| dc.description.abstract | Background: Carica papaya Linn (CP), Vernonia amygdalina Delile (VA), dihydroartemisinin (DHA) and molecular iodine (I2) were reported to have antiproliferative and cytotoxic activities on breast cancer but their anticancer effect once combined had not been explored. This study assessed the cytotoxic and synergistic effects of combining I2, DHA, methanolic extracts of CP, VA leaves against MCF-7 and MDA-MB-231 breast cancer cell lines, acute, sub-acute and sub-chronic toxicity of most efficacious combinations of CP+VA and CP+VA+DHA and phytochemical analysis of phenolic content of plants used in this study.
Methods: Cytotoxic activity and cell viability of the plant extracts and combinations were determined using trypan blue assay with peripheral blood mononuclear cells (PBMCs) and doxorubicin (DOX) as negative and positive controls respectively. The study used SigmaPlot software to obtain IC50 values and drug interactions were determined using the isobologram and combination index (CI) (Chou-Talalay method). A pilot toxicity study was carried out to determine doses for acute, sub-acute and sub-chronic toxicity studies of the two combined plant extracts and compounds in albino mice (n=20) and Wistar rats (n=36). The experiment was done based on OECD guidelines with modifications. Animals were dosed with 500, 1 000 and 2 000 mg/kg of drug combinations and observed for 24 hours, 28 and 90 days. Body weight, hematological, biochemical parameters and histopathological changes of liver and kidney were evaluated. To perform a qualitative analysis of phenolic compounds in botanicals, ultra- high-performance chromatography diode array detectorelektrospray ionization tandem mass spectroscopy (UHPLC-DAD-ESI-MS/MS) method was used and chromatographical analysis was done on a UHPLC-3000 RS system. Separation was performed on a Zorbax SB-C18 column. To isolate polyphenols for which standards are not commercially available, preparative-HPLC separation was performed. For quantitative determination of polyphenols in the plant extracts, the UHPLC-DAD method was applied. To determine the calibration curve for quantification of active compounds, commercial and non-commercial standards were used.
Statistical analysis: The IC50 values were acquired using the SigmaPlot software for analysis from the viability test data by plotting standard dose-response curves. The data from the different experiments were coded and entered into IBM SPSS Statistics 22 statistical software. The statistical procedure employed included the mean ± standard deviation (SD) and a one-sample t-test to determine whether significant mean differences exist. The mean differences were considered significant at p ≤ 0.05. In toxicity study, all values were expressed as the mean ± SD and the results were analyzed statistically using an Independent Samples t-test and one-way Analysis of Variance (ANOVA), followed by Fisher’s Least Significant Difference (LSD) comparison tests using IBM SPSS Statistics 22 statistical software . The level of significance was considered at values p ≤ 0.05.
Results: The studied plant extracts and formulations presented a different degree of efficacy against chosen breast cancer cell lines, with the combination of CP + VA having the strongest synergistic ( CI < 1 ) and cytotoxic (IC50 = 18.6 ± 2.5 μg/ml ) activity against MCF-7, and additive cytotoxic activity (CI=1) against MDA-MB-231 (IC50 = 336.9 ± 13.6 μg/ml) amongst all tested drugs. A combination of CP + VA + DHA with CI >1 and IC50 values 78.6 ± 5.2, 149.3 ± 6.4 μg/ml against MCF-7 and MDA-MB-231 respectively, was included in the further study based on its possible multi-target mechanism of action. Viability of PBMCs was significantly higher when treated with CP + VA, CP + VA + DHA (Viability (%) = 99.4 ± 0.4, 90.3 ± 2.6 respectively) in comparison to cytotoxic activity of combinations against breast cancer cells. DOX showed higher cytotoxic activity on PBMCs (Viability (%) = 73.89 ± 1.30) when compared to the effect of tested combinations. In toxicity study using animal model, drugs administration up to 5 000 mg/kg did not cause mortality ( LD50 > 5 000mg/kg) and concentrations of 2 000 mg/kg (acute study) did not exhibit toxicity. There was no significant difference in body weight between the treated and control animals. Upon administration of the drugs at the highest concentration (1 000mg/kg), hematological and biochemical analysis presented significant differences in white blood count ( CP+VA: 7.8±2.0, control 13.9±4.8) and total protein values (CP+VA+DHA: 69.5±8.3, control 86.0±5.8) at the end of the 90 days period. The 0 Day control study exhibited significant differences in albumin (24.5±3.5,control 36.5±2.1) in animals treated with CP+VA and albumin (28.0±3.6, control 33.7±1.2) and Urea/Bun parameters (8.0±0.7, control 5.9±0.1) in rats treated with CP+VA+DHA at 1 000 mg/kg at the end of the sub-chronic study. Histopathological examination of the liver and kidney did not reveal organ alterations of treated rats. Qualitative and quantitative analysis of chosen botanicals revealed the presence of phenolic compounds that have been proven to have anticancer properties. A total of six compounds was tentatively identified in CP: caffeic acid derivative, quercetin dirhamnosyl-hexoside, kaempferol dirhamnosyl-hexoside, coumaric acid derivative, quercetin rhamnosyl-hexoside, ferulic acid derivative with quercetin dirhamnosyl-hexoside being in highest amount (1.9 mg/g ). In VA a total of seven compounds: 5-O- caffeoylquinic acid, luteolin hexoside, 3,4-O- dicaffeoylquinic acid, 1,5-O- dicaffeoylquinic acid, 3,5-O- dicaffeoylquinic acid,4,5-O- dicaffeoylquinic acid, luteolin dihexoside, were found, with 3,5-O- dicaffeoylquinic acid being most abundant (27.49 mg/g).
Conclusions and recommendations; The combination of CP + VA revealed the strongest cytotoxic and synergistic activity against MCF-7 and additive activity on MDA-MB-231 cell lines respectively and had a minimal cytotoxic effect on PBMCs compared to DOX. Despite CI >1 combination of CP+VA+DHA was included in the study based on its possible multi-target mechanism of action. The cytotoxicity of most efficacious formulations was found to be more selective towards cancer cells compared to DOX and generally non-toxic in rat models with regard to the effect on body weights and histopathology, however some haematological and biochemical parameters of tested rats showed significant changes. Additionally, UHPLC-MS analysis of Ugandan origin CP and VA provided new data on their phenolic composition giving new research perspectives on the possible mechanisms of their biological activity. It is recommended that the best treatment outcomes are further explored for development of an effective herbal therapy against breast cancer. | en_US |
