Vol . 70 N o .

6 November/December 2019

JOURNAL OF COSMETIC SCIENCE

The Official Journal of the Society of Cosmetic Chemists

Contents

Page
ORIGINAL ARTICLES Assessment of the Effect of Extract Formulation of Date Palm Kernel on
Facial Skin Wrinkles: Biophysical Measurements and Digital Profilometry
Aziz Alsohaimi and Abdel-Motaal Fouda . . . . . . . . . . . . . . . . . . . . . . . 277

Electrophoretic Mobility of Some Tattoo Dyes as an Approach to Remove

Their Subcutaneous Traces
Igor Winkler, Ulyana Andrushko, and Alla Velyka . . . . . . . . . . . . . . . . 291

Effect of Palmitic Acid Conjugation on Physicochemical Properties of Peptide

KTTKS: A Preformulation Study
Seyedeh Maryam Mortazavi, Farzad Kobarfard, Howard I. Maibach,
and Hamid Reza Moghimi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

Utilization of Coffee Silverskin By-Product from Coffee Roasting Industry

through Extraction Process for the Development of Antioxidant Skin Gel
Samuel P. Kusumocahyo, Patrick Tangguh, Christina D. Annelies,
and Hery Sutanto. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

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 J. Cosmet. Sci., 70, 277–289 (November/December 2019)

Assessment of the Effect of Extract Formulation of Date Palm

Kernel on Facial Skin Wrinkles: Biophysical Measurements
and Digital Profilometry

AZIZ ALSOHAIMI and ABDEL-MOTAAL FOUDA, Dermatology,

Al Baha University Faculty of Medicine, Al Baha 65799, Saudi Arabia
(A.A.), Clinical Pharmacology and Therapeutics, Al Baha University
Faculty of Medicine, Al Baha 65799, Saudi Arabia and Faculty of
Medicine, Mansoura University, Mansoura 35516, Egypt (A.-M.F.)

Accepted for publication September 6, 2019.

Synopsis
Previous studies have shown that the date palm kernel contains plenty of phytochemicals of potential
rejuvenation benefits to skin. The aim of this study was to investigate a cream form containing date palm
kernel extract (DPKE) on facial wrinkle reduction and objective skin parameters in healthy subjects. A cream form
containing 5% DPKE was prepared and applied twice daily for 8 weeks on the facial skin of 43 volunteers.
Biophysical measurements including skin hydration, elasticity, and pigmentation as well as optical scanning
of skin surface were carried out after 4 and 8 weeks. Significant improvement in facial skin hydration,
elasticity, and melanin concentration together with reduction in the wrinkle size and depth were observed at
the two time points of measurements. In addition, DPKE cream was extremely well tolerated by the facial
skin of study participants. The work herein demonstrates and validates the use of cream form containing 5%
DPKE over placebo against fine lines and wrinkles, skin pigmentations, skin hydration, and elasticity. This
effect may be attributed to synergism of major phytochemicals and phytosterols present in DPKE.

INTRODUCTION
Skin aging involves several alterations of skin properties such as thinning, reduced elas-
ticity, increased pigmentation, and appearance of wrinkles. These changes are the result
of complex internal and external factors. Over the last decade, there has been an interest
to use herbal extracts or phytochemicals in cosmetic products to fight the aging process.
Phoenix dactylifera, commonly known as date palm, is widely cultivated across North Af-
rica and the Middle East for its edible sweet fruit. The date kernel represents 10–15% of
the total fruit weight, and studies have indicated that extracts of the date palm kernel
contains plenty of biologically active phytochemicals such as carotenoids, polyphenols
(e.g., phenolic acids, lignans, flavonoids, and isoflavones), phytosterols, and tannins (1).

Address all correspondence to Abdel-Motaal Fouda at [email protected].

277
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Many of these phytochemicals are considered potential contenders to revive tired aging
skin and to have wrinkle-combating properties if used topically for suitable duration.
However, a search in the scientific literature on the Internet yields scanty records on the
anti–skin aging properties of date palm kernel extracts (DPKE) in human subjects (2,3).
Accordingly, additional studies are necessary to boost the available reports and to estab-
lish a considerable body of evidence of anti–skin aging function of DPKE.
Assessment of the efficacy of cosmetic preparations on biophysical characteristics of human
skin such as texture, roughness, elasticity, thickness, and wrinkle depth can be achieved
by variety of techniques; the ideal method should be painless, noninvasive, and provide
the specialist with a comprehensive view of skin topography and subsurface structure.
Electronic probes with small electronic sensors are now common tools to objectively mea-
sure biophysical parameters of skin such as elasticity, hydration, and pigmentation. They
can provide clinicians with precise measurements of these parameters in a fast and eco-
nomic way (4,5). The advancement in digital technology in recent years enabled noncon-
tact measurements of skin relief via optical scanning and digital profilometric analysis
(6). The measuring principle uses scanning the surface with light of different wavelengths
and at different angles to collect multispectral optical impressions of the skin surface and
using these shape illumination 2D images to reconstruct a stereo 3D image of the skin
that can be subjected to photometric stereo analysis by specialized software (7). In this
study, we used the Antera 3D® camera (Miravex Limited, Dublin, Ireland) as a means for
objective measurement of skin surface parameters (8). The purpose of this piece of re-
search, however, was to investigate the antiaging properties of DPKE, formulated in
cream form, on the facial skin of 43 healthy volunteers. Assessment of efficacy was based
on measurement of skin biophysical parameters and skin profilometric analysis by an
Antera 3D® multispectral analyzer.

SUBJECTS AND METHODS

SUBJECTS

Forty-three healthy volunteers were enrolled in this study (11 male participants and 32
female participants, age range 39–67 years). Inclusion criteria included the absence of
connective tissue or cardiovascular diseases, nonsmokers, and absence of active skin le-
sions in the region of interest. Participants were instructed not to apply any cosmetic
product or medicinal formulation on the skin area of investigation 1 week before and
throughout the study. After fulfilling the inclusion criteria, each participant was asked to
give informed consent by a research coordinator. The study was conducted in accordance
with Al Baha University Faculty of Medicine guidelines and the Declaration of Helsinki.

PREPARATION OF DPKE CREAM

Four hundred grams of date palm kernels (Sukkary variety) were defatted by n-hexane
before being ground into a fine powder and extracted three times with 800 ml methanol
at 4°C for 24 h. After filtration and centrifugation, the resultant supernatant was concen-
trated under low pressure at 4°C for 3 h to yield the lyophilized material. A cream formula

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 DATE PALM KERNEL EXTRACT ON FACIAL SKIN WRINKLES 279

containing 5% DPKE was prepared by the method described by Meer et al. (3). In brief,
the cream was prepared as an oil-in-water disperse system by heating separately the aque-
ous phase and the oil phase containing ABIL® EM90 as a nonionic emulsifier (Cetyl PEG/
PPG-10/1 Dimethicone, Evonic Industries, Essen, Germany). The methanolic extract of
date palm kernels was added in the aqueous phase, and the aqueous phase was added drop
by drop to the oil phase at 75°C ± 1.0°C with continued stirring at 2,000 rpm. The
emulsion was cooled at room temperature, and the speed of mixer was reduced gradually
until the emulsion formed semisolid cream. The same method was used to formulate the
base (placebo) cream without adding the DPKE. The composition of the two formula-
tions is presented in Table I.
The stability tests and the physicochemical characteristics of the test and placebo creams were
performed at different time points during 8 weeks at 5 ± 0.1°C, 20 ± 0.1°C, 40 ± 0.1°C,
40 ± 0.1°C with 75% relative humidity, and 50 ± 0.1°C (9).

PATCH TEST

Before beginning the study, a patch test was performed on the skin of 12 volunteers
(mean age 46.4 years) to investigate any skin reaction. A 5-cm area was marked on both
forearms of each subject, and small amounts of both preparations were applied separately
on each forearm; then, the region was covered with surgical dressing. Positive skin reac-
tions were carefully evaluated after 1, 48, and 72–96 h. The reaction was assessed accord-
ing to the International Contact Dermatitis Research Group standard (10).

TREATMENT PROTOCOL

The region of interest was the cheek and the temple areas. Both the cream form contain-
ing DPKE and the placebo cream were coded and assigned to the right or left side of each
volunteer’s face according to a computer-generated randomization list. Participants were
asked to apply DPKE and placebo formulations separately on each side of the face twice
a day for 8 weeks. Follow-up visits took place every 1 week throughout the study.

CLINICAL ASSESSMENT

Clinical assessment of facial skin was conducted as previously described by using three-
point rating scales at baseline and at the end of the study (11). Skin roughness score: 1 =
mild, 2 = moderate, and 3 = severe; skin texture homogeneity score: 1 = slightly inhomo-
geneous, 2 = uneven, and 3 = very uneven; skin melanin pigmentation score: 1 = mild,

Table I
Cream Compositions

Phase Placebo cream Cream with DPKE

®
Oil phase Liquid paraffin (17%) ABIL EM90 (2.5%) Liquid paraffin (17%) ABIL® EM90 (2.5%)
Aqueous phase Distilled water (q.s 100%) DPKE (5.0%) Distilled water (q.s 100%)

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2 = moderate, and 3 = severe; skin redness score: 1 = mild, 2 = moderate, and 3 = severe.
Assessment was performed to study subjects by two independent dermatologists, and repeat
assessments were performed to judge the inter- and intrarater reliability, respectively.

BIOPHYSICAL MEASUREMENTS

Skin hydration, barrier function, elasticity, pigmentation, and erythema were measured
by a multiprobe adapter system (MPA system, Courage and Khazaka electronic GmbH,
Koln, Germany). Skin surface hydration was assessed by a Corneometer probe (CM825)
(12). The transepidermal water loss (TEWL) was assessed by a Tewameter probe (TM
300), which measures the density gradient of water evaporation from the skin. A micro-
processor analyses the values and expresses the evaporation rate in g/m2/h (13). A Cutom-
eter probe (MPA 850) measures skin elasticity based on the suction method. The settings
of measurements were negative pressure 500 Mbar and suction for 3 s followed by 5 s of
release. Both skin firmness and elasticity were measured (14). Skin pigmentation and
erythema were determined with a Mexameter probe (MX18). Melanin measurement was
calculated from the intensity of the absorbed and reflected light at 660 and 880 nm, respec-
tively, whereas erythema was measured at 568 and 660 nm (15). All probes were calibrated
daily using standard references, and measurements were taken at baseline, after 4 weeks,
and after 8 weeks. Values are displayed as arbitrary units (AU), and each value represents
the mean of three measurements obtained from a participant’s same area of skin.

SKIN SURFACE DIGITAL PROFILOMETRY

Skin surface profilometry was performed by an Antera 3D® multispectral analyzer

(Miravex Limited) at baseline and after 4 and 8 weeks of treatment. An Antera 3D® cam-
era uses multidirectional illumination light to acquire multiple images and reconstructs
a three dimensional image of the facial skin by the aid of computer software (8). After a
period of 20 min acclimatization in controlled ambient conditions (24°C ± 1°C with 50 ±
10% relative humidity), the camera was placed directly on the external periorbital region
over the crow’s feet lines of all subjects and optical scanning images were acquired. After
the acquisition of images, several parameters related to skin topography were measured,
including wrinkle depth, indentation index of fine lines (marginal size less than 1.5 mm),
folds (marginal size less than 2.5 mm), and wrinkles (marginal size less than 5.0 mm). In
addition, texture roughness, skin pigmentation (melanin), and redness (hemoglobin)
were also assessed. The identical area was digitally identified at each session, and values
are presented as the result of triplicate measurements from the same area.

STATISTICAL ANALYSIS

The statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL)
Data were represented as the mean ± SD. Multiple comparisons between means were
performed as appropriate by using the paired t-test or the one-way analysis of variance
(ANOVA) followed by Tukey post hoc test. In all the tests, a p-value of less than 0.05 was
considered significant.

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 DATE PALM KERNEL EXTRACT ON FACIAL SKIN WRINKLES 281

RESULTS
Only 36 subjects of 43 participants completed the study (seven male participants, age
range 41–55 years, and 29 female participants, age range 40–65 years). Reasons for drop-
ping out from follow-up visits were given as holiday time, tiredness, and other family
issues rather than any problem with the cream preparations. A preliminary patch test
revealed no skin reactions observed at 1, 24, and 96 h after removing the test materials in
all 12 subjects. The physicochemical characteristics of both the placebo and test creams,
kept at different temperatures for 8 weeks, are shown in Table II. The color was pearly
white with no changes in homogeneity or phase separation up to 50 ± 0.1°C and up to
40 ± 0.1°C with 75% relative humidity. The presence of the lipophilic emulsifier ABIL®
EM 90 stabilized the emulsions at high temperatures.

CLINICAL ASSESSMENT

Clinical assessment of facial skin appearance was conducted at baseline and after comple-
tion of the study (i.e., after 8 weeks). Based on a three-point scale evaluation, there were
statistically significant improvements in skin roughness, texture homogeneity, and melanin
pigmentation. Skin redness levels were not significantly altered in all subjects (Table III).

BIOPHYSICAL MEASUREMENTS

Measurements of objective skin parameters with a multiprobe instrument over 8 weeks

revealed progressive increases of surface hydration and elasticity together with a progres-
sive decline in both dermal pigmentation and TEWL in DPKE-treated skin compared
with placebo cream. Figure 1A illustrates changes of skin hydration at three time points
of study. At baseline, the mean values of skin hydration were 41.21 ± 10.32 and 42.10 ±
9.64 AU of placebo and DPKE creams, respectively. After 4 weeks, these values rose to
43.71 ± 11.62 and 52.92 ± 10.25 AU, respectively, and after 8 weeks, the values were
44.21 ± 10.42 and 52.23 ± 11.54, respectively (p < 0.01 vs. placebo and p < 0.001 vs.
baseline for the two time points). Figure 1B illustrates changes of TEWL at three time
points of the study. At baseline, the mean readings of TEWL were 12.23 ± 3.11 and
11.76 ± 2.92 g/m2/h of placebo and DPKE creams, respectively. After 4 weeks, these
values were 11.72 ± 2.52 and 10.43 ± 2.41 g/m2/h, respectively, and after 8 weeks, the

Table II
Physicochemical Characteristics of Placebo and DPKE Creams Kept at Different Temperatures for 8 Weeks

Color Texture Homogeneity Phase separation Immediate skin feel

5–40 ± 0.1°C White Smooth Homogeneous No Refreshing, cool, no
grittiness or greasiness
40 ± 0.1°C with 75% White Smooth Homogeneous No Refreshing, no grittiness
relative humidity or greasiness
Refreshing, no grittiness
50 ± 0.1°C White Smooth Slight liquefaction Slight
or greasiness

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Table III
Clinical Assessment Rated on a Three-Point Scale

After 8 weeks
Baseline Placebo DPKE
Skin roughness score 2.24 ± 0.81 2.17 ± 0.64 1.76 ± 0.94**,#
Skin texture homogeneity score 2.18 ± 0.76 2.01 ± 0.90 1.55 ± 0.66***,#
Skin melanin pigmentation score 2.36 ± 0.95 2.28 ± 0.74 1.90 ± 0.84*,#
Skin redness score 2.04 ± 0.84 2.11 ± 0.72 1.93 ± 0.93

Data are presented as mean ± SD of 36 subjects. Significance levels: *p < 0.05, **p < 0.01, and ***p < 0.001
versus baseline. #p < 0.05 versus placebo (ANOVA).

values were 11.53 ± 2.41 and 10.04 ± 2.73 g/m2/h, respectively (p < 0.05 vs. both pla-
cebo and baseline at the two time points).
Figure 1C and D illustrate changes of skin elasticity and firmness at three time points of
the study. At baseline, the mean values of skin elasticity were 0.84 ± 0.08 and 0.84 ±
0.09 AU of placebo and DPKE creams, respectively; after 4 weeks, these values were
0.85 ± 0.07 and 0.89 ± 0.06 AU, respectively (p < 0.05 vs. placebo and p < 0.01 vs. base-
line), and after 8 weeks, the values were 0.86 ± 0.09 and 0.94 ± 0.10 AU, respectively
(p < 0.001 vs. both placebo and baseline). At baseline, the mean firmness in the DPKE
test skin was 0.27 ± 0.08 AU, and after 4 and 8 weeks, the firmness had diminished to
0.23 ± 0.06 and 0.21 ± 0.06 AU, respectively (p < 0.01 vs. placebo). Figure 1E illustrates
changes of skin melanin density at three time points of the study. At baseline, the mean
readings of skin melanin were 273.8 ± 27.9 and 265.6 ± 26.4 AU of placebo and DPKE
creams, respectively. After 4 weeks, these values were 267.4 ± 30.2 and 260.8 ± 27.5 AU,
respectively, and after 8 weeks, the values were 275.2 ± 28.4 and 255.3 ± 26.5 AU, re-
spectively (p < 0.05 vs. both placebo and baseline). There were no significant changes in
erythema levels for both placebo and DPKE creams over 8 weeks (Figure 1F).

ANTERA 3D® MULTISPECTRAL ANALYSIS

The significant reduction in the wrinkle depth compared with pretreatment values was
confirmed by objective analysis with the Antera 3D® analyzer performed at baseline, and
after 4 and 8 weeks of treatment. Several wrinkle measurements showed significant im-
provements after 4 and 8 weeks compared with both baseline values and placebo cream.
With the medium filter, there was a significant decline in the overall size after 4 and 8
weeks by 18.4% and 28.6%, respectively (from 56.4 ± 22.2 to 46.0 ± 21.5 (p < 0.05 vs.
baseline and placebo), and 40.3 ± 22.1 AU (p < 0.01 vs. baseline and placebo, paired
t-test, Figure 2A). The maximal depth was significantly decreased by 22.5% and 29.6%,
respectively (from 0.410 ± 0.19 mm to 0.322 ± 0.18 mm and 0.284 ± 0.23 mm, respec-
tively (p < 0.05 vs. baseline and placebo for all, paired t-test, Figure 2B). Moreover, sig-
nificant improvements in the indentation index and skin roughness index were also
evident after 4 and 8 weeks; however, the reductions of these parameters were more pro-
nounced after 8 weeks, compared with baseline and placebo levels (p < 0.01 and p <
0.001, respectively, Figure 2C and D). The overall reduction in the skin color was also

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 DATE PALM KERNEL EXTRACT ON FACIAL SKIN WRINKLES 283

Figure 1. Biophysical measurements in 36 subjects at baseline and after 4 and 8 weeks of topical treatment
with 5% DPKE cream. (A) Skin hydration, (B) TEWL, (C) skin elasticity, (D) firmness, (E) melanin pigmen-
tation, and (F) erythema. Data are presented in AU as mean ± SD. Significance levels: *p < 0.05, **p < 0.01,
and ***p < 0.001 versus baseline. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus placebo (ANOVA).

evaluated during the follow-up visits using the Antera 3D. Multispectral analysis of the
skin color showed that DPKE cream statistically reduced the average concentrations of
skin melanin at the two time points of assessment compared with both the baseline and
placebo measurements (p < 0.05 for all, Figure 3). The placebo cream had no significant
effects on the overall wrinkle size, depth, indentation, and roughness indices or skin

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Figure 2. Antera 3D® analysis of wrinkle size (A) and maximal depth (B) measured in 36 subjects at baseline
and after 4 and 8 weeks of topical treatment with 5%DPKE cream. (C and D) show percentage changes in
the relative indentation index and relative roughness index, respectively. Data are presented as mean ± SD in
AU for wrinkle size and in millimeters for wrinkle depth. Significance levels: *p < 0.05, **p < 0.01, and
***p < 0.001 versus baseline. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus placebo (paired t-test).

melanin concentration at the two measured time points. Collective data obtained in both
male and female volunteers separately at baseline and after 4 and 8 weeks of treatment
with DPKE cream are presented in Table IV.

DISCUSSION
Aging involves several alterations of skin properties that result in skin thinning along
with reduced skin elasticity, increased fragility, pigmentations, and appearance of fine
lines and wrinkles. Accordingly, the search for natural and effective skin rejuvenation
remedies is an exceptionally large interest for the cosmetics industry. In this study, we
investigated the potential of topical DPKE, formulated as cream, in reducing the mani-
festations of facial skin aging in human volunteers. The work herein demonstrates and
validates the use of a cream form containing 5% DPKE over placebo against fine lines and
wrinkles, skin pigmentations, hydration, and elasticity. This improvement began to show
after 4 weeks of treatment and progressed over 8 weeks. In addition, DPKE cream was

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 DATE PALM KERNEL EXTRACT ON FACIAL SKIN WRINKLES 285

Figure 3. Percentage changes in relative melanin concentration after 4 and 8 weeks of treatment. Data are
presented as mean ± SD. Significance levels: *p < 0.05 versus baseline. #p < 0.05 versus placebo (paired t-test).

extremely well tolerated when applied daily on facial skin (no irritation, redness, burn, or
itching). This is in contrast with other topical treatments containing compounds such as
retinoic acid which provides improvements in skin appearance but at the expense of
irritating adverse effects such as skin redness and sensitivity (16). In addition to good
tolerability, DPKE is easily formulated, chemically stable, and compatible with other
formulation constituents, qualifying it to be an ideal agent for use in cosmetic products.
Skin aging is the result of a multifaceted biological phenomenon consisting of two com-
ponents: intrinsic (chronologic) aging and extrinsic aging. The pathophysiologic elucida-
tion of both components has been well documented in clinical and histologic studies (17).
Intrinsic aging is largely a genetic process in which increased production of reactive oxy-
gen species (ROS) alongside with progressive damage to mitochondrial DNA causes cell
senescence and impairment of skin repair (18). Extrinsic aging is responsible for most
skin deteriorations, and is caused by several factors, of which, exposure to ultraviolet
(UV) light (photoaging) is the most crucial, and causes DNA damage in a multiplicity of
living tissue (19). Photodamage also involves the generation of ROS that breaks the cellular
biosynthesis of collagen and glycosaminoglycans in skin along with decreased keratinocyte

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 286

Table IV
Collective Data Obtained in Both Male and Female Volunteers at Baseline and after 4 and 8 weeks of Treatment with DPKE Cream

Male volunteers (n = 7) Female volunteers (n = 29)

Baseline 4 weeks 8 weeks Baseline 4 weeks 8 weeks
Clinical assessment:
Roughness score 2.32 ± 0.83 — 1.86 ± 0.87* 1.72 ± 0.85 — 1.14 ± 0.51*
Texture homogeneity score 2.07 ± 0.53 — 1.41 ± 0.63* 2.21 ± 0.75 — 1.59 ± 0.62**
Pigmentation score 2.30 ± 0.74 — 2.08 ± 1.08 2.37 ± 0.99 — 1.83 ± 0.80*
Redness score 1.81 ± 0.77 — 2.04 ± 0.78 2.10 ± 0.85 — 1.91 ± 0.96
Biophysical measurements:
Skin hydration (AU) 43.00 ± 10.56 49.70 ± 9.83** 55.23 ± 9.63** 40.71 ± 9.60 53.74 ± 9.66** 50.93 ± 12.03**
TEWL (g/m2/h) 11.75 ± 3.32 10.24 ± 1.57 10.12 ± 1.90 12.33 ± 3.10 10.48 ± 2.54* 10.02 ± 2.90**

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Skin elasticity (AU) 0.844 ± 0.088 0.910 ± 0.057 0.976 ± 0.113* 0.836 ± 0.093 0.890 ± 0.063* 0.930 ± 0.094**
Skin firmness (AU) 0.270 ± 0.078 0.199 ± 0.047* 0.171 ± 0.061* 0.277 ± 0.084 0.236 ± 0.057* 0.218 ± 0.054**
Skin melanin density (AU) 272.9 ± 21.8 250.6 ± 20.2 264.9 ± 27.3 273.5 ± 29.7 263.4 ± 28.9 254.1 ± 26.3*
Erythema (AU)

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176.9 ± 62.8 168.6 ± 66.4 155.5 ± 74.2 167.6 ± 60.3 158.9 ± 70.4 151.7 ± 68.4
Multispectral analysis:
Wrinkle size (AU) 67.5 ± 19.5 44.0 ± 17.6 41.8 ± 15.9* 54.6 ± 22.1 46.5 ± 22.5 39.2 ± 23.1*
Maximal depth (mm)
JOURNAL OF COSMETIC SCIENCE

0.482 ± 0.117 0.315 ± 0.130 0.258 ± 0.157* 0.395 ± 0.194 0.323 ± 0.190 0.287 ± 0.225*
Relative indentation index (%) 100.0 ± 18.1 84.9 ± 16.4 78.5 ± 17.5* 100.0 ± 17.8 81.3 ± 16.5** 80.8 ± 15.4***
Relative roughness index (%) 100.0 ± 15.5 82.5 ± 13.0* 74.4 ± 12.8** 100.0 ± 15.2 75.1 ± 15.7** 67.2 ± 13.9***
Relative melanin concentration (%) 100.0 ± 13.7 92.9 ± 14.4 86.7 ± 13.5 100.0 ± 20.6 87.3 ± 20.4* 85.8 ± 25.4*

Data are presented as mean ± SD of seven male and 29 female subjects. Significance levels: *p < 0.05, **p < 0.01, and ***p < 0.001 versus baseline values (paired t-test).
 DATE PALM KERNEL EXTRACT ON FACIAL SKIN WRINKLES 287

cell turnover, resulting in grave disorganization of the dermal matrix (20). Accordingly,
there is plenty of room to explain the essentials of mechanisms underlying the benefits of
DPKE observed herein. We suggest that the anti–skin aging effect of DPKE is attributed,
at least partially, to preservation of the dermal matrix through prevention of oxidative
damage to cellular DNA. This assumption is based on data in the literature documenting
the protective role of many antioxidants against the UV-induced oxidative damage to
human skin (21). Likewise, researchers have found that date seed extract includes a myriad
of polyphenols and tocopherols with robust antioxidant capabilities. Moreover, date kernel
oil was reported to have higher oxidative stability than most vegetable oils, including olive
oil (1). These observations have led to a series of studies to investigate the notable anti-
oxidant and radical-scavenging properties of date kernel extract on human skin. Dammak
et al. (22) have demonstrated that pretreatment with date seed oil significantly amelio-
rated the expression of p53 in human skin after exposure to UV irradiation by affording
free-radical–scavenging properties. Date seed oil was also demonstrated to significantly
improve cell viability and reduce depletion of superoxide dismutase, glutathione peroxi-
dase, catalase, and lipid peroxidation in cultured human melanocytes after hydrogen per-
oxide exposure (23). The experiment was subsequently repeated on human keratinocytes
with findings analogous to those of the previous study (24). As both melanocytes and
keratinocytes are implicated in the inflammatory process of photoaging, we suggest that
these findings may explain some of the rejuvenative properties of DPKE.
Previous studies have shown that date kernel extract contains a high fraction of hydroxy-
tyrosol, one of the most powerful natural antioxidants. This compound has 10 times more
antioxidants than green tea and two times more than coenzyme Q10 (25). The extraordi-
nary scavenging activity of hydroxytyrosol has been demonstrated in several studies both
in vivo and in vitro (26,27). In its chemical structure, this compound has an extra hydroxyl
group in its benzene ring, granting it greater function as a free radical scavenger and in-
creasing its efficacy under stress conditions (25). In addition, hydroxytyrosol is an am-
phipathic, water-soluble, and fat-soluble molecule which facilitates its penetration of
cellular membranes and makes it a good transporter of substances across skin tissue (28).
Studies have also shown that hydroxytyrosol has the ability to inhibit cyclooxygenase and
lipoxygenase enzymes of arachidonic acid, reducing the oxidative corrosion characteristic
of inflammations, and stimulate the regeneration and repairing of damaged tissue (29).
Alpha (α)-tocopherol, a type of vitamin E, is another component present in date kernel
extract with significant concentration (1) and is best known for its robust antioxidant
function and good penetration into the human skin layers (30). α-tocopherol has been
used in the treatment of burns, surgical scars, and variety of skin conditions (31). Topical
formulations containing α-tocopherol have also been found to be effective in reducing
infraorbital dark haloes and wrinkles of the lower eyelids (32), although in vitro studies
revealed that α-tocopherol inhibits p53 expression in dermal tissues and protects against
the UV irradiation of cultured fibroblasts (33). α-tocopherol is, therefore, one of the most
shared ingredients in the over-the-counter treatments of skin aging.
Phytosterols and phytoestrogens are additional major phytochemicals found in the lipid
soluble fraction of the DPKE. Phytoestrogens are a group of isoflavones that can bind
both estrogen receptors ERα and ERβ (34), and they are considered to be naturally oc-
curring selective estrogen receptor modulators and potential candidates to provide a nat-
ural alternative of estrogen replacement in postmenopausal women. Studies have shown
that these phytoestrogens have favorable effects on human skin as they can minimize

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 288 JOURNAL OF COSMETIC SCIENCE

UV-induced cell damage in cultured keratinocytes, improve skin elasticity, reduce pig-
mentation and wrinkle depth, and increase the production of type 1 procollagen (35).
Moreover, the isoflavone genistein has been reported to provide significant protection
against the UV-induced damage in human dermal fibroblasts by significantly boosting
the intracellular antioxidant armamentarium in a dose-dependent way (36). Taken to-
gether, we suggest that the combination of various ingredients contained in DPKE might
provide complementary mechanisms of modulating cellular pathways involved in the
process of skin aging, and the effectiveness of these ingredients may be further potenti-
ated by synergism of their individual components.

FUNDING
This research did not receive any specific grant from funding agencies in the public, com-
mercial, or not-for-profit sectors.

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 J. Cosmet. Sci., 70, 291–298 (November/December 2019)

Electrophoretic Mobility of Some Tattoo Dyes as an

Approach to Remove Their Subcutaneous Traces

IGOR WINKLER, ULYANA ANDRUSHKO, and

ALLA VELYKA, Bucovinian State Medical University, Chernivtsi
58000, Ukraine (I.W., A.V.), Yuriy Fedkovych National University of
Chernivtsi, Chernivtsi 58012, Ukraine (U.A.)

Accepted for publication September 13, 2019.

Synopsis
The electrokinetic (ζ, zeta) potential was determined for a series of commercial tattoo pigments. A standard
experimental method involving the measuring of the level difference formed in a U-shaped tube filled with
a solution containing the dye after application of some potential difference was used to find ζ-potential
values. All of them were negative and sufficiently large to ensure electrophoretic mobility of the pigment
particles in a special gelatin-based electrophoretic bed. Gelatin-based beds, one containing a pigment
and the other without the pigment, were set side by side in a microelectrophoretic cell. The application
of relatively low potential difference (20–25 V) provoked the migration of the pigment in the gelatin
bed without pigment for as much as 10 mm after a 40-minute long electrophoresis. The intensity of the
color of the pigment did decrease noticeably. These results seem to indicate the potential applicability of the
reported method for the elimination of old and/or unwanted tattoo and of tattoo traces left after previous
manipulations.

INTRODUCTION
As tattooing becomes very popular all over the world, the necessity to remove old, no
longer needed, blurred, deformed tattoo pictures is becoming more and more widespread
too. Although “temporal” tattoo can be removed without any serious troubles, classical
subepidermal patterns are very tenacious and sometimes require more time and effort to
eliminate than was spent on their preparation.
There is a wide variety of approaches that can be used to remove or discolor the classical
tattoo. They range from the simple mechanical scraping of skin layers together with the
pigment particles fixed in them (1) to more advanced laser destruction (discoloration) of
the pigment inside the patient’s skin (1–3). It should be noted that all these approaches
are more or less traumatic and may leave traces in the form of scars, hypo/hyperpigmenta-
tion of the skin, and changes in the skin texture and may provoke its contamination or

Address all correspondence to Igor Winkler at [email protected].

291
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 292 JOURNAL OF COSMETIC SCIENCE

persisting inflammation (4,5). Even the most gentle methods may leave some pigment
traces in the form of shades or blurred spots. These problems may push patients to add
new tattoos over leftovers of the old ones to hide these cosmetic defects.
Therefore, it is obvious that the need of complete removal of old tattoo pigments with
minimal skin damage is undoubtedly acute. This need will become even greater when
tattoo carriers decide that their tattoo pictures should be removed or corrected. In this
context, we consider possible approaches that may lead to new solutions to be used in the
technologies of tattoo removal.
Organic tattoo pigments are particles usually sized from 200 nm to 5 µm (6), which are
electrophoretically mobile. Their electrokinetic (zeta) potential can be determined using
appropriate methods such as the classical measuring of the difference in liquid levels
reached in the U-shaped pipe after application of some fixed external DC-voltage during
some period of time. This method is still extensively used in many fields of applied tech-
nique, chemistry, and biology (7–9).
Then, when the determined zeta potential value is sufficient to expect any substantial
mobility of the particles, the possibility of tattoo removal can be tested further in direct
experiments involving a model of the human skin and, finally, be verified in the experi-
ments with real skin in volunteers.
Therefore, it is important to determine some typical zeta potentials of the tattoo pigments
in isotonic solutions.

MATERIALS AND METHODS

PIGMENTS

A series of unbranded dyes for tattooing (Figure 1) was purchased at an online store and
then used for the preparation of the solutions and other mixtures to investigate their
electrophoretic mobility. Only four color samples: black, white, red, and green were selected
for this investigation because they are the most popular for tattoo pictures.

PREPARATION OF PIGMENT SUSPENSIONS

All the pigments were suspended in isotonic solution (0.9% NaCl) to reach the concen-
trations described in the corresponding manuals (0.25 mL/L). As a result, four brightly
colored solutions were obtained and then each of them was placed in the U-shaped glass
tube (Figure 2, 1) for measuring its zeta potential.

DETERMINATION OF THE ζ POTENTIAL

Following the classical method of determination (10), each solution was poured into the
tube in such a way to fill it over the main faucets (Figure 2, 2) level. Then both faucets
were closed and excessive colored solution removed from the upper parts of the device
(Figure 2, 3), which then was filled with the coupling fluid (same isotonic solution of

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 ELECTROPHORETIC MOBILITY OF SOME TATTOO DYES 293

Figure 1. Samples of tattooing pigments used for investigation of the electrophoretic activity.

NaCl containing no dissolved dyes). Finally, the balancing faucet (Figure 2, 4) was opened
to equalize the coupling fluid levels in both parts of the tube.
Afterward, both extremities of the U-shaped tube were connected electrically to the
power supply through additional agar–agar bridges installed between each bend of the
tube and the power supply outputs. Then the main faucets (2) were opened and voltage
was applied between both parts of the glass tube.
Because the dye grains acquire some electric charge in the isotonic solution, they will
move toward one of the poles causing a level difference between working and coupling liq-
uids. Having measured the difference between the margins in each bend, the zeta poten-
tial can be calculated by the following formula (11):
ηhl
ζ=
Eεε0t ,
where η is the viscosity of working solution (because all working solutions were quite
diluted, it was taken as equal to the viscosity of water 1 × 10−3 Pa s); h is the final differ-
ence between the solution levels in each bend, m; l is the distance between the axes of two
parts of the U-shaped tube (0.58 m); E is the electric voltage applied; (260 V throughout
all experiments); ε0 is the standard vacuum dielectric permittivity constant (8.85 × 10−12
F/m); ε is the relative dielectric permissivity of water (equal to 81); and t is the duration
of electrophoresis (s).

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Figure 2. U-shaped tube for experimental determination of zeta potential. A difference in the liquid levels
was achieved as a result of electrophoresis. (1) Working part filled with the dye solution; (2) main faucets; (3)
coupling fluid parts and (4) balancing faucet.

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 ELECTROPHORETIC MOBILITY OF SOME TATTOO DYES 295

Figure 3. Microelectrophoresis cell with the gelatin samples: (1) raw sample; (2) the sample consisting some
dye; (3) graphite electrodes. Let us compare positions of the boundary between the colored and not-colored
samples for various gelatin/black dye samples.

The dye solution level did rise in the “positive” bend (Figure 2, right side) for all four
samples meaning that the dye grains for all colors used in the framework of our experi-
ments acquired a negative charge when dissolved in the isotonic NaCl solution.

ANALYSIS OF THE ELECTROPHORETIC MOBILITY OF PIGMENTS

Because the zeta potential of the black pigments was the largest, it was selected for the
investigations related to applicability of electrophoresis to extract samples of “real” pig-
ments samples from tissues. No human or animal tissue samples were used because of
ethical restrictions and gelatin-based mixtures were used at that stage.
Twenty-five milligrams of unbranded gelatin was dissolved in 500 mL of hot water to
obtain such mixtures. Then the solution was divided into two parts; one of them was left
for cooling and hardening, whereas the required amount of the pigment suspension was
added to the second one to make its concentration equal to 0.25 mL/L. Then it was also
left for cooling and hardening.
Parallelepipedal samples were cut out of each part and placed into a plastic microelectro-
phoretic cell (Figure 3). The cell was designed in such a way (12) that each sample was in
contact with the graphite electrodes located on the outer edges of the cell and connected
to an external electric power supply, whereas the inner sides of the samples were in con-
tact with each other ensuring transferability of the dye particles between the samples.

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Because the dye particles can move from the colored sample toward the not-colored one
under the applied external electric voltage, this process can be visualized by distortion
or shifting of the margin between the samples. In case it moves noticeably, the principal
applicability of this method to extract the tattoo dyes out of human skin tissues can be
expected.
All microelectrophoretic experiments involving gelatin samples were carried out under
the following conditions: DC voltage applied, 20–25 V; current, 5–7 mA; and process
duration, 2,400 s. It should be noted that such, or close, potential values are used regu-
larly in practice at various medical electrophoretic manipulations or biochemical investi-
gations (13).

RESULTS
All experimental results related to determination of the dyes’ zeta potentials are shown in
Table I.
As seen from the average zeta potential values given in Table I, the difference between the
potentials of various dyes was larger than the experimental error. It means that the dye
composition is an influential parameter governing the value of its electrokinetic poten-
tial. Because the zeta potential of the most popular black pigment was the largest, it
should be the most mobile and the most suitable for electrophoretic extraction. There-
fore, this pigment was selected for further experiments.
As seen from Figures 4 and 5, the black dye’s mobility was quite significant and it reached
10 mm even after the 40-min-long electrophoresis. Besides, it can be noticed that the

Table I
Experimental Data and Calculated Zeta Potentials for the Dyes

Dye Electrophoresis duration, s Level difference, m ζ, V

Red 1,200 0.020 −0.135
0.020 −0.135
0.024 −0.162
Average 0.021 −0.142 ± 0.0119
Black 1,200 0.020 −0.135
0.024 −0.162
0.022 −0.148
0.026 −0.175
0.020 −0.135
Average 0.024 −0.151 ± 0.014
Green 1,200 0.018 −0.121
0.016 −0.108
0.022 −0.148
0.020 0.135
0.026 0.175
Average 0.0204 −0.138 ± 0.0194
White 1,200 0.012 −0.0810
0.016 −0.108
0.02 −0.135
0.02 −0.135
0.014 −0.0944
Average 0.064 −0.111 ± 0.0194

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 ELECTROPHORETIC MOBILITY OF SOME TATTOO DYES 297

Figure 4. Mobility of the black dye particles after the 2,400-s electrophoresis. Left, before; right, after elec-
trophoresis. The boundary has shifted for approx. 10 mm.

color intensity of the black pigment becomes weaker after the electrophoresis. It is quite
obvious because some portions of the dye were migrating away from the sample, so its
concentration and coloring intensity should decrease.

DISCUSSION
All tested tattoo samples have revealed the negative zeta potentials, although their values
were sufficient to maintain quite significant mobility of the dye grains inside the faux
skin samples. The margin between colored and not-colored gelatin samples has moved for
about 10 mm after 40-min-long application of the 20–25 V potential difference. These
results prove that the method presented in this work looks quite promising for elimination
of the old or unwanted tattoos alone or in combination with the others. In the latter case,
it should be used at the final stage to eliminate the residual traces and shadows of the tat-
toos. Low voltages and comparatively short application time tested in the framework of
present investigation allow one to expect that the volunteer requesting the removal of the
tattoo should not experience serious discomfort because of local skin overheating during
the treatment. However, a series of thorough experiments is recommended with tattooed
animal skin to determine the exact regimes of electrophoresis before testing this approach
on human volunteers.
This result promises potential applicability of this method in technologies of elimination
of old, blurred, or unwanted tattoos.

Figure 5. Mobility of the black dye particles after the 2,400-s electrophoresis. Left, before; right, after elec-
trophoresis. The boundary has shifted for approx. 8 mm.

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nate: surface tension, ζ-potential, viscosity and emulsification, Carbohydr. Polym., 181, 56–62 (2018).
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Effect of Palmitic Acid Conjugation on Physicochemical

Properties of Peptide KTTKS: A Preformulation Study

SEYEDEH MARYAM MORTAZAVI, FARZAD KOBARFARD,

HOWARD I. MAIBACH, and HAMID REZA MOGHIMI,
Department of Pharmaceutics and Nanotechnology, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran,
1991953381 (S.M.M., H.R.M.), Department of Medicinal Chemistry,
School of Pharmacy, Shahid Beheshti University of Medical Sciences,
Tehran, Iran, 1991953381 (F.K.), Department of Dermatology, School of
Medicine, University of California, San Francisco, California, 94115
(H.I.M.), Protein Technology Research Center, Shahid Beheshti University
of Medical Sciences, Tehran, Iran, 1991953381 (H.R.M.)

Accepted for publication September 21, 2019.

Synopsis
Lys–Thr–Thr–Lys–Ser (KTTKS) minimally crosses the skin because of hydrophilicity; therefore, its palmitoyl
derivative, palmitoyl-KTTKS (Pal-KTTKS), is used in cosmetic products. In spite of this, there is insufficient
information on its physicochemical properties and the effects of palmitoylation on such properties. The aim of
this study was to investigate these properties. Such information would help appropriate formulation development.
KTTKS and Pal-KTTKS were synthesized and characterized for ultra violet (UV) absorption, structure [X-ray
diffraction (XRD)], morphology (electron microscopy), birefringence (polarized light microscopy), partitioning,
solubility, thermal behavior (melting, thermogravimetric analysis, and differential scanning calorimetry), surface
activity, critical micelle concentration (CMC, by tensiometry), and stability. KTTKS and Pal-KTTKS decomposed
at about 154 and 150°C, respectively, and did not show a melting point before decomposition. The maximum
UV absorbance of peptides was less than 200 nm. Both peptides showed birefringence, irregular flake
morphologies, and hygroscopicity. KTTKS was freely soluble in water at room temperature (logP = −1.6 ± 0.15),
indicating its hydrophilic nature. logP of Pal-KTTKS was calculated to be about 3.7, indicating a lipophilic
compound. Pal-KTTKS showed surface activity with a CMC value of 0.024 ± 0.004 mM (19.25 ± 2.9 mg/L),
whereas KTTKS did not show such surface activity. Palmitoylation demonstrated sharp peaks in the XRD
pattern of KTTKS. KTTKS and Pal-KTTKS differ mainly in terms of chemical properties and show some
similarity in physical properties. These results can be used for formulation developments.

INTRODUCTION
Skin aging prevention is an attractive issue in the cosmetic industry. Antiaging products
include retinoids, alpha hydroxy acids, moisturizers, antioxidants (such as L-ascorbic acid,
niacinamide, α-tocopherol, and ubiquinone), and peptides (1).

Address all correspondence to Hamid Reza Moghimi at [email protected] and [email protected].

299
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Based on their mechanism of action, topical peptides are classified into four categories:
signal peptides, enzyme-inhibitor peptides, neurotransmitter-affecting peptides, and car-
rier peptides (1–4). Lys–Thr–Thr–Lys–Ser (KTTKS) (see Figure 1) is a signal peptide
discovered by Katayama et al. in 1993. They demonstrated that extracellular matrix
biosynthesis in human fetal lung fibroblasts was stimulated by KTTKS as a subfragment
from C-peptides of type I procollagen, which is able to increase dermal remodeling (5).
Despite its high antiaging potential, this peptide minimally penetrates the skin. One
strategy used to overcome this problem is conjugation to a lipophilic compound such as
palmitic acid (a long-chain fatty acid containing 16 carbon atoms) (6). Palmitoyl-KTTKS
(Pal-KTTKS) (see Figure 1) (brand name MatrixylTM, Sederma Inc., Le Perray-en-Yvelines,
France) is available on the global market in antiaging formulations. Pal-KTTKS demon-
strated improved effects on reduction of skin wrinkles in a 12-week, double-blind, placebo-
controlled, clinical study on photoaged human facial skin (7). Fu et al. (8) demonstrated
that niacinamide/Pal-KTTKS/retinyl propionate products had greater effect on improve-
ment of wrinkle appearance than a 0.02% tretinoin product. Unfortunately, only few
preformulation studies exist on them. Lack of information about physicochemical proper-
ties of these compounds leads to difficulty in their formulation.
Here, KTTKS and Pal-KTTKS were synthesized, and after confirmation by mass spec-
troscopy, physicochemical properties of both peptides and, therefore, the effects of covalent
attachment of palmitic acid on KTTKS properties were investigated. To achieve these
goals, ultra violet (UV) absorption ability, structure, morphology, birefringence, thermal

Figure 1. Chemical structure of KTTKS (A) and Pal-KTTKS (B).

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 PREFORMULATION STUDIES OF PEPTIDES KTTKS AND PAL-KTTKS 301

behavior, aqueous solubility, partition coefficient, surface activity, critical micelle concen-
tration (CMC), and aqueous stabilities were evaluated. The results should be useful for
formulation development and also will provide information on palmitic acid conjugation
of peptides, possibly useful for peptides other than KTTKS.

MATERIAL AND METHOD

MATERIALS

2-chlorotrityl chloride resin (loading capacity of 1.2 mmol/g), Fmoc-Ser(tBu)-OH,

Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, and 2-(7Aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) were provided by GL Biochem
(Shanghai, China). Trifluoroacetic acid and piperidine were obtained from Exir GmbH
(Wien, Austria). Ninhydrin was purchased from BDH Chemicals (Lutterworth, England).
Isopropyl alcohol was supplied by ChemLab (Zedelgem, Belgium). Other solvents and
reagents applied in the peptide synthesis process were purchased from Sigma-Aldrich
(Dorset, United Kingdom) or Merck (Gernsheim, Germany). All materials were used
without further purification.

METHODS

Peptide synthesis and characterization. KTTKS and Pal-KTTKS were synthesized by solid-
phase peptide synthesis (SPPS) using the standard fluorenylmethyloxycarbonyl (Fmoc)
strategy in a manual glass reaction vessel (9). The 2-chlorotrityl chloride resin was ap-
plied as an insoluble support, and to protect side chain reaction during the process, amino
acids containing tert-butyloxycarbonyl (Boc) or tert-butyl side-chain protecting groups
were used. After synthesis, confirmation of peptide integrity was accomplished by mass
spectroscopy using an Agilent 6410 Triple Quad LC/MS (Agilent, Santa Clara, CA) with
an electrospray ionization interface.
Determination of UV absorbance. Aqueous solutions of KTTKS and Pal-KTTKS were pre-
pared individually. First, UV absorbance was set to zero with deionized water, and the
solvent of peptide solution was used as the blank. UV absorbance of solutions was then
measured over the wavelength range of 190–400 nm by a Cecil 2021 UV-Visible spectro-
photometer (Cecil Instrument Services, Cambridge, United Kingdom) using a quartz
cuvette with a 1-cm path length.
Evaluation of particle morphology by SEM. The morphology of KTTKS and Pal-KTTKS was
analyzed by SU3500 scanning electron microscopy (Hitachi Ltd., Tokyo, Japan) at an ac-
celerating voltage of 15 kV. Peptide powders were mounted on a stub of metal with ad-
hesive, coated with gold, and then analyzed by SEM.
Evaluation of birefringence under polarized light microscopy. Birefringence was examined using
a CETI Magnum binocular compound microscope (Medline Scientific, Oxford, United
Kingdom) with a total magnification of 100×. The glass slide of each peptide powder was
prepared and observed under cross-polarized light at ambient temperature.
Structure evaluation by X-ray diffraction (XRD). XRD patterns of KTTKS, Pal-KTTKS,
and palmitic acid were measured using an X-ray diffractometer (X’Pert Pro MPD,

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Panalytical B.V, Almelo,The Netherlands) at laboratory temperature (25°C) under the

following conditions: voltage of 40 kV, current of 40 mA, and scan range of 1–80 2θ using
Cu Kα radiation.
Partitioning and solubility investigation. The n-octanol/water partition coefficient ( Pwo) was ob-
tained using the shake flask method for KTTKS and partition coefficient and solubility of
Pal-KTTKS were computed using ACD/ChemSketch (ACD/Labs, Toronto, Canada) and
ALOGPS (VCC-LAB, Munich, Germany) programs (10–12).
In the shake flask method, n-octanol and distilled water were mutually saturated with each
other for 24 h before use. KTTKS was dissolved in water presaturated with n-octanol at
a concentration of 200 µg/mL. In the following, n-octanol, presaturated with water, was
added to the glass vials containing the aqueous solution of peptide. The glass vials were
mixed by vortex for 2 min and then placed on an orbital shaker for 1 h to achieve equili-
bration. To separate the phases, contents of the glass vials were transferred to a separation
funnel and left without shaking for 0.5 h. The phases were separated and concentrations
of peptide in the aqueous phase were measured by LC–MS, as described in the Peptide
assay by LC-MS section. The n-octanol/water partition coefficient was then calculated us-
ing equation 1 (13):
Co
logPwo  log , (1)
Cw

where Co and Cw are the concentrations of peptide in n-octanol and water after equilibra-
tion, respectively. The experiment was performed in triplicate.
Aqueous solubility of KTTKS was estimated by adding increasing amounts of peptide to
a certain volume of distilled water at room temperature.
Surface tension measurement of peptide solutions. Surface tensions of aqueous solutions of
KTTKS and Pal-KTTKS (prepared individually with concentrations of 100 µg/mL) as
well as deionized water were measured by a K100 Force tensiometer (Krüss GmbH,
Hamburg, Germany) using the Du Noüy ring method at room temperature. In addition,
the surface tensions of both peptides and also deionized water were calculated by the
ACD/ChemSketch freeware software. This software calculates the surface tension via
molar volume and parachor (14).
Determination of CMC. Aqueous solution of Pal-KTTKS was prepared using deionized
water. The CMC value was then measured fully automatically by the K100 Force tensi-
ometer (with one micro dispenser) using the Du Noüy ring method at room temperature.
The measurement was repeated three times.
Thermal behavior. Thermal behaviors of peptides were evaluated by IA9000 series capillary
melting apparatus (Cole-Parmer Instrument, Vernon Hills, IL), TGA-50, and DSC-60
(Shimadzu, Kyoto, Japan) thermal analysis systems. The melting point apparatus was
used to determine the melting points. The capillary tube was packed by gently pressing
the open end into the peptide powders. The bottom of the capillary tube was then tapped
on a hard surface so that the peptide powders packed down into the bottom of the
capillary tube. The capillary tubes containing the peptide powder were inserted into the
apparatus holder. Ramp rate of 5°C/min was adjusted. The peptide powders were then
examined through the magnifying glass of the apparatus, and changes in the powder bed
were recorded during the temperature rise.

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 PREFORMULATION STUDIES OF PEPTIDES KTTKS AND PAL-KTTKS 303

Thermogravimetric analysis (TGA) was conducted to determine peptide decomposition.

About 4–5 mg of peptide powder was weighed into an open TGA aluminum pan and
heated from ambient temperature to 595°C at a heating rate of 10°C/min under a nitro-
gen atmosphere. The experiment was performed in triplicate.
Differential scanning calorimetry (DSC) was performed for determination of peptides’
thermal characteristics. First, using indium as a standard, the DSC instrument was cali-
brated. About 3–5 mg of peptide powder was then put into the DSC aluminum pan. The
pan was sealed and thermal behavior studied at a heating rate of 10°C/min under a nitro-
gen atmosphere. The experiment was performed in triplicate. In another experiment, a
cycled heating method was used. First, the pans containing KTTKS or Pal-KTTKS were
individually heated to 100°C, and after cooling, the same pans were reheated to 165°C.
Cycled TGA experiments were also used here to interpret DSC data. In this experiment,
the pan containing peptide was heated to 100°C at a heating rate of 5°C/min and then
cooled to room temperature. The same pan was then heated again to 100°C at the same
heating rate. Thermogram was then analyzed for any weight change.
Stability studies. An aqueous solution of KTTKS was prepared at a concentration of 100
µg/mL and aliquoted into glass vials. The vials were then studied separately at 32°C in a
water bath for 48 h. Peptide concentration was then measured by LC–MS, as described in
the later section. The experiment was performed in triplicate.
Peptide assay by LC-MS. The concentration of KTTKS was quantified by a selected ion
chromatogram method using an Agilent 6410 Triple Quad LC/MS system under the fol-
lowing conditions: Capital C8-Optimal column (250 × 4.6 mm, i.e., 5 µm), isocratic
elution, mobile phase of acetonitrile (40%): 20 mM ammonium acetate solution contain-
ing 0.05% acetic acid glacial (60%), nitrogen dry gas at a temperature of 300°C and
pressure of 25 psi, injection volume of 25 µL, and flow rate of 0.6 mL/min. A mass to
charge (m/z) ratio of 564.2 was used to detect KTTKS.
Statistical analysis. SPSS Statistics software version 21.0 (IBM, Armonk, NY) was used for
data analysis. Independent sample t-test was used to determine the statistical significance
of data. Differences were considered to be significant for values of p < 0.05.

RESULTS

PEPTIDE SYNTHESIS AND CHARACTERIZATION

Peptides, which were synthesized by SPPS using the Fmoc methodology, were confirmed
by mass spectrometry. The singly charged molecular ion [M+H]+ at an m/z of 564.2 and
the doubly charged molecular ion [M+H]2+ at an m/z of 282.7 confirmed the synthesis of
KTTKS. In the case of Pal-KTTKS, [M+H]+ at an m/z of 802.3 and [M+H]2+ at an m/z
of 401.7 confirmed the synthesis of Pal-KTTKS.

DETERMINATION OF UV ABSORBANCE

Finding an appropriate assay method to quantify a drug candidate is an important step in

preformulation studies. UV spectroscopy is a simple and widely available analytical method.

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Figure 2. SEM images of peptides: (A) KTTKS and (B) Pal-KTTKS.

Hence, the UV absorbance ability of KTTKS and Pal-KTTKS was evaluated. The λmax
(wavelength of maximum) of KTTKS and Pal-KTTKS was found to be 193 and 195 nm,
respectively.

EVALUATION OF PARTICLE MORPHOLOGY BY SEM

Scanning electron microscope images of KTTKS and Pal-KTTKS are illustrated in Figure 2.
Generally, there is minimal difference between the morphology of KTTKS and Pal-KTTKS
particles. Both peptides showed irregular flake morphologies with a wide particle size
distribution.

EVALUATIONS OF BIREFRINGENCE UNDER POLARIZED LIGHT MICROSCOPY

Polarizing light microscopy images of KTTKS and Pal-KTTKS are presented in Figure 3.
As clearly seen, both peptides show birefringence under cross-polarized light, indicat-
ing their anisotropic structure. Therefore, it could be definitely said that KTTKS and
Pal-KTTKS are not amorphous and do not possess the isotropic cubic crystalline lattice
structure.

Figure 3. Polarizing light microscopy images of KTTKS (A) and Pal-KTTKS (B).

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 PREFORMULATION STUDIES OF PEPTIDES KTTKS AND PAL-KTTKS 305

STRUCTURE EVALUATION BY XRD

XRD was applied to understand the solid-state form of peptides, which is possibly the most
important state in developing a drug candidate into a drug product (15). Patterns of reflection
intensity versus 2θ are shown in Figure 4. As seen, the XRD pattern of palmitic acid
displayed an ordered structure. Peaks with the highest intensities are presented in Table I.
The XRD pattern of KTTKS shows two peaks in a wide range of Bragg’s angles: one broad
peak at 2θ = 19.5 with d-spacing (repeat distance) of 8.7 Å and another at 2θ = 10.1 with
d-spacing of 4.5 Å (Figure 4). After peptide modification with palmitic acid (Pal-KTTKS),
in addition to two peaks at 2θ = 10.6 (d-spacing = 8.3 Å) and 2θ = 19.1 (d-spacing = 4.6
Å), which are close to that of KTTKS, a peak at 2θ = 21.3 (d-spacing = 4.1 Å) appears.
In addition, three other peaks appear in the small angle area: a sharp peak at 2θ = 2.0 with
d-spacing of 44.2 Å, a peak at 2θ = 6.5 with d-spacing of 13.6 Å, and a peak at 2θ = 9.5
with d-spacing of 9.34 Å, indicating the presence of a long-range order in Pal-KTTKS.

Figure 4. XRD pattern of palmitic acid (A) and comparison of XRD patterns of KTTKS and Pal-KTTKS
(B) at ambient temperature.

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Table I
Summary of XRD Patterns of Palmitic Acid at Ambient Temperature

2θ (degree) Repeat distance (Å) Relative intensity (%)

2.6 34.6 6.2
7.5 11.7 17.5
20.5 4.3 5.9
21.7 4.1 100
24.3 3.7 18.6
30.3 2.9 4.4

PARTITIONING AND SOLUBILITY

Partition coefficient is a determining property which affects the permeation of a permeant

across the skin. The experimental logP value of KTTKS, obtained through the shake flask
method, was −1.6 ± 0.15.
We could not determine logP of Pal-KTTKS experimentally because this molecule ap-
parently self-aggregates in n-octanol–saturated water even at low concentrations (as low
as 25 µg/mL). The same problem occurred when n-hexadecane was used as the oily phase.
Lower concentrations were not used because of detection limitations. In such situations,
formulators can use different software, such as ACD/ChemSketch, to calculate logP. The
calculated logP (clogP) of Pal-KTTKS obtained here by this software was found to be
3.7 (11). Therefore, it could be concluded that the peptide conjugate Pal-KTTKS is
much more lipophilic than KTTKS (logP = −1.6) and might be a better candidate for
skin permeation than KTTKS. Such a compound might even get trapped in the lipid
domain of the stratum corneum. Such a lipophilic molecule is expected to be soluble in
lipophilic vehicles, such as ointments or oily phase of o/w and w/o emulsions and creams.
KTTKS aqueous solubility was estimated to exceed 220 mg/mL, which is considered as
freely soluble. However, we were unable to determine the solubility of Pal-KTTKS be-
cause of its complex behavior in aqueous systems possibly due to its self-aggregation and
assembly. Solubility estimation by ALOGPS revealed an aqueous solubility of 0.02 mM
(0.016 mg/mL) for Pal-KTTKS, in good agreement with CMC value obtained by the
ring method, as discussed later.

SURFACE TENSION MEASUREMENT OF PEPTIDE SOLUTIONS

Theoretical and experimental surface tensions of both peptides as well as deionized water
obtained by ACD-ChemSketch software and the ring method, respectively, are pro-
vided in Table II. The experimental surface tension of KTTKS aqueous solution was

Table II
Theoretical and Experimental Surface Tensions of Deionized Water, KTTKS, and Pal-KTTKS

Theoretical surface tension (mN/m) Experimental surface tension (mN/m) (n = 3)

Deionized water 72.2 ± 3 70.8 ± 0.05
KTTKS 63.1 ± 3 69.0 ± 2.7
Pal-KTTKS 50.1 ± 3 50.3 ± 0.4

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69.0 ± 2.7 mN/m, which is almost identical to the surface tension of water (70.8 ±
0.05 mN/m) (p > 0.05, independent sample t-test). For aqueous solution of Pal-KTTKS,
the surface tension was reduced significantly to 50.3 ± 0.4 mN/m (p < 0.05, independent
sample t-test), indicating that this peptide possesses surface activity. These data agree
with the surface tension obtained using software, which was 50.1 ± 3 mN/m.

DETERMINATION OF CMC

Because of surface activity, the CMC value of Pal-KTTKS aqueous solution was deter-
mined by the ring method and found to be 0.024 ± 0.004 mM; as will be discussed later,
such activity is expected based on the amphiphilic behavior of Pal-KTTKS.

THERMAL BEHAVIOR

Thermal behavior and stability of peptides are important issues during formulation, stor-
age, and permeation studies of these molecules. Hence, thermal behaviors of KTTKS and
Pal-KTTKS were investigated here. The results obtained from the melting apparatus
demonstrated that both peptides decomposed before melting. Their decomposition tem-
peratures were observed at 154 and 150°C for KTTKS and Pal-KTTKS, respectively.
The TGA and the first derivative TGA thermograms of KTTKS and Pal-KTTKS are
shown in Figure 5A and B and results summarized in Table III. There are three distinct
weight loss areas in TGA thermograms for both peptides. Based on the results of the
melting point apparatus, DSC (as explained in the following paragraph), and data ob-
tained from the TGA analysis, it could be said that the first weight loss area in the TGA
thermograms of KTTKS and Pal-KTTKS (25–150°C) could be probably attributed to
evaporation of water, as explained later. The second and third weight loss areas (150–250
and 250–595°C), where more weight loss has occurred, are presumably relevant to peptide

Figure 5. TGA thermograms and their first derivatives for KTTKS (A) and Pal-KTTKS (B) at a heating rate
of 10°C/min over 25–595°C (n = 3), and also heating–cooling–heating cycle TGA thermograms of KTTKS
(C) and Pal-KTTKS (D) at a heating rate of 5°C/min over 25–100°C.

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Table III
Percent Weight Loss of Peptides and the Temperature of Maximum
Weight Loss during TGA Study in the Range of 25–595°C

Weight loss (%) over three temperature ranges

Temperature of maximum
Peptide name 25–150°C 150–250°C 250–595°C weight loss (°C)
KTTKS 7.47 ± 0.84 45.34 ± 1.29 41.97 ± 1.03 233.92 ± 1.49
Pal-KTTKS 6.55 ± 0.99 33.61 ± 0.67 56.38 ± 0.67 229.26 ± 0.86

Data are represented as mean ± SD (n = 3).

decomposition. The temperature of maximum weight loss (230°C) obtained from the
first derivative TGA thermograms is almost similar for both peptides (see Table III).
DSC thermograms of KTTKS and Pal-KTTKS are illustrated in Figure 6A. As seen, both
peptides exhibit endothermic transitions with a peak of 59.8 ± 1.3°C (T1 KTTKS) for
KTTKS and 50.6 ± 4.4°C (T1 Pal-KTTKS) for Pal-KTTKS. Given the results obtained from
the melting point studies, which showed that peptides did not show any sign of melting,
these endothermic transitions could not be attributed to the melting of peptides and were
assumed here to be water-related intrinsic structural rearrangement, polymorphism, or
evaporation, as discussed later. KTTKS has another endothermic peak with low enthalpy
(T2 KTTKS) (see Figure 6A). A third endothermic transition (T3 KTTKS) starts at 152.1 ±
3.9°C in the thermogram of KTTKS. This endotherm is in good agreement with the
result of decomposition obtained from melting point studies (154°C). For Pal-KTTKS,
the second endothermic transition (T2 Pal-KTTKS), starting at 142.7 ± 0.9°C, could also be
attributed to the first decomposition of the peptide conjugate as observed in melting
point studies (150°C). Visual observations of the DSC pan after removal revealed sever
changes in the peptide appearance (change in color and texture). These changes might be
considered as signs of peptide decomposition.
To ascertain the nature of T1 KTTKS and T1 Pal-KTTKS, cycled DSC was performed. Cycled
DSC experiment indicated that T1 KTTKS and T1 Pal-KTTKS disappeared in the second heat-
ing run, whereas T2 KTTKS, T3 KTTKS, and T2 Pal-KTTKS remained unchanged (see Figure
6B). These results might show that T1 KTTKS and T1 Pal-KTTKS are related to water evapora-
tion from peptides, as discussed later.
To ensure the relationship of T1 KTTKS and T1 Pal-KTTKS to water evaporation, a cycled
TGA experiment was performed here. As shown in Figure 5C and D, TGA weight losses
for the first heating runs are 5.9% and 3.3% for KTTKS and Pal-KTTKS, respectively.

Figure 6. (A) Sample DSC thermograms of KTTKS and Pal-KTTKS at a heating rate of 10°C/min over
25–165°C and (B) DSC thermograms of preheated–cooled KTTKS and Pal-KTTKS samples at a heating rate
of 10°C/min over 25–165°C (see text for more explanation).

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In the second heating runs, weight losses were less than 1% for both peptides. After both
heating processes in TGA, the pans containing peptides were inspected visually; there
were no macroscopic changes in the appearance of peptide powders. In agreement with
the present results, some researchers (16) showed that a DSC transition of around 63.1°C
in a lyophilized synthetic trapezoid-inspired peptide fragment disappeared in the second
DSC heating run. It was concluded by investigators to be due to the presence of bound
water and subsequent water loss in the second run.

STABILITY STUDIES

Ensuring the stability of permeants during the skin permeation experiment is crucial.
The concentration of KTTKS solution was 100 µg/mL at the beginning of the stability
study. After 48 h, the concentration of KTTKS solution reached 99.3 ± 1.7 µg/mL. Thus,
it could be concluded that KTTKS remains stable at 32°C at least for 48 h. Therefore,
there is no concern about the instability of this permeant during the skin permeation
studies for at least 48 h.

DISCUSSION
KTTKS and its derivative are popular in the cosmetic industry because of their anti-
wrinkle properties, but many of their physicochemical properties remain unknown. This
study is an attempt to define the physicochemical properties of these peptides and to in-
vestigate the effects of covalent attachment of a fatty acid on peptide properties.
In the case of UV absorbance, λmax of both peptides was at less than 200 nm. KTTKS and
Pal-KTTKS have no aromatic amino acids in their structure, and the UV absorbance of
them is due to the peptide bonds. The photons are absorbed by peptide bond at the
maximum wavelength of below 210 nm (17). Note that the wavelengths below 200 nm
belong to vacuum UV (18). The UV radiation is forcefully absorbed by atmospheric oxygen
in the vacuum UV range (19). Therefore, a free oxygen environment is required to accu-
rately assay KTTKS and Pal-KTTKS if UV spectroscopy at the maximum wavelength is
to be used. Because KTTKS and Pal-KTTKS possess a broad absorption peak, it might
be said that it is possible to measure the absorption at the longer wavelength, e.g., at 205 nm,
but we noted that at this wavelength, the UV radiation is absorbed by many compounds
used in buffers (20), which are mainly applied in skin permeation studies.
In X-ray studies, both peptides showed a broad peak (hump) at around 2θ = 19° (see
Figure 4), which was absent in palmitic acid. Such a broad peak at a wide angle region,
which has also been reported for other peptides (21), might indicate that these peptides
contain either some amorphous structures or some disordered crystalline structures. Both
peptides also show peaks at 2θ  10 that indicate some long-range orders. The presence
of some degrees of orders in the structure was also confirmed by showing birefringence in
the cross-polarized light. The presence of new peaks in the XRD pattern of Pal-KTTKS,
which is not present in the pattern of KTTKS, means that the structures of these peptides
are not the same; therefore, some possible changes in the physicochemical properties of
peptides, such as solubility, dissolution rate, and flowability, are expected and should be
considered by formulators.

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Palmitic acid molecules form dimers because of OH….O hydrogen bonds and these di-
mers pack into bilayers (22,23). Palmitic acid has a small polar head group (–COOH).
Changing the head group to bulky KTTKS probably imposes some degree of disorder
into the structure. Besides this, the molecules become longer with higher d-spacing: 34.6 Å
for palmitic acid versus 44.2 Å for Pal-KTTKS. Pal-KTTKS also shows a higher
d-spacing than KTTKS that are definitely due to the presence of long chain (C16) in the
structure.
Based on the results of partitioning and solubility tests, KTTKS has a highly hydrophilic
nature (logP < 0 and high water solubility). Because higher lipophilicity is required for
good permeation through the skin (logP 0–3), KTTKS, like many other peptides such as
Ala–Ala–Pro–Val (24), tetragastrin (25), and TRH (26), is not a good candidate for skin
delivery. The results of the in vitro skin permeation study of KTTKS, which showed
KTTKS did not permeate across the skin (27), is in good agreement with the present
finding.
KTTKS is a pentapeptide without a hydrophobic tail, but Pal-KTTKS has a 16-carbon
chain (palmitic acid) as the hydrophobic tail, so Pal-KTTKS is a peptide amphiphile. The
peptide amphiphiles are capable of forming a diversity of aggregates, such as micelles,
cylindrical fibrils, sheets, and vesicles (28). Here, the results of tensiometry indicate that
Pal-KTTKS has surface activity and reduces the surface tension of water to 50.3 ± 0.4 mN/m.
The CMC of Pal-KTTKS in water was also determined by the ring method and found to
be 0.024 ± 0.004 mM. There are no reports on the CMC value of Pal-KTTKS in aqueous
solutions using the ring method. This CMC value resembles polysorbate 80 (0.02–0.03 mM)
(29), which is structurally similar to Pal-KTTKS. The CMC value could be considered as
the solubility of the individual molecules with surface activity because above this concen-
tration, the molecules are not dissolved in the monomeric forms and aggregate as micelles
(30). As said, this value for Pal-KTTKS was 0.024 ± 0.004 mM or 19.25 ± 2.9 mg/L;
thus, considering only individual molecules, it could be said this peptide amphiphile is
practically insoluble in water.
Cycled DSC and TGA results show that both peptide powders are hygroscopic. The hy-
groscopic nature of some peptides, especially when present as porous lyophilized pow-
ders, is a well-known property. On the other hand, it has been argued that peptides
containing charged amino acids (such as arginine, aspartic acid, glutamic acid, histidine,
and lysine) are hygroscopic (31). KTTKS and its derivative have two lysine amino acids,
and therefore, it is possible that they absorb water. Hence, two water-related transitions
(T1 KTTKS and T1 Pal-KTTKS) in their DSC thermograms disappear on reheating, which is in
agreement with weight losses observed in TGA thermograms.
Finally, as far as formulation is concerned, techniques such as UV, XRD, polarized light
microscopy, SEM, and thermal analysis can be used to monitor changes during pharmaceu-
tical processing, stability studies, and storage by the formulator. Some of these techniques,
e.g., DSC, can be used to determine drug–excipients interactions. During pharmaceutical
processing and storage, a formulator should be aware of the polymorphism and the pos-
sibility of change of a polymorphic form into another due to processing (such as mixing
and heating) and storage. For example, if a formulator uses these substances in the sus-
pension dosage form (the presence of solid particles in the formulation), the stability
during storage can be monitored using XRD or polarized light microscopy, as described
earlier. The same applications apply to the SEM morphologies. SEM and polarized light

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 PREFORMULATION STUDIES OF PEPTIDES KTTKS AND PAL-KTTKS 311

represent crystal habits as well. In this regard, some habits such as needle-likes might
provide some degrees of discomfort in topical applications and should be considered in
the formulation and stability studies by the formulator.

CONCLUSION
Here, KTTKS and Pal-KTTKS were synthesized and the physicochemical properties of both
peptides and, therefore, the effects of covalent attachment of palmitic acid on KTTKS
properties were investigated. In short, these peptides showed birefringence, irregular
morphology, wide particle size distribution, and no melting point before decomposition.
In addition, KTTKS was very hydrophilic in nature, and its aqueous solution remains
stable at 32°C for over 48 h. In terms of internal structure, Pal-KTTKS appeared more
ordered than KTTKS. Micelle formation of Pal-KTTKS in water occurs at low concentra-
tions. The results show that fatty acid attachment to KTTKS causes some changes in
chemical properties (such as solubility and partition coefficient), whereas physical properties
(such as morphology, thermal behavior, and birefringence) are not affected so much. These
results can be used for formulation of these peptides for topical delivery or any other drug
delivery system development. The effects of palmitic acid conjugation on KTTKS prop-
erties might be used for the preparation of palmitic acid derivatives on other peptides.

ACKNOWLEDGMENTS
This paper is a part of PhD thesis of Seyedeh Maryam Mortazavi at the School of Pharmacy,
Shahid Beheshti University of Medical Sciences (SBMU), Tehran, Iran and was financially
supported by SBMU.

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 J. Cosmet. Sci., 70, 313–325 (November/December 2019)

Utilization of Coffee Silverskin By-Product from Coffee

Roasting Industry through Extraction Process for the
Development of Antioxidant Skin Gel

SAMUEL P. KUSUMOCAHYO, PATRICK TANGGUH,

CHRISTINA D. ANNELIES, and HERY SUTANTO, Department of
Chemical Engineering, Faculty of Life Sciences & Technology, Swiss German
University, Tangerang 15143, Indonesia (S.P.K., P.T., C.D.A., H.S.)

Accepted for publication October 4, 2019.

Synopsis
Coffee roasting industries generate a by-product called coffee silverskin that is usually disposed of as waste.
The valorization of this abundant waste is necessary because of the antioxidant compounds in coffee silverskin.
In this study, coffee silverskin was extracted in different extraction conditions to obtain an extract with high
antioxidant activity and to use it as an additive for antioxidant skin gel. The extracts were characterized for
the total phenolic content by using the Folin–Ciocalteu method. The antioxidant activity was determined by
using the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay. It was found that the extraction time
and temperature strongly affected the total phenolic content and the antioxidant activity of the extracts. The
extraction at 40°C and 60 min resulted in an extract with a high total phenolic content of 31.15 ± 2.77 mg
Gallic Acid Equivalent/g coffee silverskin and a high antioxidant activity of 68.44 ± 0.76%. The extract
solution was spray-dried to produce extract powder, which was then added to a basic skin gel with different
extract concentrations. It was observed that the antioxidant activity of the gel increased with increasing
extract concentration in the gel. This result showed that coffee silverskin has great potential as a source of
antioxidants for various skin care products.

INTRODUCTION
Antioxidants are very important for the human body because they protect the human body
against reactive oxygen species, known as free radicals, which can cause oxidative reac-
tions in human cells. Although the human body has an internal defense system toward
free radicals (1), it still requires the intake of antioxidants from outside that usually can
be obtained from synthetic or natural products, such as fruits and vegetables, containing
antioxidants (2). Recently, the interest in natural products for healthy foods, beverages,
supplements, and health care products has been rising along with the increasing aware-
ness of a healthy life style (3–5).

Address all correspondence to Samuel P. Kusumocahyo at [email protected].

313
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 314 JOURNAL OF COSMETIC SCIENCE

One of the most popular and valuable natural products is coffee. Because of the high
consumption of coffee as a beverage, the coffee industry is categorized as one of the
largest industries in the world (6). Besides its good taste, coffee is beneficial for human
health because many studies have shown that coffee contains phenolic compounds such
as chlorogenic acids, which show antioxidant activity (7,8). The coffee industries also
produce a large number of by-products, such as coffee spent grounds and coffee silver-
skin (9). Coffee silverskin is produced as a by-product during the roasting process of
coffee beans. It is a thin layer surrounding the coffee bean, and it is usually still at-
tached to the coffee bean even after the depulping and dehulling process of the coffee
cherries. When the coffee beans are heated at high temperature during the roasting
process, the coffee beans will crack because of loss of moisture content, resulting in the
detachment of the coffee silverskin from the beans. Because of its lightness, the coffee
silverskin escapes from the roasting container through the roaster exhaust or cyclone,
and is usually disposed of as waste. However, recent studies on the revalorization of
coffee silverskin reported that coffee silverskin contains phenolic compounds and
shows high antioxidant activity (10–12). Bresciani et al. (13) reported that chlorogenic
acids are the sole phenolic compounds in coffee silverskin. The studies on the extrac-
tion of coffee silverskin mostly used a mixture of Robusta and Arabica coffee silverskin
because the roasting industry usually blends these two types of coffee. However, our
recent study showed that Robusta coffee silverskin showed a higher phenolic content
and, thus, a higher antioxidant activity than Arabica coffee silverskin (14). This re-
sult is in accordance with the study conducted by Farah et al. (8) who reported that
Robusta coffee beans contain a higher amount of chlorogenic acid than Arabica coffee
beans.
Rodrigues et al. (15) reported that coffee silverskin was an effective ingredient in the
improvement of skin hydration and firmness. De Hond et al. (16) studied the use of coffee
silverskin extract to protect accelerated skin aging. In spite of the high antioxidant activ-
ity of coffee silverskin and its great potential for industrial application, to the best of our
knowledge, there is no study on the development of skin care products containing coffee
silverskin extract as an antioxidant. Moreover, a proper extraction method and the opti-
mization of the extraction condition to obtain an extract of coffee silverskin with a high
phenolic content and a high antioxidant activity are not studied yet. Furthermore, the
extraction of coffee silverskin was usually conducted by maceration process, which means
the raw material is immersed in a solvent without any mechanical agitation. This conven-
tional method takes a long time to extract the phenolic compounds, and results in a low
phenolic content.
In this work, coffee silverskin of Robusta type was extracted using a hydroalcoholic
solvent with the aid of mechanical agitation at various extraction times and tempera-
tures with the purpose to maximize the phenolic content and the antioxidant activity
of the coffee silverskin extract. The extract solution was then dried using a spray
dryer to produce coffee silverskin extract in powder form. The study on the effect of
drying of the extract is important because extracts are usually produced in powder
form. Furthermore, to develop an antioxidant skin care product, a basic skin gel was
prepared and the coffee silverskin extract powder was added as an active ingredient in
the skin gel. The total phenolic content and the antioxidant activity of the skin gel
were measured to study the effect of the addition of the coffee silverskin extract to the
skin gel.

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 DEVELOPMENT OF ANTIOXIDANT SKIN GEL USING COFFEE SILVERSKIN 315

MATERIALS AND METHODS

MATERIALS

Robusta coffee silverskin was obtained from a coffee roasting company located in Bandar
Lampung, Indonesia. Technical grade ethanol (96% v/v) was purchased from PT Sumber
Abadi (Tangerang, Indonesia). Sodium carbonate (BDH, London, England), Folin–
Ciocalteu reagent (Merck, Darmstadt, Germany), gallic acid (GA) powder (Aktin Chemical,
Chengdu, China), aluminum chloride (Merck), potassium acetate (BDH), 2,2-diphenyl-
1-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO), and analytical grade ethanol
(Smart Lab, Bogor, Indonesia) were used. Chemicals to prepare the basic skin gel were
carbomer 940, propylene glycol, methylparaben, and propyl paraben, all purchased from
PT Intralab Ekatama (Bogor, Indonesia), whereas triethanolamine (TEA) and ethylene
diamine tetra acetic acid (EDTA) were purchased from PT Sumber Abadi. Distilled water
was used.

EQUIPMENT

Standard laboratory equipment was used in this work, namely, hot plate (Cimarec, Waltham,
MA), beaker glasses, Erlenmeyer flasks, magnetic stirrer, thermometer, volumetric glass
(Pyrex, Corning, NY), water bath shaker (Memmert, Schwabach, Germany), micropipette
(Eppendorf, Hamburg, Germany), micropipette tips, cuvette (Brand GMBH, Wertheim,
Germany), rotary vacuum evaporator (IKA HB 10, Shanghai, China), spray dryer (BUCHI
mini spray dryer B-290, Flawil, Switzerland), analytical balance (Ohaus PA214, Parsip-
pany, NJ), UV-Vis spectrophotometer (Genesys 10-S, Waltham, MA), vortex (Vortex-
Genie 2, St. Louis, MO), filter instrument, Whatmann filter paper 1001 125 (GE, Little
Chalfont, Buckinghamshire, UK), desiccator, moisture content analyzer (Sartorius MA35,
Goettingen, Germany), mixer (IKA Labortechnik, Staufen, Germany), pH meter (Lutron
pH-208, Taipei, Taiwan), and viscometer (Brookfield DV-E, Lorch, Germany).

EXTRACTION PROCEDURE

First, the coffee silverskin sample was rinsed with water and dried in an oven at 40°C. The
dried sample was milled using a mixer grinder to reduce the size. Then, 100 mL of water–
ethanol mixture with a weight ratio of 50:50 was poured into a glass beaker and heated using
a hot plate. The temperature was varied at 30, 40, 50, and 60°C. When the desired tem-
perature of the solvent was reached, 2 g of coffee silverskin were poured into the solvent, and
the extraction process was carried out by stirring using a magnetic stirrer at a speed of
350 rpm. Thus, the weight ratio of the coffee silverskin and water–ethanol solvent was 1:50.
The beaker was covered with aluminum foil to prevent loss of solvent because of evapora-
tion during heating. The extraction time was varied at 5, 10, 20, 30, 40, and 60 min. The
extract solution was then filtered using a filter paper and stored in a refrigerator before analysis.

ANALYSIS OF TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY

The total phenolic content was determined by using the Folin–Ciocalteu method. First,
the Folin–Ciocalteu reagent solution was made by diluting concentrated Folin–Ciocalteu

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 316 JOURNAL OF COSMETIC SCIENCE

reagent in distilled water at a ratio of 1:10. Sodium carbonate solution (7.5% w/v) was
made by diluting 7.5 g of solid sodium carbonate with 100 mL of distilled water. For the
standard curve of GA, a solution of GA was prepared by dissolving 0.1 g of solid GA in
100 mL of distilled water to obtain 1,000 mg/L of GA stock solution. Then the standard
solutions of GA were made by diluting the GA solution into concentrations of 10, 20, 50,
70, 100, 250, and 500 mg/L.
Furthermore, the coffee silverskin extract solution was diluted with a weight ratio of
1:10, and 500 µL of the diluted extract solution was mixed with 2.5 mL of the Folin–
Ciocalteu reagent solution and 2 mL of the sodium carbonate solution. The mixture was
then mixed using a vortex mixer and incubated in the dark at room temperature for 1 h.
After the incubation, the mixture was poured into a cuvette and directly checked by using
a UV-Vis spectrophotometer at 765 nm to measure the absorbance. The total phenol
content (TPC) was then calculated by using the following equation:

AbsqDF
Total Phenolic Content (mg GAE/L)  , (1)
m

where Abs is the absorbance, m is the gradient of the GA standard curve, and DF is the
dilution factor. The total phenolic content was expressed in mg of GA equivalent per liter
extract solution [mg Gallic Acid Equivalent (GAE)/L] and then converted to mg GAE/g
dry coffee silverskin (mg GAE/g CS).
To determine the antioxidant activity of the extract, the extract samples were tested using
the DPPH free radical scavenging assay. A stock solution of DPPH (250 µM) was pre-
pared by diluting 11 mg of DPPH powder in 20 mL ethanol (96% v/v). The stock solu-
tion was covered with aluminum foil and stored at a temperature of 4°C. Next, 100 µL
DPPH, 50 µL sample, and 850 µL ethanol were mixed in a test tube. For the control, 100
µL DPPH, 50 µL distilled water, and 850 µL ethanol were mixed in a test tube. Further-
more, both test tubes were wrapped with aluminum foil and stored in a dark room for 30
min. The absorbance reading was performed using a UV-Vis spectrophotometer at a
wavelength of 515 nm. The antioxidant activity was expressed as an inhibition percent-
age and calculated by using the following equation:

( Ac  As )
Antioxidant Activity %  q100%, (2)
Ac

where Ac is the absorbance of the control and As is the absorbance of the sample. The
antioxidant activity (the free radical scavenging activity) obtained by this method was
expressed as IC50 (in ppm), which means the concentration of the sample needed to in-
hibit 50% of the DPPH as the free radical.

STATISTICAL ANALYSIS

Statistical analysis of the data obtained in this work was performed by using analysis of
variance and Tukey test. The difference among data of different samples was considered
as significant if the probability was less than 0.05 (p < 0.05).

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 DEVELOPMENT OF ANTIOXIDANT SKIN GEL USING COFFEE SILVERSKIN 317

DRYING OF COFFEE SILVERSKIN EXTRACT

To produce coffee silverskin extract in powder form, the coffee silverskin extract solution
was dried using a spray dryer with an inlet temperature of 175°C and an outlet tempera-
ture of 125°C with a feed flow of 16.7 mL/min. The coffee silverskin extract powder was
then analyzed for its total phenolic content and antioxidant activity using the method as
previously described.

PREPARATION OF COFFEE SILVERSKIN SKIN GEL

A basic gel was prepared by using the cold mechanical method as described by Schmolka
(17) with some modifications. The composition of the ingredients for the basic skin gel is
shown in Table I (18).
First, Carbomer 940 was dissolved in distilled water in a glass beaker and stirred using a
mixer to obtain a gel. The mixing process was carried out at room temperature at 600 rpm
for 20 min. To reduce the bubble, the gel was kept for 24 h at room temperature. TEA
was then added to the gel and mixed. Methylparaben and propylparaben were dissolved in
propylene glycol, whereas EDTA was dissolved in distilled water by stirring. Then, these
solutions were added into the gel and stirred until a homogeneous mixture was attained.
Finally, the coffee silverskin extract powder which was already dissolved in distilled water,
was added into the gel, and stirred at room temperature for 20 min. The concentration of
the coffee silverskin extract powder in the skin gel varied at 0.125%, 0.25%, 0.5%, and
1%. The total phenolic content of the skin gel was analyzed using the method described
previously. The antioxidant activity of the skin gel was also analyzed and expressed as
IC50 value using the method described previously. The pH of the gel was measured using
a pH meter, whereas the viscosity was measured using a viscometer using spindle number
6 at a speed of 3 rpm. All measurements were carried out at room temperature.

RESULTS AND DISCUSSION

TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF COFFEE SILVERSKIN EXTRACT SOLUTION

The coffee silverskin sample was extracted using water–ethanol (50:50 w/w) as solvent at
different extraction times and temperatures. Water–ethanol mixture with a weight ratio

Table I
Composition for the Basic Skin Gel

Ingredient Composition (g/100 g water)

Carbomer 940 1
TEA 1
Propylene glycol 15
Methylparaben 0.15
Propylparaben 0.05
EDTA 0.05
Ethanol (96% v/v) 5
Perfume 0.4
Distilled water 100

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 318 JOURNAL OF COSMETIC SCIENCE

of 50:50 was chosen as the solvent because of the intermediate polarity of the phenolic
compounds Ballesteros et al. (12). The extract solutions were then analyzed for their total
phenolic content and antioxidant activity. Figures 1 and 2 show the effect of extraction
time on the total phenolic content and the antioxidant activity of the coffee silverskin
extract solution. The extraction was carried out at a constant extraction temperature of
40°C. As shown in Figure 1, a longer extraction time resulted in a higher amount of total
phenolic content of the coffee silverskin extract solution. Because phenolic compounds
are known to have antioxidant property, the antioxidant activity increased with increasing
extraction time, as shown in Figure 2. The extraction time of 60 min showed the highest
total phenolic content and antioxidant activity compared with the other extraction times.
To study the effect of extraction temperature, the extraction experiment was carried out
at different extraction temperatures at a constant extraction time of 60 min. Figures 3
and 4 show the effect of extraction temperature on the total phenolic content and the
antioxidant activity of the coffee silverskin extract solution. When the extraction tem-
perature was elevated from 30°C to 40°C, an increase in the total phenolic content was
observed. However, a further increase in temperature above 40°C did not result in a sig-
nificant increase in the total phenolic content as shown in Figure 3 (p > 0.05). A similar
phenomenon was also observed for the antioxidant activity as shown in Figure 4. An increase

Figure 1. Effect of extraction time on the total phenolic content of the coffee silverskin extract solution
(extraction temperature: 40°C).

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 DEVELOPMENT OF ANTIOXIDANT SKIN GEL USING COFFEE SILVERSKIN 319

Figure 2. Effect of extraction time on the antioxidant activity of the coffee silverskin extract solution (ex-
traction temperature: 40°C).

in the extraction temperature above 40°C did not result in a significant increase in the
antioxidant activity of the extract (p > 0.05). This result indicated that all phenolic com-
pounds were completely extracted from the coffee silverskin at an extraction temperature
of 40°C and an extraction time of 60 min. This is very interesting because an extraction
temperature of 40°C is appropriate for the industrial scale extraction process because of
the lower energy consumption for heating.

COFFEE SILVERSKIN EXTRACT POWDER

Based on the aforementioned results, coffee silverskin extract in powder form was prepared
by drying the coffee silverskin extract solution using a spray dryer. First, the extract solution
was prepared by extracting the coffee silverskin at 40°C for 60 min because this extrac-
tion condition resulted in a high total phenolic content and a high antioxidant activity.
Then, the filtered extract solution was dried using a spray dryer at an inlet temperature
of 170°C and an outlet temperature of 125°C. Figure 5 shows the picture of the extract
solution before drying and the extract powder after drying.
The measurement of the moisture content showed that the dried coffee silverskin extract
powder had a moisture content of 5.6%. The analysis of the total phenolic content using
the Folin–Ciocalteu method with GA as a standard showed that the coffee silverskin

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Figure 3. Effect of extraction temperature on the total phenolic content of the coffee silverskin extract solu-
tion (extraction time: 60 min).

extract powder had a total phenolic content of 188.83 mg GAE/g solid extract, which is
slightly lower than that of the extract solution before drying of 210.27 mg GAE/g solid
extract, as shown in Table II. The decrease in the total phenolic content after drying occurred
because the extract was treated at a high temperature of 170°C during the spray drying
process. The heating of the extract degraded some phenolic compounds, resulting in a
lower phenolic content of the dried extract. A similar phenomenon was also reported by
Orphanides et al. (19) who studied the effect of drying on the phenolic content and anti-
oxidant capacity of spearmint. The effect of drying on the antioxidant activity can be seen
in Table II. The antioxidant activity of the extract is expressed as IC50 value, which means
the concentration of the sample needed to inhibit 50% of DPPH as the free radical. Thus,
a low value of IC50 corresponds to a high antioxidant activity. As can be seen, the IC50
value of the extract powder was 433.64 ppm, higher than that of the extract solution of
358.15 ppm. This result is in accordance with the decrease in the total phenolic content
after the drying process as described earlier.

SKIN GEL CONTAINING COFFEE SILVERSKIN EXTRACT

The basic skin gel was prepared using the formulation and the method described previ-
ously. The coffee silverskin extract powder was then added to the basic skin gel and mixed

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Figure 4. Effect of extraction temperature on the antioxidant activity of the coffee silverskin extract solution
(extraction time: 60 min).

together at room temperature to obtain a homogeneous gel. The concentration of the

coffee silverskin extract powder in the skin gel varied at 0.125%, 0.25%, 0.5%, and 1%.
Figure 6 shows the physical appearance of the skin gels with different extract concentrations.

Figure 5. Coffee silverskin extract solution and powder form.

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Table II
Total Phenolic Content and Antioxidant Activity of Coffee Silverskin Extract before and after Drying
Coffee silverskin extract Coffee silverskin extract
solution (before drying) powder (after drying)
Total phenolic content 210.87 188.83
(mg GAE/g solid extract)
IC50 (ppm) 358.15 433.64

The color of the skin gel became darker when more extract powder was added to the gel.
The gel viscosity ranges between 98,300 and 109,500 cps, with pH ranges between 6.5
and 7.0.
Figure 7 shows the total phenolic content of the skin gels with various extract concen-
trations. As can be seen, the total phenolic content in the skin gel increased by adding
more coffee silverskin extract to the skin gel. The addition of the extract of more than
1% is still possible to obtain a skin gel with higher total phenolic content, but because
the color of the skin gel will become darker, the color acceptance by the users should
be considered.
To study the effect of the addition of the coffee silverskin extract on the antioxidant activ-
ity of the skin gel, an analysis using DPPH free radical scavenging assay was conducted.
It was observed that the basic skin gel that contains no coffee silverskin extract exhibited
a DPPH inhibition percentage of only 3% because of the oxidation of the bulk skin gel
by the DPPH. Interestingly, the skin gel containing 0.125% coffee silverskin extract
exhibited a DPPH inhibition percentage of 26.3%, indicating that the coffee silverskin
extract was effective as a source of antioxidants in the skin gel. Further, Figure 8 shows
the effect of the addition of the coffee silverskin extract on the antioxidant activity

Figure 6. Physical appearance of skin gels containing coffee silverskin extract at different concentrations
(left to right: F1: 0.125%, F2: 0.25%, F3: 0.5%, F4: 1%).

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Figure 7. Total phenolic content of skin gels with various concentrations of coffee silverskin extract (F1:
0.125%, F2: 0.25%, F3: 0.5%, F4: 1%).

expressed by the IC50 value of the skin gel. The IC50 value indicates the concentration of
the antioxidant, which is necessary to inhibit 50% of the DPPH as the free radical. The
IC50 is given in ppm, which means milligram skin gel per liter solution. As can be seen
in Figure 8, the addition of more coffee silverskin extract reduced the IC50 value, which
means it resulted in a skin gel with a higher antioxidant activity. The increase in the
antioxidant activity of the skin gel is correlated with the increase in the total phenolic
content of the skin gel due to the addition of the coffee silverskin extract. The statistical
analysis showed that there is a significant difference of IC50 with the variation of the
extract concentration (p < 0.05). This result indicates that the coffee silverskin extract has
great potential to be used as an additive for skin gels to obtain antioxidant-rich skin gel
products.
The pH value of a topical preparation should be within the skin pH range between 4.0
and 7.0 (20). The value of the pH should not be too acidic as it causes skin irritation and
should not be too alkaline as it may cause scaly skin. The skin gels containing coffee sil-
verskin extract showed pH values of 7.0, 6.63, 6.50, and 6.50, for the extract concentrations
of 0.125%, 0.25%, 0.5%, and 1%, respectively. The addition of more coffee silverskin
extract into the skin gel resulted in a slight decrease in the pH value. This is reasonable
because the phenolic compounds of the coffee silverskin extract are mostly chlorogenic
acids, which lead to an acidic condition of the gels. However, the pH values of the skin
gels are all still within the pH range for topical preparations.

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Figure 8. IC50 of skin gels with various concentrations of coffee silverskin extract (F1: 0.125%, F2: 0.25%,
F3: 0.5%, F4: 1%).

CONCLUSION
This study demonstrated the utilization of coffee silverskin through an optimized
extraction process to produce an extract with high total phenolic content and high
antioxidant activity. It was observed that the extraction time and temperature strongly
affected the total phenolic content and the antioxidant activity of the coffee silverskin
extract. The extraction at 40°C and 60 min resulted in an extract with a high total
phenolic content of 31.15 ± 2.77 mg GAE/g coffee silverskin and a high antioxidant
activity of 68.44 ± 0.76%. It was found that the addition of the coffee silverskin extract
powder to a basic gel resulted in a gel having antioxidant property. The antioxidant
activity of the gel increased with increasing coffee silverskin extract concentration in
the gel. The result of this study showed that coffee silverskin has great potential to be
used as a source of antioxidant and the extract can be applied as an additive for various
skin care products.

ACKNOWLEDGMENT
This work has been financially supported by the Ministry of Research, Technology and
Higher Education of the Republic of Indonesia through the Penelitian Terapan Unggulan
Perguruan Tinggi research grant program 2018.

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