Sugars, Acids, and Phenolic Compounds in Chinese Hawthorn (

Chapter 15

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Sugars, Acids, and Phenolic Compounds in Chinese Hawthorn (Crataegus spp.) Fruits of Different Origins Baoru Yang,* Pengzhan Liu, and Heikki Kallio Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland *E-mail: [email protected]

Sugars, sugar alcohols, fruit acids and phenolic compounds were analysed in fruits of different species and cultivars of hawthorn (Crataegus spp.). Glucose and fructose were the major sugars commonly present in hawthorn, whereas sucrose was found only in some varieties. The major fruit acids were citric, quinic and malic acids. Hawthorn fruits were rich in sorbitol and myo-inositol. The major phenolic compounds in hawthorn were hyperoside, isoquercitrin, other quercetin glycosides, ideain, epicatechin, B-type procyanidins with polymerization index of 2-6 and their glycosides. Significant compositional variation existed among species, cultivars and varieties of hawthorn suggesting the need for selection of optimal raw materials for specific applications.

Introduction Nutraceuticals and functional foods play an important role in the management of lifestyle-related health problems in modern societies. Compositional investigation on new potential raw materials is crucial for the development of nutraceuticals and functional foods. Fruits of hawthorn (Crataegus spp.) have been traditionally used both as food and as medicine in China (1, 2). In western countries, these fruits are increasingly popular as new raw materials for food and food supplements with targeted physiological effects (3, 4). Scientific evidence suggests beneficial effects of the fruits on sugar and lipid metabolism, cardiovascular health and immune functions (5–10). Current knowledge on the © 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

bioactive components and the mechanisms responsible for the health benefits is limited (4). The aim of the present work was to investigate the composition of hawthorn fruits with special focus on sugars, sugar alcohols, fruit acids and phenolic compounds important for the sensory properties and biological activities of the fruits. Fruits of different species, varieties and cultivars were compared.

Materials and Methods


Hawthorn Fruit Samples Altogether twenty-two samples of hawthorn fruits were included in the study including ten cultivars of the variety C. pinnatifida var. major, eight cultivars of C. brettschneideri, three forms of C. pinnatifida, and one form of C. scabrifolia. Of these samples, the fruit sample of C. scabrifolia was collected from Kunming, Yun’nan Province, China in 2007, and the rest were collected from the Chinese National Fruit Germplasm Repository, Shenyang Hawthorn Garden (Shenyang, Liaoning Province, China) during 2007 and 2008. Table I presents a summary of the samples analyzed in the current study.

Table I. Hawthorn samples analyzed in the current study C. pinnatifida var. major

C. brettschneideri

C. pinnatifida

947

Caihong

Shanzha 1

8321

Hongrou-shanlihong

Shanzha 2

Dajinxing

Hongroushanzha

Shanzha 3

Huixian-dahong

Jifu 1

Jiangou 2

Jifu 3

Mopan

Xinghong 2

Qiujinxing

Zuofu 1

Shandongdajinxing

Zuofu 2

C. scabrifolia Yun’nan shanzha

Shen78201 Zizhenzhu

The fruits were picked as optimally ripe as determined by an experienced horticulturist based on the colour, structure and flavour of the fruits. The fruits were sliced and air-dried in a cool and shady place immediately after harvesting. The samples were stored in a desiccator in a cold room with avoidance of light. Before analyses, seeds were removed from dried hawthorn fruits. The seedless fruits were milled into powder in the presence of liquid nitrogen. 276 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Reference Compounds and Reagents D-fructose, D-quinic acid, chlorogenic acid, and L-ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO). D-glucose, D-sorbitol, and the internal standard D-xylose (for sugars) were purchased from Fluka Chemie AG (Buchs, Switzerland). Malic acid and the internal standard L-tartaric acid (for acids) were purchased from Merck KGaA (Darmstadt, Germany). Sucrose, citric acid, and the internal standard D-mannitol (for sugar alcohols), methanol (HPLC grade) and formic acid were purchased from J. T. Baker B.V. (Deventer, Holland). Myo-inositol was purchased from Alexis Co. (Lausen, Switzerland). Hyperoside (quercetin-3-O-galactoside), isoquercitrin (quercetin-3-O-glucoside), indeain chloride (cyanidin-3-O-galactoside chloride), epicatechin and procyanidin B2 (epicatechin-(4β→8)-epicatechin, PA B2) were purchased from Extrasynthese (Genay, France). Ethanol was from Primalco Oy (Rajamäki, Finland), and acetone (HPLC grade) and acetonitrile (HPLC grade) from VWR International Oy (Espoo, Finland). Tri-Sil HTP reagent was purchased from Pierce Chemical Co. (Rockford, IL). The reagent was composed of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine (2:1:10).

Analysis of Sugars, Sugar Alcohols, and Fruit Acids The sugars, sugar alcohols and fruits acids were extracted from the fruit powder with MilliQ water (powder/water, 1/100, w/v) and with the aid of ultrosonication (30 min) (11). After extraction, the sample was centrifuged, and a portion (3.0 mL) of the supernatant was taken, and xylose, mannitol, and tartaric acid were added (250 µL of water solution with concentration of 0.5 g/100 mL) as the internal standards for sugars, sugar alcohols and fruit acids, respectively. After filtration of the sample, a 50 µL portion was taken and silylized with Tri-Sil HTP reagent (11). For identification of the compounds, the trimethylsily (TMS)-derivatives of the samples and the reference compounds were analyzed with a Shimadzu QP 5000 MSD GC-MS (Kyoto, Japan). The column used was DB-1MS (30 m L × 0.25 mm i.d. × 0.25 µm df) (J & W Scientific, Agilent, Folsom, CA) (11). The sugars, sugar alcohols and fruit acids were identified by comparing the retention times and the mass spectra of the sample peaks with those of the reference compounds. For quantitative analysis, the TMS-derivatives of sugars, sugar alcohols, and fruit acids were analysed with a Hewlett Packard 5890 Series II gas chromatograph (GC, Hewlett-Packard Co., Palo Alto, CA) equipped with a flame ionization detector (FID) and a Hewlett Packard 7673 auto-sampler (11). Qualitative Analysis of Phenolic Compounds Phenolic compounds were extracted from the seedless hawthorn fruit powder with 80% aqueous ethanol in an ultrasonicator bath (20 mL x 3, 15 min for each extraction). After the removal of ethanol, the extract was redissolved in 50% aqueous methanol and fractionated into 19 fractions on a polyamide column using 277 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


sequentially MilliQ water, methanol and aqueous acetone as the eluting solvents (12). The fractions were analyzed with high performance liquid chromatography combined with diod array detection (HPLC-DAD) and high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). The compounds were identified based on reference compounds, retention times, UVabsorption spectra and mass spectra. The HPLC-DAD system (Shimadzu, Kyoto, Japan) consisted of a GT-154 vacuum degasser, two LC-10AT pumps, a SIL-10A automatic injector, a CTO10A column oven, a SPD-M10A VP diode array detector (DAD), and a SCL-10A VP system controller. The system was operated using Class-VP 6.1 Workstation software. A Phenomenex Prodigy RP-18 ODS (3) column (5 µm, 250 × 4.60 mm, Torrance, CA) combined with a Phenomenex Prodigy guard column (5 µm, 30 × 4.60 mm, Torrance, CA) was used. The mobile phase consisted of water/formic acid/ (99.5/0.5, v/v) as solvent A and acetonitrile /methanol (80/20, v/v) as solvent B. The gradient programme is shown in Table II. The flow rate of the mobile phase was 1 mL/min, and the injection volume was 10 µL. Peaks were recorded at three different wavelengths: 280, 360 and 520 nm. HPLC-ESI-MS analysis was carried in a positive ion mode using a Waters Acquity Ultra Performance LC system in combination with a Waters Quattro Premier mass spectrometer (Waters Corp., Milford, MA) equipped with an ion-spray interface. The capillary voltage was set to 4.0 kV, the cone voltage 22 V, and the extractor voltage 3 V. The source temperature was 150 °C and the desolvation temperature 300 °C. Mass spectra were obtained by scanning ions between m/z 200 and 900 and between m/z 900 and 2000 (12). The column for all HPLC analysis was at room temperature (20 °C)

Table II. Mobile phase program during HPLC-DAD and HPLC-ESI-MS analysis 0-5 min

15 min

25 min

30 min

35 min

40 min

45 min

50 min

55 min

A

90%

82%

82%

75%

75%

65%

40%

90%

90%

B

10%

18%

18%

25%

25%

35%

60%

10%

10%

Quantitative Analysis of Phenolic Compounds The quantitative analysis of major phenolic compounds in aqueous ethanolic extracts of hawthorn fruits were performed with HPLC-ESI-MS in a positive ion mode using the selected-ion-recording (SIR) function (13). The ions of 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) 278 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


and PA trimers (867). The instrumentation and the instrumental parameters were the same as described for the qualitative analysis. The quantification was carried out using an external standard method. The calibration curves of chlorogenic acid, ideain, hyperoside, isoquercitrin, epicatechin, PA B2 were prepared by analysis of commercial reference compounds. In addition, a PA dimmer (PA dimer II), a PA dimer-hexoside and a PA trimer (PA trimer II) were isolated from hawthorn fruit extract by preparative HPLC and used for preparation of calibration curves for quantification of these compounds in the hawthorn samples. The calibration curve of PA trimer II was used for quantification of all the PA trimers in the hawthorn samples. Statistical Analysis Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA) and Unscrambler 9.8 (Camo Process AS, Oslo, Norway). Differences in chemical composition among the species were analyzed using one-way analysis of variance (ANOVA) and with the Games–Howell and Student–Newman–Keuls (SNK) tests. Differences reaching a confidence level of 95% were considered significant. Pearson’s correlation coefficient analysis was carried out to investigate the correlation between the contents of different phenolic compounds.

Results and Discussion Sugars, Sugar Alcohols, and Fruit Acids Fructose and glucose were the major sugars commonly found in the fruits of all the species and cultivars analyzed (11). As shown in Figure 1, the content of fructose and glucose as well as the total content of the sugars and sugar alcohols were higher in the cultivars of C. pinnatifida var. major and C. brettschneideri than in the natural forms of C. pinnatifida and C. scabrifolia. Sucrose was found only in the fruits of C. scabrifolia and three cultivars C. pinnatifida var. major, 8321, Huixiandahong, and Shandongdajinxing, at levels of 24, 21, and 11 g/100 g dry mass (DM), respectively (11). Sorbitol was abundant in practically all the samples analyzed with the highest levels (11-16 g/100 g dry mass) found in the cultivars of C. brettschneideri and the lowest level in the fruits of C. scabrifolia (about 3 g/100 g dry mass) (Figure 1). Myo-inositol was found in the hawthorn fruit samples at levels of 0.1-0.2 g/100 g dry mass (Figure 1). The exceptionally high content of sorbitol and myo-inositol may have significance in the health effects of hawthorn (14–16). Citric acid was the most abundant acid in all the samples except in C. scabrifolia, where the content of quinic acid exceeded those of citric and malic acids (11) (Figure 2). The sugar/acid ratio was higher in the cultivars of C. pinnatifida var. major and C. brettschneideri than in the natural forms of C. pinnatifida and C. scabrifolia. The highest sugar/acid ratios were found in the fruits of Jiangou 2 (13.2) and Shen78201 (11.6), two cultivars of C. pinnatifida var. major (11). Overall the hawthorn samples fell into sugar- and acid-rich groups. The cultivars Dajinxing, Jiangou2, Qiujinxing, Shen78201 and Zizhenzhu 279 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


of C. pinnatifida var. major and all the cultivars of C. brettschneideri belong to the former group and the rest to the latter (11).

Figure 1. Content of sugars and sugar alcohols in hawthorn samples of different species and varieties. For each component, values not sharing common letters in the data labels differ significantly from each other (P < 0.05) (11).

Figure 2. Content of fruit acids and sugar/acid ratio in hawthorn samples of different species and varieties. For each component, values not sharing common letters in the data labels differ significantly from each other (P < 0.05) (11). 280 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The content and composition of acids, sugars, and sugar alcohols are important quality factors affecting directly the flavor and acceptability of the fruits and berries (17). Fruit acids content may also influence the stability of phenolic compounds in the fruits and the health promotion effects of the berries (18). The high variation found in the sugar content and the sugar/acid ratio indicates considerable difference in the sensory properties and physiological effects among the cultivars and species analyzed. Two cultivars of C. pinnatifida var. major (Jiangou 2 and Shen78201) may have the best sensory properties and are most suitable for food industry due to the high S/A ratios. Most cultivars of C. brettschneideri and C. pinnatifida var. major had S/A ratio between 6–9 (Dajinxing, Quijinxing, Zizhenzhu, Caihong, Hongroushanzha, Jifu 1, Jifu 3, Xinghong 2 and Zufu 1). Those cultivars may also have good sensory profiles. All 3 forms of C. pinnatifida had quite low S/A ratio and total sugar contents. This suggest that fruits of this species have less pleasant tastes. The acid-rich cultivars, e.g. 947 and 8321 of C. pinnatifida var. major may be most efficient in promoting food digestion and improving blood circulation (18).

Profile of Phenolic Compounds Figure 3 presents the HPLC-DAD chromatogram of the raw extract of hawthorn fruit (C. pinnatifida var. major) prepared with 80% ethanol, recorded at 280 nm (12, 13). The major phenolic compounds in the extract were epicatechin, oligomeric procyanidins, glycosides of oligomeric procyanidins, phenolic acids, flavonol glycosides, and an anthocyanin. Figure 4 presents the structures of some phenolic compounds identified in the hawthorn extract.

Figure 3. HPLC-DAD chromatogram of 80% ethanolic extract of fruits of the cultivar Mopan (C. pinnatifida var. major) (12, 13). Peaks: 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). 281 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Flavonols, Chlorogenic Acid, and Ideain Two major flavonol glycosides, hyperoside (quercetin-3-O-galactoside) and isoquercitrin (quercetin-3-O-glucoside), chlorogenic acid, and ideain were identified based on UV spectra, mass spectra and reference compounds. In addition, two flavonol glycosides were tentatively identified as quercetin-di(methylpento)-hexoside and quercetin-methylpento-hexoside based on the UVand mass spectra. The content of chlorogenic acid varied in the range of 0.26-1.57 mg/g dry mass. The level of chlorogenic acid was highest in the cultivars of C. pinnatifida var. major (1.14 ± 0.28 mg/g dry mass), followed by the natural forms of C. pinnatifida (0.51 ± 0.16 mg/g dry mass). The lowest level of chlorogenic acid was found in the natural form of C. scabrifolia (0.26 mg/g dry mass) (Figure 5). Ideain was the only anthocyanin found in the hawthorn fruits. The culitvars of C. brettschneideri was richest in ideain. The fruits if C. pinnatifida contained less ideain than those of C. brettschneideri (P < 0.05) (13).

Figure 4. Structures of chlorogenic acid, ideain, epicatechin, and procyanidin B2. Ideain was not detected in Yun’nan shanzha (C. scabrifolia) at all (Figure 5). The levels of isoquercitrin were rather close among the samples (0.17-0.25 mg/g dry mass), whereas that of hyperoside varied considerably among the four groups. The cultivars of C. brettschneideri (0.53 ± 0.21 mg/g dry mass) and the natural forms of C. pinnatifida (0.42 ± 0.02 mg/g dry mass) were richer in hyperoside than the cultivars of C. pinnatifida var. major (0.26 ± 0.12 mg/g dry mass) and the natural form of C. scabrifolia (0.35 ± 0.06 mg/g dry mass) (P < 0.05, Figure 5). 282 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 5. Content of flavonols, chlorogenic acid, and ideain in hawthorn samples of different species and varieties (13). For each component, values not sharing common letters in the data labels differ significantly from each other (P < 0.05)

Flavanols and Proanthocyanidins Epicatechin and procyanidin B2 [epicatechin-(4β→8)-epicatechin, PA B2] were identified by comparing the retention times, UV- and mass spectra of the sample peaks with those of the reference compounds. Thirty six compounds were tentatively identified as B-type procyanidins and B-type procyanidin glycosides with polymerization index (PI) of 2-6 based on the UV- and mass spectra (12). To our best knowledge, this is the first report of the presence of procyanidin glycosides in hawthorn fruits. Eight most prominent compounds were quantified using single ion recording function of HPLC-ESI-MS. The [M+H]+ ions of the compounds were recorded and the peaks areas of the selected ions were used for quantification. Epicatechin was the major flavanol and PA B2 the most abundant procyanidin (Figure 6) (13). The two compounds were present at roughly equal levels (0.8712.36 mg/g dry mass). In addition to PA B2, another PA dimer (PA dimer II), a PA dimer glycoside, and three PA trimers were quantified. The content of PA dimer II varied in the range of 0.09-1.19 mg/g dry mass in the hawthorn samples analysed. The PA dimer glycoside was present at levels of 0.00-1.08 mg/g dry mass. The three PA trimers were present at levels of 0.11-2.66, 0.73-6.90, 0.01-1.24 mg/g dry mass, respectively. The contents of these compounds were higher in C. pinnatifida var. major and in C. scabrifolia than in the cultivars of C. brettschneideri and the natural forms of C. pinnatifida (P < 0.05) (Figure 6). Strong positive correlation was recognized between the content of the flavanol monomer epicatechin and and the levels of procyanidin dimers and trimers (r2 = 0.86 – 0.95, P < 0.001) (13). 283 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Accurate quantification of proanthocyanidin is often challenging due to the difficulty in identification of unknown compounds as well as the inavailability of reference compounds. For an unknown sample, the identification and quantification of epicatechin can often be achieved without much complications. The strong correlation between the content of epicatechin and those of the dimeric, trimeric and oligomeric procyanidins could be used for a fast estimation of procyanidin content based on the content of epicatechin in hawthorn samples.

Figure 6. Content of flavanol and procyanidins in hawthorn samples of different species and varieties. For each component, values not sharing common letters in the data labels differ significantly from each other (P < 0.05) (13).

Oligomeric PAs in a European hawthorn species C. laevigata fruits were quantified by a combination of solid phase fractionation and HPLC analysis. The total content of PA dimers and trimers in the fruits was 0.6 mg/g DM (19). The total content of PAs reported in the fruits of two other European hawthorn species (C. monogyna and C. oxyacantha) varied from 14 to 26 mg/g DM (20). Our results showed that the levels of PAs in Chinese hawthorn fruits were higher than that reported in C. laevigata and close to the levels found in C. monogyna and C. oxyacantha.

Conclusions Fructose and glucose were the major sugars in all the samples, whereas sucrose was present only in some samples. In all the samples analyzed, sorbitol was abundant, and myo-inositol was present. Overall, the cultivars of C. p. var. major and C. brettschneideri contained higher levels of sugars and higher sugar/acid ratio than the natural forms of C. pinnatifida and C. scabrifolia. 284 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Citric acid, quinic acid, malic acid were the major fruit acids in the hawthorn fruits analyzed, citric acid being the most abundant in the native forms of C. pinnatifida and the cultivars of C. p. var. major and C. brettschneideri and quinic acid in the natural form of C. scabrifolia. The total acid content was highest in the natural form of C. scabrifolia and lowest in the cultivars of C. brettschneideri. Cultivars of C. brettschneideri and the natural forms of C. pinnatifida were richer in flavonols but poorer in procyanidins than those of C. scabrifolia and C. pinnatifida var. major. The highest content of chlorogenic acid was found in the cultivars of C. pinnatifida var. major, and that of ideain in C. brettschneideri. The high compositional variation in the fruits among different species and cultivars of hawthorn suggests the importance of raw material selection for targeted physiological effects and proper sensory properties in industrial applications of the fruits.

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Sugars, Acids, and Phenolic Compounds in Chinese Hawthorn (

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