In Vitro and In Vivo Antimelanogenesis Effects of Leaf Essential Oil from Agathis dammara

Agathis species are widely distributed around Southeast Asia, Australasia, South Pacific islands, and etc. Traditionally, Agathis species have been used as the folk medicines, the common ethnopharmacological uses of Agathis genus are the treatments of headache and myalgia. This study aims to investigate the chemical composition of Agathis dammara (Lamb.) Rich. leaf essential oil and to explore its antimelanogenesis effect. The chemical constituents of leaf essential oil are analyzed using gas chromatography-mass spectrometry (GC-MS), the major constituents of leaf essential oil are sesquiterpenoids. The major constituents are δ-cadinene (16.12%), followed by γ-gurjunene (15.57%), 16-kaurene (12.43%), β-caryophyllene (8.58%), germacrene D (8.53%), and γ-cadinene (5.33%). As for the in vitro antityrosinase activity, leaf essential oil inhibit the tyrosinase activity of mushroom when the substrate is 3,4-dihydroxyphenylalanine (L-DOPA). Leaf essential oil prevents tyrosinase from acting as diphenolase and catalyzing L-DOPA to dopaquinone, and converting into dark melanin pigments. A. dammara leaf essential oil also exhibits the in vivo antimelanogenesis effect, leaf essential oil reduces 43.48% of melanin formation in zebrafish embryos at the concentration of 50 μg/mL. Results reveal A. dammara leaf essential oil has the potential for developing the skin whitening drug and depigmentation ingredient for hyperpigmentary disorders.


Introduction
Araucariaceae is an ancient family and widespread in the Jurassic period, distribution in both hemispheres including numerous taxa.Araucariaceae species are present in South America, Australasia, South Pacific islands, Southeast Asia and etc. Araucariaceae family contains four genera, including Agathis, Araucaria, Columbea, and Wollemia, com-monly known as kauri or dammar.The ethnopharmacological uses of the Araucariaceae family are versatile and numerous, including emollient and antiseptic properties, and to treat headache, myalgia, respiratory infections, rheumatisms, skin wounds, insomnia, and so on [1][2][3][4][5].
Genus Agathis are widely distributed in Australia, New Zealand, South Pacific islands, and Southeast Asia, the species are characterized by their brownish black bark, leathery and ovate-lanceolate leaves, circular branch scars on their trunks, and their milky resins.Agathis genus contains 18 accepted species and 4 unresolved species [1][2][3][4].To date, the timbers of Agathis spp.are recognized as good materials for processing, the timber is straight-grained, heavier and harder wood, knot free, with a fine textured and lustrous surface [3].Gum copal is harvested by tapping or cutting the living Agathis trees.Resins and gums from Agathis spp.have been used as varnishes, lacquers, adhesive, and etc. [1,5].According to the related research reports, α-pinene, β-pinene, camphene, α-copaene, α-cubebene, βcaryophyllene, δ-cadinene, allo-aromadendrene, aromadendrene, germacrene D, limonene, myrcene, sabinene, spathulenol, and 16-kaurene are the most abundant constituents found in the essential oils of Agathis species [2,3].The biological activities of genus Agathis include antibacterial, antifungal, anti-inflammatory, antileishmanial, antiplasmodial, cytotoxic activities, etc. in literatures [3,[5][6][7][8].Verma et al. investigated the antibacterial activity of resin essential oil from A. robusta (C.Moore ex F. Muell.)Bailey (Queensland Kauri), resin essential oil showed moderate activity against Staphylococcus epidermidis and good activity against S. aureus in the disc diffusion assay [5].The leaf essential oil of A. dammara (Lamb.)Rich.was also found to have significant antibacterial activity against Staphylococcus aureus (Gram positive bacterium) and Pseudomonas aeruginosa (Gram negative bacterium) in the disc diffusion method and microwell dilution assay [6]. A. atropurpurea B. Hyland bark resin exhibited effective antileishmanial activity against Leishmania amazonensis promastigotes and amastigotes [7].Resins from A. atropurpurea showed slight antifungal activity against Aspergillus niger and Rhizopus stolonifera, which are the important and widespread human and agricultural harmful pathogens [8].

Plant Material
The fresh and mature leaves (dark green) of Agathis dammara (Lamb.)Rich.were collected from the campus (25 • 01 03.0 N 121 • 32 21.1 E) of National Taiwan University, Taipei, Taiwan in March 2022.The diameter at breast height (DBH) of the tree is 31.9cm, and the height is 11.6 m.The species was identified by Dr. Chih-Chieh Yu, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.Voucher specimen (AD0322) has been kept in the laboratory of Chemical Utilization of Biomaterials, School of Forestry and Resource Conservation, National Taiwan University.

Hydrodistillation of Essential Oil
The fresh and mature leaves of A. dammara were hydrodistilled for 6 h by using the Clevenger apparatus.After the hydrodistillation, the leaf essential oil was placed in a dark glass bottle and stored in a refrigerator at 4 • C for further investigation [33][34][35].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
To investigate the chemical composition of the essential oil, the analysis of A. dammara leaf oil was carried out on a Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a DB-5 MS column (Crossbond 5% methylpolysiloxane, 30.0 m length × 0.25 mm diameter, thickness 0.25 µm; Agilent Technologies, Palo Alto, CA, USA).The oven temperature started from 60 • C for 3 min, programed at 3 • C/min to 120 • C, 5 • C/min to 240 • C for 3 min.The injector temperature was held at 250 • C, and split ratio was 10:1.The carrier gas was helium; 1.0 mL/min flow rate; ion source temperature 250 • C; mass range 50-650 amu.The constituents of essential oil were characterized by National Institute of Standards and Technology (NIST) V.2.0 and Wiley 7.0 GC-MS libraries and Kovats indexes in the reference [36].Kovats indexes of the constituents are determined by retention times of n-alkanes (C7-C30) on the DB-5MS column.The relative contents of constituents were determined by integrating the peaks on total ion chromatograms (TIC) [37,38].

Antityrosinase Assay
Tyrosinase is an essential enzyme in melanogenesis.The antimelanogeneic effect of specimen can be preliminarily determined by its tyrosinase inhibitory activity.The activity was determined by previous assays with minor modification [24,39].The specimen-buffer mixture (110 µL) was added in a 96 well microplate.The specimen was diluted in DMSO and mixed with 0.1 M potassium phosphate buffer (pH = 6.8).Kojic acid (Sigma, St. Louis, MO, USA) and arbutin (Sigma, St. Louis, MO, USA) were used as the positive controls.Subsequently, 50 µL of 200 U/mL mushroom tyrosinase (EC 1.14.18.1;Sigma, St. Louis, MO, USA) was added and mixed with the specimen-buffer mixture.After mixed with 40 µL of substrate (L-tyrosine and L-DOPA), the mixture was incubated for 10 min at room temperature.Next, the 96-well microplate was sent into SPEOTROstar, the microplate reader (BMG Labtech, Ortenberg, Germany), and the absorbance (475 nm) of each well was measured.The tyrosinase inhibitory activity was presented as inhibition rate and half maximal inhibitory concentration (IC 50 ).The equation of inhibition rate is as followed: The IC 50 was determined by concentration-response curve.The experiments were performed in triplicate.

Statistical Analysis
The statistical analysis of data obtained in the study was analyzed by SPSS (Statistical Product and Service Solutions) (Chicago, IL, USA) Version 16 with the Scheffe's multiple comparison test, a post-hoc multiple comparison method.The confidence interval was set at the level of 95%.

Chemical Constituents of A. dammara Leaf Essential Oil
In this study, leaf essential oil was hydrodistilled from the mature dark green leaves of A. dammara.The yield of leaf essential oil was 0.15 ± 0.05%, on dry matter basis of leaf. Figure 1 showed the gas chromatogram of A. dammara leaf essential oil.The constituents of A. dammara leaf essential oil were analyzed by using GC-MS, and there are 21 constituents found in the chromatogram.According to Table 1, the leaf essential oil contained sesquiterpene hydrocarbons (69.35 ± 2.18%), diterpene hydrocarbons (13.64 ± 1.46%), and oxygenated sesquiterpenes (3.10 ± 0.21%).Except for 16-kaurene (12.43 ± 1.32%), most of the major constituents were sesquiterpenoids, including δ-cadinene (16.12 ± 0.53%), γ-gurjunene (15.57± 0.49%), β-caryophyllene (8.58 ± 0.94%), germacrene D (8.53 ± 0.20%), and γ-cadinene (5.33 ± 0.16%) (Figure 2).Chen et al. analyzed the chemical constituents of A. dammara leaf essential oil, the major components were limonene (36.81%), β-bisabolene (33.43%) and β-myrcene (25.48%) [6]; the content of monoterpenoids, limonene and βmyrcene, was over 50%.The result of the study is inconsistent with our analysis of the chemical compositions of leaf essential oil.New or young leaves of A. dammara are light green color, later turn dark green after a few days, and keep dark green color.Differences between two studies of chemical constituents of leaf essential oil may be due to the maturity of leaf, collected region, environment temperature, sunlight, season, and etc.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 4 of 11 medium, and specimen diluted in 1% DMSO.After that, the plate was incubated at 28 °C for 48 h, and the 57 hpf zebrafish embryo were photographed under a stereomicroscope (Hamlet SEM-H, Taiwan) at 40× magnification.The melanin content of zebrafish embryos was quantified by ImageJ (V1.52a) and presented as inhibition rate.The calculation of inhibition is: Inhibition (%) = [1 − (Intensity specimen/Intensity control)] × 100.1-Phenyl-2-thiourea (PTU), kojic acid, and arbutin were the positive controls [24,27,40].The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) in National Taiwan University (IACUC Approval No: NTU-112-EL-00072), all zebrafish were handled by the 3R's principles of laboratory animal care and use.

Statistical Analysis
The statistical analysis of data obtained in the study was analyzed by SPSS (Statistical Product and Service Solutions) (Chicago, IL, USA) Version 16 with the Scheffe's multiple comparison test, a post-hoc multiple comparison method.The confidence interval was set at the level of 95%.

Tyrosinase Inhibitory Activity of A. dammara Leaf Essential Oil
Tyrosinase which catalyzes L-tyrosine and L-DOPA into dopaquinone and dopachrome, respectively, is the essential enzyme in melanogenesis.To inhibit the melanogenesis process, suppressing the tyrosinase activity is one of the most common and effective strategies [43].
Figure 3 shows that leaf essential oil could prevent tyrosinase from acting as diphenolase and catalyzing L-DOPA to dopaquinone.Tyrosinase inhibitory activity of A. dammara leaf essential oil presented the dose-dependent effect.The inhibition rates were 6.93 ± 1.37%, 18.36 ± 1.82%, and 28.38 ± 3.95% at the concentration of 100, 200, and 400 µg/mL, respectively.Kojic acid, positive control, presented inhibition of diphenolase.Kojic acid showed 69.15% inhibition at the concentration of 12.5 µg/mL, followed by 48.20%, 24.94%, 10.57% at the concentrations of 6.25, 3.13, and 1.56 µg/mL.The IC 50 values of leaf essential oil and kojic acid were 690.02 ± 18.85 and 6.74 ± 0.09 µg/mL, respectively (Table 2).When the substrate was L-tyrosine, no significant inhibitory activity of leaf essential oil was observed.As for positive control, kojic acid (IC 50 = 2.46 ± 0.06 µg/mL) presented the tyrosinase inhibitory activity (Table 2).Huang et al. examined the tyrosinase inhibitory activity of Vitex negundo Linn.(Lamiaceae) leaf essential oil, the inhibition rate of leaf essential oil against tyrosinase were 26.6% at the concentration of 5 mg/mL, using L-DOPA as the substrate [44].Cheraif et al. evaluated the antityrosinase activity of essential oils from six Algerian plants, Juniperus oxycedrus L. (Cupressaceae) essential oil displayed the best tyrosinase inhibitory activity with a tyrosinase inhibition rate of 39.65% at the concentration of 1 mg/mL [45].Etlingera elatior (Jack) R. M. Sm. (Zingiberaceae) leaf essential oil exhibited a moderate activity against tyrosinase, with an IC 50 value of 2.34 ± 0.04 mg/mL when using L-DOPA as the substrate [46].Salleh et al. investigated the antityrosinase effects of leaf essential oil and bark essential oil of Beilschmiedia madang Blume (Lauraceae), using L-DOPA as the substrate, inhibition rates of leaf essential oil and bark essential oil were 53.1 ± 0.2% and 51.2 ± 0.2%, respectively, at the concentration of 1 mg/mL [47].Pogostemon plectranthoides Desf.(Lamiaceae) leaf essential oils collected from three bio-geographical regions also had the antityrosinase activity.Leaf essential oils were rich in sesquiterpenes, and the active leaf essential oil possessed the tyrosinase inhibitory effect with the inhibition rate of ca.50% at the concentration of 1 mg/mL, using L-DOPA as the substrate [48].These related researches illustrated that A. dammara leaf essential oil exhibited the tyrosinase inhibition potential.

Antimelanogenesis Effect of A. dammara Leaf Essential Oil in Zebrafish
Zebrafish (D. rerio) has been a powerful and wide used model organism and the efficient alternative to other animal models in multiple researches.In the present study, zebrafish is a suitable vertebrate model for melanogenesis study because of its easily observed pigmentation and the similarity of gene sequences and organ systems [21,49,50].The antimelanogenesis effects in the zebrafish model caused by different treatments were shown in Figure 4.In control group, the patterns of pigmentation presented normal dispersion.After treated with A. dammara leaf essential oil, kojic acid, and arbutin, the zebrafish embryos displayed less amount of pigmentation.As for PTU treatment, the zebrafish embryos became transparent after 48 h incubation, inhibition rate of melanin was 98.23 ± 1.01% at the concentration of 50 µg/mL.Table 3 shows the inhibitory effect of each treatment on melanogenesis of zebrafish embryo, including leaf essential oil, arbutin, kojic acid, and PTU.The melanin contents of zebrafish embryos could decrease by 21.03%, 37.36% and 43.48% with treatment of leaf essential oil at a concentration of 12.5, 25 and 50 µg/mL, with a dose-dependent manner.In comparison with the treatment of arbutin, leaf essential oil presented higher antimelanogenesis effect at the concentrations of 25 and 50 µg/mL.Significant statistical differences (p < 0.05) were observed between the treatment of leaf essential oil (43.48%) and the treatment of arbutin (21.49%) at the concentration of 50 µg/mL.Another positive control, kojic acid, inhibited 18.53% and 20.44% of melanin production of zebrafish embryos at the concentrations of 25 and 50 µg/mL, respectively.Kojic acid exhibited the lower melanin inhibition in zebrafish embryos than leaf essential oil treatments did.Chelly et al. (2021) evaluated the antimelanogenesis activities of methanolic extracts of Rhanterium suaveolens Desf.(Asteraceae) flower, stem, and leaf, with in vitro antityrosinase inhibition assay and in vivo zebrafish embryo assay [51].Among these part extracts of R. suaveolens, flower extract exhibited the best antityrosinase effect on tyrosinase induced L-DOPA oxidation.Flower extract also possessed the statistically significant decrease of melanin formation in zebrafish embryo at the concentrations of 0.5 and 1 mg/mL.The reduction of melanin formation in zebrafish embryo was less than 50% after 48 h treatment of 1 mg/mL concentration of R. suaveolens flower extract.Dalbergia pinnata (Lour.)Prain essential oil inhibited 18.77% of melanin production of zebrafish embryos at a concentration of 30 µg/mL [52].Bletilla striata (Thunb.)Rchb.f. tuber extract reduced 18.35% and 24.39% of melanin formation of zebrafish embryos at the concentrations of 10 µg/mL and 30 µg/mL, respectively [53].The results in this study revealed that A. dammara leaf essential oil possessed a potent melanogenesis inhibition activity in zebrafish embryos.

Conclusions
For the assessment of antimelanogenesis effect of essential oil from A. dammara leaf, both in vitro and in vivo antimelanogenesis assays were conducted in this study.The chemical constituents of A. dammara leaf essential oil was dominated by sesquiterpenoids through the GC-MS analysis.The most abundant constituent was δ-cadinene (16.12%), followed by γ-gurjunene (15.57%), 16-kaurene (12.43%), β-caryophyllene (8.58%), germacrene D (8.53%), and γ-cadinene (5.33%).Among all the constituents, most of the sesquiterpenoids were the compounds with cadinane skeleton.In vitro antityrosinase assay, A. dammara leaf essential oil showed diphenolase inhibitory activity.When the substrate was L-DOPA, the IC 50 value of leaf essential oil was 690.02 µg/mL.According to the in vivo zebrafish embryo assay, leaf essential oil has shown efficacy to reduce the melanin formation in zebrafish embryos.At a concentration of 50 µg/mL, leaf essential oil, arbutin, and kojic acid inhibited 43.48%, 21.49%, and 20.44%, respectively, of melanin production of zebrafish embryos.Antimelanogenesis activity of leaf essential oil was better than those of arbutin and kojic acid in zebrafish embryos.These results revealed A. dammara leaf essential oil has in vitro and in vivo antimelanogenesis activities, and has the potential to be the skin whitening drug and depigmentation ingredient for hyperpigmentary disorders.Funding: This research was funded by Council of Agriculture, Executive Yuan, Taipei, Taiwan (111AS-1.6.1-ST-aO and 112AS-1.6.1-ST-a9).

Institutional Review Board Statement:
The zebrafish protocol was approved by the Institutional Animal Care and Use Committee (IACUC) in National Taiwan University (IACUC Approval No: NTU-112-EL-00072).Animal experiments were conducted in accordance with the 3R's principles of laboratory animal care and use.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

aFigure 2 .
Figure 2. Chemical structures of major constituents of A. dammara leaf essential oil.

Figure 2 .
Figure 2. Chemical structures of major constituents of A. dammara leaf essential oil.

Figure 3 .
Figure 3. Mushroom tyrosinase inhibitory activity of A. dammara leaf essential oil.When using L-DOPA as the substrate.(a) A. dammara leaf essential oil; (b) kojic acid.Different letters (a-d; a-e) represent significantly different at the level of p < 0.05 according to the Scheffe's test.

Figure 3 .
Figure 3. Mushroom tyrosinase inhibitory activity of A. dammara leaf essential oil.When using L-DOPA as the substrate.(a) A. dammara leaf essential oil; (b) kojic acid.Different letters (a-d; a-e) represent significantly different at the level of p < 0.05 according to the Scheffe's test.

Table 1 .
Constituents of essential oil from Agathis dammara leaves.

Table 1 .
Constituents of essential oil from Agathis dammara leaves.
a RT: Retention time; b KI: Kovats index relative to n-alkanes (C7-C30) on a DB-5MS column; c rKI: reference Kovats index from other research [36]; d MS: NIST and Wiley libraries spectra and literature; KI: Kovats index.

Table 2 .
IC 50 values of A. dammara leaf essential oil against mushroom tyrosinase.

Table 3 .
Inhibitory effect of A. dammara leaf essential oil on melanogenesis of zebrafish embryos.

Table 3 .
Inhibitory effect of A. dammara leaf essential oil on melanogenesis of zebrafish embryos.
PTU: 1-Phenyl-2-thiourea.Different letters (a-d) in the table indicate significantly different inhibition values between specimens at the level of p < 0.05 according to the Scheffe's test.