Quality Characteristics and Antioxidant Activity of Unripe Peach (Prunus Persica L. Batsch) Extracts with Distilled Water Coupled with Ultrasonication and Prethanol-A

ABSTRACT

Peach, an important fruit crop for its health benefits, is grown in different parts of the world. A huge amount of unripe peaches, which are obtained during fruit thinning or as natural fruit drop, are generally discarded as waste. It would be very fortunate for the fruit growers if the thinned out or naturally dropped peaches could be utilized as valuable resources. The objective of this study was to evaluate the quality characteristics and antioxidant potential of unripe peach of two cultivars, Changhowon Hwangdo and Kanoiwa Hakuto extracted with distilled water coupled with ultrasonication or prethanol-A, an ethanol-based food preservative. The physicochemical characteristics (pH, titratable acidity, soluble solid content, color value, mineral content, DPPH radical scavenging potential, and total phenol content) of the extracts significantly varied with cultivar, extracting solvent and/or their interaction. The results of this experiment showed that unripe peach fruits could be used as an inexpensive source of antioxidants and phenolic compounds, which could even be extracted with distilled water, and possess a big potential to be used in food and cosmetic industries.

KEYWORDS:

• Antioxidant potential
• chemical characteristic
• extracting solvent
• prethanol-A
• unripe peach

Introduction

Peach (Prunus persica L. Batsch), an economically important fruit crop, is grown in different parts of the world. It is considered as an important fruit crop for its health benefits (Ramina et al., 2008). The fruits are a good source of bioactive compounds, which possess health benefits to humans (Manzoor et al., 2012). They are reported to contain a number of phenolic compounds, including catechin, chlorogenic and neochlorogenic acids, epicatechin and derivatives of cyaniding and quercetin (Cantin et al., 2009; Cocconi et al., 2016; Tomás-Barberán et al., 2001). Among the different phenolic compounds, a considerable amount of caffeoylquinic acid, a bioactive polyphenol with significant antioxidant potential and having an beneficial effect in human health (Luo et al., 2008), was detected in the immature peach fruits (Lombardo et al., 2011). Consumption of peach fruits can provide a potential health benefit against various chronic diseases because of their good scavenging potential to the reactive oxygen species produced in human blood plasma (Tsantili et al., 2010).

A large amount of unripe peaches, which are obtained during fruit thinning or as natural drop and account for a significant percentage of the total peach production, are mostly discarded as waste. It would be very profitable for the peach growers if the thinned out or naturally dropped peaches could be utilized as valuable bioactive resources. In particular, unripe fruits are regarded as rich sources of polyphenolic compounds such as proanthocyanidin and flavonoid (Dragovic-Uzelac et al., 2007), which target cancer stem-like cells and suppress tumor organoid formation in colorectal cancer (Toden et al., 2018). The composition of polyphenols found in the unripe fruits is considerably high although the amount significantly depends on the variety and growth stage (Scordino et al., 2012). Some agro-industry by-products such as seeds, peels and pomaces of citrus fruits were investigated for their potential industrial utilization as they contain high amount of phenolic compounds (Bocco et al., 1998; Lee and Wrolstad, 2004).

A study indicated that the dropped unripe peaches were excellent sources of bioactive and antioxidant compounds, and can be explored for their health-promoting values in food products (Liu et al., 2015). Similar results were found by Kim et al. (2014a) with the unripe fruits of peach having a significant biochemical potential as a food supplement. The antioxidant potential of unripe nectarine fruits was high and decreased with storage (Baldi et al., 2012). This report reveals that unripe fruits of some species are rich source of antioxidants. Results of Kim et al. (2012a), Kim et al. (2012b) showed that unripe peach could be used for the development of natural functional cosmetics.

The availability of nutritional and functional compounds of food materials, including fruits, are highly affected by extraction method (Altemimi et al., 2016) and/or solvent variation (Kajdzanoska et al., 2011). Although there are few reports on antioxidant potentials of unripe peach, to the best of our knowledge, no report has been published on the quality characteristics and antioxidant potential of unripe peach extract with prethanol-A. Prethanol-A is an ethanol-based food preservative and has recently been used as a solvent (Le et al., 2017; Yin et al., 2017). On the other hand, distilled water is an inexpensive and widely used solvent. Extracts obtained from immature stone fruits, including peach, showed good results to prevent enzymatic browning in minimally processed peaches (Redondo et al., 2017). There are growing trends in food industries toward the development and manufacture of functional and nutraceutical products from agricultural by-products, including food waste (Kumar et al., 2017), fruit and vegetable wastes (Sagar et al., 2018; Singh and Immanuel, 2014). Considering the above background, this study was designed to investigate the quality characteristics and antioxidant potential of unripe peach extracts with distilled water and prethanol-A.

Material and Methods

Chemicals and Materials

Folin-Ciocalteu phenol reagent and DPPH were purchased from Sigma Co. (St. Louis, MO, USA). All other reagents used were of analytical grade. Unripe peach (Prunus persica L. Batsch) fruits of cultivar Kanoiwa Hakuto (KH) and Changhowon Hwangdo (CH) were obtained from Cheongdo Peach Research Institute, Gyeongsangbuk-do, Korea. KH and CH were among the widely grown peach cultivars in the region. The fruit trees were spaced 6 m between rows and 3 m between plants and were grown in vase-pergola system with three main branches. Twenty fruits were harvested from each of the 10 8-year-old trees. The fruits were manually harvested at 30 days after flowering, kept in plastic bags, transported to laboratory and stored at 4°C for 1‒2 d until analysis.

Preparation of Peach Extracts

Peach extracts of the two cultivars were prepared with two solvents, distilled water and prethanol-A at the ratios of 1:10 (w/v). In order to increase the extraction efficiency, the fruit extracts with distilled water were prepared using ultrasonic method (Garcia-Salas et al., 2010). Unripe fruit (20 g) was extracted with distilled water (200 mL) using ultrasonic (Crest Ultrasonics, Trenton, NJ, USA) for 30 min. The ultrasonic was set at a frequency of 26 kHz and a maximum peak power of 800 w and the bath temperature adjusted to 30°C during the extraction. The mixture was filtered through a filter paper (Whatman No. 1). For the extraction with prethanol-A, 200 g of fruit was extracted with 2000 mL of 50% prethanol-A (95% ethanol diluted in distilled water) for 24 h at room temperature. The mixture was filtered through a filter paper (Whatman No. 1) and condensed to 1000 mL using rotary evaporator. The extracts were stored at 4°C until further analyses.

The extracts were named based on the cultivar and solvent used as CH-DW: extract of peach cv. Changhowon Hwangdo with distilled water coupled with ultrasonication, CH-PA: extract of Changhowon Hwangdo with prethanol-A, KH-DW: extract of Kanoiwa Hakuto with distilled water coupled with ultrasonication and KH-PA: extract of Kanoiwa Hakuto with prethanol-A.

Physicochemical Parameters

The pH value of peach extract was measured using a pH Meter (Model 250; Beckman Coulter, Inc., Fullerton, CA, USA). Titratable acidity (TA) was measured following the method described by Lee et al. (2017) with some modifications. The extract sample (5 mL) was mixed with deionized water (125 mL) and titrated with 0.1 N sodium hydroxide to an endpoint pH 8.2. Soluble solid content (SSC) expressed as °Brix was measured using a hand refractometer (RX-5000α, Atago, Tokyo, Japan). All chemical measurements were replicated 3 times and mean values were reported.

Color Value Measurement

L* (lightness), a* (redness, + or greenness,–), b* (yellowness, + or blueness,–) values of peach extract were measured using a Chroma meter (Minolta CR-300, Minolta Corp., Japan). Calibration plates (Minolta calibration plate (YCIE = 94.5, XCIE = 0.3160, YCIE = 0.330)) and Hunter Lab standard plate (L* = 97.51, a* = 0.18, b* = +1.67) were used to standardize the instrument with D65 illuminant (Kim et al., 2014b). The fruit extracts were put into Petri dishes and color values were measured directly on three areas of the dishes and the average was calculated.

Determination of Total Phenol

Total phenol content was determined by Folin-Ciocalteu method (Singleton et al., 1999). A 0.79-mL of distilled water, 0.01 mL of sample extract and 0.05 mL of phenol reagent were mixed in a 1.5-mL Eppendorf tube using a vortexer. After 1 min, 0.15 mL of 20% sodium carbonate was added to the mixture and allowed to stand at room temperature for 2 h. The absorbance of reaction mixture was measured at 750 nm using a spectrophotometer (Shimadzu UV-1700, Kyoto, Japan) and the total phenol content was calculated from calibration curve, using gallic acid as a standard.

Determination of DPPH Scavenging Potential

The antioxidant potential of peach extracts was measured using DPPH free radical scavenging potential as described by Shyu and Hwang (2002). An aliquot of 0.1 mL of the sample extract was added to 2 mL of acetate buffer (0.05 M, pH 5.5), 1.9 mL of methanol and 1 mL of DPPH solution (0.5 mM). Blank contained 2 mL acetate buffer, 1.9 mL methanol and 0.1 mL of sample extract, while the control contained 2 mL of acetate buffer, 1 mL of DPPH and 2 mL of methanol. The mixture was shaken immediately after adding DPPH and allowed to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). All determinations were performed in triplicate. The inhibitory percentage of the DPPH radical by the samples was calculated as follows:

$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\left(\mathrm{\%}\right)=\left[\left({\mathrm{A}}_0-\left(\mathrm{A}-{\mathrm{A}}_{\mathrm{b}}\right)\right)/{\mathrm{A}}_0\right]\times 100$

were A₀ is the A₅₁₇ of DPPH without sample (control), A is the A₅₁₇ of sample and DPPH, and A_b is the A₅₁₇ of sample without DPPH (blank).

Determination of Mineral Composition

An aliquot of sample extract (0.5 mL) was mixed with nitric acid (15 mL) and 2 mL of distilled water was added to the mixture. Mineral composition of the sample extract was determined using an inductively coupled atomic emission spectrophotometer (ICP AES; Varian Vista Inc., Victoria, Australia) as described by Skujins (1998).

Statistical Analysis

Data were subjected to analysis of variance (ANOVA) using SAS 9.3 (SAS Institute Inc, Cary, NC, USA) and significant differences between the means were separated at 5% probability using Tukey test. Average values of triplicate measurements were considered for statistical analysis.

Results and Discussion

Chemical Composition of Unripe Peach Extracts

The value of pH, TA and SSC of peach extracts significantly (P< .05) varied with cultivar, solvent and/or their interaction (Table 1). The highest pH value was measured for the extracts obtained from cultivar Changhowon Hwangdo ‒ CH-PA (3.91) followed by CH-DW (3.77) compared to those obtained from cultivar Kanoiwa Hakuto (Table 2). On the other hand, a significantly higher TA value was observed for KH-DW (2.34) and KH-PA (2.22) compared to ‘Changhowon Hwangdo’. The SSC of KH-DW (32.0 °Brix) was significantly the highest, followed by CH-PA (29.8) and the lowest value was found for KH-PA (26.2). The variation in chemical composition of peach extracts among samples has been already observed in previous studies (Wanpeng et al., 2017; Zhao et al., 2015) which might be due to cultivar (Jung et al., 2015; Kim et al., 2014a, 2012b), extraction method (Altemimi et al., 2016) and/or solvent variation (Kajdzanoska et al., 2011).

Table 1. Analysis of variance for the effect of extracting solvents (distilled water and prethanol-A) on the different physicochemical characteristics of unripe peach extract of ‘Changhowon Hwangdo’ and ‘Kanoiwa Hakuto’

Trait/Source of variation		df	F Value	Pr > F
pH
	Solvent	1	0.51	0.496
	Cultivar	1	55.89	<.0001
	Solvent × Cultivar	1	4.58	0.0648
	Total	11
Titratable acidity
	Solvent	1	154.01	<.0001
	Cultivar	1	3658.51	<.0001
	Solvent × Cultivar	1	23.11	0.0013
	Total	11
Total soluble solid
	Solvent	1	272.4	<.0001
	Cultivar	1	2.68	0.1401
	Solvent × Cultivar	1	817.92	<.0001
	Total	11
L* (Lightness)
	Solvent	1	255.64	<.0001
	Cultivar	1	6.11	0.0385
	Solvent × Cultivar	1	50.27	0.0001
	Total	11
a* (redness)
	Solvent	1	3594.06	<.0001
	Cultivar	1	792.53	<.0001
	Solvent × Cultivar	1	2445.13	<.0001
	Total	11
b* (yellowness)
	Solvent	1	36450.9	<.0001
	Cultivar	1	1127.56	<.0001
	Solvent × Cultivar	1	9348.28	<.0001
	Total	11
DPPH scavenging potential
	Solvent	1	0.78	0.4022
	Cultivar	1	6.38	0.0354
	Solvent × Cultivar	1	0.01	0.9159
	Total	11
Total phenol
	Solvent	1	415.56	<.0001
	Cultivar	1	1263.26	<.0001
	Solvent × Cultivar	1	6.53	0.0339
	Total	11
Total mineral
	Solvent	1	121.22	<.0001
	Cultivar	1	61.59	<.0001
	Solvent × Cultivar	1	18.09	0.0028
	Total	11

df, degree of freedom.

Table 2. General chemical properties of unripe peach extracts of ‘Changhowon Hwangdo’ and ‘Kanoiwa Hakuto’ in distilled water and prethanol-A

Properties	Sample
	CH-DW	CH-PA	KH-DW	KH-PA
pH	3.77 ± 0.04^b	3.91 ± 0.07^a	3.40 ± 0.02^c	3.36 ± 0.06^c
TA (g/100 mL)	1.51 ± 0.03^c	1.26 ± 0.02^d	2.34 ± 0.01^a	2.22 ± 0.03^b
Soluble solid (°Brix)	28.1 ± 0.1^c	29.8 ± 0.3^b	32.0 ± 0.2^a	26.2 ± 0.2^d

CH-DW: peach cv. Changhowon Hwangdo extracted with distilled water (1:10 w/v), CH-PA: peach cv. Changhowon Hwangdo extracted with prethanol-A (1:10 w/v), KH-DW: peach cv. Kanoiwa Hakuto extracted with distilled water (1:10 w/v) and KH-PA: peach cv. Kanoiwa Hakuto extracted with prethanol-A (1:10 w/v).

TA, titratable acidity as lactic acid.

Data are means±SD of triplicate measurements. Values followed by different superscripts in the same row are significantly different (P< 0.0.5).

Hunter’s Color Value of Unripe Peach Extracts

Hunter’s color values of unripe peach extracts were also significantly varied with cultivar, solvent and their interaction (Table 1). Lightness values of the samples extracted with prethanol-A, CH-PA (59.73) and KH-PA (58.15) were significantly (P< .05) higher compared to those extracted with distilled water, CH-DW (51.85) and KH-DW (54.50) (Table 3). On the contrary, the redness and yellowness values of samples extracted with distilled water were significantly (P< .05) higher compared to those with prethanol-A. The variation in the Hunter’s color values of the samples might be due to the difference in cultivar Kim et al. (2012b) and/or extracting solvent (Kajdzanoska et al., 2011). Naik et al. (2017) also found that the color of the extract varied with the solvent used which might be due to differential solubility of various phytochemicals in different solvents (Alothman et al., 2009) as well as the extraction method (Altemimi et al., 2016).

Table 3. Hunter color values of unripe peach extracts of ‘Changhowon Hwangdo’ and ‘Kanoiwa Hakuto’ in distilled water and prethanol-A

Sample	Color value
	L*	a*	b*
CH-DW	51.85 ± 0.07^c	-1.57 ± 0.02^a	20.42 ± 0.03^b
CH-PA	59.73 ± 0.04^a	-2.82 ± 0.01^d	18.96 ± 0.02^c
KH-DW	54.50 ± 0.09^b	-2.46 ± 0.02^b	21.40 ± 0.02^a
KH-PA	58.15 ± 1.16^a	-2.59 ± 0.03^c	16.95 ± 0.04^d

CH-DW: peach cv. Changhowon Hwangdo extracted with distilled water (1:10 w/v), CH-PA: peach cv. Changhowon Hwangdo extracted with prethanol-A (1:10 w/v), KH-DW: peach cv. Kanoiwa Hakuto extracted with distilled water (1:10 w/v) and KH-PA: peach cv. Kanoiwa Hakuto extracted with prethanol-A (1:10 w/v).

L*, lightness (100, white; 0, black); a*, redness (–, green; +, red); b*, yellowness (–, blue; +, yellow).

Data are means±SD of triplicate measurements. Values followed by different superscripts in the same column are significantly different (P< 0.0.5).

Mineral Content of Unripe Peach Extracts

The ANOVA study showed that the total mineral contents of different peach extracts were significantly different with cultivar, solvent and their interaction (Table 1). The predominant element was potassium in all the samples (Table 4). Similar results were found in previous studies (Manzoor et al., 2012; Saidani et al., 2017). Significantly higher potassium was measured in KH-DW (6990.44 ppm) and the least was found in CH-PA (4859.09 ppm). The total mineral content of KH-DW (7387.88 ppm) was the highest followed by CH-DW (6075.45 ppm). The results revealed that high amount of mineral can be extracted with distilled water compared to prethanol-A. A previous report (Dasola et al., 2014) also stated that water could better extract mineral elements compared to ethanol. Ultrasonication produces sufficient cavitation to create shear forces to break the cell walls which increases the diffusion of cell contents into the extraction solution (Altemimi et al., 2016; Jerman et al., 2010). Ultrasonication might have further increased (Choi and Lee, 2018) the mineral content of the samples extracted with distilled water. Elements like Mg, K, and Ca, which were found in high amounts in both cultivars with both solvents, are reported to have beneficial health effects in the prevention and treatment of essential hypertension (Houston and Harper, 2008). Similarly, iron that has an important role in the prevention of anemia (Ismail et al., 2011) was also extracted in the present study with both solvents. Results of the present study revealed that unripe peach could be a potential source of mineral elements.

Table 4. Mineral contents (ppm) of unripe peach extracts of ‘Changhowon Hwangdo’ and ‘Kanoiwa Hakuto’ in distilled water and prethanol-A

Element	Sample
	CH-DW	CH-PA	KH-DW	KH-PA
Na	94.58 ± 3.69^a	71.79 ± 2.79^c	61.42 ± 2.30^d	81.39 ± 3.02^b
Mg	165.36 ± 7.89^b	130.84 ± 5.58^c	199.79 ± 0.43^a	155.68 ± 6.14^b
K	5671.08 ± 226.52^b	4859.09 ± 197.96^c	6990.44 ± 54.57^a	5322.65 ± 197.66^b
Ca	77.24 ± 0.93^c	79.32 ± 2.77^c	107.43 ± 0.67^a	91.09 ± 3.06^b
Mn	1.51 ± 0.02^a	0.79 ± 0.04^c	0.98 ± 0.01^b	0.80 ± 0.02^d
Fe	18.10 ± 0.27^a	13.71 ± 0.13^b	13.65 ± 0.07^b	11.07 ± 0.06^c
Cu	3.07 ± 0.06^b	1.78 ± 0.01^d	3.66 ± 0.01^a	2.21 ± 0.02^c
Zn	44.51 ± 0.50^a	5.84 ± 0.04^d	10.51 ± 0.05^b	8.53 ± 0.09^c
Total	6075.45	5163.16	7387.88	5673.42

CH-DW: peach cv. Changhowon Hwangdo extracted with distilled water (1:10 w/v), CH-PA: peach cv. Changhowon Hwangdo extracted with prethanol-A (1:10 w/v), KH-DW: peach cv. Kanoiwa Hakuto extracted with distilled water (1:10 w/v) and KH-PA: peach cv. Kanoiwa Hakuto extracted with prethanol-A (1:10 w/v).

Data are means±SD of triplicate measurements. Values followed by different superscripts in the same row are significantly different (P< 0.0.5).

DPPH Radical Scavenging Activity and Total Phenol Content of Unripe Peach Extracts

The DPPH radical scavenging potential of peach extracts significantly (P <.05) varied with cultivar but not with the solvent, whereas the total phenol content significantly varied with cultivar, solvent and their interaction (Table 1). The total phenol contents of ‘Kanoiwa Hakuto’ (1098.63 and 1076.76 GAE µg/mL) for distilled water and prethanol-A were higher than those of ‘Changhowon Hwangdo’ (1060.04 and 1045.75 GAE µg/mL), respectively (Table 5). The high total phenolic content for distilled water-extracted samples might be due to the effect of ultrasonication. Previous report (Rasheed et al., 2012) also showed that conventional water extraction method (without sonication) yielded significantly low amount of total phenolic compounds compared to the alcohols. Ultrasonication can increase diffusivity of the solvent and subsequently desorption and solubility of different compounds, including phenols (Jerman et al., 2010). As stated earlier, the difference in the total phenol content of peach extracts among samples might be due to cultivar and/or extraction solvent variations. Extraction methods also significantly influence the chemical compositions of the extracts (Altemimi et al., 2016). Results of this experiment showed that unripe peach could be a good source of phenolic compounds which could be extracted even with a safe extracting solvent like distilled water using an unltrasonication system. Another report also mentioned a high amount of total phenol content in immature peach fruits which remarkably decreased at ripening (Andreotti et al., 2008).

Table 5. DPPH radical scavenging activities and total phenol contents of unripe peach extracts of ‘Changhowon Hwangdo’ and ‘Kanoiwa Hakuto’ in distilled water and prethanol-A

Sample	DPPH (% inhibition)	Total phenol content (GAE µg/mL of sample)
CH-DW	93.70 ± 0.21^a	1060.04 ± 2.00^c
CH-PA	93.65 ± 0.61^a	1045.75 ± 2.11^d
KH-DW	94.03 ± 0.51^a	1098.63 ± 1.62^a
KH-PA	94.63 ± 0.30^a	1076.76 ± 1.31^b

CH-DW: peach cv. Changhowon Hwangdo extracted with distilled water (1:10 w/v), CH-PA: peach cv. Changhowon Hwangdo extracted with prethanol-A (1:10 w/v), KH-DW: peach cv. Kanoiwa Hakuto extracted with distilled water (1:10 w/v) and KH-PA: peach cv. Kanoiwa Hakuto extracted with prethanol-A (1:10 w/v).

GAE, gallic acid equivalent.

Data are means±SD of triplicate measurements. Values followed by different superscripts in the same column are significantly different (P< 0.0.5).

Conclusion

Unripe peach extracts were prepared from two cultivars, Changhowon Hwangdo (CH) and Kanoiwa Hakuto (KH) with two extraction solvents, distilled water coupled with ultrasonication and prethanol-A, a food preservative. The pH, titratable acidity, soluble solid content and color value of unripe peach extracts varied with the cultivar, solvent and/or their interaction. Mineral content of peach extracts obtained from cultivar KH was higher than that of CH, and was higher for distilled water than that for prethanol-A. Similarly, the total accumulation of phenolic compounds was found higher in KH than CH, and with distilled water than that with prethanol-A. The results of this experiment showed that unripe peach fruits, which are generally discarded as agro-byproducts, could be used in food and cosmetic industries.

Conflict of Interest

The authors declare no conflict of interest.