Chemical Constituents and Antibacterial Activity of Essential Oils of Needles of Pinus Sylvestris (Scots Pine) from South West Nigeria

Pinus sylvestris (Scots pine) is a coniferous plant that possesses different biological properties such as antiseptic, antioxidant, antifungal, and anti-inammatory. This study investigated the chemical constituents and in vitro antibacterial activities of essential oil (EO) of Pinus sylvestris (Scots pine) against thirteen bacterial species using Gas chromatography/mass spectrophotometry (GC/MS) and standard agar well diffusion assay. The EO exhibited excellent antibacterial activity inhibiting all the bacterial isolates tested with the diameter zone of inhibitions (Zi) ranging from 8–24 mm. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) ranged between 0.025mg/ml and 25.000mg/ml. The highest antibacterial activity was recorded against Micrococcus luteus NCIB 196 and Klebsiella pneumonia NCIB 418 (Zi = 24 mm), while the lowest activity was recorded against Proteus vulgaris (Zi = 8 mm). The GC/MS analysis revealed the presence of 30 chemical compounds, of which seven possess antibacterial properties. These include three oxygenated monoterpenes (α-terpineol, borneol, and fenchol), two sesquiterpenes (caryophyllene and δ-cadinene), one saturated fatty acid (palmitic acid) and monounsaturated fatty acid (oleic acids). Therefore, the results suggest the potential of P. sylvestris as an antimicrobial agent for incorporation in the treatment of pathogens. The Gas Chromatography/Mass Spectroscopy (GC/MS) technique was used to determine the chemical constituents of the EO obtained from the hydro-distillation of the needles. The GC/MS was done using HP 8060 Series II Gas chromatograph equipped with a ame ionization defector and HP-5MS (30 m x 0.25 mm x 0.25 µm) capillary column; coupled to VG Platform II Mass spectrometer to carry out mass spectrometry analysis. The temperature of the source was maintained at 180 ℃ at 300V. Ion source pressure and MS detector were held at 9.4 x 10 − 6 and 9.4 x 10 − 6 mbar, respectively. The scan cycle of the MS was 1.5 sec (scan duration of 1s and inter-scan delay of 0.5 s). Mass and scan range was set at m/z 1-1400 and 38–650, respectively, and the instrument was calibrated using heptacosauorotributyl amine (C 12 F 27 N) (CAS Number: 93792-84-8). The GC/MS column temperature was programmed as for GC, but the lm thickness of GC/MS was 0.5 µm. The sample of 1 µl was injected with a slip ratio of 1:10. All GC/MS analyses were made in the splitless mode, with helium gas as the carrier. The chemical constituents of the EO were identied and named by comparing the retention indices. The identied constituents were further conrmed by comparing the obtained mass spectra from the GC/MS analysis with the reference Library compound spectra in the database of the National Institute of Standard and Technology and published spectra [30].


Introduction
The application of antibiotics is one of the ways to control pathogenic and food spoilage bacteria that can have negative impacts on humans. However, these antibiotics are gradually failing due to several factors which include, the misuse and overuse of antibiotics without prescription for self-medication, usage of inferior antibiotics, uncontrolled application of antibiotics in animal husbandry and veterinary medicine, and dwindling rate of novel antibiotics discovery [1,2]. These factors are the major forces enhancing the development of multidrug resistance among pathogens, which is now a major global challenge both in clinical and community settings. As a survival strategy, these multidrug-resistant bacteria develop different mechanisms of resistance to antibiotics, hence making infection treatment di cult and expensive [3]. Another consequence of the global antimicrobial resistance menace is the gradual shift from orthodox medicine to herbal therapy [4,5] in an attempt to search for novel compounds that can serve as a better alternative to antibiotics.
Plants are a repository of several compounds that have useful applications in medicine and drug-making.
For instance, morphine, which is the rst natural product used for therapeutic purpose was isolated from opium (Papaver somniferum). Artemisinin from Artemisia annua, Paclitaxel from Taxus brevifolia, and Silymarin from Silybum marianum among others are some of the natural products that have been developed and sold as drugs [6,7]. Pinus koraiensis pinecone essential oil has been shown to be effective in the production of anti-tumor drugs for gastric cancer [8]. Thus, essential oils (EOs) from several plants, including coniferous plants, have antimicrobial properties [8][9][10][11][12] suggesting their use as alternatives to conventional synthetic antibiotics. Furthermore, these EOs have a low level of toxicity, side effects, and chemical diversity in terms of activity compared to synthetic chemicals [9,[13][14][15], hence making them suitable for use as medicinal ingredients [16][17][18][19]. EOs also have potential to be developed into natural antioxidant in food systems, bio-control of stored product pest and fungi growth inhibitor [20,21]. Despite their therapeutic applications, the usage of some EOs has been reported to cause side effects such as poisoning, dermatological, and neurological toxicity [10].
Pinus sylvestris (Scots pine) belongs to the family Pinaceae, originated from Eurasia and mostly found in Northern and Eastern Europe. The genus Pinus is the most common coniferous plant with about 250 species distributed worldwide [22,23]. The tree is about 25 to 40 m tall with evergreen, fragrant blue-green needle-like leaves of about 3 to 5 cm arranged alternately or spirally [24]. P. sylvestris is the most diverse of any pine species worldwide with a wide array of applications [24]. The tree is an ornamental plant with environmental value in erosion control, and as a raw material in paper-making industries [25]. P. sylvestris oil has wide arrays of medical applications due to its anti-parasitic, anti-viral, anti-allergenic, antispasmodic, anti-hyperglycemic, anti-in ammatory, and expectorant properties [26]. Moreover, the terpenic oil of P. sylvestris is useful in pharmaceutical, chemical, cosmetic, perfume industries, even as food additives and preservatives [26]. The insecticidal and larvicidal properties of P. sylvestris oil have also been reported [27].
In Nigeria, Sonibare and Olakunle reported the chemical composition and antibacterial properties of essential oil (EO) from needles of another species of Pinus, P. caribe (Caribbean pine) [22]. However, to the best of our knowledge, this report is the rst to investigate the antibacterial properties of Pinus sylvestris (Scots pine) in Nigeria, and this current study, therefore, investigated the chemical constituents and antibacterial activity of EO of P. sylvestris needles obtained from Ibadan, South Western part of Nigeria.

Plant collection
In the month of May Extraction of EO from P. sylvestris needles Sixty millilitres of distilled water were added to grounded needles of P. sylvestris in a round bottom ask. EO was extracted from the needles in Clevenger's apparatus using the hydro-distillation method under optimal operating conditions [29]. The distillation process was done until a clear distillate was obtained.
The distillates of EO were then dried over anhydrous sodium sulphate (CAS No. 7757-82-6) to remove impurities and water present in the distillates. Then, it was ltered and preserved at 4℃ for further analysis.

Chemical analysis of EO of P. sylvestris needles
The Gas Chromatography/Mass Spectroscopy (GC/MS) technique was used to determine the chemical constituents of the EO obtained from the hydro-distillation of the needles. The GC/MS was done using HP 8060 Series II Gas chromatograph equipped with a ame ionization defector and HP-5MS (30 m x 0.25 mm x 0.25 µm) capillary column; coupled to VG Platform II Mass spectrometer to carry out mass spectrometry analysis. The temperature of the source was maintained at 180℃ at 300V. Ion source pressure and MS detector were held at 9.4 x 10 − 6 and 9.4 x 10 − 6 mbar, respectively. The scan cycle of the MS was 1.5 sec (scan duration of 1s and inter-scan delay of 0.5 s). Mass and scan range was set at m/z 1-1400 and 38-650, respectively, and the instrument was calibrated using heptacosa uorotributyl amine (C 12 F 27 N) (CAS Number: 93792-84-8). The GC/MS column temperature was programmed as for GC, but the lm thickness of GC/MS was 0.5 µm. The sample of 1 µl was injected with a slip ratio of 1:10. All GC/MS analyses were made in the splitless mode, with helium gas as the carrier.
The chemical constituents of the EO were identi ed and named by comparing the retention indices. The identi ed constituents were further con rmed by comparing the obtained mass spectra from the GC/MS analysis with the reference Library compound spectra in the database of the National Institute of Standard and Technology (NIST), and published spectra [30].

Bacterial Isolates
All the thirteen bacterial isolates used in this study were culture collections obtained from Dr D. A. Akinpelu  Determination of antibacterial activities of EO of P. sylvestris needles The EO of the P. sylvestris needles was screened for antibacterial activities using the agar well diffusion method, as described by Akinpelu et al. [31]. Bacterial isolates were cultured in nutrient broth for 18 h before standardization to 10 6 CFU mL − 1 , an equivalent of 0.5 MacFarland standard was done. A loopful of the standardized bacterial culture was aseptically inoculated into 20 mL of molten Mueller Hington agar (MHA) prepared in a McCartney bottle and already cooled to 45°C. The inoculated MHA was mixed thoroughly, poured into a sterile Petri dish, and allowed to set. Sterile cork borer was used to bore two wells of 6 mm in diameter into the inoculated MHA, and 50 µL of undiluted oil sample was dispensed into one of the wells. Streptomycin (1 mg mL − 1 ) was dispensed into the second well to serve as a standard positive control. The inoculated petri dish was left on the bench at room temperature for 1 h to allow the diffusion of the oil into the medium and then incubated at 37°C for 24 h. After incubation, the diameter zones of inhibition (Zi) was measured using a ruler and recorded in millimetres (mm).

Determination of Minimum Inhibitory Concentration (MIC) and Minimum bactericidal concentration (MBC) of EO of P. sylvestris needles
The MIC of the EO was determined against tested bacterial isolates using the agar dilution method, as described by Akinpelu et al. [31]. The EO was diluted serially in two-fold to obtain the following concentrations: 25.0, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, 0.049 and 0.025 mg mL − 1 . Then 2 mL aliquot of the different concentrations of the EO were added to 18 mL of molten sterile nutrient agar using a sterile pipette. The molten nutrient agar containing the different dilutions of EO was poured into a sterile petri dish and allowed to set. An 18 h old bacterial culture already standardized using the 0.5 MacFarland standard was streaked on the dried surface of the set nutrient agar plate using a sterile loop. The plates were incubated at 37°C for 24 h and observed for the presence or absence of bacterial growth.
The MIC was taken as the lowest concentration at which no growth of the tested bacterial isolates occurred, while the MBC was taken as the next higher concentration to the MIC where no bacterial growth was observed.

Data analysis
Descriptive statistics were used with data analysis and graphs made using GraphPad Prism 7.0. (GraphPad Software, USA). All the experiments were done in triplicates, and data were expressed as arithmetic mean ± SD (standard deviation).

GC/MS Analysis of EOs of P. sylvestris
As reported in our earlier publication [27], thirty chemical constituents representing 90.50 % of the oil were identi ed from the GC/MS analysis of EO of P. sylvestris needles. Seven of the identi ed chemical constituents are known antibacterial agents (Table 1)

Antibacterial Activity
The study EO of P. sylvestris needles inhibited the growth of all the tested bacterial isolates with varied Zi ranging between 8 and 24 mm (Fig. 1). The highest antibacterial activity was observed against Klebsiella pneumoniae (NCIB 418) and Micrococcus luteus [NCIB 196], while the lowest activity was observed against Pseudomonas uorescens (NCIB 3756). The Zi observed for streptomycin, the standard antibiotic used as a control in this study ranged between 10 and 25 mm. However, streptomycin did not inhibit the growth of Escherichia coli (NCIB 86) and Corynebacterium pyogenes.

Minimum Inhibitory Concentration (MIC) and Minimum bactericidal concentration (MBC)
The MIC and MBC results of EO of P. sylvestris were presented in Fig. 2

Discussion
The EO from P. sylvestris needles obtained by hydro-distillation exhibited excellent broad-spectrum in vitro antibacterial activities against all bacterial isolates tested with the value of Zi that ranged from 8 to 24 mm. The EO compared favourably with the standard antibiotics, streptomycin with the value of Zi that ranged from 10 to 25 mm, therefore making it a potential antibacterial agent of bene cial importance. Similarly, some studies have also reported broad-spectrum activities of EO of P. sylvestris needles [32][33][34] against multidrug-resistant bacterial strains of S. aureus, enterobacterial pathogens [35,36], and antibiotic-resistant Pseudomonas spp. responsible for freshwater sh diseases (Sekiten-byo) and spoilage [27]. Hence, the EO of P. sylvestris needles can serve as an antibacterial agent for the control of multidrug-resistant pathogens and food spoilage bacteria.
The EO inhibited the growth of all the bacteria tested at an appreciable MIC ranging between 0.39 and 1.56 mg mL − 1 except for P. vulgaris, which also had the lowest Zi (8 mm). Vyas and Patil [36] obtained similar results with P. vulgaris (Zi of 4 mm, the lowest value among all the bacterial isolates tested) when the antibacterial activity of EO of P. sylvestris was examined against multidrug-resistant enterobacterial pathogens.
This study has validated the antimicrobial potential of EO derived from P. sylvestris. This might be linked to the biologically active constituents available in EO of P. sylvestris needles which enhanced the higher level of antibacterial properties reported against all the tested pathogens in this study. The result obtained from the GC-MS showed that seven antibacterial chemical constituents were present in the EO of P. sylvestris. This correlates with the result obtained by some previous researchers who identi ed seven chemical constituents in the EO of P. sylvestris [26][27][28].
Three oxygenated monoterpenes including α-terpineol (the most abundant chemical compound in the study P. sylvestris), borneol, and fenchol were identi ed as antibacterial compounds in the P. sylvestris. Antibacterial effects of oxygenated monoterpenes component of EO of P. sylvestris against a wide range of bacterial species, and their useful applications in the production of medicine and cosmetics have been reported [32]. For instance, borneol is one of the traditional Chinese medicine used to treat sore throat, ulcerations, and purulent ear discharge [35]. The presence of these oxygenated monoterpenes in P. sylvestris supports its potential as an antimicrobial agent against bacterial pathogens.
Also, hydrocarbon sesquiterpenes (caryophyllene and δ-cadinene) were observed as the antibacterial compound in the EO of P. sylvestris. Interestingly, some scientist in some part of Europe including Estonia, Lithuania and Romania has established the presence of hydrocarbon sesquiterpenes as the major component available in pine oil using GC/MS techniques. Furthermore, the presence of βcaryophyllene and δ-cadinene apart from hydrocarbon sesquiterpenes constitute the majority of the sesquiterpenes available in the EO of P. sylvestris [15,36,37]. Several scientists have established that hydrocarbon sesquiterpenes such as β-caryophyllene possess other biological properties like antiin ammatory, anticancer, antioxidant, and antifungal properties in addition to their antibacterial activities [33,38,39]. It can also inhibit bio lm-forming bacteria implicated in tooth bio lm or plaque in dogs [38], suggesting its application in veterinary medicine. The principal active constituent and antibacterial agent in Schinus molle fruits have been a rmed to possess inhibitory effect against S. pneumoniae at MIC of 31.25 mg mL − 1 . The author further utilized GC/MS to identify the presence of δ-cadinene as the active constitutes responsible for the antibacterial activity against S. pneumoniae.
During this study, the presence of oleic acid (monounsaturated fatty acid) and palmitic acid (saturated fatty acids) was also detected in the EO of P. sylvestris. It has been documented that oleic acid portends the capability to inhibits the FabI enzyme responsible for the fatty acid biosynthesis in bacteria [40], while it could also increase membrane permeability and its leakages, disruption of electron chain transport, and inhibition of the activity of bacterial enzymes [41,42]. However, oleic acid has been established to exhibit antibacterial activities against Gram-positive clinical and foodborne bacterial pathogens such as Bacillus spp., Micrococcus kristinae, Streptococcus pyogenes, Staphylococcus aureus, and Methicillin-resistant S. aureus. Gram-negative bacteria are resistant to unsaturated fatty acids because their outer membrane is highly impermeable to hydrophobic substances [34,40]. In another study, it was validated that Gram-negative bacteria were only susceptible to short-chain fatty acids (C6 and below) at high concentrations [43]. The study also reported that Gram positive-bacteria are more susceptible to fatty acid than Gramnegative bacteria, and unsaturated fatty acids like oleic acid have better antibacterial activities than saturated fatty acids like palmitic acid [43].
This study has been able to establish that the presence of oxygenated monoterpenes (α-terpineol, borneol, and fenchol), hydrocarbon sesquiterpenes (caryophyllene and δ-cadinene), and fatty acids (oleic acid and palmitic acids) might be contributing to the antibacterial activities of EO of P. sylvestris. However, future work is to understand the mechanism of action of each antibacterial components of EO of P. sylvestris and their synergistic effects on bacterial pathogens.

Conclusion
This study demonstrated that essential oil P. sylvestris needles from the South Western part of Nigeria have excellent broad-spectrum antibacterial activities, inhibiting the growth of all bacterial isolates tested.
The GC/MS analysis revealed the presence of compounds including α-terpineol, borneol, fenchol, palmitic acid, caryophyllene, oleic acid, and δ-cadinene, which may be contributing to the antibacterial activities of P. sylvestris EO. The P. sylvestris needles EO and its components is a promising antibacterial agent that can be explored on a commercial scale as a potential source of antibacterial compounds for the control of pathogens, most especially bacterial species tested in this study.