INTRODUCTION:

Immunomodulators are compounds or sets of compounds that modulate the body's immune system. These compounds boost the immune system as immunostimulators and lower the immune system as immunosuppressants to prevent inflammation^1–3. Immunomodulators are bioactive compounds that can play a role or influence the regulation of the immune system, including the innate and adaptive immune systems^4–6.

Immunostimulators are prescribed to enhance the immune response to infectious diseases, tumors, primary or secondary immunodeficiency, and changes in antibody transfer. Immunosuppressive drugs reduce the immune response to transplanted organs and treat autoimmune diseases such as pemphigus, lupus, or allergies⁷. Some compounds that have immunomodulatory properties are phenolic compounds, alkaloids, quinones, saponins, terpenoids, proteins, polysaccharides, glycolipids, glycolipids, and fatty acids¹. These bioactive compounds are mainly obtained from natural products such as plants⁸. Plants are essential and integral to medicinal and alternative systems derived from secondary metabolites⁹.

Indonesia is one of the largest biodiversity countries in the world. Plant biodiversity in Indonesia reaches around 30,000-40,000 species¹⁰. Approximately 15,000 of them have potential as medicinal plants. Unfortunately, only about 7000 species have been explored as medicinal raw materials¹¹. One of the least explored plants is the genus Marsdenia, which belongs to the family Apocyaneacea. Belincong (Marsdenia brunoniana) is rarely studied regarding its metabolite profile and pharmacological effects. This plant grows wild in East Java, Indonesia's "Tapal Kuda" area. This plant is often utilized as a vegetable by the local community¹².

Previous studies have shown that M. brunoniana leaf extract has antioxidant and antidiabetic activities with in vitro experiments¹³. In addition, the results of SPME-GC-MS analysis of the leaf and fruit of M. brunoniana showed that carbohydrates (D-mannitol) and fatty acids are the dominant compounds¹⁴. Research on fruit extracts from M. brunoniana has never been conducted. Research exploring plants as immunomodulators is very important as an alternative to preventive bacterial infectious diseases, especially in antibiotic-resistant bacteria such as Staphylococcus aureus¹⁵. Staphylococcus aureus has an antibiotic-resistant strain known as Methicillin-Resistant Staphylococcus aureus (MRSA), which has been the focus of several studies and life-threatening world issues^16,17. This study aimed to determine the phytochemical compounds of ethanol extract of M. brunoniana fruit through GC-MS analysis and determine immunomodulatory activity on S. aureus-infected mice.

MATERIALS AND METHODS:

Materials and Sample Collections:

Fresh belincong fruit (Marsdenia brunoniana) was collected in March 2023 from Alassumur Kulon Village, Kraksaan, Probolinggo, Indonesia. Belincong was identified at the Botany Laboratory, Biology Education, Jember University. The Belincong fruits used were old (6-8 weeks), dark green color, 1.5-2cm in diameter, and 9-17cm in length. Staphylococcus aureus strain ATCC 25923 was identified at the Microbiology Laboratory, Faculty of Dentistry, Jember University. Turk solution for leukocyte measurements was from Merck, Sigma-Aldrich (Germany). Giemsa solution for phagocytic activity analysis used from Merck, Sigma-Aldrich (Germany). The mouse immunoglobulin G ELISA Kit was provided by Bioassay Technology Laboratory (BT Lab) (Shanghai, China).

Plant Extraction:

The extraction method used was the maceration method. First, belincong fruit (Marsdenia brunoniana) was washed using distilled water, cut into small pieces, and air-dried. 62grams of Belincong fruit powder was soaked in 750ml of 96% ethanol, stored in a dark place in a shaker, and collected every 24hours for three days. Then, it was concentrated using a rotary evaporator at a temperature of 60°C. The extract was ready for phytochemical analysis and animal treatments.

Gass Chromatography Mass Spectrometry:

The belincong fruit extract was analyzed using GC-MS at Biosains Laboratory, Politeknik Negeri Jember. The machine of GC-MS analysis was Shimadzu GCMS-QP2010 Plus with a split injector set at a temperature of 225ºC. MS detector temperature 250ºC. Rtx-50 column (diameter 0.25mm, length 30 m and thickness 0.25μm). The detector temperature was programmed at an initial temperature of 100ºC for 20 minutes, then increased to 240ºC for 20 minutes at a 5ºC/minute speed. Helium gas was used as a carrier gas at a 3mL/minute speed.

Animal Treatments:

The animals of this study were male Balb/C strain mice aged 8-10 weeks with a body weight ranging from 25-30 grams. The ethanol extract of belincong fruit (Marsdenia brunoniana) was administered via oral gavage with 0.2ml each from the first day until the 14th day. Treatment groups were divided into five groups (KN, K-, P1, P2, and P3). The normal control group (KN) treatment given to mice was not infected with Staphylococcus aureus; they were only given distilled water. The negative control group (K-) mice were treated by being infected with Staphylococcus aureus and given distilled water. The P1 group mice were given dose I of extract (50mg/kg BW), and then infected with Staphylococcus aureus. The P2 group mice were given dose II extract (100mg/kg BW) and then infected with Staphylococcus aureus. The P3 group mice were given dose III extract (150mg/kg BW) and then infected with Staphylococcus aureus. Mice were intraperitoneally infected with S. aureus (0,5 McFarland) on the 15^th and 29^th day. Euthanized mice used a mixture of ketamine and xylazine (0.1ml per mice). The Faculty of Dentistry, University of Jember tested these experimental procedures on ethics eligibility with number: 2058/UN25.8/KEPK/DL/2023.

Phagocytic Activity Assay:

The phagocytic activity of mice was performed by counting the number of active and inactive phagocytic cells. Mice were given Staphylococcus aureus one hour before surgery. After the mice were anesthetized, a slight dissection was made on the abdomen of the mice and gave a mixture of PBS (Phosphate Buffered Saline) solution and 10% EDTA solution 1.5mL. Intraperitoneal fluid was taken using an injection and placed in a microtube. Intraperitoneal fluid was made thin on the object glass. then dried for 10 - 15 minutes. The preparations were put into methanol for 5 minutes for fixation. After fixation, the preparations were stained with 10% Giemsa for 20 minutes. Washing of the preparation was done with distilled water after Giemsa staining. Dried preparations are observed using an Optical Microscope Olympus BX53F2 (Japan) with CellSens Imaging Software with 100x magnification.

Leukocyte Number and Spleen Weight Measurements:

Mice's blood was taken from the heart’s right ventricle using a 1ml syringe and transferred into a purple vaculab tube containing EDTA. Blood samples were taken from a purple vaculab tube using a leukocyte thoma pipette and diluted with 20x Turk solution. The solution was shaken, then dropped onto the edge of the hemocytometer counting chamber, and left for 1minute to lyse the erythrocytes. Observations were used in Optical Microscope Olympus BX53F2 (Japan) with CellSens Imaging Software with 10x magnification. Leukocytes were counted from 4 large boxes in the corner of the counting room. The calculation of the number of leukocyte cells was calculated by adding up the total number of leukocytes counted, multiplied by the dilution factor, and dividing by the number of boxes counted times the area of each box times the depth of the counting chamber or by the formula:

Total number of leukocytes ×20

The number of leukocytes = --------------------------------

4 × 1mm²× 0.1 mm

The spleen is taken and washed using a Phosphate Buffer Saline (PBS) solution. The spleen was weighed using an analytical balance.

Antibody Levels:

Measurement of antibody levels with Mouse Immunoglobulin G, IgG ELISA (Enzyme-Linked Immunosorbent Assay) Kit 96 T by BT Lab Bioassay Technology Laboratory. Blood serum samples were collected by centrifugation with 3000 rpm for 10 minutes using Eppendorf Centrifuge 5810 R (USA). For the analysis of mouse blood serum samples, 25 strips were readied. A 50μl standard solution was introduced into the standard well. Subsequently, a 40μl sample was dispensed into the sample well. An anti-IgG antibody (10μl) and 50μl of streptavidin-HRP were added to the sample and standard wells. The plate was sealed and incubated at 37°C for an hour. Following incubation, the plate was washed five times with a washing buffer, with each wash involving soaking the wells with a minimum of 0.35 ml of the buffer for 30 seconds to a minute. Next, 50μl each substrate solution A and B was added to each well. The plate was again sealed and incubated in a dark environment at 37°C for 10 minutes. Finally, a 50μl stop solution was added to each well, immediately causing the blue color to turn yellow. Each well's optical density (OD value) was determined immediately using an ELISA Reader (Biorad, iMark microplate absorption reader) at 450 nm within 10 minutes after adding the stop solution.

Statistical Analysis:

All parameters were analyzed using IBM SPPS Statistic, 23 version with one-way ANOVA and Duncan test as post hoc (p < 0,05) except for phagocytosis activity using Kruskal-Wallis. The results were performed by mean ± standard deviation.

RESULTS:

Gass Chromatography and Mass Spectrometry Analysis:

Marsdenia brunoniana fruit extract (ethanol extract) has 28 volatile compounds detected by GC-MS. This extract had many fatty acids (propanoic acid, butanoic acid, butyric acid, and oleic acid). The most abundant compounds were 1,2,3-Propanetriol (CAS) Glycerol 20.26%; Benzenamine, N-methyl- (CAS) N-methylanilin 13.92%; 1,4-Benzenediol (CAS) Hydroquinone 11.48%; Piperidine, 1-methyl- (CAS) N-Methylpiperidine 10.48%. The relative percentage of the compounds is shown in Table 1.

Table 1: List of compounds detected in M. brunoniana fruit extract.

Peak	Area (%)	Name of Compound	Compound Grups
1	0.11	2-(2-Aminoethylamino)ethanol	Alcohol
2	0.46	2(3H)-Furanone, dihydro-(CAS) Butyrolactone	Lactone
3	0.11	Piperazine	Azacycloalkane
4	0.42	1-Octadecanamine, N-methyl- (CAS) N-Methyloctadecylamine	Amines
5	0.17	Erythro-1,2-dimethyl-1-methylthio-2-(dimethylamino)ethane	Trialkylamines
6	0.54	2-Cyclopenten-1-one, 3-methyl- (CAS) 3-Methyl-2-cyclopentenone	Cyclic ketone
7	3.42	1,3-Dioxane (CAS) m-Dioxane	Cyclic acetal
8	10.28	Piperidine, 1-methyl- (CAS) N-Methylpiperidine	Alkaloid
9	1.16	N-iso-butyl-N-methylamine	Amines
10	13.92	Benzenamine, N-methyl- (CAS) N-methylaniline	Aromatic amines
11	1.90	Phenol, 4-methoxy-	Phenolic
12	1.81	Cyclohexanol, 4-methyl- (CAS) 4-Methylcyclohexanol	Alcohol
13	0.07	Pyridinium, 1-(1-carboxyethyl)-, hydroxide, inner salt (CAS) 2-(N-PYRIDYL)PROPIONIC ACID BETAINE	Piridine
14	0.43	3-Ethyl-2-hydroxy-2-cyclopenten-1-one	Cyclic ketones
15	0.14	Cyclohexanone, 4-hydroxy- (CAS) 4-Hydroxycyclohexanone	Ketones
16	20.26	1,2,3-Propanetriol (CAS) Glycerol	Glycerol
17	1.20	3-METHYL-OXIRANE-2-CARBOXYLIC ACID METHYL ESTER	Oxirane
18	4.56	Benzofuran, 2,3-dihydro- (CAS) 2,3-Dihydrobenzofuran	Terpene volatile
19	0.25	1,2-Benzenediol (CAS) Pyrocatechol	Glycols
20	1.33	Butanoic acid, 2-methyl- (CAS) 2-Methylbutanoic acid	Fatty acid
21	8.76	Propanoic acid, 2-methylpropyl ester (CAS) Isobutyl propanoate	Fatty acid
22	0.84	2-AMINO-3-HYDROXY-BUTYRIC ACID	Beta-hydroxy acids
23	11.48	1,4-Benzenediol (CAS) Hydroquinone	Phenolic
24	8.47	D-ARABITOL	Polyol
25	5.98	Glycine, N-methyl-N-(1-oxododecyl)-	Amino acid
26	0.58	2-HEXADECANONE	Ketones
27	0.83	Oxacycloheptadec-8-en-2-one (CAS) Ambrettolide	Macrolides and analogues
28	0.51	9-Octadecenoic acid (Z)- (CAS) Oleic acid	Fatty acid

Phagocytic Activity Assay:

Phagocytosis is a cellular process found in many cell types capable of engulfing and removing particles with a diameter greater than 0.5 μm, such as microorganisms, foreign substances, and apoptotic cells¹⁸. Phagocytosis activity in (Figure 1) showed that active phagocytes have a large size and have a brighter cytoplasm. In comparison, inactive phagocytic cells have a relatively smaller size. The results of mice phagocytosis activity (Figure 2) showed that P2 has a higher phagocytosis activity than other M. brunoniana fruit extract treatments. The P1 and P3 groups differed significantly from the normal control treatment group (KN). The results showed the lowest phagocytosis activity was in the negative control (K-). These results determine that M. brunoniana fruit extract on 100 mg/kg BW has the same results of phagocytic activity as the normal control group (KN).

Figure 1: Phagocytic Activity on Light Microscopy Visualization 100X Magnification with Giemsa Stain. (a) Active phagocyte. (b) Inactive phagocyte

The Number of Leukocyte and Spleen Weight:

Based on the results (Table 2), the highest leukocyte number was shown by the K- group, 14060 cells/mm³, and the lowest leukocyte number was the KN group, 7880 cells/mm³, which means the normal leukocytes count in mice (2000 – 10000 cells/mm³)¹⁹. The P1 treatment group showed a leukocyte count almost close to the KN treatment group, 10390 cells/mm³. In contrast to the number of leukocytes, the results of measuring spleen weight showed that the KN treatment group had the largest weight and the K- treatment group had the slightest weight. This result showed significant differences in the KN and K- groups, but no difference exists between the P1, P2, and P3 treatment groups.

Figure 2: Effect of M. brunoniana Fruit Extract on Phagocytosis Activity.

Note: Distinct letters in each data label indicate statistically significant differences (P < 0.05). KN for the normal control group; K- for the negative control group; P1 for M. brunoniana doses 50 mg/kg BW treatment; P2 for M. brunoniana doses 100 mg/kg BW treatment; P3 for M. brunoniana doses 150 mg/kg BW treatment.

Table 2: Effect of M.brunoniana Fruit Extract on Leukocyte Number and Spleen Weight Measurements

Treatment Groups	Number of Leukocyte (cells/mm³)	Spleen Weight (mg)
KN	7880±887,1^a	630±116,6^a
K-	14060±2347,2^c	388±25,9^b
P1	10390±1806,7^ab	478±58,1^ab
P2	12250±4196,1^bc	616±233,9^ab
P3	12290±1753,4^bc	460±126,3^ab

Antibody Levels:

The results of antibody levels can be seen in Figure 3. Group P3 showed the highest antibody levels compared to other extract treatment groups. The KN, K-, P2, and P3 treatment groups did not differ significantly. Treatment groups K-, P1, P2, and P3 did not differ significantly. The KN and P1 treatment groups were significantly different. The P3 group had almost the same antibody levels as the normal control group.

Figure 3: Effect of M.brunoniana Fruit Extract on Antibody Levels.

DISCUSSION:

Based on GC-MS analysis results, the most abundant compound of M. brunoniana fruit extract was 1,2,3-Propanetriol (CAS) Glycerol. Some glycerol or its derivatives have immunomodulatory effects, such as increased cytokines and excess neutrophil migration into injured tissue^20,21. Glycerol could block toxin induction of Staphylococcus aureus by interfering with bacterial signal transduction on the bacterial cell membrane²². The second highest compound screening result is Benzenamine, N-methyl- (CAS) N-methyl aniline. This compound is toxic to humans and causes methemoglobin²³. Phenolic compounds also found in Belincong fruit extract are 1,4-benzenediol (CAS) Hydroquinone. This compound has strong antimicrobial activity²⁴, anticancer activity²⁵, and immunomodulatory activities²⁶. In addition to phenolics, an alkaloid compound in M .brunoniana fruit extract, Piperidine, 1-methyl-(CAS) N-Methylpiperidine was also detected. Piperidine compounds have antimicrobial, anticancer, anti-inflammatory²⁷, and antiviral activity²⁸. Piperidine inhibited the growth of Staphylococcus aureus²⁹ and increased total leukocytes and antibody titers³⁰. Other alkaloids have medicinal effects as anti-viral activity³¹, antioxidant activity, and antibacterial³². In addition, M. brunoniana fruit extract has Glycine, N-methyl-N-(1-oxododecyl), as an amino acid, which has immunomodulatory activityon human neutrophils³³. In Addition, M. brunoniana fruit extract has glycine as an amino acid (monomer of protein) that has immunomodulatory activity by raising antibody production³⁴. The results of these GC-MS analyses should be done through metabolomic profiling through a silico study so that the mechanism of compounds in M. brunoniana fruit extract that affect immunomodulatory activity is precise.

Based on the results that have been presented, S. aureus decreased immunomodulatory activity, such as decreased phagocytosis activity and antibody levels. This is because Protein A of S. aureus (SpA) binds to the fc part of the IgG antibody. This causes phagocytic cells to be unable to recognise S. aureus and then unable to engulf and destroy the bacteria³⁵. Protein A promoted immune suppression by binding to the Fc region of antibodies, thereby preventing normal phagocytosis³⁶. Protein A of S. aureus also has other immune evasion mechanisms, such as destroying phagocytes, preceding complement activation, and modifying B and T cell immune responses ³⁷.

This study proved that M. brunoniana fruit extract significantly affected immunomodulatory activities such as phagocytosis activity, total leukocytes, and antibody levels. Marsdenia brunoniana fruit extract has been proven to restore immunomodulatory activity, usually even though it has been twice exposed to S. aureus bacteria. Active compounds such as Benzenediol or phenolics affected increasing phagocytosis of Aβ peptides through controlling macrophage polymerization³⁸. The increase in phagocytosis activity was certainly assisted by antibodies for bacterial opsonization. Glycerol compounds in M. brunoniana fruit extract stimulated Toll-like receptor-9 (TLR-9), which induced cytokines IL-6 and IL-12³⁹. IL-6 played a role in helping B cell differentiation into plasma cells, which results in increased antibody production⁴⁰. IL-12 played a role in the differentiation of T-CD4+ cells into Th1 cells⁴¹. The cytokine IFN-γ assisted Th1 cells to activate macrophages and neutrophils⁴². T cells produce the effective proliferation of lymphokines to stimulate phagocytosis, so leukocyte production increases⁴³.

Based on the discussion of the study results, M. brunoniana fruit extract was shown to have immunomodulatory activity. Unfortunately, this study did not measure cytokine levels and cell proliferation, so the pathway explanation was incomplete. Further research should include interleukin detection and use in vitro for cell proliferation and differentiation measurements.

CONCLUSION:

Based on the results and discussion, Marsdenia brunoniana fruit extract has 28 active compounds. M. brunoniana fruit extract showed immunomodulatory effects by increasing phagocytic activity, total leukocytes, and antibody levels in infected mice. So, the Marsdenia brunoniana fruit extract has immunomodulatory activity against Staphylococcus aureus.

ACKNOWLEDGMENT:

The researcher is grateful to LP2M University of Jember for providing this study with the KeRis-DiMas research grant 2023.

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Received on 31.10.2023 Modified on 17.01.2024

Research J. Pharm. and Tech 2024; 17(8):4022-4028.

DOI: 10.52711/0974-360X.2024.00624