Effects of Domestic Cooking Methods on Polyphenols and

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Effects of Domestic Cooking Methods on Polyphenols and Antioxidant Activity of Sweet Potato Leaves Hongnan Sun, Taihua Mu,* Lisha Xi, and Zhen Song Laboratory of Food Chemistry and Nutrition Science, Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, and Key Laboratory of Agro-Products Processing, Ministry of Agriculture, No. 2 Yuan Ming Yuan West Road, Haidian District, Beijing 100193, People’s Republic of China ABSTRACT: In this study, effects of boiling, steaming, microwaving, baking, and frying on proximate composition, total and individual polyphenol contents, and antioxidant activity of sweet potato leaves were investigated. An increase of 9.44% in total polyphenol content was observed after steaming, whereas decreases of 30.51, 25.70, and 15.73% were noted after boiling, microwaving, and frying, respectively. Decreases of 63.82 and 32.35% in antioxidant activity were observed after boiling and microwaving, respectively, whereas increases of 81.40, 30.09, and 85.82% in antioxidant activity were observed after steaming, baking, and frying, respectively. Eight phenolic compounds were identified in sweet potato leaves. The correlation analysis between content of individual phenolic compounds and antioxidant activity suggested that antioxidant activity could be mainly attributed to 4,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. It was suggested that steaming is a preferred method to maintain polyphenols and antioxidant activity of sweet potato leaves. KEYWORDS: domestic cooking, phenolic acids, RP-HPLC, caffeoylquinic acids, antioxidant activity, ORAC assay

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INTRODUCTION Polyphenols, widely distributed in vegetables, are substances having many beneficial effects on human health, for example, antiarteriosclerotic, antioxidant, antimicrobial, anticarcinogenic, and antimutagenic.1 However, most vegetables are cooked by boiling, steaming, microwaving, baking, or frying before being eaten by people in most of the world. These cooking methods will cause a series of changes in the physical characteristics and chemical composition of vegetables.2,3 Perla et al. investigated the effects of several domestic cooking methods on various polyphenols and antioxidants in mature stored potato tubers; the results showed that the total phenolics, flavonoids, flavonols, anthocyanins, lutein, and antioxidant activities of mature stored potato tubers were significantly reduced by baking and microwaving treatments.4 Zhang and Hamauzu studied antioxidant components, including phenolics, ascorbic acid, and carotenoids, of broccoli florets and stems, antioxidant activity, and their changes during conventional and microwave cooking and found that antioxidant components and antioxidant activity in broccoli suffered heavy losses during the cooking.3 Oboh determined the antioxidant−phytoconstituents properties of some green leafy vegetables and the effect of blanching on some of the antioxidant constituent and property and found that blanching of vegetables, although making green leafy vegetables more palatable and less toxic, reduced their antioxidant properties drastically.5 Pellegrini et al. evaluated the effect of common cooking practices (i.e., boiling, microwaving, and basket and oven steaming) on the phytochemical content (carotenoids, chlorophylls, glucosinolates, polyphenols, and ascorbic acid) and total antioxidant capacity of three generally consumed Brassica vegetables and came to the conclusion that some domestic cooking procedures increased the bioaccessibility of polyphenols and carotenoids, © 2014 American Chemical Society

highlighting the positive role of cooking on the nutritional qualities of vegetables.6 Sweet potato leaves can be harvested several times during the year, and their yields are much higher than those of green leafy vegetables.7 Furthermore, sweet potato leaves play a role in health promotion by improving immune function, reducing oxidative stress and free radical damage, reducing cardiovascular disease risk, and suppressing cancer cell growth.8 In the previous work, we assessed the nutritional and bioactive components of leaves from 40 sweet potato cultivars and reached the conclusion that sweet potato leaves are good sources of protein, fiber, polyphenols, and minerals, and the polyphenols therein are important antioxidants.9 Sweet potato leaves are an alternative source of green leafy vegetables during their off-season and have the potential to alleviate food shortages caused by natural disasters, for example, tsunamis, floods, or typhoons.10 At the present time, domestic cooking methods are very important for human diet and health, but there is little information on the effects of cooking on the polyphenols and antioxidant activity of sweet potato leaves. Thus, the objective of the present study was to elucidate the effects of different domestic cooking methods (boiling, steaming, microwaving, baking, and frying) on the proximate composition, polyphenols, and antioxidant activity of sweet potato leaves. Received: Revised: Accepted: Published: 8982

May 18, 2014 August 23, 2014 August 24, 2014 August 24, 2014 dx.doi.org/10.1021/jf502328d | J. Agric. Food Chem. 2014, 62, 8982−8989

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Table 1. Effect of Different Domestic Cooking Methods on Proximate Composition of Sweet Potato Leaves (g/100 g DW)a cooking method raw boiling steaming microwaving baking frying a

crude protein 24.04 25.56 28.66 28.11 29.56 17.11

± ± ± ± ± ±

0.08e 0.05d 0.14b 0.13c 0.28a 0.07f

crude fat 4.39 3.19 3.32 2.90 3.38 42.92

± ± ± ± ± ±

0.12b 0.14cd 0.12c 0.06d 0.11c 0.12a

crude fiber 11.33 10.47 10.41 10.05 10.96 20.51

± ± ± ± ± ±

0.46b 0.11cd 0.25cd 0.08d 0.06bc 0.19a

ash 7.84 7.44 8.16 8.17 8.82 4.67

± ± ± ± ± ±

0.62b 0.01b 0.01ab 0.12ab 0.01a 0.09c

Values are means ± SD of three determinations. Cooking methods that were not significantly different are assigned the same letter (p > 0.05).

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the mixture. Following 30 min, absorbance was measured at 736 nm in a UV1101 spectrophotometer (Hitachi, Japan). A calibration curve consisting of chlorogenic acid (CHA) standards (Sigma-Aldrich, Inc.), ranging from 0.02 to 0.10 mg/mL, was prepared. The linear regression equation was y = 8.7671x + 0.0068 and R2 = 0.9994. TPC of sweet potato leaves was calculated according to the following equation and expressed as milligrams per gram on a DW basis (mg/g DW).

MATERIALS AND METHODS

Plant Materials. Fresh sweet potato leaves (cv. Pushu 53, a popular leaf-vegetable sweet potato cultivar in mainland China) were obtained from the Academy of Agricultural and Forestry Sciences in Hebei Province, China. All sweet potatoes were planted with standard production practices at the experimental farm of the Academy of Agricultural and Forestry Sciences in Shijiazhuang city, Hebei province, at the beginning of June 2013. The average temperatures during the growth period of 2013 were as follows: June, 26 °C; July, 26 °C; August, 31 °C; September, 23 °C; and October, 12 °C. The leaves were collected in the middle of August. Cooking Methods. The sweet potato leaves were thoroughly cleaned with tap water and wiped with tissue paper. Eighteen hundred grams was taken and divided into six portions (300 g for each application). One portion was retained raw; the others were cooked in five different methods (reported by Perla et al.,4 Palermo et al.,11 and Tudela et al.12 with slight modification) in triplicate, as given below. For boiling, sweet potato leaves (100 g) were cooked in a stainless steel pan for 2 min after distilled water (500 mL) had come to a boil. For steaming, sweet potato leaves (100 g) were placed on tray in a steam cooker (Supor Co., Ltd., Hangzhou, Zhejiang, China) covered with a lid and steamed over boiling water for 2 min under atmospheric pressure. For microwave cooking, sweet potato leaves (100 g) were placed in a glass dish and cooked in a commercial 1000 W microwave oven for 2 min. For baking, sweet potato leaves (100 g) were baked at 204 °C for 2 min in a commercial oven after the oven had come to the prescribed temperature. For frying, sweet potato leaves (100 g) were cooked in a stainless steel pan for 2 min after corn oil (50 mL) had come to 180 °C. The treated and raw samples were freeze-dried and ground in a commercial grinder and stored at −20 °C in sealed aluminum bags until analyzed. Proximate Composition. Ash, crude fat, and crude protein contents were determined according to AOAC methods.13 Ash content was determined by weighing leaf samples before and after heat treatment (550 °C for 12 h). Crude fat content was determined according to AOAC method 960.39. Crude protein was assessed by the micro-Kjeldahl method, with a nitrogen to protein conversion factor of 6.25 (AOAC method 976.05). Crude fiber was determined according to ISO method 5498:1981.14 First, a sample of leaf powder was boiled in 0.255 M sulfuric acid for 30 min. The resulting insoluble residue was filtered, washed, and boiled in 0.313 M sodium hydroxide. After the sample had been filtered and washed, it was dried at 130 ± 2 °C for 2 h. Weight loss was determined at 350 ± 25 °C. Crude fiber content was expressed relative to the dry weight (DW) of leaf powder. Total Polyphenol Content (TPC). TPC was measured by using the Folin−Ciocalteu method.15 Briefly, 1 g of leaf powder was extracted with 20 mL of 70% (v/v) ethanol for 30 min at 50 °C and subjected to ultrasonic wave treatment. Following centrifugation at 5000g for 10 min at 4 °C, the residue was re-extracted twice with 70% ethanol as described above. The supernatants were pooled, concentrated in a rotary evaporator, and freeze-dried, thereby obtaining a crude extract. The crude extract was dissolved in 100 mL of distilled water; an aliquot (0.5 mL) was mixed with 1.0 mL of Folin−Ciocalteu reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA), previously diluted 10 times, and allowed to react at 30 °C for 30 min. Subsequently, 2.0 mL of saturated Na2CO3 (10%, w/v) was added to

TPC = (A − 0.0068)/8.7671 × V /M A is the absorbance, V is the volume of the crude extract diluent (mL), and M is the mass of the tested sweet potato leaf powder on a DW basis (g). Antioxidant Activity. Antioxidant activity of sweet potato leaf powder samples was determined by using the ORAC assay, as reported by Blasa et al.16 Briefly, 1 g of leaf powder sample was extracted with 20 mL of 70% (v/v) ethanol for 30 min at 50 °C and subjected to ultrasonic wave treatment. Following centrifugation at 5000g for 10 min at 4 °C, the residue was re-extracted twice with 70% ethanol as described above. The supernatants were pooled, concentrated in a rotary evaporator, and freeze-dried, thereby obtaining a crude extract. The assay was carried out using a microplate reader (Chameleon, Hidex, Turku, Finland) equipped with a temperature-controlled incubation chamber and an automatic injection pump. Incubator temperature was set at 37 °C. The reaction mixture for hydrophilic assay was the following: 200 μL of 0.096 μM fluorescein sodium salt in 0.075 M sodium phosphate buffer (pH 7.0) and 20 μL of sample or Trolox. A calibration curve was made each time with the standard Trolox (100, 50, 25 μM). The blank was 0.075 M sodium phosphate buffer (pH 7.0). The reaction was initiated with 40 μL of 0.33 M AAPH. Fluorescence was read at 485 nm excitation and 520 nm emission until complete extinction. ORAC values were expressed as micrograms of Trolox equivalents (TE) per milligram DW of sweet potato leaves and were the means ± standard deviation (SD) of three analyses. Quantification of Phenolic Compounds by RP-HPLC. Phenolic compounds in sweet potato leaf extract were evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC, Agilent Technologies, Palo Alto, CA, USA). Detection and quantification were carried out with a four-channel gradient pump, a diode array detector, a column heater, a degasser, and an autosampler (Agilent Technologies). Separations were conducted at 30 °C on an Agilent ZORBAX Eclips Plus C-18 reversed-phase column (150 mm × 4.6 mm length, 5 μm particle size). The mobile phases were (A) 0.1% (w/v) phosphoric acid solution and (B) acetonitrile. Flow rate was 1.0 mL/min. For analysis, 2 mg of dry sweet potato leaf extract was dissolved in 10 mL of 80% (v/v) methanol, and the injection volume of the sample solution was 20 μL. The gradient program was as follows: from 20 to 65% B from 0 to 15 min, from 65 to 80% B from 15 to 15.1 min, and 80% B maintained from 15.1 to 20 min. Spectral data from 200 to 800 nm were recorded, and the polyphenol chromatograms were monitored at 326 nm. Rutin, quercetin, caffeic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid (SigmaAldrich, Inc.) were used as standards. Identification and quantitative analysis were done by comparison with standards. The amount of each phenolic compound was expressed as milligrams per gram of sweet potato leaves (DW). 8983

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Figure 1. Effect of different domestic cooking methods on total polyphenol content (A) and antioxidant activity (B) of sweet potato leaves. Values are means ± SD of three determinations. Cooking methods that were not significantly different are assigned the same letter (p > 0.05).

Table 2. Retention Time, Maximum Absorbance, and Linear Calibration Curves of Phenolic Compounds Found in Sweet Potato Leaves peak

retention time (min)

HPLC-DAD λmax (nm)

identity

1 2 3 4 5 6 7 8

1.47 1.91 2.10 2.92 4.16 4.54 4.88 6.87

326 326 326 326 326 326 326 326

5-O-caffeoylquinic acid 3-O-caffeoylquinic acid 4-O-caffeoylquinic acid caffeic acid 4,5-di-O-caffeoylquinic acid 3,5-di-O-caffeoylquinic acid 3,4-di-O-caffeoylquinic acid 3,4,5-tri-O-caffeoylquinic acid

Statistical Analyses. The results were expressed as means ± SD of three replicates. Statistical analyses were performed using SAS version 8.1 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05.

linear calibration curve y y y y y y y y

= = = = = = = =

11.372x − 0.428 9.909x + 0.286 25.894x − 17.128 28.183x − 1.211 9.208x − 7.244 18.056x − 18.405 15.353x − 12.021 6.218x − 5.158

R2 0.996 0.999 0.998 0.999 0.998 0.998 0.998 0.995

bitter melon vegetable and found that the fat content of fried sample increased from 3.6 to 67%, because frying resulted in the expulsion of water and the absorption of fat.17 Fillion and Henry have already reported that the initial composition of the food (water and fat content) has a great effect on fat uptake during frying, and plant foods, which initially have high water and low fat contents, will absorb a large amount of frying fat.18 There was no significant difference between the crude fiber content of raw sweet potato leaves and that of baked sweet potato leaves. Compared with raw sweet potato leaves, boiling, steaming, and microwaving decreased the crude fiber content of sweet potato leaves significantly, whereas frying increased the crude fiber content significantly (p < 0.05). It was suggested that boiling caused a great loss of water-soluble dietary fiber. Although sweet potato leaves were not exposed to water, a large amount of cytochylema would flow out during steaming and microwaving treatments, causing the loss of water-soluble dietary fiber. In contrast, fried samples had a higher level of fiber than raw or other cooked samples. Murniece et al. investigated the nutritional composition and energy content of several varieties of potatoes prepared by traditional cooking methods and got the similar result we did, that the fiber content of deep-fat-fried samples was higher than that of uncooked samples.19 The possible reason was that frying could lead to structural damage of vegetable cells to maximum extent, inducing great loss of other liposoluble constituents in sweet potato leaves; as a result, the fiber content was relatively increased. The ash content of sweet potato leaves cooked by boiling, steaming, and microwaving did not show significant difference when compared with that of raw sweet potato leaves. Baking

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RESULTS AND DISCUSSION Proximate Composition. The effect of cooking methods on proximate composition of sweet potato leaves is shown in Table 1. Compared with the crude protein content in raw sweet potato leaves, boiling, steaming, microwaving, and baking increased crude protein content significantly, whereas frying decreased crude protein content significantly (p < 0.05). The possible reason was that, in the processes of boiling, steaming, and baking, other constituents, for example, fat and fiber, were partially lost or destroyed, resulting in the relative increase of protein content in sweet potato leaves; in contrast, the absorption of fat through frying caused an increase of dry matter, resulting in the relative decrease of protein content in sweet potato leaves. Compared with the crude fat content in raw sweet potato leaves, the crude fat contents of sweet potato leaves cooked by boiling, steaming, microwaving, and baking were decreased significantly, whereas frying increased crude fat content significantly (p < 0.05). During boiling, steaming, and microwaving treatments, volatile and water-soluble fatty acids would be partialy lost or destroyed, which induced the decrease of fat content in sweet potato leaves. Fried samples had a higher level of fat than raw or other cooked samples, mainly due to the absorption of fat by the sweet potato leaves. Similar findings were already reported by Donya et al., who investigated the effects of cooking methods on the proximate composition of 8984

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Figure 2. Chromatograms of phenolic standards and sweet potato leaf extracts at 326 nm in HPLC. The identities of the compounds identied by numbered peaks are given in Table 2.

compared to other cooking treatments. Besides, baking induced greater loss of other constituents, for example, fat, from raw sweet potato leaves, resulting in the relative increase of ash content. On the contrary, inorganic solids partly flowed out from sweet potato leaf cells during frying treatment, causing the decrease of ash content.

increased the ash content of sweet potato leaves significantly, whereas frying decreased the ash content significantly (p < 0.05). Our results were very different from those reported by Gidamis et al., who investigated the effect of traditional cooking methods on nutrient contents in young Moringa leaves and immature pods and found a significant reduction of ash content as the result of cooking.20 Baking caused the least loss of ash, 8985

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Table 3. Contents of Individual Phenolic Compounds in Sweet Potato Leaves Treated by Different Domestic Cooking Methods (mg/g of DW)a cooking methods identity 5-O-caffeoylquinic acid 3-O-caffeoylquinic acid 4-O-caffeoylquinic acid caffeic acid 4,5-di-O-caffeoylquinic acid 3,5-di-O-caffeoylquinic acid 3,4-di-O-caffeoylquinic acid 3,4,5-tri-O-caffeoylquinic acid sum of phenolic compounds a

raw 2.58 3.06 13.55 4.62 27.23 25.02 14.18 10.68 100.92

± ± ± ± ± ± ± ± ±

boiling 0.16a 0.31a 1.36ab 0.81b 2.28ab 1.26ab 0.44b 0.00c 6.62a

1.55 1.16 11.20 0.62 19.60 18.09 12.90 10.84 75.95

± ± ± ± ± ± ± ± ±

0.11d 0.35c 0.00c 0.00c 0.13c 0.07c 0.04c 0.00bc 0.30b

steaming 1.93 1.63 14.67 0.75 29.84 26.96 17.71 11.08 104.58

± ± ± ± ± ± ± ± ±

0.16c 0.25b 0.33a 0.04c 2.88a 2.53a 1.16a 0.17a 7.52a

microwaving 2.63 1.14 13.17 0.74 24.28 23.14 14.91 10.81 90.82

± ± ± ± ± ± ± ± ±

0.22a 0.12c 0.45b 0.02c 1.47b 1.09b 0.60b 0.03c 4.01ab

baking 2.25 3.06 13.53 5.80 30.10 26.01 14.38 11.07 106.21

± ± ± ± ± ± ± ± ±

0.05b 0.06a 0.52b 0.59a 3.81a 2.80ab 1.04b 0.28ab 9.04a

frying 1.76 1.32 12.86 0.75 30.04 25.90 18.32 11.11 102.07

± ± ± ± ± ± ± ± ±

0.00cd 0.06bc 0.19b 0.02c 0.54a 0.38ab 0.08a 0.10a 0.82a

Values are means ± SD of three determinations. Cooking methods that were not significantly different are assigned the same letter (p > 0.05).

Total Polyphenols. Figure 1A shows the effect of cooking methods on TPC of sweet potato leaves. Compared with raw sweet potato leaves, the TPC of sweet potato leaves cooked by boiling, microwaving, and frying decreased significantly, whereas steaming increased the TPC of sweet potato leaves significantly (p < 0.05). There was no significant difference between the TPC of raw and baked sweet potato leaves. An increase of 9.44% in TPC was observed after steaming, whereas decreases of 30.51, 25.70, and 15.73% was noted after boiling, microwaving, and frying, respectively. The changes in TPC agreed with the findings of Wachtel-Galor et al., who investigated the effect of cooking on TPC in cauliflower and found that steaming showed the highest TPC (an increase of 45%), followed by boiling (a loss of 4%), and microwaving (a loss of 39%) showed the lowest value.21 On the other hand, Pellegrini et al. found that oven steaming was the most disadvantageous method for cooking fresh Brussels sprouts.6 The effect of cooking depends on several factors, such as the cooking procedure, degree of heating, leaching into the cooking medium, solvent used for extraction, pH, and surface area exposed to water and oxygen.21 For example, Roy et al. investigated the effect of water temperature (50−100 °C) and cooking time (10, 30, 75 min) on TPC in cabbage and found a decrease of TPC as temperature and cooking duration increased.22 Besides, different plants contain various compounds, some of which are thermally labile and some are not and, therefore, the same cooking method may have different effects on different types of plants.23 In this study, the depletion of TPC in sweet potato leaves after boiling, microwaving, and frying could be due to phenolic breakdown during cooking.24 For steaming and baking, the basis of the increased or unchanged TPC in sweet potato leaves could not be categorically stated; however, it could be attributed to the possible breakdown of complex structures liberating individual phenolics and/or phenolic degradation products, which could also react with the Folin−Ciocalteu reagent.25 Antioxidant Activity. The effect of cooking method on antioxidant activity of sweet potato leaves is shown in Figure 1B. The antioxidant activity of raw sweet potato leaves was 1.28 ± 0.07 μg Trolox equiv/mg, DW. Compared with raw sweet potato leaves, the antioxidant activity of sweet potato leaves cooked by boiling and microwaving was decreased significantly, whereas that of sweet potato leaves cooked by steaming, baking, and frying was increased significantly (p < 0.05). Decreases of 63.82 and 32.35% in antioxidant activity were noted after boiling and microwaving, respectively, which was in agreement with the loss of TPC in sweet potato leaves. Price et al. found

that only 18% of TPC was retained in broccoli after boiling, the rest being largely leached into cooking water,26 which supported the data in this study and suggested that boiling caused a decrease of antioxidant activity of sweet potato leaves. The lower antioxidant activity of microwaved samples was perhaps due to a greater thermal effect, rather than the microwaving per se.21 In contrast, increases of 81.40, 30.09, and 85.82% in antioxidant activity were observed after steaming, baking, and frying, respectively, which was not in accordance with the changes of TPC in sweet potato leaves. This was perhaps due to the production of other redox-active secondary plant metabolites or breakdown products, but was highly likely to be related to the more efficient release of other antioxidants from intracellular proteins, changes in plant cell wall structure, and matrix modifications.27 Quantification of Phenolic Compounds by RP-HPLC. It is obvious that the TPC measured by the Folin−Ciocalteu method does not give a full picture of the qualification or quantification of the phenolic compounds in the sweet potato leaf extracts. Therefore, the phenolic compounds in the sweet potato leaf extracts cooked by different methods were determined by the HPLC method, and the HPLC results are presented in Table 2 and Figure 2. Eight phenolic compounds, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, caffeic acid, 4,5-di-O-caffeoylquinic acid, 3,5-di-Ocaffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, and 3,4,5-tri-Ocaffeoylquinic acid, were identified by comparison with the retention times and UV spectra of authentic standards analyzed under identical conditions, whereas the quantitative data were calculated from their respective regression equations. On the basis of the plots of the peak area (y) versus concentration (x, mg/g, DW), the regression equations of the eight phenolic compounds detected in sweet potato leaves and their correlation coefficients (R2) were obtained and are shown in Table 2. The contents of individual phenolic compounds in sweet potato leaves treated by different cooking methods are shown in Table 3. In a comparison of the amounts of the eight phenolic compounds present in raw sweet potato leaves, 4,5-diO-caffeoylquinic acid > 3,5-di-O-caffeoylquinic acid > 3,4-di-Ocaffeoylquinic acid > 4-O-caffeoylquinic acid > 3,4,5-tri-Ocaffeoylquinic acid > caffeic acid > 3-O-caffeoylquinic acid > 5O-caffeoylquinic acid. For 5-O-caffeoylquinic acid, boiling, steaming, baking, and frying decreased its content significantly, whereas microwaving did not cause a significant change. For 3-O-caffeoylquinic acid, boiling, steaming, microwaving, and frying induced significant losses, whereas baking did not change its content significantly. 8986

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Figure 3. Correlation between the content of (a) 5-O-caffeoylquinic acid content and antioxidant activity (R = −0.16433; p = 0.7557); (b) 3-Ocaffeoylquinic acid content and antioxidant activity (R = 0.12552; p = 0.8127); (c) 4-O-caffeoylquinic acid content and antioxidant activity (R = 0.67485; p = 0.1414); (d) caffeic acid content and antioxidant activity (R = 0.01559; p = 0.9766); (e) 4,5-di-O-caffeoylquinic acid content and antioxidant activity (R = 0.90926; p = 0.0120); (f) 3,5-di-O-caffeoylquinic acid content and antioxidant activity (R = 0.86450; p = 0.0263); (g) 3,4-diO-caffeoylquinic acid content and antioxidant activity (R = 0.90267; p = 0.0137); and (h) 3,4,5-tri-O-caffeoylquinic acid content and antioxidant activity (R = 0.76352; p = 0.0773) of sweet potato leaves treated by different domestic cooking methods.

4-O-Caffeoylquinic acid was the most abundant monocaffeoylquinic acid in sweet potato leaves, and among the used cooking methods, only boiling decreased its content significantly. For caffeic acid, baking increased its content significantly, whereas the rest of the cooking methods caused significant losses of caffeic acid. Wang and Ho reported that during thermal treatment, the hydrolysis of chlorogenic acid could produce caffeic acid,28 but our result was not in accordance with it, suggesting that boiling, steaming, microwaving, and frying induced the leaching out or degradation of caffeic acid. Guillot et al. reported that under mild pyrolysis caffeic acid could be degraded into tetraoxygenated 1,3-cis- and 1,3-trans-phenyl-

indane isomers, which showed comparable antioxidant activity and were 8-fold more active than butylated hydroxytoluene (BHT),29 and this might make some contribution to the relatively high antioxidant activity of steamed and fried sweet potato leaves. 4,5-Di-O-caffeoylquinic acid was the most abundant dicaffeoylquinic acid in sweet potato leaves, and only boiling induced significant loss of 4,5-di-O-caffeoylquinic acid. For 3,5-di-O-caffeoylquinic acid, only boiling decreased its content significantly. For 3,4-di-O-caffeoylquinic acid, steaming and frying increased its content significantly, whereas boiling decreased its content significantly. For 3,4,5-tri-O-caffeoylquinic acid, steaming, baking, and frying induced significant increases, 8987

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important antioxidants. Understanding the antioxidant activity of sweet potato leaves can help human beings enhance antioxidant intake, and this work must consider the effects of cooking on antioxidant activity. The results presented here clearly demonstrated that cooking could make the antioxidant activity of cooked sweet potato leaves quite different from that of the uncooked leaves. This was perhaps due to a variety of effects, including damage, flowing out, and transformation of the constituents in sweet potato leaves. In this study, steaming was found to retain or enhance TPC and antioxidant activity of sweet potato leaves best, followed by frying, baking, microwaving, and then boiling. Cooking in water seemed to cause a leakage of polyphenols into the cooking water. The correlation between the content of individual phenolic compounds and antioxidant activity suggested that the antioxidant activity of sweet potato leaves could be mainly attributed to 4,5-di-Ocaffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-Ocaffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid.

whereas there were no significant differences between raw and other treated sweet potato leaves. The above-mentioned results indicated that most of the individual phenolic compounds measured by HPLC in sweet potato leaves showed the same general trend in their content, that is, decreases after boiling and better retention after other cooking methods. Especially, there is a common point for steamed and fried sweet potato leaves that the contents of both 3,4-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid increased significantly compared with raw samples, which is in accordance with the trend of antioxidant activity, indicating that 3,4-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid might be the main contributor to the antioxidant activity of sweet potato leaves. A sum of individual phenolic compounds detected by HPLC of 100.92 mg/g DW was obtained for raw sweet potato leaves. In steamed sweet potato leaves, the sum of individual phenolic compounds was 104.58 mg/g DW, 3.63% more than for raw samples. This value is far from the increase in antioxidant activity of 81.4% of steamed samples. The same trend could be seen for baked sweet potato leaves and fried ones. This result demonstrated a production of antioxidant compounds other than polyphenols. The interesting fact that has to be more pointed out is that these substances do not react in the Folin− Ciocalteu system, because in Figure 1A, only a slight increase of TPC has been detected in steamed sweet potato leaves in comparison with raw sweet potato leaves. It is very hard to identify which individual phenolic compound was the biggest contributor to the antioxidant activity of sweet potato leaves. Palermo et al. found that different cooking treatments caused an increase in total antioxidant activity of artichokes and ascribed this increase to the increase of 5-O-caffeoylquinic and 1,5-di-O-caffeoylquinic acid.11 However, our result was very different from theirs. To tell the relationship between individual phenolic compounds and antioxidant activity of sweet potato leaves clearly, we analyzed the correlation between the content of individual phenolic compounds and antioxidant activity of sweet potato leaves by Pearson test using SAS version 8.1 software (Figure 3). 4,5-Di-O-caffeoylquinic acid showed the biggest correlation coefficient, followed by 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4,5-tri-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, caffeic acid, and 5-O-caffeoylquinic acid. Therefore, the antioxidant activity of sweet potato leaves could be mainly attributed to the increases in the contents of 4,5-di-Ocaffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-Ocaffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. The antioxidant activity of phenolics compounds is mainly due to redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers, heavy metal chelators, and hydroxyl radical quenchers.30 The di- and tricaffeoylquinic acids have more hydroxyl groups in their molecular structure,31 making them more active than monocaffeoylquinic acids. The TPC calculated from the data obtained by HPLC analysis was lower than that estimated by using the Folin− Ciocalteu method. One of the possible reasons is that the Folin−Ciocalteu method suffers from a number of interfering substances [particularly sugars, aromatic amines, sulfur dioxide, ascorbic acid and other enediols and reductones, organic acids, and Fe(II)].32 Another possible reason is the limitation of individual phenolic compounds used as standard.33 In conclusion, sweet potato leaves are good sources of protein, fiber, and minerals, and the polyphenols therein are

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AUTHOR INFORMATION

Corresponding Author

*(T.M.) Phone: (+86) 10 62815541. Fax: (+86) 10 62815541. E-mail: [email protected]. Funding

This research was supported by Earmarked Fund for the China Agriculture Research System (CARS-11-B-19). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Academy of Agricultural and Forestry Sciences in Hebei Province for providing the sweet potato leaves used in this study.

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ABBREVIATIONS USED CHA, chlorogenic acid; CHAE, chlorogenic acid equivalents; RP-HPLC, reversed-phase high-performance liquid chromatography; SD, standard deviation; TE, Trolox equivalents; TPC, total polyphenol content

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REFERENCES

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