Studies on Chemotherapeutic Potential of Hydroethanolic Leaf Extract of Aegle marmelos on Human Lung Cancer Cell Lines

Background: Lung cancer, one of the world’s leading causes of cancer death. There is an improvement in mortality by screening of lung cancer by low dose computed tomography. Long time ago, lung cancer was a reportable disease which now became a commonest cause of death due to cancer. Discovery of medicine for various diseases is still an ongoing process. A lot of diseases are still remaining a threat without any treatment or medicine. Herbal derived medicines are preferred because of their low side effects on patients. Objective: To investigate the role of Aegle marmelos against human lung cancer cell lines (A549 cell line). Materials and Methods: A549 cell lines were procured from NCCS (National center for cell sciences) Pune, India. It was cultured and viability of the cells before and after adding the extract Original Research Article Kumar et al.; JPRI, 33(64A): 325-332, 2021; Article no.JPRI.74399 326 was analysed using the MTT assay. mRNA amplification was done using real time PCR.Statistical analysis was done using ANOVA and dunken’s multiple test. Corresponding graphs are also plotted. Results: The viability of the cells decreased from 100% to 50%. The mRNA expression of wnt and β-catenin decreased after the addition of the extract. Conclusion: The study concluded that Aegle marmelos possesses a good, novel, innovative, anticancer activity against the lung cancer cell lines A549.


INTRODUCTION
Cancer is the second leading cause for death following cardiovascular diseases in India. Herbal plants are usually used by many traditional healers to treat various symptoms and signs of diseases including fever, cold, headache, diabetes, diarrhea, and now even cancer. It is very clear that now plants have a very useful clinical effect and have an antitumor activity [1,2]. WHO has given a statistic of 70-95% of the developing world's population relying on various traditional plants based medicine systems and healthcare. Cancer cells have the ability to escape apoptosis in many ways [3,4]. The main function of anticancer drugs is to prevent it from escaping apoptosis and stop it from proliferating more. These drugs are set to induce cell apoptosis in tumour cells by having cytotoxic effect and hence remains a vital treatment for cancer [5,6,7]. A few drugs that perform the above action are 5-fluorouracil, cisplatin, etoposide, VM26, etc. [8,1]. Thus the identification of potential chemotherapeutic agents using mechanism based studies holds great promise for elucidating mechanisms and devising more specific and effective treatments for cancer related diseases [9,10,1,2,11].
One such approach of discovery is the ethnomedical data approach, where plant selection is based on prior information on the folk medicinal use of plants. It is generally known that this data substantially increases the chance of finding active plants related to random approaches [12]. The plant selected for present study -Aegle marmelos. There is no recorded data for cytotoxicity against cancer cell lines and normal cell lines for these plants [13,14]. Previous studies have concluded that the solvent extracts of the above plant-Aegle marmelos were screened for in-vitro antioxidant, antiproliferative, anti-inflammatory and anticancer activities [15,16]. This research focuses on the chemotherapeutic potential of hydro ethanolic extract of Aegle marmelos on human lung cancer cell lines. No ethical consideration involved as it is an in vitro study in the laboratory.

Inclusion criteria:
The study involved Human lung cancer cell lines for in vitro based studies and cell viability assays to evaluate anticancer activity of Aegle marmelos through Wnt-mRNA expression of A549 and β-Catenin-mRNA expression of A549.

Exclusion criteria:
The study excluded the involvement of aqueous and other solvent extracts for anticancer activity.

Cell Lines and Cell Culture
The Human Lung cancer cell line (A549) was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, CA, USA) at 37°C with 5% CO 2 .

Cell Viability by MTT Assay
Cell viability was assayed employing a modified colorimetric technique that supported the power of live cells to convert MTT, a tetrazolium compound into purple formazan crystals by mitochondrial reductases (Mosmann, 1983). Briefly, the cells (1 ×104/well) were exposed to different concentrations of Aegle marmelos extract (100-500µg/ml) with A549 cells for48 h. At the end of the treatment, 100 µl of 0.5 mg/ml MTT solution was added to each well and incubated at 37 •C for an hour. The formed formazan crystals were dissolved in dimethyl sulfoxide (100 µl) and incubated in dark for an hour. Then the intensity of the colour developed was assayed employing a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed because the percentage of control cells cultured in serum-free medium. Cell viability on top of things medium with none treatment was represented as 100%. The cell viability is calculated using the formula: red blood cell viability = [A570 nm of treated cells/A570 nm of control cells] × 100.

Statistical Analysis
The obtained data were analyzed statistically by one-way analysis of variance (ANOVA) and Duncan's multiple range test with a computerbased software (Graph Pad Prism version 5) to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan's test.

RESULTS
The viability of cancer cells which were 100% viable, after addition of Aegle marmelos extract decrease in viability based on the dosage was observed. It is observed to decreased to 50% viability when the concentration of the extract increased upto to 500 microgram (Fig. 1). It was also observed that there was a fold change over control of the Wnt-mRNA expression of A549, which decreases significantly on the addition of Aegle marmelos extract (Fig. 2). It was observed that there was a fold change over control of the β-Catenin-mRNA expression oh A549, which decreases significantly on the addition of Aegle marmelos extract (Fig. 3).

Gene Expression Analysis
Wnt mRNA expression (Fold change over control).

DISCUSSION
Many commercially proven anti cancer drugs used in modern times were initially used in crude form in traditional or folk healing practises or for other purposes [17,18]. An estimate states that 75% of 120 active plant derived compounds currently used worldwide have been through follow up studies to verify authenticity of data from folk and ethnomedical uses [19]. There is a great potential in discovery of traditional plant uses [20]. The extract with one of its phytochemicals called lupeol has increased the era gene expression in MDA -MB-231 (Eranegative breast cancer cells) thus inhibiting cell proliferation.
Plant phenols may be considered as potential compounds for selective blocking signal transduction pathways, this is in accordance with the current study where the Aegle marmelos blocks the MAPK pathway which inhibits the maturation of the Wnt-mRNA and beta-catenin mRNA [21][22][23]. The phytochemical profile of Aegle marmelos show pharmacological activity and hepatoprotective activity [24,6].
Derivatives of Aegle marmelos were found to be exhibiting strong activity in inhibiting in vitro cell growth of human K562 cells [25,26].
Extensive experimental and clinical studies prove that Aegle marmelos possess various properties which helped to play a role in prevention and treatment of many diseases [29].
The hydro alcoholic extract of Aegle marmelos exhibits strong antitumor and antioxidant activities on DLA-bearing mice [30,31].
Plant research has increased all over the world and a large body of evidence has been collected to show immense potential of medicinal plants used in various traditional systems [32,33]. Aegle marmelos has properties that are medicinal and is a hypoglycaemic extract [34,35].
Limitations of this study is, only when the study is done in in vivo conditions, exact results could be achieved but before advancements are needed to be developed , various steps of drug testing need to be approved. In vitro results have proven to be positive and this study also suggests a future study concentrating on the high significance of the extract against Aegle marmelos.

CONCLUSION
Hence, taking the entire result parameters into account, it can be concluded that Aegle marmelos hydroethanolic leaf extract possesses chemotherapeutic potential by suppressing the lung tumour growth rate. It also has hepato-renal protective effects, and thus can be targeted as a novel and safe anticancer drug against lung cancer. Hence some of the extracts of the compound,if structurally identified and characterized, may be a potent candidate for anticancer drug development.

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 AND ETHICAL APPROVAL
It is not applicable.