Cytotoxic Effect of Some Digitalis Species; a Study of Selectivity
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FABAD J. Pharm. Sci., 43(1), 25-29, 2018 RESEARCH ARTICLE Cytotoxic Effect of Some Digitalis Species; A Study of Selectivity Vahap Murat KUTLUAY *°, İclal SARAÇOĞLU* Cytotoxic Effect of Some Digitalis Species; A Study of Bazı Digitalis Türlerinin Sitotoksik Etkisi; Bir Selektivite Selectivity Çalışması SUMMARY ÖZET Our previous research, on the aerial parts and roots of three Daha önceki çalışmamızda üç farklı Digitalis türünün (D.davisiana different Digitalis species; D.davisiana Heywood, D.viridiflora Heywood, D.viridiflora Lindley ve D.grandiflora Miller) toprak üstü Lindley and D.grandiflora Miller against HEp-2 and HepG2 kısımlarının ve köklerinin HEp-2 ve HepG2 kanser hücre serileri üzerinde, cancer cell lines showed that aerial parts were more cytotoxic than toprak üstü kısımların köklere göre daha sitotoksik olduğu gösterilmiştir. roots. So for further research a comparative cytotoxic activity study Bu nedenle D.davisiana, D.viridiflora ve D.grandiflora’nın toprak üstü was performed on the aerial parts of D.davisiana, D.viridiflora kısımların üzerinde gerçekleştirilen araştırma ilerletilerek HeLa kanser and D.grandiflora against HeLa cancer cell line. 3Y1 non- hücresi üzerindeki sitotoksik etkisi incelenmiştir. Sağlıklı bir hücre cancerous cell line was used to determine the selectivity. All three olan 3Y1, selektivitenin değerlendirilebilmesi için kullanılmıştır. Her üç ekstre de HeLa hücreleri üzerinde kuvvetli sitotoksik etki göstermiş extracts showed strong cytotoxic activity on HeLa cells with IC50 values less than 15 μg/ml. However, observed cytotoxic activity on ve IC50 değerleri 15 μg/ml’nin altında hesaplanmıştır. Ekstrelerin 3Y1 hücreleri üzerinde sitotoksik etkileri daha düşük olmakla birlikte 3Y1 cells was found to be lower than that of HeLa cells with IC50 values in a range of 94.7-772.3 μg/ml. This finding indicated IC50 değerleri 94.7-772.3 μg/ml olarak hesaplanmıştır. Elde edilen bu that different Digitalis extracts showed a selective cytotoxicity bulgular Digitalis ekstrelerinin HeLa kanser hücresi üzerinde selektif against HeLa cancer cell line. sitotoksik etkili olduğunu göstermektedir. Key Words: Digitalis species, cytotoxicity, HeLa cell line, 3Y1 cell Anahtar Kelimeler: Digitalis türleri, sitotoksisite, HeLa hücre line, MTT method. serisi, 3Y1 hücre serisi, MTT metodu. Received: 09.11.2017 Revised: 24.12.2017 Accepted: 26.12.2017 * Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey ° Corresponding Author; Vahap Murat KUTLUAY 25 Kutluay, Saraçoğlu INTRODUCTION MATERIALS AND METHODS In the Flora of Turkey, the genus Digitalis Plant Material (Plantaginaceae) is represented by nine species(Davis, Voucher specimens for all plants were deposited in 1978). The genus Digitalis has been moved from Hacettepe University Faculty of Pharmacy Herbarium Scrophulariaceae family to Plantaginaceae family (HUEF). D.davisiana Heywood was collected due to the recent chemotaxonomic and phylogenetic from Antalya, Turkey (HUEF11004) in June, 2011. studies. Digitalis species are known as foxglove. Even D.grandiflora Miller (HUEF13007) and D.viridiflora there is no report for use of foxglove as a folk medicine, Lindley (HUEF13008) were collected from Kırklareli, Digitalis species have been used in treatment of Turkey in July, 2013. D.davisiana was identified by diseases since 18th century for its cardiac glycosides. Prof. Dr. Hayri Duman (Gazi University Faculty The first use of foxglove as a medicine was in 1785 of Science and Literature Department of Biology, by Withering (Goldman, 2001). Withering observed Ankara, Turkey), D.grandiflora and D.viridiflora his patients and showed that as foxglove is a toxic were identified by Dr. Z. Ceren Arıtuluk (Hacettepe plant, therapeutic index is very close. Foxglove show University Faculty of Pharmacy Department of main effect on heart and kidneys (Baytop, 1999). This Pharmaceutical Botany, Ankara, Turkey). While effect is due to the increase of sodium levels in cell D.davisiana is endemic to the south coast of Turkey, by the inhibition of Na-K ATPase pump (Katz, 1985). D.viridiflora and D.grandiflora are endemic species Foxglove also has diuretic effect depending on the for Balkans. dose (Navarro et al., 2000). Digitalis species contain Cell Lines biologically active compounds such as cardenolides, HeLa (human cervix epithelial cancer cell) cell line phenylethanoid glycosides, flavonoids, andwas provided by Prof. Dr. Nursen Basaran’s cell line anthraquinones (Ganapaty et al., 2003). Especially, collection (Hacettepe University, Faculty of Pharmacy, D.lanata and D.purpurea are very important for Department of Pharmaceutical Toxicology, Ankara), their cardioactive glycosides (Mijatovic et al., 2007). 3Y1 (rat fibroblast cell) cell were provided by the Cardiac glycosides have positive inotropic effect Health Science Research Resources Bank (Osaka, so that they are source of important drugs used for Japan). the treatment of cardiac insufficiency (Navarro Chemicals et al., 2000; Fuerstenwerth, 2014). Except these biological activities foxglove also has emetic, antiviral, Fetal bovine serum (FBS), and a minimal essential antibacterial, antifungal, cytotoxic, antiinflammatory medium with Earle’s salts (MEM‐Earle) and antibiotics and antioxidant activities (Warr et al., 1992; Lopez- (penicillin and streptomycin) were obtained from Lazaro et al., 2003; Oh et al., 2005; Benli et al., 2009; Biochrom AG (Berlin, Germany). Trypsin/EDTA Orhan et al., 2009; Jin et al., 2011). solution was purchased from Merck (Darmstadt, Germany). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- Natural products have an important role in cancer diphenyl tetrazolium bromide] was purchased from therapy. To determine cytotoxic activity is the first Sigma (St Louis, USA). step for the new anticancer drug discovery. Cytotoxic activity studies on cardiac glycosides and Digitalis Extraction extracts show promising results (Zhou et al., 1998; The same extraction method was used for all tested Lopez-Lazaro et al., 2003; Fujino et al., 2015). plants. Aerial parts of the dried plants powdered and were extracted with methanol four times. Extracts Our previous research, on the aerial parts and roots were dried by evaporating under vacuum at 40°C, of three differentDigitalis species; Digitalis davisiana and were freezedried to remove solvent residue. Heywood, D.viridiflora Lindley and D.grandiflora Then, they were dissolved in water and partitioned Miller against HEp-2 (Human larynx epidermoid with petroleum ether to discard lipophylic fractions. carcinoma) and HepG2 (Human hepatocelular Aquoeus extracts were used for cytotoxic activity carcinoma) cancer cell lines showed that aerial parts tests. were more cytotoxic than roots (Kutluay et al., 2014). So for further research a comparative cytotoxic activity Selective cytotoxic activity was determined study was performed on the aerial parts of D.davisiana, through the HeLa cancer cell line and 3Y1 D.viridiflora and D.grandiflora against HeLa (human noncancerous cell line by MTT method. Two cell lines cervix epithelial cancer cell) cell line. 3Y1 (rat fibroblast were treated with each extract in the concentration cell) cell line was used to determine the selectivity. Use range of 1-800 μg/ml. of noncancerous cell line with cancer cell line help to Cytotoxic Activity determine selectivity among different cells. Selectivity The modified version of Mosmann’s MTT between different cancer cells and non-cancerous cells [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl helps to point out drug targeting. tetrazolium bromide] method was used to determine 26 FABAD J. Pharm. Sci., 43(1), 25-29, 2018 cytotoxic activity in our research. HeLa and 3Y1 cell RESULTS AND DISCUSSION lines were used for cytotoxic activity tests. Cells were The cytotoxic activity of D.davisiana, D.grandiflora 4 seeded in a 96-well plate at a density of 3x10 cells/ml and D.viridiflora extracts tested against HeLa cell 4 for HeLa cells and 5x10 cells/ml for 3Y1 cells. MEM’s line at the concentrations between 1-400 μg/ml. The Earle medium were used for the culture. Media were results showed that all extracts have concentration supplemented with 10 % FBS, 100 U/mL penicillin dependent strong cytotoxic activity against HeLa cell and 100 μg/mL streptomycin. Cells were cultured in line (Figure 1). For D.davisiana and D.grandiflora respective culture media in a humidified 5 % CO2 in air extracts, cell viability is under 50 % even at 5 μg/ml. at 37°C for 24 hours, then were treated with different All the tested extracts showed high cytotoxicity at the concentrations of samples for the next 48 hours. After concentrations higher than 25 μg/ml. Cell viability is incubation, the cells were washed to discard sample under 10 % at high concentrations (Figure 1). solutions and replaced by 100 μl of fresh medium. D.davisiana and D.viridiflora extracts caused to 10 μl of MTT solution (5mg/ml in PBS) was added increase HeLa cell number when exposed to 1 μg/ and incubated for 4 hours. After that, 100 μl of 10 % ml concentration. This result is supported by the SDS (Sodium dodecyl sulphate) was added to each findings of Ramirez-Ortega et al. (2006). It was shown well to dissolve formazan crystals. The absorbance that digoxin and digoxigenin isolated from Digitalis was measured at 570/620 nm using microplate reader. species cause an increase in HeLa cell number when Results were given as percentage of inhibitory effect exposed lower than 10 nM concentration however treated cells to untreated cells