Supplementary Material

Chemical composition and antifungal and cytotoxicity activities of Inga laurina

(Sw.) Willd leaves

Carla de Moura Martins,1 Sérgio A. L. de Morais,2 Mário M. Martins,2 Luís C. S.

Cunha,2 Cláudio V. da Silva,3 Luiz F. Leandro,4 Carlos H. G. Martins,4 Alberto de

Oliveira,2 Francisco J. T. de Aquino,2 Evandro A. do Nascimento2 and Roberto Chang2

1Chemistry Nucleus, Goiano Federal Institute-Campus Morrinhos, BR-153, km 633,

Rural Area, 75650-000, Morrinhos-GO, Brazil.2Natural Products Research Nucleus (NuPPeN), Federal University of Uberlândia, João Naves de Ávila Avenue, 2121, Santa Mônica, 38400-902, Uberlândia-MG, Brazil.

3Institute of Biomedical Sciences-Laboratory of Trypanosomatids, Federal University of

Uberlândia, Pará Avenue, 1720, Umuarama, 38405-320, Uberlândia-MG, Brazil.4Laboratory of Research on Applied Microbiology, Franca University (UNIFRAN), Dr.

Armando Salles Oliveira Avenue, 201, University Park, 14404-600, Franca-SP, Brazil.

Correspondence should be addressed to Roberto Chang; chang@ufu.br .

All data in our manuscript is available for readers.

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Scheme S1:Flowchart for purification, identification and isolation of compounds from the leaves of I. laurina

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Figures S1 – S12: Mass spectrum of ion m/z of phenolic compounds identified in EAF from I. laurina by HPLC-ESI/MS2

4x10

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

2,2

2,4

2,6

2,8

3

-ESI Product Ion (rt: 1,382 min) Frag=175,0V CID@15,0 (169,0205[z=1] -> **) PAFMSMS15eV.d

125.0299

169.0195

Counts vs. Mass-to-Charge (m/z)110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Figure S1: Mass spectrum of ion m/z 169 by MS/MS.

2x10

0

0,51

1,5

22,5

3

3,54

4,5

5

5,56

6,5

77,5

8

-ESI Product Ion (rt: 4,490 min) Frag=175,0V CID@15,0 (305,0749[z=1] -> **) PAFMSMS15eV.d

125.0288

305.0769

167.0393

179.0437 137.0294

219.0718 261.0848

111.0528 293.6141 201.0577

Counts vs. Mass-to-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

Figure S2: Mass spectrum of ion m/z 305 by MS/MS.

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3637

38

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46

47

56

3x10

00,20,40,60,8

11,21,41,61,8

22,22,42,62,8

33,23,43,6

-ESI Product Ion (rt: 6,061 min) Frag=175,0V CID@15,0 (183,0361[z=1] -> **) PAFMSMS15eV.d

124.0224

183.0355

168.0157

Counts vs. Mass-to-Charge (m/z)110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290

Figure S3: Mass spectrum of ion m/z 183 by MS/MS.

4x10

00,10,20,30,40,50,60,70,80,9

11,11,21,31,41,51,61,71,81,9

-ESI Product Ion (rt: 8,192 min) Frag=175,0V CID@20,0 (197,0472[z=1] -> **) PAFMSMS20eV.d

124.0177

169.0147 197.0484

Counts vs. Mass-to-Charge (m/z)120 130 140 150 160 170 180 190 200 210 220 230 240

Figure S4: Mass spectrum of ion m/z 197 by MS/MS.

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5455

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78

3x10

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0,5

1

1,5

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4,5

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5,5

6

6,5

7

7,5-ESI Product Ion (rt: 9,288 min) Frag=175,0V CID@30,0 (631,0964[z=1] -> **) PAFMSMS30eV.d

479.0835 316.0244

631.0961

169.0165

Counts vs. Mass-to-Charge (m/z)150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

Figure S5: Mass spectrum of ion m/z 631 by MS/MS.

4x10

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

5,5

6

-ESI Product Ion (rt: 9,678 min) Frag=175,0V CID@20,0 (479,0856[z=1] -> **) PAFMSMS20eV.d

316.0244

479.0850

271.0270 179.0011

Counts vs. Mass-to-Charge (m/z)150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

Figure S61: Mass spectrum of ion m/z 479 by MS/MS.

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910

5x10

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2

2,2

2,4

2,6

2,8

3-ESI Product Ion (rt: 9,948 min) Frag=175,0V CID@20,0 (463,0907[z=1] -> **) PAFMSMS20eV.d

316.0241

463.0902

271.0253 179.0003

Counts vs. Mass-to-Charge (m/z)150 200 250 300 350 400 450 500 550 600 650 700 750 800

Figure S7: Mass spectrum of ion m/z 463 by MS/MS.

5x10

00,250,5

0,751

1,251,5

1,752

2,252,5

2,753

3,253,5

3,754

-ESI Product Ion (rt: 10,977 min) Frag=175,0V CID@30,0 (615,1013[z=1] -> **) PAFMSMS30eV.d

317.0316

615.0999

178.9998 463.0892

Counts vs. Mass-to-Charge (m/z)150 200 250 300 350 400 450 500 550 600 650 700 750 800

Figure S8: Mass spectrum of ion m/z 615 by MS/MS.

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4x10

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0,1

0,2

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0,6

0,7

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0,9

1

1,1

1,2

1,3

-ESI Product Ion (rt: 11,451 min) Frag=175,0V CID@20,0 (317,0321[z=1] -> **) PAFMSMS20eV.d

151.0044

179.0001

317.0317 137.0255

107.0152 165.0198 192.0076 271.0268

Counts vs. Mass-to-Charge (m/z)100 120 140 160 180 200 220 240 260 280 300 320 340 360

Figure S9: Mass spectrum of ion m/z 317 by MS/MS.

4x10

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0,1

0,2

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0,7

0,8

0,9

1

1,1

1,2

1,3

1,4

-ESI Product Ion (rt: 11,316 min) Frag=175,0V CID@20,0 (447,0955[z=1] -> **) PAFMSMS20eV.d

301.0367

447.0936

271.0265 151.0064

Counts vs. Mass-to-Charge (m/z)100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480500 520 540 560 580 600 620

Figure S102: Mass spectrum of ion m/z 447 by MS/MS.

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3x10

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5

5,5

6

-ESI Product Ion (rt: 12,143 min) Frag=175,0V CID@30,0 (599,1051[z=1] -> **) PAFMSMS30eV.d

301.0345

599.1100

169.0132 447.0954

125.0250

Counts vs. Mass-to-Charge (m/z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

Figure S113: Mass spectrum of ion m/z 599 by MS/MS.

3x10

0

0,5

1

1,5

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5,5

6

6,5-ESI Product Ion (rt: 12,245 min) Frag=175,0V CID@20,0 (301,0370[z=1] -> **) PAFMSMS20eV.d

151.0039

178.9975

121.0300 301.0373

273.0381 229.0469

Counts vs. Mass-to-Charge (m/z)100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

Figure S12: Mass spectrum of ion m/z 301 by MS/MS.

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Figures S13–S16: Mass spectrum of ion m/z of phenolic compounds identified in fractions F2, F5, F6, F7, respectively, from I. laurina by HPLC-ESI/MS2

4x10

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0,9

1

1,1

1,2

-ESI Product Ion (rt: 11,383 min) Frag=175,0V CID@20,0 (505,0968[z=1] -> **) F2 PAF MSMS 20eV.d

316.0206

505.0956

167.0346 239.0518 113.0218 463.0805 389.2486

Counts vs. Mass-to-Charge (m/z)100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

Figure S134: MS/MS spectrum of ion m/z 505.

3x10

00,250,5

0,751

1,251,5

1,752

2,252,5

2,753

3,253,5

3,754

4,254,5

-ESI Product Ion (rt: 7,314 min) Frag=175,0V CID@20,0 (495,0745[z=1] -> **) F5 PAF MSMS 20eV.d

343.0649

169.0135

495.0784

Counts vs. Mass-to-Charge (m/z)100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725

Figure S14: MS/MS spectrum of ion m/z 495.

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3x10

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1,6

1,8

2-ESI Product Ion (rt: 8,004 min) Frag=175,0V CID@30,0 (647,0871[z=1] -> **) F6 PAF MSMS 30eV.d

495.0773

343.0643

169.0129

647.0750

Counts vs. Mass-to-Charge (m/z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

Figure S15: MS/MS spectrum of ion m/z 647.

3x10

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0,2

0,4

0,6

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1,2

1,4

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1,8

2

2,2

2,4

-ESI Product Ion (rt: 8,221 min) Frag=175,0V CID@15,0 (167,0354[z=1] -> **) F7 PAF MSMS 15eV.d

108.0233

167.0348

152.0151

Counts vs. Mass-to-Charge (m/z)100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225

Figure S16: MS/MS spectrum of ion m/z 167.

Figures S17–S22: Fragmentation mechanisms of compounds identified in EAF.

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1920

Figure S17. Proposed mechanism for fragmentation of m/z 609 with loss of two

rhamnoside structures.

Figure S18. Fragmentation mechanism for the ion m/z 463.

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Figure S19: Fragmentation mechanisms of compounds identified in EAF (I-V).

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130131

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135

2324

Figure S20: Fragmentation mechanisms of compounds identified in EAF (VI-IX).

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Figure S21: Fragmentation mechanisms of compounds identified in EAF (X-XIII).

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Figure S22: Fragmentation mechanisms of compounds identified in EAF (XIV-XV).

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min

0

250

500

750

1000

1250mAU

AD1:254nm

8.09

7

Figure S23: Chromatogram and UV/Vis spectrum of fraction 4 (F4).

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250 nm0

1000

2000

3000

4000mAU

258

353

168

169

170

171

172

173

174

175

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Figure S24: 1H NMR spectra (400 MHz, DMSO-d6) of myricetin-3-O-rhamnoside

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1´´

2´;6´

8; 6

2´´3´´ 5´´

4´´

6´´

176

177

178

179

180

181

182

3132

Figure S25: 13C NMR spectra (100 MHz, DMSO-d6) of myricetin-3-O-rhamnoside

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183184185186

187

188

189

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Figure S26: HSQC contour map (DMSO-d6) of myricetin-3-O-rhamnoside in the aromatic region.

Figure S27: COSY contour map (DMSO-d6) of myricetin-3-O-rhamnoside.

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190

191192

193

194

195

196

197

198

199

200

201

202

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Figure S28: Amplification of the COSY (400 MHz, DMSO-d6) contour map in the region of glycosidic hydrogens of myricetin-3-O-rhamnoside.

Figure S29: DEPT-135 spectra (100 MHz, DMSO-d6) of myricetin-3-O-rhamnoside.

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1´´ 6 8

2´;6´

2´´;3´´;5´´;4´´6´´

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204

205206

207

208

209

210

211

212

213

214

215

216

217

3738

Table S1: 1H NMR (400 MHz, DMSO-d6,) data of myricetin-3-O-rhamnoside.

Positio

n

1H NMR

(400 MHz,DMSO-d6)

δ1; m2; J 3

COSY

1H NMR

(300 MHz, DMSO-d6)

δ1; m2; J3

[1]

6 6.20 d (2.0) H-8 6.36 d (1.8)

8 6.35 d (2.0) H-6 6.18 d (1.8)

2´ 6.89 s - 7.04 s

6´ 6.89 s - 7.04 s

1´´ 5.20 d (1.2) H-2´´ 5.18 d (1.0)

2´´ 3.98 s H-1´´e H-3´´

3.1-4.0 m3´´ 3.55 dd (2.6 e 9.3) H-2´´ e H-4´´

4´´ 3.16 m H-3´´ e H-5´´

5´´ 3.37 m H-4´´ e H-6´´

6´´ 0.84 d (6.2) H-5´´ 0.79 d (5.3)

Note: 1: chemical shift in ppm using TMS as internal standard; 2: multiplicity (s = singlet, d = doublet, dd = double doublet, m = multiplet); 3: coupling constant expressed in Hz.

Table S2: 13C NMR (100 MHz, DMSO-d6) data of myricetin-3-O-rhamnoside.

20

218

219220221222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

3940

Position δ1 δ2

(100 MHz,

DMSO-d6 DEPT-

135

HSQC1

m3

RMN 13C

(75 MHz, methanol-

d4)[1]

2 156.4 156.4 C - 158.3

3 136.5 135.1 C - 134.5

4 177.8 178.2 C - 177.9

5 161.3 161.8 C - 162.9

6 98.7 98.3 CH 6.20 d 99.9

7 164.2 166.4 C - 165.4

8 93.5 94.0 CH 6.35 d 94.3

9 157.5 158.8 C - 157.5

10 104.1 104.5 C - 105.0

1´ 119.6 121.8 C - 122.5

2´ 107.9 107.9 CH 6.89 s 109.5

3´ 145.8 146.1 C - 146.4

4´ 134.3 135.2 C - 137.1

5´ 145.8 146.1 C - 146.4

6´ 107.9 107.9 CH 6.89 s 109.5

1´´ 101.9 109.3 CH 5.20 d 102.0

2´´ 70.0 75.1 CH 3.98 s 70.3

3´´ 70.4 71.0 CH 3.55 dd 70.4

4´´ 71.3 73.7 CH 3.16 m 71.3

5´´ 70.6 74.3 CH 3.37 m 69.6

6´´ 17.5 17.0 CH3 0.84 d 17.2

Note: 1: δ in ppm using TMS as internal standard; 2: theoretical δ obtained in the ChemDrawUltra program (version 10.0); 3: multiplicity (s = singlet, d = doublet, dd = double doublet, m = multiplet).

[1] M. Ceruks, P. Romoff, O. A. Fávero, J. H. G. Lago, ‘Constituintes fenólicos polares de Schinus terebinthifolius Raddi (Anacardiaceae)’, Quim. Nova 2007, 30, 597-599.

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245

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