This credit practice report has been prepared as one of the requirements to pass the Credit Practice in the 7th academic year 2022/2023. This report has been prepared to include all the details of the project in which I participated during my internship period. The project entitled "In vitro cytoprotective study of Litsea oppositifolia stem extract against hydrogen peroxide-induced oxidative stress in HaCaT cells" is a project that aims to evaluate the protective effects of Litsea oppositifoliastem extract against ketocellular oxidative stress induced by hydrogen peroxide hydrogen (oxidative stress of human cells) line.
In Vitro Cytoprotective Study of Litsea oppositifolia Stem Extract against Hydrogen Peroxide-Induced Oxidative Stress on HaCaT Cells. Due to the nature of the process, the demand for anti-aging cosmetic products has increased, creating the need for alternatives to develop more anti-aging products. One of the plants that exert antioxidant activity is the Litsea genus, whereby several species of this genus have been recognized for their antioxidant effect.
First, a cytotoxicity test was performed on the extract to determine the toxic concentrations of the compounds, followed by cell viability measurements using the MTT assay. A cytoprotective assay was also performed to test the protective ability of the extract against damage caused by hydrogen peroxide, and cell viability of the samples was also measured by the MTT assay. To provide inter-, multi- and transdisciplinary education in the field of life sciences at an international level and to support the development of scientific and entrepreneurial thinking of students in accordance with their disciplines.
On the other hand, the Business School of the bachelor program offers four programs, which are Business and Entrepreneurship, Creative and Digital Marketing, International Applied Accounting and International Business Management.
PROJECT DESCRIPTION
Internship Project 1. Project Background
- Scope of the project
- Objectives / Aims
- Problem formulation and Proposed Solutions
However, there is still no study on the effects of the extract on cells. Investigating the cytotoxicity of Litsea oppositifoliastem extract by performing an in vitro cell viability assay on HaCaT cells. Investigation of the cytoprotective ability of Litsea oppositifoliastem extract against hydrogen peroxide (H2O2)-induced oxidative stress by performing an in vitro cell viability assay on HaCaT cells.
Investigate the cytotoxicity of Litsea oppositifolia stem extract and its cytoprotective ability to protect cells from hydrogen peroxide (H2O2)-induced oxidative stress on HaCaT cells compared to ascorbic acid, a well-known antioxidant. One genus of plants known for its antioxidant properties is the genus Litsea. Previously, DPPH assays and FRAP assays were performed to evaluate the antioxidant activity of Litsea oppositifoliastem extract.
Therefore, in this study, the antioxidant property of Litsea oppositifolia stem extract was tested by. The cytotoxicity of Litsea oppositifoliastem extract was tested on HaCaT cells by performing an MTT assay. Then, the cytoprotective effects of the extract were also tested on H2O2-induced HaCaT cells to evaluate the protective ability of the extract in alleviating the damage that was caused by excessive ROS formation.
DMEM was used during cell passaging and treatment preparation, while cDMEM was used during cell incubation. Cells were then treated in triplicate for each treatment as mapped in Figure 2.1 with the pretreatments of ascorbic acid, Litsea oppositifolia leaf and stem extracts, and H2O2, followed by another 24 hours of incubation. A 10 μL of MTT reagent was added to each of the wells after washing using 100 μL of DMEM per well.
Aluminum foil was used to cover the plate to prevent degradation of the MTT reagent. The cells were then pretreated with the pre-made Litsea oppositifolia extracts and ascorbic acid treatments according to the map in Figure 2.2 for 1 hour. Afterwards, the treatments were discarded and then replaced with a new set of treatments with the same concentration range that had been mixed with 50 μM H2O2, which was used to challenge the cells.
After incubation, 10 μL of MTT reagent was added to each well containing cells and treatments. Statistical analysis of cytotoxic and cytoprotective studies was done using GraphPad Prism 9.3.1 software.
FINDINGS
Result
Cell viability of HaCaT cells when treated with AA after 24 hours of incubation (1x104 cells/well). The data obtained were compared with the cells not treated with AA and presented as average. According to Figure 3.2, it was indicated that ascorbic acid did not show any cytotoxicity at all the concentrations used, as there was no significant difference in cell viability between the negative control (untreated cells) and the cells treated with AA.
Meanwhile, cytotoxicity assay was also performed to find the optimal concentration of H2O2 insult that would be used in the cytoprotective assay. The data were compared to the negative control consisting of cells untreated with H 2 O 2 and presented as mean. Based on Figure 3.3, it was shown that the concentration of 12.5 μM showed cytotoxicity as indicated by cell viability lower than 50%.
Meanwhile, the other concentrations also showed significantly lower cell viability compared to the negative control. However, the cell viability did not drop below 50%, making the treatments not considered cytotoxic. The results obtained from the cytoprotective assay of Litsea oppositifoliastem extract against H2O2 insults were as illustrated in Figure 3.4.
Cell viability of HaCaT cells after 1 h extract pretreatment followed by 24 h extract treatment and H2O2 insult (1x104 cells/well). The results were compared to the cells treated with 50 μM H2O2 alone (0 concentration extract) and presented as ± standard deviation of triplicate sample using one-way ANOVA. According to the findings illustrated in Figure 3.4, the extract of Litsea oppositifolia strain was suggested to exhibit cytoprotective potential at concentrations of 12.5 ppm and 25 ppm. In addition, the 50 ppm concentration also showed slightly higher cell viability compared to the 0 concentration, which may indicate that this concentration showed slight protective effects.
On the other hand, the cytoprotective ability of ascorbic acid was also tested on HaCaT cells. Cell viability of HaCaT cells after 1 h pretreatment with AA extract followed by 24 h treatment with extracts and H2O2 insult (1x104 cells/well). The results were compared to the cells treated with only 50 μM H2O2 (0 concentration of AA) and presented as mean ± standard deviation of triplicate samples using one-way ANOVA. Based on the findings illustrated in Figure 3.5, although not statistically significant, it was suggested that the protective effects from the concentration range used in the experiment were exerted at concentrations of 6.25 ppm and 12.5 ppm, which was indicated by the cell viability above. the cells treated only with 50 μM H2O2.
Analysis/Discussion
The cytotoxicity study is performed as the initial step to determine the toxicity of a tested substance, typically plant extracts or bioactive compounds isolated from plants, by analyzing the cell viability. Moreover, an extract or compound can be considered cytotoxic if the cell viability is reduced to about 50% upon treatment with the substance (Ogbole, Segun, & Adeniji, 2017). However, the formazan crystals can be dissolved in organic solvents such as isopropanol, which allows the measurement of cell viability using a spectrophotometer.
In this study, the evaluation of Litsea oppositifoliastem extract cytotoxicity was carried out by testing various concentrations of the extract on the HaCaT cells (Figure 3.1) and compared with the cytotoxicity of ascorbic acid as the standard (Figure 3.2). This study found that all concentrations of the extract and ascorbic acid used (6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm and 100 ppm) did not show any cytotoxic effects as the cell viability percentages were not significantly different from the 0 concentration. Furthermore, as illustrated in Figure 3.2, the result of the ascorbic acid did not show a discernible trend, unlike Figure 3.1, which illustrated the cytotoxicity of the Litsea oppositifolia stem extract.
In addition, the cytotoxicity of various concentrations of hydrogen peroxide (H2O2) was also tested on HaCaT cells to find the optimal concentration for the H2O2 insult of the cytoprotective agent. The cytoprotective study was also conducted to observe the protective effect of Litsea oppositifolia stem extract against H2O2-induced oxidative stress and the cell viability was measured using MTT assay. As illustrated in Figure 3.4 and Figure 3.5, it was indicated that the Litsea oppositifoliastem extract showed cytoprotective effects at concentrations of 12.5 ppm and 25 ppm compared to the cells treated only with 50 μM H2O2, while a slight cytoprotective ability was also observed. concentration of 50 ppm as indicated by the cell viability.
AA treatment in the cytoprotective assay showed significantly lower cell viability compared to other concentrations, which may indicate that 100 ppm AA treatment was toxic to the cell combined with the 50 μM H2O2 insult. Another finding by Hardaway et al. 2012), discovered that the treatment of AA and H2O2 showed lower cell viability compared to when the cells were treated with each compound independently. The mechanism why ascorbic acid decreased cell viability when combined with H2O2 was still not clearly elucidated.
In addition, the cell viability data obtained from the extract is also comparable to ascorbic acid, which is a powerful antioxidant. This was indicated by the cell survival results from the cytoprotective assay of the extract and AA, which showed cytoprotective-like cell survival at a concentration of 12.5 ppm. An important limitation of this study is that it is not possible to determine the effects of the extract on the molecular pathway associated with skin aging.
CONCLUSION AND RECOMMENDATION
SELF-REFLECTION
APPENDIX
