Solubilization of hydrophobic drugs in octanoyl-6-O-ascorbic acid micellar dispersions.

Article abstract of DOI:10.1002/jps.10180

Alkanoyl-6-O-ascorbic acid esters are easily obtained from vitamin C, and produce self-assembled aggregates in water solutions, with an inner hydrophobic pool surrounded by an external hydrophilic shell. Compared to ascorbic acid, their solubility in oils and fats is greatly enhanced, while the peculiar antioxidant activity is retained in the polar head groups of such surfactants. In virtue of their amphiphilic nature, ascorbic acid-based supramolecular systems can dissolve relevant amounts of hydrophobic, poorly water soluble chemicals such as drugs, vitamins, and so on, and at the same time they provide a suitable shield against oxidative deterioration of valuable materials. In this article we report our study on the self-assembling properties of octanoyl-6-O-ascorbic acid in water, and on the solubilization of some lipophilic molecules in its dispersions. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

Full text of DOI:10.1002/jps.10180

Solubilization of Hydrophobic Drugs in
Octanoyl-6-O-Ascorbic Acid Micellar Dispersions
SANTIAGO PALMA,1 RUBEN HILARIO MANZO,1 DANIEL ALLEMANDI,1 LAURA FRATONI,2 PIERANDREA LO NOSTRO2
1
´
´
Departamento de Farmacia, Fac. de Ciencias Quımicas, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000,
´
Cordoba, Argentina
2
`
Dipartimento di Chimica, Universita di Firenze, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
Received 21 January 2002; revised 22 February 2002; accepted 20 March 2002
ABSTRACT: Alkanoyl-6-O-ascorbic acid esters are easily obtained from vitamin C, and
produce self-assembled aggregates in water solutions, with an inner hydrophobic pool
surrounded by an external hydrophilic shell. Compared to ascorbic acid, their solubility
in oils and fats is greatly enhanced, while the peculiar antioxidant activity is retained in
the polar head groups of such surfactants. In virtue of their amphiphilic nature, ascorbic
acid-based supramolecular systems can dissolve relevant amounts of hydrophobic,
poorly water soluble chemicals such as drugs, vitamins, and so on, and at the same time
they provide a suitable shield against oxidative deterioration of valuable materials. In
this article we report our study on the self-assembling properties of octanoyl-6-O-
ascorbic acid in water, and on the solubilization of some lipophilic molecules in its
dispersions. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci
91:1810–1816, 2002
Keywords: micellar solubilization; vitamin C; ascorbic acid ester(s); micelle(s)
INTRODUCTION
nucleic acids, and cellular lipids, and induce
severe damages in biological systems.3 When
water is exposed to ionizing radiation, different
Vitamin C rapidly degrades to even slight heat-
ing, light, and action of oxidizing agents.1 In
aqueous solutions it reacts with dissolved oxygen,
behaving as an electron donor and producing de-
hydroascorbic acid (Figure 1), that eventually
further degrades.2 The C1, C2, and C3 carbon
atoms are in a tautomeric equilibrium1 as shown
in Figure 1.
Ascorbic acid and its derivatives play an impor-
tant role in many biological processes as reducing
agents or radical scavengers. Triplet excited state
carbonyl compounds react with ground state
oxygen to give the highly reactive singlet excited
molecular oxygen, that reacts with proteins,
ꢀ
ꢀ
radicals are formed, such as OH and OOH.1 The
current increment of radical species in the atmo-
sphere (particularly nitrogen oxides), the seasonal
partial loss of the ozone layer, and the increase of
the ultraviolet irradiation over the Earth, justify
the importance of using antioxidant species in
protecting terrestriallifefromfreeradicals’attack,
or in minimizing their dangerous activity.4–6
Cellular protection against singlet oxygen
damage may be granted by a number of quen-
chers, such as ascorbic acid, carotenoids and
phenols,7 notably tocopherols.8 Ascorbate behaves
as a weak singlet oxygen quencher, it is, in fact, a
better one-electron reductant than tocopherol
(E80 ¼ þ 0.23 V). Its lipophilic derivative 6-O-
ascorbyl-palmitate is a good antioxidant in model
systems and is also effective in cellular systems9
or against Epstein-Barr virus activation.10 The
presence of one or more hydrophobic chains in
Correspondence to: Pierandrea Lo Nostro (Telephone:
390554573010; Fax: 390554573036; E-mail: pln@csgi.unifi.it;
Internet: http://www.csgi.unifi.it)
Journal of Pharmaceutical Sciences, Vol. 91, 1810–1816 (2002)
ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
1810
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 8, AUGUST 2002

SOLUBILIZATION OF HYDROPHOBIC DRUGS
1811
Figure 1. Oxidation and tautomeric equilibrium of
ascorbic acid.
vitamin C-based surfactants leads to the forma-
tion of supramolecular self-assembled aggregates
in water, such as micelles, where an inner lipo-
philic core is surrounded by a hydrophilic shell
made up of the polar headgroups. Several differ-
ent amphiphilic ascorbic acid derivatives have
already been produced as esters, ethers, etc., and
their physico-chemical properties reported in the
literature.11–14 Assembled in such supramolecu-
lar structures, vitamin C derivatives may protect
degradable materials (particularly unsaturated
fats or vitamins), by confining the lipophilic solu-
tes in the micellar hydrophobic core, while the
ascorbic acid polar headgroups face the water
phase and efficiently perform their radical-
scavenger activity.
Solubilization of degradable hydrophobic mole-
cules in micellar dispersions or in microemulsions
is a valid way for enhancing their concentration
and stability in aqueous environments,15,16 and
vitamin C-based surfactants may constitute an
interesting class of amphiphilic compounds both
for their biocompatibility and for their antiradical
activity.
Figure 2. Chemical structure of octanoyl-6-O-ascor-
bic acid, anethole, carvacrol, thymol, eugenol, phena-
cetin, danthron, anthralin, and retinoic acid.
usually found in essential oils. They possess a
great and wide antibacterial and antioxidant18,19
activity, but are practically insoluble in aqueous
media.20
In this article we report a study on the physico-
chemical characterization of octanoyl-6-O-ascor-
bic acid (ASC8) micellar dispersions, and on their
capability to dissolve hydrophobic molecules:
phenacetin, danthron, anthralin, retinoic acid,
thymol, eugenol, anethole, and carvacrol. These
hydrophobic, poorly water soluble, unstable com-
pounds were selected to obtain preliminary in-
formation on the solubilizing capacity of ASC8
micellar aggregates; the evaluation of the final
formulation will be the object of future studies.
Antioxidant activity is retained in ASC8 micelles,
as DPPH tests and oxygen uptake experiments
show.
Thehydrophobicdrugsthatwetookintoconside-
ration in this study are shown in Figure 2. Phena-
cetin (PHE, p-acetophenetidide) is a neutral drug
with analgesic and antipyretics pharmacologi-
cal activities,17 danthron (DAN,1,8-dihydroxy-
anthraquinone) is a stimulant laxative,17 while
anthralin (ANT, 1,8-dihydroxy-anthrone) and
retinoic acid (RET) are used against psoriasis.17
These molecules are poorly soluble in water;
ANT and RET need to be stored in airtight
containers, protected from light. Anethole (ANE,
1-methoxy-4-(1-propenyl)benzene),carvacrol(CAR,
2-hydroxy-p-cymene), thymol (THY, 2-isopropyl-
5-methyl-phenol), and eugenol (EUG, 2-methoxy-
4-(2-propenyl)phenol) are phenolic molecules
MATERIALS AND METHODS
Octanoyl-6-O-ascorbic acid (or ascorbyl-octanoate,
ASC8) was synthesized according to the pro-
cedure already reported in the literature,13 which
involves the reaction between octanoic acid and
L-ascorbic acid in concentrated sulphuric acid at
room temperature. Purity was assessed through
TLC and elemental analysis essays. TLC (silica
gel; AcOEt þ AcOH): one spot (Rf ¼0.65). Elemen-
tal analyses: C calc. ¼ 55.62%, exp. ¼ 55.38%; H
calc. ¼ 7.34%, exp. ¼ 7.30%; m.p. ¼ 87.0–88.08C.
NMR (d, ppm; DMSO-d6): 0.85 (triplet, CH3),
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 8, AUGUST 2002

1812
PALMA ET AL.
1.23 (CH2), 1.53 (triplet, CH2), 2.31 (triplet, CH2),
4.04–4.06 (multiplet, OꢁCH2ꢁCH), 4.67 (singlet,
CH), 8.40 (C2ꢁOH), 11.11 (C3ꢁOH). UV/VIS
(lmax, nm; CH3CN): 236. IR (KBr, cmꢁ1): 3400,
2918, 2849, 1742, 1690, 1472, 1292, 1179, 1115.
The sodium salt was obtained in situ by neutra-
lization of a water solution of ASC8 with NaHCO3.
All reactants were purchased from Fluka (Milan,
Italy), and used without further purification.
Bidistilled water was purified with a MilliQ
apparatus.
Conductivity measurements were carried out
to determine the critical micellar concentration
(CMC) and the critical micellar temperature
(CMT) with a 712 Metrohn conductimeter, with
a cell constant of 0.942 cmꢁ1. The experiments
were repeated four times, according to the method
reported in the literature.21,22
The surface tension (g, mN/m) of ASC8 aqueous
solutions was measured with the Du Nou¨y ring
method at 258 and 308C. The critical micellar
concentration (CMC, mol/L) was determined from
the surface tension versus concentration plot at
the intersection point of the two straight lines
for low and high concentrations, with a thermo-
statted KSV model Sigma 70 apparatus. The
experimental error was ꢂ 1 mN/m. Surface ten-
sion experiments were repeated three times and
averaged.
was carried out with a Brookhaven Instruments
Corporation BI-2030 AT Digital Correlator.
The increment in apparent solubility of some
drugs in the presence of micellar solutions of ASC8
was determined through UV-vis spectrophoto-
metry. Aqueous dispersions of ascorbyl-octanoate
at different concentrations were saturated with
the drug, filtered, diluted (if necessary); the final
drug’s equilibrium concentration was measured
at 250 nm for phenacetin, 427 nm for danthron,
and 350 nm for both anthralin and retinoic acid,
using a Perkin-Elmer Lambda 35 instrument.
The assays were performed in triplicate, and
the data are plotted with the sample standard
deviation.
The reducing activity (RA, %) was evaluated by
measuring the absorbance at 517 nm of a DPPH
(a,a-diphenyl-b-picrylhydrazyl) solution in etha-
nol (10ꢁ4 mol/L) before (A0) and after (A20) 20 min
from the addition of an equal volume of the
sample (10ꢁ4 mol/L in ethanol or water), accord-
ing to the following formula:23
A0 ꢁ A20
RAð%Þ ¼ 100 ꢀ
A0
Reducing activity tests were repeated five
times on each sample, and the experimental error
was ꢂ 0.1%.
The lipid peroxidation study was performed
through UV spectrophotometry on a 0.1 M solu-
tion of ethyl linoleate in n-hexane;24 2.5 mL of the
solution were added to the cuvette (pure hexane
as blank reference), and a few crystals of AIBN
(recrystallized from methanol) were added to
start the oxidation of the unsaturated chain. The
reaction was monitored by measuring the absor-
bance at 234 nm, where the diene groups absorb.
Finally, 100 mL of an ethanol solution of ASC8
(10ꢁ4 M) were added to stop the lipid peroxida-
tion. The data were corrected for dilution and the
experiment was repeated twice.
Density, refractive index, and viscosity of ASC8
aqueous solutions were measured with an Anton
´
Paar DMA 4500 Densimeter, with an Abbe Re-
fractometer and with an Ubbelhode Viscometer,
respectively. All experiments were performed at
308C. The viscosity of each solution was calcu-
lated according to the following formula:
Br
t
h ¼ Art ꢁ
where h is the viscosity of the solution, A and B
the instrument constants, r is the density of the
same sample, and t the outflow time through the
viscometer capillary.
The oxygen uptake of ascorbic acid and ASC8
solutions was determined in triplicate, by using an
oxygen electrode (mod. DW1, Hansatech, United
Kingdom) at 308C.
The samples for static and dynamic light scat-
tering were filtered through 0.22 m Millipore
filters directly into cylindrical glass tubes. An
Arþ ion laser source was used, with an incident
l of 514.5 nm. Temperature was controlled with
a water circulating bath system, while filtra-
tion and circulation of the index-matching liquid
(decahydronaphthalene) were performed with a
membrane filter and a peristaltic pump. All light
scattering experiments have been performed at
a scattering angle y ¼ 908, and the data analysis
RESULTS AND DISCUSSION
The aggregation of ASC8 in water dispersions was
studied by measuring the critical micellar concen-
tration (CMC) and the critical micellar tempera-
ture (CMT) through conductivity, light scattering,
and surface tension measurements.
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SOLUBILIZATION OF HYDROPHOBIC DRUGS
1813
Figures 3a and 3b report the conductivity
values as a function of ASC8 concentration at
308C, and as a function of temperature at [ASC8]¼
5ꢀ10ꢁ2 mol/L, respectively. The breaking points in
the two plots provide a value for CMC of about
(6.1 ꢂ 0.1) ꢀ 10ꢁ3 mol/L, and for CMT of 19.38 ꢂ
0.128C (standard deviation).
Ionic surfactants’ dispersions show a critical
temperature (CMT or Krafft Temperature) above
which the surfactant solubility increases very
rapidly. This is due to the formation of self-
assembled structures from the amphiphilic mono-
mers. Above such temperature, the equilibrium
system produces a clear and homogeneous dis-
persion of self-assembled aggregates, while below
this temperature the micellar solution forms a
semicrystalline mesophase that is generally refer-
red to as ‘‘coagel’’.25,26
Figure 4 shows the surface tension of aqueous
solutions of the surfactant as a function of [ASC8],
at 258 (full circles) and 308C (white squares), res-
pectively. From the intersection point of the
two fitting lines for high and low concentrations we
determined a value for CMC of about 4ꢀ10ꢁ3 mol/L
at 258, and of 6ꢀ10ꢁ3 mol/L at 308C, respectively.
Static light scattering experiments can be used
to detect the formation of aggregated particles
in solutions, based on the refractive index dif-
ference between the oil inner micellar core and
the external medium. Measurements were per-
formed at y ¼ 908, and at 308C, as a function of
ASC8 concentration, and show that the intensity
rapidly increases for concentrations higher than
5.8 ꢀ 10ꢁ3 mol/L, confirming the CMC value
obtained from conductivity and surface tension
data. Dynamic light scattering data performed at
different angles (308 < y < 1508) indicate the pre-
sence of nearly spherical aggregates with an
˚
average hydrodynamic radius of about 26 ꢂ 1 A.
From the slope of the g versus log c plots, the
polar head group area a0 can be determined ac-
cording to the Gibbs equation for adsorption mono-
layers at the air/water interface:
ꢀ
ꢁ
ꢁ1
@g
ꢀ ¼
2:303 ꢀ n ꢀ RT @log c
T
1016
a0 ¼
NA ꢀ ꢀ
where G, R, T, and NA are the surface excess of the
surfactant, the universal gas constant, the abso-
lute temperature, and the Avogadro’s number,
respectively. n is 1 for nonionic surfactants, and
2 for 1:1 ionic amphiphiles. According to our data,
2
˚
we calculated a0 ¼ 76 at 258, and 88 A at 308C,
respectively. From previous SANS (small angle
neutron scattering) experiments27 we obtained a
2
˚
value of a0 ¼ 58 A that agrees with CPK (Corey-
Pauling-Koltun) model calculations. This differ-
ence may be ascribed to hydration of the polar
head groups, penetration of water molecules in
the hydrophobic region, micellar size polydisper-
sity, and also to the different parameters and
conditions that are used to determine a0 in the
two techniques (surface tension and SANS mea-
surements). SANS data analysis provided a value
˚
for the micellar radius of 25.4 A, that is in good
agreement with the value obtained from dynamic
light scattering in this study.
Figure 3. Conductivity measurements as a function of ASC8 concentration at 308C
(a), and as a function of temperature at [ASC8] ¼ 50 mM (b). The plot is the average over
four different sets of data; the CMT values have been determined from the intercept of
the two straight lines, and the standard deviation (error bar) has been calculated from
the four experimental results.
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1814
PALMA ET AL.
Figure 6. Retinoic acid and anthralin solubility as
a function of [ASC8]. For concentrations higher than
CMC (indicated by the arrow), the two drugs’ solu-
bilities are greatly enhanced. Error bars indicate the
sample standard deviation.
Figure 4. Surface tension of aqueous surfactant’s
solutions as a function of [ASC8], at 258 (full circles) and
308C (white squares). Error bars indicate the experi-
mental error for each data.
The apparent solubility of phenacetin, dan-
thron, anthralin, and retinoic acid was measured
as a function of [ASC8] and compared to the same
value in pure water.
Carvacrol, thymol, anethole, and eugenol are
practically insoluble in water; when shaken
with water, they produce strong turbidity and
phase separate in two layers. In the presence
of ascorbyl–octanoate micellar solutions (about
7 mM of phenol in 50 mM ASC8) they produced
opaque dispersions that did not phase separate
even after a year; this finding shows that their
dispersions are greatly stabilized by the presence
of the amphiphilic ASC8 molecules. Dynamic
light-scattering experiments on freshly prepar-
ed dispersions indicate the presence of nearly
spherical objects with an average hydrodynamic
radius of about 40 ꢂ 1 nm.
According to Figure 5, a relevant increase of
phenacetin apparent solubility is observed (about
22%) with respect to its solubility in pure water
(4.6 ꢀ 10ꢁ5 mol/L). The ASC8 concentration corres-
ponding to the initial increment in PHE solubility
agrees with the CMC values measured with dif-
ferent techniques. In ASC8 micellar dispersions,
DAN almost doubles its solubility (5.6ꢀ 10ꢁ5 mol/
L). Figure 6 shows the solubility increment of
anthralin (ANT) and retinoic acid (RET) upon
solubilization in ASC8 micellar dispersions. Res-
pect to pure water, their solubility increases of
142 times in the case of ANT, and of 53 times for
RET, in the presence of ASC8 micelles. The arrow
in Figure 6 indicates the CMC of ASC8 at 308C,
and shows that the solubility of both drugs
significantly increases only for concentrations
higher than the CMC.
To check the efficacy of ascorbyl–octanoate
aqueous dispersions against oxidative degra-
dation, we carried out some experiments that
measure the protective activity performed by
ASC8 solutions. Reducing activity was evaluated
with the DPPH method, and gave a result of about
89.3% for ASC8 and 90.3% for ascorbic acid. This
result shows that ASC8 keeps the same antiox-
idant properties of vitamin C. Reducing activity
measurements were also performed in the emul-
sified solutions containing carvacrol, thymol,
eugenol, and anetol, and provided a value higher
than 80% for all chemicals, even after 150 days
from the emulsification.
The efficiency of ASC8 in inhibiting lipid per-
oxidation in hydrophobic environments (where
ascorbic acid is not soluble and cannot work as
antioxidant) can be easily evaluated by the ability
to prevent or inhibit the oxidative degradation of
ethyl linoleate in hexane induced by a lipophilic
radical initiator, AIBN. As shown in Figure 7, the
absorbance at 234 nm increases, due to the for-
mation of conjugated double bonds in the linoleic
chain because of the radical attack. The addition
Figure 5. Phenacetin apparent solubility as a func-
tion of [ASC8]. Error bars indicate the sample standard
deviation.
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SOLUBILIZATION OF HYDROPHOBIC DRUGS
1815
reactivity of the salt presumably depends on the
fact that alkaline pH values favor the degradation
of the ascorbic ring to dehydroascorbic acid.
CONCLUSIONS
Octanoyl-6-O-ascorbic acid (ASC8) combines the
amphiphilic nature of a typical short chain sur-
factant to the antioxidant activity imparted by
the ascorbate polar headgroups. In this study
we determined the critical micellar concentration
and Krafft temperature for ASC8 in water dis-
persions, by means of conductivity, surface ten-
sion, and light scattering experiments. These data
are necessary for the characterization of this sur-
factant’s micellar solutions, and for the solubiliza-
tion of hydrophobic drugs such as phenacetin,
danthron, anthralin, and retinoic acid, whose
solubilities are greatly increases with respect to
pure water. The solubility and stability of emulsi-
fied mixtures containing some phenols (anethole,
carvacrol, thymol, and eugenol) in water disper-
sions is also enhanced by the addition of ASC8.
Furthermore, the antioxidant activity performed
by the ascorbate rings that form the hydrophilic
external shells in ASC8 aggregated structures
ensures a valid way for protecting degradable
materials that have been solubilized in the inter-
nal hydrophobic micellar core from oxidative,
radical-initiated, attack.
Figure 7. Absorbance at 234 nm after addition of
AIBN to a hexane solution of linoleic acid. The arrow
indicates the addition of ASC8, that results in the end
of the lipid’s oxidation.
of an ethanol solution of ASC8 to the sample,
indicated by the arrow in the plot, results in the
end of the degrading action, and in a constant
value of absorbance, even almost 10 h after the
addition of the ascorbyl derivative. Finally, oxy-
gen uptake experiments were performed on ASC8
dispersions and compared to ascorbic acid and
sodium ascorbate solutions at the same tempera-
ture and concentrations. Figure 8 reports the
amount of dissolved oxygen, calculated as
ꢀ
ꢁ
½O2ꢃt
½O2ꢃ0
log 100 ꢀ
;
as a function of time t. The linear fitting curves,
obtained from triplicates, indicate that ascorbyl–
octanoate is less sensitive to oxidation than as-
corbic acid and sodium ascorbate. The higher
These data can be of interest for increasing the
availability of hydrophobic drugs in water-based
micellar environments, and with a stronger resis-
tance to oxidation.
ACKNOWLEDGMENTS
Partial financial support from the Consorzio Inter-
universitario per lo Sviluppo dei Sistemi a Grande
Interfase (CSGI), Ministero dell’Istruzione, dell’
`
Universita e della Ricerca (MURST), and Consejo
´
´
Nacional de Investigaciones Cientıficas y Tecnicas
(CONICET) is greatly acknowledged.
Figure 8. Dissolved oxygen uptake as a function
of time for ASC8 (circles), ascorbic acid (squares),
and sodium ascorbate (triangles). The curves repre-
sent the linear fitting according to the expression:
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½O2ꢃt
½O2ꢃ0
log 100 ꢀ
¼ At þ B, where [O2] is the concentra-
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