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Black tea (C. sinensis) consumption is well associated with enhanced endothelial function (EF) and reduced cardiovascular (CV) risk. This clinical end...
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Compositional Variation among Black Tea Across Geographies and Their Potential Influence on Endothelial Nitric Oxide and Antioxidant Activity Paul Mark Dias,* Jayashree Changarath, Anita Damodaran,* and Manoj Kumar Joshi Unilever R&D, Bangalore, #64, Main Road Whitefield, Bangalore-560066, India S Supporting Information *

ABSTRACT: Black tea (C. sinensis) consumption is well associated with enhanced endothelial function (EF) and reduced cardiovascular (CV) risk. This clinical end benefit is endorsed to flavonoids in tea. The black tea flavonoid composition varies across geographies and may impact its health benefits. Moreover, the underlying functional species and a precise working mechanism responsible for the observed health benefit also remain to be investigated. In this Article, we investigated the effect of black teas from various geographies (WoBTs) on different working mechanisms (antioxidant potential and endothelial function) proposed to influence certain risk factors of CVH, in vitro. Pearson correlation analysis showed that the antioxidant benefits are fairly influenced by majority of tea actives such as catechins, theaflavins, thearubigins, and phenolic acids, while NO potentiating effects are mainly regulated by catechins in black tea. The data also suggest that the net vascular function benefit of black tea is majorly influenced by NO enhancement, while mildly contributed by its antioxidant benefit. KEYWORDS: nitric oxide, eNOS, reactive oxygen species, catechins, Camellia sinensis, free radical, vascular function, black tea

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INTRODUCTION Flavonoids in tea are responsible for the taste, strength, and color of black tea and have been regarded as a quality indicator of tea.1 Flavonoids are also considered to be a significant contributor toward the health benefits of tea,2 especially for vascular function. Although epidemiological and clinical evidence has been established,3,4 limited information is available on the working mechanisms contributing toward the vascular function benefits of tea. The vascular benefits of tea have been suggested mainly due to its nitric oxide (NO) promoting effect in endothelial cells,1−3,5,6 which may be complemented with its antioxidant7 and free radical scavenging properties.8 Tea flavonoids increase NO production in endothelial cells via activation of eNOS enzyme.9 The activation of eNOS depends on p38-mitogenactivated protein kinase (p38-MAPK) and ligand-independent activation of estrogen receptor-α, which leads to activation of the PI3-K/Akt pathway10 and a specific pattern of eNOS phosphorylation.9,11 In addition to the putative mechanism stimulating eNOS phosporylation and NO production, it appears that the flavonoids also activate specific signaling pathways, which increases the expression of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and peroxidases,12,13 responsible for increasing bioavailability of NO13 for its physiological function. Thus, tea flavonoids are shown to have multiple targets. Although these mechanisms contribute to increasing NO levels in endothelial cells, their free radical scavenging property also prevents oxidative modification of lipids and proteins,13,14 which can otherwise trigger prooxidant effects in endothelial cells.15,16 Thus, an in vitro investigation on whether these mechanisms individually or synergistically enhance vascular function efficacy of tea needs to be evaluated.17 © XXXX American Chemical Society

Flavonoids are the main polyphenolic constituents in tea and are classified on the basis of their flavan nucleus.18 They are mostly derivatives and/or isomers of flavones, isoflavones, flavonols, catechins, and phenolic acids.19,20 Green tea is a rich source of flavonoids, predominantly catechins (particularly epicatechin and its gallated forms like ECG, EGC, and EGCG).9,18,20 These catechins comprise 30−50% of the solids in green tea and 90% of the total flavonoids.20 In black tea, the catechins are oxidized and dimerized during fermentation and converted into theaflavins (TFs) and thearubigin polymers (TRs).20 The major portion of black tea is composed of TRs, accounting for >20% of the total solids and 70−80% of total flavonoids.9,18,20 Geography-specific compositional variations have been reported in the black teas,21 and such variations may in turn reflect the difference in delivering the functional end-benefit of these teas.3,22 The compositional variations mainly arise due to changes in the levels of catechins, theaflavins, thearubigins, and phenolic acids. In this Article, we evaluated vascular function efficacy of the representative global black teas (WoBTs) using the antioxidant activity (AOX) and eNOS activity. There is always certain limitation to an in vitro approach because the measurements made in the in vitro culture of endothelial cells may not reflect physiological endothelial function.23 Also, under conditions of ingestion, some of the components of the tested tea extract in the bioassay may not be bioavailable24 or may require metabolic transformation,25,26 for bioefficacy. However, NO production in endothelial cells has Received: April 4, 2014 Revised: June 25, 2014 Accepted: June 25, 2014

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a Phenomenx phenyl-hexyl, 5 μm, 4.6 × 250 mm HPLC column was used. The mobile phase A was 9% (volume fraction) acetonitrile, 2% (volume fraction) acetic acid with 20 μg/mL EDTA, and mobile phase B was 80% (volume fraction) acetonitrile, 2% (volume fraction) acetic acid with 20 μg/mL EDTA. Separation was carried out at 1 mL/min flow rate at 35 °C and detection at 278 nm using Agilent HPLC (1100 Series). HPLC analysis was carried out using a gradient elution. Binary gradient conditions: 0% mobile phase B for 10 min, then over 15 min, a linear gradient to 32% mobile phase B, and hold at this composition for 10 min; then reset to 0% mobile phase B to equilibrate for 10 min before next injection. Standard compounds were used to generate calibration curves. Quantification of Theaflavins (TFs) by HPLC. Theaflavins were analyzed by using the HPLC method.32 Nova-Pak C-18, 60A, 4 μm, 3.9 × 150 mm HPLC column was used. Mobile phase A was 2% (volume fraction) acetic acid, and mobile phase B was acetonitrile. Separation was carried out at 1 mL/min flow rate at 40 °C and detection at 380 nm using Shimadzu LC-10A HPLC. Standard theaflavins were used to generate calibration curves. UPLC Fingerprinting of Thearubigins. The UPLC analysis of tea infusions was done using the method developed in house. For UPLC analysis, a Phenomenex Kinetex C18, 100 × 2.1 mm, 1.7u, 100A column was used. Mobile phase A was 0.5% (volume fraction) acetic acid in water, and mobile phase B was 100% acetonitrile. Separation was carried out at 0.2 mL/min flow rate at 35 °C and detection at 350 nm using the Agilent 1290 series system. UPLC analysis was carried out using a gradient elution. Binary gradient conditions: 0% mobile phase B for 5 min, then over 50 min, a linear gradient to 25% mobile phase B, 30% mobile phase B until 55 min and coming back to 0% mobile phase B at 56 min and then to equilibrate for 4 min before next injection. The known compounds like catechins, theaflavins, chlorogenic acid, caffeine, theogalline, gallic acid, theobromine, and flavonol glycosides were identified by injecting their standards, and all of the remaining peaks that were not identified were considered as thearubigin peaks. Because thearubigins are complex compounds and their standards are not commercially available, the method was validated for repeatability of retention time (RT) and recovery of known compounds by UPLC. Determination of Free Radical Scavenging Property Using DPPH Assay. The radical scavenging capacities of black tea infusions were determined using the DPPH radical method.22 Briefly, 1 mL of 1:20 diluted infusion was added to 1 mL of 2 mM ethanolic DPPH solution in a cuvette. The mixture was shaken vigorously, and the absorbance was measured at 517 nm at 0 and 30 min. α-Tocopherol (200 μM) served as a positive control. All of the tests were performed in triplicate, and the inhibition rate was calculated according to the formula of Yen and Duh.33 Cell Culture. The human endothelial cell line (EA.hy926) was procured from American Type Culture Collection (ATCC; VA). Cells were cultured in DMEM (Sigma) supplemented with 2 mM Lglutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% vol/vol FBS (Gibco, Invitrogen). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. HUVECs were cultured in M199 supplemented with endothelial growth factor, 0.001% heparin sulfate, 10% fetal bovine serum (FBS) (v/v), 1% penicillin (w/v), and streptomycin (w/v). Cells were incubated at 37 °C in 95% humidified air and 5% CO2. After 70−80% confluence was attained, the cells were subcultured by trypsinization. Determination of Antioxidant Property of Black Tea Infusion. Tea infusion-induced antioxidant property was determined as described previously.34 Briefly, EA.hy926 (2.5 × 104) cells were seeded onto 96-well flat bottom tissue culture plates for 6 h. After adherence, cells were treated with black tea infusion for 24 h, followed by wash to remove adhered polyphenols. The cells were then loaded with DCF-DA (1 μM) for 30 min followed by a wash with serum free medium. Subsequently, cells were treated with 100 μM H2O2 for 5 min. The antioxidant capacity of cell is determined by its ability to quench H2O2 entering inside the cellular compartment. The free intracellular H2O2 was determined by its reaction with DCF dye. The fluorescence intensity was measured at excitation/emission maxima of

been shown to be a major contributor for vasodialatory mechanism,27 and actives modulating NO production in endothelial cells have been classically used to screen molecules for vascular health benefits.28 Additionally, there are also reports of some of the small molecular weight polyphenols being bioavailable29 without any biotransformation. A global database on the polyphenol levels of various black tea has been published.21 The data suggest that there is a huge variation in polyphenol levels among the black teas grown across the globe.21 There has been no attempt made to date to understand how these variations in the levels and composition of flavonoids correlate with their bioefficacy for vascular function. Also, it is not clear if bioefficacy of tea polyphenols is linked to their amount in general, or whether it is driven by specific composition of polyphenol in tea.30 In the present study, we investigated representative teas from select geographies for their influence on in vitro markers of endothelial function. Tea infusions were quantified for their polyphenolic content, and their biological functions were evaluated as free radical scavenging, intracellular antioxidant property, and NO production in endothelial cells. Further, the composition of all of these tea infusions were analyzed and correlated with investigated biological markers using the Pearson correlation. This study provides knowledge on the variability in polyphenolic content of WoBTs and their correlation to biological functions such as antioxidant property and NO enhancement properties implicated in endothelial function in vitro and therefore eventually might influence the in vivo cardiovascular benefits of tea.

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MATERIALS AND METHODS

Materials. 4-Amino-5-methylamino-2′,7′-difluororescein diacetate (DAF FM-DA) and 5-(and -6)-chloromethyl-2′,7′-dichloro-dihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) were purchased from Invitrogen (OR). DMEM, 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), α-tocopherol, N-acetyl-L-cysteine (NAC) hydrogen peroxide (H2O2), Folin−Ciocalteu’s phenol (FC) reagent, and water (HPLC grade) were purchased from Sigma Chemical Co. (MO). Unless specified, all other reagents were procured from Sigma Chemical Co. (MO). Black tea samples were procured from Unilever R&D, Colworth. Preparation of Black Tea Infusion. Tea infusions were prepared as mentioned elsewhere.21 Briefly, black tea was finely ground with mortar and pestle and passed through 0.5 mm sieve to remove tea fibers. 100 mL of boiling water (HPLC grade) was poured unto 2 g of ground powder and stirred. The content was allowed to stand for 2 min, followed by a stir. The content was filtered through 0.4 μm Whattman filter placed under vacuum. The infusion was immediately frozen in liquid nitrogen to prevent further oxidation or degradation. This process was employed to reduce variability during the extraction process as factors like particle size, fibers, and temperature play a very crucial role in the extraction process. The extraction process was standardized to maximize the yield of flavonoids and its batch to batch reproducibility. Quantification of Polyphenol in the Infusion Using the Folin−Ciocalteu (FC) Method. The total polyphenol content of the infusion was determined colorimetrically using Folin−Ciocalteu (FC) phenol reagent21 as per ISO 14502-1. Briefly, 20 μL of 1:20 diluted tea infusion was mixed with 40 μL of Na2CO3 in a 96-well microtiter plate. 50 μL of FC reagent was added after 5 min and incubated in the dark for 1 h. Reaction product was measured at 765 nm using TECAN2000. Different concentrations of gallic acid were used as standard, and polyphenols were quantified as gallic acid equivalents. Quantification of Catechin, Theobromine, and Gallic Acid by HPLC. Catechins, caffeine, theobromine (TB), and gallic acid (GA) were analyzed by ISO 14502-2:2005(E) method.31 For HPLC analysis, B

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Figure 1. Dissolved polyphenol levels in the prepared tea infusion for the global black teas (WoTs). The samples were prepared and the polyphenol were quantified by Folin−Ciocalteu (FC) phenol method using gallic acid as standard. Data are presented as mean ± SD. Statistical comparisons were made using Student t test. “*” and “**” values were statistically significant (*p < 0.05; **p < 0.01) with respect to teas with low polyphenols (#). 495/515 nm, respectively, using TECAN 2000. Cells treated with the infusion but without H2O2 served as normalization control. NAC (25 μg/mL) was used as positive control. Measurement of Intracellular Nitric Oxide (NO) in Endothelial Cells Using Flow Cytometry. Intracellular NO production was measured using flow cytometry.35 EA.hy926 (1 × 105) cells were seeded in 24-well tissue culture plates. Cells were starved for 12−14 h after adherence to reduce basal NO levels. Cells were loaded with DAF FM-DA (1 μM) for 30 min and washed twice. Subsequently, cells were stimulated with black tea infusion for 30 min, in the presence or absence of L-NAME (10 mM; 30 min preincubation). The stimulated cells were trypsinized and fixed with 2% PFA for 15 min. A population of 10 000 cells were gated and segregated on the basis of their relative fluorescence intensities using FACS Calibur (Becton Dickenson; San Diego, CA). The mean fluorescence yield was measured and compared to the respective population in untreated cells. Statistical Analyses. Data are presented as mean ± SD of at least three independent experiments (n = 3) in triplicate. Variances of mean values were statistically analyzed by the Student’s t test. p ≤ 0.01 was considered significant. Pearson correlation was carried out using Sigma stat software to observe the correlation between NO production and antioxidant property versus various ingredients in tea infusion.

antioxidant property and NO enhancement ability in endothelial cells. Polyphenol Content in Black Tea Across Geographies. Black teas originating from various geographies (Supporting Information Figure 1) were used in the study, and the tea infusions were prepared (as mentioned in M&M) and analyzed for their total polyphenol levels using Folin−Ciocalteu reagent21 and expressed as gallic acid equivalents. The data are represented in Figure 1. There was a significant variation in the extractability of polyphenols under normally used conditions for brewing tea (data not shown). These findings are similar to those reported previously.37,38 Variability of polyphenols in end cup may be the result of the inherent variation in polyphenol levels present in the tea leaves or due to their extractability.38 The major reason for variation in extractability in turn arises due to the difference in the particle size and the nature of infusion protocol. We ensured minimum variability in extraction by standardizing the protocol for infusion preparation by maintaining uniformity in granule size (0.2−0.4 mm), infusion time (3 min), cycles of stirring (8 cycles), and filtration time (1 min), so as to get reproducible polyphenol extraction. When analyzed for polyphenols levels in various tea infusions (Figure 1), Ceylon Uva (name associated with Uva province), Darjeeling, Kenya, Ceylon LGS (Ceylon, low grown semishaded), SI (South Indian) Nilgiries and Java teas contained high polyphenol levels (above 200 mg equiv GA/100 mL), while teas from Vietnam and Argentina contained the least amounts (∼100 mg equiv GA/100 mL). Chinese, Turkish, Assam, Ceylon GMD (Ceylon, good medium dust), and Sumatra teas contained intermediate polyphenols levels (125− 175 mg equiv GA/100 mL). Our data suggest that the variation in the levels of polyphenol exists in black teas (ranging from ∼100−250 mg equiv GA/100 mL/2g black tea) across geographies (WoBTs). Such variations in polyphenol levels in teas from various geographies have previously been reported.21 The difference in the polyphenol levels has been shown to be associated with the altitude in which these teas have been grown.39,40 In these studies, they demonstrated that amounts of all of the catechins increased significantly with increasing altitude. Although higher levels of polyphenols would exhibit

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RESULTS AND DISCUSSION On the basis of the extent of fermentation, tea is classified into three major types: black (maximum oxidized), green (minimum oxidized), and oolong (partially oxidized) tea. The compositional profile of flavonoids also varies with the extent of oxidation reactions;25 however, the amounts of total polyphenols in tea remain comparable, if made from the same leaf source.34 Because the water extractability of flavonoid species differs, infusions of these teas also vary in terms of their endcup composition.34 Also, composition of flavonoids differs, based on the process of tea making (orthodox (rolling tea leaves) or CTC (cut, tear, curl)),36 nature of tea variety,34 and region in which it is grown.21 This intrinsic variation of flavonoids within teas can affect both end-cup delivery of flavonoids and their composition. Considering flavonoids are the main efficacious species in tea, such variations are likely to cause differences in the extent of benefits delivered by various teas. In this Article, we investigated the polyphenol extractability of various black teas and evaluated their C

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Figure 2. DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging property of different black tea infusions normalized to polyphenol levels. The tea infusions were normalized to 25 μg/mL gallic acid equivalents. DPPH free radical scavenging property was determined as mentioned in M&M. Data represented as IC50 ± SD (μg/mL). α-tocopherol (200 μM) was used as positive control. Statistical comparisons were made using Student t test. The “#” values were statistically significant (p < 0.01) with respect to untreated control (UTC) and various tea samples. Free radical scavenging properties amongst WoT samples were not significantly different.

electron delocalization.48 However, in addition to free radical scavenging property, the intracellular antioxidant elements such as GSH and free radical reducing enzymes (SOD, catalase and peroxidase) play a crucial role in determining redox properties of cells. Such intracellular antioxidant systems prevent the uncontrolled generation of free radicals and activated oxygen species, or inhibit their reactions with biological materials, thereby preventing protein malfunction. eNOS uncoupling is one of such pathophysiological alterations observed in endothelial dysfunction.9,49 Ideally, vasodilatation measured as flow-mediated dilation (FMD) with tea intervention should be the basis of evaluation of VF in humans,6 but such measurements are resource intensive and practically not viable for investigating multiple samples.50 Therefore, physiologically relevant biomarkers such as antioxidant activity (AOX) and eNOS activity measured as increases in NO levels were chosen as representative surrogate in vitro measures of vascular function.9,49 Alteration in the in Vitro Redox Property by Various Black Teas. Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking stimulate the generation of reactive oxygen species in the endothelium.9 This increase in intracellular ROS species plays an important role in vascular damage and progression of various vascular diseases, including atherosclerosis, ischemic heart disease, hypertension, and congestive heart failure.2,9,28 Cellular mechanisms of ROS production involve sources such as the mitochondrial respiratory chain family of NADPH oxidases, xanthine oxido-reductase, or uncoupled nitric oxide synthase. All of these enzyme levels are found to be upregulated and activated in animal models of hypertension, diabetes, and in patients with sedentary lifestyle and cardiovascular risk.9,51 On the other hand, an accelerated degradation of NO (by its reaction with O2·−) is likely to occur in vascular disease, which in turn leads to eNOS uncoupling and enzyme dysfunction.52 Generation of ROS is counteracted by its destruction through antioxidant systems like super oxide dismutase, catalase, glutathione redox cycle, and ROS scavengers.12 The antioxidant benefit of tea is largely due to its ability to increase antioxidant enzymes2,3 and enhance intracellular ROS

higher biological activities, to understand the influence of composition of polyphenol on biological activity, we equalized the polyphenol levels of different teas (WoBTs) and evaluated their biological functions. Free Radical Scavenging Property of Different Black Teas. Oxidation of lipids and protein has been implicated in various diseases etiologies,41 especially in vascular pathophysiology.42,43 Mechanistically, increase in the levels of reactive species in the plasma leads to generation of oxidized lipid and proteins, which are electrophilic in nature.44 These reactive molecules (reactive lipid/protein species) interact with cellular nucleophiles such as the amino acids cysteine, lysine, and histidine, which are components of cell surface receptors. Binding of electrophilic molecules changes the conformation or activation status of receptor, resulting in altered signaling pathways44 or generation of intracellular ROS species. Also, increased levels of superoxides and peroxides react with NO produced in endothelial cells, resulting in reduced bioavailability of NO12,13,45 for its function. Consumption of phyto-chemicals rich in flavonoids is reported to increase oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), and total radical trapping parameter (TRAP) levels in plasma, which are physiological measures of free radical scavenging property in vivo.46 On the basis of this hypothesis, we used the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH)-based in vitro scavenging assay to test the ability of components to act as free radical scavengers or hydrogen donors and to evaluate the free radical scavenging property of tea phyto-chemicals. The free radical scavenging properties of different teas were evaluated. The IC50 is represented in Figure 2. All of the tested tea infusions showed good DPPH scavenging property (Supporting Information Figure 2), but at equivalent polyphenol concentrations, their efficacies were not significantly different from each other (Figure 2). Similar correlation was deduced by Anesini et al.,47 for Argentinean commercial teas. In general, the radical-scavenging property of flavonoids depends on the molecular structure and the substitution pattern of hydroxyl groups, that is, on the availability of phenolic hydrogen and on the possibility of stabilization of the resulting phenoxyl radicals via hydrogen bonding or by expanded D

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Figure 3. Antioxidant property of different black tea infusions at equivalent polyphenol levels in endothelial cells. EA.Hy926 cells were treated with different concentrations of tea infusions (5−25 μg/mL) for 24 h. N-Acetyl cystiene (NAC) was used as positive control (100% H2O2 scavenging). Cells treated with H2O2 served as negative control (0% H2O2 scavenging). Data represented for 25 μg/mL (■) and 5 μg/mL (□) with mean ± SD (n = 3). Statistical comparisons were made using the Student t test. Values were considered statistically significant (#p < 0.01) if % antioxidant capacity is above 50%. The antioxidant capacity of all WoTs was statistically significant ($p) to negative control (H2O2).

Figure 4. Pearson-correlation analysis between antioxidant property (AOX) and various components in tea infusion. (A) AOX versus total catechins; (B) AOX versus gallated catechin (EGCG+ECG+CG); (C) AOX versus total gallated polyphenols (EGCG+ECG+CG+TF4); (D) AOX versus nongallated catechins (EC+EGC+GC); (E) AOX versus gallic acid; and (F) AOX versus caffeine. The correlation graph was derived with 25 μg/mL PP of tea infusion.

scavengers such as GSH,2,9 which prevents deleterious effects of intracellular ROS. Thus, this mechanism presumes to play a major role in mediating the cardio-protective effect of tea.2,3,9,28 To evaluate the antioxidant property of these teas under cellular conditions, cells were pretreated with tea, followed by oxidant trigger, H2O2. N-Acetyl-L-cysteine (NAC) was used as positive control. The data are represented in Figure 3. The assays were performed at equated polyphenol levels to capture any effects due to compositional variations of tea infusions.

Among the tested black teas, infusions from Ceylon GMD, Nilgiries, Kenya, Assam, and Ceylon Uva showed higher antioxidant property (>50% H2O2 scavenging) (Figure 3) as compared to other teas. Because teas were tested at equated polyphenol levels, the data indicate that the chemical composition of tea infusion rather than polyphenol amount contributes to the observed antioxidant (AOX) benefit. Although we did not analyze the type of cellular antioxidant components like GSH, SOD, catalase, and other free radical scavenging enzymes, the results represent the total intracellular E

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Figure 5. Pearson-correlation analysis between antioxidant property and specific catechins in tea infusion. (A) AOX versus ECG; (B) AOX versus EC; (C) AOX versus EGCG. The correlation graph was derived with 25 μg/mL PP of tea infusion.

Figure 6. Pearson-correlation analysis between antioxidant property and specific theaflavins (TFs) and phenoli acids in tea infusion. (A) AOX versus TF1; (B) AOX versus TF2; (C) AOX versus TF3; (D) AOX versus TF4; (E) AOX versus total TFs; and (F) AOX versus theobromine (TB). The correlation graph was derived with 25 μg/mL PP of tea infusion.

scavenging capacity generated after pretreatment with tea infusion, which does represent the net effect of all of these protective enzymes. There was no significant correlation between AOX property and various tea actives (Figures 4−7). However, there was nonsignificant positive association with total catechins, gallated catechins, and gallated polyphenols, while nongallated catechins showed neutral effect (Figure 4A−D). Specifically, epicatechingallate (ECG) and epicatechin (EC) showed neutral correlation, while epigallocatechin-gallate (EGCG) showed nonsignificant positive correlation (Figure 5A−C). Among theaflavins (Figure 6A−E), TF1 (r2 = 0.12), TF3 (r2 = 0.19), and TF4 (r2 = 0.14) showed nonsignificant positive correlation, while TF3 showed neutral effect (r2 = 0.06). However, nonflavonoid polyphenol was also evaluated for their function. Gallic acid (r2 = 0.13) and caffeine (r2 = 0.13) showed nonsignificant negative correlation (Figure 4E,F), while theobromine (r2 = 0.07) showed neutral effect (Figure 6F). A large proportion of polyphenols in black tea exists as oligomeric species termed as thearubigins (TRs); however, the complete chemical characterization of these TRs is still under

investigation. Nicholai and others have reported extensive studies on SII TRs.53−55 By employing advanced analytical techniques such as electronspin transform ion cyclotron resonance mass spectroscopy (ESI-FTICR), they53 showed that TRs are comprised of several thousands of compounds, with a molecular weight ranging between 1 and 1.5KDa.18,55 They also observed a typical Gaussian hump in caffeine precipitated SII TRs,18 containing majorly trimers and tetramers of flavan-3-ols.18 Spectroscopic studies showed that TRs have basic catechin structure with benzotropolone and ortho-quinone moieties.53 The group has also investigated the possible mechanism on how these molecules are formed using MALDI-TOF studies of SII TRs. They suggest the formation of polyhydroxylated species, which are formed via oxidation and nuclieophilic addition of water and hydrogen peroxide to catechin dimer and oligomers. All of these extensive reports provide information that thearubigins have flavonoid backbone structure.53,55 Among flavonoids, flavan-3-ols (catechins) have absorption maxima at 278 nm, flavonols and chlorogenic acids at 350 nm, and oligomeric flavonoids (theaflavins) at 380 and 460 nm.56 F

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Figure 7. Pearson-correlation analysis between antioxidant property and various TRs (A−W) with specified retention time (RT) in tea infusion. The correlation graph was derived with 25 μg/mL PP of tea infusion.

During fermentation of tea, oxidation of monomeric flavonoids leads to formation of TRs. Such transformation involves oxidation followed by coupling and ring condensation reaction. These reactions would result in products with increased

conjugation and absorption at longer wavelengths. Hence, oxidized catechins or TRs should have absorption maxima also at ∼380 nm, which has been used to relatively quantify their amounts.56 The known phenolic acids, flavonol glycosides, and G

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Figure 8. Nitric oxide stimulating potential of different black tea infusions at equivalent polyphenol levels in endothelial cells. The infusions were evaluated for NO enhancement in EAHy926 cells. The cells were treated with different concentrations of tea infusions (25 μg/mL (■) and 5 μg/mL (□)) for 30 min. Data are presented as mean ± SD (n = 5). Reservetrol (10 μM) was used as positive control. Statistical comparisons were made using Student t test. “#” and “*” values were statistically significant (p < 0.01) with respect to untreated control (UTC) and Argentinean tea sample (lowest in NO enhancement).

Figure 9. Nitric oxide stimulating potential of different black tea infusions at equivalent polyphenol levels in HUVEC. The data represent NO levels in primary HUVEC at 25 μg/mL concentration of tea infusion for 30 min. The “#” values were statistically significant (p < 0.01) with respect to reservetrol ($).

property of tea. However, these actives in tea showed nonsignificant positive correlation, indicating that positive species such as catechins, TFs, and some TRs might cumulatively be responsible for the observed AOX benefit of black tea. Further work is required to evaluate the mechanisms involved in the induction of cellular antioxidant property by specific flavonoids/phenolic acids. In Vitro Efficacy of Teas To Promote Endothelial NO Production. Endothelial dysfunction (ED) significantly contributes to pathogenesis and clinical manifestation of CVD.6,9 ED is characterized by decreased endothelial NO production in endothelial cell.6,27 Black tea is reported to activate eNOS and thus enhance NO production in endothelial cells.9,28 We analyzed 13 black teas, with distinct polyphenol amounts and composition, for their NO potentiating effect after normalizing for their polyphenol concentration. The results are represented in Figure 8. Variable response to agonist stimulation has been reported in different endothelial celltypes.59 We evaluated NO production in response to various tea infusions (25 μg/mL) in primary HUVEC (Figure 9). The stimulatory response for the tested tea infusion behaved in a similar manner in both EA.hy926 (Figure 8) and HUVEC

TFs that absorb at this wavelength were not considered for TR analysis. We observed several peaks in the UPLC chromatogram (Supporting Information Figure 3A−C) after elimination of known components of black tea such as catechins, TFs, phenolic acids, and flavonol glycosides. The peaks that showed absorption at 380 nm and had retention time above 18 min were considered as thearubigin (TR) peaks. For correlation purpose, we only considered 29 major peaks that were consistently present in majority of the tea (Figure 7). The TR peaks having retention time of 18.447, 29.933, 33.441, and 34.237 (Figure 7A, E, L, and N) showed nonsignificant positive correlation with antioxidant potential, while TR peaks having retention time of 33.131 showed nonsignificant negative correlation (Figure 7K). Among the tested black teas, infusions from Ceylon GMD, Nilgiries, Kenya, Assam, and Ceylon Uva showed higher antioxidant property. This may be due to the presence of a higher amount of theobromine and gallated catechins, which are previously reported to increase expression of antioxidant enzymes.57,58 Our data suggest that none of the other actives analyzed showed significant correlation with antioxidant H

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Figure 10. Pearson-correlation analysis between nitric oxide (NO) levels and various components in tea infusion. (A) NO versus total polyphenols (PP); the circled clusters indicate quantitative correlations of PP (gray circles (rhomboid) indicate PP range from 30 to 65 μg/mL GA equiv; black circles (square) indicate PP range from 15 to 30 μg/mL GA equiv, and black dotted circles (triangle) indicate PP range from 5 to 15 μg/mL GA equiv) with NO levels. The data indicate that NO potentiating effect increases with increasing concentration of PP. The dotted box represents qualitative correlation of PP with NO level. The data indicate that some teas are efficient enough to produce NO levels (2−3-fold) at very low PP concentration. (B) NO versus total catechins. (C) NO versus gallated catechin (EGCG+ECG+CG). (D) NO versus nongallated catechin (EC+EGC +GC). The correlation graph was derived with 25 μg/mL PP of tea infusion.

Figure 11. Pearson-correlation analysis between nitric oxide levels and specific flavonoids in tea infusion. (A) NO versus gallated PP (EGCG+ECG +CG+TF4); (B) NO versus EC; (C) NO versus EGCG; (D) NO versus ECG. The correlation graph was derived with 25 μg/mL PP of tea infusion.

polyphenol levels to Kenyan teas, showed the highest NO enhancement (Figure 9). The data suggest the possibility that composition of polyphenols, besides quantity, plays a significant role in eNOS activation by various geographical teas. Several functional species in tea are reported to stimulate eNOS enzyme and enhance NO production in endothelial cells. These include

(Figure 9). Although, in general, higher polyphenol levels resulted in higher NO levels (Figure 10A), the NO potentiating effect of WoBTs did not always correlate with the polyphenol levels (Figure 10A). Tea infusions from Assam, Darjeeling, and Ceylon (UVA), which had higher polyphenols as compared to other teas, showed higher NO enhancement as compared to other teas. Darjeeling teas, while being comparable in I

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Figure 12. Pearson-correlation analysis between nitric oxide levels and specific theaflavins (TFs) and phenolic acids in tea infusion. (A) NO versus TF1; (B) NO versus TF2; (C) NO versus TF3; (D) NO versus TF4; (E) NO versus total TFs; (F) NO versus caffeine; (G) NO versus theobromine; (H) NO versus gallic acid; (I) NO versus pyrogallol. The correlation graph was derived with 25 μg/mL PP of tea infusion.

A large proportion (70%) of flavonoids in the black tea are present as TRs.17 The TRs fractions are reported to show NO enhancement in endothelial cells and dilation of mesenteric arteries.5 We evaluated the correlation between NO production and different TR peaks obtained by UPLC chromatogram. We could not find any significant positive correlation with the majority of the TR peaks (Figure 13). However, TR peak having retention time of 32.37 and 35.89 (Figure 13J and O) showed positive correlation with NO enhancement, while TR peak corresponding to retention time of 33.13 showed negative correlation (Figure 13K). From our data, it is evident that, among polyphenol, catechins (EC, EGCG, and ECG) are maximally active, while contribution by theaflavins remains miniscule. However, two of the investigated UPLC-based TR peaks showed positive correlation, while one TR peak showed negative correlation. This suggests that black tea has diverse pharmacological species, which strongly influence the NO potentiating effect in endothelial cells. Moreover, isolation, characterization, and screening of TRs would be an open area of investigation. Among the tested tea infusion, Ceylon Uva, Darjeeling, Ceylon LGS, Assam, and China tea showed significant NO potentiating effect. We observed a higher amount of gallated catechin, TFs (TF2 and TF4), and some of the positive TR species to be present in these teas, which may alone or in combination explain enhanced NO production by these teas. We also tried to understand the correlation between tea/tea ingredient-mediated increase in NO enhancement and antioxidant capacity of the cell. We observed no correlation between NO production in endothelial cell with enhanced intracellular free radical scavenging property (Figure 14; r2 = 0.01). It is reported that increased levels of ROS would

catechins,5 theaflavins,5,9,25 and flavones like quercetin, kaempferol, and rutin3,24,26 and thearubigins.5 Nonflavonoid components in tea like L-theanine,60 theobromine,61,62 and caffeine63 are also reported to contribute to this function. When analyzed for the correlation between NO production and different flavonoid components, total catechin showed significant correlation (Figure 10B; r2 = 0.68). In general, the levels of catechins in black tea varied from 0.9% to 11.5%. There was a good correlation with gallated catechins (Figure 10C; r2 = 0.61), nongallated catechins (Figure 10D; r2 = 0.64), and gallated polyphenols (Figure 11A; r2 = 0.64). This indicates that, in addition to gallated catechin, gallated polyphenols, which include gallated TFs and gallated TRs, would contribute to improve NO enhancement. There was no significant difference, however, between gallated and nongallated catechins (Figure 10C and D). Among the catechins, EC (Figure 11B; r2 = 0.71), EGCG (Figure 11C; r2 = 0.50), and ECG (Figure 11D; r2 = 0.0.65) showed significant positive correlation. We also analyzed the correlation for various TF species (Figure 12A−E). Total TFs showed mild positive correlation (r2 = 0.20), although strong positive correlation was observed for gallated TFs like TF2 (r2 = 0.32) and TF4 (TF4, r2 = 0.30), while nongallated TFs like TF1 (r2 = 0.02) and TF3 (r2 = 0.03) showed neutral effects. The amounts of such TF species are very low; hence, their contribution toward NO stimulatory effect might be negligible. We also analyzed for some of the major phenolic acids in black tea such as caffeine, theobromine, gallic acid, and pyrogallol. Caffeine (r2 = 0.50) and theobromine (r2 = 0.34) (Figure 12F,G) showed significant positive correlation, while other phenolic acids failed to show any correlation (Figure 12H,I). J

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Figure 13. Pearson-correlation analysis between NO levels and various TRs (A−X) with specified retention time (RT) in tea infusion. The correlation graph was derived with 25 μg/mL PP of tea infusion.

antioxidant and the eNOS activity of tea synergistically could contribute to the vascular benefit efficacy of specific teas. Further, spatial distribution of NO and ROS can also be crucial in NO dissipation under physiological stress. Under

dissipate NO levels and contribute toward ED. However, it needs to be further evaluated how the antioxidant activity of tea could potentiate NO bioavailability under stress conditions that would normally prevail in the in vivo situation, and both the K

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*Tel.: +91 80-39831068. Fax: +91 80-28453086. E-mail: anita. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Vikas Pawar, Unilever R&D, Mumbai for his technical help and for the critical review of data by Dr. Gurmeet Singh, Unilever R&D, Colworth and Dr. Sujatha Jayaraman, Unilever R&D, Bangalore.

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ABBREVIATIONS EC, epicatechin; ECG, epicatechingallate; EGCG, (−)-epigallocatechin-3-gallate; TF-2, theaflavin-3-gallate; TF-3, theaflavin-3′-gallate; TF-4, theaflavin-3,3′-digallate; WoBTs, world of black teas; ED, endothelial dysfunction; AOX, antioxidant property; CTC, cut tear curl

Figure 14. Pearson-correlation analysis between nitric oxide levels and antioxidant property of various tea infusions. The correlation graph was derived with 25 μg/mL PP of tea infusion.

condition of stress, ROS is generated from the mitochondria and has been shown to play a major role in NO dissipation.9,49 In our experiments, we used H2O2 as the source of ROS (external species), which could be different from the ROS generated under physiological condition. Further experimentation would be needed to dissect their specific effects. In summary, we used 13 black teas (WoBTs) representative of varied geographies, to address the contribution of quantity and composition of tea flavonoids on antioxidant and NO enhancement properties. Even though the endothelial cells were treated with tea infusions containing equated amounts of polyphenols, they induced different levels of cellular antioxidant activity and NO levels, suggesting that the composition of polyphenols in tea infusions determines the response. Statistical analysis using Pearson correlation indicated that the majority of ingredients in black tea such as catechins, TFs, TRs, and phenolic acids moderately influence antioxidant potential in endothelial cells, while its NO potentiating effect is mainly regulated by catechins in tea. Our data also suggest that the net effect of black tea on VF benefit may be due to a multitude of ingredients, majorly influencing NO enhancement, while also mildly contributed by its antioxidant benefit. Most interestingly, we also found that gallic acid and caffeine negatively affect antioxidant property, while caffeine and theobromine positively affect NO levels in endothelial cell. One of the limitations in our study was the absence of characterization of specific TRs and evaluation of their efficacy on biological function. The study was also limited to in vitro investigation using cell free and cellular systems, as these systems do not adequately address issues of bioavailability and metabolic transformation. These would be the areas in which further investigations need to be undertaken.

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ASSOCIATED CONTENT

S Supporting Information *

Figures showing illustration of geographical teas, DPPH (1,1diphenyl-2-picrylhydrazyl) free radical scavenging property of different black tea infusions in end cup, and UPLC fingerprinting for thearubigins. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +91 80-39831217. Fax: +91 80-28453086. E-mail: paul. [email protected]. L

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