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Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 62
P-ISSN: 2406-9388
E-ISSN: 2580-8303
The Effect of Vitamin C Addition on Epigallocatechin Gallate (EGCG) Stability in Green Tea Solution
Alief Putriana Rahman1, Djoko Agus Purwanto2, Isnaeni2*
1Master of Pharmaceutical Science, Faculty of Pharmacy, Universitas Airlangga, Surabaya
2Departement of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya
*Corresponding author: isna.yudi@gmail.com
Submitted: 5 Desember 2019
Accepted: 15 Januari 2020
Published: 29 Februari 2020
Abstract
Background: Epigallocatechin gallate (EGCG) is the most abundant green tea catechin with a powerful
antioxidant effect to prevent cancer cells. EGCG in green tea solution is highly susceptible to degradation, this it
is urgent to increase the stability of EGCG by vitamin C addition. Vitamin C can regenerate radical EGCG to
be normal EGCG. Objective: This research aim was to determine percent of decreased level of EGCG before
and after vitamin C addition. Methods: This paper was focused on enhancing the stability of EGCG by 1 mg
(GTVC1), 1.5 mg (GTVC2), 2 mg (GTVC3), 2.5 mg (GTVC4) and 3 mg (GTVC5) of vitamin C addition to 10 g/L
of green tea solution concentration. Evaluations of EGCG were conducted at 0 days, 1 day, 2 days, 3 days, and 4
days of storage time. EGCG was analyzed using thin layer chromatography (TLC) densitometry methods. The
stability of EGCG was determined by % of decreased EGCG. Results: Percent of loss of EGCG in GT, GTVC1,
GTVC2, GTVC3, GTVC4 and GTVC5 after 4 days storage were 19.93%, 10.89%, 21.08%, 18.18%, 28.56%, and
9.76%, respectively. Conclusion: This study showed that in 4 days storage, the decreased level of EGCG in
green tea solution with 3 mg of vitamin C addition (GTVC5) was 9.76% which was smaller than green tea
solution without vitamin C addition (GT) which EGCG decreasing 19.93%.
Keywords: EGCG, vitamin C, stability, green tea, TLC
INTRODUCTION
Tea is the most widely consumed beverage in the
world and derived from leaves of the plant Camellia
sinensis. In Indonesia, tea is one of the agricultural
main products and commodity exports. Green tea is a
type of tea that has the greatest health benefit owing to
high radical scavenging ability. Indonesia recognized
as the third-largest green tea consumption country in
Asia. It was known that about 30,000 ton green tea was
consumed annually (Kosugi, 2019).
Green tea contains a number of biologically active
compounds, which include (-)-epicatechin (EC), (-)-
epicatechin-3-gallate (ECG), (-)-epigallocatechin
(EGC), and (-)-epigallocatechin-3 gallate (EGCG)
(Rady et al., 2018).
Around 59% of the total catechin from the leaves
of the green tea is (-) epigallocatechin-3 gallate
(EGCG) (Rady et al., 2018). The radical scavenging
ability of EGCG was higher than other catechins
because EGCG has the number of hydroxyl groups
much more than others. That is why, EGCG can inhibit
or reduce cancer cells in the human body (Wu et al.,
2011). Nevertheless, the chemical structure of EGCG
makes it susceptibles to degradation. It is caused by
auto-oxidation and epimerization process. The auto-
oxidation degrades EGCG to be theasinensin A and
epimerization degrade EGCG to be GCG or
gallocatechin gallate (Krupkova et al., 2016).
The degradation of EGCG is affected by
parameters such as extraction of brewing methods,
storage temperature, and type of storage bottle. For this
research, all of the parameters were used in optimum
condition from the previous study. Ultrasonic
extraction was used for brewing the sample at
temperature 80oC (Das & Eun, 2018). Dark bottle was
used for storage to avoid the light (Zeng et al., 2017).
The temperature of storage was set at 4oC (Sigma &
Zeng et al., 2017).
The previous study explained that vitamin C or
Ascorbic acid addition could stabilize the content of
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 63
EGCG in green tea brewing. Vitamin C was found to
regenerate EGCG radicals. The interactions between
EGCG and vitamin C could be synergitic. The
synergistic effect wass considered to occur owing to
the difference of the reduction potential (Dai et al.,
2008). The reduction potential of EGCG was 430 mV
and the reduction potential of vitamin C was 282 mV
(Dai et al., 2008). Based on that, vitamin C was easier
to transfer electron and EGCG was also easier to attract
electron. EGCG degradation would attract electrons
from vitamin C easily, so EGCG radical regenerated to
normal EGCG. Oxidation of vitamin C forms oxalic
and trienoic acid (Truffault et al., 2017). Vitamin C
level of additions will give effect for pH condition of
samples. Moreover, uniformity of pH for sample could
mask the effects of vitamin C addition.
Several methods have been used to the determine
EGCG content in green tea, such as the HPLC-UV
(Fangueiro et al., 2014) and TLC-densitometry
(Abdelsalam et al., 2014). The HPLC method offer
high percentage of recovery (99-104%), however it
requires solvent and mobile phases with high purity
(HPLC grade), needs high cost, is complex to operate,
and had slower analysis time. The TLC-densitometry
can use pro analysis grade solvent and mobile phase,
low cost, simple to operate, less analysis time, and
environmentally friendly. Based on the explanation,
TLC-densitometry was chosen to analysis of EGCG
stability in green tea (Abdelsalam et al., 2014).
The aim of this study was to determine the
percentage reduction of EGCG before and after vitamin
C addition.
MATERIALS AND METHODS
Materials
Chemicals analysis
EGCG standard was purchased from Sigma
(Sigma Chemical Co., St. Louis, MO, USA). The
solvent for extraction and for mobile phases were
chloroform pro analysis (Merck), ethyl acetate pro
analysis (Fulltime), formic acid pro analysis (Merck),
acetone pro analysis (Smart-Lab). Vitamin C was
obtained from Sigma Chemical, and methanol pro
analysis (Fulltime) and the stationary phase of TLC
silica gel aluminum plate 60F-254 (Merck made in
Germany) were used. Some chemical materials for
determination of vitamin C, concentration such as
potasium iodate solution 0.002 mol L-1, starch
indicator solution 0.5%, potassium iodide solution 0.6
mol L-1 and hydrochloric acid 1 mol L-1 were also
used.
Green tea material
A commercial green tea powdered used in this
research was Meditea® (food grade) was purchased in
Surabaya, Indonesia.
Instruments
Microanalytical balance (Mattler Toledo XPE26),
analytic scale balance (Ohaus PA-214), ultrasonic bath
(Branson 3510), Burrette, Chromatography chamber
(Camag, 20 x 10 cm), pH Meter (Ohaus starter 3000),
TLC Plate (TLC Silica gel 60 F254, Merck), Camag
Linomat 5, Camag TLC Scanner 4, Camag TLC
Visualizer 2.
Methods
Vitamin C standard solution
Concentration of 500 ppm of vitamin C solution
was freshly prepared from 12.5 mg of vitamin C
standard in 25 mL of aqueous solution.
Green tea solution preparation
Distilled water was firstly heated at 80oC. As
much as 0.25 g of tea powder in six replicates were
immersed for 15 minutes in 15 mL of distilled water
(in 25 mL volumetric flask) in ultrasonic bath. After 10
minutes, 2, 3, 4, 5, and 6 mL of vitamin C (500 ppm)
were added to the sample solution and followed by
distilled water addition up to the required volume.
Then, the sample solutions were kept in dark glass
bottle at 4oC (Das & Eun, 2018).
Extraction of green tea solution
Three mL of green tea infusion was extracted
twice with 3 mL of chloroform. The remaining aqueous
layer was extracted twice with 3 mL of ethyl acetate.
The ethyl acetate layer containing EGCG was then
collected and evaporated in a fume hood.
EGCG standard solution
Pure standard of (-)-epigallocatechin gallate
(EGCG) was prepared in methanol. Six concentrations
of the calibration solution were made at the level of 50,
100, 200, 300, 400, and 500 ppm.
Maximum wavelength optimization of EGCG
The maximum wavelength of EGCG was
measured in EGCG solutions (100 and 200 ppm). The
wavelength which showed the highest absorbance was
chosen for the next analysis.
TLC condition
The chamber was previously saturated for 2 hours
with chloroform:acetone:Acetic acid (10:8:1) as a
mobile phase. Densitometric scanning was performed
with TLC scanner at λ 278 nm. The slit dimension was
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 64
4.00 x 0.30 mm. The scanning speed was 20 mm/s. The
plate was activated in an oven at 100oC for 15 minutes.
Vitamin C determination
As much as 10 mL of GT, GTVC1, GTVC2,
GTVC3, GTVC4 and GTVC5 was taken and moved
into 250 mL of conical flash and 75 mL of distilated
water, 2.5 mL of potassium iodide solution, 2.5 mL of
hydrochloric acid solution, and 0.5 mL of starch
indicator were added. And then, the sample was titrated
with potassium iodate solution until the endpoint. The
endpoint was achieved when the color of sample
changes to dark-blue. Note the volume of potassium
iodate needed.
Validation
Selectivity
The selectivity test was implemented by
determining the separation peak of EGCG and the
closest peak to it. EGCG solution (500 ppm) and
extract solution were prepared. All of the samples were
filtered using a membrane filter (0.45 μm). The EGCG
standard and extract solutions were spotted to the TLC
plate, eluted, and scanned using a densitometer. The
selectivity test parameters were a resolution (Rs), Rf,
and peak purity. The acceptance of resolution value for
Rs is ≥ 1.25, Rf between 0.3 - 0.8, and peak purity
value close to 1.0.
Linearity
The linearity test was analyzed by preparing 10
different concentration solutions of standard (500, 450,
400, 350, 300, 250, 200, 150, 100 and 50 ppm). All
EGCG standard solutions were spotted to the TLC
plates, eluted and scanned using a densitometer. A
regression line was made from the concentration of
EGCG standard (x) and area (y).
Accuracy
The accuracy test was analyzed by spiking three
different concentrations (80%, 100%, and 120%) were
made to sample. Three replicates were made for every
concentration of EGCG standard addition. All of the
samples were filtered using a membrane filter (0.45
μm). Then, all samples and EGCG standards were
spotted to the TLC plate, eluted, and scanned using a
densitometer. The sample solution without standard
addition was also analyzed. The quantity of determined
EGCG was determinate from the corresponding
calibration curve. The accuracy requirement is assessed
based on the recovery. The recovery for 1 - 10% of
EGCG in the sample was between 92 - 105% (AOAC
International, 2013).
Precision
The precision test was analyzed by spiking a 100%
concentration solution of EGCG standard to sample
solution (six replications). All of the samples and
EGCG standard were filtered using membrane filter
(0.45 μm). Then, all samples were spotted to the TLC
plate, eluted, and scanned using a densitometer. The
sample solution without standard addition was also
analyzed. EGCG was determinated from the
corresponding calibration curve. The precision
requirement is assessed the RSD value. The RSD for
1 - 10% of EGCG in the sample is < 2.7% (AOAC
International, 2013).
EGCG determination
EGCG determination was analyzed on green tea
solution without vitamin C addition (GT), green tea
with 1 mg of vitamin addition (GTVC1), green tea with
1.5 mg of vitamin addition (GTVC2), green tea with
2 mg of vitamin addition (GTVC3), green tea with
2.5 mg of vitamin addition (GTVC4) and green tea
with 3 mg of vitamin addition (GTVC5). Evaluations
of EGCG were conducted at 0 day, 1 day, 2 days, 3
days, and 4 days of storage time. All extracts were
dissolved in 2 mL of methanol p.a and filtered using
membrane filter (0.45 μm). All samples were spotted to
the TLC plate, eluted and scanned using a
densitometer. The EGCG quantity was determined
from the corresponding calibration curve.
Statistical analysis
Results of EGCG values were expressed as the
mean value ± standard deviation of three independent
experiments (n = 3). Statistical analysis was performed
by one-way analysis of variance (ANOVA) using IBM
SPSS statistics software version 24. A comparison of
means was carried out by Tukey’s multiple range
analyses at P < 0.05.
RESULT AND DISCUSSION
Extraction of green tea solution
Chloroform, a non polar solvent, was used to
remove caffeine and pigments in the green tea solution.
Ethyl acetate, a semi-polar solvent, was used to extract
EGCG of green tea. EGCG is a semi-polar compound
so that EGCG will be extracted in the ethyl acetate
layer. The ethyl acetate solution was evaporated at
room temperature because of the boiling point of ethyl
acetate is smaller than EGCG, ethyl acetate will
evaporate but not for EGCG. And then, dry extract was
dissolved with methanol. Methanol gives better
separation and more volatile than ethyl acetate.
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 65
Wavelength optimization
The maximum wavelength in 100 ppm and 200
ppm of EGCG standard solution were 278 nm. This
maximum wavelength would be used in a densitometer
scanner.
Mobile optimization
Mobile phase with different ratios were tried, such
as chloroform:acetone:formic acid (10:8:3 v/v/v),
chloroform:acetone:formic acid (10:8:2 v/v/v) and
chloroform:acetone:formic acid (10:8:1 v/v/v). The
results ware evaluated with respect to the efficiency of
separation (Rf and Rs). The optimum mobile phase
ratio was found to be chloroform:acetone:formic acid
(10:8:1 v/v/v) because the Rf of EGCG was 0.49
(acceptable value Rf = 0.3 - 0.8) and the resolution
(Rs) was 1.27 (acceptable value Rs > 1.25). The
densitogram of samples with different mobile phase
can be seen in Table 1.
Table 1. EGCG content in various samples
Time storage
EGCG Content ± SD (% w/w)
GT
GTVC1
GTVC2
GTVC3
GTVC4
GTVC5
0 day
2.568 ± 0.81
2.384 ± 0.16
2.389 ± 0.08
2.62 ± 0.35
3.027 ± 0.87
2.346 ± 0.225
1 day
2.151 ± 0.31
2.022 ± 0.03
1.667 ± 0.38
2.017 ± 0.29
1.903 ± 0.22
2.163 ± 0.06
2 days
2.125 ± 0.06
2.081 ± 0.16
2.060 ± 0.025
1.967 ± 0.026
2.061 ± 0.14
2.172 ± 0.15
3 days
2.059 ± 0.15
2.247 ± 0.19
2.409 ± 0.16
1.995 ± 0.31
1.699 ± 0.42
2.142 ± 0.05
4 days
2.056 ± 0.21
2.125 ± 0.02
1.8856 ± 0.02
2.145 ± 0.09
1.163 ± 0.05
2.117 ± 0.04
Noted: GT: green tea, GTVC1: green tea with 1 mg of vitamin C addition, GTVC2: green tea with 1.5 mg of
vitamin C addition, GTVC3: green tea with 2 mg of vitamin C addition, GTVC4: green tea with 2.5 mg of vitamin
C addition, GTVC5: green tea with 3 mg of vitamin C addition
Validation
Selectivity
Acceptance criteria for selectivity tests are a
resolution (Rs), Rf, and peak purity. The densitograms
of all the samples can be seen in Figure 1. The Rs value
of EGCG standard peak was 2.95 and the average Rs
value of EGCG peak in the samples was 1.27. The
EGCG peak of standard and in the sample have the
same Rf value (0.41). The purity of the EGCG peak
standard and sample is more than 0.99. Based on the
value of parameters, methods which was used for this
research was selective to analyze EGCG in green tea
solution.
Figure 1. Densitogram of scanned TLC plate from green tea without vitamin C addition (a), green tea with 1 mg of
vitamin C addition (b), green tea with 1.5 mg of vitamin C addition (c), green tea with 2 mg of vitamin C addition (d),
green tea with 2.5 mg of vitamin C addition (e) and green tea with 3 mg of vitamin C addition (f)
Linearity
The linearity test was carried out in a range of
EGCG concentration of 0.10276 μg/spot up to
1.0276 μg/spot. This research evaluates the linearity
using correlation coefficient (r) with acceptance criteria
r~1, relative process standard deviation value(Vxo)
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 66
B0
with acceptance criteria Vxo ≤ 5% and ANOVA
linearity testing with acceptance criteria sig ≤ 0.05.
The result of the correlation coefficient (r) was
obtained r = 0.996 which resulted in a regression line
equation y = 7363x + 622.5. The relative process
standard deviation value (Vxo) was obtained 3.48%,
and ANOVA linearity testing was obtained sig = 0.00.
So, it could be concluded that the data was linear.
Accuracy
Accuracy test was analyzed by spiking 80%, 100%
and 120% of EGCG standard to the sample and
measure the recovery. Acceptance criteria of recovery
for 1% - 10% analyte concentration is 92% - 105%
(AOAC International, 2013). The recovery of 80%,
100%, and 120% of EGCG standard addition were
99.330% ± 1.52, 103.753% ± 1.04, and 93.632% ±
0.37 respectively. The results indicated that this
method was accurate.
Precision
The precision test was analyzed by spiking 100%
of the EGCG standard (six replicates). The precision
measure RSD value. Acceptance criteria of RSD for
1% - 10% analyte concentration is ≤ 2.7% (AOAC
International, 2013). The result of RSD was 1.006%
with recovery 103.754 ± 1.04. The results suggested
that this method was precision.
Stability of EGCG value in green tea solution
Evaluation of EGCG value in all of samples was
analyzed in one TLC plate (Figure 2). EGCG value
was determined from the corresponding calibration
curve. The result of EGCG level in all samples which
were conducted at 0 day, 1 day, 2 days, 3 days and 4
days of storage can be seen in Table 1. The stability of
EGCG was determined from the percent loss of EGCG
during storage time (Table 2). Percent loss of EGCG
was calculated by the formula :
% Loss of EGCG =
Bn = EGCG value at n day storage, n = 1, 2, 3, 4 day
B0 = EGCG value at 0 day storage
Figure 2. Visualization of TLC plate after eluted. 50 ppm - 500 ppm EGCG standard (St1-St6); green tea extract (A1-
A3); green tea + vitamin C 1 mg extract (B1-B3); green tea + vitamin C 1.5 mg extract C 1.5 mg (C1-C3); green tea +
vitamin C 2 mg extract (D1-D3); green tea + vitamin C 2.5 mg extract (E1-E3) and green tea + vitamin C3 mg extract
(F1-F3)
Table 2. % Degreasing of EGCG content in all sample
Time
storage
Degreasing of EGCG content ± SD (%)
GT
GTVC1
GTVC2
GTVC3
GTVC4
GTVC5
1 day
16.23 ± 0.31
15.18 ± 0.33
30.23 ± 0.48
23.05 ± 1,35
28.56 ± 0.82
7.78 ± 0.96
2 days
17.26 ± 0.86
12.74 ± 0.96
13.76 ± 0.85
24.95 ± 0,69
31.93 ± 0.94
7.41 ± 0.55
3 days
19.85 ± 0.95
5.74 ± 0.59
-0.87 ± 0.96*
23.88 ± 0,86
43.89 ± 0.52
8.71 ± .85
4 days
19.93 ± 0.91
10.89 ± 0.92
21.08 ± 0.92
18.18 ± 0,21
28.56 ± 1.05
9.76 ± 1.04
*= increasing of EGCG content
GT: green tea, GTVC1: green tea with 1 mg of vitamin C addition, GTVC2: green tea with 1.5 mg of vitamin C
addition, GTVC3: green tea with 2 mg of vitamin C addition, GTVC4: green tea with 2.5 mg of vitamin C addition,
GTVC5: green tea with 3 mg of vitamin C addition
X 100%
B0 - Bn
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 67
The percent loss of EGCG in GT, GTVC1,
GTVC2, GTVC3, GTVC4 and GTVC5 after 4 days
storage were 19.93%, 10.89%, 21.08%, 18.18%,
28.56%, and 9.76%, respectively. There were
statistically significant differences (p < 0.05) of percent
loss of EGCG between samples.
Significant differences value show that vitamin C
addition affected to % loss of EGCG. Three mg of
vitamin C addition (GTVC5) was the best result which
prevents decreasing of EGCG in green tea solution.
GTVC5 has % loss of EGCG less then other samples.
Vitamin C can stabilize EGCG in green tea
solution. The potential reductions of vitamin C and
EGCG are 282 mV and 430 mV respectively. EGCG
potential reduction is greater than vitamin C. It caused
that EGCG is easier to attract electrons and vitamin C
is easier to release electrons (Tsao, 2015). EGCG will
be degraded to GCG or gallocatechin gallate and
theasinensin A. Vitamin C in green tea solution is
easily oxidized from diketo form to as dehydroascorbic
acid, which followed by conversion into oxalic acid
and threonic acid (Lung & Destiani, 2014). The EGCG
degradation will attract electrons from vitamin C
easily, so EGCG radical regenerated to normal EGCG.
The prediction of stability reaction between EGCG and
vitamin C can be seen in Figure 3. Oxidation of
vitamin C forms oxalic and trienoic acid (Truffault et
al., 2017). This was proofed by decreasing vitamin C
levels in green tea solution during 4 days storage. The
average of decreased level of vitamin C in GT,
GTVC1, GTVC2, GTVC3, GTVC4 and GTVC5 were
36.7%, 38.4%, 41.8%, 42.1%, 42.9%, and 47.5%,
respectively.
Electron from the degradation of vitamin C will
regenerate radical EGCG into non-radical EGCG (Dai
et al., 2008). This theory was in line with the result of
this research. The more vitamin C levels decreased, the
more EGCG radicals generated to be EGCG.
Figure 3. Prediction of EGCG stability reaction by vitamin C
CONCLUSION
The result of this study showed that during 4 days
of storage of EGCG revealed that a precentace
reduction of EGCG in green tea solution with 3 mg of
vitamin C addition (GTVC5) was 9.76%. It was
smaller than green tea solution without vitamin C
addition (GT) which decreased 19.93% of EGCG.
Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia Vol. 6 No.2 Desember 2019 68
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