Potent inhibitory action of Banana wine polyphenols on colon cancer cells

(HCT -15)

 

T. Uma Maheswari*, M. Karuppaiya, S. Subhagar, R. Rahul

Department of Horticulture, Faculty of Agriculture, Food Processing Laboratory,

Department of Chemical Engineering, Annamalai University, Annamalainagar, India -608 002.

 

ABSTRACT:

Banana is a staple starchy food consumed all over the world. It helps to prevent asthma, cancer,  high blood pressure,  diabetes,  cardiovascular disease, and digestive problems. The cell line studies were carried out with the banana wine prepared from the cultivar Dwarf Cavendish to determine the anti-cancer properties. The anti-cancer activity determination was carried over the HCT-15 colon cancer cell line. Banana wine had capability of inhibiting the cell viability of the cancerous cells. Banana wine showed its anti-cancer properties at the 10% concentration whereas the cell viability was low when compared to other concentration and control.

 

 

 

INTRODUCTION:

Wine is a world-wide used drink and mostly preferred one. Wine is produced on the basic principle of conversion sugars by yeast into ethanol and carbon-di-oxide. The main fruit source used for the production of wine is grapes for a decade. The wine is a drink preferred by the people of all ages. Banana is a staple starchy food consumed all over the world. Bananas are rich in potassium and fiber. They may help to prevent asthma, cancer, high blood pressure, diabetes, cardiovascular disease, and digestive problems. Potassium is important as it helps maintain fluid levels in the body and regulates the movement of nutrients and waste products in and out of cells. Potassium also helps muscles to contract and nerve cells to respond. It keeps the heart beating regularly and can reduce the effect of sodium on blood pressure. Potassium may reduce the risk of kidney stones forming as people age. In turn, healthy kidneys make sure that the right amount of potassium is kept in the body.

The banana growers have a major problem of low cost paid and the quick ripening of the bananas. The wastage of the banana can be minimized by utilizing the ripe bananas for production of wine. The main reason for the utilization of banana for the wine production is because of their sugar content, presence of anti-oxidants and widely consumable drink which is richer in health benefits.

 

Also, winemaking from banana is considered as an alternative of utilizing surplus and over- ripe fruits without wastage for generating additional revenues for the fruit growers. The banana wine production can also be done in smaller scale levels also. By the production of wine, the growers would get benefit and also the wastage of the bananas can be decreased. The main aim of the study is to determine the production of the banana wine and to find out the anticancer properties of banana wine.

 

MATERIALS AND METHODS:

Banana cv. Dwarf Cavendish bought from Chidambaram market was used for this study. 200gm of banana fruit with peel was taken and was cleaned with warm water. Then ‘must’ was prepared by proper mashing. 200ml of warm water was added to the must and were homogenized properly into banana juice using mixer. The banana juice was added with potassium meta-bi-sulfite to prevent the browning and to inhibit the unwanted microbial growth. Commercial baker’s yeast (Saccharomyces cerevisiae) of four grams were dissolved in warm water along with di-ammonium phosphate as yeast food and incubated in a culture propagating bottle for 48h at 30ºC. Then homogenized banana juice was taken in individual 500ml standard flask. Each standard flask was added with 4% of yeast culture. The standard flasks were locked with balloons immerse with 0.5mg/L potassium meta-bi-sulfite. The initial pH, Titrable acidity, soluble solids, specific gravity and temperature was noted. The banana juice was stored in the standard flasks for 15days with proper even shaking to complete the fermentation. Then the wine produced were transferred to pasteurized bottles and refrigerated for 30 days. The wine samples were treated against the HCT-15 colon cancer cells to determine their anticancer activity by MTT Assay method. The cell line studies were carried out at Pondicherry Centre for Bio-Sciences, Pondicherry.

 

CELL CULTURE AND MTT ASSAY:

The Colon cancer (HCT 15) cell lines were plated separately using 96 well plates with the concentration of 1×10⁴cells/well in DMEM media with 1X Antibiotic Antimycotic Solution and ^{10% fetal bovine serum (Hi-media, India) in CO}₂ ^{incubator at 37ºC with 5% CO}₂^{. The cells were} washed with 200μL of 1X PBS, and then the cells were treated with various test concentration of compound in serum free media and incubated for 24 h. The medium was aspirated from cells at the end of the treatment period. 0.5mg/mL MTT prepared in 1X PBS was added and incubated at ^{37ºC for 4 h using CO}₂ ^{incubator. After incubation period, the medium containing MTT was} discarded from the cells and washed using 200μL of PBS. The formed crystals were dissolved with 100μL of DMSO and thoroughly mixed. The development of color intensity was evaluated at 570nm. The formazan dye turns to purple blue color. The absorbance was measured at 570nm using micro plate reader.

 

RESULTS AND DISCUSSION:

The anticancer properties of the wine are studied by the MTT assay method. The MTT (3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS). The MTT tetrazolium assay technology has been widely adopted and remains popular in academic labs as evidenced by thousands of published articles. The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570nm using a plate reading spectrophotometer. A reference wavelength of 630nm is sometimes used, but not necessary for most assay conditions.

 

Viable cells with active metabolism convert MTT into a purple coloured formazan product with an absorbance maximum near 570nm. When cells die, they lose the ability to convert MTT into formazan, thus colour formation serves as a useful and convenient marker of only the viable cells. The exact cellular mechanism of MTT reduction into formazan is not well understood, but likely involves reaction with NADH or similar reducing molecules that transfer electrons to MTT. Speculation in the early literature involving specific mitochondrial enzymes has led to the assumption mentioned in numerous publications that MTT is measuring mitochondrial activity.

 

The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to solubilize the formazan product, stabilize the colour, avoid evaporation, and reduce interference by phenol red and other culture medium components. Various solubilisation methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS, and combinations of detergent and organic solvent. Acidification of the solubilizing solution has the benefit of changing the colour of phenol red to yellow colour that may have less interference with absorbance readings. The pH of the solubilisation solution can be adjusted to provide maximum absorbance if sensitivity is an issue; however, other assay technologies offer much greater sensitivity than MTT.

 

The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their metabolic activity. All of these parameters should be considered when optimizing the assay conditions to generate a sufficient amount of product that can be detected above background.

 

The cell line studies were carried out in the produced banana wine to determine the anti-cancer properties. The anti-cancer activity determination was carried over the HCT-15 colon cancer cell line. The control for the cell line was taken and given in the fig 1. The anti-cancer properties of the banana wines were determined by the cell viability and the efficiency of the cells to change its color formazan dye. The banana wine samples were taken in the concentrations of 0.63%, 1.25%, 2.50%, 5% and 10 %. The cancer cells which could not change their color using formazan dye are said to be the inhibition of growth of the cells. The results of the banana wine of the five concentrations are given in the fig 2. The triplicate OD values were noted at 570nm and the cell viability was also noted at 570nm with the values is shown in table 1&2.

 

Banana wine sample:

 

Fig 1: Control

 

Human colon adenocarcinoma HCT 15

 

Fig 2: Inhibition of growth on Morphological analysis of HCT-15 cells with different Concentration of Banana cv. Dwarf Cavendish wine

 

The color changing efficiency of the cancerous cell by intake of the banana wine sample at various concentrations such as 10%, 5%, 2.50%, 1.25% and 0.63%. The color change per concentration denotes the viability of the cancerous cells. The change in color is observed due to the utilization of the formazan dye by the cancerous cells which denotes the ability of the cells to sustain. The change of color from yellow to purple is observed and then taken for spectroscopic evaluations at 570nm to determine the cell viability percentage. The triplicate OD values of the banana wine along with % of cell viability is given in the tables 1 and 2. The graphical representation of the % of cell viability is given in fig 3. The results of wine of the five concentrations is given in the fig 3. The triplicate OD values were noted at 570nm and the cell viability was also noted at 570nm with the values in the table 1-2. The cell viability % is denoted in the graph (Fig 3). Banana wine showed its anti-cancer properties at 10% concentration where the cell viability was 80% inhibited.

 

Table 1: OD Values of banana cv. Dwarf Cavendish wine treated MTT assay at 570nm

 

Table 2: The percentage of Cell Viability with Various Concentrations of banana wine treated MTT assay at 570nm

 

Fig 3: The percentage of Cell Viability with Various Concentrations of banana wine treated MTT assay at 570nm

 

Similar results of anti-cancerous effect of herbal wine prepared from Amla, Tulsi, Ginger, Aloe vera was effective against cancer and was reported to reduce cancer chances. (Seo et al., 2009; Panda et al., 2013). Presence of phytochemicals, poly phenols and antioxidants like ascorbic acid might be the reason for anticancerous effects of banana wine (Table 3). Similar report was given by Athina (2000) as low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol‐rich foods and beverages, had reported a beneficial antiproliferative effect on breast cancer cell growth. The anticancer activity was carried out by MTT assay in banana, guava, orange and papaya showed good anticancer activity with IC50 values 31.7, 27, 95.5, and 18.5μg/ml, respectively (Sun et al., 2002; Dahham et al., 2015). Banana juice extract inhibited the proliferation of the colon cancer cell line HT-29 was explained by Praveena et al. (2018). Many fruits such as guava, banana, papaya, orange, lemon, apple, litchi possess proven medicinal activities as whole fruit, seeds, leaves, and as peels and many of them are reported to have anticancer potential such as lemon, orange, papaya, guava (Chen et al., 2010). The well ripened banana fruit has better anti-cancer property and the dark spots on ripe yellow bananas produce a substance called Tumor Necrosis Factor (TNF) that destroys cancerous tumors (Iwasawa and Yamazaki, 2009).

 

Table 3: Physicochemical Composition of Banana Juice and fermented wine

 

Banana wine can be used as a drink consisting of nutritional benefits and the post-harvest damages can be reduced. The banana wine production can also be made in small scale processes and help the producers to obtain economic growth. Banana wine is an accepted drink by all age of people as it is regularly consumed starchy food. From the study it is concluded that banana can be effectively utilized as preparation of wine. There was no change in the alcohol concentration in the wine produced on the fifteenth day produced wine and wine refrigerated for thirty days. Banana wine consisted of high amount of potassium in it. Banana wine can be utilized for its health benefits as it reduces the stress and lowers blood pressure. Considering many health problems, banana wine should be consumed in little amount due to its medicinal properties. Banana wine had capability of inhibiting the cell viability of the cancerous cells. Further researches can be carried out on the basis of the anti-cancer activity of the banana wine or the banana fruit which would be a good medicine for the cancer treatment as it is liked by the most of the people and consumed in large number. Also, winemaking from banana is considered as an alternative of utilizing surplus and over- ripe fruits for generating additional revenues for the fruit growers.

 

CONCLUSION:

Today Cancer is one of the most challenging and life-threatening health problems in the entire world. Plantains and bananas contain many bioactive compounds such as poly phenols, alkaloids, flavonoids, tannins and phenolic compounds. The use of phytochemicals may be a true therapeutic strategy for eradicating cancer cells. The banana wine showed its anti-cancer properties at the 10% concentration where the cell viability was low when compared to other concentration and control due to the Presence of phytochemicals and Poly phenol present in the Banana wine. Therefore, the banana wine had capability of inhibiting the cell viability of the cancerous cells. The utilization of banana cv. Dwarf Cavendish for the wine production is because of their high sugar content and richer in anti-oxidants for the health benefits of human being may be recommended and popularized.

 

REFERENCES:

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Received on 05.11.2019           Modified on 22.01.2020

Accepted on 11.03.2020         © RJPT All right reserved