INTRODUCTION:

Candida albicans is a pathogenic fungal microorganism that is often found in the normal flora of the oral cavity, gastrointestinal tract, and human genitourinary tract. In healthy conditions, C. albicans do not cause problems, but under certain circumstances, such as a weakened immune system or long-term use of antibiotics, this fungus can cause infection¹.

One of the most common forms of infection is oral candidiasis, or "thrush", which is characterized by excessive growth of C. albicans in the oral cavity². These infections can cause pain, white sores in the mouth, and difficulty swallowing. In addition, untreated candidiasis can worsen oral health conditions, including increasing the risk of further complications such as spreading the infection to other organs in individuals with weakened immunity³.

The metabolic response of Candida albicans in the pathogenesis of candidiasis infection is related to its ability to form biofilms and produce hydrolytic enzymes such as phospholipase and protease, which favor colonization and invasion of host tissues⁴. The biofilm formed by C. albicans provides extra protection against the immune system and antifungal drugs, making it more difficult to overcome by conventional medicine⁵. This condition triggers the repetition of various antifungal synthetic drugs, including the polyene group, such as nystatin, and the azole group, such as Fluconazole⁶. This recitation has become a significant problem in the medical field due to the limited selection of effective antifungal drugs.

The importance of this research lies in searching for alternative natural ingredients that effectively overcome C. albicans infection, especially in conditions of resistance to synthetic antifungal drugs. Moringa oleifera and Ziziphus mauritiana are plants with potential antimicrobial and antifungal activity⁷. Bioactive compounds such as flavonoids, alkaloids, and phenolics contained in these plant extracts are thought to be able to inhibit the growth of pathogenic microorganisms, including C. albicans, in a different way than synthetic drugs, thus potentially addressing drug resistance issues⁸. This research is significant in dental pharmacology because it can provide a safer and more effective alternative treatment based on natural ingredients in overcoming oral candidiasis infection. Natural ingredients such as M. oleifera and Z. mauritiana can be a more affordable solution, have fewer side effects, and support the development of more environmentally friendly herbal medicines.

This study offers novelty in using local plant extracts to assess their tolerance response and potential inhibition of C. albicans cell metabolism. The main objective of this study is to evaluate the ability of extracts of Moringa oleifera and Ziziphus mauritiana to inhibit the metabolic activity of C. albicans, especially in terms of phospholipid release, which plays an essential role in the pathogenesis of candidiasis infection. The results of this research are expected to contribute to the development of science in the field of antifungal pharmacology based on natural ingredients and provide more effective and resistant treatment options for fungal resistance.

Material and Methods:

This study used C. albicans ATCC 10231 as research subjects, seeding M. oleifera and Z. mauritiana L extracts as test materials with concentrations (50, 100, 200, and 400μg/mL), respectively. Candida albicans culture was carried out using Sabouraud Dextrose Agar (SDA) media. After aseptic inoculation, the culture is incubated at 37°C for 24-48hours. The growing colonies are then inoculated into Sabouraud Dextrose Broth (SDB) and incubated for 18-24hours to produce the mushroom suspension. The C. albicans suspension was then diluted with saline until it reached the McFarland standard of 0.5(1.5x10⁸ CFU/mL), which was measured using a spectrophotometer at a wavelength of 625nm with an absorbance value between 0.08 to 0.1(300 CFU/mL).

Plant extraction:

Extraction begins by carefully cleaning the leaves of M. oleifera and Z. mauritiana L. The leaves are then dried in the shade or in an oven at 40-50°C until completely dry. After drying, the leaves are ground until they become a fine powder. As many as 100grams of dried leaf powder are soaked in 1 liter of 70% or 96% ethanol for 3-7 days at room temperature while stirring periodically to ensure optimal extraction of active compounds. Once the soaking process is complete, the solution is filtered to separate the solid part from the liquid extract. This liquid extract is then evaporated using an evaporator or rotavapor at low temperatures to remove the ethanol solvent. The resulting concentrated extract is stored in a sterile glass bottle in a cool, dark place, such as in a refrigerator at 4°C, to maintain the stability of the active compound before being used in further testing⁹.

Growth Assay

The growth test of Candida albicans was carried out using extracts of Moringa oleifera and Ziziphus mauritiana at concentrations of 50, 100, 200, and 400 μg/mL. C. albicans cultures were prepared according to McFarland standard 0.5, then inoculated with the extract and incubated at 37°C for 24hours. The growth of microorganisms was measured using spectrophotometry at OD 625nm. Lower OD values indicate the presence of significant growth inhibition by the extract¹⁰.

Metabolic Activity Assay:

Metabolic activity tests with MTT Assay for Candida albicans affected by extracts of Moringa oleifera and Ziziphus mauritiana were performed by preparing cultures in Sabouraud Dextrose Broth (SDB), which were then incubated at 37°C for 24hours and diluted to reach the McFarland standard of 0.5. Extracts at 50, 100, 200, and 400μg/mL concentrations were added to microplates containing 100μL of culture. Negative and positive controls are also prepared. The microplate is re-incubated at 37°C for 24hours. After incubation, MTT solution is added and incubated for 4 hours to allow the formation of formazane crystals. The medium is then discarded, and the formed formazan crystals are dissolved with the addition of DMSO. Absorbance was measured at a wavelength of 570nm using a spectrophotometer, with lower absorbance values indicating a decrease in metabolic activity¹⁰.

Enzymatic Assay:

The enzymatic phospholipase test for Candida albicans, after being affected by the extracts of M. oleifera and Z. mauritiana L was carried out by preparing C. albicans cultures in Sabouraud Dextrose Broth (SDB) and incubated at 37°C for 24hours. After incubation, the culture is centrifuged to separate the cells from the supernatant. C. albicans cells were then tested on egg yolk agar media to observe phospholipase activity. Phospholipase activity is measured based on the diameter of the precipitation zone formed around the colony. The decrease in the diameter of the precipitation zone indicates the inhibition of phospholipase activity by the extract. Extracts of Moringa oleifera and Ziziphus mauritiana were tested at various concentrations (50, 100, 200, and 400μg/mL) and compared with no extract-free control to determine the effect of inhibiting phospholipase activity¹¹.

Metabolism Changes Assay:

Candida albicans cells are extracted by growing cultures in Sabouraud Dextrose Broth (SDB) incubated at 37°C for 24-48hours. After incubation, the culture is centrifuged at 3,000-5,000rpm for 10-15minutes, and the cell pellets are washed with sterile PBS. Intracellular component extraction was carried out with a chloroform-methanol mixture, then centrifuged again at 10,000-12,000rpm to separate the cell debris. Supernatants containing intracellular components are collected for analysis using FTIR (Fourier-transform infrared spectroscopy). FTIR detected phospholipids and nucleic acid deformations in C. albicans affected by M. oleifera and Z. mauritiana L extracts. Phospholipids, as an indicator of phospholipase activity, show absorption bands at 2850-2950 cm⁻¹ (C-H), 1740 cm⁻¹ (C=O), and 1250-1150 cm⁻¹ (P=O). Nucleic acid deformation is examined through absorption bands at 1700-1600 cm⁻¹ (C=O) and 1225-1250 cm⁻¹ (P=O), indicating changes in the structure of DNA or RNA. Changes in intensity in these bands indicate enzyme activity and cellular damage due to the treatment of the extract on C. albican ¹².

Hydrophobicity Assay:

Examining the surface hydrophobicity of C. albicans cells after being affected by extracts of M. oleifera and Z. mauritiana L using xylene solution began with preparing C. albicans culture. The cultures were incubated in Sabouraud Dextrose Broth (SDB) at 37°C for 24-48hours and then diluted to reach the McFarland standard of 0.5. Afterward, extracts of M. oleifera and Z. mauritiana were added at concentrations of 50, 100, 200, and 400μg/mL, and then the cultures were re-incubated at 37°C for 24 hours. After incubation, the cultures are centrifuged at 5,000rpm for 10min, and the cell pellets are washed with sterile PBS and then resuspended in 3mL of PBS. A total of 0.5mL of xylene is added to the cell suspension, left for 2minutes, and left until the water and xylene phases are separated. The water phase containing the cells was then separated, and the absorbance was measured at 600nm using a spectrophotometer. Cell hydrophobicity was calculated based on the percentage change in turbidity (OD) before and after treatment with xylene, which reflects the level of surface hydrophobicity of C. albicans cells^13,14

Statistical Analysis:

The analysis was between the treatment groups using One-Way ANOVA, while the T-test analyzed the tested extract groups. The meaning limit is p<0.05, with a value of 1 as an indicator of a strong relationship.

ReSults:

Table 1 shows that the extracts of M. oleifera and Z. mauritiana L. effectively inhibit the growth of C. albicans by increasing the concentration of extracts. At a concentration of 400μg/mL, both extracts decreased growth by up to <150CFU/mL with a growth percentage of 8%, close to the effectiveness of positive control (Fluconazole), which showed 7% growth. Both extracts showed inhibition at lower concentrations (50-200 μg/mL), but the growth percentage remained higher than at the highest concentrations. Negative control (PBS) showed the highest growth of >600 CFU/mL with a growth percentage of 35-36%. Statistical tests showed significant differences in the effect of the extract on the growth of C. albicans, with p-values of 0.011 for M. oleifera and 0.05 for Z. mauritiana.

Table 1. Growth of C. albicans under the influence of M. oleifera and Z. mauritiana L

Concentration (µg/mL)	N	*Pertumbuhan C. albicans (OD 625 nm)*								**p-value
		*M. oleifera*				*Z. mauritiana Lam*
		Mean	Std Dev	CFU/mL	Growth	Mean	Std Dev	CFU/mL	Growth
50	3	0.11	0.012	>300	22%	0.11	0.012	>300	21%	0.04
100	3	0.08	0.010	<300	16%	0.09	0.01	>300	16%
200	3	0.07	0.007	<300	13%	0.07	0.007	<300	13%
400	3	0.04	0.005	<150	8%	0.05	0.006	<150	8%
Control + (FZ)	3	0.04	0.005	<150	7%	0.04	0.005	<150	7%
Control - (PBS)	3	0.18	0.005	>600	35%	0.19	0.005	>600	36%
p-value*		0.011				0.05

* One Way ANOVA; ** T-test; FZ(Fluconazole); PBS (Phosphate Buffer Saline)

Table 2. Effect of M. oleifera and Z. mauritiana L on the activity of C. albicans metabolites

Concentration (µg/mL)	N	*Aktivitas Metabolit C. albicans (OD 570 nm)*						**p-value
		*M. oleifera*			*Z. mauritiana Lam*
		Mean	Std Dev	Effect	Mean	Std Dev	Effect
50	3	0.12	0.01	Moderate	0.13	0.011	Moderate	0.05
100	3	0.09	0.008	Hight	0.1	0.009	Moderate
200	3	0.07	0.007	Hight	0.08	0.008	Hight
400	3	0.04	0.005	Hight	0.05	0.006	Hight
Control + (FZ)	3	0.03	0.003	Hight	0.03	0.003	Hight
Control - (PBS)	3	0.18	0.015	No Effect	0.19	0.016	No Effect
p-value*		0.031			0.02
* One Way ANOVA; ** T-test; FZ(Fluconazole); PBS (Phosphate Buffer Saline)

Table 3. Effect of M. oleifera and Z. mauritiana L on phospholipase production of C. albicans

Concentration (µg/mL)	N	Fosfolipase C. albicans (mm)						**p-value
		M. oleifera			Z. mauritiana Lam
		Mean	Std Dev	%	Mean	Std Dev	%
50	3	11	1.1	26	12	1.2	20	0.15
100	3	9	0.9	40	10	1.0	33
200	3	7	0.7	53	8	0.8	46
400	3	5	0.5	66	6	0.6	60
Control + (FZ)	3	5	0.5	66	5	0.5	66
Control - (PBS)	3	15	1.5	0	15	1.5	0
*p-value		0.048			0.05
* One Way ANOVA; ** T-test; FZ(Fluconazole); PBS (Phosphate Buffer Saline)

Table 4. Inhibition of Phospholipid release of C. albicans after interaction with M. oleifera and Z. mauritiana L

Concentration (µg/mL)	N	Phospholipids C. albicans (wavelength cm-1)
		M. oleifera (%T)				Z. mauritiana Lam (%T)
		C-H (2850-2950)	C=O (1740)	P=O (1250-1150 )	% Inhibition	C-H (2850-2950)	C=O (1740)	P=O (1250-1150)	% Inhibition
50	3	88	85	83	25	87	84	82	20
100	3	78	74	72	40	77	73	71	35
200	3	65	62	58	55	64	61	57	50
400	3	45	40	37	70	44	41	36	65
Control + (FZ)	3	35	33	32	75	34	32	31	70
Control - (PBS)	3	95	97	92	0	94	96	91	0
FZ (Fluconazole); PBS (Phosphate Buffer Saline)

Table 5. Response to nucleic acid deformation of C. albicans after interaction with M. oleifera and Z. mauritiana L

Concentration (µg/mL)	N	Nucleic acid deformation of C. albicans (wavelength cm-1)
		M. oleifera (%T)			Z. mauritiana Lam (%T)
		C=O (1700-1600)	P=O (1225-1250)	% Inhibition	C=O (1700-1600)	P=O (1225-1250)	% Inhibition
50	3	88	85	25	89	86	20
100	3	77	74	40	78	75	35
200	3	65	60	55	64	60	50
400	3	44	43	70	46	44	65
Control + (FZ)	3	31	30	75	33	32	70
Control - (PBS)	3	96	91	0	96	91	0
FZ (Fluconazole); PBS (Phosphate Buffer Saline)

Table 6. Formation of C. albicans cell surface hydrophobicity after interaction with M. oleifera and Z. mauritiana L

Concentration (µg/mL)	N	Hydrophobicity C. *albicans* (OD 600 nm)						**p-value
		M. *oleifera*			Z. *mauritiana* *Lam*
		Mean	Std Dev	% Inhibition	Mean	Std Dev	% Inhibition
50	3	0.48	0.03	22	0.50	0.04	20	0.172
100	3	0.42	0.02	30	0.43	0.03	28
200	3	0.34	0.02	45	0.35	0.02	43
400	3	0.25	0.01	60	0.27	0.02	58
Control + (FZ)	3	0.12	0.01	75	0.14	0.01	73
Control - (PBS)	3	0.58	0.04	0	0.60	0.05	0
*P-Value		0.041			0.0124
* One Way ANOVA; ** T-test; FZ (Fluconazole)

DISCUSSION:

This study evaluated the potential of Moringa oleifera and Ziziphus mauritiana as antifungal agents against Candida albicans through growth tests, metabolite activity, phospholipase production, nucleic acid deformation, and cell hydrophobicity. The results showed that both extracts effectively inhibited C. albicans, with M. oleifera consistently showing more potent inhibition, especially at the highest concentration (400μg/mL). Both extracts are also close to the effectiveness of Fluconazole in inhibiting phospholipase and nucleic acid deformation. This indicates that this plant extract has the potential as a natural alternative to treating fungal infections.

Table 1 shows the effects of assay material on the growth of C.albicans; both extracts significantly inhibited the growth of C. albicans and increased concentration. Both extracts showed significant inhibition, although the growth percentage was higher than the highest concentration. This study's results align with previous findings that show that the extracts of M. oleifera and Z. mauritiana L contain bioactive compounds such as flavonoids, saponins, and phenols known to be effective antifungal agents. The bioactive compounds from plant extracts can interfere with fungal growth by inhibiting enzymatic mechanisms and the biosynthesis process of fungal cells ¹⁶. The plant extracts rich in flavonoids and phenols significantly inhibited the formation of Candida albicans biofilms and reduced their virulence¹⁷. In addition, these results reinforce research shows that M. oleifera extract has a significant antifungal effect on C. albicans through mechanisms of inhibition of cellular metabolism and enzymatic activity¹⁸.

Table 2 shows that the extracts of M.oleifera and Z. mauritiana L effectively inhibit the activity of the metabolites of Candida albicans, mainly through bioactive compounds such as flavonoids, tannins, and saponins. Flavonoids have been reported to inhibit key enzymes in the metabolism of fungal cells, reducing the ability of fungi to survive and causing infections¹⁹. In addition, tannin and saponin compounds cause damage to cell membranes, resulting in intracellular metabolite leakage and inhibiting the growth and cellular activity of C. albicans²⁰. The effectiveness of this inhibition is in line with previous research that suggests that plant extracts can be a natural alternative in overcoming resistance to synthetic antifungals such as Fluconazole ²¹.

Table 3 reports that both test materials can inhibit the production of Candida albicans phospholipase, an essential enzyme in the virulence of this fungus. This inhibition of phospholipase is significant, as the enzyme phospholipase plays a role in the degradation of host cell membranes and contributes to the spread of infection. Phospholipase is an enzyme that plays a role in the phospholipid breakdown of the host cell membrane, which allows C. albicans to invade tissues and spread disease²². Flavonoids and saponins in plant extracts are known to interfere with the structure and function of fungal cell membranes and inhibit the activity of the phospholipase enzyme, reducing the virulence of C. albicans²³. This study supports previous findings suggesting that bioactive compounds in plants have antifungal solid activity, mainly through the mechanism of inhibition of important enzymes in fungal pathogenesis²⁴. This natural extract also has the potential to be an effective therapeutic alternative to the resistance of synthetic antifungal drugs²⁵. A preview study showed that some antifungal compounds could increase the fluidity of fungal cell membranes, causing leakage of intracellular contents and ultimately inhibiting the production of enzymes necessary to maintain membrane structure, including phospholipase²⁶.

Table 4 reports that M. oleifera and Z. mauritiana L. can induce phospholipid release by Candida albicans. The antifungal compounds of both extracts can work by interfering with the phospholipids of the cell membrane of Candida albicans. Phospholipids are essential components of cell membranes, which maintain the integrity and function of the membrane and allow cells to communicate with the outside environment²⁷. The bioactive compounds in both extracts can damage phospholipid structures by disrupting hydrophobic bonds within the membrane, leading to increased membrane permeability and metabolic dysfunction of fungal cells²⁸. These disorders inhibit critical metabolic processes and weaken fungal cells, leading to cell death ²⁹.

Table 5 shows the report that extracts M. oleifera and Z. mauritiana L against the nucleic acid deformation of Candida albicans. Antifungal compounds in plant extracts, such as flavonoids, tannins, and saponins, can cause deformation in the nucleic acids of fungal cells by disrupting the stability of DNA and RNA structures³⁰. Flavonoids bind to nucleic acids, disrupting DNA replication, RNA transcription, and protein synthesis, ultimately inhibiting the growth and reproduction of fungal cells³¹. In addition, saponins can affect the permeability of cell membranes, so antifungal compounds can more easily enter cells and cause direct damage to genetic material³². Damage to the C=O and P=O groups, which are important components in DNA and RNA, suggests that these extracts can damage the essential structure of nucleic acids, resulting in significant deformation in C. albicans cells³³. This study also supports previous research that shows the potential of bioactive compounds in plant extracts as an inhibitor of the virulence of pathogenic microorganisms by damaging their genetic material³⁴.

Table 6 reports that both extracts effectively inhibit the surface hydrophobicity of Candida albicans cells, with increased inhibition as the concentration of the extract increases. Cell surface hydrophobicity is an important factor in the adhesion and biofilm formation process in Candida albicans³⁵. The bioactive compounds in plant extracts disrupt cell membrane structure and damage the lipid layer of cell membranes, disrupting hydrophobic properties that are important for cellular interactions and adhesion to the host surface³⁶. In addition, saponins are known to have natural surfactant properties capable of damaging the integrity of fungal cell membranes, disrupting hydrophobic interactions between cells, and reducing the cell's ability to form biofilms³⁷. These results suggest that plant extracts can be an effective natural alternative to prevent the development of biofilms and improve the therapeutic response to fungal infections.

Conclusion:

This study shows that the extracts of M. oleifera and Z. mauritiana L. have significant potential as antifungal agents against Candida albicans. Both extracts effectively inhibit growth, metabolite activity, phospholipase production, nucleic acid deformation, and surface hydrophobicity of C. albicans cells, especially at high concentrations (400μg/mL). Moringa oleifera tends to show slightly stronger effectiveness than Ziziphus mauritiana, although both are close to positive control effectiveness (Fluconazole). Thus, these two extracts can be developed as natural alternatives in the treatment of Candida albicans infections that are resistant to synthetic drugs, making an essential contribution to the pharmacology of dentistry and the treatment of fungal infections of the oral cavity.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

Acknowledgment:

This research is financed by the Directorate of Research, Technology, and Community Service, Ministry of Education and Culture of the Republic of Indonesia, with Master Contract Number: 094/E5/PG. 02.00.PL/2024, dated June 11, 2024, and Sub-contract Number: 664 /UN11.2.1/PG.01.03/SPK/DRTPM/2024, dated June 12, 2024

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