Food Chemistry 127 (2011) 13701377

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Food Chemistry
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Analytical Methods

Quantitative analysis of phenolic compounds in Chinese hawthorn (Crataegus spp.) fruits by high performance liquid chromatographyelectrospray ionisation mass spectrometry
Pengzhan Liu a, Heikki Kallio a,b, Deguo L c, Chuansheng Zhou c, Baoru Yang a,
a b c

Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland Department of Food Science and Engineering, Jinan University, 510632 Guangzhou, China Faculty of Horticulture, Shenyang Agricultural University, Dongling Road 120, 110161 Shenyang, China

a r t i c l e

i n f o

a b s t r a c t
Eleven major phenolic compounds (hyperoside, isoquercitrin, chlorogenic acid, ideain, epicatechin, two procyanidin (PA) dimers, three PA trimers and a PA dimer-hexoside) were quantied in the fruits of 22 cultivars/origins of three species of the Chinese hawthorn (Crataegus spp.) by HPLCESI-MS-SIR. Hyperoside (0.10.8 mg/g dry mass [DM]), isoquercitrin (0.10.3 mg/g DM), chlorogenic acid (0.21.6 mg/g DM), epicatechin (0.911.7 mg/g DM), PA B2 (0.712.4 mg/g DM), PA dimer II (0.11.5 mg/g DM), PA trimer I (0.12.7 mg/g DM), PA trimer II (0.76.9 mg/g DM), PA trimer III (0.011.2 mg/g DM) and a PA dimerhexoside (trace1.1 mg/g DM) were detected in all the samples. Ideain (0.00.7 mg/g DM) was found in all the samples except Crataegus scabrifolia. Signicant correlations between the contents of individual PA aglycons were observed (r > 0.9, P < 0.01). A strong correlation between avonols was also shown (r = 0.71, P < 0.01). Fruits of Crataegus pinnatida var. major had higher contents of PAs but lower contents of avonols compared with Crataegus brettschneideri. The fruits of C. scabrifolia contained the highest level of PA dimer-hexoside, which was present in trace amounts in the fruits of C. pinnatida. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 14 July 2010 Received in revised form 24 November 2010 Accepted 25 January 2011 Available online 31 January 2011 Keywords: Crataegus spp. Flavonoids Hawthorn Phenolics Procyanidin

1. Introduction Phenolic compounds and foodstuffs rich in phenolics have attracted increasing attention from both researchers and industry because of their potential effects on maintaining human health. Hawthorn berries (genus Crataegus, family Rosaceae) rich in phenolic compounds have been widely used as both a medical and food raw material in China and Europe (Fong & Bauman, 2002). Many studies have demonstrated the benecial effect of extracts of hawthorn fruits on the heart and blood circulation system including cardiovascular protecting and endothelium-dependent vasorelaxing effects (Kim, Kang, Kim, & Kim, 2000). Hawthorn fruit extracts also improve coronary circulation and possess hypolipidemic effects (Pittler, Schmidt, & Ernst, 2003; Quettier-Deleu et al., 2003; Schwinger, Pietsch, Frank, & Brixius, 2000; Tadic et al., 2008). The safety of the extracts of hawthorn fruits as food ingredients has been veried (Daniele, Mazzanti, Pittler, & Ernst, 2006). A standardised extract of the European hawthorn (Crataegus monogyna or Crataegus laevigata) has been used for the treatment of patients with heart failures (Holubarsch et al., 2008; Tauchert, 2002; Zapfe Jun, 2001).
Corresponding author. Tel.: +358 2 3336844; fax: +358 2 3336860.
E-mail address: baoru.yang@utu. (B. Yang). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.103

Hawthorn fruits have long been eaten in China. Commonly, it has been considered that the Chinese hawthorn comprises 18 species, of which Crataegus pinnatida Bge. and its variety Shanlihong (C. p. Bge. var. major N.E.Br.) are the most important (Zhao & Tian, 1996). In addition, the fruits of other species such as Crataegus brettschneideri (Fu hawthorn) and Crataegus scabrifolia (Yunnan hawthorn) are also commonly used as medicinal or food materials (Gao, Feng, & Qin, 1995; Zhao & Tian, 1996). Phenolics are considered among the most important bioactive compounds in Chinese hawthorn fruits (Chang, Zuo, Harrison, & Chow, 2002). Our previous study revealed the presence of more than 40 phenolic compounds in Chinese hawthorn fruits (C. p. Bge. var. major N.E.Br.). Most of these compounds belonged to B-type procyanidins (PAs) and the rest were avonol glycosides, anthocyanins or phenolic acids (Liu, Yang, & Kallio, 2010). The phenolic composition in hawthorn fruits varies among species and cultivars (Gao et al., 1995) but information of the contents of phenolics in different species of Chinese hawthorn fruits is still limited. Quantitative analysis of phenolic compounds, especially of PAs in plant materials, is a challenging task. This is largely because of a lack of commercial reference compounds and decient separation between PAs using high performance liquid chromatography (HPLC). The total contents of PAs are commonly determined by colorimetric methods without detailed information on the proles

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and contents of individual PAs (Hiermann, Kartnig, & Azzam, 1986; Rohr, Meier, & Sticher, 2000). In addition, the results are often affected by the presence of other colour compounds in the sample. Quantitative analyses of PAs have been carried out by normal phase or reversed phase HPLC with limited separation of the analytes. Normal phase HPLC separates PAs based on the degree of polymerisation (DP), but isomers with the same DP values are not separated (Gu et al., 2002; Karonen et al., 2006). In reversed phase HPLC, PA isomers with equal DP numbers can be separated, but some PAs differing in DP values can overlap (Hellstrom & Mattila, 2008; Hellstrom, Sinkkonen, Karonen, & Mattila, 2007; Karonen et al., 2007). To achieve satisfactory results, one or several solid phase extraction (SPE) columns have to be employed to fractionate the samples before HPLC analysis. Polyamide columns and Sephadex LH-20 columns are commonly used for fractionating PAs from different sources (Hellstrom & Mattila, 2008; Svedstrm, Vuorela, Kostiainen, Laakso, & Hiltunen, 2006; Svedstrm et al., 2002). The fractionation steps, though improving the separation of PAs in the subsequent HPLC analysis, make the whole quantication procedure complex and tedious. In the current study, the single ion recording function of HPLCelectrospray ionisation mass spectrometry (HPLCESI-MS-SIR) was applied to quantify the phenolic compounds in the aqueous ethanol extract of hawthorn fruits without prefractionation steps. Phenolic compounds in 22 hawthorn samples belonging to four species/varieties were determined with this signicantly simplied quantitative analysis.

Holland) and acetone (HPLC grade) and acetonitrile (HPLC grade) from VWR International Oy (Espoo, Finland).

2.4. Sample preparation Dried, seedless hawthorn fruits were milled into a ne powder with the aid of liquid nitrogen in a mortar and kept in a desiccator overnight before extraction. A sample of 1.0 g of hawthorn fruit powder was transferred into a 25 ml volumetric ask with 20 ml of 80% aqueous ethanol. The mixture was ultrasonicated for 30 min to enhance the dissolution of the phenolics into the solvent. Eighty percent aqueous ethanol was added to bring the volume to the scale and mixed thoroughly. A sample of 1.0 ml was taken and ltered through a 0.45 lm lter and analysed by HPLC. 2.5. HPLC-DADESI-MS analysis HPLC-DADESI-MS analysis was performed using a Waters Acquity Ultra Performance LC system equipped with a Waters Acquity 2996 PDA detector and in combination with a Waters Quattro Premier mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an ion-spray interface. A Phenomenex Prodigy RP-18 ODS (3) column (5 lm, 250 4.60 mm, Torrance, CA, USA) combined with a Phenomenex Prodigy guard column (5 lm, 30 4.60 mm, Torrance, CA, USA) was used. A binary solvent system was employed consisting of formic acid/water (0.5:99.5, v/v) as solvent A and acetonitrile/methanol (80:20, v/v) as solvent B. The gradient program was 05 min with 10% solvent B, 515 min with 1018% B, 1525 min with 18% B, 2530 min with 1825% B, 3035 min with 25% B, 3540 min with 2535% B, 4045 min with 3560% B, 4550 min with 6010% B and 5055 min with 10% B. The ow rate of the mobile phase was 1 ml/min, and the injection volume 5 ll. The peaks were monitored at 280 nm with PDA detection. A split joint was used after the PDA detector, directing a ow of 0.3 ml/min to the mass spectrometer and the rest to a waste bottle. The mass spectrometer was operated in a positive ion mode. The capillary voltage was set to 4.0 kV, the cone voltage to 22 V and the extractor voltage to 3 V. The source temperature was 150 C and the dissolution temperature 300 C. The HPLCESI-MS system was operated using MassLynx 4.1 software. Selected ion recording (SIR) was used for the quantitative analysis compared with the full scan method used before (Liu et al., 2010). The ions with m/z 291 (nominal mass 291.3), 303 (303.2), 355 (355.3), 449 (449.4), 579 (579.5), 741 (741.7) and 867 (867.8) were monitored. The ions presented the base peaks in the mass spectra of epicatechin (291), hyperoside (303), isoquercitrin (303), chlorogenic acid (355), ideain (449), PA dimers (579), PA dimer-hexoside (741) and PA trimers (867).

2. Materials and methods 2.1. Plant materials Hawthorn fruits of 22 cultivars and origins belonging to four species/varieties were collected in China. Among these, 21 samples were collected in the Chinese National Fruit Germplasm Repository, Shenyang Hawthorn Garden (Shenyang, Liaoning Province), including 10 cultivars of C. pinnatida Bge. var. major N.E.Br. harvested in October 2007, eight cultivars of C. brettschneideri harvested in August 2008 and three natural origins of C. pinnatida Bge. harvested in September 2008. One sample of C. scabrifolia was harvested in Kunming, Yunnan Province, China in September 2007. For each cultivar/origin, 500 g optimally ripe fruits were collected from 2 to 4 trees, from ve randomly selected collection points from different sides of each tree. All the samples were sliced and dried in a cool and shady place after harvesting. 2.2. Reference compounds Hyperoside (quercetin-3-O-galactoside), isoquercitrin (quercetin-3-O-glucoside), ideain chloride (cyanidin-3-O-galactoside chloride), epicatechin and PA B2 (epicatechin-(4b ? 8)-epicatechin) were purchased from Extrasynthese (Genay, France). Chlorogenic acid was purchased from SigmaAldrich Co. (St. Louis, MO, USA). Reference compounds of PA dimer II, PA trimer II and PA dimerhexoside were isolated from C. pinnatida Bge. var. major N.E.Br. in our laboratory by preparative HPLC, and the purity of each sample was tested with HPLC-MS. The purity of the PA dimer II and PA trimer II was more than 95%. The purity of the PA dimer-hexoside was about 80%. 2.3. Other chemical agents Ethanol was purchased from Primalco Oy (Rajamki, Finland), methanol (HPLC grade) and formic acid from J.T. Baker (Deventer,

2.6. Calibration Standard solutions of chlorogenic acid, ideain chloride, hyperoside, isoquercitrin, epicatechin, PA B2, PA dimer II, PA dimer-hexoside and PA trimer II were prepared in methanol in the concentration range of 0.010.3 mg/ml and analysed by HPLC. Five microlitres of each solution was injected into HPLC. The calibration curve of every compound was constructed using ve different concentrations by plotting the peak areas versus the concentrations. The calibration curve of the PA trimer II isolated and puried in our laboratory was used for the quantication of all the PA trimers. The purities of the compounds were taken into account when preparing the standard solution of PA dimer II, PA timer II and PA dimer-hexoside.

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2.7. Statistical analysis Statistical analyses were performed using SPSS 16.0.1 (SPSS Inc., Chicago, IL, USA) and Unscrambler 9.8 (Camo Process AS, Oslo, Norway). Differences in the chemical composition among the species were analysed using one-way analysis of variance (ANOVA) and with the GamesHowell and StudentNewmanKeuls (SNK) tests. Differences reaching a condence level of 95% were considered signicant. Pearsons correlation coefcient analysis was carried out to investigate the correlation between the contents of different phenolic compounds in hawthorn samples. Principal component analysis (PCA) was used to interpret differences between hawthorn samples based on the contents of phenolics analysed in this study. 3. Results and discussion 3.1. Quantitative analysis with HPLCESI-MS Fig. 1 presents the chromatograms of the 80% ethanolic extract of the fruits of the hawthorn cultivar Mopan (C. pinnatida. var. major), obtained by HPLC-DAD analysis at 280 nm and HPLCESIMS-SIR analysis at different m/z channels. The peaks were identied based on retention times and mass spectra. The humps under the peaks and the obvious overlapping in the HPLC-DAD chromatogram were because of insufcient separation (Liu et al., 2010). In our former study, the base peaks in the mass spectra of epicatechin, PA dimers, PA trimers, PA dimer hexoside, chlorogenic acid and ideain were found to be ions at m/z 291, 579, 867, 741, 355 and 449 ([M+H]+), respectively. The base peaks in the mass spectra of hyperoside and isoquercitrin were both at m/z 303 (protonated aglycon moieties). No fragment ions with the same m/z value and retention times as the ions above were detected in the

HPLCESI-MS from other compounds analysis of the 80% ethanolic extracts of hawthorn fruit (Liu et al., 2010). Based on the above knowledge, an HPLCESI-MS-SIR method was optimised and used to quantify the main phenolic compounds in hawthorn fruit extracts. Ions at m/z 291 (epicatechin), 579 (PA dimer), 867 (PA trimer), 741 (PA dimer-hexoside), 355 (chlorogenic acid), 449 (ideain) and 303 (hyperoside and isoquercitrin) were monitored with the SIR function of the mass spectrometer (Fig. 1). Comparing with the resolution of peaks in the HPLC-UV chromatogram at 280 nm and in the SIR chromatograms of HPLCESI-MS, the peaks of the phenolic compounds in the 80% ethanolic extracts of the hawthorn fruit were clearly better for integration in the SIR chromatograms of HPLCESI-MS, resulting in an increased accuracy of quantication (Fig. 1). Calibration curves of the PA monomer (epicatechin), dimer (PA B2) and trimer (PA trimer II) are shown in Fig. 2A (using weight concentration) and Fig. 2B (using molar concentration). Based on the linear equations presented in Fig. 2A, the relative response factors between epicatechin and PA dimers and trimers could be calculated and applied in the quantication of the dimers and trimers. With the instrumental parameters used in the current study, the correction factor of PA dimers and trimers with epicatechin was 1.87 and 4.17, respectively. By applying these correction factors, the contents of PA dimers and trimers in hawthorn fruits could be determined using epicatechin as the reference. This is especially important when reference compounds of PA dimers and trimers are unavailable. When molar concentrations (M) were used, the calibration curves of PA with different DP numbers were close to each other, and those of monomers and dimers were practically identical (Fig. 2B). Fig. 2C presents the calibration curves of two isomers of the PA dimer obtained by HPLCESI-MS-SIR analysis. The calibration

Fig. 1. HPLC-DAD and HPLCESI-MS chromatograms of the 80% ethanolic extracts of hawthorn fruits of the cultivar Mopan (C. pinnatida var. major). Peaks in HPLC-DAD: 1, ideain (14.01 min); 2, chlorogenic acid (15.33 min); 3, PA dimer-hexoside (16.03 min); 4, PA trimer I (16.43 min); 5, PA dimer I (PA B2) (17.08 min); 6, epicatechin (18.98 min); 7, PA trimer II (20.75 min); 8, unknown PA derivative (21.72 min); 9, PA tetramer (22.60 min); 10, PA trimer III (23.40 min); 11, PA dimer II (32.40 min); 12, hyperoside (33.07 min); 13, isoquercitrin (33.61 min).

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Fig. 2. Calibration curves of (A) epicatechin, PA B2 and PA trimer II (peak area vs. mass concentration) (B) epicatechin, PA B2 and PA trimer II (peak area vs. molar concentration) (C) PA dimer I (PA B2) and PA dimer II (D) chlorogenic acid (E) hyperoside (F) isoquercitrin (G) ideain in HPLCESI-MS.

curves of the isomers of PA were practically identical. The calibration curve of PA trimer II was used to calculate all the PA trimers contents of in this study because of the lack of reference compounds for PA trimer I and PA trimer III. Fig. 2DG show the calibration curves of chlorogenic acid, hyperoside, isoquercitrin and ideain, respectively, following HPLCESI-MS-SIR analysis. In the concentration range of our study, when linear regressions were used for the calculations, the R2 values of the calibration lines of all the reference compounds were between 0.986 and 0.999 and the linearity was high enough for quantication. The difculty in quantifying PAs in plants and plant products has been so far largely because of the lack of an appropriate analytical methodology and commercially available reference compounds (Svedstrm et al., 2002). Using HPLCESI-MS with the SIR function, the quantication of PAs in hawthorn fruits was signicantly simplied compared with the conventional methods based on sample fractionation by column chromatography followed by HPLC-DAD analysis.

The PA dimer-hexosides existed in trace amounts in several samples, e.g. in Huixiandahong and Caihong. The contents of hyperoside, isoquercitrin, chlorogenic acid, ideain, PA monomer (epicatechin), PA dimer I (PA B2), PA dimer II, PA trimers (I, II, III) and PA dimer-hexoside in 22 Chinese hawthorn samples were determined by the HPLCESI-MS-SIR method described above.

3.2. Phenolic contents of Chinese hawthorn fruits of different species, cultivars and origins Hyperoside, isoquercitrin, chlorogenic acid, PA monomer (epicatechin), PA dimer I (PA B2), PA dimer II, PA trimers (I, II, III) and PA dimer-hexoside were found in all the 22 samples analysed. However, ideain was not detected in Yunnan shanzha (C. scabrifolia).

3.2.1. Hyperoside, isoquercitrin, chlorogenic acid and ideain contents in hawthorn fruits Hyperoside and isoquercitrin were found to be the most abundant avonol glycosides in the extracts of hawthorn fruits in all the 22 samples. In addition, other minor avonol glycosides were detected but the peaks were too small to be integrated. Table 1 summarises the contents of avonol glycosides, chlorogenic acid and ideain in all the 22 samples analysed in this study. The total contents of avonol glycosides in the fruits varied from 0.2 to 1.1 mg/g dry mass (DM). C. brettschneideri had the highest content of avonol glycosides (0.7 mg/g DM as average content of eight cultivars in the species) among the species studied (P < 0.05). The fruits of C. pinnatida var. major contained less avonol glycosides (0.4 mg/g DM as average content of 10 cultivars in the species) than those of the other species (P < 0.05). The content of hyperoside (0.10.8 mg/g DM) was higher than that of isoquercitrin (0.10.3 mg/g DM) in almost all the samples except the cultivar Huixiandahong of C. pinnatida var. major.

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Table 1 Contentsa of avonol glycosides, chlorogenic acid and ideain in Chinese hawthorn fruits (mg/g DM). Cultivar/origin C. pinnatida var. major 947 8321 Dajinxing Huixiandahong Jiangou 2 Mopan Qiujinxing Shandongdajinxing Shen78201 Zi zhenzhu C. pinnatida var. major combined C. brettschneideri Caihong Hongroushanlihong Hongroushanzha Jifu 1 Jifu 3 Xinghong 2 Zuofu 1 Zuofu 2 C. brettschneideri combined C. pinnatida Shanzha 1 Shanzha 2 Shanzha 3 C. pinnatida combined C. scabrifolia Yunnan shanzha
a b

N 4 4 4 4 4 4 4 4 4 4 40 4 4 4 4 4 4 4 4 32 4 4 4 12 4

Hyperoside 0.26 0.01 0.12 0.01 0.24 0.01 0.08 0.00 0.32 0.02 0.19 0.01 0.26 0.01 0.22 0.01 0.43 0.01 0.48 0.06 0.26 0.12 a 0.38 0.02 0.79 0.21 0.68 0.16 0.38 0.06 0.43 0.02 0.80 0.18 0.38 0.08 0.44 0.04 0.53 0.21 b 0.37 0.00 0.48 0.04 0.43 0.03 0.42 0.05 b 0.35 0.06 a

Isoquercitrin 0.16 0.01 0.11 0.01 0.20 0.01 0.09 0.00 0.17 0.01 0.14 0.00 0.20 0.01 0.18 0.00 0.31 0.01 0.24 0.03 0.18 0.06 a 0.14 0.01 0.34 0.10 0.20 0.05 0.12 0.02 0.14 0.01 0.29 0.07 0.14 0.03 0.17 0.01 0.19 0.09 a 0.16 0.00 0.29 0.03 0.31 0.02 0.25 0.07 a 0.17 0.03 a

Flavonol totalb 0.42 0.02 0.23 0.01 0.44 0.02 0.17 0.01 0.49 0.03 0.33 0.01 0.46 0.01 0.41 0.01 0.74 0.02 0.73 0.09 0.44 0.18 a 0.52 0.02 1.13 0.31 0.87 0.20 0.51 0.07 0.57 0.03 1.08 0.24 0.52 0.11 0.61 0.06 0.73 0.29 b 0.52 0.00 0.76 0.06 0.74 0.05 0.67 0.12 b 0.52 0.09 ab

Chlorogenic acid 0.92 0.02 1.09 0.05 1.35 0.04 1.23 0.09 0.88 0.11 0.61 0.03 1.18 0.10 1.09 0.01 1.48 0.09 1.57 0.04 1.14 0.28 c 0.23 0.02 0.62 0.16 0.48 0.07 0.35 0.04 0.31 0.03 0.85 0.12 0.36 0.07 0.49 0.04 0.46 0.20 ab 0.66 0.00 0.31 0.07 0.57 0.06 0.51 0.16 b 0.26 0.05 a

Ideain 0.06 0.01 0.13 0.01 0.19 0.02 0.17 0.01 0.20 0.02 0.04 0.01 0.20 0.02 0.17 0.02 0.11 0.02 0.25 0.01 0.15 0.07 b 0.27 0.01 0.25 0.04 0.53 0.11 0.57 0.12 0.35 0.03 0.66 0.08 0.17 0.02 0.19 0.02 0.37 0.19 c 0.07 0.00 0.28 0.03 0.25 0.01 0.20 0.10 b 0a

Values (means SD) without common letters within a column are signicantly different (P < 0.05). Flavonol total includes hyperoside and isoquercitrin.

The content of chlorogenic acid varied from 0.2 to 1.6 mg/g DM. The fruits of C. pinnatida var. major contained the highest level (1.1 mg/g DM) and C. scabrifolia the lowest (0.3 mg/g DM) among the hawthorn species. Ideain was not detected in the fruits of C. scabrifolia. C. brettschneideri contained more ideain (0.4 mg/g DM) than the other species (P < 0.05). 3.2.2. PA content in hawthorn fruits A total of 36 PAs and PA derivatives, including aglycons of three dimers, three trimers, eight tetramers, four pentamers, two hexamers and two hexosides of PA monomers, seven hexosides of PA dimers, one hexoside of a PA trimer, two hexosides of PA tetramers, one hexoside of a PA pentamer and an unknown PA derivative were found in Chinese hawthorn fruits, among which epicatechin, PA dimers, PA dimer-hexosides and PA trimers were the major components representing 64% of the total peak area of PA (Liu et al., 2010). It has been indicated that PA oligomers of DP <4 are evidently partially absorbed in the gastrointestinal tract (Deprez, Mila, Huneau, Tome, & Scalbert, 2001; Holt et al., 2002). For this reason, the quantication of the low DP oligomers was emphasised. Eight PAs and PA derivatives, including a PA monomer (epicatechin), two dimers (PA B2 and PA dimer II), three trimers (PA trimers I, II and III) and a hexoside of a PA dimer were quantied in the 22 Chinese hawthorn samples. The results are presented in Table 2. The content of epicatechin in the fruits analysed in this study varied from 0.9 to 11.7 mg/g DM. Most samples contained epicatechin between 2 and 6 mg/g DM, but several samples had extremely high levels of the compound, e.g. Shandongdajinxing of C. pinnatida var. major (11.7 mg/g DM). Two major PA dimers were quantied, PA B2 being the more abundant in all 22 samples. The levels of PA B2 ranged from 0.7

to 12.4 mg/g DM and PA dimer II from 0.1 to 1.5 mg/g DM. The contents of PA B2 and epicatechin were always close to each other. Three major PA trimers were detected in hawthorn fruit samples. PA trimer II was the most abundant (0.76.9 mg/g DM) followed by PA trimer I (0.12.7 mg/g DM). The content of PA trimer III (0.011.2 mg/g DM) was always the lowest among the PA trimers. The highest level (1.1 mg/g DM) of the PA dimer-hexoside was found in C. scabrifolia fruits. In addition, the dimer-hexoside was a typical compound in the cultivars of C. pinnatida var. major, the varieties 947 and Mopan containing the highest amounts of the compound. The total contents of the eight PAs analysed in the 22 samples varied widely from 2.5 to 36.7 mg/g DM. The fruits of C. pinnatida var. major and C. scabrifolia had higher PA contents than those of the two other species (P < 0.05). Oligomeric PAs in European hawthorn C. laevigata fruits were quantied by SPE fractionation and HPLC analysis. The total contents of PA dimers and trimers in the fruits were shown to be 0.6 mg/g DM (Svedstrm et al., 2002). The total PAs in the other European hawthorn species (C. monogyna and Crataegus oxyacantha) varied from 14 to 26 mg/g DM (Gao et al., 1995). Our results showed that the levels of PAs in Chinese hawthorn fruits were higher than in C. laevigata and close to the levels found in C. monogyna and C. oxyacantha. 3.2.3. Correlation between contents of different phenolic compounds Pearsons correlation coefcients analyses were used to investigate the correlation between the contents of phenolics in Chinese hawthorn fruits. There was a positive correlation between the contents of hyperoside and isoquercitrin (Fig. 3A, R2 = 0.49, P < 0.01). Signicant positive correlations (R2 = 0.850.97, P < 0.01) were found between the content of epicatechin and the levels of

P. Liu et al. / Food Chemistry 127 (2011) 13701377 Table 2 Contentsa of PAs in Chinese hawthorn fruits (mg/g DM). Cultivar/origin C. pinnatida var. major 947 8321 Dajinxing Huixiandahong Jiangou 2 Mopan Qiujinxing Shandongdajinxing Shen78201 Zi zhenzhu C. pinnatida var. major combined C. brettschneideri Caihong Hongroushanlihong Hongroushanzha Jifu 1 Jifu 3 Xinghong 2 Zuofu 1 Zuofu 2 C. brettschneideri combined C. pinnatida Shanzha 1 Shanzha 2 Shanzha 3 C. pinnatida combined C. scabrifolia Yunnan shanzha
a b

1375

Epicatechin

PA dimer I (PA B2) 4.96 0.21 7.64 0.15 2.06 0.05 9.42 0.46 3.86 0.46 4.77 0.22 3.30 0.32 12.36 0.34 4.72 0.14 6.07 0.40 5.92 2.99 b

PA dimer II

PA dimerhexoside 0.52 0.02 0.10 0.03 0.10 0.01 0.14 0.01 Trace 0.80 0.05 0.08 0.01 0.27 0.01 0.15 0.01 0.20 0.02 0.24 0.24 b

PA trimer I

PA trimer II

PA trimer III

PA Totalb

4 4 4 4 4 4 4 4 4 4 40

4.61 0.18 7.34 0.37 1.93 0.09 8.52 0.65 3.45 0.33 3.92 0.15 3.36 0.27 11.72 0.50 3.76 0.17 5.64 0.28 5.43 2.86 b

0.69 0.03 1.00 0.03 0.28 0.01 1.19 0.07 0.52 0.05 0.68 0.03 0.41 0.03 1.54 0.08 0.64 0.12 0.77 0.06 0.77 0.37b

1.38 0.03 1.70 0.07 0.47 0.02 2.05 0.12 0.86 0.09 1.52 0.08 0.66 0.10 2.66 0.07 1.16 0.09 1.36 0.08 1.38 0.63 b

3.19 0.09 4.52 0.15 1.24 0.03 5.53 0.15 2.70 0.30 3.22 0.15 1.79 0.13 6.90 0.22 2.79 0.19 4.15 0.21 3.60 1.65 b

0.54 0.01 0.75 0.04 0.16 0.01 0.98 0.06 0.40 0.05 0.48 0.03 0.24 0.03 1.24 0.07 0.46 0.06 0.66 0.03 0.59 0.32 b

15.89 0.48 23.04 0.66 6.24 0.18 27.83 1.39 11.79 1.27 15.39 0.56 9.84 0.85 36.69 1.16 13.69 0.64 18.85 0.29 17.93 8.78 b

4 4 4 4 4 4 4 4 32 4 4 4 12 4

1.36 0.07 3.99 0.95 2.58 0.54 2.59 0.41 1.91 0.13 2.42 0.47 2.10 0.35 3.13 0.39 2.51 0.87 a 6.72 0.06 2.54 0.60 0.87 0.06 3.38 2.59 a 6.03 0.35 b

1.36 0.10 3.43 0.89 2.42 0.36 2.47 0.33 2.12 0.17 2.40 0.37 2.17 0.32 2.98 0.32 2.42 0.69 a 4.59 0.05 2.71 0.79 0.68 0.06 2.66 1.72 a 5.83 0.57 b

0.19 0.01 0.56 0.17 0.29 0.03 0.36 0.05 0.29 0.02 0.35 0.07 0.32 0.07 0.41 0.04 0.35 0.12 a 0.75 0.01 0.32 0.03 0.09 0.01 0.39 0.29 b 0.93 0.07 a

Trace 0.07 0.03 0.02 0.02 0.03 0.02 0.04 0.03 0.09 0.03 0.02 0.02 0.02 0.02 0.04 0.03 a 0.01 0.00 Trace Trace 0.00 0.01 a 1.08 0.09 c

0.23 0.02 0.62 0.17 0.45 0.12 0.47 0.06 0.43 0.06 0.49 0.11 0.47 0.06 0.54 0.07 0.46 0.13 a 0.93 0.01 0.59 0.20 0.11 0.03 0.54 0.37 a 1.44 0.19 b

1.07 0.06 2.54 0.60 2.07 0.39 1.91 0.21 1.78 0.23 1.78 0.39 1.98 0.61 2.35 0.24 1.93 0.54 a 3.67 0.07 1.93 0.17 0.73 0.05 2.11 1.26 a 3.68 0.29 b

0.10 0.01 0.37 0.12 0.18 0.01 0.24 0.04 0.19 0.04 0.23 0.04 0.27 0.11 0.32 0.04 0.24 0.10 a 0.58 0.00 0.30 0.10 0.01 0.02 0.30 0.25 a 0.62 0.07 b

4.31 0.25 11.57 2.81 8.02 1.40 8.07 1.10 6.76 0.53 7.75 1.46 7.34 1.44 9.74 1.09 7.95 2.38 a 17.25 0.06 8.40 1.69 2.50 0.19 9.39 6.39 a 19.60 1.36 b

Values (means SD) without common letters within a column are signicantly different (P < 0.05). PA total includes epicatechin, PA dimers, PA trimers and PA dimer-hexoside.

practically all PAs of different DP values except the PA dimerhexoside (Fig. 3BG). A positive correlation also existed between the contents of PA B2 and PA trimer II (Fig. 3H). Epicatechin was a monomer unit of most of the PA dimers and PA oligomers in the hawthorn fruits. A higher content of the monomeric epicatechin was in general associated with higher levels of the dimers and oligomers of the avonol. Although the absolute contents of the individual PAs differed among the samples, the ratios between procyanidins with different DP values remained constant. Therefore, it is possible to provide a rough estimation of the contents of various PAs in hawthorn fruits based on the level of monomeric epicatechin. Estimations might be based on the quantitative analysis of epicatechin and the equations presented in Fig. 3. This might provide useful compositional information with a simple analysis, especially if there is a lack of sophisticated chromatographic-mass spectrometric equipment and reference compounds. For example, if the content of epicatechin of sample 8321 and the equations between epicatechin and the other PA compounds were used for the calculation, the contents of PA B2, PA dimer II and PA trimers I, II and III were 7.52, 1.00, 1.67, 4.59 and 0.78 mg/g DM, respectively. Compared with the data acquired from experiments, the deviations are less than 3.3%. A weak negative correlation between total avonols and total PAs was also observed (R2 = 0.16, P < 0.01, Fig. 3I). 3.2.4. PCA analyses A PCA biplot was applied to illustrate the correlations between the contents of phenolic compounds of various hawthorn fruits (Fig. 4). The rst two PCs explained 80% of the variance of the data. The closer the hawthorn species/cultivars/origins lie on the plot, the more similar they are in their composition of phenolic com-

pounds. By contrast, a sample that is distant from the others can have signicantly different compositional characteristics. A species/cultivar/origin located in the vicinity of a component is typically rich in this specic component. PC1 (64%) separates the samples into two groups. The samples on the right of the biplot are relatively more abundant in PAs and less abundant in avonol glycosides than those on the left. In the PCA biplot, all the cultivars of C. brettschneideri are located on the left and almost all the samples of C. pinnatida var. major on the right, clearly demonstrating that the variety major had higher PA contents but lower levels of avonol glycosides than C. brettschneideri. PC2 (16%) explains the differences in the contents of individual phenolic compounds among the samples. The samples with high PA dimer-hexosides, e.g. Yunnan shanzha and 947, were separate from the others. The PCA biplot also shows the difference among samples within individual species. The samples of C. pinnatida var. major were scattered across a wider range than those of C. brettschneideri, suggesting a higher variation in the contents of phenolic compounds among the cultivars of C. pinnatida var. major than among those of C. brettschneideri. PCA biplot analysis can help predict the quality of fruits. Studies have shown various biological activities of oligomeric PAs (Quettier-Deleu et al., 2003; Zhang et al., 2001). Therefore, the samples lying close to the contents of PAs can have higher bio-activities than those lying far away. 4. Conclusions In this research, the simple and reliable HPLC-ESI-SIR method was optimised and applied for the quantitative analysis of phenolic

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Fig. 3. Correlations between contents of (A) hyperoside and isoquercitrin (B) epicatechin and PA B2 (C) epicatechin and PA dimer II (D) epicatechin and PA trimer I (E) epicatechin and PA trimer II (F) epicatechin and PA trimer III (G) epicatechin and PA total (H) PA B2 and PA trimer II (I) Flavonol total and PA total in Chinese hawthorn fruits.

Fig. 4. PCA biplot of phenolics in 22 Chinese hawthorn fruit samples. SLH, C. pinnatida var. major; SLH 1, 947; SLH 2, 8321; SLH 3, Dajinxing; SLH 4, Huixiandahong; SLH 5, Jiangou 2; SLH 6, Mopan; SLH 7, Qiujinxing; SLH 8, Shandongdajinxing; SLH 9, Shen78201; SLH 10, Zizhenzhu. FU, C. brettschneideri; FU 1, Caihong; FU 2, Hongroushanlihong; FU 3, Hongroushanzha; FU 4, Jifu 1; FU 5, Jifu 3; FU 6, Xinghong 2; FU 7, Zuofu 1; FU 8, Zuofu 2. SZ, C. pinnatida Bge.; SZ 1, Shanzha 1; SZ 2, Shanzha 2; SZ 3, Shanzha 3. YN (C. scabrifolia), Yunnan shanzha; CA, chlorogenic acid; Isoq, isoquercitrin; Flav Total, avonol total; Hype, hyperoside; PAD, procyanidin dimer; PAT, procyanidin trimer; PADH, procyanidin dimer-hexosides.

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compounds in Chinese hawthorn fruits. With this method, the contents of 11 major phenolic compounds were determined in 18 cultivars of C. pinnatida var. major and C. brettschneideri commonly cultivated in China and in four samples of the species C. pinnatida and C. scabrifolia of wild origins. The samples of C. pinnatida var. major differed from C. pinnatida and C. brettschneideri by higher contents of PAs (P < 0.05). The content of ideain in C. brettschneideri was typically higher compared with those in C. pinnatida var. major, C. scabrifolia and C. pinnatida (P < 0.05). C. scabrifolia contained a higher level of PA dimer-hexoside than the rest of the samples studied but no ideain (P < 0.05). Among all the species, C. pinnatida var. major contained the highest level of chlorogenic acid (P < 0.05). Based on PCA analysis, the hawthorn samples analysed fell into two groups: those rich in PAs and those rich in avonols. The compositional difference among the samples can be used to distinguish hawthorn species. Therefore, these results provide valuable information for distinguishing hawthorn fruits of different origins and evaluating their quality for industrial application. This is the rst report on the quantitative analysis of PAs in hawthorn fruits using the SIR function of HPLC-MS. Acknowledgements The authors sincerely thank Mr Li Yonghai and Mrs Xu Tao at the Sea Buckthorn Ofce, Ministry of Water Resources, China and Professor Shiyi Ou, Department of Food Science and Engineering, Jinan University, China for their assistance in collecting the samples used in the study. The work was nanced by the Centre for International Mobility (CIMO), Finland, and by the Finnish Cultural Foundation, Finland. References
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