Antifungal Analysis of Papaya Seed Extracts and Biosynthesized Silver Nanoparticles

The purpose of research is to investigate the antifungal potential of papaya seed extract and the biosynthesis of silver nanoparticles by C. papaya extract. Here we report 14 secondary metabolites from C. papaya seed extract detected by gas chromatography-mass spectroscopy. Bioactive components were majority classified as fatty acid-methyl ester, heterocyclic amides, and phenolic compounds. The proposed mechanisms of those metabolites on inducing antifungal activity were comprehensively studied. We found that the n-hexane fraction was the solely fraction to produce the silver nanoparticles. Scanning electron images presented the aggregation and evenly distribution of spherical silver nanoparticles. The XRD exhibited the crystallization of the bio-organic synthetic phase based on the specific spectrum of 2-theta at 38.9 degree with miller index [1,1,1]. The particle size analyzer also confirmed the nanoscale of synthesized materials as in the average size of 92.1 nm (Z) and 0.406 (PI). Antifungal effects were examined by disc diffusion method upon each fraction with varying concentrations of 25%, 50%, 75%, and 100% (w/w) against C. albicans . ANOVA analysis showed no significant difference among all fractions tested (p > 0.25). The antifungal activity was categorized as a moderate effect with the mean of inhibition zone ranging from 6.1 to 6.8 mm. However, the potential of papaya seed extract is relatively better than the papaya leaves extract, as previously reported. We suggest further studies on the molecular docking of the secondary metabolites against nanoparticle, and specific biomolecular analysis according to the mechanism of action.


INTRODUCTION
Fungal infectious disease such as vulvovaginal candidiasis was often caused by C. albicans (Rathod and Bu er, 2014) . In some cases, C. albicans is also found at diaper dermatitis or diaper rash in infants and children (Carr et al., 2020;Irfanti et al., 2020). The existence of antifungal resistance encourages the development of new e ective antifungal drugs, minimum side e ects, and inexpensive ones (Farahyar et al., 2020) . The papaya plant (C. papaya) has been e cacious in traditional treatment for many years. The seed and leave extract of C. papaya using methanol, and aqueous solvents have been reported for its antibacterial activity (Peter et al., 2014) . The properties of papaya seed oil have been studied previously by the extraction with petroleum ether and aqueous enzymatic methods (Puangsri et al., 2005) . A study reported that the content of 2,3,4-trihydroxytoluene in methanolic extract of the papaya seeds had shown antifungal e ects on A. avus, C. albicans, and P. citrinium (Singh and Ali, 2011) . Although a new study by Siagian et al. (2021) showed that papaya Bangkok (C. papaya L. var. Bangkok) seed extract has no antifungal activity, unfortunately, this study had no report on the phytochemical compound of the methanol extract. In response to the negative result of an investigation by Siagian et al. (2021) , we are supposed to scrutinize the absence of the extraction methods that may degrade the bioactive components so that antifungal activity was undetected (Siagian et al., 2021) . The bioactive components play essential roles in the antifungal e ect, e.g., isolated avonoids from Citrus species against C. albicans (Salas et al., 2011) . The antifungal e ects, mechanism of action, and its combination with pharmaceutical drugs have been resumed by Al Aboody and Mickymaray (2020) .
However, we still nd a research gap on the antifungal e ect of C. papaya seed extracts and non-polar extract as a natural bioreduction for AgNPs synthesis. Several pieces of research have been reported on the AgNPs biosynthesis by using C. papaya peel extract (Kokila et al., 2016) papaya seed ethanol extract (Yani and Putri, 2019) . The current literature review on the phytochemical aspects, pharmacological insights, nanoparticle synthesis, commercial production, and utilization of C. papaya waste have been resumed by Sharma et al. (2020) . The most recent study on the phytochemical screening and antimicrobial activities of methanolic and aqueous leaf extracts of C. papaya was reported by Callixte et al. (2020) . To the best of our knowledge, the comprehensive evaluation of bioactive compounds attributed to antifungal e ect altogether with the use of the papaya seed extract to fabricate the AgNPs in the non-polar fraction is still not found. Therefore, this research is aimed to contribute a more comprehensive analysis of the papaya seed metabolites, AgNPs bio-synthesis, and their antifungal potential.

Methods
Papaya seeds (1.7 kg) were dried at 70°C then macerated with 10 L solvents for three days with ethanol, ethyl acetate, water, and n-hexane simultaneously with stirring one time per day. Crude extract obtained 48.192 g (2.83%) after being treated with a rotary evaporator and followed by fractionation with water, ethanol, n-hexane, and ethyl acetate. Papaya seed extract 5 g dissolved in 80 mL with water at 70°C and placed into a separating funnel. The graded fraction of the n-hexane, ethyl acetate, and ethanol was repeated three times. Then the secondary metabolites of all fractions were analyzed by GC-MS (Buckley, 1966;Suman et al., 2016). The AgNPs were synthesized by mixing 10 mL of AgNO 3 1 mmol solution and 200 L of each fraction of papaya seed extracts. The mixture was stirred for about 30 min until homogeneous. The formation of AgNPs was observed by SEM, XRD, and PSA (Banala et al., 2015;Devanesan et al., 2021;Lohrasbi et al., 2019). For SEM analysis, the silver samples were centrifuged at 10,000 rpm for 30 min and the pellet was redispersed in 10 mL ethanol and washed 3 times with sterile distilled water. The pellet was dried in an oven and thin lms of dried samples (10 mg/mL) were prepared. XRD analysis was conducted in 2-theta region at 40 kV and a current of 30 mA with Cu K-alpha radiation. Particle size distribution was obtained using ImageJ software version 1.47v.
The Sabouraud Dextrose Agar (SDA) media was poured aseptically and solidi ed to evaluate the antifungal activity. The cotton swab was used to scratch the C. albicans suspension on the surface of the SDA media. The disc was divided into four quadrants for 25%, 50%, 75%, and 100% of samples variation and label given outside the petri dish. A paper disc with AgNPs was placed on the marked part then incubated for 24h. The clear zone was measured on the paper disc (Davis and Stout, 1971) . Statistical analysis uses Kolmogorov-Smirnov to check the normality of data and the Levene method to check homogeneity. The one-way ANOVA was applied to evaluate the signi cance of each sample.

Secondary Metabolites Analysis
To detect the metabolite compounds in the papaya seed extract, we have analyzed each fraction; ethanol, ethyl acetate, and nhexane by GC-MS, as shown in Figure 1.
Based on Figure 1, most metabolites compounds were almost detected in the ethanol and ethyl acetate fractions. These results indicate that most metabolites vary from polar to semipolar even though some fatty acids are found in the non-polar solvent in small quantities. The major compounds in the EtOH fraction are benzyl nitrile, hexadecenoic acid, methyl ester and octadecenoic acid, methyl ester. Despite their small presence, the eugenol (Abdou et al., 2021) and acetamide (Thakral and Singh, 2019) compounds are widely known as antimicrobial. An isoeugenol showed its potential inhibitor for 14--demethylase and delta-14-sterol reductase that interfered with the biosynthesis of fungal cell membrane (Medeiros et al., 2020) . Another study found that eugenol's antifungal activity is related to membrane binding and permeability changes, which leads to plasma membrane destabilization and disruption  . Moreover, lipophilic acetamide derivatives have been reported as an antimicrobial agent via EGFR protein kinase enzyme and stable interactions were similar to the co-crystallized reference ligand (Ahmed et al., 2018). Benzylamine as a variety of heterocyclic amides have been reported as excellent therapeutic e cacy and remarkable application to treat various mycoses topically (Thakral and Singh, 2019) . Because of the molecule's lipophilic character, its e ectiveness could be correlated to the interactions with phospholipids in cell membranes and permeabilization of the fungal cell wall (Mingeot-Leclercq et al., 2001) as well as the direct membrane- damaging e ect of butena ne, a benzylamine derivative, which play a signi cant role on its anticandidal activity (Iwatani et al., 1993) . Fatty acid methyl esters (FAME) group has been reported for their antifungal activity against isolates of Paracoccidioides sp. (Pinto et al., 2017) . As one of FAME's compounds, Benzylcarbamic acid-methyl ester may also contribute to the antifungal e ect (Raghunath and Viswanathan, 2014) . Another FAME compound from S. portulacastrum reported the fatty acid composition and antimicrobial activity against human pathogenic microorganisms (Chandrasekaran et al., 2011) .
Phenol, Thiocyanic acids, and Heneicosane were dominantly found in the EtOAc fraction. Phenol is active against many micro-organisms, including some fungi and viruses. Phenol may cause a change in cell surface hydrophobicity and charge, resulting in cytoplasmic content leakage (Teodoro et al., 2015) . Carvacrol and other terpenoid phenols, such as carvacrol, have antifungal activity against a variety of infections, including C. albicans (Rao et al., 2010) . Ca eic acid derivatives may interfere with the 1,3-glucan synthase, which could have a deleterious e ect on the C. albicans cell wall. According to a literature study on the antifungal activity of herbal extracts C. albicans, phenolic components such gallic acid, thymol, catechin, and other polyphenols like tannins, terpenoids, and saponins, which were the primary component, are linked to the antifungal action. The incorporation of nanoparticles is also predicted to improve the antifungal properties of natural compounds (Hsu et al., 2021) . The presence of thiocyanic acids may be attributed to antifungal activity.
In contrast, under a transmission electron microscope, C. papaya seed extract against A. avus revealed abnormal cell shape and destruction of the fungus cell wall. Still, the detail of its mechanism was unexplained (Abd El-Zaher, 2014) . The most recent study on the antifungal activity of Raphanus raphanistrum extracts against Fusarium and Pythiaceae reported that thiocyanic acid-ethyl ester was responsible for the antifungal activities (Mannai et al., 2021) . In this report, Heneicosane has the highest intensity in the EtOAc fraction, related to the antifungal activity (Chandrasekaran et al., 2011) . Other studies reported Heneicosane and further FAME's compounds from C. papaya seed extract by subjecting heat treatment to the potent antibacterial e ect (Amid and Amid, 2021) , and Pinus knot wood extracts showed antimicrobial activity in its FAME's content (Välimaa et al., 2007) .
In this study, FAME's compounds were diversely found in the EtOH, EtOAc, and n-hexane fraction, named as dodecanoic acid, methyl ester; undecanoic acid, methyl ester; tetradecanoic acid, methyl ester; hexadecenoic acid, methyl/ethyl ester; octadecenoic acid, methyl ester; heneicosane; tricosanoic  acid, methyl ester; and eicosanoic acid even though FAME were easily found in non-polar solvents due to its solubility characteristicsA study reported that fatty acid methyl esters (FAME) were found inhibit 12 clinical strains of the pathogenic fungus P. brasiliensis (Alvarez-Peral et al., 2002) . The C. oxycantha extracts and detection of likely antifungal components also showed many FAME compounds with known antifungal potential e ects (Ra q et al., 2021) . The antifungal properties of hexadecenoic acid compounds have been reported by Chandrasekaran et al. (2011) but the mechanism of action requires further research. Moreover, Alkooranee et al. (2020) reported that octadecenoic, hexadecenoic acid, and octadecadienoic acid, methyl esters, exhibited bioactivity to several phytopathogenic fungi. Furthermore, undecanoic acid has a straight chain of saturated fatty acid found in body uids, plays a role in human metabolite, and is the most fungi toxic of the decadent acid series. However, the antifungal mechanisms of undecanoic acid are not much explored. According to recent research, undecanoic acid's detrimental e ect involves modifying fungal metabolism via its e ects on the expression of fungal genes required for virulence (Rossi et al., 2021) .
To resume the bioactive compounds that correlate with antifungal activities and the proposed antifungal mechanisms as described, Table 1. We obtained that classes of heterocyclic amides, phenolics, and fatty acid methyl esters have important role in the antifungal e ects of C. papaya extracts.

Biosynthesized Silver Nanoparticles
The n-hexane fraction was the solely fraction for the biosynthesis of AgNPs; this result may be due to the nucleation process of nanoparticles induced by metabolites content which play the role as capping agents and as natural reductor for the formation of Ag + ions into Ag 0 (Zulaicha et al., 2021) . Some studies also reported that the synthesis of nanoparticles depends on pH, temperature, extract concentration, metal salt concentration, and reaction time factors (Mittal et al., 2013;Shah et al., 2015).
The XRD pattern showed ve intense peaks in the 2 (deg) at 23.09(15), 38.90(5), 42.5(8), 64.1(8), and 65.9(2) indicated that crystalline silver nanoparticles produced by conversion of Ag + to Ag 0 . The phase formation of AgNPs was con rmed with XRD analysis. However, the biosynthesized AgNPs contains of some organic components obtained from papaya seed extract as the reducing agent. As a result, the XRD analysis exhibits a broad amorphous background peak and covers most of the characteristic peaks of Ag based on the PDF le #04-0783. Fortunately, the highest peak of AgNPs is appeared at 2 theta= 38.9 degree, indicating the AgNO 3 has been reduced to AgNPs during the biosynthesis process. These results conform to the bioorganic phase crystallization of AgNPs proposed by previous studies (Banala et al., 2015;Devanesan et al., 2021;Krishnaraj et al., 2012;Singh et al., 2021).

Antifungal Activity
All fractions exhibited their most inhibitory e ect at the concentration of 100%. ANOVA analysis showed no signi cant difference among all samples tested, whereas p-value > 0.25, and the antifungal activity ranged from 6.1 to 6.8 mm, categorized as moderate e ect (Davis and Stout, 1971) . The antifungal inhibition zone has four criteria based on the diameter size of the clear zone, as de ned that very strong (> 20 mm), strong (10-20 mm), moderate (5-10 mm), and weak (< 5 mm).
Studies on the ethanolic seed extract of C. papaya described the moderate level might due to the presence of the alkaloids, avonoids, steroids, polyphenols, tannins, and saponins as antimicrobial agents (Yani and Putri, 2019) .
The non-signi cant di erence of antifungal activity between samples has been suspected by the high temperature exposed to thermolabile extract components; this might bring the degradation of components (Dewi et al., 2019) , while another reason of similar resistance triggered by AgNPs (Panáček et al., 2018) .
The intermediate e ect of papaya seed extracts in all samples is considerably less e ective than commercial antifungal Fluconazole with inhibition zone > 20 mm (Kirkpatrick et al., 1998) . However, these results are relatively better than the papaya leaves extract by cipro oxacin (Callixte et al., 2020) . In contrast to the combined e ect of the AgNPs synthesized in polar solvents could enhance the antifungal activity as reported  (Hsu et al., 2021), it is interesting to have more investigation on the non-polar extract as bioreduction agents according to the antifungal e ectiveness. Based on Table 2, we also conclude that there was no signi cant e ect of vaiation of concentration of each sample tested (p > 0.05).
The complete pictures of disc di usion methods on the inhibition zone are shown in Figure 5.
The morphological observation of the antifungal e ect showed in Figure 6.

Antifungal Mechanisms
Research by Kim et al. (2009) described that silver nanoparticles disrupt the structure of cell membranes and destroy the membrane integrity, thereby disrupting the membrane potential. Multiple ion gradients across the cytoplasmic membrane help fungal cells maintain their membrane potential. For fungal viability and the release of glucose and trehalose, appropriate preservation of intracellular components is required. Trehalose can protect proteins and biological membranes from inactivation or denaturation caused by various stress conditions, including oxidative stress, drying, dehydration, heat, cold, and toxic agents (Alvarez-Peral et al., 2002;Elbein et al., 2003). Analysis of the release of glucose and trehalose during exposure to AgNPs succeeded in breaking the membrane permeability barrier, this possibly due to disruption of the lipid membrane bilayer. By the TEM analysis, Kim et al. (2009) demonstrated Figure 6. The Morphology Observation of Antifungal Activity under A Microscope the interaction between AgNPs and membrane structure in C. albicans cells; there was a signi cant change in the membrane during exposure to AgNPs, which was recognized by cell surface deformation and cell death. A ow cytometric examination of the cell cycle revealed that AgNPs resist the cell cycle in the G2/M phase of C. albicans, which helps to explain the physiological alterations in fungal cells caused by AgNPs. These data suggest that AgNPs could inhibit several cellular processes involved in average growth.

CONCLUSIONS
In this report, the n-hexane, ethyl acetate, water, and ethanol fractions were used to extract papaya seed. Although the AgNPs were created entirely with n-hexane, the all samples exhibit moderate e ects as antifungal agents. These results are relatively better than the papaya leaves extract by cipro oxacin, as previously reported by Callixte et al. (2020) . Abundant compositions of FAME's compound, phenolics, and heterocyclicamides might be in uenced the antifungal activity. However, there was no apparent di erences between all fractions, so the presence of AgNPs caused no substantial improvement of antifungal activity. The main concern for future research is the speci c interaction between biosynthesized nanoparticles with the complexities of secondary metabolites and biomolecular aspects.

ACKNOWLEDGMENT
We thank to Universitas Prima Indonesia for supporting lab facilities and technical works.