The Australian Rainforest Rosewood: From Wood Characteristics to Chemical Profile and Biological Activity of Its Essential Oil

Abstract

Dysoxylum fraserianum (A.Juss.) Benth. (Meliaceae), commonly known as Australian rosewood, is a rare species endemic to the subtropical rainforests of New South Wales, whose hardwood is used for essential oil (EO) extraction. Despite its commercial relevance, an updated pharmacognostic characterisation of this species is lacking. This study aimed to provide an integrated analysis of the micromorphological, chemical, and biological features of D. fraserianum hardwood and its EO. Anatomical investigations revealed diffuse-porous wood and prismatic calcium oxalate crystals, while histochemical tests showed vessel occlusions with polysaccharide-rich gums, and confirmed the presence of lipophilic extractives within banded axial parenchyma cells. GC–MS and GC-FID analyses identified 52 sesquiterpenes, with ledene (12.74%), β-caryophyllene (8.43%), and δ-cadinene (7.18%) as major constituents, reflecting the chemotaxonomic traits of the Meliaceae family. The EO exhibited concentration-dependent antioxidant and anti-inflammatory activities in multiple in vitro assays and showed moderate antibacterial activity with a bacteriostatic effect against Gram-positive and Gram-negative strains. No fungicidal activity was detected against Candida albicans. These findings confirm the phytochemical uniqueness of D. fraserianum EO and support its biological relevance, offering a modern scientific basis for its potential use in pharmacological and industrial applications.

1. Introduction

Dysoxylum fraserianum (A.Juss.) Benth. (syn. Didymocheton fraserianus (A.Juss.) Mabb. & Hauenschild) is a long-lived tree of the family Meliaceae native to the subtropical dry rainforests of New South Wales, Australia. Its common name, “Australian Rosewood”, derives from the pleasant rose-like fragrance released by its freshly cut bark. D. fraserianum naturally occurs in the subtropical rainforests of New South Wales and Queensland and is currently listed as “Least Concern” on the IUCN Red List [1]. Although not classified as endangered, the species is subject to conservation and habitat management measures under regional legislation, including the Queensland Government Nature Conservation Act 1992 [2]. Consequently, Australian rosewood is used for industrial purposes, including timber for furniture manufacturing and essential oil (EO) production for perfumery, aromatherapy, and cosmetic applications, relying exclusively on naturally fallen trunks rather than harvesting living trees. From these trees, after removal of the sapwood (cream/yellow colour), only the hardwood—characterized by a more intense reddish colour—is used for steam distillation, yielding an EO that is typically viscous and exhibits a distinctive deep blue colour.

Although several anatomical studies have investigated the wood of different members of the Meliaceae family [3,4], detailed and updated anatomical information specifically referring to D. fraserianum remains very limited, with only an early report describing some structural features of this species [5]. As a result, a comprehensive micromorphological and anatomical characterization of D. fraserianum hardwood, particularly in relation to secretory tissues potentially involved in EO accumulation, is still lacking. Similarly, numerous species of the genus Dysoxylum have been extensively investigated from a phytochemical perspective, with EOs isolated from both wood and leaves being characterized in terms of their chemical composition and, in some cases, biological properties [6,7,8,9]. However, despite this growing body of literature, data specifically addressing EOs obtained from D. fraserianum are scarce and largely outdated, being mainly limited to early studies conducted several decades ago [10,11]. A modern reassessment of the phytochemical profile of D. fraserianum EO using contemporary analytical approaches is therefore still missing.

Terpene accumulation in hardwood is generally regarded as part of the chemical defense strategy of living trees against pathogens and herbivores and represents a key factor contributing to wood durability [12]. In addition, hardwood-derived terpenes are highly valued by the fragrance industry, as exemplified by santalols obtained from sandalwood [13]. It is widely recognized that secondary metabolism associated with hardwood formation involves different types of parenchyma cells and follows species-specific mechanisms, leading to the accumulation of characteristic metabolites [12]. In this context, studies on related Dysoxylum species have shown that hardwood EOs are often dominated by sesquiterpenes, such as α-muurolene, τ-muurolol, δ-cadinene, and cedrene, as reported for D. malabaricum Bedd. ex Hiern [6].

Considering that D. fraserianum remains poorly investigated from a pharmacognostic point of view, the present study was designed to provide an integrated characterization of this species by combining anatomical, phytochemical, and biological approaches. In particular, the micromorphological and anatomical features of the hardwood were investigated, with special attention to structures potentially involved in EO secretion and storage. The chemical profile of D. fraserianum EO was characterized using gas chromatographic techniques, and its biological potential was explored through the evaluation of antioxidant, anti-inflammatory, and antimicrobial activities using a panel of complementary in vitro assays.

2. Results

2.1. Anatomical and Micromorphological Studies

Hand-made transversal sections observed by light microscopy revealed a diffuse-porous wood with solitary or multiple pores, from two to four (Figure 1a,b). The presence of lipophilic substances within the cells of the banded axial parenchyma was evidenced by dark red colour with Sudan III (Figure 1a,b) and Sudan Black (Figure 1c), whereas Dragendorff reagent produced a brownish staining reaction in the same tissue (Figure 1d).

Inside the vessels, the presence of deposits (Figure 2a–c) was detected by both Light and Scanning Electron Microscopy (LM and SEM, respectively). Water mounted cross sections showed the presence of dark brown organic deposits, tentatively described as gums (Figure 2a), which turned to a pinkish-purple colour after staining with Toluidine Blue O (TBO), revealing their polysaccharide nature (Figure 2b). Analysis by SEM confirmed the presence of these deposits as electron-opaque masses (Figure 2c, white arrow).

In longitudinal sections wood was mainly characterized by numerous single prismatic crystals, which were detected in chambered axial parenchyma cells by light microscopy under polarized light (Figure 3a, red arrow). Scanning Electron Microscopy confirmed the presence and distribution of crystals and also showed vessels with simple perforations (Figure 3b white and red arrows, respectively). Moreover, SEM coupled with energy dispersive spectroscopy (SEM-EDS) allowed their classification as calcium oxalate crystals, as shown by the largest peak of calcium in the EDS spectrum (Figure 3c).

2.2. Chemical Composition of Essential Oil

The chemical composition of D. fraserianum essential oil (DFEO) is reported in

Table 1. Chemical composition of the essential oil of D. fraserianum.

N.	Compound	%	KI (A)	KI (B)
1	δ-EIemene	7.22	1235	1469
2	Silphinene	0.15	1259	1474
3	α-Ylangene	0.29	1265	1484
4	Isoledene	0.68	1266
5	α-Copaene	0.50	1269	1491
6	β-Patchoulene	0.23	1284
7	β-Elemene	0.63	1286	1591
8	α-Gurjunene	0.87	1294	1529
9	4,8-β-Epoxycaryophyllene	0.13	1300	1955
10	β-Caryophyllene	8.43	1305	1598
11	β-Gurjunene	0.50	1312	1597
12	α-Maaliene	0.26	1318
13	Aromandendrene	1.74	1323	1620
14	1,1,4,7-Tetramethyl-1a,2,3,4,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene	1.00	1325
15	Selina-5,11-diene	0.42	1327	1632
16	Guaia-6,9-diene	0.34	1329	1630
17	Isogermacrene D	1.87	1334	1665
18	α-Humulene	1.09	1338	1667
19	allo-Aromadendrene	0.40	1341	1649
20	9-epi-(E)-Caryophyllene	1.00	1344	1593
21	α-Patchoulene	1.09	1347	1658
22	γ-Gurjunene	0.37	1350	1668
23	Cadina-1(6),4-diene	0.74	1359	1659
24	γ-Muurolene	3.88	1363	1690
25	α-Amorphene	3.60	1367	1693
26	Ledene	12.74	1382	1698
27	α-Muurolene	3.57	1387	1723
28	δ-Guaiene	1.37	1391
29	γ-Cadinene	2.61	1399	1763
30	cis-Calamenene	0.42	1401	1834
31	δ-Cadinene	7.18	1406	1756
32	α-Cadinene	0.67	1417	1769
33	Selina-3,7(11)-diene	0.44	1419	1783
34	α-Calacorene	1.30	1421	1921
35	Epiglobulol	0.17	1435	2025
36	Globulol	3.16	1443	2082
37	Caryophyllenyl alcohol	1.33	1445
38	Viridiflorol	1.07	1467	2090
39	Cubeban-11-ol	3.07	1470
40	Rosifoliol	2.46	1478	2144
41	Junenol	2.45	1491
42	β-Eudesmol	2.71	1498	2238
43	Selin-6-en-4α-ol	1.29	1494
44	Cubenol	0.73	1496	2068
45	γ-Eudesmol	0.82	1498	2176
46	τ-Cadinol	4.72	1512	2151
47	δ-Cadinol	1.69	1516
48	α-Cadinol	4.88	1525	2227
49	epi-γ-Eudesmol	0.24	1532	2106
50	1,2,3,5,6,7,8,8a-Octahydro-α,α,4,7-tetramethyl-1-Naphthalenemethanol	0.63	1542
51	Eudesm-7(11)-en-4-ol	0.18	1561	2302
52	Guaiol acetate	0.15	1598
	Total	99.48
	Sesquiterpene hydrocarbons	67.18
	Oxygenated sesquiterpenes	32.30

(A, B) The Kovats retention indices are relative to a series of n-alkanes (C10–C35) on the apolar HP-5MS and the polar HP-Innowax capillary columns, respectively. The identification of all components was performed by comparing the Kovats retention indices with published data and by comparing mass spectra with those listed in the NIST 17 and Wiley 275 libraries and with published data.

. A total of 52 constituents were identified, accounting for 99.48% of the total EO composition. The volatile profile was exclusively characterized by sesquiterpenes, which were further distributed into sesquiterpene hydrocarbons (67.18%) and oxygenated sesquiterpenes (32.30%).

Among the identified compounds, ledene was the most abundant constituent (12.74%), followed by β-caryophyllene (8.43%), δ-elemene (7.22%), and δ-cadinene (7.18%). Several other sesquiterpenes were detected in appreciable amounts, including α-amorphene (3.60%), γ-muurolene (3.88%), α-muurolene (3.57%), globulol (3.16%), cubeban-11-ol (3.07%), τ-cadinol (4.72%), and α-cadinol (4.88%).

The characteristic deep blue colour of DFEO can be related to the presence of sesquiterpenes with an azulene-type skeleton. In particular, the identification of 1,1,4,7-tetramethyl-1a,2,3,4,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene (1.00%), together with a high abundance of cadinene-type sesquiterpenes, is consistent with the occurrence of blue-coloured EOs. Azulene derivatives, despite being present in relatively low amounts, are known to exhibit high chromophoric efficiency and to strongly influence oil colour. This observation is consistent with earlier reports linking azulene-type compounds to the blue coloration of certain EOs [14]. The remaining constituents were present in lower relative abundances, collectively contributing to the complexity of the sesquiterpene profile. Overall, DFEO showed a chemically homogeneous composition dominated by structurally related sesquiterpene hydrocarbons and their corresponding oxygenated derivatives, as detailed in Table 1.

2.3. Biological Activities

2.3.1. Antioxidant and Anti-Inflammatory Activities

The antioxidant and anti-inflammatory activities of DFEO were evaluated using a battery of complementary in vitro assays, and the corresponding IC₅₀ values are reported in

Table 2. Antioxidant and anti-inflammatory activities of D. fraserianum essential oil (DFEO) in comparison with reference standards. Results represent the mean of three independent experiments performed in triplicate (n = 3) and are expressed as IC₅₀ values, defined as the concentration required to inhibit 50% of the oxidant or inflammatory activity, with 95% confidence limits reported in parentheses.

Test	DFEO	RS a
	mg/mL	µg/mL
Trolox equivalent antioxidant capacity (TEAC)	1.80 (1.45–2.24)	3.78 (1.48–9.67) ***
Ferric reducing antioxidant power (FRAP)	0.73 (0.61–0.87)	3.72 (1.60–8.66) ***
Oxygen radical absorbance capacity (ORAC)	0.21 (0.13–0.36)	0.68 (0.22–82.17) ***
β-carotene bleaching (BCB)	0.61 (0.46–0.83)	0.35 (0.17–80.58) ***
Iron-chelating activity (ICA)	0.82 (0.69–0.97)	5.72 (2.32–87.13) ***
BSA denaturation assay (ADA)	2.88 (2.45–3.39)	17.05 (13.94–20.85) ***
Protease inhibitory activity (PIA)	4.64 (1.32–8.92)	28.50 (13.31–861.04) ***

^a RS, Reference standard: trolox for FRAP, TEAC and ORAC assay; BHT for β-carotene bleaching assay; EDTA for iron-chelating activity; diclofenac sodium for anti-inflammatory assays (ADA and PIA); *** p <0.001 vs. DFEO.

. In all tests, DFEO showed a clear concentration-dependent inhibitory effect, as supported by the concentration–response curves used for IC₅₀ calculation (R² ≥ 0.95).

With respect to antioxidant activity, DFEO displayed consistent inhibitory capacity across assays based on different reaction mechanisms, including electron transfer, radical scavenging, lipid peroxidation inhibition, and metal chelation. The relative performance of DFEO varied depending on the assay conditions, with particularly low IC₅₀ values observed in radical scavenging-based methods, such as the oxygen radical absorbance capacity, and the iron-chelating assay, whereas higher concentrations were required in assays conducted in more complex or lipid-rich systems, such as β-carotene bleaching.

In all assays, the reference standards showed lower IC₅₀ values expressed in the µg/mL range, as expected for pure compounds, whereas DFEO exhibited inhibitory activity in the mg/mL range. The differences between DFEO and the corresponding reference standards were statistically significant (p < 0.001; Table 2).

Regarding anti-inflammatory activity, DFEO was able to inhibit both protein denaturation and protease activity in a concentration-dependent manner. The IC₅₀ values obtained in the albumin denaturation and protease inhibition assays confirmed the capacity of DFEO to interfere with key in vitro inflammatory processes, with activity trends consistent across the two experimental models.

Overall, the biological assays highlighted a coherent antioxidant and anti-inflammatory profile for DFEO, characterized by concentration-dependent responses and assay-specific differences in inhibitory potency, as summarized in Table 2.

2.3.2. Antimicrobial Activity

The antimicrobial activity of DFEO was evaluated against selected Gram-positive and Gram-negative bacterial strains, as well as against the yeast Candida albicans. The results, expressed as minimum inhibitory concentration (MIC) for bacteria and minimum fungicidal concentration (MFC) for yeast, are summarized in

Table 3. Antimicrobial activity of D. fraserianum essential oil (DFEO) against reference microbial strains. Results are expressed as minimum inhibitory concentration (MIC) for bacterial strains and minimum fungicidal concentration (MFC) for Candida albicans, reported in µg/mL. Reference compounds were included as positive controls. Data represents the mean of three independent experiments performed in triplicate (n = 3). *** p < 0.001 vs. DFEO.

Strain	DFEO	Reference Compound
	MIC (µg/mL)
Gram-negative		Tobramycin
Pseudomonas aeruginosa ATCC 9027	500 ***	0.22 ± 0.01
Escherichia coli ATCC 10536	500 ***	0.48 ± 0.02
Gram-positive		Vancomicin
Staphylococcus aureus ATCC 6538	250 ***	0.28 ± 0.01
	MFC (μg/mL)
Yeast		Caspofungin
Candida albicans ATCC 10231	>2000 ***	0.061 ± 0.00

*** p < 0.001 vs. DFEO.

.

DFEO exhibited inhibitory activity against all the bacterial strains tested. Comparable MIC values were observed for the two Gram-negative bacteria, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 10536, both inhibited at 500 µg/mL. A lower MIC value was recorded for the Gram-positive strain Staphylococcus aureus ATCC 6538, which was inhibited at 250 µg/mL.

In contrast, DFEO did not exhibit fungicidal activity against Candida albicans ATCC 10231 at concentrations up to 2000 µg/mL, corresponding to the highest concentration tested under the experimental conditions employed.

For all bacterial strains showing growth inhibition, additional assays were performed to determine the nature of the antimicrobial effect, revealing a bacteriostatic rather than bactericidal activity.

The reference antimicrobial agents included in the assays exhibited inhibitory effects at substantially lower concentrations (Table 3), as expected for pure compounds. All differences between DFEO and the corresponding reference standards were statistically significant (p < 0.001).

Overall, DFEO demonstrated moderate antibacterial activity, with a higher level of effectiveness against the Gram-positive strain; notably, inhibitory activity was also observed against the Gram-negative bacteria tested, whereas only weak activity was detected against the yeast strain.

3. Discussion

To the best of our knowledge, the present study represents the first comprehensive and updated pharmacognostic characterization of D. fraserianum wood, including its micromorphological and anatomical features and phytochemistry of the hardwood-derived EO, laying the basis for the evaluation of its biological properties. The hardwood of D. fraserianum was characterized by a diffuse-porous wood, showing dark brown deposits occluding vessels, as previously reported by Welch [5] and similarly to those observed in another Meliaceae species, Dysoxyium spectabile (G.Forst.) Kook.f., by Patel [15]. These colored inclusions are widespread in Meliaceae and are generally described as gums, mainly composed of polysaccharides and pectins [16]. The deposition of gums inside vessels is a natural phenomenon occurring during aging and hardwood formation and plays a key role in limiting the spread of pathogens and organisms responsible for wood deterioration. Gums occluding vessels can be produced by the secretory activity of surrounding living cells, mainly ray parenchyma cells, and deposited directly into the adjacent vessel lumen [17].

Furthermore, in agreement with previous findings for most Meliaceae genera [18,19] and for other Dysoxylum species [15], abundant prismatic calcium oxalate crystals were observed in the chambered axial parenchyma cells. The presence of calcium oxalate crystals, often related to a mechanism of chemical defense [20], is considered a diagnostic feature of Meliaceae wood due to their characteristic appearance, location, and crystal type [18].

As already reported for most members of the Meliaceae family [15], the hardwood of D. fraserianum showed evident axial banded parenchyma. Although no specialized secretory tissues or ducts were detected, the micromorphological analyses highlighted the presence of intracellular oil bodies containing lipophilic substances within the banded parenchyma cells. These observations are consistent with early reports by Welch [5], who described small resinous or oily globules within parenchyma cells of D. fraserianum hardwood, suggesting that extractive accumulation rather than specialized secretory structures characterizes this species.

Histochemical staining further supported the presence of lipophilic materials in axial parenchyma cells, as demonstrated by the positive reactions obtained with Sudan III and Sudan Black [21]. Conversely, the brownish staining observed following treatment with Dragendorff’s reagent should not be interpreted as evidence of alkaloid occurrence, as no chemical data support the presence of nitrogen-containing alkaloids in D. fraserianum hardwood or essential oil. It has been clearly documented that Dragendorff’s reagent may yield non-specific reactions due to interactions between iodine-based complexes and lipophilic or resinous compounds, particularly in tissues rich in terpenoids and other extractives [21,22]. Accordingly, the Dragendorff reaction observed in this study is more plausibly attributable to non-specific interactions with lipophilic constituents rather than to true alkaloid localization and was therefore interpreted with caution [22].

Previous studies on gymnosperm hardwood have suggested a role of ray parenchyma cells in the production and accumulation of extractives [23,24,25], although the precise chemical nature of these substances could not always be confirmed by histochemical or microspectrometric approaches [25]. In angiosperms, Celedon and Bohlmann [12] demonstrated the involvement of living parenchyma cells in the hardwood of Santalum album L. (Santalaceae) in terpenoid biosynthesis, suggesting that parenchyma cells may contribute to the accumulation of secondary metabolites responsible for hardwood properties such as colour, decay resistance, and durability.

In this context, the intracellular localization of lipophilic materials observed in D. fraserianum axial parenchyma cells is consistent with a potential role of these cells in the accumulation and storage of hardwood extractives, rather than providing direct evidence of essential oil biosynthesis.

To the best of our knowledge, no recent studies have described the chemical composition of the EO of D. fraserianum. The limited information available originates from two early historical reports that addressed, albeit partially and using methodologies far from current analytical standards, the chemical composition of this species. Penfold [10] was among the first to describe distillation products of D. fraserianum wood, reporting the presence of azulenoidal compounds, cadinene-type sesquiterpenes, and a sesquiterpene referred to as “dysoxylonene”. Subsequently, Connell et al. [11] contributed to the clarification of the identity of some constituents isolated from D. fraserianum, confirming the occurrence of δ-cadinene. The presence of cadinene derivatives reported in these early studies agrees with the sesquiterpene profile observed in the present work, thus providing continuity between historical observations and modern analytical data.

Although recent literature does not provide updated information specifically for D. fraserianum, several studies on other Dysoxylum species offer valuable elements for comparison. In all cases, sesquiterpenes represent the dominant class of compounds, supporting a common phytochemical pattern within the genus. For instance, the EO of D. densiflorum Miq. was reported to be rich in sesquiterpenes, including oxygenated derivatives such as 5-hydroxy-cis-calamenene, a compound also detected in the present sample [26]. Similarly, the EO of D. cauliflorum (Turcz.) G.Perkins showed a heterogeneous composition dominated by sesquiterpenes, although accompanied by a higher proportion of monoterpenes [7]. Several sesquiterpenes identified in that study, including α-cadinol, α-copaene, α-humulene, α-muurolene, aromadendrene, cadine-1,4-diene, caryophyllene oxide, cis-calamenene, δ-cadinene, δ-elemene, globulol, β-caryophyllene, τ-cadinol, and viridiflorol, were also detected in the EO analyzed in the present study.

Comparable trends were reported for D. malabaricum, whose essential oil contained sesquiterpenes such as α-muurolene, δ-cadinene, α-copaene, α-amorphene, and τ-cadinol, together with additional non-terpenoid volatile compounds [6]. The EO of D. binectariferum (Roxb.) Hook.f. ex Bedd. was characterized by caryophyllene, α-guaiene, δ-cadinene, α-copanene and β-elemene [27], several of which are shared with the present sample.

Finally, the EO obtained from fruits of D. richii (A.Gray) C.DC. was dominated by sesquiterpene hydrocarbons and oxygenated sesquiterpenes, with δ-cadinene, germacrene D, and cadinol isomers as major components [28]. Notably, δ-cadinene and cadinol isomers (α-, δ-, and τ-cadinol) were consistently detected in the essential oil of D. fraserianum analyzed in this study.

Overall, despite the limited specific literature on D. fraserianum, the phytochemical profile of its EO appears fully consistent with the chemotaxonomic characteristics of the Meliaceae family, being dominated by sesquiterpene compounds, with a marked prevalence of cadinane, muurolane, caryophyllane, and aromadendrane skeletons [29]. The occurrence of major constituents such as ledene, β-caryophyllene, δ-elemene, δ-cadinene, α-cadinol, τ-cadinol, γ-muurolene, α-muurolene, and globulol further supports a clear chemotaxonomic affinity with other members of the genus Dysoxylum and the Meliaceae family.

The biological activities observed for DFEO can be discussed in relation to its sesquiterpene-dominated chemical profile and the experimental models adopted. Overall, DFEO exhibited consistent antioxidant and anti-inflammatory activities across multiple in vitro assays, together with a moderate antibacterial activity and a bacteriostatic mode of action. This combination of effects is frequently reported for essential oils derived from woody matrices and hardwood tissues, where secondary metabolites are thought to exert protective ecological functions [30,31].

The antioxidant activity of DFEO was demonstrated using a battery of complementary assays addressing different oxidative mechanisms, including radical scavenging, metal chelation, and inhibition of lipid peroxidation. The clear concentration-dependent behaviour observed across all assays supports the reliability of the calculated IC₅₀ values and indicates that the effects are not assay-specific artefacts but reflect a genuine antioxidant potential. Similar multi-assay approaches are recommended for the evaluation of essential oils due to their complex, multicomponent nature [30,32]. Although essential oils are generally considered weaker antioxidants than phenolic-rich extracts, sesquiterpene-rich oils have been reported to exert measurable antioxidant effects, particularly in radical scavenging and peroxyl radical-based systems such as ORAC assays [31,33]. In this context, the activity of DFEO is consistent with literature data describing the contribution of oxygenated sesquiterpenes, including cadinol isomers, globulol, and related alcohols, to antioxidant mechanisms involving hydrogen atom transfer and radical quenching [31,33]. The iron-chelating activity detected for DFEO further suggests an additional antioxidant pathway, potentially relevant in limiting metal-catalyzed oxidative reactions, as previously reported for several essential oils and terpene-rich phytocomplexes [30,34].

The anti-inflammatory activity of DFEO, evidenced by its ability to inhibit protein denaturation and protease activity in vitro, agrees with previous studies employing similar experimental models as preliminary indicators of anti-inflammatory potential [31]. These assays are widely used to assess the capacity of natural products to interfere with processes associated with inflammatory responses, such as protein destabilization and enzymatic degradation. The concentration-dependent inhibition observed for DFEO is consistent with reports describing anti-inflammatory properties of sesquiterpene-containing essential oils and isolated sesquiterpenes [35,36]. β-Caryophyllene has been extensively investigated for its anti-inflammatory effects through modulation of inflammatory mediators and signalling pathways, while cadinol derivatives and muurolane-type sesquiterpenes have also been reported to contribute to anti-inflammatory activity in different experimental systems [35,36]. Taken together, these observations support the hypothesis that the anti-inflammatory activity of DFEO arises from the combined contribution of multiple constituents rather than from a single dominant compound, in line with the well-documented additive or synergistic behaviour of EOs [30].

The antimicrobial assays further showed that DFEO exerts a moderate antibacterial activity against both Gram-positive and Gram-negative bacteria, while no fungicidal activity was observed against Candida albicans at concentrations up to the highest tested. Although DFEO was more effective against the Gram-positive strain, the observation of inhibitory effects against Gram-negative bacteria is noteworthy, as these microorganisms are generally less susceptible to essential oils due to the presence of an outer membrane acting as a permeability barrier [37,38]. The bacteriostatic, rather than bactericidal, mode of action observed for DFEO is consistent with previous reports on sesquiterpene-rich essential oils and supports a mechanism based on reversible interference with bacterial growth rather than irreversible cell damage [37,39]. Sesquiterpenes are known to interact with bacterial membranes, altering membrane fluidity and permeability and interfering with essential cellular processes without necessarily inducing rapid cell lysis, a behaviour typically associated with growth inhibition [38,39]. The lack of fungicidal activity against C. albicans further highlights the selectivity of DFEO and may be related to differences in cell wall and membrane composition between bacteria and yeasts, a trend already described for other sesquiterpene-dominated EOs [38].

From a broader perspective, the biological profile of DFEO appears coherent with its origin from hardwood tissues, where secondary metabolites are thought to play a protective role against oxidative stress, inflammation, and microbial colonization [12]. The combined antioxidant, anti-inflammatory, and antibacterial activities observed in this study therefore reflect the functional significance of sesquiterpene accumulation in hardwood and support the biological relevance of DFEO as a complex phytochemical mixture.

4. Materials and Methods

4.1. Chemicals

Sudan Black were purchased from BioGnost Ltd. (Zagreb, Croatia, EU) whereas Finefix working solution was obtained from Milestone s.r.l. (Bergamo, Italy). Sudan III, Dragendorff reagent, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2-2′-azino-bis-(3-ethyl-benzothiazolin-6-sulphonic acid) (ABTS), ammonium persulphate, potassium peroxydisulphate, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), 2-2-azobis (2-methylpropionamide) dihydrochloride (AAPH), 2,4,6-tris(2-pyridyl)- s-triazine (TPTZ), EDTA, iron(II) chloride (FeC_l2), ferrozine, β-carotene, butylated hydroxytoluene (BHT), Tween-40, linoleic acid, bovine serum albumin (BSA) without fatty acids, diclofenac sodium, Tris-HCl, casein, and trypsin type IX-S from porcine pancreas and all solvents and reagents used for GC-FID and GC-MS analyses were purchased from Merck KGaA (Darmstadt, Germany).

4.2. Plant Material and Essential Oil

From the trees growing in their natural growth habitat (Figure 4), only old, fallen trunks of D. fraserianum (A.Juss.) Benth. are used for EO extraction. The tree used in the present work was collected in May 2023, near Byron Bay (NSW, Australia) by one of the authors, Greg Trevena. The species was identified by Prof. Laura Cornara and a voucher specimen, labelled GDOR n. 64918, was deposited in the herbarium of the Natural History Museum Giacomo Doria of Genova (Italy). The timber was cut into planks, chipped into small pieces, and finally subjected to stem distillation. The distillation process was carried out at 45 °C for 8 h, giving rise to an EO with a characteristic blue colour and a subtle rose aroma.

4.3. Micromorphological and Anatomical Analyses

Dry wood samples, measuring 4 × 0.8–1 cm, were softened by immersion in boiling water for about 30 min to 1 h and then hardwood hand-made cross and longitudinal sections were obtained by using a double-edged razor blade. Fresh sections were mounted on water or stained with the polychromatic dye Toluidine Blue (TBO) to obtain wood anatomical details [40,41]. In addition, histochemical characterization of the substances present in hardwood was performed using the following histochemical stains: Sudan III, Sudan Black and Dragendorff reagent [42,43]. Observations were made with a Leica DM 2000 transmission light microscope (Leica Microsystems, Wetzlar, Germany) equipped with a ToupCam Digital Camera, CMOS Sensor 3.1 MP resolution (ToupTek Photonics, Hangzhou, China). Furthermore, polarized light was used to detect the presence and distribution of crystals.

Small specimens of hardwood were also observed using Scanning Electron Microscopy (SEM). For this purpose, samples were fixed in 70% ethanol–FineFix working solution for 24 h at 4 °C, dehydrated through a series of increasing ethanol solutions (70, 80, 90 and 100%) for 1 h each [44] and critical-point dried in CO₂ (CPD, K850 2M Strumenti s.r.l., Rome, Italy). Finally, sections of the samples were mounted on aluminum stubs using glued carbon tabs, sputter-coated with 10 nm gold [45], and observed with a Vega3 Tescan LMU SEM (Tescan USA Inc., Cranberry Twp, PA, USA) operating at an accelerating voltage of 20 kV. By Energy Dispersive X-ray Spectroscopy (EDS) (Apollo, Tescan USA Inc., Cranberry Twp, PA, USA) coupled with SEM the elemental composition of crystals was obtained [46].

4.4. Gas Chromatography with Flame Ionization Detection (GC-FID) and Gas Chromatography–Mass Spectrometry (GC-MS) Analyses

The composition of EO was studied by GC-FID and GC-MS analysis. For GC-FID analysis, a Perkin-Elmer Sigma 115 gas chromatograph (Waltham, MA, USA) equipped with a nonpolar HP-5MS fused silica capillary column (30 m × 0.25 mm i.d.; 0.25 μm film thickness) was used. For GC-MS analysis, an Agilent 6850 Series II system (Agilent, Santa Clara, CA, USA) was used, equipped with an HP-5MS fused silica capillary column (Agilent, 30 m × 0.25 mm i.d.; 0.25 μm film thickness) and coupled to an Agilent 5973 mass selective detector (Agilent, Santa Clara, CA, USA). The mass spectrometer was operated with electron impact ionization at 70 eV and an ion multiplication voltage of 2000 V. Mass spectra were acquired over a mass range of 40–500 um at a rate of five scans per second. GC-FID and GC-MS analyses were performed under the same chromatographic conditions. The injector temperature was 250 °C; the FID detector temperature was 290 °C, whereas the MS quadrupole temperature was set at 150 °C. For the chromatographic run, an oven temperature program was set as follows: initial isothermal phase at 40 °C for 5 min, then increased at a rate of 2 °C/min to 270 °C, followed by an isothermal hold at 270 °C for 20 min. GC-FID and GC-MS analyses were also performed on an HP Innowax polar column (50 m × 0.20 mm i.d.; 0.25 μm film thickness) using the same conditions to support compound identification. Helium was used as the carrier gas in the analyses at a constant flow rate of 1.0 mL/min. Components were identified by comparing their Kovats indices (KI) to those in the literature [47,48,49,50] and by careful analysis of the mass spectra against those in the NIST 17 and Wiley 257 mass libraries [51]. Kovats indices were determined relative to a homologous series of n-alkanes (C10–C35), under the same operating conditions. Relative concentrations of the components were calculated by peak area normalization. Response factors were not considered.

4.5. Antioxidant and Anti-Inflammatory Assays

The antioxidant and anti-inflammatory potential of DFEO was investigated using a panel of cell-free in vitro assays, selected to cover different reaction mechanisms and physicochemical environments relevant to oxidative stress and inflammation. All assays were performed according to validated protocols commonly employed in phytochemical and natural product research, with minor experimental adjustments [52]. DFEO was initially dissolved as a concentrated stock solution in dimethyl sulfoxide (DMSO) and subsequently diluted with the appropriate assay media to obtain the working concentrations, with the final DMSO concentration maintained at 0.1% (v/v) in all experiments. Corresponding solvent blanks were prepared and included in each assay.

The results were expressed as half maximal inhibitory concentration (IC₅₀, µg/mL) values, calculated together with their 95% confidence limits (CL) using the Litchfield and Wilcoxon method implemented in PHARM/PCS software (version 4; Consulting, Wynnewood, PA, USA).

4.5.1. Trolox Equivalent Antioxidant Capacity (TEAC) Assay

The ABTS radical cation (ABTS•⁺) was produced by reacting ABTS (1.7 mM) with potassium persulfate (4.3 mM) in a 1:5 (v/v) ratio and allowing the mixture to stand in the dark at room temperature for 12 h. Prior to analysis, the solution was diluted to obtain an absorbance of 0.70 ± 0.02 at 734 nm. Aliquots (10 µL) of DFEO, tested at increasing concentrations (0.125–2.0 mg/mL), were added to 200 µL of the ABTS•⁺ working solution. After incubation at room temperature for 6 min, the reduction in absorbance at 734 nm was measured using a Multiskan™ GO UV–Vis microplate reader (Thermo Scientific, Waltham, MA, USA). Trolox was used as a reference antioxidant.

4.5.2. Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP reagent was freshly prepared by mixing 10 mM TPTZ in 40 mM HCl, 20 mM FeCl₃·6H₂O, and 300 mM acetate buffer (pH 3.6). DFEO samples (10 µL), tested at increasing concentrations (0.063–1.0 mg/mL), were mixed with 200 µL of the FRAP reagent pre-equilibrated at 37 °C. After incubation at room temperature in the dark for 4 min, the increase in absorbance at 593 nm was recorded using the same microplate reader described for the TEAC assay. Trolox was included as a reference compound.

4.5.3. Oxygen Radical Absorbance Capacity (ORAC) Assay

ORAC measurements were carried out using fluorescein (117 nM) as the fluorescent probe and AAPH (40 mM) as the peroxyl radical generator. Briefly, DFEO aliquots (20 µL), tested over a suitable concentration range (0.025–0.40 mg/mL), were mixed with 120 µL of fluorescein solution in a 96-well microplate and pre-incubated at 37 °C for 15 min. The reaction was initiated by adding 60 µL of freshly prepared AAPH solution. Fluorescence decay was monitored every 30 s for 90 min (excitation at 485 nm, emission at 520 nm) using a FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany). Trolox served as a reference standard.

4.5.4. β-Carotene Bleaching (BCB) Assay

The antioxidant activity of DFEO was further assessed using the β-carotene–linoleic acid model system. A β-carotene emulsion was prepared by dissolving β-carotene (1 mg/mL) in ethyl acetate, followed by the addition of linoleic acid and Tween-40. After solvent evaporation, the residue was emulsified with distilled water. DFEO, tested at increasing concentrations (0.05–0.80 mg/mL), was added to the emulsion and incubated at 50 °C for 2 h. Absorbance was measured at 470 nm at regular time intervals using the same microplate reader described for the TEAC assay. Butylated hydroxytoluene (BHT) was used as a positive control.

4.5.5. Iron Chelating Activity (ICA) Assay

The iron-chelating capacity of DFEO was evaluated by mixing FeCl₂·4H₂O (2 mM) with increasing concentrations of the essential oil (0.063–1.0 mg/mL). After incubation at room temperature for 5 min, ferrozine (5 mM) was added to initiate the formation of the Fe²⁺–ferrozine complex. Following a 10 min incubation, absorbance was measured at 562 nm using the same microplate reader described for the TEAC assay, and chelating activity was calculated relative to the control. EDTA was included as a reference chelating agent.

4.5.6. Albumin Denaturation Assay (ADA)

The anti-inflammatory potential of DFEO was assessed by evaluating its ability to inhibit bovine serum albumin (BSA) denaturation. DFEO samples, tested at increasing concentrations (0.25–4.0 mg/mL), were mixed with BSA solution in phosphate-buffered saline (PBS, pH 5.3). The mixtures were incubated at 70 °C for 30 min in a shaking water bath. Absorbance was recorded at 595 nm before and after incubation using the same microplate reader described for the TEAC assay. Diclofenac sodium was used as a reference anti-inflammatory drug.

4.5.7. Protease Inhibition Assay (PIA)

Protease inhibitory activity was evaluated using trypsin as model protease and casein as substrate. DFEO samples at increasing concentrations (0.50–8.0 mg/mL) were incubated with trypsin in Tris–HCl buffer (20 mM, pH 7.5) at 37 °C for 20 min. The reaction was stopped by the addition of 2 M perchloric acid, followed by centrifugation at 3500× g for 10 min. The absorbance of the supernatant was measured at 280 nm using a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). Diclofenac sodium was included as reference inhibitor.

4.6. Antimicrobial Testing Procedure

The antimicrobial activity of DFEO was evaluated against a panel of reference microbial strains representative of both Gram-positive and Gram-negative bacteria, as well as yeast. All strains were obtained from the in-house culture collection of the University of Messina (Messina, Italy). The tested microorganisms included Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027, and Candida albicans ATCC 10231. Bacterial strains were routinely cultured in Mueller–Hinton broth (MHB; Oxoid, CM0405) and incubated at 37 °C for 24 h, whereas C. albicans was grown in Sabouraud liquid medium (SLM; Oxoid, CM0147) at 30 °C for 48 h.

The minimum inhibitory concentration (MIC) of DFEO was determined using the broth microdilution method, in accordance with the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI) M100-S22 for bacteria [53] and M27-A3 for yeasts [54]. DFEO was tested against all microbial strains over the same concentration range (0.02–2.0 mg/mL), prepared by serial dilution in the appropriate culture medium.

Microbial growth was assessed after incubation, and the MIC was defined as the lowest concentration of DFEO able to completely inhibit visible growth.

For strains showing growth inhibition, additional experiments were performed to discriminate between bacteriostatic, bactericidal, and fungicidal effects, in accordance with established microbiological criteria.

4.7. Statistical Analysis

Data are reported as mean values ± standard deviation (SD) of three independent experiments, each measured in triplicate. Statistical evaluation was performed by one-way analysis of variance (ANOVA). Tukey’s post hoc test was applied. All statistical computations were carried out using SigmaPlot software (version 12.0). A p value lower than 0.05 was considered indicative of statistical significance.

5. Conclusions

This study provides the first comprehensive pharmacognostic characterisation of D. fraserianum, integrating micromorphological, chemical, and biological analyses.

The anatomical investigation revealed features typical of the wood of Meliaceae family, including banded axial parenchyma, vessel occlusions with polysaccharide-rich gums, and the abundant presence of prismatic calcium oxalate crystals. Histochemical staining indicated the presence of lipophilic materials within parenchyma cells, supporting a model of extractive accumulation without specialised secretory ducts.

The EO distilled from the hardwood was found to be exclusively composed of sesquiterpenes, with ledene, β-caryophyllene, and δ-cadinene as major constituents, aligning with the chemotaxonomic profiles of related Dysoxylum species. Biologically, the EO exhibited a coherent profile of antioxidant and anti-inflammatory activities across multiple in vitro assays, along with moderate antibacterial efficacy and a bacteriostatic mode of action.

Together, these findings not only confirm the chemotaxonomic placement of D. fraserianum within the Meliaceae family but also highlight the potential of its hardwood essential oil as a source of bioactive sesquiterpenes. Further studies are warranted to explore its ecological significance and potential applications in phytotherapy, cosmetics, and natural product-based antimicrobial strategies.