Cytotoxic Potential of MoringaPeregrina(Moringaceae) Seeds on خط الخلايا السرطانية البشرية لسرطان الثدي

Cytotoxic Potential of MoringaPeregrina(Moringaceae) Seeds on Human Breast Cancer Cell Line

Cytotoxic Potential of MoringaPeregrina(Moringaceae) Seeds on خط الخلايا السرطانية البشرية لسرطان الثدي

Abstract: Moringa peregrinahave long been used traditionally to treat many diseases in Arabian Peninsula folklore medicine. The objective of this study was to determine the anticancer activity of Moringa peregrina seed ethanolic extract on the breast cancer cell line. MTT (3-(4,5-dimethylthiazol-2w-yl)-2,5-diphenyl tetrazolium bromide) assay was used to determine the anticancer effect of MPSE treatment at concentrations ranging from 25 to 400 ug/ml against MCF-7 breast cancer cells. The statistical values were determined employing the statistical test of one-way analysis of variance. The results show that the MPSE inhibits the proliferation of MCF-7 breast cancer cells with IC₅₀ values 13.7µg/ml. These findings suggest that MPSE has anti-proliferative effect on MCF-7 breast cancer cells can be used to treat and prevent cancer diseases, the outcomes is promising and paving the way for develop new natural drug for treatment of breast cancer disease. 

1.Introduction

Chapter one

1.    1Introduction

Moringaperegrinais a species in the family Moringaceae. Is a slender tree or shrub, can be found in Asia and Africa. Traditionally, this plant is used for the treatment of hypertension, stomach disorder, malaria, burns and diabetes (Albaayit et al., 2018; Senthilkumar et al., 2018). M. peregrina seeds contain tocopherols, flavonoid and phenolic compounds, which help to reduce cancer mortality (Koheil et al., 2018; Vijayalakshmi et al., 2013). Seeds of M. peregrina have been validating pharmacology to have Anticancer activity (Elsayed et al., 2016) as well as antidiabetic, anti-inflammatory and antimicrobial activities (Koheil et al., 2011), however, there is limited knowledge about the anti-proliferation effect of M. peregrina seed against MCF-7 cells. MCF-7 is an epidermal growth factor positive breast cancer cell line used in medical research for screening the anticancer drugs in vitro (Huang et al., 2010).

Breast cancer is the most common cancer in women after lung cancer worldwide. In Saudi Arabia and Malaysia, breast cancer was the highest type of cancer among woman (Elsayed et al., 2015). Although developments in breast cancer therapy, these chemotherapys associated side effects and often patients are concerned to use it, there have been interesting to use herbal medicine as antitumor remedy (Hadadi et al., 2018). Thus, the present study was undertaken to evaluate the activity of M. peregrina seed extract, which might provide natural drug for cancer therapeutic with less side effect to the normal system, against AU565 breast adenocarcinoma cells. 

·       1.2 Aims of study

This study aims to evaluate the cytotoxic effect of Moringaperegrina seed extracts and to detect its ability to stop the cancer progression of the most prevalent cancer types(breast cancer MCF-7) 

2. Leteritur review

Chapter Two

2.1Leteritur review

·       Plant Materials

MoringaPeregrinaEthanol Extracts Preparation (MPSE)100g of powdered dried seeds were immersed in 500 mL of ethanol for 3 days. After that the mixture was filtered by filter paper. The liquid filtrate was subject to rotary evaporator at (45°C - 50°C) in order to obtain the ethanol extract, whereas the residues were introduce to further immersion with ethanol (Albaayit et al., 2014).

·       Cell culture condition 

Human cancer cell line were used to evaluate the cytotoxic effect of ethanolic extract.

MCF-7 breast cancer cell line was purchased from ATCC (American Type Culture Collection) company and cell line cultured in DMEM (Dulbecco's Modified Eagle Medium) with 50 ml fetal bovine serum (FBS), 5 ml 2 mm L-glutamine and and 5 ml 100U penicillin / 0.1 mg/ml streptomycin penicillin/streptomycin. Cells were maintained in vented flask at 37Cº ina 5% CO2 atmosphere with 95% humidity.

·       Cell thawing

        The required cells were removed from liquid nitrogen container and dissolved rapidly in a 37°C water bath. The cells then suspended in 10 ml culture media in a 25 cm²flask and incubated 37°C in a 5% CO₂ atmosphere with 95% humidity.

·       Cell passage and seeding

Cells were maintained in 25 cm² culture flasks. When cells became confluent, media was removed, and cells washed with 3ml phosphate buffer saline (PBS), 1ml of trypsin/EDTA added to the flask and incubated for 5-10 min to detach the cells from the surface of the flask at 37°C. Thereafter, 3ml of fresh media was added to the cells with good mixing. One milliliters of cell’s suspension were maintained in 25 cm² cell culture flask with 10 ml fresh media.Cells were seeded in different densities in triplicates. By using a multichannel pipette, 100 µL of cell suspension seeded per well in 96-well plates and incubated overnight to allow for cell attachment

·       Anti-proliferative assay on MCF-7     

The cytotoxic activity of Moringa Peregrina of ethanolic extract will be determined using MTT assay. MTT (3-(4,5-dimethylthiazol-2w-yl)-2,5-diphenyl tetrazolium bromide) assay was used in order to estimate cell viability after incubation with various concentration of plant extract.

TheMCF-7 cell line  were seeded in a 96-well plate at a density of 5.0×103 cells/well, and then treated with different concentrations of MPSE ranging from 25 to 400ug/ml and incubated the plate for 48hr. 20 uL of MTT solution (0.5 mg/mL) was added to each well and incubated the plate at 37Cº for 3 hours. The purple formazan crystal was dissolved in 200 uL of dimethyl sulfoxide (DMSO) and absorbance was measured at 570 nm wavelength using a microplate reader (Tecan, Austria). Each experiment was carried out three times with triplicate wells for each concentration (Albaayit et al., 2020; Albaayit et al., 2015).

·       Statistical Analysis 

The results of cytotoxicity were assessed using the IC₅₀ value (50% cell growth inhibition) as a response parameter, which was calculated with theGraphPad prism 5.0 program by plotting cell survival against the concentrations of the natural products tested.  IC₅₀ values listed are mean ± SD (standard deviation) (µg/ml). Excel sheets was used for drawing a graph of extract concentration versus the viability percentage for the plant.

·       Results and Discussion 

In the pharmaceutical industry, natural product has become more interesting for the preparation of potential safe drug because of their wonderful medicinal properties (Albaayit et al., 2020; Al-Bahran et al., 2020; Elsayed et al., 2016). Traditionally, seeds of M. peregrina have been used to treat skin problems, abdominal pain and diabetic (Albaayit & Ozaslan, 2019; Al Dhaberi et al., 2016). Although only a few studies available on its pharmacology efficacy (Senthilkumar et al., 2018), the activity of the seed against cancer was limited interest.

Accordingly, we interest to evaluation the antiproliferative efficacy of MPSE against breast cancer cell line that may be paved as a potential cancer therapeutic drug. In the U.S. NCI plant screening program, if the IC₅₀ value (concentration causing a 50 percent cell kill) in carcinoma cells is less than 20 μg / mL for the crude extract after incubation between 48 and 72 hours, then it is mostly represented to have cytotoxic effect in vitro (Ackova, et al., 2016).  In this study, the cytotoxicity assay of MPSE on the viability of MCF-7 cells is 69%.at 400ug/ml with IC₅₀ values 13.7ug/ml Figurel.

The inhibited level in breast cancer cell (MCF-7) culture might be due to antioxidant capacity provided byMPSE as an inverse relationship between antioxidants and the growth of cancer cells (Albaayit & Maharjan,2018; Koheil et al., 2018; Saeidnia & Abdollahi, 2013). This plant has been reported to contain high amounts of flavinoid, tocopherols and phenolic compounds, which are attributed to the antiproliferative properties of extract on cancer cells (Huang et al., 2010). The present study outcome is in consist with previous study by Adebayo et al who reported that M. oleifera has a significant cytotoxic effect against the breast cancer cell.

Conclusion

In conclusion, ethanol extract of Moringa peregrina seed has cytotoxic activity against MCF-7 breast cancer cell line. This study has suggested seeds of Moringaperegrina as good anticancer agents, may be paved for further research study to understand the mechanism of action of this plant.

Further studies are recommended: to isolate and identify the major essential oil components and to test specifically their cytotoxic effect.