INTRODUCTION:

Free radicals are molecules with one or more free or unpaired electrons, making them unstable. Increased free radical production and decreased antioxidant defense can lead to oxidative stress. Oxidative stress occurs because the level of free radicals is too high and cannot be neutralized by antioxidants, so there will be an unbalanced situation between free radicals and antioxidants^1,2. Free radicals with high reactivity can cause a degenerative disease that the use of antioxidants can overcome³.

Antioxidants are molecules or compounds that are stable enough to donate their electrons or hydrogens to free radical molecules or compounds and neutralize them, thereby reducing their ability to carry out free radical chain reactions. These antioxidants mainly delay or inhibit cell damage through their free radical scavenging properties^1,4.

In an oxidative state, excess free radicals can attack cells and react with lipids, proteins and nucleic acids to cause damage to cell membranes or even damage to the cell as a whole and cause organ dysfunction. This can lead to a variety of serious diseases including cancer, heart disease, atherosclerosis, cataracts, premature aging and neurodegenerative diseases such as alzheimer's and others^5,6. Cell membranes are highly susceptible to reactive oxygen species (ROS), hence the need for antioxidant and anti-inflammatory compounds that can protect cells. Antioxidant compounds will inhibit or slow oxidation through free radical capture, while anti-inflammatory compounds stabilize the cell membrane^7,8.

Phytochemical compounds with anti-inflammatory activity are phenolics (flavonoids), alkaloids, and terpenoids. Based on the literature, flavonoids are the most widely discussed phytochemical compounds with anti-inflammatory activity. The anti-inflammatory activity of flavonoids is based on the antioxidant properties of their phenolic group⁹. One of the antioxidant testing methods used in this study is the FRAP (Ferric Reducing Antioxidant Power) method. Antioxidant activity is measured by the uptake of complex compounds. This method is known as a method that is easy to work with, cheap and fast, the reagents used are quite simple and do not use special tools to calculate the total antioxidant^10,11.

Inflammation is one type of disease that is widely treated using traditional medicine. Inflammation is a tissue response to the body's reaction that can cause cell damage. This cell damage is caused by bacteria, chemicals, mechanical trauma and physical trauma. Inflammation is common in humans and animals and is characterised by redness, heat, swelling, pain, loss of tissue function, increased permeability, increased protein and membrane denaturation^12,13.

Inflammation is the body's physiological response to infection and tissue injury. Acute inflammation is characterised by swelling/edema, heat, redness, pain, and loss of tissue function as a result of the local immune system, vascular or inflammatory cell response to infection or tissue and interleukin-1 beta (IL-1ꞵ) plays a role in the inflammatory process. Interleukin-1ꞵ is one of the mediators of inflammation, a member of the IL-1 family of cytokines that plays a strong role as a pro-inflammatory cytokine that acts as an amplifier of immune and defence reactions produced by the body, especially against infection. IL-1ꞵ is the most abundant and important pro-inflammatory cytokine in the body's defence response to infection and injury¹⁴. IL-1β is produced by activated macrophages and is involved from the early stages of connective tissue damage. IL-1β levels can be influenced by tissue inflammatory conditions related to IL-1β's function as an inducer of acute responses when inflammation occurs and tends to decrease as inflammation progresses^15,16.

The plant used in this study is songga wood (Strychnos ligustrina) from Dompu, West Nusa Tenggara. Empirically, the people of Dompu, West Nusa Tenggara use songga bark (S. ligustrina) as a toothache medicine, wound medicine, and malaria. In addition, the seeds are used to treat malaria, diarrhoea, and aches, and the roots to treat stomach pain or diarrhoea. Songga wood (S. ligustrina) has been reported as an antibacterial, anticancer agent, In addition, songga wood can be used as an analgesic, antidiabetic, tonic, febrifuge, and wound remedy¹⁷. Research on the biological activity of songga wood related to free radicals scavenger (FRAP) and a decrease in IL-1ꞵ levels (anti-inflammatory) against Songga Wood (S. ligustrina) methanol extract has never been reported, so it is necessary to conduct this research.

MATERIAL AND METHODS:

Materials:

The materials used in this study were Songga wood (S. ligustrina) extract methanol, distilled water, quinine, filter paper, Aqua Pro Injection (Otsuka®), potassium ferricyanide 1%, trichloroacetic acid 10%, FeCl₃ 0.1%, phosphate buffer (0.2M pH 6.6), methanol p.a., keragenan, standard feed, drinking water, 50mg diclofenac sodium, 0.5% Na-CMC solution, 3ml and 5 ml injection spoils, 5ml EDTA tubes (Vaculab®), ELISA Kit Reader and Rat IL-1β ELISA KIT

Equipment:

The tools used in this research are basin, analytical balance (Precisa®), glass jar, funnel, stirring rod (Pyrex®), beaker (Pyrex®), measuring cup (Pyrex®), dropper pipette, measuring pipette, porcelain cup, measuring flask, test tube (Pyrex®), micropipette (Boecof®), 3ml Eppendorf tube (Onemea®), hot plate (Stuart®), centrifuge, water bath, rotary vacuum evaporator (Rotavapor®R-300), UV-Vis spectrophotometer (Jenway 6800®), oven (Stuart®), pletismometer, jar, surgical scissors (Gesunde®), scalpel.

Determination:

Determination of songga wood was carried out with the aim of ensuring the truth and authenticity of the plants used in the study. Determination was carried out in the Biology Education laboratory, Faculty of Teacher Training and Education, Halu Oleo University with number 717/UN29.18.1/PG/2023. This research has received Ethical Clereance approval from the Halu Oleo University Animal Ethics Committee with No. 3545a/UN29.20.1.2/PG/2023.

Sample Preparation and Extraction:

The samples used in this study were 7.5kg of Songga wood (S. ligustrina) obtained from Dompu Regency, West Nusa Tenggara province, Indonesia. The obtained Songga wood simplisia powder was put in a container and extracted by maceration with methanol (3 x 24 hours) of 30 L. The filtrate obtained was concentrated with a rotary evaporator at 55⁰C until a thick extract was obtained^18–23.

Free radicals scavenger activity by FRAP Methods:

A total of 1ml of each concentration of Songga wood methanol extract stock solution and quartzetin standard solution with 6 concentration variants, namely 20, 40, 60, 80, 100, and 120ppm, added 1ml of 0.2 M phosphate buffer (pH 6.6) and 1ml of K3Fe(CN)6 1%, then incubated. Next, 1ml of 10% TCA was added, then centrifuged at 3000 rpm for 15 minutes to accelerate the precipitation process. After centrifugation, 1 mL of the top layer was taken into a test tube, then added 1 mL of distilled water and 0.5mL of FeC13 0.1%. The solution was allowed to stand for several minutes and the absorbance was measured with an ultraviolet-visible spectrophotometer (UV-Vis) at a wavelength of 582 nm^24,25.

Inhibition percentage:

The antioxidant activity of songga wood was determined by the amount of FRAP free radical uptake by calculating the percentage inhibition of solution uptake using the formula:^25,26.

Blanko absorbance - Sample absorbance

Inhibition (%) 100 = --------------------------------------

Blanko absorbance

Antioxidant activity is expressed by the IC₅₀ (50% Inhibition Concentration) value, where the measured sample concentration can reduce 50% of FRAP radicals^25,26.

Anti-inflamatory activity:

Induction of inflammation:

The creation of edema was performed on the right hind paw of rats by inducing 0.1ml of 1% carrageenan solution subplantarly. Carrageenan was chosen as an irritant to create edema because it has several advantages, namely it does not leave marks, does not cause tissue damage, and can provide a more sensitive response to anti-inflammatory drugs than other irritants so it is suitable to be chosen as an inductor to create edema²⁷.

Measurement of inflammation

Measurement of inflammation using a pletismometer, by dipping the mouse paw into a tube containing mercury solution until the limit mark. The change in volume of the solution is recorded as the specific time (Vt) of the rat's paw. The volume of edema is the difference between the volume of the rat's foot at a certain time (Vt) and the initial foot volume (Vo). Calculation of edema volume using the formula:²⁸.

Vu = Vt -Vo

Description:

Vu = Mouse paw edema volume at each time t

Vt = Volume of rat paw after 1% carrageenan induction at time t

Vo = Initial volume of rat paw before 1% carrageenan induction

Anti-inflammatory Test Based on Interleukin-1β levels:

Treatment and Rat Blood Collection and Preparation:

After the rats were induced with 1% carrageenan and experienced edema after 1hour, the rats were treated with 50, 100 and 200mg/KgBW Songga wood methanol extract as test groups, 0.5% Na-CMC as negative control and diclofenac sodium as positive control. After that, blood was taken at the 3rd hour to assess the levels of inflammatory mediator IL-1β. Blood was taken from the heart of the rats, then put in a tube containing EDTA anticoagulant²⁹. Measurement of Inteleukin 1β levels based on the Enzyme-Linked Immunosorbent Assay (ELISA Kit) protocol at absorbance with a wavelength of 450nm²⁵.

Data Analysis:

Determination of antioxidant activity of methanol extract of Songga wood was carried out using spectrophotometric method. to determine the percent inhibition and IC₅₀ value, it was analyzed by linear regression equation by plotting between concentration (x) and absorbance value obtained from measurement on spectrophotometer (y).

Inflammatory data were statistically analyzed using the SPSS program. The effect between the levels of methanol extract of songga wood with a decrease in IL-1β levels in all treatment groups and comparison controls was analyzed by One Way ANOVA (Analysis of Variance) statistical test^30–32.

RESULT AND DISCUSION:

Antioxidant activity of FRAP method:

Antioxidant activity testing using the Ferric Reducing Antioxidant Power (FRAP) method. The FRAP method is one of the antioxidant testing methods in plants with a working principle that can determine the total antioxidant content of a material based on the ability of antioxidant compounds to reduce Fe³⁺ ions to Fe²⁺ so that the antioxidant power of a compound is analogous to the ability to reduce the sample, the more the concentration of Fe³⁺ reduced by the sample to Fe²⁺, the greater the antioxidant activity of the sample. The advantage of this method is that it is simple, fast, inexpensive, and does not require large specialized equipment. Antioxidant activity was measured using methanol extract of songga wood and quercetin as standard solution. Antioxidant testing is expressed by IC₅₀ (Inhibition concentration). The amount of antioxidant activity is characterized by the IC₅₀ value, which is the concentration of sample solution required to inhibit 50% of free radicals^33,34.

Measurement of antioxidant activity of methanol extract of songga wood by FRAP method using UV-Vis spectrophotometer at wavelength of 582nm with absorbance of 0.576nm^35,36. The results of antioxidant activity measurement can be seen in Table 1.

Table 1. Antioxidant Activity of methanol extract of songga wood (S. ligustrina) by FRAP Method

Sample

IC₅₀±SD µg/mL

Category

Songga Wood

(S. ligustrina)

78.05 ± 7,73

Strong

Quersetin

7.80 ± 1,72

Very Strong

The results of antioxidant activity measurement (table 1) with 6 variations of concentration (20, 40, 60, 80, 100, and 120ppm) show that quercetin as a comparator has a very strong antioxidant activity category compared to methanol extract of songga wood with a strong category. The smaller the IC₅₀ value of a sample, the greater the ability of the compound to counteract free radicals. The value of antioxidant activity is based on previous research which states that the categories of Very strong (IC₅₀ < 50µg/mL), Strong (50-100µg/mL), Medium (101-150µg/mL), Weak (150-200µg/mL), and Very weak (IC₅₀ > 200µg/mL)^33,34.

The results of phytochemical tests on Songga wood showed that the leaves, wood, bark, and roots contained flavonoids, phenols, and tannins, both in methanol extracts as well as methanol: water and water extracts. The presence of flavonoids, phenols, and tannins in each extract of songga wood indicates that songga wood has the potential to be an antioxidant³⁷.

Antioxidants are inhibitors of the oxidation process, even at relatively small concentrations. Antioxidants are chemical components consisting of monohydroxyl or polyhydroxyl phenols. Antioxidants work in several different ways against oxidative processes, namely scavenging free radicals enzymatically or by direct chemical reactions, scavenging lipid peroxyl radicals, binding to metal ions, and repairing oxidative damage. Antioxidants are highly oxidizable or strong reductants, so they react first with free radicals rather than other molecules. Antioxidants function to add or remove one electron to neutralize ROS so that free radicals become stable and inhibit the oxidation process³⁸.

Anti-inflammatory activity:

Results of Mouse Paw Edema Examination:

Examination of edema of rat feet is done by measuring the volume of the feet using a Pletismometer. The pletismometer consists of two large and small tubes, the large tube is used to insert the foot of the experimental animal and the small tube with the transducer is a mercury tube. Measurement of edema volume with a pletismometer is based on the principle of Archimedes' law, which is that objects inserted into a liquid will exert an upward force or pressure equal to the volume moved²⁵.

Acclimatized test animals were weighed and marked on the soles of their feet and measured the initial foot volume (V0) using a plestismometer, after which they were induced with 1% carrageenan (0.1ml) intraplantarly. After 1 hour, all test animals were given 0.5% Na-CMC suspension (negative control), diclofenac sodium (positive control), and songga wood methanol extract (doses of 50, 100 and 200mg/KgBW). The average results of rat paw edema can be seen in Figure 1.

The use of carrageenan in this study because it has several advantages including not leaving marks, not damaging tissues, and providing a more sensitive response to anti-inflammatory drugs than other materials, therefore it was chosen as an inductor of edema. Carrageenan is a chemical substance that will release inflammatory mediators such as histamine and serotonin that will cause edema due to the interaction of antibodies of test animals with antigens to fight the influence of these antigens²⁷.

Figure 1. Volume of Edema before and after treatment

(significantly different with the negative control (*p < 0.05)

Figure 1 shows the difference in udder volume in each treatment. Edema volume was formed after 1 hour of carrageenan administration, this is in accordance with previously reported research which states that edema volume has been formed from 30minutes to 3hours after carrageenan induction, namely at the time of release of inflammatory mediators and lasts for 6 hours which will gradually decrease³⁹.

The results showed that the volume of rat feet in the negative control did not decrease after being given 0.5% Na-CMC solution. This indicates that Na-CMC does not have anti-inflammatory activity. This can lead to the continuous release of inflammatory mediators such as prostaglandins, histamine, bradykinin, and serotonin in the tissue after carrageenin induction²⁸. In the positive control, there was a decrease in edema volume due to the inhibition of prostaglandin synthesis in the tissue. In this study, a positive control was used, namely diclofenac sodium, a phenyl acetate derivative with the pharmacological effect of inhibiting prostaglandin synthesis. Diclofenac sodium was chosen because diclofenac sodium can reach a high enough concentration on the soles of mice that experience inflammation.

Measurement of IL-1ꞵ Levels in Wistar Male Rats:

Enzymed-Linked Immunosorbent Assay (ELISA) is one of the diagnostic test methods in the form of immunological tests that are very sensitive and used to detect or measure a substance, including antigens, antibodies, proteins, glycoproteins, and hormones. The data obtained in the form of NET median fluorescent intensity (MFI) values are converted using a regression curve and obtained cytokines in units of pg/ml.

Measurement of IL-β levels by ELISA technique was carried out by taking rat blood 1 hour after carrageenin induction. Before measuring IL-β levels, blood was taken from the normal control (without treatment). This aims to determine normal IL-β levels or without inflammation that will be compared with IL-β levels after inflammation and after administration of anti-inflammatory test samples. The time of blood collection is done when IL-β levels are at peak levels, namely in the first 30 minutes to 1hour.

The levels obtained are the initial data of IL-β levels when the inflammatory process occurs, Then each group was given their respective treatments. One hour after treatment, blood was taken again and then centrifuged to get the plasma. Blood plasma will then be read on an ELISA reader. The results of measuring IL- Iβ levels can be seen in Figure 2.

Figure 2. IL-1β levels in the treatment and normal group

(significantly different with the negative control (*p < 0.05)

Based on Figure 2, the normal group had the lowest IL-1β levels at 137.37 pg/ml. IL-1β levels will increase when there is inflammatory activity in the body. Negative control (Na-CMC) is the initial comparison of other treatment groups. The Na-CMC suspension given to the negative control does not contain active substances so that it does not have an anti-inflammatory effect. The IL-1β level value obtained in the negative control was 499.20pg/ml.

In the group of 50 mg/KgBW dose of songga wood methanol extract did not give good results at 470.30 pg/ml, this value was not significantly different from the negative control. IL-1ꞵ levels decreased when the dose of extract administration was increased. At a dose of 100 mg/KgBW, IL-1ꞵ levels were 205.99pg/ml. While the lowest IL-1ꞵ levels were at a dose of 200mg/kgBW which was able to reduce IL-1ꞵ levels to 135 pg/ml.

Anti-inflammatory activity is generally due to the presence of flavonoids, where these flavonoids can reduce the production of proinflammatory cytokines such as IL-1ꞵ⁴⁰.

Inflammation in the body occurs when the body performs a defence response to a foreign object in the form of carrageenan which will activate macrophage cells and other trigger cells, thus activating pro-inflammatory cytokines in the form of IL-1ꞵ. IL-1ꞵ levels are produced by myeloid cells and act as an essential factor for Th17 cell differentiation that can exacerbate inflammation in the blood vessel wall. In addition, IL-1β is involved in the upregulation of adhesion molecule expression by endothelial cells and macrophage activation. IL-1β exerts response effects that include accelerating the growth of target cells (lymphocytes), inducing the expression of molecules on the cell surface (ICAM-1 on endothelial cells), and releasing secondary mediators such as prostaglandins from macrophages and other cytokines⁴⁰.

In this study, statistical analysis was used to determine the anti-inflammatory activity of methanol extract of songga wood and the most effective dose to reduce IL-1β levels in rat Statistical analysis was carried out using the One Way ANOVA test then continued with the Post Hoc test. Before the One Way ANOVA test, a normality test was first carried out using the Shapiro-Wilk test to detect data homogeneity. Normally distributed and homogeneous data were tested with one-way analysis of variance (ANOVA) with a confidence level of 95% to determine whether there were differences between groups.

Based on the data normality test using the Shapiro-Wilk test, it is known that the data distribution of all groups is normally distributed (p>0.05) and based on the variance test results, the data has the same variance or homogeneous (p>0.05). Based on the One Way ANOVA test, the value obtained (p< 0.05) so that it can be interpreted that there is a significant effect of giving methanol extract of songga wood on IL-1β levels in male wistar rats after 3 hours of treatment.

CONCLUSION:

The methanol extract of songga wood (S. ligustrina) has strong antioxidant activity with IC₅₀ value of 78.05 µg/mL, and has anti-inflammatory activity with IL-1ꞵ parameter, with the most effective dose of 200 mg/KgBW (p>0.05). This study can be a reference in the discovery of new medicinal compounds from natural sources, especially as antioxidants and anti-inflammatory.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

We thank Universitas Gadjah Mada for funding and supporting this research. This research was funded by the Postdoctoral Program of Gadjah Mada University, grant no. 1688/UN1/DITLIT/PT.01.00/2024.

We would also like to thank Universitas Halu Oleo, Politeknik Bina Husada Kendari, and Universitas Muhammadiyah Kalimantan Timur for their support and cooperation in this research.

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Received on 15.06.2024 Revised on 14.10.2024

Accepted on 28.12.2024 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2620-2626.

DOI: 10.52711/0974-360X.2025.00376

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