- Email: [email protected]
Identification and characterisation of low-molecular-weight phenolic compounds in bayberry (Myrica rubra Sieb. et Zucc.) leaves by HPLC-DAD and HPLC-UV-ESIMS
Food Chemistry 128 (2011) 1128–1135
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Identification and characterisation of low-molecular-weight phenolic compounds in bayberry (Myrica rubra Sieb. et Zucc.) leaves by HPLC-DAD and HPLC-UV-ESIMS Haihua Yang a, Yiqiang Ge b, Yujing Sun a, Donghong Liu a, Xingqian Ye a,⇑, Dan Wu a a b
Department of Food Science and Nutrition, Zhejiang University, KaiXuan Road 268#, Hangzhou, CN 310029, PR China China Rural Technology Development Centre, SanLiHe Road 54#, Beijing, CN 100045, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2010 Received in revised form 19 February 2011 Accepted 30 March 2011 Available online 5 April 2011 Keywords: Bayberry Leaves Phenolic compounds HPLC-DAD HPLC-UV-ESIMS
a b s t r a c t Leaves of each of two bayberry cultivars, Biqi and Dongkui, were divided into three categories by age, namely immature, intermediate, and mature. Phenolic compounds were analysed by the methods of HPLC-DAD and HPLC-UV-ESIMS. Gallic acid and EGCG were identified positively, and 13 other compounds (flavan-3-ol monomers, prodelphinidin oligomers, and flavonol glycosides) were partially identified. Gallic acid (7.5–87.8 mg/100 g) was the only phenolic acid detected and flavan-3-ols were abundant. Myricetin deoxyhexoside (535.4–853.0 mg/100 g) was the major flavonol glycoside. Among the three categories, immature leaves of both cultivars recorded the highest level of total phenolics, irrespective of whether they were measured by the Folin–Ciocalteu method (19404.0 mg/100 g in Biqi and 19626.0 mg/100 g in Dongkui) or as the sum of individual phenolic compounds (2255.9 mg/100 g in Biqi and 1797.1 mg/100 g in Dongkui). The results showed that bayberry leaves are a potentially rich source of beneficial phenolics. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Bayberry (Myrica rubra Sieb. et Zucc.), a tree species of the family Myricaceae, has been cultivated mainly in southern China for more than 2000 years. Bayberry is also native to other countries of eastern Asia (Chen, Xu, Zhang, & Ferguson, 2004). Bayberry fruits, with their exquisite taste, flavour, and attractive red colour, are popular among local people. Bayberry is used extensively in folk medicine for a variety of ailments. Bayberry leaves, twigs, and bark have therapeutic properties and the plant has been in popular use as a traditional medicine for a long time. Herbalists recommend a warm concoction made from the root bark of bayberry as a tonic that acts as a stimulant and as an astringent. Bayberry’s astringency is believed to help in treating intestinal disorders such as the irritable bowel syndrome and mucous colitis; a paste made from powdered root bark is applied to ulcers and sores; and its infusion is even alleged to control excessive vaginal discharge. Bayberry is especially effective in the treatment of diarrhoea and, because it acts as a stomach irritant when used in large doses, bayberry bark acts as an emetic. During head colds, bayberry increases secretion of nasal mucus. When applied as a poultice, bayberry is believed to be useful in treating chronic indolent ulcers (Matsuda, Morikawa, Tao, Ueda, & Yoshikawa, 2002). Bayberry trees flush two or three times a year. Foliar growth is luxuriant and leaves remain green throughout the year. The tree ⇑ Corresponding author. Tel./fax: +86 571 86971165. E-mail addresses: [email protected], [email protected] (X. Ye). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.118
needs to be pruned every year more than once to get high fruit production. Pruning yields a mass of leaves, which are generally discarded and remain underutilized despite reports that extracts of bayberry leaves are anti-oxidative (Xia, Chen, & Wu, 2004), antimicrobial (Chen & Liu, 1999), and antiviral (Shen, Xie, Zhai, Lin, & Xie, 2004). Recent chemical investigations have brought light to several interesting chemical compounds in bayberry, of which polyphenols form a major category. Plant phenolics are aromatic secondary metabolites that are ubiquitous in the plant kingdom and comprise more than 8000 substances with highly diverse structures and molecular masses. Plant phenolics can be subdivided into classes such as phenolic acids, flavanones, flavones, flavonols, anthocyanidins, flavan-3-ols, and proanthocyanidins (Hümmer & Schreier, 2008). A substantial body of literature suggests that phenolic extracts from plants are able to prevent diseases including cancer by anti-oxidative action (Yesil-Celiktas, Sevimli, Bedir, & Vardar-Sukan, 2010) and cardiovascular diseases (Rashed, Shallan, Mohamed, Fouda, & Hanna, 2010). The polyphenols from green tea have been reported to have positive effects on anti-inflammation and anti-arthritis activities (Rashmi, Nahid, & Tariq, 2010). These polyphenolic anti-oxidants, because they have anti-microbial properties, can also be used to preserve foods (Vattem, Lin, & Shetty, 2004). However, the characterisation of phenolic compounds in bayberry has received little attention. High-performance liquid chromatography (HPLC) is the most common analytical technique for separating plant phenolics; HPLC coupled with a photodiode-array
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
detector (HPLC-DAD) provides extensive information on the structure of polyphenols in crude or partly purified plant extracts and, coupled with mass spectrometry (HPLC-MS), information that serves as a powerful aid to identify the structure. Electrospray ionisation (ESI) is a gentle, sensitive, and the most widely used method for generating ions to date (Hümmer & Schreier, 2008). The UV–vis absorption spectra of phenolic compounds enable identification of chromatographic peaks and their classification. A combination of these data with those on mass spectra and information from the literature can be used to tentatively identify the structure of polyphenols. This paper reports the results of qualitative and quantitative analysis of low molecular weight phenolics from bayberry leaves of different ages using HPLC-DAD and HPLC-UV-ESIMS. Knowledge of the precise phenolic profile of bayberry leaves may offer a scientific basis to put the underutilized bayberry leaves to good use.
1129
2.4. Extraction of phenolic compounds
2. Materials and methods
Phenolic compounds were extracted from 1 g samples of bayberry leaf powder with aqueous acetone as described above. The phenolic compounds were isolated by a method published earlier (Oszmianski & Sapis, 1989). The solvent containing the extract was treated with chloroform (15 ml) to remove acetone and compounds other than phenolics. The aqueous phase containing the phenolic compounds was recovered. Crystallized NaCl was then added until saturation point to precipitate the more polymerised tannins, which made it possible to analyse phenolics of low molecular weight. The solution was filtered and the filtrate mixed with ethyl acetate (3 20 ml). The ethyl acetate phase (60 ml) was washed with 10 ml water, concentrated under vacuum to 2 ml, and chloroform (10 ml) was added to precipitate phenolics. The suspension was filtered, the filtrate was discarded, and the residue was re-dissolved in 5 ml methanol for analysis by HPLC-DAD and HPLC-UV-ESIMS. All samples were prepared and analysed in triplicate.
2.1. Chemicals
2.5. HPLC-DAD and HPLC-UV-ESIMS analysis
Standards of gallic acid, ( )-epigallocatechin-3-O-gallate (EGCG), quercetin 3-O-glucose-rhamnoside, and Folin–Ciocalteu phenol reagent were obtained in the form of commercial samples from Sigma (St. Louis, Missouri, USA). Acetonitrile was HPLC grade. All other reagents and solvents used were of analytical grade. All solutions were prepared using distilled-deionized water.
Bayberry leaves of two cultivars, Biqi and Dongkui, growing in Xianju in Zhejiang province in south-eastern China were harvested by hand from randomly chosen plants on 6 July, 2009. Leaves of each cultivar were divided on the basis of age into three categories, namely immature, intermediate, and mature. The age of a leaf can be estimated conventionally by colour: immature leaves are bright green; mature leaves are dull and dark green; and the green of intermediate leaves is deeper than that of immature leaves but lighter than that of mature leaves. All the collected leaves were washed and then dried under vacuum at 40 °C for 12 h, ground fine, and stored at 20 °C until required.
Phenolic compounds were analysed with HPLC-DAD using an Alliance 2695 separation module (Waters) linked simultaneously to a Waters 2998 photodiode array detector (Waters). HPLC-UVESIMS analysis was performed on a Waters platform, including a Waters 1525 pump, a Waters 2487 Dual k absorbance detector, and an Esquire 3000 Pius (Bruker). Mass spectra were achieved by electrospray ionisation in the negative mode. All samples were filtered through a 0.45 lm Millipore membrane filter before injection, and a 20 ll aliquot was separated by a Zorbax SB-C18 (Agilent, USA) column (250 4.6 mm, 5 lm) protected by a guard column of the same material. The column thermostat was maintained at 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in water, v/v) and solvent B (acetonitrile). The elution profile consisted of a linear gradient comprising isocratic 10% B (5 min), 10–30% B (5–35 min), and 30–90% B (35–40 min) with a flow rate of 0.7 ml/min. After each run, the column was washed with 100% acetonitrile and equilibrated to initial conditions for 15 min. UV–Vis absorption spectra were recorded online during the HPLC analysis. The DAD detector was set to a scanning range of 200–800 nm. Phenolic acid and flavan-3-ol monomers were detected at 280 nm and flavones at 360 nm.
2.3. Determination of total phenolics
2.6. Identification and quantification of phenolic compounds
Total phenolics were extracted from samples (1 g each) of bayberry leaf powder by mixing them with 10 ml of 70% aqueous acetone for 15 min at room temperature. The process was repeated three times. After centrifuging at 2000 rpm for 10 min, the supernatant was rotary-evaporated under vacuum at 40 °C to remove the acetone and the aqueous phase diluted to 1000 ml with methanol. All samples were prepared and processed in triplicate. Total polyphenol content was determined by the Folin–Ciocalteu method (Xu et al., 2008). One millilitre of the prepared sample solution was added to a 25 ml volumetric flask filled with 9 ml distilled water. A blank sample was prepared using distilleddeionized water. Folin–Ciocalteu phenol reagent (0.5 ml) was added to the mixture and the mixture was shaken vigorously for 5 min; 5 ml of Na2CO3 solution was added to the mixture; the resulting mixture was immediately diluted to 25 ml with distilled water and mixed thoroughly; and then it was allowed to stand for 60 min before measuring its absorbance at 750 nm. The absorbance was compared with that of the prepared blank. Total polyphenol contents were expressed as milligrams per 100 g of gallic acid equivalent.
The phenolic compounds were identified mainly by their UV–Vis spectra and ESIMS spectra and by comparing with published data. If relevant standards were available, they were also identified by comparing the chromatography with the authentic standards. Gallic acid and EGCG were quantified by injecting a solution with known concentrations of standards and calculating with regression equations from the standard curves. Contents of other flavan-3-ol monomers and flavones were expressed as EGCG and quercetin 3-O-glucose-rhamnoside equivalents, respectively. The concentrations of all compounds were expressed in milligrams per 100 g of dried leaves (dry weight basis, or DW). The values were expressed as mean ± standard deviations of three replications.
2.2. Plant material
3. Results and discussion 3.1. Identification of phenolic compounds Typical HPLC profiles of Biqi and Dongkui bayberry leaves are shown in Figs. 1 and 2, respectively, and the corresponding
1130
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Fig. 1. Typical HPLC chromatogram of phenolic extract of Biqi cultivar bayberry leaves detected at 280 nm (A) and 360 nm (B). Peak numbers refer to Table 1.
UV–Vis spectra for each peak are presented in Fig. 3. Sixteen peaks (peaks 1–16, Figs. 1A and 2A) were detected at a wavelength of 280 nm and six peaks (peaks 10, 11, and 13–16, Figs. 1B and 2B) at 360 nm. These peaks in the chromatograms were classified into three groups, namely phenolic acids, flavan-3-ols, and flavonol glycosides, by comparing their UV–Vis spectra and HPLC retention times with those of the available standards. The ESIMS spectral data, UV–Vis spectral data, and tentative identification of the 16 compounds from Biqi and Dongkui bayberry leaves are shown in Table 1. 3.1.1. Phenolic acid (peak 1) The first group (phenolic acids) contained one component as indicated by peak 1 with HPLC retention time at 5.63 min. Peak 1 presented the UV kmax at 215.6 and 271.1, was symmetrical from 240 to 305 nm (with maximum symmetry around 271.1 nm), and then shifted to 325 nm. ESIMS spectra showed the molecular ion [M–H] at m/z 168.8 and a fragment ion at m/z 124.9, which corresponds to the loss of a carboxyl residue (mass unit 45) from the molecular ion. The identity of the component in peak 1 was also confirmed by adding minute amounts of gallic acid to the phenolic extract: the peaks of endogenous gallic acid in the sample and of the additional gallic acid superimposed perfectly on each other, and peak 1 was unambiguously identified as that of gallic acid. 3.1.2. Flavan-3-ols (peak 3–9, and 12) Several well-resolved chromatographic peaks of flavan-3-ols were observed under the experimental conditions mentioned earlier, which were used for HPLC-DAD and HPLC-UV-ESIMS analysis. This group of compounds corresponded to peaks 3–9 and peak 12
and was provisionally identified as comprising of four flavan-3-ol monomers, three prodelphinidin dimers, and one trimer. The dimers and trimers were all considered to be of B type based on their mass spectra. In the mass spectra of flavan-3-ol monomers, peaks 4, 5, and 9 exhibited their protonated molecular ion [M–H] at m/z 467.1, their duple molecular ion [2 M–H] at m/z 935.4, and a fragment ion at m/z 305.0, pointing to a hexosyl residue that was included in the molecule structure. Furthermore, the three peaks had almost the same UV–Vis spectrum shape with a kmax at 270 nm and a shoulder from 315 to 360 nm. Therefore, this compound was provisionally identified as (E)GC-hexoside (molecular weight = 468). Although glycosides of flavan-3-ol monomers are quite rare in plants and have never been detected in bayberry leaves so far, a previous identification of (+)-catechin 3-O-glucoside in the seed coat of lentil (Lens culinaris L.) supports the identification of compounds that corresponded to peaks 4, 5, and 9 (Dueñas, Sun, Hernández, Estrella, & Spranger, 2003). Peak 8 with a HPLC retention time of 19.59 min showed kmax at 212.0 nm and 273.5 nm, symmetry around the maximum from 258 to 290 nm, and a shoulder from 290 to 325 nm, which is typical of galloyl groups. Peak 8 also gave a molecular ion [M–H] at m/z 457.1, a duple molecular ion [2 M–H] at 915.1, and a fragment ion at m/z 304.8 corresponding to loss of a galloyl residue, and a fragment ion at m/z 168.8 (the gallic acid fragment), which indicated peak 8 to be EGCG or GCG. By comparing with the standard, this compound was positively identified as EGCG. To identify the structure of peak 3, a structure was hypothesised based on the product ions detected by ESIMS with EGC as the extension unit and EGCG as the terminal unit. Three characteristic
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
1131
Fig. 2. Typical HPLC chromatogram of phenolic extract of Dongkui cultivar bayberry leaves detected at 280 nm (A) and 360 nm (B). Peak numbers refer to Table 1.
fragmentation routes have been reported for proanthocyanidins, namely retro-Diels–Alder fission (RDA), quinone methide fission (QM), and heterocyclic ring fission (HRF) (Wu, Wang, & Simon, 2003), of which RDA was found to be the most important for elucidating the structure of dimers and to lead to the loss of ring B in the extension unit (Wu et al., 2003). Peak 3, eluting at 11.97 min, showed a molecular ion [M–H] at m/z 761.3 with fragment ions at m/z 591.2 and 423.1. The two fragment ions corresponded respectively to further loss of O-galloyl (Gu et al., 2008) and of the RDA product ion (m/z 168) (Wu et al., 2003). Based on these results, we suggested that EGC was the extension unit and EGCG was the terminal unit. Since product ions derived from fragmentation can be used only for determining the sequence of monomeric units and not for distinguishing between epimers, peak 8 was identified as (E)GC-(E)GCG. Peak 6, eluting at 16.58 min, showed a molecular ion [M–H] at m/z 1217.4 with two fragment ions at m/z 1065.3 and 913.4, which corresponded to the loss of one galloyl residue ([M–H-152] ) and two galloyl residues ([M–H-152-152] ) (Gu et al., 2008), respectively. Because there were no more fragment ions, especially the most important fragment ions to characterise trimeric proanthocyanidins produced by the cleavage of interflavanoid linkages (Gu et al., 2008), the structure of this compound could not be determined with certainty, and peak 6 was provisionally identified as a prodelphinidin trimer consisting of two (E)GCG units and one (E)GC unit. Peaks 7 and 12, the other two prodelphinidin dimers consisting of two (E)GCG units, had the same fragment ions: a molecular ion [M–H] at m/z 913.3 with two fragment ions at m/z 743.2 and
573.2, corresponding to further loss of one O-galloyl and two Ogalloyls, respectively. 3.1.3. Flavonols (peak 10, 11, and 13–16) The third group of compounds corresponded to peaks 10, 11, and 13–16. These compounds were generally eluted later than the other two groups of compounds and displayed UV–Vis spectra similar to those of rutin (quercetin 3-O-glucose-rhamnoside) with two absorption maxima at 254 and 354 nm. It is well known that flavonols exhibit two major absorption peaks in the region of 240–400 nm, in which the region of 240–280 nm (Band II) is considered to be associated with absorption due to the A-ring benzoyl system, and the region of 300–380 nm (Band I) with that due to the B-ring cinnamoyl system. Therefore, we supposed that compounds in the third group were flavonols. Peaks 10, 11, and 13 had molecular ions [M–H] at m/z 479.1, 479.1, and 463.1, respectively. All the three peaks also had a fragment ion at m/z 316.8, which is a typical mass in the negative mode of myricetin aglycone, indicating that these three compounds were myricetin derivatives. A fragment ion at m/z 927.0 of peak 13 was the double-charge molecular ion [2 M–H] . It has been reported that sugars bound to the aglycons are hexoses with a mass unit of 162, deoxyhexoses with a mass unit of 146, and pentoses with a mass unit of 132 (Määttä, Kamal-Eldin, & Törrönen, 2003). The ESIMS spectra of the three peaks indicated that their structure contained a hexosyl residue (479.1 316.8 = 162.3) or a deoxyhexosyl residue (463.1 316.8 = 146.3). Peaks 10 and 11 were therefore identified as those of myricetin hexoside and peak 13 as that of myricetin deoxyhexoside. However, peaks 10 and 11
1132
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Fig. 3. On-line UV–visible spectra of the phenolic compounds. Peak numbers correspond to those in Figs. 1 and 2 and Table 1.
had a different stereochemistry structure, and the position where these flavonols were substituted needs further study. The UV spectra of peaks 15 and 16 were very similar to those of peak 13. However, peaks 15 and 16 showed a shift of the maximum of Band II from 262 to 266.2 nm compared to peak 13. The excess of acyl
formed with hydroxybenzoic acid in peaks 15 and 16 compared to peak 13 was responsible for this shift, as has been reported earlier (Fang, Zhang, & Wang, 2007). The ESIMS data of peaks 15 and 16 revealed a molecular ion [M–H] at m/z 615.3 and a fragment ion at m/z 316.8, indicating that they were both
1133
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Table 1 Identification of individual phenolic compounds in phenolic extracts from bayberry leaves by using their UV–visible spectral characteristics, negative ions in HPLC-UV-ESIMS, and respective standards.
a
Peak No.
HPLC tR (min)
HPLC-DAD (nm)
Molecular weight
HPLC-ESIMS (m/z)
Tentative identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
5.63 8.18 11.97 13.84 15.11 16.58 17.19 19.59 21.65 22.71 23.14 24.81 25.96 30.43 33.57 34.28
216.7, 271.1 225sh, 274.5 272.3 214.4, 271.1 214.4, 271.1 272.3 273.5 212.0, 273.5 214.4, 271.1 267.6, 300sh, 355.8 269.9, 300sh, 359.9 273.5 260.4, 300sh, 349.8 255.7, 264sh, 348.6 210.9, 265.2, 295sh, 349.8 210.9, 266.2, 295sh, 349.8
170 474 762 468 468 1218 914 458 468 480 480 914 464 448 616 616
168.8, 124.9 947.0, 473.1, 318.8, 152.7 761.3, 591.2, 423.1 935.4, 467.1 935.4, 467.1 1217.4, 1065.3, 913.4 913.4, 743.2, 573.2 915.1, 457.1,304.8168.8 935.4, 467.1 479.1, 316.8 479.1, 316.8 913.3, 743.2, 573.2 927.0, 463.1, 316.8 447.1, 300.8 615.3, 316.8 615.3, 316.8
Gallic acid (stda) (Unidentified compound) (E)GC-(E)GCG (E)GC-hexoside (E)GC-hexoside 2(E)GCG + (E)GC 2(E)GCG EGCG (std) (E)GC-hexoside Myricetin hexoside Myricetin hexoside 2(E)GCG Myricetin deoxyhexoside Quercetin deoxyhexoside Myricetin deoxyhexoside-gallate Myricetin deoxyhexoside-gallate
The compound was also identified by comparing the chromatography with the authentic standards.
myricetin deoxyhexoside derivatives. Finally, peaks 15 and 16 were provisionally identified as myricetin deoxyhexoside-gallate (615.3 463 = 152.3, loss of a galloyl residue). UV spectra of peak 14 presented the same profile as that of quercetin deoxyhexoside published earlier (Sakakibara, Honda, Nakagawa, Ashida, & Kanazawa, 2003). Peak 14 also had a molecular ion [M–H] at m/z 447.1 and a fragment ion of m/z at 300.8, which indicated that a deoxyhexosyl residue had been attached to the quercetin aglycone, and was therefore identified as quercetin deoxyhexoside. This compound was detected also in bayberry fruit (Bao, Cai, Sun, Wang, & Corke, 2005; Fang et al., 2007) and bark (Wang, Liu, & Feng, 2008). Peak 2 showed a HPLC retention time of 8.18 min and also gave a UV kmax at 275.9 nm, a shoulder at 225 nm, and a very broad band shifted to 325 nm. The ESIMS spectra showed that peak 2 had a molecular ion [M–H] at m/z 473.1 and fragment ions at m/z 318.8 and 152.7. These data on the mass spectra indicated that peak 2 is a dihydromyricetin derivative. Dihydromyricetin is reported to occur in Ampelopsis grossedentata (Jiang, Dong, Zhang, Gan, & Liu, 2008). However, we could find no report of dihydromyricetin derivatives and therefore had to leave peak 2 unidentified. Isolation and NMR identification might be required to identify this compound. Chromatogram profiles of the phenolic extracts of leaves of cultivars Biqi and Dongkui were similar (Figs. 1 and 2). (E)GC-hexoside (peak 4) was not detected in mature leaves of Biqi and Dongkui; myricetin deoxyhexoside was the major phenolic compound in the phenolic extracts of leaves of Biqi and Dongkui. EGCG, a widely distributed flavan-3-ol monomer in tea leaves, was detected in leaves of bayberry (M. rubra Sieb. et Zucc.) and M. rubra var. acuminata (Yang, Chang, Chen, & Wang, 2003) although it was found neither in bayberry fruit (Fang et al., 2007; Zhou et al., 2009) nor in leaves of another unknown cultivar of bayberry (Zou & Li, 1998). Protocatechuic acid, quercetin 3-glucoside, quercetin hexosidegallate, and kaempferol hexoside were detected in bayberry fruit (Fang et al., 2007; Zhou et al., 2009), myricetin and quercetin3-O-b-D-glucoside were detected in leaves of an unknown cultivar of bayberry (Zou & Li, 1998) but the present experiment did not find all these compounds in bayberry leaves. 3.2. Quantitative analysis of total phenolics and individual phenolic compounds In the present study, the contents of total phenolics and of individual phenolics were determined by the Folin–Ciocalteu method and by HPLC-DAD, respectively. Table 2 presents the results.
Total phenolics in leaves of cultivars Biqi and Dongkui, on a dry weight basis, were 8137.5–19404.0 mg/100 g and 11566.8– 19626.0 mg/100 g, respectively. These values are intermediate between those for Nelumbo nucifera leaves (354–487 mg/g) (Huang, Ban, He, Tong, & Wang, 2010) and Stevia rebaudiana Bert. (61.5 mg/g) (Shukla, Mehta, Bajpai, & Shukla, 2009) but higher than those for bayberry pomace (27.4–47.4 mg/g) (Zhou et al., 2009). These observations suggest that bayberry leaves are rich in phenolics and have good potential as value-added products. Furthermore, immature leaves of Biqi showed the highest content of total phenolics (19404.0 mg/100 g) among all the samples, followed in that order by mature leaves (11715.5 mg/100 g DW) and intermediate leaves (8137.5 mg/100 g DW). Leaves of cultivar Dongkui showed the same pattern (Table 2), which indicated that choosing leaves of the right age is an important factor in the utilisation bayberry leaves. Immature leaves of both cultivars also had the highest content of phenolics when expressed as the sum of individual phenolic compounds (2255.9 mg/100 g in Biqi and 1797.1 mg/g in Dongkui) whereas mature leaves showed the lowest values, and leaves of intermediate age showed values of phenolics that were intermediate. The contents of phenolics expressed as the sum of individual phenolic compounds were lower than those determined by the Folin–Ciocalteu method. This difference is understandable because the Folin–Ciocalteu method tends to overestimate the contents of total phenolics since it ignores potential interference by other substances (Schieber, Keller, & Carle, 2001). Besides, there might be high-molecular-weight phenolic compounds in bayberry leaves that did not include the sum of individual phenolic compounds. Gallic acid was the only phenolic acid detected in bayberry leaves with the analytical method used in the present study. High amounts of gallic acid were noted in immature leaves of Dongkui and in leaves of the intermediate age of Biqi. The lowest value of gallic acid was recorded in leaves of the intermediate age of Dongkui. The contents of gallic acid in bayberry leaves (7.50–87.82 mg/100 g) were higher than those in bayberry pomace (2.81–9.23 mg/100 g) (Zhou et al., 2009). Compared to other polyphenols, gallic acid is very easily absorbed into the human body (Manach et al., 2005), and gallic acid was shown to have anticancer effect in vitro (Tomas-Barberan & Clifford, 2000). All flavan-3-ols and oligomeric prodelphinidins identified above were present in the leaves of all ages except that peak 4 ((E)GChexoside) was not detected in mature leaves of either cultivar. However, the leaves differed in their contents of individual constituents: in general, immature leaves of both cultivars were richer in individual flavan-3-ols and oligomeric prodelphinidins compared
1134
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Table 2 Contents of total and individual phenolic compounds (mg/100 g DW) in different bayberry leaves. Peak No.
Phenolic compounds
1 8 4 5 9 3 6 7 12
Gallic acid EGCG (E)GC-hexosidea (E)GC-hexoside (E)GC-hexoside (E)GC (E)GCG 2(E)GCG + (E)GC 2(E)GCG 2(E)GCG Total Myricetin hexosidec Myricetin hexoside Myricetin deoxyhexoside Quercetin deoxyhexoside Myricetin deoxyhexoside-gallate Myricetin deoxyhexoside-gallate Total (Unidentified compound) Sum of phenolic compounds Total phenolicsd
10 11 13 14 15 16 2
a b c d
Biqi
Dongkui
Immature
Intermediate
Mature
Immature
Intermediate
Mature
18.8 ± 0.8 228.3 ± 2.4 194.3 ± 3.1 239.2 ± 8.0 183.4 ± 6.4 76.7 ± 1.0 107.8 ± 1.0 140.3 ± 1.1 110.1 ± 0.5 1280.1 12.4 ± 0.3 7.0 ± 0.1 765.2 ± 10.5 11.3 ± 0.6 96.8 ± 3.7 7.9 ± 0.1 900.6 56.4 ± 1.6 2255.8 19404.0 ± 410.6
50.8 ± 3.7 90.7 ± 5.0 30.2 ± 0.6 45.5 ± 3.0 49.7 ± 0.4 88.2 ± 0.7 71.7 ± 0.3 102.5 ± 0.6 52.7 ± 0.9 531.2 40.0 ± 1.0 19.6 ± 0.1 583.9 ± 10.3 21.2 ± 0.3 16.1 ± 0.6 2.4 ± 0.6 683.2 30.2 ± 0.1 1295.4 8137.5 ± 42.7
27.8 ± 0.6 97.9 ± 1.4 ndb 32.6 ± 0.9 37.5 ± 0.3 93.8 ± 0.4 43.9 ± 0.1 87.9 ± 0.9 35.0 ± 0.7 428.6 43.0 ± 1.8 16.9 ± 0.4 535.4 ± 11.9 32.9 ± 1.0 12.1 ± 1.0 8.3 ± 0.5 648.6 28.3 ± 0.7 1133.1 11715.5 ± 45.3
87.8 ± 1.6 134.1 ± 7.1 33.9 ± 1.0 55.3 ± 0.3 81.1 ± 3.6 87.2 ± 0.1 77.2 ± 0.8 111.6 ± 1.4 83.9 ± 0.5 664.3 3.1 ± 0.1 2.9 ± 0.1 853.0 ± 9.3 30.3 ± 0.4 110.3 ± 5.1 15.1 ± 0.6 1014.7 30.5 ± 0.1 1797.1 19626.0 ± 325.9
7.5 ± 0.1 103.7 ± 1.5 20.7 ± 0.1 38.5 ± 0.1 37.8 ± 0.6 114.8 ± 0.4 67.2 ± 0.4 83.5 ± 0.4 37.7 ± 1.0 503.9 1.4 ± 0.2 1.0 ± 0.1 665.2 ± 3.6 27.9 ± 0.1 49.5 ± 0.2 15.8 ± 0.5 760.8 21.4 ± 0.9 1293.5 11566.8 ± 409.8
18.0 ± 0.5 120.3 ± 0.2 nd 33.3 ± 0.4 36.2 ± 0.0 116.5 ± 0.4 54.2 ± 0.3 97.6 ± 0.2 34.1 ± 0.4 492.2 0.9 ± 0.1 0.5 ± 0.1 563.5 ± 0.8 31.1 ± 0.8 26.6 ± 1.3 19.0 ± 1.0 641.6 26.3 ± 1.4 1178.0 13169.3 ± 190.6
All flavan-3-ol monomer and prodelphinidin oligomers were calculated as EGCG. nd, not detected. All flavonol glycosides were calculated as quercetin-3-O-glucoside. Extracted by 70% aqueous acetone, estimated by the Folin–Ciocalteu method and expressed as mg gallic acid/100 g dry leaves.
to leaves in the other two categories. However, the highest content of peak 3 ((E)GC-(E)GCG) was recorded in mature leaves of both cultivars, followed in that order by leaves of intermediate age and immature leaves. The results indicated that myricetin deoxyhexoside is the major constituent of all flavonol glycosides in the analysed bayberry leaves. Myricetin deoxyhexoside has also been detected in leaves of Myrica nana (Zhou & Yang, 2000) and in juice (M. rubra Sieb. et Zucc.) (Fang et al., 2007) and pomace (Zhou et al., 2009) of bayberry. We found immature leaves to be the richest and mature leaves to be the poorest source of this compound. Bayberry leaves were also rich in quercetin deoxyhexoside and myricetin deoxyhexoside-gallate (peak 15). Leaves of Biqi contained more myricetin hexoside (peak 10 and 11) and less myricetin deoxyhexosidegallate (peak 16) than those of Dongkui.
4. Conclusion The present work focused on identification and characterisation of low-molecular-weight phenolic compounds in bayberry leaves by using HPLC-DAD and HPLC-UV-ESI/MS. Two compounds (gallic acid and EGCG) were compared to the appropriate standards and identified positively; thirteen compounds were identified provisionally (three flavan-3-ol monomers, three prodelphinidin dimers, prodelphinidin trimer; two myricetin hexoside, two myricetin deoxyhexoside-gallate, and myricetin deoxyhexoside derivatives; quercetin deoxyhexoside derivative); and one compound remained unknown. Gallic acid was the only phenolic acid detected in bayberry leaves. Leaves of cultivar Biqi and Dongkui were rich in flavan-3-ols. Myricetin deoxyhexoside was the major flavonol glycoside in all leaves. Immature leaves of both cultivars showed the highest contents of total phenolics, whether determined by the Folin–Ciocalteu method or expressed as the sum of individual phenolic compounds. These results suggest that bayberry leaves are a useful source of phenolics and have the potential to contribute to human health.
Acknowledgment This project was supported by the Key Cultural Project of Innovation of Ministry Education (707034).
References Bao, J. S., Cai, Y. Z., Sun, M., Wang, G. Y., & Corke, H. (2005). Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and stability. Journal of Agricultural and Food Chemistry, 53, 2327–2332. Chen, K. S., Xu, C. J., Zhang, B., & Ferguson, I. B. (2004). Red bayberry: Botany and horticulture. Horticultural Reviews, 30, 83–114. Chen, R. R., & Liu, N. (1999). A study on inhibiting bacteria effects of Myrica nana cheval leaves, the folk medicine in Guizhou. Journal of Guizhou Normal University (Natural Science), 17, 30–32. Dueñas, M., Sun, B. S., Hernández, T., Estrella, I., & Spranger, M. I. (2003). Proanthocyanidin composition in the seed coat of lentils (Lens culinaris L.). Journal of Agricultural and Food Chemistry, 51, 7999–8004. Fang, Z. X., Zhang, M., & Wang, L. X. (2007). HPLC-DAD-ESIMS analysis of phenolic compounds in bayberries (Myria rubra Sieb. et Zucc.). Food Chemistry, 100, 845–852. Gu, H. F., Li, C. M., Xu, Y. J., Hu, W. F., Chen, M. H., & Wan, Q. H. (2008). Structural features and antioxidant activity of tannin from persimmon pulp. Food Research International, 41, 208–217. Huang, B., Ban, X. Q., He, J. S., Tong, J., & Wang, Y. W. (2010). Comparative analysis of essential oil components and antioxidant activity of extracts of Nelumbo nucifera from various areas of China. Journal of Agricultural and Food Chemistry, 58, 441–448. Hümmer, W., & Schreier, P. (2008). Analysis of proanthocyanidins. Molecular Nutrition & Food Research, 52, 1381–1398. Jiang, S. X., Dong, N. W., Zhang, J., Gan, C. L., & Liu, F. Z. (2008). Separation of myricetin and dihydromyricetin in Ampelopsis grossedentata and their inhibition of cadiomyocyte apoptosis. Journal of Harbin Medical University, 42, 4–10. Määttä, K. R., Kamal-Eldin, A., & Törrönen, A. R. (2003). High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: Ribes species. Journal of Agricultural and Food Chemistry, 51, 6736–6744. Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 81, 230S–242S. Matsuda, H., Morikawa, T., Tao, J., Ueda, K., & Yoshikawa, M. (2002). Bioactive constituents of Chinese natural medicine VII: inhibitors of degranulation in RBL-2H3 cells and absolute sterostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra. Chemical & Pharmaceutical Bulletin, 50, 208–215.
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135 Oszmianski, J., & Sapis, J. C. (1989). Fractionation and identification of some low molecular weight grape seed phenolics. Journal of Agricultural and Food Chemistry, 37, 1293–1297. Rashed, M. M., Shallan, M., Mohamed, D. A., Fouda, K., & Hanna, L. M. (2010). Hypolipidemic effect of vegetable and cereal dietary mixtures from Egyptian sources. Grasas Aceites, 61, 261–270. Rashmi, S., Nahid, A., & Tariq, M. H. (2010). Green tea polyphenol epigallocatechi3gallate: Inflammation and arthritis. Life Science, 86, 907–918. Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., & Kanazawa, K. (2003). Simultaneous determination of all polyphenols in vegetables, fruits, and teas. Journal of Agricultural and Food Chemistry, 51, 571–581. Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolics and flavonoids of apple and pear by high-performance liquid chromatography. Journal of Chromatography, A, 910, 265–273. Shen, J. G., Xie, L. Y., Zhai, M. Z., Lin, Q. Y., & Xie, L. H. (2004). Antiviral activity and chemical compositions of extracts from Myrica rubra. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 33, 441–443. Shukla, S., Mehta, A., Bajpai, V. K., & Shukla, S. (2009). In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert. Food and Chemical Toxicology, 47, 2338–2343. Tomas-Barberan, F. A., & Clifford, M. N. (2000). Dietary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80, 1024–1032. Vattem, D. A., Lin, Y.-T., & Shetty, L. K. (2004). Antimicrobial activity against select food-borne pathogens by phenolic antioxidants enriched in cranberry pomace by solid-state bioprocessing using the food grade fungus Rhizopus oligosporus. Process Biochemistry, 39, 1939–1946.
1135
Wang, D. Y., Liu, E. G., & Feng, Y. J. (2008). Study on the flavonoid constituents from the bark of Myrica rubra. Lishizhen medicine and material medical research, 19, 1149–1150. Wu, Q. L., Wang, M. F., & Simon, J. E. (2003). Determination of proanthocyanidins in grape products by liquid chromatography/mass spectrometric detection under low collision energy. Analytical Chemistry, 75, 2440– 2444. Xia, Q. L., Chen, J. C., & Wu, D. (2004). Study on scavenging efficacy of phenols extraction of Myrica rubra leaf. Food Science, 25, 80–83. Xu, G. H., Liu, D. H., Chen, J. C., Ye, X. Q., Ma, Y. Q., & Shi, J. (2008). Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chemistry, 106, 545–551. Yang, L. L., Chang, C. C., Chen, L. G., & Wang, C. C. (2003). Antitumor principle constituents of Myria rubra Var Acuminata. Journal of Agricultural and Food Chemistry, 51, 2974–2979. Yesil-Celiktas, O., Sevimli, C., Bedir, E., & Vardar-Sukan, F. (2010). Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer cell lines. Plant Foods for Human Nutrition, 65, 158–163. Zhou, S. H., Fang, Z. X., Lü, Y., Chen, J. C., Liu, D. H., & Ye, X. Q. (2009). Phenolics and antioxidant properties of bayberry (Myria rubra Sieb et Zucc.) pomace. Food Chemistry, 112, 394–399. Zou, Y. H., & Li, G. R. (1998). Analysis of flavonoid as antioxidant in Myrica rubra Leaf with reversed-phase high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 26, 531–534. Zhou, Z. H., & Yang, C. R. (2000). Phenolic constituents of the fresh leaves of Myrica nana. Acta Botanica Yunnanica, 22, 219–224.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Identification and characterisation of low-molecular-weight phenolic compounds in bayberry (Myrica rubra Sieb. et Zucc.) leaves by HPLC-DAD and HPLC-UV-ESIMS Haihua Yang a, Yiqiang Ge b, Yujing Sun a, Donghong Liu a, Xingqian Ye a,⇑, Dan Wu a a b
Department of Food Science and Nutrition, Zhejiang University, KaiXuan Road 268#, Hangzhou, CN 310029, PR China China Rural Technology Development Centre, SanLiHe Road 54#, Beijing, CN 100045, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2010 Received in revised form 19 February 2011 Accepted 30 March 2011 Available online 5 April 2011 Keywords: Bayberry Leaves Phenolic compounds HPLC-DAD HPLC-UV-ESIMS
a b s t r a c t Leaves of each of two bayberry cultivars, Biqi and Dongkui, were divided into three categories by age, namely immature, intermediate, and mature. Phenolic compounds were analysed by the methods of HPLC-DAD and HPLC-UV-ESIMS. Gallic acid and EGCG were identified positively, and 13 other compounds (flavan-3-ol monomers, prodelphinidin oligomers, and flavonol glycosides) were partially identified. Gallic acid (7.5–87.8 mg/100 g) was the only phenolic acid detected and flavan-3-ols were abundant. Myricetin deoxyhexoside (535.4–853.0 mg/100 g) was the major flavonol glycoside. Among the three categories, immature leaves of both cultivars recorded the highest level of total phenolics, irrespective of whether they were measured by the Folin–Ciocalteu method (19404.0 mg/100 g in Biqi and 19626.0 mg/100 g in Dongkui) or as the sum of individual phenolic compounds (2255.9 mg/100 g in Biqi and 1797.1 mg/100 g in Dongkui). The results showed that bayberry leaves are a potentially rich source of beneficial phenolics. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Bayberry (Myrica rubra Sieb. et Zucc.), a tree species of the family Myricaceae, has been cultivated mainly in southern China for more than 2000 years. Bayberry is also native to other countries of eastern Asia (Chen, Xu, Zhang, & Ferguson, 2004). Bayberry fruits, with their exquisite taste, flavour, and attractive red colour, are popular among local people. Bayberry is used extensively in folk medicine for a variety of ailments. Bayberry leaves, twigs, and bark have therapeutic properties and the plant has been in popular use as a traditional medicine for a long time. Herbalists recommend a warm concoction made from the root bark of bayberry as a tonic that acts as a stimulant and as an astringent. Bayberry’s astringency is believed to help in treating intestinal disorders such as the irritable bowel syndrome and mucous colitis; a paste made from powdered root bark is applied to ulcers and sores; and its infusion is even alleged to control excessive vaginal discharge. Bayberry is especially effective in the treatment of diarrhoea and, because it acts as a stomach irritant when used in large doses, bayberry bark acts as an emetic. During head colds, bayberry increases secretion of nasal mucus. When applied as a poultice, bayberry is believed to be useful in treating chronic indolent ulcers (Matsuda, Morikawa, Tao, Ueda, & Yoshikawa, 2002). Bayberry trees flush two or three times a year. Foliar growth is luxuriant and leaves remain green throughout the year. The tree ⇑ Corresponding author. Tel./fax: +86 571 86971165. E-mail addresses: [email protected], [email protected] (X. Ye). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.118
needs to be pruned every year more than once to get high fruit production. Pruning yields a mass of leaves, which are generally discarded and remain underutilized despite reports that extracts of bayberry leaves are anti-oxidative (Xia, Chen, & Wu, 2004), antimicrobial (Chen & Liu, 1999), and antiviral (Shen, Xie, Zhai, Lin, & Xie, 2004). Recent chemical investigations have brought light to several interesting chemical compounds in bayberry, of which polyphenols form a major category. Plant phenolics are aromatic secondary metabolites that are ubiquitous in the plant kingdom and comprise more than 8000 substances with highly diverse structures and molecular masses. Plant phenolics can be subdivided into classes such as phenolic acids, flavanones, flavones, flavonols, anthocyanidins, flavan-3-ols, and proanthocyanidins (Hümmer & Schreier, 2008). A substantial body of literature suggests that phenolic extracts from plants are able to prevent diseases including cancer by anti-oxidative action (Yesil-Celiktas, Sevimli, Bedir, & Vardar-Sukan, 2010) and cardiovascular diseases (Rashed, Shallan, Mohamed, Fouda, & Hanna, 2010). The polyphenols from green tea have been reported to have positive effects on anti-inflammation and anti-arthritis activities (Rashmi, Nahid, & Tariq, 2010). These polyphenolic anti-oxidants, because they have anti-microbial properties, can also be used to preserve foods (Vattem, Lin, & Shetty, 2004). However, the characterisation of phenolic compounds in bayberry has received little attention. High-performance liquid chromatography (HPLC) is the most common analytical technique for separating plant phenolics; HPLC coupled with a photodiode-array
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
detector (HPLC-DAD) provides extensive information on the structure of polyphenols in crude or partly purified plant extracts and, coupled with mass spectrometry (HPLC-MS), information that serves as a powerful aid to identify the structure. Electrospray ionisation (ESI) is a gentle, sensitive, and the most widely used method for generating ions to date (Hümmer & Schreier, 2008). The UV–vis absorption spectra of phenolic compounds enable identification of chromatographic peaks and their classification. A combination of these data with those on mass spectra and information from the literature can be used to tentatively identify the structure of polyphenols. This paper reports the results of qualitative and quantitative analysis of low molecular weight phenolics from bayberry leaves of different ages using HPLC-DAD and HPLC-UV-ESIMS. Knowledge of the precise phenolic profile of bayberry leaves may offer a scientific basis to put the underutilized bayberry leaves to good use.
1129
2.4. Extraction of phenolic compounds
2. Materials and methods
Phenolic compounds were extracted from 1 g samples of bayberry leaf powder with aqueous acetone as described above. The phenolic compounds were isolated by a method published earlier (Oszmianski & Sapis, 1989). The solvent containing the extract was treated with chloroform (15 ml) to remove acetone and compounds other than phenolics. The aqueous phase containing the phenolic compounds was recovered. Crystallized NaCl was then added until saturation point to precipitate the more polymerised tannins, which made it possible to analyse phenolics of low molecular weight. The solution was filtered and the filtrate mixed with ethyl acetate (3 20 ml). The ethyl acetate phase (60 ml) was washed with 10 ml water, concentrated under vacuum to 2 ml, and chloroform (10 ml) was added to precipitate phenolics. The suspension was filtered, the filtrate was discarded, and the residue was re-dissolved in 5 ml methanol for analysis by HPLC-DAD and HPLC-UV-ESIMS. All samples were prepared and analysed in triplicate.
2.1. Chemicals
2.5. HPLC-DAD and HPLC-UV-ESIMS analysis
Standards of gallic acid, ( )-epigallocatechin-3-O-gallate (EGCG), quercetin 3-O-glucose-rhamnoside, and Folin–Ciocalteu phenol reagent were obtained in the form of commercial samples from Sigma (St. Louis, Missouri, USA). Acetonitrile was HPLC grade. All other reagents and solvents used were of analytical grade. All solutions were prepared using distilled-deionized water.
Bayberry leaves of two cultivars, Biqi and Dongkui, growing in Xianju in Zhejiang province in south-eastern China were harvested by hand from randomly chosen plants on 6 July, 2009. Leaves of each cultivar were divided on the basis of age into three categories, namely immature, intermediate, and mature. The age of a leaf can be estimated conventionally by colour: immature leaves are bright green; mature leaves are dull and dark green; and the green of intermediate leaves is deeper than that of immature leaves but lighter than that of mature leaves. All the collected leaves were washed and then dried under vacuum at 40 °C for 12 h, ground fine, and stored at 20 °C until required.
Phenolic compounds were analysed with HPLC-DAD using an Alliance 2695 separation module (Waters) linked simultaneously to a Waters 2998 photodiode array detector (Waters). HPLC-UVESIMS analysis was performed on a Waters platform, including a Waters 1525 pump, a Waters 2487 Dual k absorbance detector, and an Esquire 3000 Pius (Bruker). Mass spectra were achieved by electrospray ionisation in the negative mode. All samples were filtered through a 0.45 lm Millipore membrane filter before injection, and a 20 ll aliquot was separated by a Zorbax SB-C18 (Agilent, USA) column (250 4.6 mm, 5 lm) protected by a guard column of the same material. The column thermostat was maintained at 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in water, v/v) and solvent B (acetonitrile). The elution profile consisted of a linear gradient comprising isocratic 10% B (5 min), 10–30% B (5–35 min), and 30–90% B (35–40 min) with a flow rate of 0.7 ml/min. After each run, the column was washed with 100% acetonitrile and equilibrated to initial conditions for 15 min. UV–Vis absorption spectra were recorded online during the HPLC analysis. The DAD detector was set to a scanning range of 200–800 nm. Phenolic acid and flavan-3-ol monomers were detected at 280 nm and flavones at 360 nm.
2.3. Determination of total phenolics
2.6. Identification and quantification of phenolic compounds
Total phenolics were extracted from samples (1 g each) of bayberry leaf powder by mixing them with 10 ml of 70% aqueous acetone for 15 min at room temperature. The process was repeated three times. After centrifuging at 2000 rpm for 10 min, the supernatant was rotary-evaporated under vacuum at 40 °C to remove the acetone and the aqueous phase diluted to 1000 ml with methanol. All samples were prepared and processed in triplicate. Total polyphenol content was determined by the Folin–Ciocalteu method (Xu et al., 2008). One millilitre of the prepared sample solution was added to a 25 ml volumetric flask filled with 9 ml distilled water. A blank sample was prepared using distilleddeionized water. Folin–Ciocalteu phenol reagent (0.5 ml) was added to the mixture and the mixture was shaken vigorously for 5 min; 5 ml of Na2CO3 solution was added to the mixture; the resulting mixture was immediately diluted to 25 ml with distilled water and mixed thoroughly; and then it was allowed to stand for 60 min before measuring its absorbance at 750 nm. The absorbance was compared with that of the prepared blank. Total polyphenol contents were expressed as milligrams per 100 g of gallic acid equivalent.
The phenolic compounds were identified mainly by their UV–Vis spectra and ESIMS spectra and by comparing with published data. If relevant standards were available, they were also identified by comparing the chromatography with the authentic standards. Gallic acid and EGCG were quantified by injecting a solution with known concentrations of standards and calculating with regression equations from the standard curves. Contents of other flavan-3-ol monomers and flavones were expressed as EGCG and quercetin 3-O-glucose-rhamnoside equivalents, respectively. The concentrations of all compounds were expressed in milligrams per 100 g of dried leaves (dry weight basis, or DW). The values were expressed as mean ± standard deviations of three replications.
2.2. Plant material
3. Results and discussion 3.1. Identification of phenolic compounds Typical HPLC profiles of Biqi and Dongkui bayberry leaves are shown in Figs. 1 and 2, respectively, and the corresponding
1130
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Fig. 1. Typical HPLC chromatogram of phenolic extract of Biqi cultivar bayberry leaves detected at 280 nm (A) and 360 nm (B). Peak numbers refer to Table 1.
UV–Vis spectra for each peak are presented in Fig. 3. Sixteen peaks (peaks 1–16, Figs. 1A and 2A) were detected at a wavelength of 280 nm and six peaks (peaks 10, 11, and 13–16, Figs. 1B and 2B) at 360 nm. These peaks in the chromatograms were classified into three groups, namely phenolic acids, flavan-3-ols, and flavonol glycosides, by comparing their UV–Vis spectra and HPLC retention times with those of the available standards. The ESIMS spectral data, UV–Vis spectral data, and tentative identification of the 16 compounds from Biqi and Dongkui bayberry leaves are shown in Table 1. 3.1.1. Phenolic acid (peak 1) The first group (phenolic acids) contained one component as indicated by peak 1 with HPLC retention time at 5.63 min. Peak 1 presented the UV kmax at 215.6 and 271.1, was symmetrical from 240 to 305 nm (with maximum symmetry around 271.1 nm), and then shifted to 325 nm. ESIMS spectra showed the molecular ion [M–H] at m/z 168.8 and a fragment ion at m/z 124.9, which corresponds to the loss of a carboxyl residue (mass unit 45) from the molecular ion. The identity of the component in peak 1 was also confirmed by adding minute amounts of gallic acid to the phenolic extract: the peaks of endogenous gallic acid in the sample and of the additional gallic acid superimposed perfectly on each other, and peak 1 was unambiguously identified as that of gallic acid. 3.1.2. Flavan-3-ols (peak 3–9, and 12) Several well-resolved chromatographic peaks of flavan-3-ols were observed under the experimental conditions mentioned earlier, which were used for HPLC-DAD and HPLC-UV-ESIMS analysis. This group of compounds corresponded to peaks 3–9 and peak 12
and was provisionally identified as comprising of four flavan-3-ol monomers, three prodelphinidin dimers, and one trimer. The dimers and trimers were all considered to be of B type based on their mass spectra. In the mass spectra of flavan-3-ol monomers, peaks 4, 5, and 9 exhibited their protonated molecular ion [M–H] at m/z 467.1, their duple molecular ion [2 M–H] at m/z 935.4, and a fragment ion at m/z 305.0, pointing to a hexosyl residue that was included in the molecule structure. Furthermore, the three peaks had almost the same UV–Vis spectrum shape with a kmax at 270 nm and a shoulder from 315 to 360 nm. Therefore, this compound was provisionally identified as (E)GC-hexoside (molecular weight = 468). Although glycosides of flavan-3-ol monomers are quite rare in plants and have never been detected in bayberry leaves so far, a previous identification of (+)-catechin 3-O-glucoside in the seed coat of lentil (Lens culinaris L.) supports the identification of compounds that corresponded to peaks 4, 5, and 9 (Dueñas, Sun, Hernández, Estrella, & Spranger, 2003). Peak 8 with a HPLC retention time of 19.59 min showed kmax at 212.0 nm and 273.5 nm, symmetry around the maximum from 258 to 290 nm, and a shoulder from 290 to 325 nm, which is typical of galloyl groups. Peak 8 also gave a molecular ion [M–H] at m/z 457.1, a duple molecular ion [2 M–H] at 915.1, and a fragment ion at m/z 304.8 corresponding to loss of a galloyl residue, and a fragment ion at m/z 168.8 (the gallic acid fragment), which indicated peak 8 to be EGCG or GCG. By comparing with the standard, this compound was positively identified as EGCG. To identify the structure of peak 3, a structure was hypothesised based on the product ions detected by ESIMS with EGC as the extension unit and EGCG as the terminal unit. Three characteristic
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
1131
Fig. 2. Typical HPLC chromatogram of phenolic extract of Dongkui cultivar bayberry leaves detected at 280 nm (A) and 360 nm (B). Peak numbers refer to Table 1.
fragmentation routes have been reported for proanthocyanidins, namely retro-Diels–Alder fission (RDA), quinone methide fission (QM), and heterocyclic ring fission (HRF) (Wu, Wang, & Simon, 2003), of which RDA was found to be the most important for elucidating the structure of dimers and to lead to the loss of ring B in the extension unit (Wu et al., 2003). Peak 3, eluting at 11.97 min, showed a molecular ion [M–H] at m/z 761.3 with fragment ions at m/z 591.2 and 423.1. The two fragment ions corresponded respectively to further loss of O-galloyl (Gu et al., 2008) and of the RDA product ion (m/z 168) (Wu et al., 2003). Based on these results, we suggested that EGC was the extension unit and EGCG was the terminal unit. Since product ions derived from fragmentation can be used only for determining the sequence of monomeric units and not for distinguishing between epimers, peak 8 was identified as (E)GC-(E)GCG. Peak 6, eluting at 16.58 min, showed a molecular ion [M–H] at m/z 1217.4 with two fragment ions at m/z 1065.3 and 913.4, which corresponded to the loss of one galloyl residue ([M–H-152] ) and two galloyl residues ([M–H-152-152] ) (Gu et al., 2008), respectively. Because there were no more fragment ions, especially the most important fragment ions to characterise trimeric proanthocyanidins produced by the cleavage of interflavanoid linkages (Gu et al., 2008), the structure of this compound could not be determined with certainty, and peak 6 was provisionally identified as a prodelphinidin trimer consisting of two (E)GCG units and one (E)GC unit. Peaks 7 and 12, the other two prodelphinidin dimers consisting of two (E)GCG units, had the same fragment ions: a molecular ion [M–H] at m/z 913.3 with two fragment ions at m/z 743.2 and
573.2, corresponding to further loss of one O-galloyl and two Ogalloyls, respectively. 3.1.3. Flavonols (peak 10, 11, and 13–16) The third group of compounds corresponded to peaks 10, 11, and 13–16. These compounds were generally eluted later than the other two groups of compounds and displayed UV–Vis spectra similar to those of rutin (quercetin 3-O-glucose-rhamnoside) with two absorption maxima at 254 and 354 nm. It is well known that flavonols exhibit two major absorption peaks in the region of 240–400 nm, in which the region of 240–280 nm (Band II) is considered to be associated with absorption due to the A-ring benzoyl system, and the region of 300–380 nm (Band I) with that due to the B-ring cinnamoyl system. Therefore, we supposed that compounds in the third group were flavonols. Peaks 10, 11, and 13 had molecular ions [M–H] at m/z 479.1, 479.1, and 463.1, respectively. All the three peaks also had a fragment ion at m/z 316.8, which is a typical mass in the negative mode of myricetin aglycone, indicating that these three compounds were myricetin derivatives. A fragment ion at m/z 927.0 of peak 13 was the double-charge molecular ion [2 M–H] . It has been reported that sugars bound to the aglycons are hexoses with a mass unit of 162, deoxyhexoses with a mass unit of 146, and pentoses with a mass unit of 132 (Määttä, Kamal-Eldin, & Törrönen, 2003). The ESIMS spectra of the three peaks indicated that their structure contained a hexosyl residue (479.1 316.8 = 162.3) or a deoxyhexosyl residue (463.1 316.8 = 146.3). Peaks 10 and 11 were therefore identified as those of myricetin hexoside and peak 13 as that of myricetin deoxyhexoside. However, peaks 10 and 11
1132
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Fig. 3. On-line UV–visible spectra of the phenolic compounds. Peak numbers correspond to those in Figs. 1 and 2 and Table 1.
had a different stereochemistry structure, and the position where these flavonols were substituted needs further study. The UV spectra of peaks 15 and 16 were very similar to those of peak 13. However, peaks 15 and 16 showed a shift of the maximum of Band II from 262 to 266.2 nm compared to peak 13. The excess of acyl
formed with hydroxybenzoic acid in peaks 15 and 16 compared to peak 13 was responsible for this shift, as has been reported earlier (Fang, Zhang, & Wang, 2007). The ESIMS data of peaks 15 and 16 revealed a molecular ion [M–H] at m/z 615.3 and a fragment ion at m/z 316.8, indicating that they were both
1133
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Table 1 Identification of individual phenolic compounds in phenolic extracts from bayberry leaves by using their UV–visible spectral characteristics, negative ions in HPLC-UV-ESIMS, and respective standards.
a
Peak No.
HPLC tR (min)
HPLC-DAD (nm)
Molecular weight
HPLC-ESIMS (m/z)
Tentative identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
5.63 8.18 11.97 13.84 15.11 16.58 17.19 19.59 21.65 22.71 23.14 24.81 25.96 30.43 33.57 34.28
216.7, 271.1 225sh, 274.5 272.3 214.4, 271.1 214.4, 271.1 272.3 273.5 212.0, 273.5 214.4, 271.1 267.6, 300sh, 355.8 269.9, 300sh, 359.9 273.5 260.4, 300sh, 349.8 255.7, 264sh, 348.6 210.9, 265.2, 295sh, 349.8 210.9, 266.2, 295sh, 349.8
170 474 762 468 468 1218 914 458 468 480 480 914 464 448 616 616
168.8, 124.9 947.0, 473.1, 318.8, 152.7 761.3, 591.2, 423.1 935.4, 467.1 935.4, 467.1 1217.4, 1065.3, 913.4 913.4, 743.2, 573.2 915.1, 457.1,304.8168.8 935.4, 467.1 479.1, 316.8 479.1, 316.8 913.3, 743.2, 573.2 927.0, 463.1, 316.8 447.1, 300.8 615.3, 316.8 615.3, 316.8
Gallic acid (stda) (Unidentified compound) (E)GC-(E)GCG (E)GC-hexoside (E)GC-hexoside 2(E)GCG + (E)GC 2(E)GCG EGCG (std) (E)GC-hexoside Myricetin hexoside Myricetin hexoside 2(E)GCG Myricetin deoxyhexoside Quercetin deoxyhexoside Myricetin deoxyhexoside-gallate Myricetin deoxyhexoside-gallate
The compound was also identified by comparing the chromatography with the authentic standards.
myricetin deoxyhexoside derivatives. Finally, peaks 15 and 16 were provisionally identified as myricetin deoxyhexoside-gallate (615.3 463 = 152.3, loss of a galloyl residue). UV spectra of peak 14 presented the same profile as that of quercetin deoxyhexoside published earlier (Sakakibara, Honda, Nakagawa, Ashida, & Kanazawa, 2003). Peak 14 also had a molecular ion [M–H] at m/z 447.1 and a fragment ion of m/z at 300.8, which indicated that a deoxyhexosyl residue had been attached to the quercetin aglycone, and was therefore identified as quercetin deoxyhexoside. This compound was detected also in bayberry fruit (Bao, Cai, Sun, Wang, & Corke, 2005; Fang et al., 2007) and bark (Wang, Liu, & Feng, 2008). Peak 2 showed a HPLC retention time of 8.18 min and also gave a UV kmax at 275.9 nm, a shoulder at 225 nm, and a very broad band shifted to 325 nm. The ESIMS spectra showed that peak 2 had a molecular ion [M–H] at m/z 473.1 and fragment ions at m/z 318.8 and 152.7. These data on the mass spectra indicated that peak 2 is a dihydromyricetin derivative. Dihydromyricetin is reported to occur in Ampelopsis grossedentata (Jiang, Dong, Zhang, Gan, & Liu, 2008). However, we could find no report of dihydromyricetin derivatives and therefore had to leave peak 2 unidentified. Isolation and NMR identification might be required to identify this compound. Chromatogram profiles of the phenolic extracts of leaves of cultivars Biqi and Dongkui were similar (Figs. 1 and 2). (E)GC-hexoside (peak 4) was not detected in mature leaves of Biqi and Dongkui; myricetin deoxyhexoside was the major phenolic compound in the phenolic extracts of leaves of Biqi and Dongkui. EGCG, a widely distributed flavan-3-ol monomer in tea leaves, was detected in leaves of bayberry (M. rubra Sieb. et Zucc.) and M. rubra var. acuminata (Yang, Chang, Chen, & Wang, 2003) although it was found neither in bayberry fruit (Fang et al., 2007; Zhou et al., 2009) nor in leaves of another unknown cultivar of bayberry (Zou & Li, 1998). Protocatechuic acid, quercetin 3-glucoside, quercetin hexosidegallate, and kaempferol hexoside were detected in bayberry fruit (Fang et al., 2007; Zhou et al., 2009), myricetin and quercetin3-O-b-D-glucoside were detected in leaves of an unknown cultivar of bayberry (Zou & Li, 1998) but the present experiment did not find all these compounds in bayberry leaves. 3.2. Quantitative analysis of total phenolics and individual phenolic compounds In the present study, the contents of total phenolics and of individual phenolics were determined by the Folin–Ciocalteu method and by HPLC-DAD, respectively. Table 2 presents the results.
Total phenolics in leaves of cultivars Biqi and Dongkui, on a dry weight basis, were 8137.5–19404.0 mg/100 g and 11566.8– 19626.0 mg/100 g, respectively. These values are intermediate between those for Nelumbo nucifera leaves (354–487 mg/g) (Huang, Ban, He, Tong, & Wang, 2010) and Stevia rebaudiana Bert. (61.5 mg/g) (Shukla, Mehta, Bajpai, & Shukla, 2009) but higher than those for bayberry pomace (27.4–47.4 mg/g) (Zhou et al., 2009). These observations suggest that bayberry leaves are rich in phenolics and have good potential as value-added products. Furthermore, immature leaves of Biqi showed the highest content of total phenolics (19404.0 mg/100 g) among all the samples, followed in that order by mature leaves (11715.5 mg/100 g DW) and intermediate leaves (8137.5 mg/100 g DW). Leaves of cultivar Dongkui showed the same pattern (Table 2), which indicated that choosing leaves of the right age is an important factor in the utilisation bayberry leaves. Immature leaves of both cultivars also had the highest content of phenolics when expressed as the sum of individual phenolic compounds (2255.9 mg/100 g in Biqi and 1797.1 mg/g in Dongkui) whereas mature leaves showed the lowest values, and leaves of intermediate age showed values of phenolics that were intermediate. The contents of phenolics expressed as the sum of individual phenolic compounds were lower than those determined by the Folin–Ciocalteu method. This difference is understandable because the Folin–Ciocalteu method tends to overestimate the contents of total phenolics since it ignores potential interference by other substances (Schieber, Keller, & Carle, 2001). Besides, there might be high-molecular-weight phenolic compounds in bayberry leaves that did not include the sum of individual phenolic compounds. Gallic acid was the only phenolic acid detected in bayberry leaves with the analytical method used in the present study. High amounts of gallic acid were noted in immature leaves of Dongkui and in leaves of the intermediate age of Biqi. The lowest value of gallic acid was recorded in leaves of the intermediate age of Dongkui. The contents of gallic acid in bayberry leaves (7.50–87.82 mg/100 g) were higher than those in bayberry pomace (2.81–9.23 mg/100 g) (Zhou et al., 2009). Compared to other polyphenols, gallic acid is very easily absorbed into the human body (Manach et al., 2005), and gallic acid was shown to have anticancer effect in vitro (Tomas-Barberan & Clifford, 2000). All flavan-3-ols and oligomeric prodelphinidins identified above were present in the leaves of all ages except that peak 4 ((E)GChexoside) was not detected in mature leaves of either cultivar. However, the leaves differed in their contents of individual constituents: in general, immature leaves of both cultivars were richer in individual flavan-3-ols and oligomeric prodelphinidins compared
1134
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135
Table 2 Contents of total and individual phenolic compounds (mg/100 g DW) in different bayberry leaves. Peak No.
Phenolic compounds
1 8 4 5 9 3 6 7 12
Gallic acid EGCG (E)GC-hexosidea (E)GC-hexoside (E)GC-hexoside (E)GC (E)GCG 2(E)GCG + (E)GC 2(E)GCG 2(E)GCG Total Myricetin hexosidec Myricetin hexoside Myricetin deoxyhexoside Quercetin deoxyhexoside Myricetin deoxyhexoside-gallate Myricetin deoxyhexoside-gallate Total (Unidentified compound) Sum of phenolic compounds Total phenolicsd
10 11 13 14 15 16 2
a b c d
Biqi
Dongkui
Immature
Intermediate
Mature
Immature
Intermediate
Mature
18.8 ± 0.8 228.3 ± 2.4 194.3 ± 3.1 239.2 ± 8.0 183.4 ± 6.4 76.7 ± 1.0 107.8 ± 1.0 140.3 ± 1.1 110.1 ± 0.5 1280.1 12.4 ± 0.3 7.0 ± 0.1 765.2 ± 10.5 11.3 ± 0.6 96.8 ± 3.7 7.9 ± 0.1 900.6 56.4 ± 1.6 2255.8 19404.0 ± 410.6
50.8 ± 3.7 90.7 ± 5.0 30.2 ± 0.6 45.5 ± 3.0 49.7 ± 0.4 88.2 ± 0.7 71.7 ± 0.3 102.5 ± 0.6 52.7 ± 0.9 531.2 40.0 ± 1.0 19.6 ± 0.1 583.9 ± 10.3 21.2 ± 0.3 16.1 ± 0.6 2.4 ± 0.6 683.2 30.2 ± 0.1 1295.4 8137.5 ± 42.7
27.8 ± 0.6 97.9 ± 1.4 ndb 32.6 ± 0.9 37.5 ± 0.3 93.8 ± 0.4 43.9 ± 0.1 87.9 ± 0.9 35.0 ± 0.7 428.6 43.0 ± 1.8 16.9 ± 0.4 535.4 ± 11.9 32.9 ± 1.0 12.1 ± 1.0 8.3 ± 0.5 648.6 28.3 ± 0.7 1133.1 11715.5 ± 45.3
87.8 ± 1.6 134.1 ± 7.1 33.9 ± 1.0 55.3 ± 0.3 81.1 ± 3.6 87.2 ± 0.1 77.2 ± 0.8 111.6 ± 1.4 83.9 ± 0.5 664.3 3.1 ± 0.1 2.9 ± 0.1 853.0 ± 9.3 30.3 ± 0.4 110.3 ± 5.1 15.1 ± 0.6 1014.7 30.5 ± 0.1 1797.1 19626.0 ± 325.9
7.5 ± 0.1 103.7 ± 1.5 20.7 ± 0.1 38.5 ± 0.1 37.8 ± 0.6 114.8 ± 0.4 67.2 ± 0.4 83.5 ± 0.4 37.7 ± 1.0 503.9 1.4 ± 0.2 1.0 ± 0.1 665.2 ± 3.6 27.9 ± 0.1 49.5 ± 0.2 15.8 ± 0.5 760.8 21.4 ± 0.9 1293.5 11566.8 ± 409.8
18.0 ± 0.5 120.3 ± 0.2 nd 33.3 ± 0.4 36.2 ± 0.0 116.5 ± 0.4 54.2 ± 0.3 97.6 ± 0.2 34.1 ± 0.4 492.2 0.9 ± 0.1 0.5 ± 0.1 563.5 ± 0.8 31.1 ± 0.8 26.6 ± 1.3 19.0 ± 1.0 641.6 26.3 ± 1.4 1178.0 13169.3 ± 190.6
All flavan-3-ol monomer and prodelphinidin oligomers were calculated as EGCG. nd, not detected. All flavonol glycosides were calculated as quercetin-3-O-glucoside. Extracted by 70% aqueous acetone, estimated by the Folin–Ciocalteu method and expressed as mg gallic acid/100 g dry leaves.
to leaves in the other two categories. However, the highest content of peak 3 ((E)GC-(E)GCG) was recorded in mature leaves of both cultivars, followed in that order by leaves of intermediate age and immature leaves. The results indicated that myricetin deoxyhexoside is the major constituent of all flavonol glycosides in the analysed bayberry leaves. Myricetin deoxyhexoside has also been detected in leaves of Myrica nana (Zhou & Yang, 2000) and in juice (M. rubra Sieb. et Zucc.) (Fang et al., 2007) and pomace (Zhou et al., 2009) of bayberry. We found immature leaves to be the richest and mature leaves to be the poorest source of this compound. Bayberry leaves were also rich in quercetin deoxyhexoside and myricetin deoxyhexoside-gallate (peak 15). Leaves of Biqi contained more myricetin hexoside (peak 10 and 11) and less myricetin deoxyhexosidegallate (peak 16) than those of Dongkui.
4. Conclusion The present work focused on identification and characterisation of low-molecular-weight phenolic compounds in bayberry leaves by using HPLC-DAD and HPLC-UV-ESI/MS. Two compounds (gallic acid and EGCG) were compared to the appropriate standards and identified positively; thirteen compounds were identified provisionally (three flavan-3-ol monomers, three prodelphinidin dimers, prodelphinidin trimer; two myricetin hexoside, two myricetin deoxyhexoside-gallate, and myricetin deoxyhexoside derivatives; quercetin deoxyhexoside derivative); and one compound remained unknown. Gallic acid was the only phenolic acid detected in bayberry leaves. Leaves of cultivar Biqi and Dongkui were rich in flavan-3-ols. Myricetin deoxyhexoside was the major flavonol glycoside in all leaves. Immature leaves of both cultivars showed the highest contents of total phenolics, whether determined by the Folin–Ciocalteu method or expressed as the sum of individual phenolic compounds. These results suggest that bayberry leaves are a useful source of phenolics and have the potential to contribute to human health.
Acknowledgment This project was supported by the Key Cultural Project of Innovation of Ministry Education (707034).
References Bao, J. S., Cai, Y. Z., Sun, M., Wang, G. Y., & Corke, H. (2005). Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and stability. Journal of Agricultural and Food Chemistry, 53, 2327–2332. Chen, K. S., Xu, C. J., Zhang, B., & Ferguson, I. B. (2004). Red bayberry: Botany and horticulture. Horticultural Reviews, 30, 83–114. Chen, R. R., & Liu, N. (1999). A study on inhibiting bacteria effects of Myrica nana cheval leaves, the folk medicine in Guizhou. Journal of Guizhou Normal University (Natural Science), 17, 30–32. Dueñas, M., Sun, B. S., Hernández, T., Estrella, I., & Spranger, M. I. (2003). Proanthocyanidin composition in the seed coat of lentils (Lens culinaris L.). Journal of Agricultural and Food Chemistry, 51, 7999–8004. Fang, Z. X., Zhang, M., & Wang, L. X. (2007). HPLC-DAD-ESIMS analysis of phenolic compounds in bayberries (Myria rubra Sieb. et Zucc.). Food Chemistry, 100, 845–852. Gu, H. F., Li, C. M., Xu, Y. J., Hu, W. F., Chen, M. H., & Wan, Q. H. (2008). Structural features and antioxidant activity of tannin from persimmon pulp. Food Research International, 41, 208–217. Huang, B., Ban, X. Q., He, J. S., Tong, J., & Wang, Y. W. (2010). Comparative analysis of essential oil components and antioxidant activity of extracts of Nelumbo nucifera from various areas of China. Journal of Agricultural and Food Chemistry, 58, 441–448. Hümmer, W., & Schreier, P. (2008). Analysis of proanthocyanidins. Molecular Nutrition & Food Research, 52, 1381–1398. Jiang, S. X., Dong, N. W., Zhang, J., Gan, C. L., & Liu, F. Z. (2008). Separation of myricetin and dihydromyricetin in Ampelopsis grossedentata and their inhibition of cadiomyocyte apoptosis. Journal of Harbin Medical University, 42, 4–10. Määttä, K. R., Kamal-Eldin, A., & Törrönen, A. R. (2003). High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: Ribes species. Journal of Agricultural and Food Chemistry, 51, 6736–6744. Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 81, 230S–242S. Matsuda, H., Morikawa, T., Tao, J., Ueda, K., & Yoshikawa, M. (2002). Bioactive constituents of Chinese natural medicine VII: inhibitors of degranulation in RBL-2H3 cells and absolute sterostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra. Chemical & Pharmaceutical Bulletin, 50, 208–215.
H. Yang et al. / Food Chemistry 128 (2011) 1128–1135 Oszmianski, J., & Sapis, J. C. (1989). Fractionation and identification of some low molecular weight grape seed phenolics. Journal of Agricultural and Food Chemistry, 37, 1293–1297. Rashed, M. M., Shallan, M., Mohamed, D. A., Fouda, K., & Hanna, L. M. (2010). Hypolipidemic effect of vegetable and cereal dietary mixtures from Egyptian sources. Grasas Aceites, 61, 261–270. Rashmi, S., Nahid, A., & Tariq, M. H. (2010). Green tea polyphenol epigallocatechi3gallate: Inflammation and arthritis. Life Science, 86, 907–918. Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., & Kanazawa, K. (2003). Simultaneous determination of all polyphenols in vegetables, fruits, and teas. Journal of Agricultural and Food Chemistry, 51, 571–581. Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolics and flavonoids of apple and pear by high-performance liquid chromatography. Journal of Chromatography, A, 910, 265–273. Shen, J. G., Xie, L. Y., Zhai, M. Z., Lin, Q. Y., & Xie, L. H. (2004). Antiviral activity and chemical compositions of extracts from Myrica rubra. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 33, 441–443. Shukla, S., Mehta, A., Bajpai, V. K., & Shukla, S. (2009). In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert. Food and Chemical Toxicology, 47, 2338–2343. Tomas-Barberan, F. A., & Clifford, M. N. (2000). Dietary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80, 1024–1032. Vattem, D. A., Lin, Y.-T., & Shetty, L. K. (2004). Antimicrobial activity against select food-borne pathogens by phenolic antioxidants enriched in cranberry pomace by solid-state bioprocessing using the food grade fungus Rhizopus oligosporus. Process Biochemistry, 39, 1939–1946.
1135
Wang, D. Y., Liu, E. G., & Feng, Y. J. (2008). Study on the flavonoid constituents from the bark of Myrica rubra. Lishizhen medicine and material medical research, 19, 1149–1150. Wu, Q. L., Wang, M. F., & Simon, J. E. (2003). Determination of proanthocyanidins in grape products by liquid chromatography/mass spectrometric detection under low collision energy. Analytical Chemistry, 75, 2440– 2444. Xia, Q. L., Chen, J. C., & Wu, D. (2004). Study on scavenging efficacy of phenols extraction of Myrica rubra leaf. Food Science, 25, 80–83. Xu, G. H., Liu, D. H., Chen, J. C., Ye, X. Q., Ma, Y. Q., & Shi, J. (2008). Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chemistry, 106, 545–551. Yang, L. L., Chang, C. C., Chen, L. G., & Wang, C. C. (2003). Antitumor principle constituents of Myria rubra Var Acuminata. Journal of Agricultural and Food Chemistry, 51, 2974–2979. Yesil-Celiktas, O., Sevimli, C., Bedir, E., & Vardar-Sukan, F. (2010). Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer cell lines. Plant Foods for Human Nutrition, 65, 158–163. Zhou, S. H., Fang, Z. X., Lü, Y., Chen, J. C., Liu, D. H., & Ye, X. Q. (2009). Phenolics and antioxidant properties of bayberry (Myria rubra Sieb et Zucc.) pomace. Food Chemistry, 112, 394–399. Zou, Y. H., & Li, G. R. (1998). Analysis of flavonoid as antioxidant in Myrica rubra Leaf with reversed-phase high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 26, 531–534. Zhou, Z. H., & Yang, C. R. (2000). Phenolic constituents of the fresh leaves of Myrica nana. Acta Botanica Yunnanica, 22, 219–224.