INTRODUCTION:

Pathogenic yeasts can infect mucous membranes included reproductive organs. Over the past 30 years, the prevalence of clinical opportunistic yeast infections has been significantly increased. Candida strains are the most common type of opportunistic yeast, capable of causing severe and even fatal infections in immunocompromised hosts¹. About more than 800 million people worldwide suffered or are suffering from fungal infections². Candida albicans are fungi found on the mucous membranes of the respiratory tract, digestive tract and female genitalia. These fungi are known to be the most dangerous of the pathogenic fungi. It is reported that 85-95% causes of vaginal discharge are Candida albicans. Vaginitis is a problem related to women's reproductive health³.

Pharmaceutical companies are interested in discovering novel medications from plants to heal microbiological infections since they are safe to use, less expensive, and more accessible to the general public^4–6. Plants with significant antimicrobial capabilities have already been documented⁷. Many studies state that therapeutic plants included a variety of phytochemical/phytotherapy ingredients have antibacterial, antifungal, antioxidant, and anticancer properties^5,8–10. Phytotherapy is defined as the therapeutic use of medicinal plants and their derivatives to prevent, relieve, or cure diseases¹¹. Young papaya seeds contain triterpenoid and saponin compounds which have potential as antimicrobial¹². The mean total tannins and phenols in young papaya seeds were higher than ripe papaya seeds^13–15. The goal of this study is to investigate the phytochemical composition of young papaya seeds and their inhibitory activity against Candida albicans, the fungal that causes vaginitis.

MATERIALS:

The materials used in this study were young papaya seeds from papaya (Carica papaya L.) Bangkok papaya varieties, 96% ethanol (solvent), Candida albicans fungi, aluminium foil, 0.9% NaCl, rotary evaporator (Buchi-R-210^®), autoclave (Presto^®), incubator (Memmert^®), hot plate (MS-H280-PRO^®), analytical balance (HWH^®), viscometer, refrigerator (Toshiba^®), blender (Philips^®), electric stirrer (Sharp^®), pH meter (Schott Instruments Lab 850^®), spirit lamp (Glass^®), clear glass jar, Biosafety cabinet, micropipette (Socorex^®), ose needle, pipette measuring, dropper pipette (Pyrex^®), Erlenmeyer 100 ml (Pyrex^®), petri dish (Glass^®), test tube (Pyrex^®), beaker 100 mL (Pyrex^®), beaker (Pyrex^®), Vortex, Erlenmeyer (Pyrex^®), Tube Rack, Roll set, stirring rod, mount, pycnometer, ruler, filler, slide, microscope and glass jar.

METHODS:

Sample Collection:

Samples of young papaya seeds were taken from Langgea village, Ranomeeto sub-district, Konawe Selatan district, Southeast Sulawesi province. Young papaya fruits were picked from the tree then split open and then taken the papaya seeds¹².

Extract preparation:

A total of 500grams of powdered young papaya seeds was macerated with 96% ethanol solution of 2000mL and the filtrate was taken by filtering, this process was carried out 3 times every 24 hours using a clear jar container. The filtrate was evaporated in a rotary evaporator at a temperature of 50°C^16–18.

Phytochemical Screening:

a. Alkaloid:

The extract was dissolved with 5mL of 2N HCl and the solution obtained was then put in a test tube. The tube was added by Dragendroff reagent as much as 3 drops. The formation of orange deposits in the tube indicated the presence of alkaloids^19,20.

b. Flavonoid:

Extract as much as 1 mL was added magnesium powder and 2 drops of HCl. A red solution was formed indicating the presence of flavonoids^19,20.

c. Tannin:

The extract was added with 1 mL of 10% Fe (III) chloride solution. If dark blue, blackish-blue or greenish-black colour formed, it indicated the presence of tannin compounds^19,20.

d. Saponin:

The extract was added to 1mL of hot water then cooled, shaken vigorously for 10 seconds. Formed steady foam for not less than 10 minutes as high as 1-10cm^19,20.

e. Terpenoid:

The terpenoid test was carried out by reacting the extract with 0.5mL of ethanol, 0.5mL of anhydrous acetic acid, and 2mL of concentrated sulfuric acid through the tube wall. The results were shown by the formation of green and blue (triterpenoids), and red or purple (steroids)^19,20.

Antifungal Activity Test of Ethanol Extract of Young Papaya Seeds:

The agar diffusion method was used to test antifungal activity²¹. This method was carried out in 2 stages, the first stage was making the basis of the test media using PDA media. A total of 10mL of sterile PDA media in a liquid state was poured into a sterile petri dish and then left for 15 minutes until it became solid. After the PDA media solidified, 4 storage devices were placed which functioned as mold holes or wells. The second stage was the manufacture of semi-solid indicator media (PDASP) by mixing 1mL of fungal suspension into 5mL of PDA media in the test tube then adding 5mL of thawed PDASP media, then homogenizing it with vortex so that the fungi are evenly distributed. The indicator medium was then poured into the petri dish around the pre-prepared storage device and then allowed to solidify. After the indicator medium had solidified, the buffer tool was then removed using sterile tweezers to form a hole (well) where the sample (ethanol extract from young papaya seeds) was inserted into each of the holes that are formed. Each well that was inserted 50μL of papaya seed samples which was dissolved in DMSO with concentrations of 1000μg/mL, 3000μg/mL, 6000 μg/mL, and 10000μg/mL respectively. Negative and positive controls were DMSO and Ketoconazole 2%. The repetition was done in triplo in the same way and incubated in incubator at 37^oC for 3x24 hours. Observed the zone of inhibition that occurred around the well then measured the diameter of the drag zone horizontally and vertically using a calliper²². Drag zone calculation formula:

The inhibition zone = The formed inhibition zone - the diameter of the wellbore

RESULTS AND DISCUSSION:

Extraction:

The samples of young papaya seeds were extracted using the maceration method with 96% ethanol as solvent. The selected ethanol was 96% because it contains 96% ethanol so that it would attract more compounds contained in young papaya seeds. By using 96% ethanol solvent, the tannin and phenol content in young papaya seeds was higher. Ethanol is universal solvent that is able to spray with large polarity from non-polar compounds to polar compounds²³. Maceration was carried out using 2000 mL of 96% ethanol for 3x24 hours, meaning that every 1x24 hours the residue and filtrate had to be separated and the solvent was changed. At this time, it was hoped that the solvent could gain as many compounds contained in the young papaya seeds as possible. The viscous extract was obtained by evaporating the solvent using Rotary Vacuum Evaporator to obtain thick brown extract. Based on the results of the evaporation process, 84.4grams of thick extract were obtained, with the yield of 16.88% of the ethanol extract of young papaya seeds.

Qualitative Phytochemical Screening:

The phytochemical screening of the extract aimed to determine the content of the compounds contained in the ethanol extract of papaya seeds. The classes of compounds tested in the study were flavonoids, alkaloids, tannins, saponins and terpenoids.

a b c

d e

Figure 1. Phytochemical screening with color reaction and precipitation methods, a. Flavonoids, b. Alkaloids, c. Tannins, d. Saponins, e.Terpenoids

Flavonoid test of the ethanol extract of papaya seeds, magnesium and HCl powder was used to produce a red solution which showed positive flavonoids and red deposits. For Alkaloid test of the ethanol extract of papaya seeds, Dragendorf reagent was used to produce orange colour which indicates positive alkaloids. Tannin test showed positive results, forming a blackish green colour when 1% FeCl₃ solution was dropped. Saponin test showed positive results, forming foam/froth for not less than 10 minutes as high as 1-10 cm. Steroid test shows positive results, namely forming a blackish-red color²⁴. The The findings of phytochemical screening revealed that the positive chemical compounds in papaya seeds were flavonoids, alkaloids, tannins, steroids and saponins²⁵.

Table 1. Phytochemical screening results of papaya seed ethanol extract (Carica papaya L.)

Compound	Phytochemical Screening
Compound	Color reaction / precipitation
Flavonoid	(+) yellowish red colour and red deposits
Alkaloid	(+) orange colour
Tanin	(+) blackish green colour
Saponin	(+) ± 3 cm foam for 10 minutes
Steroid	(+) blackish-red colour

The anti-fungal properties of young papaya seed extracts are obtained from several compounds, namely flavonoids, alkaloids, saponin tannins, and steroids. Flavonoids are the most abundant phenol group found in plants, and they have antifungal characteristics. The action mechanism of flavonoids as antifungal works by reducing the permeability of the fungus's cell wall membranes and extracellular proteins. Candida albicans^26,27.

Alkaloids work to damage the membrane by damaging the permeability of the cytoplasmic membrane. With the destruction of the permeability of the cytoplasmic membrane, the function of the membrane is disrupted, resulting in leakage of cell contents or the entry of unwanted substances into the cytoplasm, which will result in the death of the fungus²⁸.

Tannin is a polyphenol compound that can precipitate protein²⁹. If there is protein deposition in the cell wall and cytoplasm, the growth of Candida albicans cells will be disrupted and result in death in Candida albicans cells. Damage that occurs to the cell walls and cytoplasmic membranes caused by active substances in young papaya seeds, including unwanted foreign substances, enter the cells of Candida albicans. The compounds that pass through the cytoplasmic membrane will enter and hit other cell organelles such as protein membranes and mitochondria. This protein-membrane has enzyme activity such as manan synthase, chitin synthase and glucan synthase. This enzyme can catalyze the release of hydrogen from the substrate in the process of oxidation and reduction of the respiratory chain of cells in the mitochondria^29,30. If there is protein deposition in these organelles, it will cause damage to these cell organelles. The destruction of these cell organelles makes it easier for unwanted substances to enter the cell²⁹.

Saponins have soap-like properties, namely as strong surface-active compounds. Soap is a surface-active agent that can damage the permeability of the cytoplasmic membrane. The cytoplasmic membrane consists of proteins and fats which are susceptible to substances that reduce surface tension or surface-active agents³¹. The damage to the permeability of the cytoplasmic membrane allows the function of the membrane to regulate the passage of substances into and out of the cell to be disrupted, causing important organic ions such as nucleotides, coenzymes, and amino acids to seep out of the cell or the entry of unwanted substances into the cytoplasm, resulting in the death of fungal cells³². Saponin compounds have an inhibitory mechanism in which they build complex compounds with cell membranes via hydrogen bonds, destroying the permeability of the cell wall and ultimately leading to cell death³³.

Antifungal Activity Test of Young Papaya Seed Ethanol Extract Against Candida albicans:

The ethanol extract activity test of young papaya seeds was to determine activity against Candida albicans in order to see how much effect the extract had. Anti-fungal activity test was carried out using agar diffusion method (wells) because the sample in the well would diffuse on the medium that had been planted with fungi and the presence or absence of fungal growth could be observed through the clear zone formed around the well. The anti-fungal inhibition activity of young papaya seeds (Carica papaya L.) ethanol extract against Candida albicans can be seen in Figure 2.

The results of diameter measuring of inhibition area of ethanol extract of young papaya seeds (Carica papaya) are presented in Table 2.

In Figure 2 the results of the inhibitory power test showed that the extract of young papaya seeds was able to inhibit the growth of the Candida albicans which was characterized by the formation of a clear zone around the good hole. The results of measuring the diameter of the inhibition area of the ethanol extract of young papaya seeds were shown in Table 5. In this table, it could be seen that there was a change in the diameter of the inhibition area in each concentration from the lowest concentration to the high concentration. The data showed that the higher the concentration, the higher the inhibitory power, the diameter values of the inhibition area at concentrations of 0.1%, 0.3%, 0.6%, and 1% were 11mm, 12mm, 13mm, and 14mm respectively. Category of antifungal inhibitory power based on Davis and Stout's (1971) classification is as follows: The diameter of the inhibition zone is greater than 20mm, indicating that the inhibitory power is very strong, the diameter of the inhibition zone is 11-20mm means the inhibition is strong, the diameter of the zone 5-10mm of inhibition means moderate inhibition, the diameter of the inhibition zone is 0- 4 mm which means that inhibition potency is weak^34,35.

When testing the antifungal activity of the ethanol extract of young papaya seeds against Candida albicans, it showed antifungal activity in the range of 11-14mm which was categorized as strong activity. The strong antifungal activity of young papaya seeds is due to the chemical components contained in the extract of young papaya seeds showing many bioactive compounds such as flavonoids, alkaloids, saponins, and tannins. The average total tannins and phenols in young papaya seeds are higher than ripe papaya seeds¹⁵.

Tannins can form irreversible compound complexes with proline (a complete protein), where this bond has an inhibitory effect on protein synthesis for cell wall formation. Phenol works by damaging and penetrating the cell walls of Candida albicans as a result of which Candida albicans experiences damage to the cell walls. Saponins have the work of destroying the plasma membrane of fungi³⁶.

CONCLUSION:

Significant activity of Ethanol Extract of Young Papaya Seeds (Carica papaya L.) against Candida albicans sufficiently validate the medicinal uses of the plant. To conclude, the investigation of antifungal activity is required to be researched and updated as microbial drug resistance continues to endanger global health. Novel antimicrobial compounds can be derived from natural compounds like components of extract.

CONFLICT OF INTEREST:

The authors claim have no known competing financial or personal interests that could influence the findings of this study.

ACKNOWLEDGEMENTS:

The authors acknowledge their respective to dr. Mazuki Hanafi Bantayan, Rector of Institut Teknologi dan Kesehatan AVICENNA, Kemenristek-BRIN, and Universitas Halu Oleo for providing the financial support and research facilities.

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Received on 06.12.2020 Modified on 25.08.2021

Research J. Pharm. and Tech 2022; 15(9):3936-3940.

DOI: 10.52711/0974-360X.2022.00659

Fungi	Extract concentration (%)	Inhibition diameter (mm)			Inhibition zone average(mm)
Fungi	Extract concentration (%)	p1	p2	p3	Inhibition zone average(mm)
Candida albicans	0,1	11	11	11	11
	0,3	12	12	12	13
	0,6	13	13	13	13
	1	14	14	14	14
	Positive control	18	18	18	18
	Negative control	0	0	0	0