Article pubs.acs.org/JAFC
Profile and Antioxidant Activity of Phenolic Extracts from 10 Crabapples (Malus Wild Species) Nan Li,† Junling Shi,*,†,§,⊗ and Kun Wang# †
College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi Province 712100, China School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an, Shaanxi Province 710072, China ⊗ Key Laboratory for Space Bioscience and Biotechnology, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an, Shaanxi Province 710072, China # Key Laboratory of the Use of Fruit Germplasm Resources, Ministry of Agriculture/Research Institute of Pomology, Chinese Academy Agricultural Sciences, Xingcheng, Liaoning 125100, China §
ABSTRACT: Phenolic products are highly demanded by the food and cosmetics industries and consumers due to their high antioxidant activities. To evaluate the potential of crabapples (Malus wild species) in preparing phenolic extracts, fruits of 10 crabapples grown in China were separately extracted with 80% (v/v) ethanol and ethyl acetate and the phenolic profiles, polyphenol (PC) and flavonoid contents (FC), and antioxidant activities of the extracts were analyzed. Chlorogenic acid, (−)-epicatechin, rutin, hyperin, and phlorizin appeared as the major phenolic compounds in all phenolic extracts. Ethanol extracts had PC of 302.83−1265.94 mg GAE/100g and FC of 352.45−2351.74 mg RE/100g, being 4.17 and 4.49 times those obtained in ethyl acetate extracts and much higher than those previously reported in apples. Malus wild species appeared as rich sources of phenolic compounds with high antioxidant activity, especially when high chlorogenic acid and rutin contents are emphasized. KEYWORDS: wild apple, polyphenol, flavonoid, antioxidant activity
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total phenolic content than methanol, acetone, or water,16 whereas Katalinić et al.17 reported 80% (v/v) aqueous ethanol was efficient in the extraction of phenolic compounds from many plant materials. Eighty percent (v/v) aqueous methanol,18 80% (v/v) aqueous ethanol,19 and 70% (v/v) aqueous acetone20 have also been used to extract phenolic compounds from apples (Malus) and resulted in high phenolic containing extracts. From the toxicological point of view, ethanol and water are safer than methanol and other organic solvents.21 However, it should be mentioned that ethyl acetate is normally used to prepare the phenolic extracts in the assays of obtaining phenolic profiles using high-performance liquid chromatography (RP-HPLC).22−25 Furthermore, ethyl acetate is more efficacious with respect to aqueous ethanol in extracting carotenoids, and apples are known to be rich in this class of compounds. More importantly, these extraction solvents can be easily removed from the obtained phenolic extracts by evaporation, and thus there is opportunity to use these solvents to extract polyphenols for food, pharmaceutical, or cosmetics application.26 In addition, the diversity in obtained phenolic profiles is theoretically expected when different extraction solvents are used in the preparation of phenolic extracts. However, this has not been comprehensively investigated before.
INTRODUCTION Phenolic compounds are of particular interest to food processors and consumers because they play a key role in preventing oxidation processes in human.1,2 They are also highly valued as they possess high antioxidant activities.3,4 Phenolic extracts are in high demand in the cosmetics and food industries due to their many functions promoting health benefits and preventing human diseases, such as reducing the occurrence of cancer,5 anti-inflammation,6−8 decreasing blood pressure,9 resisting neurodegenerative disorders,10 modulating gut microbial composition, 11 preventing obesity,12 and protecting against acute oxidative damage.13 Polyphenols derived from natural plants are used in edible oil, ham, cakes, chocolate, and other foods to prevent spoilage caused by oxidation and inhibit the generation of nitrosamines in these foods. Apple pomace is currently developed as a major source of phenolic compounds due to its high phenolic content and widespread occurrence in the world. To make apple trees more resistant to disease and suitable for diverse environments, Malus wild species are widely used as rootstocks in apple breeding besides being of importance in ecological diversity and use as an ornamental and economic Malus germplasm resource.14 Phenolic extracts are usually prepared using different solvents according to the solubility of the major phenolic compounds in the materials. Agourram et al. found 80% (v/v) aqueous acetone was more effective than 80% (v/v) aqueous methanol and 80% (v/v) aqueous ethanol in preparing phenolic extracts from fruit and vegetable byproducts.15 However, Seo et al. found ethanol was more efficient in producing phenolic extracts from Smilax china L. leaf with higher antioxidant activity and © 2014 American Chemical Society
Received: Revised: Accepted: Published: 574
October 9, 2013 December 25, 2013 January 7, 2014 January 7, 2014 dx.doi.org/10.1021/jf404542d | J. Agric. Food Chem. 2014, 62, 574−581
Journal of Agricultural and Food Chemistry
Article
Table 1. Malus Wild Species Analyzed, Skin Color Main Features, Dimensions, and Weightsa
a
code
Chinese name
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Lijiangshandingzi Xiaojinhaitang Huaguanhaitang Donghongguo Sankuaishihaitang01 Sankuaishihaitang02 Xiaofanshanhaitang Balenghaitang Pingdinghaitang Honghaitang
Malus wild species Malus Malus Malus Malus Malus Malus Malus Malus Malus Malus
rockii Rehder xiaojinensis Cheng et Jiang coronaria (L.) Mill. prunifolia (Wild) Borkh. robusta (Carr.) Rehd. robusta (Carr.) Rehd. robusta (Carr.) Rehd. micromalus (Carr.) Rehd. robusta (Carr.) Rehd. prunifolia Mill.
skin color
dimensions (mm)
deep red yellow with small areas of bright red red with small areas of yellow deep red with small areas of yellow-green bright red with small areas of yellow deep red/green bicolor bright red with small areas of yellow bright red with small areas of yellow dark red with small areas of yellow bright pink red/bright yellow bicolor
21.8 21.0 24.2 42.7 33.3 33.4 32.0 32.5 29.8 27.2
± ± ± ± ± ± ± ± ± ±
0.5 0.7 0.3 0.9 0.7 0.5 0.4 0.5 0.7 0.9
weight (g) 4.3 3.9 7.3 39.1 12.8 16.0 13.5 14.7 11.9 9.4
± ± ± ± ± ± ± ± ± ±
0.3 0.5 0.6 1.1 0.6 0.4 0.5 0.4 0.6 0.5
Means of 3 replicates ± standard errors or standard deviation. Preparation of Phenolic Extracts. Eighty percent aqueous ethanol and ethyl acetate were separately used to extract the cooled crabapple slices according to the methods reported by Zhao et al.32 and Kong et al.33 with slight modification. Briefly, sliced and blanched crabapples were ground to a fine jam using a blender (Media, Foshan, China). A 5 g jam sample was weighed and then separately extracted with 25 mL of 80% aqueous ethanol and ethyl acetate. The extraction was conducted for 30 min at 20 °C in a Unique-1400A ultrasonic bath (Shumei, Kunshan, China) at a power of 250 W. After the extraction, centrifugation at 8000g was carried out for 10 min. The supernatant was collected, and the residue was extracted and centrifuged again two more times. The supernatants of each extraction solvent were combined and evaporated to dry powder using an R-210 vacuum rotary evaporator (Büchi, Flawil, Switzerland) at 40 °C. The dry residues were dissolved in 10 mL of 50% aqueous methanol and stored at −20 °C for measurements of polyphenol content, flavonoid content, antioxidant activity, and phenolic profiles by high-performance liquid chromatography (HPLC). Measurement of Polyphenol and Flavonoid Contents. Measurement of polyphenol content (PC) was carried out using a modified colorimetric Folin−Ciocalteu method at 765 nm.34 In summary, 0.2 mL of extract was mixed with 1.0 mL of deionized water and 1.0 mL of freshly prepared Folin−Ciocalteu reagent (diluted 10fold in water). The mixing process was performed for 5 min before 0.8 mL of 7.5% (w/v) Na2CO3 solution was added. The above mixture was incubated at 25 °C in darkness for 30 min. Absorbance was obtained using a UVmini-1240 spectrophotometer (Shimadzu, Tokyo, Japan). A standard curve was created using gallic acid. Additional dilution was carried out when the measured absorbance value outranged the linear range of the standard curve. Results were expressed as milligrams of gallic acid equivalent per 100 g of fresh weight (mg GAE/100 g). Flavonoid content (FC) was determined using the colorimetric method at 510 nm according to the method of Wolfe et al.35 with slight modification. In brief, a 0.5 mL extract sample was mixed with 1.0 mL of deionized water and 0.15 mL of a 50 g/L NaNO2 solution. After 5 min, 0.3 mL of AlCl3 (100 g/L) was added. The mixture was incubated at 25 °C for 6 min, followed by the addition of 1.0 mL 1 mol/L NaOH before absorbance reading. A standard curve was made using rutin. Additional dilution was carried out when the measured absorbance value outranged the linear range of the standard curve. Results were expressed as milligrams of rutin equivalents per 100 g of fresh weight (mg RE/100 g). Antioxidant Activity Assays. The DPPH assay was performed according to the method reported by Ma et al.36 with minor modifications. A 50 μL extract was mixed with 2.0 mL of a 6.0 × 10−5 mol/L ethanol solution containing DPPH. After incubation at 25 °C for 30 min, the absorbance at 517 nm was read. A standard solution of Trolox was used to prepare a calibration curve. Results were expressed as micromoles of Trolox equivalent antioxidant capacity per 100 g ± SD fresh weight (μmol TE/100 g). The ABTS assay was carried out according to the method described by Tai et al.37 with slight modification. ABTS radical cation (ABTS•+)
Phenolic profiles of apple fruits have been extensively studied. Apple is found to be abundant in vitamin C,27 flavonoids,9 and polyphenols and has high antioxidant capacity.4,28,29 Apple juice has been reported to have strong inhibitory activity on the proliferation of HL-60 human leukemia cells.30 Belonging to Malus species, crabapples are supposed to possess similar nutrients, particularly phenolic compounds, as apples. However, no information of phenolic content and phenolic profiles of crabapples has been found in the literature. To characterize the phenolic profile, phenolic contents, and antioxidant activity of different Malus wild species and to evaluate the potential of these species in producing phenolic extracts, 10 different crabapples grown in China were collected and analyzed. Ethanol and ethyl acetate were both used in aspects of the above characteristics to compare which is better in preparing phenolic extracts with high phenolic content and antioxidant activities.
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MATERIALS AND METHODS
Chemicals. Folin−Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzenothiazoline-6-sulfonic) (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-teramethylchroman-2-carboxylic acid (Trolox), methanol, and phosphoric acid (HPLC grade) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Gallic acid, protocatechuic acid, (+)-catechin, chlorogenic acid, caffeic acid, (−)-epicatechin, p-coumaric acid, ferulic acid, rutin, hyperin, phlorizin, and quercetin (>98%) were purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China). Ethanol, ethyl acetate, Na2CO3, NaNO2, AlCl3, NaOH, K2S2O8, CH3COONa, CH3COOH, HCl, and FeCl3 were from local reagent shops in Xi’an, China. All chemicals were of analytical grade. Water was prepared by purification with a Milli-Q water purification system (Millipore, Bedford, MA, USA). Fruit of Crabapples. Fruits of 10 wild Malus species were collected from the garden of germplasm resources in Institute of Pomology, Chinese Academy of Agricultural Sciences (Xingcheng, Liaoning provionce). No manual protection was carried out during the whole growing season. All fruits were picked by hand at their ripeness season in September 2012 and immediately cooled and stored at −20 °C before the extraction and measurement were carried out within 1 month. The cross-sectional diameter of the fruits was measured using a vernier caliper (Xigong, Jiangsu, China). The main features of the analyzed fruits are reported in Table 1. Blanching of Crabapple Fruits. The fruits of different wild Malus species were treated according to the method previously reported by McGhie et al.19 and Lin et al.31 with slight modification. In brief, 100 g of fruit was cut into slices in approximately 3 mm thickness. The slices were subjected to steam blanching for 1 min and then immediately taken out and placed in a refrigerator (Haier, Qingdao, China) for cooling. 575
dx.doi.org/10.1021/jf404542d | J. Agric. Food Chem. 2014, 62, 574−581
Journal of Agricultural and Food Chemistry
Article
was produced by reacting a 7 mmol/L ABTS solution with 140 mmol/ L K2S2O8, and the mixture was kept in the dark at room temperature for 12−16 h before use. For each assay, the above stored solution was freshly diluted with ethanol until the absorbance at 734 nm reached 0.700 ± 0.05. In the measurement, 25 μL of phenolic extract was allowed to react with 3.0 mL of ABTS•+ solution at 25 °C for 30 min. Then the absorbance was taken at 734 nm. Results wee reported as micromoles of Trolox equivalent antioxidant capacity per 100 g ± SD fresh weight (μmol TE/100 g). The FRAP assay was performed according to the method described by Ma et al.36 with slight modification. The stock solutions included 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ in 40 mmol/ L HCl, and 20 mmol/L FeCl3. A fresh working FRAP solution was prepared using the above stock solution in ratio of 25 mL of acetate buffer, 2.5 mL of TPTZ solution, and 2.5 mL of FeCl3 solution. Twenty-five microliters of phenolic extract was allowed to react with 3.0 mL of fresh FRAP solution for 10 min at 37 °C. Readings of the colored product were taken at 593 nm. Results are reported as micromoles of Trolox equivalent antioxidant capacity per 100 g ± SD fresh weight (μmol TE/100 g). Analysis of Phenolic Profiles. The phenolic profiles of phenolic extracts were measured using HPLC after a filtration through a 0.45 μm filter. The HPLC analysis was carried out using a Waters 600E HPLC system (Waters, Milford, MA, USA) equipped with a Waters 2487UV detector and a Waters Symmetry C18 reversed-phase column (250 × 4.6 mm, 5 μm). The mobile phase consisted of solvent A (methanol) and solvent B [water−phosphoric acid (pH 2.6)] using the following gradient program: 0−15 min, 15−25% solvent A; 15−25 min, 25% solvent A; 25−65 min, 25−75% solvent A; 65−70 min, 75− 15% solvent A. All of the polyphenol and flavonoid standards were prepared in 50% methanol solution. The flow rate was kept constant at 1.0 mL/min throughout the measurement. The column temperature was set at 30 °C, and the injection volume was 5 μL. The UV detection wavelength was monitored at 280 nm because the phenolic extracts had obvious absorption peaks at 280 nm, but not at 320 and 350 nm. In addition, solely reading at 280 nm was also performed in phenolic analysis by Bhaskar et al.,38 Łata et al.,39 and Du et al.40 Individual phenols and flavonoids in the sample were identified on the basis of comparison of their UV−vis spectra and retention times with the corresponding standards.
Figure 1. Polyphenol content (A) and flavonoid content (B) of 10 wild Malus species. Data are the means of three replicates ± standard deviation.
Table 2. Comparison of Polyphenol Contents and Antioxidant Activities of All Malus Wild Species Obtained in Different Solvent Extracts ethanol extractsa d
PC FCe DPPHf ABTSg FRAPh
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RESULTS AND DISCUSSION Polyphenol and Flavonoid Contents. The PC and FC values obtained from ethanol extracts were significantly (P < 0.01) higher than those from ethyl acetate extracts (Figure 1). For wild Malus species of Xiaojin (Malus xiaojinensis Cheng et Jiang), the FC value obtained from ethanol extract was nearly 14-fold that obtained from ethyl acetate extract. A similar trend was also found by NurulMarian et al.41 for Barrington racemosa leaves. Theoretically, different extraction solvents are greatly diverse in polarity, and therefore extracting abilities of different compounds are different.33,42,43 Polyphenol and flavonoid are both polar due to their polyhydroxyl groups. Normally, the less polar solvents (ether, benzene, and chloroform) are effective in extracting aglycone flavonoids and more polar solvents (acetone, ethyl acetate, alcohol, and water) are effective in extracting glycoside flavonoids.44 Also, phenolic compounds have higher solubility in higher polarity solvents,45,46 such as ethanol. Ethyl acetate is comparatively more efficacious than aqueous ethanol in extracting carotenoids.33 The higher values of PC and FC obtained in ethanol extracts than in ethyl acetate extracts illustrated that the phenolic and flavonoid compounds in crabapples are of high polarity. In detail, PC values ranged from 302.83 to 1265.94 mg GAE/ 100 g (Figure 1A) with an average of 566.58 mg GAE/100 g (Table 2) for the ethanol extracts and from 62.96 to 215.21 mg GAE/100 g (Figure 1A) with an average of 135.97 mg GAE/
566.58 965.05 2110.07 5657.71 3665.22
± ± ± ± ±
91.19 184.70 260.58 974.14 773.60
ethyl acetate extractsb
ratioc
± ± ± ± ±
4.17 4.49 6.55 4.99 6.58
135.97 214.90 321.98 1133.73 556.74
16.36 34.25 46.33 157.30 111.36
Ethanol extracts: mean values ± standard errors of 10 crabapple samples in ethanol extracts. bEthyl acetate extracts: mean values ± standard errors of 10 crabapple samples in ethyl acetate extracts. c Ratio: mean values of ethanol extracts/ethyl acetate extracts. Ratio >1 indicates that the ethanol extracts had more PC and FC and were more antioxidant. Ratio
