Supplementary material

Chemical composition, antioxidant and antimicrobial activity of the essential oil from the leaves of Macleaya cordata (Willd) R. Br.

Chun-Mei Li^1,2, Xiao-Yong Yang³, Yi-Rong Zhong³ and Jian-Ping Yu<sup>1,2, *

1</sup> Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, People’s Republic of China

² Institute of Biochemistry and Nutrition, Guizhou University, Guiyang 550025, People’s Republic of China

*Corresponding author. Jian-Ping Yu.Tel: +86 851 3856374,

The essential oil from the leaves of Macleaya cordata R.Br. obtained by hydrodistillation was analysed by gas chromatography/mass spectrometry. Sixty eight compounds consisting up to 92.53% of the essential oil were identified. Antioxidant activities of the essential oil were evaluated by using DPPH radical scavenging and β-carotene-linoleic acid assays. The essential oil showed moderate antioxidant activity. In addition, the essential oil exhibited potential antimicrobial activity against all tested microorganisms, with diameters of inhibition zones ranging from 8.7±0.5 to 17.2±1.2 mm and MIC values from 125 to 500 μg/mL. We selected the most sensitive bacterium S. aureus as model to observe of the action of essential oils of M. cordata on the membrane structure by SEM. The treated cell membranes were damaged severely. The results presented here indicate that the essential oil of M. cordata may be potential sources of antioxidant and antimicrobial agents in the future.

Keywords: Macleaya cordata, essential oil, chemical composition, antioxidant activity, antimicrobial activity.

Experimental

M. cordata were obtained in Guizhou Province of China during august 2012 and their leaves were gathered, naturally dried in the shade. The taxonomical identification of the plant was done by Prof. Haimin Liao of Guizhou University. An authenticated specimen(GZIBN20120813) of the plant was also preserved in the Institute of Biochemistry and Nutrition, Guizhou University.

Analytical grade EtOH, anhydrous Na₂SO₄, dimethyl sulphoxide (DMSO), Na₂CO₃, Linoleic acid, Tween-40, Butylated hydroxytoluene (BHT) and all cultures media were obtained from Green Ltd. (Guiyang, China). β-Carotene and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (USA). The shape of S. aureus was observed with a scanning electron microscopy(SEM)(HitachiS-3400N). Absorbance was measured using a UV-VIS spectrophotomwter (TU-1901, China).

The dried leaves of M. cordata was ground(80 mesh), then they were submitted to hydrodistillation in a Clevenger-type apparatus at 100 ℃ for 6 h as described by Roszaini et al. (2013) with minor modifications. Briefly, The oily layer obtained on top of the aqueous distillate was separated and dried over anhydrous Na₂SO₄, filtered and kept in a sealed brown glass bottle at 4 ℃ for test.

Analysis of essential oil

Gas chromatography-mass spectrometry (GC-MS) analysis was carried out on a Agilent 6890 N GC/5975MSD-SCAN (Agilent Technologies, Palo Alto, CA,USA) in the electron impact (EI) ionisation mode (70 eV) and Zebron ZB-5Msi(bonded and cross-linked 5% Phenyl-95% DiMethylpolysiloxane, 30 m length and 0.25 mm I.D., 0.25 μm film thickness) capillary column(Phenomenex, Torrance, USA). The inlet and GC/MS interface temperatures were kept at 250 ℃ and 280 ℃, respectively. The temperature of El 70 eV source was 230 ℃ with full scan (20-480m/z). The oven temperature was kept at 50℃ for 2 min, then turned to 310 ℃ at rate of 5 ℃/min kept constant temperature at 310 ℃ for 8 min. The split ratio was 1:40. The carrier gas was Helium (99.99%) at a current velocity of 1 mL/min. The essential oil sample was diluted with hexane (1/100, v/v) and were injected manually. The mass spectra of the essential oil were analysed by comparing the mass spectra of 392,086 compounds from the mass spectra of Wiley 7 N, 174,948 compounds spectra in Mass Spectra Library Nist 2002 as well as by comparison of their retention times(Oke, Aslim, Ozturk, &Altundag, 2009; Adams, 2001).

Antioxidant activity of the essential oil of M. cordata has been evaluated by two systems namely DPPH and β-carotene-linoleic acid(Oke, Aslim, Ozturk, &Altundag, 2009).

DPPH radical is a stable free radical and can easily degraded in the presence of antioxidants. The activity of antioxidant on DPPH radical scavenging was known to be owing to their hydrogen donating ability or radical scavenging activity. When an antioxidant donate a hydrogen to the radical form of DPPH, the radical form of DPPH is transformed into a stable DPPH molecule. In this reaction process, a colour change from purple to yellow, and a decrease in absorbance(Molyneux, 2004). In the DPPH assay, radical-scavenging activity of essential oils was carried out according to a published DPPH radical-scavenging activity assay method(Oke, Aslim, Ozturk, &Altundag, 2009) with minor modifications. 2 mL of essential oils at various concentrations (0.2-1.2 mg/mL)in methanol was mixed with 0.5 mL of 0.2 mM DPPH methanolic solution. The mixture was shaken intensively and kept at 37℃ for 30 min in a dark place, and BHT was selected as the positive control. The absorbance of tested solution was then determined with a ultraviolet spectrophotometer at 517 nm. The percentage inhibition of free radical DPPH (I%) was analysed with the following method: I% = [(A_NC-A_s)/A_NC] × 100. Where A_NC is the absorbance of the negative control reaction (containing all reagents except the tested samples), and A_s is the absorbance of the tested sample. Tests were implemented in triplicate and I% values were showed as means ± SD of triplicates.

β-carotene-linoleic acid bleaching method was carried out to evaluate the antioxidant activity of the essential oil. In β-carotene-linoleic acid system, β-carotene occurs fast discoloration in the absence of an antioxidant. This is because of the coupled oxidation of β-carotene and linoleic acid, which forms free radicals. The linoleic acid free radical generated upon the abstraction of a hydrogen atom from one of its diallylic methylene group attacks the highly unsaturated β-carotene molecules. As a result, β-carotene is oxidized and broken down in part, subsequently the system loses its chromophore and characteristic orange color, which is mensurated with spectrophotometrically(Kulisic, Radonic, Katalinic, &Milos, 2004). We borrowed the method described by Miraliakbari and Shahidi(Miraliakbari & Shahidi, 2008) with slight modifications. In this assay, the inhibition percentages of conjugated diene hydroperoxides arising from the linoleic acid oxidation was measured for estimating the antioxidant activity of test samples. Briefly, β-carotene-linoleic acid mixture solution was prepared as following method: 0.5 mg of β-carotene dissolved in 1 mL of CHCl₃ as the β-carotene solution preparing for following test.25 μL of linoleic acid and 200 mg of Tween-40 were added in the β-carotene solution. CHCl₃ was then evaporated using a rotary evaporator at 50℃. Then 100 mL of oxygenated distilled water were added and mix well, The samples(essential oil and BHT) were diluted in methanol to prepare different concentrations sample solution from 0.2 mg/mL to 1.2 mg/mL. 0.5mL of sample solution was added to 2.5 mL of the above mixture in test tubes. and the same volume of methanol was used as the negative control. The test tubes were kept in an electro thermostatic water Bath at 50℃ for 2 h, together with BHT as the positive control. The microplates were then placed in an incubator at 50℃ for 2 h. BHT was selected as the positive control. The absorbance was measured using an ultraviolet spectrometer at 470 nm. Inhibition percentage(I%) of samples were counted as following formula: I%=(A_{β-carotene after 2 h assay}/A_{initial β-carotene}) ×100, where A_{β-carotene after 2 h assay} is the absorbance value of samples mixture after 2 h reaction, and A_{initial β-carotene} is the absorbance of the initial mixture. Tests were implemented in triplicate and I% values were showed as means ± SD of triplicates.

The essential oil of M. cordata were individually tested against ten microorganisms. Following microbial strains were used in this report: Three Gram-positive (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 27217, and Staphylococcus haemolyticus ATCC 29213).Three Gram-negative (Ralstonia solanacarum GMI 1000, Agrobacterium tumefaciens ATCC 11158, Escherichia coli CMCC 44102), and two fungi (Aspergillus flavus ATCC 9643, Aspergillus niger CMCC 98003), two yeasts (Candida albicans ATCC 10231, Candida glabrata ATCC 15126). Bacterial strains were cultured 24 h at 37 ℃ in liquid LB medium, Fungus and yeast were cultured at 28℃ for 48 h in liquid PD medium(Wang et al., 2010). The diluted microbial suspension(10⁶ cfu/mL) was prepared for assay.

Antimicrobial activities of the essential oil of M. cordata were determined by the standard disc diffusion method(Kose, Deniz, Sarıkurkcu, Aktas, & Yavuz, 2010). The test sample was diluted in DMSO as mother liquid at an initial concentration of 10 mg/mL and sterilized after being filtered, respectively. Test microbial suspensions were applied on these media with sterilized swab. Every disc (6 mm in diameter) was injected with 10 μL of 10 mg/mL test sample, then thoes discs were placed on the petri dish one by one. DMSO was negative control. Gentamicin (20 μg/disc) was used as positive controls for bacteria and nystatin (20 μg/disc) was used as positive controls for fungi and yeasts. Antibiotic discs for suitable microorganisms were placed into the same petri dishes as positive controls. Test petri dishes with bacterial strains were incubated at 37 ℃ for 24 h, 27 ℃for 72 h for the fungi and 27 ℃ 48 h for yeasts. The diameters of inhibition zones were measured to judged antimicrobial activity and every measurement were repeated three times. Serial broth dilution method was carried out as reported by Muroi and Kubo(1996) to determine of the minimum inhibitory concentration (MIC). A set of solutions of the essential oil (concentration 1000 μg/mL to 7.8 μg/mL) were prepared by serial twofold dilutions. A suspension of each microorganism((10⁶ cfu /mL)was prepared. Test samples and 1 mL portions of the culture medium were injected the test tubes, then mixed with 20 mL of the microbial suspensions. The microbial growth was examined by turbidity after being incubated at 37 ℃ for 24 h in LB medium. Gentamicin for bacteria, nystatin for fungi and yeasts were used as standard drugs for positive control. The MIC value was the mimimum concentration of the samples yielding no visible growth.

Observation by scanning electron microscopy（SEM）

Final concentration of 100 μg/mL of the essential oils and concentration of 10⁶ cfu/mL S. aureus in LB medium was prepared. Control experiment was without essential oil. The bacterial suspension were incubated with shaking at 150 rpm at 37 ℃. After 24 h incubating, the suspension was washed with PBS buffer solution three times and then fixed with 4 % glutaraldehyde solution for 12 h at 4 ℃. After full reaction, the fixed S. aureus were then washed with 30%, 50%, 70%, 80%, 90%, and 100% of EtOH respectively. After gold spraying and freezing-drying, the shape of S. aureus cell was observed.

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Table S1. Phytochemical compositions of the essential oils from the leaves of M. cordata .