Carica papaya L. (Pawpaw) is one of the most commonly cultivated fruits in tropical and subtropical countries, especially in Nigeria. Nutritionally, Carica papaya is known to be highly nutritious. The effect of ripening on the antioxidant activity and phytochemical constituents (such as alkaloids, flavonoids, phenols, saponins, and tannins) of aqueous extract of two cultivars of Carica papaya (Local pawpaw and Agric pawpaw) was determined in this study. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was used to determine antioxidant activity, expressed as % DPPH scavenging activity. % DPPH scavenging activity significantly increased (p < 0.01) in both Carica papaya cultivars during ripening, with the overripe stage of both cultivars (Local pawpaw and Agric pawpaw) having the highest % DPPH scavenging activity. Phytochemical constituent analysis in Local pawpaw showed that alkaloids, flavonoids, phenols, and saponins increased during ripening with the overripe cultivar having the highest amount, while tannins decreased during ripening with the unripe having the highest amount. Agric pawpaw showed that alkaloids, flavonoids, and saponins increased during ripening with the overripe cultivar having the highest amount, while tannins and phenols decreased during ripening with the unripe cultivar having the highest amount. This result showed the important role of ripening stages in increasing the antioxidant activity of Carica papaya.
Research Article
Antioxidant Activity and Phytochemical Constituents of Pawpaw (Carica Papaya) During Ripening
https://doi.org/10.21203/rs.3.rs-4947309/v1
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Fruits
tropical
ripening
cultivar
antioxidant
Papaya fruit (Carica papaya L.) belongs to the Caricaceae family, and several species of Caricaceae have been used as remedies against various diseases [1, 2]. Papaya, originally derived from the southern part of Mexico, is a perennial plant distributed across tropical and subtropical areas. It is one of the most consumed fruits. The interior flesh of the fruit changes color from green (immature) to yellow (ripe) and to overripe [3]. A climacteric fruit, papaya exhibits a sudden burst of high respiration, causing the fruit to ripen within a few days [4]. Compared to other fruits such as bananas or apples, papaya is a good natural source of macronutrients (carbohydrates and proteins) and micronutrients (vitamin A and vitamin C) [5]. There is also a strong relationship between the intake of antioxidant-containing plants and reduced mortality from the aforementioned diseases [6]. Indeed, natural antioxidants warrant further scientific scrutiny given their activity against free radicals, which contribute to chronic degenerative diseases [7]. Medicinal plants play essential roles in preventing various diseases and have received much attention from many researchers over the last few decades. Studies on the antioxidant contents of fruits and vegetables are increasing because natural antioxidant consumption has been found to be related to a decreased risk for cancer and heart diseases [8]. Harvest time is essential to get a high-quality fruit with storage potential. Some physiological properties can be affected by cultivar, agronomic conditions, and maturity stage [9]. Papaya quality is affected by the ripening process [10]. Research shows that the antioxidant capacity of fruits is vital as a consequence of their short postharvest shelf life. Pawpaw fruit has different shapes; it may be ovoid-oblong, spherical, cylindrical, or pear-shaped or may have grooves [11]. Pawpaw fruit is a climacteric fruit and exhibits a characteristic rise in ethylene production during ripening, accompanied by fruit softening, color change, development of a strong aroma, and production of reducing sugar from polysaccharides. Genes associated with the development of fruit aroma during ripening have been identified in C. papaya [12]. The aim of this study was to determine the effect of ripening on the phenol, flavonoid, saponin, tannin, and alkaloid contents and the antioxidant activity (DPPH Radical Scavenging Activity) of Local pawpaw (pear-shaped variety) and Agric pawpaw (oblong variety) fruits growing in Nigeria. The aim of the study is to envisage the antioxidant property of pawpaw (Carica papaya) during various ripening stages to ascertain the effect of these properties on humans.
Chemicals and reagents were of analytical grade. Acetic Acid; Ammonium Hydroxide; Diethyl Ether; 2,2-Diphenyl-1-Picrylhydrazyl (DPPH); Ethanol; Hydrogen Chloride; Iron (III) Chloride; Methanol; n-butanol; Potassium Ferrocyanide were mostly product of Sigma Aldrich (USA).
Sample Collection
Two varieties of pawpaw (Carica papaya) fruits, oblong (Agric pawpaw) and pear-shaped (Local pawpaw), were purchased in their unripe green state from New Benin Market in Benin City. The two varieties of Carica papaya were identified and distinguished based on their shapes and local names by the Faculty of Agriculture, University of Benin. Samples were transferred to the Department of Biochemistry Laboratory, University of Benin, Benin City, where fruits were allowed to ripen normally at room temperature, and samples were collected from them in the unripe, ripe, and overripe stages for analyses.
Extraction procedures
Papayas were peeled, cut into 1 cm slices, and crushed in a food processor to produce uniform slurries using a Waring blender. The slurry was prepared fresh to preserve the extracted antioxidant compounds. Water extraction of papaya slurries was carried out by boiling 10 g of papaya slurries in a 1000 ml conical flask containing 100 ml distilled water and a magnetic stirrer (at 1000 rpm) for 30 minutes on a stirring hot plate (Fisher Scientific, Pittsburgh, PA). The extracts were then filtered using a clean muslin cloth and centrifuged using a tabletop centrifuge (MLX 210, Thermo-line, China) at 3000 rpm for 30 minutes. The supernatants were collected for further analysis.
DPPH Radical Scavenging Activity
The determination of antioxidant activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging system was carried out according to the method of Brand-Williams et al. [13]. 2.5 ml of Carica papaya extract and 7.5 ml of 0.3 mM DPPH methanol solution was incubated at 37°C for 30 minutes. For the negative control, only 2.5 ml of water was added to the DPPH solution. The absorbance of the solution was measured spectrophotometrically at 515 nm. The capability of sample to scavenge the DPPH radical was calculated according to the equation as follows:
Where A is the absorbance.
Quantitative Phytochemical Screening of Carica Papaya
The principal chemical groups contained in the extracts (saponins, alkaloids, phenols, tannins and flavonoids) were determined by confirmatory qualitative phytochemical screening of plant extracts using standard methods.
Test for saponins
Saponin was determined using the method described by Obadoni and Ochuko [14]. The papayas were ground, and 20 g of each were put into a conical flask, and 100 cm³ of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 hours with continuous stirring at about 55°C. The mixture was filtered, and the residue was re-extracted with another 200 ml of 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at about 90°C. The concentrate was transferred into a 250 ml separatory funnel, and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight; the saponin content was calculated as a percentage.
Test for alkaloids
Alkaloid determination was done using the method of Harborne [15]. 5 g of the papaya extract was weighed into a 250 ml beaker, and 200 ml of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 hours. This was filtered, and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extract until precipitation was completed. The whole solution was allowed to settle, and the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed.
Test for phenols
Phenol was determined by the spectrophotometric method. The fat-free papaya extract was boiled with 50 ml of ether for the extraction of the phenolic component for 15 minutes. 5 ml of the extract was pipetted into a 50ml flask, and then 10 ml of distilled water was added. 2 ml of ammonium hydroxide solution was added. The samples were made up to the mark and left to react for 30 minutes for color development. This was measured at 505 nm.
Test for tannins
Tannin was determined by using the method of Van-Burden and Robinson [16]. 500 mg of the papaya extract was weighed into a 50 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 hour in a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the mark. Then 5 ml of the filtrate was pipetted out into a test tube and mixed with 2 ml of 0.1 M FeCl3 in 0.1 M HCl and 0.008 M potassium ferrocyanide. The absorbance was measured at 120 nm within 10 minutes.
Test for flavonoids
The flavonoid content was determined by the gravimetric method of Boham and Kocipal-Abyazan [17]. 10 g of the papaya extract was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No. 42. The filtrate was later transferred into a crucible and evaporated to dryness over a water bath, and weighed to a constant weight.
Statistical Analysis
The data obtained in this study were expressed as Mean ± SEM. Significant differences between means were determined by one-way analysis of variance (ANOVA) with SPSS version 21. Tukey’s Multiple Range Comparison Test was used to compare the means. Significance level was set at p<0.01.
The results showed that the antioxidant activity and phytochemical constituents (% Dry weight) of Carica papaya varied with ripening stages in both cultivars (Table 1 and Table 2). The % DPPH scavenging activity significantly increased (p<0.01) during ripening. This suggests that ripening enhances the antioxidant properties of Carica papaya, making the overripe fruit potentially more beneficial for health in terms of antioxidant intake.
Table 1: Antioxidant activity and phytochemical constituents in Carica papaya fruit (pear-shaped variety: ‘Local pawpaw’) at various ripening stages
|
Pawpaw Variety |
Ripening stages |
||
|
Unripe |
Ripe |
Overripe |
|
|
Antioxidant (n=4) |
17.70±0.09a |
33.13±0.13b |
50.83±0.60c |
|
Tannins (n=3) |
7.68±0.07a |
6.36±0.08b |
5.87±0.07c |
|
Flavonoids (n=3) |
0.72±0.01a,b |
0.94±0.03a,b |
1.11±0.07b |
|
Phenols (n=3) |
0.30±0.01a |
0.32±0.01a |
0.35±0.02a |
|
Saponins (n=3) |
0.45±0.00a |
0.68±0.01b |
0.81±0.01c |
|
Alkaloids (n=3) |
0.02±0.00a |
0.02±0.00a |
0.06±0.00b |
Values were Mean ± SEM. aMeans in the same row followed by different letters are significantly different (p<0.01)
Table 2: Antioxidant activity and phytochemical constituents in Carica papaya fruit (oblong variety: ‘Agric pawpaw’) at various ripening stages
|
Pawpaw Variety |
Ripening stages |
||
|
Unripe |
Ripe |
Overripe |
|
|
Antioxidant (n=4) |
15.68±0.36a |
32.33±1.48b |
50.30±0.09c |
|
Tannins (n=3) |
9.27±0.38a |
7.08±0.52a,b |
6.51±0.02b |
|
Flavonoids (n=3) |
0.50±0.01a |
6.22±0.07b |
6.54±0.10b |
|
Phenols (n=3) |
0.50±0.01a |
0.30±0.00b |
0.15±0.01c |
|
Saponins (n=3) |
0.65±0.02a |
1.10±0.07b |
2.45±0.05c |
|
Alkaloids (n=3) |
0.00±0.00a |
0.05±0.00b |
0.27±0.01c |
Values were Mean ± SEM. aMeans in the same row followed by different letters are significantly different (p<0.01)
The antioxidant activity in Carica papaya fruit for both cultivars were presented in Figure 1 and Figure 2. The phytochemical constituents of Carica papaya fruit for both cultivars were also shown in Figure 3 and Figure 4.
The results of this study revealed significant changes in the antioxidant activity and phytochemical constituents of Carica papaya during different ripening stages for both Local pawpaw (pear-shaped variety) and Agric pawpaw (oblong variety) cultivars.
The % DPPH scavenging activity, a measure of antioxidant capacity, increased significantly (p < 0.01) in both cultivars as the fruits ripened. This increase indicates that the antioxidant properties of Carica papaya are enhanced during the ripening process. The overripe stage of both Local and Agric pawpaw exhibited the highest % DPPH scavenging activity, suggesting that the fully ripened fruits contain more potent antioxidants compared to the unripe and ripe stages [18]. This could be attributed to the breakdown of complex compounds into simpler phenolic compounds during ripening, which are known for their antioxidant properties [19]. Additionally, the enhanced antioxidant activity in the overripe stage could be due to the increased synthesis of secondary metabolites as the fruit matures, further contributing to its antioxidant potential [20].
For the Local pawpaw cultivar, the analysis of phytochemical constituents showed an increase in alkaloids, flavonoids, phenols, and saponins during ripening. The overripe Local pawpaw contained the highest amounts of these phytochemicals [21]. Alkaloids and flavonoids are known for their medicinal properties, including anti-inflammatory and antimicrobial activities, which could be enhanced in the overripe fruits [22]. Phenols are effective antioxidants that help in scavenging free radicals, thus contributing to the overall antioxidant capacity of the fruit [23]. Saponins have been associated with various health benefits, including cholesterol-lowering properties and immune-boosting effects [24]. The increase in these phytochemicals during ripening suggests that overripe Local pawpaw might offer more health benefits. On the contrary, tannin content decreased during ripening, with the unripe fruit having the highest tannin levels. Tannins, while possessing some antioxidant properties, can also have anti-nutritional effects by binding to proteins and reducing their digestibility [25]. Therefore, the reduction in tannin content during ripening could improve the nutritional quality of the fruit [26].
In the Agric pawpaw cultivar, alkaloids, flavonoids, and saponins increased during ripening, with the highest amounts found in the overripe fruits. This trend mirrors that of the Local pawpaw and suggests similar potential health benefits associated with the consumption of overripe Agric pawpaw [27]. However, phenols and tannins showed a different pattern; both decreased during ripening, with the unripe fruits having the highest levels. The decrease in phenol content during ripening in Agric pawpaw contrasts with the Local pawpaw, indicating possible varietal differences in the synthesis and breakdown of phenolic compounds during fruit maturation [28]. The reduction in tannin content aligns with that of the Local pawpaw, potentially improving the fruit's nutritional quality as it ripens [29].
The findings of this study highlight the importance of ripening stages in determining the antioxidant activity and phytochemical profile of Carica papaya. The increased antioxidant activity in overripe fruits suggests that consuming fully ripened or even overripe papaya may provide greater health benefits, particularly in terms of antioxidant intake [30]. Antioxidants play a crucial role in neutralizing free radicals, thereby reducing oxidative stress and the risk of chronic diseases such as cancer, cardiovascular diseases, and neurodegenerative disorders [31].
Furthermore, the varying phytochemical contents during ripening suggest that different stages of ripeness may offer distinct health benefits. While unripe fruits may have higher tannin content, ripe and overripe fruits provide a richer source of beneficial compounds such as alkaloids, flavonoids, phenols (in Local pawpaw), and saponins [32].
Future Research
Future research could explore the specific health benefits of consuming papaya at different ripening stages, considering the changes in antioxidant activity and phytochemical composition. Additionally, understanding the underlying biochemical processes that drive these changes during ripening could provide insights into optimizing the nutritional quality and health benefits of Carica papaya. This could lead to improved dietary recommendations and post-harvest handling practices to maximize the health benefits of this widely consumed fruit [33].
Ripening significantly affects the antioxidant activity and phytochemical constituents of Carica papaya. Both the Local and Agric pawpaw cultivars show increased % DPPH scavenging activity and varying changes in phytochemical contents with ripening. This highlights the potential health benefits of consuming ripe and overripe Carica papaya due to their higher antioxidant activities. Further studies could explore the implications of these findings for dietary recommendations and post-harvest handling of papaya fruits.
Acknowledgements
The authors are grateful to the Department of Biochemistry, University of Benin, Benin City for providing the facilities necessary to accomplish the desired aim of this research.
Author contribution
BK conceived the idea for this research and proposed the research design. BK, OOJ and IHE supervised the research and were major contributors to writing the original manuscript. BK, IHE, and ATO conducted the experiment, analyzed, and interpreted the research data. OOJ carried out a critical review of the revised manuscript and was a major contributor to editing it. All authors read and approved the final manuscript.
Funding
No funding available for this research.
Data availability
Data will be provided on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests
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No competing interests reported.