06
Control
> > > – > 4–5 >
0.25–0.5 N/A 0.06–0.5 0.02–0.3 > >4 0.5–1
0.06–0.25 0.5–3 0.1–0.25 0.06–0.25 0.1–0.5 2–3 0.5
0.03–0.5 0.5–1 0.03–0.5 0.05–0.25 0.06–0.25 2–3 0.25–1
0.2–0.5 0.5–2 0.25–0.5 0.06–1 0.25–0.5 2–3 0.5–1
0.13–1 0.5–2 0.06–0.5 0.06–1 0.5–1 4–5 0.5–1
0.13–0.5 0.5–2 0.06–0.13 0.06–1 0.1–0.5 2–3 0.1–1
0.25–0.5 0.5–2 0.13–0.25 0.13–1 0.13–0.25 2–3 0.5–1
0.06–0.25 0.5–3 0.06–0.13 0.13–0.5 0.13–0.25 2–3 0.1–1
>4
0.13–1 0.15–2 0.25–8 0.03–0.5 0.25–2 0.75–16 1.25–7
5.5 >4 >4 >4 0.25–0.5
P. squamulosum subsp. verrucosum (NS131), subsp. gracile (NS251, NS191-92-93 and NS223-24-25), subsp. ozothamnoides (P. s. ozo), subsp. aff. ozothamnoides (NS289 and NS300), subsp. coreaceum (204-05-06) and subsp. squamulosum (NS246 and NS282).
Individual discrete components corresponding with zones of antimicrobial activity were not fully resolved by TLC-bioautography, as sesquiterpenols habitually co-migrate in thin layer chromatography. However, results demonstrated that sesquiterpenols as a class were largely responsible for antimicrobial activity. Sesquiterpene alcohols have often been associated with high antifungal and antibacterial activity, particularly with components a- and b-eudesmol and bicyclogermacrene (Koroch et al., 2007). These were present in the type one and two essential oils producing the greatest bioactivity in this study. It is probable that elemol/hedycaryol is also responsible for enhanced antimicrobial ability. 3. Experimental 3.1. Collection of taxa Taxa surveyed (Table 1) are, for the first time: P. squamulosum subsp. coriaceum and subsp. verrucosum; from two subspecies that have not been hitherto examined in New South Wales populations: subsp. gracile and subsp. squamulosum; from an additional population of P. squamulosum subsp. ozothamnoides, and from populations of a putative new species currently included under subsp. ozothamnoides (subsp. aff. ozothamnoides) (Figs. 1 and 2). Aerial parts from P. squamulosum subspecies were collected from the wild in New South Wales (SL100811) (Fig. 2) or from cultivated plants of known provenance. Voucher specimens were lodged with the N.C.W Beadle Herbarium (NE), University of New England, Armidale, NSW, Australia. 3.2. Essential oil extraction and characterization Approximately 500 g of fresh foliage was removed from the twig and chopped into 0.5 mm fragments and placed into a 5 L round bottomed flask with 2.5 L of deionised distilled water. Leaves were distilled using cohobation for approximately 6 h using a 6 L mantle and the steam/oil mix condensed and collected in a 500 ml funnel. Essential oils were then separated from the hydrosol and stored in the dark at 4 °C until used. Prior to GC–MS analysis the essential oils were dried to remove hydrosol emulsions using anhydrous NaSO4 powder (0.5 g in 10 ml essential oil) for more than 24 h. Essential oils were then dissolved in dichloromethane at a ratio of 1:1000. Identification was performed by comparison of mass spectra with an electronic library database (NIST08) and confirmed by comparing arithmetic indices, calculated in our laboratory relative to linear alkanes from C8–C21 (IUPAC, 1997), with values published elsewhere (Adams, 2007). Discrepancies were resolved using NMR and by comparing mass spectra with spectra published elsewhere (Adams, 2007; NIST, 2011). Analyses were performed using an HP 6890 GC coupled to a HP 5973 mass spectrometer. An autosampler unit (HP 7673–8
positions) was used to perform 1 ll injections. Separation was accomplished with a Phenomenex ZB-5MS column (30 m 0.25 mm i.d., 0.25 lm phase thickness). Operating conditions were as follows: Injector: split ratio 25:1; Temperature: 280 °C; carrier gas: helium, 1.0-ml/min, constant flow; column temperature, 60 °C (no hold), 3 °C per min then @ 280 hold 20 min. MS was acquired at 70 eV using a mass scan range of 40–350 m/z; integration parameters: cut-off at 0.5% peak height. 3.3. Structure elucidation of squamulosone The mass spectrum of squamulosum is included as Fig. 4 in supplementary information. The structure of squamulosone was determined by exactly matching 1H (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) with literature data (Batey et al., 1971; Collins et al., 2001). In methanol [a]25D: 205°, mp 45–46 °C [lit. [a]25D: 202°, mp(1) 50–51 °C, mp(2) 45–46 °C]. 3.4. Quantification and analysis of essential oils Quantification was performed using gas-chromatography coupled with flame ionization detection (GC–FID). The relative ionization coefficient of several authentic reference standards was calculated relative to n-decane. To do this, equal masses of n-decane and authentic reference standard were dissolved in dichloromethane, and then injected into the GC–FID for analysis. n-Decane was then used as an internal standard in whole essential oils for quantification using relative ionization data calculated earlier. To do this, equal masses of essential oil and n-decane were dissolved in dichloromethane, and then injected into the GC–FID for analysis. Identification of constituents was achieved by calculating arithmetic indices relative to linear alkanes from C8–C21 and comparing with data from GC–MS identifications. Analyses were performed using a Varian 3300 gas chromatograph. Separation was accomplished with a Phenomenex ZB-5MS column (30 m 0.25 mm i.d., 0.25 lm phase thickness). Operating conditions were as follows. Injector: split turned on; temperature: 250 °C; carrier gas: helium, 4.5 ml/min, constant flow; column temperature, initially 50 °C, 50–150 °C at 5 °C/min, 150 °C hold for 10 min. Prinicipal component analysis of the data was carried out using the SPSS 20.0 statistical package (Polar Engineering and Consulting, Nikiski, IL, USA) for potential clustering of the samples based on their essential oil composition. 3.5. Antimicrobial activity Working stocks of all species were maintained on nutrient agar (NA) except Candida albicans, which used yeast extract peptone agar (YEPA). All growth media was purchased from Oxoid (Thebarton, South Australia) and prepared as per instructions.
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The minimum inhibitory concentration (MIC) of the essential oil was determined using micro-titre plates for a serial twofold broth dilution (CLSI, 2009) with the following modifications. Essential oil emulsions were prepared by vortexing a measured combination of essential oil and the appropriate broth with 0.15% w/w agar (Mann and Markham, 1998). Species were assayed in tryptone soya sloppy broth (TSSB) or yeast extract peptone sloppy broth (YEPSB) for C. albicans, produced using the same method as described above (Mann and Markham, 1998). Broth dilutions were performed using 96-well plates. Inoculation was achieved by collecting colonies from fresh working stocks and dispensing into 0.9% w/v NaCl and diluting to match a 0.5 McFarland BaSO4 Turbidity Standard (McFarland, 1907) using a spectrophotometer at 600 or 530 nm for C. albicans. To achieve a final inoculation concentration of 5 105 the adjusted saline suspension was diluted into 40 volumes of the appropriate medium and 20 ll was used to inoculate 80 ll of assay media bringing the total volume to 100 ll and reducing the essential oil concentration to the appropriate target. Following inoculation the 96-well plates were sealed using parafilm and placed into an incubator at 37 °C for approximately 20 h before dispensing 40 ll of sterile 0.2 mg/ml p-iodonitrotetrazolium dye, waiting for 1–4 h depending upon organism and examining visually for red colour development, which indicated organism growth. Experiments were repeated 4 times, varying starting concentrations to elucidate the range of MICs likely to occur from effects related to well-to-well vapour diffusion. Results are reported as a range. 3.6. TLC-bioautography Essential oils were applied undiluted using 2 ll onto aluminium backed silicon TLC plates (Merck kieselgel 60 F254). The solvent system was toluene: ethyl acetate, 90:10 by volume. The solvent chamber was allowed to equilibrate for 30 min before plates were inserted. Following separation the plates were air dried for 3 h to allow solvent to completely evaporate prior to overlay with the target organism in agar. Duplicate TLC plates were prepared and run in the same chamber; one for staining and the other for bioautographic overlay. The bioautography was performed in sterile conditions with Staphylococcus aureus (ATCC 29213) as the target organism overlay in agar (1% w/w). Nutrient agar was maintained as a liquid after autoclaving by placing into a rocking water bath at 40 °C. The agar was seeded with 100 mg/L p-iodonitrotetrazolium dye then inoculated with the test organism after matching to a 0.5 McFarland BaSO4 turbidity standard in saline. Inoculations used saline at approximately the same temperature as the agar (otherwise the organism was not viable). Seeded agar was set over the TLC plate covering a thickness of not more than 3 mm and remained for 20 h at 38 °C incubation before inspection. TLC separated components of essential oils with antibacterial activity appeared as clear zones against a red background. Acknowledgements The authors would like to thank the New South Wales National Parks and Wildlife Service for permission to collect on reserves
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under its administration; collections were made under permit SL100811. Additionally, the authors would like to express gratitude for photographs of Phebalium squamulosum specimens and support provided by Professor Jeremy J. Bruhl from the University of New England, Armidale, Australia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 10.015. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, fourth ed. Allured Publishing Corporation (ISBN 978-1-932633-21-4). Batey, I.L., Hellyer, R.O., Pinhey, J.T., 1971. The structure of squamulosone, a new sesquiterpene ketone from Phebalium squamulosum. Aust. J. Chem. 24, 2173– 2177. Brophy, J.J., Goldsack, R.J., Forster, P.I., 2005. The leaf oils of coatesia and Geijera (Rutaceae) from Australia. J. Essent. Oil Res. 17, 169–174. Brophy, J.J., Goldsack, R.J., Forster, P.I., 2006. Leaf essential oils of the Queensland species of Phebalium (Rutaceae: Boronieae). J. Essent. Oil Res. 18, 386–391. Chammorro, E.R., Ballerini, G., Sequeira, A.F., Velasco, G.A., Zalazar, M.F., 2008. Chemical composition of essential oil from Tagetes minuta L. leaves and flowers. J. Argentine Chem. Soc. 96, 80–86. Collins, D.O., Buchanan, G.O., Reynolds, W.F., Reese, P.B., 2001. Biotransformation of squamulosone by Curvularia lunata ATCC 12017. Phytochemistry 57, 377–383. CLSI, 2009. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically. Approved Standard – Eight Edition. M07–A8 29(2), 1–66. Giles, R.L., Drinnan, A.N., Walsh, N.G., 2008. Variation in Phebalium glandulosum subsp. glandulosum (Rutaceae). Aust. Sys. Bot. 21, 271–288. IUPAC, 1997. Compendium of Chemical Terminology, second ed. (the ‘‘Gold Book’’). Blackwell Scientific Publications, Oxford. XML on-line corrected version: http:// goldbook.iupac.org 2006. created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. DOI: 10.1351/goldbook. Jones, T.G.H., Smith, F.B., 1925. CCCXLIX – Olefinis terpene ketones from the volatile oil of flowering Tagetes glandulifera. Part 1. J. Chem. Soc. Trans. 127, 2530–2539. Koroch, A.R., Juliani, H.R., Zygadlo, J.A., 2007. Bioactivity of essential oils and their components. In: Berger, R.G. (Ed.), Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Springer, New York. Lassak, E.V., Southwell, I.A., 1974. Occurrence of some unusual compounds in the leaf oil of Eriostemon obovalis and Phebalium glandulosum subsp. glandulosum. Aust. J. Chem. 27, 2703–2705. Mann, C.M., Markham, J.L., 1998. A new method for determining the minimum inhibitory concentration of essential oils. J. Appl. Microbiol. 84 (4), 538–544. McFarland, J., 1907. Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Am. Med. Assoc. 14, 1176–1178. NIST, 2011. NIST Chemistry WebBook – NIST Standard Reference Database Number 69 Retrieved August, 2012. Avaialble from: . Pala-Paul, J., Copeland, L.M., Brophy, J.J., Goldsack, R.J., 2009. Essential oil composition of two new species of Phebalium (Rutaceae) from north-eastern New South Wales, Australia. Nat. Prod. Commun. 4, 983–986. Sadgrove, N.J., Telford, I.R.H., Greatrex, B.W., Dowell, A., Jones, G.L., 2013. Dihydrotagetone, an unusual fruity ketone, is found in enantiopure and enantioenriched forms in additional Australian taxa of Phebalium (Rutaceae: Boronieae). Nat. Prod. Commun. 8, 737–740. Singh, G., Singh, O.P., de Lampasona, M.P., Catalán, C.A.N., 2003. Studies on essential oils. Part 35: chemical and biocidal investigations on Tagetes erecta leaf volatile oil. Flavour Frag. J. 18, 62–65. Southwell, I.A., 1970. A new occurrence of hedycaryol, the precursor of elemol, in Phebalium ozothamnoides (Rutaceae). Phytochemistry 9, 2243–2245. Telford, I.R.H., Bruhl, J.J., 2013. Ongoing morphological investigation of Phebalium (Rutaceae) species. University of New England, Armidale NSW Australia 2351. Wilson, P.G., 1970. A taxonomic revision of the genera Crowea, Eriostemon and Phebalium (Rutaceae). Nuytsia 1, 5–55. Wilson, P.G., 2013. Phebalium. Flora of Australia, vol. 26, pp. 458-480.
