Antioxidant and Anti-Cancer Effect of Ethanolic Extract of Citrus Fruits on Hep G2 and MCF-7 Cell Lines

Background/objective: Cancer is a complex genetic disease that occurs due to mutation in genes that control apoptosis and cell growth. Uncontrolled cell growth leads to the formation of tumors. Free radical causes mutation in genes and DNA sequence, however antioxidants can stabilize these harmful effects. Citrus fruits are the rich source of antioxidants. owing to this property this Original Research Article Rasool et al.; JPRI, 33(49A): 84-90, 2021; Article no.JPRI.75724 85 study was planned to evaluate the potential of the citrus fruits for the treatment of the cancer. Aim: This study aimed to evaluate antioxidant as well anticancer potential of five different citrus strains (Citrus deliciosa, Citrus maxima, Citrus limetta, Citrus sinensis and Citrus reticulata). Methods: Peel of all the citrus fruits were collected, grinded and ethanolic extracts were prepared separately, to evaluate radical scavenging ability by employing DPPH method followed by MTT assay of the cancer cell lines to explore the anticancer potential of the extracts. Results: It was observed that citrus peels exhibited good radical scavenging activity and inhibited tumor growth. Maximum effect was produced by Citrus reticulata, and least results were obtained with Citrus sinensis. Conclusion: It was concluded that antioxidant and anti-cancer effects of citrus peels may be due to be owing to the presence of antioxidants (ascorbic acid, flavonoids, phenols, limonene). This research might open new horizon in the treatment of cancer chemotherapy.


INTRODUCTION
Cancer is a deadly disease that prevails due to mutation in genes that encodes vital cellular proteins that leads to the abnormal cellular activities [1]. There are several types of the cancer including: lung, stomach, colorectal, liver, breast, and skin cancer. In middle-and lowincome countries more than 70% of fatality is due to cancer [2].
There are several types of the cancer including genesis of the free radicals due to oxidative stress. Disturbance in equilibrium between free radicals and antioxidant defence is known as oxidative stress [3]. Free radicals cause leads to aging, cardiovascular diseases, gastric problems, cancer, diabetes, and neurodegenerative diseases, [4]. Endogenous and exogenous substances both produces free radicals such as ultraviolet radiations; γ-rays; reactive oxygen and nitrogen species are produced by neutrophils and macrophages due to inflammation; vehicle smoke; cigarette fumes and different chemicals [5]. The substances that protect the cells from harmful effects of unstable free radical are called antioxidant. To stabilize the free radicals, antioxidant act upon them and if this is not done, it may cause serious damage. Vitamins A, E, C, lycopene, beta-carotene, phytochemicals (flavonoids, phenolic compounds) are some examples of antioxidants, which helps in the neutralisation of the free radical. [ Citrus contains many phytochemicals which act as antioxidants and are of great importance. The citrus peel contains limonene, flavonoids, ascorbic acid, and carotene. Its peels as well as pulp both are useful in treating cancer, strokes and heart diseases due to the presence of vitamin C and limonene, that are known as immune modulator and antioxidants. Citrus fruits and especially flavonoids from citrus peel have formerly been recognized as compounds being used in cancer treatment [11]. Hence, currently we are evaluating antioxidant and anti-cancer activities of five citrus fruits.

Collection and Extraction of Citrus Fruits
Fresh five citrus fruits species Citrus deliciosa, Citrus maxima, Citrus limetta, Citrus sinensis and Citrus reticulata were collected during winter season from local farms of district Sargodha, Punjab, Pakistan. All samples were washed and peeled manually and cut into small pieces and then dried in shade. Dried peels were grounded into fine powder in commercial grinder and then powder was separated with the help of sieve. The grounded peel powder was then preserved in polythene bags and placed in refrigerator for further analysis [12].
Each sample of 10 g was dissolved in 100 ml of absolute ethanol in round bottom flask for three days and was shaked manually every day. The ratio of sample peels and ethanol was 1:10 (w/v). After three days of extraction, the sample solution was filtered and then solvent was removed by evaporation at room temperature. The semi solid extract was preserved in refrigerator until further investigation [13]. The percentage yield of extract was calculated using following equation:-Percentage Yield (g/100g) = weight of concentration of the extract/ weight of peel taken x 100

Determination of Antioxidant Activity (DPPH Scavenging Assay)
Scavenging activity of various citrus plant extracts against DPPH radicals was assessed according to a method described earlier [14]. Briefly, 0.1 ml of 5 mg/ml concentration of citrus extracts was added into 2.9 ml of 0.1 mM DPPHethanol solution. The resulting mixture was incubated at 25°C for 30 minutes in dark. The reduction in absorbance was noted at 517 nm. Ethanol served as control rather than antioxidant solution whereas blank solution comprised ethanol in place of DPPH. Further, ascorbic acid was taken as standard. The inhibition of DPPH radicals by the citrus peel extracts was recorded employing the following equation

MTT Assay
To evaluate cytotoxicity and viability, MTT assay was carried out. Hep G2 (Liver cancer) and MCF-7 (Breast cancer) cancer cell lines were used. Cells (1x 10 5 cells/ml) were cultured in 96 well plate containing 100 ml MEM growth medium with fetal bovine serum. The cells were incubated overnight for attachment. Then, 1 mg/ml solution of different concentrations (10 μg, 50 μg, 100 μg, 150 μg) of ethanolic extracts of peels were added and final volume was made 100 µl/well and incubated for 48 h at 5% CO 2 at 37°C. Then, 10 μl of MTT solution was added in each well to obtain final volume of 0.45 mg/ml. It was again incubated at 37°C for 3 to 4 hrs. Afterwards, all the medium including MTT solution was evaporated from wells [17]. Subsequently, 100 μl of solubilization solution was added to each well to dissolve formazan crystals. It was then mixed to make sure to solubilize completely and absorbance was measured by ELISA reader at 570 nm [18]. Untreated cells were used as negative control. The percentage of cytotoxicity was measured using the following formula Percentage cytotoxicity = 1-Absorbance of experimental well x 100 Absorbance of negative control well

Statistical Analysis
The results were explained statistically as mean ± standard error of mean (SEM). The statistical significance of the results was assessed by one way ANOVA followed by Bonferroni post hoc test was used. The analysis was carried out by using SPSS. p < 0.05 was considered a significant value.

Antioxidant Activity
The antioxidant activity of citrus extracts was evaluated via in-vitro test using DPPH assay (

Anti-cancer Activity Using MTT Method
By using MTT assay, percentage inhibition of cancer activity was recorded by measuring absorbance of ethanolic extract at different concentration on both cancer cell lines and result was compiled as in Table 3. The maximum inhibitory effect on Hep G2 and MCF-7 cell lines was obtained with Citrus reticulata and least results were shown by Citrus limetta. The most effective concentration was found to be 150 ug/ml.

DISCUSSION
Citrus fruits belonging to family Rutaceae are rich source of phytochemicals which act as antioxidants and scavenge free radicals which are responsible for many chronic diseases like cancer. Cancer is a complex genetic disease that occurs due to mutation in genes which control apoptosis and cell growth become uncontrolled and leads to tumour formation [19]. Various studies show that free radical causes mutation in genes and DNA sequence. They are not stable due to an unpaired electron due to which they hunt for and capture electrons from other molecules to become stabilize and hence, damage membranes, proteins nucleus, and DNA sequence hence alter the gene product that causes cell to escape from apoptosis and change into cancerous cell. These free radicals (HO•, •O−2) are neutralized naturally by antioxidants, which are produced naturally and guard the main cell organelles by stabilizing the harmful effects of free radicals [20]. As, citrus fruits are rich source of antioxidants so, this study included the use of citrus fruits as antioxidants for cancer treatment and to observe their effects on cancer cell lines and cancer cell growth which give cheap and easy way to control cancer beside chemotherapy and radiotherapy. The anti-cancer activity of selected citrus peels was determined by culturing cancer cell lines in growth medium and preformed MTT assay. Absorbance was measured and percentage inhibition of cells by ethanolic extract of citrus peels at different concentrations. Anti-cancer activity more than 50% is considered efficient [24]. More than 50% inhibition was shown by Citrus reticulata, Citrus maxima and Citrus deliciosa. This inhibition might be due to significant amount of antioxidants (limonene, flavonoids, ascorbic acid and carotene) present in citrus peels, which scavenged free radicals and inhibited tumour growth at specific concentrations of citrus extracts [25]. Citrus reticulata showed higher anti-cancer activity because of higher antioxidants in its peel and has high ability to scavenge free radicals.

CONCLUSION
It is concluded that peels of citrus fruits extracted by alcoholic solvent are rich source of antioxidants and play imperative role in inhibiting tumour cells at specific dosage thus preventing cancer cells to invade or spread in other parts. So, compared to synthetic antioxidants and marketed medicines, citrus antioxidants are more beneficial and have little side effects. Due to large citrus production in Pakistan, antioxidants can be obtained easily. This study provided the basis for future research on antioxidants and their use in treatment of different diseases through citrus fruits, which are the cheapest and easiest source of antioxidants as compared to radiotherapy and chemotherapy which have many side effects.

CONSENT
It is not applicable.