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Fortification of facial skin collagen efficacy by combined ascorbyl palmitate and sodium ascorbyl phosphate

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Skin care products specifically formulated for the purpose of hydrating skin by increasing collagen efficacy required to contain hydrophobic or hydrophilic antioxidants with the aim of maintaining the hydration status of the horny layer. Ascorbyl palmitate (AP) and sodium ascorbyl phosphate (SAP) are reported as lipophilic and hydrophilic antioxidants, respectively, used for skin care. Present study was aimed to assess the combined AP (in oil phase) and SAP (in aqueous phase) of a multiple emulsion (ME) for fortifying the facial skin collagen efficacy in healthy human females. Active formulation ME was applied to left and a control-placebo on the right side of face of female volunteers over 12-weeks treatment study. Instrumental measurements, Corneometer CM825, Elastometer E25 and Visioscan VC 98 with a sole UVA-light video camera were carried out for skin hydration, skin elasticity and SELS parameters (roughness (SEr), scaliness (SEsc), smoothness (SEsm) and wrinkling (SEw), respectively, at baseline visit and on each subsequent visit at specific time intervals. Combined AP and SAP of ME enhanced stratum corneum moisture content (25%), skin elasticity (10%) and SELS parameters i.e. SEr (-5%), SEsc (-12%), SEsm (+9%) and SEw (-9%) after a period of 12-weeks when equated with baseline values. In conclusion, combined treatment with lipophilic and hydrophilic combination of ascorbyl palmitate and sodium ascorbyl phosphate in a single formulation (ME) presented superior results in improving skin moisture, elasticity and SELS parameters, thus provided a way to enhance the facial skin collagen efficacy.
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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 75 No. 1 pp. 129ñ140, 2018 ISSN 0001-6837
Polish Pharmaceutical Society
The skin is fortified with a setup of lipophilic
and hydrophilic, enzymatic and non-enzymatic
antioxidant systems (1). The skin texture represents
the skin smooth surface. Many factors affect skin
texture e.g., diet, hydration, amount of collagen and
hormones. A gradual decline in skin upon chronic
sun exposure ultimately leads to skin aging (2, 3).
The skin care products have been designed to
improve the functions on the dermis to stimulate the
collagen synthesis and firmness of skin.
Antioxidants (natural or synthetic) protect human
skin against the action of free radicals from UV
radiations (4).
Vitamin C deficiency in human has been
known for centuries as scurvy. Its activity against
free radicals, role in collagen biosynthesis and its
photoprotective properties have made it potential
candidate for the treatment and prevention of skin
aging (5). Vitamin C is potent antioxidant and is
known to stimulate collagen formation (6) but has
the drawback of instability in formulations. Thatís
why deprivatives of ascorbic acid have been synthe-
sized having an action similar to ascorbic acid but
are more stable. Sodium ascorbyl phosphate and
ascorbyl esters are the stable vitamin C derivatives
(7). Ascorbyl palmitate (AP) and sodium ascorbyl
phosphate (SAP) are reported as lipophilic and
hydrophilic antioxidants, respectively, used for skin
care. Sodium ascorbyl phosphate acts as an in vivo
antioxidant. It is a stable vitamin C derivative with
hydrophilic nature that is converted into simple vita-
min C after penetration into skin, thus promotes the
development of skin (8). Ascorbyl palmitate is a
synthetic vitamin C derivative with improved chem-
FORTIFICATION OF FACIAL SKIN COLLAGEN EFFICACY BY COMBINED
ASCORBYL PALMITATE AND SODIUM ASCORBYL PHOSPHATE
HIRA KHAN1*, NAVEED AKHTAR1and ATIF ALI2
1Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, the Islamia University of
Bahawalpur, Bahawalpur 63100- Pakistan
2Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060- Pakistan
Abstract: Skin care products specifically formulated for the purpose of hydrating skin by increas-
ing collagen efficacy required to contain hydrophobic or hydrophilic antioxidants with the aim of
maintaining the hydration status of the horny layer. Ascorbyl palmitate (AP) and sodium ascorbyl
phosphate (SAP) are reported as lipophilic and hydrophilic antioxidants, respectively, used for
skin care. Present study was aimed to assess the combined AP (in oil phase) and SAP (in aqueous
phase) of a multiple emulsion (ME) for fortifying the facial skin collagen efficacy in healthy
human females. Active formulation ME was applied to left and a control-placebo on the right side
of face of female volunteers over 12-weeks treatment study. Instrumental measurements,
CorneometerÆCM825, ElastometerÆE25 and VisioscanÆVC 98 with a sole UVA-light video cam-
era were carried out for skin hydration, skin elasticity and SELS parameters (roughness (SEr),
scaliness (SEsc), smoothness (SEsm) and wrinkling (SEw), respectively, at baseline visit and on
each subsequent visit at specific time intervals. Combined AP and SAP of ME enhanced stratum
corneum moisture content (25%), skin elasticity (10%) and SELS parameters i.e. SEr (-5%), SEsc
(-12%), SEsm (+9%) and SEw (-9%) after a period of 12-weeks when equated with baseline val-
ues. In conclusion, combined treatment with lipophilic and hydrophilic combination of ascorbyl
palmitate and sodium ascorbyl phosphate in a single formulation (ME) presented superior results
in improving skin moisture, elasticity and SELS parameters, thus provided a way to enhance the
facial skin collagen efficacy.
Keywords: fortification, collagen efficacy, ascorbyl palmitate, sodium ascorbyl phosphate, com-
bined effects
129
* Corresponding author: e-mail: hirakhan464@gmail.com
130 HIRA KHAN et al.
ical stability and lipophilicity, which increased its
absorption ability through the skin (9, 10).
Multiple emulsions play an important role
when our focus is to prepare skin care products with
long-term moisture holding capacity, skin firmness
improvement and prolonged action (11). W/O/W
multiple emulsions present many interesting possi-
bilities for the controlled delivery of drug, initially
entrapped in the internal aqueous compartment and
promote the delivery of hydrophilic as well as
hydrophobic drugs (12).
Current investigation aimed to assess the com-
bined ascorbyl palmitate and sodium ascorbyl phos-
phate via a multiple emulsion (ME) for fortifying
the facial skin collagen efficacy in healthy human
females through non-invasive bioengineering tech-
niques.
MATERIALS AND METHODS
Test products
Test products (control and ME) were multiple
emulsions of W/O/W type. ME was loaded with
ascorbyl palmitate and sodium ascorbyl phosphate
as functional ingredients while control was without
active compounds. Compositions of control and
ME are shown in Table 1. Two-step emulsification
procedure was implemented for the preparation of
W/O/W multiple emulsions (control and ME) (13).
Primary W/O emulsion was prepared by heating
cetyl dimethicone copolyol plus liquid paraffin
(AP also for ME), and water plus magnesium sul-
fate to 75OC in a digital water bath (Heidolph,
Germany) and then mixing by IKA Mixing Over-
head Stirrer, Eurostar (IKA, Werke, Germany) at
2000 rpm. In secondary emulsification step, W/O
emulsion was dispersed in mixture of water plus
polysorbate-80 (SAP also for ME) at 700 rpm for
40 min to attain W/O/W multiple emulsion.
Formulated emulsions were confirmed by pho-
tomicrographs (Figs. 1a, 1b).
Each volunteer was provided with two vessels
with 50 g emulsion. The vessels with emulsions
were marked as ìright and leftî which were speci-
fied for right and left cheeks, respectively. The right
cheek was specified for control while the left cheek
was specified for active formulation (ME).
Participants were instructed to use about 1 g of con-
trol and active formulation (ME) on respective half
of the cheeks at bed time and in the morning over a
12 weeks trial period.
Measurement site
A measurement area was set on the cheek (the
sample area on the cheek; 2 x 2 cm) in such a way
that cross of the parallel line of the bottom of the
Figure 1. Photomicrograph of (a) control and (b) ME (active multiple emulsion) immediately after preparation
a b
Fortification of facial skin collagen efficacy by combined ascorbyl... 131
Figure 2. Percentage change in skin moisture after the application of active formulation ME and control
Figure 3. Percentage change in skin elasticity contents for active formulation ME and control
Figure 4. Percentage change in SELS parameters; roughness (SEr), scaliness (SEsc), smoothness (SEsm) and wrinkling (SEw) after appli-
cation of active formulation ME and control. 1, 3 and 5 represents 3 month results for control while 2, 4 and 6 represents 3 month results
for ME
132 HIRA KHAN et al.
nose and a vertical line to the inner corner of the eye
was taken a reference point.
Subjects
A sample of 11 Asian female volunteers (stu-
dents) with an age between 22-25 years (mean age:
23.5 years), non smokers, no history of dermatolog-
ical diseases or allergy to substances in test product
were recruited in this study. Principle of
Declaration of Helsinki was charted in this study.
The inclusion criteria was: no history of hypersen-
sitivity, every individual should sign the volunteer
protocol and have to come to the laboratory for
measurements on specific time intervals. The crite-
ria for exclusion were: presence of any dermatitis,
smokers and previous treatment with sunscreens,
moisturizers or anti-ageing cosmetics. Participants
were also instructed not to use any skin care prod-
ucts on the test sites 2 weeks before study and
throughout the study period of 12 weeks.
Additionally, exposure to solar radiations and use
of occlusive clothes on the test area were prohibit-
ed. Participants were restricted with use of oral nat-
ural or synthetic vitamin C supplements which may
affect the results. All the study participants were
well informed about the use of study products and
essential details of study. All volunteers completed
the study successfully.
Ethical considerations
A written informed consent was taken from all
the participants of the study. Participants were
informed about possible adverse reactions, protocols
and objectives of this study and they have the rights
to quit study without informing about such reasons.
The approval of this study was taken from the
Advanced Studies and Research Board (No. 975/AS
& RB), the Islamia University of Bahawalpur and
registered by Institutional Ethical Committee and
Board of Faculty, Faculty of Pharmacy and
Alternative Medicine (No. 2336/Dean), the Islamia
University of Bahawalpur, Bahawalpur- Pakistan.
Study protocol
The study was split face placebo controlled.
Prior to the study, a cosmetic expert examined each
volunteer for any type of skin reaction/sensitivity and
no adverse effects were ensured. An expert investiga-
tor ensured the proper conductance of CorneometerÆ
CM825, ElastometerÆE25 and Visioscan measure-
ments (Courage and Khazaka Electronic GmbH),
considering the experimentation protocols for these
skin micro-topographic techniques.
Skin moisture content determination with
CorneometerÆCM825
The measurement of the skin moisture is based
on the capacitance method which is internationally
recognized method of CorneometerÆ(14). The
device represents the values in arbitrary units by
measuring the water contents of the superficial epi-
dermal layers down to the depth of about 0.1 mm.
Moisture contents of human skin (stratum corneum
water contents) were measured with CorneometerÆ
before the application of control and active formula-
tion (ME) and then on 2nd, 4th, 6th, 8th, 10th and 12th
week of investigation period.
Skin elasticity determination with ElastometerÆ
E25
Skin ElastometerÆ allows easy and quick meas-
urement of the elastic properties of the skin (biolog-
Table 1. Composition of control and active formulation (ME).
Control (% wt.) ME (% wt.)
Primary emulsion (W/O) Primary emulsion (W/O)
Cetyl dimethicone copolyol 2.40 Cetyl dimethicone copolyol 2.40
Liquid paraffin 13.60 Liquid paraffin 13.60
Magnesium sulfate 0.56 Magnesium sulfate 0.56
- - Ascorbyl palmitate 0.5
Deionized water 63.44 Deionized water 62.69
Multiple emulsion (W/O/W) Multiple emulsion (W/O/W)
Polysorbate-80 0.8 Polysorbate-80 0.8
- - Sodium ascorbyl phosphate 0.5
Water (Q.S) 100 Water (Q.S) 100
ME stands for active multiple emulsion formulation. W/O/W = Water-in-oil-in-water, Q.S = Quantity sufficient.
Fortification of facial skin collagen efficacy by combined ascorbyl... 133
ical skin age) measured by suction. The elasticity is
expressed in % in the display. Elastometer was used
to measure the cutaneous elasticity before applica-
tion of control (C) and active formulation (ME) and
then on 2nd, 4th, 6th, 8th, 10th and 12th week of investi-
gation period.
Surface evaluation of living skin (SELS)
VisioscanÆVC 98 with a special UV light
video camera was used to measure four (SELS)
parameters i.e., roughness (SEr), scaliness (SEsc),
smoothness (SEsm) and wrinkling (SEw). SEr is the
roughness parameter which calculates the propor-
tion of dark pixels. SEsm is the index of smoothness
and is calculated from the mean width and depth of
wrinkles. SEsc is the index of scaliness of skin
which shows the level of dryness of the skin. SEw
identifies aging including wrinkles and is calculated
from the proportion of horizontal and vertical wrin-
kles (15). In our study, evaluation was done by cal-
Table 2. Values of moisture after application of control.
Volunteer Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 Week 12
1 81 66 69 59.6 83 95 103
293837966 587878
3 47.5 47.1 47.3 49 55 56.9 61
4 36 36.6 41 59.1 42 38 38
5 53.2 54.5 51 49.2 50.8 49.7 51.3
6 48 51.2 53 53 57 56 53.6
7 43 55 58.2 58 46 46 48
8 48 39.4 40 50.4 50 59.7 58.3
922362931 313432
10 84 62 54.2 62 55 57 55
11 45.2 48.9 58 69.5 68.8 68 71
Sum 600.9 579.7 579.7 606.8 596.6 638.3 649.2
Mean 54.62727 52.7 52.7 55.16364 54.23636 58.02727 59.01818
STDev 21.92488 13.97512 13.8202 10.47724 13.60024 17.5128 19.59831
SEM 6.62383 4.222091 4.175288 3.165329 4.108834 5.290876 5.920939
Table 3. Values of moisture content after application of active formulation (ME).
Volunteer Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 Week 12
1 50 69 72.4 77 71 74 74
293837966 587878
3 38.7 41 45 42 52.2 57.1 58
4 50.2 51.3 54 57 55.2 58.3 58.9
5 36.7 38.5 49 52.1 41.2 52 54
6 59 57.3 56 49 53.5 51.9 50.9
7 47.3 52.7 55.3 61.3 60.5 61.9 62
8 41.5 49 45 42.3 51.8 54.7 55
938373947 514944
10 57.2 56 61 58 60 61.5 63
11 46.7 52 55.6 55 53 49 51
Sum 516.3 556.8 591.1 610.6 615.2 635.4 636.8
Mean 46.93636 50.61818 53.73636 55.50909 55.92727 57.76364 57.89091
STDev 7.547486 9.226681 9.110903 10.92954 8.057554 7.706137 8.258869
SEM 6.62383 4.222091 4.175288 3.165329 4.108834 5.290876 5.920939
134 HIRA KHAN et al.
culating the percentage changes in different SELS
parameters (SEr, SEsc, SEsm and SEw) for a study
period of 3 months. Measurements were taken on
right and left cheeks of female adults at baseline
visit and on 1st, 2nd and 3rd month. Before any meas-
urements, all volunteers had to rest in Cosmetic Lab,
under constant environmental conditions of 22 ±
2OC and 50 ± 5% relative humidity, for at least 30
minutes Basic principle of SELS is based on evalu-
ation of an image of living skin by obtaining the pic-
ture which is finally electronically processed for
quantitative results. The measuring probe comprises
of two counter rotating halogen lamps which uni-
formly illuminate specific measuring field on the
skin. A CCD camera is fitted in the measuring head
which records the picture of skin and camera is con-
nected to the computer directly via EPP-port. Skin
smoothness, skin roughness, scaliness and wrinkles
are evaluated by the grey level distribution of the
image as an index.
Table 4. Values of elasticity (%) after application of control.
Volunteer Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 Week 12
173837575 818172
262615961 646365
351526057 545452
464626265 646265
585848686 858485
681777981 858485
763616164 666664
865636761 535859
949555554 545556
10 58 58 58 59 58 58 59
11 56 55 48 55 56 56 55
Sum 707 711 710 718 720 721 717
Mean 64.27273 64.63636 64.54545 65.27273 65.45455 65.54545 65.18182
STDev 11.44632 11.36022 11.23711 10.72465 12.54084 11.78443 11.27669
SEM 3.458102 3.43209 3.394898 3.240076 3.788774 3.56025 3.406854
Table 5. Values of elasticity (%) after application of active formulation (ME).
Volunteer Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 Week 12
172686568 708895
264677575 887475
345474248 485251
463658592 929695
564657272 606060
683909092 979595
772685872 828888
863657174 655252
963666160 616263
10 58 67 59 59 60 61 62
11 80 86 96 59 58 58 59
Sum 727 754 774 771 781 786 795
Mean 66.09091 68.54545 70.36364 70.09091 71 71.45455 72.27273
STDev 10.47334 11.30808 15.76244 13.5901 16.13691 17.25899 17.84988
SEM 3.164152 3.416339 4.762067 4.105771 4.875201 5.214196 5.392713
Fortification of facial skin collagen efficacy by combined ascorbyl... 135
Statistical analysis
All measured data for skin moisture content,
elasticity and SELS parameters were analyzed using
SPSS (version 20) software. ANOVA (analysis of
variance) and paired t-test was used for the compar-
ison of effects at baseline and after subsequent inter-
vals of treatment.
RESULTS
Skin moisture content
Active formulation (ME) studied enhanced
stratum corneum moisture content after 12-week
period of daily applications when compared with the
baseline values (Tables 2, 3). The percentage of
Table 6. Skin roughness (SEr) after the application of control.
Volunteer Zero 30 Days 60 Days 90 Days
1 0.64 0.52 0.47 0.48
2 0.2 0.13 0.11 0.16
3 0.33 0.02 0.06 0.04
4 0.1 0.04 0.11 0.05
5 0.35 0.08 0.04 0.04
6 0.11 0.33 0.36 0.39
7 0.15 0.29 0.23 0.25
8 0.71 0.8 0.7 0.5
9 0.86 0.2 0.08 0.08
10 0.06 0.09 0.08 0.09
11 0.35 0.35 0.32 0.37
Sum 3.86 2.85 2.56 795
Mean 0.350909 0.259091 0.232727 0.222727
STDev 0.272193 0.236831 0.21029 0.182214
SEM 0.082234 0.07155 0.063532 0.055049
Table 7. Skin roughness (SEr) after the application of active formulation (ME).
Volunteer Zero 30 Days 60 Days 90 Days
1 0.21 0.13 0.09 0.04
2 0.32 0.32 0.35 0.35
3 0.17 0.14 0.13 0.18
4 0.1 0.17 0.18 0.17
5 0.46 0.31 0.37 0.35
6 0.8 0.8 0.87 0.89
7 0.45 0.45 0.45 0.46
8 0.27 0.26 0.24 0.25
9 0.56 0.54 0.54 0.47
10 0.02 0.02 0.01 0.01
11 0.32 0.28 0.23 0.25
Sum 3.68 3.42 3.46 3.42
Mean 0.334545 0.310909 0.314545 0.310909
STDev 0.223086 0.219338 0.242914 0.243781
SEM 0.067398 0.066265 0.073388 0.07365
136 HIRA KHAN et al.
changes in skin moisture contents after the applica-
tion of control and ME have been shown in Figure
2. All values were measured in triplicates (n = 3).
ME produced about 25% increase in skin moisture
contents while its respective control produced 14%
increase in skin moisture contents at the end of study
period of 12 weeks. After applying the statistical
analysis (two-way ANOVA test) it was found that
ME as well as control showed significant increase in
moisture content with respect to time. With the help
of paired sample t-test analysis, significant differ-
ences in the skin moisture values were observed by
ME and control at the end of study.
Skin elasticity
Average percentage changes in skin elasticity
values after the application of control and ME have
been shown in Fig. 3. Comparison with baseline val-
Table 8. Skin scaliness (SEsc) after the application of control.
Volunteer Zero 30 Days 60 Days 90 Days
1 0.09 0.04 0.04 0.01
2 0.04 0.05 0.08 0.08
3 0.12 0.09 0.07 0.16
4 0.24 0.09 0.05 0.03
5 0.19 0.17 0.11 0.14
6 0.18 0.17 0.19 0.21
7 0.24 0.24 0.23 0.23
8 0.08 0.08 0.01 0.02
9 0.03 0.03 0.02 0.03
10 0.02 0.04 0.06 0.03
11 0.06 0.06 0.05 0.08
Sum 1.29 1.06 0.91 1.02
Mean 0.117273 0.096364 0.082727 0.092727
STDev 0.082352 0.067863 0.069151 0.079761
SEM 0.02488 0.020503 0.020891 0.024097
Table 9. Skin scalliness (SEsc) after the application of active formulation (ME).
Volunteer Zero 30 Days 60 Days 90 Days
1 0.09 0.08 0.09 0
2 0.04 0.04 0.03 0.1
3 0.15 0.11 0.1 0.12
4 0.06 0.07 0.04 0.04
5 0.07 0.08 0.08 0.09
6 0.19 0.12 0.17 0.18
7 0.93 0.8 0.7 0.2
8 0.17 0.01 0.02 0.01
9 0.43 0.36 0.36 0.17
10 0.1 0.17 0.18 0.18
11 0.15 0.14 0.13 0.12
Sum 2.38 1.98 1.9 1.21
Mean 0.216364 0.18 0.172727 0.11
STDev 0.259433 0.2253 0.199253 0.070143
SEM 0.078379 0.068066 0.060197 0.021191
Fortification of facial skin collagen efficacy by combined ascorbyl... 137
ues (Tables 4, 5) indicates that elasticity contents
were found an increase to greater extent after the
application of ME. ME caused about 10% increase
in elasticity of skin while respective control pro-
duced slight effects (2% increase) on cutaneous
elasticity. When two-way ANOVA test was applied,
control showed insignificant changes while ME pro-
duced significant changes in elasticity values with
respect to time after 12 weeks of study. With the
help of paired sample t-test, statistically significant
differences in elasticity results of ME and control
were observed.
Surface evaluation of living skin
The percentage of changes (average) in skin
roughness (SEr), scaliness (SEsc), smoothness
(SEsm) and wrinkles (SEw) were measured for sur-
face evaluation of living skin (SELS) after applica-
Table 10. Skin smoothness (SEsm) after the application of control.
Volunteer Zero 30 Days 60 Days 90 Days
1 0.09 0.08 0.09 0
2 0.04 0.04 0.03 0.1
3 0.15 0.11 0.1 0.12
4 0.06 0.07 0.04 0.04
5 0.07 0.08 0.08 0.09
6 0.19 0.12 0.17 0.18
7 0.93 0.8 0.7 0.2
8 0.17 0.01 0.02 0.01
9 0.43 0.36 0.36 0.17
10 0.1 0.17 0.18 0.18
11 0.15 0.14 0.13 0.12
Sum 2.38 1.98 1.9 1.21
Mean 18.49455 18.33182 18.18091 18.94091
STDev 2.528214 2.792847 2.224754 4.083338
SEM 0.763811 0.843761 0.672131 1.233637
Table 11. Skin smoothness (SEsm) after the application of active formulation (ME).
Volunteer Zero 30 Days 60 Days 90 Days
1 18.34 19.71 14.68 14.42
2 17.28 16.86 15.71 20.96
3 21.62 18.06 17.81 16.8
4 17.65 22.59 24.55 24.83
5 19.14 21.44 16.93 17.4
6 18.98 19.78 19.55 19.94
7 16.54 16.75 20.9 18.67
8 23.64 25.1 27.9 25.87
9 25.1 32.87 34.56 32.91
10 19.01 18.51 20.77 24.65
11 17.65 18.09 15.23 17.32
Sum 214.95 229.76 228.59 233.77
Mean 19.54091 20.88727 20.78091 21.25182
STDev 2.747659 4.711212 6.112969 5.355854
SEM 0.830108 1.423327 1.846818 1.618083
138 HIRA KHAN et al.
tion of control and ME for 12 weeks at specific time
intervals i.e. at zero hour, 1st month, 2nd month and 3rd
month of study period. Results have been shown in
Figure 4. All values were measured in triplicates (n
= 3). Obtained results indicated that smoothness val-
ues (Tables 10, 11) were increased while roughness,
scaliness (Tables 6-9) and wrinkling values (Tables
12, 13) were decreased after three months as com-
pared to baseline values. After calculating percent-
age changes in skin roughness at specific time inter-
vals in female volunteers; it was observed that the
control produced too little effects on skin roughness
index (SEr) which was seen after completion of 2nd
and 3rd month; whereas ME showed a pronounced
decrease in the skin roughness values. A pro-
nounced decrease in skin scaliness was also
observed after topical application of ME. Although
control formulation also decreased the skin scali-
Table 12. Skin wrinkling (SEw) after the application of control.
Volunteer Zero 30 Days 60 Days 90 Days
1 45.5 45 52.19 47.66
2 38.14 39.34 38.54 38.42
3 33.97 35.97 31.23 32.44
4 39.93 35.78 52.41 52.41
5 33.86 36.15 33.34 34.76
6 40.75 47.21 37.09 37.69
7 45 42.12 42.01 42.13
8 52.32 47.21 52.28 48.7
9 43.27 42.61 37.82 38.76
10 41.47 42.31 39.43 39.76
11 40.23 34.29 34.47 31.58
Sum 454.44 447.99 450.81 444.31
Mean 41.31273 40.72636 40.98273 40.39182
STDev 5.282715 4.708701 7.838022 6.753941
SEM 1.595987 1.422568 2.367982 2.040465
Table 13. Skin wrinkling (SEw) after the application of active formulation (ME).
Volunteer Zero 30 Days 60 Days 90 Days
1 33.97 37.55 40.64 42.65
2 40.29 38.14 37.78 39.87
3 36.33 37.94 38.5 36.78
4 39.93 32.14 33.41 34.43
5 54.54 39.41 33.07 49.34
6 36.15 47.21 36.65 37.21
7 47.21 46.69 45.95 44.54
8 40.91 40.89 39.79 32.41
9 50.6 50.6 53.78 35.67
10 39.93 41.23 38.17 38.87
11 37.82 34.29 35.36 35.98
Sum 457.68 446.09 433.1 427.75
Mean 41.60727 40.55364 39.37273 38.88636
STDev 6.462707 5.625576 5.967821 4.941824
SEM 1.952479 1.69957 1.802967 1.492998
Fortification of facial skin collagen efficacy by combined ascorbyl... 139
ness, but this effect was very low. After applying
two-way analysis of variance test on skin roughness,
scaling, smoothness and wrinkling values, it was
observed that control produced statistically insignif-
icant (p > 0.05) effects while ME showed significant
effects at various time intervals of three-month
study period. Paired sample t-test revealed that ME
showed significant differences during the whole
evaluation period of three months when compared
with control.
DISCUSSION
Skin moisture content
The improvement of skin hydration promotes
collagen formation (16). A dramatic increase in skin
moisture contents after application of ME contain-
ing ascorbic acid derivatives (ascorbyl palmitate and
sodium ascorbyl phosphate) was due to the reason
that ascorbyl palmitate has skin moisturizing effects
(10). In the molecule of ascorbyl palmitate with
favorable effects as an excellent skin antioxidant,
the fatty acid ester moiety is sited in 6-position and
the inorganic ester group is sited in 2-position pro-
motes its penetration into skin and promise various
advantages for skin applications including moistur-
izing potential which was found to be higher as
compared to the other hydrophilic compounds (17).
Moreover, sodium ascorbyl phosphate acts as an in
vivo antioxidant and improves hydration of skin by
promoting collagen formation. It is a stable vitamin
C derivate with hydrophilic nature that protects the
skin, promotes its development and improves its
appearance (18). Both compounds synergistically
improved the skin moisture contents.
Skin elasticity
Collagen is present in fibroblast of human der-
mis and essential for healthy firm skin (19). External
factors such as exposure to sunlight, especially UV
radiations decrease the quality and quantity of colla-
gen leading to aging skin. In the past era, topical
applications of soluble animal collagen was used to
stimulate the formation of collagen in the skin.
However, these tests were unsuccessful because col-
lagen cannot penetrate the epidermis (20, 21). We
observed that after topical application of ME (con-
taining ascorbic acid derivatives in 1% concentra-
tion), improvement of cutaneous elasticity and
hydration properties was due to high penetration
ability and synergistic antioxidant effects of ascor-
byl palmitate and sodium ascorbyl phosphate.
Ascorbyl palmitate has greater chemical stability,
lipophilicity and skin absorption ability (9). Sodium
ascorbyl phosphate is hydrolyzed by the skin cells,
which cause the in situ liberation of the ascorbic
acid (22). Ascorbic acid and its derivatives such as
ascorbyl palmitate work as potent antioxidants to
protect the skin from free radical damage and also
promote collagen production (23). Sodium ascorbyl
phosphate also has photo-protective effects and has
ability to increase collagen production in human
fibroblasts (24). As both compounds synergistically
increased the collagen production thus both can be
used safely in combination in new anti-aging prod-
ucts.
Surface evaluation of living skin
Structural changes in the skin surface around
the prominent pores, are induced not only by
changes in epidermal cells, but also by strong
anisotropy of the dermal fiber structure due to colla-
gen (25). Structural changes are evaluated by
(SELS) parameters i.e., roughness (SEr), scaliness
(SEsc), smoothness (SEsm) and wrinkling (SEw).
SEr values describe the skinís roughness.
Higher the SEr content, less will be the smoothness
of skin and vice versa. SEsc (surface evaluation of
skinís scaliness) predicts skin scaliness index.
Scaliness index is inversely related to stratum
corneum hydration level. The lower is the value of
SEsc, the higher will be skin hydration level and
vice versa. SEsm represents the surface evaluation
of skin smoothness. SEw designate the width and
number of wrinkles and relates to skin fitness. An
increase in SEw index will be the more skin wrin-
kles (15).
Thousands of natural and synthetic compounds
have been assessed for their efficiency against free
radicals. Scientists are trying to develop novel syn-
thetic antioxidants directed at retarding the effects of
free-radical-induced damage (22, 26). We have
explored the combined effects of ascorbyl palmitate
and sodium ascorbyl phosphate on SELS parameters
of skin (smoothness, roughness, scaliness and wrin-
kling) and found some interesting results. In combi-
nation, these compounds showed synergistic/com-
bined antioxidant effects as one compound is
hydrophobic (ascorbyl palmitate) and the other
(sodium ascorbyl phosphate) is hydrophilic which
might enhance their penetration into the skin. All
four parameters of SELS were improved after the
application of ME in comparison to control. ME
showed decrease in mean values of skin roughness
(SEr), scaling (SEsc), and wrinkling (SEw) which
indicated that the active formulation has anti-aging
properties. Moreover, pronounced effects were on
skin smoothness. A decline in the values of the skin
140 HIRA KHAN et al.
wrinkles (SEw) indicated the reduction in the fine
wrinkles and improvement in the texture of living
skin. Improvement of SELS parameters after topical
application of ME was due to antioxidant properties
of ascorbyl palmitate and sodium ascorbyl phos-
phate. With its antioxidant properties, ascorbyl
palmitate helped to maintain normal connective tis-
sue, promoted the synthesis of collagen and main-
tained the healthy skin and blood vessels (27).
CONCLUSION
From the study, it was observed that combined
use of ascorbyl palmitate and sodium ascorbyl phos-
phate via a multiple emulsion (ME) enhanced the
skin facial collagen by improving skin moisture,
skin elasticity and SELS parameters when compared
with baseline values. Thus, combined application of
two different lipophilic and hydrophilic nature of
antioxidants, ascorbyl palmitate and sodium ascor-
byl phosphate loaded in multiple emulsion (ME)
have potential influence on facial skin parameters,
thus provide a way to fortify collagen efficacy of
facial skin.
Acknowledgment
Author would like to thank Prof. Dr. Mahmood
Ahmad, Dean Faculty of Pharmacy and Alternative
Medicine, the Islamia University of Bahawalpur-
Pakistan for providing cosmetic laboratory services.
Conflict of interest
The authors declare that there is no conflict of
interest regarding the publication of this article.
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  • A Godic
  • Poljsak
  • M Adamic
  • R Dahmane
Godic A., Poljsak., Adamic M., Dahmane R.: Oxid. Med. Cell. Longev. 2014, 6 (2014).
  • A Sparavigna
  • R Marazzato
Sparavigna A., Marazzato R.: Skin. Res. Technol. 16, 161 (2010).
  • S O Yoon
  • C H Yun
  • A S Chung
Yoon S.O., Yun C.H., Chung A.S.: Mech. Ageing Dev. 123, 1597 (2002).
  • D Yoshihara
  • N Fujiwara
  • K Suzuki
Yoshihara D., Fujiwara N., Suzuki K.: Maturitas 67, 103 (2010).
  • A I Segall
  • M A Moyano
Segall A.I., Moyano M.A.: Int. J. Cosmet. Sci. 30, 453 (2008).