J Surfact Deterg (2019)

DOI 10.1002/jsde.12315

ORIGINAL ARTICLE

In Vitro and In Vivo Removal Efficacies of a Formulated

Pumpkin Seed Oil Makeup Remover
Anussara Setsiripakdee1 · Nattaya Lourith1,2 · Mayuree Kanlayavattanakul1,2

Received: 17 October 2018 / Revised: 28 March 2019 / Accepted: 29 May 2019

© 2019 AOCS

Abstract Pumpkin seed oil is well known for its health bene-
fits due to its high content of fatty acid constituents and tocoph-
erols. The removal efficacy of pumpkin seed oil was assessed
using UV–Vis spectroscopy. The oil was able to remove
79.92  0.07%, 41.02  0.25%, and 23.54  0.19% of foun-
dation and liquid and pen eyeliners. A stable makeup remover
was formulated using 5–15% pumpkin seed oil. Addition
of pumpkin seed oil significantly (P < 0.001) enhanced
removal ability of the base remover. The remover containing
5% pumpkin seed oil was able to remove 89.27  0.02%,
67.72  0.08%, and 41.25  0.07% of foundation, liquid,
and pen eyeliners, respectively, while those of the remover
containing 10% pumpkin seed oil were 78.24  0.02%,
66.88  0.05%, and 38.43  0.05%, and those of the remover
containing 15% pumpkin seed oil were 84.41  0.01%,
69.79  0.12%, and 41.88  0.04%, respectively. On the
other hand, removal efficiencies of the benchmark were
91.20  0.03%, 73.46  0.10%, and 54.00  0.07%, respec-
tively. The removers containing pumpkin seed oil did not cause
skin irritation as monitored by a single closed-patch test in
10 female volunteers. The remover containing 5% pumpkin
seed oil was further preference studied in 25 female volunteers
in a comparison with the benchmark. The pumpkin seed oil
remover gained a better overall preference over the benchmark
(82.29  4.17% and 80.20  8.64%; P = 0.287). Of which,
skin hydration of the developed bio-oil remover was signifi-
cantly (P < 0.001) satisfied.
* Nattaya Lourith
nattayal@mfu.ac.th
1
School of Cosmetic Science, Mae Fah Luang University, Chiang
Rai, 57100, Thailand
2
Phytocosmetics and Cosmeceuticals Research Group, Mae Fah
Luang University, Chiang Rai, 57100, Thailand
Keywords Pumpkin seed oil
Makeup remover
UV–Vis
spectroscopic analysis
Removal efficacy
Preference test
J Surfact Deterg (2019).
Introduction
Nowadays, facial makeup products are commonly used by
women and men to improve social attractiveness and
strengthen self-confidence. Makeup products are expected to
have a longer duration of wearability by consumers. Accord-
ingly, water resistance makeup products are highly in demand
despite the fact that they are harder to be removed by the com-
mon cleansing products. Makeup cleansers or removers are,
therefore, developed, and supplied into cosmetic market, of
which the efficient makeup removers containing bio-oil are
receiving high attention of the consumers (Lourith and
Kanlayavattanakul, 2014).
Pumpkin seed oil is well known for its health benefits due to
its high content of fatty acid constituents (98%) in addition to
its tocopherols (Rezig et al., 2012; Stevenson et al., 2007).
Pumpkin seed oil is, therefore, accepted as an important vegeta-
ble oil in cosmetics due to its high palmitic, stearic, oleic, and
linoleic acid contents (10–16%, 3–7%, 18–38%, and 40–62%).
Pumpkin seed oil is comparable to olive oil (7.5–20%,
0.5–3.5%, 53–86%, and 3.5–20%) and grape seed oil (7–9.5%,
3.5–5.5%, 14–44%, and 46–74%) in terms of unsaturated fatty
acids (Cosmetic Ingredient Review, 2011). These phytochemi-
cals also play an important role in cosmetic formulation
(Alvarex and Rodríguez, 2000; Kanlayavattanakul and Lourith,
2011). Nonetheless, the benefits of pumpkin seed oil in terms
of its makeup removal ability as a formulated makeup remover
have never been presented.

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Thus, pumpkin seed oil was investigated for its in vitro
makeup removal efficacy by means of UV–Vis spectroscopic
analysis, which is routinely used in quality control processes
of cosmetic industry. Furthermore, the validated analytical
method for in vitro makeup removal efficacy evaluation was
practically approved for the industrial practice (Charoennit
and Lourith, 2012; Lourith and Kanlayavattanakul, 2014).
Thereafter, makeup removers containing pumpkin seed oil in
the form of a 2 layers emulsion remover were developed. Sta-
bility and makeup removal ability were in vitro assessed in
line with the quality control practice applicable for the indus-
try. Safety was examined in human volunteers by a single
application closed-patch test, and preference of the developed
makeup remover was studied consequently in a comparison
with the benchmark. The safe and efficient innovative makeup
remover containing bio-oil, which meets the cosmetic con-
sumers’ satisfaction, is, therefore, proposed in this context.
Experimental Section
Materials
Analytical grade 95% ethanol was purchased from Labscan,
Philadelphia, Ireland. Cotton sheets were from Canton Medi-
care (Thailand). For cosmetic formulations, PEG-7 glyceryl
cocoate, sorbitan oleate, sorbeth-30 tetraoleate (Nikkol,
Japan), ethylhexyl palmitate (Inolex, USA), water, glycerin
(Namsiang, Thailand), tocopheryl acetate (BASF, Germany),
phenoxyethanol (Sharon, Israel), and fragrance (Namsiang)
were of cosmetic grade, and supplied with a certification of
analysis (COA). Pumpkin seed oil (Tropicalife, Thailand) was
certified on palmitic, stearic, oleic, and linoleic acid content
(8–15%, 3–8%, 15–40%, and 50–70%).
In vitro Cleansing Efficacy
Specificity, linearity, accuracy, and precision of the oil on the
selected makeup products, i.e., foundation (Colorstay,
Revlon, USA), liquid eyeliner (Oh my eyeliner, Etude,
Republic of Korea), and pen eyeliner (Super black eyeliner,
Mistine, Thailand) removals were examined by the published
protocol (Charoennit and Lourith, 2012). Briefly, the oil-in-
ethanol (0.080 g of pumpkin seed oil in 40 mL of 95% etha-
nol) was sonicated (2 min), filtered (Whatman no. 1), and
adjusted to 50 mL prior to absorbance patterns recorded
(Genesys 10S, Thermo, USA) for the specificity validation.
Foundation, liquid, and pen eyeliners in a serial concentration
of 0.5200–1.4200 mg mL−1 were thereafter examined at the
obtained specific wavelengths to generate the linearity of each
analysis with a correlation of determination of more than
0.995. Accuracy of the analytical condition of each pumpkin
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seed oil was consequently validated on an extraction of the
makeup (0.035 g) that was applied on the cotton sheets by
95% ethanol (10 mL × 4), filtered, adjusted to 50 mL, and
absorbance recorded prior to recovery (%) of the extracted
makeup calculation. Precision of the analytical method was
assessed by an application of each makeup product (0.035 g)
onto a clean glass (3 × 3 cm), followed by an addition of
pumpkin seed oil (0.080 g), and wiped by the cotton sheets.
The wiped cotton was thereafter extracted and spectroscopi-
cally analyzed as mentioned above.
The developed base makeup removers and those con-
taining pumpkin seed oil were in vitro makeup removal
efficacy examined by the same practice as that used for
other oils in a comparison with benchmark product delin-
eated by AOAC (2002), ICH (2005), and USFDA (2001)
guidelines for the method validation. All of the determined
data were expressed as means  RSD (relative standard
deviation).
Formulation of the Efficient and Stable Makeup
Remover Containing Pumpkin Seed Oil
A 2 layers emulsion makeup remover was developed. Sen-
sory examination during the course of the product develop-
ment was carried out on color, odor, and texture as well as
makeup removal ability by the formulator (Chuarienthong
et al., 2010). Physicochemical properties, i.e., pH (B200,
Qis, the Netherlands) and color in terms of the CIELAB
system (CM-700d, Konica Minolta, Japan) of the formula-
tions were examined.
The accelerated stability test of the formulated removers
was preliminarily carried out using a centrifugation assay at
3500 rpm for 30 min (Micromax, Thermo, USA). Those that
remained stable were included for seven cycles of heat
(45  2  C, 48 h) and cool (4  2  C, 48 h) conditions. The
base with the appropriated texture and appearance, which pas-
sed the stability test, was further incorporated with pumpkin
seed oil. Thereafter, stability of the base remover under the
same conditions was re-assessed. The stable products with
efficient in vitro removal ability against makeup products
were further examined in human volunteers.
In vivo Makeup Removal Efficacy Evaluation
Inclusion Criteria
Thai healthy female volunteers aged between 20–45 years old
who applied foundation, liquid eyeliner, and pen eyeliner for
every day makeup were enrolled in the study. All recruited
subjects were informed about the study both in writing and
verbally and signed a written consent form, which was
approved by the ethical committee of the Mae Fah Luang
University (REH-61085) prior to enrollment. All of the study
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using human volunteers was complied with the good clinical
practice and the Declaration of Helsinki.
Irritation Test
The closed-patch test of the developed makeup removers con-
taining pumpkin seed oil was performed in 10 female volun-
teers. Water was used as a negative control, whereas 0.5%
sodium lauryl sulfate was the positive control. Observation
was undertook immediately, 24, 48, and 72 h following Finn
chamber® (8 mm, SmartPractice, USA) removal. The mean
irritation index (MII) was calculated accordingly (Schnuch
et al., 2008).
Efficacy Evaluation
Twenty-five Thai female volunteers aged 20–45 years old
participated in this study. All subjects were allergy-free for
1 week prior to study enrollment. Subjects who were pregnant
or lactating or dieting were excluded from the study as well as
smoking and liquor drinking. The subjects were asked to wear
all of their makeup cosmetics as usual. A randomized single-
blind study was designed and the volunteers were requested
to apply the remover by a split-face procedure for intra-
individual comparative trials on the evening once they wanted
to remove the makeup. The volunteers were directed to drop
10 drops of the makeup remover containing pumpkin seed oil
on one palm and gently apply on one side of the moist-face
including around the eye for 10 s at which the clear liquid
remover turned to be milky solution, rinsed off with water and
gently wiped with a clean towel. The cleaned fingers and palm
were dropped with the benchmark remover and applied on
another side of the face at the same quantity and cleansing
practice. Thereafter, the subjects were asked to score the pref-
erence in terms of the remover’s appearances (color, odor,
and viscosity) and makeup removal efficacies (spreadability,
ease of makeup removal, skin hydration, and facial cleanli-
ness) in the questionnaire by means of hedonism assessment
(1–5; poor–excellent). The preference score of each parameter
was summarized and calculated into percentage.
Statistical Analysis
The parameters were compared and analyzed using IBM
SPSS statistics version 21. The paired sample t test was ana-
lyzed for the remover formulation stability. The Post Hoc test
was for in vitro cleansing efficacy evaluation. Preference
of the makeup removers was analyzed by the independent
sample t test. The significance was set at a reliability of 95%,
and expressed as the mean  RSD for in vitro makeup
removal efficacy evaluation and mean  SD for stability and
preference tests.
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Results and Discussion
Removal Efficacy of Pumpkin Seed Oil
Makeup cosmetics are basically categorized into base and
point makeup, of which foundation is the base makeup that is
mainly applied on face (Lourith and Kanlayavattanakul,
2014) and eyeliner is the most popularly used point makeup
product (Mintel, 2017). The makeup cleansing ability of
pumpkin seed oil was first validated using the UV–Vis spec-
troscopic method. The working wavelength of the foundation
was shown to be 258 nm (Fig. 1a), and the eyeliners were
220 nm (Fig. 1b, c), while λmax of pumpkin seed oil was
214 nm (Fig. 1d) confirming noninterference of the oil in
in vitro makeup removal efficacy analysis.
The analysis at a specific wavelength of each makeup
product was, therefore, undertaken for six replicates at each
concentration (Charoennit and Lourith, 2012). Specificity
in terms of the working wavelength of foundation, liquid,
and pen eyeliners was confirmed. Linearity of the makeup
product analysis as exhibited with the correlation of deter-
mination greater than 0.995 (Fig. 2) ensures precision of
this method in harmony with the validity and reliability,
which were exhibited by the relative standard deviation (%
RSD). Accuracy of this analytical method is, therefore,
confirmed and conformed to the standard guidelines
(AOAC, 2002; ICH, 2005; USFDA, 2001) as per precision
as shown in Table 1.
Pumpkin seed oil was best in cleansing of foundation
(79.92  0.07%), followed by liquid and pen eyeliners
(41.02  0.02% and 23.54  0.19%). Thereafter, pumpkin
seed oil was developed into a makeup remover in a dosage
form of a 2 layers emulsion remover. This 2 layers emul-
sion system is a popular dosage form for makeup remover
in markets including Thailand. This dosage form was,
therefore, developed for pumpkin seed oil makeup remover
in this study.
Formulation of the Efficient and Stable Makeup
Remover Containing Pumpkin Seed Oil
The base makeup removers containing PEG-7 glyceryl coco-
ate, sorbitan oleate, sorbeth-30 tetraoleate, ethylhexy palmi-
tate, water, glycerin, tocopheryl acetate, phenoxyethanol, and
fragrance were firstly formulated, and preliminary sensory
evaluated by the formulator. Those of the base removers with
appropriate texture comparable to the benchmark remover
(containing PEG-7 glyceryl cocoate, sorbitan oleate, sorbeth-
30 tetraoleate, aqua, cetyl ethylhexanoate, butylene glycol,
chlorphenesin, disodium ethylenediaminetetraacetic acid
(EDTA), and phenoxyethanol as informed in the ingredients
list of the product’s label) and adequate makeup removal abil-
ity were taken into account as the criteria during the course of
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Fig. 1 UV-spectrum of (a) foundation, (b) liquid eyeliner, (c) pen eyeliner, and (d) pumpkin seed oil

product development. The base with the greatest preference The base (F0) was consequently assessed for its in vitro
and preliminary removal results was included for stability test, makeup removal ability using the above-validated method.
and proved to be stable under the accelerated stability tests. The base was significantly (P < 0.001) better in removal

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1.5 centrifugation assay and before and after heat-cool cycles as

Foundation
shown in Fig. 3 and Table 2, although there were some shifts
Liq.eyeliner
y = 0.7549x + 0.16
Pen eyeliner R2 = 0.9964
but insignificant confirming compatibility of pumpkin seed oil
with other cosmetic ingredients.
1.0
Thereafter, in vitro makeup removal efficacy of the devel-
Absorbance

y = 0.5517x + 0.0259
R2 = 0.9977
0.5
y = 0.3697x + 0.0914
R2 = 0.9924
0.0
0 0.5 1 1.5
Concentration (mg/mL)
Fig. 2 Standard curves for removal efficacy evaluation against
makeup products
efficacy than pumpkin seed oil (Table 1). Thus, addition of
pumpkin seed oil into the developed makeup remover base
would enhance the makeup removal efficacy.
An incorporation of pumpkin seed oil into the stable and
efficient makeup remover base was trialed at 5–15% resulting
in F1-F3 (Table 2). All of the pumpkin seed oil 2 layers emul-
sion removers were light clear solutions in accordance with
their L* (0–100; black to white) and a* (− to +; green to red)
and b* (− to +; blue to yellow). pH values of the formulations
were shown to be conformed in the range of cosmetic prod-
ucts. All of the pumpkin seed oil formulations were stable
following the accelerated stability test by means of a
oped pumpkin seed oil removers was examined in a compari-
son with the benchmark 2 layers emulsion remover. Addition
of pumpkin seed oil significantly (P < 0.001) enhanced the
removal ability of the base remover, although those of the
pumpkin seed oil products were less than the benchmark one.
A greater removal efficacy of the benchmark might be
governed by a chelating agent, disodium EDTA, that addi-
tionally enhance the foaming and cleaning capabilities of cos-
metics in a dosage form of solution. The studied makeup
removers were obviously high in foundation removal ability,
followed by liquid eyeliner and pen eyeliner, respectively
(Table 1). Pen eyeliner was greater in resistance against
removal that might be because of its polymeric constituents
function as film formers with a high surface tension
preventing the wetting process in the course of removing.
Thus, the removal ability of the pumpkin seed oil remover
and the benchmark was less, additional solubilizer and/or sur-
face tension reducer for this presenting makeup remover for-
mulation would improve pen eyeliner removal ability.
In vivo Makeup Removal Efficacy Evaluation
In vivo removal efficacy of the developed makeup remover
was thereafter undertaken. Preliminary skin irritation was

Table 1 In vitro removal efficacy of pumpkin seed oil and makeup removers

Makeup Remover Absorbance Amount (mg mL−1) Removal efficacy (%recovery + RSD)
Added Found

Foundation Pumpkin seed oil 0.34  0.02 0.71  0.00 0.56  0.04 79.92  0.07
F0 0.35  0.01 0.71  0.00 0.59  0.01 83.24  0.02
F1 0.37  0.01 0.71  0.01 0.63  0.01 89.27  0.02
F2 0.33  0.01 0.70  0.00 0.55  0.01 78.24  0.02
F3 0.36  0.01 0.71  0.01 0.60  0.01 84.41  0.01
Benchmark 0.38  0.02 0.71  0.01 0.65  0.03 91.20  0.03
Liquid eyeliner Pumpkin seed oil 0.20  0.02 0.71  0.01 0.29  0.08 41.02  0.25
F0 0.27  0.04 0.71  0.01 0.48  0.11 68.54  0.25
F1 0.27  0.01 0.71  0.01 0.48  0.04 67.72  0.08
F2 0.27  0.01 0.70  0.00 0.47  0.02 66.88  0.05
F3 0.28  0.01 0.71  0.01 0.50  0.05 69.79  0.12
Benchmark 0.28  0.02 0.70  0.01 0.52  0.06 73.46  0.10
Pen eyeliner Pumpkin seed oil 0.29  0.02 0.71  0.00 0.17  0.01 23.54  0.19
F0 0.360  0.04 0.71  0.01 0.26  0.05 37.40  0.20
F1 0.38  0.02 0.70  0.00 0.29  0.02 41.25  0.07
F2 0.36  0.01 0.71  0.00 0.27  0.01 38.43  0.05
F3 0.38  0.01 0.71  0.01 0.30  0.01 41.88  0.04
Benchmark 0.45  0.02 0.71  0.01 0.38  0.03 51.00  0.07

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Table 2 The components, stability, and preference of pumpkin seed oil removers

Ingredient Formula (% w/w)

F0 F1 F2 F3

Pumpkin seed oil 0 5.0 10.0 15.0

PEG-7 glyceryl cocoate, sorbitan 35 35 35 35
oleate, sorbeth-30 tetraoleate
Ethylhexyl palmitate 42 35 30 25
Water 15.7 17.2 17.2 17.2
Glycerin 6.5 6.5 6.5 6.5
Tocopheryl acetate 0.5 0.5 0.5 0.5
Phenoxyethanol 0.5 0.5 0.5 0.5
Fragrance 0.3 0.3 0.3 0.3
Initial (or before)
pH 6.53  0.01 6.51  0.01 6.44  0.02 6.32  0.02
L* 61.10  1.06 57.11  1.25 56.86  0.84 59.89  0.82
a* 1.01  0.03 0.77  0.01 0.61  0.03 0.47  0.01
b* 0.88  0.09 1.59  0.11 2.32  0.16 3.18  0.07
After Heat-Cool 7 cycles
pH 6.03  0.02 5.96  0.01 5.95  0.03 5.96  0.02
L* 60.39  0.16 59.13  0.55 60.36  0.65 62.26  0.03
a* 0.92  0.02 0.89  0.05 0.62  0.03 0.54  0.01
b* 2.73  0.06 3.24  0.09 3.74  0.05 4.42  0.01

firstly examined. The pumpkin seed oil removers showed
none of positive skin irritation similar to the negative con-
trol, water (MII = 0). Thus, the stable and safe pumpkin
seed oil remover with the highest foundation removal
ability and appropriate eyeliner removal efficacy, although
containing the least of pumpkin seed oil content (F1), was
chosen for further evaluation using human volunteers in a
comparison with the benchmark. The pumpkin seed oil
makeup remover gained a significant (P < 0.001) prefer-
ence on skin hydration and a greater score in odor than the
benchmark, although color, viscosity, spreadability, ease of
makeup removal, and facial cleanliness were less but insig-
nificant. Nevertheless, overall preferences of the volunteers
on the pumpkin seed oil remover were better than the
benchmark one (Fig. 4). Pumpkin seed oil is rich in unsatu-
rated fatty acids supporting aesthetical benefit of the oil as
an emollient and a moisturizer (Alvarex and Rodríguez,

Overall preference

Facial cleanliness

Skin hydration

Ease of makeup removal

Spreadability

Viscosity

Odor

Color

0 20 40 60 80 100
Preference score (%)
Benchmark F1

Fig. 3 Stability of the developed makeup removers following (a) cen- Fig. 4 Preference toward pumpkin seed oil remover and the
trifugation assay and (b) before and after heat-cool cycles benchmark

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2000; Cosmetic Ingredient Review, 2011). Accordingly,
improvement of skin hydration following makeup removed
using the pumpkin seed oil remover was obviously noted
by the volunteers. In addition, a consecutive application of
this developed natural makeup remover for everyday usage
would enhance skin benefits of pumpkin seed oil’s phyto-
chemicals for instance skin hydration and delaying of skin
aging (Alvarex and Rodríguez, 2000; Kanlayavattanakul
and Lourith, 2015, 2018), respectively.
Conclusion
Pumpkin seed oil is evidentially confirmed on its makeup
removal ability. The oil efficiently removed makeup products
and was successively developed as a makeup remover. Appli-
cation of pumpkin seed oil in the cosmetic industry was
underpinned by the consumers’ attention being paid to natu-
ral, eco-friendly, and sustainable products. In addition, skin
benefits of pumpkin seed oil would be exacerbated using the
makeup remover containing pumpkin seed oil.
Acknowledgement Mae Fah Luang University is acknowledged for
facilities supports.
Conflict of Interest Authors declare that they have no conflict of
interest.
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