BackgroundCitrus sinensis peels are usually discarded as wastes; however, they are rich sources of Vitamin C, fibre, and many nutrients, including phenolics and flavonoids which are also good antioxidant agents. This study aimed to examine phytochemical composition and antioxidant capabilities of C. sinensis peel extracted conventionally with different methanol/water, ethanol/water, and acetone/water solvents.MethodsC. sinensis peels were subjected to extraction with 100%, 70% and 50% of methanol, ethanol, and acetone, respectively, as well as hot water extraction. Antioxidant activities of the peel extracts were examined via the 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity, ferric reducing antioxidant power (FRAP) assay, and oxygen radical absorbance capacity (ORAC) assay. Total phenolic content and total flavonoid content of the extracts were measured via the Folin-Ciocalteau method and the aluminium chloride colorimetric method, respectively. Phenolic acid and organic acid composition of the peel extracts were further determined via high performance liquid chromatography (HPLC) while flavonoid content was identified via ultra performance liquid chromatography (UPLC).ResultsDPPH radical scavenging activity of C. sinensis peel extracts varied from 8.35 to 18.20 mg TE/g, FRAP ranged from 95.00 to 296.61 mmol Fe(II)/g, while ORAC value ranged from 0.31 to 0.92 mol TE/g. Significant level of association between the assays was observed especially between TPC and FRAP (R-square = 0.95, P < 0.0001). TPC of various C. sinensis peel extracts ranged from 12.08 to 38.24 mg GAE/g, with 70% acetone/water extract (AEC) showing the highest TPC. TFC ranged from 1.90 to 5.51 mg CE/g. Extraction yield ranged from 0.33 to 0.54 g/g DW and tended to increase with increasing water concentration in the solvent. In the phytochemical investigation, five phenolic acids were identified using HPLC, including gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, caffeic acid and ferulic acid. A total of five organic acids including lactic acid, citric acid, L-mallic acid, kojic acid and ascorbic acid were quantified via HPLC. In addition, concentrations of six flavonoids including catechin, epigallocatechin, vitexin, rutin, luteolin and apigenin were determined via UPLC.Discussion and ConclusionPhytochemicals including phenolics and flavonoids in C. sinensis peel extracts exhibited good antioxidant properties. Among the extracts, 70% AEC with highest TPC and high TFC content showed greatest antioxidant activity in all three assays. Different phenolic acids, organic acids and flavonoids were also identified from the extracts. This study indicated that C. sinensis peels contained potential antioxidant compounds which could be exploited as value added products in the food industry.

Elephantopus scaber Linn, a traditional herb, exhibited anticancer properties, and it was cytotoxic against the monolayer estrogen receptor-positive breast cancer cell line, MCF-7, in the previous study. In order to determine the potential of E. scaber as a complementary medicine for breast cancer, this study aimed to evaluate the synergism between E. scaber and tamoxifen in cytotoxicity using MCF-7 in the form of 3-dimensional multicellular tumor spheroid (MCTS) cultures. MCTS represents a more reliable model for studying drug penetration as compared to monolayer cells due to its greater resemblance to solid tumor. Combination of E. scaber ethanol extract and tamoxifen, which were used in concentrations lower than their respective IC50 values, had successfully induced apoptosis on MCTS in this study. The combinatorial treatment showed >58% increase of lactate dehydrogenase release in cell media, cell cycle arrest at the S phase, and 1.3 fold increase in depolarization of mitochondrial membrane potential. The treated MCTS also experienced DNA fragmentation; this had been quantified by TUNEL-positive assay, which showed >64% increase in DNA damaged cells. Higher externalization of phospatidylserine and distorted and disintegrated spheroids stained by acridine orange/propidium iodide showed that the cell death was mainly due to apoptosis. Further exploration showed that the combinatorial treatment elevated caspases-8 and 9 activities involving both extrinsic and intrinsic pathways of apoptosis. The treatment also upregulated the expression of proapoptotic gene HSP 105 and downregulated the expression of prosurvival genes such as c-Jun, ICAM1, and VEGF. In conclusion, these results suggested that the coupling of E. scaber to low concentration of tamoxifen showed synergism in cytotoxicity and reducing drug resistance in estrogen receptor-positive breast cancer.