SUPPLEMENTARY MATERIAL

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1 SUPPLEMENTARY MATERIAL Chemical composition and biological activities of the essential oil from Rubus pungens var. oldhamii Yaojie Zhang, Jiajing Chen, Lizhi Wang, Jingjing Cao,and Lishan Xu* College of Chemistry and Life Science, Zhejiang Normal University,Jinhua , PR China *Corresponding author:lishan-xu, phone: , ABSTRACT This paper presents a study on chemical composition, antimicrobial, antioxidant and tyrosinase inhibitory properties of the essential oil from leaves of Rubus pungens var. oldhamii (REO). The major component of the REO is sesquiterpenes (36.04%), which consists of 1,5-Cyclooctadiene,3-(1-methylallyl)-(8CI)(17.66%), 5,6-Diethenyl -1-methylcyclohexene (12%), (+) γ-elemene (10.48%), and β-caryophyllene (8.39%).The REO showed moderately active against S.aureus, A.niger and P.glaucum, and weak antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl(DPPH) assay. Furthermore, tyrosinase inhibition was investigated against monophenolase (L-tyrosine). IC 50 values of REO and arbutin were found and mg/ml, respectively. The REO exerted potential antityrosinase activity. Our test results indicated that the REO was rich in sesquiterpenes, and also exhibited good antityrosinase activity, and moderate antimicrobial activity against pathogenic microorganisms. The REO can be used as a natural source of promising antimicrobial and tyrosinase inhibitor. Key words: Rubus pungens var. oldhamii; essential oil; chemical composition; biological activities 1. Experimental 1.1 Chemicals DPPH (2,2-dipheny-1-picryl hydrazyl), Ascorbic acid, Ethanol, DMSO were purchased from Aladdin,and Tyrosinase were purchased from Wako. All the chemicals and solvents used in this study were of analytical grade.

2 1.2 Plant material and essential oil isolation The leaf part of Rubus pungens var. oldhamii was collected from Tianmu Mountain (Hangzhou,China)during June The plant was identified by Dr. Chen Wenrong from Zhejiang Normal University. An authenticated specimen (ZNU -Ⅰ ) of the plant was also preserved in the College of Chemistry and Life Science, Zhejiang Normal University. The essential oil of Rubus pungens var. oldhamii was obtained by hydrodistillation in a Clevenger apparatus, adapted to a ml round-bottom flask. A sample of the fresh material was immersed in distilled water at a ratio of 1:10 (w/v). Extraction time was set at 120 mins, counting from the moment at which the water in the flask began to boil. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in an air tight glass container at Gas chromatography mass spectroscopy (GC MS) analysis Samples of essential oils were analysed in an Agilent GC MS apparatus equipped with an Agilent J&W GC Columns HP-5(30 m mm,0.25 μm),helium(0.8 ml/min) was used as a carrier gas. Samples were injected in the split mode at a ratio of 100:1. The injector was kept at 280 and the transfer line at 300. The column was maintained at 70 for1 min and then programmed to 180 at 3 /min and held for 15 min. The MS was operated in the EI mode at70 ev, in the m/z range The identification of the compounds was performed by comparing their retention indices and mass spectra with those found in the literature (Adams.1995) and supplemented by the Wiley & QuadLib 1607 GC MS libraries.the relative proportions of the essential oil constituents were expressed as percentages obtained by peak area normalization, all relative response factors being taken as one. The Kovat indices were determined from the retention times after co-injection with n-alkanes. 1.4 Antioxidant activity The antioxidant activity was measured in terms of radical scavenging ability using the DPPH method, as modified by Sokmen et al. (2005).A 1.0 ml of REO was mixed with 1.5 ml of 0.1 mm DPPH in ethanol. The mixture was then vortexed vigorously and allowed to stand for 30min in the dark. The absorbance of the sample was

3 measured at 517 nm against a blank. Ascorbic acid were used as positive controls and all tests were carried out in triplicate. Antioxidant activity tested of essential oils was expressed as a percentage of inhibition and it was calculated by using the following formula: DPPH(%)=[1 (absorbance of sample/absorbance of control) ] 100. Results were expressed as IC 50, it corresponds to the dose required to cause a 50%inhibition. 1.5 Tyrosinase inhibition activity Tyrosinase activity inhibition was determined for spectrophotometrically according to the method described previously (Dong et al. 2016), with minor modifications.the essential oil was firstly dissolved in DMSO and then diluted to different concentrations with ddh 2 O. Each of the sample solution(40 μl) in the test tubes, followed by the addition of 100μL of 0.44 mg/ml L-tyrosine were mixed and incubate for 5 min at 37 C. Then 10μL of mushroom tyrosinase solution (500 units/mg) was added last to the mixture. After incubation for 6 min at 37 C, and the absorbance at 475 nm was measured. ddh 2 O mixed with DMSO (40 μl ) and arbutin solution were used as the blank reference and positive control, respectively. The percentages of tyrosinase inhibition were calculated according to the following equation: Inhibition( % )=(1 OD value of sample /OD value of control) 100. Results were expressed as IC 50 (mg/ml) Antimicrobial activity Antibacterial activities of the essential oil were evaluated against the common bacteria of food by filter paper method(zhang et al.2015).compered with the McFarlandStandard,Using activated bacteria prepared a solution at a concentration of CFU/mL.After the beef-protein medium cooling down,100 µl bacterial suspension was added to each dish coated uniformly by glass spatula.then each dish put three pieces of filter paper,which were added to 20 μl ethanol diluted oil,using anhydrous ethanol as blank control.the dishes were placed in a temperature of 4 refrigerator overnight statically,then removed the dishes and placed in 37 biochemical incubator for 12 h.finally,measuring its inhibition the diameter by using

4 cross method. Using growth rate method measured the antifungal effect of the essential oil against common molds (Shen etal.2013).firstly,taking 1 ml of the essential oil uniformly coated in a condensed PDA medium,using the same amount of ethanol to replace oil as a blank control.cutting a round part of molds 9 mm in diameters and putting it in the medium,then culturing until the colony diameter more than 20 mm.according to formula (1) calculating the inhibition rate by using growth rate method. Inhibition rate (%) = [(d0-d1) / (d0-d2)] 100% (1) d0: colony diameter of blank control group (mm); d1: colony diameter of experimental group (mm); d2: bacteria patty diameter (mm). Gentamicin Sulfate and Fluconazol were used as standard antibacterial and antifungal drug respectively 2. Statistical Analysis All tests were conducted in triplicate and results are expressed as mean value ± standard deviation (SD),. Correlation and regression coefficients were performed using Statistical Package for the Social Sciences (SPSS).ANOVA was used for the analysis (LSD test) at the significant level of p-value <0.05. References Dong X, Zhang Y, He JL, Zhang S, Zeng MM, Chen J, Zheng ZP Preparation of tyrosinase inhibitors and antibrowning agents using green technology. Food Chemistry. 197: O. David Sparkman Identification of essential oil components by gas chromatography/mass spectroscopy Robert P. Adams. Journal of the American Society for Mass Spectrometry. 8: Sokmen M, Angelova M, Krumova E, Pashov S, Ivanchev S, Sokmen A In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum. Life Sciences. 76: Shen JX, Zhang LL, Wen-Shan WU, Shao-Ying WU, Rui MA, Li-Shan XU Study on antibacterial activity of extracts from Pinus elliottii against plant pathogenic fungi. Journal of Shanxi Agricultural Sciences. 41: Zhang YB, Liu XY, Jiang PP, Li WD, Wang YF Mechanism and antibacterial activity of

5 cinnamaldehyde against Escherichia coli and Staphylococcus aureus. Journal of Modern Food Science and Technology. 5: Captions list of the table and figure Table S1. The components of the essential oil obtained from R. pungens var. oldhamii Fig. S1 GC chromatogram of R. pungens var. oldhamii essential oil Fig. S2 Antioxidant activity of the essential oil of Rubus pungens var. oldhamii and Ascorbic acid measured by DPPH radical assay. Fig. S3 Tyrosinase inhibitory activity of essential oil of Rubus pungens var. oldhamii and Arbutin Fig. S4 Effects of the different concentrations of essential oil of Rubus pungens var. oldhamii on the colony diameter (mm) Fig. S5 Effects of the different concentrations of essential oil of Rubus pungens var. oldhamii on the mycelial growth rate (%) Fig. S6. Some pictures of antimicrobial activities assaies about REO

6 Table S1. The components of the essential oil obtained from R. pungens var. oldhamii No Peak No CAS Formula Constituent RetIndex Area% C6 H10 O (2E)-2-Hexenal C6 H12 O Leaf alcohol C6 H12 O (E)-2-Hexenol C6 H14 O Amylcarbinol C7 H6 O Benzaldehyde C8 H8 O Benzeneacetaldehyde C10 H18 O Linalol C9 H18 O Nonanal C11 H16 3,4-Diethenyl-3-methylcyclohexene C11 H16 5,6-Diethenyl-1-methylcyclohexene C8 H8 O3 2-Carbomethoxyphenol C11 H16 1,5-Cyclooctadiene, 3-(1-methylallyl)- (8CI) C12 H18 Naphthalene, 1,4,4a,5,6,7- hexahydro-2,3-dimethyl C15 H24 Elixene C10 H12 O2 3-Allylguaiacol C15 H24 Aglaiene C15 H24 β-elemen C15 H24 β-caryophyllene C15 H24 Humulene C15 H24 (-)-Alloaromadendrene C15 H24 Germacrene D C15 H24 (+)-γ-elemene C15 H24 γ-muurolene C15 H26 O Hedycaryol C15 H24 O Espatulenol C15 H26 O (±)-Globulol C15 H26 O β-selinenol C15 H26 O α-selinenol C15 H24 O Farnesone C18 H36 O (±)-Phytone C20 H40 O (E)-Phytol Oxygenated monoterpenes 0.28 Sesquiterpene hydrocarbons 27.42

7 % Inhibition Oxygenated sesquiterpenes 8.62 Diterpenes 4.41 other Total identified Fig. S1 GC chromatogram of R. pungens var. oldhamii essential oil 70 REO concentration (mg/ml)

8 % inhibition 100 Ascorbic acid concentration ( g/ml) Fig. S2 Antioxidant activity of the essential oil of R.pungens var. oldhamii(reo) and Ascorbic acid measured by DPPH radical assay.

9 The disc diameters of zone of inhibition(mm) Tyrosinase inhibition (%) Essention oil Arbutin Concentration (mg/ml) Fig.S3 Tyrosinase inhibitory activity of essential oil of R.pungens var. oldhamii and Arbutin Bacillus subtilis Staphylococcus aureus huifayou Concentration (mg/ml) Fig. S4 Effects of the different concentrations of essential oil of R.pungens var. oldhamii on the colony diameter (mm)

10 The disc diameters of zone of inhibition(mm) Concentration (mg/ml) Fig. S5 Effects of the different concentrations of essential oil of R. pungens var. oldhamii on the mycelial growth rate (%)

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12 Fig. S6. Some pictures of antimicrobial activities assaies about REO (All the blank control was dehydrated ethanol )