Zingiber cassumunar roxb. Extract increase the reactive oxidant level and interleukins expression in vitro

Zingiber cassumunar Roxb. (bangle) has a variety of active compounds, including curcumin and phenylbutenoid. Bangle rhizoma reported exhibiting immunomodulatory activities. This research aims to determine the mechanism of bangle extract as an immunomodulator by the secretion of Reactive Oxygen Intermediate (ROI), Nitric Oxide (NO), and interleukin (IL-10 and IL-14) expression level. Bangle extract (Zingiber cassumunar Roxb.) was made by the maceration method using 96% ethanol solvent. This research was administered in vitro using macrophage cells from male mice with Balb/C strain divided into 2 groups: normal control and treatment group (receiving 25, 50, and 100 ppm of extract). The administration of bangle extract can function as an immunomodulator by an increase of ROI in 25 and 50 ppm of the extract significantly than the control group (p <0.05), the treatment groups decrease NO level (p <0.05), it also was found to increase expression of IL-10 and IL-14 expression levels (p <0.05). Zingiber cassumunar Roxb. extract was potentially to be developed as an immunomodulator.


INTRODUCTION
The immune system defends our body against invaders, such as viruses, bacteria, and foreign bodies which are the cause of various diseases. It consists of a natural immune system (innate immunity/non-specific) and an adaptive immune system (adaptive immunity/specific) (Akrom, 2017; Baratawidjaja and Rengganis, 2014). Immunomodulators are pharmacological agents that can modulate a partial immune response that is spurred by an immune response, on the other hand, it inhibits some of the other immune system. Immunomodulators are restoring the imbalance of the disrupted immune system (Akrom, 2017).
Macrophages are professional phagocytes that act as APC and the main effectors in cellular innate and adaptive immune response (Murphy, 2012). In the body's defense mechanism against invaders, macrophages become the leading component of immune blocking. Macrophages express many surface receptors that can catch and swallow (degrade) microbes, in a process called phagocytosis (Baratawidjaja and Rengganis, 2014). Phagocytosis and reactive oxygen intermediates (ROI) are the macrophages main mechanism in destroying infected cells (Akrom et al., 2015). Activate macrophages can stimulate the proliferation and activity of T and B lymphocyte cells. Macrophage cells act as antigen-presenting cells (APC) that will activate Th-0 lymphocytes. Activated CD4 Th-0 lymphocytes will proliferate towards Th-1 or Th-2 depending on the cytokine environment and the location of the antigen. Activated T lymphocytes will release various mediators, differentiation towards Th-1 will produce proinflammatory cytokines while differentiation towards Th-2 produces anti-inflammatory cytokines (Thiery et al., 2003;Bastos et al., 2004;Akrom and Mustofa, 2017).
One of the potential plants in Indonesia is bangle (Zingiber cassumunar Roxb.) has been proven to have scientific activity as an immunomodulator. Bangle ethanol extract has been shown to have an immunomodulatory effect, indicated by an increase in the activity of macrophage cell phagocytosis, ROI secretion, and IL-10 expression, decreased NO secretion, and TNF-α production in vivo (Arini et al., 2014;Nurkhasanah et al., 2017;Fitriana et al., 2018). In vitro research, phenylbutanoid compounds from bangle rhizomes can increase phagocytic activity of macrophage cells, and inhibit NO production (Chairul et al., 2009;Nakamura et al., 2009;Kaewchoothong et al., 2012). Besides, the nhexane bangle fraction can reduce the phagocytic activity of macrophage cells, and decrease lymphocyte proliferation (Nurkhasanah et al., 2019b). In this study, we want to clarify the activity of bangle ethanol 96% extract as an immunomodulator by analyzing the secretion of ROI, NO, IL-10, and IL-14 by the macrophage.

Scientific hypothesis
Bangle has immunomodulatory activities with a mechanism of increasing ROI and NO secretion level, increasing IL-10 and IL-14 expression level, in vitro.

MATERIAL AND METHODOLOGY Material and subject
Fresh Z. cassumunar rhizome purchased from the local market, Yogyakarta, Indonesia. The sample was verified and identified in the Biology Laboratory of Universitas Ahmad Dahlan. Macrophages were obtained from the peritoneal cavity of mice Balb/c strain aged eight weeks old (20 -30 g) from the Integrated Research Laboratory of Universitas Gadjah Mada (Laboratorium Penelitian dan Pengujian Terpadu, LPPT UGM).

Preparation of Bangle Extract
Z. cassumunar extraction was carried out using the maceration method and 96% ethanol as the solvent. The maceration was done for 3 x 24 hours. The macerate was filtered, and then evaporated (rotary evaporator) for 2 hours per day in a week, and used a water bath until a thick extract was obtained.

Preparation of Test Animals
The use of test animals in this research had received ethical approval from the Commission for Research Ethics of Universitas Ahmad Dahlan with Number: 011804063. The test animals were male mice Balb/c strain aged eight weeks old.

Isolation of Macrophages
Mice were narcotized with chloroform after being fasted for 10 -12 hours. Then, mice were placed in the supine position. The mice's abdomen skin cleaned using disinfectant (70% alcohol) and dissected. The peritoneal sheath cleaned with 70% alcohol. Then, 10 mL of cold RPMI injected into the peritoneal cavity and shaken slowly for three minutes. The inner cavity pressed with two fingers and the fluid from the peritoneal cavity (the nonfatty part) drawn using an injection syringe to obtain an aspirate.
The aspirate is centrifuged at 1200 rpm, 4 °C for 10 minutes. The supernatant was removed, and the pellets (macrophages) resuspended with 1,000 μL complete medium. The number of cells counted from 10 μL macrophage suspension in a hemocytometer. The macrophage cell suspension grew in a 6-well microtiter plate (coverslip) with a density of 5 x 10 5 cells/well for the ROI and interleukin assay. And a 6-well microtiter plate with a density of 1 x 10 5 cells/well for the NO assay

Reactive Oxygen Intermediate (ROI) Secretion Assay
The 100 μL of macrophage cell suspension grew in a 6-well microtiter plate (coverslip) with a density of 5 x 10 5 cells/well. The cells incubated in a 5% CO 2 incubator at 37 °C for 15 minutes. 800 µL of complete RPMI medium added to each well, then microplates incubated overnight. The medium was removed and the sample added each well. The microplates were incubated in a 5% CO 2 incubator at 37 °C overnight.
The 50 -100 μL of NBT solution and 1 mL of PBS (containing 125 PMA) added to each well, then incubated in 5% CO 2 incubator at 37 °C for 60 minutes. The reagent was removed, dried at room temperature, and fixed with absolute methanol. After dried, applied a 2% neutral red solution to the coverslip. The percentage NBT reduction of macrophage cells, it calculated from 100 cells examined by a binocular microscope (XSZ 107 BN, Novel) and Optilab with 400x magnification (Nurkhasanah et al., 2017).

Nitric Oxide (NO) Secretion Assay
Griess Reaction Assay used for NO levels testing. Griess reagent, allow to stand for 5 -10 minutes at room temperature and protected from direct light, until the color changes. The absorbance measured using an ELISA reader at a wavelength of 550 nm (Nurkhasanah and Zulkarmen, 2014; Nurkhasanah et al., 2017).

Interleukin-10 and Interleukin-14 Expression Assay
Previously, the preparation of cell culture is the same as in ROI assay. Macrophage cells culture fixed using methanol and then washed with Phosphate Buffer Saline (PBS). The microplates soaked in 300 μL peroxidase blocking solution and washed with distilled water. Then the microplates added 20 μL protein blocking serum, incubated at humid temperature for 10 -15 minutes. Added 30 μL Interleukin-10 and Interleukin-14, incubated at room temperature then washed with PBS. Added with 30 μL of biotin, incubated at room temperature then washed with PBS. Added with 30 μL of the enzyme streptavidin-peroxidase, incubated at room temperature then washed with PBS. Added with 30 μL peroxidase substrate solution (DAB), incubated at room temperature, and washed with distilled water. Added with 100.0 μL of Mayer Hematoxylin (counterstain), incubated at room temperature then washed with distilled water. Then the microplates soaked in absolute alcohol, cleaned, and dried. The microplates dipped in xylol and dried. Then the microplates dropped with mounting media and covered using a deckglasser. After dried, observed in a binocular microscope (XSZ 107 BN, Novel) and Optilab with 400x magnification to examine the color of the cells, expression of IL-10 and IL-14 has intense brown (Javois, 1999; Nurkhasanah et al., 2019a).

Statistic analysis
All statistical analyzes performed using the SPSS version 22 program. The normality test and homogeneity test performed about the data ROI levels, NO levels, and the expression of Interleukin-10 and Interleukin-14. Then proceed with the One-way ANOVA and LSD tests (with a significance level of 0.05).
The normality test performed using the Shapiro-Wilk's test, with total data of less than 50. The variant homogeneity test performed using the Levene's test. If the results of the normality test and homogeneity test are homogeneous variance and normally distributed, then the test continued with the analysis of one way ANOVA variants, and LSD test.

RESULTS AND DISCUSSION Result of ROI Secretion Assay
The NBT reduction test (tetrazolium nitro blue reduction test, containing PMA (phorbol 12-myristate, 13-acetate)) was used to measure the ability of peritoneal macrophage cells to secrete ROI. NBT (formazan salt) will diffuse into cells, then tetrazolium succinate reductase enzyme will divide into formazan. ROI cause increased respiration and reduction of NBT by forming black formazan deposits (Leijh et al., 1986). It can be seen in Figure 2, macrophage cells are black show secrete ROI due to formazan deposition. In contrast to macrophage cells that do not secrete ROI, it looks only brown without any formazan deposits. Table 1 shows the average levels of ROI secretion in the normal control group and the treatment group concentrations of 25, 50, 100 ppm. The anti-inflammatory mechanism of this compound can be related to the ability of bangle to increase the expression of IL-10 (Fitriana et al., 2018). Increased IL-10 expression will inhibit the production of IL-12, IL-1, and TNF-α. Inhibition of IL-1 and TNF-α production can affect T-cell activation to inhibit the inflammatory reaction. IL-12 has an important role in differentiating CD4 + into Th1 cells, then Th1 cells will secrete IFN-γ to activate macrophage cells to produce ROI. The inhibition of IL-12 production will indirectly inhibit the secretion of IFN-γ in case ROI production will decrease (Bratawidjaja, 2014).

Result of NO Secretion Assay
NO is an effective antibacterial effector in the immune system. NO is a free radical synthesized by the enzyme nitric oxide synthase (NOS) through complex reactions. The main isoform expressed by macrophages is iNOS, this isoform will induce NO expression (Kil et al., 2012). In this study, NO levels were measured using a Griess Reaction Assay (colorimetric method).
The concentration of NO secreted by macrophages will be calculated in the form of nitrites. Sulfanilamide (diazotization reagent) react with nitrite (in alkaline) will form to diazonium salt, then react with N-(1-naphthyl) ethylene diamine hydrochloride (coupling reagent) to be a stable form. The final result is intensive pink color and absorbance can be measured at wavelength 550 nm using Elisa Reader (Nurkhasanah et al., 2017).
In Table 2 the average levels of NO secretion in the treatment group concentrations of 25, 50, and 100 ppm were 0.36; 0.76; and 0.47 μM. This result is significantly different (p <0.05) compared to the average NO level in the normal control group (9.26 μM). The decrease NO levels in the treatment group concentrations of 25, 50, and 100 ppm can be explained because of the results of IL-10 expression parameters. The results of IL-10 expression parameters are the treatment group 25, 50, 100 ppm has higher levels of IL-10 expression, and significantly different (p <0.05) compared to the normal control group.
The decrease NO levels were related to the results of the IL-10 parameter. In this study, the treatment group has a higher IL-10 expression than the normal group. iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (Salim et al., 2016). IL-10 (macrophage inhibitor) acts to inhibit proinflammatory cytokine production included TNF-α, IL-1, and IL-12. IL-12 has a role to stimulate IFN-γ production. Over this explanation, the active component in bangle can act as an immunomodulator by reducing NO levels (Goodyear-bruch and Pierce, 2002; Akrom, 2017).

Interleukin-10
Observation of interleukin (IL) expression was carried out by an immunocytochemical method that uses specific antibodies to detect the expression of specific proteins (antigens) in cells. This research uses indirect immunocytochemical methods, the advantage is the results obtained have a more intense color, but it requires more time in the process (Meshcer, 2017). The antigen will be bound indirectly to the primary antibody (IL-10 and IL-14) which has a role to recognize the antigen (first layer), then add a secondary antibody (biotin which binds to the enzyme streptavidin peroxidase) being the second layer. The addition of secondary antibodies is also followed by the addition of chromogen substrate (DAB or 3,3diaminobenzidine tetrahydrochloride), this substrate will be changed by enzymes so that it will form color deposits (pigments) in cells. To differentiate cells that are expressed IL-10 will have a brown color by DAB, while cells that are not expressed have a blue or purple color by Mayer hematoxylin (counterstain). Figure 3 shows the expression of IL-10 in macrophage cells treated with extract concentrations of 25, 50, and 100 ppm.
In Table 3 the average levels of IL-10 expression in the treatment group concentrations of 25, 50, and 100 ppm were 69.96%; 69.26%; and 53.29%. This result was higher and significantly different (p <0.05) compared to the average level of IL-10 expression in the normal control group (9.61%). The results obtained are suitable to those reported by other researchers that Zingiber cassumunar has the immunomodulatory activities one of activity by increase IL-10. This activity may be attributable to curcumin and phenylbutanoic as an active compound in this extract (Fitriana et al., 2018;Nurkhasanah et al., 2020).  Increasing the concentration of the test compound does not accord with an increased level of IL-10 expression because IL-10 is produced by active macrophages and Th-2 cells (Akrom, 2017). The results of the phagocytic activity parameters of macrophages also showed a decrease (%) of active phagocytic cells and phagocytosis index with an increase in the concentration of the test compound (Adhila et al.,  2019). The decrease in active macrophages will reduce the expression of IL-10 produced.

Interleukin-14
Similar to IL-10, the way to differentiate between cells expressed IL-14 will have a brown color by DAB, while cells that are not expressed have a blue or purple color by Mayer hematoxylin (counterstain). Figure 4 shows the expression of IL-14 in macrophage cells treated with extract concentrations of 25, 50, and 100 ppm.
In Table 4 the average levels of IL-14 expression in the treatment group concentrations of 25, 50, and 100 ppm were 87.44%; 70.13%; and 61.15%. This result was higher and significantly different (p <0.05) compared to the average level of IL-14 expression in the normal control group (2.16%). The administration of bangle ethanol extract showed the effect was to increase IL-14 expression. Although a decrease in IL-14 levels was seen with the administration the higher concentration of the extract. From this explanation, it can be seen that the active component of the bangle can act as an immunomodulator by improved IL-14 levels.
A previous study highlights that Zingiber cassumunar has the immunomodulatory activity which may be caused by curcumin and phenylbutanoic compound (

CONCLUSION
Bangle ethanol 96% extract decreased NO levels (in all variations of extract concentration), and increased ROI levels compared to normal control groups (at extract concentrations of 25 and 50 ppm) with a significant effect (p <0.05). Also, bangle ethanol 96% extract increased in IL-10 and IL-14 levels compared to the normal control group (in all variations of the extract concentration) with a significant effect (p <0.05). These results indicate that bangle ethanol 96% extract has an immunomodulatory effect in vitro.