INTRODUCTION:

Infectious diseases are the second cause of death worldwide after cardiovascular disease, especially in developing countries, like Indonesia with 15.6% in women and 16.7% in men, followed by cancer with 11.8% in women and 13.4% in men. The fourth cause of death is also an infectious disease, namely respiratory tract infections, with 7.4% in women and 7.1% in men¹.

According to the 2018 Riskesdas report, it was stated that the prevalence of Upper Respiratory Tract Infection (ARI), which was categorized based on the diagnosis of health workers and symptoms of ARI disease caused by Staphylococcus aureus infection in Southeast Sulawesi province, was at number 22 with a percentage of 7.5%². Some cases of infectious diseases result in decreased immune system function, such as a decreased function of phagocytic cells and T helper (CD4) lymphocytes. Currently, a health problem due to the decrease in CD4 (Cluster of Differentiation-4) in the body is the AIDS infection (Acquired Immunodeficiency Syndrome), where CD4 is the main target of HIV (Human Immunodeficiency Virus) to destroy the body's immune system³. Optimizing the function of the body's immune system is one of the efforts to prevent infection by providing immunomodulatory substances^4,5.

Immunomodulators are natural or synthetic substances that help regulate and optimize the immune system⁶. Clinically, an immunomodulator is used in patients with impaired immunity, such as cancer, HIV/AIDS, malnutrition, allergies, etc. Immunomodulators based on the source are synthetic immunomodulators and immunomodulators derived from plants. However, the use of synthetic immunomodulators often causes unwanted side effects, one of which is drugs used to restore the imbalance of the immune system, such as steroid anti-inflammatory drugs that can cause microscopic gastrointestinal bleeding, decreased platelet levels respiratory depression, immunosuppressants⁷.

CD4 is a protein molecule expressed on a subset of thymocytes and inflammatory T cells (two-thirds of peripheral T cells), monocytes, and macrophages^8,9. CD4 cells are a type of white blood cell or lymphocyte important to the immune system. CD4 cells are sometimes referred to as T cells or T4 cells (Thymocytes 4), which function as helper cells. CD4 cells activate Th1 cells (T Helper 1), which then activate macrophages to destroy microbes, so that CD4 is often used as an immunomodulator parameter. The association between macrophage phagocytic cells and CD4 is that when CD4 expression increases, CD4 as a co-activator will help Th1 activation proliferate, further releasing the cytokine Interferon-gamma, which helps macrophages in phagocytosis^10,11.

Alternative immunomodulators that can balance the body's immune function can be obtained from natural materials such as plants and marine biota^12-15. One plant that can be used as an immunomodulator is Etlingera rubroloba A.D Poulsen, which has taxonomic closeness to the Kecombrang (Etlingera elatior (Jack) R.M.Smith) plant. Based on the taxonomy, plants with the same taxon have a close relationship, especially at the family and genus levels. There may be similarities in the content of substances that have the same kinship¹⁶. The secondary metabolite content in Kecombrang consists of alkaloids, flavonoids, polyphenols, steroids, saponins, and essential oils¹⁷. According to a previous study, the ethanolic extract of Kecombrang fruit has been proven to be an immunomodulator by increasing the phagocytic activity of peritoneal macrophages in mice¹³. Another study found that methanol extract and isolate compounds from stems of Etlingera rubroloba A.D Poulsen have pharmacological activity as antioxidants and inhibitors of the xanthine oxidase enzyme^19,20.

The development of Etlingera rubroloba A.D. Poulsen plants by exploring bioactive compounds as alternative immunomodulators need to be improved. The phagocytic activity and CD4 levels have not been scientifically proven, so that researchers are interested in researching "Immunomodulatory Potential of Etlingera rubroloba A.D. Fruit Extract. Poulsen on Phagocytic Activity and CD4 Levels in Wistar Male Rats".

MATERIALS AND METHODS:

Materials:

The material used in this study was the fruit of Etlingera rubroloba A.D. Poulsen, male rat (Rattus norvegicus) Wistar strain, Staphylococcus aureus ATCC 25923, Elisa CD4 (Elabscience®) kit, 96% ethanol (Mercks®), 70% alcohol (Mercks®, methanol (Mercks®, McFarland, EDTA (Merck®), aqua pro injection (Otsu-NS®), Na-CMC 0.5% (Food Grade®), physiological NaCl (Otsu-NS®), ether (Mercks®), Giemsa 10% (Mercks®), immersion oil (Mercks®), Phosphate Buffered Saline/ PBS (Sigma®), Nutrient Agar/ NA (Merck®), and commercial Phylantii extract (Stimuno®).

Sample Preparation, Determination, Extraction and Phytochemical Screening:

The sample used in this study was the fruit of Etlingera rubroloba A.D. Poulsen taken in Punggaluku Village, Punggaluku District, South Konawe Regency, Southeast Sulawesi Province. The E. rubroloba fruit (17.5kg). Dried Simplicia powder (3.3kg) was macerated and obtained a total of 424.6grams concentrated extract with a yield value of 12.86%. The health research ethics committee has approved this research, Institute for Research and Community Service, Halu Oleo University number: 706/UN29.20/PPM/2020.

The sample was determined at the LIPI Cibinong Biology Research Center to ensure that the sample used was the fruit of Etlingera rubroloba A.D. Poulsen.

Samples were macerated for 3x24 hours with 96% ethanol. The filtrate was collected and concentrated using a rotary vacuum evaporator at a temperature of 50ºC to obtain a thick extract^13,17,18. Furthermore, phytochemical screening was carried out using the colorimetric method to determine the content of secondary metabolites, including tests for alkaloid compounds, flavonoids, saponins, tannins, and terpenoids^21,22,23.

Immunomodulator Test:

The immunomodulatory activity was assayed with parameters of phagocytic activity and CD4 levels in male Wistar rats as follows:

The experimental animals were adapted to the cage environment for 7 days to adapt the rat (Rattus norvegicus) to the new environment. Rats were placed in cages containing husks, which functioned to absorb rat droppings, and were fed and drank in each cage containing 5 rats²⁴. The mice used were healthy mice that showed normal behavior. The test animals were divided into 6 groups (n = 4) by random allocation with the number following the Federer formula consisting of normal, negative (Na-CMC 0.5%), positive (Phylantii extract 0.135mg/kgbw), extract dose I (200), dose II (300), and dose III (400) mg/kgbw²⁵. Each animal was given 3 mL orally for seven days. On the eighth day, all mice (except the normal group) were infected with 0.5 mL of Staphylococcus aureus intra-peritoneally and then allowed to stand for 1 hour^15,26. The rats were anesthetized with inhaled ether and then dissected. It was followed by taking the intraperitoneal (IP) fluid from the abdominal cavity previously added with 1 mL of Phosphate buffered saline (PBS) solution of pH 7.8. The IP fluid was smeared thinly on an object glass fixed with methanol for 5 minutes, then stained with 10% Giemsa stain, allowed to stand for 20 minutes, rinsed with running water. After the preparation was dry, it was viewed under a microscope using oil immersion with 10x–1000x magnification. The immunostimulant activity was determined by calculating the phagocytic activity of rat peritoneal macrophages. The value of phagocytic activity (SPA) is the percentage of macrophage cells that actively carry out the phagocytosis process among 100macrophage cells^12,14.

Subsequently, 1mL of blood samples were taken from the intracardial area. Blood was put into a vacutainer tube containing 0.1% EDTA anticoagulant and centrifuged at 3000rpm at 25ºC for 20 minutes, and the plasma was put into an Eppendorf tube and stored at -20˚C until the time of CD4 examination by ELISA^27,28. One-way ANOVA and post hoc Tukey analyzed measurement of CD4 levels using the sandwich ELISA method and data.

RESULT AND DISCUSSION:

The secondary metabolite compounds screening was performed to determine the compounds contained in E. rubroloba fruit²⁹. The content of the secondary metabolites in Etlingera rubroloba A.D. Poulsen fruit ethanol extract were alkaloid compounds, flavonoids, saponins, tannins, and terpenoids, as shown in Table 1 below.

Table 1. Secondary Metabolite Screening of E. rubruloba Fruit extract

Chemical Test	Reagent	Results
Flavanoid	Mg + HCl	Positive
Alkaloid	Deggendorf	Positive
saponin	H₂O + HCl 2N	Positive
Tanin	FeCl₃	Positive
Terpenoid	Liebermann Buchard	Positive

The immunomodulatory test in this study was conducted to know the immunomodulatory potential of the ethanolic extract of the fruit of Etlingera rubroloba A.D. Poulsen with parameters of macrophage cell phagocytic activity and CD4 levels (Cluster of Differentiation 4) in male Wistar rats. CD4 cells are a co-activator that can activate macrophage cell phagocytosis by activating Th1 lymphocytes, secrete interferon-gamma, and activate macrophage cells¹⁰. Thus, the higher the level of CD4 in the body, the more activated macrophages destroy microbes that enter the body. Normal levels of CD4 lymphocytes range from 600 to 1200 cells/mm3 of blood, so the lower the CD4 level, the greater the damage caused by pathogens³⁰. Testing of immunomodulatory activity in this study was carried out with three dose variations, namely the dose of 200 mg/kgBW, 300 mg/kgBW, and 400 mg/kgBW, the selection of dose variation was conducted according to the previous study¹⁴.

Increased phagocytic activity of macrophages is characterized by the shape and size of macrophages that increase in size with the highly variable extension of pseudopods, darker cytoplasmic colors. Phagosomes appear membranes that become more tortuous, lysosomes become more numerous, the Golgi apparatus enlarges, and the rough endoplasmic reticulum develops^10,14. The phagocytosis test was carried out by calculating the value of the phagocytic activity of peritoneal macrophages in mice that were actively phagocytosing in 100 cells expressed in percent activity (SPA) as shown in the following table 2 results.

Table 2. Phagocytic Activity of Macrophage

Group	Phagocytic Activity Average (%) ± SD
Negative (Na CMC 0,5%)	36,75 ± 1,71
Positive (Phylantii extract) 0,135 mg/kgbw	74,25 ± 4,03
Group 200 mg/kgbw	71,75 ± 5,68^eb
Group 300 mg/kgbw	80,5 ± 3,87^eb^↑
Group 400 mg/kgbw	70,75 ± 4,11^eb

Post Hoc Tukey test:

e : There’s no significant difference to Positive control (nilai sig <0.05)

b : There’s significant dofference to negative control (nilai sig <0.05)

↑:Increased phagocytic activity occured

The results of the Post Hoc Tukey test showed that phagocytic activity at doses of 200 and 400 (mg/kgbw) was not significantly different from the positive control group, which means that at doses of 200 and 400 (mg/kgbw), they had the same activity as the positive control. However, at a dose of 300mg/kgbw, it showed a better increase in activity compared to a dose of 200, 400 (mg/kgbw), negative control and positive control, which means that the ethanolic extract of E. rubroloba fruit has potential and effectiveness as an immunomodulator by increasing the phagocytic activity of macrophage cells.

Testing CD4 levels with Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most widely used methods to detect antibodies based on the principle of specific antigen-antibody binding^31,32. The average CD4 level measurement results using an ELISA plate reader can be seen in Figure 1.

Figure 1. The average of CD4 levels

Based on Figure 1, it can be seen that the average increase in CD4 levels in normal controls was 252.50 ng/mL, negative control was 75.62ng/mL, positive control was 167.18ng/mL, dose of 200mg/kgBB was 204.53ng/mL, a dose of 300mg/kgbw of 227.49ng/mL and a dose of 400mg/kgbw of 175.62ng/mL. In Figure 1, the average CD4 levels in the positive control group given commercial Phylantii extract was higher than the negative control group given Na-CMC, this was due to the chemical content of Phylantii extract (Phyllanthus niruri L.) such as lignans, flavonoids, alkaloids, anthraquinones, terpenes, and tannins as well as being efficacious as immunostimulators. Meanwhile, the negative control group given Na-CMC had the lowest CD4 levels because the immune defense mechanism of the test animals only came from the natural immunity of the test animals. In figure 1 can be seen that there are differences in the increase in CD4 levels. The normal control group had higher levels than the treatment group at doses of 200, 300, and 400 (mg/kgbw). It was because the normal control group was not given any treatment. In the treatment group, the CD4 levels obtained were lower than in the control group. Normally it might occur due to the infection time of S. aureus bacteria only 1 hour, where the specific immune system, in this case, T lymphocyte cells (Th/CD4) to experience activation after exposure to the pathogen that causes infection it takes ≥12 hours for the specific immune system activated. Thus, it helps macrophage phagocytic cells to carry out phagocytosis against incoming pathogens. So that when measuring CD4 levels, the dose treatment group had lower results than the normal control group because CD4 had not responded to signals sent by the non-specific immune system^12,33.

The graph of the average CD4 levels for each treatment group in Figure 1 shows a dose of 400mg/kgBW has lower CD4 levels than doses of 200 and 300 (mg/kgBW). According to a previous study, a dose of 400mg/kgbw could affect the intestinal organs this cause damage to the intestinal villi (intestinal epithelial cell surface). On the other hand, the administration of E. elatior extract with a 400mg/kg dose can cause necrosis of the digestive tract^13,34,35. Therefore, it is estimated decrease in CD4 levels in the administration of an extract at a dose of 400mg/kgbw might be due to impaired absorption of the extract in the gastrointestinal tract. Thus, the level of active compounds from the sample is small in the systemic circulation resulting in a small pharmacological effect, resulting in inactivated immune cells, especially macrophage cells, unable to perform phagocytosis of antigens. On the other hand, necrosis is a condition where the tissue experiences death, characterized by the presence of a dead nucleus caused by toxic substances from biological materials, bacteria^28,36.

At a dose of extract 200mg/kgbw and 300mg/kgbw the levels increased. The increase in CD4 levels in the test animals could be due to the presence of flavonoid chemical compounds found in the ethanolic extract of E. rubroloba fruit which is thought to improve immune system function by helping to increase differentiation and proliferation of T lymphocytes so that it will stimulate phagocytic cells to recognize antigens and carry out a phagocytic response³⁷. The phagocytic response begins with the introduction of peptides from antigens recognized by TLR 2, which then undergo signaling to be phagocytosed in macrophage cells and will be presented to the cell surface via the MHC class II pathway, which will then be responded by T helper cells to assist the activation of macrophage phagocytic cells. In carrying out phagocytosis^38,39.

The increase in the number of CD4 levels indicated that the compounds contained in the ethanolic extract of the fruit of E. rubroloba were immunostimulant. It is suspected that the flavonoid compounds in the fruit of E. rubroloba stimulate an increase in IL-2 synthesis resulting in activation and proliferation of T lymphocyte cells. Flavonol glycosides are mitogenic glycoproteins of plant origin. Mitogens are substances that induce cell mitosis. Mitogen induces thymocytes to increase transcription of interferon-γ (IFN- γ ) and tumor growth factor-β (TGF- β). The induction of IFN-γ and TGF-β transcription will increase mature T cell receptors (TCR). Mitogenic compounds also induce IL-2 synthesis. T cells exposed to mitogens will increase IL-2 mRNA expression within 4 hours and peak after 12 hours. Furthermore, IL-2 will stimulate T cell proliferation^11,40.

Tukey Post Hoc resulted that the administration of the ethanolic extract of E. rubroloba fruit in Wistar rats gave a significant difference with the negative control group and the positive control group on its ability as an immunomodulator based on CD4 level data after administration of the ethanol extract of E. rubroloba fruit at doses of 200, 300 and 400mg/kgBW. They showed a significant difference between the negative control group and the positive control group (p<0.05), so it was concluded that the three extracts had potential as immunomodulators.

These results indicate that an increase in CD4 levels can increase the phagocytic activity of phagocytic cells where an increase in CD4 levels can activate phagocytic cells such as macrophages by activating T helper-1 (Th1) cells which then secrete phagocytic activating cytokines such as Interferon-gamma which will activate macrophage cells^10,11. Thus, the higher the level of CD4 of the body, the more phagocytic cells, including macrophages activated to destroy microbes that enter the body^13,15.

CONCLUSION:

Etlingera rubroloba A.D Poulsen fruit ethanol extract has potential as an immunomodulator based on the parameters of CD4 levels in Wistar rats.

CONFLICT OF INTEREST:

None declared.

ACKNOWLEDGMENTS:

The authors thank the Pharmacy Research Laboratory of the Faculty of Pharmacy and the Laboratory of the Faculty of Medicine, University of Halu Oleo, who have facilitated the implementation of this research.

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Received on 10.07.2021 Modified on 27.11.2021

Research J. Pharm. and Tech 2022; 15(9):4067-4072.

DOI: 10.52711/0974-360X.2022.00682