Introduction

Echinacea is a perennial plant belonging to the Asteraceae family. Three out of nine species, Echinacea purpurea (L.) Moench, E. angustifolia DC., and E. pallida (Nutt.) Nutt. (EP), of the genus are currently used in therapy for their medicinal properties (McGregor, 1968). Echinacea has a long history of medicinal use for the treatment of the common cold and respiratory infections (Kindscher, 1989; Li, 1998; Chen et al., 2005; Barnes et al., 2005; Barrett, 2003). A variety of chemical compounds have been isolated and identified from the Echinacea genus, including caffeic acid derivatives (Cheminat et al., 1988; Pellati et al., 2004; Pellati et al., 2005), alkamides, acetylenes (polyacetylenes and polyenes) (Bauer et al., 1988a, b; Bauer and Remiger, 1989; Pellati et al., 2007), polysaccharides (Wagner et al., 1988), and glycoproteins (Classen et al., 2000; Thude and Classen, 2005), all of which exhibit diverse pharmacological activities. The in vitro cytotoxic and pro-apoptotic activities of the hexane root extracts from the three medicinally important Echinacea species, E. pallida, E. purpurea, and E. angustifolia, were reported (Bauer et al., 1988a, b; Bauer and Remiger, 1989; Pellati et al., 2007).

Although cytotoxic effects of E. pallida root extracted with hexane on several tumor cell lines (the human pancreatic cancer, PaCa-2 and colon cancer, COLO320 cell lines) were observed (Chicca et al., 2007), the antiproliferative effect of E. pallida root methanolic extracts (crude extract) and isolated active compounds against HeLa and C6 were not studied so far. In the present study, the antiproliferative activity of E. pallida root methanolic extract and isolated active compounds against HeLa and C6 cells were investigated.

Materials and methods

Collection of E. pallida

The plant was collected from Tokat province, identified by Dr. Oya Kacar (Uludag University), and cultivated in medicinal plants garden of Field Crops Department in Gaziosmanpasa University, during 2007–2009 vegetation periods. The roots were harvested from cultured E. pallida and dried at room condition (at 25 °C) and used for the extraction. All chemicals used were of reagent or higher grade.

Extraction and isolation of compounds

The roots were cut into small pieces and extracted successively with methanol (2.5 L) for 3 times at room temperature. The extracts were filtered through Whatman No: 2 filter paper and vacuum dried. The extract was subjected to silica gel column chromatography, affording 187 fractions. Each fraction was analyzed by TLC and GC–MS, and combined into 20 fractions according to their chromatographic profile. From these fractions, Compounds 1 and 2 (Fig. 1) were isolated by silica gel mesh 60 (0.063–0.200 mm, Merck) column chromatography and preparative TLC. The chemical structure of the compounds was determined on the basis of NMR and MS spectroscopic data.

Fig. 1
figure 1

Structures of pentadeca-(9E, 13Z)-dien-11-yn-2-one (1) and (E)-1,8-pentadecadiene (2) isolated from E. pallida roots

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Spectral analysis

Official methods were used to determine volatile part of the column chromatography by GC–MS on a Perkin Elmer Clarus 500 equipped with BPX 20 capillary column (0.25 μm ID 30 m × 250 μm), filled with 5 % Phenyl polysilphenylene-siloxane, at an ionization voltage of 70 eV. Helium was the carrier gas (1 ml min−1). The injector and detector temperatures were kept at 100 °C for 5 min and then gradually increased to 250 °C at a 5 °C/min rate, and held for 15 min. Diluted samples (1/100, v/v, in n-pentane) of 1.0 μl were injected. The hydrocarbons were identified using the molecular formula and fragmentations. The high-resolution NMR spectra (1H and 13C) were run on a Brucker Avence III spectrometer (400 MHz) and J values are given in Hz.

Cell culture and cell proliferation assay

HeLa and C6 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM, Sigma), supplemented with 10 % (v/v) fetal bovine serum (Sigma, Germany) and PenStrep solution (Sigma, Germany). Cultured cells were detached from the flasks with trypsin–EDTA (Sigma, Germany) at confluency, centrifuged, and pellet resuspended to 3 × 105 cells ml−1 in DMEM. Cells were plated in 96-well plates (COSTAR, Corning, USA) at a density of 30,000 cells/well and incubated at 37 °C with 5 % CO2 overnight for attachment. All materials including test and controls were dissolved in sterile DMSO. In each experimental set, cells were plated in triplicates and the experiment was repeated three times (n = 3). The cells were treated with crude extract and compounds were isolated at final concentrations of 5, 10, 20, 30, 40, 50, 75, and 100 μg ml−1. Controls, vehicle controls, and positive control wells were treated with culture medium, sterile DMSO, and cisplatin, respectively. Treated cells were incubated at 37 °C with 5 % CO2 for 24 h.

Cell proliferation was measured using BrdU Cell Proliferation ELISA (Roche, Germany), a colorimetric immunoassay based on BrdU incorporation into the cellular DNA according to manufacturer’s procedure. Briefly, cells were pulsed with BrdU labeling reagent for 4 h followed by fixation in FixDenat solution for 30 min at room temperature. Thereafter, cells were incubated with 1:100 dilution of anti-BrdU-POD for 1.30 h at room temperature. Finally, the immune reaction was detected by adding the substrate solution and the color developed was read at 450 nm with a microplate reader.

Statistical analysis

The results of investigation in vitro are mean ± SD of three separate experiments. Differences between groups were tested by analysis of variance (ANOVA). P values of less than 0.05 were considered statistically significant. The mean data which are significant in variance analysis were grouped with Duncan’s multiple range tests (Gomez and Gomez, 1984). All statistical analysis was performed using SPSS (Version 13.5).

Results

Isolation and characterization of active compounds from E. pallida root extract

In the present study, the fractionation of the crude methanol extract of E. pallida roots by a series of silica gel column chromatographic steps and preparative TLC resulted in the isolation of two bioactive compounds, Compound 1 and 2 (Fig. 1), which are known compounds. Compound 1 has been reported before (Pellati et al., 2006) and Compound 2 has been reported by Voaden and Jacobson (1972) but in the literature its spectral data are missing. The structure of Compound 1 was confirmed by comparison with the literature data (Pellati et al., 2006). The NMR data of the molecules (Table 1) were in perfect agreement with the structures. The compounds isolated were identified from their spectroscopic data, such as GC–MS, NMR, including (1H and 13C) and 2D (COSY, DEPT, TOCSY) techniques. According to 1H-NMR spectra of Compound 1 (Fig. 2a), methyl protons which connected with carbonyl groups were showed as singlet signals at 2.15 ppm. At 2.44 ppm, triplet signals belong to methylene protons which connected with C-3. H-13 protons were determined as doublet signals at 5.47 ppm and the protons interacted with H-14 protons as cis (J 13-14 = 10.6 Hz). In DEPT-90, four CH groups which belonged to C-8, C-9, C-13, and C-14 carbons were determined at 131.4, 124.5, 110.3, and 137.2 ppm, respectively (Fig. 2b). The DEPT-45 spectra of Compound 1 supported the presence of CH, CH2, and CH3 groups (Fig. 2b). However, at APT spectra of Compound 1 CH3 groups were exhibited as negative peaks (Fig. 2b). In 13C-NMR spectra of the compounds (Fig. 2c), the signals which belonged to carbonyl groups were obtained at 209.2 ppm. The peak carbons of C-8 and C-9 were observed at 131.4 and 124.5 ppm, respectively. The peaks which belonged to C-12 and C-11 (at 76.7, 92.9 ppm, respectively) concern with the triple bond carbons. In COSY spectra, the corelations of H-13 with H-14 were determined (at 5.47 and 5.91 ppm, respectively) (Fig. 2d). In addition to TOCSY spectra of the compounds, the correlations of H-6 with H-7 (1.40 and 2.09 ppm, respectively) were determined (at 5.47 and 5.91 ppm, respectively) (Fig. 2e).

Table 1 1H and 13C NMR spectral data of compounds 1 and 2 (400 MHz, CDCl3)
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Fig. 2
figure 2figure 2figure 2

a 1H-NMR spectra for pentadeca-(8E,13Z)-dien-11-in-2-on, b 13C, DEPT90, DEPT45, and APT spectra for pentadeca-(8E,13Z)-dien-11-in-2-on, c 13C-NMR spectra for pentadeca-(8E,13Z)-dien-11-in-2-on, d cosy spectra for pentadeca-(8E,13Z)-dien-11-in-2-on, e Tcosy spectra for pentadeca-(8E,13Z)-dien-11-in-2-on

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According to APT, DEPT135, and DEPT90 spectra of Compound 2 (Fig. 3a), four CH groups which belonged to C-1, C,2, C-8, and C-9 carbons were determined at 114.2, 139.1, 129.9, and 129.7 ppm, respectively (Fig. 3a). In APT spectra Compound 2 (Fig. 3a), CH3 groups (C-15) as positive peak were determined at 15.7 ppm. In addition to the APT spectra, ten CH2 groups which belonged to C-3, C-4, C-5, C-6, C-7, C-10, C-11, C-12, C-13, and C-14 carbons were determined at 114.2, 139.1, 129.9, and 129.7 ppm as negative peak (Fig. 3a). H-8 and H-9 protons at 5.40 ppm were determined to interact with C-8 and C-9 (129.9 and 129.7 ppm, respectively) in HETCOR spectra (Fig. 3b). C-1 carbons (at 114.2 ppm) with H-1 protons (at 5.04 ppm) and C-15 carbons (at 14.1 ppm) with H-15 protons (at 0.95 ppm) were shown to interacted in HETCOR spectra (Fig. 3b). According to 1H-NMR spectra of Compound 2 (Fig. 3c), methyl protons were showed as triplet at 0.95 ppm. H-1 [15.05 and 4.97 (dd) ppm] and H-2 [5.85 (ddt)] protons were determined in olefinic region (Fig. 3c).

Fig. 3
figure 3figure 3

a APT, DEPT135, and DEPT90 spectra for E-1.8-pentadecadien, b HETCOR spectra for E-1.8-pentadecadien, c 1H-NMR spectra for E-1.8-pentadecadien, d Hetcor spectra for E-1.8-pentadecadien

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Antiproliferative activities of crude extract and Compounds 1 and 2 against C6 and HeLa cells

For the first time, antiproliferative activity of crude extract and Compounds 1 and 2 isolated through bioassay-guided fractionation from E. pallida roots were tested on C6 and HeLa cells at 5, 10, 20, 30, 40, 50, 75, and 100 μg ml−1 for 24 h. As shown in Fig. 4, E. pallida extract and isolated Compound 1 significantly (p < 0.01) inhibited the proliferation of C6 cells compared to anticancer agent, Cisplatin. The antiproliferative activities of crude extract and Compound 1 at concentrations of 75–5 μg ml−1 were high. In addition, crude extract and Compound 1 showed a significant antiproliferative activity against HeLa cells at concentrations of 5, 10, 20, 30, and 40 μg ml−1 only (Fig. 5). As observed on C6 cells, the crude extract and Compound 1 were more effective at low concentrations against HeLa cells. However, Compound 2 had no antiproliferative activity against both cell lines at all concentrations tested.

Fig. 4
figure 4

Antiproliferative activities of E. pallida crude extract and isolated compounds against C6 cells, *p < 0.01

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Fig. 5
figure 5

Antiproliferative activities of E. pallida crude extract and isolated compounds against HeLa cells, *p < 0.01

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Discussion

Echinacea has a long history of medicinal use for the treatment of the various diseases (Kindscher, 1989; Li, 1998; Chen et al., 2005; Barnes et al., 2005; Barrett, 2003). A numerous of compounds have been isolated and identified from the Echinacea genus, including caffeic acid derivatives (Cheminat et al., 1988; Pellati et al., 2004; Pellati et al., 2005), alkamides, acetylenes (polyacetylenes and polyenes) (Bauer et al., 1988a, b; Bauer and Remiger, 1989; Pellati et al., 2007), polysaccharides (Wagner et al., 1988), and glycoproteins (Classen et al., 2000; Thude and Classen, 2005), all of which exhibit diverse pharmacological activities. The previously isolated Compound 1 was obtained for the first time from the E. pallida roots in this study. Beside the contribution to literature, it may also be used medicinally like the previously described compounds isolated from E. pallida.

Species from Echinacea genus are currently used in therapy for their medicinal properties (McGregor, 1968). In the present study, antiproliferative activity of crude extract of E. pallida and two isolated compounds, Compound 1 and 2 were investigated. Results showed that at least, crude extract and Compound 1 both have antiproliferative potential against C6 cells at concentrations of 75-5 μg ml−1 and HeLa cells at concentrations of 50-5 μg ml−1. These findings were similar to previous reports such as hexane root extracts from three medicinally important Echinacea species, E. pallida, E. purpurea, and E. angustifolia, which showed cytotoxic and pro-apoptotic activities (Bauer et al., 1988a, b; Bauer and Remiger, 1989; Pellati et al., 2007). The crude extract and compound 1 were more antiproliferative against C6 cells than HeLa cells, indicating a cell specific activity.

The antiproliferative activity of the crude extract was generally slightly higher than isolated Compound 1, indicating contribution from isolated Compound 1 and unknown compounds in the extract. Compound 1 was potent inhibitor of proliferation which is related to its chemical structure. As Compound 1, many polyphenols and flavonoids have been reported to inhibit proliferation and angiogenesis of tumor cells in vitro (Fotsis et al., 1997; Demirtas et al., 2009; Demirtas and Sahin 2013) and inhibit carcinogenesis and tumorigenesis in animal experiments (Hertog et al., 1993; Elangovan et al., 1994). Since both crude extract and Compound 1 have antiproliferative potential, it is suggested that they are likely to show in vivo activity.

In conclusion, our results demonstrated the antiproliferative effect of isolated bioactive compound from E. pallida root extract against different cancer cell lines. The crude extract itself and Compound 1 showed potent antiproliferative activity. The results suggest an anticancer property and support the ethnomedical claims for the E. pallida. In vivo studies are needed to confirm the pharmacological efficacy and safety of Echinacea pallida extract and active compound, Compound 1.