66 Dental Journal (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 Original article Fungistatic effect of Gracilaria verrucosa on phospholipase enzymes and the cell surface hydrophobicity of Candida albicans Basri A. Gani1, Ridha Andayani1, Fitri Yunita Batubara2, Ifwandi Ifwandi3, Dharli Syafriza4, Muhammad Mas’ud Herlambang1, Shiti Alya Fathirah1, Ayudia Rifki4 1Department of Oral Biology, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia. 2Department of Conservative Dentistry, Faculty of Dentistry, Universitas Sumatera Utara, Medan, Indonesia. 3Department of Prosthodontics, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia. 4Department of Pediatric Dentistry, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia. ABSTRACT Background: Candida albicans (C. albicans) was reported as a primary cause of oral candidiasis. Phospholipase enzymes and cell surface hydrophobicity (CSH) are involved in the pathogenesis of its infection. Gracilaria verrucosa (G. verrucosa) is reported to contain flavonoids, tannins, and phenolic compounds, which can inhibit the development of C. albicans. Purpose: The study analyzed the effect of the ethanol extract of G. verrucosa on the inhibition of the phospholipase enzyme and CSH of C. albicans. Methods: The G. verrucosa chemical compounds were examined by phytochemical tests and phospholipase enzyme-inhibiting egg yolk media, and a CSH assay was conducted using xylene. Results: Gracilaria verrucosa contains steroids, terpenoids, tannins/ phenolics, and flavonoids. The 25%–100% concentrations inhibit the phospholipase enzyme of C. albicans more strongly at 48 hours than at 12 hours and 24 hours (p < 0.05). Furthermore, the 100% and 75% concentrations substantially affect the inhibition of CSH of C. albicans, with a strong relationship. Conclusion: Gracilaria verrucosa has a more substantial inhibitory effect in suppressing the phospholipase enzyme and CSH of C. albicans. Keywords: Candida albicans; Gracilaria verrucosa; hydrophobicity; phospholipase enzyme Article history: Received 24 July 2023; Revised 22 January 2024; Accepted 5 February 2024; Published 1 March 2025 Correspondence: Basri A. Gani, Department of Oral Biology, Dentistry Faculty, Universitas Syiah Kuala. Jl. Hamzah Fansuri Kopelma Darussalam, Banda Aceh, 23111, Indonesia. Email: basriunoe@usk.ac.id INTRODUCTION Candida albicans (C. albicans) is a common opportunistic fungal pathogen that can cause various human infections, ranging from superficial mucocutaneous infections to severe systemic diseases.1 One of its virulence factors is the production of phospholipase enzymes, primarily phospholipase A2, which play a crucial role in the degradation of host cell membranes.2 This enzymatic activity helps the fungus invade host tissues and evade the host’s immune response.3 Increased C. albicans can occur using broad-spectrum antibiotics, corticosteroids, cytotoxic agents, and smoker isolates that can trigger oral candidiasis infections.4 Phospholipase enzymes are responsible for hydrolyzing phospholipids, the major components of cell membranes.5 Phospholipase A2 specifically targets the sn-2 position of phospholipids, releasing fatty acids and lysophospholipids.6 In the context of C. albicans, the production of phospholipase enzymes contributes to its pathogenicity by facilitating tissue invasion and nutrient acquisition.7 The phospholipase enzyme works by hydrolyzing the ester bonds of glycerophospholipids. It tends to contribute to the pathogenicity of C. albicans through the destruction of cell membranes to facilitate an invasion of host tissue.8 Researchers have classified four types of phospholipase in C. albicans, namely phospholipase A, B, C, and D. All of these phospholipase types have hydrolase activity in host cells.9 Cell surface hydrophobicity (CSH) is considered an essential non-biological factor contributing to hydrophobic interactions related to the adherence of candida cells on the mucosal host surface.10 It represents the degree to which the surface of C. albicans cells interacts with water molecules. Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 mailto:basriunoe@usk.ac.id https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 67Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 Fungi with higher hydrophobicity tend to cling to the host tissues and medical devices quickly.11 This adherence is essential in establishing fungal infections because it enables the pathogen to colonize and multiply inside the habitats of host organisms. Candida albicans regulates CSH based on the growth phase, environment, and nutrient availability.12 Changes in C. albicans pathogens are associated with patients who experience immune disorders.13 Some studies suggest the treatment of candidiasis depends on the type and virulence of the infection. Fluconazole is an effective drug for oral candidiasis, which has an excellent antifungal effect compared with other antifungal medicines.14 Unfortunately, fluconazole is not always effective because it has a secondary impact on hosts, such as nausea, vomiting, diarrhea, and stomach pain.15 Thus, alternatives continue to be sought to reduce these impacts, including using plants or natural materials to be tested for the growth and development of C. albicans, as reported by Seleem et al.,16 who study the types of natural materials for the treatment of candidiasis. Gracilaria verrucosa (G. verrucosa) is a species of red macroalgae, also known as seaweed, prevalent in coastal maritime environments. The substance contains a diverse array of bioactive compounds, several of which have been subject to scientific inquiry into their potential therapeutic use. The bioactive compounds encompass a variety of chemicals, such as polysaccharides, polyphenols, and peptides. There is a potential for specific compounds to exhibit antifungal properties, hence warranting more investigation into their potential impact on C. albicans. Gracilaria verrucosa has been reported as having antifungal potential because it contains phenol compounds widely known as antioxidants.17 The hypothesis underlying the research is that G. verrucosa may have a fungistatic (inhibitory) effect on C. albicans by targeting its phospholipase enzyme activity and altering its CSH. This inhibition could reduce the pathogen’s ability to degrade host cell membranes, limiting its invasion and virulence and impeding its ability to establish infections. Measurements of phospholipase enzyme activity and the CSH of C. albicans can provide insights into its potential fungistatic. This work aims to assess the inhibitory effects of G. verrucosa on the virulence factors of C. albicans, specifically the phospholipase enzymes and CSH activity. MATERIALS AND METHODS This research has received the ethical clearance 341/KE/ FKG/2022 from the Faculty of Dentistry, Universitas Syiah Kuala, Darussalam, Banda Aceh, Aceh, Indonesia, and the species of G. verrucosa was identified by Herbarium Laboratory, Faculty of Mathematics and Natural Science, Universitas Syiah Kuala, No. B/6665/UN11.1.8.4/ TA.00.01/2020. This test material was prepared into six different concentrations—100%, 75%, 50%, 25%, 12.5%, and 6.25%—and fluconazole (30 mg/5 mL) (Acme, Indonesia) was determined as a positive control, and the assay material concentration was prepared using the formula V1.C1−V2.V2 (where V is volume and C is concentration).18 The research materials used were C. albicans from smoker isolates obtained from the Microbiology Laboratory, Veterinary Faculty, Syiah Kuala University, Banda Aceh-Indonesia. The seaweed (G. verrucosa) was obtained from the sea of Pulo Aceh, Aceh Besar, Aceh, Indonesia, with a coordinate point of 5° 41’ 38.6” N, 95° 03’ 06.8” E. The extraction was performed at the Basic Chemistry Laboratory, Faculty of Teacher Training and Education, Syiah Kuala University, Darussalam Banda Aceh, Aceh, Indonesia. A total of 3 kg of G. verrucosa was washed thoroughly with water, drained, cut into small pieces, and dried by airing at room temperature for 3 days to remove the water content so that a constant seaweed weight was obtained. After that, small pieces of G. verrucosa seaweed were extracted using the maceration method by placing the seaweed in a jar and soaking it in a 96% ethanol solvent while stirring every day for 2 days. After that, the seaweed extract was filtered using filter paper to obtain the filtrate and residue. The extraction was repeated thrice by soaking the seaweed residue in 96% ethanol to obtain the second and third filtrates. Then, all the filtrate was collected and evaporated using a rotary evaporator at 40oC until the ethanol solvent evaporated and a thick seaweed extract was obtained.19 The chemical compounds of G. verrucosa were examined using the working principle stated by Soraya et al.20 Subsequently, the obtained extract was analyzed to determine the presence or absence of chemical constituents that could impede bacterial proliferation (Table 1). The ethanol extract analysis obtained from G. verrucosa was conducted using gas chromatography–mass spectrometry with a Shimadzu Japan QP2010 Plus gas chromatography (GC) system. The experimental setup involved the use of a fused GC column, which was coated with polymethyl silicon (0.25 nm × 50 m). The following parameters were employed in the experiment: the temperature range was 80°C–200°C; the rate of temperature increase was 5°C per min; the temperature was maintained at 200°C for 20 min; the flame ionization detector temperature was adjusted to 300°C; the injection temperature was set to 220°C; the carrier gas employed was nitrogen, flowing at a rate of 1 mL/min, with a split ratio 1:75; the pressure was 116.9 kPa; the column was 30 m long, with a diameter of 0.25 mm and a 50 mL/min flow rate. The phytochemical screening assay is reported in Table 1.18 The C. albicans CSH activity was assessed in the first stage. A 10 mL sample of C. albicans cultured in peptone media (1.5 x 108 CFU/mL) was poured into a tube and centrifuged at 7,000 rpm for 15 min supernatant. The pellet was then washed with phosphate-buffered saline (PBS) Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 68 Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 pH 7. Hydrophobicity assessment began by inserting 100 µL into a 96-well microplate. The hydrophobicity cell of C. albicans was assessed by optical density at 520 nm. Hydrophobicity inhibition was evaluated by adding 5 mL of G. verrucosa to each concentration in 1 mL of C. albicans suspension, then incubating for 24 hours at 37°C. They were then centrifuged at 7,000 rpm for 20 min. The supernatant was removed, and 1 mL of xylene (Merck KGaA, Darmstadt, Germany) was added and placed in a water bath at 37°C for 10 min. After that, a vortex was used for 30 sec to mix the suspension with xylene, then stored in a water bath at 37°C for 30 min to separate the components. Next, the residue was carefully transferred to another sterile tube, and the remaining xylene in the pipette was resuspended with 2 mL PBS pH 7.0. Then, 150 µL of suspension was applied to the 96-well microplate. The change in absorbance value of hydrophobicity is related to the ability of C. albicans to increase virulence to the knowledge of adhesion in mucosal cells. Hydrophobicity percentage index (HI) values were obtained through the following formula, wherein the first stage (the percent value of each liquid [optical density] of the liquid phase and inoculum) was obtained with the formula (percentage value of hydrophobicity OD = [OD value concentration of sample/total OD value all concentrations × 100%]): HI = (percent value of OD-inoculum + OD-HI value). The scale used was as follows: strong (65%–100%), moderate (30%–64%), and low (1%–29%).21 Sabouraud dextrose agar (Merck KGaA, Darmstadt, Germany) media was coated with 100 µL egg yolk medium and dried in sterile conditions for 30 min.22 Furthermore, 100µL of C. albicans was immersed in 1mL of G. verrucosa extract in various concentrations with fluconazole as a positive control and then adapted for 30 min at room temperature and inserted into disc paper. The homogeneity process was carried out on a shaker at a speed of 200 rpm for 15 min. Furthermore, the disc was placed on the yolk media and incubated for 12 hours, 24 hours, and 48 hours at 37°C. The zone of precipitation and the colonies’ zone refer to the inhibitory power of G. verrucosa extract against the enzyme phospholipase C. albicans. The phospholipase activity is the ratio of the colony’s diameter to the colony’s diameter added to the precipitation zone (mm) and is determined as the ratio of the diameter of the circle to the total diameter of the colony plus the zone of precipitation. Table 2. Gas chromatography–mass spectrometry analyses of chemical compounds of G. verrucosa Peak Retention time (min) Compounds Intensity (%) 1 17.345 Oxirane, decyl- 2.023 2 18.187 n-hexadecanoic acid 74.198 3 20.126 Eicosanoic acid 2.262 4 20.306 Nonanoic acid 2.084 5 20.716 Oleic acid 6.609 6 21.731 Pentadecanoic acid 1.176 7 22.862 Bicyclo[3.2.1]oct-3-en-2-one, 3,8-dihydroxy-1-methoxy-7-(7-methoxy-1,3- benzodioxol-5-yl)-6-methyl-5 2.901 8 25.373 N-(5-chloro-2-hydroxyphenyl)dodecanamide 2.048 9 27.294 Cholesta-8,24-dien-3-ol, 4-methyl-, (3.beta.,4.alpha.)- 1.542 Table 1. Phytochemical screening of G. verrucosa18 Chemical compounds Material and method Flavonoid The examination is carried out by adding Mg, 0.5 ml HCl, and amyl alcohol into a 2 mL extract. The reaction is positive if a red–purple color is formed. Tannin Tannin examination is done by mixing a 1% gelatin solution with sodium chloride into 2 mL of extract. The positive reaction wakes up, and a white precipitate is formed. Saponin Saponin examination begins by mixing HCI 0.1 into 2 mL of extract and shaking vigorously vertically for 15 sec. If persistent foam forms with a height of approximately 1 cm, then the extract is positive for containing saponin. Alkaloid The alkaloid examination is performed by dissolving hydrochloric acid in 2 mL of extract, then filtering, which includes the following: (1) Mayer’s test—Mayer’s reagent (potassium mercury iodide) is added to the extract. The formation of yellow-colored precipitates contains alkaloids; (2) Wagner’s test—Wagner’s agent (iodine in potassium iodide) is added to the extract. The formation of brown—reddish brown precipitates indicates positive alkaloids; (3) Dragendroff test—Dragendroff reagent (potassium bismuth iodide) is added to 2 mL of extract. The formation of red precipitates indicates alkaloids. Steroid Steroid and terpenoid examination is carried out by adding Carr–Price reagent to 2 mL of extract. The presence of steroid content in the extract forms a green color. Meanwhile, the terpenoid content of the extract is translated into the formation of a red color in the solution. Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 69Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 One-way analysis of variance (ANOVA) was used to analyze the results of hydrophobicity data and phospholipase enzymes from C. albicans, and the least significant difference (LSD) method was used for the post hoc test. The Shapiro–Wilk test was used for a normal distribution, and homogeneity values were determined. Data were considered normal and homogeneous if p > 0.05. The significance limit for these two data was p < 0.005. Pearson correlation was used to determine the relationship between concentration and the time required to assess the effect on hydrophobicity and phospholipase enzymes, with a strong correlation if r = 0.85–1, medium if r = 0.5–0.79, and low if r ≤ 0.5. RESULTS This study evaluates the fungistatic properties of G. verrucosa on the phospholipase enzyme and CSH of C. albicans. Furthermore, the chemical compound content of G. verrucosa and phytochemical screening are also reported as references for its fungistatic properties. Table 2 reports that there are nine chemical compounds extractable from G. verrucosa. Out of these, two chemical compounds exhibited higher values: n-hexadecanoic acid (74.198%) and oleic acid (6.61%). Table 3 shows five chemical compounds with positive (+) values in the phytochemical screening test, such as steroids, terpenoids, and tannin/ phenolic compounds. Table 4 reports the ability of G. verrucosa to inhibit the phospholipase enzyme of C. albicans. At 12 hours of incubation, none of the concentrations of G. verrucosa tested showed significant phospholipase inhibition (p = 0.069). Meanwhile, 24-hour incubation showed significant phospholipase inhibition by G. verrucosa (p = 0.047). Likewise, 48 hours showed strong phospholipase enzyme activity from all concentrations of G. verrucosa (p = 0.029). There was a significant difference in the inhibition of the C. albicans phospholipase enzyme between incubation times of 12 hours, 24 hours, and 48 hours (p = 0.031). The assessment limits for the inhibitory categories are strong (>1 mm), moderate (0.75–0.99 mm), weak (0.51–0.74 mm), and no effect (<0.05 mm).21 The LSD test shows that concentrations of 100% and 75% of G. verrucosa have good significance in inhibiting the release of the phospholipase enzyme C. albicans (Table 5). Table 6 reports the hydrophobic index of G. verrucosa towards the cell surface of C. albicans. Based on the assessment scale, 100% and 75% concentrations strongly influence changes in the hydrophobicity of the C. albicans cell surface, including positive controls. In contrast, the other concentration groups are moderate. Based on the one-way ANOVA of the hydrophobicity index of the G. verrucosa between concentration groups, there was a Table 3. Phytochemical screening of ethanol extract of G. verrucosa Chemical content Reagent Ethanol extract Observation result Alkaloid Mayer - White sediment Wegner - Brown sediment Dragendroff - Red sediment Steroid Lieberman-Burchard Test + Green / blue color Terpenoid Lieberman-Burchard Test + Red / purple Saponin Shaking + Stable foam Flavonoid 0.5 Mg and HCl + Red / purple Tannin/phenolic MgCl3 + Dark green Tabel 4. Phospholipase inhibition of C. albicans by G. verrucosa G. verrucosa (%) N Phospholipase inhibition (mm) *p-value12 h 24 h 48 h Mean SD Scale Mean SD Scale Mean SD Scale C100 3 1.02 0.00 Strong 1.11 0.00 Strong 1.28 2.12 Strong 0.031 C75 3 1.00 0.49 Strong 1.00 0.14 Strong 1.40 0.14 Strong C50 3 0.91 0.70 Moderate 1.02 0.70 Strong 1.20 0.35 Strong C25 3 0.97 0.70 Moderate 0.97 0.56 Moderate 1.15 0.42 Strong C12.5 3 0.96 0.91 Moderate 0.84 0.70 Moderate 0.95 0.00 Moderate C6.25 3 0.95 0.28 Moderate 0.93 0.58 Moderate 0.81 0.70 Moderate Fluconazole 3 0.95 0.77 Moderate 0.94 1.13 Moderate 1.04 0.84 Strong *p-value 21 0.069 0.047 0.029 * One-way ANOVA Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 70 Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 Table 6. Hydrophobicity index of C. albicans influenced by G. verrucosa G. verrucosa (%) n Hydrophobicity index (520 nm) *p-value Mean SD Frequency Index Scale C100 3 2.681 0.176 26% 68% Strong 0.041 C75 3 2.259 0.263 22% 64% Strong C50 3 1.301 0.967 13% 55% Moderate C25 3 1.364 0.346 13% 55% Moderate C12.5 3 0.311 0.053 12% 53% Moderate C6.25 3 0.301 0.054 13% 55% Moderate Fluconazole 3 2.035 0.054 20% 62% Strong * One-way analysis of variance Table 5. The least significant difference (LSD) test on the effect of G. verrucosa in inhibiting the phospholipase enzyme C. albicans G. verrucosa (%) *p-value 12 h 24 h 48 h C100 C75 0.046 0.001 0.001 C50 0.041 0.021 0.210 C25 0.439 0.011 0.471 C12.5 0.416 0.001 0.011 C6.25 0.430 0.001 0.002 Fluconazole 0.420 0.001 0.046 C75 C50 0.416 0.021 0.001 C25 0.402 0.001 0.010 C12.5 0.416 0.001 0.001 C6.25 0.339 0.001 0.001 Fluconazole 0.391 0.001 0.350 C50 C25 0.675 0.001 0.101 C12.5 0.729 0.001 0.001 C6.25 0.728 0.001 0.001 Fluconazole 0.748 0.000 0.370 C25 C12.5 0.805 0.000 0.001 C6.25 0.871 0.512 0.001 Fluconazole 0.871 0.571 0.429 C12.5 C6.25 0.717 0.012 0.617 Fluconazole 0.615 0.017 0.017 C6.25 Fluconazole 1.015 0.612 0.001 *Post hoc LSD (p < 0.05 is significant) Figure 1. Pearson correlation coefficient between inhibition of phospholipase enzyme and hydrophobicity index. The three treatment times of phospholipase enzyme inhibition strongly correlate with the hydrophobicity index of the C. albicans cell surface. Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 71Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 significant difference (p < 0.05:0.041). Based on the LSD test, Table 7 shows that concentrations of 100% and 75% have a better ability to inhibit the hydrophobicity of C. albicans cells surface. Figure 1 reports Pearson’s correlation of the role of G. verrucosa in inhibiting the formation of the phospholipase enzyme with the ability of C. albicans to form CSH. These three treatment times have a strong relationship between the inhibitory power of the phospholipase enzyme and the hydrophobicity of the C. albicans cell surface. The direction of the positive ridges indicates that G. verrucosa has a similar effect in inhibiting the phospholipase enzyme and the formation of CSH. It means that G. verrucosa can work together to cause changes in the virulence of C. albicans. DISCUSSION This study uses the ethanol extract of G. verrucosa as a test material to measure the inhibitory power of the enzyme phospholipase and CSH of C. albicans isolated from smokers’ saliva. One of the smoker isolates’ selections is expected to have high virulence properties tested for sensitivity with G. verrucosa. Candida albicans from smokers isolates had faster morphological transitions from blastospores to pseudohypha and truly hypha than C. albicans ATCC 10231 isolates.1 The chemical compounds of G. verrucosa, such as n-hexadecanoic acid and oleic acid, are reported to contain antioxidant and antifungal properties (n-hexadecanoic acid and oleic acid).23 The activities were targeted inhibitors for phospholipase A2 of C. albicans. This approach could facilitate a potential anti-inflammatory drug. The findings from the investigation on enzyme kinetics provide evidence that n-hexadecanoic acid acts as a competitive inhibitor of phospholipase A2.24 Gracilaria verrucosa contains steroid compounds, terpenoids, and tannins/phenolics, which can act as antifungal agents that can suppress the growth and development of C. albicans.25 Their compounds have different roles and properties as C. albicans virulence inhibitors. This study examined the effects of the three compounds in G. verrucosa as inhibitors of phospholipase enzyme and CSH of C. albicans smoker isolates. Furthermore, G. verrucosa can suppress the phospholipase enzyme of C. albicans. It still provides optimal effects at a concentration of 25%, especially at 48 hours of incubation time. The fact that G. verrucosa inhibits C. albicans phospholipase activity is encouraging for fungal infection treatment. The pathogenicity of C. albicans depends on its phospholipase enzyme, which breaks down host cell membranes.13 Gracilaria verrucosa at 25% is the minimum dose to block this enzyme, primarily when incubated for 48 hours. Gracilaria verrucosa may include antifungal secondary metabolites or phytochemicals. These chemicals are abundant at 25%, allowing them to interact with fungal cells. These drugs may inhibit phospholipase, preventing C. albicans from breaking down host cell membranes and reducing its pathogenicity.26 The 48-hour incubation period matters, too. Enzyme and fungal growth take time to climax. Gracilaria verrucosa can decrease phospholipase activity for a longer time by incubation for 48 hours with C. albicans. This prolonged exposure may explain the remarkable inhibitory effectiveness. Moreover, a 25% concentration may be enough to override fungal defenses. One potential approach involves the inhibition of efflux pumps or disrupting essential metabolic pathways in C. albicans.27 Implementing these measures can potentially enhance the inhibition of phospholipase in G.verrucosa. The highest concentration (100%) of G. verrucosa is most effective at inhibiting the enzyme phospholipase of C. albicans. It can be assumed that a concentration of 100% signifies the use of G. verrucosa at its full potency, hence delivering the highest quantity of bioactive components and possible inhibitory agents for combating C. albicans. The high concentration of the substance is expected to impact the fungal cells significantly, impeding their ability to evade or develop resistance against the inhibitory effects.28 Consequently, the enzyme phospholipase, which plays a pivotal role in the pathogenicity of C. albicans, experiences enhanced inhibition. Moreover, the length of incubation is of utmost importance in this procedure. Extended incubation periods offer G. verrucosa prolonged exposure to C. albicans. The long duration of exposure facilitates a continuous suppression of phospholipase activity.29 The attainment Table 7. The least squares difference (LSD) test on the effect of G. verrucosa in inhibiting hydrophobicity of C. albicans cell surface G.verrucosa (%) *p-value C100 C75 0.010 C50 0.001 C25 0.001 C12.5 0.000 C6.25 0.001 Fluconazole 0.031 C75 C50 0.010 C25 0.012 C12.5 0.000 C6.25 0.001 Fluconazole 0.041 C50 C25 0.575 C12.5 0.019 C6.25 0.011 Fluconazole 0.041 C25 C12.5 0.011 C6.25 0.017 Fluconazole 0.019 C12.5 C6.25 0.711 Fluconazole 0.015 C6.25 Fluconazole 0.001 *Post hoc LSD (p < 0.05 is significant) Copyright © 2025 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73 72 Gani et al. Dent. J. (Majalah Kedokteran Gigi) 2025 March; 58(1): 66–73 of maximum enzyme activity frequently necessitates a certain amount of time, and the inhibitory effect can be intensified by prolonging the presence of G.verrucosa. The concentration, contact time, and surface area influence natural materials, such as nanoparticles, against pathogens.30 This theory clarifies that a higher concentration of the test material used can lead to a higher ability to prevent the development of pathogens.31 This ability is correlated with G. verrucosa’s ability to disrupt the enzyme synthesis channel in the cytoplasm, thus causing cells to polarize due to the influence of some active compounds possessed by G. verrucosa. This polarization causes cells to lack nutrition and have limited oxygen uptake. It is a process to reduce the degree of virulence of C. albicans. One of the G. verrucosa compounds related to this activity is tannin.32 This compound is reported as an antifungal related to its ability to activate enzymes and interfere with intra- and extra-membrane transport of proteins.33 The highest concentration of G. verrucosa can strongly suppress the hydrophobicity activity of the cell surface of C. albicans. It can be assumed that G. verrucosa can inhibit the signaling system between pathogens when forming biofilms. Hydrophobicity is an early phase carried out by some pathogens before forming biofilms. This signaling is closely related to the degree of formation of hydrophobicity or biofilms in the pathogenesis of infections, both bacteria and fungi, such as C. albicans.34 High plant extract concentrations may promote bioactive compound –fungal cell membrane contacts, explaining its effectiveness. Many antifungals damage the cell membrane, and leakage of intracellular substances can cause cell death.35 Plant extracts may also overpower fungal defensive systems, including efflux pumps that remove antifungal drugs at higher doses. Plant extracts may hinder C. albicans from releasing antifungal chemicals, making the organism more susceptible.36 High concentrations of plant extracts may also affect C. albicans’ metabolic pathways, threatening their survival and growth. Energy generation, protein synthesis, and deoxyribonucleic acid replication can be disrupted, inhibiting fungal cell development.37 From the perspective of this study, terpenoids contained in G. verrucosa can inhibit biofilm formation, prevent adhesion, and inhibit the growth of C. albicans. Besides, terpenoids can also disrupt the morphological changes of C. albicans from blastospores to hyphae, one of the essential virulence factors in C. albicans. The consequence disturbs the synthesis of phospholipids as a source of nutrients and reactive oxygen species.38 The fungal cell wall structure’s main components are (1,3) β- and 1, 6-β glucan, chitin, and mannoprotein. (1,3) -β-glucans are factors that play a role in CSH38 and are catalyzed by the enzyme synthase (1,3) β-glucans. 1,3-β glucan synthase is a glycosyltransferase enzyme found in the plasma membrane that is responsible for the construction of the fungal cell wall.39 Therefore, when the performance of this enzyme is inhibited, the cell wall loses rigidity and causes the attachment of C. albicans to epithelial cells to be significantly weakened.40 These results indicate that the chemical compounds of G. verrucosa are more effective in inhibiting the formation of ergosterol C. albicans, where ergosterol is a plasma membrane component that plays a role in the construction of chitin, which consists of some polysaccharide components of cell walls and has an essential role in the growth of C. albicans.41 The hydrophobicity of some pathogens is known to have the property of trying to move away from non-polar molecules and approaching polar molecules.42 Therefore, it can be assumed that besides having opposite properties, G. verrucosa also has non-polar properties.43 The polarity of some compounds contained by G. verrucosa can determine the inhibitory power of the enzyme phospholipase and CSH of C. albicans.44 Both analyses demonstrated that the inhibitory capability of G. verrucosa on the hydrophobicity of C. albicans cell surfaces correlated with its ability to suppress C. albicans phospholipase activity. 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Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v58.i1.p66–73 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v58.i1.p66-73