Effect of Aegle Marmelos Hydroethanolic Leaf Extract on Expression of Antiapoptotic Markers in Human Melanoma Cells

Background: Aegle marmelos commonly known as Bael is a herbal plant. Itis from a family called Rutaceae. It has many medicinal uses: anti-diarrheal, antimicrobial, anti-viral, anti-cancer, chemopreventive, Ulcer healing and many others. Aim: To study the effect of Aegle marmelos hydroethanolic leaf extract on expression of antiapoptotic markers in human melanoma cells. Objective: The present study investigated the effect of Aegle marmelos hydroethanolic leaf extract on expression of antiapoptotic markers in human melanoma cells. Materials and Methods: DMSO and MTT chemicals were purchased from Sigma chemical Original Research Article Bhavesh et al.; JPRI, 33(47B): 594-601, 2021; Article no.JPRI.74400 595 Pvt Ltd. Trypsin EDTA, FBS, RPMI 1640 medium and PBS, Real time PCR kit was purchased from Canada. Human melanoma cell line (A375) was purchased from NCCS, Pune, India. Results: The data was analysed statistically by ANOVA and Duncan’s multiple range test with a computer based software (Graph Pad Prism version 5). The percentage of cell viability decreases with the increase in dosage of Aegle marmelos leaf extract. Conclusion: The study concluded that Aegle marmelos hydro ethanolic leaf extract a novel and innovative herbal drug has a significant effect on the expression of antiapoptotic markers in human melanoma cell lines.


INTRODUCTION
Aegle marmelos (L.) Correa (A.marmelos) is commonly called "Bael". It is a kind of tree from the family "Rutaceae" which was widely used in as Indian traditional medicinal plant because of its various medicinal properties [1]. Bael is considered among chemical diversity which has a larger number of species of plants, animals, micro-organisms.on recent discovery in cellular regeneration [2][3]. Bael grows well in drained soil in the regions of tropical and subtropical parts of Indian subcontinent of Asia [4][5][6].
In the last 20 years, plant endophytes have gained more attention towards themselves because they act as a source of secondary metabolites with a new chemical skeleton [5,7]. They are very well known for their properties of anti-proliferative, antipyretic, anti-oxidant, [8] antidiarrheal, anti-cancer [9][10]. Bael is found to have many bioactive compounds like flavonoid, quercetin, alkaloids, polysaccharides, citral, marmin, tannin and fagarine [11]. Our team has extensive knowledge and research experience that has translate into high quality publications [12][13][14][15][16].
The objective of the work reported in this article is to find out the expression of hydroethanolic leaf extract of Aegle marmelos on the human melanoma cell line [17][18]. This study targets tumor related genes like Bcl2, Bcl-xl, p53 genes [19][20].

Study design:
In vitro study.

Duration: 3 months
No ethical consideration involved as it is an in vitro study in the laboratory.

Inclusion criteria:
The study involved Human Melanoma cell lines for in vitro based studies and cell viability assays to evaluate anticancer activity of Aegle marmelos through mRNA expression of Bcl2 and mRNA expression of p53 Exclusion criteria: The study excluded the involvement of aqueous and other solvent extracts for anticancer activity.
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 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% CO2.

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 A375 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. Then the formazan formed crystals were dissolved in dimethyl sulfoxide (100 µl ) and incubated in the 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.

Gene Expression Analysis by Real Time-PCR
Samples from each group were submerged in 2 ml Trizol (Invitrogen, Carlsbad, CA, USA) for RNA extraction and stored at −80°C until further processed. cDNA synthesis was performed on 2 μg RNA in a 10 μl sample volume using Superscript II reverse transcriptase (Invitrogen) as recommended by the manufacturer. Real-time PCR array analysis was performed in a total volume of 20 μl including 1 μl cDNA, 10 μl qPCR Master Mix 2x (Takara, USA) and 9 μl ddH2O. Reactions were run on an CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) using universal thermal cycling parameters (95°C for 5 min, 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 20 sec at 72°C; followed by a melting curve: 5 sec at 95°C, 60 sec at 60°C and continued melting) [21]. For internal control purposes, melting curves were acquired for all samples. The specificity of the amplification product is decided by melting curve analysis for every primer pair [22]. The data were analyzed by comparative CT method and therefore the fold change is calculated by 2−ΔΔCT method described by Schmittgen and Livak (2008) using CFX Manager Version 2.1 (Bio Rad, USA).

Statistical Analysis
The obtained data were analyzed statistically by one-way analysis of variance (ANOVA) and Duncan's multiple range test with a computer-based 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
From the study we can infer the following. the viability of cells, cancer cells which were 100% viable, after addition of Aegle marmelos extract decreased in viability based on the dosage.It almost reached 50% when the concentration of the extract 400 to 500 micrograms. (Fig. 1).The fold changes over control of the mRNA expression of Bcl2 shows that there is a significant reduction in the mRNA expression of Bcl2 based on dosage. (Fig. 2)The fold changes over control of the mRNA expression of Bcl xL shows that there is no significant reduction in the mRNA expression of the Bcl xL based on the dosage (Fig. 3).The fold change over control of the mRNA expression of p53 shows that there is no significant reduction in the mRNA expression of the p53 based on the dosage (Fig. 4).

DISCUSSION
The cell viability of Bael leaf against A375 was determined by the MTT method. This method is based on the process of enzymatic cleavage of the tetrazolium salt into purple formazan by cellular mitochondrial dehydrogenases present in viable cells [23][24] A proportion of people in some countries depend upon traditional medicines for their health needs [25][26][27]. Novel property of extracts of Aegle marmelos, medicinal plants which are of great use with both roots,leaves and fruits in various medicinal ways [28].
As the outcome was positive, we could observe from the present study that Aegle marmelos leaf extract can be used for treating cancer, not only skin cancer but also all other cancers [34]. From the results observed above, we say that as the concentration of the drug increases there is a significant decrease in the cell viability of the cancer cells. This leaf extract acts on p53 mRNA expression [35,36]. Thus this drug can be further assessed in the future study and with clear approval it can be used on cancer patients.These natural medicines have no or less side effects in the long term effect on human life [37][38].

CONCLUSION
From the above study and the supporting articles, we can conclude that Aegle marmelos has an effect on anti apoptotic markers in human melanoma cell lines (A375), not only anticancer activity, but also other medicinal values that could be used in the future generation. In a clear view, the hydroethanolic leaf extract of Aegle marmelos on the expression of anti apoptotic markers in human melanoma cell lines has a positive effect. Thus this drug could be used for further study and can be made as an anti-cancer drug which will be useful for cancer patients.

SOURCE OF FUNDING
The present study was supported by the following agencies • Saveetha Dental College, • Saveetha Institute of Medical and Technical Science, • Saveetha University,

ACKNOWLEDGEMENT
The authors would like to thank the department of physiology and the blue lab for their kind cooperation throughout the study.