Content uploaded by Akhtar Naveed
Author content
All content in this area was uploaded by Akhtar Naveed on Feb 18, 2014
Content may be subject to copyright.
African Journal of Biotechnology Vol. 10(7), pp. 1206-1216, 14 February, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Exploring cucumber extract for skin rejuvenation
Naveed Akhtar1, Arshad Mehmood2, Barkat Ali Khan1*, Tariq Mahmood1, Haji Muhammad
Shoaib Khan1 and Tariq Saeed3
1Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur,
Pakistan.
2Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan.
3Department of Pharmacy, Punjab University Lahore, Pakistan.
Accepted 28 January, 2011
This study was designed to develop a topical skin-care cream water in oil (w/o) emulsion of 3%
cucumber extracts versus its vehicle (Base) as control and evaluates its effects on skin-melanin, skin
erythema, skin moisture, skin sebum and transepidermal water loss (TEWL). Hydroalcoholıc cucumber
(Cucumis sativus) fruit extract was entrapped in the inner aqueous phase of w/o emulsion. Base
containing no extract and a formulation containing 3% concentrated extract of C. sativus was
formulated. The odour was adjusted with few drops of lemon oil. Both the base and formulation were
stored at different storage conditions for a period of 4 weeks to predict their stability. Different stability
parameters that is: Physical stability, centrifugation and pH were monitored at different time intervals.
Both the base and formulation were applied to the cheeks of 21 healthy human volunteers for a period
of 4 weeks. The expected pharmaceutical stability of creams was achieved from 4 weeks in vitro study
period. Odour disappeared with passage of time due to volatilization of lemon oil. The base showed
insignificant (p >
> >
> 0.05) effects on all skin parameters except sebum that was not significant, whereas the
formulation showed statistically significant (p0.05) effects on skin sebum secretion. TEWL and
erythema was increased while skin melanin and skin hydration level was decreased by formulation.
However these effects were statistically insignificant (p >
> >
> 0.05). The results showed a good stability over
4 weeks of observation period of both base and formulation and the formulation has anti sebum
secretion, bleaching and moisturizing effects.
Key words: Cucumber extract, melanin, skin moisture, skin sebum, transepidermal water loss (TEWL).
INTRODUCTION
Emulsions can offer promising applications in pharma-
ceutical and cosmetic industries. There has been rene-
wed interest in the emulsion as a vehicle for delivering
drugs to the body as they frequently enhance the bio-
*Corresponding author. E-mail: barki.gold@gmail.com.
Fax: 0092629255243. Tel: 0092-333-9732578.
Abbreviations: TEWL, Transepidermal water loss; w/o, water
in oil; RH, relative humidity.
availability of the drugs (Herbert et al., 1988). Water in oil
(w/o) emulsions are employed more widely for the
treatment of dry skin and emollient applications (Magdy,
2004). Additional value can be given to these formula-
tions by including active ingredients with specific
cosmetic effects. Particularly, advantageous cosmetic
emulsion preparations are obtained when antioxidants
are used as active ingredients (Bleckmann et al., 2006).
There is a growing interest in natural antioxidants found
in plants. Many antioxidatively acting compounds are iso-
lated from natural herbs and extracts and used as
potential antioxidants in cosmetics (Naveed, 2001).
Cucumber extract can be obtained by macerating the
crushed fruit in hydro-alcoholic mixture, then filtering and
concentrating it on rotary evaporator. Extract so obtained
is rich in vitamins, especially vitamin C and A (Claudia,
1992; George, 2001), which have some cosmetic benefits
for the skin. In cosmetics, cucumber has an excellent
potential for cooling, healing and soothing to an irritated
skin, whether caused by sun, or the effects of a cuta-
neous eruption (Griere, 1992). Cucumber extract is often
used for skin problems, wrinkles, sunburn (James, 1997)
and as an antioxidant. The commercial aldehyde, trans-
cis-2, 6-nonadienal, described as a major volatile com-
pound of cucumber, was characterized as a non-
competitive inhibitor against 4-tert-butylcatechol oxidation
by mushroom tyrosinase (Fernando et al., 2003).
Cucurbitacin D and 23, 24-dihydrocucurbitacin D found in
cucumber extract are also responsible for the inhibition of
tyrosinase and melanin synthesis (Jian et al., 2005).
The purpose of this study is to develop w/o emulsion
(cream) containing the extract of cucumber and measure
its effects on different physiologic functions of skin like
melanin, erythema, skin moisture, skin sebum, pH of skin
and transepidermal water loss (TEWL).
MATERIALS AND METHODS
Identification of plant
The identification of cucumber (Cucumis Sativus) was performed at
Cholistan Institute of Desert Studies, The Islamia University of
Bahawalpur, Pakistan. The specimen was deposited in the
herbarium of Pakistan agricultural research council (PARC), the
voucher number is: pharm.3986/Feb 2001.
Materials
Cucumber was purchased locally. Paraffin oil (density 0.85) was
obtained from Merck (Germany). Abil-EM 90 (Cetyl Dimethicone
copolyol with HLB 5) was purchased from Franken Chemical
(Germany); Lemon oil was obtained from Chemoflor Manufacturing
Corporation Pakistan.
Apparatus
The apparatus used include: Centrifuge machine ( Hettich EBA 20,
Germany), cold incubator (Sanyo MIR-153, Japan), conductivity-
meter (WTW COND-197i, Germany), corneometer MPA 5, mexa-
meter MPA 5, sebumeter MPA 5, TEWA meter MPA 5 (Courage +
Khazaka, Germany), digital humidity meter (TES Electronic Corp,
Taiwan), electrical balance (Precisa BJ-210, Switzerland), homo-
genizer (Euro-Star, IKA D 230, Germany), hot incubator (Sanyo
MIR-162, Japan), pH-meter (WTW pH-197i, Germany), refrigerator
(Dawlance, Pakistan ) and rotary evaporator (Eyela, Co. Ltd.
Japan).
Preparation of emulsions
In this study, w/o emulsions were prepared by the addition of
Akhtar et al. 1207
aqueous phase to the oily phase with continuous agitation (Akhtar
et al, 2010). Oily phase consisted of paraffin oil (16%) and
surfactant ABIL-EM 90 (4%) was heated up to 75±1°C. At the same
time, aqueous phase consisting of water (q.s) was heated to the
same temperature and then the cucumber extract (3%) was added
to it. After that, aqueous phase was added to the oil phase drop by
drop. Stirring was continued at 2000 rpm by the mechanical mixer
for about 15 min until complete aqueous phase was added; 2 to 3
drops of lemon oil were added during this stirring time to give good
fragrance to the formulation. After the complete addition of the
aqueous phase, the speed of the mixer was reduced to 1000 rpm
for homogenization, for a period of 5 min, and then the speed of the
mixer was further reduced to 500 rpm for 5 min for complete
homogenization; until the emulsion cooled to room temperature.
Base was also prepared by the same above method and with same
ingredients but without cucumber extracts.
Pharmaceutical stability tests
Stability tests were performed at 8
±
0.1°C (in refrigerator),
25
±
0.1°C, 40
±
0.1°C and 40
±
0.1°C (in incubator) with 75%
relative humidity (RH). Physical characteristic of emulsions, that is,
color, creaming and liquefaction, were noted at various intervals for
28 days.
Study design for product evaluation on skin
One-sided blind study was designed with placebo control in the
month of August. 21 healthy human volunteers who signed the
informed consent, with age range 20-35 years were selected. All
the readings were performed at 21±01°C and 40±2% relative
humidity conditions.
The experiments were carried out on the cheeks of volunteers.
Patch test was performed on the forearms of each volunteer to
determine any possible reactions to the emulsions. Each volunteer
was provided with two creams. One cream was base and the other
was formulation. Each cream was marked with “right” or “left”
indicating application of that cream to the respective cheek. The
creams were applied twice a day by the volunteers themselves as
instructed for 60 days. Every individual was instructed to come on
1st, 2nd, 3rd and 4th week for the skin sebum measurements.
Ethical standards
This study was approved by the Board of Advance Study and
Research (BASR), The Islamia University of Bahawalpur and
institutional ethical committee in compliance with NIH Principles of
Laboratory Animal Care 1985. The Reference No is 1457/07.
Burchard tests (Patch tests)
Patch tests were performed on the forearms of each volunteer. The
patch (Bandage disc) for the right forearm was saturated with 1.0 g
of base while the patch for left forearm was saturated with 1.0 g of
formulation. Each was applied to the 5 cm X 4 cm marked regions
separately on each forearm. The regions were covered with the
surgical dressing after application. The patches were removed after
48 hours and the forearms were washed with physiological saline.
After 48 hours, scores were recorded for the presence of erythema
(skin redness) using a scale with 4 points from 0 to 3, where 0
stands for absence of erythema, 1 for mild erythema, 2 for mode-
rate erythema while 3 stands for severe erythema. Each volunteer
1208 Afr. J. Biotechnol.
Table 1. Score given by volunteers to base and formulation on the basis of itching/irritation.
Skin-care cream Score
0 1 2 3
Base 14 5 2 0
Formulation 16 3 2 0
was asked to note their irritation/itching towards the patches and
then assign a score from the same scale. Average score with
respect to volunteers is given in Table 1.
Panel test
Every individual was provided with a Performa preparedpreviously
to test the sensory values of cream. This form consisted of seven
parameters to be evaluated and every parameter was assigned 11
values from −5 to +5 indicating very bad to very good, respectively.
This form was asked to be completed independently by each
individual on day 28. Average points were calculated from the
points assigned by each volunteer for each question for both of the
creams, base and formulation (Figure 6).
Average points for the first question, that is, ease of application of
creams were found to be 4.00 and 4.20 for the base and
formulation, respectively. This indicated that base and
formulationcan easily be applied on the skin. Average points
regarding spread-ability were 4.40 for base and 4.10 for formulation
which meant that the formulation spread on skin better than the
base. Average points for feel on application were 3.70 for the base
and 3.30 for formulation. This indicated that base was felt well on
the skin than formulation. Average points for the sense in long-term
application of creams were 3.50 and 3.60 for the base and
formulation, respectively. This showed that formulation produced
more pleasant feeling on application to skin than base. There was
no irritation on the skin in both cases, that is, base and formulation,
as these were assigned 0.00 point for irritation by all the volunteers.
Shine on skin was 3.30 for the base and 3.20 for formulation. This
was expected since the base and formulation contained same
quantity of paraffin oil. Similarly, the formulation led to more
softness of the skin than base as the average point was 4.30 for
base and 4.60 for formulation.
It was found from paired sample t-test that there was a non-
significant difference between the average points of sensitivity for
base and formulation. It was concluded that there was no variation
between base and formulation regarding the sensory evaluation.
Both creams behaved similarly from the sensory point of view.
The percentage changes for the individual values of different
parameters, taken every week, of volunteers were calculated by the
following formula:
Percentage Change = [(A – B) / B]*100
Where, A, Individual value of any parameter of 1st, 2nd, 3rd, or 4th
week; B, zero hour value of that parameter.
Statistical analysis
The measured values obtained for different parameters (skin mois-
ture, sebum, melanin, erythema, elasticity and pH) were analyzed
using SPSS 12.0 on computer (paired samples t-test for variation
between the two preparations; two-way ANOVA for variation
between different time intervals). 5% level of significant was
applied.
RESULTS
Pharmaceutical stability tests
Organoleptic tests (color, liquefaction and phase
separation)
In this study, base and formulation were divided in to four
samples separately and these samples were kept at 8°C
in refrigerator, at 25, 40 and 40°C + 75% RH (relative
humidity) in stability chambers. They were observed
organoleptically with respect to change in color, lique-
faction and phase separation for a period of 28 days at
definite time intervals.
Centrifugation
Centrifugation tests for base and formulation kept at
different storage conditions were performed at 5000 rpm
for 10 min and phase separation was observed for 28
days at different time intervals. No phase separation after
centrifugation was found in any of the samples of base
and formulation.
Electrical conductivity
Electrical conductivity values for base and formulation
kept at different storage conditions for 28 days were
determined. No change in electrical conductivity was
found in any sample of base and formulation. The value
of electrical conductivity always remained zero.
pH tests
pH values of the base and formulation kept at different
storage conditions for 28 days have been determined and
reported in Table 2.
Dermatological tests
The percentage of changes in the measured skin-mela-
nin, skin erythema, skin moisture, skin sebum, TEWL
following the application of the base and the formulation
on the cheeks of human volunteers is demonstrated in
Akhtar et al. 1209
Table 2. pH values of base and formulation kept at 8, 25, 40 and 40°
C + 75% RH.
Time 8
°
C 25
°
C 40
°
C 40
°
C+75% RH
B F B F B F B F
0 h 5.38 5.89 5.38 5.89 5.38 5.89 5.38 5.89
12 h 5.27 5.78 5.53 5.93 5.41 5.72 5.17 5.69
24 h 5.89 5.81 5.64 5.86 5.66 5.64 5.36 5.72
36 h 5.96 5.84 5.47 5.61 5.83 5.80 5.51 5.79
48 h 5.67 5.74 5.81 5.71 5.43 5.42 5.24 5.61
72 h 5.77 5.69 5.62 5.76 5.48 5.29 5.38 5.62
7 days 5.28 5.90 5.83 5.86 5.21 5.67 4.86 5.55
14 days 5.55 5.81 5.80 5.72 4.94 5.65 4.90 5.34
21 days 5.34 5.80 5.22 5.71 4.66 5.72 4.26 5.57
28 days 5.20 5.24 4.60 5.70 4.31 5.10 4.03 5.26
Where, B, Base; F, formulation; RH, relative humidity.
-4
-2
0
2
4
6
8
10
1 2 3 4
Week period
%age change
Base Formulation
Figure 1. Percentage of change in skin melanin content after application of base and
formulation. Where, 1, 1st week; 2, 2nd week; 3, 3rd week; 4, 4th week.
Figures 1, 2, 3, 4, 5 and 6, respectively.
DISCUSSION
Color
The freshly prepared base and the formulation were ele-
gant white in color. No change occurred in color up to the
observation period of 28 days. This showed that the
emulsions were stable at different storage conditions, that
is, 8, 25, 40 and 40°C + 75% RH throughout the 28 days
study period.The observed no change in the color of base
and formulation may be attributed to different factors
contributing to emulsion stability, such as, the
components of oil phase that is, paraffin oil which is a
colorless, transparent, tasteless, non-fluorescent liquid
and is mix-ture of hydrocarbons (Henriette, 1995), Abil-
EM90 which is a clear, colorless and nontoxic liquid
emulsifier (Raymond et al., 2003). As cucumber extract
contains poly amine spermidine, which has bacterial
growth inhibitory effect (Khawola and Khuther, 1987), it
1210 Afr. J. Biotechnol.
-3
-2
-1
0
1
2
3
1 2 3 4
Week Periods
%age Change
Base Formulation
Figure 2. Percentage of change in skin erythema content after application of base
and formulation. Where, 1, 1st week; 2, 2nd week; 3, 3rd week; 4, 4th week.
-10
-5
0
5
10
15
1 2 3 4
Week Period
%age Change
Base Formulation
Figure 3. Percentage of change in skin moisture content after application of base
and formulation. Where, 1, 1st week; 2, 2nd week; 3, 3rd week; 4, 4th week.
may protect the formulation from microbial growth which
might produce such substances which are able to change
the color of the formulation during the storage time.
Liquefaction
The viscosity of emulsions plays an important role in their
flow properties and is a useful process indicator of emul-
sion quality (Nasirideen et al., 1998; Ronald and Thomas,
1994). As soon as an emulsion has been prepared, time
and temperature-dependent processes occur to affect its
separation leading to the decreased viscosity which
results in increased liquefaction (Herbert et al., 1988).
No liquefaction was observed in any of the sample of
base and formulation kept at 8 and 25°C during whole
observation period of 28 days. Slight liquefaction was
observed in the sample of base kept at 40°C on the
28thday. Liquefaction was also observed in sample of
base kept at 40°C + 75% RH from 21st day of
observation but there was no further increase in
liquefaction till the end of the study period. On the other
-40
-30
-20
-10
0
10
20
30
1 2 3 4
Week Periods
%age Change
Base Formulation
Figure 4. Percentage of change in skin sebum after application of base
and formulation. Where, 1, 1st week; 2, 2nd week; 3, 3rd week; 4, 4th
week.
-20
0
20
40
60
80
100
120
140
1 2 3 4
Week periods
%age Change
Base Formulation
Figure 5. Percentage of change in values of trans epidermal water loss
(TEWL) after application of base and formulation. Where, 1, 1st week; 2,
2nd week; 3, 3rd week; 4, 4th week.
hand, a slight liquefaction was observed in formulation
samples kept at 40°C + 75% RH on the 28th day of
observation. In w/o emulsion, the cream results from
sedimentation of water droplets and forms the lower
layer. According to the Stokes’ law, the rate of creaming
is inversely proportional to the viscosity of the dispersion
medium. So as creaming increases, the viscosity of the
base and formulation gradually decreases with increasing
temperature resulting in liquefaction (James and James,
2004).
Phase separation
Creaming is due to differences in density between the
two phases under the influence of gravity which leads to
phase separation (Derrick, 2000). Coalescence is one of
the possible mechanisms of destruction of emulsions,
which occur when the energy of adhesion between
twodroplets is larger than the turbulent energy causing
dis-persion (Abdurahman and Rosli, 2006).
The samples of base were stable at 8 and 25°C but
slight separation was observed visually at 40 and 40°C+
75% RH on the 28th day of observation. In the case of
formulation, no phase separation was observed in any of
the samples kept at 8, 25, 40 and 40°
C+ 75% RH up to
observation period of 28 days. This indicated that the
formulation was relatively more stable than base at
higher temperatures considering phase separation as a
parameter of stability. Slight phase separation in case of
Akhtar et al. 1211
1212 Afr. J. Biotechnol.
0
1
2
3
4
5
1234567
Parameters
Average Points
Base Formulation
Figure 6. Average values for panel test. Where, 1, Ease of application; 2, spreadability; 3,
sense just after application; 4, sense in long term; 5, irritation; 6, shine on skin; 7, sense of
softness.
base at higher temperatures may be attributed to the
movement of small number of surfactant molecules from
interface to the surface (Onuki, 1993) which is much
easier when the emulsion has a lower viscosity. Depen-
ding on conditions, emulsions may be more stable at
lower temperature due to increased phase viscosity
(Derrick, 2000).
Centrifugation test
Centrifugation is based on the principle of using
centrifugal force to separate two or more substances
ofdifferent density, for example, two liquids or a liquid and
a solid. In addition, it is an extremely useful tool for
evalua-ting and predicting the shelf life of emulsions
(Herbert et al, 1988).
In this study, centrifugation test was performed for the
samples of the base and formulation kept at different
storage conditions up to a period of 28 days at definite
time intervals. No phase separation on centrifugation was
seen in any of the samples up to the 28th day. This
indicated that the emulsions were stable at all the storage
conditions for 28 days. It may be said that proper homo-
genization speed during emulsion formulation prevented
the base and the formulation breakage during stress
testing (Abdurahman and Rosli, 2006).
Electrical conductivity
Conductivity differences arises when an emulsion creams
and the proportion of oil increases in the upper part of
emulsion and the proportion of water increases in the
lower part of emulsion (James et al., 2000). In this study,
conductivity test was performed for all the samples of
base and the formulation kept at different storage
conditions up to a period of 28 days at definite time
intervals. No electrical conductivity was seen in any of the
samples.
pH tests
The pH is a significant parameter as far as the effective-
ness of the cream is concerned. The pH of human skin
typically ranges from 4.5 to 6.0 (Jennifer et al., 2003) and
5.5 is considered to be average pH of the skin. Therefore,
the formulations intended for application to skin should
have pH closer to this range.
In this study, the pH of freshly prepared base and for-
mulation was 5.38 and 5.89, respectively, which is within
the range of skin pH. The pH values of the samples of
base kept at different storage conditions that is, 8, 25, 40
and 40°C+ 75% RH was found to be increase gradually in
the 1st week and then it started to decline continuously till
the 28th day with some variations. At the end of the
study, pH of the samples of base at 8, 25, 40 and 40°C+
75% RH was 5.20, 4.60, 4.31 and 4.03, respectively,
whereas pH of the samples of formulation kept at 8, 25,
40 and 40°
C + 75% RH showed gradual reduction in pH
values with slight variations with time. The pH values of
samples of formulation kept at 8, 25, 40 and 40°
C+ 75%
RH were 5.24, 5.70, 5.10 and 5.26 at 28th day, respect-
tively.
By using two-way analysis of variance (ANOVA)
technique at 5% level of significance, it was found that
the change in pH of different samples of base was not
significant at different levels of time and temperature but
there was significant difference in change of pH of
different samples of formulation at different levels of time
and temperature. When least significant difference (LSD)
test was applied to check the individual average effects of
the pH of the samples of base at different temperatures
with the passage of time by taking average pH values of
Zero hour at different temperatures as standard, it gave
non-significant changes except 3rd and 4th week where
differences were significant. Again, when LSD test was
applied to check the individual average effect of the pH of
the samples of formulation at different temperatures with
the passage of time by taking average pH values of zero
hour at different temperatures as standard, it gave
significant changes from 48th hour till the study period
except the 7th day. From LSD test it was concluded that
there was a non-significant change in pH of the samples
of base at different storage conditions but significant
changes were observed in pH of the samples of formu-
lation at different storage conditions with the passage of
time. The decrease in pH of the formulation at different
storage conditions might be due to the oxidation of
paraffin oil which produces aldehydes and organic acids
(Raymond et al., 2003).
Dermatological tests
Skin melanin
The major source of color in human skin derives from the
presence within the epidermis of specialized melanin
bearing organelles, the melanosomes. Melanosomes
synthesized by melanocytes are acquired by keratino-
cytes and transported within them to the epidermal
surface. Tanning of the human skin on exposure to
ultraviolet light results from increased amount of melanin
within the epidermis (Quevedo et al., 1975).
Polyphenol oxidase, also known as tyrosinase is widely
distributed in microorganisms, animals, and plants and is
responsible not only for browning in plants but also for
melanization in animals. The commercial aldehyde, trans,
cis-2, 6-nonadienal, described as a major volatile
compound of cucumber, was characterized as a non-
competitive inhibitor against 4- tert-butylcatechol oxide-
tion by mushroom tyrosinase (Fernando et al., 2003).
Cucurbitacin D and 23, 24-dihydrocucurbitacin D found in
cucumber extract are also responsible for the inhibition of
tyrosinase and melanin synthesis (Jian et al., 2005).
In this study, the effect of the base and the formulation
on the production of skin melanin was examined. The
amount of melanin was measured for 4 weeks at different
time intervals in each individual after application of base
and formulation and it was found that the base increased
the melanin contents in the skin irregularly till the end of
the 28th day while the formulation increased the melanin
contents in the 1st week but then decreased it gradually
throughout the study period. With the help of ANOVA
test, it was found that the base and formulation produced
a non-significant effects on skin melanin content in
volunteers. With the help of paired sample t-test, no
Akhtar et al. 1213
significant differences were observed between the
melanin effects of base and the formulation throughout
the study period.
This showed that the two creams, that is, the formu-
lation and the base, have different effects on melanin but
these differences are statistically non-significant for 28
days. It was concluded that the decreased skin melanin
content after application of formulation may be attributed
to the antioxidant activity of cucumber extract which is
rich in vitamins, especially vitamin C (Claudia, 1992) and
aldehyde, trans, cis-2,6-nonadienal, a volatile component
that causes inhibition of tyrosinase activity (Fernando et
al., 2003) and cucurbitacin D and 23, 24-dihydro-
cucurbitacin D found in cucumber which cause inhibition
of tyrosinase and melanin synthesis (Jian et al., 2005)
thus inhibiting melanogenesis.
Skin erythema
For confirming the safety of cosmetics, the important
point is that cosmetics must not cause any contact
dermatitis when applied to the skin. The cause of contact
dermatitis is not always due to cosmetic ingredients.
Even if the safety of cosmetics is verified, it is known that
environmental conditions such as temperature and
humidity, misuse by the consumer and the physical con-
ditions may all cause contact dermatitis. Skin irritation is
caused by the direct toxicity of chemicals on cells or
blood vessels in the skin and is different from contact
allergy which is caused by immune response (Naveed,
2001).
In this study, irritation was constantly monitored every
week for the base and the formulation throughout the
period of application. It was found that erythema contents
decreased in an irregular pattern after the application of
base throughout the study period. Whereas, after the
application of formulation, erythema contents were
slightly increased as the study progressed. With the help
of ANOVA test, it was found that the base and
formulation produced non-significant effects on skin
erythema at different time intervals and with the help of
paired sample t-test, it was evident that there was no
significant variation in irritation with respect to base and
formulation throughout the study period.
It was concluded that the base decreased while formu-
lation slightly increased the erythema contents of skin at
the end of study period and overall effect of formulation
on skin erythema was non-significant, so it can be used
safely without any significant skin irritation. As cucumber
extract is rich in vitamins (Claudia, 1992), it contains
vitamin A too. So the increase in the erythema contents
in case of formulation may be attributed to irritation cau-
sed by isotretinoin (Hughes et al., 1992) which is an
important metabolite of vitamin A (Kathryn, 1998). The
assumption for presence of isotretinoin is further streng-
thened by the reduction in sebum contents.
1214 Afr. J. Biotechnol.
Skin moisture content
The moisturizing treatment involves repairing the skin
barrier, retaining or increasing water content, reducing
TEWL, restoring the lipid barriers' ability to attract, hold
and redistribute water, and maintaining skin integrity and
appearance. Formulation of this study contained cucum-
ber extract as an active ingredient which is rich in
vitamins, especially vitamin C (Claudia, 1992). Vitamin C
is known to increase the collagen fibers in the dermis.
With the increase in collagen, the hydration conditions in
the dermis are improved (Padayatty and Levine, 2001). In
addition to this, extract also contains cucurbitacins, the
bitter principles of the cucurbitaceae (Gaofeng et al.,
2006) which are known for their non-specific cytotoxicity,
and very little is known about the mechanism of the effect
of cucurbitacins at the cellular and molecular level (Jian
et al., 2005). These may disrupt the super facial layer of
skin. That is, stratum corneum, thus reducing the
moisture contents.
In this study, it was found that there was a slight in-
crease in moisture values at the 1st and 2nd week after
the application of the base and a very slight decrease
was observed at the 3rd and 4th week; however after the
application of formulation, the increase in skin moisture
content was limited to the 1st week only, after this, it
decreased at the 2nd week and then remains almost
same throughout the study period. With the help of
ANOVA test, it was found that the base showed a non-
significant change with respect to the basic values
whereas the formulation showed a significant variation
throughout the study period of 28 days. By LSD test for
both base and formulation, it was found that significant
change in moisture content was observed only after the
1st week of application. Using paired sample t-test, it was
evident that insignificant differences in the moisture
values were observed after application of base and
formulation throughout the study period. The significant
reduction in moisture after application of formulation may
be due to cucurbitacin components of the cucumber, as
cytotoxicity of cucurbitacins is known before 1800 AD, but
very little is known about the mechanism of the effect of
cucurbitacins at the cellular and molecular level (Jian et
al., 2005). They may have disrupted the stratum corneum
so that water evaporated from the skin at higher rates
causing reduction in skin moisture. This argument is
further strengthened by the fact that TEWL also in-
creased significantly after application of formulation in
this study.
Skin sebum content
Sebum, the product of sebaceous glands, is a complex of
various lipids that are thought to act as an epidermal
and/or follicular lubricant (Robert et al., 2000). Sebum
production is measured using a special opalescent plastic
film, which becomes transparent when it is in contact with
sebum lipids. The device relies on a probe which presses
a piece of special film on the skin for a measured length
of time. The sebum is adsorbed on this film like ink on the
blotting paper and the film becomes transparent. The
probe is then placed into the device which radiates a light
beam onto the film. A metal mirror behind the film reflects
the beam back again through the film and then into an
instrument called a photomultiplier, which measures the
amount of light in the beam. The more sebum on the
skin, the more transparent is the film and the greater the
amount of light reflected.
In this study, the effects of the base and formulation on
the sebum contents of human cheeks were investigated.
Sebum was measured every week in all the individuals. It
was found that the base increased sebum contents in the
1st and 4th week of study period but decreased in the
2nd and 3rd week, while the formulation showed a
gradual reduction in sebum contents in a regular manner
as the study continued from the 1st to 4th week. With the
help of ANOVA test, it was evident that there was a signi-
ficant effect of base and formulation on skin sebum
throughout the study period. By applying LSD test, it was
evident that non-significant changes in sebum contents
were observed at different time intervals after application
of base while significant changes were observed after
application of formulation except the 1st week of study.
With paired sample t-test, it was found that the base and
formulation showed insignificant variations regarding the
skin sebum content after the 1st, 2nd and 3rd week while
significant variations were shown after the 4th week of
study.
It is concluded that increase in sebum contents after
the application of base may be attributed to the oily
nature of w/o emulsion having a thick viscous oily liquid,
that is, the paraffin oil (Henriette, 1995) while significant
reduction in sebum after application may be because of
isotretinoin, a natural metabolite of vitamin A (Kathryn,
1998), which is most effective in reducing sebaceous
gland size by decreasing proliferation of basal sebocytes
and in suppressing sebum production up to 90% by
inhibiting sebaceous lipid synthesis (Christos, 2006). The
assumption for presence of isotretinoin was further
strengthened by irritation observed in this study.
Trans epidermal water loss (TEWL)
TEWL is the outward diffusion of water through skin
(Jackie and Howard, 2005). TEWL is a measure of cuta-
neous barrier function and also reflects skin water
content (Ostlere et al., 1994). An increase in TEWL
reflects an impairment of the water barrier. TEWL mea-
surements allow parametric evaluation of the effect of
barrier creams against irritants and characterization of
skin functionality in clinical dermatitis and in irritant and
allergic patch test reactions. TEWL measurements can
be affected by the anatomical site, sweating, skin surface
temperature, inter- and intra-individual variation, air
convection, ambient air temperature, ambient air humidity,
and instrument related variables [Jackie, Howard, 2005).
In this study, it was found that there was increase in
TEWL values after the application of base having the
greatest value after the 1st week then gradual reduction
in loss, and after formulation, there was increase in
TEWL after the 1st, 3rd and 4th week but decrease in the
2nd week of study. With the help of ANOVA test, it was
found that changes in TEWL produced by formulation
were significant but non-significant for base during a
period of 4 week study. By applying LSD test, it was
observered that in both cases, that is, base and
formulation change in TEWL, values became significant
after the 1st week of application. Using paired sample t-
test, it was found that there was significant variation in
TEWL with respect to base and formulation after the 2nd
week of study but non-significant for other periods.
The significant increase in TEWL after application of
formulation may also be due to cucurbitacin components
of the cucumber. They may cause disruption of the
stratum corneum thus increasing the rate of water loss
from the skin. This argument was further supported by
the fact that skin moisture also decreased significantly
after application of formulation in this study.
Conclusion
This project was a preliminary step to explore the hidden
potential of cucumber for skin rejuvenation in the form of
topical cream. From our findings we concluded that a
stable topical cream (w/o emulsion) containing cucumber
extract can produce a pronounced decrease in melanin
content of the skin showing that the formulation has skin
whitening effects. The cream produced a pronounced
decrease in skin sebum content showing that the formu-
lation has anti-acne effects. The formulation was also
observed to decrease skin moisture content and increase
TEWL which strengthens the anti-acne effects. However,
further a targeted study needs to be conducted in future
in patients with freckles/melasma, acne, psoriasis as well
in people with dry wrinkled skin to evaluate this product
for the cosmetic market. Our investigations have proved
to be promising in terms of future potential applications of
cucumber extract, as skin-care products, cosmetics and/
or pharmaceutical preparations owing to these properties.
REFERENCES
Abdurahman HN, Rosli MY (2006). Stability investigation of Water-in-
Crude oil emulsion. J. Appl. Sci. 6: 2895-2900.
Akhtar N, Khan BA, Mahmood T, Parveen R, Qayum M, Anwar M,
Zaman SU, Farooq M (2010). Formulation and evaluation of
antisebum secretion effects of sea buckthorn w/o emulsion. J. Pharm.
Biol. Sci. 2: 13-17.
Akhtar et al. 1215
Bleckmann A, Kropke R, Schneider GU (2006). Preparations of the W/O
emulsion type with increased water content, and comprising cationic
polymers United States Patent. 7138128.
Christos CZ (2006). Isotretinoin Revisited: Pluripotent Effects on Human
Sebaceous Gland Cells. J. Invest. Dermatol. 126: 2154-2156.
Claudia L (1992). Nutritional aspects of fruit and vegetable consumption
Ciheam-Options Mediterranean. pp. 79-87.
Derrick R (2000). Fat crystals and emulsion stability, a review. Food
Res. Int. 3: 3-14.
Fernando GH, Mercedes JN, Juana C, Francisco GC, Josefa E (2003).
Tyrosinase Inhibitory Activity of Cucumber Compounds: Enzymes
Responsible for Browning in Cucumber. J. Agric. Food Chem. 51:
7764-7769.
Gaofeng Y, mark LW, Guoqing H, Min Y, Duo L (2006). Natural
products and anti-inflammatory activity. Asia Pacific. J. Clin. Nutr. 15:
143-152.
George M (2001). The World’s Healthiest Foods. The George Mateljan
Foundation.
Griere M A (1992). Modern Herb. Tigers Books International London, 1st
Ed. 1: 239-241.
Henriette K (1995). Paraffinum Liquidum, B.P. Liquid Paraffin. The
British Pharmaceutical Codex. 1911. at
http://www.henriettesherbal.com/.
Herbert AL, Martin MR, Gilbert SB (1988). Pharmaceutical Emulsions,
Pharmaceutical Dosage Forms: Disperse Systems, 1, Marcel Dekker,
New York and Basel. pp. 285-288.
Hughes BR, Norris JFB, Cunliffe WJ (1992). A double-blind evaluation
of topical isotretinoin 0.05%, benzoyl peroxide gel 5% and placebo in
patients with acne. Clin. Exp. Dermatol. 17: 165-168.
Jackie L, Howard M (2005). The correlation between transepidermal
water loss and percutaneous absorption: an overview. J. Cont.
Release. 103: 291-299.
James AD (1997). The Green Pharmacy. Rodale Press Emmaus,
Pennsylvania, pp. 394-459.
James S, Joseph TR, Orapin PR (2000). Remington, the science and
practices of pharmacy, Coarse dispersion, 20th ED. 1, Lipincott
Williams and Wilkins. pp. 316-334.
James SW, James BC (2004). Encyclopedia of pharmaceutical
technology. 2nd Ed. 2: 1066-1070.
Jennifer LM, Karen LC, Ibulaimu K, Philip FS, David JS (2003).
Evaluation of the effect of pH on in vitro growth of Malassezia
pachydermatis. Can. J. Vet. Res. 67: 56-59.
Jian CC, Ming C, Rui LN, Geoffrey AC, Samuel XQ (2005).
Cucurbitacins and cucurbitane glycosides: structures and biological
activities. Nat. Prod. Rep. 22: 386-399.
Kathryn MA (1998). Current therapies for the management of acne -
Tips from Other Journals. Am. Fam. Phys. 5: 13-17.
Khawola AF, Khuther DS (1987). Antimicrobial activity of the amine
fraction of cucumber (Cucumis sativus) extract. World. J. Microb.
Biotechnol. 3: 275-279.
Magdy IM (2004). Optimization of Chlorphenesin Emulgel Formulation.
AAPS J. 6: 1-7.
Naveed A (2001). Formulation and Evaluation of a Cosmetic Multiple
Emulsion system Containing Macademia Nut Oil and Two Antiaging
Agents. Dissertation for the Degree of Doctor of Philosophy (The
Department of Pharmaceutical Technology), Anadolu University. pp.
104-107.
Nasirideen S, Kas HS, Oner F, Alpar R, Hincal AA (1998). Naproxen
incorporated lipid emulsions. I. Formulation and stability studies. J.
Clin. Pharm. Ther. 23: 57-65.
Onuk A (1993). Viscosity of Emulsion with Surfactant. Europhys. Lett.
24: 151-156.
Ostlere LS, Taulor C, Baillod R, Wright S (1994). Relationship between
pruritis, transepidermal water loss and biochemical markers of renal
itch in haemodialysis patients. Nephrol. Dial. Transplant. 9: 1302-
1304.
Padayatty SJ, Levine M (2001). New insights into the physiology and
morphology of vitamin C. J. Can. Med. Assoc. 164: 353-355.
Quevedo WC, Fitzpatrick TB, Pathak MA, Jimbow K (1975). Role of
light in human skin color viariation. Am. J. Phys. Anthropol. 43: 393-
408.
1216 Afr. J. Biotechnol.
Raymond CR, Paul JS, Paul JW (2003). Dimethicone, Mineral Oil, Wax
White; Wax Yellow, Handbook of Pharmaceutical Excipients, 4th Ed,
The PhD. Washington: pp. 213-214, 395-396 and 687-690.
Robert WD, Herdt TH, Credille KM, Maier RL, Tucker KA, Davenport
GM (2000). Current Research in Canine Dermatology. The Iams
Company, Dayton, USA, pp. 10-15.
Ronald MB, Thomas FB (1994). Use of viscosity as an in-line diagnostic
for high internal phase emulsion generation US Patent 5306734.
