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

Man-Hai Liu,Bing-Huei Chen,Yi-Fen Li

doi:10.3390/plants10081664

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.

Highlights

Pomelo (Citrus grandis (Linn.) Osbeck), a vital plant possessing biological activities, is grown in more than 50 countries/regions, predominantly in China, southern Japan, Thailand, Vietnam, Indonesia, and the United States [1]
Many research groups have investigated the biological activities of chlorophyllin, a water-soluble derivative of chlorophylls shown to exert potent antimutagenic activity in prokaryotic and eukaryotic organisms in vitro [3,4] several in vivo experiments have revealed that chlorophyllin derived from rainbow trout could minimize DNA damage and liver cancer induced by aflatoxin B1 [5,6]
An adequate separation of chlorophyll b and chlorophyll a was attained with the retention time adequate separation of chlorophyll b and chlorophyll a was attained with the retention being 13.92 and 22.54 min and the retention factor of 3.35 and 6.04, respectively (Table 1)

Summary

Introduction

Pomelo (Citrus grandis (Linn.) Osbeck), a vital plant possessing biological activities, is grown in more than 50 countries/regions, predominantly in China, southern Japan, Thailand, Vietnam, Indonesia, and the United States [1]. The apoptotic pathways can be divided into intrinsic and extrinsic pathways, with the former mediated by the mitochondria and the latter by death receptors, while both pathways can lead to apoptosis via caspase activation. The intrinsic pathway can be initiated by tumor suppressors such as p53, a transcription factor activated by DNA damage, through stimulation of the expression of Bcl-2 family members such as Bax and Bad, resulting in a release of substances that reside between the inner and outer mitochondrial membranes such as cytochrome C and Smac/DIABLO. The death receptors Fas (CD95), tumor necrosis factor receptor (TNFR1 and TNFR2) and death ligands (FasL, TNF) on the cell membrane are activated when stimulated by the death signal, followed by binding of the death ligands to death receptors for formation of Fas-associated protein with death domain (FADD), leading to caspase-8 activation and formation of a death-inducing signaling complex (DISC)

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