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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 75 No. 1 pp. 97ñ105, 2018 ISSN 0001-6837
Polish Pharmaceutical Society
UV light from sun trigger free radicals leading
to visible symptoms of skin aging. Skin aging is
depicting by increased wrinkling, decline in elastic-
ity, uneven pigmentation, increased roughness and
dryness .The traditional treatment regimens com-
prised profoundly of natural products to have fresh,
supple, younger-looking skin. Antioxidant supple-
ments are very important to maintain mechanics of
human skin (1). Several classes of emulsion may be
distinguished, namely oil-in-water (O/W), water-in-
oil (W/O) and oil-in-oil (O/O). Water-in-oil emul-
sions consists of the water phase ,which is inter-
nal/dispersed phase, mixed with oil, which is con-
tinuous phase. This emulsion type is often more dif-
ficult to prepare and stabilize since it is most often
based on totally non-emulsifiers. However, recent
advances in silicon chemistry and polymer chem-
istry have allowed preparation of excellent water-in-
oil (W/O) emulsions. A real benefit of these vehicle
emulsions is that they are readily spread on to the
lipophilic skin and provide a film which is resistant
to water wash off. This is how water resistant mois-
turizing cosmeceuticals are created (2). Over the
past decade, there has been fervent interest in prod-
ucts found in nature because of their perceived safe-
ty. Skin care products are often developed from
plants. Many believe that if a product can be safely
ingested, it will also be safe for topical application.
NON-INVASIVE IN VIVO EVALUATIONS OF COSMETIC EMULSION
CONTAINING PHYTOEXTRACT OF GRAPE SEEDS IN THE TREATMENT
OF SKIN AGING BY USING NON-INVASIVE BIO-ENGINEERING
TECHNIQUES
MUHAMMAD KHURRAM WAQAS1, NAVEED AKHTAR2, AKHTAR RASUL3, AYESHA SETHI3,
KHIZAR ABBAS4and TALIB HUSSAIN1*
1Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences,
54000, Lahore, Pakistan
2Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine,
The Islamia University of Bahawalpur, 63100, Bahawalpur, Pakistan
3Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Government College University,
38000, Faisalabad, Pakistan
4Department of Pharmacy, Bahauddin Zakariya University, 60000, Multan, Pakistan
Abstract: The current study was undertaken to investigate the skin lightening and the anti-aging potential of a
cosmetic W/O emulsion containing extract of grape seeds (Vitis vinifera) versus its base as control. A single
blinded placebo controlled study was performed by using non-invasive bio-physical techniques. Formulation
comprising of 4% of the concentrated extract of grape seeds was formulated by loading in internal aqueous
phase of cosmetic emulsion and base carried no extract. The cosmetic emulsions were applied to the cheeks of
11 healthy male volunteers for duration of 12 weeks. Both base and formulation were assessed for their effects
on different skin parameters i.e., skin melanin, moisture contents, elasticity and surface evaluation of living skin
(SELS). Statistically significant (p ≤ 0.05) decrease in skin melanin contents were displayed by the formulation
and the base presented a statistically insignificant (p > 0.05) increase in skin melanin while the formulation
showed significant (p ≤0.05) and the base showed insignificant (p > 0.05) effects on skin elasticity and skin
moisture contents. There is significant decline in SELS parameters i.e. SEsc (skin scaliness), SEw (skin wrin-
kles), SEsm (skin smoothness), SEr(skin roughness) after application of formulation. Topical application of the
cosmetic emulsion containing grape seeds extract exerts potential skin antiaging and skin depigmenting effects.
Keywords: Vitis vinifera, cosmetic emulsion, skin lightening, skin elasticity, surface evaluation of living skin
(SELS), skin moisture contents
97
* Corresponding author: e-mail: talib.hussain@uvas.edu.pk; dr.talibhussain@hotmail.com; phone: +92 3006907704, +924299211449-50
Ext. 307
98 MUHAMMAD KHURRAM WAQAS et al.
In general, plant-derived, botanical cosmeceutical
products tend to be antioxidant in action since these
organisms must thrive in constant direct ultraviolet
(UV) light, the Earthís most prolific manufacturer of
free radicals (3).
The grape vine (Vitis vinifera) is indigenous to
southern Europe and Western Asia and is cultivated
today in all temperature regions of the world. Parts
of this plant are known by several trade names
throughout the world as grape seed, grape seed
extract, activin and others. The seeds of Vitis
vinifera L. (Vitaceae) and its many varieties contain
polyphenolic proanthocyanidins, which in turn can
bind to each other to form oligomers known as pro-
cyanidins (4). These procyanidins are strong antiox-
idants (compared with vitamin C and E), by inhibit-
ing lipid peroxidation, facilitate wound healing and
protect collagen and elastin from degradation.
Grape seed extract shows tyrosinase-inhibiting
activity and is used in antiaging and skin-lightening
cosmetics (5).
The purpose of the present work is to develop
a stable formulation of W/O following inclusion of
botanical extract containing grape seeds extract and
its effects were analyzed on various skin related
parameters by applying non-invasive biophysical
techniques.
MATERIALS AND METHODS
Materials
Grape seeds were obtained from a local market
of Bahawalpur, Pakistan and authenticated by the
CIDS (Cholistan Institute of Desert Plants Studies),
The Islamia University of Bahawalpur, Pakistan.
For future reference, a voucher specimen (Voucher
no. GS-LF-8-15-25) has been kept in the herbarium
at CIDS, The Islamia University of Bahawalpur,
Pakistan. Polysiloxane polyalkyl polyether copoly-
mer (ABIL EM 90) was purchased from the Franken
Chemicals, Germany, n-hexane & paraffin oil were
purchased from Merk KGaA, Darmstadt (Germany).
Ethanol and acetone were taken from BDH,
England. Distilled water was prepared in the Cos-
metics Laboratory, Department of Pharmacy, The
Islamia University of Bahawalpur, Pakistan.
METHODS
Preparation of plant extract
Grape seeds were obtained from a local mar-
ket in Bahawalpur, Pakistan. 200 grams of raisins
(dries seeds of Vitis venifera) were well ground by
a grinder and put into a glass beaker. One liter
hydroalcoholic mixture was made by mixing 70%
methanol and 30% water. This mixture was then
added to the glass beaker containing raisins. The
beaker was sealed with aluminum foil and kept at
room temperature for 72 h. The beaker was then
shaken for 10 min twice a day. After 72 h, the
macerated material of plant was passed through
16 folds of muslin cloth for coarse filtration. The
resulting filtrate was passed through a Whatman #
1 filter paper. The filtrate obtained from the pre-
vious step was evaporated with the help of rotary
evaporator under reduced pressure at temperature
of 40OC. The evaporation was carried out till the
concentrate remained one third of the original
quantity. The process of extraction was complet-
ed at this step and dark brown extract was
obtained, collected in a stoppered bottle and
refrigerated.
Figure 1. Percentage changes in skin melanin contents after application of base and formulation
Non-invasive in vivo evaluations of cosmetic emulsion containing... 99
Preparation of cosmetic emulsions
The water in oil emulsions were prepared with
little modification of the previously reported method
(6). For the formulation, oily phase composed of
paraffin oil (16%) and emulsifier Abil-EM 90 (4%)
were heated together up to 75 ± 1OC. At the same
time, distilled water (quantity sufficient to make
100%) was heated at the same temperature and then
4% grape seeds extract was added in 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 rose 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 method and with same ingredients but without
the grape seeds extract. The different samples of
base and prepared formulations were analyzed for
different in vitro characterizations to select the stable
formulation. Both base and selected formulation
was found stable after evaluating pH, centrifugation,
electrical conductivity, phase separation, organolep-
tic and physical characteristics (color, creaming and
liquefaction) and the temperature stability tests by
keeping the emulsions to storage at 8 ± 0.5OC, 25 ±
0.5OC, 40 ± 0.5OC with ambient RH (relative humid-
ity), and 40OC ± 0.5OC with 75% RH for the period
of three months already published (7).
Non-invasive techniques
The evaluation of skin microrelief parameters
was done by using Visio ScanÆVC98 (Courage and
Khazaka, Germany). The skin elasticity was meas-
ured by an elastometer (Skin ElastometerÆEM 25,
Courage and Khazaka, Germany).A skin capaci-
tance meter (CorneometerÆMPA 5, Courage and
Khazaka, Germany) was used for the determination
of skin moisture contents. Mexameter MPA 5
(Courage + Khazaka, Germany) was used for the
determination of skin melanin.
Ethical standards
This work was authorized by the Board of
Advanced Studies and Research (BASR), and its
Ethical Committee for in vivo Studies (Reference
No. 4710/Acad.), The Islamia University of
Bahawalpur, Pakistan and was directed according to
the international guidelines of Helsinki Declaration.
Patch test
To determine the skin irritation assessment, on
the first day of study patch tests were performed on
forearms of each volunteer. An area of 5 ×4 cm was
marked on both the forearms of all the volunteers
Basic values for erythema and melanin were meas-
ured with the help of mexameter. One gram of base
and formulation each were applied to the 5 ◊ 4 cm
marked regions separately on each forearm. Surgical
dressing was used to cover the marked area of right
and left forearm. After 48 h application of emulsion,
it was removed and the forearms were washed with
physiological saline and were observed for any skin
redness/irritation by a dermatologist. The measure-
Figure 2. Percentage changes in skin moisture contents after application of base and formulation
100 MUHAMMAD KHURRAM WAQAS et al.
ments of erythema and melanin were repeated on
both forearms.
Study protocol
Eleven volunteers were selected whose ages
were in between 25 and 35 years. Male volunteers
were included in this work and consent forms were
taken. Prior to the tests, a dermatologist examined
the volunteers for any serious skin disease or dam-
age especially on cheeks and forearms. Each volun-
teer was provided with the two emulsions. One
emulsion was the base (B) with no extract and other
was the active formulation (F) containing grape
seeds extract. The study was designed single blind-
ed placebo-controlled for the comparisons of two
emulsions. The investigations were achieved on
cheeks of volunteers. Each cream was marked with
ìrightî or ìleftî indicating application of that cream
to the respective cheek. They were also given
instructions regarding way of application. The vol-
unteers were advised to use an amount which can be
absorbed easily on the skin. Each volunteer applied
emulsion for the period of 12 weeks. Values for dif-
ferent parameters were taken in controlled room
temperature 25 ± 1OC and 45 ± 2% relative humidi-
ty. The site of measurement was the whole cheek.
Every volunteer was instructed to come for meas-
urement on 2nd, 4th, 6th, 8th, 10th and 12th week. Values
for each parameter was measured three times (n = 3)
and average of the values has been noted.
Mathematical analysis
The percentage changes for the individual val-
ues of different parameters, taken every week of vol-
unteers were calculated by the following formula:
Percentage change = [(A ñ B) / B] ◊ 100
where A = individual value of any parameter at 2nd,
4th, 6th, 8th, 10th and 12th week; B = zero hour value of
that parameter.
Statistical analysis
The evaluation of data was done by using
SPSS version 19.0 according to two-way ANOVA
for variation between different time intervals defin-
ing a 5% level of significance and paired samples t-
test for variation between the two preparations.
Standard error of means (SEM) was calculated for
every mean value.
RESULTS AND DISCUSSION
Skin melanin
Melanin is responsible for the diversity in
human skin tones. Darker skin does not contain
more melanocytes; the cells are simply more active.
Variation in human skin color is mainly due to the
presence of four pigments, namely, melanin, hemo-
globin, carotene and melanoid. Pigmentation of the
skin is controlled by hormones which are synthe-
sized and distributed by the pituitary gland. Tanning
of the skin when exposed to UV radiations is due to
the increase in the amount of melanin in the epider-
mis (8).
In the present study, the skin melanin content
of the volunteers was measured at specified intervals
for 12 weeks. It showed markedly different effects
of base and formulation on the skin melanin content.
The base tends to increase the melanin level where-
as the formulation gradually decreased it and shown
in Figure 1. With the help of two-way ANOVA test
Figure 3. Percentage changes in skin elasticity after application of base and formulation
Non-invasive in vivo evaluations of cosmetic emulsion containing... 101
defining a 5% level of significance, it was observed
that base insignificantly (p > 0.05) increased the
skin melanin values while in case of formulation
there was significant (p ≤0.05) decrease in skin
melanin contents with respect to time. By applying
paired sample t-test, it was observed that formula-
tion presented significant (p ≤0.05) effects with
respect to base.
The increase in melanin content by the base
may be attributed to the presence of paraffin oil. The
preparations containing paraffin oil found to
increase the melanin level in the skin when applied
topically (9). The reduction in the skin melanin con-
tent by the formulation may be credited to the proan-
thocyanidins, which are oligomers and polymers of
polyhydroxy flavan-3-ol units, such as catechin and
epicatechin, present in large amounts in the
polyphenols of red wine and grape seeds. In grape
seed extract (GSE), only the procyanidin type of
proanthocyanidins has been detected. GSE is known
as a powerful antioxidant that protects the body
from premature aging, disease, and decay. Dietary
supplements such as GSE enriched in OPCs
(oligomeric procyanidins) have been suggested to
have multiple health benefits, due to antioxidant and
other beneficial activities of the plant extract. Grape
Figure 4. An image of skin taken by VisioscanÆbefore application of formulation
Figure 5. An image of skin taken by VisioscanÆafter three months application of formulation
102 MUHAMMAD KHURRAM WAQAS et al.
seeds extract provides excellent protection against
oxidative stress and free radical-mediated tissue
injury. It has been reported that antioxidants or com-
pounds with redox properties can inhibit or delay
hyperpigmentation (10).
Topical application of GSE enhances sun pro-
tection factor in human volunteers. It has been
reported that proanthrocyanidins from GSE inhibit-
ed the activity of tyrosinases obtained from mush-
room and from B16 mouse melanoma cells and
decreased the melanin content of the cells (11). It is
demonstrated that grape seeds extract has been
effective in lightening the UV-induced pigmentation
of guinea pig skin. This effect may be related to the
inhibition of melanin synthesis by tyrosinase in
melanocytes and the ROS-related proliferation of
melanocytes (12).
Skin moisture contents
The moisturizing remedy involves renovating
the skin barrier, restoring the water contents, ability
to retain and redistribute water and sustaining the
integrity and skin complexion (13). In this study
skin moisture content of the volunteers was meas-
ured by state of the art corneometer at specified
intervals for 12 weeks. The study showed a regular
increase in the moisture content in case of formula-
tion but the effect of the base was not regular (Fig.
2). The ANOVA test showed that effect of formula-
tion was significant (p ≤0.05) whereas the base has
Figure 6. 3D image of skin taken by VisioscanÆbefore application of formulation
Figure 7. 3D image of skin taken by VisioscanÆafter 3 months of application of formulation
Non-invasive in vivo evaluations of cosmetic emulsion containing... 103
insignificant (p > 0.05) effects on the skin moisture
content with respect to time. Moreover, the paired
sample t-test proved that there had been significant
differences in moisture values at all the specified
intervals.
The increase in the skin moisture content in
case of formulation may be due to the presence of
flavonoids in grape seeds extract (14). Flavonoids
possess high affinity for the collagen and elastin
fibers which impart moisturizing effects to the skin.
Consequently, it may be used in moisturizing cos-
metic formulations and also as a complement in the
treatment of dry skin (15).
Skin elasticity
The elasticity of the skin is mainly due to the
collagen fibers which is a main constituent of the
dermis of the skin. It constitutes about 70% of
total dry weight of the skin. The collagen is vul-
nerable to the UV radiations of the sunlight which
causes structural changes in them resulting in
photo aging. Photo aging is due to the oxidative
effects of these radiations on the collagen of the
skin (16). In this study, the skin elasticity of the
volunteers was measured at specified intervals for
12 weeks. The irregular pattern in the values of
skin elasticity was observed by the base but there
was regular increase in skin elasticity after the
application of formulation throughout the study
duration shown in Figure 3. The ANOVA test
proved that the variations in values of skin elastic-
ity by formulation was significant whereas with
base it was insignificant (p ≥0.05) with respect to
time. By applying paired sample t-test it was obvi-
ous that significant differences in skin elasticity
values were noticed after application of the formu-
lation throughout the study duration.
The improvement in skin elasticity after appli-
cation of formulation is due to the presence of
proanthocyanidins (PAs) in grape seed extract
which increase the collagen synthesis and accelerate
the conversion of soluble collagen to insoluble col-
lagen during development and decrease the rate of
enzymatic degradation of collagen matrices (17).
PAs can also inhibit the catabolism of soluble
collagen in animal studies. It stimulates normal skin
fibroblast proliferation, and increase the synthesis of
extracellular matrix, including collagen and
fibronectin. PAs are natural products with polyphe-
nolic structures that have the potential to give rise to
stable hydrogen bonded structures and generate col-
lagen matrices (18).
Surface evaluation of living skin (SELS)
The radings of various parameters of SELS
e.g., Sew, , SEsc, SEsm and SEr were measured by
VisioscanÆ (VC 98, software SELS 2000, Courage
and Khazaka GmbH) before application of creams
(0 hour readings) and at 1st, 2nd and 3rd month of
study period (Table 1). SEris the roughness param-
eter which depicts the asperity of the skin and cal-
culates the gray levels above the threshold in com-
parison with the whole image. It calculates the pro-
portion of dark pixels. SEsm is the index of smooth-
ness and is calculated from mean width and depth of
wrinkles. SEsc is the index representing scaliness of
skin which shows the level of dryness of the stratum
corneum i.e., state of dehydration of the skin. It is
the number of pixels where the gray level is higher
than the threshold of SEsc. SEwidentifies aging
including wrinkles and is calculated from the pro-
portion of horizontal and vertical wrinkles (19).
Figure 5 depicts an image taken after three months
of the application of formulation. In this study it was
Table 1. The values of different parameters for both base and formulation obtained by Visioscan with standard error mean (± SEM), (n = 3).
Parameters Emulsion 0 hours 1 month 2 months 3 months
Type ± SEM ± SEM ± SEM ± SEM
SEr
Base 3.07 ± 0.10 3.08 ± 0.11 3.07 ± 0.18 3.08 ± 0.10
Formulation 3.02 ± 0.10 2.98 ± 0.08 2.93 ± 0.173 2.89 ± 0.10
SEsc
Base 1.66 ± 0.02 1.66 ± 0.044 1.66 ± 0.022 1.66 ± 0.02
Formulation 1.66 ± 0.02 1.62 ± 0.03 1.58 ± 0.05 1.53 ± 0.02
SEsm
Base 100.22 ± 2.81 100.22 ± 2.82 100.03 ± 2.4 100.82 ± 2.42
Formulation 100.22 ± 2.3 97.28 ± 3.01 94.95 ± 2.6 92.03 ± 2.01
Sew
Base 60.51 ± 1.3 60.50 ± 1.2 60.52 ± 1.3 60.49 ± 1.21
Formulation 59.45 ± 1.71 59.39 ± 0.02 58.20 ± 1.01 57.16 ± 0.05
104 MUHAMMAD KHURRAM WAQAS et al.
found that the base produced statistically insignifi-
cant (p > 0.05) effects on the roughness parameter of
skin and the formulation produced significant (p ≤
0.05) effects when ANOVA two way analysis was
performed. When paired sample t-test was applied,
significant effects were observed for active formula-
tion. Gradual decrease in the values of roughness,
scaliness, smoothness and wrinkles were observed
for the formulation. This is also obvious from
Figures 6 and 7 where the visible differences can be
seen between the peaks of the 3D image. The for-
mulation showed decrease in mean values of skin
smoothness in contrast to skin roughness which
indicates that the formulation possess anti-aging
properties. Lower values of skin scaliness showed
that the skin got hydrated with the passage of time
during treatment. It was also supported by the val-
ues obtained by CorniometerÆMPA 5 for skin
hydration. The smaller SEsc value corresponds to
higher skin moisture as treatment with moisturizing
or anti-aging formulations let the values for SEsc
go down. Over exposure to the sun light results in
many of the skin changes associated with aging
characterized by fine wrinkling and skin laxity.
Collagen is responsible for imparting the skin plia-
bility and elasticity. Decreased levels of collagen
occur in both chronologic and photoaging. As col-
lagen fibers serve as the primary structural support
of the skin, it follows logically that a reduction in
skin collagen levels would be associated with the
formation of skin wrinkles (20).
Lower values for the parameter SEw indicate
that there were less wrinkles present on the skin
which indicates that the formulation reduced the fine
wrinkles and improved the appearance of skin by
increasing the elasticity of connective tissues. It was
supported by the values obtained by Elasto-
meterÆEM 25 for skin elasticity. Different studies
have depicted that increased skin collagen reduced
fine lines and depth of wrinkles by the use of topical
products containing antioxidants. Antioxidants pro-
tect human skin from free radicals and as mentioned
previously, flavonoids are capable of increasing col-
lagen and have photo protective properties against
UV radiation (21).
The improvement in skin surface parameters
can be attributed to the phenolic compounds and fla-
vanoids present in grape seeds extract which include
catechins, epicatechin, epicatechin-3-O-gallate, pro-
cyanidins dimers (B1-B5), procyanidin C1, and pro-
cyanidin B5-3í-gallate kaempferol-3-O-glucosides,
quercetin-3-O-glucosides, quercetin, myricetin.
There is much supporting evidence present that phe-
nolic compounds contribute towards antiaging
mechanism by providing an antioxidative benefit to
skin. The antioxidant activity of phenolics is mainly
due to their redox properties, which allow them to
act as reducing agents, hydrogen donors, and oxy-
gen quenchers. They represent a class of compounds
comprising of potential candidates for prevention of
the adverse effects of UV radiation on the skin.
Grape seeds extract have been shown to exert a
much stronger oxygen free radical scavenging effect
than vitamin C and E and to prevent UVB- and
UVC-induced lipid peroxidation (22).
CONCLUSION
The study depicts that grape (Vitis vinifera)
seeds extract exhibits potent antiaging effects when
applied topically if a stable cosmetic W/O emulsion
loaded with grape seeds extract can be formulated.
The formulation was evaluated to cause an increase
in stratum corneum water contents which were
found significant in statistical analysis. This
increase in stratum corneum water contents provid-
ed a glossy appearance and protected the skin from
aging. The decline in SELS parameters represents
that the formulation containing grape seeds extract
possesses anti-wrinkle effects. Moreover the botani-
cal extract in formulation displayed no dermatolog-
ical complications so this formulation is economical
and safe for in the facial wrinkles management.
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Received: 26. 12. 2016
