Anti Depression Potential of Papaya Seed Extracts in Wistar Rat Models: a Study on Body Weights, Blood Glucose, Interleukin-6 and Malondialdehyde Levels with Force Swimming and Tail Suspension Tests

: The purpose of this study is to evaluate the potential of papaya seed extract as an antidepressant based on body weight, glucose levels, Interleukin-6, and malondialdehyde levels in treated rats. Force swimming and tail suspension tests have been conducted to measure acute stress and evaluate response behavior. Results showed that the higher concentration of papaya seed extract correlates to higher effectivity on reducing body weight and lowering glucose levels. Dose-response relationships were also observed on the inhibition effect of interleukin-6 and malondialdehyde more effective at higher extract concentrations. The experimental force swimming test and tail suspension test showed the positive effect of extract administration indicated by the shorter immobilization time. Gas chromatography analysis has confirmed the bioactive content in the extract. The main compounds such as; 9-Octadecenoic acid (Z)-methyl ester, Benzyl nitrile, Hexadecanoic acid- methyl ester, and other saponins might be responsible for the antidepressant effects of papaya seed extract. This research encourages further studies on pharmacodynamics, pharmacokinetics, and biomolecules analysis at cellular levels.


INTRODUCTION
Moderate or severe stress can lead to depression 1 and suppress the immune response 2 , such as interleukin-6. The IL-6 plays a role in stress reactions and depressive disorders, especially the physical disturbances that accompany depression in the presence of hypothalamic-pituitary-adrenal (HPA) activity 3 . The active transport systems peripherally release cytokines from the blood to the brain. The IL-6 is one of the most investigated cytokines in animal studies of Major Depressive Disorder (MDD). However, genetic studies of IL6 effects in MDD remain controversial. Increased activity of IL-6 causes depression through activation of HPA or by the influence of neurotransmitter metabolism 3,4 . The endocrine and immune systems are integrated through a bidirectional network in which hormones influence immune function, while immune responses are reflected in neuroendocrine changes.They play a role in modulating the response of the HPA at all three levels: hypothalamus, pituitary, and adrenal. Acute effects of cytokines are seen on the central nervous system, particularly the hypothalamus 5,6 . Free oxygen radicals increase due to high respiratory oxygen intake and metabolic turnover during stress. During stress, increased energy requirements caused by poor environmental conditions, backbreaking physical work, and psychological trauma (PTSD) require high oxygen intake to meet energy requirements 5 . Prolonged working hours, workload, fatigue, sleep deprivation, psychological trauma resulting oxidative injury indicated by the formation of 8-hydroxy-deoxyguanosine (8-OH-dG) as a biomarker of oxidative DNA damage 5,7 .
Herbal medicine as an alternative to cure acute stress has been known for centuries. The active metabolites contained in plant extracts may contribute to the antidepressant effects to recover the symptoms of depression. Bioactive content in plants such as alkaloids, flavonoids, saponins, tannins, and terpenoids have the ability as an antidepressant as good as tricyclic antidepressant drugs, i.e., amitriptyline 8 . Activating neurotrophic factors, such as BDNF will significantly impact neuronal function. Activating neurotransmitter systems, e.g., primarily serotonergic, noradrenergic, and dopaminergic at specific brain regions such as the hippocampus and prefrontal cortex, will activate a long-term chemical communication 8,9. .
Several metabolic compounds such as alkaloids, flavonoids, and saponins from papaya seeds have been reported recently. Some studies reported the potential of papaya seed extract as anti-diabetes 10 , lowering glucose level 11 , hypoglycemic 12 , antiobesity 13 . However, a lack of studies reported on the antidepressant effect of papaya seed extracts in rat models. Therefore, here we report an inclusive analysis of the physiological and biomolecular levels of the antidepressant effect of papaya seed extract. This report covers the effect of antidepressants on the body weights, glucose levels, MDA, IL-6 of Wistar rats under treatment with the Forced Swimming Test (FST) and Tail Suspension Test (T.S.). The efficacy of papaya seed extracts has been compared with amitriptyline 0.01 % (w/v) as a positive control.

Methods
Papaya seeds were dried, crushed, weighed 10 g, then heated in an infusion pan, mixed with 100 cc of distilled water. When the temperature reaches 90 o C let it settle for 15 min, then occasionally stir. Then filter solutions and add enough hot water to the dregs so that the required volume of infusion is 100 ml (10%). Precondition of Wistar rats was provided by feeding, namely CP 551 for one week. Wistar rats were randomly divided into six groups where each group consisted of 5 individuals: 1. Group K = Normal, no stressed, aquadest. 2. Group K-= Negative control, stress, and aquadest. 3. Group K+ = Positive control, stressed, and amitriptyline 0.01% w/v. 4. P1 = Treated with stressed and 10% extract, and amitriptyline 0.01% w/v. 5. P2 = Treated with stressed and 8% extract, and amitriptyline 0.01% w/v. 6. P3 = Treated with stressed and 6% extract, and amitriptyline 0.01% w/v.
The treated groups were placed in a stress inducer illuminated by a 60watt lamp for 30 min every day. The Force Swimming Test (FST) was conducted by placing the rats in cylindrical tanks partially filled with water, and the immobilization time gets measured 14 . The Tail Suspension Test (TST) was conducted by hanging the rat tails with tape, and the time of each mouse moving get measured 15 .
The blood collection from the heart vessels was conducted by dislocation of cervical vertebral followed by surging the chest cavity to collect 3 ml of blood from the heart. The blood was centrifuged for 10 min at 3000-4000 rpm and let precipitate to get serum layer, then put serum in a microtube and stored at -4 o C. Blood serum was then analyzed by the ELISA method. To check the IL-6, serum was inserted into the wells given with a Streptavidin-HRP and its reagents. Then incubate for 15 min at room temperature and protect from light. Determine the optical density of each well within 30 min, using a microplate reader set to 450 nm. If wavelength correction is available, then set to 540 nm or 570 nm. To check the MDA, the precipitated protein was resuspended in 2.5 ml of acetic acid and added by 3 ml of thiobarbituric acid (2g/L in 2 mol/L Na 2 SO 4 ). The solution was entirely mixed by vortex then closed and sealed with parafilm. The lid with the tube was slightly perforated with a needle. The entire solution was incubated at a temperature of 950 C for 45 min later, cooled with running water, then centrifuged at 3500 rpm for 10 min. The supernatant formed was then put into a cuvet, and its absorbance was measured at 532 nm.
Data are presented in mean ± standard deviation (mean ± S.D.). Tests for normality and homogeneity of data were carried out. If the data is normally distributed and homogeneous, then One Way Anova was applied. If the distribution data is not normal and not homogeneous, then Kruskal Wallis was applied, and also the Post Hoc test with α ≤ 0,05 was considered significant.

Phytochemistry and GCMS Analysis
The content in the papaya seed extract (Carica Papaya L.) only consists of saponin groups. Unfortunately, the alkaloids, flavonoids, tannins, steroids, and triterpenoids were indicated negative by a preliminary screening of phytochemistry tests. The organic compounds in the papaya seeds extract are presented in Figure 1. Based on the peak area (%) of the GCMS data, the main compound was 9-Octadecenoic acid (Z)-methyl ester, a kind of fatty acid methyl ester group, which known as antioxidants 16,5 . Several studies have linked antioxidant effects to phenols and their function as inhibitors of α -glucosides 17,18 . Secondly, Benzyl Nitrile was a precursor for various drugs with potential recreational use 1 . The third primary compound was Hexadecanoic acid-methyl ester, which has antioxidant and anti-inflammatory compound activity 19,20,21 . Generally, plant phenolics are considered vital components of the human diet and exhibit remarkable antioxidant activity and other health benefits 22 . Even though at a small quantity, eugenol was very well known as a component of clove essential oil and exhibited antibacterial, analgesic, anti-inflammatory, and antioxidant properties 23 .  There was a decrease for the K+ group (199.40 ± 12.985) but not significant. There was an increase in body weight in the 3rd and 4th weeks of the P2 and P3 groups. However, the weight loss in the P1 group due to the highest concentration of papaya seeds extract at 10%. Where the possibility of the number of active compounds in the content has a more significant effect, based on these data, we can conclude that the higher concentration of papaya seeds extract correlate to higher effectivity on reducing body weight in rat models.

Body Weight Analysis
In addition, a specific treatment will reduce the hypothalamic NPY expression and serum leptin in high-fat diet mice 24 . A study showed that chronic stress on repeated Force Swimming Tests reduced body weight and calorie efficiency 25 , where the pancreatic lipase enzyme shows an inhibitory effect 13 . While stressed, corticotropin hormone (CRH) will be released from neurons in the parvocellular division 26 . The corticotropin can reduce appetite 23 . Neuropeptide Y also stimulates the inhibition of incoming food (22,23 Figure 3 showed a significant effect of papaya seed extract on reducing blood sugar levels of group P1 (80.20 mg/dL ± 15.057). This effect of P1 was better than the P2 (88.80 mg/dL ± 10.86) and P3 (93.60 mg/dL ± 12.661). Papaya seeds contain saponin compounds that have similar activities to insulin. Based on these data, we can conclude that the higher concentration of papaya seed extract correlates to lower glucose levels in the rat.

Glucose Levels Analysis
Saponin could inhibit lipolysis and increase glucose uptake by adipose cells 27 . This study proposed saponins to reduce blood sugar levels with an antihyperglycemic mechanism by stimulating insulin release in pancreatic βcells 28 . This result was in line with some studies that papaya seed extract could lower blood sugar levels 29,30 . The active substance in papaya seeds may have a hypoglycemic effect that stimulates insulin release from pancreatic beta cells and somatostatin release by suppressing glucagon secretion 10 . In addition, the antioxidant could reduce blood glucose levels by improving pancreatic function (by regenerating cells) to increase insulin production 11 . The main compound, 9-Octadecenoic acid (Z)-, methyl ester, might affect and inhibit the α -glucosidase enzyme activity and minimize the blood glucose level 31 . Based on the Post Hoc test, the average group was divided into two different groups. The first group is the group of the same average that consists of group P1. The second group consisted of K, K-, K+, P2, and P3. It can be seen that the lowest average of IL-6 levels was P1 (177.3075 pg/ml) and the highest average was K-(500.9739 pg/ml). It was found that the p-value was 0.0489 or <0.05, which indicated a significant difference in IL-6 between the treatment groups. Based on these data, we can conclude that the higher concentration of papaya seed extract might correlate to lower levels of IL-6. A study by Nukina et al. (2008) has tested whether stress induces an increase in plasma IL-6 in mice. Plasma IL-6 concentrations increased after one hour of restraint stress and, after that, gradually decreased, indicating that restraint stress could increase plasma IL-6 levels. Further studies found that the induction of increased plasma IL-6 levels by restraint stress was independent of the gut microflora, the primary source of the increase being the liver during stress 32,13 . An increase in IL6 mRNA expression and a fourfold increase in circulating IL-6 levels in the rat hypothalamus was found upon application of restraint stress (3,45). A recent study using prolonged restraint stress in mice also found increased circulating expression of IL 6 mRNA 3,33 .

Analysis of Interleukin-6
Regarding the metabolite content in the papaya seed extract, we expect that the saponin groups affect T lymphocyte cells in producing cytokinins, thereby suppressing the release of IL-6 activity. While stressed, the IL-6 activity increased, whereas added by plant extract, the IL-6 decreased. In addition, 9-Octadecenoic acid (Z)-, methyl ester may affect IL-6 and play a role in immune cells. However, the process is quite complex, so that more in-depth research is needed to find out. They serve as energy sources and structural components of cell membranes, as signaling molecules and precursors for synthesizing eicosanoids and similar mediators. Another research showed that the localization and organization of fatty acids into different cellular assemblages directly influences the behavior of several proteins involved in immune cell activation, including those related to T cell response, antigen presentation, and fatty acidderived intermediates as inflammatory agents 34 . Figure 5 showed a significantly low MDA level in the P1 group compared to P2 and P3. The positive control (K+) was higher than the P1, with a mean value of 2.2996 ng/ml. Based on the Post Hoc test, it was obtained that the pvalue was 0.0464 or < 0.05. This indicated a significant difference in MDA between the treatment groups. Based on these data, we can conclude that the higher concentration of papaya seeds extract might correlate to lower levels of MDA. In our study, the compound of 9-Octadecenoic acid (Z)-methyl ester was expected to entrap the free radicals so that MDA lowered. In contrast, the increase of MDA levels was in line with oxidative stress in the body 35 . Papaya seeds have been shown to reduce blood glucose levels and contain flavonoids, Malondialdehyde alkaloids, and tannins as a source of antioxidants that can entrap free radicals. A study reported the effect of giving papaya seed extract on blood plasma MDA levels in alloxan-induced rats 36 . MDA production was caused by free radicals in the plasma membrane, which causes inflammation. Another study on binahong extract reported an increase in MDA levels at the time of administration caused by the saponin content of binahong. However, this contrasts with another result that showed a decrease in MDA levels in high-dose ethanol extract of binahong leaves, and the causative factors were not known certainly 37 .

Fig. 6. Forced Swimming Test (FST) of all groups at before and after therapy
The Force Swimming Test showed that the immobilization time in rats decreased significantly in the P1 compared to the P2 and P3 groups. The group was divided into three different groups. The first group was the same average group consisting of P1, P2, and P3. The second group had an average length of FST which consisted of P3 and K+. The third group had the same average length of FST immobilization time consisting of groups K-and K. It was seen that the lowest average FST immobilization was P1=84.20 s and P2=93.60 s. Based on these data, we can conclude that the higher concentration of papaya seeds extract might reduce the immobilization time of FST.
In this study, a group of saponins were found in papaya seeds and might affect neurobehavior. This can be seen from the behavioral test through the FST, where the immobilization time in rats decreased during the FST examination. This occurs because of the involvement of noradrenergic activity 12,35 . Nevertheless, it does not affect the activity of MAO-A and MAO-B 35,38 . In addition, saponins also affect the performance of p-regulation through the BDNF signaling pathway. Where plasma cortisol was decreased, spinal dendritic density was increased, and hippocampal neurogenesis was induced by UCMS 31,35 . Meanwhile, the 5-HT, DA, NE, and 5-HIAA also increased, and the immobilization time decreased 31,38 .

Analysis of Tail Suspension Test (TST)
The Tail  Some studies reported that saponin compounds could reduce the immobilization time; meanwhile, the level of 5-HT, DA, and N.E. and 5-HIAA were increased 31,38 . However, there was no effect on MAO-A and MAO-B activities reported 12,38 . Plasma cortisol was decreased, spinal dendritic density was increased, and hippocampal neurogenesis was induced by the UCMS 31,12 .

Proposed Mechanism of Antidepressant
Groups of saponins have been reported to reduce body weight by 20 to 30% by reducing the hypothalamic NPY expression 24 . Neuropeptide Y stimulates the inhibition of incoming food 39,24 . Saponins have insulin-like activity, and they could inhibit lipolysis and increase glucose uptake by adipose cells 29 , causing a decrease in blood glucose levels. Saponin compounds also affect neurobehavior. This effect can be seen from the behavioral test through the FST and TST, where the immobilization time in rats decreased during examinations. Some studies reported involvement of noradrenergic activity 38,40 . Here we proposed the mechanism that major compounds and saponin groups contained in papaya seed extract would activate the immune cells to lower IL-6, including those associated with T cell responses. Saponins groups were also suspected of capturing free radicals so that MDA decreased

CONCLUSION
The papaya seeds extract showed potential effects as antidepressants compared to amitriptyline in rat models. The higher concentration of extract might correlate to higher effectivity on reducing body weight, glucose level, IL-6, and MDA, and limited immobilization time of FST and TST. The phytochemical analysis confirmed the main compounds are 9-Octadecenoic acid (Z)-methyl ester, Benzyl nitrile, Hexadecanoic acid-methyl ester, and other saponins contained in the extract might affect the α -glucosidase enzyme to minimize the blood glucose. Additionally, the fatty acids may influence the behavior of several proteins involved in immune cell activation. Further studies on the immune