Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-OXIDANT COMPOSITIONS
The present inventlon relates to compositions comprising
~ an anti-oxidant solubilised in a hydrophobic solvent in
which it would not normally be soluble. In particular the
present invention relates to compositions comprising
ascorbic acid solubilised in a hydrophobic solvent in
which it would not normally be soluble.
For many applications, e.g. in the pharmaceutical
sciences, in food technology or the cosmetics industry,
it is desired ~and in certain cases essential) to empioy
anti-oxidants to limit oxidation of, for instance, an
active ingredient or food ingredient.
Anti-oxidants can be divided into two main functional
groups, the chelating agents which act by sequestering
pro-oxidant ions, such as those of transition metals,
while the second group are the free radical scavengers
(chain-breakers), which have the effect of interrupting
oxidative chain reac~ions. The latter may operate in a
hydrophilic ~normally aqueous) or a hydrophobic (e.g.
lipid) environment, depending on their solubility
characteristics. Examples of lipid-soluble, free-radical
scavengers include natural anti-oxidants such as ~-
tocopherol and ~-carotene, as well as synthetic ones,
e.g. BHA and BHT. Ascorbic acid (Vitamin C) is a water-
soluble free-radical scavenger (and thus will normally
operate in this mode only in the aqueous phase), but it
also has an important role as a chelating anti-oxidant.
Yet another, very important anti-oxidant action of
ascorbic acid is that it can interact synergistically
with ~-tocopherol, thus resulting in a greatly increased
anti-oxidant activity which exceeds the sum of the
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component anti-oxidant activities. In this relationship,
~-tocopherol functions as the primary anti-oxidant which
is able to repair a lipid free radical, thus interrupting
the oxidation chain reaction while itself being converted
to a free radical in the process. Ascorbic acid acts by
regenerating the tocopheroxyl radical, thus restoring its
anti-oxidant function. Synergistic relationships are
also known to occur between other anti-oxidant species,
but often different mechanisms apply.
A requirement for ascorbic acid/~-tocopherol synergistic
action is that in order to interact, the two species must
be able to come into close contact. This can be
difficult in view of the fact that ascorbic acid is water
soluble while ~-tocopherol is lipid soluble. Such
interactions may occur in a living cell since tocopherols
are usually present in membranes which are in intimate
contact with the aqueous cytoplasmic phase. It is
therefore possible to duplicate this type of interaction
20 in vitro using liposomes in place of biological
membranes. A further available strategy is that of using
a lipid-soluble derivative of ascorbic acid such as
ascorbyl palmitate, which does interact synergistically
with ~-tocopherol. However, ascorbyl palmitate is not
25 very soluble and can require heat to dissolve, which
paradoxically increases oxidative susceptibility of
vulnerable compounds, for example polyunsaturates, and
this therefore mitigates against its use in situations
where these compounds are required to be protected, for
example in the preparation of certain foodstuffs.
other approaches are possible, for example using
microemulsions and reverse micelles. However, these will
usually still involve the ascorb~c acid being used in a
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water solubilised form. The presence of water can, in
fact, encourage oxidation by providing a medium for
dispersing factors which can have pro-oxidant activity,
e.g. molecular oxygen, metal ions, etc. It is thus
desirable to provide the ascorbic acid, if possible, in
a water free environment. To date, no suitable method or
approach has been disclosed or suagested.
UK patent application No. 9323588.S discloses a process
by which a hydrophilic species can be solubilised in a
hydrophobic solvent in which it would not normally be
soluble The process relies on the surprising discovery
that if a hydrophilic species is mixed with an amphiphile
under certain conditions, the resultant composition will
be readily soluble in lipophilic solvents such as oils.
It has now been surprisingly found that such
compositions, comprising an anti-oxidant solubilised in
a hydrophobic solvent in which it would not normally be
soluble, are effective as anti-oxidant compositions.
Surprisingly it has been found that anti-oxidant species
retain their anti-oxidant properties in such a non-
aqueous environment.
Thus, in a first aspect the present invention provides an
anti-oxidant composition comprising at least one anti-
oxidant species solubilised in a hydrophobic solven~ in
which it would not normally be soluble.
In general, the compositions of the invention will be
anhydrous. Thus, an anti-oxidant preparation is provided
which does not contain water.
Suitably, the anti-oxidant species is selected from
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ascorbic acid, citric acid, phy~ic acid, pyrophosphate,
EDTA, transrerrin, ceruplasmln, metallothionein, albumin,
haptoglobin, cysteine, glutathione, conjugated bile
pigments (e.g. bilirubin and biliverdin), uric acid,
vanillic acid, vanillin, and Trolox.
In a preferred embodiment, the anti-oxidant is selected
from ascorbic acid, cysteine, glutathione, conjugated
bile pigments and uric acid. A particularly preferred
anti-oxidant is ascorbic acid.
In the context of the present application, "solubilised"
refers to the anti-oxidant species being held in the
hydrophobic solvent, in the absence of water, i.e.
without the need for any water to be present.
The compositions of the present invention thus provide an
anti-oxidant in a non-aqueous form to protect materials
from oxidation, since it has been surprisingly found that
such compositions are effective as a means of protecting
against oxidation notwithstanding that the anti-oxidant
is solubilised in a hydrophobic solvent.
A variety or anti-oxidants can suitably be solubilised to
produce compositions according to the invention. In
particular, the compositions of the present invention are
useful in that it is possible to provide lipid soluble
anti-oxidants, e.g. vitamin E, in combination with one or
more water soluble anti-oxidants, such as ascorbic acid,
which act synergistically with vitamin E, thus providing
an enhanced anti-oxidant composition.
Thus, in another aspect, the present invention provides
an anti-oxidant composition comprising a lipid soiuble
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anti-oxidan~ species, together with one or more anti-
oxidants solubilised in a hydropnobic solvent in which
the one or more other anti-oxidants would not normally be
soluble.
The lipid soluble anti-oxidant species can be selected
from tocopherols (e.g. ~-tocopherol), ~-carotene, d~
tocotrienol, quercetin, acacetin, BHA, BHT, TBHQ, propyl
gallate and probucol. Preferably the lipid soluble anti-
oxidant species is a tocopherol, particularly ~-
tocopherol, and the other anti-oxidant is one which can
act synergistically with ~-tocopherol, resulting in
enhanced anti-oxidant activity, e.g. ascorbic acid,
cysteine, glutathione, conjugated bile pigments or uric
acid.
Suitably the compositions of the present invention can be
prepared using the processes described in UK patent
application No. 9323588.5.
Thus, in a further aspect the present invention provides
a process for the preparation of a single phase
hydrophobic anti-oxidant preparation comprising at least
one antioxidant species solubilised in a hydrophobic
solven~ in which it would not normally be soluble, the
process comprising:
(i) associating the anti-oxidant species with an
amphiphile in a liquid medium such that, in the
liquid medium, there is no chemical interaction
between the amphiphile and the anti-oxidant species;
(ii) removing the liquid medium to leave an array
of amphiphile molecules with their hydrophilic head
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groups orientated towards the anti-oxidant species;
and optionally
(iii) providing a hydrophobic solvent around the
anti-oxidant species/amphiphile array.
Suitably, the process can be halted at the end of stage
(ii) and the resulting material can be stored under
appropriate conditions until it is needed to generate the
single-phase preparation by providing a hydrophobic
solvent.
In the context of the present invention, the term
~chemical lnteraction" relates to an interaction such as
a covalent or ionic bond or a hydrogen bond. It is not
intended to include van der Waals forces or other
interactions of that order of magnitude.
This process can also be used to produce a composition
comprising vitamin E together with one or more other
anti-oxidants which act synergistically with vitamin ~,
and which are not normally soluble in a hydrophobic
solvent, to enhance anti-oxidant activity. In this case,
vitamin ~ can be added at either or both of stages (i)
and (iii) described above.
There are numerous amphiphiles which may be used to
prepare the compositions of the present invention and
zwitterionic amphiphiles such as phospholipids are among
those which have been found to be especially suitable.
Phospholipids having a phosphatidyl choline head group
have been used with particular success and examples of
such phospholipids include phosphatidyl choline (PC)
itself, lyso-phosphatidyl choline (lyso-PC),
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sphingomyelin, derivatives of any of these, for example
y hexadecylphosphocholine or amphiphilic polymers
containing phosphoryl choline and halogenated
amphiphiles, e.g. fluorinated phospholipids. In the
present application, the terms phosphatidyl choline (PC)
and lecithin are used interchangeably. Suitable natural
lecithins may be derived from any convenient source, for
example egg and, in particular, soya. In most cases, it
is preferable to select an amphiphile which is chemically
similar to the chosen hydrophobic solvent and this is
discussed in greater detail below.
The fact that the present inventors have found
zwitterionic amphiphiles such as phospholipids to be
particularly suitable for use in the process is a further
indication of the significant differences between the
present invention and the method of Okahata et al.
Significantly, the authors of that prior art document
concluded that anionic and zwitterionic lipids were
completely unsuitable for use in their method and stated
that they obtained zero yield of their complex using
these lipids.
The hydrophobic solvent of choice will depend on the
purpose for which the composition is intended, on the
anti-oxidant species to be solubilised and on the
amphiphile. Suitable solvents include non-polar oils
such as mineral oils, squalane and squalene, long chain
fatty acids with unsaturated fatty acids such as oleic
and linoleic acids being preferred, alcohols,
particularly medium chain alcohols such as octanol and
branched long chain alcohols such as phytol, isoprenoids,
e.g. nerol and geraniol, other alcohols such as t-
butanol, terpineol, monoglycerides such as .glycerol
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monooleate (GM0), otheL ester5, e.g. ethyl acetate, amyl
acetate and bornyl acetate, diglycerides and
triglycerides, particuLarly Inedium chain triglyce;ides
and mixtures thereof, ~lllogenated analogues of any of the
S above including halo~enated oils, e.g. long chain
fluorocarbons, and iodinated triglycerides, e.g.
lipidiol. In particular, polyunsaturated oils or
saturated oils are preferred.
Optimum results are generally obtained when the
hydrophobic solvent and the amphiphile are appropriately
matched. For example, with a solvent such as oleic acid,
lyso-PC is a more effective choice of amphiphile than PC,
whereas the converse is true when the hydrophobic solvent
is a triglyceride.
In addition, in some cases it has been found to be
advantageous to add a quantity of the amphiphile to the
hydrophobic solvent before it is brought into contact
with the anti-oxidant species/amphiphile array. This
ensures that the amphiphile molecules are not st- pped
away from their positions around the anti-oxidant species
because of the high affinity of the amphiphile for the
hydrophobic solvent.
It is very much preferred that the preparations cc the
invention are optically clear and this can be moni,ored
by measuring turbidity at Visual wave lengths anG, in
some cases, by checking for ~edimentation over a period
of time.
The orientation of amphiphile molecules into an array
with their hydropnilic head groupS facing the moieties of
an anti-oxidant species can be achieved in several ways
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and examples of particularly suitable methods are
discussed in more detail below.
In a first method, an anti-oxidant species is mixed with
a dispersion of an amphiphile in a hydrophilic solvent,
such that the amphlphile molecules form an assembly in
which the hydrophilic head groups face outwards towards
the hydrophilic phase which contains the anti-oxidant
species. The hydrophilic solvent is then removed to
leave a dry composition in which the hydrophilic head
groups of the amphiphile molecules are orientated towards
the anti-oxidant species.
In this first method, it is preferred that the
hydrophilic solvent is water although other polar
solvents may be used.
The form taken by the amphiphile assembly may be
micelles, unilamellar vesicles, preferably small
unilamellar vesicles which are generally understood to
have a diameter of about 25 nm, multilamellar vesicles or
tubular scructures, for example cochleate cylinders,
hexagonal phase, cubic phase or myelin type structures.
The form adopted will depend upon the amphiphile which is
used and, for example, amphiphiles such as phosphatidyl
choline (PC) tend to form small unilamellar vesicles
whereas lyso-phosphatidyl choline forms micelles.
However, in all of these structures, the hydrophobic
tails of the amphiphile molecules face inwards towards
the centre or the structure while the hydrophilic head
groups face outwards towards the solvent in which the
anti-oxidant species is dispersed.
The weight ratio of amphiphile:anti-oxidant species will
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generally be in the region of from 1:1 to 100:1,
preferably from 2:1 to 20:1 and most preferably about 8:1
for PC and 4:1 for lyso-PC.
These ratios are preferred ratios only and, in
particular, it should be pointed out that the upper limit
is set by economic considerations which mean that it is
preferable to use the minimum possible amount of
amphiphile. The lower limit is somewhat more critical
and it is likely that ratios of 2:1 or below would only
be used in cases where the anti-oxidant species has a
significant hydrophobic portion or is exceptionally
large.
Good performance is obtained when the solvent is removed
quickly and a convenient method for the rémoval of the
solvent is lyophilisation, although other methods can be
used.
A second method for the preparation of a composition
containing an array of amphiphiles with their head groups
pointing towards the anti-oxidan~ species is to co-
solubilise the anti-oxidant species and the amphiphile in
a common solvent followed by removal of the solvent.
The solutions of the present invention may either be used
alone or they may be combined with an aqueous phase to
form an emulsion or similar two phase composition which
forms yet a further aspect of the invention.
In this aspect of the invention there is provided a two
phase composition comprising a hydrophilic phase and a
hydrophobic phase, the hydrophobic phase comprising a
preparation of an anti-oxidant species as described
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herein.
Generally, in this type of composition, the hydrophobic
phase will be dispersed in the hydrophilic phase.
The two phase compositions may be emulsions which may
either be transient or stable, depending on the purpose
for which they are required.
The average size of the emulsion particles will depend on
the exact nature of both the hydrophobic and the a~eous
phases. However, it may be in the region of 2 ~m
Dispersion of the hydrophobic preparation in the aaueous
phase can be achieved by mixing, for example either by
vigourous vortexing for a short time for example about 10
to 60 seconds, usually about 15 seconds, or by gentle
mixing for several hours, for example using an orbital
shaker.
Emulsions containing the hydrophobic preparations of the
invention can also be used in the preparation of
microcapsules. If the emulsion is formed from a gelatin-
containing aqueous phase, the gelatin can be precipitated
from the solution by coacervation by known methoàs and
will form a film around the droplets of the anti-oxidant-
containing hydrophobic phase. On removal of the
hydrophilic phase, microcapsules will remain. This
technology is known in the art, but has proved
particularly useful in combination with the preparations
of the present invention.
In other aspects the invention provides:
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(i) the use of an anti-oxidant composition of the
invention in the preparation of a pharmaceutical or
cosmetic formulation or a foodstuff;
(ii) a method for reducing oxidation of a pharmaceutical
or cosmetic formulation or foodstuff which comprises
adding an anti-oxidant composition of the invention to
the pharmaceutical or cosmetic formulation or foodstuff;
(iii) a composition comprising at least one anti-oxidant
species solubilised in a hydrophobic solvent in which it
would not normally be soluble, for use as an anti-
oxidation agent; and
(iv) the use of a composition of the invention in the
preparation of an anti-oxidation agent.
The invention will now be described with reference to the
following examples. Example 3 refers to the figures in
which:
FIGIJRE 1: shows a comparison of oxidation index
(ratio of non-saturated fatty acid remaining in oil
compared to amount of non-oxidisable interval
standard) for preparations with and without ascorbic
acid alone or in combination with ~-tocopherol.
FIG~JRE 2: shows a comparison of oxidation index for
preparations wlth and without ascorbic acid ~n the
presence of linoleic acid and linoleic acid and Soy
PC .
FIG~JRE 3: shows a comparison of oxidation index for
preparations with and without ascorbic acid alone
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and with llnolelc acid.
E~ PLE 1
2 rows of 4 small test-tubes were set up, and 0. 2ml
aliquots of 3.75, 7.5, 15 and 3OmM ascorbic acid
solutions were added to tubes 1, 2, 3 and 4 respectively,
across each row. 0.2 ml of soy PC SWs, prepared as in
Example 2, was added to each tube in the front row, and
0.2 ml of distilled water to each tube in the second row.
The tube contents were shell-frozen in liquid nitrogen
and freeze-dried overnight. To each lyophilate in the
first row was added 200mg of Miglyol 818, while 200mg of
a solution comprising lO~ soy PC in Miglyol 818 was added
to each tube in the second row. All of the mixtures were
vortexed and left for several hours to disperse. At the
end of this time, all of the first row tubes contained
clear dispersions, while those in the second row were
turbid, despite containing exactly the same components as
the corresponding tubes in the front row.
EXAMPLE 2
250mg of Soy phosphatidyl choline was dissolved in 5ml of
diethyl ether in a glass boiling tube with a ground glass
stopper. 80mg of ascorbic acid was dissolved in lml of
distilled water. 300 ~L of ascorbic acid solution was
added to the ethereal solution of phospAatidyl choline,
shaken well, stoppered, and the mixture sonicated in a
bath for two minutes to give an almost clear water-in-oil
emulsion. The ether was then removed in a rotary
evaporator at 37~C with a slight vacuum, and the residue
dried under a stream of nitrogen, followed by drying
under high vacuum at room ~emperature in a lyophiliser
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overnight. The following day, 3 ml of oleic acid was
added to the residue with gentle mixing, to give a
solution which was completely clear. The concentration
of ascorbic acid in the oil was 8mg/ml.
EXAMP~E 3
An aqueous dispersion of soy phosphatidyl choline (soy
PC) was prepared, containing 50mg/g of suspension,
flushed thoroughly with nitrogen, and sonicated in 3ml
aliquots at an amplitude of 8 microns peak to peak. Each
aliquot was subjected to a total sonication time of 4
minutes, in pulses of 30 seconds interspersed by cooling
for 30 seconds in an ice slurry bath. The resulting
opalescent dispersions of small unilamellar vesicles
(S W) were pooled and then centrifuged for ~5 minutes to
remove particles of titanium.
0.56g of S W were mixed with 0.372g of 0.5~ ascorbic acid
solution, shell-frozen and freeze-dried overnight. To the
resulting lyophilate was added 1.4g of trilinolein
containing 5~ by weight of linoleic acid and the mixture
flushed with nitrogen, vortexed briefly and left to form
a clear dispersion. This is termed the high ascorbate
dispersion. Trilinolein is a model polyunsaturated
triglyceride while linoleic acid is a solubilization
facilitator.
A trilinolein/linoleic acid/PC oil phase of similar
composition but lacking ascorbic acid was prepared by
freeze-drying 0.7g of S W and then dissolving the
lyophilate in 1.75g of the same trilinolein/linoleic acid
solution. A low ascorbate dispersion was prepared by
diluting 0.25g of the high ascorbate one with~750mg of
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the above oil phase. Into 3 small glass vials were added
aliquots of 0.6, 0.6 and 0.5mg respectively of ~-
tocopherol. For reasons of accuracy, this was added as a
O.6~ ethanolic solution, the ethanol being subsequently
removed under a stream of nitrogen. To the above vials
were then added 600mg of high ascor~ate dispersion, 600mg
of low ascorbate dispersion and 500mg of the above
ascorbate-free oil phase respectively, mixing thoroughly
to dissolve the ~-tocopherol. A tray of 7 rows of
crimpable glass gas chromatography vials were set up and
to the vials in each row were added identical 50mg
aliquots of the oil phases prepared above, according to
the following scheme.
Row no. Contents o~ each ~ial
1 Trilinolein/linoleic acid/PC oil phase
2 Hi~h ascorbate oil phase
3 Low ascorbate oil phase
4 High ascorbate oil phase + ~-tocopherol
S Low ascorbate oil phase + ~-tocopherol
6 Trilinolein/linoleic acid/PC oil phase +
~-tocopherol
7 Trilinolein/linoleic acid solution
One vial from each row was retained as a zero ~ime
control and the remainder were transrerred uncapped to a
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16
37 C incuba~or and sampled after the lncubation periods
shown overleaf. At each sampling interval, 0.Sml of iso-
octane containing 0.01~ BH~ was added to reduce further
oxidative degradation, the contents mixed, sealed and the
vials transferred to a minus 20 C freezer and stored for
up to 2 weeks prior to GC analysis. At the time of
sampling, 0.5 ml of a solution of a 0.125~ heptadecanoic
acid (a saturated, non-oxidisable, internal standard) in
iso-octane was added to each vial and mixed. The fatty
acid components in each vial were then converted to
methyl ester derivatives by standard procedures and
measured by GC. Results were expressed in terms of an
Oxidation Index, which is defined here as the percentage
of remaining 18 : 2 fatty acids (derived from trilinolein
and linoleic acid) relative to 17 : 0 fatty acids
(heptadecanoic acid).
Incubation Vial Row Number
time ( days) 1 2 3 4 5 6 7
Day zero 44.5 43.8 42.2 42.8 41.5 43.1 44.5
Day 2 41.5 n/s n/s n/s n/s n/s 28.7
Day 4 3~.7 40.4 39.0 37.8 40.0 29.8 21.7
Day 6 21.0 38.8 36.8 n/s n/s 33.7 7.2
Day 8 9.0 53.9 45.6 44.4 50.2 39.0 0.6
Day 10 5.0 51.1 31.7 n/s n/s 46.4 0.6
Day 15 0 6.6 0 45.4 45. 2 32.9 n/s
35 Day 20 0 0 0 n/s n/s 15.9 n/s
Day 30 n/s 0 0 41.5 35.9 0 n/s
Day 48 n/s n/s n/s 52.5 11. 3 n~s n/s
Day 52 n/s n/s n/s 31.7 2.3 0._ n/s
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17
Day 56 n/s n/s n/s 4.1 0.2 n/s n/s
These results are plotted in Figure 1.
EXAMPLE 4
An aqueous phospholipid dispersion was prepared
containing lOOmg soy PC/g and converted to SW as
described in Example 3. Appropriate amounts of SUV were
mixed with a~ueous 1~ ascorbic acid solutions to provide
mixtures having PC : ascorbic acid ratios o~ 13.33 : l,
and the mixtures were shell-frozen and freeze-dried. The
resulting lyophilates were mixed with refined fish oil
containing 2~ w/w of linoleic acid (as a solubilisation
enhancer) to give clear dispersions containing 1.5 or 3.0
mg ascorbic acid/g of oil phase. Control oil phases were
also prepared comprising pure fish oil, and also fish oil
containing 2% linoleic acid both in the absence and
presence of 2~ w~w of dissoived soy PC ~ie in the same
concentration as in the dispersion containing 1.5mg
ascorbic acid/g).
The two ascorbic acid-containing dispersions, and the
three oil phase controls, were each distributed as 50mg
aliquots into crimpable glass chromatography vials as in
Example 3, and again stored uncapped in a 37 C incubator,
ie under conditions for accelerated oxidation of lipids.
At appropriate intervals, samples were removed from the
incubator and lml of a solution containing lOmg BHT and
625mg heptadecanoic acid in 200ml iso-octane was added
(see Example 3 for rationale). The vials were
sealed,shaken and stored in the freezer prior to GC
analysis of the remaining fatty acids. Lipid oxidation
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18
was monitorea accoraing to the rate of disappearance of
the 22:6 polyunsaturated fatty acids. Results are shown
in Figure 2 and are again plotted as the Oxidation Index
which is the percentage of rem~'n'ng 22:6 fatty acids
relative to the rem~i n tng saturated (non-oxidising) C17:0
fatty acid (heptadecanoic acid). The time course of
oxidation of pure fish oil was effectively identical to
that for fish oil + linoleic acid and is not plotted. The
delayed oxidation of the soy PC-containing control oil
phase, may well have been due to the additional
tocopherol present in the soy PC (over and above that in
the fish oil).
EXAMPLE 5
A lyophilate of soy PC and ascorbic acid was prepared as
described in Example 4 and then mixed with sunflower oil
containing 2~ w/w of added linoleic acid to form a clear
dispersion containing 1.5mg ascorbic acid/g of oil phase.
Oil phase controls were prepared comprising pure
sunflower oil and also sunflower oil containing 2~
linoleic acid, in the absence and presence of 2~ w/w of
soy PC. 50mg aliquots of the ascorbic acid dispersion
and of the 3 oil phase controls, were incubated under
2S conditions for accelerated lipid oxidation as described
in Example 4, and were sampled periodically and analysed
in the same way. In this case however, the 18 : 2 fatty
acid (linoleic acid) content was monitored.Results are
plotted in Figure 3. The time course of oxidation of the
soy PC-containing control oil phase was effectively
identical to the equivalent control lacking PC, and was
not plotted. The reason for the accelerated oxidation of
the oil controls containing free linoleic acid, compared
with pure sunflower oil where the endogenous linoleic
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19
acia is in a conjuga~ed form, is not clear.
