The present invention relates to the use of pomegranate oil or a pomegranate oil derivative as a preservative for a lipid or lipid-containing composition. The present invention also relates to a composition comprising pomegranate oil or a pomegranate oil derivative and a lipid.

Lipids and, in particular, fatty acids, are often added to cosmetic and dermatological compositions because of their moisturizing and/or emollient properties. Lipids, such as non-conjugated fatty acids, however, can be susceptible to oxidation, particularly if stored at elevated temperatures. Compositions containing such lipids, therefore, have a tendency to degrade over time.

Preservatives may be added to such compositions to prevent or reduce their rate of degradation. An example of such a preservative is Vitamin E. Typically, Vitamin E is added at concentrations of 500 to lOOOppm and acts as an antioxidant for stabilizing the lipids in the composition.

Antioxidants have also been used as or in cosmetic compositions to impart anti-oxidant properties to the skin. Such anti-oxidants are believed to have anti-ageing properties and are typically employed in high concentrations as they are intended to impart anti-oxidant properties to the skin and not to the cosmetic composition itself.

It is among the objects of embodiments of the present invention to provide an alternative preservative for a lipid or lipid-containing composition. In particular, although not exclusively, it is among the objects of embodiments of the present invention to provide an alternative preservative for stabilizing or preserving a lipid or lipid-containing composition against oxidation, for example, at room temperature (for example from 15 to 20 ⁰ C) , or at temperatures above 40 ⁰ C.

According to a first aspect of the present invention, there is provided the use of pomegranate oil or pomegranate oil derivative as a preservative for a lipid or lipid- containing composition.

In the present invention, the pomegranate oil or pomegranate oil derivative is used as a preservative for the lipid or lipid-containing composition. The pomegranate oil or pomegranate oil preferably acts an antioxidant for stabilising the lipid or lipid-containing composition. Preferably, the pomegranate oil or pomegranate oil derivative acts as an antioxidant for stabilising the lipid or lipid-containing composition at a temperature above 40 ⁰ C. More preferably, the pomegranate oil or pomegranate oil derivative acts as an antioxidant for stabilising the lipid or lipid-containing composition at a temperature above 60 ⁰ C.

Pomegranate oil includes punicic acid or 9Z, HE, 13Z- octadeca-9, 11, 13-trienoic acid. This is a polyunsaturated fatty acid and, more specifically, a conjugated linolenic acid having three conjugated double bonds. Without wishing to be bound by any theory, the conjugated double bonds of punicic acid are believed to impart antioxidant properties to pomegranate oil and offer oxidative protection to, in particular, the fatty acids of other lipids in the composition, such as monounsaturated fatty acids e.g. palmitoleic acid, C16:1Δ ⁹ , oleic acid, C18:1Δ ⁹ , eicosenoic acid (20:1 Δ ⁹ ), erucic acid, 22:1Δ ¹³ or those containing non- conjugated fatty acids (e.g. linoleic acid, C18:2 Δ ^9>12 ; α-linolenic acid C18:3 Δ ^9>12>15 ,γ-linolenic acid C18:3 Δ ⁶ ' ⁹ ' ¹² or stearidonic acid (18:4 Δ ⁶ ' ⁹ ' ¹² ' ¹⁵ ) . The pomegranate oil or pomegranate oil derivative may also stabilise longer chain and more highly unsaturated fatty acids such as those found in pharmaceutical formulations and marine oils of fish or algal origin (e.g. arachidonic acid, C20:4 Δ ⁵ ' ⁸ ' ¹¹ ' ¹⁴ ) , eicosapentaenoic acid C20:5 Δ ⁵ ' ⁸ ' ^>14>17 ) and docosahexaenoic acid 22:6Δ ⁴ ' ⁷ ' ¹⁰ ' ¹³ ' ¹⁶ ' ¹⁹ )

Where pomegranate oil is used, it may be used in neat form or in the form of a solution or emulsion. If a solution is used, the pomegranate oil may be suspended or dissolved in a solvent, such as iso-octane, iso-hexane, n- hexane, chloroform or diethylether . If an emulsion is used, the pomegranate oil may be suspended in a liquid, such as aqueous-alcoholic mixtures. The concentration of pomegranate oil in the solution may be 1 to 10 weight %, preferably 1 to 2 weight %.

Where a pomegranate oil derivative (s) is used, it may be used in neat form or in the form of a solution or emulsion. If a solution is used, the pomegranate oil derivative (s) may be suspended or dissolved in a solvent, such as iso-octane, iso-hexane, n-hexane, chloroform or diethylether. If an emulsion is used, the pomegranate oil derivative (s) may be suspended in a liquid, such as aqueous- alcoholic mixtures. The concentration of pomegranate oil in the solution or emulsion may be 1 to 10 weight %, preferably

1 to 2 weight %

Where pomegranate oil is used, the pomegranate oil is preferably derived from a source that has been certified as organic.

Where a pomegranate oil derivative is used, the derivative is preferably derived from a natural source, such as pomegranate oil. The natural source may be one that has been certified organic. However, the pomegranate oil derivative may also be produced synthetically, although this is not preferred.

Preferably, the pomegranate oil derivative is punicic acid. More preferably, the pomegranate oil derivative consists essentially of punicic acid. The punicic acid may be extracted from natural sources, such as pomegranate oil. Alternatively, the punicic acid may be produced synthetically, although this is not preferred.

The pomegranate oil or pomegranate oil derivative may be employed in sufficient concentration to impart a preservative effect on the lipid or lipid-containing composition. Preferably, the pomegranate oil or pomegranate oil derivative may be employed in sufficient concentration to impart an antioxidant effect on the lipid or lipid- containing composition.

In one embodiment, the antioxidant effect may be determined by comparing the peroxide value (PV) of the lipid or lipid-containing composition with and without the pomegranate oil or pomegranate oil derivative. PV measures the amount of oxidising peroxide species in a sample in terms of the milliequivalents per kg (meq/kg) of sample that oxidises potassium iodide under a given set of conditions. PV may be determined by mixing the sample under test with an excess of potassium iodide and measuring the amount of iodine produced by titration, for example, with sodium thiosulphate . Standard methods include the Mylnefield standard operating procedure 040 based on the American Oil Chemists' Society protocol cdδ-53 and European Pharmacopoeia 5.0 (Method 2.5.5). Preferably, the pomegranate oil or pomegranate oil derivative is employed in sufficient concentration to reduce the PV of the lipid or lipid- containing composition by at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 40%. In a preferred embodiment, the pomegranate oil or pomegranate oil derivative is employed in sufficient concentration to reduce the PV of the lipid or lipid- containing composition by at least 50%, preferably by at least 60%, for example, by 70 to 90%. This reduction in PV may be observed at room temperature (for example from 15 to 2O ⁰ C) , and/or at a temperature of above 40 ⁰ C, preferably above 60 ⁰ C.

The weight ratio of pomegranate oil or pomegranate oil derivative to lipid is 1-10/100, preferably 1-5/100 more preferably 1-2/100. In one embodiment, the pomegranate oil or pomegranate oil derivative is used at a concentration of up to 100000 ppm, preferably up to 25000 ppm, more preferably up to 10000 ppm. The pomegranate oil or pomegranate oil derivative may be used as a preservative for any lipid. Typically, such lipids are derived from vegetable sources and include oils derived from seeds and nuts. The lipids preferably comprise fatty acid(s) and, in particular, monounsaturated fatty acids and non-conjugated fatty acids. Examples of such lipids include tri-, di- and mono-glyceride (s) . More preferably, the fatty acid is a triacylglycerol containing either monounsaturated fatty acids or at least one non- conjugated fatty acid. The non-conjugated fatty acid is selected from the group consisting of e.g. linoleic acid, C18.-2 Δ ⁹ ' ¹² ; α-linolenic acid C18:3 Δ ⁹ ' ¹² ' ¹⁵ , γ-linolenic acidC18:3 Δ ⁶ ' ⁹ ' ¹² or stearidonic acid (18:4 Δ ⁶ ' ⁹ ' ¹² ' ¹⁵ ) . The monounsaturated fatty acid may be e.g. palmitoleic acid, C16:lΔ ⁹ , oleic acid, C18:lΔ ⁹ , eicosenoic acid (20:1 Δ ⁹ ), erucic acid, 22:1 Δ . The pomegranate oil or pomegranate oil derivative may also stabilise longer chain and more highly unsaturated fatty acids such as those found in pharmaceutical formulations and marine oils of fish or algal origin (e.g. arachidonic acid, C20:4 Δ ⁵ ' ⁸ ' ¹¹ ' ¹⁴ ) , eicosapentaenoic acid C20:5 Δ ⁵ ' ⁸ ' ¹¹ ' ¹⁴ ' ¹⁷ ) and docosahexaenoic acid22:6Δ ⁴ ' ⁷ ' ¹⁰ ' ¹³ ' ¹⁶ ' ¹⁹ .

Where the pomegranate oil or pomegranate oil derivative is used in a lipid-containing composition, the composition may be a cosmetic composition, food composition, beverage, cleaning and/or pharmaceutical composition. Preferably, the composition is a cosmetic composition, such as a skin cream, skin lotion, shampoo, conditioner, body wash, lip balm, lipstick, hair gel, hair cream, or hair mousse composition. The lipid may be included in such compositions to impart moisturising or emollient properties to the composition. According to a second aspect of the present invention, there is provided a composition comprising pomegranate oil or a pomegranate oil derivative, and a lipid, wherein the weight ratio of pomegranate oil or pomegranate oil derivative to lipid is 1-10/100.

As mentioned above, the composition may be a cosmetic composition, food composition, beverage, cleaning and/or pharmaceutical composition. Preferably, the composition is a cosmetic composition, such as a skin cream, skin lotion, shampoo, conditioner, body wash, lip balm, lipstick, hair gel, hair cream, or hair mousse composition. The lipid may be included in such compositions to impart moisturising or emollient properties to the composition.

The composition of the second aspect of the invention may contain lipid in a concentration of 1-10% w/w.

The concentration of pomegranate oil or pomegranate oil derivative in the composition may be 1 to 10%, preferably 1- 2% w/w. Preferably, this concentration is not sufficient to provide any detectable anti-ageing or antioxidant effects to, for example, the skin, lips or hair.

These and other aspects of embodiments of the present invention will now be described with reference to the following Examples.

Examples Table 1

OiI type Peroxide value (Meq/kg) Time (Oh) (48h)

Borage

Neat oil 7.9+0.1 43.3±l.β

Oil + 750ppm Vitamin E 23.8±2.5

Oil + 1% pomegranate 10.2+0.4

Moringa Neat oil 2.2+0.2 30.7+2.0

Oil + 750ppm Vitamin E 25.4+2.0

Oil + 1% pomegranate 18.7+2.9

Macadamia

Neat oil O.Q±O.O 3.6±0.4

Oil + 750ppm Vitamin E 3.8+0.1

Oil + 1% pomegranate 1.9+0.1

Oils were exposed to air for up to 48h and the peroxide value determined as given below. The results in Table 1 indicate that a significant reduction in peroxides is achieved by addition of l%pomegranate oil to the sample oil. This effect is better than that achieved by the addition of a commercial antioxidant, vitamin E supplied at standard conditions of 750ppm (ppm = parts per million)

The procedure below may be used to determine the peroxide value of a lipid composition.

Procedure

Reagents and Materials

1. Acetic acid-chloroform solution (3:2, v/v)

2. Saturated ^" potassium iodide (KI) solution.

3. Sodium thiosulfate solution 0.1 N (0.1 M) or 0.01N (0.01M) .

4. Starch indicator solution (Ig in 200ml water).

Detailed method

1. Weigh accurately approximately 2.5Og of sample into 150 ml conical flask, and record weight

2. Add 30 ml acetic acid: chloroform (3:2) mixture and swirl to dissolve sample. Add 0.5 ml saturated KI solution using a dispensing pipette.

3. Stopper and allow to stand for exactly 1 minute, swirling gently approximately every 20 seconds, i.e. three times, during the minute. Immediately add 30 ml distilled water and a white stirrer bar.

4. Add 5 ml starch solution and place conical flask onto stirrer plate and turn control knob so contents stir gently.

5. Record the first burette reading.

6. Titrate with 0.01 N sodium thiosulfate, adding gradually with constant stirring to liberate iodine from the solvent layer until the blue colour just disappears. NOTE:- If the titre is more than 5ml it is necessary to repeat the analysis using 0. IN sodium thiosulfate .

7. Record second burette reading.

8. Carry out this determination in triplicate.

9. Conduct a blank titration of the reagents for every batch of analysis conducted. The blank titration should be carried out using the same concentration of sodium thiosulfate as used for the samples. The blank should not exceed 0.1 ml of 0.1N sodium thiosulfate or 0.2 ml of 0.01N sodium thiosulfate. If it does, prepare fresh reagents before proceeding with analyses. NOTE:- If more than one concentration of sodi-um thiosulfate has been -used to analyse the samples, then a blank titration must be carried out for each concentration.

Calculation of Peroxide Value

1. Calculate the peroxide value as indicated: 2. Peroxide value = (S-B) x N x 1000 wt of sample, g

(milli-equivalents peroxide per 1000 g)

where: a) B = titre of blank, ml; b) S = titre of sample, ml; c) N = normality of sodium thiosulfate solution.