Background to the invention

Fatty acids play a vital role in the biosynthesis of hormone-like substances such as prostaglandins which regulate many body functions, including heart rate, blood pressure, blood clotting, fertility and conception. Individual fatty acids are known to have some antimicrobial activity, however, insufficient to be used in a medicinal composition. The inventors have thus identified the need to investigate fatty acid combinations which together would have a synergistic antimicrobial effect as opposed to any one of the fatty acids individually as an antimicrobial preparation Likewise, individual fatty acids are known to have some anti-inflammatory activity, however, insufficient to be used in a medicinal composition. The inventors have thus identified the need to investigate fatty acid combinations which together would have a synergistic antiinflammatory effect as opposed to any one of the fatty acids used individually as an antiinflammatory preparation.

Yet further, a need was identified to find a potentiator and/or enhancement reagent for the fatty acid composition.

It is with this need in mind that the inventors thus propose the following invention.

Summary of the Invention

According to a first aspect of the invention, there is provided a fatty acid composition , wherein the composition includes the constituent fatty acids, pentadecanoic acid, oleic acid, palmitic acid and linoleic acid in a ratio other than what is commonly found in nature.

It is to be appreciated from the specification that the pentadecanoic acid, oleic acid, palmitic acid and linoleic acid constituents are all classified as fatty acids and may be saturated or unsaturated fatty acids. A constituent amount of pentadecanoic acid of greater than 25 mol% may have an increased antimicrobial effect.

The constituent amount of the pentadecanoic acid in the composition may range from 25 mol% to 66 mol%. Typically, the constituent amount of the pentadecanoic acid in the composition may range from 30 mol% to 55 mol%,

The constituent amount of the linoleic acid in the composition may range from 1 mol% to 10 mol% of the composition. Typically, the constituent amount of the linoleic acid in the composition may range from 3 mol% to 7 mol%.

The constituent amount of the oleic acid in the composition may range from 10 mol% to 40 mol%. Typically, the constituent amount of the oleic acid in the composition may range from 16 mol% to 30 mol%.

The constituent amount of the palmitic acid in the composition may range from 12 mol% to 36 mol%. Typically, the constituent amount of the palmitic acid in the composition may range from 16 mol% to 25 mol%. In a desired embodiment of the invention, the constituent amounts of the pentadecanoic acid may be 46 mol%, the linoleic acid may be 4.5 mol%, the oleic may be 22 mol% and the palmitic acid may be 18 mol%.

In another embodiment of the invention, the constituent amount of the palmitic acid in the composition may be less than the constituent amount of the oleic acid in the composition.

In yet another embodiment of the invention, the constituent amount of the linoleic acid in the composition may be lower than the constituent amounts of any one of the other three constituents.

In a further embodiment of the invention, the constituent amount of the pentadecanoic acid may be the highest constituent amount in the composition.

The constituents of the composition may be derived from nature or may be synthetic.

The fatty acids, if derived from nature, may be reptilian in origin. The reptiles from which the fatty acids may be obtained may be from the family Crocodylidae and/or or Alligatoridae.

Thus, in an embodiment of the invention, the ratio of pentadecanoic acid: oleic acid: palmitic acid: linoleic acid may be 12:5:4:1 .

According to another aspect of the invention, there is provided the use of a fatty acid composition including at least pentadecanoic acid, oleic acid, palmitic acid and linoleic acid acids substantially as described above, as an antimicrobial medicament

According to yet another aspect of the invention, there is provided the use of a fatty acid composition including at least pentadecanoic acid, oleic acid, palmitic acid and linoleic acid acids substantially as described above, in the preparation of an inflammatory medicament According to a further aspect of the invention, there is provided an anti-inflammatory composition including at least pentadecanoic acid, oleic acid, palmitic acid, linoleic acid acids and a flavonoid-type substance.

The flavonoid-type substance may be quercetin, an analogue or an equivalent thereof.

The ratio of the flavonoid-type substance to the total of the fatty acid constituent may be greater than 1 :1 .

Thus, in an example, the quercetin to fatty acid ratio may be greater than 1 :1 , and may be as high as 2:1 and as low as 1 :2.

Description of an Example of the Invention

The invention will now be described, by way of non-limiting example only, with reference to the accompanying experimental work.

In the experimental work the relative ratios of the fatty acids and the quercetin are for illustrative purposes only and the scope of the invention is not intended to be limited to these or any specific ratio's.

1. Aim of the experimental Work The aim of this project was to determine the fatty acid combination starting with the fatty acid content of a natural oil, such as a fat or oil from Alligator mississippiensis, Crocodylus siamensis, or the Nile crocodile, having anti-inflammatory and antimicrobial activities. In addition, a flavonoid-type substance (quercetin) was combined with the fatty acids to investigate a possible potentiating effect.

2. Methods

2.1 Antimicrobial susceptibility assays

2.1.1 Microbial strains

The following microorganisms were used (obtained from American Type Culture Collection - ATCC, Manassas, VA, USA):

• Staphylococcus aureus (ATCC strain 12600)

• Klebsiella pneumonia (ATCC strain 13883)

· Candida albicans (ATCC strain 10231 )

S.aureus and K.pneumoniae were cultured in Mueller-Hinton broth (optical density of 0.6-0.8 at 490 nm) and C.albicans (optical density of 0.3-0.5 at 630 nm) in Sabouraud dextrose broth. Broths were prepared according to the manufacturer's protocols.

2.1.2 Susceptibility tests

Tests were conducted in clear, sterile 96 well microtitre plates (Corning Life Sciences, Acton, MA, USA). The assay was based on the microplate method by Eloff (1998), but with modifications. Briefly, following steps were followed:

• A 100 μΙ well of appropriate broth was added.

• This was followed by a 100 μΙ well of the appropriate microbial culture.

• And finally by a 100 μΙ well of the appropriate test compound.

· The plate was incubated at 37 ^e C for 18 {S.aureus and K.pneumoniae) or 24 hours {C.albicans).

• For S. aureus and K. pneumonia, 40 μΙ well of p-INT (400 μg/ml in water) was added and incubated (37 ^e C; 15 minutes).

• Microbial growth was quantified by colourimetry (490 nm) for bacteria and optical density (690 nm) for C.albicans in a microplate reader (BioTek ELx800, Winooski,

VT, USA). Test compounds were dissolved in DMSO and the final concentration in the wells was 2 % v/v. The positive controls were neomycin (S. aureus and K.pneumoniae) and amphotericin B {C.albicans) and the final concentrations in the wells were 100 μΜ. Crocodile oil was tested at final concentrations in the wells of 0, 5, 6, 7, 8, 10 and 15 % (w/v). Individual fatty acids (pentadecanoic acid, oleic acid, palmitic acid and linoleic acid acid) were tested at final concentrations in the wells of 0.0028, 0.0049, 0.038, 0.049, 0.38 and 0.49 M. For the combined fatty acids, pentadecanoic acid (at -46 % by content) was used as the reference compound and combined with oleic acid, palmitic acid and linoleic acid (i.e pentadecanoic acid: oleic acid: palmitic acid: linoleic acid at ratio of 12:5:4:1 ).

2.2 Anti-inflammatory assays

Ethical approval (003/09/Animal) from the University of KwaZulu-Natal Animal Ethics Subcommittee was obtained prior to the investigation of croton oil-induced oedema in a mouse model. Guidelines by the University of KwaZulu-Natal Animal Ethics Subcommittee and Biomedical Resource Unit for the maintenance and treatment of laboratory animals were followed. Young adult Balb/c mice weighing 29-32 g were obtained from the Biomedical Resource Unit of the University of KwaZulu-Natal. The mice were maintained under controlled laboratory conditions with free access to food and water and with a normal day/night cycle.

For the topical anti-inflammatory assays, the study design of Lopez and co-workers (Lopez A., Sims DE., Ablett RF., Skinner RE., Leger LW., Lariviere CM., Jamieson LA., Martinez- Burnes J., Zawadzka GG. (1999). Effect of emu oil on auricular inflammation induced with croton oil in mice. American Journal of Veterinary Resonance, 60: 1558-1561 ) was essentially followed, but with modifications. For the oral anti-inflammatory assays, the study designs of Ghannadi and co-workers (Ghannadi A., Hajhashemi V., Jafarabadi H. (2005). An investigation of the analgesic and anti-inflammatory effects of Nigella sativa seed polyphenols. Journal of Medicinal Food, 8:488-493) and that of Hong and co-workers (Hong M., Zhong S., Bian R. (2001 ). Tortoise oil, turtle oil, compositions containing them, their preparation processed and uses. United States Patent 6,296,979 B1 ) were implemented, but with modifications. Equal volumes of croton oil were mixed with acetone as vehicle and applied (50 μΙ; 1 hour) onto the inner surface of the right auricle or each mouse to induce oedema. The right auricle of each mouse was treated and the left auricle was used as the negative control and was left untreated. Diclofenac (0.07 mg; 3 hours) and fatty acids (0.23 mg pentadecanoic acid; 0.081 mg palmitic acid; 0.1 1 1 mg oleic acid and 0.0225 mg linoleic acid) in combination with quercetin (0.5 mg) was topically administered in 50 μΙ acetone and the mice treated for 3 to 6 hours. The combination of fatty acids corresponds with the ratio in which they were naturally found in the Nile crocodile oil. Acetone as a vehicle has not been documented to have an anti-inflammatory effect on its own. Mice were euthanized in a CO2 chamber after treatment for 3 to 6 hours. From each mouse, left and right auricle biopsy specimens were obtained with a 6 mm biopsy punch and then weighed on an analytical balance. 2.3 Data analysis

Data are reported as the mean ± standard error of the mean of at least four to five independent experiments with duplicate measurements. For the antimicrobial assays, microbial growth was quantified as a percentage of the control without any test compound. For the anti-inflammatory assays, oedema was quantified by calculating the difference in weights of the right and left auricle biopsy specimens and expressed as a percentage of the croton oil control. Statistical comparisons were made by one-way ANOVA followed by Bonferroni's post-test for multiple comparisons, or by Student's two-tailed paired f-test to determine P values. A value of P < 0.05 was considered significant.

3. Results

3.1 Microbial Susceptibility

Fatty acids have been previously documented to possess antimicrobial activity against a variety of pathogenic microorganisms. The relatively high concentrations of linoleic acid and oleic acid may be responsible for the antimicrobial activity of crocodile oil. The antimicrobial activity of the four fatty acids (pentadecanoic acid, oleic acid, palmitic acid and linoleic acid acid) found in crocodile oil were assessed against Staphylococcus aureus, Klebsiella pneumonia and Candida albicans, however, the pentadecanoic acid content of the fatty acid composition was increased from that found in nature in crocodile fat and oil.

The antimicrobial activities of the combination are depicted in Figure 1 . Interestingly, the activity was not potentiated but rather antagonised at the higher concentration range from 0.39 M onwards against all three species. This could be due to the lower solubility of the fatty acids at higher concentrations and support the comments of Kabara and co-workers (Kabara JJ., Swieczkowski DM., Conley AJ., Truant J. P. (1972). Fatty acids and derivatives as antimicrobial agents. Antimicrobial Agents and Chemotherapy, 2:23-28.) that it is considerably difficult to test lipids due to their insolubility.

Figure 1. Microbial susceptibility to combination fatty acids. The percentage growth of S. aureus (A), K. pneumonia (B) and C. albicans (C) was measured against increasing concentrations of a mixture of pentadecanoic acid, oleic acid, palmitic acid and linoleic acid in the ratio 12:5:4:1 . The concentration (M) of the reference fatty acid, pentadecanoic acid, is indicated in each figure. Data are the mean ± standard error of the mean of duplicate measurements from 4 independent experiments. Statistical comparisons of activity were by one-way ANOVA followed by Bonferroni's post-test; ^** , P < 0.01 ; ^*** , P < 0.001 versus no fatty acid combination control.

3.2 Anti-inflammatory activity It has been previously documented that linoleic acid reduced oedema in a mouse model of acute auricular inflammation, and that the anti-inflammatory activity was due to the inhibition of the production of pro-inflammatory eicosanoids from arachidonic acid (Dobner MJ., Sosa S., Schwaiger S., Altinier G., Delia Loggia R., Kaneider NC, Stuppner H. (2004). Antiinflammatory activity of Leontopodium alpinum and its constituents. Planta Medica, 70:502- 508). The relatively high concentrations of linoleic acid may therefore be responsible for the anti-inflammatory activity of crocodile oil. Thus, the topical anti-inflammatory activities of the combined fatty acids in which the pentadecanoic acid content had been increased beyond the ratio naturally found in crocodile oil were tested in a mouse model of acute auricular contact dermatitis and the results are depicted in Figure 2.

Figure 2. The topical anti-inflammatory activity of fatty acids in combination with quercetin. Activity was measured in a mouse model of acute contact dermatitis. Diclofenac (0.07 mg; 3 hours) and fatty acids (0.23 mg pentadecanoic acid; 0.081 mg palmitic acid; 0.1 1 1 mg oleic acid and 0.0225 mg linoleic acid acid) in combination with quercetin (0.5 mg) was topically administered in 50 μΙ acetone and the mice treated for 3 to 6 hours. After treatment, the reduction in swelling of the auricular biopsies was determined and the change in weight was expressed as a percentage. Data represents the mean ± standard error of the mean of duplicate measurements from at least 4 independent experiments. Statistical comparisons of activity were by one-way ANOVA followed by Bonferroni's post-test or by Student's two- tailed paired f-test to determine P values.: P < 0.001 versus croton oil only control; ###, P < 0.001 versus diclofenac. It was previously determined that 0.07 mg diclofenac for 3 hours treatment exhibited the optimal effect. 4. Discussion

From the results above it is evident that fatty acids in this combination that are not found in nature exhibit significant antimicrobial and anti-inflammatory activities. The combination of fatty acids with quercetin showed a tendency to further increase anti-inflammatory activity, suggesting potentiation. This anti-inflammatory activity was significantly more pronounced that that of the optimal dose of diclofenac. Many microbial infections are commonly associated with inflammation and therefore this combination may be useful as a substitute for a topically applied corticosteroid.

The inventors this believe that the invention has benefits and advantages in the treatment of microbial infections and inflammatory conditions.