Biological activities of essential oil extracted from leaves of Atalantia sessiflora Guillauminin Vietnam

Introduction: The present study aimed to determine the chemical compositions and bioactivities of the essential oil of Atalantia sessiflora Guillaumin (A. sessiflora), including antibacterial, antimycotic, antitrichomonas, anti-inflammatory and antiviral effects. Methodology: The essential oil from leaves of A. sessiflora was extracted by hydrodistillation using a Clevenger apparatus. Chemical compositions of oil were identified by GC/MS. Antimicrobial and antitrichomonas activity were determined by the microdilution method; antiinflammatory and antiviral were determined by the MTT method. Results: The average yield of oil was 0.46 ± 0.01% (v/w, dry leaves). A number of 45 constituents were identified by GC/MS. The essential oil comprised four main components. The oil showed antimicrobial activities against Gram-positive strains as Staphylococcus; Gram-negative bacteria such as Klebsiella pneumoniae and Escherichia coli; and finally four Candida species. Enterococcus faecalis and Pseudomonas aeruginosa were least susceptible to the oil of A. sessiflora, as seen in their MIC and MLC values over 16% (v/v). Activity against Trichomonas vaginalis was also undertaken, showing IC50, IC90 and MLC values of 0.016, 0.03 and 0.06% (v/v) respectively, after 48 hours of incubation. The oil of A. sessiflora displayed activity against the nitric oxide generation with the IC50 of 95.94 ± 6.18 μg/mL. The oil was completely ineffective against tested viruses, ssRNA+, ssRNA-, dsRNA, and dsDNA viruses. Conclusions: This is the first yet comprehensive scientific report about the chemical compositions and pharmacological properties of the essential oil of A. sessiflora. Further studies should be done to evaluate the safety and toxicity of A. sessiflora oil.


Introduction
Atalantia Corrêa genus belong to the family Rutaceae which is composed of 17 species. The plants of this genus are found in tropical and subtropical regions of Southeast Asia and China [1,2].A. sessiflora is one of the 8 species of the genus Atalantia found in Vietnam. A. sessiflorais a shrub with green branches, spines or rarely unarmed [3]. Several species in the genus Atalantia for which the chemical compositions and bioactivities of their essential oils extractions have been well documented lately [4][5][6], yet none of such included A. sessiflora. Therefore, we aimed to identify the chemical composition and biological activities present in the essential oil of A. sessiflora which was collected in Quang Tri Province, Vietnam. To the best of our knowledge, this is the first scientific report regarding the chemical and biological analysis of essential oil in A. sessiflora.
flame-ionization detector and fitted with a 60 m × 0.25 mm, thickness 0.25 μm AT-5 fused SiO 2 capillary column (Alltech, Milan, Italy). MS analyses were carried out with an Agilent Technologies model 7820A connected with a MS detector 5977E MSD (Agilent, Santa Clara, USA). All reference standards, homologous series of n-alkanes (C 9 -C 22

Plant material
A. sessiflorawere collected from Dakrong District, Quang Tri Province, Vietnam in May 2019 (16°38′16.9″N 106°48′06.5″E) and were identified by Dr. Chinh Tien Nguyen, Vietnam National Museum of Nature. A voucher specimen (AS-03) was deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Vietnam.

Extraction of the essential oil
The leaves of A. sessiflora (5 Kg) were shredded and the essential oil was extracted by hydrodistillation using a Clevenger apparatus for 3.5 hours at normal pressure according to Vietnamese Pharmacopoeia [7]. The oil was then collected, dried by Na2SO4, and stored in sealed vials at 4 o C. The experiments were performed in triplicate.

Analysis of the essential oil
Three replicates of each sample were analyzed using a Hewlett-Packard Model 5890AGC equipped with a flame-ionization detector and fitted with a 60m × 0.25mm, thickness 0.25 μmAT-5 fused SiO 2 capillary column. Injection port and detector temperature were at 280°C. The column temperature was operated from 50 to 135°C at 5°C/minute (1 minute), 5°C/minute to 225°C (5 minutes), 5°C/minute to 260°C held for 10 minutes. The samples (0.1 μL each) generally analyzed without dilution (using 2,6-dimethylphenol as an internal standard), were injected using a split/splitless automatic injector HP 7673 and He as carrier gas. The quantification of each compound was displayed as absolute weight percentage using internal standard and response factors. The detector response factors (RFs) were determined for key components relative to 2,6dimethylphenol and assigned to other components based on similarity of functional groups and/or structures.
MS analyses were carried out with an Agilent Technologies model 7820A connected with a MS detector 5977E MSD using the same conditions and column described above. The column was connected to the ion source of the mass spectrometer. Mass units were monitored from 10 to 900 at 70 eV.
The retention indices (RI) of constituents were determined by co-injection with reference to a homologous series of n-alkanes (C 9 -C 22 ) under the same conditions to calculate the retention indexes with the generalized equation by Van del Dool and Kartz [8].
The identification of compounds was based on several methods [8]. 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.

Microbial strains and growth conditions
In the present work, a collection of 12  In order to establish the MIC and MLC of bacteria and Candida species, the broth microdilution method was used as suggested by the Clinical and Laboratory Standard Institute [9]. The inoculum was prepared by diluting colonies in salt solution at a concentration of 0.5 McFarland, and the concentration was 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. Oil concentrations were prepared by serial dilutions from 16% (v/v) to 0.0005% (v/v) and added with 0.5% Tween 80. 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 hours. MIC values were determined as the lowest concentration of the essential oil that inhibit the visible growth of the strains 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 hours 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.

Determination of anti-T.vaginalis activity
A 1% stock solution of essential oil from leaves of A. Sessiflora was prepared in Diamond's medium plus 4% of DMSO. Twofold serial dilutions of the stock solution in 100 µl of Diamond's TYM medium, ranging from 0.5% to 0.002% (v/v), were distributed in 96-well plates; 100 µl of the trichomonas suspension prepared as described above were then added to each well. The same dilutions of Diamond's TYM medium plus DMSO were used as a control. The culture plate was kept at 37 °C in a CO 2 incubator and checked after 1, 4, 24, and 48 hours. The percentage of viable T. vaginalis cells was observed by microscope. The MLC was defined as the lowest essential oil concentration at which no viable protozoa were observed. The 50% inhibitory concentration (IC 50 ) and ≥ 90% inhibitory concentration (IC 90 ) values were considered as the essential oil concentration at which 50% and ≥ 90% of T. vaginalis cells were killed, respectively. Each assay has been repeated independently at least two times [10].

Anti-inflammatory activity
Cell culture RAW 264.7 cell lines were cultured in DMEM supplemented with 2 mM L-Glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 10% FBS. Cells were maintained at 37ºC with 5% CO 2 in a humidified chamber. The cells were sub-cultured every 3-5 days with the ratio of (1:3) and incubated at 37°C under humidified 5% carbon dioxide atmosphere [11,12].

Inhibition of nitric oxide (NO) production
The inhibition of NO production was evaluated on different concentrations of essential oil of A. sessiflora (100, 20, 4, and 0.8 μg/mL). The RAW 264.7 cells were seeded at a density of 2×10 5 cells/well in 96-well plates and incubated for 24 hours at 37°C and 5% CO 2 . Then, the media of each well were aspirated and fresh FBSfree DMEM media were replaced for 3 hours. Tested samples were added carefully into each well of 96-well plates and the cultivation was continued under the same conditions. After 2 hours treatment, cells were stimulated with 1 μg/mL of LPS for 24 hours. The presence of nitrite was determined in cell culture media using commercial NO detection kit Griess Reagent System (Promega Cooperation, WI, USA). Protocols supplied with assay kit were used for the application of assay procedure. Briefly, 100 μL of cell culture medium with an equal volume of 100 μL Griess reagent: 50 μL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid, and 50 μL 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in a 96-well plate was incubated at room temperature for 10 minutes. Then the absorbance was measured at 540 nm in a microplate reader (Bio-Rad, California, USA). The amount of nitrite in the media was calculated from sodium nitrite (NaNO 2 ) standard curve. FBS-free DMEM media was used as blank sample while L-N G -monomethyl arginine citrate (L-NMMA) was used as positive control and macrophages stimulated with LPS at 1 μg/mL and untreated was used as negative control. The ability to inhibit the nitric oxide (NO) was measured at doses of 100, 20, 4, and 0.8 μg/mL, and estimated as a IC 50 which was calculated by the program Table Curve Version 4.0 [11,12].

Cells and viruses
Cell cultures were checked periodically for the absence of mycoplasma contamination with MycoTect Kit (Gibco, Waltham, Massachusetts, USA). Viruses were maintained in our laboratory and propagated in appropriate cell lines. The viruses were stored in small aliquots at -80 °C until use.

Cytotoxicity assays
Exponentially growing MT-4 cells were seeded at an initial density of 4 ×10 5 cells/mL in 96-well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G and 100 μg/mL streptomycin. BHK cells were seeded in 96-well plates at an initial density of 6×10 5 cells/mL, 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 in 96-well plates at an initial density of 1×10 6 cells/mL, in Minimum Essential Medium with Earle's salts (MEM-E), L-glutamine, 1mM sodium pyruvate and 25mg/L kanamycin, supplemented with 10% horse serum (MDBK). Vero-76 cells were seeded in 96-well plates at an initial density of 5×10 5 cells/mL, 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 in a humidified, 5% CO 2 atmosphere, in the absence or presence of serial dilutions of test compounds. The test medium used for the cytotoxic assay as well as for antiviral assay contained 1% of the appropriate serum. Cell viability was determined at 37 °C by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) method after 72 hrs for BHK, MDBK and Vero-76 or 96 hrs for MT-4 [13]. The cytotoxicity of test compounds was evaluated in parallel with their antiviral activity through the viability of mock-infected, treated cells, as monitored by the MTT method.

Antiviral assays
Essential oil's activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Compound's activity against YFV, BVDV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected at an m.o.i. of 0.01. After a 3, or 4 -day incubation at 37 °C, cell viability was determined by the MTT method [13]. 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 [14]. Briefly, monolayer of Vero-76 cells was grown overnight on 24-well plate. The cells were then infected for 2 hrs with 250 μL of proper virus dilutions to give 50-100 PFU/well. Following removal of unadsorbed virus, 500 μL of medium (D-MEM with L-glutamine and 4500 mg/L D-glucose, supplemented with 1% inactivated FBS) 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 2 (Sb-1 and VSV), 3 (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 percentage of untreated controls. Concentrations resulting in 50% inhibition (CC50 or EC 50 ) were determined by linear regression analysis.

Antimicrobial activities
The antimicrobial activities of essential oil of A. sessiflora were displayed in Table 1. The results exhibited potential antibacterial activities of the essential oil against i) Gram-positive bacterium: S. aureus (two strains) with MIC and MLC from 2 to 4% (v/v); ii) Gram-negative bacteria such as K.

Anti-Trichomonas vaginalis activity
As shown in Table 2, the essential oil obtained from leaves of A. sessiflora has a remarkable activity against T. vaginalis. Results obtained indicate a prompt effect, as demonstrated by the IC 50 after one hour of incubation (0.06%). The anti-trichomonas activity of A. sessiflora essential oil is time-dependent, reaching IC 50 , IC 90 and MLC values of 0.016, 0.03, and 0.06% (v/v) respectively, after 48 hours of incubation.

Anti-inflammatory activity
The essential oil from the leaves of A. sessiflorahave shown the ability to inhibit macrophage cells producing NO with IC 50 of 95.94 ± 6.18 µg/mL, while the IC 50 of positive control was of 7.61 ± 0.59 µg/mL ( Table 3). The anti-inflammatory effect of the oil may be attributed to the presence of high concentrations of linalool, linalyl acetate, E-βcaryophyllene and α-humulene, compounds with well documented anti-inflammatory potentials [15][16][17][18]. This is the first report about anti-inflammatory activity of essential oil from A. sessiflora.

Antiviral activity
Here, we explored the antiviral properties of leaves essential oil of A. sessiflora against RNA and DNA viruses belonging to different families, including several important human pathogens (Table 4). In order to be able to establish whether tested A. sessiflora 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 CC50 (drug concentration inhibiting cell growth by 50% referred to untreated control) was > 100µg/mL and no cell toxic effect was observed (Table 4). However, results obtained from our screening pointed out A. sessifloraessential oil was completely ineffective against the tested viruses with ED 50 values over 100µg mL -1 .

Discussion
Essential oils contain 20 to 60 compounds and nearly 85% of the essential oils are major components [19,20]. Each compound displays its own perfume and bioactivities on the human body [21]. The place of origin, climatic conditions, plant species and seasons are key factors affecting the chemical compositions of essential oils [22,23]. Some constituents are resulted from the decomposing process of the precursor components during distillation [24]. Furthermore, numerous components are generated from the hydrolysis of several compounds found in the plants [24]. In this study, the qualities and contents of the  essential oil from the leaves of A. sessiliflora from Quang Tri Province was significantly different from those in Thanh Hoa [25]. This might be attributed to the distribution of species in different geographic areas, analysis and identification methods of the constituents in the essential oil [17,18]. The leaves' essential oil of A. sessiliflora in Thanh Hoa was consisted of monoterpene hydrocarbons (40.8%) and oxygenated sesquiterpenes (27.7%), with major constituents were β-pinen (17.2%), limonen (9.7%), α-humulen (7.7%) and β-caryophyllen (7.6%). Generally, the content of essential oil from A. sessiliflora has previously been described just in one thesis [25], no more publications available about the composition of the essential oil. In comparison with previous studies in the chemical compositions of the essential oils from Atalantia species, Kathirvelu et al. [4] indicated that essential oil of Atalantia monophylla contains high content of sabinene, eugenol, 1,2-dimethoxy-4-(2methoxyethenyl)benzene, and β-asarone. This oil has also shown larvicidal and repellent activity against Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The main constituents of essential oil of Atalantia roxburghiana were p-cymene, γ-terpinene, α-pinene, and β-pinene [5]. Arun et al. [6] reported that T-cadinol, caryophyllene, and caryophyllene oxide are the main constituents in essential oil from Atalantia racemosa, while caryophyllene, decanal, and Dlimnonene found were found with large amounts in essential oil of Atalantia wightii.
In plants, essential oils play several important roles e.g. in pollination, or in a defense mechanism as a repellent or irritant. Studies have shown that essential oils include a large content of antioxidants donating hydrogen in oxidative reactions, especially in the presence of light. Essential oils were also recognized to exhibit antifungal and antibacterial properties hence preventing plants from possible pathogenic triggers [26][27][28]. Upon biotic/abiotic stress conditions, essential oils can release their chemical substituents through a range of molecular interactions [29] and each of them exhibits different mechanisms of actions on microorganism [30]. These mechanisms synergize and amplify one another to form many effective antimicrobial properties for essential oils [31]. As such, essential oils whose chemical compositions can inhibit or kill bacteria and fungi in different pathways [32]. For example, essential oils can inhibit bacteria in various approaches including triggering the degradation of bacterial cell wall, leakage of cell contents, membrane protein damage, disruption of cytoplasmic membrane, depletion of the proton motive force or coagulation of cytoplasm [30]. Additionally, the hydrophobicity of essential oils enables the permeation of essential oils into cell membrane, and thus facilitating the spillage of ions and molecules out of the cells and cell apoptosis occurs [33]. In our study, the antimicrobial activities of the essential oil could potentially stem from their main components such as linalool and E-β-caryophyllene as their antimicrobial potentials were well-reported Table 4. Cytotoxicity and antiviral activity of essential oil from A. sessiflora against representatives of ssRNA + (HIV-1, YFV, BVDV, Sb-1, CV-B4), ssRNA -(RSV, VSV), dsRNA (Reo-1), and dsDNA (HSV-1, VV) viruses.
Data represent mean values + SD for three independent determinations. For values where SD is not shown, variation among duplicate samples was less than 15%. Oil: essential oil from the leaves of A. sessiflora; RC1: Efavirenz; RC2: 2′-C-methylguanosine; RC3: Pleconaril; RC4: 6-aza-uridine; RC5: Acycloguanosine; RC6: Mycophenolic acid; RC7: 2′-C-methylcytidine. a Compound concentration (µg ml -1 ) required to reduce the proliferation of mockinfected MT-4 cells by 50%, as determined by the MTT method. b Compound concentration (µg ml -1 ) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the MTT method. c Compound concentration (µg ml -1 ) required to reduce the viability of mock-infected BHK cells by 50%, as determined by the MTT method. d Compound concentration (µg ml -1 ) required to achieve 50% protection of BHK cells from YFV or Reo-1 induced cytopathogenicity, as determined by the MTT method. e Compound concentration (µg ml -1 ) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method. f Compound concentration (µg ml -1 ) required to achieve 50% protection of MDBK cells from BVDV induced cytopathogenicity, as determined by the MTT method. g Compound concentration (µg ml -1 ) required to reduce the viability of mock-infected Vero-76 cells by 50%. as determined by the MTT method. h Compound concentration (µg ml -1 ) required to reduce the plaque number of RSV, VSV, HSV-1, VV, Sb-1, and CV-B4 by 50% in Vero-76 monolayers. * Reference Compds:CC 50 and EC 50 are in µM. [34,35]. Furthermore, the presence of many individual antibacterial 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 [36,37].
Candida is referred to a relatively common yeast in the human mucosa, present in the digestive, and reproductive tract as well as oral cavity. Approximately 80% of the human population may be exposed to the Candida infection. Candida infections usually display a broad spectrum from superficially oral thrush and vaginitis to candidemia [38]. Fluconazole is a triazole antifungal and used to treat Candida infections. Fluconazole has fungistatic effect making it an ideal condition the emergence of the resistant strains [39]. Some clinical isolates of Candida species have been resistant against fluconazole and other triazole antifungal drugs. Therefore, the demand for newly effective antifungals is urgently required [40][41][42]. Natural products have long been known as key therapeutic agents providing many potent bioactivities while exhibiting high safety profiles. Accordingly, essential oils have gained growing attention as a powerful fungal inhibitor [43]. It was reported some essential oils yield antimicrobial activity against Candida species, including tea tree [44,45], thyme, clove [43], lemongrass, geranium, cinnamon, Japanese mint, ginger grass, and motiarosha essential oils [46]. Here, the current work showed that essential oil from leaves of A. sessiflora were effective against C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis with MIC and MLC values from 0.5 to 2% (v/v).
T. vaginalis has been known as one of the most nonviral sexually transmitted infection worldwide, with an estimated 156 million cases of infections in 2016. The infection is ubiquitous, but it is mostly diffused in the depeloping countries [47,48]. T. vaginalis can cause a severe vaginitis, even if 50% of women shows no clear symptoms. Infection is associated to severe pregnancy complications, such as premature birth, premature rupture of membranes and low birth weight [49,50]. Trichomoniasis also contributes to an increasing risk of human immunodeficiency virus (HIV) infection and prostate cancer acquisition [51,52]. Metronidazole is recognized as the first-line drug option for T. vaginalis infections; nonetheless, the last few years have witnessed the new emergences of many resistant microbes very likely resulting in poor treatment efficacies [53]. To this end, plant-based products are of great interest for the treatment of T. vaginalis and other parasites attributed to relatively similar therapeutic proficiency and high safety profile. Up to now, a limited number of essential oils have been tested in vitro on T.vaginalis, including [44,[54][55][56][57][58]. Among them, Ocimum basilicum shows one of the most efficient antitrichomonas activity, with 100% inhibition of of parasitic growth at a concentration of 30 mg/ml after 24 h of incubation [59].In the present work,the authors demonstrated that essential oil of A. sessiflora display a very good inhibitory effect on T. vaginalis, with IC50 value of 0.016% (v/v) after 48 hours, corresponding to 120 µg/ml. Impressively, A. sessiflora oil against T. vaginalis was quickly exhibited only 1 h following incubation as the values of IC 50 , IC 90 and MLC were in turn at 0.06, 0.12 and 0.25% (v/v).
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 [30,[31][32][33][34]. In addition, the use of synthetic chemicals to control microorganisms is still limited due to their carcinogenic effects, acute toxicity and environmental hazards [32]. The therapeutic agents from herbal medicines have long emerged as a potential natural source for treating infectious diseases [11,36,37]. Herein, the antimicrobial, antitrichomonas, anti-inflammatory, and antiviral effects of essential oil from A. sessiflora were first studied, showing strong activity against K. pneumoniae, Candida species, and T. vaginalis.Therefore, the essential oil of A. sessifloracan be employed in the development of new anti-infectious agents thanks to its strong bactericidal effects.

Conclusions
The dried leaves essential oil of A. sessiflora after collecting from Quang Tri Province, Vietnam was composed of 45 constituents in which linalool (16.21%), E-β-caryophyllene (11.01%), ledene (8.59%), α-humulene (8.02%) were 4 main components. A. sessiflora essential oil displayed antimicrobial activities against one Gram-positive strain, S. aureus, two Gram-negative bacteria, K. pneumoniae, E. coli, and finally four Candida species. Meanwhile, E. faecalis and P. aeruginosa were the least susceptible to the essential oil of A. sessiflora.The oil also exhibited repellency against T. vaginalis and inhibited macrophage cells producing NO. It was ineffective against HIV-1, YFV, BVDV, Sb-1, CV-B4, RSV, VSV, Reo-1, HSV-1, VV viruses. Further studies should be done, such as investigating pharmacological effects of major components of A. sessiflora essential oil, testing on other cells or pathogens, studying on resistant strains, elucidating the mechanisms of action of A. sessiflora oil, and evaluating the safety and toxicity of A. sessiflora oil in animal and human before considering the development new anti-infectious agents for using in clinical settings.