INTRODUCTION:

The development of microbial biofilms is now recognized as a major mediator of infection, and it is estimated that 80% of all infections are caused by the formation of microbial biofilms¹. Candida albicans is classified as a polymorphic fungus that is a normal microbiome in humans². As normal human flora, Candida lives in the mouth and digestive tract. About 75% of healthy individuals carry Candida as part of their normal commensal oral microflora. Recently, however, infections caused by C. albicans have increased rapidly in immunocompromised patients, i.e. patients who have a weak immune response, so Candida is pathologic³. Candida albicans is a fungus that forms biofilms and C. albicans colonizes epithelial tissues in immunocompromised people and develops into an opportunistic pathogen that causes superficial infections⁴.

The pathogenicity of Candida species is associated with specific virulence factors, such as the ability to evade host defenses, adhesion and biofilm formation (on host tissue or on medical devices), and the production of tissue-damaging hydrolytic enzymes, such as proteases, phospholipases and hemolysins¹. Biofilms are now recognized as one of the main mediators of infection with an estimated 80% of infections associated with biofilm formation⁵. Candida biofilm cells form structured, coordinated and functional communities, embedded in a self-secreted extracellular matrix⁶. Biofilm production is also associated with high levels of antifungal resistance of the associated microorganisms^6,7. Moreover, the ability of candida species to form drug-resistant biofilms is an important factor in their contribution to human disease^6,7. Extracellular polymeric substances (EPS) serve as a matrix around which diverse colonies of bacteria called biofilms cling to abiotic or biotic surfaces to construct intricate structures^8–11. A single type of microorganism can produce biofilms by combining different species of bacteria, fungi, algae, and yeasts with foreign materials^12,13. Infections fungal are frequently difficult to cure because of biofilms such as vaginal yeast infections, skin yeast infections and others. Due to the fact that biofilms' cells might have up to 1000 times more antibiotic resistance than planktonic cells, they pose a considerable therapeutic obstacle⁶.

In Indonesia, the practice of traditional medicine has advanced rather quickly. Traditional medicine is now being used by the public as an alternative treatment¹⁴, Although traditional medicines derived from plants and natural ingredients also have side effects, the level of danger and risk of long-term use is much lower and has great economic value compared to chemical medicinal^15–16. The use of plants as drugs, in addition to activity tests, also needs to be done toxicity tests to ensure safety for the community. One of the plants studied is E.rubroloba AD Poulsen¹⁷.

This plant has never been reported regarding activity as an antifungal, antibiofilm and acute toxicity test using zebrafish, but several different species have been reported regarding pharmacological and biological aspects, namely E. pubescens contains the compound etlingerin and shows better antibacterial and cytotoxic activity than curcumin¹⁸, E. elatior extract has strong antioxidant and anticancer activity¹⁹, and can be used as antihyperuricemia²⁰, as an antimicrobial and antifungal^21,22, and acts as a hepatoprotective²³. Furthermore, E. brevilabrum is used to reduce cholesterol levels²⁴, E. coccinea and E. sessilantheraas antibacterial²⁵, E. rubroloba fruit as an immunomodulator²⁶, and reduce IL12 levels and TLR-2 protein expression²⁷, then E. rubroloba stem extract as an antioxidant and anti-hyperuricemia^28,29, and can also be used as an anti-inflammatory³⁰. In this study, ethanol extract of E. rubroloba AD Poulsen was tested for antifungal and antibiofilm properties as well as acute toxicity usingzebrafish. Zebrafish is a small freshwater aquarium species that is easy to raise and maintain in different environments, has a short generation time, and reproduces almost all year round³¹.

In aquatic toxicology, the fish acute toxicity test is the standard test used to determine the toxicity of chemicals on aquatic vertebrates. Acute toxicity is described by the LC₅₀value, which is the concentration of a chemical required to kill 50% of the animals tested in a 96-hour test. The LC₅₀ value is the acute toxicity value³².

MATERIAL AND METHODS:

Materials:

Ethanol extracts of E. rubroloba AD Poulsen stems, fruits, and rhizomes were employed in this work together with sterile distilled water, Sabouraud Dextrose Broth (SDB) medium, RPMI media, 1% crystal violet, Gloves, disposable and masks, biofilm-forming standard C. albicans isolate (C. albicans ATCC 10231), Nystatin, DMSO 1%, NaCl, Mc Farland Standard 0.5, PBS solution (Phosphate Buffer Saline), Zebrafish (WT AB/TL), Fertilized Zebra fish (Danio rerio) eggs.

Equipment:

The tools used in this study were autoclave (Sakura, Japan), micro pipette (Gilson, France), Laminar Air Flow, incubator (IF-2B) (Sakura, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain), analytical balance (AB2045, Switzerland), 96 well polystyrene flat-bottom microplate (Iwaki, Japan), spectrophotometry (Genesys 10 UV Scanning, 335903) (Thermo Scientific Spectronic, USA), multichannel micropipette (Socorex, Switzerland) and Leica ICC50 E - Microscope Camera (Wetzlar, Germany)

Sample Preparation and Extraction:

Stems, fruits and rhizomes of E. rubroloba were obtained from the district of Kolaka, Southeast Sulawesi, then determinated at the LIPI Research Center for Biology, Bogor Indonesia. Samples were dried at 500C, then dry sorted, then pollinated to obtain dry simplisia of 2000g each. The samples were extracted by maceration with 96% ethanol each as much as 10 L (3 x 24 hours). The filtrate obtained was then evaporated with an evaporator at 55^°C to obtain a thick extract of 100g each

Zebrafish Method acute toxicity test (LC₅₀):

Samples of E.rubroloba plant parts, namely ethanol extracts of stems, fruits and rhizomes, were selected to be tested on zebrafish eggs (Danio rerio) in 5 concentration variants, namely 625, 1250, 2500, 5000 and 10000µg/mLwith 3 repetitions. Zebrafish eggs were placed in petri dishes as many as 20 eggs each and then added to the ethanol extract of E. rubroloba at each concentration and then observed for 96 hours^33–35. Danio rerio parameters observed were contraction, survival, hatching and heart rate. These toxicological endpoints indicate the mortality rate.

Preparation of test fungi:

Sabouraud Dextrose Broth (SDB) was used to cultivate Candida albicans (ATCC 10231) for 72hours at 37°C. The microbial culture's optical density 600 (OD 600) was changed to 0.1 (corresponding to McFarland standard 0.5-1.5 × 10⁸ CFU/mL).

Antifungal testing:

Antifungal testing was carried out on a flat-bottom polystyrene microtiterplate 96 wells using the microdilution method with varying concentrations of ethanol extracts of the stem, fruit of the rhizome of E. rubroloba, namely 625, 1250, 2500, 5000, 10000 µg/mL, and Positive control, 10000µg/mL of nystatin. A microbiological suspension served as the negative control, and the solvent control was adjusted to match the solvent of the test chemical. Each microplate well was filled with RPMI medium. Following a 72-hour incubation period at 37°C, then measured the absorbance at 520 nm wavelength using a microplate reader.

Inhibition of mid-phase (24 hours) and maturation (48 hours) biofilm formation was tested by microbroth dillution method.

96-well polystyrene microtiter plates with flat bottoms are used for the test of C. albicans mono-species biofilm development (Pierce et al., 2010). Each well of the microtiter plate was filled with 100 L of the C. albicans suspension (10⁷ CFU/mL), which was then incubated at 37⁰C for 90 minutes to initiate the biofilm attachment phase³⁶.

Following incubation, non-adherent cells were removed from the plate by washing it three times with 150L of sterile aquadest. A total of 100μL of media containing ethanol extract of E. rubroloba with concentration series (625, 1250, 2500, 5000 and 10000µg/mL), was added to each washed well. Media without microbial growth was employed as the media control, while microbial suspension was used as the negative control. Nystatin 1% w/v-treated microbial suspension was employed as the positive control. After that, the plates underwent 48 hours of maturation phase biofilm formation and 24 hours of mid-phase biofilm formation at 37°C incubation³⁶.

The plate was then cleaned three times with distilled water and dried for five minutes at room temperature to remove any leftover water. The biofilm that had formed, including both dead and living cells that are also constituent components of biofilm, was colored by adding a total of 125µL of 1% crystal violet solution to each well, which was then incubated at room temperature. After the microplate had been incubating at room temperature, it was thoroughly cleaned of any leftover crystal violet, and 200µL of 96% ethanol was added to each well to dissolve the biofilm that had developed. Readings of optical density (OD) at 520nm wavelength were acquired using a microplate reader^37,38.

OD negative control mean - OD test sample mean

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

OD negative control mean

Data Analysis:

Determination of acute toxicity test values using Zebrafish on ethanol extracts of E. rubrolobastem, fruit and rhizome. The percentage of mortality was calculated based on probit analysis to obtain the LC₅₀ value. Furthermore, the Minimum Biofilm Inhibition Concentration (MBIC₅₀), which measures biofilm activity based on sample concentrations that can prevent at least 50% of biofilm development^6,36.

The data obtained were analyzed using statistics version 25 with post hoc test to determine which concentration has a difference. When the results obtained showed a difference or significant between each concentration, the value (P<0.05) was stated. Furthermore, when compared with the positive control and the results obtained were not significantly different, it was stated with a value of (P>0.05).

RESULT AND DISCUSION:

Acute Toxicity Test with Zebrafish Method (LC₅₀):

Toxicity tests are very important to be carried out on medicinal plants, as an initial step in drug safety parameters before becoming a medicinal product that can be used in humans. This is because every medicinal material has the potential to be toxic depending on the dose in the body³⁹. Acute toxicity tests were conducted on each part of the E. rubroloba plant using Zebrafish eggs. Acute toxicity test data were analyzed using probit analysis, and the results can be seen in Table 1.

Table 1: Acute Toxicity Test Results (LC₅₀)

Sample	LC₅₀ (µg/mL)	Category
Stems	3898,23	Slightly Toxic
Fruit	10310,52	Practically Non-toxic
Rhizome	4065,10	Slightly Toxic

The results in table 1, show that the stem has an LC₅₀ value of 3898.23µg/mL (Slightly Toxic category), fruit LC₅₀10310.52µg/mL (Practically Not Toxic) and rhizome LC₅₀4065.10µg/mL (Slightly Toxic). This is in accordance with the toxicity category description data, namely Extremely Toxic, (10 or less), Highly Toxic (10-100), Moderately Toxic (100-1000), Slightly Toxic (1000-10,000), Practically Non-toxic (10,000-100,000), Relatively Non-toxic (100,000)⁴⁰.

Fertilized zebrafish (Danio rerio) eggs were used to test acute toxicity (Figure 1). This fish was chosen because the development of Danio rerio has been extensively studied and information regarding the normal development of this species is available. Synthetic and herbal drugs are no exception, as the overall impact on non-target organisms has not been identified. The non-target species thought to be most harmed by their action are probably aquatic organisms. This is why fish were used in this study as subjects for acute toxicity assessment on E.rubroloba plants.

Figure 1. Zebrafish development; (a) egg (b) embryo (c) dead zebrafish (d) live zebrafish

Antifungal Activity of E.rubroloba ethanol extract against C. albicans:

This test uses ethanol extracts of stems, fruits and rhizomes of E. rubroloba plants (Figure 2) with 5 concentration variants, namely 625, 1250, 2500, 5000 and 10000µg/mL

According to this study, E. rubroloba ethanol extract has the ability to suppress the growth of C. albicansplantonic cells in all concentrations (Figure 3). Ethanol extract of E. rubroloba at a concentration of 10000µg/mL gave the greatest inhibition results, namely in the MIC stem (80.28%), fruit (81.98%), rhizome (81.53%), and nystatin as a positive control of 80.99% w/v. These results indicate that with increasing concentration, the greater the inhibitory activity, and all samples were able to inhibit above 50% except at a concentration of 625µg/mL. The results of statistical analysis using Bonferroni post hoc is to determine which concentration has a difference. The results of the data obtained (Figure 3) show that there are differences or significant between each concentration, namely 625, 1250, 2500, 5000 and 10000µg/mL.So it is stated (P <0.05). While at a concentration of 10000µg/mL compared with the positive control is not significantly different (P>0.05).

Figure 2. E. rubroloba plant (a) fruits and flowers (b)

Figure 3. Anti-fungal activity with variant concentrations in 48 hours

Antifungal activity of E. rubroloba ethanol extract against C. albicans biofilm in 24-hour phase:

According to the study's findings, which are displayed in Figure 4, the ethanol extract of E. rubroloba at a concentration of 10000µg/mL exhibits antibiofilm activity against C. albicansin the stem (77.83%), fruit (77.66%), and rhizome (78.42%), as well as nystatin as a positive control (78.62%), in the middle phase (24 hours).The results of statistical analysis using Bonferroni post hoc is to determine which concentration has a difference. The results of the data obtained (Figure 4) show that there are differences or significant between each concentration, namely 625, 1250, 2500, 5000 and 10000µg/mL.So it is stated (P<0.05). While at a concentration of 10000µg/mL compared with the positive control is not significantly different (P>0.05)

Figure 4. Antibiofilm activity of C. albicans ethanol extract of E. rubroloba 24-hour phase

In comparison to its efficacy against antifungal, the ethanol extract of E. rubroloba showed lower action in the middle of the biofilm phase. This is because the inhibition process as antibiofilm and antifungal is very different. Biofilm is a collection of microbial cells irreversibly attached to a surface and encased in a matrix of Extracellular Polymeric Substances (EPS) that it produces itself and shows phenotypic changes such as changes in growth rates and changes in gene transcription from planktonic cells or free cells^41,42.

Biofilms consist of matrix material (85% of volume) and a collection of bacterial cells (15% of volume). Extracellular Polymeric Substances (EPS) make up 50%-90% of the organic carbon of biofilms and can be considered as the main matrix material^43,44.In this phase, C. albicans has produced an EPS matrix, which is visible from the wells' bottoms and the edges of wells 96, where it is distinguished by mucus that has adhered to the wells. Moreover, the method of providing crystal violet can be used to detect the presence of biofilm, Because C. albicans has produced a biofilm and the structure of the biofilm is more complicated and organized with one another during this phase, it will be more challenging for the ethanol extract to penetrate the defense of C. albicans. As a result, activity in the 24-hour biofilm inhibition phase declines⁴⁵. This is in line with previous researchers who stated that microbial biofilms can serve as shields, allowing bacteria that create biofilms to typically withstand common antimicrobials and evade the host cell immune system. As clinical infection rates rise in host cells, biofilms form, and these biofilms eventually contribute to virulence and resistance. Biofilm formation can be stimulated by the presence of serum and saliva in the environment⁴⁶.

Antifungal activity of E. rubroloba ethanol extract against C. albicans biofilm in 48 hours phase:

According to the study's findings, which are depicted in Figure 5, the antibiofilm activity of C. albicans in the maturation phase is provided by the ethanol extract of E. rubroloba at a concentration of 10000µg/mL in the stem of 74.79%, fruit (74.63%), rhizome (74.63%), and nystatin (73.80%). The results of statistical analysis using Bonferroni post hoc is to determine which concentration has a difference. The results of the data obtained (Figure 5) show that there are differences or significant between each concentration, namely 625, 1250, 2500, 5000 and 10000µg/mL.So it is stated (P <0.05). While at a concentration of 10000µg/mL compared with the positive control is not significantly different (P>0.05). These findings suggest that E. rubroloba's ethanol extract has less activity than its planktonic and 24hour biofillm phases. This is due to the fact that during this phase, the C. albicans biofilm produces more of its EPS matrix and forms a denser, more complicated biofilm structure, as evidenced by the mucus layer that is linked to the wells ring. This makes it challenging for the ethanol extract of E. rubroloba to enter the target cells. The biofilm community established in the 48hour phase of biofilm growth is increasingly numerous and structured with one another, establishing a sort of 3-dimensional group that will communicate with one another when the 48-hour period of biofilm growth ends^47,48.

Figure 5. Antibiofilm activity of C. albicans on ethanol extract of E. rubroloba 48-hour phase

This is in accordance with the statement³that C. albicans forms its community by forming colony bonds called biofilms. Scanning electron microscopy results show that mature C. albicans biofilms contain cells in the form of yeasts and hyphae that insert and are tightly bound to extracellular material which is usually fibrous^49,50. Previous statements also explain that the availability of saliva and serum during the pre-formation of biofilms increases the attachment ability of C. albicans to host cells but has less effect on biofilm formation^51,52.

CONCLUSION:

Ethanol extracts of stems and rhizomes are slightly toxic, while fruits are practically non-toxic, and have activity as antifungal and antibiofilm. The results of this study can be a reference in the development of traditional medicine, especially antibiofilm.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:
We acknowledge the Direktorat Penelitian dan Tim Peningkatan Reputasi UGM Kepada Universities of World Class Kantor Jaminan Mutu UGM for 2022 Post Doctoral funding with grant number 5061/UNI/ DITLIT/Dit-Lit/PT.01.02/2022, with assignment letter No. 1119/UN1.P.II/KPT/ HUKOR/2022. The authors would like to thank Universitas Halu oleo, Politeknik Bina Husada Kendari and Universitas Muhammadiyah Kalimatan Timur for their cooperation in this research.

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Received on 24.07.2023 Modified on 08.09.2023

Research J. Pharm. and Tech 2024; 17(8):3613-3619.

DOI: 10.52711/0974-360X.2024.00564