|
|
|
|
SITE OF THE PhD
IN COSMETIC SCIENCE AND TECHNOLOGY
This site includes:
Titles and summaries of the thesis of the Cycles IX-XX
·Information on the activities attended by the doctors
in the academic year 99-00 up now
·Information on the activities concerning the present
academic year
·Information on the international and internal
activities
TITLES AND SUMMARIES OF THE THESIS OF THE CYCLES IX-XX
CYCLE IX
Francioli Marco
Thesis title: “Sun-protection in the cosmetic system”
Tutor: Prof. Cecilia Anselmi
Academic year: 1997/1998
Evaluation: very positive
Summary
The problem concerning
the sun-protection is still a topical problem even if in the last years the
researches have had a considerable development in this field.
The efficacy of
a sun-protection cosmetic expressed as SPF (Sun Protection Factor) is connected
with many factors as the chemical structure of the filter, its concentration,
its spreading, its penetration and its adhesiveness in relation to the skin.
Many of the
required features are determined by the composition of the sun-protection which
can be formed by oil, emulsions O/W, W/O, W/S, gels, lipogels...; thus the
importance of the emulsifier and of the emollients in determining the efficacy
of the finished product can be fully understood.
In particular
the latter have an influence on the spreadibility of the product on the
capacity of the filter substance to penetrate into the skin, on the water
resistance, on the irradiation stability and on its absorption wavelength (l).
The aim of the
research was to carry out studies on the structure-activity relationship of synthesised
sunscreens as a function of the polarity of the solvent; and to use commercial
filters to study the influence of classical and new emollients, as
a-hydroxyacids esters, on the spectrophotometrical properties of the filter and
on its chemical and photochemical stability.
Concerning the
first point, this work completes the former studies on salicylic cinnamic and
benzophenone derivatives, containing a quaternary ammonium group capable to
confer to the molecule a binding capacity with respect to the keratin of the
skin and of the hair.
The
conformational differences found (by NMR parameters and Molecular Modeling
analyses) in solvents with different polarity, among salicylic, cinnamic and
benzophenone derivatives can justify this different behaviour regarding anti
microbial activity, which is modulated by structural parameters and by vehicle.
The compounds
tested are very interesting for having a double functionality that can be
modulated according to the application demands. These molecules can considered
as part of the group of “Multiactive-Ingredients”, a field which arises
lively interest. Indeed the presence in a raw material of different activities
could reduce the number of components in the formulation of the finished
cosmetic.
Concerning the
second point, an ample study regarding the stability with irradiation and
temperature (40 °C) of four commercial filters as:
2-ethylhexyl-p-metoxycinnamate, 2-ethylhexyl -p-dimethylamminobenzoate,
2-hydroxy-4-metoxybenzophenone, 4-t-butyl-4-metoxydibenzoylmethane with several
carriers has been carried out.
To this scope, a
simple, reproducible and not very expensive method of irradiation has been
realized, using a sun spectrum-simulator, capable to simulate the conditions of
use of the product.
In all our experiments
the polarity of the carrier had an effect on the optical properties and a good
stability to the temperature (40 °C) of the filters used in classical and new
emollients (as the esters of a-hydroxy acids was observed).
The validity of
the method of irradiation allowed the evaluation of the properties of the
sun-filter in viscous carriers. The coefficients of Harkins (Spreadability) of
carriers with different polarity have been measured. They influence the
rheological properties of the sun-filters cosmetics, and are directly
correlated to their protective-capacity (SPF).
Bianchi Anna
Thesis title: “Studies towards the synthesis of AmbroxÒ”.
Tutor: Prof. Mario Guarnieri
Academic year: 1997-1998
Evaluation: positive
Summary
Grey Amber is
one of the most precious fragrances of animal origin. It’s a metabolic product
of the sperm whale (Physeter macrocephalus L.) where it accumulates as
concretions in the intestine.
The main
component of grey Amber is (-)Ambreine, triterpene odourless alcohol which by
autoxidative degradation during the ageing, under the action of air and sun,
produces volatile compounds with the unmistakable perfumed note.
One of the
components of grey Amber which has, in addition to a strong fragrance of amber,
important properties on the fixing of the odour, is a tricyclical derivative
with a labdanic structure, known as Ambrox Ò (Firmenich SA
(-)-8a-12-epoxy-13,14,15,16-ttranorlabdano) present in traces also in some
essential oils. The rarity of the natural product coupled the increase of the
request of this class of perfumes in the cosmetic field and with the
international restriction to whales hunt, has turned the interest towards
synthetical and semisynthetical choices.
My attention has
been addressed to the preparation of this molecule at the beginning as
racemate, starting with simple molecules, in a synthesis plan which is
alternative to those well-known in literature and then to the enantioselective
synthesis.
The approaches
described in the thesis are part of such topics.
The first
synthesis plan, which allows to obtain AmbroxÒ in a racemic form, can be considered competitive in
comparison with the approaches of other authors and convenient for the number
of steps and for the total yeld.
I also tried to
found a way of approach to the natural compound in a enantiomerically pure form
through a new synthesis of the bicyclic system AB of Ambrox. Once the new
synthetic plan has been designed, a chiral auxiliary has been introduced in the
starting substrate and it has been exposed to the same reactions sequence. The
obtaining in a e enantiomerically pure form of compounds used as intermediates
in the synthesis of AmbroxÒ, permits to consider this strategy a formal
synthesis of the compound.
Brambilla Andreina
Thesis title: “Isolation and characterization of
active polyphenolic molecules of vegetable origin by chromatographic mass
spectrometry techniques and evaluation of their preserving and antioxidant
activities in cosmetics ”
Tutor: Prof. Roberto Maffei Facino
Academic year: 1997/1998
Evaluation: very positive
Summary
In the operative
cosmetic field the research of new natural active principles endowed with
preserving properties and provided with less skin toxicity and/or tolerability with
respect to synthetic derivatives is an on going process.
Simple or
polymeric polyphenols are among the active vegetable principles which posses
these two important characteristics.
Ratania
(Krameria Triandra, Leguminosae) is a plant of Peru and South America. It was
introduced in Europe only at the end of the eighteenth century. The Ratania
essences are used in the folk medicine against the infection of the oral
cavity, and especially in the gingivitis, thanks to their anti-inflammatory and
anti-microbial properties. These latter have not yet been sufficiently
clarified yet.
In cosmetic
field the extracts of Ratania are used as co-antioxidant, even if till today a
rational base for their usage has not been given.
The research
activity has been divided in the following parts:
A.
Structural characterization of phenolic derivatives of Krameria Triandra
B Determination of their anti-microbial activity
C Determination of their
anti-oxidative activity
A)
The structural characterization of active principles contained in the different
fractions of the drug obtained extracting the roots with solvents of
progressive increasing polarity has been made by LC-MS and mass
spectrometry FAB-MS/MS analysis
The LC-MS
research offers a complete outline of the lipophylic components contained in
Ratania roots allowing the fast characterizaton of rataniaphenols and lignans.
The FAB-MS/MS
analysis of the most polar fractions of the drug (Butylacetate and
Ethylacetate) permitted the characterization of the hydrophilic polyphenols
components.
In particular
the FAB-MS/MS (parent scan) study of the m/z 289 ion (nucleous), of the
Butylacetate and Ethylacetate fractions, showed the presence of many ionic
species with m/z values between 400 and 800.
These ions,
produced by collision characteristic daughter spectra which allowed the
identification of glucosidic derivatives of Catechin and also
condensation products of Catechin: proanthocyanidins dimers
(Catechin-Afzelechin) and the corresponding substituded phenylpropanoids. The
spectra FAB-MS/MS (parent scan) of the ion m/z 179, have on the other hand
permitted to identify the Caffeoil derivatives “family”.
B)
Since for the lipophilic fractions (petroleum ether, choloroform, acidified chloroform)
a good anti-microbial activity concerning different strains has just been shown
by other authors (Staph. Aureus, Staph. Faecalis, Str. Faecalis,
E.coli, Proteus vulgaris, Pseud. Aureuginosa, candida albicans), the
researches have been concentrated on the evaluation of this activity on the
hydrophilic extracts (ethylacetate, butylacetate and n-butanol).
It has been
showed from our experimental data that the Ethylacetate extract produces a
degree of inhibition of the microbial growth (range of the halos of inhibition
12 mm) greater than that of the Butylacetate extract (range 9 mm).
In particular
the Ethylacetate fraction also shows a good activity towards the Streptococcus
faecalis and the Pseudomonas aeruginosa, strains on which Butylacetate extract
is ineffective. Candida albicans e Proteus vulgaris were resistant to any
tested concentration
n-butanol
extract was ineffective at all tested concentrations is not effective for all
the tested concentrations.
The
microbiological activity of the hydrophilic fractions was sensibly inferior to
that of the lipophilic fractions in which the inhibitive action on the
microbial growth is for some strains comparable to that of Ampicilline.
C The demand of new molecules able to
prevent and contrast the premature skin ageing caused by the formation of free
radicals, led us to evaluate, in the last part of this study, the
antioxidant activity .
For this
research the ethylacetate and butylacetate fractions have been considered. As
already consolidated peroxidative model was applied. This model uses
phosphatidilcholine. In particular the inhibition on the formation of the
conjugated dienes in the spreading stage of the peroxidative process has been
evaluated.
The formation of
conjugated dienes is inhibited by both fractions (ethylacetate and
butylacetate) in a dose-dipendent way starting with a concentration of 0,5 g/ml
(76% and 83% of inhibition on the conjugated dienes formation respectively;
values calculated after 18 hours from the beginning of the propagation stage).
It’s important
to underline that, while in the controls the plateau of the conjugated dienes
(77,37 nmols/ml) is reached after 18 hours from the beginning of the
peroxidative process, in the presence of the two extracts it’s possible to
observe a 30 hours gap in the conjugated dienes formation, (Lag Phase), at the
concentration of 0,5 g/ml and therefore an important delay in the reaching of
the plateau (70 hours).
The effect is
dose-dipendent: in fact at the concentration 1,5 g/ml the plateau is reached
only after 160 hours.
At all the
tested concentrations (0,5 g/ml, 1 (g/ml, 1,5 g/ml) the butylacetate extract
showed an inhibition slightly superior to that of the ethylacetate extract.
The striking
lipoperoxidative activity showed by Ratania extract, could explain and
rationalize on one hand their use in the popular medicine as anti-inflammatory
of the oral cavity, and on the other hand it could open interesting
perspectives of use in the products of the cosmetic field destined to the
prevention of the skin ageing.
CYCLE X
Bernacchi Francesca
Thesis title: “Evaluation procedures for
substances of cosmetic use (usage)”
Tutor: Prof. Cecilia Anselmi
Academic year: 1998/1999
Board of examiners: Prof. Marco Mazza
(University of Pavia)
Prof. Franco Vincieri (University of Florence)
Prof. Marcello Scalzo (University of Rome)
Evaluation: very positive
Summary
In this thesis the attention has been focused on
the sun products and on the photoprotection; the thesis has been divided in
three parts, corresponding to three different approaches to the study of these
problems. The first part is connected to the context of the VI amendment of the
law 76/768/EEC. This law has established close to the introduction of the
cosmetic dossier, that testing of raw materials and cosmetic products on
animals is not allowed. Indeed, the mutagenic and toxicological potentials in
vitro of two new synthesised organic filters of new (two different
quaternarized benzophenonic derivatives), have been evaluated by the Ames
test and the Neutral Red Uptake Test. Both products proved ineffective on the
four strains of Salmonella Typhimurium studied (TA1535, TA1537, TA98, TA100),
in the absence or in the presence of metabolic activation. This clearly
expresses the fact that the two sun filters do not produce punctiform mutations
in vitro; the two organic filters, on the contrary, showed some toxicity.
The second part
of the thesis was inspired by the introduction of the guidelines of Colipa (the
Association of the European Cosmetic Industry) for the determination of the sun
protection factor on volunteers. The method, in the attempt to implement a
protocol which, in addition to be a reference for the European cosmetic
industry, represented a first step towards the international attuning to the
subject of photoprotection, takes its way from a critical review of the
official protocols previously issued. It focuses its attention and, thus, improves,
the aspects of the methodology, that can influence the result of the
determination. Among these the introduction of the colorimetrical analysis for
the selection of the individuals undergoing the test is of paramount
importance. On the basis of these conditions, a correlation study between the
skin colour determinated instrumentally by colorimeter and the skin phototype,
determined by sun anamnesis according to the Fitzpatrick method has been
planned. The results of the analysis carried out on 557 individuals and
elaborated by a statistical method, (Kullback test and linear Bayesian
classificator) clearly showed that while it’s highly probable to succeed in
discriminating, on the basis of the colorimetrical coordinates, the individuals
belonging to extreme phototypes (I and IV), on the other hand it’s difficult to
correctly identify the individuals belonging to the intermediate phototype
classes (II and III). As the Fitzpatrick classification is based on subjective
observation, these results could show that the mistake consists in the sun
anamnesis and they could suggest an assignment of the phototype based on a
stricter method, as, for example the MED on unprotected skin.
Finally, in the
third part, a very innovative aspect of photoprotection, the instrumental
determination of the sun protection factor has been dealt with. These
measurements are carried out in a spectrophotometrical way, by the use of an
integrating sphere, comparing the quantity of radiation transmitted (within the
UV wavelengths) by an appropriate substrate during the absence and in the
presence of the product under study. The information obtained by two different
instruments have been compared: spectrophotometer with an integrating sphere,
and an instrument, available on the market, prepared for this kind of
measurements. The data obtained from market products and other products, showed
a higher precision and care that the dedicated instrument has a higher
precision accuracy than the spectrophotometer. Even if it was not possible to find
a correlation “vivo-vitro”, since for many products the “in vivo” method for
determining the sun protection factor was not known, the importance of these
determinations as pre-screening. For both planning products and finished
formulations, or as additional information which guides the dermatologist in
the “in vivo” determination (the protection factor is a necessary information
to avoid that the volunteers expose their skin to extreme and useless doses of
radiation) has been put forward. The possibility to use these determinations to
evaluate the photostability of sun formulations has also been considered.
Faiferri Laura
Thesis title: “Studies of formulations
containing vitamin A propionate”
Tutor: Prof. Mario Guarnieri
Academic year: 1998/1999
Board of examiners: Prof. Marco Mazza
(University of Pavia)
Prof. Franco Vincieri (University of Florence)
Prof. Marcello Scalzo (University of Rome)
Evaluation: very positive
Summary
Vitamin A is one of the most used active
principles in antiageing products. Several studies showed that, among
vitamin A derivatives, retinil propionate has the higher activity. Thus a study
on this molecule has been undertaken.
The aim of this
research regards the evaluation of the stability of the retinil propionate as
well as showing possible problems connected to its introduction in cosmetic
formulations.
The studies have
been carried out in standard solutions to evaluate the real stability of this
substance in comparison with other retinoids of common use, and in finished
products (oil, emulsions W/O, O/W, W/O/W) to evaluate the influence of the
formulation
The
quali-quantitative analysis has been carried out using HLPC.
The titre of the
standard solution is around 70-80% and it’s possible to note the appearance of
two degradation products which have not been identified with any known
retinoid.
The results
obtained for the formulations have pointed out a high stability of this
molecule as its titre is in the shelf-life of the product itself, within
satisfying limits (95% after conservation at room temperature and 80% after
quick ageing).
Besides, unlike
what has been observed in solution, in any formulation analysed it’s possible
to observe the formation of products of degradation. This points out that the
key reactions of the mechanism of degradation are not favoured in a structured
environment as are the emulsions.
The influence of
the oil component on the formation of lamellar structures such as to meet the
characteristics of stability typical of liquid crystals a emulsified system has
been considered. For this reason compounds with a new emulsifier (Biophilic S)
have been formulated and studied using oils with different polarity and new
multiple formulations have been developed. This latter have a special interest
for the cosmetic field as, in addition to produce a lasting and moisturizing
effect with a very good skin feeling, they permit a controlled and
time-prolonged release in the internal aqueous phase of the capsuled
substances, thus reducing their irritant potential, and moreover they allow the
partition assembling of incompatible substances.
The chemical and
rheologycal analyses pointed out that the polarity of the oils affects the
characteristics of the product. Indeed the increase in polarity of the oil
corresponds to greater stability of the emulsion; besides, it turned on that
the multiple emulsions are very sensitive to temperature. On the basis of these
results it could be possible to take into consideration the possibility of
introducing same hydrophilic polymers in the external aqueous phase to increase
the thermic stability and the elastic component of the structure so to assure
to multiple emulsions a lasting stability.
Fedeli Paola*
Thesis title: “Natural and synthetic fragrant
substances used in the cosmetic field: synthesis and evaluation”
Tutor: Prof. Cecilia Anselmi
Mulé Alessia*
Thesis
title: “Skin photo-oxidation processes, protective effect of natural
antioxidants”
Tutor: Prof. Roberto Maffei Ficino
CICLO XI
Andreassi Marco
Thesis title: “Natural raw materials and their
use in the cosmetics”
Tutor: Prof. Cecilia Anselmi
Academic year: 1999/2000
Board of examiners: Prof. Marco Mazza (University
of Pavia)
Prof. Giorgio Adembri (University of Siena)
Prof. Carlo Rossi (University of Perugia)
Evaluation: very positive
Summary
The antioxidant
substances are used in cosmetology for their capacity to neutralize free
radicals produced by different noxae, as sun radiation, chemical agents etc.
Thus their use seems to be justified in the sun products, in the antiageing
creams and in every formulation which intends to prevent anti-inflammatory
phenomena.
The chemical
world offers the cosmetologists a great number of sinthesized molecules, with
antioxidant activity, as the ascorbic acid, the alpha-tocopherol etc. However
in the last years, the idea of using natural antioxidant substances present in
vegetable food or derived from has became more and more convincing. The
possibility of using the antioxidants contained in the olive oil manufacturing
residues, and especially in the washing waters is a very attempting
possibility. The use of antioxidants contained in the olive oil manufacturing
residues, besides being favourable on economic point of view, seems to be a
very useful operation from the environmental point of view, because it assures
the recycling of products with a high demand for oxygen and as such damaging to
the agriculture.
The aim of our
research was to show the presence of phenolic compounds in the washing waters
coming from the olive oil manufacturing and their antioxidant capacity compared
to substances with well-known activity, these facts in turn will open the possibility
to use these compounds in cosmetic formulations.
In the first
part of our research the methodologies of sampling and conservation of washing
waters have been evaluated. These studies showed that the Kathon CG at the concentration
of 0,1% appeared suitable for the conservation of the product and of all
the fractions obtained, avoiding the growth of mildews and of microbial agents
able to degrade our product.
In the second
phase the presence of phenolic compounds in the samples has been controlled,
thus confirming what it was reported in literature. These tests showed that
there were no changes in the titre in polyphenols after 5 or 12 months from the
sampling. Besides, the titre in polyphenols did not reveal important changes
even the samples obtained from two different methods of separation.
In the third
phase of our research extraction tests with solvents have been made to
characterize the phenolic products contained in the isolated fractions. These
researches have also confirmed what is known in literature, that is to say the
presence of phenolic substances as 3, 4-dihydroxycinnamic, acid 3, 4-dihydroxy
benzoic, (p hydroxyphenyl) ethanol, cathecol, (3,4-dihydroxyphenyl) ethanol and
methylcathecol.
Our interest
then turned to the setting up of methods able to separate the phenolic products
by using Ionic exchange resins. The following substrates have been used:
Amberlite IRA 93 (weak basic resin), Amberlite IRA 400and Dowex 2X 200/400
(strong basic resins). These tests showed a good separation of the substances
of our interest, but with a low yield of the products stuck to the
resins. In fact, to remove the substances bounded to the resins, a quantity of
eluate about 8 times superior to the starting volume has been used.
In the final
part of the research, the samples and the isolated fractions, in which the
presence of polyphenols had been recognized, have been tested to their
efficacy. This has been verified evaluating the capacity to prevent the
hemolysis of a erithrocitic suspension in the presence an inductor, as the
Cumoxy radical. These tests have been made on all samples, using oleuropein,
tyrosol, hydroxytyrisol and vitamin E as reference substances.
The results of
this research showed that the washing waters, without the corpuscolar part and
preserved with Kathon have double anti-radical activity compared with that of
oleuropein, tyrosol and vitamin E. The concentration by evaporation increases
the antioxidant activity but not in proportion to the extent of concentration.
In fact the 8 times concentrated flowing-back shows an antioxidant capacity
only double as compared to that of the flowing-back and comparable with that of
hydroxytyrosol, product of phenolic nature, present in the washing waters. (It
was synthesised by us since it is not available on the market).
The great
antioxidant capacity of the washing waters has not been confirmed in the
samples obtained by extraction with organic solvents. Especially those obtained
by extraction with dichloromethane and ethylacetate have shown some activity.
However this activity was not reproducible and thus not precisely measurable.
In short, our
research showed that the washing waters of the olive oil manufacturing
contain many substances with an antioxidant activity. Among these
3,4-dihydroxycinnamic acid, 3,4-dihydroxy benzoic acid, (p-hydroxyphenyl)
ethanol, cathecol, (3,4-dihydroxyphenil) ethanol 4-methylcathecol whose
presence in the samples has been verified by TLC, HPLC and GCMS, must be
mentioned. The simultaneous presence of all these substances in the waters can
lead to a synergistic antioxidant activity ( the data are reported in a patent)
greater to that shown by known substances with known activity, as vitamin E.
1.
C. Anselmi, M. Andreassi, M. Centini, R. Maffei Facino, M. Carini. “Mixture
with high synergic, antioxidant and radical scavenging activity of vegetable
origin and its production process” Patent Application n.FI 2000A000223 ,
9/11/2000.
D’Agostino Rosa
Thesis title: “Evaluation of efficacy and innocuity of
new raw materials and finished cosmetic products”.
Tutor: Prof. Marina Carini
Academic Year: 1998/1999
Board of examiners: Prof. Marco Mazza (University of Pavia)
Prof. Franco Vincieri (University of Florence)
Prof. Marcello Scalzo (University of Rome)
Evaluation: Sufficient
Summary
For the
evaluation of a cosmetic product it’s possible to resort to different
methodologies based on instrumental or sensory processes, which are divided in
objective and subjective. The ambivalence of the sensory evaluation,
objective/subjective, comes from the assumption that the same sensory answer is
the result of the sum of two components. The analysis of these two components
represents the field of sensorial studies and, according to the component
considered, it will be possible to speak of hedonistic of quali-quantitative
tests. In this study the attention turned to the quali-quantitative tests whose
application in the cosmetic field is still quite limited.
Two different
kinds of products have been the object of the research: lipsticks and
emulsions. On these suitable procedures have been developed for carrying a QDA
(Quantitative Descriptive Analysis) and a Ranking test respectively. The data
obtained have been supplemented with an “in use” evaluation and in the case of
the emulsions with an instrumental evaluation.
As regards the
evaluation of innocuity the aim of this research was to comply a twofold aim:
to test a process for the objective evaluation of the tolerability of detergent
products and to use that process to evaluate to which extent possible changes
produced in the formulation of a detergent affect its skin compatibility. The
repeated arm wash test (RAWT) has been used. The process applied to the samples
showed itself as an effective instrument for the evaluation of the skin
compatibility.
Vertuani Silvia
Thesis title: “Compounds with dermo-cosmetological
application: synthesis and biological activity of new antiageing molecules with
free-radicals scavenger activity”
Tutor: Prof. Mario Guarnieri
Academic Year: 1998/1999
Board of examiners: Prof. Marco Mazza (University of Pavia)
Prof. Franco Vincieri (University of Florence)
Prof. Marcello Scalzo (University of Rome)
Evaluation: very positive
Summary
The aim of this
research was to obtain new antioxidant compounds with dermo-cosmetological
applications, deriving from the molecular alteration of vitamins with a high
antioxidant activity: ascorbic acid, (a tocopherol, retinol and carotenoids).
This research
have examined the design and synthesis of similar to ascorbic acid and to
a-tocopherol. The interest for these two vitamins has been suggested by the
wide reputation and use in cosmetic field. However along with the considerable
antiageing properties, there are problems concerning the stability of the
formulation, which represents a limit to their application. The feasibility of
modifications, whose aim was the stabilization of the vitamins and the
improvement of the distribution, to targeting of the part where the oxidative
damage could take place, has been evaluated.
The designed and
obtained molecules, can be considered as new semi-synthethic antioxidants and
as “carriers” of the corresponding vitamins. Planning these new antioxidants
there have been taken into consideration the ideal of a chain-breaker
antioxidant.
Semi-synthetic
derivatives of the ascorbic acid have been prepared, appropriately
functionalized, to allow the attachment of the ascorbic part to the
tocopherolic counterpart.
For this reason
three possible synthetic strategies have been studied. Two of these strategies
did not give the expected result, therefore they have been dropped. The third
one has produced a mixture of diastereoisomers. The obtained products have been
divided and characterized by NMR analysis and their structure has been
confirmed by molecular mechanic studies.
The antioxidant
activity in vitro of the synthetized compounds has been evaluated against
biological membranes models or against lipids isolated from membranes.
The obtained
results showed that the molecular combination of the bio-active moieties of the
ascorbic acid and of a-tocopherol, gave an increase in the activity as compared
to the simple physical mixture of the two vitamins.
The considerable
synthetic versatility of the obtained derivatives and the interesting activity
data, brought to extend this approach to other natural and non natural
antioxidants. These results are reported in a registered patent.
To fully
evaluate the potential development of these interesting molecules in the
dermato-cosmetologic field, studies on the stability in formulations on the
enzymatic hydrolysis of the conjugates, which could lead to the single
antioxidant components and on the efficacy on the protection of skin damages in
a model of erythema produced by UVB have been undertaken.
CYCLE XII
Paoli Maria Laura
Thesis title: “Molecular structure-odour
relationships”
Tutor: Prof. Cecilia Anselmi
Academic year: 1999/2000
Board of examiners: Prof. Marco Mazza (University of
Pavia)
Prof. Giorgio Adembri (University of Siena)
Prof. Carlo Rossi (University of Perugia)
Evaluation: excellent
Summary
This topic
represents the continuation of a larger research project in the field of the
olfactory perception intended to establish the structure-activity relationships
of odorous molecules.
These researches
have been addressed to the study of the molecular structure-odour relationships
concerning functionalized molecules with floral odour. The scope was trying to
give a contribution to the knowledge of the structural parameters which give to
the molecules this odour. Besides, also molecules without functional groups,
like saturated and unsaturated hydrocarbons, which have fruity odour, have been
studied.
The problem of
the structure-odour relationships has been faced studying a group of alkenes
containing a cyclohexane ring with a chain containing one or two double bonds.
In this research have been also included same cyclopentanes and cycloheptanes,
open chain hydrocarbons and bicyclic derivatives.
These compounds
have the ring of the cyclohexane and the alkylidenic group in common with the
megastigmatrienes (2,2,6-trimethyl-1-butylidencyclohexanes) discovered in the
volatile compounds of passion fruit with agreeable fruity and floral notes.
This research
permitted to point out the relations between molecular structure and corresponding
molecules odour. These molecules differs for the presence of different
nucleophilic groups: carbon-carbon double bond, oxygen atom, carbon-oxygen
double bond.
The olfactory
evaluations have put in evidence various molecules of great practical interest.
The
conformational studies, carried out by NMR analysis on floral perfumes and on
new molecules, are of great importance because they show the importance of the
structure of the molecule in its interaction with the receptor.
Calloni Maria Teresa*
Thesis title: “Application of different analytic
methodologies for the quality control of the cosmetic product”
Tutor: Prof. Roberto Maffei Facino
CYCLE XIII
Cicalò Antonella
Thesis title:
Control methods for raw materials and cosmetic formulations
Tutor: Prof. Cecilia Anselmi
Academic year 2001/2002
Board of examiners: Prof.ssa Cecilia Anselmi
(University of Siena)
Prof.ssa Paola Mura (University of Florence)
Prof. Maurizio Ricci (University of Perugia)
Evaluation: very positive
Summary
This research
had been addressed to tune up some analytic methods able to determine and to
measure some very important cosmetic ingredients in the toxicological and
medical field. Only for some of the substances included in the Enclosures of
the L.713/86 and subsequent updatings and changes, are available in literature
analytic methods which permit a simple and fast characterization. The class of
substances which have been taken into consideration in this experimental
research is that of the sun filters. A HPLC method which allows the
simultaneous determination of six common filters used for the production of
cosmetic formulations (benzophenone-3, benzophenone-4,
butylmethoxydibenzoylmethane, octyl methoxycinnamate, 4-methylbenzylidene
camphor, octyl dimethyl PABA) has been found. The method, identified starting
from matrix taken from the market and containing the substances related to this
research, has thus been applied. The aim was to verify its validity, on
artificial sample realized in laboratory and containing known amounts of the
substances to be analyzed. The parameters related to repeatability and
precision of the method have been determinated, obtaining very law values for
the indices of dispersion and very good recovering values for all the tested
substances.
In the same way
an analytical method for the determination and the dosage of nine definitely
allowed preservatives has been identified.
In literature
many contribution on the determination of analytically different preserving
substances are present, most of them are obtained after complex or involve long
procedures, or they don’t allow the contemporary determination of a great
number of substances.
A HPLC method
based on elution with binary gradient with acetonitrile and chlorate buffer for
the determination and the dosage of nine preservatives among those definitely
admitted and chosen among the most used for the production of cosmetics
(4-hydroxybenzoic acid, methylparaben, ethylparaben, propylparaben,
butylparaben, salycilic acid, dehydroacetic acid, phenoxyethanol, sorbic
acid).has been tuned up. Also in this case the method, at first tuned up on
products of the market, has been tested creating artificial products containing
the compound to be analyzed and examining its parameters of repeatability and
precision, obtaining good results concerning the recovering and the precision.
Besides the method is fast and of simple execution, thus general “cheapness” is
inherent to this method.
An other research
field was the quali-quantitative determination of the heavy metals in cosmetic
matrix. In literature analytic methodologies which provided information to
point out the content of different metals within a large variety of cosmetic
forms has not been found out. A method based on a phase of mineralization with
attack with nitric acid and 30% hydrogen peroxide as oxidant done in a
microwave oven in two consecutive steps with power increasing supply has been
accomplished. Subsequently the content in metals in the sample dilute in water
MQ has been evaluated by plasma emission photometry (ICP).
The method has
been applied to 15 different kind of cosmetic matrix (emulsions, products
cleaning, lipsticks, nail varnishes, eye shadow etc), carrying out, for each matrix
ten mineralizations on the sample and ten mineralizations for two different
levels of addition of a multistandard solution of the six metals to be
analysed. In total every sample has undergone mineralization and following
plasma reading for thirty times and in everyone the content Cu, Co, Ni, Pb, Cr,
Cd was determined. All considered 2500 determinations have been carried out.
The method showed very satisfactory values of the indexes of repeatability,
with very low values of dispersion, and very good percentage of recovery in all
the analysed matrixes.
Besides the
method foresees low times of mineralization (about 1 hour), it does not need
pretreatment of the sample, and it is of easy execution with scarce danger for
the analyst.
Finally an
analytic method for the determination and the measuring of substances with
depigmenting properties has been identified.
Among the
substances used as functional ingredients in the products destined to clear the
skin spots some substances which interfere with the synthesis mechanisms of
melanin have been used. Hydroquinone had a great success in the past, since it
has high levels of efficacy even at low doses.
Recent
legislative updating have limited the possibility of use of this substance to
products for the hair dye, reducing the highest percentage of use at 2% in the
finished product, while the use of this cosmetic ingredient has been forbidden
in the products destined to clear the skin, owing to its not negligible
toxicological potential.
The literature
relates that one of its derivatives, arbutine, (hydroquinone-b-glucoside), have
the same depigmenting characteristics (having the same action mechanism of
hydroquinone) with a high tolerability. A method in HPLC for the determination
of arbutine, of hydroquinone and of three ethers of the hydroquinone
(hydroquinone monomethylether, monopropylether and monobenzylether) included in
the enclosure II of the 713/86 has determined. The method foresees a isocratic
elution of the five analiti and it has been verified concerning the parameters
of repeatability and precision, obtaining satisfactory results.
In order to
verify if arbutine could be put in emulsions without meeting hydrolysis on the
long period, losing hydroquinone, stability tests have been carried out.
Every emulsions
has been analysed three times, and the percentage of arbutine present has been
calculated on the medium value.
Chromatograms
did not show the appearance of hydroquinone developed with hydrolysis. So the
three emulsifying systems used for the tests seem to be suitable to transmit
arbutine as depigmented active principle.
Iannuzzello Vincenza
Thesis title: “Characterization of complex thermal
matrixes. Research of active principle useful in cosmetology”
Tutor: Prof. Cecilia Anselmi
Academic year: 2003/2004
Board of examiners: Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Mario Tredici (University of Florence)
Prof. Paolo Pelosi (University of Pisa)
Evaluation: very positive
Summary
Today thermal guest, more conscious of the potential
of the spa, has guessed that the thermal therapy can be a good help to cope
with the ageing, but this aim should be pursued only by a scientific approach
able to give a reproducible and objective answer. The Italian and European
thermal researchers gave a considerable experimental contribution to the spa
giving it a new dignity and inserting it in the medical science.
In the last year
the spa has been interested in the problems of the skin carewith special
attention to the cosmetic treatments as source of comfort. In Italy about 85
thermal spa practice cosmetic and dermatological cares. In national and
international literature the attention is turned to thermal cares but the study
of the complex thermal matrixes and the research on active principles
which can come from the use of thermal auxiliaries is ignored. The aim of this
research was individuate a method of characterization of mud baths through the
process of maturation and to evaluate them from the structural and constitution
point of views. Besides the research has been addressed to the study of thermal
plankton (biogleaTM) from the chemical point of view and of its biological
activity.
The research has
been carried out on sample of thermal material coming from Saturnia spa
characterized by a swimming pool with sulphureous water with uniform
temperature (37 ˚C), which forms a little lake in a natural crater.
The mud baths
analysis has been carried out by chemical and rheologycal evaluations. The rheologycal
research showed a strong microstructural profile due to the matrix and to the
homogeneity of the clay but it is not possible to exclude that this structure
could come from the biological activity of mud bath microflora. The chemical
analysis has been carried out through extractive methods and chromatographic
analysis (TLC, HPLC, GC-MS) and it has allowed the identification of many
classes of compounds in the mud bath among which the lipophylic fraction seems
to be very important.
Concerning the biogleaTM
a study which showed some cianobacterial strains and products which could have
an interesting cosmetic functionality is now in due course.
Piccone Maria*
Thesis title: “New models for the study of the radical
scavenging activity of synthetic and natural compounds”
Tutor: Prof. Roberto Maffei Facino
Summary
The research has
been addressed firstly to the development of new, cheap and of easy execution
bioanalytical markers, for the determination of the cell damages mediated by
radical processes which are present in the membrane and intracell after
exposure to UVB. Our interest has mainly been addressed on the use of sensible
and specific fluorescent probes as: the cis-parinarico acid (PNA) for
monitoring the peroxidation of the membrane lipids, and the 2’,
7’-dichlorodihydrofluorescein diacetate (DCHF-DA) for evaluating the damage of
intracellular oxidative stress. In the same way the intracellular contents of
glutathione, the main intracellular antioxidant, has been monitored by
fluorometric determination.
It’s known that
vasodilatation, inflammation and reddening of the skin after UVB exposure are
produced by the release of NO by the activation of the isoform of NO-sintasi.
Then it has been evaluated if NO (determined indirectly as nitrite ion) could
be considered bioanalytical marker of an initial oxidative damage.
Secondly the
research has been addressed to the tune up of a cell model, in extension to the
usual membrane models, able to evaluate, by sensitive and economic procedure,
the cell oxidative stress produced by sub-erythematous doses (0,1-1 MED).
Above all the
researches have been focused on the definition of the antioxidant profile of a
lipophilic extract of ratania and of its main constituents (lignans), using
different cellular and membrane experimental models.
The tested
extract showed an antilipoperoxydant activity 20 times superior to that one of
the vitamin E, and a strong chain-breaking capability (verified on isolated rat
erythrocytes exposed to cumene hydroperoxide and on phosphatydilcholine
liposomes exposed to oxidative stress by radicals OH, respectively).
Besides the
ratania extract is able to completely inhibit, with a dose-dependent effect
(minimum effective concentration 2,5 7g/ml), the intracellular oxidative burst
induced by UVB.
According with
the results obtained using the fluorescent probe DCHF-DA, this extract is also
able to contrast with the same efficacy, the depletion of endogenous GSH.
Since
partecipation in the glutathione synthesis must be excluded it is evident that
this extract exerts on its inhibitory action towards the oxidative
intracellular burst induced by UVB, through a classical radical-scavenging
mechanism.
In conclusion,
on the basis of the results obtained in the cosmetic field it’s possible to
hypotize its use as natural antioxidant as a substitute and/or in association
with those commonly used (BHA/BHT) or in products intended for the prevention
of skin aging for its antioxidant properties against damages induced by UVB
radiations.
CYCLE XIV
Ettorre Anna
Thesis title: “Technologies and biotechnologies of
possible use in cosmetics”
Tutor: Prof. Cecilia Anselmi
Academic year 2001/2002
Board of examiners: Prof.ssa Cecilia Anselmi
(University of Siena)
Prof.ssa Paola Mura (University of Florence)
Prof. Maurizio Ricci (University of Perugia)
Evaluation: very positive
Summary
Biotechnologies have a high application
potential in cosmetics with regard above all to:
the setting up,
the optimization and the validation of in vitro systems for the evaluation of
the functionality and toxicity of cosmetic product.
Cosmetic
products, as in the pharmaceutical field, are first of all tested on animals,
the so-called test in vitro and after on man before being put on the market.
Since 30 June 2002 it will be forbidden the use of animals to test cosmetic
products.
Thus there is a
great interest in this field for the setting up, or for the optimization of
tests in vitro which are predictive and which give results to correlate with
those obtained in vivo on the animal and on man.
When the cell
undergoes a stimulus by an endogenous or exogenous agent, it can react: a)
activating defence strategies which can modify the cellular homeostasis to
survive the to underwent solicitation (ex. hypertrophy, hyperplasia); b)
producing the so-called chemiochine which are the endogenous substances
responsible of the inflammatory answer; c) if the damage is irreparable the
cell can risk a necrosis; d) the cell can also decide to kill itself.
Our aim is to
evaluate the toxicological answer to a cosmetic ingredient by the induction in
vitro on cell cultures of necrosis or apoptosis.
Apoptosis not
only is one of the most charming topics discovered till now, , but it can also
be an answer to a cell damage.
Our aim is to
make use of the study of the programmed cell death for the monitoring of cosmetic
ingredients and to understand how these ingredients can influence the cell
homeostasis and with which mechanisms for arriving to a proposal of alternative
repeatable, reliable “in vitro” methodologies to evaluate the potential
toxicity of both the ingredient and of cosmetic product.
We have chosen as raw materials to test some
preservatives not only used by cosmetic industry, but also by pharmaceutical
industry and industrially in cleansing products.
The literature
offers many microbiological tests and articles concerning dermatological
researches as patch test to point out the allergenic and irritant potential of
these substances, but the sources on researches carried out on other
experimental models as the cell cultures are few.
The most studied
and used preservatives by cosmetic industry, but also those most easily soluble
in the medium of culture, chosen for this research are imidazolidinylurea,
phenoxyethanol, a mixture made by methychloroisothiazolinone and
methylisothiazolinone (better known as Kathon GC).
The tests have
been carried out on suspended promielocitic cells lines called HL60, which
represent the strain with which many researchers undertook for the different
studies carried out on phenomenons correlated with the apoptosis. During our
tests we used short contact times with scalar concentrations of the three
preservatives. In a first screening we used as cytotoxicity test the trypan
blue and the MTT in a time range from 0 to 72 hours.
Apoptosis has
been qualitatively pointed out by the presence of the classical “ladder” of the
DNA fragmented on gel of agarose.
Quantitatively
the apoptosi has been evaluated through the analysis of the content of DNA
using cytofluorometric methodologies.
All the data
gathered during the first year confirm that these three examined preservatives
IMU, phenoxyethanol and Kathon GC can produce two different typologies of cell
death: necrosis and apoptosis. Necrosis takes place at high concentrations
while apoptosis reveals itself at low concentrations.
During the
second year of PhD we considered subtler biochemical parameters which are
involved in the cell suicide as the exposure of phosphatidilserin, Fas
expression, the decreaseof the transmembrane potential always on HL60.
At the same time
we also made the same quali-quantitative analyses on normal cells, the
linfomonocyte of peripheral blood, on which it’s also possible to evaluate the
expression of pro-inflammatory cytochine.
Our data confirm
what we have just observed on HL60, that is to say that necrosis or apoptosi
are a consequence of the cell damage dose-answer dependent.
At present we
are verifying if the data obtained with HL60 and the linfomonocyte of
peripheral blood can be compared with the answer of human and immortalised
keratinocytes which represent the best model to value the possible answer of
the skin to the cosmetic.
Catapano Katia Maria*
Thesis title: “Natural Polyphenols: analytical
characterization and study of their antioxidant activity”
Tutor: Prof. Marina Carini
Summary
In the last years the research has always been
addressed towards the experimentation of natural molecules (polyphenols) as
potential antioxidant agents as they are less toxic and have higher activity.
Helichrysum (Compositea)
includes many species differentiating in the qualitative and quantititative
composition of active principles.
Phytochemical/pharmacological
studies showed that the biological activity is connected with the flavonoids
constituents of the drug, which, owing to the strong antiradical activity, are
able to inhibit the inflammatory process. The aim of the study was to extend
the researches to the characterization of the flavonoids constituents of a
species of Helichrysum, H. stoecheas.
In particular
the object of the research was the insulation of a flavonoids polar fraction
and the identification of its main components by the combined use of
liquid chromatography and the mass spectrometry (Ion Trap Mass Spectrometry)
and using a UV-DAD detector (diode-array detector).
The radical
scavenging activity of the polar fraction of H. stoechas in different
experimental models has been also evaluated: models in homogeneous phase (DPPH,
deoxyribose test, superoxide anion test) of cell (rat erythrocytes) membrane
(phosphatilcholine liposomes, hepatic microsomes) and models to outline the
antioxidant power. Concerning the latter, the molecules studied are able to
inhibit the antioxidant processes with a dose dependent effect in the different
experimented models used.
Successively the
research has been addressed towards the evaluation of the antioxidant activity
of Elicrisio in a finished cosmetic product, an emulsion O/W formed by
lipids of everyday use in cosmetic field easily sensible to oxidation. This
formulation has been monitored from the point of view of stability.
In a second
phase the antioxidant activity of the polar fraction of H stoechas in the
finished product has been valued, by “Rancimat” method.
On the basis of
the results obtained a strong antioxidant activity can be attribuited to this
fraction, superior to that conventional synthetic antioxidants, which
associated to the antiradical activity, previously showed in experimental
models in homogeneous phase and cell models, completes and exalts its radical-scavenger
profile.
Thus the aqueous
extract of H. stoechas can be considered a potential antioxidant for the use in
the cosmetic field having a strong activity, lower toxicity and better skin
tolerability with respect to the commonly used antioxidants.
CYCLE XV
Buonocore Anna
Thesis title: "Olfactory receptor and
fragrance chemistry"
Tutor: Prof.ssa Cecilia Anselmi
Co-tutor: Prof. Hanns Hatt
Academic year: 2006/2007
Board of examiners:
Prof.ssa Cecilia Anselmi (University of Siena)
Prof. Paolo Pelosi (University of Pisa)
Prof.ssa Luisa Pistelli (University of Pisa)
Prof. Hanns Hatt (Ruhr-University-Bochum, Germany)
Dott. Domenico Montanaro (Istitute of Clinical Physiology, CNR, Pisa)
Evaluation: excellent
Summary
This research
program is an investigation project on the olfactive perception which can help
us to know better the interaction between odorous molecules and human
olfactive receptor. It could represent the key to decode the “olfactive codex”.
So the aim of this work is to build a 3D model of the human olfactive receptor
OR 17-40.
The olfaction is
still one of the less known five senses. In the last years our knowledge of the
mechanism through which the olfactive stimuli are recognised and translated in
electrical signs, is increased considerably.
Odorous
molecules are non-ionic compounds with PM lower than 300. They are usually
organic hydrophobic compounds, which may contain a limited number of functional
groups. They are not only recognized but also discriminate by our olfaction
system.
Often the odour
of a molecule can be correlate to its “oriented profile”, that is to its
shape due to its functional groups.
Indeed it is
likely that it can establish an interaction, hydrogen bond or dipole-dipole,
with the olfactive receptor thus conferring a specific “fitting” at the
molecule within the receptor itself.
Owing to the
discovery of olfactive receptors and of their functional expression. The
modelling, of a specific interaction between odorous molecule and olfactive
receptor, has found an experimental confirmation and it isn’t just a theory.
The research in
this first year was then addressed to Molecular Modelling with the “aim” of
creating a tridimensional structure of OR 17-40 receptor.
The definition
of methods to predict the tridimensional structure of a protein from its
aminoacidic sequence, starts with the comparison of the new sequence with the
protein’s sequences already characterised. Several methods of sequences
comparison are available; they use several databank and standard
computational program of alignment of sequences have been set up.
For secondary
structure predictions I have used CLUSTAL W, an alignment program that proceeds
automatically.
At this point the
sequence to be modelled is aligned with the templates, the known 3D structures
filed in the database. The result is to obtain an unrefined 3D model of the new
sequence. The model obtained should be optimised; the energy minimisation
can be useful to optimise the molecular structure, trying to eliminate
eventual steric conflict and maintaining the bond length close to their optimal
values.
Docking was the
next step.
To predict the
structure of a complex, the program requires only the atomic co-ordinates of
the two molecules; it performs an exhaustive 6-dimensional search through the
relative translation and rotations of the molecules.
The docking
results is a list of high-score (low-energy) steric fit.
Docking predict
an odour-binding pocket on the extracellular surface.
Thus, it confirm
the theory in accordance with the interaction between olfactive receptor and
odorous molecule take place on the II and the V a-helics.
The model may be
therefore useful in predicting affinities for other ligands.
The OR 17-40
model will be helpful in generating insights as experiments on this and other
olfactory receptors proceeds.
Gregorio Michela
Thesis title: “Study and evaluation of complex matrix
in cosmetic field”
Tutor: Prof. Cecilia Anselmi
Academic year: 2003/2004
Board of examiners: Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Mario Tredici (University of Florence)
Prof. Paolo Pelosi (University of Pisa)
Evaluation: very positive
Summary
The research has
been addressed to the analysis of rheological and applicatory aspects relevant
to complex matrix in cosmetic field and in particular to the evaluation of the
bioadhesive effect produced and of the functional recovery.
The biomimetical
activation is carried out by topic application of a bioadhesive film of a
liposomal phospholipid dispersion.
The
reproducibility of the test is connected to the nature, and to the stability of
the liposomal dispersion and also to its technical applicative form.
In this first part
of the research, the attention has been addressed to the setting up of a method
able to value by tensiometric system the stability of liposomal phospholipid
dispersions in aqueous medium, in a special way the influence of technological
auxiliaries, with low vapour tension, on the formation of the liposomal
vesicles and on their time stability has been studied.
The research has
been carried out on dispersions of phosphatidyl choline pharmaceutical grade in
which ethyl alcohol is used as technological.
The dimensional
and tensiometric profile of these liposomal dispersions has been evaluated by
light scattering analysis and by tensiometric system with the method of the
pendent drop and of the contact corner.
The evaluation of
ethyl alcohol has been carried out on line during the preparation process by
gascromatography.
The results
obtained showed that low-vapour tension technological auxiliaries as ethyl
alcohol influence the liposomal formation process in the initial phase of
dispersion under turbo effect. With the evaporation of the technological
auxiliary the surface free energy of the system noticeably decreases till
reaching a stable value.
At the end of
the process the liposomal dispersion shows a high tensiometric affinity for the
skin which last also in the best preferred form.
These results
have been confirmed even after long-terms stability tests.
Simeoni Silvia
Thesis title: “The influence of complexation
with cyclodextrins on the photostability and percutaneous absorption of
sunscreens”
Tutor: Prof. Dionisio Mazzotta
Academic year 2003/2004
Board of examiners: Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Mario Tredici (University of Florence)
Prof. Paolo Pelosi (University of Pisa)
Evaluation: very positive
Summary
Cyclodextrins represent one of new systems for
the application on the skin of active substances of cosmetic and pharmaceutical
interest. The three-dimensional structure of these polymers give them the
capability to include inside their hydrophobic cavity, by non-covalent bonds,
lipophylic molecules of compatible dimensions (p.m. 100-400).
This association
process cyclodextrin-host, corresponds to a molecular encapsulation able to
protect the included substance from the action of external agents (oxygen,
radiations).This aspect is particularly important for the organic sun-filters
for which photostability represents one of the essential characteristic.
Studies
previously carried out showed the influence of complexation with cyclodextrins
on the photostability of butyl-metoxydibenzoylmethane and
2-ethyhehyl-p-metoxycinnamate filters.
Literature data
show that the photostability of pharmacologic and cosmetic molecules can be
improved through their clusion in cyclodextrines. The aim of the thesis was to
study the cyclodextrin complexation to improve the chemical and photochemical
stability of two most employed sunscreens: trans 2-ethylhexylmethoxycinnmate
(trans-EHMC) and butylmethoxy dibenzoyl methane (BM-DBM).
The results
obtained indicate that the cyclodextrin inclusion complexes are effective
systems to improve the sunscreen photostability. Also, EHCM have been
encapsulated in PLGA nanoparticles. Although the photostability is also
improved in this system, the preparation is long and difficult. Therefore, the
cyclodextrin complexation is a preferable method.
Regarding
BM-DBM, the inclusion complex with hydroxypropyl-b-cycldextrin(Hp-b-CD) improves the photostability and reduces the
formation of free radicals due to irradiation, thus minimizing the damage
on biologic macromolecules.
It is also
important for an efficient activity of the sunscreens that they are not
absorbed after topic application, but remain on the skin surface. Moreover, an
absorption could produce unwelcome effects at systemic level.
The absorption
of sunscreen/CD-complex through human skin has been evaluated. Studies showed
that Hp-b-CD complex
behaves as a free sunscren. In fact, the percentage of the sunscreen on
the epidermidis and on the stratum corneous is the same; the
sulphobutylether-7-cyclodextrine (SBE7-CD) reduces the percentage of
sunscreen on epidermidis but on the stratum corneous the average is unchanged.
Regarding the
Oxybenzone/ Hp-b-CD complex, it
produces a storage of sunscreen on the surface while the SBE7-CD complex
reduces the sunscreen amount at every levels. The results obtained show that
the CD inclusion complexes are useful to modulate percutaneous absorption.
Valentini Manuela*
Thesis title: “Sunscreens and radical
scavengers”
Tutor: Prof. Cecilia Anselmi
CYCLE XVI
Menatti Silvia *
Thesis
title: “Sun-protections and radical scavenger”
Tutor: Prof. Cecilia Anselmi
Dal Bosco Camilla
Thesis title:
"Evaluation of skin moisturizing and of its modulations by TVS Skin test
(CPn)"
Tutor: Prof.
Antonio Bettero
Academic
year: 2004/2005
Board of
examiners:
Prof. Alessandro Sega (University of Siena)
Prof.ssa Patrizia Chetoni (University of Pisa)
Prof. Maurizio Ricci (University of Perugia)
Evaluation: excellent
Summary
The discovery of
the skin fluid® as a biocompatible and complementary tensiometric reference
liquid for the contact angle method allowed to modify the Owen’s model and to
obtain the normalized energetical skin polar component (CPn) using
only water as standard liquid.
CPn evaluated in
different conditions showed subjective and objective different levels of the
epidermal functional state.
From these
results, to evaluate the correlations between (a) the functional epidermal
state, (b) the specific skin reactivity and (c) the CPn skin modulation,
formulative systems able to mime the biosintetic activity of the epidermic
granular cells were developed.
Combining a
biomimetic dispersion with a suitable areosol application system, the rapid
bioadhesive effect and the prolonged CPn skin modulation were
obtained.
By working in the
stability interval of CPn, the skin reactivity and the epidermal barrier
efficiency was then evaluated in different conditions.
Preliminary
results showed significative loss of the skin barrier efficiency, particularly
on females in dipendence of their age levels.
On the basis of
these experimental evidences, the relationship between CPn and moisturizing
level of the epidermal state was investigated with the aim to discriminate
between the polar contribution of moisturizing and other skin surface components.
CYCLE XVII
Granata Paola
Thesis title: "New trends for the prevention of
skin-aging"
Tutor: Prof.ssa Cecilia Anselmi
Academic year 2005/2006
Board of
examiners Prof.ssa Cecilia Anselmi (University of Siena)
Prof. Franco F. Vincieri
(University of Florence)
Prof. Maurizio Ricci (University of Perugia)
Evaluation: excellent
Summary
Recently it has
been demonstrated that a-b unsatured aldehydes and keto-aldehydes generated as
final products of lipid peroxidation following UV exposure, and/ or present in
the environment (i.e. acrolein) are involved in the photodegenerative
processes of the skin, since they have been immunoistochemically detected in
actinic elastosis.
Until now the
biochemical pathways for HNE metabolism/detoxification in epithelial skin
constituents (keratinocytes) have not yet been identified .
Since a)
keratinocytes are potential targets of lipid peroxidation products, generated
in the skin following UV exposure, b) 4-hydroxy-2-nonenal (HNE) is one of the
more representative and reactive a,b-unsaturated aldehydes, the first aim of
this study was to evaluate the cytotoxic effects of HNE in keratinocytes and
their ability to detoxify it, by studying the metabolic pathways involved in
HNE biotransformation.
In detail the study focussed on the following points:
1) A dose dependent study of cell viability/ integrity
in presence of HNE
2) Profiling of main metabolic pathways of the
HNE in cell cultures
3) Cytotoxic effect of HNE in the
absence/presence of UVB radiations
4) Effect of UVB on the biotrasformation of HNE
The results
concerning the incubation of keratinocytes with HNE (10-140 nmoles/mg protein)
can be summarised as follows: 1) cell viability and morphology are not
significantly modified in respect to control cells until 2 h of incubation with
HNE; 2) in cell cultures a progressive and time-dependent disappearance of HNE
is observed (HPLC-UV/DAD analysis); 3) beside reduced and oxidized HNE
metabolites, four GSH-HNE conjugated have been identified and
characterised in the extracellular medium by LC-ESI/MS/MS (no metabolites
inside the cell) [1].
These findings
indicate that HNE is able to permeate keratinocyte membrane and diffuse into
the cytosol to be transformed in highly hydrophilic and unreactive conjugated
adducts with GSH which subsequently are externalized from the cell since their
high polarity.
In the second
part of the study keratinocytes were exposed to a single minimal erythemal UVB
dose (50 mJ/cm2) in the presence and in the absence of HNE.
While UVB or HNE
alone did not modify cell viability, when keratinocytes were exposed to UVB (50
mJ/cm2) and then to HNE (70 nmoles/mg protein), a significant time-dependent
loss of cell viability was observed, viability that decreases to a 10% in
respect to the controls at the last observation time (8 h after exposure to
UVB).
The synergistic
toxic effect of UVB/HNE may be due to a significant decrease of intracellular
GSH (25% less versus controls) [2] which causes a dramatic metabolic impairment
of keratinocytes for HNE metabolization. This assumption is confirmed by the
fact the extrusion in the incubation medium of the 4 GSH conjugates is reduced
by 15-25% (HPLC/MS analysis).
On the basis of
these results we can conclude that keratinocytes are able to
detoxify/metabolize a-b unsaturated aldehydes through a GSH-dependent pathway,
and that the process is extremely susceptible to oxidants, since it can be
impaired by a minimal erythemal UVB dose (1 MED).
When a significative
consumption of GSH takes place (UVB exposure), the cell becomes prone to the
attack of unmetabolized HNE; this last by reacting with bioactive
macromolecules (nucleic acids, proteins, enzymes) leads to necrotic cell death.
The results of
this study provide a rationale for the design of new cosmetic ingredients for
the prevention of skin photoaging, i.e compounds able to quench a-b unsaturated
aldehydes by mimicking the preferential sites of addition of HNE in the skin
proteins.
Carnosine
(b-alanine-L-histidine), an endogenous dipeptide present at mM concentrations
in skeletal and celebral muscle tissue, is one of these: when added to
incubation medium (20 mM) is able to prevent the synergistic toxic effect of
UVB exposure through a specific entrapping mechanism.
[1] Carini M. et al. 22th IFSCC Congress,
Edinburgh, 2002.
[2] Carini M. et al.Il Farmaco,55; 526-34, 2000.
Pulsinelli Emy
Thesis title: "Synthesis and
biological activity of ferulic acid derivatives"
Tutor: Prof.ssa Cecilia Anselmi
Academic year: 2008/2009
Board of examiners:
Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Roberto Maffei Facino
(University of Milan)
Prof. Maurizio Ricci (University
of Perugia)
Evaluation: completely deserving
Summary
Ferulic acid
occurs in rice, wheat, barley, oat, sorghum, forage, tomatoes, asparagus,
olives, peas, citrus fruits and leaves and in many other plants. Much of the
ferulic acid occurs as esters in many plants. In seeds, ferulic acid is
generally localized in the bran fraction.
Rice is one of
the three major cereals harvested by man. Brown rice is polished before eating
and 10% of its weight is discharged as rice bran. In the process of rice
salad oil extraction, waste materials are discharged as rice bran pitch, a
blackish brown waste oil with high viscosity. Ferulic acid is easily prepared
in large quantities from rice bran pitch.
In the cosmetic
field, ferulic acid was employed as UV filter, antioxidant and radical
scavenging natural agent. Unfortunately its sunscreen and antioxidant
activities are not enough high: this could be due to ferulic acid degradation
when it is exposed to air and light effects for long time.
Photochemical
stability of UV filters is important in order to retain the photoprotective
power and to reduce phototoxic and photoallergic reactions.
With the aim of
obtaining new functional cosmetic substances, we have synthesized ferulic acid
esters and we have evaluated their biological activity. Ferulic acid
derivatives have shown better antioxidant and UV filter properties and less
thermic and photochemical degradation than ferulic acid.
The research
work was articulated on esters synthesis, starting from ferulic acid with
linear and branched alkylic chain alcools, on conformational analysis, in order
to obtain tridimensional structure of each derivative, and on biological
activity study, in order to correlate the biological structure-activity
relationship.
Conformational
analysis was carried out by NMR spectroscopy and molecular modelling.
The antiradical
activity was investigated by two different methods: CumOOH Induced hemolysis
(CumOOH) and Thiobarbituric Acid Assay for Malondialdehyde (TBARS).
The first method
consists in inducing hemolysis of an erythrocyte suspension with CumOOH. The
choice of erythrocytes as model study for radical scavenging activity is based
on the fact that this cellular system presents suitable structural (high
content of polyunsaturated fatty acids) and functional (they contain
hemoglobin, potential and strong promoter of oxidation processes)
characteristics. Moreover, the membrane contains cytoskeleton proteins that,
intercalating with lipids and phospholipids, create the typical flexible
structure of the erythrocyte.
The hemolytic process
is modulated by two factors: 1) a process of lipoperoxidation on the
phospholipid bilayer, 2) a process linked to the oxidation and degradation of
cytoskeleton proteins.
The second
method consists in evaluating the lipid peroxidation on microsomes systems
obtained from rat liver. Microsomes are another source of phospholipids with
high polyunsaturated fatty acids content. Malondialdehyde is one of the
products of this lipid peroxidation which appears to be produced in relatively
constant proportion to lipid peroxidation. It is a good indicator of the rate
of lipid peroxidation, expecially in vitro.
Both methods
were used to evaluate ferulic acid activity; all derivatives were studied with
TBARS method and only carbon even number alkylic chain esters have been the
subject of investigation on erythrocyte (CumOOH).
We demonstrated
that the conformation of the molecule plays a fundamental role on the
biological activity. Branched chain molecules are less active than linear chain
isomers and the activity shows peak values for molecules with twelve and
thirteen carbon atoms alkylic chain. Conformational analysis point out that in
such molecules the alkyl chain is folded on itself, while in all other esters
alkyl chain is folded towards the aromatic ring.
In conclusion,
conformational analysis by NMR spectroscopy and molecular modelling may be a
good investigation method for understanding the structure-activity relationship
of radical scavenging molecules and furthermore, the research pointed out the
great potentiality of ferulic acid and its derivatives.
CYCLE XVIII
Borgogni Barbara
Thesis title: "Characterization and evaluation of
Saturnia SPA thermal plancton: standardizzation of application processes".
Tutor: Prof.ssa Cecilia Anselmi
Academic year: 2008/2009
Board of examiners:
Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Roberto Maffei Facino
(University of Milan)
Prof. Maurizio Ricci (University
of Perugia)
Evaluation: completely deserving
Summary
The research
activity was focused on botanical and chemical studies of algal material
collected at the Saturnia SPA, with the aim to find objective parameters for
the characterization and evaluation of bioglea and its possible application.
Bioglea is the
common definition of the thermal benthos composed of biogenic agents.
The
Saturnia SPA bioglea (BiogleaTM) is mainly composed by
cyanobacteria, a pool of oxygenic photosynthetic Gram-negative prokaryote. In
the last ten years the study of microrganisms which live in extreme conditions
has been increased, just because the estreme condition adaptation could have
allowed them to the evolution of characteristic qualities which could be
utilized in biotechnologic field (for example thermoresistent enzymes, new
bioactive compounds to use in thermal and pharmaceutical field). Studies were
carried out with the aim to characterize microbial population living in thermal
habitat, in particular the bioglea which grows up either in the thermal pool
(characterized by a natural “crater” in which flows sulphureous thermal water
at 37°C) and on mud during the maturation phase.
Studies
were carried out on samples collected at Saturnia SPA in several period of the
year; also they have been repeated during the same period in following years.
Samples
were observed by microscopy, lyophilised and extracted with solvents of
different polarity to evaluate their bioactivity. The obtained fractions were
analysed by HPLC and GC-MS. The gas-chromatography coupled to mass spectroscopy
allowed to identifie some components of bioglea and its extracts.
Saturated
and unsaturated fatty acids, (from C6-C18), were identified. Compounds with odd
number of carbon atoms are also present: saturated C15 and C17, and unsaturated
C9. The lipophilic fraction obtained from bioglea is very similar to that
observed in Saturnia SPA thermal muds collected monthly, extracted with the
same method of bioglea and analysed by GC-MS.
DPPH
and ORAC tests were performed on bioglea and its extracts to evaluate radical
scavenging activity. In particular, the ORAC data showed that bioglea is more
active than its components. Regarding lipophilic extracts (petroleum ether and
chloroform), the results obtained showed a dose-dependent effect and an highest
activity in chloroform extract as we observed using DPPH test.
The results
suggest that bioglea could represent an important source of bioactive
ingredients useful in dermocosmetological field.
Beretta Giangiacomo
Thesis title:
"Peptides in cosmetic technology: efficacy and evaluation"
Tutor: Prof.ssa Marisanna Centini
Co-tutor: Prof. Giancarlo Aldini
Academic
year: 2006/2007
Board of
examiners:
Prof.ssa Cecilia Anselmi (University of Siena)
Prof. Paolo Pelosi (University of Pisa)
Prof.ssa Luisa Pistelli (University of Pisa)
Prof. Hanns
Hatt (Ruhr-University-Bochum, Germany)
Dott. Domenico Montanaro (Istitute of Clinical Physiology, CNR, Pisa)
Evaluation:
excellent
Summary
Recent evidences
indicate the involvement of oxidative stress deriving from sunlight exposure,
pollution and aging in the production of advanced lipoperoxidation end-products
(ALEs) in body-tissues.
The ALEs are
mainly aldehyde compounds, arising by the oxidative degradation of the mono and
polyunsatured fatty acids (PUFAs, i.e. oleic, linoleic and arachidonic acids)
found in the skin.
Recently,
acrolein, a 3-C a,b-unsatured aldehyde, has been identified
as PUFAs degradation product. Acrolein is also characterized by a marked
electrophilic reactivity with respect to all the other aldehydes already
reported.
Carnosine (b-alanyl-L-histidine)
is the first molecule to which was attributed an aldehyde-sequestering ability.
Carnosine is alredy used by some cosmetics companies claimig its anti-carbonyl
(and then anti-aging) activity.
Aim of this work
was to study the interaction between acrolein and carnosine through an HPLC and
ESI-MS approach, in order to identify the molecular factors governing the
aldehyde sequestering activity of Carnosine.
The HPLC data
show that the additive interaction between carnosine and acrolein (the overall
activity of the dipeptide is almost identical to that of the sum of the
constitutive aminoacids. The ESI-MS and ESI-MS/MS data indicate a complex reaction
pathway involving several itermediates and final reaction productscontaining up
to 3 quenched acrolein molecules.
The results of
this study provides new and useful indication in order todesign new
anticarbonylation active molecules, with higher activity, wide range af action
and additional activities such as antioxidant and anti-metalprotease activity.
Miraglia Giovanna
Thesis title: "New technologies in cosmetic
products: studies and applications""
Tutor: Prof.ssa Cecilia Anselmi
Cotutor: Dott.ssa Marisanna Centini
Academic year: 2008/2009
Board of examiners:
Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Roberto Maffei Facino
(University of Milan)
Prof. Maurizio Ricci (University
of Perugia)
Evaluation: completely deserving
Summary
In the last 15
years, scientific research has developed several delivery systems most of
which, even though widely used in the pharmaceutical field, have found a
position also in the cosmetic field and are called “Novel Cosmetic Delivery
Systems”.
Among vescicular
systems, the most used are:
LIPOSOMES
MICROPARTICLES
SOLID LIPID NANOPARTICLES (SLN)
CYCLODEXTRINS
The aim of this
thesis, since photoprotection is very important, is to find a new formulated
system for sunscreens, better than traditional formulations.
The inclusion of
sunscreens in particle systems is a new and promising approach in
photoprotection, because it guarantees increased safety and efficacy.
The positive effects
of these new innovative technologies are an advantage both for the skin, which
isn’t in contact with the sunscreen, meaning reduced irritation and allergy
risk, and the filter itself, which is stable to irradiation.
Other advantages
are the best formulate characteristics: dispersion of encapsulated
sunscreen also in aqueous lotion, capability to improve the distribution on the
skin of the product or combine UVA and UVB sun filters in a single preparation.
Another important aspect could be to utilize the substantivity of some
polymeric matrices that provide prolonged permanency of the sunscreen on the
skin and resistance to water and perspiration. To this aim, we have studied
liposomes and microspheres containing Ferulic Acid and its ester derivatives.
The active compound chosen is a natural acid endowed with antioxidant, radical
scavenging and UV sunscreening activity.
Emulsion/solvent
evaporation is the technique used to prepare microspheres; after, the loading
of active compounds has been evaluated using UV spectrophotometric analysis,
while the morphological analysis has been performed using SEM.
To prepare
microspheres, we used two different polymers: polymer 1 produces microspheres
containing Ferulic Acid inside but also outside due to ionic interaction. Best
yield and loading is obtained using dodecyl ester.
Polymer 2
produces best results from a morphological and dimensional point of view, but
yield and loading don’t come up to our expectations, so the work is going on to
obtain better results.
To prepare
liposomes we used the film technique, the active loading has been evaluated
using UV spectrophotometric analysis and the morphological analysis has been
performed using TEM. It was possible to evaluate also the antioxidant activity
with DPPH and TBARS method .Also in this case, test results, both for loading
and morphology are obtained using the alkylferulates(C8-C11-C12-C13-C15
e C16) and DPSC (distearoylphosphatidylcholine).
TBARS test, to
evaluate the radical scavenging activity, showed that the C12 and C13 esters
are the most active, both alone and in liposome dispersion. Even after 24 h we
obtained a decrease of IC50 compared to values obtained after 15 min.,
indicating the delivery of the active ingredients.
The research was
carried on studying the preparation of microspheres containing commercial
sunscreens:
4-methylbenzilidene
camphor, Octylmethoxycinnamate, Butyl methoxydibenzoylmethane,
Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine, using a polymer which is
substantive for the skin.
For each of them
we have characterized the operating conditions (solvent, stirring, T….) to
obtain microspheres with desired shape, morphology and loading.
CYCLE XIX
Maggiore Maria
Thesis title: "Complexing agents in cosmetic products.
Technologies and evaluation"
Tutor: Prof.ssa Cecilia Anselmi
Academic
year: 2007/2008
Board of
examiners:
Prof.ssa Cecilia Anselmi (University of Siena)
Prof.ssa Franca Pavanetto (University of Pavia)
Prof.ssa Antonia Sacchi (University of Naples)
Evaluation:
completely deserving
Summary
Ferulic acid
(3-methoxy-4-hydroxycinnamic acid) occurs in seeds (cells or wall) of many
plant like rice, wheat, tomatoes, asparagus, olives, peas and leaves of citrus
as trans isomer. Much of the ferulic acid occurs as ester and in seeds it is
generally located in the bran.
In the cosmetic
field, ferulic acid is employed as UV filter, antioxidant and radical
scavenger. This product, listed in Japan as "positive" UV absorber, unfortunately
is not easy to use in cosmetic products, in fact in the long time it shows
functional and organoleptic modifications. This could be due to its degradation
when it is exposed to air, light and heat.
Recently,
cyclodextrins have been widely applied in the cosmetic field (fragrances,
deodorants……) and also their application in sunscreen products results very
interesting.
To this aim a
study on ferulic acid inclusion complexes preparation, with various type of
cyclodextrins, has been started.
First, g-cyclodextrin (g-CD) inclusion complex was obtained by
co-precipitation method and was characterized by DSC, XDR, 1H-NMR
and Molecular Modeling.
DSC and X-ray
diffractometric experiments give strong support to the formation of an
association complex. NMR analyses and modeling data demonstrate that ferulic
acid is embedded inside the cavity of g-CD
with the aromatic and ethylenic moieties, leaving outside the active phenolic
residue, responsible for the radical scavenging activity.
The ferulic
acid/g-CD inclusion
complex antioxidant activity, evaluated by ORAC assay, was very similar to that
of ferulic acid alone.
The g-CD complex improves emulsions photostability
evaluated through the SPF determination before and after UV irradiation
performed with a Solar Simulator with a Xenon Arc Lamp.
Picciocchi
Giuliana
Thesis title:
"Olive oil derivatives in cosmetic products"
Tutor: Dott.ssa Marisanna Centini
Summary
The concept of ecology
and the return to nature addresses research on natural compounds . Also in the
cosmetic field, research is focusing its attention on several products which
mother nature offers us either as cosmetic raw materials or as active
ingredients.
Several reasons
suggest to study olive oil and the by-products of oil refining: olive oil
is one of the plants most common in the Mediterranean basin, it has a very high
symbolic value, its leaves and fruits are very rich in active compounds that
preserve the tree by external agents and the attack of parasites.
The aim of this
study during my first year was to determine polyphenols in waste water in
sample previously purified from S. Gimignano’s oil mill.
The
concentration has been done using the Folin-Ciocalteau method.
On waste water
samples with the highest amount of polyphenols we have evaluated:
- the
concentration of oleuropein (a polyphenol presents in high content, it has antioxidant
activity higher than vitamin E but also antibacterial, antifungal,
hypoglycaemic activity) and hydroxytyrosol (an antioxidant polyphenol, derived
from enzymatic degradation of oleuropein) using HPLC analysis;
- evaluation
of radical scavenging activity using DPPH method:
- evaluation
of antioxidant activity using ORAC assay.
Together with
the study regarding the recovery of active compounds from olive oil waste
water, which is important for the recycling of compounds with high value added,
the ex-novo synthesis of a new surfactant containing olive oil fatty acids has
been started.
Scarpelli Lisa
Thesis title:
"New molecules for sun protection".
Tutor: prof.
Alessandro Sega
Summary
In recent years,
there has been an increase of interest about natural products showing UV-A
screening activity as a result of both changes due to exposition to UV
radiation, and a progressive increase in the number of people affected by skin
cancers. It is therefore necessary to devise new sunscreens in order to prevent
skin cancer and photo-aging. To this aim a new synthesis of a natural product
endowed with UV-A filtering activity1 has been developed. This products
extracted from the seaweed Aspergillus Terreus is known with the name of
Terreusinone (1).
The retro-synthetic scheme (scheme 1) goes back to two
synthetic equivalents:
- product of the reduction of an
amino acid (2)2
- 1,4-benzoquinone (3),
commercially available
Schema 1. Structural
formulas of 1, 2 e 3.
Coupling between
quinone 3 and amino alcohol 2 could be carried out
following a synthetic procedure known in the literature3:
1,4-benzoquinone was dissolved in ethanol containing 3-amino-propan-1-ol,
the reaction was stirred, in the dark, for four days at room temperature.
Purification was carried out by flash chromatography.
The chosen
starting material was L-leucinol, reduction product of L-leucine (commercially
available), because it was a structural analogue of the synthetic equivalent 2.
The next step
was the palladium-catalyzed cyclization of the functionalized quinones
previously obtained; the reaction was carried out following a synthetic
procedure known in the literature4. The palladium-source was Pd0(PPh3)3
(whose synthesis is reported in the literature)5.
The oxidant agent was MesBr.
The cyclization didn’t afford the desired bis-indolic product, but it
yielded the mono-indolic product.
These results suggested that a possible explanation for the failure of
the reaction could be related to the presence of NH groups on the starting
functionalized quinones.
So the conversion of NH groups in tert-butoxycarbammate was made.
The ciclysation
was made on the new synthesised compounds, but the mono-indolic products were
obtained again.
These new
results were rationalized in this way: the recurring failure of the
palladium-catalysed cyclization could be due to the formation of an
electron-rich indolic-system, which inactivates the system to following
cyclizations.
This leads to a
new synthetic scheme:
- oxidation of the alcohol to aldehyde
- palladium-catalysed cyclization
The oxidation
was carried out, following the procedure developed by Swern, on the N-tert-butoxycarbammate
protected products.
The next step, the
palladium-catalysed cyclization, yielded again the mono-indolic-products.
These results
can be possibly explained postulating that the failure of the
palladium-cyclization could be related to the formation of the electron-rich
mono-indolic system produced in the palladium-catalysed reaction.
New synthetic
pathway will be carried out and monoindolic system will be evaluated.
REFERENCES:
· Tet. Lett., 2003, 44, 7707-7710
· 2 JOC, 1999, 64, 9450-9458
- 3 J. Chem. Soc., 1970, 2078-2082
- 4 Tet. Lett., 1996, 37, 9203-9206
·
5 Hegedus, L. S. John Wiley & Sons, ed. 1994.
CYCLE XX
Bernardi Antonietta
Thesis title: "Multiple emulsions: formulation,
stability and cosmetic application".
Tutor: Prof.ssa Cecilia Anselmi
Academic
year: 2007/2008
Board of
examiners:
Prof.ssa Cecilia Anselmi (University of Siena)
Prof.ssa Franca Pavanetto (University of Pavia)
Prof.ssa Antonia Sacchi (University of Naples)
Evaluation:
completely deserving
Summary
The aim of my thesis is the study
of emulsion systems such as multiple emulsions in cosmetic products. Multiple
emulsions are complex and metastable systems. The droplets of the dispersed
phase contain even smaller dispersed droplets themselves. Each dispersed
globule in the double emulsion forms a vesicular structure with single or
multiple aqueous compartments separated from the aqueous phase by a layer of
oil phase compartments. Multiple emulsions can be potentially used in
pharmaceutical drug delivery, foods and cosmetics. W/O/W emulsions present many
interesting possibilities for the controlled release of drugs and cosmetics.
They can entrap substances in the inner aqueous phase, protect them from
degradation and modulate their release rate to improve product efficacy. It has
been shown that this release depends on various mechanisms. The composition of
multiple emulsions is of significant importance since the different
surfactants, along with the nature and concentration of the oil phase, will
affect the stability of the double emulsion. Much research has been done on the
nature of oils in relation to the selected emulsifiers and their influence on
the manufacturing conditions, as well as on the stability of the double
emulsion. The
emulsifiers have been found to migrate from one interface to the other and
alter the interfacial emulsifier organization. It seems that the stability could
be improved by increasing the lipophilic surfactant concentration, which could
strengthen the interfacial film. Several methods have been developed for
choosing a surfactant or mixture of surfactants in order to prepare an emulsion
of the desired type. The first method was proposed in 1949 by Griffin, who
introduced the Hydrophilic-Lipophilic Balance (HLB) number. However, this
method does not account for the effect of other formulations variables or
temperature, and is often very inaccurate [1]. Winsor’s R ratio of
interaction energies is an extremely useful theoretical concept which helps to
grasp the basic notions, thought it is not amenable to quantification [2]. In
the mid 1970s, the industrial and academic research effort targeted at
petroleum-enhanced recovery techniques by surfactant flooding resulted in
empirical correlations for attaining ultra low interfacial tension [3-5].
The correlation
for so-called ‘optimum’ formulation was found to represent the relative
contributions of the different formulation variables to a global generalized
formulation concept [6]. It was related to the free energy of transfer of a
surfactant molecule from oil to water also called the surfactant affinity
difference (SAD), ie the difference between the standard chemical potentials of
the surfactant in the oil and water phase
(1)
where Kp
is the partition coefficient of the surfactant between water and oil at a given
temperature (Co/Cw), which is experimentally attainable. The value of the
partition coefficient at optimum formulation, when the surfactant–oil–water
system exhibits three-phase behaviour, is taken as a reference (KpRef)
and the corresponding SAD value, which is called SADRef, is often
close to zero, particularly in ionic surfactant systems—although not always, as
for instance with ethoxylated non-ionics. The deviation of dimensionless SAD from
its reference has been called the hydrophilic-lipophilic deviation, HLD
[8].
(2)
HLD measures the
free energy driving force for change from the balanced ‘optimum’ formulation
state at which three-phase behaviour is exhibited.
Two important release
mechanism are often cited in the literature: the diffusion through the oily
barrier and the globules bursting that occurs either by dilution in a
hypo-osmotic solution (with regards to the internal aqueous phase).
Two formulations were prepared with different lipophilic surfactants at
different concentrations and the oily phase of each formulation has a different
value of ACN.
Tests carried
out on emulsions were microscopic analysis and macroscopic analysis: The
main properties of all formulations evaluated just after processing were very
similar. Macroscopically all the emulsions were white and homogeneous creams
with good consistency; stability studies; rheological analysis; conductometric
analysis: The analysis was carried out to measure directly the release of
electrolyte entrapped in the internal aqueous phase. The measurements
were made at 20 ± 1 °C on the diluited multiple emulsions sample under
magnetic stirring with a conductimeter (Crison). The weight fraction, b(t)
of electrolyte release into the external aqueous phase at a given time t can be
calculated from:
b = Mt/M0
(3)
where M0
is the initial incorporated mass and Mt is the mass present in
the external phase at a given time t.
Stability of
emulsions, size of aqueous and oily globules, and release mechanisms all change
according to oil and lipophilic surfactant and concentration of the latter.
Interfacial analysis was carried out on different pairs of oils and surfactants
in order to select the better system.
References:
[1] Salager JL,
Antón RE, Bricen o MI,
Choplin L, Márquez L, Pizzino A, Rodriguez M P. The emergence of formulation
engineering in emulsion making-transferring know-how from research laboratory
to plant. Polymer International 52:471-478 (2003).
[2] Winsor P A,
Solvent properties of amphiphilic compounds, Butterworth, London (1954).
Shah DO and
Schechter RS (Eds), Improved Oil Recovery by Surfactant and Polymer Flooding,
Academic Press, New York (1977).
[3] Shah DO and
Schechter RS (Eds), Improved Oil Recovery by Surfactant and Polymer Flooding,
Academic Press, New York (1977).
[4] Johansen RT
and Berg RL (Eds), Chemistry of Oil Recovery, ACS Symposium Series Nr 91,
American Chemical Society, Washington, DC (1979).
[5] Shah DO (Ed),
Surface Phenomena in Enhanced Oil Recovery, Plenum Press, New York (1981).
[6] Bourrel Mand
Schechter RS, Microemulsions and Related Systems, M Dekker, New York (1988).
[7] Marquez N,
Antón RE, Graciaa A, Lachaise J and Salager JL, Partitioning of ethoxylated
alkylphenol surfactants in microemulsion–oil–water systems. Colloids Surf A 100:225
(1995).
[8] Salager JL,
Márquez N, Graciaa A and Lachaise J, Partitioning of ethoxylated octylphenol
surfactants in microemulsion–oil–water systems. Influence of temperature and
relation between partitioning coefficient and physicochemical formulation.
Langmuir 16:553 (2000).
Lupia Simona
Thesis title:
"Delivery system for cosmetic actives".
Tutor: Dott.ssa Marisanna Centini
Summary
Thesis title: Release
systems for active compounds in cosmetics
The aim of this
thesis is to study delivery systems for active compounds in cosmetics. During
the first year, the research was focused on a formulation allowing
hair semipermanent or direct dyeing, using microencapsulated direct dyes, to
obtain a higher dye stability and light and heat resistance.
Dyes are
substances of various nature which may be added to cosmetics both for
decorative and corrector aims.
Dyes allowed
under Italian law are indicated, according to Colour Index, in the IV Annex of
713/86 law and are divided into four application fields according to
their toxicological and physical characteristics.
Dyes are
classified as:
1) Dyes
used for all cosmetics
2) Dyes
not allowed in all cosmetics except those dedicated to be applied near eyes
(for make-up and removal of make-up)
3) Dyes
allowed exclusively in cosmetics, that do not come in contact with
mucosae
4) Dyes allowed
exclusively in cosmetics coming in short contact with the skin
Regarding
hair dyes, they can be distinguished into three categories:
1) Oxidation
dyes ( or permanent)
2) Direct
dyes ( or semipermanent)
3) Temporary
dyes
This classification
is based on the stability and period of dyeing, the quality of results and the
tone variety.
Today,
most used dyes are nitroanilines, nitrophenylendiamines,
nitroaminophenols, azoquinone and anthraquinone derivatives.
The
presence of a nitro- group, being a chromophore, gives to the molecule
its dye characteristic.
The
dye employed for this research is: HC BLUE No.2
(2,2’-[4-(2-hydroxyethylamino)-3-nitrophenylimino], whose max. conc. used is
2.8% (Annex III of 713/86 Italian law and subsequent amendments). This dye has
been encapsulated in microparticle prepared with emulsification /solvent
evaporation. Spectrophotometric (to evaluate the loading), morphological
(SEM) and optical microscopic analyses ( to evaluate the size range and
morphology) were carried out.
First,
O/W emulsions have been prepared obtaining a low loading percentage. Further
experiments and improved technology afforded microspheres with 25%
loading.
Pisani Chiara
Thesis title:
"Development of strategy and protocol for cosmetic products
evaluation".
Tutor: Prof.
Lucio Andreassi
Academic year: 2008/2009
Board of examiners:
Prof.ssa Cecilia Anselmi
(University of Siena)
Prof. Roberto Maffei Facino
(University of Milan)
Prof. Maurizio Ricci (University
of Perugia)
Evaluation: completely deserving
Summary
Safety is a
fundamental requirement of cosmetic products. It is clearly recalled by the
national laws and European guidelines concerning the production and marketing
of such products, with rules sanctioning that cosmetics must be produced,
manufactured and commercialised in such way not to cause damages to human
health in current conditions of usage. Cosmetic industry is able to certify the
safety of the products by means of numerous tests: for some procedures the
employment of laboratory animals is required.
A recent
Guideline of the European Parliament and the Council (Directive 2003/15)
sanctioned that cosmetic products cannot be tested on animals. In particular,
the production and marketing will be banned after 2009 in all the Countries of
the European Union for the ingredients and products containing ingredients
tested on animals, in any country they have been produced. For ingredients
requiring tests on toxic-kinetic, reproduction toxicology and toxicity to
repeated doses, the prohibition will be actual after 2013 with possibly further
extension of deadline if alternative tests will not be developed.
Therefore, in
the last years much effort to look for alternative tests has been done.
Several in-vitro
models, which are currently used in many fields of scientific research, have
been developed. However at the present moment we do not know if they can
replace the in-vivo tests. Validation, directed to assess the reliability, in
terms of reproduction in different laboratories as well as the usefulness for
the prefixed aim, is a rather long process and can require more than ten years.
Cell cultures of human skin allow
to accomplish promising systems. Their cultivation was developed by Rheinwald e
Green (1975), who were able to obtain large amount of human keratinocytes
starting from small skin fragments. Using this method keratinocytes can
proliferate in cultures reproducing human epidermis with architecture similar
to the epidermis in vivo. A further evolution of this technique has allowed the
achievement of skin equivalents, composed of two layers, epidermis and dermis.
Two different kinds of reconstituted skin equivalents are available: a)
epidermal equivalents consisting of multi-layered differentiating human
keratinocytes grown on different synthetic matrices; b) full skin equivalents
with multi-layered, differentiating human keratinocytes grown on collagen
matrices containing fibroblasts. When such cultures are elevated at the
air-medium interface the keratinocytes of both systems develop an organized
stratum corneum, which resemble a functional barrier, but only the
keratinocytes-fibroblasts interaction induce the development of the
epidermal-dermal junction.
These composed
organotypic cultures can therefore be employed for the study of products to
apply on the skin. Really they allow a contact with single substances or with
finished products in experimental conditions similar to the ones in-vivo.
The observation of the cultures in such a way stimulated can provide elements
to evaluate the entity and nature of the phenomena after the contact with the
examined substance.
Recent studies have demonstrated that the keratinocytes are not only the
passive target of the substances that come in contact with the skin, but are
able to produce substances, the cytokines, whose delivery is thought expression
of inflammation. Therefore, the study of the culture alterations can be
integrated with the dosage in the liquid of culture of the interluekines that
are expression of the inflammation of the tissue.
After all, given
the relative facility to obtain the cultures of keratinocytes (various
companies are able to provide reconstituted human epidermis in vitro as well as
skin equivalents) it can be assumed that such cultures can replace the animals
for the toxicological studies of the cosmetic products. The organoid cultures
that are constituted of keratinocytes grown on dermal matrices seem the more
reliable model.
In collaboration
with the Skin Bank of the Department of Clinical Medicine and Immunological
Sciences – Section of Dermatological Sciences of Siena University, several
experiences have been carried out to define the best models and conditions for
the study of toxicity and activity of cosmetic products. Skin equivalents
composed of keratinocytes grown on derma-equivalents appeared a reliable system
for the study of products, whereas cultures of keratinocytes as such seem
better for the study of ingredients.
Valeria Quaranta
Thesis title:
"New technologies in photoprotection".
Tutor: Prof.ssa Cecilia Anselmi
Summary
Negative effects
of UVA radiations, believed inoffensive for long time, have been now
documented. These radiations cause cutaneous ageing and, although they
have lower energy than UV-B rays, UV-A can participate in the development
of cutaneous pathologies, because they can penetrate to the deeper layers
of the skin, where they can induce mutations in cells that are inactive replication.
They are particularly dangerous when the skin is exposed for long periods and
particularly because of the absence of physiological warning, such as the
erythema induced by UV-B rays.
Therefore,
modern sunscreens should provide protection from both UV-B and UV-A radiations
and also maintain this protection over the entire period of sun exposure.
The most common
UV-B and UV-A sunscreens are Ethylhexylmethoxycinnamate and Butyl
mehoxydibenzoyl methane.
In this PhD
thesis, we tried to find an emulsion system that could provide protection from
both UV-B and UV-A radiations and at the same time, would be endowed with an
high sunscreen photostability to ensure both efficacy and safety in
photoprotection.(the sunscreens degradation products could be toxic and
photoallergenic; also they can loose the photoprotection efficacy).
These aims were
reached including the two UV-B and UV-A sunscreens in particulate
systems, first separately and then in mixture. Microspheres, systems in which
the actives components are uniformly dispersed in a polymeric matrix,
were prepared using the emulsification/solvent evaporation technique and at
different percentages of sunscreens. Morphological (SEM),
spectrophotometric (to evaluate the loading) and dimensional analysis were carried
out.
Emulsions
containing sunscreens encapsulated in microspheres were prepared.
Spectrophotometric analysis and SPF were carried out. The photostability
of Butyl mehoxydibenzoyl methane in the microspheres was
investigated through Differential Scanning Calorimetry considering its phase
characteristics therein.
In order to
evaluate the substantivity of the sunscreens in the epidermidis, the “water
resistance test” using microspheres dispersed both in
hydroxyethylcellulose hydrogel and emulsions, according to FDA protocol,
was carried out.
CYCLE XXI
Batmangheligi-Fard
Mandana*
Thesis title:
"Safety in photoprotection"
Tutor: Prof.ssa Cecilia Anselmi
Nikolic Sasa
Thesis title:
"Formulating for efficacy"
Tutor: Prof.ssa Cecilia Anselmi
Sbano Paolo
Thesis title:
"Experimental model for the study of sin regeneration with the use of
mesenchymal stem cells"
Tutor: Prof. Lucio Andreassi
Staltari Lucia
Thesis title:
"Supramolecular and particulate systems"
Tutor: Prof.ssa Cecilia Anselmi
Stefanoni Sara
Thesis title:
"New multiacitve ingredients"
Tutor: Prof.ssa Cecilia Anselmi
CYCLE XXII
Tosi
Azzurra
Thesis title:
"Lipophilic cosmetic delivery systems"
Tutor: Prof.ssa Cecilia Anselmi
CYCLE XXIII
Khalil Mitri
Thesis title:
"Particulate systems for cosmetic applications"
Tutor: Prof.ssa Marisanna Centini
Ibrahim
Hanno
Thesis title: "Improving solar protection”
Tutor: Prof.ssa Cecilia Anselmi
CYCLE XXIV
Cristiane
Andrade Rocha
Tutor: Prof.ssa Cecilia Anselmi
Emad Hanno
Tutor: Prof.ssa Marisanna Centini
Maria Sole
Rossato
Tutor: Prof. Alessandro Sega
INFORMATION ON
THE INTERNATIONAL ACTIVITIES
COOPERATION
AGREEMENT FOR THE ESTABLISHMENT OF INTERNATIONAL RECIPROCITY LINKS WITH THE
RESEARCH DOCTORATE IN “COSMETIC SCIENCE AND TECHNOLOGY”
THE PARTIES
University of Siena
Dipartimento Farmaco Chimico tecnologico
Faculty of
Pharmacy
University of
Bochum
Dept. Cell
Physiology
Faculty of
Biology
PREAMBLE:
- the
parties agree on the fundamental importance of promoting inter-university
cooperation for the development of educational and scientific post-graduate
programmes;
- the
parties have already been collaboration on a scientific and educational level
for some time, with positive results;
- the
agreement on cooperation has been formulated in order to foster both
specialised learning and professional understanding for postgraduates;
AGREE
To sign
this agreement, which will remain valid until and unless one of the parties
will declare it expired, this to be done before October 1 of the year
preceding the year when such expiration will take place.
Art. 1
Objectives
This agreement is aimed at formalising the research
and teaching activities that the Doctoral programme on “Cosmetic Science and
Technology” , based in the Faculty of Pharmacy, University of Siena, shares of
University of Bochum.
The cooperation aims to further in high-quality
sectors the specific research interests of the doctoral programme in “Cosmetic
Science and Technology”. The scientific objectives of the programme is to
inaugurate new disciplinary sectors in an emerging field of study, and promote
the intersection of diverse research areas, such as human olfactory receptors,
odorous molecules, chemoreception, psychophysic effects of odours, odour
antagonists, molecular modelling.
The cooperation programme offers to both students and
teachers the opportunity to undertake part of their teaching and research
activity in the foreign institution with which the cooperation agreement is
established, within the framework of integrated research programmes which will
be defined each year by the two participating institutions. Such programmes
will take into account the needs which will gradually emerge within the
scientific project to which the present agreement belongs.
Specifically, the agreement with University of
Bochum aims to promote the methodological exchange between Siena and Bochum,
according to a project which has been underway in the scientific community by
the two Universities.
Art. 2 Governing
Body
The following
bodies will be responsible for managing the present agreement:
In Siena:
- the
Teaching Staff Committee of the doctoral programme in “Cosmetic Science and
Technology” proposes and verifies objectives. Members of the foreign
institution may be asked to join the Committee;
- the international
Committee for the evaluation of doctoral programmes of the University of Siena
evaluates the outcomes of the programmes.
In Bochum:
-
Department of Cell Biology (Prof. Hanns Hatt), Faculty of Biology
- International
relations office of the Rehu-University Bochum (Dez 2)
Both
institutions will define each year the educational and scientific programmes
outlined in the Preamble, and may create joint committees for the evaluation of
doctoral dissertations.
Art. 3 Validation
of study periods abroad
The Parties
agree to recognise the periods of study abroad undertaken by their respective
students. This recognition will apply to both credits and to degrees.
Art. 4
Organizzazione del Dottorato
Teaching and
seminars within the doctoral programme in “Cosmetic Science and Technology”
including first and second year modules as well as tutorial discussions
of the dissertation, are structured according to a credit system (Enclosure).
On the basis of the credit system established by the programme, each
cooperating institution will assign a credit value to both the regular
attendance of courses and seminars and the tutorial discussion of dissertation
work teachers of the relevant institution. Thanks to this credit system Italian
students spending part of the doctoral programme in one of the foreign
cooperating institutions, or, in turn, foreign students working in Siena. Will
be able to quantify the work undertaken during the exchange and have it
recognised by their home institution.
Part of the
teaching in the Siena programme will be undertake in English, in order to
enable students form the cooperation institutions better to participate to the
programme itself. All teaching undertaken in Siena by visiting members of the
cooperation institutions will be in a foreign language.
The parties
agree top create and update a website, containing all relevant information
about activities undertaken and planned, as well as teaching or research
materials relevant to the joint activities or in any other manner deemed
useful.
The doctoral
student who elect to take advantage of this exchange programme must resided for
no less that six months (not necessarily consecutive).
The doctoral
programme in “Cosmetic Science and Technology” , in order to facilitate the
presence in Siena of visiting students and teachers, can make use of the
university residence “Certosa di Pontignano” ad “Collegio di Santa
Chiara”, which the university has restored for this purpose.
Art. 5 Legal
responsibility
For personal or
material damages caused to third parties, the law of the land where these may
occur will apply.
Art. 6 Changes
to the agreement
Any change or
addition to this agreement or any supplementary agreement will have to be made
in writing, with the assent of the cooperation institutions, at least six
months in advance. Any change to the agreement will affect the activities of
the academic year following.
Both the Italian
and English texts of this agreement are equally valid. They will be approved by
the relevant bodies of the participating institutions.
Bochum,
Siena,
Ruhr-Universitat
Bochum
University of Siena
THE
RECTOR
THE RECTOR