Oral composition comprising a polyunsaturated fatty acid and salicylic acid for obtaining an antiinflammatory effect in skin

0001The present invention relates to a composition for oral consumption which, when taken orally can have benefits for the skin, and to the use of a combination of active compounds.

0002Improving the appearance and feel of human skin has received a great deal of research effort. However, the vast majority of commercially available products address this problem by acting on the exterior of the skin, the most common form being a topical skin cream. However, such topical applications have their limitations and deal primarily with the dead surface layers of the skin. It is known that certain ingredients can provide improvements in skin appearance and texture from being ingested. Such ingredients thus act from the interior of the skin and therefore can provide greater opportunities for improving the skin by accessing the living interior. Furthermore such an effect may be perceived by the general public as being more potent or medical in nature than a topical application.

0003Dietary fish oil is known to convey significant protection against UVR-induced erythema upon ingestion.

0004Our copending international application no <patcit id="pcit0001" dnum="EP2005011658W"><text>PCT/EP2005/011658</text></patcit> relates to stable consumable emulsions.

0005<nplcit id="ncit0001" npl-type="s"><text>Serhan et al, J Exp. Med., 196, no 8, October 21, 2002, 1025-1037</text></nplcit> discloses that anti-inflammatory mediators can be formed from docosahexaenoic acid (DHA) in the presence of acetylsalicylic acid (aspirin). The mechanism involved in the production of the anti-inflammatory mediators requires the acetylation of the enzyme cyclooxygenase-2 (COX-2) and the acetyl group in aspirin is therefore essential. Related effects with eicosapentaenoic acid (EPA) are described in <nplcit id="ncit0002" npl-type="s"><text>Arita et al, JEM, 201, no 5, March 7, 2005, 713-722</text></nplcit> and in <nplcit id="ncit0003" npl-type="b"><text>R&amp;D Systems, Cytokine Bulletin, November 2005 (Abingdon, UK</text></nplcit>).

0006<patcit id="pcit0002" dnum="US20020054918A"><text>US 2002/0054918</text></patcit> discloses topical compositions for treating inflammatory skin conditions. The compositions may be used together with orally administered compositions containing fish oils or aspirin.

0007<patcit id="pcit0003" dnum="US20040258645A"><text>US 2004/0258645</text></patcit> describes a personal care kit which has separate containers comprising a topical composition and a product suitable for oral consumption.

0008<patcit id="pcit0004" dnum="WO0211564A"><text>WO 02/11564</text></patcit> discloses a nutritional supplement for Type 2 diabetes. The supplements may contain fish oil or aspirin as optional active components. Compositions according to this document are said to include salicylic acid, but it is clear that the document only contemplates or intends the inclusion as an active compound of acetyl salicylic acid, i.e. aspirin, in its compositions.

0009<patcit id="pcit0005" dnum="WO0180870A"><text>WO 01/80870</text></patcit> relates to therapeutically useful compositions derived from grapefruit or cranberry. The compositions are said to be useful in the treatment of cancer and hypercholesterolemia. The cranberry extracts contain phenolic compounds and flavonoids and there is no mention of salicylic acid or its derivatives.

0010There remains a need for orally administered compositions that have improved properties for imparting benefits to the skin of the consumer.

0011<patcit id="pcit0006" dnum="US20020169209A"><text>US 2002/0169209</text></patcit> describes pharmaceutical formulations comprising at least 70% eicosapentaenoic acid or acid derivative, less than 10% docosahexaenoic acid or acid derivative and less than 10% linoleic acid or acid derivative, combined in the same dosage form or same pack with an enzyme inhibitor of COX-1 and/or COX-2, LOX or one or more of the FACL enzymes.

0012According to the invention in a first aspect, there is provided a composition which is adapted for oral consumption comprising salicylic acid, a C1 to C6 alkyl ester thereof or a salt thereof, and a polyunsaturated fatty acid, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

0013In another aspect, the invention provides a composition according to the first aspect which is in the form of a substantially homogeneous aqueous emulsion, suspension or dispersion for use in obtaining an anti-inflammatory effect in skin, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

0014The invention is based on the surprising finding of a synergistic effect between salicyclic acid and docosahexaenoic acid. This effect is particularly surprising because the disclosure in the art indicated that the acetyl group in aspirin was essential for anti-inflammatory activity. It has now been found that salicylic acid can exhibit an anti-inflammatory effect with docosahexaenoic acid when not acetylated.

0015The composition of the invention thus comprises salicylic acid or a C1 to C6 ester thereof (i.e., without an acetylated phenolic hydroxyl group) or a salt thereof and docosahexaenoic acid. It is preferred that the salicylic acid, or a C1 to C6 alkyl ester or salt thereof, and docosahexaenoic acid are present in an amount sufficient to exhibit an anti-inflammatory effect on the skin.

0016Compositions of the invention are oral compositions i.e., they are adapted for oral consumption. As such, the compositions are edible and non-toxic.

0017The composition preferably comprises one or more further components selected from antioxidants, flavouring agents, preservatives and stabilisers.

0018The composition of the invention may take any suitable form, including, for example, food products and nutritional supplements. Compositions for oral consumption include beverages, bars and other liquid and solid forms such as tablets, pills, capsules and powders (which may contain crystalline material), as well as spreads, margarines, creams, sauces, dressings, mayonnaises, ice creams, fillings, confectionaries and cereals.

0019Preferably, the compositions of the invention are in the form of a substantially homogeneous aqueous emulsion, suspension or dispersion.

0020In one embodiment of the invention, the composition of the invention is edible and is preferably water based, i.e. comprises at least 50wt% water, preferably at least 60wt% or even at least 70wt% water. The water may be added or may be derived from a natural product that contains the salicylic acid or ester thereof and/or DHA. It may be either liquid or frozen. The product thus has the sensation of being a regular water-based product and can be consumed on a regular basis as part of a consumer's normal diet. For example, it could replace a fruit juice normally consumed at breakfast time. The composition of the invention is preferably packaged as a beverage, for example in a container such as a carton or a bottle of coated paper or cardboard, glass or plastic. The container preferably has a volume of from 10 to 500 ml, such as from 20 to 100 ml.

0021In an alternative embodiment, the composition of the invention is contained in a capsule. Typically, the salicylic acid or ester thereof will then be in a more concentrated form. The capsule may be made of any suitable material well known in the art such as gelatin. The capsule is adapted to be swallowed by the consumer and typically one or two capsules will be taken from one to four times per day. Each capsule preferably comprises from 10 to 4000 mg of docosahexaenoic acid (more preferably from 10 to 3000 mg or 20 to 2000 mg or 20 to 1000 mg, even more preferably from 50 mg to 500 mg) and from 1 to 100 mg of salicylic acid or a salicylate ester, or a mixture thereof (preferably from 2 mg to 50 mg, such as from 3 mg to 30 mg or from 4 mg to 20 mg).

0022In a further contemplated embodiment, when the composition of the invention is provided as a tablet, liquid, capsule or powder, the active ingredients in the invention may be distributed across different capsules, tablets, pills or powders, so that for instance the user may be provided with one tablet, capsule, pill or powder containing the salicylic acid or salicylic acid ester/salt, and another tablet, capsule, pill or powder containing docosahexaenoic acid. In such an instance, the user is also ideally provided with instructions to take the liquids, capsules, pills or powders according to a dosage regime, which may entail taking them in the same dose, i.e. at the same time or in quick succession.

0023In yet a further embodiment, the composition of the invention may be included as one component of a complex food product. For instance, the composition may be present in a solid or gelatinous form as a filling or layer within a bar, or a similar product. The composition of the invention may therefore be included in a wide range of everyday foodstuffs, for instance in "health food" bars which could be eaten as an alternative to other food snacks.

0024Compositions of the invention comprise salicylic acid, or a C1 to C6 alkyl ester thereof. The term "alkyl" includes saturated, branched or unbranched groups. Typical alkyl groups are methyl and ethyl.

0025The salicylic acid, esters or salts thereof that are present in the compositions of the invention may and preferably do have a free (e.g. unsubstituted) phenolic hydroxyl group and do not include acetylated derivatives of salicylic acid or its esters such as aspirin. The salicylic acid, ester or salts thereof element in the claims specifically excludes aspirin. The salicylate salt may typically be sodium or potassium salicylate.

0026Compositions of the invention preferably comprise salicylic acid, a C1 to C6 ester or salt thereof, or a mixture of the acid, one or more esters and/or one or more salts, in an amount of from 0.00002 % to 1 % by weight, more preferably from 0.00004 % to 0.05 %, such as from 0.0002 % to 0.001 % by weight.

0027Preferably, compositions of the invention comprise from 0.1 to 100 mg of salicylic acid, a salicylate ester or a salicylate salt, or a mixture thereof (preferably from 1 mg to 50 mg, such as from 3 mg to 30 mg or from 4 mg to 20 mg).

0028Preferably, the salicylic acid, ester or salt thereof is present as a component of a natural product or an extract or concentrate thereof.

0029The natural product is preferably plant material, for example fruits, more preferably berries, and spices such as cumin. An example of a suitable natural product is cranberry juice; cranberry powder may also be used. Other natural sources of salicylic acid, its esters and salicylate salts are liquorice and herbs, including oregano, rosemary and thyme.

0030The compositions of the invention comprise a docosahexaenoic acid. The fatty acid may be present as a free acid, a C1 to C6 alkyl ester, a glyceride (including mono-, di- and triglycerides), or a mixture thereof. Reference herein to docosahexaenoic acid includes not only the free acid but esters, glycerides and mixtures. The DHA is preferably present in the form of a fish oil or is from a microbial source.

0031DHA is an ω-3, polyunsaturated, 22-carbon fatty acid. It is also present in abundance in certain fish (such as tuna and bluefish) and marine animal oils.

0032Typically, the amount of docosahexaenoic acid in the compositions of the invention ranges from 0.001% to 4% by weight of the composition. More preferred amounts are from 0.01% to 5% by weight, such as from 0.1% to 1% by weight.

0033When the DHA is provided by fish oil, the composition of the present invention preferably comprises from 0.2 to 10 wt% of fish oil, more preferably from 0.4 to 4 wt% fish oil. The oil typically comprises DHA and EPA. Preferably, it is made up of at least 12 wt% (such as at least 20 wt%) EPA and DHA, more preferably at least 30 wt% EPA and DHA.

0034Eicosapentaenoic acid (EPA) is one of several ω-3 fatty acids used by the body. Increased intake of EPA has been shown to be beneficial in coronary heart disease, high blood pressure, and inflammatory disorders such as rheumatoid arthritis.

0035Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), come from cold water fish such as wild salmon (not farm raised), mackerel, sardines, herring and other northern marine animals. Fish can make EPA and DHA from the ω3 essential fatty acid, alpha-linolenic acid (LNA), but get much of their EPA and DHA from brown and red algae which manufacture EPA and DHA from carbohydrates - sugar, starch, cellulose, etc.

0036More recently, brown and red algae have begun to be grown commercially for EPA and DHA. These make 10 to 14% of long-chain ω3s (on dry weight basis) and can be used as food sources of EPA and DHA-containing triglycerides.

0037One or more antioxidants are preferably present in the compositions of the invention in order to prevent or slow down the natural oxidative degradation of the polyunsaturated fatty acid. Rancid fatty acids not only have an unpleasant taste but may even have negative health effects (<nplcit id="ncit0004" npl-type="s"><text>Kubow S., "Toxicity of dietary lipid peroxidation products", Trends in Food Sciences &amp; Technology, September, 67-71 (1990</text></nplcit>)).

0038Suitable antioxidants can be selected, although not exclusively, from the following list, either singularly or in combination: TBHQ, Ascorbyl esters (e.g. ascorbyl palmitate), ascorbic acid, Tocopherols, Rosemary Extract, fruit concentrates or extracts, black or green tea extract, Propyl Gallate, essential oils or oleoresins, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), citric acid or esters, co enzyme Q10, Tocotrienols, Chelators (e.g. EDTA), Carriers, polyphenols, phenolic compounds, flavonoids, oxygen scavengers.

0039Especially preferred antioxidants are vitamins C and E. Not only are these effective antioxidants but they also have been shown to give skin benefits when consumed.

0040An amount of antioxidant should be added sufficient to prevent the polyunsaturated fatty acid from going rancid over a typical shelf-life of 6 months. Clearly the amount of antioxidant will depend on the type and activity of the antioxidant used. However, preferably the product has a weight ratio of antioxidant to polyunsaturated fatty acid of from 1:10 to 1:100 based on the antioxidant activity of vitamin C. For example, if an antioxidant with twice the activity of vitamin C was used, the weight ratio would be from 1:20 to 1:200.

0041For these purposes an antioxidant activity is as measured using an appropriate assay (e.g. Trolox equivalent antioxidant capacity).

0042The compositions of the invention may comprise a flavouring, although the addition of a flavouring may be unnecessary if the salicylic acid or ester is provided by a flavoured substance such as a fruit juice. Suitable flavouring agents may be natural or synthetic. Flavouring agents may be required to make the product more palatable for consumption.

0043The compositions preferably comprise an emulsifier, more preferably a food grade phospholipid emulsifier. The emulsifier may be required for stability of a oil-in-water emulsion. It is preferred that the phospholipid emulsifier is lecithin. Phospholipid emulsifiers are oil soluble, but the lecithin can be added to either phase prior to emulsification. Preferably it is added to the aqueous phase.

0044The emulsifier is preferably present in the composition in an amount of at least 0.01 wt%. Preferably from 0.05 to 3 wt%, more preferably from 0.1 to 1 wt% of the composition.

0045The composition of the invention may comprise other fatty acids, such as linoleic acid. Linoleic acid may be present as a component of a natural oil, such as a vegetable oil e.g., sunflower oil.

0046Another optional component of the composition is a detoxifying plant extract, such as rosemary oil.

0047The composition may also comprise carotenoids, such as in an amount of from 0.0005 to 0.1 wt%, for example from 0.002 to 0.04 wt%. The carotenoids, being oil soluble, would be comprised predominantly within the oil phase. Highly preferred carotenoids are β-carotene, and lycopene. These carotenoids provide moderate protection from UV induced erythema, thought to be due to their antioxidant functionality including scavenging of reactive oxygen species.

0048The composition may also comprise soy isoflavones (including genistein in glycosylated and/or non-glycosylated form), typically in an amount of from 0.0001% to 0.1% by weight.

0049The composition of the present invention may be made by preparing an aqueous phase and an oil phase. In general the water-soluble ingredients are put together in the aqueous phase and the oil-soluble ingredients in the oil phase. If an emulsifier is used, it is preferred that it is added to the aqueous phase.

0050The oil phase and aqueous phase are then blended together to form an emulsion.

0051In a preferred process the oil is on a powdered carrier material to assist emulsion formation.

0052The stable emulsion may then be packaged in a sealed container such as a metal, coated cardboard (e.g. tetra Pak) or plastic container. The container is then preferably sealed so as to give no headspace or a gas filled (e.g. nitrogen or carbon dioxide) headspace. This assists still further in preventing oxidation.

0053Alternatively the emulsion may be frozen and packaged and sold as a frozen consumer product.

0054The composition of the invention may have an anti-inflammatory effect in skin. Benefits of the anti-inflammatory effects in skin may include one or more of: anti-ageing effects; reduced dryness; increased firmness; increased elasticity; reduced fine lines and wrinkles; fewer spots, pimples and blemishes (including acne); clearer skin; less sensitive skin; and generally healthier skin. The skin may include the skin of the whole body, preferably of the face, neck and/or hands. The skin may also include scalp skin with benefits for hair (including reduced ageing) and scalp itch or irritation. Conveniently, the benefit can be cosmetic.

0055The composition is preferably used for its anti-ageing and/or anti-wrinkle effects. The skin of the consumer may be described as calm and/or clear and/or blemish-free.

0056The composition of the invention may be packaged together with a composition for topical application to the skin. As such the composition may be a kit of parts comprising the composition of the invention packaged separately from a composition for topical application to the skin. The kit of parts is adapted to be sold as a single article and to be used by the consumer for consumption of the oral composition and application of the topical composition to the skin. Ideally the kit of parts contains instructions to apply the topical composition and consume the oral as part of a dosage regime, which may require the user to, for example, consume the oral composition and apply the topical composition at set times of the day.

0057In a further embodiment, the kit of parts may additionally or alternatively comprise a number of separately packaged beverages, wherein the consumer will typically drink one beverage per day. Also within such a kit of parts may be found a composition for topical application to the skin, wherein the quantity of topical composition supplied is intended to provide for use by the consumer for the same number of days as beverages supplied.

0058The beverage may also be sold in a single package, from which it is intended the consumer drink a measured amount per day.

0059The topical composition may be provided in amounts which will allow for continued use of the topical composition beyond the time when the beverage has been used, similarly, more beverage may be provided so that this may continue to be consumed beyond the time when the supply of topical composition has run out.

0060Further, the kit of parts may comprise a packaged product in which different supplements are packaged together, optionally with use instructions, in order to achieve the benefits of the invention. For instance, the kit may comprise capsules, tablets pills and/or powders including separately or in combination salicylic acid, a C1 to C6 alkyl ester thereof and a salt thereof and a polyunsaturated fatty acid. It is possible that this kit would also include a topical composition.

0061A kit of parts would typically offer a product which could be used for from about 1 week to about 3 months, often about 2 weeks to about 2 months, most preferably for about 1 month.

0062Where the invention provides a composition for topical application this preferably comprises one or more anti-acne active agents.

0063Preferably, the anti-acne agent is selected from antibacterials, desquamators, keratolytics and retinoid boosters.

0064The anti-acne agents are preferably present in the topical compositions in an amount of from about 0.1% to about 20% by weight, more preferably from about 0.1% to about 10%, and most preferably from about 0.1% to about 5% by weight. Anti-acne agents may be single compounds or mixtures of two or more anti-acne agents.

0065Examples of anti-acne agents include salicylic acid, keratolytics such as sulfur, lactic acid, glycolic, pyruvic acid, urea, resorcinol, alkylresorcinols such as 4-hexylresorcinol, and N-acetylcysteine; retinoids such as retinoic acid and its derivatives (e.g., cis and trans); antimicrobials (including antibiotics and antibacterials), antifungals, antiprotozoals, and antivirals, for example, benzoyl peroxide, octopirox, erythromycin, tetracyclin, triclosan, azelaic acid and its derivatives, phenoxy ethanol and phenoxy proponol, ethylacetate, clindamycin and meclocycline, triclosan, chlorhexidine, tetracycline, neomycin, miconazole hydrochloride, parachlorometaxylenol, nystatin, tolnaftate, clotrimazole, and the like; sebostats such as flavinoids; hydroxy acids; antipruritic drugs including, for example, topically-acceptable salts of methdilizine and trimeprazine; and bile salts such as scymnol sulfate and its derivatives, deoxycholate, and cholate. The compositions may also comprise pantothenic acid or a pantothenic acid derivative, as described in <patcit id="pcit0007" dnum="US5612324A"><text>US 5,612,324</text></patcit>, the contents of which are incorporated herein by reference.

0066Preferred components of the topical composition are linoleic acid and/or salicylic acid (or a C1 to C6 ester thereof). Linoleic acid can be included in the composition as part of a natural oil, such as vegetable oil e.g., sunflower oil.

0067Also useful are non-steroidal anti-inflammatory drugs (NSAIDS). The NSAIDS can be selected from the following categories: propionic acid derivatives; acetic acid derivatives; fenamic acid derivatives; biphenylcarboxylic acid derivatives; and oxicams. All of these NSAIDS are fully described in the <patcit id="pcit0008" dnum="US4985459A"><text>U.S. Pat. No. 4,985,459 to Sunshine et al., issued Jan. 15, 1991</text></patcit>, incorporated by reference herein. Most preferred are the propionic NSAIDS including but not limited to aspirin, acetaminophen, ibuprofen, naproxen, benoxaprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen and bucloxic acid. Also useful are the steroidal anti-inflammatory drugs including hydrocortisone and the like.

0068Other examples of compounds that are useful as anti-acne agents, either alone or in combination with the anti-acne agents mentioned above, are retinoid boosters. Retinoid boosters are compounds that mimic the effect of retinoic acid on skin by enhancing the conversion of retinol or retinyl esters to retinoic acid. Retinoid boosters may be used singly or as combinations of two or more compounds. Retinoid boosters are described in <patcit id="pcit0009" dnum="WO0202074A"><text>WO 02/02074</text></patcit>, the contents of which are incorporated herein by reference. Specific retinoid boosters include, for example, ceramides, phosphatidyl choline, linoleic acid, 12-hydroxystearic acid and climbazole.

0069Optional components of the topical composition include preservatives, antioxidants, fragrances, clays, surfactants, gel-forming materials, silicones, emollients, humectants and pigments. These optional materials may be used singly or two or more of each type of materials may be used (for example, a composition may include two or more different clays). These optional components may be used in admixture e.g., a composition may contain a preservative, a fragrance and a clay.

0070Specific examples of optional components, some of which are also mentioned hereinafter, are alkyl alcohols containing from 12 to 24 carbon atoms, alkyl carboxylic acids containing from 12 to 24 carbon atoms, polyvinyl pyrrolidone, polyethylene glycol, mineral oil, polysorbates, nonionic surfactants, sorbitol, methyl cellulose, propylene glycol esters, zinc salts, titanium dioxide and mixtures thereof.

0071The following non-limiting examples illustrate the invention and do not limit its scope in any way. In the examples and throughout this specification, all percentages, parts and ratios are by weight unless indicated otherwise.

<u>EXAMPLES</u>

Example 1 - Anti-inflammatory synergistic effect between salicylic acid and DHA in human umbilical vein endothelial cells (Huvec's).

Outline of Experimental Approach

0072An <i>in vitro</i> model has been developed to investigate the impact of cytokine stress on the inflammatory status of endothelial cells. <ol id="ol0001" compact="compact"><li>a. Cells are grown in 6-well (9.5cm²) plates.</li><li>b. The cells are treated with 10 ng/ml Tumour Necrosis Factor alpha (TNF alpha).</li><li>c. Tissue culture supernatant and cell pellets were harvested at 24 hours (t24) post-TNF alpha treatment.</li><li>d. All tissue culture supernatant was assayed for Lactate Dehydrogenase (LDH), as a measure of cytotoxicity.</li><li>e. All cells were counted (Beckman Coulter Counter) and pelleted and cell lysate assayed for Intra Cellular Adhesion Molecule 1 expression (ICAM-1) and Prostaglandin E2 (PGE2).</li></ol>

Materials and Methods

Culture of Endothelial Cells

0073Huvec cells (Human umbilical vein endothelial cells, TCS Biologicals) were cultured and passaged in EGM-2 (Endothelial growth medium, Biowhittaker) supplemented with heparin, VEGF (vascular endothelial growth factor), gentamicin sulphate, ascorbic acid, HEGF (Human endothelial growth factor), hydrocortisone, HFGF-B (Human fibroblast growth factor B), R3-IGF-1 (long R insulin-like growth factor 1) and FBS (foetal bovine serum).

0074Cells were routinely plated out in 6-well tissue culture dishes, at a seeding density of about 5000 cells/cm² in 2ml complete medium/well, 24 hours before starting the experiment, and incubated at 37°C in 5% CO₂.

Addition of Test Solutions

0075Test solutions were prepared in EGM-2 containing all supplements except hydrocortisone. Endothelial cells were cells were treated for 24 hours with 10 ng/ml TNF alpha.

Harvesting Samples and Cell Number

0076Any change in cell morphology was noted before the cells were harvested. Both the tissue culture supernatant and the endothelial cells were harvested after addition of recombinant TNF alpha (t24). All tissue culture supernatants were stored at - 20°C.

00771 ml of trypsin/EDTA solution (Invitrogen 25300-054) was added to each well, and the plate incubated at 37°C until the cells detached. 50µl of this cell suspension was added to 9.95mls of Isoton II (Beckman Coulter) in an accuvette and 0.5ml of this suspension was counted twice in a Coulter Particle Counter Z1 with 140µm aperture.

0078The remaining 950 µl of original cell suspension was centrifuged at 13000 rpm in a microcentrifuge for 10 minutes. The supernatant was discarded and the cell pellet washed with 500µl of Dulbecco's PBS and centrifuged as before. The supernatant was discarded as before and cell pellet stored at -20°C prior to cell lysis. The number of cells per pellet was estimated from the Coulter Counter data.

Cytotoxicity Assay (Promega)

0079All tissue culture supernatant was examined for cytotoxicity using the Promega CytoTox 96 non-radioactive cytotoxicity assay. This assay quantitatively measures lactate dehydrogenase (LDH) released upon cell lysis and is a good indication of cell viability. 50 µl of tissue culture supernatant or control medium was added to duplicate wells of a 96-well microtitre plate. 50 µl of CytoTox reagent was added and mixed well. The plate was incubated in the dark, at room temperature, for 30 minutes. After this time 50 µl of stop solution was added to each well and the absorbance of the plate was read at 492nm. Any test samples giving an absorbance value of more than double that of the control medium was considered to be cytotoxic. No results have been included from samples that showed any signs of cytotoxicity.

Preparation of Cell lysate

0080All cell pellets were lysed on ice for 30 minutes in 1 ml cell lysis buffer per 2.5 x 10⁶ cells. The lysis buffer contained 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 6 mM sodium chloride and 0.05M Tris at pH 7.6. Protease inhibitor cocktail (1000X; Sigma P8340) was added prior to use at a level of 10 µl per ml of lysis buffer. The partially lysed cell pellets were completely homogenised with a pellet pestle and unwanted cell debris removed by centrifugation for 20 minutes at 20,000g at 4°C. The clarified cell lysate was frozen at -80°C until needed.

Total Protein Assay (Pierce)

0081The total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit. A set of eight standard solutions ranging from 0 to 1200 µg/ml protein was prepared from the supplied 2 mg/ml BSA stock solution. 10 µl of standard or cell lysate was added to duplicate wells of a flat-bottomed, 96-well microtitre plate. The reagent solution was prepared according to the kit instructions from 50 parts reagent A and 1 part reagent B. 200 µl of the final reagent was added to each well of the microtitre plate. The plate was mixed, covered and incubated at 37°C for 30 minutes and absorbance read at 562 nm. A protein standard curve was constructed and used to determine the protein concentration of each cell lysate.

ICAM-1 ELISA (R&amp;D Systems)

0082ICAM-1 protein in each cell lysate was estimated using the Human sICAM-1 DuoSet ELISA kit (R&amp;D Systems DY720) according to the manufacturer's instructions.

0083The capture antibody was diluted to a final concentration of 4 µg/ml in PBS and 100 µl was used to coat each well of a 96-well microtitre plate overnight at room temperature. The plate was then washed three times with wash buffer (0.05% Tween 20 in PBS). Each well received 300 µl of blocking buffer (1% BSA, 5% sucrose and 0.05% sodium azide in PBS); and the plate was incubated at room temperature for 1 hour before being washed as before. Each cell lysate was then diluted 1/200 in reagent diluent (1% BSA in PBS) and 100 µl added to duplicate wells of the antibody coated plate. Eight ICAM-1 standards were prepared in reagent diluent, at concentrations ranging from 0 to 1000 pg/ml, and duplicate 100 µl standards were added to the appropriate wells on the plate. A separate set of standards was routinely used for each plate. The plate was incubated at room temperature for 2 hours before being washed again. 100 µl of detection antibody, diluted to a final concentration of 100 ng/ml, was added to each well and the plate incubated at room temperature for 2 hours. The plate was washed as before. Each well received 100 µl of streptavidin-HRP conjugate dilutedl/200 in reagent diluent and the plate incubated at room temperature, in the dark, for 20 minutes. The plate was washed for the final time and 100 µl of substrate solution (1:1 mixture of colour reagent A and colour reagent B, R&amp;D Systems DY999) was added to each well. After 20 minutes incubation, at room temperature in the dark, the colour development was stopped by the addition of 50 µl of 2N sulphuric acid. The absorbance of the plates was measured at 450nm with the correction wavelength set at 570nm.

0084A standard curve was plotted of mean absorbance versus ICAM-1 concentration and the line of best fit calculated by regression analysis. The unknown concentration of ICAM-1 in the samples was calculated from this, taking the lysate dilution factor into account.

0085To normalise for differences in cell number and total protein concentration, the final result was expressed as ng ICAM-1 per mg of total protein.

Prostaglandin E2 High Sensitivity ELISA (R&amp;D Systems)

0086The PGE2 protein concentration of each tissue culture lysate was assayed using the DE2100 Human PGE2 assay (R&amp;D Systems) according to the manufacturer's instructions.

0087Eight PGE2 standards were prepared in calibrator diluent at concentrations ranging from 0 to 1000 pg/ml. 150 µl of assay diluent and 50 µl of tissue culture lysate or standard was added to duplicate wells. 50ul of PGE2 HS antibody solution was added to each well and incubated for 18-24 hours at 2-8 °C. The plate was then washed four times with wash buffer. 200 µl of pNPP substrate was added to each well and the plate incubated at room temperature for 1 hour at 37°C. 50ul of stop solution was then added to each well. The optical density of each well was determined using a microplate reader set to 405nM with wavelength correction set between 570nM and 590nM.

0088A standard curve was plotted of mean RLU versus PGE2 concentration and the line of best fit calculated by regression analysis. The unknown concentration of PGE2 protein in all the samples was estimated from this.

0089Salicylic acid (SA), acetyl salicylic acid (ASA) and DHA were purchased from Sigma Aldrich. <ul id="ul0001" list-style="none"><li><figref idref="f0001">Figure 1</figref> shows the dose response inhibition of ICAM-1 with DHA.</li><li><figref idref="f0001">Figure 2</figref> shows the synergy between salicylic acid and a fish oil (DHA) in human umbilical vein endothelial cells in terms of changes in PGE2.</li></ul>

Example 2 - Composition of the Invention

0090The following is an example of a composition of the invention. The composition can be prepared by combining the ingredients and homogenising the mixture. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="51mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><thead><row><entry valign="top"><b>Ingredient</b></entry><entry valign="top"><b>Weight %</b></entry></row></thead><tbody><row><entry>DHA</entry><entry>0.40</entry></row><row><entry>Cranberry juice (concentrate)</entry><entry>50</entry></row><row><entry>Vitamin C</entry><entry>0.17</entry></row><row><entry>Vitamin E</entry><entry>0.25</entry></row><row><entry>Lycopene</entry><entry>0.005</entry></row><row><entry>Beta-carotene</entry><entry>0.002</entry></row><row><entry>Soy isoflavones</entry><entry>0.03</entry></row><row><entry>Citric acid</entry><entry>0.18</entry></row><row><entry>Sweetener, thickener, emulsifier</entry><entry>q.v.</entry></row><row><entry>Water</entry><entry>To 100%</entry></row></tbody></tgroup></table></tables>

Example 3 - Two Part Composition of the Invention

0091A composition as described in Example 2 is packaged together with a composition for the topical treatment of skin which can have the following composition. <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><thead><row><entry valign="top"><b>Ingredients</b></entry><entry valign="top"><b>Weight %</b></entry></row></thead><tbody><row rowsep="0"><entry>Deionized Water</entry><entry>qs 100</entry></row><row rowsep="0"><entry>Ethanol (SD 40B Alcohol)</entry><entry>35.0</entry></row><row rowsep="0"><entry>Salicylic Acid</entry><entry>2.0</entry></row><row rowsep="0"><entry>n-Octyl Paraben</entry><entry>3.0</entry></row><row rowsep="0"><entry>Glycerol</entry><entry>2.0</entry></row><row rowsep="0"><entry>Disodium EDTA</entry><entry>0.005</entry></row><row><entry>Triethanolamine, 99%</entry><entry>0-1.0</entry></row></tbody></tgroup></table></tables>

Example 4 - Packaged Product of the Invention

0092The following is an alternative example of a composition of the invention. Which may be sold as a packaged product including dosage and use instructions. <ul id="ul0002" list-style="bullet"><li>The topical treatment of Example 3, intended for application twice daily;</li><li>Fish oil capsules each containing 1 gram of fish oil as a source of DHA and EPA at a recommended dosage of 4 capsules per day;</li><li>Cranberry powder as a source of salicylic acid at a recommended dosage of 6 grams per day;</li><li>Soy isoflavone capsules at a recommended dosage of 100 mg per day.</li></ul>