Phenolic contents and cellular antioxidant activity of Chinese hawthorn “Crataegus pinnatifida”

Phenolic contents and cellular antioxidant activity of Chinese hawthorn “Crataegus pinnatifida”

Food Chemistry 186 (2015) 54–62 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Phenoli...

635KB Sizes 6 Downloads 71 Views

Food Chemistry 186 (2015) 54–62

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Phenolic contents and cellular antioxidant activity of Chinese hawthorn ‘‘Crataegus pinnatifida’’ Lingrong Wen a,b, Xingbo Guo a,b, Rui Hai Liu a,c,⇑, Lijun You a,b,⇑, Arshad Mehmood Abbasi a,d, Xiong Fu a,b a

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China Center of Guangdong Food Green Processing and Nutrition Regulation Engineering Technology, South China University of Technology, Guangzhou, Guangdong Province 510640, China c Department of Food Science, Cornell University, Ithaca, NY 14853, United States d Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan b

a r t i c l e

i n f o

Article history: Received 13 January 2015 Received in revised form 6 March 2015 Accepted 7 March 2015 Available online 13 March 2015 Keywords: C. pinnatifida Phenolics Procyanidin B2 Antioxidant activity Cellular antioxidant activity

a b s t r a c t It is evident from various epidemiological studies that consumption of fruits and vegetables is essential to maintain health and in the disease prevention. Present study was designed to examine phenolic contents and antioxidant properties of three varieties of Crataegus pinnatifida (Chinese hawthorn). Shanlihong variety exhibited elevated levels of total phenolics and flavonoid contents, including free and bond phenolics. Procyanidin B2 was most abundant phenolic compound in all samples, followed by epicatechin, chlorogenic acid, hyperoside, and isoquercitrin. The free ORAC values, and free hydro-PSC values were 398.3–555.8 lmol TE/g DW, and 299.1–370.9 lmol VCE/g DW, respectively. Moreover, the free cellular antioxidant activity (CAA) values were 678–1200 lmol of QE/100 g DW in the no PBS wash protocol, and 345.9–532.9 lmol of QE/100 g DW in the PBS wash protocol. C. pinnatifida fruit could be valuable to promote consumer health. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Various epidemiological studies have demonstrated that increased consumption of plant-based whole foods, fruits and vegetables are important for good health and disease prevention, especially in the prevention of chronic diseases, which include: cancer, diabetes, cardiovascular disorders, Alzheimer disease, and age-related functional decline (Eberhardt, Lee, & Liu, 2000; Liu, 2013). Fruits and vegetables are potential sources of phytochemicals particularly phenolic compounds, which are major bioactive compounds and natural antioxidants (Sun, Chu, Wu, & Liu, 2002). In the USDA (2010) dietary intake advise people are recommended to eat at least 9 servings of fruits and vegetables per day for 2000 kcal diet. However, in China, people rarely realized the consequence of fruits and vegetables intake on daily basis. Crataegus pinnatifida (hawthorn), is a member of family Rosaceae. This species is widely distributed in Asia, Europe and North America (Kwok et al., 2013). Over 1000 species of genus Crataegus have been identified worldwide. However, C. pinnatifida ⇑ Corresponding authors at: Department of Food Science, Cornell University, Ithaca, NY 14853, United States. Tel.: +1 (607) 255 6235; fax: +1 (607) 254 4868 (R.H. Liu). College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China. Tel./fax: +86 20 87113848 (L. You). E-mail addresses: [email protected] (R.H. Liu), [email protected] (L. You). http://dx.doi.org/10.1016/j.foodchem.2015.03.017 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

and C. pinnatifida Bge. var. major N.E.Br. are common in China (Yang & Liu, 2012). Traditionally C. pinnatifida is used as Chinese medicinal herb. This species showed strong antioxidant properties (Cui, Nakamura, Tian, Kayahara, & Tian, 2006). To date, over 150 compounds, especially phenolic compounds have been identified in C. pinnatifida (Wu, Peng, Qin, & Zhou, 2014). Among these phenolic compounds, procyanidins (procyanidin B2, procyanidin B5, and procyanidin C1), flavonoids (epicatechin, hyperoside, quercetin, rutin, and isoquercitrin), and triterpenoids acid (ursolic acid, corosolic acid, oleanolic acid, and maslinic acid) are the key bioactive components of hawthorn (Chai et al., 2014; Liu, Kallio, Lu, Zhou, & Yang, 2011; Wu et al., 2014; Yang & Liu, 2012). And procyanidins and triterpenoids acid dominate in the fruits, while flavonoids are most abundant in the leaves (Wu et al., 2014; Yang & Liu, 2012). Moreover, chemicals extracted from the species or consumption of fruits from the species were reported to exhibit a variety of pharmacological effects on digestive, cardiovascular, and endocrine systems (Guo, Liu, Gao, & Shi, 2014; Li, Zhu, Guo, et al., 2013; Zhu et al., 2013), and neuro-protective (Chang et al., 2013), and have antiviral, anti-inflammatory, anticancer, and antimicrobial activities (Li, Zhu, Dong, et al., 2013; Tadic et al., 2008). Additionally, fruits of C. pinnatifida are consumed fresh or processed into canned fruits, jams, jellies, and soft drinks in food and beverages industries (Yang & Liu, 2012). Currently, different researchers have paid their attention on C. pinnatifida, because of

L. Wen et al. / Food Chemistry 186 (2015) 54–62

its health benefits and abundant bioactive compounds (Liu, Yuan, & Zhang, 2010). However, very limited information is known about cellular antioxidant activity (CAA) of different varieties of C. pinnatifida used particularly in China. The cellular antioxidant activity (CAA) assay is a newly developed approach that quantifies the antioxidant capacity of bioactive compounds in cell cultures. In this method, a probe, 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), is taken up by HepG2 cells and fluorescences when oxidized to fluorescent DCF by reactive oxygen and reactive nitrogen species. HepG2 cells treated with pure phytochemical compounds and (or) extracts show the ability of quenching peroxyl radical and inhibiting the generation of DCF (Wolfe & Liu, 2007). Comparing to other methods, the HepG2 CAA assay via probe DCFH-DA to DCF provides a better understanding on how antioxidants will be taken up, distributed and metabolized under physiological conditions? Though the extracellular antioxidant activity of C. pinnatifida extract has been studied with chemical methods, there is little information on its antioxidant properties within cell that takes bioavailability into account. Thereby, present investigation was aimed to examine the phytochemical profiles, including both free and bound phenolics and flavonoids, and antioxidant properties of three varieties of C. pinnatifida using oxygen radical absorption capacity (ORAC), hydrophilic peroxyl radical scavenging capacity (hydro-PSC), and cellular antioxidant activity (CAA) assays.

2. Materials and methods 2.1. Chemicals and reagents Ascorbic acid, gallic acid, Folin–Ciocalteu reagent, sodium borohydride (NaBH4), aluminum chloride, chloranil, catechin hydrate, vanillin, epicatechin, hyperoside, isoquercitrin, chlorogenic acid, procyanidine B2, and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Potassium hydroxide (KOH), sodium hydroxide (NaOH), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4) and sodium bicarbonate (NaHCO3) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Methanol, absolute ethyl alcohol, acetone, acetic acid, tetrahydrofuran (THF), hydrochloric acid (HCl) were obtained from Guangzhou Reagent Co. (Guangzhou, China). 2,20 -Azobis-amidinopropane (ABAP) was purchased from Aldrich (MO, USA). All reagents used were of analytical grade. Additionally, acetonitrile and methanol used for HPLC analysis was obtained from CNW Technologies Gmbh (Dusseldorf, Germany).

2.2. General method The laboratory analysis work was conducted in department B8, South campus of South China University of Technology, Guangzhou Higher Education Mega Center. All analysis was conducted in triplicate unless specifically described different.

2.3. Sample preparation Fresh fruit of three varieties of C. pinnatifida (hawthorn): Shanlihong (C. pinnatifida Bge. var. major N.E.Br.), Shanzha (C. pinnatifida Bge), and Dajinxing (C. pinnatifida Bge var. major) were harvested from commercial orchard in Shandong, China. The fruits were cleaned with distilled water, and then the pulp and seed were manually separated before use.

55

2.4. Extraction of soluble free and bound phenolic compounds Fruit pulp was cut into small pieces, and free phytochemical contents were extracted, following the method Guo, Li, Tang, and Liu (2012) with some modifications. Briefly, 25 g of sample was blended with 150 mL of chilled 80% acetone (1:6, w/v) using a Waring blender (DS-1, Shanghai Specimen and Model Factory, Shanghai, China) for 5 min and further homogenized with a homogenizer (T25, IKA Co., Staufen, Germany) for another 4 min. The mixture was then centrifuged at 3500 rpm for 10 min, then filtered through Whatman No. 2 filter paper under vacuum, and the remaining residue was extracted twice as mentioned above. The supernatants were collected and concentrated to less than 5 mL with a rotary evaporator (Hei-VAP, Heidolph, Germany) under reduced pressure at 45 °C. The extracts were reconstituted in distilled water to a final volume of 25 mL and stored at 40 °C until use. All extractions were performed in triplicate for each sample. Bound phytochemicals of fresh hawthorn pulp were extracted following the method Sun et al. (2002) with some modifications. Briefly, the residues obtained after extraction of free phytochemical contents were digested with 25 mL of 4 M NaOH at room temperature for 1 h while shaking under nitrogen. The mixture was acidified to pH 2.0 with concentrated hydrochloric acid and extracted five times with ethyl acetate. The ethyl acetate fractions were pooled and evaporated at 45 °C to dryness before reconstituting in 25 mL of water and then stored at 40 °C until use. 2.5. Determination of total phenolics The total phenolic contents were measured by colorimetric Folin–Ciocalteu method with some modifications (Singleton, Orthofer, & Lamuela-Raventos, 1999). Briefly, an aliquot (0.1 mL) of diluted fruit extracts was mixed with 0.4 mL of distilled water and 0.1 mL of Folin–Ciocalteu reagent. After incubating for 6 min, 7% Na2CO3 (1.0 mL) and 0.8 mL distilled water were added and the mixture was allowed to stand for 90 min at room temperature. The absorbance was measured at 760 nm using a Nucleic acid/ Protein analyzer (Du730, Beckman Coulter, USA). The total phenolic contents were determined as milligram gallic acid equivalents per 100 g on dry weight basis (mg GAE/100 g DW). 2.6. Determination of total flavonoids The total flavonoid contents were determined using the sodium borohydride/chloranil protocol (SBC) (Malta, Tessaro, Eberlin, Pastore, & Liu, 2013). After drying under nitrogen gas, phytochemical extracts were reconstituted in 1 mL of tetrahydrofuran/ethanol (THF/EtOH, 1:1, v/v). Catechin hydrate standard (0.3–10.0 mM) was prepared fresh in 1 mL of THF/EtOH. Each test tube (15  150 mm) with sample or standard solution was mixed with 0.5 mL of 50 mM NaBH4 solution and 0.5 mL of 74.6 mM AlCl3 solution, and was shaken in an orbital shaker at 180 rpm for 30 min at room temperature, following by shaking for another 30 min after adding an additional 0.5 mL of NaBH4 solution. Chilled acetic acid solution (2.0 mL of 0.8 M, 4 °C) was added into each test tube and kept in the dark for 15 min after thorough mixing. Then 1 mL of 20.0 mM chloranil was added into the mixture and heated at 99 °C with shaking for 60 min. The reaction solutions were cooled with tap water immediately, and volume was brought to 4 mL with methanol. After mixing with 1 mL of 16% (w/v) vanillin, the reaction solution was mixed with 2 mL of 12 M HCl and kept in the dark for 15 min. The reaction mixture were centrifuged at 2500 rpm for 10 min before measuring the absorbance at 490 nm. The standard curve of different catechin hydrate concentration was used to calculated the total flavonoid content of

56

L. Wen et al. / Food Chemistry 186 (2015) 54–62

C. pinnatifida, and the results were expressed as milligram of catechin equivalents per 100 g of dry weight (mg CE/100 g DW).

2.7. Determination of major phenolic compounds

2.8. Quantification of the total antioxidant activity by oxygen radical absorption capacity (ORAC) assay The ORAC of C. pinnatifida was determined as described by Huang, Ou, Hampsch-Woodill, Flanagan, and Prior (2002) with some modifications (Wang, Chen, Xie, Ju, & Liu, 2013). All the reagents were made fresh and dissolved in 75 mM phosphate buffer. Trolox diluted to a series of solutions of 6.25, 12.5, 25 and 5 0 lM concentrations were used as the standard. The assay was performed in the inner wells of black-walled 96-well microplates (Corning Scientific, Corning, NY, USA). Firstly, extracts dilution or Trolox (20 lL) was added in a well of the plate followed by 200 lL of fluoresce in sodium salt (0.96 lM). After the mixture was incubated for 20 min at 37 °C, ABAP

Peak intensity (AU)

Different phenolic compounds in the free phenolic extracts were identified and quantified by reverse phase HPLC, using a Waters breeze (Waters Co., Milford, MA, USA) separation module equipped with a Sunfire C18 reversed phase column (4.6  250 mm, 5 lm of particle size, Waters, USA), a binary HPLC pump (Waters 1525), an auto sampler (Waters 2707), and a Photodiode Array detector (PAD) (Waters 2998). The samples were eluted with a gradient system consisting of solvent A (0.1% trifluoroacetic acid, v/v) and solvent B (acetonitrile:methanol = 80:20, v/v) with a flow rate of 1.0 mL/min. The column temperature was maintained at 35 °C and the injection volume was 10 lL. The gradient elution program followed was: 0–5 min with 3–4% solvent B, 5–8 min with 4–10% B, 4 min with 10% B, 12–26 min with 10–14% B, 26–32 min with 14–18% B, 32–48 min with 18–20% B, 48–52 min with 20–25% B, 52–57 min with 25–60% B, 57–61 min with 60–3% B, and 4 min with 3% B. The phenolic compounds were identified from the comparison with

the retention time of standard using PDA detection at 280 and 340 nm, respectively (Liu et al., 2011), and individual phenolic content was estimated on the basis of peak area and the calibration curves of the corresponding standards. The results were expressed as mg/100 g DW. The calibration ranges of chlorogenic acid, procyanidin B2, epicatechin, hyperoside, and isoquercitrin were 1.0–7.5, 4–16, 4–20, 0.6–3.0, and 0.4–2.0 lg, respectively (Fig. 1A).

Retention time (min) Fig. 1. The chromatogram for the standards and the free extract of C. pinnatifida obtained by gradient elution on reversed phase C-18 column and PDA detector at 280 nm. A: standards; B: Shanglihong; C: Shanzha; D: Dajinxing. Compounds: 1, chlorogenic acid (r2 = 0.9998); 2, procyanidin B2 (r2 = 0.9998); 3, epicatechin (r2 = 0.9998); 4, hyperoside (r2 = 0.9993); and 5, isoquercitrin (r2 = 0.9999).

57

L. Wen et al. / Food Chemistry 186 (2015) 54–62 700 ORAC value PSC value

b

600

Antioxidant activities

ab 500 a b

400 a

a

300

200

100

0 Shanzha

Dajinxing

washed with 100 lL of PBS, followed by treating in triplicate for 1 h with 100 lL of treatment medium containing tested C. pinnatifida extracts supplemented with 50 lM DCFH-DA. After removing the treatment medium completely, cells were treated differently, one plate was washed with 100 lL of PBS (PBS wash protocol), while the other was without PBS (no PBS wash protocol). Then 100 lL of oxidant treatment medium (HBSS with 10 mM Hepes), containing 600 lM ABAP, was added into the cells, and 96-well microplate was placed in a multi-mode microplate reader (Filter Max F5, Molecular Devices, USA) at 37 °C with emission wavelength at 538 nm and excitation wavelength at 485 nm. Fluorescence generation was monitored every 5 min for 60 min. After blank subtraction and subtraction of initial fluorescence values, the area under the curve for fluorescence versus time was integrated to calculate the CAA value of each hawthorn concentration, according to the following equation:

Shanlihong

Varieties of C. pinnatifida Fig. 2. The antioxidant activities (ORAC and PSC values) of free phytochemical extracts of Chinese hawthorn fruits (bars in the same group with different letters indicated a significant difference (p < 0.05)).

(20 lL, 119 mM) was added just before analysis and the reaction was carried out at a constant temperature of 37 °C using 75 mM phosphate buffer solution (pH 7.4) as a blank. The fluorescence generation was measured every 5 min for 35 cycles using a multi-mode microplate reader (Filter Max F5, Molecular Devices, USA) with 485 nm excitation and 538 nm emission. The ORAC values were calculated from the regression equation between Trolox concentration and the net area under curve (net AUC) The results were expressed as lmol Trolox equivalents per gram of DW of hawthorn fruit (lmol TE/g DW). 2.9. Determination of the total antioxidant activity by rapid peroxyl radical scavenging capacity (PSC) assay The PSC assay was conducted as described by Adom and Liu (2005). Freshly prepared gallic acid and ascorbic acid solutions diluted in 75 mM phosphate buffer (pH 7.4) to five different concentrations, were used as standards. At first, 100 lL of each extract or standards solutions was added in each well of the 96 well plate. Then another 100 lL of DCFH dye (13.26 lM), which was prehydrolyzed with 1 mM KOH to remove the diacetate moiety just before used in the reaction, was added into each wall. After adding 50 lL of peroxyl radicals producer (ABAP, 40 mM), the reaction was performed at 37 °C for 40 min. Fluorescent was recorded at 485 nm excitation and 538 nm emission with a multi-mode microplate reader (Filter Max F5, Molecular Devices, USA). The results were calculated as lmol vitamin C equivalents per gram of hawthorn fruit on a dry weight basis (lmol VCE/g DW). 2.10. Cellular antioxidant activity (CAA) assay of C. pinnatifida extracts HepG2 cells, were cultured in growth medium (WME), containing 10 mM Hepes, 50 units/mL penicillin, 2 mM L-glutamine, 50 lg/mL streptomycin, 5 lg/mL insulin, 100 lg/mL gentamicin, 0.05 lg/mL hydrocortisone, and 5% FBS (Gibco Life Technologies, Grand Island, NY), and maintained at 37 °C in 5% CO2 in an incubator. For experiments, cells were used between 25 and 34 passages. The CAA assay protocol was conducted following the protocol described by Wang et al. (2013). Briefly, HepG2 cells were seeded at a density of 6  104 cells/well on a 96-well microplate in 100 lL of complete medium/well. After incubating for 24 h at 37 °C, the growth medium was removed, and the cells were

CAA ðunitsÞ ¼ 1 

Z

Z  CA SA

R where SA is the integrated area under the tested sample fluoresR cence versus time curve, and CA refers to the integrated area under the control in the fluorescence curve. The median effect plot of log (fa/fu) versus log (dose) was used to calculated the median effective dose (EC50) of the hawthorn extracts, where fa is the fraction affected by the treatment (CAA unit) and fu is the fraction un-affected (1  CAA unit) by the treatment. In each experiment, quercetin was used as standard, and the EC50 values were converted to CAA values, which were calculated as micromoles of quercetin equivalents per 100 g of dry weight (lmol QE/100 g DW) fruit pulp, using the mean EC50 value for quercetin from three separate experiments. 2.11. Statistical analysis Statistical analysis were performed using SPSS software 12.5 (SPSS Inc., Chicago, IL, USA) and dose–effect analysis was carried out with Calcusyn software version 2.0 (Biosoft, Cambridge, U.K.). Statistical calculations were conducted to calculate the correlation between total phenolics contents/total flavonoids contents and ORAC/PSC values. Results were subjected to ANOVA and mean difference was determined by Tukey’s multiple comparison test at p < 0.05 were considered as significant. All data were reported as the mean ± SD (n = 3). 3. Results and discussion 3.1. Results 3.1.1. Total phenolic content The free, bound, and total phenolic contents of three varieties of Chinese hawthorn are presented in Table 1. Among the three varieties analyzed, Shanlihong had the highest soluble free phenolic content, followed by Dajinxing and Shanzha. However, the trend of bound phenolic content was almost opposite, which was in a decreasing order of ‘Dajinxing’ > ‘Shanzha’ > ‘Shanlihong’. Compared with the phenolic content between free and bound phytochemical extracts, the phenolics in soluble bound form were significantly lower than that of soluble free form (p < 0.001), and highest bound phenolic content was 31.7 mg GAE/ 100 g DW (Dajinxing variety), less than 1% of the free phenolic content. The total phenolic content (soluble free + bound) was highest in Shanlihong, followed by Dajinxing and Shanzha. 3.1.2. Total flavonoid content The free, bound, and total flavonoid contents determined in the fruit samples are given in Table 1. In general the same trend was

58

L. Wen et al. / Food Chemistry 186 (2015) 54–62

Table 1 Total phenolic, total flavonoid contents and percentage contribution of free and bound fractions to the total. Varieties

Shanzha Dajinxing Shanlihong a b

Phenolics (mg GAE/100 g DW)

Flavonoids (mg CE/100 g DW)

Free

Bound

Total

Free

Bound

Total

5562 ± 137aa (99.52)b 5887 ± 152a (99.46) 6640 ± 122b (99.60)

27.3 ± 2.1a (0.48) 31.7 ± 1.9b (0.54) 26.5 ± 1.7a (0.40)

5589 ± 138a 5919 ± 152b 6667 ± 121c

3921 ± 189a (99.77) 4609 ± 124b (99.76) 6168 ± 120c (99.85)

9.3 ± 0.3a (0.23) 11.5 ± 0.6b (0.24) 8.4 ± 0.4c (0.15)

3930 ± 189a 4620 ± 124b 6177 ± 120c

Values with different letters show a significant difference (p < 0.05). Values in parentheses indicate percentage contribution to the total.

observed in free and bound flavonoids content as shown by phenolic contents. Specifically, the free flavonoid content was highest in Shanlihong, followed by Dajinxing and Shanzha. Comparatively, bound flavonoids contents were lower than free form (p < 0.001). Among bound flavonoids, Dajinxing variety exhibited highest value, followed by Shanzha, and Shanlihong. The trend of total flavonoid contents was similar to that of free flavonoid contents along with a decreasing order of ‘Shanlihong’ > ‘Dajinxing’ > ‘Shanzha’. 3.1.3. Analysis of the main individual phenolic compounds Identification of the phenolic compounds was carried out by comparing retention times with those of authentic standards. As shown in Fig. 1, five major phenolic compounds, including epicatechin, procyanidin B2, hyperoside, isoquercitrin, and chlorogenic acid, were quantified in the soluble free phytochemical extracts of fresh fruits of three varieties of C. pinnatifida by HPLC-PDA (Table 2A). The extract of Shanlihong has the highest amount of phenolics, followed by Dajinxing and Shanzha varieties, which was in agreement with the free phenolic contents. The dominant phenolics: procyanidin B2, was found to be highest in Shanlihong,

Table 2 The major phenolics of Chinese hawthorn in the present and previous work. Phenolics

Varieties Shanzha

Dajinxing

Shanlihong

(A) The major phenolics of Chinese hawthorn in the present work (mg/100 g DW) Epicatechin 488.5 ± 18.0aa 605.6 ± 14.6b 697.7 ± 14.3c Chlorogenic acid 79.5 ± 3.0a 89.4 ± 2.6b 106.2 ± 3.1c Procyanidine B2 545.3 ± 26.9a 648.8 ± 13.8b 793.2 ± 2.8c Hyperoside 28.4 ± 1.3a 22.4 ± 0.4a 49.2 ± 0.9b Isoquercitrin 13.2 ± 0.5a 11.5 ± 0.1a 23.4 ± 0.6b Phenolics

Previous results Liu et al. (2011) (mg/100 g DW)

Cui, Li, et al. (2006) (mg/100 g FW)

(B) The major phenolics of Chinese hawthorn in the previous work Epicatechin 90–1170b 69.5–246.9c Chlorogenic acid 20–160 b 11.9–54.4c Procyanidine B2 70–1240b 71.2–261.5c Hyperoside 10–80b 0.9–15.2c Isoquercitrin 10–30b 0.3–10.1c Ideain 0–70b Procyanidin dimers I 10–270b Procyanidin dimers II 10–150b Procyanidin trimer I 10–270b Procyanidin trimer II 70–690b Procyanidin trimer III 1–120b Procyanidine B5 11.8–70.6c Procyanidine C1 31.2–136.3c Oleanolic acid 3.5–22.4c Ursolic acid 64.7–117.7c a Values with different letters in a row indicated a significant difference (p < 0.05). b Contents of phenolics in the fruits of 22 cultivars/origins of three species of Chinese hawthorn (Crataegus spp.) by HPLC–ESI-MS-SIR, and expressed as mg/100 g dry weight (Liu et al., 2011). c Contents of phenolics in the matured fruits of 37 representative cultivars of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.) by HPLC, and expressed as mg/100 g fresh weight (Cui, Li, et al., 2006).

followed by Dajinxing and Shanzha with significant differences among the three varieties (p < 0.05). Similar trend was also detected for the epicatechin and chlorogenic acid contents. However, the contents of hyperoside and isoquercitrin showed different order: Shanlihong > Shanzha > Dajinxing. 3.1.4. Total antioxidant activity determined by oxygen radical absorption capacity (ORAC) assay A higher ORAC value is an indication of stronger oxygen radical absorbance capacity of the test sample. The measured values of ORAC assay for free phenolic extracts are summarized in Fig. 2. Among the three varieties, Shanlihong was found to be the most potent source of antioxidant with highest ORAC values (555.8 ± 63.7 lmol TE/g DW), followed by Dajinxing (461.3 ± 41.8 lmol TE/g DW), and Shanzha possessed lowest ORAC value of 398.3 ± 31.9 lmol TE/g DW. This order was consistent with those of free phenolic contents. Moreover, significant difference were observed in ORAC values between the varieties of Shanlihong and Shanzha (p < 0.05), but variations between Shanlihong and Dajinxing, as well as between Shanzha and Dajinxing were insignificant. Overall the total antioxidant activity of three varieties decreased as: Shanlihong > Dajinxing > Shanzha. 3.1.5. Total antioxidant activity measured by rapid peroxyl radical scavenging capacity (PSC) assay Results of total antioxidant activity determined by the HydroPSC assay for free phytochemical extracts of C. pinnatifida are displayed in Fig. 2. It was observed that Shanlihong variety exhibited elevated level for PSC (370.9 ± 22.7 lmol VCE/g DW) with a significant difference (p < 0.05), than other varieties. However, no significant difference was observed between Shanzha and Dajinxing, whose PSC values were 299.1 ± 19.3 lmol VCE/g DW, and 308.9 ± 23.2 lmol VCE/g DW, respectively. The PSC values in the three varieties were ‘Shanlihong’ > ‘Dajinxing’ > ‘Shanzha’. 3.1.6. Cellular antioxidant activity of C. pinnatifida A cellular antioxidant activity (CAA) assay was conducted to quantify the intracellular antioxidant property of Chinese hawthorn by preventing the formation of DCF using ABAP-induced peroxyl radicals in HepG2 cells. For the CAA assay, decreasing cellular fluorescence of tested sample compared to the control group indicates antioxidant capacity, because of the level of fluorescence was proportional to the degree of oxidation (Wolfe & Liu, 2007). As displayed in Fig. 3I, the kinetics of DCFH oxidation in HepG2 cells by peroxyl radicals generated from ABAP is well observed for quercetin and hawthorn phytochemical extracts. The increase in fluorescence from DCF oxidation was inhibited by quercetin and hawthorn extracts in a dose-dependent manner, as demonstrated by the curves generated from cells treated with quercetin (Fig. 3IA and B), Shanzha (Fig. 3IC and D), Dajinxing (Fig. 3IE and F), and Shanlihong (Fig. 3IG and H). The results indicated good antioxidant capacity of hawthorn phytochemical extracts regardless of whether the cells had been washed with PBS (Fig. 3IB, D, and F) or not (Fig. 3IA, C, and E).

59

L. Wen et al. / Food Chemistry 186 (2015) 54–62

no PBS wash

6e+6

0 2 5 8 12 16 20

5e+6

Fluorescence units

4e+6

3e+6

2e+6

PBS wash

5e+6

A

M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin

0 2 5 8 12 16 20

4e+6

Fluorescence units

II

1e+6

0

3e+6

2e+6

1e+6

0

0

10

20

30

40

50

60

70 0

10

20

Time (min)

2e+6

C

50

60

70

D

0 mg/mL Shanzha 1.5 mg/mL Shanzha 2.5 mg/mL Shanzha 5 mg/mL Shanzha 10 mg/mL Shanzha 15 mg/mL Shanzha 20 mg/mL Shanzha

3e+6

Fluorescence units

Fluorescence units

3e+6

40

PBS wash

4e+6

0 mg/mL Shanzha 1.5 mg/mL Shanzha 2.5 mg/mL Shanzha 5 mg/mL Shanzha 10 mg/mL Shanzha 15 mg/mL Shanzha 20 mg/mL Shanzha

4e+6

30

Time (min)

no PBS wash

5e+6

2e+6

1e+6

1e+6

0

0

0

10

20

30

40

50

60

70

0

10

20

Time (min)

30

70

F

2 mg/mL Dajinxing 4e+6

5 mg/mL Dajinxing 15 mg/mL Dajinxing 20 mg/mL Dajinxing 25 mg/mL Dajinxing 2e+6

5 mg/mL Dajinxing 10 mg/mL Dajinxing

Fluorescence units

10 mg/mL Dajinxing

3e+6

60

0 mg/mL Dajinxing

E

2 mg/mL Dajinxing

4e+6

50

PBS wash

5e+6

0 mg/mL Dajinxing

5e+6

40

Time (min)

no PBS wash 6e+6

Fuorescence unit s

B

M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin M Quercetin

3e+6

15 mg/mL Dajinxing 20 mg/mL Dajinxing

2e+6

25 mg/mL Dajinxing

1e+6

1e+6 0

0

-1e+6 0

10

20

30

40

50

60

70

0

10

20

Time (min)

no PBS wash 6e+6

3e+6

5e+6

G

50

60

0 mg/mL Shanlihong 1.5 mg/mL Shanlihong 2.5 mg/mL Shanlihong 5 mg/mL Shanlihong 10 mg/mL Shanlihong 15 mg/mL Shanlihong 20 mg/mL Shanlihong

4e+6

Fluorescence units

Fluorescence units

4e+6

40

70

PBS wash

0 mg/mL Shanlihong 1.5 mg/mL Shanlihong 2.5 mg/mL Shanlihong 5 mg/mL Shanlihong 10 mg/mL Shanlihong 15 mg/mL Shanlihong 20 mg/mL Shanlihong

5e+6

30

Time (min)

2e+6

3e+6

2e+6

H

1e+6

1e+6 0 0 -1e+6 0

10

20

30

40

50

60

70

Time (min)

0

10

20

30

40

50

60

70

Time (min)

Fig. 3. (I) Peroxyl radical-induced oxidation of DCFH to DCF in HepG2, and the inhibition of oxidation by quercetin (A, B), Shanzha extract (C, D), Dajinxing extract (E, F), and Shanlihong extract over time, using the protocol involving no PBS wash between antioxidant and ABAP treatments (A, C, E, G) and the protocol with a PBS wash (B, D, F, H), to remove antioxidants in the medium not associated with cells. The curves shown in each graph are from a single experiment (mean ± SD, n = 3). (II) Cellular antioxidant activities of C. pinnatifida expressed as CAA values (mean ± SD, n = 3, bars with different letters show a significant difference (p < 0.05)).

As shown in Table 3, the EC50 values of the three hawthorn extracts was ranged from 0.78 to 1.26 mg/mL DW in the no PBS wash protocol, which was significantly lower than that of PBS wash

protocol, whose EC50 values was ranged from 1.85 to 2.64 mg/mL DW. Among the three varieties, Shanlihong was observed to have the lowest EC50 values in the no PBS wash protocol, but the lowest

60

L. Wen et al. / Food Chemistry 186 (2015) 54–62

II

d

1400 no PBS wash PBS wash

CAA values

(µmol quercetrin equiv./g DW)

1200

c 1000

800

a a

a

600

400

b

200

0 Shanzha

Dajinxing

Shanlihong

variety of C. pinnatifida

Fig. 3 (continued)

EC50 values in PBS wash protocol was found in Dajinxing variety. While the highest EC50 values were found in the variety of Shanzha, regardless PBS wash or not. A negative correlation was found between the EC50 and CAA values, indicating a lower EC50 value is along with a higher CAA value, which was in accordance with this results (Fig. 3II). In the no PBS wash protocol, the CAA values of different tested samples was highest in Shanlihong (1200 ± 126 lmol of QE/100 g DW), followed by Dajinxing (903 ± 133 lmol of QE/100 g DW), and Shanzha (678 ± 32 lmol of QE/100 g DW) with significant differences among the three varieties (p < 0.05). However, no significant difference was observed between Dajinxing (533 ± 53 lmol of QE/ 100 g DW, the highest CAA value) and Shanlihong (521 ± 67 lmol of QE/100 g DW) in PBS wash protocol, which were significantly higher than that of Shanzha (346 ± 10 lmol of QE/100 g DW). The cellular antioxidant qualities of the phytochemical extracts were calculated for different varieties of Chinese hawthorn, based on their CAA values and total phenolic contents (Table 3). The cellular antioxidant qualities in the no PBS wash protocol varied from 2.34 of QE/100 lmol of phenolics (Shanzha) to 2.97 lmol of QE/100 lmol of phenolics (Dajinxing), which were greatly higher than that of PBS wash protocol, whose cellular antioxidant qualities were ranged from 1.20 lmol of QE/100 lmol of phenolics (Shanzha) to 1.54 lmol of QE/100 lmol of phenolics (Dajinxing). Additionally, the cellular uptake values were expressed as the percentage of antioxidant values with/without PBS wash, and the uptake rates were 51.02%, 58.99% and 43.46% in Shanzha, Dajinxing and Shanlihong phytochemical extracts, respectively. 3.2. Discussion 3.2.1. Phytochemical profiles of C. pinnatifida It has been reported that mostly fruits contain phenolic compounds in free form (Sun et al., 2002). Present findings are in

Table 3 The EC50 and cellular antioxidant quality using cellular antioxidant activity assay. Varieties

Shanzha Dajinxing Shanlihong a

EC50 (mg/mL)

Cellular antioxidant quality (lmol of QE/100 lmol of phenolics)

No PBS wash

PBS wash

No PBS wash

PBS wash

1.26 ± 0.06aa 0.86 ± 0.13b 0.78 ± 0.08b

2.64 ± 0.07a 1.85 ± 0.19b 1.90 ± 0.23b

2.34 ± 0.11a 2.97 ± 0.44a 2.88 ± 0.30a

1.20 ± 0.03a 1.54 ± 0.15b 1.34 ± 0.17ab

Values with different letters in a column show a significant difference (p < 0.05).

agreement of this as, the free phenolic content was up to 99% of total phenolics in the studied samples. Total phenolic contents in C. pinnatifida were ranged from 5589 to 6667 mg GAE/100 g DW, which were comparatively higher than fresh fruit of Crataegus monogyna Jacq. (a variety of hawthorn), containing total phenolics about 1226 ± 34 mg GAE/100 g DW (Froehlicher et al., 2009). However, Liu, Kallio, et al. (2010) determined high phenolic contents in C. pinnatifida Bge (96.9 ± 4.3 mg GAE/g DW) using microwave-assisted extraction, compare to present findings. These differences might be due to the diversity of variety and/or extraction method. It has been presented that intrinsic (genetic) variation, and extrinsic (environmental, maturity, postharvest handling and storage) factors can influence the biosynthesis of bioactive compounds in plants, which finally leads to the difference to variety (Olsson et al., 2004). Though HPLC analysis is an excellent method to measure individual flavonoid, limitation of standards for some compounds makes the use of HPLC limited. While it is more comprehensive to measure total flavonoids using the SBC assay, which is designed to quantified total flavonoids, including flavones, flavonols, flavanols, flavonones, flavononols, isoflavonoids, and anthocyanins (He, Liu, & Liu, 2008). Total flavonoid contents in the fruits of C. pinnatifida determined by SBC assay, were ranged from 3930 to 6177 mg CE/100 g DW. In the previous study, the total flavonoid contents determined by HPLC-UV assay was about 31 mg/g DW in fruits of C. pinnatifida (Yang & Liu, 2012), which was lower than present concentration. While the total flavonoid content of C. monogyna was failed to be calculated in fresh fruits extracts by aluminum chloride (AlCl3) colorimetric assay as a result of anthocyanin interference (Froehlicher et al., 2009). It seems that SBC assay may be a effective way to quantify the total flavonoid contents of C. pinnatifida fruits. The contributions of free flavonoids to free phenolics was ranged from 41.1 to 54.5%, with highest (54.4%) in Shanlihong variety (calculated on a micromole basis) indicating that flavonoids are the predominant phytochemicals in the fresh fruits of C. pinnatifida varieties which was in agreement with the results of HPLC analysis. Procyanidins, the second most abundant group of natural phenolics after lignins, are attracting attention because of their health benefits like cardiovascular protection, anti-aging, anticancer, due to their potential radical scavenging activity (Koleckar et al., 2012). Present study revealed that C. pinnatifida contains mainly flavan-3-ol monomers and dimers, such as epicatechin and procyanidin B2, which shows that procyanidins are the main active constituents of Chinese hawthorn fruits. The major phenolic compound contents reported from the previous works of the fruit of C. pinnatifida were summarized in Table 2B. Similarly, the content of epicatechin was closed to that of procyanidin B2, which was much higher than other compounds. Additionally, procyanidin dimer procyanidin B5 and trimer procyanidin C1 were also observed in Chinese hawthorn as well. Previous study showed that procyanidins in Chinese hawthorn fruits were mostly B-type procyanidins and their glycosides (Liu et al., 2011), and a significant inverse correlation was found between the procyanidin contents and the latitude of the geographical origin of the cultivars (Cui, Li, et al., 2006). It has been reported that hyperoside and isoquercitrin, two quercetin glycosides, were the most abundant flavonol glycosides in the extracts of hawthorn fruits (Jurikova et al., 2012), and hyperoside content was higher than isoquercitrin (Table 2B), which is similar to this finding. 3.2.2. Antioxidant activity of C. pinnatifida Intracellular oxidation stress is mainly caused by imbalance state between the production of reactive oxygen species (ROS) and antioxidant defense system of human body, which may lead to oxidative damage to biomolecule like DNA, lipids and protein,

L. Wen et al. / Food Chemistry 186 (2015) 54–62

thereby being a main contributor of chronic diseases, including cancer, diabetes mellitus, inflammatory diseases, aging and neurodegenerative disorders (Valko et al., 2007). It is necessary to supply enough antioxidant in the maintenance of ‘‘redox homeostasis’’ in the redox regulation to maintain good health. C. pinnatifida fruits were found to possess relatively high antioxidant activity and might be a potential source of natural antioxidants. The antioxidant activity of C. pinnatifida was determined using DPPH (Froehlicher et al., 2009; Mraihi et al., 2013; Salmanian, Mahoonak, Alami, & Ghorbani, 2014), FRAP, b-carotene/linoleic acid (Mraihi et al., 2013), and ABTS assay (Froehlicher et al., 2009). However, these methods do not take metabolism and bioavailability of antioxidant into account, and might failed to reflect the real situation of total antioxidant activity in fruits, vegetables, and other food materials (Karadag, Ozcelik, & Saner, 2009). It is important to choose the right methods to determine antioxidant activity in fruits, vegetables, and dietary supplements, because it has become an important tool used to screen foods with the greatest amount of antioxidants that exhibit benefit to human health (Guo et al., 2012). In this work, the total antioxidant activities of free phytochemical extracts were measured by ORAC and hydro-PSC methods, which were rapid and sensitive peroxyl radical scavenging capacity assay using fluorescein dyes as fluorescent probes for monitoring the reaction. Ou, Hampsch-Woodill, and Prior (2001) identified the fluorescein (FL) oxidized products induced by peroxyl radical with LC-MS technology, and found that the reaction mechanism was determined to proceed as a classic hydrogen atom transfer (HAT) mechanism which can be elucidated based on FL oxidized products. The results showed a high amount of phenolics in Chinese hawthorn fruits, which can provide enough hydrogen atom to scavenge peroxyl radicals and prevent fluorescein dyes oxidation, thereby showing high antioxidant activity. The PSC values was significantly correlated to total phenolics (R2 = 0.887, p < 0.001) and total flavonoids (R2 = 0.903, p < 0.001). These findings suggest that the phenolic compounds in hawthorn fruits might be responsible for the great peroxyl radical scavenging activity, especially the flavonoids presented in the extracts. In addition, the antioxidant activity of free phenolic extracts was comparatively better than that of bound phenolic extracts, whose ORAC and PSC values were 4.90–6.29 lmol TE/g DW, and 1.60–4.60 VCE/g DW, respectively (data not shown). These results indicated a direct relationship between total phenolic content and total antioxidant activity in phytochemical extracts of C. pinnatifida. Sun et al. (2002) reported similar phenomenon, and found that there was an obvious liner relationship between total phenolic content and total antioxidant activity in 11 fruits extracts (R2 = 0.9788, p < 0.01). Comparatively, the cellular antioxidant activity (CAA) assay, representing a marked improvement over traditional chemical antioxidant activity assays, is used for antioxidant activity determination. Due to its simulation of cellular biochemical processes which include: bio-accessibility, uptake, distribution, and metabolism of the antioxidants, CAA may be a better predictor of antioxidant behavior in biological system (Wolfe & Liu, 2007). To our knowledge this study, is the first report on the antioxidant activity of C. pinnatifida by CAA method. Results indicate that CAA values were ranged from 678 to 1200 lmol of QE/100 g DW in the no PBS wash protocol, and from 346 to 533 lmol of QE/ 100 g DW in the PBS wash protocol. The results suggest that the three varieties of C. pinnatifida show better antioxidant activity compared to other fruits (basis on fresh weight), such as wild blueberry, pomegranate, blackberry, strawberry, blueberry, cherry, apple and so on, whose CAA values ranged from 3.15 to 292 lmol of QE/100 g FW in the no PBS wash protocol, and from 3.68 to 163 lmol of QE/100 g FW in the PBS wash protocol (Wolfe et al.,

61

2008). Regardless of having the highest total phenolic content, total flavonoids content, ORAC and PSC values, Shanlihong variety did not rank first for cellular antioxidant quality, which is expressed as cellular antioxidant activity provided by 100 lmol of phenolics. This phenomenon may suggest that some compounds present in the extracts were not so effective in cellular model. Moreover, the difference between PBS wash protocol and no PBS wash protocol in this work was similar as reported in other common fruits (Wolfe et al., 2008), and this difference provide information on the degree of uptake and membrane association of the phytochemical compounds presenting in the fruit extracts, owing to their physical properties, such as polarity, solubility, and molecular size, giving them different bioavailability at the cellular, organ, and tissue (Wolfe & Liu, 2007; Wolfe et al., 2008). Previous studies indicated that flavonoids with the structure of 30 ,40 -o-dihydroxyl group in the B-ring, 2,3-double bond combined with a 4keto group in the C ring, and a 3-hydroxyl group exhibited high antioxidant activity in the CAA assay. It has been reported that compounds like quercetin, kaempferol, and EGCG showed higher CAA values, and no significant difference was found in PBS wash and no PBS wash protocols, while compounds like catechin and epicatechin showed lower CAA values along with a significant difference between PBS wash and no PBS wash protocols (the CAA value in no PBS wash protocol were much higher than that of PBS wash protocol) (Wolfe & Liu, 2008). Epicatechin and procyanidine B2, the major phenolics in the extracts of C. pinnatifida, were poorly absorbed or only loosely associated with the cell membrane, and may responsible for the lowest uptake rate and significant difference of the two protocol for CAA assay. However, recent study indicated that the presence of proanthocyanidins was correlated with the increase cell antioxidant activity of quercetin, and this enhanced effect was associated with the improvement of the solubility and stability of quercetin with proanthocyanidins addition (Zhao et al., 2015). This suggested that the high antioxidant activity in CAA assay may be because of the interaction between those procyanidins and other phytochemical compounds presented in the phytochemical extracts. 4. Conclusion This work showed a significant differences in phenolic phytochemical contents and antioxidant activity among the different C. pinnatifida varieties. The phenolics, and flavonoid contents of C. pinnatifida were mainly present in free form, and flavan-3-ol monomers and dimers, like epicatechin and procyanidin B2 were among the major phenolic compounds, and were responsible for the strong antioxidant activity of ORAC and PSC assay but not in the CAA assay. In general, C. pinnatifida has promising potential in the development of functional foods and nutritional supplements, while further investigation on other unknown phenolics and the interaction effect of different phytochemicals are worthy to be conducted in the future work. Acknowledgements The authors are grateful to the financial support from Guangdong Province Government (China) through the program of ‘‘Leading Talent of Guangdong Province (Rui-hai Liu)’’, Guangzhou Science and Technology Programm (2013J4500036), the Fundamental Research Funds for the Central Universities (No. 2013ZM0049), and the National Natural Science Foundation of China (No. 31101222). References Adom, K. K., & Liu, R. H. (2005). Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants. Journal of Agricultural and Food Chemistry, 53(17), 6572–6580.

62

L. Wen et al. / Food Chemistry 186 (2015) 54–62

Chai, W. M., Chen, C. M., Gao, Y. S., Feng, H. L., Ding, Y. M., Shi, Y., et al. (2014). Structural analysis of proanthocyanidins isolated from fruit stone of Chinese hawthorn with potent antityrosinase and antioxidant activity. Journal of Agricultural and Food Chemistry, 62(1), 123–129. Chang, C. L., Chen, H. S., Shen, Y. C., Lai, G. H., Lin, P. K., & Wang, C. M. (2013). Phytochemical composition, antioxidant activity and neuroprotective effect of Crataegus pinnatifida fruit. South African Journal of Botany, 88, 432–437. Cui, T., Li, J. Z., Kayahara, H., Ma, L., Wu, L. X., & Nakamura, K. (2006a). Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by highperformance liquid chromatography. Journal of Agricultural and Food Chemistry, 54(13), 4574–4581. Cui, T., Nakamura, K., Tian, S., Kayahara, H., & Tian, Y. (2006b). Polyphenolic content and physiological activities of Chinese hawthorn extracts. Bioscience Biotechnology and Biochemistry, 70(12), 2948–2956. Eberhardt, M. V., Lee, C. Y., & Liu, R. H. (2000). Nutrition: Antioxidant activity of fresh apples. Nature, 405(6789), 903–904. Froehlicher, T., Hennebelle, T., Martin-Nizard, F., Cleenewerck, P., Hilbert, J., Trotin, F., et al. (2009). Phenolic profiles and antioxidative effects of hawthorn cell suspensions, fresh fruits, and medicinal dried parts. Food Chemistry, 115(3), 897–903. Guo, X., Li, T., Tang, K., & Liu, R. H. (2012). Effect of germination on phytochemical profiles and antioxidant activity of mung bean sprouts (Vigna radiata). Journal of Agricultural and Food Chemistry, 60(44), 11050–11055. Guo, M., Liu, Y., Gao, Z., & Shi, D. (2014). Chinese herbal medicine on dyslipidemia: Progress and perspective. Evidence-Based Complementary and Alternative Medicine, 2014, 1–11. He, X., Liu, D., & Liu, R. H. (2008). Sodium borohydride/chloranil-based assay for quantifying total flavonoids. Journal of Agricultural and Food Chemistry, 56(20), 9337–9344. Huang, D. J., Ou, B. X., Hampsch-Woodill, M., Flanagan, J. A., & Prior, R. L. (2002). High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. Journal of Agricultural and Food Chemistry, 50(16), 4437–4444. Jurikova, T., Sochor, J., Rop, O., Mlcek, J., Balla, S., Szekeres, L., et al. (2012). Polyphenolic profile and biological activity of Chinese hawthorn (Crataegus pinnatifida Bunge) fruits. Molecules, 17(12), 14490–14509. Karadag, A., Ozcelik, B., & Saner, S. (2009). Review of methods to determine antioxidant capacities. Food Analytical Methods, 2(1), 41–60. Koleckar, V., Rehakova, Z., Brojerova, E., Kuca, K., Jun, D., Macakova, K., et al. (2012). Proanthocyanidins and their antioxidation activity. Chemicke Listy, 106(2), 113–121. Kwok, C. Y., Li, C., Cheng, H. L., Ng, Y. F., Chan, T. Y., Kwan, Y. W., et al. (2013). Cholesterol lowering and vascular protective effects of ethanolic extract of dried fruit of Crataegus pinnatifida, hawthorn (Shan Zha), in diet-induced hypercholesterolaemic rat model. Journal of Functional Foods, 5(3), 1326–1335. Li, T., Zhu, R., Dong, Y., Liu, Y., Li, S., & Chen, G. (2013b). Effects of pectin pentaoligosaccharide from hawthorn (Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet. Journal of Agricultural and Food Chemistry, 61(31), 7599–7605. Li, T., Zhu, J., Guo, L., Shi, X. L., Liu, Y. F., & Yang, X. B. (2013a). Differential effects of polyphenols-enriched extracts from hawthorn fruit peels and fleshes on cell cycle and apoptosis in human MCF-7 breast carcinoma cells. Food Chemistry, 141(2), 1008–1018. Liu, R. H. (2013). Health-promoting components of fruits and vegetables in the diet. Advances in Nutrition, 4(3), 384S–392S. Liu, P. Z., Kallio, H., Lu, D. G., Zhou, C. S., Ou, S. Y., & Yang, B. R. (2010b). Acids, sugars, and sugar alcohols in Chinese hawthorn (Crataegus spp.) fruits. Journal of Agricultural and Food Chemistry, 58(2), 1012–1019. Liu, P. Z., Kallio, H., Lu, D. G., Zhou, C. S., & Yang, B. R. (2011). Quantitative analysis of phenolic compounds in Chinese hawthorn (Crataegus spp.) fruits by high performance liquid chromatography-electrospray ionisation mass spectrometry. Food Chemistry, 127(3), 1370–1377.

Liu, J., Yuan, J., & Zhang, Z. (2010a). Microwave-assisted extraction optimised with response surface methodology and antioxidant activity of polyphenols from hawthorn (Crataegus pinnatifida Bge.) fruit. International Journal of Food Science and Technology, 45(11), 2400–2406. Malta, L. G., Tessaro, E. P., Eberlin, M., Pastore, G. M., & Liu, R. H. (2013). Assessment of antioxidant and antiproliferative activities and the identification of phenolic compounds of exotic Brazilian fruits. Food Research International, 53(1), 417–425. Mraihi, F., Journi, M., Cherif, J. K., Sokmen, M., Sokmen, A., & Trabelsi-Ayadi, M. (2013). Phenolic contents and antioxidant potential of crataegus fruits grown in Tunisia as determined by DPPH, FRAP, and beta-carotene/linoleic acid assay. Journal of Chemistry, 2013, 1–6. Olsson, M. E., Ekvall, J., Gustavsson, K., Nilsson, J., Pillai, D., Sjöholm, I., et al. (2004). Antioxidants, low molecular weight carbohydrates, and total antioxidant capacity in strawberries (Fragaria  ananassa): Effects of cultivar, ripening, and storage. Journal of Agricultural and Food Chemistry, 52(9), 2490–2498. Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry, 49(10), 4619–4626. Salmanian, S., Mahoonak, A. R. S., Alami, M., & Ghorbani, M. (2014). Phenolic content, antiradical, antioxidant, and antibacterial properties of hawthorn (Crataegus elbursensis) seed and pulp extract. Journal of Agricultural Science and Technology, 16(2), 343–354. Singleton, V. L., Orthofer, R. M., & Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin– Ciocalteu reagent. Methods in Enzymology, 299, 152–178. Sun, J., Chu, Y., Wu, X., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of common fruits. Journal of Agricultural and Food Chemistry, 50(25), 7449–7454. Tadic, V. M., Dobric, S., Markovic, G. M., Dordevic, S. M., Arsic, I. A., Menkovic, N. R., et al. (2008). Anti-inflammatory, gastroprotective, free-radical-scavenging, and antimicrobial activities of hawthorn berries ethanol extract. Journal of Agricultural and Food Chemistry, 56(17), 7700–7709. USDA. (2010). Dietary guidelines for Americans 2010. USDA human nutrition information service, vol. Hyattsville, MD, USA. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry & Cell Biology, 39(1), 44–84. Wang, L. F., Chen, J. Y., Xie, H. H., Ju, X. R., & Liu, R. H. (2013). Phytochemical profiles and antioxidant activity of Adlay varieties. Journal of Agricultural and Food Chemistry, 61(21), 5103–5113. Wolfe, K. L., Kang, X., He, X., Dong, M., Zhang, Q., & Liu, R. H. (2008). Cellular antioxidant activity of common fruits. Journal of Agricultural and Food Chemistry, 56(18), 8418–8426. Wolfe, K. L., & Liu, R. H. (2007). Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. Journal of Agricultural and Food Chemistry, 55(22), 8896–8907. Wolfe, K. L., & Liu, R. H. (2008). Structureactivity relationships of flavonoids in the cellular antioxidant activity assay. Journal of Agricultural and Food Chemistry, 56(18), 8404–8411. Wu, J., Peng, W., Qin, R., & Zhou, H. (2014). Crataegus pinnatifida: Chemical constituents, pharmacology, and potential applications. Molecules, 19(2), 1685–1712. Yang, B., & Liu, P. (2012). Composition and health effects of phenolic compounds in hawthorn (Crataegus spp.) of different origins. Journal of the Science of Food and Agriculture, 92(8), 1578–1590. Zhao, C. F., Lei, D. J., Song, G. H., Zhang, H., Xu, H., & Yu, L. J. (2015). Characterisation of water-soluble proanthocyanidins of Pyracantha fortuneana fruit and their improvement in cell bioavailable antioxidant activity of quercetin. Food Chemistry, 169, 484–491. Zhu, R., Li, T., Dong, Y., Liu, Y., Li, S., Chen, G., et al. (2013). Pectin pentasaccharide from hawthorn (Crataegus pinnatifida Bunge. var. major) ameliorates disorders of cholesterol metabolism in high-fat diet fed mice. Food Research International, 54(1), 262–268.