Preparation of Chlorophyll Nanoemulsion from Pomelo Leaves and Its Inhibition Effect on Melanoma Cells A375

Abstract

Pomelo (Citrus grandis), an important fruit crop grown in tropical and subtropical areas, is cultivated mainly in Asian countries. The dominant pigment in pomelo leaves, chlorophyll, has been reported to possess many biological activities such as antioxidant, anti-inflammation and anticancer. The objectives of this study were to determine chlorophylls in Pomelo leaves by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and to encapsulate the isolated chlorophylls from preparative column chromatography into a nanoemulsion system for elucidating the inhibition mechanism on the growth of melanoma cells A375. The results showed that chlorophyll a and chlorophyll b could be separated within 25 min by using a C18 column and a gradient ternary mobile phase of acetone, acetonitrile and methanol. Pomelo leaves mainly contained chlorophyll a (2278.3 μg/g) and chlorophyll b (785.8 μg/g). A highly stable chlorophyll nanoemulsion was prepared with the mean particle size being 13.2 nm as determined by a dynamic light scattering (DLS) method. The encapsulation efficiency of chlorophyll nanoemulsion was 99%, while the zeta potential was −64.4 mV. In addition, the chlorophyll nanoemulsion possessed high thermal stability up to 100 °C and remained stable over a 90-day storage period at 4 °C. Western blot analysis revealed that chlorophyll nanoemulsion and extract could upregulate p53, p21, cyclin B and cyclin A as well as downregulate CDK1 and CDK2 in a concentration-dependent manner for inhibition of melanoma cells A375. Furthermore, chlorophyll nanoemulsion and extract could upregulate Bax and cytochrome C and downregulate Bcl-2, leading to activation of caspase-9, caspase-8 and caspase-3 for the induction of cell apoptosis. Compared to chlorophyll extract, chlorophyll nanoemulsion was more effective in inhibiting the growth of melanoma cells A375.

Inhibition of Melanoma Cells A375 by Carotenoid Extract and Nanoemulsion Prepared from Pomelo Leaves

“…In this study, DMSO was used for cell culture, while both lecithin and Tween 80 were used for the preparation of CNs. To avoid interference of DMSO, lecithin and Tween 80 on the growth of melanoma cells A375 and fibroblast cells CCD-986SK, the tolerance of cells toward DMSO and blank nanoemulsion needs to be studied [ 22 ]. Based on a previous study by Liu, Li and Chen [ 22 ], the dose of DMSO and blank nanoemulsion was controlled at 1% to maintain the high viability of CCD-986SK and A375 cells for subsequent experiments.…”

“…All the materials dealing with cell culture including human fibroblast cells (CCD986SK) and human melanoma cells (A375), as well as reagents, media and caspase assay kits, were the same as described in a previous study [ 22 ].…”

“…A375 cells (5 × 10 6 /well) were seeded in a 6-well plate and cultured for 24 h. After cell adhesion, the medium was removed, different concentrations (5, 10 and 20 µg/mL) of CEs and CNs were added and incubated for 48 h. Then the cell-cycle distribution including the proportions of sub-G1, G0/G1, S and G2/M phases was analyzed by a flow cytometer following the same procedure as described in a previous study [ 22 ].…”

“…Initially, a 10-cm plate seeded with 5×10 5 cells was cultured for 24 h and subsequently, the medium was removed, washed with PBS and the medium replaced with 5, 10 and 20 µg/mL of CEs and CNs separately. Following the same procedure, as described in a previous study [ 22 ], the expressions of CDK1, CDK2, cyclin A, cyclin B, cytochrome-C, Bax, Bcl-2, p21 and p53 were determined.…”

“…A fluorometric assay-based kit was employed for the determination of caspase-3, caspase-8 and caspase-9 activities following the same procedures as described in a previous study [ 22 ].…”