Supplementary Information

Bioactive maca (Lepidium meyenii) alkamides are a result of traditional

postharvest drying practices

Eliana Esparzaa, Antonella Hadzicha, Waltraud Kofera, Axel Mithöferb and Eric G. Cosioa,*

aSección Química, Pontificia Universidad Católica del Perú, Avenida Universitaria 1801, San Miguel,

Lima 32, PerúbDepartment of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-

Straße 8, 07745 Jena, Germany

*Corresponding author:

Eric G. Cosio Tel. +51 1 626 2000 ext. 4218 Address: Sección Química, Pontificia Universidad

Católica del Perú, Avenida Universitaria 1801, San Miguel, Lima 32, Peru, ecosio@pucp.pe

1

Table of Contents

Page

Supplementary Materials and Methods 3

Spectral characterization of synthetic macamides 4

Mass spectral characterization of amides and fatty acids 5

Table S1. Mobile phase gradient program for the HPLC analysis described in Figs. 3 and 4 of the main text 13

Figure S1. Glucosinolates reported for maca 14

Figure S2. Analytical sequence used for the determination of maca (L. meyenii Walp.) metabolites produced during drying. 15

Figure S3. Environmental variables during the open-field drying trials 16

Figure S4. LC-ESI-MS profile of fresh lyophilized maca 17

Figure S5. LC profile of amines from maca 18

Figure S6. Phenylalanine, free phenolic acids and total phenolic acids in open-field drying process 19

Figure S7. Headspace analysis of maca VOCs 20

2

Supplementary Materials and Methods

Analysis of total and free phenolics.

The protocol described by Waterhouse (2005) was modified as follows; 50 L of sample and 50 L

of 70% methanol were placed in a plastic cuvette followed by 1.5 mL of water and 100 L of Folin

reagent, mixed, and left to react for 5 min at room temperature. Then, 300 L of a saturated

solution of sodium carbonate was added and mixed and left to react for 2 h in the dark. The

calibration curve was performed with gallic acid using stock solutions of 20, 50, 100, 200, 500 and

1000 g.mL-1 and 25 L of each solution were diluted in 75 L of 70% methanol. The absorbance

was read at 765 nm and the final concentration was expressed as mg gallic acid . g-1 dry wt.

Total phenolics were determined after basic hydrolysis of lyophilized samples using a modification

of the procedure reported by Nardini and co-workers (2002). Four mL of 2 M KOH with 1%

ascorbic acid and 10 mM EDTA were added to 0.5 mg of lyophilized maca powder and bubbled

with N2 gas in 50 mL glass culture tubes with Teflon lined screw caps, the solution was shaken in a

water bath at 70°C for 1.5 h in reduced light. After filtration, 1 mL of the solution was extracted

tree times with 0.5 mL ethyl acetate, centrifuged, and the organic phase was dried under N2 and

re-dissolved with 0.5 mL of water and analyzed as described in the previous paragraph.

Supplementary References

Nardini, M., Cirillo, E., Natella, F., Mencarelli, D., Comisso, A., Scaccini, C., 2002. Detection of

bound phenolic acids: prevention by ascorbic acid and ethylenediaminetetraacetic acid of

degradation of phenolic acids during alkaline hydrolysis. Food Chem. 79, 119-124.

Waterhouse, A.L., 2005. Determination of total phenolics. In Handbook of food analytical

chemistry: Pigments, colorants, flavors, texture, and bioactive food components; Wrolstad, R.E.,

Acree, T.E., Decker, E.A., Penner, M.H., Reid, D.S., Schwartz, S.J., Shoemaker, C.F., Smith, D.M.,

Sporns, P., Eds.; John Wiley & Sons, Hoboken, New Jersey, pp 463-470.

3

Spectral characterization of synthetic macamides (for detailed MS information see next section)

MAC 16 (11). N-benzyl hexadecanamide (palmitic acid benzylamide). Molecular formula:

C23H39NO. Formula weight: 345.56. White crystalline solid. FT-IR: 3291cm-1 N-H, 1631cm-1

C=O amide, 1553cm-1 N-C amide, 1452cm-1 N-C=O, 724cm-1 and 695cm-1 monosubstituded

aromatic ring, 2953cm-1 C-H, 2916cm-1 C-H, 2847cm-1 C-H. H1 NMR (300MHz, CDCl3) : 0,88

(t, J=6.9Hz, 3H, H=16), 1.25 (s, br, 24H, H=4-15), 1.65 (t, J=7.0Hz, 2H, H=3), 2,21 (t, J=7.7Hz,

2H, H=2), 4,43 (d, J= 7.7Hz, 2H, H=1`), 5.85 (s, br, 1H, N-H), 7.29 (m, 5H, H=3´-7´). EI-MS:

345 [M]+ (9.75), 149 [C9H11NO]+ (100), 106 [C7H8N]+ (23.03), 91 [C7H7]+ (45.34). ESI-MS:

[M+H]+ =346.3.

MAC 18:2 (12). N-benzyl-(9Z,12Z)-octadecadienamide (linoleic acid benzylamide).

Molecular formula: C25H39NO. Formula weight: 369.58. Colorless oil. FT-IR: 3285cm-1 N-H

amide, 1645cm-1 C=O amide, 1549 cm-1 N-C amide, 1454cm-1 N-C=O, 725cm-1 and 697cm-1

monosubstituded aromatic ring, 3064cm-1 and 3008cm-1 C=C-H, 2953cm-1 C-H, 2926cm-1 C-

H, 2854cm-1 C-H. H1 NMR (300MHz, CDCl3) : 2.21 (t, J=7.5 Hz, 2H, H= 2), 1.65 (m, J= 7.5 Hz,

2H, H= 3), 1.31 (s, br, 14H, H= 4-7,15-17), 2.05 (m, J=6.6 Hz, 4H, H= 8,14), 5.35 (m, 4H, H=

9,10,12,13), 2.77 (t, J=5.7 Hz, 2H , H= 11), 0.89 (t, J= 6.6 Hz, 3H ,H= 18), 4.46 (d, J=5.7 Hz,

2H , H= 1`), 7.32 (m, 5H, H= 3`-7`), 5.70 (s, br, 1H, N-H). EI-MS: 369 [M]+ (1.95), 149

[C9H11NO]+ (44.76), 106 [C7H8N]+ (58.11), 91 [C7H7]+ (100). ESI-MS: [M+H]+ =370.3.

MAC 18:3 (13). N-benzyl-(9Z,12Z,15Z) octadecatrienamide (linolenic acid benzylamide).

Molecular formula: C25H37NO. Formula weight: 367.57. Colorless oil. FT-IR: 3301 cm-1 N-H

amide, 1639cm-1 C=O amide, 1551cm-1 N-C amide, 1453cm-1 N-C=O, 3005cm-1 amide,

720cm-1 and 696cm-1 monosubstituded aromatic ring, C=C-H, 2962 cm-1 C-H, 2918 cm-1 C-

H, 2848 cm-1 C-H. H1 NMR (300MHz, CDCl3) : 2.21 (t, J=7.5Hz, 2H, H= 2), 1.65 (m, J=7.5Hz,

2H, H= 3), 1.31 (s, br, 8H, H= 4-7), 2.07 (m, 4H, H= 8,17), 5.36 (m, 6H, H= 9,10,12,13,15,16),

2.81 (t, J=5.7Hz, 4H, H= 11, 14), 0.97 (t, J=7.5Hz ,3H, H= 18), 4.45 (d, J=5.7, 2H, H= 1`), 7.33

(m, 5H , H= 3`-7`), 5.72 (s, br, 1H, N-H). EI-MS: 367 [M]+ (0.12), 149 [C9H11NO]+ (17.93), 106

[C7H8N]+ (38.21), 91 [C7H7]+ (100). ESI-MS: [M+H]+ =368.3.

4

Mass spectral information for amides and fatty acids

MAC16

Molecular Formula = C23H39NOFormula Weight = 345.56186Composition = C(79.94%) H(11.38%) N(4.05%) O(4.63%)Monoisotopic Mass = 345.303165 DaNominal Mass = 345 DaAverage Mass = 345.5619 DaM+ = 345.302616 Da [M+H]+ = 346.310441 Da

HPLC Rt=18.7min

LCMS Rt=19.2min

GCMS Rt=11.2min

GC-MS –EI

55.2

91.1

106.2

149.1

162.1

204.1345.3

+MS, 11.2min #643

0

2

4

6

5x10Intens.

100 200 300 400 500 600 m/z

5

LC-MS-ESI

346.3

368.2

+MS, 19.2min #1847

0

2

4

6

87x10

Intens.

200 300 400 500 600 m/z

NH

O

CH3

109.5123.5

137.5151.4165.4

184.4221.3

239.3

254.2

268.3

+MS2(346.3), 19.5min #1008

0.0

0.5

1.0

1.5

6x10Intens.

100 150 200 250 300 350 400 450 m/z

6

CH2+

NH

O

CH3

CH+

CH3

M + = 268.262943 Da

CH2+

CH3

M+ = 197.225829 Da

M+ = 221.225829 Da

C+

O

CH3

M + = 239.236393 Da

CH3 CH3

CH+

CH3

CH+

CH3

M + = 184.218552 Da

M+ = 165.163228 Da

M+ = 151.147578 Da

7

MAC 18:2

Molecular Formula = C25H39NOFormula Weight = 369.58326Composition = C(81.24%) H(10.64%) N(3.79%) O(4.33%)Monoisotopic Mass = 369.303165 DaNominal Mass = 369 DaAverage Mass = 369.5833 DaM+ = 369.302616 Da [M+H]+ = 370.310441 Da

HPLC RT=15.85min

LCMS RT=16.1min

GCMS RT=12.1min

GC-MS-EI

66.9

91.1

106.2149.3

162.2

216.3 260.4369.2

+MS, 12.1min #710

0.00

0.25

0.50

0.75

1.00

1.255x10

Intens.

100 200 300 400 500 600 m/z

8

LC-MS-ESI +MS

370.3

392.2

+MS, 16.1min #1541

0.00

0.25

0.50

0.75

1.00

1.258x10

Intens.

200 300 400 500 600 m/z

LC-MS-ESI +MS2

121.5 133.4149.5

161.4176.3

190.3 204.3 218.2

232.2

245.3

263.2 274.2288.2

300.2 314.2 328.3

335.1

353.3

+MS2(370.3), 16.1min #839

0

1

2

3

4

6x10Intens.

150 200 250 300 350 400 m/z

9

MAC 18:3

Molecular Formula = C25H37NOFormula Weight = 367.56738Composition = C(81.69%) H(10.15%) N(3.81%) O(4.35%)Monoisotopic Mass = 367.287515 DaNominal Mass = 367 DaAverage Mass = 367.5674 DaM+ = 367.286966 Da [M+H]+ = 368.294791 Da

HPLC RT=13.5min

LCMS RT=13.6min

GCMS RT=16.2min

GC-MS-EI

67.1

79.0

90.9

106.0

148.9

+MS, 16.2min #1037

0

20

40

60

80

100

Intens.[%]

100 200 300 400 500 600 m/z

10

LC-MS-ESI +MS

368.3

390.2

+MS, 13.6min #1499

0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

100 200 300 400 500 600 m/z

LC-MS-ESI +MS2

108.5145.4

163.3 173.3190.2

197.3

204.2218.2

232.2

243.3

261.2

272.2 286.2298.2 312.2

326.2

333.2

351.2

+MS2(368.3), 13.7min #739

0

1

2

3

4

6x10Intens.

100 150 200 250 300 350 400 m/z

11

FREE FATTY ACIDS

LC-ESI -MS

AG 18:3

172.6

277.8

345.7375.7 577.4

-MS, 12.6min #985

0

20

40

60

80

100

Intens.[%]

100 200 300 400 500 600 m/z

AG 18:2

172.6

279.5

347.4375.4

-MS, 15.0min #1172

0

20

40

60

80

100

Intens.[%]

100 200 300 400 500 600 m/z

12

Table S1. Mobile phase gradient program for the HPLC analysis described in Figs. 3 and 4 of main text.

Program time (min)

Mobile phase A %H2O*

Mobile phase B %MeOH*

Mobile phase C %ACN*

0 33 2 654 33 2 656 14 1 85

26 0 0 10035 0 0 10040 33 2 6545 33 2 65

* Containing 0.005% (v/v TFA)

13

Figure S1. Glucosinolates reported for maca (GLS, basic structure in the box) (Li et al., 2001;

Piacente et al., 2002; Yábar et al., 2011). 1: benzylglucosinolate, 14: 4-hydroxy-

benzylglucosinolate, 15: 3-hydroxy-benzylglucosinolate, 16: 3-methoxy-benzylglucosinolate, 17: 4-

methoxy-benzylglucosinolate, 18: 4-hydroxy-3-indolylmethylglucosinolate, 19: 3-

indolylmethylglucosinolate, 20: 4-methoxy-3-indolylmethylglucosinolate, 21: 4-

pentenylglucosinolate, 22: 6-methylsulfinylhexylglucosinolate. In this paper we only follow

benzylglucosinolate.

14

Figure S2. Analytical sequence used for the determination of maca (L. meyenii Walp.) metabolites

produced during drying.

15

Figure S3. Environmental variables during the open-field drying trials in the Junin highlands (4200

m altitude). Values shown are weekly averages obtained from permanent sensors placed in the

field 20 cm above the ground during the 2011 campaign from June 6th to September 20th.

16

Figure S4. LC-ESI-MS profile of desulfoglucosinolates in fresh lyophilized (upper) and field dried (lower profile) maca hypocotyls. Each sample corresponds to a 330 µg dry wt. The largest peak corresponds to benzyl desulfoglucosinolate (1), the second largest to a mix of 3- and 4-methoxybenzyl desulfoglucosinolates (16, 17, Fig. S1).

17

Figure S5. Comparison of HPLC profiles of OPA-derivatized amines in fresh lyophilized (a) and traditionally dried maca. Each sample corresponds to 267 µg dry wt. Benzylamine (7) has a retention time of 9.56 min. Detection was performed at 340 nm.

18

Figure S6. Free phenolic acid (FPhA), total phenolic acid (TPhA) and free phenylalanine (Phe) profiles during the open field drying process.

19

Figure S7. Headspace analysis chamber for maca VOCs. Closed chamber modified with three ports

with septa to assay VOCs emission from shredded hypocotyls during drying by headspace solid

phase micro-extraction (HS-SPME).

20