Hair Growth Promoting Activity of Green Tea Leaves (Camellia sinensis l.) Ethanolic Extract

Abstract

Hair loss and baldness is one of the abnormalities in the hair that often occurs in both men and women. Many cosmetic products from natural to synthetic materials have been developed to overcome these problems, yet, synthetics product is potential to give side effects, such as local irritation. In this study, green tea (Camellia sinensis L.) leaves were used as active substances. The aim of this study was to attest the hair growth promoting activity of n-hexane, water, and ethyl acetate fractions from ethanolic extract of green tea leaves. Green tea leaves contain flavonoids which can help promoting hair growth. To obtain the compound, green tea leaves were made into ethanolic extract. The extract was obtained by maceration using 70% ethanol then partitioned using n-hexane, ethyl acetate, and water, to obtain the n-hexane, ethyl acetate, and water fractions. Ethyl acetate and water fractions have been shown to contain flavonoids, so it could continue the hair growth promoting activities with concentrations of 1% and 4%. Hair growth promoting activity was performed on rabbits. The results showed that 4% of water fraction containing flavonoids had the best hair growth promoting activity.

Full text

Trad. Med. J., May-August 2019
Vol. 24(2), p 104-109
ISSN-p : 1410-5918 ISSN-e : 2406-9086
104 DOI: 10.22146/mot.44053 | Traditional Medicine Journal, 24(2), 2019
Submitted : 09-03-2019
Revised : 10-05-2019
Accepted : 15-07-2019
Hair Growth Promoting Activity of Green Tea Leaves
(Camellia sinensis l.) Ethanolic Extract
Vini Noviani1, Shelly Thauresia1, Elsa Fitria Apriani2, Partomuan Simanjuntak1*
1 Magister of Pharmaceutical Sciences, Faculty of Pharmacy, Pancasila University, Jakarta, Indonesia
2 Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Sriwijaya University, South Sumatra,
Indonesia
ABSTRACT
Hair loss and baldness are one of the abnormalities in the hair that often occurs in both men and
women. Many cosmetic products from natural to synthetic materials have been developed to overcome
these problems, yet, synthetics product is potential to give side effects, such as local irritation. In this study,
green tea (Camellia sinensis L.) leaves were used as active substances. The aim of this study was to attest
the hair growth-promoting activity of n-hexane, water, and ethyl acetate fractions from ethanolic extract of
green tea leaves. Green tea leaves contain flavonoids which can help to promote hair growth. To obtain the
compound, green tea leaves were made into ethanolic extract. The extract was obtained by maceration using
70% ethanol then partitioned using n-hexane, ethyl acetate, and water, to obtain the n-hexane, ethyl acetate,
and water fractions. Ethyl acetate and water fractions have been shown to contain flavonoids, so it could
continue the hair growth-promoting activities with concentrations of 1% and 4%. Hair growth-promoting
activity was performed on rabbits. The results showed that 4% of water fraction containing flavonoids had
the best hair growth-promoting activity.
Keywords: Green tea leaves, hair growth-promoting activity, water fraction, flavonoids.
INTRODUCTION
Hair loss and baldness are abnormalities in
the hair that occurs mostly in men and women.
This is generally caused by physiological
conditions, emotional and physical stress, lack of
nutrition, hormonal disorders and drugs (Harrison
and Bergfeld, 2009).
Hair tonic from both herbal and synthetic
cosmetic products have been developed to
overcome hair loss and baldness. Along with the
development of technology, many Indonesian
people prefer to use herbal products because they
only cause a few side effects (Thorat, 2010), unlike
synthetic products like Minoxidil which can cause
side effects such as sensitivity to the scalp
(Adhirajan et al., 2008).
Indonesia is a country that has many herbal
plants that can be used as ingredients for making
hair tonic. Green tea leaves (Camellia sinensis L) are
one of the Indonesian plants, which contain
flavonoids. Flavonoids are proven to increase the
rate of hair growth. Flavonoids are known to be the
biggest phenol compounds contained in plants in
all parts of plants such as leaves, bark, flowers,
fruit, seeds, roots, pollen and sap (Markham,
1988). Based on research conducted by Lamria,
2.5% of green tea extract in hair tonic preparations
was able to increase significantly hair growth rates
compared to 2.5% of Minoxidil (Lamria, 2013).
*Corresponding author : Partomuan Simanjuntak
Email : partomuansimanjtk@yahoo.com
Another study, green tea (Camellia sinessis L) has a
pharmacological effect of hair growth-promoting
on 50% fraction of green tea polyphenol extract
(Camellia sinesis L) given to bald rats compared to
other groups of rats which were given only water
orally. The results showed 33% of mice given
polyphenol extract showed increase hair growth
significantly for 6 months and the group of rat that
given water did not show any hair growth
(Esfandiari and Kelly, 2005).
Based on the data above, in this study,
flavonoids were separated from other compounds
found in green tea leaves. To obtain flavonoids
from green tea leaves, partition extraction was
carried out with n-hexane, ethyl acetate, and water.
The purpose of partition extraction is to separate
flavonoid compounds with other compounds
based on their level of polarity.
METHODOLOGY
Materials
Green tea leaves (Gunung Mas, Bogor,
Indonesia), 70% ethanol, 96% ethanol, ethyl
acetate and aquadest (obtained from PT Brataco
Chemical, Indonesia).
Methods
Plant Determination
Plant determination was undertaken to
ascertain the type of plant used for the study.

Hair Growth Promoting Activity of Green Tea Leaves
Traditional Medicine Journal, 24(2), 2019 105
Green tea leaves are determined in Bogoriense
herbarium, Biology Research Center LIPI,
Cibinong, Bogor.
Preparation of Simplisia
Green tea leaves are collected and sorted.
Then washed with running water until clean and
drained it. Green tea leaves are dried by oven at
temperature 400C – 500C for seven days. After
drying, the green tea leaves are smoothed with a
grinder tool to obtain a powder of simplicia.
The simplicia powder is kept in a brown bottle
tightly closed, protected from sunlight and humid
air.
Preparation of Ethanolic Extract and Their
Fractions.
The simplicia powder from green tea leaves
used is 2000 grams. One part of dry powder is
added with 5 parts of 70% ethanol. The powder is
soaked in 70% ethanol for one day and stirred
every 6 hours. The obtained macerate is separated
by filtration using flannel cloth. This process is
repeated for five days using the residue of simplisia
powder. All the macerates are collected. The
macerate was concentrated with a vacuum
evaporator at temperature of 500C and heating it
over a water bath until the solvent evaporated
perfectly or viscous extract is obtained.
The viscous extract was partitioned using n-
hexane, ethyl acetate, and water. The n-hexane and
ethyl acetate fraction was concentrated using a
rotary evaporator. The water fraction is dried
using the freeze-drying method.
Raw Material Quality Test
The quality test of the raw material consists
of specific parameters test (organoleptic
examination, soluble compounds in certain
solvents), non specific parameters (loss on drying,
determination of moisture content, determination
of ash content, determination of acid-insoluble ash
content), phytochemical test and total flavonoids
in extract (Depkes RI, 1995; Depkes RI, 2000).
Flavonoid Screening
The flavonoid screening was conducted
according to Indonesia Materia Medica (Depkes RI,
1995b) and Harborne (Harborne, 1998). This test
includes the test of color reaction (Shinoda test and
pew test) on n-hexane, ethyl acetate, and water
fractions. Also, thin layer chromatography of ethyl
acetate and water fractions. The TLC plate is
sprayed using sitroborate to detect flavonoids.
Fractions contain flavonoid were selected to hair
growth-promoting activity test.
Hair Growth Promoting Activity of The Selected
Fraction
This study is referring to method from
Tanaka et al., 1980. The dorsal of rabbits were
divided into seven areas, with a size of 2 cm x 2 cm
each, on where the test solution was applied. The
hairs on each area were shaved using a razor.
After shaving and before applying basting, rabbit's
back for treatment area was smeared with 70%
ethanol as an antiseptic. These areas are then
marked as depicted in Figure 1 as follows: P1:
2.5% ethanolic extract; P2: 1% water fraction; P3:
4% water fraction; P4: 1% ethyl acetate fraction;
P5: 4% ethyl acetate fraction; P6: Negative control
(aquadest); P7: Normal control (not smeared).
The test solution for ethanolic extract was
made by dissolving the ethanolic extract in 96%
ethanol. The test solution for water fraction was
made by dissolving the water fraction in aquadest
to get a concentration of 1% and 4%, and the test
solution for ethyl acetate fraction was made by
dissolving the ethyl acetate fraction in ethyl acetate
to get a concentration of 1% and 4%,
The test solution was administered topically
on the rabbit shaved skin, twice a day (morning
and evening) with a volume of 1 mL, for 28 days.
On day 7, 14, and 21 and 28 from each treated area
was taken randomly 6 hairs and the length of each
hair was measured. Besides the length of each hair,
hair growth rate and hair weight also were
measured. The hair growth rate is obtained from
the length of the hair.
Statistical Analysis
Kruskal-Wallis and Mann-Whitney
(ANOVA) were used to determine the statistical
significance (p < 0.05) of the differences between
the values of various group.
RESULT AND DISCUSSION
Raw Material Quality Test
The color of green tea leaves powder
obtained is green. The ethanolic extract produced
was thick and brown. The ethanolic extract
obtained was 272.49 g (yield of 13.62%).
Figure 1. The dorsal aspect of rabbits where the
position of treatment area

Partomuan Simanjuntak
106 Traditional Medicine Journal, 24(2), 2019
While the results of partition extraction from the
ethanolic extract obtained n-hexane fraction was
7.61 g (yield of 2.79%), ethyl acetate fraction was
83.77 g (yield of 30.74%) and water fraction was
21.16 g (yield of 7.76%). This indicates a more
optimal partition process using ethyl acetate as the
solvent. The characterization of the ethanolic
extract (Table I).
Flavonoid Screening
The presence of flavonoids in the n-hexane,
ethyl acetate, and water fractions was tested using
a color reaction (Shinoda and pew test) and TLC
plate. The result of color reaction (Table II).
From the results of the color reaction test
showed that the water fraction and ethyl acetate
fraction positively contained flavonoids whereas in
the n-hexane fraction the results were negative,
meaning that no flavonoids were found in the n-
hexane fraction. This is because in general
flavonoids are polar, so it would be easy to dissolve
in the fraction with a polar solvent or semi-polar
solvents such as ethyl acetate and water, so in the
non-polar solvent (n-hexane fractions) flavonoids
did not extract perfectly.
This is because in general polyphenols and
flavonoids are polar, so it would be easy to dissolve
in the fraction with a polar solvent or semi-polar so
that the fraction of n-hexane non-polar compounds
of flavonoids did not extract perfectly.
This is because in general polyphenols and
flavonoids are polar, so it would be easy to dissolve
in the fraction with a polar solvent or semi-polar so
that the fraction of n-hexane non-polar compounds
of flavonoids did not extract perfectly.
This is because in general polyphenols and
flavonoids are polar, so it would be easy to dissolve
in the fraction with a polar solvent or semi-polar so
that the fraction of n-hexane non-polar compounds
of flavonoids did not extract perfectly.
This is because in general polyphenols and
flavonoids are polar, so it would be easy to dissolve
in the fraction with a polar solvent or semi-polar so
that the fraction of n-hexane non-polar compounds
of flavonoids did not extract perfectly.
This is because in general polyphenols and
flavonoids are polar, so it would be easy to
dissolve in the fraction with a polar solvent or
semi-polar so that the fraction of n-hexane non-
polar compounds of flavonoids did not extract
perfectly.
To ensure the presence of flavonoids in the
water fraction and ethyl acetate fraction, another
qualitative test was carried out using Thin Layer
Chromatography (TLC). TLC runs using several
mobile phases and detected in 254 nm (Figure 2).
From the results of Figure 2, it was shown
that the water fraction and ethyl acetate fraction
contained flavonoids. Thus, for certain
identification of nicotine, the plate was sprayed
with a specific color Dragendorff’s reagent. This
reagent usually forms an orange-red colored
complex in the reaction with alkaloids.
Consequently, the characteristic zone of nicotine
appears as orange bands on a yellow background
of derivatized chromatographic plate (Figure 1).
The image of the TLC separation indicates that all
analyzed samples contain nicotine. Hus, for certain
identification of nicotine, the plate was sprayed
with a specific color Dragendorff’s reagent. This
reagent usually forms an orange-red colored
complex in the reaction with alkaloids.
Consequently, the characteristic zone of nicotine
appears as orange bands on a yellow background
of derivatized chromatographic plate (Figure 1).
The image of the TLC separation indicates that all
analyzed samples contain nicotine.
Thus, for certain identification of flavonoid,
the plate was sprayed with a specific color
Citroborate’s reagent. This reagent usually forms
Table I. Several characteristic of green tea ethanolic extract
Characteristics
Results
Total ash content
4.42%
Acid insoluble ash content
Not detected
Water soluble
29.02
Alcohol soluble
21.40
Table II. The result of color reaction
Compound
Fraction
N- Hexane
Ethyl Acetate
Water
Flavonoid
-
+
+
The result of (-) indicates negative flavonoids and (+) indicates positive flavonoids.

Hair Growth Promoting Activity of Green Tea Leaves
Traditional Medicine Journal, 24(2), 2019 107
a yellow colored complex in the reaction with
flavonoids and the result of the derivatized
chromatographic plate shown that the water and
ethyl acetate fraction contained flavonoids (Figure
3).
To determine the total content of flavonoids
in the water and ethyl acetate fraction, quantitative
testing was carried out using spectrophotometry.
Based on quantitative tests of total flavonoids in
water and ethyl acetate fraction, it was found that
in the water fraction the total flavonoid content
was greater than the ethyl acetate fraction,
respectively were 0.41% and 0.25%.
Furthermore, the water and ethyl acetate
fraction was selected to hair growth-promoting
activity test.
Hair Growth Promoting Activity
In the hair growth-promoting activity test,
the average hair length and weight of rabbit hair
were measured. The average hair length is shown
in Table III and Figure 4.
From Figure 4, hair growth activity was seen
at week 1. The length of the hair has increased
every week in all treatments. The normal controls
that do not receive treatment describe normal
growth, the hair growth activity is slower than
other treatments. The 4% water fraction at week 4
(day 28) has a significant hair length compared to
other treatments.
Hair weight also measured in this study. On
week 4, hair shaving was carried out on the test
area then the hair was weighed. This hair weight
parameter is used to see the effect of each
treatment on rabbit hair thickness. The results of
hair weight measurements can be seen in Table IV
and Figure 5.
The hair weight of each rabbit in each
treatment is not significantly different, only in the
treatment with a 4% water fraction between
Figure 2. The TLC separation of flavonoid in UV light detection at 254 nm
A indicate water fraction and B indicate ethyl acetate fraction. The number of 1, 2, 3, 4, 5, 6 indicates several
mobile phase. No 1 indicate n-hexane : ethyl acetate (1:1), No. 2 indicate dichloromethane : methanol (2:1),
No.3 indicate dichloromethane : methanol (5:1), No. 4 indicate dichloromethane : methanol : water (5:5:1),
No. 5 indicate dichloromethane : methanol : water (7:3:1) and No. 6 indicate dichloromethane : methanol :
water (10:3:1),
Figure 3. The TLC separation of flavonoids detection in visible light after derivatization with Citroborate’s
reagent

Partomuan Simanjuntak
108 Traditional Medicine Journal, 24(2), 2019
rabbits No. 4 with the other rabbits showed
different weight.
The data of hair length and hair weight were
analyzed statistically using ANOVA, Mann-Whitney
and Kruskal-Wallis test. The ANOVA test has
shown that the data of hair length was distributed
normally and homogeneous yet the data of hair
weight was distributed not normally and not
homogenous. Statistical analysis of hair length and
hair weight using Mann-Whitney and Kruskal-
Wallis have shown that the increase of hair length
and hair weight in treated groups is significantly
different when compared to control and negative
treated groups (P<0.05) because in the normal
group no treatment was given and in the negative
group was only smeared with aquadest. In the
group smeared with ethanolic extract showed a
significant difference with the group smeared with
water and ethyl acetate fraction, because the water
and ethyl acetate fraction containing only
Figure 4. Graph of average of hair length per week
Table III. The average of hair length result
Treatment
The average of hair length (mm)
Week
1
2
3
4
Normal control
0.3250
0.5820
1.0092
2.3242
Negative control
0.4946
0.8075
1.2538
2.6825
2.5% ethanolic extract
0.9929
1.4258
2.8250
5.0667
1% water fraction
1.1004
1.3133
3.6892
5.7763
4% water fraction
1.1617
1,5813
5.2979
10.6233
1% ethyl acetate fraction
0.7075
0.7871
2.6371
3.9938
4% ethyl acetate fraction
0.8225
0.9242
3.6558
5.7217
Figure 5. Graph of hair weight result
0
2
4
6
8
10
12
14
1 2 3 4
Average of hair length (mm)
Time (week)
Normal Control
Negative Control
2,5% Ethanolic extract
1% water fraction
0
50
100
150
200
250
300
1 2 3 4
Average of hair weight (mg)
rabbit
Normal Control
Negative Control
2,5% Ethanolic extract
1% water fraction

Hair Growth Promoting Activity of Green Tea Leaves
Traditional Medicine Journal, 24(2), 2019 109
flavonoids and other compounds according to their
polarity while ethanolic extract still contained
other compounds which can affect the activity of
the ethanolic extract. The group smeared with
water fraction showed a significant difference with
the group smeared with ethyl acetate fraction
because the flavonoid content in the water fraction
was more. The 1% water fraction with a 4% water
fraction also showed a significant difference in
significance (p<0.05), because at greater
concentrations, the flavonoid compound content
will increase so the hair growth activity will
increases.
Based on the results of the statistical study,
the 4% water fraction was given the best hair
growth activity compared the other treatment.
CONCLUSION
Water and ethyl acetate fraction of green tea
ethanolic extract contains flavonoids which have
hair growth-promoting activities. The 4% water
fraction was given the best hair growth-promoting
activity compared to ethyl acetate fraction because
the flavonoid content in the water fraction is more
than ethyl acetate fraction.
ACKNOWLEDGMENT
The authors would like to thank the
technical staff of the Pharmaceutical Research
Laboratory, Faculty of Pharmacy, Pancasila
University who have assisted in the
implementation of this research.
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Adhirajan, N., Kumar, D.V. & Gowri, C., 2008,
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Departemen Kesehatan Republik Indonesia, 1995,
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Table IV. Hair weight result
Treatment
Hair weight (mg) in rabbit
Average (mg) ±SD
R1
R2
R3
R4
Normal control
10
15
11
17
12.00±3.30
Negative control
20
25
22
24
22.33±2.22
2.5% ethanolic extract
47
60
55
55
54.00±5.38
1% water fraction
110
115
120
140
115.00±13.15
4% water fraction
115
120
129
277
121.33±78.05
1% ethyl acetate fraction
35
37
40
41
37.33±2.75
4% ethyl acetate fraction
45
50
133
82
76.00±40.47