Biological Activities of Essential Oils from Leaves of Paramignya trimera (Oliv.) Guillaum and Limnocitrus littoralis (Miq.) Swingle.

The present study aimed to determine the bioactivities of essential oils extracted from the leaves of Paramignya trimera and Limnocitrus littoralis, including cytotoxicity, antiviral, antibacterial, antimycotic, and antitrichomonas effects. Herein, it was indicated that P. trimera and L. littoralis oils showed no cytotoxicity on normal cells, namely MT-4, BHK-21, MDBK, and Vero-76. P. trimera oil (i) exhibited the strongest inhibition against Staphylococcus aureus with MIC and MLC values of 2% (v/v); (ii) showed MIC and MLC values of 8% (v/v) in Candida parapsilosis; and (iii) in the remaining strains, showed MIC and MLC values greater than or equal to 16% (v/v). On the other hand, L. littoralis oil (i) displayed the strongest inhibition against Candida tropicalis and Candida parapsilosis with 2% (v/v) of MIC and MLC; and (ii) in the remaining strains, possessed MIC and MLC greater than or equal to 16% (v/v). In addition, antitrichomonas activities of the oils were undertaken, showing IC50, IC90, MLC values, respectively, at 0.016%, 0.03%, and 0.06% (v/v) from P. trimera, and 0.03%, 0.06%, 0.12% (v/v) from L. littoralis, after 48 h of incubation. The oils were completely ineffective against ssRNA+ (HIV-1, YFV, BVDV, Sb-1, CV-B4), ssRNA- (RSV, VSV), dsRNA (Reo-1), and dsDNA (HSV-1, VV) viruses. This is the first report describing the cytotoxicity, antiviral, antibacterial, antimycotic, and antitrichomonas activities of the essential oils of P. trimera and L. littoralis.


Introduction
Essential oils, also known as volatile oils or ethereal oils, are aromatic oily liquids extracted from different parts of the plants [1] and contain a wide range of chemical compounds, mainly terpenes and terpenoids [2]. The number of identified essential oils has reached 3000, and most have been used in many applications, namely pharmaceuticals, fragrances, and the food industry [3]. Some of these have been used to treat certain organ and systemic disorders [4]. Essential oils exhibit powerful antibacterial, antifungal, antioxidant, antiviral, anti-inflammatory, and anticancer properties [5][6][7].

Antimicrobial Activities
The antimicrobial activities of essential oils from P. trimera and L. littoralis are displayed in Table 2. In general, Gram-positive bacteria were more sensitive to P. trimera oil than Gram-negative bacteria, whereas L. littoralis oil did not show any significant activity on both Gram-positive and Gram-negative strains. For Candida species, P. trimera and L. littoralis oils indicated the best antimicrobial activity against Candida parapsilosis (C. parapsilosis). Particularly, for P. trimera oil, i) it exhibited strongest inhibition against Staphylococcus aureus (S. aureus) with Minimum Inhibitory Concentrations (MIC) and Minimum Lethal Concentrations (MLC) values of 2% (v/v); ii) it showed MIC and MLC values of 8% (v/v) in C. parapsilosis; iii) the remaining strains had MIC and MLC values greater than or equal to 16% (v/v). For L. littoralis oil, i) it displayed strongest inhibition against Candida tropicalis (C. tropicalis) and C. parapsilosis with 2% (v/v) MIC and MLC; ii) the remaining strains possessed MIC and MLC more than or equal to 16% (v/v). The microbicidal or microbiostatic action is indicated via the ratio of MLC/MIC, whose value less than or equal to 4.0 shows a microbicidal effect, whilst that greater than 4.0 shows that it exerts a microbiostatic action [15,16]. Herein, the MLC/MIC ratios obtained from the experimental data were all equal to 1, proving that both P. trimera and L. littoralis essential oils showed bactericidal and fungicidal properties on the studied strains. Table 2. Antimicrobial activities (MIC and MLC) of essential oils from the leaves of P. trimera and L. littoralis.

Strains
P. trimera Oil L. littoralis Oil

Antitrichomonas Activity
As shown in Table 3, both tested essential oils have a cytotoxic activity against Trichomonas vaginalis (T. vaginalis). It can be observed that the values of 50% inhibitory concentration (IC 50 ), ≥90% inhibitory concentration (IC 90 ) and MLC values against T. vaginalis were time-dependent in the given period. Essential oils showed a weak effect after 1 h of incubation, which promptly increased over time. Both P. trimera and L. littoralis essential oils strongly affected T. vaginalis viability at 24 h with a 64-and 32-fold increase, respectively, compared to 1 h. At 48 h, the antitrichomonas activity of P. trimera and L. littoralis oils increased 2 times and 4 times more than those of 24 h, respectively. In particular, the leaves-extracted oils had IC 50 , IC 90 , and MLC values, respectively, at 0.016%, 0.03%, and 0.06% (v/v) from P. trimera, and 0.03%, 0.06%, 0.12% (v/v) from L. littoralis after 48 h.

Discussion
Essential oils comprise a myriad of more than 20 active molecules with variable low molecular weight at distinct concentrations [17]. Essential oils can exert antimicrobial properties thanks to the complex interactions between their constituents, including phenols, alcohols, aldehydes, ketones, esters, ethers, and hydrocarbons [17], or being associated with their major components [18,19]. Each substituent of the essential oil displays a variety of mechanisms of action or cellular pathways against microorganisms [1] and modulates the effects of other components present in the same essential oil [17]. Therefore, a broad range of different chemical compositions in the essential oils are likely to result in their disparate biological properties. As an example, essential oils bearing high amounts of phenolic derivatives such as carvacrol and thymol were demonstrated to exhibit strong antimicrobial effect against bacteria [1]. In addition, the abundant oxygenated monoterpenes in essential oils were reported to show higher antibacterial effect compared to those with monoterpene hydrocarbons [20]. The essential oil of P. trimera consisted of two main classes: oxygenated sesquiterpenes (41.7%) and sesquiterpene hydrocarbons (39.6%) with major constituents, namely β-caryophyllene (10.5%), β-caryophyllene oxide (9.9%), 7-epi-α-eudesmol (7.6%), and γ-muurolene (6.8%) [12]. On the other hand, the main compounds of L. littoralis oil were classified into two main categories: sesquiterpene hydrocarbons (32.3%) and monoterpene hydrocarbons (27.7%), in which myrcene (24.9%), γ-muurolene (11.0%), and oleic acid (10.3%) were found as major constituents [14]. In the present work, it can be seen that the differences of chemical compositions between P. trimera and L. littoralis essential oils, especially their main constituents ultimately induced the distinct antimicrobial activities.
Among the above-studied bacteria, the P. trimera essential oil showed enhanced sensitivity in Gram-positive bacteria than Gram-negative ones. In fact, Gram-negative bacteria are made up of more complex cell walls than those of Gram-positive bacteria, thus rendering Gram-positive bacteria more susceptible to penetration of the essential oil to inhibit the bacteria [21,22]. Herein, we indicated that the essential oil of P. trimera was more effective against S. aureus compared to Enterococcus faecalis, while L. littoralis essential oil did not show any activity towards Gram-positive bacteria. This is hypothetically attributed to the difference in chemical compositions of these oils. The antibacterial activity of P. trimera essential oil against S. aureus was likely due to its major compounds such as β-caryophyllene and β-caryophyllene oxide. According to Dahham et al. [23], β-caryophyllene from Aquilaria crassna essential oil inhibited S. aureus with an MIC value of 3 ± 0.4 (µM). Ali et al. [24] demonstrated that Teucrium yemense essential oil contains high levels of β-caryophyllene and that caryophyllene oxide has the ability to inhibit S. aureus with an MIC value of 0.156 mg/mL.
S. aureus is one of the most common causes of nosocomial infections and postsurgical wound contamination. It is also considered as one of the main etiologic contributions in food-related infections. Moreover, the development of S. aureus-resistant methicillin has gained rapidly growing attention over the last decade [25]. In the context of traditional medicine research, some studies have focused on investigating the efficacy of some essential oils against both Methicillin-sensitive and Methicillin-resistant S. aureus. It was reported that Melaleuca alternifolia oil was effective against S. aureus with MIC 90 of 0.5% (v/v) and Methicillin-resistant S. aureus with MIC 90 at 0.32% (v/v) [26].
Kwiatkowski et al. [27] indicated that Carum carvi, Pogostemon cablin, and Pelargonium graveolens essential oils showed efficiency against S. aureus with MIC values of 1.88 ± 1.03, 0.17 ± 0.08, and 0.54 ± 0.20% (v/v), respectively. The growth of the standard and clinical isolates of Methicillin-resistant S. aureus and Methicillin-sensitive S. aureus were inhibited by Zataria multiflora essential oil at concentrations of 0.55 to 1.41 µL/mL [25]. On the other hand, in the current work, P. trimera oil displayed inhibition against S. aureus with MIC and MLC of 2% (v/v) and exhibited no cell toxic effect at the same concentration, and thus P. trimera oil could be used safely at this dose. However, further testing needs to be done to study the mutagenicity, teratogenicity, or other side effects before considering using it as an agent against S. aureus.
Candida species are involved in mucocutaneous infections and are considered as one the most common causes of blood-stream infections. However, over the last decade, some clinical isolates of Candida species have been developing great resistance against the conventional use of triazole antifungal drugs such as itraconazole and fluconazole; therefore, the demand for newly effective antifungals is urgently needed [25]. Indeed, essential oils can be employed as anti-Candida agents against azole-resistant strains [28]. Vishnu et al. [28] investigated 30 essential oils against C. albicans, of which 18 were found to be effective; particularly eucalyptus and peppermint oils were proved to be the most effective. Nidhi et al. [29] reported the antimicrobial activities of olive and cinnamon oils on 100 Candida isolates, and around 50% of these Candida species exhibited sensitivity against the two studied oils. C. tropicalis and C. parapsilosis were more sensitive to the L. littoralis oil compared to the other Candida species tested, and C. parapsilosis was more sensitive to the P. trimera oil compared to the other Candida species in the present study. Given the experimental data obtained in this case and our previous works [30,31], the resistance may vary depending on the species of Candida and the essential oils. The current work indicated that L. littoralis oil was effective against C. tropicalis and C. parapsilosis with MIC and MLC of 2% (v/v), while exerting no cytotoxicity at the same concentration. Therefore, L. littoralis essential oil can be used as a potent anti-Candida agent on C. tropicalis and C. parapsilosis.
T. vaginalis is a protozoan parasite and is infective agent in human vagina, prostate gland, and urethra [32]. T. vaginalis is a flagellated parasite affecting about 156 million people each year in the world [32]. Trichomoniasis infection may cause serious health consequences, especially for women [33]. The current treatment is being based upon 5-nitroimidazole [33]. However, the emergence of resistance has limited the effectiveness of this therapy [34,35]. Therefore, it is of critical importance to search for novel pharmaceutical alternatives to treat trichomoniasis successfully in clinical settings [36]. Only a few essential oils, especially some species of the Lamiaceae family, have been investigated against Trichomonas [32,[36][37][38][39]. Particularly, the essential oil of Marrubium vulgare displayed antitrichomonas activity with an average MIC value of 291 ± 136 µg/mL after 48 h of incubation [36]. Hayam et al. [37] investigated the in vitro effects of Ocimum basilicum essential oil on T. vaginalis trophozoites and showed its MLC values of 30, 20, and 10 µg/mL after 24 h, 48 h, and 96 h of incubation, respectively. The previous study has demonstrated that low (≤1%) concentrations of two Lavandula essential oils (L. angustifolia and L. intermedia) can completely eliminate T. vaginalis in vitro [39]. After Amomum tsao-ko essential oil treatment, a series of modifications were observed in T. vaginalis cells. For example, nuclear membrane was disrupted, nuclei were dissolved, ribosomes were reduced, rough endoplasmic reticulum dilated, various vacuoles appeared, and organelles disintegrated. Furthermore, the cell membrane was partially damaged, and a leak of cytoplasmic led to cell disintegration [40]. In this work, we observed a prompt cytopathic activity of P. trimera and L. littoralis essential oils against T. vaginalis. Our results suggest further investigation since they could represent promising trichomonacidal agents.
Multidrug resistance is of great concern around the world and has caused a large number of deaths worldwide over the last decade [41]. Every day, bacteria and viruses have been advancing their resistance mechanisms against our current anti-infectious medications at a dramatic pace, making it exceptionally difficult to cope with given the available tools [34,35,42,43]. Furthermore, the use of synthetic chemicals to control microorganisms is facing several limitations due to their carcinogenic effects, acute toxicity, and environmental hazards [2]. As a result, it is of utmost importance to design novel antibiotics with high efficiency and non-toxicity [21]. To this end, plant-based therapeutic agents have gained extensive attention as a potential natural source for treating infectious diseases over the last few years [31,40,44]. For this reason, the present work, for the first time, discussed the cytotoxicity, antiviral, antibacterial, antimycotic, and antitrichomonas effects of essential oils from P. trimera and L. littoralis, showing great activity against S. aureus, C. tropicalis, C. parapsilosis, and T. vaginalis. Further research is needed on the biological effects as well as the toxicity of P. trimera and L. littoralis essential oils, before considering using them as therapeutic agents to treat infectious diseases.

Plant Material
The leaves of P. trimera and L. littoralis were collected from Hue and Quang Ngai provinces, Vietnam, respectively, in May 2019. Plant samples were identified by Dr. Chinh Tien Vu, Vietnam National Museum of Nature. Two voucher specimens (TTH-T110 and QNG-T112) were deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Vietnam.

Extraction of the Essential Oils
The leaves of P. trimera and L. littoralis were shredded, and the essential oils were hydrodistilled for 3.5 h at ambient pressure using a Clevenger-type apparatus [45]. The oils were dried on Na 2 SO 4 and stored in sealed vials, at 4 • C, ready for the biological activities test. MT-4, BHK-21, MDBK, and Vero-76 cells were seeded at 4 × 10 5 cells/mL, 6 × 10 5 cells/mL, 1 × 10 6 cells/mL, and 5 × 10 5 cells/mL, respectively. Cell cultures were treated with varying concentrations of essential oils ranging from 0.8-100 µg/mL. Equivalent DMSO concentration were also added as control and then incubated at 37 • C in a humidified, 5% CO 2 atmosphere. Cell viability was determined at 37 • C by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) method [46]. The test medium used for the cytotoxic assay as well as for antiviral assay contained 1% of the appropriate serum. DMSO was used as control in each experiment, and it was tested at the maximum concentration present in each compounds. The cytotoxicity of test compounds was evaluated in parallel with their antiviral activity.
Essential oil's activity against HIV-1 was evaluated as follow: 50 µL of RPMI-1640 containing 1 × 10 4 MT-4 cells were added to each well of flat-bottom microtitre trays, containing 50 µL of RPMI-1640 with or without serial dilutions of the essential oils. Then, 20 µL of an HIV-1 suspension containing 100 CCID 50 was added. Essential oil's activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected at an multiplicity of infection (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 oils. After a 3-or 4-day incubation at 37 • C, cell viability was measured by the MTT method [46].
Activities of essential oils and compounds 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 [47]. Briefly, Vero-76 cells were cultured in 24-well tissue culture plate and with approximately 50/100 PFU/well of virus. After 2 h on a rocker, the unadsorbed viruses were removed and replaced with 500 µL of D-MEM containing 0.75% methyl-cellulose, with serial dilutions of test products. The overlayed medium was also added to not treat wells as non-infection controls. Cultures were incubated at 37 • C 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.

Antimicrobial Activities
In the present work, a collection of 12 strains were selected, including 5 Gram-negative strains

Determination of Minimum Inhibitory Concentrations (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 [48]. 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 reading. The sensitivity test was implemented in Luria Bertani 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 and then incubated within 24 h for bacteria and 48 h for fungi at 37 • C. MIC values were determined by the lowest concentration of the essential oils in which bacterial growth is visibly inhibited after overnight incubation. In order to determine the MLC values, 10 µL was seeded on Mueller Hinton agar and Sabouraud Dextrose agar and the plates were incubated within 24 h for bacteria and 48 h for fungi 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%. A positive growth control consisting of organisms in broth and a negative sterility control consisting of uninoculated broth were included for each assay. Each experiment was performed in duplicate and repeated three times.
Essential oils from P. trimera and L. littoralis were serially diluted in 100 µL of Diamond's TYM medium from 16% to 0.002% (v/v) in 96-well plates, and 100 µL of the prepared Trichomonas suspension was added to each well. Diamond's TYM medium alone was used as a growth control. The culture plate was kept at 37 • C in a CO 2 incubator and checked after 1, 4, 24, and 48 h. The percentage of viable T. vaginalis cells was assessed by microscopic observation. The MLC was defined as the lowest essential oils concentration in which no viable protozoa were observed. The IC 50 and IC 90 values were considered as the oils concentration in which 50% and ≥90% T. vaginalis cells were killed. Each assay has been repeated independently at least two times [40].

Linear Regression Analysis and Statistical 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 (CC 50 or EC 50 ) were determined by linear regression analysis.
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.

Conclusions
The biological activities of P. trimera and L. littoralis essential oils were determined for the first time. Both oils displayed no cytotoxicity on normal cells such as MT-4, BHK-21, MDBK, and Vero-76. The P. trimera oil displayed the strongest sensitivity against S. aureus, whereas that of L. littoralis exhibited the strongest inhibition towards C. tropicalis and C. parapsilosis, with MIC and MLC values of 2% (v/v). In addition, P. trimera essential oil inhibited C. parapsilosis as observed in MIC and MLC of 8% (v/v). The oils also demonstrated inhibition against T. vaginalis with IC 50 , IC 90 , and MLC, respectively, at 0.016%, 0.03%, and 0.06% (v/v) from P. trimera and 0.03%, 0.06%, and 0.12% (v/v), respectively, from L. littoralis after 48 h of incubation. These studied essential oils were ineffective against HIV-1, YFV, BVDV, Sb-1, CV-B4, RSV, VSV, Reo-1, HSV-1, and VV viruses. Further studies should be implemented to evaluate the safety and toxicity of P. trimera and L. littoralis essential oils in humans before considering the development new anti-infectious agents for use in clinical trials.