Natural Product Research Formerly Natural Product Letters

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Chemical composition, cytotoxicity and molecular profiling of africana Lam. on human breast cancer cell line

Abeer M. Ashmawy, Iriny M. Ayoub & Omayma A. Eldahshan

To cite this article: Abeer M. Ashmawy, Iriny M. Ayoub & Omayma A. Eldahshan (2020): Chemical composition, cytotoxicity and molecular profiling of Cordia￿africana Lam. on human breast cancer cell line, Natural Product Research, DOI: 10.1080/14786419.2020.1736064 To link to this article: https://doi.org/10.1080/14786419.2020.1736064

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SHORT COMMUNICATION Chemical composition, cytotoxicity and molecular profiling of Cordia africana Lam. on human breast cancer cell line

Abeer M. Ashmawya, Iriny M. Ayoubb and Omayma A. Eldahshanb aCancer Biology Department, Biochemistry Unit, National Cancer Institute, Cairo University, Cairo, Egypt; bFaculty of Pharmacy, Pharmacognosy Department, Ain Shams University, Cairo, Egypt

ABSTRACT ARTICLE HISTORY Metabolic profiling of the hexane extract and essential oil Received 4 January 2020 of Cordia africana Lam., an economically important agroforestry Accepted 13 February 2020 species, was performed using GC-FID and GC-MS. Cytotoxicity was assessed in MCF-7 cancer cells using sulphorhodamine-B assay. KEYWORDS Cordia africana Caspase-8, BcL-2 and Ki-67 levels were evaluated using ; cytotoxicity; MCF-7; BcL-2; Ki-67; enzyme-linked immunosorbent assay. Forty-one compounds were caspase-8; apoptosis identified in the hexane extract, meanwhile, eighty compounds were annotated in the essential oil. n-Decane (9.52%), b-sitosterol (8.13%), undecane (6.32%) and palmitaldehyde (5.50 %) were the major compounds identified in the hexane extract, whereas, b-caryophyllene (32.0%), germacrene D (10.12%) and d-cadinene (7.20%) predominated in the essential oil. The half maximal inhibi- tory concentrations (IC50) of the extract and oil were 4.55 mg/mL and 12.90 mg/mL, respectively. Caspase-8 activity increased signifi- cantly in hexane extract treated cells. Furthermore, BcL-2 and Ki- 67 protein expression levels were significantly decreased. Cordia africana Lam exhibit potent cytotoxicity against breast cancer cell line, most likely through apoptosis regulation.

CONTACT Omayma A. Eldahshan [email protected] Faculty of Pharmacy, Pharmacognosy Department, Ain Shams University, African Union Organization Street, Cairo 11566, Egypt. Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1736064. ß 2020 Informa UK Limited, trading as Taylor & Francis Group 2 A. M. ASHMAWY ET AL.

1. Introduction

Globally, cancer is considered to be the second leading cause of death (Fitzmaurice et al. 2017). Breast cancer is the most common cancer among women. According to the World Health Organization, 2.1 million women are diagnosed with breast cancer every year. Moreover, breast cancer is responsible for the high- est rates of cancer deaths among women. In 2018, it was estimated that about 627,000 women died from breast cancer representing 15% of all cancer-related deaths among women (WHO 2018). Apoptosis is considered a promising target for anticancer therapy (Pfeffer and Singh 2018). Caspases are cysteine proteases that act as key mediators of apoptosis (Thornberry 1998). In cases of apoptotic cell death, caspases are activated resulting in breakdown of critical cellular substrates triggering dramatic morphological changes normally associated with apoptosis (Bratton et al. 2000). Caspase-dependent apoptosis follows intrinsic and extrinsic pathways. The intrinsic pathway arises as a consequence of DNA damage or cellular stress i.e., a signal originating within the cell. In contrast, the extrinsic pathway is elicited by stimulation of cell surface receptors, known as “death receptors” (Kominami et al. 2012). In cancer, the apoptotic pathway is inhibited through the overexpression of antiapoptotic proteins and the under-expression of proapoptotic proteins (Pfeffer and Singh 2018). Nature combats cancer by its medicinal . Naturally occurring secondary metabolites from plants offer promising new anticancer therapies that exhibit anticancer activity by activating the apoptotic pathway (Pfeffer and Singh 2018). Researchers have identified a myriad of extracts, essential oils and phytochemicals possessing promising anticancer properties (Eldahshan 2013; Ayoub et al. 2014; Al-Sayed et al. 2016; Taha and Eldahshan 2017;Elkadyand Ayoub 2018;Ashmawyetal.2019). Cordia africana Lam. (syn.: C. holstii Gurke)€ is an evergreen tree belonging to the family () (Gebreegziabher 2016). It is an important agroforestry species native to Eastern and Southern Africa (Schmidt and Mwaura 2010; Derero et al. 2011). C. africana is more valued for timber than medicinal purposes, represent- ing an economically important species (Balemie and Kebebew 2006; Regassa 2013). In folk medicine, C. africana is used for the treatment of migraine, broken bones, wounds, gastritis and constipation (Zenebe et al. 2012). Reported pharmacological activities include antibacterial (Geyid et al. 2005), anti-inflammatory and cytotoxic activities (Isa et al. 2014). Despite its applications in traditional medicine, little is known on the phytochemical profile and biological activities of C. africana. Scientific evidence addressing the cellular and molecular mechanisms is lacking. Only few reports were traced for the pharmacological activities of C. africana including antibac- terial (Geyid et al. 2005), antifungal (Moshi et al. 2007), anti-inflammatory and cytotoxic activities (Isa et al. 2014). In this study, GC-MS based metabolic profiling and cytotox- icity of C. africana leaf essential oil and hexane extract were performed. Effects on the expression levels of the molecular biomarkers caspase-8, Bcl-2 and Ki-67 were assessed to unveil the possible underlying mechanisms. To the best of our knowledge, this is the first report on the chemical profile and biological activity of the essential oil and hexane fraction of C. africana leaves. NATURAL PRODUCT RESEARCH 3

2. Results and discussion 2.1. Chemical composition of the hexane extract and essential oil

Hydrodistillation of the leaves yielded 0.016 % (w/w), colorless essential oil having a characteristic odor. Forty-one compounds were identified in the hexane extract, accounting for 70.03% of total composition. Meanwhile, 80 compounds were identified in the leaf essential oil, representing 89.45% of total oil composition. Hydrocarbons and phytosterols predominated in the hexane extract, with n-decane (9.52%), b-sitosterol (8.13%), undecane (6.32%), palmitaldehyde (5.5%), nonane (5.24%) and phytol (5.20%) representing the major constituents identified. However, sesquiterpene hydrocarbons predominated in the leaf essential oil including b-caryophyllene (32.0%), germacrene D (10.12%), d-cadinene (7.20%) in addition to phytol (5.27%). (Figures S1 and S2, Tables S1 and S2, Supplementary material).

2.2. Cytotoxicity and apoptosis

2.2.1. Determination of half maximal inhibitory concentration (IC50) Both the hexane extract and essential oil showed promising anti-proliferative activity exhibiting IC50 values of 4.55 and 12.90 mg/mL, respectively. Cytotoxicity of CA-HE was comparable to that of doxorubicin (IC50 3.83 mg/mL). Consequently, the possible underlying mechanism for the observed cytotoxic activity of the more potent CA-HE was investigated.

2.2.2. Apoptosis and proliferation Caspase-8 enzymatic activity was significantly increased in MCF-7 cells treated with

CA-HE at a dose equivalent to the IC50 value (4.55 mg/mL) (Figure S3, Supplementary material). Meanwhile, BcL-2 protein expression was significantly decreased (Figure S4, Supplementary material). Moreover, expression of the proliferative marker (Ki-67) protein expression level in MCF-7 cells was significantly reduced compared to the untreated group, P < 0.05 (Figure S5, Supplementary material). Hydrocarbons and phytosterols dominated in C. africana hexane extract (CA-HE). Phytol, an acyclic monounsaturated diterpene alcohol, induced significant anticancer activity against breast cancer at IC50 125 lg/mL (Sheeja et al. 2016). Moreover, it induced concentration-dependent cytotoxic response in many cancerous cell lines, being the most effective against the breast adenocarcinoma MCF-7 with IC50 of 8.79 mM (Pejin et al. 2014). Phytosterols possess anti-cancer effects against estrogen-dependent human breast cancer cell lines (Ju et al. 2004). Phytosterols inhibit the production of carcinogens, cancer-cell growth, invasion, metastasis, and promotes apoptosis of cancerous cells (Meric et al. 2006). b-Sitosterol treatment of breast cancer cells at 16 mM has been observed to reduce the cell growth by 50% (Awad et al. 2007). It increased activity of caspase-3 of human endothelial cells in vitro by more than 60% (Rubis et al. 2008). This could be explained through down regulation of Bcl-2. Major components of CA-EO identified include a and b-caryophyllene, germacrene D, d-cadinene and phytol. Caryophyllene significantly reduced tumors developed from orthotopically grafted colon cancer cells into nude mice (Fidyt et al. 2016). d-Cadinene 4 A. M. ASHMAWY ET AL. induced both dose and time-dependent growth inhibitory effects on OVACR-3 cell line. It induced apoptosis through chromatin condensation, cell shrinkage and rupture of nuclear membrane. In addition, it led to cell cycle arrest in sub-G1 phase (Hui et al. 2015). Results were comparable to previous studies on the essential oil composition of the leaves of various Cordia species. The essential oil of the leaves of C. leucocephala from northeast of Brazil was rich in sesquiterpenes with b-caryophyllene and bicyclogerma- crene as the major constituents identified (Diniz et al. 2008). In the same context, the essential oil composition of C. trichotoma from the northeast of Brazil showed high abundance of sesquiterpenes being dominated by a-cadinol, a-muurolol, epi- a-muurolol, and d-cadinene (de Menezes et al. 2005). The essential oil of C. verbenacea leaves from Venezuela was dominated by tricyclene, bicyclogermacrene, germacrene D and b-caryophyllene (Meccia et al. 2009). Meanwhile, the leaf essential oil of C. sebes- tena from Nigeria was dominated by oxygenated acyclic compounds and aliphatic hydrocarbons (15.5%) in addition to squalene and phytol (Adeosun et al. 2015). Various fractions and extracts of C. africana different parts (leaves, bark, stem, and fruit) were previously evaluated using brine shrimp lethality test. The methanolic extract of the bark and the chloroform fraction of the leaves exhibited potent cytotoxicity showing LD50 values of 0.0000414 and 15.653 lg/mL (Alhadi et al. 2015). In order to further assess the probable signaling pathways and the underlying molecular mechanism of the promising extract, the expression levels of caspase-8, BcL-2 protein level, and proliferative marker Ki-67 level were evaluated. Caspase-8, a 55 kDa protein, is involved in extrinsic apoptotic pathway. It plays a vital role in all physiological disorders in which apoptosis is primarily involved (Abdel-Salam et al. 2018). Bcl-2, the anti-apoptotic member of Bcl-2 family, prevents apoptosis either by sequestering caspases or by inhibiting the release of mitochondrial apoptogenic factors into the cytoplasm (Tsujimoto 1998). Ki-67 expression is strongly correlated with tumor cell proliferation and growth. It is widely used as a proliferation marker in routine pathological investigations (Li et al. 2015). In view of the above results, it can be concluded that CA-HE exerted its cytotoxic effect through apoptosis induction verified by the activation of pro-apoptotic caspase-8 and downregulation of anti-apoptotic Bcl-2 molecular biomarker. Moreover, anti-proliferative effect was confirmed by significant inhibition of the proliferative marker Ki-67 in MCF-7 compared to untreated cells.

3. Experimental

See Supplementary material.

4. Conclusions

The chemical profile of C. africana leaves hexane extract and essential oil was described, herein. CA-HE and CA-EO suppressed cell viability of MCF-7 human cancer cells. CA-HE induced apoptosis via significant elevation in pro-apoptotic caspase 8 expression level and a significant inhibition of anti-apoptotic Bcl-2 protein. Moreover, CA-HE suppressed proliferation of MCF-7 cells through downregulation of the proliferative marker Ki-67. These results provide insight on the potential role of CA-HE as a promising cytotoxic agent. NATURAL PRODUCT RESEARCH 5

Disclosure statement

The authors declare that there is no conflict of interest relevant to this study.

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