In vitro immunomodulatory activity test of Bengle rhizoma extract (Zingiber cassumunar Roxb.): phagocytic activity of macrophages and lymphocyte proliferation in mice

Immunomodulators are pharmacological agents that affect the immune system at different levels. Aside from modulating, some immunomodulators stimulate, while some others inhibit immune responses. Zingiber cassumunar Roxb. or Bengle rhizoma has been reported to exhibit immunomodulatory activities. This research was intended to determine the pharmacological effects of its extract on the phagocytic activity of macrophages and lymphocyte proliferation in vitro. It used the macrophages and lymphocytes of male BALB/c mice, which were divided into normal control and treatment group (receiving 25, 50, and 100 ppm of extract). The immunomodulatory activity test results showed that 100 ppm of Bengle rhizoma extract reduced phagocytosis in macrophages much significantly than the control group and that the treatment groups suppressed the proliferation of lymphocytes more substantially than the control group. The extract decreased the phagocytic activity of macrophages and the proliferation capacity of lymphocytes when administered at a concentration of 100 ppm.


Isolation of macrophages
After fasting for 10-12 hours, the mice were narcotized or placed in a state of narcosis with chloroform. Then, they were placed in the supine position, and the abdomen was cleaned using a disinfectant (70% alcohol). The stomach skin was dissected, and the peritoneal sheath was cleaned with 70% alcohol. Cold RPMI medium was injected into the peritoneal cavity and shaken slowly for three minutes. Afterward, the inner cavity was pressed with two fingers to remove fluid from the peritoneal cavity. Peritoneal fluid from the non-fatty part far from the intestines was drawn using an injection syringe to obtain an aspirate.
The aspirate was centrifuged at 1200 rpm, 4°C for 10 minutes. Then, the supernatant was removed, and the pellets (macrophages) were separated. The pellets were resuspended with 1,000 μL of complete medium. The number of cells was counted from 10 μL of macrophage suspension in a hemocytometer.
In the hemocytometer, four counting rooms were used because macrophages are cells with low population density. The formula used for the counting (number of cells per mL) is as follows: In the phagocytic activity test, the density of the cultured macrophages was 5x10 5 cells/well, and a 6-well microplate equipped with a coverslip was used. The cells were first incubated for 15 minutes in a 5% CO 2 incubator at 37°C. Finally, 700 μL of complete medium was added to each well, and the microplate was re-incubated for 24 hours Ulfah et al., 2017;Nurkhasanah et al., 2017.

Phagocytic activity test
After 24-hour incubation, the 6-well microplate containing macrophages was removed. Since the subsequent procedure was carried out in LAF, the complete medium in each well was discarded and replaced with 700 μL of test compound per well. The microplate was re-incubated in a 5% CO 2 incubator at 37°C for 24 hours.
In the phagocytosis test, 200 μL of latex was added into each well (2.5x10 6 density per well), and the microplate was re-incubated in a 5% CO 2 incubator at a temperature of 37ºC for one hour. The cells in each well were washed with 1,000 μL of PBS once, dried at room temperature, and fixated with 300 μL of absolute methanol for 30 seconds. After the cells were dried, 400 μL of 20% Giemsa stain was applied to each well, and the microplate was left at room temperature for 20 minutes. The remaining Giemsa was removed, and the well was washed with distilled water. After the wells were dried at room temperature, the coverslip of each well was removed. The subsequent observation involved counting the number of macrophages that had phagocyted latex particles in 100 cells (percentage) under a light microscope at 400x magnification Ulfah et al., 2017;Nurkhasanah et al., 2017.

Isolation of Lymphocytes
The spleen tissue was isolated aseptically from mice that had been narcotized with chloroform. Then, it was placed in a petri dish, washed with PBS 2-3 times, and added with 10 mL of RPMI medium to produce a lymphocyte suspension in the medium. The suspension was centrifuged at 3,200 rpm and 4°C for 10 minutes. The formed sediments were separated from the supernatant and resuspended in 3 mL of complete medium. ISSN: 2088 4559;e-ISSN: 24770256 Pharmaciana Vol. 9, No. 2, Nov 2019 The lymphocyte count was performed by placing 10.0 μL of lymphocyte suspension in a hemocytometer. Living cells with a bright and clear appearance and round shape were counted Ulfah et al., 2017.

Lymphocyte Proliferation Test
A total of 100 μL of the isolation results and lymphocyte cultures (final concentration= 1.5x10 6 cells per mL) were distributed into a 96-well microplate. Then, 50 μL of the test compound was added to each well, followed by 72 hours of incubation (for 3-day proliferation testing) in a 5% CO 2 incubator at 37°C. Afterward, 50 μL of MTT with a concentration of 5 mg/mL was added to each well, which was later incubated for 4 hours Azadmehr et al., 2016;Ulfah et al., 2017. Living cells react to MTT by forming purple formazan crystals. A reagent stopper (10% SDS) was added to each well, and then the microplate was stored at room temperature in the dark for 12 hours. Afterward, an ELISA reader was used to measure the absorbance at a wavelength of 595 nm.

Data Analysis
The normality (Shapiro-Wilk) and homogeneity tests were performed on phagocytosis activity data, i.e., % phagocytic activity and index of macrophages, and lymphocyte proliferation data, namely absorbance (ELISA test results). These tests were followed by One-way ANOVA and LSD test (with a significance level of 0.05).

RESULTS AND DISCUSSION
The part of Z. cassumunar plant used in this study was rhizome. In the extract preparation, slices of Z. cassumunar rhizomes were dried in an oven to remove water content (up to less than 10%). This drying aims to minimize the possibility of fungus and mold growth on the dried specimen to prolong its utilization without changing the active ingredients in it (Rivai et al., 2014). As for oven-drying technique, this procedure can help avoid any damages to active components. The dried specimen was then ground into a powder and macerated using 96% ethanol. The macerated mixture was then filtered, evaporated in an evaporator, and incubated in a water bath until a dense extract was obtained. This dense extract was tested for its activities or effects on the immune system.
The immune system is a defense mechanism of the body to protect itself from pathogens that can cause health problems. There are specific and non-specific immune systems (Akrom, 2013). Previous studies have reported that Z. cassumunar Roxb. is an effective immunomodulator both in increasing the phagocytic activity of macrophages and ROI secretion and decreasing NO secretion (Nurkhasanah et al., 2017;Chairul et al., 2009).

The effects of Bengle rhizoma extract on the phagocytic activity of macrophages
Antigens from macromolecules and pathogens activate phagocytosis in macrophages, which in this study was measured based on the number of macrophages that phagocyted latex. The research parameters were % activity (i.e., the percentage of active macrophages from the total number of macrophages) and the phagocytosis index (i.e., the average number of latex particles stored in 100 macrophages, that is, the number of latex that the macrophages ingested multiplied by 100 and divided by the total number of macrophages) (Nurkhasanah et al., 2017).
The process by which macrophages engulf macromolecules and pathogens and create phagosome depends on their ability to activate themselves once a foreign body, e.g., latex, is identified. The Bengle rhizoma extracts were tested for their phagocytic activity, and the test results were compared with the normal control group. These data were processed statistically using ANOVA and LSD test. The results of the phagocytic activity of macrophages are summarized in Table I and II:  Tables I and II, Bengle rhizoma extracts with concentrations of 25 ppm and 50 ppm activated phagocytosis, although not significantly different from the normal control group. In contrast, at 100 ppm, the extract decreased the activity and index of phagocytosis much more substantially than the normal control group. The phagocytic activities of macrophages, as induced by different concentrations of Bengle rhizoma extracts, are presented in Figure 1.
Another in vivo study has proven that the administration of Bengle rhizoma extract at a dose of 5 mg/20 mg BW in mice for seven days optimally increases NO and ROI secretion although it does not significantly influence the phagocytic activity of macrophages (Nurkhasanah et al., 2017). Despite the successful dose optimization in the study, the best rule of administration has not been analyzed yet.
The immunostimulant properties of the test compound is a factor that can influence the effectiveness of a compound in shaping the immune response. Based on immunostimulant properties, chemical compounds are classified into two groups, namely low molecular weight (phenols, alkaloid, saponin, and quinone) and high molecular weight (Wagner, 1999). Any substance that can induce an immune response is called an immunogen, and the molecular weight of the most potent immunogen, i.e., protein macromolecule, is 100,000 daltons. Accordingly, immunogens weighing <100,000 daltons are deemed weak; hence, tiny molecules like amino acids are not immunogenic. Phenylbutenoid is a component of Bengle rhizoma (Chairul et al., 2009). Since it is a phenolic compound, it is an immunostimulant with low molecular weight and weak immunogenic nature (Bhuiyan et al., 2008). In addition to these properties, the immunogenicity of a substance also depends on the amount of administered immunogen, as well as the way and the amount of which the substance enters into the body. The immune response can be optimized by determining the dose of immunogen, method of administration, and time of administration carefully (Bratawidjaya, 2014).

The effects of Bengle rhizoma extract on lymphocyte proliferation
The test of lymphocyte proliferation activity used the MTT assay and was based on the ability of the living cells to reduce yellow MTT (water-soluble) into formazan salt (water-insoluble). The produced formazan salts are purple, and this has a direct correlation with cell count. The microplate reader shows the absorbance that indicates the number of living cells (Mosmann, 1983;Ulfah et al., 2017).
In this study, the MTT assay was prepared for two sets of tests, namely the MTT assay for three days (MTT-3) and five days (MTT-5). It aimed to identify any differences in lymphocyte proliferation in three and five days. The ability of lymphocyte to proliferate after the addition of Bengle rhizoma extract into the wells was visible by comparing the absorbances of the test groups and the normal control group.
In the test with MTT-3 assay, the addition of various concentrations of Bengle rhizoma extract (test groups) to the cultures turned out to decrease the number of lymphocytes, while it was not the case with the normal control group. This effect was apparent from the absorbance of the cultures. The microplate reader detects absorbance values. In this case, these values are the measure of light absorption by formazan crystals-formed as the product of MTT reduction to its insoluble formazan due to succinate dehydrogenase enzyme. This enzyme is produced from the mitochondria of living cells, meaning that formazan crystals can only form in living cells. The number of living cells correlates positively with that of formazan crystal and, by extension, absorbance (Mosmann, 1983;Ulfah et al., 2017). In the MTT-5 assay, the test showed the same results as the MTT-3 assay. Contrary to the normal control group, all concentrations of Bengle rhizoma extract reduced the number of cells in the culture. The LSD test results, as seen in Table III, showed a significant difference between the normal control group (added with lymphocytes and the complete medium) and the test groups. It indicates that Bengle rhizoma extract at concentrations of 25, 50, and 100 ppm produces an effect, that is, the reduction and suppression of lymphocyte proliferation. In other words, this test compound can lower the body's immune system.
Curcumin is known to be an ingredient of Z. cassumunar. It can explain the decrease in lymphocyte proliferation in this study. Previous studies have claimed that curcumin in Curcuma xanthorrhiza has an inhibitory effect on lymphocyte proliferation in rabbit (Huang et al., 1992) and humans-where lymphocyte proliferation was induced by 0.01 and 0.05 mg/ml of PHA (Yadav et al., 2005). In another research, splenic cell antiproliferation is associated with the effects of curcumin on myelin basic protein (MBP)-specific lymphocyte T that leads to the inhibition of allergic reactions (Natrajan and Bright, 2002), confirming the results of this study.
Although the exact mechanism of the anti-proliferative effect of curcumin is not certainly known, there is a possibility that curcumin impedes proliferation by inhibiting ribonucleotide reductase and DNA polymerase activation, which are the two main enzymes in DNA synthesis or the essential processes in cell duplication in the S phase of the cell cycle (Fontecave, 1997;Sun et al., 1998).

CONCLUSION
In vitro administration of Bengle rhizoma extract (Z. cassumunar) to macrophages and lymphocytes can result in reductive effects on the body's immune system, as evident in the phagocytic activity of macrophages and the ability of lymphocyte to proliferate. At concentrations of 25 and 50 ppm, the extract can reduce the phagocytic activity of macrophages, although less significantly different than the control group. On the contrary, phagocytosis in macrophages added with 100 ppm of Bengle rhizoma extract is reduced much significantly than the control group. Also, these three concentrations successfully suppress lymphocyte proliferation.