Cytotoxicity of luteolin, a flavonoid compound isolated from Anthemis palestina

Purpose: To determine the active principle responsible for the cytotoxic effect of Anthemis palestina (Reut. ex Kotschy) Reut. ex Boiss. (Asteraceae)

Natural products and their derivatives are major resources in the drug discovery process, especially for anticancer and cytotoxic agents.This may be attributed to several features of natural products, e.g., structural diversity, potential to target many receptors, presence of wide ranges of pharmacophores, and vastness of stereochemistry.Moreover, natural products have potential for delivery through many cellular transporter systems, and they conform with Lipinski's rule of five which enhance their oral absorption from the gastrointestinal tract.In fact, with specific reference to cancer, the percent of new anticancer drugs that are either natural products or synthetic analogues of natural products, increased from 53.3 % between 1946 and 1980, to 64.9 % from 1981 to 2019 [4].Therefore, in this study, the search for cytotoxic agents that are effective against the most widely diagnosed cancer type, i.e., breast cancer, was focused on natural products.
Anthemis palestina (Reut.ex Kotschy) Reut.ex Boiss., also known as Palestine Chamomile, belongs to the Asteraceae, or the so-called Compositae family.From March to June, this annual plant grows wide in Jordan, most commonly in fields, waste grounds, and mountainous regions in Jordan Valley, Irbid, Ajloun, Jarash, Salt, and Amman.Generally, plants that belong to the genus Anthemis are rich in flavonoids, terpenes, and volatile oils.Thus, these plants usually exhibit anti-inflammatory, hepatoprotective, antioxidant, and anti-cancer properties [5].
The natives of the West Bank and Jordan employ Anthemis palestina for treating spasms, bacterial infections, inflammation and oxidative stress [6,7].Analysis of hydro-distilled essential oil obtained from A. palestina flowers revealed that it contained mainly terpenes, and results from Brine Shrimp Lethality test showed that it had cytotoxic properties [8].In addition, a study of another oil derived from hydro-distillation of A. palestina flowers revealed that it exerted antioxidant, antibacterial, and antifungal properties [6].In addition, it showed cytotoxic properties against human cervix adenocarcinoma (HeLa), human Burkitt lymphoma B (BJAB), and human colon adenocarcinoma (Caco-2) cell lines [6].Moreover, the methanolic extract of Anthemis palestina demonstrated inhibitory activities against xanthine oxidase, hormone-sensitive lipase, and α-amylase, in addition to antioxidant activities [9][10][11].Furthermore, the endophyte Chaetomium subaffine obtained from Anthemis palestina yielded four new compounds, i.e., chaetomisides A -D which showed cytotoxic activities against both ZR-75 mammary cancer and A549 pulmonary cancer cells [12].However, no phytochemical investigation has been performed to identify the compound(s) that might be responsible for the important biological activities of A. palestina, especially the cytotoxic effects.Therefore, the present work was carried out to study A. palestina from a phytochemical point of view, and to isolate the bioactive constituent that might be responsible for its cytotoxic activity.

EXPERIMENTAL Materials
Methanol (HPLC grade), acetonitrile, and hexane were purchased from LabChem (Zelienople, Pennsylvania, USA); HPLC grade ethyl acetate and dichloromethane were purchased from Honeywell (France), while luteolin standard was obtained from Sigma-Aldrich (USA).

Plant material
Anthemis palestina was collected in May 2019 from Alaluk, Zarqa Governorate, Jordan.The plant material was identified by a plant taxonomist, Prof. Dawud Al-Eisawi, Department of Biological Sciences, School of Science, The University of Jordan.A voucher specimen (201905-01-ANP) was deposited at the Department of Pharmaceutical Sciences, The University of Jordan.Additionally, the name of the plant was checked at www.theplantlist.org which was accessed in May 2022.

Extract preparation and fractionation
The dried and chopped aerial parts and roots of Anthemis palestina (300 g) were cleaned, mixed and extracted twice with 2 liters of ethyl acetate, with the aid of 2-h sonication, after which the extract was left at room temperature for 3 days.Thereafter, the extract solution was filtered and evaporated using a rotary evaporator, resulting finally in a yield of 29 g of dried extract.After that, the extract was fractionated using a gravity column (120 × 70 cm, 5 cm ID, n-silica gel).Elution was done with stepwise gradient of nhexane in ethyl acetate (i.e., 0, 10, 20, 30, 50, 70 and 100 % ethyl acetate, each at a volume of 1.5 L, yielding 10 sub-fractions.Using TLC, the eighth fraction (1.8 g) was seen to constitute the main metabolite.Therefore, it was fractionated via flash chromatography (using Silica SL 50 µm irregular 20-g cartridge), with a stepwise gradient of dichloromethane and methanol, i.e., from 100 % dichloromethane to 80 % dichloromethane and 20 % methanol over 30 min.This yielded three sub-fractions.Thereafter, 23 mg (0.08 % yield) of a pure compound, later identified as luteolin, was obtained from the second fraction using a 1.5 × 40 cm Sephadex® LH-20 column chromatography and elution with 100 % methanol.

NMR
A 5-mg specimen was solubilized in 0.60 mL of DMSO-d6 acquired from Cell Bio (Korea) and placed in an NMR tube sourced from Norell (USA).The NMR spectra were recorded on a Varian UNITY 500 FT-NMR spectrometer (Varian, Inc., Palo Alto, CA), with tetramethyl silane as an internal standard.The resultant spectra were analyzed using MestReNova 14.2.1, a software developed by Mestrelab Research (Spain).

Thin chromatography (TLC)
Thin layer chromatography (TLC) on pre-coated Merck Kiesel-gel 60 F254 0.25 mm plates was employed to pool fractions and to compare the compound obtained (luteolin) with a standard reference, using capillary tubes purchased from Hirschmann (Germany).After the TLC plates were sprayed with 10 % sulphuric acid and heated, visualization of the spots was done under ultra violet (UV) at λ of 254 nm.The solvents used are indicated in the section on Materials.The fractions were all dissolved in acetone and applied as spots on activated TLC plates prior to development of the chromatogram in a mobile phase which rose to a distance of 5 to 6 cm.Subsequently, the spots were visualized under UV light at a wavelength of 254 nm.Thereafter, the TLC plates were sprayed with a reagent containing 10 % sulfuric acid, and subsequently heated to 200 degrees Celsius.

Culture of cell lines
Parent mammary carcinoma cells MCF-7 and MDA-MB-231, and healthy fibroblasts (human normal dermal fibroblasts) were sourced from ATCC.Culturing of MCF-7 cell line was done in RPMI) 1640 medium (EuroClone, Italy), whereas the MDA-MB-231 cells were grown in Minimum Essential Medium (MEM).The fibroblasts were grown in DMEM.All the mentioned media contained glutamine (0.002 M), 10 % (v/v) heatinactivated FBS, and 1 % penicillin-streptomycin.All the cell lines were incubated as adherent single layers at 37°C in a 5 % CO2 tissue culture incubator (Memmert, Schwabach, Germany).

Cell viability assay
The anti-proliferative effects of the fractions and the isolated compound against the mentioned cell lines were determined using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, described as per Ghasemi et al [13].The MDA-MB-231, MCF-7 and fibroblast cells were plated in 96-well plates (TPP, Switzerland), with each well having approximately 8 × 10 3 cells.Samples of each cell line were either left untreated as negative control, exposed to doxorubicin as a positive control, or exposed to varying concentrations of the test samples.The test samples consisted of the crude extract and the fractions obtained after the chromatographic separation of the extract (62.2 μg/mL each), including the isolated compound, luteolin.Luteolin was used at concentrations of 0.9, 1.7, 3.4, 6.8, 13.7, 27.3, 54.7, 109.4,218.8, 437.5, 875.0, and 1750.0 µM.Untreated cell lines served as controls.Following a 72-h incubation at 37 °C in a 5 % CO2 atmosphere, the culture medium in each well was replaced with 100 μL of fresh medium containing MTT (Promega, USA).After a 3-h incubation at 37 °C, the MTT medium was discarded, and the resultant formazan crystals were dissolved in dimethyl sulphoxide (DMSO).The optical density of the solution in each well was read in a Glomax microplate reader at 560 nm.All bioassays were repeated thrice.Then, the half-maximal inhibitory concentration (IC50) of luteolin was calculated using GraphPad Prism 7 (USA).

Statistical analysis
Student's t-test was conducted using Minitab 21 (Minitab LLC, USA), and the data are presented as mean ± SD.Statistical significance was assumed for p-values less than or equal to 0.05.

Structural identification and analysis of luteolin
The isolated compound obtained was a yellowish powder.Its identification was carried out with both 1 H and 13 C NMR.Thereafter, the NMR data were compared with published data, and the isolated compound was identified as luteolin (Figure 1 and Table 1).

Figure 1: Luteolin structure
The identity of luteolin was further confirmed by direct comparison and co-elution with a standard reference sample using TLC in a solvent system of n-hexane: ethyl acetate (1:4 v: v).

In vitro cytotoxic effect of Anthemis palestina
The MTT assay was implemented to investigate the cytotoxic effects of Anthemis palestina extract and fractions, in addition to the isolated compound luteolin.The results of this assay are shown in Figures 2. Fraction 8 exhibited cytotoxic effects against both breast cancer cell lines, resulting in a total viability of less than 0.2 for each cell line.Due to its aforementioned cytotoxic effect and the fact that it contained the major metabolite, as shown in TLC, this fraction was subjected to further fractionation.In addition, it is worthy of note that fractions 1, 3, and 4 also exhibited cytotoxic activity against the tested cell lines.However, these fractions were put aside due to their high chlorophyll contents.

In vitro cytotoxic effect of luteolin
The IC50 values for the positive control, doxorubicin, and the pure compound, luteolin, were calculated based on the percent of the remaining cells in three different experiments.The IC50 values are shown in Table 2 and in the dilution curves in Figure 3.

DISCUSSION
In this phytochemical study of Anthemis palestina, a bioassay-guided fractionation approach was implemented through targeting various breast cancer cells.This led to the isolation of luteolin which was identified using 1 H and 13 C NMR, in addition to TLC.Thus, luteolin is the first pure compound to be isolated and identified from this plant.The cytotoxic effect of luteolin was established by comparison with doxorubicin which served as positive control.When the IC50 data of luteolin and doxorubicin were compared, it was shown that although luteolin exhibited good cytotoxic effect against MDA-MB-231 cells (with IC50 value of 14.91 ± 5.77 μM), it was still significantly less effective than doxorubicin.However, the LD50 value of luteolin against the other breast cancer cell line (MCF-7) was 29.28 ± 11.85 μM, indicating comparable effectiveness with doxorubicin.However, what sets luteolin apart from doxorubicin is its safety, as revealed in the IC50 values of both compounds against the normal human dermal fibroblasts (with IC50 values of 1.80 ± 0.90 μM and 51.39 ± 18.51 μM for doxorubicin and luteolin, respectively).This indicates the possibility of further investigations on luteolin as a safe anti-cancer agent.
Luteolin is a polyphenolic flavonoid compound.It is commonly found in various edible plants, including fruits, vegetables, and medicinal herbs [15].Flavonoids are usually present in the vacuoles of plant cells, and their importance for the host organisms is well-known as they serve as antioxidants, anti-inflammatory agents, detoxicants, UV-filters, signaling molecules, and defense molecules [16].In addition, flavonoids are of great benefit to humans when consumed in the diet, due to their antioxidant, anti-microbial and even estrogenic regulatory properties [16].Moreover, flavonoids exert anticancer activity in humans via modification of the activities of ROSscavenging enzymes and their role in regulation of cellular metabolism, e.g.cell cycle blockage, apoptosis induction, autophagy, and antiproliferative effects [15].
In China, plants rich in the flavonoid luteolin have been used traditionally for cancer treatment [15,17].It has been suggested that the biological activities of luteolin are functionally interrelated.For example, there is a link between its anticancer and anti-inflammatory activities [15].Furthermore, the anti-cancer property of luteolin is linked to stimulation of apoptosis, as well as suppression of cell proliferation, metastasis and angiogenesis [15].Moreover, luteolin inhibits several cell survival routes, e.g., PI3K/Akt, Nf-kB), and X-linked inhibitor of apoptosis protein (XIAP), leading to sensitization of cancer cells to induced cytotoxicity [15].Hence, it has been proposed that luteolin could function as an anticancer agent for different cancer types and also as a cancer preventive agent [15].
The in vivo and in vitro cytotoxic effects of luteolin have already been reported in different cancers, including cancers of the breast, colon, pancreas, prostate, mouth, lung, kidney, cervix, placenta, ovary, skin, liver, gastric mucosa, esophagus, and bladder [17].In MDA-MB-231 human breast cancer cells, luteolin regulated miRNAs and produced inhibition of cell cycle, tube formation, and the expression of Notch signaling-related proteins, in addition to apoptosis and cell cycle phase arrest [12,17].
On the other hand, for MCF-7 cell lines, luteolin exhibited high cytotoxicity by decreasing ROS levels, increasing the anti-apoptotic protein B cell lymphoma, inhibiting IGF-1-stimulated MCF-7 cell proliferation, blocking the cell cycle, triggering apoptosis, regulating the estrogen signaling pathway, and suppressing the p-EGFR, p-Akt, p-STAT3 and p-Erk1/2 pathways [17].These findings may explain the cytotoxic activity of luteolin against both MDA-MB-231 and MCF-7 cells as demonstrated in this work.
The consumption of biologically active molecules from the diet does indeed have a beneficial effect against several diseases, including cancer.One of these bioactive molecules is luteolin which has been shown to play therapeutic and preventive roles against various cancer types, including breast cancer [17].Hence, luteolin may be used as a complementary therapy for cancer, especially as part of a diet of edible plants that contain this flavonoid compound.It is worthy of note that luteolin has also been isolated from other species of Anthemis, namely, Anthemis chia, Anthemis cretica and Anthemis monantha [18].

CONCLUSION
This study reports the isolation of a natural product luteolin from Anthemis palestina.The isolated compound, i.e., luteolin, exhibits significant cytotoxic effect on MDA-MB-231 and MCF-7 mammary carcinoma cells, pinpointing the benefits of Anthemis palestina not only for its dietary use but also for its positive impact on health, particularly, breast cancer.

Figure 2 :
Figure 2: In vitro cytotoxic activity of crude extract (T) of Anthemis palestina and fractions (1 -10) against the 2 cell lines (a & b)

Figure 3 :
Figure 3: Dilution curves for luteolin (a -c) and doxorubicin (d) (in molar concentrations) used for the determination of IC50 values against the cell lines

Table 1 :
1H and13C NMR data for luteolin in relation to literature

Table 2 :
IC50 values of doxorubicin and luteolin against the various cell lines