Phytochemical Density of Some Promising Commercial Tea Brands

Abstract

Black Tea (Camellia sinensis) is an important source of phytochemicals with proven health benefits. The core objective of the present investigation was to characterize some promising commercial tea brands available in Pakistan for their active ingredients with special reference to flavanols. The results indicated that total phenolic contents were in the range of 26.42 to 29.74 g/100 g liquid tea, while antioxidant activity and DPPH reduction was in the range from 45 to 50 g/100 g liquid tea and 47 to 50 g/100 g liquid tea, respectively. Tea quality parameters including theaflavins, thearubingins, and theabronins were observed to be 1.71–1.79, 1.74–1.83, and 17.05–20.22 g/100 g liquid tea. Likewise, total catechins and caffeine were 1.01–1.28 and 1.22–1.25 g/100 g liquid tea, respectively. Tea extract of commercial brands containing a larger particle size exhibited better quality.

Keywords:

• Theaflavin
• Thearubingins
• Catechins
• Theabronins
• Caffeine

INTRODUCTION

Dietary phytochemicals are considered to be an effective tool to cure various human physiological disorders. Being therapeutic agents, they play a pivotal role in prevention of many ailments, particularly in life-style disorders. Moreover, their established antimutagenic effects modulate and stimulate the immune system resulting in normal functioning of the body.[1,2]

Several epidemiological studies have indicated that high intake of natural products is associated with reduced risk of a number of chronic diseases like atherosclerosis and cancer.[3–5] Such beneficial effects are attributed to compounds like flavonoids, anthocynids, and carotenoids. Flavonoids are secondary plant metabolites and contribute to the first defense line against oxidative stress, because they quench singlet oxygen radicals. The rich sources of flavonoids are fruits, vegetables, tea, and coffee.[6,7]

Black Tea (Camellia sinensis) belongs to the family Theaceae and is a fully fermented, pleasant, socially accepted, economical, and safe drink enjoyed by millions of people everyday. It is estimated that about 2.5 million metric tons of tea is manufactured annually, having a per capita consumption of 0.12 L per day. Black tea is a rich source of the polyphenols that are further subdivided into two classes: flavanols and flavonols. The flavanols are comprised of catechins, theaflavin, and thearubingins. Polyphenol oxidase is mainly responsible for catalyzing oxidation in tea leaf catechins[8] with orthoquinones to form theaflavins, that subsequently reacts with gallic acid to form epitheaflavic acid, collectively termed as thearubigins. The degree of fermentation and enzymatic oxidation bring differences in composition of various types of tea responsible for their distinguished characters.[9,10] Theaflavin and thearubingins are soluble in hot water, representing 20–35 g/100 g liquid tea of dry tea, out of which about 45 g/100 g liquid tea of the tea constituents are infused in hot water. Theaflavins comprise a group of four major constituents, namely, theaflavin, theaflavin 3-gallate, theaflavin 30-gallate, and theaflavin 3, 30-digallate. This formation occurs due to reaction between quinines derived from simple catechin and gallocatechin. They contributed towards tea brisk, astringent taste, and bright golden color to the infusion,[9] nevertheless, they are water soluble, acidic, and their rust-brown color gives richness to the taste.[11,12]

Estimation of theaflavin (TF) and thearubigins (TR) is essential to assess the tea quality along with other parameters like caffeine and theabronins.[13] In particular, theaflavins are used to assess the market value, clonel, and seasonal quality of black tea.[12] Traditional methods for the determination of polyphenols are dominated by the use of paper chromatography, column chromatography, and colorimetric measurement by using oxidation reduction properties of the molecules. Though most advanced methods like HPLC can also be used to isolate, identify, and determine individual polyphenolic compounds, calorimetric or spectrophotometer methods, however, are still the most widely used for the determination of total phenolic compounds, TF and TR, in tea due to ease of utilization, particularly in tea industries.[14,15] In Pakistan, negligible work has been carried out to evaluate the promising brands of tea with special reference to their antioxidant potential. The basic aim of the research was to explore the phytochemical density of the commercial tea brands available in the market.

MATERIALS AND METHODS

Procurement of Samples

Different commercial brands of black tea, i.e., paper packed, loose, and tea bags, were procured from the local market (Table 1). Reagents and standards were purchased from Sigma-Aldrich (Tokyo, Japan) and Merck (Darmstadt, Germany). The collected tea samples were stored at ambient temperature for further study.

Table 1 Treatment used in the study

Code of brands	Description
CB1	Commercial tea packed 1
CB2	Commercial tea packed 2
CB3	Commercial tea packed 3
LP	Loose tea
TB1	Tea bag 1
TB2	Tea bag 2

Preparation of Tea Extracts

Tea extracts were prepared using distilled water as described by Muralidharan.[16] For this purpose, 200 mL of boiling water with 2 g of tea was mixed and stirred on a magnetic stirrer (Gallan Kamp, Birmingham, England) for 10 min. The solution was filtered and the residue was washed with distilled water thrice. The tea solutions were combined, cooled, and finally diluted to 250 mL. Tea extracts were analyzed for their antioxidant potential for indices like total phenolic contents, antioxidant activity, and free radical scavenging activity (DPPH assay).

Total Phenolics

The tea solution (1 ml) was taken into a volumetric flask, then 4 mL of distilled water and 5 mL of tartrate solution (1 g of FeSO₄ and 5 g of KNaC₄H₄O₆ dissolved in 1000 mL distilled water) were added. It was diluted with 25 ml of buffer solution (23.377 g of Na₂HPO₄ in 1000 mL of distilled water and 9.078 g of KH₂PO₄ in1000 mL of distilled water in the ratio of 85 and 15% v/v). The absorbance was measured using UV/Vis spectrophotometer (CESIL CE7200, Cesil Instrumental, Cambridge, England) at 540 nm as described by Muralidharan.[16] The calculation was carried out by using the following expression:

where E is the absorbance reading of the spectrophotometer, V ₀ is total volume of the tea solution (250 mL), V ₁ is volume used for the measurement (1 mL), and W is the dry weight of the tea sample.

Total Catechins Determination

The sample (0.1 mL) was taken in a test tube containing (0.9 mL) methanol; then transfer 2.5 mL of vanillin solution (1% methanol) along with 10 mL 9 N HCl in the same tube. The solution was kept for 30 min before measuring with a UV/Vis spectrophotometer at 500 nm against the corresponding reagent methanol as blank.[17]

Antioxidant Activity

Two mg β-carotene was dissolved in 20 mL chloroform. An aliquot of (3 mL) solution was transferred in a 50-mL beaker, 40 mg of linoleic acid and 400 mg of Tween 20 were added. Chloroform was removed by purging with nitrogen. Oxygenated distilled water (100 mL) generated by bubbling air into distilled water for 1 h was added in β-carotene emulsion and mixed well by using a vortex mixer. Aliquots (3 mL) of the oxygenated β-carotene emulsion and 0.10 mL of phenolic extracts were placed in capped culture tubes and mixed vigorously. The tubes were immediately placed in a water bath and incubated at 50°C. Oxidation of β-carotene emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm after 0, 10, 20, 30, and 40 min as indicated by Taga et al.[18] The degradation rate of the extracts was calculated according to first-order kinetics using the following equation:

where ln is the natural log, a is the initial absorbance (470 nm) at time zero, b is the absorbance (470 nm) after 40 min, and t is the time (min).

Free Radical Scavenging Activity (DPPH Assay)

Extract solutions were prepared by dissolving 0.025 g of dry extract in 10 mL of ethanol. A fresh solution of DPPH^• in ethanol (6 × 10⁻⁵ M) was prepared before measurement. Approximately 3 mL of solution was mixed with 77 μL (38 or 19 μL in additional assays) extract solution in a 1-cm path length disposable microcuvette. The samples were kept in the dark for 15 min at room temperature; the decrease in absorbance was measured at 515 nm. Absorbance of a blank sample containing the same amount of ethanol and DPPH^• solution was also measured in the same fashion as described by Brand-Williams.[19] Radical scavenging activity was calculated by the following formula:

where AB is absorbance of the blank sample (t = 0 min) and AA is absorbance of the tested extract solution (t = 15 min).

Tea Quality Parameters

Tea quality parameters like theaflavin, theabrownin, and thearubingins were determined according to their respective method as described by Muralidharan.[16] Extracts were treated with different ethanolic treatments, namely, Ea, Eb, Ec, and Ed. Detail of the treatments are described herein.

Ethanolic Solution a (Ea)

Tea solution (30 mL) was mixed with ethyl acetate (30 mL) in a separating funnel, and then shook for 5 min. Part of the ethyl acetate layer (2 mL) was diluted to 25 mL with 95% (v/v) ethanol. The absorption of ethanol solution was recorded as Ea.

Ethanolic Solution b (Eb)

A portion of original tea solution (15 mL) was mixed with n-butanol (15 mL) followed by shaking for 3 min. Part of the aqueous layer (2 mL) was mixed with saturated oxalic acid (2 mL) and distilled water (6 mL) and then diluted with 25 mL (95% v/v) ethanol. The absorption of ethanol solution was recorded as Eb.

Ethanolic Solution c (Ec)

Fifteen mL of the ethyl acetate layer was mixed with an equal amount of (2.5 % w/v) NaHCO₃ followed by shaking for 30 sec. The aqueous layer was discarded and part of the ethyl acetate layer (4 mL) was diluted with 25 mL ethanol. The absorption of the ethanol solution is termed as Ec.

Ethanolic Solution d (Ed)

Two mL of the aqueous layer of Ea was mixed with saturated oxalic acid (2 mL) and distilled water (6 mL), and diluted with 25 ml (95% v/v) of ethanol. The absorption of this ethanol solution was recorded as Ed at 380 nm. The value of specific tea quality parameters was calculated by the following expressions:

where Ea, Eb, Ec, and Ed are the corresponding (absorbance) readings from the spectrophotometer and M is the % moisture content of the tea sample.

Caffeine Determination

Caffeine was also determined in tea samples following the method of Belay et al.[20] An accurate amount of sieved tea (50 mg) was dissolved in 25 mL of distilled water. The solution was stirred for 1 h using a magnetic stirrer and heated gently to remove caffeine from the solution. Finally, the absorbance of the solution was measured at 310 nm against the corresponding blank.

Statistical Analysis

Data obtained for each parameter was subjected to statistical analysis by applying a completely randomized design using Statistical Package (Statixtic V-8.1, Analytical Software Corp., USA). Significant ranges were further compared using a least significance test (LSD).[21]

RESULTS AND DISCUSSION

Total Phenolic Contents (TPC)

In plants, phenolic and polyphenolic compounds represent the main class of natural antioxidants that are directly responsible for antioxidative action.[22] Total polyphenols of black tea consist of catechins as well as oxidized products like theaflavin, thearubingins, and theabronins. Total phenolic contents effected significantly in different tea brands. The mean value for total phenolics in black tea extracts was 26.42 to 29.74 g/100 g liquid tea (Table 2). The maximum phenolic content 29.74 g/100 g liquid tea was detected in supreme packed tea (CB₂) whereas minimum 26.42 g/100 g liquid tea in Tapal packed tea (CB₃). The differences in phenolic contents were might be due to origin, processing condition and method for the adaptation of the tea leaves. The high phenolics in commercial brands indicating the high degree of fermentation show the preference of the processor for achieving the strong taste and color. The phenolics in black tea were comprised of simple polyphenols (5 g/100 g liquid tea) and oxidized poly phenols (25 g/100 g liquid tea). Most polyphenols are infused as catechins being 4.5 while the oxidized theaflavin and thearubigins are 15 g/100 g of the total volume of water extract in black tea.[21] Differences among the proportion of the polyphenols exist among the different countries. Phenolic contents ranged from 19 to 20 g/100 g liquid tea in Kenya whereas in India the phenolic contents ranged from 30 g/100 g liquid tea.[8,23, 24]

Table 2 Means (%) of some promising characteristics of various tea brands

Treatments	TPC	Antioxidant activity	DPPH	Catechins	Theaflavins	Thearubingins	Theabrownin	Caffeine
CB1	28.7 ± 0.01a	48 ± 0.01a	50.07 ± 0.07 a	1.28 ± 0.05a	1.79 ± 0.01a	1.8 ± 0.01c	19.69 ± 0.2b	1.24 ± 0.01ab
CB2	29.74 ± 0.03a	44.5 ± 0.01b	51.36 ± 0.05a	1.28 ± 0.01ab	1.763 ± 0.01c	1.81 ± 0.02bc	18.57 ± 0.5d	1.22 ± 0.01c
CB3	26.42 ± 0.02c	45.0 ± .01c	50.07 ± 0.05a	1.28 ± 0.05ab	1.76 ± 0.01b	1.83 ± 0.03ab	17.053 ± 0.03f	1.23 ± 0.01b
LP	28.06 ± 0.06b	46.03 ± 0.05d	42.03 ± 0.07c	1.28 ± 0.05bc	1.77 ± 0.02d	1.85 ± 0.01a	17.80 ± 0.01e	1.23 ± 0.02b
TB1	26.43 ± 0.01c	47.01 ± 0.06e	50.05 ± 0.06b	1.01 ± 0.01d	1.71 ± 0.01ab	1.74 ± 0.02d	20.22 ± 0.01ad	1.25 ± 0.03a
TB2	28.7 ± 0.01a	40.02 ± 0.07f	50.11 ± 0.01a	1.27 ± 0.01c	1.79 ± 0.05e	1.81 ± 0.01bc	19.17 ± 0.07c	1.23 ± 0.01b
Highly significant at P ≤ 0.01.
CB1: Commercial tea packed 1; CB2: Commercial tea packed 2; CB3: Commercial tea packed 3; LP: Loose tea; TB1: Tea bag 1; TB2: Tea bag 2.

Antioxidant Activity

Results regarding antioxidant activity of the various extracts revealed significant differences among the extracts. Means value for the antioxidant activity of the extracts (Table 2) showed that commercial brand (CB₁) exhibited the highest antioxidant activity 48 g/100 g liquid tea followed by commercial brand (TB₁) as 47.01 g/100 g liquid tea whereas lowest amount 45.00 g/100 g liquid tea was recorded in CB₃. Aforementioned results are in agreement with the early finding of Atoui et al.[25] They found that the antioxidant activity of tea varies between 41 to 70 g/100 g liquid tea in different samples. Moreover, water extracts exhibited the least antioxidant activity as compared to other solvents like methanol and ethanol as tea active molecules are less compatible with water. In another research investigation, Rusak et al.[26] observed that water gave the less yield (17.03 g/100 g liquid tea) than that of other solvents like methanol (21.25 g/100 g liquid tea). Antioxidant activity is directly associated with extraction yield as water gives the least yield, which is why less antioxidant activity was observed.

Free Radical Scavenging Activity

DPPH^• is one of the few stable and commercially available organic nitrogen radicals bearing no similarity to highly reactive and transient peroxyl radicals involved in various in vivo oxidative reactions.[27] DPPH free radical scavenging activity is one of those indicators that is important in determining antioxidant potential of selected bioactive molecules or extract. Data showing means of DPPH (Table 2) indicated that extracts differed significantly and maximum free radical scavenging activity of 51.36 g/100 g liquid tea for CB₂ followed by 50.07 g/100 g liquid tea in CB_I while minimum activity 42.03 g/100 g liquid tea for loose tea from Kenya (LP) reflects quality deterioration. The free radical g/100 g liquid tea inhibition in the range of 42 to 50 g/100 g liquid tea is in harmony with earlier results of Pilar et al.[28] as it depends upon the adopted method, nature of the solvent, and concentration of the DPPH solution.

Total Catechins

Catechins represent the unfermented fraction of the tea. promoting its health enhancing activity. Total catechins (Table 2) in Pakistani commercial brands were varied between 1.01 to1.28 g/100 g liquid tea. Maximum content (1.28 g/100 g liquid tea) was exhibited by CB₁ followed by CB₂ 1.28 g/100 g liquid tea, while loose tea (LP) contained a minimum amount 1.01 g/100 g liquid tea. Higher catechin contents of CB₁ indicated that it was less fermented as compared to other tea samples. The present results are in confirmatory within the finding of Quan et al.,[24] reflecting the value of 1 to 1.30 g/100 g liquid tea. Their study indicated the presence of an un-oxidized fraction of polyphenols. They suggested that degree of fermentation during the black tea processing determined the amount of catechins in the final product.

Tea Quality Parameters

Evaluation of tea quality parameters in respective extracts showed significant variability in quality attributes that may be due to the difference in origin and fermentation conditions. Means for various extracts revealed that CB₁ showed the highest theaflavin contents 1.79 followed by CB₂ 1.78 g/100 g liquid tea. The lowest amount was recorded in TB₁ 1.71 g/100 g liquid tea (Table 2). The differences for theaflavin in samples showed variations in storage and fermentation time of tea samples. The extracts that contained maximum theaflavin reflect prolonged storage whereas the minimum indicated their freshness and less fermentation.

The mean regarding thearubingins (Table 2) showed the highest contents (1.85 g/100 g liquid tea) in LP followed by CB₃ as 1.83 g/100 g liquid tea whereas the lowest in TB₁ with a value of 1.74 g/100 g liquid tea. As far as theabrownins contents is concerned, the highest value (20.2 g/100 g liquid tea) was observed in TB₁ while the lowest value (17.05 g/100 g liquid tea) was observed in CB₃ (Table 2). These result explicated that theaflavins, thearubingins, and theabrownins concentration were directly correlated with fermentation time that affect flavor and taste of tea brands. A similar study was conducted by Yao and Nursten[30] for the determination of tea quality parameters in different tea samples of the Australian market and proposed that theaflavins (TF), thearubigins (TR), and theabrownins (TB) are the contributory factors, determining the quality of black tea. These compounds were measured in differently packed tea samples; TF was ranging from 0.29 g/100 g liquid tea to 1.25 g/100 g liquid tea, indicating quality differences among the studied samples.

From the above results, it was assumed that tea varieties in the Pakistani market were processed considering the special market needs. They generally exhibited low levels of theaflavins due to oxidation. Using the same spectrophotometer method, Roberts and Smith[31] found that when the content of TF in a commercial tea reached 1.63 g/100 g liquid tea, tea tasters remarks were “very bright liquor and rich color,” which indicated a good quality black tea. Another study by Pilar Almajano et al.[28] in Kenyan commercial black teas, reported that the total TF ranged from 1.80 to 2.59 g/100 g liquid tea, with an average of 2.14 g/100 g liquid tea. This level of TF is much higher than that present in Pakistani tea indicating that most of the imported teas were either over-fermented black tea or underwent a relatively long period of storage resulting oxidation or decrease of TF.

Caffeine Contents

Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6 dione) belongs to a group of compounds collectively known as purine alkaloids.[7, 29] Data regarding caffeine contents for extracts (Table 2) revealed that TB₁, CB₁, and LP showed the highest caffeine content as 1.25, 1.24, and 1.23 g/100 g liquid tea, respectively, while the least caffeine contents were in the CB₂ sample 1.22 g/100 g liquid tea. In the instant study, caffeine content of different tea brands weasin the range of 1.22–1.25 g/100 g liquid tea, and is supported by the range of 1.09–1.65 g/100 g liquid tea as reported by Fujioka and Shibamoto.[32] It has been reported that the mean percentage of caffeine in different tea samples is in the range of 1.01–1.19.[20] Moreover, it was also proposed by Illy[33] that the percentage of caffeine in tea is less than 1.50 g/100 g liquid tea, indicating that Pakistani tea samples are risk free as this trait is below the threatening level.

CONCLUSIONS

Total polyphenols of black tea from Pakistani markets are close to those present in Kenyan tea. The variations in phenolics are due to different processing and storage conditions as well as the trends of companies towards the consumer's demands, however, the role of packaging material cannot be denied. The result of a recent investigation indicates that total polyphenols could be used as one of the quality tools for tea processing and marketing. Catechins, TF, TR, and TB from Pakistani markets are at par with Kenyan tea. Caffeine contents were also in the permissible limit. It is concluded that investigated commercial brands were safe for use, however, further research is needed to explore the health claims of such samples.