JCS Complete Volume 70 No 5

Vol . 70 N o .
5 September/October 2019
JOURNAL OF COSMETIC SCIENCE

The Official Journal of the Society of Cosmetic Chemists
Contents
Page
ORIGINAL ARTICLES Titanium Dioxide and Zinc Oxide Nanoparticles in Sunscreens:
A Review of Toxicological Data
Maja Vujovic and Emilija Kostic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Comparative Assessment of Biological Activities of Mistletoes for

Cosmetic Applications: Viscum Album Var. Coloratum (Kom.) Ohwi
and Loranthus Tanakae Franch. & Sav.
Sung-Up Choi, Sung Tae Kim, Dong Gyun Han, Young-Ha Hwang,
Koo Yeon Lee, Dong Uk Kim, Kwan Hyung Cho, Sang Yeob Park,
Hee-Cheol Kim, Seong-Bo Kim, and Dong-Jin Jang . . . . . . . . . . . . . . . . 235
In Vitro Penetration of Petrolatum in Stratum Corneum from Bodywash

Formulation
Apipa Wanasathop, Zhanquan Shi, Q. Ching Stella, Karl S. Wei,
Peter B. Styczynski, Chuiying Li, and S. Kevin Li . . . . . . . . . . . . . . . . . . 247
Does Salt and Mineral Content of Dead Sea Mud Affect Its Irritation
Potential: A Laser Doppler Flowmetry Study
Saja Hamed, Abdel-Majeed Almalty, Hatim S. Alkhatib,
Nabil N. Al-Hashimi, and Duha Hamed . . . . . . . . . . . . . . . . . . . . . . . . 259
Purchased for the exclusive use of nofirst nolast (unknown)

From: SCC Media Library & Resource Center (library.scconline.org)
J. Cosmet. Sci., 70, 223–234 (September/October 2019)
Titanium Dioxide and Zinc Oxide Nanoparticles in

Sunscreens: A Review of Toxicological Data
MAJA VUJOVIC and EMILIJA KOSTIC, Faculty of Medicine,

Department of Pharmacy, University of Nis, 18000 Nis, Serbia (M.V., E.K.),
Institute of Forensic Medicine in Nis, 18000 Nis, Serbia (M.V., E.K.)
Accepted for publication June 24, 2019.
Synopsis
The positive effects of sunlight have been known for many years, and the negative ones, too. Sunscreens are
physical and chemical UV absorbers. Nanotechnology has developed nanoparticles of physical blockers:
titanium dioxide (TiO2) and zinc oxide (ZnO). Their smaller diameter and increased bioreactivity are the
focus of many toxicological studies. The usage of sunscreens has increased around the world, so all toxicological
aspects should be carefully considered. There are in vitro and in vivo studies: studies on animal and human
skin; investigations of potential genotoxicity and cytotoxicity; generation of reactive oxygen species;
penetration; skin irritation; acute, subchronic, and chronic toxicity; and carcinogenesis. The experimental
conditions of these studies differ from study to study, but most authors agree that there is no penetration of
nanoparticles into viable skin layers. Risk-benefit analysis of TiO2 and ZnO nanoparticles (NPs) usage in
sunscreens strongly indicates that potential risks are vastly outweighed over the benefits. Because of the
results of some authors indicating possible penetration through damaged skin, further studies should be
conducted, primarily addressed on skin penetration mechanisms.
INTRODUCTION
UV radiation can cause harmful effects on the skin. UVC, and partly UVB, can be absorbed
by molecular oxygen to produce ozone. Stratospheric ozone absorbs UV rays below 290 nm,
but UVB and UVA rays reach the human skin and cause metabolic and biological
reactions (1).
Although many patients and physicians believe that regular use of sunscreens provides
protection from skin cancer, this protective effect has been confirmed only in the cases of
squamous cell carcinoma and actinic keratoses, but for basal cell carcinoma and malig-
nant melanoma, the results are inconclusive (2–4).
In the United States, skin cancer is the most common form of cancer. Annually, more than
one million cases are diagnosed in the form of squamous cell and basal cell carcinoma,
both associated with UV radiation. The incidence of melanoma is rising significantly.
Address all correspondence to Maja Vujovic at majavujovic1@gmail.com and Emilija Kostic at

emilija293@gmail.com.
223
224 JOURNAL OF COSMETIC SCIENCE
More people are diagnosed with skin cancer each year in the United States than all other
cancers combined (5,6).
SUNSCREENS
The negative effects of sunlight, sunburns, photoaging, and skin cancers are reduced by the
use of sunscreens. Sunscreens should provide protection against the adverse effects of both
UVB and UVA radiation. Compounds that have the ability to protect from UV radiation
are classified into two groups: organic and inorganic blockers. Minerals such as zinc oxide
(ZnO) and titanium dioxide (TiO2) are often used as inorganic physical sun blockers.
TiO2 and ZnO have been used as ingredients for sunscreen formulations for more than 20
years. Their mechanism of actions involves absorption, reflection, and reflecting and the
scattering of UV sunlight (7).
The disadvantage of microsized ZnO and TiO2 particles is their poor dispersive proper-
ties, resulting in a white color that is not cosmetically appealing (8).
NANOPARTICLES
The rapid development of nanotechnology has resulted in an increasing number of nano-

material-based products. Because of their physicochemical properties, nanomaterials have
found an important role in cosmetics. When particles become smaller than 100 nm (the
optimal light scattering size), visible light is transmitted across the particles. This avoids
the cosmetically undesired opaqueness of inorganic sunscreens and makes the application
of cosmetic products based on nanoparticles (NPs) commercially attractive, without re-
ducing UV-blocking effectiveness (9).
Possible adverse effects have been considered. Because the surface area to volume ratio of
particles increases as the particle diameter decreases, NPs may be more (bio) reactive than
normal bulk materials. This is the reason why safety of cosmetic products containing NPs
has been frequently discussed. Many scientists and research institutes mainly focus on
various kinds of toxicological and skin penetration studies. However, safety also concerns
the physicochemical properties of sunscreen ingredients to be taken up by the skin in
both the absence and presence of light.
With the widespread use and the potential for TiO2 or ZnO NPs exposure, concerns have
focused on their possible resorption.
COSMETIC REGULATION
International Cooperation on Cosmetic Regulation defines a nanomaterial in cosmetics as

an insoluble intentionally manufactured ingredient with one or more dimensions ranging
from 1 to 100 nm in the final formulation. In addition, the nanomaterial must be suffi-
ciently stable and persistent in biological media to disable potential interactions with
biosystems (10).
In 2012, the International Organization for Standardization underlined that the physico-
chemical characterization of nanomaterials was critical for the identification of test mate-
rials before toxicological assessment (11).

TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES IN SUNSCREENS 225
Important physicochemical parameters for their characterization are particle composi-

tion, size/particle size distribution, surface charge, solubility/dispersibility, aggregation/
agglomeration state, shape, surface area, and surface chemistry (12).
INSTRUMENTAL METHODS FOR THE CHARACTERIZATION OF TIO2 AND ZNO NPS
Sunscreen preparations with micronized TiO2 and/or ZnO are complex and opaque, so
NPs detection and characterization are complicated. Tyner has evaluated the ability of 20
analytical methods to detect TiO2 and ZnO NPs in unmodified commercial sunscreens
(13). Variable-pressure scanning electron microscopy, laser scanning confocal microscopy,
X-ray diffraction, and atomic force microscopy were considered applicable and compli-
mentary for NPs characterization in sunscreens. Guidelines on the safety assessment of
nanomaterials in cosmetics from the Scientific Committee on Consumer Safety suggested
the use of at least two methods, of which one should be electron microscopy, preferably
high-resolution transmission electron microscopy, to determine the size of nanomaterial
particles (14).
TOXICOLOGICAL CONCERNS
There are conclusive results that titanium can cause lung cancer after inhalation, so it is
the reason for increased concerns about potential toxicity after dermal applications. Tita-
nium is classified into group 2B of carcinogens. Organized, accurate, and detailed studies
must be conducted to give information about dermal permeation, local effects, and even-
tually generalized effects. It is necessary because sunscreens are applied to the skin in
some countries during the whole year, in large amounts, on almost the whole skin area.
DERMAL PENETRATION OF ZNO AND TIO2
There are few parameters that must be considered during the research of dermal perme-
ation of substance. One of them is the characteristics of the studied substance. Theoreti-
cally, only those materials with an adequate log P coefficient (octanol/water partition
coefficient) and low molecular weight (<ca. 500) can penetrate the intact human skin
through the stratum corneum (SC).
The second parameter is the skin itself. The SC represents the outermost layer of the skin
and plays an important role in protecting the human organism against penetration by
xenobiotics. It should be emphasized that although cosmetics and sunscreens containing
ZnO and TiO2 are normally used on healthy skin, injuries to the skin can occur under
certain circumstances (physical force or sunburn), which can cause enhancement of re-
sorption. This is the reason why skin penetration studies of TiO2 and ZnO particles are
usually investigated in vivo and in vitro with both intact skin and stripped skin which
mimics an injured skin (15).
The ingredients of sunscreen formulation must also be known. The biopharmaceutical
characteristics are not the same comparing O/W emulsions, W/O emulsions, silicone-based
emulsions, or aerosol sprays. Researchers investigating dermal absorption should under-
line all properties and circumstances of the experiment.

ABSORPTION OF TIO2 AND ZNO—IN VITRO STUDIES
Many authors reported the penetration of NPs from sunscreens onto the human skin. Hu-
man epidermal penetration of a transparent TiO2 and ZnO sunscreen formulation was
determined using Franz-type diffusion cells and electron microscopy to verify the loca-
tion of NPs in exposed membranes. In most studies, no particles could be detected in the
lower layers of SC or viable epidermis by electron microscopy, suggesting that minimal
nanoparticle penetration occurs through the human epidermis. Thus, some researchers
have concluded that NPs penetrate to 13 layers into the UVB-damaged SC, whereas only
seven layers in the intact skin. The experiment conditions and effects are shown in Table I.
ABSORPTION OF TIO2 AND ZNO—ANIMAL SKIN
Gamer et al. reported the dermal absorption of ZnO and TiO2 particles through the skin
of domestic pigs. In addition, the results show that microfine ZnO particles were not able
to penetrate the porcine-dermatomed skin preparations (20).
Wu et al. (21) and Adachi et al. (22) have concluded that after prolonged application,
NPs can penetrate through the SC and they can be located in the deep layer of the epider-
mis. Wu et al. investigated the penetration and toxicity of TiO2 NPs after in vivo animal
(BALB/c hairless mice) dermal application. After 60 d of dermal exposure in hairless
mice, TiO2 NPs not only penetrated the skin but also reached different tissues and in-
duced pathological lesions in several major organs. In addition, they found TiO2 NPs in
the mouse brain without inducing any pathological changes (21).
Recently, Adachi et al. (23) found signs of irritant dermatitis with focal parakeratosis in
the SC and epidermal spongiosis after applying uncoated TiO2 NPs for a long time.
The experiment conditions and results of other researchers are shown in Table II.
Table I
Absorption of TiO2 and ZnO in vitro
Type of NPs Formulation Experimental system Effects Ref.

ZnO NPs Transparent Franz-type diffusion No particles could (16)
formulation cells be detected in
the lower SC
w/o emulsion Franz-type diffusion No particles could (17)
cells on the excised be detected in
porcine skin the lower SC
TiO2 NPs Suspensions (1.0 g/L), Franz cells using Not detectable in receiving (18)
24 h intact and solutions for
needle-abraded both intact and damaged
human skin skin
TiO2 and ZnO NPs 1) 10% coated TiO2 in Skin in flow-through 1) TiO2 in w/o penetrated (19)
w/o, 2) 10% coated diffusion cells deeper in UVB-damaged SC;
TiO2 o/w, 3) 5% 2) penetrated 13 layers into
coated ZnO in o/w, UVB-damaged SC, whereas
and 4) 5% uncoated only seven layers in normal;
ZnO in o/w and 3) and 4) were localized
to the upper one to two SC
layers

ABSORPTION OF TIO2 AND ZNO—INTACT AND DAMAGED HUMAN SKIN
Many studies showed that there was no penetration of TiO2 and ZnO NPs. Also, they
have shown that particle shape and formulation have no significant impact on penetration
through the SC in in vivo studies. The experiment conditions and results are shown in
Table III.
Gulson et al. have conducted the study to detect possible amounts of ZnO in blood and
urine. The experiment was assessed under real-life conditions. Sunscreen was applied
to the skin of volunteers for 5 d. Blood and urine levels of 68Zn from 68ZnO particles in
sunscreens increased in all subjects over the period of exposure, with significantly
higher levels of 68Zn in females exposed to a sunscreen containing NPs of 68ZnO than
in females exposed to larger 68ZnO particles and males exposed to particles of both
sizes (27).
Tan et al. (28) have researched if there is a difference in resorption of NPs in elderly pa-
tients. There was no significant difference in the level of TiO2 in dermis compared with
the control group.
Bennat et al. found that TiO2 NPs are able to penetrate through hair follicles or pores,
but no closer information is given on the fate of those particles. This result can show the
importance of permeation through hair follicles (29).
Zvyagin et al. investigated the distribution of topically applied ZnO on excised human
skin. The lack of penetration of these NPs suggests that safety concerns are not objective
and evidence based (26).
Presently, only a few studies have been conducted with TiO2 or ZnO NPs applied to the
damaged skin and on what normally happens when sunscreens are reapplied to sunburned
skin. UVB-damaged skin slightly enhanced TiO2 NPs or ZnO NPs penetration in sun-
screen formulations, but no transdermal absorption was detected (19).
Table II
Dermal Absorption of TiO2 and ZnO NPs; Animal Skin
Type of NPs Formulation Skin type Effect Ref.

TiO2 and ZnO NPs Sunscreen UVB sunburned Slightly enhanced penetration, (19)
formulation skin pigs but no detection of transdermal
25 µL resorption
TiO2, ZnO NP o/w emulsion Normal and UVB- UVB-damaged skin slightly (19)
24 and 48 h sunburned skin enhanced penetration, but
pigs no transdermal absorption was
detected
Ultrafine TiO2 10% W/O Hairless rat skin Neither penetrate viable cell (22)
emulsion layers nor biologically cause
any cellular changes
Ultrafine TiO2 10% W/O Dorsal skin of No evidence of penetration after (21)
emulsion hairless Wistar the subchronic exposure
2, 4, 8 week Yagi rats
Micronized TiO2 Human skin Do not penetrate through the (23)
transplanted intact epidermal barrier
to immuno-
deficient mice

Table III
Absorption of TiO2 and ZnO on Human Skin in vivo
Type of NPs Formulation Skin type Effect Ref.

TiO2 (20 nm) w/o emulsion, 5 h Human skin No penetration (24)
Micronized TiO2, w/o emulsion, 6 h Human forearms Particle shape and (25)
hydrophobic formulation
100 nm, amphiphilic nonsignificant impact
10–15 nm, and on penetration
hydrophilic 20 nm
Micronized TiO2 w/o emulsion Older patients’ skin, The insignificant level in (25)
(10–50 nm) 59–85 years and dermis higher than
9–31 d in control
ZnO (26–30 nm) w/o emulsion, Human skin Remained in the SC (26)
68
ZnO (19 nm) w/o emulsion, 5 d Human skin Small increases 68Zn in blood (27)
and urine
TiO2 (20 nm) w/o emulsion and Hairy skin May be able to penetrate (28)
aqueous through hair follicles and
suspension pores
TiO2, ZnO w/o emulsion Excited human skin Remained on the surface (29)
TiO2, ZnO NP w/o emulsion Mechanically, Very small particles to cross (29)
physically, and to the SC increases relative
chemically to control
damaged skin
TiO2 NP w/o emulsion Healthy and Deeply penetrated to psoriatic
(30)
psoriatic skin relative to the normal skin
Pincheiro et al. have investigated the difference in resorption of ZnO through healthy
and psoriatic skin. In psoriatic skin, the fragility of the SC seemed to facilitate the
penetration of the NPs, although they did not reach living cell layers. However, the
desquamation of the SC hindered the adequate distribution of the cream along the skin
surface (30).
SKIN IRRITATION BY TOPICAL APPLICATION OF ZNO AND TIO2 AND STUDIES OF ACUTE TOXICITY
Effects of ZnO NPs and TiO2 NPs, and their mixtures on skin corrosion and irritation
were investigated by using in vitro 3D human skin models (KeraSkin™), and the results
were compared with those of an in vivo animal test. The results provide the evidence that
ZnO NPs and TiO2 NPs and their mixture are “nonirritant” and “noncorrosive” to the
human skin by a globally harmonized classification system. In vivo test using animals may
be replaced by an alternative in vitro test (31).
The potential effects of photosensitization and photo irritation of ZnO on the human skin
were also discussed. There was no evidence of any positive findings in two photo irritation
studies and two photosensitization studies after topical application on the intact human
skin. Furthermore, in a review of photoprotection, Lautenschlager et al. reported that
neither TiO2 nor ZnO NPs possess skin irritation or sensitization properties when used
in sunscreens on humans (32).
Using acute dermal irritation studies in rabbits and local lymph node assay in mice
(CBA/JHsd), Warheit et al. concluded that water solution of TiO2 NPs used in different

concentrations from 0% to 100% applied for three consecutive days were not a dermal
sensitizer or skin irritant (33).
In a 14-d toxicity study, TiO2 NPs applied topically to rat skin (Wistar) induced short-
term toxicity at the biochemical level (34). Enzymes for which concentrations increased
are lactate dehydrogenase, lipid peroxidase, serum glutamic pyruvic transaminase, and
serum glutamic oxaloacetic transaminase. Depletion in the levels of catalase and glutathi-
one S-transferase (GST) activity was detected. They concluded that short-term exposure
to TiO2 NPs can cause hepatic and renal toxicity in rats. It should be underlined that the
doses used in these studies are high (14, 28, 42, and 56 mg/kg) and humans are not ex-
posed to those high concentrations (35).
INFLUENCE OF TIO2 AND ZNO ON ROS GENERATION AND POTENTIAL CYTOTOXICITY
Results of the recent studies provided the information that both ZnO and TiO2 NPs can
generate reactive oxygen species (ROS): superoxide anions, hydroxyl radicals, and singlet
oxygen (36,37). The mechanism of the reaction is UV-induced photocatalysis. ROS can
damage cellular components and macromolecules, and ultimately cause cell death if pro-
duced in excess or if they are not neutralized by antioxidant defenses. ROS derived from
the photocatalysis of NPs are cytotoxic to a variety of cell types (38).
Sayes et al. have investigated the difference between two crystal forms of TiO2 NPs in
producing ROS. They reported that anatase NPs generated more ROS than rutile after
UV irradiation. It has been concluded that TiO2 anatase has a greater toxic potential than
TiO2 rutile. Also, anatase ROS production does not occur under ambient light conditions
(39).
A study by Lewicka et al. (40) reported a greater generation of ROS by ZnO NPs than
TiO2 NPs.
The cytotoxicity of TiO2 NPs was demonstrated in keratinocytes, using different tests
and exposures, with or without UV exposure, but many in vivo experiments on animals
did not confirm this effect (41–43).
Cytotoxicity studies on HaCaT cells gave an important result that TiO2 NPs induce cy-
totoxic effects only at very high concentrations after 7 d (44).
In vitro toxicity was also observed. Vinaredell et al. used the EpiSkin model, to determine
the differences between ZnO and ZnO NPs. Formulations with ZnO and ZnO NPs were
first applied for 15 min and for 24 h, but cytotoxic effects were not observed. The per-
centage of viability of the treated cells was around 100% for all ZnO materials, regardless
of their size (45).
Kiss et al. investigated in vivo penetration and effects on cell viability of TiO2 on human
skin transplanted to immunodeficient mice. They demonstrated that with TiO2 NPs,
there was no penetration through the skin, but when exposed directly to cell culture in
vitro, they have significant effects on cell viability (23).
Liu et al. have conducted an important study. During the PC12 cells treatment with different
concentrations of TiO2 NPs, the viability of cells was significantly decreased in the peri-
ods of 6, 12, 24, and 48 h, showing a significant dose effect and time-dependent manner.
The number of apoptotic PC12 cell increased with the increasing concentration of TiO2
NPs (35) (Table IV).

GENOTOXICITY
TiO2 and ZnO NPs were investigated for their potential genotoxicity in in vitro and in vivo
test systems. No genotoxicity was observed in vitro (Ames’ Salmonella gene mutation test
and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micro-
nucleus test and Comet DNA damage assay in lung cells from rats exposed by inhalation) (46).
The SCCS (2012) comprehensive review of ZnO NPs revised both in vitro and in vivo
studies on photo-mutagenicity/genotoxicity and concluded that there is no definite evi-
dence to claim if ZnO NPs pose a mutagenic/genotoxic, phototoxic, or photomutagenic/
genotoxic risk to humans (47).
CARCINOGENESIS
There are reports that dermal application of noncoated rutile TiO2 does not exhibit a
promoting effect on UVB-induced skin carcinogenesis in rats. Xu et al. researched c-Ha-
ras proto-oncogene transgenic rats, which are sensitive to skin carcinogenesis, and their
wild-type siblings were exposed to UVB radiation. Their back skin is shaved twice weekly
for 10 weeks. On the shaved area, a suspension of 100 mg/ml TiO2 NPs was applied. In
the observed groups, the tumor incidence was not different (48).
Sagawa et al. have reached the same conclusion, after studying the promoting effect of
silicone-coated TiO2 NPs suspended in silicone oil and noncoated TiO2 NPs suspended
in Pentalan 408 on a two-stage skin chemical carcinogenesis model (49).
Newman et al. also suggested that TiO2 NPs are not carcinogenic to the skin. However,
the authors emphasized that further studies for the safety evaluation of the TiO2 NPs in
sunscreens must be performed to simulate real-world conditions, particularly in sun-
burned skin and under UV exposure (50).
Table IV
Cytotoxicity of TiO2 NPs in vivo
Type of NPs Cells Effect Ref.

TiO2 NPs < 20 nm Human HaCaT and Induction of the mitochondrial “common (41)
keratinocytes deletion” in HaCaT cells following exposure
to TiO2 NPs, which strongly suggests a
ROS-mediated cytotoxic and genotoxic
potential of NPs.
TiO2, 25 nm Immortalized keratinocyte Increase production of ROS, the toxicological (43)
dispersion in cells and HaCaT cells effects can be simplified into six events
serum-free
medium
TiO2 NPs Keratinocyte cells Alter the calcium homeostasis and induced a (42)
decrease in cell proliferation associated with
early keratinocyte differentiation, without any
indication of cell death.
TiO2 NPs (anatase, Human keratinocyte and Induced ROS resulted in oxidative stress in these (44)
rutile, and HaCaT cells cells by reducing SOD and increasing MDA
anatase–rutile) levels and damage HaCaT cells
sizes (4, 10, 21,
25, and 60 nm)
UVA radiation

The lack of penetration through the epidermis is considered as the main reason for the
absence of skin carcinogenesis–promoting effects.
INTERACTIONS OF SUNSCREENS WITH OTHER SUBSTANCES
Sunscreens are not only dermal preparations applied on the skin. Many cosmetic prepara-
tions, dermocosmeceutic, and dermal preparations are applied every day. It is important
to investigate potential interactions between sunscreens containing NPs, to find out
whether sunscreens enhance or block resorption. Effects of drugs applied to the skin for
many diseases can be modulated and disturbed. Peire et al. have researched interactions
with amphotericin. TiO2 NPs can modulate the transdermal permeation of the ampho-
tericin. The main reason is the superficial chemistry of TiO2 (51).
ROS generated by TiO2 and ZnO NPs can increase skin permeability. Transdermal drug
and other substances (dyes, pesticides, and toxins) penetration can be favored or reduced
by modulating the TiO2 surface charge (coating) and its oxidative potential (crystalline
phases), so the enhancer effect of TiO2 NPs can be adjusted and converted up or down-
ward (52,53).
People are encouraged to use sunscreens when they are exposed to sunlight, and it is often
on fields where pesticides are applied. The dermal penetration of the herbicide
2,4-dicholorophenoxyacetic acid (2,4-D) is enhanced by the formulations containing chem-
ical UV absorbers, the absorbers themselves, and the insect repellent DEET. Brand et al.
investigated whether commercially available sunscreens containing TiO2 or ZnO enhance
the transdermal absorption of pesticides. For in vitro studies, hairless mouse skin was
used. In vitro permeability studies were performed with the pesticides: parathion, mala-
thion, 2,4-D, and paraquat. The data demonstrate that there was significant penetration
enhancement of malathion, parathion, and paraquat when compared with controls. The
difference between ZnO and TiO2 was noticed because ZnO can interfere with 2,4-D
penetration and TiO2 had no effect. Although, the risk-benefit analysis gives the recom-
mendation for using sunscreens (54).
CONCLUSION
There are conclusive pieces of evidence that TiO2 and ZnO NPs do not penetrate through
intact and healthy human skin, but further studies are necessary to confirm their penetra-
tion through damaged and sensitive skin. Of paramount importance is the finding that
most studies do not demonstrate NPs’ skin penetration and that no significant concentra-
tions are found in layers of viable cells. It must be emphasized that cytotoxic and patho-
logical outcomes are presented in studies using high concentrations of NPs, which are
impossible to be used for human purpose.
Results of in vivo–in vitro and human–animal studies should be cautiously extrapolated.
Some studies have given conclusive pieces of evidence about the potential of NPs to in-
duce ROS in vitro, which largely mediate NP-induced cytotoxicity and genotoxicity, but
the important real-situation information is that NPs used in sunscreens have modified
the surface so it has less possibility to produce ROS, even after UV exposure. Sunscreens
also contain antioxidants to neutralize generated ROS, and endogenous antioxidants can

protect against oxidative stress. These are important factors for the prevention of possible
harmful effects of ROS.
Risk-benefit analysis of TiO2 or ZnO NPs usage in sunscreens strongly indicates that
potential risks are vastly outweighed by the benefits. Sunscreens afford protection against
UV-induced skin damages, such as photoaging and, importantly, skin cancer.
Possibilities of artificial intelligence, especially machine learning, as one of the most pow-
erful technique, must be applied in further studies in the prediction of the mechanism of
penetration and toxicity.
REFERENCES
(1) M. Norval, R. M. Lucas, A. P. Cullen, F. R. de Gruijl, J. Longstreth, Y. Takizawa, J. C. van der Leun, The
human health effects of ozone depletion and interactions with climate change, Photochem. Photobiol. Sci.,
10, 199–225 (2011).
(2) J. C. van der Pols, G. M. Williams, N. Pandeya, V. Logan, A. C. Green, Prolonged prevention of squamous
cell carcinoma of the skin by regular sunscreen use, Cancer Epidemiol. Biomarkers Prev., 15, 2546–2548
(2006).
(3) A. Green, R. MacLennan, and V. Siskind, Common acquired naevi and the risk of malignant melanoma,
Int. J. Cancer, 35, 297–300 (1985).
(4) M. E. Burnett and S. Q. Wang, Current sunscreen controversies: a critical review, Photodermatol. Photoimmunol.
Photomed., 27, 58–67 (2011).
(5) T. H. Keegan, L. A. Ries, R. D. Barr, A. M. Geiger, D. V. Dahlke, B. H, Pollock, W. A. Bleyer, Com-
parison of cancer survival trends in the United States of adolescents and young adults with those in
children and older adults, Cancer, 122, 1009–1016 (2016).
(6) American Cancer Society, Cancer Facts and Figures, 2018, accessed January 26, 2018, https://www.cancer.
org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2018/
cancer-facts-and-figures-2018.pdf.
(7) T. G. Smijs and S. Pavel, Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their
safety and effectiveness, Nanotechnol. Sci. Appl., 4, 95–112 (2011).
(8) X. Wu and R. H. Guy, Applications of nanoparticles in topical drug delivery and in cosmetics, J. Drug
Deliv. Sci. Technol., 19, 371–384 (2009).
(9) N. Durán, S. S. Guterres, and O. L. Alves, Nanotoxicology: Materials, Methodologies, and Assessments
(Springer-Verlag, New York, 2014).
(10) International Cooperation on Cosmetic Regulation, Report of the ICCR Joint Ad Hoc Working Group on
Nanotechnology in Cosmetic Products: Criteria and Methods of Detection - ICCR-4. From the Federal Register
Online via the Government Publishing Office, Washington 2010.
(11) International Organization of Standardization, Nanotechnologies–Guidance on Physico-Chemical Characterization
of Engineered Nanoscale Materials for Toxicologic Assessment (Geneva, Switzerland, 2012).
(12) FDA, Guidance for Industry: Safety of Nanomaterials in Cosmetic Products, https://www.fda.gov/
media/83957/download accessed February 5, 2019.
(13) K. M. Tyner, A. M. Wokovich, W. H. Doub L. F. Buhse, L. P. Sung, S. S. Watson, N. Sadrieh, Compar-
ing methods for detecting and characterizing metal oxide nanoparticles in unmodified commercial sun-
screens, Nanomedicine 4, 145–159 (2009).
(14) P. J. Lu, S. C. Huang, Y. P. Chen, L. C. Chiueh, D. Y. Shih, Analysis of titanium dioxide and zinc oxide
nanoparticles in cosmetics, J. Food Drug Anal., 23, 587–594 (2015).
(15) H. Shi, R. Magaye, V. Castranova, J. Zhao, Titanium dioxide nanoparticles: a review of current toxicological
data, Part Fibre Toxicol., 10, 15 (2013).
(16) S. E. Cross, B. Innes, M. S. Roberts, T. Tsuzuki, T. A. Robertson, P. McCormick, Human skin penetration
of sunscreen nanoparticles: in-vitro assessment of a novel micronized zinc oxide formulation, Skin Pharmacol.
Physiol., 20, 148–154 (2007).
(17) F. Pflücker, H. Hohenberg, E. Hölzle, T. Will, S. Pfeiffer, R. Wepf, W. Diembeck, H. Wenck, H. Gers-
Barlag, The outermost stratum corneum layer is an effective barrier against dermal uptake of topically
applied micronized titanium dioxide, Int. J. Cosmet. Sci., 21, 399–411 (1999).

(18) A. S. Dussert, E. Gooris, and J. Hemmerle, Characterization of the mineral content of a physical sunscreen
emulsion and its distribution onto human stratum corneum, Int. J. Cosmet. Sci., 19, 119–129 (1997).
(19) N. A. Monteiro-Riviere, K. Wiench, R. Landsiedel, S. Schulte, A. O. Inman, J. E. Riviere, Safety evalua-
tion of sunscreen formulations containing titanium dioxide and zinc oxide nanoparticles in UVB sun-
burned skin: an in vitro and in vivo study, Toxicol. Sci., 123, 264–280 (2011).
(20) A. O. Gamer, E. Leibold, and B. van Ravenzwaay, The in vitro absorption of microfine zinc oxide and
titanium dioxide through porcine skin, In Vitro Toxicol., 20, 301–307 (2006).
(21) J. Wu, W. Liu, C. Xue, S. Zhou, F. Lan, L. Bi, H. Xu, X. Yang, F. D. Zeng, Toxicity and penetration of
TiO2 nanoparticles in hairless mice and porcine skin after subchronic dermal exposure, Toxicol. Lett.,
191, 1–8 (2009).
(22) K. Adachi, N. Yamada, Y. Yoshida, O. Yamamoto, Subchronic exposure of titanium dioxide nanopar-
ticles to hairless rat skin, Exp. Dermatol., 22, 278–283 (2013).
(23) K. Adachi, N. Yamada, K. Yamamoto, Y. Yoshida, O. Yamamoto, In vivo effect of industrial titanium
dioxide nanoparticles experimentally exposed to hairless rat skin, Nanotoxicology, 4, 296–306 (2010).
(24) B. Kiss, T. Biro, G. Czifra, B. I. Tóth, Z. Kertész, Z. Szikszai, A. Z. Kiss, I. Juhász, C. C. Zouboulis,
J. Hunyadi, Investigation of micronized titanium dioxide penetration in human skin xenografts and
its effect on cellular functions of human skin-derived cells, Exp. Dermatol., 17, 659–667 (2008).
(25) A. Mavon, C. Miquel, O. Lejeune, B. Payre, P. Moretto, In vitro percutaneous absorption and in vivo
stratum corneum distribution of an organic and a mineral sunscreen, Skin Pharmacol. Physiol., 20, 10–20
(2007).
(26) A. V. Zvyagin, X. Zhao, A. Gierden, W. Sanchez, J. A. Ross, M. S. Roberts, Imaging of zinc oxide
nanoparticle penetration in human skin in vitro and in vivo, J. Biomed. Opt., 13(6), 064031 (2008).
(27) B. Gulson, H. Wong, M. McCall, P. Casey, J. Trotter, M. McCulloch, G. Greenoak, J. Stauber, Dermal
absorption of ZnO nanoparticles in sunscreen using the stable isotope approach, Toxicol. Lett., 180, S222
(2008).
(28) M. H. Tan, C. A. Commens, L. Burnett, P. J. Snitch, A pilot study on the percutaneous absorption of
microfine titanium dioxide from sunscreens, Australas J. Dermatol., 37, 185–187 (1996).
(29) C. Bennat and C. C. Müller-Goymann, Skin penetration and stabilization of formulations containing
microfine titanium dioxide as physical UV filter, Int. J. Cosmet. Sci., 22, 271–283 (2000).
(30) T. Pinheiro, J. Pallon, L. C. Alves, A. Veríssimo, P. Filipe, J. N. Silva, R. Silva, The influence of corneocyte
structure on the interpretation of permeation profiles of nanoparticles across skin, Nucl. Instrum. Methods
Phys. Res., B260, 119–123 (2007).
(31) J. Choi, H. Kim, J. Choi, S. M. Oh, J. Park, K. Park, Skin corrosion and irritation test of sunscreen
nanoparticles using reconstructed 3D human skin model, Environ. Health Toxicol., 29, e2014004 (2014).
(32) S. Lautenschlager, H. C. Wulf, and M. R. Pittelkow, Photoprotection, Lancet, 370, 528–537 (2007).
(33) D. B. Warheit, R. A. Hoke, C. Finlay, E. M. Donner, K. L. Reed, C. M. Sayes, Development of a base
set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management,
Toxicol. Lett., 171, 99–110 (2007).
(34) J. Unnithan, M. U. Rehman, F. J. Ahmad, M. Samim, Aqueous synthesis and concentration-dependent
dermal toxicity of TiO2 nanoparticles in Wistar rats, Biol. Trace Elem. Res., 143, 1682–1694 (2011).
(35) S. Liu, L. Xu, T. Zhang, G. Ren, Z. Yang, Oxidative stress and apoptosis induced by nanosized titanium
dioxide in PC12 cells, Toxicology, 267, 172–177 (2010).
(36) R. Li, Y. Zhenquan, and M. A. Trush, Defining ROS in biology and medicine, React. Oxyg. Species, 1(1),
9–21 (2016).
(37) R. K. Shukla, V. Sharma, A. K. Pandey, S. Singh, S. Sultana, A. Dhawan, ROS-mediated genotoxicity
induced by titanium dioxide nanoparticles in human epidermal cells, Toxicol. In Vitro, 25, 231–241
(2011).
(38) A. Manke, L. Wang, and Y. Rojanasakul, Mechanisms of nanoparticle-induced oxidative stress and
toxicity, Biomed. Res. Int., 1, 1-15 (2013).
(39) C. M. Sayes, R. Wahi, P. A. Kurian, Y. Liu, J. L. West, K. D. Ausman, D. B. Warheit, V. L. Colvin, Correlat-
ing nanoscale titani structure with toxicity: a cytotoxicity and inflammatory response study with
human dermal fibroblasts and human lung epithelial cells, Toxicol. Sci., 92, 174–185 (2006).
(40) Z. A. Lewicka, W. W. Yu, B. L. Oliva, E. Quevedo, C. V. L. Colvin, Photochemical behavior of nanoscale
TiO2 and ZnO sunscreen ingredients, J. Photochem. Photobiol. A Chem., 263, 24–33 (2013).
(41) A. Jaeger, D. G. Weiss, L. Jonas, R. Kriehuber, Oxidative stress-induced cytotoxic and genotoxic ef-
fects of nano-sized titanium dioxide particles in human HaCaT keratinocytes, Toxicology, 296, 27–36
(2012).

(42) E. Fink, Repeat photopatch test for photoxicity and photoallergy of HR 96/104702 and HR 96/104702
VeK in healthy adult male and female volunteers, part 1, 1997, Company Study No: 9066/lib.
(43) J. Chan, T. Ying, Y. F. Guang, L. X. Lin, T. Kai, Z. Y. Fang, Y. X. Ting, L. F. Xing, Y. Y. Ji, In vitro toxic-
ity evaluation of 25-nm anatase TiO2 nanoparticles in immortalizedkeratinocytecells, Biol.Trace Elem.
Res., 144, 183–196 (2011).
(44) M. Crosera, A. Prodi, M. Mauro, M. Pelin, C. Florio, F. Bellomo, G. Adami, P. Apostoli, G. De
Palma, M. Bovenzi, M. Campanini, F. L. Filon, Titanium dioxide nanoparticle penetration into the
skin and effects on HaCaT cells, Int. J. Environ. Res. Public Health, 12, 9282–9297 (2015).
(45) M. P. Vinardell, H. Llanas, L. Marics, M. Mitjans, In vitro comparative skin irritation induced by nano
and non-nanozinc oxide, Nanomaterials (Basel), 7, 56 (2017).
(46) R. Landsiedel, L. Ma-Hock, B. Van Ravenzwaay, M. Schulz, K. Wiench, S. Champ, S. Schulte, W.
Wohlleben, F. Oesch, Gene toxicity studies on titanium dioxide and zinc oxide nanomaterials used for
UV-protection in cosmetic formulations, Nanotoxicology, 4, 364–381 (2010).
(47) S. Hackenberg and N. Kleinsasser, Dermal toxicity of ZnO nanoparticles: a worrying feature of sunscreen?
Nanomedicine, 7, 461–463 (2012).
(48) J. Xu, Y. Sagawa, M. Futakuchi, K. Fukamachi, D. B. Alexander, F. Furukawa, Y. Ikarashi, T. Uchino,
T. Nishimura, A. Morita, M. Suzui, H. Tsuda, Lack of promoting effect of titanium dioxide particles on
ultraviolet B-initiated skin carcinogenesis in rats, Food Chem. Toxicol., 49, 1298–1302 (2011).
(49) Y. Sagawa, M. Futakuchi, J. Xu, K. Fukamachi, Y. Sakai, Y. Ikarashi, T. Nishimura, M. Suzui, H. Tsuda,
A. Morita, Lack of promoting effect of titanium dioxide particles on chemically-induced skin carcinogenesis
in rats and mice, J. Toxicol. Sci., 37, 317–327 (2012).
(50) M. D. Newman, M. Stotland, and J. I. Ellis, The safety of nanosized particles in titanium dioxide– and
zinc oxide–based sunscreens, J. Am. Acad. Dermatol., 61, 685–692 (2009).
(51) E. Peira, F. Turci, I. Corazzari, D. Chirio, L. Battaglia, B. Fubini, M. Gallarate, The influence of surface
change and photo-reactivity on skin permeation enhancer property of nano-TiO2 in ex vivo pig skin
model under indoor light, Int. J.Pharm., 467, 90–99 (2014).
(52) X. J. Cai, A. Woods, P. Mesquida, A. Jones, Assessing the potential for drug–nanoparticle surface interactions
to improve drug penetration into the skin, Mol. Pharm., 13, 1375–1384 (2016).
(53) R. P. Moody, B. Nadeau, and I. Chu, In vitro dermal absorption of pesticides: VI. In vivo and in vitro
comparison of the organochlorine insecticide DDT in rat, Guinea pig, pig, human and tissue-cultured
skin, Toxicol. In Vitro, 8, 1225–1232 (1994).
(54) R. M. Brand, J. Pike, R. M. Wilson, A. R. Charron, Sunscreens containing physical UV blockers can
increase transdermal absorption of pesticides, Toxicol. Ind. Health, 19, 9–16 (2003).

Comparative Assessment of Biological Activities of

Mistletoes for Cosmetic Applications: Viscum
Album Var. Coloratum (Kom.) Ohwi and
Loranthus Tanakae Franch. & Sav.
SUNG-UP CHOI, SUNG TAE KIM, DONG GYUN HAN,

YOUNG-HA HWANG, KOO YEON LEE, DONG UK KIM,
KWAN HYUNG CHO, SANG YEOB PARK, HEE-CHEOL KIM,
SEONG-BO KIM, and DONG-JIN JANG, Food and Pharmaceutical
Science, Dongnam Health University, Suwon, 50834, South Korea
(S.-U.C.), Department of Pharmaceutical Engineering, Inje University,
Gimhae 50834, South Korea (S.T.K., D.G.H., Y.-H.H., D.U.K.,
D.-J.J.), Institute of Digital Anti-Aging Healthcare, Inje University,
Gimhae 50834, South Korea (S.T.K., H.-C.K., D.-J.J.), Department of
Bio-health Technology, Kangwon National University, Kangwon 24341,
South Korea (K.Y.L.), Department of Pharmacy, Inje University, Gimhae
50834, South Korea (K.H.C.), Samyang Biopharmaceuticals Corporation,
Seongnam 13488, South Korea (S.Y.P.), CJ CheilJedang,
Life Ingredient & Material Research Institute, Suwon 16495,
South Korea (S.-B.K.)
Accepted for publication June 25, 2019.
Synopsis
Mistletoes, hemiparasites, contain many components with various biological activities and have been used in
cosmetics industry. Loranthacease (1,000 species) and Viscaceae (550 species) have the most dominant species
in mistletoes (nearly 1,600 species). It can be expected that the biological activities vary from species to
species; therefore, we have tested Viscum album var. coloratum (Kom.) Ohwi (belonging to Santalaceae) and
Loranthus tanakae Franch. & Sav. (belonging to Loranthacease) for a comparative study of their cosmetic
properties, including antioxidant, antimelanogenic, and antiwrinkle activities. As results, the ethanol extract
of L. tanakae had higher phenolic content and showed effective antioxidant activity and elastase inhibition.
Meanwhile, the ethanol extract of V. album more effectively inhibited tyrosinase. Comparing with ethanol
extracts, the water extracts of both mistletoes showed lower biological efficacy than the ethanol extracts or no
significant effect. Thus, these results show that different extracts of mistletoe have different levels of biological
activities, presumably because of the differences in their phytochemical profiles and because of the different
extraction methods used.
Address co-correspondence to Dong-Jin Jang at djjang@inje.ac.kr and Seong-Bo Kim at seongbo.kim@cj.net.

Sung-Up Choi and Sung Tae Kim contributed equally to this work as co-first authors.
235
INTRODUCTION
Mistletoes are semiparasitic plants that grow on deciduous trees, such as oak, pine, and
elm. They have been widely used in traditional medicine in Asia, Africa, and Europe (1).
Mistletoe extracts containing polyphenols, lectins (2), alkaloids (3), viscotoxins (4), and
polysaccharides (5) have various biological properties, such as antioxidant (6), antitumor
(7), antimicrobial (8), antiviral (9), and immunomodulatory activities (10,11). In addition
to herbal medicines, mistletoes have been used in dietary supplements (12) and can be
used as a cosmetic ingredient (13). Mistletoe extracts contain various phenolic derivatives
such as phenolic acids and flavonoids (14), which have been attractive ingredients for
cosmetics (15). For example, Viscum album (CAS No. 84929-55-5 in EU) contains secondary
metabolites with antioxidant properties (11). In practice, V. album (CAS No. 84929-55-5
in EU) has already been registered as a cosmetic ingredient in the Cosmetic Ingredient
Database. In Korea, mistletoe is considered a potent biological ingredient. V. album has
been studied and is listed in the Korean Cosmetic Ingredient Dictionary, which implies
that there is a great degree of interest for cosmetic and cosmeceutical applications.
Four species and four genera of the two families of mistletoe (Santalaceae and Lorantha-
ceae) are distributed across the Korean Peninsula (16). The representative species of San-
talaceae and Loranthaceae are V. album var. coloratum (Kom.) Ohwi (V. album) and Loranthus
tanakae Franch. & Sav. (L. tanakae), respectively. The former is relatively well-studied than
the latter regarding various aspects, as it is more widely distributed globally. Most stud-
ies using Korean mistletoe V. album have evaluated its medicinal properties (17,18), al-
though a few studies have focused on the biological, nutritional, and cosmeceutical
aspects (19,20). The latter has rarely been studied, in spite of its effective biological activ-
ity (21). To date, except for therapeutic approaches, mistletoes have been rarely studied
despite their great potential in health and cosmetic industries. More importantly, thus
far, there has been no comparative study among the different species of Korean mistletoes
although their compositions are dependent on species.
We aimed to investigate the potential effects of two different types of mistletoe extracts
as a cosmetic/cosmeceutical ingredient through a comparative study of V. album belong-
ing to Santalaceae and L. tanakae belonging to Loranthaceae because different species can
have a vast range of biological effects. Our study can provide a rational basis, depending
on the species and extraction methods, for biological as well as cosmetic applications.
MATERIALS AND METHODS
PREPARATION OF MISTLETOE EXTRACTS
Both V. album and L. tanakae were provided by Prof. Kooyeon Lee from the Institute of
Bioscience and Biotechnology, Kangwon National University. These mistletoes were au-
thenticated by Prof. Yi Sung Shim and Dr. Bo Duk Lee, University of Seoul. In brief, V.
album was collected from Jeongseon, Kangwon province, whereas L. tanakae was collected
from Mt. Seorak, Kangwon province, South Korea. Both samples were dried naturally
and extracted using double-distilled water (ddH2O) or ethanol (100% EtOH). 2-Diphenyl-
1-picrylhydrazyl (DPPH), mushroom tyrosinase, L-tyrosine, N-succinyl-Ala-Ala-Ala-p-
nitroanilide, porcine pancreatic elastase (PPE), potassium ferricyanide, trichloroacetic

ASSESSMENT OF BIOLOGICAL ACTIVITIES OF MISTLETOES FOR COSMETIC APPLICATIONS 237
acid, and iron chloride were purchased from Sigma-Aldrich (St. Louis, MO). All other
chemicals used were of analytical grade.
TOTAL PHENOLIC CONTENT ASSAY
The total content of phenolic compounds in the mistletoe extracts were quantitatively
determined by using the Folin–Denis method (22). In brief, 200 µL of the mistletoe ex-
tract was mixed with 200 µL of Folin–Denis reagent. After incubation for 3 min at room
temperature, 400 µL of 2M Na2CO3 and 200 µL of ddH2O were added to each sample.
Subsequently, the mixture was allowed to stand for 30 min in the dark. The total content
of phenolic compounds in the mixture was measured at 725 nm by using a microplate
spectrophotometer (Synergy HT; BIOTEK Instruments, Winooski, VT).
DPPH FREE RADICAL SCAVENGING ACTIVITY ASSAY
Free radical scavenging activity of the mistletoe mixture is based on DPPH radical scav-
enging, which is popular for evaluation of antioxidant activity of natural product. In a
given experiment, DPPH is used as a stable free radical, the color of which is changed
from purple to yellow when scavenged. Antioxidants in mistletoe extracts react with
DPPH and reduce it to DPPH-H, which can be evaluated by the degree of discoloration
depending on the scavenging potential of antioxidants in terms of hydrogen donating
ability. Based on the assay, the experiment was carried out as follows. Briefly, 100 µL of
0.2 mM DPPH was first added to 100 µL of mistletoe extract. Subsequently, the mixture
was incubated for 10 min in the dark, and the absorbance was measured at 517 nm using
microplate spectrophotometer (Synergy HT; BIOTEK). The antioxidant activity can be
easily tested and relatively compared because this reaction is basically stoichiometric with
respect to the number of hydrogen atoms absorbed. The results were calculated by using
the following equation: free radical scavenging activity (%) = [1 − (experimental value −
blank)/control] × 100 (%).
REDUCING POWER ASSAY
For evaluating the antioxidant activity of mistletoes, a reducing power assay was performed
by using the Oyaizu method (23). In brief, 100 µL of 0.2 M sodium phosphate buffer (pH
6.6) was mixed with 100 µL of the mistletoe extract. Subsequently, 100 µL of 10% (w/v)
potassium ferricyanide solution was added to the mixture. The mixture was incubated for
20 min at 50°C. Next, 100 µL of 10% (w/v) trichloroacetic acid was added and the mixture
was centrifuged at 13,400 g. The solution in the upper layer was collected and allowed to
react with 100 µL of 0.1% iron chloride solution and the absorbance was measured at 700
nm by using microplate spectrophotometer (Synergy HT; BIOTEK).
TYROSINASE INHIBITION ASSAY
For evaluating antimelanogenesis, a tyrosinase inhibition assay was performed, as tyrosinase

plays an important role in melanogenesis. According to previous literatures, we chose

mushroom tyrosinase, which is frequently used because of its commercial availability

(24,25). In brief, the test substance was dissolved in 0.1 M sodium phosphate buffer (pH 6.8).
The reaction mixture was prepared with different concentrations of each mistletoe extract
and phosphate buffer (200 µL) was mixed with 20 µL of the extract. Next, 20 µL of mushroom
tyrosinase (2,000 U/mL; Sigma-Aldrich) and 20 µL of 1.5 mM L-tyrosinase were added.
The reaction mixture was incubated for 10 min at 37°C. The absorbance was measured at
490 nm by using a microplate spectrophotometer (Synergy HT; BIOTEK). Tyrosinase
inhibition activity was expressed as the percentage inhibitory of tyrosinase, calculated as
{[1 − (experimental value − blank)/control × 100(%)]}, where experimental value is the
absorbance of the sample wells, blank is the absorbance of the sample wells without tyrosinase,
and control is the absorbance of the control wells with tyrosinase but without sample.
ELASTASE INHIBITION ASSAY
PPE was assayed via a spectrophotometric method with the substrate N-succinyl-(Ala)3-
p-nitroanilide, which was prepared using 50 mM Tris–Cl buffer (pH 8.6). The reaction
mixture was incubated for 10 min at 25°C, which contained 100 µL of the mistletoe ex-
tract at different concentrations, 100 µL of N-succinyl-(Ala)3-p-nitroanilide, and 50 µL of
elastase (0.6 U/mL). The absorbance was measured at 410 nm by using a microplate
spectrophotometer (Synergy HT, BIOTEK). Elastase inhibition activity was expressed as
the percentage inhibitory of elastase, calculated as {[1 − (experimental value − blank)/
control × 100(%)]}, where experimental value is the absorbance of the sample wells,
blank is the absorbance of the sample wells without elastase, and control is the absorbance
of the control wells with elastase but without sample.
STATISTICS
Experimental data were expressed as mean ± standard deviation and were analyzed by
GraphPad Prism (GraphPad Software, San Diego, CA). Statistical significance was deter-
mined based on p values. Statistically significant differences between the groups were deter-
mined by the Student’s t-test; p values  0.05 were considered statistically significant.
RESULTS AND DISCUSSION
TOTAL CONTENT OF PHENOLIC COMPOUNDS
Previous studies have shown that mistletoes have high levels of phenolic compounds
among various bioactive components, which have antioxidant activities (26). Similar to
other mistletoes, Korean mistletoes also contain considerable amounts of phenolic com-
pounds (27). To determine the amount of each phenolic compound in V. album and L.
tanakae, the Folin–Denis method was used (Table I). Because the amount of polyphenols
may vary depending on the extraction solution, both mistletoes were extracted using dif-
ferent solutions: ddH2O and 100% ethanol, as described in the section of preparation of
mistletoe extracts, Materials and Methods. The water extracts of V. album and L. tanakae
contained 93.58 ± 2.85 mg/g and 139.83 ± 3.20 mg/g, of phenolic compounds, respec-

Table I
Total Amounts of Phenolic Compounds in V. album and L. tanakae
Total amounts of phenolic

Types of mistletoe extract compounds (mg/g)
V. album (Viscumalbum var. coloratum (Kom.) Ohwi Water extract 93.58 ± 2.85
Ethanol extract 160.81 ± 6.28
L. tanaka (Loranthus tanakae Franch. & Sav.) Water extract 139.83 ± 3.20
Ethanol extract 233.19 ± 4.30
tively, whereas each ethanol extract of V. album and L. tanakae contained 160.81 ± 6.28
mg/g and 233.19 ± 4.30 mg/g, respectively (Table I). These data show that L. tanakae has
a higher amount of phenolic compounds (about 1.45–1.49 times) than V. album. In par-
ticular, ethanol is more effective in extracting phenolic compounds from mistletoes than
water. The V. album ethanol extract had 1.72 times more phenolic compounds than the
water extract. Alternatively, the L. tanakae ethanol extract had 1.67 times more phenolic
compounds than the water extract. Thus, the total amount of phenolic compounds in
mistletoes was different depending on their species and extracting solutions. Based on
these results, we presumed that mistletoe extracts have antioxidant activity because phe-
nolic compounds in natural extracts have important antioxidant properties, leading to
prevention and treatment of skin diseases (28). To further clarify this, DPPH scavenging
activity assay and reducing power analysis were additionally performed.
DPPH FREE RADICAL SCAVENGING ACTIVITY
To assess the antioxidant activity of the mistletoe extracts, DPPH free radical scavenging
assay was used, as described in the Materials and Methods section. Using the DPPH free
radical scavenging assay, it was determined that stable DPPH radicals encounter a pro-
ton-donating substrate in the sample, which lead to radical scavenging. Four mistletoe
extracts (water and ethanol extracts of V. album or L. tanakae, respectively) were evaluated
with concentrations ranging from 100 to 1,000 µg/mL. L-Ascorbic acid was used as the
standard, which is a well-known antioxidant agent. As shown in Figure 1, the ethanol
extracts showed a relatively high scavenging effect compared with the water extracts of
both mistletoes, except for the ethanol extract of V. album (100 µg/mL). Its scavenging
effect (%) was above 87%, which was effective, compared with that of L-ascorbic acid.
Interestingly, the ethanol extract of L. tanakae also showed a high scavenging efficacy
(85.52%) even at low concentrations (100 µg/mL) compared with the ethanol extract of
V. album (48.48%), which indicates that the extract of L. tanakae may contain more natural
components with antioxidant properties. As concentration of mistletoes increases, scaveng-
ing effects were slightly increased and saturated. Above 300 µg/mL, both mistletoe ex-
tracts with EtOH showed statistically similar effects. To complement this, reducing
power analysis was additionally performed.
REDUCING POWER ANALYSIS
The antioxidant activities of both mistletoes were also evaluated using the widely used re-
ducing power assay (Figure 2). Using this assay, it was determined that reducing antioxidant

Figure 1. DPPH scavenging activity using the DPPH scavenging assay, V. album and L. tanakae were com-
pared in the range of 100–1,000 µg/mL. L-ascorbic acid was used as a standard.
power measures the reducing ability against ferric ion, which indicates the ability of hy-
drolysates to donate an electron to the free radical in the extracts. Experimentally, the
reduction ability is determined by measuring the colored complex at 700 nm as described
in the Materials and Methods section. The yellow color of the sample changes to a color
between green and blue, depending on the reducing power of each sample in the presence
of reducers (antioxidants). The antioxidants reduce the Fe3+/ferricyanide complex to the
ferrous form (Fe2+), which was monitored at 700 nm. Figure 2 shows the reducing power
of the V. album and L. tanakae extracts: V. album water extract, V. album ethanol extract,
L. tanakae water extract, and L. tanakae ethanol extract. Ascorbic acid was used as the
standard. The absorbance at 700 nm increased in a dose-dependent manner, which did not
correlate well with the DPPH free radical scavenging assay (Figure 1). In the experiment,
Figure 2. Reducing power analysis using the reducing power assay, V. album and L. tanakae were compared
in the range of 100–1,000 µg/mL. L-ascorbic acid was used as a standard.

the ethanol extract of L. tanakae showed a higher absorbance than that from V. album. Fur-
thermore, the water extract of L. tanakae showed a higher absorbance than that from V.
album. Based on the absorbance, the ethanol extract of L. tanakae showed a similar antioxidant
activity to that of ascorbic acid in the range of 300 to 1,000 µg/mL. Ascorbic acid had a
marginally higher reducing power but was statistically not significant. Taken together,
our results demonstrate that the antioxidant activity depends on the species and extrac-
tion conditions. In addition, antioxidant components as well as the phenolic compounds
might be present in these extracts, considering the slight differences in the result values.
INHIBITION OF TYROSINASE
Figure 3 shows the inhibition of tyrosinase activity by the mistletoe extracts of V. album
and L. tanakae in the range of 100 to 1,000 µg/mL. Each measurement was compared
with that of arbutin, a glycosylated hydroquinone-inhibiting tyrosinase, as the standard.
The ethanol extracts of both mistletoes inhibited tyrosinase, whereas their water extracts
showed no significant inhibition. The ethanol extracts of both mistletoes had a lower
inhibitory effect on tyrosinase than arbutin; the ethanol extract of V. album showed a
higher inhibitory effect. When 1,000 µg/mL of the extract was used, the inhibitory effect
was 35.96%, which was similar to the value obtained when arbutin was used at 500 µg/mL.
These results show that the ethanol extracts contain more natural compounds with anti-
tyrosinase activity (e.g., tyrosinase inhibitor) and the extract of V. album is more effective
than that of L. tanakae. In comparison, the water extracts of V. album and L. tanakae did not
show any significant effects, whereas their ethanol extracts inhibited tyrosinase. Physiologi-
cally, tyrosinase, a glycoprotein in melanosomes, plays an important role in melanogene-
sis; tyrosinase is involved in the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine
(L-DOPA) and the subsequent conversion to L-DOPA-quinone (29). The maintenance of
melanogenesis is critical because a high level of tyrosinase activity causes hyperpigmenta-
tion disorders, such as melasma, lentigo, and drug/UV-induced hyperpigmentation (30).
Even if not diseases, abnormal hyperpigmentation is rarely preferred aesthetically. Skin
lightening (in Asia)/brightening (in West), through removal of dark spots or uneven skin
Figure 3. Inhibition of tyrosinase activity using the tyrosinase inhibitory assay, V. album and L. tanakae were
compared in the range of 100–1,000 µg/mL. Arbutin was used as a standard.

tone due to the overproduction of melanin, has been the recent area of interest of in the
cosmetic industry (31). In light of this, the suppression of tyrosinase activity is a potential
strategy for inhibiting hyperpigmentation, and mistletoe extract could be useful in in-
hibiting tyrosinase activity, although bioactives with hypopigmentation effect are different
depending on types as well as extracting methods.
INHIBITION OF ELASTASE
Elastase inhibitory effect was analyzed using the elastase inhibition assay, as described
in Experimental. Ursolic acid was used as the standard. Both extracts of V. album and
L. tanakae showed considerably low efficacies with respect to inhibition of elastase at
100 µg/mL; however, there were no significant differences among the test extracts. At
concentrations above 300 µg/mL, the ethanol extract of L. tanakae effectively inhibited
elastase, which was even higher than the efficacy of ursolic acid at 300 and 500 µg/mL
(p < 0.05). Both extracts of L. tanakae were comparable with those of V. album at 100–
1,000 µg/mL. The ethanol extract of L. tanakae showed a higher inhibitory effect than the
water extract of L. tanakae. The effects of their ethanol extracts were 3.2, 2.4, and 1.9
times higher than those of their water extracts at 300, 500, and 1,000 µg/mL, respec-
tively. Our findings revealed that L. tanakae demonstrated a higher inhibitory effect on
elastase activity than V. album. In a biological system, elastase, a protease secreted by fi-
broblast, reduces the elasticity and tortuosity of dorsal skin by breaking down elastic fibers.
An increase in elastase activity can cause various diseases, such as cystic fibrosis and psoriasis,
and delay wound healing and trigger wrinkle formation through the cleavage of collagen
and elastic fibers (32). Therefore, the inhibition of elastase can prevent these diseases as
well as the damage of elastic fibers in the skin, leading to reduction in wrinkle formation.
As such, the inhibition of key enzymes, such as tyrosinase and elastase, is important in
biomedical and esthetic aspects. As described, it is meaningful that inhibition efficacy of
mistletoe extracts on tyrosinase and elastase (Figures 3 and 4, respectively). Based on their
inhibitory effects, both V. album and L. tanakae are attractive natural sources with numer-
ous biological activities, which could be applied to various fields such as cosmetics and
cosmeceuticals.
Figure 4. Inhibition of elastase activity using the elastase inhibitory assay, V. album and L. tanakae were
compared in the range of 100–1,000 µg/mL. Ursolic acid was used as a standard.

Recently, the cosmetic market has been emerged and driven toward natural, organic ingre-
dients because of consumers’ concerns about synthetic ingredients. A number of these phy-
tochemicals are today being developed, used, or considered for antiaging effects in spite
of synthetic ingredient developed. For example, although synthetic antioxidants have
been reported to be effective for inhibiting oxidation, they have been restricted because
of possible health risks and toxicity (33). Also, cosmetic market and consumers still con-
tinue to demand natural cosmetic ingredient (34). Thus, the use of natural ingredients
from botanicals accomplishes for preventing degradation of natural ingredients in cos-
metic product and for protecting the skin cells from being damaged as well as aged (35).
Like other phytochemicals, a previous report demonstrated that mistletoe extract could
be a safe natural antioxidant and antimicrobial agent in food products (36), which are also
useful biological activities as cosmetic ingredients. In light of this, in our study, we be-
lieve mistletoe extracts can provide potential biological function in cosmetic products to
beautify and maintain the physiological balance of our skin.
CONCLUSIONS
In this study, we evaluated biological activities of mistletoes in aspect of cosmetics. Both
V. album and L. tanakae showed antioxidant activities and inhibitory effects on tyrosinase
and elastase, which are important for preventing skin aging. Depending on mistletoe
family and extracting solvent, they showed different levels of these activities because of
different phytochemical profiles, suggesting the importance of choosing cosmetic ingre-
dients. Our findings suggest that appropriate mistletoes and extraction conditions should
be carefully chosen to broaden cosmetic and cosmeceutical applications.
DISCLOSURE
The authors declare no conflict of interest in this work.
ACKNOWLEDGMENTS
This research was supported by the Ministry of Trade, Industry and Energy (MOTIE),
KOREA, through the Education Program for Creative and Industrial Convergence (Grant
Number N0000717).
REFERENCES
(1) M. I. Choudhary, S. Maher, A. Begum, A. Abbaskhan, S. Ali, and A. Khan, Characterization and anti-
glycation activity of phenolic constituents from Viscum album (European Mistletoe), Chem. Pharm. Bull.
(Tokyo), 58, 980–982 (2010).
(2) H. Franz, P. Ziska, and A. Kindt, Isolation and properties of three lectins from mistletoe (Viscum album
L.), Biochem. J., 195, 481–484 (1981).
(3) T. A. Khwaja, J. C. Varven, S. Pentecost, and H. Pande. Isolation of biologically active alkaloids from
Korean mistletoe Viscum album, coloratum, Experientia, 36, 599–600 (1980).
(4) S. Romagnoli, R. Ugolini, F. Fogolari, G. Schaller, K. Urech, M. Giannattasio, L. Ragona, and H. Molinari,
NMR structural determination of viscotoxin A3 from Viscum album L., Biochem. J., 350, 569–577 (2000).

(5) E. A. Mueller and F. A. Anderer, A Viscum album oligosaccharide activating human natural cytotoxicity
is an interferon γ inducer, Cancer Immunol. Immunother., 32, 221–227 (1990).
(6) S. Zorofchian Moghadamtousi, M. Hajrezaei, H. Abdul Kadir, and K. Zandi, Loranthus micranthus Linn.:
biological activities and phytochemistry. Evid. Based Complement. Alternat. Med., 2013, 273712 (2013).
(7) S. Y. Han, C. E. Hong, H. G. Kim, and S. Y. Lyu, Anti-cancer effects of enteric-coated polymers con-
taining mistletoe lectin in murine melanoma cells in vitro and in vivo, Mol Cell Biochem., 408, 73–87
(2015).
(8) S. N. Kang, Ethanol extracts from mistletoe (Viscum album L.) act as natural antioxidants and antimicro-
bial agents in uncooked pork patties during refrigerated storage, Asian-Australas J. Anim. Sci., 29,
109–118 (2016).
(9) J. Nazaruk and P. Orlikowski, Phytochemical profile and therapeutic potential of Viscum album L., Nat.
Prod. Res., 30, 373–385 (2016).
(10) A. Büssing, Immune modulation using mistletoe (Viscum album L.) extracts Iscador, Arzneimittelforsc-
hung, 56, 508–515 (2016).
(11) E. ÖnayUçar, A. Karagöz, and N. Arda, Antioxidant activity of Viscum album ssp., Fitoterapia, 77, 556–
560 (2006).
(12) K. Kado, A. Forsyth, P. R. Patel, and J. A. Schwartz, Dietary supplements and natural products in
breast cancer trials, Front. Biosci. (Elite Ed)., 1, 546–567 (2012).
(13) A. S. Ribeiro, M. Estanqueiro, M. B. Oliveira, and J. M. S. Lobo, Main benefits and applicability of
plant extracts in skin care products, Cosmetics, 2, 48–65 (2015).
(14) S. Vicas, J. Prokisch, O. D. Rugina, and C. Socaciu, Hydrophillic and lipophilic antioxidant activities
of mistletoe (Viscum album) as determined by FRAP method, Not. Bot. Horti Agrobot. Cluj Napoca, 37,
112–116 (2009).
(15) O. V. Zillich, U. Schweiggert-Weisz, P. Eisner, and M. Kerscher, Polyphenols as active ingredients for
cosmetic products, Int. J. Cosmetic Sci., 37, 455–464 (2015).
(16) C. S. Kim, S. Y. Kim, B. Y. Sun, and J. S. Yi, A review of the taxonomic and ecological characteristics
of Korean mistletoe types (Viscum, Korthalsella, Loranthus and Taxillus), Korean J. Pl. Taxon., 43, 81–89
(2013).
(17) T. J. Yoon, Y. C. Yoo, O. B. Choi, M. S. Do, T. B. Kang, S. W. Lee, I. Zuma, and J. B. Kim, Inhibitory
effect of Korean mistletoe (Viscum album coloratum) extract on tumour angiogenesis and metastasis of
haematogenous and non-haematogenous tumour cells in mice, Cancer Lett., 97, 83–91 (1995).
(18) J. Y. Lee, J. Y. Kim, Y. G. Lee, S. E. Byeon, B. H. Kim, M. H. Rhee, A. Lee, M. Kwon, S. Hong, and
J. Y. Cho, In vitro immunoregulatory effects of Korean mistletoe lectin on functional activation of mono-
cytic and macrophage-like cells, Biol. Pharm. Bull., 30, 2043–2051 (2007).
(19) B. K. Kim, M. J. Choi, K. Y. Park, and E. J. Cho, Protective effects of Korean mistletoe lectin on radi-
cal-induced oxidative stress, Biol. Pharm. Bull., 33, 1152–1158 (2010).
(20) S. Y. Kim, E. J. Yang, Y. K. Son, J. H. Yeo, and K. S. Song, Enhanced anti-oxidative effect of fermented
Korean mistletoe is originated from an increase in the contents of caffeic acid and lyoniresinol, Food
Funct., 7, 2270–2277 (2016).
(21) Y. K. Kim, Y. S. Kim, S. U. Choi, and S. Y. Ryu. Isolation of flavonol rhamnosides from Loranthus
tanakae and cytotoxic effect of them on human tumor cell lines, Arc. Pharm. Res., 27, 44–47 (2004).
(22) M. Oyaizu, Studies on products of browning reactions: antioxidative activities of products of browning
reaction prepared from glucosamine, Jpn. J. Nutr., 44, 307–315 (1986).
(23) S. I. Rahmawati, K. Ishimaru, D. Hou, and N. Hayashi, Antioxidant activity and phenolic content of
mistletoe extracts following high-temperature batch extraction, Food Sci. Technol. Res., 20, 201–206
(2014).
(24) T. Pillaiyar, M. Manickam, and V. Namasivayam, Skin whitening agents: medical chemistry perspective
of tyrosinase inhibitors, J. Enzyme Inhib. Med. Chem., 32, 403–425 (2017).
(25) R. Uchida, S. Ishikawa, and H. Tomoda, Inhibition of tyrosinase activity and melanine pigmentation by
2-hydroxytyrosol, Acta Pharm. Sin. B, 4, 141–145 (2014).
(26) T. Jang, Y. Yoo, Y. Hwang, H. Kim, and H. Woo, Total polyphenol content and antioxidative activities
of mistletoe (Viscum album) extracts by supercritical carbon dioxide, J. Korean Soc. Food Sci. Nutr., 39,
20–24 (2010).
(27) M. S. Padda and D. H. Picha, Methodology optimization for quantification of total phenolics and indi-
vidual phenolic acids in sweet potato (Ipomoea batatas L.) roots, J. Food Sci., 72, C412–C416 (2007).
(28) M. Działo, J. Mierziak, U. Korzun, M. Preisner, J. Szopa, and A. Kulma, The potential of plant pheno-
lics in prevention and therapy of skin disorders, Int. J. Mol. Sci., 17, 160 (2016).

(29) S. Y. Lee, N. Baek, and T. Nam, Natural, semisynthetic and synthetic tyrosinase inhibitors, J. Enzyme
Inhib. Med. Chem., 21, 1–13 (2016).
(30) B. H. Bin, S. T. Kim, J. Bhin, T. R. Lee, and E. G. Cho, The development of sugar-based anti-melanogenic
agents, Int. J. Mol. Sci., 17, 583 (2016).
(31) R. Sarkar, P. Arora, and K. V. Garg, Cosmeceuticals for hyperpigmentation: what is available? J. Cutan.
Aesthet. Surg., 6, 4–11 (2013).
(32) N. Tsuji, S. Moriwaki, Y. Suzuki, Y. Takema, and G. Imokawa, The role of elastases secreted by fibro-
blasts in wrinkle formation: implication through selective inhibition of elastase activity, Phytochem.
Photobiol., 74, 283–290 (2001).
(33) B. Sun and M. Fukuhara, Effects of co-administration of butylated hydroxytoluene, butylated hydroxy-
anisole and flavonoids on the activation of mutagens and drug-metabolizing enzymes in mice, Toxicology,
122, 61–72 (1997).
(34) T. Hansen, M. S. Risborg, and C. D. Steen, Understanding consumer purchase of free-of cosmetics: a
value-driven TRA approach, J. Consum. Behav., 11, 477–486 (2012).
(35) A. Ribeiro, M. Estanqueiro, M. Oliveira, and J. Sousa Lobo, Main benefits and applicability of plant
extracts in skin care products, Cosmetics, 2, 48–65 (2015).
(36) S. Kang, Ethanol extracts from mistletoe (Viscum album L.) act as natural antioxidants and antimicrobial
agents in uncooked pork petties during refrigerated storage, Asian Australas. J. Anim. Sci., 29, 109–118
(2016).

In Vitro Penetration of Petrolatum in Stratum Corneum from

Bodywash Formulation
APIPA WANASATHOP, ZHANQUAN SHI, Q. CHING STELLA,

KARL S. WEI, PETER B. STYCZYNSKI, CHUIYING LI, and
S. KEVIN LI, Division of Pharmaceutical Sciences, James L. Winkle
College of Pharmacy, University of Cincinnati, Cincinnati, Ohio
(A.W., Z.S., S.K.L.), Procter & Gamble Co., Mason,
Ohio (Q.C.S., K.S.W., P.B.S., C.L.)
Accepted for publication July 16, 2019.
Synopsis
Petrolatum is a mixture of hydrocarbons that is widely used as a moisturizer. It is incorporated in bodywash
formulations to help hydrate and maintain healthy skin appearance. The aim of this study was to investigate
skin deposition and penetration of petrolatum from an experimental bodywash system consisting of
petrolatum in vitro. Experiments were performed using cadaver split-thickness skin and Franz diffusion cells.
Radiolabeled 14C-dotriacontane (C32-alkane) was used as a model permeant for petrolatum. The bodywash
was applied on the skin and subsequently rinsed. At predetermined time points, the skin was wiped to
remove the residual material on the surface, and tape-stripping was performed. Petrolatum was observed to
deposit from the bodywash when applied on split-thickness skin with simulated rinsing. Petrolatum then
penetrated into the stratum corneum and was detected at the depth of 12 tape-stripping and in the epidermis.
The bodywash formulation could provide significant deposition and penetration of petrolatum into the
stratum corneum at 1–72 hours postapplication.
INTRODUCTION
Bodywash is a widely used cosmetic product and is directly applied to the human skin.
Its purpose is to cleanse and condition the skin. The active ingredients in bodywash are
surfactants that remove dirt and soil on the skin. Surfactants also remove protective oils
that are naturally present. This can lead to dry and uncomfortable skin after washing (1).
Therefore, moisturizing bodywashes are formulated to improve skin hydration and main-
tain a healthy skin surface condition.
Petrolatum is a semisolid widely used in cosmetics as a moisturizer. It is a complex mixture
of hydrocarbons obtained from dewaxing paraffinic residual oil (2). It was suggested that
petrolatum can accelerate the skin surface barrier recovery and replace intercellular lipids
by filling in the stratum corneum interstices (3). Petrolatum was also found to induce
Address all correspondence to Apipa Wanasathop at wanasaaa@mail.uc.edu.

247
expression of key barrier differential markers such as filaggrin and loricrin and upregulate
antimicrobial peptides and innate immune genes, modulating skin antimicrobial activity
(4). Petrolatum in bodywash formulation could therefore benefit the function of the skin
surface barrier.
The aim of this in vitro study was to investigate skin deposition and penetration of petro-
latum into the stratum corneum from a bodywash system consisting of petrolatum. The
experiments were performed with human cadaver skin in Franz diffusion cell in vitro.
Radiolabeled 14C-dotriacontane (C32-alkane) was the model permeant for petrolatum
that was mixed with the bodywash before the study. After bodywash application and
rinse-off from the skin surface, the amount of petrolatum deposited on the skin was de-
termined by the amount in the rinse-off solution and mass balance. Tape-stripping was
then performed to determine the amount and depth of petrolatum penetration into the
stratum corneum. The epidermis was separated from the dermis by dissection to deter-
mine the amounts of petrolatum in these skin layers.
MATERIALS AND METHODS
MATERIALS
14
C-dotriacontane (specific activity 10 mCi/mmol) was purchased from American Radio-
labeled Chemicals (Saint Louis, MO). 3H-water (specific activity 1 mCi/g) was purchased
from Moravek Biochemicals (Brea, CA). Ultima Gold scintillation cocktail was purchased
from PerkinElmer (Waltham, MA). Glyceryl monooleate was purchased from BASF Cor-
poration (Florham Park, NJ). Petrolatum was purchased from Sonneborn (Petrolia, PA).
An experimental bodywash base without petrolatum and glyceryl monooleate was obtained
from the Procter & Gamble Co. (P&G). Hexane and bovine serum albumin, fraction V
(BSA) were purchased from Fisher Scientific (Fair Lawn, NJ). Ethanol was purchased from
Pharmco-Aaper (Shelbyville, KY). Polysorbate 20 (Tween 20) was purchased from
Uniqema (Wilmington, DE). Sodium azide (NaN3) was purchased from Acros Organics
(Morris Plains, NJ). Phosphate-buffered saline (PBS: 0.01 M phosphate buffer, 0.0027 M
potassium chloride, and 0.137 M sodium chloride), pH 7.4, was prepared using PBS
tablets and deionized water as described by the manufacturer (MP Biomedicals, Solon,
OH) and preserved using 0.02% NaN3.
FRANZ DIFFUSION CELL SETUP
Posterior torso split-thickness cadaver skin (thickness ~0.01–0.04 cm) from eight skin
donors was obtained from the New York Firefighters Skin Bank (New York, NY) in pack-
ages at −80°C. The ages of the skin donors were between 45 and 70 years. The split-
thickness skin was thawed in PBS according to the instructions on the skin packages and
stored at −20°C until use. Before use, the skin sample was cut into 1.5 × 1.5 cm pieces
and equilibrated in PBS at room temperature for 2 h. The vertical Franz diffusion cell
used in this study had a diffusional area of 0.71 cm2. The fully hydrated skin sample was
mounted on the cell between the donor and receptor chambers, with the stratum corneum
side of the skin facing upward to the environment. The dermis side of the skin sample

IN VITRO PENETRATION OF PETROLATUM IN STRATUM CORNEUM 249
was in contact with 5 mL receptor medium, which consisted of PBS containing 2% w/v
BSA and 0.02% NaN3. The use of BSA in the receptor chamber increased the solubility
of lipophilic compounds in the chamber and resembled the condition of dermis in vivo for
the penetration study (5). Each diffusion cell was placed on a thermostated heating and
stirring module and maintained at 37°C at the receptor, resulting in a stratum corneum
surface temperature of 34 ± 1°C. A micro magnetic stir bar was placed in the receptor
chamber to ensure stirring throughout the experiments. The relative humidity level in
the room during the penetration experiments was approximately 20–30%. The skin sam-
ple was equilibrated for 2 h, followed by a prescreening water permeability assay to con-
firm the skin integrity before the penetration experiments. Briefly, 0.15 mL of 3H-water
was added onto each skin in the diffusion cells. Five minutes postdosing, cotton swabs
were used to remove excess 3H-water from the donor chambers. After 1 h, 2-mL samples
from the receptor chamber were collected in scintillation vials and the solutions were
mixed with 10 mL scintillation cocktail and analyzed using a liquid scintillation counter
(Beckman Coulter LS 6500 multipurpose scintillation counter, Fullerton, CA). Skin sam-
ples with water permeation values greater than 1.6 µL/cm2 were discarded. Following the
prescreening procedure, the receptor solution was replaced with fresh receptor medium
twice to remove the residual radioactivity. The skin sample was then allowed to equili-
brate in the heating and stirring module overnight.
FINITE DOSE SKIN PENETRATION STUDY WITH SPLIT-THICKNESS SKIN SAMPLES
Bodywash preparation. The experimental bodywash was prepared by mixing the bodywash
base (ingredients include water, sodium trideceth sulfate, sodium chloride, cocamidopro-
pyl betaine, trideceth-3, fragrance, guar hydroxypropyltrimonium chloride, sodium ben-
zoate, xanthan gum, disodium ethylenediaminetetraacetic acid, citric acid, sodium hydroxide,
acrylates/C10-30 alkyl acrylate crosspolymer, methylchloroisothiazolinone, and methyl-
isothiazolinone) with 9.8% petrolatum, 0.2% glyceryl monooleate, and 14C-dotriacontane
(C32 alkane, as the model permeant for petrolatum). Specifically, 29.4 mg of petrolatum
and 0.6 mg glyceryl monooleate were mixed with 30 µL hexane containing the radiola-
beled dotriacontane in a small glass vial by hand using a stainless steel spatula for 3 min.
The mixture was then left in a fume hood to allow hexane evaporation for approximately
50 min. After that, 270 mg bodywash base was added to the mixture and mixed by the
spatula for 3 min. The size of petrolatum particles in the mixture was examined by light
microscopy to ensure uniform sizes in the range of 0.10 ± 0.05 mm. The final concentra-
tion of petrolatum was 9.8% as particles dispersed in the bodywash base with 10 µCi
14
C-dotriacontane as the tracer in 300 mg bodywash.
Applying and rinsing method. To simulate a skin washing condition, 5 µL dose of body-
wash was applied to the stratum corneum side of the skin sample using a positive dis-
placement pipette. The bodywash was spread evenly on the skin with a glass rod (5 mm
diameter, with a smooth ground glass flat tip). The flat surface of the glass rod tip was
moved over the skin in circular motion for 30 s. After the bodywash was applied and
left on the skin for another 30 s, i.e., a total of 1-min contact, 0.5 mL water was added
to the donor chamber of the Franz diffusion cell. A pipette was used to create water
motion in the donor chamber by pipetting the water “in and out” of the chamber three
times over 10 s (approximately one “in and out” action every 3 s). Immediately after
rinsing, the solution in the donor chamber was removed and placed into a collection

vial. This rinsing step was repeated three times to simulate a washing scenario in the
rinse-off protocol. After the rinsing step, the pipette tip used in this step was rinsed
twice with 0.5 mL hexane and twice with 0.5 mL water. The used glass rod was also
rinsed twice with 1 mL hexane and twice with 2 mL water. The rinse-off solution, the
pipette tip rinsing solution, and glass rod rinsing solution were then combined and
mixed with scintillation cocktail and analyzed using the liquid scintillation counter.
To check the weight and amount of 14C-dotriacontane in the applied dose, 5 µL of the
bodywash was placed in a container and weighed with an analytical balance, and the
content in the container was mixed with scintillation cocktail and analyzed using the
liquid scintillation counter.
Sample collection and tape-stripping. In the penetration study, 2-mL samples of receptor medium
were collected at 1, 24, or 72 h postapplication of the bodywash. After sampling, the dif-
fusion cells were disassembled and each skin sample was wiped twice with Whatman
filter paper soaked with 0.5% Tween 20 in PBS and once with filter paper soaked with
70%/30% ethanol/deionized water (v/v) to remove the remaining bodywash on the skin
surface. After the filter wipes, tape-stripping was performed to remove a portion of the
stratum corneum. To determine the penetration profile of petrolatum in the stratum cor-
neum, tape-stripping was performed up to 12 times at 1, 24, or 72 h postapplication of
the bodywash: in the 1-h experiments, five times tape-stripping were applied, and in
selected skin samples, 12 times tape-stripping were applied; in the 24 and 72 h experi-
ments, 5 times tape-stripping were applied. For the tape-stripping, the edges of the skin
were covered with adhesive tapes (fixing tapes), leaving a central square hole of 1 × 1 cm2
(the available area for tape-stripping). Standard D-Squame® disc (Cuderm Corporation,
Dallas, TX), a diameter of 2.2 cm and an area of 3.8 cm2, was pressed onto the skin for
5 s using the D-Squame® pressure instrument, and the D-Squame® disc was quickly re-
moved. After tape-stripping, the skin was cut to isolate the diffusion region of the
epidermis using a cork borer, and the epidermis was separated from the remaining
skin using a pair of forceps. Each skin section was dissolved separately in 1 mL Solvable
(PerkinElmer Life and Analytical Sciences, Boston, MA): each tissue was mixed with
Solvable in a scintillation vial and kept in an oven at 50°C overnight for the solubiliza-
tion of the sample. The donor chamber was washed with hexane to remove the residual
cosmetic ingredients on its surface. The receptor collections, filter paper wipes, tapes
from tape-stripping, solubilized skin sections, and donor chamber wash solutions were
mixed with scintillation cocktail and analyzed separately using the scintillation counter.
The samples were discarded when one of the following occurred: tearing of the skin be-
fore Tape 3 in the tape-stripping procedure or an outlier from the statistical analysis in an
experiment.
DATA ANALYSIS
Figure 1A summarizes the experimental procedure in the present study. The net applied dose
of dotriacontane was the remaining amount of dotriacontane on the skin before the rinse-
off and was calculated by the difference between the total applied dose and the amount
remaining on the glass rod (i.e., the net applied dose of dotriacontane = the total applied
dose of dotriacontane minus the amount in the glass rod rinse). The total applied dose of
dotriacontane was determined by mixing 5-µL dose of bodywash directly with the scintil-
lation cocktail. The % applied dose in each compartment was calculated by the amount

Figure 1. (A) Schematic diagram of the experimental procedure to evaluate petrolatum deposition on the
skin surface and its penetration into the stratum corneum from the bodywash formulation, and (B) the
amount of petrolatum measured in different compartment of the rinsed-off solution, the net applied dose, and
the total 5-µL applied dose in the experiments.
of dotriacontane in the compartment normalized by the total applied dose of dotriacon-

tane (total applied radioactivity in 5 µL from the pipette). The amount of petrolatum
normalized by the skin area was calculated by the amount of dotriacontane in each com-
partment, specific activity of dotriacontane for petrolatum, weight of petrolatum in the
5-µL dose bodywash formulation, and the diffusion area of the Franz diffusion cell (i.e.,
the amount of petrolatum in µg = the amount of dotriacontane divided by the specific
activity of dotriacontane and 0.71 cm2). Total recovery was calculated using mass balance.
Figure 1B summarizes the compartments to be investigated in the present petrolatum
penetration study. All experiments were performed with at least four skin donors for each
condition.

STATISTICAL ANALYSIS
The means ± standard errors of the means (SEM) of the data are presented. Data analyses
and statistical tests (one-way Analysis of variance, ANOVA) were performed using
Microsoft Excel (Redmond, WA) and GraphPad InStat (San Diego, CA), respectively,
and a difference of p < 0.05 was considered statistically significant. Outlier data points
(~9% of the data) of the amounts of dotriacontane (as % applied dose) in the tapes and
epidermis in each set of experiments were identified using an outlier test (Outlier
calculator, GraphPad Software, La Jolla, CA).
RESULTS
DEPOSITION AND PENETRATION OF PETROLATUM INTO THE STRATUM CORNEUM
The average weight of bodywash formulation (5 µL) applied in vitro on the skin (a diffu-
sion area of 0.71 cm2) was 2.38 ± 0.05 mg (mean ± SEM, n = 54 total samples, from n =
3 in each set of experiments). Dotriacontane was used as the model compound for the
penetration of petrolatum into the stratum corneum. Table I shows that the majority,
61–66% (mean values), of the dotriacontane in the bodywash formulation was removed
by the rinse-off protocol. Approximately 8–14% (mean values) of the dotriacontane in
the bodywash remained on the skin surface that was removed by the filter wipes before the
tape-stripping evaluation. Figure 2 shows the amounts of dotriacontane as % applied dose
and amounts of petrolatum (µg/cm2) in the first five tapes after the application of the
bodywash system and rinsing. The amounts of dotriacontane in the stratum corneum,
collected by tape-stripping, decreased from 2.9% ± 0.3% in the first tape to 0.33% ± 0.05%
(mean ± SEM) in the fifth tape at 1 h postapplication. At 24 and 72 h after bodywash
TABLE I
The Amount of Dotriacontane (Petrolatum Probe) as Percent of Bodywash Net Applied Dose
Recovered from Different Compartments at 1, 24, and 72 h after Application and Rinsing
in the Skin Penetration Study. Mean ± SEM, n = 7–11
Compartment 1h 24 h 72 h
Rinse-offa 61 ± 5 61 ± 4 66 ± 5
Skin surface wipe 14 ± 1 8±1 10 ± 1
Tape-strippingb 8±1 9±1 10 ± 1
Epidermis below Tape 5 (cut out epidermis) 0.7 ± 0.1 3.0 ± 0.4 5±1
Remaining skin (after tape-stripping and cut out epidermis) 0.8 ± 0.2 1.6 ± 0.3 2.0 ± 0.3
Buffer in receptor 0 ± 0c 0 ± 0c 0 ± 0c
Donor washd 10 ± 2 10 ± 2 9±1
Total 95 ± 5 92 ± 3 103 ± 4
Range (82–129) (84–104) (87–112)
a
Rinse-off solution was collected immediately after rinsing, which was performed at 1 min after dosing.
b
Amounts of dotriacontane on the fixing tapes used in the tape-stripping procedure are less than 1% applied
dose of dotriacontane.
c
Mean values of dotriacontane in the receptor chamber <0.1% applied dose.
d
Residual dotriacontane on the glass surface of the donor chamber. The residual amount was determined by
washing the donor chamber cap with hexane and collecting the wash solution. This amount was only used for
recovery calculation.

Figure 2. Amount of dotriacontane (% applied dose) and calculated petrolatum amount (µg/cm2) in each
tape, from Tape 1 to 5, at 1 h (circles), 24 h (diamonds), and 72 h (triangles) after bodywash application and
rinsing. Mean ± SEM (n = 8 for 1 h, n = 7 for 24 h, and n = 11 for 72 h). * denotes significant difference
between the data at 1 and 72 h (p < 0.05, ANOVA). ** denotes significant difference between the data at 1
and 24 h and between the data at 1 and 72 h (p < 0.05, ANOVA).
application and rinsing, the amount versus tape number profiles of dotriacontane (from
Tape 1 to Tape 5: 3.1% ± 0.4% to 0.66% ± 0.07% and 2.7% ± 0.3% to 0.74% ± 0.11%
at 24 and 72 h, respectively, mean ± SEM) were similar to that at 1 h with slightly larger
amounts of dotriacontane in the later tapes (Tapes 4 and 5) compared with the 1 h data.
PENETRATION DEPTH OF PETROLATUM INTO THE STRATUM CORNEUM
A more complete stratum corneum penetration profile of dotriacontane was obtained by

using up to 12 tapes in the tape-stripping at 1 h after bodywash application and rinsing.
The amounts of dotriacontane in this profile are shown in Figure 3 as % applied dose.
Because of the small amounts of dotriacontane in the deeper layers of the stratum cor-
neum, the later tapes (Tapes 7–12) were combined in pairs: Tapes 7 and 8, Tapes 9 and
10, and Tapes 11 and 12. Although only a small % of dotriacontane was found in Tapes
7–12, the data support the hypothesis that petrolatum can penetrate into the deeper layers
of the stratum corneum after bodywash formulation application and rinsing.
PENETRATION OF PETROLATUM INTO THE DEEPER SKIN LAYERS
Figure 4 shows the amount of dotriacontane beyond the first five tapes in the epidermis
(stratum corneum and viable epidermis excluding the first five tape-stripping) as % ap-
plied dose. Considerable amount of dotriacontane was found in the deeper layers in the
stratum corneum and epidermis. The % applied dose of dotriacontane in the deeper layers
of epidermis at 1 h after application and rinsing was 0.78% ± 0.17% (mean ± SEM), cor-
responding to 2.6 ± 0.6 µg/cm2 petrolatum. This amount tripled at 24 h to 2.3% ± 0.3%
and then increased to 3.9% ± 0.4% applied dose at 72 h. The amounts of dotriacontane
recovered in the dermis (remaining skin in Table I) were 0.8% ± 0.2% at 1 h, 1.6% ±
0.3% at 24 h, and 2.0% ± 0.3% at 72 h (mean ± SEM). Together, these results suggest

Figure 3. Amount of dotriacontane (% applied dose) in each tape, from Tape 1 to 12, at 1 h after bodywash
application and rinsing. Mean ± SEM (n = 7 for Tapes 1–8, n = 5 for Tapes 9–10, and n = 4 for Tapes 11–12).
The amounts between Tape 1, Tape 2, and other tapes are considered significantly different (p < 0.05, ANOVA).
that the dotriacontane and petrolatum remaining on the skin after bodywash application and
rinsing were able to penetrate the stratum corneum into the deeper skin layers over time.
PENETRATION OF PETROLATUM THROUGH THE SPLIT-THICKNESS SKIN AND TOTAL PERCENT

RECOVERY
No dotriacontane was detected in PBS in the receptor samples, suggesting that the pet-
rolatum cannot penetrate through the dermis to reach the systemic circulation at a sig-
nificant quantity under the condition studied. The total % recovery of dotriacontane at
Figure 4. Amount of dotriacontane (% applied dose) and calculated petrolatum amount (µg/cm2) in the
stratum corneum and epidermis beyond the 5 tape-stripping (i.e., epidermis minus the first 5 tapes) at 1, 24,
and 72 h after bodywash application and rinsing. Mean ± SEM (n = 8 for 1 h, n = 7 for 24 h, and n = 11 for
72 h). * denotes significant difference compared with the data at 1 h (p < 0.05, ANOVA). ** denotes sig-
nificant difference compared with the data at 1 h (p < 0.01, ANOVA).

all time points is summarized in Table I. The average total recovery was between 92%
and 103% (range: 82–129%).
DISCUSSION
APPLYING AND RINSE-OFF METHOD
A previous study investigated the penetration of petrolatum into the stratum corneum
after skin application (6). The differences between the previous Franz diffusion cell and
tape-stripping study and the present study include the methodology (with and without
simulated rinsing) and the use of the bodywash formulation. In the present study, the
bodywash formulation was applied to the in vitro skin and spread in circular motions
using a smooth ground flat surface glass rod. A rinse-off protocol was then applied to
remove the bodywash from the in vitro skin surface. Nguyen et al. (7) studied the meth-
ods to simulate rubbing of a gel topical formulation on skin and found that using the
vertical glass rod method resulted in spreading of the gel to the entire area and facili-
tated a rapid onset of product delivery at 4 h. The glass rod method was the method of
choice for applying topical formulations in skin penetration studies in vitro. Using the
present method of bodywash application and rinsing, a significant portion of petrola-
tum was deposited on the skin. Particularly, the rinse-off data in Table I show that
approximately 1/3 of the petrolatum in the bodywash formulation was deposited on the
skin after the rinse-off protocol. Approximately 1/3 of the petrolatum in the bodywash
remaining on the skin (~1/9 of the total petrolatum in the bodywash) was removed by
the filter wipes at the end of the penetration study. The relatively large amount of pet-
rolatum deposited on the skin surface after rinsing (i.e., ~1/3 of the dose of petrolatum)
could lead to better petrolatum penetration into the stratum corneum with the body-
wash formulation.
PENETRATION OF PETROLATUM INTO THE STRATUM CORNEUM
In a previous study with a heat-separated human epidermal membrane, when petrola-

tum was directly deposited on the skin surface using a volatile solvent, hexane, a trace
amount of petrolatum (<2% applied dose) was found to penetrate the membrane into
the receptor chamber of the diffusion cell at 72 h postapplication and no detectable
penetration of petrolatum was found in the receptor chamber when split-thickness skin
was used (<0.05% applied dose, estimated from the detection limit) (6). This result
indicates that petrolatum could penetrate across the epidermis but not the split-thick-
ness dermis, possibly because of the high lipophilicity of petrolatum. This also suggests
that petrolatum likely would not penetrate across the dermis and enter the systemic
circulation from each application under the in vivo condition. Petrolatum is a mixture
of C12–C85 long-chain aliphatic hydrocarbons (8). It was suggested that highly lipo-
philic compounds such as petrolatum can accumulate in the stratum corneum (9). It
was also suggested that the shorter chain hydrocarbons in petrolatum were able to dis-
solve and penetrate into the deeper layers of the epidermis, whereas the longer chain
hydrocarbons were “trapped” in the upper layers. 14C-dotriacontane is a C32 alkane and
is one of the main components of petrolatum (6). It was used as the radiolabeled probe

(i.e., a model permeant) of the petrolatum in the present study. The study results show
that dotriacontane was mainly found in the upper layers of the stratum corneum (e.g.,
Tapes 1 and 2) and was traceable in the stratum corneum deeper layers (e.g., in 12 tape-
stripping study, Figure 3). The petrolatum detected in the stratum corneum from tape-
stripping could be related to the ridges and furrows (10) and/or desquamated layers of
the skin. A comparison of these tape-stripping results with those from petrolatum de-
position using a volatile solvent in a previous study (6) shows that the amounts of
petrolatum (as % applied dose) penetrated into the stratum corneum from the solvent
deposition method were ~5× larger than those from the bodywash. The difference is
likely attributed to the (a) different dosing methods, (b) different amounts of petrola-
tum deposited on the skin surface possibly relating to the dosing methods, (c) different
data analyzes including the number of compartments used in the calculations, and/or
(d) skin-to-skin variability. Despite these differences, when the data were compared
based on the total amounts of petrolatum penetrating the stratum corneum, taking
into account the differences due to water rinsing, skin wiping, and donor chamber re-
covery wash, the two skin deposition methods provided similar % amount penetration
profiles in the stratum corneum.
In addition to the present in vitro study, previous clinical data have suggested that the
penetration of petrolatum into the stratum corneum is related to the effect of glycerol
monooleate (11). Glycerol monooleate is a glyceryl fatty acid ester that has a cis C=C
double bond at the C9 position. It is widely used in cosmetic products as an emulsifier and
absorption enhancer. The Food and Drug Administration (FDA) has classified this ingredient
as generally recognized as safe in the FDA Inactive Ingredient Guide. It is considered
nontoxic, biodegradable, and biocompatible (12). The study concluded that petrolatum
bodywash with glyceryl monooleate clinically improved skin appearance, skin surface
barrier function, and hydration in humans compared with bodywashes without glyceryl
monooleate (11). The results in the present study support a possible relationship between
petrolatum skin penetration and the observed clinical benefits, and the increase in petro-
latum penetration depth by glyceryl monooleate in the stratum corneum (6) could be one
of the factors leading to these benefits. Further studies will be needed to explore the rela-
tionship and mechanism of petrolatum and glyceryl monooleate.
CONCLUSION
The bodywash formulation with petrolatum could provide significant deposition and
penetration of petrolatum into the stratum corneum (~0.03–0.05 mg/cm2 from 5 µL
bodywash formulation) at 1–72 h postapplication. Specifically, petrolatum was observed
to deposit from the bodywash when applied on the split-thickness skin with simulated
rinsing. The petrolatum then penetrated into the stratum corneum and was detected at
the depth of 12 tape-stripping and further in the epidermis.
ACKNOWLEDGMENTS
This study was financially supported by Procter & Gamble Co. (Mason, Ohio). The
authors are grateful to Dr. Gerald B. Kasting for his invaluable discussion and expert
guidance.

REFERENCES
(1) A. A. Scafidi, Body wash composition to impart conditioning properties to skin, United States Patent,
US 5,683,683 (1997).
(2) T. Petry, D. Bury, R. Fautz, M. Hauser, B. Huber, A. Markowetz, S. Mishra, K. Rettinger, W. Schuh,
and T. Teichert, Review of data on the dermal penetration of mineral oils and waxes used in cosmetic
applications, Toxicol. Lett., 280, 70–78 (2017).
(3) R. Ghadially, L. Halkier-Sorensen, and P. M. Elias, Effects of petrolatum on stratum corneum structure
and function, J. Am. Acad. Dermatol., 26, 387–396 (1992).
(4) T. Czarnowicki, D. Malajian, S. Khattri, J. Correa da Rosa, R. Dutt, R. Finney, N. Dhingra, P. Xiangyu,
H. Xu, Y. D. Estrada, X. Zheng, P. Gilleaudeau, M. Sullivan-Whalen, M. Suaréz-Fariñas, A. Shemer, J.
G. Krueger, and E. Guttman-Yassky, Petrolatum: barrier repair and antimicrobial responses underlying
this “inert” moisturizer, J. Allergy Clin. Immunol., 137, 1091–1102 (2016).
(5) D. P. Alma, E. Liggeri, C. Delucca, and G. Calabrese, Prediction of skin permeation of highly lipophilic
compounds; in vitro model with a modified receptor phase, Int. J. Pharm., 70(3), 219–223 (1991).
(6) R. Intarakumhaeng, Z. Shi, A. Wanasathop, Q. C. Stella, K. S. Wei, P. B. Styczynski, C. Li, E. Smith,
and S. K. Li, In vitro skin penetration of petrolatum and soybean oil and effects of glyceryl monooleate,
Int. J. Cosmet. Sci., 40, 367–376 (2018).
(7) H. X. Nguyen, A. Puri, and A. K. Banga, Methods to simulate rubbing of topical formulation for in
vitro skin permeation studies, Int. J. Pharm., 519, 22–23 (2017).
(8) I. Jakasa, S. Kezic, and P. J. Boogaard, Dermal uptake of petroleum substances, Toxicol. Lett., 235,
123–139 (2015).
(9) M. A. Bolzinger, S. Briançon, J. Pelletier, and Y. Chevalier, Penetration of drugs through skin, a com-
plex rate-controlling membrane, Curr. Opin. Colloid Interface Sci., 17, 156–165 (2012).
(10) R. G. Van der Molen, F. T. Spies, J. M. Van’t Noordende, E. Boelsma, A. M. Mommaas, and H. K.
Koerten, Tape stripping of human stratum corneum yields cell layers that originate from various depths
because of furrows in the skin, Arch. Dermatol. Res., 289, 514–518 (1997).
(11) Q. C. Stella, S. K. Li, R. Intarakumhaeng, Z. Shi, G. B. Kasting, K. S. Wei, C. Li, E. Smith, and R.
Spruell, The effect of glyceryl monooleate on lipid penetration kinetics and profile in stratum corneum
for skin enhancement in appearance, hydration and barrier function, J. Am. Acad. Dermatol., 76, AB251
(2017).
(12) S. Milak and A. Zimmer, Glycerol monooleate liquid crystalline phases used in drug delivery systems,
Int. J. Pharm., 478, 569–587 (2015).

Does Salt and Mineral Content of Dead Sea Mud Affect Its
Irritation Potential: A Laser Doppler Flowmetry Study
SAJA HAMED, ABDEL-MAJEED ALMALTY,

HATIM S. ALKHATIB, NABIL N. AL-HASHIMI,
and DUHA HAMED, Faculty of Pharmaceutical Sciences, Hashemite
University, Zarqa 13115, Jordan (S.H., N.A.-H.), Faculty of Allied
Health Sciences, Hashemite University, Zarqa 13115, Jordan (A-M.A.),
School of Pharmacy, University of Jordan, Amman, Jordan (H.S.A.), College
of Arts & Sciences, Winthrop University, Rock Hill, South Carolina (D.H.)
Accepted for publication July 22, 2019.
Synopsis
The aim of the study was to investigate skin microcirculation, flux, and temperature changes induced by the
application of Dead Sea mud (DSM) formulas with different mud salts and mineral contents using laser
Doppler flowmetry. Instrumental analysis of eight over-the-shelf DSM products and four different samples of
nonformulated Dead Sea mud were carried out to determine their contents of various salts and elements,
including K, Na, Cl, Mg, Mn, Ca, SO3, SiO2, Al, Br, Fe, Hg, Cr, Co, Ni, Cu, Zn, As, Cd, Pb, and Sr. Three
DSM samples with different levels of salts were then used to study the influence of salt content on skin
irritation potential using laser Doppler flowmetry. Fifteen healthy nonsmoking females aged 18–45 years
participated in the study. Subjects were randomly assigned to either “Salted” mud group (n = 5), “As is” mud
group (n = 5), or “Over-the-Shelf” mud group (n = 5). Five circular areas were marked on the ventral aspect
of each forearm. One forearm was assigned randomly for mud treatment and the other forearm was untreated.
Ten milliliters of mud was applied on the assigned forearm and left for 30 minutes. Two reading protocols
were designed and used to study the effects of tested type of mud on skin blood flux and temperature during
mud application (protocol 2) as well as before and after mud removal (protocol 1). All types of tested mud
were not associated with a significant measurable elevation in skin temperature and skin blood flow. All types
of Dead Sea mud did not cause detectable microcirculatory and skin temperature changes regardless of their
different mineral and salts contents.
INTRODUCTION
Dead Sea water is the richest natural mineral source in the world with the main elements
being chlorine, magnesium, sodium, calcium, potassium, and bromine (1).
The beneficial role of salt and mineral content of Dead Sea water in the treatment of various
skin problems has been shown in several published studies. The anti-inflammatory activity
Address all correspondence to Saja Hamed at hamedsh@hu.edu.jo.
259
of Dead Sea salt solution has been demonstrated in a number of clinical studies in patients
with psoriasis (2) and atopic dermatitis (3). In addition, the clinically proven enhance-
ment of skin barrier properties and the anti-inflammatory effect of Dead Sea salt have also
been studied at the cellular level. Schempp et al. (4), demonstrated that magnesium ions
inhibit the antigen-presenting function of human epidermal Langerhans cells both in vivo
and in vitro. Furthermore, magnesium and potassium ions have been shown to have a
specific inhibitory effect on the uncontrolled proliferation of psoriatic dermis cells grown
in tissue culture (5).
The therapeutic efficacy of Dead Sea products can be attributed to the penetration of
minerals into the skin where it affects its biochemical processes. Penetration of salts
through healthy and damaged epidermis was observed in healthy volunteers as well as
psoriatic patients after bathing in Dead Sea or in simulated bath-salt solutions for 4
weeks (6).
Besides Dead Sea water and salts, Dead Sea mud is a popular over-the-shelf product in the
Jordanian market. It is an unconventional treatment modality that is used at the Dead
Sea shore of Jordan. Tourists and local visitors of the Dead Sea intentionally apply mud
directly onto their skin for a period of time to achieve different perceived cosmetic and
therapeutic benefits.
In addition, mud pack therapy is widely used in rehabilitation centers and spas to allevi-
ate joint pain. In such cases, the mud is preheated to 50oC and then applied to the affected
area while the patient is covered with a woolen blanket protected by a film of plastic (7).
The Dead Sea mud is believed to be a rich source of various minerals and salts (8). The
physical and chemical characteristics of 24 Dead Sea mud samples collected from differ-
ent locations on the eastern shore of Dead Sea has shown that mud sample’s composition
varied because of different phenomena taking place at different sampling points (8).
Although the effects of Dead Sea salts on various skin biochemical processes were previ-
ously investigated, there are no reports, to date, that assessed, quantitatively, the effect of
different levels of minerals and salts on skin microcirculation and temperature as surro-
gate indices of irritation.
The objective of this study was to investigate the safety of Dead Sea mud preparations
with varied levels of minerals and salts and determine their irritation potential in human
volunteers using the laser Doppler method. Laser Doppler method allows noninvasive,
objective, and quantitative measurement of microvascular blood perfusion and skin tem-
perature and can be used to discriminate between irritant and nonirritant substances and
to detect early stages in the development of an irritant reaction before it is visible (9).
MATERIAL AND METHODS
SAMPLES
Eight samples of over-the-shelf Dead Sea mud from popular Jordanian brands of personal
care products were purchased, two different samples of raw Dead Sea mud were gener-
ously provided by Numeira Mixed Salts & Mud Company Ltd. (Amman, Jordan). Raw
Dead Sea mud samples were treated in our laboratory by removing stones and made
spreadable either by mixing with distilled water or Dead Sea water. Raw mud samples

DEAD SEA MUD & CUTANEOUS BLOOD FLOW 261
mixed with distilled water were called “As is” mud and the one mixed with Dead Sea
water was called “Salted” mud. Then, both were autoclaved for 15 min at 121 C.
Samples were sent to the Royal Scientific Society Instrumental Analysis Laboratories
(Amman, Jordan) for analysis of their salts and element contents. Laboratory tests and
analyses performed included moisture content; loss on ignition (BS EN 196-2); total Al,
K, Ca, Cr, Co, Ni, Cu, Zn, As, Cd, Pb, Li, Sr, Na, Mg, Mn, Fe, and SiO2 (SOP No.
3/01/04-005, atomic absorption spectroscopy); total Hg and As (SOP No. 3/01/04-006,
atomic absorption spectroscopy); total Br (ASTM D-3869-79, gravimetric analysis); and
Cl water soluble (BS EN 196-2).
SUBJECTS
Fifteen healthy, normotensive, nonsmoking female volunteers aged 18–45 years partici-
pated in the study after giving their informed consent. Subjects with any lesions on their
forearms or receiving any local or systemic treatments were excluded from the study.
The study protocol was officially approved by the Hashemite University Institutional
Review Board. Subjects were randomly assigned to either “Salted” mud group (n = 5),
“As is” mud group (n = 5), or “Over-the-Shelf” mud group (n = 5). Each subject served as
her own control.
INSTRUMENT
Skin blood flow and temperature were monitored pre-, during, and postmud application
using moorVMS-LDF dual channel—laser Doppler and temperature monitoring (Perimed®
PF4001, wavelength 82 nm, Perimed, Moor, UK) and recorded by the system software
with a 3-s time constant downstream from a broadband filter (12 MHz).
Laser Doppler is a gold standard for dynamic microvascular blood flow assessment. Laser
Doppler flowmetry (LDF) provides continuous noninvasive measurements of microvascu-
lar perfusion in terms of flux, which represents the movement of erythrocytes through the
microvasculature (10). It was used to assess the effect of local treatment in improving skin
microcirculation, thus improving the healing of skin ulcers (11) and to assess the effect of
a topical product on microcirculation (12). In addition, LDF data correlate well with vi-
sual scores of patch test reaction and is used in dermatology to predict irritancy and to
assess topical products such as anti-inflammatory, vasoactive drugs, and detergent barrier
creams (10).
A dual channel laser Doppler monitor (VMS-LDF2) with two (2) laser probes for simul-
taneous recording of skin microcirculation and skin temperature was used. VMS-LDF2
was computer-controlled with software (MoorVMS-PC V2.2). The sampling depth of the
probe is around 1 mm. Low-power laser light is transmitted via the optic probe to the
tissue, and the light is scattered by the tissue and moving blood cells and their frequency
is Doppler-broadened. Some of the scattered light is collected by the optic probe and
transmitted to a photodetector. The data are electronically processed by the system software
to produce the laser Doppler (blood flow) flux signal. The probes were calibrated just
before the start of the study according to the manufacturer’s guidelines. Perfusion values
were measured for each tested time points at a sample rate of 33 recordings per second

and were analyzed by averaging over 1-min intervals for protocol 1 and averaging over 10
min before and 30 min during intervals for protocol 2 using the manufacturer software.
STUDY DESIGN
The participants were randomly assigned into three groups of five subjects each. Each
group (n = 5) was used to test one of the aforementioned mud types. According to the
published guidelines for measurement of cutaneous blood flow (13), the subjects were
asked not to drink caffeinated beverages, not to eat a high-calorie meal at least 2 h before
the experiment, and not to exercise at least 24 h before the experiment. The subjects were
asked to relax for 20 min in a supine position while their forearms are relaxed and in an
extension position in a controlled temperature and a controlled relative humidity room.
Lighting conditions in the room were kept constant by closing window blinds and turn-
ing off ceiling lights, and there was typically sufficient light to be able to read. Five cir-
cular areas were marked on the ventral aspect of each forearm (Figure 1). One forearm was
Figure 1. Five circular areas were marked on the ventral aspect of each forearm. Areas 1–4 were used for
protocol 1 and area 5 for protocol 2. For protocol 1; laser Doppler readings (flux and temperature) were taken
for areas 1–4 at baseline before mud application and then directly and 15 min after mud removal.

assigned randomly for mud treatment and the other forearm was untreated. All mud
samples were left at room temperature for 24 h before application.
Ten milliliter of mud was gently applied using feeding syringe on the assigned forearm
and left for 30 min. Then, the mud was gently removed and washed using warm tap wa-
ter. The untreated forearm was also washed in a similar fashion to exclude the effect of
washing on skin temperature and blood flow.
Two reading protocols were designed and used to study the effects of tested types of mud
on skin blood flux and temperature during mud application as well as before and after
mud removal.
Protocol 1 (Figure 1): laser Doppler readings (Flux and temperature) were taken for areas
1–4 at baseline before mud application and then directly and 15 min after mud removal.
A ring of a double-sided tape was applied to hold the laser Doppler probes in place and
to allow the removal and reattachment of the probes without applying pressure or other-
wise disturbing the microvascular structure under each site. The procedure was simulta-
neously performed on the left and right forearms using the dual channel monitor with
two probes.
Perfusion values for each circular area at the tested time point were measured over 1-min
intervals using the manufacturer software, and the mean flux and skin temperature for
each area at each time point were used for data analysis.
Protocol 2 (Figure 2): a ring of a double-sided tape was applied to hold the laser Doppler
probes in place on areas numbered 5 on both forearms simultaneously. Baseline readings
of the microvascular blood perfusion and skin temperature were performed for areas num-
bered 5 for 10 min before mud application (Figure 2) and then 10 ml of mud was gently
Figure 2. Protocol 2; laser Doppler probes were fixed simultaneously on areas 5 on both forearms and base-
line readings were recorded over 10 min before mud application and then during the whole mud treatment
time for both the treated and untreated forearms.

applied using feeding syringe on the assigned forearm and readings were recorded during
the whole mud treatment time (i.e., 30 min). The mean flux and skin temperature for the
baseline readings and for the 30 min during mud treatment readings were used for data
analysis.
DATA ANALYSIS
The flux and skin temperatures at different time points for each area were summarized as
mean ± SEM for both the treated and untreated forearms. The three groups of Dead Sea
mud were treated as one group (n = 15) and the statistical analysis was performed using
the software Minitab© (LLC, State College, PA).
Multiple paired t-tests were used, and its use was justified by the normality test for the
differences between the treated and the untreated forearms.
The paired t-tests were used to compare the flux and the temperature measurements’ dif-
ference for each area (of the five circular areas) between the treated and untreated fore-
arms. No significant differences were found between the treated and untreated forearm
measurements for both the flux and the temperatures for any of the five circular areas.
Furthermore, comparison between treated and untreated forearms for each mud type (n = 5)
was performed using nonparametric testing, and no significant differences were found for
the variable flux and temperatures for each mud type.
All statistical tests were performed with 5% level of significance.
RESULTS AND DISCUSSION

The chemical analysis results of the Dead Sea mud are presented in Table I. Samples 1–4
are native Dead Sea mud samples that were treated in our laboratories. Samples 1 and 2
were mixed with distilled water and then autoclaved. Samples 3 and 4 were mixed with
Dead Sea water and then autoclaved. Samples 5–12 were over-the-shelf Dead Sea mud
products available in the Jordanian market.
Potassium levels (%) ranged from 0.23 for RV Body Mud to 0.79 for “As is” mud-1.
Calcium contents (%) ranged from 11.59 for BS Mud Mask up to 18.3 for “As is” mud-1.
Sodium levels (%) ranged from 0.53 to 1.86. The highest levels were for AT Body Mud
and “As is” mud-1 (1.86% and 1.85%, respectively) and the lowest for RV Body Mud.
Water-soluble chloride levels (%) ranged from 0.68 to 11.24. The highest levels were for
“Salted” mud-1 and 2 (11.1% and 11.24%, respectively) and the lowest level was for RV
Facial Mask (0.68%). Manganese levels were below 0.03% for all tested mud samples.
Aluminum levels (%) ranged from 0.41 to 0.7 and were highest for BS Mud Mask and
lowest for NC Body Mud. Iron (Fe) contents ranged from 0.56% for NC Body Mud to
1.7% for BS Mud Mask. Magnesium levels (%) ranged from 0.82 to 3.57 and were the
highest in salted mud samples (3.22% and 3.57%) and lowest for RV Facial Mask
(0.82%).
Results show that “Salted” mud were the richest in magnesium and chloride ions as com-
pared with “As is” mud and other processed “Over-the-Shelf” mud. Sodium contents were
almost similar between “As is” mud-1 and “Salted” mud-1 and between “As is” mud-2
and “Salted” mud-2. We have noticed that for mud produced from the same company,

Table I
Chemical Compositions (%) of Different Dead Sea Mud Samples. Samples 1–4 Were
Native Dead Sea Mud Treated at Our Laboratory. Samples 5–12 were “Over-the-Shelf”
Mud Bought from the Market. Mud Samples Used in the Clinical Study are in Gray-Shaded Rows
Moisture
Mud K Ca Na Cl Mg Mn Al Fe SO3 SiO2 L.O.I content
1 “As is” mud-1 0.79 18.3 0.78 9.62 2.96 0.01 0.55 0.66 0.61 19.78 33.55 28.26
2 “As is” mud-2 0.61 16.49 1.85 8.77 2.72 0.03 0.6 0.71 1.08 16.32 45.93 30.05
3 “Salted” mud-1 0.72 17.52 0.84 11.1 3.57 0.02 0.44 0.62 0.52 17.26 35.28 26.64
4 “Salted” mud-2 0.59 14.46 1.72 11.24 3.22 0.03 0.6 0.81 0.3 14.54 50.3 31.77
5 RV Body Mud 0.23 13.27 0.53 0.71 0.93 0.02 0.64 0.77 0.27 25.5 40.51 37.22
6 RV Facial Mask 0.25 13.77 0.56 0.68 0.82 0.02 0.57 0.75 < 0.1 24.72 40.07 35.49
7 BO Body Mud 0.5 14.05 1.31 7.14 2.22 0.02 0.6 0.77 0.73 14.12 51.58 34.68
8 BS Mud Mask 0.43 11.59 1.15 4.94 1.8 0.03 0.7 1.7 0.39 29.1 34.19 39.73
9 AT Body Mud 0.57 15.71 1.86 4.16 2.97 0.03 0.64 0.8 0.61 15.13 47.57 32.78
10 AT Facial Mask 0.59 15.38 1.76 4.13 2.87 0.03 0.42 0.61 0.68 15.42 47.14 32.07
11 NC Body Mud 0.46 12.59 1.31 3.07 1.86 0.02 0.41 0.56 0.41 19.15 49.94 29.35
12 NC Facial Mask 0.47 12.78 1.25 5.72 1.89 0.02 0.49 0.65 0.84 21.73 45.31 26.64
there were no tangible differences in mineral and salt levels between mud designed for
the face and mud designed for the body.
Based on their mineralogical contents, we have chosen three muds for the clinical study:
“Salted” mud-2, “As is” mud-2, and RV Body Mud (Table 1, gray-shaded rows). The
“Salted” mud, “As is” mud and RV Body Mud contain the following amounts (%) of K:
0.59, 0.61, and 0.23; Na: 1.72, 1.85, and 0.53; Cl: 11.24, 8.77, and 0.71; and Mg: 3.22,
2.72, and 0.93, respectively.
RV Body Mud contained the lowest levels (%) of K (0.23), Na (0.53), Cl (0.71), and Mg
(0.93).
Changes in mean skin blood flow (flux) and skin temperature during the different proto-
cols for each tested group (n = 5) are presented as mean ± SEM in Figures 3 and 4 and
Tables II and III. In addition, changes in mean skin blood flow (flux) during the different
protocols for the three groups of Dead Sea mud combined together (n = 15) are presented
in Figure 5.
LDF has been in clinical use since 1977; it is characterized by its unique capacity for
noninvasive measurement of local cutaneous blood flow with its moment-to-moment
variability (14).
The effect of Dead Sea mud on skin microcirculation according to protocol 1 and 2 is
represented in Figures 3–5.
Before applying Dead Sea mud, resting skin blood flow values and skin temperatures for
both treated and untreated (control) forearms were statistically similar.
There were no statistically significant differences between the flux of treated forearms
directly after mud removal as well as 15 min posttreatment for both “As is” mud and RV
Body Mud group compared with the control forearms at each time points as shown in
Figure 3A and C.
Interestingly, salted Dead Sea mud group showed slight increase in flux directly after
mud removal (Δ 1.94), which was maintained 15 min posttreatment (Δ = 2.16) compared

Figure 3. The effect of each type of Dead Sea mud on skin blood flow, according to study protocol 1; di-
rectly and 15 min after mud removal as compared with untreated control forearm. Mean (n = 5) ± SEM.
with control forearms at each time points as shown in Figure 3B. However, this slight
increase was not statistically significant (p > 0.05).
The results from study protocol 1 were confirmed by study protocol 2 as shown in Figure 4.
In this protocol, the effect of Dead Sea mud on skin microcirculation were monitored
during the whole treatment period (i.e., 30 min) by the dual channel LDF for both treated
and control forearms, simultaneously.
Resting skin blood flow and skin temperature readings (i.e., baseline) were monitored for
10 min before applying Dead Sea mud and were statistically similar for both treated and
untreated (control) forearms.
Applying Dead Sea mud for 30 min did not result in a statistically significant increase in
skin microcirculation for all types of Dead Sea mud compared with control untreated
forearm as shown in Figure 4.
The slight increase in skin microcirculation for salted Dead Sea mud–treated forearms
compared with untreated forearms was also shown in study protocol 2 in which salted
Dead Sea mud caused a slight, insignificant increase in Flux (Δ 1.28) compared with
control untreated forearm as shown in Figure 4B.
The effect of the three types of Dead Sea mud combined together as one group (n = 15)
regardless of their salt content according to both protocols is presented in Figure 5.

Figure 4. The effect of each type of Dead Sea mud on skin blood flow, according to study protocol 2; during
mud application (i.e., 30 min) compared with untreated control forearm. Mean (n = 5) ± SEM.
Dead Sea mud application did not cause an increase in skin blood flow directly and 15
min postapplication compared with both baseline readings as well as to readings of skin
blood flow of the untreated (control) forearms at the same time points as shown in Figure
5A. These findings were confirmed with protocol 2, where applying Dead Sea mud for 30
min did not result in a statistically significant increase in skin microcirculation compared
with baseline readings as well as compared with control untreated forearm as shown in
Figure 5B.
Table II
The Effect of Dead Sea Mud on Skin Temperature, According to Study Protocol 1,
Skin Temperature before Mud Application, Directly, and 15 min after Mud Removal as
Compared with Untreated Control Forearm. Mean (n = 5) ± SEM
Skin temperature
Baseline Direct 15 min after
Treated arm Control Treated arm Control Treated arm Control
“As is” mud 28.11 (0.12) 28.02 (0.20) 24.61 (0.20) 25.29 (0.35) 25.32 (0.14) 26.08 (0.17)
“Salted” mud 28.03 (0.53) 27.95 (0.74) 25.04 (0.27) 26.04 (0.31) 26.29 (0.09) 26.98 (0.11)
RV Body Mud 28.75 (0.33) 29.06 (0.44) 25.61 (0.31) 26.93 (0.34) 26.68 (0.10) 27.95 (0.17)

Table III
The Effect of Dead Sea Mud on Skin Temperature, According to Study Protocol 2,
Skin Temperature Baseline Value before Mud Application (i.e., for 10 min) and
during Mud Application (i.e., for 30 min) Compared with Untreated Control Forearm.
Mean (n = 5) ± SEM
Skin temperature
Baseline 30 min
Treated arm Control Treated arm Control
“As is” mud 27.95 (0.42) 28.02 (0.49) 26.35 (0.41) 27.17 (0.51)
“Salted” mud 28.61 (0.29) 29.24 (0.39) 27.17 (0.28) 28.49 (0.38)
RV Body Mud 29.26 (0.43) 29.58 (0.48) 27.31 (0.42) 28.81 (0.51)
The effect of Dead Sea mud on skin temperature was in good agreement with the effect
of Dead Sea mud on skin microcirculation. Resting skin temperatures before applying
Dead Sea mud were statistically similar for both treated and untreated (control) forearms
in both study protocols. In addition, all types of Dead Sea mud did not result in a sig-
nificant increase in skin temperature as compared with untreated control forearms at all
tested time points as shown in Tables II and III.
The slight increase in skin blood flow for salted Dead Sea mud–treated forearms as com-
pared with control forearms was not associated with any measurable increase in skin
temperature directly and 15-min posttreatment in study protocol 1 (Table II) and during
the 30-min treatments in study protocol 2 (Table III) compared with control forearm at
the mentioned time points.
Erythema is an important consequence of skin irritation. It is documented that erythema
cannot be detected by visual observation until the skin blood flow has at least tripled (15).
In addition, skin temperature is closely associated with cutaneous blood flow (16). All
types of Dead Sea mud did not cause significant increase in skin blood flow and skin tem-
perature during as well as 15 min postapplication.
It is noteworthy to mention that, in both protocols and for both treated and untreated
forearms, readings for skin blood flow (Figure 5) were higher at baseline than readings
Figure 5. The effect of Dead Sea mud combined together on skin blood flow, according to study protocol 1
(A) and according to study protocol 2; during mud application (i.e., 30 min) (B) compared with untreated
control forearm. Mean (n = 15) ± SEM.

during mud application as well as directly and 15 min posttreatment. This confirms that
Dead Sea mud application for 30 min did not increase skin blood flow as compared with
baseline values.
However, because cutaneous microcirculation is a physiological parameter that is subject
to dynamic regulation and rapid variation and influenced by many environmental and
individual factors, it is important to compare flux readings simultaneously at the same
time point in a contralateral site (13). Taking this in consideration, and after confirming
that baseline flux and temperature readings were stable and comparable for each circular
area in both treated and control forearms, we compared flux and skin temperature read-
ings of the treated forearms with their simultaneously recorded values of the control
forearms for each circular area at each measured time points in both protocols.
CONCLUSION
Our two clinical studies showed the lack of a significant increase in skin blood flow and
skin temperature during, as well as 15 min postapplication of all types of Dead Sea mud;
“Salted” mud, “As is” mud, and “Over-the-Shelf” mud.
We may conclude that the increase in skin blood flow and temperature advertised by the
manufacturer after mud application is not attributed to skin irritation rather, it could be
attributed to the direction of use suggested by the manufacturer. The manufacturer direct
mud user to warm the mud before application. This warming may enhance skin blood
flow and skin temperature as it is well documented that heat increases skin blood flow in
healthy subjects (17).
This LDF study confirms the mildness of all Dead Sea mud regardless of their salt con-
tent. DSM mildness previously confirmed in our laboratory by the lack of any marked
differences in skin erythema as well as skin pH in mud-treated forearms compared to
baseline values (18).
REFERENCES
(1) Z. Ma’or, S. Yehuda, and W. Voss, Skin smoothing effects of Dead Sea minerals: comparative profilomet-
ric evaluation of skin surface, Int. J. Cosmet. Sci., 19(3), 105–110 (1997).
(2) S. Halevy, H. Giryes, M. Friger, N. Grossman, Z. Karpas, B. Sarov, and S. Sukenik, The role of trace
elements in psoriatic patients undergoing balneotherapy with Dead Sea bath salt, Isr. Med. Assoc. J.,
3(11), 828–832 (2001).
(3) M. Harari, J. Shani, V. Seidl, and E. Hristakieva, Climatotherapy of atopic dermatitis at the Dead Sea:
demographic evaluation and cost-effectiveness, Int. J. Dermatol., 39(1), 59–69 (2000).
(4) C. M. Schempp, H. C. Dittmar, D. Hummler, B. Simon-Haarhaus, J. Schulte-Monting, E. Schopf, and
J. C. Simon, Magnesium ions inhibit the antigen-presenting function of human epidermal Langerhans
cells in vivo and in vitro. Involvement of ATPase, HLA-DR, B7 molecules, and cytokines, J. Investig.
Dermatol., 115(4), 680–686 (2000).
(5) J. Shani, R. Sharon, R. Koren, and Z. Even-Paz, Effect of Dead-Sea brine and its main salts on cell
growth in culture, Pharmacology, 35(6), 339–347 (1987).
(6) J. Shani, S. Barak, D. Levi, M. Ram, E. R. Schachner, T. Schlesinger, H. Robberecht, R. Van Grieken,
and W. W. Avrach, Skin penetration of minerals in psoriatics and Guinea-pigs bathing in hypertonic
salt solutions, Pharmacol. Res. Commun., 17(6), 501–512 (1985).
(7) D. Poensin, P. H. Carpentier, C. Fechoz, and S. Gasparini, Effects of mud pack treatment on skin
microcirculation. Joint Bone Spine, 70(5), 367–370 (2003).

(8) A. Khlaifat, O. Al-Khashman, and H. Qutob, Physical and chemical characterization of Dead Sea mud,
Mater. Charact., 61(5), 564–568 (2010).
(9) V. Zuang, C. Rona, G. Archer, and E. Berardesca, Detection of skin irritation potential of cosmetics by
non-invasive measurements, Skin Pharmacol. Appl. Skin Physiol., 13(6), 358–371 (2000).
(10) V. Rogiers, M. Balls, D. Basketter, E. Berardesca, C. Edwards, P. Elsner, J. Ennen, J. L. Leveque, M.
Loden, P. Masson, J. Parra, M. Paye, G. Pierard, L. Rodrigues, H. Schaefer, D. Salter, and V. Zuang, The
potential use of non-invasive methods in the safety assessment of cosmetic products, Altern. Lab. Anim.,
27(4), 515–537 (1999).
(11) G. Belcaro, M. R. Cesarone, B. M. Errichi, A. Di Renzo, S. Errichi, A. Ricci, G. Gizzi, M. Dugall, M.
Cacchio, I. Ruffini, F. Fano, G. Vinciguerra, and M. G. Grossi, Improvement of microcirculation and
healing of venous hypertension and ulcers with Crystacide: evaluation with a microcirculatory model,
including free radicals, laser Doppler flux, and PO2/PCO2 measurements, Angiology, 58(3), 323–328
(2007).
(12) H. M. Hafner, S. R. Thomma, M. Eichner, A. Steins, and M. Junger, The influence of Emla cream on
cutaneous microcirculation, Clin. Hemorheol. Microcirc., 28(3), 121–128 (2003).
(13) A. Bircher, E. M. de Boer, T. Agner, J. E. Wahlberg, and J. Serup, Guidelines for measurement of cuta-
neous blood flow by laser Doppler flowmetry. A report from the Standardization Group of the European
Society of Contact Dermatitis, Contact Derm., 30(2), 65–72 (1994).
(14) A. M. Schabauer and T. W. Rooke, Cutaneous laser Doppler flowmetry: applications and findings, Mayo
Clin. Proc., 69(6), 564–574 (1994).
(15) J. E. W., “Assessment of Erythema: A Comparison Between the Naked Eye and Laser Doppler Flow-
metry,” in Current Topics in Contact Dermatitis, P. J. Frosch, J. M. Lachapelle, R. J. G. Rycroft, and R. J.
Scheper. Eds. (Springer, Berlin, Heidelberg, 1989), pp. 549–553.
(16) E. H. Page and N. H. Shear, Temperature-dependent skin disorders, J. Am. Acad. Dermatol., 18(5 Pt 1),
1003–1019 (1988).
(17) E. B. Lohman, G. S. Bains, T. Lohman, M. DeLeon, and J. S. Petrofsky, A comparison of the effect of a
variety of thermal and vibratory modalities on skin temperature and blood flow in healthy volunteers,
Med. Sci. Monit., 17(9), Mt72–Mt81 (2011).
(18) S. Hamed and A. Almalty, Skin tolerance of three types of Dead Sea mud on healthy skin: a short term
study, J. Cosmet. Sci., 69(4), 269–278 (2018).


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Vol . 70 N o .

JOURNAL OF COSMETIC SCIENCE

Comparative Assessment of Biological Activities of Mistletoes for

In Vitro Penetration of Petrolatum in Stratum Corneum from Bodywash

Purchased for the exclusive use of nofirst nolast (unknown)

Titanium Dioxide and Zinc Oxide Nanoparticles in

MAJA VUJOVIC and EMILIJA KOSTIC, Faculty of Medicine,

Accepted for publication June 24, 2019.

Address all correspondence to Maja Vujovic at majavujovic1@gmail.com and Emilija Kostic at

The rapid development of nanotechnology has resulted in an increasing number of nano-

International Cooperation on Cosmetic Regulation defines a nanomaterial in cosmetics as

Purchased for the exclusive use of nofirst nolast (unknown)

Important physicochemical parameters for their characterization are particle composi-

INSTRUMENTAL METHODS FOR THE CHARACTERIZATION OF TIO2 AND ZNO NPS

DERMAL PENETRATION OF ZNO AND TIO2

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ABSORPTION OF TIO2 AND ZNO—IN VITRO STUDIES

ABSORPTION OF TIO2 AND ZNO—ANIMAL SKIN

Type of NPs Formulation Experimental system Effects Ref.

Purchased for the exclusive use of nofirst nolast (unknown)

ABSORPTION OF TIO2 AND ZNO—INTACT AND DAMAGED HUMAN SKIN

Type of NPs Formulation Skin type Effect Ref.

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Type of NPs Formulation Skin type Effect Ref.

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INFLUENCE OF TIO2 AND ZNO ON ROS GENERATION AND POTENTIAL CYTOTOXICITY

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Type of NPs Cells Effect Ref.

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INTERACTIONS OF SUNSCREENS WITH OTHER SUBSTANCES

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Purchased for the exclusive use of nofirst nolast (unknown)

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Purchased for the exclusive use of nofirst nolast (unknown)

Comparative Assessment of Biological Activities of

SUNG-UP CHOI, SUNG TAE KIM, DONG GYUN HAN,

Accepted for publication June 25, 2019.

Address co-correspondence to Dong-Jin Jang at djjang@inje.ac.kr and Seong-Bo Kim at seongbo.kim@cj.net.

MATERIALS AND METHODS

PREPARATION OF MISTLETOE EXTRACTS

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TOTAL PHENOLIC CONTENT ASSAY

DPPH FREE RADICAL SCAVENGING ACTIVITY ASSAY

REDUCING POWER ASSAY

TYROSINASE INHIBITION ASSAY

For evaluating antimelanogenesis, a tyrosinase inhibition assay was performed, as tyrosinase

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mushroom tyrosinase, which is frequently used because of its commercial availability

ELASTASE INHIBITION ASSAY

RESULTS AND DISCUSSION

TOTAL CONTENT OF PHENOLIC COMPOUNDS

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Total amounts of phenolic

DPPH FREE RADICAL SCAVENGING ACTIVITY

REDUCING POWER ANALYSIS

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Purchased for the exclusive use of nofirst nolast (unknown)

Purchased for the exclusive use of nofirst nolast (unknown)

Purchased for the exclusive use of nofirst nolast (unknown)

Purchased for the exclusive use of nofirst nolast (unknown)

Purchased for the exclusive use of nofirst nolast (unknown)

Purchased for the exclusive use of nofirst nolast (unknown)

In Vitro Penetration of Petrolatum in Stratum Corneum from

APIPA WANASATHOP, ZHANQUAN SHI, Q. CHING STELLA,