A DPPH· Kinetic Approach on the Antioxidant Activity of Various Parts and Ripening Levels of Papaya (Carica papaya L.) Ethanolic Extracts

Papaya fruits (Carica papaya L.) are valuable both as food, including concentrates and mixed beverages and in traditional medicine. The goal of the study was to evaluate the antioxidant activity of various parts of unripe and ripe papaya fruit from the DPPH· kinetics point of view. Peel, pulp, seed, and seed-pulp of unripe and ripe papaya fruits (¼ and >¾ level of ripening) were extracted with ethanol and monitored at 517 nm in the presence of DPPH·. The radical scavenging capacity (RSC) at various time ranges and DPPH· reaction rates for specific time intervals were determined. The highest RSC values were obtained for papaya pulp extracts, consistently higher for the ripe samples in comparison with the unripe ones (86.4% and 41.3%). The DPPH· rates significantly differ for the unripe and ripe papaya extracts, especially for the first time range. They are more than double for the ripe papaya. These values were 2.70, 4.00, 3.25, 2.75 μM/s for the peel, pulp, seed, seed-pulp extracts from the ripe papaya and only 1.00, 1.65, 1.40, 1.80 μM/s for the unripe samples. DPPH· kinetic approach can be useful for a fast and simple evaluation of the overall antioxidant properties of fruit extracts.


Introduction
Papaya is the generic name of the tree and fruit of Carica papaya L., which belongs to the genus Carica, botanical family Caricaceae. It originates from Central and South America but is cultivated in all tropical regions. Papaya had various traditional and ethnic applications, both in the food and medicinal fields, such as culinary (cooked unripe fruit, raw ripe papaya, salads, savory dishes), non-alcoholic and alcoholic beverages (papaya concentrates, mixed beverages, papaya wine), food ingredients (papaya extracts for instant paya fruit at levels of 0.31-0.80, 0.15-1.2/1.7, and 63-73.7/25.1-58.6 mg/100 g dry sample (Hawaiian/Mexican), respectively [26,27].
The main goal of the study was to emphasize the DPPH· kinetics on the discrimination between papaya ethanolic extracts, especially regarding the level of ripening. This approach was for the first time performed for such extracts and can be useful as a fast and simple evaluation method of the overall antioxidant properties of fruit extracts.

Radical Scavenging Capacity of Unripe and Ripe Papaya Extracts
Papaya fruit contains various compounds having antioxidant properties, which were widely studied (e.g., ascorbic acid, myricetin from flavonoid class, or cinnamic acid derivatives) [1,5,[24][25][26][27]. The present study was only focused on the overall antioxidant activity of ethanolic extracts obtained from unripe and ripe papaya parts, using the DPPH· method. The free radical DPPH· has a maximum absorbance at 517 nm, where no significant absorbance appears for other antioxidant compounds (or other biologically active compounds) from fruit extracts [33,[36][37][38]. Consequently, the monitoring of the absorbance of the mixture of DPPH· solution and papaya extract allows evaluating the RSC of the last through the reaction of DPPH· radical with the antioxidant compounds from the extract. The absorbance of the reaction products (neutral DPPH-H and degradation compounds resulted from the antioxidant compounds from the extract) is not significant at the measuring wavelength (517 nm). They present absorbance at lower wavelength values than 450 nm, according to previous studies [36][37][38]. As a result of the consumption of DPPH· during the interaction with the papaya extract, the absorbance of the mixture at 517 nm linearly decreases with the increase of the RSC, according to Equation (1) (see Section 3.3).
Representative RSC values for papaya extracts were obtained at the beginning of the antioxidant activity determination (after 5 s of DPPH·-antioxidant compound reaction), as well as at 1, 5, and 15 min of reaction (Table 1, Figures S1 and S2). The RSC versus Time plot differs for the papaya peel extracts in comparison with pulp, seed, and seed-pulp extracts, for both unripe and ripe cases (see also Tables S1-S4 for the p-values). The RSC values have Plants 2021, 10, 1679 4 of 14 a slower increase for the peel extracts. This behavior can be observed by comparison of the RSC values at the beginning and the end of the antioxidant activity evaluation. They are more than sixteen times higher at 15 min in comparison with values at 5 s in the case of peel extract from the unripe papaya (code Lu). For comparison, the RSC value for the peel extract from the ripe papaya (code Lr) is almost two times higher at the end of the analysis. In all other cases, RSC values are less than 1.3 times higher at the end of the analysis, in comparison with the beginning of antioxidant activity determination (1.17-1.37 and 1.02-1.26 times higher for unripe and ripe cases, respectively). However, the overall RSC data after 15 min of analysis reveals the highest values for all extracts from the ripe papaya parts, especially for the pulp extract (86.44 and 68.1% for the ripe and unripe papaya pulp extracts, respectively; p < 0.02). The differences are also statistically significant for the other papaya extracts (Table S4): 68.10 and 39.95% for the ripe and unripe papaya peel extracts (p < 0.075), 74.76 and 32.76% for the ripe and unripe papaya seed extracts (p < 0.05), 60.66 and 41.05% for the ripe and unripe papaya seed-pulp extracts (p < 0.10), respectively. These differences are more obvious at the intermediate time intervals, such as at 5 s (p < 0.04 for all cases, especially for the ripe and unripe papaya pulp extracts, p < 0.005, Table S1), as well as at 1 min (p < 0.09, Table S2). These RSC values were compared with those obtained for standard compounds such as propyl gallate (PG), caffeic acid (CA), or tert-butylated hydroxyanisole (BHA). Ethanolic solutions of standard compounds at various concentrations were subjected to the same DPPH· technique and RCS values at the same time ranges were recorded (according to the work of Ivanovici et al. [37] and Table S5). The RSC values for the ripe papaya pulp extracts are similar to those obtained for PG solution at a concentration of 0.2 mM (RSC of 83.32% at 15 min, Table S5). On the other hand, the corresponding extract obtained from unripe papaya pulp has similar RSC values to the 0.1 mM PG solution (RSC of 44.86%). Moreover, the extracts obtained from the unripe papaya pulp from the seeds region (code SPu, RSC of 41.05%) have similar antioxidant activity behavior such as the same 0.1 mM PG solution. This behavior suggests the involvement of similar antioxidant structures in the DPPH· radical scavenging (i.e., myricetin from papaya and the standard compound propyl gallate, Figure 1). The monitoring of the antioxidant activity was also performed for other fruit extracts such as pomegranate or kiwi [36,38]. The highest RSC values in the case of pomegranate extracts were obtained for red and white peel ethanolic extracts (48.94-64.99% after 15 min; Table S6), while for kiwi fruit the best results were obtained for the peel extracts obtained using 60% ethanol solution (84.1% after 15 min; Table S7). Galang et al. [2] evaluated the antioxidant activity of the methanol-water fractionated extracts obtained from unripe C. papaya L. and compared with ascorbic acid as the standard antioxidant compound. The RSC values after 30 min were almost as high as for the standard compound in the case of the last fractions (five fractions of a total of twelve having RSC values of 91-96%, in comparison with ascorbic acid, with an RSC value of 98%). An increase of the antioxidant activity with the ripening of papaya fruit was also observed [7,39]. The antioxidant activity of papaya seeds extracts was evaluated for various extraction techniques, i.e., subcritical water extraction (SWE) and conventional Soxhlet technique [22]. The antioxidant activity was expressed as the DPPH· concentration required to reduce the absorbance by 50%, EC50. It was observed that the increase of extraction temperature in SWE from 70 to 150 °C determines the decrease of the EC50 values from 4.1 to 1.67 μg/mL, while the conventional technique provided an intermediate value of 3.74 μg/mL. Higher values for the DPPH· inhibitory concentration 50% of papaya seeds methanol extracts (1.0 mg/mL) were obtained by Salla et al. [23]. In another study performed by Zhou et al. [40], the best results on antioxidant activity of papaya seed extracts were obtained if more hydrophobic solvents were used. Thus, ethyl acetate provided the best results and some phenolic compounds such as p-hydroxybenzoic and vanillic acids were isolated and identified in these extracts (as antioxidant compounds among already known ones, such as tocopherols). In conclusion to these comparisons, the present study reveals similar antioxidant activities with some of the above-mentioned extracts. Thus, ripe papaya peel and pomegranate white peel extracts have similar antioxidant activity behavior. The same observation can be done for the ripe papaya pulp and kiwi peel extracts. This behavior suggests the presence of the same type of antioxidants in these extracts, which react with DPPH· radical.
Further, the monitoring of the antioxidant activity through the DPPH· kinetics of radical scavenging of papaya extracts was studied.

DPPH· Reaction Kinetics in the Presence of the Unripe and Ripe Papaya Extracts
The reaction of the stable radical DPPH· with various antioxidant compounds from the papaya extracts is presented in Figure 2. Antioxidant compounds such as ascorbic acid, ferulic acid, and myricetin react with DPPH· in order to generate the neutral compound DPPH-H (2,2-diphenyl-1-pycrylhydrazine) and other radical intermediates, which are further transformed in neutral degradation compounds [41,42]. Only DPPH· radical has significant absorbance at 517 nm, while the neutral compounds do not absorb in this region. The maximum absorbance of DPPH· is shifted to a much lower value after these The monitoring of the antioxidant activity was also performed for other fruit extracts such as pomegranate or kiwi [36,38]. The highest RSC values in the case of pomegranate extracts were obtained for red and white peel ethanolic extracts (48.94-64.99% after 15 min; Table S6), while for kiwi fruit the best results were obtained for the peel extracts obtained using 60% ethanol solution (84.1% after 15 min; Table S7). Galang et al. [2] evaluated the antioxidant activity of the methanol-water fractionated extracts obtained from unripe C. papaya L. and compared with ascorbic acid as the standard antioxidant compound. The RSC values after 30 min were almost as high as for the standard compound in the case of the last fractions (five fractions of a total of twelve having RSC values of 91-96%, in comparison with ascorbic acid, with an RSC value of 98%). An increase of the antioxidant activity with the ripening of papaya fruit was also observed [7,39]. The antioxidant activity of papaya seeds extracts was evaluated for various extraction techniques, i.e., subcritical water extraction (SWE) and conventional Soxhlet technique [22]. The antioxidant activity was expressed as the DPPH· concentration required to reduce the absorbance by 50%, EC 50 . It was observed that the increase of extraction temperature in SWE from 70 to 150 • C determines the decrease of the EC 50 values from 4.1 to 1.67 µg/mL, while the conventional technique provided an intermediate value of 3.74 µg/mL. Higher values for the DPPH· inhibitory concentration 50% of papaya seeds methanol extracts (1.0 mg/mL) were obtained by Salla et al. [23]. In another study performed by Zhou et al. [40], the best results on antioxidant activity of papaya seed extracts were obtained if more hydrophobic solvents were used. Thus, ethyl acetate provided the best results and some phenolic compounds such as p-hydroxybenzoic and vanillic acids were isolated and identified in these extracts (as antioxidant compounds among already known ones, such as tocopherols). In conclusion to these comparisons, the present study reveals similar antioxidant activities with some of the above-mentioned extracts. Thus, ripe papaya peel and pomegranate white peel extracts have similar antioxidant activity behavior. The same observation can be done for the ripe papaya pulp and kiwi peel extracts. This behavior suggests the presence of the same type of antioxidants in these extracts, which react with DPPH· radical.
Further, the monitoring of the antioxidant activity through the DPPH· kinetics of radical scavenging of papaya extracts was studied.

DPPH· Reaction Kinetics in the Presence of the Unripe and Ripe Papaya Extracts
The reaction of the stable radical DPPH· with various antioxidant compounds from the papaya extracts is presented in Figure 2. Antioxidant compounds such as ascorbic acid, ferulic acid, and myricetin react with DPPH· in order to generate the neutral compound DPPH-H (2,2-diphenyl-1-pycrylhydrazine) and other radical intermediates, which are further transformed in neutral degradation compounds [41,42]. Only DPPH· radical has significant absorbance at 517 nm, while the neutral compounds do not absorb in this region. The maximum absorbance of DPPH· is shifted to a much lower value after these reactions (<450 nm [41,42]). On the other hand, papaya extracts do not have significant absorbance at 517 nm (e.g., ascorbic acid has a maximum absorbance in the UV region at 243 nm, ferulic Plants 2021, 10, 1679 6 of 14 acid at 291 and 321 nm, while myricetin has a maximum absorbance at 328 nm [43][44][45]). Consequently, the monitoring of the absorbance at 517 nm for the DPPH· and papaya extract mixture allows to determine the actual DPPH· concentration. Considering the variation of DPPH· concentration in a specific time range, the overall mean DPPH· reaction rate can be calculated (Equations (2) and (3); see Section 3.4).
Plants 2021, 10, x FOR PEER REVIEW 6 of 14 reactions (<450 nm [41,42]). On the other hand, papaya extracts do not have significant absorbance at 517 nm (e.g., ascorbic acid has a maximum absorbance in the UV region at 243 nm, ferulic acid at 291 and 321 nm, while myricetin has a maximum absorbance at 328 nm [43][44][45]). Consequently, the monitoring of the absorbance at 517 nm for the DPPH· and papaya extract mixture allows to determine the actual DPPH· concentration. Considering the variation of DPPH· concentration in a specific time range, the overall mean DPPH· reaction rate can be calculated (Equations (2) and (3); see Section 3.4).
Generally, the reaction of DPPH· with the antioxidant compounds from papaya fruit extracts exhibits a decreasing of the absorbance at 517 nm that has three specific time ranges: 0-30 s, where the antioxidant compounds with higher reactivity interact with DPPH· (e.g., ascorbic acid), 30-80 s and 80-900 s, corresponding to DPPH· reaction with antioxidant compounds having lower reactivity (e.g., compounds with hindered hydroxyl groups such as in the case of ferulic acid or flavonoids, as well as the synthetic standard compound, BHA). These regions are also revealed in the Conc. (DPPH·) versus Time plots ( Figures S3-S10). Correlations on these regions provide the mean variation of DPPH· concentration (decrease) on the specific time range, which means the overall mean DPPH· reaction rates (vmean(1-3), Equation (3), Figure 3). All DPPH· reaction rates, expressed as μM/s, are presented in Table 2. Generally, the reaction of DPPH· with the antioxidant compounds from papaya fruit extracts exhibits a decreasing of the absorbance at 517 nm that has three specific time ranges: 0-30 s, where the antioxidant compounds with higher reactivity interact with DPPH· (e.g., ascorbic acid), 30-80 s and 80-900 s, corresponding to DPPH· reaction with antioxidant compounds having lower reactivity (e.g., compounds with hindered hydroxyl groups such as in the case of ferulic acid or flavonoids, as well as the synthetic standard compound, BHA). These regions are also revealed in the Conc. (DPPH·) versus Time plots (Figures S3-S10). Correlations on these regions provide the mean variation of DPPH· concentration (decrease) on the specific time range, which means the overall mean DPPH· reaction rates (v mean(1-3) , Equation (3), Figure 3). All DPPH· reaction rates, expressed as µM/s, are presented in Table 2.
Plants 2021, 10, x FOR PEER REVIEW 6 of 14 reactions (<450 nm [41,42]). On the other hand, papaya extracts do not have significant absorbance at 517 nm (e.g., ascorbic acid has a maximum absorbance in the UV region at 243 nm, ferulic acid at 291 and 321 nm, while myricetin has a maximum absorbance at 328 nm [43][44][45]). Consequently, the monitoring of the absorbance at 517 nm for the DPPH· and papaya extract mixture allows to determine the actual DPPH· concentration. Considering the variation of DPPH· concentration in a specific time range, the overall mean DPPH· reaction rate can be calculated (Equations (2) and (3); see Section 3.4). Generally, the reaction of DPPH· with the antioxidant compounds from papaya fruit extracts exhibits a decreasing of the absorbance at 517 nm that has three specific time ranges: 0-30 s, where the antioxidant compounds with higher reactivity interact with DPPH· (e.g., ascorbic acid), 30-80 s and 80-900 s, corresponding to DPPH· reaction with antioxidant compounds having lower reactivity (e.g., compounds with hindered hydroxyl groups such as in the case of ferulic acid or flavonoids, as well as the synthetic standard compound, BHA). These regions are also revealed in the Conc. (DPPH·) versus Time plots (Figures S3-S10). Correlations on these regions provide the mean variation of DPPH· concentration (decrease) on the specific time range, which means the overall mean DPPH· reaction rates (vmean(1-3), Equation (3), Figure 3). All DPPH· reaction rates, expressed as μM/s, are presented in Table 2      There are significant differences between the DPPH· reaction rate values in the first time range. Thus, v 1 is 1.53-2.70 times higher in the case of the ripe papaya extracts, in comparison with the corresponding unripe fruit extracts, the highest difference being obtained for the peel extracts (2.70 µM/s for the ripe papaya peel extract and only 1.00 µM/s for the unripe case, Table 2). Slightly lower differences were also obtained for pulp and seed extracts (4.00 µM/s and 3.25 µM/s for the ripe papaya extracts, 1.65 µM/s and 1.40 µM/s for the corresponding unripe fruit extracts, respectively; Table 2). However, the absolute DPPH· reaction rates are higher in these last cases. Differences between DPPH· reaction rate values for the unripe and ripe papaya extracts are statistically significant in all cases. P-values were lower than 0.043 (except for SPu and SPr cases, p < 0.098), with the lowest value for the papaya pulp extracts (p < 0.016; see Tables S8-S10 in Supplementary Material File for all p-values). On the contrary, DPPH· reaction rates for the last two time ranges were not relevant for the differentiation between the unripe and ripe papaya extracts. The mean reaction rate in the second time range was five to twelve times lower (<0.34 µM/s) than the corresponding v 1 values, while v 3 was much lower (values of 0.001-0.050 µM/s). Kinetic results on the papaya fruit extracts are in agreement with the kinetic data for both standard antioxidant compounds and other fruit extracts [36][37][38]. Regarding the standard antioxidant compounds such as PG, CA, and BHA, the DPPH· reaction rates on similar time ranges were in the same region. Thus, the highest v 1 values were obtained for CA and PG solutions at concentrations of 1 mM (2.50 and 1.90 µM/s, but for the time range of 0-60 s, respectively; Table S11), which are close to the v 1 values for the ripe papaya extracts. On the other hand, unripe papaya extracts have similar kinetic behavior with the standard compounds PG, CA, and BHA at concentrations of 0.1, 0.2/0.1, and 1.0 mM, respectively (1.20-1.30 µM/s, Table S11). Higher v 1 values, which are close to the corresponding DPPH· reaction rate values for the ripe papaya extracts, were also obtained for other fruit extracts, such as pomegranate red and white peel and pulp extracts (2.43-3.03 µM/s for the time range of 0-30 s, Table S12) [38].

Materials
Papaya (Carica papaya L.) fruits were purchased from the local market (Timişoara, Romania) in the autumn of 2019. Fruits were imported from South America and two types of papaya fruits were selected, according to the ripening level: unripe papaya fruit with 1 4 level of ripening and ripe papaya fruit with > 3 4 level of ripening ( Figure 4). The level of ripening was evaluated according to the works of Calvache et al. and Ikram et al. [1,5], the unripe samples having a maturity stage of "2" (25-50% yellow surface, surrounded by light green), while the ripe papaya had a maturity stage of "4" (>75% yellow surface). The fresh samples (two fruits for every type of papaya) were manually separated into four parts: peel (the exterior of the fruit, codes "Lu" and "Lr" for the unripe and ripe papaya), pulp (the main core part, without the seeds region, codes "Pu" and "Pr" for the unripe and ripe fruit), seed (without the surrounding pulp, codes "Su" and "Sr" for the unripe and ripe fruit) and seed-pulp (the pulp from the seeds region, codes "SPu" and "SPr" for the unripe and ripe papaya). Papaya samples were stored at 4 • C until extraction. A representative image for these parts of papaya fruit used in this study is presented in Figure 4.
¼ level of ripening and ripe papaya fruit with >¾ level of ripening (Figure 4). The level of ripening was evaluated according to the works of Calvache et al. and Ikram et al. [1,5], the unripe samples having a maturity stage of "2" (25-50% yellow surface, surrounded by light green), while the ripe papaya had a maturity stage of "4" (>75% yellow surface). The fresh samples (two fruits for every type of papaya) were manually separated into four parts: peel (the exterior of the fruit, codes "Lu" and "Lr" for the unripe and ripe papaya), pulp (the main core part, without the seeds region, codes "Pu" and "Pr" for the unripe and ripe fruit), seed (without the surrounding pulp, codes "Su" and "Sr" for the unripe and ripe fruit) and seed-pulp (the pulp from the seeds region, codes "SPu" and "SPr" for the unripe and ripe papaya). Papaya samples were stored at 4 °C until extraction. A representative image for these parts of papaya fruit used in this study is presented in Figure  4.

Figure 4.
Representative image for the papaya parts used in this study: peel (the exterior of the fruit, codes "Lu" and "Lr" for the unripe and ripe papaya), pulp (the main core part, without the seeds region, codes "Pu" and "Pr" for the unripe and ripe fruit), seed (without the surrounding pulp, codes "Su" and "Sr" for the unripe and ripe fruit) and seed-pulp (the pulp from the seeds region, codes "SPu" and "SPr" for the unripe and ripe papaya).

Obtaining Papaya Fruit Extracts
The extracts of papaya fruit were obtained by solid-liquid extraction using mild conditions for protecting antioxidant compounds against degradation. Thus, the fresh samples were well ground in a mortar at room temperature and 5.0 g were weighed for every extraction. The extraction was performed in a 150 mL sealed flask in the presence of 20 mL ethanol 96% by intermittent agitation at 25 (±1) °C for 120 h. The extract was then filtered and the solid residue was washed with ethanol. The filtrate was diluted with ethanol at a final volume of 25 mL for the antioxidant activity evaluation and kinetic studies. Extracts were obtained as duplicates from two different fruits for every type of papaya, unripe and ripe (four parts: peel, pulp, seeds, and pulp-seeds). All extracts were made in parallel: 2 × 4 extracts for unripe papaya (two unripe fruits, maturity stage of "2") and 2 × 4 extracts for ripe papaya (two ripe fruits, maturity stage of "4"). For some cases, spectrophotometric monitoring cannot be performed up to the end of measurements (i.e., sample duplicates of Lu and Pr cannot be included in the evaluation; see Table 1).

Figure 4.
Representative image for the papaya parts used in this study: peel (the exterior of the fruit, codes "Lu" and "Lr" for the unripe and ripe papaya), pulp (the main core part, without the seeds region, codes "Pu" and "Pr" for the unripe and ripe fruit), seed (without the surrounding pulp, codes "Su" and "Sr" for the unripe and ripe fruit) and seed-pulp (the pulp from the seeds region, codes "SPu" and "SPr" for the unripe and ripe papaya).

Obtaining Papaya Fruit Extracts
The extracts of papaya fruit were obtained by solid-liquid extraction using mild conditions for protecting antioxidant compounds against degradation. Thus, the fresh samples were well ground in a mortar at room temperature and 5.0 g were weighed for every extraction. The extraction was performed in a 150 mL sealed flask in the presence of 20 mL ethanol 96% by intermittent agitation at 25 (±1) • C for 120 h. The extract was then filtered and the solid residue was washed with ethanol. The filtrate was diluted with ethanol at a final volume of 25 mL for the antioxidant activity evaluation and kinetic studies. Extracts were obtained as duplicates from two different fruits for every type of papaya, unripe and ripe (four parts: peel, pulp, seeds, and pulp-seeds). All extracts were made in parallel: 2 × 4 extracts for unripe papaya (two unripe fruits, maturity stage of "2") and 2 × 4 extracts for ripe papaya (two ripe fruits, maturity stage of "4"). For some cases, spectrophotometric monitoring cannot be performed up to the end of measurements (i.e., sample duplicates of Lu and Pr cannot be included in the evaluation; see Table 1).

Evaluation of the Antioxidant Activity by DPPH· Method
The antioxidant activity was evaluated through the radical scavenging capacity (RSC) of the papaya extracts in the presence of DPPH· [36][37][38]. The absorbance of a mixture of 0.5 mL papaya extract, 0.5 mL 0.1 mM DPPH· ethanolic solution, and 2 mL ethanol was monitored for 900 s at 517 nm in a 10 mm length quartz cuvette, using a CamSpec M501 single-beam Scanning UV-Visible spectrophotometer (CamSpec Ltd., Cambridge, United Kingdom). The "Time Scan" module was used. Actual RSC t values were obtained using Equation (1), where A t and A 0 stand for the absorbance of the papaya extract and DPPH· solution at the reaction time t and t = 0, respectively (Equation (1)).

Evaluation of the DPPH· Reaction Rates
The actual DPPH· concentration was determined by means of a calibration curve obtained for DPPH· solutions in the range of 0-300 µM. The following DPPH· calibration curve was obtained (Equation (2)).

Evaluation of the Antioxidant Activity by DPPH· Method
The antioxidant activity was evaluated through the radical scavenging capacity (RSC) of the papaya extracts in the presence of DPPH· [36][37][38]. The absorbance of a mixture of 0.5 mL papaya extract, 0.5 mL 0.1 mM DPPH· ethanolic solution, and 2 mL ethanol was monitored for 900 s at 517 nm in a 10 mm length quartz cuvette, using a CamSpec M501 single-beam Scanning UV-Visible spectrophotometer (CamSpec Ltd., Cambridge, United Kingdom). The "Time Scan" module was used. Actual RSCt values were obtained using Equation (1), where At and A0 stand for the absorbance of the papaya extract and DPPH· solution at the reaction time t and t = 0, respectively (Equation (1)). (1)

Evaluation of the DPPH· Reaction Rates
The actual DPPH· concentration was determined by means of a calibration curve obtained for DPPH· solutions in the range of 0-300 μM. The following DPPH· calibration curve was obtained (Equation (2)).

Statistical Analysis
RSC and DPPH· reaction rate values are expressed as mean (±standard deviation, SD). Correlations between dependent and independent variables were performed by linear regression analysis (DPPH· concentration and reaction rate as dependent variables, as well as absorbance or time as independent variables). Linear regression analysis also provides standard error values for regression coefficients, standard error of estimate (s), correlation and determination coefficients (r 2 and r 2 adjusted ), Fisher (F), and p-values. Significant differences of the values for the RSC of unripe and ripe papaya, various parts, were obtained by Fisher LSD (least significant difference) test. The parametrization was sigmarestricted, while the confidence limit and significance level were set at 0.95 and 0.05, respectively. The one-way ANOVA and Multiple Linear Regression modules in Statistica 7.1 software (StatSoft, Inc., Tulsa, OK, USA) were used for statistical analysis.

Conclusions
A DPPH· kinetic approach for discriminating between the unripe and ripe papaya (Carica papaya L.) fruit ethanolic extracts was proposed for the first time. The radical scavenging capacity clearly differs for the ripe and unripe papaya extracts for all studied fruit parts, i.e., peel, pulp, seed, and pulp from the seed region. By far, the highest antioxidant activity was observed for the ripe papaya pulp extract, which is 1.2-1.4 times higher than the other ripe papaya extracts at the end of antioxidant activity monitoring. Comparing these radical scavenging capacity values for the ripe and unripe papaya extracts, the values for the first cases are consistently higher (1.5-2.3 times higher for the ripe cases, with the highest difference for the extracts of the seeds). The DPPH· reaction rates are significantly different in the case of interaction with the antioxidant compounds from the ripe and unripe papaya extracts for the beginning of the reaction. The highest overall mean DPPH· reaction rates were obtained in the case of the ripe papaya pulp extracts. They can be compared with the DPPH· reaction behavior of standard antioxidant compounds resembling those occurring in the papaya fruit (according to other studies; the quantification of specific antioxidant compounds was not the goal of the present study). These similarities were obtained for gallate and hydroxycinnamic acid moieties, which resembles the antioxidant flavonoids (e.g., myricetin) and caffeic/ferulic acids found in papaya fruit. In conclusion, kinetics approach on the first time range of DPPH· reaction can be useful for a fast and simple evaluation of the overall antioxidant properties of fruit extracts designed for food, pharmaceutical or cosmetic applications (such as nonalcoholic and alcoholic beverages, food supplements, antimicrobial, anti-inflammatory or antioxidant products, anti-aging, anti-acne, and natural skincare products) as well as for the differentiation and evaluation of the level of ripening of papaya used for obtaining the hydrophilic extracts.