AU700084B2 - Anti-oxidant compositions - Google Patents

Description

WO 96/17899 PCT/GB95/02888 1 ANTI-OXIDANT COMPOSITIONS The present invention 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 employ 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 reactions. 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 atocopherol and B-carotene, as well as synthetic ones, e.g. BHA and BHT. Ascorbic acid (Vitamin C) is a watersoluble 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 a-tocopherol, thus resulting in a greatly increased anti-oxidant activity which exceeds the sum of the WO 96/17899 PCT/GB95/02888 2 component anti-oxidant activities. In this relationship, a-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/a-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 a-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 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 a-tocopherol. However, ascorbyl palmitate is not 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 ascorbic acid being used in a water solubilised form. The presence of water can, in fact, encourage oxidation by providing a medium for dispersing factors which can have prooxidant 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 suggested.

UK patent application No. 9323588.5 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.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The present invention provided an anti-oxidant composition comprising at least one anti-oxidant species solubilised in a hydrophobic solvent in which it would normally be soluble.

•"25 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 9* WO 96/17899 PCT/GB95/02888 4 ascorbic acid, citric acid, phytic acid, pyrophosphate, EDTA, transferrin, ceruplasmin, metallothionein, albumin, haptoglobin, cysteine, glutathione, conjugated bile pigments 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 of 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 soluble anti-oxidant species, together with one or more anti-oxidants solubilised in a hydrophobic 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 a-tocopherol), p-carotene, dp-tocotrienol, quercetin, acacetin, BHA, BHT, TBHQ, propyl gallate and probucol. Preferably the lipid soluble antioxidant species is a tocopherol, particularly a-tocopherol, and the other antioxidant is one which can act synergistically with a-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.

The present invention also provides a process for the preparation of a single phase hydrophobic anti-oxidant preparation comprising at least one anti-oxidant species solubilised in a hydrophobic solvent in which it would not normally be soluble, the process comprising: 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

*U*

U

U

*o *oo WO 96/17899 PCT/GB95/02888 6 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 interaction" 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 E, and which are not normally soluble in a hydrophobic solvent, to enhance anti-oxidant activity. In this case, vitamin E 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), WO 96/17899 PCT/GB95/02888 7 sphingomyelin, derivatives of any of these, for example 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 tbutanol, terpineol, monoglycerides such as glycerol WO 96/17899 PCT/GB95/02888 8 monooleate (GMO), othel esters, e.g. ethyl acetate, amyl acetate and bornyl acetate, diglycerides and triglycerides, particularly medium chain triglycerides and mixtures thereof, halogenated analogues of any of the above including halogenated 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 stripped 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 of the invention are optically clear and this can be monitored by measuring turbidity at visual wave lengths and, in some cases, by checking for sedimentation over a period of time.

The orientation of amphiphile molecules into an array with their hydrophilic head groups facing the moieties of an anti-oxidant species can be achieved in several ways WO 96/17899 PCT/GB95/02888 9 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 amphiphile 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 structures, 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 of 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 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 removal 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 antioxidant species is to co-solubilise the anti-oxidant species and the amphiphile in a common solvent followed by removal of the solvent.

Thus, in a further aspect the present invention is directed to a process for the preparation of an anhydrous single phase composition comprising an anti-oxidant and an amphiphile in a hydrophobic solvent comprising the steps of: either mixing the anti-oxidant with dispersion of an amphiphile in water, wherein the amphiphile forms micelles in the water and wherein the mixing gives a micellar solution; or ,ii mixing an amphiphile with water, wherein the amphiphile forms vesicles in the water, treating the mixture in such a way as to form a dispersion of small unilamellar vesicles and adding the anti-oxidant to the dispersion; or co-solubilizing the anti-oxidant and an amphiphile in a common solvent; or 30 emulsifying a solution of the amphiphile in a hydrophobic solvent with a solution of the anti-oxidant in a hydrophilic solvent; (ii) removing the solvent or solvents to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the antioxidant species and wherein there is no chemical interaction between the 35 amphiphile and the anti-oxidant; and (iii) providing a hydrophobic solvent around the antioxidant/amphiphile array.

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 WO 96/17899 PCT/GB95/02888 11 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 aqueous phases. However, it may be in the region of 2 Am Dispersion of the hydrophobic preparation in the aqueous phase can be achieved by mixing, for example either by vigourous vortexing for a short time for example about 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 gelatincontaining aqueous phase, the gelatin can be precipitated from the solution by coacervation by known methods and will form a film around the droplets of the anti-oxidantcontaining 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: WO 96/17899 PCT/GB95/02888 12 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 antioxidation 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: FIGURE 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 a-tocopherol.

FIGURE 2: shows a comparison of oxidation index for preparations with and without ascorbic acid in the presence of linoleic acid and linoleic acid and Soy

PC.

FIGURE 3: shows a comparison of oxidation index for preparations with and without ascorbic acid alone WO 96/17899 PCT/GB95/02888 13 and with linoleic acid.

EXAMPLE 1 2 rows of 4 small test-tubes were set up, and 0.2ml aliquots of 3.75, 7.5, 15 and 30mM ascorbic acid solutions were added to tubes 1, 2, 3 and 4 respectively, across each row. 0.2 ml of soy PC SUVs, 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 10% 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 Iml of distilled water. 300 AL of ascorbic acid solution was added to the ethereal solution of phosphatidyl 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 0 C with a slight vacuum, and the residue dried under a stream of nitrogen, followed by drying under high vacuum at room temperature in a lyophiliser WO 96/17899 PCT/GB95/02888 14 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.

EXAMPLE 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 (SUV) were pooled and then centrifuged for 15 minutes to remove particles of titanium.

0.56g of SUV 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 SUV 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 WO 96/17899 PCT/GB95/02888 the above oil phase. Into 3 small glass vials were added aliquots of 0.6, 0.6 and 0.5mg respectively of atocopherol. For reasons of accuracy, this was added as a 0.6% ethanolic solution, the ethanol being subsequently removed under a stream of nitrogen. To the above vials were then added 600mg of high ascorbate dispersion, 600mg of low ascorbate dispersion and 500mg of the above ascorbate-free oil phase respectively, mixing thoroughly to dissolve the a-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 aliquots of the oil phases prepared above, according to the following scheme.

Row no. Contents of each vial 1 Trilinolein/linoleic acid/PC oil phase 2 High ascorbate oil phase 3 Low ascorbate oil phase 4 High ascorbate oil phase a-tocopherol Low ascorbate oil phase a-tocopherol 6 Trilinolein/linoleic acid/PC oil phase a-tocopherol 7 Trilinolein/linoleic acid solution One vial from each row was retained as a zero time control and the remainder were transferred uncapped to a WO 96/17899 PCT/GB95/02888 16 37 C incubator and sampled after the incubation periods shown overleaf. At each sampling interval, 0.5ml of isooctane containing 0.01% BHT 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 31.7 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 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 0.3 n/s WO 96/17899 PCT/GB95/02888 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 100mg soy PC/g and converted to SUV as described in Example 3. Appropriate amounts of SUV were mixed with aqueous 1% ascorbic acid solutions to provide mixtures having PC ascorbic acid ratios of 13.33 1, 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 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 dissolved soy PC (ie in the same concentration as in the dispersion containing ascorbic acid/g).

The two ascorbic acid-containing dispersions, and the three oil phase controls, were each distributed as 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 Iml of a solution containing 10mg 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 WO 96/17899 PCT/GB95/02888 18 was monitored according 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 remaining 22:6 fatty acids relative to the remaining 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 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 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 WO 96/17899 1 9 acid is in a conjugated form, is not clear.

PCT/GB95/02888

Claims (16)

1. A process for the preparation of an anhydrous single phase composition including an anti-oxidant and an amphiphile in a hydrophobic solvent including the steps of: either mixing the anti-oxidant with dispersion of an amphiphile in water, wherein the amphiphile forms micelles in the water and wherein the mixing gives a micellar solution; or mixing an amphiphile with water, wherein the amphiphile forms vesicles in the water, treating the mixture in such a way as to form a dispersion of small unilamellar vesicles and adding the anti-oxidant to the dispersion; or co-solubilizing the anti-oxidant and an amphiphile in a common solvent; or emulsifying a solution of the amphiphile in a hydrophobic solvent with a solution of the anti-oxidant in a hydrophilic solvent; (ii) removing the solvent or solvents to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the anti- oxidant species and wherein there is no chemical interaction between the amphiphile and the anti-oxidant; and (iii) providing a hydrophobic solvent around the anti-oxidant/amphiphile array.

2. The process as claimed in claim 1, wherein the anti-oxidant species is ascorbic acid, phytic acid, pyrophosphate, EDTA, transferrin, ceruplasmin, 25 metallothionein, albumin, haptoglobin, cysteine, glutathione, a conjugated bile pigment, uric acid, vanillic acid, vanillin or trolox.

3. The process as claimed in claim 1 or claim 2, wherein a lipid soluble anti-oxidant is added at either or both of stages and (iii).

4. The process as claimed in claim 3, wherein the anti-oxidant acts synergistically with vitamin E and wherein the lipid soluble anti-oxidant is vitamin E. The process as claimed in claim 4, wherein the anti-oxidant is ascorbic acid, cysteine, glutathione, a conjugated bile acid or uric acid.

6. The process as claimed in any one of claims 1 to 5, wherein the 35 amphiphile is a phospholipid.

7. The process as claimed in claim 6, wherein the phospholipid has a phosphalidyl choline head group.

8. The process as claimed in claim 7, wherein the phospholipid is phosphatidyl choline lyso-phosphatidyl choline (lyso-PC), sphingomyelin, a derivative of one of the above such as hexadecyl phosphocholine or an amphiphile polymer containing phosphoryl choline.

9. The process as claimed in any one of claims 1 to 8, wherein the hydrophobic solvent includes a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, diglyceride, medium chain triglyceride or long chain triglyceride. The process as claimed in claim 9, wherein the amphiphile includes PC and the hydrophobic solvent is a triglyceride or wherein the amphiphile comprises lyso-PC and the hydrophobic solvent is oleic acid.

11. The process as claimed in any one of claims 1 to 10, wherein the process includes step i(a) or i(b) and wherein the weight ratio of amphiphile to hydrophilic species is from 1:1 to 100:1.

12. The process as claimed in any one of claims 1 to 11, wherein the process includes step i(a) or i(b) and wherein the water is removed by lyophilisation.

13. The process as claimed in any one of claims 1 to 9, which includes step i(c) or i(d) and wherein the weight ratio of amphiphile to hydrophilic species is from about 1:1 to 50:1.

14. The process as claimed in any one of claims 1 to 9 or 13, wherein the 25 process includes step i(d) and wherein the emulsion is a water-in-oil 25 emulsion. The process as claimed in any one of claims 1 to 9, 13 or 14, wherein *the process includes step i(d) and wherein, in step i(d) the hydrophobic solvent is a low boiling point organic solvent such as diethyl ether.

16. A two-phase composition including a hydrophilic phase and a 30 hydrophobic phase, wherein the hydrophobic phase includes a composition prepared by a process as claimed in any one of claims 1 to

17. The composition as claimed in claim 16, wherein the hydrophilic phase is continuous.

18. The composition as claimed in claim 16 or claim 17 which is an 35 emulsion.

19. The use of a composition as claimed in any one of claims 16 to 18 in the preparation of a pharmaceutical or cosmetic formulation or a foodstuff. A process for the preparation of an anhydrous single phase composition substantially as hereinbefore described with reference to Examples 1 and 2. DATED this 30th day of October 1998 CORTECS LIMITED Patent Attorneys for the Applicant: F.B. RICE CO. o•