Oral anti-parasitic composition

Abstract

The present invention relates to a composition for use as an oral anti-parasitic, the composition comprises one or more of an essential oil which contains gamma-terpinene, and/or carvacrol, and/or thymol, and/or terpinenol, and/or eucalyptol and/or eugenol, an essential oil of genus Cinnamomum, Eugenia, Eucalyptus or a source of saponin. It also relates to a method of preventing or treating parasitic infection in an animal, the method comprising orally administering to said animal a composition comprising one or more of an essential oil which contains gamma-terpinene, and/or carvacrol, and/or thymol, and/or terpinenol, and/or eucalyptol and/or eugenol, an essential oil of genus Cinnamomum, Eugenia, Eucalyptus or a source of saponin.

Claims

1. A pet consumable comprising a single oral anti-parasitic composition, the single oral anti-parasitic composition consisting of a source of saponin, an essential oil of oregano and an essential oil from genus Cinnamomum or from genus Eugenia, wherein the essential oil of oregano and the essential oil from genus Cinnamomum or Eugenia are present in the anti-parasitic composition in a ratio of from 4:1 to 1:4, and wherein the anti-parasitic composition exhibits a greater proliferation inhibition with respect to Neospora caninum or Toxoplasma gondii than the combined proliferation inhibition of the individual components.

2. The pet consumable of claim 1, wherein the source of saponin contains a steroid sapogenin.

3. The pet consumable of claim 1, wherein the consumable is a foodstuff, and wherein the foodstuff comprises 70% to 90% moisture.

4. The pet consumable of claim 1, wherein the consumable is in the form of a liquid.

5. The pet consumable of claim 1, wherein the consumable is a foodstuff, and wherein the foodstuff comprises 5% to 15% moisture.

6. The pet consumable of claim 5, wherein the foodstuff comprises 10-40% protein or 5-40% fat.

7. The pet consumable of claim 6, wherein the foodstuff is in the form of a biscuit.

8. The pet consumable of claim 5, wherein the consumable is in the form of a tablet.

9. An anti-parasitic composition, consisting of an essential oil of oregano and an essential oil from genus Cinnamomum or genus Eugenia, and optionally, a source of saponin, wherein the essential oil of oregano and the essential oil from genus Cinnamomum or Eugenia are present in the anti-parasitic composition in a ratio of from 4:1 to 1:4, and wherein the anti-parasitic composition exhibits a greater proliferation inhibition with respect to Neospora caninum or Toxoplasma gondii than the combined proliferation inhibition of the individual components.

10. The pet consumable of claim 9, wherein the single oral parasitic composition consists of an essential oil of oregano and an essential oil from genus Cinnamomum or genus Eugenia.

11. A method of preventing or treating parasitic infection in an animal, the method comprising orally administering to said animal an anti-parasitic composition consisting of a source of saponin, an essential oil of oregano and an essential oil from genus Cinnamomum, or from genus Eugenia, wherein the essential oil of oregano and the essential oil from genus Cinnamomum or Eugenia are present in the anti-parasitic composition in a ratio of from 4:1 to 1:4 and wherein the composition exhibits a greater proliferation inhibition with respect to Neospora caninum or Toxoplasma gondii than the combined proliferation inhibition of the individual components.

Description

(1) The invention will now be further described by way of reference to the following Examples and Figures, which are provided for the purpose of illustration only and are not to be construed as being limiting on the invention.

(2) FIG. 1: shows the 7 solutions of essential oils and source of saponin used. Each solution has been labeled A through to H.

(3) FIG. 2A: shows a schematic diagram of the proliferation protocol followed. FIG. 2B shows fluorescence microscopy of HFF cells infected by different parasitical inoculums of Toxoplasma gondii.

(4) FIG. 3: shows results of a test of inhibition of the proliferation of Neospora caninum parasite in the presence of different essential oils at different concentrations. Summary of results and IC50 are set out in Table 1.

(5) FIG. 4: shows comparative results of IC50 of 7 essential oils (oregano (E) tested at 50 ppm and 100 ppm) and the cell viability.

(6) FIG. 5: shows results from a test of inhibition of proliferation of Toxoplasma gondii with a cocktail of essential oils EB (Oregano and Clove).

EXAMPLES

(7) The study aimed to determine an anti-parasitic activity from seven essential oils on the intracellular microorganism Neospora caninum, but also combinations of these drugs on Toxoplasma gondii. The work focused on the capacity of proliferation of the parasite in the presence of these solutions containing the essential oils and invasion after pre-treatment.

(8) Seven solutions of essential oils and one solution with a source of saponin were tested (see FIG. 1: A to H).

(9) In order to solubilize the essential oils, the oils were diluted in dimethyl sulfoxide (DMSO) and then spread in the cell culture medium (DMEM) that was supplemented with 10% fetal calf serum (D10). DMSO was also used at a final concentration not exceeding 0.1%. Separate cultures of both parasites, Toxoplasma gondii and Neospora caninum, were prepared in vitro in human cell type HFF (Human Foreskin Fibroblasts), grown at 37° C. under 5% CO2 in a D10 medium. Both cultures were similar, the inoculum used for the various tests were the same, 105 parasites per well. It was observed that growing Neospora caninum is slower than that of Toxoplasma gondii.

Example 1: Proliferation Assay of Neospora caninum Parasite in the Presence of Essential Oils

(10) Proliferation of the parasite in the cells is measured by counting the specific radioactivity of the incorporated tritiated uracil and compared to the control without essential oil (100% proliferation). The test is performed in 24-well plates and the whole test lasts about 24 hours. The positive control is achieved using pyrimethamine (ICSO: 0.1 μg/mL). FIG. 3 shows ICSO of each essential oil solution tested at different concentrations in the presence of Neospora caninum.

(11) Table 1 (below) summarises the results from example 1 in FIG. 3. Values represent inhibition of proliferation. The bracketed results show the ICSO of each essential oil tested.

(12) TABLE-US-00001 Essential 12.5 25 50 100 Oil ppm ppm ppm ppm A [43.7%] 84.3%   95.3% 94.5% (Cinnamon) B   21.0% [58.7%] (Clove) C  4.6%  −3.1% [42.2%] (Winter savory) D  6.0%   [43.8%] 41.1% (Thyme) E 26.7%   [53.9%] 75.9% (Oregano) F  −1.6% 23.9% (Eucalyptus) G    7.9% 18.7% (Tea tree) Essential 200 400 800 Oil ppm ppm ppm A (Cinnamon) B 78.0% (Clove) C 85.2% (Winter savory) D (Thyme) E (Oregano) F 15.0%   20.7% 69.5% (Eucalyptus) G 11.6%   24.5% (Tea tree)

Example 2

(13) The inhibition of proliferation was measured by incorporating tritiated uracil in both Toxoplasma gondii and Neospora caninum and the viability of HFF cells was assessed using MTT assay. The results are shown in FIG. 4.

(14) FIG. 4 shows for each essential oil, having a concentration of proliferation inhibition properties for each of the parasites while maintaining cell viability HFF near 80%.

(15) It can be observed that oils having a pesticidal activity, the IC50 concentrations obtained from Toxoplasma gondii are the same as for Neospora caninum. However, F and G oils have little or no action on the inhibition of the proliferation of Neospora caninum.

Example 3: Proliferation Assay of Toxoplasma gondii in the Presence of a Set of Combinations of Essential Oils

(16) The set of essential oils combinations are: AB, AE, AD and BE.

(17) For each combination a 3 time 3 factorial design was drawn. The theoretical IC50 is adjusted to the measured inhibition proliferation rate and then is used as the basis to make dilutions and to make the subsequent 9 different ratios of the 2 essential oils combined in a cocktail.

(18) ##STR00001##

(19) For each association, 9 tests of the inhibition of proliferation of Toxoplasma gondii were performed (all data not shown). Again, the inhibition of proliferation was measured by incorporation of tritiated uracil.

(20) Cocktail BE (see FIG. 5): Shows the synergistic effect of cocktail BE on Toxoplasma gondii proliferation.

(21) Essential oil B at 100 ppm alone corresponds to an IC58 (58% of proliferation inhibition). Essential oil E at 75 ppm alone corresponds to an IC88 (88% of proliferation inhibition). Assuming an independent relationship between the two oils on proliferation inhibition, a cocktail of essential oil B at IC58/2=IC29 with the essential oil E at IC88/4=IC22 leads to an expected additional effect of IC22+IC29=IC51 (51% of proliferation inhibition). The measured of proliferation inhibition of such a cocktail (B at IC22 combined to E at IC29) revealed a surprising measurable proliferation inhibition of 87% (IC87). This highly significant difference clearly highlights a synergistic effect of the cocktail BE on Toxoplasma gondii proliferation.

(22) Bilan Cocktail

(23) Table 2 (below) includes all the results on the inhibition of the proliferation of Toxoplasma gondii in the presence of the 36 batches of essential oils.

(24) TABLE-US-00002 IC 50/4 IC 50/4 IC 50/4 IC 50/2 IC 50/2 IC 50/4 IC 50/2 IC 50 IC 50/4 IC 50/2 AB 16.4% 34.3% [63.5%] 16.7% 39.3% AE 45.5% [53.3%] [85.7%] 23.1% [53.5%] BE 27.7% [52.1%] [79.6%] [87.2%] (93.4%) AD 19.4%  0.3% 31.9% [16.0%] 20.2% IC 50/2 IC 50 IC50 IC 50 IC 50 IC 50/4 IC 50/2 IC 50 AB [63.2%] 47.6% [75.2%] [82.4%] AE [86.0%] [57.0%] [67.0%] (91.9%) BE [81.5%] [73.8%] [85.2%] (96.4%) AD [53.5%] [54.1%] [60.0%] [69.6%]

(25) Values represent inhibition of proliferation. Bracketed results show the combinations of inhibiting between 50-90% of the proliferation of the parasite. The results in parentheses shows those for inhibiting the proliferation of more than 90%.

CONCLUSION

(26) Various tests on the two parasites have yielded highly similar results. The combination of essential oils has in some cases increased the capacity of inhibiting the proliferation of the compounds. This suggests that essential oils can act synergistically. This is particularly true for the combinations AE (data not shown) and BE, where the measured effect of the cocktail is significantly higher than the expected additional effect of both essential oil taken alone. It is also important to note that all items tested had no cytotoxic effects on HFF cells (monolayer integrity).