Chemical Profiling of Some Promising Black Tea Brands With Special Reference To Cup Quality
Abstract
Present research was conducted in different available tea brands for
exploring their phytochemical profiling with special consideration of
tea cup quality. Research design was based upon the extraction of
antioxidant with varied concentration of methanol and optimization of
extraction criteria. Higher extraction yield was noted for 20 minutes as
compared to 10 minutes. As a function of extraction time, various tea
quality parameters were increased. The values regarding cup quality
indicated that T1 (commercial brand 1) hold highest theaflavin as 1.84%
in contrast with T4 (loose pack) that contained lowest as 1.78%.
Whereas, T5 had highest level of theabronin (20.23%) indicating strong
color and brightness of the extract but lessen its allied health
benefits. In general tea extract, which was under study, exhibited good
antioxidant and free radical scavenging activity ranging from 39.36 to
44.87 and 47.56-70.01%, respectively. Caffeine content was seemed to be
in safe limit (1.17- 1.39%).
Abbreviations: TFs : Theaflavins; EI :
Ethanolic Solution; TPC: Total Phenolic Contents; Completely CRD:
Randomized Design; LSD: Least Significance Test
Introduction
Enhanced association between nutrition and health with inclination to
avoid maladies rather than cure and concept of diet-based therapy have
led to the concept of nutraceuticals. Now nutraceuticals are considered
as a mainstay in domain of diet- based therapies. Phytochemicals are in
attention for a long time for discoveries ofthe nutraceuticals [1]
Researchers and scientists divert their attention towards the
discoveries of those phytochemicals that are used in daily diet as well
as not expensive. In that sense certain discoveries are appeared in the
horizon of nutraceuticals and black tea and its constituents is one of
the important components of these strategies to mitigate and cure
various diseases primarily due to its antioxidant potential. Tea brewed
from the plant Camellia sainensis is consumed in different parts
of the world as green, black or oolong tea. However, black tea occupies
about 80% of the world tea production, while rest is contributed by
green and oolong tea [2,3]. During the production of black tea, about
75% of major polyphenols of the tea (catechins) undergo enzymatic
transformation consisting of oxidation and partial polymerization by
polyphenoloxidize that catalyze the reaction and transforming tea
inheriting antioxidant into distinguished black tea compounds,
nevertheless some can remain as an original state as catechins.
The resulting black tea composition depends upon the technological
procedures [3]. Black tea pytochemistry like the other medicinal plant
also inheriting with some prominent constitute but also accompanied by
some components that proves a thereat when used in an excessive amount.
Major components are catechins, theaflavins, thearubigins, caffeine and
flavonols Bech-Larsen, Scholderer, et al. Black tea parental composition
is strongly resembles with other teas but its divine components make it
an alternative source for incorporation into diet based therapies [4].
Major polyphenols in black tea are theaflavins, Thearubingins and
theabronin along with unoxidized catechins [5]. Theaflavins (TFs) are
dimeric polyphenols produced during production of tea. Due to strong
antioxidant properties, there is growing interest in the food, beverages
and healthcare industries in producing and incorporation of theaflavins
in various products [6], produced during the oxidative fermentation of
tea leaf. Thearubingins (TRs) are the high molecular weight compounds
that are responsible for the tea brisk and astringent and present almost
in double amount as the theaflavins [7]. They also give body to the
brewed tea.
TRs are larger in size and darker in color than TFs [1]. TRs are a
heterogeneous group of phenolic pigments with relative molecular masses
in the range 700-40,000 Da .The content of TFs in black tea is 0.3-2% on
dry weight basis whilest the TRs fraction comprises 10-20% of black
teas and together with TFs contribute to the tea brew characteristics
such as color, strength and body [8]. Black tea flavonols included
quercetin, kaempferol and myricetin. They make up 2-3% of the
water-soluble extract in tea. These flavonol have proved effective
against cancer and also inhibit the growth of malignant cell [9-11]. Cup
quality of tea is referred by the amount of selected parameters that
contributed the pharmacological and health associated properties of the
tea [12]. Health benefits of tea cannot be understood without
polyphenols. Tea polyphenols are associated with some key health
benefits like anticancer, anti-mutagenesis and antidiabetic properties.
Moreover, they are effectively used for the treatment of obesity,
artherocesrolises and heart diseases [13].
In the guide line of theses finding there is a possibility for cup
quality with the use of polyphenolic assay and other related attributes
like total phenolic, antioxidant activity, theaflavin, therubingin and
theabronins along with total catechins and caffeine can effectively
describe the cup quality [14]. Tea active ingredient extraction is very
crucial stage so optimization of extraction condition is a key factor
[13]. The extraction process can be carried out by using different
solvents like water, methanol, ethanol, and acetonitrle. Water exhibited
least extraction yield as contract to others [14]. Time and temperature
have a great importance in this contest, generally increase in time and
temperature proved beneficial for extraction efficiency. Multi-step
extraction with gradually increase time yields better as compared to
single step extraction. Extraction decreased when it extended prolonged
in a single step due to the destruction of the poly phenols [15].
Present research was carried out to explore the extraction efficiency of
polyphenols with respect to the time variation using methanol as
solvent and it is correlated with tea cup quality to set its role in the
modern life style.
Material and Method
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 (Sigma-Aldrich Tokyo, Japan)
and Merck (Merck KGaA, Darmstadt, Germany). The collected tea samples
were stored at ambient temperature for further study.
Preparation of Tea Extracts
Tea brands were subjected to extraction by using methanol as solvent
according to specified method given by [14]. Tea sample (5g) from each
brand was extracted with methanol (100mL) on a water bath at 60°C for 10
and 20 minutes. Extracts were filtered by using vacuum filtration
assembly and solvent was recovered by rotary vacuum evaporator (EYELA,
N-N series, Japan) at 40°C. The extract of each sample was weighted to
determine the yield of soluble constituents and stored at 4°C until
further use.
Analysis of Tea Extracts
Tea extracts were analyzed for their antioxidant potential for
indices like total phenolics, antioxidant activity and free radical
scavenging activity (DPPH assay) by following respective procedures. The
brief description of each method is given below.
Total Phenolics
Tea solution (1mL) was taken into a volumetric flask then added 4mL distilled water and 5mL of tartrate solution (1g of FeSO4 and 5 g of KNaC4H4O6 dissolved in 1000mL distilled water). It was diluted with 25mL buffer solution (23.377 g of Na2HPO4 in 1000mL of distilled water and 9.078 g of KH2PO4
in 1000 mL of distilled water in the ratio of 85% and 15% v/v). The
absorbance was measured using UV/Vis spectrophotometer (CESIL CE7200,
Japan) at 540nm as described by [14].The calculation was carried out by
using following expression. Total polyphenols (%) = 0.3914EV0/ V1/W
[15-17].
Where as
E = The absorbance reading of the spectrophotometer
V0 = Total volume of the tea solution (250mL)
V1 = Volume used for the measurement (1mL)
W = The dry weight of the tea sample.
Total Catechins
The sample (0.1mL) was taken in a test tube containing methanol
(0.9mL); transfer 2.5mL of vanillin solution (1% methanol) along with
10mL 9N HCl in the same tube. The solution was kept for 30 minutes
before measuring with UV/Vis spectrophotometer at 500nm against the
corresponding reagent methanol as blank [18].
Antioxidant Activity
β-carotene (2mg) was dissolved in 20mL chloroform. Aliquot of (3mL)
solution was transferred into 50 mL beaker and 40mg linoleic acid and
400mg Tween 20 were added. Chloroform was removed by purging with
nitrogen. Oxygenated distilled water (100mL) generated by bubbling air
into distilled water for 1 hour was added in ß-carotene emulsion and
mixed well by using vortex mixer. Aliquots (3mL) of the oxygenated
ß-carotene emulsion and 0.10mL of phenolic extracts were placed in
capped culture tubes and mixed vigorously. The tubes were immediately
placed in a water bath and incubated at 500C. Oxidation of
ß-carotene emulsion was monitored spectrophotometrically by measuring
absorbance at 470nm after 0, 10, 20, 30 and 40 minutes as indicated by
[16].The degradation rate of the extracts was calculated according to
first order kinetics using following equation
In(a/b) x 1/t = sample degradation rate
ln = The natural log
a = The initial absorbance (470 nm) at time zero
b = The absorbance (470 nm) after 40 min
t = The time (min)
Free Radical Scavenging Activity (DPPH assay)
Extract solutions were prepared by dissolving 0.02 5g of dry extract
in 10 mL of ethanol. A fresh solution of DPPH •in ethanol (6x10-5 M) was
prepared before measurement. Approximately 3mL of solution was mixed
with 77μL (38 or 19 μL in additional assays) extract solution in 1 cm
path length disposable microcuvette. The samples were kept in dark for
15 minutes at room temperature; the decrease in absorbance was measured
at 515nm. Absorbance of blank sample containing the same amount of
ethanol and DPPH• solution was also measured in the same fashion as
describe by [17]. Radical scavenging activity was calculated by the
following formula
Reduction of absorbance (%) = [(AB - AA) / AB] x 100
AB = Absorbance of blank sample (t = 0 min)
AA = Absorbance of tested extract solution (t = 15 min)
Tea Quality Parameters
Tea quality parameters like theaflavin, theabrownin and thearubingins
were determined according to their respective method (Muralidharan)
[14] Extracts were treated with different ethanolic treatments namely
EI, EII, EIII and EIV as described below
Ethanolic Solution I (EI): Tea solution (30mL) was mixed with
ethyl acetate (30mL) in a separating funnel, shake for 5 minutes. Part
of the ethyl acetate layer (2mL) was diluted to 25mL with 95% (v/v)
ethanol. The absorption of ethanol solution was recorded as EI.
Ethanolic Solution II (EII): A portion of original tea
solution (15mL) was mixed with n-butanol (15 mL) followed by shaking for
3 minutes. Part of the aqueous layer (2mL) was mixed with saturated
oxalic acid (2mL) and distilled water (6 mL) and then diluted with 25mL
(95% v/v) ethanol. The absorption of ethanol solution was recorded as
EII.
Ethanolic Solution III (EIII): 15mL of the ethyl acetate layer was mixed with equal amount of (2.5% w/v) NaHCO3
followed by shaking for 30 sec. The aqueous layer was discarded and
part of ethyl acetate layer (4mL) was diluted with 25mL ethanol. The
absorption of ethanol solution was recorded as EIII.
Ethanolic Solution IV (E IV): Aqueous layer of Ea (2mL) was
mixed with saturated oxalic acid (2 mL) and distilled water (6mL) and
diluted with 25ml (95% v/v) ethanol. The absorption of this ethanol
solution was recorded as EIV at 380 nm.
The value of specific tea quality parameters was calculated by following expression. Theaflavin % = TF% 2.25EIII /1-M
Thearubingin % = TR% 7.06 (2EI+2EIV-2EII-EIII) /1-M
Theabronin % = TB% 7.06*2Eb/1-M
Where EI, EII, EIII and EIV are the corresponding (absorbance)
readings taken with spectrophotometer, whereas, M is the moisture
content of the tea sample
Caffeine Determination
Caffeine was determined in tea samples following the method of [17].
An accurately amount of sieved tea (50mg) was dissolved in (25mL) of
distilled water. The solution was stirred for one hour using magnetic
stirrer and heated gently to remove caffeine from the solution. Finally,
the absorbance of the solution was measured at 310nm against the
corresponding blank.
Statistical Analysis
Data obtained for each parameter was subjected to statistical
analysis by applying completely randomized design (CRD) using
Statistical Package (Statixtic V-8.1). Significant ranges were further
compared using least significance test (LSD) [18].
Result and Discussion
Extraction Efficiency
Choice for selecting an extraction method based its ability to
preserve the bioactive compounds and extract most of these otherwise at
least if not all the desired compounds. Wet extractions involve solid
material in direct contact with a liquid solvent [19]. During the
extraction, organic solvents diffuse into the solid material and
solubilize compounds with similar polarity. The nature of the solvent
used will determine the types of chemicals likely to be extracted from
the plant. Organic solvents for extractions include polar solvents such
as water, ethanol, methanol and acetone to nonpolar solvents such as
dichloromethane and hexane.
The extraction yield differs significantly with respect to extraction
time. The results regarding the means (Table 2) for extraction yield
varies from 21.3 to 25.7% for 10 minutes extraction time. T5 (tea bag 1)
showed maximum yield (25.7%) while T1 that was the commercial packed 1
exhibited the minimum yield (21.3%). Extraction yield increased
prominently when extraction time was increased. The yield for 20 minutes
extracts was varied between 25 to 31% and maximum found in T5 (TB1) as
31%. Extraction efficiency was increased by increase the time of
extraction however, percent extraction depend on type of solvent [14].
In another study it was found that methanol was an effective solvent for
the extraction of antioxidant [19].
Total Phenolic Contents (TPC)
Phenolics and polyphenolic compounds constitute the main class of
natural antioxidants present in plants and may contribute directly to
antioxidative action [20]. Means for TPC depicted (Table 3) that extract
ranged from 39.36% to 44.87 %. T2 (CB2) commercial brand 2 showed the
highest (44.87%) TPC contents whilst, lowest was exhibited by T4 (LP)
loose pack as 39.36% of 10 minutes extracts. Highest value for 20
minutes extract (44.95 %) in T2 (CB2) followed by T1 (CB1) as 42.36%.
The lowest amount (40.51%) was recorded in T4 (LP). This is might be due
to the presence of more moisture as compared to the others. In another
study total phenolic contents of different tea samples were found to be
in the range of 14 to 45 %, highlighted the effect of extraction time
and solvent. This increase in the TPC contents emphasize the importance
of solvent and methanol proved better from water probably due to its
nature [21]. Another study conducted in Australian supermarket on
different tea samples with water showed the total polyphenols in black
teas ranged from 14% to 34%, in of different brands extracted with water
these differences are might be due to solvent , extraction time and
temperature Wright et al. A hypothesis was developed by [22] explained
the role of temperature and time in the extraction that was proved true
by the [14] ;extraction time is a key factor for determination the
extraction efficiency which effect all its attributes
Antioxidant activity
Antioxidant activity of different tea extract of 10 minutes
extraction time revealed (Table 3) that T1 (CB1) showed the highest
amount for that trait (52.02%) followed by T2 (CB2) as 49.39%. The
lowest amounts (40.02%) was noted in T6 (TB2). The same relation was
found in extracts of 20 minutes among them T1 (CB1) showed the highest
antioxidant activity (54.02%) followed by T2 (CB2) as 49.39%. The lowest
activity (40.04%) was recorded in T6 (TB2). Extraction for antioxidant
activity from tea was dependent upon the choice of solvent and methanol
was found to be beneficial in this regard [22]. also found the
antioxidant activity of black tea methanolic extracts at higher time
gives higher antioxidant activity as compared with water and methanol at
shorter times. In an early study, the antioxidant status of different
tea samples were estimated and found that methanolic extracts of green,
oolong and black tea showed antioxidant activity ranged from 50 to 80%
accordingly green tea exhibited maximum activity [19].
DPPH Free Radical Scavenging Activity
It is one of the stable and commercially available organic radicals
bearing no similarity to the highly reactive and transient peroxyl
radicals involved in various oxidative reactions in vivo. DPPH free
radical scavenging activity indicates antioxidant potential of selected
bioactive molecules [23]. Mean for DPPH free radical scavenging activity
of different tea brands at 10 minutes extracts (Table 3) ranged from
47.56 to 59.11%. Maximum free radical inhibition off 59.11% in T2 (CB2)
followed by 55.6% in T1 (CB1) and minimum was observed as 47.56% in T5
(TB1). While means for 20 minutes extracts ranged from 60.48 to
70.01%.Maximum inhibition 70.01% was found in T3 (CB3) followed by 68.42
% in T1 (CB1) while the minimum activity was found 60.48% in T6 (TB2). A
project that was conducted for evaluation of time and temperature for
extracting the antioxidant and it was revealed that enhanced temperature
and time implicit a positive effect for that trait. Methanol used as
solvent for that regards in another study and found that work better
than the other [19,22].
Cup Quality
Cup quality reflects health promoting parameters of the tea in the
single serving of the cup. The quality of tea brands highly depend upon
this parameters. This is a healthy attribute that determined the quality
of the brand. Tea bioactive molecules are theaflavin, thearubingin,
theabronin and total catechins as well as the caffeine. The brief
discussion concerning these parameters is described below.
Theaflavin
Theaflavin contributed towards the taste, brisk and astringent to the
tea along with bright golden color. Theaflavin levels generally depend
upon the amount of fermentation, genetic variations and climatic
condition of the growing region so its level varied in the different
cultivator. Mean for theaflavin in commercially available brands in
Pakistan (Table 4) showed highest level (1.83%) in T1 (CB1) whereas
lowest (1.71%) was detected in T4 (Lp) in 10 minutes extraction time.
Results for 20 minutes extracts followed the similar trend like the
earlier extraction condition with enhanced yield. Maximum recovery of
theaflavin was recorded in T1 as 1.84% in contrast with T4 that yielded
minimum (1.80%). Highest level of theaflavin in T1 reflects its bright
color and full fermentation, which is co-related with its strong taste.
However, theaflavin amount also dependent upon initial moisture content.
The effect of extraction temperature exhibited a pronounced effect upon
solubilization of theaflavin. Extraction time along with temperature is
a prime factor affecting the concentration of theaflavin in the final
extracts. Lowest level in T4 might be due to its packaging condition
loose packaging caused more moisture content that result in low
Theaflavin contents. Another reason might be due to over fermentation of
the samples that was required by their specific buyer. Difference in
the Theaflavin contents may be due to the difference in their
permeability that proved a driving forced for extracting this active
compound Theaflavin from them.
Low TF content in black tea may be due to over-fermenting or long
periods of storage [24]. The information derived from the earlier study
used to predicting black tea country of origin based on polyphenols
composition because geographical area of tea production may contribute
to the pattern of individual theaflavin composition in black tea [25].
In another study it was proved that the theaflavin holds a strong effect
on color and brightness of the tea more over, it was also suggested
that the liquor brightness was due to the theaflavin and their
constitute so it was noteworthy to quantification of theaflavins or
enrichment for the production of the tea of strong taste and color Ali
et al. Solvent extraction with methanol for 20 minute proved better then
water but effect of temperature cannot be denied as well as time of
extraction holds significant position in this contest so extraction time
increase exhibited marked increase in their extraction efficiency as
compared with that water and methanol for 10 minute. The influence of
extraction time on extraction efficiency was observed in earlier study
that was designed to determine the effect of varying temperature on the
methanolic extraction. Results depicted the enhanced yield as the time
of extraction was increased for theaflavin thearubingins and theabrownin
as compared to shorter time. The ranged of 30% at shorter time while
33% at longer time was observed [1].
Thearubingins
Thearubingins are a group of compounds formerly recognized as
insoluble fractions, SI and SII, of ethyl acetate extraction. TR was
further separated by butanol into soluble and insoluble TR that
contributed the reddish colour and richness in taste, totally termed as
body to black tea. Thereby, it is associated with quality and other
desirable liquor characters [26]. Results regarding the means for
methanolic extracts at 10 min extraction time (Table 4) indicating the
highest thearubingin value (1.87 %) in T2 (CB2) while, lowest (1.78%) in
T5 (TB1). In contrary, when extraction time was increased it reflected
the maximum amount in T2 as 1.89% and minimum (1.78%) in T5 (TB1). These
result suggested the suitable temperature along with time of extraction
were an influenced factors for extracting the tea active molecules.
Highest level containing extract showed less brightness and darker look
with contrast to extract containing fewer amounts Martin et al.proved a
relation between liquor brightness and thearubingins and they inference a
hypothesis that high level of thearubingins reduce the tea brightness.
They also performed a hedonic evaluation for authenticity for this
possibility proved that relation successfully.
Theabronin
Theabronin is a member of purine allkolids along with caffeine.
Generally the amount of theabronin should be less in good quality tea.
The mean values for the theabronin (Table 5) showed the same trend that
its value increased when extraction time was increased. The extracts of
10 minutes varies from 17.06-19.70% and heights claimed by T1 (CB1) as
19.70% followed by T2 as 18.58% and lowest depicted in T3 (CB3)
as17.06%. The maximum theabronin (21.22 %) was found in T5 (TB1%) and
minimum was reflected in T4 (LP) as 18.10 %. The amount of theabronin
has a negative effect on the tea. TB endows tea liquor and leaf with a
dark brown color, which has a negative effect on tea quality. The
information that derived from analysis of theabronin used as objective
method in the determination of black tea quality [25]. The extracts that
contain the heights level of theabronin exhibited dark red color and
strong appearance that was misleading with the quality. The highest
level may be due to the origin, cultivar, climatic conditions and
processing properties Amount of the theabronin and caffeine is a
function of the extraction conditions and solvent used for tea active
ingredient extraction. There is positive correlation for methanolic
extraction as compared to the other solvents [27].
Total catechins content
Catechin represents the unfermented product of black tea remained
unchanged during the fermentation. The amount of total catechin holds
prime importance for health enhancing properties of tea. Data
representing the methanolic extract of 10min extraction time in Table 4
indicating the peak value for this trait T2 as 1.30% followed by (1.29%)
in T3 while lowest recorded in T6 (TB2) as 1.27% . Data regarding 20min
extraction time disclosed the highest value of (1.36%) in T1 (CB1)
whereas lowest noted for the T5 (TB1) as 1.30%. The increment of
catechins was observed in the infusion of different samples of black and
green tea extracts with solvents like methanol ethanol and acetonitrile
as compared to water [26]. Enhanced extraction yield was obtained when
extraction time was increased that revealed in the data represented
(Table 5). The maximum catechin content in T1 represented its low degree
of fermentation, which are responsible for its less thearubingins and
color which is a marker of its quality. When the time of extraction
increase by the same solvent the extraction efficiency was also
increased for tea catechins [28].
Caffeine
Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6 dione) belongs to
a group of compounds collectively known as purine alkaloids [20,21].
Data regarding caffeine contents for extracts (Table 5) revealed that T6
(TB1) showed the highest caffeine content as 1.28 whilst the least
caffeine contents were in T2 (CB2) sample 1.20 %. Extracts for 20
minutes showed maximum caffeine yield in T2 (1.34%) and T3 exhibited
minimum (1.17%). In the instant study caffeine content of different tea
brands were in the range of 1.22-1.25 g/100 g liquid tea, are supported
by the range of 1.09-1.65% as reported by [29]. It has been reported the
mean percentage of caffeine in different tea samples is in the range of
1.011.19. Moreover, it was also proposed by [30] that the percentage of
caffeine in tea is less than 1.50 %, indicating that Pakistani tea
samples are risk free as this trait is below the threatening level.
Conclusion
Black tea phytochemicals that contributed towards its cup quality in
Pakistan are in close proximity with international market. Methanolic
extracts exhibited the more yield as a function of time. Cup quality of
T1 was greater among the others while, tea bag of same brands showed
fewer amounts of phytochemicals due to their permeability. Difference
between the key quality parameters might be due to the processing
technology, fermentation and storage period [31-35]. The result of
recent investigation indicates that methanol provide a mean for
optimization of cup quality, may be used as one of the quality tools for
tea processing and marketing. It is concluded that investigated
commercial brands were safe as for as their composition is concerned.
However, safety evaluation of these methanolic extracts will be carried
out by animal modeling.
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