This report was prepared as part of an internship project conducted at the Indonesian International Institute for Life Sciences (i3L) in the Department of Biomedicine. It was a pleasant and memorable experience to be able to learn and be part of a research project conducted at the Indonesian International Institute of Life Sciences (i3L). The intern would also like to express his deep gratitude to the Department of Biomedicine, Indonesian International Institute of Life Sciences (i3L) as the host institution and for providing the intern with an outstanding opportunity.
This report summarizes a total of 52 days of internship project at the Indonesia International Institute for Life-Sciences (i3L). The project was carried out under the Biomedicine Department at the Indonesia International Institute for Life-Sciences (i3L).
Product of the Host Institution / Company
Currently, i3L has two specialization streams that focus on human diseases, which are infectious diseases and tumor biology, two major public health areas in Indonesia. The scope of the Biomedicine program includes the analysis of human biological samples, the conduct of research into human and animal diseases, the design and discovery of new therapeutic strategies, such as the development of vaccines and other medical treatments in private industry or government agencies.
Scope of the project
Finally, the fecal sample was plated using the surface stable count method on agar plates to count the number of colonies (CFU) and each sample was compared with bacterial growth before bacterial infection, after bacterial infection, before treatment, day 1 and 3. after treatment.
Objectives/Aims
- Components Extraction of Pangium edule
- Determination of Extracts
- Klebsiella pneumoniae Isolate Preparation
- Conditioning/acclimatization of Normal and Klebsiella-infected Mice
- Treatment Injection and Sample Collection
- Miles Misra and CFU Counting
The formed solution was added with 100 ml of chloroform and again mixed with hexane so that the solution forms two characteristic layers. The extract formed was then re-concentrated with a rotary evaporator at a temperature of 50℃ for about 10 min. The solution was then centrifuged at 2000 x g for 30 min, and the supernatant obtained from the solution was reconcentrated to reach the volume of 2 ml using a rotary evaporator.
3 ml of HCl acid was added to the resulting solution, it was re-extracted with the addition of 15 ml of diethyl ether and concentrated again with a rotary evaporator at 40 °C for about 10 minutes. Next, 1 ml of 50% methanol was added to the solution to redissolve the phenolic acid bound to the glycoside. The resulting solution is separated from chloroform with a separatory funnel and the solution is then placed in a UV spectrophotometer and its concentration is measured at a wave of 470 nm.
On the other hand, the phenolic acid determination was performed by preparing standard curves using gallic acid. The result of light absorption from the solution can be calculated in the total phenol content. Similar to the alkaloid batch, the phenolic acid treatment group used 8% phenolic acid for the treatment.
Positive treatment was given to mice numbers 1 to 3, glycoside bound phenolic acid was given to mice numbers 4 to 6, free bound phenolic acid was given to mice numbers 7 to 9, and negative treatment was administered to mice numbers 9 to 12.
FINDINGS 3.1 Result
Extraction and Determination
Based on Table 2, the absorbance value of glycoside-linked PA content from the extract was approx. 0.803, while the absorbance value of free bound PA content from the extract was 0.352. For comparison, the standard curve for gallic acid was used in the range from 0.01 to 0.1 mg/ml (see Appendix 2). From this value, it can be determined that the concentration of glycoside bound PA from the extract was 0.0754 mg/ml, while the concentration of free bound PA was 0.01063 mg/ml.
Therefore, both the glycosidically bound and free bound PA samples were selected for further analysis because they show the best concentration and exhibit almost similar concentration to gallic acid. After the determination of both extracts, alkaloid and phenolic acid were made at 8% concentration and compared with ciprofloxacin which served as positive control and DMSO as negative control. Then, the comparison of the anti-infective effect of the extract would be seen through the viable count method by injecting the extracts orally into mice and observing their faecal samples on NA plates to count CFU.
Mice Weight
Based on Figure 3, there were no significant body weight changes/losses in the mean data even after 24-48 hours post-treatment administration. In addition, from day 8-10, the average weight of the mice was seen to increase gradually for the negative treatment, stable for the alkaloid treatment and only the positive treatment showed a decrease. Statistical analysis was also performed by two-way ANOVA (see Table 3) and it shows no significant changes between positive control and negative control mice weight from day 1 to day 10, but on day 11 there is a significant difference with a P value of about 0.01.
On the other hand, the comparison between mice treated with the alkaloid and the negative treatment shows no significant changes in the body weight of the mice from the first day to the last day. Based on the average data, all treated mice lost weight during day 1 post-infection, but the average mouse weight continued to increase over the following days for all treatments. Statistical analysis was also performed by two-way ANOVA and showed no significant changes among all treatments compared to the negative control.
The CFU observation of batch 1 as shown in Table 5 consists of the data before the treatment, after the elimination of normal gut microbiome using kanamycin, after K. From the data in Table 5 it can be seen that the colonies on the plate a massive decrease after the administration of kanamycin treatment to mice compared to before treatment. In addition, the fecal samples from treated mice did not show any significant changes in bacterial growth, as they continued to grow excessively and considered as.
Similar to the first series, the colonies formed after the administration of kanamycin are significantly reduced.
Analysis/Discussion
- Extraction of Pangium edule Components
- Weight Measurement
- Anti-infective Activity of Pangium edule Extracts
15 device would also increase the probability of thermal degradation of the fat to be obtained after the extraction process, as well as the loss and degradation of solvent due to high temperature (Zhang, Lin. According to Boeing et al. 2014), the combination of methanol and water was the most effective solvent for phenolic extraction because they increase solvation. The higher the solvation interaction, this will result in a better interaction of the hydrogen bonds between the polar sites of the phenolic acid and the solvent.
In addition, a study conducted by Shahidi & Niazmand (2021), stated that another method that can be used to extract phenolic acid to obtain a good yield is the use of subcritical water extraction where the temperature will be set at 374 ℃ while water pressure is 22.1 mPa, causing hydrogen bonds and plant ions to change. The weight measurement of mice was one of the indicators to evaluate the effect of bacterial infection and treatments on mice. 2017, Klebsiella infection can cause loss of appetite, however, Klebsiella itself is an opportunistic bacterium that commonly causes nosocomial infection and is commensal to the gut, which may be one of the reasons the infection did not occur.
When the treatments were administered to the mice, most of the results were still TMTC, and only during the first day of ciprofloxacin administration were there still some countable colonies. Furthermore, the CFU observation of batch 2 (see Table 6) corresponds to the first batch, where the colonies formed after the administration of kanamycin show a significant decrease. The results did not find the importance of the extracts as anti-infective compounds, and one of the reasons is that the antimicrobial properties of each plant can lead to different results.
However, the negative result obtained in this project does not indicate that the extracts obtained have no significant effect or ineffective because there are several factors that can affect the extracts obtained, which are the extraction processes and also the presence of other compounds (Ncube et al., 2008).
CONCLUSION AND RECOMMENDATION
The internship research project gave the intern a valuable insight to work on an interesting project in a laboratory in which the intern had an opportunity to put his knowledge into practice and carry out procedures described in the protocol . During the internship, the intern learned how to listen to specific instructions, analyze data and also think critically. The internship was such a growing experience for the intern as it provides many lessons that the intern gained from the internship project.
For example, the internship project significantly improved the intern's knowledge of working in a scientific background and also left the intern with laboratory skills that would be very useful throughout the intern's career because it includes a laboratory constraint project. Therefore, the trainee had to learn to plan ahead, work precisely and react to results. In addition, through laboratory work, it involved developing the ability to efficiently and accurately perform the correct technique in each procedure described in the protocol, while maintaining good communication with colleagues.
The intern also learned that communication is very important when working as a team and also how to communicate and respond to the results. Perhaps most importantly, the intern also experienced failure during laboratory work that did not work as planned. However, the intern thought that the failure is not a bad thing as it encourages the intern to learn and respond positively to any kind of failure which improves his problem solving skills.
Thus, the internship project brought so many benefits to the intern, both soft skills and hard skills that will surely advance his careers as a successful scientist and a person. Evaluation of solvent effect on the extraction of phenolic compounds and antioxidant capacities from the berries: application of principal component analysis.
