INTRODUCTION:

In a local culture in Indonesia, there is a term for traditional medicine known as “jamu”, which is a decoction of plants consumed to maintain health. Jamu is used not only to cure the symptoms of disease, but also to protect someone from disease. The use of other forms of herbal medicine in Indonesia, such as Obat Herbal Terstandar that have been pre-clinically tested and Fitofarmaka that have been clinically tested, is increasing in Indonesia. This phenomenon is not only found in Indonesia but is also seen in the use of herbal medicines throughout the world. Plant-based treatment have become a popular alternative to complement existing standard therapies¹.

In a plant, various chemical compound components can modulate the immune system and can be used as immunomodulators. Immunomodulators can be defined as substances that influence the immune system by regulating immune system molecules such as cytokines, hormones, neurotransmitters, and other peptides, where these substances can stimulate or inhibit immune responses^2,3,4,5. One plant that is known to have been used empirically in the treatment of autoimmune disease in Indonesian society is Cymbopogon citratus or lemongrass. Cymbopogon citratus (DC.) Stapf is a perennial herbaceous plant indigenous to India and widely planted in tropical and subtropical areas^1,6,7. This plant is known by several local names, such as lemongrass (England), Sereh dapur (Indonesia), Sakumau (Malaysia), and Ta-khrai (Thailand)⁸. Lemongrass is an annual plant that morphologically forms a broad section consisting of several blades that grow from rhizomes connected by a fibrous root system. The leaves are used as traditional medicine in the form of herbal tea, while the stems are used as a kitchen spice to add aroma to dishes. The essential oil is used empirically to treat disorders of the gastrointestinal tract, central nervous system, as an antispasmodic, anti-inflammatory, analgesic, antipyretic, diuretic, and sedative^2,9,10.

Various studies have examined the activity of lemongrass and shown that this plant has antioxidant, antibacterial, antifungal, anti-inflammatory, antidiabetic, antiseptic, insecticidal, anticarcinogenic, antinociceptive, and antiherpetic activities against HSV-1^11,12,13,14. As stated above, in Indonesia, the decoction of lemongrass stems is believed to be able to treat autoimmune diseases. However, because empirical use is not based on definitive doses and benefits, this research attempts to determine definite doses and benefits that can be used to facilitate future use. Apart from that, this research also aims to develop immunomodulatory therapy derived from natural ingredients and its application in one model of autoimmune disease, namely rheumatoid arthritis.

MATERIALS AND METHODS:

Plant Materials:

C. citratus stems were obtained from Aceh Besar, Aceh Province, Indonesia. The obtained plant identity was determined at the Biological Research Center of the National Research and Innovation Agency in Bogor, West Java Province, Indonesia. The stem parts were then dried and processed into powder.

Extraction:

Lemongrass stem extraction was performed using 96% ethanol solvent using the reflux method (1:10). The reflux was repeated 3 times each for 2hours. The extract obtained was then concentrated using a Buchi rotary evaporator at a a speed of 50rpm and temperature of 40°C. The thick extract was then evaporated on a water bath at 65°C to obtain a concentrated extract.

Animal:

The test animals used in this study were female BALB/c mice (6-8weeks) for testing immunomodulatory activity and female Wistar rats (6-8weeks) for testing anti-rheumatoid arthritis activity. This animal testing has received ethical permission from the Animal Experimentation Ethics Committee of the Institut teknologi Bandung with numbers KEP/I/2022/IX/ H050922MF/AISD and KEP/I/2022/IX/ H050922MF/AAKS.

Characterization and Phytochemical Screening:

The dried powder of lemongrass stems and the ethanol extract of lemongrass was characterized and screened for phytochemicals content. Screening for phytochemical content includes the examination of flavonoids, tannins, saponins, alkaloids, and steroids/triterpenoids. Meanwhile, the characterization parameters examined include ash content, loss on drying, water-soluble content, ethanol-soluble content, residual solvent content, and aflatoxin contamination.

Determination of Total Phenols and Total Flavonoids:

The total phenol and flavonoid content in the extract were examined using the Folin-Ciocalteu reagent and the Aluminum chloride colorimetric method^15,16.

Determination of Antioxidant Activity:

The antioxidant activity of the extract was determined using the DPPH method with slight modifications. Vitamin C was used as a standard and IC50 was determined^17,18.

Immunomodulatory Activity Test:

To test immunomodulatory activity, mice were divided into 6 groups, namely the test extract group with doses of 375, 750, and 1500mg/kg bw, methylprednisolone 15 mg/kg bw, and the control group. The extract dose was determined based on the empirical use of lemongrass stems (750mg/kg bw) where we also carried out tests at doses below and above that dose. There were two type of tests carried out, the non-specific immune response test (carbon clearance test), and the specific immune response test (antibody titer test and delayed-type hypersensitivity test).

Non-specific Immune Response Test:

This test was carried out by examining carbon clearance which determines the phagocytic activity of reticuloendothelial cells^19,20,21. The test material was administered orally once a day for 7 consecutive days. On the 8th day, 0.1mL/10g bw of colloidal carbon suspension (Pelican-17 Black Ink in 1% gelatin in sterile saline solution) was injected through the tail vein of the mice. A total of 25μL of mouse blood was taken through the tail at 0, 5, and 15 minutes after carbon injection. The blood taken was lysed in 3mL of 1% acetic acid. Transmittance and Optical Density (OD) were measured using a spectrophotometer at a wavelength of 675nm. The carbon elimination rate (K) is calculated using the formula K = (ln OD1-lnOD2)/(t2-t1) where OD1 is the absorbance at 0 minutes, and OD2 is the absorbance at 15 minutes. The phagocytic index was then calculated as the ratio of K sample/K control.

Specific Immune Response Assay:

Determination of Antibody Titers as a Humoral Immune Response:

Determination of antibody titers is carried out using the hemagglutination method. This test was carried out by injecting sheep red blood cells (SRBC) as an immunogen. Animals were immunized using 10% SRBC in PBS (0.1mL/10 g bw, i.p) on the third day after administration of the test material. On the eighth day, animal blood was taken, and serum was separated to determine primary antibody titers^22,23,24. On day 8, animals were re-immunized with SRBC to determine secondary antibody titers. On the 15th day, animal blood was taken, and serum was separated for examination of secondary antibody titers. The highest dilution showing 50% hemagglutination is expressed as an antibody titer.

Determination of Cellular Immune Response (Delayed-Type Hypersensitivity Test):

Animals were immunized with 10% SRBC in PBS (0.1 mL/10g bw, i.p) on the third day after administration of the test material. On the eighth day, the thickness of the mice's hind feet was measured using a plethysmometer (t0), then the same feet were injected using SRBC (50μL, i.d). Foot sole thickness was measured again at 24 and 48 hours after the antigen challenge. Changes in the thickness of the soles of the test animals reflect cellular immune responses^22,25.

Anti-inflammatory Activity Test using the Rheumatoid Arthritis Model:

Rheumatoid arthritis was induced in animals by injecting 0.05mL of Complete Freund's Adjuvant (CFA) into the sole of the left hind foot intradermally²⁶. CFA-induced mice then experienced chronic inflammation. After inflammation occurred, the mice were divided into four groups, namely the control group, dexamethasone 0.1mg/kg bw, extract dose 750mg/kg bw, and 1500 mg/kg bw. The development of inflammation on the paw was recorded every 5 days including the volume of the paw, joint circumference, and thickness of the paw. The data obtained was then determined to be statistically meaningful.

RESULTS:

The plant used in this research was identified in the Herbarium Bogoriense in West Java, Indonesia and declared to have the species name Cymbopogon citratus (DC.) Stapf. with the identification number B-368/V/DI.05.07/1/2022.

Table 1. Characteristic of plant and ethanol extract of C. citratus

Parameter	C. citratus
Parameter	Plant	Extract
Total Ash Content (% w/w)	8.60	8.50
Loss on Drying (%w/w)	8.53	12.5
Water Soluble Content (%w/w)	10.53	56.65
Ethanol Soluble Content (%w/w)	16.70	61.25
Solvent residual level (%)	Not tested	0.02
B1 and B2 Aflatoxin Contamination	Not tested	Undetected
G1 and G2 Aflatoxin Contamination	Not tested	Undetected

Table 2. Phytochemical content of Plant and Ethanol Extract of C. citratus

Group	C. citratus
Group	Plant	Extract
Flavonoid	+	+
Phenol	+	+
Alkaloid	-	-
Tannin	-	-
Saponin	-	-
Steroid/Triterpenoid	+	+
GC-MS result	Selin-6-en-4.alpha.-ol; Geranic acid; N-Methyl-1-adamantaneacetamide; Oplopanone; Santalcamphor

Table 3. Total Phenolic and Flavonoid Content of EECC

Parameter	EECC
Phenolic content (g GAE/100 g)	7.00±0.24
Flavonoid content (g QE/100 g)	2,09±0.07

Table 4. Antioxidant Activity

Sample	IC₅₀ (µg/mL)	AAI (Antioxidant Activity Index)
Ethanol Extract C. citratus (EECC)	193.07	0.258
Ascorbic Acid	5.10	9.800

Table 5. Phagocytic index

Groups	Dose (mg/kg BW)	Phagocytic Index (K)	Category (Wagner, et.al. 2002)
Methylprednisolone	15	0.921	Immunosuppressant
EECC	375	0.933	Immunosuppressant
	750	1.029	Immunosuppressant
	1500	0.836	Immunosuppressant
Control	-	1	-

Table 6. Humoral antibody response

Group	Dose (mg/kg BW)	Primary Antibody Titer	Secondary Antibody Titer
Control	-	1:1024	1:1024
EECC	375	1:1024	1:1024
	750	1:1024	1:1024
	1500	1:1024	1:1024
Methylprednisolone	15	1:512	1:1024

Figure 1. Organ Index after 7 days administration of extracts and methylprednisolone

Figure 2. Paw volume of Delayed Type Hypersensitivity Test. * p<0.05 vs control

Figure 3. Percent inflammation of paw volume in rheumatoid arthritis animal model treated with EECC

Figure 4. Reduction of inflammation in joint circumference in rheumatoid arthritis animal model treated with EECC

DISCUSSION:

Compounds that can act as immunomodulators can influence the immune response by regulating the activity of immune system cells by increasing or decreasing the production or release of various molecules such as hormones, cytokines, neurotransmitters, and other peptides². These compounds can come from various sources, such as from secondary metabolites contained in plants. The polyphenol content in plants, especially tannins and flavonoids, has been reported to have antioxidant, anti-inflammatory, and immune system modulation activities, both in vitro and in vivo¹². Medicinal plants, which contain many polyphenolic compounds, have been used as a complement to standard therapy because they are considered to have better safety. The World Health Organization (WHO) has emphasized the need to guarantee the quality of medicinal plants through characterization and standardization processes²⁷. Considering that C. citratus can be developed into a phytopharmaceutical in the future, phytochemical characterization and screening were carried out on the dry stem powder and ethanol extract (tables 1 and 2).

Based on the phytochemical screening, it is known that the ethanol extract of C. citratus contains flavonoids, phenols, and steroids/triterpenoids. These results were also confirmed through content analysis using GC-MS. The results obtained are in line with another where the presence of phenolic acids, monoterpenes, and sesquiterpenes was found in the C. citratus extract (table 2)^7,28.

Phenolic compounds are compounds in the antioxidant group that function as scavengers of free radicals. Flavonoids, which are part of the phenolic compounds, have been known to have antioxidant activity and have great benefits on human health and nutrition¹⁵. The results of examining the total phenol and flavonoid content of the ethanol extract of C. citratus (EECC) found that the phenol content was greater than the flavonoids (table 3). In the antioxidant activity test using the DPPH method, EECC still had antioxidant activity, although it was lower than ascorbic acid as a comparison (table 4). The results may differ from the results of other studies. Still, this difference can be assumed to occur due to differences in soil conditions, air, planting, and harvest factors where the C. citratus used in this study grows. Differences in antioxidant activity can also occur due to plant drying factors, extraction time, and temperature. Based on one study, dried kitchen lemongrass has the lowest antioxidant activity at various boiling times when compared to fresh kitchen lemongrass²⁹. The primary mechanism of the antioxidant function of flavonoids is their ability to donate electrons, which enables them to scavenge free radicals. Many flavonoids and other phenolic compounds are recognized as antioxidants not only due to their free radical scavenging activity but also because they chelate metals, thereby enhancing antioxidant capacity³⁰. Antioxidants can neutralize free radicals through single electron transfer in which is the basis for testing the antioxidant activity of the DPPH method³¹.

The immunomodulatory activity test of C. citratus carried out in this study was based on the empirical use of stem decoction as a medicine to treat autoimmune diseases. The immunomodulatory tests carried out include tests for non-specific and specific immune responses. The non-specific immune response test described through the carbon clearance test is used to measure the activity of drugs or plant secondary metabolite compounds in the reticuloendothelial system (RES) which could swallow and digest foreign particles. As an antigen, carbon ink colloidal suspension is used. During the experiment, carbon ink was injected directly into the bloodstream to be eliminated by RES through the process of phagocytosis. Through this test, around 90% of carbon particles will be eliminated by phagocytic cells in the liver and the remainder by phagocytic cells in the spleen^21,23. This elimination process can also involve the complement system, where the antigen interacts directly with C3 or through the role of antibodies or C3b as opsonin, which results in the elimination of the antigen²⁵. In this carbon removal method, the carbon elimination rate is said to be low if the phagocytic index is <1. This means that the drug or test substance being examined reduces the activity of phagocytic cells and can be said to have immunosuppressant activity. Meanwhile, if the phagocytic index is >1, then the test material is said to have immunostimulant activity³². Test results on EECC and methylprednisolone showed that at all doses extra and methylprednisolone had immunosuppressive activity as seen from the phagocytic index value of less than 1 (table 5).

We also examined the index of organs involved in the immune system such as the liver, spleen, and thymus. These organ indexes can reflect the level of moderation of immunity³³. The liver and spleen indices obtained in this experiment showed differences with the control group, although not significantly different. The liver and spleen are two organs rich in phagocytic cells. Meanwhile, the thymus is a primary lymphoid organ, where T cell maturation occurs through the production of thymic hormones. A decrease in the spleen index in the group given EECC indicates suppression of the nonspecific immune response (Figure 1).

The effect of EECC administration on the humoral immune response was concluded by determining antibody titers. In this test, B cells, which also act as antigen-presenting cells, interact with the injected antigen which then displays the antigen in a complex with MHC Class II which activates TCD4 cells. Through the secretion of cytokines by TCD4 cells in the co-stimulation process, these cells then proliferate and differentiate into cells that produce antibodies. The antibodies produced then bind to the antigen, neutralize it, or form cross-links which create a complex that is easily phagocytosed³⁵. The humoral immune response occurs with the formation of antibodies in response to foreign particles such as proteins and polysaccharides. In this study, the foreign particle used was SRBC. B lymphocytes control this immune response, producing antibodies when they encounter foreign objects³⁶. In this study, we observed that there was no difference in antibody titers between the group given EECC and the control group, which means that EECC has no activity to modulate the humoral immune response (table 6).

Meanwhile, to examine the activity of the cellular immune response, we examined the delayed-type hypersensitivity (DTH) response. DTH response is a reaction that commonly occurs in type IV allergic responses where this reaction is mediated by T lymphocytes. After sensitization using SRBC, the antigen will activate T lymphocytes, leading to the appearance of local swelling. Therefore, the degree of swelling is an inflammatory response that functions as an indicator of the effectiveness of the cellular immune response³². The results of the DTH test in this study showed that the three doses of EECC tested had immunosuppressive activity as indicated by a decrease in the volume of the soles of the mice's feet 48 hours after SRBC injection (figure 2). It indicated a decrease in T lymphocyte activity which plays a role in eliminating the antigen given in the challenge process.

Due to the results of immunomodulatory tests showing that the three doses used were immunosuppressive, we tried to look further at the activity of this extract in autoimmune animal models, where we used rheumatoid arthritis animal models in the next stage. One of the clinical manifestations of rheumatoid arthritis is chronic inflammation. After the injection of CFA into the paws of mice on day 0, the paws (volume, thickness, and joint circumference) were observed every 5 days. On the 35th day, animals that had been induced were divided into 4 groups (control, EECC doses of 750 mg/kg bw and 1500 mg/kg bw, and Dexamethasone). After administration of the test material, the inflammation that occurred in the soles of the mice's feet decreased (figures 3, 4, and 5).

Inflammation is a complex process involving humoral and cellular responses through various mediators. Swelling occurs in the affected area as antigen is contained in that area. Regardless of the fact that inflammation can assist in the healing process following exposure to antigens, chronic inflammation can affect the quality of life of patients³⁴. Chronic inflammation is one of the causes of death worldwide and is associated with various pathologies such as cancer, diabetes, cardiovascular, neurodegenerative, and autoimmune diseases. In inflammatory processes, macrophages play an important role in providing direct defense against foreign particles. When activated by compounds that cause inflammation, such as lipopolysaccharide (LPS), macrophages will produce several pro-inflammatory mediators such as prostaglandin E2 (PGE2) and nitric oxide (NO). Other research shows that C. citratus extract can inhibit the production of nitrite, PGE2, and the expression of induced Nitrite Oxide Synthase (iNOS) produced by macrophages. It is suspected that this inhibition process occurs due to the polyphenol content in the extract³⁷. Meanwhile, the polyphenol-rich fraction of C.citratus extract has inhibitory activity on the expression of TNF-α, a cytokine that produces various inflammatory effects. Deregulation of pro-inflammatory cytokines is important in the treatment of several diseases such as chronic inflammation and autoimmune disease³⁸. In another study, water extract from dried leaves of C. citratus could reduce inflammation in the soles of rats injected with carrageenan. Presumably the steroid and terpenoid content in this plant is responsible for its anti-inflammatory activity¹⁴. In other in vitro experiments, C. citratus extract showed antioxidant and anti-inflammatory activity by reducing the production of Il-1β and IL-6. Meanwhile, in tests using mouse skin dendritic cells, C. citratus showed anti-inflammatory activity by inhibiting NO production and expression of induced NO synthase (iNOS) induced by LPS³⁹ The study we conducted also examined TNF-α levels from the serum of rheumatoid arthritis animals that had been treated with the extract and comparator. The results showed a decrease in TNF-α levels, although it was not significantly different compared to the control group.

CONCLUSION:

Based on the results of this study, it can be concluded that the ethanol extract of C. citratus has potential as an immunosuppressant and anti-inflammatory, especially at doses of 750 and 1500mg/kg bw. This dose is equivalent to 4-8 stems of C. citratus. The results of preclinical trials on the ethanol extract of C. citratus support the empirical use of lemongrass in the community as a treatment for autoimmune diseases.

ACKNOWLEDGEMENT:

The authors would like to thank Higher Education Directorate General for the PPMI grant received from Institut Teknologi Bandung; and Center for Higher Education Fund, Ministry of education, culture, research, and technology of the Republic of Indonesia.

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Received on 30.05.2024 Revised on 11.09.2024

Accepted on 19.11.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1718-1724.

DOI: 10.52711/0974-360X.2025.00246

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