Cytotoxic Potential of Ficus palmata Extracts on Lung Cancer Cell Lines (A549)

Cancer accounts for 10 million deaths globally whereas the treatments available either have several side effects or non-effective due to multi-drug resistant property of cancer cells for long term use. Therefore, it is utmost important to find safe therapeutic drugs that have cytotoxic potentials against cancer cells. Medicinal plants are believed to have lesser side effects with huge therapeutic efficacy. One such species is Ficus palmata which is known to have several medicinal properties and great antioxidant potentials. Earlier it was reported that methanolic extract of leaves (FPLM) and aqueous extract stembark (FPBA) of this plant have decent antioxidant properties. Therefore, the anticancer potentials of these plant extracts have been evaluated in addition of hydroxyl radical scavenging property. The results shown that FPBA represents higher antioxidant (IC50 value= 242.46±11.26 μg/ml) and anticancer effects against lung cancer (A549) cell lines (IC50 value= 69.74±2.12) whereas this plant extract have lower toxicity (IC50 value= 249.61±7.31) on normal cell lines (3T3-L1) which indicate that FPBA can be a potential therapeutic option for cancer and oxidative stress. The study concluded that the stem bark aqueous extract having therapeutic properties against lung cancer. It is to be recommended that the bioactive compound responsible for its therapeutic properties need to explore against cancer cell by evaluating various other parameters such as apoptosis, cell cycle arrest, and toxic effect on cell morphology.


INTRODUCTION
Cancer is the major health concern which accounts for around more than 70% of deaths globally specially in middle-and low-income countries. Approximately 10 million deaths worldwide in 2020 was due to cancer, and the major cause of these deaths was specially lung cancer that accounts for 1.80 million deaths [1][2][3]. Lung cancer mortality rate is higher than the breast, colon and prostate cancer might be due to higher metastasis potential, and difficulties in diagnostic such as delayed in diagnosis [2,4]. The diagnosis of lung cancer is not easy because it cannot be easily seen or felt, and it's also harder to realize by the patient until there are problematic symptoms exhibited, like chest pain, persistent cough, shortness of breath. According to a report the median time spent from the onset of symptoms to the initiation of treatment is around 138 days, this delay in diagnosis might be the hurdle for the early treatment of disease which can increase the mortality rate due to lung cancer [5]. Recently there are novel approaches reported for the detection and characterization of lung cancer based on artificial neural network and machine learning model which may be beneficial in prediction and detection of lung cancer [6,7]. Despite having several therapeutic interventions, and discoveries, still there are requirements of novel approaches for therapeutic potentials with lower side effects due to multi-drug resistance, several side effects and lack of selectivity [8][9][10][11][12]. Lung cancer mostly results from extensive use of smoking, tobacco exposure and decrease in physical activity which can increase the oxidative stress, atherosclerosis and induce the cancer [13]. To target these issues, there is a need to investigate for the better medicinal options to minimize the pathophysiological causes by reducing the oxidative stress and cure the lung cancer. Therefore, there is a requirement to investigate for the novel therapeutics from natural resources due their easy availably, economic, and lower side effects in prolonged use [14].
Medicinal plants such as Ficus species demonstrate better antioxidant potentials against cigarette smoke induce oxidative stress, and treat several diseases by inhibiting key regulatory enzymes during pathogenesis [16,20,21,23,30]. Moreover, it has been reported that fruits, leaves and stem-bark extracts of Ficus palmata have strong reducing property and enzyme inhibition potentials [23,32]. Hammoud & Shalaby [33] showed that leaves and fruits of F. palmata exhibited protection against diabetic induced liver and pancreas damage [34]. Several parts of F. palmata was also explored for their phytochemicals profiling, antimicrobial, nanoparticle's preparation, and toxicological effects which indicates its potential role designing novel nutraceuticals and associated products [23,32,35]. Studies also confirmed the safety of F. palmata extracts that it does not affect the RAW 264.7 murine macrophages cell viability on up to 100 μg/mL [36].
Despite these several medicinal properties and safety of this plant, still there is very less information for its potential against cancer cells specially lung cancer. Therefore, it has been hypothesized that the leaves and stem bark extracts can be a potential therapeutic cure for lung cancer due to its high antioxidant nature and several medicinal properties. In this study the anticancer properties of leaf methanol extract and bark aqueous extract of F. palmata (FPLM and FPBA, respectively) have been explored by MTT assay for cytotoxic ability and hydroxyl radical scavenging assay has been performed to analyze the antioxidant potential.

MATERIALS AND METHODS
All the chemicals such as deoxyribose, ferric chloride, EDTA, citric acid, hydrogen peroxide, trichloroacetic acid, thiobarbituric acid, hydrochloric acid (HCL), methanol, Dulbecco's modifications of eugal's medium, DMSO, and MTT used in this study were of analytical grade and procured from Sigma-Aldrich (St. Louis, MO, USA).

Plant Material Preparation
Ficus palmata (Family: Moraceae) is a shrub or tree of up to 10-meter height and favorable to grow in cold region of desert area. The fresh leaves and stem bark of plant F. palmata were collected from mountain area of hail region, Qassim province, Saudi Arabia. Plant materials were rinsed with tap water and shed-dried at room temperature for fifteen days after that it was grinded to make dry powder. The 10 grams of dry powder of plant leaves was extracted with 100ml of methanol (MeOH) solvent and 10 grams of dried stembark was extracted with 100ml of Aqueous solvent in Soxhlet apparatus for 12 hours. The extracts were evaporated using Rotatory evaporator and the remaining residue were stored at -20 ℃ for further analysis.

Hydroxyl radical quenching assay
The hydroxyl radical quenching effect of plant extracts was estimated according to the method of Halliwell et al. [37] with some modification [24]. Briefly, 3mM of deoxyribose, 0.1mM of ferric chloride, 0.1mM of EDTA, 0.1mM of citric acid and 4mM of hydrogen peroxide was prepared individually in 20 mM of phosphate buffer, pH 7.4. The 200µl of varying concentration of plant extracts were mixed with 200µl of each of the above prepared solvents and then this mixture was kept for incubation (1 hour) at 37 ˚C. Further the ice-cold trichloroacetic acid (0.2 ml, 15% w/v) and thiobarbituric acid (0.2 ml, 1% w/v in 0.25N HCL) was combined in reaction mixture and this mixture was kept in boiling water bath for thirty minutes. The mixture was refrigerated to cool for few minutes then its absorbance was measured at 532 nm. The percentage of hydroxyl radical scavenging potential was determined by using the formula given below and IC50 was calculated as described earlier: penicillin/streptomycin (250 U/ml), gentamycin (100 μg/ml) and amphotericin B (1 mg/ml). the cell line was incubated at physiological temperature (37 °C) with 5% CO2 in humidified atmosphere to achieve growth confluency for 24 hours.

MTT assay
To verify the cytotoxic impact of F. palmata extracts, cell viability experiment was performed with the conventional MTT-reduction assay with slight modifications [38]. Briefly, 3T3-L1 cells, and A-549 cell lines earlier seeded in a 96-well plate at the density of 5 × 103 Cells/well were taken and media was removed and replaced with varying concentrations of plant extract mixed in 0.5% DMSO and media (31, 62.5, 125, 250, 500 μg/ml) in triplicate and incubated for 48 hours. After 48 hours of incubation cell were treated with MTT (10 μl of 5 mg/ml) and incubated at 37 °C for another 4 h. Absorbance of the formazan product formation was read at 540 nm wavelength using VICTOR Nivo Multimode Microplate Reader, Perkin Elmer. The results were given as mean of three independent experiments. The percentage growth inhibition was calculated using the following formula mentioned below: % Inhibition = (Absorbance of the control-Absorbance of sample/Absorbance of control) × 100

Statistical Analysis
All the samples were analyzed in triplicate and the results were expressed as mean ± S.D. IC 50 value were calculated by Origin version 6.0 Professional software and the results were evaluated by using one-way analysis of variance (ANOVA) and two tailed Student's t-test. Nonsignificant (ns), significantly different *P<0.05, **P<0.01 vs 0 µg/ml.

Cytotoxic potential of F. palmata extracts against 3T3-L1, and A549 cells
The toxicity potential of FPLM and FPBA extracts on lung cancer and normal cell lines, respectively were assessed, and reported in Fig 2 and 3

DISCUSSION
Oxidative stress induced by excessive release of free radicals has been reported to stimulate cellular destruction and further leads to various diseases including cancer [11,12,39]. Since, secondary metabolites from natural products have been suggested for the quenching of free radicals and balance the redox process, therefore it is valuable to evaluate the antioxidant properties of plant extracts. Excessive production of hydroxyl radicals (•OH) are believed to causes macromolecules damage specially DNA which can ultimately results in various illness [40,41]. The medicinal plants having hydroxyl radical scavenging properties have direct role in their antioxidant potentials [42]. Our results are in agreement with previously published report [23], where it was established that F. palmata leaves methanolic extracts showed better antioxidant effects and have IC 50 value for hydroxyl radical scavenging (≥ 250 µg/ml) which is nearly equivalent to the result of this study. Previously it has been reported that fruits of F. palmata showed antioxidant activity due their higher flavonoids content [32]. Alqasoumi et al. [35] reported that F. palmata aerial part was having germanical acetate, psoralene, bergapten, vanillic acid and psoralenoside which can be responsible for its reducing properties and other therapeutic potentials, moreover Iqbal et al. [23] found very similar compounds in stembark aqueous extract of F. palmata which was believed to be responsible for their antioxidative properties.
It has also been noticed from these results that FPBA extract have significantly higher toxic against cancer cell lines and lower toxic against normal cell lines. This may be because of its higher antioxidant activity [23,34]. Our results are in correspondence with previous report that extracts possess strong antioxidant have higher cytotoxic activity against cancer cell lines [43].
Along with this current study several other studies reported the various medicinal aspects of F. palmata which suggests that this underexplored plant have strong therapeutic potential and can also be used as an anticancer drug [23,32,[34][35][36]. The limitations of this study that the bioactive compounds screening, have not been performed, only one parameter for cytotoxicity (MTT assay) has been done, and the cytomorphological assay was not performed.

CONCLUSION
The work represented in this study confirms that FPBA have strong antioxidant potential and anticancer property against lung cancer (A549).
There is an opportunity to explore the anticancer properties by touching various aspects such as apoptosis, cell cycle arrest, and pathways targeted by the bioactive compounds of FPBA extract.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
The author would like to thank Deanship of Scientific Research, Majmaah University for providing the facilities to conduct this work. The author would also like to thank Dr. Danish Iqbal for his continuous support and suggestions.

COMPETING INTERESTS
Author has declared that no competing interests exist.