ENVIRONMENT FRIENDLY GREEN CHEMICALS

J. Appl. Cosmetol. 18, 51-63 (April-July 2000)
ENVIRONMENT FRIENDLY GREEN CHEMICALS
P. Morganti', G. Fabrizi', F.Guarneri' and C. Bruno•
l .President/Director, R. & D - Mavì Sud S.r.l., Viale dell'Industria l, 04011 Aprilia (LT), ltaly
Dept. of Internal Medicine, Aesthetic Medicine Training School , University of Rome "Tor Vergata", ltaly
2.Dermatological Dpt. Catholic University of Rome
3.R.&D l.S.C.D.
4 Physiology lnstitute, University of Urbino, ltaly
Received: June, 1999, Presented at the Persona/ Care lngredients Asia C onference,
Tahiland, 8-1 O March 2000.
Key words: gelatin, glycine, PCA chitosan, polysaccharides, biomaterials, glycolic acid, skin
roughness, skin dryness, skin hydrat ion.
Summary
Polysaccharides are the ma in source of green biomolecules used as biomaterials in phar macology
and cosmeti c de rmato logy.
The industriai interest about these molecules is continuously increasing and that's due to the different c haracteristics they proved to have: absence or near-absence of antigenicity, inability to cause
a n inflammatory reaction and high ability to enhance stroma l-cell colonization and g lycoprotein
sy nthesis when e mployed in ski n tissue repair and high environ men t compatibi lity.
T he aim of this study was to evaluate the property of innovative and patented glico-chi tosa n a nd
PCA chitosan ge ls to increase ski n hydration and e lasticity, and to evaluate its protective and anti- irritation pote ntia l efficacy.
The two-month tria! was a randomi zed doubl e-bli nd-placebo-controlled study carried out on 60 dry
ski nned female volunteers, aged 32-43 with a moderate xerosis of grade 5 accordi ng to Dahl. Surface li pids, skin hydrati on and TEWL were detected by the 3C System®(Dermotech, l taly). Sk in e lastic ity and softness were evaluated by the Dermaflex® A (Cortex Technology, De nmark). lrritation
potential was detected by SOS Challenge test a nd Pyrexal Erythema test evaluati ng the intensity of
ski n redness by Chromameter<» C 200.
T he treatment by this green gels induced a significant and progressive improvement in skin hydrati on (fro m +30% to +80%, p<0 .005), skin surface li pids (fro m + I 0% to +60%, p<0 .005) and skin
e lastic ity (+ 14%, p<0.005) and a contemporary decrease of TEWL (from -50% to - 70%, p<0.005)
and free radicals (-25 %, p<0.005) compared to the baseline values.
The most and unexpected property was the high anti- inflammatory acti vity recorded.
The improvement, that starts to be evident fro m the first week of treatment, shows how these gels
cou ld be conside red as usefu l means to improve skin hydration and elasticity of subjects affected by
skin xerosis due to environmental or pathological reasons.
No side effects were observed during the study period.
Riassunto
I polisaccaridi rappresentano la maggiore sorgente di molecole "verdi" utili come biomateri ali di
51
Envlfonment Friendfy Green Chem1cofs
uso farmacologico e cosmetico.
L'i nteresse industriale nei confronti di queste molecole è in continuo aumento date le loro particolari
caratteristiche: assenza quasi totale nel provocare effetti antigenici, o processi infiammatori, capacità elevata di incrementare la colonizzazione delle cellule stromali e la sintesi delle glicoproteine,
se utilizzate per la riparazione tissutale, ed alta compatibilità con l'ambiente .
Scopo di questo studio è di valutare le proprietà innovative quali idratanti ed elasticizzanti cutanei
ad attività antinfiammatoria di diversi gel chitosanici trattati con miscele di gelatina-glicina o PCA
gelatina.
E' stato condotto uno studio a doppio ceco su 60 pazienti volontari di sesso femminile di età tra i 32
ed i 43 a nni con cute particolarmente secca e disidratata di grado 5 secondo la classificazione Dahl.
I lipidi di superficie, l'idratazione cutanea e la TEWL sono state controllate utilizzando il 3C
System®(Dermotech, Italia)
L'elasticità cutanea è stata controllata mediante l'utilizzazione del Dermaflex®A, mentre effetto antii rritante è stato determinato con la metodica dell'SDS e del Pirexal e controllato anche con l'utilizzazione del Chromameter®C200.
Il trattamento con questi gel "verdi" ha indotto un incremento statisticamente significativo dell'idratazione cutanea (dal + 30 ali'+ 80%, p<0,005) dei lipidi di superficie (dal+ IO al + 60%, p<0,005),
della elasticità cutanea (+ 14% p<0,005) ed una contemporanea diminuzione della TEWL (dal - 50
al - 70%,p<0,005) e dei radicali liberi (-25%, p<0,005) rispetto ai valori iniziali. Il risultato più sorprende nte ed inaspettato è stata l'attività antiinfiammatoria riscontrata dei prodotti controllati. I miglioramenti, evidenti fin dalla prima settimana di trattamento dimostrano come questi gel possano
essere utilizzati per migliorare l'idratazione e l 'elasticità della cute di persone affette da xerosi cutanea provocata anche da cause patologiche in atto.
Non sono stati osservati effetti secondari di alcun genere dopo il periodo di trattamento.
52
P.Morgont1. G Fobnz1. F.Guornen and e Bruno
INTRODUCTION
Polysaccha rides a re the main source of green
bio-molecules for use as biomaterials in pharmacology and cosmeti c dermatology. ( 1-11 )
The industri ai interest about these molecules is
co ntinuously increasing because of the interesting characteristics they proved to have: absence or near-absence of a ntigenicity, inability to
cause an inflammatory reaction and high ability
to enhance stromal-cell colonization and glycoprotein synthesis, when e mployed in skin tissue
repair and hi gh environment compatibility.
These cha racteristics have been highlighted partic ul arly in the naturally occurring chitosan and
chitosan derivatives (4- 11 ). These green compounds because of the ir biocompatibility with
the huma n body have been employed extensive ly in th e developme nt of a nioni c/cationic
polysacc haride matrices for mi c roparticulate
drug delivery ( 12- 15).
They seem also to be immuno-potentiating, considering their simil ariti es with hyaluronic acid
through the common N-ace tylglucosamine sugar, and showed to be mucoadhesive.
As matter of fact, chitosans previde an attracti ve muco-adhesiveness because of their polycationic comple mentarity to the polyanionic mucins, whic h constitute the key macromolecular
component of muc us ( 16, 17).
Moreover their topica! application seems also to
minimi ze scarring a nd improve skin healing
process (1 8-22).
The formul ation of this gel is based on the exclusive usage of a new chitosan derivati ve solubilized with g lycolic acid and/or with PCA (pyrrolidon carboxylic acid), and subsequently treated with a patented mixture of gelatin-glycine
additi vated with sodium methyl hydroxylglycinate (30).
The exclu sive use of raw materials of natural
origin, allows to define "green" the gel obtained , also beca use completely and of pro ved
eco-co mpatibility. In fact the presence of this
preservati ve compound, as methylene glycol, in
this c hitosan-based formulation does not provoke liberation of free formaldehyde at skin leve! because of the strong presence of groups
NH2 of chitosan. Therefore from two moles of
chitosan and one of hydroxylmethyl glycinate
and one of water we obtain:
RNH-CHr OH +
2 Chit-NH 2
Hydroxymethyl glycine
_ . Chit-NH-CHrNH-Chit+ H20 + RNH2
reticulated chitosan + Water+ glycine
Where the reacti on goes only versus the formation of reticulated chitosan. Differently it happens rn most of me thylols where it seems to
establish an eq uilibrium between the methylol
and methylene glycol in solution:
AIM
The aim of this study was to evaluate the prope rty of an innovati ve glyco-c hitosa n and/or
PCA-chitosan gel to increase hydration, e lasticity and antioxidant potential of the skin , to restore the skin lipid content and eventually to decrease inflammati on and skin irritation pote ntial
alle viating sy mptoms of dry skin during a 8
weeks treatment course.
(+H20)
~
CH 20
lii
(-H 0)
HO-CH2 - OH
methylene glycol
(+ RNH2)
.. .
(- R-NH2)
R-NH-CHz-OH +H 20
N-methylol + water
53
Envlfonment Fnendly Green Chem1cots
MATHERIALS ANO METHODS
EXPERIMENTAL DESIGN
MATERIALS
PRE - TEST
For thi s study was used a g lycochi tosan gel as
base (glycolic acid and ch itosan), and/or a PCA
chitosa n (pyrrol idon carboxylic acid and chitosan) preserved by a derivative of glyc ine, fundamenta l a minoacid in cuta neous biology. T hese
three products, being of natural origi n, could be
s urely classified as "green compou nds" (2 1-23)
METHOD
SUBJECTS IN THE STUDY
T he two-month tria! was a random ized doublebl ind-placebo-contro ll ed study carried out fo r 8
weeks on 60 dry skinn ed female volun teers,
aged 32-43 wi th a moderate xeros is of g rade 5
according to Dahl (24). Surface lipids, s kin hydratio n and TEWL (25,26) were detected by the
3C System® (Dermotech, Italy). Ski n elastic ity
and softness were evaluated by the De rmafl ex®
A (Cortex Technology, Denmark). (27) Antiox idant potential was evalua ted by measure me nt of
s kin hydroperoxides (28) meanwhil e irritation
potenti al and the anti-i nflammato ry activity were investigated both by the SDS challenge tests
and the Pyrexal Erythe ma test (29), using a lso
the C hromameter®C 200.
Before starting the experiment, the erythema tes t was done in a dermatologica! office in order
to check if the ge l would perfor m a lso an eventual anti-infla mmato ry act ivity. T h is experime ntation was tested on the volunteers' back of
20 people onl y, a week before starting the study,
according to the methods re ported.
No vo lunteers used othe r top ica! treatments
w ithin the two weeks or syste mic drug of diet
s upple ments within the 4 weeks before starting
the s tud y. A li the s ubjects gave their writte n
informed consent in accordance with the ethics
of cosmetics experimentation.
The quantity of sodium-hydroxymethyl glycinate was always consta nt for each sample and was
prev iously mixed with the chitosan solution.
Neither the operator no r the subjects were ab le
to ide ntify the ass ig ned product.
The gel was applied twi ce daily a li over the face
and on both the forearms. The treated a reas were always cleaned w ith the s upplied c leansing
emulsion (Mav igen® latte). Fifteen days before
starting the study a li other cosmeti cs were d isco ntinued.
M easurements were performed at the I " day
TEST
T he s ubjects were randomly divided into six g roups of I O people each and received respectively:
I
GEL
A
glyco-chi tosan neutralized with gelati n-glycine at pH 4.8
Il
GEL
B
glyco-chitosan neutralized with glycine/sodium glycinate at pH 4.8
111
GEL
e
glyco-chitosan neutralized with gelatin at pH 4.8
IV
GEL
o
PCA-chitosan neutralized with gelatin-glycine at pH 4.8
V
GEL
E
PCA-chitosan neutralized with glycine/sodi um-glycinate al pH 4.8
VI
GEL
F
PCA-chitosan neutralized with gelatin at pH 4.8
54
P.Morganti, G. Fabrizi, F.Guarnen and C. Bruno
(baseline) and at days 10'', 20,30,40,50,60'h (end
of trea tment), and day 90'h always in the morning be tween 8 and l l a.m.
3C SYSTEM METHODOLOGV
Quantitative mea sure ments of skin hydra tion ,
s urface lipids and TEWL were performed accordi ng to C a rdill o a nd Morga nti met hod (25 )
before the l st day (base line) a nd a fte r d ays
I 0 ,2 0 ,30 ,40,50,60 (e nd of treatm e nt) and 90
days of treatment, a lways in the morning from 8
and 11 a.m. on skin cleaned the night before.
This co mputeri zed methodology collects up to
I O/ 15 measure me nts o ver 25 second sampling
period and records the mean va lues, automatically sta ndardi z in g the e nvironme nta l conditions (RH = 50% t = 22°C).
To alleviate the possibil ity of the patient phys iologic state, the othe r maj or factor-influe nc ing
rate of wate r loss, it was asked to rest in the testing room for 30 minutes before measure me nts.
Possible site -to-site variation was e li minated by
rando m selection of treated sites.
Skin hydration was assessed by measuri ng tota!
capacitance of the horny layer and the values
are ex pressed in 3C arbitrary units; skin !ipids,
o b serv ed by a s p ec ia l fro s te d p la st ic fo il
(l cm 2), are measured photo metrically and expressed as µ g/cm 2 ; TEWL was measured by a
s pecia l 3C probe. It c ons ists of a cylindr ical
o pen c hamber measuring syste m, d ia meter 14
mm., height I O mm. and two sensor units, conta ining a thin capacitance film transducer placed
at 3 and 7 mm. distance from the skin.
T EWL is calculated digitally as g/m 2 h.
The instrume nt probe was a lways he ld pe rpendi cular to the skin surface a nd allowed to equ ilibrate for 20 sec.
Ali the obtained results are expre ssed a s mea n
values of the measurements performed on fou r
different right or le ft skin sites (cheek, forehead,
chin and nose).
The obtained results are reported on fig. 1,2 and 3.
SKIN HYORATION AFTER A lWO MONTH TOPICAL TREATMENTWITH GLYCO.CHITOSAN OR
PCA·CHITOSAN GEL NEUTRALIZEO WITH OIFFERENT AMNOACIO MXTURE
n =60
t
=22 •c
RH
=50%
• STA RTlNG
:POINT
90
80
·-
w
(/)
70
et
w
ir
u
60
z
50
~
40
~
o
et
o
ir
>z
30
:i:
se
(/)
20
1o
~
OAYS
20
30
....._GEL
e
e
-;.11-G E l
A ( G lyco -C h ilo sa n w i h G el atin -G ly cfn n)
_._G EL
FIG. 1
10
(Glyco. C h lto s;, n wi1h Gela tin )
(G lyc o- C hilosan w lhGlyctne)
40
50
60
90
~GE l F ( PC A-C h lto sa n w lth G elatl n )
GE LE (P CA-C h ito sanw it hGly ck1 e)
-:>-GEL O (PCA -Ch ito san w ithGel a1i n-G ly cin e)
All p vaJues are highly significant(p < 0.005) as baseline vaJues and as to groups
238
55
Envlfonment Friendly Green Chemrcols
SUPERFICIAL SKIN LIPIDS AFTER A TWO MONTH TOPICAL TREA TMENTWITH GLYCO·
CHITOSAN OR PCA·CHITOSAN GEL NEUTRALIZED WITH DIFFERENT AMNOACID M XTURE
=60
n
=22·c
t
RH
=50%
• STARTING
:POINT
60
50
w
(I)
40
<(
w
o::
(.)
30
~
(I)
o
ii:
20
:::i
z
i:
(I)
10
'*'
DAYS
10
20
30
40
50
60
90
~GEL F (PC A- C hltosa n wi1h Go l atln )
~ G EL C (Glyco. C h l1o sa n wll h Ge latin )
G El E (PC A -Chltosa n w tth G ly cin e)
-.fr-G EL B (G lyco- C ll itosa n w lh G ly cìn e)
-
"""*"""G E L A (G l yco -C lt ito sa n w l:h G ehHin .e; lycln e)
- 0 - G El O (P C A -C hilo sa n w it h G efatin..G lyck1 e)
All p values are highly significant (p < 0.005) as baseline values and as to group_
s
FIG. 2
TEWL after repeated SOS application on the forearm skin pre-treated with Glyco-
Chitosan or PCA-Chitosan neutralized with a different aminoacid mixture.
eomoanson between s1tes untreated
100
n
=60
t
=22 ·e
RH
=50%
90
80
70
.-
60
50
ns
40
~ ~
n<:>
30
20
10
o
-
-
GEL A
(GlycoChitos an
\'Jith Gelatm-
Glycine
56
-
ns
A
I
Untreated SDStreated
1Control
2 Contro!
FIG. 3
-
,--A---,
-
rt__ GEL D (PCA-
GELB
Chitos an
(Glycowith Gelatin- Chitosan
Glycine)
with
Glycine)
-
-
-
>---
-
~
~
-
-
GEL E (PCA·
GEL C
GEL F (PCAG lycoChitosan
Chito san
\•Jith
Chitosan with Ge latin)
Gl>fcine) with Gelatin)
All p values are hlghly significant (p < 0.005) as baseline
PMorgant1. G FabrlZI. F.Guarnen and C Bruno
SOS CHALLENGE TEST
moved after 4 hours and reapplied to the same
sites for two consecutive days for 4 hours each.
After l hour and 24 hours of remava! of the last
set of patches, the skin reaction was graded and
evaluated by TEWL and Chromameter® measurements.
The intensity of the irritation was assessed by
visual scoring chromametry and TEWL measurements 5 hours a nd 24 hours after application
of the patches.
Thi s test is generally assessed to evaluate protective and anti-irritation potential efficacy of
the studied products. Thereafter, one occlusive
patch with 20% sodium dodecyl sulfate (S DS)
solution were applied in a randomized way to
left or right forearm of each subject for 3 days
and different hours before and after 8 weeks of
twice da ily treatme nt with the different kind of
gels used.
Two SDS treated and untreated sites were left as
contro I.
There was no further application of the studi ed
gels after removal of the last patch. Patches were app lied to the pre-marked fo rearm areas, re-
( O = no-erythema; 0.5 = equivocai reaction; l
= slig ht erythema; 2 = mod e rate, uniform
erythema; 3 = intense; 4 = fiery redness with
edema.)
The obtained results are repo1ted on fig. 3 and 4.
Chrornarneter® values a* ofthe forearrn skin after repeated SOS applications.
Differences to baseline value of sites treated with Glyco-Chitosan or PCA-Chitosan gels
7
[a*]
.../T~~~~~~-n_
e~
ut~ra
~l~
a~
ed
:::....:..:
w~
ff~
h~d~fffi~e~re~n~t=a~n1=m~o=ac~1=a~n~11='xt~u~re~.~~~~~~~
n 60
t 22 •e RH 50%
=
=
=
ns
6,5
ns
5,5
ns
SOStretted
control
GELA(Glyco -
GELO(PCA-
GELB(Glyco-
GELE(PCA-
GELCGlyco-
GELF(PCA -
Chit o un with
Chito san wilh
Chitosan with
Chito sa n wit h
Chitos a n with
C hitos•n with
Gclatin -Glyclne
Gcla t in-
Glycinc)
Glycine)
Gelatin)
Geletin)
G lycinc)
A and Dare highly significant vs Band E or e and F (p < 0.005) and as contrai
FIG. 4
57
EnVlfonmenf Fflendly Green Chem1cots
SKIN ELASTICITY
SKIN HYDROPEROXIDES
Skin e levatio n (elastic ity) was evaluated e lectronically (according to G .Gniadeck and Serup
27) by measu ring electric capacitance between
skin surface and the electrode placed on the top
of the s uction chamber o n the left or right forearm s of the treated vol unteers (s uc ti on 300
mbar, suction period 20 s, number of cycles 5).
Measure me nts were performed o n I " day (base1ine) and at 10 ",20,30,40,50,60 days (end of
treat111e nt) and at 90 days a lways in the morning between 8 and 11 a.111.
The obtained res ults are re ported on Fig.5
Free radicals were evaluated according to Pugliese (28).
A g lass cyl in der 111eas uring 51111. in diameter
was placed on the skin and held snug ly, extracting the lipids by two different aliquots of 51111.
portion of acetone. The lipid residue, dried under nitrogen st ream , was e111ul sifi ecl with sod iu m dodecyl sul fate, acetic acicl and thioba rbituric ac id. Th e supernatant extracted , read a t
531 n111 by a spectrophotometer, gave the concentration o f lipi d peroxides as MBA prec ursors.
Meas ure ments were performed on I q day (baseli ne) and at 20,40,60 1" day (end of treat111e nt)
and at 90 day always in the mo rning between 8
and 11 a. m.
The obtai ned resu lts are reported on Fig.6
1
1
"
1
"
1
"
SKIN ELASTICITY AFTER A TWO MONTH TOPICAL TREAlMENTWITH GLYCO-CHITOSAN OR
PCA-CHITOSAN GEL NEUTRALIZEO WITH OIFFERENT AMNOACIO MXTURE
n
=60
t
=22 •e
RH
=50%
w
(/)
ci:
10
w
a:
u
~
>I-
u
~
(/)
ci:
..J
w
.,e
OAYS
10
20
30
4--GEL C (Glyco - Chltosan vllth Gclalln )
_._GEI. B (Gtyco . Chltosan w i h G ty c lnc)
......... G EL A (Cilyco .Chilosa n w lh G elafn..G lyclne)
FIG. 5
58
40
~ GEL
50
60
F (P C A. Ch ilo sa n with G elatln)
G E L E (P C A - Chlto sa n wilh G lycin e)
- .:-G EL O (P C A .ChltosanwilhGelatin-Glyc"1e)
Ali p values are highly significant (p < 0.005) as basellne values and as to groups
90
P.Morgonti, G Fobriz1, F.Guomen o n d C. Bruno
ACETONE EXTRACTED LIPID PEROXIDE FROM FOREARM SKIN OF XEROTIC PEOPLE AFTER A
TWO MONlH TOPICAL TREATMENTWITH GLYCO-CHITOSAN OR PCA-CHITOSAN GEL
NEUTRALIZED WITH DIFFERENT AMNOACID MXTURE
MDA
mm/100 mglliquid
0.9
n
=60
t
=22 •e
RH
=50%
1"~~~~~~~~~~~~-=-'----=..:.._..::..::.:.:_~~~~~~~~~~~
Il
20
40
GO
90
DAYS
~GE L
e (G lyco -C h ito sa n w ith G ela tin )
_._GEL
e
(Glyco - C hitosan wilh G ly clne)
-+- G E L A (G l yco - C hi1osan with Gelatin -G lycin c)
FIG. 6
- e -GE L f ( P C A -Chitosa n withGelatin)
GEL D (PC A - C ll l1on n w lt h Ge l atin .G ly c inc)
~ G EL E PCA-Chitosan wlth G~clne)
Ali p values are highly significant (p < 0.005) as baseline values and as to groups
ERYTHEMA TEST
This inflammation is obtained injecting intracutaneously O. I ml. Pyrexal (lipo-polysaccharide
from sa lmo nella abortus equii)) into dorsal skin
of the vo luntee r s ubjects, accordi ng to H e ilmeyer and Hiemejer (29)
These bac terial pyrogens induce an inflammation. The morpholog ic sig ns take the form of a
sharply defined erythema whose surface area is
me as ured over time. Simultaneous application
of an anti-inflammatory c re am inhibits its
spread. By thi s injection 8 vials were induced in
each s ubject. These were then topicall y treated
in randomized s uccession with 0.2 ml. of the 6
different preparations and covered with transparent film.
One of these areas re mained untreated to serve
as a contro] and another was treated by 0.2 ml.
of betamethasone va lerate O. I o/o and covered also with transparent fi lm.
fn this way the erytherna is visible at ali times
and can be measured through the film. The exte nt of the erythema was determined 6,8, 10 and
12 hours after application, the maxim um (a) and
minimum (b) diameter be ing mea sured and the
s urface area calcul ated by the elliptic formu la:
p-axbxp
4
The efficacy was classified by determining the
sum of the erythema surface areas from the 4th
to the l 2th hour. A small area means that the
preparation is hi g hly effective. The comparati ve
evaluation was performed w ith the a id of bifactorial analysis of variance
The obtained res ults are reported on Fig. 7
59
Environment Fnendly Green Chemica/s
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Author Address:
Pierfrancesco Morganti
Via Innocenzo XL 41 - 00 165 Rome ltaly
Tel. +39.6.9286261
Fax +39.06.928 1523
E-mail: info@mavicosmetics. it
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