The Determination of In vitro Antioxidant and Cytotoxic Activities of Resin Obtained from Cilician Fir

In this study, the antioxidant and cytotoxic activities of resin obtained from the Cilician Fir plant were evaluated. This resin has antioxidant activity according to 2,2′-Azino-bis-(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) radical scavenging assay. The in vitro cytotoxic activity of the resin was investigated against a panel of human cancer cells (MDA-MB-231, Hep G2, PC-3, U-87, MCF-7, HT-29) with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for 48 h. Normal human lung fibroblast cells (WI-38) were used as healthy cells. The results indicated that the in vitro cytotoxic activity of the resin depends on the cell line type and concentration of the resin. According to the IC50 values, the resin has the most cytotoxic activity on endometrial adenocarcinoma cancer cells (IC50=8.94 ± 0.03 μg mL-1) compared to other cancer cells. The results also indicated that Ishikawa endometrial adenocarcinoma cells, which have Selectivity Index (SI) value >2, have the most sensitivity against the resin. This study provides the first evidence that the resin inhibits the different cancer cells' growth. Research Article Article History Received : 16.04.2020 Accepted : 31.05.2020

medicines such as colds, bronchitis, stomach-ache, dyspepsia, and tuberculosis , Yeşilada et al. 1995, Singh et al. 2000, Wu et al. 2016. It is also known to have antioxidant, antibacterial and antitumor activities (Handa et al. 2013, Hasegawa et al. 1987, Lavoie et al. 2013. Abies cilicica is one of the native fir species in Turkey, with two subspecies of A. cilicia subsp. isaurica and A. cilicia subsp. cilicica. Abies cilicica subsp. cilicica is known as Cilician Fir in the Mediterranean region of Turkey. Abies cilicica subsp. cilicica does not have resinous buds and has hairy young shoots (Davis 1967).
Plant medicines and natural products are currently widely used as traditional medicine, and because of the side-effects of some chemical drugs are increasing in importance and attracting more attention (Pohanka 2011). It has been known for decades that plant extracts obtained from medicinal and aromatic plants have antioxidant, antimicrobial and anticancer effects (Yeomans 1996, Do 2004, Kunyanga et al. 2012. Some active components of plant extracts can prevent oxidative tissue damage caused by oxygen free radicals (Waris and Ahsan 2006, Evans et al. 2004, Silva et al. 2006 and in this regard, the antioxidant uptake into the body plays an important role in preventing various diseases such as cancer and cardiovascular diseases and in delaying the aging process (Albayrak et al. 2010).
Plant chemicals have primary and secondary metabolites. Although primary metabolites are needed for plants because of their role in basic cell metabolism, secondary metabolites have no effect on the plant's primary metabolism. Secondary metabolites are generally produced for defense against ecological conditions. Medicinal and aromatic plants often have organic compounds such as oils, resins, tannins, natural rubbers, waxes and dyes (Camarda et al. 2011).
Currently and in the future, it is expected that there will be more research into the chemical or biologically active constituents. The aim of this study was to focus on the antioxidant and cytotoxic activities of the resin obtained from Cilician Fir (Abies cilicica (Antoine & Kotschy) Carrière) as a biologically active component to find a new active ingredient against cancer.

MATERYAL ve METOD
This study was conducted in the laboratories of CUTAM (Cumhuriyet University Advanced Technology Research Center), Cumhuriyet University, Sivas in 2019. Fir plants were collected from the wild flora of Kahramanmaraş (Göksun district, Tekir plateau (1400m)) in Turkey. The experiments were carried out in completely randomized design with three replications.

Preparation of Extracts
The resin that accumulating and collected on tree bark, was used as the study material ( Figure 1). 10 gr resin was soaked in 20 mL ethanol for 24 h with intermittent agitation. At the end of this process, a uniform solution was obtained. The obtained extracts were analyzed by GC-MS The DPPH radical scavenging activity of the extract was examined using the Clarke method (2013). This method was slightly modified as follows. 20 μL of test solution was mixed with 180 μL of DPPH solution and placed in a 96-well plate. The plates were left in the dark for 15 min, then absorbance was read with an ELISA reader at 540 nm. Gallic acid solution prepared with DMSO instead of test resin as standard and DMSO as the control were run in parallel. All the experiments were made in parallel in three groups and the results were evaluated. The standard average error (SEM) was calculated. The results are expressed as % DPPH sweeping effect using the following equation.
1 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assay The method of Chun et al. (2005) was used to determine the ABTS scavenging activity of the plant extract. On a 96-well plate, 50 μL resin solution was mixed with 100 μL ABTS solution. Then, the mix of ABTS solution and resin solution was kept for 10 minutes (room temperature), and absorbance was read at 734 nm.

Total phenol content
The total amount of phenol was performed according to Clarke method (2013). After experiment, the total phenol quantities were calculated from the absorbance values of the samples (Clarke et al. 2013).

Total flavonoid content
The aluminum chloride colorimetric method was used to determine total flavonoid content in the extract. The quantities of total flavonoids were calculated as mg equivalent of quercetin over dry weight of extract (Yang et al. 2011).

Cell culture
The MTT assay was done against MDA-MB-231 and MCF-7 breast, HepG2 liver, Ishikawa endometrial, PC-3 prostate, U-87 glioblastoma, HT-29 colon cancer cells to determine the in vitro cytotoxic activity of the resin. The resin was also applied to , WI-38 healthy cells to investigate selectivity of the resin between cancer cells and healthy cells. MDA-MB-231, MCF-7, HT-29 and PC-3, cells were cultured in the DMEM medium. HepG2, Ishikawa, U-87, and WI-38 cell lines were cultured in EMEM medium. 10% FBS and 1% antibiotics solution were added in DMEM and EMEM medium. All cells were plated of 1x10 5 cells mL -1 (each well 100 µL) in 96-well plates and incubated one day.
The resin was dissolved in DMSO and 1 μl of the different concentration (1-10 000 µg mL -1 ) of the resin was added and the incubated of 48 h of incubation. Culture medium and sterile DMSO (0.5% v/v) were added controls and negative control wells. After 48 hrs, 10 μL of MTT (5 mg mL -1 in PBS) in PBS was added and the plates were incubated for 2-3 h. After, 100 μL of DMSO was added to each well and the plates were incubated for 15 min at RT with agitation. The absorbance values were at 570 nm on an ELISA reader (Biotek, Epoch, USA).

Statistical analysis
All in vitro cytotoxic activity experiments were carried out in triplicate (n=9) and the results were expressed as mean ± SEM. Data were analyzed using one-way analysis of variance and Dunnett's multiple comparisons test. Differences were considered statistically significant at *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. The IC50 were determined by statistical software, GraphPad Prism7 (GraphPad Software, San Diego, CA, USA).

Chemical composition
The Cilician fir ethanol extracts were analyzed with GC-MS and the chemical composition of the plant was evaluated. According to the data obtained, the major component of the Cilician fir was determined to be αpinene (50.43%). The other components determined were 2-β-pinene (20.17%), l-limonene (11.53%), 3,4-Dihydrothenyl-[3,4,B]-5-Carboxythiol (9.22%) and Butanoic acid, 3,7-dimethyl-2,6-octadienyl ester (8.64%) (  Figure 2. According to the data obtained, the scavenging effect of the extract on ABTS radical increased linearly with increasing concentration from 0.1 to 2.0 mg ml -1 , although lower than the gallic acid that was the standard. According to Broznic et al. 2 Total phenol and flavonoid content Phenolic compounds are generally used for protection from oxidative damage for living creatures (Duthie et al., 1997;Skaper et al., 1997). There is a relationship between antioxidants and flavonoids/phenolic acids. In this respect, these components are important for antioxidant activity properties (Saddiqe et al. 2010). In this study, flavonoids and phenolic acids were examined and the results are given in Figure 3. According to the results obtained from the ethanol extract of Cilician fir, while the total flavonoid content was determined as 94,85054 ± 4.67 µg, the total phenolic content was found to be 308,8282 ± 4.83 µg.
The results showed that the ethanol extract of the resin of Cilician fir has phenolic substances.  According to Table 2, the resin had similar cytotoxic activity toward PC-3, U-87 and MCF-7 cancer cells with IC50 values of 23.0 ± 0.05, 30.6 ± 0.02, and 38.4 ± 0.02 µg mL -1 , respectively. The results also indicated that the resin was more cytotoxic on HT-29 colorectal adenocarcinoma cells than on MDA-MB-231 breast cancer cells.
These results also showed that Ishikawa endometrial 3 adenocarcinoma cells had the lowest IC50 value (8.94 ± 4 0.03 µg mL -1 ). As the resin did have larger IC50 values 5 for healthy cells (18.32 ± 0.03 µg mL -1 ) compared to the 6 Ishikawa endometrial adenocarcinoma cells (8.94 ± 7 0.03 µg mL -1 ), the resin was seen to be more effective 8 on endometrial adenocarcinoma cancer cells.

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In order to select the most sensitive cancer cell line, the selectivity index (SI) of the resin was calculated. The calculated SI value for the resin was found to be >2 toward endometrial adenocarcinoma cancer cells,  It has been measured that the effects on gene expression levels of ICAM-1, P-selectin, VCAM1, monocyte adhesion, and trans endothelial migration for the two in vitro models. They were also determined the total phenolic and total flavonoid contents of the extract. Another study, researchers found to be essential oil of the resin from Abies cilicica subsp. and against a few bacteria and fungi and found encouraging results (Kızıl et al., 2001).

CONCLUSION
Despite many researches on cancer, which is one of the deadliest diseases of today, there is still no complete treatment method. For these reasons, the antioxidant and cytotoxic activity values of resin obtained from the Cilician Fir plant were evaluated. According to obtained data, the resin has been found to have cytotoxic activity on endometrial adenocarcinoma cancer cells (IC50=8.94 ± 0.03 µg mL -1 ) and antioxidant activity.