In vitro Antimicrobial Activity of Essential Oil Extracted from Leaves of Leoheo domatiophorus Chaowasku, D.T. Ngo and H.T. Le in Vietnam.

:The present study aimed to determine the antimicrobial activity and chemical composition of leaves-extracted essential oil of Leoheo domatiophorus Chaowasku, D.T. Ngo and H.T. Le (L. domatiophorus), including antibacterial, antimycotic, antitrichomonas and antiviral effects. The essential oil was obtained using hydrodistillation, with an average yield of 0.34 ± 0.01% (v/w, dry leaves). There were 52 constituents as identified by GC/MS with available authentic standards, representing 96.74% of the entire leaves oil. The essential oil was comprised of three main components, namely viridiflorene (16.47%), (-)-δ-cadinene(15.58%) and γ-muurolene (8.00%). The oil showed good antimicrobial activities against several species: Gram-positive strains: Staphylococcus aureus (two strains) and Enterococcus faecalis, with Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC) values from 0.25 to 1% (v/v); Gram-negative strains such as Escherichia coli (two strains), Pseudomonas aeruginosa (two strains) and Klebsiella pneumoniae, with MIC and MLC values between 2% and 8% (v/v); and finally Candida species, having MIC and MLC between 0.12 and 4% (v/v).Antitrichomonas activity of the oil was also undertaken, showing IC50, IC90 and MLC values of 0.008%, 0.016% and 0.03% (v/v), respectively, after 48h of incubation. The essential oil resultedin being completely ineffective against tested viruses, ssRNA+ (HIV-1, YFV, BVDV, Sb-1, CV-B4), ssRNA- (hRSVA2, VSV), dsRNA (Reo-1), and dsDNA (HSV-1, VV) viruses with EC50 values over 100 µg/mL. This is the first, yet comprehensive, scientific report about the chemical composition and pharmacological properties of the essential oil in L. domatiophorus.


Introduction
Medicinal plants have received a great deal of scientific attention over the past decades due to their low toxicity, cost-effectiveness, and promising pharmacological properties [1]. A wide range of

Extraction of the Essential Oil
The leaves of L. domatiophorus (5 kg) were shredded and the essential oil was hydrodistilled for 3.5 h at ambient pressure using a Clevenger-type apparatus [19]. The extraction yields (based on three replications) were calculated as percentages on dry material. The oil was dried on Na 2 SO 4 and stored in sealed vials, at 4 • C, ready for the chemical analysis.

Analysis of the Essential Oil
Three repeats of sample were examined by the using of a Hewlett-Packard Model 5890A GC furnished with a flame-ionization detector and fitted with a 60 m × 0.25 mm, thickness 0.25 µm AT-5 fused SiO 2 capillary column. The injector and detector temperatures were the same (280 • C). The column temperature was programmed from 50 to 135 • C at 5 • C/min (1 min), 5 • C/min to 225 • C (5 min), 5 • C/min to 260 • C, held for 10 min. The oil (0.1 µL each) was analyzed without dilution (using 2,6-dimethylphenol as an internal standard) and injected by a split/splitless automatic injector HP 7673, using He as a carrier gas. The percentage of each compound was referred to absolute weight using internal standard and response factors. The detector response factors (RFs) that were determined for key constituents relative to 2,6-dimethylphenol and given to other components based on the correspondence of functional groups and/or structures.
MS analyses were carried out using an Agilent Technologies model 7820A associated with a MS detector 5977E MSD (Agilent), at the same conditions used for GC analyses. The column was linked to the ion source of the mass spectrometer. Mass units were monitored from 10 to 900 at 70 eV.
The retention indices (RI) of single compound were determined by co-injection with a homologous series of n-alkanes (C 9 -C 22 ) under the same conditions to (the retention indexes was calculate with the generalized equation by Van del Dool and Kartz [20]).
Data were processed for ANOVA by means of the software MSTAT-C and mean separation was performed by application of the LSD test at p ≤ 0.05 level of significance.

Antimicrobial Activity
Herein, there were 12 bacterial strains being selected, including 5

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC)
In order to establish the MIC and MLC of bacteria and Candida species, the broth dilution method was employed, as reported by the Clinical and Laboratory Standard Institute [21]. The inoculum was prepared by diluting colonies in salt solution at a concentration of 0.5 McFarland, then confirmed at a wavelength of 530 nm by a spectrophotometric. The sensitivity test was implemented in LB broth and RPMI-1640 medium using 96-well plates. The oil solutions were diluted to a range of concentrations from 16% (v/v) to 5×10 −4 % (v/v). After shaking, 100 µL of each oil dilution and 100 µL of bacterial/yeast suspension at a concentration of 10 6 CFU/mL were added to each well, then incubated at 37 • C for 24 to 48 h. MIC values were determined by the lowest concentration of the essential oil in which bacterial growth is visibly inhibited after overnight incubation. In order to determine the MLC value, 10 µL were seeded on Mueller Hinton agar and Sabouraud Dextrose agar and the plates were incubated for 24 to 48 h at 37 • C. Minimal lethal concentration (MLC) was considered as the lowest concentration that reduces the viability of the initial microbial inoculum, by ≥99.9%. Each experiment was performed in duplicate and repeated three times.

Cultivation of Trichomonas Vaginalis
Trichomonas vaginalis were axenically cultured in vitro by daily passages in Diamond's TYM medium, and modified by adding 20% FBS, 300 IU/mL penicillin G, 300 µg/mL streptomycin at 37 • C in a 5% carbon dioxide atmosphere. The medium was well-observed and renewed on a daily basis to remove any miscellaneous matter [22]. Viable trophozoites were counted in a hemocytometer. T. vaginalis was harvested at the mid logarithmic phase with more than 95% viable cells, by centrifugation at 500 rpm for 10 min. A standard inoculum of 2 × 10 5 cells/mL was prepared [23]. L. domatiophorus essential oil was diluted in Diamond's TYM medium from 2% to 0.002% (v/v). 100 µL of microbial culture was added to 100 µL at each concentration of different samples in 96-well plates. Sterile distilled water was used as a growth control. The culture plate was placed in a regular 37 • C incubator and examined after 1, 4, 24 and 48 h. Viable T. vaginalis cells were identified and counted upon microscopy, based on the morphology and motility. The MLC was defined as the lowest essential oil concentration in which no motile organism was observed. The IC 50 and IC 90 values were considered as the oil concentration, in which 50% and ≥90% T. vaginalis cells were killed. A positive growth control, consisting of organisms in broth, and a negative sterility control consisting of uninoculated broth, were included for each assay. Each assay was repeated independently at least twice [23]. The following viruses were purchased from the American Type Culture Collection (ATCC), with the exception of Human Immunodeficiency Virus type-1 (HIV-1) and yellow fever virus (YFV):IIIB laboratory strain of HIV-1, was obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983); yellow fever virus (YFV) (strain 17-D vaccine (Stamaril Pasteur J07B01)); bovine viral diarrhoea virus (BVDV) (strain NADL (ATCC VR-534)); coxsackie type B4 (CV-B4) (strain J.V.B. (ATCC VR-184)); human enterovirus C (poliovirus type-1 (Sb-1) (Sabin strain Chat (ATCC VR-1562)); vesicular stomatitis virus (VSV) (lab strain Indiana (ATCC VR 1540)); human respiratory syncytial virus (hRSV) (strain A2 (ATCC VR-1540)); reovirus type-1 (Reo-1) (simian virus 12, strain 3651 (ATCC VR-214)), vaccinia virus (VV) (vaccine strain Elstree-Lister (ATCC VR-1549)); human herpes 1 (HSV-1) (strain KOS (ATCC VR-1493)).

Cells and Viruses
Cell cultures were checked periodically for the absence of mycoplasma contamination with MycoTect Kit (Gibco). The virus stocks were maintained in our laboratory and propagated in appropriate cell lines and aliquots were stored at −80 • C until use.

Cytotoxicity Assays
Exponentially growing MT-4 cells were seeded in 96-well plates, at a density of 4 × 10 5 cells/mL in RPMI-1640 medium, 100 units/mL penicillin G and 100 µg/mL streptomycin and supplemented with 10% fetal bovine serum (FBS). BHK-21 cells were seeded at 6 × 10 5 cells/mL in 96-well plates, in Minimum Essential Medium with Earle's salts (MEM-E), L-glutamine, 1mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% fetal bovine serum (FBS). MDBK cells were seeded at 1 × 10 6 cells/mL in 96-well plates, in Minimum Essential Medium with Earle's salts (MEM-E), L-glutamine, 1 mM sodium pyruvate and 25mg/L kanamycin, supplemented with 10% horse serum. Vero-76 cells were seeded at an initial density of 5 × 10 5 cells/mL in 96-well plates, in Dulbecco's Modified Eagle Medium (D-MEM) with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then incubated at 37 • C with an atmosphere of 5% CO 2 , in the absence or presence of serial dilutions of test essential oil. The medium employed for the cytotoxic assay, as well as for the antiviral assay, contained 1% of the appropriate serum. Cell viability was verified at 37 • C by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) method after 72 h for BHK-21, MDBK and Vero-76 or 96 h for MT-4 [24]. The cytotoxic activity of leaves' essential oil of L. domatiophorus was evaluated in parallel with its antiviral activity, through the viability of mock-infected, treated cells, as determined by the MTT method. Essential oil's activity against YFV, and Reo-1 was based on the inhibition of virus-induced cytopathogenicity in BHK-21 cells, acutely infected at an m.o.i. of 0.01. Essential oil's activity against BVDV was based on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected at an m.o.i. of 0.01. Briefly, BHK and MDBK cells were seeded in 96-well plates, at a density of 5 × 10 4 and 3 × 10 4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 • C in a humidified CO 2 (5%) atmosphere. Cell monolayers were then infected with 50 µL of a proper virus dilution in MEM-E, then 50 µL of medium, with or without serial dilutions of the essential oil, were added. After a 3, or 4 -day incubation at 37 • C, cell viability was measured by the MTT method [24]. Compound's activity against CV-B4, Sb-1, VV, VSV, hRSV A2 and HSV-1 was determined by plaque reduction assays in infected cell monolayers, as described previously [25]. Briefly, the monolayer of Vero-76 cells was grown overnight on a 24-well plate. The cells were then infected for 2 h with 250 µL of proper virus dilutions, to give 50-100 PFU/well. After the incubation period, the unadsorbed virus was removed, 500 µL of D-MEM containing 0.75% methyl-cellulose, with serial dilutions of test products, were added. The overlayed medium was also added to not treat wells as non-infection controls.
Cultures were incubated at 37 • C for a period of 2 (Sb-1 and VSV), 3 days (CV-B4, hRSV A2, VV, and HSV-1), and then fixed with PBS containing 50% ethanol and 0.8% crystal violet, washed and air-dried. The number of plaques in the control (no inhibitor) and experimental wells were then counted.

Linear Regression Analysis
The extent of cell growth/viability and viral multiplication, at each drug concentration tested, were expressed as percentages of untreated controls. Concentrations resulting in 50% inhibition (CC 50 or EC 50 ) were determined by a linear regression analysis using data from three independent experiment performed in duplicate.

Extraction Yield and Chemical Composition of Essential Oil
In L. domatiophorus leaves, the average yield of hydrodistilled essential oil was of 0.34 ± 0.01%, calculated on a dry weigh of three samples. The obtained essential oil was a pale, yellow liquid with odor and lighter than water. The GC/MS analysis indicated that the leaves' essential oil contained 52 constituents representing 96.74% of the total oil content ( Table 1). The main classes of compounds in this oil were sesquiterpene hydrocarbons (74.04%), oxygenated sesquiterpenes (22.01%). The constituents accounted for higher amounts in the leaves oil of L. domatiophorus were viridiflorene (16.47%), (-)-δ-cadinene (15.58%) and γ-muurolene (8.00%). The other components found at lower concentration were α-muurolene (5.45%), γ-cadinene (5.18%), (+)-aromadendrene (3.82%), α-cadinol (3.59%) and globulol (3.10%). In addition, the oil also contained one aldehyde, nonanal (0.02%) and one alcohol, cis-α-ambrinol (0.51%), having a non-terpenic structure. To the best of our knowledge, this is the first scientific report about chemical composition of the essential oil from L. domatiophorus . Data are the mean of three replicates ± SD. a Retention index (Kovalts) relative to n-alkanes (C 9 -C 22

Antimicrobial Activities
The antimicrobial activities of essential oil in L. domatiophorus were displayed in Table 2. The results exhibited potential antibacterial activities of the essential oil against i) Gram-positive bacterium: S. aureus (two strains) with MIC of 0.25% (v/v) and MLC of 0.5% (v/v), and E. faecalis with MIC and MLC were both 1% (v/v); Gram-negative bacteria such as E. coli (two strains) with MIC and MLC from 2 to 8% (v/v), P. aeruginosa (two strains) having MIC and MLC between 2 and 4% (v/v), and K. pneumoniae with MIC and MLC both equivalent to 4% (v/v); Candida species showed MIC and MLC as follows: C. albicans, with MIC and MLC were both 4% (v/v), C. glabrata, with MIC and MLC were both 2% (v/v), C. tropicalis, with MIC and MLC of 1% and C. parapsilosis, with MIC and MLC both equivalent to 0.12% (v/v).

Antitrichomonas Activity
As shown in Table 3, the leaves essential oil of L. domatiophorus showed potential in vitro antitrichomonas activity. Essential oil of L. domatiophorus indicated a remarkable antitrichomonas activity against T. vaginalis, and the values IC 50 , IC 90 , MLC were time-dependent at 1, 4, 24 and 48 h. The obtained results suggested the efficacy of the essential oil after 1 h of incubation, and trichomonas was strongly inhibited after 24h. In particular, the effect of the agent increased, the values IC 50 , IC 90 and MLC of L. domatiophorus essential oil from leaves were 0.008, 0.016, and 0.03% (v/v), respectively, after 48 h of incubation.

Antiviral Activity
Here, we explored the antiviral properties of leaves' essential oil of L. domatiophorus against a broad spectrum of RNA (Herpesviridae). In order to be able to establish whether test L. domatiophorus oil were endowed with selective antiviral activity, their cytotoxicity was evaluated in parallel assays with uninfected cell lines. In vitro cytotoxicity was measured based on cell proliferation and viability. The CC 50 (drug concentration inhibiting cell growth by 50% referred to untreated control) was > 100 µg/mL and no cell toxic effect was observed (Table 4). However, results obtained from our screening pointed out L. domatiophorus essential oil was completely ineffective against the tested viruses with EC 50 values over 100 µg mL −1 .
The Annonaceae family was documented by Jussieu in 1789 [13]. Their essential oils predominantly constituted monoterpenes, sesquiterpenes, and alkaloids, especially isoquinoline alkaloids [13]. Several compositions of essential oils have been commonly reported, for example α-pinene, β-pinene, limonene, p-cymene, β-caryophyllene and caryophyllene oxide [14]. Nonetheless, the components of essential oils can vary upon specific species and their distributions in distinct geographic regions [35,36]. In the present work, the main components of L. domatiophorus essential oil was reported, such as (-)-δ-cadinene, γ-muurolene and α-cadinol. These compounds were also found with high concentration in essential oils of some species belonging to Annonaceae family, namely Anaxagoma dolichocarpa [14], Annona salzmannii [37], Annona muricata [38], Xylopia pynaertii [39] and Cananga odorata [40]. Additionally, the main ingredients in essential oil of Xylopia frutescens were viridiflorene and δ-cadinene [41], while such of Xylopia laevigata were δ-cadinene and γ-muurolene [42]. Even though some of these volatile compounds can be present in essential oils of other families, the similarity of the main constituents in essential oils of species in the same family suggested that they have an important chemotaxonomic relevance [42].
Moreover, L. domatiophorus essential oil exhibited the strongest antimicrobial activity against S. aureus and Candida species, which was observed to be in line with previous studies on antimicrobial activity of Annonaceae species. Several work demonstrated that A. salzmannii, A. senegalensis and X. aethiopica essential oils could inhibit S. aureus [43][44][45], while such of A. vepretorum and X. aethiopica possessed the inhibiting capacity towards Candida species [44,46]. The biological activities of essential oils, mainly focused on the antibacterial, antifungal and antioxidant activities [1,3]. Only a few essential oils, especially some species of the Lamiaceae family, have been investigated against Trichomonas [47][48][49][50][51]. Notably, our work on the antitrichomonas activity of L. domatiophorus oil, for the first time, demonstrated an effective inhibition against T. vaginalis.
Until now, the efficacy of some essential oils has been explored against S. aureus and C. parapsilosis. A report of 105 clinical isolates showed Melaleuca alternifolia oil against S. aureus with MIC 90 of 0.5% (v/v) [52]. A later study was conducted on 100 clinical isolates of methicillin-resistant S. aureus found the MIC 90 of M. alternifolia oil at 0.32% (v/v) [52]. The antimicrobial activity against S. aureus of Carum carvi, Pogostemon cablin and Pelargonium graveolens essential oils were observed [53]. The MIC values of C. carvi, P. cablin and P. graveolens oils were 1.88 ± 1.03; 0.17 ± 0.08 and 0.54 ± 0.20% (v/v) respectively [53]. Herein however, it can be seen that L. domatiophorus oil displayed higher inhibition against S.aureus with MIC and MLC of 0.25% and 0.5% (v/v) respectively, compared to that of M. alternifolia, C. carvi, P. cablin and P. graveolens oils. According to Mondello et al, M. alternifolia oil inhibited Candida strains with MIC ranging from 0.03% to 0.25% [54]. On the contrary, L. domatiophorus essential oil displayed promising inhibition against Candida species, especially in C. parapsilosis with MIC and MLC values of 0.12% (v/v). As a result, L. domatiophorus oil is very likely to become an alternative therapeutic agent for infectious diseases, yet further microbiological tests and clinical trials should be assessed. Table 4. Cytotoxicity and antiviral activity of essential oil from L. domatiophorus against representatives of ssRNA + (HIV-1,YFV, BVDV, Sb-1, CV-B4), ssRNA − (hRSV A2, VSV), dsRNA (Reo-1), and dsDNA (HSV-1, VV) viruses.  Several types of plant-extracted compounds may display antimicrobial activities. They are synthesized to protect the plants from external pathogens [55]. Under particular biotic/abiotic stress conditions, their chemical constituents are released via a plethora of molecular interactions [56]. Each composition exhibits various mechanisms of actions against bacteria [57], thus exerting different effects on the ultimate antimicrobial properties of essential oils [58]. In other words, essential oils containing different chemical compositions are prone to disrupt bacteria in different pathways [55]. Additionally, hydrophobicity plays a crucial role in essential oils, contributing to an increased permeation through the cell membrane. This is likely to result in the spillage of ions and molecules, and consequently to cell death [59]. In general, Gram-positive bacteria are more sensitive to essential oils than Gram-negative bacteria because the cell wall of Gram-positive bacteria is less complex than Gram-negative ones [58,60]. In our study, the antimicrobial activities of the essential oil could potentially stem from their main components, such as (-)-δ-cadinene. Several studies have demonstrated that plants containing (-)-δ-cadinene as the main compound display good antibacterial activity [61][62][63][64]. In addition, the presence of many individual antimicrobial components in the essential oil is more likely to produce a synergy, and that amplifying the antimicrobial activities of the essential oil as a whole [35,65].

Cell Lines and
Multidrug resistance has become a huge challenge of healthcare worldwide, remaining one of the leading causes of human mortality over the past few years [66]. Bacteria, fungi and parasites have consecutively been developing numerous resistant mechanisms against current antibiotics, hampering the success of anti-infectious therapies, and thus leaving severe consequences on patients' health [67][68][69][70]. In addition, the use of synthetic chemicals to control microorganisms is still limited due to their carcinogenic effects, acute toxicity and environmental hazards [55]. Hence, a demand for new antibiotics is urgently raised among scientific community to address multidrug resistance [58]. The therapeutic agents from herbal medicines have long emerged as a potential natural source for treating infectious diseases [23,71]. Herein, the antimicrobial, antitrichomonas and antiviral effects of essential oil from L. domatiophorus were first studied, showing strong activity against the studied strains, in particular S. aureus, E. faecalis, Candida species and T. vaginalis. There by, the essential oil of L. domatiophorus can be employed in the development of new anti-infectious agents, thanks to its strong bactericidal effects.

Conclusions
The fresh leaves essential oil of L. domatiophorus after collecting from Thua Thien Hue Province, Vietnam was composed of 52 constituents in which viridiflorene (16.47%), (-)-δ-cadinene (15.58%) and γ-muurolene (8.00%) were three main components. L. domatiophorus essential oil displayed antimicrobial activities against two Gram-positive strains, S. aureus and E. faecalis, with MIC and MLC values from 0.25 to 1% (v/v); three Gram-negative bacteria, E. coli, P. aeruginosa and K. pneumoniae, with MIC and MLC values between 2 and 8% (v/v); and finally Candida species, having MIC and MLC between 0.12 and 4% (v/v). The oil also exhibited repellency against T. vaginalis with IC 50 , IC 90 and MLC values of 0.016, 0.03 and 0.06% (v/v). It was ineffective against HIV-1, YFV, BVDV, Sb-1, CV-B4, hRSVA2, VSV, Reo-1, HSV-1, VV viruses. Further studies should be done to evaluate the safety and toxicity of L. domatiophorus essential oil in animals, before considering the development of new anti-infectious agents for use in clinical trials.

Data Availability:
The data used to support the findings of this study are available from the corresponding author upon request.