Study on the Anti-cancer Potential of Aegle marmelos Fruit Extract Pro and Anti Apoptotic Molecule in Human Melanoma Cell Line-A375

Aegle marmelos is also known as bael which is commonly found in south East Asia and Indian-sub continent. The origin of bael is India. Bael is also known as the golden apple, Bengal-quince in India. In the ancient medical system, Aegle marmelos play an important role and its extract is also useful in inflammation, diabetes, cancer and asthma. The leaves are used for anti-inflammatory, nervous disorder, control blood sugar and fruit is used to treat antiviral, anti-diabetics, and brain and heart tonic. In addition, studies have proved that bael is used for the treatment and prevention of cancer. The main aim of this study is to assess the anti-cancer potential of Aegle marmelos fruit extract pro and anti apoptotic molecules in human melanoma cell line-A375. In the present study, Human Melanoma A375 cells will be produced, grown and will be passed in different culture flasks, Original Research Article Venkatakarthikeswari et al.; JPRI, 33(59A): 287-294, 2021; Article no.JPRI.78938 288 then RNA isolation was done and by reverse transcriptase process, RNA gets converted into cDNA. This cDNA will be used for the amplification of growth factor beta using gene specific primers by commercially available real time PCR kit. The anticancer potential effect was found in 400μg/ml, as the concentration increases, the cell viability is decreased. The current study explains the potential application of bael in pharmacological and medicinal uses in near future.


INTRODUCTION
According to modern Ayurvedic sources, Aegle marmelos belong to the Rutaceae family and are morphologically deciduous, 6-9 metres in height with trifoliate leaves, and bark is shallow, furrowed with spines, short flowers, slightly pear shaped fruits [1]. About 80% of the world population depends on traditional medicines for health needs. Bael fruits possess various nutrients like vitamin C, B1, B2, B3, proteins and minerals like calcium, potassium [2]. Modern allopathic medicine has developed many cost diagnostic methodologies which also cause sideeffects like constipation, diarrhea [3]. Availability of natural products which have low side effects and are highly effective [4]. Bael possesses a broad range of therapeutic effects like antiulcerative, hepatoprotective, cardioprotective, radioprotective effects [5].
Cancer is a painful and aggressive killer disease. Nowadays 60% of drugs used for the treatment of cancer have been isolated from natural products [6]. Cancer is mainly caused by genetic mutation in DNA [7]. Nowadays various methods used to treat cancer like chemotherapy may cause side effects like hair loss. Malignant Melanoma is also known as skin cancer which is developed from melanocytes that produce melanin [8]. There are four types of melanoma cancer-superficial spreading, nodular, lentigo maligna and acral lentiginous [9]. In male it commonly occurs on the backside of their body and for females it occurs on legs [10]. Symptoms are changes in skin colour, increase in mole size, itchiness. The drugs used in melanoma cancer do not kill all cancer cells and the cells which survey are responsible for drug resistance and increases mortality rate. The advantage of traditional medicine is being cost effective, easily available [11]. The ripened fruit is useful for the treatment of anti-viral, treatment of rectum inflammation. The unripened fruit is useful for astringent dysentery [12]. The essential oil extract from bael is useful for several biological activities like radiation protection, chemoprevention, antipyretic. The role of apoptotic is regulated by (BCL-2, BCL-XL) [13] and pro-apoptotic is regulated by (BID, BIM) [14][15].
The main aim of this study is to assess the anticancer potential of Aegle marmelos fruit extract pro and anti apoptotic molecules in human melanoma cell line-A375.

Cell Lines and Cell Culture
The Human Melanoma cell line (A375) was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in DMEM 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% CO2.

Cell Viability by MTT Assay
Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert MTT, a tetrazolium compound into purple formazan crystals by mitochondrial reductases (Mosmann, 1983). Briefly, A375 Human Melanoma cell lines (1 ×104/well) were exposed to different concentrations of Aegle Marmelos fruit extract(100-500µg) with A549 cells for 48 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 crystals formed were dissolved in dimethyl sulfoxide (100 µl) and incubated in dark for an hour. Then the intensity of the color developed was assayed using a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed as the percentage of control cells cultured in serum-free medium. Cell viability in the control medium without any treatment was represented as 100%. The cell viability is calculated using the formula: % 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 tests with 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 point finding showed that the Aegle marmelos fruit induces cytotoxicity in A375 cells. M-RNA expression of anti-apoptotic molecules such as BCL-2, BCL-XL found to be decreased in 300 and 400 μg/ml treated cells (Fig. 1)

DISCUSSION
Oncologists square measure typically face a huge drawback in deciding the effective doses of chemo and radiation therapies to attenuate the biological science and genetic damages on the healthy cells [13]. Most importantly one of the most vigorous cancers is melanoma cancer with a high mortality rate [35]. These cells reproduce faster than normal cells and migrate to various parts of the human body easily [36]. From the study of Tripti singh, Berberis vulgaris of A375 and Hs294 human melanoma cells has significance of (P<0.01-0.001) in A375 cells which has no significant effect on cell viability [37] whereas in this current study the significance is at P<0.05 in A375 cells. From the study of Ayoub khalfaui, Algerian Asphodelus tenuifolius A375 melanoma cells in which the decrease in the percentage of cell viability is significant when the concentration of the extract is 50µg/ml [38].
In malignant melanoma cells, S. frutescens extract is effective in inducing apoptosis. The viability of A375 and Colo-800 human melanoma cells was lowered by S. frutescens extract in a dose-and time-dependent manner [28] Significant increases in the expression of the CASP9 and FASLG genes in response to S. frutescens extract suggest that the extract may trigger apoptosis through both intrinsic and extrinsic mechanisms [39]. In this study, the percentage of cell viability is significant when the concentration of the extract is 400µg/ml. The chemical composition and relative quantity of biological active components determine the cytotoxicity of the plant extracts tested on A431 cells [9]. The A375 cell line was sensitive, but not as sensitive as the A431 cell line [40]. In the present study, 500µg treated A375 cells showed reduced mRNA expression and the extract showed potent anticancer activity.
This study clearly indicates that plant extract has a significant role in modulation of intrinsic apoptotic signaling in melanoma cells which might be due to presence of marmelosin, tannins present in bael. In accordance with present studies on various cancer cell lines have demonstrated that bael and their bio-chemical compounds showed anti-proliferative activity in vitro and in vivo condition. This study only dealt with anticancer and anti proliferative activity of fruit extract alone. Further studies required to check other parts of the plant and also proceed in molecular techniques to confirm the activity.

CONCLUSION
From this study concluded that Aegle marmelos may be used as a potential anti-cancer, antiproliferation activity. Further studies need to be analysed on signaling molecules involved in cancer progression in order to accretion potential of Aegle marmelos.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.