Review of the Leaf Essential Oils of the Genus Backhousia Sens. Lat. and a Report on the Leaf Essential Oils of B. gundarara and B. tetraptera

A review of the leaf oils of the 13 species now recognised in the genus Backhousia is presented. This review carries on from, and incorporates data from, an earlier (1995) review of the then recognised eight species. The leaf oils of two new species of Backhousia, B. gundarara and B. tetraptera are reported for the first time. B. gundarara contains a mixture of mono- and sesquiterpenes, with α-pinene (14%) and spathulenol (11%) being the main members. In B. tetraptera, the principal component of the mainly terpenoid leaf oil is myrtenyl acetate (20–40%). The review also incorporates the two species of the genus Choricarpia, which have been subsumed into Backhousia, viz. B. leptopetala and B. subargentea. Due to its history in Backhousia, Syzygium anisatum, which has been transferred out of Backhousia, is included in the review for historical reasons.


Introduction
Backhousia Hook. & Harv. is a genus currently comprised of 13 species, within the Family Myrtaceae, sub family Myrtoideae [1]. It is now the sole member of Tribe Backhousieae. It was first described in Curtis' Botanical Magazine in 1845 by William Jackson Hooker and William Henry Harvey. The species so named was Backhousia myrtifolia Hook. & Harv. As the authors report: "This very pretty greenhouse shrub, its conspicuous petalois calycine segments giving the idea at first sight of large corollas to flowers, was found by Mr. James Backhouse in the Illawara (sic) district of New South Wales; and, not being referable to any Myrtaceous Genus yet described, Mr. Harvey and myself are anxious to dedicate it to our mutual friend now mentioned, who, amidst his various and arduous labors of love during a voyage to, and journeyings in, various parts of Australia and South Africa, still found leisure to collect and to describe in manuscript many interesting plants, which his previous botanical acquirements enabled him to do with great judgement" [2].
The species in this genus grow as aromatic shrubs or trees (5-25 m tall), with leaves 3-12 cm long and 1-6 cm wide, which are arranged opposite to each other. The genus is represented, with one exception, as endemic to the rain forests and forests of eastern Australia (New South Wales and Queensland). Recently, one species has been identified from the Kimberley region of Western Australia.
The first report of leaf oils from the genus was in 1888, by the firm Schimmel & Co of Miltitz, Germany, who reported that the leaf oil of B. citriodora was almost exclusively (95%) citral [3].
The leaf oil of B. myrtifolia, from which the genus was named, was investigated by Penfold in 1922 [4]. Later work by Penfold et al. in 1953 [5] showed the existence of second collection was made by G. and N. Sankowsky, also in the upper region of the Prince Regent River in 2003. It is known only from these two locations, in the Kimberley region of Western Australia. The leaf oil sample described below originates from the Sankowsky collection. This species is the only species of Backhousia not to occur naturally on the east coast of Australia, although it is growing at Tolga in north Queensland from cuttings taken during the Sankowsky collection. The leaf oil of B. gundarara, produced in 0.3% yield (w/w fresh leaf), contained a mixture of mono-and sesquiterpenes in approximately equal amounts. Additionally, present were six aromatic compounds (from their mass spectra) totalling approx. 9% of the oil. These remain unidentified at the moment. The main monoterpenes were the hydrocarbons α-pinene (13.6%), limonene (3.6%), and p-cymene (1.2%). The oxygenated monoterpenes were not as plentiful, with the principal members being terpinene-4-ol (1.5%) and α-terpineol (1.2%). Of the sesquiterpenes, the major compounds were the alcohols globulol (6.1%), viridiflorol (3.3%) and spathulenol (11.1%). Of the hydrocarbons the main compounds were aromadendrene (1.6%), viridiflorene (1.2%) and an unknown sesquiterpene hydrocarbon (unknown X), whose mass spectrum is given in the footnotes to Table 1 (2.1%). Additionally, present in the oil was what is suspected, from mass spectrum and linear retention index (LRI) data, of being 2,4,6-trimethoxytoluene (0.4%). A detailed list of compounds identified in the oil is set out in Table 1 below, and a Total Ion Current (TIC) trace of the leaf oil from B. gundarara on a polar column is given in Figure 1.

Backhousia tetraptera Jackes
Backhousia tetraptera Jackes is a newly described species growing in gullies on Mount Stuart, Townsville, Qld, at an altitude of about 500 m. It occurs as a population of 170-180 trees. A second site has recently been found at Clement State Forest, near Rollingstone, Qld. It is a tree, usually growing 5-8 m in height, but can grow up to 15 m. It has leaves 5.5-9 cm in length and 1.5-3.8 cm in width. Oil glands are rather sparse in the leaves, and it was not surprising that the oil yield on steam distillation was low, 0.1-0.2%, w/w fresh weight leaves.
Three collections of B. tetraptera foliage were available for steam distillation: one individual tree, a bulk of 3 trees and a sample grown from seed from an individual tree. The oils obtained from the three samples were similar. There were a considerable number of monoterpenes present in this oil, though sesquiterpenes were also well represented. The main component was the ester myrtenyl acetate (24-46%). This ester was accompanied by lesser amounts of α-pinene (3.7-3.8%), linalool (5.0-8.9%), myrtenol (0.5-2.1%) and α-terpineol (0.3-0.7%).

Backhousia tetraptera Jackes
Backhousia tetraptera Jackes is a newly described species growing in gullies on Mount Stuart, Townsville, Qld, at an altitude of about 500 m. It occurs as a population of 170-180 trees. A second site has recently been found at Clement State Forest, near Rollingstone, Qld. It is a tree, usually growing 5-8 m in height, but can grow up to 15 m. It has leaves 5.5-9 cm in length and 1.5-3.8 cm in width. Oil glands are rather sparse in the leaves, and it was not surprising that the oil yield on steam distillation was low, 0.1-0.2%, w/w fresh weight leaves.
Three collections of B. tetraptera foliage were available for steam distillation: one individual tree, a bulk of 3 trees and a sample grown from seed from an individual tree. The oils obtained from the three samples were similar. There were a considerable number of monoterpenes present in this oil, though sesquiterpenes were also well represented. The main component was the ester myrtenyl acetate (24-46%). This ester was accompanied by lesser amounts of α-pinene (3.7-3.8%), linalool (5.0-8.9%), myrtenol (0.5-2.1%) and α-terpineol (0.3-0.7%).
Five chemical varieties have tentatively been suggested in this species. These were:
The structures of these compounds are shown in Figure 3. The composition of the different varieties are given in Table 3 below.

Backhousia bancroftii F.M.Bailey
Backhousia bancroftii F.M.Bailey is a rainforest tree growing up to 25 m in height [37]. It occurs in the Cook and north Kennedy pastoral districts of tropical Queensland. The oil yield from this species was poor (0.03-0.10%) based on fresh leaves. Before the analyses by Brophy et al. [29] the only report on the leaf oil was in 1939, where Lahey and Jones found very poor oil yields and sesquiterpenes as the principal components, with α-pinene and esters as minor constituents [8].
Brophy et al. [29] found that the principal components of the oils of this species were terpenes (mainly sesquiterpenes), alkyl derivatives (alcohols and esters, mainly acetates) and aromatic compounds. There was significant between-tree variation in the oils.
In all but one of the trees examined, the main components were alkyl acetates: in the majority of trees, it was octyl acetate (33-62%), but in one bulk sample it was decyl acetate, with another tree containing approximately equal amounts of decyl-and dodecyl acetates and the corresponding alcohols. In all the oil samples, octyl-, decyl-, dodecyl-and tetradecyl acetates and the corresponding alcohols were significant components, between them accounting for the majority of the leaf oil. There were also small amounts of higher esters identified by mass spectrometry.
Terpenes were very minor components, two trees containing α-pinene, but all trees contained small amounts of sesquiterpenes (both hydrocarbons and oxygenated). In all cases they were individually <3%.
The two principal aromatic compounds identified in the oil of all trees were 2,4,6trimethoxy-3-methylacetophenone and 6-hydroxy-2,4-dimethoxy-3-methyl-acetophenone = bancroftinone (5), shown in Figure 3. This latter novel compound is related in ring substitution to isobaeckeol [38]. In one cultivated tree of unknown origin, it accounted for 85% of the leaf oil, but in natural stands it accounted for trace-3%. 2,4,6-Trimethoxy-3methylacetophenone accounted for 23% of the oil in one tree, but usually was present in the range 0.1-3.9%.
A list of the compounds identified in the oils of this species is given in Table 4. Compounds identified at the level of formulae only have been omitted, but a complete list can be found in [29] 2.5. Backhousia citriodora F.Muell.
Backhousia citriodora F.Muell. is a small-medium sized rainforest tree, endemic to Queensland, Australia. It occurs in the Sunshine coast region of Queensland near Eumondi, Maroochydore, Noosa and Woondon, in the ranges west of Mirriamvale, in the Mackay, Whitsunday, Townsville regions, and near Herberton, Queensland [25]. Several populations have been reduced to isolated trees through land clearing.
The leaf oil of B. citriodora was first described by the firm of Schimmel & Co of Miltitz, Germany in 1888 [3]. It was reported to be almost entirely (95%) citral. This was confirmed in 1923 by Penfold [22]. It has since become the source of a commercial industry for supply of geranial/neral. This is detailed in a recent comprehensive review in a sister publication by Southwell [26]. The presence of (Z)-iso-citral (11), (E)-iso-citral (12) and exo-isocitral (10) in these oils has been confirmed by Doimo [39]. The oil yield is 1.1-3.2% (w/w fresh leaf).
In 1950, Mr. J. R. Archbold, who was collecting from natural stands of the species near Miriam Vale, about 300 km north west of Maryborough, QLD, noticed slight differences in the odour of the oil produced by some trees in the area, indicating that a different type of oil was being produced by some plants in the area. An examination of the oils from single tree sampled by Penfold et al., indicated that the oil from these trees, which were morphologically indistinguishable from other Backhousia citriodora trees, contained-L citronellal (62-80%) [23][24][25]. The trees in question were found scattered throughout a rocky hillside area of about 2 hectares. The variant trees were located in 2 pockets, each containing about 12 trees, of which about half were the variants (one tree being about 27 m in height and slightly over 2 m in girth at breast height). Nothing else was published on this citronellal variant for about the next 50 years. As part of a systematic breeding project to produce clones of B. citriodora with greater percentage of citral, 16 open pollinated families were selected. From this trial, 3 trees, out of 272 sampled, gave the L-citronellal oils. As part of this trial, a re-examination of the parent trees in the population at Noosa, QLD, from which the parent trees of the 3 citronellal producing offspring had originally been obtained, was undertaken. It was found that 1 tree was producing the L-citronellal oil. Breeding trials from this 1 tree were then undertaken [25].
The composition of the leaf oils from both chemotypes are given in Table 5. This is based on the oil composition obtained from the 3 clones taken from the open pollenated trees in the breading trials for the L-citronellal chemotype and from commercially harvested material of the citral chemotype [23,26]. The oil yield from the L-citronellal chemotype was 1.8-3.2% (w/w dry weight). The structures of the numbered compounds are given in Figure 3. Backhousia enata A.J.Ford is a relatively recently described species [40,41]. It is a large shrub or tree growing to 5-15 m in height, with a trunk diameter up to 20 cm diameter at breast height. It occurs in northeastern Queensland, where it is endemic to the 'Wet Tropics' and is currently confined to the Tully River catchment area. It inhabits notophyll vine forest/rainforest on soil derived from rhyolite and basalt. In 2007, there were less than 200 individuals known.
The leaf oil of B. enata bears no similarity to that of B. myrtifolia, its nearest morphological relative, whose leaf oil is dominated by the aromatic ethers, elemicin, isoelemicin, methyl eugenol or methyl isoeugenol.

Backhousia hughesii C.T.White
Backhousia hughesii C.T.White is a tree growing up to 30 m in height. It grows in the Atherton tablelands (in the Cook pastoral district) of Queensland [33]. Early work on the leaf oil of this species by Jones and Lahey, published in 1938 [7], showed that it contained mostly D-α-pinene and D-β-pinene. Brophy et al. [29] in 1995, who examined the oil of this species from 3 populations, found that the oil contained mainly sesquiterpenes rather than monoterpenes. One tree contained 12% of α-pinene, but all others examined contained <5%. The oil yield (on a fresh weight basis) was 0.13-0.45%. In contrast to other Backhousia species, there appeared to be only one chemotype.

Backhousia kingii Guymer
Backhousia kingii Guymer is a relatively recently described species [30]. It is a tree growing up to 20 m and is endemic to subcoastal, central eastern Queensland in the Leichhardt, Wide Bay and Burnett pastoral districts [33]. It grows in noto-or microphyll semi evergreen vine thickets in an altitude range of 0-400 m above sea level.
As part of the survey of the oils of Backhousia, Brophy et al. were able to confirm the presence of the three chemotypes (elemicin, methyl eugenol, and methyl isoeugenol) [29], but in the trees available were not able to confirm the isoelemicin chemotype, first recorded by Penfold in 1953, in the trees they examined.
The analyses of samples of the methyl eugenol methyl isoeugenol, and elemicin chemotypes, together with the isoeugenol chemotype, taken from Penfold et al. [5] are listed in Table 10. The oil yields obtained by Brophy et al. in 1995 [29] were in the range 1.0-2.2% (on fresh weight basis), although 1 tree of the elemicin chemotype gave a yield of 0.5%. The oil yields obtained by Penfold et al. and Hellyer et al. were lower, despite the fact that they were measured on a dry weight basis (0.1-0.7%).   As can be seen from Table 10, one aromatic ether dominated the oil from each chemotype. The compound is accompanied by a large number of terpenes (usually sesquiterpenes). Several compounds that were identified only at the formula level have not been included here, but can be found in [29]. Structures of numbered compounds are given in Figure 3.

Backhousia oligantha A.R.Bean
Backhousia oligantha A.R.Bean, called Backhousia sp. (Dicot Pilferer 12671) in a previous publication [29], is a small tree growing to a height of 4 m, but is often multi-stemmed, forming a low groundcover. It is found in semi-evergreen microphyll vine thicket near Biggenden in the Wide Bay pastoral district of south-east Queensland [29,43].
The leaf oil also contained a homologous series of both alkanols and their corresponding acetates. The series commenced octanol (0.2%) and continued to tetradecanol (0.4%), with the principal members being decanol (2.2%) and dodecanol (8.2%). The alkyl acetates were present, with the odd numbered members being present in lesser amounts compared to the even numbered members. The alkyl acetates present corresponded to the alkanols found, the principal members being decyl acetate (1.5%) and dodecyl acetate (8.0%). Several propionate esters were also detected (decyl-and dodecyl-), but were present in amounts of less than (0.4%). Backhousia sciadophora F.Muell. is a tree attaining a height of 30 m, and occurs in drier rainforest gorges and steep slopes from Dungog (NSW) to Nambour (QLD) [42,43]. The oil was first reported on by Penfold in 1924 [27]. He reported that the oil from this species contained D-α-pinene (80-85%), the remainder of the oil being sesquiterpenoid.

Syzygium anisatum (Vickery) Craven & Biffin
Syzygium anisatum (Vickery) Craven & Biffin (syn Backhousia anisata) is a fairly dense glabrous foliage tree that can reach 50 m in height and have a circumference of 4 m. It inhabits rainforests in a few places in the Bellingen and Nambucca valleys of northern New South Wales [42]. In its natural state, it is regarded as a rare and endangered species [16,45]. The species, since then, has had two changes of name as its taxonomy has been reinvestigated, passing through Anetholea anisata (Vickery) Peter G., Wilson [20], and finally being placed in Syzygium anisatum (Vickery) Craven & Biffin [19]. Due to its long history in Backhousia, its leaf oils are still considered here in this review.
McKern was the first to analyse the oil of B. anisata and found it to contain anethole at about 60% of the oil, the oil yield being 0.5% [15]. Brophy and Boland [16] reported that two chemotypes existed, having an oil yield of 1.3-2.0% (w/w fresh leaf) for both chemotypes of this species. The methyl chavicol (18) chemotype was found in approximately 25% of the trees examined (9 trees, including 1 bulk of 3 trees). Blewitt and Southwell [18], in a later and more widespread survey, found that the methyl chavicol (18) chemotype was approximately 1: 4.7 of the E-anethole (19) chemotype. They found that three of the ten sites sampled contained both chemotypes occurring within meters of each other. Southwell et al. also found that a few trees contained approx. equal amounts of both E-anethole and methyl chavicol [17].

Discussion
In their 2012 paper [1], Harrington et al. argued that "there were four strongly supported clades containing two to four taxa, with no support for relationships among clades, and the relationships of Backhousia bancroftii and B. citriodora remain unresolved". They also state that on the analyses of the DNA data "The current distribution of Backhousia is inferred to be largely due to the contraction of Australian rainforests in the Neogene". This is supported by Figure 2 in their paper [1].
From this diagram, it might be expected that species grouped together might have similar leaf oils, and that the closer together the species were grouped, the more similar the leaf oils of the species might be.
Examining the dendrogram, Figure 4, there appear to be a significant number of species where their close proximity is also reflected in the leaf oils. Thus B. leptopetala and B. subargentea, species that have been transferred from the genus Choricarpia, (and in the dendrogram are still mentioned as species of Choricarpia) do possess similar leaf oils, which are heavily based on monoterpenes, with α-pinene, limonene, and 1,8-cineole being prominent compounds in both species. There are, however, other compounds, present in small amounts, that do differ between the species.
Backhousia kingii was relatively recently split form B. sciadophora [30]. Both species possess similar leaf oils, in which monoterpenes predominate, with α-pinene and limonene being prominent components and sesquiterpenes being only minor components.
Backhousia hughesii and B. gundarara do not, however, follow this line, with B. hughesii having an oil rich in sesquiterpenes, with β-elemene and β-bisabolene being the major components. B. gundarara, (Backhousia sp. Prince Regent in Figure 4) while possessing major amounts of globulol, viridiflorol, spathulenol and other sesquiterpene hydrocarbons, also contains considerable amounts of α-pinene, limonene and other monoterpene hydrocarbons. It also contains a series of, as yet, unidentified aromatic compounds, whose mass spectra are given in the footnote to Table 1.
Of the three species in the clade containing B. myrtifolia, B. enata and B. tetraptera, B. myrtifolia stands out distinctively because of the presence of the aromatic ethers, methyl eugenol, E-methyl isoeugenol, elemicin and E-isoelemicin, as a principal component in its leaf oil, vastly overshadowing any other terpenoid components. The other two species contain mainly monoterpenoid leaf oils, with the B. enata oil being dominated by α-pinene and sabinene, while in the case of B. tetraptera (Backhousia sp. Mt. Stuart in Figure 4), the major components were myrtenyl acetate and linalool. B. citriodora, whose leaf oil is dominated by either citral or L-citronellal, stands apart from the other members of this clade. subargentea, species that have been transferred from the genus Choricarpia, (and in drogram are still mentioned as species of Choricarpia) do possess similar leaf oils are heavily based on monoterpenes, with α-pinene, limonene, and 1,8-cineole bein inent compounds in both species. There are, however, other compounds, present amounts, that do differ between the species. In the clade containing B. bancroftii, B. angustifolia and B. oligantha, B. bancroftii has an "unresolved" morphological relationship to the other two species [1], but the contents of its leaf oil, containing major amounts of alkyl acetates and alcohols, is a lot more closely related to the oils of B. oligantha, which also contains significant amounts of these compounds. These two species are the only species of Backhousia to contain the alkyl esters and alcohols in any quantity. B. bancroftii also contains varying amounts (trace to 23%) of 2,4,6-trimethoxy-3-methylacetophenone and bancroftinone (5) (trace->80%), not present in any other species of Backhousia.

Conclusions
The relationship of the leaf oils of a species of Backhousia to that species' place in the dendrogram (Figure 4) is rather problematic. Two species (B. bancroftii, B. oligantha) possess similar oils, containing a series of alkanols and their corresponding acetate esters, rare in the oils of Backhousia, though B. bancroftii bears an 'unresolved' relationship to B. oligantha. In other cases, e.g., B. kingii, B. sciadophora, the leaf oils are very similar and, in fact, B. kingii was split from B. sciadophora on morphological grounds. The two species which, in terms of classes of compounds, are most similar, B. myrtifolia, containing di-or tri-methoxy-allyl or -propenyl benzene, and Syzygium anisatum, containing methoxy-allyl or-propenyl benzene, are no longer in the same genus. It would appear that with our present knowledge, it would be wise to not place too much reliance on the relative grouping of the species when considering their leaf oils: more research on the genes directing the syntheses of these components is required.