This report is dedicated to the completion of the credit internship program in the Department of Biomedicine of the Indonesia International Institute for Life Sciences (i3L). This internship program is done as one of the graduation requirements and to familiarize the author with laboratory skills and knowledge, especially the in vivo study. This research was conducted under the supervision of the Department of Biomedicine in i3L and was funded by PT Indofood Sukses Makmur Tbk through the Indofood Riset Nugraha (IRN) program.
This report provides the summary, analysis, results and discussion of the experiment as well as opens a promising window for future research and applications exclusively in healthcare fields. One of the causes of diarrhea is Salmonella typhi infection and it is one of the leading causes of death worldwide, causing more than one hundred thousand deaths each year. Hepatocellular carcinoma (HCC) is one of the primary cancers with a high mortality rate worldwide due to late diagnosis and complicated molecular pathology of the disease.
Exploiting miRNAs as a potential biomarker for HCC diagnosis and understanding one of the molecular pathological pathways via LncRNA-miRNA interaction may become an opportunity to increase disease prognosis. Some of the potential downregulated miRNAs were also found, where miRNA-4764-5p was the most downregulated in HCC and HCV patient serum as a result with lncRNA-RP11-156p1.3, which results in cancer progression via continuous TNF-α and NF - KB signaling. The clinicopathological data extraction also found that the majority of patients come with lower grading/staging, as the study's concern is to observe possible miRNA for early HCC detection.
However, both specializations in the department focus on treatment and drug development.
PROJECT DESCRIPTION 1. Internship Project
In this experiment, the alkaloid and phenolic acid (PA) compounds from PE seeds are tested for Salmonella typhimurium infection in laboratory-bred BALB/c mice. This study is conducted to observe whether alkaloid and phenolic acid compounds from PE seed extract have an anti-infective property against S. In the finding of naturally derived compounds used in the treatment of Salmonella serovar typhimurium infection, alkaloid and phenolic acid extracted from PE seed was made by evaporation.
The injection of bacteria and the treatment of alkaloid and phenolic acid were administered orally, the feces of the mice were dropped onto agar plates using the surface viable counting method to be counted for the colonies. The anti-infective properties of the compound were investigated by comparing the colonies formed from the feces before bacterial infection, after bacterial infection, before treatment, day 1 after treatment and day 3 after treatment. The aim of this wet-lab study is to prove the anti-infective property of alkaloid and phenolic acid of PE seed extract in vivo.
However, this white layer was discarded and the extract was mixed with 10 ml of 10% ammonia, filtered with filter paper, then mixed with 2 grams of sodium sulfate for about one hour to remove moisture from the extract. Quantification of the extract was done by obtaining a standard curve using 0.3% atropine sulfate (appendix 1). The solution was then placed in the rotary evaporator until it reached 10 ml of pure atropine solution.
The phenolic acid extract was mixed with the Folin–Ciocâlteu reagent and then measured with a UV spectrophotometer at 725 nm. The total content of phenols in the extract was compared with the absorbance of the naturally occurring phenolic acid, gallic acid. These mice were observed in two series, the first series was the alkaloid treatment and the second series was the phenolic acid treatment.
After the injection of the bacteria, it was confirmed that no mice experience abnormal breathing or other stress-related reactions. 24 hours after the injection of bacteria, the feces were collected and cultured on nutrient agar for an additional 24 hours to observe the success of the infection. Positive treatment was given to mice number 1 to 3, glycosidically bound phenolic acid was given to mice number 4 to 6, freely bound phenolic acid was given to mice number 7 to 9, and negative treatment was given to mice number 9 to 12.
FINDINGS
On the other hand, there were also no significant changes in the body weight of the mice in the alkaloid treatment group compared to the negative control group. However, fats and terpenes are still in the solution, to remove them completely hexane was added to the mixture. The fats were then completely separated, but there were other secondary metabolites such as saponin, alkaloids, flavonoids and quinones in the seed extract (Silalahi, Fachriyah . & Wibawa, 2018).
The alkaloid was found in the aqueous layer, while the other secondary metabolites remained in the organic layer. It was also affected by factors such as temperature, polarity and particle size (Yi et al., 2012). The presence of water was useful as the extraction enhancer, it increases the solvation of the solvent and the solute (Do et al., 2014).
In addition, to increase the amount of extracted PA and to reduce the extraction time, a method called subcritical water extraction may be carried out in the future. It is a new method which uses hot water under pressure to retain the water in the liquid phase, resulting in the hydrogen bonds and ions in the plants being disrupted and the phenolic acid compound will be separated from the water. The purpose of the production of freely bound PA is thus that PA does not physically and chemically interact with other molecules inside the body (Rocchetti et al., 2022).
On the other hand, injection of ciprofloxacin as a positive control showed no significant changes in body weight, which may be due to the inability of the antibiotic to clear the typhoid infection due to ciprofloxacin resistance (Wattal et al., 2008). In the PA group, all tested variables showed no significance to the negative control group. Antibiotic cannot cure typhoid infection due to bacterial resistance (Wattal et al., 2008).
Before treatment and infection, the number of CFU was too high to count due to the existence of normal microbiota in the gut of mice (Wang et al., 2019). It is known that the administration of kanamycin can disrupt the existence of normal intestinal microbiota, and can even cause long-term health consequences (Ramirez et al., 2020). At 24 h and 72 h post-infection, injection of ciprofloxacin as a positive control showed no change in CFU counts, although ciprofloxacin has well-known efficacy against Gram-negative bacteria, including S.
Identical results were observed in the alkaloid and PA treatment group, 24 and 72 hours after treatment the colonies were too numerous to count. Nevertheless, the normal intestinal microbiota can grow in the middle of the observation, because one of the normal intestinal bacteria, Escherichia coli, can grow in a very short time under an optimal condition, this can cause bacteria other than S.
CONCLUSION AND RECOMMENDATION
Self-Reflection
I have been able to improve my laboratory skills and knowledge by applying techniques and methods that I have never done before. I learned about how compounds were extracted, mice treatment, mice treatment, oral gavage, absorption measurement, miles misra, and also the things associated with bacterial infection, especially Salmonella typhi infection. During this internship project I also gained confidence in working and collaborating with other people to achieve one goal.
I had no experience before joining this project, but I would like to express my gratitude to my seniors, Adrian Wangsawijaya and Albert Tee, who equip me with the skills and technique required in this project. In the near future, I believe that all the things I learned in this internship project will be useful in my professional career.
Atropine Sulfate Standard Curve
Gallic Acid Standard Curve
Mice Weight Before the Infection of Batch 1
Mice Weight After the Infection and Treatment of Batch 1
Mice Weight Before the Infection and Treatment of Batch 2
Mice Weight After the Infection and Treatment of Batch 2
Mice stool mixed with PBS
Bacterial isolate
Pomelo, the fattest
Evaluation of solvent effect on the extraction of phenolic compounds and antioxidant capacities from the berries: application of principal component analysis. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity 33 of Limnophila aromatica. The Mouse Intestinal Bacterial Collection (miBC) provides host-specific insight into cultured diversity and functional potential of the gut microbiota.
Antifungal activity and major bioactive compounds of pangium edule seed water extract against Aspergillus flavus. KAJIAN ACTIVITY ANTI-BACTERIAL EXTRACT BIJI PANGI (Pangium edule Reinw) TERHADAP Staphylococcus Aureus, Bacillus Cereus, Pseudomonas Aeruginosa AND Escherichia Coli SECARA IN VITRO. Clinical significance of bacteriostatic versus bactericidal mechanisms of action in the treatment of gram-positive bacterial infections.
Global, regional and national age-sex mortality for 282 causes of death in 195 countries and territories a systematic analysis for the Global Burden of Disease Study 2017. Use of Pangium edule alkaloid seed extract to overcome contamination with foodborne pathogenic bacteria Staphylococcus aureus, Escherichia coli , Salmonella typhimurium and Klebsiella pneumoniae, 40. Isolation of alkaloid compounds from ethanol extract of Rimpang Galang Merah (Alpinia purpurata (Vielli) K. Schum) and production of nanoparticles from its alkaloid extract.
Antibacterial activity of phenolic acid extracted from Pangium edule against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae as a foodborne pathogen, 38. Estimates of the global, regional and national morbidity, mortality and etiologies in a195 countries in diarrhea systematic analysis for the Global Burden of Disease Study 2016. High level of ciprofloxacin resistance in Salmonella enterica isolated from blood.
Simultaneous quantification of important flavonoids in "Bawanghua", the edible flower of Hylocereus undatus using pressurized liquid.
