Thiotagetin A, a new cytotoxic thiophene from Tagetes minuta

Abstract Phytochemical investigation of the n-hexane fraction of the methanolic extract of Tagetes minuta L. (Asteraceae) aerial parts afforded a new thiophene derivative: thiotagetin A (3), together with β-sitosterol (1) and stigmasterol (2). The structure of the new thiophene was identified by UV, IR, 1D (1H and 13C), 2D (1H–1H COSY, HSQC and HMBC) NMR and HRESIMS spectral data. Compound 3 displayed cytotoxic activity against KB and MCF7 cancer cell lines with ED50 values of 2.03 and 3.88 μg/mL, respectively, compared to adriamycin (0.26 and 0.07 μg/mL, respectively).


Introduction
Thiophenes which represent a unique class of natural products, are heterocyclic aromatic compounds based on a five-membered ring, containing one sulphur and four carbon atoms. They are commonly found in Asteraceae (Ibrahim et al. 2016).
They often exhibit a wide variety of biological activities such as antimicrobial, antiviral, HIV-1 protease inhibitor, antileishmanial, nematicidal, insecticidal, phototoxic and anticancer (Ibrahim et al. 2016). The genus Tagetes includes about 56 species (Soule 1993). Several species of this genus have been investigated as possible sources of different chemical compounds with high pharmaceutical and nutritional values (Vasudevan et al. 1997;Shahzadi et al. 2010). Tagetes minuta L. commonly known as marigold, is widely grown from temperate to tropical regions of the world in a wider range of climatic conditions (Shahzadi & Shah 2015). It has been used as a remedy for several medical aliments such as colds, respiratory inflammations, stomach problems, catarrh, wounds, cuts, calluses and bunions (Gillij et al. 2008;Rahimi et al. 2010;Maity et al. 2011). Moreover, it showed a wide range of biological properties, including α-amylase inhibitor, antimicrobial, anti-spasmodic, anti-parasitic, anti-septic, insecticidal, sedative, anti-inflammatory and acaricidal (Tereschuk et al. 1997;Shahzadi et al. 2010;Andreotti et al. 2013;Ibrahim et al. 2015). Previous chemical investigations of T. minuta growing in Saudi Arabia revealed the presence of thiophenes and flavonoids (Al-Musayeib et al. 2014;Ibrahim et al. 2015). In the present study, a new thiophene derivative: thiotagetin A (3), along with β-sitosterol (1) and stigmasterol (2) were isolated from the aerial parts of T. minuta ( Figure 1). Herein, the isolation and structure elucidation as well as cytotoxic activity of compound 3 have been reported.
The known compounds were identified on the basis of co-TLC with authentic samples, as well as analysis of the spectroscopic data and comparison of their data with those in the literature to be: β-sitosterol (1) (Mohamed & Ibrahim 2007) and stigmasterol (2) (Mohamed et al. 2009).
In the current study, the cytotoxic effect of the new thiophene derivative 3 was evaluated, using MTT assay towards KB (epidermoid carcinoma) and MCF7 (hormone-dependent breast carcinoma) cancer cell lines.
Several thiophenes have been found to exhibit cytotoxic effects against a wide range of human cancer cell lines (Ibrahim et al. 2016). Cancer chemoprevention by the use of natural agents that can reverse, suppress or prevent carcinogenic progression, has become an appealing strategy to combat the dogma associated with increasing cancers cases worldwide (Tsao et al. 2004). Cytotoxicity assay is an appropriate method for screening new substances within a short time in order to determine the cytotoxicity on the cancer cells (Alley et al. 1988). MTT assay has been described as rapid, simple, and reproducible method, which is widely used in the screening of anticancer drugs and to measure the cytotoxic properties (Ferrari et al. 1990).
MCF-7 was established as the most commonly used breast cancer cell line in the world in 1973 at the Michigan Cancer Foundation (Soule et al. 1973). Its popularity is largely due to its exquisite hormone sensitivity through expression of oestrogen receptor (ER), making it an ideal model to study hormone response (Levenson & Jordan 1997). While, KB cell line is a human oral epithelial-like cell most commonly used as a model system in studies of biological activity of antineoplastic agents (DiRienzo et al. 2002). Yen et al. (1994) reported that these cells are sensitive or supersensitive in the presence of antineoplastic agents (Yen et al. 1994).
It is of interest that compound 3 was found to display potent and moderate activity towards KB (ED 50 2.03 μg/mL) and MCF7 (ED 50 3.88 μg/mL) cancer cell lines, respectively compared to adriamycin (0.26 and 0.07 μg/mL, respectively).
Hence, thiophene derivatives could be a rich source of potential entities in search of new generation of anticancer compounds. However, the inconsistence of their in vivo activities needs further systemic study to be evaluated. Also, more studies need to emphasise onto their therapeutic potential by providing new insights into their molecular mechanisms for cancer prevention to optimise the efficacy and safety of this series of compounds.

Plant material
T. minuta L. aerial parts were collected in March 2014 from Al-Baha city, Saudi Arabia. The identification of the plant was achieved on the basis of its morphological features and the library database (Collenette 1999). It was confirmed by Dr Nahed Morad, Faculty of Science, King Abdulaziz University, Saudi Arabia. A voucher specimen (TM-11-2014) was archived at the Department of Natural Products and Alternative Medicine herbarium, King Abdulaziz University, Saudi Arabia.

Conclusion
A new thiophene derivative (3), along with two known compounds (1 and 2) have been isolated from the aerial parts of T. minuta. Their structures were determined on the basis of extensive spectroscopic data analysis. Compound 3 displayed cytotoxic activity towards KB and MCF7 cancer cell lines.

Supplementary material
The general experimental procedures, extraction and isolation of compounds, and cytotoxicity assay, as well as NMR spectra of compound 3 are available as Supplementary material.

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