A. Sánchez-Medina et al. / Phytochemistry 70 (2009) 765–772
771
(
3
30 m  0.25 mm  0.25
00 °C at 6 °C/min; injection temperature 350 °C; carrier gas, He
at 1 ml/min. Compounds 1–4 and the 5:4 mixture of 5 and 6 gave
-xylose, -arabinose, -rhamnose and -glucose at t 9.48, 9.52,
0.19 and 12.20 min, respectively. In addition, 3 and the 5:4 mix-
l
m), oven temperature program, 180–
European Collection of Animal Cell Cultures (Porton Down,
Salisbury, UK). The medium used for the maintenance of the cell
line and for the in vitro bioassay was Dulbecco’s modified eagle’s
medium (Sigma–Aldrich), supplemented with 10% of heat inacti-
D
L
L
D
R
1
vated foetal bovine serum (Sigma–Aldrich), 2.5
lg/ml amphoteri-
ture of 5 and 6 gave
D
-apiose at t
R
9.27 min (identical to authentic
cin B, 30 g/ml streptomycin and 30 IU/ml penicillin. The
l
standards).
modified MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide) (Sigma–Aldrich) assay was used to detect the cel-
3
.5. Saponin 1
lular viability (Habtemariam, 1995). Briefly, cells were harvested
3
(
5 Â 10 cells/well) in 100
l
l in 96-well plates. After 24 h incuba-
1
13
Amorphous white powder. H and C NMR: see Tables 1 and 2;
tion at 37 °C and 5% CO
2
to allow cell establishment, different
À
ESI-MS (negative mode) m/z: 1531 [M À H] ; ESI-MS (positive
concentrations of compounds or extracts made with 1:3 or 1:2
serial dilutions were added. The starting concentration was
100 lg/ml for pure compounds and 500 lg/ml for crude extracts
or fractions. Four replicates were set up for each experiment and
the experiment was repeated four times (n = 4). Samples were left
in contact with cells for 24 h and cell viability was assessed by
+
+
mode) m/z: 1555 [M + Na] ; MS/MS of m/z 1555 [M + Na] m/z:
+
8
1
67 [(M + Na) À (3 Â 132) À (2 Â 146)] , 711 [(M + Na) À (2 Â
+
+
62) À 520] , 579 [(M + Na) À (2 Â 162) À 520 À 132] , 563 [(M +
+
Na) À (2 Â 162) À 520 À 146] .
3.6. Saponin 2
adding 10 ll of MTT solution (5 mg/ml) during the last 4 h of
incubation. The absorbance associated with formazan-blue forma-
tion was then measured using a Thermo Labsystems-Multiskan
EX ELISA reader at 570 nm. Calculation of IC50 values (concentra-
tion required to kill 50% of cells) was carried out using the corre-
sponding Microsoft Excel formula for correlation and regression.
Significant differences (95% confidence) between fractions as the
fractionation steps advanced were obtained using the GraphPad
Prism 5.01 software. The positive control, podophyllotoxin, gave
an IC50 value of 29 ± 5 nM against RAW 264.7 cells under the
same experimental conditions.
Amorphous white powder. H and 13C NMR: see Tables 1 and 2;
1
À
ESI-MS (negative mode) m/z: 1369 [M À H] ; ESI-MS (positive
+
+
mode) m/z: 1393 [M + Na] ; MS/MS of m/z 1393 [M + Na] m/z:
+
+
7
5
5
1
11 [(M + Na) À 162 À 520] , 579 [(M + Na) À 162 À 520 À 132] ,
+
63 [(M + Na) À 162 À 520 À 146] ,
433 [(M + Na) À 162 À
+
20 À 132 À 146] ,
287
[(M + Na) À 162 À 520 À 132 À (2 Â
+
+
46)] , 155 [(M + Na) À 162 À 520 À (2 Â 132) À (2 Â 146)] .
3
.7. Saponin 3
1
13
Amorphous white powder. H and C NMR: see Tables 1 and 2;
Acknowledgments
À
ESI-MS (negative mode) m/z: 1531 [M À H] ; ESI-MS (positive
+
mode) m/z: 1555 [M + Na] .
The authors thank Mr. Filogonio May-Pat, from the Centro de
Investigación Científica de Yucatán, for his help and assistance in
the identification and collection of the plant material, as well as
in the preparation of the voucher specimen, Ms Gilda Erosa-Rejón
for technical assistance, Dr. Andrew P. Mendham (University of
Greenwich) for carrying out preliminary NMR experiments, Dr.
Geoffrey Kite (Royal Botanic Gardens, Kew) for help with sugar
analysis, and the MRC Biomedical NMR Centre (National Institute
for Medical Research, Mill Hill, London, UK) for access to higher-
field NMR instrumentation. L.M.P.-R. wishes to thank the British
Council (Higher Education Link MXC/991) and FOMIX-Yucatán
3
.8. Saponin 4
Amorphous white powder. 1H and 13C NMR: identical to lit.
(Eskander et al., 2005; compound 2); ESI-MS (negative mode)
À
+
m/z: 1353 [M À H] ; ESI-MS (positive mode) m/z: 1377 [M + Na] .
3.9. Saponins 5 and 6
Amorphous white powder comprising 5:4 mixture of 5 and 6.
1
13
1
H and C NMR of glycosidic resonances: see Table 3; H NMR
OD, assignments identical for 5 and 6): d 2.06,
(
Project No. 66262) for partial financial support. A.S. thanks the
of aglycone (CD
3
Mexican Council for Science and Technology (CONACYT) for the
award of a scholarship for fees and living expenses during his
PhD studies.
1
5
2
.18 (2 Â m, H-1a,b), 4.34 (br m, H-2), 3.58 (m, H-3), 1.33 (m, H-
), 4.47 (br m, H-6), 1.82, 1.56 (2 Â m, H-7a,b), 1.65 (m, H-9),
.12, 1.98 (2 Â m, H-11a,b), 5.42 (br m, H-12), 1.84, 1.41 (2 Â m,
H-15a,b), 4.49 (br m, H-16), 3.08 (dd, J = 14.4, 4.1 Hz, H-18), 2.27,
1
.06 (2 Â m, H-19a,b), 1.90, 1.16 (2 Â m, H-21a,b), 1.90, 1.79
),
), 0.89 (s, 29-
OD, assignments
identical for 5 and 6): d 46.8 (C-1), 71.4 (C-2), 83.8 (C-3), 44.1 (C-
), 49.0 (C-5), 68.7 (C-6), 41.5 (C-7), 40.0 (C-8), 48.8 (C-9), 37.3
References
(
1
2 Â m, H-22a,b), 3.72, 3.43 (2 Â m, H-23a,b), 1.31 (s, 24-CH
3
Durán, R., Trejo-Torres, J.C., Ibarra-Manríquez, G., 1998. Endemic phytotaxa of the
peninsula of Yucatán. Harvard Papers in Botany 3, 263–314.
Durán, R., Campos, G., Trejo, J.C., Simá, P., May-Pat, F., Juan-Qui, M., 2000. Listado
Florístico de la Península de Yucatán. Centro de Investigación Científica de
Yucatán, A.C. Mérida, Yucatán.
Duus, J.Ø., Gotfredsen, C.H., Bock, K., 2000. Carbohydrate structural determination
by NMR spectroscopy: modern methods and limitations. Chemical Reviews 100,
4589–4614.
.63 (s, 25-CH
3
), 1.05 (s, 26-CH
3
), 1.34 (s, 27-CH
3
13
CH
3
), 0.98 (s, 30-CH
3
); C NMR of aglycone (CD
3
4
(
(
C-10), 24.6 (C-11), 124.2 (C-12), 144.0 (C-13), 43.5 (C-14), 36.4
C-15), 74.6 (C-16), 50.5 (C-17), 42.3 (C-18), 47.7 (C-19), 31.4 (C-
Eskander, J., Lavaud, C., Abdel-Khalik, S.M., Soliman, H.S.M., Mahmoud, I.I., Long, C.,
2
1
0), 36.5 (C-21), 31.8 (C-22), 65.6 (C-23), 16.4 (C-24), 19.3 (C-25),
9.1 (C-26), 27.4 (C-27), 177.1 (C-28), 33.4 (C-29), 25.2 (C-30);
2005. Saponins from the leaves of Mimusops laurifolia. Journal of Natural
Products 68, 832–841.
À
À
ESI-MS (negative mode) m/z: 1369 [M À H] (5), 1501 [M À H]
Eskander, J., Lavaud, C., Pouny, I., Soliman, H.S.M., Abdel-Khalik, S.M., Mahmoud, I.I.,
2006. Saponins from the seeds of Mimusops laurifolia. Phytochemistry 67,
1793–1799.
Espadas-Manrique, C., Durán, R., Argáez, J., 2003. Phytogeographic analysis of taxa
endemic to the Yucatán Peninsula using geographic information systems, the
domain heuristic method and parsimony analysis of endemicity. Diversity and
Distributions 9, 313–330.
+
(6); ESI-MS (positive mode) m/z: 1393 [M + Na] (5), 1525
+
[
M + Na] (6).
3.10. Cytotoxicity bioassay by MTT
Fuentes-García, A.G., 2001. Evaluacíon de la actividad biológica en extractos de
plantas nativas de la península de Yucatán. BSc Thesis, Universidad Autonoma
de Yucatán, Mérida, Yucatán, México.
The murine macrophage-like cell line RAW 264.7 was used for
the cell viability assays. The cell line was purchased from the