Anticancer Activity of Leaf Hydro Ethanolic Extract of Aegle marmelos in Human Lung Cancer Cell Mediated through Caspase-3 and Caspase-9 mRNA Expression

Background: Aegle marmelos (AE) is a medicinal plant that comes under the rutaceae family and the plant was used in the past for treating many diseases and illness symptoms. The plant has many effects such as anti-diarrhoeal, antimicrobial, antiviral, radioprotective, anticancer, chemopreventive, antipyretic, ulcer healing, antigenotoxic, diuretic, antifertility and antiinflammatory properties. Aim: To know the anticancer activity of hydroethanolic leaf extract of Aegle marmelos over lung cancer cells treated with caspase 3 and caspase 9 mRNA expression. Materials and Methods: The required chemicals were collected mainly from Canada. The lung Original Research Article Sukanth et al.; JPRI, 33(58B): 336-343, 2021; Article no.JPRI.74348 337 cancer cells (A549) were collected from NCCS pune and then RNA was extracted from the cells and then the study was conducted after treating it with caspase 3 and caspase 9 mRNA expression. The cells were treated with many dosage of hydroethanolic extract of Aegle marmelos and the cell viability was noted. Results: The study reported that extract of Aegle marmelos has a great anticancer activity about 1 fold change over rate of 1.7 for cells treated with caspase 3 and a fold change over of 1 in caspase 9 treated lung cancer cells. Conclusion: The study concluded an innovative finding that the hydroethanolic leaf extract of Aegle marmelos has a great anticancer activity against lung cancer cells treated with caspase 3 and caspase 9 mRNA expression.


INTRODUCTION
Cancer is one of the deadliest diseases that is more prevalent over the growing world and the death due to cancer increases each and every day and there are many solutions available for this deadly disease but as we get cured from the cancer we get affected by the side effects of the treatments [1]. There should be some alternative for these side effects causing medicines and the natural answers for this question [2].
Nature never fails to fascinate us with its power, and nature gives us everything we need, in the same way we get the solution for cancer from nature itself [3,4]. In nature we have many medicinal herbs and trees that we did not discover until now and if we do that we would get almost solutions for all the problems we face in the present days [5,6]. One such medicinal herb is Aegle marmelos commonly known as bael tree, contains many medicinal effects such as antidiarrheal [7], antimicrobial, antiviral, radioprotective, chemopreventive, antipyretic, ulcer healing, antigenotoxic, diuretic, antifertility and anti-inflammatory properties [8].
This old herb was used in olden days by our ancestors to cure many diseases [9]. Now interestingly this plant is found to have anticancer properties also and if this property of the plant is used wisely we could create a potential anticancer drug [9,10]. The aim of this study is to know the anticancer of Aegle marmelos over lung cancer cells treated with caspase 3 and caspase 9 mRNA expression [11,12].
Aegle marmelos was used in the countryside part of India as dried pulp in summer drinks as it helps in overcoming sunstroke. The Bale leaves are also used in the preparations of salads. Bale fruit absorbs toxins produced by bacteria and other pathogens in the intestine and hence helps in treating dysentery [13]. The bale leaves are also used in ayurvedic medicine to treat loss of appetite. Our team has extensive knowledge and research experience that has translate into high quality publications [14][15][16][17][18][19][20][21][22][23].
The oil extracted from its fruits and leaves are used to cure respiratory disorders. As it had an anti-inflammatory effect the fruit was used for the cure of tuberculosis [14].

Cell Viability by MTT Assay
Cell viability was assayed with a modified colorimetric technique that is based on the power of live cells to convert MTT, a tetrazolium compound into purple formazan crystals by mitochondrial reductases (Mosmann, 1983). Briefly, the cells (1 ×10 4 /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. Then the formed crystals were dissolved in dimethyl sulfoxide (100 µl) and incubated in the dark for an hour. Then the intensity of the color developed was assayed with a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed in the form of percentage of control cells cultured in serumfree medium. Cell viability over the control medium with no treatment was represented as

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 ddH 2 O. 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). For the purpose of quality control, melting curves were acquired for all samples. The specificity of the amplification product was decided by melting curve analysis for every primer pair. The data were analyzed by comparative CT method and the fold change is calculated by 2−ΔΔCT method described bySchmittgen and Livak (2008) using CFX Manager Version 2.1 (Bio Rad, USA).

Cell lines and cell culture
Human Lung cancer cell line (A549) was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in DMEM 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 .

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 cell viability of the lung cancer cells (A549) when treated with hydro ethanolic leaf extract of Aegle marmelos was discussed in Fig. 1. The cell viability showed a decrease with increase in concentration of the leaf extract of AE and at 400 and 500 microgram of leaf extract the cell viability reduced notably. The effect of Aegle marmelos on the caspase 3 mRNA treated lung cancer cells in the form of fold changeover was discussed in Fig. 2. It showed that as the expression is positive, there is an increase in the fold change as the cell viability decreases. The effect of Aegle marmelos on caspase 9 mRNA expression treated lung cancer cells in the form of fold over change was discussed in Fig. 3. It showed that as the expression is positive the fold change increases as the cell viability decreases.

DISCUSSION
From the results obtained within the limit of study it could be seen that the hydroethanolic leaf extract has anticancer activity over lung cancer cells(A549) treated with caspase 3 and caspase 9 mRNA expression [24,25]. Phytochemical studies shows that fruits and leaves of the medicinal herb Aegle marmelos contains many phytochemical compounds like flavonoids, tannins and carotenoids which are the main reason behind the anticancer and other medicinal properties of the Aegle marmelos [26] [27]. The anticancer activity is not only seen toward the lung cancer cells but also many other cells such as breast cancer cell lines (MCF7) and melanoma cancer cells [28] [29] The anticancer activity of the Aegle marmelos could be even seen in the Swiss albino mice [30][31][32][33].
The anticancer activity of the plant is mainly due to the free radical scavengers that occur in the phytochemical aspects [24,25,34]. The lung cancer cells at initial stage remain actively dividing and then when the hydroethanolic extract of the Aegle marmelos is added the cell viability starts to decrease slowly as the over proliferation of the cell is stopped by the plant extract [35]. The cell viability reduces to the most at the concentration of 400 and 500 micrograms of the hydroethanolic extract and this dosage was taken as the dosage for the conduction of the MTT assay [27,36].
The anticancer activity of the Aegle marmelos now is tested only with the in vitro samples for now but in future the hydroethanolic extract of Aegle marmelos could be tested with the in vivo samples and if positive results are acquired [35,37], it could be used as an effective anticancer drug [30][31][32].
The anticancer drugs used at present in chemotherapy even though give a great result, it also gives many side effects that could not be avoided [38,39]. This could be stopped by bringing in a nearly effective natural remedy that could solve the problems nearly as effectively as the present synthetic drugs but does not give any side effects [40,41]. The anticancer activity of the natural plants that are found could be used to solve this problem [42]. The study was time consuming and costly hence it was hard to conduct and complete the research [43].

CONCLUSION
From the results gathered from the study and experiment it is clear that the hydroethanolic extract of the Aegle marmelos has anticancer activity against the lung cancer cells (A549) mediated through caspase 3 and caspase 9 mRNA expression. This property could be used in future with further in vivo studies and experiments.
The present project is supported by · Saveetha Institute of Medical and Technical sciences · Saveetha Dental College and Hospitals, · Saveetha University and · Uma maheswari fire works.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.