Studies on chemical constituents and anti-hepatoma effects of essential oil from Annona squamosa L. pericarps

Abstract Annona squamosa L. fruit played great anti-neoplastic activities. Its pericarps were discarded as waste. In this study, essential oil extracted from A. squamosa L. pericarps (APEO) was obtained by hydrodistillation and analysed by GC–MS. Furthermore, the anti-hepatoma activities and the underlying mechanism of the oil were firstly described. A total of 59 compounds were identified by Gas chromatography–mass spectrometry (GC–MS). The major compound in the oil was (−)-spathulenol (32.51%). The APEO demonstrated anti-hepatoma activity against SMMC-7721 hepatoma cell line with IC50 lower than 55 μg/mL. At the same time, nucleus shrinkage or broken were found in cells incubated with APEO through fluorescent microscope. In addition, pro-apoptosis and cell cycle arrest effects were confirmed by flow cytometry analysis.


Introduction
Hepatocellular carcinoma (HCC) is now the third leading cause of cancer deaths worldwide (Kessler et al. 2015). Among different treatments, chemotherapy is still the important means to control HCC (Zou et al. 2015), while the cardio and renal toxicities of chemotherapy affect patient's health seriously (Kintzel 2001;Minotti 2010). Currently, there is growing interest on natural anti-tumour materials, which prevent the proliferation of tumours.
The genus Annona is an important genus of the Annonaceae family and various researches on plants in this genus have been conducted (Lage et al. 2014;Rout & Kar 2014;Santos Pimenta et al. 2014;Orozco-Castillo et al. 2016). Custard apple (Annona squamosa Linn.), one of Annona species, is a famous and popular fruit. Its seed, one kind of folk medicine, is widely used to treat 'malignant sore' (cancer) in southern China (Miao et al. 2016). Pericarps of the fruit are always discarded as waste. Previous studies have shown the anti-neoplastic activity of pericarps (Joy & Remani 2008). Essential oil is the important component from natural products (Lukas et al. 2015;Ghribi et al. 2016;Zeng et al. 2016). Previous researches focused on essential oil from leaves and barks of A. squamosa L. (Chavan et al. 2006;Thang et al. 2013;Meira et al. 2015). However, there is no report with chemical constituents and the anti-neoplastic activity of APEO. This work aims to identify the chemical constituents of APEO, investigate the anti-proliferation effect on SMMC-7721 cells and explore the underlying mechanism of action in vitro. The biological activities are researched for the first time.

Gas chromatography-mass spectrometry analysis of APEO
The crude APEO was light yellow with a yield of 0.26% (w/w). Upon Gas chromatographymass spectrometry (GC-MS) analysis, lots of peaks appeared on total ion chromatogram. Fifty-nine compounds were identified. The sesquiterpenes were more abundant in the APEO. (−)-spathulenol, one of the sesquiterpenes, nearly accounted for one-third (32.51%) of the oil. n-Hexadecanoic acid accounted for 9.41%.

Cell viability
The human SMMC-7721hepatoma cells, seeded in 96-well plate, were incubated with different doses of the essential oil. 48 h later, the cytotoxicity was assessed by MTT assay. The cell viabilities were relative to oil concentrations, with IC 50 value of 54.81 μg/mL. When oil concentration was 3.1 μg/mL, the inhibition ratio was still more than 40% (43.3%).

Apoptosis analysis
The significant apoptosis was found in fluorescent microscope assay (Figure 1). In the control group, every cell nucleus was uniform on cell size and fluorescence intensity. Amidst cells incubated with APEO, the condensed bright and broken nuclei were readily observed. The presence of apoptotic cells in medicated groups was further confirmed by flow cytometry. The results indicated that the number of apoptotic cells in the medicated group increased significantly compared with that of the control group. APEO caused dose-dependent apoptosis.

Cell phase distribution analysis
The cell phase distribution assay showed that SMMC-7721 cells treated with APEO were arrested at G1 phase pronouncedly. Furthermore, dramatic reductions in the S phase and G2 phase populations were observed. 74.76% of cells were arrested after treatment with 40 μg/mL APEO.

Conclusion
The essential oil was extracted by hydrodistillation and identified by GC-MS analysis. It showed the obvious anti-tumour activity against SMCC-7721 cells. Pro-apoptosis and cell cycle arrest were observed in fluorescent microscope and flow cytometry assays. Fifty-nine compounds were identified from the APEO. (−)-spathulenol was the most ample compound in the sesquiterpenes, which was first isolated from plants in 1976 (Juell et al. 1976). (−)-spathulenol was a common ingredient in essential oil of different plants (Manzo et al. 2015;Rezaie et al. 2015;Liu et al. 2016;Xiao et al. 2016). All the essential oil have similar activities, such as anti-tumour and anti-bacterial effects (Manzo et al. 2015;Moricz et al. 2015). Little research on activities of (−)-spathulenol has been reported until now. According to the study in this article, (−)-spathulenol might be one of the possible active compounds in APEO. Further studies are ongoing in order to undiscover active compounds and the pro-apoptosis signal pathways.

Disclosure statement
No potential conflict of interest was reported by the authors.