Journal of Food Science and Technology

, Volume 49, Issue 3, pp 356–361

Comparative studies on the physicochemical and antioxidant properties of different tea extracts

Authors

    • Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and TechnologyTianjin University
  • Yu Zhang
    • Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and TechnologyTianjin University
  • Xueming Lu
    • Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and TechnologyTianjin University
  • Zhishuang Qu
    • Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and TechnologyTianjin University
Original Article

DOI: 10.1007/s13197-011-0291-6

Cite this article as:
Chen, H., Zhang, Y., Lu, X. et al. J Food Sci Technol (2012) 49: 356. doi:10.1007/s13197-011-0291-6

Abstract

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.

Keywords

Tea polyphenolsComparative studiesPhysicochemical propertiesAntioxidant activityHPLC-MS

Introduction

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

Materials

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.

RP-HPLC analysis

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.

Statistical analysis

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.

Results

Physicochemical analysis

Oxidation change of the physicochemical properties of tea extracts is shown in Table 1. There were similar solubility of GTP and BTP. Amongst all tea extracts, the solubility for HOP was the highest and that for POP was the lowest. The pH values of the four polyphenol extracts were similar. All extracts had an acidic pH, ranging from 4.8 to 5.3. The UV spectra (190–340 nm) of tea extracts showed two absorption maxima, namely λmax1 and λmax2, which were similar for each extracts. Overall, the range of λmax1 was 232–233 nm, whereas λmax2 ranged from 269–273 nm. All tea extracts were compared for their total polyphenol contents, in terms of gallic acid equivalents. HOP showed relatively greater reactivity to the Folin-Ciocalteu reagent, while POP was the least reactive. The total polyphenol content in tea extracts was in the following order: HOP > GTP > BTP > POP.
Table 1

Physicochemical Characteristics of four different tea polyphenol extractsa

Characteristic

GTPb

BTPb

HOPb

POPb

Solubility (mg/ml)

13.8

13.5

15.6

5.3

UVλmax(nm)

232, 271

232, 272

233, 269

232, 269

pH (1.0 mg/ml aqueous solutions)

5.3

4.9

5.1

4.9

Total polyphenols (mg of gallic acid/g of extract)

381.1

300.1

501.1

226.3

aValues are the mean of three analyses;

bGTP: Tea polyphenol extracts from green tea; BTP: Tea polyphenol extracts from black tea; HOP: Hydrogen peroxide oxidized tea polyphenols; POP: Potassium hexacyanoferrate oxidized tea polyphenols

DPPH radical scavenging effects

DPPH radical is a stable free radical and is commonly used as a substrate to evaluate antioxidant activity (Kaur et al. 2008). The scavenging effects of four tea extracts and vitamin C on DPPH free radicals were shown in Fig. 1. All of the four samples, GTP, BTP, HOP and POP, exhibited a strong ability to quench DPPH radicals. Stronger scavenging effects were observed with increasing concentrations used in the test (from 1.7 μg/ml to 13.3 μg/ml). The IC50 value of GTP, BTP, HOP, POP and Vitamin C were 1.7, 2.3, 1.4, 2.5 and 2.1 μg/ml, respectively. Although no significant difference (P > 0.05) was observed between the scavenging rate of HOP and vitamin C at a relatively high concentration, the DPPH radical-scavenging activity of HOP at low concentrations (1.7, 3.3, 6.7 and 13.3 μg/ml) was significantly higher (P < 0.05) than that of vitamin C at the same concentration. This indicated that HOP was a good antioxidant with strong DPPH radical-scavenging activity.
https://static-content.springer.com/image/art%3A10.1007%2Fs13197-011-0291-6/MediaObjects/13197_2011_291_Fig1_HTML.gif
Fig. 1

DPPH scavenging activities of four different tea polyphenol extracts. Each value is the mean ± SD of triplicate measurements. GTP: Tea polyphenol extracts from green tea; BTP: Tea polyphenol extracts from black tea; HOP: Hydrogen peroxide oxidized tea polyphenols; POP: Potassium hexacyanoferrate oxidized tea polyphenols. bP < 0.05 compared to the group of Vc

Ferric reducing power of tea extracts

Ferric reducing power of tea extracts is shown in Fig. 2. All the tea polyphenol extracts showed good FRP potentials. GTP, BTP, HOP and POP had dose-dependent reducing power with the coefficient R2 = 0.9615, 0.9301, 0.9336 and 0.9595, respectively. Although weaker than reference antioxidant vitamin C, HOP had the strongest activity in FRP activities among the four tea polyphenol extracts. The activity order of the four tea polyphenol extracts for FRP potential were HOP > GTP > BTP > POP.
https://static-content.springer.com/image/art%3A10.1007%2Fs13197-011-0291-6/MediaObjects/13197_2011_291_Fig2_HTML.gif
Fig. 2

Ferric Reducing Power of four tea polyphenol extracts. Each value is the mean ± SD of triplicate measurements. GTP: Tea polyphenol extracts from green tea; BTP: Tea polyphenol extracts from black tea; HOP: Hydrogen peroxide oxidized tea polyphenols; POP: Potassium hexacyanoferrate oxidized tea polyphenols

Identification of chromatographic peaks

Figure 3 showed the LC-DAD chromatograms of HOP, which was the most bioactive extract in the four tea polyphenol extracts. UV-visible characteristics and LC/MS/MSn data were given in Table 2. Based on the LC-MS data and the identity of tea polyphenols previously established (Del Rio et al. 2004, Lee et al. 2005, Krishnan and Maru 2006), peaks 2, 3, 5, 6, 7, 8, 10, 11 and 13 were identified as gallic acid, 5-galloylquinic acid, (−)-gallocatechin, (+)-catechin, (−)-epigallocatechin, HHDP-galloylglucose, (−)-epigallocatechin- 3-gallate, (−)-epicatechin, (−)-gallocatechin-3-gallate, (−)-epicatechin-3-gallate, respectively. Peaks 12 and 15 were the polymeric polyphenols which was identified as (−)-gallocatechin-3-gallate dimer and EGCG quinone dimer (Tanaka et al. 2005). Other major peaks in the chromatograms, 8, 16 and 17 of HOP, were the glycoside conjugate that were identified as HHDP-galloylglucose, quercetin-3-galactoside and kaempferol-3-rutinoside. The positive ion mass spectra recorded molecular ions at m/z 195 (peak 9) was identified as caffeine. Peaks 14 were partially identified as HHDP derivatives.
https://static-content.springer.com/image/art%3A10.1007%2Fs13197-011-0291-6/MediaObjects/13197_2011_291_Fig3_HTML.gif
Fig. 3

Profile of HPLC-DAD chromatograms at 273 nm of hydrogen peroxide oxidized tea polyphenols (HOP). 1, unidentified; 2, gallic acid; 3, 5-galloylquinic acid; 4, unidentified; 5, (−)- gallocatechin; 6, (+)- catechin; 7, (−)-epigallocatechin; 8, HHDP-galloylglucose; 9, caffeine; 10, (−)- epigallocatechin-3-gallate; 11, (−) – epicatechin; 12, (−)-gallocatechin-3-gallate Dimer; 13, (−)-epicatechin-3-gallate; 14, HHDP derivatives; 15, EGCG quinone Dimer;16, quercetin-3-galactoside; 17, kaempferol-3-rutinoside

Table 2

Identification of compounds in HOPa by using their spectral characteristics in LC-DAD, negative ions and positive ions in LC-MS and MSn

peak

tR (min)

UVdata (nm)

[M-H] (m/z)

MS2/MS3 (m/z)

tentative identification

1

2.7

210, 270

961

791,747,639

unidentified

2

4.1

210, 272

169

gallic acid

3

5.1

210, 275

343

191,169

5-galloylquinic acid

4

5.5

210, 270

946

775,731,623,301

unidentified

5

8.2

210, 270

305

221,179

(−)- gallocatechin

6

19.8

210, 275

289

245,205

(+)- catechin

7

20.3

210, 270

305

261,219,179

(−)-epigallocatechin

8

22.0

210, 265

633

463,301,275

HHDP-galloylglucose

9

29.4

210, 270

195b

caffeine

10

31.5

225, 275

457

331,193,169

(−)- epigallocatechin-3-gallate

11

33.9

210, 275

289

245,203,123

(−) - epicatechin

12

40.1

225, 275

883

457, 331,193,169

(−)-gallocatechin-3-gallate Dimer

13

46.8

210, 275

441

331, 289, 169

(−)-epicatechin-3-gallate

14

48.9

220, 275

635

465, 447,313,169

HHDP derivatives

15

59.6

275, 360

929

EGCG quinone Dimer

16

60.1

270, 370

463

301

quercetin-3-galactoside

17

62.2

270, 350

593

447,285

kaempferol-3-rutinoside

aHOP: Hydrogen peroxide oxidized tea polyphenols.

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.

Discussion

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.

Acknowledgements

This research is funded by the National Natural Science Foundation of China to H. Chen (Grant No. 30600470).

Copyright information

© Association of Food Scientists & Technologists (India) 2011