The antimicrobial activity and essential oil constituents of the leaves and trunks of Aquilaria banaensis P.H.Hô (Thymelaeceae) from Vietnam

Abstract The aim of the present paper was to report the chemical constituents and antimicrobial activity of essential hydrodistilled from the leaves and trunk of Aquilaria banaensis P.H.Hô (Thymelaeceae) from Vietnam. The essential oils were analysed comprehensively for their constituents by using Gas chromatography coupled with Mass spectrometry (GC/MS). The antimicrobial activity was determined by agar well diffusion and broth microdilution methods. The leaf essential oil comprised mainly of sesquiterpenes while fatty acids constitutes the bulk of the trunk essential oil. The main constituents of the leaf essential oil were β-caryophyllene (17.11%), α-selinene (10.99%), α-humulene (8.98%), β-selinene (8.01%), β-guaiol (6.69%) and β-elemene (5.65%). However, hexadecanoic acid (48.46%), oleic acid (19.80%) and tetradecanoic acid (5.32%) were the major compounds identified in the trunk essential oil. The trunk essential oil displayed antimicrobial activity against Staphylococcus aureus, with the minimum inhibitory concentration (MIC) value of about 256.0 µg/mL. Graphical Abstract


Introduction
Agar (Aquilaria) tree belongs to the family Thymelaeaceae.There are 17 Aquilaria species distributed around the world (Wang et al. 2021).Agarwood is the resinous heartwood of agar tree, comparatively light and pale colored, and are produced in response to natural infections, artificial inductions or injury-induced defense (Akter et al. 2013).Healthy Aquilaria tree does not produce agarwood.The healthy wood is white, soft, even-grained and not having a perfumed smell, as compared with the dark, hard and heavy scented characteristics resin-impregnated agarwood (Chen and Rao 2022).Investigations found that four Aquilaria spp.namely, A. crassna Pierre ex.Lecomte, A. baillonii Pierre ex.Lecomte, A. banaensis P. H. Hô., and A. rugosa K. Le-Cong & Kessler, were known in Vietnam (Thuy et al. 2019;Hai et al. 2021).
A variety of volatile and non-volatile extracts and phytochemicals from agarwood has obvious pharmacological uses and has always been an interesting topic among researchers and scientists (Wang et al. 2021;Chen and Rao 2022).For example, the sesquiterpene β-caryophyllene isolated from the essential oil of A. crassna had an inhibitory effect on the growth rate of Gram-positive bacteria (Dahham et al. 2015).The volatile oil extracted from A. crassna had antibacterial activity, especially against methicillin-resistant Staphylococcus aureus (Li et al. 2014).The compound, 5-deoxylongiferol isolated from Chinese agarwood had an antibacterial effect on S. aureus and Ralstonia solanacearum (Wang et al. 2016).The essential oil of A. crassna exhibited potent cytotoxic activity against MIA PaCa-2 cells with an IC 50 (11 ± 2.18 µg/ mL) (Dahham et al. 2016).The essential oils of A. crassna (Dahham et al. 2016) and A. malaccensis (Ibrahim et al. 2011) have strong anticancer activity towards human colon HCT 116 cancer cells.
Aquilaria banaensis P. H. Hô. is a species endemic to Vietnam (Thuy et al. 2019;Hai et al. 2021;Chen and Rao 2022).The plant was firstly described by Pham Hoang Ho in 1986 in Da Nang of Vietnam (Ho 1999;Thuy et al. 2019).A. banaensis is a small tree or shrub that grows up to 2-3 m tall.It has ovate to obovate leaves that are 5-10 × 1.6-3.5 cm in size.The leaves are glabrous with a midrib that is sparsely apprised and hairy beneath.The leaf has 20-25 pairs of veins and petioles are 6-7 mm.The plant gives flowers from May to October (Nha et al. 2019).According to the IuCN Red List 2017 of threatened species, A. banaensis is vulnerable to extinction under criteria D1 (Philip and Yvette 2017).Till moment, no information could be seen on the phytochemical constituents of the volatile and non-volatiles of A. banaensis.In addition, there is first of reports on the biological and pharmacological activities of any extracts and compounds of A. bananensis.
In continuation of our research on the phytochemical constituents and biological potentials of Vietnamese plants (Tuan et al. 2021;2022), we report herein for the first-time chemical compositions and antimicrobial activity of essential oils from the leaves and trunks of A. bananensis.

Chemical composition of the essential oil
The yields of essential oils, based on the fresh weight (w/w) from the leaves and trunks of A. banaensis were 0.03% and 0.04%, respectively.The hydrodistilled essential oils from these plant parts are a dark-yellow liquid with a characteristic odor and density lighter than water.The analysis of GC-MS spectral indicated that the number of identified constituents in the essential oils from the leaves and trunks of this plant species 22 and 17, respectively, accounting for 97.37% and 97.33% of the oil contents (Table S1).In the chemical composition of the leaf oil, monoterpene compounds were not identified in the essential oil.The sesquiterpene hydrocarbons (70.82%) and oxygenated sesquiterpenes (24.92%) are the main classes of compounds identified in the essential oil.Phytol (1.63%) was the only diterpene of the essential oil.The main constituents of the leaf essential oil were β-caryophyllene (17.11%), α-selinene (10.99%), α-humulene (8.98%), β-selinene (8.01%), β-guaiol (6.69%) and β-elemene (5.65%).There is significant amount of germacrene D (4.93%), caryophyllene oxide (4.68%), bulnesol (3.84%), α-cadinol (3.78%), and δ-cadinene (3.71%).
The quantitative and qualitative components of both essential oils from the leaves and trunks of A. banaensis were different from each other.The observed compositional variation in both essential oils may be due to the different part of the plants, which stored different phytochemicals.The high content of sesquiterpene and fatty acids in the essential oils of A. banaensis was in agreement with previous findings that the major components of agarwood are terpenoids, mainly sesquiterpenes and diterpenes, as well as fatty acids (Tajuddin and Yusoff 2010;Thuy et al. 2019;Mohamad Hamdan et al. 2020;Ngan et al. 2020;Hezelin elayana et al. 2021).Although no report could be seen on the volatile contents of A. banaensis, notable sesquiterpenes such as neopetasone, dihydroagarofuran-15-al, β-agarofuran, kessane, kusnol, dihydrokaranone, valerianol jinkoeremol among others that are characteristics of previously analysed essential oils of some other Aquilaria species (Tajuddin and Yusoff 2010;Thuy et al. 2019;Mohamad Hamdan et al. 2020;Ngan et al. 2020;Hezelin elayana et al. 2021) were not identified in the present study.Moreover, 3-phenyl-2-butanone and α-cubebene, the main compounds of A. malaccensis and A. subintegra were conspicuously absent in A. banaensis (Hashim et al. 2014).However, n-hexadecanoic acid and oleic acid, the main compounds of the trunk essential oil of A. banaensis was also found in significant in the leaf essential oils of A. crassna from Vietnam (Thuy et al. 2019), and Laos (Ngan et al. 2020), as well as A. sinensis from China (Chen et al. 2011).These variations in the compositional patterns of Aquilaria essential oils may be explained by the nature of the plant parts, differences in the ecological and climatic conditions at the points of collection among others, handling conditions and methods of analysis among others.

Antimicrobial activity of the essential oil
The trunk essential oil displayed antimicrobial activity against Staphylococus aureus with the minimum inhibitory concentration (MIC) value of about 256.0 µg/mL and IC 50 value of 153.7 (Table S2). the trunk essential oil exhibited moderate action against, Bacillus subtilis, Lactobacillus fermentum, Escherichia coli, Salmonella enteric, Pseudomonas aeruginosa and Candida albicans, with MIC value in the range > 256.0 µg/mL.The leaf essential oil did not show meaningful and considerable activity towards the tested microorganisms.This is the first report on the antimicrobial activity of essential oils of A. banaensis.Accordingly, 19 substances with MIC values < 100 μg/ mL were considered to be of good antimicrobial activity, while MIC values from 500 − 100 μg/mL are considered as moderate activity.In addition, MIC values from 1000 − 500 μg/mL are said to be of weak activity while MIC values above 1000 μg/mL are considered inactive.Thus, essential oils from trunks of A. banaensis possessed moderate activity against S. aurues (Holetz et al. 2002).
The essential oil of A. banaensis exhibited selective antimicrobial activity in agreement with data for other species.Previously essential oils from other Aquilaria species have shown varying antibacterial and antifungal activities.Like A. banaensis, the volatile oil extracted from A. crassna exerted antibacterial activity, especially against methicillin-resistant S. aureus (Li et al. 2014).Results showed that β-caryophyllene, a major compound of essential oil of A. crassna demonstrated selective antibacterial activity against S. aureus (MIC 3 ± 1.0 µM) and more pronounced anti-fungal activity than kanamycin (Dahham et al. 2015).essential oils of A. sinensis were more potent inhibitors of Lasiodiplodia theobromae, Fusarium oxysporum, and Candida albicans (Zhang et al. 2014).The essential oils of A. sinensis had better inhibition activities towards Gram-positive bacterial strains Bacillus subtilis (MIC, 0.195-0.78mg/mL) and S. aureus (MIC 0.195-3.125mg/mL), compared to Gram-negative bacteria strain of E. coli (MIC, 6.25-12.5 mg/mL) (Chen et al. 2011).The essential oils of A. crassna from Thailand produced by different extraction methods had antimicrobial activities against S. aurues and C. albicans, but were not sensitive to E. coli at maximum concentration of the study, 2 mg/mL (Penpun et al. 2009).The essential oils of A. malaccensis from Bangladesh only showed moderate to weak sensitivity against E. coli, S. aureus, and Salmonella vibrio (Hoque et al. 2018).
Previous studies have demonstrated that the antimicrobial activity of essential oils can vary either because of their major compounds or the synergies between the major and some minor constituents.This result can be explained by the presence of secondary metabolites.It was therefore surprising that the leaf essential oil of A. banaensis with high content of β-caryophyllene has no antimicrobial activity against studied pathogens.β-caryophyllene possessed considerable antibacterial activity towards several pathogens including S. aureus (Dahham et al. 2015).On the other hand, some other compounds identified in the trunk essential oils including hexadecanoic acid, oleic acid and tetradecanoic acid were previously reported to show antimicrobial actions against a varying number of microorganisms.The n-hexadecanoic acid showed that moderate antibacterial activity against S. aureus, B. subtilis, E. coli, and K. pneumonia (Ganesan et al. 2022).A synergistic effect oleic acid and linoleic acid produced potent antibacterial activity against S. aureus and Micrococcus kristinae (Dilika et al. 2000).Oleic acid is found to have antibacterial activity, particularly in inhibiting the growth of several Gram-positive bacterial species (Stenz et al. 2008).Tetradecanoic acid contributed immensely to the antimicrobial activity of extracts from Allium hookeri (Kima et al. 2016).

material
leaves (500 g) and trunk (500 g) of A. banaensis were collected from A Luoi district, Thua Thien Hue province in February 2022 (GPS: 16°28'1.10″N 107°20'3.35e).Botanical identification was defined by Dr. Tuan Anh Le (Mientrung Institute for Scientific Research, Vietnam National Museum of Nature, VAST, Vietnam).A voucher specimen (AB-T135) was stored in the laboratory of Forestry Faculty, Hue university of Agriculture and Forestry, Hue university, Vietnam.

Hydrodistillation of the essential oils
In the hydrodistillation process, the leaves and trunk of A. banaensis were separately hydrodistilled using a Clevenger-type apparatus as described previously (Tuan et al. 2021;2022).The distillation time was 3 h at normal pressure.The essential oils were treated with anhydrous sodium sulfate to remove moist content and kept in the refrigerator at 4 °C for further analysis.each experiment was repeated in triplicate.

Analysis of the essential oils
The chemical composition of the essential oil was analyzed using GCMS-QP2010 Plus system (Shimadzu, Kyoto, Japan) equipped with equity-5 capillary column (30 m × 0.25 mm, 0.25 µm film thickness) and coupled with a mass spectrometer (MSD QP2010 Plus) used for GC/MS analysis.The oil was diluted with n-hexane in a ratio of 1:100, and 1 µL was used for analysis.The analytical conditions were as follows: carrier helium (1.5 mL/min), injector temperature of 280 °C, interface temperature of 280 °C, and a column temperature programmed from 60 °C (2 min hold) to 240 °C at 3 °C/min (10 min hold) and then increased to 280 °C at 5 °C/min (40 min hold).Samples were injected using a split-less mode.The MS conditions were as follows: ionization voltage 70 eV; acquisitions scan mass range of 45-500 (m/z) at a sampling rate of 1.0 scan/s.The retention indices (RI) of constituents were determined by co-injection with reference to a homologous series of n-alkanes (C 8 -C 38 ) under the same conditions.
The identification of components was carried out by comparison of their RI values with those in the literature (Adams 2017).Further identification was performed by comparison of their mass spectra with those from NIST 11 and WILeY 7 stored in the GC/MS database.Quantification was performed using the relative peak area percentage.
A modified method of broth microdilution was used to determine the minimum inhibitory concentration (MIC) values of essential oils (Hadacek and Greger 2000;Cos et al. 2006).The concentrations of the essential oils were dissolved DMSO and sterilized distilled water to a range of 4-10 concentrations with the highest concentration at 256 µg/mL.The experiment was carried out in sterile 96-well microtiter plates.each well containing 10 µL sample received an addition of 190 µL of bacterial suspension 5 × 10 5 CFu/mL and fungi suspension 1 × 10 3 CFu/mL.DMSO was used as a negative control while Ampicillin, Cefotaxime and Nystatine were employed as positive controls against bacteria and fungi, respectively.The experiment was conducted in triplicate.The minimum inhibitory concentration values were determined to be the lowest concentration of the essential oil of A. banaensis which there was no microorganism growth.

Conclusions
In summary, this study provides information on the chemical composition and antimicrobial activity of A. banaensis leaf and trunk essential oils.The major components identified from the essential oils of A. banaensis were different from each other.The compositions comprised of β-caryophyllene, α-selinene, α-humulene, β-selinene, β-guaiol and β-elemene in the leaf, while hexadecanoic acid, oleic acid and tetradecanoic acid were identified in the trunk.The trunk essential oil also exhibits potential antimicrobial activity against S. aureus with MIC value of 256.0 μg/mL.Therefore, the present result opens up a possibility for further exploitation of A. banaensis essential oil.