Chemical and Antimicrobial Analyses of Juniperus chinensis and Juniperus seravschanica Essential Oils and Comparison with Their Methanolic Crude Extracts

Juniperus chinensis and Juniperus seravschanica are commonly used in the traditional folk medicine to treat microbial infection. In this study, the essential oils obtained from the leaves of J. chinensis growing in Malaysia and J. seravschanica growing in Oman were analysed by head space-solid phase microextraction-gas chromatography mass spectrometry (HS-SPME-GC-MS) and screened for antimicrobial activities against Escherichia coli (NCTC 10418), Pseudomonas aeruginosa (NCTC 10662), Bacillus subtilis ATCC6059, Micrococcus luteus (ATCC 9341), Staphylococcus aureus (NCTC 6571), and methicillin-resistant S. aureus (MRSA; ATCC 33591). To compare the antimicrobial activities of extracts using different extraction methods, methanol extraction was performed to obtain crude extracts from the leaves of J. chinensis and J. seravschanica for antimicrobial analysis. The HS-SPME-GS-MS analysis of the essential oils from the leaves of J. chinensis and J. seravschanica identified 37 and 36 components, respectively. Essential oils from these two species had distinctive chemical component profiles, with α-pinene (27.2%) as the major component of J. chinensis essential oil, while dl-limonene (45.2%) constitutes the major component of J. seravschanica essential oil. Essential oils of these two species shared only six similar terpenoids compounds: α-pinene, β-pinene, γ-elemene, sabinene, elemol, and 3-cyclohexen-1-ol. Overall, the essential oils showed antimicrobial activities against all the six bacterial strains tested, with the highest antagonistic activity against M. luteus and B. cereus; while, methanolic crude extracts showed the highest activities against S. aureus and MRSA strains. The methanolic crude extracts demonstrated significantly higher antibacterial activity against the Gram-positive bacteria (p < 0.005); while, the essential oils of Juniperus did not show significant differences between Gram-positive and Gram-negative bacteria. Future studies are needed to investigate the active compounds present in the essential oils and methanolic crude extracts that confer the selectivity in the antimicrobial activity.


Introduction
Asia has a large number of medicinal plants that have been used traditionally as folk medicine. In fact, of the 70,000 species of medicinal plants used in traditional medicine globally, a huge proportion are actually originated from Asia. Of these Asian traditional plants, Juniperus tree is one of the most popular and extensively studied medicinal plants. Juniperus belongs to the Cupressaceae family consisting of approximately 70 species that are widely distributed throughout the Northern Hemisphere. Some of the most popular species includes J. chinensis, J. excelsa, and J. communis. Research studies of the various species of Juniperustree revealed a wide range of unique medicinal properties such as antimicrobial [1], anticancer [2] antioxidant [3], antitumor [4], antidiabetic [5], antimicrobial [6], and many other therapeutic properties [7].
Juniperus seravschanica, which is also commonly known as Pashtun juniper, is widely distributed in the central Asia from the southern Kazakhstan through Kyrgyzstan, Tajikistan, eastern Uzbekistan, and Turkmenistan to northern and eastern Afghanistan, northern Pakistan, and Kashmir, as well as in the mountains of southeastern Iran and the Al Hajar Mountains of Oman [8]. e Pashtun juniper is used by the Himalayans to treat a range of diseases including abdominal cramps, diarrhoea, asthma, headache, gonorrhoeae, and leucorrhoea [9]. e Lebanese and Turkish use J. seravschanica to treat asthma, cough, common cold, bronchitis, pneumonia, throat inflammation and tuberculosis, urinary tract inflammations, rheumatism, and to remove renal and gall bladder stones [9].
On the other hand, J. chinensis, which is also known as the Chinese juniper, is commonly found in many regions in the east and southeast Asia including China, Taiwan, Myanmar, Japan, Korea, and Malaysia [10]. e stems of J. chinensis is used in the Chinese medicine to treat parasitic skin problems and rheumatism; the fruit is used in the treatment of convulsions, excessive sweating, and hepatitis; and the root is used to treat burns and scalds [11].
is study aims to determine and compare the chemical compositions of the essential oils obtained from the leaves of the Omani J. seravschanica and the Malaysian J. chinensis. e antimicrobial activity of the essential oils and methanolic crude extracts from the leaves of the Omani J. seravschanica and the Malaysian J. chinensis were also determined.

Plant Materials.
Two types of plant species were examined in this study, namely, Juniperus seravschanica and Juniperus chinensis. J. seravschanica was from Oman, while J. chinensis was from Malaysia. e leaves of J. seravschanica were collected from the tree grown in the Jabel Akhdar Mountains of northern Oman).
is plant was previously thought to belong to the Persian juniper population (J. excelsa subsp. polycarpos) but has been recently confirmed as the Zeravshan juniper J. seravschanica [8]. It is one of the dominant species native to the montane woodlands of Oman [12]. e specimen was authenticated at the Herbarium of the Plant Unit, Biology Department, Sultan Qaboos University, Oman, with voucher code Al-Dhafri, Kh. s.n. e leaves of Juniperus chinensis were collected from the trees grown in the University of Malaya campus located in Kuala Lumpur, Malaysia. e leaves of J. chinensis are needle shaped when young and scaly when mature [13]. For this particular plant specimen used in this study, the leaves were spiny and had a maximum diameter of 7.5 cm. e identity of the plant was confirmed by a botanist, Dr. M. Sugumaran, of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia. A voucher of the specimen was deposited at the University of Malaya Herbarium with the code number KLU047364.

Essential Oils Extraction.
Essential oil was extracted from the leaves of J. chinensis and J. seravschanica via using the modified steam distillation method developed by Masango [14]. In brief, 500 g of leaves sample was placed in the distillation flask that was connected to the steam generator and a condenser to collect the essential oil. After that, the water was heated to 100°C to produce steam to volatilize the essential oils from the leave samples. e volatilization process was conducted for about 5 hours. en, the mixture was collected and separated using a separatory funnel, in which the essential oils that settled at the bottom layer of the separatory funnel were collected in a clean bottle for analysis.

Preparation of Methanol Extracts.
Leaves of the respective plants (J. chinensis and J. seravschanica) were washed with sterile water and then sun-dried for 48 h. e sun-dried leaves were then grinded to fine powder with a stainless-steel blender. After that, each powdered sample (30 g) was macerated in 500 mL of 80% methanol (Sigma-Aldrich, St. Louis, MO, USA) for 3 days at 4°C to extract the phytochemicals presence in the leaf samples [3]. e liquid extract was subsequently filtered and concentrated using a rotary vacuum evaporator (BUCHI Rotavapor, Switzerland), at 40°C. e resulting semisolid extracts which had a yield of about 15% (w/w) was then freeze-dried with a freeze dryer (Flexi-Dry MP, FTS Systems, China) into the dry finepowdered sample, which was subsequently dissolved in sterile distilled water to an extract stock solution at a concentration of 32 mg/mL. e extract stock solutions were kept at −80°C until tested.

Head Space-Solid Phase Microextraction-Gas Chromatography Mass Spectrometry Analysis.
e essential oils extracted from J. seravschanica and J. chinensis were analysed using the head space-solid phase microextraction-gas chromatography mass spectrometry (HS-SPME-GC-MS) method. e solid phase microextraction fibre was exposed to the headspace of the extracted essential oils, and the sample fibre was then inserted to the injection port of a Shimadzu gas chromatograph model GC-MS-QP/5050A, equipped with a quadrupole mass spectrometer and a J&W Scientific DB-5MS (5% phenyl/95% dimethylpolysiloxane) fused silica capillary column (30 m × 0.25 mm I.D. x 0.25 mm film thickness). e oven temperature was programmed to increase from 51°C to 251°C at 3°C/min. Injector and interface temperatures were kept at 275°C and 300°C, respectively. Helium was used as the carrier gas with a linear velocity of 44.6 cm/s, column flow rate of 1.5 mL/min, and total flow rate of 36 ml/min. e split ratio was 1 : 21. Mass spectra were continuously recorded over the mass range 35-501 amu. e MS operating parameters were as follows: ionization voltage 70 eV and scan rate 500 amu/s. e mass spectra obtained were compared to those recorded in the computer MS library (Wiley 229,000 database). e percentage composition was determined by using the single area percentage method without considering corrections for response factors.

Antimicrobial Assay.
Bacteria strains used in this study were revived from glycerol stock cultures maintained in the laboratory. e Gram-positive bacteria used were Bacillus subtilis (ATCC6059), Staphylococcus aureus (NCTC 6571), Micrococcus luteus (ATCC 9341), and methicillin-resistant S. aureus (ATCC 33591); while, the Gram-negative bacteria used were Pseudomonas aeruginosa (NCTC 10662) and Escherichia coli (NCTC 10418). e bacteria were subsequently subcultured on nutrient agar (BDH Media, London, United Kingdom), and the purity of each strain was determined prior to the antibacterial assay. e antimicrobial activities of the methanolic extracts of J. seravschanica and J. chinensis and the essential oil of J. seravschanica and J. chinensis were evaluated against the six strains of bacteria. e bacterial inoculums were prepared from overnight broth culture in physiological saline (0.8% of NaCl) to reach the turbidity equivalent to 0.5 McFarland standard (∼10 8 cfu/mL) and further diluted to 10 6 cfu/mL. Muller-Hinton agar (MH agar; from Sigma-Aldrich, USA) were poured in Petri dishes, solidified, and surface dried before inoculation. e extracts were dissolved in deionized water to obtain a final concentration of 10 mg/mL, and the suspension was filter-sterilized using the 0.22 μm membrane filter. Sterilized 6 mm filter paper discs were impregnated with 10 μL of the respective extracts and were placed on inoculated MH agar plates that had been inoculated with the test bacteria. All the plates were incubated at 37°C for 24 h and were examined for zones of growth inhibition. e diameters of the zones of inhibition obtained after incubation were measured in millilitres for all the test organisms. e tests were performed in triplicates. Tetracycline 30 μg (Oxoid Ltd, Ontario, USA) and filter paper disc soak in sterile distilled water were used as the positive and negative controls in all experiment, respectively.

Statistical Analysis.
Descriptive statistical analysis of the antibacterial data and comparison of significant differences among the plant extracts and bacterial strains were conducted using IBM SPSS Statistics version 26 (IBM, New York, USA).

Chemical Compositions of J. seravschanica and J. chinensis Essential Oils.
e HS-SPME-GCMS analysis of the essential oils extracted from the leaves of J. seravschanica and J. chinensis contained 37 and 36 volatiles components, respectively (Table 1). Essential oils of J. seravschanica and J. chinensis had very different chemical components make up. In total, 67 chemical compounds were detected in the essential oils of both Juniperus species. Most of these compounds have previously been reported to present in the essential oils of the leaves of the Juniperus plant from Iran. e chemical composition of the Juniperus leaf essential oils could be classified into five different classes of components: monoterpenes (MT), monoterpenoids (MTO), sesquiterpenes (ST), sesquiterpenoids (STO), and other, nonterpene components (NT) ( Table 1). Diterpene was not detected in these essential oils.

Selective Inhibition of J. seravschanica and J. chinensis Essential Oils and Methanolic Crude Extracts against Gram-
Positive and Gram-Negative Bacteria. Overall, the methanolic extracts of J. seravschanica showed the strongest antibacterial activity against the six bacterial strains tested (zone of inhibition = 14.3 ± 3.0 mm) compared with the methanolic extracts of J. chinensis (zone of inhibition = 12.6 ± 2.0 mm) and F. bruguieri (zone of inhibition = 12.4 ± 1.7 mm). In comparison, the essential oils of both J. seravschanica (zone of inhibition = 12.0 ± 2.5 mm) and J. chinensis (zone of inhibition = 11.9 ± 2.4 mm) demonstrated a weaker antibacterial activity based on the disc diffusion method ( Table 2).
It was noteworthy to point out that the methanolic extracts were found to have significantly higher antibacterial activity against the Gram-positive bacteria compared with the Gram-negative bacteria (p < 0.005); while, the essential oils of Juniperus did not show significant differences between Gram-positive and Gram-negative bacteria (Figure 1). A number of studies have also reported that plant extracts from Juniperus leaf [17] and fruit [18] could have a higher bactericidal activity against Gram-positive bacteria [17,18]. Although another study from Oman that assess the antimicrobial activity of leaf extracts of Omani Juniperus excels reported only moderate antimicrobial activity of the extracts, reexamination of the data published indicates that the extracts seem to demonstrate a better activity against Gram-positive bacteria [19]. Similar scenario was also observed in extracts from other types of plants. Based on the study conducted by Rumolo and coworkers [20], Aleurites  e observed lesser inhibitory effect against the Gramnegative bacteria was not surprising, as in general, these bacteria are more resistant than Gram-positive ones [21]. ese differences may be attributed to the fact that the cell wall in Gram-positive bacteria consists of a single layer, whereas the Gram-negative bacterial cell wall is a multilayered structure and quite complex [22], with an outer membrane consisting of a hydrophilic surface rich in lipopolysaccharide molecules [23]. In addition, the periplasmic space contains enzymes that could degrade any exogenous molecules and prevent the entry of inhibitors, including antibiotic molecules [24]. Gram-positive bacteria do not possess this type of outer membrane and cell wall structure. erefore, antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane and produce a leakage of the cytoplasm and its coagulation [22].

Antibacterial Activity of Juniperus Methanolic Crude Extracts against Methicillin-Resistant Staphylococcus aureus.
Methanolic crude extract of J. seravschanica demonstrated better antibacterial activity against MRSA compared with the methanolic extracts of J. chinensis (Table 2). Methanolic crude extract of J. seravschanica showed high antibacterial activity against both methicillin-sensitive S. aureus (MSSA) and MRSA, with their inhibition zone more or less similar (Table 2). On the other hand, the methanolic extracts of J. chinensis and essential oils of J. seravschanica and J. chinensis showed a decreased activity against MRSA when compared with the MSSA strain tested (Table 1).
Staphylococcus aureushas been identified as one of the major pathogens causing community-acquired and nosocomial infections [25]. It could cause a wide range of diseases in human, including skin infections, sepsis, osteomyelitis, pneumonia, and even death. Staphylococcus aureus is an opportunistic pathogen that presents in the nose and skin of about 30% and 20% of healthy adults, respectively [26]. MRSA is S. aureus strain that has acquired multidrug resistance to beta-lactam antibiotic that is commonly used to treat bacterial infection [27]. erefore, due to its resistance to the broadspectrum beta-lactam antibiotics, MRSA infections are difficult to treat and pose a higher public health risk. As such, the increased effort has been focusing on searching for an alternative treatment for MRSA infection. Numerous studies have evaluated the antibacterial activity of plant compounds against MRSA. Research by Martz and coworkers [28] on the antibacterial activity of Juniperus communis methanolic extracts showed a satisfactory antibacterial activity against both MSSA and MRSA. Nitta and coworkers [29] evaluated the efficacy of plant extracts from 181 species of tropical and subtropical plants in inactivation of MRSA and found that the Shorea hemsleyana bark and Cyphostemma bainesii root extracts that contained stilbene derivatives demonstrated an excellent anti-MRSA activity. Other than that, essential oils of Eucalyptus globulus, ymus vulgaris, Clerodendrum serratum, Terminalia arjuna, and Coridothymus capitatus showed good capability to inhibit growth of MRSA [7][8][9][10][11].
e better antibacterial activity of the methanolic leaf extract of J. seravschanica against MRSA as observed in this study requires more in-depth investigation to identify the active compounds that contribute to its inhibitory actions against MRSA.

Conclusions
In conclusion, J. seravschanica from Oman and J. chinensis from Malaysia, Juniperus trees of similar genus but different species as well as from different geographical locations yielded essential oils with variable chemical compositions but exert minimal differences in its antimicrobial activities. Overall, the Juniperus essential oils demonstrated antimicrobial activities against all the six Gram-positive and Gramnegative bacteria tested. On the other hand, different extraction methods yielded extracts with different antimicrobial capabilities. While the essential oils showed minimal selectivity in its antimicrobial activities against Gram-positive and Gram-negative bacteria, the methanolic crude extracts showed higher inhibitory activities against Grampositive bacteria tested in this study. Also, the methanolic extract of J. seravschanica leaves showed a potential anti-MRSA capability. It is evident from this study that despite variation in the chemical compositions, both the J. chinensis and J. seravschanica essential oils demonstrated broadspectrum antimicrobial activities against both Gram-positive and Gram-negative bacteria. Nevertheless, further studies are required to reveal the mechanisms of actions of the Juniperus essential oils against the bacteria.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Disclosure e findings presented in this study are part of the PhD project of Mr. Al-Dhafri Khamis S.
is study was conducted at both the University of Malaya, Malaysia, and Sultan Qaboos University, Oman.