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SUPPLEMENTARY MATERIAL

Phytochemical and biological studies of constituents from roots of Salacia

crassifolia (Celastraceae)

Josana Pereira dos Santosa, Willian Xerxes Coelho Oliveiraa, Sidney A. Vieira-Filhob,

Rafael C. G. Pereiraa, Grasiely Faria de Souzaa, Viviane Alves Gouveiac, Adriano de

Paula Sabinod, Fernanda C. G. Evangelistad, Jacqueline Aparecida Takahashia, Marília

A. F. Mouraa, Filipe B. Almeidae, and Lucienir Pains Duartea,*,

aDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal de

Minas Gerais, 31270-901 Belo Horizonte – MG, Brasil

bDepartamento de Farmácia, Universidade Federal de Ouro Preto, Campus Morro do

Cruzeiro, 39401-089 Ouro Preto – MG, Brasil

cDepartamento de Microbiologia, Instituto de Ciências Biológicas, Universidade

Federal de Minas Gerais, 31270-901 Belo Horizonte – MG, Brasil

dFaculdade de Farmácia, Universidade Federal de Minas Gerais, 31270-901 Belo

Horizonte – MG, Brasil

eDepartamento de Química Inorgânica, Universidade Federal Fluminense, Campus

Valonguinho, 24020-150 Niterói – RJ, Brasil

*e-mail: lucienir@gmail.com

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Figure 1S. Photos of S. crassifolia: leaves, branches and fruits (Left), green and ripe

fruit (Center) and a cross-section of the root (Right), highlighting the red color

attributed to the presence of quinonamethides, mainly tingenone and pristimerine

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Figure 2S. Infrared spectrum of compound 2 (KBr)

Figure 3S. HR-ESI-MS spectrum of 2

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Figure 4S. 1H NMR spectrum (400 MHz, CDCl3 + Pyridine-d5) of 2

Figure 5S. Expansion of 1H NMR spectrum (400 MHz, CDCl3 + Pyridine-d5) of 2

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Figure 6S. 13C NMR spectrum (100 MHz, CDCl3 + Pyridine-d5) of 2

Figure 7S. DEPT-135 spectrum (100 MHz, CDCl3 + Pyridine-d5) of 2

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Figure 8S. HSQC contour map (400 MHz, CDCl3 + Pyridine-d5) of 2

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Figure 9S. HMBC contour map (400 MHz, CDCl3 + Pyridine-d5) of 2

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Figure 10S. Expansion of the HMBC contour map (400 MHz, CDCl3 + Pyridine-d5) of

2

9

.

Figure 11S. COSY contour map (400 MHz, CDCl3 + Pyridine-d5) of 2

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Figure 12S. HR-ESI-MS spectrum of 10

Figure 13S. 1H NMR spectrum (400 MHz, CDCl3) of 10

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Figure 14S. 13C NMR spectrum (100 MHz, CDCl3) of 10

Figure 15S. 13C NMR (100 MHz, CDCl3) DEPT-135 spectrum of 10

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Figure 16S. HSQC contour map (400 MHz, CDCl3) of 10

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Figure 17S. HMBC contour map (400 MHz, CDCl3) of 10

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Figure 18S. COSY contour map (400 MHz, CDCl3) of 10

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Figure 19S. NOESY contour map (400 MHz, CDCl3) of 10

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Figure 20S. Differential NOE spectrum of 10 (400 MHz, CDCl3) after irradiation of

hydrogen H-9

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Figure 21S. Differential NOE spectrum of 10 (400 MHz, CDCl3) after irradiation of

hydrogen H-8

Figure 22S. Differential NOE spectrum of 10 (400 MHz, CDCl3) after irradiation of

hydrogen H-14

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Description of crystal structure of 10

The crystal structure of 10 was unambiguously obtained from single crystal X-

ray diffraction experiments. For data collection, a single crystal obtained from ethanol

slow evaporation and irradiated with Mo Kα radiation (0.71073 Å). The compound

crystallized in the non-centrosymmetric orthorhombic space group P212121, which

indicated the obtained crystals were enantiomerically pure. Figure 2 shows the

representation of structure 10 demonstrating it is a molecule build up by four

condensed rings. The central ring is a seven-membered unit (C1 and C5-C10), with all

carbon atoms in sp3 hybridization. It has attached an epoxide oxygen atom (O1),

forming a three-member ring with two adjacent carbon atoms (C9 and C10) from the

central ring. Right on the following carbons on this inner ring, from each side, are

found two five-membered rings that end in the next carbon of the central ring. One of

these lateral rings is a lactam (C7, C8, C11, C12 and O3) and the other one is a

cyclopentane (C1-C5), both in envelope conformation and the carbon atom out of plane

are shared with the inner ring (C5 and C7). All side rings have methyl substituents, in

the epoxide the methyl (C14) is on C10, in the lactam (C13) is on C11 and in the

cyclopentane (C15) is on C4. Also, at the cyclopentane is present a hydroxyl group (O2)

in C3, which interact with other closest hydroxyl groups leading to a supramolecular

chain formed by hydrogen bonds (See Figure 2b).

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Figure 23S. 1H NMR spectrum (400 MHz, CDCl3) of 1

Figure 24S. 13C NMR spectrum (100 MHz, CDCl3) of 1

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Figure 25S. DEPT-135 spectrum (100 MHz, CDCl3) of 1

Figure 26S. 1H NMR spectrum (400 MHz, CDCl3) of 3

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Figure 27S. 13C NMR spectrum (100 MHz, CDCl3) of 3

Figure 28S. DEPT-135 spectrum (100 MHz, CDCl3) of 3

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Figure 29S. 1H NMR spectrum (400 MHz, CDCl3) of 4

Figure 30S. 13C NMR spectrum (100 MHz, CDCl3) of 4

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Figure 31S. DEPT-135 spectrum (100 MHz, CDCl3) of 4

Figure 32S. 1H NMR spectrum (400 MHz, CDCl3) of 5

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Figure 33S. 13C NMR spectrum (100 MHz, CDCl3) of 5

Figure 34S. DEPT-135 spectrum (100 MHz, CDCl3) of 5

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Figure 35S. 1H NMR spectrum (400 MHz, CDCl3) of 6

Figure 36S. 13C NMR spectrum (100 MHz, CDCl3) of 6

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Figure 37S. DEPT-135 spectrum (100 MHz, CDCl3) of 6

Figure 38S. 1H NMR spectrum (400 MHz, CDCl3) of 7

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Figure 39S. 13C NMR spectrum (100 MHz, CDCl3) of 7

Figure 40S. DEPT-135 spectrum (100 MHz, CDCl3) of 7

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Figure 41S. 1H NMR spectrum (400 MHz, CDCl3) of 8

Figure 42S. 13C NMR spectrum (100 MHz, CDCl3) of 8

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Figure 43S. DEPT-135 spectrum (100 MHz, CDCl3) of 8

Figure 44S. 1H NMR spectrum (400 MHz, CDCl3 + Pyridine-d5) of 9

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Figure 45S. 13C NMR spectrum (100 MHz, CDCl3 + Pyridine-d5) of 9

Figure 46S. DEPT-135 spectrum (100 MHz, CDCl3 + Pyridine-d5) of 9.

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Figure 47S. 1H NMR spectrum (400 MHz, CDCl3 + Pyridine-d5) of 11

Figure 48S. 13C NMR spectrum (100 MHz, CDCl3 + Pyridine-d5) of 11

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Figure 49S. DEPT-135 spectrum (100 MHz, CDCl3) of 11

Figure 50S. 1H NMR spectrum (400 MHz, MeOD) of 12

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Figure 51S. 13C NMR spectrum (100 MHz, MeOD) of 12

Figure 52S. DEPT-135 spectrum (100 MHz, MeOD) of 12

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Figure 53S. 1H NMR spectrum (400 MHz, MeOD) of 13

Figure 54S. 13C NMR spectrum (100 MHz, MeOD) of 13

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Figure 55S. DEPT-135 spectrum (100 MHz, MeOD) of 13

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Table 1S. Main crystallographic data of compound 10

Compound 1

Formula C15H22O4

Fw/g mol–1 266.32

T/K 298

λ/Å 0.71073

Crystal System Prthorhombic

Space group

a/Å 5.6629(2)

b/Å 8.4104(3)

c/Å 29.4251(9)

V/Å3 1401.44(8)

Z 4

ρ/mg m–3 1.262

μ/mm–1 0.090

F(000) 576

Crystal size/mm3 0.33×0.20×0.06

Reflections collected (Rint) 49585 (0.0251)

Unique Reflections 2863

Reflections with I ≥ 2σ(I) 2458

Goodness-of-fit on F2 1.046

Ra, wRb 0.0441, 0.1193

Ra, wRb (all data) 0.0532, 0.1243

Larg. diff. peak and hole/e Å–3) 0.284, –0.245

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Table 2S. Concentration of Salacia crassifolia root samples used in toxicity tests on

Caenorhabditis elegans

Sample

Name

Stock solution

(mg mL-1)

Final test

concentration

(µg mL-1)

3 Cariopristimerin 10 200

5 Pristimerin 10 200

8 20-Hydroxy-20-epi-

tingenone

10 200

9 6-oxo-pristimerol 10 200

12 4`-O-metilepigalocatechin 10 200

EHE Hexane extract 40 800

EC Chloroform extract 40 800

ACE Acetone extract 40 800

EMet Metanol extract 40 800

Extracts: Hexane (EHE), chloroform (EC), ethyl acetate (ACE) and methanol (EMet).

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Table 3S. Values of the median inhibitory concentration (IC50) of cell growth obtained

for the isolated compounds of Salacia crassifolia roots

Sample IC50 (μg/mL ± SD) of the cells*

THP-1 K562 MDA-MB-231 WI-26 VA4

3 47.20 ± 1.85 48.05 ± 2.95 38.10 ± 2.10 62.32 ± 2.90

5 30.55 ± 1.30 46.25 ± 2.75 40.70 ± 1.75 58.74 ± 2.62

8 50.70 ± 2.95 55.12 ± 3.05 64.60 ± 3.15 72.70 ± 3.30

9 46.16 ± 2.82 41.60 ± 2.53 39.10 ± 2.05 49.08 ± 2.25

Etoposide 12.04 ± 2.32 9.11 ± 1.33 12.00 ± 0.63 8.63 ± 0.13

Citarabine 12.70 ± 1.20 ND ND 58.70 ± 3.76

Imatinib

mesylate ND 10.50 ± 1.05 ND 69.63 ± 3.13

P value <0.05a <0.05b <0.05c <0.05d

>0.05e

*Values presented as the medium ± standard deviation (SD)). a = THP-1, citarabine and

etoposide versus all tested samples; b = K562, Imatinib and etoposide versus all tested

samples; c = MDA-MB-231, etoposide versus all tested samples; d: WI-26VA4,

etoposide versus all tested samples / citarabine versus SC08, SC09, SC17 and SC14/

Imatinib versus SC03, SC05, SC09, SC15, SC16 and SC18; e = WI-26VA4, citarabine

versus SC03, SC05, SC15, SC16 and SC18/ Imatinib versus SC08, SC17 and SC14.

THP-1 = acute myeloid leukemia cells (ATCC-TIB-202), K562 = chronic myeloid

leukemia cells (ATCC-CRL-3344) and MDA-MB-231 = breast carcinoma cells

(ATCC-HTB-26). The cytotoxicity of the samples against the Wi-26VA4 lineage

[healthy cells from lung fibroblasts, (ATCC-CCL-75)] was used to establish the

selectivity index (SI). ND = not detected.