Investigation of in vitro cytotoxic, mutagenic and anti-mutagenic effects of shirazolide extracted from Jurinea leptoloba

Abstract Shirazolide is an elemanolide isolated from Jurinea leptoloba DC. The aim of this study was to determine the cytotoxic, mutagenic and anti-mutagenic properties of shirazolide from J. leptoloba DC in vitro. Cytotoxicity was measured using a modified MTT (3-(4,5-di methyl thiazol-2-yl)-2,5-di phenyltetrazolium bromide) assay on normal human lymphocytes and tumor HeLa cells, showing that the cytotoxicity of shirazolide is much higher for HeLa cells than for normal lymphocytes. Mutagenic and anti-mutagenic activities of shirazolide were evaluated using the Salmonella typhimurium tester strains TA98 and TA100 showing anti-mutagenic properties against the former strain under metabolic activation.


Introduction
The genus Jurinea (tribe Cynareae, subtribe Carduinea) with about 250 species is distributed in Asia and Europe (Rustaiyan & Ganji 1988). Several species of Jurinea have been investigated chemically, and the presence of sesquiterpenes, especially elemanolides and melampolides, were reported . The extract of the aerial parts of Jurinea leptoloba DC., afforded, in addition to several germacranolides albicolide, pectorolide, salonitenolide, jurinelloide and its derivatives, four melampolides, two elemanolides, glucopyranoside and dihydrosyringenin . The less polar fractions of the extract of J. leptoloba afforded an elemanolide, namely shirazolide identified by the characteristic signals of protons of two vinylic groups in the 1 H NMR spectrum ( Figure 1) . Several studies in vitro have shown the inhibitory activity of sesquiterpene lactones against cells derived from human carcinoma (Fernandes et al. 2008). Chemical studies showed that the cytotoxic activity is due to the reaction of α,β-unsaturated carbonyl structures of the sesquiterpene lactones with thiols such as cysteine (Fernandes et al. 2008). The structure of shirazolide is valued for two reasons. First, shirazolide can undergo a Cope rearrangement due to a 1,5-diene unit. Secondly, as α,β-unsaturated carbonyl group it may undergo many nucleophilic reactions. Bioassay screening studies have shown that the majority of the sesquiterpene lactones can be highly cytotoxic toward human cancer cells (Fischedick et al. 2013). Hence, the sesquiterpene lactone shirazolide with some unsaturated groups can be a candidate as anticancer or antimutagen agent. Previously, shirazolide and other phytochemicals from J. leptoloba have not been characterised with respect to its biological, toxicological and pharmacological activities. Therefore, the aims of this study were to determine in vitro the anticancer, mutagenic and antimutagenic effects of shirazolide extracted from J. leptoloba.

Cytotoxicity assay
The cytotoxic effects of shirazolide were tested using the human tumor cell line (HeLa) and lymphocyte cells by a modified MTT assay (Table S1). At a concentration of 28 μg/mL, shirazolide inactivated HeLa cells by 82%, with a 50% cytotoxicity concentration of 2.8 μg/mL (y = 1.2706x + 46.44, r 2 = 0.99). Shirazolide was toxic to HeLa cells at very low concentrations.
On the other hand, at a concentration of 5600 μg/mL, shirazolide inactivated lymphocytes by 40% (y = 0.0027x + 25.154, r 2 = 0.987). The IC 50 values for HeLa and lymphocyte cells were obtained to be 2.80 and 9202.22 μg/mL, respectively (Table S1). Thus, shirazolide exerted a highly significant cytotoxic effect on the HeLa. The obtained results suggest that the cytotoxicity of shirazolide on human cancer cells is much higher than that seen in normal human lymphocytes. Depending on type and concentration of natural products, they exhibit cytotoxic effects on living cells but are usually non-genotoxic (Bakkali et al. 2008). The unique structures of natural products cannot be obtained easily by chemical synthesis. Interest in medicinal plant investigation has increased in recent years, especially for the treatment of cancer (Bayala et al. 2014). Cancer chemo-prevention is defined as the use of chemicals or dietary components to block, inhibit, or reverse the development of cancer in normal or pre-neoplastic tissue. Some studies have been pointed that sesquiterpene lactones inhibit tumor growth by selective alkylation of growth-regulatory biological macromolecules, such as DNA and key enzymes, which control cell division, thereby inhibiting a variety of cellular functions, which leads cells into apoptotic death (Fernandes et al. 2008;Rozenblat et al. 2008). Sesquiterpene lactones caused cell-cycle arrest at G 2 /M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. These changes explain the ability of the sesquiterpene lactones to induce G 2 /M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that sesquiterpene lactones might be developed as new agents for melanoma treatment (Rozenblat et al. 2008). A large number of potential chemo-preventive agents have been identified, and they function by mechanisms directed to all major stages of carcinogenesis (Hou et al. 2007). Based on previous studies, two new elemanolides, epivernodalol and lasiopulide, were isolated from alcoholic extract of Vernonia lasiopus (koul et al. 2003). Both isolated elemanolides were shown in vitro cytotoxicity against human cancer cell lines in culture (koul et al. 2003).

Mutagenicity and antimutagenicity test
The concentration of shirazolide (1.2 mg/plate) was selected based on a preliminary toxicity test. The colonies were counted to determine the mutagenic and antimutagenic potencies of this compound. Mutagenesis and antimutagenesis effects of shirazolide against Salmonella typhimurium strains TA98 and TA100, with (+S9) and without (−S9) metabolization was determined and listed in Table S2. Shirazolide showed antimutagenic effects at the concentration of 1.2 mg/plate by the bacterial reverse mutation assay against chemical mutagens in S. typhimurium strains. On the other hand, mutagenesis experiments showed that shirazolide has moderate mutagenic activity in the absence of metabolic activation S9. The Ames test is used to detect mutagenic and anti-mutagens activities and is a widely accepted method for identifying different chemicals and drugs that can cause gene mutations (Maron & Ames 1983). There is a relationship between mutagenesis and carcinogenesis. According to the obtained results, the best conditions for the appearance of antimutagenic activity was observed with shirazolide at 1.2 mg/plate against 2-aminoanthracene-induced mutagenicity in strain S. typhimurium TA98 in the presence of metabolic activation (S9). In the presence of chemical mutagens, shirazolide showed resistance against all the used mutagens in both strains of S. typhimurium TA100 and TA98, but with different percentages. It showed better resistance against sodium azide and 2-aminoantracene in both strains of S. typhimurium TA100 and TA98. From the mentioned results, it can be concluded that the antimutagenic activity of shirazolide in the presence of metabolic activation was higher than its mutagenic activity. To our knowledge, there was no research carried out on the mutagenic and antimutagenic effects of shirazolide before.

Conclusion
In the present study, shirazolide was found to be particularly cytotoxic to HeLa cells. Despite the limitations of all in vitro investigations with respect to in vivo impact, the obtained results are very promising as far as antineoplastic chemotherapy is concerned. This appears to be a good basis for more detailed future studies. The results obtained with human cells and with S. typhimurium suggest that shirazolide may be exploited as a natural anticancer and antimutagenic agent.