EFFECTS OF WHOLE FRUIT EXTRACT OF ELAEAGNUS ANGUSTIFOLIA L. ON GLIOBLASTOMA CELL LINES

were identified using high-performance liquid chromatography (HPLC). The MTT and LDH assays were performed to evaluate the cytotoxicity effect of the extract and the determination of cell death. Wound-healing assay and colony formation analysis were employed for migration and proliferation evaluation. Based on HPLC analysis, the main flavonoid components of the extract were included rutin and apigenin. The results demonstrated that EA extract at the dose of 125 to 2000 µg/ml and 61.5 to 2000 µg/ml inhibited C6 and U87 cells' viability and induced significant cell cytotoxicity at both 48 and 72 h incubation times. Besides, EA extract significantly inhibited cell migration and colony formation at 250 to 1000 µg/ml concentration. Overall, the results showed that EA extract could inhibit several stages in glioblastoma carcinogenesis in vitro. Therefore, it can be suggested as an anticancer in the clinical treatment approaches of glioblastoma cancer. on two types of glioblastoma cell lines, the EA extract can decrease cell survival and increase tumor cell death in a dose-dependently manner. Our study also shows that the extract signiﬁcantly inhibits the migration and proliferation of C6 and U87 glioblastoma cancer cells. Rat C6 glioblastoma cell line was initially induced in Wistar rats by N, N’-nitroso-methyl urea. This cell is very close to glioblastoma multiforme (GBM) morphologically when injected into rats' brains. Another cell line was U87, which is a cell line decreased by increasing the E. angustifolia extract concentration. Also, a realistic cytotoxicity effect was observed on cell lines after the determined incubation time. The E. angustifolia extract proved wound healing activity on evaluated glioblastoma cells. The colony proliferation rate and colony size were inhibited in treating the cancer cells by E. angustifolia extract. This study showed that E. angustifolia has a good potential for alleviating and inhibiting glioblastoma cancer. Considering the beneficial therapeutic properties of E. angustifolia it could be proposed as a safe medicinal plant to manage glioblastoma cancer. Further clinical trials are recommended to discover probable effects in various cancer.

The rat C6 and U87 human glioblastoma cell lines were provided by the Pasteur Institute of Iran (National Cell Bank of Iran, Tehran, Iran).

Figure 1
The photo of Elaeagnus. angustifolia L fruit

Extract preparation
The EA whole fruit powder (10 g) was macerated in ethanol (70% v/v) at 1:10 volume for 48 h in a shaker incubator. The solution was filtered using filter paper (Whatman no: 1), and the ethanol was evaporated in a rotary evaporator. The final residual extract was oven-dried at 40℃ for 24 h, and then the dried extract was scrubbed from the surface of glass plates and used for the next analysis.

HPLC analysis
Phenolic compounds and flavonoids of the extract were determined using highperformance liquid chromatography (HPLC) apparatus (waters 2695, USA) equipped with a PDA detector (waters 996, USA). Millennium32 software was used for data acquisition and integration. The used column was a C18-Waters (15 cm×4.6 mm). Solvent A was methanol, and solvent B was distilled water in a gradient manner when the flow rate was 1 ml min -1 . The temperature was adjusted at 25℃ and the wavelength at 195-400 nm. The quantification was performed using the linear calibration curves of standard compounds.

Cell Culture
The rat C6 and U87 human glioblastoma cell lines were cultured in DMEM-F12 culture media containing heat-treated fetal bovine serum, penicillin, and streptomycin by a monolayer manner (Gibco, Grand Island, USA). Then, they were placed in an incubator with a humidified atmosphere of 95% air and 5% CO2 (37℃).

MTT Assay
Cell viability was measured via MTT assay kit (Sigma-Aldrich). Briefly, the glioblastoma cell lines were adjusted at 10 4 cells/well and cultured (24 h) in 96well plates. The cells were transfer to prepared mediums containing determined EA extract concentrations and then incubated for 24, 48, and 72 h. In the next step, the MTT solution was poured into wells, and incubated (37℃ for 4 h). The formazan crystals formed were dissolved in 100 µ1 acid/alcohol (0.04NHCl in isopropanol) by mixing. A microplate reader was used to determine the optical density of samples (570 nm) (Sun, Liu et al. 2018). The evaluation of cell viability was performed in comparison to the non-treated cell.

Cytotoxicity assay
A cytotoxicity detection kit containing lactate dehydrogenase (LDH) was used to analyze cytotoxicity activity (Roche Applied Science, Germany). First, 10 4 cells were cultured in wells (24 h) and then inoculated to a prepared medium (containing EA extract concentrations). For low and high control, the cells were cultured in a medium. After 48 and 72 h, 100 μL of Triton X-100 solution was poured into high control wells and mixed thoroughly to destroy the cell membranes. Afterward, the plates were centrifuged (10 min at 250 g), and the 100 μL of supernatants were relocated to another flat-bottom plate. In the next step, 100 μL of the reagent of LDH kit placed into each well with incubation at 21℃ (Ghanghareh and Zare 2020). The absorbance was monitored in ELISA reader by 490 nm wavelength. The cytotoxicity was calculated as : Cytotoxicity (%) = experimental value − low control high control − low control × 100

Wound-healing assay
The wound-healing was evaluated to indicate cell migration ( After replacing the medium with PBS, the wound gap photos were taken using a digital camera on a microscope fitted state. The wound width was determined in six random zones and processed using Image J software (National Institutes of Health, Bethesda, MD, USA). Wound closure distance was quantified according to:

Colony formation assay
To assay the colony formation (Helderman, Löke et al. 2020, Jabeen, Sharma et al. 2020) the C6 and U87 cells were plated in 6-well plates (2×10 3 ) and exposed to 250 , 500, and 1000 µg/ml of EA extract as treatment groups and cell lines without treatment were considered as the control group for 72 h. The medium was refreshed by 3 days up to grow new colonies. At the cell numbers greater than 30, then washed by PBS and placed in paraformaldehyde. Before washing and drying, the cells were stained with crystal violet. The counting of cells was done on colonies containing more than 50 cells using a microscope. The colony formation ratio was determined according to the following equation: Plate colony formation inhibitory ratio = number of colonies treated with EA number of cells inoculated × 100

Statistical Analysis
Statistical analyses were done by SPSS 16.0 and GraphPad Prism version 5.0 software. Comparisons between treatment groups were performed using variance test analysis (ANOVA) followed by post hoc Student's t-tests, and the results were stated as means ± SEM. Differences were considered significant when P ≤ 0.05.

Determination of phytochemical compounds of the extract
HPLC analyzed the phytochemical compounds of EA whole fruit extract are summarized in Table 1. The major compounds of EA extract were rutin and apigenin. These compounds are belonging to the flavonoid class act as core compounds in the pharmacological activities of EA extract. Also, the rutin and apigenin are such plant pigments that are found in certain fruits and vegetables.

Cell viability
The effect of EA extract on cell viability in glioblastoma cancer C6, and U87 cells was quantitatively evaluated using the MTT assay. Treatment with EA extract at the dose of 125 to 2000 µg/ml and 61.5 to 2000 µg/ml inhibited C6 and U87 cells' viability, respectively (Fig. 2). The IC50 values of EA for C6 and U87 cells were 541.6 and 247 µg/ml, respectively.

Figure 2
Effect of EA extract on C6 and U87 cells viability in 48 and 72 h expressed as mean ± SD of three replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p<0.00001 represent significant differences between treatment groups and control group (zero concentration of extract))

Cytotoxic Effect
The release of LDH is a marker for cell death. We, therefore, assessed whether treatment of cells with EA extract resulted in LDH release. The effects of a range of EA extract concentrations (0, 31.25, 62.5, 125, 250, 500, and 1000 µg/ml) on cell death in glioblastoma lines are shown in Fig. 3. The results revealed that a dose of 1000 at 48 h culture and 62.5,250, 500, and 1000 µg/ml at 72 h culture resulted in a significant induction of death of the C6 cell line. In the U87 cell line, the concentration of 31.25 to 1000 µg/ml induced remarkable cell cytotoxicity at both 48 and 72 h incubation times, although this response is not dose-dependent.

Figure 3
Cytotoxic effect of EA extract on C6 and U87 cells in 48 and 72 h expressed as mean ± SD of three replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p<0.00001 represent significant differences between treatment groups and control group (zero concentration of extract))

Cell migration
The effect of different doses (250, 500, and 1000 µg/ml) of EA extract on cell migration is quantified using the average distance of scratch width between its edges. Fig. 4A showed after making a scratch and comparing the migrating cells after 24 h in the no-treatment group, and it was observed that the wounding space between C6 cell layers was almost complete. The wound closure distance is shown in the column bar in Fig. 4B. An observed dose-dependent effect in wound closure distance was observed in the presence of EA extract relative to untreated control cells by 24 hours.

Figure 4 (A)
The changes in cell migration in C6 and U87 cells treated with EA extract for 24 h; (B) The wound width of C6 and U87 cells treated with EA extract. The data are presented as mean ± SD of three replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p<0.00001 represent significant differences between treatment groups and control group (zero concentration of extract)

Colony formation
The effects of EA extract on tumor cell clonogenicity in C6, and U87 cells are illustrated in Fig. 5. As can be seen, the extract not only could notably inhibit the colony formation but also cause a significant decrease in colony formation number ratio in both cell lines. The culture of the C6 cell line with 1000,500 and 250 µg/ml resulted in diminished colony size significantly. In the U87 cell line, the concentration of 1000 and 500 µg/ml induced a remarkable colony size decreasing. These results reflect the ability of EA extract to prevent the tumor cell from dividing. Considering the effect of EA fruit extract on two types of glioblastoma cell lines, the EA extract can decrease cell survival and increase tumor cell death in a dosedependently manner. Our study also shows that the extract significantly inhibits the migration and proliferation of C6 and U87 glioblastoma cancer cells. Rat C6 glioblastoma cell line was initially induced in Wistar rats by N, N'-nitroso-methyl urea. This cell is very close to glioblastoma multiforme (GBM) morphologically when injected into rats' brains. Another cell line was U87, which is a cell line  Overall, our findings confirmed the antiproliferative and cytotoxic potency of EA fruit extract on C6 and U87 (rat and human) cancer cells. However, it is necessary to discover the main mechanism underlying these results, particularly the related molecular pathways. Routine cancer treatment protocols, including surgery, chemotherapy, and radiation therapy, cannot reduce mortality and are accompanied by side effects as disadvantages. Therefore, several attempts have been focused on finding impressive therapeutic agents to reduce the unfavorable adverse effects. In this regard, natural compounds and food supplements with potential anticancer properties could apply as safe alternatives.

CONCLUSION
The efficiency assessment of the extract of E. angustifolia fruit revealed a significant effect on viability, cytotoxicity, migration, and colony formation of C6 and U87 glioblastoma cancer cell lines. Based on the phytochemical analysis, the major bioactive compounds of E. angustifolia extract was flavonoids including rutin and apigenin. The viability of both C6 and U87 were notably decreased by increasing the E. angustifolia extract concentration. Also, a realistic cytotoxicity effect was observed on cell lines after the determined incubation time. The E. angustifolia extract proved wound healing activity on evaluated glioblastoma cells. The colony proliferation rate and colony size were inhibited in treating the cancer cells by E. angustifolia extract. This study showed that E. angustifolia has a good potential for alleviating and inhibiting glioblastoma cancer. Considering the beneficial therapeutic properties of E. angustifolia it could be proposed as a safe medicinal plant to manage glioblastoma cancer. Further clinical trials are recommended to discover probable effects in various cancer.