This report is subject to the successful completion of the credit internship program at the Indonesia International Institute for Life Sciences (i3L). The aim of this study is to investigate the immunomodulatory effect of Gynura divaricata extract on the immune cell population of mice. The scope of this study includes characterization of total phenolic and flavonoid content of Gynura divaricata extract, as well as evaluation of Gynura divaricata immunomodulatory properties for mouse innate immune system.
Therefore, a direct study is needed to investigate the immunomodulatory properties of Gynura divaricata extract in enhancing the innate immune system. Immunomodulatory effect of Gynura divaricata extract on mouse immune cell population Indonesian International Institute of Life Sciences (i3L). Therefore, the aim of this research is to investigate the immunomodulatory effect of Gynura divaricata extract on the immune cell population of mice.
Furthermore, the immunostimulatory effect of Gynura divaricata extract on the immune cell population of mice was then examined through the leukocyte count assay in which the treatments were administered through the oral gavage technique. The finding shows that Gynura divaricata has a selective immunostimulatory effect on immunocompromised mice caused by infection, but not on irritated mice caused by gavage treatment or control.
INTRODUCTION
Description about the company
Description of department
The Department of Biomedicine at i3L is focused on research and development that combines the study of human well-being with the study of therapeutic interventions.
Product of the Host Institution/Company
PROJECT DESCRIPTION
Internship Project
- Project Background
- Scope of the Project
- Objectives/Aims
- Problem Formulation and Proposed Solutions
The aim of the project is to characterize the total phenolic and flavonoid content of Gynura divaricata extract as well as to evaluate the effect of oral administration of Gynura divaricata extract on the immune cell population of mice. The objective of this project is to investigate the immunomodulatory effect of Gynura divaricata extract on the immune cell population of mice through oral administration of sewage. Therefore, people tend to limit the use of synthetic immunomodulators and prefer to use herbal alternatives such as Gynura divaricata.
However, there has been no direct research examining the immunomodulatory properties of Gynura divaricata. Therefore, this research suggested several solutions that could be used to examine the immunomodulatory potential activity of Gynura divaricata extract in mice. The total flavonoid content of Gynura divaricata leaves was measured using a colorimetric method using aluminum chloride with quercetin as a standard.
The immunostimulatory effect of Gynura divaricata extract on the innate immune system was analyzed using leukocyte count assay. Each group of treatment consisted of 6 mice for the first group of treatment and 4 mice for the second group of treatment.
FINDINGS
Result
- Extraction and Characterization of Gynura divaricata Leaves
- Differential Mice Condition from 2 Batches of Mice
- Distribution of Mice Body Weight
- Mice Survival Rate
- Immunostimulatory Effect of Gynura divaricata Extract toward Mice Immune Cell
All treatment groups were statistically compared with the negative control using two-way ANOVA analysis. For the first group, it can be seen that the average body weight of mice from most treatment groups increased slightly during the first and second week of pretreatment, although it was not significant compared to the negative control based on two-way ANOVA analysis . (Figure 2A; Appendix 8). According to statistical analysis using two-way ANOVA (Appendix 9), all treatment groups showed significant increases in mean body weight during the second week of pre-treatment compared to the negative control.
In addition, the average body weight of mice was significantly reduced for all treatment groups except the untreated group compared to negative control beginning the first week of post-treatment (between weeks 6 and 7). During the second week of post-treatment, the untreated mice and mice treated with a higher dose of stimulo could significantly increase the average body weight compared to the negative control (Appendix 9). According to the statistical analysis using one-way ANOVA (Appendix 10), mice treated with stimulo and G.
Mouse survival rate for batch 2. Untreated negative control Stimuno 12h Stimuno 24h extract 12h extract 24h. A) Survival rate graph for batch 1 treatment (n=6) and (B) Survival rate graph for batch 2 treatment (n=4). All treatment groups were compared statistically with the negative control using one-way ANOVA analysis. Statistical analysis by one-way ANOVA also showed that there was no significant difference between mice treated with negative control and mice treated with lower dose of stimulo and G.
On the other hand, all treatment groups in batch 2 treatment also showed higher leukocyte counts compared to the negative control, except for mice treated with a higher dose of G. All treatment groups were statistically compared with the negative control using one-way ANOVA. With this in mind, different types of leukocytes, including agranulocytes (lymphocytes and monocytes) and granulocytes (neutrophils, basophils and eosinophils), were examined to observe the status of mice from all treatment groups compared to the negative control.
However, from the first batch of treatment, it can be seen that the number of lymphocytes, monocytes, neutrophils, basophils and eosinophils did not show any significant difference compared to the negative control due to the limitation of the sample number (Figure 5a and 5c Appendix 19) . For the second batch of treatment, it can be seen that the percentage of each leukocyte type also showed no significant difference compared to the negative control (Figure 5b). On the other hand, the number of monocytes, neutrophils, basophils and eosinophils showed no significant difference compared to the negative control (Appendix 20).
Analysis/Discussion
On the other hand, the condition of the mice in the second batch was better because no defective mice were found. Infections can reduce food intake, limit nutrient absorption and cause direct nutritional losses, explaining the decrease in pretreatment body weight of mice in the first batch of mice (Stephensen, 1999 and DeBoer et al., 2017). Nevertheless, all treatment groups are still within the normal body weight distribution, according to The Jackson Laboratory.
In addition, low survival rate found in the negative control in the first batch of treatment may also be due to the negative effect of CMC-Na. On the other hand, as previously mentioned, the condition of the mice in the second batch is better compared to the first batch, as no immunocompromised mice were found. Theoretically, the immunostimulating effect can be observed through the increase in the hematological parameters, specifically in the leukocyte count (Sahu et al., 2010).
The finding in this study shows that mice treated with CMC-Na as a negative control have different leukocyte counts between the first and second treatment groups, although both showed a net reduction in leukocyte counts compared to the other groups. The decrease in the number of leukocytes in the negative control group from both groups indicates the absence of the immunostimulatory effect of the negative control group. Since the mice of group 1 were immunocompromised or infected, the survival rate of the negative control group is decreased in the first group compared to the second group, although both do not show any immunostimulatory effect on the number of leukocytes compared to the groups of others.
Regarding the cell count, however, the number of lymphocytes was significantly higher in the untreated mice as well as mice treated with a lower dose of stimulo and G. In addition, the number of lymphocytes was higher in the second batch of negative control group compared to the first batch. Based on the finding, it can be seen that the percentage of neutrophils was still in the normal levels, which is around Nugroho, 2018).
However, a higher number of neutrophils are found in the negative control group compared to others, and this may occur as a response to irritants caused by gavage or CMC-Na. This may indicate the presence of sick mice or the possibility of infection, which then explains the condition of the immunocompromised mice in the first batch (Rosenberg et al., 2012). Based on the findings, it can be seen that the percentage of basophils from all treatment groups was still in the normal range of basophils, which is between 0-1.5% (Nugroho, 2018).
CONCLUSION AND RECOMMENDATION
SELF REFLECTION
Gynura divaricata extract characterization standard curve. a) Flavonoid characterization standard curve measured at 510 nm wavelength using UV-Vis spectrophotometer; (b) phenolic characterization standard curve measured. Statistical analysis for group 1 mice mean body weight vs age analyzed using two-way ANOVA. Statistical analysis for group 2 mice mean body weight vs age analyzed using two-way ANOVA.
Statistical analysis for the percentage of each type of leukocyte in the first treatment series, analyzed by two-way ANOVA. Statistical analysis for the percentage of each type of leukocyte in the second treatment series, analyzed by two-way ANOVA. Statistical analysis for the number of each type of leukocyte in the first treatment series, analyzed by two-way ANOVA.
Statistical analysis for the number of each leukocyte type in the second batch of treatment, analyzed by two-way ANOVA. Evaluation of immunomodulatory activity of the alkaloid fraction of Trichopus zeylanicus gaertn in laboratory animals. Determination of chemical composition, total phenol content, total flavonoid content and in vitro antioxidant capacity of various organic extracts of Gynura crepioides leaves grown in Malaysia.
Immunomodulatory activity of a combination of nanoparticles of Allium sativum, Curcuma mangga and Acorus calamus on the leukocyte profile of mice.
