Analytical, Nutritional and Clinical MethodsHPLC-DAD-ESIMS analysis of phenolic compounds in bayberries (Myrica rubra Sieb. et Zucc.)
Introduction
Bayberry (ssMyrica rubra Sieb. et Zucc.) is the name of a tree growing wildly or under cultivated conditions in southern China for more than 2000 years (Chen, 1996). Bayberry fruits, with its special sweet, sour taste and exquisite flavor, have been traditionally used to deal with gastric intestinal problems, such as diarrhea and gastroenteritis in China (Chen, 1996, Li, 2001). The fruits of most cultivars ripen in the hot and raining seasons of June or July and can only be stored fresh with attractive dark red color for 3 days at 20–22 °C and 9–12 days at 0–2 °C (Xi & Zheng, 1993). In order to reach a more widespread and longer consumption, bayberry fruits are often processed into juice and juice concentrate, in which cultivar Biqi is often used because of its big production and excellent quality. However, the color instability and haze formation during juice processing and storage bothered food technologists for years (Chen et al., 1994, Li et al., 2002, Zhong, 2002).
Phenolic compounds are major constituents of fruits and play an important role in the nutritional, organoleptic and commercial properties of the fruits and their derived products. According to the epidemiological studies, the intake of flavonoids is inversely correlated with the risk of coronary heart disease (Hertog et al., 1993, Hertog et al., 1997), stroke (Keli, Hertog, Feskens, & Kromhou, 1996) and cancer (Hollman & Katan, 1999). Polyphenols have been demonstrated to act as antioxidants and are assumed to contribute to the beneficial health effects of fruits and vegetables (Tomás-Barberán & Robins, 1997). Bayberry juice (Pan, 2002) and jam (Amakura, Umino, & Tonogai, 2000b) have also been shown to exert strong antioxidant activities in vitro.
Moreover, phenolic compounds are implicated in the color degradation in blueberry juice (Kader et al., 1997, Kader et al., 1998), and the formation of hazes and sediments in fruit juices and alcoholic beverages (Beveridge, 1997, Siebert, 1999). Anthocyanins, as one major class of phenolics in colored fruits and vegetables, are easily oxidized and susceptible to degradative reactions during various steps of processing. The major anthocyanin in bayberry fruits has been identified as cyanidin 3-glucoside, which represents above 95% of the total pigments (Ye, Chen, & Su, 1994). One of the degradation mechanisms of cyanidin 3-glucoside is enzymatic oxidation, but this ortho-diphenolic anthocyanin is a poor substrate of polyphenoloxidase (PPO), and degrades by coupled oxidation involving the enzymatically generating o-quinone with partial regeneration of the o-diphenolic cosubstrate (Kader et al., 1998, Peng and Markakis, 1963). Phenolic compounds, such as chlorogenic acid (Kader et al., 1997), caffeoyltartaric acid (Sarni, Fulcrand, Souillol, Souquet, & Cheynier, 1995), catechol (Peng & Markakis, 1963), gallic acid (Prabha & Patwardhan, 1986) and catechin (Wesche-Ebeling & Montgomery, 1990), are good substrates for PPO. Unfortunately, up to date, data of phenolic acids and other kinds of phenolics from bayberry fruits are terribly scarce. The main flavonols in bayberry fruits were identified as myricetin, quercetin, and kaempferol, and they might be existed as glycoside forms in the fruits Amakura et al., 2000a, Amakura et al., 2000b. Ellagic acid was also detected in bayberry fruits by the work of Amakura et al. (2000a).
High performance liquid chromatography coupled with a photodiode-array detector and a mass spectrometry (HPLC-DAD-MS) provides powerful and economical tool for polyphenol analysis in crude plant extracts. HPLC-DAD provides extensive information on polyphenol structures (Alonso-Salces et al., 2001, Price et al., 1999) while HPLC–MS provides information about the polyphenol molecular weight and the molecular structure from its fragmentation data (Mämmelä et al., 2000, Queiroz et al., 2002). This hyphenated technique gives a precise idea of plant constituents and has been widely and successfully used in the identification of phenolic compounds in foodstuffs (Alonso-Salces et al., 2004, Amaral et al., 2004, Määttä et al., 2003, Owen et al., 2003, Tomás-Barberán et al., 2001).
Knowledge of the precise phenolic composition of bayberry varieties may contribute to a better understanding of their influence on the quality and diversity of bayberry products, such as bayberry juice and wine. The aim of our project is to learn the mechanisms of color degradation and haze formation during bayberry juice processing and storage, in which phenolics are supposed to involve. The purpose of the present work is to determine the phenolics except anthocyanins in bayberry fruits using a HPLC-DAD-ESIMS method, since anthocyanins have been analysed by other authors (Pan, 2002, Ye et al., 1994).
Section snippets
Chemicals and solvents
Pure standards of gallic acid, protocatechuic acid, (+)-catechin, ellagic acid, and quercetin 3-glucoside were purchased from Sigma (St. Louis, MO, USA) and Fluka (Buchs, Switzerland). Standards were dissolved in methanol. Working solutions we prepared daily by appropriate dilution with methanol to make the concentration from 1.2 to 120.0 mg/L. Acetonitrile and methanol (HPLC grade), ethyl acetate, acetic acid, formic acid and hydrochloric acid (analytical grade) were purchased from Shanghai
Identification of the chromatographic peaks
Efficient separations of bayberry phenolics found in the ethyl acetate extracts were achieved using the reversed-phase C-18 column. Fig. 1 was the HPLC chromatogram from cultivar Xiangshan. Peaks in the chromatograms were classified into two groups: hydroxybenzoic acids and flavonol glycosides, by comparison of their UV–Vis spectra and HPLC retention times to those of the available standards. Mass spectra from the negative ionization mode were also used for identification of these phenolic
Conclusion
For the first time, a hyphenated technique of HPLC coupled to photodiode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESIMS) was successfully used in determination of phenolic compounds from bayberry cultivars of Xiangshan, Biqi and Dongkui. Three phenolic compounds were identified (gallic acid, protocatechuic acid, and quercetin 3-glucoside) and seven others tentatively identified (two myricetin hexoside and two myricetin deoxyhexoside derivatives; quercetin hexoside
Acknowledgements
The authors thank Haitong Food Group Corporation Limited for providing bayberry fruits and Tao Guangjun, Qing Fang, Analysis Center of Southern Yangtze University, for the HPLC-DAD-ESIMS technical assistance.
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