Antimicrobial Activities of Sesquiterpene-Rich Essential Oils of Two Medicinal Plants, Lannea egregia and Emilia sonchifolia, from Nigeria

Lannea egregia (Anacardiaceae) and Emilia sonchifolia (Asteraceae) are plants used in traditional medicine in southwestern Nigeria. The essential oils from the leaves of L. egregia and E. sonchifolia were obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry. Both essential oils were dominated by sesquiterpenoids. The major components in L. egregia leaf essential oil were α-panasinsen (34.90%), (E)-caryophyllene (12.25%), α-copaene (11.39%), and selina-4,11-diene (9.29%), while E. sonchifolia essential oil was rich in γ-himachalene (25.16%), (E)-caryophyllene (15.72%), and γ-gurjunene (8.58%). The essential oils were screened for antimicrobial activity against a panel of bacteria and fungi and displayed minimum inhibitory concentrations ranging from 156 μg/mL to 625 μg/mL. Based on these results, either L. egregia or E. sonchifolia essential oil may be recommended for exploration as complementary antibacterial or antifungal agents.


Introduction
Medicinal plants are widely used in treatment of diseases, and this has encouraged researchers to investigate plants that are of pharmacological value with potential therapeutic application in the management of human health [1]. Many ethnomedicinal plants have been investigated and reported to possess antiviral [2], anticancer [3], antiprotozoal [4], antibacterial [5], antifungal [6], anti-inflammatory [7], antioxidant [8] and other biocidal activities [9][10][11]; hence, their usefulness in folk medicine for treatment of various diseases has given credence to the application of the ethnopharmacological approaches for drug discovery.

Results and Discussion
As far as we are aware, there have been no published reports on essential oils from Lannea species, so essential oil compositional comparisons at the genus level are not possible. Both α-copaene and (E)-caryophyllene are common essential oil components, including the Anacardiaceae (see, for example [35,36]). Selin-4,11-diene, on the other hand, is relatively uncommon in the family, but has been observed in Sclerocarya birrea leaf essential oil [37] and Haematostaphis barteri leaf essential oil [38]. Likewise, α-panasinsen is a rare volatile component in the Anacardiaceae, but detected as an aroma component of Mangifera indica cv. Alphonso [39] and Sclerocarya birrea subsp. caffra [40] fruits.

Emilia sonchifolia
Hydrodistillation of the leaves of E. sonchifolia yielded a pale-yellow essential oil (0.46%). A total of 62 constituents, 97.60% of E. sonchifolia volatile oil, were identified by GC-MS. The volatile oil composition is displayed in Table 3 Table 3.
The essential oil of the aerial parts of E. sonchifolia from Belagavi, Karnataka, India, has been reported [41]. The essential oil from India was also dominated by sesquiterpene hydrocarbons (67.6%), but with a remarkably different composition. The major components in the essential oil from India were γ-muurolene (32.1%) and (E)-caryophyllene (22.7%). γ-Muurolene was not observed in the essential oil from Nigeria, while γ-himachalene, γ-gurjunene, and germacrene D were not reported in the essential oil from India. Both caryophyllene oxide and palmitic acid were found in the essential oil from India (1 .1% and 1.2%, respectively). Apparently, the geographical separation of these two sam- The essential oil of the aerial parts of E. sonchifolia from Belagavi, Karnataka, India, has been reported [41]. The essential oil from India was also dominated by sesquiterpene hydrocarbons (67.6%), but with a remarkably different composition. The major components in the essential oil from India were γ-muurolene (32.1%) and (E)-caryophyllene (22.7%). γ-Muurolene was not observed in the essential oil from Nigeria, while γ-himachalene, γ-gurjunene, and germacrene D were not reported in the essential oil from India. Both caryophyllene oxide and palmitic acid were found in the essential oil from India (1.1% and 1.2%, respectively). Apparently, the geographical separation of these two samples has a profound effect on the phytochemistry.
Both L. egregia and E. sonchifolia essential oils were dominated by sesquiterpene hydrocarbons, with (E)-caryophyllene abundant in both oils. α-Copaene, abundant in L. egregia essential oil, was found to be only 1.5% in E. sonchifolia oil. Selina-4,11-diene and α-panasinsen were major components in L. egregia essential oil but were not detected in the essential oil of E. sonchifolia. Likewise, γ-himachalene, abundant in E. sonchifolia essential oil, was not detected in the essential oil of L. egregia.

Antimicrobial Activity
The leaf essential oils of L. egregia and E. sonchifolia were screened for antibacterial and antifungal activity against a panel of microorganisms (Table 4). It has been suggested that essential oils having MIC values < 100 µg/mL show very strong activity, those with MIC of 101-500 µg/mL show strong activity, 500 µg/mL < MIC < 1000 µg/mL are moderately active, and above 1000 µg/mL are inactive [42,43]. Thus, the essential oils in this study can be considered strongly active. It is not readily apparent which essential oil components are responsible for the activities; most sesquiterpenes have not been individually screened for antimicrobial activity. However, three of the major components, β-pinene, linalool, and (E)-caryophyllene, were also screened in this work and these compounds showed activities similar to the essential oils themselves. The observed antimicrobial activities are consistent with some of the ethnobotanical uses of these two plants, and based on these results, either L. egregia or E. sonchifolia essential oil may be recommended for exploration as antibacterial or antifungal agents. Table 4. Antibacterial and antifungal activities (MIC, µg/mL) of Lannea egregia and Emilia sonchifolia essential oils from southwest Nigeria.

Plant Materials
Leaves of Lannea egregia and Emila sonchifolia were collected directly from source plants in two locations in southwestern states in Nigeria in the month of August, 2019. and FHI 112546, respectively) have been deposited. The leaves L. egregia and E. sonchifolia were manually removed, chopped, air-dried in the laboratory for 7-10 days, pulverized using an electric blender, and stored in polyethene containers until ready for use.

Isolation of Essential Oils
A sample (450 g each) of L. egregia leaves and E. sonchifolia leaves was subjected to hydrodistillation thrice in an all-glass Clevenger-type apparatus. Each sample of L. egregia and E. sonchifolia, respectively, was mixed with water in a ratio of 2:6. The mixture was hydrodistilled for 3-4 h with constant stirring until no additional oil was observed to be distilled. For each plant species, the essential oils were combined, dried over anhydrous sodium sulfate to eliminate traces of water, and stored in a sealed vial under refrigeration (4 • C) prior to analysis.

Gas Chromatography-Mass Spectrometry
The leaf essential oils of L. egregia and E. sonchifolia were analyzed using gas chromatography-mass spectrometry (GC-MS) as previously described by us [38]: Shimadzu GCMS-QP2010 Ultra, ZB-5 ms GC column, GC oven temperature 50 • C-260 • C (2 • C/min), 1-µL injection of 5% solution of each essential oil dissolved in CH 2 Cl 2 (split mode, 30:1). Each essential oil sample was injected three times. Retention indices (RI) were calculated in comparison with a homologous series of n-alkanes. Compounds were identified by comparison of the MS fragmentation and retention indices with those in the databases [44][45][46][47] and with matching factors >90%. Quantification was done by external standard method. Calibration curves of representative compounds from each class were drawn and used for quantification.

Antibacterial and Antifungal Screening
The essential oils were screened for antimicrobial activity against a panel of bacteria ( [48,49] as previously reported by us [38]. Serial dilutions of the essential oils (2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 µg/mL) in appropriate media (cationadjusted Mueller Hinton broth for bacteria and yeast-nitrogen base growth medium for fungi) were carried out in 96-well microtiter plates. Microorganisms (1.5 × 10 8 CFU/mL for bacteria and 7.5 × 10 7 CFU/mL for fungi) were added to the 96-well plates, which were incubated for 24 h at 37 • C for bacteria and 35 • C for fungi. Minimum inhibitory concentrations (MIC) were determined to be the lowest concentrations without turbidity. Gentamicin (Sigma-Aldrich, St. Louis, MO) was the positive antibacterial control, amphotericin B (Sigma-Aldrich, St. Louis, MO) was the positive antifungal control, and dimethylsulfoxide (DMSO) was used as the negative control (50 µL DMSO diluted in 50 µL broth medium, and then serially diluted as above). (-)-β-Pinene, (±)-linalool, (E)-caryophyllene, and caryophyllene oxide (Sigma-Aldrich, St. Louis, MO) were also individually screened for activity.

Conclusions
The essential oils of Lannea egregia and Emilia sonchifolia, medicinal plants collected from southwestern Nigeria, were found to be rich in sesquiterpenoids. Both essential oils exhibited antibacterial and antifungal activities that are consistent with traditional uses of the plants. While sesquiterpene hydrocarbons were the predominant chemical class in both essential oils, it is not apparent which individual components may be responsible for the antimicrobial activity. It is likely, however, that synergistic effects are also responsible for the activities of the components. Nevertheless, the essential oils may be recommended for further exploration as complementary antimicrobial agents.