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ISSN - 0975-7058
Special Issue (October)
USING HAIR GROWTH ACTIVITY, PHYSICAL STABILITY, AND SAFETY TESTS TO STUDY HAIR
TONICS CONTAINING ETHANOL EXTRACT OF LICORICE (GLYCYRRHIZA GLABRA LINN.)
SHEILA MEITANIA UTAMI, JOSHITA DJAJADISASTRA, FADLINA CHANY SAPUTRI*
Department of Herbal, Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia. Email: fadlina.chany@farmasi.ui.ac.id
Received: 21 April 2017, Revised and Accepted: 18 August 2017
ABSTRACT
Objective: The purpose of this study was to determine the safety, physical stability, and hair growth activity of ethanol extract of licorice.
Methods: In this study, 2.5%, 5%, and 10% licorice extract was formulated into a hair tonic as a tonic is easier to use and is not sticky like a semisolid
dosage. The hair growth activity test was conducted by rubbing the hair tonic preparations on rabbit’s backs; subsequently, the hair length, hair
thickness, hair weight, and hair density were measured. Quantitative analysis of glycyrrhizic acid from the licorice ethanol extract with a ultraviolet
spectrophotometer showed a level of about 156.65 mg/g or 15.665%. The physical stability test was performed on samples of the tonic stored at low
(4±2°C), room (25±2°C), and high (40±2°C) temperature, and a cycling test was also performed. The safety test was performed using an eye irritation
test that employed the Hen’s egg test–chorioallantoic membrane (HET-CAM) method and a skin irritation test that employed the patch test method.
Results: The hair tonics containing 5% and 10% licorice extract had an equivalent activity of hair growth and even better than the positive control
containing 2% minoxidil. The physical stability test showed that the licorice extract hair tonic has good physical stability. The results of the safety test
showed no skin irritation, whereas the HET-CAM test showed that the hair tonic containing licorice extract showed mild eye irritation.
Conclusions: Licorice ethanol extract hair tonic solutions in concentrations of 2.5%, 5%, and 10% had hair growth activity similar to that of the
positive control (minoxidil). They have a good physical and chemical stability, also safe for topical use, except the 2.5% licorice ethanol extract hair
tonic solution which caused mild eye irritation.
Keywords: Licorice, Hair growth activity, Glycyrrhizic acid, Mild irritation.
INTRODUCTION
Hair protects against environmental factors, such as harsh temperatures
and ultraviolet (UV) light. Similar to how eyelashes protect the eye from
dust and nose hair protects the nose by filtering inhaled hair, hair also
protects the scalp against irritants. Hair also has a role in perspiration
and temperature regulation and has sensitive tactile senses [1]. Hair has
a growth and loss cycle that differs between each strand [2]. Although
hair loss is part of the natural hair cycle, an increase in the quantity
and frequency of hair loss results in balding. This increased quantity
and frequency of hair loss usually stem from stress, food consumption,
hormonal disturbances, and the side effects of medications.
The use of shampoo and conditioner is usually not enough as a hair
treatment to counter hair loss because hair is a vital cell that needs
high care and maintenance to stay healthy. Therefore, hair tonics can
be used for hair treatment [3]. Hair tonics are used to thicken hair or to
stimulate hair growth in people experiencing balding or hair loss [4].
In general, hair tonics contain irritants, such as alcohol, camphor,
capsicum, cantharidin, cinnamon, garlic, vitamin, ginseng, or nicotinic
acid [3]. Currently, most Indonesians choose to use herbal products due
to their minimal side effects compared with synthetic products, such as
minoxidil, which has side effects such as scalp sensitivity [5].
Licorice has been used in Ayurveda and Chinese medicine for its anti-
hair loss effect and its ability to restore dry hair. Its main components,
glyceric acid and its derivatives, are suspected to produce an anti-hair
loss effect by inhibiting the production of dihydrotestosterone (DHT)
through the suppression of 5-alpha-reductase enzyme activity, which
subsequently increases hair growth [6-8]. A study by Khalaf et al.
(2010) on methanol extract from licorice showed that there were eight
components with phytoestrogen activity (seven isoflavone components
and one coumestan component): Daidzein, daidzin, genistein, glycitein,
formononetin, ononin, and coumestrol. The phytoestrogen components
contained in licorice (isoflavone, daidzin, genistein, and glycitein) act as
active components in hair loss prevention by blocking the production
of DHT (a DHT blocker) and a β-sitosterol compound, which is a
hair regrowth promoter [9]. Glabridin and glabrene are flavonoids
contained in licorice that is suspected to have estrogen-like activity. One
study proved that petroleum ether extracted from licorice roots had the
potential to act as an agent to promote hair growth in female white rats
(Rattus norvegicus) [10].
The glyceric acid compound and its derivatives and phytoestrogen in
licorice have been suspected to promote hair growth. However, a study
on the activity of the compounds derived from licorice on hair growth has
not been conducted. This study was expected to produce raw materials
for a hair growth product with minimum side effects. The study involved
experimentation and observation of the hair growth of New Zealand
rabbits. This study also uses a stability and safety test using the patch
test and Hen’s egg test–chorioallantoic membrane (HET-CAM).
MATERIALS AND METHODS
Materials
This study used the following materials: Licorice extract (PT Tri Rahardja
Javaplant, Karanganyar, Indonesia), 2% minoxidil (Regrou®, obtained
from PT Surya Dermato Medica Laboratories, Surabaya, Indonesia),
96% ethanol (PT Brataco, Jakarta, Indonesia), distilled water,
propylene glycol (PEG) (PT Brataco, Jakarta, Indonesia), Microcare®,
PEG-40 hydrogenated castor oil and water, BHT (PT Brataco, Jakarta,
Indonesia), 2N hydrochloride acid (HCl), Veet® cream (PT Reckitt
Benckiser, Bogor, Indonesia), Lieberman-Burchard reagent, Mayer
reagent, Dragendorf reagent, 50% methanol, magnesium (Mg), dense
HCl, Chloroform (CHCl3), sulfuric acid (H2SO4)p.a., sodium hydroxide
(NaOH), acetate anhydride acid, Iron(III) chloride (FeCl3) solution,
0.4 M lead (II) acetate, isopropanol, anhydrous sodium sulfate, molish
LP, and standard glyceric acid (Sigma Aldrich, Nucleos, Singapore).
Research Article
PTMDS 2017 | The 1st Physics and Technologies in Medicine and Dentistry Symposium
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijap.2017.v9s1.20_25
45
Production of licorice ethanol extract
First, 5 L of ethanol was added to 500 g of dry licorice extract powder
and macerated at room temperature for approximately 48 hrs. Then,
after the filtrate and residue were separated, the filtrate was condensed
using a rotary evaporator at 60°C or lower and was macerated again
for 12 hrs by adding 3 L of ethanol and filtering it. This procedure
was repeated thrice to obtain a combination filtrate. Next, 1 L of 70%
ethanol was added and was maintained at a cool temperature for at
least 48 hrs. The residue and filtrate were separated through filtration.
The obtained filtrate was condensed to produce licorice ethanol extract.
This procedure was expected to attract 3-4% or more glyceric acid as its
main active component [11].
Determination of the glyceric acid concentration
A standard solution was produced by dissolving 10 mg of standard
glyceric acid in 100 mL of distilled water to obtain a master solution
of 100 ppm. The master solution was diluted into solutions of differing
concentrations, and the maximum wavelength and absorption scores
of the solutions were calculated using a UV spectrophotometer.
A calibration curve was made from the absorption scores.
A licorice extract sample solution was produced by dissolving 1 g
of the licorice extract sample in 100 mL of distilled water to obtain a
master solution of 1000 ppm. The solution was then diluted, and the
absorption scores were calculated using a UV spectrophotometer with
the maximum wavelength that had been previously determined based
on the calculations of the glyceric acid standard solution. The obtained
absorption scores were added to a formula on the calibration curve
to obtain the concentration of glyceric acid contained in the licorice
extract sample.
Production of the hair tonic
The formula was divided into three groups of differing licorice extract
concentrations: 2.5%, 5%, and 10%. The materials were weighed
according to the formula. In the oil phase, 1.2% PEG-40 hydrogenated
castor oil was mixed with a little ethanol. In another container, 0.5%
BHT, 0.1% menthol, and 0.5% microcare were dissolved with ethanol,
PEG was added little by little, and the solution stirred until it was
thoroughly mixed. The oil phase, the licorice extract solution, and
the water phase were mixed thoroughly, and the volume of the mixed
solution was increased by adding distilled water.
Evaluation of the hair tonic
Organoleptic examination
A visual examination of the odor, shape, and color of the produced hair
tonic was conducted.
Viscosity test
To test the hair tonic’s viscosity, 250 ml of the tonic was poured into a
Becker glass, and a spindle 1 was inserted to a previously determined
limit. Measurements were conducted with a Brookfield viscometer at
speeds of 5, 10, 20, 50, and 100 rpm. The obtained data were plotted to
sliding pressure (dyne/cm2) and sliding speed.
pH measurement
The measurement of the pH of the solutions was conducted using a pH
meter, and the pH was measured once every 1 weeks for 12 weeks.
Testing the physical stability of the hair tonic
The physical stability of the hair tonic tested in this study included its
odor, color, homogeneity, pH, and viscosity. The hair tonic’s physical
stability was evaluated at room (25±2°C), warm (40±2°C), and cold
(4±2°C) temperatures every 2 weeks for 12 weeks. A cycling test was
also conducted (six cycles) [12].
Hair growth activity test
This study used male New Zealand rabbits from Balai Penelitian Hewan
Ternak, Ciawi, that weighed 2-2.5 kg. Tanaka et al. modification was
used to measure hair growth [13]. The hairs on the rabbits’ backs
were divided into 5 ×10 cm test areas. After the rabbits’ backs were
shaved, six squares were made: Three squares on the left side and three
squares on the right side of the rabbits’ backs with measurements of
2.2 cm × 2.5 each. Every test area was marked with a marker. Each test
involved the application of a different treatment. The rabbits were left
for 24 hrs before the experiment. 1 mL of each sample was applied
twice a day for 6 weeks. The first day of the application was considered
day 0. Hair growth was measured based on hair length, density, weight,
and thickness.
Safety test
The safety test was conducted using the HET-CAM as an alternative to the
mucous membrane safety test method (for eye irritation) to avoid the
use of animals. The safety test was conducted by 20 volunteers who
were 17-30 years old [14]. The volunteers were chosen by inclusion
and exclusion criteria for participation in the study.
RESULTS
Determination of the concentration of glyceric acid in licorice
ethanol extract
The determination of glyceric acid in licorice ethanol extract was done
using a Hitachi U-2910 UV spectrophotometer with glyceric acid from
Sigma-Aldrich as a standard. Distilled water was used as a solvent
because of its dissolving properties and its ready availability [15].
The standard glyceric acid solution was diluted into concentrations
of 10 ppm, 20 ppm, 30 ppm, 40 ppm, and 50 ppm. The maximum
wavelength measurement, which was 256.5 nm, was then obtained
with the UV spectrophotometer, and the absorption scores of each
concentration were determined to be 0.140, 0.289, 0.433, 0.606, and
0.729, respectively. The obtained absorption scores were plotted with
the concentration to obtain a calibration curve formula.
The licorice extracts weighed 1 g, and the sample concentration of the
standard was 313.3 µg/ml. Finally, the glyceric acid concentration in the
licorice extract, 156.65 mg/g or 15.665%, was obtained.
Hair growth activity test
Hair length parameter
Hair length was measured every week from the 1st week of application
to the 6th week. The measurement was done by randomly taking 10
strands of hair from each test area and measuring their length using a
digital caliper. The mean hair length of each week’s samples is shown
in Table 1.
The results of the normality distribution test (Shapiro–Wilk test) and
the homogeneity test (Levene test) on the mean length of the hair in
each test group each week showed that the distribution of data was
normal and homogeneous every week (6 weeks in total). Therefore,
analysis of variance (ANOVA) testing was performed. The results of the
ANOVA test showed that there was no significant difference between the
test groups in weeks 4, 5, and 6. To find whether there was a significant
difference between the test groups, a least significant difference (LSD)
test was conducted. In week 2, the normality distribution test showed
that the data were normally distributed. However, the homogeneity test
showed that the data were not homogenous, so a non-parametric data
analysis was conducted using the Kruskal–Wallis test.
Hair weight parameter
Hair weight was measured on the 42nd day (the end of week 6) by
pulling all the hair from a 1×1-cm area in each test area and weighing
the hair. The weights of the samples are shown in Table 2.
The mean weights of the normal control, negative control, positive
control, formula 1, formula 2, and formula 3 groups were 93.7±8.88 mg,
97.26±6.15 mg, 115.3±15.00 mg, 114.56±7.34 mg, 130.66±11.38 mg,
and 118.52±8.50 mg, respectively. Statistical analysis was needed
to assess whether there was a difference between the weights of
Utami et al.
Int J App Pharm, Vol 9, Suppl 1, 2017,
46
hair. Based on a normality distribution test (Shapiro–Wilk test) and
a homogeneity test (Levene test) of the mean weights of the hair, the
data distribution was normal and homogeneous. The results of the
LSD test showed that the normal and negative controls did not show a
significant difference, meaning that the activity of the negative control
on the weight of the hair was similar to that of the normal control.
The three formulas showed a significant difference when they were
compared with the negative control. However, formulas 1 and 3 were
not significantly different from the positive control, whereas formula 2
differed significantly from the positive control.
Hair density parameter
Hair density was measured by counting the amount of hair per cm2 area
on the 42nd day (the end of week 6). The results are shown in Table 3.
The mean amounts of hair of the normal control, negative control, positive
control, formulas 1-3 groups were 1202±49, 1300±102, 1502±82,
1457±263, 1621±286 and 1322±146, respectively. Statistical analysis
was needed to assess whether there was a difference between the hair
densities. The normality distribution test (Shapiro–Wilk Test) and the
homogeneity test (Levene Test) on the mean amounts of hair showed
that the data distribution was normal and homogeneous. The LSD test
showed that the normal control and negative control did not have a
significant difference, meaning that the normal control and negative
control resulted in similar hair densities. When the positive control
was compared with the results of formulas 1-3, the three formulas did
not differ significantly from the positive control. This result shows that
formulas 1-3 showed hair densities similar to that of the positive control.
However, formula 2 differed significantly from formula 3.
Parameter of hair thickness
Hair thickness was assessed by measuring the thickness of hair strands
(diameter) at week 1 and week 6 using a scanning electron microscope.
The results are shown in Table 4. The diameters of the hair strands
of the normal control, negative control, positive control, formulas 1-3
groups in week 1 were 11.00±0.96 µm, 16.75±0.73 µm, 64.63±1.57 µm,
67.32±4.64 µm, 31.80±3.14 µm, and 71.72±3.39 µm, respectively.
The diameters of the hair strands in week 6 were 47.83±9.22 µm,
109.72±3.87 µm, 119.12±5.88 µm, 90.35±5.06 µm, 60.19±7.61 µm, and
112.65±.24 µm, respectively.
Evaluation of the hair tonic
Measurement of viscosity and rheology
The viscosity of the hair tonic was measured at weeks 0 and 12, and the
solutions were stored at temperatures of 4±2°C, 25±2°C, and 40±2°C.
The obtained data were then plotted with sliding pressure (dyne/cm2)
and sliding speed (rpm) until a flow property (rheology) was obtained.
The measurement of the viscosity at week 12 showed that there was no
change in the viscosity of the hair tonic. The rheogram showed that the
flow type from the hair tonic was Newton.
Physical stability test of the hair tonic
The three hair tonic formulas were stable at the low (4±2°C), room
(25±2°C), and high (40±2°C) temperatures. The cycling test of the hair
tonic formulas resulted in neither a physical change nor a separation
of phases.
Measurement of pH
The pH of the three hair tonic formulas was stable at around 5.6-5.9.
Safety test
HET chloriallantoic
The HET-CAM can observe hemorrhaging, lysis, and coagulation.
Hemorrhaging is defined as bleeding or blood leaving the circulatory
system. Lysis is the absence or the disappearance of small, fine
arteries after a hemorrhage. Coagulation, which can happen intra- or
extra-cellular, is the clotting of blood, which can appear a dark stain
and which may happen as a reaction of opacifying or turbidity of
the CAM.
Table 1: The mean length of rabbit hair each week
Test group Treatment Mean hair length (mm) ± SD
M1 M2 M3 M4 M5 M6
Group 1 Normal control 5.13±1.42 5.79±2.26 10.18±3.26 13.70±1.86 16.26±1.92 16.37±5.69
Group 2 Negative control 7.82±4.07 9.25±6.56 12.98±5.71 17.23±5.61 19.45±3.49 19.87±7.92
Group 3 Positive control (2% minoxidil) 9.23±3.84 10.18±7.27 13.57±6.06 19.22±5.20*,** 21.45±4.81*,** 22.80±6.46
Group 4 Formula 1 (2.5% licorice) 5.81±4.51 7.45±3.73 11.64±4.17 15.27±0.93 20.40±5.01 16.24±4.50
Group 5 Formula 2 (5% licorice) 6.78±4.28 8.86±4.73 12.80±5.35 18.57±2.98 21.70±5.31 22.78±5.66
Group 6 Formula 3 (10% licorice) 8.85±5.27 11.30±8.63 16.67±6.90 21.66±5.02* 24.45±4.86* 29.18±8.97*,**
SD: Standard deviation. *Significantly different (p<0.05) from the normal control, **significantly different (p<0.05) from the negative control
Table 2: The mean weight of rabbit hair in week 6
Test
group
Treatment Mean hair
weight (mg)±SD
Group 1 Normal control 93.7±8.88
Group 2 Negative control 97.26±6.15
Group 3 Positive control 115.3±15.00*,**
Group 4 Formula 1 (2.5% licorice extract) 114.56±7.34*,**
Group 5 Formula 2 (5% licorice extract) 130.66±11.38*,**
Group 6 Formula 3 (10% licorice extract) 118.52±8.50*,**
SD: Standard deviation. *Significantly different (p<0.05) from the normal
control, **significantly different (p<0.05) from the negative control
Table 3: The mean amount of hair at week 6
Test group Treatment Mean amount
of hair±SD
Group 1 Normal control 1202±49
Group 2 Negative control 1300±102
Group 3 Positive control 1502±82*,**
Group 4 Formula 1 (2.5% licorice extract) 1457±263*,**
Group 5 Formula 2 (5% licorice extract) 1621±286*,**
Group 6 Formula 3 (10% licorice extract) 1322±146
SD: Standard deviation. *Significantly different (p<0.05) from the normal
control, **significantly different (p<0.05) from the negative control
Table 4: The mean hair diameter at week 6
Test
group
Treatment Mean hair
diameter (µm)±SD
Week 1 Week 6
Group 1 Normal control 11.00±0.96 47.83±9.22
Group 2 Negative control 16.75±0.73 109.72±3.87
Group 3 Positive control 64.63±1.57 119.12±5.88
Group 4 Formula 1 (2.5% licorice
extract)
67.32±4.64 90.35±5.06
Group 5 Formula 2 (5% licorice
extract)
31.80±3.14 60.19±7.61
Group 6 Formula 3 (10% licorice
extract)
71.72±3.39 112.65±6.24
Utami et al.
Int J App Pharm, Vol 9, Suppl 1, 2017,
47
The results of the HET-CAM showed that, during the 5 minutes of
observation time, a lysis and coagulation reaction did not occur for
the three test groups. According to the irritation score obtained, the
negative control was non-irritative, whereas the positive control
and the 2.5% licorice ethanol extract were irritative. The irritative
properties contained in the hair tonic solution stemmed from the
properties of PEG and menthol, which might be slightly irritative to
the eye.
Patch test
The safety test for skin irritation was conducted with a single closed
patch test for 48 hrs and was attended by 20 volunteers who filled
the inclusion criteria. The solutions used for the irritation safety test
were a basic hair tonic and a 2.5% licorice ethanol extract hair tonic
solution. Before treatment, the volunteers signed an informed consent
form. A dose of 0.1 mL of the two solutions of hair tonic was applied to
the upper back of each volunteer, which was observed after 30 minutes,
24 hrs, and 48 hrs to assess whether there were any erythema or edema
reactions.
The results of the primary irritation index (PII) scoring for the basic
hair tonic and 10% licorice ethanol extract showed that the PII was
0.125, so the irritation was classified as insignificant (0-0.4). Based on
the results, the basic hair tonic and the 10% licorice ethanol extract
were safe for topical use.
DISCUSSION
Based on the hair growth activity test, formula 1 (2.5% licorice ethanol
extract), formula 2 (5% licorice ethanol extract), and formula 3 (10%
licorice ethanol extract) had hair growth activity as compared to the
activity of positive control, minoxidil. However, formulas 2 and 3
showed significantly better hair growth activity than that of the positive
control. For hair weight, formulas 1 and 3 had results similar to that of
the positive control. However, formula 2 (5% licorice ethanol extract)
had better results than the positive control.
Based on the mean amount of hair, formula 2 had the best results of
the three formulas. The bigger and rougher texture of the hair strands
seen in subjects to which formulas 1 and 3 were applied might cause
the amount of hair per cm2 to be less than that seen in formula 2. The
hair density parameter has shown that the positive control and the
three formulas stimulate hair follicles through the growth of new
hair. Based on the graphic seen in attachment 49 and Table 4, an
increase in the diameter (thickness) of each hair strand between week
1 and week 6 was evident. The difference in hair diameter between
the test groups was also evident. The positive control group had the
largest hair strands according to diameter, followed by the formula 3,
formula 1, negative control, and normal control groups. Based on the
four hair growth parameters, the three licorice ethanol extract hair
tonic formulas had activities similar to the positive control. However,
formulas 2 and 3 had significantly better hair growth activity than the
positive control.
The mechanism of action or chemical compound responsible for the
hair growth activity of licorice ethanol extract might be glyceric acid
and its derivatives and glycyrrhizic acid and its derivatives, which are
suspected to have anti-hair loss effects resulting from the inhibition
of the production of DHT through suppression of 5-alpha-reductase
activity [8,16,17]. According to phytochemistry identification, licorice
ethanol extract contains flavonoid and terpenoid, which can increase
hair growth by strengthening the capillary walls of the small blood
vessels that supply the hair follicles and by increasing blood flow to
hair follicles [18]. Alkaloids can enlarge hair strands, increasing the
food supply and nutrition to hair [19]. Saponin is a chemical compound
mostly found in licorice extracts that can stimulate hair growth in
alopecia (balding) cases caused by hormones or genetics. Saponin can
form bubbles, meaning it can clean dirt from the skin and can also act
as a counterirritant. Saponin can increase peripheral blood circulation,
thereby increasing hair growth [20]. Moreover, the phytoestrogen
compounds (isoflavone daidzin, genistein, and glycitein) contained in
licorice are the active ingredients in preventing hair loss by blocking the
formation of DHT (a DHT blocker), and the β-sitosterol compound acts
as a hair regrowth promoter [9].
Based on the results obtained from this research, the licorice extract
hair tonic accelerated the active phase of hair growth, prolonged the
active phase, and stimulated hair follicles, leading to larger strands
of rabbit hair. Based on the stability test, which studied the solutions’
color, odor, and pH stability, the hair tonic solutions showed good
physical stability when stored at low (4±2°C), room (25±2°C), and
high (40±2°C) temperatures. The safety test, which examined the skin
irritation of the hair tonic solutions containing 10% licorice ethanol
extract on 20 volunteers, showed that the solutions were safe for skin.
However, the eye irritation test showed that the 2.5% licorice ethanol
extract hair tonic solutions were irritative to the eyes.
CONCLUSION
Licorice ethanol extract hair tonic solutions in concentrations of
2.5%, 5%, and 10% had hair growth activity similar to that of the
positive control (minoxidil) according to the hair length and hair
thickness parameters. The 5% and 10% licorice ethanol extract
hair tonic solutions had better hair weight and density than that of
the positive control. However, the 5% hair tonic solution had worse
hair density results than the positive control. The concentration of
glycyrrhizic acid contained in the licorice ethanol extract used as
an active ingredient in the hair tonic solutions was 1456.65 mg/g
or 5.665%. The hair tonic solutions containing 2.5%, 5%, and 10%
licorice ethanol extract showed physical and chemical stability
when stored at low (4±2 °C), room (28±2°C), and high (40±2°C)
temperatures for 12 weeks. The HET-CAM and patch safety test
showed that the 10% licorice ethanol extract hair tonic solution was
safe for topical use. However, the 2.5% licorice ethanol extract hair
tonic solution was irritative to the eye.
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