Iraqi J Pharm Sci, Vol.31( 2 ) 2022 Detection of epicatechin in tea leaves by TLC and HPLC
DOI: https://doi.org/10.31351/vol31iss2pp304-312
304
Detection of Epicatechin in Camellia sinensis Leaves by Thin Layer
Chromatography and High Performance Liquid Chromatography
Techniques
Ruaa Mohammed Ibrahim *,1 and Zahraa Suhail Nassir**
*Department of Pharmacognosy and medicinal plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq.
Abstract
The current study performed in order to detect and quantify epicatechin in two tea samples of Camellia
sinensis (black and green tea) by thin layer chromatography (TLC) and high performance liquid chromatography
(HPLC). Extraction of epicatechin from black and green tea was done by using two different methods: maceration
(cold extraction method) and decoction (hot extraction method) involved using three different solvents which are
absolute ethanol, 50% aqueous ethanol and water for both extraction methods using room temperature and direct
heat respectively. Crude extracts of two tea samples that obtained from two methods were fractionated by using
two solvents with different polarity (chloroform and ethyl acetate). Qualitative and quantitative determinations of
epicatechin in tea samples were investigated. Epicatechin identification was made by utilizing preliminary
chemical tests and TLC. This identification was also boosted by HPLC and the quantity of epicatechin was
determined in all ethyl acetate fractions of two tea samples. This research revealed the existence of epicatechin in
black and green tea according to TLC and HPLC. Aqueous ethanol 50% was the best solvent for extraction of
epicatechin from leaves of tea. Quantitative estimation of epicatechin by HPLC revealed that ethyl acetate fraction
of DGTAE contains the higher concentration of epicatechin than other analyzed fractions. Conclusion, tea is an
excellent source of catechins particularly epicatechin that possessed various pharmacological effects.
Keywords: Black and green tea, TLC, HPLC, Epicatechin.
بواسطة كروماتوغرافيا الطبقة الرقيقة الشايأوراق في Epicatechin مادةالكشف عن
والكروماتوجرافيا السائلة عالية األداء
** زهراء سهيل ناصر و 1*، رؤى محمد ابراهيم
. العراقبغداد، ، كلية الصيدلة، جامعه بغداد، والنباتات الطبية فرع العقاقير *
الخالصة
كمية الكشف أجل من الحالية الدراسة أجريت بواسطة( واألخضر األسود الشاي) شايال من عينتين في epicatechinوقياس
واألخضر األسود الشاي من epicatechin استخالص تم(. HPLC) األداء عالية السائلة والكروماتوجرافيا( TLC) الرقيقة الطبقة كروماتوغرافيا
على تشتمل مختلفة مذيبات ثالثة باستخدام( الساخن االستخالص طريقة) االغالء و( البارد االستخالص طريقة) النقع: مختلفتين طريقتين باستخدام
الخام المستخلصات تجزئة تم. التوالي على المباشرة والحرارة الغرفة حرارة درجة باستخدام والماء المائي اإليثانول من ٪ 50 و ، المطلق اإليثانول
التحديدات في التحقيق تم .(االثيل وخالت الكلوروفورم) مختلفة قطبية لهما مذيبين باستخدام طريقتين من عليهما الحصول تم الشاي من لعينتين
تعزيز تم. TLC و األولية الكيميائية االختبارات استخدام خالل من epicatechin تحديد تم. الشاي في عينات epicatchin لمادة والكمية النوعية
خالت جميع في epicatechin كمية تحديد ثم HPLC بواسطة أيًضا التحديد هذا مادة وجود عن البحث هذا كشف . الشاي لعينتي اإلثيل بقات
epicatechin لـ وفقًا واألخضر األسود الشاي في TLC و HPLC .مادة الستخالص مذيب أفضل٪ 50 بنسبة المائي اإليثانول يعتبرepicatechin
من أعلى تركيز على يحتوي DGTAE من اإلثيل بقات خالت أن HPLC بواسطة epicatechin لمادة الكمي التقدير أظهر. الشاي أوراق من
. مختلفة دوائية تأثيرات تمتلك التي epicatechin وخاصة االكسدة لمضادات ممتاز مصدر هو الشاي ، الخالصة. تحليلها تم التي األخرى االجزاء
والكروماتوجرافيا السائلة عالية األداء، كروماتوغرافيا الطبقة الرقيقةالشاي األسود و األخضر، حية :الكلمات المفتا
Introduction
Camellia sinensis (L.) is tea plant belongs
to the family Theaceae and implants in about thirty
countries the whole world (1). It is originating in
China and later distributed to Japan and India, then
to Russia and Europe (2).
Tea plants are woody shrubs of medium size
with height reach to 1.8 meters (3).
Its leaves are pointed and oval at the tip with length
5-10 centimeters. Its flowers are fragrant, white, of
diameter 4 centimeters; contain 5 petals. The fruits
are as three-angled capsule that contain three seeds (3)
(figure 1). While its leaf buds and leaves are utilized
to make tea. It has a good taste, attractive aroma, and
health-reinforcing effects. These advantages made
tea one of the most common beverages in the world.
(4)
1Corresponding author E-mail:
Received: / /
Accepted: / /
Iraqi Journal of Pharmaceutical Science
https://doi.org/10.31351/vol31iss2pp304-312
Iraqi J Pharm Sci, Vol.31(2) 2022 Detection of epicatechin in tea leaves by TLC and HPLC
305
There are three types of tea taken from
Camellia sinensis which are either not fermented
like white and green tea, or partially fermented like
oolong and red tea, and completely fermented like
black tea, those compositions of those types are
influenced by the process of fermentation(5).
The most widely used is black and green
tea that are obtained from the same species of plant
(C. sinensis L.) but varying in their organoleptic
taste, appearance, chemical contents and flavor
attributed to their respective process of
fermentation(2).
The chemical composition of tea leaves
includes alkaloids (theobromine, caffeine,
theophylline), polyphenols (flavonoids and
catechins), polysaccharides, volatile oils, amino
acids, vitamins (like vitamin C), lipids, inorganic
elements (like fluorine, manganese and aluminum),
and other (2).
Figure 1. Photo of Camellia sinensis plant (5)
Camellia sinensis has been shown to
possessed pharmacological and physiological
activities such as anticancer (6), antioxidant (7), anti-
inflammatory (8), antibacterial (9), antidiabetic (10),
hepatoprotective activity (11), and others. These
activities are due to polyphenolic compounds mostly
catechins and flavonoids that are considered as the
major active components. Recently catechins are the
most significantly studied, including epicatechin
(figure 2), catechin, epigallocatechin gallate and
epicatechin gallat (12).
Therefore; the aim of this study was
qualitative and quantitative determination of
epicatechin in two tea samples of Camellia Sinensis
(black and green tea).
Figure 2. Chemical structure of epicatechin (1)
Material and Method
Chemicals and reference standards
The reference standard and chemicals that
utilized in this work are listed with their purity
percentage in table 1.
Table 1. Chemicals and standard used with assay
percentage
Chemicals Assay percentage
Acetic acid glacial 99.8- 100.5 %
Acetone 99.9%
Chloroform 99.8%
Dioxan 98%
Epicatechin standard 99.9%
Ethanol 99.7-100%
Ethyl acetate 99%
Formic acid 88%
Methanol 99.5%
n-butanol 99.7%
Toluene 99.5%
Instruments used for the conduction of the study
The instruments that utilized in this work are
summarized with their manufacture in table 2.
Table 2. Instruments used and their manufacture
Instrument Manufacturer
Electrical sensitive
balance
Sartorius/ Germany
HPLC system
(Shimadzu, 2010 CHT)
Shimadzu/Japan
Oven: Memmert 854 Buchi /Germany
Rotatory evaporator:
Buchi rotatory
evaporator attached to
vacuum pump
Buchi/ Germany
Ultraviolet light
(DESAGA
HEIDELBERG) of 254
nm and 366 nm wave
lengths
DESAGA/Germany
Plant materials
Dried green and black tea leaves obtained
from the local market and identified by
Pharmacognosy and medical plants department in
collage of Pharmacy\ Baghdad university and
authenticated by prof. Dr. Sukaena Abbas\
Department of Biology\ College of the Science
\University of Baghdad.
Extraction of plant material
A- Extraction by hot method (decoction)
15 gm of dried leaves from both tea samples were
put in conical flask and extracted by direct heat (70-
80 °C) for 20 mins, using three different solvents
involved absolute ethanol, 50% aqueous ethanol and
water (150ml of each) for extraction. (13)
B- Extraction by cold method (maceration)
15 gm of dried leaves from each tea sample are
placed in beaker and macerated for 5 days at room
temperature in 3 solvents which are ethanol, 50%
aqueous ethanol and water (150ml of each).
After that, filtration was done for crude
extracts obtained by both methods and concentrated
by a rotary evaporator. The crude extracts were
Iraqi J Pharm Sci, Vol.31(2) 2022 Detection of epicatechin in tea leaves by TLC and HPLC
306
hanging in water, then partitioning with chloroform
(CHCl3) and ethyl acetate by separating funnel in
each solvent for 3 times to get their respective
fractions rich with catechins and then concentrated (13)
as shown in figure 3.
Preliminary phytochemical investigation
Ethyl acetate fractions were undergoing
phytochemical analysis for flavonoids and phenolic
acid. Two chemical tests are utilized to detect their
presence (14).
Phenolic acid test: in a test tube, few gm of
ethyl acetate fraction were hanging in distilled water
(1ml), then add 5% ferric chloride (few drops) and
observed the color, a deep green to black coloration
confirm the existence of phenolic acids.
Flavonoid test: Few gm of ethyl acetate
fraction were dissolved in 80% ethanol (20ml) and
filtered, then take 1ml of the filtrate and dissolved in
1% potassium hydroxide (2ml) in a test tube, and
observed the color. A yellow color confirms the
existence of flavonoids.
Identification of epicatechin by TLC
Ethyl acetate fractions were analyzed by
TLC for the presence of epicatechin, using plates of
silica gel GF254 (20 x 10cm, 250 μm thickness);
development occur in 20cm x 10cm double tank
glass chamber presaturated for 30 minutes with
different mobile phases as follow: (13,15)
• S1: toluene: chloroform: acetone: formic acid (8: 4:
3: 3)
• S2: toluene: dioxan: acetic acid (9.2: 4 :0.6)
• S3: chloroform: ethyl acetate: formic acid (5: 4: 1)
• S4: n-butanol: acetone: acetic acid (5: 5: 3)
• S5: chloroform: acetone: formic acid (75:16.5:8.5)
The detection was done by utilizing UV
light at wavelength 254 nm and then calculated Rf
value.
Qualitative and Quantitative estimations of
epicatechin by HPLC
Identification and quantitative
determination of epicatechin in ethyl acetate
fractions were made by using HPLC (Shimadzu,
2010 CHT) with UV detector in which identification
was performed by comparing the time of retention
of analyzed fraction with that of reference standards
at same chromatographic conditions with reverse-
phase C18 column (TARGA) (250 × 4.6 mm, 5 𝜇) and
the temperature of column was kept at 30∘C. Mobile
phase of HPLC consist of solvent A prepared by
dissolving 0.1mL of orthophosphoric acid in 0.9L of
grade water of HPLC and complete the volume to 1.0
L with water and then filtered by filter membrane
(0.45 𝜇m) and degassed for 3 min in a sonicator, and
solvent B which is acetonitrile. By gradient elution,
mobile phase was developed at 0.01min 11% B; at 30
min 25% B; at 35-39 min 100% B; and at 40-50 min
11%B. The rate of flow of mobile phase was 1 ml/min
and the volume of injection was 15 𝜇l. The
wavelength of detection was 280 nm. (16)
Quantitative determination of epicatechin was
performed by using calibration curve in which serial
dilutions (10, 20, 30, and 40 ppm) from stock
solution of epicatechin standard (10 ppm) was
prepared.
Figure 3. Systematic scheme for extraction of crude catechins (13)
Iraqi J Pharm Sci, Vol.31(2) 2022 Detection of epicatechin in tea leaves by TLC and HPLC
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Results and Discussion
Extraction of catechins
In this work, the variation in yield
percentages (%w/w) of ethyl acetate fraction of two
tea samples was determined as shown in table 3.
Table 3. The yield percentages of ethyl acetate
fractions of green and black tea.
According to our work, the highest
percentage of yield was obtained from maceration
method of green tea (GT) which was (1.796) while for
the decoction method of GT gave the highest yield
which was (1.683), when we used 50% aqueous
ethanol as solvent in two extraction method. And the
best solvent for extraction of epicatechin is 50%
aqueous ethanol.
Preliminary phytochemical investigation
Preliminary screening of phytochemicals
confirmed that phenolic acid and flavonoids are
present in ethyl acetate fractions of two tea samples.
These chemical compounds may be in charge for
different medicinal properties.
Identification of epicatechin by TLC
The TLC results for all ethyl acetate
fractions of two tea samples indicated the presence
of epicatechin in which the color and Rf value of
epicatechin in analyzed fraction were identical to the
epicatechin standard and identified as epicatechin,
as shown in table 4 and figures 4-6.
Figure 4. TLC chromatogram of ethyl acetate fractions of green and black tea, ST: epicatechin standard, 1:
MGTA, 2: MGTE, 3: MGTAE, 4: MBTA, 5: MBTE, 6: MBTAE, 7: DGTA, 8: DGTE, 9: DGTAE, 10:
DBTA, 11: DBTE, 12: DBTAE, using S1 as a solvent system.
Analyzed fraction Percentage yield
(%w/w)
Decoction green tea in
ethanol (DGTE)
1.005
Decoction green tea in
distilled water (DGTA)
1.233
Decoction green tea in
aqueous: ethanol (1:1)
(DGTAE)
1.683
Decoction black tea in
ethanol (DBTE)
0.718
Decoction black tea in
distilled water (DBTA)
1.119
Decoction black tea in
aqueous: ethanol (1:1)
(DBTAE)
1.437
Maceration green tea in
ethanol (MGTE)
1.147
Maceration green tea in
distilled water (MGTA)
0.933
Maceration green tea in
aqueous: ethanol (1:1)
(MGTAE)
1.796
Maceration black tea in
ethanol (MBTE)
0.932
Maceration black tea in
distilled water (MBTA)
0.859
Maceration black tea in
aqueous: ethanol (1:1)
(MBTAE)
1.753
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Figure 5. TLC chromatogram of ethyl acetate fractions of green and black tea, ST: epicatechin standard, 1:
MGTA, 2: MGTE, 3: MGTAE, 4: MBTA, 5: MBTE, 6: MBTAE, 7: DGTA, 8: DGTE, 9: DGTAE, 10:
DBTA, 11: DBTE, 12: DBTAE, using S2 as a solvent system.
Figure 6. TLC chromatogram of ethyl acetate fractions of green and black tea, ST: epicatechin standard, 1:
MGTA, 2: MGTE, 3: MGTAE, 4: MBTA, 5: MBTE, 6: MBTAE, 7: DGTA, 8: DGTE, 9: DGTAE, 10:
DBTA, 11: DBTE, 12: DBTAE, using S3 as a solvent system.
The Rf values of epicatechin in all ethyl
acetate fractions with its standard in the best three
mobile phases were calculated as shown in table 4.
Table 4. The Rf values of epicatechin in all ethyl acetate fractions with epicatechin standard in best three
mobile phases.
Mobile phase S1 S2 S3
St. 0.387 0.333 0.289
DGTE 0.387 0.326 0.282
DGTA 0.387 0.333 0.289
DGTEA 0.380 0.319 0.275
DBTE 0.373 0.326 0.282
DBTA 0.373 0.319 0.275
DBTEA 0.373 0.326 0.289
MGTE 0.387 0.304 0.275
MGTA 0.387 0.290 0.275
MGTEA 0.380 0.304 0.268
MBTE 0.373 0.312 0.275
MBTA 0.373 0.319 0.275
MBTEA 0.373 0.333 0.282
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Qualitative and quantitative estimation of
epicatechin by HPLC
In this study, identification and quantitative
determination of epicatechin in all ethyl acetate
fractions of two tea samples were made by HPLC.
The epicatechin peak in ethyl acetate fractions was
detected by comparing the UV spectra and retention
time (min) with that of standard at same conditions
as given in figures 7-9.
Figure 7. HPLC chromatogram for epicatechin standard
Figure 8. HPLC chromatogram of ethyl acetate fraction of A: DGA, B: MGA, C: DGAE, D: MGAE, E:
DGE, F: MGE.
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310
Figure 9. HPLC chromatogram of ethyl acetate fraction of G: DBA, H: MBA, I: DBAE, J: MBAE, K: DBE,
L: MBE .
For quantitative determination, the curve of
calibration was drawn using area under the curve
(AUC) vs. four levels of concentration of
epicatechin standard. An equation of straight line
was got from which the analyte concentration was
determined in each ethyl acetate fraction as given in
figure 10.
The epicatechin was quantified in all ethyl
acetate fractions and the results indicated that ethyl
acetate fraction of DGTEA showed the highest
concentration of epicatechin (1104.34ppm) when
compared to other analyzed fraction. The ethyl
acetate fraction of MGTA showed the smaller
concentration of epicatechin (35.32ppm).
The concentrations of epicatechin in ethyl
acetate fractions of green and black tea are listed in
Table 5.
Iraqi J Pharm Sci, Vol.31(2) 2022 Detection of epicatechin in tea leaves by TLC and HPLC
311
Figure 10. HPLC Calibration curve of epicatechin
Table 5. The concentration of epicatechin in ethyl
acetate fractions of green and black tea.
Conclusion
Camellia sinensis (tea) is a widely used
beverage. Tea contains several useful substances for
human health. It is an excellent source of catechins
(bioactive molecule) including epicatechin, catechin,
epigallocatechin gallate, and epicatechin gallate. The
50% aqueous ethanol is better solvent for extraction
of epicatechin from green and black tea than water
and ethanol. The qualification of ethyl acetate
fractions by TLC and HPLC indicated that
epicatechin was found in two tea samples. The HPLC
method is appropriate for the determination of
epicatechin content in green and black tea samples.
There was a difference in the concentration of
epicatechin between the analyzed fractions but the
content of epicatechin was higher in the ethyl acetate
fraction of DGTAE when compared with other
fraction. It is suggested that the pharmacological
activities of plant as an anticancer and antioxidant
should be studied.
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Analyzed fraction Concentration of
epicatechin (ppm)
DGTE 344.22
DGTA 97.41
DGTAE 1104.34
DBTE 163.66
DBTA 247.25
DBTAE 748.47
MGTE 81.82
MGTA 35.32
MGTAE 924.09
MBTE 129.88
MBTA 161.06
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