Chemical composition and cytotoxic activity of Garcinia atroviridis Griff. ex T. Anders. essential oils in combination with tamoxifen

Abstract Garcinia atroviridis Griff. ex T. Anders. is used as a medication agent in folkloric medicine. The present study was to examine the chemical composition of the stem bark and leaf of G. atroviridis as well as their cytotoxic effects against MCF-7 cells. The constituents obtained by hydrodistillation were identified using GC-MS. The stem bark oil (EO-SB) composed mainly the palmitoleic acid (51.9%) and palmitic acid (21.9%), while the leaf oil (EO-L) was dominated by (E)-β-farnesene (58.5%) and β-caryophyllene (16.9%). Treatment of MCF-7 cells using EO-L (100 μg/mL) caused more than 50% cell death while EO-SB did not induce cytotoxic effect. EO-L has stimulated the growth of BEAS-2B normal cells, but not in MCF-7 cancerous cells. The IC50 of EO-L in MCF-7 and BEAS-2B cells were 71 and 95 μg/mL, respectively. A combination treatment of EO-L and tamoxifen induced more cell death than the treatment with drug alone at lower doses.


Introduction
The genus Garcinia is the biggest genus in the family of Clusiaceae, with over 400 species in the tropics of Africa and Asia, and about 50 species found in the lowland and mountains of Peninsular Malaysia (Corner 1952;Pangsuban et al. 2009). The various plant parts (fruits, leaves, flowers, stem and bark) of Garcinia species have been used to treat abdominal pain, dysentery, diarrhoea, suppuration, infections, leucorrhoea, chronic ulcer and gonorrhoea in traditional ways (Jamila et al. 2016). Garcinia atroviridis Griff. ex T. Anders. is a medium-sized fruit tree found wild in the forest of Peninsular Malaysia (Tan et al. 2013). In folkloric medicine, G. atroviridis has been used as a postpartum medication agent to treat earache, throat irritation, cough, dandruff and some stomach ache associated with pregnancy (Tan et al. 2013).
Human breast cancers are mainly dependent on oestrogens, via interaction with specific oestrogen receptors (ERs), in which about 70% breast cancers are classified as ER positive. In regard to the mechanisms of breast carcinogenesis, oestrogens are involved in the differentiation and proliferation of normal epithelial cells of the breast, via the alpha and beta ERs. The ability of activated ERs to induce cell proliferation is central to its roles in breast cancer (Renoir et al. 2013;Yue et al. 2013). Chemotherapy is the mainstream method of breast cancer treatment, where, tamoxifen is the most-prescribed anti-oestrogen therapy for treating invasive and non-invasive ER-positive patients (Obiorah and Jordan 2011;Jordan 2014). Despite its great efficaciousness, the long-term use of chemotherapy can lead to a number of undesired adverse side effects and drug resistance via several different mechanisms; therefore, improved treatment strategies of chemotherapeutic agents with synthetic or natural origins are urgently needed. In this study, we report the chemical composition of the stem bark and leaf of G. atroviridis as there is no information regarding their constituents in the literature. At the same time, the cytotoxic effect of the volatiles was reported for the first time. Table 1 lists the constituents identified in the stem bark and leaf of G. atroviridis Griff. ex T. Anders., their relative peak areas and the experimental retention indices on the HP-5 column. In total, fourteen compounds, constituting 90.4 and 97.1% of the sample by peak area %, were identified in the total volatiles of the stem bark and the leaf, respectively. Fatty acid was the dominant chemical class in the stem bark oil (EO-SB), accounting for 79.8% of the sample, although this figure was largely due to palmitoleic acid (51.9%) and palmitic acid (21.9%). With regard to the leaf oil (EO-L), it yielded an oil rich in sesquiterpenes (86.3%), dominating by (E)-β-farnesene (58.5%) and β-caryophyllene (16.9%) ( Figure S1).

Composition of essential oils
Comparison of the composition of the EO-SB and EO-L, it showed marked differences in the constituents present. Palmitoleic acid, an unsaturated fatty acid, is found in high content in the EO-SB of G. atroviridis. It is being previously reported in the fatty acid composition from the mesocarps of Hippophae rhamnoides, constituted mainly palmitoleic acid (47.8%) and palmitic acid (29.3%) (Cakir 2004). On the other hand, the EO-L was dominated by (E)β-farnesene. It is an acyclic sesquiterpene olefin, often involved in chemical communication, particularly in the avoidance of predation in aphids (Su et al. 2015). Results of the present study showed that the EO-L of G. atroviridis contained a much higher amount of (E)-β-farnesene compared to that of Nepalese chamomile oil (42.2%) (Satyal et al. 2015), rhizomes oil in Alpinia galanga (8.4%), (Raina et al. 2014) and leaf oil in Tephrosia densiflora (3.6%) (Noudogbessi et al. 2012). In our previous study, we have demonstrated that the volatile constituents of G. atroviridis fruits was comprised of mainly sesquiterpenoids (Tan et al. 2013). It is interesting that the volatile constituents of the fruits are markedly different from those in the stem bark while resembled those identified in the leaf (Tan et al. 2013).

Cytotoxic activity
The EO-L and EO-SB of G. atroviridis were tested in vitro using an MTT assay to explore their potential inhibitory effect on the growth of MCF-7 human breast cancer cell line. The growth-inhibitory effects of EO-L and EO-SB within 24 h and up to 72 h of incubation period were measured. After 24-72 h post-treatment, 10-100 μg/mL concentrations of EO-L induced evidently anti-proliferative effects. Treatment of MCF-7 cells at the highest concentrations of EO-L (100 μg/mL) caused more than 50% cell death as early as 24 h. At this concentration, EO-L also caused the lowest cell viability with 16%. As shown in Figure S2A, EO-L treatment exhibited its anti-proliferative effect in MCF-7 cells in a dose-and time-dependent manner. On the contrary, treatment of MCF-7 cells with EO-SB ( Figure S2B) did not induce pronounced cytotoxic effect. The highest level of cytotoxicity noted was less than 30% at a concentration of 100 μg/mL, and remain plateau throughout the experiment.
According to a study conducted by Afoulous et al. (2013), the essential oil of Cedrelopsis grevei leaves which constituted mainly (E)-β-farnesene (27.67%) was shown to be active against the cell line MCF-7.On the other hand, β-caryophyllene, one of the major constituents of essential oils, was shown to exhibit moderate cytotoxic effect against MCF-7 (Dahham et al. 2015). However, the reduction of MCF-7 cell line proliferation is increased when β-caryophyllene is used with its isomers than when used separately (Fidyt et al. 2016). In our results, (E)-β-farnesene and β-caryophyllene were found to be 58.5 and 16.9%, respectively, and the cytotoxic activity of the EO-L might be due to the synergic effects of the sesquiterpenes in the oil.
In this study, the constant IC 50 value of EO-L was graphically established based on the dose-response curves obtained from each incubation period. The IC 50 values obtained following EO-L treatment of MCF-7 cells is shown in Figure S3A. In MCF-7 cells, the curve was decreased against the treatment duration, which eventually reached a constant effect and produced a constant IC 50 value of 71 μg/mL. In comparison to MCF-7 cancerous cells, the IC 50 curve of EO-L exponentially increased against the treatment duration in BEAS-2B lung normal cells, which produced an IC 50 value of 95 μg/mL ( Figure S3B), indicating EO-L had stimulated the growth of BEAS-2B lung normal cells, but not in MCF-7 breast cancerous cells.
Tamoxifen, by actions, binds to the ER and blocks oestrogen, resulting in growth inhibition of the cancerous cells (Lu et al. 2012). In this study, MCF-7 cells were further treated with EO-L (10-100 μg/mL), tamoxifen (10-100 μM) and the combination of EO-L with the tamoxifen for 24, 48 and 72 h of incubation period. As shown in Figures S4A-S4C, tamoxifen causes dose-and time-dependent cytotoxicity where maximum cytotoxic effect (more than 90% cell death, p < 0.001) occurred at concentration of 50-100 μM. Tamoxifen alone at 10 μM concentration, did not elicit any significant inhibitory effects in MCF-7 cells, while about 10-30% (p < 0.05) of growth inhibition was observed at 25 μM concentration within 24 h post-treatment. Combination treatment (EO-L + tamoxifen) induced more cell death than treatment with drug alone, particularly at lower doses (10-25 μM). Combined treatment of tamoxifen at 10 μM with EO-L (10 μg/mL) had induced 30-46% (p < 0.05) of growth inhibition within 72 h post-treatment. Similarly, treatment with 25 μM tamoxifen in the presence of EO-L killed about 60-88% (p < 0.01) of MCF-7 cells within 72 h post-treatment. These data demonstrate that EO-L co-treatment reduces tamoxifen dosage to inhibit the growth of MCF-7 cells, compared to the tamoxifen alone. CompuSyn analysis ( Figure S5) further showed a significant synergy for 10-25 μg/mL of EO-L in combination with ≤ 25 μM tamoxifen, which consistently showed CI values < 1.

Conclusions
In conclusion, the EO-L from G. atroviridis, constituted mainly (E)-β-farnesene was found to show anti-proliferative effect on MCF-7 cells. This study has convinced that EO-L is more potent than EO-SB in inhibiting the growth of MCF-7 ER-positive breast cancer cell line in time-dependent and concentration-dependent profiles. It is worthy to note that this is the first study to report the essential oils from the leaf of G. atroviridis which enhanced the anti-proliferative effect of the lower dosage of tamoxifen, and hence could be considered as a potential chemotherapeutic agent in breast cancer.

Supplementary material
Supplementary materials relating to this article are available online.

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