New Labdanes with Antimicrobial and Acaricidal Activity: Terpenes of Callitris and Widdringtonia (Cupressaceae)

In spite of the evidence for antimicrobial and acaricidal effects in ethnobotanical reports of Callitris and Widdringtonia, the diterpene acids from Widdringtonia have never been described and no comparison to the Australian clade sister genus Callitris has been made. The critically endangered South African Clanwilliam cedar, Widdringtonia wallichii (syn. W. cedarbergensis), of the Cederberg Mountains was once prized for its enduring fragrant timbers and an essential oil that gives an aroma comparable to better known Mediterranean cedars, predominantly comprised by widdrol, cedrol, and thujopsene. In South Africa, two other ‘cedars’ are known, which are called W. nodiflora and W. schwarzii, but, until now, their chemical similarity to W. wallichii has not been investigated. Much like Widdringtonia, Callitris was once prized for its termite resistant timbers and an ‘earthy’ essential oil, but predominantly guaiol. The current study demonstrates that the essential oils were similar across all three species of Widdringtonia and two known non-volatile diterpene acids were identified in leaves: the pimaradiene sandaracopimaric acid (1) and the labdane Z-communic acid (2) with a lower yield of the E-isomer (3). Additionally, in the leaves of the three species, the structures of five new antimicrobial labdanes were assigned: 12-hydroxy-8R,17-epoxy-isocommunic acid (4), 8S-formyl-isocommunic acid (5), 8R,17-epoxy-isocommunic acid (6), 8R-17R-epoxy-E-communic acid (7), and 8R-17-epoxy-E-communic acid (8). Australian Callitris columellaris (syn. C. glaucophylla) also produced 1 and its isomer isopimaric acid, pisiferal (9), and pisiferic acid (10) from its leaves. Callitris endlicheri (Parl.) F.M.Bailey yielded isoozic acid (11) as the only major diterpene. Diterpenes 4–6, pisiferic acid (10), spathulenol, and guaiol (12) demonstrated antimicrobial and acaricidal activity.


Introduction
Southern African Widdringtonia were once prized for their timbers and have been heavily exploited over the course of more than a century. The most pronounced impact has been on the now critically endangered W. wallichii Endl. ex Carrière (=W. cedarbergensis J.A.Marsh) [1], which was favoured for its durable fragrant timber [2,3]. Unlike other species in the genus, it does not re-sprout after fire [3,4] and so its conservation status is now critical. South African Widdingtonia has long been recognised as closely alligned with Australian Callitris, with the two described as clade sisters [14]. Due to the extreme disjunction between the two genera, it has been suggested that they form part of a relict group with many extinct members [15]. Unlike Widdringtonia, Australian Callitris is still widespread despite the historical popularity of the timbers for their termite resistance. The most common species are C. columellaris F. Muell (coastal cypress pine), C. endlicheri (Parl.) F.M.Bailey (black cypress), C. glaucophylla Joy Thomps. & L.A.S.Johnson (white cypress) and C. intratropica (R.T.Baker & H.G.Sm.) Silba (blue cypress). The latter two, C. glaucophylla and C. intratropica, are not recognized as distinct outside of Australia, which is classified as synonyms of C. columellaris by The Plant List. Nevertheless, chemical characterization of various extractives from these geographically segregated taxa demonstrates consistent chemical divergence [16]. In the current study, specimens of C. glaucophylla are referred to as C. columellaris to remain consistent with the international recommendation. South African Widdingtonia has long been recognised as closely alligned with Australian Callitris, with the two described as clade sisters [14]. Due to the extreme disjunction between the two genera, it has been suggested that they form part of a relict group with many extinct members [15]. Unlike Widdringtonia, Australian Callitris is still widespread despite the historical popularity of the timbers for their termite resistance. The most common species are C. columellaris F. Muell (coastal cypress pine), C. endlicheri (Parl.) F.M.Bailey (black cypress), C. glaucophylla Joy Thomps. & L.A.S.Johnson (white cypress) and C. intratropica (R.T.Baker & H.G.Sm.) Silba (blue cypress). The latter two, C. glaucophylla and C. intratropica, are not recognized as distinct outside of Australia, and are classified as synonyms of C. columellaris by The Plant List. Nevertheless, chemical characterization of various extractives from these geographically segregated taxa demonstrates consistent chemical divergence [16].
Antibiotics 2020, 9, 173 3 of 16 In the current study, specimens of C. glaucophylla are referred to as C. columellaris to remain consistent with the international recommendation.
Essential oils from timbers of Callitris predominantly consist of guaiol [17], along with other sesquiterpenes such as azulenes [18], which are responsible for the blue colour of material referred to as C. intratropica essential oil (guaiazulene, chamazulene). Costols [18] and Antibiotics 2020, 9, x 3 of 16 Essential oils from timbers of Callitris predominantly consist of guaiol [17], along with other sesquiterpenes such as azulenes [18], which are responsible for the blue colour of material referred to as C. intratropica essential oil (guaiazulene, chamazulene). Costols [18] and ƴ-lactones [19] are also present and aid termite resistance [20]. Guaiol and azulenes have higher relative abundances in the essential oils, while costols and ƴ-lactones are more strongly represented in solvent extracts [20] due to their higher boiling points. A recent study of Callitris spp. also reported diterpene acids using mass spectrometry on extracts from the resins [21].
As with all members of Cupressaceae, leaf essential oils from Callitris and Widdringtonia are significantly different from heartwoods [10,16] with monoterpenes predominating in leaf oils and sesquiterpenes predominating in heartwood timber oils. A study on Callitris leaves by gas chromatography reported only one diterpene, pisiferal [16], but the major non-volatile diterpenes from Callitris have remained hitherto unidentified, as is the case for Widdringtonia. Both Widdringtonia and Callitris have been extensively milled for timber, which resulted in continued interest in valorisation of the saw dust waste [11,22]. Strangely, no similar interest has been expressed for leaves that are also a by-product of the logging process. In the current study, we have investigated the chemistry of leaves and, in some cases, we have contrasted between leaves and branch/twig pulp. Traditional use reports describe topical therapeutic applications of leaf extracts of C. columellaris and cone extracts (clear hard gum) of Widdringtonia spp. for ailments consistent with microbial or parasitic (ectoparasitic) infection [16,23]. Thus, isolated compounds from Callitris and Widdringtonia have also been examined for antimicrobial and acaricidal activity, as knowledge in the area is currently lacking [22].
-lactones [19] are also present and aid termite resistance [20]. Guaiol and azulenes have higher relative abundances in the essential oils, while costols and 3 of 16 ers of Callitris predominantly consist of guaiol [17], along with other es [18], which are responsible for the blue colour of material referred il (guaiazulene, chamazulene). Costols [18] and ƴ-lactones [19] are also ance [20]. Guaiol and azulenes have higher relative abundances in the d ƴ-lactones are more strongly represented in solvent extracts [20] due A recent study of Callitris spp. also reported diterpene acids using mass the resins [21]. Cupressaceae, leaf essential oils from Callitris and Widdringtonia are eartwoods [10,16] with monoterpenes predominating in leaf oils and g in heartwood timber oils. A study on Callitris leaves by gas ly one diterpene, pisiferal [16], but the major non-volatile diterpenes itherto unidentified, as is the case for Widdringtonia. Both Widdringtonia nsively milled for timber, which resulted in continued interest in aste [11,22]. Strangely, no similar interest has been expressed for leaves the logging process. In the current study, we have investigated the ome cases, we have contrasted between leaves and branch/twig pulp. be topical therapeutic applications of leaf extracts of C. columellaris and of Widdringtonia spp. for ailments consistent with microbial or parasitic ]. Thus, isolated compounds from Callitris and Widdringtonia have also ial and acaricidal activity, as knowledge in the area is currently lacking Widdringtonia on, only leaves and branches (two-inch circumference) of Widdringtonia study, as heartwood harvesting is destructive. A series of known (1-3) (4-8) have been identified in extracts from leaves and other aerial parts e terpenes consistent with those identified in earlier phytochemical Widdringtonia ( Table 2). The overwhelmingly higher representation of leaves is surprising, but consistent across all specimens and species Since diterpenes were not isolated in the earlier studies that focused on e diterpenes are restricted to branching parts and sesquiterpenes are e heartwood. s 1-12 in % (g/g). C.e-Callitris endlicheri, C.c-C. columellaris, W.s-.c-W. wallichii, W.n-W. nodiflora. (L)-Leaves, (Tw)-Twigs, (Ti)racopimaric acid (1), Z-communic acid (2), E-communic acid (3), 12mmunic acid (4), 8S-formyl-isocommunic acid (5), 8R,17-epoxy-7-epoxy-E-communic acid (7), 8R-17-epoxy-Z-communic acid (8), pisiferal ic acid (11), and guaiol (12).
-lactones are more strongly represented in solvent extracts [20] due to their higher boiling points. A recent study of Callitris spp. also reported diterpene acids using mass spectrometry on extracts from the resins [21].
As with all members of Cupressaceae, leaf essential oils from Callitris and Widdringtonia are significantly different from heartwoods [10,16] with monoterpenes predominating in leaf oils and sesquiterpenes predominating in heartwood timber oils. A study on Callitris leaves by gas chromatography reported only one diterpene, pisiferal [16], but the major non-volatile diterpenes from Callitris have remained hitherto unidentified, as is the case for Widdringtonia. Both Widdringtonia and Callitris have been extensively milled for timber, which resulted in continued interest in valorisation of the saw dust waste [11,22]. Strangely, no similar interest has been expressed for leaves that are also a by-product of the logging process. In the current study, we have investigated the chemistry of leaves and, in some cases, we have contrasted between leaves and branch/twig pulp. Traditional use reports describe topical therapeutic applications of leaf extracts of C. columellaris and cone extracts (clear hard gum) of Widdringtonia spp. for ailments consistent with microbial or parasitic (ectoparasitic) infection [16,23]. Thus, isolated compounds from Callitris and Widdringtonia have also been examined for antimicrobial and acaricidal activity, as knowledge in the area is currently lacking [22].

Chemistry of South African Widdringtonia
For reasons of conservation, only leaves and branches (two-inch circumference) of Widdringtonia were extracted in the present study, as heartwood harvesting is destructive. A series of known (1)(2)(3) and new labdane derivatives (4-8) have been identified in extracts from leaves and other aerial parts (Table 1), along with volatile terpenes consistent with those identified in earlier phytochemical studies on essential oils from Widdringtonia ( Table 2). The overwhelmingly higher representation of diterpenes in the extracts of leaves is surprising, but consistent across all specimens and species investigated in Widdringtonia. Since diterpenes were not isolated in the earlier studies that focused on heartwood, it is likely that the diterpenes are restricted to branching parts and sesquiterpenes are obtained in higher yield in the heartwood. Table 1.
-Pi-OH Car-Ox F-Ac B-Ac Th-ene Spath The most abundant diterpenes are already well known from Cupressaceae with sandaracopimaric acid (1) being the dominant terpene in our extracts, which is followed by Z-communic acid (2) ( Table 1). The name sandaracopimaric acid has its etymology in the product 'sandarac', historically derived from the Moroccan species Tetraclinis articulata (Vahl) Mast., which is another member of Cupressaceae [24]. The communic acid isomers are evidently familiar with the fruits of Juniperus communis L. [25], which is popularly known for its essential oil wherein the acids are absent due to higher boiling points.

E-(7)
Z-(8) 13  In most plant parts, the spiroepoxy diterpenes were of low relative abundance, except for in the cones of W. nodiflora, which were richer in the new diterpenes as compared to 1 and 2. Furthermore, the cones yielded over 1% w/w spathulenol in flash chromatography. Spathulenol (Table 5) is an antimicrobial sesquiterpene [26] that demonstrates enhanced activity against skin pathogens when encapsulated (Table 6). An encapsulation effect similar to that derived from inclusion in the chemically diverse gum that exudes from the cones. Spathulenol is also present at lower concentrations in the essential oils of other plant parts ( Table 5). The pronounced chemical difference of the cones compared to other plant parts provides the first insight into why these are chosen above other organs in therapeutic applications consistent with antimicrobial outcomes. Antimicrobial testing provided further testimony to this (Table 6). See Section 2.3 for more details. antimicrobial sesquiterpene [26] that demonstrates enhanced activity against skin pathogens when encapsulated (Table 6). An encapsulation effect similar to that derived from inclusion in the chemically diverse gum that exudes from the cones. Spathulenol is also present at lower concentrations in the essential oils of other plant parts ( Table 5). The pronounced chemical difference of the cones compared to other plant parts provides the first insight into why these are chosen above other organs in therapeutic applications consistent with antimicrobial outcomes. Antimicrobial testing provided further testimony to this (Table 6). See section 2.3 for more details.
inantly consist of guaiol [17], along with other onsible for the blue colour of material referred zulene). Costols [18] and ƴ-lactones [19] are also zulenes have higher relative abundances in the strongly represented in solvent extracts [20] due ris spp. also reported diterpene acids using mass ential oils from Callitris and Widdringtonia are monoterpenes predominating in leaf oils and er oils. A study on Callitris leaves by gas eral [16], but the major non-volatile diterpenes is the case for Widdringtonia. Both Widdringtonia ber, which resulted in continued interest in no similar interest has been expressed for leaves In the current study, we have investigated the ntrasted between leaves and branch/twig pulp. pplications of leaf extracts of C. columellaris and or ailments consistent with microbial or parasitic unds from Callitris and Widdringtonia have also ity, as knowledge in the area is currently lacking nches (two-inch circumference) of Widdringtonia arvesting is destructive. A series of known (1-3) ied in extracts from leaves and other aerial parts with those identified in earlier phytochemical ). The overwhelmingly higher representation of ut consistent across all specimens and species not isolated in the earlier studies that focused on cted to branching parts and sesquiterpenes are   inantly consist of guaiol [17], along with other onsible for the blue colour of material referred zulene). Costols [18] and ƴ-lactones [19] are also zulenes have higher relative abundances in the strongly represented in solvent extracts [20] due ris spp. also reported diterpene acids using mass ential oils from Callitris and Widdringtonia are monoterpenes predominating in leaf oils and er oils. A study on Callitris leaves by gas eral [16], but the major non-volatile diterpenes is the case for Widdringtonia. Both Widdringtonia ber, which resulted in continued interest in no similar interest has been expressed for leaves In the current study, we have investigated the ntrasted between leaves and branch/twig pulp. pplications of leaf extracts of C. columellaris and or ailments consistent with microbial or parasitic unds from Callitris and Widdringtonia have also ity, as knowledge in the area is currently lacking nches (two-inch circumference) of Widdringtonia arvesting is destructive. A series of known (1-3) ied in extracts from leaves and other aerial parts with those identified in earlier phytochemical ). The overwhelmingly higher representation of ut consistent across all specimens and species not isolated in the earlier studies that focused on cted to branching parts and sesquiterpenes are   inantly consist of guaiol [17], along with other onsible for the blue colour of material referred zulene). Costols [18] and ƴ-lactones [19] are also zulenes have higher relative abundances in the strongly represented in solvent extracts [20] due ris spp. also reported diterpene acids using mass ential oils from Callitris and Widdringtonia are monoterpenes predominating in leaf oils and er oils. A study on Callitris leaves by gas eral [16], but the major non-volatile diterpenes is the case for Widdringtonia. Both Widdringtonia ber, which resulted in continued interest in no similar interest has been expressed for leaves In the current study, we have investigated the ntrasted between leaves and branch/twig pulp. pplications of leaf extracts of C. columellaris and or ailments consistent with microbial or parasitic unds from Callitris and Widdringtonia have also ity, as knowledge in the area is currently lacking nches (two-inch circumference) of Widdringtonia arvesting is destructive. A series of known (1-3) ied in extracts from leaves and other aerial parts with those identified in earlier phytochemical ). The overwhelmingly higher representation of ut consistent across all specimens and species not isolated in the earlier studies that focused on cted to branching parts and sesquiterpenes are  In most plant parts, the spiroepoxy diterpenes were of low relative abundance, except for in the cones of W. nodiflora, which were richer in the new diterpenes as compared to 1 and 2. Furthermore, the cones yielded over 1% w/w spathulenol in flash chromatography. Spathulenol (Table 5) is an antimicrobial sesquiterpene [26] that demonstrates enhanced activity against skin pathogens when encapsulated ( Table 6). An encapsulation effect similar to that derived from inclusion in the chemically diverse gum that exudes from the cones. Spathulenol is also present at lower concentrations in the essential oils of other plant parts ( Table 5). The pronounced chemical difference of the cones compared to other plant parts provides the first insight into why these are chosen above other organs in therapeutic applications consistent with antimicrobial outcomes. Antimicrobial testing provided further testimony to this (Table 6). See section 2.3 for more details.   (1), Z-communic acid (2), 12-hydroxy-8R,17-epoxy-isocommunic acid (4), 8S-formyl-isocommunic acid (5), 8R,17-epoxy-isocommunic acid (6), 8R-17-epoxy-E-communic acid (7), 8R-17-epoxy-Z-communic acid (8), and pisiferic acid (10 Essential oils from leaves and stem pulp (Table 5) of W. nodiflora and W. schwartzii were chemically similar to essential oils from aerial parts of W. wallichii described in other studies [10]. While the essential oils of branches displayed high chemical consistency with that reported for the heartwood, the yields obtained by us were still lower than the reported yields in heartwood.

Chemistry of Callitris
Essential oils from leaves of the two species of Callitris sampled in the current study (C. endlicheri and C. columellaris) have been previously characterised [16,27]. Furthermore, timber essential oils and solvent extracts have also been studied [18]. Thus, in the current study, an examination of solvent extracted (non-volatile) components from the leaves was undertaken to gratify this neglected area of research in order to make a comparison to Widdringtonia. Unlike Widdringtonia, interspecific differences in the Genus were pronounced. Major diterpenes in the leaves of C. columellaris were pisiferal (9) and pisiferic acid (10), whereas a single diterpene was evident in C. endlicheri leaves, being isoozic acid (11), which is the isomer of the more widespread ozic acid in other species of Callitris [21]. In addition, sandaracopimaric acid (1) was identified in both species, but at lower concentrations as compared to Widdringtonia. The study by Simoneit et al. [21] reported 2 and 3 extensively in most other species of Callitris, which is similar to the pattern evident in Widdringtonia, but these diterpenes were not identified in the two species of Callitris in the current study. Nevertheless, mass spectral analysis can be used to verify if they occur in trace amounts. The identification of 12-hydroxycallitrisic acid in the resin of C. baileyi C.T. White by Simoneit et al. [21] is interesting since it only differs from pisiferic acid by the positioning of the carboxyl group.
The species sampled for the current study (C. endlicheri & C. columellaris) were geographically disjunct from those studied by Simoneit et al. [21]. Within Australia, C. columellaris is treated as three distinct species, as previously mentioned [28]. Because Simoneit et al. [21] did not identify the abietanes pisiferal (9) or pisiferic acid (10), it is likely that they sampled the actual C. columellaris cultivated from the coastal south-east Qld genepool, which grows in the sand dunes. The specimens of C. columellaris sampled for the current study were all taken from the inland temperate regions and represent the taxa that is widely recognized in Australia as C. glaucophylla. In the current study, we have chosen to recognize these interior specimens as a distinct chemotype.
Nevertheless, the current study constitutes the first appearance of the abietanes pisiferal (9) and pisiferic acid (10) in Callitris. Otherwise, they are familiar to Cupressaceae, first isolated from the etymologically related Japanese species Chamaecyparis pisifera (Siebold and Zucc.) Endl. [29]. The authors considered the discovery of pisiferic acid (10) in C. columellaris leaves fortuitous since it has been recognised as having significant commercial potential in the context of preservatives and topical antimicrobial therapy. It yields at 0.1% w/w from the leaves and is sustainably harvested, which ensures that trees are able to recover without long-term negative effects. Since 10 has a carboxylic acid moiety, it can be enriched using an organic/base partition using aqueous ammonia extraction and then neutralised with HCl to create a composition of 40-50% purity.
In the interest of confirming chemical similarity of C. columellaris over a wide geographic range, the specimens were sampled from several locations in Qld and NSW, which all displayed similar yields of 10. Specimens collected from as far south as Peak Hill (central NSW) to as far north as Blackall (north Qld) all yielded 10. In contrast, C. endlicheri was considerably variable, but a full report of the chemical character is beyond the scope of this study. Variation can be explained by the disjunct nature of its distribution since it is restricted to rocky slopes and plains, gorges, and other geographical features that are not widespread. This creates barriers to gene spread and inevitably leads to chemical divergence.
The butadiene moiety of compounds 4-6 had characteristic broad proton singlets in the olefinic region (4: δ H 5.11, 5.21, 5.23 and 5.43 ppm) and were seen in the HSQC (heteronuclear single quantum coherence) spectrum to be part of two methylidenes. In the HMBC (heteronuclear multiple bond correlation) spectrum, these protons interacted with the neighbouring olefin ( Table 3). The overlap of 13 C shifts to isocommunic acid [30] showed strong agreement (Supplementary Files, Table S1) after allowing for the electronic influences of the oxidised and adjacent carbons. For example, the C12-OH shift on 4 altered most of the 13 C shifts in the butadiene moiety, but, on 5 and 6, the shifts from C13 to C16 had <1 ppm difference as compared to isocommunic acid.
HRESIMS of 4 gave a molecular ion peak at m/z 357.2030 [M + Na] + , which is consistent with a molecular formula of C 20 H 30 O 4 . This only differs from isocommunic acid by the addition of two oxygen atoms. This molecular formula gives an index of hydrogen deficiency (IHD) of 6 with four olefinic carbons (δ C 115.0, 115.5, 136.6 & 145.9 ppm) and a resonance in the acid region (δ C 182.3). This indicated a tricyclic molecule. Relative to isocommunic acid, 4 does not have the C8-C17 methylidene, but instead displays two resonances consistent with an epoxide (C8 δ C 60.3 ppm & C17 δ C 50.4 ppm). The HSQC spectrum was used to assign the two oxirane protons at 2.82 and 2.60 ppm to C17 at δ C 50.4 ppm. The HMBC spectrum indicated that these oxirane protons have a 1 H-13 C interaction with the quaternary C8 position, and, in the COSY spectra, a 4 J 1 H-1 H coupling was observed between the downfield oxirane proton (δ H 2.82 ppm) and one of the C7 protons (δ H 1.92 ppm). Similar chemical shifts were observed in several other labdanes [31][32][33][34][35][36] with a spiro-epoxy group at C8. Another downfield resonance was observed at δ C 83.8 ppm, which is consistent with an allylic alcohol, with the position established as C12 using the HMBC spectrum. A 1 H-13 C interaction from H16/H16' to C12 was observed, and the reverse interaction, H12 to the olefinic resonance C16 was also seen. These observations led to the structure of 12-hydroxy-8R,17-spiroepoxy-isocommunic acid for 4 with the 2D NMR spectra being consistent with the proposed structure ( Table 3). The configuration of C12 could not be unambiguously determined due to free rotation in the chain. In the 2D NOESY (nuclear overhauser effect spectroscopy) spectrum, a through-space interaction from the downfield C17 oxirane proton (δ H 2.82 ppm) to one of the C11 protons and the C20 methyl group were seen. However, this was insufficient to assign the configuration at C12. The replacement of the C8-C17 methylidene with an oxirane also presumably affects the chemical shifts throughout the spectrum of 4 relative to isocommunic acid (Supplementary: Tables S1 and S2) [37].
Determining the configuration of spiroepoxy groups can be challenging due to the small differences in chemical shifts and the similar position for the oxiranyl hydrogens in the two isomers. Bastard et al. [32] have reported that the 13 C shift at C17 of 8-spiroexpoxy labdanes is further downfield in the 8R configuration (which they refer to as α-configuration) with values ranging from δ C 50-51 ppm. This contrasts with the 8S configuration with C17 shifts of δ C 48-49 ppm. The oxirane protons are also affected with H17 shifting on the 8R epimer ranging from δ H 2.55-2.60 ppm and δ H 2.70-2.82 ppm in contrast with δ H 2.20-2.30 ppm and δ H 2.42-2.70 ppm on the 8S epimer. The difference is believed to be a consequence of equatorial vs. axial shielding effects [32]. By comparing chemicals shifts for eight labdanes with the 8R-configuration and five with the 8S-configuration (Supplementary: Tables S3-S5 and Figures S1-S3), the relationship appears to hold. While it is possible that the 13 C NMR chemical shift of C17 on the 8S epimer can be shifted due to the C11-C16 moiety [36], the oxiranyl protons in 4 demonstrated shifts within the predicted range for the 8R configuration.
HRESIMS of 5 detected a molecular ion peak at m/z 341.2094 [M + Na] + , which is consistent with a molecular formula of C 20 H 30 O 3 and an IHD of 6. The 13 C NMR shifts in the butadiene moiety (C13-C16) very closely and coincided with those reported for isocommunic acid with ∆δ < 0.6 and the compound differed from 4 due to the absence of the C12-OH. The 13 C NMR spectrum of 5 lacked the oxiranyl signals and contained a C17 aldehyde with signals at δ C 205.2 ppm and δ H 9.56. The H17 aldehyde resonance coupled to H8 in the COSY spectrum. The configuration at C8 is also believed to be S as the NOESY spectrum showed that H8 interacted with H9, and there was an interaction between H17 at δ H 9.56 ppm and H11 at δ H 1.18 ppm, which supports the cis configuration. Thus, 5 was tentatively assigned as 8S-formyl-isocommunic acid and HMBC couplings corroborated this (Table 3).
Similar to 5, the 13 C shifts on the butadiene moiety of 6 were consistent with isocommunic acid (Supplementary: Table S1). HRESIMS of 6 detected a molecular ion peak at m/z 319.2258 [M + H] + , which is consistent with a molecular formula of C 20 H 30 O 3 and an IHD of 6. Aside from the butadiene moiety, the 13 C and 1 H spectrums of 6 closely resembled that of 4 (Table 3), which indicates a structure differing only from isocommunic acid by the spiroepoxy moiety. By examination of the HMBC couplings on 6 ( Table 3), the structure was assigned as 8R,17-epoxy-isocommunic acid.
HRESIMS of 7 and 8 gave a molecular ion peak at m/z 341.2081 [M + Na] + for both compounds, which is again consistent with a molecular formula of C 20 H 30 O 3 and an IHD of 6. Out of the new compounds, only 7 and 8 were demonstrated to be derivatives of the communic acid cis/trans isomers [38]. The 1 H and 13 C spectra of both isomers (Table 4) very closely matched that of 6 ( Table 3) with the exception of the butadiene chain, where the second methylidene in 6 was not observed in 7 or 8. In contrast, the olefinic carbon at C13 was substituted with a methyl group (C16), which gave a 3H singlet in the 1 H NMR spectrum in the olefinic methyl region (7: δ H 1.68 ppm, 8: δ H 1.77 ppm). Since the most significant differences between 7 and 8 were on the side chain moiety, it was established that they differed via isomerism at C13. A key diagnostic chemical shift at C14 can be used to distinguish between the isomers, with δ H 6.30 indicating the E-isomer and δ H 6.77 ppm for the Z-isomer [38]. In the current study, the H14 in 7 was observed at δ 6.33 ppm, and, on 8, H14 was observed at δ 6.76 ppm ( Table 4). The 13 C shifts of C14 are also diagnostic with δ C 141.8 ppm seen for the E-isomer and δ C 133.9 ppm for the Z-isomer [38], which were identical to the 13 C chemical shifts for C14 on 7 and 8, respectively. Thus, 7 was assigned as 8R-17-epoxy-E-communic acid and 8 as 8R-17-epoxy-Z-communic acid.
A detailed summary of 13 C NMR shifts for derivatives of labdane C4 acid esters are given by Barrero and Altarejos [33] and labdane C4 dimethyl derivatives by Bastard et al. [32]. However, a more comprehensive comparison to data in the current manuscript is provided in the Supplementary Files.

Antimicrobial and Acaricidal Activity
Regrettably, due to the low yield and instability of the spiroexpoxy communic acid derivatives (4)(5)(6)(7)(8), acaricidal activities were not measured for these compounds. However, the major diterpenes of Widdringtonia displayed very modest acaricidal activity using ticks as our model organism (Table 7, IC50 = 84-482 µg/mL). Although LC 50 values within the range of 90-500 µg/mL are included in Table 7, only values lower than 25 µg/mL were considered interesting. In this regard, values for pisiferic acid were only moderate, but the treatments with noteworthy activity included guaiol (12) (6.9-15.1 µg/mL) and the sesquiterpene rich essential oil from Widdringtonia timber (15.5-39.8 µg/mL). Table 7. Acaricidal activity against tick species for compounds. Sandaracopimaric acid (1), Z-communic acid (2), pisiferal (9), pisiferic acid (10), guaiol (12), and essential oil from W. nodiflora timber (EO-W.n). LC 99 values were extrapolated using probit analysis. Guaiol is one of the predominating sesquiterpenes in the timber of all Callitris and may be considered important in the termite resistance [39], but this is the first account of activity against Acari. Since this outcome was also evident using essential oils from timbers of Widdringtonia, it may be feasible to correlate termite resistant timbers to resistance against Acari in general. This generates more questions about possible insect repellent activity and may translate to the ethnopharmacological context where topical applications may have alleviated ectoparasitic problems.
The antimicrobial activity of guaiol is moderate with values as low as 120-250 µg/mL against Gram-positive bacteria. The diterpenes 1 and 2 were not active at the starting concentrations used, but the spiroepoxy diterpenes were moderate to interesting, in particular 4 and 5 with MIC values ranging from 43-400 µg/mL against Gram-positive strains and 160 µg/mL against P. aeruginosa (Table 6).
Moderate MIC values for spathulenol and guaiol were demonstrated, which ranged from 100-250 µg/mL against Gram-positives and 300 µg/mL against both Gram-negative organisms. All of these compounds are of high relative abundance in the cones of Widdringtonia with the yield of spathulenol at 1% by mass (Table 2). Thus, these specialised metabolites evidently provide the pharmacological basis of the traditional use of the clear, hard gum in applications consistent with antimicrobial or acaricidal activity.
The activity of 10 (pisiferic acid) was reiterated here, with results not unlike those reported previously [40], by inhibiting Gram-positive bacteria at concentrations as low as 50 µg/mL, but not Gram-negative bacteria at the concentrations tested (Table 6). In the current study, 10 was then screened against an MRSA strain and it was clear that the activity was consistently 50 µg/mL.

General Discussion
It is strange that both Callitris and Widdringtonia had not, until recently, had diterpenes reported, despite evident widespread occurrence and high relative abundance in leaves. This may be explained by the general trend in previous studies of the two genera to focus almost exclusively on solvent extractables from heartwood timber or volatiles from timber and leaves. In the current study, it was demonstrated that these diterpenes are not restricted to leaves, as they are also present in twigs and timber from young thin branches. Future research may demonstrate much higher yields from the cones, which has been tentatively observed in the current study.
The pronounced chemical difference between heartwood and aerial parts of the species may have ecological connotations for the genera, particularly since part of the termite and fungal resistance of the heartwood timbers. In the current study, termiticidal activities were not measured and acaricidal assays were conducted merely to address questions of a commercial interest. Nevertheless, the broad-spectrum activity of guaiol (from heartwood of Callitris) and widdrol/cedrol (from timbers of Widdringtonia) gives impetus to further investigate in this regard. Watanabe et al. [39] demonstrated that most of the essential oil components in timbers of Callitris repelled termites, which included components in the costol group, such as costic acid, eudesmols, collumellarin, and guaiol [39]. No similar studies have been conducted on essential oils from Widdringtonia.

Ticks Collection
Tick specimens were collected from the Indian states. Fully engorged females of Rhipicephalus sanguineus sensu lato, Rhipicephalus (Boophilus) microplus, and Haemaphysalis bispinosa were collected from dogs, cattle, and goats, respectively, from Assam state, North-east India. Rhipicephalus (Boophilus) annulatus and Hyalomma dromedarii fully engorged females were collected from Coimbatore, Tamil Nadu state and Ludhiana, Punjab state (South and North India, respectively). All specimens were taken back to the laboratory of Acarology, Department of Entomology, Assam Agricultural University, and then identified with the help of the taxonomical key previously described by Hoogstraal [41] and Geevarghese and Mishra [42]. Only females with healthy status and with no acaricide application history were selected to be used in the bioassay tests.

Adult Immersion Test
A total of 270 fully engorged females from each species (1650 individuals for all the used ticks) were washed twice with distilled water and dried on filter paper (Whatman, Kent, UK). Following the adult immersion tests (AIT) [43], preliminary concentrations of 950 and 5 µg/mL from each compound or mixture were prepared using methanol as a solvent and evaluated to determine the concentration sequence side-by-side with a control group using only methanol. Ten individuals of three replicates of each concentration were evaluated then left in large glass vials (Borosil, Mumbai, India) in a darkened incubator (Scigenics, Chennai, India). The mortality rates were observed after one week as the treatments require seven days [44]. A series of descending concentrations of 900, 800, 500, 300, 150, 50, and 10 µg/mL were then evaluated using the same method of the bioassay.
Probit analysis was then performed to determine the LC 50 , LC 99 , Slope, X 2 , and fiducial limit values using POLO-PC (LeOra, Berkeley, CA, USA) based on the mortality rates of the investigated ticks.

Botanical Material, Extraction, Isolation, and Nuclear Magnetic Resonance (NMR) Assignment
Leaves and thick branches were harvested from Widdringtonia specimens growing in Pretoria Botanic Gardens, South Africa or from private land in the Cape. Leaves from Callitris were harvested from remote locations in New South Wales, Australia. Voucher specimens were lodged either in the University of Johannesburg Herbarium (JRAU) or the NE Beadle Herbarium at the University of New England, Armidale, NSW, Australia.
Pulverised leaves or branches were extracted in dichloromethane. Compounds were isolated in flash chromatography over silica gel 60 (Merck) using 10%-20% ethyl acetate made up with pet ether. Compounds 1 and 2 eluted with 15% EtOAc in pet ether while all others are eluted at 20% EtOAc.
1D and 2D NMR spectra were generated on a Bruker Avance 500 MHz spectrometer using standard pulse sequences. Sandaracopimaric acid (1) spectra were matched to those provided in earlier studies of diterpenes from Juniperus [45,46]. The isomers of communic acid (Z-and E-) (2, 3) were matched to spectra by Fang et al. [47] and Olate et al. [38]. Spectra of the abietanes pisiferol/pisiferal (9) and pisiferic acid (10) were matched to the spectra by Pati and Mukherjee [48] and Pal et al. [49], respectively. 1 H spectra for isoozic acid (11) was matched to what was provided by Martins et al. [50] and 13 C assignments are provided here for the first time. The spectra of guaiol (12) was matched to spectra by Raharivelomanana et al. [51].

Determination of Minimum Inhibitory Concentrations (MIC)
The minimum inhibitory concentration (MIC) method described by Eloff [52], which is the same as that used by Clinical Laboratory Standards Institute [53], was used to determine the susceptibility of test pathogens to compounds. The organisms were Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 29213 & a methicillin resistant strain), Pseudomonas aeruginosa (ATCC 27703), Bacillus subtilis (University of New England strain), and Escherichia coli (ATCC 25922). The positive control used was tetracycline.

GC-MS, HRESIMS
Relative abundances of essential oil components and esterified diterpene acids were studied using gas chromatography with mass spectrometric detection (GC-MS). GC-MS analyses were performed using an Agilent Technologies 7890A GC-System coupled with an Agilent 5975C mass selective detector (triple-Axis detector, Agilent Technologies, Wilmington, DE, USA). An autosampler unit (Agilent Technologies 7693-100 positions) held samples. Separation of 1-µL injections used an HP-5MS Agilent column (30 m × 250 µm × 0.25 µm). Operating conditions were as follows: injector split ratio 25:1, temperature 250 • C, carrier gas helium, 1.0 mL/min, and constant flow. Column temperature was 50 • C (no hold) and 5 • C per minute. Then, at 280 • C, it was held at 5 min. Mass fragmentation patterns were acquired at −70 eV using a mass scan range of m/z 30-400.
Primary identifications were performed by comparison of mass spectra with an electronic library database [54] and confirmed using arithmetic indices, calculated relative to n-alkanes, when compared with values published in Adams [55] by visual comparison against mass spectral images [55]. Semi-quantification was achieved by the GC-MS operating software, using data with a minimum peak area of 0.1%, by calculating the area under the curve.
HRESIMS spectra were recorded using an AB Sciex 5600 TripleTOF mass spectrometer in positive mode.