Chemical composition, antioxidant activity and cytotoxicity on tumour cells of the essential oil from flowers of Magnolia grandiflora cultivated in Iran

Abstract Magnolia grandiflora (Magnoliaceae) is an evergreen tree with fragrant and showy flowers native to southeastern USA but widely cultivated all over the world and used in cosmetics industry in treatment of skin diseases. Here, we report on the chemical analysis of the essential oil obtained from flowers of plants cultivated in Iran, together with the evaluation of its antioxidant and cytotoxic activities. The essential oil composition was dominated by bioactive sesquiterpenes, namely β-elemene, bicyclogermacrene, germacrene D and (E)-caryophyllene. The oil exhibited moderate radical scavenging activity towards the radical, and mild non-selective inhibitory effects against A375, MDA-MB 231 and T98 G tumour cell lines. The latter were influenced by the presence of the anticancer β-elemene. These results provided new insights for potential application of M. grandiflora volatile oil in the pharmaceutical and cosmetics industry where only the non-volatile magnolol and honokiol have hitherto been fully exploited.


Introduction
The Magnolia family (Magnoliaceae), considered to be among the most primitive flowering plants, consists of 12 genera and over 200 species in the world (Glimn-Lacy and Kaufman, . Magnolia species have been used in Asian and North American traditional medicine for centuries and many of them have great economic importance as natural sources of aroma and bioactive compounds (Seth 2003;Schühly et al. 2009;Lee et al. 2011). Magnolia grandiflora L., commonly known as Southern Magnolia, is an evergreen tree with fragrant flowers, distributed throughout the areas of the south-eastern United States from Virginia south to central Florida, Texas and Oklahoma (Lim 2014). The species is widely cultivated in subtemperate areas across the world, as well as in Iran (Mozaffarian 2005;Lim 2014). M. grandiflora is used in traditional medicine as a stimulant, diaphoretic, anti-inflammatory and antiseptic agent as well as pain control and anxiety, and nervous disturbances (Squires et al. 1999;Khare 2008). Different parts of M. grandiflora contain biologically active components, such as alkaloids, sesquiterpene lactones, sesquiterpenoids, biphenyls, neolignans and volatile compounds (Hong et al. 2007;Schühly et al. 2009;Mohamed et al. 2010;Baez et al. 2012;Davé et al. 2012). Recent findings have demonstrated the biological properties of many identified components in M. grandiflora, including anti-tyrosinase , anti-inflammatory (Kim and Cho 2008;Feltenstein et al. 2004), anti-allergic (Niitsuma et al. 2001), antiviral (Lan et al. 2012), antimicrobial (Chang et al. 1998), cardioprotective (Lee et al. 2001;Ho and Hong 2012) and antitumour (Bai et al. 2003;Marin and Mansilla 2010;Chilampalli et al. 2011) activities. Currently, M. grandiflora is widely used in cosmetics because of its claimed anti-inflammatory and anti-acne effects, and most of the studies have been focused on the biological activity of its biphenols honokiol and magnolol (Shen et al. 2010;Mukherjee et al. 2011). However, further studies are needed on its essential oil in order to evaluate its potential application in pharmaceutics and cosmetics (Lim 2014;Raut and Karuppayil 2014). Odoriferous flowers of M. grandiflora are a valuable source of essential oil (Azuma et al. 1996;Baez et al. 2012;Davé et al. 2012). Essential oils are known to be comprised primarily of monoterpenoids and sesquiterpenoids that are formed by mevalonate and methyl erythritol phosphate (MEP) pathways and also, phenylpropanoids by the shikimate pathway (Lim 2014). Phytochemical studies on M. grandiflora demonstrated that the oil constituents belong to different chemical classes with a noteworthy qualitative and quantitative variation in composition (Azuma et al. 1997;Garg and Kumar 1999;Baez et al. 2012;Davé et al. 2012;Farag and Al-Mahdy 2013). β-Caryophyllene (Garg and Kumar 1999), cyclocolorenone, bicyclogermacrene, germacrene D, β-elemene (Davé et al. 2012), (E)-β-ocimene, geraniol and β-elemene (Baez et al. 2012) are the most reported constituents of M. grandiflora flowers essential oil. This variability could be due to the effects of genetic factors, environmental conditions, developmental stages of flowers, methods of extraction, etc. (Morshedloo et al. 2015).
Essential oils, as natural antioxidants, exhibited effective activity against cancerous cell proliferation in many studies and supposed to have potential anticancer activities for prevention and therapeutic strategies (Raut and Karuppayil 2014). A recent study on seed essential oil of M. grandiflora growing in China demonstrated that the oil has a strong antioxidant activity (Luo et al. 2012). M. grandiflora flowers' essential oil exhibited significant antioxidant activity against DPPH free radical and was active against human breast and lung carcinoma cell lines (Farag and Al-Mahdy 2013).
Many studies have reported that β-elemene, one of the main components of M. grandiflora essential oil, inhibits proliferation of tumour cells by inducing apoptosis and arresting cell cycle at G2/M stage (Yuan et al. 1999;Qian and Qin 1999;Zou et al. 2001;Wang et al. 2005;Li et al. 2009). According to clinical trials, β-elemene can be well tolerated by cancer patients with low toxicity and low rate of drug resistance development, making this compound an important chemotherapeutic agent (Li et al. 2009).
In the present study, the essential oil from M. grandiflora flowers cultivated in Iran was analysed to provide a more comprehensive profile of the chemical composition of floral scent. The study also evaluated the antioxidant activity and cytotoxicity on human tumour cells by the DPPH, ABTS, FRAP and MTT assays.
The major volatile component of M. grandiflora, β-elemene, is a natural sesquiterpene occurring in the rhizome of Curcuma species, which are widely used in the Traditional Chinese Medicine in the treatment of cardiovascular diseases and cancer (Lu et al. 2012).
Thus, our results proved that M. grandiflora exhibits a chemical polymorphism depending on the geographic origin of the samples.

Antioxidant activity
We analysed the total of phenol content in M. grandiflora essential oil, estimated as gallic acid equivalent and low values were observed in the oil (2.01 ± 0.4 mg/g, Table 2). Higher phenolic content is possible to obtain from M. grandiflora flower extracts that contain bioactive compounds, such as tannins and flavonoids . The antioxidant activity of the essential oil from flowers of M. grandiflora was reported in Table 2 and from the data we can observe that the oil exhibited different reactivity towards the different radicals used in the assays. The relatively stable nitrogen-centred free radical DPPH· is ubiquitously used to measure the scavenging ability of different phytochemicals, extracts or essential oils and their antioxidant effects on DPPH radical depend from their hydrogen donating ability. (Dastmalchi et al. 2007). Minor radical scavenging power was observed for M. grandiflora essential oil against DPPH radical (1270 times lower than Trolox). This negligible antioxidant activity could be due to the reduced ability of terpene compounds to donate a hydrogen atom as previously reported by Mata et al. (2007). Thus, the fact that the essential oil assayed contains high concentrations of sesquiterpene and monoterpene hydrocarbons (about 64%) could be responsible for the low DPPH reactivity observed. Although the DPPH· free radical is ubiquitously used to estimate the potential free radical scavenging activity of natural products, the ABTS ⋅+ free radical is commonly used when issues of solubility of interference arise and the use of DPPH-based assay becomes inappropriate (Dorman and Hiltunen 2004). The ABTS ⋅+ assay measures the scavenging of free radicals as the discolouration of the ABTS ⋅+ blue reactant. The decrease in ABTS ⋅+ concentration is linearly dependent on the antioxidant concentration, including Trolox as a calibration standard. As reported in Table  2, moderate antioxidant power resulted from the value of scavenging activity towards ABTS ⋅+ radical (TEAC = 95.02 ± 0.4 μmol TE/g), with a reactivity that was only 18 times less active of Trolox. Moreover, the M. grandiflora essential oil seems to act also as moderate reducing agent (FRAP activity, TEAC = 24.4 ± 2.3 μmol TE/g). The observed antioxidant activity of M. grandiflora essential oil is comparable to that reported for the essential oil isolated from  (Bajpai et al. 2009), or from the flower extract of M. grandiflora itself , Guerra-Boone et al. 2013. The antioxidant activity of the extract can be given by the presence of the biphenols honokiol and magnolol. Among the different essential oil components of M. grandiflora, β-elemene (14.33%) showed antioxidant activity and this property stimulated great interest on the antioxidant potential of β-elemene derivatives (Chen et al. 2014). Lower antioxidant activity was reported for bicyclogermacrene (10.33%) and germacrene D (7.59%) (Farag and Al-Mahdy 2013). Thus, the contribution of minor components present in the oil to the whole antioxidant activity observed is possible.

Cytotoxicity effects
The effects of M. grandiflora essential oil on the proliferative response of the A375, MDA-MB 231 and T98G cell lines have been analysed in vitro by treating the cells with different concentrations of the essential oil and significant decrease in cell lines proliferation was observed. The results of cytotoxic tests have been shown in Table 3. IC 50 values were about 35 μg/mL for all cell lines tested, indicating that the essential oil had no cytotoxic selectivity. The cytotoxic activity of the essential oil can be attributed to the presence of β-elemene, bicyclogermacrene, germarene D, β-pinene and (E)-caryophyllene. These compounds have been demonstrated to have cytotoxic activity against tumour cell lines (Venditti et al. 2013;Woguem et al. 2014;Capello et al. 2015). The same β-elemene, tested in our assay system, showed significant cytotoxic activity (Table 3). Notably, it was more active on A375 (IC 50 of 15 μg/mL) than T98G (20.26 μg/mL) and MDA-MB 231 (23.26 μg/mL) cells. β-Elemene is known for its anticancer activities exerted on different tumours, such as brain, liver, breast, lung and leukaemia in both in vitro and in vivo experiments. Its main mechanisms of action are the induction of cell cycle arrest and apoptosis, the inhibition of ROS through the function of anti-lipid oxidisation and suppression of telomerase activity. Furthermore, its effects are exploited to synergise the anticancer activity of conventional chemotherapeutic agents (Adio 2009).
Our results were not in accordance with those reported by Farag and Al-Mahdy (2013) on the cytotoxicity of M. grandiflora flower essential oil. In that study, the IC 50 values of the essential oil on A549 and MDA-MB 231 cells were higher than 200 μg/mL. We hypothesise that this difference in cytotoxicity may depend on the different chemical profile observed between the two oils.

Conclusions
The essential oil from flowers of M. grandiflora cultivated in Iran proved to be a rich source of bioactive sesquiterpenes, such as β-elemene, bicyclogermacrene, germacrene D and (E)-caryophyllene. These compounds are known as anti-inflammatory and anticancer agents. Furthermore, the essential oil was a moderate radical scavenger against the ABTS radical and showed cytotoxicity against skin-related ailments such as melanoma. On this basis, the M. grandiflora essential oil may be of interest for the pharmaceuticals and cosmetics industry beyond the non-volatile compounds honokiol and magnolol.