Figure 1. Identification of the most-abundant phytochemicals in GLE

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Figure 1. Identification of the most-abundant phytochemicals in GLE Figure 1. Identification of the most-abundant phytochemicals in GLE. (A) LC–UV/MS (TIC(+)) comparison of the GLE, FEF and AEF profiles. (B) Quantitation of rutin, quercetin-3-glucuside (Q-3-G), quercetin and kaempferol was performed employing a calibration curve using pure standards. From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figure 2. Graviola leaf phytochemicals inhibit proliferation of human prostate cancer cells. (A) Determination of IC<sub>50</sub> of GLE and its constituents. PC-3 cells were treated with GLE, AEF, FEF and rutin at various concentrations for 48h. The percentage cell proliferation was measured by MTT assay. (B) Bar-graphical representation of IC<sub>50</sub> values (mentioned above) of GLE, AEF, FEF and rutin tested in PC-3 cells. (C) GLE and its constituents affect the cell viability of PC-3 cells. Bar graphical representation of percent cell viability of PC-3 cells treated with GLE, AEF and FEF as demonstrated by trypan blue exclusion assay. (D) GLE and its constituents inhibit clonogenic capacity of prostate cancer cells. Bar-graphical representation and photograph of crystal violet-stained surviving colonies from control and GLE-, AEF- and FEF-treated groups. Values and error bars shown in the graphs represent Mean ± SD. (*P < 0.05, compared with controls). From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figure 3. Dietary feeding of GLE showed inhibition of human prostate tumor xenograft growth in nude mice. Male nude mice were subcutaneously injected with 10<sup>6</sup> PC-3-luc cells. (Ai) Bioluminescent images (representative one animal per group) depicting tumor progression over 4 weeks. (Aii) Quantitation of radiance (photons/s/cm<sup>2</sup>/sr) measured from tumors of vehicle- and GLE-, AEF-, FEF- and rutin-fed mice. (B) Tumor-volume (in mm<sup>3</sup>). (C) Tumor weight comparison along with photographic images of excised tumors. (D) Body weight comparison of vehicle and treatment groups. (*P < 0.05; two-way analysis of variance, compared with controls). From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figure 4. Plasma concentration-time profiles of GLE and FEF (100mg/kg bw). The plasma concentration-time profile of (A) rutin and (B) Q-3-G were quantitated in blood samples collected at various time points (0.08, 0.16, 0.25, 0.5, 1, 2, 4 and 6h) following oral administration of GLE and FEF. Error bars refer to Mean ± SD (*P < 0.05). From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figure 5. Oral PK parameters of GLE and FEF Figure 5. Oral PK parameters of GLE and FEF. (A) Peak plasma concentration of rutin and Q-3-G (C<sub>max</sub>), (B) area under the concentration-time curve (AUC<sub>last</sub>) for rutin and Q-3-G determined using plasma samples collected at various time points (0.08, 0.16, 0.25, 0.5, 1, 2, 4 and 6h) for GLE and FEF. From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figure 6. In vitro cellular uptake of flavonoids Figure 6. In vitro cellular uptake of flavonoids. (A) Comparison of cellular concentrations of rutin in the case of GLE and FEF treatments. (B) Concentration values of rutin in the case of GLE and FEF treatment for 4, 8 and 12h. Values and error bars refer to Mean ± SD (*P < 0.05). From: Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer Carcinogenesis. 2015;36(6):656-665. doi:10.1093/carcin/bgv046 Carcinogenesis | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.