Cytotoxic Podophyllotoxin Type-Lignans from the Steam Bark of Bursera fagaroides var. fagaroides

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED50 = 9.6 × 10−2 μg/mL), PC-3 (ED50 = 2.5 × 10−1 μg/mL), MCF-7 (ED50 = 6.6 μg/mL), and HF-6 (ED50 = 7.1 × 10−3 μg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5′-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED50 = 1.0 × 10−5 μg/mL), and KB (ED50 = 1.0 × 10−5 μg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.

In this paper we report on the antitumor and potent cytotoxic activities of the hydroalcoholic extract (HA) of the steam bark of B. fagaroides var. fagaroides. Purification of this extract by bioassay-guided chromatographic methods afforded seven podophyllotoxin-type lignans, which showed important cytotoxic activities against KB (nasopharyngeal), HF-6 (colon), MCF-7 (breast), and PC-3 (prostate) cancer cell lines with ED 50 values comparable to those displayed by camptothecin, podophyllotoxin and etoposide used as positive controls.

Results and Discussion
The intraperitoneal administration of 50 and 100 mg/Kg of the hydroalcoholic extract of the bark of B. fagaroides var. fagaroides (HA), on mice inoculated with 2 × 10 4 L5178Y lymphoma cells/mouse, showed an increase on the survival time ( Figure 1). Mice with 2 × 10 4 L5178Y cells usually die within 30 days without treatment. When treated with the dose of 100 mg/Kg of HA extract over 15 days, the survival was significantly prolonged (p < 0.001) compared with the control groups. Median survival time for the group without HA treatment was of 29 days, while for those that received the dose of 50 and 100 mg/Kg, this time increased to 35 and 38 days, respectively. The survival of the 100 mg/Kg group was 50% better than the 50 mg/Kg group (p < 0.05). The best response was observed with the 100 mg/Kg/day dose, where the survival of treated mice was significantly prolonged (p < 0.001) compared with the placebo and control groups. This dose cured 26% of the treated mice. Survival for more than 60 days without a tumor was considered to be a 'cure' [31]. On the other hand, this extract also significantly inhibited the proliferation of KB (ED 50 = 9.6 × 10 −2 µg/mL), PC-3 (ED 50 = 2.5 × 10 −1 µg/mL), HF-6 (ED 50 = 7.1 × 10 −3 µg/mL) and MCF-7 (ED 50 = 6.6 µg/mL) tumor cell lines (Table 1).

Figure 1.
Antitumor activity of Bursera fagaroides var. fagaroides HA extract evaluated for the survival rate of mice with lymphoma L5178Y, comparing the control groups (without or placebo treatments) and versus treated groups with 50 and 100 mg/Kg/day/15 days (n = 10). All of them by Kaplan-Maier estimation of survival were different (p < 0.001). Bioassay-guided isolation procedures, using the activity against KB, HF-6, MCF-7, and PC-3 cancer cell lines were carried out to define active components in this plant. Figure 2 shows the chromatographic fractionation of the HA extract monitored by the cytotoxic activity against PC-3 cells. Chromatographic fractionation of the HA extract afforded four fractions, two of which (F-1 and F-2), displayed potent cytotoxic activity against the four tested cell lines, principally against PC-3, both with ED 50 values (1 × 10 −5 μg/mL) greater than that displayed by the therapeutic drugs camptothecin (0.96 μg/mL), and etoposide (5.6 × 10 −3 μg/mL) used as positive controls (Table 1). Chromatographic purification of F-1 afforded β-peltatin-A-methylether (2), 5′-desmethoxy-βpeltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5); while purification of F-2 afforded podophyllotoxin (1), burseranin, (6), and acetyl podophyllotoxin (7), which were identified by comparing their spectroscopic data with those previously described in the literature. The purity of isolated compounds was determined to be above of 95%, based on the peak areas of their HPLC chromatograms, as well as by their 1 H-NMR spectra. The structures of these compounds are shown in Figure 3.
The cytotoxic activity of podophyllotoxin (1), desoxypodophyllotoxin (5) and their congeners is well known [49][50][51][52]. Some structure-activity relationship studies, using several podophyllotoxin analogues, showed that the core structure of deoxypodophyllotoxin (5) is responsible for this cytotoxicity. The extra methoxy group on the 6-position in 5′-desmethoxy-β-peltatin-A-methylether (3) significantly changed the in vitro cytotoxicity when compared to desoxypodophyllotoxin (5). Compounds 4 and 6 which do not have the core structure of 5, displayed less potent activity against KB, MCF-7 and HF-6, and were more selective against PC-3 cell line (Table 1). Results here obtained confirm the structure-activity relationships previously described, and provide new cytotoxic data for compounds 3, 4, 6 and 7 that complement the knowledge of this type of compounds.
Although the cytotoxicity of podophyllotoxin (1) and desoxypodophyllotoxin (5) is well known, the cytotoxicity of 3, 4, 6, and 7 against the four cell lines tested, and of 2 against PC-3, MCF-7, and HF-6, is reported here for the first time.

General
NMR spectra were acquired on a Varian Unity NMR spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C nuclei. Chemical shifts are listed in parts per million (ppm), referenced to CDCl 3 and were made on the basis of 1 H-1 H gCOSY, 1 H-1 H TOCSY, NOESY, gHSQC and gHMBC spectral analysis as required. NMR experiments performed in CDCl 3 are referenced to Me 4 Si (0 ppm). FABMS spectra in a matrix of m-nitrobenzyl alcohol or glycerol were recorded on a JEOL JMX-AX 505 HA mass spectrometer. All reagents and solvents used were analytical grade. Optical rotations were acquired with a Perkin-Elmer 241MC polarimeter (10 cm, 1 mL cell) at the sodium D line. High Performance Liquid Chromatography (HPLC) was performed using a Waters Delta Prep 4000 Module System equipped with a Waters 717 plus Autosampler and 996 Photodiode Array Detector (Waters Co., Milford, MA, USA), and a Xterra prep C18 column (5 μm, 7.8 × 100 mm) with MeOH-H 2 O (52:48) as the isocratic eluent system, UV detection at 215 nm and a flow rate of 1 mL/min.

Plant Material
The bark of B. fagaroides var. fagaroides (H.B.K.) Engl. was collected in the village of Capula between Zacapu and Quiroga, Michoacán, México. Its identification was made at the herbarium of the Instituto Mexicano del Seguro Social (registration number-12 051 IMSSM) and the Institute of Botany, University of Guadalajara (IBUG-140 748), México.

Spectral Data
Podophyllotoxin ( with 10% fetal bovine serum, 5,000 units/mL penicillin, 5 mg/mL streptomycin, 7.5% NaHCO 3 , and cultured in a 96-well microtiter plate (10 4 cells/mL, 190 μL/well) at 37 °C in a 5% CO 2 -air atmosphere (100% humidity). The cells at the log phase of growth were treated in triplicate (n = 3) with different concentrations of the test compounds (0.16, 0.8, 4 and 20 μg/mL), and incubated for 72 h. The cell concentration was determined by protein analysis. The optical density was measured at 590 nm with an ELISA-Reader (Molecular Devices, SPECTRA max plus 384). Results were expressed as the concentration that inhibits 50% of control growth after the incubation period (IC 50 ). The values were estimated from a semi-log plot of the extract concentration (μg/mL) against the percentage of viable cells. Camptothecin, etoposide, and podophyllotoxin were included as positive standards.

Antitumor Activity
Male BALB/c mice (6-8 weeks old, 22-26 g) were provided by the Centro de Investigación Biomedica de Occidente (CIBO-IMSS). A lymphoma L5178Y cell line was used derived from a thymic lineage (haplotype H-2d) tumor induced in DBA/2 mouse by methyl-cholanthrene adopted to an ascetic form, and maintained by intraperitoneal (i.p.) transplantation of 10 × 10 6 cells/mouse every 15 days in syngenic BALB/c mice [31]. For this study, all procedures involving animals were performed according to protocols approved by NOM-062-ZOO-1999. Animals were inoculated i.p. with 0.1 mL of suspension of fresh ascitic fluid, containing L5178Y lymphoma (2 × 10 4 ) cells/mouse on day zero. Treatment with HA extract started 24 h after inoculation at doses of 50 or 100 mg/kg oral rout/day during 15 days, each group containing five mice and were observed during 60 days.

Statistical Analysis
The results were analyzed using one-way ANOVA followed by Kaplan-Meier estimation of survival and Cox's regression through the statistical package SPSS V.15.

Conclusions
Bioassay-guided isolation of the hydroalcoholic extract obtained from the steam bark of B. fagaroides var. fagaroides identified a family of seven related lignans, among which podophyllotoxin (1) and acetyl podophyllotoxin (7) are described by the first time in Bursera. The presence of podophyllotoxin (1), together with six other related lignans in the cytotoxic extract of B. fagaroides var. fagaroides is noteworthy. In summary the cytotoxic and antitumor activities observed for B. fagaroides var. fagaroides are ascribable to the lignans present in this extract. Investigation of the podophyllotoxin-related lignans obtained from B. fagaroides var. fagaroides may lead to new cytostatic compounds, which could serve as the basis for new anti-tumor drugs.