Chemical Investigation and Therapeutic Significance of Essential Oils of Nigerian Plumeria acuminata Ait.

MATERIALS AND METHODS

Study area: This study was carried out from May, 2017 to September, 2018.

Plant preparation and extraction of essential oils: The plant was collected at Masaba Road, University of Ibadan (GSP Coordinates: 7°23'0.2"N/3°54'28.8"E) and authenticated at Forestry Research Institute of Nigeria where a voucher specimen (FHI 112495) was deposited. The leaves and roots were air-dried and pulverized while the flowers were used fresh due to their perishable nature. The 3 plant parts were subjected to hydrodistillation separately in an all-glass modified clevenger apparatus for 3 hrs according to the British Pharmacopeia specification as described by Alade et al.¹⁰. The isolated volatile oils were dried over anhydrous sodium sulphate. The oils were stored in sealed vials and kept in the refrigerator before analysis and bioassay.

Analysis of the essential oils: The essential oils were analyzed through Gas Chromatography-Mass Spectrometry (GC-MS) using a Shimadzu GC-MS-QP2010 equipped with a ZB-5 fused silica capillary column (Phenomenex, Torrance, California, USA) with a (5% phenyl)-polydimethylsiloxane stationary phase and a film thickness of 0.25 μm, operated in the electron-impact mode of 70 eV, scan rate (3.0 scan s^–1) and scan range (40-400 atomic mass units). The injector’s initial temperature was 50°C increased at a rate of 2°C min^–1 to 260°C. A 5% w/v solution of each sample in CH₂Cl₂ was prepared and 0.1 μL was injected with a splitting mode of 30:1 using helium as carrier gas with a column head pressure of 552 kPa and flow rate of 1.37 mL min^–1.

Identification of constituents was based on a comparison of their mass spectra with the literature and those of reference compounds stored in the home database library¹⁰. The relative percentage of each constituent was calculated by the integration of GC peak areas.

Toxicity test: The essential oils were subjected to a brine shrimp lethality assay in other to evaluate their toxicity. The assay was carried out using the method described by Hamidi et al.¹¹ with a slight adjustment. Artemia salina eggs (brine shrimp) were hatched into larvae after 48 hrs of soaking them in seawater under sunlight. Ten nauplii were added into three replicates of 10, 100 and 1000 ppm solution of essential oils dissolved in Dimethylsulphoxide (DMSO). After 24 hrs at room temperature, the numbers of survived and dead larvae were counted and the values were subjected to Probit regression analysis to evaluate their toxicity in terms of lethality concentration (i.e., LC₅₀), which is the concentration that kills 50% of the larvae¹².

Antimicrobial assay: The essential oils were tested against seven bacteria (Escherichia coli, Staphylococcus aureus, Salmonella typhi, Leclercia adecarboxylata, Morganella morganii, Citrobacter freundii and Klebsiella pneumoniae) and 3 fungi (Aspergillus niger, Fusarium solani and Candida albicans) using agar well diffusion method. All microbes were clinical isolates obtained from University College, Ibadan, Nigeria. Diluted overnight cultures (10^–2 CFU mL^–1) of the selected microorganisms were inoculated each into a sterile agar inside the Petri dish. Bacteria and fungi strains were maintained on Mueller-Hinton agar and Sabouraud dextrose agar, respectively. Wells of uniform diameter were created in the seeded agar plates using an 8 mm cork borer. The wells were labelled according to the number of the essential oil samples (dissolved in DMSO) with their serial dilutions (1, 10 and 100 μg mL^–1). The oil samples were then introduced into each well accordingly and allowed to diffuse into the seeded agar for 1 hr before incubating at 37°C for 24 hrs for bacteria and at 25-32°C for 48 hrs for fungi after which the inhibition zones were observed and recorded. Each assay was carried out in triplicate and the results were reported as Mean±Standard deviation. Gentamycin (10 μg mL^–1) and ketoconazole (200 μg) were used as positive references, respectively for bacteria and fungi.

DPPH free radical-scavenging assay: The DPPH free radical-scavenging ability of the essential oils was studied to assess the antioxidant properties of the oils using the method described by Saleh et al.¹³ with some modifications. In this assay, 1.5 mL of different concentrations of all the essential oil samples (5, 25 and 100 mg mL^–1) was separately mixed with 1.5 mL of 0.2 mM DPPH using methanol as solvent. These were incubated in the dark for 20 min at room temperature. The absorbance at 517 nm was recorded as A_(sample) using CE 2021, 2000 series double beam UV-Vis spectrophotometer. The absorbance of the blank, A_(blank) was also recorded using the same procedure but without essential oil. Each experiment was carried out in triplicate and the free radical-scavenging activities of the oils were calculated as percentage inhibition using the formula:

The values of the percentage inhibitions were plotted against the observed concentrations using Microsoft excel to obtain the IC₅₀ value which was the concentration required for 50% inhibition¹⁴. Activities of the oils were compared with control antioxidants butylated hydroxyanisole (BHA) and α-tocopherol.

RESULTS

Chemical compositions of the essential oils: The percentage yields of the leaf, flower and root essential oils of P. acuminata were 0.18, 0.13 and 0.08%, respectively. Table 1 shows the retention indices (RI) of the compounds present in the oils in order of their elution from a ZB-5 fused silica capillary column with their percentage composition (peak area). The percentage composition was obtained by the ratio of the individual area (TIC: Total ion count) of the component with the total TIC area of all the peaks and then multiply by 100. The major components in the leaf essential oil were linalool (16.1%), phytol (6.8%), (E)-β-ionone (6.1%) and (E)-hexadecatrienal (5.4%). Linalool was also an abundant component of the floral essential oil (12.6%), but the fatty aldehydes nonanal (20.5%) and eicosanal (10.6%) were also abundant. In contrast, the root essential oil was dominated by β-eudesmol (43.0%), palmitic acid (27.6%) and 11-hydroxy-(8E)-dodecenoic acid lactone (14.9%). The gas chromatograms of the essential oils are given in Fig. 1-3. The x-axis of the chromatograms represents the retention time (minutes) which is the time taken by each constituent to reach the mass spectrometer detector from the injector. The y-axis shows the relative abundance of the oils’ constituents which quantifies the amount of ion produced relative to the total ion count of all peaks. The classes of compounds identified include monoterpenes, sesquiterpenes, diterpenes and apocarotenoids. In addition, alkanes, aldehydes, alcohol, ketones, amide, ester, fatty acid and aromatic compounds of non-terpene derivatives were present. The compounds identified are shown in Table 1 and Fig. 1-3.

Toxicity against Artemia salina: The ability of the essential oils to kill Artemia salina larvae in many cases translates to toxicity and is correlated to the acute oral toxicity assay in mice¹². The lethality of the volatile oils was found to be concentration-dependent because maximum mortalities (100%) were observed at 1000 ppm and it decreased as the concentration decreased. The toxicity of the oils against brine shrimps in terms of Lethal Concentration (LC₅₀) obtained from the mortality rate of the shrimps is presented in Table 2. Log-Probit analysis of the brine shrimp lethality data revealed median Lethal Concentrations (LC₅₀) of 6.90, 23.20 and 61.22 ppm, respectively, for the leaf, floral and root essential oils.

Values of LC₅₀ less than 100 ppm are known to be toxic, values between 100-500 ppm are moderately toxic and 500-1000 ppm are less toxic while above 1000 ppm are non-toxic. High toxicity against brine shrimps also shows that the tested samples contain biologically active constituents.

Fig. 1:

Gas chromatogram of Plumeria acuminata leaf essential oil

Fig. 2:

Gas chromatogram of Plumeria acuminata root essential oil

Fig. 3:

Gas chromatogram of Plumeria acuminata flower essential oil

Antimicrobial activity: The account of the antibacterial and antifungal activities of both essential oils, standard drugs and the solvent (DMSO) used are given in Table 3 and 4, respectively. Their inhibitory activities were reported as inhibition zone diameter. The essential oils exhibited concentration-dependent activity while the solvent do not affect the tested microbes. The antibacterial activity of the oils at 100 μL mL^–1 against E. coli, S. typhi, S. aureus and L. adecarboxylata was lower than that of gentamycin except M. morganii, C. freundii and K. pneumoniae which were resistant to the reference compounds but susceptible to the oils. Only the leaf essential oils demonstrated better antifungal effects than ketoconazole at 100 μL mL^–1.

Free-radical scavenging activity: The free radical-scavenging activity of the leaves, flowers and root essential oils of P. acuminata compared with the reference compounds α-tocopherol and butylated hydroxyanisole is given in Table 5. The free-radical scavenging assay measures the ability of the essential oils to donate proton or electron to free radicals and this is characterized by IC₅₀. The lower the IC₅₀ the higher the scavenging activity and the higher the antioxidant property of the samples. The median Inhibitor Concentrations (IC₅₀) for DPPH radical-scavenging activities were 695, 441 and 582 μg mL^–1, respectively, for the leaf, floral and root essential oils, respectively and were much less active than the positive controls α-tocopherol (IC₅₀ = 81.6 μg mL^–1) or BHA (IC₅₀ = 45.1 μg mL^–1).

DISCUSSION

A total of 26 components were identified in the leaf oil, 85 in the flower and 12 in the root essential oil representing 70.0, 99.8 and 97.3% of the total oil content, respectively. The leaf oil consisted more of terpenes (46.5%) than the non-terpenes (23.5%). The major constituents were linalool (16.1%), (E)-β-ionone (6.1%) and phytol (6.8%). The flower essential oil on the other hand had more non-terpene (72.7%) than terpenes (26.5%) components. The main components were nonanal (20.5%), eicosanal (10.6%), linalool (12.6%) and nonadecane (8.6%). The root essential oil, like the flower oil, had a good proportion of terpene, principally oxygenated sesquiterpenes and an appreciable amount of fatty acid. The most abundant components in the root volatile oil were β-eudesmol (43.0%), palmitic acid (27.6%) and 11-hydroxy-(8E)-dodecenoic acid lactone (14.9%). Palmitic acid is a useful ingredient in food, cosmetics, soaps and industrial mould release agents’ production¹⁵. Most of the terpenes and non-terpene compounds identified as major or minor constituents in the P. acuminata oils are used in perfumery. Examples are nonanal, linalool, geraniol and citronellol.

In the previous study from Nigeria, the chemotype detected in appreciable quantity were linalool (13.2%) and nonanal (9.6%) in the leaf oil, limonene (9.1%), caryophyllene oxide (7.9%) and (E, E)-α-farnesene (6.6%) in the flower oil⁶. Most of these compounds were present in small quantities in the present study.

Information from other geographical locations on the essential oils from Plumeria species was a little different from the chemotype reported from Nigeria. Some of the main components reported were absent and those present were in small quantity^5,16,17. Phenylethyl benzoate, methyl phenylacetate, benzyl salicylate, and benzyl benzoate found as prominent constituents in Egyptian, Malaysian and Indian flower oils were not detected in our study. Factors like geographical area, maturity, seasonal variation and extraction procedure might have contributed to the variation in the chemical profiles of these oils.

The oils were potent against the brine shrimps with LC₅₀ less than 100 ppm¹⁸. The leaf oil was most toxic (LC₅₀ = 6.9070 ppm) followed by the flower (23.1982 ppm) and then the root oil (61.2245 ppm). The toxicity of the leaf oil could likely be due to the synergistic effects of some of its constituents, which likely are absent in other oil samples. The leaf volatile oil may possess antitumor activity. Negative control DMSO does not affect the brine shrimps.

All the essential oils demonstrated good antimicrobial activities with the leaf oil displaying the highest activity. Comparing the antibacterial activities of the oils with the standard drug, gentamycin showed no activity against M. morganii, C. freundii and K. pneumoniae but the microbes were inhibited by all the oil samples except for the root oil which showed no activity against K. pneumoniae. In addition, S. typhi was also resistant to only the root oil. The leaf essential oil also showed a higher inhibition zone (22.1±0.1 mm) at 100 μg mL^–1 against C. albicans when compared to that of ketoconazole (21.0±0.0 mm) at 200 μg. Only F. solani was susceptible to the root oil. Extracts of the plant have been previously reported as good antimicrobial agents^2,7.

Linalool, β-eudesmol, phytol and palmitic acid which were major constituents in the oils and have shown good antimicrobial activities from the previous works^19-21, might have improved the antimicrobial potentials exhibited by the oils. Synergistic effects of minor constituents like caryophyllene oxide, limonene and α-terpineol can likewise contribute to the oils’ activities.

The essential oils exhibited lower activity with IC₅₀ values higher than that of reference compounds. The higher the IC₅₀ values the lower the scavenging activity. Although linalool, α-terpineol, geraniol, thymol, limonene, α-pinene, phytol and caryophyllene, which have a good record of free radical-scavenging activities in the literature²² were found in appreciable quantity in the oils, it is expected of the oils to have high ability to scavenge free-radicals. Therefore, antagonistic effects of other constituents could likely have contributed to the low activity displayed by the essential oils²³. Crude extracts from the plant demonstrated good free-radical scavenging activity^2,7.

CONCLUSION

The essential oils hydrodistilled from the leaf, flower and root of Plumeria acuminata showed commendable antimicrobial activities and toxicity against brine shrimp. The results from this study suggest that the essential oils of P. acuminata, especially the leaf oil, might be an alternative source of antimicrobial and antitumor agents. However, further investigations to determine the minimum inhibitory concentrations and the toxicity of the oils against human cell lines will be carried out.

SIGNIFICANCE STATEMENT

This study discovered new antibiotics of plant origin that could be beneficial to the pharmaceutical and cosmetic industries.

ACKNOWLEDGMENT

The authors are grateful to Dr. Olufunmi Ayeni of the Department of Pharmaceutical Microbiology of the University of Ibadan, Nigeria, for the assistance she rendered during the antimicrobial assay.