Article pubs.acs.org/JAFC
Phenylethanoid Glycoside Profiles and Antioxidant Activities of Osmanthus fragrans Lour. Flowers by UPLC/PDA/MS and Simulated Digestion Model Yirong Jiang,† Shuqin Mao,† Weisu Huang,§ Baiyi Lu,*,† Zengxuan Cai,# Fei Zhou,† Maiquan Li,† Tiantian Lou,† and Yajing Zhao† †
College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Laboratory of Quality & Safety Risk Assessment for Agro-products on Storage and Preservation, Zhejiang University, Hangzhou 310058, China § Department of Applied Technology, Zhejiang Economic & Trade Polytechnic, Hangzhou 310018, China # Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China ABSTRACT: Variations of phenylethanoid glycoside profiles and antioxidant activities in Osmanthus fragrans flowers through the digestive tract were evaluated by a simulated digestion model and UPLC/PDA/MS. Major phenylethanoid glycosides and phenolic acids, namely, salidroside, acteoside, isoacteoside, chlorogenic acid, and caffeic acid, were identified in four cultivars of O. f ragrans flowers, and the concentration of acteoside was the highest, being up to 71.79 mg/g dry weight. After simulated digestion, total phenylethanoid glycoside contents and antioxidant activities were significantly decreased. Acteoside was identified as decomposing into caffeic acid, whereas salidroside was found to be stable during simulated digestion. According to Pearson’s correlation analysis, acteoside contents showed good correlations with antioxidant activities during simulated digestion (R2 = 0.994, P < 0.01). In conclusion, acteoside was the major contributor to the antioxidant activity of O. f ragrans flowers, and salidroside was considered as the major antioxidant compound of O. f ragrans flowers in vivo. KEYWORDS: Osmanthus fragrans Lour. flower, simulated digestion model, antioxidant capacity, UPLC/PDA/MS, phenylethanoid glycoside, acteoside, salidroside
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INTRODUCTION Osmanthus fragrans (Thumb.) Lour., generally known as sweetscented osmanthus, is a common edible flower in China. It has long been consumed as an additive to Chinese traditional foods, such as sweet Osmanthus cake, Osmanthus tea, and scented Osmanthus jam.1 Chinese traditional medicine named Ben Cao Gang Mu claimed that O. f ragrans flowers had stasisremoving and phlegm-reducing effects. In recent research, various biological activities have been attached to O. f ragrans Lour. flowers, including free radical scavenging,2 neuroprotection,3 and tyrosinase activity inhibition.4 In addition, a clinical trial showed that the consumption of an O. f ragrans Lour. flower beverage for 7 days (one bottle per day) may improve the antioxidant levels in healthy individuals.5 Thus, O. f ragrans Lour. flowers showed a great potential to be food materials for natural antioxidants. More than 20 kinds of phytochemicals were identified in O. f ragrans Lour. flowers, including acteoside, ligustroside, salidroside, rutin, (+)-phillygenin, phillyrin, (−)-phillygenin, taxiresinol, (−)-olivil, and so on.6−9 However, information is lacking on the major antioxidant compounds of O. f ragrans Lour. flowers. Digestion plays an essential role in the effect of natural antioxidants in the human body. There are great differences in the bioactivities of antioxidants between in vitro and in vivo. Variation of antioxidants during digestion could be generally affected by many factors, such as body tempeature, pH value, and digestive enzymes.10 To simulate the effects of digestion, an in vitro digestion model was developed by Versantvoort et al.11 considering the conditions of gaseous environment, pH value, © XXXX American Chemical Society
reaction temperature, and gastrointestinal enzymes. This model had been applied to study macronutruient decomposition in humans with milk matrix.12 Paolella et al.13 applied this model to characterize the released peptide fraction of different drycured ham maturation times and to analyze the phenols in microencapsulated blueberry anthocyanin.14 Cellular antioxidant activity (CAA) assay is a novel approach to the evaluation of antioxidant activity, considering the biological factors, such as uptake, metabolism, and localization of antioxidant compounds within cells.15 Combined with the CAA assay, simulated digestion model is more likely to authentically evaluate biological activities of natural antioxidants in vivo.16 This study aimed to determine the major antioxidant compound of O. f ragrans Lour. flower by UPLC/PDA/MS and a simulated digestion model and to investigate the variations of phenylethanoid glycoside properties and antioxidant capacities of O. f ragrans Lour. flower during simulated digestion. In addition, the antioxidant capacities of O. fragrans Lour. flower were evaluated by DPPH• assay, ABTS•+ assay, ferric reducing antioxidant power (FRAP), and CAA assay. Special Issue: Phytochemicals in Food (ISPMF 2015) Received: July 15, 2015 Revised: August 25, 2015 Accepted: August 31, 2015
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DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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kV, a capillary temperature of 350 °C, a cone voltage of 30.0 V, and a sheath gas flow rate of 540 L/h.21 Phenylethanoid glycosides and related compounds were quantified using the following standards: acteoside, salidroside, isoacteoside, caffeic acid, and chlorogenic acid. The results were expressed as milligrams per gram of dry weight. Chemical Antioxidant Activity Assays. DPPH• assay was performed as described by Bao et al.22 with slight modifications. Briefly, 3.9 mL of 0.1 mM DPPH• solution (dissolved in methanol) was added to 100 μL of sample. The mixture was well mixed by a vortex. The reaction for scavenging DPPH• radicals was performed in the dark at room temperature for 1 h. The absorbance was measured at 517 nm. The results were expressed as milligrams of Trolox equivalent (TE) per gram of dry weight. ABTS•+ assay was performed as described by Re et al.23 with little modification. The ABTS•+ reagent was prepared by mixing 7.4 mM ABTS•+ solution and 2.6 mM potassium persulfate solution. The solution was left to react in the dark for 12 h at room temperature. The reagent was diluted with distilled water until the absorbance reached 0.700 ± 0.025 at 734 nm. The final ABTS•+ reagent was prepared fresh daily. Up to 100 μL of samples at proper concentrations was added to 3.0 mL of ABTS•+ reagent in the dark for 1 h at room temperature. The absorbance was measured at 734 nm. The results were expressed as milligrams of TE per gram of dry weight. A modified FRAP assay was performed as described by Benzie et al.24 The FRAP reagent consisted of 10 mL of 2,4,6-tri(2-pyridyl)1,3,5-triazine solution (10 mM TPTZ in 40 mM HCl), 10 mL of ferric chloride solution (20 mM), and 100 mL of acetate buffer (300 mM, pH 3.6). The FRAP reagent was prepared fresh daily. Each sample (100 μL) was mixed with 3 mL of the FRAP reagent and allowed to react for 4 min at 37 °C, after which absorbance was measured at 593 nm. FRAP values were calculated by the standard curve of ferrous sulfate solution (0.1−1 mM) and were expressed as micromoles of Fe(II) equivalent per gram of dry weight. Cellular Antioxidant Activity Assay. CAA assay was performed as described by Wolfe and Liu.15 HepG2 cells were grown at 37 °C in an incubator with 5% CO2. The cells were used between passages 12 and 35. Cytotoxicity values of the samples were measured and expressed as median cytotoxic doses (CC50). Briefly, HepG2 cells were seeded at a density of 6 × 104 cells per well on black 96-well plates with 100 μL of growth medium. After 24 h of incubation at 37 °C, the medium was removed, and the cells were washed with 100 μL of PBS. The cells were treated for 1 h with medium containing 100 μL of samples at different concentrations or standard phytochemicals (e.g., quercetin) or with 25 μL of 1 M DCFH-DA. The treatment media were removed, and the cells were washed with PBS. Subsequently, 100 μL of 1 M AAPH solution dissolved in Hank’s balanced salt solution (HBSS) was added. The plates were read every 2 min for 1 h at an emission wavelength of 538 nm and at an excitation wavelength of 485 nm. Each plate included control and blank wells with triplicate parallel repetitions. The control wells contained cells treated with oxidant and DCFH-DA, whereas the blank wells contained cells treated with HBSS and dye. The results were expressed as micromoles of quercetin equivalent (QE) per 100 g of dry weight. Statistical Analysis. All of the experiments were performed in triplicate, and the results were expressed as the mean ± SD. Statistical analysis was performed using SPSS 19 and Excel 2007. Means were compared using ANOVA, and statistical significance was set at P < 0.05. Retention rate = value of phytochemical content (or antioxidant activity) after digestion/value of phytochemical content (or antioxidant activity) before digestion × 100% (formula 1). In the CAA assay, the median effective dose (EC50) was the concentration at which fa/f u = 1, where fa is the fraction affected (CAA unit) and f u is the fraction unaffected (1 − CAA unit) by the treatment. Absorptivity = CAA value with PBS wash/CAA value without PBS wash × 100% (formula 2). Correlations between phenylethanoid glycoside contents and antioxidant capacities were analyzed by Pearson’s correlation analysis, and statistically significant differences were expressed as ∗ (P < 0.05) and ∗∗ (P < 0.01).
MATERIALS AND METHODS
Sample Preparation. Four cultivars of O. f ragrans Lour. flowers (O. f ragrans var. thunbergii, O. fragrans var. latifolius, O. f ragrans var. aurantiacus, and O. f ragrans var. semperf lorens) were collected at Lin’an (Hangzhou, China) in October 2013. Flowers from the four cultivars were freeze-dried and pulverized into powders separately. The prepared samples were then stored at −20 °C for further use. Chemicals. Chlorogenic acid (purity > 99%), caffeic acid (purity = 98%), quercetin (purity > 95%), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), 2,7-dichlorofluorescin diacetate (DCFH-DA), 2,2-azobis(2-methylpropionamidine) dihydrochloride (granular, AAPH), fluorescein, and Folin−Ciocalteu reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals used for simulated digestion, including α-amylase, pepsin, parenzyme, lipase, bile salts, and uric acid, were obtained from Aladdin Industrial Co. (Shanghai, China). Acteoside (purity > 99%), salidroside (purity = 97%), and isoacteoside (purity = 98%) were obtained from Yuanye Biotechnology Co. (Shanghai, China). UPLCgrade acetonitrile and methanol were obtained from ACS (Houston, TX, USA). All other chemicals and reagents were of analytical grade. Phytochemical Extraction. The phytochemicals in O. f ragrans flowers were extracted using a modified method described by Wolfe et al.17 Each sample (1 g) was soaked in 80% aqueous acetone (15 mL) for 12 h at 40 °C. The mixtures were filtered by vacuum pump. The filtrates were concentrated at 40 °C and subsequently diluted to a final volume of 10 mL with distilled water. The extracts were stored at −20 °C for the evaluation of antioxidant activities within 30 days. Simulated Digestion Model. A simulated digestion model was modified from the model developed by Versantvoort et al.11 Briefly, saliva with α-amylase was added to a buffer system at pH 6.8 ± 0.2 with sodium and potassium. Gastric juice containing pepsin was added to a buffer system at a final pH of 2.00 ± 0.02 with sodium, potassium, and calcium. Duodenal juice containing parenzyme and lipase was added to a buffer system at pH 8.1 ± 0.2. Bile juice with bile salt was added to a buffer system at pH 8.2 ± 0.2. The prepared samples (1 g each) were incubated with 3 mL of saliva for 5 min and were mixed with 6 mL of gastric juice for 120 min, followed by 6 mL of duodenal juice and 3 mL of bile juice for another 120 min. All incubations were performed at 37 °C on a rotating wheel. The ethanol was added after digestion to ensure the inactivation of enzymes. Mixtures were filtered by vacuum pump. The filtrates were concentrated at 45 °C and then diluted to 10 mL with distilled water. A controlled trial was operated without prepared samples to improve accuracy. The digesta of O. f ragrans flowers were stored at −20 °C for the evaluation of antioxidant activities within 30 days. Total Phenylethanoid Glycoside Content. Ultraviolet spectroscopy was used to determine the total phenylethanoid glycoside content, as described by Lu and Mei.18 The samples were diluted with methanol to a suitable concentration. The absorbance of the diluent was measured at 334 nm. The results were expressed as milligrams of acteoside equivalent per gram of dry weight. Phenylethanoid Glycoside Profiles. Phenylethanoid glycosides presenting in the O. f ragrans flowers were identified using a modified method described by Gruz et al.19 Analysis was performed on UPLC (ACQUITY, Waters, Milford, MA, USA) equipped with a PDA detector (ACQUITY) and mass spectrometer (Xevo TQ, Waters). The BEH-C18 Symmetry column (150 mm × 2.1 mm; 1.7 μm) was maintained at 40 °C. Mobile phase A consisted of 1% acetic acid− water, whereas mobile phase B consisted of acetonitrile (flow rate = 0.2 mL/min). The gradient elution profiles were as follows: from 94 to 90% A for 0−10 min; from 90 to 80% A for 10−35 min; 80% A for 35−45 min; from 80 to 0% A for 45−48 min; 0% A for 48−49 min; from 0 to 94% A for 49−52 min; and 94% A for 52−55 min.20 The injection volume was 5 μL, and UPLC/PDA was set from 200 to 400 nm. A Z-spray electrospray ionization source was operated in negativeion mode. The full-scan mass spectra ranged from m/z 50 to 850. The parameters of mass spectrometer were set at a capillary voltage of 3.2 B
DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION Phenylethanoid Glycoside Contents. Total phenylethanoid glycoside contents in O. f ragrans flowers after simulated digestion are listed in Table 1, ranging from 33.57
caffeic acid, and chlorogenic acid, were identified and quantified in O. f ragrans flowers. As shown in Table 2, salidroside and acteoside were major phenylethanoid glycosides in O. fragrans flowers after simulated digestion, with contents from 4.730 to 16.57 mg/g dry wieght (DW) and from 2.08 to 3.43 mg/g DW, respectively. Compared with total phenylethanoid glycoside contents in O. f ragrans flowers before simulated digestion, it declined significantly (P < 0.05) after simulated digestion. Up to a 73.88% reduction in total phenylethanoid glycoside contents was determined through simulated digestive tract. Phenylethanoid glycoside profiles of O. f ragrans flowers before and after simulated digestion are shown in Figure 1, taking O. f ragrans var. thunbergii as an example. In fact, the losses in phenylethanoid glycoside contents do not happen in all types of phenylethanoid glycosides in O. f ragrans flowers. As shown in Table 2, the content of acteoside significantly decreased during simulated digestion, whereas salidroside was stable, resulting in no significant variation in its content during simulated digestion (P > 0.05). As reported by Huang et al., free phenolic acid contents of Chinese bayberry fruits were higher in the digesta than in the extracts.25 In nature, most phenolic acids are present in the form of glycoside esters or bound to the food matrix; resulting from digestive juice or pH values, phenolic acids could release from the food and transform into free form. Contrary to phenolic acids, most phenylethanoid glycosides in the shape of glycoside ester itself would be hydrolyzed or enzymolyzed during simulated digestion.26 In addition, the content of caffeic
Table 1. Total Phenylethanoid Glycoside Contents in O. fragrans Flower Extract before and after Simulated Digestion total phenylethanoid glycosides contents (mg AE/g DW) sample O. f ragrans var. thunbergii O. f ragrans var. latifolius O. f ragrans var. aurantiacus O. f ragrans var. semperf lorens
beforea 115.56 92.66 128.51 130.57
± ± ± ±
11.58ab 2.24b 8.97a 2.28a
aftera
RRb (%)
± ± ± ±
35.47 39.75 26.12 37.07
40.99 36.83 33.57 48.40
0.53c 2.71c 0.73c 1.64c
a
Values with different letters are significantly different (P < 0.05). bRR, retention rate.
to 48.40 mg acteoside equiv/g dry weight. O. f ragrans var. semperf lorens had the highest phenylethanoid glycoside content of 48.40 mg acteoside equiv/g dry weight after simulated digestion, followed by O. f ragrans var. thunbergii, O. f ragrans var. latifolius, and O. f ragrans var. aurantiacus in order. According to UV spectra and mass spectra of phytochemicals presented in Figures 1 and 2, phenylethanoid glycosides and phenolic acids, namely, salidroside, acteoside, isoacteoide,
Figure 1. UPLC-PDA chromatogram of O. f ragrans flower extract before(A) and after (B) simulated digestion. O. f ragrans var. thunbergii was taken as an example. C
DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Mass spectrum of phytochemical compounds in O. f ragrans flowers.
acid in our results significantly increased after simulated digestion. Qi et al. had demonstrated that acteoside was degraded during simulated digestion and caffeic acid was considered to be one of its decomposition products.27 These partly demonstrated the difference between acteoside and salidrside in their stabilities during simulated digestion.
Acteoside was a kind of glycoside ester appearing sensitive and fragile during simulated digestion, whereas salidroside was a kind of glycoside without ester bond that remained stable during simulated digestion. Chemical Antioxidant Activities. DPPH• and ABTS•+ assays were primary approaches for radical-scavenging assay D
DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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and can be used in both organic and aqueous solvent systems. These assays were widely used to test the free radicalscavenging ability of various samples.28 Two radical-scavenging activities of O. fragrans flowers are presented in Figure 3. After
Values in a horizontal row with different letters are significantly different (P < 0.05). bRR, retention rate, representing the ratio of reserved phytochemical contents after simulated digestion.
93.55 3.58 3.24 23.08 628.38
Figure 3. Chemical antioxidant activities of O. f ragrans flowers before and after simulated digestion.
simulated digestion, DPPH values of O. f ragrans flowers ranged from 19.25 to 37.06 mg TE/g DW. ABTS values ranged from 79.79 to 128.04 mg TE/g DW. To synthetically evaluate the antioxidant capacity, the FRAP assay was used to measure the reducing potential of O. f ragrans flowers, of which the FRAP values are presented in Figure 3 as well. The results showed that the ferric ion-reducing power of O. f ragrans flowers after simulated digestion was in the range from 183.59 to 240.63 μmol Fe(II)/g DW. Among four cultivars of O. fragrans flowers, O. f ragrans var. aurantiacus had the highest radicalscavenging activities and ferric ion-reducing power after simulated digestion. The variation of antioxidant capacities during simulated digestion was similar to the variation of phenylethanoid glycoside contents in O. f ragrans flowers. The antioxidant
a
0.22b 0.03c 0.01e 0.00e 0.03b ± ± ± ± ± 11.03 2.48 0.06 0.12 0.93 0.32b 2.19a 0.50b 0.01a 0.01d ± ± ± ± ± 11.79 69.25 1.85 0.52 0.15 103.05 4.78 1.48 17.24 742.11 0.48a 0.02c 0.01e 0.00f 0.01a ± ± ± ± ± 16.57 3.43 0.06 0.05 1.41 0.40a 5.20a 0.30a 0.00d 0.01c ± ± ± ± ± 16.08 71.79 4.05 0.29 0.19 100.21 6.74 8.57 14.71 1337.48 ± ± ± ± ± ± ± ± ± ± 10.41 40.73 0.49 0.46 0.13
0.21c 6.78b 0.00d 0.02b 0.02d
10.14 2.08 0.02 0.05 1.44 ± ± ± ± ± salidroside acteoside isoacteoside chlorogenic caffeic
0.48c 0.09c 0.00e 0.00f 0.29a
4.72 32.78 0.70 0.34 0.08
0.40d 2.99b 0.05c 0.01c 0.01e
4.73 2.21 0.07 0.05 1.07 ± ± ± ± ± 97.41 5.11 4.08 10.87 1107.69
0.45d 0.29c 0.01e 0.00f 0.04b
aftera beforea aftera
O. fragrans var. aurantiacus
beforea aftera
O. f ragrans var. latifolius
beforea aftera beforea compound
O. f ragrans var. thunbergii
RRb (%)
RRb (%)
sample
Table 2. Phytochemical Contents (mg/g DW) in O. f ragrans Flower Extract before and after Simulated Digestion
RRb (%)
O. f ragrans var. semperf lorens
RRb (%)
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DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 3. Cellular Antioxidant Activities of O. f ragrans Flower Extract before and after Simulated Digestion PBS wash EC50a (mg/mL)
samples O. f ragrans var. thunbergii O. f ragrans var. latifolius O. f ragrans var. aurantiacus O. f ragrans var. semperf lorens
before after before after before after before after
45.32 149.07 64.52 161.76 45.35 155.78 47.65 112.78
± ± ± ± ± ± ± ±
no PBS wash
CAA valuea,b
5.56c 12.88a 5.97c 8.33a 5.90c 15.58a 4.94c 13.05b
14.60 5.82 11.90 5.33 16.12 7.95 16.36 6.69
± ± ± ± ± ± ± ±
1.06ab 0.48c 1.29b 0.80c 1.01a 0.59c 1.94a 0.87c
RRc (%) 39.86 44.79 49.32 40.89
EC50a (mg/mL) 15.28 42.26 18.81 48.80 14.84 32.15 15.64 35.09
± ± ± ± ± ± ± ±
1.93c 5.23ab 2.88c 3.37a 1.58c 3.11a 1.43c 2.99b
CAA valuea,b 71.04 25.84 58.15 24.32 74.89 31.35 78.13 30.33
± ± ± ± ± ± ± ±
5.44a 3.80c 5.61b 2.89c 6.40ab 2.91c 7.32a 4.73c
RRc (%)
absorptivityd (%)
CC50 (mg/mL)
36.37
20.55 22.52 20.46 21.92 21.52 25.36 20.94 22.06
>20 >20 >20 >20 >20 >20 >20 >20
41.82 41.86 38.82
a Values in a vertical column with different letters are significantly different (P < 0.05). bThe unit of CAA value is expressed as μmol quercetin equivalent per 100 g dry weight. cRR, retention rate, representing the ratio of reserved cellular antioxidant activity values after simulated digestion. d Absorptivity represents the ratio of cellular antioxidant activities absorbed by cells.
Table 4. Correlation Coefficients between Antioxidant Activity Values and Phenylethanoid Glycosides Profiles of O. f ragrans Flowersa total compounds TPGC DPPH values ABTS values FRAP values CAA values with PBS wash CAA values without PBS wash a
b
0.934** 0.929** 0.971** 0.964** 0.980**
individual compounds salidroside
chlorogenic acid
caffeic acid
acteoside
isoacteoside
0.456 0.450 0.274 0.314 0.232
0.788* 0.790* 0.844** 0.890** 0.919**
−0.380 −0.376 −0.510 −0.521 −0.574
0.974** 0.943** 0.995** 0.953** 0.952**
0.867** 0.826** 0.857** 0.754* 0.727*
*, P < 0.05; **, P < 0.01. bTPGC, total phenylethanoid glycosides contents.
Rosae Rugosae flower, which were lower than those of Paeonia suffruticosa and Rosa chinensis. Changes in CAA values during simulated digestion were also similar to the variation of phenylethanoid glycoside contents. According to the data in the PBS wash protocol, CAA values were significantly lower after simulated digestion (P < 0.05). As reported by Huang et al.,25 the CAA values of bayberry fruit digesta were higher than those of their extracts, in which the main phytochemicals were phenolic acids. Given the differences in phytochemical constitutions, their different properties, such as sensitivities, accounted for the diversity in CAA values during simulated digestion. According to the retention rate (formula 1), 39.86−49.32% of CAA values in the PBS wash protocol was retained after simulated digestion, which was higher than that in the no PBS wash protocol. This pointed out that natural antioxidants that can be taken up by cells were much more stable than antioxidants attached to the cellular membrane. As for absorptivity (formula 2), the treatment of simulated digestion could enhance the absorptivity of CAA values in O. f ragrans flowers, indicating that antioxidant compounds after simulated digestion could be absorbed by cells much more easily. Correlations between Phenylethanoid Glycoside Contents and Antioxidant Activities. Pearson’s correlation analysis was used to evaluate the correlations between phenylethanoid glycoside contents and antioxidant capacities, as shown in Table 4. Total phenylethanoid glycoside contents were correlated significantly with each antioxidant activity assay, ranging from 0.929 to 0.980 (P < 0.01). As for individual compounds, contents of acteoside showed the best correlation with antioxidant activities, with correlation coefficients from 0.943 and 0.995 (P < 0.01). Therefore, acteoside was considered to be the major antioxidant component in O. f ragrans flowers. In previous research, antioxidant activities of
capacities of O. f ragrans flowers were significantly lower after simulated digestion (P < 0.05). Without the treatment of simulated digestion, DPPH values of O. f ragrans flowers ranged from 49.74 to 85.99 mg TE/g DW. ABTS values were much higher than DPPH values, ranging from 160.11 to 260.25 mg TE/g DW. As reported by Wu et al.,4 the antioxidant capacities of O. f ragrans flowers in the DPPH• and ABTS•+ assays were 304.9 mg vitamin C equiv/g of extract and 516.3 mg TE/g of extract, respectively. The ferric ion-reducing power of O. f ragrans flowers was in the range from 959.81 to 1759.54 μmol Fe(II)/g DW. Only 11.68−58.80% of antioxidant capacities in O. f ragrans flowers could be maintained after simulated digestion. Digestion of phytochemicals had an important effect on the in vivo antioxidant properties. Cellular Antioxidant Activity. To further evaluate the activities, we measured CAA values (presented in Table 3) of O. f ragrans flowers. As shown in Table 3, all of the CC50 values of cultivars were >20 mg/mL, which indicated that none of these cultivars were cytotoxic; hence, the results revealed below could provide insight into the real-life application of these compounds. CAA values were measured by two protocols, namely, PBS wash and no PBS wash. The PBS wash protocol demonstrated the activities of antioxidants taken up in cells, whereas the no PBS wash protocol contained the antioxidants that were not only absorbed by cells but also closely associated with the cellular membrane. After simulated digestion, the EC50 values of four cultivars of O. f ragrans flowers ranged from 112.78 to 161.76 mg/mL in the PBS wash protocol, higher than those in the no PBS wash protocol. CAA values in the PBS wash protocol of O. f ragrans flowers were 5.33−7.95 μmol QE/ 100 g, among which O. f ragrans var. aurantiacus had the highest CAA values after simulated digestion. Compared with a previous study on CAAs of 10 common edible flowers,29 the CAA values in O. f ragrans flowers were similar to those of Flos F
DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry O. f ragrans flower extracts were commonly attributed to phenols and flavonoids.2,4 Within the scope of our knowledge, the main contributor of antioxidant activities in O. f ragrans flowers had not been reported. Results in this paper gave evidence of the main antioxidant compound, which was likely to be acteoside. Conclusion. In this paper, phenylethanoid glycoside profiles and antioxidant activities of O. f ragrans flowers were evaluated by UPLC/PDA/MS and simulated digestion model. Acteoside and salidroside were considered as two major phenylethanoid glycosides contributing to the great antioxidant capacities of O. f ragrans flowers. After simulated digestion, the total phenylethanoid glycoside contents and antioxidant activities were significantly decreased (P < 0.05). Acteoside was identified as decomposing into caffeic acid after simulated digestion, and the content of acteoside declined significantly. However, salidroside was found to be stable during simulated digestion, which showed the potential of bioactive effects in vivo. According to Pearson’s correlation analysis, acteoside contents showed the significantly best correlations with antioxidant activities (R2 = 0.995, P < 0.01), whereas salidroside contents did not correlate with antioxidant activities. The results indicated that acteoside was the major contributor to the antioxidant capacity of O. f ragrans flowers; salidroside was stable during simulated digestion and could be considered as a potential bioactive component in the human body.
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AUTHOR INFORMATION
Corresponding Author
*(B.L.) Mail: Yuhangtang Road 866#, Hangzhou 310058, Zhejiang, China. Phone/fax: +86-571-88982665. E-mail: bylu@ zju.edu.cn. Funding
This work was supported financially by the Zhejiang Provincial Natural Science Foundation of China (No. R15C200002), the Foundation of Fuli Institute of Food Science, Zhejiang University, and the Special Project of Agricultural Product Quality Safety Risk Assessment (No. GJFP201502-3), Ministry of Agriculture, China. Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.5b03474 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
