LWT - Food Science and Technology 79 (2017) 260e266

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LWT - Food Science and Technology

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Different thermal drying methods affect the phenolic profiles, their bioaccessibility and antioxidant activity in tomentosa (Ait.) Hassk berries

** Guanghe Zhao a, b, Ruifen Zhang a, b, , Lei Liu a, Yuanyuan Deng a, Zhencheng Wei a, * Yan Zhang a, Yongxuan Ma a, Mingwei Zhang a, a Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/ Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510610, b College of Food Science & Technology, Huazhong Agricultural University, Wuhan 430070, China article info abstract

Article history: Extended shelf life and convenient industrial application have led to increasingly wide use of drying Received 22 August 2016 technology in and vegetables processing. However, drying process usually has a negative effect on Received in revised form the active ingredients in fruits and vegetables. In this study, the effects of three thermal drying methods 19 November 2016 (hot air drying (HD), microwave drying (MD) and combined microwave-hot-air-drying (CD)) on phenolic Accepted 14 January 2017 profiles, their bioaccessibility and antioxidant activity in Rhodomyrtus tomentosa berries were estimated. Available online 17 January 2017 The total phenolics, flavonoids and anthocyanins contents of CD-berries were significantly higher than those of HD- and MD-ones. CD-berries had higher contents of individual phenolics and showed stronger Keywords: Rhodomyrtus tomentosa berries antioxidant activity than HD- and MD-ones. Conversely, the bioaccessibility of phenolics in HD-berries Drying methods was remarkably higher than that of MD- and CD-ones, although the latter 2 provided more bio- Phenolics accessible phenolics than the former after in vitro gastrointestinal digestion. Thus, combined microwave- Antioxidant activity hot-air-drying (CD) is a more suitable drying technique for Rhodomyrtus tomentosa berries to maintain Bioaccessibility their phenolics and antioxidant activity. © 2017 Published by Elsevier Ltd.

1. Introduction downy rose-myrtle, is a belonging to family. It originated from South-East , and mainly distributed in China, Epidemiological studies have shown that consumption of fruits , , Indonesia, and Vietnam (Lai et al., 2015; Liu, and vegetables may offer protection against various chronic dis- Guo, & Sun, 2012). According to a preliminary statistics, the pro- eases, including cardiovascular disease, type 2 diabetes, cancers, duction of Rhodomyrtus tomentosa berries is approximately 100 and may impart other health benefits (Barrett & Lloyd, 2012; Del thousand tons per year in China. It has long been used to treat Rio et al., 2013; Plaza et al., 2011). Increasing evidences have diarrhoea, dysentery, traumatic hemorrhage and also to strengthen shown that these health-promoting properties of fruits and vege- immunity (Agro Forestry Tree Database, 1992; Do, 2011; Geetha, tables are mainly due to their phytochemical constituents, espe- Sridhar, & Murugan, 2010; Institute of Chinese Medicine, 2010). cially phenolics (Crozier, Jaganath, & Clifford, 2009; Noratto, Porter, As a good source of phenolics, till date, more than 19 individual Byrne, & Cisneros-Zevallos, 2009). phenolics, including anthocyanins, phenolic acids, flavonols, ella- Rhodomyrtus tomentosa (Ait.) Hassk, commonly known as gitannins, and stilbenes, have been identified in Rhodomyrtus tomentosa berries (Cui et al., 2013; Lai et al., 2013; Liu et al., 2012; Wu et al., 2015). Furthermore, Rhodomyrtus tomentosa berries * Corresponding author. have been reported strong antioxidant activities and abundant ** Corresponding author. Sericultural & Agri-Food Research Institute, Guangdong supply of nutrients (Cui et al., 2013; Huang, Cai, Corke, & Sun, 2010; Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Lai et al., 2015; Wu et al., 2015). In view of the excellent nutritional Agriculture/Guangdong Key Laboratory of Agricultural Products Processing, fl Guangzhou 510610, China. properties and delicious avor, Rhodomyrtus tomentosa berries E-mail addresses: [email protected] (R. Zhang), [email protected] became a popular regional . (M. Zhang). http://dx.doi.org/10.1016/j.lwt.2017.01.039 0023-6438/© 2017 Published by Elsevier Ltd. G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266 261

However, due to short maturity and soft texture, Rhodomyrtus HD: Samples were dried at 70 C in a DHG-9240A electro- tomentosa berries can be easily infected by mold. The timely drying thermal constant-temperature dry box (Shanghai Keelrein instru- of ripe Rhodomyrtus tomentosa berries can effectively prolong their ment Co.Ltd.) for 12 h. storage time and contribute to their usage in food and nutraceutical MD: Samples were dried at 136 W in a MG823LA3-NR domestic production. Traditionally, Rhodomyrtus tomentosa berries are dried microware oven (Midea Group Co. Ltd., China) for 4 h (heat for 300 s in sunlight or shade, which is time consuming, and worse than that then stop for10 s, and again). is mustiness, a common problem due to prolonged drying duration. CD: Samples were dried at 136 W in a household microware Till date, the main industrial methods for fruits and vegetables mentioned above for 2.5 h (heat for 300 s then stop for10 s, and drying include hot air drying, microwave drying and freeze drying. again), then dried at 70 C in a electrothermal constant- Freeze drying is considered as the best drying method for main- temperature dry box as mentioned above for 1 h. taining the maximum amount of phenolic components in fruits but Water contents of the dried samples were approximately 5%. All high equipment cost limits its large scale application. Drying pro- dried Rhodomyrtus tomentosa berries were grinded into powder cesses may have positive or negative effects on phenolic compo- with a FW80 disintegrator (Tianjin Taisite Instrument Co., LTD) (80 nents in fruits and vegetables products compared to their fresh mesh) and stored in refrigerator at 20 C in sealed polypropylene counterparts. In previous research, hot air drying increased bags for further analysis. phenolic contents in tomato at 60e80 Cby13e29% (Chang, Lin, Chang, & Liu, 2006), whereas decreased it in murta berries at 2.3. Extraction of phenolics 40e80 C by more than 70% (Rodríguez et al., 2014). In addition, microwave drying increased phenolic contents in apple slices by The phenolics were extracted by adding 50 mL of 80% acetone 5e31% at 300 W (Tarko, Duda-Chodak, & Tuszunski, 2009), but into 1.0 g of Rhodomyrtus tomentosa berries powder (Liu et al., _ decreased it in goldenberry up to 60% at 160 W (Izli,Yıldız, Ünal, 2015). The mixture was shaken using a SHA-C constant tempera- Isık, & Uylas¸er, 2014). It was observed that drying process can ture oscillation water-bath (Changzhou Aohua apparatus Co.Ltd., have variable effects on fruit's phenolics and mainly depend on the Changzhou, China) at 200 rpm at room temperature for 24 h. The type of drying process and fruit variety. Besides the effects on supernatants were removed after centrifugation at 6800g for phenolics, drying process may alter the microstructure of fruits and 10 min and concentrated at 45 C by using a rotary evaporator (N- subsequently affect the bioaccessibility of their phenolics after 1200B, Eyela, Japan). The concentrated extract was then recon- gastrointestinal digestion (Aydin & Gocmen, 2015; Bondaruk, stituted to a final volume of 10 mL with methanol and stored Markowsk, & Błaszczak, 2007; Caparino et al., 2012). at 20 C until further analysis. Till date, researches on Rhodomyrtus tomentosa berries were focused on the estimation of nutritional, phytochemical profile, and 2.4. Content of total phenolics, flavonoids, and anthocyanins antioxidant activity. Little is known about the effects of drying methods on health-promoting properties of Rhodomyrtus tomen- 2.4.1. Total phenolic content tosa berries. Therefore, this study aims to evaluate the effects of The total phenolic content was analyzed by Folin-Ciocalteu three thermal drying methods (hot air drying (HD), microwave colorimetric method as previously reported by Dewanto, Wu, drying (MD) and combined microwave-hot-air-drying (CD)) on Adom, & Liu, (2002). Total phenolic content was expressed as mg phenolic profiles, their bioaccessibility and antioxidant activity in of gallic acid equivalents (GAE) per g dry weight (DW) of sample. Rhodomyrtus tomentosa berries. The results presented in this study could provide scientific basis for choosing the right drying process 2.4.2. Total flavonoids content of Rhodomyrtus tomentosa berries. The total flavonoids content was determined using the aluminium chloride colorimetric method described by Ti 2. Materials and methods et al.(2014). Total flavonoids content was expressed as mg of rutin equivalents (RE) per g dry weight (DW) of sample. 2.1. Chemical and reagents 2.4.3. Total anthocyanins content 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH$), Folin & Cio- The quantification of total anthocyanins was evaluated by the calteu's phenol reagent, 2,4,6-tripyridyl-s-triazine (TPTZ), 6- pH differential method described by Lee, Durst, and Wrolstad hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), (2005). The anthocyanin content was expressed as mg of cyani- 2,20-Azobis-(2-amidinopropane) dihydrochloride (ABAP) and 30,60- din-3-O-glucoside equivalents (CGE) per g dry weight (DW) of Dihydroxy-spiro[isobenzofuran-1(3H),9’-(9H)-xanthene]-3-one sample. disodium salt (FL) were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Ascorbic acid, gallic, syringic, ferulic acid, proto- 2.5. HPLC determination of phenolic compositions catechuic acid, cyanidin-3-O-glucoside, rutin, quercetin, picea- tannol and resveratrol were purchased form Aladdin Reagents Phenolic compositions were determined according to a method (Shanghai, China). All other chemicals used were of analytical reported by Ti et al.(2014) and Lai et al. (2013) with some modifi- grade. cation. In brief, all samples were analyzed on an Agilent 1260 HPLC system (Waldbronn, Germany) equipped with a DAD detector and 2.2. Drying processes autosampler, using a 250 4.6 mm, 5 mm Agilent Zorbox SB-C18 column (Palo Alto, CA, USA). The conditions used were: column Fresh mature Rhodomyrtus tomentosa berries were purchased temperature 25 C; injection volume 20 mL; total HPLC run time from a local market in Hezhou city, Guangxi Zhuang Autonomous 55 min; mobile phase, acetonitrile (solution A) & 0.4% acetic acid Region, P. R. China. Samples were quickly sent to the laboratory. aqueous solution (solution B). The mobile phase was programmed Unblemished and mature fruits were selected for further treat- as follows: 0e40 min, solution B 5e25%; 40e45 min, solution B ment. About 200 g of Rhodomyrtus tomentosa berries were spread 25e35%; 45e50 min, solution B 35e50%; 50e55 min, solution B 50- evenly on a microwave ovenable polypropylene tray and dried by 5%. The mobile phase was pumped at a constant flow rate of 1.0 mL/ different methods as follows: min. Simultaneous monitoring was set at 280 nm for quantification 262 G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266 of phenolic acids (gallic acid, syringic acid, protocatechuic acid and (6 mM pyrogallol in 10 mM HCL). The absorbance (Ai) of the ferulic acid), at 320 nm for stilbenes (resveratrol and piceatannol), mixture was measured at 325 nm (UNICO 2100, Shanghai, China) at 360 nm for flavonoids (quercetin) and at 520 nm for anthocya- immediately and every 30 s until the reaction time reached 5 min. nins (cyanidin-3-glucoside, a flavonoid also), respectively. Before The sample solution was replaced by methanol to get the absor- analysis, all samples were filtered through a 0.25 mm membrane bance of the reagent blank solution (A0). Pyrogallol was replaced by - filter (Millipore, Billerica, MA, USA). Samples were quantified by methanol to get the absorbance of sample background (Aj). The O2$ comparing the retention times with the known authentic scavenging ability was calculated using the following equation: standards. scavenging activity (%) ¼ {[A0-(Ai-Aj)]/A0}*100. Ascorbic acid was used as a standard reference. Results were 2.6. Estimation of antioxidant capacity expressed as mg of ascorbic acid equivalent (mg AE) per g DW of sample. 2.6.1. DPPH· radical-scavenging capacity assay $ The DPPH radical-scavenging capacity assay was carried out by 2.7. Bioaccessible phenolics and phenolic bioaccessibility the method of Gao, Wu, and Yu (2012). with some modifications. fl Brie y, phenolic extracts were diluted with methanol to a suitable To determin bioaccessible phenolics, an in vitro gastrointestinal concentration. Three milliliters of the diluted extract was mixed digestion method was used as described by Vitali, Dragojevi, and $ with 3 mL DPPH ethanol solution (0.16 mmol/L) in a 10 mL Sebecic (2009) with slight modifications. In brief, 30 mL of colorimetric tube, and incubated at 25 C for 15 min. The absor- distilled water and 1.5 mL of pepsin (20 g/L in 0.1 mol/L HCl) were bance (Ai) of the mixture was measured at 517 nm (UNICO 2100, mixed with 1.5 g of Rhodomyrtus tomentosa berries powder, and the $ Shanghai, China). The DPPH ethanol solution was replaced by pH was adjusted to 2.0 using 6 mol/L HCl. The mixture was incu- distilled water to get the absorbance of sample background (Aj). The bated at 37 C in a shaking water bath for 1 h. At the end of incu- sample solution was replaced by methanol to get the absorbance of bation time, pH was adjusted to 7.2 with 1 mol/L NaHCO3 to stop $ the reagent blank solution (A0). The DPPH radical-scavenging ca- gastric digestion. Then, 7.5 mL of bile/pancreatin solution (2 g/L of pacity was calculated using the following equation: scavenging pancreatin and 12 g/L of bile salt in 0.1 mol/L NaHCO3) and 7.5 mL of ¼ activity (%) {[A0-(Ai-Aj)]/A0}*100. Standard curve was constructed NaCl/KCl (120 mmol/L NaCl and 5 mmol/L KCl) were added to by using rutin as standard. Final results were expressed as mg RE/g simulate the intestinal digestion for a period of 2.5 h at 37 Cina DW of sample. shaking water bath. The final mixture was centrifuged at 5000 g for 10 min and supernatant was used for determination of bio- 2.6.2. Ferric reducing antioxidant power (FRAP) accessible phenolics using Folin-Ciocalteu method and expressed fi The FRAP assay was carried out according to a modi ed method as gallic acid equivalents (mg of GAE/g DW). Bioaccessibility of of Benzie and Strain (1996). The working solution consisted of phenolics was also calculated as the percentage of bioaccessible $ 25 mL 300 mM acetate buffer (3.1 g of CH3COONa 3H2O and 16 mL phenolics to the total phenolics in Rhodomyrtus tomentosa berries. of CH3COOH per liter of buffer solution, pH 3.6), 2.5 mL 10 mM TPTZ solution (3.12 g TPTZ per liter of 40 mM HCl), and 2.5 mL 20 mM 2.8. Statistical analyses FeCl3$6H2O solution, incubated at 37 C for 10 min before use. A 0.03 mL aliquot of the above extract was diluted with 0.09 mL All drying experiments and analyses were performed in tripli- distilled water. The diluted extract reacted with 0.9 mL of the cate. Results were expressed as mean ± SD. Statistical analyses were working solution for 30 min in dark at room temperature. Absor- performed by using the IBM SPSS Statistic Version 20.0 (SPSS Inc., bance was measured at 593 nm using a Shimadzu UV-1800 spec- Chicago, IL, USA). Data were subjected to ANOVA followed by trometer. Standard curve was built by using Trolox and results were Duncan to identify differences between values. Statistical signifi- expressed as mg Trolox equivalents (TE) per g DW of sample. cance was considered at p < 0.05. 2.6.3. Oxygen radical antioxidant capacity (ORAC) The oxygen radical absorbance capacity (ORAC) assay was car- 3. Results and discussion ried out according to a method reported by Zhang, Zhang, Zhang, and Liu (2010) with slight modifications. Determination was per- 3.1. Effects of drying methods on the phytochemical contents of formed by using black-walled 96-well plates (Corning Scientific, Rhodomyrtus tomentosa berries Corning, NY, USA). The extracts were diluted with 75 mM phos- phate buffer (pH 7.4). Each well contained 20 mL of a diluted extract Table 1 shows the total phenolics, flavonoids and anthocyanins or of the Trolox standard (range ¼ 6.25e50 mM) and 200 mLof contents of Rhodomyrtus tomentosa berries dried by HD, MD and fluorescein (final concentration 0.96 mM). The mixture was incu- CD, separately. The total phenolic contents ranged from 9.88 to bated at 37 C for 20 min.Then, 20 mL of 119 mM ABAP was added to 15.57 mg GAE/g DW, which was significantly lower than those re- each well by using a multichannel pipette. Fluorescence intensity ported by Huang et al. (2010) (24.00 ± 0.40 mg GAE/g DW) and Lai was measured by using a Fluoroskan Ascent FL plate-reader (Thermo Labsystems, Franklin, MA, USA) at a wavelength of Table 1 485 nm for excitation and 538 nm for emission for 35 cycles every Effect of drying methods on the contents of phenolic compounds in Rhodomyrtus 4.5 min. ORAC activity was expressed as mM Trolox equivalents per tomentosa berries. g DW of sample. Drying methods Total phenolic Total flavonoid Total anthocyanins (mg GAE/g DW) (mg RE/g DW) (mg CGE/g DW) - 2.6.4. Superoxide radical (O2· ) scavenging activity HD 9.88 ± 0.23c 8.52 ± 0.21c 0.35 ± 0.04c Superoxide radical scavenging ability was determined by the MD 13.50 ± 0.72b 13.34 ± 0.42b 0.50 ± 0.02b method of Stefan and Gudrun (1974). Briefly, reaction mixtures CD 15.57 ± 0.64a 15.51 ± 0.20a 0.65 ± 0.35a containing 0.5 mL sample solution at a suitable concentration and ± Note: Values are expressed as means SD of triplicates. Values with different letters 4.5 mL 50 mM Tris-HCl buffer (pH 8.2) were put in 25 C bath for in the same column are significantly different (p < 0.05). HD, hot air drying; MD, 20 min, and subsequently mixed with 70 mL pyrogallol solution microwave drying; CD, combined microwave-hot-air-drying. G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266 263 et al. (2015) (49.21 ± 0.35 mg GAE/g DW) respectively. The 3.3. Effects of drying methods on the antioxidant activity of discrepancy may be mainly attributed to freeze drying method Rhodomyrtus tomentosa berries adopted previously, which can usually maintain much more phe- nolics in dried fruits. As shown in Table 1, CD reserved more total To date, there are few reports about the antioxidant activity of phenolics in berries than HD and MD markedly, this might be due the Rhodomyrtus tomentosa whole fruit. Owing to multiple mech- to more loss of phenolics in HD- and CD-berries for longer drying anisms of antioxidant activity (Çam, His¸ il, & Durmaz, 2009; Mao, time or higher temperature. MD-berries maintained more total Pan, Que, & Fang, 2006; Perez-Jim enez et al., 2008), four methods phenolics than HD-samples significantly, as is consistent with the were employed to assess the antioxidant capacityof Rhodomyrtus _ results of Hamrouni-Sellami et al. (2013); Izli, Yıldız, Ünal, Isık, and tomentosa berries dried by different methods in our study. As - Uylas¸ er (2014); Tarko et al. (2009). It can be assumed that, on one shown in Table 3, DPPH radical scavenging capacity, FRAP and O2$ hand, less oxidation reaction occurred in MD-berries duo to shorter radical scavenging capacity of Rhodomyrtus tomentosa ranged from time of exposure to oxygen and lower enzyme activities induced by 1.54 to 2.96 mg RE/g DW, from 6.38 to 8.73 mg TE/g DW and from higher temperature (Arsian & Ozcan, 2010; Yang, Chen, Zhao, & 7.01 to 9.79 mg AE/g DW, respectively. The ORAC of Rhodomyrtus Mao, 2010). While, on the other hand, microwave drying may tomentosa berries ranged from 222.85 to 309.16 mmol TE/g DW, accelerate the release of bound phenolics covalently linked to the which was significantly lower than that (431.17 ± 14.56 mmol TE/g macromolecules such as dietary fiber or protein through rupture of DW) reported by Lai et al., (2015), but still higher than those re- fruit tissue (Boateng, Verghese, Walker, & Ogutu, 2008; Hayat et al., ported in commonly consumed fruits such as apple, banana, 2010; Omwamba & Hu, 2010). blackberry, blueberry, grape, kiwifruit, mango, and orange The total flavonoids contents of Rhodomyrtus tomentosa berries (8.13e92.09 mmol TE/g DW) (Wu et al., 2004). in this study ranged from 8.52 to 15.51 mg RE/g DW, which was FRAP of CD-berries was significantly higher than that of MD- significantly higher than the results (5.21 ± 0.20 mg RE/g DW) re- berries. Conversely, CD-berries showed lower DPPH radical scav- - ported by Wu et al., (2015) in air dried berries. The total anthocy- enging capacity than MD-berries. But ORAC and O2$ radical scav- anins contents of Rhodomyrtus tomentosa berries dried with enging capacity of MD-berries was equivalent to those of CD- different methods ranged from 0.35 to 0.65 mg CGE/g DW. These berries. HD-berries showed the lowest antioxidant activity for all results were consistent with those reported by Cui et al., (2013), but the assays used in this study. In general, microwave drying was significantly lower than the results (1.60 mg CGE/g DW) reported beneficial to maintain or improve the antioxidant ability of Rho- by Lai et al.(2013). The contents and composition of anthocyanins domyrtus tomentosa non-nutritional antioxidants compared with are greatly influenced by genetic and environmental conditions, other thermal drying methods (Benlloch-Tinoco, Moraga, Camacho, _ like UV radiation and temperature. The difference in cultivars, & Martínez-Navarrete, 2013; Izli et al., 2014; Tarko et al., 2009). pedoclimatic condition, as well as drying methods used may explain the discrepancy in anthocyanin contents. As shown in 3.4. Effects of drying methods on the phenolic bioaccessibility of Table 1, berries dried by CD maintained much higher content of Rhodomyrtus tomentosa berries total flavonoids and anthocyanins than HD- and MD-dried samples, which was consistent with the changes in total phenolic contents. The current data as well as the previous literature have proved Rhodomyrtus tomentosa berries as rich source of phenolics. How- ever, consumption of fruits rich in phenolics does not usually mean 3.2. Effects of drying methods on phenolic profiles of Rhodomyrtus that high amount of active components will be absorbed and used tomentosa berries by human body (Manach, Williamson, Morand, Scalbert, & Remesy, 2005; Palafox-Carlos, Ayala-Zavala, & Gonzalez-Aguilar, 2011). This Detection of total phenolic contents may deviate due to non- can generally be explained by the differences in bioaccessibility and phenolic reducing substances, such as reducing sugar and ascorbic bioavailability of phenolics from fruits and other foods, which acid. Therefore, changes in individual phenolic compounds can directly influence their biological function in vivo. The bio- more accurately reflect the effects of different drying methods on accessibility of phenolic compounds from fruits and other foods can phenolic contents in Rhodomyrtus tomentosa berries. For this pur- be defined as the amount of the phenolics released from its food pose, 8 representative individual phenolics including 4 phenolic matrix after digestion and available for intestinal absorption. It can acids (gallic acid, syringic acid, ferulic acid and protocatechuic acid), calculated as the percentage of released phenolics to the total 2 flavonoids (cyanidin-3-O- glucoside and quercetin) and 2 stil- phenolics in food. benes (resveratrol and piceatannol) were quantified in dried Rho- So far, there are few studies involved phenolic bioaccessibility of domyrtus tomentosa berries by HPLC (Fig. 1). As shown in Table 2, Rhodomyrtus tomentosa berries and its products. In this study, the contents of eight individual phenolics were significantly phenolic bioaccessibility of Rhodomyrtus tomentosa berries was different in berries dried with 3 different methods. The contents are investigated in order to assess them as sources of accessible phe- significantly lower than those in freeze-dried Rhodomyrtus nolics. As shown in Table 4, the bioaccessible phenolics after gastric tomentosa berries reported by Lai et al.(2013) and Cui et al., (2013). digestion (GD) and gastrointestinal digestion (GID) ranged from Gallic acid and cyanidin-3-O-glucoside contents in MD-berries 2.99 to 5.50 mg GAE/g DW and from 5.84 to 7.05 mg GAE/g DW, was significantly higher than that in HD- and CD-berries, while respectively. The release of total phenolics in MD- and CD-berries syringic acid, ferulic acid, protocatechuic acid and piceatannol was mainly occurred in stomach. Similar results were found for contents of CD-berries were significantly higher than those in HD- apple phenolics reported by Bouayed, Hoffmann, and Bohn (2011), and MD-berries. Resveratrol contents of MD- and CD-berries were citrus phenolics and red bayberry phenolics reported by Chen et al. not significantly difference, but higher than that of HD-berries. (2014), whereas, phenolics in HD-berries was released in stomach Additionally, quercetin contents of HD- and CD-berries were not and small intestine to an equal extent. Phenolic bioaccessibility in significantly different, but higher than that of MD-berries. These Rhodomyrtus tomentosa berries after gastrointestinal digestion results suggest that CD was the most suitable method among the ranged from 45.24 to 59.15%, was higher than that of black carrot tested ones for the preservation of individual phenolics, which was (Kamiloglu, Pasli, Ozcelik, Camp, & Capanoglu, 2015), but signifi- consistent with the effects of drying methods on the total phenolic cantly lower than that of apples (Bouayed et al., 2011; Peng, Cong, & contents stated earlier. Liu, 2016) and grapes (Tagliazucchi, Verzelloni, Bertolini, & Conte, 264 G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266

Fig. 1. HPLC chromatogram of phenolic standards (280 nm) (A) and a crude extract from Rhodomyrtus tomentosa berries dried by combined microwave-hot-air-drying (280 nm & 520 nm) (B & C): (1), gallic acid, (2), protocatechuic acid, (3), cyanidin-3-O-glucoside, (4), syringic acid, (5), ferulic acid, (6), piceatannol, (7), resveratrol, (8), quercetin.

Table 2 Effect of drying methods on the contents of individual phenolic compounds in Rhodomyrtus tomentosa berries.

Individual phenolic contents HD MD CD

Gallic acid (mg/g DW) 112.7 ± 6.6c 297.0 ± 17.8a 207.7 ± 8.0b Syringic acid (mg/g DW) 32.9 ± 2.1b 37.2 ± 3.2b 44.4 ± 3.6a Ferulic acid (mg/g DW) 127.8 ± 6.9c 179.5 ± 10.7b 204.5 ± 7.3a Protocatechuic acid (mg/g DW) 21.2 ± 2.0b 14.56 ± 0.9c 30.4 ± 1.9a Cyanidin-3-O-glucoside (mg/g DW) 60.5 ± 10.7b 97.4 ± 14.6a 73.8 ± 3.3b Quercetin (mg/g DW) 6.5 ± 1.4a 3.5 ± 0.1b 6.6 ± 0.4a Resveratrol (mg/g DW) 19.5 ± 3.0b 35.0 ± 7.8a 28.7 ± 4.2ab Piceatannol (mg/g DW) 242.6 ± 7.2c 477.9 ± 47.3b 851.3 ± 80.8a

Note: Values are expressed as means ± SD of triplicates. Values with different letters in the same line are significantly different (p < 0.05). HD, hot air drying; MD, microwave drying; CD, combined microwave-hot-air-drying.

Table 3 Effect of drying methods on the antioxidant activity of Rhodomyrtus tomentosa berries.

- Drying methods DPPH radical scavenging capacity FRAP ORAC O2$ radicalscavenging (mg RE/g DW) (mg TE/g DW) (mmol TE/g DW) capacity (mg AE/g DW)

HD 1.54 ± 0.05c 6.38 ± 0.20c 222.85 ± 29.31b 7.01 ± 0.00b MD 2.96 ± 0.14a 8.02 ± 0.10b 309.16 ± 17.16a 9.79 ± 0.41a CD 2.54 ± 0.14b 8.73 ± 0.50a 303.20 ± 49.31a 9.69 ± 1.35a

Note: Values are expressed as means ± SD of triplicates. Values with different letters in the same column are significantly different (p < 0.05). HD, hot air drying; MD, mi- crowave drying; CD, combined microwave-hot-air-drying.

Table 4 Effects of drying methods on the phenolic bioaccessibility of Rhodomyrtus tomentosa berries.

Drying methods Total phenolic content Bioaccessible phenolics after GD Bioaccessible phenolics after GID Phenolic bioaccessibility after GID (%) (mg GAE/g DW) (mg GAE/g DW) (mg GAE/g DW)

HD 9.88 ± 0.23c 2.99 ± 0.07c 5.84 ± 0.17b 59.15 ± 1.43a MD 13.50 ± 0.72b 5.17 ± 0.19b 7.05 ± 0.02a 52.33 ± 2.79b CD 15.57 ± 0.64a 5.50 ± 0.09a 7.03 ± 0.03a 45.24 ± 1.72c

Note: Values are expressed as means ± SD of triplicates. Values with different letters in the same column are significantly different (p < 0.05). Bioaccessibility was calculated as the percentage of total phenolic content. HD, hot air drying; MD, microwave drying; CD, combined microwave-hot-air-drying.

2010). The difference may be due to different food matrix, phenolic digestion was found in MD-and CD-berries, however, the highest profiles and sample preparation. The highest content of bio- total phenolic bioaccessibility value (59.15%) was observed in HD- accessible phenolics (7.05 mg GAE/g DW) after gastrointestinal berries. Presumably, drying affected the microstructure of G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266 265

Rhodomyrtus tomentosa berries, and consequently altered their bioavailability and effects on health. Natural Product Reports, 26,1001e1043. phenolic bioaccessibility. Cui, C., Zhang, S. M., You, L. J., Ren, J. Y., Luo, W., Chen, W. F., et al. (2013). Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification The in vitro gastrointestinal digestion procedure in this study did of the major anthocyanins. Food Chemistry, 139,1e8. not include colonic fermentations. Only 45.24e59.15% of phenolics Del Rio, D., Rodriguez-Mateos, A., Spencer, J. P. E., Tognolini, M., Borges, G., & in Rhodomyrtus tomentosa berries were released in our study, while Crozier, A. (2013). Dietary (poly)phenolics in human health: Structure, bioavailability, and evidence of protective effects against chronic disease. An- the indigestible fraction may be released by the metabolism of tioxidants & Redox Signaling, 18, 1818e1892. abundant microflora in colon. Therefore, further investigation is Dewanto, V., Wu, X., Adom, K. K., & Liu, R. H. (2002). Thermal processing enhances needed to undertand phenolics bioaccessibility of Rhodomyrtus the nutritional value of tomatoes by increasing total antioxidant activity. Journal of Agricultural and Food Chemistry, 50,3010e3014. tomentosa berries by a suitable in vitro colonic model. Do, T. L. (2011). SIM. In A. Ertr (Ed.), Medicine and remedies of Vietnam (16th ed., p. 434). Hanoi: Vietnam: Thoi Dai Publication House. 4. Conclusion Gao, Q. H., Wu, C. S., & Yu, J. G. (2012). Textural characteristic, antioxidant activity, sugar, organic acid, and phenolic profiles of 10 promising jujube (Ziziphus jujuba Mill.) selections. Journal of Food Science, 77,C1218eC1225. The effects of three thermal drying methods on phenolic pro- Geetha, K. M., Sridhar, C., & Murugan, V. (2010). Antioxidant and healing effect of files, their bioaccessibility and antioxidant activity of Rhodomyrtus aqueous alcoholic extract of Rhodomyrtus tomentosa (Ait.) Hassk on chronic gastric ulcers in rats. Journal of Pharmacy Research, 3, 2860e2862. tomentosa berries were investigated in this study. It was concluded Hamrouni-Sellami, I., Rahali, F. Z., Rebey, I. B., Bourgou, S., Limam, F., & Marzouk, B. that CD-berries contained more phenolics, higher antioxidant ac- (2013). Total phenolics, flavonoids, and antioxidant activity of sage (Salvia tivity and more bioaccessible phenolics than MD- and HD-ones officinalis L.) plants as affected by different drying methods. Food and Bioprocess Technology, 6, 806e817. although the bioaccessibility of phenolics in HD-berries was the Hayat, K., Zhang, X., Farooq, U., Abbas, S., Xia, S., Jia, C., et al. (2010). Effect of mi- highest. crowave treatment on phenolic content and antioxidant activity of citrus mandarin pomace. Food Chemistry, 123, 423e429. Huang, W. Y., Cai, Y. Z., Corke, H., & Sun, M. (2010). Survey of antioxidant capacity Acknowledgements and nutritional quality of selected edible and medicinal berries plants in . Journal of Food Composition and Analysis, 23,510e517. We sincerely appreciate Dr. Sher Ali Khan in our lab for his kind Encyclopedia on contemporary medicinal plants: Rhodomyrtus tomentosa (Downy Rose Myrtle). (2010). Institute of Chinese Medicine. http://www.hkjcicm.org/ help in language editing. This work was supported by the National cm-database/plants/detail_e.aspx?herb_id¼489 (Accessed 24 September 2011). _ Natural Science Foundation of China (31571828), the Group Pro- Izli, N., Yıldız, G., Ünal, H., Isık, E., & Uylas¸ er, V. (2014). Effect of different drying gram of Natural Science Foundation of Guangdong Province methods on drying characteristics, colour, total phenolic content and antioxi- dant capacity of goldenberry (Physalis peruviana L.). International Journal of Food (2016A030312001) and Guangdong Provincial Science and Tech- Science and Technology, 49,9e17. nology Project (2016B070701012, 2016A040403081). Kamiloglu, S., Pasli, A. A., Ozcelik, B., Camp, J. V., & Capanoglu, E. (2015). Influence of different processing and storage conditions on in vitro bioaccessibility of polyphenols in black carrot jams and marmalades. Food Chemistry, 186,74e82. References Lai, T. N. H., Andre, C., Rogez, H., Mignolet, E., Nguyen, Y. B. T., & Larondelle, Y. (2015). Nutritional composition and antioxidant properties of the sim berries (Rhodo- Agro Forestry Tree Database. (1992). A tree species reference and selection and se- myrtus tomentosa). Food Chemistry, 168,410e416. lection guide. Rhodomyrtus tomentosa. http://www.worldagroforestrycentre. Lai, T. N. H., Herent, M., Quetin-Leclerc, J., Nguyen, T. B. T., Rogez, H., Larondelle, Y., org/sea/Products/AFDbase/af/asp/SpeciesInfo.asp?SpID-18093 (Accessed 23 et al. (2013). Piceatannol, a potent bioactive stilbene, as major phenolic September 2011). component in Rhodomyrtus tomentosa. Food Chemistry, 138, 1421e1430. Arsian, D., & Ozcan, M. M. (2010). Study the effect of sun, oven and microwave Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric drying on quality of onion slices. LWT - Food Science and Technology, 43, anthocyanin pigment content of berries juices, beverages, natural colorants, 1121e1127. and wines by the pH differential method: Collaborative study. Journal of AOAC Aydin, E., & Gocmen, D. (2015). The influences of drying method and metabisulfite Internationl, l88, 1269e1278. pre-treatment on the colour, functional properties and phenolic acids contents Liu, G. L., Guo, H. H., & Sun, Y. M. (2012). Opimization of the extraction of antho- and bioaccessibility of pumpkin flour. LWT-Food Science and Technology, 60, cyanins from the fruit skin of Rhodomyrtus tomentosa (Ait.) Hassk and identi- 385e392. fication of anthocyanins in the extract using high-performance liquid Barrett, D. M., & Lloyd, B. (2012). Advanced preservation methods and nutrient chromatography-electrospray ionization-mass spectrometry(HPLC-ECI-MS). retention in fruits and vegetables. Journal of the Science of Food and Agriculture, International Journal of Molecular Sciences, 13, 6292e6302. 92,7e22. Liu, L., Guo, J. J., Zhang, R. F., Wei, Z. C., Deng, Y. Y., Guo, J. J., et al. (2015). Effect of Benlloch-Tinoco, M., Moraga, G., Camacho, M. M., & Martínez-Navarrete, N. (2013). degree of milling on phenolic profiles and cellular antioxidant activity of whole Combined drying technologies for high-quality kiwiberries powder production. brown rice. Food Chemistry, 185,318e325. Food and Bioprocess Technology, 6, 3544e3553. Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 measure of ‘‘antioxidant power’’: The FRAP assay. Journal of Clinical and Scien- bioavailability studies. American Journal of Clinical Nutrition, 81, 230Se242S. tific Research, 239,70e76. Mao, L. C., Pan, X., Que, F., & Fang, X. H. (2006). Antioxidant properties of water and Boateng, J., Verghese, M., Walker, L. T., & Ogutu, S. (2008). Effect of processing on ethanol extracts from hot air-dried and freeze dried daylily flowers. European antioxidant contents in selected dry beans (Phaseolus spp. L.). LWT - Food Sci- Food Research and Technology, 222, 236e241. ence and Technology, 41, 1541e1547. Noratto, G., Porter, W., Byrne, D., & Cisneros-Zevallos, L. (2009). Identifying peach Bondaruk, J., Markowsk, M., & Błaszczak, W. (2007). Effect of drying conditions on and plum polyphenols with chemopreventive potential against estrogen- the quality of vacuum-microwave dried potato cubes. Journal of Food Engi- independent breast cancer cells. Journal of Agricultural and Food Chemistry, 57, neering, 81, 306e312. 5119e5126. Bouayed, J., Hoffmann, L., & Bohn, T. (2011). Total phenolics, flavonoids, anthocya- Omwamba, M., & Hu, Q. (2010). Antioxidant activity in barley (Hordeum vulgare L.) nins and antioxidant activity following simulated gastro-intestinal digestion grains roasted in a microwave oven under conditions optimized using response and dialysis of apple varieties: Bioaccessibility and potential uptake. Food surface methodology. Journal of Food Science, 75,C66eC73. Chemistry, 128,14e22. Palafox-Carlos, H., Ayala-Zavala, J. F., & Gonzalez-Aguilar, G. A. (2011). The role of Çam, M., His¸ il, Y., & Durmaz, G. (2009). Classification of eight pomegranate juices dietary fiber in the bioaccessibility and bioavailability of fruit and vegetable based on antioxidant capacity measured by four methods. Food Chemistry, 112, antioxidants. Journal of Food Science, 76,R6eR15. 721e726. Peng, M. X., Cong, Y. L., & Liu, D. (2016). Evaluation of phenolics, flavonoids and Caparino, O. A., Tang, J., Nindo, C. I., Sablani, S. S., Powers, J. R., & Fellman, J. K. (2012). antioxidant activity of apples following simulated gastro-intestinal digestion. Effect of drying methods on the physical properties and microstructures of Modern Food Science and Technology (Chinese), 32,122e128. þ296. mango (Philippine ‘Carabao’ var.) powder. Journal of Food Engineering, 111, Perez-Jimeneza, J., Arranza, S., Taberneroa, M., Díaz-Rubioa, M. E., Serranob, J., ~ 135e148. Gonib, I., et al. (2008). Updated methodology to determine antioxidant capacity Chang, C., Lin, H., Chang, C., & Liu, Y. (2006). Comparisons on the antioxidant in foods, oils and beverages: Extraction, measurement and expression of properties of fresh, freeze-dried and hot-air-dried tomatoes. Journal of Food results. Food Research International, 41,274e285. Engineering, 77,478e485. Plaza, L., Crespo, I., de Pascual-Teresa, S., de Ancos, B., Sanchez-Moreno, C., ~ Chen, G. L., Chen, S. G., Zhao, Y. Y., Luo, C. X., Li, J., & Gao, Y. Q. (2014). Total phenolic Munoz, M., et al. (2011). Impact of minimal processing on orange bioactive contents of 33 fruits and their antioxidant capacities before and after in vitro compounds during refrigerated storage. Food Chemistry, 2, 646e651. digestion. Industrial Crops and Products, 57,150e157. Rodríguez, K., Ah-Hen, K., Vega-Galvez, A., Lopez, J., Quispe-Fuentes, I., Lemus- Crozier, A., Jaganath, I. B., & Clifford, M. N. (2009). Dietary phenolics: Chemistry, Mondaca, R., et al. (2014). Changes in bioactive compounds and antioxidant 266 G. Zhao et al. / LWT - Food Science and Technology 79 (2017) 260e266

activity during convective drying of murta (Ugni molinae T.) berries. Interna- raw materials and dietary fibre on the selected nutritional and functional tional Journal of Food Science and Technology, 49, 990e1000. properties of biscuits. Food Chemistry, 114, 1462e1469. Stefan, M., & Gudrun, M. (1974). Involvement of the superoxide anion radical in the Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. autoxidation of pyrogallol and a convenient assay for superoxide dismutase. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in European Journal of Biochemistry, 47, 469e474. the United States. Journal of Agricultural and Food Chemistry, 52, 4026e4037. Tagliazucchi, D., Verzelloni, E., Bertolini, D., & Conte, A. (2010). In vitro bio- Wu, P. P., Ma, G. Z., Li, N. H., Deng, Q., Yin, Y. Y., & Huang, R. Q. (2015). Investigation of accessibility and antioxidant activity of grape polyphenols. Food Chemistry, in vitro and in vivo antioxidant activities of flavonoids rich extract from the 120, 599e606. berries of Rhodomyrtus tomentosa (Ait.) Hassk. Food Chemistry, 173,194e202. Tarko, T., Duda-Chodak, A., & Tuszunski, T. (2009). The influence of microwaves and Yang, J., Chen, J., Zhao, Y., & Mao, L. (2010). Effects of drying processes on the selected manufacturing parameters on apple chip quality and antioxidant ac- antioxidant properties in sweet potatoes. Agricultural Sciences in China, 9, tivity. Journal of Food Processing and Preservation, 33,676e690. 1522e1529. Ti, H. H., Li, Q., Zhang, R. F., Zhang, M. W., Deng, Y. Y., Wei, Z. C., et al. (2014). Free and Zhang, M. W., Zhang, R. F., Zhang, F. X., & Liu, R. H. (2010). Phenolic profiles and bound phenolic profiles and antioxidant activity of milled fractions of different antioxidant activity of black rice bran of different commercially available vari- indica rice varieties cultivated in southern China. Food Chemistry, 159,166e174. eties. Journal of Agricultural and Food Chemistry, 58, 7580e7587. Vitali, D., Dragojevi, I. V., & Sebecic, B. (2009). Effects of incorporation of integral