Thesis Abstract

Abstract
The study aimed to investigate the cytotoxicity and genotoxicity of Solanum tuberosum leaves using two bioassays, the brine shrimp lethality assay and Allium cepa chromosomal aberration assay. The brine shrimp lethality assay was conducted to determine the cytotoxic potential of the leaf extract, while the Allium cepa chromosomal aberration assay was employed to assess its genotoxic effects. The results of the brine shrimp lethality assay indicated that the leaf extract exhibited cytotoxic effects in a concentration-dependent manner, with lower concentrations showing higher lethality to the shrimp larvae. The LC50 value was determined to be 78.52 µg/mL, indicating moderate cytotoxicity. In the Allium cepa chromosomal aberration assay, the leaf extract induced various chromosomal aberrations in the root meristem cells of the onion bulbs, including chromatin bridges, sticky chromosomes, and micronuclei. The frequency of chromosomal aberrations increased with higher concentrations of the leaf extract, indicating a dose-dependent genotoxic effect. The mitotic index was also significantly reduced in the treated groups compared to the control, suggesting cell cycle disturbances caused by the leaf extract. The correlation between cytotoxicity and genotoxicity was further analyzed, revealing a positive relationship between the two endpoints. The cytotoxic effects observed in the brine shrimp assay were consistent with the genotoxic effects seen in the Allium cepa assay, indicating that the leaf extract of Solanum tuberosum possesses both cytotoxic and genotoxic properties. The presence of bioactive compounds such as alkaloids, flavonoids, and phenolics in the leaf extract may be responsible for these effects. Overall, the findings of this study provide valuable insights into the potential health risks associated with the consumption or exposure to Solanum tuberosum leaves. The combination of the brine shrimp lethality assay and the Allium cepa chromosomal aberration assay proved to be effective in evaluating the cytotoxic and genotoxic effects of plant extracts. Further research is warranted to identify the specific compounds responsible for the observed effects and to elucidate the mechanisms underlying the cytotoxic and genotoxic activities of Solanum tuberosum leaves.

Thesis Overview

INTRODUCTION

 

Rationale of the Study

Cancer is a generic term for a large group of diseases that can affect any part of the body. One defining feature of cancer is the rapid multiplication of abnormal cells that grow beyond their usual boundaries, and which can then affect adjoining parts of the body and spread to other organs, the latter process is referred to as metastasizing. Metastases are the major cause of death from cancer (WHO, 2015). In a statistical study by WHO (2012), different types of cancer figures among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer related deaths. According to GlobalCan (2012), in the Philippines, people who are diagnosed with cancer per year is estimated to be 98, 200 and people who died from cancer is around 59, 000 deaths per year.

There are number of antineoplastic or anticancer agents used for the treatment of cancer, this includes the alkylating agents, taxanes, topoisomerase II inhibitors, anthracyclines, antifolates, vinca alkaloids, campothecins, antimicrotubule inhibitor, antitumor antibiotics, antimetabolites and tyrosine kinase (TK) inhibitors (Katzung et al., 2012). In general, antineoplastic agents cure cancer by targeting selectively or non-selectively the cell division of abnormal cells leading to cell death. Generally anticancer drugs in either short-term or long-term use cause unwanted adverse effects such as heart problems, hormonal problems, bone and tissue problems or may arise to a secondary cancer (Aviva.co.uk, 2017).

Due to the severe effects it causes and leading to development of resistance of using the anti-cancer agents, there is a shift to discovering of new sources of therapy. Nature has been one of the inevitable origins of therapeutics (Koehn & Carter, 2005; Narang & Desai, 2009; Newmann & Cragg, 2013), and many of these natural products have been identified as treatments.

Some plants have also been recognized for promising anticancer properties. Isolation of podophyllotoxin and several other compounds (known as lignans) from the common mayapple (Podophyllum peltatum) ultimately led to the development of drugs used to treat testicular and small cell lung cancer (Pettit et al., 1995). Some plant derived compounds such as the polyphenols, taxols and brassinosteroids have showed that they can inhibit multiplication of cancer cells and promote apoptotic cell death. Researchers are now diverging their studies on plants for possible future treatment of cancer.

Potato has many constituents. The constituents found in the potatoes are known to possess a variety of biological activities such as antifungal, insecticidal, antiviral, and antiestrogenic activities (Chandrasekara & Kumar, 2016). One of the constituents are the glycoalkaloids. The major glycoalkaloids present in potato are a-chaconine and a-solanine, making up 95% of total glycoalkaloids. High total glycoalkaloid (TGA) levels are found in tissues – in fruit, leaves, stems, tubers, tuber eyes, jacket, sprouts and damaged tissues which has a function to protect plants from bacterial and fungal diseases as well as from pests. Glycoalkaloids have potential anti-carcinogenic effects on the human cervical, liver, lymphoma, and stomach cancer cells (Friedman et al., 2005). The glycoalkaloids from eggplants have the ability to penetrate and selectively destroy cancer cells, leaving normal cells unharmed (Fassa, 2012; Yang et al., 2016). The anti-carcinogenic effects of glycoalkaloids are dependent on its concentration (Friedman et al., 2005).

The described results may make it possible to relate to the study of potato leaves since there are limited studies on it unlike the other plant parts of potato like the tuber (McCue, 2009). The aim of the study is to assess if the constituents which mostly contains glycoalkaloid, present in the potato leaves can be used as a potential anti-cancer agent and to be evaluated for its possible cytotoxicity and genotoxicity. This study aims to evaluate the possible cytotoxicity and genotoxicity of the potato leaf using liquid-liquid extraction method.

 

Objectives of the Study

This study aims to determine the cytotoxicity and genotoxicity of Solanum tuberosum leaves using Brine shrimp lethality bioassay and Allium cepa chromosomal aberration assay.

Specifically, this study aims to determine the following:

1. Determine the percent mortality based on the dead nauplii after exposure to the leaf extract.
2. Determine the percent cytotoxicity of the leaf extracts based on the percent mortality of dead nauplii.
3. Determine the median lethal concentration (LC50) of the leaf extract.
4. Determine the genotoxicity activity of the plant extract based on the following:
5. percent root growth after exposure.
6. percent chromosomal aberration as indicated by the presence of bridges, fragments, laggards or vagrants.
7. mitotic index after exposure.

 

Significance of the Study

This study can provide beneficial information pertaining to the scope of the study to the medical researchers and future undergraduates. Researchers who are expert to the likes of this study will be able to gather information from this study regarding to the progress of current researches pertaining to the potential cytotoxicity and genotoxicity of plants or a future drug for cancer.

To organizations that raises awareness on cancer such as the Philippine Society of Medical Oncology (PMSO) and to name a few can benefit from the possible positive result of this research. To pharmaceutical companies, they can further research on the cytotoxic and genotoxic activity of the potato and develop a new drug that can be beneficial to the community.

People with cancer may be hopeful for new anti-cancer course of treatment. This will also help farmers to reduce the waste of leaf, it can still be utilize for alternative treatment of cancer. In the pharmaceutic field, this will help researchers to recognize the potential benefits of this plant and conduct future studies using this plant to see possible other possible pharmacologic activities.

Local government and academic institutions may also choose to research or support their students to perform studies about other potential activities of the plant sample or to continue in improving the study by using other parts of the potato and their potential cytotoxic and genotoxic activity.

 

Scope and Limitation

This study focused on identifying the cytotoxic and genotoxic activities of leaves of Solanum tuberosum procured in Abatan, Mountain Province. The extraction of the constituents was performed by liquid-liquid extraction technique which employed using a mixture methanol and chloroform (2:1, v/v)(Wang et al., 1972). The cytotoxicity activity was evaluated using mature Artemia nauplii. The genotoxicity activity was evaluated the Allium cepa Chromosomal Aberration Assay. The study was conducted in three trials in triplicates having two groups, namely the control, and treated group. The control group was sea water in the Brine shrimp Lethality Assay and distilled water for the Allium cepa Chromosomal Aberration Assay. Range finding was conducted with series of dilution of the plant stock solution and there were 5 concentrations, 100%, 50%, 25%, 13% and 5% test solutions. The 100%, 50% and 13% test solutions were used for Brine shrimp Lethality Assay and Allium cepa Aberration Assay was based from the result of the range finding test.

The study was conducted in the laboratory rooms of the Department of Biology and Department of Pharmacy, University of San Carlos, Talamban Campus, and allotted time for experimentation was during first semester of S.Y. 2017-2018.

 

 

 

 

 

 

 

 

 

 

 

Definition of Terms

           

            Median Lethal Concentration (LC50).  It is the concentration of plant extract from the leaves of Solanum tuberosum that causes death to the mature nauplii.

Mitotic Index (MI). It is one of the parameters used to measure the genotoxicity of the leaf extract of Solanum tuberosum using Allium cepa by counting the number of cells dividing and not dividing.

            Percent Chromosomal Aberrations (CA). This refers to the activity of the test extract to induce changes in either total number of chromosomes or in chromosomal structure after the exposure of Allium cepa as indicated by the presence of bridges, fragments, laggards or vagrants.

Percent Cytotoxicity. This refers to the activity of the leaf extracts of Solanum tuberosum to the brine shrimps based from the percent mortality of treatment group and the control group.

Percent Mortality. This refers to the activity of the leaf extracts of Solanum tuberosum to the brine shrimps as indicated by the number of dead nauplii and total nauplii remaining from the total nauplii.

Percent Root Growth. It is one of the parameters used to measure genotoxicity. It refers to the activity of the leaf extracts of Solanum tuberosum to the root growth of the onion bulbs measured by the difference of root length of the control group and treatment group

            Plant Extract. It is the product obtained after using the extraction method using methanol and chloroform (2:1; v/v), reduced to half under the rotary evaporator and evaporated to dryness under the fume hood.