Cosmetics and Toiletries 08-2017

July/August 2017
Face Value
of SPF Testing
Testing
Folates for Anti-pollution
Next-gen
Solutions
UV Protection
Preserving
Microbiome
Harmony
WANT MORE?
www.CosmeticsandToiletries.com
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Cover Story Contents
July/August 2017 | Volume 132, number 7
6 Editors Note
Youve Been Training for This
8 Advisor Insight
Pollen: A Particulate Concern for Skin
72 Ad Index
Market Intelligence
10 Tech Launches
34 Regulatory
12 The Face Value of SPF Testing

Rethinking Sun Protection Claims
by C. Flower, Ph.D., and E. Meredith, Ph.D.
Research
16 Dermal-epidermal
Separation, Part III
Heat and Mechanical Methods
by Y. Zou and H.I. Maibach, Ph.D.
24 Preserving Microbiome Harmony

Antimicrobial Peptides Balance Skin Health
by T. Alkazaz, et al.
24 34 Natural Folate in Human Skin

A New Approach to UV Damage Protection
50
by S.W. Bailey and J.E. Ayling
Testing
50 When the Dust Settles
Keratinocyte Differentiation is the
Anti-pollution Solution
by A.-F. Clay, et al.
58 How Damaged is Hair?

Part III: Better Defining the Problem
by T.A. Evans, Ph.D.
2 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017
CT170708_TOC_Masthead_irv.indd 2 6/26/17 10:39 AM

30
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and GMP stability verified. 15 eye shadows evaluated for an
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For over 40 years, our team of experts have proven that before beauty there must be
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Analytical / Clinical / Microbiology / Photobiology / Safety / Consulting
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Editors note | C&T
Contents EDITORIAL
Director Jo-El M. Grossman
Managing Editor Rachel L. Grabenhofer | 1-630-344-6072/rgrabenhofer@allured.com
Assistant Editors Jennifer Novoseletsky | 1-630-344-6045/jnovoseletsky@allured.com
Brooke Schleehauf | 630-344-6032/bschleehauf@allured.com
Lisa Schryver | 630-344-6068/lschryver@allured.com
Digital/Social Media Editor Audrey Latimer | 1-630-344-6067/alatimer@allured.com
Research Analyst Nicole Urbanowicz | 1-630-344-6053/nurbanowicz@allured.com
ADVERTISING SALES
Business Development Manager/
C&T Summit Exhibits
& Sponsorships Tom Harris | 1-201-445-4702/tharris@allured.com
Business Development Manager
Fragrance Paige Crist | 1-630-344-6060/pcrist@allured.com
Coordinator Kasia Smialkowski | 1-630-344-6025/ksmialkowski@allured.com
AUDIENCE DEVELOPMENT
Marketing Specialist Marie Galvan
Marketing Specialist Alyssa Derby
58
Customer Service 1-888-355-5962/customerservice@cosmeticsandtoiletries.com
DESIGN
Graphic Design Manager Lisa Hede
Graphic Designer James Fergus
Formulating
Production Manager Bryan Crowe
EVENTS
Group Show Director Maria Prior | 1-630-344-6065/mprior@allured.com
68 Sponsored: Stronger hair for Show Manager Brittany Peck | 1-630-344-6073/bpeck@allured.com
the socially conscious
by BASF
CORPORATE
Partner & President Janet Ludwig
Partner & CEO George Fox
70 Skin Health and Wellness Formulary Controller Linda Getner
Digital Products Director Rose Southard
Executive Assistant Maria Romero
Digital Edition Exclusives

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Vol. 132, No. 7 | July/August 2017
CT170708_TOC_Masthead_fcx.indd 4 6/26/17 4:48 PM

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Editors Note | C&T
Youve Been Training for This

Its the solstice today (I wrote this in June), the height of outdoor engagement and
merriment. Like many, our family is planning a summer vacationboating and swimming on
a lake with friends for an entire week. Moments like these are what skin care formulators have
been training for. What do I mean?
As your consumer, what greater exposure will I have to the elements? Ill certainly put
sunscreen (and deodorant) to the test under sweat, water and UV/vis/IR. No doubt my hair
color will be tasked, and moisturizer, wrinkle cream and skin-soothers like aloe (hopefully not
much) will enter the equation. Not to mention the lake, with its own microbial inhabitants and
possible pollutants; thus, body washes, too.
None of this will stop the fun, although as my family and friends plan our travel route and
meals, Ill be focused on packing the right personal care products. But I have full confidence in
your formulas because you read Cosmetics & Toiletries.
That brings me to this edition. Here we explore a novel approach to UV protection: fortifying
skins natural folate pool, which protects against free radical damage (see Page34). We
examine what happens after the dust settles on skin, in a test method to scan
promising anti-pollution solutions (see Page 50). We also learn how natural
antimicrobial peptides preserve not just products, but harmony
within skins microbiome (see Page24).
And three must be our lucky number this month, as we take
a look at internal hair damage (see Page 58) and skin separation
techniques (see Page16); both of which are part III in their
respective series.
As I pack our bags for vacation, I thank you for your skill and
effort to make these products (and our trip) possible. Theres no
way Id want to spend hours in the sun, sweating and burning, then Rachel L. Grabenhofer
return to a rental house with 10 other sore and smelly people for C&T Managing Editor
days in a row. You make it not just bearable but beautiful.
CT170708_Editors_Note_fcx.indd 6 6/26/17 10:45 AM

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Scientific Advisory industry Insight | C&T
Board
Pollen: A Particulate Concern

for Skin
Eric Abrutyn
TPC2 Advisors Ltd.
Zoe Diana Draelos, M.D.

Dermatology In an interview with Tom Mammone, Ph.D., of the
Consulting Services
Este Lauder Companies, surprising findings were
Angela R. Eppler, Ph.D. revealed about the effects of pollen on skin. Following is
Pfizer Consumer Healthcare
an excerpt from our exclusive podcast.
C&T : Describe your findings about pollen. How

Trefor Evans, Ph.D.
TA Evans LLC
does it affect skin?
S. Peter Foltis
Advisor: Our work on pollen actually stems from

LOral
Mindy Goldstein, Ph.D. work weve been doing for several years on PM2.5
Atlantic Coast Media Group or particular matter pollution. In that research, we
found particulates are very damaging to the skin
Shuzo Ishidate, Ph.D.
Shiseido Research Center barrier and trigger an inflammatory cascade, so we
started to take apart particulate pollution to look at
Karl Laden, Ph.D. the different elements. One of the larger-sized compo-
Alpa Cosmetics
nents we found was pollen.
Prithwiraj Maitra, Ph.D. Pollen runs the gamut from 2-10 microns in size
Johnson & Johnson depending on the species but its a major component
at certain times of the year, especially allergy season.
Jennifer Marsh, Ph.D.
We took purified pollen and treated artificial skin
Procter & Gamble
with it and saw really similar results to what we see
Marc Pissavini, Ph.D. for pollutionbarrier damage and inflammatory
Coty-Lancaster response. This is what you see with sunexposure.
Luigi Rigano, Ph.D.

Industrial Consulting Research C&T: Whats driving your interest in this area?
Sylvianne Schnebert, M.D. Advisor: Particulate pollution is a growing con-
LVMH Recherche cern. Im really surprised by this because originally,
we thought it would be narrowly based in areas of
Ron Sharpe
Amway the world that still burn a lot of coal. But what Ive
noticed in many cities around the world, like Paris
Leslie C. Smith, Ph.D. and New York, are pollution alerts [and calls to action
Consultant
to share a ride and reduce vehicle emissions].
David C. Steinberg
I think pollen fits within a whole category of
Steinberg & Associates elements we were not aware of that could
damage skin, and our consumers really want
Peter Tsolis to know how to protect themselves. Not
The Este Lauder Companies Tom Mammone, Ph.D.
just from the sun, but anything else they Vice President, Skin Physiology
Russel Walters, Ph.D. think in the future will damage their skin. and Pharmacology,
C&T

: What steps or treatments might
Johnson & Johnson
The Este Lauder Companies
Claudie Willemin Research and Clinique Labs
mitigate this impact? Company
LOral
Shuliang Zhang, Ph.D. Advisor: Weve looked at several different path-

Unilever ways and one thing weve discovered is antioxidants
can alleviate some of the damage. This
was surprising because the proteases
released from pollen were not thought Want More?
to have a free radical element. But I For more insight from Mammone and others,
log onto www.CosmeticsandToiletries.com
think they probably trigger free radical
formation in the skin.
CT170708_Advisor_Insight_fcx.indd 8 6/27/17 1:27 PM

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Technology Launches
Male Anti-balding Active Activated Charcoal
Photo Credit: Vantage
Photo Credit: Silab
Silab is addressing male baldness with Hairgenyl (INCI: Vantage is responding to pollution with Lipobead
Yeast Extract), an anti-hair loss active ingredient that is Detox with Charcoal (INCI: Mannitol (and)
rich in biopeptides, supports the biological activity of Cellulose (and) Iron Oxides (and) Charcoal
the dermal papilla and stimulates hair follicle growth. Powder (and) Caprylic/Capric Triglyceride (and)
According to various tests, the preservative-free powder Hydroxypropyl Methylcellulose), which helps
acts on androgenetic alopecia by correcting the abnormal protect, cleanse and detoxify skin. This ingredient
expression of both signaling molecules and epigenetics. consists of a small black bead incorporating
Additional effects include an increase in mitochondrial activated charcoal, which provides several key
fusion, which boosts the capacity for energy production benefits for masks, facial cleansers, scrubs, body
used for growth of the hair follicle. washes, shampoos and/or hand soaps.
www.silab.fr www.vantagespecialties.com
Botanical Relaxer Holistic Skin Renewal
Photo Credit: Givaudan
IBR Ltd. has introduced IBR-CalmDeAge (INCI: Glycerin (and) Givaudan launched Revivyl (INCI: Propanediol (and)
Water (aqua) (and) Phoenix Dactylifera (Date) Seed Extract), a Orobanche Rapum Extract), a holistic skin renewal
natural ingredient that relaxes skin and conditions hair. This accelerator. Tested in vitro, in vivo and ex vivo, the ingredient
botanical inspired by ancient date seedhelps slow cell shows six holistic effects on the skin: protection of epidermis
proliferation and the effects of environmental aging factors with stem cells, reactivation of cellular metabolism, stimulation of
its anti-aging properties. Free of preservatives, IBR-CalmDeAge epidermis cell differentiation, reinforcement of skin barrier,
also provides antioxidant protection, relief from rosacea and reactivation of natural exfoliation and active protection of
reduction of dark circles. skin microflora.
www.ibrweb.com www.givaudan.com
CT170708_Tech_Launches_fcx.indd 10 6/26/17 10:56 AM

Regulatory | C&T
KEY POINTS
Consumer safety could be at risk if SPF values
are not accurately represented on product
labels, providing inconsistant results in
the industry.
Formulators must use corroborating evidence

to ensure that tested SPF values ring true,
based on dependable products.
EU Regulatory Update
The Face Value

of SPF Testing
Rethinking Sun Protection Claims
W
Chris Flower, Ph.D.
Cosmetic, Toiletry &
Perfumery Association,
London
hen I was in school, complex
calculations were carried out using
log tables, though we were permit-
ted to use slide rules for quick
calculations where the necessary
level of precision was achieved.
It wasnt that we didnt have personal computerswe didnt even have
pocket calculators! Neither had yet been developed. However, what we
Reproduction in English or any other language of

12 | www.CosmeticsandToiletries.com all or part of this article is strictly prohibited. Vol. 132, No. 7 | July/August 2017
2017 Allured Business Media.
CT1706_Regulatory_Flower_fcx.indd 12 6/27/17 1:33 PM

We need to be able to spot a result that is
simply unexpected, not always taking the
numbers at face value, and be prudent before
basing decisions on them.
did have was the ability to look at the answer degree of protection they have become accus-
to see whether it made sense; we knew roughly tomed to and again may be harmed.
what the answer should look like.
Technology now means that individuals have SPF Revisited
access to levels of computing power that would In the EU and across most of the world, the
have been unimaginable only a generation ago, measurement of SPF is based on an in vivo test
but with that has come an increasing tendency method. This involves exposing human skin to
to take the output of such technology at face UV light to determine the exposure necessary
value. The term face value originates from to cause minimal reddening, applying product
money, and is the value given to an essentially and then exposing the protected area to see
worthless piece of metal or paper simply by the exposure now needed to cause minimal
virtue of a number present on its face. Yet if I reddening. The multiple of "protected" dose
were to place the same number on a page from over "unprotected" dose gives the SPF. Simply,
my notebook, no one would take that at face if protected skin requires 15 times the dose of
value, but would recognize it as worthless. UV to cause minimal reddening compared to
This is the skill we need to cultivate, particu- unprotected skin, the SPF is 15.
larly now that the efficacy of cosmetic products Although this sounds relatively straight-
is supported by evermore complex technology forward, there are many variables that need
making detailed measurements of many param- to be controlled to ensure a consistent and
eters, which are then analyzed for statistical reproducible test methodfurther variables in
significance. We need to be able to spot a result the conduct of the test means the data needs
that is simply unexpected, not always taking the to be analyzed appropriately in order to ensure
numbers at face value, and be prudent before the result is statistically robust. Such variables
basing decisions on them. include the UV source itself, the assessment
Of course, that outlier may be a false of minimal reddening, the quantity of product
impression of high efficacy or a false impres- applied and how it is applied, and the biological
sion of poor efficacy, but in either case it variability between individuals.
needs to be spotted, investigated and, if There is still the possibility of a rogue result,
necessary,corrected. despite method protocol attempts to control
This is especially important in the case of all of these variables as far as possible per
sun protection products where consumer safety International Organization for Standardization
relies on the labeled SPF value being an accu- (ISO) standard testing. It might be a statisti-
rate representation of product performance in
protecting against UVB rays. If the product fails
to deliver on its claim, a consumer will not get
the protection they expect and may be burned
by the sun as a result. In 53% of women surveyed, SPF was a part of their
Conversely, if a product provides sig- daily routine, while only 33% reported wearing it
nificantly greater protection than it claims, when they expected to be exposed to the sun.
consumers using that product will gain a false
impression of the degree of protection provided
by the declared SPF. If they then change to Source: Global Cosmetic Industry
another product or brand with the same or (www.GCImagazine.com)
similar SPF declaration, they will not obtain the
Vol. 132, No. 7 | July/August 2017 Cosmetics & Toiletries | 13
CT1706_Regulatory_Flower_fcx.indd 13 6/26/17 11:07 AM

cally robust value, but it is still rogue. Therefore, it is
essential to not take this result at face value but to call
on corroborating evidence. This is why the on-pack SPF
number is not based on the final in vivo SPF test alone.
Testing Based On Success

Corroborating evidence usually comes from the
skill and knowledge of the formulator, who calls upon
past experience from developing the product to ensure
sufficient and appropriate UV filters are included and
well-dispersed; that the product is stable; and that appli-
cation produces an even spread of product that remains
in place on the skin. Other desired attributes will also
be incorporated and influence the final outcome. During
product development, the formulator will test the new
mix in comparison with tried and trusted existing prod-
ucts to ensure the desired protection level is delivered,
and will adjust the formulation as necessary. Thus,
when the product is subjected to a full in vivo SPF test
according to the ISO method, the developer has a good
idea of the intended outcomeand, from past experi-
encethe likely variation in the expected result.
For example, in testing an SPF 30 product, some
individuals within the panel will exhibit higher or lower
results than SPF 30 during the test, but all must be
within a specified percentage of the mean for the test to
be statistically valid. However, separate to that, corrobo-
rating evidence is needed against which one can judge
whether or not the SPF obtained from a statistically
valid test makes practical sense. If a repeat test on the
same or similar product shows wildly different results,
the operator and/or the formulator should be question-
ing the situation.
In those circumstances, there would be a thorough
review of the whole development program, a re-
appraisal of the control products used as benchmarks
and a re-examination of the manufacturing records of
the test batch to identify why the unexpected discrep-
ancy arose. Certainly, it would be incorrect simply to
label the SPF 30 product as SPF 20 or SPF 50 based
solely on one SPF test taken at face value.
Conclusion
While there are variables in the in vivo SPF test
method, it is simple biology. The ISO in vivo SPF test
method is dependable when the results are used in
conjunction with a body of supporting information on
the product.
Of course, we must not forget that sun protection
products should also protect against UVA rays, which is
determined in the EU using the ISO invitro UVA test
methodthe results of which should consistently
parallel the SPF level.
CT1706_Regulatory_Flower_fcx.indd 14 6/26/17 11:08 AM

Research | C&T
KEY POINTS
Readers previously expressed interest in
understanding the most efficient and reliable
methods to separate the epidermis and
dermis for basic skin investigation.
This third and final part in our series focuses

on heat and mechanical techniques.
A Dermatological View
Dermal-epidermal
Separation, Part III Heat and Mechanical Methods
T
Ying Zou
Shanghai Skin Disease
Hospital, Shanghai, and
University of California,
San Francisco his column concludes a three-part
series on techniques for epidermal-
Howard I. Maibach, M.D. dermal separation (see Figure 1). If
University of California, focuses on heat and mechanical tech-
San Francisco niques and describes the advantages
and disadvantages of each. Previous
installments reviewed enzyme digestion options and the use
of acid, alkalis and natural salts.

CT170708_Research_Maibach_fcx.indd 16 6/26/17 11:19 AM

There is need for techniques by which the
epidermis can be separated from the dermis
via purely mechanical forces, to avoid
chemical or thermal damage.
Heat Separation (compared to fresh skin). After heating at 60C

for 1 min, neither epidermis nor the stratum cor-
Epidermal-dermal separation by heat (see
neum exhibited positive histochemical staining
Table 3) was introduced in 1942 by Baum-
for esterases activity. Therefore, heat separation
berger et al. Since increasing temperatures can
should not be used to study the permeation of
soften collagen fibers, temperatures much lower
ester-containing permeants.
than the boiling point of water may be used to
separate the epidermis from the dermis.
For example, after exposure for 2 min on
Mechanical Methods
a slide warming table at 50C, the epidermis The methods above have the disadvantage
was found to peel readily and completely using of altering, to some degree, the physical and/
forceps. Complete division occurred with ease or chemical integrity of one or both layers and
at 49.2C, after incompleteness at 48.2C. With may therefore be unsatisfactory for studies that
temperatures above 51C, detachment dif- require an isolatedbut otherwise intactepi-
ficulty occurred. Felsher4 detected an influence dermis or dermis. There is a need for techniques
of salts on the subsequent separation of the by which epidermis can be separated from the
epidermis by heat. Heat separation is facilitated dermis via purely mechanical forces, to avoid
by ions, which can promote collagen swelling chemical or thermal damage.
ordepression.
In the studies of Sonderga et al.18 and Wad-
Stretching
skov et al.,19 separation of the epidermis from Van Scott22 stretched the skin and removed
the dermis in normal human skin by heat was the epidermis as a continuous sheet by means of
reported possible after 10 min at 60C. a razor blade or scalpel. When skin is stretched,
Kassis et al.20 analyzed epidermal and the epidermis slips off easily using this proce-
dermal prostaglandins by heat separation. They dure and can be rapidly removed. Gilbert23 and
kept frozen biopsy specimens in a water bath Sputt24 improved the technique so skin only
for various times and found that heating at needed stretching to a relaxed length. However,
60C for 1 min produced a distinct separation these methods require comparatively large surgi-
of epidermis from dermis. As there was no cal necropsy specimens, and when scraped off
significant change in Prostaglandin E1 (PGE1) with a scalpel, may lack a basal cell layer.
activity after 1 min of heating, this heat separa-
tion technique seemed a reliable and quick
method for PG analysis of skin.
Esterases are important for topical or trans- The skin care sector is on track to reach
dermal delivery of drugs since these enzymes $130 billion in sales in 2019, with facial skin
may catalyze the hydrolysis of pharmaceuticals care and actives accounting for a large
containing ester bonds. Lau et al.21 detected majority of the segment.
esterase activity in fresh and heat-separated
porcine ear skin. Esterase activity was visibly
diminished in common heating separated skin Source: Global Cosmetic Industry
(www.GCImagazine.com)
CT170708_Research_Maibach_fcx.indd 17 6/27/17 1:50 PM

Suction the dermis along the dermal-epidermal junc-
As with unidirectional stretching, dermal- tion (DEJ) during dry cupping. Blank et al.26
epidermal separation has been achieved by also separated the epidermis from the dermis
the application of pressure gradients causing with vacuum via bullae formation, which
multidirectional distension. In 1878, Unna25 demonstrated DEJ separation histologically.
described the separation of the epidermis from This approach therefore became a mechani-
Figure 1. Diagram of action site of dermal-epidermal separation methods
Table 3. Heat Separation
Temperature Duration
Year Author Ideal Solution Detail
(C) Time
1 1942 Baumberger, J5 50 2 min / /
/
56 45 min / Immersed in salt
2 1947 Felsher, Z4 56 1 min 2N Sodium Chloride solution for 4 hr,
56 1.5 min 2N Potassium Chloride subsequently to heat
treatment
3 1968 Sonderga, J18 60 10 min Water bath /
4 1978 Wadskov, S 19
60 10 min Water bath /
5 1982 Kassis, V et al.20 60 1 min Water bath /
6 1995 Ohata, Y et al. 28
56 30 sec PBS /
Esterase activity
7 2012 Lau, WM et al.21 60 1 min /
reduced

The separation method
must be appropriate
for the experimental
project. No single
method appears
superior for
all purposes.
cal method to separate the epidermis. In vivo of the dermis was minimal. Thus, the suction
separation of the complete human epidermis by epidermis appeared useful for tissue culture
suction was accomplished in 1964. and biochemical analyses. Today, this method is
Kiistala et al.27 found that suction at a widely applied in dermatological research and
pressure of 20 cm Hg applied to intact human practice for epidermal grafting2931 for creating
healthy skin produced blisters within 2 hr, the standardized wound-healing models32, 33 and
roof of which consisted solely of epidermis and for studying morphological, physiological or
included the basal cell layer. They constructed a pharmacological phenomena.34, 35
device that produced standard suction blisters
of preselected size and number on human skin Overall Pros and Cons
without discomfort. In approx. 2 hr, suction Many techniques relying on different mecha-
at 150 mm Hg28 permitted separation of the nisms have been described in this series.
epidermis from cadavers and certain animals. Heat is simple, yet tedious. Although
The blister roof consisted of viable, full- the heat process has been used successfully,
thickness epidermis and the clear fluid was a chemical reagents are effective but disturb
non-inflammatory transudate. the electrolyte cellular equilibrium. Digestion
No scarring resulted and the suction trauma by enzymes gives complete separation but it

Table 4. Comparison of Separation Methods
Separation
Year Author Results Conclusion
Methods
Epidermal tissues are not
Epidermal tissues treated not killed killed by both methods
but temporarily loosened Marked oxygen
NH4OH
1 1942 Baumberger, J5 Oxygen consumption rate by heat: consumption by both
Heat
23% by NH4OH 14% compared methods
with method of razor blade Heat technique is
superior as water-free
Sulfhydryl values obtained after
Enzyme
NH4OH method similar to that of NH4OH separation
Heat
2 1960 Ogura, R et al.36 mechanical technique appeared
NH4OH
Heat and enzymes alter the results procedure of choice
Mechanical
significantly
Bullous pemphigoid antigen
(BPA) remained associated with
epidermis of all specimens with
a 1:320 or greater dilution except
Only trypsin effectively
Trypsin warm PBS separation
degrades the
1M NaCl Antigenicity of basement
3 1983 Woodley et al.37 molecule, making the
PBS membrane heparin sulfate
antibody binding site
Suction proteoglycan lost after trypsin
unrecognized
separation
Laminin, type IV and V collagen
remained with dermis by all
methods
1M NaCl resulted split within
lamina lucid, with intact
hemidesmosomes and swollen
mitochondria. No other
degenerative features
1M NaCl was the most
Suction Thermolysin developed intra-
reproducible, convenient
4 1991 Willsteed et al.38
1M NaCl epidermal separation in 4 of 5
and reliable separation
Thermolysin specimens
method
Suction caused split through the
lamina lucina, associated with
hemidesmosome disruption and
formation of cytoplasmic vacuoles
within keratinocytes
Antigen molecules (PV, PF, BP and
Heat-separation
EBA) can be detected after these
EDTA should apply as
methods at either the cell surface
5 1995 Ohata, Y et al.39 Dispase standard substrate for
or BMZ, except for negative
Heat immunoblotting for the
reactivity with BPAG2 in dispase-
shorter preparation time
treated epidermis
Trypsin was unsuitable
Initial rate of soluble protein:
Physical scraping was
EDTA EDTA > heat > scraping > trypsin
less a consideration for
Trypsin Initial rate of tissue:
difficulty in determining
Heat EDTA > heat > scraping > trypsin
6 1994 Fort et al.40 cross-contamination of
Mechanical Microscopic examination
layers occurred
(stretching confirmed not known to the extent
Heat separation slightly
and scraping) of contamination of dermis after
inferior to EDTA
scraping
EDTA was favorite
CT170708_Research_Maibach_fcx.indd 20 6/27/17 1:52 PM

Table 4. Comparison of separation methods (Continued)
Separation
Year Author Results Conclusion
Methods
5869% and 8992% less
Separation with 3.8%
Ammonium kallikrein 12 mRNA detected after
ammonium thiocyanate
Thiocyanate, treating with heat and diapase
was optimal choice
7 2007 Trost, A et al.41 Dispase than with ammonium thiocyanate,
for high-quality RNA
Heat respectively
isolation from human
1M NaCl No kallikrein detected after 1M
skin
NaCl treating
No significant difference between

total cell counts, dead cell counts,
and percent of alive cells of four
methods: NaBr(4N) method yielded
the most cells (total and alive);
NaBr trypsin method produced the most
NaBr(4N) method yields
(2N and 4N), dead cell counts
8 2011 Maharlooei 42
more alive cells and less
NH4OH A significant higher number of
toxicity
Trypsin alive cell counts from NaBr(4N)
method compared with the others
Immunocytochemical staining
of cytokeratin and melanosome
antibodies showed no difference
between four methods
destroys important components. Meanwhile, Dermo-epidermal separation is an essential

mechanical division necessitates a relatively technique for immunoblotting studies and for
large tissue piece but has the advantage in that the diagnostic immunofluorescence of autoim-
chemical changes do not occur. Many inves- mune bullous diseases. To study the localization
tigations have compared these methods and of basement membrane components after
documented their different influences on the different separation methods, Woodley et al.37
structure and function of skin biochemistry. separated adult human skin using four meth-
Baumberger, when comparing NH4OH ods. Only the basement membrane heparan
and heat separation methods, found that sulfate proteoglycan was trypsin-sensitive.
epidermal tissues treated were not killed but Willsteed et al.38 examined the electron
only temporarily loosened, and there was a microscopic appearance of specimens separated
marked decrease in oxygen consumption by the by three methods, concluding that 1M NaCl
epidermis by both methods. However, the heat remains the most convenient and reproducible
technique may be superior because the tissue means of inducing dermal-epidermal separation
is not brought into water and there is no loss of through the lamina lucida.
material (see Table 4). Ohata et al.39 compared the autoantigens
Quantitative determinations of stratum for bullous skin diseases by immunoblotting
corneum sulfhydryl and disulfide concentration using different dermal-epidermal separation
revealed relatively consistent values in certain techniques; the results suggest that EDTA-,
skin diseases. Various methods of separation of dispase- and heat-separated skin were similar
epidermal sulfhydryl showed its values obtained for detecting various autoantigens but heat
after ammonium separation resembled those separation is preferable because the prepara-
following mechanical separation, while heat tion time is shorter.
and enzymes appeared to alter the results To compare the effects of epidermal-dermal
significantly and were deemed not satisfactory.36 separation techniques of hairless mouse skin,

the most suitable method was determined by cosmetic research based on this methodology
kinetic studies in homogenate medium.40 Based and predictability will lead to greater utiliza-
on data of the effects of separation methods tion, as more refined knowledge should lead to
on soluble protein yield and dermal enzymatic enhanced research integrity.
activity using DBMTX as a substrate, EDTA
treatment appears a favorable method for References
epidermal separation. 4. Z Felsher, Studies on the adherence of the epidermis to the
To obtain rapid, high-quality epidermal- corium, J Invest Dermatol 8 35-47 (1947)
specific RNA from human skin, Trost et al.41 18. J Sndergaard, H Zachariae, Epidermal histamine, Arch Klin
investigated the effect of dermal-epidermal Exp Dermatol, 233 323328 (1968)
separation methodsincluding ammonium 19. S Wadskov, J Sndergaard, Determination of cyclic-amp

in heat-separated human epidermal tissue, Acta Derm
thiocyanate, dispase, heat and 1M NaCland Venereol, 58 191195 (1978)
concluded that the fast chemical separation 20. V Kassis, J Sndergaard, Heat-separation of normal
method with 3.8% ammonium thiocyanate is human-skin for epidermal and dermal prostaglandin analy-
the optimal choice for producing high-quality sis, Arch Dermatol Res, 273 301306 (1982)
RNA isolation from human skin. 21. WM Lau, KW Ng, K Sakenyte, CM Heard, Distribution of
esterase activity in porcine ear skin, and the effects of freez-
Epidermis separation is also useful in ing and heat separation, Int J Pharm 433 1015 (2012)
autologous skin transplantation. The first step 22. EJ Van Scott, Mechanical separation of the epidermis from
in isolating epidermal cells is to separate it from the corium, J Invest Dermatol 18 377379 (1952)
dermis. Recently, several methods for epidermis 23. D Gilbert, PD Mier, TE Jones, An improved technic for the
isolation and epidermal cell suspension were isolation of epidermis from human skin, J Invest Dermatol
40 165167 (1963)
compared to select the optimum one for clinical
24. D Sprutt, An improved technic for the isolation of epidermis
use. NaBr (4N) method is considered as the from larger specimens of human skin, J Invest Dermatol 42
least toxic and the most viable cells produced.42 285 (1964)
25. P Unna, Zur Anatomie der Blasenbildung an der menschli-
Conclusion chenHaut, Vjschr Dermatol Syphil 5 3 (1878)
26. IH Blank, OG Miller, A method for the separation of the epi-
In conclusion, the separation method must
dermis from the dermis, J Invest Dermatol 15 910 (1950)
be appropriate for the experimental project,
27. U Kiistala, KK Mustakallio, In-vivo separation of epidermis
and no single method appears superior for all by production of suction blisters, Lancet 7348 14441445
purposes. Future comparative investigation (1964)
should benefit the integrity of skin care, and

28. U Kiistala, Suction blister device for separation of viable epi- 36. R Ogura, JM Knox, AC Griffin, Separation of epidermis
dermis from dermis, J Invest Dermatol 50 129137 (1968) for the study of epidermal sulfhydryl, J Invest Dermatol 35
29. S Gupta, S Shroff, Modified technique of suction blistering 239243 (1960)
for epidermal grafting in vitiligo, Int J Dermatol 38 306309 37. D Woodley, D Sauder, MJ Talley, M Silver, G Grotendorst, E
(1999) Qwarnstrom, Localization of Basement-Membrane Compo-
30. R Falabella, Suction blister device for separation of viable nents after Dermal-Epidermal Junction Separation, J Invest
epidermis from dermis, Clin Exp Dermatol 29 105106 Dermatol 81 149153 (1983)
(2004) 38. EM Willsteed, BS Bhogal, AB Das et al., An Ultrastructural
31. J Li, WW Fu, ZZ Zheng, QQ Zhang, Y Xu, L Fang, Suction Comparison of Dermo-Epidermal Separation Techniques, J
blister epidermal grafting using a modified suction method Cutan Pathol 18 812 (1991)
in the treatment of stable vitiligo: a retrospective study, 39. Y Ohata, T Hashimoto, T Nishikawa, Comparative study
Dermatol Surg 37 9991006 (2011) of autoantigens for various bullous skin diseases by
32. J Kottner, K Hillmann, S Fimmel, S Seite, U Blume-Peytavi, immunoblotting using different dermo-epidermal separation
Characterisation of epidermal regeneration in vivo: a 60-day techniques, Clin Exp Dermatol 20 454458 (1995)
follow-up study, J Wound Care 22 395400 (2013) 40. JJ Fort, AK Mitra, Effects of epidermal/dermal separation
33. V Czaika, A Alborova, H Richter et al., Comparison of methods and ester chain configuration on the bioconversion
transepidermal water loss and laser scanning microscopy of a homologous series of methotrexate dialkyl esters in
measurements to assess their value in the characterization dermal and epidermal homogenates of hairless mouse skin,
of cutaneous barrier defects, Skin Pharmacol Physiol. 25 Int J Pharm 102 241247 (1994)
3946 (2012) 41. A Trost, JW Bauer, C Lanschuetzer et al., Rapid, high-
34. JJ Levy, J Vonrosen, J Gassmuller, RK Kuhlmann, L quality and epidermal-specific isolation of RNA from human
Lange, Validation of an in-vivo wound-healing model for skin, Exp Dermatol 16 185190 (2007)
the quantification of pharmacological effects on epidermal 42. MK Maharlooei, AA Mohammadi, A Farsi, I Ahrari, A Attar, A
regeneration, Dermatology 190 136141 (1995) Monabati, A comparison between different existing methods
35. IG Panoutsopoulou, G Wendelschafer-Crabb, JS Hodges, used to separate epidermal cells from skin biopsies for
WR Kennedy, Skin blister and skin biopsy to quantify autologous transplantation, Indian J Dermatol 56 666669
epidermal nerves A comparative study, Neurology 72 (2011)
12051210 (2009)

Research | C&T
KEY POINTS
In personal care, indiscriminate microbial
destruction by traditional preservatives can
unintentionally alter the thriving ecosystem
that is the skin's microbiome.
As an alternative, antimicrobial peptides

were explored for their effects on the skin's
microbial species using 16S rRNA and a
novel comparative approach.
Preserving
Microbiome
Harmony
all or part of this article is strictly prohibited.
CT170708_Rsrch_Alkazaz_fcx.indd 24 6/26/17 11:33 AM

Antimicrobial Peptides
Balance Skin Health
Tia Alkazaz, Maureen Danaher, Jennifer Goodman,

Erica Segura and Durant Scholz
Active Micro Technologies, Lincolntown, NC USA
peptides were explored for effects on the skin's
J
microbial species.
In relation, novel research analyzing activ-
ity of the histone deacetylase (HDAC) enzyme
has concluded some naturally derived antimi-
crobials are capable of destroying pathogenic
bacteria while maintaining commensal
microflora on the skinsupporting the balance
ust as every individual has of the microbiome and promoting overall
a distinct fingerprint, they skin health. HDAC expression was therefore
also have their own unique used as an indicator to compare effects on the
microbiome that is an skins microbiome of traditional biocides vs.
accumulation of microbial naturalantimicrobials.
community inhabitants. This Indeed, the application of topical antimi-
serves in host defense. Commensal microflora crobials altered levels of HDAC expression and
on the skin, for example, are responsible decreased the population of the microbiome.
for maintaining skin health by restoring While this research suggested HDAC is a
immunity and communication with the channel of communication between microflora
lymphaticsystem.1 and the skin, the messenger of the microbial
However, in personal care, indiscriminate cross-talk has yet to be determined.
microbial destruction, as traditional preserva- As will be shown, a conventional approach
tives often employ, can unintentionally alter was taken to analyze effects in species of
the thriving ecosystem that is the skin's micro- the skins microbiome. Specifically, effects
biome. Here, as an alternative, antimicrobial were assessed with the application of three
Vol. 132, No.

Reproduction 7 | July/August
in English 2017
or any other language or part of this article is strictly prohibited. 2017 Allured Business Media.
of all Cosmetics & Toiletries | 25

The individuality of each person's microbiome
makes it difficult to establish trends within a
group of subjects.
antimicrobial peptides: Leuconostoc radish as well as the method of bacterial identification

root ferment filtrate, Lactobacillus ferment, and employed here.
Lactobacillus (and) Cocos nucifera (coconut) The species of microorganisms investigated
fruit extract. These were compared with a were: Staphylococcus sp., Corynebacterium sp.,
negative control (water) and a positive control Propionibacterium sp., Streptococcus sp. and
(triclosan). The microbiome population was Aerobacillus sp. S. epidermidis is one of the most
determined by DNA extraction, 16S ribosomal common and abundant microbes on the skin,
RNA (rRNA) polymerase chain reaction (PCR) and may have a similar mutual relationship
amplification and sequencing. with skin as most flora function in the gut.2
A less conventional approach was taken C.jeikeium offers epidermal protection via a
regarding the panel size and evaluation during mutualistic relationship with the host and is
this study. While large subject panels allow for thought to be more beneficial than harmful to
trend recognition between subjects, with the skin.2 P. acnes is often associated with detrimen-
individuality of each persons microbiome in tal effects such as acne but it is well-established
mind, it would be difficult to establish trends that both healthy and acne-prone patients are
within a group of subjects. Examining the colonized with the bacterium. S. pyrogenes
nasolabial folds of each subject can isolate has been found in vitro to serve the host in
the geographic location of the microbiome, a protective role by secreting pore-forming
although the person-to-person variation of toxins to promote wound healing.2 Finally,
microflora is still uncontrollable. Thus, patterns Aerobacillus sp. has the ability to grow and
in microbial change on each test subject were producepolysaccharides.3
evaluated individually. The beneficial role of microbes on the skin
surface, unlike digestive probiotics, remains
Skin Microbes and relatively untapped aside from the known roles
the 16S rRNA Method of these species in protection against other
pathogenic and opportunistic invaders.
To understand the significance of the present
The method of bacterial identification
study, it is first crucial to recognize some of the
for this study was16S rRNA sequencing, an
common microorganisms located on the skin
overview of which is given in Figure 1. This is a
common amplicon sequencing method used to
identify and compare bacteria present within
complex microbiomes and environments. As
the name suggests, the technique focuses on the
The probiotic trend is gradually making analysis of ribosomal RNA.
its way into skin care, and along with Ribosomes are complex structures found in
dermocosmetics, is among the leading the cells of all living organisms that play a role
growthareas ahead for the industry. in protein synthesis. Prokaryotic ribosomes con-
sist of two subunits, a large and a small subunit,
the latter of which 16S rRNA is a part. The 16S
Source: Global Cosmetic Industry rRNA gene contains hyper variable regions that
(www.GCImagazine.com) provide a species-specific signature, which is
useful for the bacterial identification process.4

Ribosomal RNA is conserved in cells, and The amplified rRNA genes are then cloned
distantly related organisms have remarkably and sequenced. Comparison of the sequenced
similar portions of 16S rRNA sequences. 16S rRNA with those in the genetic sequence database
rRNA gene sequencing is commonly used to allows for the identification of phylogenetic
identify diversities in bacterial microorganisms groups.5 The 16S rRNA-based taxonomic char-
and study phylogenetic relationships between acterization can provide information on the
them.4 There are several advantages to using microbial population present in a given environ-
ribosomal RNA in molecular techniques such ment, and how its relative distribution may
as gene sequencing. These include the pres- differentially evolve under an applied treatment
ence of ribosomes and ribosomal RNA in all over time (see Figure 1).
cells, and the 16S rRNA gene being highly
conserved in nature and large enough to use for Materials and Methods
informaticspurposes.4 A DNA extraction, 16S rRNA PCR ampli-
The analysis of rRNA genes begins by fication and sequencing study of the skin was
isolating a sample of bacteria, followed by the conducted to evaluate the microbiome population
extraction of bacterial DNA. The bacterial DNA present on facial skin and respective changes in
undergoes PCR amplification using primers microbial populations after two weeks of product
that specifically code for the 16S rRNA gene application. Participants (n = 15) were separated
fragment. Amplification produces a popula- into five blinded treatment groups, each applying
tion of rRNA gene fragments of equal size, one of the following products twice daily to the
determined by the specific primers used. This lateral nasal folds: Leuconostoc radish root fer-
population of rRNA gene fragments is still ment filtrate, Lactobacillus ferment, Lactobacillus
considered to be representative of the natural (and) Cocos nucifera (coconut) fruit extract, water
microbialpopulation.5 ortriclosan.
Figure 1. Overview of bacterial identification by 16S rRNA sequencing

Application to the lateral nasal folds was application to serve as a reference for the
performed by rubbing a pre-moistened swab normal microbial presence on each participants
back and forth across the treatment area for a skin. Each untreated skin swab sample was
total of 60 sec. Consistent pressure was applied taken using a sterile swab pre-moistened with
to the treatment area to ensure substantial sterile salinesolution.
recovery of the microbial population. Untreated As noted, treatments were then applied
skin swab samples were taken prior to product twice daily for a period of two weeks and new
Figure 2. Sampling of phylogenetic tree taxonomy at timepoint 1
Figure 3. Sketch of phylogenetic tree at timepoint 1

Taxonomic units do not always provide
specific species but they serve as effective
indicators for bacterial diversity.
samples were taken from each participant to underwent taxonomic clustering and analysis.
analyze population differences after product The resultant usable reads were clustered into
application. One week after the conclusion of Organizational Taxonomic Units (OTUs)b. OTUs
product treatments, the last round of samples classify closely distinct microbial organisms
was taken from each participant to analyze the from sequences via DNA homology, although
populations present after treatment ceased. In in some cases, they may only read genus or a
total, 45 samples were stored in 15-mL conical higher levels of taxonomy. It is important to
tubes and frozen at -20C immediately after note that OTUs do not always provide specific
sampling. These samples were submitteda for species for each sequence but these units still
DNA extraction, 16S rRNA PCR amplification serve as effective indicators of the bacterial
and sequencing analysis. diversity on skin.
The amplicons obtained from PCR ampli- Taxonomic clustering and analysis was
fication from each sample were collected in performed after sequencing the samples to allow
equimolar proportions into a single pool for for the generation of a phylogenetic tree. Quality
sequencing. After sequencing, the samples assurance analysis was performeda for base call-
ing quality and in-depth analysis was performed
a
Genomics Laboratory at the David H. Murdoch Research Institute
(DHMRI) b
Silva OTU Reference Database for ARB software

to analyze the size and
nature of 16S rRNA. The
data obtained from the
readings met the quality
assurance specifica-
tions to create scoring
reads for alignment and
database searching.
The usable reads were
blasted against the OTU
reference databaseb to
generate OTU abun-
dance results, creating
phylogenetic trees and
multiple alignments.
These results were used
to calculate diversity
estimates based on
the abundance of
microorganism genus
in each sample. Overall,
the analysisa showed a
diverse population with
an abundant presence
Figure 4. Changes in microbiome population for participant 1, of Propionobacterium
treated with Leuconostoc radish root ferment filtrate sp., Staphylococcus
sp., Aeribacillus sp.,
Streptococcus sp. and
Corynebacterium sp.
Microbiome
Results
As stated, this study
determined the micro-
bial population present
on skin and any changes
to this population after
two weeks of varying
product treatments. The
microbiome population
was determined after
sampling the skin area
treated with sterile
swabs pre-moistened
with sterile sodium chlo-
ride solution followed
by DNA extraction, 16S
rRNA PCR amplification
and sequencing. During
the treatment, one of
Figure 5. Changes in microbiome population for participant 6, the participants showed
treated with Lactobacillus ferment high sensitivity to the
positive control, triclo-

san, and for this reason
discontinued further
treatment.
The DNA extracted
from the samples
taken before treatment
application began and
after the bioinformatic
analysisa showed a
diverse population,
previously mentioned,
as well as different
populations known
as transient and/or
opportunistic invaders,
such us Escherichia
sp, Pseudomonas sp.,
Vibrio sp., Clostridium
sp., Neisseria sp.,
etc. Examples of a
phylogenetic tree and
a phylogenetic tree tax-
onomy obtained during
this study are shown in
Figure 6. Changes in microbiome population for participant9,
Figures 2 and 3. treated with Lactobacillus (and) Cocos nucifera (coconut)
Lactobacillus fruitextract
ferment decreased
all bacteria genera,
whereas Leuconostoc
radish root ferment
filtrate and Lacto-
bacillus (and) Cocos
nucifera (coconut)
fruit extract increased
the beneficial bacteria
Staphylococcus and
Corynebacterium,
and decreased Propi-
onibacterium. After
the conclusion of
topical treatments,
Propionibacterium
sp. reappeared on the
skin of all participants
using antimicrobial
peptidetreatments.
The ben-
eficial bacteria
Staphylococcus sp.,
and Corynebacterium
sp. continued increas- Figure 7. Changes in microbiome population for participant 12,
ing after completion
treated with triclosan
of the treatments.

Figure 8. Changes in microbiome population for participant 15, treated with water
Examples of changes in microbiome popula- Propionibacterium sp. Lactobacillus ferment

tion observed during this study are shown in decreased all bacteria genera found in par-
Figures48. Patterns in microbial change on ticipants, compared with triclosan as positive
each test subject were evaluated individually, as control and water as negative control. After the
noted, since the microbiome of each participant conclusion of the topical treatments, Propi-
is unique. nobacterium sp. reappeared in the skin of the
participants treated with antimicrobial peptides.
Discussion The beneficial bacteria Staphylococcus sp.
DNA extraction, 16S rRNA PCR amplifica- and Corynebacterium sp. continued increasing
tion and sequencing for the current study were after completion of the treatment. By increas-
conducted in part to satisfy the NIH Human ing the populations of beneficial bacteria and
Microbiome Project: Microbiome Analysis and decreasing the population of Propionibacterium
Sample Collection, and the following State- sp., i.e, the commensal bacteria associated with
ments of Worka: ALCLLC-105 DNA Extraction the development of acne, this study demon-
from Skin Swabs, ALCLLC-106 16S Sequencing strates the potential of natural antimicrobials to
on DNA Extracted from Skin Swabs. promote a balanced skin microbiome.
Under the conditions of this in vivo human
skin microbiome assay, two of the antimicrobial Conclusion
peptides increased the beneficial bacteria in The complexity of the skin microbiome is a
participants while decreasing the presence of relatively new area of research in the cosmetic

industry. Since the discovery of microbes on the human
body, information on the microbial population has been
reported; however, the role of microbial population in
skin health is yet to be fully elucidated. This study adds
to the understanding of several dominant microorgan-
isms such as Propionibacterium and Staphylococcus
spp., which are common components of the skin
microbiome. It also provides insight on the effect of
topical application of antimicrobial peptides on the
microflora of skin.
While traditional preservatives may introduce
inhibiting factors that directly influence the balance
of skin's microbiome, the use of natural antimicrobial
peptides may serve as a novel approach to maintain-
ing and promoting healthy skin microbiota. However,
further metagenomics analysis is necessary to reveal
the complex functions and interactions between these
antimicrobial peptides and the skin microbiome, as
well as between microorganisms within the skin micro-
biome. Further research in this area could provide
more comprehensive approaches to the development
of topical products that consider the integral contribu-
tions of the skin microbiome.
References
1. D Erturk-Hasdemir and D and Kasper, Resident commensals shaping
immunity, Current Opinion in Immunology 25(4) 450-455 (2013)
2. A Cogen, V Nizet and R Gallo, Skin microbiota: A source of disease or
defence? Brit J Dermatology 442-455 (2008)
3. J Ouyang et al, Paenibacillus thiaminolyticus: A new cause of human
infection, inducing bacteremia in a patient on hemodialysis, Annals of
Clinical and Laboratory Science 38(4) 939-400 (2008)
4. JM Janda and L Abott, 16S rRNA gene sequencing for bacterial
identification in the diagnostic laboratory: Pluses, perils and pitfalls,
JClin Microbio 45(9) 2761-2764 (2007)
5. KH Wilson RB Blitchinton and RC Greene, Amplification of bacterial
16S ribosomal DNA with polymerase chain reaction, JClin Microbio
28(9) 1942-1946 (1990)
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Research | C&T
KEY POINTS
The predominant natural folate in skin,
5-MTHF, is intrinsically stable under UV but
can degrade if photosensitizers are present.
Sub-micromolar 5-MTHF protects DNA against
oxidation, and ascorbate levels in skin help
maintain this folate.
Diminished folate levels impact the rate of
DNA damage, thus this article explores how
reinforcing skin folates prior to UV exposure
could protect against DNA damage.
NaturalinFolate
Human Skin
all or part of this article is strictly prohibited.
CT170708_Rsrch_Bailey_fcx.indd 34 6/27/17 2:02 PM

A New Approach to
UV Damage Protection
Steven W. Bailey and June E. Ayling

University of South Alabama, Mobile, AL USA
by pigmentation. It is well-known that darker-

skinned individuals have a much lower risk of
T
skin cancer than lighter-skinned individuals.
However, the penetration of UVB into the
basal layers of the epidermis is required for the
photo-conversion of 7-dehydrocholesterol to
pre-vitamin D3.
It has been hypothesized that the need
for adequate vitamin D production drove the
he risk for developing evolution of lighter skin color in early human
skin cancer is strongly populations that migrated from Africa into
related to sun exposure.1 northern latitudes, where annual UV exposure
Both UVB and the more is less intense.20 On the other hand, those hav-
deeply penetrating UVA ing little melanin who have moved closer to the
components of solar radia- equator, such as in northern Australia, experi-
tion stimulate the progression of melanoma.2 enced especially higher rates of skincancer.
Moreover, the lamps used in modern tanning In addition to pigmentation, DNA repair
beds, though predominately UVA, are often mechanisms are another class of inherent
more intense than in natural sunlight. Indeed, protection aimed at preventing permanent
squamous and basal cell carcinoma and radiation damage. DNA lesions can be induced
melanoma have been associated with tanning by UVA in cell cultures and human skin via a
bed use.3-7 number of processes. One such process and a
Several mechanisms inherent to the skin major contributor is the formation of bypy-
help mitigate the damage caused by UV radia- rimidine photoproducts such as cyclobutane
tion. The first is the physical block provided pyrimidine dimers (CPD).8-10 For the UVA
Vol.Reproduction
132, No.in7English
| July/August 2017 of all or part of this article is strictly prohibited. 2017 Allured Business Media.
or any other language Cosmetics & Toiletries | 35
CT170708_Rsrch_Bailey_fcx.indd 35 6/26/17 11:59 AM

Of the various forms of DNA damage, double
strand breaks may be the most problematic.
band, CPD are believed to arise both from direct CPD, are a second line of defense against the
absorption by DNA and photosensitization harmful effects of radiation on DNA. However,
reactions.9, 11, 12 DNA repair essentially equates to trying to close
The next most prevalent contributor to DNA the barn door after the horse has escaped.
damage is the formation of 8-oxo-deoxyguano- Yet another inherent mechanism is the
sine (8-oxo-dGua), although the ratio of this to interception of reactive species generated by
CPD has been reported to be dependent on the UV, and work in our lab has identified a specific
cell type. This oxidized base can arise from the entity capable of doing so even prior to their
action of singlet oxygen,13, 14 hydroxyl radicals interacting with DNA: folate.24
or type-I photosensitized one-electron transfer.9 In considering the role of human pigmenta-
Strand breaks can be produced by additional tion, it was previously suggested that darker
oxidation of DNA containing 8-oxo-dGua.9, 15 skin could not only protect DNA, but also slow
Although it is still debated,9 several laboratories the photodegradation of the folate pool.19, 20
have provided evidence for the formation of Stemming the loss of folate in regions having
both replication-dependent and replication- intense UV radiation also helped to prevent
independent double-strand breaks by UVA birth defects and/or decrease the risk for
mediated by reactive oxygen species (ROS).16 cancer.20, 21 However, much of the initial concern
Of the various forms of DNA damage, double about the impact of light on folate was based on
strand breaks may be the most problematic.17 the well-known photolability of folic acid. This
These can be repaired by homologous recom- synthetic form is not a participant in physiologi-
bination by cells during the S or G2 phases of cal, folate-dependent, one-carbon metabolism
mitosis, or by non-homologous end joining (see Figure 1) unless converted by the slow
(NHEJ), mostly during the G1 phase. However, action of dihydrofolate reductase.22
since NHEJ does not match against a homolo- Thus, since little was known about the pho-
gous segment and does not always have the tochemistry of natural folates, we investigated
exact single strand overhang required to restore them primarily using 5-methyltetrahydrofolate
the original sequence, this method is especially (5-MTHF). This folate is the most abundant
prone to errors found in cancers.18 form in blood and many tissues, especially
These and other activities such as base exci- the skin.23 The present work not only demon-
sion repair, which can replace 8-oxo-dGua, and strated the circumstances under which this
nucleotide excision repair, which can replace folate is or is not degraded by light, but also
revealed a new mechanism for the protection
of biomolecules, especially in the aqueous
intracellularcompartment.
As consumers actively seek out effective and

Folate Stability to UV and
reliable sun care, two trends that will grow Photosensitization
the market through 2020 include product The stability of 5-MTHF under both UVA and
innovation and the use of nanotechnology. UVB was tested. However, even in the absence
of light, 5-MTHF in aqueous solutions is suscep-
tible to metal catalyzed reactions with molecular
Source: Global Cosmetic Industry oxygen. Correctly assessing its photostability
(www.GCImagazine.com) therefore requires working with metal-free buf-
fer, in which only approximately 1% is lost over

the course of 80 min at near-ambient tempera- bon metabolism participates in the biosynthesis
ture in the dark. Irradiation with 2.3 mW/cm2 of the purine bases and also thymidylate; and a
of UVA or 2.2 mW of UVB (filtered to remove depleted folate pool (see Figure 1) could hinder
UVC) induced a first order loss of 5-MTHF at DNA repair, as has been demonstrated in cell
0.0004min-1 and 0.0008 min-1, respectively (see culture, and lead to non-reversible damage.27
Figure2). The slightly higher rate caused by Regulation of DNA transcription also is, in part,
UVB may be related to its extinction maximum mediated by methylation reactions that are
at 290 nm when at a neutral pH. dependent on S adenosylmethionine, the supply
While direct photo-degradation is negligible of which can in turn be influenced by the level
in a physiological context, 5-MTHF was dis- of 5-MTHF. Both hyper- and hypomethylation of
covered to be decomposed in the presence of DNA have been reported in skin cancer as well
photosensitizers such as pterin-6-carboxylic acid as other cancers.28, 29
with UVA, or rose bengal with visible light.24
This has subsequently been investigated in vitro Folate Effect on
using several naturally occurring photosensitiz- UV-induced DNA Damage
ers: riboflavin, uroporphyrin and conjugated While investigating whether 5-MTHF might
bilirubin.25 Other endogenous photosensitizers exacerbate UV-mediated DNA damage, another
in skin include trans-urocanic acid, tryptophan previously unknown aspect of the photochem-
oxidation products, pyridinoline collagen cross- istry of this natural folate was revealed. It had
links and melanin precursors.26 been reported that folic acid can catalyze UVA
The potential loss of folate within skin could oxidation of DNA particularly targeting G-G
have multiple effects. Folate dependent one-car- repeats to form 8-oxo-dGua.30 The main culprit
Figure 1. The overall role of canonical folate metabolism is the removal of a one carbon unit
from various sources, such as amino acids or formate, and transferring this (sometimes in a
different oxidation state) to construct another molecule, such as S-adenosylmethionine or
nucleic acid bases. Our studies show that 5-MTHF can also defeat photosensitization reactions.

is pterin-6-carboxylc acid (PCA), arising from sis. As expected from the work of Hirakawa et
the photolysis of the folic acid C9-N10 bond al.,30 exposure of supercoiled DNA to 4 J/cm2 of
yielding p-aminobenzoic acid and initially, UVA in the presence of folic acid converted a
pterin-6-carboxaldehyde; the latter being subse- large percentage to the linear form.
quently oxidized to PCA. However, a similar experiment carried
Both pterin scission products are much more out with the same concentration of 5-MTHF
efficient photosensitizers than folic acid itself.31 produced no more relaxed or circular DNA than
This can be seen in the accelerating self-destruc- was present as a contaminant of the original
tion of folic acid during photolysis with UVA, plasmid preparation (see Figure 3a). Thus, this
which was about three-fold less intense than natural folate does not initiate photosensitized
the UVA used to examine the loss of 5-MTHF cleavage of DNA even in concentrations exceed-
(see Figure 2). Thus, the idea that nature might ing those found in skin.
have selected a form of folate more resistant to The big surprise was the result of an experi-
photosensitization reactions than synthetic folic ment that included both folic acid and 5-MTHF,
acid was explored. where no DNA cleavage was observed. More-
The fragmentation of plasmid supercoiled over, so long as the concentration of 5-MTHF
DNA (PBR 322) was used to examine the was maintained (by constant infusion with a
photosensitization of folates by UV.24 When this syringe pump) at 500 nMa level equivalent to
is nicked by a single strand break, it is converted that found in the epidermis23no degradation
into a relaxed circular form: a double strand of the folic acid was detected. In other words,
break opens the DNA altogether to generate a both the photo cleavage of the folic acid and
linear form. The supercoiled, relaxed and linear that of DNA were blocked.
forms can all be separated by gel electrophore- This effect was observed with other sensitiz-
Figure 2. Photo-decay of 5-MTHF with UVA or UVB and folic acid with UVA, in pure and
metal-free aerobic buffer (pH 7.4)

The big surprise was
when both folic acid and 5-MTHF
were included and no DNA
cleavage was observed.
ers as well. For example, the more potent PCA, Next, the effect of oxygen concentration
with both UVA (see Figure 3b) and UVB filtered on the loss of 5-MTHF in photosensitization
to remove any UVC content (data not shown). reactions was assessed. Results revealed that
high oxygen paradoxically suppresses 5-MTHF
Mechanism of 5-MTHF with degradation by photosensitizers (see Figure 5).24
Photosensitizers This result can be understood by an additional
The mechanism(s) by which 5-MTHF exerts activity by the natural folate: rapid quenching of
its influence on photosensitization were then the excited state of the photosensitizer. Thus, in
explored. Competition reactions were performed high oxygen the excited photosensitizer is forced
with 5-MTHF and varied azide, a potent (4.5 to produce more singlet oxygen than at lower
108 M-1s-1in water) and fairly selective singlet concentrations. The decreased rate of 5-MTHF
oxygen scavenger. The reactions showed that loss is due to the quenching action being even
an azide concentration twenty-fold higher faster (probably diffusion limited) than the 1O2
than 5-MTHF was needed to decrease the scavenging action (see Figure 6).
rate of folate loss by half (see Figure 4). This The initial products of both the scavenging
implies 5-MTHF is an exceptionally avid singlet and quenching activities of 5-MTHF in vitro
oxygenscavenger.24 can be largely, but not completely, regenerated

Figure 3. Effect of UVA irradiation on strand breaks in plasmid supercoiled DNA in the
presence of: a) folic acid, 5-MTHF or both in combination; and b) the folic acid photo-decay
product PCA, or this plus 5-MTHF
Figure 4. Photolysis of 5-MTHF in varied competing azide concentrations showing the

former to be a 20-fold more effective scavenger of singlet oxygen

Figure 5. High dissolved oxygen concentration inhibits the decay of 5-MTHF when
irradiated with UVA in the presence of a photosensitizer
by ascorbic acid. With

rose bengal stain as the
photosensitizer, which has
well-characterized singlet
oxygen production, in the
presence of 100% O2, the
initial rate of decrease in
5-MTHF was slowed by
more than five-fold, with a
2 mM initial concentration
of sodium ascorbate (see
Figure 7).
One of the initial
products of 5-MTHF oxida-
tion is its radical cation,
which can be rapidly
reduced by ascorbate.
Since the ascorbate itself
in this in vitro experiment
is not being regenerated, Figure 6. High oxygen (3O2) concentration drives the
its concentration also partitioning of excited photosensitizers (PS*) toward singlet
decreases with time. The oxygen (1O2) formation, with which 5-MTHF less reacts
ascorbate concentration in quickly than directly with the PS*
human skin is fairly high,

particularly in the epidermis (~4 mM).32 Of course, this experiment with
rose bengal, while demonstrating the regenerative capacity of ascorbate in
general, may not fully reflect what happens to intact skin when exposed to
UV (see following studies with exvivo human skin).
The two activities of 5-MTHF for both scavenging singlet oxygen and
quenching the excited state of photosensitizers can serve to block damag-
ing photo-oxidation in skin. Other natural quenchers of photo-excited
states such as the carotenoids are known,26 but these are active in the lipid
compartment, e.g., membranes. Conversely, folates are water-soluble and
more in position to protect polynucleotides in the aqueous compartment.
Therefore, low skin folate levels might exacerbate the rate of DNA degrada-
tion as well as limit its repair.
The risk for developing squamous cell carcinoma in individuals having
a history of skin cancer has been reported to be two-fold lower in those
with a high intake of green leafy vegetables. This was believed to be due
to the maintenance of the genetic integrity by the higher folate intake.33
Our work24 shows this could also be due to the photoantioxidative activity
of5-MTHF.
UV Degradation of
Skin Folate
DNA damage in skin due to UV irradiation is well-documented15, 34, 35
but nothing was known about the direct effect of UV on skin folates.36, 37
Most studies of the changes in blood folate concentrations following UVA
or solar exposure in humans have found no decrease in serum and/or red
cell concentrations38, 39except for one group of subjects receiving folic
acid supplements. And, as established, folic acid is more photolabile, so it
may have been destroyed by light.38
Figure 7. Vitamin C (sodium ascorbate) at a

concentration found in skin slows the loss of 5-MTHF during
photosensitization reactions.

The lamps used in tanning beds, although
predominantly UVA, are often more intense than
natural sunlight.
Recently, a negative correlation between red unexposed section of skin subjected to the same
cell folate and cumulative exposure to sunlight conditions, decreased by approx. 30% after
in Australia was reported.40 In another earlier 187J/cm2. This is similar to the maximal daily
study, the treatment of light-skinned patients UVA irradiation at sea level near the equator.
with a combination of UVA and methoxsalen This decrease of total folate content
showed lower serum folate levels than in healthy (210pmol/g tissue) from epidermal samples
subjects.19 Experiments examining blood signifies an overall loss of approx. 120 nmoles,
samples, however, do not reveal changes specific calculated using the thickness of the sections
to skin folates, and folate concentrations in taken and irradiation of the entirety of 1.8 m2 of
human epidermis and especially dermis are low adult skin. Although this is more than what is
in comparison with other tissues, such as the typically present in plasma, it is a small portion
liver and kidneys.23 of the folates present in throughout the entire
To reveal the potential vulnerability of skin body (60 to 225 mols).42, 43
folates, ex vivo experiments were performed Therefore, given the depletion of total folate
exposing fresh whole human skin to UVA. Here, observed in this study using a single dose of
the total folate levels in the epidermis (but not UVA, one might expect a transient impact
dermis) of light-colored skin were significantly on plasma folate concentrations which, after
degraded during exposure to 13 mW/cm2 of reaching homeostasis with the other organs,
UVA; i.e., less than most tanning beds (see would not produce a measurable change in
Figure 8).41 The total folate levels, relative to an overall folate status. Moreover, it is unlikely the

entire skin area would be exposed simultane- shown to enhance reactive oxygen species in
ously to the full solar irradiance, and therefore reconstituted skin.46 Therefore, the pressure
would not receive as large an average dose as in on skin folate levels might not be limited to
thisstudy. UVwavelengths.
On the other hand, many individuals even The ability to observe transient plasma
in developed countries who are neither users of folate depletion due to UVA depends on irradi-
supplements nor subject to folic acid fortifica- ance, length of exposure time, percentage of
tion have been reported to consume less than whole body coverage, and the rate at which the
250 g (570 nmols) per day.44, 45 Therefore, loss from plasma is restored by tissues having
even at half the exposure used here, folate high folate content, e.g., the liver. This latter
loss could be meaningful for such people who rate is unknown but it has been reported that
are exposed every day and who also have an serum folate measured immediately after either
especially low intake and/or high need, such as low-flux hemodialysis or on-line hemofiltration/
duringpregnancy. hemodiafiltration did not change in comparison
Thus, continuous exposure to the intensity of with the concentration just before treatment;
UVA radiation that can occur near the equator although ascorbate levels were substantially
may put enough pressure on folate stores to give decreased by these procedures.47
darker skin an advantage, especially when high This suggests a rapid restoration of the
folate-containing foods are not readily available. equilibrium between plasma folate and liver and
Indeed, in samples of black skin, no loss of total kidney stores, which would be consistent with
folate was observed, consistent with the hypoth- the observed effects of transient UVA treatment
esis that dark skin color helps to protect against on systemic plasma folate concentrations
degradation of this essential nutrient.19, 20 especially given the lower doses used in most
Furthermore, photosensitizers can also previous human studies.38, 39 For example, in a
be activated by visible light, which has been study of individuals having Fitzpatrick skin type
Figure 8. Loss of: a) total folate and b) 5-MTHF; and c) gain of tetrahydrofolate (THF)
plus 5,10 methylene-THF upon UVA irradiation of fresh whole ex vivo white human skin
as a percentage of unexposed control

II, subjects were exposed to 16 J/cm2 of UVA per total folate loss (see Figure 8b). This apparent
session (~9% of the dose in the current study). discrepancy between total folate and 5-MTHF,
Here, no change in serum folate was detected which is by far the most predominant folate
30 min after the first session.39 Another study, form in this layer, is explained by the substantial
in which lightly dressed Japanese students were four fold (approx.) increase in the epidermal
exposed to sunlight having 19 J/cm2 of UVA, also concentration of tetrahydrofolic acid (THF) and/
did not find a decrease in total plasma folate.38 or 5,10 methylene-THF (see Figure 8c), which
On the other hand, it is not clear how quickly in skin unexposed to UV light, is only a minor
the epidermis reestablishes its own folate home- component of the folate pool in this layer.23 This
stasis after UVA exposure. It has been reported loss occurs despite the high concentration of
that 50% of the vitamin A depleted from the ascorbate in human skin (see above).
epidermis of rabbits by UVA was replenished However, UV radiation also depletes reduced
from blood circulation within one day of the ascorbate in the skin.49 Therefore, the part of the
exposure, while full restoration required more loss of 5-MTHF that is not due to conversion to
than one week.48 Therefore, understanding the tetrahydrofolate may be related to the disap-
restoration kinetics of the more water-soluble pearance of sufficient ascorbate to regenerate
folate is important for predicting the risks of the initial 5-MTHF photo-oxidation product.
UVA exposure; especially when a transient Moreover, the previously reported loss of
loss could occur just at the time when folate is ascorbate may, in turn, be in large part due to its
needed to support DNA repair and methylation. regeneration of skin folate.
The changes to individual components of The conversion and re-organization of a
the epidermal folate pool are complex. The substantial fraction of the epidermal pool of
rate of loss specifically for 5-MTHF in the 5-MTHF into THF and/or 5,10-methylene-THF
epidermis, i.e., 56% after exposure to 187 J/ in white skin is surprising. Direct light driven
cm2 UVA, occurred at nearly double the rate of demethylation of 5-MTHF, a two-electron
Figure 9. Skin folate is directly related to blood folate. The major form of folate in the
epidermis is 5-MTHF. Note the break in the curve since the epidermis contains an order of
magnitude more folate than the dermis.

Several mechanisms of protection against the
effects of UV are afforded by maintaining skin
folate at optimum levels.
process, would not seem to be very likely. The 5-MTHF stores into these other folate forms
only known enzymatic mechanisms for removal may be beneficial in assisting in DNA synthesis
of the 5-methyl group are the actions of methio- and repair to counteract the damage occurring
nine synthasewhich converts homocysteine to DNA during UV exposure.27
and 5-MTHF into methionine and THF in a On the other hand, it has been reported that
B12-dependent processand the reverse activity MTHFR polymorphisms are risk factors for skin
of 5,10-methylenetetrahydrofolate reductase cancer54, 55 associated with DNA hypomethyl-
(MTHFR), which normally produces 5-MTHF ation.56 Those with the C677T polymorphism
using NADPH (see Figure 1). were recently reported to be more susceptible
Although MTHFR can make THF from to the effects of solar exposure on blood folate.40
5-MTHF in the presence of a suitable electron Thus, it appears that both 5-MTHF and other
acceptor in vitro, this activity is generally con- forms of reduced folates may influence the
sidered not to be physiologically important due integrity and function of skin DNA important
to the normally high ratio of NADPH to NADP for preventing skin cancer.
entirely favoring the forward direction.50 There- In addition to preventing photosensitiza-
fore, the continuing consumption of 5-MTHF tion reactions, the decreased availability of
by methionine synthase would appear to be the epidermal 5-MTHF found in response to UVA
major factor contributing to the appearance irradiation could also influence methylation
of THF/5,10-methylene-THF. However, the reactions. Gene-specific hypermethylation has
normally high percentage of 5-MTHF in the been observed in skin cancers as well as, though
epidermis23 suggests that one-carbon addition less frequently, global hypomethylation.28 A
followed by MTHFR rapidly converts THF back paradoxical relation between folate deficiency
to 5-MTHF in the absence of UV irradiation. In and increased methylation29 has been suggested
this context, it is interesting to note the greater due changes in intracellular S adenosylmethio-
susceptibility of subjects having the thermola- nine and S adenosyl-homocysteine levels.57
bile C677T polymorphism of MTHFR to loss of
blood folate with exposure to solar UV.40 Effects of Simulated UVA
The apparent decreased recycling of THF UVA irradiance in tanning beds varies
back to 5-MTHF observed could be due to the considerably among manufacturers, and is often
activation of NADPH oxidase demonstrated several times higher than that of mid-day, sum-
in human keratinocytes after UVA irradiation, mer sun in mid latitudes. The intensity is greater
which leads to decreased levels of NADPH.51 than that used for this study and may include
The concentration of NADPH in human epider- hot spots, such as in the region surrounding the
mis52 is around 100 M and its Km for human head, that are much higher than the average.58
MTHFR is 30 M.53 Therefore, substantial Although a 20 min to 30 min session may not be
UV-induced depletion of NADPH could lead to enough to elicit a detectable decrease in plasma
lower MTHFR activity, and promote accumula- levels, local depletion and rearrangement of the
tion of 5,10-methylene-THF or THF, which are epidermal folate pool may occur. The extent and
measured together in the HPLC assay employed. persistence of these changes, whether from solar
In any event, the increase in 5,10-methylene- or artificial exposure, depends on the as-yet
THF/THF would be useful for stimulating unknown rate of re-equilibration with circulat-
biosynthesis of thymidylate and purine nucleo- ing folate.
tide bases, respectively (see Figure 1). In this Laser capture microdissection has demon-
view, the conversion of a portion of epidermal strated UVA signature mutations in the basal

layer of the epidermis, whereas damage due to the
more readily absorbed UVB is deposited closer to the
surface.59 The basal layer, which contains keratinocyte
stem cells and melanocytes, is also a likely location for
most of the epidermal folate pool, and thus may suffer
the largest depletion. On the other hand, although some
UVA penetrates the dermis,60 no significant change was
seen in the 5-MTHF, THF/5,10-methylene-THF or total
folate measurements in this layer even with the longest
exposure. It is still possible that the outer portion of the
dermis suffered some folate loss and/or interconversion,
but that this might have been made unobservable by the
homogenization of the entire dermis.
Conclusion
Several mechanisms of protection against the effects
of UV are afforded by maintaining skin folate at opti-
mum levels. The concentrations of both total folate and
5-MTHF have been shown to be linearly correlated with
blood folate, measured in U.S. subjects either as serum
total folate or as shown in Figure 9 as serum 5-MTHF.23
Despite the U.S. folic acid fortification program, many
people have a folate status approaching deficiency.
Moreover, folate levels in countries lacking fortification,
such as Europe, have even lower folate levels, and may
be especially susceptible to UVradiation.
One disadvantage of relying on supplements con-
sumed just before exposure to the sun or indoor tanning
is that the increased folate intake may not have an
immediate impact on the skin. In this regard, investiga-
tions on the rate of uptake of topical 5-MTHF into skin
appear to be promising.
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rahydrofolate in human skin. Am J Clin Nutr 98:42-8 edge in Nutrition (Bowman B., Russell R, eds), Washington,
24. Offer T, Ames BN, Bailey SW et al. (2007) 5-Methyltetrahy- DC: International Life Sciences Institute, 278-301
drofolate inhibits photosensitization reactions and strand 43. Lin Y, Dueker SR, Follett JR et al. (2004) Quantitation of in
breaks in DNA. FASEB J 21:2101-7 vivo human folate metabolism. Am J Clin Nutr 80:680-91
25. Juzeniene A, Thu Tam TT, Iani V, Moan J (2009) 5-Methyl- 44. Roman Vias B, Ribas Barba L, Ngo J, et al. (2011) Pro-
tetrahydrofolate can be photodegraded by endogenous jected prevalence of inadequate nutrient intakes in Europe.
photosensitizers. Free Radic Biol Med 47:1199-204 Ann Nutr Metab 59(2-4):84-95.
26. Wondrak GT, Jacobson MK, Jacobson EL (2006) Endoge- 45. Elmadfa I, Meyer A, Nowak V et al. (2009) European Nutri-
nous UVA-photosensitizers: Mediators of skin photodamage tion and Health Report 2009. Forum Nutr 62:1-405
and novel targets for skin photoprotection. Photochem 46. Liebel F, Kaur S, Ruvolo E et al., (2012) Irradiation of skin
Photobiol Sci 5:215-37 with visible light induces reactive oxygen species and
27. Williams JD, Jacobson MK (2010) Photobiological implica- matrix-degrading enzymes. J Invest Dermatol 32:1901-7
tions of folate depletion and repletion in cultured human 47. Fehrman-Ekholm I, Lotsander A, Logan K et al. (2008)
keratinocytes. J Photochem Photobiol B 99:49-61 Concentrations of vitamin C, vitamin B12 and folic acid in
28. Van Doorn R, Gruis NA, Willemze R et al. (2005) Aberrant patients treated with hemodialysis and on-line hemodiafiltra-
DNA methylation in cutaneous malignancies. Semin Oncol tion or hemofiltration. Scand J Urol Nephrol 42:74-80
32:479-87 48. Berne B, Nilsson M, Vahlquist A (1984) UV irradiation and
cutaneous vitamin A: An experimental study in rabbit and
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49. Shindo Y, Witt E, Han D et al. (1994b) Dose-response effects of
acute ultraviolet irradiation on antioxidants and molecular markers
of oxidation in murine epidermis and dermis. J Invest Dermatol
102:470-5
50. Green JM, Ballou DP, Matthews RG (1988) Examination of the role of
methylenetetrahydrofolate reductase in incorporation of methyltetra-
hydrofolate into cellular metabolism. FASEB J 2:42-7
51. Valencia A, Kochevar IE (2008) Nox1-based NADPH oxidase is the
major source of UVA-induced reactive oxygen species in human
keratinocytes. J Invest Dermatol 128:214-22
52. Hammar H (1975) Epidermal nicotinamide adenine dinucleotides in
psoriasis and neurodermatitis (Lichen simplex hypertrophicus). Arch
Dermatol Res 252:217-27
53. Suormala T, Gamse G, Fowler B (2002) 5,10-Methylenetetrahydro-
folate reductase (MTHFR) assay in the forward direction: Residual
activity in MTHFR deficiency. Clin Chem 48:835-43
54. Han J, Colditz GA, Hunter DJ (2007) Polymorphisms in the MTHFR
and VDR genes and skin cancer risk. Carcinogenesis 28:390-7
55. Lesiak A, Norval M, Wodz-Naskiewicz K et al. (2011) An enhanced
risk of basal cell carcinoma is associated with particular polymor-
phisms in the VDR and MTHFR genes. Exp Dermatol 20:800-4
56. Laing ME, Cummins R, O'Grady A et al. (2010) Aberrant DNA
methylation associated with MTHFR C677T genetic polymorphism in
cutaneous squamous cell carcinoma in renal transplant patients. Br
J Dermatol 163:345-52
57. Jhaveri MS, Wagner C, Trepel JB (2001) Impact of extracellular folate
levels on global gene expression. Mol Pharmacol 60:1288-95
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40:485-94
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Testing | C&T
KEY POINTS
Urban pollution including particulate matter
(PM) can affect the skin barrier by inducing
numerous cell stresses.
This article describes in vitro, ex vivo and
invivo models to examine the effects of PM
in skin, with particular interest in a botanical
extract to mitigate the described effects.
When the
Dust Settles Reproduction in English or any other language of
CT170708_Test_Clay_fcx.indd 50 6/26/17 2:03 PM

Keratinocyte
Differentiation is the
Anti-pollution Solution
Anne-Franoise Clay, Valre Busuttil, Isabelle Imbert,

Christophe Capallere, Laurine Bergeron, Catherine
Serre, Christelle Plaza, Gilles Oberto, Karine Cucumel,
Jean-Marie Botto and Nouha Domloge
Ashland Specialty Ingredients, Vincience,
Nice Sophia-Antipolis, France
D
PM-containing compounds, e.g., heavy metals
(HMs) and polycyclic aromatic hydrocarbons
(PAHs), ranging in size from the nano- to
the micrometer. As this article explains, to
study these effects and develop preventative
solutions, in vitro and ex vivo models were
designed using PM to mimic urban pollution
aily, the human body exposure in human skin biopsies, three-dimen-
encounters many sional (3D) reconstructed human epidermises
stresses to which (RHEs) and cultured normal human keratino-
the skin acts as the cytes (NHK).
first external bar- Beyond the effect of PM on epidermal
rier. Environmental differentiation and barrier function, possible
pollution is one stressor and is present in many epigenetic impacts were investigated as well
forms, including volatile organic compounds, as their consequences in vivo on skin protein
cigarette smoke, ozone as well as particulate carbonylations. A botanical ingredient was
matter (PM). This persistent presence of urban then developed and tested for its ability to
pollution has become increasingly problematic shield against PM.
in large cities worldwide. It affects not only
the quality and beauty of skin, but also barrier Materials and Methods
function; these deleterious effects have been In vitro, ex vivo: The in vitro and ex vivo
studied and demonstrated.1 tests described employed both fine suspended
In human skin, the major pollutants to particles of < 10 m (PM10) and small particles
which detrimental effects can be linked are of < 2.5 m (PM2.5)smaller than cutaneous
Vol. 132, No.

Reproduction 7 | July/August
in English 2017
or any other language or part of this article is strictly prohibited. 2017 Allured Business Media.
of all Cosmetics & Toiletries | 51

Caspapse 14 was assessed since this enzyme
is required for filaggrin degradation into
natural moisturizing factors.
pores. After exposure to these stressors, the (PM10) and small particles (PM2.5), i.e., smaller
integrity of the skins barrier function was than cutaneous pores, which can penetrate
evaluated using specific biomarkers or probes, the skin and directly induce damage. These
claudin-1, hyaluronic acid, epidermal lipids and PM were applied separately to NHK in culture
Lucifer Yellow skin penetration in skin biopsies, pre-treated or not with the botanical ingredi-
RHEs and cultured NHKs. ent to evaluate cell stress induced in vitro, as
Test ingredient: A sustainable botani- measured by lactate dehydrogenase (LDH)
cal ingredient derived from Schinus molle release (see Figure1a-b; SEM = standard
(Brazilian peppertree) extracta, which is rich error of the mean). The in vitro results indicate
in bioflavonoids such as quercitrin and mique- the NHK treated with the botanical extract
lianin, was then developed and evaluated for its were better protected from stress induced by
activitiesmore particularly, for its potential to both PM10 and PM2.5 exposure, compared with
protect the quality of the skin exposed to PM. untreatedkeratinocytes.
Clinical study: In addition, twenty volun-
teers participated in a double-blind, eight-week Results:
study of the effects of PM on epigenetics and the Differentiation Markers
potential for the extract to mitigate them. The
After in vitro treatment of keratinocytes
arms of the subjects were exposed to cigarette
with the botanical ingredient, the expression
smoke, to simulate air pollutant exposure, for a
levels of genes including long-non-coding RNAs
determinate amount of time. Skin samples were
(lncRNAs) specifically involved in skin renewal
then collected by tape-stripping and skin protein
and barrier functions were assessed by quantita-
carbonylations were analyzed. The effects of the
tive real-time PCR (qPCR) in keratinocytes.2,3
extract on carbonylations also were assessed.
Of particular interest were differentiation
antagonizing non-protein coding RNA (DANCR)
Results: Keratinocytes and tissue differentiation-inducing non-protein-
As noted, air pollution is associated with coding RNA (TINCR) (see Figure 2a-b).
the presence of both fine suspended particles Results showed DANCR decreased with epi-
dermal differentiation whereas TINCR increased
a
Elixiance (INCI: Propanediol (and) Water (aqua) (and) with epidermal differentiation. This profile is
Bioflavonoids)
similar to that observed in normal epidermal
differentiation. Thus, we conclude the botanical
ingredient positively modulated tissue differen-
Pollution can wreak havoc on skin health but a tiation in vitro, leading to an improvement in
recent study found that 66% of Americans still the skin barrier function.
do not protect their skin against pollution
exposing a market opportunity. Results: Barrier Biomarkers
To evaluate the effects of the botanical ingre-
dient as a pollution shield, key markers were
Source: Global Cosmetic Industry
again evaluated via qPCR, including E-cadherin,
(www.GCImagazine.com) involucrin and transglutaminase-1, which
are known to play a role in skin cohesion and
mechanical resistance to stress. Caspase 14 also

was assessed since this enzyme is required for Results: Lipid Expression
filaggrin degradation into natural moisturizing
To evaluate and visualize the effects of the
factors (NMF) and is associated with healthy-
botanical ingredient on skin lipid content, Nile
looking skin (see Figure 2c).
red staining was used in vitro on NHK and
The mRNA levels of involucrin, trans-
ex vivo in skin biopsies. Nile red binds to and
glutaminase-1, caspase 14 and E-cadherin,
fluoresces intensely in the presence of skins
markers of skin barrier function, were sig-
hydrophobic epidermal lipids including cho-
nificantly increased in vitro in keratinocytes
treated with the botanical
ingredient at 0.1% for
48hr, compared with
untreatedcells.
Results:
Tight Junction
To compare the
effects of the botanical
ingredient vs. placebo
on the skin barrier, the
expression of claudin-1,
an important protein
involved in the integrity
of tight junctions, was
assessed in human
skin biopsies (see
Figure3a). The ex
vivo results showed
a significant increase
of claudin-1 expres-
sion (+128.2%) in skin
biopsies treated with
the botanical ingredi-
ent, compared with
theplacebo.
Results:
Moisturization
To keep skin mois-
turized and protect
against environmental
stress, levels of hyal-
uronic acid (HA), a
hallmark of youthful-
looking skin, also were
important to evaluate
(see Figure 3b). Ex vivo
results showed a signifi-
cant increase (+58.9%) Figure 1. LDH activity measurement in NHK treated or not with
in HA expression in skin 0.1% extract twice daily for 48 hr prior to exposure or not to: PM10 at
biopsies treated with
250 g/mL or 500 g/mL once for 24 hr (a); or PM2.5 at 100 g/mL
the botanical ingredient,
compared with placebo. for 24 hr (b)

lesterol sulfate, phospholipids, sphingomyelin treated with the botanical ingredient at 0.1%
and glucosylceramideskey components for for 48 hr, compared with untreated cells (see
barrierfunction. Figure4a). Ex vivo results also showed a
In vitro results showed a significant increase significant increase in lipid expression (+41%) in
in lipid expression (+102%) in keratinocytes biopsies treated with the botanical in the same
manner, compared with
placebo (see Figure4b).
Results:
Barrier
Reinforcement
Changes to skin barrier
functioning after SDS stress
deterioration were next
assessed on RHEs treated
with either placebo or the
botanical ingredient at 1%
for 48 hr, and were then
submitted to 0.15% SDS
stress for a period of 3 hr
(see Figure 5). A significant
decrease in passive dye dif-
fusion was observed (-16%)
in the RHE pre-treated with
the botanical ingredient,
compared with RHE treated
only with placebo.
Results:
Clinical Efficacy
Finally, to test the ability
of the botanical extract to
shield against atmospheric
pollution, protein carbon-
ylations were evaluated in
vivo in 20 Asian volunteers
between the ages of 36 and
65 years.
The volunteers applied
a cream containing 1% of
the botanical extract or its
placebo to each of their
forearms. After eight weeks,
the subjects forearms were
exposed to cigarette smoke
to mimic air pollution;
samples were then collected
and protein carbonylation
Figure 2. qPCR evaluation of DANCR (a) TINCR (b) and involucrin, levels were analyzed. A
transglutaminase-1, caspase 14 and E-cadherin mRNA levels (c) in significant decrease in
keratinocytes after 48 hr treatment or not twice daily with 0.1% botanical protein carbonylations
was observed in forearms

The botanical ingredient
positively modulated tissue
differentiation in vitro,
leading to improved skin
barrier function.
a) b)
Figure 3. Claudin-1 (a) and HA immunofluorescent staining (b) in skin biopsies after twice daily, 48-hr
treatment with placebo or 1% botanical; 20 objective; green = claudin-1 or HA staining; blue = nucleus
staining using DAPI

treated with the botanical extract, compared PM. Beneficial modulations of two epigenetic
with the placebo side, providing positive lncRNA biomarkers involved in tissue dif-
indication of the extracts protective activity ferentiation also were observed in vitro with
(see Figure 6). application of the botanical ingredient.
Furthermore, skin barrier protection mark-
Conclusion ers as well as epidermal lipid content were
Protecting skin against urban pollution, enhanced in vitro or ex vivo following applica-
especially PM, is an important factor to tion of the botanical ingredient. Taken together,
reducing skin damage and the subsequent these in vitro tests demonstrate the benefit of
appearance of the visible signs of aging. In this ingredient for preserving the skins barrier
vitro, the application of a botanical ingredient function ex vivo.
on primary keratinocytes is shown here to Finally, positive in vivo effects of the botani-
protect against stress induced by exposure to cal ingredient against protein carbonylations
a) b)
Figure 4. Quantification of Nile red staining after 48 hr treatment twice daily with 1% botanical
ingredient in NHK (a) or skin biopsies (b)
Figure 5. Quantification of Lucifer Yellow penetration via the skin barrier after treatment and SDS stress

Figure 6. Variation of stratum corneum carbonylated protein (SCCP);
*= significant per student t-test; n = 20 +/- SEM
verified its ability to guard against air pollution C&T Daily Newsletter
damage to skin, and by doing so, to maintain Get the latest from Cosmetics & Toiletries
skin health and subsequent signs of aging. delivered straight to your inbox everyday!
http://www.CosmeticsandToiletries.com/newsletter
References
1. TL Pan et al, The impact of urban particulate pollution on
skin barrier function and the subsequent drug absorption, J
Dermatol Sci 78(1) 51-60 (2015)
2. M Ketz et al, Suppression of progenitor differentiation C&T Webcasts
requires the long noncoding RNA ANCR, Genes Dev 26(4) Find current and upcoming webcasts at
338-43 (2012) www.CosmeticsandToiletries.com
3. M Ketz, et al, Control of somatic tissue differentiation by the
long non-coding RNA TINCR, Nature 493(7431) 231-5
(2013)

Testing | C&T
KEY POINTS
Completing our three-part series, this final
piece on hair damage provides insight on new
ideas and strategies for problematic hair.
By identifying elements at the heart of

technical issues in hair, product developers will
uncover options to consider for improving its
health and appearance.
Testing Tactics in Hair
How
Damaged
is Hair?
Part III: Better Defining
the Problem
T
Trefor A. Evans, Ph.D.
TRI-Princeton Princeton,
New Jersey
he term hair damage is tossed around our
industry ubiquitously and haphazardly. It
represents a decidedly generic term insinuat-
ing that hair properties have been negatively
altered in some way, shape or form relative to
the initial, freshly grown state. Yet, the causes
and materialization of these changes are plentiful. A small sampling
of irksome symptoms might include undesirable tactile properties,
visual dullness, an increased propensity for breakage and/or an

CT170708_Testing_Evans_irv.indd 58 6/26/17 2:42 PM

Heat, high pH relaxers and formaldehyde-
based Brazilian keratin treatments all
compromise hairs tensile properties without
notable cysteic acid formation.
unruly and unmanageable disposition. Possible color, while seemingly leaving behind micro-
instigators, on the other hand, might involve scopic voids.
grooming-related abrasion and fatiguing, Oxidative side reactions with hair protein
extreme temperatures, environmental battering, deplete strength-supporting cystine disulfide
chemical treatments and others. bonds by conversion to cysteic acid.
There is frequently a desire to measure the Individual hair fibers become weaker and
degree to which hair has become damaged, more prone to breakage.
often for the purpose of evaluating the aggres- The dry state modulus increases, leaving
siveness of known deleterious treatments or individual fibers stiffer.
to try and quantify an ability to protect and The lipid layer on the very outside sur-
possibly even "repair" the hair. But the above face of a hair fiberthe f-layeris removed,
discourse shows how this is not a trivial task. changing its interaction with water and possibly
Hair damage is a convoluted and multifaceted leading to slower hair drying.
concept; a fact that is perhaps lost and there- Enhanced swelling in combination with
fore not properly accepted and appreciated by a more hydrophilic character can lead to
the industry's use of such an imprecise, broad increased fiber water content.
and humdrum descriptor. Lipid structure becomes weakened and
Our industry needs a more precise means components are more easily removed.
for describing the vast number of ways by This quickly becomes a lengthy listand it
which the hair structure can be altered or is by no means exhaustive. On top of this, daily
compromised. Clearly, the consumer word activities and maintenance routines add extra
damage will not go away but only by adequately repetitiveand often exacerbatedstimuli for
describing the problem can better solutions be further structural wear and tear.
conceived. The final article in this trilogy1, 2 on To this end, now consider what happens to
hair damage further explores how this might hair during simple washing:
beaccomplished. Fibers swell, putting strain on the cuticle.
Swollen fibers are then subjected to inter-
The Scope of the Problem fiber abrasion and friction, while likely causing
To illustrate the problem, consider what cuticle wear.
happens to hair upon exposure to a simple,
skeletal hydrogen peroxide bleaching treatment:
Penetration of the solution between cuticle
scales will weaken the inter-cuticular cement
and leave this protective structure more suscep- According to Klines Cosmetics & Toiletries USA
tible to deterioration. report, the value of the 2016 U.S. hair care
Oxidative conditions, in combination market was about $13 billion at the retail level,
with elevated pH, will irreversibly increase the up 0.6% from 2015.
swelling capacity of fibers and place additional
strain on the cuticle structure each time the
hair is wetted.
Source: Global Cosmetic Industry
Degradation and dissolving of melanin (www.GCImagazine.com)
pigment granules leads to lightening of the hair

Bending and twisting of fibers could initi- These thought exercises are useful in
ate localized cuticle uplift. identifying elements at the heart of technical
Fibers encounter various tugging and issues but at the same time they produce a
fatiguing forces while in a decidedly weaker, rather daunting number of factors to potentially
plasticized state. consider. A means of further categorization
Lipids and degraded protein may be might be based on their materialization into
leached from the hair. commonly heard consumer concerns and
Grooming, heat styling and all other hair issues such as: sensorial-compromised hair,
care habits and practices, in a similar vein, lead strength-compromised hair and manageability-
to their own lengthy lists. compromised hair.
Sensorial-
compromised
Hair
Sensorial-compro-
mised hair likely starts
with cuticle breakdown
where uplifted, irregular
and eroded cuticle tiles
impart a rough feel
while also producing a
dulled appearance (see
Figure1). This state
likely occurs primar-
ily due to mechanical
manipulation.
During grooming,
fibers slide past each
other in a highly chaotic
manner, generating con-
Figure 1. Cuticle uplifting during hair fiber bending siderable frictional and
abrasive forces that wear
on the cuticle structure.
Diminishing surface
properties generate still
higher friction and a self-
perpetuating cycle is set
up (see Figure 2) with
correspondingly mount-
ing sensorial negatives.
Chemical treatments
seemingly exacerbate
this process via weak-
ening of the cuticular
intercellular cement.
Another potential
contributor to senso-
rial negatives could be
increased fiber stiffness,
which seemingly arise
Figure 2. Self-perpetuating cycle for hair surface damage due to all manners of
insults. The previous

article in this series2 showed how bleaching conventional mechanical testing experiments
raises the dry state modulus of hair (i.e., fiber reveal how the underlying tensile properties
stiffness) but further testing demonstrates of single fibers can be diminished by a variety
how this state can also transpire after other of insults, which act to alter the chemistry and
chemical treatments (i.e., perms, formaldehyde- structure of proteins within the internal cortical
based Brazilian keratin treatments, etc.). structure of the hair.
Similarly, Figure 3 shows the same occurrence As an aside, here there may also be desire
in heat straightened hair, while an especially to specify the nature of these transformations.
pronounced effect seemingly arises as a conse- For example, perms, bleaches, permanent color
quence of UV exposure. treatments and UV exposure all involve an
The mechanism for this unexpected oxidative stimulus whereby cystine concentra-
alteration of hairs mechanical properties is still tion is depleted with commensurate formation
unclear, but this notable changein concert of cysteic acid. This new species can be identi-
with declining surface propertieswould seem fied relatively easily in hair by spectroscopic
to be behind consumer language such as rough, measurement, and acts a marker for oxidative
coarse, dry, straw-like, lacking in smoothness or modification of the protein. Accordingly, it
softness, etc. becomes evident that heat, high pH relaxers
and formaldehyde-based Brazilian keratin treat-
Strength-compromised Hair ments all compromise hairs tensile properties
To the consumer, the primary symptom of without notable cysteic acid formation. In
strength-compromised hair is likely a height- short, different reagents transform the protein
ened degree of hair breakage during everyday chemistry and structure in different ways.
habits and practices. As has been shown,2 In the laboratory, we tend to study the
Figure 3. Effect of heat straightening on the dry-state Young's modulus of hair

Previously unrecognized factors can have a
sizableand possibly dominantimpact on
the rate at which hair fibers break during
everyday grooming.
influence of these various insults on hair but as this column will cover, mitigating
individually, but consumers frequently mix and factors exist.
match such practices while also periodically
reapplying treatments to recreate desired looks Manageability-
and touch-up new growth. It is surprising to compromisedHair
learn just how weak hair can become when Manageability-compromised hair might
harvested from the heads of some individuals. begin with an issue as simple as difficulty
Instinctively, diminished mechanical in brushing or combing, yet factors already
strengthirrespective of the stimuluswould described can also be conceptualized as
seemingly make hair more prone to breakage contributors. Increased fiber stiffness might

Figure 4. Wet combing results for conditioned and unconditioned hair
Figure 5. Ability for conditioned hair to reduce breakage in a repeated grooming

experiment (2,000 brush strokes)

Manageability-compromised hair might begin with an issue as
simple as difficulty in brushing or combing.
add to an unruly disposition where hair is more 1. Repair/reversal of induced changes,

difficult to style. The combination of increased 2. Removal of negative symptoms, and/or
stiffness and higher inter-fiber friction suggests 3. Reducing the occurrence of
hair would not move in a fluid, flowing manner. negativeeffects.
Broken fibers will protrude at different Repair/reversal: The repair of anything
lengths from others and produce undesirable generally constitutes a sizable undertaking, so
frizzies. Often these fragmented tips will fray perhaps it is again worth noting the magnitude
into unsightly split ends. The ability to create of the problems at hand. In a nutshell, cuticle
sleek, aligned styles is hindered and might tiles become battered, eroded and torn free
manifest as a general perception of frizz.3 from their underlying cement, while chemical
reactions have altered the fundamental protein
Forward-looking Strategies composition and structure. Solutions to these
With problems identified, forward-looking issues are not readily apparent.
strategies become apparent. Improved hair One possible exception to this line of think-
health and appearance might be obtained by: ing might involve hairs lipid structure. The

The alleviation of sensory negatives, the
cessation of breakage levels and/or the
mitigation of manageability issues will return
hair to a desired state.
function and importance of these components short, while the hair structure still undergoes
is also not well-understood. Hairs structure is all of the issues described earlier, the consumer
generally considered to consist of around 90% does not encounter problems.
protein and only 10% lipid. Not surprisingly Surface lubrication also produces a sizable
then, the majority of scientific papers in the benefit in terms of reducing hair breakage.
literature on this topic focus on the protein. To illustrate, Figure 5 shows results from
Lipid components are widely believed to be a standard repeated grooming experiment4
leached from the hair during everyday wash where hair tresses were brushed 2,000 times
and care practices but the evidence is not with subsequent counting of broken fibers.
overwhelming. If true, maybe replenishment of Conventional tensile experiments demonstrate
these materials could be possible wherein simi- no changes in the fundamental mechanics of
lar materials could diffuse back into the hair. It conditioner-treated hair, but the benefits in
is not known what symptoms are causedand terms of anti-breakage are undeniable.
therefore could possibly be reversedby this Evolving theory is showing how previously
process, but on paper at least, this seems more unrecognized factors can have a sizableand
feasible than repair of protein structures. possibly dominantimpact on the rate at which
Removing negative symptoms: True fiber hair fibers will break during everyday groom-
repair would seem to necessitate feats of micro- ing.5 High on this list is the magnitude of the
engineering but in the eyes of consumers at fatiguing (brushing) forces. As shown, surface
least, a comparable end result can be obtained lubrication greatly lowers these forces and the
by elimination of offending symptoms. The tendency for breakage is hugely curtailed. The
alleviation of sensorial negatives, the cessation consumer again encounters a reprieve.
of worrisome breakage levels and/or the mitiga- Reducing the occurrence: An old adage
tion of manageability issues will all return an suggests an ounce of prevention is equal to a
individuals hair properties to a desired state. pound of cure. Yet, long term abstention from
Our industry relies on conventional con- coloring, heat styling or other well-entrenched
ditioner products to achieve many of these yet deleterious habits and practices cannot real-
goals. The deposition of a highly lubricious istically be expected. Quite simply, the benefits
and esthetically-pleasing surface coating can outweigh the negatives.
effectively mask many consequences of degrad- Again, surface lubrication comes to the
ing cuticle structure. To illustrate, Figure 4 rescue. The ability to reduce friction and
shows instrumental wet combing results for abrasion provides a means of interrupting
different hair types in both an untreated and the self-perpetuating cycle to surface damage
conditionedstate. shown in Figure 2. Similarly, lower fatiguing
Combing forces rise dramatically for uncon- forces greatly limit fiber breakage and help
ditioned hair as a consequence of chemical protect against split end formation.
treatments with increasing severity. Yet, in each
instance, washing with a leading commercial Summary
conditioner returns these forces to a level The goal of this trilogy of articles has been
commensurate with conditioned virgin hair. In to highlight the complexity by which hairs

structure can be modified by everyday habits to provide some specificity by focusing on the
and practices. To most, this is simply damage, regions of the hair where changes predomi-
but use of this vague and lackluster descriptor nantly manifest, i.e., surface or bulk. Another
sidesteps any technical comprehension. reasonable strategy might involve classification
In this specific article, outside of the intro- based on the nature of the insult. Here, it is now
ductory section, I have purposely avoided using suggested that combining both of these ideas
this generic term. Unquestionably, this exercise might represent the most useful approach.
represented somewhat of an ordeal and ably The expression grooming-related surface
demonstrated the casual nature with which it is abrasion provides a detailed description of
tossed around. But in doing so, I found myself cause and manifestation. To the hair scientist,
searching for alternate words and phrases, this implies a degree of cuticle wearchipping,
which inevitably involved deeper contempla- cracking, upliftingbut likely with minimal
tion of underlying processes. We are reminded impact on the cortex. This provides a distinc-
that protein chemistry can change, structural tion from heat styling-related surface abrasion,
building blocks can be altered and in some which possibly provides a different manifesta-
cases degraded, and certain components might tion of surface attrition, but also with a degree
progressively be leached away. of internal structure alteration.
Lists of possible occurrences quickly become Similarly, bleaching related cortex
sizable and there is perhaps a desire to subdi- modification denotes oxidative chemical
vide or categorize a dauntingly large problem. modification of the hair protein, which will
The first two articles in this series attempted be accompanied by now well-known changes

in fiber properties (i.e., lower cystine levels, reactivity. They can however be formulated in
increased cysteic acid levels, diminished wet ways to produce the surface lubrication that
state tensile properties, enhanced dry-state effectively masks and mitigates so many issues.
stiffness, etc.). The specificity of this description
again distinguishes from different chemistries References
associated with, for example, heat styling 1. TA Evans, How Damaged is Hair? Part I: Surface Damage,
related cortex modification or relaxer-related Cosm & Toil 132(4) 38-48 (Apr 2017)
cortexmodification. 2. TA Evans, How Damaged is Hair? Part II: Internal Damage,
Taking this one step further, consumer- Cosm & Toil 132(6) 36-45 (Jun 2017)
perceived issues could also be incorporated. 3. TA Evans, Defining and Controlling Frizz, Cosm & Toil 130(4)
46-53 (2015)
For example: strength compromised hair as a
4. TA Evans, Measuring Hair Strength, Part II: Fiber Breakage,
result of bleaching related cortex modification; or Cosm & Toil 128(12) 854-859 (Dec 2013)
sensorial compromised hair as a result of surface 5. TA Evans, A unifying theory for visualizing the causes of hair
damage caused by grooming wear and tear. breakage and subsequent strategies for mitigation, J Cos
While undoubtedly a bit of a mouthful, these Sci 68 137-140 (2017)
expressions clearly define the issues at hand in

terms of the symptom, the manifestation and
the cause and, in doing so, allow for strate-
gies to be conceived for repairing, masking or
limiting each problem. For instance, returning
to a previous example, a solid strategy for truly
repairing strength-compromised hair as a
C&T Daily Newsletter
result of bleaching related cortex modification
Get the latest from Cosmetics & Toiletries
might be to introduce new cross-linking bonds.
delivered straight to your inbox everyday!
The tactic is a sound one but it represents
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proposition, as described earlier, might be to
use surface lubrication to mask the symptom.
These strategies, in turn, point to the types
of molecules that couldand possibly equally C&T Webcasts
as important, could notproduce the desired
outcome. New bonding necessitates reactive www.CosmeticsandToiletries.com
chemistry together with all its accompanying
worries. Conventional cosmetic ingredients
surfactants, oils, polymershave no such

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Formulating | C&T
Skin Health and Wellness Formulary

Skin Care Skin Care
POREFECTION GEL-CREAM BALANCE AND CLEAR
Lucas Meyer Cosmetics CREAM
With resurfacing action, this formula reduces the Ashland Specialty Ingredients
appearance of pores and wrinkles, features a cooling and
For oily skin with clogged pores, this formula helps to
melting texture, and is preservative-free.
prevent the development of acne with mild exfoliation
Ingredient Focus: Miniporyl (INCI: Isopentyldiol (and) to balance sebum and remove pore-cloggingimpurities.
Trifolium Pratense (Clover) Flower Extract); a picture-
perfect pore minimizer that improves keratinocyte
differentiation, inhibits 5-alpha-reductase, inhibits
Skin Care
sebum accumulation in sebocytes and improves skin DIAPER LOTION
contraction properties
PROTECTIVE SPRAY
Lubrizol Advanced Materials, Inc.
Skin Care This mild and PEG-free sprayable diaper lotion
RESURFACING SEARUM is designed for babies. Pemulen EZ-4U polymeric
Lucas Meyer Cosmetics emulsifier is combined with Glucate DO emulsifier,
a vegetable-derived ingredient, to enable efficient
This highly concentrated serum is based on seawater and emulsion stabilization at pH 5.0-5.8, with suspension
exhibits high compatibility with electrolytes. With a 2% of tapioca starch and Acticel 12 microcrystalline
oil phase, it gives a silicone-like skin feel. cellulose powder. Ion-Moist molecular film provides an
immediate and long-lasting moisturizing effect thanks
Ingredient Focus: Siligel (INCI: Xanthan Gum (and) to ingredients from the natural moisturizing factor and
Lecithin (and) Sclerotium Gum (and) Pullulan); a gelling Actiphyte Calendula GL, with anti-inflammatory and
agent that provides silicone-like skin feel and a high soothing benefits.
resistance to electrolytes; designed to revolutionize the
use of gums as it does not require pre-dispersion; natural
solution with both efficiency and aesthetics
Skin Care
CERAMIDE-INDUCING
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LIFE ESSENCE Seiwa Kasei Co., Ltd.
The Hallstar Company This formulation contains Amitose 3LGA
(3-Laurylglyceryl Ascorbate), which activates the
Life Essence contains Olos Life Olobooster to preserve
synthesis of ceramideskey to the skin barrier.
optimal energy levels in cells, and Eurol WHT to make
Amitose 3LGA also increases glutathione for
skin look and feel fresh, radiant and healthy. The
antioxidative benefits and reinforces the skin barrier
combination of ingredients helps protect and heal the
function, bringing out skins essential power.
skin from damage caused by free radicals found in the
environment, leaving it brighter, balanced and hydrated.
The light texture is easily absorbed, delivering moisture
and nourishment. Olos, based in France, is a recently C&T Skin Health and Wellness Formulary
acquired part of Hallstar.
Visit CosmeticsandToiletries.com to view the
complete formulas.
http://www.CosmeticsandToiletries.com
CT170708_Form_Skin_Formulary_fcx.indd 70 6/27/17 4:39 PM

New York City
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Advertiser Index | C&T
July/August 2017 | Volume 132, number 7
Angus Chemical Company Ikeda Corp. Sytheon Ltd.

C4 14 7
info@angus.com info@ikeda-america.com info@sytheonltd.com
www.angus.com www.ikeda-corp.co.jp www.sytheonltd.com
Arista Industries, Inc. Lucas Meyer Cosmetics Vantage Specialty

33 15 66
info@aristaindustries.com info@lucasmeyercosmetics.com Ingredients, Inc.
www.aristaindustries.com www.lucasmeyercosmetics.com marketing.pc.us@vantagegrp.com
www.vantagegrp.com
BASF Micro Powders, Inc.

68 C2
yvonne.specht@basf.com mpi@micropowders.com Wacker Chemie AG
9
www.carecreations.basf.com www.mpipersonalcare.com www.wacker.com
Bio-Botanica, Inc. MilliporeSigma Welch Holme & Clark Co., Inc.

C3 67 42
www.bio-botanica.com sigmaaldrich.com/cosmeticstandards www.welch-holme-clark.com
Consumer Product Testing Co. Reed Exhibitions/

3 71
sales@cptclabs.com in-cosmetics North America
www.cptclabs.com in-cosmeticsnorthamerica.com
Grant Industries Silab

1 47
info@grantinc.com silab@silab.fr
www.grantinc.com www.silab.fr
(p. 49)
Ichimaru Pharcos Co. Ltd.
5
gifu@ichimaru.co.jp Sinerga
www.ichimaru.co.jp 11
info@sinerga.it
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CT170708_Advertiser_Index_fcx.indd 72 6/26/17 3:46 PM

Market Intelligence | C&T
KEY POINTS
Growth in the skin care market is anticipated to
slow, with pockets of strength in naturals and
dermocosmetics.
Seizing and disrupting niche categories

provides new opportunities but depends on the
categorys concentration in a given market.
Skin Care Slows

But Naturals and Dermocosmetics Prevail*
W
*Adapted with permission from Global Cosmetic Industry; GCImagazine.com/business/marketing/How-Beauty-Will-Grow-in-2017-and-Beyond-414901203.html.
Nicholas Micallef
Euromonitor International
hile sentiment on the beauty
and personal care industrys
performance in 2016 points to a
vibrant color cosmetics cat-
egory, skin care is anticipated to
slow down, albeit with pockets
of strength in segments such as naturals and dermocosmetics. This
is highly indicative of several prevailing trends, described here.

Vol. 132, No. 7 | July/August 2017 all or part of this article is strictly prohibited. Cosmetics & Toiletries | DE1
CT170708_Mrkt_Rprt_fcx.indd 9 6/29/17 2:18 PM

Wellness Cuts Through Benefits Set You Apart
the Clutter Claiming to be natural and clean is not
Evolving consumer patterns across multiple enough, however, because consumers are not
industries are opening new avenues to beauty easily convinced. As certain claims, such as
players. For example, a holistic approach anti-pollution, proliferate, brands must stand
to a healthier lifestyle is being manifested out with tangible evidence or they risk being
in consumers choices of food, alcohol con- dismissed as far-fetched. Ultimately, a products
sumption, sports and exercise, and greater performance is most crucial, and what sets a
attention to personal hygiene and appearance brand apart could be how visible its benefits are
through the use of preventative beauty prod- to the consumer; for example, the improvement
ucts. More consumers are basing purchasing of dark spots or acne breakouts.
decisions on a products wellness priority,
formulation and prominence in an otherwise
Probiotic Skin Care
clutteredmarketplace. Meanwhile, the beauty market is witnessing
New market entrants are exploiting this the launch of relatively bold concepts, leverag-
scenario with unique offerings and the devel- ing success achieved in other industries. One
opment of cross-category and inter-industry example is probiotic-based skin care, which is
product innovations. This evolution sums up inspired by probiotic supplements and yogurts.
why niche labels, dermocosmetics and clean U.S. brand Mother Dirt pioneered the first
label products are challenging legacy power skin care range formulated with live cultures,
brands, as consumers, in particular those in reported to restore the skins bacterial equilib-
saturated Western markets, seek authentic rium. Other niche brands later came to the fore,
options. including Esse and Yun Priobiotherapy, promis-
ing to take skin care to a new level.
Active Beauty Big players are already taking note as they
Beauty players also are exploiting the seek revenues when opportunities arise. For
thriving sportswear arena as the wellness drive example, Johnson & Johnson announced a
extends to fashion. For example, Birchbox partnership with S-Biomedic, a start-up biotech
launched the Arrow line in 2016, while Sweat firm specializing in probiotic cosmetics, and
Cosmetics specializes in sweat-proof permeable has plans to launch its first product, an acne
mineral makeup. Others, such as Mio Skincare, skin recovery solution. Under the agreement,
are more specific to users activities, with lines S-Biomedic receives help from Johnson &
such as the Workout Wonders and Liquid Yoga. Johnson while supporting the latters skin care
strategy, which is also geared toward dermocos-
Consumer Scrutiny as a metics, as evidenced in the 2016 acquisition
of Neostrata.
Source of Novelty
Consumers are more discerning than ever, Seizing and Disrupting
especially regarding product formulations. In
virtually all categories, legacy brands are falling
Niche Categories
out of favor, replaced by those professing to be Niche brands have motivated several acqui-
natural, organic, chemical-free and green, and sitions, such as LOrals recent purchase of
products that instill a sense of clean living with AcneFree, CeraVe and Ambi; or Unilevers string
the use of feweringredients. of premium skin care acquisitions, including
This is leading to a new wave of products Murad and Dermalogica. These acquisitions
under the green/clean label, as demand for natu- show swift efforts on the part of multinationals
rally derived ingredients surges and consumers to seize ripe opportunities, especially when
become more informed about the origin, segments such as dermocosmetics and doctor
processing and overall social and environmen- brands remaindynamic.
tal footprint of a product. Some consumers also The scope for niche acquisitions is depen-
reject products based on unsustainable ingre- dent on a categorys level of concentration in a
dients, e.g., those derived from limited sources given market. For example, a high concentra-
such as the Dead Sea. tion for oral care in Western Europe opens up
DE2 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

Extensions into dermocosmetics
could see an upsurge as brands
expand equity.
opportunities to for niche brands to disrupt the such as Mother Dirt, are perhaps too new but
category; as with the U.S. subscription-based beauty players will watch them closely over the
oral care brand Quip. This is similar to Bull- next few years.
dog in the United Kingdom and Dollar Shave Finally, extensions in the dermocosmetic
Club in the United States, which have spurred arena could see an upsurge as brands expand
growth in mens toiletries in their markets and their equity. This explains LOrals acquisi-
hold potential to disrupt elsewhere. tion of IT Cosmetics and Johnson & Johnsons
In contrast, in a fragmented market sce- acquisition of Neostrata; the latter could raise
nario, such as baby care in Asia-Pacific and the companys profile in color cosmetics via
color cosmetics in North America, new entrants skin-friendlymakeup. Other hybrid brands such
have a tougher time disrupting. Thus, the focus as Glossier, born out of an online community
more likely should shift toward acquiring niche blog, and Niod Photography Fluid, a high-tech
brands that facilitate specialization, such as foundation that Photoshops the wearer, could
personalized solutions and uniquely sourced be up for grabs as big players seek ventures
product ingredients, to maintain the competi- with a unique proposition to grow and generate
tive appeal. fast revenues.
Acquisition Targets
Dermocosmetics are set to exert a greater
influence on the beauty industry, and additional
C&T Online
opportunities will open up as healthy aging is
placed at the core of consumer lifestyles. This Find related content at
will be spear-headed by millennials, who put
greater focus on preventative skin care through
the use of facial cleansers, masks and moistur-
izers, which are anticipated to gain in vigor
from 20152020, with global CAGR prospects
of 2.4%, 8.1% and 3.5%, respectively; exceeding C&T Webcasts
their growth from 20102015.
Facial masks are one category where com- www.CosmeticsandToiletries.com
panies will seek acquisitions due to its dynamic
outlook and the categorys prevention-based
direction. Probiotic-based skin care brands,
Vol. 132, No. 7 | July/August 2017 Cosmetics & Toiletries | DE3

Regulatory | C&T
KEY POINTS
As air pollution climbs worldwide, targeted
personal care products are becoming more
desirable to prevent premature aging and
protect skin health.
Formulators have their eyes on efficacy and

product variations to best suit mulitiple skin
types and ethnicities.
Worldwide Regulatory Review
Rising Temperatures for

the Anti-Pollution Market
T
What Does Global Warming Mean for Personal Care?
Karen Yarussi-King
he global environment is changing. Whether
Global Regulatory
you believe in global warming, climate change
Associates, Inc.,
or neither, air pollution does not appear to be
Raleigh, N.C. USA
getting any better around the worldand the
personal care industry is paying attention.
According to the Intergovernmental Panel
on Climate Change (IPCC), Global carbon emissions from fossil
fuels have significantly increased since 1900. Since 1970, CO2 emis-
sions have increased by about 90%, with emissions from fossil fuel
combustion and industrial processes contributing about 78% of the
total greenhouse gas emissions increase from 1970 to 2011.1

DE4 | www.CosmeticsandToiletries.com all or part of this article is strictly prohibited. Vol. 132, No. 7 | July/August 2017
CT170708_Regulatory_Yarussi_fcx.indd 54 6/29/17 5:06 PM

Formulating global anti-pollution products
may pose challenges for three reasons: types
of air pollution, ethnicity-related aging and
regional consumer skin care routines.
How is this issue impacting the cosmetics and keratin in skin. But the visible signs of
industry? Asian consumers are driving the pollution-induced aging may vary by ethnicity.
demand for anti-pollution products, as indi- Since efficacy is the driver, it will be neces-
cated by the majority of launches occurring in sary to develop regional skin care. It is believed
Asian countriesaccording to research by Min- the first visible sign of aging in Asian skin
tel, 38% of new products in this category were is hyperpigmentation, while Caucasian skin
introduced in the Asia-Pacific market between experiences fine lines and wrinkles. A few
January and October in 2016. European and studies have been conducted to ascertain the
American consumers are also beginning to impact by ethnicity but no consensus has been
recognize the impact of pollution on skin, with reached as to the cause and effects of pollution
14% of U.S. consumers agreeing pollution is a on specific skin types. Controlling for the effects
driving factor in their skin health.2 of UV-induced photoaging, the synergistic
Consumers are looking for efficacy in the effects of regional-specific air pollutants and the
anti-pollution space as with all new beauty absence of well-developed test methods will be
trends. Thus, it is expected that the opportu- a challenge for ethnicity-related research in the
nity for anti-pollution product introductions future. This, of course, will also impact claims
will grow over the next few years. However, substantiation across all product categories.
formulating global anti-pollution products may The variation in regional skin care routines
pose challenges for three reasons: types of air will also drive new product introductions and
pollution, ethnicity-related aging and regional key marketing initiatives. It is well-established
t
consumer skin care routines. that Asian consumers prefer a multi-step,
Burning of fossil fuels, industrial processes, personalized skin care regimen. Whitening,
energy production, cigarette smoke and the UV protection and moisturization will be the
use of volatile organic compounds (VOCs) are primary purchase drivers for this region.
key sources of air pollution such as soot, dust, In Europe and North America, consum-
smoke, dirt and liquid particulate matter. As ers prefer simple two or three-step skin care
one can imagine, the complex mixture of these routines that emphasize science-based, anti-
pollutants varies by region and country. For-
mulating a product that specifically addresses
the needs of a region may be critical to the
success of this type of product. To do this, the
industry will need to understand the key air
pollutants by region and the synergistic effects
of the combinations on the skin, then formulate
Women are more likely to protect skin from
efficacious products accordingly.
pollutants than men, with 62% of female
Some consensus has been reached on the
consumers showing interest in the category
general impact of air pollution on skin. Most
experts agree pollution can break down the skin
compared to 55% of men.
barrier, exacerbating existing skin problems
such as hyperpigmentation; photoaging; oily, Source: Global Cosmetic Industry
sensitive or dehydrated skin; eczema; atopic (www.GCImagazine.com)
dermatitis; and premature loss of collagen
Vol. 132, No. 7 | July/August 2017 Cosmetics & Toiletries | DE5

Opportunities for anti-pollution
raw materials and products will be
the future of this industry.
aging and natural product claims. The trend for U.S. would pull out of the Paris Agreement.
natural beauty and healthier products is a sig- Irregardless, each remaining country will deter-
nificant driver for growth across all categories. mine how to meet the goals of the agreement to
One thing is certainas awareness of the help reduce global warming.
link between air pollution and premature aging The top carbon dioxide emitters pledged
of the skin grows, opportunities for anti- to reduce greenhouses gases as follows: China
pollution raw materials and products will be (20.09%), the United States (17.89%, originally),
the future of this industry. the European Union (12.06%), India (4.1%),
the Russian Federation (7.53%), Japan (3.79%)
State of the Issue and Canada (1.95%). That said, only the United
Many major cities in Asia, as reported by States ($3 billion, originally), the European
various media outlets, seem to be suffering the Union ($4.6 billion), Japan ($1.5 billion) and
most from air pollution. Yet western cities like Canada ($277 million) pledged money to the
Paris, London, Los Angeles, Mexico City and United Nations Green Climate Fund, and the
Rome are also seeing a rise in smog and air emission reductions are voluntary with no
pollution. The U.S. Environmental Protection mandated level or time frame.
Agency (EPA) reported, In 2011, the top carbon As stated, air pollution and global warming
dioxide (CO2) emitters were China, the United do not appear to be improving. Thankfully
States, the European Union, India, the Russian science and consumers are rallying to save not
Federation, Japan and Canada. This further only our own skin but our environment.
reinforces and contextualizes the global nature
of this problem. References
The Paris Agreementor Paris Climate 1. ipcc.ch/report/ar5/wg3/ (Accessed June 14, 2017)
Accordhas brought more attention to the 2. mintel.com/press-centre/beauty-and-personal-care/
problem of air pollution, specifically green- over-a-third-of-all-anti-pollution-beauty-products-were-
house gases. Representatives from 196 nations launched-in-asia-pacific-in-2016-mintel-reports (Accessed
June 14, 2017)
adopted the language of the agreement on
December 12, 2015; although President Donald
Trump announced on June 1, 2017, that the
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24 Preserving Microbiome Harmony

24 34 Natural Folate in Human Skin

58 How Damaged is Hair?

2 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

CT170708_TOC_Masthead_irv.indd 2 6/26/17 10:39 AM

30 SPF Sunscreen. FDA, ISO

One lab behind the results.

Tested by science. Approved by humanity.

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Digital Edition Exclusives

DE4 Rising Temperatures for

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Anti-pollution Natural Cosmetic Ingredient

UV rays aren t the only reason of

The leading natural ingredient manufacturer for the cosmetic industry

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Youve Been Training for This

6 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

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Preserving Collagen Protecting Collagen

Product: Standardized edible fruit extract of Terminalia chebula; Globally approved

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Pollen: A Particulate Concern

Zoe Diana Draelos, M.D.

C&T : Describe your findings about pollen. How

Advisor: Our work on pollen actually stems from

Luigi Rigano, Ph.D.

Shuliang Zhang, Ph.D. Advisor: Weve looked at several different path-

8 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

CT170708_Advisor_Insight_fcx.indd 8 6/27/17 1:27 PM

For more information, visit www.wacker.com/belsil www.wacker.com/socialmedia

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Photo Credit: Silab

Botanical Relaxer Holistic Skin Renewal

Photo Credit: Givaudan

10 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

CT170708_Tech_Launches_fcx.indd 10 6/26/17 10:56 AM

Formulators must use corroborating evidence

The Face Value

Rethinking Sun Protection Claims

Reproduction in English or any other language of

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Vol. 132, No. 7 | July/August 2017 Cosmetics & Toiletries | 13

CT1706_Regulatory_Flower_fcx.indd 13 6/26/17 11:07 AM

Testing Based On Success

14 | www.CosmeticsandToiletries.com Vol. 132, No. 7 | July/August 2017

CT1706_Regulatory_Flower_fcx.indd 14 6/26/17 11:08 AM

This third and final part in our series focuses

Reproduction in English or any other language of

CT170708_Research_Maibach_fcx.indd 16 6/26/17 11:19 AM

Heat Separation (compared to fresh skin). After heating at 60C

Vol. 132, No. 7 | July/August 2017 Cosmetics & Toiletries | 17

CT170708_Research_Maibach_fcx.indd 17 6/27/17 1:50 PM

Figure 1. Diagram of action site of dermal-epidermal separation methods

Table 3. Heat Separation

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No significant difference between