Acknowledgment was also given to all the team members and faculty members who participated in the Gynura divaricata project and who helped me during this research project. The internship report contains an overview of the internship institution and the activities, tasks, and projects the author worked on during the internship. Gynura divaricata has been widely used as a traditional Chinese medicine to treat a variety of ailments.
MTT test was used to evaluate the cytotoxicity of the crude ethanolic extract in human cell line (HEK-293). This institution was founded in 2014 and currently has 2 faculties: the School of Life Sciences and the School of Business (iSB). In the life science faculty, i3L offers undergraduate degree programs such as Biomedicine, Biotechnology, Bioinformatics, Food Sciences and Nutrition, Food Technology, and Pharmacy.
They are also encouraged to participate in the discovery and development of new therapeutic strategies, both in private industry and government agencies. The department has the vision to bridge the research gap between human health and drug development through research and innovation; and the mission to create a community of Biomedicine experts who can contribute to the development of communities around the world, especially in the Biomedical science field, and disseminate innovations and research results in Biomedicine, both nationally and internationally. Some research has been done to study human diseases, food innovation and nutrition, bioproducts and information technology in life sciences.
In addition, i3L has published a scientific journal called Indonesian Journal of Life Sciences (IJLS), which contains original research articles, review articles and short communications covering all aspects of life sciences.
PROJECT DESCRIPTION
- Internship Project 1. Project Background
- Scope of the project
- Objectives / Aims
- Problem formulation and Proposed Solutions 1. Problem Formulation
- Characterization of flavonoid and phenolic contents of G. divaricata crude extract
- Cytotoxicity testing in mammalian cells (MTT Assay) Cell Culture
- Statistical analysis
The measurement of the flavonoid and phenol content of the extract was made using aluminum chloride (AlCl3) and the Folin-Ciocalteu colorimetric method. MTT assay was also performed to examine the cytotoxicity of the extract against human cell lines (HEK-293). One of the most common cytotoxicity tests performed in cell toxicity studies is the MTT assay.
A rotary evaporator was used to remove the solvent from the crude mixture, yielding a thick semi-dry paste of the crude extract. The crude extract was placed in an evaporating dish and further heated on a hot plate at a temperature of 80°C until the weight of the extract was stable. For the crude extract solution, 30 mg of the crude extract was diluted with 20% aqueous methanol solution (v/v) in a 10 ml volumetric flask.
Six ml of each concentration of the standard dilutions, as well as 6 ml of the sample solution, were transferred to individual test tubes. The measurement of the crude extract sample was done in triplicate and the absorbance was compared with the calibration curve. A stock solution of 1000 ppm was made by mixing 10 mg of gallic acid with 10 ml of methanol.
For the sample solution, 100 mg of the crude extract was diluted with 20% methanol in water (v/v) in a 10 ml volumetric flask. One ml of each concentration of the standard solution, as well as 1 ml of the sample solution, were placed individually in a test tube. The contents of the vial were transferred to a 15-ml centrifuge tube containing 1 ml of incomplete DMEM medium and centrifuged at 12,000 rpm for 5 minutes.
Then the supernatant was removed and 1 ml of complete DMEM was added to the centrifuge tube. Then 1 ml of FBS or incomplete medium was added to the flask to wash the cells. The contents of the flask were transferred to a 15 ml centrifuge tube and centrifuged at 12,000 rpm for 5 minutes.
FINDINGS
Results
- Extraction of G. divaricata leaves
- Cytotoxicity testing in mammalian cells (MTT Assay)
The absorbance of the sample was compared with the standard curve to obtain the flavonoid content of the extract. The flavonoid content was calculated using the absorbance data and the equation from the quercetin standard curve. The absorbance of the sample was compared with the equation from the gallic acid standard curve to obtain the phenolic content of the extract.
Phenolic content was calculated using absorbance data and the equation from the gallic acid standard curve. The obtained absorbance result of the MTT assay was later used to determine the percentage of cell viability and the IC50 value, which is shown in Figure 4.
Analysis/Discussion
- Extraction of G. divaricata leaves
- Cytotoxicity testing in mammalian cells
The reason for the low yield may be due to the extraction method, which was performed only once in this study, and the difference in temperature. In the second batch of extraction, the ethanol from the first batch was reused, as this step was commonly performed in several plant extraction studies to reduce the amount of waste and solvents required (Gong et. al., 2014; Donno et. al., 2020 ). Extraction efficiency is affected by various factors such as temperature, solvent type, extraction duration, and solid-to-liquid ratio, of which the solvent has a strong influence on the extraction yield and chemical composition of the extracts (Choung et al. 2014).
Furthermore, according to Saptarini & Wardati (2020), the effectiveness of the reflux method depends on the effective diffusion and solubility of secondary metabolites. Some parameters that were suspected to have a role in the amount of TFC and TPC are ethanol concentration, temperature and plant-to-solvent ratio. In the case of TPC, changes in solvent concentration led to modifications in polarity and solubility, resulting in changes in extraction yield.
In contrast, a study by Chen et. divaricata has low cytotoxic activity against HepG2 cells at a concentration of 100 mg/ml. Cell processing was also done by different people in each of the replicates. However, the results were not as expected, as the doxorubicin-treated cells were found to have the highest absorbance among the others, indicating that this resulted in the highest cell viability.
It is strongly suspected that the bright red colored property of DOXO interferes with the formazan dye in the measurement of metabolic activity, resulting in misleading measurements (Luis et al., 2019). The first 2 MTT runs showed an upward trend in the IC50 chart. In addition, it was also possible that the extract used in the first 2 trials had not been properly diluted, so that the amount of extract was insufficient to exert its effect on the cells.
Thus, for the third experiment, the assay was performed by one person and the stock solution of the extract was made again and became the positive control. supernatant after MTT incubation, which may play a role in the difference in IC50. 2021) also mentioned that discrepancies between variables make it difficult to compare the measured OD values between different studies. The use of the MTT assay to test plant extract is also known to result in false-positive results for viability. 2017), plant extracts have interfering effects with the MTT compound, where even in the case of total cell death it can still give rise to increases in viability. The inaccuracy of the assay was often caused by the reduction of the formazan dye, which primarily depends on cell metabolism and sometimes reflects cell viability (Ghasemi et. al., 2021).
CONCLUSION AND RECOMMENDATION
SELF-REFLECTION
During this internship project, one of the things I understood is that learning takes on many faces. From performing basic maintenance of cultures to performing synchronization and assays, my knowledge of biomedical theories has been transformed into a series of practical techniques and skills that I can now implement in real research scenarios, all thanks to my internship. I got a chance to learn how to communicate and build relationships with the people I worked with.
Fortunately, I can overcome those problems with the help of my friends and the people I work with. From this internship I also learned that everything has to go through trial and error to achieve success.
APPENDICES
Mapping for MTT Assay, concentration in mg/ml
Obtained samples’ absorbance of cytotoxicity evaluation (MTT Assay) MTT Assay Absorbance
Identification of phenolic compounds, antioxidant activity and anti-cancer effects of the extract obtained from the shoots of Ornithogalum narbonense L. Avoiding the interference of doxorubicin with MTT measurements on the MCF-7 breast cancer cell line. High-dose chemotherapy and autologous bone marrow support as consolidation after standard-dose adjuvant therapy for high-risk primary breast cancer.
Morphological identification and in vitro cytotoxic evaluation of crude extracts of Jak-Na-Rai (Gynura divaricata (L.) DC.).
