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SUPPLEMENTARY MATERIAL

Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesteraseactivity of essential oil fromEugeniabrasiliensis Lam.

Diogo Alexandre Sieberta, Adrielli Tenfena, Celina Noriko Yamanakaa,Caio Maurício Mendes de Cordovaa, Dilamara Riva Scharfb, Edésio Luiz Simionattob, Michele Debiasi Albertona,*



aDepartamento de Ciências Farmacêuticas, Universidade Regional de Blumenau, Brazil; bInstituto de Pesquisas Tecnológicas, Universidade Regional de Blumenau, Brazil

*Corresponding author. Address: Rua São Paulo, 2171. Blumenau-SC, Brazil. CEP 89030-000. E-mail: michele@furb.br. Telephone: +55 47 33217318.



Abstract

This study describes the seasonal composition and the antibacterial, antioxidant and anticholinesterase activity of the essential oil from E. brasiliensis leaves. Analysis by GC allowed the identification of 40 compounds. It was observed that the monoterpenes varied more (42 %) than the sesquiterpenes (14 %), and that the monoterpene hydrocarbons suffered the greatest variation throughout the year (64 %). Major compounds were spathulenol in the spring (16.02 ± 0.44 %) and summer (18.17 ± 0.41 %), τ-cadinol in the autumn (12.83 ± 0.03 %) and α-pinene (15.94 ± 0.58 %) in the winter. Essential oils were tested for its antibacterial activity, and best result was obtained from the autumn oil, with MIC = 500 μg.mL-1againstS. saprophyticus and P. aeruginosa. Antioxidant activity was evaluated using DPPH, lipid peroxidation and iron reducing power assays, as well as the anticholinesterase activity. Both tests showed a weak performance from the essential oils.



Keywords: Eugenia brasiliensis; essential oil; antibacterial; antioxidant; anticholinesterase.

Experimental

Chemicals


All chemicals were of analytical grade. 2,2-diphenyl-1-picrylhydrazyl (DPPH), linoleic acid(≥ 99 %), β-carotene(≥ 97 %), 3,5-Di-tert-4-butylhydroxytoluene (BHT, ≥ 95 %), acetylcholinesterase (AChE) type VI-S, from electric eel(lyophilized powder, 411 U.mg-1 protein), 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB, ≥ 99 %), acetylthiocholine iodide (AChI, ≥ 98 %), galanthamine hydrobromide(≥ 96 %), ascorbic acid(≥ 99 %),tris[hydroxymethyl]aminomethane (Tris buffer),bovine serum albumin(BSA, ≥ 96 %), dimethylsulfoxide (DMSO), Tween-80, ferric chloride(FeCl3, ≥ 99 %) and potassium ferricyanide [K3Fe(CN)6, ≥ 99 %]were acquired from Sigma-Aldrich®(Steinheim,Germany).

Plant material


Leaves ofE. brasiliensiswere collected in October 2012 and February, May and July 2013 for extraction of essential oil from all the four seasons, in Florianópolis (27°36'13.65"S, 48°31'14.75"W), Santa Catarina state, Southern Brazil. Professor Dr. Daniel de Barcellos Falkenberg from the Botany Department of Universidade Federal de Santa Catarina identified the samples. A voucher specimen was deposited at the herbarium FLOR under number 34675.

Extraction procedure


Essential oils of freshleaves were obtained by hydrodistillation for 4 h in a modified Clevenger-type apparatus, in the proportion of 1 g of leaves to 10 mL of distilled and deionized water. After extraction, the essential oils were dried with sodium sulfate and stored at a low temperature.

GC-FIDand GC-MS Analysis


All reagents were of analytical grade. The qualitative analysis were performed by gas chromatography coupled with mass spectrometry (GC-MS) in a Shimadzu® GCMS-QP2010 Plus chromatograph (nonpolar capillary column RTx®-5MS: 30 m x 0.25 mm x 0.25 μm), and the quantitative analysis by gas chromatography coupled with flame ionization detector (GC-FID) in a Shimadzu® GC-FID2010 chromatograph (nonpolar capillary column OV®-5: 30 m x 0.25 mm x 0.25 μm), being this the quantitative analysis performed in triplicate. The oven temperature program was 60 °C for 5 min, with increase of 3 °C per each min until 240 °C remaining in this temperature for 5 min. Helium was used as carrier gas (flow rate of 1 mL.min-1), with the temperature of the injector of 250 °C (1:20 split) and an ion source of 70 eV on the MS, with interface at 280 °C. Identification of the essential oilcomponents was based upon their retention indexes (in comparison with an homologous series of alkanes from C8 to C19), and by comparison of these and their mass spectral patterns with those reported in the literature (Adams 2007; Silva et al. 1999) and stored in the MS library NIST 2008 (National Institute for Standards and Technology) database. Since the standard for the positive confirmation were not used in the identification of the compounds, they must be considered only tentative identified.

The quantitativevariation of the constituents of the essential oils in the seasons was analysed using descriptive statistics, where the coefficient of variation was calculated for each compound. The compounds whose concentration could not be measured were considered as traces and for purposes of calculation equal to 0 %. The coefficient of variation was calculated for the compounds present in at least two samples. As criterion of analysis, changes from 0 to 15 % were considered low, 15 to 30 % moderate and above 30 % considered high (Löesch& Stein 2011). Data for the geographic region were provided by CIRAM/EPAGRI (Florianópolis-SC) and used for qualitative correlations between phytochemicals and climatic data.


Evaluation of antibacterial activity

Microorganisms and medium


The Laboratory of Clinical Microbiology from Universidade Regional de Blumenau (FURB) provided the bacterial strains. Tests were evaluated against gram-positive bacteria Staphylococcus aureus (ATCC 25923) and S. saprophyticus (ATCC 15305) and against gram-negative bacteria Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Mollicutes strains Mycoplasma mycoides subsp. capri (NCTC 10137) andUreaplasma urealyticum (ATCC 27618) were also assessed.

For the growth of bacterial strain, Müller-Hinton broth was used for S. aureus, S. saprophyticus, E. coli and P. aeruginosa, U10 broth (Shepard, 1974) was used for U. urealyticumand SP4 broth to M. mycoides subsp. capri (Velleca, 1979).


Determination of minimum inhibitory concentration (MIC)


The antibacterial activity of the essential oils was evaluated by determination of the minimum inhibitory concentration (MIC). The microdilution broth assay was performed in sterile 96-well microplates, as recommended by the Clinical and Laboratory Standards Institute (CLSI 2012) for cell-wall bacteria and Bebear& Robertson(1996) for mollicutes.

The essential oils were properly prepared and transferred to each microplate well with the appropriate culture medium, in order to obtain a twofold serial dilution of the original extract in a 10 % H2O/dimethyl sulfoxide (DMSO) solution, obtaining sample concentrations ranging between 1000 μg.mL-1 to 7.81 μg.mL-1. The inoculum containing 104 to 105 microorganisms per mLwere then added to each well. A number of wells were reserved in each plate to test for sterility control (no inoculum added), positive control (gentamycin or ciprofloxacin), inoculum viability (no extract added), and the DMSO inhibitory effect.



The microplates were incubated at 37 °C ± 1 °C for 24 or 48 hours (depending on the bacterium). Thereafter, growth of mollicutes strains was detected by observing the colour change in the medium. For cell-wall bacteria, a methanolic solution of triphenyl tetrazolium chloride (5 mg.mL-1) was added into each well, and the presence of a reddish bacterial “button” observedat the bottom of each well. The MIC was defined as the lowest concentration of the essential oil able to inhibit bacterial growth.

Evaluation of antioxidant activity

Determination of DPPH free radical scavenging activity


The assay for the determination of antioxidant activity using the DPPH free radical is based on the method described by Cavin et al. (1998), with slight modifications. Two mL of a DPPH solution (0.004 % in methanol) was added to 1 mL of the test solutions at concentrations from 500 to 62.5 ug.mL-1. The mixture was shaken and allowed to stand 30 min in the dark. The absorbance of the solutions was observed in a UV-Vis spectrophotometer (517 nm). For each sample, a blank solution (1 mL of test solution + 2 mL methanol) was used in order to discount the possible interference of the extract. BHT was used as a positive control and a solution of 2 mL of DPPH and 1 mL of methanol was used as negative control. Inhibition of DPPH radical was calculated by the following formula: DPPH scavenging effect (%) = (A0 – A1/A0) x 100;where A0 and A1 are the absorbance at 30 minutes of the negative control and the sample, respectively. The antiradical activity was expressed as IC50 (ug.mL-1), the extract dose required to cause a 50 % decrease of the absorbance at 517 nm. A lower IC50 value corresponds to a higher antioxidant activity. The test were performed in triplicate.

Determination of the inhibitor potential of the lipid peroxidation


The inhibition of lipid peroxidation was assessed by the model of β-carotene/linoleic acid (Mokbel& Hashinaga 2006). An emulsion was prepared with 3.0 mg of β-carotene, 1.0 mL of CHCl3, 45 mg of linoleic acid and 215 mg of Tween-80. Chloroform was removed in a rotary evaporator under the temperature of 45 °C. To the resulting mixture, 6.0 mL of distilled water were added under stirring, to prepare the emulsion. The emulsion was then dissolved to 100 mL with 0.01 M of hydrogen peroxide. Aliquots of this emulsion (4 mL) were transferred to tubes containing 0.2 mL of the test solutions (essential oils at 1,000 μg.mL-1, in methanol). As a positive control, a solution of BHTat a concentration of 1000 μg.mL-1 was used. Negative control containing 0.2 mLof methanol and 4.0 mL of the above emulsion was also prepared. All tubes were placed in a water bath at 50 °C, and the absorbance of the solutions were determined at time zero and every 30 min in a spectrophotometer at 470 nm, until the discoloration of the tube containing the negative control (180 min). A blank emulsion was prepared as described above but without the presence of β-carotene. The antioxidant activity (inhibitionof the lipid peroxidation in percentage - ILP %) was calculated using the following formula: ILP (%) = 100 [1 – (A0 – At) / (A00 – A0t)]; where A0 and At are the absorbance of the sample at time zero and after 180 min, respectively, and A00 and A0t are the absorbance of the negative control at time zero and after 180 min.

Determination of iron reducing power


The assay for the analysis of antioxidant activityby determination of the reduction potential was based on the method of Price & Butler, proposed by Waterman & Mole (1994), with adaptations. To the 100 μL of the test solutions (essential oils,diluted in methanol at a concentrationof 1,000 μg.mL-1) were added 8.5 mLof deionized water. Then, 1 mLof a 0.1 M FeCl3 solution was added, and after 3 min, 1mL of a potassium ferricyanide 0.08M solution was mixed. After 15 min, the reading of the solution absorbance was performedin a spectrophotometer at 720 nm.A blanksolution was preparedaccording to the above procedure, without adding the sample. Astandard curve was performed using acid ascorbic solutions in concentrations rangingfrom 100 to 1000 μg.mL-1 (y = 0.0019 x + 0.0698, R2 = 0.9967). The reduction potentialwas expressed in mg of ascorbic acid (AA) per g of sample (mg AA.g-1). Analysis were performed in triplicate.

Acetylcholinesterase inhibition


The enzymatic activity was measured using an adaption of the method described by Mata et al. 2007. Briefly, 325 μL of 50 mM Tris-HCl buffer (pH 8), 100 μL of a buffer solution of sample (0.1 mg.mL-1in methanol) and 25 μL of AChE solution containing 0.28 U.mL-1 (50 mM Tris-HCl, 0.1% BSA) were incubated for 15 min. Subsequently, 75 μL of a solution of AChI (0.023 mg.mL-1 in water) and 475 μL of DTNB (3mM in Tris-HCl) were added and the final mixture incubated for another 30 min at room temperature. Absorbance of the mixture was measured at 405 nm. A control mixture was prepared, using 100 μL of a solution similar to the sample mixture but with methanol instead of sample. Inhibition (%) was calculated as follows: I (%) = 100 – (Asample/Acontrol) x 100; where Asample is the absorbance of the sample containing the reactant and Acontrol the absorbance of the reaction control. Tests were carried out in triplicate. The sample concentration providing 50% inhibition (IC50) was obtained by plotting the inhibition percentage against the sample solution concentrations. Galanthamine was used as the positive control.Table S1. Compounds identified (%) in the essential oil of leaves from E. brasiliensis in the four seasons of the year.

Compounds

Rta

Relative Concentration (%)

AIb

CVe

Spring

Summer

Autumn

Winter

Ec

Ld




α-pinene

7.24

1.77 ± 0.18

9.05 ± 0.37

5.14 ± 0.15

15.94 ± 0.58

928

932

76

β-pinene

8.97

2.98 ± 0.22

7.35 ± 0.17

4.42 ± 0.15

11.24 ± 0.30

971

974

56

Mircene

9.60

t

0.26 ± 0.01

0.15 ± 0.03

0.34 ± 0.01

986

988

79

p-cimene

11.09

0.31 ± 0.01

0.55 ± 0.08

0.08 ± 0.02

0.47 ± 0.01

1020

1020

59

Limonene

11.28

0.50 ± 0.05

0.76 ± 0.21

0.71 ± 0.01

1.30 ± 0.05

1024

1024

42

Eucaliptol

11.41

3.11 ± 0.10

2.53 ± 0.21

1.65 ± 0.02

2.67 ± 0.05

1026

1026

25

E-β-ocimene

12.22

t

0.34 ± 0.01

0.26 ± 0.04

0.37 ± 0.01

1044

1044

69

γ-terpinene

12.69

0.29 ± 0.01

0.32 ± 0.01

0.32 ± 0.01

0.49 ± 0.02

1054

1054

26

Terpinolene

14.10

t

0.36 ± 0.01

0.35 ± 0.01

0.48 ± 0.01

1083

1086

70

Linalool

14.68

1.46 ± 0.20

1.28 ± 0.05

1.18 ± 0.05

1.09 ± 0.03

1096

1095

14

4-terpineol

18.35

0.99 ± 0.05

0.84 ± 0.02

0.47 ± 0.06

0.55 ± 0.02

1173

1174

34

α-terpineol

19.02

3.48 ± 0.14

3.66 ± 0.18

2.72 ± 0.03

2.59 ± 0.02

1187

1186

17

Myrtenil acetate

25.21

0.46 ± 0.02

0.35 ± 0.01

0.35 ± 0.02

0.28 ± 0.01

1318

1324

21

α-cubebene

26.19

t

t

0.25 ± 0.01

0.15 ± 0.01

1341

1345

122

α-copaene

27.37

0.69 ± 0.01

0.52 ± 0.01

0.83 ± 0.06

0.56 ± 0.01

1368

1374

22

E-cariofilene

29.25

4.57 ± 0.14

3.82 ± 0.10

8.65 ± 0.06

5.65 ± 0.07

1415

1417

37

Aromadendrene

30.05

0.47 ± 0.07

0.43 ± 0.01

0.50 ± 0.06

0.40 ± 0.04

1434

1439

10

α-humulene

30.66

1.13 ± 0.04

0.95 ± 0.03

1.74 ± 0.02

1.21 ± 0.02

1449

1452

27

Allo-aromadendrene

30.97

0.48 ± 0.02

0.42 ± 0.05

0.79 ± 0.06

0.54 ± 0.03

1456

1458

29

β-cadinene

31.52

0.32 ± 0.01

t

t

t

1470

1472f



γ-muurolene

31.63

0.52 ± 0.03

0.45 ± 0.01

0.41 ± 0.01

0.34 ± 0.01

1472

1478

18

Germacrene D

32.24

0.34 ± 0.03

0.27 ± 0.01

t

t

1487

1484

117

Biciclogermacrene

32.45

1.67 ± 0.08

1.10 ± 0.05

3.86 ± 0.01

2.12 ± 0.03

1492

1500

54

α-muurolene

32.60

0.44 ± 0.07

0.43 ± 0.08

0.45 ± 0.03

0.29 ± 0.01

1496

1500

19

γ-cadinene

33.15

0.99 ± 0.04

0.77 ± 0.04

1.53 ± 0.03

0.86 ± 0.01

1510

1513

26

δ-cadinene

33.54

4.44 ± 0.08

3.38 ± 0.08

5.81 ± 0.02

3.87 ± 0.07

1520

1522

24

α-calacorene

34.29

t

0.43 ± 0.10

t

t

1539

1544



E-nerolidol

35.14

1.56 ± 0.08

0.47 ± 0.08

0.55 ± 0.03

0.38 ± 0.04

1561

1561

74

Spathulenol

35.77

16.02 ± 0.44

18.17 ± 0.41

8.10 ± 0.04

8.31 ± 0.09

1577

1577

41

Caryophyllene oxide

35.79

4.08 ± 0.38

t

0.87 ± 0.05

1.41 ± 0.02

1578

1582

111

Globulol

35.97

5.54 ± 0.40

5.03 ± 0.85

7.87 ± 0.06

4.55 ± 0.12

1582

1590

26

Viridiflorol

36.25

3.02 ± 0.15

2.56 ± 0.24

2.24 ± 0.06

1.51 ± 0.06

1589

1592

27

Rosifoliol

36.65

1.77 ± 0.03

1.30 ± 0.19

2.91 ± 0.01

1.90 ± 0.02

1600

1600

26

1,10-di-epi-cubenol

37.12

1.89 ± 0.01

1.76 ± 0.13

1.15 ± 0.01

0.99 ± 0.02

1612

1618

31

1-epi-cubenol

37.65

7.02 ± 0.13

7.46 ± 0.41

4.83 ± 0.03

5.16 ± 0.08

1627

1627

22

Cis-cadin-4-en-7-ol

37.76

t

t

t

0.30 ± 0.01

1630

1635



τ-cadinol

38.15

15.30 ± 0.09

12.60 ± 0.24

12.83 ± 0.03

10.38 ± 0.17

1640

1638

16

δ-cadinol

38.32

2.89 ± 0.17

2.16 ± 0.15

2.35 ± 0.02

1.80 ± 0.02

1645

1644

20

α-cadinol

38.64

6.04 ± 0.12

5.47 ± 0.21

5.41 ± 0.03

4.27 ± 0.05

1654

1652

14

Khusinol

39.76

0.76 ± 0.05

0.93 ± 0.03

0.51 ± 0.04

0.59 ± 0.01

1675

1679

27

Monoterpene hydrocarbons

5.84 ± 0.09

18.99 ± 0.11

11.43 ± 0.05

30.63 ± 0.12

 

 

64

Oxygenated monoterpenes

9.49 ± 0.10

8,65 ± 0.09

6.36 ± 0.04

7.18 ± 0.03







18

Total monoterpenes

15.33 ± 0.10

27.63 ± 0.10

17.79 ± 0.05

37.81 ± 0.08







42

Sesquiterpene hydrocarbon

16.07 ± 0.05

12.97 ± 0.05

24.82 ± 0.03

16.00 ± 0.03







29

Oxygenated sesquiterpenos

65.89 ± 0.17

57.93 ± 0.27

49.62 ± 0.03

41.55 ± 0.05







20

Total sesquiterpenes

81.96 ± 0.11

70.90 ± 0.15

74.44 ± 0.03

57.55 ± 0.04







14

TOTAL

 

97.29 ± 0.11

98.53 ± 0.13

92.24 ± 0.04

95.36 ± 0.06

 

 

 

Identified compounds listed by order of elution in nonpolar column (RTx®-5MS).

aRt = Retention time in minutes.

bAI = Arithmetic index.

cE = Experimental data.

dL = Adams 2007.

eCV = Coefficient of variation in %.

fSilva et al. 1999.

t = Traces, not quantified.

Table S2. Climatic conditions in the region and seasons of collection.


 

Spring

Summer

Autumn

Winter

Total rainfall (mm)

285

578

332

429

Number of rainy days

41

43

26

30

Absolute maximum temperature (°C)

39

33

32

31

Minimum absolute temperature (°C)

15

17

8

4

Average temperature (°C)

23

24

20

17

Average relative humidity (%)

77

76

78

80

Source: CIRAM/EPAGRI.

Table S3. Antibacterial activity of essential oil from E. brasiliensis.






MIC (µg.mL-1)




Spring

Summer

Autumn

Winter

S. saprophyticus (ATCC 15305)

1,000

1,000

500

500

S. aureus (ATCC 25923)

> 1,000

> 1,000

> 1,000

> 1,000

E. coli (ATCC 25922)

> 1,000

> 1,000

1,000

> 1,000

P. aeruginosa (ATCC 27853)

> 1,000

> 1,000

500

1,000

M. mycoides subs. capri (NCTC 10137)

> 1,000

> 1,000

> 1,000

> 1,000

U. urealyticum (ATCC 27618)

> 1,000

> 1,000

> 1,000

> 1,000

Table S4. Results for the antioxidant and anticholinesterase assaysof essential oil from E. brasiliensis.






Spring

Summer

Autumn

Winter

Positive controla

DPPH (EC50– μg.mL-1)

> 500

> 500

> 500

> 500

17.2

ILP (%)

14.05

1.67

1.71

5.50

94.58

RP (mg AA.g-1)

60.37

61.95

94.32

67.21



AChE (IC50– μg.mL-1)

> 1,000

> 1,000

> 1,000

> 1,000

6,93

aPositive control: BHT, for antioxidants tests; galantamine, for AChE inhibition test.

References


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Bebear C, Robertson J. 1996. Molecular and Diagnostic Procedures in Mycoplasmology. 1st ed. San Diego: Academic Press.

Cavin A, Hostettmann K, Dyatmyko W, Potterat O. 1998. Antioxidant and lipophilic constituents of Tinospora crispa. Planta Med. 64: 393–396.

CLSI. 2012. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Second Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute, (M100-S22/ v. 32, n. 3).

Löesch C, Stein CE. 2011. Estatística descritiva e teoria das probabilidades [Descriptive statistics and probability theory]. 2nd ed. Blumenau: Edifurb.

Mata AT, Proença C, Ferreira AR, Serralheiro MLM, Nogueira JMF, Araújo MEM. 2007. Antioxidant and antiacetylcholinesterase activities of five plants used as Portuguese food spices. Food Chem. 103: 778–786.

Mokbel MS, Hashinaga F. 2006. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 94: 529–534.

Shepard MC. 1974. Standard fluid medium U10 for cultivation and maintenance of Ureaplasma urealyticum. Int. J. Syst. Bacteriol. 24: 160–171.

Silva MHL, Andrade EHA, Zoghbi MGB, Luz AIR, Silva JD, Maia JGS. 1999. The essential oils of Lantana camara L. occurring in North Brazil. Flavour Fragr J. 14: 208–210.

Velleca WM, Bird BR, Forrester FT. 1979. Laboratory diagnosis of Mycoplasma infections: course. Atlanta: U.S. Department of Health.



Waterman PG, Mole S. 1994. Analysis of phenolic plant metabolites. Oxford: Blackwell Scientific.

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