Immunomodulatory Activities of Sambucus javanica Extracts in DMBA-Exposed BALB/c Mouse

Purpose: Accumulating evidence shows the genus of Sambucus exerts a broad spectrum of medicinal potencies such as anticancer, antiviral, antibacterial, and antidiabetes. Based on the previous studies, we hypothesized that bioactive compounds of Sambucus might alter several biological systems, including the immune system. Therefore, this study extensively aimed to evaluate the immunomodulatory activities of Sambucus javanica extracts in 7,12-dimethylbenz[a] anthracene (DMBA)-treated BALB/c mouse. Methods: The experimental mice were orally administrated with 2.8 mg.kg-1 BW of DMBA for ten times within a month. After that, the mice were treated by S. javanica berries and leaves extracts for 2 weeks. Subsequently, the inflammation rate was evaluated by using flow cytometry analysis, whereas the necrosis incidences were observed by hematoxylin & eosin staining. Results: Based on the results, we found the expression of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-ɣ) were increased however after treated by S. javanica berries and leaves extracts were significantly decreased. In the same way, necrosis incidence was increased in the DMBA-treated group however it was diminished with S. javanica extracts treatment. Conclusion: Together, these results suggested that S. javanica extracts have immunomodulatory activities to suppress inflammation and reduce necrosis incidence in experimental mice.

evaporated to elucidate the ethanol as the solvent. Finally, the extracts were carried out at 4°C room storage for further experiment.

Immunostaining and flow cytometry
Flow cytometry analysis was performed to evaluate inflammation rate in an experimental animal through measuring the relative number of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-ɣ). Specifically, isolated spleen was homogenized and was stained using several antibodies such as anti-CD4, anti-TNF-α, and anti-IFN-ɣ antibody (Biolegend). Together with that, FACS Calibur TM were employed to evaluate the expression of CD4 + TNF-α + and CD4 + IFN-ɣ + . 14,15

Tissue preparation and hematoxylin & eosin staining
As similar as the previous study, the staining procedures started by specimen deparaffinization with xylol and ethanol respectively. After that, the tissue section of liver and renal cortex were stained with hematoxylin and eosin for 3 minutes. Stained samples then cleaned and dehydrated by using tap water and xylol respectively. Finally, each sample was mounted with malinol and was covered by cover glass. 16

Statistical analysis
The student t test was used to review and compare the statistical difference between two different groups. Thus, in this study, a value of P<0.05 was considered significant between two different groups. The data were expressed as means ± standard deviation.

Results and Discussion
Inflammation rate which evaluated by measuring the relative number of TNF-α and IFN-ɣ was analyzed by flow cytometry after treatment of DMBA or S. javanica berries and leave extracts in experimental mice. According to the analyzed results, the relative number of TNF-α and IFN-ɣ which produced by CD4 helper T-cells was increased by DMBA treatment. Otherwise, the treatment of S. javanica berries and leave extracts significantly decreased the inflammation rate ( Figure 1).
The histological analysis performed to assess the effect of DMBA and bioactive compounds of S. javanica after induction in an experimental animal and to clarify the  Representative microphotograph of necrosis incidence in renal cortex and liver tissues from experimental mice (HE Staining, M=400×). The DMBAtreated mice showed more numbers of necrosis incidence comparing to the S. javanica extracts treatment. Moreover, the treatment group in this study were vehicle; DMBA 2.8 mg.kg -1 BW; BD1, DMBA 2.8 mg.kg -1 + berries extracts 200 mg.kg -1 BW; BD2, DMBA 2.8 mg.kg -1 + berries extracts 400 mg.kg -1 BW; BD3, DMBA 2.8 mg.kg -1 + berries extracts 800 mg.kg -1 BW; LD1, DMBA 2.8 mg.kg -1 + leaves extracts 200 mg.kg -1 BW; LD2, DMBA 2.8 mg.kg -1 + leaves extracts 400 mg.kg -1 BW; LD3, DMBA 2.8 mg.kg -1 + leaves extracts 800 mg.kg -1 BW. Semi-quantitative measurement was applied to cluster the samples as high, moderate, or low of necrotic cells incidence. The bars are the frequencies of necrotic cells incidence in lung samples of mice lung cancer model. necrosis incidence in each group of treatment. In this present study, we applied semi-quantitative analysis which group the necrosis incidence into three parts, i.e. high, medium, and low incidence. Based on the experiment, DMBA promoted necrosis incidence in the liver, however it reduced after the animal model treated by S. javanica extracts. Similar to that, necrosis incidences in the renal cortex were promoted by DMBA induction compared to S. javanica berries or leave extracts (Figure 2).
DMBA is a member of polycyclic aromatic hydrocarbons that abundantly found in the environment as a part of pollutant. DMBA has multiple adverse effects and act as a toxic and carcinogenic agent by inducing the production of reactive oxygen species (ROS) resulting in the increasing level of lipid peroxidation, promoting DNA damage, and depleting the antioxidant system of the cell. 17,18 Currently, we observed the immunomodulation effects of S. javanica extracts toward DMBA-administrated mouse by evaluating the inflammation rate and necrosis incidence ( Figure 3).
In this present study, we revealed that S. javanica extracts can reduce the relative number of TNF-α. Consistent with that, IFN-ɣ also suppressed after the treatment of the extract in an experimental model ( Figure 1). Together, these results suggest that bioactive compounds of S. javanica might reduce inflammation status in DMBAadministrated mouse. Identically, several reports also showed the anti-inflammatory effects of phenolic acids and flavonoid from medicinal plants crude extracts such as Gymnema sylvestre and Alternanthera tenella. 19,20 Other investigations about Sambucus plant, commonly S. nigra and S. ebulus, determined that their bioactive compounds have been effectively used as antioxidant, antiviral, and antidiabetes. 12,13,21 Molecular mechanism showed that polyphenols such as luteolin and quercetin suppressed lipopolysaccharide expression which in turn promoted the activation of mitogen-activated protein kinases signalling pathway including p38 and extracellular signal-regulated kinases pathways, therefore, reducing the production of proinflammatory cytokines and leukocyte. 5,22 Both liver and renal are the most important excretory organs which play a crucial role in detoxification of some toxic metabolic waste products. DMBA induced hepatorenal dysfunction in rats. According to Dakrory et al and Sharma et al, several hallmarks for nephrotoxicity such as plasma level of urea, creatinine, and uric acid were increased in DMBA-treated rats. 23,24 Similarly, in the liver case, DMBA showed the increasing number of liver malondialdehyde (MDA) and the decreasing number of glutathione, glutathione-S-transferase, superoxide dismutase, and catalase which all of those compounds would worsen the condition of the liver. [23][24][25] According to our observation, we found that DMBA treatment enhances the necrosis incidence both in the liver and renal cortex tissues. Whereas, the S. javanica extracts dramatically decreased the number of necrosis incidence ( Figure 2). According to these results, we implied that bioactive compounds bearing in S. javanica exert their effects to reduce necrosis incidence in experimental mice. Comparatively, an investigation reported that Punica granatum which contains rich antioxidants including tannins and flavonoids had been known to decrease oxidative stress in DMBA-administrated rats. 26 Another study related to the medicinal plant also showed that Moringa oleifera crude extract has a protective effect in the liver and renal tissue against DMBA toxicity. 24

Conclusion
Based on the above results, it suggests that S. javanica extracts decreased the inflammation rate in experimental mice. Moreover, the extracts also induced the decline of necrosis incidence in both liver and renal tissues in DMBA-administrated BALB/c mice. Finally, emphasizing the observations about the medicinal properties of S. javanica is still needed for a future therapeutic strategy against other diseases.

Ethical Issues
All experiment aspects of this study have been evaluated by Brawijaya University, Research Ethics Committee with legal number 312-KEP-UB.

Conflict of Interest
We declare there is no conflict of interest in this study.