Introduction

Antioxidants provide protection to human against infections and degenerative diseases by inhibiting and scavenging free radicals [1]. The natural occurrence of antioxidants is directly related to phenolic contents of fruits and vegetables. Thus, extensive research efforts are directed towards natural antioxidants originated from plants due to safe therapeutics [2].

Black carrots (Daucuscarota ssp. sativus) originating from Turkey, Middle and Far East, have very high anthocyanin contents [3] which are the most abundant group of pigments responsible for the red, blue, and purple colors of vegetables and fruits [4] and are of prime importance to human well-being as they are well distinguished for their role in health promotion and aging-related diseases including cancer, cardio- and cerebrovascular diseases [5–10]. Additionally, extracts of black carrots gain importance alternatives to synthetic colorants being less toxic, more heat and pH stable food colorant [11].

It is reported that black carrots have high anthocyanin content up to 1750 mg/kg fresh weight [8] and mainly consist of acylated cyanidin-based anthocyanins namely cyanidin 3-sinapoyl-xylosyl-glucosyl-galactoside (27.5 %) and cyanidin 3-feruloyl-xylosyl-glucosyl-galactoside (13.5 %) [8, 11]. The anthocyanin derivatives in black carrots proved to exhibit antioxidant and anti-inflammatory activities [9, 12]. Especially, acylated cyanidin-based anthocyanins reduce NF-κB activation in endothelial cells and decrease nitric oxide production in macrophages [12]. Anthocyanin-rich extract from black carrot significantly inhibited the growth of HT-29 colorectal adenocarcinoma and HL-60 promyelocytic leukaemia cells in vitro [7]. Since carrots constitute one of the main components of human nutrition and attract attention to be used as functional food due to their high anthocyanin content [10], carrot callus cultures were also reported to possess antioxidant activity [13].

In this study, different medium compositions were investigated in order to increase the anthocyanin content of black carrot callus cultures. Thereafter, extracts of black carrot obtained by cell culture and harvested from fields were screened and compared in terms of antioxidant and cytotoxic activities against MCF-7, SK-BR-3, MDA-MB-231, HT-29, PC-3, Neuro 2A cancer cell lines, and VERO normal cell line.

Materials and Methods

Plant Material

Black carrot specimens were purchased from a local market in Kutahya. The specimens were dried at 30 °C and stored in the cold room (4 °C).

Materials and Reagents

Murashige Skog (MS) basal salt mixture (Catalog No: M5524), MS vitamin power (Catalog No: M7150), kinetin (Catalog No: K0753), 2,4-dichlorophenoxyacetic acid (2,4-D) (Catalog No: D7299), MTT [3′-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] and etoposide (Catalog No: E1383) were purchased from Sigma-Aldrich (St Louis, MO, USA). Folin-Ciocalteu’s reagent, 2,2-diphenyl-1-picrylhydrazylhydrate (DPPH), and sodium carbonate were obtained from Sigma, whereas gallic acid was from Fluka. The HPLC grade organic solvents, ethanol and methanol, were purchased from Merck. All other chemicals were of analytical grade purity. Nanopure water was prepared by using in-house nanopure water system (Sartorius Arium 611, Goettingen, Germany).

Plant Material and Sterilization

Black carrot seeds were soaked in an ethanol solution 70 % (v/v) for 5 min, then surface-sterilized in a 0.5 % (v/v) sodium hypochlorite solution for 7 min and subsequently, washed with sterile water three times and then allowed to germinate for 2 weeks at 25 ± 2 °C on MS solid medium with 8 g/L of agar and 30 g/L sucrose. Hypocotyl explants without apical meristems were removed from the seedlings, cut into 0.5 cm segments and placed on MS solid medium with 0.1–0.5-1 mg/L of 2,4-D, 30 g/L of sucrose and 8 g/L of agar for callus initiation (pH: 5.8). Finally, the explants were incubated at 25 ± 2 °C under continuous light of 2000 lux.

In vitro Callus Cultures of Black Carrot

After four weeks of inoculation explants were swelled and induction of callus started. Calli were transferred to new MS medium supplemented with 1 mg/L 2,4 D + 0.1 mg/L kinetin, 1 mg/L 2,4 D + 0.5 mg/L kinetin, 0.5 mg/L 2,4 D + 0.1 mg/L kinetin and 0.5 mg/L 2,4 D + 0.5 mg/L kinetin. They were subcultured on the same medium four times with intervals of two weeks. The calli collected from each vessel were gently pressed on filter paper to remove excess water and their fresh weights were recorded.

Preparation of Extracts

Dried samples weighing 1 g were extracted ultrasonically (Ultrasonic LC30, Elma) with 15 ml ethanol for 15 min. The extraction was performed for five times, subsequently filtered and the filtrates were evaporated to dryness under vacuum at 50 °C (SpeedVac Concentrator, Savant SPD 121P, Thermo Scientific) and the extracts were stored at −20 °C before use.

Cancer Cell Lines

The cancer cells MCF7, SK-BR-3, MDA-MB-231, HT-29, PC-3, Neuro 2A cancer cell lines and VERO normal cell line were obtained from American Cell Culture Collection (ATCC Mannassas, VA). The cells were cultured in RPMI 1640 or DMEM-Ham’s F12 supplemented with 10 % fetal bovine serum, L-glutamine, (2 mmol/L), penicillin (100 U/ml) and streptomycin (100 μg/ml) and maintained at 37 °C with 5 % CO2 in a humidified atmosphere.

Cytotoxic Activity Assay

The cytotoxic activities of the extracts and doxorubicin (positive control, 10 μg/ml) on various cancer cells were measured by MTT assay. Cells in exponential growth phase were placed in 96-well plates to make 6000 cells/wells and sample solutions were added at concentrations ranging from 100 to 6.25 μg/ml in each well, subsequently incubated for 48 h. Negative control was treated with 0.1 % methanol. Quantities of blue formazan product were measured as triplicates at 570–690 nm using a microplate reader (Versamax, Tunable Microplate Reader, USA). Cytotoxicity was determined according to percent cell viability.

Total Phenol Assay

The total phenols in the extracts were determined by Folin-Ciocalteu method as described in Akay et al. [14] and given as gallic acid equivalent (GAE) per gram of extract.

Radical Scavenging Activity

Radical scavenging activities of the extracts were determined by using 1 mM methanolic solution of DPPH• as described in Yesil-Celiktas et al. [15].

Statistics

Statistical analyses of the data were performed by Student’s t-test. A probability value of P ≤ 0.05 was considered to denote a statistically significant difference, and P ≤ 0.01 was also used to show the power of the significance. Data are presented as mean values ± S.E.M. (Standard Error of the Mean)

Results and Discussion

Calli of black carrot induced from stem explants on a nutrient medium supplemented with two different concentrations of 2,4-D which is a synthetic auxin and kinetin, a type of cytokinin, a class of plant hormone that promotes cell division were tested in terms of antioxidant and cytotoxic activities. With respect to biomass production, MS1 and MS3 accumulated the lowest amount of biomass (24.08 and 29.89 g, respectively), whereas MS2 and MS4 accumulated the highest amounts (157.82 and 191.21 g, respectively) among the callus samples.

Cytotoxic activities of the black carrot extracts, together with doxorubicin as positive control were analyzed by MTT assay and IC50 values are provided in Table 1. All investigated extracts displayed dose dependent inhibition profile in terms of cell viability in MCF-7 cells (Fig. 1a). MS1, MS3 and MS2, MS4 were evaluated jointly in order to develop an understanding of the possible effects of kinetin addition to plant cell culture medium on cell viability. This might have two facets; possible differences in terms of secondary metabolite profiles of the callus biomass due to hormone usage and in chemical composition due to extraction methods employed and type of solvents used. At a concentration of 25 μg/ml, MS1 and MS3 exhibited the same cell viability value of 46 %, whereas MS2 and MS4 showed values of 55 and 57 %, respectively. The results suggest that the increase in kinetin concentration in plant cell culture medium did not have an effect on cytotoxicity considering 5-fold higher kinetin concentration in MS3 and MS4 in comparison to MS1 and MS2. Therefore, it can be concluded that increasing kinetin concentration might not be responsible for the cytotoxic activity. IC50 values of MS1 and MS3 were similar (48.60, 31.37 μg/ml), whereas values of MS2 and MS4 (148.12, 119.26 μg/ml) were significantly higher than the other samples implying a poorer inhibiting capability containing lower concentrations of 2,4-D. Consequently, a 2-fold increase in concentration of 2,4-D resulted in more than 3-fold decrease in IC50 values suggesting that 2,4-D might have enhanced cytotoxicity.

Table 1 IC50 values (μg/ml), total phenol content (TPC mg GAE/g extract), and radical scavenging activities (RSA %) of extracts obtained from plant cell culture (MS1, MS2, MS3, MS4), fields (NBC) and doxorubicin as positive control
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Fig. 1
figure 1

Dose-dependent cytotoxic activity of extracts obtained from black carrot callus cultures (MS1: 1 mg/L 2,4 D-0.1 mg/L kinetin; MS2: 0.5 mg/L 2,4D-0.1 mg/L kinetin; MS3: 1 mg/L 2,4 D-0.5 mg/L kinetin; MS4: 0.5 mg/L 2,4 D-0.5 mg/L kinetin) and natural black carrot (NBC) on MCF7 (a), MDA-MB-231 (b) and SK-BR-3 (c) tumor cells treated with 6.25, 12.5, 25 and 50, 100 μg/mL doses of the extracts. “NC” denotes negative control, whereas “PC” to positive control

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Extracts showed dose dependent activity in MDA-MB-231 (Fig. 1b). At a concentration of 100 μg/ml, MS1 and MS3 exhibited cell viability values of 56 and 43 %, whereas MS2 and MS4 showed values of 60 and 52 %, respectively. IC50 values of MS1 and MS4 were similar (114.39 and 109.25 μg/ml, respectively), whereas values of MS2 and MS3 (130.96 and 93.19 μg/ml, respectively) were significantly different probably due to the 2-fold higher concentration of 2,4-D which is in accordance with the results of other breast cancer cell lines.

Extracts showed dose dependent activity in SK-BR-3 (Fig. 1c). At a concentration of 25 μg/ml, MS1 and MS3 exhibited cell viability values of 54 and 49 %, whereas MS2 and MS4 showed values of 74 and 133 %, respectively. IC50 values of MS1 and MS3 were similar (38.74 and 39.01 μg/ml, respectively), whereas values of MS2 and MS4 (78.01 and 103.51 μg/ml, respectively) were significantly higher than the other samples confirming the enhancement capability of 2,4-D.

Among the breast cancer cell lines, MCF-7 and SK-BR-3 turned out to be the most susceptible cell lines inhibited by MS1 and MS3. It is reported that lack of estrogen receptor (ER) and presence of vimentin (VIM) is associated with poor prognosis in human breast cancer [16]. Especially MS1 and MS3 showed significant IC50 values against the ER+/VIM- and ER-/VIM- cell lines of MCF-7 and SK-BR-3, whereas were not that effective against MDA-MB-231 being a ER-/VIM + cell line for which new therapeutics urgently need to be developed.

Extracts showed a similar dose dependent inhibition profile in terms of cell viability in HT-29 cells (Fig. 2a). All four samples exhibited similar cell viability values between 43 and 54 % at a concentration of 100 μg/ml. IC50 values of MS1 and MS2 were similar (85.57, 74.36 μg/ml), whereas values of MS3 and MS4 were significantly different. Jing et al [17] reported a GI50 value of 68.5 μg/ml for purple carrot extract which is in accordance with our findings. In particular, IC50 values of MS1 and MS3 in HT-29 cells were comparatively higher than the values recorded for MCF-7 cells.

Fig. 2
figure 2

Dose-dependent cytotoxic activity of black carrot callus extracts (MS1: 1 mg/L 2,4 D-0.1 mg/L kinetin; MS2: 0.5 mg/L 2,4D-0.1 mg/L kinetin; MS3: 1 mg/L 2,4 D-0.5 mg/L kinetin; MS4: 0.5 mg/L 2,4 D-0.5 mg/L kinetin) and natural black carrot (NBC) on HT-29 (a), PC-3 (b), Neuro-2A tumor cells (c) and VERO cells (d) treated with 6.25, 12.5, 25 and 50, 100 μg/mL doses of the extracts

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Extracts showed dose dependent activity in PC-3 cell lines (Fig. 2b). Cell viabilities were between 58 and 77 % at a concentration of 100 μg/ml. Although IC50 values of the samples were quite high, a similar trend was observed in terms of the capability of 2,4-D contributing to the inhibitory effect of the extracts.

Black carrot extracts displayed the highest cytotoxic activities against Neuro-2A cell lines which are associated with brain cancer (Fig. 2c). Cell viability values ranged between 38 and 46 % at a concentration of 6.25 μg/ml. The cell viability of extract obtained from black carrot harvested from fields was also in the same range. However, a significantly high IC50 value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of neuroblastoma without causing negative effects to normal healthy cells (Fig. 2d). In a recent study, pretreatment with soybean seed anthocyanins reduced the cytotoxicity of H2O2 and inactivated apoptosis signal-regulating kinase proteins suggesting that anthocyanins have brain neuroprotective effects against oxidative stress by inhibiting the activation of ASK1–JNK/p38 pathways and scavenging intracellular ROS [18]. Considering that delphinidin and cyanidin or their derivatives are the major compounds in both black soybean seed and black carrot, a similar ROS mechanism might be responsible for the high inhibition effect observed in Neuro-2A cell line. On the other hand, MS1, MS3 and MS4 were cytotoxic toward normal cell line VERO as were in the case of cancer cell lines.

Dry extracts were also used to determine total phenol content (TPC) and radical scavenging activities (RSA) (Table 1). Although all samples were found to have higher TPC in comparison to natural black carrot (NBC) extract, significantly higher TPC was observed in MS3 samples (381.08 ± 10.9 mg GAE/g extract) which contained 1 mg/L 2,4 D and 0.5 mg/L kinetin in culture medium. To investigate the effect of 2,4 D on TPC there was no significant differences observed even as TPC values were higher at lower concentrations of 2,4-D. This negative effect have been observed when compared with biomass production, whereas at the lowest concentration of 2,4 D the highest amounts (157.82, 191.21 g) accumulated among the callus samples. These results are in agreement with those of Moneruzzaman and coworkers [19] where the effect of 2,4-D treatment on phenol and flavonoid content was the highest with lower concentrations of 2,4-D.

Significant changes in the phenol contents of callus samples were also observed with kinetin treatments. The highest TPC values were attained with the highest concentrations of kinetin. Among the callus samples MS1 containing low concentrations of kinetin at culture medium, produced the lowest amount of TPC (50.00 ± 3.19 mg GAE/g extract), while MS3 containing high concentrations of kinetin at culture medium exhibited the highest amount (381.08 ± 10.9 mg GAE/g extract). These findings confirmed the results of another study [20] where the effect of kinetin on TPC was investigated.

The highest antioxidant activities were achieved with MS3 and MS1 containing same concentrations of 2,4 D and 5-fold higher kinetin. However, at low concentrations of 2,4 D, samples exhibited comparatively lower radical scavenging capacities. It is also worth to mention that RSA values were lower at high concentrations of kinetin. The lowest RSA were observed in MS4 containing a low concentration of 2,4 D and high concentration of kinetin. Consequently, high concentrations of 2,4 D in the medium enhanced antioxidant activities of plant cell culture extracts.

Conclusion

The findings confirm the superiority of black carrot extracts possessing high antiproliferative effects in particular to certain cancer cell lines and antioxidant activity. With a holistic evaluation, MS1 and MS3 were the most potent cytotoxic extracts against MCF-7, SK-BR-3 and Neuro-2A cancer cell lines, together with normal cell line VERO. In order to overcome the negative effects against the normal cell line, studies can be conducted to improve therapeutic index. Considering our results along with the preliminary studies, we suggest that black carrot extract alone or in combination with the anticancer drugs may offer a good strategy for the treatment of a variety of human cancers that are resistant to chemotherapy and worthwhile for further studies.