beverages Article Total Polyphenol Content and Antioxidant Capacity of Tea Bags: Comparison of Black, Green, Red Rooibos, Chamomile and Peppermint over Different Steep Times Riley Cleverdon 1,2,†, Yasmeen Elhalaby 1,2,†, Michael D. McAlpine 1,2 ID , William Gittings 1,2 and Wendy E. Ward 1,2,* ID 1 Department of Kinesiology, Faculty of Applied Health Sciences, Walker Complex RM 290, Brock University, 1812 Sir Issac Brock Way, St. Catharines, ON L2S 3A1, Canada; [email protected] (R.C.); [email protected] (Y.E.); [email protected] (M.D.M.); [email protected] (W.G.) 2 Centre for Bone & Muscle Health, Brock University, 1812 Sir Issac Brock Way, St. Catharines, ON L2S 3A1, Canada * Correspondence: [email protected]; Tel.: +1-905-688-5550 (ext. 3024) † These authors contributed equally to this work. Received: 13 December 2017; Accepted: 19 January 2018; Published: 12 February 2018 Abstract: Globally, traditional and herbal teas are a prominent dietary source of polyphenols, and represent a class of bioactive molecules that are closely associated with a variety of health benefits. Most consumers prepare tea using tea bags, although there is little information about whether this production step alters the content of the final product. The study purpose was to investigate the effect of steep time and tea type on the polyphenol content and predicted antioxidant capacity of commercially available tea bag products, including Green, Orange Pekoe, Red Roiboos, Peppermint, and Chamomile. Total polyphenol content (TPC), antioxidant capacity (1,1-diphenyl-2-picrylhydrazyl inhibition), and total predicted antioxidant capacity were measured in aliquots sampled every minute for 10 min. Polyphenols were extracted into solution in a nonlinear fashion, with ~80–90% of the TPC appearing within 5 min of tea bag immersion. Moreover, a significant range in TPC values was observed between products, with true teas containing at least two-fold greater polyphenol content than the herbal varieties. Our results are consistent with previous work using loose-leaf tea products and demonstrates that tea bag products are an effective source of polyphenols that may offer health benefits relating to their constituent antioxidant activity. Keywords: tea; polyphenol; antioxidant; steep time; Camellia sinensis; Aspalathus linearis; Mentha piperita; Matricaria chamomilla 1. Introduction Tea, particularly green and black, is increasingly being studied for its potential role in the prevention and attenuation of a wide variety of diseases [1–6]. Given that the term tea is often used erroneously to also refer to rooibos and herbal beverages that do not originate from Camellia sinensis, it is important to acknowledge that these beverages are most accurately referred to as tisanes while recognizing that both tea and tisanes all have polyphenols, a common component that is believed to mediate health benefits. However, the polyphenol profile is markedly different among teas and tisanes and this may in part explain the diverse potential biological activities that includes antiosteoporotic, antioxidant, antiatherosclerotic, antiallergic antifibrotic, hypolipidemic, hypocholesterolemia, anti-obesity, antiviral, antimutagenic, antimicrobial, anticarcinogenic, antidiabetic, and neuroprotective effects [1–8]. Beverages 2018, 4, 15; doi:10.3390/beverages4010015 www.mdpi.com/journal/beverages Beverages 2018, 4, 15 2 of 13 Rooibos, green or red, is derived from Aspalathus linearis, with red rooibos being the oxidized form, while common herbal teas such as peppermint and chamomile are from Mentha piperita and Matricaria chamomilla, respectively [7–11]. There is a strong interest in rooibos as it does not contain caffeine and many herbal tisanes are also caffeine-free, whereas a single cup of green or black tea from the Camellia sinensis plant contains approximately 30–50 mg of caffeine per serving while a serving of brewed coffee contains 135 mg [12]. To put this in perspective, Health Canada’s recommendations suggest no adverse effects in adults with caffeine intakes of up to 400 mg [12]. In studying the health benefits of tea consumption in the laboratory setting, it is important to understand how steep time alters total polyphenol content (TPC) and antioxidant capacity, and to also prepare tea used in laboratory experiments in a similar manner as a consumer. Two considerations include the steep time as well as whether loose tea or a commercial tea bag is used. The effect of steep time on polyphenol content and antioxidant activity in eight commercially available and popular loose teas has been previously reported by our group [13]. Steep time was studied from 1 to 10 min at 1-min intervals, thus mimicking steep times often used by consumers. This study showed that over 50% of polyphenols were released in the initial 5 min of steep time and that TPC increased in a nonlinear relationship for all tea types tested [13]. Interestingly, longer steep time was not associated with greater antioxidant capacity (measured as the percent inhibition of the free radical DPPH) when the polyphenol content was standardized, but tea types did differ in their antioxidant capacity. Specifically, the green and black teas resulted in greater DPPH inhibition compared to rooibos (green or red) or herbals (peppermint or chamomile). However, when differences in TPC were considered, one but not both black teas studied and both green teas as well as peppermint had a higher predicted total antioxidant capacity than the rooibos (red, green) and chamomile tisanes. The finding of peppermint having a higher antioxidant capacity may have been due to smaller leaf size, and thus increased surface area that may have resulted in the higher TPC with longer steep time. Importantly, the aforementioned study investigated samples from loose-leaf teas only; however, it is unclear if these findings represent the steep time-polyphenol concentration relationship that might exist when bagged teas are brewed under similar conditions. Others have reported that particle size and use of packaging in a tea bag modulates polyphenol content [14,15]. In a study of black tea, tea from tea bags showed a significantly higher level of polyphenols than loosely-packed brands after a 5-min steep time [15]. Moreover, for bagged tea, TPC was significantly lower after a 1 min versus a longer steep time of 3, 4 or 5 min [15]. Tea in tea bags is widely available and in recent years has become an increasingly common choice for consumers. For example, in the UK, the UK Tea and Infusions Association reports an increase in use of teabags has increased from 5% in 1960 to 96% in 2007 [16]. The present study further develops the experimental paradigm from our previous study in which we showed that steep time altered TPC in a variety of loose-leaf teas and tisanes. Given the widespread and increasingly prevalent usage of tea bags by consumers, the objective of this study was to determine how steep time altered TPC and antioxidant activity of commercially-available tea bags that contained black and green teas, as well as red rooibos and popular herbals (chamomile, peppermint). 2. Materials and Methods The methods used in the current experiments have been described in detail previously [13], and only minor changes have been made to the apparatus to allow the steeping of tea prepared from tea bags, which requires a larger vessel than that required for loose-leaf products. Tea samples consisted of Orange Pekoe (OP) or Green (G) teas, or Red Rooibos (RR), Peppermint (P), or Chamomile (C): all products were purchased from a local supplier and prepared according to the manufacturers recommendations (see Tables1 and A3 for tea product details). All experiments for each tea were performed from a single batch of product. Tea samples were steeped using a tea mass to distilled water ratio of 0.04 g/mL in a specially prepared glassware apparatus with a Liebig condenser (Thermo Fisher Scientific, Burlington, ON, Canada) to prevent loss of volume by vaporization (see Figure1). Samples Beverages 2018, 4, 15 3 of 13 (100 µL) were extracted at 1-min intervals for 10 min, after which they were microfiltered (0.2 µm) and immediately analyzed for polyphenol content. The boiling vessel used in the present study was larger than that used for our previous work with loose leaf teas [13], and therefore rather than independent tea batches steeped to each time point, we utilized a repeated-measures design where small aliquots were removed from the 500 mL vessel for each of the time points to reduce inter-batch variability in the study design. Beverages 2018, 4, x Table 1. Origin and description of teabag samples. 3 of 13 ◦ Commonwhich they Name were Scientificmicrofiltered Name (Variety)(0.2 µm) and Tea imme Typediately Steep analyzed Temperature for polyphenol ( C) Tea content. Mass Per BagThe (g) boilingOrange Pekoevessel used inCamellia the present sinensis study was Blacklarger Tea than that used for 96◦ Cour previous work 3.07 with± 0.01 loose ◦ leafGreen teas Tea [13], and thereforeCamellia sinensisrather than independGreen Teaent tea batches 79steepedC to each time 2.07 point,± 0.02 we Red Roiboos Aspalathus linearis Red Rooibos 96◦C 2.08 ± 0.01 utilizedChamomile a repeated-measuresMatricaria chamomilla design where smallHerbal aliquots were removed 96◦C from the 500 mL 1.21 vessel± 0.01 for eachPeppermint of the time pointsMentha to reduce piperita inter-batch variabilityHerbal in the study96 design.◦C 1.94 ± 0.01 FigureFigure 1. Experimental 1. Experimental apparatus apparatus for