Chemical composition, antibacterial and antifungal activities of Cinamomum bejolghota bark oil from Thailand

Article history: Received on: 09/02/2017 Accepted on: 24/03/2017 Available online: 30/04/2017 The volatile constituents of Cinnamomum bejolghota bark essential oil were investigated by using gas chromatography-mass spectrometry (GC-MS). Thirty-six volatile constituents were identified with the major components being 1,8-cineole, γ-terpineol, borneol and terpinen-4-ol. Essential oil of C. bejolghota bark was firstly screened for their antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, as well as, Colletotrichum sp. fungi using disc diffusion method. Minimal inhibitory concentration (MIC) of C. bejolghota bark oil was further analyzed by microdilution. Essential oil of C. bejolghota bark was most effective against bacteria with MIC ranging from 31.25-62.50 μg/mL, whereas inhibition against fungal pathogens was moderate, with MIC of 125500 μg/mL. The strong antimicrobial activity of C. bejolghota bark oil was correlated mainly to 1,8-cineole, γ-terpineol, borneol, terpenen-4-ol and linalool.


INTRODUCTION
Volatile components of essential oils are mainly represented by terpenoids, phenylpropanoids or benzenoids, fatty acid derivatives and amino acid derivatives (Dudareva et al., 2006). Volatile components of essential oils possess potential antimicrobial and insecticidal activities against pathogens including those causing human pathogenic diseases and crop spoilage in agriculture (Singh & Maurya, 2005). Use of essential oils as antimicrobial agents is environmentally safe and economical. In addition, essential oils from various parts of plants are widely used for gargles in throat infection, skin care (Gutiérrez et al., 2008), beauty treatments (Price, 2003), herbal medicines (Schultz et al., 2001), aromatherapy (Price, 2003), cosmetics (Tisserand & Young, 2013) and perfumery applications (Nielsen & Rios, 2000). Essential oil of Cinnamomum plant, belonging to the Lauraceae family, is obtained from its leaves and barks and is widely used as a flavoring agent in food, as well as for cosmetic and pharmaceutical applications (Sudmoon et al., 2014). The essential oil of Cinnamomum genus plants contained the great antimicrobial (Ooi et al., 2006), antifungal (Giordani et al., 2006), antiinflammatory (Miguel, 2010) and antixidant (Jayaprakasha et al., 2003) properties. Cinamomum bejolghota (Buch.-Ham.) is a medicinal plant, apply as the treatment of a cough, cold, toothache, liver complaints (Rao, 1979). The plant is widely distributed in China, Vietnam, Sri Lanka, Madagascar, India and East of Thailand (Li et al., 2013). Baruah et al., 1997 reported linalool as a major volatile in essential oil of C. bejolghota leaf and panicle cultivated in India, whereas α-terpineol and E-nerolidol were found as the main components in its stem bark oil. Conversely, high amounts of 1,8-cineole and α-terpineol were detected in essential oil of C. bejolghota bark collected from different areas in India (Choudhury et al., 1998). Only few studies have identified volatile profiles of C. bejolghota essential oil, though there is no previous study reporting the antimicrobial and antifungal properties of C. bejolghota oil. The aim of this study was to investigate the chemical composition of C. bejolghota oil from Thailand, to provide baseline data on its antibacterial and antifungal properties, and to predict its usefulness as a natural antimicrobial and antifungal agent in postharvest processing.

Plant material
Stem bark of C. bejolghota (Buch.-Ham.) was collected in April 2015 from Trat province, Eastern Thailand and air dried for 7 days. Voucher herbarium specimen (MFLU No. 10000) of the 1-year old plant was identified and deposited at the Mae Fah Luang University Botanical Garden, Chiang Rai, Thailand.

Extraction of essential oil and chemical composition analysis
One hundred grams of C. bejolghota dried bark were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus. The essential oils were dried using anhydrous sodium sulfate. The chemical composition of C. bejolghota essential oil was carried out on a Hewlett Packard model HP6890 gas chromatograph (GC) (Agilent Technologies, Palo Alto, CA, USA) equipped with an HP-5MS (5% phenylpolymethylsiloxane) capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm; Agilent Technologies, USA) employed with an HP model 5973 mass selective detector (MS). The oven temperature was programmed at an initial temperature of 60 °C prior ramping at a 3 °C/min until a maximum of 200 °C was reached. The temperatures of the injection and detection steps were set at 250 and 280 °C, respectively. Helium was used as the carrier gas with a flow rate of 1 mL/min. The EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 29-300. The electron multiplier voltage was 1150 V. The ion source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. One microliter of C. bejolghota essential oil was dissolved in n-hexane (1:100 v/v) prior to injection into the GC-MS system with a split ratio of 1:200. Identification of essential oil composition was accomplished by comparison between their relative retention indices (RI) to C 8 -C 16 n-alkanes, and using a comparison of the mass spectra of individual components with the reference mass spectra in the W8N08 and NIST08 databases, and published literature. Quantification of all identified components was investigated by using a percent relative peak area.

Antibacterial activity assay
Antibacterial activities of C. bejolghota bark oil were investigated against 6 bacterial pathogens representing three Gram-negative bacteria (Salmonella typhimurium TISTR292, Pseudomonas aeruginosa TISTR781 and Escherichia coli TISTR780) and three Gram-positive bacteria (Staphylococcus aureus TISTR1466, Bacillus subtilis TISTR008 and B. cereus TISTR687). All bacterial pathogens were obtained from the Thailand Institute of Scientific and Technological Research, Thailand. The antibacterial activities of C. bejolghota essential oil were determined by using a disc diffusion assay (Ross et al., 2013). Each bacterial strain was cultured in tryptic soy agar medium at 37 °C which the single colony was collected and further adjusted to 0.5 McFarland standard. Subsequently, the bacteria were swabbed on a Mueller Hinton agar medium plate by using sterilized cotton. Essential oil of C. bejolghota bark was diluted by two-fold dilution method with dichloromethane to perform the final concentrations of 1000, 500, 250, 125, 62.50 31.25, 7.81 and 3.91 μg/mL, respectively. Twenty microliters of C. bejolghota bark oil with different concentrations were loaded into a 6 mm-diameter sterile paper disc (Whatman TM , USA) and then placed on Mueller Hinton agar medium plate. All plates were incubated at 37 °C for 24 h. The inhibition zone diameter of different C. bejolghota oil concentrations was measured in millimeters. Minimum inhibitory concentration (MIC) values inhibiting bacterial growth were also determined. Penicillin was used as positive control in this study. All experiments were performed in triplicate.

Antifungal activity assay
The plant pathogenic fungi used in this study were obtained from the Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand including Collectotrichum asianum MFULCC10-0286, C. fruticola MFLUCC10-0288, C. tropica MFLUCC11-0114, and C. magna MFLUCC12-0713. The antifungal activities of C. bejolghota essential oil were determined by using the disc diffusion method (Murray et al., 1995). Essential oil of C. bejolghota bark was prepared by using two-fold dilution method at final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 7.81 and 3.91 μg/mL. Initially, all pathogenic fungi were cultured on potato dextrose agar (PDA) media and incubated at 30 °C for 1 week. A plug of 1-week old fungal culture (6 mm diameter) of each strain was placed on the center of PDA medium plates. Ten microliters of different essential oil concentrations were individually loaded into 6 mmdiameter sterile paper disc (Whatman TM , USA) and then placed on plates containing a plug of fungal culture. The plates were incubated at 30 °C for a week. The mycelial fungal growth inhibition of each fungal strain was calculated according to the following equation: Percentage of inhibition (%) = 100 [(1-radical growth of treatment (mm)/radical growth of control (mm)]. All experiments were performed in triplicate. In addition, MIC values inhibiting mycelial fungal growth were also determined.

Data analysis
The experiments were performed in triplicate and are reported as mean ± standard deviation. Quantitative variations were analyzed by one-way ANOVA (at P<0.05). Duncan's Multiple Range test combined with the Statistical Analysis System (Sas, 1990) was used to study the differences among samples.

RESULTS AND DISCUSSION
The extraction yield of C. bejolghota bark oil was 1.01%v/v with pale yellow color. Thirty-six volatile components were detected in the essential oil of C. bejolghota bark, accounting for 97.96% of the total oil composition. Oxygenated monoterpene and monoterpene were considered as the major compounds as shown in Table 1. The major constituent was 1,8-cineole (40.24%), followed by γ-terpineol (15.41%), borneol (7.86%), terpinen-4-ol (7.55%) and α-pinene (6.58%), respectively (Adams, 1995;König et al., 1999). Volatile compounds represented aromatic profile of Cinnamomum plant were also detected such as Z-cinnamaldehyde, α-amyl cinnamyl alcohol, E-isoamyl cinnamate, E-2-hexyl cinnamaldehyde, benzyl cinnamate and phenethyl cinnamate. The volatile profiles in this study differed from the study of Baruah et al., 1997 andChoudhury et al., 1998 whereby α-terpineol and E-nerolidol were identified as the principle components in bark oil of C. bejolghota. The high variation of essential oil components between locations could be due to differences in the time of harvesting and extraction method (Heywood, 2002). Extrinsic variables based on geographic origin include climatic and soil-growth conditions, both of which may cause environmental stress and variability of chemical composition (Vokou et al., 1993). The antibacterial activities of C. bejolghota bark oil in terms of inhibition zone diameter and MIC are demonstrated in Table 2. The most sensitive bacterial strain was E. coli TISTR780 followed by P. aeruginosa TISTR781, S. aureus TISTR1466, B. subtilis TISTR008, S. typhimurium TISTR292 and B. cereus TISTR687. The MIC of C. bejolghota bark oil against various bacterial species ranged between 31.25 and 62.25 µg/mL. The antifungal properties of C. bejolghota bark oil against four postharvest pathogenic fungi and MIC values are shown in Table  3. The C. bejolghota bark oil displayed the strongest antifungal activity against C. asianum, with MIC of 125 µg/mL, while the MIC values against other postharvest pathogenic fungi ranged between 250 and 500 µg/mL. Although antimicrobial activities of Cinnamomum spp. essential oils have been widely reported, the effectiveness of C. bejolghota bark oil on pathogenic species has been less studied. The mechanisms of the antimicrobial action of essential oil from plants are still not clearly understood. Terpenoids are major components of essential oil possessing hydrophobic and hydrophilic parts with different functional groups. This enables terpenoids to simply transport across bacterial or fungal cell walls and interact with the microbes (Burt, 2004;Koroch et al., 2007). The antimicrobial activity of C. bejolghota bark oil may be correlated to the diversity of its bioactive compounds. These include 1,8-cineole (comprising 40.24% of the oil) and γ-terpineol (15.41%) in the essential oil, both of which have potent antibacterial and fungicidal activities (Carson et al., 2002;Hendry, Worthington et al., 2009;Wang et al., 2012). Mahboubi and Kazempour, 2009 reported that antibacterial activity of whole essential oil was greater than that obtained from major components alone. The great antimicrobial activity of C. bejolghota bark oil could be also contributed to a combination of minor components including linalool, borneol, isoborneol, α-pinene, β-pinene and camphor (Koutsoudaki et al., 2005;Santoyo et al., 2005;Sivropoulou et al., 1997). The strong antimicrobial activity against E. coli and B. cereus is particularly interesting, because both microbes are classified as human pathogens. According to the Advisory Committee on Dangerous Pathogens, both bacteria belong to the second hazard group of biological agents which pose risk to human health. Moreover, growth inhibition of these bacteria is important because of their role in food contamination. In addition, strong antimicrobial activity was significant against C. asianum with 30.86% growth inhibition. Therefore, essential oil of C. bejolghota bark is a potential antibacterial and antifungal agent that may find wider applications in food industry and postharvest processing.

CONCLUSIONS
The present study indicated that essential oil obtained from the stem bark of C. bejolghota is rich in oxygenated monoterpenes, mainly 1,8-cineole, which constitutes 40.24% of the total oil composition. Biological evaluation revealed that the C. bejolghota bark oil possesses strong antibacterial and antifungal activities. C. bejolghota oil may be viewed as a bioactive natural product with cosmetic or postharvest production applications.