Cytotoxic profile activities of ethanolic and methanolic extracts of chicory plant (Cichorium intybus L.)

ABSTRACT This study aims to highlight the antioxidant and cytotoxic effects of Cichorium intybus L.. The cytotoxic activity for cancer cell lines was evaluated by MTT assay. Total phenol and flavonoid content were examined using chemical analysis methodology. The highest content of phenols and flavonoids have occurred in the methanolic extract (1162.29 ± 103.4 µg.g−1 GAE D.W, 167.47 ± 5.83 µg QE.g−1 D.W), respectively, followed by ethanolic extract (153.83 ± 10.25 µg GAE.g`1 D.W, 36.56 ± 5.95 µgQE.g−1 D.W), respectively. The cytotoxic effect of methanolic extract on MCF-7 cell line was achieved at 500 µg.mL−1. The results showed that methanolic extract possessed potent cytotoxic activity while ethanol extract possessed antioxidant activity. In conclusion, natural plant extract might be prevent and working as a defense by enhancing the immune system against cancer.


Introduction
Breast cancer has surged worldwide, and is the second reason for female mortality (Hortobagyi et al., 2005), especially in Americans and Asians countries (Jemal et al., 2008). Various therapy methods have been applied to decline the percentage of the mortality caused by breast cancer (Kim & Choi, 2013).
Acute myeloid leukemia (AML) is a highly heterogeneous disease; AML cells is the most common acute leukemia in adults. The incidence of AML was ranged from 3 to 5 patients per 100,000 of the population in the United States (Yamamoto & Goodman, 2008).
Oxidative damage is attributed to free radical compounds, which eventually leads to worsening diseases especially cancer (Miller, Rigelhof, Marquart, Prakash, & Kanter, 2000). Antioxidant compounds have the ability to prevent or delay the oxidative stress through free radical trap (Halliwell, Gutteridge, & Cross, 1992).
Medicinal plants have been used in folk medicine for thousands of years as natural products which play a key role in pharmaceutical biology. According to the World Health Organization (WHO), the active ingredients of anticancer drugs have been extracted from natural products (Siegel, Miller, & Jemal, 2015). In addition, the current drugs share fully or some parts of structures with natural compounds.
The current treatments of cancer showed severe side effects, so the use of natural products such as Vinorelbine and Paclitaxel with chemotherapy gave promising results (Jose & Rao, 2006).
This plant has many anticancer compounds which showed antiproliferative activity against some cancer cell lines (Zhang, Yan, Wang, & Liu, 2016;Zhou et al., 2010). Therefore, this work aims to study the cytotoxic activity of ethanolic, methanolic extracts of C. intybus against both MCF-7 cell line and AML cells. The extracts were also evaluated for their antioxidant capacities. The separation and fractionation of active ingredient from ethanolic extract was processed using TLC technique.

Sample collection and authentication
Fresh leaves of C. intybus L were collected from Faculty of Agriculture farm in March 2017, and the plant was identified and authenticated at department of Vegetable Crops, Faculty of Agriculture -Cairo University, Giza Egypt. methanol were added sequentially and soaked for 24 h at a refrigerator at 10ºC. Ethanol and methanol extracts gave the highest phenolic content and antioxidant activity, so we decline the other extracts. The ethanol extract was further applied to thin-layer chromatography (TLC) technique to separate active ingredients using silica gel 60 F254 aluminum sheets 20 × 20 (Merck, Darmstadt, Germany), by using system solvents petroleum ether: ethyl acetate (1:1v/v). The fractions were detected by UV lamp at 240 nm and scratched. The scratched fractions were dissolved in ethanol 80%, filtered and evaporated to dryness and stored at −20°C until used.
2.3. Determination of total phenolic content (Y = 0.0036x-0.007 at R 2 = 0.9897) The total phenol (TP) of both extracts was estimated (Singleton & Rossi, 1965). 200 µL of extract or standard gallic acid (20-240 mg.L −1 ) was added to 3 mL distilled water and then 250 µL Folin-Ciocalteu reagent/2 min was added. Then 750 μL sodium carbonate 20% was added and the mixture was made up to 5 mL with distilled water. The mixture was incubated for 2 h and the absorbance was measured at 750 nm.

DPPH radical scavenging activity
Quantitative measurement of % inhibition was recorded (Burits & Bucar, 2000). 1 mL of DPPH solution (0.002%) was added with 1 mL of plant extracts at different concentrations, and butylated hydroxyl toluene (BHT) was used as positive control. The absorbance was measured after 30 min at 517 nm. The percentage of DPPH radical scavenging was estimated using the following formula: where A 0 is the control absorbance and A 1 is the sample absorbance. IC 50 value was calculated using the dose inhibition curves (Y = 0.0332x +9.041 at R 2 = 0.5296 for ethanolic extract, Y = 0.0541x +9.806at R 2 = 0.9046 for methanolic extract).
2.6. Reducing power activity (y = 0.0292x, at R 2 = 0.9936) The reducing power of extracts was determined according to Dorman, Kosar, Kahlos, Holm, and Hiltunen (2003). 500 µL of standard or plant extracts at different concentrations was mixed with 500 µL phosphate buffer and then 500 µL potassium ferricynide was added. The mixture was incubated at 50°C for 20 min. Then 500 µL trichloroacetic acid (TCA) was added to the mixture. 1400 µL of supernatant was taken and then 200 µL FeCl 3 was added and absorbance was measured at 700 nm. Reducing power was expressed as gallic acid equivalent.

Cytotoxic activity
The cell line MCF-7 was obtained from VACSERA, AML cells from Kasr El-Ainy Giza, Egypt. Two cancer cells were maintained in RPMI media with 10% fetal bovine serum and streptomycin. The cell viability was determined by MTT assay (Wilson, 2000). Cells (MCF-7only) were seeded overnight in 96-well culture plates at a density of 2 × 10 4 cells/well with 200 µl media. Cells (MCF-7 and AML) were treated with extracts at different concentrations. After 24 h of incubation, 20 µl of 5 mg/mL MTT was added to each well and incubated at 37ºC for 4 h. DMSO (100 µL) was added to wells to solubilize formazan crystals. The absorbance was measured at 630 nm (Wilson, 2000).

Statistical analysis
Experimental results are shown as mean ± standard deviation (SD) of three replications. Data were analyzed with analysis of variance (ANOVA), Duncan test, using ASSISTAT Version 7.7 beta. The IC 50 values were calculated from linear regression analysis.

DPPH radical scavenging activity
In the present work, the DPPH radical scavenging assay was used for evaluation of the antioxidant activity of C.
There was eventually a higher antioxidant power recorded by the methanol extract as compared to the ethanolic extract and characterized by the calculated IC 50 values 742.96 µg.mL −1 and 1098.90 µg.mL −1 , respectively.

Reducing power activity
The reducing power of the ethanolic and methanolic extracts of C. intybus was estimated relative to that of the gallic. The reducing power was 58.24 ± 4.30 in the methanolic extract and 29.77 ± 0.90 in ethanol extract at 1000 µg.mL −1 as GAE. It was clear that the methanolic extract possesses a higher reducing power than ethanolic extracts with IC 50 (814.54 and 1719.59 µg. mL −1 ), respectively (Table 2). There is a dose relation between the reducing power and the concentration of the both extracts.

Cytotoxic activity
In this study, the cytotoxic activities of methanol and ethanol extracts of C. intybus were tested against MCF-7 cell line and AML cells (Figures 2 and 3) at incubation period of 24 h. A wide range of extract concentrations were used ranging from 250 to 750 µg.mL −1 of extracts. The data revealed that both extracts had cytotoxic activities against both cancer cell types. The results of the cytotoxic effect of ethanol extract showed the moderate effect at 750 µg.mL −1 (83.98 ± 1.80%) against AML cells in (Figure 2), while the methanol extract recorded 80.71 ± 3.10% at 500 µg.mL −1 . There was no significant difference of cytotoxic activity between two extracts at different concentrations.

Discussion
It is observed from the results of this study that the highest phenolic and flavonoid content of chicory extracts exhibited high antioxidant activities. Previous publications found that methanolic extract of C. intybus possessed the highest content of phytochemicals (Jasim, 2018). It was reported that the presence of phenolic compounds such as coumaric, protocatechuic, vanillic, p-hydroxybenzoic, ferulic, syringic and caffeic (Malik, Pirzadah, Tahir, & Rehman, 2017;Mansouri, Embarek, Kokkalou, & Kefalas, 2005) was thought to contribute in their antiradical potency which appeared in method of DPPH assay (Hsu, Coupar, & Ng, 2006).
According to Jabłońska-Trypuć et al. (2018), cichoric acid showed the highest percent of DPPH radical inhibition, and the scavenging mechanisms for antioxidant of flavonoids were detected (Pai et al., 2015). Therefore, the reducing power activity of chicory extracts was recognized due to the presence of phenolic and flavonoid compounds percentage (Mansouri et al., 2005).
Additionally, both extracts of chicory recorded cytotoxic activity against MCF-7 cell line and AML cells which indicated the importance of natural crude extract for cancer reduction.
For more scientific evidences regarding the cytotoxic effect of chicory extracts against cancer cell lines from molecular point of view (Bøe et al., 1991). This molecular mechanism involves many caspase genes, for example, caspase-3 which has been reported to cleave a number of substrates response to DNA strand breaks leading to apoptosis (Mancini et al., 1998;Nicholson & Thornberry, 1997).
The present findings have recorded a tremendous relation between antioxidant, cytotoxic activities of methanolic extracts of C. intybus. Oxidative stress is a main factor for cancer progression and treatment. In addition, many signaling pathways that are responsible for carcinogenesis can also control reactive oxygen species (ROS) production and ROS downstream mechanisms in which the excess of ROS levels can trigger cancer initiation by enhancing DNA mutation and pro-oncogenic signaling. The majority of the natural active antioxidant compounds, such as flavonoids,  Data represent mean ± SD (n = 3). Significant at P < 0.05.
phenolic, vitamins C and E, are known to have a vital role in the cytotoxic activity (Al-Snafi, 2016). The current study displayed the highly effective action of the ethanolic and methanolic extracts of C. intybus against two cancer cell lines. Hence, it could be anticipated that compounds derived from C. intybus can be used for treatment as cancer chemosensitive agents.

Conclusion
The objective of this work was to study the antioxidant and cytotoxic activities of two extracts of C. intybus. The data revealed that the ethanolic and methanolic extracts have cytotoxic and antioxidant activities. Hence, it was anticipated that C. intybus extracts can be a useful pharmaceutical material to protect or treat cancer cells. Two fractions of ethanolic extracts exhibited anticancer activity more than 50%. Therefore, the results of the current study also suggest that inclusion of anticancer-rich extract or fractions of C. intybus can be used as a dietary supplement. Further studies will be conducted using these plant extracts to identify the molecular mechanism of cytotoxic effect. The expression of some apoptotic and anti-apoptotic genes using RT-PCR is under investigation to know the possible mechanism of cytotoxic effect of C. intybus extracts against MCF-7 cell line and AML cells.