Evaluation of Phenolic Content and Antioxidant Activity of Aqueous Extracts of Three Carica papaya Varieties Cultivated in Senegal

The aqueous extracts of different parts (old leaves (OL), young leaves (YL), peels (PE) and delipidated seed residues (DS)) of three varieties of papaya are studied. Extraction conditions are optimized: an extraction time of 20 minutes, a temperature of 70 ̊C and a plant material/water mixture of 1% give the best yield of polyphenol. The amount of polyphenols, flavonoids, saponins and proanthocyanins of each aqueous extract was investigated. Antioxidant activities are measured using two different methods (DPPH and ABTS). The delipidated seeds (DS) of V1 have the highest total phenolic content (TPC = 72.56 ± 3.16 mg GAE/g) while they have the lowest total flavonoid content (TFC = 0.22 ± 0.01). With regard to saponins, the PE of V3 is much richer in saponins (194.03 ± 15.78 mg AeE/g) than all the other extracts studied. The OL of V2 and PE of V1 contain the most proanthocyanidins with very similar values of 2.51 ± 0.03 mg CE/g and 2.53 ± 0.34 mg CE/g respectively. The study of the antioxidant activities of the extracts showed a correlation between the amount of polyphenols and IC50. DPPH OL and YL V2, which are rich in polyphenols, have the lowest IC50 of 0.072 mg/ml and 0.080 mg/ml respectively, whereas for ABTS we have PE of V1 that is very rich in polyphenols which has the smallest IC50 value of 0.218 mg/ml.


Introduction
Metabolic and respiration processes generate oxidation reactions in living.During these metabolic reactions, the use of oxygen can require highly reactive oxygen forms (ROS) such as free radicals or peroxidised ( .
Since the process of onset of ROS is natural, one must try to control their concentrations in the human body in particular.For this purpose it is necessary to provide supplements able to neutralize them without generating harmful species.This is possible with antioxidants such as polyphenols [9] [10] [11] [12].Polyphenols are a set of chemical compounds such as phenolic acids, coumarin, flavonoids, anthocyanin, tannin, lignin, which are widespread in the plant kingdom and derived from the secondary metabolism of plants [13] [14] [15] [16].
Synthetic antioxidants can also be used but unfortunately, their use is very restricted [17] [18] [19] [20].For several years natural antioxidants from fruit and vegetable plants have been widely examined to understand their role in the reduction of ROS in cells [11] [21] [22] [23].Usually, only fruits and vegetables were consumed for their bioactive components but investigations showed that other organs of the plant (leaves, bark, peelings, roots...) can contain bioactive chemical species, in particular polyphenolic compounds [24] [25] [26].Thus these organs which constituted a problem of waste management can be used advantageously to extract the bioactive species which have a rather important added value.In this context, Carica papaya known for its nutritional and therapeutics interests offers an advantageous opportunity.In fact, leaves are used as to relieve nerve pain, asthma attacks or sugar's levels control [27].The seeds are used in the treatment of ulcers [28], diabetes [29], hypertension [30], hypercholesterolemia [31] and liver diseases.
The principal aim of this study is to optimize the extraction of polyphenols in the different parts (old leaves (OL), young leaves (YL), peels (PE) and defatted seeds (DS)) of three varieties of Carica papaya.Secondary, the different categories of molecules (polyphenols, flavonoids...) of interest are quantified and the antioxidant activities of the three varieties are studied according to the different parts.

Plant Material
Three varieties of Carica papaya L. were harvest at the Sébikhotane protected forest (region of Dakar in Senegal 14˚43'14.4"N,17˚08'16.4"W):Ordinary (V1), Red Lady (V2) and Sunrise (V3).Old (OL) and young (YL) leaves, the seed (DS) and the peels (PE) from mature fruits were collected.The ordinary variety produces round fruits with a yellowish flesh while Sunrise ones are oblong with red have been finely ground using a grinder and stored at 4˚C for later use.The seeds were ground and defatted with hexane and the residue (DS: defatted seed) was air dried and stored at 4˚C for further uses.

Extraction Process
Extraction process was done according to the procedure used by Vuong et al. [32].TPC is used as response value.For the determination of optimum temperature leaves were crushed and 0.5 g of powder is extracted in 50 ml of distillate water at variable temperatures (50˚C, 60˚C, 70˚C, 80˚C and 90˚C) for 20 minutes using an agitated water bath whose temperature is well controlled.This optimal temperature is used to determine the optimum extraction duration.
Then 0.5 g of papaya leaves' powder was extracted with 50 ml of water at the optimum temperature with varying extraction times (5, 10, 20 and 30 min).The optimum temperature and the optimum duration are used to determine the optimal ratio in the range of 0.5 -1.0 -1.25 -2.5 -3.25 and 5.0 g with 50 ml of water.For further study, the optimal values of these three parameters are used.
Prior the extraction, the seeds were defatted by hexane.Then the extract was filtered through a Whatmann filter paper N˚1.The extract obtained was stored at 4˚C for further use.

Determination of the Total Phenolic Content
The assay was due according to Mohdaly et al. [33] with some modifications.A test sample of 200 µl final (several dilution were made) was mixed with 150 µL of Folin-Ciocalteu reagent, 600 µl of Na 2 CO 3 20% and 2.32 ml of distillate water.
After 1h of incubation in the darkness at room temperature, the absorbance was read at 760 nm with a Perkin-Elmer UV/Visible spectrophotometer Lambda 365.
Gallic acid (GA) was used as a standard.The results were expressed as mg GAE/g ± Standard deviations.

Determination of the Flavonoids Contents
According to the method of Ordoñez et al. [34], 2.5 ml of sample were mixed with 2.5 ml of an ethanolic solution of AlCl 3 .After 1 h of incubation in the darkness at room temperature, the absorbance was read at 425 nm with a Perkin-Elmer UV/Visible spectrophotometer Lambda 365.Quercetin (Q) was used as a standard.The results were expressed as mg QE/g ± Standard deviations.

Determination of Saponins Content
The procedure developed by Vuong et al. [32] was used with a little modifiction.0.5 ml of sample (dilutions were made if they are required) were mixed with 0.5 ml of a vanillin ethanolic solution and 5 ml of H 2 SO 4 at 72%.The samples were incubated at 70˚C in a water bath during 10 minutes.After that they were cooled slowly at room temperature.The absorbance was measured at 560 nm with a Perkin-Elmer UV/Visible Spectrophotometer Lambda 365.Aescin was used as a standard and results were expressed as mg AeE/g ± Standard deviations.

Determination of Proanthocyanidins
The proanthocyanidin content was determined according to the procedure described by Li et al ( 2006) [35].To 0.5 mL of diluted sample, 3 mL of 4% (w/v) vanillin is added before adding this mixture to 1.5 mL of concentrated HCl.This mixture is incubated at room temperature for 15 min before reading the absorbance at 500 nm.Catechin (C) was used as standard and the results are expressed as mg CE/g ± Standard deviations.

DPPH Free Radical Scavenging
Before determining the IC50 we have to determine the TEAC (Trolox Equivalent Antioxidant Capacity) according to Akhtar et al. [36] with modifications.

ABTS Free Cation Radical Scavenging
Like DPPH assay, we have to determine the TEAC and the IC50.The assay developed by Thaipong et al. [37]

Statistical Analyses
All the measurements were carried out in triplicate.The mean values and stan-Food and Nutrition Sciences dard deviations were calculated and the data were expressed as mean ± SD.
Xlstat 2019 software was used for the data and statistical analysis.Differences were considered significant at the p < 0.05 level based on Duncan's new multiple range test.

Optimisation of the Extraction
Under the described experimental conditions, Figure 1  reach a maximum at 70˚C which will be considered as the optimum.This observation is consistent with that reported by Vuong et al. [32].Similarly, the extractability of the polyphenols increases and reaches a maximum at 20 minutes.
Beyond this extraction time, there is a degradation of the yield of polyphenols.
Therefore, the optimal temperature of 70˚C and the extraction time of 20 minutes are chosen to study the influence of the ratio on material plant/water (w/v).It is observed in Figure 1(c) that the extraction efficiency is better for the 1/100 ratio.This result seem to be logical as it is reported by Gertenbach et al. [38] that the lower the plant/water ratio (w/v), the higher is the rate of extraction.Indeed, there is a concentration gradient between the phenolic compounds inside the foliar particles and the ones located on the surface, thus leading to an acceleration of the extraction process at high dilution.
For the present work the extractions are carried out at a temperature of 70˚C for 20 minutes in a vegetable/water ratio of 1/100 to quantify total phenolic, total flavonoid, saponins and proanthocyanidins.

Total Phenolic and Total Flavonoid Content from Three Varieties of Carica papaya
The results of the quantitative determination of the polyphenol contents of the  It is observed that the defatted seeds of V1 are twice as rich in polyphenols as V3 ones and that it is three times richer than that of V2 which is poor in polyphenols.In V2 the polyphenols are found in the leaves whereas in V1 the seeds contain almost as many polyphenols as all the other parts of the plant combined.For V3 the polyphenols are fairly homogeneously distributed in the leaves and seeds.It should be noted that for varieties V2 and V3 the total polyphenol content (TPC) of young leaves and the old leaves are comparable contrary to V1 for which the TPC of the young leaves is higher than the TPC of the old leaves.The three varieties studied in this work have a TPC that is higher than Carica studied by Vuong et al. [32].
Leaves of a variety of C. papaya, cultivated in Pakistan [39], give TPC value of 49.94 ± 0.60 mg AeE/g which is less than the values found for OL and YL of V2 and higher than those for OL and YL of V1 and V3.The TPC (32.23 ± 0.64 mg AeE/g) of the peels of the Pakistan's variety is greater than those of the three varieties used in this study while the seeds' TPC (27.94 ± 0.09 mg AeE/g) is very low compared to the TPC value of the DS of V1 and V3.For all three varieties, flavonoid levels are low (Table 1).This can be explained Table 1.Total phenolic and total flavonoid content of three varieties of Carica papaya.by the low solubility of flavonoids in water which is a very polar solvent.Indeed, the flavonoids which are low polar molecules are more soluble in the organic solvents and little and medium polar solvents.For all varieties, flavonoid levels of OL and YL are comparable and range between 1.10 mg QE/and 1.50 mg QE/g.These values are lower than the value reported in the literature (3.33 mg/g) for young leaves of the papaya variety which growth in Malaysia [40].The TFC of aqueous extracts of defatted seeds (0.22 mg QE/g -1.01 mg QE/g) are greater than the TFC of aqueous extract of the seeds of the Malaysian variety [40].The TFC of PE are between 0.23 mg QE/g and 0.35 mg QE/g and are higher than the value reported (0.056 mg/g) for the Carica papaya L. var solo 8 variety that is cultivated in Ivory Coast [41].

Saponins Content of Three Varieties of Carica papaya
The  2).This is consistent with the fact that saponins are slightly polar are more compatible with solvents which are less polar than water.These results are in accordance with those reported for the Carica studied by Vuong et al. [32].

Proanthocyanidins Content of Aqueous Extracts
Owing to their low polarity the proanthocyanidins extraction yields in water are low (Table 3).Legend: Ordinary (V1), Red Lady (V2) and Sunrise (V3).OL: Old leaves, YL: Young leaves; PE: Peels and DS: defatted seeds.Values with different letters in superscript in column showed the significant differences at the significance level of p-value of 0.05 or 0.001*.
parts of the plant For V1 the proanthocyanidins quantities are regularly distributed between the different parts of the plant: 2.23 ± 0.15 mg CE/g -2.53 ± 0.34 mg CE/g (Table 3).

Antioxidant Capacity and IC50
Since phenolic compounds are known for their ability to trap free radicals a study of their antioxidant activity and IC50 was carried out for the three varieties of C. papaya.32].Other organic solvents such as the extracts of n-hexane, dichloromethane, n-butanol, ethyl acetate, methanol and ethanol showed a significant difference in scavenging activity between the different parts of Carica papaya [32] [39].According to study of Asghar et al. [39], leaf ethanol extract shows the highest free radical scavenging of DPPH compared to seed and peel extracts.Extracts of n-hexane and n-butanol give the lowest DPPH free radical scavenging probably due to the difference in polarity of the solvents and extracted compounds.
Owing to the quantitative presence of polyphenols and flavonoides in the aqueous extracts of the different studied parts of the plant and the antioxidant activities presented by these extracts, it is conceivable to make formulations in the form of infusion for human consumption.Indeed polyphenols are good substrat for protection against oxidative stress.All these parts of the papaya plant considered as waste can find in this sector a potential valuation at low cost.

Conclusion
In Senegal, Carica papaya is a widespread tree and several varieties are grown in various regions during all seasons.While only the papaya pulp is consumed in Senegal, this work shows that polyphenols, flavonoids, proanthocyanidins and saponins are present in other organs such as seed, leaves and peels.Their optimal aqueous extraction conditions are reminiscent of the conditions for obtaining infusions.These observations offer interesting prospects for the development of new product in food and nutrition area.
Firstly, 0.1014 mM of fresh DPPH in methanol was prepared.Trolox was used as a standard; dilutions were made to have a final volume of 200 µl.The different extracts or Trolox solutions (200 µl) were mixed with 3.8 ml of DPPH.After 30 minutes of incubation in the dark at room temperature we read the absorbance at 517 nm.Next, we made adequate dilution to determine the IC50.Results were expressed as µg TrE/g ± Standard deviations.DPPH scavenging activity was determined by calculating the percentage of inhibition: summarizes the variation of polyphenols extraction according the temperature (a), the duration (b) and the ratio (c).It shows that the temperature, the time and the ratio parameters have an influence on the yield of the extraction of polyphenols during the aqueous extraction of papaya leaves.The yield increases with the temperature to

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Gaye et al., juicy and sweet.The Red Lady fruits are also oblong with a sweet-fruited cultivar, but they are three to four times larger than the fruit of the Sunrise variety.Samples were rinsed with water and distillate water and were air-dried free for 3 days.After this process, they were dried at 55˚C during 48 hours in an oven.Then, the different organs of C. papaya L. (OL, YL and PE) DOI: 10.4236/fns.2019.103021278 Food and Nutrition Sciences flesh, fragrant
all the other studied parts of the plant whereas the OL are the poorest saponins content (49.73 ± 0.92 mg AeE/g) unlike to the other two varieties (Table

Table 2 .
Saponins content in different organs of three varieties of Carica papaya.
The proanthocyanidins content of OL of the three varieties are comparable and are in the range 2.19 -2.51 mg CE/g and are slightly greater than those found for YL (1.57-2.33 mg CE/g).Peels (PE) and defatted seeds Legend: Ordinary (V1), Red Lady (V2) and Sunrise (V3).OL: Old leaves, YL: Young leaves; PE: Peels and DS: defatted seeds.Values with different letters in superscript in column showed the significant differences at the significance level of p-value of 0.05 or 0.001*.

Table 3 .
Proanthocyanidins content in different organs of three varieties of Carica papaya.

Table 4
[43]arizes their results.DPPH ., which is a stable radical, is widely used to evaluate free radical scavenging ability[42][43].Globally, the results show that V2 has a better antioxidant capacity than V1 and V3 which antioxidant's activities seem similar.The

Table 4 .
The antioxidant capacity (DPPH and ABTS) and IC50 in different organs of three varieties of Carica papaya.values are different with respectively 46.70 ± 2.66 mg TrE/g and 27.89 ± 0.75 mg TrE/g for V2, 56.83 ± 4.61 mg TrE/g and 39.60 ± 1.19 mg TrE/g for V3.The lowest antioxidant capacity value for YL was found for V3 with 47.97 ± 0.86 mg TrE/g versus 57.26 ± 3.10 mg TrE/g for V1.In previous studies, the antioxidant activities of Carica papaya extracts reported are lowest than our results A. A. Gaye et al.