Supplementary Material

Seasonal variation in the chemical compositon of two chemotypes of Lippia alba

Angélica Ferraz Gomes a, Maiara Prates Almeida a, Mateus Freire Leite a,d, Stefan

Schwaiger b, Hermann Stuppner b, Maria Halabalaki b,e, Juliano Geraldo Amaral a, Jorge

Maurício David*c.

a Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, 45029-094

Vitória da Conquista – BA, Brazil

b Institute of Pharmacy, Pharmacognosy, CMBI, University of Innsbruck, 6020

Innsbruck, Austria

c Instituto de Químicam, Universidade Federal da Bahia, 40170-280 Salvador – BA,

Brazil

d Universidade Federal de Alfenas, Alfenas - MG, Brazil.

e Laboratory of Pharmacognosy and Natural Products Chemistry, School of Pharmacy,

University of Athens, Greece.

* Corresponding author: E-mail: jmdavid@ufba.br tel: +55 71 32836864

Development of Chromatographic Conditions

The HPLC profiles of leaves were analyzed in a previous validated method

using Gemini C18 110A 3 µ 150x 4.6mm column at 45°C, flow 0.4 ml/min; gradient

solvent system consisting of mixture 0.1% acetic acid and 0.9% formic acid (v/v)

aqueous phase and acetonitrile (0-98%) during 45 minutes. After this result, it was

tested 13 different columns following in the same method: YMC – Pack Pro C18 150x

4.6 mm ID S-3 µm, 12 nm; Phenomenex Hyperclone 3 µ ODS C18 120A 150 x 4.60

mm; Waters X Terra RP18 3.5 µm 150 x 4.6 mm; Phenomenex Synergi 4µ Fusion –

RP 80 150 x 4.6 mm; Phenomenex Aqua 5µ C18 125A 250 x 4.6 mm; Agilent Eclipse

XDB C18 8 x 100 mm 6.5 µm; YMC – Pack Pro C18 RS 150 x 4.6 s-3µm 8 nm;

Phenomenex Synergi Max RP 80A 150 x 4.6 4 micron; Phenomenex Luna C8 100A

150 x 3 mm 3 µm; YMC – Triart Phenyl 150 x 3.0 mm S-3 µm, 12 nm; Phenomenex

Kinetex Phenyl - Hexyl 100A 100 x 2.1 mm and,YMC – Pack Pro C18 RS 150 x 4.6 s-

3µm 8 nm. For the next steps of development we selected the column which presented

the best separation among them.

During further investigations, the chromatographic parameters (mobile phase,

flow rate, volume injection, temperature and, elution program) were optimized in order

to provide better separation of components in a shorter run time. For all the tested

conditions mixture of the extracts containing A and B chemotypes (1:1) was used for

the purpose of the separation method was suitable for both.

After optimizing such parameters described, the method was applied in the

identical equipment but with MS detector in order to confirm the identity and peak

purity of analytes. Therefore, all analytical markers were well resolved and could be

assigned by comparison of their retention times, online-UV-spectra of the respective

standards, and by LC-MS experiments (ESI, alternating mode).

Figure S1 – Chromatogram of mixture of geneposidic acid (1), 8-epi-loganin (2), mussaenoside (3), chrysoeriol – 7-O-diglucuronide (4), tricin-7-O-diglucuronide (5), acteoside (6) and isoacteoside (7), except luteolin-7-O-glucuronide, apigenin-7-O- glucuronide and tricin-7-O-glucuronide)

Table S1: Chemical profile quali and quantitative of Lippia alba´s chemotype extracts

(µg mg-1)

Chemotype A B C D E F GGeneposidic acid 10.296 ±

0.0066.745 ± 0.002

2.901 ± 0.002

10.617 ± 0.002

13.218 ± 0.012

12.038 ± 0.028

4.819 ± 0.004

8-epi-Loganin - 10.890 ± 0.003

- - 5.854 ± 0.003

4.772 ± 0.003

2.261 ± 0.001

Mussaenoside - 20.394 ± 0.018

4.933 ± 0.004

6.397 ± 0.003

6.952 ± 0.004

6.233 ± 0.007

14.199 ± 0.008

Chrysoeriol 7-O-diglucuronide

8.517 ± 0.001

7.462 ± 0.001

- - - - -

Tricin 7-O-diglucuronide

28.264 ± 0.002

16.156 ± 0.002

- 9.987 ± 0.002

- - 10.653 ± 0.002

Acteoside - - - 189.765 ± 0.035

- - 0.628 ± 0.003

Isoacteoside - - - 15.060 ± 0.001

- - -

Apigenin 7-O-glucoside

- - - - 7.759 ± 0.002

8.038 ± 0.009

-

Tricin 7-O-glucuronide

3.768 ± 0.001

2.984 ± 0.001

1.822 ± 0.002

- 7.063 ± 0.002

6.891 ± 0.002

1.593 ± 0.001

Table S2: Comparison between quantitative analyses found by Timóteo et al., 2015

Substance Chemotype Range concentration (µg mg-1) *

Range concentration (µg mg-1) **

8-epi-Loganin Carvone 2 - 18 12.11 – 13.32Linalool - 18.25 – 15.47

Geneposidic acid Carvone 2 - 13 11.12 – 11.67Linalool 3 - 36 13.21 – 14.45

Chrysoeriol 7-O-diglucuronide Carvone 6 – 61 47.77 – 59.58Linalool 11 - 66 45.72 – 58.11

Tricin 7-O-diglucuronide Carvone 9 – 215 155.9 – 182.76Linalool 12 - 181 128.03 – 168.36

Acteoside Carvone 12 - 231 51.13 – 135.58Linalool 28 - 226 18.45 – 97.23

* present work** Results from Timóteo and co-workers (2015)

Table S3: Recoveries of the developed method

Compound Initial concentration

(µg mg-1)

Spike(µg mg-1)

Final concentration

(µg mg-1)

% Recovery RSD %

Acteoside 59.004 11.71 70.298 99.41 0.4623.71 81.967 99.10 0.6347.43 105.521 99.14 0.38

Geneposidic acid

5.686 1.13 6.694 98.21 0.352.26 7.839 98.65 0.484.53 10.388 101.68 0.84

RSD - relative standard deviation

Table S4: Obtained data of precision of the optimized HPLC method expressed in area

peaks for known solutions of the compound sample mixture references.

Reference coumpond

Concentration (µg ml-1)

1° Day

2° Day

Average 1° Day

2° Day

Average

High RSD %

RSD%

RSD% Low RSD%

Tricin-7-O-diglucuronide

266.898 1.14 1.95 1.55 0.793 0.71 0.75

Acteoside 96.886 2.66 2.55 2.60 0.771 0.78 0.77Isoacteoside 151.141 0.41 1.69 1.05 1.011 1.09 1.05Geneposidic acid

28.617 0.68 1.00 0.84 0.980 1.39 1.19

8-epi-loganin 29.096 1.66 1.713

1.69 1.754 1.17 1.46

Mussaenoside 51.636 0.66 1.233

0.95 0.400 0.45 0.43

RSD - relative standard deviation

min0 10 20 30 40 50

mAU

0

50

100

150

200

DAD1 E, Sig=240,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\GENEPOSIDIC ACID\GENEPOSIDIS ACID 0_7A.D)

Figure S2: HPLC/DAD of geneposidic acid isolated from L. alba extracts (λ= 240 nm)

373.5

747.4

-MS, 7.5min #327

0

1

2

3

4

56x10

Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S3: Negative ESIMS of geneposidic acid

93.3

121.1

149.0

177.0

261.1339.2

397.2

501.0 551.1611.2

655.1

773.2

+MS, 7.4min #322

0

1

2

3

6x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S4: Positive ESIMS of geneposidic acid

Figure S5:1H NMR spectra of geneposidic acid [600 MHz, CD3OD, δ (ppm)]

Figure S6:13C NMR spectra of geneposidic acid [150 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

0

20

40

60

80

100

DAD1 E, Sig=240,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\EPI LOGANIN\EPILOGANIN 0_73A.D)

Figure S7: HPLC/DAD of 8-epi-loganin isolated from L. alba extracts (λ= 240 nm)

435.4

479.1

-MS, 14.9min #646

0.0

0.5

1.0

1.5

2.0

2.5

3.06x10

Intens.

200 400 600 800 1000 1200 1400 m/z Figure S8: Negative ESIMS of 8-epi-loganin

71.5 149.1 201.0 279.1 351.2

391.3

517.3 590.6 1017.5

+MS, 14.6min #633

0

2

4

6

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S9: Positive ESIMS of 8-epi-loganin

Figure S9:1H NMR spectra of 8-epi-loganin [600 MHz, CD3OD, δ (ppm)]

Figure S10:13C NMR spectra of 8-epi-loganin [150 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

0

20

40

60

80

100

120

140

160

DAD1 E, Sig=240,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\MUSSAENOSIDEO\MUSSAENOSIDEO 0_6B.D)

Figure S11: HPLC/DAD of mussaenoside isolated from L. alba extracts (λ= 240 nm)

435.3

779.2

-MS, 17.1min #741

0.0

0.2

0.4

0.6

0.8

1.07x10

Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S12: Negative ESIMS of mussaenoside

85.3161.0

209.1

261.0 313.2

359.2

391.3

552.8 621.5

795.3

915.1 1042.7

+MS, 16.8min #726

0

1

2

3

4

5

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S13: Positive ESIMS of mussaenoside

Figure S14:1H NMR spectra of mussaenoside [600 MHz, CD3OD, δ (ppm)]

Figure S15:13C NMR spectra of mussaenoside [150 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

0

25

50

75

100

125

150

175

DAD1 D, Sig=325,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\ACTEOSIDE\ACTEOSIDE 0_6C.D)

Figure S16: HPLC/DAD of acteoside isolated from L. alba extracts (λ= 325 nm)

623.5

-MS, 24.4min #1058

0

1

2

3

6x10Intens.

200 400 600 800 1000 1200 1400 m/z Figure S17: Negative ESIMS of acteoside

Figure S18:1H NMR spectra of acteoside [600 MHz, CD3OD, δ (ppm)]

Isoacteosídeo

C29H36O15

MM = 624Sólido amorfo amarelado

min0 10 20 30 40 50

mAU

0

20

40

60

80

100

120

DAD1 D, Sig=325,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\ISOACTEOSIDE\ISOACTEOSIDE 0_7A.D)

Figure S19: HPLC/DAD of iso-acteoside isolated from L. alba extracts (λ= 325 nm)

623.4

-MS, 26.9min #1168

0

1

2

3

4

6x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S20: Negative ESIMS of iso-acteoside

Figure S21:1H NMR spectra of iso-acteoside [600 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

0

20

40

60

80

DAD1 B, Sig=254,4 Ref=550,4 (ANGELICA\C...BRATION NEW\CURVE APIGENIN 2016-03-22 11-34-48\APIGENIN 0_7B.D)

Figure S21: HPLC/DAD of apigenin 7-O-glucuronide isolated from L. alba extracts (λ= 254 nm)

445.2

668.2

891.4

-MS, 31.2min #1361

0.0

0.5

1.0

1.5

2.0

2.5

6x10Intens.

200 400 600 800 1000 1200 1400 m/z Figure S22: Negative ESIMS of apigenin 7-O-glucuronide

447.2

+MS, 31.4min #1368

0.0

0.2

0.4

0.6

0.8

1.0

8x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S23: Positive ESIMS of apigenin 7-O-glucuronide

Figure S24:1H NMR spectra of apigenin 7-O-glucuronide [600 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

0

20

40

60

80

100

DAD1 B, Sig=254,4 Ref=550,4 (ANGELICA\CALIBRATION NEW\LUTEOLIN\LUTEOLIN 0_7C.D)

Figure S25: HPLC/DAD of luteolin 7-O-glucoside isolated from L. alba extracts (λ= 254 nm)

447.3

561.0

895.1

-MS, 25.7min #1113

0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

200 400 600 800 1000 1200 1400 m/z Figure S26: Negative ESIMS of luteolin 7-O-glucoside

391.2

449.2

+MS, 25.8min #1120

0.00

0.25

0.50

0.75

1.00

1.25

7x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S27: Positive ESIMS of luteolin 7-O-glucoside

Figure S28:1H NMR spectra of luteolin 7-O-glucoside [600 MHz, CD3OD, δ (ppm)]

min0 10 20 30 40 50

mAU

-10

-5

0

5

10

15

20

25

DAD1 B, Sig=254,4 Ref=550,4 (ANGELICA\C...ION NEW\CURVE TRICIN DIGLUCURONIDE\TRICIN DIGLUCURONIDE 0_7B.D)

Figure S29: HPLC/DAD of tricin 7-O-glucoronide isolated from L. alba extracts (λ= 254 nm)

681.2

-MS, 35.4min #1522

0.0

0.5

1.0

1.5

2.0

2.5

3.0

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S30: Negative ESIMS of tricin 7-O-glucoronide

391.2

683.1

+MS, 35.4min #1525

0

1

2

3

4

6x10Intens.

200 400 600 800 1000 1200 1400 m/z

Figure S31: Positive ESIMS of tricin 7-O-glucoronide

Figure S32:1H NMR spectra of tricin 7-O-glucoronide [600 MHz, DMSO-d6, δ (ppm)]