Comparative studies on the physicochemical and antioxidant properties of different tea extracts
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- Chen, H., Zhang, Y., Lu, X. et al. J Food Sci Technol (2012) 49: 356. doi:10.1007/s13197-011-0291-6
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Tea is one of the most popular drinks next to water. Tea polyphenol is one of the main bioactive constituents of tea with health functions. In order to find the most bioactive tea polyphynols, polyphenol extracts from green tea, black tea and chemical oxidation products of green tea extracts were comparatively studied on the physicochemical and antioxidant properties. Results showed physicochemical and antioxidant properties of polyphenol extracts changed greatly after the chemical oxidation. Hydrogen peroxide induced oxidation products (HOP) possessed the highest antioxidant ability among the four tea polyphenol extracts. Thirteen phenolic compounds and one alkaloid in HOP were identified by reversed phase high-performance liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (RP-HPLC-DAD-ESI-MS). Hydrogen peroxide induced oxidation of tea polyphenol extracts could improve the antioxidant activity and could be used to produce antioxidants for food industry.
KeywordsTea polyphenolsComparative studiesPhysicochemical propertiesAntioxidant activityHPLC-MS
Tea (Camellia sinensis L.) was originally used as a beverage thousands of years ago. It was a complex mixture containing multiple compounds from simple phenolics, catechins to polysaccharides and complex thearubigins, many of which had been reported to have health functions such as antiallergic (Suzuki et al. 2000), antioxidative (Jhoo et al. 2005), antimutagenic and anticarcinogenic (Standley et al. 2001), antiatherosclerotic (Yokozawa et al. 2002), antibacterial (Yildirim et al. 2000), and hypoglycemic (Zhou et al. 2007) activities.
Tea has been consumed for centuries in the forms of unfermented (green tea), semi-fermented (oolong), and fermented (black and pu-erh or red) by ancient cultures for its medicinal properties (Lin et al. 2008). The content and constituents of the tea polyphenols are different in different forms. There are four major tea catechins existed in green tea because there was no enzymatic oxidation in the process procedure of green tea. While during the fermentation process of black tea, four major tea catechins originally contained in fresh leaves are enzymatically oxidized and converted to various oxidation products comprising black tea polyphenols. Because the enzymatic oxidation procedure produces a complex and small amount of oxidation products, only little oxidation products had been studied (Sava et al. 2001, Tanaka et al. 2005). An alternative approach, using a model oxidation system, had thus been developed for the study of polyphenolic compounds. For example, oxidation in model systems has been carried out both chemically by using ferrous sulphate (Oszmianski et al. 1996) or hydrogen peroxide (Zhu et al. 2000) and enzymatically with polyphenoloxidase (Guyot et al. 1996). But until now there are no reports on the comparative studies on the polyphenol extracts from green tea, black tea and chemical oxidation products of green tea extracts.
This paper aimed to comparatively study the physicochemical and antioxidant properties of the polyphenol extracts from green tea, black tea and chemical oxidation products of green tea extracts with hydrogen peroxide and potassium hexacyanoferrate respectively. The constituents of the most bioactive tea polyphenol extract among the four extracts were determined by HPLC-DAD-ESI-MS analysis.
Materials and methods
Green tea and black tea were produced in Huangshan Mountain, Anhui, China. They were purchased from the local market in Tianjin, P.R.China. 1,1- Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St, Louis, MO, USA). HPLC solvents were obtained from Concord Technology Co., Ltd (Tianjin, P.R.China). All other chemicals and reagents were purchased locally and were of analytical grade.
Extract isolation from green tea and black tea
One hundred grams of dry green tea or black tea leaves was extracted for 1 h with 1500 ml of boiling water. After cooling to 45 °C, the aqueous infusion was concentrated using vacuum rotary evaporator (RE-52CS Rotary Evaporator, Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) and then extracted with chloroform and ethyl acetate for three times (1:1, v/v), respectively. The ethyl acetate extract was vacuum dried and lyophilized to obtain the main polyphenol extract. The yields of tea polyphenol extracts were 10.2 g and 9.0 g for green tea (named as GTP) and black tea (named as BTP), respectively.
Chemical oxidation of green tea extracts
Chemically oxidation of green tea extracts by hydrogen peroxide was done according to (Li and Xie 2000). Briefly, 10% of the green tea polyphenol extract with aquatic solution as substrate was mixed with 2.5% (v/v) of hydrogen peroxide, and reacted at 45 °C for 6 h with stirring at medium velocity. This is followed by ethyl acetate (1:1, v/v) extraction for three times. The hydrogen peroxide oxidized tea polyphenols (named as HOP) was obtained by drying the ethyl acetate extract in vacuum and freeze-dried. Chemical oxidation of green tea extracts by potassium hexacyanoferrate was done according to Wan et al. (1997) and the potassium hexacyanoferrate oxidized tea polyphenols (named as POP) were obtained.
Physicochemical analysis of tea extracts
Solubility of different tea extracts was measured by employing AOAC (1984) methods. The pH was recorded with a Schott pH-meter (Model CG840, Schott Instruments, Mainez, Germany) at the concentration of 1.0 mg/ml in aqueous solution of each tea polyphenol extracts. UV absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Total phenolics in tea extracts were determined according to the method reported by Chen (1997) using gallic acid as standard. Contents of total phenolics in GTP, BTP, HOP and POP were expressed as gallic acid equivalents in milligrams per g tea polyphenol extract weight (mg GAE/g TEW). The results were averages of triplicate analyses.
Scavenging effects on DPPH radicals
Scavenging effects of the extracts on DPPH radicals were determined according to Duan et al. (2006). Hundred microliters of various concentrations of tea extracts was mixed with 2900 μl DPPH solution (120 μM) in ethanol and incubated at 37 ˚C for 30 min in the dark. The absorbance was recorded at 517 nm. Scavenging effects of extracts GTP, BTP, HOP and POP on DPPH free radicals in percentage (%) was calculated and ascorbic acid was used as positive controls. All tests were carried out in triplicate.
Measurement of ferric reducing power (FRP)
FRP potential of the extracts was determined according to Gow-Chin Yen et al. (1995). The reaction solutions (0.1 ml) containing different concentrations of samples in 0.2 M PBS (pH 6.6) were mixed with 30 mM aqueous potassium hexacyanoferrate solution (0.7 ml). After incubating at 50 °C for 20 min, 10% trichloroacetic acid (2.0 ml) was added and centrifuged at 3000 × g for 10 min. The supernatant (1.0 ml) was mixed with 1.7 mM aqueous FeCl3 (3.0 ml) and absorbance at 700 nm was determined. The ascorbic acid was used as the control.
Sample of HOP (the most active extract) was analyzed by RP-HPLC. A rapid resolution, 1200 series HPLC system with a quaternary SL pump, well plate autosampler, thermostat and heated column compartment and diode array detector (Agilent Techologies, Palo Alto, CA, USA) was used. A Zorbax 80SB-C18 column (150 × 2.1 mm, 3.5 μm) (Agilent Techologies, Palo Alto, CA, USA) was used for the separation of phenolic compounds. Sample HOP was prepared at a concentration of 1.0 mg/ml in water, and filtered through a 0.45 μm polytetrafluoroethylene (PTFE) filter (Whatman, Florham Park, NJ). Sample was injected into a 20 μl injection loop and the column was maintained at 40 °C. Gradient elution was performed with water/0.1% formic acid (solvent A) and MeOH (solvent B) at a constant flow rate of 200 μl/min. An increasing linear gradient (v/v) of solvent B was applied: (t (min),%B): (0, 5), (50, 30), (70, 100). Chromatograms were recorded at 273 nm, with peak scanning between 200 and 600 nm.
Qualitative Analysis by HPLC-DAD/ESI-MSn
Sample of HOP was analyzed by HPLC-DAD/ESI-MSn. An angilent 6310 ion trap mass spectrometer fitted with an electrospray interface coupled online to HPLC system as described above was used. Experiments were performed in negative and positive ion modes. Scan range and scan rate were 150–2000 and 13000 m/z/sec, respectively. The drying gas temperature was 300 °C. High spray voltage was set at 3500 V. Nitrogen was used as the dry gas at a flow rate of 12 l/min. MS/MS and MS3 were carried out using helium as the target gas. Identifications were achieved on the basis of the ion molecular mass, MSn, and UV-visible spectra.
Values were expressed as means±standard deviation (SD) of three replicates, and Student’s test was used for the statistical analysis. The values were considered to be significantly different when the P value was less than 0.05.
Physicochemical Characteristics of four different tea polyphenol extractsa
pH (1.0 mg/ml aqueous solutions)
Total polyphenols (mg of gallic acid/g of extract)
DPPH radical scavenging effects
Ferric reducing power of tea extracts
Identification of chromatographic peaks
Identification of compounds in HOPa by using their spectral characteristics in LC-DAD, negative ions and positive ions in LC-MS and MSn
(−) - epicatechin
331, 289, 169
EGCG quinone Dimer
Unidentified Peaks in HOP
There are still some constituents corresponding to the peaks that we are unable to identify according to the LC-DAD and LC-MS data (Table 2). Peaks 1 and 4 displayed a UV spectrum similar to that of gallic acid, and there was a molecular ion at m/z 169 in the MSn analysis. So they were gallic acid conjugates but no reference data were available for further confirmation. Isolation and NMR identification are needed for an unequivocal structural elucidation of all unidentified peaks.
Two chemical oxidation methods (H2O2 induced polyphenol chemical oxidation and potassium hexacyanoferrate induced chemically oxidation) were adopted to obtain two types of tea polyphenol extracts, HOP and POP. Compared to extracts from original green tea and black tea, there were great changes on the physicochemical properties. Antioxidant assays of scavenging abilities on DPPH free radicals and ferric reducing power showed that H2O2-induced chemical oxidation of tea polyphenol extracts had the highest antioxidant ability which was in accordance with the total polyphenol content.
In this study, hydrogen peroxide induced oxidation of tea polyphenol extracts could improve the antioxidant activity. Thirteen phenolic compounds and one alkaloid in HOP were identified by reversed phase high-performance liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (RP-HPLC-DAD-ESI-MS). Among the constituents identified in HOP, (−)-Epigallocatechin-3-gallate (EGCG), was still the major compound, which is typical in Camellia species. Two dimers of catechin identified in HOP suggested that the extract was only partially oxidized. The higher antioxidant activity of HOP compared to that of GTP indicated that the polymerization of tea polyphenols could improve the antioxidant activities. These results agree with the previous report (Li and Xie 2000), in which the antioxidant activity of tea catechin oxypolymers was equal to or even more notable than that of tea catechin. The mechanism might due to the increase of solubility and the conformation change of the partially oxidized tea polyphenol, which might make it more easily to bind free radicals (Ding et al. 2005). The detailed structural analysis and evaluation of the bioactivities mechanism of the partially oxidized tea polyphenols will need further study.
In conclusion, it can be stated that the polyphenol extracts from green tea, black tea and chemical oxidation products of green tea extracts with hydrogen peroxide and potassium hexacyanoferrate respectively were found to have different physicochemical and antioxidant properties. Our finding indicated that H2O2 induced oxidation of tea extracts could improve the antioxidant activities and this method could be used to produce antioxidants for food industry.
This research is funded by the National Natural Science Foundation of China to H. Chen (Grant No. 30600470).