INTRODUCTION:
Breast cancer is the most common cancer among women, according to global cancer statistics, with approximately 1.7 million new cases and nearly 522,000 related deaths globally in 20121. Hormonal status, hormonal therapy, lifestyle, smoking, drinking alcohol, dietary habits, and predisposing genetic factors in women such as BRCA-1, BRCA-2, and P53 mutation are all high-risk factors for breast cancer progression2.
Several synthetic chemotherapeutic agents, including Cisplatin, Carboplatin, Taxol, Paclitaxel, Fluorouracil (5-FU), and Mitomycin, are currently used for the treatment of breast cancer, but long-term administration of these anticancer drugs leads to massive systemic side effects and the development of multiple drug resistance in breast cancer tissues3. Systemic chemotherapy using clinically approved anti-cancer medications results in poor response, severe toxicity, and the development of multidrug resistance4. Conventional chemotherapy agents, unfortunately, have several disadvantages, such as non-specific target, resulting in systemic toxic effects, and harmful to normal cells, resulting in chronic toxicity with manifestations such as alopecia, mucositis, and thrombocytopenia5. Therefore, researchers focused on the validation of some natural anticancer agents in order to deliver an effective cancer treatment without any treatment-related complications6. Plants are an important source of exceptional anticancer agents such as vincristine, doxorubicin, and paclitaxel; thus, eating fruits and leafy vegetables has been shown to reduce the risk of cancer; therefore, much attention is now being paid to the development of chemopreventive anticancer agents derived from medicinal plants7,8. Several studies have reported that isolated plant-derived anti-cancer agents from medicinal plants have high effectiveness and few side effects when used to moderate their potential anti-cancer activity9.
Rumex abyssinicus (R. abyssinicus) is a perennial weed plant belonging to a family of Polygonaceae10. It is a large annual herb up to 4 m high; it grows in tropical Africa, particularly in Central and Eastern Africa, more commonly grown in cultivated lands11. It is commonly known as meqmeqo in both Tigrigna and Amharic in Ethiopia12. In Ethiopia, traditionally the roots of R. abyssinicus are used for the treatment of various types of diseases such as hypertension, hepatitis, malaria, gonorrhoea, constipation, neuralgia, rheumatism, migraine, ear problems, rabies, scabies, wound, typhus, diabetes12. Behailu et.al.,13 reported that root extract of R. abyssinicus has shown in vitro anticancer activity against prostate, brain and in leukaemia cancer cell lines. Therefore, in the current study, the researchers aimed to evaluate in vitro anticancer activities of the organic root extracts of R. abyssinicus on MCF-7 Breast cancer lines.
MATERIALS AND METHODS:
Plant material collection and authentication:
The roots of R.abyssinicus were collected in December 2019 in Axum, Tigray region's Central zone, and the plant was authenticated by the Department of Biology, Addis Ababa University, and it can be deposited in Ethiopia's National Herbarium.
Preparation of plant extract:
According to the study conducted by Das et.al.,in 201014 the root was washed with tap water to remove dirt and impurities then it was dried at 25oC in shade and optimal ventilation for two weeks or 15 days. The root was ground into powders for further processing using an electrical grinder, and the powder was stored in a plastic container. The powdered root samples were subjected to successive extraction with Diethyl ether, chloroform, ethyl acetate, and ethanol using a Soxhlet apparatus. During this extraction method, the extraction starts from a low polar solvent (Diethyl ether) to a high polar solvent (ethanol) to ensure that a wide polarity range of compounds could be extracted. During extraction, solvents diffused into the plant material and solubilised.
Extraction obtained
Percentage of yield = ------------------------------ x 100
Total amount of material
Cell culture and MTT assay:
The Breast Cancer cell line (MCF-7) 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 (Himedia, India) in CO2 incubator at 37˚C with 5% CO2. The cells were washed with 200 μL of 1X PBS, and then the cells were treated with various test concentration of compound in serum free mediaand incubated for 24 hrs. 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 37˚C for 4 h using CO2 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 microplate reader15.
% Growth Inhibiton = Mean OD of individual Test Group \Mean OD of control Group ×100
All experiments were performed in triplicate (n=3) and the results were expressed as Mean±SD and Statistical differences between the Mean±SD of the anticancer activity (% cell viability) between the control (without plant extract) and experimental group was assessed by Excel 2007 software and Graph Pad Prism 7.0.The data was analyzed by One way ANOVA and Tukey Post Hoc test with the level of significance set *p values <0.05, **p values <0.01, ***p values <0.01
RESULTS AND DISCUSSION:
The colour, consistency and percentage yield of R. abyssinicus roots:
The root extracts of R. abyssinicus showed the highest percentage of yield in chloroform (3.26g/100g), diethyl ether (2.8g/100g), ethanol (1.005g/100g) and ethyl acetate (0.57g/100g) solvents respectively as shown in table 1.The highest percentage of yield obtained in chloroform which indicated that the roots of R. abyssinicus contained non-polar phytoconstituents. Obtained results were disagreement with Limenew Abate and Birhanu Ayalew16reported that the roots of R. abyssinicus showed highest percentage of yield in polar solvent methanol (1.62g/20g), followed by chloroform (0.6g/20g), ethyl acetate (0.57g/20g) and diethyl ether (0.5g/20g) by maceration extraction method.
Table 1: Colour, consistency and percentage yield (w/w) of different organic solvent root extract of R. abyssinicus
|
Solvent |
Colour |
Consistency |
Percentage of yield (g/100g) |
|
Root extract of R. abyssinicus |
|||
|
Diethyl Ether |
Green |
Sticky |
2.8 |
|
Chloroform |
Red |
Sticky |
3.26 |
|
Ethyl acetate |
Dark red |
Sticky |
0.57 |
|
Ethanol |
Green |
Non-Sticky |
1.005 |
Qualitative phytochemical analysis:
The qualitative phytochemical analysis of diethyl ether, chloroform, ethyl acetate and ethanolic root extract of R. abyssinicus were showed in the table 2, all the tested four organic root extracts of R. abyssinicus contained alkaloids, flavonoids, phenolic compounds and terpenoids. The diethyl ether root extract of R. abyssinicus shown that presence of phytoconstituents such as alkaloids, flavonoids, phenolic compounds, terpenoids, quinines and remaining amino acid and proteins phytoconstituents cardiac glycosides, saponins, steroids were absent (table 2). The chloroform root extracts of R. abyssinicus indicated that the presence of phytoconstituents such as amino acid and protein, alkaloids, flavonoids, phenolic compounds, terpenoids, quinines, coumarins and remaining phytoconstituents such as cardiac glycosides and saponins were absent.
Table 2: Qualitative phytochemical analysis of different organic solvents crude root extract of R. abyssinicus.
|
Phytoconstituents |
Solvent extracts |
|||
|
Diethyl ether |
Chloroform |
Ethyl acetate |
Ethanol |
|
|
Alkaloids |
+ |
+ |
+ |
++ |
|
Flavonoids |
+ |
+++ |
++ |
++ |
|
Phenolic compounds |
+ |
+++ |
++ |
++ |
|
Cardiac glycosides |
- |
- |
- |
+ |
|
Terpenoids |
+ |
++ |
+ |
+ |
|
Saponins |
- |
- |
+ |
+ |
|
Steroids |
- |
+ |
+ |
+ |
|
Quinones |
+ |
++ |
- |
++ |
|
Coumarins. |
- |
++ |
+ |
++ |
|
Amino acid and protein |
+ |
++ |
++ |
++ |
“+” indicates presence and “-” indicates absence of Phytoconstituents
“+” Minimum; ++: Moderate +++ Maximum
The ethyl acetate root extract of R. abyssinicus indicated that the presence of phytoconstituents such as amino acid and protein, alkaloids, flavonoids, phenolic compounds, terpenoids, saponins, coumarins, steroids and remaining phytoconstituents such as cardiac glycosides and quinones, were absent (table 2). The ethanolic root extracts of R. abyssinicus result indicated that presence of all phytoconstituents such as amino acid and protein, alkaloids, flavonoids, phenolic compounds, cardiac glycosides, terpenoids, saponins, coumarins, quinones and steroids (table 2). In agreement to this finding Limenew Abate and Birhanu Ayalew16 were reported that the methanolic root extract of R. abyssinicus showed the presence of flavonoids, tannins and phenolic compounds and glycosides. Moreover Hadera et al.,17 reported that the organic leaf extract of same genus medicinal plant R. nervosus shown the presence of secondary metabolites such as alkaloids, flavonoids, tannins/phenols, cardio glycosides, terpenoids and saponins. Alkaloids and flavonoids have been reported to have been reported to possess anticancer activities against different cell lines18.
In vitro cytotoxicity effect of root extracts of R. abyssinicus on MCF-7 cell lines.
Diethyl ether root extract of R. abyssinicus has shown inhibitory in vitro cytotoxicity effect against MCF-7 breast cancer cell lines. The results as shows in figure 1, at concentration 25 µg/m, the percentage of growth inhibition found to be 3.97 % and 500 µg/mL and the percentage of growth inhibition found to be 50.04 % on of MCF-7 breast cancer cell lines. Moreover, the cell viability of MCF-7 breast cancer cell lines were decreased with increasing concentration of diethyl ether root extract of R. abyssinicus with IC50 value of 503 ±13.28µg/mL. Diethyl ether root extract of R. abyssinicus at 250 and 500 µg/mL concentrations exhibited 40.18 and 50.04 % inhibitory effects on MCF-7 breast cancer cell lines proliferation, which significantly difference with control group **p values <0.01, however the lowest concentrations 25, 50 and 100µg/mL concentrations indicated that less significant *P values <0.05 compared with control group. The dose depended in vitro cytotoxicity effect of diethyl ether root extract R. abyssinicus on MCF-7 breast cancer cell lines due to presence of secondary metabolites such as alkaloids, flavonoids, phenolic compounds, terpenoids, quinones, and coumarins.
Figure 1: The effect of diethyl ether root extract of R. abyssinicus on cell viability of MCF-7 breast cancer cell lines each bar represents the Mean ± SD ( n=3). *P values <0.05, **p values <0.01, ***p values <0.001as compared with the control value.
The changes in cell morphology and number of MCF-7 breast cancer cell lines after treatment with different concentrations of (25 to 500 μg/mL) diethyl ether root extract of R. abyssinicus were changed compared with control. Sajuthi 19 reported that organic fractions of Gynura pseudochina shown significant in vitro anticancer activity against MCF-7 breast cancer cell lines due to presence of alkaloids and terpenoids.
Chloroform root extract of R. abyssinicus has shown inhibitory in vitro cytotoxicity effect against MCF-7 breast cancer cell lines. The results given in figure 2, shows at concentration 25 µg/mL the percentage of growth inhibition found to be 21.43% and 500 µg/mL the percentage of growth inhibition found to be 81.25 % on of MCF-7 breast cancer cell lines. Moreover the cell viability of MCF-7 breast cancer cell lines were significantly decreased with increasing concentration of diethyl chloroform root extract of R. abyssinicus with IC50 value of 56.71±4.73 µg/mL.
As shown in the figure-3, chloroform root extract of R. abyssinicus at 250 and 500 µg/mL concentrations exhibited 66.6 and 81.25 % inhibitory effects on MCF-7 breast cancer cell lines proliferation, which more significantly difference with control group ***p values <0.001, however the lowest concentrations 25, 50 and 100µg/mL concentrations indicated that significant **p values <0.01 compared with control group. The dose depended promising in vitro cytotoxicity effect of chloroform root extract of R. abyssinicus on MCF-7 breast cancer cell lines due to presence of maximum number of secondary metabolites such as alkaloids, flavonoids, phenolic compounds, terpenoids, quinines and coumarins.
Figure 2: The effect of chloroform root extract of R. abyssinicus on cell viability of MCF-7 breast cancer cell lines each bar represents the Mean ± SD ( n=3). *P values <0.05, **p values <0.01, ***p values <0.001as compared with the control value.
After 24 hrs of treatment with chloroform, root extract normal cell morphology was distorted in MCF-7 breast cancer cell lines compared with control. From the microscopic studies it was cleared that chloroform root extract of R. abyssinicus treatment can induce death and severe morphological changes in MCF-7 breast cancer cell lines.
Ethyl acetate root extract of R. abyssinicus has
shown inhibitory in vitro cytotoxicity effect against MCF-7 breast
cancer cell lines. The results given in figure 3 shows at concentration of 25
µg/mL the percentage of growth inhibition found to be7.39 % and at 500 µg/mL
concentration, the percentage of growth inhibition found to be 69.88 % on MCF-7
breast cancer cell lines. Moreover, the cell viability of MCF-7 breast cancer
cell lines were significantly decreased with increasing concentration of ethyl
acetate root extract of R. abyssinicus with IC50 value of 78.78
34.3µg/mL.
As shown in the figure 3, ethyl acetate root extract of R. abyssinicus at concentrations from 50-500 µg/mL concentrations exhibited in range of 22.45 to 69.88 % inhibitory effects on MCF-7 breast cancer cell lines proliferation, which was shown significantly **p values <0.01 difference with control group, however the lowest concentrations 25 µg/mL concentrations indicated that significant *P values <0.05 compared with control group. The dose depended promising in vitro cytotoxicity effect of ethyl acetate root extract of R. abyssinicus on MCF-7 breast cancer cell lines due to presence of secondary metabolites such as alkaloids, flavonoids, phenolic compounds, terpenoids, quinines and coumarins.
Figure 3: The effect of ethyl acetate root extract of R. abyssinicus on cell viability of MCF-7 breast cancer cell lines each bar represents the Mean ± SD ( n=3). *P values <0.05, **p values <0.01, ***p values <0.001as compared with the control value.
After 24 hrs of treatment with ethyl acetate root extract normal cell morphology of was distorted in MCF-7 breast cancer cell lines compared with control. From the microscopic studies it was cleared that ethyl acetate root extract of R. abyssinicus treatment can induce death and severe morphological changes in MCF-7 breast cancer cell lines.
Ethanolic root extract of R. abyssinicus has shown inhibitory in vitro cytotoxicity effect against MCF-7 breast cancer cell lines. The results given in figure 4shows at concentration 25 µg/mL the percentage of growth inhibition found to be moreover the cell viability of MCF-7 breast cancer cell lines were significantly decreased with increasing concentration 15.67 % and 500 µg/mL and the percentage of growth inhibition found to be 74.60 % on of MCF-7 breast cancer cell lines with IC50 value of 71.38±4.12µg/mL. As shown in the figure.7, ethanolic root extract of R. abyssinicus at concentration 500 µg/mL concentrations exhibited 74.60 % inhibitory effects on MCF-7 breast cancer cell lines proliferation, which was shown more significantly ***p values <0.001 difference with control group, however the lowest concentrations 250 and 100 µg/mL indicated that significant **p values <0.01 compared with control group and 25 and 50 µg/mL. In vitro cytotoxicity effect of ethanolic root extract of R. abyssinicus on MCF-7 breast cancer cell lines due to presence of all tested concentrations shown less significant *P values <0.05 compared with control group. The dose depended promising insecondary metabolites such as alkaloids, flavonoids, phenolic compounds, terpenoids, quinines coumarins and cardiac glycosides.
Figure 4: The effect of ethanolic root extract of R. abyssinicus on cell viability of MCF-7 breast cancer cell lines each bar represents the Mean ± SD ( n=3). *P values <0.05, **p values <0.01, ***p values <0.001as compared with the control value
After 24 hrs of treatment with ethanolic root extract normal cell morphology of was distorted in MCF-7 breast cancer cell lines compared with control. From the microscopic studies it was cleared that ethanolic root extract of R. abyssinicus treatment can induce death and severe morphological changes in MCF-7 breast cancer cell lines.
The in vitro cytotoxicity effect of R. abyssinicus root diethyl ether , chloroform, ethyl acetate and ethanolic extracts at the concentrations of (25, 50, 100, 250, and 500µg/ml) were shown in the figure 5indicated that the R. abyssinicus root extracts exhibited in vitro cytotoxicity on MCF-7 human breast cancer cell lines in the range of 3.97% to 81.25% with the significant cytotoxicity effect was shown by chloroform root extract of R. abyssinicus against MCF-7 human breast cancer cell lines.
Figure 5: The Comparative effect of organic root extracts of R. abyssinicus on cell viability of MCF-7 breast cancer cell lines each bar represents the mean ± SD ( n=3).
CONCLUSION:
According to the current findings, the presence of secondary metabolites such as alkaloids, flavonoids, tannins and phenolic compounds, terpenoids, quinines, and coumarins in R.abyssinicus organic root extract has shown significant in vitro cytotoxicity activity on MCF-7 breast cancer cell lines.
ACKNOWLEDGMENTS:
The authors sincerely thanks to the National herbarium, Department of Biology, Addis Ababa University, Ethiopia for authentication of medicinal plant.
FINANCIAL SUPPORT:
This project work was funded by Ministry of Education, Ethiopia
CONFLICT OF INTEREST:
The authors confirm that this content has no conflict of interest.
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Received on 06.01.2022 Modified on 08.06.2023
Accepted on 04.12.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(11):5410-5415.
DOI: 10.52711/0974-360X.2023.00876