INTRODUCTION:

The liver plays an important role in the human body, which is responsible for a series of functions that help metabolism, immunity, digestion, detoxification, and vitamin storage, which is the center of the body's metabolism, such as drug and toxin/poison metabolism, which results in liver damage^1,2.

This unhealthy lifestyle causes a person to experience a decrease in endurance, so they are susceptible to viral infections such as viral hepatitis, which can cause liver damage. This disease causes disturbances in the liver due to the onset of edema or necrosis so that liver function is damaged^3,4. However, several other factors cause liver damage, such as chemical exposure. One substance that can cause oxidative stress in the liver is carbon tetrachloride (CCl4). CCl₄ can cause damage to the liver caused by free radicals; CCl₄ requires metabolic activation mainly by cytochrome P450 enzymes in the liver. The activation will convert carbon tetrachloride into more toxic metabolites, which can cause liver damage in experimental animals and humans. Excessive formation of free radicals will lead to oxidative stress, which can cause liver disorders^5,6.

Liver disease is inflammation caused by infections such as viruses, bacteria, or toxic substances when the liver cannot perform its detoxification function properly. The most common cause of liver disease is viral infection, which can be spread through feces, parenteral fluids, perineal, and other routes. Toxic substances from narcotics, alcohol, poisons, and side effects from the use of hepatotoxic drugs^7,8

Liver abnormalities or damage are characterized by an increase in several enzymes secreted by the liver, such as alkaline phosphatase and bilirubin. Bilirubin is derived from the breakdown of heme due to the destruction of red blood cells by reticuloendothelial cells. Excessive accumulation of bilirubin in the skin, sclera, and mucous membranes causes a yellow color called jaundice. The normal bilirubin level in serum ranges from 0.3-1.0mg/dl. Elevated serum bilirubin levels can cause jaundice^9,10. Besides bilirubin, another indication of liver damage is increased alkaline phosphatase. Alkaline phosphatase (ALP) is an enzyme that indicates hepatocellular and hepatobiliary abnormalities. This enzyme is present in bone, liver, and placenta. ALP in liver cells is found in sinusoids and bile duct membranes. A high increase in ALP value indicates that the bile ducts are obstructed. ALP enzyme examination can determine the normal 30-115IU/L value. An abnormal rise in ALP values may indicate liver or bone disease. ALP enzyme activity is used to assess cholestasis function. This enzyme is present in bone, liver, and placenta. ALP in liver cells is found in sinusoids and bile duct membranes, whose release is facilitated by bile salts, and ALP is found in osteoblasts. ALP levels depend on age and gender. ALP activity more than four times the upper limit of the reference value leads to abnormalities towards hepatobiliary rather than hepatocellular^11,12.

Handling liver disease is currently carried out in various ways, including by administering hepatitis vaccines, avoiding contact with blood or body fluids from other people, and can be prevented by taking drugs that work as hepatoprotectors. In addition to hepatoprotectors and antibacterials, this study also conducted antifungal testing against Candida albicans. Candida albicans is classified as a polymorphic fungus, which is a normal microbiome in humans^13,14. As normal human flora, candida lives in the mouth and digestive tract. About 75% of healthy individuals carry Candida yeast as part of their normal commensal oral microflora. However, in recent years, infections caused by C. albicans have increased rapidly in immunocompromised patients, those with weakened immune responses, where candida is pathologic^15,16.

Songga Wood (Strychnos ligustrina) is a plant species that belongs to the Loganiaceae family; this plant is endemic to West Nusa Tenggara (NTB) but is also found in several areas, including Roti, Kalimantan, Timor, Bali, Pasuruan, Banyuwangi and in Meru Betiri National Park. This plant can grow at 1-1500 m altitude above sea level. Previously reported research results from phytochemical screening of songga wood, including flavonoids, alkaloids, tannins, brucine, striknin, and steroids/triterpenoids. Naturally, tannins and flavonoids can be antioxidants and antibacterial and anti-inflammatory agents. The community has empirically used this plant as an analgesic, anti-inflammatory, antibacterial, fever, antidiabetic, and snake bite medicine. Several studies have been reported regarding the biological activity of Strychnos ligustrina and other species, namely Strychnos lucida as antimicrobial, antioxidant and anticancer¹⁷, Strychnos ligustrina leaf extract as antimicrobial in acnes^18,19, and Strychnos lucida as antimalarial^20–22, Research on songga wood with antibacterial, anti-fungal, and hepatoprotective activities has never been reported. Hence, it is necessary to explore its biological activities further so that it becomes a source of information in the development of traditional medicines.

MATERIAL AND METHODS:

Materials:

The materials that will be used in this study are ethanol extract of Songga Wood (Strychnos ligustrina) of 1 Kg, S.aureus, E. Coli, Candida albicans, mice (Mus musculus), curcumin, carbon tetrachloride (CCl₄), distilled water, Na CMC 0.5%, ALP and bilirubin reagents, NaCl 0.9% physiological, and mice feed ingredients.

Equipment:

The tools used in this study are a rotary vacuum evaporator (Stuart), analytical balance (Precisa®), measuring cup (Pyrex), hot plate (Stuart), beaker (Pyrex), oven (Gallenkamp Civilah-Australia), Glory 127 Spectrophotometer, EDTA tube, microcentrifuge tube, drip pites, stirring rod, jar, spoit, and cannula.

Determination:

Determination of songga wood was carried out to ensure the truth and authenticity of the plants used in the study. Determination was conducted 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 Clearance approval from the Halu Oleo University Animal Ethics Committee with No. 3545a/UN29.20.1.2/PG/2023.

Sample Preparation and Extraction:

A total of 2.5kg of Songga wood was collected from Dompu Regency, West Nusa Tenggara, then the sample was determined at the Biology Education Study Programme Laboratory, Faculty of Teacher Training and Education (FKIP), Halu Oleo University. Samples were cleaned, dried in direct sunlight, then made into powder, then extracted with 96% ethanol (10L, 3 x 24hours), using the maceration method. The filtrate obtained was evaporated with an evaporator (50⁰C), and a thick extract was obtained^23–27.

Antibacterial Activity Test of Songga Wood ethanol extract against Staphylococcus aureus and Escherichia coli:

The antibacterial activity test was carried out by microdilution method using a 96-well polystyrene flat-bottom microtiter plate with a concentration series of 0.125, 0.25, 0.5, and 1% b/v. The control used was drug control using chloramphenicol 1% b/v. The growth control was a microbial suspension, and the solvent control was adjusted to the solvent of the test compound. BHI media bacterial suspension was put into each microplate well and then incubated at 37⁰C for 24 hours. The microplate was read using a microplate reader at a wavelength of 595nm.

Antifungal testing:

Antifungal testing was performed on polystyrene flat-bottom microtiter plate 96 wells using the microdilution method with varying concentrations of ethanol extracts of songga wood, namely 0.125, 0.25, 0.5, 1%, and nystatin (1%) as a positive control. The microbiological suspension served as a negative control, and the solvent control was adjusted to match the solvent of the test chemical. Each microplate well was filled with Roswell Park Memorial Institute (RPMI) media. After an incubation period of 72 hours at 37°C, the absorbance was measured at a wavelength of 520 nm using a microplate reader^26,28

OD _{Negative control mean} - OD _{Test sample mean}

% Inhibition = ------------------------------------- x 100%

OD _{Negative control mean}

Hepatoprotector Test:

Animal Treatment:

The test animals were divided into 6 groups, with each group consisting of five mice, then induced CCl₄ with a dose of 1ml/kgBW, except for the normal group:

a. Group I (Normal), experimental animals were not given treatment and only given food and drink.

b. Group II (negative control), 0.5% NaCMC

c. Group III (positive control), cavicur drug at a dose of 52mg/kgBW

d. Group IV, ethanol extract of Songga wood (Strychnos ligustrina) at a dose of 26mg/kgBW

e. Group V, ethanol extract of Songga wood (Strychnos ligustrina) at a dose of 52mg/kgBW

f. Group VI, ethanol extract of Songga wood (Strychnos ligustrina) at a dose of 104mg/kgBW

Before being induced by CCl₄, for the treatment group, mice were given ethanol extract of songga wood; for positive control, cavicur was given at a dose of 52 mg/kg BW, and negative control was given NaCMC 0.5% for 14 consecutive days except for the normal group. Furthermore, on the 15th day, all groups except the normal group were induced with CCl₄ at a dose of 1 mL/kgBW, which was injected intraperitoneally. Blood sampling was done 24hours after CCl₄ induction. Mice were anesthetized using chloroform and then dissected for intracardial blood sampling. Blood samples taken are inserted in a centrifuge tube and then centrifuged at 3500rpm for 15minutes to obtain clear blood plasma. Plasma is collected and accommodated in a microtube. Plasma that has been separated is ready to be used for examination of bilirubin and ALP levels.

Billirubin level measurement:

A total of 1000μL of serum bilirubin was reacted with 100μL of the sample at 37⁰C Mixed and the absorbance with a spectrophotometer at a wavelength of 365nm after one minute.

Alkaline Phosphatase levels Measurement:

A total of 1000μL of ALP reagent was reacted with 100 μL of the sample at 37⁰C Mixed and the absorbance using a spectrophotometer at a wavelength of 365nm after one minute.

Data Analysis:

Minimum inhibitory concentration (MIC₅₀), which measures antibacterial and antifungal activity based on the concentration of the sample, can prevent at least 50% of bacterial and fungal growth.

The data obtained were analyzed using the SPSS version 25 statistical program with post hoc tests. These tests were conducted to determine which concentrations had differences, providing a more detailed understanding of the data. If the results show a difference or significance between each concentration, it is stated with a value (P < 0.05). Furthermore, compared with the positive control, the results are not significantly different; it is indicated with a value of (P>0.05). The hepaprotector test was analyzed using the one-way ANOVA method to determine whether or not there was an effect of ethanol extract of Songga wood on increasing hepatoprotective activity or to see the effectiveness of the sample compared to the positive control^29,30

RESULT AND DISCUSSION:

Antibacterial Activity of Songga Wood ethanol extract against Staphylococcus aureus and Escherichia coli:

Antibacterial activity testing was carried out by microdilution with 4 concentration variations, namely 0.125, 0.25, 0.5, and 1%. The results showed that the ethanol extract of songga wood had an inhibitory effect on planktonic cells at all concentrations; this can be seen from the percent inhibition given at each concentration. The highest inhibitory activity of ethanol extract against Staphylococcus aureus bacteria at 1% concentration is 84.46% and Chloramphenicol 85.68% as a positive control (Figure1)^31–33. Furthermore, the inhibitory activity against Escherichia coli at 1% concentration is 87.01% and chloramphenicol 86.38% as a positive control (Figure 2).

These results indicate that with increasing concentration, the inhibitory activity is greater, and all concentrations used are able to inhibit above 50% except at a concentration of 0.125%. The data results show differences or significance between each concentration, namely 0.125, 0.25, 0.5, and 1%. So, it is stated (P <0.05). While the 1% concentration compared to the positive control was not significantly different (P>0.05). These results illustrate that ethanol extract of songga wood has antibacterial activity against gram-positive bacteria (Staphylococcus aureus) and negative bacteria (Escherichia coli).

Figure 1. Antibacterial activity against Staphylococcus aureus in 24 hours

Figure 2. Antibacterial activity against Escherichia coli in 24 hours

Antifungal Activity of Songga Wood ethanol extract against C. albicans:

Antifungal activity testing was carried out by microdilution with 4 concentration variations, namely 0.125, 0.25, 0.5, and 1%. The results showed that the ethanol extract of songga wood can suppress the growth of C. albican splantonic cells at all concentrations (Figure 3). Songga wood ethanol extract at a concentration of 1% gave the greatest inhibition results, namely MIC (84.94%) and nystatin as a positive control of 86.45% b/v. These results indicate that with increasing concentration, the inhibitory activity is greater, and all concentrations used were able to inhibit above 50% except at a concentration of 0.125%^28,33. The results of the data obtained (figure 3) show differences or significance between each concentration, namely 0.125, 0.25, 0.5, and 1%. So, it is stated (P<0.05). Meanwhile, the 1% concentration compared to the positive control is not significantly different (P>0.05).

Figure 3. Antifungal activity with variant concentrations in 48 hours

Hepaprotector Activity:

The ethanol extract of songga wood used consists of 3 dose variations, namely 26, 52, and 104 mg/kgBW. Test animals were given the extract for 14 consecutive days orally once a day. This study used Carbon tetra chloride (CCl4) as an inducer of liver damage at a dose of 1 ml/kgBW. Each group of test animals was induced by CCl₄ on day 14, except the normal group. CCl₄ is one of the compounds that can cause liver damage characterized by acute inflammation of liver cells. The initial stage of damage to liver cells is in the form of hydrophic degeneration, then continues into fatty degeneration, before finally, the cells experience death or necrosis³⁴.

Alkaline phosphatase (ALP) levels:

Alkaline phosphatase (ALP) is a liver enzyme in the blood. Increased activity of this enzyme indicates cellular leakage and loss of liver cells' integrity of the plasma membrane. If the liver is damaged, the level of ALP enzyme in the blood will increase, exceeding the normal level of ALP enzyme (10.5-27.6 U/L). The average ALP levels after treatment can be seen in Figure 4.

Figure 4. ALP levels (U/L) after treatment.

Based on Figure 4. shows that the normal group has the lowest ALP levels compared to all treatment groups. The measurement results of ALP levels in the normal group are still in the range of normal ALP levels (10.5-27.6 U/L). This is because the ALP levels of the normal group have not been influenced by anything. The natural antioxidants in the body can still neutralize radicals in normal body conditions. In contrast, the average ALP levels in the negative control group had the highest ALP levels compared to all treatment groups because Na-CMC does not have liver-protecting activity, so when exposed to CCl₄, there is an expansion of the ALP enzyme and shows a significant difference to the normal group. This indicates that CCl₄ induction can damage the liver by producing oxidative stress conditions that will cause cell membrane damage and liver disease^34,35.

The positive control group, given Curcuma xanthorrhiza Roxb, showed that the mean ALP produced significantly differed from that of the negative control group. This is because the cavicur tablet preparation contains 20 mg Curcuma xanthorrhiza Roxb extract, which contains curcumin compounds that can protect liver function, treat jaundice, and increase appetite. The compound has been widely used as a comparator drug (positive control) to test hepatoprotector activity. Curcumin compounds from Curcuma xanthorrhiza Roxb extract have a hepatoprotective mechanism of action that can prevent liver cell damage by capturing superoxide ions and breaking the ion chain between superoxide (O₂-) which, in turn, this lipid peroxidation process will prevent hepatic damage mediated by antioxidant enzymes, namely Superoxide Dismutase (SOD) where the SOD enzyme will convert O₂- into less toxic products. In addition to this mechanism, the mechanism of curcumin in preventing hepatic cell damage is also by increasing glutathione S-transferase (GST) and inhibiting several proinflammatory factors such as nuclear factor-ĸB (NF-kB) and profibrotic cytokines.

The statistical analysis of ALP levels of mice using Tukey's Post Hoc test showed that the negative control group compared to the normal group had a significant value of p<0.05, meaning that the negative control group had a significant difference from the normal control group. This is because the negative control is only given 0.5% Na-CMC, which does not protect the liver induced by CCl4.

In the extract treatment group (doses of 26, 52 and 104 mg/kgBW) had significantly different results p<0.05 against the negative group. This means that the three extract treatment groups have hepatoprotective activity with parameters to reduce ALP levels. When compared with the positive control group, all doses of extract have a significant value of p < 0.05, meaning that there is a significant difference, or it can be said that songga wood extract does not have the same effectiveness as the positive control.

Bilirubin Level:

Excessive increase of bilirubin in the skin, sclera, and mucous membranes can cause a yellow color called jaundice. Normal Billirubin levels range from (0.10-0.90 mg/dl)^9,10. The value of Bilirubin levels after treatment can be seen in Figure 5.

Figure 5. Bilirubin levels (U/L) after treatment

Based on Figure 5. the normal group has the lowest average bilirubin level compared to all treatment groups. The measurement results of normal group bilirubin levels are still in the range of normal bilirubin levels (10-0.90mg/dl). This is because the normal group bilirubin levels have not been affected by anything, and the natural antioxidants in the body can still neutralize radicals in normal conditions. At the same time, the average bilirubin levels in the negative control group had the highest bilirubin levels compared to all treatment groups. They showed a significant difference from the normal group. This indicates that CCl₄ induction can damage the liver by producing oxidative stress conditions that cause cell membrane damage and liver disease^36–38.

The results of the average bilirubin levels after administering songga wood ethanol extract doses of 26, 52, and 104mg/kgBw were significantly different from the negative group. In addition, judging from the vulnerability of normal bilirubin levels in mice, the extract treatment group has average bilirubin levels that are still in normal conditions so that Songga wood plant extract can be used as a hepatoprotective or liver damage protector. Plant extracts will increase total protein and albumin synthesis to accelerate the regeneration process and protect liver cells. Therefore, increased bilirubin levels in animals given the extract indicate hepatoprotective activity.

The results of Tukey's Post Hoc test showed that the negative control group Na-CMC 0.5% compared with the positive control cavicur and with the treatment group showed a significant 0.00(p<0.05), which means significantly different, which indicates that CCl₄ managed to do mice liver damage. This is also due to the negative control only given Na-CMC 0.5%, which does not protect the liver induced by CCl₄. At the same time, the normal control group was not significantly different from the positive group, with a sig value of p>0.05. This indicates that cavicur has hepatoprotective activity in reducing bilirubin levels.

The extract dose group (doses of 26, 52, and 104 mg/kgBW) had significantly different results from the negative group (p<0.05), so the extract has hepatoprotective activity with the parameter of decreasing bilirubin levels. Compared with the positive control group, all doses of the extract have a significant value of p<0.05, meaning that there is a significant difference, or it can be said that Songga wood extract does not have the same effectiveness as the positive control.

CONCLUSION:

The ethanol extract of songga wood has antibacterial activity (Staphylococcus aureus, Escherichia coli), antifungal (Candida albicans), and as a hepatoprotector (P<0.05) based on Alkaline phosphatase (ALP) and bilirubin parameters. This study is a reference in the discovery of new drugs sourced from natural materials.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

Our gratitude goes to the Ministry of Education, Culture Research and Technology, Directorate of Higher Education, Research and Technology for the research funding support provided by the contract for the Implementation of the BOPTN Program for fiscal year 2024 with number 049/E5/PG.02.00.PL/2024 Dated 11 June 2024. We also thank Halu Oleo University for its support so that the implementation of the research can be carried out correctly.

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Received on 10.09.2024 Revised on 12.01.2025

Accepted on 16.03.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4377-4383.

DOI: 10.52711/0974-360X.2025.00627

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