SUPPLEMENTARY MATERIAL

Unprecedented New Nonadecyl para-Hydroperoxycoumaroate isolated

from Erythrina excelsa and its Cytotoxic Activity

Guy M. N. Kwamoua, Louis P. Sandjoa,*, Victor Kueteb,c, Anaelle

A. K. Wandjaa, Simplice B. Tankeob, Thomas Efferth**c and

Augustin E. Nkengfacka

aDepartment of Organic Chemistry, University of Yaoundé I, P. O. Box 812 Yaoundé,

CameroonbDepartment of Biochemistry, Faculty of Science, University of Dschang, P. O. Box 67

Dschang CamerooncDepartment of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,

University of Mainz, Staudinger weg 5, 55128 Mainz, Germany

Corresponding author:

Dr. Louis P. Sandjo E-mail: plsandjo@yahoo.fr

Prof. Thomas Efferth Phone: +49-6131-3925751; Fax: +49-6131-

39-23752; E-mail: efferth@uni-mainz.de

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Abstract: A new unprecedented cinnamate derivative (1) was

obtained from Erythrina excelsa (Leguminosae) and identified as

nonadecyl para-hydroperoxycoumaroate. This compound was isolated

together with three known compounds namely lupeol (2), mixture

of Sitosterol and Stigmasterol (3), and Isoneorautenol (4).

Their structures were established on the base of NMR and mass

spectroscopic data in conjunction to those reported in the

literature. Compound 1 was evaluated for its capability of

inhibiting cancer cell lines and a panel of microbial strains

growth. It turned out that 1 is moderately to significantly

cytotoxic against six cancer cell lines and shows weak to no

antimicrobial activity.

Keywords: Anticancer activity; Drug resistance; para-

Hydroperoxycoumaroate; Fabaceae.

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Figure S1 HRMS-ESI spectrum

Figure S2 1H NMR spectrum (CDCl3, 400 MHz)

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cyclohexane

Figure S3 13C NMR spectrum (CDCl3, 100 MHz)

Figure S4 COSY spectrum (CDCl3)

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Figure S5 HSQC spectrum (CDCl3)

Figure S6 HMBC spectrum (CDCl3)

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Figure S7 NOESY spectrum (CDCl3)

Figure S8 Enlarged NOESY spectrum (CDCl3)

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Figure S9 LC-ESI MSChromatogram og compound 1

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Figure S10: Key HMBC correlations of compound 1

Figure S11: NOESY and COSY correlations of compound 1

Figure S12: proposal of biosynthetic pathway of compound 1

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Figure S13: IR spectrum of compound 1

1. Antibacterial assay

The studied microorganisms included reference (ATCC) and

multidrug resistant strains of Pseudomonas aeruginosa (PA01),

Klebsiella pneumoniae (KP55, KP63), Escherichia coli (ATCC8739, AG100,

AG102), and Enterobacter aerogenes (ATCC13048, EA3, EA27, CM64)

obtained from the American Type Culture Collection. They were

maintained on agar slant at 4 oC and sub-cultured on a fresh

appropriate agar plates 24 h prior to any antimicrobial test.

Nutrient Agar and Sabouraud Glucose Agar were used for the

activation of bacteria and fungi respectively. The Mueller

Hinton Broth (MHB) was used for the MIC and MMC

determinations. The Mueller Hinton Agar (MHA) was also used

for the determination of the MMC on these species (Kuete et

al., 2011). Tetracycline (Sigma-Aldrich, St. Quentin

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Fallavier, France) was used as reference antibiotics (RA)

respectively against bacteria. p-Iodonitrotetrazolium chloride

(INT, Sigma-Aldrich) was used as microbial growth indicator

(Mativandlela et al., 2006). The MIC determinations on

bacteria was conducted using rapid INT colorimetric assay

according to described methods (Mativandlela et al., 2006)

with some modifications. Briefly, the test sample was first of

all dissolved in 10% (v/v) DMSO/MHB to give a final

concentration of 512 µg/mL and serially diluted twofold to

obtain concentration ranges. 100 µL of each concentration was

added in a well (96-well microplate) containing 95 µL of MHB

and 5 µL of inoculum (standardized at 1.5×106 CFU/mL by

adjusting the optical density to 0.1 at 600 nm SHIMADZU UV-

120-01 spectrophotometer) (Tereschuk et al., 1997). The final

concentration of DMSO in the well was less than 3%

(preliminary analyses with 3% (v/v) DMSO do not alter the

growth of the test organisms). The negative control well

consisted of 195 µl of MHB and 5 µl of the standard inoculum

(Zgoda and Porter 200). The plates were covered with a sterile

plate sealer, then agitated to mix the contents of the wells

using a plate shaker and incubated at 37 oC for 24 h. The assay

was repeated three times in triplicate. The MIC of samples was

detected following addition (40 µL) of 0.2 mg/mL p-

iodonitrotetrazolium chloride and incubation at 37 oC for 30

min (Mativandlela et al., 2006). Viable microorganisms reduced

the yellow dye to a pink colour. MIC was defined as the lowest

sample concentration that prevented this change and exhibited

complete inhibition of bacterial growth (Kuete et al., 2008).

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2. Cytotoxicity Assay

The resazurin reduction assay (O’Brien et al., 2000) was

performed to assess the cytotoxicity of compound 1 and

doxorubicin towards various sensitive and drug-resistant

cancer cell lines, including the leukemia CCRF-CEM and

CEM/ADR5000, breast MDA-MB231, colon HCT116, glioblastoma

U87MG and hepatocarcinoma HepG2. The assay is based on the

reduction of the indicator dye, resazurin, to the highly

fluorescent resorufin by viable cells. Non-viable cells

rapidly lose their metabolic capacity to reduce resazurin and,

thus, do not produce fluorescent signals anymore. Briefly,

adherent cells were detached by treatment with 0.25%

trypsin/EDTA (Invitrogen, Darmstadt Germany) and an aliquot of

1 × 104 cells was placed in each well of a 96-well cell culture

plate (Thermo Scientific, Langenselbold, Germany) in a total

volume of 200 μL. Cells were allowed to attach overnight and

then were treated with different concentrations of compounds.

For suspension cells, aliquots of 2 × 104 cells per well were

seeded in 96-well-plates in a total volume of 100 μL. The

studied compound was immediately added in varying

concentrations in an additional 100 μL of culture medium to

obtain a total volume of 200 μl/well. After 72 h, resazurin

(Sigma-Aldrich, Schnelldorf, Germany) (20 μL, 0.01% w/v) in

distilled H2O was added to each well and the plates were

incubated at 37 °C for 4 h. Fluorescence was measured on an

Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany)

using an excitation wavelength of 544 nm and an emission

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wavelength of 590 nm. Each assay was done at least twice with

six replicates each. The viability was evaluated based on a

comparison with untreated cells. IC50 values represent the

compound concentrations required to inhibit 50% of cell

proliferation and were calculated from a calibration curve by

linear regression using Microsoft Excel.

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