Antifungal Activity and Major Bioactive Compounds of Water Extract of Pangium edule Seed against Aspergillus flavus

Pangium edule seeds are widely used as spices in Southeast Asia in a fresh and fermented form and are reported to have active compounds for food preservation. However, scientific data on the active compounds of P. edule seed that can prevent the growth of toxigenic Aspergillus flavus have not been widely reported. This research subjected to determine the antifungal activity and identify the active compounds of water extract of old and fermented seed of P. edule against A. flavus. The water extract was compared to the extracts obtained by multilevel maceration using 50% ethanol, ethyl acetate, and n-hexane as solvents. Alkaloid, saponin, phenolic compound, flavonoid, triterpenoid, and glycoside were detected qualitatively in the crude extracts. The water extract showed the best activity to suppress the growth of A. flavus, determined by the agar dilution method, with the minimum inhibitory concentration (MIC) of 12.5 and 25 mg/mL for old and fermented seed, respectively. The water extracts showed a moderate toxicity with LC50 of 100-500 μg/mL, determined by the brine-shrimp toxicity test. After fractionation using 3 kDa molecular-weight (MW) cut-off ultrafiltration membrane, two fractions, i.e., fraction with MW < 3 kDa and >3 kDa, were obtained. The fraction with MW < 3 kDa showed the best antifungal activity with the MIC of 6.25 and 12.5 mg/mL for old and fermented seed, respectively. LC-MS/MS profile showed that different compounds belong to fatty acid, amino acid, glycoside, and peptide were found as major active compounds in the fractionated water extract. The principal compounds and partial least-square analysis, however, suggested that fatty acid and glycoside are responsible for the antifungal activity. Hence, this study concluded that the water extract of P. edule seed had promising antifungal activity against A. flavus which was due to presence of particular compounds belong to fatty acid and glycoside.


Introduction
Pangium edule is a traditional medicinal plant which its fruitseed is often used as spices in Southeast Asian countries, including Indonesia. The fresh, old, and fermented seeds are widely used in different culinary recipes. The fermentation process of P. edule seed usually done by buried the boiled P. edule seeds in the ground for 40 days covered with husk ashes [1]. After fermentation, the seeds turn to a darkbrown product with slightly mushy texture.
Some studies have shown that P. edule seeds contain glycoside, unsaturated fatty acids, tannins, phenolic compounds, and alkaloid which have an inhibitory effect on the microbial growth [2][3][4][5]. Traditionally, the Indonesian people use P. edule seed to prolong the shelf-life of fishery products and using it as a fermentation medium [6]. Fresh and fermented P. edule seeds that have also been reported can be used as potential preservative in fresh fish by inhibiting the growth of pathogenic and histamine-producing microorganisms in fish [2,7,8]. The preservation of fishery products with P. edule seed suggested that P. edule seed has potential antifungal activity.
Previous studies have been conducted to assess the antifungal activity of P. edule seed against some fungi [9,10]. Study using fresh seed showed that the water extract of fresh P. edule seed with concentrations of 20, 40, 60, 80, and 100% (v/v) could inhibit the growth of Rhizoctonia sp. The optimum inhibition was showed by 100% (v/v) water extract, with inhibition of 39.34%. This study also indicated that 30% (v/v) water extract of fresh P. edule seed could inhibit the growth of Cylindrocladium sp. [9]. Furthermore, another study reported that methanolic-extract of fresh P. edule seed with a concentration of 15% could optimally inhibit the growth of Aspergillus flavus [10]. However, the data on the antifungal activity and the identified active compounds of water extract of P. edule against A. flavus are still limited. A. flavus has been known as aflatoxin producer, a toxin that can affect the mechanism of the liver in humans, mammals, and poultry, thus becoming a factor causing liver cancer [11,12]. A. flavus that has been reported is also found in the preserved fishery products, such as semidried and salted fish products [13,14]. Hence, this research is aimed at determining the antifungal activity of water extract of old and fermented seed of P. edule against toxigenic A. flavus. The fractionated extracts by ultrafiltration membrane were also studied, and the major active compounds were identified by LC-MS/MS.

Sample Collection and Preparation.
Old (dark-brown color) P. edule seeds were collected from Padang Pariaman, Padang, West Sumatra. Fermented P. edule seeds were collected from Nganjuk, East Java. Both samples were collected in 2019. The collected seeds were peeled manually and airdried for 2 days. The dried seeds were then crushed to obtain powdered P. edule seed. The powdered sample were kept in plastic bags and transported to the laboratory for analysis.  [16], and carbohydrate content (by difference). Color analysis was performed using a Chromameter (Konica Minolta, CR-300).
2.3. Sample Extraction. The extraction process was carried out by multilevel maceration methods using water, 50% ethanol, ethyl acetate, and n-hexane as solvents. Seventy-five grams of both P. edule seed powders, i.e., old and fermented seed, was macerated using orbital shaker (New Brunswick, Germany) for 72 hours at a weight ratio of the sample, and the volume of solvent was 1 : 4. The extracts obtained from maceration processes were filtered using Whatman grade 1 filter paper and concentrated using vacuum rotary evaporator (Buchi, Switzerland) at high pressure at 50-60°C. The extracts that have been obtained then be stored in a freezer with a temperature of -15 to -20°C until it was used for analysis.
2.4. Qualitative Phytochemical Screening. The P. edule crude seed extracts were used to perform the phytochemical analysis. The various natural compounds present in the extracts were determined qualitatively using the methods for the detection of alkaloid, saponin, tannin, phenolic, flavonoid, triterpenoid, steroid, and glycoside [17,18].
2.5. Determination of Antifungal Activity. Pure culture of Aspergillus flavus was maintained on slant of potato dextrose agar (PDA, Oxoid, UK) at 4°C. Prior to the testing, A. flavus was subcultured by transferring a loop of cells onto slant of PDA medium and incubated for five days at 28°C, and the spores were harvested in sterilized saline solution (NaCl 0.85%) [19]. The crude extracts that have been obtained was dissolved in 100% dimethyl sulfoxide (DMSO, Merck, Germany) and prepared as stock solutions, and serial twofold dilutions were performed. The final concentrations of the extracts ranged from 8 to 250 mg/mL. The antifungal activity of the extracts against A. flavus was measured using the agar dilution method [20,21]. These extracts were diluted 1 : 10 in plates containing melted PDA medium to obtain the test concentrations from 0.8 to 25 mg/mL and a final DMSO concentration 10%. The harvested spores of A. flavus were then inoculated into these plates, after the medium had solidified, in 32 spots. The plates were incubated at 28°C for three days. Dimethyl sulfoxide (DMSO) 100% was tested as a negative control. The MIC value was read as the lowest concentration showing 100% growth inhibition. Each assay was conducted in three replicates.
2.6. Fractionation of Water Extract of P. edule Seed. Fractionation was performed according to Andayani et al. [22], with slight modification. The water extract was dissolved in DMSO to obtain an extract concentration of 500 mg/mL. The extract solution was then centrifuged (Hermle Z383-K, Germany) at 1600 × g for 30 minutes at room temperature and filtered using a glass microfiber filter (Whatman GF/A Glass Circles, 1.6 μm pore size, 110 mm, UK). Then, the extract solution was filtered again using nylon membrane filter (nylon 25 mm; 0.22 μm pore size, Aijiren, China). Each of 20 mL of extract solution obtained was fractionated using the 3 kDa MWCO (molecular weight cut off) ultrafiltration membrane (Amicon Ultra-15 3 K Centrifugal Filter Unit, Ultracel-3 regenerated cellulose membrane, Merck Millipore, USA) for 30 minutes at 3900 × g, 28°C. Two fractions were obtained, the fraction with molecular weight (MW) of <3 kDa and >3 kDa. Each fraction was adjusted to the final volume of 20 mL using DMSO. Antifungal activity was tested for the both fractions by agar dilution method. The fraction which had the best inhibitory activity was defined as the fraction that showed the lowest MIC [20,21]. International Journal of Food Science sample was performed on a C18 column (100 mm × 2:1 mm; 1.5 μm, Accucore C18, Thermo Fisher Scientific, USA). The mobile phase consisted of water containing 0.1% formic acid for solvent A and acetonitrile containing 0.1% formic acid for solvent B. The gradient elution was 0-1 min (5% B), 1-25 min (5-95% B), 25-28 min (95% B), and 28-30 min (5% B).
2.8. Brine Shrimp Lethality Assay. Artemia salina Leach eggs were hatched in artificial sea water by dissolving 27 g commercial sea-salt in 3 L of water. After 48 hours, the shrimps matured as nauplii and were ready for the assay. The brine shrimp lethality test was carried out on the water extract of P. edule seed using the standard procedure [23,24]. Ten milligrams of the extract was dissolved in 1 mL of water to give an extract concentration of 10 mg/mL as stock solution.
Concentrations of 1 mg/mL, 500 μg/mL, 100 μg/mL, and 10 μg/mL were prepared by making a serial dilution from the stock solution. A suspension of nauplii, containing 10 nauplii, was added into each test tube, and the number of dead nauplii was counted after 24 hours. Each assay was conducted in three replicates. Lethal concentration (LC 50 ) was determined using the probit analysis method by SPSS 24.
2.9. Statistical Analysis. Data generated from the antifungal activity analysis were processed with SPSS 24 to perform one-way analysis of variance (ANOVA) using Tukey's multiple comparisons. Differences were considered statistically significant when p < 0:05. The data were expressed by means ± standard deviation. The categorization and contribution of compounds identified by LC-MS/MS analysis were analyzed by principal component analysis (PCA) and partial least-square regression (PLSR) using XLSTAT.

Proximate and Color of Seed Powders and the Yield of
Extraction. The fermented P. edule seed showed higher moisture and protein content but lower in carbohydrate content (p < 0:05) than the old P. edule seed ( Table 1). The differences were most likely attributed to the fermentation process, where the breakdown of chemical components occurs naturally. Microorganisms that are involved in the spontaneous fermentation process of P. edule seed thought to have amylolytic properties, indicated by the decrease in carbohydrate content in the fermented seeds. A previous study reported that fresh P. edule seed contains about 52% water (wet basis), 16% fat, 18% carbohydrate, and 13% protein [8].
The L * value ranges between 0 and 100, with 0 pointing black/dark color and 100 pointing to white/light. The more positive the a * value, the sample tends to be red, while the more negative the a * value, the sample tends to be green. The more positive the b * value, then the sample tends to be yellow, while the more negative the b * value, the sample tends to be blue. As shown in Table 1, the color of P. edule seed powders had a low brightness intensity and was a mixture of red and blue since it had positive a * value and negative b * value. Brightness intensity in old P. edule seed (23.70) was significantly higher than that of fermented P. edule seed (18.92) (p < 0:05).
The P. edule seeds were peeled manually and air-dried for 2 days before the extraction which yielded 24% dry material. The dried seeds were then crushed to obtain powdered P. edule seed, and 75 g of both powdered seeds was extracted using the maceration method. The multilevel extraction process using the maceration method was chosen because this method can produce large amounts of extract and avoid chemical changes due to heating [25]. The extraction of old and fermented P. edule seed with water solvent resulted in the highest yield extract value, i.e., 16.71% and 19.67%, respectively ( Figure 1). However, the yield of ethyl acetate extract was relatively high due to the oil in P. edule seed was also extracted. Proximate analysis showed that the fat or oil content of old and fermented P. edule seed was relatively high, i.e., 42.38% and 41.83%, respectively. The oil in P. edule seed could be extracted when the seeds were macerated using ethyl acetate solvent since oil can dissolve in ethyl acetate. It causes the ethyl acetate extract not be able to fully dried by evaporative process.
Heruwati et al. [8] reported that the extract-yield of fresh P. edule seed using water, 50% ethanol, and n-hexane was 2.46%, 2.72%, and 0.54%, respectively. On the other hand, extraction of fermented P. edule seed using water, 50% ethanol, and n-hexane gave the yield of 7.72, 10.26, and 0.56%, respectively. It shows that the results of both studies showed same trend, i.e., the polar solvent gave high yield.

Qualitative Phytochemicals of Seed Extracts.
Alkaloid was present in water and ethanolic seed extracts, while tannin and steroid were absent in all P. edule crude seed extracts ( Table 2). All extracts contain saponin, phenolic compounds, flavonoid, and triterpenoid as well as glycosides. Phytochemicals are nonnutritive chemical compounds in plants that have protective or preventive properties against disease which are beneficial to human health [26].
These results have some similarities with previous study. Alkaloid was also found in crude extract of fermented P. edule seeds using water, 70% ethanolic, acetone, and nhexane as solvents, while saponin, flavonoid, and tannin 3 International Journal of Food Science were found in water and ethanolic extracts [27]. Triterpenoid and steroid were not found in all P. edule seed crude extracts. Furthermore, phenolic compounds and condensed-tannin in mature P. edule seed were found in water and ethanolic extracts as reported by Makagansa et al. [5]. The differences may be caused by the origin of P. edule seeds and the extraction process [28].

Antifungal Activity of Extracts and Fractionated Extract.
Detectable growth of A. flavus by agar-dilution method was observed after three days incubation on plate without extract (control), while with 25 mg/mL water extract of fermented P. edule seed the growth was apparently inhibited ( Figure 2). As shown in Figure 2, higher concentration of the P. edule crude seed extracts resulted in fewer and smaller visible mold spots on the plate, indicating that fewer fungal spores were able to germinate and 100% DMSO used as negative control showed no activity, indicated by visible growth of all mold spots (32 mold spots). Many studies have been used the agar dilution method to assess the antifungal activity of plant extracts. The main advantage of this method is provision of uniform and stable dispersion of extracts when they are incorporated into the agar medium. Another advantages of agar dilution method include the allowing of the qualitative and quantitative determinations to be carried out together; accurate determination of minimum inhibitory concentrations (MICs); allowing the determination of the antimicrobial resistance level, and potential to extend the antimicrobial concentration as far as required [29,30].
The result showed that the concentration of the P. edule crude seed extracts and the type of solvent affected the growth inhibition of A. flavus (Table 3). The growth of A. flavus was reduced with the increase of crude extract concentration, indicated by a decrease on the number of visible mold spots growing on the plate. This reduction, however, only occurred on the plates with water and 50% ethanolic extracts. No inhibition was observed on the plates with ethyl acetate and n-hexane extracts. The water extract of old and fermented seed was found as the two-best inhibitor to the growth of A. flavus (p < 0:05).
Furthermore, the fractions that were obtained from water extract of old and fermented seed, i.e., the fraction with MW of <3 kDa and MW of >3 kDa, also showed antifungal activity against A. flavus ( Table 3). The fraction with MW < 3 kDa showed a stronger antifungal activity than the fraction with MW > 3 kDa. This fraction also exhibited a relatively stronger antifungal activity compared to the unfractionated one, indicated by a lower concentration that was needed in achieving 100% inhibition.    International Journal of Food Science inhibition, was only determined for water extract and its fraction with MW < 3 kDa (Table 4). As shown in Table 3, apparent mold spots were still found on all plates of the other extracts in a concentration between 0.8 mg/mL and 25 mg/mL. The MIC 50 and MIC 90 , that indicated, respectively, ≥50% and ≥90% inhibition, were also determined to facilitate the comparison between antifungal activity levels of P. edule seed extracts [20,31]. As described in Table 4, the MIC shown by the water extract of old seed was lower than that of fermented seed. The fraction with MW of <3 kDa of old seed, however, showed the best inhibitory activity than the other samples (p < 0:05), indicated by the lowest MIC. The results are in line with the previous studies which indicated that P. edule seed extracted using polar solvents could inhibit the growth of A. flavus [10]. Some studies have shown that low MW compounds have a contribution to antifungal activity [32][33][34][35].

Identified Compounds in Fractionated Extract.
The antifungal compound profiles of fraction < 3 kDa of old and fermented seeds which found as the two-best inhibitor for A. flavus were analyzed using LC-MS/MS instrument ( Figure 3). Thirteen major compounds were detected on the chromatogram and identified based on the retention time and MS data (molecular mass, m/z value of the MS/MS fragments) ( Table 5). Based on the results of this analysis, the chemical profiles of fraction < 3 kDa of water extract of old and fermented P. edule seeds were identified as fatty acids (peaks 2, 3, and 6), amino acids (peaks 1, 4, 7, 8, and 9), glycoside (peak 5), and peptides (peaks 10-13). Furthermore, the description of the fraction < 3 kDa of water extract of old and fermented P. edule seeds towards the chromatogram peaks of the LC-MS/MS and its antifungal activity was visualized using principal component analysis (PCA) (Figure 4). F1 and F2 represented 99.72% of total data variance, with F1 covered for 78.73% of the data.  Numbers are means of three replications. Numbers followed by different letters in each row of P. edule seed show a significant difference (p < 0:05) within different extract concentration and also between control.

International Journal of Food Science
The PCA score plot discriminated OSE1 and OSE2 from other samples by separate cluster on the positive score value of F1, which showed the similar characteristics as antifungal activity (Figure 4(a)). The loading plot (Figure 4(b)) of PCA result showed that the most discriminatory constituents in OSE1 and OSE2 were peaks 2, 5, and 6 which presented in   International Journal of Food Science  7 International Journal of Food Science high amounts in OSE1 and OSE2 compared to in FSE1 and FSE2. Loading plot also showed that peaks 12 and 13 suggested as discriminant compounds in FSE1, while FSE2 formed a separate cluster with peaks 10 and 11 as discriminant compounds.
Contribution and correlation of each peak to the antifungal activity were described by variable importance in projection (VIP) scores and correlation coefficient (r) using partial least squares regression (PLSR) analysis. The result of PSLR indicated that peaks 2, 5, and 6 were contributed to the antifungal activity of water extract of old P. edule seed (VIP score > 1) and showed a positive correlation to antifungal activity (r > 0:90) (Figure 4(b)). Meanwhile, peaks 1, 3, 4, 7, 8, and 9 were contributed to the antifungal activity of water extract of fermented P. edule seed (VIP score > 1) but showed a negative correlation to antifungal activity (r < 0). Moreover, the result of PSLR also indicated that peaks 10, 11, 12, and 13 were not contributed to the antifungal activity of water extract of old and fermented P. edule seed. The different compounds present in the different extracts cannot be described by the result as shown in Table 2.
Based on the LC-MS/MS chromatogram in Figure 3, from the three peaks that contributed and are positively correlated on antifungal activity (peaks 2, 5, and 6), there were two peaks that were detected and identified in both water extract of old P. edule seed (OSE) and water extract of fermented P. edule seed (FSE), i.e., peaks 5 and 6 which were classified as glycoside and fatty acid. Peak 2 which classified as fatty acid was only detected and identified in water extract of old P. edule seed. This indicated that the peaks (compounds) responsible for the antifungal activity of the water extract of old P. edule seed are peaks 2, 5, and 6, while the peaks (compounds) responsible for the antifungal activity of the water extract of fermented P. edule seed are only peaks 5 and 6.
Overall, the PCA and PSLR analysis revealed that peptides were not contributed to the antifungal activity of the fraction < 3 kDa of water extract of old and fermented seeds. All of contributed peaks in this study classified as fatty acid, amino acid, and glycoside compounds (Table 5). Amino acids showed a negative correlation to the antifungal activity of the fraction < 3 kDa of water extract of old and fermented seeds, while fatty acids and glycoside showed a positive correlation to its antifungal activity. Therefore, both fatty acids and glycoside were compounds that are responsible for the antifungal activity of the fraction < 3 kDa of water extract of old and fermented P. edule seeds. Previous studies have been reported that fatty acids showed antifungal activity against pathogenic fungi, such as Candida albicans, Aspergillus flavus, and Rhizopus nigricans [36,37]. Fatty acids induce fungal cell inhibition through cell membrane which is a major target for these compounds. Fatty acids can increase the membrane fluidity and cause a generalized disorganization of the cell membrane. It leads to conformational changes in membrane proteins which will result in leakage of the intracellular components and cell death [38,39].
Moreover, the PCA and PSLR results also showed that glycosides contributed to antifungal activity of water extract of old and fermented P. edule seed. Glycosides have been reported to have antifungal activity against T. mentagrophytes, C. albicans, B. cinerea, A. alternata, A. flavus, Aspergillus niger, Aspergillus ochraseus, Aspergillus versicolor, Penicillium funiculosum, Rhizoctonia cerealis, and Trichoderma viride [40][41][42][43]. Zhang et al. [44] revealed that mechanism of antifungal properties of glycosides was damaging the plasma membrane and cause the leakage of cytoplasmic materials which leads to cell death.
The toxicity test of the seed water extracts against A. salina larvae has not been reported before, though Sudjana et al. [46] and Simanjuntak et al. [47] have described the toxicity of P. edule seed extract with other solvents. The methanolic and chloroform extracts of P. edule seed had LC 50 of 274.26 μg/mL and 916.13 μg/mL, respectively, while the nhexane extract was nontoxic since it had LC 50 > 1000 μg/mL [46]. Furthermore, the ethanolic extract of fresh P. edule seed was also considered as nontoxic against A. salina larvae (LC 50 > 1000 μg/mL) as reported by Simanjuntak et al. [47]. The differences in the LC 50 value can be due to different type of P. edule used which cause differences in the secondary metabolites produced. In addition, different extraction methods and solvents also affect the dissolved active compounds, causing differences in biological activities of same plant species [48,49].
Previous studies reported that LC 50 of the BSLT and the LD 50 of the acute oral toxicity assay in animal models has a positive correlation [45,50]. According to Parra's research, the brine shrimp LC 50 < 10 μg/mL has LD 50 100-1000 mg/kg; LC 50 < 20 μg/mL has LD 50 1000-2500 mg/kg; L C 50 > 25 μg/mL has LD 50 2500-8000 mg/kg [50]. We can assume that the LD 50 of oral acute toxicity for water extract International Journal of Food Science of old and fermented of P. edule seed also will be more than 2500 mg/kg, because the LC 50 of BSLT is 468 and 244 μg/mL, respectively. The chemical labeling and classification of acute systemic toxicity based on oral LD 50 are recommended by the Organization for Economic Co-operation and Development (OECD) as follows: ≤ 5 mg/kg, very toxic; 5-50 mg/kg, toxic; 50-500 mg/kg, harmful; and 500-2000 mg/kg, no label [51]. Hence, this finding showed that the water extract of P. edule seed could be developed as an antifungal or preservative agent since the oral LD 50 suggests that the extract is nontoxic.

Conclusions
This study found that the water extract fraction with MW < 3 kDa of old P. edule seed showed the best inhibitory activity against A. flavus, followed by the fraction with MW < 3 kDa of fermented seed. These fractions showed a stronger antifungal activity than the unfractionated water extract, indicated by the lower MIC. The responsible compounds for the antifungal activity of water extract of old and fermented seed were classified into the same group, i.e., fatty acid and glycoside. Purification and confirmation of the antifungal activity of the purified compound were included in the further study.

Data Availability
All datasets used to support the findings of this study are available upon reasonable request from the corresponding author.

Disclosure
The main results of this article have been published as a master thesis in IPB repository and have been approved to republish the data. International Journal of Food Science