The internship report is a part of the credit internship as the research conducted for the credit internship program in the Department of Biomedicine of the International Institute of Life Sciences Indonesia (I3L). Thus, the main research carried out in this project is to evaluate the quality and safety level of propolis extract for promising medicines for the cure of COVID-19. EXTRACT STANDARDIZATION AND ACUTE TOXICITY STUDY OF PROPOLIS EXTRACT TO MICE AS A POTENTIAL TREATMENT OF COVID-19.
Body weight variation of 300 mg/kg BW propolis-treated mice during the first preliminary study (Blue) and the second preliminary study (Red). Comparison of changes in the appearance of mice during the first preliminary study and the second preliminary study. However, there is a need to ensure the safety level of propolis before using it for medicinal purposes.
This study aims to investigate the standardization of propolis extract and to determine the safety dose of propolis extract to mice by performing an acute toxicity study. The standardization of propolis extract was performed according to the standard of the Indonesian Food and Drug Administration, while the in vivo acute toxicity study was performed according to the guidelines of the Organization for Economic Cooperation and Development. Thus, the result showed that propolis extract met the standardization criteria, and the acceptance dose of propolis extract for mice is 300 mg/kg bw, which is included in category 4 of the classification of the global harmonized system.
Indonesian Journal of Life Sciences (IJLS) is a journal published by Indonesia International Institute for Life Sciences Press (i3L Press) as a product of research and development of life sciences in i3L.
PROJECT DESCRIPTION
Afterwards, the in vivo study must also be conducted to evaluate the safety of the propolis extract towards the organism (Maheshwari & Shaikh, 2016). Acute toxicity study is an in vivo study conducted to evaluate the adverse effects of the extract on an organism. This internship report was carried out to further perform extract standardization of propolis extract as well as perform the acute toxicity study of propolis test against mice.
The density of water was determined by filling the pycnometer with water and the total weight of the pycnometer and water was recorded. The determination of the density of the propolis extract was done by introducing the propolis extract into the empty pycnometer. The jar was then placed in the desiccator to cool down to the temperature of the kettle.
Then it was placed in the heating mantle with a temperature of 110oC to 150oC and subjected to a heating process until the mixture boiled. Then 15 ml of the agar solution was poured into a petri dish in BSC and kept until solidified. Acute toxicity study was performed with a fixed dose method of the Propolis extract according to the OECD guideline.
In the beginning of the study, the mice were fasted overnight before the treatment, meanwhile water was still given to the mice. During the study, the mice were given five grams of food and water every day, the temperature of the animal room is 22oC, also the light-dark cycle is divided into 12 hours of light and 12 hours of darkness. The main study was carried out to further investigate the adverse effect of propolis extract as well as to determine the toxicity classification of the propolis extract.
Propolis extract was administered at 10% of body weight with a concentration of 300 mg/kg body weight. Observation of the propolis acute toxicity study in mice was also performed by monitoring the body weight of propolis-treated mice before the propolis test dose for 14 days, and at the end of the observation period, the surviving propolis-treated mice will be sacrificed. . All propolis-treated mice (including mice that died during the observation period and propolis-sacrificed mice) were necropsied to observe changes in the pathology of each mouse tested and the organ index of several mouse organs (e.g., liver , spleen, intestine, kidney, lung) were calculated using the following calculation,.
Thus, several decisions can be made during the test: to continue the study at a high dose when the starting dose shows no toxicity symptoms, to continue the study at a lower dose when the starting dose causes the death of more than two mice, or to stop the test at the initial dose when the initial dose shows toxicity symptoms and/or causes the death of one mouse and can be classified in the GHS classification, the classification system based on the subject's death and toxicity symptoms. The p-value of p<0.05 is considered significant changes in the organ index of the mice.
FINDINGS 3.1. Result
Body image of the mice used during the first preliminary study showed inflammation in the right side of the mice's body. The main study Propolis Acute Toxicity Study on rats was conducted twice, the first study and the second main study with the same dose, which is 300 mg/kg BW. During the first main study, it can be seen that the body weight of the control mice remains constant with slight changes in the body weight of the mice.
Both main studies showed that no abnormalities were shown in body autopsy during the first and second main studies for the control mice and the mice treated with 300 mg/kg bw propolis extract. Figure 6 shows the comparison of necropsy of mice from mouse 5 from the first main study, mouse 4 as representative of propolis-treated mice necropsy, as well as mouse 20 as representative of the control group. Furthermore, the gross pathology was used for the organ index of the mice, which must be obtained to further check the difference between control mice and propolis-treated mice (Figure 7). Furthermore, the organ index of the propolis-treated mice and control mice was obtained to check the changes in the organ index of propolis-treated mice (Figure 8).
In vivo studies of propolis extract were done to further check the safety level of the propolis extract. Thus, according to the OECD Regulation (2002), because one mouse from the preliminary study showed toxic symptoms as a result of oral ingestion of 300 mg/kg BW propolis extract, 300 mg/kg BW was used for the main acute toxicity study study of propolis mice. Based on Figure 5, the mean variation in body weight of mice during the first pivotal study does not show significant changes in body weight of the CMC-Na treated mice (control mice).
However, the second main study does not show significant changes in the body weight of the control and propolis-treated mice (Figure 5). Based on Figure 7, there are no significant changes in organ index for several organs such as liver, spleen, kidney and lungs in the mice. However, the intestinal organ index shows a significant difference between control mice and treated mice.
However, the second main study shows no significant difference of organ index of control mice and mice treated with propolis extract (Figure 8). Furthermore, the necropsy results of one of the rats from the first main study of the 300 mg/kg BW extract-treated group (Figure 6) show the presence of a blister-like intestine and putrid organ. Based on the necropsy results of mice, propolis does not cause any significant changes in the organ of mice treated with propolis.
Thus, the main reason for the bubble-like intestinal presence in one of the mice from the first main study with 300 mg/kg extract is more likely due to the infection in the mice due to the presence of pathogens in the environment of mice. Thus, the cause of the bubble-like intestine in one of the mice from the first main study at 300 mg/kg bw may be due to bacterial or parasitic infection.
CONCLUSION AND RECOMMENDATION
The internship project is a great opportunity for me to grow, because during the internship I learn a lot of knowledge, especially technical and soft skills that I did not acquire in the classroom before. During the internship, I learned a lot about the common mistakes that can happen during an in vivo study and the unpredictable events that always happened in laboratory work. However, during my internship, I learned that mistakes should be taken as hidden knowledge that can benefit me in life after graduation.
Our task as researchers is to search for the hidden gems in this vast universe with our little understanding. This leads me to believe that there will be many opportunities to come during the research. The solution is still there, but we have to overcome a lot of trial and error to reach the solution.
It may be difficult at first, but this step will influence human knowledge for a better life. So I look forward to my future development and will remind myself that everything will shine when the right time has come. Contamination of herbal medicines marketed in Kaduna metropolis with selected pathogenic bacteria. African Journal of Traditional, Complementary and Alternative Medicine.
Review of bee propolis (propolis) biological properties and toxicity. Food and chemical toxicology: an international journal published for British Industrial. 2017). European Union Reference Laboratory for Alternatives to Animal Testing. In silico investigation of potential inhibitors for head protease and spike protein of SARS-CoV-2 in propolis. Toxicity studies of drugs and chemicals in animals: an overview. Bulgarian Journal of Veterinary Medicine,20(4).
