Evaluation of Chemical Composition and Anti‐Staphylococcal Activity of Essential Oils from Leaves of Two Indigenous Plant Species Litsea leytensis and Piper philippinum from Leyte, Philippines

1. Introduction

Indigenous plants, especially endemic species, are less explored as sources of new phytochemicals, including those found in essential oils (EOs), with potential industrial uses [1,2]. Most of these plants are found in specific regions known as biodiversity hotspots. The Philippines, a tropical archipelago located in Southeast Asia, is considered one of the world’s best biodiversity hotspots, with more than 5,800 species of endemic plants, including EO-bearing flora [3]. Various EOs isolated from the numerous native plants of the Philippines have been recognised worldwide for their commercial and economic importance, and many of them are currently utilised in the cosmeceutical and pharmaceutical industries. For example, EO distilled from the resin of Canarium luzonicum, commonly known as Manila elemi, is used as a fragrance in soap and perfumes, and as a base for liniments [4]. Moreover, other economically valuable EOs can also be obtained from plant species commonly belonging to the Annonaceae, Burseraceae, Lauraceae, Piperaceae, and Zingiberaceae families, among others. However, the EO chemical composition of some species belonging to these families has not yet been analysed. One typical example is Litsea leytensis Merr. (Lauraceae), a species endemic in the Philippines commonly known as batikuling or bitokling. It is a medium-sized tree that inhabits low- and medium-altitude forests in Luzon and the Eastern Visayas regions [5]. Light to medium-weight wood from the tree is used locally for pattern making, ceilings, and carving due to its scent that naturally repels termites, ants, and woodworms [6]. Another example of a native Philippine plant is Piper philippinum Miq. (Piperaceae), which is a woody climber distributed throughout the country in thickets and forests at low and medium altitudes [7]. With the exception of bioactive lignans isolated from P. philippinum [8], there is currently no information about the chemistry and biological activity of both these species. To determine the chemical composition of the volatile compounds present in indigenous Philippine plants, gas chromatography and mass spectrometry (GC-MS) analysis of the EOs from the leaves of L. leytensis and P. philippinum was performed. Additionally, in vitro anti-staphylococcal susceptibility testing of these two EOs was performed, in both the liquid and vapour phases.

2. Results and Discussion

Hydrodistillation of the leaves L. leytensis and P. philippinum produced light, yellow-coloured EOs with yields (v/w) of 0.14% and 0.77% on a dry plant weight basis, respectively. The EOs did not show any antibacterial activity against S. aureus in the liquid nor vapor phases (MICs >1,024 μg/mL). The complete results of the chemical analysis and the composition of L. leytensis and P. philippinum EOs is provided in Table 1 and Table 2.

^aRI = retention indices. Obs. = retention indices determined relative to a homologous series of n-alkanes (C₈–C₄₀) using a HP-5MS column. Lit. = literature RI values [9,10]. ^bCl. = chemical classification; A – Aldehydes, DH – Diterpene hydrocarbons, E – Esters, K – Ketones, MH – Monoterpene hydrocarbons, O – Others, OD – Oxygenated diterpenes, OM – Oxygenated monoterpenes, OS – Oxygenated sesquiterpene, SH – Sesquiterpene hydrocarbons, ^cRelative peak area percentage as the mean of three measurements. ^dIdentification method: MS = Mass spectrum was identical to that of National Institute of Standards and Technology Library (ver. 2.0.f), RI = the retention index matching literature database; Std = constituent identity confirmed by co-injection of authentic standards. ^eIdentification on DB-WAX was confirmed based on the MS spectrum. NA = RI values not available in the literature.

Based on the GC-MS analysis using HP-5MS/DB-WAX columns, a total of 68/61 and 54/47 compounds were identified in the samples of L. leytensis and P. philippinum, representing 93.81/91.65% and 89.48/89.29% of total contents, respectively. Analysis revealed that monoterpenes and sesquiterpenes were the predominant chemical classes within the major constituents of the tested EOs. For L. leytensis, 1.81/1.49% monoterpenes and 89.41/84.77% sesquiterpenes were identified, respectively, using HP-5MS/DB-WAX columns, while 7.23/10.73% monoterpenes and 81.31/73.95% sesquiterpenes were identified for P. philippinum. Caryophyllene oxide (15.70/16.76%) was the main component of L. leytensis EO, followed by β-caryophyllene (11.04/11.12%) and α-copaene (8.85/8.87%). All three compounds are known to exhibit various biological activities. For example, analgesic, antibacterial, anticancer, antifungal, and antioxidant effects were reported for β-caryophyllene [11,12]. Likewise, caryophyllene oxide has analgesic and anti-inflammatory properties [12,13]. Besides its antioxidant and neuroprotective activities [14], α-copaene is a common attractant to insect pests such as the Mediterranean fruit fly [15] and redbay ambrosia beetles [16]. According to the results of our analysis, the chemical composition of L. leytensis EO significantly differs from that of L. cubeba, which is the most important species of the genus economically. Its EO is used in the perfume industry as a commercial source of citral [17]. Although the chemistry of the EO from the leaf of L. cubeba vary significantly depending on the geographical origin of the sample, with 1,8-cineole or linalool being the main constituents [17], its chemical composition differs significantly from that of L. leytensis leaf EO. Previously published analyses of EOs from other species of the Litsea genus suggest that the chemical composition of L. deccanensis left EO is more similar to that of L. leytensis, as it also contains β-caryophyllene and caryophyllene oxide as the main components [18,19].

The chemical analysis showed that ishwarane (26.98/24.89%) is a major component of P. philippinum leaf EO, followed by nerolidol (10.14/10.42%) and 3-ishwarone (6.84/2.46%). The chemical profile of P. nigrum, an economically and industrially the most important species of the genus, differs therefore significantly from P. philippinum EO [20,21,22,23]. Nevertheless, leaf EO of other Piper species, such as P. arboretum, P. aduncum, and P. guadianum, also contain a significant amount of δ-cadinene, caryophyllene, and nerolidol [22]. Nerolidol is an economically important sesquiterpene since it is predominantly utilised as a fragrance component in the perfume industry [24] with known antifungal, antimalarial, and antiparasitic activities [25,26,27]. On the other hand, ishwarane is a rare sesquiterpene among the species of Piper genus [28], which is only found in the leaf EO of P. fulvescens [29] and P. alatipetiolatum [30], as well as in the fruit EO of P. guineense [31]. Ishwarane was reported to have antifungal activity against Cladosporium cladosporioides [28]. 3-Ishwarone, another rare sesquiterpene detected in P. philippinum, was also found in the leaf EO of Peperomia oreophila [32] and Peperomia scandens [33].

The separation of EOs using GC on stationary phases of different polarity provides an analytical tool useful for various applications, the most notable being the confirmation of specific isomers [34]. Effective separation of several isomers from the two plant EOs in the current study was conducted using non-polar HP-5MS and polar DB-WAX columns. Using this approach, isomeric cadinenes detected in L. leytensis EO were identified, whereas α- and γ-cadinene were found on HP-5MS, and δ-cadinene was found on DB-WAX, only. In the case of P. philippinum EO, effective separation was achieved for α-and β-copaene on both columns, and their corresponding isomer ylangene was found only when using DB-WAX. Moreover, the polar DB-WAX column offered additional information regarding the components of each EO sample, such as the detection of hexadecenoic acid in P. philippinum EO.

3. Materials and Methods

3.1. Chemicals and Reagents

Camphene (CAS 79-92-5), β-caryophyllene (CAS 87-44-5), geraniol (CAS 106-24-1), α-caryophyllene (CAS 6753-98-6), linalool (CAS 126-91-0), methyl octanoate (CAS 111-11-5), myrcene (CAS 123-35-3), α-pinene (CAS 7785-70-8), β-pinene (CAS 18172-67-3) were used as analytical standards. Furthermore, n-alkanes (ranging from C₈ to C₄₀) were used as calibration standards. With the exception of n-hexane (CAS 110-54-3; Merck, Darmstadt, Germany), which was used as a solvent for the preparation of analytical EOs samples, all other chemicals were obtained from Sigma-Aldrich (Prague, Czech Republic).

3.2. Plant Material

The leaves of L. leytensis and the aerial parts of P. philippinum were collected during January 2019 at the base of Mount Pangasugan, Leyte Island, Philippines. The plants were authenticated at the Faculty of Tropical AgriSciences of the Czech University of Life Sciences Prague (CZU) by ethnobotanist Ladislav Kokoska, and at the Jose Vera Santos Memorial Herbarium of the College of Science of the University of the Philippines by plant taxonomist Edwino S. Fernando. Voucher specimens were deposited at CZU, in the herbarium of the Department of Botany and Plant Physiology, Faculty of Agrobiology, Food and Natural Resources: 02576KBFRB (L. leytensis) and 02576KBFRB (P. philippinum). For EO extraction, separate air-dried plant samples were homogenised (Grindomix GM 100, Retsch, Haan, Germany). Residual moisture content was analysed in triplicate using a Scaltec SMO 01 (Scaltec Instruments, Gottingen, Germany) at 130 °C.

3.3. Hydrodistillation

The EOs from L. leytensis and P. philippinum were extracted via hydrodistillation from air-dried plant material. A Clevenger-type apparatus (Merci, Brno, Czech) was used to extract the EO from the material placed in 1 L of distilled water for 3 h, according to the European Pharmacopoeia [35]. After distillation, the EOs were stored in sealed glass vials at 4 °C until analysed.

3.4. Bacterial Strain and Culture Media

Staphylococcus aureus standard strain ATCC 29213 was cultivated in Mueller-Hinton broth and agar, both purchased from Oxoid (Basingstoke, UK). The pH of the broth was equilibrated to 7.6 with Trizma base (Sigma-Aldrich).

A stock culture of S. aureus was cultivated at 37 °C for 24 h prior to susceptibility testing. The turbidity of the bacterial suspension was then adjusted to 0.5 McFarland standard, using a Densi-La-Meter II (Lachema, Brno, CZ), to obtain a final concentration of 10⁷ CFU/mL. Susceptibility of the bacterium to oxacillin (86.3%, CAS 7240-38-2; Sigma-Aldrich) was utilized as a positive antibiotic control [36].

3.5. Antimicrobial Assay

The antibacterial potential of the plant EOs was assessed, in both the liquid and vapor phase, using the broth microdilution volatilization method [37]. Standard 96-well immune plates with flanged lids designed to reduce evaporation (SPL Life Sciences, Naechon-Myeon, KR) were utilized. First, 30 μL agar was pipetted into each flange, except the outermost flanges, and inoculated with 5 μL of the bacterial suspension. Secondly, samples of the EOs were dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich) at a maximum concentration of 1% and diluted in broth medium. Serial dilutions were prepared from the samples of both EOs (seven two-fold dilutions), starting at 1,024.00 μg/mL. A 96-pin multi-blot replicator (National Institute of Public Health, Prague, CZ) was used to inoculate the plates with the bacterial suspension. Wells containing inoculated and non-inoculated broth were used as growth and purity controls simultaneously. Lastly, the plates and lids were fastened together to ensure an air-tight fit using clamps (Lux Tool, Prague, CZ) and handmade wooden pads and incubated at 37 °C for 24 h. The minimum inhibitory concentrations (MICs) were evaluated by visual assessment. A metabolically active bacterial colony was coloured with thiazolyl blue tetrazolium bromide dye (Sigma-Aldrich) at a concentration of 600.00 μg/mL. When the colour changed from yellow to purple (relative to the colours in the control wells and flanges), the endpoint (MIC value) was recorded in the broth and agar. The MIC values (μg/mL) were the lowest concentrations capable of inhibiting bacterial growth, compared with the compound-free control. The negative control containing 1 % of DMSO did not inhibit the growth of the strain tested, neither in the broth or agar media. All experiments were performed in triplicate, in three independent experiments, and the results are expressed as median/modal MICs values.

3.6. GC-MS Analysis

GC-MS analysis was used to determine the main components if the EOs. An Agilent GC-7890B dual-column/dual detector gas chromatograph was utilised, equipped with an Agilent 7693 autosampler, two columns: a fused-silica HP-5MS column (30 m × 0.25 mm, film thickness 0.25 µm, Agilent 19091s-433) and a DB-WAX column (30 m × 0.25 mm, film thickness 0.25 µm, Agilent 122–7132), and a flame ionisation detector (FID) coupled with a single quadrupole mass selective detector Agilent MSD-5977B (Agilent Technologies, Santa Clara, CA, USA).

Helium was used as a carrier gas (1 ml/min) and the injector temperature for both columns was set at 250 °C. The oven temperature was increased after 3 min, from 50 to 280 °C for both columns. After an isothermic period of 3 min, a heating rate of 3 °C/min was used until 120 °C, after which 5 °C/min was utilised until 250 °C. This was followed by a 5 min holding time at 250 °C, after which the heating rate increased to 15 °C/min until 280 °C. An isothermic period of 20 min followed. EOs were diluted to a concentration of 20 µL/mL in n-hexane, and, subsequently, 1 µL of each sample was injected into the GC MS in a split mode (split ratio 1:50). The mass detector conditions were as follows: ionisation energy 70 eV, ion source temperature 230 °C, scan time 1 s, and mass range 40–600 m/z. Identification of the constituents was performed by comparing their retention indices (Ris), retention times (RTs), and spectra with those in the National Institute of Standards and Technology Library ver. 2.0.f (NIST, USA) [9,10], as well as against authentic standards (Sigma-Aldrich) and with the literature. The RIs were calculated using the RTs of n-alkanes series (ranging from C₈ to C₄₀) for compounds separated on HP5-MS column. The relative percentage of the EOs components were determined on both columns using FID.

4. Conclusions

In summary, this is the first report of the chemical composition of EOs hydrodistilled from the aerial parts of two indigenous Philippine plant species, namely L. leytensis (Lauraceae) and P. philippinum (Piperaceae), which were analysed using GC-MS equipped with two columns of differing polarity. Sesquiterpenoids, namely caryophyllene oxide, α-copaene and β-caryophyllene (L. leytensis) and 3-ishwarone, ishwarane, and nerolidol (P. philippinum), were the predominant classes of compounds identified in both EOs. These results provide new knowledge on the chemical composition of leaf EOs from these two indigenous plant species found in the Philippines, which belong to two commercially important genera. Nevertheless, the assessment of the antibacterial activity of the EOs showed no growth-inhibitory effect on S. aureus. However, the presence of bioactive compounds in the EOs suggests their potential for future investigation, which should focus on the determination of their biological effects.