Pharmaceutical Biology

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Effect of Citrullus colocynthis. on Hair Growth in

Albino Rats

R.K. Roy, Mayank Thakur & V.K. Dixit

To cite this article: R.K. Roy, Mayank Thakur & V.K. Dixit (2007) Effect of Citrullus
colocynthis. on Hair Growth in Albino Rats, Pharmaceutical Biology, 45:10, 739-744, DOI:
10.1080/13880200701585709

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Pharmaceutical Biology
2007, Vol. 45, No. 10, pp. 739–744
Effect of Citrullus colocynthis on Hair Growth in Albino Rats
R.K. Roy, Mayank Thakur, and V.K. Dixit
Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar (M.P.), India
Abstract
Citrullus colocynthis Schrad (Cucurbitaceae) is a tradition-
ally acclaimed hair tonic in Ayurveda (the traditional
Indian system of medicine). Studies were therefore
undertaken to evaluate petroleum ether and ethanol
extracts of C. colocynthis for their effect on hair growth
in albino rats. The extracts incorporated into oleaginous
ointment base were applied topically on shaved denuded
skin of albino rats. The time required for initiation of
hair growth as well as completion of hair growth cycle
was recorded. Minoxidil 2% solution was applied topi-
cally and served as the standard. Hair growth initiation
time was significantly reduced to half on treatment with
the petroleum ether extracts compared with untreated
control animals. The time required for complete hair
growth was also considerably reduced. The treatment
was successful in bringing a greater number of hair
follicles (> 70%) to anagenic phase than standard
minoxidil (67%). The result of treatment with 2 and
5% petroleum ether extracts were comparable with the
standard minoxidil.
Keywords: Alopecia, bitter apple, Citrullus colocynthis,
hair growth.
Introduction
Hair loss is a dermatologic disorder that has been recog-
nized for more than 2000 years. It is common throughout
the world and has been estimated to affect nearly 2% of
the world population (Olsen, 1993; Bertolino, 2000).
Apart from metabolic and hereditary causes, alopecia
has also been observed as a major side effect of anti-
cancer, immunosuppressant, and many others drug
treatments. Currently, minoxidil (Goodman & Gilman,
1996) (useful in both male and female pattern baldness)
Accepted: July 3, 2007.
Address correspondence to: Prof. V.K. Dixit, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar (M.P.) 470 003,
India. E-mail: vkdixit2011@rediffmail.com
DOI: 10.1080/13880200701585709
and finasteride (useful in male pattern baldness) (Libecco
& Bergfeld, 2004) are two U.S. FDA approved synthetic
drugs finding concomitant use for treatment of andro-
genic alopecia, but their side effects have reduced their
usage.
Natural products are unequivocally advocated in the
cosmetic and hair care industries and about 1000 plant
extracts have been examined for hair care usage.
Proanthocynidine from grape seeds and b-sitosterol in
saw palmetto have shown remarkable hair growth–
promoting activity (Takahashi et al., 1998). There are
many products available in the market, which are
prepared by combination of one or more herbal drugs
and find acceptability as hair tonic, hair growth
promoter, hair conditioner, hair cleansing agent, anti-
dandruff agents, and for the treatment of alopecia and
lice infection (Saraf et al., 1991; Roy et al., 2007).
The traditional system of medicine in India (Ayurveda
and Unani) recommends numerous herbal drugs for hair
growth promotion. However, lack of sound scientific evi-
dence limits their usage. Citrullus colocynthis Schrad
(Cucurbitaceae) is a herb that has been recommended
in traditional literature as a hair growth promoter
(Mukerji, 1953). The dried pulp of the unripe but full-
grown fruit freed from the rind constitutes the drug,
colocynth of commerce, and is used for treating loss of
hair (Anon, 1992). The oil from the seeds of the plant
has been reported and is used by ethnic tribes for con-
trolling premature falling and graying of hairs (Kirtikar
& Basu, 2003; Anon, 1998). Bhavprakash, an Ayurvedic
treatise, mentions the use of the drug for the treatment of
‘‘Indralupta’’, that is, the drug used in the treatment of
hair loss. The Sanskrit name ‘‘Indrayan’’ probably sig-
nifies the attributed hair growth rejuvenating property
of the drug (Chunekar & Hota, 2002). The drug was
included in the Indian Pharmaceutical Codex and Martin-
dales Extra Pharmacopoeia.
# 2007 Informa Healthcare USA, Inc.
740 R.K. Roy et al.
Besides its use as a hair growth promoter, C. colo-
cynthis is used for variety of purposes, viz, as a hypo-
glycemic (Al-Ghaithi et al., 2004), antifertility agent
(Chaturvedi et al., 2003), and anti-inflammatory agent
(Memon et al., 2003).
In the current study, we examined the effect of ethanol
and petroleum ether extracts of C. colocynthis on hair
growth initiation and promotion.
Materials and Methods
Plant material and preparation of extracts
C. colocynthis fruits were procured in the month of
November from the local market at Sagar (M.P., India)
and authenticated by comparing the characteristics in the
Indian Pharmaceutical Codex (Mukerji, 1953). A voucher
specimen for the plant has been deposited in the her-
barium at the Department of Pharmaceutical Sciences,
Dr. H.S. Gour University, Sagar. The plant material
was dried under the sun, and moderately coarse powder
(all the particles passed through a sieve of 710 mm and
not more than 40% through a sieve of 250 mm) of the
drug was used for the purpose of extraction. All the
chemicals and reagents used were of analytical grade.
The dried coarse powder (500 g) of C. colocynthis was
fed in to Soxhlet extractor and extracted initially with
petroleum ether (60–80
C) until completely exhausted.
The marc was further extracted in a Soxhlet extractor
with ethanol (95%) until completely exhausted. Removal
of solvent under reduced pressure gave yields of
4.7% w=w and 4.1% w=w for petroleum ether and
ethanol extracts, respectively.
Characterization of extracts
Thin-layer chromatography was performed on precoated
silica gel-G plates (10  10) (E. Merck, Darmstadt,
Germany) for characterization of the extracts. Chloro-
form:methanol:water (7:2.1:0.9 v=v) gave best resolution
for ethanol extract. Petroleum ether extract was best
resolved in toluene:ethyl acetate (84:16 v=v) as mobile
phase (Wagner & Bladt, 1992). The spots were visualized
using 50% ethanol H2SO4 as derivatizing agent.
Animals
Wistar strain albino rats of either sex weighing between
120 and 150 g were fed standard diet and water ad libi-
tum. The animals were housed at room temperature
(24  2
C) on a normal day-night cycle (0600 h to
1800 h). All animal experimentation was carried out after
approval of the protocol by the Institutional Ethical
Committee of Dr. H.S. Gour University. The guidelines
of CPCSEA, India, were strictly followed.
Preparation of samples
The petroleum ether and ethanol extracts were incorpor-
ated into ointment base in concentrations of 2% and 5%
(w=w), respectively. Ointments were made following
the procedure given in the Pharmacopoeia of India
(Anonymous, 1996). The extracts were then incorporated
in the prepared oleaginous base.
Treatment
Animals were divided into groups of six rats each. The
following treatment was given to animals of different
groups:
Group I: Vehicle only and served as control.
Group II: Topical application of 2% petroleum ether
extract in ointment base.
Group III: Topical application of 5% petroleum ether
extract in ointment base.
Group IV: Topical application of 2% ethanol extract in
ointment base.
Group V: Topical application of 5% ethanol extract in
ointment base.
Group VI (standard): Topical application 2% alcohol
solution of minoxidil (Mintop, Dr. Reddys Labora-
tory, Hyderabad, India).
Hair on the dorsal sides of the rats was removed using
hair clippers and electric shavers. A marketed hair
remover (Anne French) was also used to ensure complete
denual of a 6 cm2 area (Roy et al., 2006).
Toxicity studies
The ethanol and petroleum ether extracts were applied
on the surface of denuded skin for 7 days at 5% concen-
tration and observation made for skin rashes, inflam-
mation, or allergic reactions (Adhirajan et al., 2003).
Thus, the prepared extracts were safe for topical admin-
istration. Permission from the institutional ethical
committee was obtained for animal experimentation.
Statistical analysis
Data are reported as mean  SEM. Statistical analysis of
data was carried out by one-way ANOVA comparing all
test groups versus control followed by Dunnett’s test
using Instat v 2.1 software.
Qualitative hair growth study
Qualitative hair growth was evaluated by visual obser-
vation of two parameters: (a) hair growth initiation
time, that is minimum time to initiate perceptible hair
growth, and (b) hair growth completion time, that is,
 Hair growth and Citrullus 741

minimum time taken to cover the denuded skin region Table 1. Effect of Citrullus colocynthis Schrad extracts on
with new hair completely (Adhirajan et al., 2001). Hair qualitative hair growth.
growth initiation and completion time was recorded for
Hair growth (days)
each group of animals.
Treatment Initiation Completion
Sample no. (topical) time time
Quantitative hair growth study
1. Control (vehicle only) 12
0.82 24
1.02
The method reported by Uno (1991) was followed for the 2. Standard (2% minoxidil 6
0.41 20
1.08
quantitative evaluation of C. colocynthis extract. One rat solution)
from each group was euthanized after 10, 20, and 30 days 3. Petroleum ether extract 5
0.28 18
0.56
of treatment, skin biopsies were taken from the shaved (2% ointment)
area, and specimens preserved in 10% formalin. The spe- 4. Petroleum ether extract 4
0.12 18
0.42
cimens were fixed on paraffin wax, and blocks were pre- (5% ointment)
5. Ethanol extract 11
0.46 24
1.34
pared for microtomy. After fixation, vertical sections of
(2% ointment)
the skin were cut with the help of a semiautomatic rotary
6. Ethanol extract 10
0.68 24
1.21
microtome (Remi-1871, Mumbai, India). The sections (5% ointment)
were stained with hematoxylin and eosin. The number
of hair follicles per mm area of skin and ratio of hair fol- Values are mean
SEM, n ¼ 6.

licles in different cyclic phases, that is, anagen (active p < 0.05.

growth phase) and telogen (resting phase), were determ- p < 0.01.

ined using a microscope. Hair folliculogram was pre- p < 0.001, significance versus control.
pared by observing growth cycle of 100 hairs and
length of hair follicle (Uno & Kurata, 1993).
the minoxidil (2%)-treated group, which were short
and silky, and the hairs of the ethanol extract treated–
animals were coarse and rough as well (Fig. 1).
Results The results clearly show that the petroleum ether
extract was successful in reducing the time taken for hair
Qualitative studies on hair growth
growth initiation and completion. The results further
Hair growth initiation and completion time was con- suggest that petroleum ether extract of C. colocynthis
siderably reduced upon treatment with petroleum has better activity compared with minoxidil as far as hair
ether extract of C. colocynthis. In the control group growth initiation and completion is concerned.
animals, hair growth was initiated in the denuded area
in the second week, whereas it was noted in the
Quantitative studies on hair growth
first week in petroleum ether extract–treated groups
and minoxidil-treated groups. Hair growth was A considerable difference in cyclic phase of hair growth
initiated on the fourth day with 5% ointment of pet- was observed in groups treated with minoxidil and pet-
roleum ether extract of C. colocynthis. Hair growth roleum ether extract of C. colocynthis. In vehicle-treated
initiation was recorded on the fifth day with 2% oint- control group animals, most of the hair follicles are in
ment of C. colocynthis, whereas it was on the sixth telogenic phase, only one or two are in catagenic phase
day in minoxidil-treated standard group. Although hair (PM I). In the ethanol extract–treated group, a similar
growth initiation time was reduced in 2% and 5% etha- scenario is visible with most of the follicles in telogenic
nol extract–treated groups, it was only marginal and phase and only few in anagenic phase with no anagenic
not as pronounced as in the petroleum ether extract– hair follicles (PM II). There is a complete reversal of
treated group (Table 1). the picture in the petroleum ether extract–treated and
The time taken for complete hair growth on shaved minoxidil-treated groups where the majority of hair
area was also significantly affected with the petroleum follicles are in anagenic phase with only a few catagenic
ether extract of C. colocynthis. Complete hair growth hair follicles. Telogenic follicles are almost negligible
was observed on the 18th day with 2% and the 16th (PM III–IV).
day with 5% petroleum ether extract treatment. With An appreciable increase was noted in percent anagenic
standard minoxidil, complete hair growth was observed population (Table 2). An anagenic population of
on the 19th day. In ethanol extract and vehicle control 47
0.3% was found in vehicle-treated control group,
group animals, complete hair growth was noted after whereas, with 2% petroleum ether extract, it was
24 days. It was further observed that in the petroleum 72
0.2%, and with 5% petroleum ether extract treat-
ether extract–treated group, the texture of hair was ment, 75
0.2% hair follicles were found in anagenic
coarse, rough, and hard, compared with the hairs of phase. With minoxidil treatment, 67
0.5% hair follicles
742 R.K. Roy et al.

Figure 1. Qualitative effect of extracts of Citrullus colocynthis Schrad on hair growth.

were observed in anagenic phase, whereas treatment with
ethanol extract also improved population of anagenic
hair follicles, but it was not as appreciable as petroleum
ether extract treatment (Table 2).
Length of hair follicles
Treatment with 2% and 5% petroleum ether extract of
C. colocynthis had a remarkable effect on the length of
hair follicle. In the control group, only 34  0.4% had
average length of 0.5 mm, whereas in extract-treated
groups, 46  0.3% and 48  0.1% hair population
with more than 0.5 mm was observed with 2% and
5% extract treatment, respectively. The results of treat-
ment were comparable with the minoxidil group, where
49  0.1% hair population had length of 0.5 mm and
above (Fig. 2).
Discussion and Conclusions
Topical application with the petroleum ether extract of
C. colocynthis fruit reduced the time required for hair
growth initiation and was superior to standard (minoxidil
2%) solution. The quality of hair in the petroleum ether
extract–treated group was coarse, rough, and hard, whereas
the minoxidil-treated group.
Petroleum ether extract treatment caused premature
switching of hair follicle from resting telogenic phase to
active anagenic phase. It also helped the treated animals
to retain anagenic hair follicles. Thus, percent hair
follicle population in anagenic phase exhibited marked
improvement over the control group.
Petroleum ether extract was the best in inducing hair
growth initiation as well as completion. Minoxidil treat-
ment was the next to follow. The study thus confirms that
the petroleum ether extract (5%) treatment is on par
with 2% minoxidil treatment in hair growth in rats.

Table 2. Hair growth population (anagen=telogen ratio) after treatment with extracts of Citrullus colocynthis Schrad.

Percentage of hair follicles (%)

After 10 days After 20 days After 30 days

T=A ratio
Treatmenta Telogen Anagen Telogen Anagen Telogen Anagen

Control (vehicle) 58  0.4 42  0.4 55  0.4 45  0.4 53  0.3 47  0.3 1.21

Standard (2% minoxidil) 54  0.3 46  0.3 37  0.9 62  0.9 33  0.5 67  0.5 0.49
2% pet. ether extract 52  0.3 48  0.3 37  0.1 63  0.1 28  0.2 72  0.2 0.38
5% pet. ether extract 46  0.4 54  0.4 34  0.2 66  0.2 25  0.2 75  0.2 0.33
2% ethanol extract 55  0.5 45  0.5 50  0.6 50  0.6 46  0.8 54  0.8 0.85
5% ethanol extract 54  0.4 46  0.5 46  0.2 54  0.2 43  0.6 57  0.6 0.75

Values are % mean  SEM.

T=A, telogenic=anagenic ratio after 30 days of treatment.
a
All extracts were applied topically.

p < 0.05 (considered significant).
 Hair growth and Citrullus
Figure 2. Percent frequency distribution of hair follicle population after 30 days of treatment with C. colocynthis.
The remarkable improvement in length of hair follicles also
supports the hair growth–promoting effects of the herb.
Androgenetic alopecia (AGA) is a dihydrotestosterone
(DHT)-mediated process, characterized by continuous
miniaturization of androgen reactive hair follicles and
accompanied by perifollicular fibrosis of follicular units
in histologic examination (Yoo et al., 2006). Retention
of late anagenic follicles as well as an increase in follicular
length and prevention of their miniaturization may there-
fore be attributed due to 5a-reductase inhibitory activity.
However, detailed investigations in this regard are neces-
sary to explore the possibility of this mechanism.
The periodic transformation of hair follicles from telo-
gen to anagen phase in petroleum ether extract–treated
group is a healthy validation of hair growth potential
of C. colocynthis. During the treatment, the secondary
germ associated with aggregated dermal papilla cells in
telogen follicles augmented, and their continuous growth
and differentiation may have resulted in construction of
new anagenic follicles (Tanaka et al., 1980).
The current study validates the ethnomedical use of this
plant for hair loss treatment. Further utilization of the pet-
roleum ether extract and its incorporation in a formulation
are warranted for commercial utilization of C. colocynthis.
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