Synergistic interactions among flavonoids and acetogenins in Graviola (Annona muricata) leaves confer protection against prostate cancer

Abstract

Phytochemical complexity of plant extracts may offer health-promoting benefits including chemotherapeutic and chemopreventive effects. Isolation of 'most-active fraction' or single constituents from whole extracts may not only compromise the therapeutic efficacy but also render toxicity, thus emphasizing the importance of preserving the natural composition of whole extracts. The leaves of Annona muricata, commonly known as Graviola, are known to be rich in flavonoids, isoquinoline alkaloids and annonaceous acetogenins. Here, we demonstrate phytochemical synergy among the constituents of Graviola leaf extract (GLE) compared to its flavonoid-enriched (FEF) and acetogenin-enriched (AEF) fractions. Comparative quantitation of flavonoids revealed enrichment of rutin (~7-fold) and quercetin-3-glucoside (Q-3-G, ~3-fold) in FEF compared to GLE. In vivo pharmacokinetics and in vitro absorption kinetics of flavonoids revealed enhanced bioavailability of rutin in FEF compared to GLE. However, GLE was more effective in inhibiting in vitro prostate cancer proliferation, viability and clonogenic capacity compared to FEF. Oral administration of 100mg/kg bw GLE showed ~1.2-fold higher tumor growth-inhibitory efficacy than FEF in human prostate tumor xenografts although the concentration of rutin and Q-3-G was more in FEF. Contrarily, AEF, despite its superior in vitro and in vivo efficacy, resulted in death of the mice due to toxicity. Our data indicate that despite lower absorption and bioavailability of rutin, maximum efficacy was achieved in the case of GLE, which also comprises of other phytochemical groups including acetogenins that make up its natural complex environment. Hence, our study emphasizes on evaluating the nature of interactions among Graviola leaf phytochemcials for developing favorable dose regimen for prostate cancer management to achieve optimal therapeutic benefits.

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.

Figure 2.

Graviola leaf phytochemicals inhibit proliferation of human prostate cancer cells. (A) Determination of IC₅₀ 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 IC₅₀ 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).

Figure 3.

Dietary feeding of GLE showed inhibition of human prostate tumor xenograft growth in nude mice. Male nude mice were subcutaneously injected with 10⁶ PC-3-luc cells. (Ai) Bioluminescent images (representative one animal per group) depicting tumor progression over 4 weeks. (Aii) Quantitation of radiance (photons/s/cm²/sr) measured from tumors of vehicle- and GLE-, AEF-, FEF- and rutin-fed mice. (B) Tumor-volume (in mm³). (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).

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).

Figure 5.

Oral PK parameters of GLE and FEF. (A) Peak plasma concentration of rutin and Q-3-G (C _max), (B) area under the concentration-time curve (AUC _last) 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.

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).