Limonene and eucalyptol rich essential oils with their antimicrobial activity from the leaves and rhizomes of Conamomum vietnamense N.S. Lý & T.S. Hoang (Zingiberaceae)

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Introduction
The genus Conamomum Ridl. is a small genus in the Zingiberaceae family with only 12 accepted species worldwide, mainly occurring in evergreen lowland and montane forests in Southeast Asian countries, such as Vietnam, Cambodia, Thailand, Malaysia, Singapore, Sumatra and Borneo (Boer et al. 2018;Luu et al. 2019;Ngoc-Sam et al. 2022). Before 2018, the genus Conamomum had been a synonym of the genus Amomum Roxb., however, based on the combination of molecular (nrITS and matK) and morphological evidence, the botanist De Boer proposed Conamomum as a separate genus (Boer et al. 2018). To date, there have been a limited number of studies on the essential oils of Conamomum species and their bioactivities. Conamomum rubidium (Lamxay & N.S.Lý) Škorničk. & A.D.Poulsen was described and studied essential oil before the combination of the genus. Therefore, relevant documents mentioned this species under the old name Amomum rubidum. C. rubidium leaves mainly contained 1,8-cineole (37.7%), δ-3-carene (19.5%) and limonene (16.3%) while the stem part was found to contain δ-3-carene (21.9%), limonene (17.8%) and β-phellandrene (14.6%). The oils were also shown to inhibit the growth of Candida albicans and Fusarium oxysporum with MIC values of 50 μg/mL (Le et al. 2021). The essential oil prepared from rhizome was found to have similar components to that of stem, specifically β-phellandrene (16.1%), limonene (14.4%), and δ-3carene (13.9%). The rhizome oil exhibited larvicidal activity against Aedes aegypti (LC 90 values of 31.44 μg/mL and 31.03 μg/mL (Huong et al. 2020), and antimicrobial activities against Escherichia coli and Fusarium oxysporum with MIC values of 50 μg/mL (Le et al. 2019). The newest species of this genus, C. vietnamense ( Fig. 1) was described in Tay Nguyen (Central Highlands), Vietnam (Ngoc-Sam et al. 2022). The plant is most similar to C. odorum Luu, Tran & Tran and C. rubidium, except for well-developed stilt roots, elliptic leaf blades, narrowly ovate bracts, abaxially pubescent bracteoles, longer calyx with two truncate lobes, broadly obovate to orbicular glabrous labellum, longer filament, and glabrous style (Ngoc-Sam et al. 2022). In this study, we report for the first time chemical composition of essential oil prepared from C. vietnamense leaves and rhizomes and their antimicrobial activity.

Plant materials
The fresh leaves and rhizomes of Conamomum vietnamense were collected from Loc Bac, Bao Lam District (11°47'31.9"N, 107°35'47.2"E), Lam Dong Province, Vietnam in September 2022. The plant was identified by the first author based on the protologue (Ngoc-Sam et al. 2022), and a voucher specimen (HC015) was deposited at the Institute of Applied Technology, Thu Dau Mot University.

Preparation of essential oil
The scraps of leaves (500 g) and rhizomes (500 g) of Conamomum vietnamense were hydro-distilled for 3 h (beginning from the water boiling point) using a Clevenger-type apparatus, according to the Vietnamese Pharmacopoeia (The Committee of Vietnamese Pharmacopoeia 2017). Then, the obtained essential oil was removed from all water traces with Na 2 SO 4 and stored in sealed glass vials at 4 °C. The oil yield (%) was calculated by dividing the weight (g) of the essential oil over the weight (g) of the fresh samples.

GC-MS analysis
The essential oils of Conamomum vietnamense were analyzed for their constituents via Gas chromatographic/ mass spectrometry using an Agilent GC-7980 linked to an Agilent MS 5977C system working in EI mode, with an HP-5MS UI column (30 m × 0.25 mm id. × 0.25 μm film thickness, Agilent Technologies). System installation: carrier gas was Helium (flow rate of 1 mL/min); injection volume: 1 μL, split ratio 1:50; the temperature was programmed from 60 °C (kept for 1 min) to 240 °C (kept for 4 min) at a rate of 4 °C/min; injector temperature: 300 °C, MS Quad temperature: 150 °C, transfer line temperature: 300 °C, MS source: 230 °C; ionization energy: 70 eV, and mass range: 50-550 amu (2.0 scan/s). Identification of the oil components was performed based on comparing their mass spectra value to that of the NIST14 library and then confirmed by comparing retention indices with reference to a homologous series of n-alkanes. The percentage of the relative peak area was used for quantification.

Antimicrobial assay
The anti-microbial bioassays were performed on Gram-positive bacterial strains (Enterococcus faecalis ATCC 299212, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579), and Gram-negative bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 13076), and a pathogenic yeast (Candida albicans ATCC 10231). All strains were provided by National Institute for Food Control (No. 65 Pham Than Duat Street, Mai Dich Ward, Cau Giay District, Ha Noi, Vietnam). The antimicrobial activity of the essential oil prepared from the leaves and rhizomes of C. vietnamense was assessed by microdilution broth susceptibility assay method, which strictly followed the standards issued by the Clinical and Laboratory Standards Institute (CLSI supplement M100, 2020) (Clinical and Laboratory Standards Institute 2020). Luria Bertani Agar (LBA) and Sabouraud Dextrose Agar (SDA) were used to culture bacteria and yeast on 96-well plates, respectively. Streptomycin and cycloheximide were used as positive controls for bacteria and yeast, respectively, at the concentration range of 2-256 μg/mL, which were provided by the Institute of Drug Quality Control in Ho Chi Minh City, Vietnam. The experimental design was conducted as follows: (1) the essential oils were diluted in 10% DMSO and prepared in eight different concentrations (2,4,8,16,32,64,128, 256 µg/mL). The bacteria and fungi (yeast) were standardized with a concentration of 2×10 5 CFU/mL. The mixtures of microbiological and tested samples were incubated at 37 °C/18-24h for bacteria and at 35-37 °C/36-48h for yeast. After 24h, the minimum inhibitory concentration (MIC) value was observed and preliminarily determined as the lowest reagent concentration that completely inhibited microbial growth after 24h of culture. Additionally, the IC 50 values were also calculated based on the turbidity measurement data by Bioteck spectrophotometer (USA) and RawData software (Brussels, Belgium) (Huong et al. 2021(Huong et al. , 2022. The experiments were performed in triplicate.
The microbial inhibition rate expressed as a percentage of inhibition was calculated using the following formula (Huong et al. 2021(Huong et al. , 2022

Results and discussion
The yields of the hydrodistilled essential oils from the leaves and rhizomes of Conamomum vietnamense were 0.43% and 0.37% (w/w, fresh weight), respectively. The chemical composition of essential oils was analyzed and presented in Table 1, whereby there were a total of 52 (in the rhizome oil) and 28 (in the leaf oil) identifiable compounds, representing 99.35% and 99.83% of the volatile content. As can be seen from Table 1, the major compositions of both oil samples were almost similar. Specifically, it is noticeable that eucalyptol was a dominant component in rhizome and leaf oil samples with 40.47% and 49.49% of the total content, respectively. Following that, limonene was identified as the second-highest oil component with contents of 18.74% and 26.20%. Two monoterpenes, α-pinene and α-phellandrene, were also present in both oils with similar percentages (3.30-4.91%). GC-MS chromatograms of leaf and rhizome oil samples from C. vietnamense can be found in Figs 2, 3. The two oil samples were then evaluated for their antimicrobial activities against several bacterial and fungal strains. The investigated strains included Enterococcus faecalis (causing endocarditis, sepsis, urinary tract infections, and meningitis), Staphylococcus aureus (causing bacteremia, infective endocarditis), Bacillus cereus (causing food-borne illnesses with symptoms of nausea, vomiting, and diarrhea), Escherichia coli (causing diarrhea, urinary tract infections, respiratory illnesses, and pneumonia), Pseudomonas aeruginosa (causing pneumonia, urinary tract infections, and wound, ear, and bloodstream infections), Salmonella enterica (food-borne pathogen causing gastroenteritis), and Candida albicans (causing hospital-acquired infections, vaginal yeast infection, and bloodstream infection). The bioassays used streptomycin and cycloheximide as positive controls ( Table 2). As for antibacterial properties, the leaf oil of C. vietnamense displayed moderate antibacterial effects against E. faecalis with MIC value of 32 µg/mL and weak activities against S. aureus (MIC 128 µg/mL), B. cereus (MIC 128 µg/mL), E. coli (MIC 256 µg/mL), P. aeruginosa (MIC 128 µg/mL), S. enterica (MIC 128 µg/mL). Meanwhile, the rhizome oil exhibited more   The oil samples were also tested against a fungal strain, C. albicans. As a result, the leaf and rhizome oils showed potential activities with MIC values of 32 and 64 µg/mL, respectively, which were comparable to that of the positive control, cycloheximide (MIC 32 µg/mL). Eucalyptol and limonene were the two main components of C. vietnamense leaf and rhizome essential oils, which occupied 75.69 and 59.21% of the total oil content, respectively. Eucalyptol, or 1,8-cineol, is a bicyclic terpenoid, which naturally occurs in many plants, especially Eucalyptus species with high-content (60-97.32%) (Mączka et al. 2021). The oxygenated monoterpene was shown to possess potential antimicrobial activities. Furthermore, eucalyptol was also shown to synergistically inhibit the growth of methicillin-resistant Staphylococcus aureus strains with chlorhexidine gluconate and amoxicillin/ clavulanic acid combination (Merih and Reşat 2017;Hriouech et al. 2020). Further studies indicated that eucalyptol might affect quorum sensing, a chemical signal-based system of communication between microorganisms (Sybiya Vasantha Packiavathy et al. 2012), and change the shape and size of bacterial cell (Li et al. 2014). Limonene, a well-known monoterpene with lemon-like odor, has been found mostly in the essential oil of Citrus plants. The compound showed moderate antimicrobial activities against various types of bacteria and fungi with MIC values ranging between 1 and 20 mg/mL dependent on bioassays. Limonene was found to affect cell respiration and electron chain activity, which leads to cellular energy depletion and intracellular ROS accumulation (Thielmann and Muranyi 2019). Therefore, the high content of eucalyptol and limonene in prepared essential oils partly explained the antimicrobial activities against investigated bacterial and fungal strains in this study.

Conclusion
The essential oils were prepared from C. vietnamense leaves and rhizomes by hydrodistillation method with yields of 0.43% and 0.37% (w/w, fresh weight), respectively. The chemical composition of both oil samples was identified by GC-MS analysis, indicating that eucalyptol and limonene were the two dominant components (40.47-49.49% and 18.74-26.20%, respectively). The essential oils were then evaluated for their antimicrobial activities. The leaf oil was the most active against E. faecalis and C. albicans with MIC values of 32 µg/mL while the rhizome oil showed potential inhibitory effects on E. faecalis (MIC 32 µg/mL), B. cereus (MIC 64 µg/mL), S. enterica (MIC 64 µg/mL), and C. albicans (MIC 64 µg/mL). The study results showed that the essential oil of C. vietnamense can be a resource for eucalyptol and limonene and possessed potential antimicrobial effects. Remarkably, this is the first report on the chemical constituents and antimicrobial activity of the essential oil prepared from Conamomum vietnamense.