With increasing evidence that reactive oxygen species (ROS) induce oxidative stress during the development of various chronic and degenerative diseases, such as cancer, cardiovascular disease, diabetes mellitus, and aging, radical-scavenging antioxidants for preventing oxidative stress-related diseases have received great attention [1
]. The increased demand for new, natural antioxidant foods has generated an interest in edible flowers in recent years [2
Tree peony (Paeonia suffruticosa Andr.
) is native to China with more than 1600 years’ history of cultivation, and is considered as a traditional ornamental and medicinal plant [5
]. The flower color of tree peony is diverse with nine categories that include red, white, green, pink, blue, purple, black, yellow, and dual colors [5
]. The antioxidant activity is attributed mainly to anthocyanins in the purple, pink, and red categories [6
], and flavonoids in the yellow and white categories [6
]. In China, the flowers of tree peony are used as herbal medicines for the treatment of diseases mainly related to irregular menstruation and dysmenorrhea in women [9
]. Some of the flowers have also been used to make a variety of delicious folk foods since the Song dynasty (A.D. 960–1279), such as teas, cakes, casseroles, and drinks, owing to the presence of nutrients including proteins, microelements, and vitamins [6
]. However, until now, only the flower of one white category, named Fengdan Bai
(FDB), was officially approved as a new food material by China’s Ministry of Health in 2013. Following the approval, different foods made from the petals, stamens, or the whole flower of FDB have become popular for their flavor and health function. In order to expand the utilization of the approved edible flowers as functional food material, two polysaccharides were purified from the petals of FDB and characterized in our previous study [10
Various phytochemicals, including phenolics [11
], stilbenes [13
], and terpenoids [14
], were identified in tree peony in the previous studies. The phytochemical species and contents in tree peony differ according to the cultivars and organs involved [6
]. Tree peony seeds exhibit strong antioxidant activity and phenolic compounds have been verified as the most important antioxidants [11
]. However, the phenolic profiles of the edible tree peony flower FDB have not been identified and the antioxidant properties have not yet been characterized. In addition to free radical scavenging capacity, cellular oxidative models have been widely used to investigate the potential mechanisms of phytochemicals involved in human health [20
]. In the present study, following extraction and identification of phenolics in the petal, stamen, calyx, and ovary of the tree peony flower (cultivar FDB), the 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+
) radical scavenging activities, and oxygen radical absorbance capacity (ORAC) were determined. Furthermore, an oxidative damage model of Caco-2 cells was established by hydrogen peroxide (H2
) treatment, and the mechanisms of the extracts on the recovery of the Caco-2 cells from oxidative damage were revealed via examining the antioxidant system and the tight junction protein expression.
2. Materials and Methods
2.1. Tree Peony Flower Samples
The edible tree peony flowers (Fengdan Bai) were collected in full blossom from the campus of Henan University of Science & Technology, China in mid April 2018. The whole flower was freeze-dried and different organs, including petal, stamen, calyx, and ovary, were separated and milled to powder in an electric mill (Micro-Mill, Bel-Art Products Co., Wayne, NJ, USA) with a 60 mesh screen. The ground samples were stored at −20 °C before extraction.
Folin-Ciocalteu reagent, 2,2′-azobis-(2-methylpropionamidine) dihydrochloride (AAPH), 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), fluorescein, HPLC grade methanol, and MS grade methanol were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Standards of gallic acid, methyl gallate, rutin, and quercetin were also purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hexanes, sodium hydroxide, hydrochloric acid, and sodium carbonate were purchased from Fisher Scientific (Ottawa, ON, Canada). Hydrogen peroxide (H2O2) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Hank’s balanced salt solution (HBSS), dulbecco’s modified eagle medium /F12 (DMEM/F12), anti-anti and fetal bovine serum (FBS) T-75 culture flasks, Transwell Permeable supports (0.6 cm2, 0.4 μm pore size), and cell culture plate were purchased from Invitrogen, Fisher Scientific (Ottawa, ON, Canada).
2.3. Sample Preparation
The extraction of phenolic compounds was conducted according to the method reported earlier [21
] with some modifications. The different organ parts of FDB were defatted twice with hexanes and then extracted three times with 80% methanol for 1 h at room temperature. The supernatants were combined and dried under vacuum at 38 °C, and reconstituted in 50% methanol for use as crude extracts.
2.4. Determination of Total Phenolic Content and Total Flavonoid Content
Determination of total phenolic content (TPC) was based on the method described by Xiang, et al. [22
] using a 96-well ELX800 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) and gallic acid as the standard. TPC was expressed as milligrams of gallic acid equivalents per gram (g) of the sample (mg GAE/g). Total flavonoid content (TFC) was determined as described by Biney and Beta [23
] using rutin as the standard. The results were expressed as milligrams of rutin equivalents per gram (g) of the sample (mg RE/g).
2.5. Determination of Antioxidant Capacity In Vitro
DPPH and ABTS•+
radical scavenging activities of the four parts of the flower were measured as described by Xiang, et al. [24
] using a 96-well ELX800 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Oxygen radical absorbance capacity (ORAC) was measured as described by Qiu, et al. [25
]. The results were expressed as micromole Trolox equivalents per gram of the sample (μmol TE/g).
2.6. Identification and Quantification of Phenolic Compounds by HPLC- Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF-MS2)
An HPLC (Waters 2695) equipped with a photodiode array (PDA) detector (Waters 996) and autosampler (717 plus, Waters) coupled to a quadrupole time-of-flight mass spectrometer (Q-TOF-MS) (Micromass, Waters Corp., Milford, MA, USA) was employed for LC and mass spectrometric analyses (LC–MS). Samples were eluted through a 150 mm × 4.6 mm, Gemini 5 μm C18
110A column (Phenomenex, Torrance, CA, USA) with a binary mobile phase consisting of A (water with 0.1% formic acid) and B (methanol with 0.1% formic acid). Separation of phenolic compounds was achieved by our previously reported method [26
]. The elution procedure was as follows: 0 min 4% B, 18 min 18% B, 35 min 30% B, 58 min 42% B, and 70 min 60% B. Compounds were identified by comparing retention time (RT) and UV spectra information and confirmed by Q-TOF-MS/MS.
The Q-TOF-MS was calibrated for the negative mode through the mass range of 100–1500 with the resolution of 5000, according to the method previously reported [26
]. Full mass spectra were recorded using a capillary voltage of 1.45 kV and a cone voltage of 30 V. The flow rates of cone gas (He) and desolvation gas (N2
) were 45 and 900 L/h, respectively. The desolvation gas temperature and ion source temperature were set at 250 °C and 120 °C, respectively, while the collision energies of 20, 30, and 45 V were used to acquire the MS2
The individual phenolic compounds were separated under the same HPLC conditions described above, and quantification was based on the area of the peak at wavelengths of 280 nm for gallic acid and methyl gallate derivatives, and 350 nm for flavonoid glycosides. The compounds without authentic standards were measured semi-quantitatively according to the method previously reported [8
]. Gallic acid was used for quantification of gallic acid, galloyl hexose, and gallotannins, and the contents of individual gallic acid derivatives were expressed as mg GAE/100 g. Methyl gallate (MGA) was used for quantification of methyl gallate and methyl digallates, and the contents of individual methyl gallate derivatives were expressed as mg methyl gallate equivalents per 100 g of the sample. Quercetin was used for quantification of all the flavonoid glycosides, and the individual flavonoid glycosides were expressed as mg quercetin equivalents per 100 g of the sample.
2.7. Cell Culture
The non-transformed neonatal human colon cancer cells (Caco-2) were supplied by the American Type Culture Collection (ATCC, Manassas, VI, USA). Cell culture was conducted according the previous study [27
]. Culture medium was replaced every 2−3 d, and then treated with trypsin (Fisher Scientific, Ottawa, ON, Canada) and seeded into a 96-well, 12-well, or 24-well plate. Cells were treated with H2
and total phenolic extract (TPE) after six days (100% confluent).
Oxidative damage was induced by H2O2. For TPE treatment, a stock solution containing 20 mg/mL of phenolic compound in dimethyl sulfoxide (DMSO, 0.1%) was freshly prepared and diluted in the culture medium. To eliminate the influence of DMSO, equal levels of DMSO were added to all of the experimental groups. The treatments were as follows: (A) prevention: cells were treated with TPE for 1 h, and then 0.8 and 2.0 mM H2O2 were added for 1 h treatment, respectively. (B) Protection: cells were treated with TPE for 1 h, and then TPE plus 0.8 and 2.0 mM H2O2 were added for 1 h treatment, respectively. (C) Remedy: cells were treated with 0.8 and 2.0 mM H2O2 1 h, and then treated with TPE containing 0.5, 1.0, 10, 50, and 100 μg/mL of the phenolic compound for 4 h.
2.8. Cell Viability Assay
Cell viability was measured according to Yang et al. [27
]. Briefly, Caco-2 cells were seeded into 96-well plates at a density of 2 × 104
/mL and cultured in the culture medium for 6 d (100% confluent). After different treatments described above, the cells were washed once with phosphate belanced solution (PBS). A 100 μL fresh medium containing 10% WTS-1 was added and the cells were incubated for 1.5 h. The absorbance at 450 nm was measured using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek, Kobashigawa LC, Tokyo, Japan). Cell viability was presented as a percentage of untreated, control cells.
2.9. Transepithelial Electrical Resistant (TEER) Measurement
TEER measurements were performed using the method of Yang et al. [27
2.10. Reactive Oxygen Species (ROS) Assay
Cells were cultured onto coverslips (24-well plate, Fisher Scientific) for two days and then treated with 2 mM of H2O2 for 1 h, and followed by TPE containing 1.0 μg/mL of the phenolic compound from stamen for 4 h (0.5 mL). At the end of treatment, 0.5 mL DCFH-DA solution (10 µM in medium) was added and cells were incubated for 30 min at 37 °C continuously. Cells were then fixed using 4% paraformaldehyde (PFA) for 15 min at room temperature. Cells were then washed twice with PBS and mounted using Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc. Burlingame, CA, USA). The images were taken by a Zeiss Fluorescence Microscope (Carl-Zeiss Ltd., Toronto, ON, Canada).
2.11. Glutathione (GSH) Content Determination
Total GSH and glutathiol (GSSG) contents were determined using a Glutathione Colorimetric Detection Kit (Catalog Number: EIAGSHC, Invitrogen, Carlsbad, CA, USA) according to the instructions. Cells were cultured in 12-well plates and treated with 2 mM of H2O2 for 1 h and then treated with 1.0 μg/mL of TPE from stamen.
2.12. RNA Extraction and Real-Time PCR
After being treated with 2 mM of H2
for 1 h and then treated with 1 μg/mL of TPC from the stamen, cells were washed with PBS once. Total RNA was extracted from Caco-2 cells using Trizol reagent (Invitrogen, Fisher Scientific; Ottawa, ON, Canada) following the manufacturer’s protocol. The, 1 μg of total RNA was reverse transcribed into cDNA using the iScriptTM cDNA Synthesis kit (Bio-Rad, Laboratories Ltd., Montreal, QC, Canada) following the manufacturer’s instruction. Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad) on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Laboratories Ltd.). The reference gene was β-actin. The primers for real-time PCR analysis were designed with Primer-Blast based on the published cDNA sequence in the GenBank. The detected genes and sequences of primers are listed in Table S1
2.13. Immunofluorescent Staining
Immunofluorescent staining assay was conducted according to Yang et al. [27
] with minor modification. Cells were cultured onto coverslips (24-well plate, Fisher Scientific) for four days and treated with 2 mM of H2
for 1 h, and then treated with 1.0 μg/mL of TPC from stamen.
2.14. Statistical Analysis
The data were analyzed using GraphPad Prism 7.0 (San Diego, CA, USA). The differences of mean values among treatments were determined using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant differences (HSD) test at p < 0.05 significance level. The results are presented as mean ± standard deviation (SD).
The TPEs of the four organs showed similar phenolic profiles (Figure 1
). In total, twenty-one phenolic compounds were identified and quantified (Table 1
and Table 2
). Contrary to the TFC obtained using colorimetric methods, the contents of individual flavonoid glycosides determined using HPLC and the sum values of TPE from the ovary and calyx are far lower than those of the petal and stamen. This result is likely attributed to more protein and sugar in the ovary and calyx. High levels of identifiable flavonoid glycosides were found in the petal and stamen. Li et al. [8
] identified and quantified 26 flavonoid glycosides in the petals of six yellow tree peony flower cultivars. In this study, eriodictyol-O
-glucoside, isosalipurposide, luteolin-7-O
-neohesperidoside, and kaempferol-3-O
-glucoside were identified and quantified in the petal, stamen, ovary, and calyx of FDB flower; however, no isosalipurposide was detected in ovary and calyx. Isosalipurposide was found at a relatively higher level in the stamen, which in turn was reported to have very high antioxidant activity owing to the hydroxyl group on position 4 of the B ring of this chalcone derivative [8
Given the only approved edible tree peony flower cultivar, all four of the organs of the FDB flower exhibited high ORAC, DPPH, and ABTS•+
radical scavenging activities, which can be attributed to their phenolic compounds. Both the ovary and calyx presented higher TPC and TFC (Figure 2
) than other organs, while the ovary gave the highest DPPH and ABTS•+
radical scavenging activities (Table 3
). These findings suggest that these two—often overlooked—organs could also be used as antioxidant food ingredients. In this study, the petal had the highest ORAC values, which were not consistent with values obtained using the DPPH and ABTS•+
assays (Table 3
). The electron transfer mechanism prevails in the latter, while ORAC is based on the hydrogen–atom transfer mechanism [28
Usually, the occurrence of intestinal diseases is associated with a defective barrier function caused by the injury from oxidation [29
]. Hence, preventing oxidative damage and repairing the intestinal barrier should be effective to prevent intestinal diseases occurrence. It has been proven that phytochemicals could reduce the production of cellular ROS. In the present study, an oxidative damage model of Caco-2 cells was established using H2
induction, aiming to evaluate the therapeutic effects of TPE on intestinal oxidative damage. Interestingly, at a low concentration of H2
(0.8 mM), TPE addition increased the viability (Figure 3
B,C). However, only H2
(0.8 mM) and TPE (from 0 to 100 μg/mL) treatment showed no impact on cell viability (Figure S1
), indicating that the reaction between H2
and TPE might produce some unknown components with antioxidant activity or could stimulate cell metabolism. A higher concentration of H2
(2.0 mM) contributed to the death of cells (Figure 3
A) and the positive effect of TPE could no longer be displayed (Figure 3
B). Cell viability was increased when the cells were washed with PBS after treatment with 2.0 mM of H2
and then treatment with TPE (Figure 3
C). The effect of TPE from the petal, stamen, calyx, and ovary of the tree peony flower on cell viability after H2
treatment is depicted in Figure 3
D. TPE from the stamen was more effective for recovering the cell viability compared with that from the other three organs. However, TPC (Figure 2
) and free radical scavenging ability (Table 3
) of the stamen were relatively low, likely owing to the method used for presenting the data. The total phenol content and free radical scavenging ability were calculated by the dry weight of the flower, while the cell study was conducted with a consistent concentration of TPE (1.0 μg/mL). These findings also indicated that TPE from the stamen likely contains more components that assisted in combating oxidative damage. Compared with the other three organs, phenolics identified from the stamen included a high content of methyl digallate (8.05% of total), penta-O
-galloyl-glucose (15.38% of total identified phenolics), and a relatively higher level of isosalipurposide (Table 2
). Hence, it was demonstrated that these three phenolic components might be responsible for the relatively high antioxidant activity.
-induced oxidative stress triggered an imbalanced redox state and excessive ROS accumulation in Caco-2 cells, indicating that H2
treatment induced oxidative damage to the cells. However, a follow-up TPE treatment for 4 h significantly decreased ROS level (Figure 4
A), indicating that TPE from the stamen of the tree peony flower had a high ability for ROS scavenging (Table 3
). GSH is an important component of the biological redox system; therefore, its total content and the ratio of reduced GSH to GSSG can partially reveal the status of redox. H2
treatment decreased total GSH content, but did not affect GSSG content (Figure 4
B), indicating that H2
treatment initiated the consumption of reduced GSH. However, follow-up TPE treatment decreased GSSG content, although the total GSH content was unaltered. Hence, the ratio of reduced GSH to GSSG increased. This was evidence of TPE playing an important role in the direct scavenging of ROS, causing decreased consumption of reduced GSH (Figure 4
C). In addition, TPE treatment up-regulated the mRNA abundance of GPX-Px and SOD (Figure 4
D), which are the critical antioxidant enzymes [30
]. However, HO-1 expression was not affected. This observation illustrated that TPE did not stimulate the Nrf-2 pathway in which HO-1 expression acts as the downstream signal transmitting messenger [31
The intact gut barrier is essential to prevent cell damage and restore cellular function caused by intestinal oxidative stress [32
]. The TEER value is an important indicator to evaluate the integrity and tightness of the barrier formed by epithelial cells [36
]. The barrier function of Caco-2 cells was measured until there was the formation of the stable layer. The H2
treatment lowered the TEER value, suggesting an increase in cell permeability [37
], and a defective barrier function of cells. TEER values increased after 4 h intervention with TPE, indicating that the barrier function of Caco-2 cells was significantly enhanced. It was shown that phytochemicals from apples could enhance the TEER value of Caco-2 cells [38
], and this could potentially be attributed to the antioxidant effects of TPE from the tree peony flower. In the present study, the mRNA level of tight junction proteins including ZO-1, CLDN1, CLDN3, and occludin was determined (Figure 5
treatment increased the mRNA expression of CLDN3 and occludin. When compared with the sole H2
treatment, TPE enhanced the mRNA abundance of ZO-1, CLDN3, and occludin, but did not affect the CLDN1 mRNA level in the Caco-2 cells. This could be attributed to the different sensitivity of gene expression to H2
]. In addition, the morphology of the cytoskeleton and tight junction was visualized by β-actin and ZO-1 immunofluorescent staining. H2
treatment disorganized the β-actin fibrin and diffused ZO-1, indicating that H2
caused oxidative damage to the cell membrane monolayer. The addition of TPE obviously repaired cell structure and stabilized morphological characteristics of ZO-1 (Figure 5
C). These findings indicated that TPE could regulate the tight junction protein mRNA expression and repair the structure of ZO-1 protein to enhance the barrier function of Caco-2 cells.