1531 ISSN 0326-2383

KEY WORDS: Fomes officinallis, Glutamic acid, Neuromuscular preparation, Rice protein.
* Author to whom correspondence should be addressed. E-mail: yoko.franco@prof.uniso.br
Latin American Journal of Pharmacy
(formerly Acta Farmacutica Bonaerense)
Lat. Am. J. Pharm. 30 (8): 1531-5 (2011)
Regular Article
Received: April 3, 2011
Revised version: July 4, 2011
Accepted: July 8, 2011
Naturally Occurring Ingredients as Potential Antiaging Cosmetics
Monique N. SANTANA, Gustavo M.C. OLIVEIRA,
Valquria M.H. YOSHIDA, Maricene SABHA & Yoko OSHIMA-FRANCO *
University of Sorocaba, Rodovia Raposo Tavares km 92.5,
18023-000 Sorocaba/SP, Brazil
SUMMARY. The criteria adopted for establishing whether a determined substance has potential as a cos-
metic constituent are based on the present legislation of each country. In this study, natural antiaging con-
stituents as Fomes officinalis, rice protein and glutamic acid were pharmacologically evaluated using neu-
romuscular preparation. These constituents induced a neuromuscular blockade, individually and also in
mixture, simulating a Botox

, but not, dimethylaminoethanol-effect. The pharmacological knowledge is

beneficial since the real effect of each ingredient becomes apparent, increasing the consumers confidence
on the antiaging cosmetic.
INTRODUCTION
The interest in noninvasive topical treatments
cosmetics and antiaging products, to reverse
the effects of photoaging on human skin and
marketed as an over-the-counter has generated
billions of dollars in commerce, as exemplified
by retinoic acid, hydroxy acids and vitamin
creams common constituents in cosmetics aim-
ing to reduce wrinkles and improve skin ap-
pearance
1
.
The categorization and regulation of cosmet-
ics will depend upon how product claims are
presented to the public
2
. In Brazil, these prod-
ucts are regulated by Anvisa (National Health
Surveillance Agency) according to Collegiate Di-
rectory Resolution n 211/05, that recognizes the
safety and the efficacy of technical data from
chemical ingredients, which, in turn, must be
available for inspection by authorities, at the site
of manufacture or importation of enterprises,
mainly those for register approval as grade 2
(potential hazard) products
3
. According to An-
visa, any methodology to determine the prod-
ucts efficacy must follow the good practices of
manufacture recommendations and have a solid
scientific and rational basis containing detailed
experimental protocol and reproducible results.
Usually, it is the manufacturers responsibility to
carry on the in vivo and/or in vitro assays ac-
cording to the National Committee for Ethics in
Research (Conselho Nacional de tica em
Pesquisa - CONEP) Resolution no 196/96
4
.
However, this is a global concern and this is-
sue was appropriately mentioned by Lintner et
al. Cosmetic ingredients previously considered
inert have the potential to provide a biologic
effect to the skin. In a cosmetic formulation, the
boundary between an active and inert ingre-
dient may be obscured. For this reason, the cos-
metic distributor must find a nonambiguous
method to demonstrate the efficacy of a new in-
gredient. For a product to be successful in the
market, its benefits must be clearly communicat-
ed to the consumer, and the consumer must be
satisfied with the product performance
5
.
Hence, there is a need to develop scientific
studies to assess the real efficacy of these anti-
aging products and validation of their claims
1532
SANTANA M.N., OLIVEIRA G.M.C., YOSHIDA V.M.H., SABHA M. & OSHIMA-FRANCO Y.
(reactions or interactions) using clinical testing
6
,
although it can be difficult to demonstrate the
effect of a cosmetic on the skin, since there are
no placebos, because anything that is applied to
the skin will have an effect. According to
Manela-Azulav et al.
7
the cosmetic dermatology
is an area in constant growing, reason by which
it is necessary the use of objective methods for
validating the scientific results. Nowadays, in
vitro release studies, permeation and retention
have been used to prove the ability of a certain
product to reach its place of action. For exam-
ple the Franzs cell diffusion through artificial
membrane (cellulose acetate) or natural (human
skin, swine skin, mouse hairless skin). The tech-
niques used in most of these studies are:
histopathologic, immunohistochemical, morfom-
etry, stereology, digital photography, biometry,
optical profilometry and confocal microscopy.
In this work, the naturally occurring as rice
protein and Fomes officinalis or glutamic acid,
isolately or in mixture, were assayed by myo-
graphic technique using mouse phrenic-di-
aphragm muscle
8
for showing their pharmaco-
logical potential as antiaging cosmetics.
MATERIALS AND METHODS
Cosmetic ingredients
Fomes officinalis, rice protein and Fomes of-
ficinalis + rice protein + glutamic acid mixture
are liquids with colors ranging yellow to color-
less; and glutamic acid (L-glutamic acid
monosodium salt monohydrate) is a white pow-
der. All the ingredients were kindly donated by
Vital Especialidades Dermocosmticas Ltda, So
Paulo, Brazil.
Animals
Male Swiss white mice (26-32 g) were sup-
plied by Anilab-Animais de Laboratorio
(Paulinia, So Paulo, Brazil). The animals were
housed at 25 3 C, on a 12 h light/dark cycle,
with ad libitum access to food and water. This
project (protocol number A029/CEP/2009) was
approved by the institutional Ethics Committee
from Vale do Paraba University (UNIVAP), and
the experiments were within the guidelines of
the Brazilian College for Animal Experimenta-
tion. All efforts were made to minimize the ani-
mal amount and suffering.
Mouse nerve phrenic-diaphragm (NPD)
preparation
The nerve phrenic-diaphragm was obtained
from anesthetized mice using halothane and
sacrificed by exsanguinations
8
. The diaphragm
was removed and mounted under a 5 g tension
in a 5 mL organ bath containing aerated Tyrode
nutritive solution (control) of the following com-
position (mM): NaCl, 137; KCl, 2.7; CaCl
2
, 1.8;
MgCl
2
, 0.49; NaH
2
PO
4
, 0.42; NaHCO
3
, 11.9; and
glucose, 11.1. After calibration with 95% O
2
/5%
CO
2
, the solution pH was 7.0. The preparations
were stimulated indirectly with supramaximal
stimuli (4 x threshold, 0.06 Hz, 0.2 ms) deliv-
ered from a stimulator (model ESF-15D, pur-
chased from Vechio FD, Ribeiro Preto, Brazil)
to the nerve through bipolar electrodes. Isomet-
ric twitch tension was recorded with a force dis-
placement transducer (cat. 7003, Ugo Basile),
coupled to a physiograph 2-Channel Recorder
Gemini (cat. 7070, Ugo Basile) via a Basic
Preamplifier (cat. 7080, Ugo Basile). Organ
preparations were allowed to stabilize for at
least 20 min before the addition of the following
substances: Tyrode solution (control); Fomes of-
ficinalis (5 L, 10 L, 50 L, 100 L, 200 L and
500 L; n = 6, 5, 6, 4, 6 and 5; respectively); rice
protein (10, 25, 100, 250, 500 and 600 L; n = 8,
9, 6, 4, 11 and 7; respectively); glutamic acid (2,
3, 8 and 10 mg; n = 8, 10, 9 and 5, respectively)
and Fomes officinalis + rice protein + glutamic
acid mixture (25, 100, 200 and 800 L, n = 5
each).
Data obtained from myographic registers
All myographic registers resulting from
above protocols have their twitch amplitudes
measured every 10 min and compared to initial
control (before cosmetic ingredients addition),
which means 100% (Fig. 1).
Figure 1. A typical myographic register from mouse
nerve phrenic-diaphragm preparation, under indirect
stimuli. Initial twitches (before arrow) mean the con-
trol, which amplitude represents 100%. Every 10 min
the amplitude was measured and converted to per-
centage. W, washing. Arrow, time addition (zero
time). Tension, 5 g. Register kindly assigned by Adri-
ana Cristina Werner and Mirile Cristina Ferraz from
our research group.
1533
Latin American Journal of Pharmacy - 30 (8) - 2011
Statistical methods
Each pharmacological protocol was repeated
at least four times. The results were expressed
as the mean S.E.M. Statistical analysis was per-
formed applying Students t-test. A value of p <
0.05 was considered significant.
RESULTS AND DISCUSSION
The criteria for selecting any substance as
cosmetic ingredient are based on actual legisla-
tion and are considerably well established by
commercial distributors. Here, using the electro
myographic technique applied on mice isolated
preparations, we demonstrated the pharmaco-
logical effects of natural active ingredients from
antiaging cosmetics as Fomes officinalis, rice
protein, glutamic acid and all these mixed ingre-
dients (Figs. 2-5 exhibit the dose-response ef-
fects, respectively). This technique shows phar-
macological activities as paralysis or facilitatory
effect simulating Botox

- or dimethy-
laminoethanol-like (DMAE) effects, respectively.
Fomes officinalis (VILL. ex FR.) is a wood
rotting fungus which is found on the trunks of
living or dead coniferous trees in the northern
region of China and in the Pacific Northwest
United States, Canada and Europe. It is tradi-
tionally used in Chinese Uigur prescription to
treat cough and asthma
9,10
. The fruit bodies of
F. officinalis are also used for the treatment of
gastric cancer, rheumatism and hydropsia
11
,
and more recently as an active ingredient of an-
tiaging cosmetics marketed by Vital Especiali-
dades

. In Figure 2, all concentration of F. of-

ficinalis applied to the bath containing the iso-
Figure 2. Mouse nerve phrenic-diaphragm prepara-
tion, indirect stimuli. Dose-response curve of Fomes
officinalis. Each point represents the mean S.E.M.
of the number of experiments showed in the legend.
*p < 0.05 (at 30 min until the end of experiments). W,
washing. Dot line, Tyrode control.
lated preparation induced a partial blockade,
meaning it is statistically significant when com-
pared to the Tyrode solution (*p < 0.05 at 30
min until the end of experiment), but not
among them. This partial paralysis is interrupted
when fresh Tyrode is replaced to the bath (W),
showing the recovery of muscle contraction ma-
chinery.
Rice protein from Oryza sativa L. seeds has
also been applied as active ingredient in antiag-
ing cosmetics, due to its permeability to pene-
trate the skin. The skin consists of two distinct
layers, the dermis and epidermis. The first one is
a thicker, deeper layer of skin underlying the
epidermis, and is mainly composed of connec-
tive tissues such as collagen and elastic fibers; it
also contains proteins, nerves, blood vessels,
lymph and muscles. Among these, collagen fiber
is the main component of the extracellular ma-
trix (ECM) as the representative connective tis-
sue that comprises about 90% of the dermis; col-
lagen has a direct influence on skin tension
12
.
Maintenance of collagen structure is related to
the intrinsic aging and photo-aging processes of
the skin
13,14
. Thus, an interesting strategy fo-
cused on protection against skin aging could be
the inhibition of enzyme activity which disinte-
grates the ECM proteins
15
.
Rice protein is classified as an enzyme in-
hibitor, which mechanism of action is to inhibit
the matrix metalloprotease (MMP) activity and
to induce the expression of hyaluronan synthase
2 gene in keratinocytes
16
. When added into the
bath containing the isolated biological prepara-
tion, rice protein induced neuromuscular block-
ade in a dose-dependent manner (Fig. 3), which
is reversible after washing and replacing it with
fresh Tyrode solution.
The pharmacological effect of glutamic acid
on the isolated phrenic nerve-diaphragm prepa-
ration is showed in Figure 4. Glutamic acid
causes a dose-dependent neuromuscular block-
ade in corroboration with previous studies
17
.
A dose-response curve was also performed
with the mixture containing the cosmetic ingre-
dients (Fomes officinalis, rice protein and glu-
tamic acid). As showed by each ingredient, iso-
lately, the mixture reproduced the same phar-
macological profile (Fig. 5).
Considering that a cosmetic formulation fol-
lows the criteria with respect to the percuta-
neous/dermal absorption process according to
the World Health Organization
18
, i.e. the pas-
sage of compounds across the skin, the phrenic
nerve-diaphragm muscle is an ideal model for
searching the pharmacological effects of any
1534
SANTANA M.N., OLIVEIRA G.M.C., YOSHIDA V.M.H., SABHA M. & OSHIMA-FRANCO Y.
substance. Although in this study, the sub-
stances used as examples showed only a unique
effect (neuromuscular blockade), not all sub-
stances exhibit this profile. For example,
dimethylaminoethanol (DMAE) when applied to
the same biological preparation induced 30 % of
a facilitatory effect (increase of the muscular
contraction amplitude)
19
.
Figure 3. Mouse nerve phrenic-diaphragm prepara-
tion, indirect stimuli. Dose-response curve of Rice
protein. Each point represents the mean S.E.M. of
the number of experiments showed in legend.
*p<0.05 among the curves (at 30 min until the end of
experiments). # p<0.05 between 250 L and 500 L
(at 30 min until the end of experiments). W, washing.
Dot line, Tyrode control.
Figure 4. Mouse nerve phrenic-diaphragm prepara-
tion, indirect stimuli. Dose-response curve of Glutam-
ic acid. Each point represents the mean S.E.M. of
the number of experiments showed in the legend.
There was no significant difference, neither between
2 and 3 mg, nor between 8 and 10 mg, although they
all presented significant difference from Tyrode con-
trol (dot line) at 20 min until the end of the experi-
ments (*p<0.05). The doses of 2 and 3 mg glutamic
acid were significantly different from 8 and 10 mg (#p
<0.05). There was a certain recovery of muscular con-
traction after washing the nerve with fresh nutrient
solution (W).
Figure 5. Mouse nerve phrenic-diaphragm preparation,
indirect stimuli. Dose-response curve of the (F. offici-
nalis + Rice protein + Glutamic acid) mixture. Each
point represents the mean S.E.M. of the number of
experiments showed in the legend. There was no sig-
nificant difference between 25 L, 100 L and 200 L,
but high dose of 800 L led to a complete paralysis.
*p<0.05 compared to control (dot line). There was a
certain recovery of muscular contraction after washing
the nerve with fresh nutrient solution (W).
As demonstrated here, this biological prepa-
ration allows discriminating the pharmacological
Botox

- or dimethylaminoethanol- like (DMAE)

effects or even whether determined cosmetic
acts at the synaptic cleft level, just like an anti-
cholinesterasic agent does. Besides, the comple-
mentary use of electrophysiological technique
also permits: 1) to propose the mechanism of
action as seen with Botox

20
, 2) to predict if its
action occurs at pre- and/or post- synaptic sites
as seen with DMAE, that acts either pre- (on
cholinergic nerve ending increasing the neuro-
transmitter quanta release) or post- (on muscle
fiber increasing the muscular contraction
strength) synaptically
21
. Neuromuscular in vitro
preparation also provides supplementary analyt-
ical benefits (Fig. 6) as mentioned above
(histopathologic, immunohistochemical, mor-
phometry, etc.). The resulting muscle can be
submitted to treatments and further analyzed by
light microscopy or any other microscopy tech-
nique.
The use of neuromuscular preparation is an
interesting model of antiaging cosmetics evalua-
tion, since the real effect of each active ingredi-
ent used in cosmetic formulations becomes ap-
parent, by a consolidated and validated tech-
nique. In this sense, our pharmacological results
about cosmetic ingredients naturally occurring
as rice protein and Fomes officinalis until now
concealed, can aggregate therapeutical value to
1535
Latin American Journal of Pharmacy - 30 (8) - 2011
cosmetic formulation. Our findings show that
these two constituents induce a Botox

-like ef-
fect, by an unclear mechanism of action, with
no possibility of being a cosmetic as firm as
DMAE. When glutamic acid is added to the for-
mulation it reproduces the same effect. It was
possible to observe the sinergistic effect from all
associated compounds, even in small amounts,
although the commercial formulation did not re-
veal the quantities of each compound. Howev-
er, we agree with the established by the Basic
criteria for the in vitro assessment of dermal ab-
sorption of cosmetic ingredients, that states that
the purpose of dermal absorption studies of
cosmetic ingredients is to obtain qualitative
and/or quantitative information on the amounts
that may enter, under in-use conditions, into the
systemic compartment of the human body.
These quantities can then be taken into consid-
eration to calculate the margin of safety using
the NOAEL (No observed adverse effect level)
of an appropriate repeated dose toxicity study
with the respective substance
22
.
Aknowledgements. Fapesp Proc. 2004/09705-8;
07/53883-6; 08/52643-4, Students from Apprentice
Scientist Program/FDA (M.N.S. and G.C.M.O) and
Probic/Uniso (M.C.F., A.C.W. and G.A.A.S.).
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