Chemical Characterization, Antioxidant, Antimicrobial, and Antibiofilm Activities of Essential Oils of Plumeria alba (Forget-Me-Not)

Essential oils are known to possess many biological properties such as antimicrobial and antioxidant activities. Plumeria alba flowers are used in traditional remedies for diarrhea, cough, fever, and asthma treatment. This work evaluated the chemical composition and the biological activities of essential oils obtained from the flowers and leaves of Plumeria alba. The essential oils were extracted using the Clevenger-type apparatus and characterized using GC-MS. In the flower essential oil, a total of 17 compounds were identified, with linalool (23.91%), α-terpineol (10.97%), geraniol (10.47%), and phenyl ethyl alcohol (8.65%) being abundant. In the leaf essential oil, a total of 24 compounds were identified, with benzofuran, 2,3-di, hydro-(3.24%), and muurolol (1.40%) being present. Antioxidant activities were assessed using hydrogen peroxide scavenging, phosphomolybdenum, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assays. Antimicrobial activities were assessed through a microdilution assay. The essential oil showed antimicrobial activity against test microorganisms with minimum inhibitory concentrations ranging from 25.0 to 50.0 mg/mL. Biofilm inhibition ranged from 27.14 ± 1.0 to 58.99 ± 0.6 mg/mL. The essential oil exhibited total antioxidant capacities which ranged from 17.5 μg/g AAE to 83 μg/g AAE in the phosphomolybdenum assay. The IC50 values in the DPPH and hydrogen peroxide radical scavenging assays for both flowers and leaves ranged from 18.66 μg/mL to 38.28 μg/mL. Both essential oils also displayed good antibiofilm activities, with the concentration required for half-maximal inhibition of biofilm formation being ∼60 mg/mL for both oils. This study shows that essential oils of Plumeria alba possess good antioxidant and antimicrobial activities and could be used as a source of natural antioxidants and antimicrobial agents.


Introduction
Essential oils are predominantly volatile and odorous fractions isolated from plants. Tese oils usually impart a distinctive and often diagnostic odor to plants. Essential oils have been isolated from various plant parts such as leaves, fowers, and stems (peppermint and lavender), fruits (anise), bark (cinnamon), and seeds (nutmeg). Plants store these oil components in the glandular cells or pockets, which release them with aroma when squeezed or pressed. In plants, essential oils are mostly devoid of cellulose, glycerides, sugars, tannins, salts, and minerals. Conventional methods such as steam distillation, mechanical expression (physical crushing of essential oil glands situated in fruit rinds or outermost waxy layers of fruit's peel), microwaveassisted extraction, solvent extraction, or enfeurage (transfer of the essential oil from fower petals to fat) are usually employed in the extraction of essential oils [1]. Essential oils yields are usually low, often ranging between 0.05 and 18.0% [2]. Te yield obtained from the extraction of essential oils depends on various factors, such as the extraction method utilized, environment, climate, soil conditions, time of harvesting, and postharvest handling before isolation [3].
Due to their varied application in food, pharmaceutical, and cosmetics industries, essential oils have become the focus of intense investigations. Te pharmaceutical and therapeutic properties of some plants have been attributed to their essential oils and the compounds that make up these oils [4]. In many essential oils, monoterpenes, sesquiterpenes, and aromatic and aliphatic compounds are the principal chemical components. It has been reported that essential oils containing mainly aldehydes or phenols, such as cinnamaldehyde, citral, carvacrol, eugenol, or thymol, are characterized by impressive antibacterial activity [5]. Essential oils with antioxidant, antibioflm, antitumor, and anti-infammatory properties have also been reported [6]. Te activity of the given essential oil is usually a function of the chemical components present in the oil. Since essential oil composition is afected by cultivation conditions such as soil types and geographical locations and environmental factors like climate and habitat conditions, plants of the same species from diferent countries may have diferent chemical compositions and diferent chemical properties [3]. Due to these, investigations of the essential oils from diferent plants in diferent locations have become very important.
Te genus Plumeria (Apocynaceae) contains a large number of shrubs and fowering trees which are grown throughout the tropics. Plumeria alba, locally known as forget-me-not in Ghana, is an essential member of the Plumeria genus. It is indigenous to South America and primarily cultivated for its blossoms and fragrant fowers [7]. Ethnomedicinally, diferent parts of forget-me-not are used in treating several diseases, such as abdominal tumors, leprosy, rheumatism, and malaria. Te latex of the leaf and stem is used to treat skin diseases like ulcers, scabies, and herpes [8]. Te bark of P. alba is used as a plaster for challenging tumors, and the fruit is known as a natural source of antioxidants that can reduce free radical-mediated diseases such as diabetes, cancer, and coronary heart disease [9]. Reports on essential oils from P. alba from various countries indicate varying chemical compositions. Lawal et al. in Nigeria identifed 43 compounds as the constituents of P. alba fower essential oil. Gas chromatography-mass spectrometry (GC/MS) analysis showed that the Nigerian oil was rich in sesquiterpene hydrocarbons (25.2%) with benzyl salicylate (33.98%) as the major constituent [10]. In India, 46 volatile constituents have been characterized in the P. alba fower essential oil, accounting for 98.1% of the total essential oil constituents. Esters were the major compound class (48.6%) identifed in that study [11]. Other studies have focused on the biological activities of essential oils from P. alba. Liu et al. studied the antimicrobial, antioxidant, and antipyretic-analgesic efects of P. alba fower essential oil and indicated that the bioactivity may be due to the presence of major constituents such as benzyl salicylate, benzyl benzoate, germacrene B, and linalool in the essential oil [12].
Since essential oils isolated from P. alba plant parts in diferent countries possess varying chemical constituents (qualitative and quantitative) and diferent biological activities, likely as a result of the diferences in chemical composition, the fower and leaf essential oils of Ghanaian cultivars of P. alba were studied in this work. Gas chromatography-mass spectrometry was used to identify the essential oils' chemical constituents, whereas standard in vitro assays were used to evaluate the antioxidant, antimicrobial, and antibioflm activities of oils. In the fower and leaf essential oils, 17 and 24 compounds, respectively, were identifed, with linalool (fower) and hexadecane (leaf ) being the major components. Both oils possessed appreciable antioxidant and antimicrobial activities. Again, both oils were able to inhibit bioflm formation in Pseudomonas aeruginosa.

Essential Oil Extraction.
Fresh fowers were washed with distilled water. Afterwards, the fowers were subjected to steam distillation for 4 hours in a modifed Clevenger-type setup. Fresh leaves were also washed and extracted similarly. Te essential oils were recovered and treated with anhydrous sodium sulphate to remove traces of water. Essential oils were stored at 4°C until used in further analysis [13]. Te yield of essential oils was calculated with respect to the fresh weight of the plant material before distillation (expressed as the percentage w/w of the fresh material).

Chemical Composition Analysis Using GC-MS.
Chemical constituents of the essential oils were assayed on a PerkinElmer GC Clarus 580 gas chromatograph interfaced with a PerkinElmer (Clarus SQ 8 S) mass spectrometer. A DB-5 (ZB-5HTMS; 5% diphenyl/95% dimethylpolysiloxane) fused capillary column with dimensions of 30 × 0.25 mm ID × 0.25 μm DF was used. Te GC oven program and the mass spectrometer conditions were the same as reported in previous works [14,15].

Identifcation of Compounds.
Te constituents of the essential oils were identifed by matching the mass spectra obtained to mass spectra databases of the National Institute of Standard and Technology (NIST) and Wiley. Where available, mass spectra present in the published literature were also used [16]. Quantitation of essential oil components was by normalization of the peak area of each constituent.

Antioxidant Activity.
Various assays were used to evaluate the antioxidant activity of the fower and leaf essential oils of P. alba. Te assays employed included the inhibition of lipid peroxidation, hydrogen peroxide, and 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and phosphomolybdenum assays.

Inhibition of Lipid Peroxidation (Tiobarbituric Acid
Reactive Substance, TBARS) Assay. Tis assay was performed according to methods described by Nartey [15,17]. Te oxidizable substrate used in this assay was the egg yolk, which is known to be rich in lipids. Butylated hydroxytoluene (BHT) was used as the positive control. Te percentage inhibition of lipid peroxidation was calculated, and IC 50 values (extract concentration required to achieve 50% inhibition of lipid peroxidation) were obtained from a graph of % inhibition against concentration.

Phosphomolybdenum Assay.
In the phosphomolybdenum (PM) assay, essential oils of diferent concentrations were prepared in dimethyl sulfoxide (DMSO). Five millimeters of the PM reagent made up of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate was added to 0.5 mL of each test sample in a test tube. Te test tube was shaken and then incubated at 95°C for 90 min. After the reaction mixture had cooled to room temperature, the absorbance at 695 nm was measured against a blank solution. Ascorbic acid was used as a standard in this experiment [18,19].

Hydrogen Peroxide-Scavenging Assay.
Efective hydrogen peroxide-scavenging activity of the essential oils was assayed by adding to a test tube 0.5 mL of 1 mM ferrous ammonium sulphate, followed by 0.13 mL of 5 mM H 2 O 2 and 3 mL of essential oil or a standard drug at varying concentrations. Each test tube was incubated in the dark for 5 min at room temperature. After that, 3 mL of 1 mM 1,10phenanthroline was added to each mixture, and the test tube was shaken to ensure a uniform mixture. Te mixture was then incubated for 10 min at room temperature. Absorbances of the mixtures were taken at 510 nm. Water was used in place of the essential oil in the blank. Ascorbic acid and gallic acid were used as standard drugs. Te amount of hydrogen peroxide scavenged was obtained from: where A test is the absorbance of the test sample and A control is the absorbance of the blank. Te IC 50 values were obtained from a graph of % hydrogen peroxide scavenged against concentration [20].

DPPH Radical-Scavenging
Assay. Te 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical-scavenging activities of the fower and leaf essential oils of P. alba were evaluated according to a reported method [21,22]. Methanol was used in place of the essential oils in the negative control set ups. Ascorbic acid was used as a standard drug. Te percent inhibition of DPPH free radicals was calculated from the absorbance of the control (Ac) and that of the test (At) from: Te IC 50 values were obtained from a graph of % inhibition against concentration.

Minimum Inhibitory Concentration Determination.
Te minimum inhibitory concentrations (MIC) of the fower and leaf essential oils were determined using the broth dilution method. In the broth dilution assay, two-fold serial dilution of the essential oil or standard antibiotic (ciprofoxacin) was prepared in sterile 96-well microtiter plates. In each plate, 100 μL of two-fold serial dilution of essential oil was transferred into the wells. To each well was added 100 μL of double-strength nutrient broth containing an inoculum size of ∼2.0 × 10 5 CFU/mL. After incubation for 24 hours at 37°C, 20 μL of 1.25 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide solution (MTT) was added to each well and further incubated for 30 min at 37°C. Te MIC was determined as the lowest concentration of the essential oil or antibiotic that completely inhibited the growth of the organism in microdilution wells as detected by the absence of the purple coloration after MTT addition during a 24hour incubation period at 37°C. All tests were performed in triplicate [23].

Bioflm Inhibition.
Bioflm inhibition was investigated in P. aeruginosa. A 96-well sterile microtiter plate was flled with 100 μL of various concentrations (MIC and sub-MIC) of essential oils. Gentamicin was used as a standard drug and water as a negative control. Bacterial suspension of P. aeruginosa was adjusted to the 0.5 McFarland standard in sterile saline and subsequently inoculated in double-strength nutrient broth to an inoculum size of ∼2.0 × 10 5 CFU/mL. An aliquot of the broth suspension (100 μL) was inoculated into each well to a fnal volume of 200 μL. Te plate was incubated at 37°C for 24 hours. After the incubation period was over, the contents of the plates were emptied and then washed 3 times with deionized water to remove loosely attached cells. Te wells were stained with 0.1% (v/v) crystal violet followed by elution with 30% (v/v) glacial acetic acid. Tis was transferred into a new sterile plate. Absorbance was measured at 595 nm (BioTeK ® Synergy H1 multimode microplate reader, Germany). Bioflm inhibition was estimated from equation (2) [24]. Biochemistry Research International 2.8. Data Analysis. All experiments were conducted in triplicate, and data were presented as a mean ± standard deviation. Statistical analyses were performed in GraphPad Prism 6.0 for Windows. Where applicable, a p value <0.05 was considered to be statistically signifcant.

Results and Discussion
Essential oils are generally present in trace amounts in plant epidermic cells, secretory cells, canals, and cavities [1]. Varied compounds present in essential oils contribute to the fragrance of the oil. Te major compound present in an essential oil may range from 20 to 85% and may contribute greatly to the aroma and pharmacological activity of the essential oil. Compounds present in trace amounts may also act in synergy to elicit specifc biological efects [15]. Steam distillation of the fowers and leaves of P. alba yielded 0.09% and 0.05% of essential oils, respectively, with both being pale yellow in color. Te yield of the fower essential oil is slightly higher than the 0.05% yield as reported by Sahoo et al. in India [11]. An earlier investigation on the leaf and fower essential oil of P. alba from Nigeria revealed yields of 0.12% and 0.23%, respectively, with both being signifcantly greater than the yields obtained in this study [10]. Variations in essential oil yields depend on the method of extraction, age of plant, variety, and plant-growing conditions [3]. Tese factors may all have played a role in the variations in essential oil yields reported.
Te total ion chromatograms (TICs) of the fower and leaf essential oils are shown in Figures 1 and 2, respectively, with the composition data of both oils shown in Table 1 (fower essential oil) and Table 2 (leaf essential oil). Te compounds present in the fower essential oil were generally primary alcohols, terpenes, terpenoids, ketones, esters, and saturated fatty acids, with terpene and terpenoids dominating. Linalool, α-terpineol, and geraniol were the major components present, with their individual compositions being 23.91%, 10.97%, and 10.47% of the total composition, respectively, as shown in Table 1. For the leaf essential oil, 24 compounds were identifed and the compounds were mainly carboxylic acids and hydrocarbons. Te most abundant component in the leaf essential oils was hexadecane ( Table 2). Hexadecane has been found in essential oils from a variety of plants, including Phlomis lurestanica [25] and Cassia Fistula [26]. In contrast to this study, Lawal identifed acyclic monoterpenoids and aldehydes as the main constituents of the leaf oil of P. alba in Nigeria [10]. Interestingly, all of the major components of the P. alba leaf essential oil reported by Lawal were absent in the leaf essential oil in this study (Table 3). Despite using the same method of extraction, both the yield and composition of the essential oil varied. Tis variation could be attributed to the diference in geographical locations since essential oil composition is afected by cultivation conditions such as soil types and geographical locations and some environmental factors [3]. Due to these reasons, we suspected that their biological activities may difer since compositions were diferent.
Essential oils exhibit various biological properties including antioxidant, antimicrobial, and anti-infammatory. Antioxidants can function by preventing active oxidant generation or scavenging, quenching, and elimination of active oxidants [29]. A formidable challenge in food processing is lipid oxidation. Peroxidation of lipids produces unwanted side products such as aldehydes and peroxides. Oxidized lipid-derived aldehydes are very stable and toxic and have been implicated in a number of human diseases such as carcinogenesis, cardiovascular diseases, neurodegeneration, and aging [30]. In foods, these compounds impart poor sensory properties to the food and contribute signifcantly to reducing their shelf life. Ultimately, the products of lipid peroxidation enhance food spoilage [31]. Compounds capable of inhibiting lipid peroxidation are therefore desired as they could be used as food preservatives. Te TBARS assay was used in assessing lipid peroxidation. Tis test detects malondialdehyde (MDA), a split product of an endoperoxide of unsaturated fatty acids produced by lipid substrate oxidation [31]. In the lipid peroxidation assay, the IC 50 value of the fower essential oil was 1943 ± 0.9 μg/mL, while that of the leaf essential oil was 3069 ± 0.9 μg/mL. Te standard, BHT, also recorded an IC 50 value of 9.238 ± 1.7 μg/ mL (Table 4). Te results imply that the foral essential oil is a better inhibitor of lipid peroxidation than the leaf essential oil. Youdim et al. in 2000 evaluated the antioxidant activity of thyme essential oil and observed that components in the essential oil inhibited lipid peroxidation in the order as follows: p-carvacrol > c-terpinene > myrcene > linalool > pcymene > limonene > 1,8-cineole > α-pinene [32]. Linalool showed a moderate lipid peroxidation inhibition activity and may be a major contributor to the observed inhibition of lipid peroxidation by the fower essential oil of P. alba because of its high content in the oil.
Te total antioxidant capacity of the essential oil of P. alba fower, as determined from the PM assay, was 57 μg/g AAE, whereas that of the leaf essential oil was 87 μg/g AAE. In the hydrogen peroxide-scavenging activity test, the IC 50 of the fower essential oil was 370.5 μg/mL and that of the P. alba leaf essential oil was 476 μg/mL. In the case of the DPPH assay, the concentrations, at which 50% of DPPH radicals were scavenged, were 1014 ± 0.6 and 2798 ± 1.1 μg/ mL for the fower and leaf essential oils, respectively (Table 4). Te fower essential oil possessed better scavenging activity than the leaf essential oil, whereas the leaf essential oil performed better in the PM assay. Both essential oils could be described as possessing moderate antioxidant activities. Some reports have shown that essential oils possess antioxidant activity, which plays a vital role in neutralizing free radicals and benefting human health [29,30,33]. Te antioxidant evaluation of the organic solvent leaf extract of P. alba by Siang and et al. revealed moderate antioxidant activity in the DPPH assay, where the IC 50 was found to be 23.96 mg/mL [34]. Essential oils are therefore better radical scavengers than the organic solvent extracts of the same plant. Te antioxidant action of essential oils could be due to the presence of compounds such as linalool, geraniol, and α-terpineol in their composition since these compounds are known for their antioxidant properties. Linalool is 9    a principal component in most essential oils with several biological activities which include antioxidant, antiinfammatory, antibacterial, and antiplasmodial activity [35][36][37]. Te presence of linalool in the fower essential oil as the most abundant component and its complete absence in the leaf essential oil could be a major contributor to the observed patterns in antioxidant capability. Te antimicrobial activity of plant essential oils and extracts has formed the basis for many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine, and natural therapies [6,38]. Essential oils from various plants have been reported to have impressive antimicrobial properties [6,14,39]. Essential oils from the fowers and leaves of P. alba were screened against 7 bacteria and 1 fungus. Antimicrobial activity was observed for both Gram-negative and Gram-positive bacteria. However, the essential oils exhibited no antifungal activity at the concentrations used. Te results of the antimicrobial susceptibility test (Table 5) show that the MICs of the essential oils against all tested microorganisms ranged between 12.50 and 50 mg/mL. In general, the fower essential oil exhibited better antimicrobial activity when compared to the leaf essential oil. E. coli was the most susceptible organism to the fower essential oil at an MIC of 25 mg/mL. Te growth of B. subtilis, S. aureus, E. faecalis, and P. aeruginosa was also inhibited at 50 mg/mL by the fower essential oil. E. coli and E. faecalis were susceptible to the leaf essential oil at an MIC of 12.5 mg/mL in both cases. Kumari et al. investigated the antifungal activity of fower essential oils from P. alba in India. Te results showed that fower essential oil was highly active against Aspergillus niger, Candida albicans, and Penicillium chrysogenum [40]. Tis was diferent from what was observed in this study where no activity was observed against Candida albicans at the concentrations used. Tis observation could be attributed to the diference in essential oil composition.
It has been reported that essential oils from the fower of P. alba were efective against S. aureus and B. subtilis [41]. Another report on the activity of the petals of P. alba highlighted a signifcant antimicrobial capacity against pathogenic E. coli, one of the most common bacteria with pathogenic strains [42]. In our study, E. coli was the most susceptible organism for both essential oils from the fower and leaf of P. alba, with the leaf essential oil having a lower MIC than that of the fower essential oil. In general, the fower and leaf essential oils from P. alba had a moderate antimicrobial activity against the tested microorganisms. According to several authors, Gram-negative bacteria appear to be less sensitive to the action of many other plant essential oils [43]. Tis higher resistance among Gram-negative bacteria could be due to the diferences in the cell membrane of these bacterial groups. Indeed, the external membrane of Gram-negative bacteria renders their surfaces highly hydrophilic [44], whereas the lipophilic ends of the lipoteichoic acids of the cell membrane of Gram-positive bacteria may facilitate penetration by hydrophobic compounds [45,46]. Hence, essential oils that can target both bacteria classes will signifcantly help fght against antimicrobial resistance. Interestingly, in this study, both essential oils of P. alba appeared to be active against the tested Gram-positive and Gram-negative bacteria.
Bioflm formation is one of the several strategies utilized by bacteria to evade host immune action and the efects of antimicrobial agents. Bioflm formation has been identifed as a plausible strategy used by most bacteria to establish  Present study Hexadecane (11.06%), octadecane (9.72%), tetradecane (8.32%), hexacosane (7.85%), eicosane (7.15%), phytol (6.83%), docosane (6.26%), and 2-methoxy-4-vinyl phenol (5.76%) infection in host cells [47]. P. aeruginosa is a model bioflmforming organism that has been well characterized. In this study, P. aeruginosa was used for the frst time to examine the bioflm inhibition potential of the essential oils from P. alba. Because bacterial growth is essential during bioflm development, viable bacteria are needed to develop bioflm in order not to alter bioflm development. For this reason, subinhibitory concentrations of essential oils were used to allow assessment of antimicrobial agents on bioflm formation. Both fower and leaf essential oils displayed good inhibitory efects on bioflm formation, with percent inhibitions ranging from 80 to 41.8% from MIC to sub-MIC doses. At MIC/16, both fower and leaf essential oils inhibited bioflm formation by >40% (Table 6). Te concentrations of essential oil, which were determined to afect half-maximal inhibition of bioflm formation (BIC 50 ), were 58.99 mg/mL and 60.06 mg/mL, respectively, for fower and leaf essential oils. Tus, the fower essential oil performed better as an antibioflm agent than the essential oil of the leaf. Tis was similar to the observation in the antimicrobial studies against P. aeruginosa. Extracts of P. alba have been shown to possess antibioflm capabilities. Te minimum bioflm inhibitory concentration exhibited by the ethyl acetate extracts of P. alba against P. aeruginosa was determined to be 60 μg/mL [48]. Tus, both extracts and essential oils from the P. alba plant do possess antibioflm capabilities, although at varying potencies. Several studies have documented the bioflm inhibitory capabilities of essential oils. Essential oils isolated from Spondias mombin, Averrhoa carambola and Chrysophyllum albidum have been reported to possess bioflm inhibition capabilities [14,15,17]. Previous studies have also reported the antibioflm activities of compounds such as linalool, geraniol, and α-terpineol [49]. Te antibioflm potential of the P. alba essential oils could be due to the presence of some of these compounds.

Conclusion
Te extraction and characterization of essential oils from the fower and leaves of P. alba were successful. Terpenoids and terpenes were found in high concentrations in the foral oil, while acids and hydrocarbons were most prevalent in the leaf oil. Both essential oils showed moderate antioxidant and antimicrobial properties, making them potential sources for food preservation and processing, as well as for use in the pharmaceutical and cosmetic industries due to their ability to inhibit bioflm formation. Data are represented as a mean ± standard deviation. * TAC: total antioxidant capacity; * TBARS: thiobarbituric acid reactive substance assay; * ND: not determined (compound not used in that experiment).  78.08 ± 6.5 BIC 50 (mg/mL) 58.99 ± 0.6 60.06 ± 0.9 27.14 ± 1.0 Data are represented as a mean ± standard deviation. Concentrations of essential oils or gentamicin used were based on their minimum inhibitory concentration (MIC) against P. aeruginosa. * Gentamicin was in μg/ mL doses.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
Te authors declare no conficts of interest.