Red Ginger's Anti-Anxiety Effect on BALB/c Strain Mice (Mus musculus) Pro-Inflammatory and Anti-Inflammatory Measurements as Anxiety Model

There is a correlation between the occurrence of anxiety and the production of inflammatory mediators, and red ginger rhizome is a well-known herbal product with a high content of phenolic and flavonoid compounds that can be used as anti-inflammatories and antioxidants. The aim of study to evaluate the effect of red ginger as antianxiety in mice (Mus musculus) BALB/c strain by measuring levels of TNF-α, IL-6 and IL-10. Anxiety model mice were carried out by giving treatment with the Forced Swimming Test (FST) for 7 days then assessed by carrying out the Elevated Plus Maze for Mice (EPM) test for one day. After the treatment, the anxiety mice model was made, followed by administration of red ginger ethanol extract therapy for 14 days. The distribution of the experimental animal model groups was divided into control groups (KN, K, K+) and treatment groups (P, P2, P3).There was a significant difference the decreased of the TNF-α levels at the all of treatment groups with red ginger rhizome extract (P1, P2, P3) compared with the control groups (KN, K) (p<0.05), the significantly decreased of IL-6 levels in the three doses treatment group (P1, P2, P3) compared to the control group (K, K+) (p <0.05) and an increase in IL-10 levels in the 50 mg treatment group compared to group K -, statistically not significant (p>0.05).In overall, this study suggests that FST stimulation will create anxiety symptoms and behavior as well as impact cytokine levels, namely elevated levels of TNF-α and IL-6. Giving red ginger ethanol extract has the potential to be researched further for reducing anxiety symptoms because it can block pro-inflammatory cytokines by significantly decreased levels of TNF-α, IL-6, and increased IL-10 cytokines a brief abstract about your paper’s subject of study.


Introduction
Anxiety is the most commonly reported psychiatric condition, causing a wide range of psychological effects on mental health crises, including factors related to work, finance, micro and macroeconomics, crime, bullying, and social isolation pressures, all of which are thought to be factors in the rise of anxiety disorders 1 .Anxiety is defined by awareness, such as nervousness and fear, and physiological sensations, such as sweating and palpitations, which generate feelings of discomfort and unjustified fear and are generally accompanied by autonomic symptoms 2 .Anxiety feelings are comparable to those of fear, but anxiety differs in that it responds to unreal danger and predicts approaching danger that may or may not occur 3 .The high prevalence of anxiety disorders and the need for anxiety treatment continue to cause many side effects, particularly cardiovascular, gastrointestinal, and hepatotoxic, resulting in complications such as seizures, suicide, sexual dysfunction, weight gain, hypo sodium, insomnia, and hypersomnia, even in patients with cardiovascular problems and diseases of the brain nerves 4 .The Increased inflammatory activity is regarded to be a crucial mediator in the development of anxiety and therefore it was necessary to measure the levels of pro-inflammatory cytokines, such as Tumor Necrotic Factor-α (TNF-α), interleukin-6 (IL-6), anti-inflammatory interleukin-10 (IL-10), and inflammatory markers systemic C-Reactive Protein (C-RP) 5 .Pharmacological interventions with drugs such as using Selective Serotonin Reuptake Inhibitors (SSRIs) are the first line of treatment for anxiety disorders, various classes of antidepressant drugs include Selective Serotonin Norepinephrine Reuptake Inhibitors (SNRIs), Tricyclic Anti-Depressants (TCAs) and Monoamine Oxidase Inhibitors (MAOIs), anxiolytics such as benzodiazepines, non-benzodiazepine hypnotics, also used in the treatment of anxiety disorders and depression, However, the use of medicinal compounds in the treatment of various disease causes many side effects and costs quite a lot of money if these drugs are used long term 6,7 , so an alternative to the use of herbs is sought, which is quite easy to obtain and inexpensive, regular use or regular consumption does not cause significant side effects, so the selection of herbal ingredients can be considered for safety as a therapeutic treatment 8 .
Red ginger is a variant of the species Zingiber officinale that is cultivated in Indonesia and Malaysia.Because red ginger has many benefits, particularly as an anti-inflammatory, antimicrobial, antioxidant, antiulcer, anti-clotting, anticancer, and immunostimulant 9 .The aim of the study is to determine the effect of red ginger ethanol as antianxiety in male mice induced into anxiety with Force Swimming Test (FST) and Elevated Plus Maze for Mice (EPM) as evaluation by measuring the duration and measuring the number of open and closed mice arms that entered in anxiety model mice, then measuring molecular proteins such as TNF-α, IL-6 as inflammatory mediators and IL-10 as an anti-inflammatory mediator.This FST treatment lasted 7 days and was followed by Elevated Plus Maze for Mice (EPM) for one day.

Materials and Methods
After generating anxiety mouse models, therapy with red ginger ethanol extract was given for 14 days.

Examination of Molecular Protein
All mice were sampled by withdrawing blood from an orbital vein (4ml/ mouse), blood was collected in serum separator tubes (SST), and samples allowed to clot overnight at 4 degrees Celsius before centrifugation for 15 minutes at 1000×g.The serum was removed for assay immediately.The measurement of molecular protein such as TNF-α, CRP, IL6 and IL 10 with ELISA methods in laboratory of the Institute of Biosciences, University of Brawijaya Malang.

Statistical Analysis:
The statistical analysis was carried out using SPSS version 20.0 using analysis of variance (ANOVA) test and to be continued by post hoc LSD analysis and Kruskal-Wallis and to be continued by Mann Whitney analysis.P-values less than 0.05 (p < 0.05) were considered statistically significant 10 .

Results
The results of the extract's phytochemical screening found the presence of flavonoids, phenolic, saponins, alkaloids and steroids/terpenoids and no tannins were found and for determining of the levels of secondary metabolites from the ethanol extract of red ginger obtained flavonoid levels of 43.97 mg QE/g extract, phenol levels of 97.

The Effect of TNF-α Levels on Anxiety Model Mice Treated
The examination of TNF-α levels in the serum, there was a significant difference in average TNF-α levels between the two groups, namely the control and treatment groups (with a significance level of p<0.05).Furthermore, the results showed that TNFα levels tended to significantly decrease in the treatment group at three different dose levels (P1, P2, P3) when compared to the control group (referred to as KN and K-in the study).In addition, through the post hoc analysis test, it can also be observed that there is a significant difference in the mean TNF-α levels between the untreated control group (K-) and the treated control group (K+), as well as between the treated groups at all doses (P1, P2, P3) and the untreated control group (K-) as shown in table 4.

The Effect of IL-6 Levels on Anxiety Model Mice Treated
The measurement of IL-6 levels in the control and treatment groups of mice, found IL-6 levels in the KN group 23,168 (23,159-23,179) pg/ml, K-group 23,199 (23,171-23,547) pg/ml, K+ group 23,186 (23,178-23,202) pg /ml, treatment group P1 23.185 (23.181-23.225)pg/ml, P2 23.172 (23.161-23.228)pg/ml, P3 23.177 (23.168-23.179)pg/ml, there was a significant difference in.A decrease in IL-6 levels was found in the treatment group at the three doses (50,100,200) mg compared to the control group (K-, K+).With post hoc test analysis found a significant difference in mean IL-6 levels in the normal group  developed in the 1970s by Roger Porsolt as a rapid detection of behavior for antidepressant drugs and it is currently regarded the gold standard of animal testing for depression 12 .
The period of time required for the Elevated Plus Maze test was considerably shorter and the number of open arms was lower in the FST treatment group compared to the control group.According to the habit of avoiding open arms is thought to be the outcome of increased anxiety.This EPM test has high predictive validity when used to screen for anxiolytic medications 13 .
In the study, we found that there was a significant difference in the duration of the opened and closed arms in the control group and the treatment group (p <0.05),We found that there was the shorter duration of the opened arms in the treatment group with a dose of 50 and 100 mg (p<0.005)compared to the three control groups, while the duration of closed arms was found to be longer in the three treatment groups (P1, P2, P3), but the longest duration was the treatment group with a dose of 100 mg.
There was no statistically significant difference (P>0.05) between the treatment group with a dose of 100 and the KN and K-groups although the fact that there were less open arms in the treatment group.The treatment group with a dose of 100 was found to have less closed arms than the control group K-, the K+ group, but more closed arms than the KN group, and this difference was statistically significant.The present study is consistent with the research 14 .
Other studies state that the EPM test has weaknesses such as variables that cannot be controlled, such as existing procedural differences, and that it is difficult to replicate for more specific results.The Porsolt forced swimming test, anxiety in a bright room, light/dark and open room transition tests, and open field testing are other procedures that can be utilized for anxiety conditioning 15 .
Several studies have found that depression and anxiety are associated with increased peripheral inflammatory markers such as C-RP, IL-6, and TNF-α, as well as blood cytokines 16 .Previous research has revealed that inflammation affects brain areas associated with anxiety, particularly the amygdala, insula, and anterior cingulate cortex, which appears to be caused by cytokine effects on monoamines and glutamate 17 .Other research stated that CRP that as biomarker that severe cases in infection of COVID-19 18 .Psychological stress affects the immune system in many ways.The effect of stress on the immune system varies and can be individual specific 19 .The most frequently observed response is immune suppression, especially when stress is chronic and persistent.Several studies have suggested that there is a relationship between TNF-α secretion and anxiety 20 .In this study there was a significant difference in TNF-α levels in the control group and the treatment group (p<0.05).In the use of ethanol extract of red ginger rhizome, there was a significant decrease in TNF-α levels at doses of 50 mg and 200 mg (p<0.05).Previous research stated that potential therapeutic strategies for mood disorders are compounds that can reduce TNF-α or compounds that are selectively anti-TNF-α 21 .
Studies show that of the many cytokines, IL-6 is one of the most studied cytokines in terms of its relation to depression 22 .Evidence has been carried out from various studies either through experimental animals or clinically showing that there is an increase in peripheral and central cytokines and IL-6 plays an important role in stress reactions and depressive disorders, and increased release of IL-6 is a factor associated with the prognosis of Major Depression Disorder and It is also a therapeutic response, and may contribute to various depressive symptoms 23 .In this study, a decrease in IL-6 levels was found in the treatment group at doses of 100 and 200 mg compared to the control group, so the red ginger extract used can reduce anxiety with evidence of decreased IL-6 in the treatment group and statistically found a significant decreased with the control group (p<0.05).Pro-inflammatory and anti-inflammatory cytokines are in a balanced state in healthy people, with IL-6 mediating the early stages of inflammation and then triggering the release of IL-10, which has immunoregulatory effects and suppresses inflammation, that pro-inflammatory cytokines play an important role in intercellular communications 24 .In individuals in good health, there is a regulated balance of pro-and anti-inflammatory cytokines, such as IL-6, which mediates the initial phase of the inflammatory process and then causes the production of IL-10, which has immunoregulatory effects and overcomes inflammation 25 .These findings clearly show that the immune system can respond immediately to inflammation and then return to a homeostatic state.The immunoregulatory balance between pro-and antiinflammatory cytokines is a crucial mechanism in anxiety-related inflammatory diseases.In this case, it might be argued that depressed patients have lower levels of IL-10 and higher levels of proinflammatory cytokines.The decreased of IL-10 levels will prevent inflammation repair, impair pro-inflammatory inhibition in undamaged tissue, and allow the adverse development of acute to chronic inflammation 26 .Giving red ginger in this study showed that there is a repair mechanism that responds to inflammation to maintain homeostasis.This was proved by an increase in IL-10 levels in the group that received therapy at both doses of 50 mg/kg, 100 mg/kg BW, and 200 mg/kg BW against pro-inflammatory cytokines such as TNF-α and IL-6, as it is well known that treatment increases IL-10 levels 27 .

Conclusion
In general, this study indicates that FST stimulation will cause anxiety and cause anxiety symptoms and behavior and affect cytokine levels, namely increased levels of TNF-α and IL-6.Giving red ginger can inhibit pro-inflammatory cytokines by markedly decreasing levels of TNF-α, IL-6 and increasing IL-10 cytokines and has the potential to be studied further to reduce anxiety symptoms.

Red ginger rhizome (Zingiber officinale Rosc.
Elevated Plus Maze for Mice (EPM).The purpose of this EPM test is to count the number of open and closed arms in mice that enter the labyrinth, as well as the time it takes for the arms to open and close.

Table 1 . EPM Assessment of FST treatment in the mice group (Number of open and closed arms)
22mg GAE/g extract and anti-oxidant levels (IC50) of 38.86 ppm.In this study, involves various groups: a normal control group (KN) with no treatment, a negative control group (K-) treated with 0.5% CMC-Na suspending agent, a positive control group (K+) treated with sertraline 50 mg, converted to 0.13 mg for 20-gram mice and 0.195 mg for 30-gram mice.but in the FST treatment with an assessment of the number of closed arms a significant difference was found in the control group and the treatment group, p<0.05, and with the post hoc One Way ANOVA test analysis found significant differences in the number of closed arms in the normal control group (KN) with the (K-) group, the (K+) group, and the treatment group (P1, P3), in the (K+) group there was a difference significantly the number of closed arms in the P2 group, P1 group there is a significant difference in the number of closed arms with the P2 group and P2 group there is a significant difference in the number of open arms in the P3 group, p<0.05.

Table 2 . EPM assessment of the FST treatment in the mice group (Duration time of open arms) Groups Median (min -max) p value Post Hoc
While the (EPM) test on the assessment of the duration of time required for closed arms in mice was significantly different in the treatment group compared to the control group, p <0.05., with the Man Whitney post hoc test, found a significant difference duration of open arms in the KN group 2023, 20(6 Suppl.):2363-2372https://dx.doi.org/10.21123/bsj.2023.9035P-ISSN:2078-8665-E-ISSN:2411-7986 Baghdad Science Journal with the K-, K+ and P2 treatment groups, p<0.05.We can see on the table3.

Table 5 . IL-6 levels in the anxiety model mice
Effect of IL-10 Levels in Anxiety Model MiceIL-10 levels showed a increased in the group of treated anxiety mice (P1) compared to the group of the control group (K-), but statistically there was no significant difference as shown in tablebelow