SUPPLEMENTARY MATERIAL

Transcription

1 SUPPLEMENTARY MATERIAL Chemical composition and antimicrobial activities of essential oil from Wedelia urticifolia growing wild in Hunan Province, China Junpeng Hu a,mengmeng Jia a,liang Zhu a * a School of Food Science and Engineering, South China University of Technology, Wushan Road 381, Guangzhou, , China *Corresponding author: School of Food Science and Engineering, South China University of Technology, Wushan Road 381, Guangzhou, , China; zhuliang@scut.edu.cn The essential oil obtained from W. urticifolia growing in Hunan Province, China, was analyzed for the first time by capillary GC and GC-MS. A total of 67 constituents, representing % in essential oil were identified. The major constituents of the oil were: α-pinene (8.85 %), limonene(6.38 %), carvacrol (6.15 %), caryophyllene (6.08 %), spathulenol (5.49 %), sabinene (5.36 %), camphor (4.34 %). Antimicrobial potential of oil against bacterial strains (Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis, and Staphylococcus aureus), yeast strains (Hansenula anomala and Saccharomy cescerevisiae) and molds (Aspergillus niger, Chaetomium globosum, Mucor racemosus, and Monascus anka) was determined by disc diffusion method and broth micro dilution method, respectively. The oil exhibited promising antimicrobial effect as a diameter of zones of inhibition ( mm). Minimum inhibitory concentration values of oil were ranged μg/ml. Keywords:Wedelia urticifolia; Essential oil; Chemical composition; Antimicrobial activity

2 Experimental Section Plant material:wedelia urticifolia plants were collected from the Heng Mountains, Hunan Province, China on August 2010, and identified by Dr. Xun Gong. The voucher specimen (No ) was deposited in the Kuming institute of Botany, Chinese Academy of Sciences. The plants were dried in the shade (at room temperature). Isolation of the essential oil: The air-dried plant materials (1000 g) of W. urticifolia was chopped and subjected to hydrodistillation for 5 h using a Clevenger type apparatus. The obtained oils were dried over hydrous sodium sulphate for 24 h, filtered, and then stored at 4 C in sealed brown glass vials until tested. GC-FID Analysis: An Agilent HP-6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with an HP-5 5% phenylmethylsiloxane capillary column (30 m 0.25 mm i.d., 0.25 μm film thickness) and equipped with an FID detector was used for the GC-FID analysis. Helium gas at a constant flow rate of 1 ml/min was used as the carrier gas. The injector and mass transfer line temperatures were set at 250 and 280 C, respectively. The essential oil solution (1 μl) in hexane was injected and analysed under the following column conditions: initial column temperature at 40 C for 1 min, which was then increased to 250 C at a 3 C/min heating ramp, and then subsequently kept at 250 C for 20 min. GC-MS analysis: Quantitative and qualitative analysis of the essential oil was performed using a GC-MS system (Agilent Technologies, Palo Alto, CA, USA) equipped with a HP-5 MS fused silica capillary column (30 m 0.25 mm i.d., 0.25 μm film thickness). For GC-MS detection, an electron ionisation system with a 70 ev ionisation energy was used. Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. The injector and mass transfer line temperatures were set at 250 and 280 C, respectively. Essential oil solution (1 μl) in hexane was injected and then analysed under the following column conditions: initial column temperature at 40 C for 1 min, which was then increased to 250 C at a 3 C/min heating ramp, and then subsequently kept at 250 C for 20 min. The Kovats indices

3 were calculated for all volatile components using a homologous series of n-alkanes (C 8 C 25 ) on the HP-5 MS column. The major oil components were identified via coinjection with standards (whenever possible) and confirmed through the Kovats indices using the Wiley (V.7.0) and National Institute of Standards and Technology (NIST) V.8.0 GC MS library. The relative concentration of each compound in the essential oil was quantified based on the peak area integrated in the analysis program. Identification of compounds:the essential oils components were identified by comparing their spectra and retention indices (Kovat Index) with those of the authentic samples and literature values(bajalan et al. 2017, Rao et al. 2014, Zhu et al. 2005) Antimicrobial activity: The in vitro antimicrobial activities of the essential oil were evaluated against a panel that included laboratory control strains obtained from the China Centre for Type Culture Collection (CCTCC). These strains are two Gram-negative bacteria (Pseudomonas aeruginosa CCTCC AB93066 and Escherichia coli CCTCC AB91112), two Gram-positive bacteria (Bacillus subtilis CCTCC AB92068 and Staphylococcus aureus CCTCC AB91053), two yeast strains (Hansenula anomala CCTCC AY92046 and Saccharomy cescerevisiae CCTCC AY92042), and four moulds (Aspergillus niger CCTCC AF91004, Chaetomium globosum CCTCC AF , Mucor racemosus CCTCC AF 93209, and Monascus anka CCTCC AF93208). All strains were maintained on an agar slant at 4 C. The bacterial strains were cultured in a Muller-Hinton broth (MHB) at 37 C for 24 h, whereas the yeast strains were cultured on a Sabouraud dextrose agar (SDA) at 28 C for 48 h. The fungal strains were cultured on SDA at 28 C for 120 h prior to testing. The disc diffusion method was used to determine the antimicrobial activities of the essential oils. Petri plates were prepared by pouring 20 ml MBH or SDA and allowing the solution to solidify. The plates were then dried, and 0.1 ml of the standardised inoculum containing 10 6 to 10 7 CFU/ml of the bacterial suspension was poured, uniformly spread, and allowed to dry for 5 min. A Whatman No. 1 sterile filter paper disc (6 mm diameter) was impregnated with 1000 μg/disc of the essential oils. Negative controls were prepared using the same solvent employed used to

4 dissolve the samples. The standard reference antibiotics, namely, streptomycin and tetracycline (10 μg/disc) were used as the positive controls for the test bacteria. The plates were incubated for bacteria at 37 C for 24 h, for yeasts at 28 C for 48 h, and for fungi at 28 C for 120 h. The antimicrobial activity was evaluated by measuring the diameter of the zones of inhibition against the test organisms(li et al. 2016, Sureshkumar et al. 2007). The experiments were repeated in triplicate and the results are expressed as average values. The minimum inhibitory concentration (MIC) values of the essential oils against the test microorganisms were determined using the broth micro dilution method. Dilutions of the essential oils, ranging from 0.25 to 1000 µg/ml, were prepared in MHB or SDA. Exactly 0.5 MacFarland standard suspensions of the test microorganisms were inoculated in the tubes. A control test was also performed using inoculated broth or agar supplemented only with dimethyl sulphoxide under identical conditions, with streptomycin as the reference. The bacteria were incubated at 37 C for 24 h, the yeast strains at 28 C for 48 h, and fungi at 28 C for 120 h. Statistical analysis: All tests were performed in triplicate, and the results were calculated as the mean ± SD. Reference Bajalan I, Rouzbahani R, Pirbalouti AG, Maggi F Variation in chemical composition and antibacterial activity of the essential oil of wild populations of Phlomis olivieri. Chem Biol Li SF, Ding JY, Li YT, Hao XJ, Li SL Antimicrobial Diterpenoids of Wedelia trilobata (L.) Hitchc. Molecules. Apr 07;21:457. Rao BRR, Syamasundar KV, Patel RP Chemical Profile Characterization of L. Essential Oils From South India Through GC-FID and GC-MS Analyses. Journal of Essential Oil Bearing Plants.17: Sureshkumar S, Kanagasabail R, Sivakumar T, Chandrasekar MJN, Thiruvenkatasubramaniam R, Thenmozhi S Antimicrobiological studies on different essential oils of Wedelia species (W. chinensis, W. trilobata and W. biflora) and Eclipta alba (Asteraceae). Asian J Chem.19:

5 Zhu S, Lu X, Xing J, Zhang S, Kong H, Xu G, Wu C Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography mass spectrometry for the analysis of tobacco essential oils. Anal Chim Acta.545:

6 Table S1. Chemical composition of the essential oil of W. urticifolia. Peak no. RI a Components % RA b Identification Methods c Monoterpene hydrocarbons α-thujene 0.28 MS, RI α-pinene 8.85 MS, RI, Co Camphene 1.81 MS, RI Sabinene 5.36 MS, RI, Co β-pinene 1.22 MS, RI β-myrcene 1.03 MS, RI α-phellandrene 2.35 MS, RI α-terpinene 0.37 MS, RI Carene 0.48 MS, RI Limonene 6.38 MS, RI, Co β-phellandrene 1.26 MS, RI, Co β-ocimene 0.46 MS, RI γ-terpinene 2.35 MS, RI Oxygenated monoterpenes Eucalyptol 1.28 MS, RI Lavandulol 0.37 MS, RI Linalool oxide 0.65 MS, RI Linalool 1.25 MS, RI trans-pinocarveol 0.63 MS, RI trans-sabinol 0.46 MS, RI Camphor 4.34 MS, RI, Co Borneol 0.67 MS, RI Terpinene-4-ol 0.54 MS, RI α-terpineol 1.56 MS, RI Carvone 0.64 MS, RI Geraniol 0.53 MS, RI

7 Sesquiterpene hydrocarbons α-cubebene 1.57 MS, RI α-copaene 0.44 MS, RI β-bourbonene 0.83 MS, RI β-elemene 0.64 MS, RI α-cedrene 1.05 MS, RI α-gurjunene 0.36 MS, RI Caryophyllene 6.08 MS, RI, Co β-cedrene 0.63 MS, RI γ-elemene 1.12 MS, RI α-humulene 1.25 MS, RI trans-β-farnesene 1.46 MS, RI γ-muurolene 2.64 MS, RI Germacrene D 1.78 MS, RI Bicyclogermacrene 0.46 MS, RI α-muurolene 0.65 MS, RI trans-α-farnesene 1.37 MS, RI δ-cadinene 0.34 MS, RI Selina-3,7(11)-diene 0.36 MS, RI Germacrene B 0.25 MS, RI Oxygenated sesquiterpenes trans-nerolidol 1.03 MS, RI Spathulenol 5.49 MS, RI, Co Caryophyllene oxide 2.39 MS, RI Guaiol 0.56 MS, RI Cedrol 0.54 MS, RI Isospathulenol 0.38 MS, RI α-muurolol 1.24 MS, RI α-cadinol 0.67 MS, RI

8 Pogostol 0.46 MS, RI β-eudesmol 0.58 MS, RI α-bisabolol 1.67 MS, RI Phenylpropanoids p-cymene 2.35 MS, RI, Co Methyl thymyl ether 0.82 MS, RI Carvacrol 6.15 MS, RI, Co Eugenol 1.33 MS, RI Methyl eugenol 1.45 MS, RI Others Hexen-1-ol 0.32 MS, RI Heptanal 0.56 MS, RI Octen-3-ol 0.44 MS, RI Bornyl acetate 1.26 MS, RI Isophytol 0.32 MS, RI n-hexadecanoic acid 0.27 MS, RI Total identified (%) 98.68

9 Table S2. Zones of growth inhibition (mm) and minimum inhibitory concentrations of essential oil of W. urticifolia against the growth of microorganisms. Microorganism Diameter of zones of inhibition Essential oil a Standard b MICs of Essential oil c SM TC Pseudomonas aeruginosa 19.3 ± ± ± Escherichia coli 18.6 ± ± ± Bacillus subtilis 24.9 ± ± ± Staphylococcus aureus 24.6 ± ± ± Hansenula anomala 22.2 ± ± ± Saccharomy cescerevisiae 22.0 ± ± ± Aspergillus niger 18.1 ± ± ± Chaetomium globosum 17.5 ± ± ± Mucor racemosus 16.8 ± ± ± Monascus anka 17.3 ± ± ± Diameter of inhibition zones (mm) including the diameter of disc (6 mm), values are given as mean ± SD of triplicate experiment. a Diameter of inhibition zones of essential oil (tested volume 1000 μg/disc). b Standard antibiotics: SM, streptomycin; TC, tetracycline (tested volume 10 μg/disc). c MIC, Minimum inhibitory concentration (values in μg/ml).