Effect Of Aegle marmelos on IgM And IgG Antibody Titers in Rattus

Aegle marmelos is a traditional plant with many therapeutic benefits; for example, it is used as an immunomodulatory agent. Literature studies reveal many functional and bioactive compounds, such as alkaloids, phenolics, coumarins, flavonoids, terpenoids, and other antioxidants. Based on its chemical content, Aegle marmelos could be an immunomodulatory agent because it can stimulate the production of antibodies against foreign antigens, increasing IgM and IgG. This study aims to determine the effect of Aegle marmelos filtrate on IgM and IgG titers in Rattus norvergicus induced (sheep red blood cells) SRBC 2%. In this study, 30 rats were divided into five groups: negative control received standard diets, positive control received SRBC 2% injection, P1 received SRBC 2% + Aegle marmelos filtrate 25%, P2 received SRBC 2% + Aegle marmelos filtrate 50%, and P3 received SRBC 2% + Aegle marmelos filtrate 75%. The results showed that administration of Aegle marmelos filtrate could increase IgM titers at the highest dose of 75%, although it was not statistically significant. Meanwhile, Aegle marmelos filtrate does not affect IgG titers in SRBC 2% compared to treatment groups.


INTRODUCTION
The immune system was first discovered because of the desire to prevent the spread of disease and develop better treatments for the sick (McComb et al., 2019).The first antibody formed in the primary immune response is Immunoglobulin M (IgM), produced when the antigen first enters the body.IgM will appear on the fifth day of infection.However, follicular B cells undergo isotype switching upon antigen exposure, producing IgG (Sathe & Cusick, 2022).Antibody production can be seen by examining agglutination obtained from red blood cells taken from various components of microorganisms, such as bacterial or animal proteins, using the hemagglutination method, which is the clumping of red blood cells caused by antibodies to antigens on the surface of red blood cells (Agustina et al., 2022).Disease manifestations due to pathogens depend on the ability of the immune system to achieve disease resistance.An alternative that can be given to optimize the work of the immune system is to use herbal plants (Paul et al., 2020).
Several studies have revealed the beneficial application of herbs as natural additives, which have the potential to induce immune responses and increase resistance to disease or pathogen infection (Wangkahart et al., 2022).The efficacy of herbal plants in helping to prevent various diseases has long been studied globally, but information related to how strong the research evidence regarding the potential of herbal plants in improving the body's immune system is still limited (Suciady et al., 2021).
Aegle marmelos (maja plant) comes from the Rutaceae family, one of the traditional and nutrient-rich plants.This plant has many therapeutic benefits, one of which is as an immunomodulatory agent (Savita et al., 2021).Literature studies reveal the presence of many functional and bioactive injection, blood was retaken for examination of IgG titer using the hemagglutination method.

Hemagglutination Test
The serum that has been obtained is heated in a water bath at 56°C for 30 minutes.
Serum dilution is done in multiples of two, namely ⅟2, ⅟4, ⅟8, ⅟16, ⅟32, ⅟64, ⅟128, ⅟256, ⅟512, ⅟1024 and so on.The diluted serum is then incubated for one hour at room temperature, and hemagglutination will be formed.The observation is based on the spread of SRBC at the bottom of the tube, which can be seen uprightly.

Data Analysis
Titer data was statistically processed using the Kruskal Wallis test After being transformed with 2 log(titer)+1 (Indrisari, 2018).

IgM Titer Test Result
The  However, no adverse reaction occurred in the low-dose group.The highest IgM titer production in the filtrate dosing with a concentration of 75% is likely due to the highest the dose is given, the more it will trigger antibody production so that the bond between the antigen and antibody is also higher (Perkasa et al., 2016) Image 2. Distribution of converted hemagglutination titers (IgG) in all groups.

CONCLUSION
The administration of Aegle marmelos filtrate to Rattus norvegicus affected the

T
Wallis test results obtained a pvalue of 0.010 <0.05, which means that giving Aegle marmelos filtrate affects the production of IgG antibody titers.Mann-Whitney further test showed a significant difference (p<0.05) in group K-with K+, P1, P2, and P3.However, there was no difference between the K+ and all treatment groups (p>0.05).In testing the IgG titer, the K+ group still has the highest mean antibody titer compared to the treatment groups.It occurs because the K+ group has produced more elevated antibodies in the primary immune response, and antibodies continue to fight secondary infections due to second exposure to SRBC (Restuati & Gultom, 2013).The inability of Aegle marmelos filtrate to increase IgG production in groups P1, P2, and P3 could be due to secondary metabolites, such as terpenoids, that inhibit lymphocyte proliferation., In vivo studies show that the administration of limonene contained in terpenoids can suppress the production of Th1 and Th2 mediated by CD4+ and CD8+ this terpenoid content causes the administration of Aegle marmelos filtrate in groups P1, P2, and P3 to be unable to increase IgG production due to the suppression of the immune system (immunosuppressant).However, the shortcoming of this study is that reuse should have tested not test the content of secondary metabolites contained in Aegle marmelos fruit.In addition, technical errors during the study may inevitably lead to misinterpretation.The researcher believes that the lack of secondary immune response is due to the reduced antigenic properties of the SRBC used.In this study, the SRBC used was SRBC that had been stored in the freezer for more than ten days.The researcher's assumption is also reinforced by Oktari & Mulyati's research (2022) that red blood cells are best used when they do not pass through a storage period of more than four days because it can cause contamination so that there will be two populations of antigens, namely antigens contained in the blood and foreign antigens that stimulate antigen-antibody reactions on the part of antibodies contained in the sample against foreign antigens so that mixed reactions occur can cause false negative and false positive responses (Oktari & Mulyati, 2022).