Plant extracts were prepared with two solvents, distilled water and methanol ACS grade (cat. no. 6501-04, CAS no. 67-56-1; Anhui Fulltime Specialized Solvent & Reagent Co., Ltd.). Whole papayas with medium ripeness were used. All fruits were washed with distilled water, cut into small pieces, and dried until excess water was evaporated. A total of 75 g fruits were boiled with distilled water (1:2) for 30 min at 80˚C. The mixture was cooled and filtered with a Buchner funnel using Whatmann filter paper no. 1 three times to remove solid residue. The papaya extract was kept in a fridge at 4˚C until further experiments. For the methanol extraction, the fruits were separated and dried at 70˚C overnight. The dried fruit was ground to a soft powder using a blending machine. A total of 10 g fruits was weighed into a sterile Erlenmeyer flask, then 100 ml 70% methanol was added and left for 72 h at room temperature on the shaker. Filtered extract was evaporated at 40˚C using a rotary evaporator. The crude extract was stored at 4˚C for further processing (15). Bioactive compound analysis was conducted on both extracts using qualitative phytochemical tests, including phenolic, tannin, flavonoid, saponin, triterpenoid, steroid and alkaloid tests described by Ehiowemwenguan et al (16).

NP synthesis was conducted as described by Bayrami et al (17) and Dmochowska et al (18) with modifications. A total of ~20 ml papaya extract was diluted in 80 ml distilled water. Then, 6.42 mg zinc nitrate [Zn(NO₃)2.6H₂O, Sigma Aldrich; Merck KGaA; cat. no. 228737-100G] was added and stirred using a magnetic stirrer for 10 min. A total of 5 M NaOH was added until the pH reached 12. The solution was oven-dried at 60˚C for 1 h or until white precipitate was formed. The precipitate was rinsed with distilled water and ethanol (3:1) after supernatant was decanted. The pellet was centrifuged at 4,025 x g for 20 min at room temperature and incubated in an oven at 60˚C for 24 h. Then, using a crucible cup, the pellet was furnaced at 400˚C for 2 h, producing a white powder of NPs. The product was stored in a hermetic tube for testing and characterization.

The structure of synthesized ZnO NPs was determined by SEM analysis (JSM 6510 LA, JEOL Ltd.) using gold (99.9%) coating with sputtering for 90 sec at room temperature, and the size of particles was analyzed using ImageJ software version 1.53t (ImageJ.org). The optical absorption spectra of ZnO NPs were recorded using a UV-visible spectrophotometer (Shimadzu Corporation; cat. no. UV-1900) at a range of 200-600 nm. Diffraction patterns were determined by XRD at 1˚/min with two angles from 20 to 80˚ (Bruker D8 Advance). Cu-K radiation (1.54060 Å) was operated at 40 kV and 40 mA. The results of XRD were analyzed using OriginLab version 2023b (originlab.com) to identify the type, morphology and crystal size of the measured particles. The diffraction peak maximum was observed at the 101 plane and the crystallite size was determined using Scherrer's physical formula as follows: D=0.94 λ/βcosθ where D is crystallite size, is the X-ray wavelength, and is the full width at half the maximum of the peak (19). Functional groups and compound classes of papaya extract and synthesized ZnO NPs were identified using FTIR (Shimadzu Corporation; Prestige 21) at room temperature with frequencies of 400-4,000 cm^-1 (14).

Toxicity evaluations were conducted to identify the lethality of extract and ZnO NPs in fish embryos for 96 h. The mortality, abnormality, and hatching rate were assessed according to standard procedure by OECD Fish Embryo Acute Toxicity Test (FET) no. 236(20).

Adult wild-type zebrafish (n=30, 4-5 months, 0.4-0.6 g) purchased from a local breeder from Bogor, Indonesia were maintained under standard laboratory conditions. Zebrafish were maintained in a temperature-controlled room at 28̊C with a 14 h light/10 h dark cycle in 12 L tanks with aerator. The zebrafish were fed three times/day with commercial pellets. The eggs of zebrafish were obtained 4-5 h post-fertilization (hpf) from breeding adult fish in a 1:2 female: male ratio and analyzed under stereo microscope (magnification 1.575x) to separate the viable eggs. A total of ~20 viable embryos were transferred to each well of a 24-well plate with 2 ml zebrafish culture medium (5 mM NaCl; 0,17 mM KCl; 0,33 mM CaCl2; 0,33 mM MgSO4, Sigma Aldrich). ZnO NPs synthesized from distilled water [papaya aqueous extract (PAE); 0.01, 0.10, 1.00, 10.00 and 100.00 mg/l] and methanol extract [papaya methanolic extract (PME); 1.25, 2.50, 5.00, 10.00 and 20.00 mg/l] were dispersed in distilled water before being added to the wells. Negative controls (zebrafish culture medium) were used to compare with positive controls (3,4-dichloroaniline) and treated groups, while internal plate controls (also in zebrafish culture medium) were used for checking the quality of the embryosaccording to standard procedure (20). The embryos were incubated for 96 h at 27±1˚C and observed every 24 h for toxicity evaluation. Toxicity evaluation comprised mortality, malformation, and hatching rate. The mortality and hatching rate were expressed as the number of dead embryos or eggs hatched compared with the control group. Abnormalities were analyzed by observing coagulation of embryos, lack of somite formation, pericardial/cardial edema and non-detachment of the tail. The probit analysis were used to calculate the LC50 dosage which is a method to analyze the relationship between the test compound/treatment and the response (mortality) in a binominal manner (21), The zebrafish embryos used for toxicity evaluation were euthanized using excess clove oil >100 ppm (22). All research procedures were approved by Research Ethics Commission, Padjadjaran University (approval no. 1026/UN6.KEP/EC/2022).

A total of ~40 zebrafish larvae 96 hpf from each treatment (lethal concentration 50 (LC50) and untreated control group were used for total RNA isolation using Quick-RNA^™ MiniPrep Plus (Zymo Research Corp.), according to the manufacturer's instructions. The RNA was quantified using NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies Inc.). cDNA strand was then synthesized from total RNA templates using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The synthesized cDNA was stored at -20˚C for further experiments.

cDNA amplification was performed using SYBR Green Mastermix (GoTaq^® qPCR Master Mix; cat. no. A6001; Promega Corporation). Targeted genes associated with the immune response in zebrafish were IL-10, IL-1, and TNF-α. The housekeeping gene EF1α was used to normalize results. Primer sequences are listed in Table I. DNA amplification was performed according to the manufacturer's instructions using 5 µl 2X PCR premix, 1 µl primer mix, 2 µl nuclease-free water and 2 µl DNA template (nuclease-free water was used as negative control). Thermocycling conditions were as follows: Initial denaturation at 95˚C for 1 min, followed by 40 cycles (30 sec denaturation at 95˚C, 30 sec annealing at 58˚C and 45 sec extension at 72˚C ) and 1 min final extension at 60˚C. Amplification and quantification were performed with CFX96 Biorad system (Biorad) and Quantstudio^™ 1 RT-PCR, for analysis using QuantStudio Design and Analysis Software version 1.5.2 (Applied Biosystem, Thermo Fisher Scientific). Expression was calculated by normalizing Cq values of the target gene to the Cq value of the housekeeping gene (ΔCq) and normalized to untreated control (ΔCq untreated-ΔCq treated) (23).

Data are presented as the mean ± standard error of the mean and statistical significance of differences between groups was analyzed by performing one-way ANOVA followed by Tukey's post hoc test. For assessing mortality and hatching rate, a two-way ANOVA followed by Tukey's post hoc test with dosage was utilized. Data were obtained from three independent experiments. All the statistical analysis was performed using IBM Corp. SPSS Statistics 29.1 for Windows. P<0.05 was considered to indicate a statistically significant difference.

PAE and PME underwent preliminary phytochemical screening test to identify the bioactive compounds involved in synthesis of nanomaterials. Key phytochemical components of PME included phenolic compounds, tannins, saponins and triterpenoids, whereas PAE only contained triterpenoid (Table II).

ZnO NPs were synthesized using both PAE and PME, as corroborated by absorption bands at 387.4 and 364.0 nm using UV-Vis spectroscopy, respectively (Fig. 1A). Both bands are characteristic of ZnO NP (1,2). Furthermore, particles were also characterized using XRD, revealing typical hexagonal wurtzite structure of ZnO NPs based on the diffraction angles at 31.641, 34.291, 36.271, 47.481, 56.471, 62.801, 66.351, 67.711, 69.101 and 76.831, corresponding to the reflection planes of 110, 002, 101, 102, 110, 103, 200, 112, 201 and 202, respectively (Fig. 1B). The sharp and narrow diffraction peaks were in accordance with Joint Committee on Powder Diffraction Standards card no. 36-1451, which is a compiled database of diffraction patterns of various high quality powder, confirming the pure crystallite form of the ZnO hexagonal phase (wurtzite structure) (24). The diffraction peak maximum was observed at the 101 plane and. Mean crystallite size of ZnO NP PAE and PME was 13 and 12 nm, respectively. To visualize the structure of ZnO NPs, particles were also subjected to SEM imaging. ZnO NP synthesized had mean particle sizes of 198 and 152 nm for PAE and PME, respectively (Fig. 1C). The particles exhibited nanoflower morphology and surface structure, with only slight variations in thickness.

To identify the chemistry of the compounds from PAE and PME involved in the formation of ZnO NPs, analyses based on FTIR spectra were conducted (Fig. 1D). For PAE, the vibration bands were observed at 586.36, 777.31 and 817.82 (C-H), 1056.99 (C-O stretch of alcohols), 1409.96 (OH bend of phenol), 1622.13 (C=C stretching alkene), 2933.73 (C-H stretching of methylene) and 3423.65 cm^-1 (O-H stretching of alcohols). For PME, the bands were recorded at 514.99, 628.79, 777.31, 819.75 and 866.04 (C-H vibrations), 700.16 (C-C vibrations), 921.97 (-CH=CH₂ vinyl terminal), 1,076.28 and 1,265.30 (C-O stretch of alcohols and phenol), 1,436.97 (C-H bend of methylene), 1,635.64 (C=C stretching alkene), 2,854.65 and 2,924.09 (C-H stretching of methylene) and 3,410.15 cm^-1 (O-H stretching of alcohols). The functional groups identified by FTIR analysis were consistent with the phytochemical screening which indicated the presence of phenolic compounds, tannins, saponins and triterpenoids.

FTIR analysis of ZnO-PME revealed vibration bands at 422.41, 902.69, 1,382.96 and 1,620.21 (C=C stretching alkene), 2,372.44 (N-H component), 2,931.8 (C-H stretching of methylene) and 3,404.36 cm^-1 (O-H stretching of alcohols). For ZnO-PAE, bands were observed at 414.7, 896.9, 1,016.49, 1,382.96 and 1,622.13 (C=C stretching alkene), 2,368.59 (N-H component), 2,931.8 (C-H stretching of methylene) and 3,425.58 cm^-1 (O-H stretching of alcohols). The vibration bands between 400 and 600 cm^-1 were attributed to the Zn-O group due to vibration of Zn and O atoms in ZnO (25,26).

Healthy zebrafish embryos (6 hpf) were used to assess the toxicity in terms of mortality, hatching rate and malformation. Mortality is defined as the number of zebrafish embryos that died during observation, while hatching rate is defined as the number of zebrafish embryos that hatched from their chorion. Finally, malformation was considered to be a common abnormality that occurs in pericardial edema and yolk sac edema.

Mortality rate. The mortality rate of zebrafish exposed to ZnO NPs synthesized from PAE and PME was observed for 96 h (Fig. 2). Both types of ZnO NP showed a tendency for higher concentrations to cause significant mortality in zebrafish, with similar results for the positive control (3,4-dicholoroaniline) after 96 hpf. The highest concentration of ZnO NP PAE, 100 mg/l (Fig. 2A), led to the death of all zebrafish embryos at 96 h of observation. ZnO NP PME (Fig. 2B) at 20 mg/l showed mortality after 48 hpf. The concentration of 10 mg/l in both ZnO NPs showed a fairly high mortality, in which half of zebrafish embryos died. On the other hand, lower concentrations displayed similar results to the control group. For ZnO NP PAE, concentrations of 0.1 and 0.01 mg/l showed similar results at all time points. Similarly, the results obtained from the lowest concentration of ZnO NP PME which is 1.25 mg/l indicated similar results with that of the control group. The 96-h LC50 values estimated by probit analysis for ZnO NP PAE and PME were 8.246 and 6.568 mg/l, respectively.

Hatching rate. The hatching rate of zebrafish embryos was also investigated for 96 h. At 24 h, no embryos had hatched and the hatching rate significantly increased at 48 h (Fig. 3). In the control group, normal embryos hatched at 48-72 h. The hatching rate in all treatments using both ZnO NPs at low concentrations had the same results as the control group at 96 h of observation. (ZnO NP PAE, 0.10 and 0.01; PME: 1.25 and 2.50 mg/l; Fig. 3A and B, respectivey). The higher concentrations showed a significantly decreased hatching rate, with 100 PAE and 20 mg/l PME preventing all hatching. The concentration at 10 mg/l for both types of ZnO NP yielded a lower hatching rate compared with the positive control, showing that exposure to ZnO NP >10 mg/l led to inhibition of the development of the zebrafish embryo. ZnO NP exerted embryonic toxicity in a dose- and time-dependent manner.

Malformation. After exposure to ZnO NP PAE and PME, larvae showed several abnormalities or morphological alterations typical of metal oxide NP-induced toxicity, such as coagulation of the embryo and pericardial edema (20). Fish embryo acute toxicity tests have four core endpoints: i) Coagulation of fertilized eggs; ii) lack of somite formation; iii) non-detachment of the tail bud and iv) lack of heartbeat (20). Zebrafish embryos exposed to 20 mg/l ZnO NP PAE and PME showed coagulation after 48 hpf (Fig. 4A). Coagulation occurred at 24 hpf (Fig. 4A), indicating early death, and in later developmental stages, where the general development was delayed and the body typically started coagulating from the tail and the yolk sac.

In addition to the endpoints, other observations were recorded as lethal or sublethal endpoints (27). Malformations were identified (arrows) at the spine and sac yolk compared with the control group (Fig. 4B, C and F) following treatment with 5 mg/l ZnO NP PME and 10 mg/l ZnO NP PME and PAE, indicating a toxic effect on embryonic development. Zebrafish embryos exhibited decreased eye size and the formation of pericardial edema at 72 hpf following exposure to 10 mg/l ZnO NP PME (Fig. 4D). However, lower concentrations of ZnO NP PAE and 1.25 and 2.5 mg/l ZnO NP PME showed no significant difference compared with the control (Fig. 4E).

LC50 of synthesized ZnO NPs upregulated transcripts of IL-1 and -10 and TNF-α (Fig. 5). TNF-α and IL-1 and -10 mRNA expression levels in whole zebrafish larvae were stable in controls but changed following exposure to 8.246 mg/l ZnO NP PAE and 6.568 mg/l PME for 96 h. The proinflammatory cytokine TNF-α mRNA expression levels following ZnO NP PME exposure were higher than following ZnO NP PAE exposure, while the highest IL-1 mRNA expression was observed following exposure to ZnO-PAE. IL-10 mRNA expression, an anti-inflammatory cytokine, was observed to be upregulated after exposure to both ZnO NP PAE and PME. This may indicate an immunomodulatory response after exposure to ZnO NPs (28).