Rev. Med. Chir. Soc. Med. Nat., Iaşi 2016 vol. 120, no. 2 PHARMACY ORIGINAL PAPERS CATECHINS PROFILE, CAFFEINE CONTENT AND ANTIOXIDANT ACTIVITY OF CAMELLIA SINENSIS TEAS COMMERCIALIZED IN ROMANIA V.S. Luca*, Ana-Maria Stan, Adriana Trifan, Anca Miron, Ana Clara Aprotosoaie University of Medicine and Pharmacy Grigore T. Popa -Iaşi Faculty of Pharmacy Department of Pharmacognosy * Corresponding author. E-mail : simon-vlad.v.luca@d.umfiasi.ro CATECHINS PROFILE, CAFFEINE CONTENT AND ANTIOXIDANT ACTIVITY OF CA- MELLIA SINENSIS TEAS COMMERCIALIZED IN ROMANIA (Abstract). Aim: Catechins profile, caffeine content and antioxidant activity of different green tea and white tea samples commercialized on the Romanian market were investigated. Material and methods: Five green tea samples and five white tea samples commonly available on the Romanian market were processed by infusion and the lyophilisates of infusions were analyzed. Total phenolic content was determined using the Folin-Ciocalteu method. Catechins and caffeine profile was analyzed by RP-HPLC-DAD (Agilent Eclipse XDB-C18 column, binary mobile phase (A) 3% acetic acid and (B) methanol). In vitro antioxidant activity was assessed by free radical scavenging and ferrous ion chelating assays. Results and discussions: Total phenolic content ranged between 44.73±0.63 and 63.57±0.45 GAE% in green tea samples and between 9.69±0.90 and 52.99±0.45 GAE% in white tea samples. RP-HPLC-DAD analysis allowed the identification of epigallocatechin gallate (45.18-118.58 mg/g lyophilisate) and caffeine (47.79-108.07 mg/g lyophilisate) in all tea samples; epicatechin was detected in all samples (5.04-31.04 mg/g lyophilisate) except for two white teas infusions. Green tea samples scavenged DPPH radical and chelated ferrous ion with EC 50 =9.68±0.02-16.11±0.02 g/ml and 10.91±0.04-18.65±0.03 g/ml, respectively. For white teas, EC 50 values varied between 9.50±0.02-20.95±0.02 g/ml in DPPH assay and 12.49±0.03-20.32±0.07 g/ml in ferrous ion chelating assay. Conclusions: This study showed a large variability in the content of catechins and caffeine and in the antioxidant capacity of both green and white tea samples. Keywords: CATECHINS, CAFFEINE, GREEN TEA, WHITE TEA, ANTIOXIDANT. Tea is the second most consumed beverage in the world. All types of tea come from the leaves of Camellia sinensis (L.) Kuntze (Theaceae), a plant originally native to China, Thailand, Laos and Vietnam (1). Based upon the degree of fermentation, tea can be classified into unfermented tea (white tea and green tea), semi fermented tea (oolong tea) and fully fermented tea (black tea and Pu-erh tea) (2). Tea provides complex chemical substances, like: flavanols (catechins), flavonols and flavones glycosides, proantocyanidols, bisflavanols, theaflavins, thearubigins, theasinensins, caffeine, amino acids and minerals. Catechins are the representative class of compounds from green and white tea, with epigallocatechin-3-o-gallate (EGCG) as the most abundant constituent 457
V.S. Luca et al. (50-80% of the total catechins). Among polyphenols, catechins are primarily responsible for almost all the beneficial effects of tea: antioxidant, chemopreventive, neuroprotective, radioprotective, antimicrobial, anti-aging, hypoglycemiant, and hypolipemiant. Caffeine is the main purine alkaloid of tea with CNS stimulant effects (3).The chemistry and quality of tea are significantly influenced by the environmental conditions of the regions where the plants grow, sampling time, cultivation and processing practices. Therefore, numerous studies have reported a considerable variability among different tea types and brands (2). The aim of this study was to analyze the catechins profile, caffeine content, and antioxidant activity of different green and white tea samples commercialized on the Romanian market. MATERIAL AND METHOD Chemicals Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid, and methanol were purchased from Merck (Darmstadt, Germany); Folin-Ciocalteu s phenol reagent, 3-(2- pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4 -disulfonic acid monosodium salt (ferrozine), and epicatechin (EC) were purchased from Fluka (Steinheim, Germany); iron (II) sulfate heptahydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate, acetic acid, gallic acid, and epigallocatechin gallate (EGCG) were obtained from Sigma- Aldrich (Steinheim, Germany); caffeine was supplied by Carl Roth (Karlsruhe, Germany). Plant material Five green tea samples and five white tea samples of different origins (China, India, Vietnam), of different varieties (Bi Luo Chan, Darjeeling, Fanning, Pai Mu Tan), and in different packages were purchased from a local market in Iasi, Romania (tab. I). Voucher samples are deposited in the Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa Iaşi, Romania. Green teas White teas TABLE I Sample numbers, varieties, origins and types of package Sample Variety Origin Type of package GT1 BI LUO CHAN China Bulk GT2 DARJEELING India (packaged in Austria) Bulk GT3 FANNING Vietnam Sachet GT4 Unknown China Bulk GT5 BI LUO CHAN China Bulk WT1 PAI MU TAN China (packaged in Austria) Bulk WT2 PAI MU TAN China (packaged in Czech Republic) Bulk WT3 Unknown China (packaged in Canada) Sachet WT4 PAI MU TAN China (packaged in Austria) Sachet WT5 PAI MU TAN China Bulk Bi Luo Chun is one of the most famous ten Chinese teas originated in Dong Ting Mountain (Jiangsu Province). Its specific flavor is determined among other things by various fruit trees (apricot, peach, plum) which are found on tea plantations. After 458
Catechins profile, caffeine content and antioxidant activity of Camellia sinensis teas commercialized in Romania harvest, the leaves are manually sorted, treated with heat (190-200 C) for 3-5 min, and then rolled. Darjeeling tea is obtained from plantations located in the region with the same name in West Bengal, India, and it is distinguished by its unique flavor. Plant material consists only of the first two leaves from the top or from the apical buds. The processing of leaves is achieved by exposing them to a hot air flow and subsequent mechanical twisting. In the case of Pai Mu Tan tea ( white peony ), after harvest, tea leaves are sun-dried or dried in closed rooms, without any further technological intervention (4). Extraction 2 g of tea leaves were placed into boiled water (200 ml) and incubated at 80-90 C for 3 min. This condition was similar to an actual brewing condition for a cup of tea. After cooling, each sample solution was filtered through Whatman filter papers. Each extract was concentrated under reduced pressure at < 40 C (Büchi R-210 rotary evaporator, Büchi V-850 vacuum controller, Büchi V-700 vacuum pump, Büchi B-490 heating bath, Büchi, Switzerland) and then freeze-dried (Alpha 1-2 LD Plus freeze-dryer, Christ, Germany). Total phenolic content Total phenolic content was estimated using the Folin-Ciocalteu assay. The results were expressed as g gallic acid equivalents (GAE)/100 g extract (5). RP-HPLC-DAD analysis Reversed-phase high performance liquid chromatography (RP-HPLC) analysis was conducted on an Agilent 1200 HPLC system (Agilent Technologies, PaloAlto, CA, USA) equipped with a DAD detector. The chromatographic separations were performed on an Agilent Eclipse XDB C-18 (150 mm x 4.6 mm, i.d. 5 μm) column, with column temperature set at 20 C. The binary mobile phase consisted of (A) 3% acetic acid and (B) methanol; the flow rate was 1 ml/min; the detection wavelength was set at 280 nm. The gradient elution profile started with 0% B; then B was gradually increased to 5% at 10 min, to 10% at 35 min, to 50% at 45 min, maintained at 50% at 50 min, and decreased back to 0% at 55 min [6]. Catechins and caffeine were identified by comparing their UV spectra and retention times with those of commercial standards. EGCG, EC and caffeine were quantified on the basis of their calibration curves (0.125-1 mg/ml), and the results were expressed as mg compound/g lyophilisate. The calibration curves were: for EGCG: A=1227.3c-69.206, R 2 =0.9997; for EC: A=504.72 c+19.928, R 2 =0.9998; for caffeine: A=1790.4c+9.114, R 2 =1. DPPH radical scavenging assay DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was performed according to Malterud et al (7, 8, 9). Epicatechin was used as positive control. The radical-scavenging activity (%) was calculated as follows: %=[(absorbance of DPPH solution - absorbance of DPPH solution with sample after 5 min)/absorbance of the DPPH solution] 100. Ferrous ion chelating ability assay This assay was done according to Dinis et al (10). Epicatechin was the positive control. The percent inhibition of Fe 2+ - ferrozine complex formation was calculated according to the following equation: % inhibition=[(absorbance of control - absorbance of sample)/absorbance of control] 100. Statistical analysis All experiments were conducted in triplicate. The results were expressed as mean 459
V.S. Luca et al. ± standard deviation (SD). The EC 50 values were calculated by linear interpolation between values above and below 50% activity. RESULTS AND DISCUSSION Phenolic content and profile The total phenolic content ranged between 44.73±0.63 and 63.57±0.45 g GAE% in green tea samples and between 9.69±0.90 and 52.99±0.45 g GAE% in white tea samples, respectively (fig. 1). Green tea samples contained higher amounts of polyphenols compared to white tea samples. RP-HPLC-DAD analysis revealed the presence of EGCG in all infusions; EC was detected in all samples except for two white tea samples (WT1 and WT5) (fig. 2). Also, caffeine was identified in all tea samples. EGCG was the major catechin in tea samples; it has been found in concentrations between 49.85 and 118.58 mg/g lyophilisate in green teas. In white tea samples, EGCG concentrations were lower (45.18-85.01 mg/g lyophilisate), and four samples had a concentration of less than 65 mg EGCG/g lyophilisate (fig. 3). Although green tea contained higher amounts of EGCG, the variability of its content was much higher than in white tea samples. This may be due to the fact that commercial samples of green tea were more heterogeneous (Darjeeling, Fanning, Bi Luo Chun) while white tea samples were predominantly Pai Mu Tan. Compared to EGCG, the EC content values were much lower (5.04-31.04 mg/g lyophilisate) (fig. 4); GT4 green tea sample contained the highest concentration of EC. The content of EC can vary up to 6 times between the two types of tea. Except WT2 sample that contained 27.79 mg EC/g lyophilisate, all other white tea samples showed lower concentrations of EC (5.04-8.76 mg/g lyophilisate) compared to green teas; therefore, white tea was characterized by a more pronounced heterogeneity. Regarding caffeine content, all samples of green tea had at most 65.19 mg/g lyophilisate (GT3); in white tea, the amounts of caffeine were found to be higher (67.44-108.07 mg/g lyophilisate) (fig. 5). Fig. 1. Total phenolic content of tea samples Fig. 2. RP-HPLC-DAD of tea samples (1-caffeine, R t =29.49 min; 2-EGCG, R t =32.69 min; 3-EC, R t =37.36 min; GT-green tea, WT-white tea) 460
Catechins profile, caffeine content and antioxidant activity of Camellia sinensis teas commercialized in Romania Fig. 3. EGCG concentration of tea samples Fig. 4. EC concentration of tea samples Fig. 5. Caffeine concentration of tea samples DPPH radical scavenging assay Both types of tea scavenged DPPH radical in a concentration dependent manner; at the concentration of 41.66 g/ml, all samples with the exception of WT2 showed a DPPH radical scavenging activity higher than 90% which was similar to that of the positive control (EC) (tab. II). At the concentration of 10.41 g/ml, DPPH radical scavenging activity of samples decreased reaching values lower than 50%. Therefore, at low concentrations we noticed a greater heterogeneity of DPPH radical inhibitory activity: between 30.26% and 64.15% for green tea samples, and between 23.28% and 53.55% for white tea samples, respectively. DPPH scavenging activity of white tea samples was lower than that of green teas. The samples of white tea showed a greater variability of free radical scavenging activity (EC 50 =9.50±0.02-20.95±0.02 g/ml) than that of green tea (EC 50 =9.68±0.02-16.11±0.02 g/ml) (tab. II). Ferrous ion chelating ability assay Ferrous ion chelating abilities of both types of tea samples increased with concentration. At the concentration of 41.66 μg/ml, all tea samples showed the highest values of ferrous ion chelating activity (77.79±0.20%-92.26±0.19% for white teas and 88.35±0.21-99.31±0.16% for green teas, respectively) (tab. III). Overall, green tea samples were more active as ferrous ion chelating agents (EC 50 =10.91±0.04-18.65±0.03 g/ml) than white teas (EC 50 =12.49±0.03-20.32±0.07 g/ml). Also, some of the samples, either green tea or white tea, were even more potent than the positive control, EC (EC 50 =15.44±0.01 g/ml) (tab. III). 461
V.S. Luca et al. Sample/ Positive control TABLE II DPPH scavenging activity of tea samples DPPH scavenging activity (%)±SD 41.66 μg/ml 20.83 μg/ml 10.41 μg/ml EC50±SD (μg/ml) GT1 94.59±0.06 73.52±0.04 47.7±0.11 11.14±0.01 GT2 94.82±0.11 90.92±0.08 64.15±0.08 9.68±0.02 GT3 94.29±0.03 77.33±0.08 47.19±0.12 10.92±0.01 GT4 92.87±0.04 78.40±0.14 30.26±0.16 16.11±0.02 GT5 91.65±0.08 69.95±0.11 33.63±0.11 15.39±0.01 WT1 93.42±0.08 57.89±0.09 34.97±0.11 14.87±0.01 WT2 78.98±0.10 42.67±0.05 23.28±0.04 20.95±0.02 WT3 93.25±0.04 80.02±0.06 41.99±0.13 12.92±0.02 WT4 92.65±0.18 68.89±0.08 53.55±0.15 9.50±0.02 WT5 93.97±0.12 81.83±0.05 49.30±0.21 10.93±0.02 EC 95.64±0.08 94.93±0.08 60.24±0.03 8.33±0.01 Sample/ Positive control TABLE III Ferrous ion chelating activity of tea samples Ferrous ion chelating activity (%)±SD 41.66 μg/ml 20.83 μg/ml 10.41 μg/ml EC50±SD (μg/ml) GT1 98.12±0.16 63.34±0.21 40.91±0.10 12.97±0.03 GT2 96.49±0.14 80.17±0.12 40.17±0.20 13.28±0.05 GT3 99.31±0.16 69.80±0.16 48.28±0.16 10.91±0.04 GT4 88.35±0.21 49.14±0.04 22.15±0.01 18.65±0.03 GT5 88.67±0.20 58.89±0.02 32.63±0.11 16.12±0.05 WT1 78.39±0.04 52.46±0.08 28.52±0.06 19.77±0.04 WT2 77.79±0.20 45.64±0.08 24.17±0.19 20.32±0.07 WT3 88.92±0.13 58.50±0.20 41.70±0.04 13.21±0.02 WT4 84.81±0.10 67.69±0.09 38.91±0.11 13.59±0.02 WT5 92.26±0.19 55.15±0.09 43.59±0.08 12.49±0.03 EC 94.56±0.08 71.73±0.02 21.93±0.06 15.44±0.01 CONCLUSIONS In our study, samples of green tea were characterized by a richer content in total polyphenols (that can be almost 5 times higher) compared to white teas. EGCG and EC were found in higher concentrations in green teas. EGCG content showed a higher degree of variability in green tea samples 462
Catechins profile, caffeine content and antioxidant activity of Camellia sinensis teas commercialized in Romania while EC content exhibited a great heterogeneity in white teas. The highest concentrations of caffeine, exceeding 65 mg/g lyophilisate, were identified in white teas samples. Overall, green tea samples were more active as free radical scavengers and ferrous ion chelating agents than white tea samples. REFERENCES 1. Harbowy ME, Balentine DA. Tea chemistry. Crit Rev Plant Sci 1997; 16: 415-480. 2. Pereira VB, Knor FJ, Vellosa JCR, Beltrame FL. Determination of phenolic compounds and antioxidant activity of green, black and white teas of Camellia sinensis (L.) Kuntze, Theaceae. Rev Bras Plantas Med 2014; 16: 490-498. 3. Aprotosoaie C, Stănescu U. Alcaloizi-substanţe biogene de interes terapeutic. Iaşi: Editura "Gr. T. Popa" U.M.F. Iaşi, 2010. 4. Heiss ML, Heiss RJ. The story of tea: a cultural history and drinking guide. New York: Ten Speed Press, 2007. 5. Singleton VL, Rossi JA. Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult 1965; 16: 144-158. 6. ISO 14502-2. Determination of substances characteristic of green tea and black tea-part 2: Content of catechins in green tea - Method using high-performance liquid chromatography. 2005. 7. Malterud KE, Farbrot TL, Huse AE, Sund RB. Antioxidant and radical scavenging effects of anthraquinones and anthrones. Pharmacology 1993; 47: 77-85. 8. Bedreag CF, Trifan A, Vasincu A, Miron SD, Aprotosoaie AC, Miron A. In vitro screening of Crataegus succulenta extracts for free radical scavenging and 15-lipoxygenase inhibitory activities. Rev Med Chir Soc Med Nat Iaşi 2014; 118(2): 544-550. 9. Zavastin DE, Mircea C, Aprotosoaie AC, Gherman S, Hăncianu M, Miron A. Armillaria mellea: phenolic content, in vitro antioxidant and antihyperglycemic effects. Rev Med Chir Soc Med Nat Iaşi 2015; 119(1): 273-280. 10. Dinis TCP, Madeira VMC, Almeida MLM. Action of phenolic derivates (acetaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys 1994; 315: 161-169. 463