Phytochemical analysis, antiproliferative and antioxidant activities of Chrozophora tinctoria: a natural dye plant

Abstract Context:Chrozophora tinctoria (L.) A. Juss. (Euphorbiaceae) is known as ‘dyer’s-croton’ and used to obtain dye substances. Recently, natural antioxidants and colorants have been of interest because of their safety and therapeutic effects. Objective: This study investigates the antiproliferative and antioxidant activities of the various extracts and fractions from C. tinctoria and analyzes their phytochemical contents. Materials and methods: The aerial parts of C. tinctoria were extracted with water, ethyl acetate, n-butanol, and methanol/chloroform. Phenolic compounds and other constituents of the extracts were analyzed by HPLC/TOF-MS. The ethyl acetate extract (EA) was fractionated by flash chromatography. The extracts, fractions, and major phenolic compounds were investigated for their antiproliferative activities on human cervical adenocarcinoma (HeLa) cell line at the concentrations of 5–100 μg/mL by using BrdU ELISA assay during 24 h of incubation. DPPH radical scavenging activities (5–150 μg/mL) and total phenolic contents of the samples were also evaluated. Results: 4-Hydroxybenzoic acid (268.20 mg/kg), apigenin-7-glucoside (133.34 mg/kg), and gallic acid (68.92 mg/kg) were the major components of EA. CT/E-F6 (IC50 = 64.59 ± 0.01 μg/mL) exhibited the highest antiproliferative activity. CT/E-F2 (IC50= 14.0 ± 0.0 μg/mL) and some fractions displayed higher radical scavenging activity compared to synthetic antioxidant BHT (IC50 = 23.1 ± 0.0 μg/mL). Among the main phenolics, gallic acid exhibited the highest antiproliferative and radical scavenging abilities (IC50 < 5 μg/mL). Conclusion: In this study, we have determined the biologically active fractions and their high effects may be attributed to the presence of gallic acid.

Plants have always played an important role as a source of drugs (Shakhatreh 2013). Recently, natural antioxidants and colorants present in foods have attracted interest because of their safety and potential nutritional and therapeutic effect (Esp ın et al. 2000). Several synthetic dyes have been banned because they cause allergy-like symptoms or are carcinogens. Moreover, naturally derived colorants are also used in the cosmetic industry due to lack of side effects, UV protection, and anti-aging properties (Chengaiah et al. 2010).
The bioassay guided fractionation of this plant species has not previously been reported. The aim of this study was (i) to evaluate antiproliferative activities of the various extracts against HeLa cell line by BrdU Elisa assay (ii) to analyze the phytochemicals of the extracts by HPLC-TOF/MS, (iii) to fractionate the active extract by flash chromatography, (iv) to investigate antiproliferative and antioxidant activities of these fractions by using complementary assays.

Plant materials
Chrozophora tinctoria materials were collected from Karali village (Elaz{ g-Turkey) in August 8, 2012 (20 km from Elaz{ g to Baskil, west of Karali village, fields, 1000-1200 m). The identification of plant materials were confirmed by taxonomist in the Department of Biology, Bingol University, Turkey. A voucher specimen (Behçet 8259b) was deposited at the Herbarium of the Biology Department, Bingol University, Turkey.

Preparation of plant extracts
The dried aerial parts of C. tinctoria were powdered in a mill. For water extraction, the plant material (50 g) was boiled with 1 L of distilled water for 2 h and filtered through Whatman No. 1 filter paper. The aqueous extract was subsequently extracted with ethyl acetate (EA) and n-butanol (n-BuOH). The plant residue was finally extracted with 1:1 (v/v) methanol/chloroform (MeCh). The organic layers of EA, n-BuOH and MeCh were concentrated under reduced pressure.

Fractionation of the ethyl acetate extract by flash chromatography
The ethyl acetate extract (10 g) was dissolved in methanol (20 mL, HPLC grade) and mixed with silica gel (10 g). The solvent was evaporated and the solid silica-extract mixture was subjected to flash chromatography column (Combiflash V R Rf, Teledyne Isco, Lincoln, NE) using hexane, CHCl 3 -hexane (1:1, v/ v) and methanol. Fractions were combined with TLC (using ethyl acetate-methanol solvent system (1:1, v/v)) visualized under UV light (254 nm) and cerium (IV) sulfate (2 g cerium (IV) sulfate was dissolved in 100 mL of 15% H 2 SO 4 ).

Antiproliferative effect by BrdU ELISA assay
The tested samples and 5-fluorouracil (5-FU; positive control) were dissolved in dimethyl sulfoxide (DMSO). Then the stock solution was diluted with Dulbecco's modified eagle medium (DMEM). DMSO concentration was below 0.1% in stock solutions. HeLa cell line was grown in DMEM supplemented with 10% of fetal bovine serum (FBS) and 2% of penicillin-streptomycin. The medium was changed twice a week.
The extracts, fractions, and major phenolic compounds were investigated for their antiproliferative activities against HeLa cell line by using BrdU ELISA assay (Ceyhan et al. 2013;Karakus et al. 2013). Cultured cells were grown in 96-well plates (COSTAR, Corning, NY) at a density of 3 Â 10 4 cells/well. In each experimental set, the cells were plated in triplicates and replicated twice. The cell lines were exposed to eight concentrations of the samples and 5-FU for 24 h at 37 C in a humidified atmosphere of 5% CO 2 . The cells were than incubated for overnight before applying the BrdU Cell proliferation ELISA assay reagent (Roche, Germany), according to manufacturer's procedure. The amount of cell proliferation was assessed by determining the A450 nm of the culture media after addition of the substrate solution by using a microplate reader (Awareness Chromate, Palm City, FL). Results were reported as percentage of the inhibition of cell proliferation, where the optical density measured from vehicle-treated cells was considered to be 100% of proliferation. All assays were repeated twice. Percentage of inhibition of cell proliferation was calculated as follows: (1-A treatments/A vehicle control) Â 100. Differences between groups were determined by ANOVA method (p < 0.01). IC 50 value was determined using ED50plus vol. 1 software (developed by Mario H. Vargas, Bethesda, MD).

DPPH radical scavenging assay
Free radical scavenging activity of the samples was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to method of Blois (1958). The sample solutions were added to 0.004% methanol solution of DPPH. The mixture was shaken vigorously and left to stand at room temperature for 30 min in the dark. The absorbance was measured at 517 nm against a blank by a spectrophotometer (Rayleigh UV-2601, BRAIC Co. Ltd., Beijing, China). Scavenging of DPPH radical was calculated according to the formula: BHT was used as a positive control.

Determination of total phenolic contents
Total phenolic contents of the extracts were determined using the modified Folin-Ciocalteu method as described by Singleton and Rossi (1965). The sample solutions were mixed with 0.2 mL of 50% Folin-Ciocalteu reagent and allowed to react for 3 min and 1 mL of aqueous solution of 2% Na 2 CO 3 was added. At the end of the 45 min incubation at room temperature, absorbance of each mixture was measured at 760 nm. The same procedure was also applied to the standard solutions of gallic acid. Total phenolic contents were expressed as lg gallic acid equivalents per mg of the fractions/extract.
For quantitative analysis, 22 standard components were analyzed in the extracts (Table 1). 4-Hydroxybenzoic acid (268.20 mg/kg), apigenin-7-glucoside (133.34 mg/kg), and gallic acid (68.92 mg/kg) were the major components of ethyl acetate extract. The major component of the water extract and BuOH extract was determined as gallic acid (13.98 and 31.12 mg/kg, respectively), while the major component of MeCh extract was determined as apigenin-7-glucoside (13.68 mg/kg). The maximum number of phenolic compounds was determined in EA (18), followed by BuOH (13) and water (9) extracts. All of the phenolic compounds except gentisic acid, chlorogenic acid and catechin were higher amounts in EA extract than the other extracts. On the other hand, naringenin, and protocatechuic acid ethyl ester were not found in all of the extracts. Hashim et al. (1990) found acacetin, luteolin and apigenin glycosides in Chrozophora species. Apigenin and quercetin glycosides (Noori 2012) and the acylated flavone glucosides have been reported from C. tinctoria (Delazar et al. 2006).

Antiproliferative effects of the samples from C. tinctoria
In this study, antiproliferative effects of the samples from C. tinctoria were examined on HeLa cell lines at the eight concentrations (5,10,20,30,40,50,75 and 100 lg/mL). EA extract exhibited higher antiproliferative effect than the BuOH extract at the concentration of 100 lg/mL (Figure 3). The potency of inhibitions (at 100 lg/mL) against HeLa cells were: 5-FU > EA extract > BuOH extract.
The antiproliferative effects of EA extract and the fractions that were collected as UV peak from the extract were examined on HeLa cell lines at the eight concentrations (5-100 lg/mL). CT/E-F6 (IC 50 ¼ 64.59 ± 0.01 lg/mL) had higher activity against HeLa cells compared to other fractions, EA extract and 5-FU (standard compound) ( Table 2). The potency of inhibition (at 100 lg/mL) against HeLa cells were: CT/E-F6 > 5-FU > CT/E- The antiproliferative effects of EA extract and fractions that did not have a UV peak from the extract, were examined on HeLa cell lines at the eight concentrations (5-100 lg/mL). The potency of inhibitions (at 100 lg/mL) against HeLa cells were: (Table 2).
Whereas no literature data were available for C. tinctoria, other plants of the genus Chrozophora were evaluated for their antiproliferative effects on human cell lines. Mothana et al. (2011) investigated the inhibitory effect of the methanol and hot aqueous extracts of Chrozophora oblongifolia (Delile) A.Juss. ex Spreng against human urinary bladder carcinoma (5637) and human breast cancer (MCF-7) cell lines. In that study, the hot aqueous extract exhibited moderate effect against 5637 cell line (IC 50 ¼ 298 ± 3 lg/mL).
4-Hydroxybenzoic acid (4-HBA), gallic acid (GA), and apigenin-7-glucoside (A-7-G) were found to be the main components of the EA. Therefore, the antiproliferative effects of these components of C. tinctoria were examined on HeLa cell lines. GA exhibited the highest activity against HeLa cells (IC 50 < 5 lg/mL) ( Table 4). The potency of inhibitions (at 100 lg/mL) against HeLa cells were found as GA> 5-FU > A-7-G> 4-HBA.
Some studies have been reported on the antiproliferative effects of the gallic acid and it was found that gallic acid had remarkable effects on some cancer cell lines including HeLa by inducing apoptosis and activating caspases (Ji et al. 2009;You et al. 2010You et al. , 2011. DPPH radical scavenging activity and total phenolic content of the samples from C. tinctoria DPPH radical scavenging activities of the EA extract, fractions, and BHT are given in Table 3. Lower IC 50 values indicate higher free radical scavenging activity. The highest free radical scavenging activity was obtained from CT/E-F2 with the lowest IC 50 value of 14.0 ± 0.0 lg/mL, followed by CT/E-F4 (16.2 ± 0.5 lg/mL) and CT/E-F6 (17.3 ± 0.1 lg/mL). CT/E-F2, CT/E-F4, CT/E-F6, CT/E-F7 (18.6 ± 0.4 lg/mL), CT/E-F8 (17.8 ± 0.4 lg/mL) and CT/ E-F5 (18.7 ± 0.0 lg/mL) exhibited higher radical scavenging activity than the reference compound BHT (IC 50 ¼ 23.1 ± 0.0 lg/mL). Figure 4 shows the dose-response curves of the DPPH radical scavenging activities of the fractions from C. tinctoria. DPPH radical scavenging abilities increase with the increased concentration of the samples. IC 50 value of EA extract was determined as 27.3 ± 0.4 lg/mL. Shahwar et al. (2010) investigated that DPPH radical scavenging activity of the four extracts (pethroleum ether, chloroform, ethyl acetate, methanol/n-butanol) from C. tinctoria and they observed the best activity in the ethyl acetate extract. This result was similar to that observed in our present study. Sharifi-Rad et al. (2015) observed a noticeable radical scavenging effect in the leaf methanol extract of C. tinctoria (IC 50 ¼ 26.4 ± 0.2 lg/mL).
4-Hydroxybenzoic acid, gallic acid, and apigenin-7-glucoside were the main components of EA. Therefore, the radical scavenging effects of these components were studied at the concentrations of 5-150 lg/mL. DPPH scavenging activity of the tested compounds decreased in the order of gallic acid > apigenin-7-glucoside >4-hydroxybenzoic acid (Table 4). Gallic acid exhibited very strong radical scavenging ability (IC 50 < 5 lg/mL). The radical-scavenging activities of phenolic acids depend on the number of hydroxyl moieties attached to the aromatic ring of the benzoic or cinnamic acid molecule (Karamac et al. 2005).
In the study of Karamac et al. (2005), the radical-scavenging activities of the phenolic acids were found in the order of gallic > gentisic > syringic > caffeic > protocatechuic > sinapic > ferulic > isoferulic > vanillic > p-coumaric > o-coumaric > m-coumaric > salicylic > p-hydroxybenzoic. Gallic acid, with three hydroxyl groups, was observed to be the most active phenolic acid. Zhao et al. (2015) reported that DPPH free radical scavenging activities of some compounds from Lavandula angustifolia Mill. (Lamiaceae) and they were determined the IC 50 value of radical scavenging activity of apigenin 7-O-b-D-glucoside as 103.42 lg/mL. In our study, IC 50 value of apigenin-7-glucoside was determined as 134.7 lg/mL.

Conclusions
In this study, we have successfully determined the biologically active fractions (CT/E-F2 and CT/E-F6) from ethyl acetate extract of C. tinctoria. Their high antiproliferative and antioxidant effects may be attributed to the presence of gallic acid. The results showed that C. tinctoria could be used as a natural source in food, cosmetics and pharmaceuticals industries. Further studies are needed to isolate and identify the compounds from these active fractions and also to evaluate in vivo biological activities of the isolated compounds.