Influence of Drying Treatments on Polyphenolic Contents and Antioxidant Properties of Raw and Ripe Papaya (Carica papaya L.)

Abstract

Studies were undertaken to evaluate the effects of drying treatments (oven and freeze drying) on polyphenols and antioxidant compounds of raw (green) and ripe papaya extracted with ethanol and distilled water. The total phenolics varied between 21.1 and 217.3 mg GAE equivalent/g (fresh weight). Freeze-dried, ripened papaya extracts exhibited significantly higher tannin (9.58 and 6.0 mg CAE/g fresh weight) and flavonoid levels (8.40 and 3.64 mg CAE/g fresh weight) in ethanolic and aqueous extracts, respectively. Additionally, freeze-dried, ripened papaya showed higher inhibition of DPPH (91.8 and 93.0%) and ABTS radicals (87.1 and 97.5% fresh weight) in ethanolic and aqueous extracts, respectively. Similar results were observed for total antioxidant capacity, wherein freeze-dried, ripened papaya extracts exhibited higher levels (1315 and 2167 mg AAE/g fresh weight, respectively). As enhanced antioxidant activities were observed in raw and ripened papaya on freeze drying, it can be further explored for producing papaya–based, value-added products to benefit both consumers and producers.

Keywords:

• Papaya
• Phenolic compounds
• Antioxidant activity
• Raw papaya
• Ripe papaya
• Freeze drying
• Oven drying

INTRODUCTION

Fresh fruits encompass rich amounts of bioactive and nutraceutically valued compounds, which renders potential health benefits. Epidemiological data available have shown the effectiveness of consuming fresh fruits and their juice on overcoming certain degenerative diseases including cancer, cardiovascular diseases, aging, arthritis, and others.[1,2]

The papaya plant (Carica papaya, Caricaceae) produces fruits throughout the year and is widely cultivated in the tropical regions of the world that include: India, Bangladesh, Malaysia, Australia, Indonesia, Philippines, Sri Lanka, South Africa, and parts of tropical America.[3] In Malaysia, papaya fruit is highly popular among the general population and is consumed on a daily basis as a part of the normal basal diet. Also, Malaysia is one of the main exporting countries of papaya to the EU market.[4] Ripe papaya fruits are widely consumed for their unique taste as well as for their high nutritional value (vitamins A and C, minerals, carotenoids, and sugars). Ripe papaya is also used as an ingredient in jellies, purees, fruit bars, and for preparation of sweet dishes or desserts. Consumption of ripe papaya fruit or their juice is believed to overcome certain digestive disorders, relieve colon infections (attributed to the presence of papain enzyme), and is recommended as a remedy for food poisoning. In traditional medicines, the juice is reported to be effective for treating warts, cancer, tumors, and indurations of the skin.[5]

Similar to ripe papaya, even raw papaya (green or unripe papaya) have multiple uses. They are used as a vegetable for cooking purposes, for making pickles, or sometimes eaten directly. A popular, tasty and spicy snack item of Thailand ‘Som-tum Thai’, is prepared by using green papaya salad mixed with dried shrimp and peanuts. The raw papaya possesses high amounts of carbohydrates, starch, cardenolides, and saponins.[5] The cardenolides present in raw papaya have a rich therapeutic value and are used in the treatment of congestive heart failure.

Papaya fruits (both ripe and green) are rich in latex, which is considered to be a source of cysteine endo-peptidases.[3] Additionally, papaya latex has high amounts of proteolytic enzymes that have displayed high in vivo efficacy against the gastrointestinal worm: Heligmosomoides polygyrus.[6] The raw or green papaya has been reported to exhibit anti-sickling activity, which has been useful for preventing the sickling of hemoglobin in sickle cell disease patients.[7] The potential wound healing effects of raw papaya are also being reported by Anuar et al.[8]

Postharvest storage of ripe papaya is highly difficult as they form a part of perishable horticultural commodity. It has been reported that papaya fruits can be maintained intact only up to 3 weeks under conditioned storage conditions (10°C at 90–95% relative humidity [RH]).[4] Hence, this is a major hindrance considering the export market demands worldwide. One of the promising methods of preserving quality of agriculture produce is dehydration or drying.[9] Drying of exotic tropical fruits, such as durian, guava, jackfruit, mango, and figs, have been reported to preserve the overall nutritional qualities and retain the concentration of bioactive compounds.[10] Based on the above facts, the prime objective of this study was to compare the effects of drying treatments (freeze and oven drying) and extracting solvents (ethanol and aqueous) on the status of polyphenols and antioxidants in raw and ripened papaya fruits. It is envisaged that the results obtained in the present study might not only provide adequate details on the overall status of antioxidants in papaya, but can also be useful for preserving the quality, extending the shelf life (aimed for export purpose) and be beneficial to the agro industry and health conscious consumers.

MATERIALS AND METHODS

Materials

The papaya fruits used in the present study were collected from a single plant source, growing in a natural plot in Penang, Malaysia. The fruits (raw or naturally ripe) collected were of equal maturity (physiologically) with regard to uniformity in shape, size, color, and stage of ripeness. The raw fruits had green colored skin (100%) with a white inner pulp, while the skin of the ripe fruits was yellow in color with yellow to dark orange colored pulp. The criteria reported by Santamaría-Basulto et al.[11] were used to determine the maturity indices of the fruit. After harvest, the fruits were surface cleaned with a clean and dry cotton cloth followed by washing in water at 48°C for 10 min. Only healthy fruits without any apparent physical damage or insect infestation were selected for the work. Further, the fruits (raw or ripe) were peeled separately (using a sterilized stainless steel knife), seeds removed, and the pulp was cut into small cubes (2 × 2 cm) followed by sample preparation.

Sample Preparation for Analysis

For evaluation of antioxidant activities, the pulp portions of the papaya (both raw and naturally ripe) were subdivided into three portions: one portion for direct extraction and another two portions for freeze drying and oven drying. For direct extraction, the cut cubes of the pulp portion (10 g) were used directly for blending in a kitchen mixer (Panasonic, MX-898 M, Selangor, Malaysia) with the respective solvents (100 ml of 100% ethanol or pure distilled water). For freeze drying, cut cubes (2 × 2 cm) of papaya were freeze dried for 24 h (freeze dryer, Christ, Alpha 1-2 LD Plus, Hamburg, Germany; temperature, −50°C; vacuum pressure, 0.12 Torr) followed by grinding into a fine powder (30 mesh size; 0.595 mm) using a kitchen mixer (Panasonic, MX-898 M). To obtain oven dried samples, separately cut cubes of papaya were dried in a hot air oven at 60°C for 24 h (Afos Dryer, Model Mini, CK 80520, Afos, West Hessle, UK), followed by grinding into a fine powder (30 mesh size; 0.595 mm) with the same kitchen mixer, after appropriate cleaning and drying. The extraction of samples (oven and freeze dried) for antioxidant assays was performed by mixing a known quantity of samples (10 g) with 100 ml of ethanol or distilled water followed by blending at high speed (for 5 min) in a kitchen mixer (Panasonic, MX-898 M). Furthermore, all the sample extracts obtained were filtered under suction by using Whatman No. 1 filter paper followed by centrifugation at 2000 × g for 15 min (Model 4000, KUBOTO, Tokyo, Japan). The supernatant obtained was filtered through a 0.45-μm membrane and used for further analysis.

Determination of Antioxidant Compounds

The Folin–Ciocalteu assay method was employed for measuring the total phenolics in the sample extracts.[12] A suitable calibration curve was prepared using a standard solution of gallic acid and the absorbance was measured at 765 nm using a UV-visible spectrophotometer (Shimadzu UV-160A, Kyoto, Japan). All the results were expressed as mg gallic acid equivalents (GAE) per gram fresh weight of sample. The tannin concentration in the extracts was determined based on the vanillin-HCl method.[13] A standard calibration curve was prepared using catechin (2.5–100 μg/ml) and tannins were expressed as mg catechin equivalent (CAE) per gram fresh weight of sample. The total flavonoids contents were determined based on the colorimetric method described by Sakanaka et al.[14] The results were expressed as mg of (+)-catechin equivalent (CAE) per gram of fresh weight of sample. The standard calibration curve was prepared using catechin in the range of 2.5–100 μg/ml.

Determination of Antioxidant Activity

DPPH and ABTS⁺ radical scavenging activity

The DPPH and ABTS⁺ radical scavenging activity of ethanolic and aqueous extracts were measured by employing the method described by Blois[15] and Re et al.,[16] respectively. The results were expressed according to the following formula: percent inhibition of DPPH and ABTS⁺ radicals = [(Acontrol – Asample)/Acontrol] × 100, where Acontrol is the absorbance of the DPPH solution without extract and Asample is the absorbance of the sample with DPPH solution.

Phosphomolybdenum assay

The antioxidant capacity of papaya extracts were evaluated by the formation of phosphomolybdenum complex, based on the method described by Prieto et al.[17] A suitable calibration curve was prepared using a standard solution of ascorbic acid (25–300 μg/ml) and the antioxidant activity was expressed relative to that of ascorbic acid (mg AAE AO/g fresh weight of sample).

Chemicals and Reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), Folin–Ciocalteu reagent, potassium persulfate, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), vanillin, sodium carbonate, aluminium chloride hexahydrate, sodium acetate, gallic acid, quercetin, catechin, and butylated hydroxytoluene were purchased from Sigma Aldrich (Munich, Germany). All the solvents used in the study were of analytical grade and purchased from Fisher Scientific (M) Sdn. Bhd, Malaysia.

Statistical Analysis

Results obtained in this study are represented as mean values (n = 3) ± standard deviation (SD). One way analysis of variance (ANOVA) was performed and the significant differences obtained between the mean values were determined by Tukey's pair-wise comparison test at a significance level of P < 0.05. The statistical analyses were conducted using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

Total Phenolics, Tannins, and Flavonoids

Results on the total phenolics, tannins, and flavonoids are shown in Table 1. Polyphenolic compounds present in plants act as potential antioxidants and are active free radical scavengers.[1,18] The basic antioxidant mechanisms of these compounds are dependent on their hydrogen donating and metal ion chelating abilities. In this study, the total phenolics varied between 21.1 (in ripe ethanolic oven dry sample) up to 217.3 mg GAE/g (fresh weight) in freeze-dried aqueous extracts. These results clearly indicate that both the raw and ripe papaya possess a high phenolic content, which needs to be tapped to exploit for potential health benefits. However, it should be noted that the potential health benefits of dietary phenolic compounds also depend largely on their absorption capacity and metabolism.

Table 1 Total phenolics, tannin and flavonoids content of ethanol, and aqueous extracts of raw and ripe papaya obtained after different drying treatments. ¹

		Total phenolics ^2		Total tannins ^3		Total flavonoids ^3
Treatment	Nature of fruit	Ethanol	Aqueous	Ethanol	Aqueous	Ethanol	Aqueous
Normal (control/untreated)	Raw	44.8 ± 1.00^b	37.9 ± 1.28^a	1.25 ± 0.44^a	2.30 ± 0.22^ab	1.48 ± 0.20^a	2.24 ± 0.19^b
	Ripe	65.0 ± 2.25^d	75.7 ± 2.43^b	3.75 ± 0.34^b	3.36 ± 0.41^bc	2.86 ± 0.19^c	3.10 ± 0.31^c
Oven dry	Raw	52.4 ± 1.27^c	104.7 ± 0.92^c	1.67 ± 0.38^a	4.12 ± 1.18^c	3.04 ± 0.08^c	2.37 ± 0.25^b
	Ripe	21.1 ± 0.32^a	119.1 ± 2.32^d	1.58 ± 0.41^a	2.03 ± 0.36^ab	1.91 ± 0.12^b	1.01 ± 0.12^a
Freeze dry	Raw	70.2 ± 2.30^e	157.6 ± 2.75^e	2.00 ± 0.24^a	1.79 ± 0.23^a	1.63 ± 0.14^ab	1.12 ± 0.16^a
	Ripe	141.7 ± 3.21^f	217.3 ± 0.51^f	9.58 ± 0.65^c	6.00 ± 0.55^d	8.40 ± 0.23^d	3.64 ± 0.18^d
^1All the values are expressed on fresh weight basis. All the data are the mean ±SD of three replicates; mean value followed by different letters in the same column differs significantly from each other (P < 0.05).
^2Milligram gallic acid equivalents/g fresh weight of sample.
^3Milligram catechin equivalents/g fresh weight of sample.

With regard to tannins, only freeze-dried ripe papaya extracts showed significantly higher tannin content (9.58 and 6.0 mg CAE/g fresh weight of sample in ethanol and aqueous extracts, respectively) compared to other samples. Tannins are high molecular weight compounds, which constitute the third important group of phenolics and occur either as hydrolysable or condensed tannins. Based on the available reports, tannins can exhibit high bioactivity as well as antibacterial, antiviral, antiparasitic, and anticancer activities.[19] Results on the total flavonoids revealed significant differences between the drying treatments as well as between the extracting solvents used. The freeze-dried ripe papayas had the highest total flavonoids content (8.40 and 3.64 mg CAE/g fresh weight of sample in ethanol and aqueous extracts, respectively), while the lowest levels were recorded in raw papaya samples (control/untreated) and ripe oven dried samples (1.48 and 1.01 mg CAE/g fresh weight). Flavonoids are low molecular weight phenolic compounds, which are powerful antioxidants present in most plants.[20] These compounds are capable of retarding lipid oxidation by inhibiting lipoxygenase activity and under in vitro conditions, can act as scavengers of active oxygen species and as chelators of metal ions. Hence, the presence of flavonoids even at threshold levels in papaya is expected to have some health beneficial effects on human health.

DPPH, ABTS Radical Scavenging Activity, and Antioxidant Capacity

Results of DPPH and ABTS radical scavenging activities and on the antioxidant capacity are depicted in Table 2. Overall, the DPPH and ABTS radical scavenging activity were significantly higher in the freeze-dried ripe papaya sample extracts compared to untreated and oven dried samples. Also, the solvents used for extraction significantly affected the radical scavenging ability, wherein mixed results were observed for each type of treatment. The free radical scavenging activity is one of the main mechanisms by which most of the plant antioxidants act and the activity of which is determined by their scavenging potential of synthetic radicals, such as DPPH and ABTS in organic solvents, at room temperature.

Table 2 DPPH, ABTS radical scavenging activity, and total antioxidant capacity of ethanolic and aqueous extracts of raw and ripe papaya obtained after different drying treatments. ¹

		Antioxidant activity ^3
		DPPH assay ^2		ABTS assay ^2		Phosphomolybdenum assay ^3
Treatment	Nature of fruit	Ethanol	Aqueous	Ethanol	Aqueous	Ethanol	Aqueous
Normal (control/untreated)	Raw	64.2 ± 1.40^b	44.0 ± 1.38^b	27.0 ± 1.44^b	42.5 ± 3.56^a	628.1 ± 15.76^a	793.7 ± 5.63^a
	Ripe	81.9 ± 1.34^d	85.6 ± 1.58^d	44.5 ± 1.54^d	72.7 ± 2.40^c	837.5 ± 12.76^d	1086.5 ± 6.43^b
Oven dry	Raw	80.4 ± 0.45^d	60.2 ± 1.41^c	38.2 ± 2.93^c	87.2 ± 2.01^d	826.7 ± 16.03^cd	2054.5 ± 21.61^e
	Ripe	60.6 ± 0.70^a	29.8 ± 0.56^a	21.7 ± 1.04^a	68.2 ± 1.53^b	744.5 ± 12.81^b	1977.7 ± 16.46^d
Freeze dry	Raw	71.1 ± 0.71^c	87.5 ± 0.19^d	53.1 ± 0.62^e	86.0 ± 0.79^d	803.1 ± 9.85^c	1756.1 ± 21.72^c
	Ripe	91.8 ± 0.25^e	93.0 ± 0.98^e	87.1 ± 0.64^f	97.5 ± 1.35^e	1315.7 ± 13.71^e	2167.5 ± 14.00^f
^1Values are expressed on fresh weight basis. All data are the mean ±SD of three replicates; mean values followed by different letters in the same column differ significantly from each other (P < 0.05).
^2Percentage inhibition of DPPH and ABTS^+ radical by 1 ml of extract (fresh weight).
^3Milligram ascorbic acid equivalent antioxidant capacity/g fresh weight of sample.

Results on DPPH radical scavenging assay revealed the highest percent inhibition in the freeze-dried ripe papaya sample extracts (91.8 and 93.0% in ethanol and aqueous extracts, respectively) over other samples under study. A similar trend was observed for ABTS radical scavenging assay, wherein freeze-dried ripe papaya samples showed the highest value of 87.1 and 97.5% in ethanolic and aqueous extracts, respectively.

Antioxidant capacity of a plant extract is evaluated based on the reduction of Mo (VI) to Mo (V), which is a green colored complex formed by an antioxidant compound present in the extract, which in turn is directly dependent on the amount of polyphenols and flavonoids present.[17] In this study, almost all the samples exhibited higher values for total antioxidant capacity, which ranged between 628 to 2167 mg AAE/g fresh weight of the sample in ethanol and aqueous extracts, respectively. From the above results, it was evident that irrespective of maturity, both the raw and ripe papaya extracts exhibit rich antioxidant activities.

Effect of Extracting Solvents

The extraction and recovery of polyphenols and antioxidant compounds from a given plant material entirely depends on the compounds solubility in the extracting solvent, polarity of the solvent, viscosity, and vapor pressure.[2,21] In the present study, the extractability of phenolics and antioxidant compounds varied significantly between the extracting solvents (Tables 1 and 2). Previously, solvents such as ethanol, methanol, acetone, and ethyl acetate have been routinely employed for extraction and recovery of polyphenolic compounds from fruit samples.[22] However, it should be noted that each of these solvents might provide varied results for a particular plant produce, thus rendering it difficult to conclude the exact amount/quantity of compounds present in a given sample. Accordingly, our results on polyphenols or antioxidant compounds might vary from some of the previous reports in papaya fruits.[22,23] In the present study, we used ethanol and water for extraction purposes. This is due to the fact that, apart from being the most commonly used solvent for extraction of bioactive compounds from plant materials; ethanol and water find high acceptability for human consumption too.[24]

Effects of Drying

Overall, compared to oven drying, freeze drying of papaya was found to be the most effective drying method, which either retained or enhanced the bioactive compounds. Previously, freeze drying has been reported to be a better and highly efficient method for preserving large molecular weight compounds, such as condensed tannins, compared to oven drying.[25] Additionally, freeze-dried fruits (such as Barbados cherry, guava, mango, papaya, and pineapple) have been reported to preserve some of the organoleptical properties or sensory attributes like color, flavor, and taste more effectively than non freeze-dried fruits.[26] Our results on the oven dried papaya sample extracts are comparable to some of the earlier reports, wherein oven drying or application of heat has been shown to reduce the antioxidant compounds. Larrauri et al.[27] have reported increased drying temperatures (100 and 140°C) to significantly reduce total extractable polyphenols (18.6 and 32.6%) and condensed tannins (11.1 and 16.6%) accompanied by a decrease in the antioxidant activity (by 28 and 50%) in ‘red grape pomace peels’. Oven drying of marion berries, strawberries, and corn at a high temperature range has been reported to show significant reductions in polyphenols, condensed tannins, and antioxidant activity, while freeze-dried samples consistently had a higher level of total phenolics content.[28] Schieber et al.[29] reported that during heat treatments, loss of macromolecules like flavonoids occur, which is attributed to the harsh drying conditions, mainly, the temperature and time duration employed.

Also, our results on the observed enhancement in the phenolic compounds as well as antioxidant capacity on oven drying compared to untreated/normal samples are on par with the some of the earlier reports, wherein mild heating has been shown to enhance some of the bioactive compounds.[30] This increase has been attributed to the enhanced activity of phenylalanine ammonia-lyase activity (on mild heating), a key enzyme in the synthesis of phenolic compounds in plant tissues or to the enhanced extractability by the solvents used. Also, the non-enzymatic inter-conversion between phenolic molecules and the availability of precursors of phenolic molecules might have contributed to the observed increase on oven heating. Earlier, the increase in the activities has also been correlated to enhanced generation and accumulation of Maillard-type antioxidants.

CONCLUSIONS

Even though the best possible way to get an abundance of natural antioxidants is consuming the fresh fruits or their juice, considering the perishable nature of papaya, the best option would be to choose drying methods and also freeze drying, which has been proved to be the best method as observed in this study. From the obtained results of this study, it was evident that opting for drying treatments tends to enhance some of the bioactive compounds in papaya. Future research needs to be initiated to assess the rehydration capacity of these dried papayas on the overall qualities. Studies are also warranted to evaluate the shelf life stability as well as retention of nutritional and sensory qualities in dried papayas. Once these parameters exhibit positive outcomes, then development of value-added products can be attempted on a pilot scale to benefit both producers and the consumers.