INTRODUCTION:

Candida albicans are normal flora in human, including oral cavity, that is not dangerous in the oral cavity. In healthy people, there are around 40-60% Candida albicans detected in oral cavity and gastrointestinal tract. However several factors such as immune system disorders or the use of drugs can cause these to become pathogenic^1–4. This fungus can cause oral candidiasis and periodontal disease as an opportunistic infection in the oral cavity^5,6. Clinical findings of oral candidiasis in the oral cavity include pseudomembranous candidiasis, erythematous candidiasis, chronic hyperplastic candidiasis, median rhomboid glossitis, denture stomatitis and angular cheilitis⁵.

About 50% of the normal population has (carries) Candida albicans as normal flora in the oral cavity, especially on the posterior dorsum of the tongue¹. Candida albicans can transition into a pathogenic state when the host's immune system is compromised or reduced such as AIDS and diabetes mellitus^7–9. According to a study, oral candidiasis is frequently observed in individuals with acquired immunodeficiency syndrome (AIDS), with reported prevalence reaching as high as 95%¹⁰. The latest data in 2022 regarding the cumulative number of AIDS patients in Indonesia was 137, 397 cases. In cancer patients, it is reported that around 7-52% of oral candidiasis cases involve head and neck cancer¹¹.

The main treatment for oral candidiasis currently is with topical and systemic antifungal drugs¹². The most frequently used topical drugs are nystatin, miconazole, ketoconazole and clotrimazole troches¹. At a further stage, patients can be given oral medication, namely fluconazole, ketoconazole, itraconazole and several other azole groups^13,14. With the potential for good inhibitory power, this type of azole drug has side effects in patients when used. Side effects that can occur due to the use of nystatin in the oral cavity are a burning sensation or pain in the oral cavity, changes in taste on the tongue, redness and itching in the treated area. Fluconazole has side effects of headache, nausea and vomiting, diarrhea, dizziness, dry mouth and redness of the skin^15,16.

Perceptions about safety as well as negative side effects arising from conventional medicines are strong reasons for people to switch back to traditional medicines or herbal medicines. According to the Food and Drug Supervisory Agency of the Republic of Indonesia (2014), traditional medicine refers to ingredients or mixtures of ingredients from plants, animals or minerals that have been used for treatment for generations based on experience. Most traditional medicines today consist of a mixture of plant ingredients and are better known as herbal medicines.

Papaya is a tropical plant that grows widely in Indonesia because papaya plants can grow in various seasons. Total papaya production in 2011 ranked 6th in fruit production in Indonesia. Due to its abundant availability in Indonesia, papaya plants are easy to obtain¹⁷.

Papaya plants are known to have many benefits because they contain phytochemicals, enzymes, polysaccharides, vitamins and minerals^18,19. A phytochemical analysis test of papaya seeds showed positive results for the content of alkaloids, flavonoids, tannins, saponins, phenols, and steroids²⁰. On the papaya leaves, phytochemical examination showed positive results for the content of flavonoids, alkaloids, tannins and saponins²¹. Comparison between content seeds and papaya leaves showed that papaya leaves were superior in flavonoid content, but the alkaloid content was found to be higher in the seeds²². Previous research has also been carried out papaya leaves upon Candida albicans and papaya seeds against Candida albicans has been proven to inhibit colony growth^23–25. Other research also proved that Carica papaya seeds have a great potential as antimicrobial and antifungal²⁶.

Even though it has many benefits, in Indonesian society the papaya plant is still underutilized²⁷. This research aims to prove the antifungal potential of combination of papaya seed and leaf extracts to inhibit the growth of Candida albicans.

MATERIALS AND METHODS:

Materials:

The study employed an experimental laboratory design with a control group, conducted between July and September 2023 at the Testing Service Unit of Universitas Airlangga, the Research Center of the Faculty of Dental Medicine, Universitas Airlangga, and the Laboratory of the Faculty of Pharmacy, Universitas Airlangga. Ethical clearance was granted by the Ethics and Law Committee of the Faculty of Dental Medicine, Universitas Airlangga, Surabaya. (1074/HRECC. FODM/VIII/2023).

The materials used in this research include: papaya leaves, papaya seeds, label paper, paper disc, cotton swab sticks, filter paper, Sabouraud Dextrose Agar (SDA) and Sabouraud Dextrose Broth (SDB). The tools needed for this research include: incubator, petri dish, Erlenmeyer, oscillator, spirit, test-tube, test-tube rack, measuring cup, oven, volume pipette, vernier calliper, Buchner funnel, air flow laminate, blender, autoclave, anaerobic jar, stirrer/magnetic stirrer, rotary evaporator, hotplate stirrer.

The papaya fruit and leaves used in this research were obtained from California papaya plantations in Cianjur, West Java and extracted at The Airlangga University Testing Service Unit. The combination extract of papaya seeds and leaves was a solution obtained by macerating a combination of crushed papaya seeds and leaves in 96% ethanol solvent. Candida albicans was a fungi taken from the fungi stock at The Research Center of Faculty of Dental Medicine, Universitas Airlangga.

The papaya seeds and leaves that will be used are subjected to a plant taxonomic validation test by observing morphological characteristics and measuring them. The plant determination test was carried out at Batu Herbal Materia Medica Laboratory UPT.

Processing and making a combination of papaya seed and leaf extracts:

The seeds are separated from the fruit and dried. Drying was carried out using the freeze-drying method for 3 days. Next, the papaya seeds are ground using a blender, weighed 100grams and set aside.

At the same time papaya leaves were separated from the stems and dried by freeze drying for 2 days. Once dry, the papaya leaves are ground using a blender, weigh 100 grams of papaya leaf powder and set aside.

Extraction was carried out by mixing 100grams of papaya seed powder and 100grams of papaya leaf powder. Followed by maceration of a combination of papaya seeds and leaves using 96% ethanol solvent, then left for 3 days (with stirring). The final step was to separate the maceration solution from the dregs, so that they obtained a combination of papaya seed and leaf extract solution. The mixture of extracts was subsequently evaporated with a rotary evaporator to eliminate the residual 96% ethanol solvent present in the extract.

Phytochemistry Test:

To verify the antifungal content contained in papaya seeds and leaves, phytochemical testing was carried out. The ingredients that will be tested are flavonoids, saponins and alkaloids. Flavonoids were tested by dissolving the extract with Mg (Magnesium) and HCl. If the results show a dark red to orange coloured solution, then the extract was positive for containing flavonoids. Saponin was tested by dissolving the extract with HCl, if the results show the solution was foamy for at least 10 minutes, then the extract was positive for containing saponin. Alkaloids are tested by dissolving the extract with Dragendroff's reagent. If the solution produces a white or creamy precipitate then the extract was positive for containing alkaloids^28,29.

Research Group:

The research samples were divided into 8 groups, namely: Negative control group (K-) which was given Aquades; The positive control group (K+) was given Fluconazole 100mg; Treatment group 1 (P1) was given a combination of 80% papaya seed and leaf extract; Treatment group 2(P2) was given a combination of 40% papaya seed and leaf extract; Treatment group 3(P3) was given a combination of 20% papaya seed and leaf extract; Treatment group 4(P4) was given a combination of 10% papaya seed and leaf extract; Treatment group 5 (P5) was given a combination of 5% papaya seed and leaf extract; Treatment group 6(P6) was given a combination of 2.5% papaya seed and leaf extract. Each group carried out 8 replications.

Candida albicans Dilution Series:

The first was making the inoculum by preparing 8 sterilized test tubes. Next, 10mL of distilled water was added to the first tube, 200mg Fluconazole tablets were added to the second tube, dissolved in 10mL of distilled water, 2mL of SDA was given to the third tube and 8 mL of pure extract to get a concentration of 80%. The 4th to 8th tubes were given 5mL of SDA each.

Serial Dilution begins by taking 5mL of the 3rd tube solution (80%) and transferring it into the 4th tube. Stir until the solution is homogeneous. Taken back from the 4th tube (40%) as much as 5 mL and transferred into the 5th tube. Stir until the solution is homogeneous and so on until the 8th tube.

After completing the serial dilution, it was then incubated for 24 hours at 37℃. Next, the inoculum is taken with a micropipette and then poured onto agar medium, to test its antifungal power. The same procedure was carried out 8 times ¹⁶.

Data collection in this research was carried out after treatment was given to a sample.

Based on the results of further difference tests with the Mann Whitney test in the table, the differences between K(+) and K(-); K(+) and P3; K(+) and P4; K(+) and P5; K(+) and P6; K(-) and P1; K(-) and P2; K(-) and P3; K(-) and P4; K(-) and P5; K(-) and P6 Next, there are also differences between P1 and P3; P1 and P4; P1 and P5; P1 and P6. There is a difference between P2 and P3; P2 and P4; P2 and P5; P2 and P6. There is a difference between P3 and P4; P3 and P5; P3 and P6.

This shows that the combination extract seeds and leaves Papaya has antifungal potential against growth of Candida albicans which is indicated by the greater the concentration, the more it inhibits fungal growth.

DISCUSSION:

Candida albicans, a yeast-like fungus, serves as a prominent illustration of an opportunistic pathogen responsible for an array of superficial and systemic infections in humans. Among the over 150 Candida species identified, approximately 20 are recognized for causing infections in humans. Candida albicans stands out as the primary culprit behind candidiasis and primary fungal infections in both adult and pediatric populations³⁰. Candida albicans commonly resides as part of the normal microbial flora in the human oral cavity and typically remains harmless in individuals with a healthy immune system. However, in immunocompromised individuals, it can lead to disease, presenting with a spectrum of clinical symptoms ranging from mild to severe. This fungus is also present on the skin, mucous membranes, and in the genital and gastrointestinal tracts of humans³¹. In the oral cavity, Candida albicans primarily colonized sites such as the mucosa (including the buccal mucosa and palate), saliva, oral prosthetics, and dentures. The fungal overgrowth of Candida albicans in the oral cavity can lead to oral candidiasis, which is a fungal infection characterized by various oral manifestations³².

Oral candidiasis is a common condition referred to as oral thrush or sores including infections of the tongue and several parts of the mucosa in the oral cavity. Oral candidiasis is characterized by fungal overgrowth and invasion of superficial tissue^7,33. In addition, oral candidiasis can be associated with changes in other systemic conditions such as diabetes mellitus, hypoparathyroidism, and immunodeficiency diseases. Chronic cases of oral candidiasis can be found commonly in AIDS patients³⁴.

The human body has a natural defense against fungal infections. In the oral cavity there is saliva which contains histatin. Histatin, which is also supported by the process of exfoliating epithelial cells from mucosal surfaces, helps prevent fungal infections of Candida albicans. In a non-pathogenic state, Candida albicans attach inadequately to its host cells, but environmental factors in the oral cavity force this fungus to increase its ability to attach and become pathogenic. This ability to survive is called a virulence factor. These virulence factors include adhesion, hyphae formation, and biofilm formation³⁵.

The cell wall of Candida albicans composed of chitin, glucan, and mannoprotein³⁶. In its life cycle, there are three morphological forms, namely yeast, pseudohyphae and hyphae. These three forms will differentiate the morphology, function and growth potential of the fungus Candida albicans. The shape of this fungus is influenced by the host's environment. At low pH (<6) this fungus is found in the form of yeast, while at high pH (>7) this fungus is found to form hyphae³⁷.

Currently there is an increase in the use of herbal-based medicines, this is because herbal medicines have effectiveness that is not much different compared to chemical medicines, apart from that herbal medicines also have minimal side effects. Several studies have proven that herbal plants have anti-inflammatory, antibacterial, antifungal, antiviral, insecticidal, anticancer and cytotoxic effects. One of the plants used as a basic ingredient for herbal medicine is papaya. The parts of the papaya plant that are used are the papaya leaves and seeds.

The purpose of this study was to demonstrate the antifungal efficacy of a blend of extracts derived from papaya seeds and leaves (Carica papaya L). This was achieved by determining the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) against Candida albicans.

In this study, phytochemical tests were carried out and flavonoid levels were found to be 4.05%; saponin 5.08%; alkaloids 11.21%; polyphenols 4.82%; and triterpenoid 2.88%. This combination of papaya seeds and leaves produces much higher concentration levels compared to previous research by Nduche (2019) and Kingsely (2017)^22,38. Phytochemical research on papaya leaves by Nduche (2019) showed flavonoid levels of 0.67%; alkaloids 11.1% and saponins 0.53%. Phytochemical research on papaya seeds by Kingsley (2017) showed flavonoid levels of 0.49%; alkaloids 2.67% and saponins 0.35%. Nduche (2019) and Kingsely (2017) stated that the highest levels of flavonoids were found in the leaves of the papaya plant and the highest alkaloids were found in the papaya seeds, this is in accordance with the phytochemical results carried out by researchers^22,38.

To find out the MIC and MFC, a dilution test is carried out. The dilution test results of a combination of papaya seed and leaf extracts produced MIC at a concentration of 20% and MFC at a concentration of 40%. Previous research by Ameliala (2013) with concentrations of 100%, 50%, 25% and 12.5% showed that papaya leaf extract has MIC against Candida albicans at 50% concentration²³. Other research related to papaya seeds conducted by Dea (2016) with concentrations of 20%, 10% and 5% showed that papaya seed extract had the best inhibitory concentration against Candida albicans at a concentration of 20%²⁴. This shows that compared to Amelia's (2013) research, this research has a better MIC. This happens because the flavonoids, saponins and alkaloids contained in the combination of papaya seed and leaf extract have much higher levels, resulting in better inhibitory levels compared to leaf extract alone. However, compared with research by Dea (2016), it shows the same MIC. This can happen because the concentration interval used in Dea's (2016) research is different from that used in this research. Overall, the combination of papaya seed and leaf extract has better antifungal potential compared to leaf and seed extract alone.

Growth of Candida albicans which is inhibited is influenced by the contents contained in papaya seed and leaf extracts, namely flavonoids, saponins and alkaloids. Flavonoids are secondary metabolites of polyphenols found naturally in various fruits and vegetables³⁹. Flavonoids typically have a structural foundation composed of a chain of 15 carbons, linking two aromatic rings together through a 3-carbon bridge chains⁴⁰. Based on specific modifications of the central carbon ring, these flavonoids can be divided into discrete sub-groups. There are six subclasses, namely flavonols, flavanols, isoflavones, flavones, flavanones, and anthocyanidins⁴¹.

Flavonoids in conjunction with Candida albicans have the effect of damaging the cell plasma membrane. The flavonoid subclass, namely flavonols, contains compounds called quercetin and carvacrol. Both are flavonoid compounds which have activity as growth inhibitors of Candida albicans. Mechanism of action of quercetin in inhibiting growth of Candida albicans is by damaging the mannoproteins found in the cell walls of Candida albicans. Alkyl groups (methyl, ethyl, propyl and isopropyl) can increase growth inhibitory activity of Candida albicans because the alkyl group is an electron driver which can make the benzene ring more reactive in damaging mannoproteins in the cell walls of Candida albicans. Apart from that, the alkyl group makes quercetin more non-polar so it interacts more easily with the cell walls of Candida albicans which consists of carbohydrates, β-glucan, chitin and mannan. Candida albicans relies heavily on its cell walls to interact with its host. When the cell wall of Candida albicans is damaged, then growth becomes stunted⁴¹.

Saponins are a group of natural plant products that have many benefits. Saponins play a role in ecology including plant defense against diseases that attack them and herbivorous species. Some saponins are also important medicines for humans. The literature indicates that saponins exhibit various biological functions and medicinal properties, including but not limited to hemolytic activity, anti-inflammatory effects, antibacterial, antifungal, antiviral, insecticidal, anticancer, cytotoxic, and molluscicidal actions⁴².

Saponins are natural bioorganic compounds characterized by at least one glycosidic bond (C-O-sugar bond) at C-3, linking the aglycone to the sugar chain. Upon hydrolysis, saponins yield two components: the aglycone and the sugar moiety. Isolated saponins typically exist as solid amorphous substances with a high molecular weight, containing 27 to 30 carbon atoms in the non-saccharide portion. This non-saccharide segment, referred to as genin, sapogenin, or aglycone, constitutes the hydrocarbon skeleton devoid of a sugar chain. Based on the sapogenin type, saponins can be categorized into three primary classes: triterpenoid saponins, steroid saponins, and alkaloid saponins⁴³.

One of the saponin compounds is teasaponin⁴⁴. Teasaponin can inhibit hypha proliferation and formation of Candida albicans. Hyphal growth is characterized by the formation of specific genes, namely RAS1, ALS3, HWP1, CDC35, EFG1, ECE1. This saponin has been shown to downregulate this gene by decreasing CAMP⁴⁵.

Alkaloids represent a group of organic compounds comprising nitrogen bases, primarily produced as secondary metabolites within plants and fungi, exhibiting diverse biological activities. These compounds are commonly distributed throughout various plant components, including flowers, seeds, leaves, twigs, roots, and bark²⁵. Based on the presence of a basic heterocyclic core, alkaloids are divided into tropane alkaloids, pyrrolizidine alkaloids, piperidine alkaloids, quinolines alkaloids, isoquinoline alkaloids, indoline alkaloids, steroidal alkaloids, imidazole alkaloids, purine alkaloids, and pyrrolidine alkaloids⁴⁶.

Alkaloids can damage the cell membranes of Candida albicans. Damage to cell membranes causes leakage of micromolecules such as K ions+ and Na+, nucleic acids, and intercellular proteins. This causes the cells to lose their function and the cells will adapt to lysis or apoptosis. The impact of exposure to Candida albicans by alkaloids can be seen in an electron microscope which shows the shape of Candida albicans which shriveled, changed shape, and broke after being given saponin⁴⁷.

When compared with fluconazole 100 mg, the antifungal power of the combination of papaya leaf and seed extract is still lower, but in several aspects the combined extract of papaya leaves and seeds also has advantages, namely that it is a herbal ingredient that does not cause side effects and the raw material is easy to obtain.

CONCLUSION:

The combination of papaya leaf and seed extract can be used as an alternative ingredient for treating oral candidiasis and implemented in health products such as mouthwash, toothpaste, topical medicine and others.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 28.08.2024 Revised on 08.02.2025

Accepted on 11.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5655-5662.

DOI: 10.52711/0974-360X.2025.00817

Component	Concentration
Saponin	4,05 %
Flavonoid	5,08%
Alkaloid	11,21%
Triterpenoid	2,88%
Polyphenol	4,82%

Replication	Number of Colonies
Replication	K(+)	K(-)	P1(80%)	P2 (40%)	P3 (20%)	P4 (10%)	P5 (5%)	P6 (2.5%)
1.	-	165	-	-	12	22	142	157
2.	-	162	-	-	11	25	151	159
3.	-	168	-	-	12	26	154	163
4.	-	152	-	-	10	21	148	153
5.	-	161	-	-	9	24	152	158
6.	-	166	-	-	15	30	155	161
7.	-	169	-	-	16	24	150	159
8.	-	155	-	-	14	23	147	153

Treatment	Mean±SD	Significancy
Control (+)	-	p = 0,00*
Control (-)	162.25±6.09
P1 (80%)	-
P2 (40%)	-
P3 (20%)	12.38±2.45
P4 (10%)	24.38±2.77
P5 (5%)	149.88± 4.19
P6 (2.5%)	157.88 ± 3.52

Treatment	K(+)	K(-)	P1 (80%)	P2 (40%)	P3 (20%)	P4 (10%)	P5 (5%)	P6 (2.5%)
K (+)		0,000*	1,000	1,000	0,000*	0,000*	0,000*	0,000*
K (-)	0,000*		0,000*	0,000*	0,001*	0,001*	0,002*	0,103
P1 (80%)	1,000	0,000*		1,000	0,000*	0,000*	0,000*	0,000*
P2 (40%)	1,000	0,000*	1,000		0,000*	0,000*	0,000*	0,000*
P3 (20%)	0,000*	0,001*	0,000*	0,000*		0,001*	0,001*	0,001*
P4 (10%)	0,000*	0,001*	0,000*	0,000*	0,001*		0,001*	0,001*
P5 (5%)	0,000*	0,002*	0,000*	0,000*	0,001*	0,001*		0,003*
P6 (2.5%)	0,000*	0,103	0,000*	0,000*	0,001*	0,001*	0,003*